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Mechanisms of potassium permeation in the In Vitro perfused cortical collecting duct of the rabbit

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Mechanisms of potassium permeation in the In Vitro perfused cortical collecting duct of the rabbit
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Zhou, Xiaoming
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English
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xvii, 150 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Electric potential ( jstor )
Enzymes ( jstor )
Excretion ( jstor )
Histamines ( jstor )
Kidneys ( jstor )
Potassium ( jstor )
Rabbits ( jstor )
Rats ( jstor )
Secretion ( jstor )
Sodium ( jstor )
Department of Physiology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Physiology -- UF ( mesh )
Kidney Tubules, Collecting -- metabolism ( mesh )
Potassium -- metabolism ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1992.
Bibliography:
Includes bibliographical references (leaves 137-149).
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Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Xiaoming Zhou.

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University of Florida
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MECHANISMS OF POTASSIUM PERMEATION IN THE IN VITRO PERFUSED CORTICAL COLLECTING DUCT OF THE RABBIT












By

XIAOMING ZHOU













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
1992





























To Xiaofang, Martin, my parents, and my brother.













TABLE OF CONTENTS


ACKNOWLEGEMENTS ............................ v

LIST OF TABLES ................................ viii

LIST OF FIGURES ................................ x

KEY TO ABBREVIATIONS .......................... xiii

ABSTRACT .................................... xv

CHAPTER 1 INTRODUCTION ....................... 1
1.1 Background ................. ... 1
1.2 Potassium Secretion by the Cortical Collectin Duct ..... 4 1.3 Potassium Absorption by the Cortical Collectmg Duct ... 10 1.4 The Aims and Objectives of the Present Studies ....... 13 CHAPTER 2 GENERAL METHODOLOGY ................. 23

2.1 In Vitro Microperfusion .................... 23
2.2 Flameless Atomic Absorption Spectrophotometry ........ 25 2.3 Statistical Analyses ........................ 26

CHAPTER 3 EFFECT OF 10% CO2 ON Rb EFFLUX .......... 28

3.1 Introduction ............................28
3.2 Methods and Materials ......................30
3.3 Results ........ ....................... 31
3.4 Discussion ............................. 38
3.5 Summary .............................. 48

CHAPTER 4 EFFECT OF BARIUM, AMILORIDE AND OUABAIN
ON Rb EFFLUX ............................ 70

4.1 Introduction ............................70
4.2 Methods and Material ......................72
4.3 Results .............................. 72
4.4 Discussion ............................. 76
4.5 Summary .............................. 79

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CHAPTER 5 EFFECTS OF LUMINAL SODIUM ON Rb EFFLUX 88

5.1 Introduction ............................88
5.2 Methods and Material ......................88
5.3 Results .............................. 89
5.4. Discussion .............................. 94
5.5 Summary .............................. 97
CHAPTER 6 EFFECT OF ANGIOTENSIN II, HISTAMINE, AND
CARBACHOL. ............................. 111
6.1 Introduction l..........................111
6.2 Methods and Material .................... 111
6.3 Results ............................. 112
6.4 Discussion ........................... 114
6.5 Summary ............................. 115

CHAPTER 7 EFFECT OF Na AND K INTAKE ON SERUM AND
URINE Na AND K LEVELS AND URINE OUTPUT .... 122

7.1 Introduction ...........................122
7.2 Methods and Materia.l .....................122
7.3 Results .............................. 122
7.3 Discussion ............................ 123

CHAPTER 8 SUMMARY AND CONCLUSIONS .............. 127

REFERENCES .................................. 137

BIOGRAPHICAL SKETCH .......................... 150


















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ACKNOWLEDGEMENTS


I wish to express my gratitude and appreciation to Dr. Charles S. Wingo, the chairman of my supervisory committee, for his instrumental guidance, productive encouragement, invaluable support and kind care throughout the fulfillment of this work. His tremendous contribution to my scientific knowledge, professional career, my personality and personal life are beyond my ability to acknowledge. I will benefit from his tradition of dedication, criticism, responsibility, preciseness and carefulness in both of my academic activities and private life for a very long time to come. I am indebted to Dr. George A. Gerencser, the co-chair of my committee for his vital role in my doctoral education. His thoughtful instruction was invaluable during the course of my studies. His understanding and friendship are greatly appreciated.

I extend my sincere thanks to the members of my supervisory committee, Drs. Lal C. Garg, Bruce R. Stevens and Brian D. Cain for their very helpful suggestions and criticism. They have provided me with alternative interpretations and additional insight that have required additional studies. I am grateful to Scott Straub and Amy Wall for their expert technical help when I was learning in vitro microperfusion. The personal friendship with Scott Straub and Harold Suellen established during past four years is warmly appreciated. I sincerely appreciate Dr. Frances Armitage and Wei Ueberschaer for their important role


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in this research and for their help with my English. The discussions of my research results with them and with Drs. Kevin Curran and Mitchell Hebert have been productive and enjoyable. I am also grateful to the faculty members in the Department of Physiology, especially Drs. M. Ian Phillips, Colin Sumners, Sidney Cassin, Melvin Fregly and Charlie Wood for their advice and encouragement. In addition, I wish to thank Drs. C. Craig Tisher, Christopher Wilcox, Kirsten Madson, William Welch, and I David Weiner in the Division of Nephrology, Hypertension and Transplatation for their concern and advice. I wish to thank Ann Crawford in depth for her excellent and courteous help in preparation of this thesis as well as other manuscripts and abstracts. I am always grateful to have the manuscripts back from Ann before Friday so that I have something to work with during the weekend. My sincere thanks should be presented to Robert Fleming for his constant concern and support. My special thanks should be offered to Janice Dolson not only for her expert help with my learning some special functions of Wordperfect, but also for her friendly understanding and support during the past three years. I also appreciate Ms. Ginny Young for her secretarial assistance to Dr. Wingo regarding my education. My deep thanks are extended to Miss. B. J. Streetman, Victoria La Placa, Pia Jacobs and Gayle Butters and Mr. Kevin Fortin for their necessary role in my doctoral education. The constant concern and support from my fellow students in the Department of Physiology, especially Hong-gen Chen and Jian Kane, and Jenny Zhang in the Department of Pharmacology and Therapeutics are warmly appreciated.
I am greatly indebted to my wife Xiaofang, my father Ouibao Zhou, my


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mother Suping Bu and my brother Xiaolin for their love, consideration, backing and understanding. Xiaofang has unselfishly devoted her life to the family. This dissertation is especially dedicated to Xiaofang and Martin at this special time, although it is impossible to express my gratitude to them adequately in words. The birth of Martin in the final stage of my doctoral education not only has increased my excitement for the present studies, but also has inspired me to aim at higher quality of professional and private life in order for me to carry out the honor, duty, desire, and role model as a father. Without the fundamental and instrumental education of my childhood by my parents, it would be unimaginable for me to be able to choose the present way of pursuing my life.

























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LIST OF TABLES


Table 3-1. Composition of solution ................... 50

Table 3-2. Effect of luminal 0.1% DMSO on KRb and VT ....... 51

Table 3-3. Time control for KRb and VT ............... 52

Table 3-4. Effect of 10% CO2 on VT in the presence colchicine 53

Table 3-5. Effect of 10% CO on V in the tubules
pretreated with 0.5aM MA PTAM and W-7 ....... ..54

Table 3-6. Effect of 10% CO2 on Ksb and VT by the normal
CCD in the presence anY absence of luminal Ba .... 55 Table 4-1. Composition of solutions .................. 80

Table 4-2. Effect of DMSO in the absence of luminal Na
and luminal 1.5 mM Na .... .................. 81
Table 4-3. Effect of luminal 10aM SCH28080 or 0.1 mM ouabain
on VT in the absence of luminal Na ............. 82
Table 5-1. Composition of solution ................... 99

Table 5-2. Effect of removal of luminal Na in the absence
of luminal Ba, and of luninal Ba addition in the
absence of luminal Na ................... 101
Table 5-3. Effect of luminal Ba addition in the presence
of luminal Na, and of luminal Na removal in the
presence of luminal Ba. ...................... 102
Table 5-4. Effect of removal of luminal Na in the
presence of 4 mM Ba. ....................... 103
Table 5-5. Effect of luminal Na removal of in the presence of
both 2mM Ba and 10AM SCH28080 ............ 104

Table 5-6. Effect of luminal SCH28080 on VT in the absence and

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presence of luminal Na ...................... 105

Table 5-7. Effect of SCH28080 in the presence of 20 mM K . 106
Table 6-1. Composition of solution ...................... 116

Table 6-2. Effect of histamine on VT in the presence of
luminal 3 mM Ba .......................... 117
Table 6-3. Effect of 0.1 mM carbachol ................... 118

Table 7-1. Effect of low Na and low K on excretion of
urine, Na and K ........................... 125
Table 7-2. Effect of low Na and low K diet, and low K diet on
serum Na and K levels ................... 126































ix













]LIST OF FIGURES

Figure 1-1. Distribution of potassium in the body............. 16

Figure 1-2. Segmental analysis of tubule potassium transport . .. 17

Figure 1-3. Urinary potassium excretion as a function of
plasma potassium concentration in control animals
and in alkaotic and acidotic conditions .. .. .. .. ....18
Figure 1-4. Effect of different types of acid-base disturbances
on potassium secretion by distal tubule .. .. .. .. ....19

Figure 1-5. The cellular models of the principal cell and the
intercalated cell. .. .. .. .. .. .. .. .. .. .....20
Figure 1-6. Cell model of epithelial sodium and potassium
transport .. .. .. .. .. .. ... .. .. .. .. .....21
Figure 1-7. The chemical structure of SCH28080 .. .. .. .. .. ..22

Figure 2-1. The cortical collecting duct perfused in vitro .. .. .. ..27

Figure 3-1. Effect of 10% CO2 on KR, and VT in the absence
of SCH28080. .. .. ... .. .. .. .. .. .. ..56
Figure 3-2. The predicted voltage-mediated increase in KR, and
observed increase in K.~ following exposure
to 10% CO2. .. .. .. .. .. .. .. .. .. ....57
Figure 3-3. Effect of 10% CO2 on KR, and VT in the presence
of SCH28080. .. .. .. .. .. .. .. .. .. .. ..58
Figure 3-4. Effect of 10% CO2 and 0.1 mM methazolamide on K
and VT in the absence of SCH28080 .. .. .. .. .. ..59
Figure 3-5. Effect of 10% CO2 and 0.1 mM methazolanude on P
and VT in the presence of SCH28080. .. .. .. .. ..60
Figure 3-6. Effect of simultaneous exposure to 10% CO2 and
0.1 mM methazolamide on KR, and VT ...........61


x








Figure 3-7. Effect of 0.1 mM methazolanmide on K, and
VT in the presence of 5% CO2 . . . . . . . 62
Figure 3-8. Effect of simultaneous exposure to 10% CO2 and
0.5 mM colchicine and exposure to colchicine
after 10% CO2 on K ................... 63
Figure 3-9. Effect of 10% CO on Kn by the tubules pretreated
with 0.5 u&M MAPTAM and -7 ................ 64
Figure 3-10. Effect of 10% CO2 on Kn by the tubules pretreated
with peritubular 3 mM Ba . . . . . . . . 65
Figure 3-11. The dose-response curve of Ba on K . . . . . 66
Figure 3-12. The CCD was swollen following exposure
to 10% CO2 . ...................... 67
Figure 3-13. The simultaneous presence of 10% CO2 and
colchicine prevented the cell swelling . . . . . 68
Figure 3-14. Effect of 10% CO2 on cell swelling in
the presence of MAPTAM . . . . . . . . 69
Figure 4-1. Effect of luminal 1 mM amiloride on KR, in the
absence of luminal Ba and in the presence of luminal Ba, and effect of SCH28080 on Kn in
the presence of luminal 1 mM amiloride
and 2 mM Ba ........................ 83
Figure 4-2. Effect of luminal 1 mM amiloride on VT in the
absence of luminal Ba and in the presence of luminal Ba, and effect of SCH28080 on VT in
the presence of luminal 1 mM amiloride
and 2 mM Ba ........................ 84
Figure 4-3. Effect of peritubular 0.1 mM ouabain on KR
in the absence of luminal SCH28080 and in the
presence of luminal SCH28080 . . . . . . . 85
Figure 4-4. Effect of peritubular 0.1 mM ouabain on Kn
in the absence of luminal SCH28080 and in the
presence of luminal SCH28080 . . . . . . . 86
Figure 4-5. Effect of luminal SCH28080 or ouabain on Kn . . 87
Figure 5-1. Effect of lumen Na removal on K and VT in the
presence of luminal 10 yM SCH280 . . . . . 107


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Figure 5-2. Inhibition of K4 by luminal 10 AM SCH28080 in the
p resence of luminunal Na and in the absence of
lumninal Na .......................... 108
Figure 5-3. Effect of SCH28080 on Kl in the presence of
ambient 0.5 mM K or 20 mM K . . . . . . 109
Figure 5-4. Effect of SCH28080 on VT in the presence of
ambient 0.5 mM K or 20 mM K . . . . . . 110
Figure 6-1. Effect of angiotensin II on K, and VT in
the presence of luminal 3 mM Ba and absence
of luminal SCH28080 .................... 119
Figure 6-2. Effect of angiotensin II on K and VT in
the presence of luminal 10 SCH28080 and
absence of luminal Ba .................... 120
Figure 6-3. Effect of histamine on K, in the presence
of luminal 3 mM Ba .................... 121

























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KEY T O ABBREVIATIONS


Ang H angiotensin H

ANOVA analysis of variance

ATP adenosine, triphosphate

Ba barium
Ca calcium
CCD the cortical collecting duct

CD the collecting duct

CO, carbon dioxide
H proton

HEPES (N-[2-hydroxyethyllpiperazine-N-[2-ethanesulfonic acid]
K potassium

KRb 86Rb lumen-to-bath efflux coefficient

KN. 22Na lumen-to-bath efflux coefficient

MAPTAM Bis-(2-amino-5-methyl-phenoxy)-ethane-NNN',N'-tetraacetic

acidtetraacetoxymethyl ester N-(6-aminohexyl)-5-chloro-lnaphthaIene-sulfonamide Na sodium

Rb rubidium
SCH28090 (3-tyanomethyl-2-8-phenylmethoxy)iniidazol[1,2-a]pyridine


xiii








TMA tetramethylammonium

VT transepithelial voltage
W-7 N-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide





































idv













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
MECHANISMS OF POTASSIUM PERMEATION IN THE IN
VITRO PERFUSED CORTICAL COLLECTING DUCT OF THE RABBIT By
Xiaoming Zhou
May 1992
Chairman: Charles S. Wingo, M.D.
Major Department: Physiology
These studies were designed to examine the mechanisms of potassium
(K) permeation across rabbit cortical collecting duct (CCD) assessed as 6Rb lumen-to-bath efflux coefficient (Km,, or Rb efflux). This segment is primarily responsible for potassium secretion, but recent studies have demonstrated potassium absorption by the CCD.
The first set of studies was designed to examine whether acute peritubular acidosis (10% CO2) stimulates rubidium (Rb) efflux, and whether this stimulation is dependent on the presence of functional H-K-ATPase, carbonic anhydrase, microtubules, the increase of intracellular calcium activity, calmodulin, and basolateral potassium conductance in the cortical collecting duct from K-restricted rabbits. Exposure to 10% CO2 substantially stimulated Rb efflux, and this stimulation was totally abolished by 1014M SCH28080, a specific inhibitor of H-K-ATPase. After stimulation of Rb efflux by 10% CO,


XV







subsequent addition of methazolamide or colchicine failed to affect Rb efflux. However, simultaneous exposure to 10% and methazolamide or colchicine prevented the stimulation of Rb efflux. Treatment with MAPTAM, W-7, and peritubular barium blocked the stimulatory effect of 10% CO2. The data are consistent with the following conclusions, at least during K restriction: 1) an existing H-K-ATPase mediates the stimulatory effect of 10% CO2 on Rb efflux; 2) carbonic anhydrase and microtubules are not necessary for maintaining H-KATPase activity, but is required for activation of H-K-ATPase by 10% C02; 3) the effect 10% CO. is dependent on the increase in intracellular calcium activity and presence of basolateral barium-sensitive exit mechanism.
The second set of the studies was mainly to examine the effect of luminal amiloride on Rb efflux. Amiloride significantly increased Rb efflux, and this effect was fully blocked by luminal barium, suggesting that a Ba-sensitive K conductance mediates the effect of amiloride.
The third set of the studies was to examine whether removal of luminal sodium had similar effect as amiloride. In contrast to the effect of amiloride, the stimulation of Rb efflux by sodium removal was only inhibited by simultaneous presence of barium and SCH28080, indicating that the effect of Na removal is in part via the H-K-ATPase. To test whether Na acts as a partial agonist of the H-K-ATPase we examined the effect of SCH28080 on Na efflux. In the presence of luminal amiloride and an ambient K concentration of 0.5 mM, SCH28080 significantly inhibited Na efflux, whereas this effect was abolished when similar experiments were performed in the presence of 20 mM ambient K. In conclusion: 1) both Ba-sensitive K conductance and H-K-ATPase


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mediate the effect of sodium removal; 2) sodium competes with K for transport via the H-K-ATPase.









































xvii













CHAPTER 1
INTRODUCTION

1.1 Background
Potassium (K) is one of the most abundant cations in the body and plays an important role in a variety of physiological functions. Cell growth, division, volume regulation, and acid-base balance depend on a high intracellular K activity. Reduction in K intake induces hypertension and supplement of K intake ameliorates hypertensive symptoms. High potassium concentration in cells and low concentration in the extracellular fluid contribute to the electrical properties of both excitable and nonexcitable tissures. Changes in either intracellular or extracellular K activity result in modification of both intracellular and extracellular pH. Potassium affects muscle metabolism, in broad term, by four different ways: by regulation of muscle blood flow during exercise; by its role in carbohydrate metabolism; by its effect on insulin secretion; and by its direct effect on muscle itself.
The performance of these vital functions by potassium is dependent on a variety of regulatory mechanisms that maintain K homeostasis. Figure 1-1 (Giebisch et al., 1981) schematically depicts the distribution of potassium ion in the body. As can be seen, most of potassium is located within cells, especially within muscle cells, with smaller amount in liver and erythrocytes. Potassium is the most abundant intracellular cation. Only 2% of body's potassium is distributed in the extracellular compartment, normally at


I








2
concentrations varying between 3.5 to 5 mM. Therefore, the loss or gain of an amount of potassium equivalent to 1% of total body potassium from the extracellular fluid may halve or double the potassium in plasma and the concentration ratio of potassium across the plasma membrane. This could substantially affect the electrical polarization of both excitable and nonexcitable tissue and be potentially lethal. In contrast, translocation of similar amount of potassium into or from the intracellular compartment only has a minimal effect on the concentrations. Therefore, the mechanisms controlling extracellular K must be more sensitive than those responsible for control of the intracellular potassium content. In fact, because of the highly regulated control mechanisms, potassium intake can increase over 20-fold with little change in body potassium content. Maintenance of this homeostasis reguires a balance between intake and excretion, so-called external potassium balance. The main route for potassium intake is the intestinal absorption which is not subject to specific control mechanisms. There are, however, three mechanisms that maintain the level of extracellular potassium within a critically narrow range.
1. When potassium intake increases, potassium can be translocated from the extracellular compartment to the intracellular fluid so that only a small fraction of any potassium added remains in the extracellular fluid. Conversely, extracellular potassium losses can be alleviated by shifting this ion from the cellular pool to the extracellular fluid. This distribution of potassium between extracellular and intracellular fluids, so-called internal potassium balance, is regulated by a variety of hormones such as aldosterone, catecholamines and insulin and by acid-base balance.








3
2. The colon has a capacity for potassium secretion and absorption which may be stimulated when the kidney's ability to handle potassium balance is compromised.
3. The kidney plays the key role and is ultimately for the appropriate response to changes in potassium excretion to match potassium intake. As shown in Figure 1-2 (Giebisch et al., 1981), in an individual ingesting 100 mmol of K per day, the proximal tubules absorbs up to 70% of filtered potassium which is approximately 600 mmol per minute. The Loop of Henle absorbs additional 22% of filtered K When the luminal fluid deliveries to the early of distal tubule, only less than 10% of filtered K, approximately 50 mM, are left. Potassium secretion commences in the initial collecting duct and continues along the cortical collecting duct. The collecting duct (CD) is the final segment to determine net K excretion (Giebisch et al., 1981; Wright and Giebisch, 1992). In this case, the collecting duct transports net 50 mmol K into the urine. Therefore, 100 mmol potassium appears in the final urine, and the balance between potassium intake and output is maintained.
1) Proximal tubule. Our present state of knowledge about the role of the proximal tubule in the regulation of potassium excretion is largely derived from in vivo free-flow micropuncture of tubules accessible at the kidney surface (Giebisch et al., 1981). This segment absorbs the largest amount of potassium, which is mediated by both active and passive processes. However, the exact mechanisms explaining K absorption remain to be elucidated. The K concentration in samples of fluid from the proximal tubule has been found to be close to the concentration in plasma, suggesting that the rate of reabsorption of K along the proximal convoluted tubule is rather rigidly coupled to sodium








4
and water absorption. A direct comparison of extent of fluid absorption with the range of potassium concentration in the collected fluid samples has indicated that under normal condition, similar fractions of water, sodium, and potassium are reabsorbed between the glomerulus and the last accessible part of the proximal tubules (Beck et al., 1973; Diezi et al., 1976).

2) L&pgL o Hnle. Approximately 5 to 15 % of the amount of potassium filtered reaches the early distal tubule of superficial nephron, and that this amount of potassium is not subject to a significant changes in spite of that the amount of K in the final urine may be changed as much as 200-fold (Malnic et al., 1964; Malnic et al., 1971).

3) Cofleting duct. A great deal of evidence has demonstrated that K excretion is not dependent on K filtration but rather dependent on K secretion (blood-to-lumen) (Wright, 1987). The recent studies have suggested that K excretion is also dependent on K reabsorption (lumen-to-blood) by the collecting duct (Mujais and Katz, 1992). Because this segment is the final tuning site for potassium excretion, the amount of K that appeares in the urine is determined by net balance between potassium secretion and absorption processes which are critically regulated according to the different needs of the body. High K intake stimulates K secretion, whereas low K intake increases K absorption by this segment (Mujais and Katz, 1992; Wingo, 1987; Wright and Giebisch 1992).

1.2 Potassium Secretion by the Cortical Collecting Duct

Berliner and Kennedy (1948) and Mudge et al. (1948) were the first to dissociate K excretion from K filtration from their clearance studies. They reported that kidneys regularly responded to certain experimental manipulations by excreting K at the rate exceeding the rate of K filtration, suggesting that








5
potassium secretion is an important factor determining potassium excretion. Subsequently, Davidson and his associates (1958) performed another elegant studies in order to gain insight into the mechanisms of K secretion. They observed that when the glomerular filtration rate was reduced by about 30%, sodium excretion decreased by 80%, and potassium excretion decreased by 50%. In contrast, if the rate of sodium excretion was maintained at a high level by infusing salt and administering diuretics, then similar reduction in the glomerular filtration rate did not reduce potassium excretion. These data were taken as a clear evidence that potassium excretion is independent of potasium ifitration and that potassium secretion is related to the amount of Na delivery. Since the early work of Granthanm et al. (1970), the cortical collecting duct has been firmly established as the main site of potassium secretion. During past several decades, tremendous efforts have been devoted to understand K secretory mechanisms, and this process has been relatively well characterized.

It has been believed that at least four systemic factors affect primarily potassium secretion: potassium intake and total body potassium content; acidbase balance; sodium intake and extracellular fluid volume; chloride and noncliloride anions delivery (Wright, 1987). Generally speaking, the observations that increases in potassium intake and in total body potassium content stimulate potassium secretion have been attributable to the increase plasma K and circulating level of aldosterone. Changes in acid-base balance affects potassium excretion mainly because they modify distal flow rate, intracellular and tubular pH, and the anion composition of luminal fluid of distal tubule. Increases in sodium intake and in extracellular fluid volume promote potassium secretion (although they supress plasma aldosterone concentration) mainly by increasing








6
the driving force for K secretion and the flow rate of fluid passing the cortical collecting duct. Reduction of chloride level in the distal luminal fluid stimulates potassium secretion by stimulating potassium and chloride co-transport mechanism. Elevated concentrations of nonchloride anions in arterial plasma generally increase potassium secretion through modification of the anion composition of distal tubule fluid. Because of the relevance to the present studies, this section wil be limited to review the relationship of acid-base disturbances and sodium absorption with potassium secretion and the current knowledge of the cellular model underlying potassium secretion.

Perhaps, Toussaint and Vereerstraeten (1962) first examined the relationship between acid-base balance and potassium secretion using clearance methods. The results are shown in Figure 1-3, in which the rate of renal potassium excretion is plotted as a function of plasma potassium concentrations at three different pH levels. It is clear that metabolic alkalosis stimulates and acute metabolic acidosis depresses the urinary excretion of potassium. The site of the alteration of potassium secretion during acid-base disturbances has been investigated more definitively by several approaches including free-flow micropuncture (Malnic, 1971) and in vitro microperfusion (Boundxy et al., 1976). It is known that infusion of bicarbonate, or administration of carbonic anhydrase inhibitor, or hyperventilation of animals stimulates K secretion along the distal convoluted tubule (Kubota et al., 1983). In contrast, both acute metabolic and respiratory acidosis decreases potassium secretion in the distal tubule (Giebisch and Stanton, 1979; Malic et al., 1971). Boundry et al. (1976) have demonstrated that potassium secretion by the cortical collecting duct was reduced when perfused in vitro by lowing luniinal pH. The development of








7
electrophysiology techniques, especially the patching-clamp techniques, has extended our understanding of mechanisms explaining the relationship between acid-base disturbance and K secretion at the cellular level. Potassium secretion involves the translocation of this ion across the double-membrane. First, potassium is actively uptaken to cell by Na-K-ATPase located in the basolateral membrane. Second, potassium passively exits out of the cell to lumen in part through K-conductive pathway. Potassium conductance is pH sensitive. Decrease in pH has been shown to inhibit this conductive pathway (Stanton et al., 1982; O'Neil, 1983). Recently, low-conductance K channels have been identified in the apical membrane of the cortical collecting duct (Frindt and Palmer, 1989; Wang et al., 1990). The characteristics of this type of channels have made it to be a strong candidate for potassium conductance. Reducing pH also decreases open probability of this type of channels (Wang et al., 1990). Acidosis has been shown to inhibit Na-K-AT~ase and reduce intracellular potassium activity (Giebisch, 1987). Inhibition of Na-K-ATPase and reduction of intracellular potassium activity inhibit K secretion by reducing the driving force for K exit out of the apical membrane (Giebisch, 1987; Wingo, 1984; Wright and Giebisch, 1992). Figure 1-4 summarizes the effect of different types of acid-base disturbances on potassium secretion obtained from micropuncture studies in the rat.
The idea that potassium secretion was coupled to sodium absorption was proposed on the basis of three major observations made in the late of fifties and the early of sixties. (1) In 1958, Davidson et al. (1958) reported their observations that sodium delivery drastically stimulated potassium secretion, particularly when sodium excretion was initially maintained at low level; (2)








8
Adrenal steroids increased both sodium absorption as well as potassium secretion (Sansom and O'Neil, 1985 & 1986); and (3) there was no evidence suggesting that tubular anion secretion could account for the amount of potassium secreted. In the in vitro perfused tubules, the experimental minipulations expected to abolish Na absorption such as removal of luminal Na, blocking Na entry by amiloride, and inhibiting basolateral Na exit by ouabain decreased K secretory flux (Stokes, 1981; Wingo, 1984). These studies not only reproduced the in vivo observations, but also provided the clear evidence of dependence of potassium secretion on sodium transport. The advance of technology has permitted us to understand the effect of sodium on potassium secretion at the cellular level. In all of the epithelial cells, sodium movement across the apical membrane are passive and driven by sodium concentration gradient and electrical potential gradient. The sodium concentration gradient is maintained by Na-K-ATPase located at the basolateral membrane. Sodium entry to the cortical collecting duct from the apical membrane is not exceptional. This sodium permeability is stimulated by circulating level of aldosterone (Sansom and O'Neil, 1985) and inhibited by administration of amiloride, a potassiumsparing diuretics (O'Neil and Boulpaep, 1979). The prevailing explainations for dependence of potassium secretion on sodium are listed as following (Wright and Giebisch, 1992): first, Na entry to the cell depolarizes apical membrane pontential, the depolarization of apical membrane potential generates a diffusion potential that favors K secretion; second, Na-K-ATPase is essential to maintain intracellular K activity above its electrochemical equilibrium, which is critical for potassium secretion, sodium entry into cell would provide the appropriate stimulus for increased activity of the basolateral Na-K-ATPase.








9
Two major cell types have been identified in the CCD (Figure 1-5). The majority cell type is the principal cell which is believed to be responsible for K secretion (Wright and Giebisch, 1992). The minority cell is the intercalated cell. More and more evidence suggests that this type of cell participates in proton secretion and potassium absorption (Mujais and Katz, 1992). Current concepts of the principal cell mode are based on the double-membrane model originally proposed by Koefoed-Johnsen and Ussing (1958). Its essential features and some modifications are summarized in Figure 1-6. (1) The apical membrane of cell is highly and selectively permeable to Na and K; (2) the basolateral cell membrane is highly potassium selective, (3) the only active transport operation is the ATP-driven sodium-potassium exchange pump in the basolateral cell membrane. A still growing fund of information derived from the efforts of many investigators, especially Drs. Berliner and Giebisch, and their coworkers, during past several decades has significantly contributed to the conversion of this simple original model into the more complex cell model that endows the tubular cells with capacity of potassium secretion. The current knowledge of principal cell with respect to K secretion is summarized in Figure 1-5.
1. Sodium (Na) enters the cell from luminal fluid passively through amiloride-sensitive Na conductive pathway. The sodium permeability plays an important role in potassium secretion as discussed before.
2. A Ba-sensitive K conductance is present in the apical membrane. This highly K-selective pathway provides an important route for potassium secretion from cell to lumen. The K-conductance accounts for the majority of conductance in the apical membrane of the principal cell, and is stimulated after administration of mineralocorticoids or increase in K intake, and inhibited








10
by acidification or by lowing K intake. This transport step has been extensively demonstrated to be passive in nature. Accordingly, lowing the potassium concentration in the lumen or increasing lumen-negative potential through external current application stimulates potassium secretion by steeping the electrochemical potential difference between cytoplasma and luminal fluid.
3. Evidence based on in vitro microperfusion study is consistant with potassium secretion in part through K-Cl cotransport (Ellison et al, 1985; Wingo, 1989). Reduction of chloride concentration in the perfusate stimulates potassium secretion. This component of potassium secretion is not associated with alterations in the transepithelial voltage and, importantly, is not blocked by barium.
4. Na-K-ATPase located in the basolateral membrane is the main transport element. This active uptake step responds to changes in acid-base disturbances, changes in plasma potassium level, and alterations in circulating level of plasma aldosterone. The increase in the plasma level of K or aldosterone stimulates Na-K-ATPase activity, and acidosis inhibits the activity of this enzyme.
1.3 Potassium Absorption by the Cortical Collecting Duct
In contrast to potassium secretion, little is known about the mechanisms of K absorption. As early as 1954, Spargo (1954) reported that the first mophologic changes during K depletion were in the collecting duct with a hyperplastic lesion developing in the inner red medulla, suggesting that collecting duct might participate in K conservation. Ten years later, from the micropuncture studies, Malnic, Klose and Giebisch (1964) demonstrated that during K depletion urinary fractional excretion of K was less than fractional








11
delivery of K to the late of distal nephron of superficial nephrons. This is the first physiological evidence of involvement of the collecting duct in active K absorption in response to K depletion. The postulate for active potassium absorption was based on the conditions which were characterized by complete absence of net potassium secretion, yet persistence of a distinctly lumen-negative transepithelial voltage. In the absence of other transport mechanisms, potassium should be secreted to lumen down its electrochemical gradient. To counterbalance the potassium secretion, an active force must move potassium out of the lumen. This suggests that an active potassium absorptive mechanism is present in the apical cell membrane, and the segment traditionally conceived as K-secretory also plays K conserving roles, especially when the balance of K requires it. Linas et al. (1979) studied the adaptation of the rat to a low K diet and found that the recovery of distally injected "K was significantly reduced 72 hours after exposure to the diet. Wingo (1987) has shown that K absorption was increased by the in vitro perfused outer medullary collecting duct dissected from K-restricted rabbits and provided the direct evidence substantiating the role of the collecting duct in K conservation. The further observations from mophological studies have demonstrated that the intercalated cells of the collecting duct undergo hypertrophy and ultrastructural changes during K restriction and acidosis, suggesting that these cells are involved in K absorption and H secretion (Hansen et al., 1980; Ordonez and Spargo, 1976; Stanton et al., 1981). However, the mechanisms underlying these observations remain unclear. The discovery of K-ATPase in rat and rabbit kidney made by Doucet and Marsy in 1987 (1987) has seeded more light on this poorly understood component. ATP hydrolysis by this enzyme is K-dependent and








12
reaches the maximal activity with 1 mM K (Km about 0.2-0.4 mM). Sodium, choline, chloride and sulfate do not stimulate ATP hydrolysis by this enzyme. Ouabain, a specific inhibitor of Na-K-ATPase does not inhibit the activity of this enzyme, indicating that this enzyme is different from Na-K-ATPase. In contrast, omeprazole, an inhibitor of gastric H-K-ATPase, inhibits ATP hydrolysis by this enzyme, suggesting that this enzyme is pharmacologically similar to gastric H-K-ATPase. Because vanadate also inhibits this enzyme, K-ATPase has been classified as E1-E2 type ATPase. The activity of this enzyme is proportional to the density of intercalated cells, highest in the connecting duct, intermediate in the cortical collecting duct, and lowest in the outer medullary collecting duct, and not detectable in all other nephron segments. Hydrolysis of ATP by this enzyme in rat kidney increased by 100-200% following adaptation of the animals to a low K diet. In 1988, Garg and Narang (1988) reported a similar K-ATPase was also present in the rabbit collecting duct from their fluorometric mnicroassay studies. This enzyme shares similar distribution pattern in the kidney as to that reported By Doucet and Marsy. This enzyme is inhibited not only by omeprazole and vanadate, but also by SCH28080, an imidazopyridine derivative shown in Fig. 1-7 which has been extensively studied in the gastric system and demonstrated to be a specific inhibitor of gastric H-KATPase (Beil et al., 1986 & 1987 & 1988; Dantzig et al., 1991; Mendlein and Sachs, 1990; Scott et al., 1987). Chronic hypokalemia stimulates the activity of this enzyme, and chronic hyperkalemia suppresses this enzyme (Garg and Narang, 1989). In 1989, Wingo (1989) observed that omeprazole inhibited both net K absorptive flux and total CO2 flux in the in vitro microperfused medullary collecting duct without significantly changing the transepithelial








13
voltage, providing the functional evidence that an H-K-ATPase is present in this segment. The results from microenzymatic assay indicate that K-ATPase is stimulated by K and by Rb in a similar fashion. The apparent stoichiometry of this enzyme is 1 Rb:1 ATP. Adaptation of animals to a low K diet not only increases ATP hydrolysis, but also Rb uptake by this enzyme. SCH28080 has been demonstrated to inhibit initial Rb uptake in the rat collecting duct (Cheval et al., 1991) and proton (H) secretion in the amphibian nephron (Planelles et al., 1991). Although at the molecular level whether renal H-K-ATPase is similar to gastric H-K-ATPase, or colonic H-K-ATPase has not been reconciled, several investigators have expressed mRNA of a putative H-K-ATPase in the kidneys (Gifford et aL, 1991; Okusa et al., 1990), and this expression is enhanced by respiratory acidosis. By using mouse monoclonal antibodies against hog gastric H-K-ATPase, Wingo et al. (1991) revealed diffuse cytoplasmic staining indicating H-K-ATPase immunoreactivity in intercalated cells in the cortical collecting duct and outer medullary collecting duct of both rat and rabbit. The percentage of H-K-ATPase immunoreactive cells has been demonstrated to correspond to the percentage of intercalated cell except in rat CCD in which the percentage of positive staining is less than the percentage of intercalated cells.
Besides it has been well characterized in the gastric gland, a similar HK-ATPase has been also identified in colonic epithelium (Kaunitz and Sachs, 1986; Suzuki and Kaneko, 1987 & 1989) and turtle urinary bladder (Sharma et al., 1991). The evidence obtained from functional studies suggests that H-KATPase may be also present in the amphibian jejunum (Imon and White, 1984), smooth muscle cell and the Manduca sexta embryonic cell line CHE (English and Kantley, 1985).








14
1.4 The Aims and Objectives of the Present Studies
The discovery of H-K-ATPase is a novel approach to characterize K absorption. Its significance in both understanding basic K transport mechanism and clinical implications may significantly extend our present knowledge. This dissertation attempts to characterize potassium permeation by the CCD with emphasis of H-K-ATPase in the conditions which are close to in vivo without equivocating the interpretation of the results. In vitro micropersion technique is able to serve this dual-purposes. Potassium permeation was evaluated as MRb lumen-to-bath efflux coefficient (Kn, or Rb efflux). The H-K-ATPase activity was assessed as SCH28080-sensitive Ku or SCH28080-sensitive Rb efflux. The specific objectives are summarized as following:
1. Because H-K-ATPase secretes proton in exchange of K absorption, we will determine whether acute peritubular acidosis (10% CO2) stimulates Rb efflux, whether this stimulation is dependent on the presence of functional H-KATPase. We will also identify the intracellular and basolateral mechanisms explaining the effect of 10% CO2 on Rb efflux.
2. Because luminal amiloride and peritubular ouabain has been shown to inhibit net K excretion, we will examine whether luminal amiloride and peritubular ouabain increases Rb efflux, and whether the effects of amiloride and ouabain are mediated by K-conductance or by H-K-ATPase. Because of the indirect evidence suggesting that an apical Na-K-ATPase may participate in potassium conservation, we will identifiy the roles of H-K-ATPase and Na-KATPase in mediation of Rb efflux.
3. Because removal of luminal Na produces similar effect on ion transport in many cases as the addition of luminal amiloride, we will examine








15
whether Na removal stimulates Rb efflux, whether the effect of Na removal is mediated by K-conductance or by H-K-ATIase or both. We will also determine the mechanism underlying the effect of Na removal on H-K-ATPase.
Additional efforts have been made to study hormonal regulation of this pump. The results of metabolic studies on a low Na, low K diet, and a low K diet are also reported1.

































All of the studies were funded by the Department of Veterans Affairs







16












100 mEq /day GI intake
MUSCLE :
CELLS


65 Eq
Extracellular
Liver 200 mEq fluid
calls

RBC 235 mEq Urine Stool
92 mEq/day 8 mEq /day
















Figure 1-1. Distribution of potassium in the body, including routes of acquisition and excretion (Giebisch et al, 1981).












17






















P"MINAL DISTA %.
TuwLE ow-co"p-lis TUGULt








MUM

mccoLLA mrt"
z






Loop or *emu





























F 7- m 'W ys f tbule P' m u mp ti Ao,, md,,at,
L gPget tmnZbulan Omme nt (tGebmsht:t alo 981).








18












NORMAL .
N; TM











PLASMA 9 CONCENTRATION .Cq/I













Figure 1-3. Urinary potassium excretion as a function of plasma potassium concentration in control animals and in alkalotic and acidotic conditions (Toussaint and Vereerstraeten, 1962).








19













Control 24%

Metabolic acidosis 0%

Respiratory acidosis 6%

Metabolic alkalosis 64%

Respiratory alklosis -" 39%

Metabolic + Respiratory 75%
elkolosis
Metabolic olkalosis *27%
Respiratory acidosis





















Figure 1-4. Effect of different types of acid-base disturbances on potassium secretion by distal tubule (Giebisch et al, 1981).







20







LUMEN CELL BLOOD




---C I Na Principal cell








K H Na Intercalated cell












Figure 1-5. The cellular models of the principal cell (top) and the intercalated ceU (bottom).







21














spcim baotratwo



-- 2K*
























Figure 1-6. Cell model of epithelial sodium and potassium transport (Giebisch, 1987).






22










N CH2 CN N CH3
0

CH2


0
SCH 28080










Figure 1-7. The chemical structure of SCH2S88.













CHAPTER 2
GENERAL METHODOLOGY


2.1 In Vitro Microperfusion
In vivo conditions. Female New Zealand White rabbits were maintained on a regular diet (Ralston Purina), or a low K (K-restricted) diet (0.25% K, TD 87433, Teklad, Madison, WI) as appropriate and allowed free access to tap water. The exposure time to the low K diet was at least for 4 days before experimentation. The majority of experiments were performed on the Krestricted rabbits unless indicated. Rabbits were adapted to the K-restricted diet in order to enhance the signals.
In vitro methods. Standard in vitro microperfusion methods (Burg et al, 1966) as modified in this laboratory (Wingo, 1984 & 1985) were used. Briefly, rabbits were decapitated, the left kidney was quickly removed, and 1- to 2-mm slices were placed in a chilled petri dish containing an artificial ultrafiltrate of plasma. Dissection proceeded superficially from the corticomedullary junction. Tubules were transferred to a thermostatically controled chamber (37oC), and the two ends of the tubule were aspirated into holding pipettes (Figure 2-1). The perfusing pipette was advanced 100 um beyond the holding pipette, and transepithelial voltage (VT) was continuously monitored by means of Ag/AgC1 electrodes and a FD-223 high-impedence electrometer (World Precision Instruments, Sarasota, FL). The bath solution was continuously exchanged at the rate of 0.64 mlmin"n. The perfusate contained 50 uCi of [methoxy-3H]inulin

23








24
exhaustively dialyzed according to the method of Schafer et al. (1974). The equilibration time between two periods was 30 minutes unless indicated. Effluent fluid was collected into a constant-volume pipette for measurement of volume flux, isotopic flux and net chemical flux. Volume flux was determined from timed collections of the effluent fluid using the equation Jv = ([cpm./cpmi-1]VoV)/L. where Jv is the net volume absorption in nanoliters per millimeter per minute, cpmo and cpmj are the [3]-inulin counts per minute per nanoliter in the collected and the perfused fluid, respectively, V. is the rate of fluid collection in nanoliters per minute, and L is the tubule length in millimeters. In all experiments, the percent of [3I]-inulin leak was less than 5% or the experiment was discarded. In most tubules the leak rate was less than 2%. Isotopic "Rb was used because it has been shown to be a qualitative marker of K efflux (134), and is transported by the renal H-K-ATPase quantitatively similar to K (14). The "Rb lumen-to-bath efflux (Kas) was determined by the disappearance of "Rb from the perfusate according to the following equation:

V Rbi-RbRbi + Rb.


where Rbi and Rbo are the "Rb counts per minute per nanoliter in the perused and collected fluid, respectively. The Na efflux rate coefficient (KN) was calculated by a similar equation. Counts for 3H, "Rb and 'Na were measured by a liquid scintillation counter (LS-7800, Beckman Instruments, Irvine, CA). The overlap of "Rb or nNa counts in the 3H channel was corrected as previously described (Zhou and Wingo, 1992). Net chemical flux was determined








25
by the equation Jx = (VjL)*([XP-[XIo) where V. and L are as before and [X]i and [X]o are the sodium or potassium concentration of the perfusate and collectate, respectively. At least three and generally four collections were obtained for measurement of flux. The passive paracellular K flux was calculated according to the Goldman flux equation kb MI ex[VT& /(RT)]
VT*

where PK is the paracellular K permeability, [K]b is the bath K concentration, [K], the mean luminal K concentration, and z, F, R, and T have their usual meaning. All chemicals were analytical grade or the highest available purity.
- inulin, "Rb and zNa were obtained from New England Nuclear (Boston, MA).
2.2 Flameless atomic absorption speetrophotometry
Flameless atomic absorption spectrophotometry described by Wingo et al (1987) was used for the analysis of sodium and potassium concentrations in the perfusate and collectate in order to determine net sodium and potassium flux. Briefly, the standards and samples were placed on a clean silver platform, freshly polished, and immersed under 0.5 cm of water-equilibrated paraffin oil. The diluent was prepared from 100 ;1 Ultrex (J. T. Baker chemical Co.) and 100 ml ultra-high-purity (18 Mohm) water ("18 Mhom water," Continental Water System) in a clean polypropylene container. To prevent loss of the analyte, the samples were slowly dried before pyrolysis and atomization (951 AA/AE spectrophotometer and IL 655 furnance atomizer, Allied Analytic, Waltham, MA). Peak height integration started at the end of the pyrolysis and continues for 8 seconds in the single beam mode at a scale expansion of 1.00 for both








26
sodium and potassium. The first atomization step of 500 degree of centigrade to 1500 degree of centigrade in "0" second allows a rapid increase in temperature during atomization. The instrumental conditions for analysis of Na and K were set as followings : 589.6 and 766.5 nm, bandwidth; 0.15 and 1.0 mm, lamp current; 8.0 and 7.0 mamp. N2 gas pressure was 12.0 psi. The spectrophotometer was allowed to warm up for at least one hour and then "auto zeroed" by atomizing without sample introduction into the furnance. The diluted samples were delivered into a pyrolytically coated graphite cuvette in the furnance with a 1- to 10-Al Eppendorf digital pipette. The exact volume chosen for a given analysis was determined by repetitive runs of the high and low standards and adjusting the delivery volume for an optimum absorption signal from the sodium and potassium channels. Each sample was analyzed for Na and K a minimum of three times; generally four to six runs were employed.

2.3 Statistical analyses
Data are expressed as mean standard error. Statistical analyses were performed by paried t test for the experiments containing two periods and by analysis of variance (ANOVA) for repeated measures of the experiments containing more than two periods. Post hoc comparisons were made by the Ryan-Einot-Gabriel-Welch F test. The null hypothesis was rejected at the 0.05 level of significance.








27








































Figure 2-1. The cortical collecting duct perfused in vitro.













CHAPTER 3
EFFECT OF 10% CO2 ON Rb EFFLUX


3.1 Introduction
Substantial evidence indicates that an H-K-ATPase is present at the apical membrane of the cortical collecting duct (Cheval et aL, 1991; Doucet and Marsy, 1987; Garg and Narang, 1988; Gifford et al., 1991; Okusa et al., 1990; Planelles et al., 1991; Wingo, 1989; Wingo et al., 1989 & 1990; Wingo and Straub, 1991; Wingo and Zhou, 1990; Zhou and Wingo, 1992). This enzyme has been demonstrated to be responsible for potassium absorption in exchange of proton secretion, especially during K-restriction (Cheval et al., 1991; Wingo, 1989; Zhou and Wingo, 1992). Komatsu and Garg (1991) have shown that metabolic acidosis increases ATP hydrolysis by this enzyme, suggesting that acidbase balance regulates the activity of this enzyme. Perrone and McBride (1988) have demonstrated that 10% carbon dioxide (CO2) increases rubidium absorption in colon, possibly mediated by a related pump. However, whether respiratory acidosis stimulates one of the physiologic functions of the renal H-K-ATPase, i.e. K absorptive flux, is unknown. Nevertheless, to our knowledge, the direct relationship between acidosis and K absorptive flux has not been demonstrated in the CCD. Therefore, the first objective of this study is to examine whether respiratory acidosis induced by 10% CO2 increases K absorption, and whether this increment is dependent on the presence of a functional H-K-ATPase in the in vitro microperfused CCD from K-restricted rabbits.

28








29
The second issue is the dependence of proton secretion in the collecting duct on carbonic anhydrase. Carbonic anhydrase has been identified in the CCD (Dobyan and Bulger, 1982). Carbonic anhydrase catalyzes the hydration of CO2 and an increase in pCO2 results in a decrease intracellular pH (Cannon et al., 1985). The decrement in intracellular pH is a direct stimulus for H secretion (Cannon et al., 1985; Schwartz and Al-Awqati, 1985; Stetson and Steinmetz, 1983 & 1986; Van Adelsberg and AI-Awqati, 1986). Because H-K-ATPase participates in H secretion, it is plausible that the enhancement of K absorption resulted from the activation of H-K-ATPase by 10% CO2 is carbonic anhydrasedependent. However, several investigators have demonstrated that acidification by the collecting duct is in part independent of carbonic anhydrase (Cogan et al., 1979; DuBose and Lucci, 1983; Frommer et al., 1984; Laski, 1987; McKinney and Davidson, 1988). Sharma et al. (1991) have suggested that not all H-K-ATPase activity is dependent on the function of carbonic anhydrase. Therefore, the second objective of this study is to evaluate the role of carbonic anhydrase in the stimulation of H-K-ATPase-mediated K absorption and the role of carbonic anhydrase in maintaining basal rate of K absorptive flux in the Krestricted CCD.
The third issue is the intracellular mechanisms involved in the effect of 10% CO2. Exocytotic insertion of H-K-ATPase in response to several stimuli has been demonstrated in gastric gland (Forte et al., 1981). This process is cytoskelton-dependent. The majority of exocytosis is also calcium (Ca)-dependent, more importantly, exocytotic fusion of H-ATPase has been shown to be Camediated in the turtle urinary bladder (Schwartz and Al-Awqati, 1985 & 1986; Van Adelsberg and AI-Awqati, 1986). Therefore, the third objective of the








30
present studies is to evaluate the role of microtubules, intracellular calcium activity, and calmodulin in the stimulation of Rb efflux (presumably activation of H-K-ATPase) by 10% CO2.
The fourth issue is the mechanism explaining basolateral K exit. In the rabbits adapted to a normal diet the minority cells (presumably intercalated cells) exhibit quite low basolateral K conductance (Muto et al., 1987). However, Schlatter and Schafer (1987) have identified that a Ba-sensitive K conductance is present in the basolateral membrane of both principal cells and intercalated cells of the CCD of rats. If a similar pathway is present in the K-restricted rabbits, this pathway could mediate K absorption. Therefore, the fourth objective of this study is to examine whether a Ba-sensitive pathway is present in the basolateral membrane of the CCD, and whether this pathway mediates the stimulation of K absorption by 10% CO2 in the K-restricted rabbits.
The final issue to be addressed by the present studies is whether 10% CO2 stimulates Rb efflux in the CCD dissected from normal rabbits.

3.2 Methods and Materials
The tubules was dissected in an artificial ultrafiltrate of plasma (in mM; Na 145; K 5; Cl 112; HCO3 25; Ca 1.8; P04 2.3; Mg 1.0; SO4 1.0; acetate 10; glucose 8; and alanine 5) gassed with 5% CO2 containing additional 5% vol/vol fetal calf serum. The bath solutions were gassed with 5% CO2 (pH = 7.4 0.0) or 10% CO2 (pH = 7.1 0.0) as appropriate. The electrolyte and nonelectrolyte concentrations of the bath solution were identical to the dissection solution unless indicated. In the peritubular Ba study, the composition of bath were (in mM): Na 135; K 5; Cl 115.4; HCO3 25; Ca 1.2; Mg 1.0; acetate 10; glucose 8; alanine 5; Ba 3; and mannitol 4.5. In the Ba








31
dose-response study, the composition of bath were (in mM): Na 135; K 5; Cl 106.4; HCO3 25; Ca 1.2; Mg 1.0; PO4 1.5; acetate 10; glucose 8; alanine 5; mannitol 26.5. The equilibration time between two periods was 30 minutes unless indicated. The perfusate was identical to bath except for the absence of fetal calf serum and gassed only with 5% CO2 unless indicated. The composition of the perfusate for peritubular Ba study were (in mM): Na 138; K 5; Cl 109.4; HCO3 25; Ca 1.2; Mg 1.0; acetate 10; glucose 8; alanine 5; mannitol 9; and P04 1.5. Because Ba precipitates with phosphate, HEPES (N-2Hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffer was used as the perfusate in the Ba dose-response study. Warden et al 0() have shown that Rb efflux by the CCD perfused with HEPES-buffer is not significantly different from that perfused with bicarbonate-buffer. Total three perfusates listed in Table 3-1 were used in this set of studies.
SCH28080 (gift of Dr. James Kaninsky, Schering Corporation, Bloomfield, NJ) was dissolved in dimethyl sulfoxide (DMSO) and applied to the perfusate with final concentration of 10 pM. Methazolamide (American Cyanamide Company, Pearl River, NY), Bis-(2-amino-5-methyl-phenoxy)-ethane-N,N,N',N'tetraacetic acid tetraacetoxymethyl ester (MAPTAM, Sigma, St. Louis, MO), and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7, Sigma, St. Louis, MO) were also dissolved in DMSO and applied to bath with final concentration of 0.1 mM, 0.5pM, and 0.5pM, respectively. Colchicine (Sigma, St. Louis, MO) was dissolved in 0.9% NaCl solution and applied to bath with the final concentration of 0.5mM. The final concentration of 0.1 % DMSO was present in bath during basal period of methazolamide sets of experiments.

3.3 Results








32
To examine whether acute respiratory acidosis stimulates Rb efflux, we perfused the CCD with symmetrical Ringer's bicarbonate solution gassed with 5% CO2 (5% CO2 period) or 10% CO2 (10% CO2 period). In our initial studies we allowed one hour equilibration after obtaining basal flux rates prior to a second period of measurement in the presence of 10% CO2. As shown in Figure 3-1, 10% CO2 substantially increased Kp, from 93.1 23.8 nmsec' to 249 60.2 nmsec1 (P < 0.05, n = 7). Concomitantly the transepithelial voltage (VT) became more lumen-positive from -2.4 1.3 mV to -1.2 0.9 mV (P < 0.05, n = 7, Fig. 3-1). The greater lumen-positive voltage could enhance paracellular Rb passive efflux. To examine whether the change in VT accounted for the effect of 10% CO2 on Rb efflux, we calculated the voltagemediated Rb efflux by Goldman flux equation. As shown in Figure 3-2, the change in VT can not explain for the increase in Kft by 10% CO2. Voltagemediated Rb efflux as predicted by Goldman flux equation was only 10.6 3.7 nm-sec"1, whereas the increment of 156 58.4 nmsec1 was observed. To examine whether H-K-ATPase mediated the effect of 10% CO2 on Rb efflux we repeated the experiments under the identical conditions except for the presence of luminal 10 AM SCH28080. Luminal SCH28080 totally abolished the stimulatory effect of 10% CO2 on Rb efflux (76.4 15.1 umec1 vs 76.8 13.3 nmsec-1, n = 5, Fig. 3-3), although VT became more lumen-positive during 10% CO2 period (-14.6 9.6 mV vs -10.7 9.1 mV, P < 0.01, n = 5, Fig. 3-3). In additional experiments, there was no evidence to suggest that 0.1% DMSO affected either Kb or VT (Table 3-2). These data suggest that an H-K-ATPase, not the change in VI, mediates the enhancement of Rb efflux by 10% CO2. To examine the time course of the stimulation of Rb efflux by 10%








33
CO2, we perfused the CCD in the absence of luminal SCH28080. After measurement of the basal rate of Rb efflux (5% CO2 period), effluent fluid was collected from 30 to 120 minutes after exposure to 10% CO2. To evaluate the role of carbonic anhydrase in the stimulation of Rb efflux by 10% C02, 0.1 mM methazolamide was added to the bath from 120 to 180 minutes after exposure to 10% CO2. We were not only able to reproduce the previous stimulation of Kiu, by 10% CO2, but also observed this effect at the earliest collection time, i.e., 30 to 60 minutes later after the tubule exposure to 10% CO2. This effect was persistent for the entire 10% CO2 period. However, subsequent addition of methazolamide did not significantly inhibit Kpb (72.4 + 11.8 nmsec1, 5% CO2 period; 121 29.9 nmnsee4, 135 343 nmsec1, and 133 29.0 nmsecd1, 30-60 minutes, 60-90 minutes, 90-120 minutes after exposure to 10% C02, respectively; 133 29.0 nmsec1, methazolamide period, n = 6, Fig. 3-4). During the methazolamide period VT was significantly more lumen-negative (-3.0 2.2 mV, 5% CO2 period; -2.4 23 mV, -1.3 1.8 mV, and -0.8 1.6 mV, 30-60 minutes, 60-90 minutes, and 90-120 minutes after exposure to 10% CO2, respectively; -7.6 4.2 mV, methazolamide period, n = 6, Fig. 3-4). When the experiments were repeated under same conditions except for the presence of luminal 10pM SCH28080, SCH28080 totally blocked the enhancement of Rb efflux in response to 10% CO2 and methazolamide had no significant effect on Rb efflux under these conditions (90.8 16.5 nm-sec1, 5% CO2 period; 80.5 12.3 nm-sec", 83.9 14.3 nmsec", and 78.7 10.9 nmsec 1, 30-60 minutes, 60-90 minutes, and 90-120 minutes after exposure to 10% CO2, respectively; 81.9 13.5 nmsec-1, methazolamide period, n = 7, Fig. 3-5), although VT became more lumen-negative during methazolamide period








34
(-4.5 3.1 mV 5% CO2 period; -3.2 2.5 mV, -2.0 2.8 mV, and -0.8 2.6
mV, 30-60 minutes, 60-90 minutes, and 90-120 minutes after exposure to 10% CO2, respectively; -7.8 3.2 mV, methazolamide, n = 7, Fig. 3-5). These data not only confirm that an H-K-ATPase mediates the stimulatory effect of 10% CO2 on Km,, but also suggest that an existing H-K-ATPase mediates this effect. Again, the parallel VT responses to 10% CO2 regardless of the presence or absence of SCH28080 indicate that the changes in VT do not explain the effect of 10% CO2 on Rb efflux. The lack of inhibitory effect of methazolamide on K by subsequent addition after 10% CO2 implies that carbonic anhydrase may not be necessary to maintain the activation of H-K-ATPase by 10% CO2. In the presence of luminal SCH28080, methazolamide did not reduce Rb efflux either, suggesting that methazolamide had no effect on the other pathways of K permeation. To examine whether carbonic anhydrase is required for initiating the stimulation of Rb efflux by 10% C02, we simultaneously exposed the CCD to methazolamide and 10% CO2 after measurement of the basal rate of Rb efflux. Under these conditions we were not able to detect the stimulatory effect of 10% CO2 on Rb efflux (98.6 14.1 nm-ec"1 vs 86.2 16.5 nm'sec"1, n = 6, Fig. 3-6), although methazolamide significantly made VT more lumennegative from 13 0.9 mV to -4.1 1.2 mV (P < 0.005, n = 6, Fig. 3-6). Time-control experiments demonstrated that perfusion time did not significantly affect Kpb or VT (Table 3-3). These data suggest that carbonic anhydrase is necessary for initiating the stimulatory effect of 10% CO2 on Rb efflux. To examine whether methazolamide inhibits the basal rate of Rb efflux, we perfused the CCI in the presence of 5% CO2 throughout the experiments instead of changing to 10% CO2. As shown in Figure 3-7, methazolamide did








35
not significantly inhibit Kg (60.4 10.1 nmsec' vs 59.1 10.6 nmsec', n = 6), although the effect of methazolamide on VT was reproducible (-12.9 2.7 mV vs -27.1 4.8 mV, P < 0.05, Fig. 3-7, n = 6). This suggests that the basal Rb efflux may be dependent on methazolamide-insensitive pathway.
Exocytotic insertion of H-K-ATPase in response to several stimuli has been demonstrated in gastric gland (Forte et al, 1981). This process is cytoskelton-dependent. To examine whether microtubules mediate the effect of 10% CO2 on Rb efflux, the tubules were simultaneously exposed to 10% CO2 and 0.5 mM colchicine, an inhibitor of tubulin polymerization. Colchicine totally blocked the stimulatory effect of 10% CO2 on Rb efflux (85.0 15.0 nmsec1 vs 81.3 13.6 nm-sec-', n=6, Fig. 3-8), indicating that the stimulation of Rb efflux is dependent on intact microtubule function. Meanwhile, colchicine had no significant effect on VT (-12.9 4.9 mV vs -15.6 5.0 mV, n=6, Table 34). To further test the insertion hypothesis, we examined the effect of colchicine after stimulation of Kn by 10% CO2. Under these conditions, colchicine had no inhibitory effect on Rb effiux (72.5 7.9 nmsec', 5% CO2 period; 97.3 10.1 nmsec1, 10% CO2 period; vs 110 13.2 nmsec-1, 10% CO2 plus colchicine period', 'p<0.05 compared with 5% CO2 period, no significant difference between 10% CO2 and 10% CO2 plus colchicine period, n=6, Fig. 3-8). Voltage was significantly altered by this maneuver (-11.2 7.1 mV, 5% CO2 period; -16.9 9.4 mV, 10% CO2 period'; -17.4 9.5 mV, 10% CO2 plus colchicine period', p<0.05 as compared with 5% CO2 period, n=6, Fig. 3-3). These data are consistant with the postulation that exocytotic process mediates the effect of 10% CO2 on Rb efflux.








36
Because the majority of exocytosis is Ca-dependent, more importantly, exocytotic fusion of H-ATPase has been shown to be Ca-mediated in the turtle urinary bladder (Schwartz and Al-Awqati, 1985 & 1986; Van Adelsberg and AlAwqati, 1986), to examine whether the increase in intracellular Ca activity mediates the effect of 10% CO2, the CCD was perfused in the presence of 0.5mM MAPTAM, an intracellular Ca buffer, throughout the experiments. In this case 10% CO2 failed to stimulate Rb efflux (89.5 19.5 nmsec1 vs 85.6 23.6 nm-sec1, n=6, Fig 3-9). In addition, VT became more lumen negative during 10% CO2 period (-15.7 5.9 mV vs -23.9 6.8 mV', "p<0.05, n=6, Table 3-5). To evaluate the role of calmodulin in the stimulation of KP by 10% CO2, the CCD was perfused in the presence of 0.5M W-7, an
antaganizer of calmodulin, throughout the experiments. In the presence of W-7, Rb efflux was significantly reduced (89.3 14.0 nmsec1 vs 65.1 13.0 nm'sec1, p<0.05, n=6, Fig. 3-9), suggesting that the stimulation of Rb efflux by 10% CO2 is dependent on the functional calmodulin. Transepithelial voltage was not significantly altered (-12.2 3.9 mV vs -14.4 3.5 mV, Table 3-5).
Because Schlatter and Schafer (1987) have identified a Ba-sensitive K conductance located in the basolateral membrane of both principal cells and intercalated cells of the CCD of K replete rats, although the intercalated cells of the K replete rabbits exhibits a little Ba-sensitive K conductance (Muto et al., 1987). To examine whether a Ba-sensitive pathway is present in the Krestricted CCD of rabbits, and whether this pathway mediates the stimulation of Rb efflux by 10% CO2, we perfused the CCD in the presence of 3mM Ba in bath. Pretreatment with Ba totally abolished the stimulation of Ka, by 10% CO2 (73.0 8.2 nmsec"1 vs 70.9 8.3 nm-secs, n = 6, Fig. 3-10) without








37
significantly affecting VT (-2.2 3.0 mV vs -0.0 1.7 mV, n = 6, Fig. 3-10). This indicates that K conductance is present in the basolateral membrane under present conditions, and this conductive pathway mediates the stimulatory effect of 10% CO2 on Rb effilux.
To test whether normal CCD has the similar response to 10% CO2 as the K-restricted CCD, we examined the effect of 10% CO2 on the CCD dissected from regular dietary rabbits. Because the apical Ba-sensitive Kconductance has been demonstrated to participate in Rb efflux (Warden et aL, 1989; Wingo and Zhou, 1990) also because the decrease pH has been shown to inhibit this conductive pathway (Boudry et al., 1976; Wang et al., 1990), in order to eliminate the overshadowing effect of 10% CO2 on K-conductance and H-K-ATPase, the tubules were perfused in the presence of luminal Ba. To find out what concentration of Ba maximally inhibits Rb efflux, the first set of experiments was designed to examine the dose-response of Ba with the perfusates of solution A, solution B, and solution C designated as 0 mM Ba, 2 mM Ba, and 4 mM Ba periods, respectively. The order of these three periods were rotated according to balanced Latin-square design (Cochran and Cox, 1957) to control for time-dependent effect on Rb efflux and the order of the periods did not significantly affect Rb efflux. As shown in Fig. 3-11, 2 mM Ba is sufficient enough to inhibit Kp, (76.3 14.6 nm.sec1, 0 mM Ba period; 32.9 4.4 nmesec1, 2 mM Ba period; and 32.1 9.3 nm.sec1, 4 mM Ba period). The next set of experiments was performed in the presence of luminal 2 mM Ba to examine the effects of 10% CO2 and 0.1 mM methazolamide. 10% CO2 and methazolamide had no significant effect on Rb efflux (48.1 7.2 nmesec"1, 5% CO2 period; 42.5 8.3 nmsec-1, exposure to 10% CO2 from 30 to 60








38
minutes; 44.6 7.5 nm.sec"1, exposure to 10% CO2 from 60 to 90 minutes; and 44.5 9.2 nm.sec", 10% CO2 plus methazolamide period, n=6, Table 3-6), although VT was significantly was significantly changed following exposure to 10% CO2 and methazolamide (-35.0 11.6 mV, 5% CO2 period; -44.2 14.0 mV, exposure to 10% CO2 from 30 to 60 minutes; -46.9 14.7 mV, exposure to 10% CO2 from 60 to 90 minutes; and -50.7 14.5 mV, 10% CO2 plus methazolamide period, n=6, Table 3-6). Because Oberleithner and his associates (1990) have demonstrated that H-K-ATPase requires K conductance for its function, and Ba secondarily inhibits H-K-ATPase in Madin Darby Canine Kidney (MDCK) cell, the CCD was alternatively perfused in the absence of luminal Ba. As shown in Table 3-6, 10% CO2 did not have trend to stimulate Rb efflux (73.1 37.9 nmsec"1, 5% CO2 period; 66.9 40.1 nmsec-, exposure to 10% CO2 from 60 to 90 minutes; and 64.4 32.2 nmsec-, exposure to 10% CO2 from 90 to 120 minutes, n=2, Table 3-6).

3.4 DjcQn
3.4.1 Effect of 10% CO2
To our knowledge, the present studies provide the direct evidence that respiratory acidosis (10% CO2) stimulates K absorptive flux, assessed as 86Rb lumen-to-bath effiux coefficient (Kab), in the CCD of K-restricted rabbits. Moreover, this enhanced Keb appears mediated by an H-K-ATPase. The rapid response to 10% CO2 suggests that the stimulus increases either the H-KATPase units in the apical membrane or the kinetics of this pump. Exocytosis of H-K-ATPase to the apical membrane in response to the several stimuli have been demonstrated in the gastric gland (Forte et al., 1981). In addition, CO2 has been shown to be lack of major effects on kinetics of H pumps in turtle








39
urinary bladder (Schwartz and AI-Awqati, 1986). The present studies are consistent with the hypothesis that 10% CO2 stimulates the exocytotic insertion of H-K-ATPase to the apical membrane of the collecting duct. 10% CO2 stimulates H secretion in gastric gland (Kidder and Montgomery, 1974). Wingo (1989) and other investigators (Planelles et al., 1991) have shown that renal HK-ATPase is responsible not only for K absorption, but also for H secretion. Indeed, microcatheterization studies in rats with acute respiratory acidosis demonstrated that acidification was augmented prior to the inner medullary collecting duct (Bengele et al., 1984). From the in vitro microperfusion studies, Laski and Kurtzman (1990) have observed that total CO2 flux was significantly enhanced by the CCD after the rabbits were exposed to elevated CO2 tension chamber, indicating that the CCD increases its acidification rate in response to hypercapnia. More direct evidence supporting the positive role of CO2 in acidification comes from McKinney and Davidson (1988) in vitro perfusing tubule study. They have found that total CO2 absorption was profoundly increased by the CCD following exposure to 15% CO2 for at least 20 minutes. On the other hand, by acute reduction of peritubular pCO2 from 40 to less than 14 mmHg, Jacobson (1984) has shown that bicarbonate absorption significantly decreased by medullary collecting duct. However, some studies were not able to demonstrate stimulatory effect of CO2 on acidification (Breyer et al., 1986; Lucci et al., 1982). We have no mechanism to reconcile these data. It is pertinent to ask whether the H-K-ATPase, or H-ATPase, or both, mediate this effect. The application of the specific inhibitor for each enzyme would shed more light on this question. Nevertheless, exocytotic insertion process has been proposed to be a strong candidate to mediate this enhancement in








40
acidification by increase in tension of CO2 (McKinney and Davidson, 1988). The observations made at cellular level further strengthes such a hypothesis H-K-ATPase has been localized by inmunocytochemistry to the intercalated cell in the CCD and outer medullary collecting duct (Wingo et al, 1989 & 1990). Madsen and Tisher (1983) have quantified that respiratory acidosis increases the surface density of the apical membrane concomitantly with decrease in the number of tubulovesicular profiles in the apical region of the intercalated cell of rats. Schwartz and AI-Awqati (1985), using fluorescent dextran assay, have directly demonstrated that CO2 stimulates exocytotic fusion of the vesicles in the proximal straight tubule and collecting duct of rabbits. Turtle urinary bladder has many transport characteristics similar to collecting duct. Exposure of CO2 also increases H secretion in this epithelia, although H-ATPase has been interpreted to be responsible in part for this stimulation (Cannon et al., 1985; Schwartz and AI-Awqati, 1986; Steinmetz, 1986; Stetson and Steinmetz, 1983 & 1986; Van Adelsberg and Al-Awqati, 1986). However, recent studies have also demonstrated the presence of K-ATPase activity in this epithelia (Husted and Steinmetz, 1981; Sharma et al., 1991). Exocytosis of the pumps has been repeatedly shown to mediate this response (Cannon et al., 1985; Schwartz and AI-Awqati, 1986; Steinmetz, 1986; Stetson and Steinmetz, 1983; Van Adelsberg and A1-Awqati, 1986).
The cell swelling has been observed following exposure to 10% CO2 (Figure 3-12), whereas the change in morphology of the tubules was not able to be observed if the stimulatory effect of 10% CO2 on Rb efflux was inhibited either by colchicine (Figure 3-13) or by MAPTAM (Figure 3-14). These observations suggest that the renal H-K-ATPase participates in cell volume








41
regulation. Two possible mechanisms may explain the effect of H-K-ATPase on cell volume. 10% CO2 stimulates H-K-ATPase thereby increasing K/Rb entry from the apical membrane. It has been well known that acidosis inhibits K conductance or K channels (Boundry et al., 1976; Wang et al., 1990; Wright and Giebisch, 1992). It is possible that 10% CO2 inhibits the K exit at the basolateral membrane. The intracellular K level increases from the these two additive effects. As a result, the osmolarity gradient is established. Alternatively, the renal H-K-ATPase can transport Na as demonstrated in Chapter 5. 10% CO2 stimulates H-K-ATPase presumably increasing Na entry from the apical membrane. Acidosis inhibits Na-K-ATPase thereby inhibiting Na exit at the basolateral membrane (Wright and Giebisch, 1992). As a result, the osmolarity gradient is established.
As shown in Figure 3-1, each individual tubule responded to 10% CO2 differently. Some tubule had big response in terms of Rb efflux stimulation upon exposure to 10% CO2, whereas others did not. Rb efflux response to the stimulus is not correlated with voltage response. Whether the variation in KRI response to 10% CO2 is due to the different populations of the CCD or the biological variation among the tubules is not clear. In fact, 20% CCD comes from the juxtamedullary nephron, whereas 80% CCD comes from the superficial nephron. Whether these two different populations of the CCD have different physiological functions in terms of potassium transport remain unknown.

3.4.2 Effect of Methazolamide
Exposure to CO2 has been demonstrated to decrease intracellular pH, and this results in increase intracellular Ca level in turtle urinary bladder, although the effect of CO2 on intracelular Ca activity varies in different cell








42
types (Cannon et al., 1985). Exocytotic fusion of H pumps is pH- and Cadependent in turtle urinary bladder. In this tissue decrease in tracellular pH results in an increase in intracellular Ca activity by CO2 stimulates this insertion process (Cannon et al., 1985; Van Adelsberg and AI-Awqati, 1986). Acetazolamide, another inhibitor of carbonic anhydrase, has been shown to alkaline intracellular pH and decrease intracellular Ca activity (Van Adelsberg and A1-Awqati, 1986). The increase in intracellular pH and decrease in intracellular Ca level prevents the exocytotic insertion of acidic cytoplasmic vesicles containing H pumps into the apical membrane of turtle urinary bladder (Cannon et al., 1985; Schwartz and Al-Awqati, 1986; Steinmetz, 1986; Stetson and Steinmetz, 1983; Van Adelsberg and Al-Awqati, 1986). Consistent with the insertion hypothesis for H-K-ATPase, simultaneous exposure to 10% CO2 and methazolamide prevented the stimulatory effect of 10% CO2 on Rb efflux presumably because the alkalinization of intracellular pH by methazolamide inhibited the insertion cascade, whereas after 10% COz enhanced Rb efflux presumably activated the exocytotic process of H-K-ATPase, subsequent addition of methazolamide had no detectable inhibition on Rb efflux (Fig. 3-4). In addition, methazolamide did not significantly affect Rb efflux when the CCD was perfused in the presence of 5% CO2 throughout the experiments. This suggests that methazolamide-insensitive H source could be as a substrate for HK-ATPase. Recently, the observation made by Sharma et al. (1991) suggests that not all H-K-ATPase depends on carbonic anhydrase-originated H in turtle urinary bladder. They have demonstrated that 1 mM acetazolamide applied to the serosal solution only reduced proton secretion to 30% of control, the subsequent addition of SCH28080 caused H secretion fall to zero. When








43
SCH28080 was given first, proton secretion decreased to 70% of control, and subsequent addition of acetazolamide reduced proton secretion to zero. Carbonic anhydrase-dependent acidification has been repeatedly demonstrated in the proximal tubule (Chan et aL., 1983; Cogan et al., 1979; DuBose and Lucci, 1983). In contrast, whether all of acidification in the collecting duct is carbonic anhydrase-dependent remains controversial. Several groups have demonstrated that the collecting duct is able to maintain almost completely normal acidification rate after administration of acetazolamide both in vivo and in vitro, suggesting that a substantial carbonic anhydrase-independent H source is present in the collecting duct (Cogan et al., 1979; DuBose and Lucci, 1983; Frommer et al., 1984; Laski, 1987; McKinney and Davidson, 1987). However, the inhibitory effect of acetazolamide on acidification in the collecting duct has been reported by other investigators (Lombard et al., 1983). It is difficult to reconcile these observations at present time. Carbonic anhydrase-independent acidification may not be an unique phenomenon only observed in collecting duct. As early as in 1958, Durbin and Heinz (1958) have shown that carbonic anydrase is not essential for H secretion in gastric gland. Acidification by turtle urinary bladder has been also reported to be in part independent of carbonic anhydrase (Schilb and Brodsky, 1966; Schwartz et al., 1972). However, the sources of these protons remain speculative. At least two attractive mechanisms have has been proposed to explain the carbonic anbydraseindependent H sources which could be from ATP hydrolysis as suggested for gastric H-K-ATPase (Ljungstrom et al., 1984), or non-catalyzed hydration of CO2 (Maren, 1974).








44
Methazolamide has been repeatedly demonstrated to make VT more lumen-negative in the present studies which is consistant with other reports(Koeppen, 1989; Koeppen and Helman, 1982; Lombard et al., 1983; McKinney and Davidson, 1987). However, the mechanisms underlying this effect is not clear. This effect could be due to inhibition of electrogenic H secretion or influence of anion transport (Koeppen, 1989; Koeppen and Helman, 1982), or both.
3.4.3 Effects of Colchicine. MAPTAM. and W-7
Much of our knowledge about the role of microtubules, intracellular Ca, and calmodulin in the stimulation of H secretion (presumebly in part via H-KATPase) is derived from the observations made in the turtle urinary bladder and collecting duct. Pretreatment with colchicine inhibits exocytotic fusion of proton pumps to the apical membrane and the increase in the apical surface density and proton secretion by the increase in CO2 tension in the turtle urinary bladder, whereas lumicolchicine, which is structually similar to colchicine but does not bind to tubulin, had no effect (Stetson and Steinmetz, 1983). More directly, Schwartz and Al-Aqwati (1985) demonstrated that colchicine prevented the insertion process of proton pumps on exposure to CO2 in the proximal tubule and cotical and outer medullary collecting duct. McKinney and Davidson (1988) later reported that colchicine inhibited total CO2 absorption in response to 15% CO2 at least by the inner strip of the outer medullary collecting duct, whereas lumicolchicine had no effect. Recently, Brown et al. (1991) have shown that colchicine scatters the proton pumps in the cytoplasma of the renal epithelial cells. The soluble GTP-binding proteins have been suggested to be in control of the direction of insertion (Bourne, 1988; Hall, 1990). If H-K-ATPase








45
is involved in the stimulation of H secretion upon exposure to CO2, colchicine may also inhibit K absorption. Based on these observations and hypothesis, we selectively minupulated the conditions which were expected to inhibit microtubule functions at two different stages. When the CCD was simultaneous exposed to 10% CO2 and colchicine, colchicine inhibited the stimulation of Rb efflux by 10% CO2. In contrast, after Rb efflux increased by 10% C02, subsequent addition of colchicine had no significant effect. These results are consistant with the dependence of the increased Rb efflux on exocytosis of H-KATPase, which is mediated by the rearrangement of microtubule. The voltage became more lumen-negative during 10% CO2 and 10% CO2 plus colchicine periods, which is in contrast to the initial studies. We have no precise explaination for this observation but speculations. The baseline of voltage of these two sets of experiments was more lumen-negative than that of the previous studies. This sugggests that the tubules used in these two sets of experiments characterize more similarly to normal tubules in which voltage became more lumen-negative upon exposure to 10% CO2.
Intracellular calcium is respobsible for a variety of cellular events to numerous stimuli such as activation of gastric H-K-ATPase elicited by carbachol and gastrin and of H-ATPase in turtle urinary bladder induced by carbon dioxide (Forte et al., 1981). It has been believed that many Ca responses result from protein phosphorylation or dephosphorylation through the reactions with calmodulin. For instance, calmodulin regulating Ca-dependent microtubule assembly and disassembly has been illustrated in several types of cells (Marcum et al., 1978). A great deal of demonstrations have been evidently suggestive that exocytotic fusion of acidic vesicles containing H-ATPase is Ca-dependent process








46
in turtle urinary bladder (Cannon et al., 1985; Schwartz and Al-Awqati, 1986; Van Adelsberg and AI-Awqati, 1986). Exposure to CO2 increases intracellular Ca activity in tissue (Cannon et al., 1985). This elicits whole cascade of insertion process. When the increase in intracellular Ca activity is minimized, either by chelation of extracellular Ca (Van Adelsberg and Al-Awqati, 1986), or by buffering intracellular Ca (Cannon et al., 1985; McKinney and Davidson, 1988), the excytosis and the increased proton secretion are mitigated both in the turtle urinary bladder and the collecting duct. A body of evidence is also consistant with a mediator role for calmodulin in the stimulation of H secretion resulting from exposure to CO2. The increment in total CO2 absorption upon exposure to CO2 by the inner strip of outer medullary collecting duct is markedly attenuated by the potent antaganizer of calmodulin (W-7) but not by a structurally related analogue with less calmodulin inhibitory effect (W-5) (Dytko and Arruda, 1985; McKinney and Davidson, 1988). In view of these observations, we have chosen two maneuvers expected to be associated with alterations in intracellular Ca or calmodulin activity. Pretreatment with 0.5,uM MAPTAM blocked the stimulatory effect of Rb efflux by 10% CO2. The result is presumably due to the failure of intracellular ionized Ca level to rise. Moreover, pretreatment with 0.5MAM W-7 totally abolished the increase in Rb efflux by 10% CO2. However, the significant decrease in Rb efflux was observed during W-7 period. The mechanisms explaining this phenomenon remain to be elucidated. W-7 may have additional inhibition of Rb efflux mediated by K conductance. The important point here is that W-7 prevented the increase in Rb efflux induced by 10% CO2, providing the clear evidence indicating the role of calmodulin in this response. MAPTAM significantly made voltage more








47
lumen negative, whereas W-7 only had a trend. Many factors contribute and influence voltage. The mechanisms of these observations remain speculutive. It is possible that MAPTAM and W-7 inhibit electrogenic H-ATPase, which has been demonstrated in the turtle urinary bladder (Dytko and Arruda, 1985; Schwartz and Al-Awqati, 1985 & 1986; Van Adelsberg and Al-Awqati, 1986).
3.4.4 Effects of Basolateral Ba
on K-restricted CCD and 10% CO2 on Normal CCD
A large Ba-sensitive K conductance has been identified in the basolateral membrane of both principal cell and intercalated cell of rat (Schlatter and Schafer, 1987). However, electrophysiological studies conducted by Muto et al. (1987) suggest that little K conductance is present in the basolateral membrane of intercalated cell of K-replete rabbit CCD. Our present studies indicate that a Ba-sensitive pathway is present in the basolateral membrane of K-restricted CCD, and this pathway participates in K/Rb absorptive process. Alternatively, it may reflect an effect of Ba on non-conductive K exit as proposed by Greger and Schalatter (1983) for the thick ascending limb of Henle. It is reasonable to posit that under K-restriction, this pathway exerts its function to meet the need for K conservation, whereas this pathway may not function under K replete conditions. Consistantly with such a hypothesis, 10% CO2 had no significant effect on Rb efflux by normal CCD regardless of the presence or absence of luminal Ba. Under K-replete condition, the main function of H-KATPase may be secret proton, whereas the absorbed K by this enzyme leaks back to the lumen because of the functionally closed basolateral K exit pathway. Rats conserve K more efficiently than rabbits. Whether the species-difference in conservation of K is due to the basolateral K exit is unknown at present time. The alternative possibility is that the stimulatory effect of 10% CO2 on








48
Rb efflux through H-K-ATPase is overshadowed by the inhibitory effect on Rb efflux via K-conductive pathway when the tubules were perfused in the absence of luminal Ba. Ba may secodary inhibit H-K-ATPase when the CCD was perfused in the presence of luminal Ba. The precedent suggestion is derived from Oberleithner et al. (1990) observation made in MDCK cell. They have shown that Ba and omeprazole inhibit short-circuit current and acidification of the surface of the apical side of the dome. The effect of Ba is attributable to inhibition of K recycle at the apical membrane thereby inhibits H-K-ATPase in this type of cells.

3.5 Suma
In summary, these studies demonstrate that 10% CO2 profoundly stimulates Rb efflux, and this stimulation was totally abolished by SCH28080, suggesting that an H-K-ATPase mediates this process in the K-restricted CCD. The rapid response to 10% CO2 implies that this maneuver increases the luminal activity of existing H-K-ATPase pump units. The parallel changes in VT regardless of the absence or presence of SCH28080 reflect that the change in VT does not account for the effect of 10% CO2 on Rb efflux. The subsequent addition of methazolamide after 10% CO2 had no significant effect on Rb efflux, whereas simultaneous exposure of the tubules to 10% CO2 and methazolamide prevented the enhancement of Rb efflux by 10% CO2, suggesting that carbonic anhydrase is not necessary for maintaining activation of H-KATPase by 10% CO2, but is necessary for initiating activation of H-K-ATPase. Colchicine, MAPTAM, and W-7 inhibit the stimulation of Rb efflux by 10/%CO2, implying that activation of H-K-ATPase is dependent on the functional microtubules, increase in intracellular Ca activity, and functional calmodulin and








49
posssiblly mediated by exocytotic process. Basolateral Ba totally blocked the enhancement of Rb efflux by 10% CO2, indicating that a Ba-sensitive exit pathway mediates the stimulation of K., by 10% CO2.








50

Table 3-1. Composition of solutions (in mM)


Solution A Solution B Solution C
Na 135 135 135
K 5 5 5
Cl 110.4 114.4 114.4
Ca 1.2 1.2 1.2
Mg 1 1 1
Acetate 10 10 10
Glucose 8 8 8
Alanine 5 5 5
HEPES 10 10 10
Gluconate 23 23 23
Ba 0 2 4
TMA 4 4 0
Osmol (m Osm) 317.6 322.6 321.6








51
Table 3-2. Effect of luminal 0.1% DMSO on K, and VT


Basal DMSO
Ks,, (nmsec-1) 81.4 9.0 83.0 13.7
VT (mny) -8.3 3.4 -5.8 4.1

DMSO, dinethylsufoide (n=6).









52
Table 3-3. Time control for K, and VT

Time after
decapitation 100-150 150-200 200-250 250-300
(in minutes)
Km (nmsec-) 82.1 13.0 83.6 11.7 106 8.8 100 15.1
VT (my) 4.2 3.4 3.9 3.1 5.5 + 2.6 5.4 2.3

(n=5).








53
Table 3-4. Effect of 10% CO2 on VT in the presence of colchicine.

10% CO2 +
5% CO, Colchicine
VT (mV) (n= 7) -12.9 4.9 -15.6 5.0
10% CO2 +
5% CO, 10% CO0, Colchicine
VT (mV) (n=7) -11.2 7.1 -16.9 9.4a -17.4 9.5b
b p< 0.05, vs 5% CO2 period; b p< 0.01, vs 5% CO2 period.








54
Table 3-5. Effect of 10% CO2 on VT in the tubules pretreated with 0.5 uM MAPTAM and W-7.

MAPTAM
5% CO, 10% CO,
VT (mY) (n=6) -15.7 5.9 -23.9 6.8"
W-7
5% CO, 10% CO,
VT (mV) (n=6) -12.2 3.9 -14.4 3.5
p<0.05, vs 5% CO2 period.








55
Table 3-6. Effect of 10 CO2 on the normal CCD in the presence and absence of luminal Ba

With Ba (n=6)
10% CO,(30- 10% CO2(60- 10%CO2+Me5% CO, 60 minutes) 90 minutes) thazolazmide
Ktb (nmsec1) 48.1 72 42.5 83 44.6 7.5 44.5 9.2
VT (mV) -35.0 11.6 -44.2 + 14.0 -46.9 14.7' -50.7 14.5
Without Ba (n=2)
10% C02(60- 10% CO2(905% CO, 90 minutes) 120 minutes)
KRb (nmsec1) 73.1 + 37.9 66.9 + 40.1 64.4 + 32.2
VT (mV) -33.4 0.4 -32.5 0.2 -20.4 + 10.4
a p<0.05, vs the 5% CO2 period.









56


p<0.05 600 500 400 soo

A


200 100


5% 2 0 C002


S p<0.05 1

2





I-I
-2



-4




-10
5% 02 10% 002



Figure 3-1. Effect of 10% CO2 on Kn (top) and VT (bottom) in the absence of SCH28080 (n = 7). The tubules were allowed equilibrating for one hour before starting collection of sample during 10% CO2 period. Kn., Rb lumento-bath efflux coefficient. V., transepithelial voltage.









57














350300

.. 250- Measured

a 200E
S150
S100- ----- --- --- Predicted


50

0
5% CO, 10% CO2
















Figure 3-2. The predicted voltage-mediated increase in Kn and observed increase in K, following exposure to 10% CO2. Kn, "Rb lumen-to-bath efflux coefficient.











58





NS

140

120

100

so.









-2
40

20


0 5% CO2 10% CO2








-2

-10 -12
.144
-14 .16
-18
-20
-22
-24


.48
-W0
-52
-54

*58

5% CO2 10% CO2








Figure 3-3. Effect of 10% C02 on Kn (top) and VT (bottom) in the presence of SCH28080 (n =5). The tubules were allowed equilibrating for one hour before starting collection of sample during 10% CO2 period. Kn, 'Rb lumento-bath efflux coefficient Vr, transepithelial voltage.











59









18*a 160140

,' 120100





40.

20

0 5%6CO2 0-0mn W60 m1. 90120 min Uhaalaide

10% CO2






2

0 --- - - - - - - - - - - - - - -



-







-10

-12
5%6CO2 0-0ml n 60-0 min 90-120 min MethazolaMld

10% CO2






Figure 3-4. Effect of 10% CO2 and 0.1 mM methazolamide on Kb and VT in the absence of SCH28080 (n = 6). *P < 0.05 compared with 5% CO perid. 'P = 0.06 compared with 60-90 minute period. IP < 0.05 compare with90-120 minute penod. K~b, 'Rb lumen-to-bath efflux coefficient. VT, transepitbelial voltage.









60




12D0







40.

M


5% Co2 30-60 win go-1nli 90-120 -On "MelWulud 10%0 CO2






2-


-2 .- \








-121
5% CO2 W0-0 owni 60-B min 90120 nU 8OIn IVAC02





Figure 3-5. Effect of 10% CO and 0.1 mM methazolamnide on KR,, (top) V (bottom) in the presence of SOi2080(n = 7). -P < 0.05 compared with; CO, period. P < 0.01 compared with 30-60 minute period. bp < 0.001 compared with 90-120 minute period. Kft, t6Rb lumen-to-bath efflux coefficient. V1, transepithelial voltage.












61








14012D0







402D


S*%C02 I0%C02
Methazoemide








6 r- P<.O-6 S

4

2



S-2

.41







5% C02 10%/ CO2
Methazolamido








Figure 3-6. Effect of simultaneous exposure to 10% C02 and 0.1 mM
methazolanide on Kp, (top) and VT (bottom) (n =6). Kft, MRb lumen-tobath efflux coefficient. VI,- transepiflielial voltage.










62











NS 1w









840,


20'


01 5%6025% C02 5% CO2 Mthu@Iamido













.25

-10.



-30' -35

..L
5% C02 5% C02









Figure 3-7. Effect of 0.ImM methazolamide on K~b (top) and VT (bottom) in the presence Of 5% C2 (n =6). K~b 3Rb ue-obteflxcfiin. V-D transepithefial voltage. i~ ue-obt flxcefcet










63




r NS
140 120

~100 8a0






20

0
5% C02 10% C02
COIChicIne




120 100

~so
E


402D 0
5% CO2 10%C02 10% C02
COICI1r1.



Figure 3-8. Effect of simultaneous exposure to 10% CO2 and 0.5 rm
colchicine (top) and exposure to colchicine after 1OC02 (bottom) on KP, (n = 6). 86Rb lumen-to-bath efflux coefficient.











64






I NS 200 180 160
C'140
120







40


5% C02 10% CO2
MAPTAM MAPTAM





1<060

140 120







40.





5% CO2 10% C02










Figure 3-9. Effect of 10% CO2 on K,, by the tubules pretreated with 0.5gM MAPTAM (top) and W-7 (bottoni)(n = 6). Km,, 86Rb lumen-to-bath efflux coefficient.











65




f NS
100






840




40, 20

0 5%C 02 10%'CO2
3 mM Ba 3 mMBa


r- NS ----4

2
E-- ----- -1
I-2


-4


-10 -12


5% CO2 10% C02
3 mM Ba 3 mM Ba





Figure 3-10. Effect of peritubular 3 mM Ba on K (top) and VT (bottom) (n = 6). Kft, MRb lumen-to-bath efflux coefficient. Vtransepithelial voltage.









66













100, 90, 807060


&40'
30
20, PCO.05
10, 0
0mM Ba 2mM Ba 4mM Ba
















Figue 311.The dose-response curve of Ba on K~b. Kp,, 86Rb lumen-to-bath








67








































Figure 3-12. The CCD was swollen following expusure to 10% CO2. Top: 5% C02 period, Bottom: 10% CO2.








68









































Figure 3-13. The simultaneous presence of 10% CO and colchicine prevented the cell swelling. Top: 5% CO2 period, Bottom: 10% CO2 plus colchicine period.








69











































Figure 3-14. Effect of 10% CO, on cell swelling in the presence of MAPTAM. Top: 5% CO, period, Bottom: 10% CO,.











CHAPTER 4
EFFECT OF BARIUM, AMILORIDE AND OUABAIN ON Rb EFFELUX


4.1 Introduction
There is general agreement that potassium (K) secretion by the cortical collecting duct (CCD) involves active translocation of K across the basolateral membrane via Na-K-ATPase and passive exit across the apical membrane, in part via a K-conductive pathway (Muto et al., 1988; Stokes, 1981; Warden et al., 1989; Wingo, 1984). Administration of luminal barium, an inhibitor of Kconductance, or amiloride, an inhibitor of conductive Na entry, or peritubular ouabain, an inhibitor of Na-K-ATPase, inhibits net K secretion (O'Neil and Boulpaep, 1979; Stoner et al., 1974; Wingo, 1984 & 1985). These facts suggest that K secretion is largely dependent on K exit through an Ba-sensitive pathway and Na entry through an amiloride-sensitive pathway at the apical membrane, and K uptake through Na-K-ATPase at the basolateral membrane.
Additionally, luminal amiloride has been demonstrated to substantially stimulate lumen-to-bath tracer K efflux, or tracer Rb efflux, used as a marker of K efflux in the in vitro microperfused CCD of both normal rabbits and DOCA-treated rabbits (Stokes, 1981; Warden et al., 1989). This effect depends on a Ba-sensitive K conductance. Therefore, the first objective of this studies is to examine whether Ba inhibits tracer Rb efflux, and whether the effect of amiloride on Rb efflux is preserved in K-restricted CCD.




70








71
Ouabain profoundly increases K absorption in gastric gland, and this stimulation has been attributed to H-K-ATPase (Reenstra et al., 1986). H-KATPase has been also identified in colonic epithelium which has many transport characteristics similar to the collecting duct (Kaunitz and Sachs, 1986). Serosal ouabain has been shown to not only inhibit K secretion, but also stimulate K absorption by this tissue (Halm and Frizzell, 1986; Turnamian and Binder, 1989). Therefore, the second objective of this studies is to address the issue whether peritubular ouabain increases Rb efflux, and whether this increment is mediated by an H-K-ATPase in the K-restricted CCD.
However, the issue of whether K absorption by the CD proceeds via an H-K-ATPase is not completely clear. During K restriction, Na-K-ATPase activity increases in the rat CD, although this increase in Na-K-ATPase activity is not accompanied by an increased [3H]-ouabain binding in intact tubules. However, in permeabilized tubules, [3H]-ouabain binding does increase proportionately to the increase in Na-K-ATPase activity. These observations led Hayashi and Katz (1987) to hypothesize that Na-K-ATPase was sequestered in an inaccessible site, either an intracellular compartment or the luminal membrane. If Na-K-ATPase is present at the luminal membrane, it could participate in K absorption. In addition, mucosal ouabain has been shown to inhibit active K absorption in the rat and guinea pig colon (Perron and McBride, 1988; Sweiry and Binder, 1990; Suzuki and Kaneko, 1987 & 1989) and in the turtle bladder (Husted and Steinmetz, 1981). Thus, the third objective of the present studies was to determine whether a functional H-K-ATPase or a functional Na-K-ATPase was present at the apical membrane of the CCD of K-restricted rabbits.








72
4.2 Methods and Material
The tubules were dissected with Solution A (Table 4-1) containing additional 5% vol/vol feral calf serum. The bath solution which is identical to dissection solution was used throughout all sets of experiments. Three different perfusates (Solution A, Solution B or Solution C) were used as appropriate. All solutions were gassed to pH 7.4 with 95% 02 and 5% COz. Amiloride was a gift of Merck Sharp & Dohme (Rahway, NJ) and was dissolved in the perfusate directly. SCH28080 (gift of Dr. James Kaninsky, Schering Corporation, Bloomfield, NJ) was dissolved in dimethyl sulfoxide (DMSO). Ouabain was dissolved in 0.9% NaCl solution.

43 Results
4.3.1 Protocol 1
To examine whether amiloride enhances K, in the K-restricted animals, the first set of experiments was performed in the absence of luminal Ba. Thus, eight tubules were perfused with solution A, or solution A plus 1 mM amiloride designated as basal and amiloride periods, respectively. The order of the periods was rotated and there was no evidence of time-dependent effect on Kn or VT. In the absence of luminal Ba, luminal 1 mM amiloride significantly increased I from 90.7 14.3 nmsec' (basal period) to 119 20.6 nmsec' (amiloride period, P < 0.05, Figure 4-1), and VT from 0.6 1.5 mV (basal period) to 4.7 0.9 mV (amiloride period, P < 0.05, Figure 4-2). To examine whether the effect of amiloride on Rb efflux was mediated by Ba-sensitive pathway, the second set of experiments was performed in the presence of luminal 2 mM Ba, because in view of the voltagedependent nature of Ba inhibition, Rb efflux in the K-restricted CCD should be maximally inhibited by 2 mM Ba at which has been demonstrated to maximally inhibit








73
Ka by normal CCD. To determine whether tracer Rb efflux via the H-K-ATPase was demonstrable in the presence of a maximum pharmacologic concentration of amiloride, 10 MM SCH28080 was applied to the perfusate containing 1 mM amiloride and 2 mM Ba during the third period. Thus, nine tubules were perfused with solution B, solution B plus amiloride, and solution B plus amiloride and SCH28080 designated as the basal, amiloride, and amiloride plus SCH28080 periods, respectively. The order of the periods was rotated according to balanced Latin-square design (Cochran and Cox, 1957) and there was no evidence of a time-dependent effect on K, or VT. As shown in Figure 4-1, in the presence of luminal Ba, 1 mM amiloride failed to stimulate K, (81.2 7.1 nmsec-', basal period vs. 91.2 10.2 nmsec-, amiloride period) in spite of significant increase in VT from 1.8 1.5 mV during basal period to 8.5 1.5 mV during the aminloride period (P < 0.05, Figure 4-2). The observation that Ba inhibits the stimulatory effect of amiloride on Kn, indicates that a Basensitive pathway mediates the effect of amiloride on Rb efflux. The parallel VT response to amiloride regardless of the absence or presence of Ba suggests that VT can not account for the effect of amiloride on Rb efflux. Moreover, in the presence of luminal 1 mM amiloride, SCH28080 still significantly inhibited Rb efflux from 91.2 10.2 nmsec"1 (amiloride period) to 68.3 8.9 nmsec- 1 (amiloride plus SCH28080 period, P < 0.05, Figure 4-1). These data demonstrate that SCH28080 inhibits a pathway of Rb efflux which is unaffected by a maximum pharmacologic concentration of amiloride and insensitive to 2 mM luminal Ba. SCH28080 had no significant effect on VT (8.5 1.5 mV, amiloride period; vs 12.1 2.5 mV, amiloride plus SCH28080 period, P = NS, Figure 4-2).








74
43.2 Protocol 2
To examine whether the basolateral ouabain stimulates Rb efflux which is mediated by an H-K-ATPase, seven tubules were perfused with Solution B in the absence of SCH28080 and presence of Ba. After basal K, and VT were measured, 0.1 mM ouabain was added to the bath. As shown in Figure 4-3, basolateral ouabain significantly increased K, from 69.8 11.1 nmsec"1 (basal period) to 95.9 18.7 nmnsec-' (ouabain period, P < 0.05). The greater lumenpositive VT induced by ouabain did not reach the 0.05 level of significance (-2.3 2.3 mV, basal period; 8.6 3.3 mV, ouabain period, P = 0.08, Figure 4-4). In contrast, six additional tubules were studied under identical conditions except that 10 uM SCH28080 was present in the perfusate. Perfusion with SCH28080 totally abolished the effect of ouabain on Rb efflux (69.8 14.0 nmsec-1 during basal period vs 69.6 14.1 nmsec'1 during ouabain period, P = NS, Figure 4-3), although ouabain significantly increased VT from -1.5 1.1 mV (basal period) to 2.9 0.4 mV (ouabain period, P < 0.01, Figure 4-4). Moreover, analysis of the individual changes in VT and changes in Ku in response to ouabain demonstrated no relationship compatible with a voltagedmediated change in paracellular Rb efflux. In fact, the largest changes in VT were associated with some of the smallest changes in Kn. These observations suggest that the enhancement of Ku, by basolateral ouabain is mediated by HK-ATPase.
43.3 Protocol 3



2 This lack of a significant effect of onabain on VT can be attributed to the fact that one tubule had a much larger voltage response to ouabain than that the other six (from -15 mV during the basal period to 27 mV during the ouabain period). Were this tubule excluded from the analysis, the effect of ouabain on voltage would be significant.








75
This set of experiments was designed to identify the role of a primary active K pumps in K absorption, was conducted in the absence of luminal Na, because removal of luminal Na enhances Rb efflux (Wingo and Zhou, 1990). To examine whether H-K-ATPase mediates Rb efflux, 10 pM of SCH28080 was added to the perfusate during the SCH28080 period. To examine the role of a luminal Na-K-ATPase, 0.1 mM of ouabain was added to the perfusate during the ouabain period (no SCH28080 present). Thus, nine tubules were perfused with Solution C, or Solution C plus 10 pM SCH28080, or Solution C plus 0.1 mM ouabain and these periods were designated as basal, SCH28080, and ouabain periods, respectively. The order of the periods was rotated according to balanced Latin square design (Cochran and Cox, 1957) to control for time-dependent effects. There was no evidence that the order of the periods affected either KR, or VT (P = NS). SCH28080 significantly decreased Rb efflux by 39 8.0%, from 106 12.1 nmsec" (basal period) to 65.4 11.2 nm-sec"1 (SCH28080 period, P < 0.05, Figure 4-5). However, a maximal pharmacological dose of ouabain (0.1 mM) did not significantly affect KR, (106 12.1 nmsec-1, basal period; 84.5 8.84 nmsec-1, ouabain period, P = NS, Figure 4-5). Ouabain reduced Rb efflux only 10 14% as calculated by the mean of the percent inhibition observed in the individual experiments. Vehicle experiments demonstrated that neither DMSO nor NaCl at the same concentration to that used in the experimental protocol affected Rb efflux or voltage (Table 4-2). These data demonstrate that under the present experimental conditions a luminal H-K-ATPase, not a luminal Na-K-ATPase, represents a major pathway for Ba-insensitive Rb efflux. The transepithelial voltages were not








76
significantly altered during this maneuver (-0.4 1.1 mV, basal period, 0.1
1.3 mV, SCH28080 period; and 0.4 2.1 mV, ouabain period, Table 4-3).

4.4 Discuss
4.4.1 Effect of Lumen Ba
Barium is a rapidly reversible inhibitor of K channels and K conductance (Van Driessche and Zeiske, 1980). The present studies demonstrate that Basensitive K permeation is present even in K-restricted rabbits. Warden et al (1989) demonstrated that Rb behaves qualitatively similar to K and concluded that there is no evidence to suggest that these ions are transported by different mechanisms. Ba-resistant Rb efflux is small in the CCD from normal rabbits (Warden et al, 1989 and Fig. 3-11), whereas in the K-restricted rabbit Bainsensitive Rb efflux is relatively larger suggesting that K restriction increases a Ba-insensitive pathway. Since the Ba-sensitive pathway has been attributed to Ba-sensitive K channels which should mediate K secretion (Frindt and Palmer, 1989; Koeppen and Helman, 1982; Wang et al., 1990; Warden et al., 1989), the smaller Ba-sensitive pathway observed in the present study compared with that in normal rabbit is in agreement with earlier studies indicating that a reduction in K intake decreases K secretion by the CCD (O'Neil and Helman, 1977; Schwartz and Burg, 1978; Wingo et al., 1982).

4.4.2 Effect of Amiloride
It is generally accepted that both and amiloride-sensitive Na conductance and a Ba-inhibitable K conductance are present in the apical membrane of the principal cells of the CCD (Koeppen et al., 1983; Koeppen and Giebisch, 1988; Muto et al., 1988; O'Neil and Helman, 1977; Sansom et al., 1987; Sauer et al., 1990). Stokes (1984) has observed that the amiloride-induced increase in K








77
permeation is significantly larger than could be accounted for by the changes in voltage alone. Recently, the increment in K permeation by amiloride has been shown to be largely, via a conductive pathway in the CCD of both normal and desoxycorticosterone-treated rabbits (Warden et al., 1989). The findings that amiloride significantly increased K, in the absence of luminal Ba (Figure 4-1), whereas amiloride failed to increase KRb in the presence of luminal Ba (Figure 4-1) are consistent with these observations. Moreover, in the presence of luminal 1 mM amiloride and 2 mM Ba, SCH28080 was still able to be demonstrated to significantly inhibit Rb efflux. These data show that SCH28080 inhibits a pathway of Rb efflux which is unaffected by a maximum pharmacologic concentration of amiloride and insensitive to 2 mM luminal Ba.

4.4.3 Effect of Peritubular Ouabain
H-K-ATPase activity may be regulated by intracellular potassium activity, and this may explain the effect of ouabain on Rb efflux. Thus, a reduction in intracellular K activity may stimulate the activity of the enzyme, whereas an increase in intracellular K activity may inhibit enzyme activity. In support of this hypothesis Koelz et al. (1981) have found that a decrease in intracellular K activity to 60 mM stimulates the gastric H-K-ATPase as measured by aminopyrine accumulation. Lorentzon et al. (1988) have demonstrated that a decrease in intracellular K activity stimulates phosphoenzyme formation and thereby increases the enzyme activity. Accordingly, maneuvers that result in a decrease in intracellular K activity should stimulate K absorption. In gastric glands intracellular K activity was significantly reduced following addition of ouabain (Koelz et al., 1981) and ouabain substantially increased K absorption in the gastric mucosa (Reenstra et al., 1986). Subsequent addition of omeprazole








78
significantly decreased K absorption, suggesting that the effect of ouabain on K absorption is mediated by H-K-ATPase. Ouabain significantly decreases intracellular K level of both principal cells and intercalated cells in the CCD (Sauer et al., 1989) and increases Rb efflux when SCH28080 is absent in the perfusate (Figure 4-3) whereas this maneuver fails to affect Rb efflux when SCH28080 is present in the perfusate (Figure 4-3). These data further support such a hypothesis.
The decrease in intracellular potassium concentration increasing H-KATPase may not unique phenomenon only obverved with peritubular ouabain. Several investigators have shown that the intracellular K level in the distal tubule was reduced by adaptation of the animals to a low K diet (Beck et al., 1982; Linas et al., 1979). Both incerase in SCH28080-sensitive ATP hydrolysis and Rb efflux were observed in the CCD of rats adapted to K-restricted diet as compared with those on a regular diet, suggesting that H-K-ATPase activity is enhanced under these conditions (Cheval et al., 1991; Doucet and Marsy, 1987).
4.4.4 Effects of Luminal SCH28080 and Ouabain
In the absence of luminal Na, luminal 10 pM SCH28080 significantly reduced K. by 39%, whereas luminal 0.1 mM ouabain did not significantly affect Ku,. These observations suggest that under the present experimental conditions, H-K-ATPase mediates in part Ba-insensitive Rb/K efflux and a ouabain-sensitive luminal Na-K-ATPase is not a major pathway for Rb/K absorption. Were an apical Na-K-ATPase mediating K absorption, the greatest degree of inhibition would be expected with luminal ouabain, whereas only the effect of SCH28080 was significant. The present demonstration is consistant with








79
the observations made through microenzymatic assay from which ouabain has no significant effect on ATP hydrolysis by H-K-ATPase in both rat and rabbit CCD (Doucet and Marsy, 1987; Garg and Narang, 1988). It should be emphasized that the inhibition of K absorption by ouabain has been only observed in the colonic epithelium and the distal tubule of rat and turtle urinary bladder. These observations are in contrast to the findings in the bullfrog (Ganser and Forte, 1973) and rabbit stomach (Gunther et al., 1987) and colon (Gustin et al., 1981; Halm and Frizzell, 1986) in which the H-KATPase is relatively resistant to inhibition by ouabain. Therefore, the speciesdifference should be appreciated.

4.5 Summar
In summary, amiloride increased Rb efflux, and this increment was inhibited by Ba, implying that a K-conductive pathway mediates the effect of amiloride. Basolateral ouabain increased Rb efflux in the absence of luminal SCH28080, but this same maneuver failed to affect K. when SCH28080 was present in the perfusate, reflecting that an apical H-K-ATPase mediates the enhancement of Rb efflux following inhibition of basolateral Na-K-ATPase. Furthermore, only luminal SCH28080 not luminal ouabain was shown to inhibit Rb efflux, suggesting that a functional H-K-ATPase, not a functional Na-KATPase, participates in Rb efflux.








80
Table 4-1. Composition of solutions (in mM)

Solution A Solution B Solution C
Na 135 135
Choline 135
K 5 5 5
Cl 106.4 110.4 106.4
HCO3 25 25 25
Ca 1.2 12 1.2
Ba 2
Mg 1 1 1
Phosphate 1.5 1.5 1.5
Acetate 10 10 10
Glucose 8 8 8
Alanine 5 5 5
Mannitol 26.5 20.5 26.5
Osmal (m Osm) 324.6 324.6 324.6








81
Table 4-2. Effect of DMSO in the absence of luminal Na and luminal 1.5 mM

Basal DMSO 1.5 mM Na
Ku (nmesec1) 84.2 18.5 82.6 17.0 87.6 14.9
VT (mV) 2.8 5.4 0.6 2.8 1.7 3.3
(n=6).








82
Table 4-3. Effect of luminal 10pM SCH28080 or 0.1 mM ouabain on VT in the absence of luminal Na


Basal SCH28080 Ouabain
VT (mV) -0.4 1.1 0.1 13 0.4 2.1
(n=9).








83















9.0






Fiue41 ffc flmna M oie nVi heasneo

InminalU -a(on=8adi h rsnc flmnlIm a(ide



9) fet fSH8N n i tepeeneo -ia M mlrd










Fiue41 feto uia Mamiloride onrod were 15575msdin the absence of fZa 25.0 6.3 nrsec' (in the presence of luminal Ba) calculated by Goldman flux equation. P. 1 x 101 nmsec-1. Kft, "Rb lumen-to-bath efflux coefficient.




Full Text
119
o
i
Basal
ANG II
p<0.01
Figure 6-1. Effect of angiotensin II on Kr,, (top) and Vj. (bottom, n=6) in the
presence of luminal 3mM Ba and absence of luminal SCH28080. K^, Rb
lumen-to-bath efflux coefficient. Vt, transepithelial voltage.


KEY TO ABBREVIATIONS
Ang II
ANOVA
ATP
Ba
Ca
CCD
CD
co2
H
HEPES
K
KRb
KNa
MAPTAM
Na
Rb
SCH28080
angiotensin II
analysis of variance
adenosine triphosphate
barium
calcium
the cortical collecting duct
the collecting duct
carbon dioxide
proton
(N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]
potassium
Rb lumen-to-bath efflux coefficient
Na lumen-to-bath efflux coefficient
Bis-(2-amino-5-methyl-phenoxy)-ethane-N,N,N,N-tetraacetic
acidtetraacetoxymethyl ester N-(6-aminohexyl)-5-chloro-l-
naphthalene-sulfonamide
sodium
rubidium
(3-cyanomethyl-2-8-phenylmethoxy)imidazol[l,2-a]pyridine
xiii


22
SCH 28080
Figure 1-7. The chemical structure of SCH28080.


75
This set of experiments was designed to identify the role of a primary
active K pumps in K absorption, was conducted in the absence of
luminal Na, because removal of luminal Na enhances Rb efflux (Wingo and
Zhou, 1990). To examine whether H-K-ATPase mediates Rb efflux, 10 /jM of
SCH28080 was added to the perfusate during the SCH28080 period. To examine
the role of a luminal Na-K-ATPase, 0.1 mM of ouabain was added to the
perfusate during the ouabain period (no SCH28080 present). Thus, nine tubules
were perfused with Solution C, or Solution C plus 10 /jM SCH28080, or
Solution C plus 0.1 mM ouabain and these periods were designated as basal,
SCH28080, and ouabain periods, respectively. The order of the periods was
rotated according to balanced Latin square design (Cochran and Cox, 1957) to
control for time-dependent effects. There was no evidence that the order of the
periods affected either K^, or VT (P = NS). SCH28080 significantly decreased
Rb efflux by 39 8.0%, from 106 12.1 nm-sec'1 (basal period) to 65.4
11.2 nm-sec'1 (SCH28080 period, P < 0.05, Figure 4-5). However, a maximal
pharmacological dose of ouabain (0.1 mM) did not significantly affect KRb (106
12.1 nm-sec-1, basal period; 84.5 8.84 nm-sec-1, ouabain period, P = NS,
Figure 4-5). Ouabain reduced Rb efflux only 10 14% as calculated by the
mean of the percent inhibition observed in the individual experiments. Vehicle
experiments demonstrated that neither DMSO nor NaCl at the same
concentration to that used in the experimental protocol affected Rb efflux or
voltage (Table 4-2). These data demonstrate that under the present experimental
conditions a luminal H-K-ATPase, not a luminal Na-K-ATPase, represents a
major pathway for Ba-insensitive Rb efflux. The transepithelial voltages were not


83
*4.09
Figure 4-1. Effect of luminal 1 mM amiloride on in the absence of
luminal Ba (top, n = 8)and in the presence of luminal 2 mM Ba (middle, n
= 9). Effect of SCH28080 on in the presence of luminal 1 mM amiloride
and 2 mM Ba (bottom, n = 9). Voltage-mediated paracellular K^, during
amiloride periods were 15.5 7.5 nm-sec^in the absence of luminalBa) and
25.0 6.3 nm-sec'1 (in the presence of luminal Ba) calculated by Goldman flux
equation. PK = 1 x 10"5 nm-sec'1. K^, Rb lumen-to-bath efflux coefficient.


in this research and for their help with my English. The discussions of my
research results with them and with Drs. Kevin Curran and Mitchell Hebert
have been productive and enjoyable. I am also grateful to the faculty members
in the Department of Physiology, especially Drs. M. Ian Phillips, Colin Sumners,
Sidney Cassin, Melvin Fregly and Charlie Wood for their advice and
encouragement. In addition, I wish to thank Drs. C. Craig Tisher, Christopher
Wilcox, Kirsten Madson, William Welch, and I David Weiner in the Division
of Nephrology, Hypertension and Transplatation for their concern and advice.
I wish to thank Ann Crawford in depth for her excellent and courteous help
in preparation of this thesis as well as other manuscripts and abstracts. I am
always grateful to have the manuscripts back from Ann before Friday so that
I have something to work with during the weekend. My sincere thanks should
be presented to Robert Fleming for his constant concern and support. My
special thanks should be offered to Janice Dolson not only for her expert help
with my learning some special functions of WordPerfect, but also for her
friendly understanding and support during the past three years. I also appreciate
Ms. Ginny Young for her secretarial assistance to Dr. Wingo regarding my
education. My deep thanks are extended to Miss. B. J. Streetman, Victoria La
Placa, Pia Jacobs and Gayle Butters and Mr. Kevin Fortin for their necessary
role in my doctoral education. The constant concern and support from my
fellow students in the Department of Physiology, especially Hong-gen Chen and
Jian Kane, and Jenny Zhang in the Department of Pharmacology and
Therapeutics are warmly appreciated.
I am greatly indebted to my wife Xiaofang, my father Quibao Zhou, my
vi


Table 5-1. Composition of solutions (in mM)
Solution A
Solution B
Solution C
Solution D
Solution E
Solution F
Na
135
135
135
Choline
135
135
K
5
5
5
5
5
5
Cl
106.4
106.4
110.4
110.4
115.4
114.4
HC03
25
25
25
25
25
25
Ca
1.2
1.2
1.2
1.2
1.2
1.2
Ba
2
2
4
4
Mg
1
1
1
1
1
1
Phosphate
1.5
1.5
1.5
1.5
Acetate
10
10
10
10
10
10
Glucose
8
8
8
8
8
8
Alanine
5
5
5
5
5
5
Mannitol
26.5
26.5
26.5
26.5
4.5
7.5
TMA
132
Osmolarity
(mOsm)
324.6
324.6
324.6
324.6
313.3
313.3


79
the observations made through microenzymatic assay from which ouabain has
no significant effect on ATP hydrolysis by H-K-ATPase in both rat and rabbit
CCD (Doucet and Marsy, 1987; Garg and Narang, 1988). It should be
emphasized that the inhibition of K absorption by ouabain has been only
observed in the colonic epithelium and the distal tubule of rat and turtle
urinary bladder. These observations are in contrast to the findings in the
bullfrog (Ganser and Forte, 1973) and rabbit stomach (Gunther et al., 1987)
and colon (Gustin et al., 1981; Halm and Frizzell, 1986) in which the H-K-
ATPase is relatively resistant to inhibition by ouabain. Therefore, the species-
difference should be appreciated.
4.5 Summary
In summary, amiloride increased Rb efflux, and this increment was
inhibited by Ba, implying that a K-conductive pathway mediates the effect of
amiloride. Basolateral ouabain increased Rb efflux in the absence of luminal
SCH28080, but this same maneuver failed to affect Km, when SCH28080 was
present in the perfusate, reflecting that an apical H-K-ATPase mediates the
enhancement of Rb efflux following inhibition of basolateral Na-K-ATPase.
Furthermore, only luminal SCH28080 not luminal ouabain was shown to inhibit
Rb efflux, suggesting that a functional H-K-ATPase, not a functional Na-K-
ATPase, participates in Rb efflux.


45
is involved in the stimulation of H secretion upon exposure to C02, colchicine
may also inhibit K absorption. Based on these observations and hypothesis, we
selectively minupulated the conditions which were expected to inhibit
microtubule functions at two different stages. When the CCD was simultaneous
exposed to 10% C02 and colchicine, colchicine inhibited the stimulation of Rb
efflux by 10% C02. In contrast, after Rb efflux increased by 10% C02,
subsequent addition of colchicine had no significant effect. These results are
consistant with the dependence of the increased Rb efflux on exocytosis of H-K-
ATPase, which is mediated by the rearrangement of microtubule. The voltage
became more lumen-negative during 10% C02 and 10% C02 plus colchicine
periods, which is in contrast to the initial studies. We have no precise
explanation for this observation but speculations. The baseline of voltage of
these two sets of experiments was more lumen-negative than that of the
previous studies. This sugggests that the tubules used in these two sets of
experiments characterize more similarly to normal tubules in which voltage
became more lumen-negative upon exposure to 10% C02.
Intracellular calcium is respobsible for a variety of cellular events to
numerous stimuli such as activation of gastric H-K-ATPase elicited by carbachol
and gastrin and of H-ATPase in turtle urinary bladder induced by carbon
dioxide (Forte et al., 1981). It has been believed that many Ca responses result
from protein phosphorylation or dephosphorylation through the reactions with
calmodulin. For instance, calmodulin regulating Ca-dependent microtubule
assembly and disassembly has been illustrated in several types of cells (Marcum
et al., 1978). A great deal of demonstrations have been evidently suggestive that
exocytotic fusion of acidic vesicles containing H-ATPase is Ca-dependent process


MECHANISMS OF POTASSIUM PERMEATION IN THE IN VITRO
PERFUSED CORTICAL COLLECTING DUCT OF THE RABBIT
By
XIAOMING ZHOU
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
1992


47
lumen negative, whereas W-7 only had a trend. Many factors contribute and
influence voltage. The mechanisms of these observations remain speculutive. It
is possible that MAPTAM and W-7 inhibit electrogenic H-ATPase, which has
been demonstrated in the turtle urinary bladder (Dytko and Arruda, 1985;
Schwartz and Al-Awqati, 1985 & 1986; Van Adelsberg and Al-Awqati, 1986).
3.4.4 Effects of Basolateral Ba
on K-restricted CCD and 10% CO, on Normal CCD
A large Ba-sensitive K conductance has been identified in the basolateral
membrane of both principal cell and intercalated cell of rat (Schlatter and
Schafer, 1987). However, electrophysiological studies conducted by Muto et al.
(1987) suggest that little K conductance is present in the basolateral membrane
of intercalated cell of K-replete rabbit CCD. Our present studies indicate that
a Ba-sensitive pathway is present in the basolateral membrane of K-restricted
CCD, and this pathway participates in K/Rb absorptive process. Alternatively,
it may reflect an effect of Ba on non-conductive K exit as proposed by Greger
and Schalatter (1983) for the thick ascending limb of Henle. It is reasonable
to posit that under K-restriction, this pathway exerts its function to meet the
need for K conservation, whereas this pathway may not function under K
replete conditions. Consistantly with such a hypothesis, 10% C02 had no
significant effect on Rb efflux by normal CCD regardless of the presence or
absence of luminal Ba. Under K-replete condition, the main function of H-K-
ATPase may be secret proton, whereas the absorbed K by this enzyme leaks
back to the lumen because of the functionally closed basolateral K exit pathway.
Rats conserve K more efficiently than rabbits. Whether the species-difference
in conservation of K is due to the basolateral K exit is unknown at present
time. The alternative possibility is that the stimulatory effect of 10% C02 on


CHAPTER 7
EFFECT OF Na AND K INTAKE ON SERUM AND URINE Na
AND K LEVELS AND URINE OUTPUT
7.1 Introduction
It has been widely documented that low K and low Na intakes decrease
urinary output of Na and K, plasma level of K, and disturb acid-base balance.
To examine whether these observations arers
reproducible in our system, we conducted the metabolic studies.
7.2 Methods and Material
Two different diets (TD 87317 and TD 87433, Teklad, Madison, WI)
were used as appropriate. Blood was drawn from central ear artery. Urinary
sample was collected as appropriate. The concentrations of sodium and
potassium were measured by the auto-analyzer (ASTRA 880, Beckman,
Somerset, NJ).
7.3 Results
7.3.1 Protocol 1
To examine whether restrictions of Na and K intake decrease plasma
level and urinary excretion of Na and K and increase urine output, the rabbits
were adapted to a low Na (0% Na) and low K (0.25% K) diet (TD 87317) for
nine days. Urinary Na and K level and urine output were measured each day.
The plasma levels of Na and K were measured on 9th day of adaptation to
this diet. As shown in Table 7-1, there was a tendancy in decrease in urinary
122


74
4.3.2 Protocol 2
To examine whether the basolateral ouabain stimulates Rb efflux which
is mediated by an H-K-ATPase, seven tubules were perfused with Solution B
in the absence of SCH28080 and presence of Ba. After basal ^Rb and Vx were
measured, 0.1 mM ouabain was added to the bath. As shown in Figure 4-3,
basolateral ouabain significantly increased Kg,, from 69.8 11.1 nm-sec"1 (basal
period) to 95.9 18.7 nm-sec"1 (ouabain period, P < 0.05). The greater lumen
positive Vx induced by ouabain did not reach the 0.05 level of significance (-2.3
2.3 mV, basal period; 8.6 3.3 mV, ouabain period, P = 0.08, Figure 4-4)2.
In contrast, six additional tubules were studied under identical conditions except
that 10 /rM SCH28080 was present in the perfusate. Perfusion with SCH28080
totally abolished the effect of ouabain on Rb efflux (69.8 14.0 nm-sec"1
during basal period vs 69.6 14.1 nm-sec'1 during ouabain period, P = NS,
Figure 4-3), although ouabain significantly increased Vx from -1.5 1.1 mV
(basal period) to 2.9 0.4 mV (ouabain period, P < 0.01, Figure 4-4).
Moreover, analysis of the individual changes in Vx and changes in KRb in
response to ouabain demonstrated no relationship compatible with a voltaged-
mediated change in paracellular Rb efflux. In fact, the largest changes in Vx
were associated with some of the smallest changes in Kr,,. These observations
suggest that the enhancement of Kr,, by basolateral ouabain is mediated by H-
K-ATPase.
4.3.3 Protocol 3
2 This lack of a significant effect of ouabain on VT can be attributed to the fact that one tubule
had a much larger voltage response to ouabain than that the other six (from -15 mV during the
basal period to 27 mV during the ouabain period). Were this tubule excluded from the analysis,
the effect of ouabain on voltage would be significant.


21
Figure 1-6. Cell model of epithelial sodium and potassium transport (Giebisch,
1987).


105
Table 5-6. Effect
of luminal Na
of luminal SCH28080 on Vx in the
absence and presence
135 mM Na
Basal
SCH28080
VT (mV) (n=6)
-2.8 2.7
-1.0 1.2
No Na
Basal
SCH28080
VT (mV) (n=4)
-5.7 9.5
-6.6 5.5


TMA
VT
W-7
tetramethylammonium
transepithelial voltage
N-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide
xiv


73
Krb by normal CCD. To determine whether tracer Rb efflux via the H-K-ATPase was
demonstrable in the presence of a maximum pharmacologic concentration of
amiloride, 10 pM SCH28080 was applied to the perfusate containing 1 mM
amiloride and 2 mM Ba during the third period. Thus, nine tubules were
perfused with solution B, solution B plus amiloride, and solution B plus
amiloride and SCH28080 designated as the basal, amiloride, and amiloride plus
SCH28080 periods, respectively. The order of the periods was rotated according
to balanced Latin-square design (Cochran and Cox, 1957) and there was no
evidence of a time-dependent effect on K^, or VT. As shown in Figure 4-1, in
the presence of luminal Ba, 1 mM amiloride failed to stimulate KRb (81.2 +
7.1 nm-sec'1, basal period vs. 91.2 1012 nm-sec'1, amiloride period) in spite
of significant increase in VT from 1.8 1.5 mV during basal period to 8.5
1.5 mV during the amiloride period (P < 0.05, Figure 4-2). The observation
that Ba inhibits the stimulatory effect of amiloride on K,y, indicates that a Ba-
sensitive pathway mediates the effect of amiloride on Rb efflux. The parallel
VT response to amiloride regardless of the absence or presence of Ba suggests
that VT can not account for the effect of amiloride on Rb efflux. Moreover,
in the presence of luminal 1 mM amiloride, SCH28080 still significantly
inhibited Rb efflux from 91.2 10.2 nm-sec'1 (amiloride period) to 68.3 8.9
nm-sec'1 (amiloride plus SCH28080 period, P < 0.05, Figure 4-1). These data
demonstrate that SCH28080 inhibits a pathway of Rb efflux which is unaffected
by a maximum pharmacologic concentration of amiloride and insensitive to 2
mM luminal Ba. SCH28080 had no significant effect on VT (8.5 1.5 mV,
amiloride period; vs 12.1 2.5 mV, amiloride plus SCH28080 period, P = NS,
Figure 4-2).


69
Figure 3-14. Effect of 10% C02 on cell swelling in the presence of
MAPTAM. Top: 5% C02 period, Bottom: 10% C02.


82
Table 4-3. Effect of luminal IOjiM SCH28080 or 0.1 mM ouabain on VT in
the absence of luminal Na
Basal
SCH28080
Ouabain
VT (mV)
-0.4 1.1
0.1 1.3
0.4 2.1
(n=9).


103
Table 5-4. Effect of removal of luminal Na in the presence of 4 mM Ba
Basal
No Na
^Rb
(nm-sec1)
60.4 14.4
106.5 14.3'
Vi
isa
-9.6 5.3
-5.5 2.0
Km,, Rb lumen-to-bath efflux coefficient; Vt> transpithelial voltage (n=4).
p < 0.005.


97
SCH28080 on Rb efflux in the presence of 20 mM K. The results from this set
of experiments suggests that the lack of effect of SCH28080 on Na efflux in
the presence of 20 mM K is not due to the lack of inhibitory effect of
SCH28080 on H-K-ATPase (Table 5-7). These data suggest that Na competes
with K for absorption via the H-K-ATPase. In contrast, microenzymatic assay
has shown that ATP hydrolysis by this enzyme is not Na-dependent (Doucet
and Marsy, 1987; Garg and Narang, 1988). The different results between the
previous studies and present studies may be reconciled by different
measurement. In the previous studies ATP hydrolysis was detected in the
permeabolized tubules in the absence of K, whereas in the present studies Rb
efflux was measured in the intact tubules. It is not unlikely that H-K-ATPase
need certain amount of K to maintain its activity. More importantly, Na entry
to the cell is down its electrochemical potential. H-K-ATPase may operate in
a fashion similar to Na-H antiport when this enzyme transports Na. In this
case, H-K-ATPase operates like a secondary active transporter and may not
need ATP hydrolysis to supply energy. Moreover, SCH28080 was still able to
inhibit Rb efflux even in the presence of a maximum pharmacologic dose of
amiloride (Figure 4-1) demonstrating that H-K-ATPase is pharmacologically
distinguishable from a Na-H antiporter.
5.5 Summary
The increase in Rb efflux by luminal Na removal was only fully blocked
by the simultaneous presence of luminal Ba and SCH28080 indicating that both
Ba-sensitive K conductance and H-K-ATPase mediate the effect of lumen Na
removal. The potential clinical significance of the present observations includes
an additional mechanism to explain the impairment in net K secretion when


42
types (Cannon et al., 1985). Exocytotic fusion of H pumps is pH- and Ca-
dependent in turtle urinary bladder. In this tissue decrease in tracellular pH
results in an increase in intracellular Ca activity by C02 stimulates this insertion
process (Cannon et al., 1985; Van Adelsberg and Al-Awqati, 1986).
Acetazolamide, another inhibitor of carbonic anhydrase, has been shown to
alkaline intracellular pH and decrease intracellular Ca activity (Van Adelsberg
and Al-Awqati, 1986). The increase in intracellular pH and decrease in
intracellular Ca level prevents the exocytotic insertion of acidic cytoplasmic
vesicles containing H pumps into the apical membrane of turtle urinary bladder
(Cannon et al., 1985; Schwartz and Al-Awqati, 1986; Steinmetz, 1986; Stetson
and Steinmetz, 1983; Van Adelsberg and Al-Awqati, 1986). Consistent with the
insertion hypothesis for H-K-ATPase, simultaneous exposure to 10% C02 and
methazolamide prevented the stimulatory effect of 10% C02 on Rb efflux
presumably because the alkalinization of intracellular pH by methazolamide
inhibited the insertion cascade, whereas after 10% C02 enhanced Rb efflux
presumably activated the exocytotic process of H-K-ATPase, subsequent addition
of methazolamide had no detectable inhibition on Rb efflux (Fig. 3-4). In
addition, methazolamide did not significantly affect Rb efflux when the CCD
was perfused in the presence of 5% C02 throughout the experiments. This
suggests that methazolamide-insensitive H source could be as a substrate for H-
K-ATPase. Recently, the observation made by Sharma et al. (1991) suggests
that not all H-K-ATPase depends on carbonic anhydrase-originated H in turtle
urinary bladder. They have demonstrated that 1 mM acetazolamide applied to
the serosal solution only reduced proton secretion to 30% of control, the
subsequent addition of SCH28080 caused H secretion fall to zero. When


52
Table 3-3. Time
control for K,^
and VT
Time after
decapitation
(in minutes)
100-150
150-200
200-250
250-300
Kr,, (nm-sec'1)
82.1 13.0
83.6 11.7
106 8.8
100 + 15.1
VT (mV)
4.2 3.4
3.9 + 3.1
5.5 2.6
5.4 2.3
(n=5).


36
Because the majority of exocytosis is Ca-dependent, more importantly,
exocytotic fusion of H-ATPase has been shown to be Ca-mediated in the turtle
urinary bladder (Schwartz and Al-Awqati, 1985 & 1986; Van Adelsberg and Al-
Awqati, 1986), to examine whether the increase in intracellular Ca activity
mediates the effect of 10% C02, the CCD was perfused in the presence of
0.5mM MAPTAM, an intracellular Ca buffer, throughout the experiments. In
this case 10% C02 failed to stimulate Rb efflux (89.5 19.5 nm-sec'1 vs 85.6
23.6 nm-sec'1, n=6, Fig 3-9). In addition, Vx became more lumen negative
during 10% C02 period (-15.7 5.9 mV vs -23.9 6.8 mV, 'p<0.05, n=6,
Table 3-5). To evaluate the role of calmodulin in the stimulation of KRb by
10% C02, the CCD was perfused in the presence of 0.5/iM W-7, an
antaganizer of calmodulin, throughout the experiments. In the presence of W-7,
Rb efflux was significantly reduced (89.3 14.0 nm-sec'1 vs 65.1 13.0
nm-sec'1, p<0.05, n=6, Fig. 3-9), suggesting that the stimulation of Rb efflux
by 10% C02 is dependent on the functional calmodulin. Transepithelial voltage
was not significantly altered (-12.2 + 3.9 mV vs -14.4 3.5 mV, Table 3-5).
Because Schlatter and Schafer (1987) have identified a Ba-sensitive K
conductance located in the basolateral membrane of both principal cells and
intercalated cells of the CCD of K replete rats, although the intercalated cells
of the K replete rabbits exhibits a little Ba-sensitive K conductance (Muto et
al., 1987). To examine whether a Ba-sensitive pathway is present in the K-
restricted CCD of rabbits, and whether this pathway mediates the stimulation
of Rb efflux by 10% C02, we perfused the CCD in the presence of 3mM Ba
in bath. Pretreatment with Ba totally abolished the stimulation of KRb by 10%
C02 (73.0 8.2 nm-sec'1 vs 70.9 8.3 nm-sec'1, n = 6, Fig. 3-10) without


86
p 0.08 "1
Figure 4-4. Effect of basolateral 0.1 mM ouabain on Vx in the absence of
luminal SCH28080 (top, n = 7) and in the presence of luminal 10 uM
SCH28080 (bottom, n = 6). Ouabain had qualitatively similar effects on VT
both in the absence and in the presence of SCH28080 although this effect was
only significant at the 0.05 level in the presence of luminal SCH28080. VT,
transepithelial voltage.


136
ambient K level, suggesting that Na competes with K for transport via
the H-K-ATPase, SCH28080 significantly reduced Rb efflux in the
presence of 20 mM K, indicating that 20 mM K does not demolish the
effect of this agent on renal H-K-ATPase;
12. Effect of SCH28080 on Rb efflux was demonstrable in the presence of
maximal pharmacological dose of amiloride, implying that H-K-ATPase
is pharmacologically distinguishable from a Na-H antiporter;
13. Angiotensin II inhibited Rb efflux, and this effect was prevented by Ba,
indicating that Ang II inhibits Ba-sensitive K conductane;
14. Neither histamine nor carbachol stimulated Rb efflux or net K
absorption, suggesting that histamine and acetylcholine are not the first
messagers activating renal H-K-ATPase.


17
Figure 1-2. Segmental analysis of tubule potassium transport. Arrows indicate
the direction of net transtubular movement (Giebisch et al., 1981).


CHAPTER 4
EFFECT OF BARIUM, AMILORIDE AND
OUABAIN ON Rb EFFELUX
4.1 Introduction
There is general agreement that potassium (K) secretion by the cortical
collecting duct (CCD) involves active translocation of K across the basolateral
membrane via Na-K-ATPase and passive exit across the apical membrane, in
part via a K-conductive pathway (Muto et al., 1988; Stokes, 1981; Warden et
al., 1989; Wingo, 1984). Administration of luminal barium, an inhibitor of K-
conductance, or amiloride, an inhibitor of conductive Na entry, or peritubular
ouabain, an inhibitor of Na-K-ATPase, inhibits net K secretion (ONeil and
Boulpaep, 1979; Stoner et al., 1974; Wingo, 1984 & 1985). These facts suggest
that K secretion is largely dependent on K exit through an Ba-sensitive pathway
and Na entry through an amiloride-sensitive pathway at the apical membrane,
and K uptake through Na-K-ATPase at the basolateral membrane.
Additionally, luminal amiloride has been demonstrated to substantially
stimulate lumen-to-bath tracer K efflux, or tracer Rb efflux, used as a marker
of K efflux in the in vitro microperfused CCD of both normal rabbits and
DOCA-treated rabbits (Stokes, 1981; Warden et al., 1989). This effect depends
on a Ba-sensitive K conductance. Therefore, the first objective of this studies
is to examine whether Ba inhibits tracer Rb efflux, and whether the effect of
amiloride on Rb efflux is preserved in K-restricted CCD.
70


140
Garg, L. C., and N. Narang. Ouabain-insensitive K-adenosine triphosphatase in
distal nephron segments of the rabbits. J. Clin. Invest. 81:1204-1208,
1988.
Garg, L. C., and N. Narang. Suppression of ouabain-insensitive K-ATPase
activity in rabbit nephron segments during chronic hyperkalemia. Renal
Physiol. Biochem. 12:295-301, 1989.
Giebisch, G. Cell models of potassium transport in the renal tubule. In:
Current topics in membranes and transport vol 28, Potassium transport:
physiology and pathophysiology, pp 133-183. (Ed. by Bronner and
Kleinzeller). Academic Press. San Diego, CA, 1987.
Giebisch. G., and B. Stanton. Potassium transport in the nephron. Annu. Rev.
Physiol. 41:241-256, 1979.
Giebisch, G., G. Malnic, and R. W. Berliner. Renal transport and control of
potassium excretion. In:The Kidney (Ed by Brenner and Rector), pp 408-
439, W. B. Saunders Company, Philadelphia, 1981.
Gifford, J. D., M. W. Ware, S. Crowson, and G. E. Shull. Expression of a
putative rat distal colonic H, K-ATPase mRNA in rat kidney:Effect of
respiratory acidosis. J. Am. Soc. Nephrol. 2:700, 1991.
Gluck, S., C. Cannon, and Q. Al-Awqati. Exocytosis regulates urinary
acidification in turtle bladder by rapid insertion of H pumps into the
luminal membrane. Proc. Natl. Acad. Sci. USA 79:4317-4331, 1982.
Grantham, J. J., M. B. Burg, and J. Orloff. The nature of transtubular Na and
K transport in isolated rabbit renal collecting tubules. J. Clin. Invest.
49:1815-1826, 1970.
Greger, R., and E. Schalatter. Properties of the basolateral membrane of the
cortical thick ascending limb of Henles loop of rabbit kidney. A model
for secondary active chloride transport. Pfiigers Arch. 396:325-334, 1983.
Gunther, R. D., S. Bassilian, and E. C. Rabn. Cation transport in vesicles
from secreting rabbit stomach. J. Biol. Chem. 262:13966-13972, 1987.
Gustin, M. G, and D. B. P. Goodman. Isolation of brush-border membrane
from the rabbit descending colon epithelium. Partial characterization of
a unique K+-activated ATPase. J. Biol. Chem. 256: 10651-10656, 1981.
Haddy, F. J. Sodium-potassium pump in low-renin hypertension. Ann. Intern.
Med. 93:781-784, 1983.
Hall, A The cellular functions of small GTP-binding proteins. Sci. 249:635-
640, 1990.


90
plus Ba period (0 mM Na and 2 mM Ba). The order of the periods was
rotated and there was no evidence of a time-dependent effect on KRb. Addition
of 2 mM luminal Ba significantly inhibited Kju, from 103 + 17.6 nm-sec'1 (basal
period) to 70.3 16.6 nm-sec'1 (Na plus Ba period, P < 0.05, Table 5-3).
Luminal Na removal significantly stimulated K^, in the presence of 2 mM
luminal Ba from 70.3 16.6 nm-sec'1 (Na plus Ba period), to 97.7 16.6
nm-sec'1 (no Na plus Ba period, P < 0.05, Table 5-3). VT was not significantly
affected by Ba addition or luminal Na removal [3.1 4.5 mV (basal period),
-0.7 3.6 mV (Na plus Ba period), and 4.7 2.1 mV (No Na plus Ba
period)] (Table 5-3). To determine whether K conductance was completely
blocked by 2 mM Ba, we repeated the experiments under the maximal
pharmacologic dose of barium which is 4 mM. To examine whether the effect
of Na removal was unique only for choline substitution, sodium was replaced
with TMA in this set of experiment. Thus, four tubules were perfused with
Solution E (basal period) and Solution F (No Na period). The order of periods
was rotated to control for time-depedent effect. There is no evidence that the
order of the periods affected either Rb efflux or voltage. As shown in Table
5-4, Removal of luminal Na with TMA substitution still significantly increased
Rb efflux (60.4 14.4 nm-sec'1 vs 106.5 14.3 nm-sec'1) without significantly
affecting voltage (-9.6 5.3 mV vs -5.5 2.0 mV). These data indicate that
stimulation of Ku, following luminal Na removal is mediated in part by a Ba-
insensitive pathway, and the effect of Na removal on Rb efflux is not only
unique for choline substitution but also for TMA substitution.
53.2 Protocol 2


19
Control
Metabolic acidosis
Respiratory acidosis
Metabolic alkalosis
Respiratory alkalosis
Metabolic + Respiratory
alkalosis
Metabolic alkalosis +
Respiratory acidosis
Figure 1-4. Effect of different types of acid-base disturbances on potassium
secretion by distal tubule (Giebisch et al, 1981).


126
Table 7-2. Effect of low Na and low K diet, and low K diet on serum Na
and K level (in mM)
Low Na and Low K diet
(n=5)
Na level
K level
Basal
140.5 0.7
4.8 0.1
On the diet
140.4 3.2
2.8 0.2'
Low K diet
(n=5)
On the diet
142.4 1.8
2.8 0.1
p<0.05, vs the basal.


145
ONeil, R. G., and S. I. Helman. Transport characteristics of renal collecting
tubules: influences of DOCA and diet. Am. J. Physiol. 233:F544-F558,
1977.
ONeil, R. G., and S. C. Sansom. Electrophysiological properties of cellular and
aracellular conductive pathways of the rabbit cortical collecting duct.
. Membr. Biol. 82: 281-295, 1984.
Ordonez, M. G., and B. H. Spargo. The morphologic relationship of light and
dark cells of the collecting tubule in potassium-depleted rats. Am. J.
Pathol. 84:317-322, 1976.
Omt, D. B., and R. L. Taimen. Demonstration of an intrinsic renal adaptation
for K conservation in short-term K depletion. Am. J. Physiol. 245:F329-
F338, 1983.
Perrone, R. D., and D. E. McBride. Aldosterone and PCO, enhance rabidium
absorption in rat distal colon. Am. J. Physiol. 254:G898-G906, 1988.
Peterson, L. N., and F. S. Wright. Effect of sodium intake on renal potassium
excretion. Am. J. Physiol. 233:F225-F234, 1977.
Planelles, G., T. Anagnostopoulos, L. Cheval, and A. Doucet. Biochemical and
functional characterization of H+-K+-ATPase in distal amphibian
nephron. Am. J. Physiol. F806-F812, 1991.
Reenstra, W. W., J. D. Bettencourt and J. G. Forte. Active K+ absorption by
the gastric mucosa: inhibition by omeprazole. Am. J. Physiol.
250:G455-G460, 1986.
Sabatini, S., and N. A. Kurtzman. Pathophysiology of the renal tubular
acidoses. Semin. Nephrol. 11:202-211, 1991.
Sansom, S., S. Muto, and G. Giebisch. Na-dependent effects of DOCA on
cellular transport properties of CCD from ADX rabbits. Am. J. Physiol.
253:F753-F759, 1987.
Sansom, S. C., and R. G. ONeil. Mineralocorticoid regulation of apical cell
membrane Na and K transport of the cortical collecting duct. Am. J.
Physiol. 248:F858-F863, 1985.
Sansom, S. C, and R. G. ONeil. Effects of mineralocorticoids on transport
properties of cortical collecting duct basolateral membrane. Am. J.
Physiol. 251:F743-F757, 1986.
Sauer, M., A. Drge, K. Thurau, and F. X. Beck. Effect of ouabain on
electrolyte concentrations in principal and intercalated cells of the
isolated perfused cortical collecting duct. Pfliigers Arch. 413: 651-655,
1989.


29
The second issue is the dependence of proton secretion in the collecting
duct on carbonic anhydrase. Carbonic anhydrase has been identified in the CCD
(Dobyan and Bulger, 1982). Carbonic anhydrase catalyzes the hydration of C02
and an increase in pC02 results in a decrease intracellular pH (Cannon et al.,
1985). The decrement in intracellular pH is a direct stimulus for H secretion
(Cannon et al., 1985; Schwartz and Al-Awqati, 1985; Stetson and Steinmetz,
1983 & 1986; Van Adelsberg and Al-Awqati, 1986). Because H-K-ATPase
participates in H secretion, it is plausible that the enhancement of K absorption
resulted from the activation of H-K-ATPase by 10% C02 is carbonic anhydrase-
dependent. However, several investigators have demonstrated that acidification
by the collecting duct is in part independent of carbonic anhydrase (Cogan et
al., 1979; DuBose and Lucci, 1983; Frommer et al., 1984; Laski, 1987;
McKinney and Davidson, 1988). Sharma et al. (1991) have suggested that not
all H-K-ATPase activity is dependent on the function of carbonic anhydrase.
Therefore, the second objective of this study is to evaluate the role of carbonic
anhydrase in the stimulation of H-K-ATPase-mediated K absorption and the role
of carbonic anhydrase in maintaining basal rate of K absorptive flux in the K-
restricted CCD.
The third issue is the intracellular mechanisms involved in the effect of
10% C02. Exocytotic insertion of H-K-ATPase in response to several stimuli has
been demonstrated in gastric gland (Forte et al., 1981). This process is
cytoskelton-dependent. The majority of exocytosis is also calcium (Ca)-dependent,
more importantly, exocytotic fusion of H-ATPase has been shown to be Ca-
mediated in the turtle urinary bladder (Schwartz and Al-Awqati, 1985 & 1986;
Van Adelsberg and Al-Awqati, 1986). Therefore, the third objective of the


13
voltage, providing the functional evidence that an H-K-ATPase is present in this
segment. The results from microenzymatic assay indicate that K-ATPase is
stimulated by K and by Rb in a similar fashion. The apparent stoichiometry of
this enzyme is 1 Rb:l ATP. Adaptation of animals to a low K diet not only
increases ATP hydrolysis, but also Rb uptake by this enzyme. SCH28080 has
been demonstrated to inhibit initial Rb uptake in the rat collecting duct (Cheval
et al., 1991) and proton (H) secretion in the amphibian nephron (Planelles et
al., 1991). Although at the molecular level whether renal H-K-ATPase is similar
to gastric H-K-ATPase, or colonic H-K-ATPase has not been reconciled, several
investigators have expressed mRNA of a putative H-K-ATPase in the kidneys
(Gifford et al., 1991; Okusa et al., 1990), and this expression is enhanced by
respiratory acidosis. By using mouse monoclonal antibodies against hog gastric
H-K-ATPase, Wingo et al. (1991) revealed diffuse cytoplasmic staining indicating
H-K-ATPase immunoreactivity in intercalated cells in the cortical collecting duct
and outer medullary collecting duct of both rat and rabbit. The percentage of
H-K-ATPase immunoreactive cells has been demonstrated to correspond to the
percentage of intercalated cell except in rat CCD in which the percentage of
positive staining is less than the percentage of intercalated cells.
Besides it has been well characterized in the gastric gland, a similar H-
K-ATPase has been also identified in colonic epithelium (Kaunitz and Sachs,
1986; Suzuki and Kaneko, 1987 & 1989) and turtle urinary bladder (Sharma et
al., 1991). The evidence obtained from functional studies suggests that H-K-
ATPase may be also present in the amphibian jejunum (Imon and White, 1984),
smooth muscle cell and the Manduca sexta embryonic cell line CHE (English
and Kantley, 1985).


121
100
g 60 -

20 -
0J 1
Basal
1 1 1
Histamine 30-60 min 60-90 min
No Histamine
Figure 6-3. Effect of histamine on in the presence of luminal 3mM Ba
(n=5). K^,, Rb lumen-to-bath efflux coefBcient.


Table 5-3. Effect of luminal Ba addition in the presence of luminal Na, and of luminal Na removal in the
presence of luminal Ba
Basal
Na plus Ba
No Na + Ba
(135 mM Na OmMBa)
(135 mM Na 2mM Ba)
(0 mM Na 2mM Ba)
KRb (nm-sec"1)
103 17.6
70.3 16.6a
97.7 16.6b
VT (mV)
3.1 4.5
-0.7 3.6
4.7 2.1
a p<0.05, compared with basal period. b p<0.05, compared with Na plus Ba period (n = 6). Tubules were
perfused with Solution A (basal period), Solution D (Na plus Ba period) and Solution C (no Na plus Ba
period). KRb, 86Rb lumen-to-bath efflux coefficient; VT, transepithelial voltage.
O
ro


54
Table 3-5. Effect of 10% CO, on VT in the tubules pretreated with 0.5 mM
MAPTAM and W-7.
MAPTAM
5% CO,
10% CO,
VT (mV) (n=6)
-15.7 5.9
-23.9 6.8
W-7
5% CO,
10% CO,
VT (mV) (n=6)
-12.2 3.9
-14.4 3.5
p<0.05, vs 5% C02 period.


2
concentrations varying between 3.5 to 5 mM. Therefore, the loss or gain of an
amount of potassium equivalent to 1% of total body potassium from the
extracellular fluid may halve or double the potassium in plasma and the
concentration ratio of potassium across the plasma membrane. This could
substantially affect the electrical polarization of both excitable and nonexcitable
tissue and be potentially lethal. In contrast, translocation of similar amount of
potassium into or from the intracellular compartment only has a minimal effect
on the concentrations. Therefore, the mechanisms controlling extracellular K
must be more sensitive than those responsible for control of the intracellular
potassium content. In fact, because of the highly regulated control mechanisms,
potassium intake can increase over 20-fold with little change in body potassium
content. Maintenance of this homeostasis reguires a balance between intake and
excretion, so-called external potassium balance. The main route for potassium
intake is the intestinal absorption which is not subject to specific control
mechanisms. There are, however, three mechanisms that maintain the level of
extracellular potassium within a critically narrow range.
1. When potassium intake increases, potassium can be translocated from
the extracellular compartment to the intracellular fluid so that only a small
fraction of any potassium added remains in the extracellular fluid. Conversely,
extracellular potassium losses can be alleviated by shifting this ion from the
cellular pool to the extracellular fluid. This distribution of potassium between
extracellular and intracellular fluids, so-called internal potassium balance, is
regulated by a variety of hormones such as aldosterone, catecholamines and
insulin and by acid-base balance.


Figure 3-7. Effect of 0.1 mM methazolamide on and
VT in the presence of 5% C02 62
Figure 3-8. Effect of simultaneous exposure to 10% C02 and
0.5 mM colchicine and exposure to colchicine
after 10% C02 on Kr,, 63
Figure 3-9. Effect of 10% C02 on KRb by the tubules pretreated
with 0.5 pM MAPTAM and W-7 64
Figure 3-10. Effect of 10% C02 on by the tubules pretreated
with peritubular 3 mM Ba 65
Figure 3-11. The dose-response curve of Ba on Kr,, 66
Figure 3-12. The CCD was swollen following exposure
to 10% C02 67
Figure 3-13. The simultaneous presence of 10% C02 and
colchicine prevented the cell swelling 68
Figure 3-14. Effect of 10% C02 on cell swelling in
the presence of MAPTAM 69
Figure 4-1. Effect of luminal 1 mM amiloride on Krj, in the
absence of luminal Ba and in the presence of
luminal Ba, and effect of SCH28080 on KRb in
the presence of luminal 1 mM amiloride
and 2 mM Ba 83
Figure 4-2. Effect of luminal 1 mM amiloride on VT in the
absence of luminal Ba and in the presence of
luminal Ba, and effect of SCH28080 on VT in
the presence of luminal 1 mM amiloride
and 2 mM Ba 84
Figure 4-3. Effect of peritubular 0.1 mM ouabain on Kr,,
in the absence of luminal SCH28080 and in the
presence of luminal SCH28080 85
Figure 4-4. Effect of peritubular 0.1 mM ouabain on K^,
in the absence of luminal SCH28080 and in the
presence of luminal SCH28080 86
Figure 4-5. Effect of luminal SCH28080 or ouabain on KRb .... 87
Figure 5-1. Effect of lumen Na removal on K^ and VT in the
presence of luminal 10 /iM SCH28080 107
xi


REFERENCES
Beck, F., A. Dorge, J. Mason, R. Rick, and K. Thurau. Element concentrations
of renal and hepatic cells under potassium depletion. Kidney Int.
22:250-256, 1982.
Beck, L. H., D. Senesky, and M. Goldberg. Sodium-independent active
potassium reabsorption in proximal tubule of the dog. J. Clin. Invest.
52:2641-2645, 1973.
Beil, W., I. Hackbarth, and E. F. Sewing. Mechanism of gastric antisecretory
effect of SCH28080. Br. J. Pharmacol. 88:19-23, 1986.
Beil, W., U. Staar, P. Schiinemann and K. F. Sewing. Omeprazole, SCH28080
and Doxepin differ in their characteristics to inhibit H + /K+-ATPase
driven proton accumulation by parietal cell membrane vesicles. Biochem.
Pharmacol. 37: 44874493, 1988.
Beil, W., U. Staar, and K. F. Sewing. SCH28080 is a more selective inhibitor
than SCH32651 at the K+ site of gastric K+/H+-ATPase. Eu. J.
Pharmacol. 139: 349-352, 1987.
Bengele, H. H., M. L. Graber, and E. A. Alexander. 1983. Effects of
respiratory acidosis on acidification by medullary collecting duct. Am.
J. Physiol. 244:F89-F94, 1984.
Berliner, R. W., and T. J. Kennedy, Jr. Renal tubular secretion of potassium
in the dog. Proc. Soc. Exp. Biol. Med. 67:542-545, 1948.
Boudry, J. F., L. C. Stoner, and M. B. Burg. Effect of acid lumen pH on
potassium transport in renal cortical collecting tubules. Am. J. Physiol.
230:239-244, 1976.
Bourne, H. R. Do GTPases direct membrane traffic in secretion? Cell
53:669-671, 1988.
Brauneis, U., P. M. Vassilev, S. J. Guinn, G. H. Williams, and D. L. Tillotson.
Ang n blocks potassium currents in zona glomerulosa cells from rat,
bovine, and human adrenals. Am. J. Physiol. 260:E772-E779, 1991.
Breyer, M. D., J. P. Kokko, and H. R. Jacobson. Regulation of net
bicarbonate transport in rabbit cortical collecting tubule by peritubular
137


95
pathway mediates the effect of amiloride. These data provide clear evidence
that lumen Na removal has more generalized effect on Rb efflux than lumen
amiloride addition. Several investigators have shown that removal of Na results
in a more lumen-positive VT (ONeil and Boulpaep, 1979), whereas addition of
Ba makes VT more lumen-negative (Frindt and Palmer, 1989; ONeil, 1985).
However, appreciable transepithelial voltage changes were not observed in the
present study. The discrepancy may be due to the different measurement of
VT and the different diets used in the present studies. In the previous
experiments, the instantaneous voltage changes following removal of Na (ONeil
and Boulpaep, 1979) and addition of Ba (Frindt and Palmer, 1989; ONeil,
1985) were monitored, whereas in the present study, we measured the average
voltage over the time of the sample collection. Equally important, all previous
experiments were conducted on K-replete animals (ONeil and Boulpaep, 1979;
Frindt and Palmer, 1989; ONeil, 1985), whereas the present results were
performed in K-restricted rabbits. The plasma aldosterone level is reduced
during K-restriction (Wingo, 1987) and it is generally accepted that K intake
and mineralocorticoid levels have significant effects on Na and K transport
(Muto et al, 1988; ONeil and Helman, 1977; Sansom and ONeil, 1985 & 1986;
Schwartz and Burg, 1978; Wingo et al., 1982). Moreover, removal of luminal
Na does not significantly affect VT in the CCD from adrenalectomized rabbits
(Wingo, 1985). Thus, one explanation for the lack of an effect of luminal Na
removal on VT in the present experiments is the significantly reduced
mineralocorticoid activity.
5.4.2 Interaction Between Na and K


38
minutes; 44.6 7.5 nm^sec'1, exposure to 10% C02 from 60 to 90 minutes; and
44.5 9.2 nmsec'', 10% C02 plus methazolamide period, n=6, Table 3-6),
although VT was significantly was significantly changed following exposure to
10% C02 and methazolamide (-35.0 11.6 mV, 5% C02 period; -44.2 14.0
mV, exposure to 10% C02 from 30 to 60 minutes; -46.9 14.7 mV, exposure
to 10% C02 from 60 to 90 minutes; and -50.7 14.5 mV, 10% C02 plus
methazolamide period, n=6, Table 3-6). Because Oberleithner and his associates
(1990) have demonstrated that H-K-ATPase requires K conductance for its
function, and Ba secondarily inhibits H-K-ATPase in Madin Darby Canine
Kidney (MDCK) cell, the CCD was alternatively perfused in the absence of
luminal Ba. As shown in Table 3-6, 10% C02 did not have trend to stimulate
Rb efflux (73.1 37.9 nm-sec"1, 5% C02 period; 66.9 40.1 nm-sec"1,
exposure to 10% C02 from 60 to 90 minutes; and 64.4 32.2 nm-sec"1,
exposure to 10% C02 from 90 to 120 minutes, n=2, Table 3-6).
3.4 Discussion
3.4.1 Effect of 10% CO-,
To our knowledge, the present studies provide the direct evidence that
respiratory acidosis (10% C02) stimulates K absorptive flux, assessed as Rb
lumen-to-bath efflux coefficient (K^), in the CCD of K-restricted rabbits.
Moreover, this enhanced appears mediated by an H-K-ATPase. The rapid
response to 10% C02 suggests that the stimulus increases either the H-K-
ATPase units in the apical membrane or the kinetics of this pump. Exocytosis
of H-K-ATPase to the apical membrane in response to the several stimuli have
been demonstrated in the gastric gland (Forte et al., 1981). In addition, C02
has been shown to be lack of major effects on kinetics of H pumps in turtle


CHAPTER 2
GENERAL METHODOLOGY
2.1 In Vitro Microperfusion
In vivo conditions. Female New Zealand White rabbits were maintained
on a regular diet (Ralston Purina), or a low K (K-restricted) diet (0.25% K,
TD 87433, Teklad, Madison, WI) as appropriate and allowed free access to tap
water. The exposure time to the low K diet was at least for 4 days before
experimentation. The majority of experiments were performed on the K-
restricted rabbits unless indicated. Rabbits were adapted to the K-restricted diet
in order to enhance the signals.
In vitro methods. Standard in vitro microperfusion methods (Burg et al,
1966) as modified in this laboratory (Wingo, 1984 & 1985) were used. Briefly,
rabbits were decapitated, the left kidney was quickly removed, and 1- to 2-mm
slices were placed in a chilled petri dish containing an artificial ultrafiltrate of
plasma. Dissection proceeded superficially from the corticomedullary junction.
Tubules were transferred to a thermostatically controlled chamber (37 C), and
the two ends of the tubule were aspirated into holding pipettes (Figure 2-1).
The perfusing pipette was advanced 100 nm beyond the holding pipette, and
transepithelial voltage (VT) was continuously monitored by means of Ag/AgCl
electrodes and a FD-223 high-impedence electrometer (World Precision
Instruments, Sarasota, FL). The bath solution was continuously exchanged at
the rate of 0.64 mimin'1. The perfusate contained 50 ttCi of [methoxy-3H]inulin
23


49
posssiblly mediated by exocytotic process. Basolateral Ba totally blocked the
enhancement of Rb efflux by 10% C02, indicating that a Ba-sensitive exit
pathway mediates the stimulation of by 10% C02.


To Xiaofang, Martin, my parents, and my brother.


134
angotension II was the signal activating the renal H-K-ATPase. However, the
experimental results did not prove the hypothesis with the serendipitous
discovery that Ang II reduced Rb efflux, and this effect was dependent on a
Ba-sensitive K conductance. The renal H-K-ATPase has been demonstrated to
be similar to gastric H-K-ATPase pharmacologically and molecular biologically.
Because histamine and acetylcholine are the potent stimulator of gastric H-K-
ATPase, it is reasonable to investigate whether these observations are
reproducible in the cortical collecting duct. Under our experimental conditions,
neither histamine nor carbachol stimulated Rb efflux or net K absorption,
suggesting that histamine and acetylcholine are not the first messager for renal
H-K-ATPase.
In conclusion:
1. Exposure to 10% C02 dramatically stimulated Rb efflux within 30
minutes, and this effect was totally blocked by luminal SCH28080,
suggesting that an existing H-K-ATPase mediates the stimulatory effect
of peritubular acidosis on Rb efflux;
2. After Rb efflux was stimulated by 10% C02, subsequent addition of
metbazolamide did not inhibit Rb efflux, whereas simultaneous exposure
to 10% C02 and methazolamide abolished the effect of 10% C02,
implying that carbonic anhydrase is not necessary for maintaining H-K-
ATPase activity, but is required for activation of H-K-ATPase by 10%
C02;
3. Only simultaneous exposure to 10% C02 and colchicine prevented the
stimulation of Rb efflux by 10% C02, reflecting that activation of H-K-
ATPase depends on intact microtubule function;


124
The primary purpose of the studies is to determine whether low salt and
low K intake affect serum Na and K level and urinary output of these cations
in our system. The dramatical reduction in Na intake for nine days did not
significantly altered serum Na level. However, both urinary Na concentration
and total Na excretion significantly decreased. In contrast to rabbit handling Na,
the moderate restriction of K intake significantly reduced the serum and urinary
K level and total K output. The serum K level was virtually same on the low
K diet as compared with on low Na and low K diet, suggesting the
hypokalemic effect by restriction of K intake is independent of Na intake. In
addition, urine output increased upon adaptation to the low Na and low K diet
because low Na intake contracts extracellular volume, and low K intake impair
urine concentration mechanism. All of these data are consistant with the
previous reports (Dahl et al, 1972; Garg et al, 1982; Kirkendall et al, 1976).
No further attempts are made to explore the mechanism for explanation of
these observations, because it is impossible to clearly explain this very
complicate issue by the present amount of data.


26
sodium and potassium. The first atomization step of 500 degree of centigrade
to 1500 degree of centigrade in "0" second allows a rapid increase in
temperature during atomization. The instrumental conditions for analysis of Na
and K were set as followings : 589.6 and 766.5 nm, bandwidth; 0.15 and 1.0
mm, lamp current; 8.0 and 7.0 mamp. N2 gas pressure was 12.0 psi. The
spectrophotometer was allowed to warm up for at least one hour and then
"auto zeroed" by atomizing without sample introduction into the fumance. The
diluted samples were delivered into a pyrolytically coated graphite cuvette in the
fumance with a 1- to 10-m1 Eppendorf digital pipette. The exact volume chosen
for a given analysis was determined by repetitive runs of the high and low
standards and adjusting the delivery volume for an optimum absorption signal
from the sodium and potassium channels. Each sample was analyzed for Na and
K a minimum of three times; generally four to six runs were employed.
2.3 Statistical analyses
Data are expressed as mean standard error. Statistical analyses were
performed by paried t test for the experiments containing two periods and by
analysis of variance (ANOVA) for repeated measures of the experiments
containing more than two periods. Post hoc comparisons were made by the
Ryan-Einot-Gabriel-Welch F test. The null hypothesis was rejected at the 0.05
level of significance.


55
Table 3-6. Effect of 10 C02 on the normal CCD in the presence and absence
of luminal Ba
With Ba
(n=6)
5% CO,
10% CO2(30-
60 minutes)
10% CO2(60-
90 minutes)
10%CO2+Me-
thazolazmide
Krs
(nm'sec'1)
48.1 7.2
42.5 8.3
44.6 7.5
44.5 9.2
vT
(mV)
-35.0 + 11.6
-44.2 14.0
-46.9 14.7
-50.7 14.5
Without Ba
(n=2)
5% CO,
10% CO2(60-
90 minutes)
10% CO2(90-
120 minutes)
K*,
(nm-sec'1)
73.1 37.9
66.9 40.1
64.4 32.2
Yn
(mV)
-33.4 0.4
-32.5 0.2
-20.4 10.4
p<0.05, vs the 5% C02 period.


6
the driving force for K secretion and the flow rate of fluid passing the cortical
collecting duct. Reduction of chloride level in the distal luminal fluid stimulates
potassium secretion by stimulating potassium and chloride co-transport
mechanism. Elevated concentrations of nonchloride anions in arterial plasma
generally increase potassium secretion through modification of the anion
composition of distal tubule fluid. Because of the relevance to the present
studies, this section will be limited to review the relationship of acid-base
disturbances and sodium absorption with potassium secretion and the current
knowledge of the cellular model underlying potassium secretion.
Perhaps, Toussaint and Vereerstraeten (1962) first examined the
relationship between acid-base balance and potassium secretion using clearance
methods. The results are shown in Figure 1-3, in which the rate of renal
potassium excretion is plotted as a function of plasma potassium concentrations
at three different pH levels. It is clear that metabolic alkalosis stimulates and
acute metabolic acidosis depresses the urinary excretion of potassium. The site
of the alteration of potassium secretion during acid-base disturbances has been
investigated more definitively by several approaches including free-flow
micropuncture (Malnic, 1971) and in vitro microperfusion (Boundry et al., 1976).
It is known that infusion of bicarbonate, or administration of carbonic anhydrase
inhibitor, or hyperventilation of animals stimulates K secretion along the distal
convoluted tubule (Kubota et al., 1983). In contrast, both acute metabolic and
respiratory acidosis decreases potassium secretion in the distal tubule (Giebisch
and Stanton, 1979; Malnic et al., 1971). Boundry et al. (1976) have
demonstrated that potassium secretion by the cortical collecting duct was
reduced when perfused in vitro by lowing luminal pH. The development of


48
Rb efflux through H-K-ATPase is overshadowed by the inhibitory effect on Rb
efflux via K-conductive pathway when the tubules were perfused in the absence
of luminal Ba. Ba may secodary inhibit H-K-ATPase when the CCD was
perfused in the presence of luminal Ba. The precedent suggestion is derived
from Oberleithner et al. (1990) observation made in MDCK cell. They have
shown that Ba and omeprazole inhibit short-circuit current and acidification of
the surface of the apical side of the dome. The effect of Ba is attributable to
inhibition of K recycle at the apical membrane thereby inhibits H-K-ATPase in
this type of cells.
3.5 Summary
In summary, these studies demonstrate that 10% C02 profoundly
stimulates Rb efflux, and this stimulation was totally abolished by SCH28080,
suggesting that an H-K-ATPase mediates this process in the K-restricted CCD.
The rapid response to 10% C02 implies that this maneuver increases the
luminal activity of existing H-K-ATPase pump units. The parallel changes in
VT regardless of the absence or presence of SCH28080 reflect that the change
in VT does not account for the effect of 10% C02 on Rb efflux. The
subsequent addition of methazolamide after 10% C02 had no significant effect
on Rb efflux, whereas simultaneous exposure of the tubules to 10% C02 and
methazolamide prevented the enhancement of Rb efflux by 10% C02, suggesting
that carbonic anhydrase is not necessary for maintaining activation of H-K-
ATPase by 10% C02, but is necessary for initiating activation of H-K-ATPase.
Colchicine, MAPTAM, and W-7 inhibit the stimulation of Rb efflux by
10%CO2, implying that activation of H-K-ATPase is dependent on the functional
microtubules, increase in intracellular Ca activity, and functional calmodulin and


66
Figure 3-11. The dose-response curve of Ba on Kjy,. K^, Rb lumen-to-bath
efflux coefficient.


mediate the effect of sodium removal; 2) sodium competes with K for transport
via the H-K-ATPase.
XVII


UNIVERSITY OF FLORIDA
3 1262 08554 8088


sec-1)
87
i NS
i p<0.05 ,
BASAL SCH28080 OUABAIN
Figiire 4-5. Effect of luminal SCH28080 and luminal ouabain on Kh (n=9).
Kr¡,> Rb lumen-to-bath efflux coefBcient.


16
MUSCLE
is:cells::
100 mEq /doy w-r Gl intoke
Liver
cells
200
RBC 235 mEq £|
Figure 1-1. Distribution of potassium in the
acquisition and excretion (Giebisch et al, 1981).
body, including routes of


147
Stanton, B. A., W. B. Guggino, and G. Giebisch. Acidification of the
basolateral solution reduces potassium conductance of the apical
membrane. Fed. Proc. Fed. Am. Soc. Exp. Biol. 41:1006, 1982.
Steinmetz, P. R. Cellular organization of urinary acidification. Am. J. Physiol.
251:F173-F187, 1986.
Stetson, D. L., and P. R. Steinmetz. Role of membrane fusion in C02
stimulation of proton secretion by turtle bladder. Am. J. Physiol.
245:013-020, 1983.
Stetson, D. L., and P. R. Steinmetz. Correlation between apical
intramembrane particles and H+ secretion rates during C02 stimulation
in turtle bladder. Pflger Arch. 407:580-584, 1986.
Stetson, D. L., J. B. Wade, and G. Giebisch. Morphologic alterations in the rat
medullary collecting duct following potassium depletion. Kidney Int.
17:45-56, 1980.
Stokes, J. B. Potassium secretion by cortical collecting tubule: relation to
sodium absorption, luminal sodium concentration, and transepithelial
voltage. Am. J. Physiol. 241:F395-F402, 1981.
Stokes, J. B. Pathways of K permeation across the rabbit cortical collecting
tubules: effect of amiloride. Am. J. Physiol. 246: F457-F466, 1984.
Stoner, L. C., M. B. Burg, and J. Orloff. Ion transport in cortical collecting
tubule, effect of amiloride. Am. J. Physiol. 227(2): 453-459, 1974.
Sweiry, J. H., and H. J. Binder. Active potassium absorption in rat distal
colon. J. Physiol. 423: 155-170, 1990.
Suzuki, Y., and K. Kaneko. Acid secretion in isolated guinea pig colon. Am.
J. Physiol. 253:G155-G164, 1987.
Suzuki, Y., and K. Kaneko. Ouabain-sensitive H-K exchange mechanism in the
apical membrane of guinea pig colon. Am. J. Physiol. 256:G979-G988,
1989.
Taimen, R. L., and L. Gerrits. Response of the renal K+-conserving mechanism
to kaliuretic stimuli: evidence for a direct kaliuretic effect by
furosemide. J. Lab. Clin. Med. 107:176-184, 1986.
Toussaint, C., and P. Vereerstraeten. Effects of blood pH changes on
potassium excretion in the dog. Am. J. Physiol. 202:768-772, 1962.
Tumamian, S. G., and H. J. Binder. Regulation of active sodium and
potassium transport in the distal colon of the rat: role of the
aldosterone and glucocorticoid receptors. J. Clin. Invest. 84:1924-1929,
1989.


108
p < 0.05 1
Figure 5-2. Inhibition of by luminal 10 /iM SCH28080 in the presence of
luminal Na (top, n=6) and m the absence of luminal Na (bottom, n = 4). In
both cases SCH28080 significantly inhibited Kr,,. K^, Rb lumen-to-bath efflux
coefficient.


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
MECHANISMS OF POTASSIUM PERMEATION IN THE IN
VITRO PERFUSED CORTICAL COLLECTING DUCT OF THE RABBIT
By
Xiaoming Zhou
May 1992
Chairman: Charles S. Wingo, M.D.
Major Department: Physiology
These studies were designed to examine the mechanisms of potassium
(K) permeation across rabbit cortical collecting duct (CCD) assessed as Rb
lumen-to-bath efflux coefficient (Krj,, or Rb efflux). This segment is primarily
responsible for potassium secretion, but recent studies have demonstrated
potassium absorption by the CCD.
The first set of studies was designed to examine whether acute
peritubular acidosis (10% C02) stimulates rubidium (Rb) efflux, and whether
this stimulation is dependent on the presence of functional H-K-ATPase,
carbonic anhydrase, microtubules, the increase of intracellular calcium activity,
calmodulin, and basolateral potassium conductance in the cortical collecting duct
from K-restricted rabbits. Exposure to 10% C02 substantially stimulated Rb
efflux, and this stimulation was totally abolished by 10uM SCH28080, a specific
inhibitor of H-K-ATPase. After stimulation of Rb efflux by 10% C02,
xv


14
1.4 The Aims and Objectives of the Present Studies
The discovery of H-K-ATPase is a novel approach to characterize K
absorption. Its significance in both understanding basic K transport mechanism
and clinical implications may significantly extend our present knowledge. This
dissertation attempts to characterize potassium permeation by the CCD with
emphasis of H-K-ATPase in the conditions which are close to in vivo without
equivocating the interpretation of the results. In vitro micropersion technique is
able to serve this dual-purposes. Potassium permeation was evaluated as Rb
lumen-to-bath efflux coefficient (Kpj, or Rb efflux). The H-K-ATPase activity
was assessed as SCH28080-sensitive or SCH28080-sensitive Rb efflux. The
specific objectives are summarized as following:
1. Because H-K-ATPase secretes proton in exchange of K absorption,
we will determine whether acute peritubular acidosis (10% C02) stimulates Rb
efflux, whether this stimulation is dependent on the presence of functional H-K-
ATPase. We will also identify the intracellular and basolateral mechanisms
explaining the effect of 10% C02 on Rb efflux.
2. Because luminal amiloride and peritubular ouabain has been shown
to inhibit net K excretion, we will examine whether luminal amiloride and
peritubular ouabain increases Rb efflux, and whether the effects of amiloride
and ouabain are mediated by K-conductance or by H-K-ATPase. Because of the
indirect evidence suggesting that an apical Na-K-ATPase may participate in
potassium conservation, we will identifiy the roles of H-K-ATPase and Na-K-
ATPase in mediation of Rb efflux.
3. Because removal of luminal Na produces similar effect on ion
transport in many cases as the addition of luminal amiloride, we will examine


20
LUMEN
CELL
BLOOD
Principal cell
Intercalated cell
Figure 1-5. The cellular models of the principal cell (top) and the intercalated
cell (bottom).


128
Because renal H-K-ATPase has been demonstrated to be involved in
proton secretion, and metabolic acidosis stimulates ATP hydrolysis by this
enzyme, our first serial of studies were designed to examine whether acute
respiratory acidosis induced by the presence of peritubular 10% C02 and
presumably intracellular acidosis stimulates Rb efflux, and whether this
stimulation is dependent on the presence of functional H-K-ATPase. These
studies also extended to investigate the roles of carbonic anhydrase,
microtubules, intracellular Ca activity, calmodulin, and basolateral K conductance
in these maneuvers. Time-control experiments demonstrated that perfusion time
did not significantly affect transepithelial voltage and Rb efflux. However, when
the CCD was exposed to 10% C02 for 60 minutes Rb efflux increased from
93.1 23.8 nm sec'1 (5% C02 period) to 249 + 60.2 nm-sec'1 (10% C02
period, p<0.05). When the experiments were repeated under identical conditions
except for the presence of the H-K-ATPase inhibitor SCH28080, 10% C02
failed to increase Rb efflux (76.4 + 15.1 nm-sec'1, 5% C02 period vs 76.8
13.3 nm-sec'1, 10% C02 period). This suggests that the effect of 10% C02 is
dependent on H-K-ATPase. To examine the time course of this effect we
conducted additional experiments. We were not only able to reproduce the
stimulatory effect of 10% C02 on Rb efflux, but also observed this effect at
the earliest collection time (30-60 minutes after exposure to 10% C02). Again,
SCH28080 (10/jM) totally abolished the Rb response, suggesting that an existing
H-K-ATPase mediates the stimulatory effect of 10% C02 on Rb efflux.
However, whether de novo H-K-ATPase synthesis is involved in this process can
not be ruled out by the present experiments. Carbonic anhydrase catalyzes
hydration of C02 which result in decrease in intracellular pH. The decrease in


59
Figure 3-4. Effect of 10% C02 and 0.1 mM methazolamide on KRb and VT
in the absence of SCH28080 (n = 6). 'P < 0.05 compared with 5% CO,
period. aP = 0.06 compared with 60-90 minute period. P < 0.05 compared
with 90-120 minute period. K^, ^Rb lumen-to-bath efflux coefficient. VT,
transepithelial voltage.


78
significantly decreased K absorption, suggesting that the effect of ouabain on K
absorption is mediated by H-K-ATPase. Ouabain significantly decreases
intracellular K level of both principal cells and intercalated cells in the CCD
(Sauer et al., 1989) and increases Rb efflux when SCH28080 is absent in the
perfusate (Figure 4-3) whereas this maneuver fails to affect Rb efflux when
SCH28080 is present in the perfusate (Figure 4-3). These data further support
such a hypothesis.
The decrease in intracellular potassium concentration increasing H-K-
ATPase may not unique phenomenon only obverved with peritubular ouabain.
Several investigators have shown that the intracellular K level in the distal
tubule was reduced by adaptation of the animals to a low K diet (Beck et al.,
1982; Linas et al., 1979). Both incerase in SCH28080-sensitive ATP hydrolysis
and Rb efflux were observed in the CCD of rats adapted to K-restricted diet
as compared with those on a regular diet, suggesting that H-K-ATPase activity
is enhanced under these conditions (Cheval et al., 1991; Doucet and Marsy,
1987).
4.4.4 Effects of Luminal SCH28080 and Ouabain
In the absence of luminal Na, luminal 10 qM SCH28080 significantly
reduced Kr, by 39%, whereas luminal 0.1 mM ouabain did not significantly
affect Kr!,. These observations suggest that under the present experimental
conditions, H-K-ATPase mediates in part Ba-insensitive Rb/K efflux and a
ouabain-sensitive luminal Na-K-ATPase is not a major pathway for Rb/K
absorption. Were an apical Na-K-ATPase mediating K absorption, the greatest
degree of inhibition would be expected with luminal ouabain, whereas only the
effect of SCH28080 was significant. The present demonstration is consistant with


46
in turtle urinary bladder (Cannon et al., 1985; Schwartz and Al-Awqati, 1986;
Van Adelsberg and Al-Awqati, 1986). Exposure to C02 increases intracellular
Ca activity in tissue (Cannon et al., 1985). This elicits whole cascade of
insertion process. When the increase in intracellular Ca activity is minimized,
either by chelation of extracellular Ca (Van Adelsberg and Al-Awqati, 1986),
or by buffering intracellular Ca (Cannon et al., 1985; McKinney and Davidson,
1988), the excytosis and the increased proton secretion are mitigated both in
the turtle urinary bladder and the collecting duct. A body of evidence is also
consistant with a mediator role for calmodulin in the stimulation of H secretion
resulting from exposure to C02. The increment in total C02 absorption upon
exposure to C02 by the inner strip of outer medullary collecting duct is
markedly attenuated by the potent antaganizer of calmodulin (W-7) but not by
a structurally related analogue with less calmodulin inhibitory effect (W-5)
(Dytko and Arruda, 1985; McKinney and Davidson, 1988). In view of these
observations, we have chosen two maneuvers expected to be associated with
alterations in intracellular Ca or calmodulin activity. Pretreatment with 0.5qM
MAPTAM blocked the stimulatory effect of Rb efflux by 10% C02. The result
is presumably due to the failure of intracellular ionized Ca level to rise.
Moreover, pretreatment with 0.5/rM W-7 totally abolished the increase in Rb
efflux by 10% C02. However, the significant decrease in Rb efflux was observed
during W-7 period. The mechanisms explaining this phenomenon remain to be
elucidated. W-7 may have additional inhibition of Rb efflux mediated by K
conductance. The important point here is that W-7 prevented the increase in
Rb efflux induced by 10% COj, providing the clear evidence indicating the role
of calmodulin in this response. MAPTAM significantly made voltage more


107
0
Figure 5-1. Effect of lumen Na removal in the presence of luminal 10 nM
SCH28080 on K^, (top) and on (bottom, n = 10). Removal of luminal Na
significantly stimulated from 76.7 7.3 nm-sec'1 to 101 10.1 nm-sec'1
but had no significant effect on VT (-4.3 13 mV vs -1.7 1.0 mV). K^,,
tRb Iumen-to-bath effiux coefficient. transepithelial voltage.


Table 3-4. Effect of 10% C02 on VT in the presence of colchicine.
53
5% CO,
10% co2 +
Colchicine
VT (mV) (n=7)
-12.9 4.9
-15.6 5.0
5% CO,
10% CO,
10% co2 +
Colchicine
VT (mV) (n=7)
-11.2 7.1
-16.9 9.4"
-17.4 9.5b
' p< 0.05, vs 5% C02 period;
b p< 0.01, vs 5% C02 period.


113
p<0.05, n=6, Fig. 6-2). Taken together, this suggests that inhibition of Rb efflux
by Ang n depends on the Ba-sensitive K conductive pathway.
6.3.2 Protocol 2
To examine whether histamine stimulates Rb efflux, the experiments were
performed in the absence of luminal SCH28080 and presence of luminal 3 mM
Ba with Solution A as bath and Solution B as perfusate. 10 mM histamine had
no significant effect on Rb efflux, and the observation from 30 to 90 minutes
later after the removal of histamine indicates that perfusion time did not affect
Rb efflux (77.9 13.8 nm.sec'1, basal period; 79.5 + 16.8 nm.sec'1, histamine
period; 73.3 20.9 nm.sec'1, 30-60 minutes after histamine; and 80.9 20.8
nm.sec'1, 60 to 90 minutes after histamine, n=5, Fig. 6-3). The voltage has not
been significantly changed by these maneuvers (-11.8 5.8 mV, basal period; -
11.6 7.4 mV, histamine period; -9.2 7.5 mV, 30 to 60 minutes after
histamine; and -7.6 + 6.3 mV, 60 to 90 minutes after histamine, n=5, Table
6-2). This suggests that histamine did not stimulate H-K-ATPase under the
present experimental conditions. Carbachol has been shown to cause natriuresis.
Inhibition of Na transport by removed of luminal Na and addition of peritubular
ouabain has been demonstrated to stimulate Rb efflux mediated by H-K-
ATPase. The next set of experiments was designed to examine whether
carbachol affects both Na and K transport with Solution C as bath and
perfusate). For the technical convenience, the net chemical flux was measured
using flameless atomic absorption spectrophotometer during this study. As shown
in Table 6-3, although carbachol significantly stimulated Na absorptive flux from
5.0 2.2 pmol/mm/min to 11.5 2.3 pmol/mm/min, the magnitude of this
stimulation is quite small and has no physiological significance. The effect of


CHAPTER 5
EFFECTS OF LUMINAL SODIUM ON Rb EFFLUX
5.1 Introduction
Luminal Na removal has been demonstrated to inhibit K secretion
similarly to that observed with luminal amiloride and peritubular ouabain
(Stokes, 1981; Wingo, 1985). It is possible that luminal Na affects Rb efflux
which is mediated by K-conductive pathway, or H-K-ATPase, or both. This
hypothesis is further substantiated by the observation made in rat colon that Na
removal stimulates the effect of 10% C02 on K absorption possibly by a closely
related H-K-pump (Perron and McBride, 1988). These data suggest that luminal
sodium concentration may influence H-K-ATPase activity in the CD. Therefore,
the present studies aim at examination of the lumen Na removal on Rb efflux
and the mechanisms underlying this effect.
5.2 Methods and Material
The tubules were dissected with Solution A (Table 5-1) containing
additional 5% vol/vol feral calf serum. The bath solution which is identical to
dissection solution was used throughout all sets of experiments except the study
of Na efflux. All solutions were gassed to pH 7.4 with 95% 02 and 5% C02.
Liquid junction potentials were corrected as previously described (Wingo, 1989).
The overlap of Rb or Na counts in the 3H channel was corrected as
previously described (Zhou and Wingo, 1992). SCH28080 (gift of Dr. James
Kaninsky, Schering Corporation, Bloomfield, NJ) was dissolved in dimethyl
88


Table 3-2. Effect of luminal 0.1% DMSO on KRb and VT
51
Basal
DMSO
^Rb
(nm-sec1)
81.4 9.0
83.0 13.7
VT
(mV)
-8.3 3.4
-5.8 4.1
DMSO, dimethylsufoxide (n=6).


92
To further examine whether H-K-ATPase mediates in part the increase
in Rb efflux following removal of luminal Na, we examined the effect of
SCH28080 both in the presence (Solution D) and in the absence (Solution C)
of luminal Na with 2 mM Ba in the perfusate throughout the experiment. In
each case K^, and VT were measured in the absence of luminal SCH28080
(basal period) and in the presence of luminal SCH28080 (10 jiM, SCH28080
period). The order of the periods was rotated to control for time-dependent
effects. There was no evidence to suggest that the order of the periods affected
Rb efflux and voltage (P = NS). As shown in Figure 5-2, in the presence of
luminal Na, SCH28080 significantly reduced K*. from 78.3 12.0 nm-sec"1
during the basal period to 67.2 10.8 nm-sec'1 during the SCH28080 period
(P < 0.05) without significantly affecting Vx (-2.8 2.7 mV vs -1.0 1.2 mV,
Table 5-6). In the absence of luminal Na, SCH28080
significantly decreased KRb from 122 + 30.3 nm-sec'1 to 69.4 22.0 nm-sec'1
(Figure 5-2, P < 0.05) without significantly affecting Vx (5.7 9.5 mV vs 6.6
5.5 mV, Table 5-6). The percentage inhibition of K^, by SCH28080 was
significantly less in the presence of luminal Na (14.7 5.0%) as compared with
the absence of luminal Na (48.2 8.2%, P < 0.01). These experiments confirm
that 1) H-K-ATPase is responsible for Rb/K absorption in the rabbit CCD, and
2) an increase in Rb/K absorption following removal of luminal Na is in part
via an H-K-ATPase.
5.3.4 Protocol 4
The effect of luminal Na removal on Rb efflux is consistent with the
hypothesis that Na acts as a partial agonist at the luminal K binding site and
competes with K for transport via the renal H-K-ATPase. Such a hypothesis


118
Table 6-3. Effect of carbachol
Basal
Carbachol
^Na
(pmol/mm/min)
5.0 2.2
11.5 2.3'
JK
(pmol/mm/min)
-3.1 0.4
-1.6 0.5
VT
isY)
1.4 1.5
1.3 1.7
p<0.05, vs the basal period (n=6).


15
whether Na removal stimulates Rb efflux, whether the effect of Na removal is
mediated by K-conductance or by H-K-ATPase or both. We will also determine
the mechanism underlying the effect of Na removal on H-K-ATPase.
Additional efforts have been made to study hormonal regulation of this
pump. The results of metabolic studies on a low Na, low K diet, and a low
K diet are also reported1.
1 All of the studies were funded by the Department of Veterans Affairs


144
McKinney, T. D., and K. K. Davidson. Bicarbonate transport in collecting
tubules from outer stripe of outer medulla of rabbit kidneys. Am. J.
Physiol. 253:F816-F822, 1987.
McKinney, T. D., and K. K. Davidson. Effects of respiratory acidosis on -
HC03 transport by rabbit collecting duct. Am. J. Physiol. 255:F656-
F665, 1988.
McMurray, J., and A. D. Struthers. Effects of angiotensin II and atrial
natriuretic peptide alone and in combination on urinary water and
electrolyte excretion in man. Clin. Sri. 74:419-425, 1988.
Mendlein, J., and G. Sachs. Interaction of a K+-competitive inhibitor, a
substituted imidazo[l,2a]-pyridine, with the phospho- and
dephosphoenzyme forms of H, K-ATPase. J. Biol. Chem. 265:5030-5036,
1990.
Mudge, G. H., J. Foulks, and A. Gilman. The renal excretion of potassium.
Proc. Soc. Exp. Biol. Med. 67:545-547, 1948.
Mujis, S. K., and A. I. Katz. Potassium deficiency. In:The Kidney The
Physiology and Pathophysiology, pp 2249-2278, Raven Press, New York,
1992.
Mujis, S. K., S, Kauffman, and A. I. Katz. Angiotensin II binding sites in
individual segments of the rat nephron. 77:315-318, 1986.
Muto, S., G. Giebisch, and S. Sansom. Effects of adrenalectomy on CCD:
evidence for differential response of two cell types. Am. J. Physiol.
253:F742-F752, 1987.
Muto, S., S. Sanson, and G. Giebisch. Effects of a high potassium diet on
electrical properties of cortical collecting ducts from adrenalectomized
rabbits. J. Clin. Invest. 81:376-380, 1988.
Oberleithner, H., W. Steigner, S. Silbemag, U. Vogel, G. Gstraunthaler, and W.
Pfaller. Madin-Darby canine kidney cells HI. Aldosterone stimulates an
apical H/K pump. Pfluger Arch. 416:540-547, 1990.
Okusa, M. D., R. Unwin, F. S. Wright, G. Giebisch, and M. J. Caplan. H-K
ATPase mRNA expression in rat kidney. Kidney Int. 37: 568, 1990.
ONeil, R. G. Voltage-dependent interaction of barium and cesium with the
potassium conductance of the cortical collecting duct apical cell
membrane. J. Membr. Biol. 74: 165-173, 1985.
ONeil, R. G., and E. L. Boulpaep. Effect of amiloride on the apical cell
membrane cation channels of a sodium-absorbing, potassium-secreting
renal epithelium. J. Membr. Biol. 50: 365-387, 1979.


135
4. Pretreatment with MAPTAM and W-7 blocked the effect of 10% C02
on Rb efflux, suggesting that the changes in intracellular Ca activity and
calmodulin mediate the effect of respiratory acidosis;
5. Pretreatment with peritubular Ba abolished the effect of 10% C02,
indicating that the stimulation of Rb efflux is dependent on the presence
of functional barium-sensitive basolateral exit mechanism;
6. Luminal barium inhibitied Rb efflux, indicating that Ba-sensitive K
conductance is present in the K-restricted CCD;
7. Luminal Ba fully inhibited the stimulatory effect of amiloride on Rb
efflux, suggesting that a Ba-sensitive K conductance mediates the effect
of amiloride;
8. Basolateral ouabain stimulated Rb efflux, and this effect was totally
inhibited by SCH28080, implying that the stimulation of Rb efflux by
ouabain is via H-K-ATPase mechanism;
9. Only luminal SCH28080, not luminal ouabain, inhibited Rb efflux,
suggesting that a luminal H-K-ATPase, not a luminal Na-K-ATPase,
mediates Rb efflux;
10. Removal of luminal Na stimulated Rb efflux, and
this effect was not totally inhibited by SCH28080 or Ba alone, only
simultaneous presence of these two agents fully blocked the effect
oonducf Na removal, indicating that both Ba-sensitive K conductance and
H-K-ATPase mediate the enhancement of K^, following lumen Na
removal;
SCH28080 reduced Na efflux in the presence of low ambient K
concentration, whereas the effect of SCH28080 was inhibited by high
11.


72
4.2 Methods and Material
The tubules were dissected with Solution A (Table 4-1) containing
additional 5% vol/vol feral calf serum. The bath solution which is identical to
dissection solution was used throughout all sets of experiments. Three different
perfusates (Solution A, Solution B or Solution C) were used as appropriate. All
solutions were gassed to pH 7.4 with 95% 02 and 5% C02. Amiloride was a
gift of Merck Sharp & Dohme (Rahway, NJ) and was dissolved in the perfusate
directly. SCH28080 (gift of Dr. James Kaninsky, Schering Corporation,
Bloomfield, NJ) was dissolved in dimethyl sulfoxide (DMSO). Ouabain was
dissolved in 0.9% NaCl solution.
4.3 Results
4.3.1 Protocol 1
To examine whether amiloride enhances ^Rb in the K-restricted animals, the
first set of experiments was performed in the absence of luminal Ba. Thus,
eight tubules were perfused with solution A, or solution A plus 1 mM amiloride
designated as basal and amiloride periods, respectively. The order of the periods
was rotated and there was no evidence of time-dependent effect on KKb or VT.
In the absence of luminal Ba, luminal 1 mM amiloride significantly increased K^, from
90.7 14.3 nmsec1 (basal period) to 119 20.6 nm-sec'1 (amiloride period, P <
0.05, Figure 4-1), and VT from 0.6 1.5 mV (basal period) to 4.7 0.9 mV
(amiloride period, P < 0.05, Figure 4-2). To examine whether the effect of amiloride
on Rb efflux was mediated by Ba-sensitive pathway, the second set of experiments was
performed in the presence of luminal 2 mM Ba, because in view of the voltage-
dependent nature of Ba inhibition, Rb efflux in the K-restricted CCD should be
maximally inhibited by 2 mM Ba at which has been demonstrated to maximally inhibit


64
(XO.05
Figure 3-9. Effect of 10% C02 on KRb by the tubules pretreated with 0.5mM
MAPTAM (top) and W-7 (bottom)(n = 6). Kr,,, Rb lumen-to-bath efflux
coefficient.


115
glomerulosa cells have shown that K channels are reversibly blocked when Ang
II was added to the bath solution. These observations are compatible with the
results of the present studies.
6.4.2 Effect of Histamine and Carbachol
The present data do not support the positive role of histamine and
carbachol in stimulation of renal H-K-ATPase, although these two hormones are
the potent stimulators of gastric H-K-ATPase (Forte et al, 1981; Koelz et al,
1981). Many possibilities could be proposed to explain these observations.
However, either no receptors of the hormones in the CCD or the different
signal transduction of the renal H-K-ATPase from the gastric H-K-ATPase is
most plausible.
6.5 Summary
Angiotensin II inhibited Rb efflux mediated by a Ba-sensitive
conductance, suggesting that Ang II inhibit K secretion. The stimulatory effect
of histamine and carbachol on Rb efflux or K absorptive flux was not observed
in the present studies, implying that under the present conditions these
hormones do not stimulate H-K-ATPase.


31
dose-response study, the composition of bath were (in mM): Na 135; K 5; Cl
106.4; HC03 25; Ca 1.2; Mg 1.0; P04 1.5; acetate 10; glucose 8; alanine 5;
mannitol 26.5. The equilibration time between two periods was 30 minutes
unless indicated. The perfusate was identical to bath except for the absence
of fetal calf serum and gassed only with 5% C02 unless indicated. The
composition of the perfusate for peritubular Ba study were (in mM): Na 138;
K 5; Cl 109.4; HC03 25; Ca 1.2; Mg 1.0; acetate 10; glucose 8; alanine 5;
mannitol 9; and P04 1.5. Because Ba precipitates with phosphate, HEPES (N-2-
Hydroxyethylpiperazine-N-2-ethanesulfonic acid)-buffer was used as the perfusate
in the Ba dose-response study. Warden et al () have shown that Rb efflux by
the CCD perfused with HEPES-buffer is not significantly different from that
perfused with bicarbonate-buffer. Total three perfusates listed in Table 3-1 were
used in this set of studies.
SCH28080 (gift of Dr. James Kaninsky, Schering Corporation, Bloomfield,
NJ) was dissolved in dimethyl sulfoxide (DMSO) and applied to the perfusate
with final concentration of 10 nM. Methazolamide (American Cyanamide
Company, Pearl River, NY), Bis-(2-amino-5-methyl-phenoxy)-ethane-N,N,N,N-
tetraacetic acid tetraacetoxymethyl ester (MAPTAM, Sigma, St. Louis, MO), and
N-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide (W-7, Sigma, St. Louis, MO)
were also dissolved in DMSO and applied to bath with final concentration of
0.1 mM, 0.5pM, and 0.5pM, respectively. Colchicine (Sigma, St. Louis, MO) was
dissolved in 0.9% NaCl solution and applied to bath with the final
concentration of 0.5mM. The final concentration of 0.1 % DMSO was present
in bath during basal period of methazolamide sets of experiments.
3.3 Results


96
The stimulatory effect of luminal Na removal on Rb efflux via H-K-
ATPase may be similar to the mechanism proposed for a closely related K-
ATPase in the colonic epithelium. Although the pharmacologic characteristics
of this putative pump were not characteristic of the gastric H-K-ATPase, Sweiry
and Binder (1990) demonstrated that Na absorptive flux, like K-absorptive flux,
was inhibited by orthovanadate and mucosal ouabain when mucosal and serosal
K concentration was 0.6 mM. However, when K level was 5.2 mM in the
mucosal and serosal solutions, both drugs had less effect on Na absorption.
The authors interpreted the data as evidence that mucosal Na competed with
mucosal K for uptake across the apical membrane. Although the putative
pump described by Sweiry and Binder was relatively resistant to SCH28080 but
sensitive to mucosal ouabain, it should be noted that a similar K-absorptive
mechanism in the rabbit colon is not inhibited by ouabain (Gustin and
Goodman, 1981; Halm and Frizzell, 1986). Thus species-specific differences in
the pharmacologic sensitivity of K-absorbing pumps may be more important than
previously appreciated. In support of such a hypothesis, SCH28080 significantly
inhibited Na efflux when the tubules were perfused in the presence of 0.5 mM
K (Figure 5-3), whereas the effect of SCH28080 was not observed when the
tubules were perfused in the presence of 20 mM K (Figure 5-3). Because
SCH28080 has ben shown to compete with K at K binding site of gastric H-K-
ATPase, and high K concentration abolishes the inhibitory effect of SCH28080
on gastric H-K-ATPase (Keeling et al., 1988; Mendlein and Sachs, 1990), to
determine whether the lack of inhibition of KNa by SCH28080 in the presence
of 20 mM K is due to potassium diminishing Na efflux or due to K
demolishing the inhibitory effect of SCH28080, we examined the effect of


Table 3-1. Composition of solutions (in mM)
50
Solution A
Solution B
Solution C
Na
135
135
135
K
5
5
5
Cl
110.4
114.4
114.4
Ca
1.2
1.2
1.2
Mg
1
1
1
Acetate
10
10
10
Glucose
8
8
8
Alanine
5
5
5
HEPES
10
10
10
Gluconate
23
23
23
Ba
0
2
4
TMA
4
4
0
Osmol (m Osm)
317.6
322.6
321.6


56
p<0.05 1
*f^c^uo"oAon^?eCt 0L^^LC02 on Kr,, (top) and VT (bottom) in the absence
or 5LH28U8U (n = 7). The tubules were allowed equilibrating for one hour
before stating collection of sample during 10% C02 period. Kr,,, Rb lumen-
to-bath efflux coefficient. transepithelial voltage.


68
Figure 3-13. The simultaneous presence of 10% C02 and colchicine prevented
the cell swelling. Top: 5% C02 period, Bottom: 10% C02 plus colchicine
period.


58
p<0.01
Figure 3-3. Effect of 10% C02 on Krj, (top) and VT (bottom) in the presence
of SCH28080 (n =5). The tubules were allowed equilibrating for one hour
before starting collection of sample during 10% C02 period. K^, Rb lumen-
to-bath efflux coefficient VT, transepithelial voltage.


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.
Charles S. Wingo, Chairman/
Associate Professor of Medicine and
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.
Professor 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.
LclX C .
Lai C. Garg
Processor of Pharmacology and
Therapeutics


89
sulfoxide (DMSO). Amiloride was a gift of Merck Sharp & Dohme (Rahway,
NJ) and was dissolved in the perfusate directly.
5.3 Results
5.3.1 Protocol 1
To examine whether lumen Na removal stimulates Rb efflux as lumen
amiloride addition, the first set of experiments was designed to evaluate the
effect of luminal Na removal on K,^ in the absence of luminal Ba and the
degree of inhibition of Kr, by Ba in the absence of luminal Na. Six tubules
were perfused with each of the following: solution A designated the basal
period (135 mM Na and 0 mM Ba); solution B designated the no Na period
(0 mM Na and 0 mM Ba); and solution C designated the no Na plus Ba
period (0 mM Na and 2 mM Ba). The order of the periods was rotated and
there was no evidence of a time dependent effect on Rb efflux. Luminal Na
removal significantly stimulated Kr,, (Table 5-2) from 96.8 22.2 nm-sec"1
(basal period) to 127 20.0 nm-sec'1 (no Na period, P < 0.005). Luminal
addition of 2 mM Ba significantly inhibited KRb from 127 20.0 nm-sec'1 (no
Na period) to 90.9 23.0 nm-sec'1 (no Na plus Ba period, P < 0.005). In
contrast to Rb efflux, VT was not significantly different during the basal, the
no Na, or the no Na plus Ba periods (6.3 2.2 mV, 6.4 1.9 mV and 7.2
2.1 mV respectively, Table 5-2). The second set of experiments examined the
degree of inhibition of Rb efflux by Ba when Na is present in the perfusate
and the effect of luminal Na removal on K,^ in the presence of luminal Ba.
Six tubules were perfused with each of the following: solution A designated the
basal period (135 mM Na and 0 mM Ba), solution D designated the Na plus
Ba period (135 mM Na and 2 mM Ba), and solution C designated the no Na


57
Figure 3-2. The predicted voltage-mediated increase in KRb and observed
increase in KRb following exposure to 10% C02. Kr,,, Rb lumen-to-bath efflux
coefficient.


85
I NS 1
Figure 4-3. Effect of basolateral 0.1 mM ouabain on KRb in the absence of
luminal SCH28080 (top, n = 7) and in the presence of luminal 10 mM
SCH28080 (top, n = 6). In the absence of luminal SCH28080, ouabain
significantly increased KRh, whereas in the presence of luminal SCH28080, the
effect of ouabain on was abolished. Kg,,, Rb lumen-to-bath efflux
coefficient.


116
Table 6-1. The composition of solutions (in mM).
Solution A
Solution B
Solution C
Na
138
135
145
K
5
5
5
Cl
109.4
115.4
112
HC03
25
25
25
Ca
1.2
1.2
1.8
Mg
1
1
1
Acetate
10
10
10
Glucose
8
8
8
Alanine
5
5
5
Ba
Phosphate
1.5
3
2.3
so4
Mannitol
9
4.5
1
Osmol (m Osm)
313.1
313.1
316.1


3
2. The colon has a capacity for potassium secretion and absorption
which may be stimulated when the kidneys ability to handle potassium balance
is compromised.
3. The kidney plays the key role and is ultimately for the appropriate
response to changes in potassium excretion to match potassium intake. As
shown in Figure 1-2 (Giebisch et al., 1981), in an individual ingesting 100 mmol
of K per day, the proximal tubules absorbs up to 70% of filtered potassium
which is approximately 600 mmol per minute. The Loop of Henle absorbs
additional 22% of filtered K. When the luminal fluid deliveries to the early of
distal tubule, only less than 10% of filtered K, approximately 50 mM, are left.
Potassium secretion commences in the initial collecting duct and continues along
the cortical collecting duct. The collecting duct (CD) is the final segment to
determine net K excretion (Giebisch et al., 1981; Wright and Giebisch, 1992).
In this case, the collecting duct transports net 50 mmol K into the urine.
Therefore, 100 mmol potassium appears in the final urine, and the balance
between potassium intake and output is maintained.
1) Proximal tubule. Our present state of knowledge about the role of
the proximal tubule in the regulation of potassium excretion is largely derived
from in vivo free-flow micropuncture of tubules accessible at the kidney surface
(Giebisch et al., 1981). This segment absorbs the largest amount of potassium,
which is mediated by both active and passive processes. However, the exact
mechanisms explaining K absorption remain to be elucidated. The K
concentration in samples of fluid from the proximal tubule has been found to
be close to the concentration in plasma, suggesting that the rate of reabsorption
of K along the proximal convoluted tubule is rather rigidly coupled to sodium


Table 7-1. Effect of low Na and low K diet on excretion of urine, urinary Na, and K
Days on
the diet
¡fon
Uv
(ml)
(mBq)
T
(mfeq)
0
65.612.5
120.221.9
92.315.3
5.20.4
11.73.1
1
28.412.3
78.1 16.6
75.221.2
1.5 0.4
5.1U
2
22.35.6
46.010.2
105.035.6
1.70.4
3.70.8
3
23.07.1
46.213.0
76.5 32.8
0.8 0.3
1.80.8
4
25.86.4
28.22.9
98.226.9
1.803
2.60.5
5
13.62.7
33.67.1
275.889.7
2.80.7
6.21.2
6
8.3 2.1
26.96.5
103.464.5
0.60.3
1.60.7
7
9.5 3.3
33.29.4
131.247.0
0.60.2
2.2 0.7
8
11.34.2
32.69.2
67.632.0
0.40.1
1.00.3
9
15.96.1
36.210.0
30.86.7
0.30.1
0.80.2
there was a tendancy in decrease in urinary Na level one day after the animals were exposed to the diet.
Urinary Na concentration was significantly reduced from 2nd day on the diet (at least p < 0.05), and no further
reduction in Na level was observed for the entire period on the diet. However, the rabbits excreted K differently
from Na. A significant reduction in urinary K level (at least p<0.01) was observed one day after the animals
were on the diet, and a further reduction in K concentration (at least p<0.05) was observed four days after the
rabbits were on the diet. The total urinary excretion of Na and K was significantly reduced one day after the
diet (at least p<0.01). The total excreted Na and K were further reduced eight days after the diet because the
urine output decreased. Moreover, there was a trend in increase in urine output after animals were adapted to
the diet. However, this effect did not reach the significant level until five days later. Urine excretion was
dramatically reduced after the rabbits were exposed to the diet for eight to nine days. (n=5).
Up^aj and Upg, the concentrations of urinary Na and K, respectively. Uv, urinary output. Tn. and tk. total
urinary excretion of Na and K, respectively.


40
acidification by increase in tension of C02 (McKinney and Davidson, 1988).
The observations made at cellular level further strengthes such a hypothesis
H-K-ATPase has been localized by immunocytochemistry to the intercalated cell
in the CCD and outer medullary collecting duct (Wingo et al., 1989 & 1990).
Madsen and Tisher (1983) have quantified that respiratory acidosis increases the
surface density of the apical membrane concomitantly with decrease in the
number of tubulovesicular profiles in the apical region of the intercalated cell
of rats. Schwartz and Al-Awqati (1985), using fluorescent dextran assay, have
directly demonstrated that C02 stimulates exocytotic fusion of the vesicles in the
proximal straight tubule and collecting duct of rabbits. Turtle urinary bladder
has many transport characteristics similar to collecting duct. Exposure of C02
also increases H secretion in this epithelia, although H-ATPase has been
interpreted to be responsible in part for this stimulation (Cannon et al., 1985;
Schwartz and Al-Awqati, 1986; Steinmetz, 1986; Stetson and Steinmetz, 1983 &
1986; Van Adelsberg and Al-Awqati, 1986). However, recent studies have also
demonstrated the presence of K-ATPase activity in this epithelia (Husted and
Steinmetz, 1981; Sharma et al., 1991). Exocytosis of the pumps has been
repeatedly shown to mediate this response (Cannon et al., 1985; Schwartz and
Al-Awqati, 1986; Steinmetz, 1986; Stetson and Steinmetz, 1983; Van Adelsberg
and Al-Awqati, 1986).
The cell swelling has been observed following exposure to 10% C02
(Figure 3-12), whereas the change in morphology of the tubules was not able
to be observed if the stimulatory effect of 10% C02 on Rb efflux was inhibited
either by colchicine (Figure 3-13) or by MAPTAM (Figure 3-14). These
observations suggest that the renal H-K-ATPase participates in cell volume


109
Figure 5-3. Effect of SCH28080 on Kfj, in the presence of ambient 0.5 mM
K (top) and in the presence of ambient 20 mM K (bottom). In the presence
of 0.5 mM K, SCH28080 significantly decreased KNa from 47.6 4.8 nm-sec'1
to 35.0 6.8 nm-sec'1 (n = 6), whereas in the presence of 20 mM K
SCH28080 failed to inhibit KNa (33.3 + 5.0 nm-sec'1 vs 30.6 5.2 nm-sec'1, n
= 8). KNa, Na lumen-to-bath efflux coefficient.


CHAPTER 1
INTRODUCTION
1.1 Background
Potassium (K) is one of the most abundant cations in the body and
plays an important role in a variety of physiological functions. Cell growth,
division, volume regulation, and acid-base balance depend on a high intracellular
K activity. Reduction in K intake induces hypertension and supplement of K
intake ameliorates hypertensive symptoms. High potassium concentration in cells
and low concentration in the extracellular fluid contribute to the electrical
properties of both excitable and nonexcitable Assures. Changes in either
intracellular or extracellular K activity result in modification of both intracellular
and extracellular pH. Potassium affects muscle metabolism, in broad term, by
four different ways: by regulation of muscle blood flow during exercise; by its
role in carbohydrate metabolism; by its effect on insulin secretion; and by its
direct effect on muscle itself.
The performance of these vital functions by potassium is dependent on
a variety of regulatory mechanisms that maintain K homeostasis. Figure 1-1
(Giebisch et ah, 1981) schematically depicts the distribution of potassium ion
in the body. As can be seen, most of potassium is located within cells,
especially within muscle cells, with smaller amount in liver and erythrocytes.
Potassium is the most abundant intracellular cation. Only 2% of bodys
potassium is distributed in the extracellular compartment, normally at
l


41
regulation. Two possible mechanisms may explain the effect of H-K-ATPase on
cell volume. 10% C02 stimulates H-K-ATPase thereby increasing K/Rb entry
from the apical membrane. It has been well known that acidosis inhibits K
conductance or K channels (Boundry et al., 1976; Wang et al., 1990; Wright
and Giebisch, 1992). It is possible that 10% C02 inhibits the K exit at the
basolateral membrane. The intracellular K level increases from the these two
additive effects. As a result, the osmolarity gradient is established. Alternatively,
the renal H-K-ATPase can transport Na as demonstrated in Chapter 5. 10%
C02 stimulates H-K-ATPase presumably increasing Na entry from the apical
membrane. Acidosis inhibits Na-K-ATPase thereby inhibiting Na exit at the
basolateral membrane (Wright and Giebisch, 1992). As a result, the osmolarity
gradient is established.
As shown in Figure 3-1, each individual tubule responded to 10% C02
differently. Some tubule had big response in terms of Rb efflux stimulation
upon exposure to 10% C02, whereas others did not. Rb efflux response to the
stimulus is not correlated with voltage response. Whether the variation in Kjy,
response to 10% C02 is due to the different populations of the CCD or the
biological variation among the tubules is not clear. In fact, 20% CCD comes
from the juxtamedullary nephron, whereas 80% CCD comes from the superficial
nephron. Whether these two different populations of the CCD have different
physiological functions in terms of potassium transport remain unknown.
3.4.2 Effect of Methazolamide
Exposure to C02 has been demonstrated to decrease intracellular pH,
and this results in increase intracellular Ca level in turtle urinary bladder,
although the effect of C02 on intracellular Ca activity varies in different cell


VT(mV) KRb (nm-sec-1)
65
20 -
1 1
5% C02 10% CO2
3 mM Ba 3 mM Ba
NS
Figure 3-10. Effect of peritubular 3 mM Ba on (top) and Vj (bottom)
(n = 6). Kr,,, Rb lumen-to-bath efflux coefficient. VT, transepithelial voltage.


24
exhaustively dialyzed according to the method of Schafer et al. (1974). The
equilibration time between two periods was 30 minutes unless indicated. Effluent
fluid was collected into a constant-volume pipette for measurement of volume
flux, isotopic flux and net chemical flux. Volume flux was determined from
timed collections of the effluent fluid using the equation Jv =
([cprr^/cpm,-!] -VJ/L, where Jv is the net volume absorption in nanoliters per
millimeter per minute, cpm0 and cpnij are the [3H]-inulin counts per minute per
nanoliter in the collected and the perfused fluid, respectively, Vc is the rate of
fluid collection in nanoliters per minute, and L is the tubule length in
millimeters. In all experiments, the percent of [3H]-inulin leak was less than 5%
or the experiment was discarded. In most tubules the leak rate was less than
2%. Isotopic Rb was used because it has been shown to be a qualitative
marker of K efflux (134), and is transported by the renal H-K-ATPase
quantitatively similar to K (14). The Rb lumen-to-bath efflux (K^,) was
determined by the disappearance of Rb from the perfusate according to the
following equation:
KRb
Yu. :Rb*-Rb*
L Rbl + Rb
where Rb¡ and Rbc are the Rb counts per minute per nanoliter in the
perfused and collected fluid, respectively. The Na efflux rate coefficient (KNl)
was calculated by a similar equation. Counts for 3H, Rb and Na were
measured by a liquid scintillation counter (LS-7800, Beckman Instruments, Irvine,
CA). The overlap of Rb or Na counts in the 3H channel was corrected as
previously described (Zhou and Wingo, 1992). Net chemical flux was determined


33
C02, we perfused the CCD in the absence of luminal SCH28080. After
measurement of the basal rate of Rb efflux (5% C02 period), effluent fluid was
collected from 30 to 120 minutes after exposure to 10% C02. To evaluate the
role of carbonic anhydrase in the stimulation of Rb efflux by 10% C02, 0.1
mM methazolamide was added to the bath from 120 to 180 minutes after
exposure to 10% C02. We were not only able to reproduce the previous
stimulation of KRb by 10% C02, but also observed this effect at the earliest
collection time, i.e., 30 to 60 minutes later after the tubule exposure to 10%
C02. This effect was persistent for the entire 10% C02 period. However,
subsequent addition of methazolamide did not significantly inhibit Kr,, (72.4
11.8 nm-sec'1, 5% C02 period; 121 29.9 nm-sec'1, 135 34.3 nm-sec'1, and
133 29.0 nm-sec'1, 30-60 minutes, 60-90 minutes, 90-120 minutes after
exposure to 10% C02, respectively; 133 29.0 nm-sec'1, methazolamide period,
n = 6, Fig. 3-4). During the methazolamide period VT was significantly more
lumen-negative (-3.0 + 2.2 mV, 5% C02 period; -2.4 2.3 mV, -1.3 1.8 mV,
and -0.8 1.6 mV, 30-60 minutes, 60-90 minutes, and 90-120 minutes after
exposure to 10% C02, respectively; -7.6 4.2 mV, methazolamide period, n =
6, Fig. 3-4). When the experiments were repeated under same conditions
except for the presence of luminal 10qM SCH28080, SCH28080 totally blocked
the enhancement of Rb efflux in response to 10% C02 and methazolamide had
no significant effect on Rb efflux under these conditions (90.8 16.5 nm-sec"1,
5% C02 period; 80.5 12.3 nm-sec'1, 83.9 14.3 nm-sec'1, and 78.7 10.9
nm-sec'1, 30-60 minutes, 60-90 minutes, and 90-120 minutes after exposure to
10% C02, respectively; 81.9 13.5 nm-sec'1, methazolamide period, n = 7, Fig.
3-5), although VT became more lumen-negative during methazolamide period


141
Halm, D. R., and R. A. Frizzell. Active K transport across rabbit distal colon:
relation to Na absorption and Cl secretion. Am. J. Physiol. 25LC252-
C267, 1986.
Hansen, G. T., C. C. Tisher, and R. R. Robinson. Response of the collecting
duct to disturbances of acid base and potassium balance. Kidney Lnt.
17:326-337, 1980.
Harris, P. J., and L. G. Navar. Tubular transport response to angiotensin.
Am. J. Physiol. 248:F621-F630, 1985.
Hayashi, M., and A. I. Katz. The kidney in potassium depletion. I. Na-K-
ATPase activity and [3H] ouabain binding in MCT. Am. J. Physiol.
252:F437-F446, 1987.
Hunter, M., A. G. Lopes, E. Boulpalp, and G. Giebisch. Regulation of single
potassium ion channels from apical membrane of rabbit collecting tubule.
Am. J. Physiol. 251:F725-F733, 1986.
Husted, R. F., and P. R. Steinmetz. Potassium absorptive pump at the luminal
membrane of turtle urinary bladder. Am. J. Physiol. 241 (Renal Fluid
Electrolyte Physiol. 10): F315-F321, 1981.
Imon, M. A., and J. F. White. Association between HCOa absorption and K
uptake by Amphiuma jejunum: relations among HC03 absorption, luminal
K, and intracellular K activity. Am. J. Physiol. 246:G732-744, 1984.
Jacobson, H. R. Medullary collecting duct acidification: effect of potassium,
HC03 concentration and pC02. J. Clin. Invest. 84:2107-2114, 1984.
Kaunitz, J. D., R. D. Gunther, and G. Sachs. Characterization of an
electrogenic ATP and chloride-dependent proton translocating pump from
rat renal medulla. J. Biol. Chem. 260: 11567-11573, 1985.
Kaunitz, J. D., and G. Sachs. Identification of a vanadate-sensitive potassium-
dependent proton pump from rabbit colon. J. Biol. Chem. 261:
14005-14010, 1986.
Keeling, D., J. A. Taylor, and C. Schudt. The binding of a K competitive
ligand, 2-Methyl,8-(phenylmethoxy)imidazo(l,2-a)pyridine 3-Acetonitrile, to
the gastric (H + K)-ATPase. J. Biol. Chem. 264:5545-5551, 1989.
Khuri, R. N., M. Wiederholt, N. Strieder, and G. Giebisch. Effects of flow
rate and potassium intake on distal tubular potassium transfer. Am. J.
Physiol. 228: 1249-1261, 1975.
Kidder IH, G. W., and C. W. Montgomery. 1974. CO diffusion into frog
gastric mucosa as rate-limiting factor in acid secretion. Am. J. Physiol.
227:300-304.


67
Figure 3-12. The CCD was swollen following expusure to 10% C02. Top: 5%
C02 period, Bottom: 10% C02.


32
To examine whether acute respiratory acidosis stimulates Rb efflux, we
perfused the CCD with symmetrical Ringers bicarbonate solution gassed with
5% C02 (5% C02 period) or 10% C02 (10% C02 period). In our initial
studies we allowed one hour equilibration after obtaining basal flux rates prior
to a second period of measurement in the presence of 10% C02. As shown
in Figure 3-1, 10% C02 substantially increased K,^ from 93.1 23.8 nm-sec'1
to 249 60.2 nm sec'1 (P < 0.05, n = 7). Concomitantly the transepithelial
voltage (Vx) became more lumen-positive from -2.4 + 1.3 mV to -1.2 0.9 mV
(P < 0.05, n = 7, Fig. 3-1). The greater lumen-positive voltage could enhance
paracellular Rb passive efflux. To examine whether the change in VT
accounted for the effect of 10% C02 on Rb efflux, we calculated the voltage-
mediated Rb efflux by Goldman flux equation. As shown in Figure 3-2, the
change in VT can not explain for the increase in K^, by 10% C02. Voltage-
mediated Rb efflux as predicted by Goldman flux equation was only 10.6 3.7
nm-sec"1, whereas the increment of 156 + 58.4 nm-sec'1 was observed. To
examine whether H-K-ATPase mediated the effect of 10% C02 on Rb efflux
we repeated the experiments under the identical conditions except for the
presence of luminal 10 qM SCH28080. Luminal SCH28080 totally abolished the
stimulatory effect of 10% C02 on Rb efflux (76.4 15.1 nm-sec'1 vs 76.8
13.3 nm-sec'1, n = 5, Fig. 3-3), although VT became more lumen-positive
during 10% C02 period (-14.6 9.6 mV vs -10.7 9.1 mV, P < 0.01, n =
5, Fig. 3-3). In additional experiments, there was no evidence to suggest that
0.1% DMSO affected either or VT (Table 3-2). These data suggest that an
H-K-ATPase, not the change in Vp mediates the enhancement of Rb efflux by
10% C02. To examine the time course of the stimulation of Rb efflux by 10%


71
Ouabain profoundly increases K absorption in gastric gland, and this
stimulation has been attributed to H-K-ATPase (Reenstra et al., 1986). H-K-
ATPase has been also identified in colonic epithelium which has many transport
characteristics similar to the collecting duct (Kaunitz and Sachs, 1986). Serosal
ouabain has been shown to not only inhibit K secretion, but also stimulate K
absorption by this tissue (Halm and Frizzell, 1986; Tumamian and Binder,
1989). Therefore, the second objective of this studies is to address the issue
whether peritubular ouabain increases Rb efflux, and whether this increment is
mediated by an H-K-ATPase in the K-restricted CCD.
However, the issue of whether K absorption by the CD proceeds via an
H-K-ATPase is not completely clear. During K restriction, Na-K-ATPase
activity increases in the rat CD, although this increase in Na-K-ATPase activity
is not accompanied by an increased [3H]-ouabain binding in intact tubules.
However, in permeabilized tubules, [3H]-ouabain binding does increase
proportionately to the increase in Na-K-ATPase activity. These observations led
Hayashi and Katz (1987) to hypothesize that Na-K-ATPase was sequestered in
an inaccessible site, either an intracellular compartment or the luminal
membrane. If Na-K-ATPase is present at the luminal membrane, it could
participate in K absorption. In addition, mucosal ouabain has been shown to
inhibit active K absorption in the rat and guinea pig colon (Perron and
McBride, 1988; Sweiry and Binder, 1990; Suzuki and Kaneko, 1987 & 1989) and
in the turtle bladder (Husted and Steinmetz, 1981). Thus, the third objective
of the present studies was to determine whether a functional H-K-ATPase or
a functional Na-K-ATPase was present at the apical membrane of the CCD of
K-restricted rabbits.


138
pH, carbon dioxide tension, and bicarbonate concentration. J. Clin.
Invest. 77:1650-1660, 1986.
Brown, D., I. Sabolic, and S. Gluck. Colchicine-induced redistribution of proton
pumps in kidney epithelial cells. Kidney Int. 40, suppl.33:s-79-s-83, 1991.
Burg, M., J. Grantham, M. Abramon, and J. Orloff. Preparation and
study of fragments of single rabbit nephrons. Am. J. Physiol.
210:1293-1298, 1966.
Cannon, C., J. van Adelsberg, S. Kelly, and Q. Al-Awqati. Carbon-dioxide-
induced exocytotic insertion of H+ pumps in turtle-bladder luminal
membrane: role of cell pH and calcium. Nature 314:443-446, 1985.
Chan, Y. L., B. Biagi, and G. Giebisch. Control mechanisms of bicarbonate
transport across the rat proximal convoluted tubule. Am. J. Physiol.
242:F532-F543, 1982.
Cheval, L., C. Barlet-Bas, C. Khadouri, E. Feraille, S. Marsy, and A. Doucet.
K+-ATPase-mediated Rb+ transport in rat collecting tubule: modulation
during K+ deprivation. Am. J. Physiol. 260:F800-F805, 1991.
Cochran, W. G., and G. M. Cox. Experimental Design (2nd ed.). New York:
Wiley, 1957, p. 117-121.
Cogan, M. G., D. A Maddox, D. G. Wamock, E. T. Lin, and F. C. Rector,
Jr. Effect of acetazolamide on bicarbonate reabsorption in the proximal
tubule of the rat. Am. J. Physiol. 237:F447-F454, 1979.
Dahl, L. K., G. Leitl, and M. Heine. Influence of dietary potassium and
sodium/potassium molar ratios on the development of salt hypertension.
J. Exp. Med. 136:318-330, 1972.
Dantzig, A H., P. L. Minor, J. L. Garrigus, D. S. Fukuda and J. S. Mynderse.
Studies on the mechanism of action of A80915A, a semi-naphthoquinone
natural product, as an inhibitor of gastric (H-K)-ATPase. Biochem.
Pharmacol. 42:2019-2026, 1991.
Davidson, D. G., N. G. Levinsky, and R. W. Berliner. Maintenance of
potassium excretion despite reduction of glomerular filtration during
sodium diuresis. J. Clin. Invest. 37:548-555, 1958.
Diezi, J., P. Michoud, A Gmadchamp, and G. Giebisch. Effects of
nephrectomy on renal salt and water transport in the remaining kidney.
Kidney Int. 10:450-462, 1976.
Dobyan, D. C., and R. E. Bulger. Renal carbonic anhydrase. Am. J. Physiol.
243:F311-F324, 1982.


11
delivery of K to the late of distal nephron of superficial nephrons. This is the
first physiological evidence of involvement of the collecting duct in active K
absorption in response to K depletion. The postulate for active potassium
absorption was based on the conditions which were characterized by complete
absence of net potassium secretion, yet persistence of a distinctly lumen-negative
transepithelial voltage. In the absence of other transport mechanisms, potassium
should be secreted to lumen down its electrochemical gradient. To
counterbalance the potassium secretion, an active force must move potassium
out of the lumen. This suggests that an active potassium absorptive mechanism
is present in the apical cell membrane, and the segment traditionally conceived
as K-secretory also plays K conserving roles, especially when the balance of K
requires it. Linas et al. (1979) studied the adaptation of the rat to a low K
diet and found that the recovery of distally injected 42K was significantly
reduced 72 hours after exposure to the diet. Wingo (1987) has shown that K
absorption was increased by the in vitro perfused outer medullary collecting duct
dissected from K-restricted rabbits and provided the direct evidence
substantiating the role of the collecting duct in K conservation. The further
observations from mophological studies have demonstrated that the intercalated
cells of the collecting duct undergo hypertrophy and ultrastructural changes
during K restriction and acidosis, suggesting that these cells are involved in K
absorption and H secretion (Hansen et al., 1980; Ordonez and Spargo, 1976;
Stanton et al., 1981). However, the mechanisms underlying these observations
remain unclear. The discovery of K-ATPase in rat and rabbit kidney made by
Doucet and Marsy in 1987 (1987) has sheded more light on this poorly
understood component. ATP hydrolysis by this enzyme is K-dependent and


subsequent addition of methazolamide or colchicine failed to affect Rb efflux.
However, simultaneous exposure to 10% and methazolamide or colchicine
prevented the stimulation of Rb efflux. Treatment with MAPTAM, W-7, and
peritubular barium blocked the stimulatory effect of 10% C02. The data are
consistent with the following conclusions, at least during K restriction: 1) an
existing H-K-ATPase mediates the stimulatory effect of 10% C02 on Rb efflux;
2) carbonic anhydrase and microtubules are not necessary for maintaining H-K-
ATPase activity, but is required for activation of H-K-ATPase by 10% C02; 3)
the effect 10% C02 is dependent on the increase in intracellular calcium
activity and presence of basolateral barium-sensitive exit mechanism.
The second set of the studies was mainly to examine the effect of
luminal amiloride on Rb efflux. Amiloride significantly increased Rb efflux, and
this effect was fully blocked by luminal barium, suggesting that a Ba-sensitive
K conductance mediates the effect of amiloride.
The third set of the studies was to examine whether removal of luminal
sodium had similar effect as amiloride. In contrast to the effect of amiloride,
the stimulation of Rb efflux by sodium removal was only inhibited by
simultaneous presence of barium and SCH28080, indicating that the effect of
Na removal is in part via the H-K-ATPase. To test whether Na acts as a
partial agonist of the H-K-ATPase we examined the effect of SCH28080 on Na
efflux. In the presence of luminal amiloride and an ambient K concentration
of 0.5 mM, SCH28080 significantly inhibited Na efflux, whereas this effect was
abolished when similar experiments were performed in the presence of 20 mM
ambient K. In conclusion: 1) both Ba-sensitive K conductance and H-K-ATPase
xvi


CHAPTER 6
EFFECT OF ANGIOTENSIN II, HISTAMINE,
AND CARBACHOL
6.1 Introduction
Angiotensin II is a potent stimulator of proton secretion and solute
transport by the proximal tubule (Harris and Navar, 1985; Liu and Cogan, 1987;
Mujis et al., 1986; Schuster et al., 1984). Mujaris et al. (1986) have
demonstrated specific angiotensin II binding in the collecting duct, yet a
functional role for this hormone in the collecting duct has not been examined.
Angiotensin II has been shown to be antikaliuretic in human when infused at
the concentrations that do not elicit systemic or renal hemodynamic changes
(McMurray and Struthers, 1988). This observation is consistant with hypothesis
that Ang II stimulate H-K-ATPase. Alternatively, Ang II may inhibit K
secretion. Therefore, The first studies aim at identification of whether Ang II
increases H-K-ATPase activity, or decreases K secretion, or both.
The renal H-K-ATPase has been demonstrated to be pharmacologically
(Wingo, 1989), or even molecular biologically similar to the gastric H-K-ATPase
(Okusa et al., 1990). Because histamine and carbachol substantially stimulate the
gastric H-K-ATPase (Forte et al., 1981; Koelz et al., 1981), the second studies
aim at examination of whether these two hormones exhibit the similar effect
on renal H-K-ATPase.
6.2 Methods and Material
111


12
84
Figure 4-2. Effect of luminal 1 mM amiloride on VT in the absence of
luminal 2 mM Ba (top, n = 8) and in the presence of luminal 2 mM Ba
(middle, n = 9). Effect of SCH28080 on VT in the presence of luminal 1 mM
amiloride and 2 mM Ba (bottom, n = 9). In both cases (top & middle),
amiloride significantly increased VT (0.6 1.5 mV vs 4.7 + 0.9 mV, in the
absence of luminal Ba; 1.8 + 1.5 mV vs 8.5 1.5 mV, in the presence of
luminal Ba). Again, SCH28080 had no significant effect on VT (8.5 1.5 mV
vs 12.1 2.5 mV). Vj, transepithelial voltage.


presence of luminal Na 105
Table 5-7. Effect of SCH28080 in the presence of 20 mM K . 106
Table 6-1. Composition of solution 116
Table 6-2. Effect of histamine on VT in the presence of
luminal 3 mM Ba 117
Table 6-3. Effect of 0.1 mM carbachol 118
Table 7-1. Effect of low Na and low K on excretion of
urine, Na and K 125
Table 7-2. Effect of low Na and low K diet, and low K diet on
serum Na and K levels 126
IX


120
pcO.05
Figure 6-2. Effect of angiotensin II on K^, (top) and VT (bottom, n=6) in the
iresence of luminal IOmM SCH28080 and absence of luminal Ba. Kr,,, Rb
umen-to-bath efflux coefficient. VT, transepithelial voltage.


35
not significantly inhibit K^, (60.4 10.1 nm -sec'1 vs 59.1 10.6 nm-sec'1, n
= 6), although the effect of methazolamide on VT was reproducible (-12.9
2.7 mV vs -27.1 + 4.8 mV, P < 0.05, Fig. 3-7, n = 6). This suggests that the
basal Rb efflux may be dependent on methazolamide-insensitive pathway.
Exocytotic insertion of H-K-ATPase in response to several stimuli has
been demonstrated in gastric gland (Forte et al, 1981). This process is
cytoskelton-dependent. To examine whether microtubules mediate the effect of
10% C02 on Rb efflux, the tubules were simultaneously exposed to 10% C02
and 0.5 mM colchicine, an inhibitor of tubulin polymerization. Colchicine totally
blocked the stimulatory effect of 10% C02 on Rb efflux (85.0 + 15.0 nm-sec'1
vs 81.3 13.6 nm-sec'1, n=6, Fig. 3-8), indicating that the stimulation of Rb
efflux is dependent on intact microtubule function. Meanwhile, colchicine had
no significant effect on VT (-12.9 4.9 mV vs -15.6 5.0 mV, n = 6, Table 3-
4). To further test the insertion hypothesis, we examined the effect of colchicine
after stimulation of KRb by 10% C02. Under these conditions, colchicine had
no inhibitory effect on Rb efflux (72.5 7.9 nm-sec'1, 5% C02 period; 97.3
10.1 nm-sec'1, 10% C02 period'; vs 110 + 13.2 nm-sec'1, 10% C02 plus
colchicine period, p<0.05 compared with 5% C02 period, no significant
difference between 10% C02 and 10% C02 plus colchicine period, n=6, Fig.
3-8). Voltage was significantly altered by this maneuver (-11.2 7.1 mV, 5%
C02 period; -16.9 9.4 mV, 10% C02 period; -17.4 9.5 mV, 10% C02 plus
colchicine period, p<0.05 as compared with 5% C02 period, n = 6, Fig. 3-3).
These data are consistant with the postulation that exocytotic process mediates
the effect of 10% C02 on Rb efflux.


27
Figure 2-1.
The cortical collecting duct perfused in vitro.


100
Table 5-1. Composition of solutions (in mM, continue)
Solution G
Solution H
Na
28
28
Choline
102
82.5
K
0.5
20
Cl
106.9
106.9
HCOj
25
25
Ca
1.2
1.2
Mg
1
1
Phosphate
1.5
1.5
Acetate
10
10
Glucose
8
8
Alanine
5
5
Mannitol
26.5
26.5
Osmolarity (mOsm)
298.6
298.6


131
pathway is also present in the basolateral membrane of K-restricted CCD of
rabbit, this pathway may mediate the effect of 10% C02. To test this
hypothesis, the tubules was perfused in the presence of peritubular barium.
Pretreatment with Ba totally blocked the stimulation of Rb efflux by 10% COz
(73.0 8.2 nm-sec'1 vs 70.9 8.3 nm-sec'1), implying the efect of 10% C02
is dependent on the presence of functional barium-sensitive basolateral exit
mechanism.
Amiloride substantially increases K permeation by normal and DOCA-
treated CCD. This effect has been eluciated to be dependent on Ba-sensitive
K conductance. To examine whether the effect of amiloride is persistant in the
K-restricted CCD, the first set of experiments in this set of studies was
performed in the absence of luminal Ba. Luminal addition of amiloride (1 rnM)
significantly increased the Rb lumen-to-bath efflux coefficient from 90.7 14.3
nm-sec'1 to 119 20.6 nm-sec'1 (P < 0.05). Because 2 mM Ba has been
shown to sufficient enough to block Ba-sensitive pathway, the second set of
experiments was repeated under identical conditions except for the presence of
2 mM Ba. The effect of amiloride was fully blocked by the presence of Ba,
suggesting that, like in normal and DOCA-treated CCD, a Ba-sensitive K
conductance mediates the stimulation of by amiloride. Ouabain has been
demonstrated to increase K absorption in gastric gland. This effect has been
attributed to gastric H-K-ATPase. The second purpose of this serial of studies
is to examine whether the effect of ouabain is reproducible in the cortical
collecting duct. Addition of 0.1 mM ouabain to the bath increased KRb (69.8
11.1 nm-sec'1 vs 95.9 18.7 nm-sec'1, P < 0.05). However, this effect of
ouabain on Kr,, was totally abolished by perfusion of the CCD in the presence


148
Van Adelsberg, J., and Q. Al-Awqati. Regulation of cell pH by Ca+2-mediated
exocytotic insertion of H+-ATPase. J. Cell. Biol. 102:1638-1645, 1986.
Van Driessche, W., and W. Zeiske. Ba+ +-induced conductance fluctuations of
spontaneously fluctuating K+ channels in the apical membrane of frog
skin (Rana temporaria). J. Membr. Biol. 56: 31-42, 1980.
Walter, S. J., A. C. Shore, and D. G. Shirley. Effect of potassium depletion
on renal function in rat. Clin. Sci. 75: 621-628, 1988.
Wang, T., and Y. L. Chan. Mechanism of angiotensin II action on proximal
tubular transport. J. Pharmacol. Exp. Tier. 252:689-695, 1990.
Wang, T., and Y. L. Chan. The role of phosphoinositide turnover in mediating
the biphasic effect of angiotensin II on renal tubular transport. J.
Pharmacol. Exp. Ther. 256:309-317, 1991.
Wang, W., J. Geibel, and A Cassola. and G. Giebisch. Ca-dependent signal
pathway links apical K secretory channel to basolateral Na-K-ATPase in
rat cortical collecting duct (CCD). J. Am. Soc. Nephrol. 2(3):754, 1991.
Wang, W., and G. Giebisch. Protein kinase a (PKA) and protein kinase c
(PKC) modulate the apical small-conductance K channel of rat cortical
collecting duct (CCD). J. Am. Soc. Nephrol. 1 (4):694, 1990.
Wang, W., A. Schwab, and G. Giebisch. Regulation of small-conductance K
channel in apical membrane of rat cortical collecting tubule. Am. J.
Physiol. 259:F494-F502, 1990.
Warden, D. H., M. Hayashi, V. L. Schuster, and J. B. Stokes. K+ and Rb+
transport by the rabbit CCD: Rb+ reduces K+ conductance and Na+
transport. Am. J. Physiol. 257: F43-F52, 1989.
Wingo, C. S. Effect of ouabain on K secretion in cortical collecting tubules
from adrenalectomized rabbits. Am. J. Physiol. 247:F588-F595, 1984.
Wingo, C. S. Cortical collecting tubule potassium secretion: effect ofamiloride,
ouabain, and luminal sodium concentration. Kidney Int. 27: 886-891,
1985.
Wingo, C. S. Potassium transport by medullary collecting tubule of rabbit:
effects of variation in K intake. Am. J. Physiol. 253: F1136-F1141, 1987.
Wingo, C. S. Potassium secretion by the cortical collecting tubule: effect of
ouabain and Cl gradients. Am. J. Physiol. 256:F306-F313, 1989.
Wingo, C. S. Active proton secretion and potassium absorption in the rabbit
outer medullary collecting duct: functional evidence for proton-potassium
- activated adenosine triphosphatase. J. Clin. Invest. 84: 361-365, 1989.


106
Table 5-7. Effect of SCH28080 in the presence of 20 mM K
Basal
SCH28080
Kr,
(nmsec')
97.9 12.0
79.2 9.2
VT
(mV)
6.0 1.5
5.8 1.5


98
distal Na delivery is reduced. A decrease of net K secretion following
reduction in luminal sodium concentration (Stokes, 1981; Wingo, 1985) may
reflect in part enhanced K-absorptive flux (lumen-to-blood) as well as decreased
K secretory flux (blood-to-lumen). Indeed, in K-depleted animals, the delivery
of Na to the distal tubule and collecting duct is markedly reduced (Khuri et
al., 1975; Walter et al., 1988). This would provide a better environment for H-
K-ATPase to function optimally in order to conserve K whereas an increase in
distal Na delivery would promote a kaliuresis (Tannen and Gerrits, 1986). The
presence of SCH28080-sensitive Na efflux is reduced by increasing ambient K
concentration suggesting that Na competes with K for transport via the H-K-
ATPase. These observations may have direct relevance to the observed Na
retension that accompanies severe K depletion. Furthermore, the inhibition of
KRb by SCH28080 in the presence of 1 mM amiloride indicates that an H-K-
ATPase is pharmacologically distinguishable from a Na-H exchanger.


81
Table 4-2. Effect of DMSO in the absence of luminal Na and luminal 1.5 mM
Basal
DMSO
1.5
mM Na
Kr,
(nmsec'!)
84.2 18.5
82.6 17.0
87.6
14.9
(V)
2.8 5.4
0.6 2.8
1.7
3.3
(n=6).


44
Methazolamide has been repeatedly demonstrated to make VT more
lumen-negative in the present studies which is consistant with other
reports(Koeppen, 1989; Koeppen and Helman, 1982; Lombard et al., 1983;
McKinney and Davidson, 1987). However, the mechanisms underlying this effect
is not clear. This effect could be due to inhibition of electrogenic H secretion
, or influence of anion transport (Koeppen, 1989; Koeppen and Helman, 1982),
or both.
3.4.3 Effects of Colchicine. MAPTAM. and W-7
Much of our knowledge about the role of microtubules, intracellular Ca,
and calmodulin in the stimulation of H secretion (presumebly in part via H-K-
ATPase) is derived from the observations made in the turtle urinary bladder
and collecting duct. Pretreatment with colchicine inhibits exocytotic fusion of
proton pumps to the apical membrane and the increase in the apical surface
density and proton secretion by the increase in C02 tension in the turtle
urinary bladder, whereas lumicolchicine, which is structually similar to colchicine
but does not bind to tubulin, had no effect (Stetson and Steinmetz, 1983). More
directly, Schwartz and Al-Aqwati (1985) demonstrated that colchicine prevented
the insertion process of proton pumps on exposure to C02 in the proximal
tubule and cotical and outer medullary collecting duct. McKinney and Davidson
(1988) later reported that colchicine inhibited total C02 absorption in response
to 15% C02 at least by the inner strip of the outer medullary collecting duct,
whereas lumicolchicine had no effect. Recently, Brown et al. (1991) have shown
that colchicine scatters the proton pumps in the cytoplasma of the renal
epithelial cells. The soluble GTP-binding proteins have been suggested to be in
control of the direction of insertion (Bourne, 1988; Hall, 1990). If H-K-ATPase


112
The tubules were dissected with Solution A (Table 6-1) containing
additional 5% vol/vol fetal calf serum. Three bathes and perfusates were used
in these studies as approriate. All of solutions were gased to pH 7.4 with 5%
C02 and 95% 02. Angiotensin n, histamine and carbachol were dissolved in
0.9% NaCl solution and applied to bath with the final concentrations of 0.1
nM, 10 mM, and 0.1 mM, respectively.
6.3 Results
6.3.1 Protocol 1
To examine whether Ang n enhances Rb efflux, the first set of
experiments was performed in the absence of luminal SCH28080. To eliminate
possible inhibition of K conductance by Ang II which might overshadow the
stimulatory effect of Ang II on Rb efflux, 3 mM Ba was present in the
perfusate. Three tubules were perfused with Solution A as bath, the other three
were perfused with same solution except for absence of phosphate. Thus, six
tubules were perfused with Solution B (Table 6-1) as the perfusate and Solution
A as the bath. Under these conditions, 0.1 nM angiotensin II failed to stimulate
Rb efflux (64.5 11.2 nm.sec'1 vs 54.5 10.5 run.sec'1, n=6, Fig. 6-1),
suggesting that angiotensin II has no significant effect on K absorptive flux. Ang
II significantly increased lumen-negative voltage (-0.7 2.9 mV vs -10.4 3.8
mV, n = 6, p<0.01, Fig. 6-1). Alternatively, to examine whether Ang II inhibits
K conductance, the second set of experiments was conducted in the absence of
luminal Ba and presence of luminal SCH28080 with Solution A as perfusate
and bath, 0.1 nM Ang II significantly reduced Rb efflux (97.7 16.5 nm.sec'1
vs 72.4 14.3 nm.sec'1, p<0.05, n=6, Fig. 6-2). The voltage became more
lumen-negative during angiotensin II period (0.9 2.6 mV vs -7.0 2.7 mV,



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81,9(56,7< 2) )/25,'$


132
of luminal SCH28080 (10 pM) (69.8 14.0 nm-sec'1, basal period; 69.6 14.1
nm-sec'1, ouabain period), indicating that, similar to gastric gland, basolateral
ouabain addition stimulates Rb (K) absorption via H-K-ATPase mechanism. The
issue whether luminal Na-K-ATPase or luminal H-K-ATPase mediates K
absorption has not been completely solved yet. Therefore, the third set of
experiments of these studies aims at determination of the role of a primary
active K pump in K absorption. We examined the action of known inhibitors
of H-K-ATPase (SCH28080) and Na-K-ATPase (ouabain) on KRb. Luminal
SCH28080 (10 uM) significantly reduced K^, by 39 8.0% (P < 0.05), whereas
luminal ouabain (0.1 mM) reduced KRb by 10 14% (P = NS), suggesting that
a luminal H-K-ATPase, not a luminal Na-K-ATPase, mediates Rb efflux.
Removal of luminal Na produces the similar effect on ion transport as
amiloride addition in many cases. It is reasonable to examine the effect of
lumen Na removal on Rb efflux. As predicted, Rb efflux was significantly
enhanced by this maneuver. However, this effect was not abolished by luminal
Ba. Only simultaneous addition of luminal Ba and SCH28080 (10 tM) fully
inhibited the increase in KRb upon luminal Na removal (68.8 9.8 nm-sec'1
vs 73.3 10.0 nm-sec'1, P = NS), indicating that the effect of luminal Na
removal is more generalized than amiloride, and mediated in part via H-K-
ATPase. To exploit these observations further, additional experiments were
conducted to examine the effect of SCH28080 on Rb efflux in the presence and
the absence of luminal Na. In the presence of luminal Na, SCH28080 (10 pM)
reduced Kg,, by 15 5.0%. However, in the absence of luminal Na, SCH28080
(10 pM) decreased Rb efflux by 48 8.2%. The percentage inhibition of Rb
efflux by SCH28080 was significantly greater in the absence of luminal Na than


12
reaches the maximal activity with 1 mM K (K^ about 0.2-0.4 mM). Sodium,
choline, chloride and sulfate do not stimulate ATP hydrolysis by this enzyme.
Ouabain, a specific inhibitor of Na-K-ATPase does not inhibit the activity of
this enzyme, indicating that this enzyme is different from Na-K-ATPase. In
contrast, omeprazole, an inhibitor of gastric H-K-ATPase, inhibits ATP hydrolysis
by this enzyme, suggesting that this enzyme is pharmacologically similar to
gastric H-K-ATPase. Because vanadate also inhibits this enzyme, K-ATPase has
been classified as Ej-E2 type ATPase. The activity of this enzyme is
proportional to the density of intercalated cells, highest in the connecting duct,
intermediate in the cortical collecting duct, and lowest in the outer medullary
collecting duct, and not detectable in all other nephron segments. Hydrolysis of
ATP by this enzyme in rat kidney increased by 100-200% following adaptation
of the animals to a low K diet. In 1988, Garg and Narang (1988) reported a
similar K-ATPase was also present in the rabbit collecting duct from their
fluorometric microassay studies. This enzyme shares similar distribution pattern
in the kidney as to that reported By Doucet and Marsy. This enzyme is
inhibited not only by omeprazole and vanadate, but also by SCH28080, an
imidazopyridine derivative shown in Fig. 1-7 which has been extensively studied
in the gastric system and demonstrated to be a specific inhibitor of gastric H-K-
ATPase (Beil et al., 1986 & 1987 & 1988; Dantzig et al., 1991; Mendlein and
Sachs, 1990; Scott et al., 1987). Chronic hypokalemia stimulates the activity of
this enzyme, and chronic hyperkalemia suppresses this enzyme (Garg and
Narang, 1989). In 1989, Wingo (1989) observed that omeprazole inhibited both
net K absorptive flux and total C02 flux in the in vitro microperfused
medullary collecting duct without significantly changing the transepithelial


77
permeation is significantly larger than could be accounted for by the changes
in voltage alone. Recently, the increment in K permeation by amiloride has
been shown to be largely, via a conductive pathway in the CCD of both normal
and desoxycorticosterone-treated rabbits (Warden et al., 1989). The findings that
amiloride significantly increased in the absence of luminal Ba (Figure 4-1),
whereas amiloride failed to increase KRb in the presence of luminal Ba (Figure
4-1) are consistent with these observations. Moreover, in the presence of luminal
1 mM amiloride and 2 mM Ba, SCH28080 was still able to be demonstrated
to significantly inhibit Rb efflux. These data show that SCH28080 inhibits a
pathway of Rb efflux which is unaffected by a maximum pharmacologic
concentration of amiloride and insensitive to 2 mM luminal Ba.
4.4.3 Effect of Peritubular Ouabain
H-K-ATPase activity may be regulated by intracellular potassium activity,
and this may explain the effect of ouabain on Rb efflux. Thus, a reduction in
intracellular K activity may stimulate the activity of the enzyme, whereas an
increase in intracellular K activity may inhibit enzyme activity. In support of this
hypothesis Koelz et al. (1981) have found that a decrease in intracellular K
activity to 60 mM stimulates the gastric H-K-ATPase as measured by
aminopyrine accumulation. Lorentzon et al. (1988) have demonstrated that a
decrease in intracellular K activity stimulates phosphoenzyme formation and
thereby increases the enzyme activity. Accordingly, maneuvers that result in a
decrease in intracellular K activity should stimulate K absorption. In gastric
glands intracellular K activity was significantly reduced following addition of
ouabain (Koelz et al., 1981) and ouabain substantially increased K absorption
in the gastric mucosa (Reenstra et al., 1986). Subsequent addition of omeprazole


91
To examine whether H-K-ATPase mediates this enhancement in Ba-
insensitive Rb efflux, we perfused the CCD in the presence of luminal 10 /M
SCH28080 throughout the experiment and examined the effect of Na removal
on Rb efflux. Thus, ten tubules were perfused with solution A, or solution B
designated as the basal and no Na periods, respectively. The order of the
periods was rotated and there was no evidence of time-dependent effect on K,y,
or VT. In the presence of luminal SCH28080 (without luminal Ba), removal of
luminal Na stimulated KRb from 76.7 7.3 nnrsec'1 (basal period) to 101
10.1 nnrsec"1 (no Na period, P < 0.05, Figure 5-1). Again, removal of luminal
Na had no significant effect on VT (-4.3 1.3 mM, basal period; vs -1.7 1.0
mM, no Na period, Figure 5-1). These data indicate that SCH28080 did not
fully inhibit the increase in Rb efflux following removal of luminal Na. To
examine whether both a Ba-sensitive K conductance and H-K-ATPase mediate
the enhancement of Rb efflux, we examined the effect of luminal Na removal
during perfusion of the CCD in the presence of both 2 mM Ba and 10 pM
SCH28080. Thus, six tubules were perfused with solution D, or solution C
designated as basal and no Na periods, respectively. As shown in Table 5-5,
only the simultaneous presence of luminal Ba and SCH28080 inhibited the
stimulation of Rb efflux by luminal Na removal (68.8 9.8 nnrsec"1, basal
period; vs 73.3 10.0 nnrsec"1, no Na period, P = NS). Removal of luminal
Na had no significant effect on VT (-1.8 3.2 mV, basal period, vs -0.4 2.4
mV, no Na period, Table 5-5). These data indicate that both K-conductive and
H-K-ATPase pathways mediate the enhancement of Rb efflux following lumen
Na removal.
5.3.3 Protocol 3


139
Doucet, A., and S. Marsy. Characterization of K-ATPase activity in distal
nephron: stimulation by potassium depletion. Am. J. Physiol. 253:F418-
F423, 1987.
DuBose, Jr., T. D., and A. Bidani. Kinetics of C02 exchange in the kidney.
Ann. Rev. Physiol. 50:653-667, 1988.
DuBose, Jr., T. D., and M. S. Lucci. Effect of carbonic anhydrase inhibition
on superficial and deep nephron bicarbonate reabsorption in the rat.
J. Clin. Invest. 71:55-65, 1983.
DuBose, Jr., T. D., L. R. Pucacco, and M. S. Lucci. Miropuncture
determination of pH, pC02, and total C02 concentration in accessible
structures of the rat renal cortex. J. Clin. Invest. 64:476-482, 1979.
Durbin, R. P., and R. Heinz. Electromotive chloride transport and gastric acid
secretion in the frog. J. Gen. Physiol. 41:1035-1047, 1958.
Dytko, G., and J. A. L. Arruda. CO, stimulation of H secretion by turtle
bladder:role of exocytosis, intracellular pH and calcium (abstr). Kidney
Int. 27:280, 1985.
Ellison, D. H., H. Velazquez, F. S. Wright. Stimulation of distal potassium
secretion by low lumen chloride in the presence of barium. Am. J.
Physiol. 248:F638-F649, 1985.
English, L. H., and L. C. Cantley. Delta endotoxin inhibits Rb uptake, lowers
cytoplasmic pH and inhibits a K-ATPase in Manduca sexta CHE cells.
J. Membr. Biol. 85:199-204, 1985.
Forte, J. G., J. A. Black, T. M. Fortes, T. E. Machen, and J. M. Wolosin.
Ultrastructural changes related to functional activity in gastric oxyntic
cells. Am. J. Physiol. 241:G349-G358, 1981.
Frindt, G., and L. G. Palmer. Ca-activated K channels in apical membrane of
mammalian CCT and their role in K secretion. Am. J. Physiol.
252:F458-F467, 1987.
Frindt, G. and L. G. Palmer. Low conductance K channels in apical membrane
of rat cortical collecting tubule. Am. J. Physiol. 256:F143-F151, 1989.
Frommer, J. P., M. E. Laski, D. E. Wesson, and N. A. Kurtzman.
Intemephron heterogeneity for carbonic anhydrase-independent
bicarbonate reabsorption in the rat. J. Clin. Invest. 73:1034-1045, 1984.
Ganser, A. L., and J. G. Forte. K-stimulated ATPase in purified microsomes
of bullfrog oxyntic cells. Biochim. Biophys. Acta 307:169-180, 1973.
Garg, L. C., S. Mackie, and C. C. Tisher. Effect of low potassium-diet on Na-
K-ATPase in rat nephron segments. Pfluger Arch. 394:113-117, 1982.


129
intracellular pH stimulates H-K-ATPase which is evidently regulated by acid-base
balance. Therefore, it is logical to evaluate the role of carbonic anhydrase in
response to 10% C02. After stimulation of Rb efflux by 10% C02, subsequent
addition of 0.1 mM methazolamide failed to affect Rb efflux. However,
simultaneous exposure to 10% C02 and methazolamide prevented the
stimulation of Rb efflux (98.6 + 14.1 nm-sec'1 vs 86.2 16.5 run-sec"1). In
addition, methazolamide had no significant effect on Rb efflux when the CCD
was perfused continuously in the presence of 5% C02. The data are consistent
with the conclusions that carbonic anhydrase is not necessary for maintaining
H-K-ATPase activity, but is required for activation of H-K-ATPase by 10% C02.
What is the mechanism explaining these observations? Exocytotic insertion of
the acidic vesicles containing proton pumps has been demonstrated to mediate
the stimulation of proton secretion by 10% C02 in turtle urinary bladder which
has many transport characteristics similar to the collecting duct. This exocytotic
process is pH- and Ca-dependent, and decrease in pH resulting in increase in
intracellular Ca activity stimulates this process. Acetazolamide increases
intracellular pH and decreases intracellular Ca activity, which conceivablly inhibit
insertion process. This exocytotic process is also dependent on the presence of
functional calmodulin and microtubule. Current knowledge of the cellular
mechanisms explaining the insertion of proton pumps in the turtle urinary
bladder is that exposure to C02 decreases intracellular pH, this process is
mediated by carbonic anhydrase, the decrease in intracellular pH increases
intracellular Ca activity, Ca combinds with calmodulin in order to amplify its
signal, the complex of Ca-calmodulin causes the rearrengement of microtubules,
the rearrengement of microtubules translocates the proton pumps from cytosol


62
Figure 3-7. Effect of O.lmM methazolamide on ^Rb (top) and VT (bottom) in
the presence of 5% C02 (n = 6). Rb lumen-to-bath efflux coefficient.
V-r, transepithelial voltage.


BIOGRAPHICAL SKETCH
Xiaoming Zhou was bom in Shanghai city, China. He obtained his
Bachelor of Medicine degree with honor from Nanjing College of Traditional
Chinese Medicine in February of 1982 and subsequently practiced pharmacy for
two-and-half years. In August of 1984 he entered the graduate program in
biochemistry at China Pharmaceutical University and was directed by Dr. Q.
Chen, studying the inhibitory effect of anthraquinone derivatives on Na-K-
ATPase derived from the rabbit renal medulla. In July, 1987, he was awarded
a Master of Science degree in biochemistry. He entered the United States as
a graduate student in the Department of Physiology, College of Medicine at
University of Florida in October, 1987. Since matriculation of his primary
interest has been the mechanisms of renal potassium transport, especially the
renal H-K-ATPase, a K absorptive pump recently described by Dr. Charles S.
Wingo, he formally joined Dr. Wingos laboratory in January of 1988 to study
this enzyme using primarily the technique of in vitro microperfusion. His
research work has been recognized with several awards both regionally and
nationally. He anticipates receiving his Ph.D. degree in physiology in May of
1992, and will persue the post-doctoral training in the laboratory of Dr. Douglas
M. Fambrough at The Johns Hopkins University studying cation-transporting
ATPase at molecular level.
150


mother Suping Bu and my brother Xiaolin for their love, consideration, backing
and understanding. Xiaofang has unselfishly devoted her life to the family. This
dissertation is especially dedicated to Xiaofang and Martin at this special time,
although it is impossible to express my gratitude to them adequately in words.
The birth of Martin in the final stage of my doctoral education not only has
increased my excitement for the present studies, but also has inspired me to
aim at higher quality of professional and private life in order for me to carry
out the honor, duty, desire, and role model as a father. Without the
fundamental and instrumental education of my childhood by my parents, it
would be unimaginable for me to be able to choose the present way of
pursuing my life.
vii


CHAPTER 3
EFFECT OF 10% C02 ON Rb EFFLUX
3.1 Introduction
Substantial evidence indicates that an H-K-ATPase is present at the
apical membrane of the cortical collecting duct (Cheval et al., 1991; Doucet and
Marsy, 1987; Garg and Narang, 1988; Gifford et al., 1991; Okusa et al., 1990;
Planelles et al., 1991; Wingo, 1989; Wingo et al., 1989 & 1990; Wingo and
Straub, 1991; Wingo and Zhou, 1990; Zhou and Wingo, 1992). This enzyme
has been demonstrated to be responsible for potassium absorption in exchange
of proton secretion, especially during K-restriction (Cheval et al., 1991; Wingo,
1989; Zhou and Wingo, 1992). Komatsu and Garg (1991) have shown that
metabolic acidosis increases ATP hydrolysis by this enzyme, suggesting that acid-
base balance regulates the activity of this enzyme. Perrone and McBride (1988)
have demonstrated that 10% carbon dioxide (C02) increases rubidium absorption
in colon, possibly mediated by a related pump. However, whether respiratory
acidosis stimulates one of the physiologic functions of the renal H-K-ATPase,
i.e. K absorptive flux, is unknown. Nevertheless, to our knowledge, the direct
relationship between acidosis and K absorptive flux has not been demonstrated
in the CCD. Therefore, the first objective of this study is to examine whether
respiratory acidosis induced by 10% C02 increases K absorption, and whether
this increment is dependent on the presence of a functional H-K-ATPase in the
in vitro microperfused CCD from K-restricted rabbits.
28


94
to 79.2 + 9.2 mn-sec'1 ( SCH28080 period). These data demonstrate that Na
can be transported via the renal H-K-ATPase by competing with K. In either
of the cases, SCH28080 had no significant effect on VT (in the first set, 0.5
mM K, 16.7 5.2 mV, basal period; vs 18.1 6.3 mV, SCH28080 period,
Figure 5-4, and in the second set, 20 mM K, 5.9 + 0.7 mV basal period; vs
6.4 0.8 mV, SCH28080 period, Figure 5-4, in the third set, 6.0 + 1.5 mV,
basal period; vs 5.8 + 1.5 mV SCH28080 period, Table 5-7), suggesting that
10 /iM SCH28080 had no significant effect on an electrogenic process.
5.4 Discussion
5.4.1 Effect of Lumen Na Concentration
The application of 2 mM luminal Ba produced significant inhibition of
Rb efflux regardless of the presence or absence of luminal Na (Table 5-2 and
5-3). Removal of luminal Na produced a highly significantly increase in K^,
when Ba was absent in the lumen (Table 5-2), and this effect can not be
blocked by either Ba (Table 5-3) or SCH28080 (Figure 5-1) alone. Only the
combined application of Ba and SCH28080 in the perfusate inhibited the
stimulatory effect of Na removal (Table 5-5), suggesting that both a Ba-sensitive
K conductance and H-K-ATPase mediate the enhancement of Rb efflux
following removal of luminal Na, at least during K restriction. Such a hypothesis
is further substantiated by the subsequent observations. When the CCD was
perfused with 2 mM Ba, SCH28080 produced a greater degree of inhibition of
Kjy, in the absence of luminal Na than in the presence of luminal Na. These
results imply that Rb efflux is stimulated in part via an H-K-ATPase following
removal of luminal Na. On the other hand, 2 mM Ba inhibited the stimulatory
effect of amiloride on (Figure 4-1), suggesting that only a Ba-sensitive K


149
Wingo, C. S., G. B. Bixler, C. H. Park, and S. G. Straub. Picomole analysis
of alkili metals by flameless atomic absorption spectrophotometry.
Kidney Int. 31:1225-1228, 1987.
Wingo, C. S., K. M. Madsen, A. Smolka, and C. C. Tisher. Structural-
functional evidence for H-K-ATPase in intercalated cells of rabbit
cortical collecting duct. Clin. Res. 37: 27A, 1989.
Wingo, C. S., K. M. Madsen, A Smolka, and C. C. Tisher. H-K-ATPase
immunoreactivity in cortical and outer medullary collecting duct. Kidney
Int. 38: 985-990, 1990.
Wingo, C. S., D. W. Seldin, J. P. Kokko, and H. R. Jacobson. Dietary
modulation of active potassium secretion in the cortical collecting tubule
of adrenalectomized rabbits. J. Clin. Invest. 70: 579-586, 1982.
Wingo, C. S. and S. Straub. Ouabain-insensitive Cl absorption by the cortical
collecting duct (CCD) is mediated by H-K-ATPase. J. Am. Soc.
Nephrol. (ASN program and abstracts, 24th Annual Meeting) 2(3):755,
1991.
Wingo, C. S., and X. Zhou. Mechanisms of K permeation by the rabbit
cortical collecting duct. J. Am. Soc. Nephrol. 1(4):695, 1990.
Wright, F. S. Overview:renal potassium transport along the nephron. ImCurraet
topics in membranes and transport vol 28, Potassium transport:physiology
and pathophysiology (Ed. by Bronner and Kleinzeller). pp99-113,
Academic Press, San Diego, CA, 1987.
Wright, F. S., and G. Giebisch. Regulation of potassium excretion. In:The
Kidney Physiology and Pathophysiology, pp. 2209-2247. (Seldin and
Giebisch Ed.). Raven Press, New York, 1992.
Zhou, X., and C. S. Wingo. H-K-ATPase enhancement of Rb efflux by the
cortical collecting duct. Am. J. Physiol. 1992. (in press).


80
Table 4-1. Composition of solutions (in mM)
Solution A
Solution B
Solution C
Na
135
135
Choline
135
K
5
5
5
Cl
106.4
110.4
106.4
HC03
25
25
25
Ca
1.2
1.2
1.2
Ba
2
Mg
1
1
1
Phosphate
1.5
1.5
1.5
Acetate
10
10
10
Glucose
8
8
8
Alanine
5
5
5
Mannitol
26.5
20.5
26.5
Osmal (m Osm)
324.6
324.6
324.6


104
Table 5-5. Effect of luminal Na removal in the presence of both 2 mM Ba
and 10 mM SCH28080
Basal
No Na
^Rb
(nm-sec'1)
68.8 9.8
73.3 10.0
Vn
(mV)
-1.8 3.2
-0.3 2.4
Kiy,, Rb lumen-to-bath efflux coefficient; VT, transpithelial voltage (n=6).


143
Linas, S. L., L. N. Peterson, R. J. Anderson, G. A. Aisenbery, F. R. Simon,
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of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa.
Biochim. Biophys. Acta 769:220-230, 1984.
Lombard, W. E., J. P. Kokko, and H. R. Jacobson. Bicarbonate transport in
cortical and outer medullary collecting tubules. Am. J. Physiol.
244:F289-F296, 1983.
Lorentzon, P., G. Sachs and B. Wallmark. Inhibitory effects of cations on the
gastric H+, K+-ATPase: a potential-sensitive step in the K+ limb of the
pump cycle. J. Biol. Chem. 263: 10705-10710, 1988.
Lucci, M. S., L. R. Pucacco, N. W. Carter, and T. D. DuBose, Jr. Evaluation
of bicarbonate transport in rat distal tubule: effects of acid-base status.
Am. J. Physiol. 243:F335-F341, 1982.
Madsen, K. M., and C. C. Tisher. Cellular response to acute respiratory
acidosis in rat medullary collecting duct. Am. J. Physiol. 245:F670-F679,
1983.
Malnic, G., R. M. Klose, and Giebisch. Micropuncture study of renal potassium
excretion in the rat. Am. J. Physiol. 206:674-686, 1964.
Malnic, G., M. deMello-Aires, and G. Giebisch. Potassium transport across
renal distal tubule during acid-base disturbances. Am. J. Physiol.
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Manalan, A. S., and C. B. Lee. Calmodulin. Adv. Cyclic Nucleotide protein
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Mario, W. J. Ion transport studies with H-K-ATPase-rich vesicles:implications
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248:G595-G607, 1985.


110
NS
Figure 5-4. Effect of SCH28080 on Vj in the presence of ambient 0.5 mM
K (top, n = 6) and in the presence of ambient 20 mM K (bottom, n = 8).
In either of cases SCH28080 had no significant effect on VT (16.7 5.2 mV
vs 18.1 6.3 mV, in the presence of ambient 0.5 mM K; 5.9 0.7 mV vs 6.4
0.8 mV, in the presence of ambient 20 mM K). V-p, transepithelial voltage.


142
Kirkendall, W. M., W. F. Connor, F. Abboud, S. P. Rastogi, and T. A.
Anderson. The effect of dietary sodium chloride on blood pressure,
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characterization of the rabbit cortical collecting duct. Am. J. Physiol.
244:F35-F47, 1983.
Koeppen, B., and S. L. Helman. Acidification of luminal fluid by rabbit cortical
collecting perfused in vitro. Am. J. Physiol. 242:F521-F531, 1982.
Koeppen, B., and G. Giebisch. Cellular electrophysiology of
potassium transport in the mammalian cortical collecting tubule. Pfliigers
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Komatsu, Y., and L. Garg. Stimulation of ouabain-insensitive
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Kubota, T., B. A. Biagi, and G. Giebisch. Effects of acid-base disturbances on
basolateral membrane potential and intracellular potassium activity in the
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Kurtzman, N. A. Disorders of distal acidification. Kidney Int. 38:720-727,
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Laski, M. E. Total C02 flux in isolated collecting tubules
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respiratory acidosis induced in vivo. Am J. Physiol. 258:F15-F20, 1990.


146
Sauer, M., A. Flemmer, K. Thuran and F. X. Beck. Sodium entry routes in
principal and intercalated cells of the isolated perfused cortical collecting
duct. Pfliigers Arch. 416: 88-93, 1990.
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transepithelial potential differences, and ionic permeability properties in
mammalian superficial proximal straight tubules. J. Gen. Physiol. 64:
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of bicarconate ion in turtle bladders. Am. J. Physiol. 210:997-1008, 1966.
Schlafter, E., and J. A. Schafer. Electrophysiological studies in principal cells
of rat cortical collecting tubules: DH increase the apical membrane
Na-conductance. Pfliigers Arch. 409: 81-92, 1987.
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stimulates sodium transport in rabbit proximal convoluted tubules. J.
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containing H pumps in isolated perfused proximal and collecting tubules.
J. Clin. Invest. 75:1638-1644, 1985.
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by exocytosis and endocytosis. Ann. Rev. Physiol. 48:153-161, 1986.
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ACKNOWLEDGEMENTS
I wish to express my gratitude and appreciation to Dr. Charles S. Wingo,
the chairman of my supervisory committee, for his instrumental guidance,
productive encouragement, invaluable support and kind care throughout the
fulfillment of this work. His tremendous contribution to my scientific knowledge,
professional career, my personality and personal life are beyond my ability to
acknowledge. I will benefit from his tradition of dedication, criticism,
responsibility, preciseness and carefulness in both of my academic activities and
private life for a very long time to come. I am indebted to Dr. George A.
Gerencser, the co-chair of my committee for his vital role in my doctoral
education. His thoughtful instruction was invaluable during the course of my
studies. His understanding and friendship are greatly appreciated.
I extend my sincere thanks to the members of my supervisory committee,
Drs. Lai C. Garg, Bruce R. Stevens and Brian D. Cain for their very helpful
suggestions and criticism. They have provided me with alternative interpretations
and additional insight that have required additional studies. I am grateful to
Scott Straub and Amy Wall for their expert technical help when I was learning
in vitro microperfusion. The personal friendship with Scott Straub and Harold
Snellen established during past four years is warmly appreciated. I sincerely
appreciate Dr. Frances Armitage and Wei Ueberschaer for their important role
v


18
Figure 1-3. Urinary potassium excretion as a function of plasma potassium
concentration in control animals and in alkalotic and acidotic conditions
(Toussaint and Vereerstraeten, 1962).


4
and water absorption. A direct comparison of extent of fluid absorption with
the range of potassium concentration in the collected fluid samples has
indicated that under normal condition, similar fractions of water, sodium, and
potassium are reabsorbed between the glomerulus and the last accessible part
of the proximal tubules (Beck et al., 1973; Diezi et al., 1976).
2) Loop of Henle. Approximately 5 to 15 % of the amount of
potassium filtered reaches the early distal tubule of superficial nephron, and that
this amount of potassium is not subject to a significant changes in spite of that
the amount of K in the final urine may be changed as much as 200-fold
(Malnic et al., 1964; Malnic et al., 1971).
3) Collecting duct. A great deal of evidence has demonstrated that K
excretion is not dependent on K filtration but rather dependent on K secretion
(blood-to-lumen) (Wright, 1987). The recent studies have suggested that K
excretion is also dependent on K reabsorption (lumen-to-blood) by the collecting
duct (Mujis and Katz, 1992). Because this segment is the final tuning site for
potassium excretion, the amount of K that appeares in the urine is determined
by net balance between potassium secretion and absorption processes which are
critically regulated according to the different needs of the body. High K intake
stimulates K secretion, whereas low K intake increases K absorption by this
segment (Mujis and Katz, 1992; Wingo, 1987; Wright and Giebisch 1992).
1.2 Potassium Secretion bv the Cortical Collecting Duct
Berliner and Kennedy (1948) and Mudge et al. (1948) were the first to
dissociate K excretion from K filtration from their clearance studies. They
reported that kidneys regularly responded to certain experimental manipulations
by excreting K at the rate exceeding the rate of K filtration, suggesting that


114
carbachol on K flux was not significant (-3.1 + 0.4 pmol/mm/min vs -1.6 0.5
pmol/mm/min), suggesting that carbachol has no significant effect on H-K-
ATPase.
6.4 Discussion
6.4.1 Effect of Angiotensin II
In human subject, infusion of angiotensin II cause antikaliuretic at the
concentration which does not elicit systemic and renal hemodinamic changes
(McMurray and Strothers, 1988). This effect may be attributed to increase K
absorption, or inhibit K secretion, or both. The present studies have shown that
Ang II at the physiological concentration has no significant effect on Rb efflux,
indicating that Ang II has little effect on K absorptive process. On the other
hand, Ang II significantly inhibited Rb efflux which was mediated by K-
conductive pathway. The present results are compatible with other studies made
at cellular level. Angiotensin II has been shown to activate protein kinase C
and increase intracellular Ca activity. Wang et al. (Wang et al., 1991; Wang
and Giebisch, 1990) have provided the evidence that protein kinase C and
intracellular Ca decrease the open probability of the low conductance of K
channel of the collecting duct from their patching-clamp studies. Because this
type of K-conductance (Frindt and Palmer, 1989; Wang et al., 1990), not Ca-
activated K channel (Frindt and Palmer, 1987; Hunter et al., 1986), has been
proposed to be a strong candidate for K secretion, This suggests that Ang II
inhibit K secretion. In addition, Brauneis et al. (1991) have recently
demonstrated that angiotensin II blocks outward potassium currents in zona
glomerolosa cells from rat, bovine, and human adrenals using whole cell patch
clamp. With use of cell-attached mode, single-channel recordings in bovine zona


Figure
5-2.
Inhibition of Kj^ by luminal 10 /nM SCH28080 in the
presence of luminal Na and in the absence of
luminal Na
108
Figure
5-3.
Effect of SCH28080 on Kr,, in the presence of
ambient 0.5 mM K or 20 mM K
, 109
Figure
5-4.
Effect of SCH28080 on VT in the presence of
ambient 0.5 mM K or 20 mM K
, 110
Figure
6-1.
Effect of angiotensin II on Km, and VT in
the presence of luminal 3 mM Ba and absence
of luminal SCH28080
. 119
Figure
6-2.
Effect of angiotensin II on Kw and VT in
the presence of luminal 10 mM SCH28080 and
absence of luminal Ba
120
Figure
6-3.
Effect of histamine on in the presence
of luminal 3 mM Ba
121
xii


34
(-4.5 3.1 mV 5% C02 period; -3.2 2.5 mV, -2.0 2.8 mV, and -0.8 2.6
mV, 30-60 minutes, 60-90 minutes, and 90-120 minutes after exposure to 10%
C02, respectively; -7.8 3.2 mV, methazolamide, n = 7, Fig. 3-5). These data
not only confirm that an H-K-ATPase mediates the stimulatory effect of 10%
C02 on Krj,, but also suggest that an existing H-K-ATPase mediates this effect.
Again, the parallel VT responses to 10% C02 regardless of the presence or
absence of SCH28080 indicate that the changes in Vx do not explain the effect
of 10% C02 on Rb efflux. The lack of inhibitory effect of methazolamide on
KRb by subsequent addition after 10% C02 implies that carbonic anhydrase may
not be necessary to maintain the activation of H-K-ATPase by 10% C02. In the
presence of luminal SCH28080, methazolamide did not reduce Rb efflux either,
suggesting that methazolamide had no effect on the other pathways of K
permeation. To examine whether carbonic anhydrase is required for initiating
the stimulation of Rb efflux by 10% C02, we simultaneously exposed the CCD
to methazolamide and 10% C02 after measurement of the basal rate of Rb
efflux. Under these conditions we were not able to detect the stimulatory
effect of 10% C02 on Rb efflux (98.6 14.1 nnrsec"1 vs 86.2 16.5 nm-sec'1,
n = 6, Fig. 3-6), although methazolamide significantly made VT more lumen
negative from 1.3 0.9 mV to -4.1 1.2 mV (P < 0.005, n = 6, Fig. 3-6).
Time-control experiments demonstrated that perfusion time did not significantly
affect Kr,, or VT (Table 3-3). These data suggest that carbonic anhydrase is
necessary for initiating the stimulatory effect of 10% C02 on Rb efflux. To
examine whether methazolamide inhibits the basal rate of Rb efflux, we
perfused the CCD in the presence of 5% C02 throughout the experiments
instead of changing to 10% C02. As shown in Figure 3-7, methazolamide did


117
Table 2. Effect of histamine on VT in the presence of luminal 3mM Ba
Recovery
Recovery
Basal
Histamine
(30-60 min)
(60-90 min)
VT (mV)
-11.8 5.8
-11.6 7.4
-9.2 7.5
-7.6 6.3


5
potassium secretion is an important factor determining potassium excretion.
Subsequently, Davidson and his associates (1958) performed another elegant
studies in order to gain insight into the mechanisms of K secretion. They
observed that when the glomerular filtration rate was reduced by about 30%,
sodium excretion decreased by 80%, and potassium excretion decreased by 50%.
In contrast, if the rate of sodium excretion was maintained at a high level by
infusing salt and administering diuretics, then similar reduction in the glomerular
filtration rate did not reduce potassium excretion. These data were taken as a
clear evidence that potassium excretion is independent of potasium filtration and
that potassium secretion is related to the amount of Na delivery. Since the
early work of Grantham et al. (1970), the cortical collecting duct has been
firmly established as the main site of potassium secretion. During past several
decades, tremendous efforts have been devoted to understand K secretory
mechanisms, and this process has been relatively well characterized.
It has been believed that at least four systemic factors affect primarily
potassium secretion: potassium intake and total body potassium content; acid-
base balance; sodium intake and extracellular fluid volume; chloride and
nonchloride anions delivery (Wright, 1987). Generally speaking, the observations
that increases in potassium intake and in total body potassium content stimulate
potassium secretion have been attributable to the increase plasma K and
circulating level of aldosterone. Changes in arid-base balance affects potassium
excretion mainly because they modify distal flow rate, intracellular and tubular
pH, and the anion composition of luminal fluid of distal tubule. Increases in
sodium intake and in extracellular fluid volume promote potassium secretion
(although they supress plasma aldosterone concentration) mainly by increasing


(nnvsec*1)
61
o
5% C02
10%CO2
Methazolamide
Figure 3-6. Effect of simultaneous exposure to 10% C02 and 0.1 mM
methazolamide on KRb (top) and VT (bottom) (n = 6). KRb, Rb lumen-to-
bath efflux coefficient. Vr, transepithelial voltage.


10
by acidification or by lowing K intake. This transport step has been extensively
demonstrated to be passive in nature. Accordingly, lowing the potassium
concentration in the lumen or increasing lumen-negative potential through
external current application stimulates potassium secretion by steeping the
electrochemical potential difference between cytoplasma and luminal fluid.
3. Evidence based on in vitro microperfusion study is consistant with
potassium secretion in part through K-Cl cotransport (Ellison et al, 1985;
Wingo, 1989). Reduction of chloride concentration in the perfusate stimulates
potassium secretion. This component of potassium secretion is not associated
with alterations in the transepithelial voltage and, importantly, is not blocked
by barium.
4. Na-K-ATPase located in the basolateral membrane is the main
transport element. This active uptake step responds to changes in acid-base
disturbances, changes in plasma potassium level, and alterations in circulating
level of plasma aldosterone. The increase in the plasma level of K or
aldosterone stimulates Na-K-ATPase activity, and acidosis inhibits the activity of
this enzyme.
1.3 Potassium Absorption bv the Cortical Collecting Duct
In contrast to potassium secretion, little is known about the mechanisms
of K absorption. As early as 1954, Spargo (1954) reported that the first
mophologic changes during K depletion were in the collecting duct with a
hyperplastic lesion developing in the inner red medulla, suggesting that collecting
duct might participate in K conservation. Ten years later, from the
micropuncture studies, Malnic, Klose and Giebisch (1964) demonstrated that
during K depletion urinary fractional excretion of K was less than fractional


8
Adrenal steroids increased both sodium absorption as well as potassium
secretion (Sansom and ONeil, 1985 & 1986); and (3) there was no evidence
suggesting that tubular anion secretion could account for the amount of
potassium secreted. In the in vitro perfused tubules, the experimental
minipulations expected to abolish Na absorption such as removal of luminal Na,
blocking Na entry by amiloride, and inhibiting basolateral Na exit by ouabain
decreased K secretory flux (Stokes, 1981; Wingo, 1984). These studies not only
reproduced the in vivo observations, but also provided the clear evidence of
dependence of potassium secretion on sodium transport. The advance of
technology has permitted us to understand the effect of sodium on potassium
secretion at the cellular level. In all of the epithelial cells, sodium movement
across the apical membrane are passive and driven by sodium concentration
gradient and electrical potential gradient. The sodium concentration gradient is
maintained by Na-K-ATPase located at the basolateral membrane. Sodium entry
to the cortical collecting duct from the apical membrane is not exceptional. This
sodium permeability is stimulated by circulating level of aldosterone (Sansom
and ONeil, 1985) and inhibited by administration of amiloride, a potassium
sparing diuretics (ONeil and Boulpaep, 1979). The prevailing explainations for
dependence of potassium secretion on sodium are listed as following (Wright
and Giebisch, 1992): first, Na entry to the cell depolarizes apical membrane
pontential, the depolarization of apical membrane potential generates a diffusion
potential that favors K secretion; second, Na-K-ATPase is essential to maintain
intracellular K activity above its electrochemical equilibrium, which is critical for
potassium secretion, sodium entry into cell would provide the appropriate
stimulus for increased activity of the basolateral Na-K-ATPase.


LIST OF TABLES
Table 3-1. Composition of solution 50
Table 3-2. Effect of luminal 0.1% DMSO on and VT 51
Table 3-3. Time control for KRb and Vx 52
Table 3-4. Effect of 10% COa on VT in the presence colchicine 53
Table 3-5. Effect of 10% CO, on VT in the tubules
pretreated with 0.5/jM MAPTAM and W-7 54
Table 3-6. Effect of 10% C02 on KRb and VT by the normal
CCD in the presence andabsence of luminal Ba . . 55
Table 4-1. Composition of solutions 80
Table 4-2. Effect of DMSO in the absence of luminal Na
and luminal 1.5 mM Na 81
Table 4-3. Effect of luminal 10pM SCH28080 or 0.1 mM ouabain
on VT in the absence of luminal Na 82
Table 5-1. Composition of solution 99
Table 5-2. Effect of removal of luminal Na in the absence
of luminal Ba, and of luminal Ba addition in the
absence of luminal Na 101
Table 5-3. Effect of luminal Ba addition in the presence
of luminal Na, and of luminal Na removal in the
presence of luminal Ba 102
Table 5-4. Effect of removal of luminal Na in the
presence of 4 mM Ba 103
Table 5-5. Effect of luminal Na removal of in the presence of
both 2mM Ba and 10iM SCH28080 104
Table 5-6. Effect of luminal SCH28080 on VT in the absence and
viii


r
Table 5-2. Effect of removal of luminal Na in the absence of luminal Ba, and of luminal Ba addition in the
absence of luminal Na
Basal
No Na
No Na + Ba
(135 mM Na OmMBa)
(0 mM Na OmM Ba)
(OmM Na 2mM Ba)
Kr,, (nm-sec'1)
96.8 22.2
127 20.0a
90.9 23.0b
VT (mV)
6.3 2.2
6.4 1.9
7.2 2.1
a p< 0.005, compared with basal period. b p< 0.005, compared with no Na period (n = 6). Tubules were perfused
with Solution A (basal period), Solution B (no Na period) and Solution C (no Na plus Ba period). KRb, 86Rb
lumen-to-bath efflux coefficient; VT, transepithelial voltage.
O


CHAPTER 5 EFFECTS OF LUMINAL SODIUM ON Rb EFFLUX 88
5.1 Introduction 88
5.2 Methods and Material 88
5.3 Results 89
5.4. Discussion 94
5.5 Summary 97
CHAPTER 6 EFFECT OF ANGIOTENSIN II, HISTAMINE, AND
CARBACHOL Ill
6.1 Introduction Ill
6.2 Methods and Material Ill
6.3 Results 112
6.4 Discussion 114
6.5 Summary 115
CHAPTER 7 EFFECT OF Na AND K INTAKE ON SERUM AND
URINE Na AND K LEVELS AND URINE OUTPUT .... 122
7.1 Introduction 122
7.2 Methods and Material 122
7.3 Results 122
7.3 Discussion 123
CHAPTER 8 SUMMARY AND CONCLUSIONS 127
REFERENCES 137
BIOGRAPHICAL SKETCH 150
IV


123
Na level one day after the animals were exposed to the diet. Urinary Na
concentration was significantly reduced from 2nd day on the diet (at least
p<0.05), and no further reduction in Na level was observed for the entire
period on the diet. However, the rabbits excreted K differently from Na. A
significant reduction in urinary K level (at least p<0.01) was observed one day
after the animals were on the diet, and a further reduction in K concentration
(at least p<0.05) was observed four days after the rabbits were on the diet.
The total urinary excretion of Na and K was significantly reduced one day after
the diet (at least p<0.01). The total excreted Na and K were further reduced
eight days after the diet because the urine output decreased. Moreover, there
was a trend in increase in urine output after animals were adapted to the diet.
However, this effect did not reach the significant level until five days later.
Urine excretion was dramatically reduced after the rabbits were exposed to the
diet for eight to nine days. The serum Na level was not significantly altered
nine days after the diet. In contrast, the serum K level significantly decreased
after the same length of time adapted to the diet.
7.3.2 Protocol 2
Because the majority of our experiments were performed in the rabbits
adapted on a low K diet (0.25% K, TD 87433) with normal amount of Na, to
examine whether this diet causes hypokalemia, the rabbits were exposed on the
diet averagely for 10 days. Under these conditions, serum Na concentration did
not significantly changed (142.4 1.8 mM). However, serum K level was
significantly reduced (2.8 0.1 mM).
7.4 Discussion


37
significantly affecting VT (-2.2 3.0 mV vs -0.0 1.7 mV, n = 6, Fig. 3-10).
This indicates that K conductance is present in the basolateral membrane under
present conditions, and this conductive pathway mediates the stimulatory effect
of 10% C02 on Rb effllux.
To test whether normal CCD has the similar response to 10% C02 as
the K-restricted CCD, we examined the effect of 10% C02 on the CCD
dissected from regular dietary rabbits. Because the apical Ba-sensitive K-
conductance has been demonstrated to participate in Rb efflux (Warden et al.,
1989; Wingo and Zhou, 1990) also because the decrease pH has been shown
to inhibit this conductive pathway (Boudry et al., 1976; Wang et al., 1990), in
order to eliminate the overshadowing effect of 10% C02 on K-conductance and
H-K-ATPase, the tubules were perfused in the presence of luminal Ba. To find
out what concentration of Ba maximally inhibits Rb efflux, the first set of
experiments was designed to examine the dose-response of Ba with the
perfusates of solution A, solution B, and solution C designated as 0 mM Ba,
2 mM Ba, and 4 mM Ba periods, respectively. The order of these three periods
were rotated according to balanced Latin-square design (Cochran and Cox,
1957) to control for time-dependent effect on Rb efflux and the order of the
periods did not significantly affect Rb efflux. As shown in Fig. 3-11, 2 mM Ba
is sufficient enough to inhibit K^, (76.3 14.6 nmsec"\ 0 mM Ba period; 32.9
4.4 nnwsec"1, 2 mM Ba period; and 32.1 9.3 nm^sec'1, 4 mM Ba period).
The next set of experiments was performed in the presence of luminal 2 mM
Ba to examine the effects of 10% C02 and 0.1 mM methazolamide. 10% C02
and methazolamide had no significant effect on Rb efflux (48.1 7.2 nmsec\
5% C02 period; 42.5 8.3 nm-sec'1, exposure to 10% C02 from 30 to 60


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
Associate Professor 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
qualify, as a dissertation for the degree of Doctor of Philosophy.
Brian D. Cain
Assistant Professor of Biochemistry
and Molecular Biology


43
SCH28080 was given first, proton secretion decreased to 70% of control, and
subsequent addition of acetazolamide reduced proton secretion to zero. Carbonic
anhydrase-dependent acidification has been repeatedly demonstrated in the
proximal tubule (Chan et al., 1983; Cogan et al., 1979; DuBose and Lucci,
1983). In contrast, whether all of acidification in the collecting duct is carbonic
anhydrase-dependent remains controversial. Several groups have demonstrated
that the collecting duct is able to maintain almost completely normal
acidification rate after administration of acetazolamide both in vivo and in vitro,
suggesting that a substantial carbonic anhydrase-independent H source is present
in the collecting duct (Cogan et al., 1979; DuBose and Lucci, 1983; Frommer
et al., 1984; Laski, 1987; McKinney and Davidson, 1987). However, the
inhibitory effect of acetazolamide on acidification in the collecting duct has been
reported by other investigators (Lombard et al., 1983). It is difficult to
reconcile these observations at present time. Carbonic anhydrase-independent
acidification may not be an unique phenomenon only observed in collecting
duct. As early as in 1958, Durbin and Heinz (1958) have shown that carbonic
anhydrase is not essential for H secretion in gastric gland. Acidification by
turtle urinary bladder has been also reported to be in part independent of
carbonic anhydrase (Schilb and Brodsky, 1966; Schwartz et al., 1972). However,
the sources of these protons remain speculative. At least two attractive
mechanisms have has been proposed to explain the carbonic anhydrase-
independent H sources which could be from ATP hydrolysis as suggested for
gastric H-K-ATPase (Ljungstrom et al., 1984), or non-catalyzed hydration of C02
(Maren, 1974).


93
would predict that the H-K-ATPase could transport Na when luminal K
concentration is sufficiently low. Thus, we tested the effect of SCH28080 on
aNa lumen-to-bath efflux coefficient (KNa) when ambient K level was 0.5 mM
and compared these results to similar studies when ambient K concentration was
20 mM K. To inhibit conductive Na efflux, all studies were performed in the
presence of 0.1 mM luminal amiloride. Thus, in the first set of experiments, six
tubules were perfused with solution G, or solution G plus 10 fiM SCH28080
designated as basal and SCH28080 periods, respectively. The order of the
periods was rotated and there was no evidence of time-dependent effect on KNa
or VT. The second set of experiments was conducted under exactly the same
conditions except for the presence of 20 mM K (perfused with solution H). In
the presence of 0.5 mM K, SCH28080 significantly decreased KNa from 47.6
4.8 nm-sec'1 (basal period) to 35.0 6.8 run-sec'1 (SCH28080 period, P <
0.005, n = 6, Figure 5-3). In contrast, in the presence of 20 mM K, SCH28080
had no significant effect on KNa (33.3 5.0 nm-sec'1, basal period; vs 30.6
5.2 nm-sec'1, SCH28080 period, P = NS, n = 8, Figure 5-3). Because
SCH28080 has ben shown to compete with K at K binding site of gastric H-K-
ATPase, and high K concentration abolishes the inhibitory effect of SCH28080
on gastric H-K-ATPase (Keeling et al, 1988; Mendlein and Sachs, 1990), to
identify that the lack of inhibition of KNa by SCH28080 in the presence of 20
mM K is due to potassium diminishing Na efflux or due to K demolishing the
inhibitory effect of SCH28080, the third set of experiments was designed to
examine the effect of SCH28080 on Rb efflux in the presence of 20 mM K.
Thus, eight tubules were perfused with Solution F. As shown in Table 5-7,
SCH28080 significantly reduced Kjy, from 97.9 + 12.0 nm.sec'1 (basal period)


39
urinary bladder (Schwartz and Al-Awqati, 1986). The present studies are
consistent with the hypothesis that 10% C02 stimulates the exocytotic insertion
of H-K-ATPase to the apical membrane of the collecting duct. 10% C02
stimulates H secretion in gastric gland (Kidder and Montgomery, 1974). Wingo
(1989) and other investigators (Planelles et al., 1991) have shown that renal H-
K-ATPase is responsible not only for K absorption, but also for H secretion.
Indeed, microcatheterization studies in rats with acute respiratory acidosis
demonstrated that acidification was augmented prior to the inner medullary
collecting duct (Bengele et al., 1984). From the in vitro microperfusion studies,
Laski and Kurtzman (1990) have observed that total C02 flux was significantly
enhanced by the CCD after the rabbits were exposed to elevated C02 tension
chamber, indicating that the CCD increases its acidification rate in response to
hypercapnia. More direct evidence supporting the positive role of C02 in
acidification comes from McKinney and Davidson (1988) in vitro perfusing
tubule study. They have found that total C02 absorption was profoundly
increased by the CCD following exposure to 15% C02 for at least 20 minutes.
On the other hand, by acute reduction of peritubular pC02 from 40 to less
than 14 mmHg, Jacobson (1984) has shown that bicarbonate absorption
significantly decreased by medullary collecting duct. However, some studies
were not able to demonstrate stimulatory effect of C02 on acidification (Breyer
et al., 1986; Lucci et al., 1982). We have no mechanism to reconcile these data.
It is pertinent to ask whether the H-K-ATPase, or H-ATPase, or both, mediate
this effect. The application of the specific inhibitor for each enzyme would
shed more light on this question. Nevertheless, exocytotic insertion process has
been proposed to be a strong candidate to mediate this enhancement in


30
present studies is to evaluate the role of microtubules, intracellular calcium
activity, and calmodulin in the stimulation of Rb efflux (presumably activation
of H-K-ATPase) by 10% C02.
The fourth issue is the mechanism explaining basolateral K exit. In the
rabbits adapted to a normal diet the minority cells (presumably intercalated
cells) exhibit quite low basolateral K conductance (Muto et al., 1987). However,
Schlatter and Schafer (1987) have identified that a Ba-sensitive K conductance
is present in the basolateral membrane of both principal cells and intercalated
cells of the CCD of rats. If a similar pathway is present in the K-restricted
rabbits, this pathway could mediate K absorption. Therefore, the fourth objective
of this study is to examine whether a Ba-sensitive pathway is present in the
basolateral membrane of the CCD, and whether this pathway mediates the
stimulation of K absorption by 10% C02 in the K-restricted rabbits.
The final issue to be addressed by the present studies is whether 10%
C02 stimulates Rb efflux in the CCD dissected from normal rabbits.
3.2 Methods and Materials
The tubules was dissected in an artificial ultrafiltrate of plasma (in mM;
Na 145; K 5; Cl 112; HC03 25; Ca 1.8; P04 2.3; Mg 1.0; S04 1.0; acetate 10;
glucose 8; and alanine 5) gassed with 5% C02 containing additional 5% vol/vol
fetal calf serum. The bath solutions were gassed with 5% C02 (pH = 7.4
0.0) or 10% C02 (pH = 7.1 0.0) as appropriate. The electrolyte and
nonelectrolyte concentrations of the bath solution were identical to the
dissection solution unless indicated. In the peritubular Ba study, the
composition of bath were (in mM): Na 135; K 5; Cl 115.4; HC03 25; Ca 1.2;
Mg 1.0; acetate 10; glucose 8; alanine 5; Ba 3; and mannitol 4.5. In the Ba


9
Two major cell types have been identified in the CCD (Figure 1-5). The
majority cell type is the principal cell which is believed to be responsible for
K secretion (Wright and Giebisch, 1992). The minority cell is the intercalated
cell. More and more evidence suggests that this type of cell participates in
proton secretion and potassium absorption (Mujis and Katz, 1992). Current
concepts of the principal cell mode are based on the double-membrane model
originally proposed by Koefoed-Johnsen and Ussing (1958). Its essential features
and some modifications are summarized in Figure 1-6. (1) The apical membrane
of cell is highly and selectively permeable to Na and K; (2) the basolateral cell
membrane is highly potassium selective, (3) the only active transport operation
is the ATP-driven sodium-potassium exchange pump in the basolateral cell
membrane. A still growing fund of information derived from the efforts of many
investigators, especially Drs. Berliner and Giebisch, and their coworkers, during
past several decades has significantly contributed to the conversion of this "
simple original model into the more complex cell model that endows the
tubular cells with capacity of potassium secretion. The current knowledge of
principal cell with respect to K secretion is summarized in Figure 1-5.
1. Sodium (Na) enters the cell from luminal fluid passively through
amiloride-sensitive Na conductive pathway. The sodium permeability plays an
important role in potassium secretion as discussed before.
2. A Ba-sensitive K conductance is present in the apical membrane.
This highly K-selective pathway provides an important route for potassium
secretion from cell to lumen. The K-conductance accounts for the majority of
conductance in the apical membrane of the principal cell, and is stimulated
after administration of mineralocorticoids or increase in K intake, and inhibited


LIST OF FIGURES
Figure 1-1. Distribution of potassium in the body 16
Figure 1-2. Segmental analysis of tubule potassium transport .... 17
Figure 1-3. Urinary potassium excretion as a function of
plasma potassium concentration in control animals
and in alkalotic and acidotic conditions 18
Figure 1-4. Effect of different types of acid-base disturbances
on potassium secretion by distal tubule 19
Figure 1-5. The cellular models of the principal cell and the
intercalated cell 20
Figure 1-6. Cell model of epithelial sodium and potassium
transport 21
Figure 1-7. The chemical structure of SCH28080 22
Figure 2-1. The cortical collecting duct perfused in vitro 27
Figure 3-1. Effect of 10% CO, on and VT in the absence
of SCH28080 56
Figure 3-2. The predicted voltage-mediated increase in KRb and
observed increase in Kh following exposure
to 10% C02 57
Figure 3-3. Effect of 10% CO, on and VT in the presence
of SCH28080 58
Figure 3-4. Effect of 10% C02 and 0.1 mM methazolamide on Kr,,
and VT in the absence of SCH28080 59
Figure 3-5. Effect of 10% C02 and 0.1 mM methazolamide on
and VT in the presence of SCH28080 60
Figure 3-6. Effect of simultaneous exposure to 10% C02 and
0.1 mM methazolamide on K^, and VT 61
x


133
in the presence of luminal Na (P < 0.01), suggesting that the presence of
luminal Na reduces Rb (K) absorption by an H-K-ATPase-mediated mechanism.
These observations are consistant with the hypothesis that Na may compete with
K for transport via an H-K-ATPase. To test whether Na acts as a partial
agonist of the H-K-ATPase we examined whether Na could be transported by
H-K-ATPase. In the presence of luminal amiloride and an ambient K
concentration of 0.5 mM, SCH28080 (10 ixM) significantly inhibited the Na
lumen-to-bath efflux coefficient (KNa) from 47.6 4.8 nm-sec"1 to 35.0 6.8
nm-sec'1 (P < 0.005), whereas the effect of SCH28080 on Na efflux was
abolished when similar experiments were performed in the presence of 20 mM
ambient K (33.3 5.0 nm-sec"1 vs 30.6 5.2 nm-sec"1, P = NS). The lack of
effect of SCH28080 on Na efflux in the presence of 20 mM K is not
attributable to the lack of inhibitory effect of SCH28080 on H-K-ATPase,
because SCH28080 significantly reduced Rb efflux when the experiments were
repeated under exactly same conditions. Moreover, in the presence of luminal
1 mM amiloride, 10 SCH28080 significantly decreased KRb from 91.2
10.2 nm-sec'1 to 68.3 8.9 nm-sec"1 (P < 0.05). These data suggest that Na
competes with K for transport via the H-K-ATPase, and H-K-ATPase is
pharmacologically distinguishable from a Na-H antiporter.
In the gastric pland, the stimulatory effects of acetylcholine and gastrin
on the gastric H-K-ATPase are mediated by intracellular Ca. Angiotensin II
increases intracellular Ca. Angiotensin II binding has been demonstrated in the
collecting duct. Moreover, angiotensin II has been shown to be antikaliuretic in
human when infused at the concentrations that do not elicit systemic or renal
hemodynamic changes. In view of these observation, we hypothesized that


130
to the apical membrane. In view of these obsercations, we postulate that the
stimulation of Rb efflux by 10% C02 depends on fusion of H-K-ATPase to the
apical membrane of the cortical collecting duct. This hypothesis is consistant
with the effect of methazolamide. After Rb efflux was stimulated by 10% C02
(presumably activated insertion process), subsequent addition of methazolamide
failed to inhibit Rb efflux, whereas simultaneous exposure to 10% C02 and
methazolamide abolished the effect of 10% C02 (presumably prevented insertion
initiation by alkalinization of intracellular pH). However, the proton sources
originated from methazolamide-insensitive pathway remain to be identified.
Exocytosis of proton pumps in turtle urinary bladder is microtubule-dependent.
To further test the insertion hypothesis, and also to examine whether insertion
of H-K-ATPase in the cortical collecting duct shares the similar characteritics
as the insertion of proton pumps in the turtle urinary bladder, we selectively
manipulated several experimental conditions which were expected to influence
the functions of intracellular calcium, calmodulin, and microtubules. As
predicted, buffering intracellular Ca activity with MAPTAM and antaganizing
calmodulin with W-7 prevented the stimulatory effect of 10% C02. Similarly to
methazolamide, only simultaneous exposure to 10% C02 and colchicine blocked
the stimulation of Rb efflux by 10% C02. Taken together, these studies suggest
that the stimulation of Rb efflux upon exposure to 10% C02 is dependent on
the fusion of H-K-ATPase to the apical membrane of the tubules, and that this
exocytotic process is mediated by intracellular pH and Ca, calmodulin, and
microtubules. The next question remaining to be answered is the basolateral K
exit mechanism. A Ba-sensitive K conductance has been identified in the
basolateral membrane of both principal and intercalated cell of rats. If a similar


TABLE OF CONTENTS
ACKNOWLEGEMENTS v
LIST OF TABLES viii
LIST OF FIGURES x
KEY TO ABBREVIATIONS xiii
ABSTRACT xv
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Potassium Secretion by the Cortical Collecting Duct 4
1.3 Potassium Absorption by the Cortical Collecting Duct ... 10
1.4 The Aims and Objectives of the Present Studies 13
CHAPTER 2 GENERAL METHODOLOGY 23
2.1 In Vitro Microperfusion 23
2.2 Flameless Atomic Absorption Spectrophotometry 25
2.3 Statistical Analyses 26
CHAPTER 3 EFFECT OF 10% C02 ON Rb EFFLUX 28
3.1 Introduction 28
3.2 Methods and Materials 30
3.3 Results 31
3.4 Discussion 38
3.5 Summary 48
CHAPTER 4 EFFECT OF BARIUM, AMILORIDE AND OUABAIN
ON Rb EFFLUX 70
4.1 Introduction 70
4.2 Methods and Material 72
4.3 Results 72
4.4 Discussion 76
4.5 Summary 79
iii


25
by the equation Jx = (VyL^flX^-pcy where V0 and L are as before and
[X]j and [X]0 are the sodium or potassium concentration of the perfusate and
collectate, respectively. At least three and generally four collections were
obtained for measurement of flux. The passive paracellular K flux was
calculated according to the Goldman flux equation
j> tF [K]b [K]i cxpfVT xF/(RT)1
= ^ -vT-pK 1 exptVt zF/(RT)j
where PK is the paracellular K permeability, [K]b is the bath K concentration,
[K], the mean luminal K concentration, and z, F, R, and T have their usual
meaning. All chemicals were analytical grade or the highest available purity.
3H inulin, Rb and Na were obtained from New England Nuclear (Boston,
MA).
2.2 Flameless atomic absorption spectrophotometry
Flameless atomic absorption spectrophotometry described by Wingo et al
(1987) was used for the analysis of sodium and potassium concentrations in the
perfusate and collectate in order to determine net sodium and potassium flux.
Briefly, the standards and samples were placed on a clean silver platform,
freshly polished, and immersed under 0.5 cm of water-equilibrated paraffin oil.
The diluent was prepared from 100 nl Ultrex (J. T. Baker chemical Co.) and
100 ml ultra-high-purity (18 Mohm) water ("18 Mhom water," Continental Water
System) in a clean polypropylene container. To prevent loss of the analyte, the
samples were slowly dried before pyrolysis and atomization (951 AA/AE
spectrophotometer and IL 655 fumance atomizer. Allied Analytic, Waltham,
MA). Peak height integration started at the end of the pyrolysis and continues
for 8 seconds in the single beam mode at a scale expansion of 1.00 for both


60
120 -
100 -
20-
0* I | | T I
5% C02 30-60 min 60-90 min 90-120 min Methazolamide
10% C02
10%CO2
Figure 3-5. Effect of 10% CO, and 0.1 mM methazolamide on Kr,, (top) VT
(bottom) in the presence of SCH28080 (n = 7). *P < 0.05 compared with 5%
C02 period. P < 0.01 compared with 30-60 minute period. bP < 0.001
compared with 90-120 minute period. Krj,, Rb lumen-to-bath efflux coefficient.
Vts transepithelial voltage.


CHAPTER 8
SUMMARY AND CONCLUSIONS
Potassium is pivotal for a variety of physiological functions such as acid-
base balance, metabolism, cell volume regulation, blood pressure control, and
the electrical properties of both excitable and nonexcitable cells. The carrying
out of these vital functions by potassium is critically dependent on K
homeostasis which is primarily accomplished by kidneys in the long-term.
Potassium excretion is independent of K filtration. The collecting duct is the
final tuning segment determining the amount of K appeared in the urine. The
cortical collecting duct is capable of secreting K and absorbing K. The balance
between these two processes contribute largely to determination of net
potassium excretion. During past several decades, the potassium secretory
mechanism has been extensively studied and relatively well understood. However,
potassium absorptive mechanism remains to be elucidated. The discovery of H-
K-ATPase in the collecting duct is a novel approach to understand K
absorption. The present dissertation attempts to characterize this pump, mainly
assessed as SCH28080-sensitive Rb lumen-to-bath efflux coefficient, in the in
vitro perfused CCD of the rabbits with emphasizing the effects of acute
peritubular acidosis (10% C02), luminal amiloride addition and Na removal, and
peritubular ouabain on this enzyme. Additional investigations have been made
to study hormonal regulation of this pump.
127


7
electrophysiology techniques, especially the patching-clamp techniques, has
extended our understanding of mechanisms explaining the relationship between
acid-base disturbance and K secretion at the cellular level. Potassium secretion
involves the translocation of this ion across the double-membrane. First,
potassium is actively uptaken to cell by Na-K-ATPase located in the basolateral
membrane. Second, potassium passively exits out of the cell to lumen in part
through K-conductive pathway. Potassium conductance is pH sensitive. Decrease
in pH has been shown to inhibit this conductive pathway (Stanton et al., 1982;
ONeil, 1983). Recently, low-conductance K channels have been identified in the
apical membrane of the cortical collecting duct (Frindt and Palmer, 1989; Wang
et al., 1990). The characteristics of this type of channels have made it to be
a strong candidate for potassium conductance. Reducing pH also decreases open
probability of this type of channels (Wang et al., 1990). Acidosis has been
shown to inhibit Na-K-ATPase and reduce intracellular potassium activity
(Giebisch, 1987). Inhibition of Na-K-ATPase and reduction of intracellular
potassium activity inhibit K secretion by reducing the driving force for K exit
out of the apical membrane (Giebisch, 1987; Wingo, 1984; Wright and Giebisch,
1992). Figure 1-4 summarizes the effect of different types of acid-base
disturbances on potassium secretion obtained from micropuncture studies in the
rat.
The idea that potassium secretion was coupled to sodium absorption was
proposed on the basis of three major observations made in the late of fifties
and the early of sixties. (1) In 1958, Davidson et al. (1958) reported their
observations that sodium delivery drastically stimulated potassium secretion,
particularly when sodium excretion was initially maintained at low level; (2)


76
significantly altered during this maneuver (-0.4 1.1 mV, basal period, 0.1
13 mV, SCH28080 period; and 0.4 2.1 mV, ouabain period, Table 4-3).
4.4 Discussion
4.4.1 Effect of Lumen Ba
Barium is a rapidly reversible inhibitor of K channels and K conductance
(Van Driessche and Zeiske, 1980). The present studies demonstrate that Ba-
sensitive K permeation is present even in K-restricted rabbits. Warden et al
(1989) demonstrated that Rb behaves qualitatively similar to K and concluded
that there is no evidence to suggest that these ions are transported by different
mechanisms. Ba-resistant Rb efflux is small in the CCD from normal rabbits
(Warden et al, 1989 and Fig. 3-11), whereas in the K-restricted rabbit Ba-
insensitive Rb efflux is relatively larger suggesting that K restriction increases
a Ba-insensitive pathway. Since the Ba-sensitive pathway has been attributed to
Ba-sensitive K channels which should mediate K secretion (Frindt and Palmer,
1989; Koeppen and Helman, 1982; Wang et al., 1990; Warden et al., 1989), the
smaller Ba-sensitive pathway observed in the present study compared with that
in normal rabbit is in agreement with earlier studies indicating that a reduction
in K intake decreases K secretion by the CCD (ONeil and Helman, 1977;
Schwartz and Burg, 1978; Wingo et al., 1982).
4.4.2 Effect of Amiloride
It is generally accepted that both and amiloride-sensitive Na conductance
and a Ba-inhibitable K conductance are present in the apical membrane of the
principal cells of the CCD (Koeppen et al., 1983; Koeppen and Giebisch, 1988;
Muto et al., 1988; ONeil and Helman, 1977; Sansom et al., 1987; Sauer et al.,
1990). Stokes (1984) has observed that the amiloride-induced increase in K


63
Figure 3-8. Effect of simultaneous exposure to 10% C02 and 0.5 mM
colchicine (top) and exposure to colchicine after 10%CO2 (bottom) on Kr,, (n
= 6). Rb lumen-to-bath efflux coefficient.


This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirement for the degree of Doctor of Philosophy.
Dean, College of Medicine
,j.. e.

Dean, Graduate School
/ V-