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Independent regulation of alanine and arginine transport in human intestinal epithelial cell line Caco-2

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Title:
Independent regulation of alanine and arginine transport in human intestinal epithelial cell line Caco-2
Creator:
Pan, Ming, 1963-
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Language:
English
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vii, 272 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Caco 2 cells ( jstor )
Cell growth ( jstor )
Cell membranes ( jstor )
Epithelial cells ( jstor )
Incubation ( jstor )
Kinetics ( jstor )
Neutral amino acids ( jstor )
Protein synthesis ( jstor )
Receptors ( jstor )
Alanine -- metabolism ( mesh )
Alanine -- physiology ( mesh )
Arginine -- metabolism ( mesh )
Arginine -- physiology ( mesh )
Biological Transport, Active -- physiology ( mesh )
Caco-2 Cells ( mesh )
Cell Differentiation ( mesh )
Cycloheximide -- pharmacology ( mesh )
Department of Physiology thesis Ph.D ( mesh )
Diffusion -- physiology ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Physiology -- UF ( mesh )
Epidermal Growth Factor -- pharmacology ( mesh )
Phorbol Esters -- pharmacology ( mesh )
Transforming Growth Factor alpha -- pharmacology ( mesh )
Translation, Genetic -- physiology ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 262-271).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Ming Pan.

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University of Florida
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University of Florida
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Copyright Ming Pan. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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49673956 ( OCLC )
028070221 ( ALEPH )

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INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2



















By

MING PAN


















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1993




































This dissertation is dedicated to my wife Jun, and my parents.















ACKNOWLEDGEMENTS


I would like to thank Dr. Bruce Stevens, chairman of my supervisory committee, for his tremendous support in both my professional and personal life. Words can hardly express my deep appreciation. Working under his supervision has been a wonderful experience.

I would also like to thank members of my supervisory committee, Dr. Edward Copeland, Dr. George Gerencser, Dr. Michael Kilberg, and Dr. Souba for their support and valuable suggestions.

I would like to express my appreciation to Dr. Colin Sumners for providing the cell culture equipment through out my study. I would also like to thank Ms. Tammy Gault, for teaching me cell culture technique and sharing the cell culture equipment.

I would like to take this opportunity to thank all the faculty members of the Physiology Department for making graduate study an enjoyable experience.

Finally, I specially thank my wife and my family for their unconditional support and great family value.







iii
















TABLE OF CONTENTS



ACKNOWLEDGEMENTS. ................ iii

ABSTRACT. . . ........ .. . ... ... vi

CHAPTERS

1 INTRODUCTION ................... 1

1.1 Introduction ........ ........ 1
1.2 Amino Acid Absorption in the
Small Intestine . . . ..... . . 2
1.3 The Human Intestinal Epithelial Cell
Line (Caco-2 Cell Line) ... . . 20
1.4 The Objective and Aims of the
Present Study . . ..... . . 22

2 GENERAL METHODOLOGY ....... . . . . 25

2.1 Caco-2 Cell Culturing. ......... 25
2.2 Caco-2 Monolayer Transport .... .... 31
2.3 Monolayer Transport in Caco-2 Cells
Grown on Porous Filters. .. . . . 35
2.3 Statistical Analysis .. . . . . 39

3 CLASSIFICATION OF ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE ..... 40

3.1 Introduction . . . ...... . . 40
3.2 Methods and Materials. ..... ... 43
3.3 Results. . . . . . . . . . 44
3.4 Discussion . . . . . . . . 50
3.5 Summary . . . . . . . . 58

4 CLASSIFICATION OF ARGININE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE . . . 113

4.1 Introduction ... ..... . . . .. 113
4.2 Methods and Materials. ... ...... . . 115
4.3 Results. . . . . . . . . . 116
4.4 Discussion ................ 120
4.5 Summary. . . . . . . . . . 123


iv









5 THE EFFECTS OF INDIVIDUAL AMINO ACID ON THE
SYSTEM B AND SYSTEM y TRANSPORT ACTIVITIES . 156

5.1 Introduction .............. 156
5.2 Methods and Materials ..... . . .. 157
5.3 Results. . . . . . . . ... 158
5.4 Discussion . ............ . 165
5.5 Summary. ................. 170

6 THE EFFECTS OF PEPTIDE GROWTH FACTORS ON THE
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES . 199

6.1 Introduction ................ 199
6.2 Methods and Material ... .. . ... 202
6.3 Results. .......... ....... 204
6.4 Discussion ................. 206
6.5 Summary. ............ ..... 209

7 THE EFFECTS OF PHORBOL ESTERS ON THE SYSTEM B
AND SYSTEM y TRANSPORT ACTIVITIES . . . 223

7.1 Introduction ......... ..... 223
7.2 Methods and Materials. .... . . . 224
7.3 Results. ..... . . . . . . ... 225
7.4 Discussion ................ . . 231
7.5 Summary. ..... . . . . . . . 233

8 SUMMARY AND CONCLUSIONS ........ . . 258

8.1 Summary. ..... . . . . . . . 258
8.2 Conclusions............ . . . 260

REFERENCE LIST. ............... .. ... 262

BIOGRAPHICAL SKETCH .......... . ..... 272




















v














Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2 By

Ming Pan

May 1993

Chairperson: Bruce R. Stevens
Major Department: Physiology

Membrane transporter systems serving arginine (Na'independent system y+) and alanine (Na -dependent system B) were investigated in the human intestinal Caco-2 cell line. The uptake kinetics were different for each transport system. For each system, the Va, was greater in undifferentiated cells compared to differentiated cells, while the K, values were each unaffected by cell differentiation status. Amino acid substrates unique to System y+ acutely stimulated only system y activity, while substrates unique to system B only stimulated system B activity. For each transport system, the ranking of amino acid stimulation was directly correlated with the degree of competitive analogue inhibition (assessed by Dixon analysis). The prolonged substrate induction of system B activity, but not system y+ activity, was prevented by the protein synthesis inhibitor cycloheximide. Peptide growth vi








factors epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-) each stimulated system B and system y' activity following a lag period of several hours. EGF/TGFe activation was abolished by cycloheximide, or by inhibitors of protein kinase C. Phorbol esters stimulated system B and system y+ activity following a lag period of several hours, and this stimulation was prevented by cycloheximide and inhibitors of protein kinase C. For each transport system, EGF, TGF-, and phorbol ester increased the V,, but not the K,. Together these data suggest that (1) Caco-2 epithelial differentiation status is associated with regulation of amino acid transport; (2) amino acid transporter system B and system y are regulated independently; (3) amino acid substrates upregulate their own transporter's activity via transstimulation or by a mechanism involving de novo protein synthesis; (4) EGF and TGF- likely activated protein kinase C in the up-regulation of system B and system y+ activity via a de novo protein synthesis mechanism.

















vii













CHAPTER 1
INTRODUCTION


1.1 Introduction


Amino acid transport by the small intestine is a vital process involved in whole-body nitrogen balance. Intestinal absorption studies have received considerable attention during the past 35 years, primarily focussing on description of the site of absorption within the mucosa, classifications of uptake pathways based on substrate selectivity, and phenomenological kinetic mechanisms of transporter function using the universal principles of membrane transport (Hopfer, 1987; Munck, 1981; Stevens et al., 1984, Stevens, 1992a,b). Past and current studies were conducted in a variety of species and at various stages of development. These studies have included patients and intact animals with inborn errors of transport (Desjeux et al., 1980), perfused intestines, isolated membranes from the brush border and basolateral surfaces (Stevens et al., 1982, 1984; Mircheff et al, 1879, 1980), and enterocytes freshly isolated from mucosa (Reiser and Christiansen, 1971a,b,c), or grown as in vitro cell lines (Pan et al., 1991).

This project concentrates on amino acid absorption from the outer environment; the role of the intestine in inter1








2

organ amino acid flow is beyond the scope of this project. The small intestine is unique in extracting amino acids, in contrast to other internal organs. The enterocyte amino acids transport systems, especially those at the brush border membrane are unique in substrate and modes of regulation. Unlike the amino acid transport substrate adaptive downregulation universally observed in many internal organs, substrates in the small intestine up-regulate their transporter activities in vivo studies (Diamond, 1991; Stevens, 1992a,b).


1.2 Amino Acid Absorption in the Small Intestine


Like the membrane transport processes in internal organs, small intestinal amino acid transport has been studied qualitatively and quantitatively based on uptake phenomena and non-equilibrium thermodynamic principles.

The small intestinal mucosa separates the outer environment from the internal milieu. The intestinal mucosa is composed of a single layer of historically polarized epithelial cells (enterocytes) which are joined by a tight junction to form a continuous layer. Amino acid transport across the mucosa is mainly a trans-cellular phenomenon. The epithelial villous cells are responsible for amino acid absorption (Munck, 1981; Wilson, 1962). Along the crypt-villus axis the enterocytes originate from stem cells within the crypt. The undifferentiated immature cells rapidly proliferate








3

and migrate up the villi to become the mature villous cells. The well-developed enterocytes stay at the villus tip for several days, and then are shed away to the intestinal lumen. The location of greatest individual amino acid absorption differs among species along the oral-aboral axis of the small intestine (Diamond, 1991; Stevens, 1992a,b). The lumen to blood absorption involves the movement of amino acid through a series of aqueous and membrane compartments (Hopfer, 1987; Stevens, 1992a,b). Each compartment acts as a barrier which influences the overall amino acid movement across all the compartments. The brush border apical membrane of these enterocytes is the initial active step regulating the flow of amino acids from the intestinal lumen into the cell cytosol.


1.2.1 Lumen to Blood Amino Acid Movement


Beginning in the lumen, amino acids travel through an unstirred water layer, the apical membrane, the cytosol, the basolateral membrane compartments, and finally capillary endothelium. Each compartment determines the "real" amino acid concentration reaching the following compartment.

The unstirred water layer is about 50 Am thick layer and is composed of water and mucous/glycocalyx. Amino acids diffuse across the unstirred water layer. The amino acid that diffuses across the unstirred layer then reach the apical or brush border membrane. This plasma membrane is a bilayer phospholipid structure separating the cell cytosol environment








4

from the lumen. Amino acids cross surface by a simple passive diffusion plus some carrier-mediated transport mechanisms. In some instances, the amino acid is transported via a secondary active transport mechanism against its own electrochemical gradient across the membrane. The accumulated amino acids in the cytosol then exit via passive diffusion or/and carriermediated transport mechanism at the basolateral membranes. Each of the barriers can influence the rate of the amino acid absorption: the "true concentration" of the amino acids reaching the brush border membrane is determined by the amount of substrate within the unstirred water layer, rather than the bulk phase concentration present in the lumen. The brush border membrane and the basolateral membrane possess biochemically and histologically different structures. Some of the amino acid transport systems at the brush border membrane are not found in the basolateral membrane and the other internal organs (Stevens, 1992a,b). On the other hand, the basolateral membrane possesses many of the same transporters as the membrane found at other internal organs.


1.2.2 Modes of the Amino Acid Transport in the Small Intestine


The membrane amino acid transport movement is classified into two general categories: simple passive diffusion and carrier-mediated transport. The carrier-mediated transport is further divided to Na -independent facilitated transport and Na+-dependent secondary active transport








5

mechanisms.

In the case of the simple passive diffusion, the membrane electrochemical gradients and permeability coefficients of the amino acid govern the direction and rate of the passive amino acid movement across the membrane.

In the case of the Na+-independent carrier-mediated transport mechanism, amino acid is carried by its specific transporter across the membrane, directed by the electrochemical acting across the membrane. In the case of charged amino acids such as arginine, the negative membrane potential can drive it against its chemical gradient.

In the case of the secondary active transport, a series of spatially separated events occur that couple energy derived from ATP hydrolysis to solute flux. The Na'/K-ATPase at the basolateral membrane creates electrochemical Na' and K+ gradients across the basolateral and the apical membranes. The Na+/amino acid cotransporters at the apical membrane bind to amino acids, and utilize this Na+ electrochemical energy (AANa) for concentrative uptake of amino acids across the apical membrane. The accumulated amino acids inside the enterocytes exit via the Na -independent facilitated transporters and the passive diffusion at the basolateral membranes.

A kinetic model of Na/amino acid transport describing the secondary active transport has been recently examined Stevens (Stevens, 1992a) as a paradigm for all Na+-dependent systems.








6

This model describes an prefer-ordered mechanism with the Na* activator ion binding preferentially first to the cis transporter conformation, and this binding increases the affinity for amino acid binding to the cis side of the transporter. Amino acid then binds to transporter. The ciscomplex isomerizes to place Na and amino acid on the trans side, and the substrates are released to the cytoplasm by either a random or ordered sequence. The trans transporter conformation isomerizes back to cis-transporter conformation. The overall rate-limiting step is the isomerization of the two transporter forms (cis and trans). In the absence of trans amino acid, the influx is predicted by: (Stevens & Wright, 1987): jA = (JA.ax -[A])/K3 + [A], where jA = amino acid flux, J',ax = maximal flux rate, and the apparent affinity K, for solute A is a function of both YN, (the apparent dissociation constant for dissociation and binding):

K, = ((KINa/Na])n + (nKa/J[Na]) + 1) KA

where KA is the apparent amino acid-carrier dissociation constant, and n is the Hill coefficient describing the number of Na ions coupled to movement of each amino acid molecule. Note that regulation of the transporter activity could conceivably occur by modifying J,,x (i.e, activity of the functional transporter), or apparent K, (which includes the Na affinity and substrate modulation).


1.2.3 The Amino Acid Transport Systems in the Small Intestinal Membrane








7

Christensen and colleagues developed the original criteria to discriminate different amino acid transport systems in mammalian cells (Christensen, 1975, 1984, 1985 & 1990) through substrate specificity, ion-dependency, transport kinetics, and numerous other characteristics. Many facilitated and Na'-dependent secondary active transport systems such as Systems A, ASC, L and y which were first described in nonepithelial cell were found in many cell types including intestinal epithelial cells (Kilberg et al., 1993; Stevens, 1992a).

Much work has been conducted in the amino acid transport system classification at the tissue, cellular, and membrane vesicle levels in the intestine (Munck, 1981; Stevens et al., 1984; Hopfer, 1987; Stevens, 1992a,b). The major tools for the membrane transport system classification are (1) substrate preference; (2) ion-dependency; (3) substrate uptake kinetics,

(4) patterns of analogue cross inhibition of amino acids, and

(5) specific renal and intestinal inborn amino acid malabsorption syndromes (Stevens et al., 1984; Wright et al., 1986, Kilberg et al, 1993). Certain amino acids transported by a single transporter are used to test for the presence of the characteristic transporter. For example, methylaminoisobutyric acid (MeAIB) and pipecolic acid are thought to be transported only through the System A and the System IMINO, respectively (Christensen, 1975; Stevens & Wright 1985, 1987; Wright 1985). Due to variations among the








8

animal species, the stages of development, the tissue studied, and the methods used in amino acid transport systems studies, many different systems have been reported in the small intestine (Munck, 1981; Stevens et al., 1984; Hopfer, 1987; Stevens, 1992a,b). With some conflicts, there is a similarity in the amino acid transport systems among different species. The functionally and biochemically distinct brush border membrane and basolateral membrane possess different transport systems. The compiled membrane transport systems at the brush border membrane and basolateral membrane will be discussed individually.

Several distinctive transport systems are found only at the brush border membrane (Kilberg et al., 1993; Stevens et al., 1982, 1984, Stevens, 1992a,b). One of these is System B. System B is described as a strictly Na-dependent system broadly selective for the dipolar (neutral) amino acid alanine, serine, cysteine, glutamine, and interacts with 2amino-2-norbornanecar-boxylic acid (BCH) and threonine. At first it was named System NBB "Neutral Brush Border" (Stevens et al, 1982, 1984; Stevens, 1992a,b). System B is characteristically similar to System Bo,+ described in blastocyte (Van Winkle et al., 1985). Both System B and System BO,+ broadly transport dipolar amino acids, except that System BO,+ interacts with cationic amino acids, while System B is not interactive with cationic amino acids. System B has been reported to exist in the apical membrane of rabbit (Stevens et








9

al, 1984), pig (Maenz et al., 1992), dog (Bulus, 1989), human fetal (Malo, 1991), lower vertebrate small intestines (Ahearn et al., 1991), and in the undifferentiated and differentiated enterocytic Caco-2 cells (Pan et al, 1991; Souba et al., 1992). Another distinct amino acid transport system found only at the intestinal apical membrane is System IMINO (Stevens & Wright, 1985, 1987; Wright et al, 1985; Stevens, 1992a,b). System IMINO is also a strictly Na -dependent system highly selective to heterocyclic imino acid such as proline and pipecolate. System IMINO uptake has been reported in many species intestine (Ahearn et al., 1991; Karasov et al., 1986, 1987; Moe et al., 1987; Munck, 1983; Stevens et al., 1984, 1992a,b).

Nat-dependent transport Systems A and ASC (Christensen et al., 1965; Oxender et al., 1963; Kilberg et al., 1981, 1993), serve dipolar amino acid in the non-epithelial cell membrane, and reportedly exist in the guinea pig apical membrane (Del Castillo & Muniz, 1991; Hayashi et al., 1980). But up to date, no definite test has been able to discriminate them from the System B or System IMINO. Other Na+-dependent systems, XAG serving D-aspartate and glutamate, 0 serving P-alanine and taurine were also reported in the intestinal apical membrane (Hofper, 1987; Munck, 1990, 1992; Miyamoto et al., 1990a,b). Some studies also suggested possible existence of a System N for glutamine transport (Salloum et al., 1990, 1991).

There are three Na-independent transport systems at the








10

intestinal apical membrane. One is System L (Christensen et al, 1963, 1969, 1975) which transports large neutral amino acids and favors lipophilic substrates such as phenylalanine, leucine, and BCH. System L excludes #-alanine. A second is System y+ (Christensen, 1964, 1966) which prefers cationic amino acids such as lysine and arginine, although it tolerates the substrate combination of sodium plus neutral amino acids such as homoserine. A third is System bo,+ (Van Winkle et al, 1985, 1987, and 1988) which serves neutral amino acids and cationic amino acid, and interacts with BCH.

Recent cloning of cDNA encoding System y+ (Kim et al., 1991; Wang et al., 1991) provides a breakthrough in the membrane transport systems studies. It is possible to study membrane transport systems using the traditional phenomenological method as well as more advanced molecular biological methods. The finding of the same protein serving as both the System y+ transporter and a retrovirus receptor not only make the possible molecular studies of membrane amino acid transporters, but the physiological or pathological relationships among the nutrient absorption and cell functions in healthy and disease states. Recent cloning of cDNAs NAA/D2, rBAT, and F4 for putative regulatory protein for Systems bo,+, y+ or Bo,+ (Betran et al., 1992; Magagnin et al, 1992; Tate et al., 1992; Wells et al., 1992a,b) were also reported.

All amino acids passively diffuse across the apical membrane with their permeability rates directly proportioned








11

to their hydrophobicity. The order of permeability diffusion coefficients is phenylalanine > P-alanine > mannitol > alanine > MeAIB > proline > glycine > lysine (Stevens et al., 1982,1984). At high luminal amino acid concentration, the passive diffusion may be the predominant transport ways in the intestine. The carrier-mediated transport systems may be the favored route at lower concentrations.

In contrast to the apical membrane, all the basolateral membrane amino acid transport systems studied also exist in other non-epithelial membrane. These include Systems ASC and A, and Nat-independent Systems y+ and L, plus simple passive diffusion. The characteristics of these systems are as the same as those described in the apical membrane.


1.2.4 The Ontogenetic Developments of the Amino Acid Transport


Developmental studies of amino acid transport in the small intestine of various species has demonstrated that the timing and class of amino acid transporter appearance differs not only among the animal species but also at the various development stages of the same specie (Buddington & Diamond, 1989, 1990). Both herbivores and omnivores prefer high protein diet in their youth, a period when the absorption of essential amino acids is high. Because of some amino acids are in higher demand in adults making these conditionally essential amino acids. This is the case for arginine in puppies. On the oralanal axis, one dramatic change is in colon, whereby adults








12

colon only transports electrolytes and water, but neonatal and fetal colon possesses many amino acid transport systems. On the crypt-villous axis, the youth cope with the higher amino acid transport load by creating a large surface area that increase nutrient uptake non-specifically. The enterocyte turnover rate of the villus tip is slower, the crypt cell migration is greater, and the crypt cells possess transport ability, all of which contributes to the increased mass of intestine possessing transport activity. Two mechanisms were proposed (Buddington & Diamond, 1989, 1990) to explain the control of amino acid transporter expression during animal development: (1) an external control mechanism by dietary changes in substrate or by some growth factors in diet (e.g. epidermal growth factor stimulating transport by enterocyte, paracrine/autocrines, secreted by salivary gland or Bruners glands or from food source such as milk); (2) an internal genetically hard-wired control mechanism that controls change independent of external environment.


1.2.5 Requlation of Intestinal Amino Acid Transport


The intestinal membrane amino acid transport systems are regulated by various factors, such as the animal development regulation discussed above, certain physiological states like pregnancy, or certain pathological states like disease diabetes, hyperthyroidism. Much attention was given to the regulation of transport activity at certain stage of








13

development by systemically circulating factors like hormones, or by the luminal composition like transporter substrates. The intestinal apical membrane amino acid transport regulation by transport substrate has been studied in vivo as described below.

Unlike other internal organs, in vivo studies show that the activities of the intestinal amino acid transporters are up-regulated by the dietary substrates they transport (Stevens, 1992a,b; Salloum et al 1990; Sharrer et al, 1981; Stein et al, 1987; Ferraris et al, 1988a,b; Diamond & Karasov, 1987; Ferraris & Diamond, 1989; Diamond, 1991). The substrates' unique pattern of up-regulation, their amplitude, and selectivity of each system's activity indicated that individual amino acid transporters are regulated independently by dietary substrates. Non-essential caloric amino acids upregulate their transporter activities with increasing substrate. The essential, but potentially toxic amino acids regulate their transporter systems' activities in a different pattern. That is, at lower substrate concentration, the transporter activity decreases as substrate concentration increase; at higher concentration the transport activity increases as substrate concentration increases. A similar pattern is observed for sugar and dietary carbohydrate. The transport of non-essential amino acids is increased more by the dietary protein than that of the more toxic essential amino acids. This supports the notion that the absorption of








14

glucose, caloric and catabolic amino acids, and essential amino acids (possibly toxic if in excess amount) is regulated independently in vivo, which provides needed nutrients for the entire organism and which prevents substrate toxicity (Diamond, 1991). The mechanism of this induction has not been addressed hereafter.

Amino acids differ in their potencies to induce the same transporter. Although substrates generally make good inducers of their own transporters, there are some discrepancies (Levine, 1991; Diamond, 1991) between the inducers and transported substrates: transport unrelated amino acid is the best inducer.

The change in substrate-related transport activity is a relatively slow process. An increase in the luminal substrate level induces an reversible transport uptake capacity increase over the existing absorbing capacity by 2- to 10- fold within 24 hours. The substrate-specific up-regulation of nutrient absorption is directly related to the level of these substrate in the intestinal lumen. Lowering substrate levels causes the intestinal absorption capacity to decrease back down to the baseline level that appears to be genetically hard-wired (reviewed by Diamond, 1991). The down-regulation is a slow process (eg. 3 days for proline transporter in mouse).

Two mechanisms were proposed to explain the substraterelated intestinal amino acid uptake activity. The first is mucosal hyperplasia resulting in nonspecific uptake increase








15

(Laganiere et al, 1986; Diamond, 1991). Nontoxic nutrient exposure can induce a non-specific hyperplasia of the epithelium (increase epithelial cell numbers and size) and lengthen the villi to provide more absorbing capacity for all nutrients (Laganiere et al, 1986). The second explanation is that individual transporter activities are selectively increased as a result of the modification of transporter or/and increase the copies of transporters (James et al., 1987; Stein et al., 1987; Diamond et al., 1987; Scharrer et al., 1981) by the exposure of specific transport substrate.

In addition to the substrate regulation of transport activity, often conditions such as corticosteroid treatment and the conditions related to diabetes, hyperthyroidism, neoplasia, and pregnancy and lactation can induce intestinal mucosal hyperplasia (James et al., 1987; Levine, 1991).

In addition to absorbing nutrients for whole body needs, the small intestine enterocytes also require amino acids for their own proliferation, growth and differentiation. Epithelial cells rapidly turnover as enterocytes continuously migrate up from the immature proliferating crypt cells to become mature villous enterocytes along the crypt villous axis. The supply of amino acids by membrane transport may be the rate-limiting step in the rapidly proliferating and protein synthesizing in undifferentiated cells (Seitz et al, 1989). Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-) also stimulate epithelia proliferation and








16

growth (Carpenter & Wahl, 1990). The relationship between the cell proliferation and the membrane amino acid transport is not clear.

Substrates not only regulate their absorptive activity, but are also vital to enterocyte health. Glutamine, transported via intestinal System B (Souba et al., 1992), is essential in preserving the intestinal mucosa (Souba, 1990). Glutamine deficiency cause impairment of intestinal mucosal barrier function (Souba et al, 1990). In this sense, glutamine regulates its transport activity through preserving a healthy state, in addition to its direct regulation of transport activity.


1.2.6 Molecular and Cellular Models of Amino Acid Transport Regulation


Several amino acid transport regulation models have been proposed for nonintestinal cell types. However, the knowledge of intestinal membrane amino acid transport regulation is still lacking.

One model for substrate adaptive regulation of System A in hepatocytes was proposed by Kilberg (1986). The model is based on the assumption that the rate of repressing System A independent of substrate concentration. The System A transporter protein synthesis process is controlled at the transcriptional level as a consequence of the equilibrium between positive and negative regulating factors: in the absence of extracellular amino acid and/or in the presence of








17

hormone, System A associated protein synthesis is stimulated (depressed), while in the presence of elevated intracellular amino acid levels, System A-associated protein synthesis is repressed by a regulatory protein.

Another model for adaptive regulation of System A in CHOK, cells (chinese hamster ovary cell) was proposed by Englesberg group (1986). This model suggested that System A is regulated by at least two regulatory genes, R1 and R2. R1 produces an apopressor/inactivator (apo-ri) that is in equilibrium with a repressor/inactivator (ri). The elevated transported amino acids shift the apo-ri to ril which inhibits the transcription of the gene encoding System A transporter, and converts the existing transporter to an inactive state. R2 produces a constitutive repressor r2 which also negatively regulates the gene A. Insulin binds to its receptor and through an unknown pathway converts r2 to its inactive form. The absence of transported substrate and the presence of insulin have a synergistic effect on stimulating System A activity.

One study in a kidney cell line (MDCK) indicates the involvement of protein kinase C in System A regulation (Dawson & Cook, 1987).

Even though System A adaptive regulation was intensively studied, the molecular and cellular mechanisms of intestinal transport regulation of any amino acid are still unknown. The recent cloning of the System A cDNA (Kong et al., 1993) will








18

encourage regulation studies.


1.2.7 The Effects of Peptide Growth Factors in the Small Intestine


As members of the peptide growth factors family, Epidermal Growth Factor (EGF) and/or Transforming Growth Factor (TGF) each stimulate cell proliferation, protein synthesis, and cell differentiation in many cell types including the intestinal epithelial cells (Morrisset & Solomon, 1991; Carpenter & Wahl, 1990).

EGF is a 53 amino acid polypeptide, while TGF- is a 48 amino acid peptide. TGF- is structurally and biological functionally similar to the that of EGF. EGF is normally present in the intestine lumen from endogenous secretions from the salivary glands, the small intestinal Brunner's glands, autocrine/paracrine sources from the mucosa, or from exogenous sources such as milk and colostrum (Gaull et al.,1985; Britton, 1988; Potter, 1989). The sites for the EGF secretion to blood stream is unknown.

EGF and/or TGF- binds to the same EGF receptor in the plasma membrane, which is a member of the tyrosine kinase receptor family. The activated growth factor-receptor complex immediately phosphorylates the receptor itself and phosphorylates other substrates such as erb B2, ras oncogen, polyoma middle T antigen, or phospholipase C (PLC). The activated PLC alters inositol phospholipid metabolism leading to a elevated level of diacylglycerol (DAG) (Berridge, 1985;








19
Edelman et al, 1987; Klip & Douen, 1989), which activates intracellular protein kinase C. PK-C activates a series of unresolved mechanisms that ultimately result in cell division, proliferation and differentiation (Saier et al., 1988).

EGF receptors appear at both the luminal and basolateral membranes at a density gradient greater in immature crypt cells and less dense in villous enterocytes along the crypt -> villous axis (Hidalgo et al., 1989). This parallels the high proliferation rate in undifferentiated crypt cells (Pamukcum & Owens, 1991). Two-thirds of the EGF receptors are reportedly in the basolateral membrane (Reviewed by Brand, 1990). EGF/TGF- stimulate small intestinal epithelial proliferation, protein synthesis, and crypt cell maturation and migration toward villous tip cells. Recently, EGF receptor mRNA was identified in developing intestinal epithelial cells (Koyama & Podolsky, 1989). The EGF receptors reportedly existed at the apical and basolateral membranes of the human intestinal epithelial Caco-2 cell line (Hidalgo et al., 1989), with higher density in the undifferentiated cells compared to the differentiated cells. Two-thirds of the receptors expressed at the basolateral membrane. The Y, of the EGF receptors is 0.67 nM in Caco-2 cells (Hidalgo et al., 1989). Experiment data in our laboratory indicate that functionally EGF or TGF- each stimulates Caco-2 cell alanine and arginine transporter activities with similar potency when applied to either brush border or basolateral surfaces.








20

Even though the structure and biological functions of EGF and TGF- have been widely studied, the EGF/TGF- effects on intestinal amino acid transport has not been addressed.


1.3 The Human Intestinal Epithelial Cell Line (Caco-2 Cell Line)

The established intestinal epithelial cell line Caco-2 is derived from human colon adenocarcinoma cells (Fogh et al., 1977). It was originally used for in vitro colonic tumor studies.

Caco-2 cells can been grown on both solid plastic and porous filters for many sub-cultural generations. When grown on a solid surface, the Caco-2 cells form a confluent monolayer with tight junction and dome formation. Under normal cell culture conditions the confluent cells undergo a spontaneous enterocytic differentiation process (Pinto et al., 1983; Rousset et al., 1985). The biochemical and historical characteristics of the undifferentiated cells resemble those of the immature enterocytes, while the differentiated cells resemble the mature small intestinal epithelial cells. The differentiated Caco-2 cells become polarized, forming brush border apical membranes complete with peptide and carbohydrate hydrolases normally found as small intestinal apical marker enzymes. The enzymes include sucrase-isomaltase, lactase, trehalase, aminopeptidedase N, dipeptidylpeptidase IV, yglutamyltranspeptidase, and alkaline phosphatase (Pinto et al., 1983; Hauri et al., 1985; Rousset et al., 1985). The








21

undifferentiated sub-confluent cells are morphologically and biochemically equivalent to immature crypt cells, and differentiated post-confluent cells are undistinguished both morphologically and enzymatically from mature villus tip enterocytes (Hidalgo, 1988, 1989, 1990). The Caco-2 undifferentiated sub-confluent -> differentiated postconfluent state developmental steps mimic the enterocytes crypt -> villous maturation process. Many studies favorably recognize the Caco-2 cell line as an ideal in vitro analog of normal small intestine enterocytes (Zweibaum et al., 1983, 1991).

Organic solute transport studies on Caco-2 cells have revealed the same characteristics as those from other in vitro and in vivo small intestinal preparations (Blairs et al, 1987; Mohrmann et al, 1986; Nicklin, 1992). A few studies have been conducted regarding glutamine and proline transport characteristics in Caco-2 (Nicklin et al., 1992; Souba et al., 1992). These studies paralleled to those in other intestinal preparations.

In addition to characteristics indistinguishable from enterocytes, Caco-2 cells excel in providing a well-controlled homogenous population over a prolonged life span during cell development and differentiation. Uncontrolled adverse systemic factors found in vivo preparation are eliminated in the cell culture systems, so that the effect of a single variable can be studied in an unbiased setting. The Caco-2 cell line makes








22

it possible to study the nutrient transport and associated regulation over the enterocytes' entire developmental period. Nonetheless, Caco-2 cells are not entirely normal small intestinal epithelial cells. However, until the normal small intestinal epithelial cell model is established, the Caco-2 cell line provides the best in vitro human intestinal enterocyte model.


1.4 The Objective. Hypothesis, and Aims of the Present Study


1.4.1 The Objective


The overall objective of this in vitro study is to investigate the cellular basis of amino acid transport regulation in undifferentiated and differentiated states of a human intestinal epithelial cell line (Caco-2 cell line). This project concerns independent transporters serving structurally distinct amino acid substrates in the apical membrane of Caco2 cells.


1.4.2 The Hypothesis


The hypothesis is that alanine and arginine are independently transported by discrete transporter systems in the Caco-2 apical membrane, and that the transporter activities are independently regulated in mature enterocytes and during enterocyte development. Dipolar L-alanine is transported via Na+-dependent secondary active transport System B, while cationic L-arginine is transported via Na+-








23

independent Systems y in Caco-2 cells grown on solid surface or on porous membrane filters. We further hypothesize that the membrane's constitutive activities for System B and System y each decrease over time during Caco-2 enterocyte differentiation and development. The activity of each transport system can be up-regulated above the constitutive level by two categories of regulating agents: (i) substrate analogues served by each transporter, and (ii) the peptide growth factors epidermal growth factors (EGF) and transforming growth factor (TGF-). Finally, we hypothesize that upregulation of transporter activities occurs in two phases: an acute phased characterized by protein-synthesis-independent substrate trans-stimulation, and a chronic prolonged phase that likely involves protein kinase C and de novo protein synthesis.


1.4.3 The Specific Aims


Aim 1: To kinetically classify the alanine and arginine transport systems in the Caco-2 apical membrane and to examine the changes in the constitutive baseline transporter capacities of the sodium-dependent alanine transporter (System B) and the sodium-independent arginine transporter (System y+) during the Caco-2 epithelial development and differentiation.

Aim 2: To examine the acute and the prolonged phases of individual amino acid substrates in increasing System B and System y+ transporter capacities, in undifferentiated and








24

differentiated states.

Aim 3: To examine the roles of peptide growth factors TGF- and EGF and protein synthesis in changing System B and System y activities in undifferentiated and differentiated states.

Aim 4: To examine the role of cellular protein kinase C in regulation of System B and System y by substrate or TGF=/EGF.














CHAPTER 2
GENERAL METHODOLOGY


2.1 Caco-2 Cell Culturing


The human intestinal epithelial Caco-2 cell is derived from human colon adenocarcinoma cells. The cells can be grown as a monolayer on both porous filters and plastic. Under normal cell culture conditions, Caco-2 cells can be subcultured for many generations. Some labs reports 90 or more passages. Caco-2 cell growth on the plastic surface is dependent on cell density. Cells divide horizontally, and cell attachment does not stop the cell growth as normally seen in cell culture. The attached cells continuously divide at a lower rates. Days later (depends on cell density, with higher cell density having a shorter turnover), the attached cells become confluent. The confluent state is represented by the cell to cell tight junction and by dome formation. The dome is caused by the unidirectional transport and trapping of water and electrolyte cross cell monolayer. Unique in the Caco-2 cells, the confluent Caco-2 cell undergo a spontaneous enterocytic differentiation process without changing cell culture conditions. The differentiating cells start to polarize by forming apical and basolateral membranes, with expression of the normal small intestinal epithelial cell 25








26

apical membrane marker enzymes on the cell membrane. To date, the biochemical and histological tests indicate that differentiated Caco-2 cells are quite similar to, but exactly like normal small intestinal epithelial cells. The timing and cellular characteristics associated with the differentiation process of the Caco-2 cells resemble those of the normal crypt to villous cell development. Caco-2 cells are a human colon tumor transformed cell line having 106 chromosomes. The unmistakable similarity of the histological and biochemical characteristics makes the Caco-2 cell line a ideal model for the in vitro analogue of the normal adult intestinal epithelium.

The confluency and differentiation of Caco-2 cell states are cell-attachment dependent. After trypsinization, the attached polarized cells detached to become single nonpolarized cells, and lose their differentiated characteristics. Whether this process is a de-differentiation, or simply a turning off of existing differentiation expression, is still debatable. The daughter cells of these de-differentiated or undifferentiated cells then undergo another un-differentiation-confluency-differentiation process. Regardless the states of parent cells, newly divided daughter cells are undifferentiated. To ensure that the majority of cells are at the undifferentiated state, we have used cells only in their relative early generations (#19-50). The limited time of subculturing also reduce the possibility of mutation.








27

Cell culture studied have their advantages and limitations. On the positive side, cell culture provide a uniform environment. It is a relatively simple and straight forward preparation without the adverse effect of in vivo preparation. The experimental conditions are controllable. On the negative side, the cell line is not entirely normal cells. Furthermore cell culture conditions are not those of the in vivo physiological conditions, and there is the possibility of mutation. The cell conditions after subculturing may be different from the in vivo state. Despite the limitation of the cell culture, the Caco-2 cells are still considered to be an excellent model for adult intestinal epithelial study (Pinto et al., 1983; Hidalgo et al., Zwebaum et al., 1991)

For my study, transport studies in Caco-2 cells were performed in both the undifferentiated (age day 2-3) and the differentiated (age day 8-9) cells of the same subcultured batch of cells. In some cases in other cell ages (mentioned in text). Cell culture techniques are based on established procedures (Hidalgo et al., 1989; Blairs et al., 1987; Mohrmann et al., 1986; Pinto et al., 1983) and our modifications. The Caco-2 cells used for the present experiments were between the cell sub-cultured passages #19 #50.


2.1.1 Materials

The established human intestinal epithelial cell line








28

Caco-2 was obtained from American Type Culture Collection, Rockville, MD. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, sodium bicarbonate, penicillin, streptomycin, non-essential amino acids, Trypsin/EDTA, and Dimethyl sulfoxide(DMSO) were from Sigma Co., St. Louis, MO. The 6-well Falcon tissue culture dishes and 100 mm tissue culture dishes were obtained from the Fisher Scientific, Pittsburgh, PA. The 0.2 AM medium filters were from Millipore Co. Bedford, MA. The 0.4 Am 24 mm Costar's Transwell-COL collagen treated microporous membrane filters (Catalog # 3425) were from Costar Co. Cambridge, MA. [3H]-Alanine, [3H]-arginine, [3H]-glutamine and [3H]-Threonine were obtained from Amersham Co., Arlinton Heights, IL. [3H]-=-methyl aminoisobutyric acid was from American radiolabled chemicals Inc., St. Louis, MO. NaC1, choline Cl, KC1, MgSO4, KH2PO4, CaC12, NaOH, and N-(2hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES)/tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma Co., St.Louis, MO. Liquiscint scintillation fluid was from National Diagnostics, Atlanta, GA. The protein assay reagent was obtained from Bio-Rad Lab, Richmond, CA. Glacial acetic acid was from Fisher Scientific, Pittsburgh, PA. The scintillation counter and spectrophotometer were from Beckman, Irvine, CA.


2.1.2 Caco-2 Cell Culture


The Caco-2 cells were routinely grown on the 100 mm








29

falcon tissue culture dishes in 15 ml Sigma's Dulbecco's Modified Eagle Medium (DMEM; Sigma Co, St. louis, MO) containing 4.5 g/l glucose and 0.584 g/l glutamine, and supplemented with: 10% fetal bovine serum (Sigma Co, heat untreated catalog # F 4884)), 3.7% sodium bicarbonate, 100 IU/ml penicillin, 100 Ag/ml streptomycin (Sigma Co., St louis, MO) and 1% non-essential amino acids (Sigma Co. St. Louis, MO). The original seeding cell density was 3 x 105 cells/ml, cells were counted using a hemacytometer. Cells were grown in a humidified incubator at 370C in 10% C02/90% 02. The day of seeding was designated as day 0. The growth medium was changed and cells were inspected daily.

2.1.2.1 Caco-2 cell subculturing

For the sub-culturing cells, cells four days old on the 100 mm dish were taken out of the incubator. The growth medium was aspirated, and cells were washed once with 37*C isotonic calcium-free saline solution containing 0.05% trypsin and 0.02% ethylenediamine-tetraacetic acid (EDTA; Sigma Co. St.Louis, MO) and immersed in 10 ml the same solution for 5 minutes in the sterile hood. The cell/trypsin mixture was dispersed with a narrow tip glass pipette (Fisher Scientific, Pittsburgh, PA) and the trypsin reaction was stopped by adding DMEM with 10% FBS. Then the dispersed cells were sedimented in a sterile conical centrifuge tube (Corning, Corning, NY) at 1000 x g for 5 minutes, and the supernatant was removed. Next, growth medium was added to re-suspend the cells (using a








30

narrow tip glass pipette) until cells were separated. Possible cell clumps were allowed to settle for a few minutes at 1 x g, and only the top layer of medium containing single clumped cells was used for sub-culture as confirmed by microscope examination. Cells then were seeded in three ways: (1) seeded in the 100 mm dish at a cell density of 3 x 105 cells/ml for future sub-culture purpose, (2) seeded in the 35 mm Falcon tissue culture dishes (6-well clusters; Becton Dickinson, Lincoln park, NJ)) at a density of 1.93 x 105 cell/ml for subsequent transport experiments, and (3) seeded in 24 mm porous filter at a cell density of 1.93 x 105 cells/ml for subsequent transport experiments. All cell culture procedures were performed under sterile conditions in a hood. All solutions used in cell culturing were filter-sterilized (0.2 AM membrane filter; Nalge, Rochester, NY; Millipak 20, Millipore Co, Bedford, MA), or/and autoclave sterilized. The growth medium, including the medium in the both upper and lower chambers of porous filter (Costar Co, Cambridge, MA), was changed daily. The cultures are inspected daily (using a phase contrast microscope) to monitor cell growth (dome formation, absence of contamination, etc).

2.1.2.2 Freezing Caco-2 cells

Four-day-old Caco-2 cells grown in the 100 mm dishes were trypsinized and centrifuged as described in above subculturing section. Cells were then re-suspended in 4*C DMEM containing 10% FBS and 5% dimethyl sulfoxide (DMSO; Sigma Co, St.Louis,








31

MO). One milliliter of the cell/medium mixture (10 million cells/ml) was then transferred into a sterile glass ampule or a Nunc plastic tube (Inter Med, Denmark), which was later sealed in a sterile hood. The sealed ampules and the Nunc tubes were immersed into a 4*C methyl alcohol freezing tank (Fisher Scientific, Pittsburgh, PA). The freezing tank was then placed into a -70*C freezer for 72 hours before the ample/tube were transferred into liquid nitrogen storage.

2.1.2.3 Re-thawing frozen Caco-2 cells

Sealed ampules containing the frozen Caco-2 cells from the American Type Culture Collection or liquid nitrogen storage were immediately immersed into a 37*C water bath until the ampule content was completely thawed. And the sealed ampules were immersed into 70% (v/v) alcohol for a minute. The following procedures were then performed inside a sterile hood. The ampules containing cells were opened and cells were transferred and suspended in 37*C DMEM containing 10% FBS. The cells were then sedimented in a conical centrifuge tube at 1000 x g for 5 minutes, and seeded in the 100 mm cell culture dishes following the procedures described in the above subculturing section. The re-thawing cells were grown for at least two subculturing generations before being used in any experiment.


2.2 Caco-2 Cell Monolayer Transport


The Caco-2 cell form a monolayer on both the plastic








32
surface and porous filters. For the pre-confluent cells, the junctions among cells are loose, and cell membrane has not polarize yet. The cell uptake may involve the membrane, excluding the portion attached to the plastic surface. For the differentiated state, cells have already polarized with basal membranes attached to plastic surface or filter; lateral membranes are formed beneath the tight junctions which connect the apical membranes. The apical surface faces the outer environment. Organic solutes enter cells through the apical membrane, so that the para-cellular pathway is minimal. This has been confirmed by [3H]-inulin extracellular studies (Arturson et al., 1992).

The membrane transport of amino acid is a bi-directional process. The measured transport activity is the net influx of transport equivalent to vectorial difference between the two unidirectional fluxes. The rate of the net is therefore determined by the total flux during a period of time during which the flux is linear. In the case of monolayer transport, the rate of the membrane transport of amino acid is therefore equal to the rate of net accumulation of amino acid within the cells over a period of time. We measured the total accumulation of amino acid and the time at which the amount accumulation is linear proportional to the accumulation time. The mode and characteristics of membrane transport of amino acid are determined by the Menten-Michaelis kinetic analysis.

The amino acid transport experiments were performed on








33

the 35 mm falcon dishes (6 well clusters) at the cell passages #17 #50.


2.2.1 Caco-2 Cell Monolaver Transport


The amino acid uptake experiments were performed at room temperature (22.05C 1.0*C). Cells were taken out of the incubator. Then the growth medium was aspirated, and cells were rinsed three times with uptake buffer (22.5C) containing 137 mM NaCl (or choline Cl), 10 mM HEPES/Tris (pH 7.4), 4.7 mM KC1, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 2.5 mM CaCl2. The uptake was initiated at time = 0 by adding 1 ml uptake buffer plus 0.01 -10 mM [3H]-alanine (2 gCi/ml, isotope was dried in nitrogen gas first and was then re-hydrolyzed in the uptake buffer), 0.005 5 mM [3H]-Arginine (2 ACi/ml), or other isotopes and concentrations as mentioned below into the cell monolayer. For the System B regulation experiments, 1 mM unlabeled MeAIB was also added to the uptake buffer to block possible alanine uptake via the System A. For the arginine uptakes, 10 mM unlabeled leucine was added to the uptake buffer to block the System bc, During the uptake procedures, the cell cultures were continuously shaken by an orbital shaker (1 Hz). The uptakes were stopped by aspirating the uptake buffer, and then adding 2 ml ice-cold uptake buffer (lacking substrate) immediately to the cells. The ice-cold buffer is aspirated, cells were rinsed two more times using the same ice-cold buffer. Isotope was extracted from the cells








34

by adding 1 ml 1N NaCI to the cells. After overnight extraction (continuous shaking), a 200 Al aliquot of NaOH extract was added to 10 ml Liquiscint scintillation fluid which was then neutralized with 200 Al glacial acetic acid. Radioactivity (counts per minute, CPM) was measured in the Beckman scintillation counter with quench normalized using the "H-number" method. Because the nonspecifically adhering label was < 1 % of the total counts per minute (CPM) associated with the cell uptake, the double label 14C-inulin space measurement was not performed for subsequent experiments. The protein content in the NaOH extract was measured by the Bio-Rad protein assay. The rate of amino acid uptake activity was equivalent to the initial linear slope of the uptake time course. All subsequent uptake experiments were conducted within the linear period at a uptake period < 10 minutes, with the 0 minute points serving as blanks. The amino acid uptake rates are expressed as nmole amino acid/mg cell protein/minute. The sodium-dependent alanine transport is equivalent to measuring total alanine uptake in NaCl buffer and in choline Cl buffer, and then subtracting the difference.


Bio-Rad Protein Assay


The Bio-Rad protein assay reagent was diluted 1 : 4 with de-ionized water. Fifty microliter cell/NaOH mixture was added into the diluted reagent (5 ml), with 50 Ml 1.4 mg/ml standard y-globulin as the protein reference (plus additional 50 ul 1








35

N NaOH) and the 5 ml diluted reagent plus 50 Al 1 N NaOH as the blank. The samples and reagents were mixed well and stabilized for 15 minutes. The protein absorbance was measured at wavelength of 595 nm by using the spectrophotometer. The sample protein contents were then calculated by: sample protein = (sample absorbance x 1.4 mg/ml) + (standard protein absorbance).


2.2.2 Radioactivity Measurement


Cell/NaOH aliquot (200 Al) was added into a 20 ml vial and was neutralized by adding 200 Al glacial acetic acid. Liquiscint scintillation cocktail (10 ml) was added to the mixture. A 200 Al sample of uptake buffer (containing a known specific activity of isotope), 200 Al lN NaOH, and 200 ml glacial acetic acid were added together with 10 ml liquiscint in the 20 ml vials. The vials were then placed into the Beckman scintillation counter. The [3H]-radioactivity was obtained as counts per minute. Uptake was subsequently calculated as nmole amino acid/mg protein/minute.


2.3 Monolaver Transport In Caco-2 Grown On Porous Filters


As mentioned above, the Caco-2 cells can be grown on either the plastic surface or porous filters. The confluent cell monolayer on porous filters provide additional dimensions to the membrane transport studies. The confluent cell monolayer forms a barrier separating the upper chamber and








36

lower chamber of the filters. The confluency is determined by measuring the electrical resistance across the cell layer. The apical membrane is accessible via upper chamber and the basolateral membrane is accessible to the lower chamber. Using cell monolayers grown on plastic we measured only the flux of amino acid across the apical membrane to cell cytosol. By using the porous filter, we can not only measure the flux from apical to cytosol, but also flux from basal to cytosol, and therefore the trans-cellular fluxes from apical to basal or basal to apical side. Thus we can integrate the events at basal or apical surfaces, including regulatory receptors, and site sites of transport. The membrane transport characteristics were determined by kinetic analysis in both basolateral and apical membranes.


2.3.1 Transport In Caco-2 Grown On Porous Filters


The uptake experiments were performed at room temperature (22.5C 1.0*C). The Caco-2 cell culture was taken out of the incubator. The trans-epithelial resistance was measured using a trans-epithelial open circuit potential difference apparatus (world precision instrument Inc, New Haven, CT), confluent cells with a resistance 2 300 n cm2 were used for uptake studies. The growth medium in both the upper and lower chambers was aspirated, and the cells were rinsed three times with uptake buffer (room temperature) containing 137 mM NaCl (or choline Cl), 10 mM HEPES/Tris (PH 7.4), 4.7 mM KC1, 1.2 mM








37

MgSO4, 1.2 mM KH2PO4, and 2.5 mM CaCl2. These experiments were conducted in collaboration with Dr. S. Smith of BurroughsWellcome company (Research triangle, NC).

For the measurements of the apical-cellular-basolateral amino acid movement, the amino acid uptake was initiated at the time = 0 by adding 1 ml uptake buffer plus [3H]-alanine or [3H]-arginine into the apical side (the upper chamber), with the basolateral side exposed to 3 ml uptake buffer (lacking amino acids) in the lower chamber. During the uptake period, the cell cultures were continuously shaken by an orbital shaker (1 Hz). The uptakes were stopped by aspirating the uptake buffer, taking the filters out of lower chamber, and adding 2 ml ice-cold uptake buffer (lacking substrate) immediately to the upper chamber. The buffer was aspirated and cells were rinsed by the ice-cold buffer three times. Isotope trapped inside the cells was extracted from the cells by adding 2 ml 1 N NaOH to the cells (filters were first cut out off the dishes). After overnight extraction (continuous shaking), a 200 p1 aliquot of NaOH extract was added to 10 ml Liquiscint scintillation fluid which was neutralized with 200 p1 glacial acetic acid. Isotope trapped in the lower chamber was measured by transferring 200 p1 of this buffer to 10 ml Liquiscint. The radioactivity was counted in the Beckman scintillation counter.

For the measurement of basolateral-cellular-apical movement, uptake was initiated at time = 0 by immersing








38

filters (with 1 ml uptake buffer with no substrate in the upper chamber) into 3 ml uptake buffer plus [3H]-alanine or [3H]-arginine in the lower chamber. During the uptake period, the cell cultures were continuously shaken by the orbital shaker (1 Hz). The uptake was stopped by removing the filters out of the lower chamber, and removing the upper chamber immediately. The filters were rinsed three time with ice-cold buffer. Isotope trapped in the cells and in the upper chamber buffer was measured separately as described above.

For the measurement of the apical- basal trans-cellular amino acid movement, the uptake was initiated at time = 0 by adding 1 ml uptake buffer plus [3H]-alanine or [3H]-arginine to the apical side and immersing the filter into 3 ml buffer (lacking substrate) in the lower chamber. Uptakes were stopped by removing filters from the lower chamber. The isotope accumulated in the lower chamber was the measured as described above.

For the measurement of the basal-apical trans-cellular movement, the uptake was initiated at time = 0 by placing the filter (with 1 ml uptake buffer with no substrate in the upper chamber) into 3 ml buffer containing [3H]-alanine or [3H]arginine in the lower chamber. During the uptake period, the cell cultures were continuously shaken (1 Hz). The uptakes were stopped by taking out the buffer from the upper chamber. The isotope accumulated in the upper chamber was measured in scintillation counter as described above.








39

2.3 Statistical Analysis


All experiments were conducted in triplicate (including the 0 time blanks, and the uptakes in Na and choline buffers) and all experiments were confirmed in at least two differently seeded generations of cells. Curve fitting was conducted by non-linear regression analysis. Values were reported as the mean + SE. ANOVA used for statistical analysis with Duncan's test, the level of significant p & 0.05.














CHAPTER 3
CLASSIFICATION OF THE ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE


3.1 Introduction


The characterization of membrane amino acid transport was pioneered by Christensen several decades ago (Christensen et al., 1952). Since that time, the criteria to assess a membrane amino acid transport systems in mammalian membranes have indeed substrate specificity, ion-dependency, initial uptake rate kinetics, patterns of analogue cross-inhibition of amino acid uptake, and exclusive substrates transported through specific transport systems. The development concept of the Na'-gradient-driven, secondary active solute transport (Crane et al., 1962) was an another important addition to describing the transport phenomena. In the past several years, there have been reports of cloning cDNA encoding several amino acid transporters (Kilberg et al., 1993), and cloning of possible regulatory proteins for Systems L, bO,+ or y+. There has no cloning, antibody production, nor purified transporter protein reported for major alanine transport systems in intestine. The phenomenological criteria remain the sole tools to classify alanine transport systems. The sole exception is the recent cloning of System A (Kong et al. 1993), although System A


40








41

represents a minor pathway in intestine (described below).

The major membrane transport Systems A, ASC, BO' B, L, and asc for the dipolar amino acid L-alanine have been intensively studied in various tissues. (Oxender et al., 1963; Christensen et al., 1963; Stevens et al., 1982; Van Winkle et al., 1985, 1987, 1988; Van Winkle & Campione, 1990; Kilberg et al, 1993; Stevens, 1992a,b). Among these alanine transport systems, only System B was originally described as an unique in intestinal epithelial cell transporter (Stevens et al., 1982, 1984). The others systems were first described for nonepithelial cells (Stevens, 1992a,b).

System A is a strictly Na'-dependent system which is broadly selective for most dipolar (neutral) amino acids (Christensen et al., 1963). System A is found in many tissue membranes. One specific feature of System A is that the nonmetabolized MeAIB is a specific substrates for System A. AIB serves as less specific substrate. System A regulatory properties have been intensively investigated in hepatocytes and other tissue. Several regulation mechanisms of System A activity were proposed (Kilberg, 1986; Engleberg, 1986; Dawson & Cook, 1987). System A reportedly exists at the apical and basolateral membrane of guinea pig intestinal epithelia (Hayashi et al., 1980; Del Castillo et al., 1991).

System ASC is another Nat-dependent transport system serving 3- and 4-carbon neutral amino acids, exemplified by alanine, serine and cysteine (Kilberg et al, 1981). System ASC








42

is distinct from System A in that System ASC does not transport MeAIB. System ASC is also found in many tissue. System ASC reportedly exists at guinea pig intestinal epithelial apical membrane (Hayashi et al., 1980).

System Bo,+, first described in mouse blastocytes (Van Winkle et al., 1985), is a Na+-dependent transport system that transports both the cationic and neutral amino acids. System Bo', is expressed in many tissues.

The strictly Na4-dependent transport System B exclusively found in the apical membrane of the epithelial cells. It was first described in rabbit jejunum apical membrane vesicles as a broadly selective system serving neutral amino acids (Stevens et al., 1982, 1984, Stevens, 1992a,b). It was originally named System Neutral Brush Border (NBB), and later was renamed as "System B" (Stevens, 1992a). System B substrate selectivity is similar to the System Bo,+ in that both are Na dependent, as it possesses a broad selectivity for most dipolar amino acids. Both Systems B and Bo,+ interact with the bicyclo-amino acids 2-amino-2-norbornanecar-boxylic acid (BCH) or BCO. However, System B 0,+ is inhibited by cationic amino acids such as lysine and arginine, while System B is not interactive with cationic amino acids. System B may possible be a variant of System Bo,+.

The Na -independent System L was shown to exist in the apical and basolateral membrane of many cell types. System L is a broadly selective system serving neutral amino acids,








43

cysteine, phenylalanine, and BCH.

System bo,+, a Na+-independent analogue of System Bo,+ serving neutral and cationic amino acid such as lysine and arginine. The substrate scope of System bo,+ is similar to System Bo,+. System bo,+ exists in many cell types, but it has been previously reported in the intestinal membranes.

Only a few organic solute transport studies in the Caco-2 cells have been reported (Blais et al., 1987; Hidalgo et al., 1988; Mohrmann et al., 1986; Souba et al., 1992, Nicklin, 1992). Each of the reports showed the similarity of solute transport characteristics in Caco-2 cells and intestinal epithelial cell.

Inasmuchas alanine transport systems were not described for Caco-2 culture, our study began by describing the alanine transport systems in the Caco-2 monolayer.


3.2 Methods And Materials


3.2.1 Methods


The [3H]-alanine uptake experiments were performed in both the pre-confluent (day 2 3) and confluent (day 8 -9) cells. The basic uptake procedures were as described above (chapter 2). Special uptake conditions are presented below where appropriate.








44

3.2.2 Materials


The materials were as the same as discussed in the chapter 2.


3.3 Results


3.3.1 Alanine Uptake Time Course


The 50 AM and 5 mM [3H]-alanine uptakes were measured at during various times (0 45 minutes) in the uptake media containing 137 mM NaCI or 137 mM choline Cl. The 50 AM alanine uptake on day 2 cells was shown at Fig. 3-1. The accumulation in the NaCl medium was greater at any point than that in the in choline Cl medium suggesting a Na+-activation phenomenon. The initial alanine accumulation in the Caco-2 monolayers was linear up to 15 minutes (at both [3H]-alanine concentrations of 50 AM and 5 mM). Since the transport activity was expressed as the alanine uptake rate measured during the linear period, the uptake period of 0 10 minutes was chosen for all the subsequent uptake measurements. The rate was equal to total accumulation divided by the time period.


3.3.2 Alanine Uptake Rates at Various Caco-2 Cell Ages

The 50 MM [3H]-alanine uptake rates were measured at various Caco-2 cell ages ranging from 1 day old to 35 days old in both the NaCl and choline Cl media (Fig. 3-2). At each cell age, the total alanine uptake rate in NaCl medium was








45

consistently greater than that in the choline Cl medium. This difference was greater in the younger cells, compared to confluent cells.

Alanine uptake rates in both the NaCl and choline Cl media decreased as cell age increased. The uptake rates in NaCl medium decreased rapidly over a period of several days (5 4 days old), and maintained steady after differentiation (2 9 day old). The decrease in alanine uptake in choline Cl medium was less dramatic than that in the NaCl medium, with the rate consistently decreasing throughout the cell ages.

In a separate study, the 24 hours proliferation rates of Caco-2 cells (2 day old 14 days old) were measured by incubating the cells in [3H]-thymidine medium for 24 hours. The amount of thymidine incorporated into the cells during the period represented the cells relative proliferation rate. The thymidine incorporation into the Caco-2 cells deceased as cell age advanced (Fig. 3-3).

The pattern alanine uptake rates at various cell ages was coincident with the cell proliferation rates.


3.3.3 Ion-dependency


The uptake of 50 pM [3H]-alanine was measured in the uptake media containing 137 mM NaCl, 137 mM choline Cl, 137 mM KC1, or 137 mM LiC1. The total alanine uptake rate in the NaCl medium was 8-fold greater than that in either the choline Cl, KCl and LiCl media at [alanine] = 50 AM (Fig. 3-4). The total








46

alanine uptake rates in the choline Cl, KC1, or LiC1 media were not significantly different. Alanine uptake in the NaCl uptake medium was not significantly different from that in the medium containing sodium gluconate.

These data indicated that alanine uptake activation was strictly Na+-dependent. Other cations such as Li or K+ could not substitute Na+ in activating alanine transport. Chloride was not required for the Na+-activation.

For all the subsequent experiments, the Na+-dependent fraction of alanine total uptake was obtained by subtracting the uptake measured in choline medium from the total uptake measured in sodium medium.


3.3.4 The Effect of pH on Alanine Uptake


The uptake of 50 AM alanine in both the NaCl and choline Cl uptake media was measured at various media pH ranging from pH 6.0 to pH 8.5. HEPES and Tris were used to adjust media pH. The total alanine uptake rates in both the NaCl and choline Cl media increased steadily as the medium pH increased (pH = 6.1,

7.4, and 8.5) (Fig. 3-5).


3.3.5 Na+-Activation of Alanine Uptake in Caco-2 Cells


The uptake of 50 MM alanine was measured in the uptake media containing NaCl ranging from [NaCl] = 0 mM to 137 mM (choline Cl as substitute). The total alanine uptake rates at both day 2 and day 9 cells increases as media NaCl








47

concentration increase. The uptake rates as a function of NaC1 concentrations gave a hyperbolic shape (Fig. 3-6). The nonlinear regression analyses of the Hill equation gave the same Na+-activation Hill coefficient (n = 1) at each cell age, while the V3ax was greater in day 2 cells than in day cells. The same Hill coefficient (n = 1) indicates that one Na* binds to transporter coupled with each alanine molecule transported, in cells 2 days and 9 days old.

These data indicated that the alanine uptake capacity was greater in day 2 cells. Therefore, the difference in transport capacity between day 2 and day 9 cells was not due to the transport system's affinity for Na+-activation coefficient changes.


3.3.6 Alanine Uptake Kinetics

The alanine uptake in uptake media containing 137 mM NaCl or containing choline Cl was measured at various [H]-alanine concentrations ranging from 10 AM to 5 mM at the cell ages of day 2 and day 9. The kinetics at day 2 and day 9 cells were shown as uptake rates measured as a function of alanine concentration (Fig. 3-7; Fig. 3-8). The kinetics in either the NaCl or choline Cl medium each displayed both saturable and non-saturable components, indicating multiple transport systems were involved. At each alanine concentration, the uptake rate was higher in the NaCl medium.

In the choline Cl media, alanine uptake occurred via two








48

pathways: a saturable Na+-independent carrier-mediated system and non-saturable simple passive diffusion. In NaC1 media, besides the two pathways discussed in the choline Cl medium, an additional saturable Na+-dependent system exists.

For the non-saturable component, the passive permeability coefficient (P) describing the relation J = P [Ala] was constant at 0.53 .08 Aliter/(mg protein)/min regardless of the cell differentiation states.

For the saturable components, non-linear regression analyses of Na+-dependent alanine transport kinetics gave K, = 164 26.1 Amole alanine and V,,x = 2.79 0.21 nmole/mg protein/min for the day 2 cells. For 9 day old differentiated cells, the K, was 159.0 13.6 Amole alanine and V..x was 0.512 0.03 nmole/mg protein/min (Fig. 3-9). Regarding the Na+independent alanine uptake system (tentatively, System L), the activity decreased from Vmax = 1.85 + 0.25 nmole/mg/min in the undifferentiated (day 2) cells to V,ax = 0.38 + 0.017 nmole (mg protein)-' min"' in the differentiated (day 9) cells. The System L apparent K for alanine was unaffected by cell age (differentiated cell K, = 1.10 + 0.19 mM vs. differentiated cells K, = 1.02 + 0.007 mM alanine).

The kinetics revealed that alanine uptake capacity was higher in day 2 cells than that in day 9 cells, and therefore the difference was only a V,,x effect. The affinities of both the saturable components were not affected by cell ages. The non-saturable passive diffusion was not affected by cell age.








49

3.3.7 The Analoque Cross-inhibition Pattern


The 50 AM [3H]-alanine uptake rates were measured in media containing 137 mM NaCi and 137 mM choline Cl plus 5 mM single amino acid analogues (natural amino acids, BCH, MeAIB, AIB, and f-alanine with 5 mM mannitol as control).

For the Na -independent alanine transport system, alanine uptake was strongly inhibited by phenylalanine, alanine, leucine, threonine, serine, glutamine, asparagine, cysteine, and BCH, and weakly by MeAIB, AIB, and glycine. Lysine, and glucose did not inhibit (Fig. 3-10 & Fig. 3-11).

For the Na -dependent alanine transport system, the uptake activity was inhibited by 5 mM amino acid analogues (natural AAs plus BCH, MeAIB, and P-alanine) was shown in Fig. 3-12. The Na+-dependent [3H] alanine transport was strongly inhibited by threonine, glutamine, serine, cysteine, and asparagine. Weaker inhibition was elicited by glycine, phenylalanine, leucine and the bicyclo amino acid BCH. MeAIB and cationic amino acids elicited <10% inhibition. Dixon inhibition analyses indicated that the glutamine inhibition was classic competitive inhibition, while the MeAIB affect was un-competitive (Figs. 3-13 3-15). Proline, glycine, and phenylalanine gave high Ki values (Figs. 3-16 3-21).

The pattern and degree of amino acid analogue inhibition of the Na+-dependent alanine uptake was identical at both cell ages, suggesting that the same transporter system was operative regardless of the cell age (Fig. 3-22).








50

In a separate study, the 50 MM [3H]-MeAIB uptake rates were < 5% of the same concentration of alanine, suggesting a minimal contribution by System A in our Caco-2 cells line.


3.3.8 Alanine Uptake on Porous Filters


Uptake of alanine (50 MM) into the apical and basolateral surfaces of confluent Caco-2 monolayer grown on porous filters were measured. The Caco-2 cell monolayer confluency was determined by measuring the trans-cellular resistance, with the trans-epithelial resistance > 300 n x cm2 was considered confluent. The apical compartment to the basal compartment of 50 AM alanine uptake was measured (Fig. 3-23). The majority of alanine across the apical membrane was accumulated inside the cells rather than transport across to the basal side (Fig. 324).

Alanine (50 MM) uptake at the basal membrane to cytosol and apical compartment was also measured (Fig.3-25, 3-26, and 3-27). The uptake in NaCl medium was greater than that in the choline Cl medium, indicating a Na*-activation event.


3.4 Discussion

The alanine uptake in Caco-2 monolayer at the different cell ages was studied. The alanine uptake activity was different at various cell age, indicating the possible regulation of cell development. There were several pathways for alanine uptake. By using the membrane transport system








51

classification criteria, we classified the alanine transport systems in Caco-2 cells, as discussed below.


3.4.1 Alanine Uptake Activity vs Cell Ages

Both the Nad-dependent and Nat-independent alanine uptake activities decreased as cell age increased at the alanine concentration of 50 AM (over the cell age span of 1 35 days) (Fig. 3-2). What were the mechanisms underlie this cell development regulation? There were several possible mechanisms that could underlie this development-related regulation. Nonspecific membrane potential or other membrane property change could cause a non-specific driving force alteration, permeability of the membrane could change, or specific functional change of specific transport systems could occur. Each of these possibilities was explored.

The Na/glucose cotransport activity on Caco-2 monolayer has been reported to increased with cell age (Blais et al., 1987). The opposite direction of alanine uptake activity and Na/glucose activity with cell age rule (Fig. 3-23) out the possibility that the age-associated transport effect was due to the non-specific membrane electrochemical gradient which may associated with cell age. Therefore the non-specific driving force was not likely to be involved in the mechanism.

In terms of membrane properties at different cell ages, our kinetics studies gave the same diffusion permeability coefficients in day 2 and day 9 cells, even though the alanine








52

uptake rates were several fold higher in the day 2 cells (Fig. 3-2). The alanine uptake change over the cell ages was therefore only a portion of saturable carrier-mediated uptake. The membrane permeability was then unlikely to be involved in the regulation mechanism.

As discussed above, the alanine uptake rates decreased with advancing cell age, while Na/glucose cotransport increased with cell age (Fig. 3-23). This opposite direction of transport activity suggests that the function of alanine and glucose were not the same in cell development. Alanine was not solely for caloric purpose. In the light of the cell proliferation rate decrease with the cell age increase (Fig. 3-3), the reduced alanine uptake may be due to the lowered requirement for amino acids, but not for glucose. These data also indicated that the Na/glucose and alanine uptake in Caco2 cells were independently regulated by the cell differentiation and development.


3.4.2 Classification of the Alanine Transport Systems


There were three alanine transport pathways in Caco-2 monolayers for alanine at both cell stages (the undifferentiated and the differentiated stages): a simple passive diffusion, a Na+-independent system, and a Na+dependent system.

3.4.2.1 Simple Passive diffusion

The same passive permeability coefficient measured in








53
both the day 2 and day 9 cells suggested that the Caco-2 cell development did not alter the membrane permeability to alanine. The diffusion rates of alanine across the cell membrane at certain alanine concentrations were the same at either cell ages. The passive diffusion contribution at 50 AM alanine uptake was minimal, less than 1% of total uptake in NaCl. At higher alanine concentrations, the passive diffusion contribution was greater. At [alanine] = 5 mM, passive diffusion contributes 90% of total alanine uptake in NaCl.

3.4.2.2 Na -indeDendent transport system is System L

One saturable Na+-independent alanine transport system existed in both the day 2 and day 9 cells. The observed activity decreased with the advancing cell age (Fig. 3-2). The activity decrease was coincident with the cell proliferation rates (Fig. 3-3). The activity was possibly regulated by cell proliferation requirements.

The Na -independent alanine transport kinetics in both the 2 day old and 9 day old cells revealed that the transport activity Vx was higher in the day 2 cells. The transport apparent affinity K, was the same at both cell ages. These kinetic parameters strongly indicate that the transport capacity was greater in the day 2 cells; the activity change was a Vax effect, not 1K effect, suggesting the presence of same transport system in both differentiated and undifferentiated states. The activity change was likely a change of copies of functional transport units in the membrane








54

instead of some modification of transporter affinities.

The amino acid analogue inhibition patterns on both the day 2 and day 9 cells were similar in that phenylalanine, leucine, BCH, and alanine strongly inhibited the alanine uptake, while MeAIB, glycine and lysine were weak inhibitors (Figs. 3-10, 3-11). These inhibition patterns strongly resemble that of the System L. The non-interaction with lysine ruled out the possibility of the System bo,+. The strong BCH inhibition suggesting the unlikely System asc.

Based on the kinetic characteristics, the pH sensitivity, and the cross-inhibition pattern, We conclude that the Na+independent alanine uptake is via the System L at both the day

2 and day 9 cells.

3.4.2.3 Na+-dependent alanine transport System B

The alanine uptake in the Caco-2 cells was strongly Na+dependent in either day 2 old and day 9 old cells (Fig. 3-4). The Na+-dependent portion was more than 85% of the total alanine uptake in NaCl medium at 50 AM alanine (Fig. 3-4). No other monovalent cationic K+ or Li substitute for the Na in activating the alanine uptake. Furthermore, the system was not activated concomitantly by Cl1.

One of the important aspect in classifying transport systems was the cross-inhibition profile. The amino acid analogue inhibition pattern of the Na/alanine uptake for both the day 2 and day 9 cells was similar: the Na/alanine transport was strongly inhibited by threonine, serine,








55

glutamine, cysteine, and asparagine; Weaker inhibition was elicited by glycine, phenylalanine, leucine and BCH. MeAIB and cationic amino acids elicited < 10% inhibition (Fig. 3-12 & 322). We can compare this inhibition pattern with the amino acid inhibition patterns of the known Na/alanine Systems A, ASC, B, and Bo,+. System A is a strictly Na -dependent system selective for dipolar amino acids including alanine. Many neutral amino acids competitively inhibit Na/alanine transport via System A. One special aspect of System A is its unique ability to transport MeAIB. In our inhibition study, MeAIB blocked less than 10% of the Na/alanine transport activity (Fig. 3-12). Dixon analysis revealed that the MeAIB inhibition was a non-competitive inhibition (Fig.3-15). These combined data exclude System A as a major transport system in Caco-2 cells.

System ASC, the Na -dependent system serves short-chain neutral amino acids alanine, serine, and cysteine. In our study, serine, cysteine strongly inhibited alanine/Na uptake. However, phenylalanine and glycine, two competitive inhibitors of System ASC did not inhibit the Na/alanine transport in our study, as it would for the classic System ASC (Figs. 3-12, 318-21). Based on this and the similarity of our data to System B (discussed below), we exclude System ASC as the transport system. Because the characteristics were very close, however, definite classification is not possible without more precise test methods such as cDNA probes or antibodies.








56

Another Nat-dependent alanine transport system is System BO,+ which serves both the neutral amino acids and cationic amino acids. The only evidence that does not support existence of System Bo,+ was that cationic amino acids arginine and lysine did not inhibit Na/alanine uptake in our study (Figs. 3-12 & 3-22). Thus, it is unlikely that System Bo,+ exists in Caco-2 cells.

The final Na/alanine system candidate System B described first for intestinal epithelial cells (Stevens et al. 1984). System B has only been found in the epithelial cells of vertebrate and invertebrates (Stevens, 1992). The substrate selectivity of System B is very similar to System Bo,+ except that System B does not interact with cationic amino acids (Figs 3-12 & 3-22). Na/alanine uptake was strongly inhibited by of neutral amino acids serine, threonine, cysteine, weakly by glycine and phenylalanine, interaction with BCH, and was interactive with cationic amino acids arginine and lysine in our studies (Figs. 3-12 & 3-22). The amino acid analogue inhibition pattern supports the existence of System B. The apparent affinity K1 = 159 pmole alanine in Caco-2 was similar to the System B report elsewhere (Stevens et al., 1982) (Fig. 3-9). Furthermore, the pH sensitivity (Fig. 3-5) and the Na+activation Hill coefficient (n = 1) (Fig. 3-6) further support the case for System B. Based on the our evidence (Figs. 3-4 through 3-9, and 3-11 through 3-22), we conclude that the Na/alanine transport system in the Caco-2 cells was likely








57

System B.

The System B transport activity decreases as cell age increases (Fig. 3-2). The decrease in activity was coincident with the decrease of cell proliferation rates with cell ages (Fig. 3-3). The proliferation rate may be related to the cell requirement for amino acid. In contrast to the Na/alanine transport activity, the Na/glucose activity increases as cell age advances (Blais et al., 1987) (Fig. 3-23). These opposing changes in activity for Na+-dependent solute transport as a function of cell ages excludes the likelihood that the amino acid transport was regulated by non-specific membrane electrochemical potential effects. Furthermore these data suggest that cell development is associated with the independent regulation of amino acid and glucose transport systems. We tested the Na/alanine transport characteristics in two different cell states, the undifferentiated state (day 2 cells) and differentiated state (day 9 cells). The amino acid analogue inhibition pattern, pH sensitivity, and Na activation Hill number were the same for both the day 2 and day 9 cells (Figs. 3-4, 3-5, and 3-12). The transport kinetics gave a V,,x on the day 2 that was greater than for day 9 cells, while the apparent affinity K, was the same on both cell ages (Fig. 3-9). All the transport characteristics of the Na+dependent alanine transport (except V.ax) were identical in both the undifferentiated and differentiated states, suggesting that the same transporter system was operative








58

regardless of the cell age. The kinetics also indicated that the transport capacity was greater in the day 2 cells, and that the activity difference between the two days was like caused by the change in functional transporter units expressed in the membrane, rather than modification of existing transporter affinity.


3.5 Summary

Alanine is transported in Caco-2 cell by a Na -dependent transport System B, a Nat-independent transport System L, and simple passive diffusion. These same systems were operative in both the undifferentiated and differentiated cell states. The passive diffusion coefficient was not affected by cell development. The alanine transport Systems B and L activities are down-regulated as the cell develops, coincident with the cell proliferation rates. The decrease in transport activities are likely caused by the decrease in copies of functional transporter units, rather than modification of existing transporter affinity for substrate or ions.















Fig. 3-1. Alanine uptake time course

The uptake of alanine (50 MM and 5 mM) was measured in uptake media containing NaCl and choline Cl in day 2 and day 9 cells. The alanine uptake in NaCl media was greater than that in the choline Cl media at any point (except t = 0, p < 0.05, n = 6). The uptake values in this figure and subsequent experiments were expressed as mean standard error (SE). The data shown were from the alanine (50 AM) in day 2 cells, with similar data obtained in the day 9 cells and at other alanine concentrations.












501

e In NaCl
c 40 0 In Choline Cl E 40

0
E 30
C







c 10 O
d






o *
-Y
20
00








0 5 10 15 20 25 30 35 40 45

Time, minutes
O



a'













Fig. 3-2. Alanine uptake at various cell aces

The uptake of alanine (50 AM) was measured in NaCl and choline Cl media over cell ages of 1 35 days old. At any cell age, the alanine uptake in the NaCl was greater than that in the choline Cl media (p < 0.05, n = 6), even though the difference margin was smaller in the older cells. The Na'-dependent alanine uptake decreased with the advancing cell age, while the Na'independent alanine uptake also decreased at less extent.













1 .0 I I I I I I


+
e Na -dependent bo .75 in Choline CI



0
5 0.5




-- .25





I i I I I I i I I i I

S0 1 2 3 4 5 6 7 8 9 10 16 30 Cell age, days














Fig. 3-3. Caco-2 cell proliferation rates at various cell aces

The 24 hour incorporation rates of [3H]-thymidine into various cell ages were measured. The cells had been incubated in serum-free DMEM for 24 hours prior to the measurements. The blank control value = 1012 CPM, and the incubation medium value = 3.74 x 105 CPM.













80 .4- 70
0

60


50 m 40 0 30
'I




10


E-e 0
0 2 4 6 8 10 12 14

Cell age, days














Fig. 3-4. Alanine uptake ion-dependency

The uptake of alanine (50gM) was measured in uptake media containing 137 mM NaCl, 137 mM choline Cl, 137 mM KC1, or LiC1. The uptake in NaCl media in both the day 3 and day 8 cells was greater than that in either those in choline Cl, KC1, or LiC1 media (p < 0.05, n = 6). Uptake in choline Cl, KC1, or LiC1 media was not significantly different (p > 0.05, n = 6).

















-1 -1
Alanine uptake nmol mg min

0 0 0 0 0 0




CHO





K CIO







CHO Co
Li Na











99














Fig. 3-5. The effect of pH on alanine uptake

The uptake of alanine (50M) in day 3 and day 8 cells was measured at various medium pH (at pH = 6.1, 7.4, and 8.4). The uptake rates were higher in more alkaline media.











0.5

Na-dep
0.4



0.3 day 3 a 0.2 -day 8



0.1 day 3 & 8



6.0 6.5 7.0 7.5 8.0 8.5

pH














Fig. 3-6. Nat-activation of alanine uptake

The uptake of total alanine (50 AM) in day 2 and day 9 cells was measured in media containing various NaCl concentrations ([NaCl] = 0 137 mM, choline Cl substituted NaC1). The Non-linear regression of these data gave the same Na -activation Hill coefficient of n = 1 for both the day 2 and day 9 cells.











wuJ '[ON]

oCL 0oL 001 0 09 ot oz 0 0 0"0



o .
9*




0 o



0 I9 00


-o3
I ID 9C~o














Fig. 3-7. Alanine uptake kinetics in day 2 cells

The alanine (10 gM 5 mM) uptake was measured in the day 2 cells. The figure showed the total alanine uptake rates in NaCl, choline Cl media, or Na -dependent alanine uptake rate as a function of alanine concentrations. The curve contained nonsaturable and saturable components.














+
c V Na -dependent E total in CHO
0 total in NoCl
6
E
0 5
0
E
c 4


a- 3 Q
2 i
c 2 c
C
0
0

0 100 200 300 400 500 600 700 800 900 1000

[Alanine], pM














Fig. 3-8. Alanine uptake kinetics in day 9 cells

The uptake of alanine (10 AM 5 mM) was measured in NaCl and choline Cl media. The total uptake in NaCl and choline Cl media, and Na -dependent uptake were showed as a functional of alanine concentrations. The curves showed non-saturable and saturable components.












1.5 I I I

C +
-- V No -dependent E 0 total in CHO
S total in NaCl

E
1.0

0
E
C



4-' 0.5
vO


C17
0 *V



000.0
0.0 c I
0 100 200 300 400 500 600 700 800 900 1000

[Alanine], pM














Fig. 3-9. Eadie-Hofstee transformation of Na -dependent alanine
uptake kinetics in day 2 and day 9 cells

The Na -dependent alanine (10 gM 5 mM) uptake of fig. 3-7 was expressed as alanine uptake as a function of alanine uptake/alanine concentration. Non-linear regression of these data gave a straight line, indicating a single transport system. The Vax values (the interception of the line and the y axis) were Vax = 3.1 0.21 nmole/mg/min for day 2 cells, and Vax = 0.51 nmole/mg/min for day 9 cells. The K, values (the negative slope of the line) were K, = 167 26.1 mole alanine for day 2 cells, and K, = 159.0 13.6 mole alanine for day 9 cells.












5.0
0 day 2
-4 [ day 9
4.0



3.0



o 2.0
o

O
1.0



0.0
0 5 10 15 20
-1 -1
J/[Ala], jiLiter mg mm














Fig. 3-10. Na -independent alanine uptake inhibition pattern in day
3 cells

Alanine (50 MM) uptake in choline Cl medium was measured in day 3 cells, with 5 mM single amino acid present in the uptake media. The Na -independent portion was the difference between the total uptake in choline Cl media and passive diffusion.


















Alanine uptake, nmole mg min

I I I I Cont Ala Asp BCH Cys GIn Leu Ser
Thr Phy AIB
Pro Gly MeAIB Lys Glu
1 I I I














Fig. 3-11. Na -independent alanine uptake inhibition pattern in day
9 cells
















-1 -1
Alanine uptake, pmole mg min
0 --, N N 0 01 0 .n I I I
Cont

Ala Asp Gin Phy
Ser Thr BCH

Leu
MeAIB
Pro Gly
AIB Cys
Lys Glu
I I I I







08














Fig. 3-12. Na -dependent alanine uptake inhibition pattern in day
3 cells

Alanine (50 AM) uptake in day 3 cells was measured in NaCl and choline Cl uptake media containing single 5 mM amino acid. The Na -dependent portion was shown.























-1 -1
Alanine uptake, nmole mg min

0 0 0 0 0




Control Cysteine, HCI Cysteine, free base Cysteine, free base + DTT Cysteine, HCI + DTT Serine
Asparagine Glutarnmine Alanine Threonine Methionine Leucine O pXXXXo Glycine
X Tryptophon
XO Valine Histidine XU)-1 Isoleucine 3X Proline AIB
XX X Phenylalanine Arginine Tyrosine SMeAIB BCH
DTT
Cystin
S Lysine














Fig. 3-13. Dixon analysis of Na+-dependent alanine uptake with
glutamine as inhibitor

Alanine (25 AM, 50 AM, and 100 AM) uptake in NaCI and choline Cl media was measured with various concentration of glutamine (10 AM 5 mM) presented in uptake media. The dixon plot gave a Ki of 35 MM glutamine.












0.4

0
E

c O.3
E [Ala]=25)uM

-._
0
0.2
E O
[Ala]=50pM S0.1


[Ala]= 1 OOM

-50 -25 0 25 50 75 100 = [Glutamine], .LM














Fig. 3-14. Replot of the slopes of Dixon plot with glutamine as
inhibitor

The slopes of dixon plot shown at Fig 3-13 were shown as a function of (corresponding alanine concentrations)"'. Non-linear regression of these data intercepted 0. The combination of Fig. 313 and this figure indicated that glutamine was a competitive inhibitor for System B.












0.12 I I


0.10 Glutamine Ki=35 pM


0.08


a
o 0.06 In


0.04 0.02


0.00 I I I
0 1 2 3 4
4
1 /[Ala], 10 L/mole













Fig. 3-15. Dixon analysis of Na -dependent alanine uptake with MeAIB
as inhibitor

Alanine (25 M, 50 AM and 100 AM) uptake was measured with various concentrations of MeAIB (10 AM 5 mM) in uptake media. Non-linear regression of these data were parallel, indicating MeAIB was not a competitive inhibitor.












0 4.8
E


E 3.6 0
[Ala]=25uM
()
42.4

0) [Ala]=50uM
E

1.2
--D [Ala]= 100uM



I I I I I11
-10 -8 -6 -4 -2 0 2 4 6 8 10
[MeAIB], mM













Fig. 3-16. Dixon analysis of Na+-dependent alanine uptake with
proline as inhibitor

Alanine (25 AM, 50MM, and 100 AM) uptake was measured with various concentrations of proline (10 AM 5 mM) in uptake media. Ki = 7.1 mM proline.













_ 12
0
E


E

c
Q



O [Ala]=25uM
S6.0



o *) [Al]= 50uM


[Ala]= 100uM

1 I fI ~ [ Li 1 I I 10 -8 -6 -4 -2 0 2 4 6 8 10 [Proline], mM













Fig. 3-17. Replot of the slopes of Dixon plot with proline as
inhibitor

The slopes of figure 3-16 dixon plot were shown as a function of 1/[alanine]. Non-linear regression of these data was through the interception of x axis and y axis. These data combined with fig. 3-16 suggested that proline was a weak competitive inhibitor for the Na -dependent alanine uptake.













0.010

I = Proline

0.008



0.006
CL
C1.
0

0.004



0.002



0.000
0 1 2 3 4 1/[Alonine], 104 L/mol














Fig. 3-18. Dixon analysis of Na+-dependent alanine uptake with
glycine as inhibitor

Alanine (25 gM, 50 gM, and 100 AM) uptake was measured with various concentrations of glycine (10 AM 5 mM) in uptake medium. The Ki value was about 5.5 mM glycine.




Full Text
36
lower chamber of the filters. The confluency is determined by
measuring the electrical resistance across the cell layer. The
apical membrane is accessible via upper chamber and the
basolateral membrane is accessible to the lower chamber. Using
cell monolayers grown on plastic we measured only the flux of
amino acid across the apical membrane to cell cytosol. By
using the porous filter, we can not only measure the flux from
apical to cytosol, but also flux from basal to cytosol, and
therefore the trans-cellular fluxes from apical to basal or
basal to apical side. Thus we can integrate the events at
basal or apical surfaces, including regulatory receptors, and
site sites of transport. The membrane transport
characteristics were determined by kinetic analysis in both
basolateral and apical membranes.
2.3.1 Transport In Caco-2 Grown On Porous Filters
The uptake experiments were performed at room temperature
(22.5C 1.0C). The Caco-2 cell culture was taken out of the
incubator. The trans-epithelial resistance was measured using
a trans-epithelial open circuit potential difference apparatus
(world precision instrument Inc, New Haven, CT), confluent
cells with a resistance > 300 cm2 were used for uptake
studies. The growth medium in both the upper and lower
chambers was aspirated, and the cells were rinsed three times
with uptake buffer (room temperature) containing 137 mM NaCl
(or choline Cl), 10 mM HEPES/Tris (PH 7.4), 4.7 mM KCl, 1.2 mM


Alanine uptake nmol mg min
o o o o o
ro go £*
99
Q'O


Arginine uptake, nmole
149


Slope
1 /[Ala], 10 L/mole


166
the 24 arginine incubation returned to control level after the
3 hours starvation.
5.4 Discussion
system B and system y+ activities were up-regulated by
their own substrates, in contrast to substrate repression of
transport activities found in other internal organs. System
B and System y+ were regulated independently.
5.4.1 Short-Term System B Activity Regulation bv its
Substrates
System B activity was up-regulated when cells were
incubated with individual substrates. As shown in the Fig. 5-
2, only the alanine, cystine, serine, threonine, and glutamine
(that are transported by system B) induced the system B
transport activity. Those non-system B substrates such as
MeAIB, proline, lysine, arginine, and phenylalanine did not
effect on System B activity. These data strongly suggest that
the increase in System B activity following short-term amino
acid exposure was a specific regulation. As will be discussed
later in the following sections, System y+ activity was up-
regulated only by its own substrates. The fact that the System
B substrates alanine, serine, cysteine, threonine did not
inhibit nor induce System y+ transport activity, and that,
the System y+ substrates lysine and arginine did not interfere
System B transport nor induce the System B activity, indicated


48
pathways: a saturable Na+-independent carrier-mediated system
and non-saturable simple passive diffusion. In NaCl media,
besides the two pathways discussed in the choline Cl medium,
an additional saturable Na+-dependent system exists.
For the non-saturable component, the passive permeability
coefficient (P) describing the relation J = P [Ala] was
constant at 0.53 + .08 /xliter/(mg protein)/min regardless of
the cell differentiation states.
For the saturable components, non-linear regression
analyses of Na+-dependent alanine transport kinetics gave KB =
164 2 6.1 /zm ole alanine and VBax = 2.79 0.21 nmole/mg
protein/min for the day 2 cells. For 9 day old differentiated
cells, the K, was 159.0 13.6 jumle alanine and VBax was 0.512
0.03 nmole/mg protein/min (Fig. 3-9). Regarding the Na+-
independent alanine uptake system (tentatively, System L) the
activity decreased from VBax = 1.85 0.25 nmole/mg/min in the
undifferentiated (day 2) cells to Viax = 0.38 + 0.017 nmole (mg
protein)'1 min'1 in the differentiated (day 9) cells. The
System L apparent K for alanine was unaffected by cell age
(differentiated cell K, = 1.10 + 0.19 mM vs. differentiated
cells K = 1.02 + 0.007 mM alanine).
The kinetics revealed that alanine uptake capacity was
higher in day 2 cells than that in day 9 cells, and therefore
the difference was only a VBax effect. The affinities of both
the saturable components were not affected by cell ages. The
non-saturable passive diffusion was not affected by cell age.


Fig. 3-10. Na+-independent alanine uptake inhibition pattern in day
3 cells
Alanine (50 /M) uptake in choline Cl medium was measured
in day 3 cells, with 5 mM single amino acid present in the uptake
media. The Na+-independent portion was the difference between the
total uptake in choline Cl media and passive diffusion.


Fig. 7-11. The effect of chelervthrine on the TPA-induced System
v* activity
System y+ arginine uptake was measured in cells (9 days
old) which had been incubated in DMEM, 0.5 /M TPA, 6.6 /lxM CHE,
or 6.6 /nM CHE plus 0.5 /uM TPA. Arginine uptake was stimulated by
TPA incubation. The TPA stimulation effect was blocked by the CHE
in the TPA incubation medium (p < 0.05, n = 6 ). Similar results
were obtained in day 2 cells.


Cell age, days
239


228
TPA/actinomycin D had no affect on System B activity (Fig. 7-
5).
7.3.6 Phorbol Ester Stimulation of System B Activity vas via
PKC Activation
The Caco-2 cells were pre-incubated with 0.5 iM TPA in
the serum-free medium, 6.6 /M chelerythrine or 50 nM
calphostin C in the medium for 24 hours prior to the uptake
experiments. The 50 iM alanine System B uptake activity was
measured. The system B activity increased 2 fold following the
TPA incubation alone. The TPA/chelerythrine or the
TPA/calphostin C incubation did not affect System B activity
(Fig. 7-6). H-7 isomer did not alter the System B activity nor
the effect of TPA.
7.3.7 The Effect of Phorbol Ester on the System B Transport
Kinetics
The Caco-2 cells were pre-incubated with 0.5 juM TPA in
the serum-free medium for 24 hours prior to the uptake
experiments. The system B alanine transport kinetics ([3H]-
alanine = 1 iM 5 mM) were then measured (Figs. 7-7) The VBax
of day 2 cells was significantly increased 2 fold by TPA (Viax
= 3.05 nmole/mg/min in the control cells, VBax = 5.9
nmole/mg/min with the TPA treatment) The VBax of day 9 cells
was increased 3 fold by TPA incubation (VBax =0.5 nmole/mg/min
in the control cells, Vmax = 1.65 nmole/mg/min with the TPA
treatment) The Km (K, = 160 juM alanine) was the same


Fig. 3-15. Dixon analysis of Na+-dependent alanine uptake with MeAIB
as inhibitor
Alanine (25 /xM, 50 /xM and 100 /xM) uptake was measured with
various concentrations of MeAIB (10 /xM 5 mM) in uptake media.
Non-linear regression of these data were parallel, indicating MeAIB
was not a competitive inhibitor.


Fig. 6-6. The effect of CHX on the chronic TGF^/EGF-induced
System v+ activity
System y+ arginine (5 juM) uptake in cells (2 days old) was
stimulated by 48 hours TGF or EGF incubation. CHX (10 mM) in the
TGF/EGF incubation blocked this TGF/EGF stimulation effect (p < 0.05,
n = 6) .


17
hormone, System A associated protein synthesis is stimulated
(depressed), while in the presence of elevated intracellular
amino acid levels, System A-associated protein synthesis is
repressed by a regulatory protein.
Another model for adaptive regulation of System A in CHO-
cells (Chinese hamster ovary cell) was proposed by
Englesberg group (1986). This model suggested that System A is
regulated by at least two regulatory genes, R1 and R2. R1
produces an apopressor/inactivator (apo-ri) that is in
equilibrium with a repressor/inactivator (ri). The elevated
transported amino acids shift the apo-ri to ril which inhibits
the transcription of the gene encoding System A transporter,
and converts the existing transporter to an inactive state. R2
produces a constitutive repressor r2 which also negatively
regulates the gene A. Insulin binds to its receptor and
through an unknown pathway converts r2 to its inactive form.
The absence of transported substrate and the presence of
insulin have a synergistic effect on stimulating System A
activity.
One study in a kidney cell line (MDCK) indicates the
involvement of protein kinase C in System A regulation (Dawson
& Cook, 1987).
Even though System A adaptive regulation was intensively
studied, the molecular and cellular mechanisms of intestinal
transport regulation of any amino acid are still unknown. The
recent cloning of the System A cDNA (Kong et al., 1993) will


21
undifferentiated sub-confluent cells are morphologically and
biochemically equivalent to immature crypt cells, and
differentiated post-confluent cells are undistinguished both
morphologically and enzymatically from mature villus tip
enterocytes (Hidalgo, 1988, 1989, 1990). The Caco-2
undifferentiated sub-confluent -> differentiated post
confluent state developmental steps mimic the enterocytes
crypt -> villous maturation process. Many studies favorably
recognize the Caco-2 cell line as an ideal in vitro analog of
normal small intestine enterocytes (Zweibaum et al., 1983,
1991).
Organic solute transport studies on Caco-2 cells have
revealed the same characteristics as those from other in vitro
and in vivo small intestinal preparations (Blairs et al, 1987;
Mohrmann et al, 1986; Nicklin, 1992). A few studies have been
conducted regarding glutamine and proline transport
characteristics in Caco-2 (Nicklin et al., 1992; Souba et al.,
1992). These studies paralleled to those in other intestinal
preparations.
In addition to characteristics indistinguishable from
enterocytes, Caco-2 cells excel in providing a well-controlled
homogenous population over a prolonged life span during cell
development and differentiation. Uncontrolled adverse systemic
factors found in vivo preparation are eliminated in the cell
culture systems, so that the effect of a single variable can
be studied in an unbiased setting. The Caco-2 cell line makes


Alanine uptake, nmole mg hr (Day
CD
102


Slope
4
1/[Alanine], 10 L/mol


200
160
120
80
40
0
i i i i i 1 1 1 1 1 r
J 1 1 i i i i i i i i i i i i I
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Cell age, day
251


50
In a separate study, the 50 mM [3H]-MeAIB uptake rates
were < 5% of the same concentration of alanine, suggesting a
minimal contribution by System A in our Caco-2 cells line.
3.3.8 Alanine Uptake on Porous Filters
Uptake of alanine (50 /iM) into the apical and basolateral
surfaces of confluent Caco-2 monolayer grown on porous filters
were measured. The Caco-2 cell monolayer confluency was
determined by measuring the trans-cellular resistance, with
the trans-epithelial resistance > 300 n x cm2 was considered
confluent. The apical compartment to the basal compartment of
50 alanine uptake was measured (Fig. 3-23). The majority of
alanine across the apical membrane was accumulated inside the
cells rather than transport across to the basal side (Fig. 3-
24) .
Alanine (50 /M) uptake at the basal membrane to cytosol
and apical compartment was also measured (Fig.3-25, 3-26, and
3-27). The uptake in NaCl medium was greater than that in the
choline Cl medium, indicating a Na+-activation event.
3.4 Discussion
The alanine uptake in Caco-2 monolayer at the different
cell ages was studied. The alanine uptake activity was
different at various cell age, indicating the possible
regulation of cell development. There were several pathways
for alanine uptake. By using the membrane transport system


Alanine uptake, per cent
N)
Cn
cn
o
-^i
cn
o
o
K>
cn
ai
o
'vj
cn
|\J K>
O N>
o cn
Control
h TGFa
EGF
CHX
H CHX+TGFa
CHX+EGF
J i i L
J L
9TZ
250


Fig. 4-1. Arginine uptake time course
The uptake of arginine (5 /M and 1 xnM) was measured in
choline Cl and NaCl uptake media in the day 2 and day 9 cells.
The total arginine accumulation inside the cells was measured at
various time periods (0 30 minutes) At each point, the total
arginine uptake in choline Cl medium was > 90% of those in the
NaCl medium. The data shown was from the arginine (5 /liM) uptake
in day 2 cells, similar results were obtained in other cell ages
(day 9) and other arginine concentrations (1 mM) Na+-independent
pathway was the major uptake mechanism in Caco-2 cells.


46
alanine uptake rates in the choline Cl, KC1, or LiCl media
were not significantly different. Alanine uptake in the NaCl
uptake medium was not significantly different from that in the
medium containing sodium gluconate.
These data indicated that alanine uptake activation was
strictly Na+-dependent. Other cations such as Li+ or K+ could
not substitute Na+ in activating alanine transport. Chloride
was not required for the Na+-activation.
For all the subsequent experiments, the Na+-dependent
fraction of alanine total uptake was obtained by subtracting
the uptake measured in choline medium from the total uptake
measured in sodium medium.
3.3.4 The Effect of pH on Alanine Uptake
The uptake of 50 M alanine in both the NaCl and choline
Cl uptake media was measured at various media pH ranging from
pH 6.0 to pH 8.5. HEPES and Tris were used to adjust media pH.
The total alanine uptake rates in both the NaCl and choline Cl
media increased steadily as the medium pH increased (pH =6.1,
7.4, and 8.5) (Fig. 3-5).
3.3.5 Na*-Activation of Alanine Uptake in Caco-2 Cells
The uptake of 50 xM alanine was measured in the uptake
media containing NaCl ranging from [NaCl] = 0 mM to 137 mM
(choline Cl as substitute). The total alanine uptake rates at
both day 2 and day 9 cells increases as media NaCl


Fig. 2-28. Alanine basal to apical trans-cellular uptake
in cells grown on porous filters


206
6.3.2 The EGF/TGF<* Pulse Stimulation Effects
The Caco-2 cells were pre-incubated with TGF (20 ng/ml)
or EGF (100 ng/ml) for 2 hours, and incubated in the serum-
free medium (lacking TGF/EGF) for the remaining 46 hours. The
System B activity was not increased by the TGF/EGF pulse
treatments.
6.3.3 The TGF/EGF Effect on the System v* Activity
The Caco-2 cells were pre-treated with TGF (20 ng/ml) or
EGF (100 ng/ml) in serum-free medium for various length of
time (0-48 hours) Like the System B, the System y+ activity
was increased by TGF/EGF only after 30 hours of continuous
incubation. At 48 hours of incubation, TGF increased the
System y+ arginine (5 xM) uptake by 80% and EGF increased the
arginine uptake by 70% (Fig. 6-2) The addition of IOjliM
cycloheximide or 6.6 /lxM chelerythrine in the TGF or EGF
incubation medium blocked the TGF/EGF stimulation effects
(Figs. 6-3, 6-4). The addition of 50 nM calphostin C also
blocked the TGF/EGF effects. H-7 (later disclaimed as a
defective H-7 isomer by Sigma) did not have any effect on the
System y+ activity.
6.3.4 The TGF/EGF Pulse Effect on System v+
The Caco-2 cells were pre-incubated with TGF (20 ng/ml)
or EGF (100 ng/ml) for 2 hours, and then incubated in the same
serum-free medium (lacking TGF/EGF) for the remaining 46


UNIVERSITYOFFIORIDA
3 1262 08554 8302


226
every 6 hours. The 50 /iM alanine system B uptake activity was
measured immediately after each incubation point. The system
B alanine uptake activity was stimulated by a prolonged TPA
incubation (> 8 hours). The stimulation effect increased
steadily up to at least 24 hours (Fig. 7-1). At incubation
times less than 8 hours, there was no TPA effect on the system
B activity.
The system L alanine uptake was not stimulated by TPA
incubation (0-24 hours). Due to the increase cell protein
synthesis, the system L alanine uptake per mg protein actually
decreased.
7.3.2 TPA Pulse Effect on System B Activity
The Caco-2 cells were pre-treated with 0.5 /iM TPA for 0 -
2 hours, and then were washed and incubated in the serum-free
medium (lacking TPA) for the remaining time period (22 hours).
The total incubation time was 24 hours, including the TPA
incubation plus the following non-TPA incubation. System B
activity was then measured immediately after the total
incubation. Unlike their continuous incubation counterparts,
the pulse incubation did not affect the system B activity.
7.3.3 Dose Response for Phorbol Ester (TPA) Stimulation of
System B
The Caco-2 cells were pre-incubated in the serum-free
medium with various concentration of TPA (1 pM 10 /liM) for 24
hours prior to the uptake experiments. The 50 iM alanine


23
independent Systems y+ in Caco-2 cells grown on solid surface
or on porous membrane filters. We further hypothesize that the
membrane's constitutive activities for System B and System y+
each decrease over time during Caco-2 enterocyte
differentiation and development. The activity of each
transport system can be up-regulated above the constitutive
level by two categories of regulating agents: (i) substrate
analogues served by each transporter, and (ii) the peptide
growth factors epidermal growth factors (EGF) and transforming
growth factor (TGF). Finally, we hypothesize that up-
regulation of transporter activities occurs in two phases: an
acute phased characterized by protein-synthesis-independent
substrate trans-stimulation, and a chronic prolonged phase
that likely involves protein kinase C and de novo protein
synthesis.
1.4.3 The Specific Aims
Aim 1: To kinetically classify the alanine and arginine
transport systems in the Caco-2 apical membrane and to examine
the changes in the constitutive baseline transporter
capacities of the sodium-dependent alanine transporter (System
B) and the sodium-independent arginine transporter (System y+)
during the Caco-2 epithelial development and differentiation.
Aim 2: To examine the acute and the prolonged phases of
individual amino acid substrates in increasing System B and
System y+ transporter capacities, in undifferentiated and


259
B and System y+ transport activities. A wide variety of other
growth factors and hormones were without effect. The relative
transport capacities of Systems B and y+ paralleled the
activation or inhibition of protein kinase C. Furthermore,
inhibition of protein kinase C or inhibition of protein
synthesis each prevented the EGF/TGF activation of Systems B
or y+.
In addition to the biochemical and histological
similarities of Caco-2 cells and normal small intestinal
enterocytes, the Caco-2 apical membrane also possesses the
same alanine and arginine transport systems found in the small
intestinal epithelial brush border membrane. The transport
activities changes that occur during Caco-2 cell development
also resemble those found in the small intestinal cells. The
independent regulation of the stated alanine and arginine
transporters by their transported substrates, was strikingly
consistent with that measured in vivo.
This study provides a better understanding of the
mechanism of small intestinal nutrient absorption, provides
information concerning enterocytic development, and helps our
understanding of the fundamentals physiology of epithelial
transporter regulation. This project provides an excellent in
vitro model for future studies of intestinal regulation of
nutrient absorption in states of health and disease, including
adenocarcinoma development.


Fig. 3-23. Na*-dependent alanine and Na*-dependent glucose uptake at
various cell ages
Na+-dependent alanine (50 /M) uptake and Na+-dependent -
methyl-glucoside uptake (Blais et al., 1987) was shown as a
function of Caco-2 cell ages. The alanine uptake decreased, while
the glucose uptake increased with advancing cell age. 100% alanine
uptake = 0.5 nmole/mg/min; 100% -methyl-glucoside uptake = 0.12
nmole/mg/min.


118
described in the chapter 2 general methodology section. The
uptake experiments with special treatments will be mentioned
below where appropriate.
4.3 Results
4.3.1 Arginine Uptake Time Course
The uptakes of 5 /M and 1 mM [SH]-arginine were measured
in the Caco-2 monolayer (cell ages day 2 and day 9) at
increasing times (0 30 minutes) in uptake media containing
137 mM NaCl or 137 mM choline Cl. A representative time course
for 5 iM arginine uptake in day 2 cells is shown (Fig. 4-1) .
During the course of uptake, the [3H]-arginine accumulated
inside cells in NaCl medium was not different from that in the
choline Cl medium. This indicated that the arginine uptake was
mainly a Na+-independent phenomenon. The initial arginine
accumulation in the cells was linear during the initial 10
minutes (at both the [3H]-arginine concentrations of 5 /M and
1 mM) The initial arginine uptake rate that represent the
transport activity was obtained by dividing the total arginine
accumulation by the uptake time period (within the linear
accumulation limit). The uptake period of 0 5 minutes was
chosen for all the subsequent uptake measurements to ensure
the uptake rates represented the true initial arginine
transport.


39
2.3 Statistical Analysis
All experiments were conducted in triplicate (including
the 0 time blanks, and the uptakes in Na+ and choline buffers)
and all experiments were confirmed in at least two differently
seeded generations of cells. Curve fitting was conducted by
non-linear regression analysis. Values were reported as the
mean + SE. ANOVA used for statistical analysis with Duncan's
test, the level of significant p < 0.05.


169
availability. The specific up-regulation of transporter units
could be an evolutionary adaptation which permitted animals
to effectively adapt to their changing environment
surroundings, provided that no toxic effects would occur.
5.4.3 System v* Activity Induced bv its Own Substrate
System y+ activity was induced after the cells were
exposed to only the system y+ substrates (Fig. 5-10). The
inductive potency of these amino acids was directly related
to the potency of these amino acids in inhibiting the system
y+ transport by analogue cross-inhibition. In other words,
the amino acids which were transported by system y+ induced
the system y+ activity (Figs. 4-6,7; Fig. 5-10). As we
discussed in above, only the System y+ substrate induced the
System y+ activity, and only System B substrates induced
System B activity; there was no interference between the two
systems. System B and System y+ are specifically and
independently regulated.
For the acute phase of System y+ stimulation, the
activity was not sensitive to cycloheximide. This rules out
a mechanism of new transport protein synthesis. There are two
acute activation mechanisms, trans-stimulation and trans
location. Our kinetic study unveiled changes in both the Vmax
and K,,, for the substrate-induced System y+, supporting the
trans-stimulation theory. Future studies of acute stimulation
should investigate phosphorylation event and measurement using


224
Many protein kinase C inhibitors have been developed, and
are classified according to their working mechanisms.
Inhibitors such as H-7 and calphostin C bind to the regulatory
side, while inhibitors such as chelerythrine bind to the
catalytic subunit. Either of above step will block the PKC
activation.
Calphostin C blocks the binding of phorbol ester to the
PKC when photo-activated by a fluorescent light. Calphostin C
is exceptionally selective for PKC, with inhibitory
concentration much less than that required to inhibit protein
kinase A or other protein tyrosine kinases (Kobayashi et al.,
1989; Bruns et al., 1991).
Chelerythrine inhibits protein kinase C activity at a
concentration over 100 times lower than that for inhibition of
protein kinase A, protein tyrosine kinases, or the Ca++-
calmodulin-dependent protein kinases (Herbert et al., 1990).
Therefore, both the calphostin C and chelerythrine are
excellent protein kinase C inhibitor with high selectivity.
7.2 Methods And Materials
7.2.1 Pre-treatment with Phorbol Esters
The Caco-2 cells were washed three time with the serum-
free-medium, and incubated in the dark or under fluorescent
light with the same medium containing: (1) control group,
serum-free DMEM plus the same amount of DMSO as appeared in
the phorbol esters (DMSO was < 0.5% of the medium volume) (2)


Slope
1/[Alanine], 10 L/mol
100


150
day 9 cells
O day 2 cells
CD
E
_CD
O
E
Q_
a
o_
D
C
c
CD
<
120
60
30 L
6.0
6.6
o
o
7.2 7.8
PH
-#
8.4
131


119
4.3.2 Arginine Uptake Rates Decrease with Caco-2 Cell Age
The 5 juM [SH]-arginine uptake rates in choline medium
were measured at various Caco-2 cells age from cell ages of
1 day old to 14 days old. The arginine uptake in the choline
Cl uptake media decreased while the cell age increased (Fig.
4-2). The decline in arginine uptake rates was more rapid in
the pre-confluent cells than for the older cells. The decrease
in arginine uptake was paralleled the decrease in alanine
uptake with increasing cell age (Fig. 3-2). As we discussed
in Chapter 3, the cell proliferation rates also decreased with
advancing cell ages (Fig. 3-3). The arginine uptake change and
the alanine uptake change with cell ages may well be due to
the decrease of cell proliferation rates at older cells. As
discussed below, the passive diffusion coefficient for
arginine uptake was the same in day 2 and day 9 cells,
suggesting the decrease in arginine uptake with cell age was
due to a mechanism other than diffusion.
4.3.3 The Effect of pH on Arginine Uptake
The Na+-independent arginine uptake was measured in
choline Cl medium at pH 6.4 8.4 (HEPES/Tris buffers). The
arginine uptake in both the day 2 and day 9 cells was
unaffected by uptake buffer pH changes (Fig. 4-3).


162
to bind alanine and/or Na+
5.3.6 System B Activity Increased by Chronic Alanine Exposure
was Dependent on Protein Synthesis and PKC activation
The Caco-2 cells were washed with the depletion medium,
and incubated in the same medium containing 0, 0.1, 1, or 10
mM alanine, 10 /M cycloheximide or 6.6 /M chelerythrine Cl
for 24 hours. The medium was changed every 6 hours. The 50 /liM
alanine system B activity was then measured. The system B
activity was increased by various alanine exposures, with
greater alanine concentrations causing a greater stimulation
effect. The alanine stimulation was partially blocked by
cycloheximide or chelerythrine (Fig. 5-7 & Fig. 5-8).
Caco-2 cells were then pre-incubated with alanine for 24
hours, as described above, and the cells were then incubated
in the depletion medium for 3 hours before System B activity
measurement. System B activity was increased by exposure to
alanine, and this increase was then completely blocked by
cycloheximide or chelerythrine.
5.3.7 System y+ Activity Decreased in Starved Caco-2 Cells
The Caco-2 cells were washed three times with the
depletion medium, and then incubated in the same medium 1
mM L-arginine or D-arginine for various length of time (30
seconds to 48 hours) in the 37C incubator. One millimole
mannitol was used as control. The medium was changed every 6


Fig. 4-5. Eadie-Hofstee transformation of Na+-independent arginine
uptake kinetics in day 2 and day 9 cells
The arginine (0.1 /M 1 mM) uptake rates described in the
fig. 4-4 was showed as a function of Jarg/[arg]. The Na+-independent
portion was the difference of total arginine uptake in choline Cl
medium and the passive diffusion at the [arginine]. Non-linear
regression of the data gave straight lines, indicating a single
transport system in each of these day cells. Viax was 430
pmole/mg/ml in day 2 cells, and Vax = 340 pmole in day 9 cells.
The Km values were K = 31 nmole arginine for day 2 cells, and
Kb = 37 /mole arginine in day 9 cells.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Wiley W. ouba, Jr.
Associate Professor of Surgery
This dissertation was submitted to the Graduate Faculty
of the College of Education and to the Graduate School and was
accepted as partial fulfillment of^the requirements for the
degree of Doctor of Philosophy.
May, 1993
Dean, College of
Medicine
Dean, Graduate School


CHAPTER 5
THE EFFECT OF INDIVIDUAL AMINO ACIDS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
5^1 Introduction
Intestinal epithelial cells encounter various amino acids
concentrations in the lumen at various times. The luminal
concentrations of amino acids depend on the timing of food
intake and on food composition. In contrast to the cells of
many internal organs, the small intestine adaptively up-
regulates its amino acid transport capacity in response to the
increase of amino acids concentrations exposed (Stevens, 1991,
1992a,b; Salloum et al., 1990; Scharrer et al., 1981; Stein
et al., 1987; Ferraris et al. 1988a,b; Diamond & Karasov,
1987; Ferraris & Diamond, 1989).
The transport activity of system A in the hepatocytes and
MDCK cells increased upon prolonged amino acid starvation.
Addition of amino acids to the amino acid deficient medium
resulted in a loss of system A activity (Kilberg et al., 1985;
Boerner & Saier, 1985; Bracy et al., 1985). Substrate
regulation of the system A activity involves a short-term
cycloheximide-insensitive mechanism and a long-term
cycloheximide-sensitive mechanism (Boerner & Saier, 1985).
Whether the short-term inhibition is involved a trans-
156


cr>
E
_ o
E
c
CD
O
Q_
D
CP
L_
<
_J
127


47
concentration increase. The uptake rates as a function of NaCl
concentrations gave a hyperbolic shape (Fig. 3-6). The non
linear regression analyses of the Hill equation gave the same
Na+-activation Hill coefficient (n = 1) at each cell age,
while the VBax was greater in day 2 cells than in day cells.
The same Hill coefficient (n = 1) indicates that one Na+ binds
to transporter coupled with each alanine molecule transported,
in cells 2 days and 9 days old.
These data indicated that the alanine uptake capacity was
greater in day 2 cells. Therefore, the difference in transport
capacity between day 2 and day 9 cells was not due to the
transport system's affinity for Na+-activation coefficient
changes.
3.3.6 Alanine Uptake Kinetics
The alanine uptake in uptake media containing 137 mM NaCl
or containing choline Cl was measured at various [3H]-alanine
concentrations ranging from 10 /xM to 5 mM at the cell ages of
day 2 and day 9. The kinetics at day 2 and day 9 cells were
shown as uptake rates measured as a function of alanine
concentration (Fig. 3-7; Fig. 3-8). The kinetics in either the
NaCl or choline Cl medium each displayed both saturable and
non-saturable components, indicating multiple transport
systems were involved. At each alanine concentration, the
uptake rate was higher in the NaCl medium.
In the choline Cl media, alanine uptake occurred via two


Fig. 4-2. Arginine uptake at various cell ages
Arginine (5 /xM) uptake was measured in choline Cl uptake
medium over the cell ages of 2 14 days old. The uptake rates
decreased as cell age increased, with rapid decrease in the pre
confluent cells (< 6 days old) .


Fig. 7-8. Eadie-Hofstee transformation of TPA-induced System B
activity kinetics
The kinetic data in Fig. 7-7 was re-plotted as a function
of Jala vs. J/[ala]. TPA increased the Vax in both day 2 and day
9 cells, while the K was not affected.


Fig. 5-6. The acute effect of amino acids on MeAIB uptake
Na+-dependent MeAIB (2.5 x 10'8 M) uptake was measured in
cells (2 days old) after cells had been incubated in salt medium,
or salt medium plus 1 mM MeAIB, 1 mM alanine, or 1 mM serine for
3 hours. The MeAIB incubation inhibited the MeAIB uptake (p <
0.05, n = 3), while alanine or serine incubation had not effect
on the MeAIB uptake (p > 0.05, n = 3).


DIXON PLOT
Inhibitor = [Ornithine], mM
141


Fig. 4-13. Arginine apical to cytosol and apical to basal uptake
in cells grown on porous filters
The arginine (5 /xM) movement from the apical side to
cytosol and apical to basal in 30 minutes was measured.


P6
15.0


ACKNOWLEDGEMENTS
I would like to thank Dr. Bruce Stevens, chairman of my
supervisory committee, for his tremendous support in both my
professional and personal life. Words can hardly express my
deep appreciation. Working under his supervision has been a
wonderful experience.
I would also like to thank members of my supervisory
committee, Dr. Edward Copeland, Dr. George Gerencser, Dr.
Michael Kilberg, and Dr. Souba for their support and valuable
suggestions.
I would like to express my appreciation to Dr. Colin
Sumners for providing the cell culture equipment through out
my study. I would also like to thank Ms. Tammy Gault, for
teaching me cell culture technique and sharing the cell
culture equipment.
I would like to take this opportunity to thank all the
faculty members of the Physiology Department for making
graduate study an enjoyable experience.
Finally, I specially thank my wife and my family for
their unconditional support and great family value.
iii


Arginine uptake, pmole mg min
i
>
N>
cn
o
cn
O
cn
o
O
o
O
O
o
zzz
300


58
regardless of the cell age. The kinetics also indicated that
the transport capacity was greater in the day 2 cells, and
that the activity difference between the two days was like
caused by the change in functional transporter units expressed
in the membrane, rather than modification of existing
transporter affinity.
3.5 Summary
Alanine is transported in Caco-2 cell by a Na+-dependent
transport System B, a Na+-independent transport System L, and
simple passive diffusion. These same systems were operative in
both the undifferentiated and differentiated cell states. The
passive diffusion coefficient was not affected by cell
development. The alanine transport Systems B and L activities
are down-regulated as the cell develops, coincident with the
cell proliferation rates. The decrease in transport activities
are likely caused by the decrease in copies of functional
transporter units, rather than modification of existing
transporter affinity for substrate or ions.


4
from the lumen. Amino acids cross surface by a simple passive
diffusion plus some carrier-mediated transport mechanisms. In
some instances, the amino acid is transported via a secondary
active transport mechanism against its own electrochemical
gradient across the membrane. The accumulated amino acids in
the cytosol then exit via passive diffusion or/and carrier-
mediated transport mechanism at the basolateral membranes.
Each of the barriers can influence the rate of the amino acid
absorption: the "true concentration" of the amino acids
reaching the brush border membrane is determined by the amount
of substrate within the unstirred water layer, rather than the
bulk phase concentration present in the lumen. The brush
border membrane and the basolateral membrane possess
biochemically and histologically different structures. Some of
the amino acid transport systems at the brush border membrane
are not found in the basolateral membrane and the other
internal organs (Stevens, 1992a,b). On the other hand, the
basolateral membrane possesses many of the same transporters
as the membrane found at other internal organs.
1.2.2 Modes of the Amino Acid Transport in the Small Intestine
The membrane amino acid transport movement is
classified into two general categories: simple passive
diffusion and carrier-mediated transport. The carrier-mediated
transport is further divided to Na+-independent facilitated
transport and Na+-dependent secondary active transport


Fig. 5-10. The acute effect of amino acids on System v+ activity
System y+ arginine (5 ^M) uptake was measured in cells (2
days old) which had been incubated in salt medium, or salt medium
plus 1 mM of various amino acids for 3 hours. The System y+
arginine uptake was stimulated by 180% by system y+ substrates
lysine, ornithine, or arginine. Non-system y+ substrates alanine,
serine etc did not stimulated the arginine uptake. Similar results
were also obtained in 9 days old cells.


268
Moolenaar, W. H., Bierman, A. J., Tilly, B. C., Verlaan, I.,
Defize, L. H., Ullrich, A., and Schelessinger, J. 1988.
A point mutation at the ATP-binding site of the EGF
receptor abolishes signal transduction. EMBO J. 8:707-710
Munck, B.G. 1981. Intestinal absorption of amino acids. In:
Physiology of the Gastrointestinal Tract, edited by
Johnson-LR. Raven Press, New York, pp 1097-1122
Oxender, D.L. and Christensen, H.N. 1963. Distinct mediating
systems for the transport of neutral amino acids ny the
Ehrlich cell. J. Biol. Chem. 238:3686-99
Pan, M., Neu, J., Stevens, B.R. 1991. Regulation of alanine
transport in human intestinal epithelial cell line Caco-
2. FASEB J. 5(4):A760
Ray, P. Moy, F.J., Montelione, G.T., Liu, J.F., Narang, S.A.,
Scheraga, H.A., Wu, R. 1988. Structure-function studies
of murine epidermal growth factor: expression and
site-directed mutagenesis of epidermal growth factor
gene. Biochemistry. 27(19): 7289-95
Reiser, S. and Christiansen, P.A. 1971a. The properties of the
preferential uptake of L-leucine by isolated intestinal
epithelial cells. Biochim. Biophys. Acta. 225(1): 123-39
Reiser, S. and Christiansen, P.A. 1971b. Inhibition of amino
acid uptake by ATP in isolated intestinal epithelial
cells. Biochim. Biophys. Acta. 233(2): 480-4
Reiser, S. and Christiansen, P.A. 1971c. Stimulation of basic
amino acid uptake by certain neutral amino acids in
isolated intestinal epithelial cells. Biochim. Biophys.
Acta. 241(1): 102-13
Rousset, M. Laburthe, M. Pinto, M, Chevalier, G. ,
Rouyer-Fessard, C. Dussaulx, E. Trugnan, G. Boige, N. ,
Brun, J.L., Zweibaum, A. 1985. Enterocytic
differentiation and glucose utilization in the human
colon tumor cell line Caco-2: modulation by forskolin. J.
Cell. Physiol. 123(3): 377-85
Saier, M.H. Jr., Daniels, G.A. Boerner, P. Lin, J. 1988.
Neutral amino acid transport systems in animal cells:
potential targets of oncogene action and regulators of
cellular growth. J. Membr. Biol. 104(1): 1-20


54
instead of some modification of transporter affinities.
The amino acid analogue inhibition patterns on both the
day 2 and day 9 cells were similar in that phenylalanine,
leucine, BCH, and alanine strongly inhibited the alanine
uptake, while MeAIB, glycine and lysine were weak inhibitors
(Figs. 3-10, 3-11). These inhibition patterns strongly
resemble that of the System L. The non-interaction with lysine
ruled out the possibility of the System b0,+ The strong BCH
inhibition suggesting the unlikely System asc.
Based on the kinetic characteristics, the pH sensitivity,
and the cross-inhibition pattern, We conclude that the Na+-
independent alanine uptake is via the System L at both the day
2 and day 9 cells.
3.4.2.3 Na*-deoendent alanine transport System B
The alanine uptake in the Caco-2 cells was strongly Na+-
dependent in either day 2 old and day 9 old cells (Fig. 3-4).
The Na+-dependent portion was more than 85% of the total
alanine uptake in NaCl medium at 50 /xM alanine (Fig. 3-4) No
other monovalent cationic K+ or Li+ substitute for the Na+ in
activating the alanine uptake. Furthermore, the system was not
activated concomitantly by Cl'.
One of the important aspect in classifying transport
systems was the cross-inhibition profile. The amino acid
analogue inhibition pattern of the Na/alanine uptake for both
the day 2 and day 9 cells was similar: the Na/alanine
transport was strongly inhibited by threonine, serine,


29
falcon tissue culture dishes in 15 ml Sigma's Dulbecco's
Modified Eagle Medium (DMEM; Sigma Co, St. louis, MO)
containing 4.5 g/1 glucose and 0.584 g/1 glutamine, and
supplemented with: 10% fetal bovine serum (Sigma Co, heat
untreated catalog # F 4884)), 3.7% sodium bicarbonate, 100
IU/ml penicillin, 100 ng/nil streptomycin (Sigma Co., St louis,
MO) and 1% non-essential amino acids (Sigma Co. St. Louis,
MO). The original seeding cell density was 3 x 105 cells/ml,
cells were counted using a hemacytometer. Cells were grown in
a humidified incubator at 37C in 10% CO2/90% 02. The day of
seeding was designated as day 0. The growth medium was changed
and cells were inspected daily.
2.1.2.1 Caco-2 cell subculturina
For the sub-culturing cells, cells four days old on the
100 mm dish were taken out of the incubator. The growth medium
was aspirated, and cells were washed once with 37C isotonic
calcium-free saline solution containing 0.05% trypsin and
0.02% ethylenediamine-tetraacetic acid (EDTA; Sigma Co.
St.Louis, MO) and immersed in 10 ml the same solution for 5
minutes in the sterile hood. The cell/trypsin mixture was
dispersed with a narrow tip glass pipette (Fisher Scientific,
Pittsburgh, PA) and the trypsin reaction was stopped by adding
DMEM with 10% FBS. Then the dispersed cells were sedimented in
a sterile conical centrifuge tube (Corning, Corning, NY) at
1000 x g for 5 minutes, and the supernatant was removed. Next,
growth medium was added to re-suspend the cells (using a


Fig. 3-9. Eadie-Hofstee transformation of Na+-dependent alanine
uptake kinetics in day 2 and day 9 cells
The Na+-dependent alanine (10 /M 5 mM) uptake of fig.
3-7 was expressed as alanine uptake as a function of alanine
uptake/alanine concentration. Non-linear regression of these data
gave a straight line, indicating a single transport system. The Viax
values (the interception of the line and the y axis) were Vn)ax =
3.1 0.21 nmole/mg/min for day 2 cells, and Vaax = 0.51
nmole/mg/min for day 9 cells. The Km values (the negative slope
of the line) were Km = 167 26.1 /mole alanine for day 2 cells,
and Km = 159.0 13.6 /mole alanine for day 9 cells.


Time, minutes
O


9
al, 1984), pig (Maenz et al., 1992), dog (Bulus, 1989), human
fetal (Malo, 1991), lower vertebrate small intestines (Ahearn
et al., 1991), and in the undifferentiated and differentiated
enterocytic Caco-2 cells (Pan et al, 1991; Souba et al.,
1992). Another distinct amino acid transport system found only
at the intestinal apical membrane is System IMINO (Stevens &
Wright, 1985, 1987; Wright et al, 1985; Stevens, 1992a,b).
System IMINO is also a strictly Na+-dependent system highly
selective to heterocyclic imino acid such as proline and
pipecolate. System IMINO uptake has been reported in many
species intestine (Ahearn et al., 1991; Karasov et al., 1986,
1987; Moe et al., 1987; Munck, 1983; Stevens et al., 1984,
1992a,b).
Na+-dependent transport Systems A and ASC (Christensen et
al., 1965; Oxender et al., 1963; Kilberg et al., 1981, 1993),
serve dipolar amino acid in the non-epithelial cell membrane,
and reportedly exist in the guinea pig apical membrane (Del
Castillo & Muniz, 1991; Hayashi et al., 1980). But up to date,
no definite test has been able to discriminate them from the
System B or System IMINO. Other Na+-dependent systems, XAG'
serving D-aspartate and glutamate, 0 serving fi-alanine and
taurine were also reported in the intestinal apical membrane
(Hofper, 1987; Munck, 1990, 1992; Miyamoto et al., 1990a,b).
Some studies also suggested possible existence of a System N
for glutamine transport (Salloum et al., 1990, 1991).
There are three Na-independent transport systems at the


231
did not affect activity (Fig. 7-11).
7.3.14 The Effect of Phorbol Ester on the System y* Transport
Kinetics
The Caco-2 cells were pre-treated with TPA in the serum-
free medium for 24 hours prior to the uptake experiments, and
the system y+ arginine transport kinetics were measured ([3H]
arginine = 0.1 /xM 1 mM) The VBax was increased by TPA in
both the day 2 and day 9 cells (VBax = 777 pmole/min/min in day
2 cell with VBax = 1111 pmole/mg/min in TPA treated day 2
cells, VBax = 541 pmole/mg/min in day 9 cells and VBax = 720
pmole/mg/min with TPA treatment). The K, of 43.3 and 55 /xM
arginine was the same regardless the cell age and the TPA
incubation (Fig. 7-12).
7.4 Discussion
The phorbol ester TPA stimulated both System B and System
y+ activities. The TPA effects on transporter activities were
similar in terms of the potency, the onset of stimulation, and
the dependency of de novo protein synthesis.
TPA diffuses through cell membrane and directly binds to
the protein kinase C regulatory domain, the endogenous
diacylglycerol binding site. TPA possesses structural
similarities with diacylglycerol, and both activate protein
kinase C. TPA can acutely activate protein kinase C, or
chronically inactivate PKC by depleting the cell of active
membrane-bound form of PKC.


30
narrow tip glass pipette) until cells were separated- Possible
cell clumps were allowed to settle for a few minutes at 1 x g,
and only the top layer of medium containing single clumped
cells was used for sub-culture as confirmed by microscope
examination. Cells then were seeded in three ways: (1) seeded
in the 100 mm dish at a cell density of 3 x 105 cells/ml for
future sub-culture purpose, (2) seeded in the 35 mm Falcon
tissue culture dishes (6-well clusters; Becton Dickinson,
Lincoln park, NJ)) at a density of 1.93 x 105 cell/ml for
subsequent transport experiments, and (3) seeded in 24 mm
porous filter at a cell density of 1.93 x 105 cells/ml for
subsequent transport experiments. All cell culture procedures
were performed under sterile conditions in a hood. All
solutions used in cell culturing were filter-sterilized (0.2
MM membrane filter; Nalge, Rochester, NY; Millipak 20,
Millipore Co, Bedford, MA), or/and autoclave sterilized. The
growth medium, including the medium in the both upper and
lower chambers of porous filter (Costar Co, Cambridge, MA),
was changed daily. The cultures are inspected daily (using a
phase contrast microscope) to monitor cell growth (dome
formation, absence of contamination, etc).
2.1.2.2 Freezing Caco-2 cells
Four-day-old Caco-2 cells grown in the 100 mm dishes were
trypsinized and centrifuged as described in above subculturing
section. Cells were then re-suspended in 4C DMEM containing
10% FBS and 5% dimethyl sulfoxide (DMSO; Sigma Co, St.Louis,


[Phenylalanine].
3
ai
i
cr>
Gj
O
Oj
a>
IV)
Oi
86
4.8


Fig. 4-7. Arginine Na*-independent uptake inhibition pattern in day
3 and day 9 cells
Na+-independent arginine (5 /xM) uptake rates with 5 mM
amino acid in uptake media were measured in both the day 3 and
day 9 cells. The uptake rates in the day 3 cells were shown as
a function of the uptake rates in the day 9 cells. The degree of
arginine uptake inhibited by amino acids was similar in both cell
ages.


56
Another Na+-dependent alanine transport system is System
B0,+, which serves both the neutral amino acids and cationic
amino acids. The only evidence that does not support existence
of System B0,+ was that cationic amino acids arginine and
lysine did not inhibit Na/alanine uptake in our study (Figs.
3-12 & 3-22) Thus, it is unlikely that System B0,+ exists in
Caco-2 cells.
The final Na/alanine system candidate System B described
first for intestinal epithelial cells (Stevens et al. 1984).
System B has only been found in the epithelial cells of
vertebrate and invertebrates (Stevens, 1992). The substrate
selectivity of System B is very similar to System B0,+ except
that System B does not interact with cationic amino acids
(Figs 3-12 & 3-22). Na/alanine uptake was strongly inhibited
by of neutral amino acids serine, threonine, cysteine, weakly
by glycine and phenylalanine, interaction with BCH, and was
interactive with cationic amino acids arginine and lysine in
our studies (Figs. 3-12 & 3-22). The amino acid analogue
inhibition pattern supports the existence of System B. The
apparent affinity K, = 159 mole alanine in Caco-2 was similar
to the System B report elsewhere (Stevens et al., 1982) (Fig.
3-9). Furthermore, the pH sensitivity (Fig. 3-5) and the Na+-
activation Hill coefficient (n = 1) (Fig. 3-6) further support
the case for System B. Based on the our evidence (Figs. 3-4
through 3-9, and 3-11 through 3-22), we conclude that the
Na/alanine transport system in the Caco-2 cells was likely


121
4.3.5 Amino Acid Analogue Cross-inhibition
The Na+-independent arginine uptake activities in choline
Cl media which contained single amino acid analogues (5 mM
each of the natural amino acids, ornithine, homoserine plus
sodium, or D-arginine) were measured at both the day 2 and
day 9 cells. The pattern and degree of the amino acid analog
inhibition for the Na+-independent arginine transport was
identical in both cell states, suggesting that same
transporter system was operative regardless of the cell age
(Fig. 4-7). The Na+-independent [3H]-arginine transport was
strongly inhibited only L-lysine, L-arginine, ornithine, and
histidine; weaker inhibitors were D-arginine, D-lysine,
homoserine (in Na+ buffer), tryptophan, and methionine; the
amino acids which inhibited less than 20 % arginine uptake
included alanine, BCH, phenylalanine threonine, serine,
asparagine, valine, homoserine (in choline Cl media) and
leucine (Fig. 4-6). The analogue cross-inhibition patterns
were consistent with that of system y+ (White, 1985) Dixon
analysis of [SH]-arginine uptake inhibited by ornithine,
homoserine, and D-arginine revealed that ornithine was a
classic competitive inhibitor, while the homoserine was a
weaker inhibitor, and D-arginine showed an uncompetitive weak
inhibition effects (Fig. 4-8 to 4-12).


BIOGRAPHICAL SKETCH
Ming Pan, born on February 5, 1963 in ShaoGuan City of
the GuangDong Province, People's Republic of China. His father
is Jiaan Pan, mother is FengQun Chen. He went to elementary
and middle schools in ShaoGuan city. In 1980, after graduating
from high school, he went to Sun Yat-Sen University of Medical
Sciences studying medicine, specializing in surgery. He
graduated from medical school with a Medical Bachelor (M.B)
degree in 1986. He then worked in the YueBei People's Hospital
in ShaoGuan as a surgeon during 1986-1987. He came to United
States of America in 1987. He entered the Ph.D program in the
Department of physiology, College of Medicine, University of
Florida in May 1988. Under the guidance of Dr. Bruce R.
Steven, he has been studying the amino acid transport
regulation in the small intestine. He has completed all the
degree requirements and successfully defended his
dissertation. He is expected to graduate and receive a Doctor
of Philosophy degree in Physiology in May 1993.
272


227
System B uptake activity was then measured immediately after
each incubation. System B alanine uptake activity was
stimulated at concentrations of [TPA] > 10 nM. A peak
stimulation of 2 fold was observed at [TPA] = 1/xM, and the
stimulation effect was attenuated at [TPA] = 10 /M (Fig. 7-2) .
7.3.4 Phorbol Ester Stimulated the System B Activity
Regardless the Cell Age
The Caco-2 cells (1 day old through 35 days old) were
pre-incubated with 0.5 /M TPA in the serum-free medium for 24
hours prior to the uptake experiments. The system B activity
was stimulated at least 2 fold by TPA at all cell ages (Fig.
7-3) At each cell ages, System L uptake was not significantly
altered by TPA.
7.3.5 The TPA Stimulation of System B Activity Involved De
Novo Protein Synthesis
Caco-2 cells were pre-incubated in serum-free medium
containing 0.5 iM TPA, 10 M CHX for 24 hours prior to the
uptake experiments. The 50 /M alanine System B uptake activity
was stimulated 2 fold by the TPA incubation. The addition of
CHX in the TPA incubation medium blocked the stimulation
effect. CHX alone did not significantly affect System B
activity (Fig. 7-4).
Caco-2 cells were also pre-incubated with 0.5 /lxM TPA in
serum-free medium, 0.5 /M actinomycin D for 24 hours. System
B activity increased following TPA exposure. The incubation in


[Proline],
l/JAla (m<3 Pro^e'n) m'n nm0le
- cn m
O O cn
O c c
c s <:
06


163
hours to ensure that the amino acid concentration was constant
and the possible autocrine accumulation was eliminated. The
system y+ transport activities were measured immediately after
each incubation period. The 5 /nM arginine system y+ uptake
decreased as the incubation time increased, and reached the
lowest level at about 3 hours where they stayed 48 hours. The
declining system y+ activity was partially prevented by
exposure to L-arginine or D-arginine (at lesser degree) (Fig.
5-9) The 3 hours depletion incubation was chosen for the
subsequent experiments.
5.3.8 System v* Activity was Stimulated bv Acute Amino Acid
Exposure
The Caco-2 cells were washed three time with the
depletion medium, and incubated in the same medium containing
1 mM individual amino acids (1 mM mannitol as control, all
cysteine solution also contained 1 mM dithiothreitol in the
case 1 mM dithiothreitol was used as control) for 3 hours.
The System y+ activity was measured immediately after each
incubation. The System y+ activity was increased two-fold by
the system y+ substrates lysine, arginine, ornithine. System
y+ non-substrates proline, BCH, and alanine did not affect
the System y+ activity A pattern emerged such that these
amino acids which weakly inhibited System y+ activity also
weakly stimulated System y+ activity (Fig. 5-10). These data
suggested that the system y+ activity was specifically


5
mechanisms.
In the case of the simple passive diffusion, the membrane
electrochemical gradients and permeability coefficients of the
amino acid govern the direction and rate of the passive amino
acid movement across the membrane.
In the case of the Na+-independent carrier-mediated
transport mechanism, amino acid is carried by its specific
transporter across the membrane, directed by the
electrochemical acting across the membrane. In the case of
charged amino acids such as arginine, the negative membrane
potential can drive it against its chemical gradient.
In the case of the secondary active transport, a series
of spatially separated events occur that couple energy derived
from ATP hydrolysis to solute flux. The Na+/K+-ATPase at the
basolateral membrane creates electrochemical Na+ and K+
gradients across the basolateral and the apical membranes. The
Na+/amino acid cotransporters at the apical membrane bind to
amino acids, and utilize this Na+ electrochemical energy
(A/Na) for concentrative uptake of amino acids across the
apical membrane. The accumulated amino acids inside the
enterocytes exit via the Na+-independent facilitated
transporters and the passive diffusion at the basolateral
membranes.
A kinetic model of Na/amino acid transport describing the
secondary active transport has been recently examined Stevens
(Stevens, 1992a) as a paradigm for all Na+-dependent systems.


Cell age, day
Arginine uptake, pmole mg min
K> > cn co O
o o o o o o
621
120


Fig. 7-5. The effect of actinomvcin D on the TPA-induced System
B activity
System B alanine (50 /M) uptake was measured in cells (3
days old) which had been incubated in 0.5 /M TPA 0.5 /jlM
actinomycin D for 24 hours prior to the uptake measurements. TPA
alone stimulated the System B alanine uptake (p < 0.05, n = 6),
while actinomycin D in the tPA medium blocked the TPA's
stimulation effect (p < 0.05, n = 6). Similar results were
obtained in 9 days old cells.


Fig. 6-5. The effects of chelervthrine on the chronic TGF^/EGF-
System v+ activity
System y+ arginine (5 /zM) uptake was measured after cells
old) had been incubated in DMEM, TGF, or EGF, 6.6 fiM CHE
hours. TGF** and EGF each stimulated the arginine uptake, CHE
TGF/EGF incubation media blocked this stimulation effect (p <
= 6) .
induced
(2 days
for 48
in the
0.05, n


Alanine uptake, nmole mg min
o
bo
o
4^
O
O)
O
D
TPZ


43
cysteine, phenylalanine, and BCH.
System b0,+, a Na+-independent analogue of System B0,+
serving neutral and cationic amino acid such as lysine and
arginine. The substrate scope of System b0,+ is similar to
System B0,+. System b0,+ exists in many cell types, but it has
been previously reported in the intestinal membranes.
Only a few organic solute transport studies in the Caco-2
cells have been reported (Blais et al., 1987; Hidalgo et al.,
1988; Mohrmann et al., 1986; Souba et al., 1992, Nicklin,
1992). Each of the reports showed the similarity of solute
transport characteristics in Caco-2 cells and intestinal
epithelial cell.
Inasmuchas alanine transport systems were not described
for Caco-2 culture, our study began by describing the alanine
transport systems in the Caco-2 monolayer.
3.2 Methods And Materials
3.2.1 Methods
The [3H]-alanine uptake experiments were performed in
both the pre-confluent (day 2-3) and confluent (day 8 -9)
cells. The basic uptake procedures were as described above
(chapter 2) Special uptake conditions are presented below
where appropriate.


Arginine uptake, nmole mg
o
b
OJ
b
CD
b
Basal to Apical
Basal to Apical
(+ 0.5 mM Lysine)
Basal to
Cell Accu
mulation
Basal to Cell Accumulation
(+ 0.5 mM Lysine)
gsi
9.0


Fig. 4-15. Arginine basal to cytosol and basal to apical uptake
The arginine (5 /iM) uptake (30 minutes) at basal side in
choline Cl medium 0. 5 mM lysine was measured. Data shown were
the total arginine in the apical side and inside the cells at 30
minute uptake.


Fig. 6-4. The chronic effect of TGF^ or EGF on System v* activity
System y+ arginine (5 /M) uptake was measured in day 2 cells which
had been incubated in DMEM, TGF0', or EGF in DMEM for 48 hours. TGF and
EGF each stimulated the System y+ arginine uptake (p < 0.05, n = 6).
Similar results were obtained in 9 days old cells.


Fig. 5-5. Kinetics of the acute alanine-stimulated System B
activity
System alanine (10 /M 5 mM) uptake was measured in cells
(day 2) which had been incubated in salt medium, DMEM, 5 mM
alanine in salt medium. In the salt incubation, VBax = 0.67
nmole/mg/min and K, = 150 /mole alanine; in the DMEM and alanine
incubation, VmiX = 2.9 nmole/mg/min and K,, = 390 /mole alanine.


Fig. 3-19. Replot of the slopes of Dixon plot with glycine as
inhibitor
The slopes of the dixon plots at Fig. 3-18 were shown as
a function of l/[alanine]. Non-linear regression of these data was
through 0 point of both axis. These data combined with fig. 3-18
suggested that glycine was a weak competitive inhibitor of the Na+-
dependent alanine uptake.


203
acetic acid vesicle as used in the EGF/TGF treatment (< 0.5%
of the medium volume) (ii) treatment with EGF/TGF, DMEM plus
various concentration of (EGF/TGF is diluted from stocks in
0.1 M acetic acid stored at 4C), and (iii) treatment with
EGF/TGF with additional agents, DMEM plus EGF/TGF plus agent
specified in the text, with each specified agent in DMEM as
control. Cell were exposed to the treatments up to 48 hours.
Cvcloheximide (CHX) treatments. The cells were treated
with: (i) control group, serum-free medium, (2) treatment with
CHX, DMEM plus 10 100 /M CHX (CHX was prepared in aqueous
solutions made the day of the experiment), (iii) treatment
with CHX and other agents, DMEM plus 10 100 /xM CHX with
specified agent added in to the medium, internal control used
DMEM plus specified agent. The treatments were for various
lengths of time.
Chelerythrine Cl experiments. The cells were treated
with: (i) control group, only serum-free DMEM, (ii) treatment
with chelerythrine, serum-free DMEM plus chelerythrine
(chelerythrine Cl was diluted from stocks in H20 stored at -
20C), (iii) treatment with chelerythrine Cl and specified
agents, DMEM plus chelerythrine plus specified agent, with
DMEM plus specified agent as internal control.
Calphostin C treatments. Cells were treated with: (i)
control group serum-free DMEM plus the same amount of DMSO as
appeared in the calphostin C solution (<1% of the medium
volume), (ii) treatment with calphostin C, serum-free DMEM


Fig 5-11. The effect of CHX on the acute arginine-stimulated
System v* activity
System y+ arginine (5 /xM) uptake was measured after cells
(2 days old) had been incubated in salt, 1 mM arginine, 50 /M
CHX, or 1 mM arginine plus 50 /M CHX for 3 hours. The arginine
uptake was stimulated by the arginine incubation (p < 0.05, n =
6), CHX had no effect on the induction (p 0.05, n = 6).
Similar results were observed in 9 day old cells.


Fig. 3-7. Alanine uptake kinetics in day 2 cells
The alanine (10 /iM 5 mM) uptake was measured in the day
2 cells. The figure showed the total alanine uptake rates in NaCl,
choline Cl media, or Na+-dependent alanine uptake rate as a
function of alanine concentrations. The curve contained non
saturable and saturable components.


Fig. 7-1. TPA System B stimulation time course
System B alanine (50 /xM) uptake was measured in day 3
cells which had been incubated in DMEM or 1 /xM TPA for various
periods of time (0 24 hours). Continuous exposure (> 8 hours)
to TPA resulted in a increase in alanine uptake.


99
90


230
uptake activity was stimulated up to 2 fold by TPA at all cell
ages (Fig. 7-9).
7.3.11 The TPA Stimulation of System y* Activity Involved De
Novo Protein Synthesis
The Caco-2 cells were pre-incubated in serum-free medium
with 0.5 iM TPA for 24 hours, 50 jiM or 20 /M cycloheximide
including in the incubation medium for various windows of time
(first 6 hours, second 6 hours, third 6 hours, fourth 6 hours,
first 12 hours, first 18 hours, and the entire 24 hour
period) Fig. 7-10 shows that the 5 /xM arginine system y+
uptake activity was stimulated 50% by TPA incubation, but the
stimulation was not retarded by each of the 6 hour CHX
incubation. However, the first 12 and 18 hour periods of
CHX/TPA incubation blocked the TPA stimulation of System y+
activity. The absolute uptake activity was decreased following
24 hours of CHX incubation, but due to the greater decrease in
cell protein, the activity per mg protein was not affected.
7.3.13 The Phorbol Ester Stimulation of System v* Activity was
Inhibited by Specific Inhibitor of Protein Kinase C
The Caco-2 cells were pre-incubated with TPA in the
serum-free medium, 6.6 /M chelerythrine or 50 nM calphostin
C for 24 hours prior to the uptake measurements. System y+
activity was increased by TPA alone. The TPA/chelerythrine or
TPA/calphostin C combination incubation did not stimulate
System y+ activity, and chelerythrine or calphostin C alone


TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
1.1 Introduction 1
1.2 Amino Acid Absorption in the
Small Intestine 2
1.3 The Human Intestinal Epithelial Cell
Line (Caco-2 Cell Line) 20
1.4 The Objective and Aims of the
Present Study 22
2 GENERAL METHODOLOGY 25
2.1 Caco-2 Cell Culturing 25
2.2 Caco-2 Monolayer Transport 31
2.3 Monolayer Transport in Caco-2 Cells
Grown on Porous Filters 35
2.3Statistical Analysis 39
3 CLASSIFICATION OF ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE 4 0
3.1 Introduction 4 0
3.2 Methods and Materials 4 3
3.3 Results 44
3.4 Discussion 50
3.5 Summary 58
4 CLASSIFICATION OF ARGININE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE 113
4.1 Introduction 113
4.2 Methods and Materials 115
4.3 Results 116
4.4 Discussion 120
4.5 Summary 12 3
iv


122
4.3.6 Arginine Uptake in Caco-2 Cells on Porous Filters
Trans-epithelial arginine (5 xM) uptake was measured from
apical to basal sides in confluent Caco-2 cells (14 days old).
The intact confluent monolayers with trans-cellular resistance
> 300 ft cm2 were used. The arginine trans-cellular rate from
apical side to basal side was steady during a 30 minute period
(Fig. 4-13). Cellular accumulation of arginine during 30
minutes was 5 times greater than the apical to basal trans-
cellular movement (Fig. 4-14).
Arginine (5 xM) movement from basal side to cellular and
apical side was also measured in intact confluent Caco-2 cell
monolayers. The basal-apical side transport rate was steady
for up to 30 minutes (Fig. 4-15). During a 30 minute
incubation, the [3H]-arginine trapped in the cellular
compartment was 5 times greater than amount of arginine across
basal-apical compartment (Fig. 4-16).
4.4 Discussion
The arginine uptake activity across Caco-2 cell monolayer
was studied at various cell stages of development. By using
classic transport system criteria, we classified the arginine
transport systems in the Caco-2 in the undifferentiated state
(day 2) and differentiated state (day 9).


157
inhibition mechanism or other regulatory mechanism is still
inconclusive. The long-term regulation involves a
transcriptional and translational regulatory mechanism. Two
regulatory mechanisms have proposed to explain the system A
substrate regulation (Kilberg, 1986; Englesberg et al, 1986).
Substrate regulation of amino acid transport systems of
intestinal epithelial cells ha been investigated in whole
animals fed various diets (Ferraris & Diamond, 1989; Ferraris
et al., 1988a,b). Animal feeds with high protein or high amino
acid diet increased their transport of non-essential amino
acids, such as aspartate. Essential amino acid transport
activity decreased in animals on high amino acid diets. In
general, the transporter substrates are often the best
inducers of uptake capacity, but sometimes the best inducers
are un-related to the transport systems (Stein et al., 1987;
Diamond & Karasov, 1987; Scharrer et al., 1981). The up-
regulation of the intestinal amino acids uptake occurs over
0.5 1 day, whereas the reversal of the up-regulated level
to the original level takes several days.
The mechanism of intestinal substrate induction of
transport has not been addressed at cellular level. In this
study, we explored the effect of individual amino acids to
increase the activity of System B and System y+ in both short
term and long-term exposures.


min
O 4 8 12 16 20
Jarg/fAri3] MLiter mg
. -1
min
257


CHAPTER 1
INTRODUCTION
1.1 Introduction
Amino acid transport by the small intestine is a vital
process involved in whole-body nitrogen balance. Intestinal
absorption studies have received considerable attention during
the past 35 years, primarily focussing on description of the
site of absorption within the mucosa, classifications of
uptake pathways based on substrate selectivity, and
phenomenological kinetic mechanisms of transporter function
using the universal principles of membrane transport (Hopfer,
1987; Munck, 1981; Stevens et al., 1984, Stevens, 1992a,b).
Past and current studies were conducted in a variety of
species and at various stages of development. These studies
have included patients and intact animals with inborn errors
of transport (Desjeux et al., 1980), perfused intestines,
isolated membranes from the brush border and basolateral
surfaces (Stevens et al., 1982, 1984; Mircheff et al, 1879,
1980), and enterocytes freshly isolated from mucosa (Reiser
and Christiansen, 1971a,b,c), or grown as in vitro cell lines
(Pan et al., 1991).
This project concentrates on amino acid absorption from
the outer environment; the role of the intestine in inter-
1


Fig. 7-3. Effect of TPA on System B activity at various cell ages
System B alanine (50 /M) uptake in cells (2 days
days old) which had been incubated in 0.5 /jM TPA for 24
prior to the uptake experiments.
to 35
hours


Fig. 5-1. System B activity in cells incubated in depletion medium
with or without alanine
System B alanine (50 nK) uptake was measured in Caco-2
cells which had been incubated in depletion salt medium ( 1 mM
alanine) for various period of time (0 6 hours). At incubation
period > 1 hour, the System B alanine uptake was significantly
greater in cells incubated in salt plus 1 mM alanine than that
incubated in salt only medium (p < 0.05, n = 6) Data shown were
obtained in day 2 cells, with similar results obtained in day 9
cells.


264
Diamond, J.M., Karasov, W.H. 1987. Adaptive regulation of
intestinal nutrient transporters. Proc. Natl. Acad. Sci.
U. S. A. 84(8): 2242-5
Edelman, A.M., Blumenthal, D.K., Krebs, E.G. 1987. Protein
serine/threonine kinases. Annu. Rev. Biochem. 56: 567-613
Ellis, C., Moran, M., McCormick, F., and Pawson, T. 1990. EGF
stimulates GAP in vitro. Nature 311:377-381.
Englesberg, E., Moffett, J. Perier, F. 1986. Mechanism of
regulation of amino acid transport in CH0-K1 cells and
the insulin connection. Federation Pro. 45(10):2441-2443
Fava, R. A. and Cohen, S. 1984. Isolation of a calcium-
dependent 35 kilodalton substrate for the EGF
receptor/kinase from A-431 cells. L. Biol. Chem 258:2636-
2645
Ferraris, R.P., Diamond, J., Kwan, W.W. 1988a. Dietary
regulation of intestinal transport of the dipeptide
carnosine. Am. J. Physiol. 255(2 Pt 1): G143-50
Ferraris, R.P., Kwan, W.W., Diamond, J. 1988b. Regulatory
signals for intestinal amino acid transporters and
peptidases. Am. J. Physiol. 255(2 Pt 1): G151-7
Ferraris, R.P., Diamond, J.M. 1989. Specific regulation of
intestinal nutrient transporters by their dietary
substrates. Annu. Rev. Physiol. 51: 125-41
Frexes-Steed, M., Warner, M.L., Bulus, N., Flakoll, P. 1990.
Abumrad-NN. Role of insulin and branched-chain amino
acids in regulating protein metabolism during fasting.
Am. J. Physiol. 258(6 Pt 1): E907-17
Gaull, G.E., Wright, C.E., Isaacs, C.E. 1985. Significance of
growth modulators in human milk. Pediatrics. 75(1 Pt 2):
142-5
Gerencser, G.A., Stevens, B.R. 1989. Energetics of
sodium-coupled active transport mechanisms in
invertebrate epithelia. Am. J. Physiol. 257(3 Pt 2):
R461-72
Glenney, J.R. Jr., Chen, W.S., Lazar, C.S., Walton, G.M. ,
Zokas, L.M., Rosenfeld, M.G., Gill, G.N. 1988.
Ligand-induced endocytosis of the EGF receptor is blocked
by mutational inactivation and by microinjection of
anti-phosphotyrosine antibodies. Cell. 52(5): 675-84


J j nmole mg min
1 M 04
boo
9


165
5.3.11 System v* Activity Increased by Arginine Exposure was
a Kinetic Modification Effect
Caco-2 cells were washed three times with the depletion
medium, and incubated in the same medium containing 1 mM
arginine (1 mM mannitol as control) for 3 hours. The system
y+ uptake kinetics were measured over the [SH]-arginine
concentration ranging from 0.1 mM to 1 mM. The kinetics showed
that both the Vmax and K,,, of the system y+ activity was
increased by the arginine pre-incubation (Fig. 5-13 & Fig. 4-
14) .
5.3.12 System v* Activity Increased by Arginine Chronic
Exposure was not a Protein Synthesis-Dependent Process
The Caco-2 cells were washed with the depletion medium,
and incubated in the same medium containing 0, 0.1, 1.0, or
10 mM arginine 10 /M cycloheximide or 6.6 /xM chelerythrine
for 24 hours. The medium was changed every 6 hours. The 5 iM
arginine system y+ activity was increased 7 fold by the
arginine exposure. The degree of arginine uptake increased by
the 0.1, 1.0 or 10 mM arginine incubation was the same.
Cycloheximide or chelerythrine in the incubation medium did
not block the system y+ activity which was increased by the
arginine incubation (Fig. 5-12).
In another study, Caco-2 cells pre-incubated with 1 mM
arginine for 24 hours were then incubated in depletion medium
lacking arginine for 3 hours. System y+ activity increased by


Alanine uptake, nmole mg
NJ
O
. -1
min
zvz
150


161
medium did not affect the alanine uptake, nor did the CHX in
the alanine incubation medium affect the increase of alanine
uptake (Fig. 5-4).
5.3.4 The System B Activity Increase bv Substrate Acute
Exposure was Reversible
The Caco-2 cell were washed and incubated in the
depletion medium containing 1 mM alanine (mannitol as control)
for 3 hours as described above. The cells were then washed
three times with the depletion medium, and incubated in the
depletion medium (lacking amino acids) for 3 hours. The 50 /xM
alanine system B activity was increased 2 fold after
incubation for 3 hours with alanine, and returned to the
control level after the additional 3 hours in alanine-free
depletion medium incubation.
5.3.5 The System B Activity Increased bv its Substrate
Exposure Involved Kinetic Modifications
The Caco-2 cells were washed three time with the
depletion medium, and incubated in the same medium 1 mM
alanine or DMEM for 3 hours. The kinetics of the system B
transport activity ([SH]-alanine concentration = 1 /xM 5 mM)
showed that the alanine incubation resulted in a 2 fold Vmax
increase and plus an increase of K,^ (Fig. 5-5) The DMEM
incubation also resulted in a increase of Vmax and I^. These
data indicated that the system B activity increase is likely
involved in the modification of the transport system affinity


Fig. 7-4. The effect of CHX on the TPA-induced alanine uptake
Alanine (50 iM) uptake was measured in cells (3 days old
and 9 days old) which had been incubated in 0.5 juM TPA 10 /lxM
CHX for 24 hours. TPA stimulated the Na+-dependent System B alanine
uptake (p < 0.05, n = 6). CHX in the TPA incubation medium
blocked this TPA's stimulation effect (p < 0.05, n = 6). The
alanine uptake in choline Cl medium was not affected by TPA (p
< 0.05, n = 9) Data shown were obtained from 3 days old cells,
with similar results observed in 9 days old cells.


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REFERENCE LIST
Akiyama, T. Saito, T. Ogawara, H. Toyoshima, K. and
Yamamoto, T. 1988. Tumor promoter and epidermal growth
factor stimulate phosphorylation of the c-erbB-2 gene
production in MKN-7 human adenocarcinoma cells. Mol.
Cell. Biol 8:1019-1026
Betran, J., Werner, A., Moore, M.L., Stange, G. Markovich, D.
1992. Expression cloning of a cDNA from rabbit kidney
cortex that induces a single transport system for cystine
and dibasic and neutral amino acids. Proc. Natl. Acad.
Sci. USA. 89:5601-5
Bruns, R. F, Smith, D. R, and Nakono, K. 1991. Inhibition of
protein kinase C by calphostin C is light-dependent.
Biochem. Biophy. Res. Comm. 176:288-293
Buddington, R.K., Chen, J.W. Diamond, J.M. 1987. Genetic and
phenotypic adaptation of intestinal nutrient transport to
diet in fish. J. Physiol. Lond. 393: 261-81
Buddington, R.K., Chen, J.W., Diamond, J.M. 1991. Dietary
regulation of intestinal brush-border sugar and amino
acid transport in carnivores. Am. J. Physiol. 261(4 Pt
2): R793-801
Buddington, R.K., Diamond, J.M. 1987. Pyloric ceca of fish: a
"new" absorptive organ. Am. J. Physiol. 252(1 Pt 1):
G65-7 6
Buddington, R.K., Diamond, J.M. 1989. Ontogenetic development
of intestinal nutrient transporters. Annu. Rev. Physiol.
51: 601-19
Buddington, R.K., Diamond, J.M. 1990. Ontogenetic development
of monosaccharide and amino acid transporters in rabbit
intestine. Am. J. Physiol. 259(4 Pt 1): G544-55
Campa, M.J., Kilberg, M.S. 1989. Characterization of neutral
and cationic amino acid transport in Xenopus oocytes. J.
Cell. Physiol.141(3): 645-52
262


Fig. 3-1. Alanine uptake time course
The uptake of alanine (50 /M and 5 mM) was measured in
uptake media containing NaCl and choline Cl in day 2 and day 9
cells. The alanine uptake in NaCl media was greater than that in
the choline Cl media at any point (except t = 0, p < 0.05, n =
6) The uptake values in this figure and subsequent experiments
were expressed as mean standard error (SE) The data shown were
from the alanine (50 /uM) in day 2 cells, with similar data
obtained in the day 9 cells and at other alanine concentrations.


Fig. 6-1. The effects of chronic TGF, or EGF System B activity
System B alanine (50 /M) uptake was measured in cells (2 days old
and 9 days old), after cells had been incubated in DMEM, 20 ng/ml TGF,
or 100 ng/ml EGF for 48 hours. TGF0' or EGF each stimulated the alanine
uptake (p < 0.05, n = 12).


33
the 35 mm falcon dishes (6 well clusters) at the cell passages
#17 #50.
2.2.1 Caco-2 Cell Monolayer Transport
The amino acid uptake experiments were performed at room
temperature (22.5C 1.0C). Cells were taken out of the
incubator. Then the growth medium was aspirated, and cells
were rinsed three times with uptake buffer (22.5C) containing
137 mM NaCl (or choline Cl) 10 mM HEPES/Tris (pH 7.4) 4.7 mM
KC1, 1.2 mM MgS04, 1.2 mM KH2P04, and 2.5 mM CaCl2. The uptake
was initiated at time = 0 by adding 1 ml uptake buffer plus
0.01 -10 mM [3H]-alanine (2 jiCi/ml, isotope was dried in
nitrogen gas first and was then re-hydrolyzed in the uptake
buffer), 0.005 5 mM [3H]-Arginine (2 Ci/ml) or other
isotopes and concentrations as mentioned below into the cell
monolayer. For the System B regulation experiments, 1 mM
unlabeled MeAIB was also added to the uptake buffer to block
possible alanine uptake via the System A. For the arginine
uptakes, 10 mM unlabeled leucine was added to the uptake
buffer to block the System b0,+. During the uptake procedures,
the cell cultures were continuously shaken by an orbital
shaker (1 Hz). The uptakes were stopped by aspirating the
uptake buffer, and then adding 2 ml ice-cold uptake buffer
(lacking substrate) immediately to the cells. The ice-cold
buffer is aspirated, cells were rinsed two more times using
the same ice-cold buffer. Isotope was extracted from the cells


Alanine uptake, per cent
-*-*-*-* N> N>
NJCn^lOMOl-JON)
ocnomocnocnocn
VTZ
250


nmole mg min


15
(Laganiere et al, 1986; Diamond, 1991). Nontoxic nutrient
exposure can induce a non-specific hyperplasia of the
epithelium (increase epithelial cell numbers and size) and
lengthen the villi to provide more absorbing capacity for all
nutrients (Laganiere et al, 1986). The second explanation is
that individual transporter activities are selectively
increased as a result of the modification of transporter
or/and increase the copies of transporters (James et al.,
1987; Stein et al., 1987; Diamond et al., 1987; Scharrer et
al., 1981) by the exposure of specific transport substrate.
In addition to the substrate regulation of transport
activity, often conditions such as corticosteroid treatment
and the conditions related to diabetes, hyperthyroidism,
neoplasia, and pregnancy and lactation can induce intestinal
mucosal hyperplasia (James et al., 1987; Levine, 1991).
In addition to absorbing nutrients for whole body needs,
the small intestine enterocytes also require amino acids for
their own proliferation, growth and differentiation.
Epithelial cells rapidly turnover as enterocytes continuously
migrate up from the immature proliferating crypt cells to
become mature villous enterocytes along the crypt villous
axis. The supply of amino acids by membrane transport may be
the rate-limiting step in the rapidly proliferating and
protein synthesizing in undifferentiated cells (Seitz et al,
1989). Epidermal growth factor (EGF) and transforming growth
factor-alpha (TGF) also stimulate epithelia proliferation and


26
apical membrane marker enzymes on the cell membrane. To date,
the biochemical and histological tests indicate that
differentiated Caco-2 cells are quite similar to, but exactly
like normal small intestinal epithelial cells. The timing and
cellular characteristics associated with the differentiation
process of the Caco-2 cells resemble those of the normal crypt
to villous cell development. Caco-2 cells are a human colon
tumor transformed cell line having 106 chromosomes. The
unmistakable similarity of the histological and biochemical
characteristics makes the Caco-2 cell line a ideal model for
the in vitro analogue of the normal adult intestinal
epithelium.
The confluency and differentiation of Caco-2 cell states
are cell-attachment dependent. After trypsinization, the
attached polarized cells detached to become single non
polarized cells, and lose their differentiated
characteristics. Whether this process is a de-differentiation,
or simply a turning off of existing differentiation
expression, is still debatable. The daughter cells of these
de-differentiated or undifferentiated cells then undergo
another un-differentiation-confluency-differentiation process.
Regardless the states of parent cells, newly divided daughter
cells are undifferentiated. To ensure that the majority of
cells are at the undifferentiated state, we have used cells
only in their relative early generations (#19-50). The limited
time of subculturing also reduce the possibility of mutation.


Fig. 3-16. Dixon analysis of Na+-dependent alanine uptake with
proline as inhibitor
Alanine (25 /zM, 50/zM, and 100 /zM) uptake was measured with
various concentrations of proline (10 /zM 5 mM) in uptake media.
Ka = 7.1 mM proline.


57
System B.
The System B transport activity decreases as cell age
increases (Fig. 3-2). The decrease in activity was coincident
with the decrease of cell proliferation rates with cell ages
(Fig. 3-3). The proliferation rate may be related to the cell
requirement for amino acid. In contrast to the Na/alanine
transport activity, the Na/glucose activity increases as cell
age advances (Blais et al., 1987) (Fig. 3-23). These opposing
changes in activity for Na+-dependent solute transport as a
function of cell ages excludes the likelihood that the amino
acid transport was regulated by non-specific membrane
electrochemical potential effects. Furthermore these data
suggest that cell development is associated with the
independent regulation of amino acid and glucose transport
systems. We tested the Na/alanine transport characteristics in
two different cell states, the undifferentiated state (day 2
cells) and differentiated state (day 9 cells). The amino acid
analogue inhibition pattern, pH sensitivity, and Na+-
activation Hill number were the same for both the day 2 and
day 9 cells (Figs. 3-4, 3-5, and 3-12). The transport kinetics
gave a VBax on the day 2 that was greater than for day 9 cells,
while the apparent affinity K, was the same on both cell ages
(Fig. 3-9) All the transport characteristics of the Na+-
dependent alanine transport (except VBax) were identical in
both the undifferentiated and differentiated states,
suggesting that the same transporter system was operative


Fig. 6-2. The effect of CHE on the chronic TGF^/EGF-induced System
B activity
System B alanine (50 /M) uptake was measured after cells had been
incubated in DMEM, TGF or EGF, 6.6 /xM CHE for 48 hours. TGFoc or EGF
each stimulated the alanine uptake, CHE in the TGF or EGF medium
blocked this stimulative effect (p < 0.05, n = 6)


Fig. 3-22. Na*-dependent alanine uptake inhibition pattern in both
day 3 and day 9 cells
Na+-dependent alanine uptake rates with various 5 mM amino
acid in uptake media were measured in both the day 3 and day 9
cells. The uptake in the day 3 cells was shown as a function of
the uptake in the day 9 cells. As shown in the figure, the degree
of alanine uptake inhibited by amino acids was similar in the day
3 and day 9 cells. Symbol keys: X = MeAIB, B = BCH, J = Cystine,
U = AIB, Z = control, A = Ala, C = Cysteine, F = Phe, G = Gly,
H = His, I = lie, L = Leu, M = Met, N = Asn, P = Pro, Q =
Gin, R = Arg, S = Ser, T = Thr, V = Val, W = Trp, Y = Tyr.


208
effect on the System B and System y+ activities was likely
inhibiting new protein synthesis rather than cytotoxic effect.
Prolonged and continuous exposure to TGF/EGF was
required for the System B and System y+ activity stimulation.
The mechanism for the delay is unknown. In the light of a
System A regulatory mechanism proposed by Engleberg (1986),
the System B and System y+ could be regulated by at least two
groups of regulatory forces which are always present and are
in equilibrium. TGF/EGF shift the balance to the stimulatory
side, resulting in synthesis of transporter protein or other
regulatory proteins, while the negative regulatory force
always tries to bring the balance back.
As we discussed above in the introduction section,
TGF/EGF bind to the EGF receptor and the TGF/EGF-receptor
complex phosphorylates many substrates of the EGF receptor.
The substrates may include the receptor itself, the ras
GTPase-activating protein or GAP, PI-3 kinase, and PLCyl.
Which signal pathway did TGF/EGF participate in activating
Systems B and y+ activities? The inhibition of the TGF/EGF
stimulation effect on the alanine and arginine transport by
the specific protein kinase C inhibitors chelerythrine and
calphostin C suggested that the protein kinase C activation
was involved in the process. Chelerythrine CL specifically
inhibits PKC by acting at PKC's catalytic subunits, while
calphostin C binds at PKC's regulatory subunits (Tamaoki et
al., 1990). We have found that the H-7 isomer defectively


Alanine uptake, per cent
ZTZ
250


Alanine uptake, nmole mg min
o o o o o o o
M OJ 4^ Ln CD kj
SPZ


Alanine uptake, nmole mg min
o ro io go
en cn b cn b
981


% Stimulation of Arginine Uptake
-4^ M hO
o o o o
>

*
1.
to
K>
to
-p
CD
00
o
NO
-P-
CD
00
o
ro
o
O
O
o
O
o
o
o
o
o
o
iiii
Cysteine
Proline
Phenylalanine
BCH
Aspartic acid
Control (mannitol)
^ Glycine
Methionine
Tyrosine
Valine
^ Threonine
^ Serine
Alanine
<2h Cystine
hE
Asparagine
D-Arginine
Leucine
Glutamine
Homoserine
Histidine
0^-l Medium + Amino acid mixture
C^KKKK^CKK^OOOOOOOOOOfl Arginine
yTXX)>CkXKK>CK>0 Lysine
J I
061


Fig. 7-7. The effect of TPA on System B activity kinetics
System B alanine (10 /M 5 mM) uptake kinetics were
measured in cells (2 days old and 9 days old) which had been
incubated in DMEM or 0.5 uM TPA for 24 hours. System B uptake
was plotted as a function of alanine concentration.


124
inhibit the arginine uptake in the choline Cl media,
consistent with the cross inhibition pattern described for
System y+. The kinetics and pH insensitivity, combined with
the inhibition patterns together strongly indicated that the
Na+-independent carrier-mediated arginine transport system in
Caco-2 cells was System y+.
System b0,+ is another Na+-independent transporter of
arginine, and is the counterpart of the system B0,+ The weak
inhibition of arginine uptake by the neutral amino acids
alanine and leucine indicated the an unlikely major
involvement of system bD,+ in the current passages of our Caco-
2 cells (passages # 18 50) The non-inhibition effect of
alanine, BCH, Phenylalanine, and leucine excludes the possible
involvement of systems L, or asc.
Over the arginine concentration of 1 /M to 1 mM, the
arginine uptake in NaCl medium was not different from that in
the choline Cl. These data suggested that the arginine uptake
in the Caco-2 cells was mainly a diffusion plus Na+-
independent system y+ transport event; no Na+-dependent
transport phenomenon is involved.
The Na+-independent carrier-mediated system, affinity
characteristics, inhibition patterns, and pH insensitivity
were the same in both the undifferentiated day 2 and
differentiated day 9 cells. Only the Vmax value was higher in
day 2 cells compared to day 9 cells (Figs. 4-3 4-12). These
combined data indicated that arginine was transported through


Alanine uptake, nmole mg min
o k> c-j -f>- cn
Cont
Ala
Asp
BCH
Cys
Gin
Leu
Ser
Thr
Phy
AIB
Pro
Gly
MeAlB
Lys
Glu
8


en
£
_cu
o
£
Q.
cn
1000
900
800
700
600
500
400
300
200
100
0
day 9
O day 2
O
0
o'
/
o
100
200
300
[Arginine], fiW\
400
5
500
133


Fig. 3-3. Caco-2 cell proliferation rates at various cell ages
The 24 hour incorporation rates of [3H]-thymidine into
various cell ages were measured. The cells had been incubated in
serum-free DMEM for 24 hours prior to the measurements. The blank
control value = 1012 CPM, and the incubation medium value = 3.74
x 105 CPM.


T
T
8
c
E
cn
E
_qj
o
E
c
CD
o
-+-J
Cl
Z5
CD
C
'c
_o
<
7
6
5
4
3
2
T Na dependent
total in CHO
O total in NaCI
0


o
00* ^
0 100
I I I I
200 300 400 500
[Alanine]
600 700 800 900 1000


16
growth (Carpenter & Wahl, 1990). The relationship between the
cell proliferation and the membrane amino acid transport is
not clear.
Substrates not only regulate their absorptive activity,
but are also vital to enterocyte health. Glutamine,
transported via intestinal System B (Souba et al., 1992), is
essential in preserving the intestinal mucosa (Souba, 1990) .
Glutamine deficiency cause impairment of intestinal mucosal
barrier function (Souba et al, 1990). In this sense, glutamine
regulates its transport activity through preserving a healthy
state, in addition to its direct regulation of transport
activity.
1.2.6 Molecular and Cellular Models of Amino Acid Transport
Regulation
Several amino acid transport regulation models have been
proposed for nonintestinal cell types. However, the knowledge
of intestinal membrane amino acid transport regulation is
still lacking.
One model for substrate adaptive regulation of System A
in hepatocytes was proposed by Kilberg (1986). The model is
based on the assumption that the rate of repressing System A
independent of substrate concentration. The System A
transporter protein synthesis process is controlled at the
transcriptional level as a consequence of the equilibrium
between positive and negative regulating factors: in the
absence of extracellular amino acid and/or in the presence of


225
treatment with phorbol esters, serum-free DMEM plus various
concentration of TPA or PDBU (1 pM 10 iM) (TPA and PDBU
were diluted from DMSO stock solution and stored at -20C),
(3) treatment with phorbol esters and other agents, DMEM plus
phorbol ester plus additional other chemicals such as
cycloheximide, chelerythrine, calphostin C, with DMEM plus
other agent as an internal control, chelerythrine and
calphostin C were from LC Services Co., Woburn, MA.). The
cells were incubated for various periods of time in the 37C
incubator. The medium was changed very 6 hours. For the
calphostin C incubation, a 20 watt fluorescent light was
placed in the incubator. The details of each incubation are
explained below.
7.2.2 Pre-treatment with Dibutvrvl Cvclic-AMP (dcAMP)
The Caco-2 cells were washed three times with the serum-
free medium, and incubated with the same medium with 0.5 /M
dcAMP for various times (0-24 hours) The medium was changed
every 6 hours.
7.3 Results
7.3.1 The Phorbol Ester (TPA) Stimulation of the System B
Activity Time Course
The Caco-2 cells were pre-incubated with 0.5 /M TPA in
the serum-free medium for various length of time (0-24
hours) prior to the uptake experiments. The medium was changed


CHAPTER 2
GENERAL METHODOLOGY
2.1 Caco-2 Cell Culturing
The human intestinal epithelial Caco-2 cell is derived
from human colon adenocarcinoma cells. The cells can be grown
as a monolayer on both porous filters and plastic. Under
normal cell culture conditions, Caco-2 cells can be
subcultured for many generations. Some labs reports 9 0 or more
passages. Caco-2 cell growth on the plastic surface is
dependent on cell density. Cells divide horizontally, and cell
attachment does not stop the cell growth as normally seen in
cell culture. The attached cells continuously divide at a
lower rates. Days later (depends on cell density, with higher
cell density having a shorter turnover), the attached cells
become confluent. The confluent state is represented by the
cell to cell tight junction and by dome formation. The dome is
caused by the unidirectional transport and trapping of water
and electrolyte cross cell monolayer. Unique in the Caco-2
cells, the confluent Caco-2 cell undergo a spontaneous
enterocytic differentiation process without changing cell
culture conditions. The differentiating cells start to
polarize by forming apical and basolateral membranes, with
expression of the normal small intestinal epithelial cell
25


207
hours. System y+ arginine uptake was unaffected by this
treatment.
6.4 Discussion
The peptide growth factors TGF and EGF both stimulated
the System B and System y* activities in the Caco-2 cells at
both the undifferentiated and differentiated cells.
In addition to being a the slow process requiring many
hours (>30 hours), the TGF/EGF effect was cycloheximide-
sensitive. These combined data suggest that it is unlikely
that the TGF/EGF effect was caused by rapid phosphorylation
of transporter protein. A de novo protein synthesis process
was likely involved in the TGF/EGF stimulation of System B
and System y+. But whether the synthesized protein was the
transporter protein, regulatory protein, or other protein was
unknown. Future studies using a System y+ antibody probe may
provide a more precise answer. Normally, de novo protein
synthesis can occurs within several hours, and it is not clear
why TGF/EGF took more than 30 hours to show their effect. A
cascade of regulatory processes may be involved, in addition
to protein synthesis.
The protein content and cell numbers of the 48 hour CHX-
treated cells was comparable to the pre-CHX-treatment level.
The viability of CHX-treated cells was >99%. Compared to the
control group (only DMEM treatment), the CHX-treated cells had
40% less protein and 40% less cells. So the CHX's inhibitory


Fig. 4-3. The effect of pH on arginine uptake
Arginine (5 /M) uptake in day 2 and day 9 cells was
measured in choline Cl medium, with various medium pH (pH 6.1,
7.4, or 8.4). The pH was adjusted by using 10 mM HEPES and 10
mM Tris buffer. The arginine uptake was not affected by the medium
pH.


-1
Alanine uptake, pmole mg min
cn
b
o
Cn
IV)
O
M
cn
Cont
Ala
Asp
Gin
Phy
Ser
Thr
BCH
Leu
MeAlB
Pro
Gly
AIB
Cys
Lys
Glu
09


Fig. 5-14. Kinetics of acute arginine- or DMEM-stimulated System
v* activity
System y+ arginine (0.1 /uM 1 mM) uptake kinetics were
measured after cells had been incubated in DMEM or DMEM plus 1
mM arginine for 3 hours. For the DMEM incubation, VBax = 1.05
nmole/mg/min and K = 39 /nmole arginine; for the arginine/DMEM
incubation, VBax = 22.7 nmole/mg/min and KB = 79 /nmole arginine.


159
alanine also decreased as incubation progress, however, the
decrease was less than that in cells incubated in depletion
medium alone. The 50 /M alanine System B uptake rate in the
alanine-incubated cells was 50% higher than that in the
depletion-incubated cells (Fig. 5-1).
5.3.2 System B Activity was Activated by Acute Amino Acid
Exposure
The Caco-2 cells were incubated in the depletion medium
containing 1 mM individual amino acids for 3 hours. Mannitol
(1 mM) was the control. Cysteine solutions contained 1 mM
dithiothreitol, with the 1 mM dithiothreitol solution serving
as control. System B activities were measured immediately
after each incubation. In comparison to the cells incubated
in the control depletion medium alone, the increase in System
B activity in cells incubated in solution containing amino
acids gave a pattern of stimulation that matched the ranking
of System B substrates (described in the chapter 3) That was,
alanine, serine, glutamine, threonine and cysteine each
increased the system B activity by 1.5 2 fold; the weaker
stimuli (which weakly inhibited system B activity in the
cross-inhibition study (Fig. 3-12)) were amino acids such as
histidine, glycine, and valine. Finally phenylalanine,
leucine, lysine, arginine, and MeAIB, which did not inhibit
system B activity in cross-inhibition study, did not
stimulated the system B activity (Fig. 5-2).


INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2
By
MING PAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993

This dissertation is dedicated to my wife Jun, and my
parents.

ACKNOWLEDGEMENTS
I would like to thank Dr. Bruce Stevens, chairman of my
supervisory committee, for his tremendous support in both my
professional and personal life. Words can hardly express my
deep appreciation. Working under his supervision has been a
wonderful experience.
I would also like to thank members of my supervisory
committee, Dr. Edward Copeland, Dr. George Gerencser, Dr.
Michael Kilberg, and Dr. Souba for their support and valuable
suggestions.
I would like to express my appreciation to Dr. Colin
Sumners for providing the cell culture equipment through out
my study. I would also like to thank Ms. Tammy Gault, for
teaching me cell culture technique and sharing the cell
culture equipment.
I would like to take this opportunity to thank all the
faculty members of the Physiology Department for making
graduate study an enjoyable experience.
Finally, I specially thank my wife and my family for
their unconditional support and great family value.
iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
1.1 Introduction 1
1.2 Amino Acid Absorption in the
Small Intestine 2
1.3 The Human Intestinal Epithelial Cell
Line (Caco-2 Cell Line) 20
1.4 The Objective and Aims of the
Present Study 22
2 GENERAL METHODOLOGY 25
2.1 Caco-2 Cell Culturing 25
2.2 Caco-2 Monolayer Transport 31
2.3 Monolayer Transport in Caco-2 Cells
Grown on Porous Filters 35
2.3Statistical Analysis 39
3 CLASSIFICATION OF ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE 4 0
3.1 Introduction 4 0
3.2 Methods and Materials 4 3
3.3 Results 44
3.4 Discussion 50
3.5 Summary 58
4 CLASSIFICATION OF ARGININE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE 113
4.1 Introduction 113
4.2 Methods and Materials 115
4.3 Results 116
4.4 Discussion 120
4.5 Summary 12 3
iv

5 THE EFFECTS OF INDIVIDUAL AMINO ACID ON THE
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES 156
5.1 Introduction 156
5.2 Methods and Materials 157
5.3 Results 158
5.4 Discussion 165
5.5 Summary 170
6 THE EFFECTS OF PEPTIDE GROWTH FACTORS ON THE
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES 199
6.1 Introduction 199
6.2 Methods and Material 2 02
6.3 Results 204
6.4 Discussion 206
6.5 Summary 2 09
7 THE EFFECTS OF PHORBOL ESTERS ON THE SYSTEM B
AND SYSTEM y+ TRANSPORT ACTIVITIES 223
7.1 Introduction 223
7.2 Methods and Materials 224
7.3 Results 225
7.4 Discussion 231
7.5 Summary 233
8 SUMMARY AND CONCLUSIONS 258
8.1 Summary 258
8.2 Conclusions 260
REFERENCE LIST 262
BIOGRAPHICAL SKETCH 272
V

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2
By
Ming Pan
May 1993
Chairperson: Bruce R. Stevens
Major Department: Physiology
Membrane transporter systems serving arginine (Na+-
independent system y+) and alanine (Na+-dependent system B)
were investigated in the human intestinal Caco-2 cell line.
The uptake kinetics were different for each transport system.
For each system, the V>ax was greater in undifferentiated cells
compared to differentiated cells, while the Kra values were
each unaffected by cell differentiation status. Amino acid
substrates unique to System y+ acutely stimulated only system
y+ activity, while substrates unique to system B only
stimulated system B activity. For each transport system, the
ranking of amino acid stimulation was directly correlated with
the degree of competitive analogue inhibition (assessed by
Dixon analysis). The prolonged substrate induction of system
B activity, but not system y+ activity, was prevented by the
protein synthesis inhibitor cycloheximide. Peptide growth
vi

factors epidermal growth factor (EGF) and transforming growth
factor-alpha (TGF) each stimulated system B and system y+
activity following a lag period of several hours. EGF/TGF
activation was abolished by cycloheximide, or by inhibitors of
protein kinase C. Phorbol esters stimulated system B and
system y+ activity following a lag period of several hours,
and this stimulation was prevented by cycloheximide and
inhibitors of protein kinase C. For each transport system,
EGF, TGF, and phorbol ester increased the Vnax but not the Km.
Together these data suggest that (1) Caco-2 epithelial
differentiation status is associated with regulation of amino
acid transport; (2) amino acid transporter system B and system
y+ are regulated independently; (3) amino acid substrates up-
regulate their own transporter's activity via trans
stimulation or by a mechanism involving de novo protein
synthesis; (4) EGF and TGF likely activated protein kinase C
in the up-regulation of system B and system y+ activity via a
de novo protein synthesis mechanism.
Vll

CHAPTER 1
INTRODUCTION
1.1 Introduction
Amino acid transport by the small intestine is a vital
process involved in whole-body nitrogen balance. Intestinal
absorption studies have received considerable attention during
the past 35 years, primarily focussing on description of the
site of absorption within the mucosa, classifications of
uptake pathways based on substrate selectivity, and
phenomenological kinetic mechanisms of transporter function
using the universal principles of membrane transport (Hopfer,
1987; Munck, 1981; Stevens et al., 1984, Stevens, 1992a,b).
Past and current studies were conducted in a variety of
species and at various stages of development. These studies
have included patients and intact animals with inborn errors
of transport (Desjeux et al., 1980), perfused intestines,
isolated membranes from the brush border and basolateral
surfaces (Stevens et al., 1982, 1984; Mircheff et al, 1879,
1980), and enterocytes freshly isolated from mucosa (Reiser
and Christiansen, 1971a,b,c), or grown as in vitro cell lines
(Pan et al., 1991).
This project concentrates on amino acid absorption from
the outer environment; the role of the intestine in inter-
1

2
organ amino acid flow is beyond the scope of this project. The
small intestine is unique in extracting amino acids, in
contrast to other internal organs. The enterocyte amino acids
transport systems, especially those at the brush border
membrane are unique in substrate and modes of regulation.
Unlike the amino acid transport substrate adaptive down-
regulation universally observed in many internal organs,
substrates in the small intestine up-regulate their
transporter activities in vivo studies (Diamond, 1991;
Stevens, 1992a,b).
1.2 Amino Acid Absorption in the Small Intestine
Like the membrane transport processes in internal organs,
small intestinal amino acid transport has been studied
qualitatively and quantitatively based on uptake phenomena and
non-equilibrium thermodynamic principles.
The small intestinal mucosa separates the outer
environment from the internal milieu. The intestinal mucosa is
composed of a single layer of historically polarized
epithelial cells (enterocytes) which are joined by a tight
junction to form a continuous layer. Amino acid transport
across the mucosa is mainly a trans-cellular phenomenon. The
epithelial villous cells are responsible for amino acid
absorption (Munck, 1981; Wilson, 1962). Along the crypt-villus
axis the enterocytes originate from stem cells within the
crypt. The undifferentiated immature cells rapidly proliferate

3
and migrate up the villi to become the mature villous cells.
The well-developed enterocytes stay at the villus tip for
several days, and then are shed away to the intestinal lumen.
The location of greatest individual amino acid absorption
differs among species along the oral-aboral axis of the small
intestine (Diamond, 1991; Stevens, 1992a,b). The lumen to
blood absorption involves the movement of amino acid through
a series of aqueous and membrane compartments (Hopfer, 1987;
Stevens, 1992a,b). Each compartment acts as a barrier which
influences the overall amino acid movement across all the
compartments. The brush border apical membrane of these
enterocytes is the initial active step regulating the flow of
amino acids from the intestinal lumen into the cell cytosol.
1.2.1 Lumen to Blood Amino Acid Movement
Beginning in the lumen, amino acids travel through an
unstirred water layer, the apical membrane, the cytosol, the
basolateral membrane compartments, and finally capillary
endothelium. Each compartment determines the "real" amino acid
concentration reaching the following compartment.
The unstirred water layer is about 50 nm thick layer and
is composed of water and mucous/glycocalyx. Amino acids
diffuse across the unstirred water layer. The amino acid that
diffuses across the unstirred layer then reach the apical or
brush border membrane. This plasma membrane is a bilayer
phospholipid structure separating the cell cytosol environment

4
from the lumen. Amino acids cross surface by a simple passive
diffusion plus some carrier-mediated transport mechanisms. In
some instances, the amino acid is transported via a secondary
active transport mechanism against its own electrochemical
gradient across the membrane. The accumulated amino acids in
the cytosol then exit via passive diffusion or/and carrier-
mediated transport mechanism at the basolateral membranes.
Each of the barriers can influence the rate of the amino acid
absorption: the "true concentration" of the amino acids
reaching the brush border membrane is determined by the amount
of substrate within the unstirred water layer, rather than the
bulk phase concentration present in the lumen. The brush
border membrane and the basolateral membrane possess
biochemically and histologically different structures. Some of
the amino acid transport systems at the brush border membrane
are not found in the basolateral membrane and the other
internal organs (Stevens, 1992a,b). On the other hand, the
basolateral membrane possesses many of the same transporters
as the membrane found at other internal organs.
1.2.2 Modes of the Amino Acid Transport in the Small Intestine
The membrane amino acid transport movement is
classified into two general categories: simple passive
diffusion and carrier-mediated transport. The carrier-mediated
transport is further divided to Na+-independent facilitated
transport and Na+-dependent secondary active transport

5
mechanisms.
In the case of the simple passive diffusion, the membrane
electrochemical gradients and permeability coefficients of the
amino acid govern the direction and rate of the passive amino
acid movement across the membrane.
In the case of the Na+-independent carrier-mediated
transport mechanism, amino acid is carried by its specific
transporter across the membrane, directed by the
electrochemical acting across the membrane. In the case of
charged amino acids such as arginine, the negative membrane
potential can drive it against its chemical gradient.
In the case of the secondary active transport, a series
of spatially separated events occur that couple energy derived
from ATP hydrolysis to solute flux. The Na+/K+-ATPase at the
basolateral membrane creates electrochemical Na+ and K+
gradients across the basolateral and the apical membranes. The
Na+/amino acid cotransporters at the apical membrane bind to
amino acids, and utilize this Na+ electrochemical energy
(A/Na) for concentrative uptake of amino acids across the
apical membrane. The accumulated amino acids inside the
enterocytes exit via the Na+-independent facilitated
transporters and the passive diffusion at the basolateral
membranes.
A kinetic model of Na/amino acid transport describing the
secondary active transport has been recently examined Stevens
(Stevens, 1992a) as a paradigm for all Na+-dependent systems.

6
This model describes an prefer-ordered mechanism with the Na+
activator ion binding preferentially first to the cis
transporter conformation, and this binding increases the
affinity for amino acid binding to the cis side of the
transporter. Amino acid then binds to transporter. The cis-
complex isomerizes to place Na+ and amino acid on the trans
side, and the substrates are released to the cytoplasm by
either a random or ordered sequence. The trans transporter
conformation isomerizes back to cis-transporter conformation.
The overall rate-limiting step is the isomerization of the two
transporter forms (cis and trans) In the absence of trans
amino acid, the influx is predicted by: (Stevens & Wright,
1987): JA = (JAax ~ [ A]) /K + [A], where JA = amino acid flux,
Jaiax = maximal flux rate, and the apparent affinity K, for
solute A is a function of both KNa (the apparent dissociation
constant for dissociation and binding):
K. = ((K^/ENa])" + (nK^/ENa]) + 1) KA
where KA is the apparent amino acid-carrier dissociation
constant, and n is the Hill coefficient describing the number
of Na+ ions coupled to movement of each amino acid molecule.
Note that regulation of the transporter activity could
conceivably occur by modifying JBax (i.e, activity of the
functional transporter), or apparent K, (which includes the Na+
affinity and substrate modulation).
1.2.3 The Amino Acid Transport Systems in the Small Intestinal
Membrane

7
Christensen and colleagues developed the original
criteria to discriminate different amino acid transport
systems in mammalian cells (Christensen, 1975, 1984, 1985 &
1990) through substrate specificity, ion-dependency, transport
kinetics, and numerous other characteristics. Many facilitated
and Na+-dependent secondary active transport systems such as
Systems A, ASC, L and y+ which were first described in non-
epithelial cell were found in many cell types including
intestinal epithelial cells (Kilberg et al., 1993; Stevens,
1992a).
Much work has been conducted in the amino acid transport
system classification at the tissue, cellular, and membrane
vesicle levels in the intestine (Munck, 1981; Stevens et al.,
1984; Hopfer, 1987; Stevens, 1992a,b). The major tools for
the membrane transport system classification are (1) substrate
preference; (2) ion-dependency; (3) substrate uptake kinetics,
(4) patterns of analogue cross inhibition of amino acids, and
(5) specific renal and intestinal inborn amino acid
malabsorption syndromes (Stevens et al., 1984; Wright et al.,
198 6, Kilberg et al, 1993) Certain amino acids transported by
a single transporter are used to test for the presence of the
characteristic transporter. For example, -
methylaminoisobutyric acid (MeAIB) and pipecolic acid are
thought to be transported only through the System A and the
System IMINO, respectively (Christensen, 1975; Stevens &
Wright 1985, 1987; Wright 1985). Due to variations among the

8
animal species, the stages of development, the tissue studied,
and the methods used in amino acid transport systems studies,
many different systems have been reported in the small
intestine (Munck, 1981; Stevens et al., 1984; Hopfer, 1987;
Stevens, 1992a,b). With some conflicts, there is a similarity
in the amino acid transport systems among different species.
The functionally and biochemically distinct brush border
membrane and basolateral membrane possess different transport
systems. The compiled membrane transport systems at the brush
border membrane and basolateral membrane will be discussed
individually.
Several distinctive transport systems are found only at
the brush border membrane (Kilberg et al., 1993; Stevens et
al., 1982, 1984, Stevens, 1992a,b). One of these is System B.
System B is described as a strictly Na+-dependent system
broadly selective for the dipolar (neutral) amino acid
alanine, serine, cysteine, glutamine, and interacts with 2-
amino-2-norbornanecar-boxylic acid (BCH) and threonine. At
first it was named System NBB "Neutral Brush Border" (Stevens
et al, 1982, 1984; Stevens, 1992a,b). System B is
characteristically similar to System B0,+ described in
blastocyte (Van Winkle et al., 1985). Both System B and System
B0,+ broadly transport dipolar amino acids, except that System
B0,+ interacts with cationic amino acids, while System B is not
interactive with cationic amino acids. System B has been
reported to exist in the apical membrane of rabbit (Stevens et

9
al, 1984), pig (Maenz et al., 1992), dog (Bulus, 1989), human
fetal (Malo, 1991), lower vertebrate small intestines (Ahearn
et al., 1991), and in the undifferentiated and differentiated
enterocytic Caco-2 cells (Pan et al, 1991; Souba et al.,
1992). Another distinct amino acid transport system found only
at the intestinal apical membrane is System IMINO (Stevens &
Wright, 1985, 1987; Wright et al, 1985; Stevens, 1992a,b).
System IMINO is also a strictly Na+-dependent system highly
selective to heterocyclic imino acid such as proline and
pipecolate. System IMINO uptake has been reported in many
species intestine (Ahearn et al., 1991; Karasov et al., 1986,
1987; Moe et al., 1987; Munck, 1983; Stevens et al., 1984,
1992a,b).
Na+-dependent transport Systems A and ASC (Christensen et
al., 1965; Oxender et al., 1963; Kilberg et al., 1981, 1993),
serve dipolar amino acid in the non-epithelial cell membrane,
and reportedly exist in the guinea pig apical membrane (Del
Castillo & Muniz, 1991; Hayashi et al., 1980). But up to date,
no definite test has been able to discriminate them from the
System B or System IMINO. Other Na+-dependent systems, XAG'
serving D-aspartate and glutamate, 0 serving fi-alanine and
taurine were also reported in the intestinal apical membrane
(Hofper, 1987; Munck, 1990, 1992; Miyamoto et al., 1990a,b).
Some studies also suggested possible existence of a System N
for glutamine transport (Salloum et al., 1990, 1991).
There are three Na-independent transport systems at the

10
intestinal apical membrane. One is System L (Christensen et
al, 1963, 1969, 1975) which transports large neutral amino
acids and favors lipophilic substrates such as phenylalanine,
leucine, and BCH. System L excludes 0-alanine. A second is
System y+ (Christensen, 1964, 1966) which prefers cationic
amino acids such as lysine and arginine, although it tolerates
the substrate combination of sodium plus neutral amino acids
such as homoserine. A third is System b0,+ (Van Winkle et al,
1985, 1987, and 1988) which serves neutral amino acids and
cationic amino acid, and interacts with BCH.
Recent cloning of cDNA encoding System y+ (Kim et al.,
1991; Wang et al., 1991) provides a breakthrough in the
membrane transport systems studies. It is possible to study
membrane transport systems using the traditional
phenomenological method as well as more advanced molecular
biological methods. The finding of the same protein serving as
both the System y+ transporter and a retrovirus receptor not
only make the possible molecular studies of membrane amino
acid transporters, but the physiological or pathological
relationships among the nutrient absorption and cell functions
in healthy and disease states. Recent cloning of cDNAs NAA/D2,
rBAT, and F4 for putative regulatory protein for Systems b0,+,
y+ or B0,+ (Betran et al., 1992; Magagnin et al, 1992; Tate et
al., 1992; Wells et al., 1992a,b) were also reported.
All amino acids passively diffuse across the apical
membrane with their permeability rates directly proportioned

11
to their hydrophobicity. The order of permeability diffusion
coefficients is phenylalanine > 0-alanine > mannitol > alanine
> MeAIB > proline > glycine > lysine (Stevens et al.,
1982,1984). At high luminal amino acid concentration, the
passive diffusion may be the predominant transport ways in the
intestine. The carrier-mediated transport systems may be the
favored route at lower concentrations.
In contrast to the apical membrane, all the basolateral
membrane amino acid transport systems studied also exist in
other non-epithelial membrane. These include Systems ASC and
A, and Na+-independent Systems y+ and L, plus simple passive
diffusion. The characteristics of these systems are as the
same as those described in the apical membrane.
1.2.4 The Ontogenetic Developments of the Amino Acid Transport
Developmental studies of amino acid transport in the
small intestine of various species has demonstrated that the
timing and class of amino acid transporter appearance differs
not only among the animal species but also at the various
development stages of the same specie (Buddington & Diamond,
1989, 1990) Both herbivores and omnivores prefer high protein
diet in their youth, a period when the absorption of essential
amino acids is high. Because of some amino acids are in higher
demand in adults making these conditionally essential amino
acids. This is the case for arginine in puppies. On the oral-
anal axis, one dramatic change is in colon, whereby adults

12
colon only transports electrolytes and water, but neonatal and
fetal colon possesses many amino acid transport systems. On
the crypt-villous axis, the youth cope with the higher amino
acid transport load by creating a large surface area that
increase nutrient uptake non-specifically. The enterocyte
turnover rate of the villus tip is slower, the crypt cell
migration is greater, and the crypt cells possess transport
ability, all of which contributes to the increased mass of
intestine possessing transport activity. Two mechanisms were
proposed (Buddington & Diamond, 1989, 1990) to explain the
control of amino acid transporter expression during animal
development: (1) an external control mechanism by dietary
changes in substrate or by some growth factors in diet (e.g.
epidermal growth factor stimulating transport by enterocyte,
paracrine/autocrines, secreted by salivary gland or Bruners
glands or from food source such as milk) ; (2) an internal
genetically hard-wired control mechanism that controls change
independent of external environment.
1.2.5 Regulation of Intestinal Amino Acid Transport
The intestinal membrane amino acid transport systems are
regulated by various factors, such as the animal development
regulation discussed above, certain physiological states like
pregnancy, or certain pathological states like disease
diabetes, hyperthyroidism. Much attention was given to the
regulation of transport activity at certain stage of

13
development by systemically circulating factors like hormones,
or by the luminal composition like transporter substrates. The
intestinal apical membrane amino acid transport regulation by
transport substrate has been studied in vivo as described
below.
Unlike other internal organs, in vivo studies show that
the activities of the intestinal amino acid transporters are
up-regulated by the dietary substrates they transport
(Stevens, 1992a,b; Salloum et al 1990; Sharrer et al, 1981;
Stein et al, 1987; Ferraris et al, 1988a,b; Diamond & Karasov,
1987; Ferraris & Diamond, 1989; Diamond, 1991). The
substrates' unique pattern of up-regulation, their amplitude,
and selectivity of each system's activity indicated that
individual amino acid transporters are regulated independently
by dietary substrates. Non-essential caloric amino acids up-
regulate their transporter activities with increasing
substrate. The essential, but potentially toxic amino acids
regulate their transporter systems' activities in a different
pattern. That is, at lower substrate concentration, the
transporter activity decreases as substrate concentration
increase; at higher concentration the transport activity
increases as substrate concentration increases. A similar
pattern is observed for sugar and dietary carbohydrate. The
transport of non-essential amino acids is increased more by
the dietary protein than that of the more toxic essential
amino acids. This supports the notion that the absorption of

14
glucose, caloric and catabolic amino acids, and essential
amino acids (possibly toxic if in excess amount) is regulated
independently in vivo, which provides needed nutrients for the
entire organism and which prevents substrate toxicity
(Diamond, 1991). The mechanism of this induction has not been
addressed hereafter.
Amino acids differ in their potencies to induce the same
transporter. Although substrates generally make good inducers
of their own transporters, there are some discrepancies
(Levine, 1991; Diamond, 1991) between the inducers and
transported substrates: transport unrelated amino acid is the
best inducer.
The change in substrate-related transport activity is a
relatively slow process. An increase in the luminal substrate
level induces an reversible transport uptake capacity increase
over the existing absorbing capacity by 2- to 10- fold within
24 hours. The substrate-specific up-regulation of nutrient
absorption is directly related to the level of these substrate
in the intestinal lumen. Lowering substrate levels causes the
intestinal absorption capacity to decrease back down to the
baseline level that appears to be genetically hard-wired
(reviewed by Diamond, 1991). The down-regulation is a slow
process (eg. 3 days for proline transporter in mouse).
Two mechanisms were proposed to explain the substrate-
related intestinal amino acid uptake activity. The first is
mucosal hyperplasia resulting in nonspecific uptake increase

15
(Laganiere et al, 1986; Diamond, 1991). Nontoxic nutrient
exposure can induce a non-specific hyperplasia of the
epithelium (increase epithelial cell numbers and size) and
lengthen the villi to provide more absorbing capacity for all
nutrients (Laganiere et al, 1986). The second explanation is
that individual transporter activities are selectively
increased as a result of the modification of transporter
or/and increase the copies of transporters (James et al.,
1987; Stein et al., 1987; Diamond et al., 1987; Scharrer et
al., 1981) by the exposure of specific transport substrate.
In addition to the substrate regulation of transport
activity, often conditions such as corticosteroid treatment
and the conditions related to diabetes, hyperthyroidism,
neoplasia, and pregnancy and lactation can induce intestinal
mucosal hyperplasia (James et al., 1987; Levine, 1991).
In addition to absorbing nutrients for whole body needs,
the small intestine enterocytes also require amino acids for
their own proliferation, growth and differentiation.
Epithelial cells rapidly turnover as enterocytes continuously
migrate up from the immature proliferating crypt cells to
become mature villous enterocytes along the crypt villous
axis. The supply of amino acids by membrane transport may be
the rate-limiting step in the rapidly proliferating and
protein synthesizing in undifferentiated cells (Seitz et al,
1989). Epidermal growth factor (EGF) and transforming growth
factor-alpha (TGF) also stimulate epithelia proliferation and

16
growth (Carpenter & Wahl, 1990). The relationship between the
cell proliferation and the membrane amino acid transport is
not clear.
Substrates not only regulate their absorptive activity,
but are also vital to enterocyte health. Glutamine,
transported via intestinal System B (Souba et al., 1992), is
essential in preserving the intestinal mucosa (Souba, 1990) .
Glutamine deficiency cause impairment of intestinal mucosal
barrier function (Souba et al, 1990). In this sense, glutamine
regulates its transport activity through preserving a healthy
state, in addition to its direct regulation of transport
activity.
1.2.6 Molecular and Cellular Models of Amino Acid Transport
Regulation
Several amino acid transport regulation models have been
proposed for nonintestinal cell types. However, the knowledge
of intestinal membrane amino acid transport regulation is
still lacking.
One model for substrate adaptive regulation of System A
in hepatocytes was proposed by Kilberg (1986). The model is
based on the assumption that the rate of repressing System A
independent of substrate concentration. The System A
transporter protein synthesis process is controlled at the
transcriptional level as a consequence of the equilibrium
between positive and negative regulating factors: in the
absence of extracellular amino acid and/or in the presence of

17
hormone, System A associated protein synthesis is stimulated
(depressed), while in the presence of elevated intracellular
amino acid levels, System A-associated protein synthesis is
repressed by a regulatory protein.
Another model for adaptive regulation of System A in CHO-
cells (Chinese hamster ovary cell) was proposed by
Englesberg group (1986). This model suggested that System A is
regulated by at least two regulatory genes, R1 and R2. R1
produces an apopressor/inactivator (apo-ri) that is in
equilibrium with a repressor/inactivator (ri). The elevated
transported amino acids shift the apo-ri to ril which inhibits
the transcription of the gene encoding System A transporter,
and converts the existing transporter to an inactive state. R2
produces a constitutive repressor r2 which also negatively
regulates the gene A. Insulin binds to its receptor and
through an unknown pathway converts r2 to its inactive form.
The absence of transported substrate and the presence of
insulin have a synergistic effect on stimulating System A
activity.
One study in a kidney cell line (MDCK) indicates the
involvement of protein kinase C in System A regulation (Dawson
& Cook, 1987).
Even though System A adaptive regulation was intensively
studied, the molecular and cellular mechanisms of intestinal
transport regulation of any amino acid are still unknown. The
recent cloning of the System A cDNA (Kong et al., 1993) will

18
encourage regulation studies.
1.2.7 The Effects of Peptide Growth Factors in the Small
Intestine
As members of the peptide growth factors family,
Epidermal Growth Factor (EGF) and/or Transforming Growth
Factor (TGF) each stimulate cell proliferation, protein
synthesis, and cell differentiation in many cell types
including the intestinal epithelial cells (Morrisset &
Solomon, 1991; Carpenter & Wahl, 1990).
EGF is a 53 amino acid polypeptide, while TGF is a 48
amino acid peptide. TGF is structurally and biological
functionally similar to the that of EGF. EGF is normally
present in the intestine lumen from endogenous secretions from
the salivary glands, the small intestinal Brunner's glands,
autocrine/paracrine sources from the mucosa, or from exogenous
sources such as milk and colostrum (Gaull et al.,1985;
Britton, 1988; Potter, 1989). The sites for the EGF secretion
to blood stream is unknown.
EGF and/or TGF binds to the same EGF receptor in the
plasma membrane, which is a member of the tyrosine kinase
receptor family. The activated growth factor-receptor complex
immediately phosphorylates the receptor itself and
phosphorylates other substrates such as erb B2, ras oncogen,
polyoma middle T antigen, or phospholipase C (PLC). The
activated PLC alters inositol phospholipid metabolism leading
to a elevated level of diacylglycerol (DAG) (Berridge, 1985;

19
Edelinan et al, 1987; Klip & Douen, 1989), which activates
intracellular protein kinase C. PK-C activates a series of
unresolved mechanisms that ultimately result in cell division,
proliferation and differentiation (Saier et al., 1988).
EGF receptors appear at both the luminal and basolateral
membranes at a density gradient greater in immature crypt
cells and less dense in villous enterocytes along the crypt ->
villous axis (Hidalgo et al., 1989). This parallels the high
proliferation rate in undifferentiated crypt cells (Pamukcum
& Owens, 1991). Two-thirds of the EGF receptors are reportedly
in the basolateral membrane (Reviewed by Brand, 1990).
EGF/TGF stimulate small intestinal epithelial proliferation,
protein synthesis, and crypt cell maturation and migration
toward villous tip cells. Recently, EGF receptor mRNA was
identified in developing intestinal epithelial cells (Koyama
& Podolsky, 1989). The EGF receptors reportedly existed at the
apical and basolateral membranes of the human intestinal
epithelial Caco-2 cell line (Hidalgo et al., 1989), with
higher density in the undifferentiated cells compared to the
differentiated cells. Two-thirds of the receptors expressed at
the basolateral membrane. The K, of the EGF receptors is 0.67
nM in Caco-2 cells (Hidalgo et al., 1989). Experiment data in
our laboratory indicate that functionally EGF or TGF each
stimulates Caco-2 cell alanine and arginine transporter
activities with similar potency when applied to either brush
border or basolateral surfaces.

20
Even though the structure and biological functions of EGF
and TGF have been widely studied, the EGF/TGF effects on
intestinal amino acid transport has not been addressed.
1.3 The Human Intestinal Epithelial Cell Line
(Caco-2 Cell Line)
The established intestinal epithelial cell line Caco-2 is
derived from human colon adenocarcinoma cells (Fogh et al.,
1977) It was originally used for in vitro colonic tumor
studies.
Caco-2 cells can been grown on both solid plastic and
porous filters for many sub-cultural generations. When grown
on a solid surface, the Caco-2 cells form a confluent
monolayer with tight junction and dome formation. Under normal
cell culture conditions the confluent cells undergo a
spontaneous enterocytic differentiation process (Pinto et al.,
1983; Rousset et al., 1985). The biochemical and historical
characteristics of the undifferentiated cells resemble those
of the immature enterocytes, while the differentiated cells
resemble the mature small intestinal epithelial cells. The
differentiated Caco-2 cells become polarized, forming brush
border apical membranes complete with peptide and carbohydrate
hydrolases normally found as small intestinal apical marker
enzymes. The enzymes include sucrase-isomaltase, lactase,
trehalase, aminopeptidedase N, dipeptidylpeptidase IV, y-
glutamyltranspeptidase, and alkaline phosphatase (Pinto et
al., 1983; Hauri et al., 1985; Rousset et al., 1985). The

21
undifferentiated sub-confluent cells are morphologically and
biochemically equivalent to immature crypt cells, and
differentiated post-confluent cells are undistinguished both
morphologically and enzymatically from mature villus tip
enterocytes (Hidalgo, 1988, 1989, 1990). The Caco-2
undifferentiated sub-confluent -> differentiated post
confluent state developmental steps mimic the enterocytes
crypt -> villous maturation process. Many studies favorably
recognize the Caco-2 cell line as an ideal in vitro analog of
normal small intestine enterocytes (Zweibaum et al., 1983,
1991).
Organic solute transport studies on Caco-2 cells have
revealed the same characteristics as those from other in vitro
and in vivo small intestinal preparations (Blairs et al, 1987;
Mohrmann et al, 1986; Nicklin, 1992). A few studies have been
conducted regarding glutamine and proline transport
characteristics in Caco-2 (Nicklin et al., 1992; Souba et al.,
1992). These studies paralleled to those in other intestinal
preparations.
In addition to characteristics indistinguishable from
enterocytes, Caco-2 cells excel in providing a well-controlled
homogenous population over a prolonged life span during cell
development and differentiation. Uncontrolled adverse systemic
factors found in vivo preparation are eliminated in the cell
culture systems, so that the effect of a single variable can
be studied in an unbiased setting. The Caco-2 cell line makes

22
it possible to study the nutrient transport and associated
regulation over the enterocytes' entire developmental period.
Nonetheless, Caco-2 cells are not entirely normal small
intestinal epithelial cells. However, until the normal small
intestinal epithelial cell model is established, the Caco-2
cell line provides the best in vitro human intestinal
enterocyte model.
1.4 The Objective, Hypothesis, and Aims of the Present Study
1.4.1 The Objective
The overall objective of this in vitro study is to
investigate the cellular basis of amino acid transport
regulation in undifferentiated and differentiated states of a
human intestinal epithelial cell line (Caco-2 cell line). This
project concerns independent transporters serving structurally
distinct amino acid substrates in the apical membrane of Caco-
2 cells.
1.4.2 The Hypothesis
The hypothesis is that alanine and arginine are
independently transported by discrete transporter systems in
the Caco-2 apical membrane, and that the transporter
activities are independently regulated in mature enterocytes
and during enterocyte development. Dipolar L-alanine is
transported via Na+-dependent secondary active transport
System B, while cationic L-arginine is transported via Na+-

23
independent Systems y+ in Caco-2 cells grown on solid surface
or on porous membrane filters. We further hypothesize that the
membrane's constitutive activities for System B and System y+
each decrease over time during Caco-2 enterocyte
differentiation and development. The activity of each
transport system can be up-regulated above the constitutive
level by two categories of regulating agents: (i) substrate
analogues served by each transporter, and (ii) the peptide
growth factors epidermal growth factors (EGF) and transforming
growth factor (TGF). Finally, we hypothesize that up-
regulation of transporter activities occurs in two phases: an
acute phased characterized by protein-synthesis-independent
substrate trans-stimulation, and a chronic prolonged phase
that likely involves protein kinase C and de novo protein
synthesis.
1.4.3 The Specific Aims
Aim 1: To kinetically classify the alanine and arginine
transport systems in the Caco-2 apical membrane and to examine
the changes in the constitutive baseline transporter
capacities of the sodium-dependent alanine transporter (System
B) and the sodium-independent arginine transporter (System y+)
during the Caco-2 epithelial development and differentiation.
Aim 2: To examine the acute and the prolonged phases of
individual amino acid substrates in increasing System B and
System y+ transporter capacities, in undifferentiated and

24
differentiated states.
Aim 3: To examine the roles of peptide growth factors
TGF* and EGF and protein synthesis in changing System B and
System y* activities in undifferentiated and differentiated
states.
Aim 4: To examine the role of cellular protein kinase C
in regulation of System B and System y+ by substrate or
TGF/EGF.

CHAPTER 2
GENERAL METHODOLOGY
2.1 Caco-2 Cell Culturing
The human intestinal epithelial Caco-2 cell is derived
from human colon adenocarcinoma cells. The cells can be grown
as a monolayer on both porous filters and plastic. Under
normal cell culture conditions, Caco-2 cells can be
subcultured for many generations. Some labs reports 9 0 or more
passages. Caco-2 cell growth on the plastic surface is
dependent on cell density. Cells divide horizontally, and cell
attachment does not stop the cell growth as normally seen in
cell culture. The attached cells continuously divide at a
lower rates. Days later (depends on cell density, with higher
cell density having a shorter turnover), the attached cells
become confluent. The confluent state is represented by the
cell to cell tight junction and by dome formation. The dome is
caused by the unidirectional transport and trapping of water
and electrolyte cross cell monolayer. Unique in the Caco-2
cells, the confluent Caco-2 cell undergo a spontaneous
enterocytic differentiation process without changing cell
culture conditions. The differentiating cells start to
polarize by forming apical and basolateral membranes, with
expression of the normal small intestinal epithelial cell
25

26
apical membrane marker enzymes on the cell membrane. To date,
the biochemical and histological tests indicate that
differentiated Caco-2 cells are quite similar to, but exactly
like normal small intestinal epithelial cells. The timing and
cellular characteristics associated with the differentiation
process of the Caco-2 cells resemble those of the normal crypt
to villous cell development. Caco-2 cells are a human colon
tumor transformed cell line having 106 chromosomes. The
unmistakable similarity of the histological and biochemical
characteristics makes the Caco-2 cell line a ideal model for
the in vitro analogue of the normal adult intestinal
epithelium.
The confluency and differentiation of Caco-2 cell states
are cell-attachment dependent. After trypsinization, the
attached polarized cells detached to become single non
polarized cells, and lose their differentiated
characteristics. Whether this process is a de-differentiation,
or simply a turning off of existing differentiation
expression, is still debatable. The daughter cells of these
de-differentiated or undifferentiated cells then undergo
another un-differentiation-confluency-differentiation process.
Regardless the states of parent cells, newly divided daughter
cells are undifferentiated. To ensure that the majority of
cells are at the undifferentiated state, we have used cells
only in their relative early generations (#19-50). The limited
time of subculturing also reduce the possibility of mutation.

27
Cell culture studied have their advantages and
limitations. On the positive side, cell culture provide a
uniform environment. It is a relatively simple and straight
forward preparation without the adverse effect of in vivo
preparation. The experimental conditions are controllable. On
the negative side, the cell line is not entirely normal cells.
Furthermore cell culture conditions are not those of the in
vivo physiological conditions, and there is the possibility of
mutation. The cell conditions after subculturing may be
different from the in vivo state. Despite the limitation of
the cell culture, the Caco-2 cells are still considered to be
an excellent model for adult intestinal epithelial study
(Pinto et al., 1983; Hidalgo et al., Zwebaum et al., 1991)
For my study, transport studies in Caco-2 cells were
performed in both the undifferentiated (age day 2-3) and the
differentiated (age day 8-9) cells of the same subcultured
batch of cells. In some cases in other cell ages (mentioned in
text). Cell culture technigues are based on established
procedures (Hidalgo et al., 1989; Blairs et al., 1987;
Mohrmann et al., 1986; Pinto et al., 1983) and our
modifications. The Caco-2 cells used for the present
experiments were between the cell sub-cultured passages #19 -
#50.
2.1.1 Materials
The established human intestinal epithelial cell line

28
Caco-2 was obtained from American Type Culture Collection,
Rockville, MD. Dulbecco's Modified Eagle Medium (DMEM), fetal
bovine serum, sodium bicarbonate, penicillin, streptomycin,
non-essential amino acids, Trypsin/EDTA, and Dimethyl
sulfoxide(DMSO) were from Sigma Co., St. Louis, MO. The 6-well
Falcon tissue culture dishes and 100 mm tissue culture dishes
were obtained from the Fisher Scientific, Pittsburgh, PA. The
0.2 /iM medium filters were from Millipore Co. Bedford, MA. The
0.4 jum 24 mm Costar's Transwell-COL collagen treated
microporous membrane filters (Catalog # 3425) were from Costar
Co. Cambridge, MA. [3H]-Alanine, [3H]-arginine, [3H]-glutamine
and [3H]-Threonine were obtained from Amersham Co., Arlinton
Heights, IL. [3H]--methyl aminoisobutyric acid was from
American radiolabled chemicals Inc., St. Louis, MO. NaCl,
choline Cl, KC1, MgS04, KH2P04, CaCl2, NaOH, and N-(2-
hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)
(HEPES)/tris(hydroxymethyl)aminomethane (Tris) were obtained
from Sigma Co., St.Louis, MO. Liguiscint scintillation fluid
was from National Diagnostics, Atlanta, GA. The protein assay
reagent was obtained from Bio-Rad Lab, Richmond, CA. Glacial
acetic acid was from Fisher Scientific, Pittsburgh, PA. The
scintillation counter and spectrophotometer were from Beckman,
Irvine, CA.
2.1.2 Caco-2 Cell Culture
The Caco-2 cells were routinely grown on the 100 mm

29
falcon tissue culture dishes in 15 ml Sigma's Dulbecco's
Modified Eagle Medium (DMEM; Sigma Co, St. louis, MO)
containing 4.5 g/1 glucose and 0.584 g/1 glutamine, and
supplemented with: 10% fetal bovine serum (Sigma Co, heat
untreated catalog # F 4884)), 3.7% sodium bicarbonate, 100
IU/ml penicillin, 100 ng/nil streptomycin (Sigma Co., St louis,
MO) and 1% non-essential amino acids (Sigma Co. St. Louis,
MO). The original seeding cell density was 3 x 105 cells/ml,
cells were counted using a hemacytometer. Cells were grown in
a humidified incubator at 37C in 10% CO2/90% 02. The day of
seeding was designated as day 0. The growth medium was changed
and cells were inspected daily.
2.1.2.1 Caco-2 cell subculturina
For the sub-culturing cells, cells four days old on the
100 mm dish were taken out of the incubator. The growth medium
was aspirated, and cells were washed once with 37C isotonic
calcium-free saline solution containing 0.05% trypsin and
0.02% ethylenediamine-tetraacetic acid (EDTA; Sigma Co.
St.Louis, MO) and immersed in 10 ml the same solution for 5
minutes in the sterile hood. The cell/trypsin mixture was
dispersed with a narrow tip glass pipette (Fisher Scientific,
Pittsburgh, PA) and the trypsin reaction was stopped by adding
DMEM with 10% FBS. Then the dispersed cells were sedimented in
a sterile conical centrifuge tube (Corning, Corning, NY) at
1000 x g for 5 minutes, and the supernatant was removed. Next,
growth medium was added to re-suspend the cells (using a

30
narrow tip glass pipette) until cells were separated- Possible
cell clumps were allowed to settle for a few minutes at 1 x g,
and only the top layer of medium containing single clumped
cells was used for sub-culture as confirmed by microscope
examination. Cells then were seeded in three ways: (1) seeded
in the 100 mm dish at a cell density of 3 x 105 cells/ml for
future sub-culture purpose, (2) seeded in the 35 mm Falcon
tissue culture dishes (6-well clusters; Becton Dickinson,
Lincoln park, NJ)) at a density of 1.93 x 105 cell/ml for
subsequent transport experiments, and (3) seeded in 24 mm
porous filter at a cell density of 1.93 x 105 cells/ml for
subsequent transport experiments. All cell culture procedures
were performed under sterile conditions in a hood. All
solutions used in cell culturing were filter-sterilized (0.2
MM membrane filter; Nalge, Rochester, NY; Millipak 20,
Millipore Co, Bedford, MA), or/and autoclave sterilized. The
growth medium, including the medium in the both upper and
lower chambers of porous filter (Costar Co, Cambridge, MA),
was changed daily. The cultures are inspected daily (using a
phase contrast microscope) to monitor cell growth (dome
formation, absence of contamination, etc).
2.1.2.2 Freezing Caco-2 cells
Four-day-old Caco-2 cells grown in the 100 mm dishes were
trypsinized and centrifuged as described in above subculturing
section. Cells were then re-suspended in 4C DMEM containing
10% FBS and 5% dimethyl sulfoxide (DMSO; Sigma Co, St.Louis,

31
MO). One milliliter of the cell/medium mixture (10 million
cells/ml) was then transferred into a sterile glass ampule or
a Nunc plastic tube (Inter Med, Denmark), which was later
sealed in a sterile hood. The sealed ampules and the Nunc
tubes were immersed into a 4C methyl alcohol freezing tank
(Fisher Scientific, Pittsburgh, PA). The freezing tank was
then placed into a -70C freezer for 72 hours before the
ample/tube were transferred into liquid nitrogen storage.
2.1.2.3 Re-thawing frozen Caco-2 cells
Sealed ampules containing the frozen Caco-2 cells from
the American Type Culture Collection or liquid nitrogen
storage were immediately immersed into a 37C water bath until
the ampule content was completely thawed. And the sealed
ampules were immersed into 70% (v/v) alcohol for a minute. The
following procedures were then performed inside a sterile
hood. The ampules containing cells were opened and cells were
transferred and suspended in 37 C DMEM containing 10% FBS. The
cells were then sedimented in a conical centrifuge tube at
1000 x g for 5 minutes, and seeded in the 100 mm cell culture
dishes following the procedures described in the above
subculturing section. The re-thawing cells were grown for at
least two subculturing generations before being used in any
experiment.
2.2 Caco-2 Cell Monolayer Transport
The Caco-2 cell form a monolayer on both the plastic

32
surface and porous filters. For the pre-confluent cells, the
junctions among cells are loose, and cell membrane has not
polarize yet. The cell uptake may involve the membrane,
excluding the portion attached to the plastic surface. For the
differentiated state, cells have already polarized with basal
membranes attached to plastic surface or filter; lateral
membranes are formed beneath the tight junctions which connect
the apical membranes. The apical surface faces the outer
environment. Organic solutes enter cells through the apical
membrane, so that the para-cellular pathway is minimal. This
has been confirmed by [3H]-inulin extracellular studies
(Arturson et al., 1992).
The membrane transport of amino acid is a bi-directional
process. The measured transport activity is the net influx of
transport equivalent to vectorial difference between the two
unidirectional fluxes. The rate of the net is therefore
determined by the total flux during a period of time during
which the flux is linear. In the case of monolayer transport,
the rate of the membrane transport of amino acid is therefore
equal to the rate of net accumulation of amino acid within the
cells over a period of time. We measured the total
accumulation of amino acid and the time at which the amount
accumulation is linear proportional to the accumulation time.
The mode and characteristics of membrane transport of amino
acid are determined by the Menten-Michaelis kinetic analysis.
The amino acid transport experiments were performed on

33
the 35 mm falcon dishes (6 well clusters) at the cell passages
#17 #50.
2.2.1 Caco-2 Cell Monolayer Transport
The amino acid uptake experiments were performed at room
temperature (22.5C 1.0C). Cells were taken out of the
incubator. Then the growth medium was aspirated, and cells
were rinsed three times with uptake buffer (22.5C) containing
137 mM NaCl (or choline Cl) 10 mM HEPES/Tris (pH 7.4) 4.7 mM
KC1, 1.2 mM MgS04, 1.2 mM KH2P04, and 2.5 mM CaCl2. The uptake
was initiated at time = 0 by adding 1 ml uptake buffer plus
0.01 -10 mM [3H]-alanine (2 jiCi/ml, isotope was dried in
nitrogen gas first and was then re-hydrolyzed in the uptake
buffer), 0.005 5 mM [3H]-Arginine (2 Ci/ml) or other
isotopes and concentrations as mentioned below into the cell
monolayer. For the System B regulation experiments, 1 mM
unlabeled MeAIB was also added to the uptake buffer to block
possible alanine uptake via the System A. For the arginine
uptakes, 10 mM unlabeled leucine was added to the uptake
buffer to block the System b0,+. During the uptake procedures,
the cell cultures were continuously shaken by an orbital
shaker (1 Hz). The uptakes were stopped by aspirating the
uptake buffer, and then adding 2 ml ice-cold uptake buffer
(lacking substrate) immediately to the cells. The ice-cold
buffer is aspirated, cells were rinsed two more times using
the same ice-cold buffer. Isotope was extracted from the cells

34
by adding 1 ml IN NaCl to the cells. After overnight
extraction (continuous shaking) a 200 fil aliquot of NaOH
extract was added to 10 ml Liquiscint scintillation fluid
which was then neutralized with 200 nl glacial acetic acid.
Radioactivity (counts per minute, CPM) was measured in the
Beckman scintillation counter with quench normalized using the
"H-number" method. Because the nonspecifically adhering label
was < 1 % of the total counts per minute (CPM) associated with
the cell uptake, the double label 14C-inulin space measurement
was not performed for subsequent experiments. The protein
content in the NaOH extract was measured by the Bio-Rad
protein assay. The rate of amino acid uptake activity was
equivalent to the initial linear slope of the uptake time
course. All subsequent uptake experiments were conducted
within the linear period at a uptake period < 10 minutes, with
the 0 minute points serving as blanks. The amino acid uptake
rates are expressed as nmole amino acid/mg cell
protein/minute. The sodium-dependent alanine transport is
equivalent to measuring total alanine uptake in NaCl buffer
and in choline Cl buffer, and then subtracting the difference.
Bio-Rad Protein Assay
The Bio-Rad protein assay reagent was diluted 1 : 4 with
de-ionized water. Fifty microliter cell/NaOH mixture was added
into the diluted reagent (5 ml), with 50 zl 1.4 mg/ml standard
y-globulin as the protein reference (plus additional 50 nl 1

35
N NaOH) and the 5 ml diluted reagent plus 50 /il 1 N NaOH as
the blank. The samples and reagents were mixed well and
stabilized for 15 minutes. The protein absorbance was measured
at wavelength of 595 nm by using the spectrophotometer. The
sample protein contents were then calculated by:
sample protein = (sample absorbance x 1.4 mg/ml) + (standard
protein absorbance).
2.2.2 Radioactivity Measurement
Cell/NaOH aliquot (200 1) was added into a 20 ml vial
and was neutralized by adding 200 /il glacial acetic acid.
Liquiscint scintillation cocktail (10 ml) was added to the
mixture. A 200 /il sample of uptake buffer (containing a known
specific activity of isotope) 200 /il IN NaOH, and 200 ml
glacial acetic acid were added together with 10 ml liquiscint
in the 20 ml vials. The vials were then placed into the
Beckman scintillation counter. The [3H]-radioactivity was
obtained as counts per minute. Uptake was subsequently
calculated as nmole amino acid/mg protein/minute.
2.3 Monolayer Transport In Caco-2 Grown On Porous Filters
As mentioned above, the Caco-2 cells can be grown on
either the plastic surface or porous filters. The confluent
cell monolayer on porous filters provide additional dimensions
to the membrane transport studies. The confluent cell
monolayer forms a barrier separating the upper chamber and

36
lower chamber of the filters. The confluency is determined by
measuring the electrical resistance across the cell layer. The
apical membrane is accessible via upper chamber and the
basolateral membrane is accessible to the lower chamber. Using
cell monolayers grown on plastic we measured only the flux of
amino acid across the apical membrane to cell cytosol. By
using the porous filter, we can not only measure the flux from
apical to cytosol, but also flux from basal to cytosol, and
therefore the trans-cellular fluxes from apical to basal or
basal to apical side. Thus we can integrate the events at
basal or apical surfaces, including regulatory receptors, and
site sites of transport. The membrane transport
characteristics were determined by kinetic analysis in both
basolateral and apical membranes.
2.3.1 Transport In Caco-2 Grown On Porous Filters
The uptake experiments were performed at room temperature
(22.5C 1.0C). The Caco-2 cell culture was taken out of the
incubator. The trans-epithelial resistance was measured using
a trans-epithelial open circuit potential difference apparatus
(world precision instrument Inc, New Haven, CT), confluent
cells with a resistance > 300 cm2 were used for uptake
studies. The growth medium in both the upper and lower
chambers was aspirated, and the cells were rinsed three times
with uptake buffer (room temperature) containing 137 mM NaCl
(or choline Cl), 10 mM HEPES/Tris (PH 7.4), 4.7 mM KCl, 1.2 mM

37
MgS04, 1.2 mM KH2P04, and 2.5 mN CaCl2. These experiments were
conducted in collaboration with Dr. S. Smith of Burroughs-
Wellcome company (Research triangle, NC).
For the measurements of the apical-cellular-basolateral
amino acid movement, the amino acid uptake was initiated at
the time = 0 by adding 1 ml uptake buffer plus [3H]-alanine or
[3H]-arginine into the apical side (the upper chamber), with
the basolateral side exposed to 3 ml uptake buffer (lacking
amino acids) in the lower chamber. During the uptake period,
the cell cultures were continuously shaken by an orbital
shaker (1 Hz) The uptakes were stopped by aspirating the
uptake buffer, taking the filters out of lower chamber, and
adding 2 ml ice-cold uptake buffer (lacking substrate)
immediately to the upper chamber. The buffer was aspirated and
cells were rinsed by the ice-cold buffer three times. Isotope
trapped inside the cells was extracted from the cells by
adding 2 ml 1 N NaOH to the cells (filters were first cut out
off the dishes). After overnight extraction (continuous
shaking), a 200 nl aliquot of NaOH extract was added to 10 ml
Liquiscint scintillation fluid which was neutralized with 200
/I glacial acetic acid. Isotope trapped in the lower chamber
was measured by transferring 200 il of this buffer to 10 ml
Liquiscint. The radioactivity was counted in the Beckman
scintillation counter.
For the measurement of basolateral-cellular-apical
movement, uptake was initiated at time = 0 by immersing

38
filters (with 1 ml uptake buffer with no substrate in the
upper chamber) into 3 ml uptake buffer plus [3H]-alanine or
[3H]-arginine in the lower chamber. During the uptake period,
the cell cultures were continuously shaken by the orbital
shaker (1 Hz). The uptake was stopped by removing the filters
out of the lower chamber, and removing the upper chamber
immediately. The filters were rinsed three time with ice-cold
buffer. Isotope trapped in the cells and in the upper chamber
buffer was measured separately as described above.
For the measurement of the apical- basal trans-cellular
amino acid movement, the uptake was initiated at time = 0 by
adding 1 ml uptake buffer plus [3H]-alanine or [3H]-arginine
to the apical side and immersing the filter into 3 ml buffer
(lacking substrate) in the lower chamber. Uptakes were stopped
by removing filters from the lower chamber. The isotope
accumulated in the lower chamber was the measured as described
above.
For the measurement of the basal-apical trans-cellular
movement, the uptake was initiated at time = 0 by placing the
filter (with 1 ml uptake buffer with no substrate in the upper
chamber) into 3 ml buffer containing [3H]-alanine or [3H]-
arginine in the lower chamber. During the uptake period, the
cell cultures were continuously shaken (1 Hz) The uptakes
were stopped by taking out the buffer from the upper chamber.
The isotope accumulated in the upper chamber was measured in
scintillation counter as described above.

39
2.3 Statistical Analysis
All experiments were conducted in triplicate (including
the 0 time blanks, and the uptakes in Na+ and choline buffers)
and all experiments were confirmed in at least two differently
seeded generations of cells. Curve fitting was conducted by
non-linear regression analysis. Values were reported as the
mean + SE. ANOVA used for statistical analysis with Duncan's
test, the level of significant p < 0.05.

CHAPTER 3
CLASSIFICATION OF THE ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE
31 Introduction
The characterization of membrane amino acid transport was
pioneered by Christensen several decades ago (Christensen et
al., 1952). Since that time, the criteria to assess a membrane
amino acid transport systems in mammalian membranes have
indeed substrate specificity, ion-dependency, initial uptake
rate kinetics, patterns of analogue cross-inhibition of amino
acid uptake, and exclusive substrates transported through
specific transport systems. The development concept of the
Na+-gradient-driven, secondary active solute transport (Crane
et al., 1962) was an another important addition to describing
the transport phenomena. In the past several years, there have
been reports of cloning cDNA encoding several amino acid
transporters (Kilberg et al., 1993), and cloning of possible
regulatory proteins for Systems L, b0,+ or y+. There has no
cloning, antibody production, nor purified transporter protein
reported for major alanine transport systems in intestine. The
phenomenological criteria remain the sole tools to classify
alanine transport systems. The sole exception is the recent
cloning of System A (Kong et al. 1993), although System A
40

41
represents a minor pathway in intestine (described below).
The major membrane transport Systems A, ASC, B0,+, B, L,
and asc for the dipolar amino acid L-alanine have been
intensively studied in various tissues. (Oxender et al., 1963;
Christensen et al., 1963; Stevens et al., 1982; Van Winkle et
al., 1985, 1987, 1988; Van Winkle & Campione, 1990; Kilberg et
al, 1993; Stevens, 1992a,b). Among these alanine transport
systems, only System B was originally described as an unique
in intestinal epithelial cell transporter (Stevens et al.,
1982, 1984). The others systems were first described for non-
epithelial cells (Stevens, 1992a,b).
System A is a strictly Na+-dependent system which is
broadly selective for most dipolar (neutral) amino acids
(Christensen et al., 1963). System A is found in many tissue
membranes. One specific feature of System A is that the non-
metabolized MeAIB is a specific substrates for System A. AIB
serves as less specific substrate. System A regulatory
properties have been intensively investigated in hepatocytes
and other tissue. Several regulation mechanisms of System A
activity were proposed (Kilberg, 1986; Engleberg, 1986; Dawson
& Cook, 1987). System A reportedly exists at the apical and
basolateral membrane of guinea pig intestinal epithelia
(Hayashi et al., 1980; Del Castillo et al., 1991).
System ASC is another Na+-dependent transport system
serving 3- and 4-carbon neutral amino acids, exemplified by
alanine, serine and cysteine (Kilberg et al, 1981). System ASC

42
is distinct from System A in that System ASC does not
transport MeAIB. System ASC is also found in many tissue.
System ASC reportedly exists at guinea pig intestinal
epithelial apical membrane (Hayashi et al., 1980).
System B0,+, first described in mouse blastocytes (Van
Winkle et al., 1985), is a Na+-dependent transport system that
transports both the cationic and neutral amino acids. System
B0,+ is expressed in many tissues.
The strictly Na+-dependent transport System B exclusively
found in the apical membrane of the epithelial cells. It was
first described in rabbit jejunum apical membrane vesicles as
a broadly selective system serving neutral amino acids
(Stevens et al., 1982, 1984, Stevens, 1992a,b). It was
originally named System Neutral Brush Border (NBB), and later
was renamed as "System B" (Stevens, 1992a). System B substrate
selectivity is similar to the System B0,+ in that both are Na+-
dependent, as it possesses a broad selectivity for most
dipolar amino acids. Both Systems B and B0,+ interact with the
bicyclo-amino acids 2-amino-2-norbornanecar-boxylic acid (BCH)
or BCO. However, System B 0,+ is inhibited by cationic amino
acids such as lysine and arginine, while System B is not
interactive with cationic amino acids. System B may possible
be a variant of System B0,+.
The Na+-independent System L was shown to exist in the
apical and basolateral membrane of many cell types. System L
is a broadly selective system serving neutral amino acids,

43
cysteine, phenylalanine, and BCH.
System b0,+, a Na+-independent analogue of System B0,+
serving neutral and cationic amino acid such as lysine and
arginine. The substrate scope of System b0,+ is similar to
System B0,+. System b0,+ exists in many cell types, but it has
been previously reported in the intestinal membranes.
Only a few organic solute transport studies in the Caco-2
cells have been reported (Blais et al., 1987; Hidalgo et al.,
1988; Mohrmann et al., 1986; Souba et al., 1992, Nicklin,
1992). Each of the reports showed the similarity of solute
transport characteristics in Caco-2 cells and intestinal
epithelial cell.
Inasmuchas alanine transport systems were not described
for Caco-2 culture, our study began by describing the alanine
transport systems in the Caco-2 monolayer.
3.2 Methods And Materials
3.2.1 Methods
The [3H]-alanine uptake experiments were performed in
both the pre-confluent (day 2-3) and confluent (day 8 -9)
cells. The basic uptake procedures were as described above
(chapter 2) Special uptake conditions are presented below
where appropriate.

44
3.2.2 Materials
The materials were as the same as discussed in the
chapter 2.
3.3 Results
3.3.1 Alanine Uptake Time Course
The 50 /M and 5 mM [3H]-alanine uptakes were measured at
during various times (0-45 minutes) in the uptake media
containing 137 mM NaCl or 137 mM choline Cl. The 50 /M alanine
uptake on day 2 cells was shown at Fig. 3-1. The accumulation
in the NaCl medium was greater at any point than that in the
in choline Cl medium suggesting a Na+-activation phenomenon.
The initial alanine accumulation in the Caco-2 monolayers was
linear up to 15 minutes (at both [3H]-alanine concentrations
of 50 M and 5 mM) Since the transport activity was expressed
as the alanine uptake rate measured during the linear period,
the uptake period of 0 10 minutes was chosen for all the
subsequent uptake measurements. The rate was equal to total
accumulation divided by the time period.
3.3.2 Alanine Uptake Rates at Various Caco-2 Cell Ages
The 50 iM [3H]-alanine uptake rates were measured at
various Caco-2 cell ages ranging from 1 day old to 35 days old
in both the NaCl and choline Cl media (Fig. 3-2) At each cell
age, the total alanine uptake rate in NaCl medium was

45
consistently greater than that in the choline Cl medium. This
difference was greater in the younger cells, compared to
confluent cells.
Alanine uptake rates in both the NaCl and choline Cl
media decreased as cell age increased. The uptake rates in
NaCl medium decreased rapidly over a period of several days (<
4 days old), and maintained steady after differentiation (> 9
day old). The decrease in alanine uptake in choline Cl medium
was less dramatic than that in the NaCl medium, with the rate
consistently decreasing throughout the cell ages.
In a separate study, the 24 hours proliferation rates of
Caco-2 cells (2 day old 14 days old) were measured by
incubating the cells in [3H]-thymidine medium for 24 hours.
The amount of thymidine incorporated into the cells during the
period represented the cells relative proliferation rate. The
thymidine incorporation into the Caco-2 cells deceased as cell
age advanced (Fig. 3-3).
The pattern alanine uptake rates at various cell ages was
coincident with the cell proliferation rates.
3.3.3 Ion-deoendencv
The uptake of 50 M [3H]-alanine was measured in the
uptake media containing 137 mM NaCl, 137 mM choline Cl, 137 mM
KC1, or 137 mM LiCl. The total alanine uptake rate in the NaCl
medium was 8-fold greater than that in either the choline Cl,
KC1 and LiCl media at [alanine] = 50 /M (Fig. 3-4) The total

46
alanine uptake rates in the choline Cl, KC1, or LiCl media
were not significantly different. Alanine uptake in the NaCl
uptake medium was not significantly different from that in the
medium containing sodium gluconate.
These data indicated that alanine uptake activation was
strictly Na+-dependent. Other cations such as Li+ or K+ could
not substitute Na+ in activating alanine transport. Chloride
was not required for the Na+-activation.
For all the subsequent experiments, the Na+-dependent
fraction of alanine total uptake was obtained by subtracting
the uptake measured in choline medium from the total uptake
measured in sodium medium.
3.3.4 The Effect of pH on Alanine Uptake
The uptake of 50 M alanine in both the NaCl and choline
Cl uptake media was measured at various media pH ranging from
pH 6.0 to pH 8.5. HEPES and Tris were used to adjust media pH.
The total alanine uptake rates in both the NaCl and choline Cl
media increased steadily as the medium pH increased (pH =6.1,
7.4, and 8.5) (Fig. 3-5).
3.3.5 Na*-Activation of Alanine Uptake in Caco-2 Cells
The uptake of 50 xM alanine was measured in the uptake
media containing NaCl ranging from [NaCl] = 0 mM to 137 mM
(choline Cl as substitute). The total alanine uptake rates at
both day 2 and day 9 cells increases as media NaCl

47
concentration increase. The uptake rates as a function of NaCl
concentrations gave a hyperbolic shape (Fig. 3-6). The non
linear regression analyses of the Hill equation gave the same
Na+-activation Hill coefficient (n = 1) at each cell age,
while the VBax was greater in day 2 cells than in day cells.
The same Hill coefficient (n = 1) indicates that one Na+ binds
to transporter coupled with each alanine molecule transported,
in cells 2 days and 9 days old.
These data indicated that the alanine uptake capacity was
greater in day 2 cells. Therefore, the difference in transport
capacity between day 2 and day 9 cells was not due to the
transport system's affinity for Na+-activation coefficient
changes.
3.3.6 Alanine Uptake Kinetics
The alanine uptake in uptake media containing 137 mM NaCl
or containing choline Cl was measured at various [3H]-alanine
concentrations ranging from 10 /xM to 5 mM at the cell ages of
day 2 and day 9. The kinetics at day 2 and day 9 cells were
shown as uptake rates measured as a function of alanine
concentration (Fig. 3-7; Fig. 3-8). The kinetics in either the
NaCl or choline Cl medium each displayed both saturable and
non-saturable components, indicating multiple transport
systems were involved. At each alanine concentration, the
uptake rate was higher in the NaCl medium.
In the choline Cl media, alanine uptake occurred via two

48
pathways: a saturable Na+-independent carrier-mediated system
and non-saturable simple passive diffusion. In NaCl media,
besides the two pathways discussed in the choline Cl medium,
an additional saturable Na+-dependent system exists.
For the non-saturable component, the passive permeability
coefficient (P) describing the relation J = P [Ala] was
constant at 0.53 + .08 /xliter/(mg protein)/min regardless of
the cell differentiation states.
For the saturable components, non-linear regression
analyses of Na+-dependent alanine transport kinetics gave KB =
164 2 6.1 /zm ole alanine and VBax = 2.79 0.21 nmole/mg
protein/min for the day 2 cells. For 9 day old differentiated
cells, the K, was 159.0 13.6 jumle alanine and VBax was 0.512
0.03 nmole/mg protein/min (Fig. 3-9). Regarding the Na+-
independent alanine uptake system (tentatively, System L) the
activity decreased from VBax = 1.85 0.25 nmole/mg/min in the
undifferentiated (day 2) cells to Viax = 0.38 + 0.017 nmole (mg
protein)'1 min'1 in the differentiated (day 9) cells. The
System L apparent K for alanine was unaffected by cell age
(differentiated cell K, = 1.10 + 0.19 mM vs. differentiated
cells K = 1.02 + 0.007 mM alanine).
The kinetics revealed that alanine uptake capacity was
higher in day 2 cells than that in day 9 cells, and therefore
the difference was only a VBax effect. The affinities of both
the saturable components were not affected by cell ages. The
non-saturable passive diffusion was not affected by cell age.

49
3.3.7 The Analogue Cross-inhibition Pattern
The 50 /M [3H]-alanine uptake rates were measured in
media containing 137 mM NaCl and 137 mM choline Cl plus 5 mM
single amino acid analogues (natural amino acids, BCH, MeAIB,
AIB, and /3-alanine with 5 mM mannitol as control) .
For the Na+-independent alanine transport system, alanine
uptake was strongly inhibited by phenylalanine, alanine,
leucine, threonine, serine, glutamine, asparagine, cysteine,
and BCH, and weakly by MeAIB, AIB, and glycine. Lysine, and
glucose did not inhibit (Fig. 3-10 & Fig. 3-11).
For the Na+-dependent alanine transport system, the
uptake activity was inhibited by 5 mM amino acid analogues
(natural AAs plus BCH, MeAIB, and /3-alanine) was shown in Fig.
3-12. The Na+-dependent [3H] alanine transport was strongly
inhibited by threonine, glutamine, serine, cysteine, and
asparagine. Weaker inhibition was elicited by glycine,
phenylalanine, leucine and the bicyclo amino acid BCH. MeAIB
and cationic amino acids elicited <10% inhibition. Dixon
inhibition analyses indicated that the glutamine inhibition
was classic competitive inhibition, while the MeAIB affect was
un-competitive (Figs. 3-13 3-15). Proline, glycine, and
phenylalanine gave high K values (Figs. 3-16 3-21).
The pattern and degree of amino acid analogue inhibition
of the Na+-dependent alanine uptake was identical at both cell
ages, suggesting that the same transporter system was
operative regardless of the cell age (Fig. 3-22).

50
In a separate study, the 50 mM [3H]-MeAIB uptake rates
were < 5% of the same concentration of alanine, suggesting a
minimal contribution by System A in our Caco-2 cells line.
3.3.8 Alanine Uptake on Porous Filters
Uptake of alanine (50 /iM) into the apical and basolateral
surfaces of confluent Caco-2 monolayer grown on porous filters
were measured. The Caco-2 cell monolayer confluency was
determined by measuring the trans-cellular resistance, with
the trans-epithelial resistance > 300 n x cm2 was considered
confluent. The apical compartment to the basal compartment of
50 alanine uptake was measured (Fig. 3-23). The majority of
alanine across the apical membrane was accumulated inside the
cells rather than transport across to the basal side (Fig. 3-
24) .
Alanine (50 /M) uptake at the basal membrane to cytosol
and apical compartment was also measured (Fig.3-25, 3-26, and
3-27). The uptake in NaCl medium was greater than that in the
choline Cl medium, indicating a Na+-activation event.
3.4 Discussion
The alanine uptake in Caco-2 monolayer at the different
cell ages was studied. The alanine uptake activity was
different at various cell age, indicating the possible
regulation of cell development. There were several pathways
for alanine uptake. By using the membrane transport system

51
classification criteria, we classified the alanine transport
systems in Caco-2 cells, as discussed below.
3.4.1 Alanine Uptake Activity vs Cell Ages
Both the Na+-dependent and Na+-independent alanine uptake
activities decreased as cell age increased at the alanine
concentration of 50 /xM (over the cell age span of 1 35 days)
(Fig. 3-2) What were the mechanisms underlie this cell
development regulation? There were several possible mechanisms
that could underlie this development-related regulation. Non
specific membrane potential or other membrane property change
could cause a non-specific driving force alteration,
permeability of the membrane could change, or specific
functional change of specific transport systems could occur.
Each of these possibilities was explored.
The Na/glucose cotransport activity on Caco-2 monolayer
has been reported to increased with cell age (Blais et al.f
1987). The opposite direction of alanine uptake activity and
Na/glucose activity with cell age rule (Fig. 3-23) out the
possibility that the age-associated transport effect was due
to the non-specific membrane electrochemical gradient which
may associated with cell age. Therefore the non-specific
driving force was not likely to be involved in the mechanism.
In terms of membrane properties at different cell ages,
our kinetics studies gave the same diffusion permeability
coefficients in day 2 and day 9 cells, even though the alanine

52
uptake rates were several fold higher in the day 2 cells (Fig.
3-2) The alanine uptake change over the cell ages was
therefore only a portion of saturable carrier-mediated uptake.
The membrane permeability was then unlikely to be involved in
the regulation mechanism.
As discussed above, the alanine uptake rates decreased
with advancing cell age, while Na/glucose cotransport
increased with cell age (Fig. 3-23). This opposite direction
of transport activity suggests that the function of alanine
and glucose were not the same in cell development. Alanine was
not solely for caloric purpose. In the light of the cell
proliferation rate decrease with the cell age increase (Fig.
3-3) the reduced alanine uptake may be due to the lowered
requirement for amino acids, but not for glucose. These data
also indicated that the Na/glucose and alanine uptake in Caco-
2 cells were independently regulated by the cell
differentiation and development.
3.4.2 Classification of the Alanine Transport Systems
There were three alanine transport pathways in Caco-2
monolayers for alanine at both cell stages (the
undifferentiated and the differentiated stages): a simple
passive diffusion, a Na+-independent system, and a Na+-
dependent system.
3.4.2.1 Simple Passive diffusion
The same passive permeability coefficient measured in

53
both the day 2 and day 9 cells suggested that the Caco-2 cell
development did not alter the membrane permeability to
alanine. The diffusion rates of alanine across the cell
membrane at certain alanine concentrations were the same at
either cell ages. The passive diffusion contribution at 50 ;xM
alanine uptake was minimal, less than 1% of total uptake in
NaCl. At higher alanine concentrations, the passive diffusion
contribution was greater. At [alanine] = 5 mM, passive
diffusion contributes 90% of total alanine uptake in NaCl.
3.4.2.2 Na*-independent transport system is System L
One saturable Na+-independent alanine transport system
existed in both the day 2 and day 9 cells. The observed
activity decreased with the advancing cell age (Fig. 3-2). The
activity decrease was coincident with the cell proliferation
rates (Fig. 3-3). The activity was possibly regulated by cell
proliferation requirements.
The Na+-independent alanine transport kinetics in both
the 2 day old and 9 day old cells revealed that the transport
activity VBax was higher in the day 2 cells. The transport
apparent affinity K, was the same at both cell ages. These
kinetic parameters strongly indicate that the transport
capacity was greater in the day 2 cells; the activity change
was a Vaax effect, not K, effect, suggesting the presence of
same transport system in both differentiated and
undifferentiated states. The activity change was likely a
change of copies of functional transport units in the membrane

54
instead of some modification of transporter affinities.
The amino acid analogue inhibition patterns on both the
day 2 and day 9 cells were similar in that phenylalanine,
leucine, BCH, and alanine strongly inhibited the alanine
uptake, while MeAIB, glycine and lysine were weak inhibitors
(Figs. 3-10, 3-11). These inhibition patterns strongly
resemble that of the System L. The non-interaction with lysine
ruled out the possibility of the System b0,+ The strong BCH
inhibition suggesting the unlikely System asc.
Based on the kinetic characteristics, the pH sensitivity,
and the cross-inhibition pattern, We conclude that the Na+-
independent alanine uptake is via the System L at both the day
2 and day 9 cells.
3.4.2.3 Na*-deoendent alanine transport System B
The alanine uptake in the Caco-2 cells was strongly Na+-
dependent in either day 2 old and day 9 old cells (Fig. 3-4).
The Na+-dependent portion was more than 85% of the total
alanine uptake in NaCl medium at 50 /xM alanine (Fig. 3-4) No
other monovalent cationic K+ or Li+ substitute for the Na+ in
activating the alanine uptake. Furthermore, the system was not
activated concomitantly by Cl'.
One of the important aspect in classifying transport
systems was the cross-inhibition profile. The amino acid
analogue inhibition pattern of the Na/alanine uptake for both
the day 2 and day 9 cells was similar: the Na/alanine
transport was strongly inhibited by threonine, serine,

55
glutamine, cysteine, and asparagine; Weaker inhibition was
elicited by glycine, phenylalanine, leucine and BCH. MeAIB and
cationic amino acids elicited < 10% inhibition (Fig. 3-12 & 3-
22). We can compare this inhibition pattern with the amino
acid inhibition patterns of the known Na/alanine Systems A,
ASC, B, and B0,+. System A is a strictly Na+-dependent system
selective for dipolar amino acids including alanine. Many
neutral amino acids competitively inhibit Na/alanine transport
via System A. One special aspect of System A is its unique
ability to transport MeAIB. In our inhibition study, MeAIB
blocked less than 10% of the Na/alanine transport activity
(Fig. 3-12). Dixon analysis revealed that the MeAIB inhibition
was a non-competitive inhibition (Fig.3-15). These combined
data exclude System A as a major transport system in Caco-2
cells.
System ASC, the Na+-dependent system serves short-chain
neutral amino acids alanine, serine, and cysteine. In our
study, serine, cysteine strongly inhibited alanine/Na uptake.
However, phenylalanine and glycine, two competitive inhibitors
of System ASC did not inhibit the Na/alanine transport in our
study, as it would for the classic System ASC (Figs. 3-12, 3-
18-21) Based on this and the similarity of our data to System
B (discussed below), we exclude System ASC as the transport
system. Because the characteristics were very close, however,
definite classification is not possible without more precise
test methods such as cDNA probes or antibodies.

56
Another Na+-dependent alanine transport system is System
B0,+, which serves both the neutral amino acids and cationic
amino acids. The only evidence that does not support existence
of System B0,+ was that cationic amino acids arginine and
lysine did not inhibit Na/alanine uptake in our study (Figs.
3-12 & 3-22) Thus, it is unlikely that System B0,+ exists in
Caco-2 cells.
The final Na/alanine system candidate System B described
first for intestinal epithelial cells (Stevens et al. 1984).
System B has only been found in the epithelial cells of
vertebrate and invertebrates (Stevens, 1992). The substrate
selectivity of System B is very similar to System B0,+ except
that System B does not interact with cationic amino acids
(Figs 3-12 & 3-22). Na/alanine uptake was strongly inhibited
by of neutral amino acids serine, threonine, cysteine, weakly
by glycine and phenylalanine, interaction with BCH, and was
interactive with cationic amino acids arginine and lysine in
our studies (Figs. 3-12 & 3-22). The amino acid analogue
inhibition pattern supports the existence of System B. The
apparent affinity K, = 159 mole alanine in Caco-2 was similar
to the System B report elsewhere (Stevens et al., 1982) (Fig.
3-9). Furthermore, the pH sensitivity (Fig. 3-5) and the Na+-
activation Hill coefficient (n = 1) (Fig. 3-6) further support
the case for System B. Based on the our evidence (Figs. 3-4
through 3-9, and 3-11 through 3-22), we conclude that the
Na/alanine transport system in the Caco-2 cells was likely

57
System B.
The System B transport activity decreases as cell age
increases (Fig. 3-2). The decrease in activity was coincident
with the decrease of cell proliferation rates with cell ages
(Fig. 3-3). The proliferation rate may be related to the cell
requirement for amino acid. In contrast to the Na/alanine
transport activity, the Na/glucose activity increases as cell
age advances (Blais et al., 1987) (Fig. 3-23). These opposing
changes in activity for Na+-dependent solute transport as a
function of cell ages excludes the likelihood that the amino
acid transport was regulated by non-specific membrane
electrochemical potential effects. Furthermore these data
suggest that cell development is associated with the
independent regulation of amino acid and glucose transport
systems. We tested the Na/alanine transport characteristics in
two different cell states, the undifferentiated state (day 2
cells) and differentiated state (day 9 cells). The amino acid
analogue inhibition pattern, pH sensitivity, and Na+-
activation Hill number were the same for both the day 2 and
day 9 cells (Figs. 3-4, 3-5, and 3-12). The transport kinetics
gave a VBax on the day 2 that was greater than for day 9 cells,
while the apparent affinity K, was the same on both cell ages
(Fig. 3-9) All the transport characteristics of the Na+-
dependent alanine transport (except VBax) were identical in
both the undifferentiated and differentiated states,
suggesting that the same transporter system was operative

58
regardless of the cell age. The kinetics also indicated that
the transport capacity was greater in the day 2 cells, and
that the activity difference between the two days was like
caused by the change in functional transporter units expressed
in the membrane, rather than modification of existing
transporter affinity.
3.5 Summary
Alanine is transported in Caco-2 cell by a Na+-dependent
transport System B, a Na+-independent transport System L, and
simple passive diffusion. These same systems were operative in
both the undifferentiated and differentiated cell states. The
passive diffusion coefficient was not affected by cell
development. The alanine transport Systems B and L activities
are down-regulated as the cell develops, coincident with the
cell proliferation rates. The decrease in transport activities
are likely caused by the decrease in copies of functional
transporter units, rather than modification of existing
transporter affinity for substrate or ions.

Fig. 3-1. Alanine uptake time course
The uptake of alanine (50 /M and 5 mM) was measured in
uptake media containing NaCl and choline Cl in day 2 and day 9
cells. The alanine uptake in NaCl media was greater than that in
the choline Cl media at any point (except t = 0, p < 0.05, n =
6) The uptake values in this figure and subsequent experiments
were expressed as mean standard error (SE) The data shown were
from the alanine (50 /uM) in day 2 cells, with similar data
obtained in the day 9 cells and at other alanine concentrations.

50
40
30
20
10
0
1 1 1 I 1 I
In NaCI *
[)
O In Choline Cl
-
* *

1
r
5
O
O
_

0
o
1 1 1 1 1 1
1 L
5 10 15 20 25 30 35 40 45
Time, minutes
<7>
O

Fig. 3-2. Alanine uptake at various cell ages
The uptake of alanine (50 /xM) was measured in NaCl and
choline Cl media over cell ages of 1 35 days old. At any cell
age, the alanine uptake in the NaCl was greater than that in the
choline Cl media (p < 0.05, n = 6), even though the difference
margin was smaller in the older cells. The Na+-dependent alanine
uptake decreased with the advancing cell age, while the Na+-
independent alanine uptake also decreased at less extent.

Alanine uptake, nmole mg min
Cell age, days
30

Fig. 3-3. Caco-2 cell proliferation rates at various cell ages
The 24 hour incorporation rates of [3H]-thymidine into
various cell ages were measured. The cells had been incubated in
serum-free DMEM for 24 hours prior to the measurements. The blank
control value = 1012 CPM, and the incubation medium value = 3.74
x 105 CPM.

Cell age, days
Thymidine 10 CPM (mg protein)
^Moj-f^cncri^joo
ooooooooo

Fig. 3-4. Alanine uptake ion-dependency
The uptake of alanine (50jUM) was measured in uptake media
containing 137 mM NaCl, 137 mM choline Cl, 137 mM KC1, or LiCl.
The uptake in NaCl media in both the day 3 and day 8 cells was
greater than that in either those in choline Cl, KC1, or LiCl
media (p < 0.05, n = 6). Uptake in choline Cl, KC1, or LiCl
media was not significantly different (p > 0.05, n = 6) .

Alanine uptake nmol mg min
o o o o o
ro go £*
99
Q'O

Fig. 3-5. The effect of pH on alanine uptake
The uptake of alanine (50/iM) in day 3 and day 8 cells was
measured at various medium pH (at pH = 6.1, 7.4, and 8.4). The
uptake rates were higher in more alkaline media.

99
90

Fig. 3-6. Na*-activation of alanine uptake
The uptake of total alanine (50 /M) in day 2 and day 9
cells was measured in media containing various NaCl concentrations
([NaCl] = 0 137 mM, choline Cl substituted NaCl). The Non-linear
regression of these data gave the same Na+-activation Hill
coefficient of n = 1 for both the day 2 and day 9 cells.

[Na], mM
'si
o

Fig. 3-7. Alanine uptake kinetics in day 2 cells
The alanine (10 /iM 5 mM) uptake was measured in the day
2 cells. The figure showed the total alanine uptake rates in NaCl,
choline Cl media, or Na+-dependent alanine uptake rate as a
function of alanine concentrations. The curve contained non
saturable and saturable components.

T
T
8
c
E
cn
E
_qj
o
E
c
CD
o
-+-J
Cl
Z5
CD
C
'c
_o
<
7
6
5
4
3
2
T Na dependent
total in CHO
O total in NaCI
0


o
00* ^
0 100
I I I I
200 300 400 500
[Alanine]
600 700 800 900 1000

Fig. 3-8. Alanine uptake kinetics in day 9 cells
The uptake of alanine (10 /iM 5 mM) was measured in NaCl
and choline Cl media. The total uptake in NaCl and choline Cl
media, and Na+-dependent uptake were showed as a functional of
alanine concentrations. The curves showed non-saturable and
saturable components.

i i r
cn
E
_0)
o
E
c
CD
o
Cl
Z3
CD
C
c
_o
<
1.0 -
0.5
V Na -dependent
O total in CHO
total in NaCI
.*
0.0
v
Iw
0
V
,o
V
O
O
V
o
0 100 200 300 400 500 600 700 800 900 1000
[Alanine], /jlM

Fig. 3-9. Eadie-Hofstee transformation of Na+-dependent alanine
uptake kinetics in day 2 and day 9 cells
The Na+-dependent alanine (10 /M 5 mM) uptake of fig.
3-7 was expressed as alanine uptake as a function of alanine
uptake/alanine concentration. Non-linear regression of these data
gave a straight line, indicating a single transport system. The Viax
values (the interception of the line and the y axis) were Vn)ax =
3.1 0.21 nmole/mg/min for day 2 cells, and Vaax = 0.51
nmole/mg/min for day 9 cells. The Km values (the negative slope
of the line) were Km = 167 26.1 /mole alanine for day 2 cells,
and Km = 159.0 13.6 /mole alanine for day 9 cells.

J j nmole mg min
1 M 04
boo
9

Fig. 3-10. Na+-independent alanine uptake inhibition pattern in day
3 cells
Alanine (50 /M) uptake in choline Cl medium was measured
in day 3 cells, with 5 mM single amino acid present in the uptake
media. The Na+-independent portion was the difference between the
total uptake in choline Cl media and passive diffusion.

Alanine uptake, nmole mg min
o k> c-j -f>- cn
Cont
Ala
Asp
BCH
Cys
Gin
Leu
Ser
Thr
Phy
AIB
Pro
Gly
MeAlB
Lys
Glu
8

Fig. 3-11. Na*-independent alanine uptake inhibition pattern in day
9 cells

-1
Alanine uptake, pmole mg min
cn
b
o
Cn
IV)
O
M
cn
Cont
Ala
Asp
Gin
Phy
Ser
Thr
BCH
Leu
MeAlB
Pro
Gly
AIB
Cys
Lys
Glu
09

Fig. 3-12. Na*-dependent alanine uptake inhibition pattern in day
3 cells
Alanine (50 /iM) uptake in day 3 cells was measured in NaCl
and choline Cl uptake media containing single 5 mM amino acid. The
Na+-dependent portion was shown.

82
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Fig. 3-13. Dixon analysis of Na+-dependent alanine uptake with
glutamine as inhibitor
Alanine (25 juM, 50 /M, and 100 /M) uptake in NaCl and
choline Cl media was measured with various concentration of
glutamine (10 /M 5 mM) presented in uptake media. The dixon
plot gave a Ki of 35 /M glutamine.

[Glutamine], ¡jM
0.4

Fig. 3-14. Replot of the slopes of Dixon plot with glutamine as
inhibitor
The slopes of dixon plot shown at Fig 3-13 were shown as
a function of (corresponding alanine concentrations)'1. Non-linear
regression of these data intercepted 0. The combination of Fig. 3-
13 and this figure indicated that glutamine was a competitive
inhibitor for System B.

Slope
1 /[Ala], 10 L/mole

Fig. 3-15. Dixon analysis of Na+-dependent alanine uptake with MeAIB
as inhibitor
Alanine (25 /xM, 50 /xM and 100 /xM) uptake was measured with
various concentrations of MeAIB (10 /xM 5 mM) in uptake media.
Non-linear regression of these data were parallel, indicating MeAIB
was not a competitive inhibitor.

0
o
§

Fig. 3-16. Dixon analysis of Na+-dependent alanine uptake with
proline as inhibitor
Alanine (25 /zM, 50/zM, and 100 /zM) uptake was measured with
various concentrations of proline (10 /zM 5 mM) in uptake media.
Ka = 7.1 mM proline.

[Proline],
l/JAla (m<3 Pro^e'n) m'n nm0le
- cn m
O O cn
O c c
c s <:
06

Fig. 3-17. Replot of the slopes of Dixon plot with proline as
inhibitor
The slopes of figure 3-16 dixon plot were shown as a
function of 1/[alanine]. Non-linear regression of these data was
through the interception of x axis and y axis. These data combined
with fig. 3-16 suggested that proline was a weak competitive
inhibitor for the Na+-dependent alanine uptake.

Slope
4
1/[Alanine], 10 L/mol
4

Fig. 3-18. Dixon analysis of Na+-dependent alanine uptake with
glycine as inhibitor
Alanine (25 /xM, 50 /xM, and 100 /xM) uptake was measured
with various concentrations of glycine (10 /xM 5 mM) in uptake
medium. The value was about 5.5 mM glycine.

P6
15.0

Fig. 3-19. Replot of the slopes of Dixon plot with glycine as
inhibitor
The slopes of the dixon plots at Fig. 3-18 were shown as
a function of l/[alanine]. Non-linear regression of these data was
through 0 point of both axis. These data combined with fig. 3-18
suggested that glycine was a weak competitive inhibitor of the Na+-
dependent alanine uptake.

Slope
4
1/[Alanine], 10 L/mol

Fig. 3-20. Dixon analysis of Na+-dependent alanine uptake with
phenylalanine as inhibitor
Alanine (25 /M, 50 /liM, and 100 juM) uptake was measured
with various concentrations of phenylalanine in uptake media. Non
linear regression of these data intercepted at x axis, indicating
a non-competitive inhibition profile.

[Phenylalanine].
3
ai
i
cr>
Gj
O
Oj
a>
IV)
Oi
86
4.8

Fig. 3-21. Replot of the slopes of Dixon plot with phenylalanine
as inhibitor
The slopes of fig. 3-20 were shown as a function of
1/[alanine]. Non-linear regression of these data intercepted at y
axis, these data combined with fig. 3-20 suggested that
phenylalanine was not a competitive inhibitor of Na+-dependent
alanine uptake.

Slope
1/[Alanine], 10 L/mol
100

Fig. 3-22. Na*-dependent alanine uptake inhibition pattern in both
day 3 and day 9 cells
Na+-dependent alanine uptake rates with various 5 mM amino
acid in uptake media were measured in both the day 3 and day 9
cells. The uptake in the day 3 cells was shown as a function of
the uptake in the day 9 cells. As shown in the figure, the degree
of alanine uptake inhibited by amino acids was similar in the day
3 and day 9 cells. Symbol keys: X = MeAIB, B = BCH, J = Cystine,
U = AIB, Z = control, A = Ala, C = Cysteine, F = Phe, G = Gly,
H = His, I = lie, L = Leu, M = Met, N = Asn, P = Pro, Q =
Gin, R = Arg, S = Ser, T = Thr, V = Val, W = Trp, Y = Tyr.

Alanine uptake, nmole mg hr (Day
CD
102

Fig. 3-23. Na*-dependent alanine and Na*-dependent glucose uptake at
various cell ages
Na+-dependent alanine (50 /M) uptake and Na+-dependent -
methyl-glucoside uptake (Blais et al., 1987) was shown as a
function of Caco-2 cell ages. The alanine uptake decreased, while
the glucose uptake increased with advancing cell age. 100% alanine
uptake = 0.5 nmole/mg/min; 100% -methyl-glucoside uptake = 0.12
nmole/mg/min.

100
80
60
40
20
0
i i i i i i i i r
a methyl-
20
104

Fig. 3-24. Apical to basal trans-cellular alanine uptake in
cells grown on porous filters
Alanine (50 /zM) uptake was measured in cells grown on
porous filters. Data shown were the amount of [3H]-alanine
transported across cell monolayer from the apical chamber to the
basal chamber, in both NaCl and choline Cl uptake media.

Alanine uptake, nmole mg protein
2.0
1 1 1
Apical to basal
NaCI
O Choline Cl
1
1
o
1
o

o

O

i
)
o

-

k 1 1 1
1
1
l_
1.5
1.0
0.5 -
0.0'
o
10 15 20
Time, minutes
25 30
106

Fig. 3-25. Alanine apical to basal or apical to cytosol uptake in
cells grown on filters
The total amount of 50 /jM alanine transported from apical
side to basal side, and apical side to cytosol in 30 minute was
measured.

Alanine uptake, nmole mg ( 30 min)
o nj oo o
801

Fig. 3-26. Alanine uptake across basal membrane in cells grown
on porous filters
The total alanine (50 /M) uptake from the basal compartment
to cytosol and apical compartments was measured in NaCl and choline
Cl media. The amount of basal to cell uptake = the amount of
alanine accumulated inside the cells (shown in Fig. 3-26) plus the
amount of alanine accumulated in the apical chamber (shown in Fig.
3-27) .

Time, minutes
O

Fig. 3-27. Alanine basal to cytosol uptake in cells
grown on porous filters
Alanine (50 n M) basal to cytosol uptake across
membrane was measured in NaCl and choline Cl media.
basal

en
E
_a>
o
E
c
D
-i1
Q_
Z5
C
c
_D
<
112

Fig. 2-28. Alanine basal to apical trans-cellular uptake
in cells grown on porous filters

2.0
en
E
_CL)
o
E
c
O
Q_
Z5
CD
C
c
o
<
Basal to apical
NaCL
O Choline CL
0.5
0
O
10
0.0 0
0
Time,
o
O
o
o
15 20 25 30
minutes
114

CHAPTER 4
CLASSIFICATION OF THE ARGININE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE
4.1 Introduction
The cationic amino acid arginine has attracted much
attention during the past years. Not only does arginine
possess many important physiological functions, such as blood
pressure regulation and urea synthesis, but also its transport
systems have unique characteristics. The cloning of system y+
cDNA marked a breakthrough in the amino acid transport study
(Kim et al., 1992). The membrane transport systems for
cationic amino acid arginine and lysine have been studied in
the past decades, and arginine transport systems has been
classified into three systems, a Na+-dependent System B0,+ and
two Na+-independent Systems, y+ and b0,+ (Oxender et al., 1963;
Van Winkle et al., 1985, 1987, 1988).
The Na+-dependent transport System B0,+ first described
in the blastocyte ( Van Winkle et al., 1985) transports both
the neutral amino acids and cationic amino acids. System B0,+
has not been reported for intestinal cells. The similar System
B transporter with characteristics of Na+-dependent neutral
amino acid uptake is exclusively found in intestinal apical
membrane, and is considered a variant of System B0,+ (Stevens,
115

116
1992a,b). The most predominant feature that distinguishes
System B from System B0,+ is that latter transport arginine and
other cationic amino acids.
The Na+-independent cationic amino acid transport System
b0,+ has the similar transport characteristics except the Na+-
dependency.
System y+, originally described in the Ehrlich cell,
(Christensen, 1964), reportedly exists in variety of cell
types including the intestinal epithelial cells (Christensen,
1975, 1990; Stevens, 1992a,b; Munck, 1981; Hopfer, 1987;
White, 1985; Segal et al., 1967; Kilberg et al., 1993). System
y+ Differs from other Na+-independent neutral amino acid
transport Systems L and asc, in that System y+ has a relative
narrow substrate scope selectively serving cationic amino
acids. System y+ activity is not sensitive to pH. Even though
System y+ is a Na+-independent facilitated transport system,
it can transport these cationic substrate against a
concentration gradient because of the positive charges
possessed by the cationic substrates and the negative
electrical PD across the plasma membrane. Unlike System b0,+ ,
neutral amino acids does not interact with System y+ activity
in Na-free medium. But in the presence of Na+ neutral amino
acids such as homoserine form a surrogate substrate that can
competitively inhibit the System y+ activity. In the
intestinal apical membrane, System y+ is the predominant
transport system for the cationic amino acids transport.

117
The cloning of system y+ cDNA has opened a new chapter
for membrane amino acid transport study. The discovered system
y+ cDNA also codes for the murine ecotropic retrovirus
receptor on cell plasma membrane. System y+ cDNA was
successfully expressed in oocytes. Injection of this cDNA into
oocyte results in a increase of typical system y+ activity
(Kim et al., 1991; Wang et al., 1991).
The cDNA encoding NAA/D2, rBAT and 4F2 peptides from rat
or rabbit kidney have been expressed in oocytes and apparently
increase activity of endogenous System b0,+ B0,+ activities.
These cDNA fragments have been suggested to encode possible
regulatory subunits of the system y+, b0,+ or B0,+ (Bertrn et
al., 1992; Magagnin et al., 1992; Tates et al., 1992; Wells
et al., 1992a,b).
To examine the arginine transport systems in the Caco-2
cells, we conducted a series of phenomenological studies to
define the arginine transport systems. At the time of this
study, we did not possess the system cDNA probes for y+, rBAt,
NAA/D2, or 4F2.
4.2 Methods and Materials
4.2.1 Methods
The [3H]-arginine uptake experiments were performed in
both the pre-confluent (day 2-3) cells and the confluent
(day 8 9) cells. The basic uptake procedures were as

118
described in the chapter 2 general methodology section. The
uptake experiments with special treatments will be mentioned
below where appropriate.
4.3 Results
4.3.1 Arginine Uptake Time Course
The uptakes of 5 /M and 1 mM [SH]-arginine were measured
in the Caco-2 monolayer (cell ages day 2 and day 9) at
increasing times (0 30 minutes) in uptake media containing
137 mM NaCl or 137 mM choline Cl. A representative time course
for 5 iM arginine uptake in day 2 cells is shown (Fig. 4-1) .
During the course of uptake, the [3H]-arginine accumulated
inside cells in NaCl medium was not different from that in the
choline Cl medium. This indicated that the arginine uptake was
mainly a Na+-independent phenomenon. The initial arginine
accumulation in the cells was linear during the initial 10
minutes (at both the [3H]-arginine concentrations of 5 /M and
1 mM) The initial arginine uptake rate that represent the
transport activity was obtained by dividing the total arginine
accumulation by the uptake time period (within the linear
accumulation limit). The uptake period of 0 5 minutes was
chosen for all the subsequent uptake measurements to ensure
the uptake rates represented the true initial arginine
transport.

119
4.3.2 Arginine Uptake Rates Decrease with Caco-2 Cell Age
The 5 juM [SH]-arginine uptake rates in choline medium
were measured at various Caco-2 cells age from cell ages of
1 day old to 14 days old. The arginine uptake in the choline
Cl uptake media decreased while the cell age increased (Fig.
4-2). The decline in arginine uptake rates was more rapid in
the pre-confluent cells than for the older cells. The decrease
in arginine uptake was paralleled the decrease in alanine
uptake with increasing cell age (Fig. 3-2). As we discussed
in Chapter 3, the cell proliferation rates also decreased with
advancing cell ages (Fig. 3-3). The arginine uptake change and
the alanine uptake change with cell ages may well be due to
the decrease of cell proliferation rates at older cells. As
discussed below, the passive diffusion coefficient for
arginine uptake was the same in day 2 and day 9 cells,
suggesting the decrease in arginine uptake with cell age was
due to a mechanism other than diffusion.
4.3.3 The Effect of pH on Arginine Uptake
The Na+-independent arginine uptake was measured in
choline Cl medium at pH 6.4 8.4 (HEPES/Tris buffers). The
arginine uptake in both the day 2 and day 9 cells was
unaffected by uptake buffer pH changes (Fig. 4-3).

120
4.3.4 Arginine Uptake Kinetics
Arginine uptake in the choline Cl media was measured at
various arginine concentrations ranging from [arginine] =0.1
/iM to [arginine] = 1 mM for both day 2 and day 9 cells. The
uptake kinetics are shown as the uptake rates plotted as a
function of arginine concentrations (Fig. 4-4). The kinetics
studies of the arginine (concentration range 1 /xM 1 mM)
transport in the choline Cl buffer indicated that there was
a non-saturable component and Na+-independent saturable
component. The Eadie-Hofstee transformation gave one non
saturable component as passive diffusion and a single Na+-
independent carrier system (Fig. 4-5).
The non-saturable component was simple passive diffusion.
The diffusion coefficient for both the day 2 and day 9 cells
was the same, P = 1.1 /iliter/mg protein/min.
For the single carrier-mediated system, the non-linear
regression analyses of the Eadie-Hofstee transformation of the
kinetics data gave a Vmax = 430 pmole/mg protein/min and =
31 /mole arginine for the day 2 cells, while at the day 9
cells the Vmax = 340 pmole/mg protein/min and = 37 /mole/mg
protein/min. These kinetic data suggested that the same
transport system was operative at the two cell ages, and the
activity difference was a Vmax effect reflecting a change in
the functional transporter units.

121
4.3.5 Amino Acid Analogue Cross-inhibition
The Na+-independent arginine uptake activities in choline
Cl media which contained single amino acid analogues (5 mM
each of the natural amino acids, ornithine, homoserine plus
sodium, or D-arginine) were measured at both the day 2 and
day 9 cells. The pattern and degree of the amino acid analog
inhibition for the Na+-independent arginine transport was
identical in both cell states, suggesting that same
transporter system was operative regardless of the cell age
(Fig. 4-7). The Na+-independent [3H]-arginine transport was
strongly inhibited only L-lysine, L-arginine, ornithine, and
histidine; weaker inhibitors were D-arginine, D-lysine,
homoserine (in Na+ buffer), tryptophan, and methionine; the
amino acids which inhibited less than 20 % arginine uptake
included alanine, BCH, phenylalanine threonine, serine,
asparagine, valine, homoserine (in choline Cl media) and
leucine (Fig. 4-6). The analogue cross-inhibition patterns
were consistent with that of system y+ (White, 1985) Dixon
analysis of [SH]-arginine uptake inhibited by ornithine,
homoserine, and D-arginine revealed that ornithine was a
classic competitive inhibitor, while the homoserine was a
weaker inhibitor, and D-arginine showed an uncompetitive weak
inhibition effects (Fig. 4-8 to 4-12).

122
4.3.6 Arginine Uptake in Caco-2 Cells on Porous Filters
Trans-epithelial arginine (5 xM) uptake was measured from
apical to basal sides in confluent Caco-2 cells (14 days old).
The intact confluent monolayers with trans-cellular resistance
> 300 ft cm2 were used. The arginine trans-cellular rate from
apical side to basal side was steady during a 30 minute period
(Fig. 4-13). Cellular accumulation of arginine during 30
minutes was 5 times greater than the apical to basal trans-
cellular movement (Fig. 4-14).
Arginine (5 xM) movement from basal side to cellular and
apical side was also measured in intact confluent Caco-2 cell
monolayers. The basal-apical side transport rate was steady
for up to 30 minutes (Fig. 4-15). During a 30 minute
incubation, the [3H]-arginine trapped in the cellular
compartment was 5 times greater than amount of arginine across
basal-apical compartment (Fig. 4-16).
4.4 Discussion
The arginine uptake activity across Caco-2 cell monolayer
was studied at various cell stages of development. By using
classic transport system criteria, we classified the arginine
transport systems in the Caco-2 in the undifferentiated state
(day 2) and differentiated state (day 9).

123
4.4.1 The Decrease in Arginine Uptake Activity as Cells Age
The Caco-2 total arginine uptake rates in choline Cl
medium decreased as the cells aged (Fig. 4-2). Furthermore,
the arginine passive permeability diffusion coefficient in
both the undifferentiated day 2 cells and differentiated day
9 cells was the same. The decrease in transport activity with
cell age was therefore due to the non-diffusion portion of
uptake. In the light of the reduced proliferation rates
characteristic of the older cells, the decrease in System y+
activity with the cell age increase may be associated with the
cell's reduced requirement for amino acids.
4.4.2 Classification of Arginine Transport Systems
The kinetic data (Fig. 4-4 & Fig. 4-5) indicated that
there was Na+-independent transport system plus simple passive
diffusion at both cell differentiation stages.
The simple passive diffusion coefficients were constant
at different cell ages, suggesting that the cell aging was not
associated with the diffusion changes.
The amino acid analogue inhibition pattern of the
carrier-mediated arginine uptake was strongly inhibited only
by lysine, ornithine, and histidine. Homoserine in choline Cl
medium has weak inhibition effects, but its inhibition effect
was enhanced by the presence of NaCl in the uptake media. The
neutral amino acids alanine, phenylalanine, leucine did not

124
inhibit the arginine uptake in the choline Cl media,
consistent with the cross inhibition pattern described for
System y+. The kinetics and pH insensitivity, combined with
the inhibition patterns together strongly indicated that the
Na+-independent carrier-mediated arginine transport system in
Caco-2 cells was System y+.
System b0,+ is another Na+-independent transporter of
arginine, and is the counterpart of the system B0,+ The weak
inhibition of arginine uptake by the neutral amino acids
alanine and leucine indicated the an unlikely major
involvement of system bD,+ in the current passages of our Caco-
2 cells (passages # 18 50) The non-inhibition effect of
alanine, BCH, Phenylalanine, and leucine excludes the possible
involvement of systems L, or asc.
Over the arginine concentration of 1 /M to 1 mM, the
arginine uptake in NaCl medium was not different from that in
the choline Cl. These data suggested that the arginine uptake
in the Caco-2 cells was mainly a diffusion plus Na+-
independent system y+ transport event; no Na+-dependent
transport phenomenon is involved.
The Na+-independent carrier-mediated system, affinity
characteristics, inhibition patterns, and pH insensitivity
were the same in both the undifferentiated day 2 and
differentiated day 9 cells. Only the Vmax value was higher in
day 2 cells compared to day 9 cells (Figs. 4-3 4-12). These
combined data indicated that arginine was transported through

125
the same System y+ in both the day 2 and day 9 cells. The
kinetic parameters (Fig. 4-5) strongly suggested that the
change in transport capacity during cell development was
likely due to the number of copies of functioning transport
units in the apical membrane (per cell mass) rather than
modification of characteristics of existing transporters.
The arginine uptake measurements in the Caco-2 monolayer
grown on the porous filters showed that the majority of
uptaked arginine accumulated inside the cells. Arginine exits
from the cytosol across either the basal membrane or apical
membrane to the extracellular media at a much slow rate than
the arginine transport across the membrane from outside to
inside rates.
4.5 Summary
Arginine is transported in Caco-2 cells by passive
diffusion and System y+. System y+ behaves with the same
kinetic characteristics operative in both the undifferentiated
and differentiated states. The System y+ capacity is down-
regulated during the cell development, while the diffusion
coefficient is not affected. The system y+ activity decrease
is coincident with the declining cell proliferation rate. The
decrease in System y+ activity is likely caused by the
decrease of number of copies of functional transporter units,
rather than the modification of existing transport affinity
for substrate.

Fig. 4-1. Arginine uptake time course
The uptake of arginine (5 /M and 1 xnM) was measured in
choline Cl and NaCl uptake media in the day 2 and day 9 cells.
The total arginine accumulation inside the cells was measured at
various time periods (0 30 minutes) At each point, the total
arginine uptake in choline Cl medium was > 90% of those in the
NaCl medium. The data shown was from the arginine (5 /liM) uptake
in day 2 cells, similar results were obtained in other cell ages
(day 9) and other arginine concentrations (1 mM) Na+-independent
pathway was the major uptake mechanism in Caco-2 cells.

cr>
E
_ o
E
c
CD
O
Q_
D
CP
L_
<
_J
127

Fig. 4-2. Arginine uptake at various cell ages
Arginine (5 /xM) uptake was measured in choline Cl uptake
medium over the cell ages of 2 14 days old. The uptake rates
decreased as cell age increased, with rapid decrease in the pre
confluent cells (< 6 days old) .

Cell age, day
Arginine uptake, pmole mg min
K> > cn co O
o o o o o o
621
120

Fig. 4-3. The effect of pH on arginine uptake
Arginine (5 /M) uptake in day 2 and day 9 cells was
measured in choline Cl medium, with various medium pH (pH 6.1,
7.4, or 8.4). The pH was adjusted by using 10 mM HEPES and 10
mM Tris buffer. The arginine uptake was not affected by the medium
pH.

150
day 9 cells
O day 2 cells
CD
E
_CD
O
E
Q_
a
o_
D
C
c
CD
<
120
60
30 L
6.0
6.6
o
o
7.2 7.8
PH
-#
8.4
131

Fig. 4-4. Arginine uptake kinetics
Arginine (0.1 /M 1 mM) uptake was measured in choline
Cl medium in day 2 and day 9 cells. The total uptake rates at
each arginine concentration were showed as a function of arginine
concentration. The shape of the kinetic curves indicated the
existence of both non-saturable and saturable components.

en
£
_cu
o
£
Q.
cn
1000
900
800
700
600
500
400
300
200
100
0
day 9
O day 2
O
0
o'
/
o
100
200
300
[Arginine], fiW\
400
5
500
133

Fig. 4-5. Eadie-Hofstee transformation of Na+-independent arginine
uptake kinetics in day 2 and day 9 cells
The arginine (0.1 /M 1 mM) uptake rates described in the
fig. 4-4 was showed as a function of Jarg/[arg]. The Na+-independent
portion was the difference of total arginine uptake in choline Cl
medium and the passive diffusion at the [arginine]. Non-linear
regression of the data gave straight lines, indicating a single
transport system in each of these day cells. Viax was 430
pmole/mg/ml in day 2 cells, and Vax = 340 pmole in day 9 cells.
The Km values were K = 31 nmole arginine for day 2 cells, and
Kb = 37 /mole arginine in day 9 cells.

600
500
400
300
200
100
0
day 2
O day 9
Jarg//[Arg]- . -1
min
135

Fig. 4-6. Arginine Na+-independent uptake inhibition pattern in day
3 cells
Arginine (5 /xM) uptake in choline Cl medium was measured
in day 3 cells, with 5 mM single amino acid present in the
uptake medium. Similar results were obtained in day 9 cells.

-1 -1
Arginine uptake, pmole mg min
o
o
ho
o
Ul
O
O
i i 1
Control
LArg
DArg
<>WOO^^
L-Lys
D Lys
Orn
Homoser
Homeser (Na)
His
OH
Ala
,0£KXXXXXXXXXX>CKXXXXXXXX) BCH
<> Pro
Phy
^XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX1
Asp
Thr
4WXX><^XXX^^
Val
<>ww<^^
Ser
Leu
^><><><><><^^
Glu
4^^^
Met
Try
1 1 1
LZI

Fig. 4-7. Arginine Na*-independent uptake inhibition pattern in day
3 and day 9 cells
Na+-independent arginine (5 /xM) uptake rates with 5 mM
amino acid in uptake media were measured in both the day 3 and
day 9 cells. The uptake rates in the day 3 cells were shown as
a function of the uptake rates in the day 9 cells. The degree of
arginine uptake inhibited by amino acids was similar in both cell
ages.

Arginine uptake (pmol/mg/min
in undifferenentiated cells
Competitive
Inhibitors
X MeAlB
Z control
J Homoserine
plus Na
R Arg
K Lys
0 Ornithine
A Ala
F Phe
H His
L Leu
M Met
P Pro
Arginine uptake (pmol/mg/min)
in differentiated cells
139

Fig 4-8. Dixon analysis of Na+-independent arginine uptake with
ornithine as inhibitor
Arginine (o.5 nM, 5 ijlM, and 50 ¡iM) uptake was measured
with various concentration of ornithine (1 /M 1 mM) in choline
uptake medium. Non-linear regression gave a Kx = 80 iM ornithine.

DIXON PLOT
Inhibitor = [Ornithine], mM
141

Fig. 4-9. Replot of the slopes of Dixon plot with ornithine
as inhibitor
The slopes of fig. 4-8 were shown as a function of
1[arginine]. Non-linear regression of these data was through 0 of
the axis, indicating that ornithine was a competitive inhibitor of
the arginine uptake.

Slope
1/[Arginine], 10 L/Mole
2.0

Fig. 4-10. Dixon analysis of Na*-independent arginine uptake with
homoserine as inhibitor
Arginine (0.5 juM, 5 /zM, and 50 /zM) uptake was measured
with various concentration of homoserine (1 juM -1 mM) in choline
Cl uptake medium. Non-linear regression of these data gave a Kt
of 570 /zM for homoserine.

0.5
0.4
Z
-0.5 0.0
[Homoserine],
-1.0
0.5
mM
§
f
1.0
[Arg] = 0.5/i.M
[Arg] = 5/u.M
145

Fig. 4-11. Dixon analysis of Na+-independent L-arcrinine uptake with
D-arginine as inhibitor
Arginine (0.5 /xM, 5 /xM, and 50 /xM) uptake was measured
with various concentrations of D-arginine in choline Cl uptake
medium. Non-linear regression of these arginine uptake indicated
that D-arginine was not inhibitor of arginine uptake.

*p*

Fig. 4-12. Arginine apical to basal trans-cellular uptake
in cells grown on porous filters
Arginine (5 /M) uptake from the apical chamber to the
basal chamber was measured in choline Cl medium 0.5 mM L-lysine
as inhibitor.

Arginine uptake, nmole
149

Fig. 4-13. Arginine apical to cytosol and apical to basal uptake
in cells grown on porous filters
The arginine (5 /xM) movement from the apical side to
cytosol and apical to basal in 30 minutes was measured.

-1
Arginine uptake, nmole mg
o ui cn
boo
1ST

Fig. 4-14. Arginine basal to apical trans-cellular uptake
Arginine (5 /liM) uptake from the basal side to the apical
side was measured at various time periods.

153

Fig. 4-15. Arginine basal to cytosol and basal to apical uptake
The arginine (5 /iM) uptake (30 minutes) at basal side in
choline Cl medium 0. 5 mM lysine was measured. Data shown were
the total arginine in the apical side and inside the cells at 30
minute uptake.

Arginine uptake, nmole mg
o
b
OJ
b
CD
b
Basal to Apical
Basal to Apical
(+ 0.5 mM Lysine)
Basal to
Cell Accu
mulation
Basal to Cell Accumulation
(+ 0.5 mM Lysine)
gsi
9.0

CHAPTER 5
THE EFFECT OF INDIVIDUAL AMINO ACIDS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
5^1 Introduction
Intestinal epithelial cells encounter various amino acids
concentrations in the lumen at various times. The luminal
concentrations of amino acids depend on the timing of food
intake and on food composition. In contrast to the cells of
many internal organs, the small intestine adaptively up-
regulates its amino acid transport capacity in response to the
increase of amino acids concentrations exposed (Stevens, 1991,
1992a,b; Salloum et al., 1990; Scharrer et al., 1981; Stein
et al., 1987; Ferraris et al. 1988a,b; Diamond & Karasov,
1987; Ferraris & Diamond, 1989).
The transport activity of system A in the hepatocytes and
MDCK cells increased upon prolonged amino acid starvation.
Addition of amino acids to the amino acid deficient medium
resulted in a loss of system A activity (Kilberg et al., 1985;
Boerner & Saier, 1985; Bracy et al., 1985). Substrate
regulation of the system A activity involves a short-term
cycloheximide-insensitive mechanism and a long-term
cycloheximide-sensitive mechanism (Boerner & Saier, 1985).
Whether the short-term inhibition is involved a trans-
156

157
inhibition mechanism or other regulatory mechanism is still
inconclusive. The long-term regulation involves a
transcriptional and translational regulatory mechanism. Two
regulatory mechanisms have proposed to explain the system A
substrate regulation (Kilberg, 1986; Englesberg et al, 1986).
Substrate regulation of amino acid transport systems of
intestinal epithelial cells ha been investigated in whole
animals fed various diets (Ferraris & Diamond, 1989; Ferraris
et al., 1988a,b). Animal feeds with high protein or high amino
acid diet increased their transport of non-essential amino
acids, such as aspartate. Essential amino acid transport
activity decreased in animals on high amino acid diets. In
general, the transporter substrates are often the best
inducers of uptake capacity, but sometimes the best inducers
are un-related to the transport systems (Stein et al., 1987;
Diamond & Karasov, 1987; Scharrer et al., 1981). The up-
regulation of the intestinal amino acids uptake occurs over
0.5 1 day, whereas the reversal of the up-regulated level
to the original level takes several days.
The mechanism of intestinal substrate induction of
transport has not been addressed at cellular level. In this
study, we explored the effect of individual amino acids to
increase the activity of System B and System y+ in both short
term and long-term exposures.

158
5.2 Methods and Materials
Caco-2 cells were incubated in depletion medium for 2
hours, and then incubated in the same medium containing
various concentration of alanine, arginine, or other amino
acids cycloheximide (10 iM or 50 /xM) for various windows of
time (30 seconds up to 48 hours) in the 37"C incubator. The
incubation medium was changed every 8 hours to ensure that
amino acid concentration were constant, and to remove possible
build up of autocrine.
The depletion medium contained 0.265 g/L CaCl2, 0.0001
g/L Ferric Nitrate, 0.09767 g/L MgS04, 0.4 g/1 NaHCOs, 6.4 g/L
NaCl, 0.109 g/L NaH2P04, 4.5 g/L glucose, 0.0159 g/L phenol
Red-Na, 0.004 g/L choline chloride, 0.004 g/L folic acid,
0.0072 g/L myo-inositol, 0.004 g/L niacinamide, 0.004 g/L D-
pantothenic acid, 0.004 g/L pyridoxaHC1, 0.0004 g/L
riboflavin, and 0.004 g/L thiamineHC1.
5.3 Results
5.3.1 System B Activity Decrease in Starved Caco-2 Cells
The system B alanine uptake rate declined as early as 15
minutes following depletion medium incubation, and reached the
lowest level at 2 hours, where they remained steady for at
least 48 hours. The system B alanine uptake rate in the Caco-
2 cells which were incubated in depletion medium plus 1 mM

159
alanine also decreased as incubation progress, however, the
decrease was less than that in cells incubated in depletion
medium alone. The 50 /M alanine System B uptake rate in the
alanine-incubated cells was 50% higher than that in the
depletion-incubated cells (Fig. 5-1).
5.3.2 System B Activity was Activated by Acute Amino Acid
Exposure
The Caco-2 cells were incubated in the depletion medium
containing 1 mM individual amino acids for 3 hours. Mannitol
(1 mM) was the control. Cysteine solutions contained 1 mM
dithiothreitol, with the 1 mM dithiothreitol solution serving
as control. System B activities were measured immediately
after each incubation. In comparison to the cells incubated
in the control depletion medium alone, the increase in System
B activity in cells incubated in solution containing amino
acids gave a pattern of stimulation that matched the ranking
of System B substrates (described in the chapter 3) That was,
alanine, serine, glutamine, threonine and cysteine each
increased the system B activity by 1.5 2 fold; the weaker
stimuli (which weakly inhibited system B activity in the
cross-inhibition study (Fig. 3-12)) were amino acids such as
histidine, glycine, and valine. Finally phenylalanine,
leucine, lysine, arginine, and MeAIB, which did not inhibit
system B activity in cross-inhibition study, did not
stimulated the system B activity (Fig. 5-2).

160
In a separate study, 1 mM alanine was added to Caco-2
cells which had been incubated in the depletion medium for 3
hours. Within 5 minutes the system B activity was increased
compared to the control level. After 2 hours in depletion
incubation, cells were incubated in the alanine metabolism
inhibitor aminooxy acetic acid (AOA, 2.5 mM) The system B
alanine uptake rate increased only of 7 hours AOA incubation
(Fig. 5-3).
MeAIB uptake was measured in Caco-2 cells which had been
incubated in depletion medium, 1 mM alanine, 1 mM serine or
1 mM MeAIB for 3 hours. The MeAIB (2.5xl08 M) uptake was not
affected by the incubation with added 1 mM alanine or serine.
MeAIB incubation decreased the MeAIB uptake (Fig. 5-6) .
The Na+-independent system L alanine uptake was also
measured in above experiment conditions. The system L alanine
uptake was increased by alanine, serine, glycine, and was not
affected by MeAIB and proline.
5.3.3 Short-Term Activation of System B Activity did not
Involve Protein-Svnthesis
The Caco-2 cells were washed three times with the
depletion medium, and incubated with the same medium
containing 1 mM alanine 50 /zM cycloheximide for 3 hours.
The 50 /M alanine system B activity was measured. The system
B alanine uptake was increased 2 folds by the alanine
incubation alone. The cycloheximide alone in the incubation

161
medium did not affect the alanine uptake, nor did the CHX in
the alanine incubation medium affect the increase of alanine
uptake (Fig. 5-4).
5.3.4 The System B Activity Increase bv Substrate Acute
Exposure was Reversible
The Caco-2 cell were washed and incubated in the
depletion medium containing 1 mM alanine (mannitol as control)
for 3 hours as described above. The cells were then washed
three times with the depletion medium, and incubated in the
depletion medium (lacking amino acids) for 3 hours. The 50 /xM
alanine system B activity was increased 2 fold after
incubation for 3 hours with alanine, and returned to the
control level after the additional 3 hours in alanine-free
depletion medium incubation.
5.3.5 The System B Activity Increased bv its Substrate
Exposure Involved Kinetic Modifications
The Caco-2 cells were washed three time with the
depletion medium, and incubated in the same medium 1 mM
alanine or DMEM for 3 hours. The kinetics of the system B
transport activity ([SH]-alanine concentration = 1 /xM 5 mM)
showed that the alanine incubation resulted in a 2 fold Vmax
increase and plus an increase of K,^ (Fig. 5-5) The DMEM
incubation also resulted in a increase of Vmax and I^. These
data indicated that the system B activity increase is likely
involved in the modification of the transport system affinity

162
to bind alanine and/or Na+
5.3.6 System B Activity Increased by Chronic Alanine Exposure
was Dependent on Protein Synthesis and PKC activation
The Caco-2 cells were washed with the depletion medium,
and incubated in the same medium containing 0, 0.1, 1, or 10
mM alanine, 10 /M cycloheximide or 6.6 /M chelerythrine Cl
for 24 hours. The medium was changed every 6 hours. The 50 /liM
alanine system B activity was then measured. The system B
activity was increased by various alanine exposures, with
greater alanine concentrations causing a greater stimulation
effect. The alanine stimulation was partially blocked by
cycloheximide or chelerythrine (Fig. 5-7 & Fig. 5-8).
Caco-2 cells were then pre-incubated with alanine for 24
hours, as described above, and the cells were then incubated
in the depletion medium for 3 hours before System B activity
measurement. System B activity was increased by exposure to
alanine, and this increase was then completely blocked by
cycloheximide or chelerythrine.
5.3.7 System y+ Activity Decreased in Starved Caco-2 Cells
The Caco-2 cells were washed three times with the
depletion medium, and then incubated in the same medium 1
mM L-arginine or D-arginine for various length of time (30
seconds to 48 hours) in the 37C incubator. One millimole
mannitol was used as control. The medium was changed every 6

163
hours to ensure that the amino acid concentration was constant
and the possible autocrine accumulation was eliminated. The
system y+ transport activities were measured immediately after
each incubation period. The 5 /nM arginine system y+ uptake
decreased as the incubation time increased, and reached the
lowest level at about 3 hours where they stayed 48 hours. The
declining system y+ activity was partially prevented by
exposure to L-arginine or D-arginine (at lesser degree) (Fig.
5-9) The 3 hours depletion incubation was chosen for the
subsequent experiments.
5.3.8 System v* Activity was Stimulated bv Acute Amino Acid
Exposure
The Caco-2 cells were washed three time with the
depletion medium, and incubated in the same medium containing
1 mM individual amino acids (1 mM mannitol as control, all
cysteine solution also contained 1 mM dithiothreitol in the
case 1 mM dithiothreitol was used as control) for 3 hours.
The System y+ activity was measured immediately after each
incubation. The System y+ activity was increased two-fold by
the system y+ substrates lysine, arginine, ornithine. System
y+ non-substrates proline, BCH, and alanine did not affect
the System y+ activity A pattern emerged such that these
amino acids which weakly inhibited System y+ activity also
weakly stimulated System y+ activity (Fig. 5-10). These data
suggested that the system y+ activity was specifically

164
stimulated by its own substrates.
In another study, Caco-2 cells were incubated in the
depletion medium for 3 hours, and 1 mM arginine was added to
the medium. System y+ arginine uptake increased as early as 5
minutes arginine incubation.
5.3.9 System v+ Activity Increased by Acute Arginine Exposure
did not Involve Protein Synthesis
The Caco-2 cells were washed and incubated in the
depletion medium containing 1 mM arginine (1 mM mannitol as
control) 50 nK. cycloheximide or 0.5 /ig/m 1 actinomycin D for
3 hours. The system y+ activity increased by the arginine
exposure was not blocked by the cycloheximide or actinomycin
D in the incubation medium (Fig. 5-11).
5.3.10 System v+ Activity Increased bv Acute Arginine Exposure
was Reversible
Caco-2 cells (2 days and 9 days old)were washed and
incubated in the depletion medium 1 mM arginine for 3 hours.
The cells which had been incubated with arginine were then
washed three time with the depletion medium and incubated in
the depletion medium 1 mM arginine for another 3 hours.
System y+ activity was increased following a 3 hours arginine
incubation, and the increased was diminished after the cells
were then incubated in depletion medium (lacking arginine)
for 3 hours.

165
5.3.11 System v* Activity Increased by Arginine Exposure was
a Kinetic Modification Effect
Caco-2 cells were washed three times with the depletion
medium, and incubated in the same medium containing 1 mM
arginine (1 mM mannitol as control) for 3 hours. The system
y+ uptake kinetics were measured over the [SH]-arginine
concentration ranging from 0.1 mM to 1 mM. The kinetics showed
that both the Vmax and K,,, of the system y+ activity was
increased by the arginine pre-incubation (Fig. 5-13 & Fig. 4-
14) .
5.3.12 System v* Activity Increased by Arginine Chronic
Exposure was not a Protein Synthesis-Dependent Process
The Caco-2 cells were washed with the depletion medium,
and incubated in the same medium containing 0, 0.1, 1.0, or
10 mM arginine 10 /M cycloheximide or 6.6 /xM chelerythrine
for 24 hours. The medium was changed every 6 hours. The 5 iM
arginine system y+ activity was increased 7 fold by the
arginine exposure. The degree of arginine uptake increased by
the 0.1, 1.0 or 10 mM arginine incubation was the same.
Cycloheximide or chelerythrine in the incubation medium did
not block the system y+ activity which was increased by the
arginine incubation (Fig. 5-12).
In another study, Caco-2 cells pre-incubated with 1 mM
arginine for 24 hours were then incubated in depletion medium
lacking arginine for 3 hours. System y+ activity increased by

166
the 24 arginine incubation returned to control level after the
3 hours starvation.
5.4 Discussion
system B and system y+ activities were up-regulated by
their own substrates, in contrast to substrate repression of
transport activities found in other internal organs. System
B and System y+ were regulated independently.
5.4.1 Short-Term System B Activity Regulation bv its
Substrates
System B activity was up-regulated when cells were
incubated with individual substrates. As shown in the Fig. 5-
2, only the alanine, cystine, serine, threonine, and glutamine
(that are transported by system B) induced the system B
transport activity. Those non-system B substrates such as
MeAIB, proline, lysine, arginine, and phenylalanine did not
effect on System B activity. These data strongly suggest that
the increase in System B activity following short-term amino
acid exposure was a specific regulation. As will be discussed
later in the following sections, System y+ activity was up-
regulated only by its own substrates. The fact that the System
B substrates alanine, serine, cysteine, threonine did not
inhibit nor induce System y+ transport activity, and that,
the System y+ substrates lysine and arginine did not interfere
System B transport nor induce the System B activity, indicated

167
that the System B and System y+ activities were specifically
and independently regulated only by their own substrates.
The fact that the System B activity could be induced by
alanine within minutes and the increase was reversible,
suggests that the increase in System B activity after the
short-term substrate incubation could be due to: (1) a tran-
stimulation of the transport systems, or/and (2) a trans
location of the transport units from cytosol to membrane. The
insensitivity of cycloheximide or actinomycin D eliminated the
possible involvement of de novo protein synthesis or new RNA
synthesis mechanisms. Kinetic are valuable in identifying the
trans-stimulation and the trans-location mechanisms. For a
pure trans-location of transporter units, the only kinetic
parameter that would change is Vmax without alteration. For
the trans-stimulation mechanism, both the Vmax and the would
be changed, and indeed in our kinetic studies, both the Vmax
and were changed, favoring the notion that the acute
activity increase was due to a trans-stimulation. We can not
determine whether both the trans-stimulation and trans
stimulation were involved. In the future, by using the
membrane vesicles pre-loaded with various concentrations of
alanine, we will be able to determine if a trans-stimulation
is involved. The only way to determine a role for tran-
location is to probe transporter protein level using
antibodies.
The System L activity increased by its own substrate

168
during short-term exposure, and this also likely involves a
trans-stimulation phenomenon.
5.4.2 System B Activity Induced bv Chronic Alanine Exposure
The cycloheximide- and chelerythrine-sensitive System B
activity increased with the chronic alanine incubation. This
differed from the short-term regulation, and suggested that
de novo protein synthesis was involved, and that protein
kinase C activation was involved. Because of the
unavailability of specific probes such as antibodies or cDNA's
at the time of this writing, it is not possible to determine
whether the newly synthesized protein was the transporter
protein, transporter regulatory protein, or some other
regulatory protein. We predicted that the chronic activation
effect would be a Vmax effect without the modification of
or Hill number. In other words, the chronic alanine incubation
stimulated the synthesis of System B transport-associated
protein, resulting in a increase in functional transporter
units. Alanine is a caloric amino acid and a carbon chain
precursor for many metabolic intermediates, in addition to
being non-toxic. The specific transporter activity stimulated
by alanine was consistent with the observed in vivo up-
regulation of intestinal epithelial amino acid transport
(Diamond, 1991). The trans-stimulation provides an immediate
safety margin at any given moment so that the cells can
extract the maximum amount of nutrient from limited

169
availability. The specific up-regulation of transporter units
could be an evolutionary adaptation which permitted animals
to effectively adapt to their changing environment
surroundings, provided that no toxic effects would occur.
5.4.3 System v* Activity Induced bv its Own Substrate
System y+ activity was induced after the cells were
exposed to only the system y+ substrates (Fig. 5-10). The
inductive potency of these amino acids was directly related
to the potency of these amino acids in inhibiting the system
y+ transport by analogue cross-inhibition. In other words,
the amino acids which were transported by system y+ induced
the system y+ activity (Figs. 4-6,7; Fig. 5-10). As we
discussed in above, only the System y+ substrate induced the
System y+ activity, and only System B substrates induced
System B activity; there was no interference between the two
systems. System B and System y+ are specifically and
independently regulated.
For the acute phase of System y+ stimulation, the
activity was not sensitive to cycloheximide. This rules out
a mechanism of new transport protein synthesis. There are two
acute activation mechanisms, trans-stimulation and trans
location. Our kinetic study unveiled changes in both the Vmax
and K,,, for the substrate-induced System y+, supporting the
trans-stimulation theory. Future studies of acute stimulation
should investigate phosphorylation event and measurement using

170
isolated membrane vesicles. Unlike System B, the System y+
activity increased by chronic arginine exposure was not
sensitive to cycloheximide or chelerythrine. The chronic
arginine incubation did not induced a new transporter
associated protein synthesis, and furthermore protein kinase
C was not involved. This is consistent with the findings in
other in vivo intestinal studies that showed that the
intestinal essential lysine/arginine transport was not up-
regulated in a long-term feeding related to the potential
toxic effect of these essential amino acids. The transport
capacity is determined by the factors such as the genetic
hard-wiring or by the cell's needs other than the
environmental availability. The trans-stimulation of the
transporters provides the flexibility for maximum nutrient
extraction at any given time within the safety margin.
5.5 Summary
The System B and System y+ activities are up-regulated
independently and specifically by only the substrates they
transport. The system B is regulated in two phases: an acute
trans-stimulation phase, and a chronic de novo protein
synthesis- and protein kinase C-dependent phase. The System
y+ activity is only regulated by a substrate trans
stimulation. The difference between the System B and System
y+ substrate regulation may reflect the intrinsic properties
of the transported substrates.

Fig. 5-1. System B activity in cells incubated in depletion medium
with or without alanine
System B alanine (50 nK) uptake was measured in Caco-2
cells which had been incubated in depletion salt medium ( 1 mM
alanine) for various period of time (0 6 hours). At incubation
period > 1 hour, the System B alanine uptake was significantly
greater in cells incubated in salt plus 1 mM alanine than that
incubated in salt only medium (p < 0.05, n = 6) Data shown were
obtained in day 2 cells, with similar results obtained in day 9
cells.

Incubation time, minutes
172

Fig. 5-2. The acute effect of amino acids on System B activity
System B alanine (50 uM) uptake was measured in cells
which had been incubated in DMEM salt medium, 1 mM amino acid
for 3 hour. Amino acids alanine, serine, glutamine, cysteine, and
threonine transported by System B induced the System B alanine
uptake. Non-System B substrates such as phenylalanine, proline,
arginine, and MeAIB did not induced the System B activity. 100%
alanine uptake = 0.28 nmole/mg/min.

o
o
% of Alanine Transport
(Na+ -
- dependent)
N>
O
O
400
300
h
&
Mannitol
Phe
AOA
Pro
MeAlB
Arg
His
Gly
Val
Thr
Cys+DTT
Ala
GLN
Ser
DMEM
Ala+AOA
VLX
500

Fig. 5-3. The effect of AOA on System B activity
System B alanine (50 /M) uptake was measured in cells
which had been incubated in salt medium, 1 mM alanine, 2.5 mM
(aminooxy) acetic acid (AOA), or 1 mM alanine + 2.5 mM AOA for
various periods of time (2 7 hours). Alanine alone and alanine
plus AOA incubation stimulated the System B activity at each
incubation period (p < 0.05, n = 6). AOA alone did not affect
the System B activity after 2 and 4 hours incubation, but showed
its stimulation effect after 7 hours incubation (p < 0.05, n =
6) .

Alanine
Incubation
T
T
ALA+AOA
control
4 5 6 7
time, hour

Fig. 5-4. The Effect of CHX on the acute alanine-stimulated System
B activity
System B alanine (50 /M) uptake was measured in cells
which had been incubated in salt medium (with or without 1 mM
alanine) 50 /M cycloheximide in the incubation medium for 3
hours. The CHX incubation did not block the alanine induced System
B alanine uptake (p < 0.05. n = 9) .

en
o
Alanine uptake, nmole mg min
- M gj <_n
o o o o o o
Contri
CHX
Ala
Ala
+CHX

Fig. 5-5. Kinetics of the acute alanine-stimulated System B
activity
System alanine (10 /M 5 mM) uptake was measured in cells
(day 2) which had been incubated in salt medium, DMEM, 5 mM
alanine in salt medium. In the salt incubation, VBax = 0.67
nmole/mg/min and K, = 150 /mole alanine; in the DMEM and alanine
incubation, VmiX = 2.9 nmole/mg/min and K,, = 390 /mole alanine.

3.0
en
E
Q)
O
E
o
o
D
2.5
2.0
1.5
1.0
0.5
Jala/[alQn¡ne]
1 1
DMEM
O 5 mM ala
DMEM salt
-1 -1
/Liter mg min
180

Fig. 5-6. The acute effect of amino acids on MeAIB uptake
Na+-dependent MeAIB (2.5 x 10'8 M) uptake was measured in
cells (2 days old) after cells had been incubated in salt medium,
or salt medium plus 1 mM MeAIB, 1 mM alanine, or 1 mM serine for
3 hours. The MeAIB incubation inhibited the MeAIB uptake (p <
0.05, n = 3), while alanine or serine incubation had not effect
on the MeAIB uptake (p > 0.05, n = 3).

-14
-1
MeAlB uptake, 10 mole mg min
O O 1 N)
b ai en
Control
MeAlB
Alanine
Serine
j L
281

Fig. 5-7. The effect of chelervthrine on the chronic alanine-
induced System B activity
System B alanine (50 /M) uptake was measured in cells (2
days old) which had been incubated in salt medium, salt medium
plus various concentrations of alanine (0.1, 1.0, or 10 mM) with
or without 6.6 /lM chelerythrine in the incubation medium. The
alanine incubation stimulated the System B alanine uptake (p <
0.05, n = 6) the stimulation was partially blocked by CHE (p <
0.05, n = 6) Similar results were obtained in 9 days old cells.

Alanine uptake, nmole mg min
o o ^ ro
b bi b bi
*8T

Fig. 5-8. The effect of cvcloheximide on the chronic alanine-
induced System B activity
System B alanine (50 juM) uptake was measured in cells (2
days old) which had been incubated in salt medium (with or without
10 mM alanine) 10 juM CHX for 24 hours. The System B alanine
uptake was stimulated by alanine incubation (p < 0.05, n = 6),
and this stimulation was blocked by CHX (p < 0.05, n = 6).
Similar results were obtained in 9 days old cells.

Alanine uptake, nmole mg min
o ro io go
en cn b cn b
981

Fig. 5-9. System v* activity in cells incubated L-arginine or
D-arginine
System y+ arginine (5 /liM) uptake was measured in cells (2
days old) which had been incubated in salt medium, 1 mM arginine
in salt, or 1 mM D-arginine in salt for various periods of time
(1 12 hours). The System y+ arginine uptake was greatly
stimulated by arginine incubation, D-arginine marginally increased
the arginine uptake. Similar result were also obtained in 9 day
s old cells.

160
140
120
100
80
60
40
20
0
T
II I I I I L_
1 2 3 4 5 6 7 8 9 10 1 1 12
Incubation time, hours
13
188

Fig. 5-10. The acute effect of amino acids on System v+ activity
System y+ arginine (5 ^M) uptake was measured in cells (2
days old) which had been incubated in salt medium, or salt medium
plus 1 mM of various amino acids for 3 hours. The System y+
arginine uptake was stimulated by 180% by system y+ substrates
lysine, ornithine, or arginine. Non-system y+ substrates alanine,
serine etc did not stimulated the arginine uptake. Similar results
were also obtained in 9 days old cells.

% Stimulation of Arginine Uptake
-4^ M hO
o o o o
>

*
1.
to
K>
to
-p
CD
00
o
NO
-P-
CD
00
o
ro
o
O
O
o
O
o
o
o
o
o
o
iiii
Cysteine
Proline
Phenylalanine
BCH
Aspartic acid
Control (mannitol)
^ Glycine
Methionine
Tyrosine
Valine
^ Threonine
^ Serine
Alanine
<2h Cystine
hE
Asparagine
D-Arginine
Leucine
Glutamine
Homoserine
Histidine
0^-l Medium + Amino acid mixture
C^KKKK^CKK^OOOOOOOOOOfl Arginine
yTXX)>CkXKK>CK>0 Lysine
J I
061

Fig 5-11. The effect of CHX on the acute arginine-stimulated
System v* activity
System y+ arginine (5 /xM) uptake was measured after cells
(2 days old) had been incubated in salt, 1 mM arginine, 50 /M
CHX, or 1 mM arginine plus 50 /M CHX for 3 hours. The arginine
uptake was stimulated by the arginine incubation (p < 0.05, n =
6), CHX had no effect on the induction (p 0.05, n = 6).
Similar results were observed in 9 day old cells.

200

Fig. 5-12. The effect of CHX and chelervthrine on the chronic
arginine-induced system v+ activity
System y+ arginine (5 /M) uptake was measured after cells
(2 days old) had been incubated in salt medium, salt medium plus
various concentrations of arginine (1, or 10 mM) 10 juM CHX or
6.6 /liM CHE for 24 hours. The arginine uptake was increased by
arginine incubation, CHX or CHE did not block this arginine
induction.

Control
7
c
400
Je
350
mg
300
CD
O
E
250
Cl
200
O
-t11
Q.
150
13
CD
C
100
"c
'en
50
<
0
[Arg]
E £
O
ww 01
+ CHX
T
+ CHE
o
194

Fig. 5-13. Kinetics of acute arginine-stimulated System v* activity
in salt medium
System y+ arginine (0.1 /M 1 mM) uptake kinetics were
measured after cells (2 days old) had been incubated in salt
medium or 1 mM arginine in this medium for 3 hours. For the
cells incubated with salt only, the VBax = 0.25 nmole/mg/min and
Km = 31 mole arginine? for the arginine incubation group, VBax =
2.75 nmole/mg/min and Km = 81 /mole arginine.

nmole mg min
0 5 10 15 20 25 30 35
JArg/[Ar9]> ^Liter m9 min
196

Fig. 5-14. Kinetics of acute arginine- or DMEM-stimulated System
v* activity
System y+ arginine (0.1 /uM 1 mM) uptake kinetics were
measured after cells had been incubated in DMEM or DMEM plus 1
mM arginine for 3 hours. For the DMEM incubation, VBax = 1.05
nmole/mg/min and K = 39 /nmole arginine; for the arginine/DMEM
incubation, VBax = 22.7 nmole/mg/min and KB = 79 /nmole arginine.

/[Arg], /Liter
JArg, nmole mg min
C_
>
"1
02)
3
lQ
3
Z)
O O N) N)
en en cn
861
3.0

CHAPTER 6
THE EFFECTS OF PEPTIDE GROWTH FACTORS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
6.1 Introduction
Epidermal growth factor (EGF), is a member of the growth
factor family, which has been intensively studied over the
past 30 years (Hernandez-Sotomayor & Carpenter, 1992). The
protein structure, gene expression, biological function, and
the molecular regulation of EGF and the EGF receptor are well
understood. Transforming growth factor-alpha (TGF) is
structurally similar to EGF (Montelione et al., 1988, 1989;
Caver et al., 1986; Mayo et al., 1989). The structure of EGF
and TGF<* is reported to be related to their functions
(Capenter & Wahl, 1990).
EGF/TGF command their function through a binding to the
EGF membrane receptor. The EGF receptor is a glycoprotein
composed of three major domains: an extracellular hormone
binding domain, a hydrophobic transmembrane region, and a
cytoplasmic domain. TGF also binds to the same EGF receptor.
EGF receptor belongs to the tyrosine kinase family. The
EGF/TGF binding to the EGF receptor induces a rapid
reversible changes in the receptor tyrosine kinase activity
causing an auto-phosphorylation of the EGF receptor and the
199

200
phosphorylation of other receptor's substrates. The tyrosine
kinase activity is essential for the EGF receptor biological
activity (Chen et al., 1987; Glenney et al., 1988; Honnerger
et al., 1987; Moolenaar et al., 1988)). The EGF receptor
substrates include; PLC-yl (Margolis et al., 1989;
Meisenhelder et al., 1989; Wahl et al., 1989), GAP (Ellis et
al., 1990; Molloy et al., 1990), lipocortin I (Fava & Cohen,
1984), c-erbB-2 (Akiyama et al., 1988; Stern & Kemps, 1988),
and PI-3 kinase (Whittman et al., 1988).
One action of the activated EGF receptor kinase is to
phosphorylate the phospholipase C (PLC) which hydrolyzes the
phosphatidylinositol-4,5-biphosphate to produce inositol-
1, 4 5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 and
DAG stimulate the increases in the intracellular free Ca2+ and
protein kinase C activity, respectively. Protein kinase C then
causes various biological responses, mainly gene-expressed
related mechanisms through a series unknown pathways.
The EGF biological responses are of two types. The first
concerns a rapid signal transduction via the receptor tyrosine
auto-phosphorylation, acting in concert with the calcium
released from the intracellular stores, and leading to the
phospholinositide hydrolysis, with subsequent formation of
diacylglycerol (Carpenter et al., 1979; Moolenaar et al.,
1984; Morris et al., 1984; Johnson et al., 1986; Wahl &
Carpenter, 1988a; Smith et al., 1983; Serreo, 1987). The
second mechanism involves long term biological responses such

201
as stimulation of cell proliferation, inhibition of
differentiation, stimulation of oocyte maturation, stimulation
of vasoconstriction. (Raymond et al., 1986; Centrlla, 1987;
Reilly et al., 1987; Kim et al., 1987; Downs et al. 1988;
Berk et al., 1985; Muramatsu et al., 1985, 1986). Depending on
the cell type and the physiological circumstances, EGF exerts
many biological functions (Carpenter & Wahl, 1990). One of the
most dramatic biological effects of EGF is the regulation of
cell growth and differentiation, especially in epithelial
cells and tissues (Carpenter & Wahl, 1990).
The EGF receptors were reportedly found at developing
intestinal epithelial cells and the human Caco-2 cell line. In
the Caco-2 cell line there is a greater density of receptors
in less differentiating cells. Two-thirds of the EGF receptors
appear on the basolateral membrane, while the one-third on the
apical membrane. The K, of the EGF receptor in the Caco-2 cell
is 0.6 juM EGF (Hidalgo et al., 1989; Koyama & Podolsky, 1989).
EGF is normally present in the intestinal mucosa and in
the lumen. The major sources are from the submaxillary gland
secretion, from the Brunner's glands of the duodenum, from the
jejunal/ileal mucosa, and from exogenous sources such as milk
(which contains 40 -400 ng/ml EGF). EGF and TGF are extremely
stable in the presence of the gastric acid and the intestinal
digestive enzymes (Britton et al., 1989; Potter, 1990). The
secretary sources for the EGF in blood stream are still
unknown.

202
Insulin, glucagon, adenosine induce System A transport
activity via a transcription and translation mechanism in
hepatocyte (La Cam & Freychet, 1978; Cariappa & Kilberg, 1990;
Kiyokawa et al., 1991).
Despite the understanding of many EGF biological
activities, the effect of EGF on the intestinal absorption of
amino acids has not been explored.
6.2 Methods And Materials
6.2.1 Methods
6.2.1.1 Treatments with EGF/TGF<*. cvcloheximide,
chelervthrine, and H-7/calphostin C
The protocols to treat Caco-2 cells were basically the
same for each of the various agents. Serum-free DMEM was
prepared by supplementing Sigma's DMEM with non-essential
amino acids, penicillin and streptomycin, but not serum.
Treatments began with a 2 hour preincubation of cells in the
depletion medium. The cells were then exposed to various
buffers for various times described below. Every 6 hours,
buffers were removed and replenished with the same buffers to
ensure a constant agent concentration and to eliminate a
possible autocrines released into the medium from the cells.
Caco-2 cells remained healthy during at least 48 hours
exposure to our depletion medium.
Experiments with EGF/TGF. The cell treatments were: (i)
control group, serum-free DMEM plus the same amount of 0.1 M

203
acetic acid vesicle as used in the EGF/TGF treatment (< 0.5%
of the medium volume) (ii) treatment with EGF/TGF, DMEM plus
various concentration of (EGF/TGF is diluted from stocks in
0.1 M acetic acid stored at 4C), and (iii) treatment with
EGF/TGF with additional agents, DMEM plus EGF/TGF plus agent
specified in the text, with each specified agent in DMEM as
control. Cell were exposed to the treatments up to 48 hours.
Cvcloheximide (CHX) treatments. The cells were treated
with: (i) control group, serum-free medium, (2) treatment with
CHX, DMEM plus 10 100 /M CHX (CHX was prepared in aqueous
solutions made the day of the experiment), (iii) treatment
with CHX and other agents, DMEM plus 10 100 /xM CHX with
specified agent added in to the medium, internal control used
DMEM plus specified agent. The treatments were for various
lengths of time.
Chelerythrine Cl experiments. The cells were treated
with: (i) control group, only serum-free DMEM, (ii) treatment
with chelerythrine, serum-free DMEM plus chelerythrine
(chelerythrine Cl was diluted from stocks in H20 stored at -
20C), (iii) treatment with chelerythrine Cl and specified
agents, DMEM plus chelerythrine plus specified agent, with
DMEM plus specified agent as internal control.
Calphostin C treatments. Cells were treated with: (i)
control group serum-free DMEM plus the same amount of DMSO as
appeared in the calphostin C solution (<1% of the medium
volume), (ii) treatment with calphostin C, serum-free DMEM

204
plus various concentrations of calphostin C (calphostin C was
diluted from stock DMSO solution stored at -20C), (i i i)
treatment with calphostin C and other agent, serum-free DMEM
plus calphostin C plus specified agent, with DMEM plus
specified agent as internal control. These groups were
continuously exposed to a 20 watt fluorescent light in the
37C humidified incubator to photo-activate the calphostin C.
H-7 treatments. The cells were treated with: (i) control
group, serum-free medium plus the same amount of DMSO as
appeared in H-7 (<1% of the medium volume) (ii) treatment
with H-7, serum-free DMEM plus H-7 (H-7 was diluted from a
DMSO stock solution stored at -20C), (iii) treatment with H-7
and other agents, serum-free DMEM plus H-7 plus the specified
agent, with DMEM plus the specified agent as internal control.
6.2.2 Materials
TGF (human recombinant) and EGF (human recombinant) were
obtained from Promega Co., Madison, WI. Chelerythrine Cl and
Calphostin C were from LC services Co., Woburn, MA. H-7,
cycloheximide, medium and other chemicals were from Sigma Co.,
St. Louis, MO.

205
6.3 Results
6.3.1 The Effect of EGF/TGF on System B Activity
The Caco-2 cells were pre-incubated with 20 ng/ml or 100
ng/ml EGF in the serum-free medium for various length of time
(0 48 hours) The medium was changed every 6 hours. The
System B activities were measured at 5 minutes, 10 minutes, 30
minutes, 1, 2, 4, 8, 12, 18, 24, 30, 41, and 48. System B
activities were not affected by TGF/EGF at the incubation
time less than 30. After 48 hours of continuous incubation,
TGF increased the System B activity by 75%, while EGF
stimulated at least 57% (Fig. 6-1) Cycloheximide (10 /M) or
6.6 /iM chelerythrine in the incubations medium blocked the
System B activity increased by TGF/EGF (Fig* 6-1).
In separate experiments, the Caco-2 cells were pre
treated with insulin, glucagon, dexamethasone, or TGF¡3 for 0 -
48 hours. The System B activity was not affected by either
insulin, glucagon, or dexamethasone. In contrast to TGF, TGF¡3
(>18 hours continuous incubation) inhibited the System B
activity.
In separate experiments, other protein kinase C
inhibitors H-7 and calphostin C were added to the TGF<*/EGF
incubation media. Calphostin C (50 nM) blocked the TGF/EGF
System y+ stimulation effect, while the H-7 (200 ;uM) did not
have any effect.

206
6.3.2 The EGF/TGF<* Pulse Stimulation Effects
The Caco-2 cells were pre-incubated with TGF (20 ng/ml)
or EGF (100 ng/ml) for 2 hours, and incubated in the serum-
free medium (lacking TGF/EGF) for the remaining 46 hours. The
System B activity was not increased by the TGF/EGF pulse
treatments.
6.3.3 The TGF/EGF Effect on the System v* Activity
The Caco-2 cells were pre-treated with TGF (20 ng/ml) or
EGF (100 ng/ml) in serum-free medium for various length of
time (0-48 hours) Like the System B, the System y+ activity
was increased by TGF/EGF only after 30 hours of continuous
incubation. At 48 hours of incubation, TGF increased the
System y+ arginine (5 xM) uptake by 80% and EGF increased the
arginine uptake by 70% (Fig. 6-2) The addition of IOjliM
cycloheximide or 6.6 /lxM chelerythrine in the TGF or EGF
incubation medium blocked the TGF/EGF stimulation effects
(Figs. 6-3, 6-4). The addition of 50 nM calphostin C also
blocked the TGF/EGF effects. H-7 (later disclaimed as a
defective H-7 isomer by Sigma) did not have any effect on the
System y+ activity.
6.3.4 The TGF/EGF Pulse Effect on System v+
The Caco-2 cells were pre-incubated with TGF (20 ng/ml)
or EGF (100 ng/ml) for 2 hours, and then incubated in the same
serum-free medium (lacking TGF/EGF) for the remaining 46

207
hours. System y+ arginine uptake was unaffected by this
treatment.
6.4 Discussion
The peptide growth factors TGF and EGF both stimulated
the System B and System y* activities in the Caco-2 cells at
both the undifferentiated and differentiated cells.
In addition to being a the slow process requiring many
hours (>30 hours), the TGF/EGF effect was cycloheximide-
sensitive. These combined data suggest that it is unlikely
that the TGF/EGF effect was caused by rapid phosphorylation
of transporter protein. A de novo protein synthesis process
was likely involved in the TGF/EGF stimulation of System B
and System y+. But whether the synthesized protein was the
transporter protein, regulatory protein, or other protein was
unknown. Future studies using a System y+ antibody probe may
provide a more precise answer. Normally, de novo protein
synthesis can occurs within several hours, and it is not clear
why TGF/EGF took more than 30 hours to show their effect. A
cascade of regulatory processes may be involved, in addition
to protein synthesis.
The protein content and cell numbers of the 48 hour CHX-
treated cells was comparable to the pre-CHX-treatment level.
The viability of CHX-treated cells was >99%. Compared to the
control group (only DMEM treatment), the CHX-treated cells had
40% less protein and 40% less cells. So the CHX's inhibitory

208
effect on the System B and System y+ activities was likely
inhibiting new protein synthesis rather than cytotoxic effect.
Prolonged and continuous exposure to TGF/EGF was
required for the System B and System y+ activity stimulation.
The mechanism for the delay is unknown. In the light of a
System A regulatory mechanism proposed by Engleberg (1986),
the System B and System y+ could be regulated by at least two
groups of regulatory forces which are always present and are
in equilibrium. TGF/EGF shift the balance to the stimulatory
side, resulting in synthesis of transporter protein or other
regulatory proteins, while the negative regulatory force
always tries to bring the balance back.
As we discussed above in the introduction section,
TGF/EGF bind to the EGF receptor and the TGF/EGF-receptor
complex phosphorylates many substrates of the EGF receptor.
The substrates may include the receptor itself, the ras
GTPase-activating protein or GAP, PI-3 kinase, and PLCyl.
Which signal pathway did TGF/EGF participate in activating
Systems B and y+ activities? The inhibition of the TGF/EGF
stimulation effect on the alanine and arginine transport by
the specific protein kinase C inhibitors chelerythrine and
calphostin C suggested that the protein kinase C activation
was involved in the process. Chelerythrine CL specifically
inhibits PKC by acting at PKC's catalytic subunits, while
calphostin C binds at PKC's regulatory subunits (Tamaoki et
al., 1990). We have found that the H-7 isomer defectively

209
manufactured by Sigma Co as H-7 did not influence the TGF<*/EGF
effect on System B and System y+ activities. The phospholipase
Cy (PLC) was likely a major intermediate phosphorylated by
TGF/EGF in the System B and System y+ transport activation.
PLC hydrolysizes phosphatidylinositol-4,5-biphosphate (PIP2) ,
generating the intracellular second messenger diacylglycerol
(DAG) which is an endogenous activator of protein kinase C.
Further study of the PIP2 level, diacylglycerol level, and the
inositol hydrolysis in cells will provide the information
concerning details of the pathways. It is still unclear if the
PLC pathway is the only pathway for this stimulation.
Because the protein kinase C inhibitors also inhibited
general protein synthesis, it is likely that the protein
kinase C activation precedes the de novo protein synthesis
associated with the System B and System y+ activation.
It is notable that the Na+-independent alanine System L
transport was not stimulated by the TGF/EGF incubation. In
fact, due to the stimulation effect of TGF/EGF on cell
protein synthesis, the System L activity per mg protein
actually decreased. These findings support the notion that
System B and System y+ activation is a selective event.
Unlike the transport substrate regulation we discussed in
the preceding chapter, TGF/EGF stimulated both the System B
and System y+ transport activities. This phenomenon may be
associated with the stimulation effect of TGF/EGF on the
epithelial proliferation, cell growth, or mitogenesis.

210
TGF/EGF increased the needs of cells for amino acids for
their growth.
6.5 Summary
Prolonged, continuous exposure to peptide growth factors
TGF or EGF stimulates System B and System y+ activities in
Caco-2 cell in both the undifferentiated and differentiated
states. The stimulatory effect involves a de novo protein
synthesis process. Whether this involves the transporter
protein or a regulatory protein is not clear. The
intracellular protein kinase C activation is involved in the
pathway of TGF/EGF activation of the System B and System y+
transport activities, thereby suggesting the possibility of a
role for PLC-y phosphorylation.

Fig. 6-1. The effects of chronic TGF, or EGF System B activity
System B alanine (50 /M) uptake was measured in cells (2 days old
and 9 days old), after cells had been incubated in DMEM, 20 ng/ml TGF,
or 100 ng/ml EGF for 48 hours. TGF0' or EGF each stimulated the alanine
uptake (p < 0.05, n = 12).

Alanine uptake, per cent
ZTZ
250

Fig. 6-2. The effect of CHE on the chronic TGF^/EGF-induced System
B activity
System B alanine (50 /M) uptake was measured after cells had been
incubated in DMEM, TGF or EGF, 6.6 /xM CHE for 48 hours. TGFoc or EGF
each stimulated the alanine uptake, CHE in the TGF or EGF medium
blocked this stimulative effect (p < 0.05, n = 6)

Alanine uptake, per cent
-*-*-*-* N> N>
NJCn^lOMOl-JON)
ocnomocnocnocn
VTZ
250

Fig. 6-3. The effectof CHX on the chronic TGF^/EGF-induced
System B activity
System B alanine uptake was stimulated by 48 hours of continuous
TGF0' or EGF incubation. CHX (10 juM) in the TGF or EGF medium blocked
the stimulation effects (p < 0.05, n = 12). Data shown were from day 2
cells, with similar results obtained in day 9 cells. 100% alanine uptake
= 0.8 nmole/mg/min.

Alanine uptake, per cent
N)
Cn
cn
o
-^i
cn
o
o
K>
cn
ai
o
'vj
cn
|\J K>
O N>
o cn
Control
h TGFa
EGF
CHX
H CHX+TGFa
CHX+EGF
J i i L
J L
9TZ
250

Fig. 6-4. The chronic effect of TGF^ or EGF on System v* activity
System y+ arginine (5 /M) uptake was measured in day 2 cells which
had been incubated in DMEM, TGF0', or EGF in DMEM for 48 hours. TGF and
EGF each stimulated the System y+ arginine uptake (p < 0.05, n = 6).
Similar results were obtained in 9 days old cells.

Arginine uptake, pmole mg min

l
ro
M
cn
o
cn
o
cn
o
O
o
o
o
o
QTZ
300

Fig. 6-5. The effects of chelervthrine on the chronic TGF^/EGF-
System v+ activity
System y+ arginine (5 /zM) uptake was measured after cells
old) had been incubated in DMEM, TGF, or EGF, 6.6 fiM CHE
hours. TGF** and EGF each stimulated the arginine uptake, CHE
TGF/EGF incubation media blocked this stimulation effect (p <
= 6) .
induced
(2 days
for 48
in the
0.05, n

A -1-1
Arginine uptake, pmole mg min
o
cn
O
O
o
Cn
o
o cn
o o
Control
CHE
H EGF
EGF + CHE
TGFa
TGFa + CHE
300

Fig. 6-6. The effect of CHX on the chronic TGF^/EGF-induced
System v+ activity
System y+ arginine (5 juM) uptake in cells (2 days old) was
stimulated by 48 hours TGF or EGF incubation. CHX (10 mM) in the
TGF/EGF incubation blocked this TGF/EGF stimulation effect (p < 0.05,
n = 6) .

Arginine uptake, pmole mg min
i
>
N>
cn
o
cn
O
cn
o
O
o
O
O
o
zzz
300

CHAPTER 7
THE EFFECTS OF PHORBOL ESTERS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
7.1 Introduction
The tumor promotors phorbol 12-myristate 13-acetate
(TPA), or phorbol 12,13-dibutyrate (PDBU), have long been
known for their effects in promoting mitogenesis and cell
growth. The phorbol ester effect is through a series of an
intracellular cascade processes that is initiated by protein
kinase C activation. The lipophilic phorbol esters diffuse
through the cell membrane as a substitute for DAG in directly
activating the intracellular protein kinase C. Even though
there have been reports that phorbol esters are not specific
protein kinase C activators, and that they may activate other
processes, phorbol esters are widely used to activate protein
kinase C.
Protein kinase C has two subunits: a regulatory subunit,
and a catalytic subunit. Diacylglycerol and phorbol esters,
bind to the regulatory subunit causing a structure change
which activates or inactivates the kinase. Once the protein
kinase C is activated, the catalytic subunit bind to its
substrate and catalyzes it. Both steps are essential for the
biological functions.
223

224
Many protein kinase C inhibitors have been developed, and
are classified according to their working mechanisms.
Inhibitors such as H-7 and calphostin C bind to the regulatory
side, while inhibitors such as chelerythrine bind to the
catalytic subunit. Either of above step will block the PKC
activation.
Calphostin C blocks the binding of phorbol ester to the
PKC when photo-activated by a fluorescent light. Calphostin C
is exceptionally selective for PKC, with inhibitory
concentration much less than that required to inhibit protein
kinase A or other protein tyrosine kinases (Kobayashi et al.,
1989; Bruns et al., 1991).
Chelerythrine inhibits protein kinase C activity at a
concentration over 100 times lower than that for inhibition of
protein kinase A, protein tyrosine kinases, or the Ca++-
calmodulin-dependent protein kinases (Herbert et al., 1990).
Therefore, both the calphostin C and chelerythrine are
excellent protein kinase C inhibitor with high selectivity.
7.2 Methods And Materials
7.2.1 Pre-treatment with Phorbol Esters
The Caco-2 cells were washed three time with the serum-
free-medium, and incubated in the dark or under fluorescent
light with the same medium containing: (1) control group,
serum-free DMEM plus the same amount of DMSO as appeared in
the phorbol esters (DMSO was < 0.5% of the medium volume) (2)

225
treatment with phorbol esters, serum-free DMEM plus various
concentration of TPA or PDBU (1 pM 10 iM) (TPA and PDBU
were diluted from DMSO stock solution and stored at -20C),
(3) treatment with phorbol esters and other agents, DMEM plus
phorbol ester plus additional other chemicals such as
cycloheximide, chelerythrine, calphostin C, with DMEM plus
other agent as an internal control, chelerythrine and
calphostin C were from LC Services Co., Woburn, MA.). The
cells were incubated for various periods of time in the 37C
incubator. The medium was changed very 6 hours. For the
calphostin C incubation, a 20 watt fluorescent light was
placed in the incubator. The details of each incubation are
explained below.
7.2.2 Pre-treatment with Dibutvrvl Cvclic-AMP (dcAMP)
The Caco-2 cells were washed three times with the serum-
free medium, and incubated with the same medium with 0.5 /M
dcAMP for various times (0-24 hours) The medium was changed
every 6 hours.
7.3 Results
7.3.1 The Phorbol Ester (TPA) Stimulation of the System B
Activity Time Course
The Caco-2 cells were pre-incubated with 0.5 /M TPA in
the serum-free medium for various length of time (0-24
hours) prior to the uptake experiments. The medium was changed

226
every 6 hours. The 50 /iM alanine system B uptake activity was
measured immediately after each incubation point. The system
B alanine uptake activity was stimulated by a prolonged TPA
incubation (> 8 hours). The stimulation effect increased
steadily up to at least 24 hours (Fig. 7-1). At incubation
times less than 8 hours, there was no TPA effect on the system
B activity.
The system L alanine uptake was not stimulated by TPA
incubation (0-24 hours). Due to the increase cell protein
synthesis, the system L alanine uptake per mg protein actually
decreased.
7.3.2 TPA Pulse Effect on System B Activity
The Caco-2 cells were pre-treated with 0.5 /iM TPA for 0 -
2 hours, and then were washed and incubated in the serum-free
medium (lacking TPA) for the remaining time period (22 hours).
The total incubation time was 24 hours, including the TPA
incubation plus the following non-TPA incubation. System B
activity was then measured immediately after the total
incubation. Unlike their continuous incubation counterparts,
the pulse incubation did not affect the system B activity.
7.3.3 Dose Response for Phorbol Ester (TPA) Stimulation of
System B
The Caco-2 cells were pre-incubated in the serum-free
medium with various concentration of TPA (1 pM 10 /liM) for 24
hours prior to the uptake experiments. The 50 iM alanine

227
System B uptake activity was then measured immediately after
each incubation. System B alanine uptake activity was
stimulated at concentrations of [TPA] > 10 nM. A peak
stimulation of 2 fold was observed at [TPA] = 1/xM, and the
stimulation effect was attenuated at [TPA] = 10 /M (Fig. 7-2) .
7.3.4 Phorbol Ester Stimulated the System B Activity
Regardless the Cell Age
The Caco-2 cells (1 day old through 35 days old) were
pre-incubated with 0.5 /M TPA in the serum-free medium for 24
hours prior to the uptake experiments. The system B activity
was stimulated at least 2 fold by TPA at all cell ages (Fig.
7-3) At each cell ages, System L uptake was not significantly
altered by TPA.
7.3.5 The TPA Stimulation of System B Activity Involved De
Novo Protein Synthesis
Caco-2 cells were pre-incubated in serum-free medium
containing 0.5 iM TPA, 10 M CHX for 24 hours prior to the
uptake experiments. The 50 /M alanine System B uptake activity
was stimulated 2 fold by the TPA incubation. The addition of
CHX in the TPA incubation medium blocked the stimulation
effect. CHX alone did not significantly affect System B
activity (Fig. 7-4).
Caco-2 cells were also pre-incubated with 0.5 /lxM TPA in
serum-free medium, 0.5 /M actinomycin D for 24 hours. System
B activity increased following TPA exposure. The incubation in

228
TPA/actinomycin D had no affect on System B activity (Fig. 7-
5).
7.3.6 Phorbol Ester Stimulation of System B Activity vas via
PKC Activation
The Caco-2 cells were pre-incubated with 0.5 iM TPA in
the serum-free medium, 6.6 /M chelerythrine or 50 nM
calphostin C in the medium for 24 hours prior to the uptake
experiments. The 50 iM alanine System B uptake activity was
measured. The system B activity increased 2 fold following the
TPA incubation alone. The TPA/chelerythrine or the
TPA/calphostin C incubation did not affect System B activity
(Fig. 7-6). H-7 isomer did not alter the System B activity nor
the effect of TPA.
7.3.7 The Effect of Phorbol Ester on the System B Transport
Kinetics
The Caco-2 cells were pre-incubated with 0.5 juM TPA in
the serum-free medium for 24 hours prior to the uptake
experiments. The system B alanine transport kinetics ([3H]-
alanine = 1 iM 5 mM) were then measured (Figs. 7-7) The VBax
of day 2 cells was significantly increased 2 fold by TPA (Viax
= 3.05 nmole/mg/min in the control cells, VBax = 5.9
nmole/mg/min with the TPA treatment) The VBax of day 9 cells
was increased 3 fold by TPA incubation (VBax =0.5 nmole/mg/min
in the control cells, Vmax = 1.65 nmole/mg/min with the TPA
treatment) The Km (K, = 160 juM alanine) was the same

229
regardless the age and the TPA treatment (Fig. 7-8).
7.3.8 Phorbol Ester Up-Regulated the System v* Uptake Activity
The Caco-2 cells were pre-incubated with 0.5 /xM TPA in
serum-free medium for various length of time (0-24 hours)
prior to the uptake experiments. The System y+ activity was
stimulated by TPA only after a prolonged incubation; at least
8 hours were required for the effect. The TPA stimulation
effect increased steadily up to at least 24 hours. A 24 hours
TPA incubation period was chosen for the subsequent TPA
experiments.
7.3.9 The TPA Pulse Effect on System v* Activity
The Caco-2 cells were pre-incubated with 0.5 /xM TPA in
the serum-free medium for various periods of time (0-2
hours), then re-incubated in the serum-free medium (lacking
TPA) for the remaining periods prior to the uptake
measurements. The total incubation time was 24 hours,
including the pulse TPA incubation plus the following non-TPA
incubations. The TPA pulse treatments alone did not affect the
system y+ activity.
7.3.10 TPA/s Effect on the System v* Activity at Various Cell
Ages
The Caco-2 cells (1 day old 14 day old) were pre
treated with 0.5 /xM TPA in the serum-free medium for 24 hours
prior to the arginine uptake experiments. The 5 /xM arginine

230
uptake activity was stimulated up to 2 fold by TPA at all cell
ages (Fig. 7-9).
7.3.11 The TPA Stimulation of System y* Activity Involved De
Novo Protein Synthesis
The Caco-2 cells were pre-incubated in serum-free medium
with 0.5 iM TPA for 24 hours, 50 jiM or 20 /M cycloheximide
including in the incubation medium for various windows of time
(first 6 hours, second 6 hours, third 6 hours, fourth 6 hours,
first 12 hours, first 18 hours, and the entire 24 hour
period) Fig. 7-10 shows that the 5 /xM arginine system y+
uptake activity was stimulated 50% by TPA incubation, but the
stimulation was not retarded by each of the 6 hour CHX
incubation. However, the first 12 and 18 hour periods of
CHX/TPA incubation blocked the TPA stimulation of System y+
activity. The absolute uptake activity was decreased following
24 hours of CHX incubation, but due to the greater decrease in
cell protein, the activity per mg protein was not affected.
7.3.13 The Phorbol Ester Stimulation of System v* Activity was
Inhibited by Specific Inhibitor of Protein Kinase C
The Caco-2 cells were pre-incubated with TPA in the
serum-free medium, 6.6 /M chelerythrine or 50 nM calphostin
C for 24 hours prior to the uptake measurements. System y+
activity was increased by TPA alone. The TPA/chelerythrine or
TPA/calphostin C combination incubation did not stimulate
System y+ activity, and chelerythrine or calphostin C alone

231
did not affect activity (Fig. 7-11).
7.3.14 The Effect of Phorbol Ester on the System y* Transport
Kinetics
The Caco-2 cells were pre-treated with TPA in the serum-
free medium for 24 hours prior to the uptake experiments, and
the system y+ arginine transport kinetics were measured ([3H]
arginine = 0.1 /xM 1 mM) The VBax was increased by TPA in
both the day 2 and day 9 cells (VBax = 777 pmole/min/min in day
2 cell with VBax = 1111 pmole/mg/min in TPA treated day 2
cells, VBax = 541 pmole/mg/min in day 9 cells and VBax = 720
pmole/mg/min with TPA treatment). The K, of 43.3 and 55 /xM
arginine was the same regardless the cell age and the TPA
incubation (Fig. 7-12).
7.4 Discussion
The phorbol ester TPA stimulated both System B and System
y+ activities. The TPA effects on transporter activities were
similar in terms of the potency, the onset of stimulation, and
the dependency of de novo protein synthesis.
TPA diffuses through cell membrane and directly binds to
the protein kinase C regulatory domain, the endogenous
diacylglycerol binding site. TPA possesses structural
similarities with diacylglycerol, and both activate protein
kinase C. TPA can acutely activate protein kinase C, or
chronically inactivate PKC by depleting the cell of active
membrane-bound form of PKC.

232
In the last chapter, we discussed that TGF/EGF activated
the System B and System y+ activities by generating the
intracellular second messenger diacylglycerol, which activates
protein kinase C. In this chapter, we used phorbol ester TPA
to directly activate the cellular protein kinase C, bypassing
the signal pathway between the TGF/EGF to the PKC.
In stimulating the System B and System y+ activities, the
onset of the TPA stimulation was slow (Fig. 7-1) Onset of
activation was observed only after 8 hours, and 24 hours were
needed to significantly stimulate the transport activities.
Continuous TPA exposure was necessary to stimulate the
transport activities.
Cycloheximide and actinomycin D blocked the TPA's
stimulation effect on the System B and System y+ activities,
suggesting that a transcriptional and a translational control
mechanism was involved. Whether the gene expression and the de
novo protein synthesis involved the transporter protein per
se, or other regulatory proteins, is not clear. Further
molecular study using the cDNA probes would give a more
precise answer.
Even though we did not have molecular probe for the
present study, the kinetics studies of the transport
activities are still useful tools. TPA increased both the
system B and system y+ transport Vaax without changing the
other kinetic parameters. The increase in V>ax without K
changing, strongly suggests that increases in System B and

233
System y+ activities by TPA were due to the increase in copies
of functional transporter units, rather than due to
modification of the transport affinity.
To further confirm that the effect of TPA was a protein
kinase C activation phenomenon, we used the specific protein
kinase C inhibitors chelerythrine Cl and calphostin C. As
shown in the results, these PKC inhibitors blocked the TPA's
effect on the System B and System y+ activities. These data
indicated that the TPA stimulation of transport activities
involved protein kinase C activation, not protein kinase C
inhibition.
When we compare the System B and System y+ transport
activities that were stimulated by TGF/EGF (chapter 6) and
TPA (this chapter), we can see that each system's transporter
characteristics were very similar. These data further support
the notion that TGF/EGF stimulate the system B and system y+
activities by engaging protein kinase C.
7.5 Summary
Phorbol ester TPA activates System B and System y+, but
not System L, activities throughout all cell ages. The
activation involves transcription and translation process, and
likely is mediated via protein kinase C. The phorbol ester
stimulation of System B and System y+ results in an increase
in system's V,ax without affecting the corresponding K,.

Fig. 7-1. TPA System B stimulation time course
System B alanine (50 /xM) uptake was measured in day 3
cells which had been incubated in DMEM or 1 /xM TPA for various
periods of time (0 24 hours). Continuous exposure (> 8 hours)
to TPA resulted in a increase in alanine uptake.

Uptake (10 mol/mg/hr)
o 4 8 12 16 20 24
Incubation Time (hours)
235

Fig. 7-2. Dose curve of the TPA System B stimulation
System B alanine (50 M) uptake was measured in cells
which had been incubated in various concentration of TPA (10'12 -
10'5 M) for 24 hours.

Alanine uptake, nmole mg min
-r' /*-
1.2 -
1.0 -
0.8 -
0.6 -
0.4 -
0.2
n i i r
+
Na dependent
+
Na independent
-

i r
f
- oo
-12 -11 -10 -9 -8 -7 -6
TPA Concentration, log[M]
-5
237

Fig. 7-3. Effect of TPA on System B activity at various cell ages
System B alanine (50 /M) uptake in cells (2 days
days old) which had been incubated in 0.5 /jM TPA for 24
prior to the uptake experiments.
to 35
hours

Cell age, days
239

Fig. 7-4. The effect of CHX on the TPA-induced alanine uptake
Alanine (50 iM) uptake was measured in cells (3 days old
and 9 days old) which had been incubated in 0.5 juM TPA 10 /lxM
CHX for 24 hours. TPA stimulated the Na+-dependent System B alanine
uptake (p < 0.05, n = 6). CHX in the TPA incubation medium
blocked this TPA's stimulation effect (p < 0.05, n = 6). The
alanine uptake in choline Cl medium was not affected by TPA (p
< 0.05, n = 9) Data shown were obtained from 3 days old cells,
with similar results observed in 9 days old cells.

Alanine uptake, nmole mg min
o
bo
o
4^
O
O)
O
D
TPZ

Fig. 7-5. The effect of actinomvcin D on the TPA-induced System
B activity
System B alanine (50 /M) uptake was measured in cells (3
days old) which had been incubated in 0.5 /M TPA 0.5 /jlM
actinomycin D for 24 hours prior to the uptake measurements. TPA
alone stimulated the System B alanine uptake (p < 0.05, n = 6),
while actinomycin D in the tPA medium blocked the TPA's
stimulation effect (p < 0.05, n = 6). Similar results were
obtained in 9 days old cells.

Alanine uptake, nmole mg
NJ
O
. -1
min
zvz
150

Fig. 7-6. The effects of chelrvthrine on the TPA-induced System
B activity
System B alanine (50 fM) uptake was measured in day
9 cells which had been incubated 0.5 /M TPA 6.6
chelerythrine for 24 hours. The TPA stimulation of alanine uptake
was blocked by the CHE (p < 0.05, n = 6).

Alanine uptake, nmole mg min
o o o o o o o
M OJ 4^ Ln CD kj
SPZ

Fig. 7-7. The effect of TPA on System B activity kinetics
System B alanine (10 /M 5 mM) uptake kinetics were
measured in cells (2 days old and 9 days old) which had been
incubated in DMEM or 0.5 uM TPA for 24 hours. System B uptake
was plotted as a function of alanine concentration.

6.0
Y 5.0
c
tac
s
0
O
4.0
3.0
2.0
ct
<
-3 1.0
O o
o

o
o
2
day 2 TPA

Day 2 W/o TPA
Day 9 TPA
Day 9 w/o TPA
o
i i i
4 6 8
[Ala], 10~4M
O
10
247

Fig. 7-8. Eadie-Hofstee transformation of TPA-induced System B
activity kinetics
The kinetic data in Fig. 7-7 was re-plotted as a function
of Jala vs. J/[ala]. TPA increased the Vax in both day 2 and day
9 cells, while the K was not affected.

nmole mg min

Fig. 7-9. The effect of TPA on System v+ activity at various cell
ages
System y+ arginine (5 /M) uptake was measured in various
ages of cells which had been incubated in DMEM or DMEM plus 0.5
MM TPA for 24 hours.

200
160
120
80
40
0
i i i i i 1 1 1 1 1 r
J 1 1 i i i i i i i i i i i i I
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Cell age, day
251

Fig. 7-10. The effect of CHX on TPA-induced System v* activity
System y+ arginine (5 /M) uptake was measured in day 2
cells which had been incubated in 0.5 /xM TPA for 24 hours with
or without 50 /xM CHX in the first 12 hours incubation. TPA alone
incubation stimulated the arginine uptake, and this stimulation was
blocked by CHX in the first 12 hours TPA incubation (p < 0.05,
n = 3) .

Arginine uptake, pmole mg min
1 NO hO
t'OUl'-JONJCri'vlON)
ocnocnocnocnocn
I l I l I l 1 1 1
Control
CHX
* TPA
TPA + CHX
j i i L
Z9Z
250

Fig. 7-11. The effect of chelervthrine on the TPA-induced System
v* activity
System y+ arginine uptake was measured in cells (9 days
old) which had been incubated in DMEM, 0.5 /M TPA, 6.6 /lxM CHE,
or 6.6 /nM CHE plus 0.5 /uM TPA. Arginine uptake was stimulated by
TPA incubation. The TPA stimulation effect was blocked by the CHE
in the TPA incubation medium (p < 0.05, n = 6 ). Similar results
were obtained in day 2 cells.

Arginine uptake, per cent
-* > M NJ
cn o cn o cn
o o o o o o
300

Fig. 7-12. The effect of TPA on System v+ transport kinetics
System y+ arginine (0.5 /M 1 mM) uptake was measured in
cells (2 days old and 9 days old) which had been incubated in
DMEM or 0.7 mM TPA for 24 hours. TPA increased the VMX in both
day 2 and day 9 cells without affecting the k.

min
O 4 8 12 16 20
Jarg/fAri3] MLiter mg
. -1
min
257

CHAPTER 8
SUMMARY AND CONCLUSIONS
8.1 SUMMARY
We have studied amino acid transport systems
independently serve alanine or arginine in the apical membrane
of the human intestinal epithelial cell line, Caco-2. We have
investigated the in vitro cellular mechanisms that underlie
the independent regulation of these systems.
The structurally different substrates L-alanine and L-
arginine are transported separately by unique transport
pathways. The pathways serving alanine are: System B, System
L, and simple passive diffusion. The pathways for arginine are
System y+ and simple passive diffusion. The transport
activities of System B, System y+, and System L were each-down
regulated in parallel with advancing Caco-2 cell development
and differentiation. System B and System y+ activities were
each actually trans-stimulated by their own substrate. System
B capacity was chronically up-regulated by its own substrates
through a mechanism that involved protein kinase C and de novo
protein synthesis. Simple passive diffusion of each substrate
was unaffected by any cellular conditions that affected the
carrier-mediated transport.
The peptide growth factors EGF and TGF stimulated System
258

259
B and System y+ transport activities. A wide variety of other
growth factors and hormones were without effect. The relative
transport capacities of Systems B and y+ paralleled the
activation or inhibition of protein kinase C. Furthermore,
inhibition of protein kinase C or inhibition of protein
synthesis each prevented the EGF/TGF activation of Systems B
or y+.
In addition to the biochemical and histological
similarities of Caco-2 cells and normal small intestinal
enterocytes, the Caco-2 apical membrane also possesses the
same alanine and arginine transport systems found in the small
intestinal epithelial brush border membrane. The transport
activities changes that occur during Caco-2 cell development
also resemble those found in the small intestinal cells. The
independent regulation of the stated alanine and arginine
transporters by their transported substrates, was strikingly
consistent with that measured in vivo.
This study provides a better understanding of the
mechanism of small intestinal nutrient absorption, provides
information concerning enterocytic development, and helps our
understanding of the fundamentals physiology of epithelial
transporter regulation. This project provides an excellent in
vitro model for future studies of intestinal regulation of
nutrient absorption in states of health and disease, including
adenocarcinoma development.

260
8.2 Conclusions
(1) Caco-2 epithelial differentiation status is
associated with regulation of amino acid transport.
(2) Amino acid transporters System B and System y+
independently serve alanine and arginine, and are regulated
independently.
(3) Amino acid substrates up-regulate their own
transporters' activities via trans-stimulation, or chronically
by a mechanism involving de novo protein synthesis.
(4) Chronic up-regulation of System B or System y+
capacities by de novo protein synthesis is activated by
EGF/TGF acting through a protein kinase C pathway.
The Proposed Amino Acid Transport Regulation Mechanism in
Caco-2 Cells;

261
Amino Acid Transport Regulation
in Caco-2 Cells

REFERENCE LIST
Akiyama, T. Saito, T. Ogawara, H. Toyoshima, K. and
Yamamoto, T. 1988. Tumor promoter and epidermal growth
factor stimulate phosphorylation of the c-erbB-2 gene
production in MKN-7 human adenocarcinoma cells. Mol.
Cell. Biol 8:1019-1026
Betran, J., Werner, A., Moore, M.L., Stange, G. Markovich, D.
1992. Expression cloning of a cDNA from rabbit kidney
cortex that induces a single transport system for cystine
and dibasic and neutral amino acids. Proc. Natl. Acad.
Sci. USA. 89:5601-5
Bruns, R. F, Smith, D. R, and Nakono, K. 1991. Inhibition of
protein kinase C by calphostin C is light-dependent.
Biochem. Biophy. Res. Comm. 176:288-293
Buddington, R.K., Chen, J.W. Diamond, J.M. 1987. Genetic and
phenotypic adaptation of intestinal nutrient transport to
diet in fish. J. Physiol. Lond. 393: 261-81
Buddington, R.K., Chen, J.W., Diamond, J.M. 1991. Dietary
regulation of intestinal brush-border sugar and amino
acid transport in carnivores. Am. J. Physiol. 261(4 Pt
2): R793-801
Buddington, R.K., Diamond, J.M. 1987. Pyloric ceca of fish: a
"new" absorptive organ. Am. J. Physiol. 252(1 Pt 1):
G65-7 6
Buddington, R.K., Diamond, J.M. 1989. Ontogenetic development
of intestinal nutrient transporters. Annu. Rev. Physiol.
51: 601-19
Buddington, R.K., Diamond, J.M. 1990. Ontogenetic development
of monosaccharide and amino acid transporters in rabbit
intestine. Am. J. Physiol. 259(4 Pt 1): G544-55
Campa, M.J., Kilberg, M.S. 1989. Characterization of neutral
and cationic amino acid transport in Xenopus oocytes. J.
Cell. Physiol.141(3): 645-52
262

263
Carpenter, G., King, L. Jr., Cohen, S. 1979. Rapid enhancement
of protein phosphorylation in A-431 cell membrane
preparations by epidermal growth factor. J. Biol. Chem
254:4884-4891
Carpenter, G., Wahl, M.I. 1990. The epidermal growth factor
family. In: Peptide growth factors and their receptors I,
edited by Sporn, M.B. and Roberts, A.B, SpringerVerlag,
Germany. pp69-172
Carver, J. A, Cooke, R. M. Esposito, G., Campbell, I. D. ,
Gregory, H. and Sheard, B. 1986. A high resolution H NMR
study of the solution structure of human epidermal growth
factor, FEBS Lett 205:77-81.
Chen, W.S., Lazar, C.S., Poenie, M. Tsien, R.Y., Gill, G.N.,
Rosenfeld, M.G. 1987. Requirement for intrinsic protein
tyrosine kinase in the immediate and late actions of the
EGF receptor. Nature. Aug 27-Sep 2; 328(6133): 820-3
Christensen, H.N. 1984. Organic ion transport during seven
decades. Biochim. Biophys. Acta 779:225-69
Christensen, H.N. 1985. On the strategy of kinetic
discrimination of amino acid transport systems. J. Membr.
Biol. 84:97-103
Christensen, H.N. 1990. Role of amino acid transport and
countertransport in nutrition and metabolism. Physiol.
Rev.70:43-77
Christensen, H.N., Riggs, T.R., Fischer, H., Palatine, I.M.
1952. Amino acid concentration by a free cell neoplasm:
Relations among amino acids. J. Biol. Chem. 198:1-22
Christensen, H.N., Oxender, D.L., Liang, M., Vatz. K.A. 1965.
The use of N-methylation to direct the route of mediated
transport of amino acids. J. Biol. Chem. 240:3609-16.
Christensen, H.N., Liang, M. 1966. Transport of diamino acids
into the Enrlich cells. J. Biol. Chem. 241:5542-51
Dawson, W.D., Cook, J.S. 1987. Parrallel changes in amino acid
transport and protein kinase C localization in LLC-PK1
cells treated with TPA or diradylglycerols. J. Cell.
Physiol. 132:104
Diamond, J.M. 1991. Evolutionary design of intestinal nutrient
absorption:enough but not too much. NIPS. 6:92-96

264
Diamond, J.M., Karasov, W.H. 1987. Adaptive regulation of
intestinal nutrient transporters. Proc. Natl. Acad. Sci.
U. S. A. 84(8): 2242-5
Edelman, A.M., Blumenthal, D.K., Krebs, E.G. 1987. Protein
serine/threonine kinases. Annu. Rev. Biochem. 56: 567-613
Ellis, C., Moran, M., McCormick, F., and Pawson, T. 1990. EGF
stimulates GAP in vitro. Nature 311:377-381.
Englesberg, E., Moffett, J. Perier, F. 1986. Mechanism of
regulation of amino acid transport in CH0-K1 cells and
the insulin connection. Federation Pro. 45(10):2441-2443
Fava, R. A. and Cohen, S. 1984. Isolation of a calcium-
dependent 35 kilodalton substrate for the EGF
receptor/kinase from A-431 cells. L. Biol. Chem 258:2636-
2645
Ferraris, R.P., Diamond, J., Kwan, W.W. 1988a. Dietary
regulation of intestinal transport of the dipeptide
carnosine. Am. J. Physiol. 255(2 Pt 1): G143-50
Ferraris, R.P., Kwan, W.W., Diamond, J. 1988b. Regulatory
signals for intestinal amino acid transporters and
peptidases. Am. J. Physiol. 255(2 Pt 1): G151-7
Ferraris, R.P., Diamond, J.M. 1989. Specific regulation of
intestinal nutrient transporters by their dietary
substrates. Annu. Rev. Physiol. 51: 125-41
Frexes-Steed, M., Warner, M.L., Bulus, N., Flakoll, P. 1990.
Abumrad-NN. Role of insulin and branched-chain amino
acids in regulating protein metabolism during fasting.
Am. J. Physiol. 258(6 Pt 1): E907-17
Gaull, G.E., Wright, C.E., Isaacs, C.E. 1985. Significance of
growth modulators in human milk. Pediatrics. 75(1 Pt 2):
142-5
Gerencser, G.A., Stevens, B.R. 1989. Energetics of
sodium-coupled active transport mechanisms in
invertebrate epithelia. Am. J. Physiol. 257(3 Pt 2):
R461-72
Glenney, J.R. Jr., Chen, W.S., Lazar, C.S., Walton, G.M. ,
Zokas, L.M., Rosenfeld, M.G., Gill, G.N. 1988.
Ligand-induced endocytosis of the EGF receptor is blocked
by mutational inactivation and by microinjection of
anti-phosphotyrosine antibodies. Cell. 52(5): 675-84

265
Grasset, E., Pinto, M. Dussaulx, E., Zweibauxn, A., Desjeux,
J.F. 1984. Epithelial properties of human colonic
carcinoma cell line Caco-2: electrical parameters. Am. J.
Physiol. 247(3 Pt 1): C260-7
Grasset, E., Bernabeu, J., Pinto, M. 1985. Epithelial
properties of human colonic carcinoma cell line Caco-2:
effect of secretagogues. Am. J. Physiol. 248(5 Pt 1) :
C410-8
Herbert, J. M, Smith, D. B, and Satoma, G. 1990. Chelerythrine
is a potent and specific inhibitor of protein kinase C.
Bichem. Biophys. Res. Comm. 172:993-999.
Hernandez-Sotomayor, S.M.T., Carpenter, G. 1992. Epidermal
growth factor receptor:elements of intracellular
communication. J. Memb. Biol. 128:81-89.
Hidalgo, I.J., Kato, A., Borchardt. 1989. Binding of epidermal
growth factor by human colon carcinoma cell (Caco-2)
monolayers. Biochim. Biophy. Res. Comm. 160(1):317-324
Honegger, A.M., Dull, T.J., Felder, S., Van Obberghen, E.,
Bellot, F., Szapary, D., Schmidt, A., Ullrich, A.,
Schlessinger, J. 1987a. Point mutation at the ATP binding
site of EGF receptor abolishes protein-tyrosine kinase
activity and alters cellular routing. Cell. 51(2):
199-209
Honegger, A.M., Szapary, D., Schmidt, A., Lyall, R. Van
Obberghen, E., Dull, T.J., Ullrich, A., Schlessinger,
J. A. 1987b. Mutant epidermal growth factor receptor with
defective protein tyrosine kinase is unable to stimulate
proto-oncogene expression and DNA synthesis. Mol. Cell.
Biol. 7(12): 4568-71
Hopfer, U. 1987. Membrane transport mechanism for hexoses and
amino acids in the small intestine. In: Physiology of the
Gastrointestinal Tract, edited by Johnson-LR. Raven
Press, New York, pp 1499-1526
Jumarie, C., Malo, C. 1991. Caco-2 cells cultured in
serum-free medium as a model for the study of enterocytic
differentiation in vitro. J. Cell. Physiol. 149(1): 24-33
Karasov, W.H., Diamond, J.M. 1983. Adaptive regulation of
sugar and amino acid transport by vertebrate intestine.
Am. J. Physiol. 245(4): G443-62

266
Karasov, W., Solberg, D., Carter, S., Hughes, M., Phan, D.,
Zollman, F., Diamond, J. 1986. Uptake pathways for amino
acids in mouse intestine. Am. J. Physiol. 251(4 Pt 1):
G501-8
Karasov, W.H., Solberg, D.H., Diamond, J.M. 1987. Dependence
of intestinal amino acid uptake on dietary protein or
amino acid levels. Am. J. Physiol. 252(5 Pt 1): G614-25
Kilberg, M.S. 1986. Amino acid transport in eukaryotic cells
and tissues. Federation Pro. 45(10): 2438-2440
Kilberg, M.S., Handlogten, M.E., Christensen, H.N. 1981.
Characteristics of system ASC for transport of neutral
amino acids in the isolated rat hepatocyte. J. Biol.
Chem. 256(7): 3304-12
Kilberg, M.S., Bracy, D.S., Handlogten, M.E. 1986. Substrate
regulation of hepatic system A transport activity after
induction by substrate starvation or glucagon. Fed. Proc.
45(10): 2438-41
Kilberg, M.S, Stevens, B.R., Novak, D.A. 1993. Recent advances
in mammalian amino acid transport. Annu. Rev. Nutr.
13:137-65
Kim, J.W., Closs, E.I., Albritton, L.M., Cunningham, J.M.
1991. Transport of cationic amino acids by the mouse
ecotropic retrovirus receptor [see comments]. Nature.
352(6337): 725-8
Klip, A., Douen, A.G. 1989. Role of kinases in insulin
stimulation of glucose transport. J. Membr. Biol. 111(1):
1-23
Koboyashi, E, Mituta, M, and Suzuki, H. 1989. Calphostin C, a
novel microbial compound, is a highly potent and specific
inhibitor of protein kinase C. Bichem. Biophy. Res. Comm.
159:548-553
Koyama, S.Y., Podolsky, D.K. 1989. Differential expression of
transforming growth factors alpha and beta in rat
intestinal epithelial cells. J. Clin. Invest. 83(5):
1768-73
Laganiere, S., Maestracci, D., Berteloot, A. 1986. Adaptation
of intestinal sugar and amino acid transport to gastric
hyperalimentation. Clin. Invest. Med. 176-85

267
Magagnin, S., Bertrn, J., Werner, A., Markovich, D., Biber,
J., Palacin, M. Murer, H. 1992. Poly(A)+ RNA from rabbit
intestinal mucosa induces b0,+ and y+ amino acid
transport activities in Xenopus laevis oocytes. J. Biol.
Chem. 267(22): 15384-90
Malo, C. 1991. Multiple pathways for amino acid transport in
brush border membrane vesicles isolated from the human
fetal small intestine. Gastroenterology. 100(6): 1644-52
Margolis, B. L., Rhee, S. G., Felder, S. Mervic, M., Lyall R.,
Levitzki, A., Zilberstein, A., and Schlessinger. 1989.
EGF induces tyrosine phosphorylation of phospholipase C-
II: A potential mechanism for EGF receptor signaling.
Cell 57:1101-1107
Mayo, K.H., Cavalli, R.C., Peters, A.R., Boelens, R., Kaptein,
R. 1989. Sequence-specific lH-n.m.r. assignments and
peptide backbone conformation in rat epidermal growth
factor. Biochem. J. 257(1): 197-205
Meisenhelder, J., Suh, P. G., Rhee, S. G., Hunter, T. 1989.
Phospholipase C-y is a substrate for the PDGF and EGF
receptor protein-kinases in vivo and in vitro. Cell
57:1109-1122
Mircheff, A.K., Sachs, G., Hanna, S.D., Labiner, C.S., Rabn,
E., Douglas, A.P., Walling, M.W., Wright, E.M. 1979.
Highly purified basal lateral plasma membranes from rat
duodenum. Physical criteria for purity. J. Membr. Biol.
50(3-4): 343-63
Mircheff, A.K., van Os, C.H., Wright, E.M. 1980. Pathways for
alanine transport in intestinal basal lateral membrane
vesicles. J. Membr. Biol. 52(1): 83-92
Molloy, C. J., Bottaro, D. P., Fleming, T. P., Marshall, M.
S., Gibbs, J. B., Aaronson, S. A. 1990. GAP is a possible
epidermal growth factor receptor substrate. Nature
342:711-714
Montelione, G.T., Winkler, M.E., Burton, L.E., Rinderknecht,
E. Sporn, M.B., Wagner, G. 1989. The solution
sequence-specific 1H-NMR assignments and identification
of two small antiparallel beta-sheets in structure of
recombinant human transforming growth factor alpha. Proc.
Natl. Acad. Sci. USA. 86(5): 1519-23
Moolenaar, W. H., Tertoolen, L. G. J., de Laat, S. W. 1984.
Rapid increase of intracellular DAG level in A-431 cells.
J. Biol. Chem. 259:8060-8069

268
Moolenaar, W. H., Bierman, A. J., Tilly, B. C., Verlaan, I.,
Defize, L. H., Ullrich, A., and Schelessinger, J. 1988.
A point mutation at the ATP-binding site of the EGF
receptor abolishes signal transduction. EMBO J. 8:707-710
Munck, B.G. 1981. Intestinal absorption of amino acids. In:
Physiology of the Gastrointestinal Tract, edited by
Johnson-LR. Raven Press, New York, pp 1097-1122
Oxender, D.L. and Christensen, H.N. 1963. Distinct mediating
systems for the transport of neutral amino acids ny the
Ehrlich cell. J. Biol. Chem. 238:3686-99
Pan, M., Neu, J., Stevens, B.R. 1991. Regulation of alanine
transport in human intestinal epithelial cell line Caco-
2. FASEB J. 5(4):A760
Ray, P. Moy, F.J., Montelione, G.T., Liu, J.F., Narang, S.A.,
Scheraga, H.A., Wu, R. 1988. Structure-function studies
of murine epidermal growth factor: expression and
site-directed mutagenesis of epidermal growth factor
gene. Biochemistry. 27(19): 7289-95
Reiser, S. and Christiansen, P.A. 1971a. The properties of the
preferential uptake of L-leucine by isolated intestinal
epithelial cells. Biochim. Biophys. Acta. 225(1): 123-39
Reiser, S. and Christiansen, P.A. 1971b. Inhibition of amino
acid uptake by ATP in isolated intestinal epithelial
cells. Biochim. Biophys. Acta. 233(2): 480-4
Reiser, S. and Christiansen, P.A. 1971c. Stimulation of basic
amino acid uptake by certain neutral amino acids in
isolated intestinal epithelial cells. Biochim. Biophys.
Acta. 241(1): 102-13
Rousset, M. Laburthe, M. Pinto, M, Chevalier, G. ,
Rouyer-Fessard, C. Dussaulx, E. Trugnan, G. Boige, N. ,
Brun, J.L., Zweibaum, A. 1985. Enterocytic
differentiation and glucose utilization in the human
colon tumor cell line Caco-2: modulation by forskolin. J.
Cell. Physiol. 123(3): 377-85
Saier, M.H. Jr., Daniels, G.A. Boerner, P. Lin, J. 1988.
Neutral amino acid transport systems in animal cells:
potential targets of oncogene action and regulators of
cellular growth. J. Membr. Biol. 104(1): 1-20

269
Salloum, R.M., Souba, W.W., Fernandez, A., Stevens, B.R. 1990.
Dietary modulation of small intestinal glutamine
transport in intestinal brush border membrane vesicles of
rats. J. Surg. Res. 48(6): 635-8
Salloum, R.M., Stevens, B.R., Souba, W.W. 1991. Adaptive
regulation of brush-border amino acid transport in a
chronic excluded jejunal limb. Am. J. Physiol. 261(1 Pt
1): G22-7
Souba, W.W., Klimberg, V.S., Plumley, D.A., Salloum, R.M,
Flynn, T.C., Bland, K.I., Copeland, E.M. 3d. 1990. The
role of glutamine in maintaining a healthy gut and
supporting the metabolic response to injury and
infection. J. Surg. Res. 383-91
Souba, W. W., Pan, M., and Stevens, B. R. 1992. Kinetics of
the Sodium-dependent glutamine transporter in human
intestinal cell confluent monolayers. Bioch. Biophy. Res.
Comm. 188 (2) :746-753
Stein, E.D., Chang, S.D., Diamond, J.M. 1987. Comparison of
different dietary amino acids as inducers of intestinal
amino acid transport. Am. J. Physiol. 252(5 Pt 1) :
G626-35
Stelzner, M. Buddington, R.K., Phillips, J.D., Diamond, J.M.,
Fonkalsrud, E.W. 1990a. Changes in mucosal nutrient
transport in small and large ileal reservoirs after
endorectal ileal pullthrough. J. Surg. Res. 49(4): 344-55
Stelzner, M. Fonkalsrud, E.W., Buddington, R.K., Phillips,
J.D., Diamond, J.M. 1990b. Adaptive changes in ileal
mucosal nutrient transport following colectomy and
endorectal ileal pull-through with ileal reservoir. Arch.
Surg. 125(5): 586-90
Stern, D. F. and Kemps, M. P. 1988. EGF-stimulated tyrosine
phosphorylation of pl85neu: a potential model for receptor
interactions. EMBO J. 7:995-1001
Stevens, B.R. Amino acid transport in intestine. 1992a. In:
Mammalian Amino Acid Transport, edited by Kilberg, M.S.
and Haussinger, D., Plenum Press, New York, 149-163
Stevens, B.R. 1992b. Vertebrate intestine apical membrane
mechanisms of organic nutrient transport. Am. J. Physiol.
263:R458-63

270
Stevens, B.R., Ross, H.J., Wright, E.M. 1982. Multiple
transport pathways for neutral amino acids in rabbit
jejunal brush border vesicles. J. Membr. Biol. 66(3):
213-25
Stevens, B.R., Kaunitz, J.D., Wright, E.M. 1984. Intestinal
transport of amino acids and sugars: advances using
membrane vesicles. Annu. Rev. Physiol. 46: 417-33
Stevens, B.R. and Wright, E.M. 1985. Kinetic model of the
brush-border proline/sodium (IMINO) cotransporter. Ann.
N. Y. Acad. Sci. 456: 115-7
Stevens, B.R., Kempner, E.S., Wright, E.M. 1986. Radiation
inactivation probe of membrane-bound enzymes:
gamma-glutamyltranspeptidase, aminopeptidase N, and
sucrase. Anal. Biochem. 158(2): 278-82
Stevens, B.R., Wright, E.M. 1987. Kinetics of the intestinal
brush border proline (Imino) carrier. J. Biol. Chem.
262(14): 6546-51
Tate, S.S., Yan, N.N., Udenfriend, S. 1992. Expression cloning
of a Na(+)-independent neutral amino acid transporter
from rat kidney. Proc. Natl. Acad. Sci. USA. 89(1): 1-5
Van Winkle, L.J. 1988. Amino acid transport in developing
animal oocytes and early conceptuses. Biochim. Biophys.
Acta. 947(1): 173-208
Van Winkle, L.J., Campione, A.L., Gorman, J.M. 1988.
Na+-independent transport of basic and zwitterionic amino
acids in mouse blastocysts by a shared system and by
processes which distinguish between these substrates. J.
Biol. Chem. 263(7): 3150-63
Van Winkle, L.J. and Campione, A.L. 1990. Functional changes
in cation-preferring amino acid transport during
development of preimplantation mouse conceptuses.
Biochim. Biophys. Acta. 1028(2): 165-73
Van Winkle, L.J., Campione, A.L., Gorman, J.M., Weimer, B.D.
1990. Changes in the activities of amino acid transport
systems b0,+ and L during development of preimplantation
mouse conceptuses. Biochim. Biophys. Acta. 1021(1): 77-98
Wahl, M. I., Nishibe, S., Pann-Ghill S., Rhee, S. G. ,
Carpenter, G. 1989. Epidermal growth factor stimulates
tyrosine phosphorylation of phospholipase c-II
independently of receptor internalization and
extracellular calcium. Proc. Natl. Acad. Sci. USA.
86:1568-1572

271
Wang, H., Kavanaugh, M.P., North, R.A., Kabat, D. 1991.
Cell-surface receptor for ecotropic murine retroviruses
is a basic amino-acid transporter [see comments]. Nature.
352(6337): 729-31
Wells, R.G., Hediger, M.A. 1992a. Cloning of a rat kidney cDNA
that stimulates dibasic and neutral amino acid transport
and has sequence similarity to glucosidases. Proc. Natl.
Acad. Sci. USA. 89(12): 5596-600
Wells, R.G. Lee, W.S., Kanai, Y., Leiden, J.M., Hediger, M.A.
1992b. The 4F2 antigen heavy chain induces uptake of
neutral and dibasic amino acids in Xenopus oocytes. J.
Biol. Chem. 267(22): 15285-8
White, M. F. 1985. The transport of cationic amino acids
across the plasma membrane of mamalian cells. Biochem.
Biophys. Acta. 822:355-74
Whittman, M. Downes, C. P. Keeler, T. Cantley, L. 1988.
Epidermal growth factor receptor stimulates Pi-s kinase.
Nature 332:644-647
Wice, B.M., Trugnan, G., Pinto, M. Rousset, M. Chevalier,
G., Dussaulx, E., Lacroix, B. Zweibaum, A. 1985. The
intracellular accumulation of UDP-N-acetylhexosamines is
concomitant with the inability of human colon cancer
cells to differentiate. J. Biol. Chem. 260(1): 139-46
Zweibaum, A., Triadou, N., Kedinger, M., Augeron, C., Robine,
R. Leon, S., Pinto, M. Rousset, M. Haffen, K. 1983.
Sucrase-isomaltase: a marker of foetal and malignant
epithelial cells of the human colon. Int. J. Cancer.
32(4): 407-12

BIOGRAPHICAL SKETCH
Ming Pan, born on February 5, 1963 in ShaoGuan City of
the GuangDong Province, People's Republic of China. His father
is Jiaan Pan, mother is FengQun Chen. He went to elementary
and middle schools in ShaoGuan city. In 1980, after graduating
from high school, he went to Sun Yat-Sen University of Medical
Sciences studying medicine, specializing in surgery. He
graduated from medical school with a Medical Bachelor (M.B)
degree in 1986. He then worked in the YueBei People's Hospital
in ShaoGuan as a surgeon during 1986-1987. He came to United
States of America in 1987. He entered the Ph.D program in the
Department of physiology, College of Medicine, University of
Florida in May 1988. Under the guidance of Dr. Bruce R.
Steven, he has been studying the amino acid transport
regulation in the small intestine. He has completed all the
degree requirements and successfully defended his
dissertation. He is expected to graduate and receive a Doctor
of Philosophy degree in Physiology in May 1993.
272

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Bruce R. Stevens, Chair
Associate Professor
Physiology
of
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosor
Edward M. Cop
Professor of
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
George
Professoi
Gerencser
of Physiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Michael S. Kilb*
Professor of Bi ochis try
and Molecular Biology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Wiley W. ouba, Jr.
Associate Professor of Surgery
This dissertation was submitted to the Graduate Faculty
of the College of Education and to the Graduate School and was
accepted as partial fulfillment of^the requirements for the
degree of Doctor of Philosophy.
May, 1993
Dean, College of
Medicine
Dean, Graduate School

UNIVERSITYOFFIORIDA
3 1262 08554 8302



271
Wang, H., Kavanaugh, M.P., North, R.A., Kabat, D. 1991.
Cell-surface receptor for ecotropic murine retroviruses
is a basic amino-acid transporter [see comments]. Nature.
352(6337): 729-31
Wells, R.G., Hediger, M.A. 1992a. Cloning of a rat kidney cDNA
that stimulates dibasic and neutral amino acid transport
and has sequence similarity to glucosidases. Proc. Natl.
Acad. Sci. USA. 89(12): 5596-600
Wells, R.G. Lee, W.S., Kanai, Y., Leiden, J.M., Hediger, M.A.
1992b. The 4F2 antigen heavy chain induces uptake of
neutral and dibasic amino acids in Xenopus oocytes. J.
Biol. Chem. 267(22): 15285-8
White, M. F. 1985. The transport of cationic amino acids
across the plasma membrane of mamalian cells. Biochem.
Biophys. Acta. 822:355-74
Whittman, M. Downes, C. P. Keeler, T. Cantley, L. 1988.
Epidermal growth factor receptor stimulates Pi-s kinase.
Nature 332:644-647
Wice, B.M., Trugnan, G., Pinto, M. Rousset, M. Chevalier,
G., Dussaulx, E., Lacroix, B. Zweibaum, A. 1985. The
intracellular accumulation of UDP-N-acetylhexosamines is
concomitant with the inability of human colon cancer
cells to differentiate. J. Biol. Chem. 260(1): 139-46
Zweibaum, A., Triadou, N., Kedinger, M., Augeron, C., Robine,
R. Leon, S., Pinto, M. Rousset, M. Haffen, K. 1983.
Sucrase-isomaltase: a marker of foetal and malignant
epithelial cells of the human colon. Int. J. Cancer.
32(4): 407-12


A -1-1
Arginine uptake, pmole mg min
o
cn
O
O
o
Cn
o
o cn
o o
Control
CHE
H EGF
EGF + CHE
TGFa
TGFa + CHE
300


11
to their hydrophobicity. The order of permeability diffusion
coefficients is phenylalanine > 0-alanine > mannitol > alanine
> MeAIB > proline > glycine > lysine (Stevens et al.,
1982,1984). At high luminal amino acid concentration, the
passive diffusion may be the predominant transport ways in the
intestine. The carrier-mediated transport systems may be the
favored route at lower concentrations.
In contrast to the apical membrane, all the basolateral
membrane amino acid transport systems studied also exist in
other non-epithelial membrane. These include Systems ASC and
A, and Na+-independent Systems y+ and L, plus simple passive
diffusion. The characteristics of these systems are as the
same as those described in the apical membrane.
1.2.4 The Ontogenetic Developments of the Amino Acid Transport
Developmental studies of amino acid transport in the
small intestine of various species has demonstrated that the
timing and class of amino acid transporter appearance differs
not only among the animal species but also at the various
development stages of the same specie (Buddington & Diamond,
1989, 1990) Both herbivores and omnivores prefer high protein
diet in their youth, a period when the absorption of essential
amino acids is high. Because of some amino acids are in higher
demand in adults making these conditionally essential amino
acids. This is the case for arginine in puppies. On the oral-
anal axis, one dramatic change is in colon, whereby adults


Fig. 6-3. The effectof CHX on the chronic TGF^/EGF-induced
System B activity
System B alanine uptake was stimulated by 48 hours of continuous
TGF0' or EGF incubation. CHX (10 juM) in the TGF or EGF medium blocked
the stimulation effects (p < 0.05, n = 12). Data shown were from day 2
cells, with similar results obtained in day 9 cells. 100% alanine uptake
= 0.8 nmole/mg/min.


160
140
120
100
80
60
40
20
0
T
II I I I I L_
1 2 3 4 5 6 7 8 9 10 1 1 12
Incubation time, hours
13
188


CHAPTER 7
THE EFFECTS OF PHORBOL ESTERS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
7.1 Introduction
The tumor promotors phorbol 12-myristate 13-acetate
(TPA), or phorbol 12,13-dibutyrate (PDBU), have long been
known for their effects in promoting mitogenesis and cell
growth. The phorbol ester effect is through a series of an
intracellular cascade processes that is initiated by protein
kinase C activation. The lipophilic phorbol esters diffuse
through the cell membrane as a substitute for DAG in directly
activating the intracellular protein kinase C. Even though
there have been reports that phorbol esters are not specific
protein kinase C activators, and that they may activate other
processes, phorbol esters are widely used to activate protein
kinase C.
Protein kinase C has two subunits: a regulatory subunit,
and a catalytic subunit. Diacylglycerol and phorbol esters,
bind to the regulatory subunit causing a structure change
which activates or inactivates the kinase. Once the protein
kinase C is activated, the catalytic subunit bind to its
substrate and catalyzes it. Both steps are essential for the
biological functions.
223


CHAPTER 8
SUMMARY AND CONCLUSIONS
8.1 SUMMARY
We have studied amino acid transport systems
independently serve alanine or arginine in the apical membrane
of the human intestinal epithelial cell line, Caco-2. We have
investigated the in vitro cellular mechanisms that underlie
the independent regulation of these systems.
The structurally different substrates L-alanine and L-
arginine are transported separately by unique transport
pathways. The pathways serving alanine are: System B, System
L, and simple passive diffusion. The pathways for arginine are
System y+ and simple passive diffusion. The transport
activities of System B, System y+, and System L were each-down
regulated in parallel with advancing Caco-2 cell development
and differentiation. System B and System y+ activities were
each actually trans-stimulated by their own substrate. System
B capacity was chronically up-regulated by its own substrates
through a mechanism that involved protein kinase C and de novo
protein synthesis. Simple passive diffusion of each substrate
was unaffected by any cellular conditions that affected the
carrier-mediated transport.
The peptide growth factors EGF and TGF stimulated System
258


123
4.4.1 The Decrease in Arginine Uptake Activity as Cells Age
The Caco-2 total arginine uptake rates in choline Cl
medium decreased as the cells aged (Fig. 4-2). Furthermore,
the arginine passive permeability diffusion coefficient in
both the undifferentiated day 2 cells and differentiated day
9 cells was the same. The decrease in transport activity with
cell age was therefore due to the non-diffusion portion of
uptake. In the light of the reduced proliferation rates
characteristic of the older cells, the decrease in System y+
activity with the cell age increase may be associated with the
cell's reduced requirement for amino acids.
4.4.2 Classification of Arginine Transport Systems
The kinetic data (Fig. 4-4 & Fig. 4-5) indicated that
there was Na+-independent transport system plus simple passive
diffusion at both cell differentiation stages.
The simple passive diffusion coefficients were constant
at different cell ages, suggesting that the cell aging was not
associated with the diffusion changes.
The amino acid analogue inhibition pattern of the
carrier-mediated arginine uptake was strongly inhibited only
by lysine, ornithine, and histidine. Homoserine in choline Cl
medium has weak inhibition effects, but its inhibition effect
was enhanced by the presence of NaCl in the uptake media. The
neutral amino acids alanine, phenylalanine, leucine did not


Fig. 3-20. Dixon analysis of Na+-dependent alanine uptake with
phenylalanine as inhibitor
Alanine (25 /M, 50 /liM, and 100 juM) uptake was measured
with various concentrations of phenylalanine in uptake media. Non
linear regression of these data intercepted at x axis, indicating
a non-competitive inhibition profile.


[Glutamine], ¡jM
0.4


Fig. 4-10. Dixon analysis of Na*-independent arginine uptake with
homoserine as inhibitor
Arginine (0.5 juM, 5 /zM, and 50 /zM) uptake was measured
with various concentration of homoserine (1 juM -1 mM) in choline
Cl uptake medium. Non-linear regression of these data gave a Kt
of 570 /zM for homoserine.


266
Karasov, W., Solberg, D., Carter, S., Hughes, M., Phan, D.,
Zollman, F., Diamond, J. 1986. Uptake pathways for amino
acids in mouse intestine. Am. J. Physiol. 251(4 Pt 1):
G501-8
Karasov, W.H., Solberg, D.H., Diamond, J.M. 1987. Dependence
of intestinal amino acid uptake on dietary protein or
amino acid levels. Am. J. Physiol. 252(5 Pt 1): G614-25
Kilberg, M.S. 1986. Amino acid transport in eukaryotic cells
and tissues. Federation Pro. 45(10): 2438-2440
Kilberg, M.S., Handlogten, M.E., Christensen, H.N. 1981.
Characteristics of system ASC for transport of neutral
amino acids in the isolated rat hepatocyte. J. Biol.
Chem. 256(7): 3304-12
Kilberg, M.S., Bracy, D.S., Handlogten, M.E. 1986. Substrate
regulation of hepatic system A transport activity after
induction by substrate starvation or glucagon. Fed. Proc.
45(10): 2438-41
Kilberg, M.S, Stevens, B.R., Novak, D.A. 1993. Recent advances
in mammalian amino acid transport. Annu. Rev. Nutr.
13:137-65
Kim, J.W., Closs, E.I., Albritton, L.M., Cunningham, J.M.
1991. Transport of cationic amino acids by the mouse
ecotropic retrovirus receptor [see comments]. Nature.
352(6337): 725-8
Klip, A., Douen, A.G. 1989. Role of kinases in insulin
stimulation of glucose transport. J. Membr. Biol. 111(1):
1-23
Koboyashi, E, Mituta, M, and Suzuki, H. 1989. Calphostin C, a
novel microbial compound, is a highly potent and specific
inhibitor of protein kinase C. Bichem. Biophy. Res. Comm.
159:548-553
Koyama, S.Y., Podolsky, D.K. 1989. Differential expression of
transforming growth factors alpha and beta in rat
intestinal epithelial cells. J. Clin. Invest. 83(5):
1768-73
Laganiere, S., Maestracci, D., Berteloot, A. 1986. Adaptation
of intestinal sugar and amino acid transport to gastric
hyperalimentation. Clin. Invest. Med. 176-85


170
isolated membrane vesicles. Unlike System B, the System y+
activity increased by chronic arginine exposure was not
sensitive to cycloheximide or chelerythrine. The chronic
arginine incubation did not induced a new transporter
associated protein synthesis, and furthermore protein kinase
C was not involved. This is consistent with the findings in
other in vivo intestinal studies that showed that the
intestinal essential lysine/arginine transport was not up-
regulated in a long-term feeding related to the potential
toxic effect of these essential amino acids. The transport
capacity is determined by the factors such as the genetic
hard-wiring or by the cell's needs other than the
environmental availability. The trans-stimulation of the
transporters provides the flexibility for maximum nutrient
extraction at any given time within the safety margin.
5.5 Summary
The System B and System y+ activities are up-regulated
independently and specifically by only the substrates they
transport. The system B is regulated in two phases: an acute
trans-stimulation phase, and a chronic de novo protein
synthesis- and protein kinase C-dependent phase. The System
y+ activity is only regulated by a substrate trans
stimulation. The difference between the System B and System
y+ substrate regulation may reflect the intrinsic properties
of the transported substrates.


0
o
§


Alanine uptake, nmole mg min
o o ^ ro
b bi b bi
*8T


200
phosphorylation of other receptor's substrates. The tyrosine
kinase activity is essential for the EGF receptor biological
activity (Chen et al., 1987; Glenney et al., 1988; Honnerger
et al., 1987; Moolenaar et al., 1988)). The EGF receptor
substrates include; PLC-yl (Margolis et al., 1989;
Meisenhelder et al., 1989; Wahl et al., 1989), GAP (Ellis et
al., 1990; Molloy et al., 1990), lipocortin I (Fava & Cohen,
1984), c-erbB-2 (Akiyama et al., 1988; Stern & Kemps, 1988),
and PI-3 kinase (Whittman et al., 1988).
One action of the activated EGF receptor kinase is to
phosphorylate the phospholipase C (PLC) which hydrolyzes the
phosphatidylinositol-4,5-biphosphate to produce inositol-
1, 4 5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 and
DAG stimulate the increases in the intracellular free Ca2+ and
protein kinase C activity, respectively. Protein kinase C then
causes various biological responses, mainly gene-expressed
related mechanisms through a series unknown pathways.
The EGF biological responses are of two types. The first
concerns a rapid signal transduction via the receptor tyrosine
auto-phosphorylation, acting in concert with the calcium
released from the intracellular stores, and leading to the
phospholinositide hydrolysis, with subsequent formation of
diacylglycerol (Carpenter et al., 1979; Moolenaar et al.,
1984; Morris et al., 1984; Johnson et al., 1986; Wahl &
Carpenter, 1988a; Smith et al., 1983; Serreo, 1987). The
second mechanism involves long term biological responses such


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2
By
Ming Pan
May 1993
Chairperson: Bruce R. Stevens
Major Department: Physiology
Membrane transporter systems serving arginine (Na+-
independent system y+) and alanine (Na+-dependent system B)
were investigated in the human intestinal Caco-2 cell line.
The uptake kinetics were different for each transport system.
For each system, the V>ax was greater in undifferentiated cells
compared to differentiated cells, while the Kra values were
each unaffected by cell differentiation status. Amino acid
substrates unique to System y+ acutely stimulated only system
y+ activity, while substrates unique to system B only
stimulated system B activity. For each transport system, the
ranking of amino acid stimulation was directly correlated with
the degree of competitive analogue inhibition (assessed by
Dixon analysis). The prolonged substrate induction of system
B activity, but not system y+ activity, was prevented by the
protein synthesis inhibitor cycloheximide. Peptide growth
vi


6.0
Y 5.0
c
tac
s
0
O
4.0
3.0
2.0
ct
<
-3 1.0
O o
o

o
o
2
day 2 TPA

Day 2 W/o TPA
Day 9 TPA
Day 9 w/o TPA
o
i i i
4 6 8
[Ala], 10~4M
O
10
247


261
Amino Acid Transport Regulation
in Caco-2 Cells


34
by adding 1 ml IN NaCl to the cells. After overnight
extraction (continuous shaking) a 200 fil aliquot of NaOH
extract was added to 10 ml Liquiscint scintillation fluid
which was then neutralized with 200 nl glacial acetic acid.
Radioactivity (counts per minute, CPM) was measured in the
Beckman scintillation counter with quench normalized using the
"H-number" method. Because the nonspecifically adhering label
was < 1 % of the total counts per minute (CPM) associated with
the cell uptake, the double label 14C-inulin space measurement
was not performed for subsequent experiments. The protein
content in the NaOH extract was measured by the Bio-Rad
protein assay. The rate of amino acid uptake activity was
equivalent to the initial linear slope of the uptake time
course. All subsequent uptake experiments were conducted
within the linear period at a uptake period < 10 minutes, with
the 0 minute points serving as blanks. The amino acid uptake
rates are expressed as nmole amino acid/mg cell
protein/minute. The sodium-dependent alanine transport is
equivalent to measuring total alanine uptake in NaCl buffer
and in choline Cl buffer, and then subtracting the difference.
Bio-Rad Protein Assay
The Bio-Rad protein assay reagent was diluted 1 : 4 with
de-ionized water. Fifty microliter cell/NaOH mixture was added
into the diluted reagent (5 ml), with 50 zl 1.4 mg/ml standard
y-globulin as the protein reference (plus additional 50 nl 1


Fig. 5-9. System v* activity in cells incubated L-arginine or
D-arginine
System y+ arginine (5 /liM) uptake was measured in cells (2
days old) which had been incubated in salt medium, 1 mM arginine
in salt, or 1 mM D-arginine in salt for various periods of time
(1 12 hours). The System y+ arginine uptake was greatly
stimulated by arginine incubation, D-arginine marginally increased
the arginine uptake. Similar result were also obtained in 9 day
s old cells.


202
Insulin, glucagon, adenosine induce System A transport
activity via a transcription and translation mechanism in
hepatocyte (La Cam & Freychet, 1978; Cariappa & Kilberg, 1990;
Kiyokawa et al., 1991).
Despite the understanding of many EGF biological
activities, the effect of EGF on the intestinal absorption of
amino acids has not been explored.
6.2 Methods And Materials
6.2.1 Methods
6.2.1.1 Treatments with EGF/TGF<*. cvcloheximide,
chelervthrine, and H-7/calphostin C
The protocols to treat Caco-2 cells were basically the
same for each of the various agents. Serum-free DMEM was
prepared by supplementing Sigma's DMEM with non-essential
amino acids, penicillin and streptomycin, but not serum.
Treatments began with a 2 hour preincubation of cells in the
depletion medium. The cells were then exposed to various
buffers for various times described below. Every 6 hours,
buffers were removed and replenished with the same buffers to
ensure a constant agent concentration and to eliminate a
possible autocrines released into the medium from the cells.
Caco-2 cells remained healthy during at least 48 hours
exposure to our depletion medium.
Experiments with EGF/TGF. The cell treatments were: (i)
control group, serum-free DMEM plus the same amount of 0.1 M


265
Grasset, E., Pinto, M. Dussaulx, E., Zweibauxn, A., Desjeux,
J.F. 1984. Epithelial properties of human colonic
carcinoma cell line Caco-2: electrical parameters. Am. J.
Physiol. 247(3 Pt 1): C260-7
Grasset, E., Bernabeu, J., Pinto, M. 1985. Epithelial
properties of human colonic carcinoma cell line Caco-2:
effect of secretagogues. Am. J. Physiol. 248(5 Pt 1) :
C410-8
Herbert, J. M, Smith, D. B, and Satoma, G. 1990. Chelerythrine
is a potent and specific inhibitor of protein kinase C.
Bichem. Biophys. Res. Comm. 172:993-999.
Hernandez-Sotomayor, S.M.T., Carpenter, G. 1992. Epidermal
growth factor receptor:elements of intracellular
communication. J. Memb. Biol. 128:81-89.
Hidalgo, I.J., Kato, A., Borchardt. 1989. Binding of epidermal
growth factor by human colon carcinoma cell (Caco-2)
monolayers. Biochim. Biophy. Res. Comm. 160(1):317-324
Honegger, A.M., Dull, T.J., Felder, S., Van Obberghen, E.,
Bellot, F., Szapary, D., Schmidt, A., Ullrich, A.,
Schlessinger, J. 1987a. Point mutation at the ATP binding
site of EGF receptor abolishes protein-tyrosine kinase
activity and alters cellular routing. Cell. 51(2):
199-209
Honegger, A.M., Szapary, D., Schmidt, A., Lyall, R. Van
Obberghen, E., Dull, T.J., Ullrich, A., Schlessinger,
J. A. 1987b. Mutant epidermal growth factor receptor with
defective protein tyrosine kinase is unable to stimulate
proto-oncogene expression and DNA synthesis. Mol. Cell.
Biol. 7(12): 4568-71
Hopfer, U. 1987. Membrane transport mechanism for hexoses and
amino acids in the small intestine. In: Physiology of the
Gastrointestinal Tract, edited by Johnson-LR. Raven
Press, New York, pp 1499-1526
Jumarie, C., Malo, C. 1991. Caco-2 cells cultured in
serum-free medium as a model for the study of enterocytic
differentiation in vitro. J. Cell. Physiol. 149(1): 24-33
Karasov, W.H., Diamond, J.M. 1983. Adaptive regulation of
sugar and amino acid transport by vertebrate intestine.
Am. J. Physiol. 245(4): G443-62


260
8.2 Conclusions
(1) Caco-2 epithelial differentiation status is
associated with regulation of amino acid transport.
(2) Amino acid transporters System B and System y+
independently serve alanine and arginine, and are regulated
independently.
(3) Amino acid substrates up-regulate their own
transporters' activities via trans-stimulation, or chronically
by a mechanism involving de novo protein synthesis.
(4) Chronic up-regulation of System B or System y+
capacities by de novo protein synthesis is activated by
EGF/TGF acting through a protein kinase C pathway.
The Proposed Amino Acid Transport Regulation Mechanism in
Caco-2 Cells;


Alanine uptake, nmole mg min
Cell age, days
30


158
5.2 Methods and Materials
Caco-2 cells were incubated in depletion medium for 2
hours, and then incubated in the same medium containing
various concentration of alanine, arginine, or other amino
acids cycloheximide (10 iM or 50 /xM) for various windows of
time (30 seconds up to 48 hours) in the 37"C incubator. The
incubation medium was changed every 8 hours to ensure that
amino acid concentration were constant, and to remove possible
build up of autocrine.
The depletion medium contained 0.265 g/L CaCl2, 0.0001
g/L Ferric Nitrate, 0.09767 g/L MgS04, 0.4 g/1 NaHCOs, 6.4 g/L
NaCl, 0.109 g/L NaH2P04, 4.5 g/L glucose, 0.0159 g/L phenol
Red-Na, 0.004 g/L choline chloride, 0.004 g/L folic acid,
0.0072 g/L myo-inositol, 0.004 g/L niacinamide, 0.004 g/L D-
pantothenic acid, 0.004 g/L pyridoxaHC1, 0.0004 g/L
riboflavin, and 0.004 g/L thiamineHC1.
5.3 Results
5.3.1 System B Activity Decrease in Starved Caco-2 Cells
The system B alanine uptake rate declined as early as 15
minutes following depletion medium incubation, and reached the
lowest level at 2 hours, where they remained steady for at
least 48 hours. The system B alanine uptake rate in the Caco-
2 cells which were incubated in depletion medium plus 1 mM


o
o
% of Alanine Transport
(Na+ -
- dependent)
N>
O
O
400
300
h
&
Mannitol
Phe
AOA
Pro
MeAlB
Arg
His
Gly
Val
Thr
Cys+DTT
Ala
GLN
Ser
DMEM
Ala+AOA
VLX
500


/[Arg], /Liter
JArg, nmole mg min
C_
>
"1
02)
3
lQ
3
Z)
O O N) N)
en en cn
861
3.0


49
3.3.7 The Analogue Cross-inhibition Pattern
The 50 /M [3H]-alanine uptake rates were measured in
media containing 137 mM NaCl and 137 mM choline Cl plus 5 mM
single amino acid analogues (natural amino acids, BCH, MeAIB,
AIB, and /3-alanine with 5 mM mannitol as control) .
For the Na+-independent alanine transport system, alanine
uptake was strongly inhibited by phenylalanine, alanine,
leucine, threonine, serine, glutamine, asparagine, cysteine,
and BCH, and weakly by MeAIB, AIB, and glycine. Lysine, and
glucose did not inhibit (Fig. 3-10 & Fig. 3-11).
For the Na+-dependent alanine transport system, the
uptake activity was inhibited by 5 mM amino acid analogues
(natural AAs plus BCH, MeAIB, and /3-alanine) was shown in Fig.
3-12. The Na+-dependent [3H] alanine transport was strongly
inhibited by threonine, glutamine, serine, cysteine, and
asparagine. Weaker inhibition was elicited by glycine,
phenylalanine, leucine and the bicyclo amino acid BCH. MeAIB
and cationic amino acids elicited <10% inhibition. Dixon
inhibition analyses indicated that the glutamine inhibition
was classic competitive inhibition, while the MeAIB affect was
un-competitive (Figs. 3-13 3-15). Proline, glycine, and
phenylalanine gave high K values (Figs. 3-16 3-21).
The pattern and degree of amino acid analogue inhibition
of the Na+-dependent alanine uptake was identical at both cell
ages, suggesting that the same transporter system was
operative regardless of the cell age (Fig. 3-22).


Fig. 4-14. Arginine basal to apical trans-cellular uptake
Arginine (5 /liM) uptake from the basal side to the apical
side was measured at various time periods.


13
development by systemically circulating factors like hormones,
or by the luminal composition like transporter substrates. The
intestinal apical membrane amino acid transport regulation by
transport substrate has been studied in vivo as described
below.
Unlike other internal organs, in vivo studies show that
the activities of the intestinal amino acid transporters are
up-regulated by the dietary substrates they transport
(Stevens, 1992a,b; Salloum et al 1990; Sharrer et al, 1981;
Stein et al, 1987; Ferraris et al, 1988a,b; Diamond & Karasov,
1987; Ferraris & Diamond, 1989; Diamond, 1991). The
substrates' unique pattern of up-regulation, their amplitude,
and selectivity of each system's activity indicated that
individual amino acid transporters are regulated independently
by dietary substrates. Non-essential caloric amino acids up-
regulate their transporter activities with increasing
substrate. The essential, but potentially toxic amino acids
regulate their transporter systems' activities in a different
pattern. That is, at lower substrate concentration, the
transporter activity decreases as substrate concentration
increase; at higher concentration the transport activity
increases as substrate concentration increases. A similar
pattern is observed for sugar and dietary carbohydrate. The
transport of non-essential amino acids is increased more by
the dietary protein than that of the more toxic essential
amino acids. This supports the notion that the absorption of


52
uptake rates were several fold higher in the day 2 cells (Fig.
3-2) The alanine uptake change over the cell ages was
therefore only a portion of saturable carrier-mediated uptake.
The membrane permeability was then unlikely to be involved in
the regulation mechanism.
As discussed above, the alanine uptake rates decreased
with advancing cell age, while Na/glucose cotransport
increased with cell age (Fig. 3-23). This opposite direction
of transport activity suggests that the function of alanine
and glucose were not the same in cell development. Alanine was
not solely for caloric purpose. In the light of the cell
proliferation rate decrease with the cell age increase (Fig.
3-3) the reduced alanine uptake may be due to the lowered
requirement for amino acids, but not for glucose. These data
also indicated that the Na/glucose and alanine uptake in Caco-
2 cells were independently regulated by the cell
differentiation and development.
3.4.2 Classification of the Alanine Transport Systems
There were three alanine transport pathways in Caco-2
monolayers for alanine at both cell stages (the
undifferentiated and the differentiated stages): a simple
passive diffusion, a Na+-independent system, and a Na+-
dependent system.
3.4.2.1 Simple Passive diffusion
The same passive permeability coefficient measured in


Cell age, days
Thymidine 10 CPM (mg protein)
^Moj-f^cncri^joo
ooooooooo


Incubation time, minutes
172


en
o
Alanine uptake, nmole mg min
- M gj <_n
o o o o o o
Contri
CHX
Ala
Ala
+CHX


2.0
en
E
_CL)
o
E
c
O
Q_
Z5
CD
C
c
o
<
Basal to apical
NaCL
O Choline CL
0.5
0
O
10
0.0 0
0
Time,
o
O
o
o
15 20 25 30
minutes
114


269
Salloum, R.M., Souba, W.W., Fernandez, A., Stevens, B.R. 1990.
Dietary modulation of small intestinal glutamine
transport in intestinal brush border membrane vesicles of
rats. J. Surg. Res. 48(6): 635-8
Salloum, R.M., Stevens, B.R., Souba, W.W. 1991. Adaptive
regulation of brush-border amino acid transport in a
chronic excluded jejunal limb. Am. J. Physiol. 261(1 Pt
1): G22-7
Souba, W.W., Klimberg, V.S., Plumley, D.A., Salloum, R.M,
Flynn, T.C., Bland, K.I., Copeland, E.M. 3d. 1990. The
role of glutamine in maintaining a healthy gut and
supporting the metabolic response to injury and
infection. J. Surg. Res. 383-91
Souba, W. W., Pan, M., and Stevens, B. R. 1992. Kinetics of
the Sodium-dependent glutamine transporter in human
intestinal cell confluent monolayers. Bioch. Biophy. Res.
Comm. 188 (2) :746-753
Stein, E.D., Chang, S.D., Diamond, J.M. 1987. Comparison of
different dietary amino acids as inducers of intestinal
amino acid transport. Am. J. Physiol. 252(5 Pt 1) :
G626-35
Stelzner, M. Buddington, R.K., Phillips, J.D., Diamond, J.M.,
Fonkalsrud, E.W. 1990a. Changes in mucosal nutrient
transport in small and large ileal reservoirs after
endorectal ileal pullthrough. J. Surg. Res. 49(4): 344-55
Stelzner, M. Fonkalsrud, E.W., Buddington, R.K., Phillips,
J.D., Diamond, J.M. 1990b. Adaptive changes in ileal
mucosal nutrient transport following colectomy and
endorectal ileal pull-through with ileal reservoir. Arch.
Surg. 125(5): 586-90
Stern, D. F. and Kemps, M. P. 1988. EGF-stimulated tyrosine
phosphorylation of pl85neu: a potential model for receptor
interactions. EMBO J. 7:995-1001
Stevens, B.R. Amino acid transport in intestine. 1992a. In:
Mammalian Amino Acid Transport, edited by Kilberg, M.S.
and Haussinger, D., Plenum Press, New York, 149-163
Stevens, B.R. 1992b. Vertebrate intestine apical membrane
mechanisms of organic nutrient transport. Am. J. Physiol.
263:R458-63


117
The cloning of system y+ cDNA has opened a new chapter
for membrane amino acid transport study. The discovered system
y+ cDNA also codes for the murine ecotropic retrovirus
receptor on cell plasma membrane. System y+ cDNA was
successfully expressed in oocytes. Injection of this cDNA into
oocyte results in a increase of typical system y+ activity
(Kim et al., 1991; Wang et al., 1991).
The cDNA encoding NAA/D2, rBAT and 4F2 peptides from rat
or rabbit kidney have been expressed in oocytes and apparently
increase activity of endogenous System b0,+ B0,+ activities.
These cDNA fragments have been suggested to encode possible
regulatory subunits of the system y+, b0,+ or B0,+ (Bertrn et
al., 1992; Magagnin et al., 1992; Tates et al., 1992; Wells
et al., 1992a,b).
To examine the arginine transport systems in the Caco-2
cells, we conducted a series of phenomenological studies to
define the arginine transport systems. At the time of this
study, we did not possess the system cDNA probes for y+, rBAt,
NAA/D2, or 4F2.
4.2 Methods and Materials
4.2.1 Methods
The [3H]-arginine uptake experiments were performed in
both the pre-confluent (day 2-3) cells and the confluent
(day 8 9) cells. The basic uptake procedures were as


Fig. 3-13. Dixon analysis of Na+-dependent alanine uptake with
glutamine as inhibitor
Alanine (25 juM, 50 /M, and 100 /M) uptake in NaCl and
choline Cl media was measured with various concentration of
glutamine (10 /M 5 mM) presented in uptake media. The dixon
plot gave a Ki of 35 /M glutamine.


Fig. 7-6. The effects of chelrvthrine on the TPA-induced System
B activity
System B alanine (50 fM) uptake was measured in day
9 cells which had been incubated 0.5 /M TPA 6.6
chelerythrine for 24 hours. The TPA stimulation of alanine uptake
was blocked by the CHE (p < 0.05, n = 6).


Fig. 3-14. Replot of the slopes of Dixon plot with glutamine as
inhibitor
The slopes of dixon plot shown at Fig 3-13 were shown as
a function of (corresponding alanine concentrations)'1. Non-linear
regression of these data intercepted 0. The combination of Fig. 3-
13 and this figure indicated that glutamine was a competitive
inhibitor for System B.


209
manufactured by Sigma Co as H-7 did not influence the TGF<*/EGF
effect on System B and System y+ activities. The phospholipase
Cy (PLC) was likely a major intermediate phosphorylated by
TGF/EGF in the System B and System y+ transport activation.
PLC hydrolysizes phosphatidylinositol-4,5-biphosphate (PIP2) ,
generating the intracellular second messenger diacylglycerol
(DAG) which is an endogenous activator of protein kinase C.
Further study of the PIP2 level, diacylglycerol level, and the
inositol hydrolysis in cells will provide the information
concerning details of the pathways. It is still unclear if the
PLC pathway is the only pathway for this stimulation.
Because the protein kinase C inhibitors also inhibited
general protein synthesis, it is likely that the protein
kinase C activation precedes the de novo protein synthesis
associated with the System B and System y+ activation.
It is notable that the Na+-independent alanine System L
transport was not stimulated by the TGF/EGF incubation. In
fact, due to the stimulation effect of TGF/EGF on cell
protein synthesis, the System L activity per mg protein
actually decreased. These findings support the notion that
System B and System y+ activation is a selective event.
Unlike the transport substrate regulation we discussed in
the preceding chapter, TGF/EGF stimulated both the System B
and System y+ transport activities. This phenomenon may be
associated with the stimulation effect of TGF/EGF on the
epithelial proliferation, cell growth, or mitogenesis.


42
is distinct from System A in that System ASC does not
transport MeAIB. System ASC is also found in many tissue.
System ASC reportedly exists at guinea pig intestinal
epithelial apical membrane (Hayashi et al., 1980).
System B0,+, first described in mouse blastocytes (Van
Winkle et al., 1985), is a Na+-dependent transport system that
transports both the cationic and neutral amino acids. System
B0,+ is expressed in many tissues.
The strictly Na+-dependent transport System B exclusively
found in the apical membrane of the epithelial cells. It was
first described in rabbit jejunum apical membrane vesicles as
a broadly selective system serving neutral amino acids
(Stevens et al., 1982, 1984, Stevens, 1992a,b). It was
originally named System Neutral Brush Border (NBB), and later
was renamed as "System B" (Stevens, 1992a). System B substrate
selectivity is similar to the System B0,+ in that both are Na+-
dependent, as it possesses a broad selectivity for most
dipolar amino acids. Both Systems B and B0,+ interact with the
bicyclo-amino acids 2-amino-2-norbornanecar-boxylic acid (BCH)
or BCO. However, System B 0,+ is inhibited by cationic amino
acids such as lysine and arginine, while System B is not
interactive with cationic amino acids. System B may possible
be a variant of System B0,+.
The Na+-independent System L was shown to exist in the
apical and basolateral membrane of many cell types. System L
is a broadly selective system serving neutral amino acids,


Arginine uptake, per cent
-* > M NJ
cn o cn o cn
o o o o o o
300


Fig. 4-4. Arginine uptake kinetics
Arginine (0.1 /M 1 mM) uptake was measured in choline
Cl medium in day 2 and day 9 cells. The total uptake rates at
each arginine concentration were showed as a function of arginine
concentration. The shape of the kinetic curves indicated the
existence of both non-saturable and saturable components.


Fig. 5-8. The effect of cvcloheximide on the chronic alanine-
induced System B activity
System B alanine (50 juM) uptake was measured in cells (2
days old) which had been incubated in salt medium (with or without
10 mM alanine) 10 juM CHX for 24 hours. The System B alanine
uptake was stimulated by alanine incubation (p < 0.05, n = 6),
and this stimulation was blocked by CHX (p < 0.05, n = 6).
Similar results were obtained in 9 days old cells.


Uptake (10 mol/mg/hr)
o 4 8 12 16 20 24
Incubation Time (hours)
235


-1 -1
Arginine uptake, pmole mg min
o
o
ho
o
Ul
O
O
i i 1
Control
LArg
DArg
<>WOO^^
L-Lys
D Lys
Orn
Homoser
Homeser (Na)
His
OH
Ala
,0£KXXXXXXXXXX>CKXXXXXXXX) BCH
<> Pro
Phy
^XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX1
Asp
Thr
4WXX><^XXX^^
Val
<>ww<^^
Ser
Leu
^><><><><><^^
Glu
4^^^
Met
Try
1 1 1
LZI


18
encourage regulation studies.
1.2.7 The Effects of Peptide Growth Factors in the Small
Intestine
As members of the peptide growth factors family,
Epidermal Growth Factor (EGF) and/or Transforming Growth
Factor (TGF) each stimulate cell proliferation, protein
synthesis, and cell differentiation in many cell types
including the intestinal epithelial cells (Morrisset &
Solomon, 1991; Carpenter & Wahl, 1990).
EGF is a 53 amino acid polypeptide, while TGF is a 48
amino acid peptide. TGF is structurally and biological
functionally similar to the that of EGF. EGF is normally
present in the intestine lumen from endogenous secretions from
the salivary glands, the small intestinal Brunner's glands,
autocrine/paracrine sources from the mucosa, or from exogenous
sources such as milk and colostrum (Gaull et al.,1985;
Britton, 1988; Potter, 1989). The sites for the EGF secretion
to blood stream is unknown.
EGF and/or TGF binds to the same EGF receptor in the
plasma membrane, which is a member of the tyrosine kinase
receptor family. The activated growth factor-receptor complex
immediately phosphorylates the receptor itself and
phosphorylates other substrates such as erb B2, ras oncogen,
polyoma middle T antigen, or phospholipase C (PLC). The
activated PLC alters inositol phospholipid metabolism leading
to a elevated level of diacylglycerol (DAG) (Berridge, 1985;


210
TGF/EGF increased the needs of cells for amino acids for
their growth.
6.5 Summary
Prolonged, continuous exposure to peptide growth factors
TGF or EGF stimulates System B and System y+ activities in
Caco-2 cell in both the undifferentiated and differentiated
states. The stimulatory effect involves a de novo protein
synthesis process. Whether this involves the transporter
protein or a regulatory protein is not clear. The
intracellular protein kinase C activation is involved in the
pathway of TGF/EGF activation of the System B and System y+
transport activities, thereby suggesting the possibility of a
role for PLC-y phosphorylation.


Fig. 3-25. Alanine apical to basal or apical to cytosol uptake in
cells grown on filters
The total amount of 50 /jM alanine transported from apical
side to basal side, and apical side to cytosol in 30 minute was
measured.


CHAPTER 6
THE EFFECTS OF PEPTIDE GROWTH FACTORS ON
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES
6.1 Introduction
Epidermal growth factor (EGF), is a member of the growth
factor family, which has been intensively studied over the
past 30 years (Hernandez-Sotomayor & Carpenter, 1992). The
protein structure, gene expression, biological function, and
the molecular regulation of EGF and the EGF receptor are well
understood. Transforming growth factor-alpha (TGF) is
structurally similar to EGF (Montelione et al., 1988, 1989;
Caver et al., 1986; Mayo et al., 1989). The structure of EGF
and TGF<* is reported to be related to their functions
(Capenter & Wahl, 1990).
EGF/TGF command their function through a binding to the
EGF membrane receptor. The EGF receptor is a glycoprotein
composed of three major domains: an extracellular hormone
binding domain, a hydrophobic transmembrane region, and a
cytoplasmic domain. TGF also binds to the same EGF receptor.
EGF receptor belongs to the tyrosine kinase family. The
EGF/TGF binding to the EGF receptor induces a rapid
reversible changes in the receptor tyrosine kinase activity
causing an auto-phosphorylation of the EGF receptor and the
199


125
the same System y+ in both the day 2 and day 9 cells. The
kinetic parameters (Fig. 4-5) strongly suggested that the
change in transport capacity during cell development was
likely due to the number of copies of functioning transport
units in the apical membrane (per cell mass) rather than
modification of characteristics of existing transporters.
The arginine uptake measurements in the Caco-2 monolayer
grown on the porous filters showed that the majority of
uptaked arginine accumulated inside the cells. Arginine exits
from the cytosol across either the basal membrane or apical
membrane to the extracellular media at a much slow rate than
the arginine transport across the membrane from outside to
inside rates.
4.5 Summary
Arginine is transported in Caco-2 cells by passive
diffusion and System y+. System y+ behaves with the same
kinetic characteristics operative in both the undifferentiated
and differentiated states. The System y+ capacity is down-
regulated during the cell development, while the diffusion
coefficient is not affected. The system y+ activity decrease
is coincident with the declining cell proliferation rate. The
decrease in System y+ activity is likely caused by the
decrease of number of copies of functional transporter units,
rather than the modification of existing transport affinity
for substrate.


19
Edelinan et al, 1987; Klip & Douen, 1989), which activates
intracellular protein kinase C. PK-C activates a series of
unresolved mechanisms that ultimately result in cell division,
proliferation and differentiation (Saier et al., 1988).
EGF receptors appear at both the luminal and basolateral
membranes at a density gradient greater in immature crypt
cells and less dense in villous enterocytes along the crypt ->
villous axis (Hidalgo et al., 1989). This parallels the high
proliferation rate in undifferentiated crypt cells (Pamukcum
& Owens, 1991). Two-thirds of the EGF receptors are reportedly
in the basolateral membrane (Reviewed by Brand, 1990).
EGF/TGF stimulate small intestinal epithelial proliferation,
protein synthesis, and crypt cell maturation and migration
toward villous tip cells. Recently, EGF receptor mRNA was
identified in developing intestinal epithelial cells (Koyama
& Podolsky, 1989). The EGF receptors reportedly existed at the
apical and basolateral membranes of the human intestinal
epithelial Caco-2 cell line (Hidalgo et al., 1989), with
higher density in the undifferentiated cells compared to the
differentiated cells. Two-thirds of the receptors expressed at
the basolateral membrane. The K, of the EGF receptors is 0.67
nM in Caco-2 cells (Hidalgo et al., 1989). Experiment data in
our laboratory indicate that functionally EGF or TGF each
stimulates Caco-2 cell alanine and arginine transporter
activities with similar potency when applied to either brush
border or basolateral surfaces.


41
represents a minor pathway in intestine (described below).
The major membrane transport Systems A, ASC, B0,+, B, L,
and asc for the dipolar amino acid L-alanine have been
intensively studied in various tissues. (Oxender et al., 1963;
Christensen et al., 1963; Stevens et al., 1982; Van Winkle et
al., 1985, 1987, 1988; Van Winkle & Campione, 1990; Kilberg et
al, 1993; Stevens, 1992a,b). Among these alanine transport
systems, only System B was originally described as an unique
in intestinal epithelial cell transporter (Stevens et al.,
1982, 1984). The others systems were first described for non-
epithelial cells (Stevens, 1992a,b).
System A is a strictly Na+-dependent system which is
broadly selective for most dipolar (neutral) amino acids
(Christensen et al., 1963). System A is found in many tissue
membranes. One specific feature of System A is that the non-
metabolized MeAIB is a specific substrates for System A. AIB
serves as less specific substrate. System A regulatory
properties have been intensively investigated in hepatocytes
and other tissue. Several regulation mechanisms of System A
activity were proposed (Kilberg, 1986; Engleberg, 1986; Dawson
& Cook, 1987). System A reportedly exists at the apical and
basolateral membrane of guinea pig intestinal epithelia
(Hayashi et al., 1980; Del Castillo et al., 1991).
System ASC is another Na+-dependent transport system
serving 3- and 4-carbon neutral amino acids, exemplified by
alanine, serine and cysteine (Kilberg et al, 1981). System ASC


INDEPENDENT REGULATION OF ALANINE AND ARGININE TRANSPORT
IN HUMAN INTESTINAL EPITHELIAL CELL LINE CACO-2
By
MING PAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993


Fig. 3-11. Na*-independent alanine uptake inhibition pattern in day
9 cells


Alanine uptake, nmole mg protein
2.0
1 1 1
Apical to basal
NaCI
O Choline Cl
1
1
o
1
o

o

O

i
)
o

-

k 1 1 1
1
1
l_
1.5
1.0
0.5 -
0.0'
o
10 15 20
Time, minutes
25 30
106


-14
-1
MeAlB uptake, 10 mole mg min
O O 1 N)
b ai en
Control
MeAlB
Alanine
Serine
j L
281


Fig. 3-6. Na*-activation of alanine uptake
The uptake of total alanine (50 /M) in day 2 and day 9
cells was measured in media containing various NaCl concentrations
([NaCl] = 0 137 mM, choline Cl substituted NaCl). The Non-linear
regression of these data gave the same Na+-activation Hill
coefficient of n = 1 for both the day 2 and day 9 cells.


12
colon only transports electrolytes and water, but neonatal and
fetal colon possesses many amino acid transport systems. On
the crypt-villous axis, the youth cope with the higher amino
acid transport load by creating a large surface area that
increase nutrient uptake non-specifically. The enterocyte
turnover rate of the villus tip is slower, the crypt cell
migration is greater, and the crypt cells possess transport
ability, all of which contributes to the increased mass of
intestine possessing transport activity. Two mechanisms were
proposed (Buddington & Diamond, 1989, 1990) to explain the
control of amino acid transporter expression during animal
development: (1) an external control mechanism by dietary
changes in substrate or by some growth factors in diet (e.g.
epidermal growth factor stimulating transport by enterocyte,
paracrine/autocrines, secreted by salivary gland or Bruners
glands or from food source such as milk) ; (2) an internal
genetically hard-wired control mechanism that controls change
independent of external environment.
1.2.5 Regulation of Intestinal Amino Acid Transport
The intestinal membrane amino acid transport systems are
regulated by various factors, such as the animal development
regulation discussed above, certain physiological states like
pregnancy, or certain pathological states like disease
diabetes, hyperthyroidism. Much attention was given to the
regulation of transport activity at certain stage of


53
both the day 2 and day 9 cells suggested that the Caco-2 cell
development did not alter the membrane permeability to
alanine. The diffusion rates of alanine across the cell
membrane at certain alanine concentrations were the same at
either cell ages. The passive diffusion contribution at 50 ;xM
alanine uptake was minimal, less than 1% of total uptake in
NaCl. At higher alanine concentrations, the passive diffusion
contribution was greater. At [alanine] = 5 mM, passive
diffusion contributes 90% of total alanine uptake in NaCl.
3.4.2.2 Na*-independent transport system is System L
One saturable Na+-independent alanine transport system
existed in both the day 2 and day 9 cells. The observed
activity decreased with the advancing cell age (Fig. 3-2). The
activity decrease was coincident with the cell proliferation
rates (Fig. 3-3). The activity was possibly regulated by cell
proliferation requirements.
The Na+-independent alanine transport kinetics in both
the 2 day old and 9 day old cells revealed that the transport
activity VBax was higher in the day 2 cells. The transport
apparent affinity K, was the same at both cell ages. These
kinetic parameters strongly indicate that the transport
capacity was greater in the day 2 cells; the activity change
was a Vaax effect, not K, effect, suggesting the presence of
same transport system in both differentiated and
undifferentiated states. The activity change was likely a
change of copies of functional transport units in the membrane


229
regardless the age and the TPA treatment (Fig. 7-8).
7.3.8 Phorbol Ester Up-Regulated the System v* Uptake Activity
The Caco-2 cells were pre-incubated with 0.5 /xM TPA in
serum-free medium for various length of time (0-24 hours)
prior to the uptake experiments. The System y+ activity was
stimulated by TPA only after a prolonged incubation; at least
8 hours were required for the effect. The TPA stimulation
effect increased steadily up to at least 24 hours. A 24 hours
TPA incubation period was chosen for the subsequent TPA
experiments.
7.3.9 The TPA Pulse Effect on System v* Activity
The Caco-2 cells were pre-incubated with 0.5 /xM TPA in
the serum-free medium for various periods of time (0-2
hours), then re-incubated in the serum-free medium (lacking
TPA) for the remaining periods prior to the uptake
measurements. The total incubation time was 24 hours,
including the pulse TPA incubation plus the following non-TPA
incubations. The TPA pulse treatments alone did not affect the
system y+ activity.
7.3.10 TPA/s Effect on the System v* Activity at Various Cell
Ages
The Caco-2 cells (1 day old 14 day old) were pre
treated with 0.5 /xM TPA in the serum-free medium for 24 hours
prior to the arginine uptake experiments. The 5 /xM arginine


263
Carpenter, G., King, L. Jr., Cohen, S. 1979. Rapid enhancement
of protein phosphorylation in A-431 cell membrane
preparations by epidermal growth factor. J. Biol. Chem
254:4884-4891
Carpenter, G., Wahl, M.I. 1990. The epidermal growth factor
family. In: Peptide growth factors and their receptors I,
edited by Sporn, M.B. and Roberts, A.B, SpringerVerlag,
Germany. pp69-172
Carver, J. A, Cooke, R. M. Esposito, G., Campbell, I. D. ,
Gregory, H. and Sheard, B. 1986. A high resolution H NMR
study of the solution structure of human epidermal growth
factor, FEBS Lett 205:77-81.
Chen, W.S., Lazar, C.S., Poenie, M. Tsien, R.Y., Gill, G.N.,
Rosenfeld, M.G. 1987. Requirement for intrinsic protein
tyrosine kinase in the immediate and late actions of the
EGF receptor. Nature. Aug 27-Sep 2; 328(6133): 820-3
Christensen, H.N. 1984. Organic ion transport during seven
decades. Biochim. Biophys. Acta 779:225-69
Christensen, H.N. 1985. On the strategy of kinetic
discrimination of amino acid transport systems. J. Membr.
Biol. 84:97-103
Christensen, H.N. 1990. Role of amino acid transport and
countertransport in nutrition and metabolism. Physiol.
Rev.70:43-77
Christensen, H.N., Riggs, T.R., Fischer, H., Palatine, I.M.
1952. Amino acid concentration by a free cell neoplasm:
Relations among amino acids. J. Biol. Chem. 198:1-22
Christensen, H.N., Oxender, D.L., Liang, M., Vatz. K.A. 1965.
The use of N-methylation to direct the route of mediated
transport of amino acids. J. Biol. Chem. 240:3609-16.
Christensen, H.N., Liang, M. 1966. Transport of diamino acids
into the Enrlich cells. J. Biol. Chem. 241:5542-51
Dawson, W.D., Cook, J.S. 1987. Parrallel changes in amino acid
transport and protein kinase C localization in LLC-PK1
cells treated with TPA or diradylglycerols. J. Cell.
Physiol. 132:104
Diamond, J.M. 1991. Evolutionary design of intestinal nutrient
absorption:enough but not too much. NIPS. 6:92-96


Fig. 3-26. Alanine uptake across basal membrane in cells grown
on porous filters
The total alanine (50 /M) uptake from the basal compartment
to cytosol and apical compartments was measured in NaCl and choline
Cl media. The amount of basal to cell uptake = the amount of
alanine accumulated inside the cells (shown in Fig. 3-26) plus the
amount of alanine accumulated in the apical chamber (shown in Fig.
3-27) .


Arginine uptake (pmol/mg/min
in undifferenentiated cells
Competitive
Inhibitors
X MeAlB
Z control
J Homoserine
plus Na
R Arg
K Lys
0 Ornithine
A Ala
F Phe
H His
L Leu
M Met
P Pro
Arginine uptake (pmol/mg/min)
in differentiated cells
139


24
differentiated states.
Aim 3: To examine the roles of peptide growth factors
TGF* and EGF and protein synthesis in changing System B and
System y* activities in undifferentiated and differentiated
states.
Aim 4: To examine the role of cellular protein kinase C
in regulation of System B and System y+ by substrate or
TGF/EGF.


204
plus various concentrations of calphostin C (calphostin C was
diluted from stock DMSO solution stored at -20C), (i i i)
treatment with calphostin C and other agent, serum-free DMEM
plus calphostin C plus specified agent, with DMEM plus
specified agent as internal control. These groups were
continuously exposed to a 20 watt fluorescent light in the
37C humidified incubator to photo-activate the calphostin C.
H-7 treatments. The cells were treated with: (i) control
group, serum-free medium plus the same amount of DMSO as
appeared in H-7 (<1% of the medium volume) (ii) treatment
with H-7, serum-free DMEM plus H-7 (H-7 was diluted from a
DMSO stock solution stored at -20C), (iii) treatment with H-7
and other agents, serum-free DMEM plus H-7 plus the specified
agent, with DMEM plus the specified agent as internal control.
6.2.2 Materials
TGF (human recombinant) and EGF (human recombinant) were
obtained from Promega Co., Madison, WI. Chelerythrine Cl and
Calphostin C were from LC services Co., Woburn, MA. H-7,
cycloheximide, medium and other chemicals were from Sigma Co.,
St. Louis, MO.


45
consistently greater than that in the choline Cl medium. This
difference was greater in the younger cells, compared to
confluent cells.
Alanine uptake rates in both the NaCl and choline Cl
media decreased as cell age increased. The uptake rates in
NaCl medium decreased rapidly over a period of several days (<
4 days old), and maintained steady after differentiation (> 9
day old). The decrease in alanine uptake in choline Cl medium
was less dramatic than that in the NaCl medium, with the rate
consistently decreasing throughout the cell ages.
In a separate study, the 24 hours proliferation rates of
Caco-2 cells (2 day old 14 days old) were measured by
incubating the cells in [3H]-thymidine medium for 24 hours.
The amount of thymidine incorporated into the cells during the
period represented the cells relative proliferation rate. The
thymidine incorporation into the Caco-2 cells deceased as cell
age advanced (Fig. 3-3).
The pattern alanine uptake rates at various cell ages was
coincident with the cell proliferation rates.
3.3.3 Ion-deoendencv
The uptake of 50 M [3H]-alanine was measured in the
uptake media containing 137 mM NaCl, 137 mM choline Cl, 137 mM
KC1, or 137 mM LiCl. The total alanine uptake rate in the NaCl
medium was 8-fold greater than that in either the choline Cl,
KC1 and LiCl media at [alanine] = 50 /M (Fig. 3-4) The total


Fig. 3-12. Na*-dependent alanine uptake inhibition pattern in day
3 cells
Alanine (50 /iM) uptake in day 3 cells was measured in NaCl
and choline Cl uptake media containing single 5 mM amino acid. The
Na+-dependent portion was shown.


44
3.2.2 Materials
The materials were as the same as discussed in the
chapter 2.
3.3 Results
3.3.1 Alanine Uptake Time Course
The 50 /M and 5 mM [3H]-alanine uptakes were measured at
during various times (0-45 minutes) in the uptake media
containing 137 mM NaCl or 137 mM choline Cl. The 50 /M alanine
uptake on day 2 cells was shown at Fig. 3-1. The accumulation
in the NaCl medium was greater at any point than that in the
in choline Cl medium suggesting a Na+-activation phenomenon.
The initial alanine accumulation in the Caco-2 monolayers was
linear up to 15 minutes (at both [3H]-alanine concentrations
of 50 M and 5 mM) Since the transport activity was expressed
as the alanine uptake rate measured during the linear period,
the uptake period of 0 10 minutes was chosen for all the
subsequent uptake measurements. The rate was equal to total
accumulation divided by the time period.
3.3.2 Alanine Uptake Rates at Various Caco-2 Cell Ages
The 50 iM [3H]-alanine uptake rates were measured at
various Caco-2 cell ages ranging from 1 day old to 35 days old
in both the NaCl and choline Cl media (Fig. 3-2) At each cell
age, the total alanine uptake rate in NaCl medium was


Fig. 5-4. The Effect of CHX on the acute alanine-stimulated System
B activity
System B alanine (50 /M) uptake was measured in cells
which had been incubated in salt medium (with or without 1 mM
alanine) 50 /M cycloheximide in the incubation medium for 3
hours. The CHX incubation did not block the alanine induced System
B alanine uptake (p < 0.05. n = 9) .


Fig. 3-24. Apical to basal trans-cellular alanine uptake in
cells grown on porous filters
Alanine (50 /zM) uptake was measured in cells grown on
porous filters. Data shown were the amount of [3H]-alanine
transported across cell monolayer from the apical chamber to the
basal chamber, in both NaCl and choline Cl uptake media.


27
Cell culture studied have their advantages and
limitations. On the positive side, cell culture provide a
uniform environment. It is a relatively simple and straight
forward preparation without the adverse effect of in vivo
preparation. The experimental conditions are controllable. On
the negative side, the cell line is not entirely normal cells.
Furthermore cell culture conditions are not those of the in
vivo physiological conditions, and there is the possibility of
mutation. The cell conditions after subculturing may be
different from the in vivo state. Despite the limitation of
the cell culture, the Caco-2 cells are still considered to be
an excellent model for adult intestinal epithelial study
(Pinto et al., 1983; Hidalgo et al., Zwebaum et al., 1991)
For my study, transport studies in Caco-2 cells were
performed in both the undifferentiated (age day 2-3) and the
differentiated (age day 8-9) cells of the same subcultured
batch of cells. In some cases in other cell ages (mentioned in
text). Cell culture technigues are based on established
procedures (Hidalgo et al., 1989; Blairs et al., 1987;
Mohrmann et al., 1986; Pinto et al., 1983) and our
modifications. The Caco-2 cells used for the present
experiments were between the cell sub-cultured passages #19 -
#50.
2.1.1 Materials
The established human intestinal epithelial cell line


116
1992a,b). The most predominant feature that distinguishes
System B from System B0,+ is that latter transport arginine and
other cationic amino acids.
The Na+-independent cationic amino acid transport System
b0,+ has the similar transport characteristics except the Na+-
dependency.
System y+, originally described in the Ehrlich cell,
(Christensen, 1964), reportedly exists in variety of cell
types including the intestinal epithelial cells (Christensen,
1975, 1990; Stevens, 1992a,b; Munck, 1981; Hopfer, 1987;
White, 1985; Segal et al., 1967; Kilberg et al., 1993). System
y+ Differs from other Na+-independent neutral amino acid
transport Systems L and asc, in that System y+ has a relative
narrow substrate scope selectively serving cationic amino
acids. System y+ activity is not sensitive to pH. Even though
System y+ is a Na+-independent facilitated transport system,
it can transport these cationic substrate against a
concentration gradient because of the positive charges
possessed by the cationic substrates and the negative
electrical PD across the plasma membrane. Unlike System b0,+ ,
neutral amino acids does not interact with System y+ activity
in Na-free medium. But in the presence of Na+ neutral amino
acids such as homoserine form a surrogate substrate that can
competitively inhibit the System y+ activity. In the
intestinal apical membrane, System y+ is the predominant
transport system for the cationic amino acids transport.


600
500
400
300
200
100
0
day 2
O day 9
Jarg//[Arg]- . -1
min
135


6
This model describes an prefer-ordered mechanism with the Na+
activator ion binding preferentially first to the cis
transporter conformation, and this binding increases the
affinity for amino acid binding to the cis side of the
transporter. Amino acid then binds to transporter. The cis-
complex isomerizes to place Na+ and amino acid on the trans
side, and the substrates are released to the cytoplasm by
either a random or ordered sequence. The trans transporter
conformation isomerizes back to cis-transporter conformation.
The overall rate-limiting step is the isomerization of the two
transporter forms (cis and trans) In the absence of trans
amino acid, the influx is predicted by: (Stevens & Wright,
1987): JA = (JAax ~ [ A]) /K + [A], where JA = amino acid flux,
Jaiax = maximal flux rate, and the apparent affinity K, for
solute A is a function of both KNa (the apparent dissociation
constant for dissociation and binding):
K. = ((K^/ENa])" + (nK^/ENa]) + 1) KA
where KA is the apparent amino acid-carrier dissociation
constant, and n is the Hill coefficient describing the number
of Na+ ions coupled to movement of each amino acid molecule.
Note that regulation of the transporter activity could
conceivably occur by modifying JBax (i.e, activity of the
functional transporter), or apparent K, (which includes the Na+
affinity and substrate modulation).
1.2.3 The Amino Acid Transport Systems in the Small Intestinal
Membrane


Fig. 5-12. The effect of CHX and chelervthrine on the chronic
arginine-induced system v+ activity
System y+ arginine (5 /M) uptake was measured after cells
(2 days old) had been incubated in salt medium, salt medium plus
various concentrations of arginine (1, or 10 mM) 10 juM CHX or
6.6 /liM CHE for 24 hours. The arginine uptake was increased by
arginine incubation, CHX or CHE did not block this arginine
induction.


Fig. 5-2. The acute effect of amino acids on System B activity
System B alanine (50 uM) uptake was measured in cells
which had been incubated in DMEM salt medium, 1 mM amino acid
for 3 hour. Amino acids alanine, serine, glutamine, cysteine, and
threonine transported by System B induced the System B alanine
uptake. Non-System B substrates such as phenylalanine, proline,
arginine, and MeAIB did not induced the System B activity. 100%
alanine uptake = 0.28 nmole/mg/min.


CHAPTER 4
CLASSIFICATION OF THE ARGININE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE
4.1 Introduction
The cationic amino acid arginine has attracted much
attention during the past years. Not only does arginine
possess many important physiological functions, such as blood
pressure regulation and urea synthesis, but also its transport
systems have unique characteristics. The cloning of system y+
cDNA marked a breakthrough in the amino acid transport study
(Kim et al., 1992). The membrane transport systems for
cationic amino acid arginine and lysine have been studied in
the past decades, and arginine transport systems has been
classified into three systems, a Na+-dependent System B0,+ and
two Na+-independent Systems, y+ and b0,+ (Oxender et al., 1963;
Van Winkle et al., 1985, 1987, 1988).
The Na+-dependent transport System B0,+ first described
in the blastocyte ( Van Winkle et al., 1985) transports both
the neutral amino acids and cationic amino acids. System B0,+
has not been reported for intestinal cells. The similar System
B transporter with characteristics of Na+-dependent neutral
amino acid uptake is exclusively found in intestinal apical
membrane, and is considered a variant of System B0,+ (Stevens,
115


Fig. 5-13. Kinetics of acute arginine-stimulated System v* activity
in salt medium
System y+ arginine (0.1 /M 1 mM) uptake kinetics were
measured after cells (2 days old) had been incubated in salt
medium or 1 mM arginine in this medium for 3 hours. For the
cells incubated with salt only, the VBax = 0.25 nmole/mg/min and
Km = 31 mole arginine? for the arginine incubation group, VBax =
2.75 nmole/mg/min and Km = 81 /mole arginine.


7
Christensen and colleagues developed the original
criteria to discriminate different amino acid transport
systems in mammalian cells (Christensen, 1975, 1984, 1985 &
1990) through substrate specificity, ion-dependency, transport
kinetics, and numerous other characteristics. Many facilitated
and Na+-dependent secondary active transport systems such as
Systems A, ASC, L and y+ which were first described in non-
epithelial cell were found in many cell types including
intestinal epithelial cells (Kilberg et al., 1993; Stevens,
1992a).
Much work has been conducted in the amino acid transport
system classification at the tissue, cellular, and membrane
vesicle levels in the intestine (Munck, 1981; Stevens et al.,
1984; Hopfer, 1987; Stevens, 1992a,b). The major tools for
the membrane transport system classification are (1) substrate
preference; (2) ion-dependency; (3) substrate uptake kinetics,
(4) patterns of analogue cross inhibition of amino acids, and
(5) specific renal and intestinal inborn amino acid
malabsorption syndromes (Stevens et al., 1984; Wright et al.,
198 6, Kilberg et al, 1993) Certain amino acids transported by
a single transporter are used to test for the presence of the
characteristic transporter. For example, -
methylaminoisobutyric acid (MeAIB) and pipecolic acid are
thought to be transported only through the System A and the
System IMINO, respectively (Christensen, 1975; Stevens &
Wright 1985, 1987; Wright 1985). Due to variations among the


3.0
en
E
Q)
O
E
o
o
D
2.5
2.0
1.5
1.0
0.5
Jala/[alQn¡ne]
1 1
DMEM
O 5 mM ala
DMEM salt
-1 -1
/Liter mg min
180


Fig 4-8. Dixon analysis of Na+-independent arginine uptake with
ornithine as inhibitor
Arginine (o.5 nM, 5 ijlM, and 50 ¡iM) uptake was measured
with various concentration of ornithine (1 /M 1 mM) in choline
uptake medium. Non-linear regression gave a Kx = 80 iM ornithine.


3
and migrate up the villi to become the mature villous cells.
The well-developed enterocytes stay at the villus tip for
several days, and then are shed away to the intestinal lumen.
The location of greatest individual amino acid absorption
differs among species along the oral-aboral axis of the small
intestine (Diamond, 1991; Stevens, 1992a,b). The lumen to
blood absorption involves the movement of amino acid through
a series of aqueous and membrane compartments (Hopfer, 1987;
Stevens, 1992a,b). Each compartment acts as a barrier which
influences the overall amino acid movement across all the
compartments. The brush border apical membrane of these
enterocytes is the initial active step regulating the flow of
amino acids from the intestinal lumen into the cell cytosol.
1.2.1 Lumen to Blood Amino Acid Movement
Beginning in the lumen, amino acids travel through an
unstirred water layer, the apical membrane, the cytosol, the
basolateral membrane compartments, and finally capillary
endothelium. Each compartment determines the "real" amino acid
concentration reaching the following compartment.
The unstirred water layer is about 50 nm thick layer and
is composed of water and mucous/glycocalyx. Amino acids
diffuse across the unstirred water layer. The amino acid that
diffuses across the unstirred layer then reach the apical or
brush border membrane. This plasma membrane is a bilayer
phospholipid structure separating the cell cytosol environment


100
80
60
40
20
0
i i i i i i i i r
a methyl-
20
104


14
glucose, caloric and catabolic amino acids, and essential
amino acids (possibly toxic if in excess amount) is regulated
independently in vivo, which provides needed nutrients for the
entire organism and which prevents substrate toxicity
(Diamond, 1991). The mechanism of this induction has not been
addressed hereafter.
Amino acids differ in their potencies to induce the same
transporter. Although substrates generally make good inducers
of their own transporters, there are some discrepancies
(Levine, 1991; Diamond, 1991) between the inducers and
transported substrates: transport unrelated amino acid is the
best inducer.
The change in substrate-related transport activity is a
relatively slow process. An increase in the luminal substrate
level induces an reversible transport uptake capacity increase
over the existing absorbing capacity by 2- to 10- fold within
24 hours. The substrate-specific up-regulation of nutrient
absorption is directly related to the level of these substrate
in the intestinal lumen. Lowering substrate levels causes the
intestinal absorption capacity to decrease back down to the
baseline level that appears to be genetically hard-wired
(reviewed by Diamond, 1991). The down-regulation is a slow
process (eg. 3 days for proline transporter in mouse).
Two mechanisms were proposed to explain the substrate-
related intestinal amino acid uptake activity. The first is
mucosal hyperplasia resulting in nonspecific uptake increase


i i r
cn
E
_0)
o
E
c
CD
o
Cl
Z3
CD
C
c
_o
<
1.0 -
0.5
V Na -dependent
O total in CHO
total in NaCI
.*
0.0
v
Iw
0
V
,o
V
O
O
V
o
0 100 200 300 400 500 600 700 800 900 1000
[Alanine], /jlM


Fig. 4-6. Arginine Na+-independent uptake inhibition pattern in day
3 cells
Arginine (5 /xM) uptake in choline Cl medium was measured
in day 3 cells, with 5 mM single amino acid present in the
uptake medium. Similar results were obtained in day 9 cells.


Arginine uptake, pmole mg min
1 NO hO
t'OUl'-JONJCri'vlON)
ocnocnocnocnocn
I l I l I l 1 1 1
Control
CHX
* TPA
TPA + CHX
j i i L
Z9Z
250


Fig. 4-9. Replot of the slopes of Dixon plot with ornithine
as inhibitor
The slopes of fig. 4-8 were shown as a function of
1[arginine]. Non-linear regression of these data was through 0 of
the axis, indicating that ornithine was a competitive inhibitor of
the arginine uptake.


51
classification criteria, we classified the alanine transport
systems in Caco-2 cells, as discussed below.
3.4.1 Alanine Uptake Activity vs Cell Ages
Both the Na+-dependent and Na+-independent alanine uptake
activities decreased as cell age increased at the alanine
concentration of 50 /xM (over the cell age span of 1 35 days)
(Fig. 3-2) What were the mechanisms underlie this cell
development regulation? There were several possible mechanisms
that could underlie this development-related regulation. Non
specific membrane potential or other membrane property change
could cause a non-specific driving force alteration,
permeability of the membrane could change, or specific
functional change of specific transport systems could occur.
Each of these possibilities was explored.
The Na/glucose cotransport activity on Caco-2 monolayer
has been reported to increased with cell age (Blais et al.f
1987). The opposite direction of alanine uptake activity and
Na/glucose activity with cell age rule (Fig. 3-23) out the
possibility that the age-associated transport effect was due
to the non-specific membrane electrochemical gradient which
may associated with cell age. Therefore the non-specific
driving force was not likely to be involved in the mechanism.
In terms of membrane properties at different cell ages,
our kinetics studies gave the same diffusion permeability
coefficients in day 2 and day 9 cells, even though the alanine


168
during short-term exposure, and this also likely involves a
trans-stimulation phenomenon.
5.4.2 System B Activity Induced bv Chronic Alanine Exposure
The cycloheximide- and chelerythrine-sensitive System B
activity increased with the chronic alanine incubation. This
differed from the short-term regulation, and suggested that
de novo protein synthesis was involved, and that protein
kinase C activation was involved. Because of the
unavailability of specific probes such as antibodies or cDNA's
at the time of this writing, it is not possible to determine
whether the newly synthesized protein was the transporter
protein, transporter regulatory protein, or some other
regulatory protein. We predicted that the chronic activation
effect would be a Vmax effect without the modification of
or Hill number. In other words, the chronic alanine incubation
stimulated the synthesis of System B transport-associated
protein, resulting in a increase in functional transporter
units. Alanine is a caloric amino acid and a carbon chain
precursor for many metabolic intermediates, in addition to
being non-toxic. The specific transporter activity stimulated
by alanine was consistent with the observed in vivo up-
regulation of intestinal epithelial amino acid transport
(Diamond, 1991). The trans-stimulation provides an immediate
safety margin at any given moment so that the cells can
extract the maximum amount of nutrient from limited


Slope
1/[Arginine], 10 L/Mole
2.0


2
organ amino acid flow is beyond the scope of this project. The
small intestine is unique in extracting amino acids, in
contrast to other internal organs. The enterocyte amino acids
transport systems, especially those at the brush border
membrane are unique in substrate and modes of regulation.
Unlike the amino acid transport substrate adaptive down-
regulation universally observed in many internal organs,
substrates in the small intestine up-regulate their
transporter activities in vivo studies (Diamond, 1991;
Stevens, 1992a,b).
1.2 Amino Acid Absorption in the Small Intestine
Like the membrane transport processes in internal organs,
small intestinal amino acid transport has been studied
qualitatively and quantitatively based on uptake phenomena and
non-equilibrium thermodynamic principles.
The small intestinal mucosa separates the outer
environment from the internal milieu. The intestinal mucosa is
composed of a single layer of historically polarized
epithelial cells (enterocytes) which are joined by a tight
junction to form a continuous layer. Amino acid transport
across the mucosa is mainly a trans-cellular phenomenon. The
epithelial villous cells are responsible for amino acid
absorption (Munck, 1981; Wilson, 1962). Along the crypt-villus
axis the enterocytes originate from stem cells within the
crypt. The undifferentiated immature cells rapidly proliferate


Fig. 4-12. Arginine apical to basal trans-cellular uptake
in cells grown on porous filters
Arginine (5 /M) uptake from the apical chamber to the
basal chamber was measured in choline Cl medium 0.5 mM L-lysine
as inhibitor.


Fig. 3-5. The effect of pH on alanine uptake
The uptake of alanine (50/iM) in day 3 and day 8 cells was
measured at various medium pH (at pH = 6.1, 7.4, and 8.4). The
uptake rates were higher in more alkaline media.


10
intestinal apical membrane. One is System L (Christensen et
al, 1963, 1969, 1975) which transports large neutral amino
acids and favors lipophilic substrates such as phenylalanine,
leucine, and BCH. System L excludes 0-alanine. A second is
System y+ (Christensen, 1964, 1966) which prefers cationic
amino acids such as lysine and arginine, although it tolerates
the substrate combination of sodium plus neutral amino acids
such as homoserine. A third is System b0,+ (Van Winkle et al,
1985, 1987, and 1988) which serves neutral amino acids and
cationic amino acid, and interacts with BCH.
Recent cloning of cDNA encoding System y+ (Kim et al.,
1991; Wang et al., 1991) provides a breakthrough in the
membrane transport systems studies. It is possible to study
membrane transport systems using the traditional
phenomenological method as well as more advanced molecular
biological methods. The finding of the same protein serving as
both the System y+ transporter and a retrovirus receptor not
only make the possible molecular studies of membrane amino
acid transporters, but the physiological or pathological
relationships among the nutrient absorption and cell functions
in healthy and disease states. Recent cloning of cDNAs NAA/D2,
rBAT, and F4 for putative regulatory protein for Systems b0,+,
y+ or B0,+ (Betran et al., 1992; Magagnin et al, 1992; Tate et
al., 1992; Wells et al., 1992a,b) were also reported.
All amino acids passively diffuse across the apical
membrane with their permeability rates directly proportioned


Fig. 3-8. Alanine uptake kinetics in day 9 cells
The uptake of alanine (10 /iM 5 mM) was measured in NaCl
and choline Cl media. The total uptake in NaCl and choline Cl
media, and Na+-dependent uptake were showed as a functional of
alanine concentrations. The curves showed non-saturable and
saturable components.


Fig. 3-2. Alanine uptake at various cell ages
The uptake of alanine (50 /xM) was measured in NaCl and
choline Cl media over cell ages of 1 35 days old. At any cell
age, the alanine uptake in the NaCl was greater than that in the
choline Cl media (p < 0.05, n = 6), even though the difference
margin was smaller in the older cells. The Na+-dependent alanine
uptake decreased with the advancing cell age, while the Na+-
independent alanine uptake also decreased at less extent.


-1
Arginine uptake, nmole mg
o ui cn
boo
1ST


28
Caco-2 was obtained from American Type Culture Collection,
Rockville, MD. Dulbecco's Modified Eagle Medium (DMEM), fetal
bovine serum, sodium bicarbonate, penicillin, streptomycin,
non-essential amino acids, Trypsin/EDTA, and Dimethyl
sulfoxide(DMSO) were from Sigma Co., St. Louis, MO. The 6-well
Falcon tissue culture dishes and 100 mm tissue culture dishes
were obtained from the Fisher Scientific, Pittsburgh, PA. The
0.2 /iM medium filters were from Millipore Co. Bedford, MA. The
0.4 jum 24 mm Costar's Transwell-COL collagen treated
microporous membrane filters (Catalog # 3425) were from Costar
Co. Cambridge, MA. [3H]-Alanine, [3H]-arginine, [3H]-glutamine
and [3H]-Threonine were obtained from Amersham Co., Arlinton
Heights, IL. [3H]--methyl aminoisobutyric acid was from
American radiolabled chemicals Inc., St. Louis, MO. NaCl,
choline Cl, KC1, MgS04, KH2P04, CaCl2, NaOH, and N-(2-
hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)
(HEPES)/tris(hydroxymethyl)aminomethane (Tris) were obtained
from Sigma Co., St.Louis, MO. Liguiscint scintillation fluid
was from National Diagnostics, Atlanta, GA. The protein assay
reagent was obtained from Bio-Rad Lab, Richmond, CA. Glacial
acetic acid was from Fisher Scientific, Pittsburgh, PA. The
scintillation counter and spectrophotometer were from Beckman,
Irvine, CA.
2.1.2 Caco-2 Cell Culture
The Caco-2 cells were routinely grown on the 100 mm


232
In the last chapter, we discussed that TGF/EGF activated
the System B and System y+ activities by generating the
intracellular second messenger diacylglycerol, which activates
protein kinase C. In this chapter, we used phorbol ester TPA
to directly activate the cellular protein kinase C, bypassing
the signal pathway between the TGF/EGF to the PKC.
In stimulating the System B and System y+ activities, the
onset of the TPA stimulation was slow (Fig. 7-1) Onset of
activation was observed only after 8 hours, and 24 hours were
needed to significantly stimulate the transport activities.
Continuous TPA exposure was necessary to stimulate the
transport activities.
Cycloheximide and actinomycin D blocked the TPA's
stimulation effect on the System B and System y+ activities,
suggesting that a transcriptional and a translational control
mechanism was involved. Whether the gene expression and the de
novo protein synthesis involved the transporter protein per
se, or other regulatory proteins, is not clear. Further
molecular study using the cDNA probes would give a more
precise answer.
Even though we did not have molecular probe for the
present study, the kinetics studies of the transport
activities are still useful tools. TPA increased both the
system B and system y+ transport Vaax without changing the
other kinetic parameters. The increase in V>ax without K
changing, strongly suggests that increases in System B and


Fig. 3-17. Replot of the slopes of Dixon plot with proline as
inhibitor
The slopes of figure 3-16 dixon plot were shown as a
function of 1/[alanine]. Non-linear regression of these data was
through the interception of x axis and y axis. These data combined
with fig. 3-16 suggested that proline was a weak competitive
inhibitor for the Na+-dependent alanine uptake.


31
MO). One milliliter of the cell/medium mixture (10 million
cells/ml) was then transferred into a sterile glass ampule or
a Nunc plastic tube (Inter Med, Denmark), which was later
sealed in a sterile hood. The sealed ampules and the Nunc
tubes were immersed into a 4C methyl alcohol freezing tank
(Fisher Scientific, Pittsburgh, PA). The freezing tank was
then placed into a -70C freezer for 72 hours before the
ample/tube were transferred into liquid nitrogen storage.
2.1.2.3 Re-thawing frozen Caco-2 cells
Sealed ampules containing the frozen Caco-2 cells from
the American Type Culture Collection or liquid nitrogen
storage were immediately immersed into a 37C water bath until
the ampule content was completely thawed. And the sealed
ampules were immersed into 70% (v/v) alcohol for a minute. The
following procedures were then performed inside a sterile
hood. The ampules containing cells were opened and cells were
transferred and suspended in 37 C DMEM containing 10% FBS. The
cells were then sedimented in a conical centrifuge tube at
1000 x g for 5 minutes, and seeded in the 100 mm cell culture
dishes following the procedures described in the above
subculturing section. The re-thawing cells were grown for at
least two subculturing generations before being used in any
experiment.
2.2 Caco-2 Cell Monolayer Transport
The Caco-2 cell form a monolayer on both the plastic


Fig. 3-21. Replot of the slopes of Dixon plot with phenylalanine
as inhibitor
The slopes of fig. 3-20 were shown as a function of
1/[alanine]. Non-linear regression of these data intercepted at y
axis, these data combined with fig. 3-20 suggested that
phenylalanine was not a competitive inhibitor of Na+-dependent
alanine uptake.


Fig. 3-27. Alanine basal to cytosol uptake in cells
grown on porous filters
Alanine (50 n M) basal to cytosol uptake across
membrane was measured in NaCl and choline Cl media.
basal


22
it possible to study the nutrient transport and associated
regulation over the enterocytes' entire developmental period.
Nonetheless, Caco-2 cells are not entirely normal small
intestinal epithelial cells. However, until the normal small
intestinal epithelial cell model is established, the Caco-2
cell line provides the best in vitro human intestinal
enterocyte model.
1.4 The Objective, Hypothesis, and Aims of the Present Study
1.4.1 The Objective
The overall objective of this in vitro study is to
investigate the cellular basis of amino acid transport
regulation in undifferentiated and differentiated states of a
human intestinal epithelial cell line (Caco-2 cell line). This
project concerns independent transporters serving structurally
distinct amino acid substrates in the apical membrane of Caco-
2 cells.
1.4.2 The Hypothesis
The hypothesis is that alanine and arginine are
independently transported by discrete transporter systems in
the Caco-2 apical membrane, and that the transporter
activities are independently regulated in mature enterocytes
and during enterocyte development. Dipolar L-alanine is
transported via Na+-dependent secondary active transport
System B, while cationic L-arginine is transported via Na+-


267
Magagnin, S., Bertrn, J., Werner, A., Markovich, D., Biber,
J., Palacin, M. Murer, H. 1992. Poly(A)+ RNA from rabbit
intestinal mucosa induces b0,+ and y+ amino acid
transport activities in Xenopus laevis oocytes. J. Biol.
Chem. 267(22): 15384-90
Malo, C. 1991. Multiple pathways for amino acid transport in
brush border membrane vesicles isolated from the human
fetal small intestine. Gastroenterology. 100(6): 1644-52
Margolis, B. L., Rhee, S. G., Felder, S. Mervic, M., Lyall R.,
Levitzki, A., Zilberstein, A., and Schlessinger. 1989.
EGF induces tyrosine phosphorylation of phospholipase C-
II: A potential mechanism for EGF receptor signaling.
Cell 57:1101-1107
Mayo, K.H., Cavalli, R.C., Peters, A.R., Boelens, R., Kaptein,
R. 1989. Sequence-specific lH-n.m.r. assignments and
peptide backbone conformation in rat epidermal growth
factor. Biochem. J. 257(1): 197-205
Meisenhelder, J., Suh, P. G., Rhee, S. G., Hunter, T. 1989.
Phospholipase C-y is a substrate for the PDGF and EGF
receptor protein-kinases in vivo and in vitro. Cell
57:1109-1122
Mircheff, A.K., Sachs, G., Hanna, S.D., Labiner, C.S., Rabn,
E., Douglas, A.P., Walling, M.W., Wright, E.M. 1979.
Highly purified basal lateral plasma membranes from rat
duodenum. Physical criteria for purity. J. Membr. Biol.
50(3-4): 343-63
Mircheff, A.K., van Os, C.H., Wright, E.M. 1980. Pathways for
alanine transport in intestinal basal lateral membrane
vesicles. J. Membr. Biol. 52(1): 83-92
Molloy, C. J., Bottaro, D. P., Fleming, T. P., Marshall, M.
S., Gibbs, J. B., Aaronson, S. A. 1990. GAP is a possible
epidermal growth factor receptor substrate. Nature
342:711-714
Montelione, G.T., Winkler, M.E., Burton, L.E., Rinderknecht,
E. Sporn, M.B., Wagner, G. 1989. The solution
sequence-specific 1H-NMR assignments and identification
of two small antiparallel beta-sheets in structure of
recombinant human transforming growth factor alpha. Proc.
Natl. Acad. Sci. USA. 86(5): 1519-23
Moolenaar, W. H., Tertoolen, L. G. J., de Laat, S. W. 1984.
Rapid increase of intracellular DAG level in A-431 cells.
J. Biol. Chem. 259:8060-8069


*p*


35
N NaOH) and the 5 ml diluted reagent plus 50 /il 1 N NaOH as
the blank. The samples and reagents were mixed well and
stabilized for 15 minutes. The protein absorbance was measured
at wavelength of 595 nm by using the spectrophotometer. The
sample protein contents were then calculated by:
sample protein = (sample absorbance x 1.4 mg/ml) + (standard
protein absorbance).
2.2.2 Radioactivity Measurement
Cell/NaOH aliquot (200 1) was added into a 20 ml vial
and was neutralized by adding 200 /il glacial acetic acid.
Liquiscint scintillation cocktail (10 ml) was added to the
mixture. A 200 /il sample of uptake buffer (containing a known
specific activity of isotope) 200 /il IN NaOH, and 200 ml
glacial acetic acid were added together with 10 ml liquiscint
in the 20 ml vials. The vials were then placed into the
Beckman scintillation counter. The [3H]-radioactivity was
obtained as counts per minute. Uptake was subsequently
calculated as nmole amino acid/mg protein/minute.
2.3 Monolayer Transport In Caco-2 Grown On Porous Filters
As mentioned above, the Caco-2 cells can be grown on
either the plastic surface or porous filters. The confluent
cell monolayer on porous filters provide additional dimensions
to the membrane transport studies. The confluent cell
monolayer forms a barrier separating the upper chamber and


Fig. 5-3. The effect of AOA on System B activity
System B alanine (50 /M) uptake was measured in cells
which had been incubated in salt medium, 1 mM alanine, 2.5 mM
(aminooxy) acetic acid (AOA), or 1 mM alanine + 2.5 mM AOA for
various periods of time (2 7 hours). Alanine alone and alanine
plus AOA incubation stimulated the System B activity at each
incubation period (p < 0.05, n = 6). AOA alone did not affect
the System B activity after 2 and 4 hours incubation, but showed
its stimulation effect after 7 hours incubation (p < 0.05, n =
6) .


[Na], mM
'si
o


en
E
_a>
o
E
c
D
-i1
Q_
Z5
C
c
_D
<
112


Slope
4
1/[Alanine], 10 L/mol
4


Fig. 7-2. Dose curve of the TPA System B stimulation
System B alanine (50 M) uptake was measured in cells
which had been incubated in various concentration of TPA (10'12 -
10'5 M) for 24 hours.


20
Even though the structure and biological functions of EGF
and TGF have been widely studied, the EGF/TGF effects on
intestinal amino acid transport has not been addressed.
1.3 The Human Intestinal Epithelial Cell Line
(Caco-2 Cell Line)
The established intestinal epithelial cell line Caco-2 is
derived from human colon adenocarcinoma cells (Fogh et al.,
1977) It was originally used for in vitro colonic tumor
studies.
Caco-2 cells can been grown on both solid plastic and
porous filters for many sub-cultural generations. When grown
on a solid surface, the Caco-2 cells form a confluent
monolayer with tight junction and dome formation. Under normal
cell culture conditions the confluent cells undergo a
spontaneous enterocytic differentiation process (Pinto et al.,
1983; Rousset et al., 1985). The biochemical and historical
characteristics of the undifferentiated cells resemble those
of the immature enterocytes, while the differentiated cells
resemble the mature small intestinal epithelial cells. The
differentiated Caco-2 cells become polarized, forming brush
border apical membranes complete with peptide and carbohydrate
hydrolases normally found as small intestinal apical marker
enzymes. The enzymes include sucrase-isomaltase, lactase,
trehalase, aminopeptidedase N, dipeptidylpeptidase IV, y-
glutamyltranspeptidase, and alkaline phosphatase (Pinto et
al., 1983; Hauri et al., 1985; Rousset et al., 1985). The


This dissertation is dedicated to my wife Jun, and my
parents.


201
as stimulation of cell proliferation, inhibition of
differentiation, stimulation of oocyte maturation, stimulation
of vasoconstriction. (Raymond et al., 1986; Centrlla, 1987;
Reilly et al., 1987; Kim et al., 1987; Downs et al. 1988;
Berk et al., 1985; Muramatsu et al., 1985, 1986). Depending on
the cell type and the physiological circumstances, EGF exerts
many biological functions (Carpenter & Wahl, 1990). One of the
most dramatic biological effects of EGF is the regulation of
cell growth and differentiation, especially in epithelial
cells and tissues (Carpenter & Wahl, 1990).
The EGF receptors were reportedly found at developing
intestinal epithelial cells and the human Caco-2 cell line. In
the Caco-2 cell line there is a greater density of receptors
in less differentiating cells. Two-thirds of the EGF receptors
appear on the basolateral membrane, while the one-third on the
apical membrane. The K, of the EGF receptor in the Caco-2 cell
is 0.6 juM EGF (Hidalgo et al., 1989; Koyama & Podolsky, 1989).
EGF is normally present in the intestinal mucosa and in
the lumen. The major sources are from the submaxillary gland
secretion, from the Brunner's glands of the duodenum, from the
jejunal/ileal mucosa, and from exogenous sources such as milk
(which contains 40 -400 ng/ml EGF). EGF and TGF are extremely
stable in the presence of the gastric acid and the intestinal
digestive enzymes (Britton et al., 1989; Potter, 1990). The
secretary sources for the EGF in blood stream are still
unknown.


factors epidermal growth factor (EGF) and transforming growth
factor-alpha (TGF) each stimulated system B and system y+
activity following a lag period of several hours. EGF/TGF
activation was abolished by cycloheximide, or by inhibitors of
protein kinase C. Phorbol esters stimulated system B and
system y+ activity following a lag period of several hours,
and this stimulation was prevented by cycloheximide and
inhibitors of protein kinase C. For each transport system,
EGF, TGF, and phorbol ester increased the Vnax but not the Km.
Together these data suggest that (1) Caco-2 epithelial
differentiation status is associated with regulation of amino
acid transport; (2) amino acid transporter system B and system
y+ are regulated independently; (3) amino acid substrates up-
regulate their own transporter's activity via trans
stimulation or by a mechanism involving de novo protein
synthesis; (4) EGF and TGF likely activated protein kinase C
in the up-regulation of system B and system y+ activity via a
de novo protein synthesis mechanism.
Vll


0.5
0.4
Z
-0.5 0.0
[Homoserine],
-1.0
0.5
mM
§
f
1.0
[Arg] = 0.5/i.M
[Arg] = 5/u.M
145


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Bruce R. Stevens, Chair
Associate Professor
Physiology
of
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosor
Edward M. Cop
Professor of
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
George
Professoi
Gerencser
of Physiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Michael S. Kilb*
Professor of Bi ochis try
and Molecular Biology


nmole mg min
0 5 10 15 20 25 30 35
JArg/[Ar9]> ^Liter m9 min
196


Alanine
Incubation
T
T
ALA+AOA
control
4 5 6 7
time, hour


Control
7
c
400
Je
350
mg
300
CD
O
E
250
Cl
200
O
-t11
Q.
150
13
CD
C
100
"c
'en
50
<
0
[Arg]
E £
O
ww 01
+ CHX
T
+ CHE
o
194


Fig. 4-11. Dixon analysis of Na+-independent L-arcrinine uptake with
D-arginine as inhibitor
Arginine (0.5 /xM, 5 /xM, and 50 /xM) uptake was measured
with various concentrations of D-arginine in choline Cl uptake
medium. Non-linear regression of these arginine uptake indicated
that D-arginine was not inhibitor of arginine uptake.


8
animal species, the stages of development, the tissue studied,
and the methods used in amino acid transport systems studies,
many different systems have been reported in the small
intestine (Munck, 1981; Stevens et al., 1984; Hopfer, 1987;
Stevens, 1992a,b). With some conflicts, there is a similarity
in the amino acid transport systems among different species.
The functionally and biochemically distinct brush border
membrane and basolateral membrane possess different transport
systems. The compiled membrane transport systems at the brush
border membrane and basolateral membrane will be discussed
individually.
Several distinctive transport systems are found only at
the brush border membrane (Kilberg et al., 1993; Stevens et
al., 1982, 1984, Stevens, 1992a,b). One of these is System B.
System B is described as a strictly Na+-dependent system
broadly selective for the dipolar (neutral) amino acid
alanine, serine, cysteine, glutamine, and interacts with 2-
amino-2-norbornanecar-boxylic acid (BCH) and threonine. At
first it was named System NBB "Neutral Brush Border" (Stevens
et al, 1982, 1984; Stevens, 1992a,b). System B is
characteristically similar to System B0,+ described in
blastocyte (Van Winkle et al., 1985). Both System B and System
B0,+ broadly transport dipolar amino acids, except that System
B0,+ interacts with cationic amino acids, while System B is not
interactive with cationic amino acids. System B has been
reported to exist in the apical membrane of rabbit (Stevens et


Alanine uptake, nmole mg ( 30 min)
o nj oo o
801


120
4.3.4 Arginine Uptake Kinetics
Arginine uptake in the choline Cl media was measured at
various arginine concentrations ranging from [arginine] =0.1
/iM to [arginine] = 1 mM for both day 2 and day 9 cells. The
uptake kinetics are shown as the uptake rates plotted as a
function of arginine concentrations (Fig. 4-4). The kinetics
studies of the arginine (concentration range 1 /xM 1 mM)
transport in the choline Cl buffer indicated that there was
a non-saturable component and Na+-independent saturable
component. The Eadie-Hofstee transformation gave one non
saturable component as passive diffusion and a single Na+-
independent carrier system (Fig. 4-5).
The non-saturable component was simple passive diffusion.
The diffusion coefficient for both the day 2 and day 9 cells
was the same, P = 1.1 /iliter/mg protein/min.
For the single carrier-mediated system, the non-linear
regression analyses of the Eadie-Hofstee transformation of the
kinetics data gave a Vmax = 430 pmole/mg protein/min and =
31 /mole arginine for the day 2 cells, while at the day 9
cells the Vmax = 340 pmole/mg protein/min and = 37 /mole/mg
protein/min. These kinetic data suggested that the same
transport system was operative at the two cell ages, and the
activity difference was a Vmax effect reflecting a change in
the functional transporter units.


153


5 THE EFFECTS OF INDIVIDUAL AMINO ACID ON THE
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES 156
5.1 Introduction 156
5.2 Methods and Materials 157
5.3 Results 158
5.4 Discussion 165
5.5 Summary 170
6 THE EFFECTS OF PEPTIDE GROWTH FACTORS ON THE
SYSTEM B AND SYSTEM y+ TRANSPORT ACTIVITIES 199
6.1 Introduction 199
6.2 Methods and Material 2 02
6.3 Results 204
6.4 Discussion 206
6.5 Summary 2 09
7 THE EFFECTS OF PHORBOL ESTERS ON THE SYSTEM B
AND SYSTEM y+ TRANSPORT ACTIVITIES 223
7.1 Introduction 223
7.2 Methods and Materials 224
7.3 Results 225
7.4 Discussion 231
7.5 Summary 233
8 SUMMARY AND CONCLUSIONS 258
8.1 Summary 258
8.2 Conclusions 260
REFERENCE LIST 262
BIOGRAPHICAL SKETCH 272
V


Fig. 7-10. The effect of CHX on TPA-induced System v* activity
System y+ arginine (5 /M) uptake was measured in day 2
cells which had been incubated in 0.5 /xM TPA for 24 hours with
or without 50 /xM CHX in the first 12 hours incubation. TPA alone
incubation stimulated the arginine uptake, and this stimulation was
blocked by CHX in the first 12 hours TPA incubation (p < 0.05,
n = 3) .


Fig. 7-9. The effect of TPA on System v+ activity at various cell
ages
System y+ arginine (5 /M) uptake was measured in various
ages of cells which had been incubated in DMEM or DMEM plus 0.5
MM TPA for 24 hours.


Fig. 7-12. The effect of TPA on System v+ transport kinetics
System y+ arginine (0.5 /M 1 mM) uptake was measured in
cells (2 days old and 9 days old) which had been incubated in
DMEM or 0.7 mM TPA for 24 hours. TPA increased the VMX in both
day 2 and day 9 cells without affecting the k.


270
Stevens, B.R., Ross, H.J., Wright, E.M. 1982. Multiple
transport pathways for neutral amino acids in rabbit
jejunal brush border vesicles. J. Membr. Biol. 66(3):
213-25
Stevens, B.R., Kaunitz, J.D., Wright, E.M. 1984. Intestinal
transport of amino acids and sugars: advances using
membrane vesicles. Annu. Rev. Physiol. 46: 417-33
Stevens, B.R. and Wright, E.M. 1985. Kinetic model of the
brush-border proline/sodium (IMINO) cotransporter. Ann.
N. Y. Acad. Sci. 456: 115-7
Stevens, B.R., Kempner, E.S., Wright, E.M. 1986. Radiation
inactivation probe of membrane-bound enzymes:
gamma-glutamyltranspeptidase, aminopeptidase N, and
sucrase. Anal. Biochem. 158(2): 278-82
Stevens, B.R., Wright, E.M. 1987. Kinetics of the intestinal
brush border proline (Imino) carrier. J. Biol. Chem.
262(14): 6546-51
Tate, S.S., Yan, N.N., Udenfriend, S. 1992. Expression cloning
of a Na(+)-independent neutral amino acid transporter
from rat kidney. Proc. Natl. Acad. Sci. USA. 89(1): 1-5
Van Winkle, L.J. 1988. Amino acid transport in developing
animal oocytes and early conceptuses. Biochim. Biophys.
Acta. 947(1): 173-208
Van Winkle, L.J., Campione, A.L., Gorman, J.M. 1988.
Na+-independent transport of basic and zwitterionic amino
acids in mouse blastocysts by a shared system and by
processes which distinguish between these substrates. J.
Biol. Chem. 263(7): 3150-63
Van Winkle, L.J. and Campione, A.L. 1990. Functional changes
in cation-preferring amino acid transport during
development of preimplantation mouse conceptuses.
Biochim. Biophys. Acta. 1028(2): 165-73
Van Winkle, L.J., Campione, A.L., Gorman, J.M., Weimer, B.D.
1990. Changes in the activities of amino acid transport
systems b0,+ and L during development of preimplantation
mouse conceptuses. Biochim. Biophys. Acta. 1021(1): 77-98
Wahl, M. I., Nishibe, S., Pann-Ghill S., Rhee, S. G. ,
Carpenter, G. 1989. Epidermal growth factor stimulates
tyrosine phosphorylation of phospholipase c-II
independently of receptor internalization and
extracellular calcium. Proc. Natl. Acad. Sci. USA.
86:1568-1572


38
filters (with 1 ml uptake buffer with no substrate in the
upper chamber) into 3 ml uptake buffer plus [3H]-alanine or
[3H]-arginine in the lower chamber. During the uptake period,
the cell cultures were continuously shaken by the orbital
shaker (1 Hz). The uptake was stopped by removing the filters
out of the lower chamber, and removing the upper chamber
immediately. The filters were rinsed three time with ice-cold
buffer. Isotope trapped in the cells and in the upper chamber
buffer was measured separately as described above.
For the measurement of the apical- basal trans-cellular
amino acid movement, the uptake was initiated at time = 0 by
adding 1 ml uptake buffer plus [3H]-alanine or [3H]-arginine
to the apical side and immersing the filter into 3 ml buffer
(lacking substrate) in the lower chamber. Uptakes were stopped
by removing filters from the lower chamber. The isotope
accumulated in the lower chamber was the measured as described
above.
For the measurement of the basal-apical trans-cellular
movement, the uptake was initiated at time = 0 by placing the
filter (with 1 ml uptake buffer with no substrate in the upper
chamber) into 3 ml buffer containing [3H]-alanine or [3H]-
arginine in the lower chamber. During the uptake period, the
cell cultures were continuously shaken (1 Hz) The uptakes
were stopped by taking out the buffer from the upper chamber.
The isotope accumulated in the upper chamber was measured in
scintillation counter as described above.


Fig. 3-18. Dixon analysis of Na+-dependent alanine uptake with
glycine as inhibitor
Alanine (25 /xM, 50 /xM, and 100 /xM) uptake was measured
with various concentrations of glycine (10 /xM 5 mM) in uptake
medium. The value was about 5.5 mM glycine.


Alanine uptake, nmole mg min
-r' /*-
1.2 -
1.0 -
0.8 -
0.6 -
0.4 -
0.2
n i i r
+
Na dependent
+
Na independent
-

i r
f
- oo
-12 -11 -10 -9 -8 -7 -6
TPA Concentration, log[M]
-5
237


Fig. 5-7. The effect of chelervthrine on the chronic alanine-
induced System B activity
System B alanine (50 /M) uptake was measured in cells (2
days old) which had been incubated in salt medium, salt medium
plus various concentrations of alanine (0.1, 1.0, or 10 mM) with
or without 6.6 /lM chelerythrine in the incubation medium. The
alanine incubation stimulated the System B alanine uptake (p <
0.05, n = 6) the stimulation was partially blocked by CHE (p <
0.05, n = 6) Similar results were obtained in 9 days old cells.


CHAPTER 3
CLASSIFICATION OF THE ALANINE TRANSPORT
SYSTEMS IN THE CACO-2 CELL MEMBRANE
31 Introduction
The characterization of membrane amino acid transport was
pioneered by Christensen several decades ago (Christensen et
al., 1952). Since that time, the criteria to assess a membrane
amino acid transport systems in mammalian membranes have
indeed substrate specificity, ion-dependency, initial uptake
rate kinetics, patterns of analogue cross-inhibition of amino
acid uptake, and exclusive substrates transported through
specific transport systems. The development concept of the
Na+-gradient-driven, secondary active solute transport (Crane
et al., 1962) was an another important addition to describing
the transport phenomena. In the past several years, there have
been reports of cloning cDNA encoding several amino acid
transporters (Kilberg et al., 1993), and cloning of possible
regulatory proteins for Systems L, b0,+ or y+. There has no
cloning, antibody production, nor purified transporter protein
reported for major alanine transport systems in intestine. The
phenomenological criteria remain the sole tools to classify
alanine transport systems. The sole exception is the recent
cloning of System A (Kong et al. 1993), although System A
40


50
40
30
20
10
0
1 1 1 I 1 I
In NaCI *
[)
O In Choline Cl
-
* *

1
r
5
O
O
_

0
o
1 1 1 1 1 1
1 L
5 10 15 20 25 30 35 40 45
Time, minutes
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160
In a separate study, 1 mM alanine was added to Caco-2
cells which had been incubated in the depletion medium for 3
hours. Within 5 minutes the system B activity was increased
compared to the control level. After 2 hours in depletion
incubation, cells were incubated in the alanine metabolism
inhibitor aminooxy acetic acid (AOA, 2.5 mM) The system B
alanine uptake rate increased only of 7 hours AOA incubation
(Fig. 5-3).
MeAIB uptake was measured in Caco-2 cells which had been
incubated in depletion medium, 1 mM alanine, 1 mM serine or
1 mM MeAIB for 3 hours. The MeAIB (2.5xl08 M) uptake was not
affected by the incubation with added 1 mM alanine or serine.
MeAIB incubation decreased the MeAIB uptake (Fig. 5-6) .
The Na+-independent system L alanine uptake was also
measured in above experiment conditions. The system L alanine
uptake was increased by alanine, serine, glycine, and was not
affected by MeAIB and proline.
5.3.3 Short-Term Activation of System B Activity did not
Involve Protein-Svnthesis
The Caco-2 cells were washed three times with the
depletion medium, and incubated with the same medium
containing 1 mM alanine 50 /zM cycloheximide for 3 hours.
The 50 /M alanine system B activity was measured. The system
B alanine uptake was increased 2 folds by the alanine
incubation alone. The cycloheximide alone in the incubation


Fig. 3-4. Alanine uptake ion-dependency
The uptake of alanine (50jUM) was measured in uptake media
containing 137 mM NaCl, 137 mM choline Cl, 137 mM KC1, or LiCl.
The uptake in NaCl media in both the day 3 and day 8 cells was
greater than that in either those in choline Cl, KC1, or LiCl
media (p < 0.05, n = 6). Uptake in choline Cl, KC1, or LiCl
media was not significantly different (p > 0.05, n = 6) .


205
6.3 Results
6.3.1 The Effect of EGF/TGF on System B Activity
The Caco-2 cells were pre-incubated with 20 ng/ml or 100
ng/ml EGF in the serum-free medium for various length of time
(0 48 hours) The medium was changed every 6 hours. The
System B activities were measured at 5 minutes, 10 minutes, 30
minutes, 1, 2, 4, 8, 12, 18, 24, 30, 41, and 48. System B
activities were not affected by TGF/EGF at the incubation
time less than 30. After 48 hours of continuous incubation,
TGF increased the System B activity by 75%, while EGF
stimulated at least 57% (Fig. 6-1) Cycloheximide (10 /M) or
6.6 /iM chelerythrine in the incubations medium blocked the
System B activity increased by TGF/EGF (Fig* 6-1).
In separate experiments, the Caco-2 cells were pre
treated with insulin, glucagon, dexamethasone, or TGF¡3 for 0 -
48 hours. The System B activity was not affected by either
insulin, glucagon, or dexamethasone. In contrast to TGF, TGF¡3
(>18 hours continuous incubation) inhibited the System B
activity.
In separate experiments, other protein kinase C
inhibitors H-7 and calphostin C were added to the TGF<*/EGF
incubation media. Calphostin C (50 nM) blocked the TGF/EGF
System y+ stimulation effect, while the H-7 (200 ;uM) did not
have any effect.


37
MgS04, 1.2 mM KH2P04, and 2.5 mN CaCl2. These experiments were
conducted in collaboration with Dr. S. Smith of Burroughs-
Wellcome company (Research triangle, NC).
For the measurements of the apical-cellular-basolateral
amino acid movement, the amino acid uptake was initiated at
the time = 0 by adding 1 ml uptake buffer plus [3H]-alanine or
[3H]-arginine into the apical side (the upper chamber), with
the basolateral side exposed to 3 ml uptake buffer (lacking
amino acids) in the lower chamber. During the uptake period,
the cell cultures were continuously shaken by an orbital
shaker (1 Hz) The uptakes were stopped by aspirating the
uptake buffer, taking the filters out of lower chamber, and
adding 2 ml ice-cold uptake buffer (lacking substrate)
immediately to the upper chamber. The buffer was aspirated and
cells were rinsed by the ice-cold buffer three times. Isotope
trapped inside the cells was extracted from the cells by
adding 2 ml 1 N NaOH to the cells (filters were first cut out
off the dishes). After overnight extraction (continuous
shaking), a 200 nl aliquot of NaOH extract was added to 10 ml
Liquiscint scintillation fluid which was neutralized with 200
/I glacial acetic acid. Isotope trapped in the lower chamber
was measured by transferring 200 il of this buffer to 10 ml
Liquiscint. The radioactivity was counted in the Beckman
scintillation counter.
For the measurement of basolateral-cellular-apical
movement, uptake was initiated at time = 0 by immersing


55
glutamine, cysteine, and asparagine; Weaker inhibition was
elicited by glycine, phenylalanine, leucine and BCH. MeAIB and
cationic amino acids elicited < 10% inhibition (Fig. 3-12 & 3-
22). We can compare this inhibition pattern with the amino
acid inhibition patterns of the known Na/alanine Systems A,
ASC, B, and B0,+. System A is a strictly Na+-dependent system
selective for dipolar amino acids including alanine. Many
neutral amino acids competitively inhibit Na/alanine transport
via System A. One special aspect of System A is its unique
ability to transport MeAIB. In our inhibition study, MeAIB
blocked less than 10% of the Na/alanine transport activity
(Fig. 3-12). Dixon analysis revealed that the MeAIB inhibition
was a non-competitive inhibition (Fig.3-15). These combined
data exclude System A as a major transport system in Caco-2
cells.
System ASC, the Na+-dependent system serves short-chain
neutral amino acids alanine, serine, and cysteine. In our
study, serine, cysteine strongly inhibited alanine/Na uptake.
However, phenylalanine and glycine, two competitive inhibitors
of System ASC did not inhibit the Na/alanine transport in our
study, as it would for the classic System ASC (Figs. 3-12, 3-
18-21) Based on this and the similarity of our data to System
B (discussed below), we exclude System ASC as the transport
system. Because the characteristics were very close, however,
definite classification is not possible without more precise
test methods such as cDNA probes or antibodies.


Arginine uptake, pmole mg min

l
ro
M
cn
o
cn
o
cn
o
O
o
o
o
o
QTZ
300


167
that the System B and System y+ activities were specifically
and independently regulated only by their own substrates.
The fact that the System B activity could be induced by
alanine within minutes and the increase was reversible,
suggests that the increase in System B activity after the
short-term substrate incubation could be due to: (1) a tran-
stimulation of the transport systems, or/and (2) a trans
location of the transport units from cytosol to membrane. The
insensitivity of cycloheximide or actinomycin D eliminated the
possible involvement of de novo protein synthesis or new RNA
synthesis mechanisms. Kinetic are valuable in identifying the
trans-stimulation and the trans-location mechanisms. For a
pure trans-location of transporter units, the only kinetic
parameter that would change is Vmax without alteration. For
the trans-stimulation mechanism, both the Vmax and the would
be changed, and indeed in our kinetic studies, both the Vmax
and were changed, favoring the notion that the acute
activity increase was due to a trans-stimulation. We can not
determine whether both the trans-stimulation and trans
stimulation were involved. In the future, by using the
membrane vesicles pre-loaded with various concentrations of
alanine, we will be able to determine if a trans-stimulation
is involved. The only way to determine a role for tran-
location is to probe transporter protein level using
antibodies.
The System L activity increased by its own substrate


200


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233
System y+ activities by TPA were due to the increase in copies
of functional transporter units, rather than due to
modification of the transport affinity.
To further confirm that the effect of TPA was a protein
kinase C activation phenomenon, we used the specific protein
kinase C inhibitors chelerythrine Cl and calphostin C. As
shown in the results, these PKC inhibitors blocked the TPA's
effect on the System B and System y+ activities. These data
indicated that the TPA stimulation of transport activities
involved protein kinase C activation, not protein kinase C
inhibition.
When we compare the System B and System y+ transport
activities that were stimulated by TGF/EGF (chapter 6) and
TPA (this chapter), we can see that each system's transporter
characteristics were very similar. These data further support
the notion that TGF/EGF stimulate the system B and system y+
activities by engaging protein kinase C.
7.5 Summary
Phorbol ester TPA activates System B and System y+, but
not System L, activities throughout all cell ages. The
activation involves transcription and translation process, and
likely is mediated via protein kinase C. The phorbol ester
stimulation of System B and System y+ results in an increase
in system's V,ax without affecting the corresponding K,.


164
stimulated by its own substrates.
In another study, Caco-2 cells were incubated in the
depletion medium for 3 hours, and 1 mM arginine was added to
the medium. System y+ arginine uptake increased as early as 5
minutes arginine incubation.
5.3.9 System v+ Activity Increased by Acute Arginine Exposure
did not Involve Protein Synthesis
The Caco-2 cells were washed and incubated in the
depletion medium containing 1 mM arginine (1 mM mannitol as
control) 50 nK. cycloheximide or 0.5 /ig/m 1 actinomycin D for
3 hours. The system y+ activity increased by the arginine
exposure was not blocked by the cycloheximide or actinomycin
D in the incubation medium (Fig. 5-11).
5.3.10 System v+ Activity Increased bv Acute Arginine Exposure
was Reversible
Caco-2 cells (2 days and 9 days old)were washed and
incubated in the depletion medium 1 mM arginine for 3 hours.
The cells which had been incubated with arginine were then
washed three time with the depletion medium and incubated in
the depletion medium 1 mM arginine for another 3 hours.
System y+ activity was increased following a 3 hours arginine
incubation, and the increased was diminished after the cells
were then incubated in depletion medium (lacking arginine)
for 3 hours.


32
surface and porous filters. For the pre-confluent cells, the
junctions among cells are loose, and cell membrane has not
polarize yet. The cell uptake may involve the membrane,
excluding the portion attached to the plastic surface. For the
differentiated state, cells have already polarized with basal
membranes attached to plastic surface or filter; lateral
membranes are formed beneath the tight junctions which connect
the apical membranes. The apical surface faces the outer
environment. Organic solutes enter cells through the apical
membrane, so that the para-cellular pathway is minimal. This
has been confirmed by [3H]-inulin extracellular studies
(Arturson et al., 1992).
The membrane transport of amino acid is a bi-directional
process. The measured transport activity is the net influx of
transport equivalent to vectorial difference between the two
unidirectional fluxes. The rate of the net is therefore
determined by the total flux during a period of time during
which the flux is linear. In the case of monolayer transport,
the rate of the membrane transport of amino acid is therefore
equal to the rate of net accumulation of amino acid within the
cells over a period of time. We measured the total
accumulation of amino acid and the time at which the amount
accumulation is linear proportional to the accumulation time.
The mode and characteristics of membrane transport of amino
acid are determined by the Menten-Michaelis kinetic analysis.
The amino acid transport experiments were performed on