Citation
Characterization and regulation of renal vasopressin receptors

Material Information

Title:
Characterization and regulation of renal vasopressin receptors
Creator:
Steiner, Martin, 1952-
Publication Date:
Language:
English
Physical Description:
ix, 132 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Binding sites ( jstor )
Cells ( jstor )
Hormones ( jstor )
Hydrolysis ( jstor )
Hypertension ( jstor )
Kidneys ( jstor )
Plasmas ( jstor )
Rats ( jstor )
Receptors ( jstor )
Vasopressin receptors ( jstor )
Binding Sites -- drug effects ( mesh )
Hypertension, Renal -- chemically induced ( mesh )
Muridae ( mesh )
Receptors, Cell Surface -- physiology ( mesh )
Vasopressins -- physiology ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 118-131).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Martin Steiner.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
18721353 ( OCLC )
ocm18721353
0030608887 ( ALEPH )

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Full Text














CHARACTERIZATION AND REGULATION OF RENAL
VASOPRESSIN RECEPTORS








By




MARTIN STEINER


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





















This dissertation is dedicated to my parents, Lillian and Murry Steiner.

















ACKNOWLEDGEMENTS

I wish to express my most sincere gratitude and appreciation to

the chairman of my supervisory committee, Dr. M. Ian Phillips, for

his friendship, guidance and support and the confidence in me that he

exhibited. Sincere thanks are extended to the members of my

committee, Drs. Steven R. Childers, Mohan K. Raizada and Colin

Sumners, for helpful advice and invaluable criticism throughout this

project. Thanks go to the many faculty members in the Departments

of Physiology and Pharmacology, but especially Drs. Melvin J. Fregly,

Bruce R. Stevens, Lal C. Garg and Stephen P. Baker, who offered me

guidance, suggestions or the use of their facilities. Thanks also go

to my friends and colleagues, past and present, Dr. Rod Casto, Elaine

M. Richards and Dr. Klaus Hermann, for continued encouragement.

The expert secretarial skills of Carolyn Gabbard are greatly

appreciated. And a very special note of gratitude is extended to

Birgitta Stenstrom Kimura, for expert technical assistance and, more

importantly, for her kindness the last 4 1/2 years.
















TABLE OF CONTENTS


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

KEY TO ABBREVIATIONS ................. vi

ABSTRACT .................. ... viii

CHAPTER 1 BACKGROUND. .. 1

Synthesis, Transport and Release of
Vasopressin . .. 1
Control of Vasopressin Secretion 2
Biological Actions of AVP in the
Periphery .... ..... 4
Vasopressin in the Central Nervous
System .. 11
Vasopressin Receptors. .. 15
Significance. .................. 23

CHAPTER 2 CHARACTERIZATION OF 3H-VASOPRESSIN
BINDING SITES IN RENAL TUBULAR
BASOLATERAL MEMBRANES ... 25

Introduction . 25
Materials and Methods ... 26
Results. . 30
Discussion . 34

CHAPTER 3 DEHYDRATION-INDUCED DOWNREGULATION OF
RENAL TUBULAR VASOPRESSIN
RECEPTORS. .. 39

Introduction . 39
Materials and Methods ... 41
Results . 44
Discussion . 50

CHAPTER 4 REGULATION OF VASOPRESSIN BINDING IN
MINERALOCORTICOID-DEPENDENT
HYPERTENSION 54

Introduction . 54
Materials and Methods ... 57
Results . 60
Discussion . 71









CHAPTER 5 EFFECTS OF MINERALOCORTICOIDS ON
VASOPRESSIN RECEPTORS AND ADENYLATE
CYCLASE-COUPLED RESPONSES IN LLC-PK1
CELLS .. .

Introduction .
Materials and Methods .. .....
Results .. .
Discussion .

CHAPTER 6 INTRACELLULAR SECOND MESSENGERS IN
LLC-PKI CELLS: A DUAL TRANSMEMBRANE
SIGNALING SYSTEM .. .....


Introduction .
Materials and Methods
Results. .
Discussion .....

CHAPTER 7 SUMMARY .


. 93
. 97
. 103

. 110


REFERENCES .................. .... 118

BIOGRAPHICAL SKETCH. ................. .132














KEY TO ABBREVIATIONS

ACTH adrenocorticotropic hormone

ADH antidiuretic hormone

ANF atrial natriuretic factor

ANG II angiotensin II

ANOVA analysis of variance

ATCC American Tissue Culture Collection

ATP adenosine triphosphate

AVP arginine vasopressin

BSA bovine serum albumin

cAMP cyclic adenosine 3',5'-monophosphate

cGMP cyclic guanosine 3',5'-monophosphate

CNS central nervous system

CPM counts per minute

DAME D-ala-met-enkephalinamide

DDAVP deamino-D-arginine vasopressin

DG diacylglycerol

DMEM Dulbecco's modified Eagle's medium

DMSO dimethylsulfoxide

DOCA desoxycorticosterone acetate

DPM disintegrations per minute

EDTA ethylenediaminetetraacetic acid

EGTA ethyleneglycoltetraacetic acid

FBS fetal bovine serum

GTP guanosine triphosphate









IBMX isobutylmethylxanthine

IP inositol phosphate

KIU kallikrein inhibitory units

KRB Kreb's Ringer's buffer

LVP lysine vasopressin

MDCK Madine-Darby canine kidney

NTS nucleus of the tractus solitarius

OXY oxytocin

PBS phosphate-buffered saline

PI phosphatidyl inositol

PMSF phenylmethylsulfonyl fluoride

PNPP p-nitrophenyl phosphate

PVN paraventricular nucleus

RIA radioimmunoassay

RPM revolutions per minute

SEM standard error of the mean

SHR spontaneously hypertensive rat

SON supraoptic nucleus

TFA trifluoroacetic acid

WKY Wistar-Kyoto
















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


CHARACTERIZATION AND REGULATION
OF RENAL VASOPRESSIN RECEPTORS


By

Martin Steiner

April, 1988


Chairman: M. Ian Phillips
Major Department: Physiology


Vasopressin receptors were characterized on basolateral

membranes of renal tubular epithelia and their capacity for regulation

was studied during alterations in fluid balance. Membranes were

obtained from rat kidney and prepared by differential and density

gradient centrifugation techniques. Radioreceptor methodologies were

used to demonstrate that binding was rapid, reversible, specific and

saturable. The binding site displayed a pharmacological profile for

the V2 receptor subtype, based on competition studies with selective

V1 and V2 antagonists.

Vasopressin receptors were impacted by the physiological state

of the animal. Water-deprived animals displayed significantly elevated

plasma vasopressin levels. As a consequence vasopressin binding sites

were downregulated. Conversely, the administration of the

viii









mineralocorticoid desoxycorticosterone acetate (DOCA) to rats

resulted in the increased expression of tubular vasopressin receptors

and the development of DOCA-salt hypertension. This was associated

with an increase in adenylate cyclase activity in the basolateral

membrane preparations. These data provide a cellular basis for the

increased tubular sensitivity to vasopressin and its fluid-retaining and

sodium-reabsorbing properties that are characteristic of this volume-

dependent model of hypertension. The heightened expression of

vasopressin receptors appears to result from an increased

transcriptional rate of protein synthesis induced by the

mineralocorticoid DOCA.

Desoxycorticosterone acetate (DOCA) was ineffective in

regulating vasopressin receptors or adenylate cyclase activity in LLC-

PKI cells, a cell line of renal epithelial origin. This coincides with a

lack of mineralocorticoid receptors in this particular cell strain.

Signal transduction mechanisms were also evaluated and vasopressin

stimulated cyclic AMP formation and phosphatidyl inositol hydrolysis,

suggesting that these cells possess both V1 and V2 receptor subtypes.

This demonstrates that one hormone can modulate cellular activity by

interacting with multiple effector systems.

These studies demonstrate that renal vasopressin receptors are

influenced by the physiological state of the animal. Similarly,

receptor-coupled signal transduction mechanisms are subject to

modification, in vitro. Alterations in vasopressin receptors or

receptor-mediated processes may provide a cellular basis for

pathophysiological conditions associated with fluid and electrolyte

balance.















CHAPTER 1
BACKGROUND

Arginine8-vasopressin (AVP) is an ancient hormone. It is one of

the most phylogenetically conserved, presumably because of its impor-

tance for water balance in early invertebrates (Sawyer, 1977).

Scientifically, it is an old hormone. Oliver and Schaefer (1895) first

described the rise in systemic blood pressure following the infusion of

a whole pituitary extract. The renal antidiuretic actions of the

hormone were first noted by Starling and Verney (1924). A wealth of

information has accumulated on the physiological actions of

vasopressin since these pioneering studies. It is now clear that

vasopressin not only serves as the principal hormone governing fluid

homeostasis, thereby requiring the antidiuretic hormone (ADH)

nomenclature, but also plays a role in cardiovascular control. In

addition, various central nervous system (CNS) effects have been

ascribed to the hormone.

Synthesis. Transport and Release of Vasopressin

Arginine -vasopressin and oxytocin both are closely related

nonapeptides, characterized by cysteine residues linked by disulfide

bridges. Often classified as posterior pituitary hormones, the

hormones are actually produced within specialized neurons in the

hypothalamus. Whereas the biologically active molecules are small

molecular weight peptides, they are synthesized, transported and

stored in secretary granules in association with specific carrier

proteins or neurophysins. Sachs and Takabatake (1964) first

1











demonstrated that the vasopressin-neurophysin molecule is formed as

part of a common precursor hormone. The precursor molecule is

synthesized in the neuronal cell body by a ribosomal mechanism and

packaged into secretary granules by the Golgi apparatus. The

vesicles are transported by axoplasmic flow toward the nerve terminal

in the neural lobe of the pituitary where they are stored. Gainer et

al. (1977) have shown that the prohormone undergoes post-

translational processing during axonal transport, suggesting that the

granules must contain both the precursor molecule and enzymes

responsible for its modification. The contents of the secretary

granules (hormone, neurophysin, and residual peptide fragments) are

released upon depolarization of the nerve terminal. This exocytotic

process involves a calcium-dependent fusion of the secretary vesicle

with the nerve terminal membrane, followed by an opening of the

vesicle and an emptying of its contents.

Control of Vasopressin Secretion

It is reasonable to expect that the release of vasopressin from

the neural lobe of the pituitary gland should be influenced by factors

pertaining to vasopressin's role in fluid homeostasis and circulatory

control. Furthermore, regulation of vasopressin secretion would be

expected to affect the activity of the paraventricular (PVN) and

supraoptic nuclei (SON). Indeed, the activity of these structures is

influenced by both osmotic and non-osmotic stimuli.

The pioneering study of Verney (1947) was the first to

demonstrate that centrally located structures which were osmo-

sensitive, affected the secretion of vasopressin. These structures are

so exquisitely sensitive that changes in plasma osmolality as small as











a few milliosmoles, which represents a change of only 1%, can effect

the release of vasopressin. Ten years later, experiments conducted

by Jewell and Verney (1957) localized the osmoreceptors to the

basomedial forebrain. The exact location of Verney's osmoreceptors

has been the subject of considerable controversy. While some have

claimed that the magnocellular neurons of the PVN and SON are

themselves osmosensitive (Leng, 1980), it is now believed that

osmosensitive cells within the organum vasculosum of the lamina

terminalis may account for the osmotically-induced alterations in

vasopressin secretion (Thrasher et al., 1982). This same group has

also laid to rest the argument over whether the mechanism for the

control of vasopressin release involves osmosensitive neurons or

sodium receptors located on the cells within the organum vasculosum

laminas terminalis (Thrasher et al., 1980). The neural connections

which relay information between cells in this circumventricular region

and the PVN and SON have been described by Miselis (1981).

Non-osmotic control of vasopressin release is probably more

important for cardiovascular regulation. Thus, hypovolemia,

hemorrhage or decreased cardiac output will elicit the secretion of

AVP, for the purpose of maintaining circulatory homeostasis. This

type of control arises from high pressure baroreceptors in the carotid

sinus and aortic arch and low pressure volume receptors in the left

atrium and pulmonary veins. The afferent impulses of this

neurohumoral arc are carried by the ninth and tenth cranial nerves

to their respective nuclei in the brainstem. Multisynaptic pathways

then transmit this information to the PVN and SON of the











hypothalamus. The osmotic and non-osmotic control of vasopressin

secretion has been reviewed by Schrier et al. (1979).

The synaptic inputs to the PVN and SON of the hypothalamus

which are involved in the control of vasopressin secretion are less

well understood (Morris, 1983). The nature of the neurotransmitters

and neuromodulators involved in these processes has only recently

begun to be addressed (Sladek, 1983). Angiotensin II, another peptide

hormone involved in blood pressure control and fluid homeostasis, has

been shown to stimulate the release of vasopressin, following

intracerebroventricular administration (Phillips, 1987). Cholinergic

parasympathetic pathways subserve both the cardiovascular- and

volume-related functions of AVP release. The ascending

noradrenergic pathways from the medullary cardiovascular centers

appear to exert a tonic inhibitory affect on vasopressin release. In

addition, dopamine and gamma-aminobutyric acid also are inhibitory.

The actions of other putative mediators of vasopressin release has

recently been reviewed by Sklar and Schrier (1983).

Biological Actions of AVP in the Periphery

Since the original observation of a vasopresssin-induced pressor

response by Oliver and Schaefer (1895), numerous other biological

effects of vasopressin have subsequently been noted. Many of the

observations pertain to the fluid-retaining and cardiovascular

properties of the hormone, or are in concert with other systems that

subserve these functions.

Renal Effects

It is now clear that vasopressin acts at multiple sites within the

kidney to affect renin release, glomerular filtration, NaCI transport








5

and water reabsorption. Vasopressin is a potent inhibitor of renin

secretion. Subpressor doses of AVP decrease plasma renin levels. It

is thought that vasopressin acts directly on the juxtaglomerular cells,

as the effect can be demonstrated in the isolated kidney and occurs

with no demonstrable effect on renal hemodynamics (Vander, 1968).

Arginine8-vasopressin reduces the glomerular ultrafiltration

permeability coefficient (Kf) and may decrease the glomerular

filtration rate (Ichikawa and Brenner, 1977). While the mechanism by

which AVP decreases the Kf is not clear, Ausiello et al. (1980) have

shown that vasopressin stimulates the contraction of glomerular

mesangial cells grown in culture. Therefore, it is possible that

mesangial contraction reduces the surface area available for

glomerular ultrafiltration.

The next demonstrable action of vasopressin occurs at the thick

ascending Limb of Henle. Morel et al. (1976) made the first

observation of vasopressin-sensitive adenylate cyclase activity in both

the medullary and cortical portions of the thick ascending limb. The

concentration of AVP necessary to stimulate adenylate cyclase

activity in the thick ascending limb is one or two orders of

magnitude higher than that needed in more distal tubular segments.

The physiological significance of this finding remained in doubt until

Hebert et al. (1981a) found that AVP, in pico-molar concentrations,

stimulated NaCI transport out of the tubular lumen into the

interstitium. This suggests a role for vasopressin in the maintenance

of the corticomedullary interstitial osmotic gradient.

The major target for the action of vasopressin is the distal

segment of the collecting tubule. Here vasopressin, through a cAMP-










dependent mechanism, can exert its well known antidiuretic effect, by

altering the permeability of the tubular membrane to water (Grantham

and Burg, 1966). The cellular events beyond cAMP formation include

stimulation of cAMP-dependent protein kinase in the cytosol and

protein kinase A-dependent phosphorylation of the apical membrane,

resulting in an increased permeability of the apical membrane to

water (Figure 1-1). At present, the precise mechanism by which

protein kinase activation, calcium activation and microtubule-

microfilament assembly result in an alteration in membrane

permeability remain unclear, despite the well-established antidiuretic

effect of the hormone. The most favored hypothesis to emerge,

however, is that vasopressin induces, by some conformational change,

the formation of aqueous channels in the apical membrane (Gluck and

Al-Awqati, 1980). This is supported by the appearance of ADH-

induced intramembraneous particle aggregates in the luminal

membrane. The number of aggregates and the cumulative area of

membrane occupied by the particles are directly related to the

magnitude of ADH-induced changes in water permeability (Muller et

al., 1980).

Cardiovascular Effects

Although an elevation of blood pressure was the first

physiological effect noted following the administration of vasopressin,

the cardiovascular actions of vasopressin have generally been

regarded as physiologically unimportant. This is somewhat surprising

as vasopressin has been shown to be one of the most potent

vasoconstrictive agents (Altura and Altura, 1984). In addition, even

the antidiuretic action of the hormone contributes to the regulation
















A


TUBULAR CELL PERITUBULAR
LUMEN FLUID




?In*hitid by prostalondins
4 Co, omes?
HAdewnlote cycloS Rptr



lick AWP

Apical Mmb 5'AP Bolers l Manhbe
Microfitmnes














Figure 1-1. Model of antidiuretic hormone (ADH)-stimulated
adenylate cyclase cascade in renal tubular epithelial cell. ADH
combines with receptor in basolateral membrane. Interaction of
hormone and receptor stimulates adenylate cyclase, resulting in the
formation of cyclic AMP and the activation of cAMP-dependent
protein kinases. These enzymes act to phosphorylate microtubules,
microfilaments or integral proteins in the apical membrane. While the
mechanism of increased apical permeability to water remains unclear,
the most widely accepted model postulates the formation of aqueous
channels (Gluck and Al-Awqati, 1980).











of extravascular volume and exerts hemodynamic effects even at

subpressor doses (Cowley et al., 1984b). Guyton and others propose

that the antidiuretic action of the hormone can even produce

hypertension under certain conditions (Manning et al., 1979). The

cellular basis for this phenomenon is alluded to in Chapter 4 of this

dissertation.

Vasopressin has been shown to exert direct negative chrono-

tropic and inotropic effects in vitro (Nakashima et al., 1982). The

physiological significance of these direct cardiac effects remains in

question as exceedingly high concentrations of vasopressin were

necessary to elicit the observed effects.

Vasopressin is a potent vasoconstrictor, in vivo and in vitro. In

vivo, administration of vasopressin leads to an increase in total

peripheral resistance from the vasoconstriction of a variety of

vascular beds. The skin, muscle and splanchnic circulations are the

most sensitive, followed by the mesenteric and coronary beds. The

renal, hepatic and cerebral circulations are unaffected by AVP (Liard,

1984). The vasoconstrictive potencies of several pressor substances

and a number of vasopressin analogues has recently been evaluated in

an isolated smooth muscle preparation and AVP was determined to be

the most potent agent tested, although the in vitro activity did not

exactly parallel the in vivo activity (Altura and Altura, 1984).

However, when vasopressin is infused into conscious intact animals,

blood pressure does not normally rise unless exceedingly high plasma

levels of vasopressin are attained (>100 pg/ml). Although AVP-

induced elevations in total peripheral resistance might occur, arterial

pressure is not drastically affected because it is strongly buffered by











the baroreceptor reflex. Furthermore, vasopressin actually tends to

enhance the baroreflex by increasing vagal tone and inhibiting

sympathetic outflow following i.v. administration, apparently through

an interaction with the cardiovascular centers in the brainstem

(Cowley et al., 1984a). The role of vasopressin in the central control

of the circulation is addressed later in this chapter.

Because the pressor effects of vasopressin are normally obscured

by the baroreflex, the physiological importance of AVP in

cardiovascular function has often been ignored. Nevertheless,

vasopressin plays an important role in the maintenance of blood

pressure under conditions of dehydration (Schwartz and Reid, 1983),

when circulatory volume is compromised as in the case of hemorrhage

(Zerbe et al., 1982), in a situation of adrenal insufficiency (Schwartz

et al., 1983) or when the vasoregulatory reflex mechanisms are

interrupted. In each of these cases, plasma levels of vasopressin rise

in excess of the concentration necessary for maximal antidiuresis.

That vasopressin is essential for maintaining circulatory homeostasis

in even a moderately compromised condition is evident in the findings

of Aisenbrey et al. (1981). In that study, rats were dehydrated for a

period of 72 hours and then challenged with a vascular vasopressin

blocker. Whereas the blocker had no intrinsic cardiovascular activity

nor did it affect blood pressure in normally hydrated animals, blood

pressure fell precipitously in the dehydrated rats. In a related study,

Brattleboro rats, which are unable to synthesize vasopressin, were

unable to maintain their blood pressure when subjected to dehydration

(Woods and Johnston, 1983). Collectivelly, these data lend support

for a physiological role for vasopressin in circulatory control.











Vasopressin is not a hormone unto itself. It is reasonable to

expect that AVP should interact with other regulatory systems to

acheive homeostasis. This is most clear in terms of the cardio-

vascular effects of vasopressin. Vasopressin, the renin-angiotensin

system and the autonomic nervous system all interact, to the point of

redundancy in maintaining circulatory control. As noted previously,

vasopressin can inhibit the secretion of renin from the kidney

(Vander, 1968). Conversely, angiotensin has been shown to stimulate

the release of AVP (Phillips, 1987). Vasopressin and the autonomic

nervous system interact extensively within the brain to acheive

circulatory control, as is discussed in a subsequent section. In the

periphery, vasopressin potentiates the vasoconstrictor action of nor-

epinephrine and sympathetic nervous stimulation (Bartlestone and

Nasmyth, 1965). Conversely, blockade of the sympathetic nervous

system greatly accentuates the pressor effect of vasopressin (Pullan

et al., 1978). A methodical study by Gavras et al. (1982) provides

experimental evidence which supports the interaction of these three

effector systems in the maintenance of blood pressure.

Other Biological Effects

While the renal and cardiovascular actions of vasopressin are

perhaps the best known, other biological effects have recently been

attributed to vasopressin. The central effects of vasopressin in

cardiovascular regulation will be discussed in a subsequent section.

Acting as a neuropeptide, AVP has been ascribed a role in

thermoregulation, nociception, memory and behavior. Another

neuroendocrine action of vasopressin is to cause the release of ACTH

from the anterior pituitary, either by acting directly or by











potentiating the effect of corticotropin releasing factor (De Wied,

1983). A metabolic action has been attributed to vasopressin, by

virtue of its glycogenolytic effect in the liver (Michell et al., 1979).

Steroidogenic effects have been attributed to vasopressin in the

Leydig cells of the testes and the glomerulosa cells of the adrenals

(Nicholson et al., 1984). It has also been shown that vasopressin is

involved in platelet aggregation (Haslam and Rosson, 1972).

Vasopressin in the Central Nervous System

Vasopressineraic Proiections

Vasopressin (and oxytocin) are synthesized in neurosecretory

neurons located within the paraventricular and supraoptic nuclei of

the hypothalamus. It was originally thought that oxytocin was

exclusively formed in the SON and that vasopressin was produced

only in the PVN. It is now clear that both peptides are synthesized

in separate and topographically distinct populations of neurons within

each nucleus (Swanson and Sawchenko, 1983). Each nucleus is

characterized cytoarchitecturally by the presence of large

magnocellular neurons and small, round parvocellular neurons. It has

been known since the work of Cajal (1894) that the magnocellular

neurons of the SON project to the neural lobe of the pituitary.

While morphologically quite similar, it was not recognized until forty-

two years later that the magnocellular neurons of the PVN also

project to the posterior pituitary (Ingram et al., 1936). Although the

findings of Cajal remain unchanged, histological evidence concerning

vasopressinergic projections from the PVN has been greatly expanded.

Magnocellular projections from the PVN have been traced to the

external lamina of the median eminence (Diedrickx et al., 1976).











Maraka et al. (1981) have suggested that vasopressin released into

this capillary portal zone may affect the synthesis and release of

ACTH and other adenohypophyseal hormones.

Parvocellular neurons of the PVN, which are morphologically and

topographically distinct from magnocellular neurons, have recently

been shown to project to neural lobe of the pituitary (Swanson and

Kuypers, 1980). Vasopressinergic fibers from the hypothalamus are

reported to innervate the lateral septum in the forebrain (Sofroniew

and Weindl, 1978). More extensive descending projections have been

traced to the intermediolateral column of the spinal cord, the nucleus

tractus solitarius and the dorsal vagal complex in the brainstem

(Buijs, 1978; Swanson and Kuypers, 1980) and the locus coeruleus

(Sofroniew, 1980). Despite the complex organization of the PVN,

Swanson et al. (1980), using retrograde labeling techniques, have

convincingly demonstrated that separate neurons project to each of

the areas described above.

Afferent Proiections to the PVN

The development of histofluorescence methods lead to the

demonstration that the PVN and the SON contained one of the

highest densities of noradrenergic terminals in all of the brain

(Carlsson et al., 1962). By a variety of anterograde and retrograde

labeling techniques, the origins of these synaptic inputs have been

traced to Al and C1 cell groups of the ventral medulla, the A2 and

C2 cell groups in the medial NTS and the A6 cell group in the locus

coeruleus. The parvocellular neurons of the PVN are preferentially

innervated by noradrenergic axons from the medulla and locus

coeruleus (A2 and A6) while the Al area of the ventrolateral medulla











projects to magnocellular and parvocellular neurons of the PVN and

SON. In addition to rostral projections from the brainstem, the PVN

receives numerous inputs from diencephalic and telencephalic

structures. The efferent connections of the PVN and the complex

organization of this hypothalamic nucleus have been elegantly

reviewed by Swanson and Sawchenko (1983).

The ascending catecholaminergic pathways and descending

vasopressinergic pathways provide an anatomical basis for the

feedback loops which enable vasopressin to influence autonomic and

cardiovascular function and in turn, allow for adrenergic control of

vasopressin release. This latter point may account for the tonic

inhibitory influence that oc-adrenergic stimuli have on vasopressin

secretion (Kimura et al., 1981).

Role of AVP in Central Cardiovascular Regulation

The diversity of afferent and efferent projections of the PVN

has lead Swanson and Sawchenko (1980) to view this vasopressin-

synthesizing site as a visceral effector nucleus, capable of influencing

the secretion of hormones from the anterior and posterior lobes of

the pituitary and neural activity in the sympathetic and

parasympathetic divisions of the autonomic nervous system. The

vasopressinergic innervation of certain brainstem and forebrain nuclei

tends to suggest that AVP plays an important role in the central

control of circulation, exclusive of its well known vasoconstrictive

action in the periphery.

One potential role of AVP in the central control of

cardiovascular regulation involves its ability to modulate neuronal

activity of the NTS, which constitutes the anatomical substrate of the











baroreceptor reflex. The NTS receives information from afferent

baroreceptor fibers originating in the carotid sinus and aortic arch.

The NTS influences cardiovascular function in three ways: (a) it

relays baroreceptor activity to other brainstem areas to modulate

spinal preganglionic neuronal activity; (b) it relays baroreceptor and

chemoreceptor activity to the hypothalamus, which serves to regulate

and integrate autonomic and cardiovascular responses; and (c) it

participates in the regulation of fluid and electrolyte homeostasis by

influencing the release of AVP (Reis et al., 1984). Experimental

evidence tends to support the hypothesis that bloodborne AVP

enhances the sensitivity of the baroreceptor efferent pathway.

Courtice et al. (1984) have electrophysiological data which show that

AVP enhances the baroreflex by increasing parasympathetic vagal tone

and depressing barosympathetic outflow. In addition, Brattleboro rats,

which are deficient in vasopressin, have a depressed baroreflex, which

can be corrected by intravenous infusions of vasopressin (Imai et al.,

1983). Though somewhat paradoxical, centrally administered AVP

acts in an opposite manner to peripherally administered AVP.

Intracerebroventricular administration of AVP elcits a pressor

response, tachycardia, increased sympathetic outflow and increased

levels of plasma catecholamines (Unger et al., 1984). Matsuguchi et

al. (1982) reported that the microinjection of AVP directly into the

NTS caused increases in arterial pressure and heart rate. Berecek et

al. (1984) reported similar findings following microinjections of AVP

into the locus coeruleus.

In addition to the reciprocal neuroanatomical pathways between

the PVN and NTS that are described above, functional interactions











have also been noted. Lesions of the PVN prevent the elevation in

arterial pressure and heart rate associated with aortic baroreceptor

deafferentation (Zhang et al., 1983). Employing electrophysiological

techniques, Ciriello and Calaresu (1980) have shown that the NTS and

the dorsal motor nucleus of the vagus relay cardiovascular

information directly to the PVN. In the same study it was also

shown that electrical stimulation of the PVN lead to an increase in

arterial pressure and heart rate and inhibited the reflex bradycardia

evoked by stimulation of the carotid sinus nerve. Conversely,

destruction of the Al region, the group of noradrenergic cell bodies

thought to tonically inhibit AVP secretion, results in a condition of

fulminating hypertension, marked by high levels of vasopressin in the

plasma (Blessing et al., 1982).

Thus, a feedback loop appears to exist between the PVN and the

NTS which serves to modulate the ability of the baroreflex to

regulate arterial pressure. Since the NTS is the primary afferent

relay for baroreceptor fibers and the dorsal motor nucleus of the

vagus contains vagal cardioinhibitory preganglionic neurons,

projections to these structures from the PVN appear to participate in

the central control of cardiovascular regulation. Vasopressin also

appears to influence other sites in the central nervous system

involved in circulatory control, such as the locus coeruleus. A

vasodepressor function has been attributed to the action of

vasopressin in the dentate gyrus of the hippocampus (Versteeg et al.,

1984).









16

Vasopressin Receptors

Current dogma holds that all peptide or protein hormones

initiate their actions by binding to membrane-bound receptors located

on the cell surface of the target tissues. The interaction of the

hormone with its receptor causes the activation of one of a number

of second messenger systems (cAMP, cGMP, PI). Depending on the

tissue and receptor involved, this can result in the activation of

enzymes, mobilization of intracellular stores of calcium (which, in

turn can affect enzyme activity), phosphorylation of membrane

proteins or the activation of ion channels, leading to the appropriate

biological response.

AVP interacts with at least two subclasses of vasopressin

receptors, depending on the tissue in question. Michell et al. (1979)

formally designated these different subtypes as V1 and V2 vasopressin

receptors on the basis of the effector systems to which these

receptors are coupled. Elucidation of the subtypes has been greatly

facilitated by the synthesis of structurally distinct vasopressin

analogues which bind differentially to the V1 or V2 receptor.

Development of these analogues has resulted in the design of specific

agonists and antagonists for each of the receptor subtypes (Sawyer et

al., 1981).

V1 receptors are coupled to phospholipase C and the hydrolysis

of phosphatidyl inositol (PI) with the subsequent formation of inositol

phosphates (IP) and diacylglycerol (DG). Inositol phosphates have

been shown to increase the mobilization of intracellular stores of

calcium. Diacylglycerol on the other hand, activates the enzyme

protein kinase C. V2 receptors are coupled to adenylate cyclase and











the formation of cAMP. Both VI and V2 receptors are functionally

linked to their respective effector systems through guanine nucleotide

regulatory proteins (G proteins). Thus, the antidiuretic action of

vasopressin is mediated through V2-type receptors while the

vasoconstrictive, glycogenolytic and steroidogenic actions of AVP are

mediated by Vl-type receptors, based on structure-activity

relationship studies with vasopressin analogues or by directly

determining the second messenger. A novel vasopressin receptor

(Vlb) which displays a pharmacological profile that does not clearly

fall into one category or the other has been reported to mediate the

effects of AVP in the anterior pituitary (Antoni, 1984). Furthermore,

recent evidence suggests that at least one tissue may possess both VI

and V2 receptor subtypes (Chapter 6).

Kidney

Evidence for the binding of vasopressin to hormone-responsive

cells in the kidney was first suggested by the autoradiographic

studies of Darmady et al. (1960). Specific binding of 3H-AVP to

plasma membranes of the renal medulla was subsequently reported by

two independent laboratories (Campbell, et al., 1972; Bockaert et al.,

1973). Neither study succeeded in localizing the binding to plasma

membranes of renal tubular origin nor were they able to distinguish

between basolateral and apical membranes. However, Bockaert et al.

(1973) and others (Nakahara et al., 1978) were able to demonstrate

the correlation between receptor occupancy and the activation of

adenylate cyclase. These studies were consistent with the finding

that cAMP and vasopressin-induced changes in membrane permeability

were only noted following application to the peritubular side of











isolated renal tubules (Grantham and Burg, 1966). It may be

concluded that these vasopressin binding sites constitute the

receptors which mediate the antidiuretic action of the hormone in the

distal tubular collecting segments of the nephron. Since then,

vasopressin binding sites have been characterized on a variety of

membranes prepared from porcine, bovine, rat and human kidneys

(Jard, 1983). A more convincing argument for the epithelial

localization of vasopressin binding sites comes from the finding of

Roy and Ausiello (1981a). Using tissue culture techniques, these

investigators were the first to characterize vasopressin receptors in

LLC-PKI cells. This cell line is derived from tubular elements of the

pig kidney (Hull et al., 1976). Both intact cells and renal membranes

all appear to possess a single class of high affinity binding sites (KD

= 1-25 nM).

All vasopressin-sensitive renal tissue need not be of epithelial

origin, however. Vasopressin receptors have been characterized on

glomerular mesangial cells (Jard et al., 1987) and appear to mediate

the vasopressin-induced contraction of this tissue (Ausiello et al.,

1980). This action may account for the alteration in glomerular

ultrafiltration observed with vasopressin in the intact glomerulus.

Vasopressin binding sites in intact glomeruli has recently been

reported (Stoeckel et al., 1987). Thus, caution must be employed

when interpreting vasopressin binding studies conducted in the kidney

by autoradiography or when using crude membrane preparations.

Vasculature

In order to effect the cardiovascular actions of vasopressin

mediated through its vasoconstrictive properties, it follows that











vasopressin binding sites must be present on the smooth muscle cells

of the resistance vasculature. This observation has proven difficult

due to the high degree of cellular heterogeneity of the vascular wall

and technical problems encountered in preparing pure plasma

membrane fractions derived from smooth muscle cells. These

obstacles have apparently been overcome as judged by the report by

Schiffrin and Genest (1983) describing vasopressin binding in a

mesenteric artery membrane preparation. AVP binding has also been

demonstrated in aortic smooth muscle cells in primary culture (Penit

et al., 1983) and in an established smooth muscle cell line (A-10

cells) (Stassen et al., 1987). These receptors, like those in renal

tissue, exhibit nanomolar affinity constants and a single class of

binding sites. Whereas renal tubular vasopressin receptors are

coupled to adenylate cyclase activation, those in the vasculature

stimulate the hydrolysis of phosphatidyl inositol and the mobilization

of intracellular stores of calcium, as a means of signal transduction

(Aiyar et al., 1986).

Central Nervous System

Vasopressin receptors in the brain have been characterized using

traditional biochemical techniques and their distribution analyzed by

means of autoradiography (Dorsa et al., 1983). This group found that

the receptor behaves kinetically as those found in the periphery.

Furthermore, they reported that the NTS in the brainstem displayed a

high level of binding. Other investigators have confirmed this

finding and extended their observations to include AVP binding in the

hippocampus and lateral septum (Biegon et al., 1984). These findings

are consistent with the functional, behavioral and immunocytochemical











data which contend that vasopressin acts in the brain as a neuro-

transmitter or neuromodulator. The presence of AVP receptors in the

regions mentioned above parallel the vasopressinergic pathways

involved in central cardiovascular regulation.

The nature of the effector system coupled to the vasopressin

receptor in the brain still remains in question. Competition of 3H-

vasopressin binding with vasopressin analogues suggests that the

receptor resembles the vascular or VI subtype (Cornett and Dorsa,

1985; Audigier and Barberis, 1985), although no direct measurements

of vasopressin-stimulated PI hydrolysis have been reported. However,

a presynaptic membrane phosphoprotein known as B50 which is a

substrate for protein kinase C, is phosphorylated upon the addition of

AVP to rat hippocampal synaptic membranes (Hinko and Perlmutter,

1987). These data tend to suggest that vasopressin acts through Vj-

type receptors in neuronal membranes to cause the formation of

inositol phosphates and diacylglycerol. DG may activate protein

kinase C which in turn, phosphorylates integral membrane proteins.

Given the importance of calcium in the process of synaptic

transmission, the actions of IP cannot be excluded either.

Other Tissues

A burgeoning number of studies have recently reported the

presence of vasopressin receptors in a variety of tissues. 3H-

vasopressin binding and its relation to glycogen phosphorylase activity

was reported by Cantau et al. (1980). Activation of this enzyme and

its resulting glycogenolytic action is mediated by V1 receptors

coupled to PI hydrolysis (Michell et al., 1979). In the anterior

pituitary gland, vasopressin receptors were first characterized by











Lutz-Bucher and Koch (1983). Antoni (1984) reported that the ligand

specificity of pituitary vasopressin receptors was distinct from

previously characterized V1 and V2 receptors although no

measurements of the second messengers were conducted. Gaillard et

al. (1984) however, did report that vasopressin failed to influence

adenylate cyclase activity in pituitary membrane fractions, but did

potentiate CRF-stimulated cAMP accumulation. Balla et al. (1985)

described the binding and signal transduction mechanisms for

vasopressin in membranes prepared from rat adrenal capsules (chiefly

zona glomerulosa). Displacement studies suggested that the receptor

was of the V1 variety and incorporation studies indicated that AVP

promoted 32P uptake into PI. This effect was blocked by V1

antagonists as was AVP-stimulated PI hydrolysis. The net effect of

vasopressin binding in isolated zona glomerulosa cells was the

stimulation of aldosterone secretion. Whereas vasopressin stimulates

steroid biosynthesis in the adrenals, it inhibits testicular Leydig cell

steroidogenesis. Receptors which mediate this effect were described

by Meidan and Hsueh (1985). Relative binding potencies of selective

agonists and antagonists suggest that it is a V1 subtype. Finally, a

number of cellular elements in the bloodstream are reported to

possess vasopressin receptors. Binding sites on platelets were

described by Berrettini et al. (1982). Mononuclear phagocytes were

shown by Block et al. (1981) to possess vasopressin receptors.

Although AVP has been shown to cause platelet aggregation, the

function of vasopressin receptors in monocytes is unknown. Table 1-

1 summarizes the current state of knowledge concerning vasopressin

receptors in biological tissues and their significance.

















TABLE 1-1.
BIOLOGICAL ACTIONS OF ARGININE8-VASOPRESSIN.


Tissue Action Receptor Subtype Reference



Brain Neuromodulator. VI DeWied, 1983
cardiovascular
control, nociception,
thermoregulation

Anterior ACTH release Vlb Antoni, 1984
pituitary

Platelets Aggregation 7 Berrettini et
al., 1982

Mononuclear 7 ? Block et al.,
phagocytes 1981

Blood vessels Vasoconstriction V1 Schiffrin &
Genest, 1983

Liver Glycogenolysis V1 Michell et al.,
1979

Adrenal Steroidogenesis V1 Balla et al.,
glomerulosa 1985
cells

Testes Inhibits androgen V1 Meidan &
biosynthesis Hsueh, 1985


Juxtaglo- Inhibits renin ? Vander, 1968
merular cells secretion

Glomerular Contraction V1 Jard et al.,
mesangial cells 1987

Renal tubular Solute, ion V2 Morel et al.,
epithelium transport 1987









23

Significance

Myriad biological functions have now been attributed to

vasopressin. The preponderance of evidence suggests that its major

action pertains to its role in circulatory and fluid homeostasis. This

is achieved in a concerted manner, through fluid and electrolyte

reabsorption, vasoconstriction, modulation of the baroreflex and

central regulation of cardiovascular centers.

The actions of vasopressin can be controlled in a number of

ways. One is to control vasopressin secretion itself. The levels of

circulating AVP are regulated by osmotic and non-osmotic

mechanisms. Endocrine feedback systems also act to control AVP

release. For example, dehydration will cause an osmotically-mediated

release of vasopressin but, in addition, will activate the renin-

angiotensin system in the brain, which in turn will stimulate the

release of AVP.

The action of vasopressin can also be affected at the target

tissue. This usually implies some type of modulation of hormone

receptor or post-receptor modification. The concept of hormone

receptor regulation has been the subject of several recent reviews

(Catt et al., 1979; Roth and Taylor, 1982; Lefkowitz et al., 1984).

Receptor regulation can occur by "homologous" or "heterologous"

mechanisms. The former implies that a hormone can modulate the

cell membrane content of its own specific receptor. Alternatively,

receptor number may remain the same but the system is desensitized

through an uncoupling of the receptor from the signal transducing

elements, often by covalent modification of the receptor.

Heterologous regulation suggests that other hormones or factors can











regulate the receptor of a second hormone. For example, a steroid

hormone may affect the expression of a peptide hormone receptor

through transcriptional events. In any case, regulation of receptors

can affect the cellular sensitivity to hormonal stimulation. For the

antidiuretic hormone, this is of tanamount importance in its ability to

maintain fluid homeostasis.

The studies delineated in this dissertation were conducted to

(a) characterize antidiuretic hormone receptors in the kidney, (b)

study their physiological regulation in vivo, by altering the state of

fluid balance, ranging from a condition of dehydration to a volume-

expanded model of hypertension, and to (c) establish a "parallel"

system in vitro, to examine receptor regulation and signal

transduction mechanisms at the cellular level. Hopefully, this

information will expand the current state of thinking about the

mechanisms of action of arginine vasopressin and its role in fluid and

electrolyte homeostasis and circulatory control.
















CHAPTER 2
CHARACTERIZATION OF 3H-VASOPRESSIN BINDING
SITES IN RENAL TUBULAR BASOLATERAL MEMBRANES

Introduction

Arginine8-vasopressin in most species is the mammalian anti-

diuretic hormone. It acts upon distal tubular segments of the

nephron to promote fluid reabsorption by altering the permeability of

the tubular luminal membrane to water. Vasopressin-stimulated

activation of the enzyme adenylate cyclase initiates the biochemical

cascade responsible for generating transepithelial net fluxes of ions

and solutes.

The presence of vasopressin binding sites in the kidney was

described as early as 1960 (Darmady et al.) and autoradiographic

reports of vasopressin receptors continue up to the present day

(Stoeckel et al., 1987). Early studies which utilized traditional

biochemical techniques to characterize vasopressin receptors in plasma

membranes prepared from renal medullary tissue did so in somewhat

crude preparations which may have been contaminated with vascular

and non-epithelial elements (Campbell et al., 1972; Bockaert et al.,

1973). These types of studies, therefore, fail to distinguish between

vasopressin-sensitive cells of tubular epithelial origin from cells which

respond to AVP located in the glomerulus and the renal vasculature.

Nor do these studies allow one to localize the binding specifically to

the basolateral as opposed to the apical region of hormone-sensitive

epithelia. Although it is well-established that the vasopressin-

25











sensitive adenylate cyclase system is present on the peritubular side

of tubular epithelia (Grantham and Burg, 1966), the characterization

of vasopressin receptors localized specifically to the basolateral

membrane has not been previously described. This study was

conducted to determine the characteristics of vasopressin binding for

a basolateral membrane preparation of rat renal tubular epithelia.

Materials and Methods

Animals and Chemicals

Male Sprague-Dawley rats weighing 200-250g were obtained from

the Animal Resource Facility at the University of Florida. Rats were

housed individually in 20x18x24 cm wire mesh cages, exposed to a

12:12 hour lightdark cycle, provided a standard Purina rat chow diet

and allowed free access to tap water.

Percoll and synthetic peptides were purchased from Sigma

Chemical Co. (St. Louis). d(Et)2Tyr(Et)D-AVP (VI) and d(CH2)5D-

Ile2-Ile4-AVP (V2) vasopressin analogues were generous gifts of Dr.

M. Manning of the Medical College of Ohio in Toledo. Tritiated 8-L-

arginine [phenylalanine-3,4,5-3H]-AVP (3H-AVP) used in receptor

binding studies was purchased from New England Nuclear Corp.

(Boston) with a specific activity of 70 Ci/mmol. Other reagents were

obtained from Sigma, unless otherwise indicated.

Membrane Preparation

Animals were sacrificed by decapitation. Renal basolateral

membranes were prepared according to the density gradient

centrifugation method of Boumendil-Podevin and Podevin (1983), with

slight modification. Kidneys were removed, decapsulated and rinsed

in a chilled buffer containing 250 mM sucrose, 2 mM Tris-HEPES, 0.1











mM PMSF, pH 7.4. The tissue was minced and then homogenized

with 5 strokes of a Dounce homogenizer followed by 5 strokes of a

teflon pestle homogenizer at a setting of 800 rpm. The resulting

homogenate was centrifuged at 1,000 x g for 10 minutes at 4C. The

pellet (PI) is discarded and the supernatant (SI) centrifuged at 22,000

x g for 15 minutes at 4C. The supernatant (S2) is discarded, leaving

a triple-layered pellet (P2). The upper two layers of the pellet were

removed by gentle vortexing and homogenized in 10 ml of the Tris-

HEPES-sucrose buffer without PMSF. Percoll (1.2 ml) is added and

mixed, then centrifuged at 40,000 x g for 35 minutes at 4*C. The F1

band (Basolateral membranes, density = 1.037 g/ml) is aspirated and

diluted in 5 volumes of 2 mM Tris-HEPES, 85 mM KCI, 85 mM

sucrose, pH 7.4. Percoll was removed from the mixture by

ultracentrifugation at 60,000 x g for 15 minutes at 4*C. The

supernatant is decanted, the membranes are gently washed away from

the Percoll pellet, resuspended in Tris-HEPES-sucrose buffer with

PMSF and stored in liquid N2. An outline of this membrane

preparation is depicted in Figure 2-1. Protein content was

determined with the Folin reagent, using bovine serum albumin as the

standard, according to the method of Lowry et al. (1951).

Determination of Na+K-ATPase Activity

The Na+K+-ATPase activity was determined by measuring the K+-

stimulated, ouabain-sensitive p-nitrophenylphosphate (PNPP) hydrolysis

at pH 7.8, based on the method of Yoshida et al. (1969). Briefly, the

assay buffer was composed of 50 mM Tris-HCI (pH 7.8), containing 5

mM MgCl2, 2.5 mM EDTA, 50 mM KCI and 5 mM PNPP. The reaction

was carried out in the presence and absence of ouabain (1 mg/ml) by


















Kidney tissue homogenized in 2 mM Tris-HEPES, 250 mM Sucrose buffer,
pH 7.4 containing 0.1 mM PMSF.


Homogenate IK x g, 10'


Si 22K x g, 15'


P2 (upper two layers of "triple" pellet)
I
10% Percoll Gradient- 40K x g, 35'


F1 = Basolateral Membrane (1.037g/ml)


F2 = Brush Border Membrane (1.041g/ml)


F3 = Mitochondrial Membrane (1.067g/ml)
















Figure 2-1. Scheme for preparation of basolateral membranes from
renal tubular epithelia. Kidneys from male Sprague Dawley rats
homogenized in Tris-Sucrose buffer containing the protease inhibitor,
PMSF. Membranes prepared by density gradient and differential
centrifugation, as described in "Methods" section.











the addition of 10 p1 membranes (100 pg protein) to 0.5 ml of the

assay buffer. The mixtures were incubated for 2-10 minutes at 37'C.

The reaction was stopped by the addition of 3.5 ml IN NaOH and

absorbance was read at 410 nm. Net Na+K+-ATPase activity was

determined by subtracting the PNPP hydrolysis activity in the

presence of ouabain from the activity in the absence of ouabain.

Specific activity was calculated based on a molar extinction

coefficient c = 15,400 M- cm 1, and final values were expressed as

units/mg protein (where 1 unit = 1 pmol PNPP hydrolyzed per

minute).

Vasopressin Receptor Assay

Membranes, corresponding to 100-200 pg protein were suspended

in 300 p of 100 mM Tris-HCI buffer containing 5 mM MgC12, 1 mM

EGTA and 0.1% BSA, pH 7.3 at 20"C. The incubation buffer

contained the peptidase inhibitors aprotinin (1000 K.I.U./ml), leupeptin

(1 pg/ml) and pepstatin A (1 pg/ml), to prevent proteolytic

degradation of the peptides or radioligand. For saturation studies,

triplicate samples were incubated for 45 minutes at 20"C with

concentrations of 3H-AVP ranging from 50 pM to 5,000 pM. Non-

specific binding was determined in the presence of 5 pM unlabelled

AVP. Competitive inhibition by various peptides was assessed in the

presence of 1.0 nM 3H-AVP. The incubation was terminated by the

addition of 1 ml ice-cold Tris-buffer and the bound tracer was

rapidly separated from the unbound moiety by centrifugation at 10,000

x g for 2.5 minutes at 4*C. The membrane pellet was dissolved in

100 p of formic acid and transferred to a minivial containing 4 ml of

liquid scintillation fluid (Liquiscint; National Diagnostics).











Radioactivity was determined in an LKB LS counter. Samples were

corrected for variations in efficiency of counting by an internal

program and expressed as disintegrations per minute (dpm). Based on

the radioactivity and protein in each sample, values are reported as

fmoles bound/mg protein. Specific binding was calculated as the

difference between total binding and non-specific binding.

Analysis

Data from saturation and competition experiments were analyzed

using iterative curve fitting programs. Data are reported as the

mean S.E.

Results

The activity of Na+K+-ATPase, a marker enzyme for the

basolateral membrane was markedly enriched in the F1 fraction,

which displayed a 3.25-fold increase in activity over that of the P2

pellet and a 10-fold increase when compared to the homogenate

(Table 2-1). A two-fold enrichment of AVP binding per unit protein

was achieved in the BL membrane preparation compared to the P2

pellet. A tritiated ligand was chosen for these experiments since

iodine destroys the biological activity of the vasopressin molecule

(Dorsa et al., 1983). The binding of 3H-AVP to renal basolateral

membranes was linearly related to the amount of protein (i.e.,

membrane) present in the assay (Figure 2-2). Association and

dissociation of 3H-AVP is illustrated in Figure 2-3. Equilibrium

binding of the ligand occurred within 30 minutes and was stable for

up to 2 hours. Upon equilibrium, binding was shown to be reversible

by the addition of 1 uM AVP. Specific binding, determined in the

presence of an excess amount of AVP was 80-85% of total binding.


















TABLE 2-1.
K+-STIMULATED, OUABAIN-SENSITIVE Na+-K+ ATPase ACTIVITY



Fraction Units*/Mg Protein (x 10-3) Purification



H 0.08 + .04 IX

P2 0.26 + .01 3.25X

FI 0.80 .01 10X



Potassium-stimulated, ouabain-sensitive Na+-K+-ATPase activity.
Activity determined by measuring K+-stimulated, ouabain-sensitive p-
nitrophenylphosphate (PNPP) hydrolysis at pH 7.8. Values are
expressed as units/mg protein, where 1 unit = 1 pmole PNPP
hydrolyzed per minute. H, homogenate. P,, 224,000g pellet. F1,
basolateral membrane fraction. *(1 unit = 10-6 moles/min.)



















100



90



80



70



60



50



40



30



20


100 200 300 400 500
ug Protein










Figure 2-2. 3H-vasopressin binding as a function of basolateral
membrane protein concentration. Receptor binding protocol described
in "Methods" section. Protein concentration determined by the
method of Lowry et al. (1951).




















Association


1 uM AVP Disssociatlon
L


Total


o150

NSB





15 30 s45 0 75 a0 s05 120
minutes
















Figure 2-3. 3H-vasopressin (3H-AVP) binding in renal tubular
basolateral membranes as a function of time. Receptor binding
protocol described in "Methods" section. Association: Total binding
determined at 1 nM [3H-AVP]. Non-specific binding (NSB) determined
in the presence of 1 nM [3H-AVP] + 1 ApM AVP. Total NSB =
Specific, which represents 80-85%. Dissociation: At equilibrium
binding (1 hr), 1 nM [3H-AVP] is dissociated from receptor site by
the addition of 1 pM unlabeled AVP.











Specificity of the receptor for related AVP analogues and non-

related peptides is illustrated in Figure 2-4. The data show that

lysine vasopressin (LVP), a vasopressin peptide differing from AVP by

only one amino acid in the number 8 position, is some 13 times less

effective in displacing the ligand than AVP (IC50: AVP = 0.74 nM vs.

LVP 9.7 nM). Furthermore, the synthetic vasopressin analogue

d(CH2)5D-Ile2-Ile4-AVP, a specific tubular (V2) antagonist is some

3,700 times more effective in displacing the ligand than is the

vascular (V1) antagonist d(Et)2Tyr(Et)D-AVP (IC50: V2 0.0039 nM

vs. V1 = 14.7 nM). At concentrations of 1 pM, oxytocin, angiotensin

II and D-ala-met-enkephalinamide were incapable of displacing even

50% of the ligand. A representative saturation isotherm for 3H-AVP

binding to membranes is depicted in Figure 2-5. Transformation of

the data for Scatchard analysis is also represented in Figure 2-5

(insert). Analysis of the data indicates that the maximal binding

capacity (Bmax) for vasopressin receptors on the membranes is -220

fmol/mg protein and the affinity constant for the receptors is 0.61

nM.

Discussion

Several studies have demonstrated the presence of vasopressin

receptors in the kidney (Campbell et al., 1972; Bockaert et al., 1973;

Dorsa et al., 1983; Stoeckel et al., 1987). The present study reports

the preparation of Na+K+-ATPase-enriched renal tubular epithelial

basolateral membranes and the characterization of 3H-AVP binding in

these membranes. This preparation allows one to separate, by

differential and density gradient centrifugation, tubular epithelial

membranes possessing vasopressin receptors linked to adenylate


































Compound IC50 (nM)
o6




20
V V2 0.0039

-AVP 0.74



asar / i f H e (HAV
S4ox



20



SNG II

.001 01 .1 1 10 100 1000
[Peptldel (nM)







Figure 2-4. Specificity of the vasopressin receptor in renal tubular
basolateral membranes. Competition of 3H-vasopressin (3H-AVP)
binding at 1 nM by vasopressin analogues and unrelated peptides.
Receptor binding protocol described in "Methods" section. V1
antagonist, d(Et)2Tyr(Et)D-AVP; V2 antagonist, d(CH2)5D-Ile2-Ile4-
AVP; AVP, arginine vasopressin; LVP, lysine vasopressin; OXY,
oxytocin; Ang II, angiotensin II; DAME, D-ala-met-enkephalinamide.


































S150
K 0 61 nM
E 3





100 y-






50

r 0.96

50 100 150 200 250
Bound (fmol/mg PI


0.5 1.0 1.5 2.0 2.5 3.0
['H-AVP] (nM)







Figure 2-5. Saturation isotherm and Scatchard plot (insert) of 3H-
vasopressin (3H-AVP) binding in renal tubular basolateral membranes.
Concentration of 3H-AVP ranged from 50 pM to 3,000 pM. Specific
binding determined in the presence of 3 uM unlabeled AVP. Details
described in "Methods" section.











cyclase (V2) from vascular endothelial and glomerular mesangial cells

possessing vasopressin receptors coupled to the hydrolysis of

phosphatidyl inositol (V1). Recent evidence suggests that certain

renal epithelial tissue (LLC-PKI cells) possess both V1 receptors

(Garg et al., 1988) and V2 receptors (Lester et al., 1985).

The basolateral membrane preparation yielded a 3.25-fold

increase in Na+K+-ATPase activity with respect to the P2 pellet and a

10-fold increase compared to the homogenate. Vasopressin binding

sites per unit protein doubled in the basolateral preparation over that

in the P2 fraction. The discrepancy may be explained in terms that

while basolateral membranes may be enriched, not all of the

membranes may possess AVP receptors due to their heterogeneous

distribution along the renal tubule (Morel et al., 1987). Scatchard

analysis revealed that a single class of high affinity AVP receptors is

present on these basolateral membranes. Vasopressin binding was

rapid, reversible, specific and displayed a pharmacological profile

characteristic of V2 (adenylate cyclase-coupled) vasopressin receptors.

The maximal binding capacity determined in the present study (-220

fmol/mg P) is somewhat less than the value of 300 fmol/mg P

obtained by Rajerison et al. (1977) but is in close agreement with the

value of 228 fmol/mg P reported by Cornett and Dorsa (1985). This

variation is not entirely surprising, however, given the differences in

membrane preparation and other variations in the protocol.

In summary, these data demonstrate the presence of high

affinity receptors for arginineg-vasopressin on basolateral membranes

prepared by differential and density gradient centrifugation from rat

renal tubular epithelia. These data are consistent with and









38

complement other studies that have examined the vasopressin-sensitive

adenylate cyclase system in the kidney and its coupling to V2-type

receptors (Roy et al., 1975; Rajerison et al., 1977; Butlen et al., 1978).















CHAPTER 3
DEHYDRATION-INDUCED DOWNREGULATION OF RENAL
TUBULAR VASOPRESSIN RECEPTORS

Introduction

Several endocrine systems act to maintain fluid and electrolyte

balance. Alterations of fluid homeostasis influence plasma levels of a

number of hormones. This is particularly evident during dehydration.

This condition leads to elevated circulating levels of vasopressin

(Aisenbrey et al., 1981; Woods and Johnston, 1983), angiotensin II

(Di Nicolantonio and Mendelsohn, 1986) and endogenous opiate

peptides (Mata et al., 1977). Plasma levels of atrial natriuretic

peptide on the other hand tend to be lower, which is consistent with

the physiological role of this hormone (Ogawa et al., 1987). In turn,

these hormones act upon their respective receptors in proportion to

the response required to re-establish volume homeostasis. Paramount

to this task is the action of arginine vasopressin, exerting its well-

known antidiuretic and vasoconstrictive effects, through interactions

with V2 and V1 receptors, respectively.

Peptide hormone receptors can be regulated by the concentration

of hormone to which they are exposed. This can often lead to a

condition described as "downregulation." This phenomenon results

from withdrawal of cell surface membrane receptors by an

internalization process. Reduced cellular sensitivity to hormonal

stimulation can also occur by an uncoupling of the existing receptors

from the signal-transducing effector system. The most well-











documented examples of these types of regulation come from the

elegant studies by Lefkowitz et al. (1984) involving f-adrenergic

receptors. His group has demonstrated examples of "homologous"

regulation by receptor-specific agonists such as isoproterenol and

"heterologous" regulation, where stimulation by a drug or hormone

leads to a change in sensitivity to another unrelated drug or

hormone. They found that thyroid status could have a profound

effect on both the number and coupling of oc-adrenergic and 8-

adrenergic receptors. Presumably, this control of adrenergic

receptors was exerted at the level of gene transcription. Adrenergic

receptors are by no means unique in terms of their susceptibility to

regulation. Insulin receptors too, undergo homologous regulation by

insulin (Gavin et al., 1974). Insulin receptors also undergo

heterologous regulation, under the action of f-adrenergic agents

(Pessin et al., 1983). Additional examples and more insight into the

phenomena of receptor regulation may be gained from the recent

reviews by Roth and Taylor (1982) and Poste (1984).

The cellular sensitivity to vasopressin can be drastically reduced

following chronic stimulation by the hormone. When the isolated

toad bladder, an epithelial model for the mammalian renal tubule, is

stimulated repetitively with high concentrations of the neuro-

hypophyseal hormone, the osmotic water flow across the bladder

diminishes progressively (Eggena, 1981). Renal medullary plasma

membranes incubated with high concentrations of vasopressin display

reduced hormone-sensitive adenylate cyclase activity (Roy et al.,

1976). Furthermore, vasopressin-elicited desensitization of adenylate

cyclase activity has been shown in renal membranes prepared from











rats injected with pharmacological doses of vasopressin (Rajerison et

al., 1977). AVP-induced downregulation of vasopressin receptors and

a concomitant desensitization of vasopressin-sensitive adenylate

cyclase activity has been demonstrated in a renal epithelial cell line

(Lester et al., 1985).

Thus, vasopressin can affect the cellular sensitivity of its target

tissue by regulating its own receptor. Because dehydration is a

condition characterized by high circulating levels of AVP, the present

study was conducted in order to determine whether vasopressin

receptors are commensurately regulated under conditions of water

deprivation.

Materials and Methods

Animals

Male Sprague-Dawley rats (n=6 per group) weighing 200-250g

were obtained from the Animal Resource Facility at the University of

Florida. Rats were housed individually in 20x18x24 cm wire mesh

cages, exposed to a 12:12 hour light:dark cycle, provided a standard

Purina rat chow diet and allowed free access to tap water. The

dehydrated experimental group was deprived of water for 72 hours

prior to sacrifice.

Chemicals

Tritiated 8-L-arginine [phenylalanine-3,4,5-3H]-AVP ([3H]-AVP)

used in receptor binding studies was purchased from New England

Nuclear Corp. (Boston) with a specific activity of 70 Ci/mmol.

Iodinated vasopressin (1 I-AVP), specific activity of 2200 Ci/mmol,

was also obtained from New England Nuclear for use in the

vasopressin radioimmunoassay (RIA). AVP antibody was purchased











from Amersham (Arlington Hts., IL) while AVP standard was obtained

from Peninsula Labs (Belmont, CA). Other reagents were obtained

from Sigma, unless otherwise indicated.

Membrane Preparation and Plasma Collection

Animals were sacrificed by decapitation and trunk blood was

collected. A 100 pl sample was obtained separately for the

determination of plasma osmolality while the remainder was collected

in chilled tubes containing 0.3 M EDTA (50 pl/ml) for the

determination of plasma AVP concentration. Blood samples were

centrifuged at 3,000 x g for 10 minutes at 4C, the plasma removed

and stored at -20*C. Renal basolateral membranes were prepared by

modifying the density gradient centrifugation method of Boumendil-

Podevin and Podevin (1983), according the description provided in

Chapter 2.

Vasooressin Receptor Assay

Membranes, corresponding to 100-200 pg protein were suspended

in 300 pl of 100 mM Tris-HCI buffer containing 5 mM MgCl2, 1 mM

EGTA and 0.1% BSA, pH 7.3 at 20'C. Triplicate samples were

incubated for 45 minutes at 20*C with concentrations of 3H-AVP

ranging from 50 pM to 5,000 pM. Non-specific binding was

determined in the presence of 5 pM unlabelled AVP. Further details

of the receptor binding protocol are described in Chapter 2.

RIA for Vasopressin

Plasma samples for the determination of AVP were thawed and

purified using SepPak C18 cartridges (Waters). Briefly, the

purification process is as follows: Cartridges are pre-wetted with 5 ml

methanol followed by 5 ml 1% trifluroacetic acid (TFA). A 1 ml











plasma sample is acidified with 1% TFA and applied to the column

dropwise. The cartridge was rinsed with 5 ml 1% NaCI/l% TFA, then

AVP was eluted with 4 ml methanol:H20.TFA (80:19:1). The samples

were evaporated under a light stream of air then reconstituted in 0.5

ml of assay buffer (50 mM phosphate buffer, pH 7.4 containing 0.2%

bovine serum albumin and 10 mM EDTA). The RIA was carried out

by incubating 100 pil samples or known amounts of standard (0.1-20 pg

AVP) with 25 pl AVP antiserum and 25 pl 125I-AVP (corresponding to

3,000 cpm) for 24 hours at 4C. Free ligand was sequestered by the

addition of dextran-coated charcoal and centrifugation at 2,000 x g

for 15 minutes. The supernatant containing the antibody-bound

ligand was aspirated and counted in a Beckman 5500 gamma counter

at an efficiency of 79% for 125I. Values are expressed as pg AVP.

Cross-reactivity with related peptides is <1%. Recovery of AVP for

the extraction procedure was determined to be >90%. Sensitivity of

the assay was 0.15 pg per tube.

Plasma Osmolalitv

Plasma osmolality was determined using a Westcor model 5500C

vapor pressure osmometer. Data are expressed as mosmols/kg H20.

Analysis

Data from saturation and competition experiments were analyzed

using iterative curve fitting programs. Data are reported as the

mean + S.E. Statistical significance of the data was assessed using

one-way analysis of variance and Student's t-test. A level of p<.05

was considered to be significant.









44

Results

The state of hydration of the rats was evaluated by comparing

the urine ouput and osmolality, plasma osmolality and plasma

vasopressin concentration among the two groups. Urine output in the

normally hydrated control group was 10.61.1 ml/24 hr while that of

the dehydrated group was 1.90.8 ml/24 hr (p<.05 vs. control) (Figure

3-1A). Urine osmolality was significantly elevated among the

dehydrated animals (2789169 mosmol/kg) when compared to control

values (1447225 mosmols/kg) (p<.05) (Figure 3-1B). Similarly, plasma

osmolality was raised from control values of 288 0.8 mosmol/kg to

299 1.7 mosmol/kg among the water deprived group (p<.05) (Figure

3-2A). Vasopressin values were determined from the standard curve

shown in Figure 3-3. In response to the heightened osmolality of the

extracellular fluid, plasma vasopressin levels were commensurately

elevated. AVP concentrations were raised 11.5-fold, from a value of

0.210.02 pg/ml in the plasma of the control animals to 2.40.3 pg/ml

in the plasma of the dehydrated animals (p<.05) (Figure 3-2B).

3H-vasopressin binding was determined in renal tubular epithelial

basolateral membranes prepared from the kidneys of the control and

dehydrated animals. A representative saturation isotherm for 3H-

AVP binding is depicted in Figure 3-4. Transformation of the data

for Scatchard analysis is represented in Figure 3-5. Analysis of the

data between the two groups indicates a significant reduction in the

number of vasopressin receptors on the membranes from the

dehydrated animals (Control B = 184+15 fmol/mg protein vs.
max
Dehydrated B = 1142.5 fmol/mg protein) (p<.01). No significant
max
difference was observed in the affinity of the receptor between the


















A

















CONTROL DOMDRATE


CONTROL DEHYDRATED


Figure 3-1. Urine output and urine osmolality in control and
dehydrated rats. A: urine output (Uv). B: urine osmolality (Uosm).
Osmolality determined by vapor pressure osmometry. Error bars
represent S.E.M. *p<.05 vs. control.


































n-6


n-6


Control Dehydrated


n-6


Control Dehydrated


Figure 3-2. Plasma osmolality and plasma vasopressin concentrations
in control and dehydrated rats. A: Plasma osmolality, determined by
vapor pressure osmometry. B: Plasma AVP determined by RIA. Error
bars represent S.E.M. *p<.05 vs. control. AVP, arginine vasopressin;
RIA, radioimmunoassay.


300




E
0
E


S290
E

0

E
o



280





























o 60

50

40

30


0.1 1.0 5.0
pg AVP


Figure 3-3. Standard curve for arginine vasopressin (AVP). Values
determined by radioimmunoassay. Details described in "Methods"
section.



























Control



S150




0
'Z 100 Dehydrated






I ,,o t








I3H]-AVP (nM)







Figure 3-4. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
water-replete and water-deprived (72 hr) rats. Details of binding
protocol described in "Methods" section of Chapter 2. Error bars
represent S.E.M. of triplicate determinations.































* Control


o Dehydrated


Bma(fmoll/mg P) KD (nM)


S 184-15 0.61,.04


: 114-2.5 0.64 .03
(n 5)


3 -







2 2














r- 96


50 100 150 200 250
Bound (fmols/mg Protein)









Figure 3-5. Scatchard analyses of 3H-vasopressin binding. Binding
determined in basolateral membranes prepared from kidneys of water-
replete and water-deprived (72 hr) rats. Analyses of data in Figure
3-4. *p<.05 vs. control.









50

two groups (Control KD = 0.61+.04 nM vs. Dehydrated KD = 0.64+.03

nM).

Discussion

Alterations in fluid balance can affect not only the hormones

themselves which participate in its regulation but also the receptors

which mediate the biological responses of the hormones. Thus,

dehydration causes a reduction in K-opiate receptor binding in the

neurohypophysis (Brady and Herkenham, 1987) and a decrease in

angiotensin II binding in the adrenal medulla (Hwang et al., 1986).

Alternatively, receptors for atrial natriuretic factor are increased in

the kidney and adrenal gland, in response to reduced circulating

levels of the hormone (Lynch et al., 1986).

These data in the present study show that V2-type vasopressin

receptors located on renal tubular epithelial basolateral membranes

are modulated when fluid homeostasis is compromised by dehydration.

A significant reduction in the number of vasopressin receptors (Bax)

was noted in the renal tubular basolateral membranes prepared from

the water-deprived animals. There was no difference between the

two groups with respect to the affinity (KD) of the receptor for the

hormone. Water deprivation for 72 hours significantly elevated

plasma AVP concentrations 11.5-fold. The magnitude of this increase

is comparable to the response observed by Woods and Johnston (1983)

in rats dehydrated for a similar period of time. Thus, it would

appear that the receptors are sensitive to the circulating levels of

vasopressin. This is supported by the study of Shewey and Dorsa

(1986) who reported finding no difference in the level of 3H-AVP

binding in renal medullary membranes prepared from heterozygous











Brattleboro rats and their chronically dehydrated homozygous

counterparts, which are unable to synthesize AVP. This would

suggest that the reduction in vasopressin receptor density observed in

the present study is attributable to the elevated plasma levels of

vasopressin, rather than some nonspecific effect of dehydration.

Homologous downregulation of vasopressin receptors has been

reported in a renal epithelial cell line (Lester et al., 1985) and in the

toad urinary bladder (Eggena and Ma, 1986), following treatment with

exogenous vasopressin. The phenomenon of downregulation results

from an increased rate of internalization when the hormone

concentration has been greatly enhanced (Catt et al., 1979). The

"anatomical correlate" of downregulation has been described by Kirk

(1987). He has elegantly shown by morphometric analysis that

retrieval or internalization of the basolateral membrane occurs in the

cells of the rabbit cortical collecting tubule when induced by a

vasopressin-stimulated transcellular water flow. He suggests that

membrane internalization could reduce the sensitivity of the cortical

collecting tubule cells to vasopressin if AVP receptors are present on

the retrieved membrane. While not addressed in the present study,

the issue of adenylate cyclase responsiveness has been examined in

conjunction with the downregulation of AVP receptors. Rajerison et

al. (1977) showed in the rat that vasopressin-sensitive adenylate

cyclase activity in membrane preparations from the rat kidney medulla

is greatly reduced after the infusion of pharmacological doses of AVP

to the animals.

In the present study, it would seem odd that an animal in dire

need of water conservation would lose AVP receptors as the need











became greater. An explanation for this may be that the kidney

possesses "spare" receptors (Ariens et al., 1960). Baddouri et al.

(1984) noted a downregulation of vasopressin receptors in the kidney

of the water deprived jerboa, Jaculus orientalis. Water-deprived

animals in that study displayed a five-fold elevation in plasma AVP

levels and a 36% reduction in 3H-lysine vasopressin binding to renal

medullary membranes. Despite reduced vasopressin binding, the

kidneys of some of these desert rodents still retained the capacity to

significantly concentrate urine under these conditions. The latter

finding suggests that "spare" receptors may have been involved in the

regulatory phenomenon. A receptor reserve has also been noted in

other adenylate cyclase-linked tissues as well (Flores et al., 1974).

Alternatively, reduced binding without a decrement in biological

responsiveness may occur if those receptors which were

downregulated are "silent", or not coupled to an effector system.

Maack et al. (1987) have recently shown that the overwhelming

majority (>90%) of receptors for atrial natriuretic factor that are

present in the kidney are biologically silent. That is to say, they do

not mediate any of the known renal effects of the hormone. The

authors speculate that these silent receptors may serve as "peripheral

storage clearance binding sites, acting as a hormonal buffer system to

modulate plasma levels of atrial natriuretic factor". The physiological

significance of silent or spare receptors and their regulation remains

to be explored.

In summary, the present study shows that V2 vasopressin

receptors are differentially expressed in basolateral membrane

preparations from the kidneys of water-replete and water-deprived









53

rats. Scatchard analyses indicate that the difference in binding is

due to a reduction of receptor numbers on the membranes prepared

from the dehydrated animals. These results show that AVP receptor

downregulation can occur in response to physiologically elevated

levels of circulating vasopressin. Such a condition could ultimately

affect the antidiuretic action of the hormone.















CHAPTER 4
REGULATION OF VASOPRESSIN BINDING
IN MINERALOCORTICOID-DEPENDENT HYPERTENSION

Introduction

The role of vasopressin in the etiology of hypertension has

received considerable attention, much of it being reviewed recently by

McNeill (1983) and Johnston (1985). The pressor action of vaso-

pressin was the first physiological response attributed to this

posterior pituitary substance (Oliver and Schafer, 1895) and the

vasoconstrictive properties of the hormone are now well-documented

both in vivo (Liard, 1984) and in itro (Altura and Altura, 1984).

One line of evidence which points to a relationship between

vasopressin and high blood pressure is that circulating levels of

vasopressin are elevated in several models of hypertension. Crofton

et al. (1978) noted higher plasma vasopressin concentrations in the

spontaneously hypertensive rat. Similar observations were noted in

renovascular (one kidney,one clip) (Pullan et al., 1978) and volume-

expanded (DOCA-salt) (Mohring et al., 1977; Crofton et al., 1979)

models of hypertension. While the circulating levels of vasopressin

are significantly increased in all of the above studies, the plasma

concentrations are, nonetheless, below the threshold for pressor

activity. This discrepancy can be resolved, however, by invoking

increased vascular reactivity toward vasopressin. Such an enhanced

pressor sensitivity to AVP has been noted in the SHR (Crofton et al.,

1978) and in DOCA-salt hypertensive rats (Crofton et al., 1980).











Added support for the involvement of vasopressin in the pathogenesis

of DOCA-salt hypertension comes from the study of Friedman et al.

(1960). These investigators noted that the administration of AVP

accelerated the development of DOCA-salt hypertension. The most

telling piece of evidence however, which implicated vasopressin in

DOCA-salt hypertension was the finding of Crofton et al. (1979) who

reported that Brattleboro rats, which are unable to synthesize AVP,

failed to develop DOCA-salt hypertension.

The development of selective antagonists to the pressor and

antidiuretic effects of vasopressin greatly facilitated our

understanding of the involvement of vasopressin in hypertension.

Crofton et al. (1979) found that the administration of a selective VI

(vascular) antagonist caused an acute fall in blood pressure in DOCA-

salt hypertensive rats. Similarly, Mohring et al. (1977) were able to

lower the blood pressure by the injection of a highly specific

vasopressin antibody. Hofbauer et al. (1984) attenuated the

development of DOCA-salt hypertension by the simultaneous infusion

of V1 antagonists. Collectively, these studies suggest that

vasopressin may contribute to the development of hypertension by

virtue of its vasoconstrictive properties. Conversely, other

investigators (Rascher et al., 1983; Yamamoto et al., 1984; Filep et

al., 1987) have been unable to reduce the high blood pressure

associated with DOCA-salt hypertension by the administration of VI

blockers.

A renal locus in the pathogenesis of DOCA-salt hypertension has

been largely overlooked until recently. Saito and Yajima (1982)

observed that when Brattleboro rats were treated with DDAVP, a









56

selective tubular (V2) agonist, DOCA-salt hypertension developed. A

similar phenomenon was observed by Woods and Johnston (1981),

using renovascular models of hypertension in Brattleboro rats.

Whereas Hofbauer et al. (1984) attenuated the development of DOCA-

salt hypertension with V1 blockers as noted above, the simultaneous

infusion of a combined VI/V2 antagonist completely abolished the rise

in blood pressure. These studies suggest a tubular site of action for

vasopressin in the development of DOCA-salt hypertension.

The role of the kidney in the pathogenesis of hypertension has

been a major focus of Guyton and his colleagues (Guyton et al.,

1972). The action of vasopressin in this context was underscored in

the findings of Manning et al. (1979) and Cowley et al. (1984b) who

reported that subpressor doses of vasopressin can contribute to a rise

in arterial pressure, which is dependent on the fluid-retaining

properties of the hormone. In addition, it has been shown that

vasopressin stimulates sodium reabsorption in the medullary thick

ascending limb of the mouse (Hebert et al., 1981a) and the cortical

collecting tubule of the rat (Reif et al., 1986). The latter authors

also showed that the effects of vasopressin on sodium reabsorption

are augmented in DOCA-treated rats.

Thus, it appears that there is an interaction between vasopressin

and mineralocorticoids at the level of the renal tubule. Increased

activity of the salt and fluid transport processes would lend itself to

a volume-expanded state, with the potential for raising arterial

pressure. The question of how vasopressin activity is increased in

DOCA-treated animals remains unanswered. This study will address

the question at the cellular level. Vasopressin receptors and











vasopressin-stimulated adenylate cyclase activity will be measured in

basolateral membranes prepared from renal tubular epithelia in normo-

tensive and DOCA-salt hypertensive rats.

Materials and Methods

Chemicals

3H-argininie-vasopressin (3H-AVP), 125I-arginine-vasopressin

(125I-AVP) and 3H-3',5'-cyclic adenosine monophosphate (3H-cAMP)

were purchased from New England Nuclear Corporation (Boston).

Peptides, cAMP, protein kinase, desoxycorticosterone acetate (DOCA),

protein kinase, and the regenerating reagents for adenylate cyclase

stimulation (theophylline, creatine phosphokinase, phosphocreatinine,

ATP and GTP) were all obtained from Sigma Chemical Company (St.

Louis). Vasopresssin antibody was purchased from Amersham

(Arlington Heights, IL) and Liquiscint liquid scintillation fluid from

National Diagnostics (New Jersey).

Animals

Thirty male Sprague Dawley rats weighing 175-225 g were

obtained from the Animal Resource facility at the University of

Florida. Rats were housed in a climate-controlled room and caged

individually in 20x18x24 cm wire mesh cages. They were provided a

standard Purina rat chow diet and allowed free access to tap water.

The rats were divided into five groups containing six rats per group.

Under chloral hydrate anesthesia (0.4 g/kg body weight, i.p.), animals

in Group I were implanted subcutaneously with two silastic capsules,

5 cm long, containing DOCA. They were unilaterally nephrectomized

using a retroperitoneal approach and provided with a 1% NaCI

drinking solution in lieu of water. Group II animals received 1% NaCI











drinking solution and was unilaterally nephrectomized according to

the procedures described above, but received no DOCA implants.

Animals in Group III received DOCA implants, tap water to drink and

their kidneys remained intact. Those in Group IV were implanted

with DOCA capsules, received 1% NaCI to drink, but their kidneys

were left intact. Animals in Group V received no special treatment

and were provided with tap water to drink. Blood pressures were

recorded by tail cuff plethysmography prior to surgery and once

weekly, thereafter. Values are expressed as mm Hg.

Membrane Preparation and Plasma Collection

After four weeks of treatment, animals were sacrificed by

decapitation. Trunk blood was collected in chilled tubes containing

0.3 M EDTA (50 pl/ml blood) for the determination of plasma

vasopressin concentration. Blood samples were processed according to

the procedure described in Chapter 3. Renal basolateral membranes

were prepared by modifying the density gradient centrifugation

method of Boumendil-Podevin and Podevin (1983), according to the

description in Chapter 2. For vasopressin receptor binding assays,

membranes were stored in liquid N2 until use. For adenylate cyclase

measurements, membranes were used immediately.

Vasopressin Receptor Assay

Binding studies were conducted according to the procedure

described in Chapter Two. Briefly, membrane samples were incubated

in triplicate at 20'C for 45 minutes with concentrations of 3H-AVP

ranging from 50 pM to 2,500 pM. Non-specific binding was

determined in the presence of 2.5 pM unlabelled AVP.











Vasooressin Radioimmunoassav

Plasma vasopressin concentrations were determined according to

the method described in Chapter 3.

Adenvlate Cvclase Stimulation

Basal and vasopressin-stimulated adenylate cyclase activity in

renal basolateral membranes was performed according to the following

protocol: Basal samples (membranes corresponding to 100 pg protein)

were suspended in a total volume of 150 1l of 50 mM Tris buffer, pH

7.4, containing 138 pg ATP, 39 pg GTP, 150 pg MgCl2, 38 pg EGTA,

150 pg BSA, and a regenerating system consisting of 228 pg theo-

phylline, 60 pg creatine phosphokinase and 100 fpg phosphocreatine.

Agonist-stimulated samples were prepared as above but were incubated

in the presence of AVP ranging in concentration from 0.1 nM to

10 pM. Triplicate samples were incubated at 37'C for 10 minutes.

The reaction was stopped by the addition of 300 pl of 100 mM Tris

buffer containing 5 mM EDTA, pH 7. The samples were boiled for 5

minutes and then centrifuged at 3,000 rpm for 5 minutes. Super-

natants were removed at stored at -20'C for subsequent determination

of cAMP formation.

cAMP Determination

The cellular cAMP content was determined by the competitive

protein binding assay as modified by Baker et al. (1986). Samples for

cAMP determination were reconstituted in 200 pl of 25 mM Tris

buffer, pH 7.0. 10 pl of each sample were incubated at 4'C for 60

minutes in a total volume of 200 pl Tris buffer containing 25,000 cpm

of 3H-cAMP, 42 pg BSA, 280 pg theophylline and the reaction

initiated by the addition of 24 pg of cAMP-dependent protein kinase.











At the end of the incubation, 70 p1 of a hydroxyapatite suspension

(50% w/v) was added to each tube, vortexed and allowed to sit for

six minutes. The contents of the tube were then filtered under

vacuum through Whatman GF/C filters, then washed with 12 ml of 10

mM Tris buffer at pH 7.0. Filters were removed and transferred to

scintillation vials containing 1 ml of 0.3N HCI. After the hydroxy-

apatite had dissolved (-10 min), 9 ml of liquid scintillation fluid

(Liquiscint) was added and each vial counted. Non-specific binding

was determined in the presence of 10-5M CAMP. The amount of

cAMP present in each sample was calculated from a standard curve

determined with known amounts of unlabeled cAMP.

Analysis

Data from saturation studies were analyzed using iterative curve

fitting programs. Statistical significance was assessed by one-way

analysis of variance. The post-hoc analysis between treatment pairs

was performed by Duncan's method. A "p" value of 0.05 or less was

considered an acceptable level of significance.

Results

The combination of desoxycorticosterone acetate treatment and

increased sodium intake precipitates the development of the well-

characterized syndrome of DOCA-salt hypertension. Figure 4-1

depicts the rapid development of high blood pressure in rats

administered the mineralocorticoid and offered 1% NaCI to drink.

Those animals in group I with a reduced renal mass became

hypertensive most rapidly and attained the highest arterial pressure

(1765 mm Hg). Even those animals with intact kidneys but provided

a similar regimen (Group IV) became hypertensive (1548 mm Hg)
























180
I

170


160
IV

E 150
E

14 -


o 130 -


120 11


110 II
V

100 -

0 1 2 3 4
Time (weeks)






Figure 4-1. Arterial pressure in control and DOCA-treated rats.
Blood pressures were determined prior to experiment and once per
week thereafter by tail cuff plethysmography. Arrow indicates start
of treatment. Error bars represent S.E.M. (n = 6 per group except V,
where n = 4). *p<.05 vs. V and II. See "Methods" section for key to
group designations.











after only 4 weeks of treatment. This contrasts to the animals in

group III who received only DOCA and displayed an arterial pressure

of 1217 mm Hg. This value was not significantly different from the

values observed in animals in Groups V (1088 mm Hg) and II (1106

mm Hg), receiving no treatment or 1% NaCI and unilateral

nephrectomy, respectively.

3H-vasopressin binding in renal epithelial basolateral membranes

prepared from the DOCA-salt hypertensive rats (Group I) and their

appropriate controls (Group II) revealed a higher level of binding in

membranes from the hypertensive group (Figure 4-2). As depicted in

Figure 4-3, Scatchard analysis of these data indicate that this higher

level of binding is due to an increased number of binding sites

(Group I 30729 fmoles/mg protein vs. Group II 17917 fmoles/mg

protein) [p<.05], rather than a change in affinity (Group I = 0.59.11

nM vs. Group II = 0.54.09 nM) [p>.05]. This represents a 71%

increase over control values. Table 4-1 summarizes the binding data

for the other experimental groups. It can been noted that a higher

Bmax is also evident for the hypertensive animals in Group IV

(28133 fmoles/mg protein). This too, is significantly higher than the

values for Group II (p<.05). Furthermore, there were no significant

differences between the values in Group II and the untreated animals

in Group V (Bmax = 19623 fmoles/mg protein; KD = 0.67.12) [p>.05].

Binding (Bmax = 19114 fmoles/mg protein; KD = 0.63.04) in

membranes prepared from the animals receiving DOCA only (Group

III) was not significantly different from either of the control groups

(II or V).


































O- 200
a
E

o o



I
.0


O DOCA NaCI 1K (I)

* ---- NaCI 1K (II)


(3H-AVPI (nM)





Figure 4-2. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Details of binding protocol described in "Methods" section of
Chapter 2. Error bars represent S.E.M. of triplicate determinations.
DOCA, desoxycorticosterone acetate; Na, 1% NaCI; 1K, one kidney.
































S(0) DOCA NaCI 1K (


(6) ---- NSCI 1K (I


Bmax (fmoles/mg P) KD (nM)


I) 307 29 0.59 .11

II) 179 17 0.54 .09
(n-4)













r-0.993


0 100 200 300

Bound (fmoles/mg P)










Figure 4-3. Scatchard analyses of 3H-vasopressin binding. Binding
determined in basolateral membranes prepared from kidneys of DOCA-
salt hypertensive rats and normotensive sham-operated control rats.
Analyses of data in Figure 4-2. *p<.05 vs. control. Abbreviations are
as in Figure 4-2.



















TABLE 4-1.
VASOPRESSIN BINDING IN DOCA-TREATED RATS.


Treatment Bma KD n
Group (fmoles/mg P) (nM)


I 307 29* 0.59 .11 4

II 179 17 0.54 .09 4

III 191 14 0.63 .04 3

IV 281 33* 0.62 t .14 2

V 196 23 0.67 .12 3



* p < .05 vs. Groups II and V









66

Basal and vasopressin-stimulated cyclic AMP formation was

measured in freshly prepared membranes from the various groups.

The values were derived from the standard curve depicted in Figure

4-4. Figure 4-5 represents the production of cAMP in membranes

prepared from animals in the DOCA-salt hypertensive group (I) and

the corresponding control group (II). Whereas basal values did not

differ from each another, membranes from the hypertensive animals

were stimulated to a greater degree than membranes from the control

group. The enhanced sensitivity to vasopressin is evidenced by a

shift toward the left for the curve depicting Group I values.

Subtracting basal values, the calculated ED50's for the hypertensive

and control group are 2x10 M and 8.5xl-10 M, respectively.

Table 4-2 summarizes the basal and vasopressin-stimulated cyclic AMP

production in membranes of the other experimental groups. Basal

values did not differ from one another but a greater level of

stimulation and an enhanced sensitivity toward vasopressin were also

noted in membranes from the hypertensive animals in Group IV.

Finally, plasma vasopressin levels were determined in the

animals of the various groups and those data are presented in Table

4-3. Values for the non-hypertensive animals in Groups II and V

ranged from 1.20.2 pg/ml to 1.80.2 pg/ml, respectively. Plasma

vasopressin concentrations were significantly elevated among the

hypertensive animals. Measured values in Groups I and IV were

6.10.8 pg/ml and 8.44.5 pg/ml, respectively. This represents a five-

fold elevation in plasma vasopressin concentrations between the

hypertensive and normotensive animals.










67














100





so
80
60



S40



20


0 l 1 1 1 ...
1 2.5 5 10 25 50 100 250 500
cyclic AMP (pmoles)




















Figure 4-4. Standard curve for cyclic AMP. Values for cyclic AMP
determined by competitive binding assay, using 3H-cAMP. Details of
procedure described in "Methods" section. cAMP, adenosine 3'5'-
cyclic monophosphate.
























400 0- 0
Oo 0


0



E 300

i:





200
E 0





100
o DOCA NSCI 1K (1)

---- NaCI IK (II)




0 11 10 9 8 7 6 5
[AVPI (-log M)




Figure 4-5. Basal and vasopressin-stimulated cyclic AMP formation.
Cyclic AMP production by basolateral membranes prepared from
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Membrane preparation described in "Methods" section of
Chapter 2. Procedures for the stimulation and determination of
cyclic AMP described in "Methods" section. Abbreviations are as in
Figure 4-2 except AVP, arginine vasopressin; cAMP, adenosine 3'5'-
cyclic monophosphate.









69






TABLE 4-2.
VASOPRESSIN STIMULATED cAMP FORMATION IN DOCA SALT
HYPERTENSIVE RATS




Group Basal Maximal Stimulation IC50
pmoles/10 min./mg P pmoles/10 min./mg P


1 72 14 410 + 20 2xl0-1OM

II 61 + 12 355 + 10 8xl0-1OM

III 64 + 07 360 + 10 7xlO-1OM

IV 65 10 400 + 12 2.5x0l-1OM

V 58 15 345 15 8.5xlO-1OM


















TABLE 4-3.
PLASMA AVP LEVELS IN DOCA-SALT
HYPERTENSIVE RATS


Group [AVP] (pg/ml)


I 6.1 0.8

II 1.8 0.2

III 1.9 0.3

IV 8.4 4.5

V 1.2 0.2









71

Discussion

The role of vasopressin in the development of DOCA-salt

hypertension appears to be unequivocal. Blood pressure will not

increase in this model unless vasopressin is present. This was

demonstrated most elegantly by the use of Brattleboro rats, animals

which are genetically incapable of synthesizing AVP (Crofton et al.,

1979). What is unclear is the mechanism or mechanisms which

underlie the etiology of the blood pressure increase. Various target

tissues have been implicated to account for the hypertensive actions

of vasopressin. A central site of action has been suggested by the

study of Chen et al. (1986). These investigators reported heightened

vasopressinergic activity in DOCA-salt hypertensive rats. This

augmentation in hypothalamic release of vasopressin was exclusive of,

and proceeded the well-known increase in peripheral AVP levels or

elevation of blood pressure. The authors suggested that this

alteration in central vasopressinergic activity may affect blood

pressure by modulation of the cardiovascular centers in the brain.

Studies with vasopressor antagonists suggest a vascular site of action.

Crofton et al. (1979) reported that the acute administration of VI

blockers lowered blood pressure in animals with established DOCA-salt

hypertension. Hofbauer et al. (1984) found that the chronic

administration of V1 blockers during the DOCA-salt regimen

attenuated the development and magnitude of hypertension associated

with this model. Other studies however, tend to refute these

findings (Rascher et al., 1983; Yamamoto et al., 1984; Filep et al.,

1987).









72

A tubular site of action for vasopressin in the development of

DOCA-salt hypertension is suggested by the study of Saito and

Yajima (1982). Using Brattleboro rats, these investigators observed

that DOCA-salt hypertension would develop following the chronic

administration of DDAVP, a V2-specific tubular agonist. These

findings are consistent with the hypothesis of Cowley et al. (1984)

that subpressor doses of vasopressin can contribute to a rise in

arterial pressure, by virtue of the fluid-retaining properties of the

hormone. Indeed, plasma levels of vasopressin tend to be raised in

DOCA-salt hypertension. If the tubular sensitivity to vasopressin is

concommitantly increased, as is the vascular sensitivity (Berecek et

al., 1982), then these processes may lend themselves to the

development of hypertension.

The present study shows that V2 vasopressin receptors in

basolateral membranes prepared from renal tubular epithelia in DOCA-

salt treated rats are significantly elevated relative to tissue from

non-DOCA treated animals. Animals treated with DOCA-salt and

having a reduced renal mass (Group I) displayed a 56% increase in

binding with respect to tissue from untreated animals (Group V) and

a 71% increase compared to uninephrectomized animals receiving 1%

NaCI (Group II). Those DOCA-salt hypertensive rats with intact

kidneys (Group IV) displayed increases of 43% and 56%, respectively.

The present finding that mineralocorticoids modulate the expression

of vasopressin receptors in the kidney is consistent with the study of

Rajerison et al. (1974). These authors observed that adrenalectomy

resulted in a reduction of 3H-lysine vasopressin binding in a renal

medullary membrane preparation. A concomitant lowering of











vasopressin-stimulated adenylate cyclase activity was also noted.

Replacement therapy with aldosterone restored the hormone binding

capacity and adenylate cyclase activity to control levels. In the

present study, vasopressin-stimulated cAMP production was

commensurately increased in membranes prepared from the DOCA-salt

hypertensive rats. Furthermore, the effective concentration which

caused half-maximal stimulation (ED50) was reduced. Thus, an

increased sensitivity to vasopressin is suggested on the basis of

adenylate cyclase activity, although no change in the affinity

constant of the receptor was observed. Using microdissected renal

tubules, Pettinger et al. (1986) have observed a similar increase in

the sensitivity of adenylate cyclase in response to vasopressin in

tubules obtained from DOCA-salt treated rats. This response was

specific for vasopressin and occurred only in the segment of the

cortical collecting tubule. The phenomenon was observed as early as

five days post-treatment, a period which preceded the elevation of

blood pressure.

The mechanism of vasopressin receptor up-regulation associated

with DOCA-salt hypertension is not clear. It might have been

expected that vasopressin binding would decrease in the face of

elevated circulating levels of AVP, as was observed in Chapter 3.

However, the magnitude of change in plasma vasopressin levels in the

present study was only half of what occurred with dehydration.

Clearly, factors other than homologous regulation must be invoked.

As a steroid, DOCA probably acts by increasing the transcriptional

rate for the vasopressin receptor protein. Increased expression of

other tubular basolateral membrane proteins is also observed with











DOCA treatment. Garg et al. (1981) reported that Na-K-ATPase

activity was markedly elevated in the cortical collecting duct of

rabbits treated with DOCA. Similar results were observed by El

Mernissi et al. (1983) and Mujais et al. (1985). Mineralocorticoids

can affect the expression of receptors in other tissues as well.

Wilson et al. (1986) observed a greater density of angiotensin II

receptors in brain tissue. Angiotensin II receptors are also increased

in vascular smooth muscle tissue in DOCA-salt hypertensive rats

(Schiffrin et al., 1983). These findings correlate quite nicely with

the increased dipsogenic and pressor responsiveness to Ang II in

DOCA-salt treated animals. Not all receptors are modulated by

mineralcorticoids, however, Alpha2-adrenergic receptors were not

significantly altered in membrane fractions prepared from DOCA-salt

hypertensive and normotensive rats (Fukuda et al., 1983). These

authors did observe differences however, in 3H-yohimbine binding

between spontaneously hypertensive rats and the their normotensive

counterparts, Wistar-Kyoto rats.

How might an up-regulation of renal tubular vasopressin

receptors contribute to the development of DOCA-salt hypertension?

Vasopressin regulates fluid reabsorption in the distal nephron and also

stimulates sodium reabsorption in at least two distinct sites: the

medullary thick ascending limb (Hebert et al., 1981a) and in the

cortical collecting tubule (Reif et al., 1986). A relationship between

vasopressin-stimulated cAMP production and Na-K-ATPase activity,

the enzyme involved in active transport of sodium, has been

demonstrated by Tomita et al. (1987). As shown in the present study,

the increased density of AVP receptors appears to render the tubule









75

more sensitive to vasopressin, reflected by the enhanced AVP-

stimulated production of cAMP. Reif et al. (1986) have shown that

chronic DOCA treatment enhances vasopressin-stimulated sodium

reabsorption in the cortical collecting tubule. Treatment with DOCA

also increases Na-K-ATPase activity in distal segments of the nephron

(Garg et al., 1981). Thus, the increased concentration of plasma

vasopressin noted in the present study coupled with an enhanced

sensitivity to AVP in renal tubules may lead to an exaggerated

retention of salt and fluids, resulting in an expanded blood volume.

Extracellular volume expansion has been noted in at least some stage

of the development of DOCA-salt hypertension (Villamil et al., 1982;

Tajima et al., 1983).

Taken together, the present findings and the above studies

suggest that the development of DOCA-salt hypertension may be

caused, in part, by an increased tubular sensitivity to vasopressin.

Increased activity of vasopressin-mediated processes would lend itself

to a volume-expanded state, a condition necessary to prevent hyper-

natremia. Accordingly, the resultant blood pressure elevation could

be viewed as a compensatory mechanism in response to an increase in

cardiac output, secondary to hypervolemia and aimed at the

restoration of fluid and electrolyte balance.















CHAPTER 5
EFFECTS OF MINERALOCORTICOIDS ON VASOPRESSIN
RECEPTORS AND ADENYLATE CYCLASE-COUPLED RESPONSES
IN LLC-PK1 CELLS

Introduction

Although vasopressin can act on the kidney and affect renin

secretion, glomerular filtration and tubular transport, a detailed

understanding of how AVP or other hormones affect these processes

at the cellular level has been difficult to ascertain. In large part,

this is due to the complexity of the kidney and the veritable myriad

of potential target tissues. One attempt to solve this problem has

been to study the action of vasopressin on isolated glomeruli,

vascular tissue or discretely dissected tubular segments. While

somewhat more refined than an isolated whole kidney, this approach

still ignores the heterogeneity of cell types within anatomically

distinct regions. In order to circumvent these potential problems,

one technique that has been applied to answer many of the questions

concerning the cellular actions of vasopressin in the kidney is cell

culture. The use of primary cultures and cell lines has been utilized

successfully in a number of studies, ranging from transport physiology

to hormonal regulation. Handler (1986) has recently written an

elegant review on the use of kidney cells in culture and succinctly

summarizes the wide-ranging studies amenable to these techniques.

The primary objective for using cell culture techniques in this

study was to establish an in vitr system in which to evaluate the

effects of mineralocorticoids on vasopressin binding, in order to











compare these results with those observed in vivo that are associated

with mineralocorticoid-dependent hypertension. To this end,

vasopressin binding was characterized in LLC-PKI cells (ATCC # CL

101), a continuous cell line of renal epithelial origin, established by

Hull et al. (1976).

Materials and Methods

Chemicals

Tritiated 8-L-arginine [phenylalanine-3,4,5-3H]-vasopressin (3H-

AVP) and tritiated 3',5'-cyclic adenosine monophosphate (3H-cAMP)

were purchased from New England Nuclear Corporation (Boston).

Desoxycorticosterone acetate (DOCA), arginine and lysine vasopressin

(AVP, LVP), oxytocin (oxy), angiotensin II (ang I), D-ala-met-

enkephalinamide (DAME), cAMP and cAMP-dependent protein kinase

were all purchased from Sigma Chemical Co. (St. Louis). Dulbecco's

Modified Eagle's Medium and fetal bovine serum were obtained from

GIBCO (Long Island, NY). All other reagents were obtained from

Sigma, unless otherwise indicated.

Cell Culture

LLC-PKI cells (ATCC # CL 101) were routinely grown as mono-

layers in plastic 75 cm2 Corning flasks using Dulbecco's Modified

Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum

(FBS), streptomycin [100 jg/ml], penicillin [100 units/ml], and

NaHCO3 [44 mM]. Cell cultures were maintained at 37'C in an

atmosphere of 95% 02 and 5%C02. Dome formation typically appeared

after several days of growth. Dome formation in epithelial cell

cultures is acceptable presumptive evidence of the following processes

that are required for its expression: functional plasma membrane











polarization, formation of occluding junctions and vectorial trans-

epithelial active ion transport (Lever, 1985). At confluence, cells

were subcultured as follows: the medium was removed and the cells

washed with once with trypsin-EDTA solution (0.05% trypsin, 0.2%

EDTA) for 10-15 minutes. The cells were centrifuged (500 x g, 5

min), resuspended in DMEM + 10% FBS and plated at a density of 2-

2.5 x 104 cells/cm2 in flasks as above for continued passage or in 35

mm plastic Falcon petri dishes and utilized upon achieving confluence.

Vasopressin Receptor Binding Assay

The vasopressin binding assay was carried out directly in the

dish, on the monolayer. Twenty four hours prior to the experiment,

the growth medium was removed and replaced with serum-free DMEM.

At the time of the experiment, the medium was aspirated from the

plates and the monolayer washed once with phosphate-buffered saline

(PBS). PBS was composed of NaCI (8g/L), KCI (0.2g/L), Na2HP04

(1.15g/L) and KH2PO4 (0.2g/L). Binding was initiated by the addition

of 0.5 ml of the reaction buffer (PBS containing 0.8% bovine serum

albumin, I Jg/ml aprotinin, I fg/ml pepstatin A, I pg/ml leupeptin,

pH 7.2) containing 3H-AVP. Non-specific binding was determined in

the presence of a 1000-fold excess of non-radioactive AVP. Studies

were conducted in triplicate using 3H-AVP at a concentration of 2.5

nM, for 30 minutes at 20C, unless indicated otherwise and repeated

at least once. For dissociation studies, the reaction medium was

removed after 60 minutes and replaced with buffer containing 2.5 pM

AVP. Saturation studies were performed with concentrations of 3H-

AVP ranging from 0.5-20 nM. Binding was terminated by rapidly

aspirating the reaction mixture from the cells and washing twice with











ice-cold PBS. The cells were dissolved by the addition of 0.5 ml

0.1N NaOH, scraped free from the plate with a rubber policeman,

transferred to a vial containing 10 ml of liquid scintillation fluid

(Liquiscint), and counted for 5 minutes in an LKB liquid scintillation

counter.

Stimulation of Adenvlate Cvclase

Twenty four hours prior to the experiment, the growth medium

was removed and replaced with serum-free medium. At the time of

the experiment, the medium was aspirated and the cells washed once

with PBS. The "control" buffer consisted of serum-free DMEM

containing 11 g/ml isobutyl-methyl xanthine (IBMX), a phospho-

diesterase inhibitor and 0.1 mg/ml aprotinin, a serine protease

inhibitor. The "stimulation" buffer consisted of the components

described above, plus AVP in concentrations ranging from 1 nM to

1 pM. One ml of the control or stimulation buffer was added in

triplicate to the plates and incubated at 37"C for 20 minutes. The

reaction was terminated by rapidly aspirating the reaction mixtures,

washing the monolayer once with PBS and adding 1 ml of boiling IN

HCI. The cells were scraped with a rubber policeman and the

contents transferred into a 4 ml polypropylene tube with a Pasteur

pipette. Samples were centrifuged at 3000 rpm for 5 minutes. The

supernatants were removed and blown to dryness under a stream of

air, before being stored at -20"C, for subsequent determination of

cAMP formation. The pellets were saved for protein determination,

according to the method of Lowry et al. (1951).











cAMP Determination

The cellular cAMP content was determined by the competitive

protein binding assay as modified by Baker et al. (1986), as described

in detail in Chapter 4.

DOCA Treatment of Cell Cultures

Desoxycorticosterone acetate (DOCA) was dissolved in

dimethylsulfoxide (DMSO) at a concentration of 1 mg/ml. This stock

concentration was initially diluted with PBS and subsequent dilutions,

ranging from 1 pg/ml to 10 pg/ml were made in serum-free DMEM.

The solutions were sterilized by passing them through 0.2 micron

mesh Nalgene filters. Growth medium was aspirated off of the cells

and replaced with DMEM containing DOCA. Cells were treated for 1-

24 hours with DOCA concentrations ranging from 1 pg/ml to 1 pig/ml.

Results

Binding of 3H-AVP to monolayers of LLC-PKI cells increased

rapidly over time and attained equilibrium after 45 minutes. At this

time, specific binding, represented -75% of total 3H-AVP bound.

Upon equilibrium, binding was shown to be reversible by the removal

of the radioligand and the addition of buffer containing unlabeled

vasopressin. As with association, dissociation was rapid and reached

levels equivalent to non-specific binding after 45 minutes (Figure 5-

1). The specificity of the receptor was determined by competition of

3H-AVP binding with a variety of related and unrelated peptides.

Lysine8-vasopressin and arginine8-vasopressin were equally effective

(IC50 = 2.1 nM) in terms of their ability of displacing the radioligand

from the binding sites on the cells. On the other hand, oxytocin,

angiotensin II and D-ala-met-enkephalinamide were ineffective even at


































50


NSB

25


S Association Dissociation


10 20 30 45 0 10 20 30 45 60
Time (min)














Figure 5-1. 3H-vasopressin (3H-AVP) binding in LLC-PKI cells as a
function of time. Receptor binding protocol described in "Methods"
section. association: Total binding determined at 2.5 nM [3H-AVP].
Non-specific binding (NSB) determined in the presence of 2.5 nM [3H-
AVP] + 2.5 pM unlabeled AVP. Total NSB Specific (75%).
Dissociation: At equilibrium binding (1 hr), total 3H-AVP binding is
dissociated from receptor by the addition of 2.5 pM unlabeled AVP.











the highest concentration (10-6 M) in displacing 3H-AVP (Figure 5-2).

Using increasing concentrations of 3H-AVP, binding sites on the

surface of the cells appear to be saturable (Figure 5-3). Scatchard

analysis of the saturation isotherm data reveals a single class of high

affinity binding sites (KD = 2.98 nM). This is in good agreement

with the value of 2.1 mM observed for the IC50 for vasopressin. The

maximal binding capacity (Bmax) was calculated to be 155 fmoles/mg

protein (Figure 5-3, insert). Vasopressin stimulated cAMP

accumulation in a dose-dependent manner with LLC-PKi cells (Figure

5-4) which was abolished by pre-treatment with V2 antagonists.

Treatment of the cell cultures with DOCA, either over varying

periods of time or with varying concentrations, failed to alter the

binding of 3H-AVP (Figure 5-5). While binding was significantly

reduced at 1 Mg/ml DOCA, this effect can be attributed to the "toxic"

effect of the mineralocorticoid, as cell viability was adversely

affected. Basal and vasopressin-stimulated cyclic AMP formation in

LLC-PK1 cells were not significantly affected by DOCA treatment

(Figure 5-6).

Discussion

The structural and functional heterogeneity of the kidney is not

particularly amenable to the study of hormonal action at the cellular

level. One attempt to resolve this dilemma has been the use of

tissue cultures of specific cellular elements in the kidney. These

techniques have facilitated our understanding of vasopressin action at

targets as diverse as glomerular mesangial cells (Bonventre et al.,

1986) and tubular epithelial cells (Tang and Weinberg, 1986). In the

present study, the interaction of vasopressin with its receptor was









83










100



80

80 -



/ LVP o

60 / AVP *
OXY *

ANG II o

40 DAME *






20






10 9 8 7 6

[Peptide] (-log M)






Figure 5-2. Specificity of vasopressin receptor in LLC-PK5 cells.
Competition of 3H-vasopressin (3H-AVP) binding with vasopressin
analogues and unrelated peptides. Receptor binding protocol
described in "Methods" section. Abbreviations are as in Figure 2-4.
































S 6

o100 5 I KD 2.98 nM

4




50 I

0 94

50 100 150
Bound (fmoilmg P


5 10 15 20
13H-AVPI (nM)


















Figure 5-3. Saturation isotherm and Scatchard plot (insert) of 3H-
vasopressin (3H-AVP) binding in LLC-PKi cells. Concentration of
3H-AVP ranged from 500 pM to 20,000 pM. Specific binding was
determined using a thousand-fold excess of unlabeled AVP. Details of
the binding procedure are described in the "Methods" section.























2000


1500-


10001


I I I I I I I l l i j i


Basal 9 8 7

[AVPI (-log M)


6 6 + V V2
blocked blockI


Figure 5-4. Basal and vasopressin-stimulated cyclic AMP formation in
LLC-PKi cells. Procedures for the stimulation and determination of
cyclic AMP are described in the "Methods" section. AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate; V, blocker,
d(CH,)sD-Ile'-tle4-AVP.

































80


o0

S
40


S2 1 4 24 1 4 24 1 4 224 1 4 24 4 24

hr hr hr hr hr hr hr hr hr hr hr hr hr hr hr hr hr hr
0 ,- I- I I I L-__L- I _
Control 1 pg/ml 10 pg/ml 100 pg/ml 1000 pg/ml 1 jg/ml

[DOCAl




















Figure 5-5. Effect of DOCA on 3H-vasopressin (3H-AVP) binding in
LLC-PKI cells. Cells were treated for 1-24 hours with DOCA ranging
in concentration from 1 pg/ml to 1 pg/ml. Binding was assessed at
3.0 nM [3H-AVP], with details provided in the "Methods" section.
DOCA, desoxycorticosterone acetate. *p.05 vs. control.





























1s500


10001-


B Basal

S Stimulated (10 nM AVPI


Control


10
DOCA (pg/mil


100 1000


Figure 5-6. Effect of DOCA on basal and vasopressin-stimulated
cAMP production in LLC-PK1 cells. Basal cAMP was measured in the
absence of AVP. Stimulated values were assessed in the presence of
10 nM AVP. Details of procedures are found in the "Methods"
section. DOCA, desoxycorticosterone acetate; AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate.









88

examined in a homogeneous population of tubular epithelial cells and

the influence of mineralocorticoids on AVP binding and adenylate

cyclase activity was determined.

Binding of 3H-arginine vasopressin to LLC-PK1 cells was rapid,

reversible and specific. The receptor displayed a high affinity (2-3

nM) for arginine vasopressin and the closely related moiety, lysine

vasopressin, but for no other peptide, including oxytocin. This

finding differs somewhat from that reported by Roy and Ausiello

(1981a) who found that the IC50 for AVP was more than an order of

magnitude greater than that for LVP. This may be due to differences

in the radioligands used between the two studies. Roy and Ausiello

utilized a tritiated lysine vasopressin compound with a specific

activity of 8.5 Ci/mmol, whereas the tritiated vasopressin analogue

used in the present study contained arginine in the number 8 position

of the peptide and had a specific activity eight times greater (70

Ci/mmol). Variability in the phenotypic expression of the cells from

passage to passage must also be considered when interpreting any

result. Scatchard analysis performed on the saturation data in the

present study revealed a single class of binding sites with an

apparent Bmax of 155 fmol/mg protein. This finding too differs from

those of Roy and Ausiello (1981a). They reported a nonlinear plot,

indicative of heterogeneity in the population of binding sites.

Comparison of the maximal binding capacities for the high affinity

sites in each of the studies is difficult, as Roy and Ausiello (1981a)

expressed their data on the basis of cell number, rather than per mg

of protein. However, the Bm of 155 fmols/mg protein calculated in
max









89

the present study is in close agreement with the value of 166

fmoles/mg protein, as determined by Jans et al. (1987).

Roy and Ausiello (1981b) found that cells suspended in

hypertonic saline or treated with EDTA exhibited a homogeneous

population of binding sites. They proposed a hypothesis for

vasopressin-receptor interactions involving a hormone-induced

alteration in binding affinity or so-called "receptor transition". In

other words, rather than postulating two independent binding sites, as

a nonlinear Scatchard plot might suggest, they believe in an

interconversion of receptor states of different affinity. Why a

similar situation was not observed in the present study is unclear.

The binding sites characterized in the present study apparently

represent vasopressin receptors, as vasopressin was shown to

stimulate cAMP production in a dose-dependent manner. Agonist-

induced stimulation resulted in a sixteen-fold increase over basal

levels. This effect was completely abolished by pretreatment of the

cells with a V2 antagonist. The values for basal and AVP-stimulated

cAMP levels are in good agreement with those reported by Lester et

al. (1985) and Skorecki et al. (1987).

The synthetic mineralocorticoid DOCA, had no demonstrable

effect on either vasopressin binding or cAMP production in LLC-PKI

cells in this study. This contrasts with the observations noted in

vivo, as described in Chapter 4. In that study, a significant increase

was found in the vasopressin-stimulated adenylate cyclase activity and

number of vasopressin binding sites on tubular epithelial membranes

prepared from the kidneys of rats chronically treated with DOCA and

NaCI.









90

It may be appropriate to consider several possibilities to account

for the lack of mineralocorticoid effect noted in the LLC-PK) cells.

The most obvious explanation is that this particular cell line does not

possess receptors for mineralocorticoids, whereas cytosolic and

nuclear binding sites for mineralocorticoids have been reported for A6

cells (Handler et al., 1981) and MDCK cells (Luden et al., 1978). No

such reports have appeared in the literature for LLC-PKI epithelial

cells. This is consistent with the observation that LLC-PKI cells,

unlike the A6 and MDCK epithelial cell lines, are relatively refractory

to the effects of aldosterone, at least with respect to transepithelial

Na+ transport (Meier and Insel, 1985).

Perhaps the most pertinent evidence concerning the lack of

effect of mineralocorticoids in LLC-PKI cells comes from a study by

Roy et al. (1980). These investigators examined the regulation of

vasopressin receptors in LLC-PKIL cells, a subline of the parent

LLC-PK1 strain. These cells are characterized by possessing only 5%

as many vasopressin receptors as the parent cell line. They reported

that fetal bovine serum or insulin could markedly stimulate the

expression of receptors in these cells. In contrast, thyroid hormone,

dexamethasone and aldosterone were ineffective in evoking a change

in receptor number.

Alternatively, methodological considerations must not be

overlooked. DOCA was only soluble in organic solvents such as

absolute ethanol or DMSO. Dilution of the DOCA-containing solvents

with aqueous buffers often resulted in the precipitation of the

steroid. Thus, it is impossible to say with any degree of certainty

exactly how much DOCA was applied to the cell cultures. On the









91

other hand, at high doses (>1 pg/ml), viability of the cells became a

significant factor. One study reported that 3H-DOCA receptor

binding was inhibited by a broad class of protease inhibitors (Baker

and Fanestil, 1977). Whether mineralocorticoids exert any

physiological effects in LLC-PK1 cells remains in question, but it is

entirely possible that the use of protease inhibitors in the present

study may have antagonized the effect of the steroid.

Thus, vasopressin receptors are expressed in vivo on renal

tubular epithelial membranes and in vitro on renal epithelial cells.

Both receptors are coupled to adenylate cyclase. In addition, both

receptors are susceptible to regulation by vasopressin (Chapter 3 and

Lester et al., 1985). It appears however, that the receptors respond

differentially to the effects of mineralocorticoids. The question of

whether mineralocorticoids directly affect the expression of

vasopressin receptors at the cellular level remains unanswered using

LLC-PKI cells as a model. The question may be more suitably

addressed using a primary culture of epithelial cells prepared from rat

collecting tubules.




Full Text
3
a few milliosmoles, which represents a change of only 1%, can effect
the
release
of
vasopressin. Ten years
later,
experiments
conducted
by
Jewell
and
Verney (1957) localized
the
osmoreceptors
to the
basomedial forebrain. The exact location of Verneys osmoreceptors
has been the subject of considerable controversy. While some have
claimed that the magnocellular neurons of the PVN and SON are
themselves osmosensitive (Leng, 1980), it is now believed that
osmosensitive cells within the organum vasculosum of the lamina
terminalis may account for the osmotically-induced alterations in
vasopressin secretion (Thrasher et al., 1982). This same group has
also laid to rest the argument over whether the mechanism for the
control of vasopressin release involves osmosensitive neurons or
sodium
receptors
located on
the cells within
the
organum
vasculosum
laminas
terminalis
(Thrasher
et al., 1980).
The
neural
connections
which relay information between cells in this circumventricular region
and the PVN and SON have been described by Miselis (1981).
Non-osmotic control of vasopressin release is probably more
important for cardiovascular regulation. Thus, hypovolemia,
hemorrhage or decreased cardiac output will elicit the secretion of
AVP, for the purpose of maintaining circulatory homeostasis. This
type of control arises from high pressure baroreceptors in the carotid
sinus and aortic arch and low pressure volume receptors in the left
atrium and pulmonary veins. The afferent impulses of this
neurohumoral arc are carried by the ninth and tenth cranial nerves
to their respective nuclei in the brainstem. Multisynaptic pathways
then transmit this information to the PVN and SON of the


8
of extravascular volume and exerts hemodynamic effects even at
subpressor doses (Cowley et al., 1984b). Guyton and others propose
that the antidiuretic action of the hormone can even produce
hypertension under certain conditions (Manning et al., 1979). The
cellular basis for this phenomenon is alluded to in Chapter 4 of this
dissertation.
Vasopressin has been shown to exert direct negative chrono
tropic and inotropic effects in vitro (Nakashima et al., 1982). The
physiological significance of these direct cardiac effects remains in
question as exceedingly high concentrations of vasopressin were
necessary to elicit the observed effects.
Vasopressin is a potent vasoconstrictor, in vivo and in vitro. In
vivo, administration of vasopressin leads to an increase in total
peripheral resistance from the vasoconstriction of a variety of
vascular beds. The skin, muscle and splanchnic circulations are the
most sensitive, followed by the mesenteric and coronary beds. The
renal, hepatic and cerebral circulations are unaffected by A VP (Liard,
1984). The vasoconstrictive potencies of several pressor substances
and a number of vasopressin analogues has recently been evaluated in
an isolated smooth muscle preparation and AVP was determined to be
the most potent agent tested, although the in vitro activity did not
exactly parallel the in vivo activity (Altura and Altura, 1984).
However, when vasopressin is infused into conscious intact animals,
blood pressure does not normally rise unless exceedingly high plasma
levels of vasopressin are attained (>100 pg/ml). Although A VP-
induced elevations in total peripheral resistance might occur, arterial
pressure is not drastically affected because it is strongly buffered by


15
have also been noted. Lesions of the PVN prevent the elevation in
arterial pressure and heart rate associated with aortic baroreceptor
deafferentation (Zhang et al.t 1983). Employing electrophysiological
techniques, Ciriello and Calaresu (1980) have shown that the NTS and
the dorsal motor nucleus of the vagus relay cardiovascular
information
directly to the
PVN.
In the same
study
it was also
shown
that
electrical stimulation of the PVN lead
to an
increase in
arterial
pressure and heart
rate and inhibited the
reflex
bradycardia
evoked
by
stimulation of
the
carotid sinus nerve.
Conversely,
destruction
of the Aj region, the
group of noradrenergic
cell bodies
thought
to
tonically inhibit
AVP
secretion, results
in a
condition of
fulminating hypertension, marked by high levels of vasopressin in the
plasma (Blessing et al., 1982).
Thus, a feedback loop appears to exist between the PVN and the
NTS which serves to modulate the ability of the baroreflex to
regulate arterial pressure. Since the NTS is the primary afferent
relay for baroreceptor fibers and the dorsal motor nucleus of the
vagus contains vagal cardioinhibitory preganglionic neurons,
projections to these structures from the PVN appear to participate in
the central control of cardiovascular regulation. Vasopressin also
appears to influence other sites in the central nervous system
involved in circulatory control, such as the locus coeruleus. A
vasodepressor function has been attributed to the action of
vasopressin in the dentate gyrus of the hippocampus (Versteeg et al.,
1984).


CHAPTER 1
BACKGROUND
Arginine**-vasopressin (AVP) is an ancient hormone. It is one of
the most phylogenetically conserved, presumably because of its impor
tance for water balance in early invertebrates (Sawyer, 1977).
Scientifically, it is an old hormone. Oliver and Schaefer (1895) first
described the rise in systemic blood pressure following the infusion of
a whole pituitary extract. The renal antidiuretic actions of the
hormone were first noted by Starling and Verney (1924). A wealth of
information has accumulated on the physiological actions of
vasopressin since these pioneering studies. It is now clear that
vasopressin not only serves as the principal hormone governing fluid
homeostasis, thereby requiring the antidiuretic hormone (ADH)
nomenclature, but also plays a role in cardiovascular control. In
addition, various central nervous system (CNS) effects have been
ascribed to the hormone.
Synthesis, Transport and Release of Vasopressin
Arginine** vasopressin
and
oxytocin
both
are closely
related
nonapeptides, characterized
by
cysteine
residues
linked by
disulfide
bridges. Often classified as posterior pituitary hormones, the
hormones are actually produced within specialized neurons in the
hypothalamus. Whereas the biologically active molecules are small
molecular weight peptides, they are synthesized, transported and
stored in secretory granules in association with specific carrier
proteins or neurophysins. Sachs and Takabatake (1964) first
I


Ill
profound physiological consequences. These range from diabetes
insipidus, a condition characterized by an inability to synthesize
vasopressin and manifested clinically by polydipsia and polyuria, to
the syndrome of inappropriate ADH secretion wherein circulating
levels of vasopressin are dramatically elevated, promoting fluid
retention and often resulting in hyponatremia.
Conversely, the biological activity of vasopressin can be affected
by modifying the sensitivity of the target tissue to the hormone.
AVP initiates a biological response, be it central, renal, vascular or
otherwise, by interacting with plasma membrane-bound receptors
located on the cells of the target tissue. These receptors are
integral proteins which transduce the principal (hormone) signal into
a cellular response
by activation
of
"second messenger"
systems.
Currently, vasopressin
receptors
have
been
classified
into two sub-
types (Michell et al.,
1979).
The
Vl
receptor is
coupled
to the
activation of phosopholipase C, resulting in the hydrolysis of
phosphatidyl inositol and the generation of inositol phosphates (IP)
and diacylglycerol (DG). DG is an endogenous activator of protein
kinase C, an enzyme which causes the phosphorylation of numerous
cells proteins. IP acts to cause the release of calcium from intra
cellular stores, which in turn, can affect the activity of calcium-
dependent enzymes (Berridge, 1987). Vj receptors have been
characterized in smooth muscle tissue where they mediate the
vasoconstrictive actions of the hormone. In addition Vj sites have
also been reported on glomerular mesangial cells and in gonadal, liver
and adrenal tissue (Chapter 1). In addition, it now apprears that Vj-
type receptors are also present in epithelial cells of renal origin


115
findings indicate that transporting-epithelial tissue in the kidney
display an increased sensitivity toward vasopressin, as indicated by
the enhanced formation of A VP-stimulated cyclic AMP. The increased
fluid and sodium-retaining properties of the hormone lend themselves
to the development of hypertension, exclusive of its vasopressor
activity. The
role of
the
kidneys
in the pathophysiology
of
hypertension is
supported
by
Guyton
et al. (1972) and
the
observations of Manning et al. (1979) and Cowley et al. (1984).
Regulation of vasopressin receptors in vitro has been reported
by Roy et al. (1980) and Lester et al. (1985). The lack of a
demonstable effect of DOCA in LLC-PK] cells in the present study
may reflect an absence of receptors for mineralocorticoids in this cell
line. This is supported by the findings of Roy et al. (1980) who were
unable to modulate receptor expression by aldosterone in LLC-PK jl
cells, a subline of the parent LLC-PK j strain. This contrasts with
the ability of mineralocorticoids to exert effects on physiological
processes in A^ and MDCK epithelial cell lines (Meier and Insel,
1985). Methodological considerations should not be overlooked
however, when interpreting the results of DOCA-treated LLC-PK \
cells.
Perhaps the most exciting finding in this series of studies is
that vasopressin is coupled to two distinct receptor subtypes in LLC-
PK i cells. This exciting discovery would redefine the long-held
dogma that VI receptors are present on a variety of tissues, but that
transport-epithelia possess only V2 receptors, linked to adenylate
cyclase. The present study demonstrated that indeed, vasopressin-
stimulated cyclic AMP formation does occur in these cells. By


37
cyclase (V2) from vascular endothelial and glomerular mesangial cells
possessing vasopressin receptors coupled to the hydrolysis of
phosphatidyl inositol (V[). Recent evidence suggests that certain
renal epithelial tissue (LLC-PK] cells) possess both V[ receptors
(Garg et al., 1988) and V2 receptors (Lester et al., 1985).
The basolateral membrane preparation yielded a 3.25-fold
increase in Na+K+-ATPase activity with respect to the P2 pellet and a
10-fold increase compared to the homogenate. Vasopressin binding
sites per unit protein doubled in the basolateral preparation over that
in the P2 fraction. The discrepancy may be explained in terms that
while basolateral membranes may be enriched, not all of the
membranes may possess AVP receptors due to their heterogeneous
distribution along the renal tubule (Morel et al., 1987). Scatchard
analysis revealed that a single class of high affinity AVP receptors is
present on these basolateral membranes. Vasopressin binding was
rapid, reversible, specific and displayed a pharmacological profile
characteristic of V2 (adenylate cyclase-coupled) vasopressin receptors.
The maximal binding capacity determined in the present study (~220
fmol/mg P) is somewhat less than the value of 300 fmol/mg P
obtained by Rajerison et al. (1977) but is in close agreement with the
value of 228 fmol/mg P reported by Cornett and Dorsa (1985). This
variation is not entirely surprising, however, given the differences in
membrane preparation and other variations in the protocol.
In summary, these data demonstrate the presence of high
affinity receptors for arginine-vasopressin on basolateral membranes
prepared by differential and density gradient centrifugation from rat
renal tubular epithelia. These data are consistent with and


33
Figure 2-3. 3H-vasopressin (3H-AVP) binding in renal tubular
basolateral membranes as a function of time. Receptor binding
protocol described in "Methods section. Association: Total binding
determined at 1 nM [3H-AVP]. Non-specific binding (NSB) determined
in the presence of 1 nM [3H-AVP] + 1 /iM AVP. Total NSB =
Specific, which represents 80-85%. Dissociation: At equilibrium
binding (1 hr), 1 nM [3H-AVP] is dissociated from receptor site by
the addition of 1 pM unlabeled AVP.


120
Boumendil-Podevin, E.F., and R.A. Podevin. Isolation of baso-lateral
and brush-border membranes from rabbit kidney cortex.
Biochim. Biophys. Acta. 735:86-94, 1983.
Brady, L.S., and M. Herkenham. Dehydration reduces receptor binding
in the neurohypophysis of the rat. Brain Res. 425:212-217, 1987.
Buijs, R.M. Intra- and extra-hypothalamic vasopressin and oxytocin
pathways in the rat. Pathways to the limbic system, medulla
oblongata and spinal cord. Cell. Tiss. Res. 192:423-435, 1978.
Burnatowska-Hledin, M.A. and W.S. Spielman. A pertussin toxin-
sensitive vasopressin Vj receptor in rabbit cortical collecting
tubule (RCCT) cells. Kidney Int. 33:257, 1988 (Abstract).
Butlen, D., G. Guillon, R.M. Rajerison, S. Jard, W.H. Sawyer and M.
Manning. Structural requirements for activation of vasopressin-
sensitive adenylate cyclase, hormonal binding and antidiuretic
action. Effects of highly potent analogues and competitive
inhibitors. Mol. Pharmacol. 14:1006-1017, 1978.
Cajal, S. Ramon y, Algunas contribuciones al concimento de los
ganglios del encfalo. III. Hypophysis. An. Soc. Esp. Hist. Rat.
Ser. 23:195-237, 1894.
Campbell, B.J., G. Woodward and V. Borberg. Calcium-mediated inter
actions between the antidiuretic hormone and renal plasma
membranes. J. Biol. Chem. 247:6167-6175, 1972.
Cantau, B., S. Keppens, H. DeWulf and S. Jard. 3H-AVP binding to
isolated rat hepatocytes and liver membranes: Regulation by GTP
and relation to glycogen phosphorylase activation. J. Recept.
Res. 1:137-168, 1980.
Carlsson, A., B. Falck and N.A. Hillarp. Cellular localization of brain
monoamines. Acta. Physiol. Scand. 56(Suppl. 196):l-27, 1962.
Catt, K.J., J. Harwood, G. Aguilera and M.L. Dafau. Hormonal
regulation of peptide receptors and target cell response. Nature
(London) 280:109-116, 1979.
Ciriello, J., and F.R. Calaresu. Role of the paraventricular and
supraoptic nuclei in central cardiovascular regulation in the cat.
Am. J. Physiol. 239:R137-R142, 1980.
Cornett, L.E., and D.M. Dorsa. Vasopressin receptor subtypes in
dorsal hindbrain and renal medulla. Peptides 6:85-89, 1985.
Courtice, G.P., T.E.K. Wong, E.R. Lumbers and E.K. Potter.
Excitation of the cardiac vagus by vasopressin in mammals. J.
Physiol. (London) 354:547-556, 1984.


44
Results
The state of hydration of the rats was evaluated by comparing
the urine ouput and osmolality, plasma osmolality and plasma
vasopressin concentration among the two groups. Urine output in the
normally hydrated control group was 10.611.1 ml/24 hr while that of
the dehydrated group was 1.90.8 ml/24 hr (p<.05 vs. control) (Figure
3-1 A). Urine osmolality was significantly elevated among the
dehydrated animals (2789169 mosmol/kg) when compared to control
values (1447225 mosmols/kg) (p<.05) (Figure 3-IB). Similarly, plasma
osmolality was raised from control values of 288 0.8 mosmol/kg to
299 1.7 mosmol/kg among the water deprived group (p<.05) (Figure
3-2 A). Vasopressin values were determined from the standard curve
shown in Figure 3-3. In response to the heightened osmolality of the
extracellular fluid, plasma vasopressin levels were commensurately
elevated. AVP concentrations were raised 11.5-fold, from a value of
0.21 0.02 pg/ml in the plasma of the control animals to 2.40.3 pg/ml
in the plasma of the dehydrated animals (p<.05) (Figure 3-2B).
^H-vasopressin binding was determined in renal tubular epithelial
basolateral membranes prepared from the kidneys of the control and
dehydrated animals. A representative saturation isotherm for
AVP binding is depicted in Figure 3-4. Transformation of the data
for Scatchard analysis is represented in Figure 3-5. Analysis of the
data between the two groups indicates a significant reduction in the
number of vasopressin receptors on the membranes from the
dehydrated animals (Control Bmax = 184+15 f mol/mg protein vs.
Dehydrated Bmax = 114+2.5 fmol/mg protein) (p<.01). No significant
difference was observed in the affinity of the receptor between the


CHAPTER 7
SUMMARY
Arginine^-vasopressin, a neurohypophyseal hormone, is
synthesized
as
a prohormone in
the
supraoptic and
paraventricular
nuclei of
the
hypothalamus, transported
and
modified
axonally and
stored in
nerve
terminals within
the
neural
lobe of
the posterior
pituitary gland. An extrahypothalamic distribution of vasopressinergic
fibers also innvervates multiple regions of the central nervous system,
with an especially dense field of terminals found in the brainstem.
The peripheral and central vasopressin systems act, by vastly
different mechanisms, to maintain fluid balance and circulatory
homeostasis. Centrally, vasopressin can affect circulatory control by
modulating the activity of the baroreceptor reflex and more directly,
by affecting sympathetic and parasympathetic outflow. In the
periphery vasopressin is a pressor substance, acting on resistance
vessels and causing vasoconstriction. More importantly, vasopressin
represents the principal hormone of fluid balance, acting at discrete
tubular sites in the kidney to promote the reabsorption of water and
electrolytes, principally sodium. Therefore, it is not surprizing that
the regulation of vasopressin synthesis and release should be
governed by both osmotic stimuli, mediated by centrally-located
osmosensitive cells and non-osmotic stimuli, sensed by volume-
sensitive mechanoreceptors in the heart and peripheral vessels.
Alterations in the synthesis or release of the hormone can have
110


65
TABLE 4-1.
VASOPRESSIN BINDING IN DOCA-TREATED RATS.
Treatment
Group
Bmax
(fmoles/mg P)
Kp
(nM)
n
I
307 29*
0.59 .11
4
II
179 17
0.54 .09
4
III
191 14
0.63 .04
3
IV
281 33*
0.62 .14
2
V
196 23
0.67 .12
3
p < .05 vs. Groups II and V


23
Significance
Myriad biological functions have now been attributed to
vasopressin. The preponderance of evidence suggests that its major
action pertains to its role in circulatory and fluid homeostasis. This
is achieved in a concerted manner, through fluid and electrolyte
reabsorption, vasoconstriction, modulation of the baroreflex and
central regulation of cardiovascular centers.
The actions of vasopressin can be controlled in a number of
ways. One is to control vasopressin secretion itself. The levels of
circulating
AVP are
regulated
by
osmotic
and
non-osmotic
mechanisms.
Endocrine
feedback
systems
also act
to
control AVP
release. For example, dehydration will cause an osmotically-mediated
release of vasopressin but, in addition, will activate the renin-
angiotensin system in the brain, which in turn will stimulate the
release of AVP.
The
action of vasopressin can also
be
affected at the target
tissue.
This usually implies some type
of
modulation of
hormone
receptor
or post-receptor modification.
The
concept of
hormone
receptor
regulation has been the subject
of
several recent
reviews
(Catt et al., 1979; Roth and Taylor, 1982; Lefkowitz et al., 1984).
Receptor regulation can occur by "homologous" or "heterologous"
mechanisms. The former implies that a hormone can modulate the
cell membrane content of its own specific receptor. Alternatively,
receptor number may remain the same but the system is desensitized
through an uncoupling of the receptor from the signal transducing
elements, often by covalent modification of the receptor.
Heterologous regulation suggests that other hormones or factors can


58
drinking solution and was unilaterally nephrectomized according to
the procedures described above, but received no DOCA implants.
Animals in Group III received DOCA implants, tap water to drink and
their kidneys remained intact. Those in Group IV were implanted
with DOCA capsules, received 1% NaCl to drink, but their kidneys
were left intact. Animals in Group V received no special treatment
and were provided with tap water to drink. Blood pressures were
recorded by tail cuff plethysmography prior to surgery and once
weekly, thereafter. Values are expressed as mm Hg.
Membrane PreparatiQn and Plasma Collection
After four weeks of treatment, animals were sacrificed by
decapitation. Trunk blood was collected in chilled tubes containing
0.3 M EDTA (50 /xl/ml blood) for the determination of plasma
vasopressin concentration. Blood samples were processed according to
the procedure described in Chapter 3. Renal basolateral membranes
were prepared by modifying the density gradient centrifugation
method of Boumendil-Podevin and Podevin (1983), according to the
description in Chapter 2. For vasopressin receptor binding assays,
membranes were stored in liquid N2 until use. For adenylate cyclase
measurements, membranes were used immediately.
Vasopressin Receptor Assay
Binding studies were conducted according to the procedure
described in Chapter Two. Briefly, membrane samples were incubated
in triplicate at 20C for 45 minutes with concentrations of -^H-AVP
ranging from 50 pM to 2,500 pM. Non-specific binding was
determined in the presence of 2.5 /xM unlabelled AVP.


43
plasma sample is acidified with 1% TFA and applied to the column
dropwise. The cartridge was rinsed with 5 ml 1% NaCl/1% TFA, then
A VP was eluted with 4 ml methanol:H20:TFA (80:19:1). The samples
were evaporated under a light stream of air then reconstituted in 0.5
ml of assay buffer (50 mM phosphate buffer, pH 7.4 containing 0.2%
bovine serum albumin and 10 mM EDTA). The RIA was carried out
by incubating 100 /xl samples or known amounts of standard (0.1-20 pg
125
AVP) with 25 /il AVP antiserum and 25 /I I-AVP (corresponding to
3,000 cpm) for 24 hours at 4C. Free ligand was sequestered by the
addition of dextran-coated charcoal and centrifugation at 2,000 x g
for 15 minutes. The supernatant containing the antibody-bound
ligand was aspirated and counted in a Beckman 5500 gamma counter
125
at an efficiency of 79% for I. Values are expressed as pg AVP.
Cross-reactivity with related peptides is <1%. Recovery of AVP for
the extraction procedure was determined to be >90%. Sensitivity of
the assay was 0.15 pg per tube.
Plasma Osmolality
Plasma osmolality was determined using a Westcor model 5500C
vapor pressure osmometer. Data are expressed as mosmols/kg H2O.
Analysis
Data from saturation and competition experiments were analyzed
using iterative curve fitting programs. Data are reported as the
mean + S.E. Statistical significance of the data was assessed using
one-way analysis of variance and Students t-test. A level of p<.05
was considered to be significant.


100
co
Q)
(D
a:
i
i
D
0)
CO
k_
O
a
o
o
c
Figure 6-3. Dose-dependent stimulation of 3H-inositol phosphate
release in LLC-PKj cells. Vasopressin (A VP), in concentrations
ranging from 1 nM to 10 /iM, were used to stimulate the release of
3H-inositol phosphate. Results are expressed as in Figure 6-2.
Details of the procedure are found in the "Methods" section.


99
Time (min)
Figure 6-2. Time dependence of 3H-inositol phosphate release in
LLC-PKj cells. Control represents Krebs-Ringers buffer. Vasopressin
(AVP)-stimulated release was conducted at a concentration of 10 pM
AVP. Results are expressed as 3H-inositol incorporated into the
phosphatidyl inositol pool. Details of the procedure are found in the
"Methods section.


96
buffer II. In this way, the buffers simulated the osmolality and
composition of the renal medullary interstitium.
IncorpQ ration of 3H.-jngsitpl into Memferang PhpSBhainQSitides and
Release of 3H-inositol Phosphates
The method for measurement of phosphoinositide (PI) hydrolysis
in
LLC-PK] cells
was
similar
to that
described
by Gonzales
and
Crews (1984) for
brain
slices.
LLC-PK]
cells were incubated
with
0.1
/M ^H-inositol
(Specific
activity =
16.0
Ci/mmol) in
the
appropriate buffer (I to IV) for 3 hours at 37C in a shaking water
bath. The cells were then washed with buffer of the same
osmolality (except that it did not contain -ll-inositol) and divided
into several aliquots. In each aliquot, more of the same buffer but
containing 20 mM LiCl was added. LiCl inhibits dephosphorylation of
inositol phosphates (IP), and has been used to amplify hormone-
dependent PI responses in numerous tissues including LLC-PKj cells
(Berridge, 1987; Kinoshita et al., 1987).
To
determine the
hormone-stimulated release
of
3H-IP, the
reaction
was
started by
adding vasopressin
or other
agents and
incubating
the
samples at
37C in a shaking
water
bath. During
incubation
the
buffers
were kept saturated
with
02:C02 (95:5)
mixture.
After
one hour,
the reaction was terminated
by
the addition
of chloroform:methanol (1:2 v/v) mixture. The organic and aqueous
layers were separated by centrifugation.
Quantitation of Inositol Phosphates (IP) Released
In order to eliminate the variation due to differences in number
of cells or 3H-inositol incorporation in each sample, values are
expressed as 3H-inositol phosphates released as a percentage of the
total amount of 3H-inositol incorporated in each sample. To do this,


105
elicit an increase in cytosolic calcium in these cells (Tang and
Weinberg, 1986). Why the concentrations of vasopressin necessary to
stimulate these second messenger systems are higher than circulating
levels of vasopressin in vivo is unclear. However, it should be noted
that in certain tissues (kidney, urinary bladder), activation of only a
small fraction of hormone receptors is necessary to produce maximal
physiological responses.
Only arginine- and lysine-vasopressin were effective in
stimulating PI hydrolysis in LLC-PKj cells. The muscarinic agonist
carbachol and angiotensin II were without effect in this cell system.
This differs from the findings of Garg et al. (1986 and unpublished
observations) in which the cholinergic agent and peptide were shown
to stimulate PI hydrolysis in kidney slices. This suggests that the
stimulation of PI hydrolysis in LLC-PKj cells by vasopressin is
hormone-specific.
The nature of the receptors which mediate the vasopressin-
induced release of inositol phosphates from the membranes of LLC-
PKj cells remains to be clarified. Two major subtypes of vasopressin
receptors have been characterized, based on the effector system to
which they are coupled. Vj receptors, which mediate the
glycogenolytic action in hepatocytes and the vasoconstrictor action in
smooth muscle cells, are coupled to the hydrolysis of phosphatidyl
inositol and the generation of inositol phosphates and diacylglycerol.
V2 receptors on the other hand, mediate the antidiuretic action of
the hormone and are linked to adenylate cyclase and the production
of cAMP (Michell et al., 1979). Based on these criteria, it would
appear that the vasopressin-induced release of inositol phosphates in


4
hypothalamus. The osmotic and non-osmotic control of vasopressin
secretion has been reviewed by Schrier et al. (1979).
The synaptic inputs to the PVN and SON of the hypothalamus
which are involved in the control of vasopressin secretion are less
well understood (Morris, 1983). The nature of the neurotransmitters
and neuromodulators involved in these processes has only recently
begun to be addressed (Sladek, 1983). Angiotensin II, another peptide
hormone involved in blood pressure control and fluid homeostasis, has
been shown to stimulate the release of vasopressin, following
intracerebroventricular administration (Phillips, 1987). Cholinergic
parasympathetic pathways subserve both the cardiovascular- and
volume-related functions of AVP release. The ascending
noradrenergic pathways from the medullary cardiovascular centers
appear to exert a tonic inhibitory affect on vasopressin release. In
addition, dopamine and gamma-aminobutyric acid also are inhibitory.
The actions of other putative mediators of vasopressin release has
recently been reviewed by Sklar and Schrier (1983).
Biological Actions of AVP in the Periphery
Since the original observation of a vasopresssin-induced pressor
response by Oliver and Schaefer (1895), numerous other biological
effects of vasopressin have subsequently been noted. Many of the
observations pertain to the fluid-retaining and cardiovascular
properties of the hormone, or are in concert with other systems that
subserve these functions.
Renal Effects
It is now clear that vasopressin acts at multiple sites within the
kidney to affect renin release, glomerular filtration, NaCl transport


90
It may be appropriate to consider several possibilities to account
for the lack of mineralocorticoid effect noted in the LLC-PKj cells.
The most obvious explanation is that this particular cell line does not
possess receptors for mineralocorticoids, whereas cytosolic and
nuclear binding sites for mineralocorticoids have been reported for A$
cells (Handler et al., 1981) and MDCK cells (Luden et al., 1978). No
such reports have appeared in the literature for LLC-PKj epithelial
cells. This is consistent with the observation that LLC-PKj cells,
unlike the A$ and MDCK epithelial cell lines, are relatively refractory
to the effects of aldosterone, at least with respect to transepithelial
Na+ transport (Meier and Insel, 1985).
Perhaps the most pertinent evidence concerning the lack of
effect of mineralocorticoids in LLC-PKj cells comes from a study by
Roy et al. (1980). These investigators examined the regulation of
vasopressin receptors in LLC-PKj ^ cells, a subline of the parent
LLC-PKj strain. These cells are characterized by possessing only 5%
as many vasopressin receptors as the parent cell line. They reported
that fetal bovine serum or insulin could markedly stimulate the
expression of receptors in these cells. In contrast, thyroid hormone,
dexamethasone and aldosterone were ineffective in evoking a change
in receptor number.
Alternatively, methodological considerations must not be
overlooked. DOCA was only soluble in organic solvents such as
absolute ethanol or DMSO. Dilution of the DOCA-containing solvents
with aqueous buffers often resulted in the precipitation of the
steroid. Thus, it is impossible to say with any degree of certainty
exactly how much DOCA was applied to the cell cultures. On the


103
Vj and V2 blockers by themselves caused a small but statistically
non-significant stimulation of PI hydrolysis (data not shown).
The effect of increasing the osmolality of the incubation medium
was assessed on vasopressin-stimulated PI hydrolysis. These results
are presented in Figure 6-6. Raising the osmolality to 600 mosmol/kg
H2O had no significant effect on AVP-evoked PI release. However,
when the osmolality was increased further, to 900 and 1200
mosmol/kg H2O, vasopressin-stimulated PI hydrolysis was significantly
reduced
with respect
to the
isoosmotic
buffer
(p<.05).
Altering the
osmotic
environment
had no
significant
effect
on the
buffer-evoked
release of PI.
Discussion
The vast preponderance of evidence suggests that vasopressin
causes the transepithelial transport of fluid and solutes in mammalian
renal tubules and amphibian urinary bladder by stimulating adenylate
cyclase which leads to the activation of cAMP-dependent protein
kinases. Vasopressin-stimulated cAMP production has also been
demonstrated in LLC-PKj cells, epithelial cells of renal epithelial
origin (Goldring et al., 1978; Chapter 5). The experiments conducted
in the present study represent the first documentation of vasopressin-
stimulated hydrolysis of phosphatidyl inositol in cells of renal
epithelial origin.
The effect of AVP-stimulated PI hydrolysis was time- and dose-
-8
dependent, with the minimum effective dose being 10 M. While this
concentration of vasopressin is generally higher than that required to
stimulate adenylate cyclase activity in these cells (Goldring et al.,
1978; Ausiello et al., 1987), the value is identical to that required to


123
Gluck, S., and Q. Al-Awqati. Vasopressin induces aqueous channels in
luminal membrane of toad bladder. Nature 284:531-532, 1980.
Goldring, S.R., J.M. Dayer, D.A. Ausiello and S.M. Krane. A cell
strain cultured from porcine kidney increases cyclic AMP
content upon exposure to calcitonin or vasopressin. Biochem.
Biophys. Res. Comm. 83:434-440, 1978.
Gonzales, R.A., and F.T. Crews. Characterization of the cholinergic
stimulation of phosphoinositide hydrolysis in rat brain slices. J.
Neurosci. 4:3120-3127, 1984.
Grantham, J.J., and M.B. Burg. Effect of vasopressin and cyclic AMP
on permeability of isolated collecting tubules. Am. J. Physiol.
211:255-259, 1966.
Guyton, A.C., T.G. Coleman, A.W. Cowley, K.W. Scheel, R.D. Manning
and R.A Norman. Arterial pressure regulation: overriding
dominance of the kidneys in long-term regulation and
hypertension. Am. J. Med. 52:584-594, 1972.
Handler, J.S. Studies of kidney cells in culture. Kidney Int. 30:208-
215, 1986.
Handler, J.S., A.S. Preston, F.M. Perkins and M. Matsumura. The
effect of adrenal steroid hormones on epithelia formed in culture
by A6 cells. Ann. N.Y. Acad. Sci. 372:442-454, 1981.
Haslam, R.J., and G.M. Rosson. Aggregation of human blood platelets
by vasopressin. Am. J. Physiol. 233:958-967, 1972.
Hebert, S.C., R.M. Culpepper and T.E. Andreoli. NaCl transport in
mouse medullary thick ascending limb. I. Functional nephron
heterogeneity and ADH-stimulated NaCl cotransport. Am. J.
Physiol. 241:F412-431, 1981a.
Hebert, S.C., R.M. Culpepper and T.E. Andreoli. NaCl transport in
mouse medullary thick ascending limb. III. Modulation of the
ADH effect by peritubular osmolality. Am. J. Physiol. 24EF443-
451, 1981b.
Hinko, A., and A.F. Pearlmutter. Effects of arginine vasopressin on
protein phosphorylation in rat hippocampal synaptic membranes.
J. Neurosci. Methods 17:71-79, 1987.
Hofbauer, K.G., S.C. Mah, H.P. Baum, H. Hanni, J.M. Wood and J.
Kraetz. Endocrine control of salt and water excretion: The role
of vasopressin in DOCA-salt hypertension. J. Cardiovasc.
Pharmacol. 6(Suppl.):184-191, 1984.
Horster, M.F. and M. Stopp. Transport and metabolic functions in
cultured renal tubule cells. Kidney Int. 29:46-53, 1986.


35
Figure 2-4. Specificity of the vasopressin receptor in renal tubular
basolateral membranes. Competition of 3H-vasopressin (3H-AVP)
binding at 1 nM by vasopressin analogues and unrelated peptides.
Receptor binding protocol described in "Methods" section. Vj
antagonist, d(Et)2Tyr(Et)D-AVP; V2 antagonist, d(CH2)5D-Ile2-Ile4-
AVP; A VP, arginine vasopressin; LVP, lysine vasopressin; OXY,
oxytocin; Ang II, angiotensin II; DAME, D-ala-met-enkephalinamide.


64
Bound (fmoles/mg P)
Figure 4-3. Scatchard analyses of 3H-vasopressin binding. Binding
determined in basolateral membranes prepared from kidneys of DOCA-
salt hypertensive rats and normotensive sham-operated control rats.
Analyses of data in Figure 4-2. *p<.05 vs. control. Abbreviations are
as in Figure 4-2.


CHAPTER 6
INTRACELLULAR SECOND MESSENGERS IN LLC-PKj CELLS:
A DUAL TRANSMEMBRANE SIGNALING SYSTEM
Introduction
It is now well-documented that vasopressin receptors are
integral components in the plasma membrane of LLC-PKj cells, a
continous cell line of renal epithelial origin (Ausiello et al., 1987).
Vasopressin interacts with these receptors to initiate the sequence of
intracellular events that ultimately lead to the transepithelial
transport of fluid and solutes, in a manner not unlike that seen in
the distal collecting segments of the nephron. As in the kidney,
vasopressin receptors in LLC-PKj cells display characteristics of the
V2 subtype and are coupled to adenylate cyclase, resulting in a
hormone-specific stimulation of cAMP accumulation (Goldring et al.,
1978; Roy and Ausiello, 1981a). Recently, it was reported that
vasopressin stimulated transient increases in cytosolic calcium in
LLC-PKi cells (Tang and Weinberg, 1986). This action is not unlike
the vasopressin-stimulated calcium fluxes noted in hepatocytes
(Michell et al., 1979) and smooth muscle cells (Aiyar et al., 1986). In
these tissues however, vasopressin exerts its action through V j
receptors, which are coupled to phospholipase C and the hydrolysis of
phosphatidyl inositol (PI). This leads to the generation of inositol
phosphates (IP) and diacylglycerol (DG). Inositol triphosphate (IP3) is
thought to be involved in the mobilization of calcium from
intracellular organelles such as the endoplasmic reticulum whereas DG
92


CHAPTER 5 EFFECTS OF MINERALOCORTICOIDS ON
VASOPRESSIN RECEPTORS AND ADENYLATE
CYCLASE-COUPLED RESPONSES IN LLC-PKj
CELLS 76
Introduction 76
Materials and Methods 77
Results 80
Discussion 82
CHAPTER 6 INTRACELLULAR SECOND MESSENGERS IN
LLC-PKj CELLS: A DUAL TRANSMEMBRANE
SIGNALING SYSTEM 92
Introduction 92
Materials and Methods 93
Results 97
Discussion 103
CHAPTER 7 SUMMARY 110
REFERENCES 118
BIOGRAPHICAL SKETCH 132
v


CHAPTER 5
EFFECTS OF MINERALOCORTICOIDS ON VASOPRESSIN
RECEPTORS AND ADENYLATE CYCLASE-COUPLED RESPONSES
IN LLC-PKj CELLS
Introduction
Although vasopressin can act on the kidney and affect renin
secretion, glomerular filtration and tubular transport, a detailed
understanding of how AVP or other hormones affect these processes
at the cellular level has been difficult to ascertain. In large part,
this is due to the complexity of the kidney and the veritable myriad
of potential target tissues. One attempt to solve this problem has
been to
study
the action of
vasopressin on
isolated
glomeruli,
vascular
tissue
or discretely dissected tubular
segments.
While
somewhat
more
refined than an
isolated whole kidney, this
approach
still ignores the heterogeneity
of cell types
within anatomically
distinct
regions.
In order to
circumvent these
potential
problems,
one technique that has been applied to answer many of the questions
concerning the cellular actions of vasopressin in the kidney is cell
culture. The use of primary cultures and cell lines has been utilized
successfully in a number of studies, ranging from transport physiology
to hormonal regulation. Handler (1986) has recently written an
elegant review on the use of kidney cells in culture and succinctly
summarizes the wide-ranging studies amenable to these techniques.
The primary objective for using cell culture techniques in this
study was to establish an in vitro system in which to evaluate the
effects of mineralocorticoids on vasopressin binding, in order to
76


102
12
A B
A) Buffer
B) AVP [10 7M]
C) AVP + V1 antagonist
D) AVP + V2 antagonist
* p<.05 vs A.C.D
C D
Figure 6-5. Antagonism of vasopressin-stimulated 3H-inositol
phosphate release in LLC-PKj cells. Vasopressin at a concentration
of 100 nM, stimulated the release of 3H-inositol phosphate. This
effect was totally abolished by pretreatment with equimolar amounts
of the Vj antagonist and attenuated by the V2 antagonist. Results
are expressed as in Figure 6-2. Details of the procedure are found
in the "Methods" section. Abbreviations for the Vj and V2
antagonists are as in Figure 2-4. AVP, arginine vasopressin.


49
Bound (fmols/mg Protein)
Figure 3-5. Scatchard analyses of 3H-vasopressin binding,
determined in basolateral membranes prepared from kidneys of
replete and water-deprived (72 hr) rats. Analyses of data in
3-4. *p<.05 vs. control.
Binding
water-
Figure


11
potentiating the effect of corticotropin releasing factor (De Wied,
1983). A metabolic action has been attributed to vasopressin, by
virtue of its glycogenolytic effect in the liver (Michell et al., 1979).
Steroidogenic effects have been attributed to vasopressin in the
Leydig cells of the testes and the glomerulosa cells of the adrenals
(Nicholson et al., 1984). It has also been shown that vasopressin is
involved in platelet aggregation (Haslam and Rosson, 1972).
Vasopressin in the Central Nervous System
Vasooressinergic Projections
Vasopressin (and oxytocin) are synthesized in neurosecretory
neurons located within the paraventricular and supraoptic nuclei of
the hypothalamus. It was originally thought that oxytocin was
exclusively formed in the SON and that vasopressin was produced
only in the PVN. It is now clear that both peptides are synthesized
in separate and topographically distinct populations of neurons within
each nucleus (Swanson and Sawchenko, 1983). Each nucleus is
characterized cytoarchitecturally by the presence of large
magnocellular neurons and small, round parvocellular neurons. It has
been known since the work of Cajal (1894) that the magnocellular
neurons of the SON project to the neural lobe of the pituitary.
While morphologically quite similar, it was not recognized until forty-
two years later that the magnocellular neurons of the PVN also
project to the posterior pituitary (Ingram et al., 1936). Although the
findings of Cajal remain unchanged, histological evidence concerning
vasopressinergic projections from the PVN has been greatly expanded.
Magnocellular projections from the PVN have been traced to the
external lamina of the median eminence (Diedrickx et al., 1976).


80
cAMP Determination
The cellular cAMP content was determined by the competitive
protein binding assay as modified by Baker et al. (1986), as described
in detail in Chapter 4.
POCA Treatment of Cell Cultures
Desoxycorticosterone acetate (DOCA) was dissolved in
dimethylsulfoxide (DMSO) at a concentration of 1 mg/ml. This stock
concentration was initially diluted with PBS and subsequent dilutions,
ranging from 1 pg/ml to 10 /xg/ml were made in serum-free DMEM.
The solutions were sterilized by passing them through 0.2 micron
mesh Nalgene filters. Growth medium was aspirated off of the cells
and replaced with DMEM containing DOCA. Cells were treated for 1-
24 hours with DOCA concentrations ranging from 1 pg/ml to 1 /g/ml.
Results
Binding of ^H-AVP to monolayers of LLC-PKj cells increased
rapidly over time and attained equilibrium after 45 minutes. At this
time, specific binding, represented ~75% of total -^H-AVP bound.
Upon equilibrium, binding was shown to be reversible by the removal
of the radioligand and the addition of buffer containing unlabeled
vasopressin. As with association, dissociation was rapid and reached
levels equivalent to non-specific binding after 45 minutes (Figure 5-
1). The specificity of the receptor was determined by competition of
^H-AVP binding with a variety of related and unrelated peptides.
Lysine^-vasopressin and arginine^-vasopressin were equally effective
(IC^q = 2.1 nM) in terms of their ability of displacing the radioligand
from the binding sites on the cells. On the other hand, oxytocin,
angiotensin II and D-ala-met-enkephalinamide were ineffective even at


Plasma Osmolality (mOsm/kg)
46
A B
Figure 3-2. Plasma osmolality and plasma vasopressin concentrations
in control and dehydrated rats. A: Plasma osmolality, determined by
vapor pressure osmometry. B: Plasma AVP determined by RIA. Error
bars represent S.E.M. *p<.05 vs. control. AVP, arginine vasopressin;
RIA, radioimmunoassay.


101
Figure 6-4. Specificity of agonist-stimulated 3H-inositol phosphate
release in LLC-PKj cells. Peptides and the muscarinic agonist
carbachol were used, in the concentrations indicated to stimulate 3H-
inositol phosphate release. Results are expressed as in Figure 6-2.
Details of the procedure are found in the "Methods" section. A VP,
arginine vasopressin; LVP, lysine vasopressin; CARB, carbachol; ANG
II, angiotensin II.


87
DOCA [pg/ml]
Figure 5-6. Effect of DOCA on basal and vasopressin-stimulated
cAMP production in LLC-PKj cells. Basal cAMP was measured in the
absence of AVP. Stimulated values were assessed in the presence of
10 nM AVP. Details of procedures are found in the "Methods"
section. DOCA, desoxycorticosterone acetate; AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate.


83
[Peptide] (-log M)
Figure 5-2. Specificity of vasopressin receptor in LLC-PKj cells.
Competition of 3H-vasopressin (3H-AVP) binding with vasopressin
analogues and unrelated peptides. Receptor binding protocol
described in "Methods" section. Abbreviations are as in Figure 2-4.


73
vasopressin-stimulated adenylate cyclase activity was also noted.
Replacement therapy with aldosterone restored the hormone binding
capacity and adenylate cyclase activity to control levels. In the
present study, vasopressin-stimulated cAMP production was
commensurately increased in membranes prepared from the DOCA-salt
hypertensive rats. Furthermore, the effective concentration which
caused half-maximal stimulation (ED50) was reduced. Thus, an
increased sensitivity to vasopressin is suggested on the basis of
adenylate cyclase activity, although no change in the affinity
constant of the receptor was observed. Using microdissected renal
tubules, Pettinger et al. (1986) have observed a similar increase in
the sensitivity of adenylate cyclase in response to vasopressin in
tubules obtained from DOCA-salt treated rats. This response was
specific for vasopressin and occurred only in the segment of the
cortical collecting tubule. The phenomenon was observed as early as
five days post-treatment, a period which preceded the elevation of
blood pressure.
The mechanism of vasopressin receptor up-regulation associated
with DOCA-salt hypertension is not clear. It might have been
expected that vasopressin binding would decrease in the face of
elevated circulating levels of AVP, as was observed in Chapter 3.
However, the magnitude of change in plasma vasopressin levels in the
present study was only half of what occurred with dehydration.
Clearly, factors other than homologous regulation must be invoked.
As a steroid, DOCA probably acts by increasing the transcriptional
rate for the vasopressin receptor protein. Increased expression of
other tubular basolateral membrane proteins is also observed with


CHARACTERIZATION AND REGULATION OF RENAL
VASOPRESSIN RECEPTORS
By
MARTIN STEINER
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
1988

This dissertation is dedicated to my parents, Lillian and Murry Steiner.

ACKNOWLEDGEMENTS
I wish to express my most sincere gratitude and appreciation to
the chairman of my supervisory committee, Dr. M. Ian Phillips, for
his friendship, guidance and support and the confidence in me that he
exhibited. Sincere thanks are extended to the members of my
committee, Drs. Steven R. Childers, Mohan K. Raizada and Colin
Sumners, for helpful advice and invaluable criticism throughout this
project. Thanks go to the many faculty members in the Departments
of Physiology and Pharmacology, but especially Drs. Melvin J. Fregly,
Bruce R. Stevens, Lai C. Garg and Stephen P. Baker, who offered me
guidance, suggestions or the use of their facilities. Thanks also go
to my friends and colleagues, past and present, Dr. Rod Casto, Elaine
M. Richards and Dr. Klaus Hermann, for continued encouragement.
The expert secretarial skills of Carolyn Gabbard are greatly
appreciated. And a very special note of gratitude is extended to
Birgitta Stenstrom Kimura, for expert technical assistance and, more
importantly, for her kindness the last 4 1/2 years.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS üi
KEY TO ABBREVIATIONS v i
ABSTRACT viii
CHAPTER 1 BACKGROUND 1
Synthesis, Transport and Release of
Vasopressin 1
Control of Vasopressin Secretion 2
Biological Actions of AVP in the
Periphery 4
Vasopressin in the Central Nervous
System 11
Vasopressin Receptors 15
Significance 23
CHAPTER 2 CHARACTERIZATION OF 3H-VASOPRESSIN
BINDING SITES IN RENAL TUBULAR
BASOLATERAL MEMBRANES 25
Introduction 25
Materials and Methods 26
Results 30
Discussion 34
CHAPTER 3 DEHYDRATION-INDUCED DOWNREGULATION OF
RENAL TUBULAR VASOPRESSIN
RECEPTORS 39
Introduction 39
Materials and Methods 41
Results 44
Discussion 50
CHAPTER 4 REGULATION OF VASOPRESSIN BINDING IN
MINERALOCORTICOID-DEPENDENT
HYPERTENSION 54
Introduction 54
Materials and Methods 57
Results 60
Discussion 71
iv

CHAPTER 5 EFFECTS OF MINERALOCORTICOIDS ON
VASOPRESSIN RECEPTORS AND ADENYLATE
CYCLASE-COUPLED RESPONSES IN LLC-PKj
CELLS 76
Introduction 76
Materials and Methods 77
Results 80
Discussion 82
CHAPTER 6 INTRACELLULAR SECOND MESSENGERS IN
LLC-PK.! CELLS: A DUAL TRANSMEMBRANE
SIGNALING SYSTEM 92
Introduction 92
Materials and Methods 93
Results 97
Discussion 103
CHAPTER 7 SUMMARY 110
REFERENCES 118
BIOGRAPHICAL SKETCH 132
v

KEY TO ABBREVIATIONS
ACTH
adrenocorticotropic hormone
ADH
antidiuretic hormone
ANF
atrial natriuretic factor
ANG II
angiotensin II
ANOVA
analysis of variance
ATCC
American Tissue Culture Collection
ATP
adenosine triphosphate
AVP
arginine vasopressin
BSA
bovine serum albumin
CAMP
cyclic adenosine 3\5'-monophosphate
cGMP
cyclic guanosine 3',5'-monophosphate
CNS
central nervous system
CPM
counts per minute
DAME
D-ala-met-enkephalinamide
DDAVP
deamino-D-arginine vasopressin
DG
diacylglycerol
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
dimethylsulfoxide
DOCA
desoxycorticosterone acetate
DPM
disintegrations per minute
EDTA
ethylenediaminetetraacetic acid
EGTA
ethyleneglycoltetraacetic acid
FBS
fetal bovine serum
GTP
guanosine triphosphate
vi

IBMX
isobutylmethylxanthine
IP
inositol phosphate
K.IU
kallikrein inhibitory units
KRB
Kreb’s Ringer’s buffer
LVP
lysine vasopressin
MDCK
Madine-Darby canine kidney
NTS
nucleus of the tractus solitarius
OXY
oxytocin
PBS
phosphate-buffered saline
PI
phosphatidyl inositol
PMSF
phenylmethylsulfonyl fluoride
PNPP
p-nitrophenyl phosphate
PVN
paraventricular nucleus
RIA
radioimmunoassay
RPM
revolutions per minute
SEM
standard error of the mean
SHR
spontaneously hypertensive rat
SON
supraoptic nucleus
TFA
trifluoroacetic acid
WKY
Wistar-Kyoto
vii

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
CHARACTERIZATION AND REGULATION
OF RENAL VASOPRESSIN RECEPTORS
By
Martin Steiner
April, 1988
Chairman: M. Ian Phillips
Major Department: Physiology
Vasopressin receptors were characterized on basolateral
membranes of renal tubular epithelia and their capacity for regulation
was studied during alterations in fluid balance. Membranes were
obtained from rat kidney and prepared by differential and density
gradient centrifugation techniques. Radioreceptor methodologies were
used to demonstrate that binding was rapid, reversible, specific and
saturable. The binding site displayed a pharmacological profile for
the V2 receptor subtype, based on competition studies with selective
V] and V2 antagonists.
Vasopressin receptors were impacted by the physiological state
of the animal. Water-deprived animals displayed significantly elevated
plasma vasopressin levels. As a consequence vasopressin binding sites
were downregulated. Conversely, the administration of the
viii

mineralocorticoid desoxycorticosterone acetate (DOCA) to rats
resulted in the increased expression of tubular vasopressin receptors
and the development of DOCA-salt hypertension. This was associated
with an increase in adenylate cyclase activity in the basolateral
membrane preparations. These data provide a cellular basis for the
increased tubular sensitivity to vasopressin and its fluid-retaining and
sodium-reabsorbing properties that are characteristic of this volume-
dependent model of hypertension. The heightened expression of
vasopressin receptors appears to result from an increased
transcriptional rate of protein synthesis induced by the
mineralocorticoid DOCA.
Desoxycorticosterone acetate (DOCA) was ineffective in
regulating vasopressin receptors or adenylate cyclase activity in LLC-
PKj cells, a cell line of renal epithelial origin. This coincides with a
lack of mineralocorticoid receptors in this particular cell strain.
Signal transduction mechanisms were also evaluated and vasopressin
stimulated cyclic AMP formation and phosphatidyl inositol hydrolysis,
suggesting that these cells possess both Vj and V2 receptor subtypes.
This demonstrates that one hormone can modulate cellular activity by
interacting with multiple effector systems.
These studies demonstrate that
renal
vasopressin
receptors
are
influenced by
the physiological state
of
the animal.
Similarly,
receptor-coupled
signal transduction
mechanisms are
subject
to
modification, in
vitro.
Alterations
in
vasopressin
receptors
or
receptor-mediated
processes
may provide
a cellular
basis
for
pathophysiological
conditions
associated
with
fluid and electrolyte
balance.
IX

CHAPTER 1
BACKGROUND
Arginine**-vasopressin (AVP) is an ancient hormone. It is one of
the most phylogenetically conserved, presumably because of its impor¬
tance for water balance in early invertebrates (Sawyer, 1977).
Scientifically, it is an old hormone. Oliver and Schaefer (1895) first
described the rise in systemic blood pressure following the infusion of
a whole pituitary extract. The renal antidiuretic actions of the
hormone were first noted by Starling and Verney (1924). A wealth of
information has accumulated on the physiological actions of
vasopressin since these pioneering studies. It is now clear that
vasopressin not only serves as the principal hormone governing fluid
homeostasis, thereby requiring the antidiuretic hormone (ADH)
nomenclature, but also plays a role in cardiovascular control. In
addition, various central nervous system (CNS) effects have been
ascribed to the hormone.
Synthesis, Transport and Release of Vasopressin
Arginine** - vasopressin
and
oxytocin
both
are closely
related
nonapeptides, characterized
by
cysteine
residues
linked by
disulfide
bridges. Often classified as posterior pituitary hormones, the
hormones are actually produced within specialized neurons in the
hypothalamus. Whereas the biologically active molecules are small
molecular weight peptides, they are synthesized, transported and
stored in secretory granules in association with specific carrier
proteins or neurophysins. Sachs and Takabatake (1964) first
I

2
demonstrated that the vasopressin-neurophysin molecule is formed as
part of a common precursor hormone. The precursor molecule is
synthesized in the neuronal cell body by a ribosomal mechanism and
packaged into secretory granules by the Golgi apparatus. The
vesicles are transported by axoplasmic flow toward the nerve terminal
in the neural lobe of the pituitary where they are stored. Gainer et
al. (1977) have shown that the prohormone undergoes post-
translational processing during axonal transport, suggesting that the
granules must contain both the precursor molecule and enzymes
responsible for its modification. The contents of the secretory
granules (hormone, neurophysin, and residual peptide fragments) are
released upon depolarization of the nerve terminal. This exocytotic
process involves a calcium-dependent fusion of the secretory vesicle
with the nerve terminal membrane, followed by an opening of the
vesicle and an emptying of its contents.
Control of Vasopressin Secretion
It is reasonable to expect that the release of vasopressin from
the neural lobe of the pituitary gland should be influenced by factors
pertaining to vasopressin’s role in fluid homeostasis and circulatory
control.
Furthermore,
regulation
of
vasopressin secretion
would
be
expected
to affect the
activity
of
the paraventricular
(PVN)
and
supraoptic
nuclei (SON).
Indeed,
the
activity of these
structures
is
influenced by both osmotic and non-osmotic stimuli.
The
pioneering study
of
Verney (1947)
was
the first to
demonstrate that
centrally
located structures
which
were osmo-
sensitive,
affected
the secretion
of vasopressin.
These
structures are
so exquisitely sensitive that changes in plasma osmolality as small as

3
a few milliosmoles, which represents a change of only 1%, can effect
the
release
of
vasopressin. Ten years
later,
experiments
conducted
by
Jewell
and
Verney (1957) localized
the
osmoreceptors
to the
basomedial forebrain. The exact location of Verney’s osmoreceptors
has been the subject of considerable controversy. While some have
claimed that the magnocellular neurons of the PVN and SON are
themselves osmosensitive (Leng, 1980), it is now believed that
osmosensitive cells within the organum vasculosum of the lamina
terminalis may account for the osmotically-induced alterations in
vasopressin secretion (Thrasher et al., 1982). This same group has
also laid to rest the argument over whether the mechanism for the
control of vasopressin release involves osmosensitive neurons or
sodium
receptors
located on
the cells within
the
organum
vasculosum
laminas
terminalis
(Thrasher
et al., 1980).
The
neural
connections
which relay information between cells in this circumventricular region
and the PVN and SON have been described by Miselis (1981).
Non-osmotic control of vasopressin release is probably more
important for cardiovascular regulation. Thus, hypovolemia,
hemorrhage or decreased cardiac output will elicit the secretion of
AVP, for the purpose of maintaining circulatory homeostasis. This
type of control arises from high pressure baroreceptors in the carotid
sinus and aortic arch and low pressure volume receptors in the left
atrium and pulmonary veins. The afferent impulses of this
neurohumoral arc are carried by the ninth and tenth cranial nerves
to their respective nuclei in the brainstem. Multisynaptic pathways
then transmit this information to the PVN and SON of the

4
hypothalamus. The osmotic and non-osmotic control of vasopressin
secretion has been reviewed by Schrier et al. (1979).
The synaptic inputs to the PVN and SON of the hypothalamus
which are involved in the control of vasopressin secretion are less
well understood (Morris, 1983). The nature of the neurotransmitters
and neuromodulators involved in these processes has only recently
begun to be addressed (Sladek, 1983). Angiotensin II, another peptide
hormone involved in blood pressure control and fluid homeostasis, has
been shown to stimulate the release of vasopressin, following
intracerebroventricular administration (Phillips, 1987). Cholinergic
parasympathetic pathways subserve both the cardiovascular- and
volume-related functions of AVP release. The ascending
noradrenergic pathways from the medullary cardiovascular centers
appear to exert a tonic inhibitory affect on vasopressin release. In
addition, dopamine and gamma-aminobutyric acid also are inhibitory.
The actions of other putative mediators of vasopressin release has
recently been reviewed by Sklar and Schrier (1983).
Biological Actions of AVP in the Periphery
Since the original observation of a vasopresssin-induced pressor
response by Oliver and Schaefer (1895), numerous other biological
effects of vasopressin have subsequently been noted. Many of the
observations pertain to the fluid-retaining and cardiovascular
properties of the hormone, or are in concert with other systems that
subserve these functions.
Renal Effects
It is now clear that vasopressin acts at multiple sites within the
kidney to affect renin release, glomerular filtration, NaCl transport

5
and water reabsorption. Vasopressin is a potent inhibitor of renin
secretion. Subpressor doses of AVP decrease plasma renin levels. It
is thought that vasopressin acts directly on the juxtaglomerular cells,
as the effect can be demonstrated in the isolated kidney and occurs
with no demonstrable effect on renal hemodynamics (Vander, 1968).
Arginine^-vasopressin reduces the glomerular ultrafiltration
permeability coefficient (Kf) and may decrease the glomerular
filtration rate (Ichikawa and Brenner, 1977). While the mechanism by
which AVP decreases the Kf is not clear, Ausiello et al. (1980) have
shown that vasopressin stimulates the contraction of glomerular
mesangial cells grown in culture. Therefore, it is possible that
mesangial contraction reduces the surface area available for
glomerular ultrafiltration.
The next demonstrable action
of vasopressin occurs at
the
thick
ascending Limb
of Henle. Morel et al. (1976)
made
the
first
observation of vasopressin-sensitive
adenylate cyclase
activity
in
both
the medullary and
cortical portions
of the thick ascending limb.
The
concentration of
AVP necessary
to stimulate
adenylate
cyclase
activity in the
thick ascending
limb is one or
two
orders of
magnitude higher
than that needed
in more distal
tubular
segments.
The physiological significance of this finding remained in doubt until
Hebert et al. (1981a) found that AVP, in pico-molar concentrations,
stimulated NaCl transport out of the tubular lumen into the
interstitium. This suggests a role for vasopressin in the maintenance
of the corticomedullary interstitial osmotic gradient.
The major target for the action of vasopressin is the distal
segment of the collecting tubule. Here vasopressin, through a cAMP-

6
dependent mechanism, can exert its well known antidiuretic effect, by
altering the permeability of the tubular membrane to water (Grantham
and Burg, 1966). The cellular events beyond cAMP formation include
stimulation of cAMP-dependent protein kinase in the cytosol and
protein kinase A-dependent phosphorylation of the apical membrane,
resulting in an increased permeability of the apical membrane to
water (Figure 1-1). At present, the precise mechanism by which
protein kinase activation, calcium activation and microtubule-
microfilament assembly result in an alteration in membrane
permeability remain unclear, despite the well-established antidiuretic
effect of the hormone. The most favored hypothesis to emerge,
however, is that vasopressin induces, by some conformational change,
the formation of aqueous channels in the apical membrane (Gluck and
Al-Awqati, 1980). This is supported by the appearance of ADH-
induced intramembraneous particle aggregates in the luminal
membrane. The number of aggregates and the cumulative area of
membrane occupied by the particles are directly related to the
magnitude of ADH-induced changes in water permeability (Muller et
al., 1980).
Cardiovascular Effects
Although an elevation of blood pressure was the first
physiological effect noted following the administration of vasopressin,
the cardiovascular actions of vasopressin have generally been
regarded as physiologically unimportant. This is somewhat surprising
as
vasopressin
has been
shown
to be
one of
the
most potent
vasoconstrictive
agents
(Altura and
Altura,
1984).
In
addition, even
the
antidiuretic
action
of
the hormone
contributes
to
the regulation

7
A
Figure 1-1. Model of antidiuretic hormone (ADH)-stimulated
adenylate cyclase cascade in renal tubular epithelial cell. ADH
combines with receptor in basolateral membrane. Interaction of
hormone and receptor stimulates adenylate cyclase, resulting in the
formation of cyclic AMP and the activation of cAMP-dependent
protein kinases. These enzymes act to phosphorylate microtubules,
microfilaments or integral proteins in the apical membrane. While the
mechanism of increased apical permeability to water remains unclear,
the most widely accepted model postulates the formation of aqueous
channels (Gluck and Al-Awqati, 1980).

8
of extravascular volume and exerts hemodynamic effects even at
subpressor doses (Cowley et al., 1984b). Guyton and others propose
that the antidiuretic action of the hormone can even produce
hypertension under certain conditions (Manning et al., 1979). The
cellular basis for this phenomenon is alluded to in Chapter 4 of this
dissertation.
Vasopressin has been shown to exert direct negative chrono¬
tropic and inotropic effects in vitro (Nakashima et al., 1982). The
physiological significance of these direct cardiac effects remains in
question as exceedingly high concentrations of vasopressin were
necessary to elicit the observed effects.
Vasopressin is a potent vasoconstrictor, in vivo and in vitro. In
vivo, administration of vasopressin leads to an increase in total
peripheral resistance from the vasoconstriction of a variety of
vascular beds. The skin, muscle and splanchnic circulations are the
most sensitive, followed by the mesenteric and coronary beds. The
renal, hepatic and cerebral circulations are unaffected by A VP (Liard,
1984). The vasoconstrictive potencies of several pressor substances
and a number of vasopressin analogues has recently been evaluated in
an isolated smooth muscle preparation and AVP was determined to be
the most potent agent tested, although the in vitro activity did not
exactly parallel the jn vivo activity (Altura and Altura, 1984).
However, when vasopressin is infused into conscious intact animals,
blood pressure does not normally rise unless exceedingly high plasma
levels of vasopressin are attained (>100 pg/ml). Although A VP-
induced elevations in total peripheral resistance might occur, arterial
pressure is not drastically affected because it is strongly buffered by

9
the baroreceptor reflex. Furthermore, vasopressin actually tends to
enhance the baroreflex by increasing vagal tone and inhibiting
sympathetic outflow following i.v. administration, apparently through
an interaction with the cardiovascular centers in the brainstem
(Cowley et al., 1984a). The role of vasopressin in the central control
of the circulation is addressed later in this chapter.
Because the pressor effects of vasopressin are normally obscured
by the baroreflex, the physiological importance of AVP in
cardiovascular function has often been ignored. Nevertheless,
vasopressin plays an important role in the maintenance of blood
pressure under conditions of dehydration (Schwartz and Reid, 1983),
when circulatory volume is compromised as in the case of hemorrhage
(Zerbe et al., 1982), in a situation of adrenal insufficiency (Schwartz
et al., 1983) or when the vasoregulatory reflex mechanisms are
interrupted. In each of these cases, plasma levels of vasopressin rise
in excess of the concentration necessary for maximal antidiuresis.
That vasopressin is essential for maintaining circulatory homeostasis
in even a moderately compromised condition is evident in the findings
of Aisenbrey et al. (1981). In that study, rats were dehydrated for a
period of 72 hours and then challenged with a vascular vasopressin
blocker. Whereas the blocker had no intrinsic cardiovascular activity
nor did it affect blood pressure in normally hydrated animals, blood
pressure fell precipitously in the dehydrated rats. In a related study,
Brattleboro rats, which are unable to synthesize vasopressin, were
unable to maintain their blood pressure when subjected to dehydration
(Woods and Johnston, 1983). Collectivelly, these data lend support
for a physiological role for vasopressin in circulatory control.

10
Vasopressin is not a hormone unto itself. It is reasonable to
expect that AVP should interact with other regulatory systems to
acheive homeostasis. This is most clear in terms of the cardio¬
vascular effects of vasopressin. Vasopressin, the renin-angiotensin
system and the autonomic nervous system all interact, to the point of
redundancy in maintaining circulatory control. As noted previously,
vasopressin
can
inhibit the
secretion
of
renin
from
the kidney
(Vander, 1968).
Conversely,
angiotensin
has
been
shown
to stimulate
the release
of
AVP (Phillips,
1987).
Vasopressin
and
the autonomic
nervous system interact extensively within the brain to acheive
circulatory control, as is discussed in a subsequent section. In the
periphery, vasopressin potentiates the vasoconstrictor action of nor¬
epinephrine and sympathetic nervous stimulation (Bartlestone and
Nasmyth, 1965). Conversely, blockade of the sympathetic nervous
system greatly accentuates the pressor effect of vasopressin (Pulían
et al., 1978). A methodical study by Gavras et al. (1982) provides
experimental evidence which supports the interaction of these three
effector systems in the maintenance of blood pressure.
Other Biological Effects
While the renal and cardiovascular actions of vasopressin are
perhaps the best known, other biological effects have recently been
attributed to
vasopressin.
The
central
effects of vasopressin in
cardiovascular
regulation will
be
discussed
in a subsequent
section.
Acting as a
neuropeptide,
AVP
has
been ascribed a
role in
thermoregulation, nociception, memory and behavior. Another
neuroendocrine action of vasopressin is to cause the release of ACTH
from the anterior pituitary, either by acting directly or by

11
potentiating the effect of corticotropin releasing factor (De Wied,
1983). A metabolic action has been attributed to vasopressin, by
virtue of its glycogenolytic effect in the liver (Michell et al., 1979).
Steroidogenic effects have been attributed to vasopressin in the
Leydig cells of the testes and the glomerulosa cells of the adrenals
(Nicholson et al., 1984). It has also been shown that vasopressin is
involved in platelet aggregation (Haslam and Rosson, 1972).
Vasopressin in the Central Nervous System
Vasooressinergic Projections
Vasopressin (and oxytocin) are synthesized in neurosecretory
neurons located within the paraventricular and supraoptic nuclei of
the hypothalamus. It was originally thought that oxytocin was
exclusively formed in the SON and that vasopressin was produced
only in the PVN. It is now clear that both peptides are synthesized
in separate and topographically distinct populations of neurons within
each nucleus (Swanson and Sawchenko, 1983). Each nucleus is
characterized cytoarchitecturally by the presence of large
magnocellular neurons and small, round parvocellular neurons. It has
been known since the work of Cajal (1894) that the magnocellular
neurons of the SON project to the neural lobe of the pituitary.
While morphologically quite similar, it was not recognized until forty-
two years later that the magnocellular neurons of the PVN also
project to the posterior pituitary (Ingram et al., 1936). Although the
findings of Cajal remain unchanged, histological evidence concerning
vasopressinergic projections from the PVN has been greatly expanded.
Magnocellular projections from the PVN have been traced to the
external lamina of the median eminence (Diedrickx et al., 1976).

12
Maraka et al. (1981) have suggested that vasopressin released into
this capillary portal zone may affect the synthesis and release of
ACTH and other adenohypophyseal hormones.
Parvocellular neurons of the PVN, which are morphologically and
topographically distinct from magnocellular neurons, have recently
been shown to project to neural lobe of the pituitary (Swanson and
Kuypers, 1980). Vasopressinergic fibers from the hypothalamus are
reported to innervate the lateral septum in the forebrain (Sofroniew
and Weindl, 1978). More extensive descending projections have been
traced to the intermediolateral column of the spinal cord, the nucleus
tractus
solitarius and
the
dorsal
vagal complex in
the
brainstem
(Buijs,
1978; Swanson
and
Kuypers, 1980) and the
locus
coeruleus
(Sofroniew, 1980).
Despite
the
complex organization
of
the PVN,
Swanson et al. (1980), using retrograde labeling techniques, have
convincingly demonstrated that separate neurons project to each of
the areas described above.
Afferent Projections to the PVN
The development
of histofluorescence
methods lead to
the
demonstration that the
PVN and
the SON
contained one of
the
highest densities of
noradrenergic
terminals
in all of the
brain
(Carlsson et al., 1962). By a variety of anterograde and retrograde
labeling techniques, the origins of these synaptic inputs have been
traced to A\ and Cj cell groups of the ventral medulla, the A2 and
Ci cell groups in the medial NTS and the A$ cell group in the locus
coeruleus. The parvocellular neurons of the PVN are preferentially
innervated by noradrenergic axons from the medulla and locus
coeruleus (A2 and A$) while the A\ area of the ventrolateral medulla

13
projects to magnocellular and parvocellular neurons of the PVN and
SON. In addition to rostral projections from the brainstem, the PVN
receives numerous inputs from diencephalic and telencephalic
structures. The efferent connections of the PVN and the complex
organization of this hypothalamic nucleus have been elegantly
reviewed by Swanson and Sawchenko (1983).
The ascending catecholaminergic pathways and descending
vasopressinergic pathways provide an anatomical basis for the
feedback loops which enable vasopressin to influence autonomic and
cardiovascular function and in turn, allow for adrenergic control of
vasopressin
release.
This
latter point
may
account
for the tonic
inhibitory
influence
that
oc-adrenergic
stimuli
have
on vasopressin
secretion (Kimura et al., 1981).
Role of AVP in Central Cardiovascular Regulation
The diversity of afferent and efferent projections of the PVN
has lead Swanson and Sawchenko (1980) to view this vasopressin-
synthesizing site as a visceral effector nucleus, capable of influencing
the secretion of hormones from the anterior and posterior lobes of
the pituitary and neural activity in the sympathetic and
parasympathetic divisions of the autonomic nervous system. The
vasopressinergic innervation of certain brainstem and forebrain nuclei
tends to suggest that AVP plays an important role in the central
control of circulation, exclusive of its well known vasoconstrictive
action in the periphery.
One potential role of AVP in the central control of
cardiovascular regulation involves its ability to modulate neuronal
activity of the NTS, which constitutes the anatomical substrate of the

14
baroreceptor reflex. The NTS receives information from afferent
baroreceptor fibers originating in the carotid sinus and aortic arch.
The NTS influences cardiovascular function in three ways: (a) it
relays baroreceptor activity to other brainstem areas to modulate
spinal preganglionic neuronal activity; (b) it relays baroreceptor and
chemoreceptor activity to the hypothalamus, which serves to regulate
and integrate autonomic and cardiovascular responses; and (c) it
participates in the regulation of fluid and electrolyte homeostasis by
influencing the release of A VP (Reis et al., 1984). Experimental
evidence tends to support the hypothesis that bloodborne AVP
enhances the sensitivity of the baroreceptor efferent pathway.
Courtice et al. (1984) have electrophysiological data which show that
AVP enhances the baroreflex by increasing parasympathetic vagal tone
and depressing barosympathetic outflow. In addition, Brattleboro rats,
which are deficient in vasopressin, have a depressed baroreflex, which
can be corrected by intravenous infusions of vasopressin (Imai et al.,
1983). Though somewhat paradoxical, centrally administered AVP
acts in an opposite manner to peripherally administered AVP.
Intracerebroventricular administration of AVP elcits a pressor
response, tachycardia, increased sympathetic outflow and increased
levels of plasma catecholamines (Unger et al., 1984). Matsuguchi et
al. (1982) reported that the microinjection of AVP directly into the
NTS caused increases in arterial pressure and heart rate. Berecek et
al. (1984) reported similar findings following microinjections of AVP
into the locus coeruleus.
In addition to the reciprocal neuroanatomical pathways between
the PVN and NTS that are described above, functional interactions

15
have also been noted. Lesions of the PVN prevent the elevation in
arterial pressure and heart rate associated with aortic baroreceptor
deafferentation (Zhang et al.t 1983). Employing electrophysiological
techniques, Ciriello and Calaresu (1980) have shown that the NTS and
the dorsal motor nucleus of the vagus relay cardiovascular
information
directly to the
PVN.
In the same
study
it was also
shown
that
electrical stimulation of the PVN lead
to an
increase in
arterial
pressure and heart
rate and inhibited the
reflex
bradycardia
evoked
by
stimulation of
the
carotid sinus nerve.
Conversely,
destruction
of the Aj region, the
group of noradrenergic
cell bodies
thought
to
tonically inhibit
AVP
secretion, results
in a
condition of
fulminating hypertension, marked by high levels of vasopressin in the
plasma (Blessing et al., 1982).
Thus, a feedback loop appears to exist between the PVN and the
NTS which
serves to
modulate
the
ability of the baroreflex
to
regulate arterial pressure.
Since
the
NTS is the
primary afferent
relay for baroreceptor fibers and
the
dorsal motor
nucleus of
the
vagus contains vagal
cardioinhibitory preganglionic neurons,
projections to
these structures from
the
PVN appear
to participate
in
the central
control of
cardiovascular
regulation.
Vasopressin
also
appears to
influence other sites
in
the central
nervous system
involved in
circulatory
control, such
as the locus
coeruleus.
A
vasodepressor
function
has been
attributed to
the action
of
vasopressin in
the dentate gyrus of
the
hippocampus
(Versteeg et
al.,
1984).

16
Vasopressin Receptors
Current
dogma
holds
that all peptide or protein
hormones
initiate their
actions
by
binding to membrane-bound receptors
located
on the cell
surface
of
the
target tissues. The interaction
of the
hormone with its receptor causes the activation of one of a number
of second messenger systems (cAMP, cGMP, PI). Depending on the
tissue and receptor involved, this can result in the activation of
enzymes, mobilization of intracellular stores of calcium (which, in
turn can affect enzyme activity), phosphorylation of membrane
proteins or the activation of ion channels, leading to the appropriate
biological response.
AVP interacts with at least two subclasses of vasopressin
receptors, depending on the tissue in question. Michell et al. (1979)
formally designated these different subtypes as Vj and V2 vasopressin
receptors on the basis of the effector systems to which these
receptors are coupled. Elucidation of the subtypes has been greatly
facilitated by the synthesis of structurally distinct vasopressin
analogues which bind differentially to the V] or V2 receptor.
Development of these analogues has resulted in the design of specific
agonists and antagonists for each of the receptor subtypes (Sawyer et
al., 1981).
V1 receptors are coupled to phospholipase C and the hydrolysis
of phosphatidyl inositol (PI) with the subsequent formation of inositol
phosphates (IP) and diacylglycerol (DG). Inositol phosphates have
been shown to increase the mobilization of intracellular stores of
calcium. Diacylglycerol on the other hand, activates the enzyme
protein kinase C. V2 receptors are coupled to adenylate cyclase and

17
the formation of cAMP. Both Vj and V2 receptors are functionally
linked to their respective effector systems through guanine nucleotide
regulatory proteins (G proteins). Thus, the antidiuretic action of
vasopressin is mediated through V'2-type receptors while the
vasoconstrictive, glycogenolytic and steroidogenic actions of AVP are
mediated by Vj-type receptors, based on structure-activity
relationship studies with vasopressin analogues or by directly
determining the second messenger. A novel vasopressin receptor
(Vib) which displays a pharmacological profile that does not clearly
fall into one category or the other has been reported to mediate the
effects of AVP in the anterior pituitary (Antoni, 1984). Furthermore,
recent evidence suggests that at least one tissue may possess both V]
and V2 receptor subtypes (Chapter 6).
Kidney
Evidence for the binding of vasopressin to hormone-responsive
cells in the kidney was first suggested by the autoradiographic
studies of Darmady et al. (1960). Specific binding of ^H-AVP to
plasma membranes of the renal medulla was subsequently reported by
two independent laboratories (Campbell, et al., 1972; Bockaert et al.,
1973). Neither study succeeded in localizing the binding to plasma
membranes of renal tubular origin nor were they able to distinguish
between basolateral and apical membranes. However, Bockaert et al.
(1973) and others (Nakahara et al., 1978) were able to demonstrate
the correlation between receptor occupancy and the activation of
adenylate cyclase. These studies were consistent with the finding
that cAMP and vasopressin-induced changes in membrane permeability
were only noted following application to the peritubular side of

18
isolated renal tubules (Grantham and Burg, 1966). It may be
concluded that these vasopressin binding sites constitute the
receptors which mediate the antidiuretic action of the hormone in the
distal tubular collecting segments of the nephron. Since then,
vasopressin binding sites have been characterized on a variety of
membranes prepared from porcine, bovine, rat and human kidneys
(Jard, 1983). A more convincing arguement for the epithelial
localization of vasopressin binding sites comes from the finding of
Roy and Ausiello (1981a). Using tissue culture techniques, these
investigators were the first to characterize vasopressin receptors in
LLC-PKj cells. This cell line is derived from tubular elements of the
pig kidney (Hull et al., 1976). Both intact cells and renal membranes
all appear to possess a single class of high affinity binding sites (Kj)
= 1-25 nM).
All vasopressin-sensitive renal tissue need not be of epithelial
origin, however. Vasopressin receptors have been characterized on
glomerular mesangial cells (Jard et al., 1987) and appear to mediate
the vasopressin-induced contraction of this tissue (Ausiello et al.,
1980). This action may account for the alteration in glomerular
ultrafiltration observed with vasopressin in the intact glomerulus.
Vasopressin binding sites in intact glomeruli has recently been
reported (Stoeckel et al., 1987). Thus, caution must be employed
when interpreting vasopressin binding studies conducted in the kidney
by autoradiography or when using crude membrane preparations.
Vasculature
In order to effect the cardiovascular actions of vasopressin
mediated through its vasoconstrictive properties, it follows that

19
vasopressin binding sites must be present on the smooth muscle cells
of the resistance vasculature. This observation has proven difficult
due to the high degree of cellular heterogeneity of the vascular wall
and technical problems encountered in preparing pure plasma
membrane fractions derived from smooth muscle cells. These
obstacles have apparently been overcome as judged by the report by
Schiffrin and Genest (1983) describing vasopressin binding in a
mesenteric artery membrane preparation. AVP binding has also been
demonstrated in aortic smooth muscle cells in primary culture (Penit
et al., 1983) and in an established smooth muscle cell line (A-10
cells) (Stassen et al., 1987). These receptors, like those in renal
tissue,
exhibit
nanomolar affinity
constants
and
a
single
class of
binding
sites.
Whereas renal
tubular
vasopressin
receptors are
coupled
to
adenylate cyclase activation,
those
in
the
vasculature
stimulate
the
hydrolysis of phosphatidyl inositol
and
the
mobilization
of intracellular stores of calcium, as a means of signal transduction
(Aiyar et al., 1986).
Central Nervous System
Vasopressin receptors in the brain have been characterized using
traditional biochemical techniques and their distribution analyzed by
means of autoradiography (Dorsa et al., 1983). This group found that
the receptor behaves kinetically as those found in the periphery.
Furthermore, they reported that the NTS in the brainstem displayed a
high level of binding. Other investigators have confirmed this
finding and extended their observations to include AVP binding in the
hippocampus and lateral septum (Biegon et al., 1984). These findings
are consistent with the functional, behavioral and immunocytochemical

20
data which contend that vasopressin acts in the brain as a neuro-
transmitter or neuromodulator. The presence of AVP receptors in the
regions mentioned above parallel the vasopressinergic pathways
involved in central cardiovascular regulation.
The nature of the effector system coupled to the vasopressin
receptor in the brain still remains in question. Competition of ^H-
vasopressin binding with vasopressin analogues suggests that the
receptor resembles the vascular or V \ subtype (Cornett and Dorsa,
1985; Audigier and Barberis, 1985), although no direct measurements
of vasopressin-stimulated PI hydrolysis have been reported. However,
a presynaptic membrane phosphoprotein known as B50 which is a
substrate for protein kinase C, is phosphorylated upon the addition of
AVP to rat hippocampal synaptic membranes (Hinko and Perlmutter,
1987).
These data
tend
to
suggest that
vasopressin acts through Vi¬
type
receptors in
neuronal
membranes
to cause the formation of
inositol
phosphates
and
diacylglycerol.
DG may activate
protein
kinase
C which in
turn,
phosphorylates
integral membrane
proteins.
Given
the importance
of
calcium in
the process of
synaptic
transmission, the actions of IP cannot be excluded either.
Other Tissues
A burgeoning number of studies have recently reported the
presence of vasopressin receptors in a variety of tissues. ^H-
vasopressin binding and its relation to glycogen phosphorylase activity
was reported by Cantau et al. (1980). Activation of this enzyme and
its resulting glycogenolytic action is mediated by Vj receptors
coupled to PI hydrolysis (Michell et al., 1979). In the anterior
pituitary gland, vasopressin receptors were first characterized by

21
Lutz-Bucher and Koch (1983). Antoni (1984) reported that the ligand
specificity of pituitary vasopressin receptors was distinct from
previously characterized Vj and V2 receptors although no
measurements of the second messengers were conducted. Gaillard et
al. (1984) however, did report that vasopressin failed to influence
adenylate cyclase activity in pituitary membrane fractions, but did
potentiate CRF-stimulated cAMP accumulation. Balia et al. (1985)
described the binding and signal transduction mechanisms for
vasopressin in membranes prepared from rat adrenal capsules (chiefly
zona glomerulosa). Displacement studies suggested that the receptor
was of the V\ variety and incorporation studies indicated that A VP
32
promoted P uptake into PI. This effect was blocked by V\
antagonists as was AVP-stimulated PI hydrolysis. The net effect of
vasopressin binding in isolated zona glomerulosa cells was the
stimulation of aldosterone secretion. Whereas vasopressin stimulates
steroid biosynthesis in the adrenals, it inhibits testicular Leydig cell
steroidogenesis. Receptors which mediate this effect were described
by Meidan and Hsueh (1985). Relative binding potencies of selective
agonists and antagonists suggest that it is a V\ subtype. Finally, a
number of cellular elements in the bloodstream are reported to
possess vasopressin receptors. Binding sites on platelets were
described by Berrettini et al. (1982). Mononuclear phagocytes were
shown by Block et al. (1981) to possess vasopressin receptors.
Although A VP has been shown to cause platelet aggregation, the
function of vasopressin receptors in monocytes is unknown. Table 1-
1 summarizes the current state of knowledge concerning vasopressin
receptors in biological tissues and their significance.

22
TABLE 1-1.
BIOLOGICAL ACTIONS OF ARGININE8-VASOPRESSIN.
Tissue
Action
Receptor Subtype
Reference
Brain
Neuromodulator:
cardiovascular
control, nociception,
thermoregulation
Vl
DeWied, 1983
Anterior
pituitary
ACTH release
Vlb
Antoni, 1984
Platelets
Aggregation
?
Berrettini et
al„ 1982
Mononuclear
phagocytes
?
7
Block et al.,
1981
Blood vessels
Vasoconstriction
Vl
Schiffrin &
Genest, 1983
Liver
Glycogenolysis
Vl
Michell et al.,
1979
Adrenal
glomerulosa
cells
Steroidogenesis
Vl
Balia et al.,
1985
Testes
Inhibits androgen
biosynthesis
Vl
Meidan &
Hsueh, 1985
Juxtaglo¬
merular cells
Inhibits renin
secretion
?
Vander, 1968
Glomerular
mesangial cells
Contraction
Vl
Jard et al.,
1987
Renal tubular
epithelium
Solute, ion
transport
v2
Morel et al.,
1987

23
Significance
Myriad biological functions have now been attributed to
vasopressin. The preponderance of evidence suggests that its major
action pertains to its role in circulatory and fluid homeostasis. This
is achieved in a concerted manner, through fluid and electrolyte
reabsorption, vasoconstriction, modulation of the baroreflex and
central regulation of cardiovascular centers.
The actions of vasopressin can be controlled in a number of
ways. One is to control vasopressin secretion itself. The levels of
circulating
AVP are
regulated
by
osmotic
and
non-osmotic
mechanisms.
Endocrine
feedback
systems
also act
to
control AVP
release. For example, dehydration will cause an osmotically-mediated
release of vasopressin but, in addition, will activate the renin-
angiotensin system in the brain, which in turn will stimulate the
release of AVP.
The
action of vasopressin can also
be
affected at the target
tissue.
This usually implies some type
of
modulation of
hormone
receptor
or post-receptor modification.
The
concept of
hormone
receptor
regulation has been the subject
of
several recent
reviews
(Catt et al., 1979; Roth and Taylor, 1982; Lefkowitz et al., 1984).
Receptor regulation can occur by "homologous" or "heterologous"
mechanisms. The former implies that a hormone can modulate the
cell membrane content of its own specific receptor. Alternatively,
receptor number may remain the same but the system is desensitized
through an uncoupling of the receptor from the signal transducing
elements, often by covalent modification of the receptor.
Heterologous regulation suggests that other hormones or factors can

24
regulate the receptor of a second hormone. For example, a steroid
hormone may affect the expression of a peptide hormone receptor
through transcriptional events. In any case, regulation of receptors
can affect the cellular sensitivity to hormonal stimulation. For the
antidiuretic hormone, this is of tanamount importance in its ability to
maintain fluid homeostasis.
The studies delineated in this dissertation were conducted to
(a) characterize antidiuretic hormone receptors in the kidney, (b)
study their physiological regulation in vivo, by altering the state of
fluid balance, ranging from a condition of dehydration to a volume-
expanded model of hypertension, and to (c) establish a "parallel"
system ¿n vitro. to examine receptor regulation and signal
transduction mechanisms at the cellular level. Hopefully, this
information will expand the current state of thinking about the
mechanisms of action of arginine vasopressin and its role in fluid and
electrolyte homeostasis and circulatory control.

CHAPTER 2
CHARACTERIZATION OF 3H-VASOPRESSIN BINDING
SITES IN RENAL TUBULAR BASOLATERAL MEMBRANES
Introduction
Arginine^-vasopressin in most species is the mammalian anti¬
diuretic hormone. It acts upon distal tubular segments of the
nephron to promote fluid reabsorption by altering the permeability of
the tubular luminal membrane to water. Vasopressin-stimulated
activation of the enzyme adenylate cyclase initiates the biochemical
cascade responsible for generating transepithelial net fluxes of ions
and solutes.
The presence of vasopressin binding sites in the kidney was
described as early as 1960 (Darmady et al.) and autoradiographic
reports of vasopressin receptors continue up to the present day
(Stoeckel et al., 1987). Early studies which utilized traditional
biochemical techniques to characterize vasopressin receptors in plasma
membranes prepared from renal medullary tissue did so in somewhat
crude preparations which may have been contaminated with vascular
and non-epithelial elements (Campbell et al., 1972; Bockaert et al.,
1973). These types of studies, therefore, fail to distinguish between
vasopressin-sensitive cells of tubular epithelial origin from cells which
respond to AVP located in the glomerulus and the renal vasculature.
Nor do these studies allow one to localize the binding specifically to
the basolateral as opposed to the apical region of hormone-sensitive
epithelia. Although it is well-established that the vasopressin-
25

26
sensitive adenylate cyclase system is present on the peritubular side
of tubular epithelia (Grantham and Burg, 1966), the characterization
of vasopressin receptors localized specifically to the basolateral
membrane has not been previously described. This study was
conducted to determine the characteristics of vasopressin binding for
a basolateral membrane preparation of rat renal tubular epithelia.
Materials and Methods
Animals and Chemicals
Male Sprague-Dawley rats weighing 200-250g were obtained from
the Animal Resource Facility at the University of Florida. Rats were
housed individually in 20x18x24 cm wire mesh cages, exposed to a
12:12 hour light:dark cycle, provided a standard Purina rat chow diet
and allowed free access to tap water.
Percoll and synthetic peptides were purchased from Sigma
Chemical Co. (St. Louis). d(Et)2Tyr(Et)D-AVP (Vj) and d(CH2)5D-
Ile^-Ile^-AVP (V2) vasopressin analogues were generous gifts of Dr.
M. Manning of the Medical College of Ohio in Toledo. Tritiated 8-L-
arginine [phenylalanine-3,4,5-^H]-AVP (^H-AVP) used in receptor
binding studies was purchased from New England Nuclear Corp.
(Boston) with a specific activity of 70 Ci/mmol. Other reagents were
obtained from Sigma, unless otherwise indicated.
Membrane Preparation
Animals were sacrificed by decapitation. Renal basolateral
membranes were prepared according to the density gradient
centrifugation method of Boumendil-Podevin and Podevin (1983), with
slight modification. Kidneys were removed, decapsulated and rinsed
in a chilled buffer containing 250 mM sucrose, 2 mM Tris-HEPES, 0.1

27
mM PMSF, pH 7.4. The tissue was minced and then homogenized
with 5 strokes of a Dounce homogenizer followed by 5 strokes of a
teflon pestle homogenizer at a setting of 800 rpm. The resulting
homogenate was centrifuged at 1,000 x g for 10 minutes at 4°C. The
pellet (P]) is discarded and the supernatant (Sj) centrifuged at 22,000
x g for 15 minutes at 4°C. The supernatant (S¿) is discarded, leaving
a triple-layered pellet (P2). The upper two layers of the pellet were
removed by gentle vortexing and homogenized in 10 ml of the Tris-
HEPES-sucrose buffer without PMSF. Percoll (1.2 ml) is added and
mixed, then centrifuged at 40,000 x g for 35 minutes at 4°C. The F |
band (Basolateral membranes, density = 1.037 g/ml) is aspirated and
diluted in 5 volumes of 2 mM Tris-HEPES, 85 mM KC1, 85 mM
sucrose, pH 7.4. Percoll
was removed
from the
mixture
by
ultracentrifugation at 60,000
x g for
15
minutes at
4°C.
The
supernatant is decanted, the
membranes
are
gently washed
away
from
the Percoll pellet, resuspended in Tris-HEPES-sucrose buffer with
PMSF and stored in liquid N2. An outline of this membrane
preparation is depicted in Figure 2-1. Protein content was
determined with the Folin reagent, using bovine serum albumin as the
standard, according to the method of Lowry et al. (1951).
Determination of Na+K+-ATPase Activity
The Na+K+-ATPase activity was determined by measuring the K+-
stimulated, ouabain-sensitive p-nitrophenylphosphate (PNPP) hydrolysis
at pH 7.8, based on the method of Yoshida et al. (1969). Briefly, the
assay buffer was composed of 50 mM Tris-HCl (pH 7.8), containing 5
mM MgCl2, 2.5 mM EDTA, 50 mM KCI and 5 mM PNPP. The reaction
was carried out in the presence and absence of ouabain (1 mg/ml) by

28
Kidney tissue homogenized in 2 mM Tris-HEPES, 250 mM Sucrose buffer,
pH 7.4 containing 0.1 mM PMSF.
Homogenate-» IK x g, 10’
t
S, 22K x g, 15’
1
P2 (upper two layers of "triple” pellet)
l
10% Percoll Gradient-» 40K x g, ;
Fj = Basolateral Membrane (1.037g/ml)
Fj = Brush Border Membrane (1.041g/ml)
F3 = Mitochondrial Membrane (1.067g/ml)
Figure 2-1. Scheme for preparation of basolateral membranes from
renal tubular epithelia. Kidneys from male Sprague Dawley rats
homogenized in Tris-Sucrose buffer containing the protease inhibitor,
PMSF. Membranes prepared by density gradient and differential
centrifugation, as described in "Methods" section.

29
the addition of 10 /¿I membranes (100 fig protein) to 0.5 ml of the
assay buffer. The mixtures were incubated for 2-10 minutes at 37°C.
The reaction was stopped by the addition of 3.5 ml IN NaOH and
absorbance was read at 410 nm. Net Na+K+-ATPase activity was
determined by subtracting the PNPP hydrolysis activity in the
presence of ouabain from the activity in the absence of ouabain.
Specific activity was calculated based on a molar extinction
coefficient e = 15,400 M_1cm_1, and final values were expressed as
units/mg protein (where 1 unit = 1 fimol PNPP hydrolyzed per
minute).
Vasopressin Receptor Assay
Membranes, corresponding to 100-200 fig protein were suspended
in 300 fi\ of 100 mM Tris-HCl buffer containing 5 mM MgCl2, 1 mM
EGTA and 0.1% BSA, pH 7.3 at 20°C. The incubation buffer
contained the peptidase inhibitors aprotinin (1000 K.I.U./ml), leupeptin
(1 /ig/ml) and pepstatin A (1 /¿g/ml), to prevent proteolytic
degradation of the peptides or radioligand. For saturation studies,
triplicate samples were incubated for 45 minutes at 20°C with
concentrations of ^H-AVP ranging from 50 pM to 5,000 pM. Non¬
specific binding was determined in the presence of 5 /¿M unlabelled
AVP. Competitive inhibition by various peptides was assessed in the
presence of 1.0 nM ^H-AVP. The incubation was terminated by the
addition of 1 ml ice-cold Tris-buffer and the bound tracer was
rapidly separated from the unbound moiety by centrifugation at 10,000
x g for 2.5 minutes at 4°C. The membrane pellet was dissolved in
100 fi\ of formic acid and transferred to a mini vial containing 4 ml of
liquid scintillation fluid (Liquiscint; National Diagnostics).

30
Radioactivity was determined in an LKB LS counter. Samples were
corrected for variations in efficiency of counting by an internal
program and expressed as disintegrations per minute (dpm). Based on
the radioactivity and protein in each sample, values are reported as
f moles bound/mg protein. Specific binding was calculated as the
difference between total binding and non-specific binding.
Analysis
Data from saturation and competition experiments were analyzed
using iterative curve fitting programs. Data are reported as the
mean + S.E.
Results
The activity of Na+K+-ATPase, a marker enzyme for the
basolateral membrane was markedly enriched in the F\ fraction,
which displayed a 3.25-fold increase in activity over that of the P2
pellet and a 10-fold increase when compared to the homogenate
(Table 2-1). A two-fold enrichment of A VP binding per unit protein
was achieved in the BL membrane preparation compared to the P2
pellet. A tritiated ligand was chosen for these experiments since
iodine destroys the biological activity of the vasopressin molecule
(Dorsa et
al.,
1983).
The
binding of ^H-AVP
to renal basolateral
membranes
was
linearly
related to the amount
of protein (i.e.,
membrane)
present in
the
assay (Figure 2-2).
Association and
dissociation
of
3h-avp
is
illustrated in Figure
2-3. Equilibrium
binding of the ligand occurred within 30 minutes and was stable for
up to 2 hours. Upon equilibrium, binding was shown to be reversible
by the addition of 1 /iM AVP. Specific binding, determined in the
presence of an excess amount of AVP was 80-85% of total binding.

31
TABLE 2-1.
K+-STIMULATED, OUABAIN-SENSITIVE Na+-K+ ATPase ACTIVITY
Fraction
Units*/Mg Protein (x 10
Purification
H
0.08 ± .04
IX
P2
0.26 + .01
3.25X
Fl
0.80 ± .01
10X
Potassium-stimulated, ouabain-sensitive Na+-K+-ATPase activity.
Activity determined by measuring K.+-stimulated, ouabain-sensitive p-
nitrophenylphosphate (PNPP) hydrolysis at pH 7.8. Values are
expressed as units/mg protein, where 1 unit = 1 pmole PNPP
hydrolyzed per minute. H, homogenate. P2, 224,000g pellet. Fj,
basolateral membrane fraction. *(1 unit = 10~6 moles/min.)

32
ug Protein
Figure 2-2. 3H-vasopressin binding as a function of
membrane protein concentration. Receptor binding protocol
in "Methods" section. Protein concentration determined
method of Lowry et al. (1951).
basolateral
described
by the

33
Figure 2-3. 3H-vasopressin (3H-AVP) binding in renal tubular
basolateral membranes as a function of time. Receptor binding
protocol described in "Methods" section. Association: Total binding
determined at 1 nM [3H-AVP]. Non-specific binding (NSB) determined
in the presence of 1 nM [3H-AVP] + 1 /xM AVP. Total - NSB =
Specific, which represents 80-85%. Dissociation: At equilibrium
binding (1 hr), 1 nM [3H-AVP] is dissociated from receptor site by
the addition of 1 /xM unlabeled AVP.

34
Specificity of the receptor for related A VP analogues and non-
related peptides is illustrated in Figure 2-4. The data show that
lysine vasopressin (LVP), a vasopressin peptide differing from AVP by
only one amino acid in the number 8 position, is some 13 times less
effective in displacing the ligand than AVP (IC50: AVP = 0.74 nM vs.
LVP = 9.7 nM). Furthermore, the synthetic vasopressin analogue
d(CH2)5D-Ile2-Ile4-AVP, a specific tubular (V2) antagonist is some
3,700 times more effective in displacing the ligand than is the
vascular (Vj) antagonist d(Et)2Tyr(Et)D-AVP (IC50: V2 = 0.0039 nM
vs. Vj = 14.7 nM). At concentrations of 1 /xM, oxytocin, angiotensin
II and D-ala-met-enkephalinamide were incapable of displacing even
50% of the ligand. A representative saturation isotherm for ^H-AVP
binding to membranes is depicted in Figure 2-5. Transformation of
the data for Scatchard analysis is also represented in Figure 2-5
(insert). Analysis of the data indicates that the maximal binding
capacity (^max) for vasopressin receptors on the membranes is ~220
f mol/mg protein and the affinity constant for the receptors is 0.61
nM.
Discussion
Several studies have demonstrated the presence of vasopressin
receptors in the kidney (Campbell et al., 1972; Bockaert et al., 1973;
Dorsa et al., 1983; Stoeckel et al., 1987). The present study reports
the preparation of Na+K+-ATPase-enriched renal tubular epithelial
basolateral membranes and the characterization of ^H-AVP binding in
these membranes. This preparation allows one to separate, by
differential and density gradient centrifugation, tubular epithelial
membranes possessing vasopressin receptors linked to adenylate

35
Figure 2-4. Specificity of the vasopressin receptor in renal tubular
basolateral membranes. Competition of 3H-vasopressin (3H-AVP)
binding at 1 nM by vasopressin analogues and unrelated peptides.
Receptor binding protocol described in "Methods" section. Vj
antagonist, d(Et)2Tyr(Et)D-AVP; V2 antagonist, d(CH2)5D-Ile2-Ile4-
AVP; A VP, arginine vasopressin; LVP, lysine vasopressin; OXY,
oxytocin; Ang II, angiotensin II; DAME, D-ala-met-enkephalinamide.

36
[3H-AVP] Figure 2-5. Saturation isotherm and Scatchard plot (insert)) of 3H-
vasopressin (3H-AVP) binding in renal tubular basolateral membranes.
Concentration of 3H-AVP ranged from 50 pM to 3,000 pM. Specific
binding determined in the presence of 3 /¿M unlabeled AVP. Details
described in "Methods" section.

37
cyclase (V2) from vascular endothelial and glomerular mesangial cells
possessing vasopressin receptors coupled to the hydrolysis of
phosphatidyl inositol (V[). Recent evidence suggests that certain
renal epithelial tissue (LLC-PK] cells) possess both V | receptors
(Garg et al., 1988) and V2 receptors (Lester et al., 1985).
The basolateral membrane preparation yielded a 3.25-fold
increase in Na+K+-ATPase activity with respect to the P2 pellet and a
10-fold increase compared to the homogenate. Vasopressin binding
sites per unit protein doubled in the basolateral preparation over that
in the P2 fraction. The discrepancy may be explained in terms that
while basolateral membranes may be enriched, not all of the
membranes may possess AVP receptors due to their heterogeneous
distribution along the renal tubule (Morel et al., 1987). Scatchard
analysis revealed that a single class of high affinity AVP receptors is
present on these basolateral membranes. Vasopressin binding was
rapid, reversible, specific and displayed a pharmacological profile
characteristic of V2 (adenylate cyclase-coupled) vasopressin receptors.
The maximal binding capacity determined in the present study (~220
fmol/mg P) is somewhat less than the value of 300 fmol/mg P
obtained by Rajerison et al. (1977) but is in close agreement with the
value of 228 fmol/mg P reported by Cornett and Dorsa (1985). This
variation is not entirely surprising, however, given the differences in
membrane preparation and other variations in the protocol.
In summary, these data demonstrate the presence of high
affinity receptors for arginine®-vasopressin on basolateral membranes
prepared by differential and density gradient centrifugation from rat
renal tubular epithelia. These data are consistent with and

38
complement other studies that have examined the vasopressin-sensitive
adenylate cyclase system in the kidney and its coupling to V2-type
receptors (Roy et al., 1975; Rajerison et al., 1977; Butlen et al., 1978).

CHAPTER 3
DEHYDRATION-INDUCED DOWNREGULATION OF RENAL
TUBULAR VASOPRESSIN RECEPTORS
Introduction
Several endocrine systems act to maintain fluid and electrolyte
balance. Alterations of fluid homeostasis influence plasma levels of a
number of hormones. This is particularly evident during dehydration.
This condition leads to elevated circulating levels of vasopressin
(Aisenbrey et al.,
1981;
Woods and Johnston,
1983),
angiotensin II
(Di Nicolantonio
and
Mendelsohn, 1986)
and
endogenous opiate
peptides (Mata et
al.,
1977). Plasma levels of
atrial natriuretic
peptide on the other hand tend to be lower, which is consistent with
the physiological role of this hormone (Ogawa et al., 1987). In turn,
these hormones act upon their respective receptors in proportion to
the response required to re-establish volume homeostasis. Paramount
to this task is the action of arginine vasopressin, exerting its well-
known antidiuretic and vasoconstrictive effects, through interactions
with V2 and Vj receptors, respectively.
Peptide hormone receptors can be regulated by the concentration
of hormone to which they are exposed. This can often lead to a
condition described as "downregulation." This phenomenon results
from withdrawal of cell surface membrane receptors by an
internalization process. Reduced cellular sensitivity to hormonal
stimulation can also occur by an uncoupling of the existing receptors
from the signal-transducing effector system. The most well-
39

40
documented examples of these types of regulation come from the
elegant studies by Lefkowitz et al. (1984) involving /9-adrenergic
receptors. His group has demonstrated examples of "homologous"
regulation by receptor-specific agonists such as isoproterenol and
"heterologous" regulation, where stimulation by a drug or hormone
leads to a change in sensitivity to another unrelated drug or
hormone. They found that thyroid status could have a profound
effect on both the number and coupling of oc-adrenergic and p-
adrenergic receptors. Presumably, this control of adrenergic
receptors was exerted at the level of gene transcription. Adrenergic
receptors are by no means unique in terms of their susceptibility to
regulation. Insulin receptors too, undergo homologous regulation by
insulin (Gavin et al., 1974). Insulin receptors also undergo
heterologous regulation, under the action of ^-adrenergic agents
(Pessin et al., 1983). Additional examples and more insight into the
phenomena of receptor regulation may be gained from the recent
reviews by Roth and Taylor (1982) and Poste (1984).
The cellular sensitivity to vasopressin can be drastically reduced
following chronic stimulation by the hormone. When the isolated
toad bladder, an epithelial model for the mammalian renal tubule, is
stimulated repetitively with high concentrations of the neuro¬
hypophyseal hormone, the osmotic water flow across the bladder
diminishes progressively (Eggena, 1981). Renal medullary plasma
membranes incubated with high concentrations of vasopressin display
reduced hormone-sensitive adenylate cyclase activity (Roy et al.,
1976). Furthermore, vasopressin-elicited desensitization of adenylate
cyclase activity has been shown in renal membranes prepared from

41
rats injected with pharmacological doses of vasopressin (Rajerison et
al., 1977). AVP-induced downregulation of vasopressin receptors and
a concomitant desensitization of vasopressin-sensitive adenylate
cyclase activity has been demonstrated in a renal epithelial cell line
(Lester et al., 1985).
Thus, vasopressin can affect the cellular sensitivity of its target
tissue by regulating its own receptor. Because dehydration is a
condition characterized by high circulating levels of AVP, the present
study was conducted in order to determine whether vasopressin
receptors are commensurately regulated under conditions of water
deprivation.
Materials and Methods
Animals
Male Sprague-Dawley rats (n=6 per group) weighing 200-250g
were obtained from the Animal Resource Facility at the University of
Florida.
Rats were
housed individually in 20x18x24
cm
wire mesh
cages, exposed to a
12:12 hour light:dark cycle,
provided
a standard
Purina rat
chow diet
and allowed free access
to tap
water. The
dehydrated
experimental
group was deprived of
water
for
72 hours
prior to sacrifice.
Chemicals
Tritiated 8-L-arginine [phenylalanine-3,4,5-^H]-AVP ([^HJ-AVP)
used in
receptor binding studies
was purchased
from
New
England
Nuclear
Corp. (Boston) with a
specific
activity
of
70
Ci/mmol.
lodinated
125
vasopressin ( I-AVP),
specific
activity
of
2200
Ci/mmol,
was also
obtained from New
England
Nuclear
for
use
in the
vasopressin
radioimmunoassay (RIA). AVP antibody
was
purchased

42
from Amersham (Arlington Hts., IL) while AVP standard was obtained
from Peninsula Labs (Belmont, CA). Other reagents were obtained
from Sigma, unless otherwise indicated.
Membrane Preparation and Plasma Collection
Animals were sacrificed by decapitation and trunk blood was
collected. A 100 yl sample was obtained separately for the
determination of plasma osmolality while the remainder was collected
in chilled tubes containing 0.3 M EDTA (50 y 1/ml) for the
determination of plasma AVP concentration. Blood samples were
centrifuged at 3,000 x g for 10 minutes at 4°C, the plasma removed
and stored at -20°C. Renal basolateral membranes were prepared by
modifying the density gradient centrifugation method of Boumendil-
Podevin and Podevin (1983), according the description provided in
Chapter 2.
Vasopressin Receptor Assay
Membranes, corresponding to 100-200 yg protein were suspended
in 300 yl of 100 mM Tris-HCl buffer containing 5 mM MgCl2, 1 mM
EGTA and 1
3.1%
BSA, pH
7.3 at
20°C.
Triplicate
samples
were
incubated for
45
minutes
at 20”C
with
concentrations
of 3H-
-AVP
ranging from
50
pM to
5,000
pM.
Non-specific
binding
was
determined in the presence of 5 yM unlabelled AVP. Further details
of the receptor binding protocol are described in Chapter 2.
R1A for Vasopressin
Plasma samples for the determination of AVP were thawed and
purified using SepPak Cjg cartridges (Waters). Briefly, the
purification process is as follows: Cartridges are pre-wetted with 5 ml
methanol followed by 5 ml 1% trifluroacetic acid (TFA). A 1 ml

43
plasma sample is acidified with 1% TFA and applied to the column
dropwise. The cartridge was rinsed with 5 ml 1% NaCl/1% TFA, then
A VP was eluted with 4 ml methanol:H20:TFA (80:19:1). The samples
were evaporated under a light stream of air then reconstituted in 0.5
ml of assay buffer (50 mM phosphate buffer, pH 7.4 containing 0.2%
bovine serum albumin and 10 mM EDTA). The RIA was carried out
by incubating 100 /xl samples or known amounts of standard (0.1-20 pg
125
AVP) with 25 /il AVP antiserum and 25 /xl I-AVP (corresponding to
3,000 cpm) for 24 hours at 4°C. Free ligand was sequestered by the
addition of dextran-coated charcoal and centrifugation at 2,000 x g
for 15 minutes. The supernatant containing the antibody-bound
ligand was aspirated and counted in a Beckman 5500 gamma counter
125
at an efficiency of 79% for I. Values are expressed as pg AVP.
Cross-reactivity with related peptides is <1%. Recovery of AVP for
the extraction procedure was determined to be >90%. Sensitivity of
the assay was 0.15 pg per tube.
Plasma Osmolality
Plasma osmolality was determined using a Westcor model 5500C
vapor pressure osmometer. Data are expressed as mosmols/kg H2O.
Analysis
Data from saturation and competition experiments were analyzed
using iterative curve fitting programs. Data are reported as the
mean + S.E. Statistical significance of the data was assessed using
one-way analysis of variance and Student’s t-test. A level of p<.05
was considered to be significant.

44
Results
The state of hydration of the rats was evaluated by comparing
the urine ouput and osmolality, plasma osmolality and plasma
vasopressin concentration among the two groups. Urine output in the
normally hydrated control group was 10.611.1 ml/24 hr while that of
the dehydrated group was 1.9±0.8 ml/24 hr (p<.05 vs. control) (Figure
3-1 A). Urine osmolality was significantly elevated among the
dehydrated animals (2789±169 mosmol/kg) when compared to control
values (1447±225 mosmols/kg) (p<.05) (Figure 3-IB). Similarly, plasma
osmolality was raised from control values of 288 ± 0.8 mosmol/kg to
299 ± 1.7 mosmol/kg among the water deprived group (p<.05) (Figure
3-2 A). Vasopressin values were determined from the standard curve
shown in Figure 3-3. In response to the heightened osmolality of the
extracellular fluid, plasma vasopressin levels were commensurately
elevated. AVP concentrations were raised 11.5-fold, from a value of
0.21 ±0.02 pg/ml in the plasma of the control animals to 2.4±0.3 pg/ml
in the plasma of the dehydrated animals (p<.05) (Figure 3-2B).
^H-vasopressin binding was determined in renal tubular epithelial
basolateral membranes prepared from the kidneys of the control and
dehydrated animals. A representative saturation isotherm for
AVP binding is depicted in Figure 3-4. Transformation of the data
for Scatchard analysis is represented in Figure 3-5. Analysis of the
data between the two groups indicates a significant reduction in the
number of vasopressin receptors on the membranes from the
dehydrated animals (Control Bmax = 184+15 f mol/mg protein vs.
Dehydrated Bmax = 114+2.5 fmol/mg protein) (p<.01). No significant
difference was observed in the affinity of the receptor between the

45
Figure 3-1. Urine output and urine osmolality in control and
dehydrated rats. A: urine output (Uv). B: urine osmolality (Uosm).
Osmolality determined by vapor pressure osmometry. Error bars
represent S.E.M. *p<.05 vs. control.

Plasma Osmolality (mOsm/kg)
46
A
B
Figure 3-2. Plasma osmolality and plasma vasopressin concentrations
in control and dehydrated rats. A: Plasma osmolality, determined by
vapor pressure osmometry. B: Plasma AVP determined by RIA. Error
bars represent S.E.M. *p<.05 vs. control. AVP, arginine vasopressin;
RIA, radioimmunoassay.

47
pg AVP
Figure 3-3. Standard curve for arginine vasopressin (AVP). Values
determined by radioimmunoassay. Details described in "Methods"
section.

48
Figure 3-4. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
water-replete and water-deprived (72 hr) rats. Details of binding
protocol described in "Methods" section of Chapter 2. Error bars
represent S.E.M. of triplicate determinations.

49
Bound (fmols/mg Protein)
Figure 3-5. Scatchard analyses of 3H-vasopressin binding,
determined in basolateral membranes prepared from kidneys of
replete and water-deprived (72 hr) rats. Analyses of data in
3-4. *p<.05 vs. control.
Binding
water-
Figure

50
two groups (Control Kjj = 0.61+.04 nM vs. Dehydrated Kjj = 0.64+.03
nM).
Discussion
Alterations in fluid balance can affect not only the hormones
themselves which participate in its regulation but also the receptors
which mediate the biological responses of the hormones. Thus,
dehydration causes a reduction in /c-opiate receptor binding in the
neurohypophysis (Brady and Herkenham, 1987) and a decrease in
angiotensin II binding in the adrenal medulla (Hwang et al., 1986).
Alternatively, receptors for atrial natriuretic factor are increased in
the kidney and adrenal gland, in response to reduced circulating
levels of the hormone (Lynch et ah, 1986).
These data in the present study show that V2-type vasopressin
receptors located on renal tubular epithelial basolateral membranes
are modulated when fluid homeostasis is compromised by dehydration.
A significant reduction in the number of vasopressin receptors (£*max)
was noted in the renal tubular basolateral membranes prepared from
the water-deprived animals. There was no difference between the
two groups with respect to the affinity (K q) of the receptor for the
hormone. Water deprivation for 72 hours significantly elevated
plasma AVP concentrations 11.5-fold. The magnitude of this increase
is comparable to the response observed by Woods and Johnston (1983)
in rats dehydrated for a similar period of time. Thus, it would
appear that the receptors are sensitive to the circulating levels of
vasopressin. This is supported by the study of Shewey and Dorsa
(1986) who reported finding no difference in the level of 1-AVP
binding in renal medullary membranes prepared from heterozygous

51
Brattleboro
rats and
their
chronically dehydrated
homozygous
counterparts,
which are
unable
to synthesize AVP.
This would
suggest that
the reduction
in vasopressin receptor density
observed in
the present study is attributable to the elevated plasma levels of
vasopressin, rather than some nonspecific effect of dehydration.
Homologous downregulation of vasopressin receptors has been
reported in a renal epithelial cell line (Lester et al., 1985) and in the
toad urinary bladder (Eggena and Ma, 1986), following treatment with
exogenous vasopressin. The phenomenon of downregulation results
from an increased rate of internalization when the hormone
concentration has been greatly enhanced (Catt et al., 1979). The
"anatomical correlate" of downregulation has been described by Kirk
(1987). He has elegantly shown by morphometric analysis that
retrieval or internalization of the basolateral membrane occurs in the
cells of the rabbit cortical collecting tubule when induced by a
vasopressin-stimulated transcellular water flow. He suggests that
membrane internalization could reduce the sensitivity of the cortical
collecting tubule cells to vasopressin if AVP receptors are present on
the retrieved membrane. While not addressed in the present study,
the issue of adenylate cyclase responsiveness has been examined in
conjunction with the downregulation of AVP receptors. Rajerison et
al. (1977) showed in the rat that vasopressin-sensitive adenylate
cyclase activity in membrane preparations from the rat kidney medulla
is greatly reduced after the infusion of pharmacological doses of AVP
to the animals.
In the present study, it would seem odd that an animal in dire
need of water conservation would lose AVP receptors as the need

52
became greater. An explanation for this may be that the kidney
possesses "spare" receptors (Ariens et al., 1960). Baddouri et al.
(1984) noted a downregulation of vasopressin receptors in the kidney
of the water deprived jerboa, Jaculus orientalis. Water-deprived
animals in that study displayed a five-fold elevation in plasma AVP
levels and a 36% reduction in ^H-lysine vasopressin binding to renal
medullary membranes. Despite reduced vasopressin binding, the
kidneys of some of these desert rodents still retained the capacity to
significantly concentrate urine under these conditions. The latter
finding suggests that "spare" receptors may have been involved in the
regulatory phenomenon. A receptor reserve has also been noted in
other adenylate cyclase-linked tissues as well (Flores et ah, 1974).
Alternatively, reduced binding without a decrement in biological
responsiveness may occur if those receptors which were
downregulated are "silent", or not coupled to an effector system.
Maack et ah (1987) have recently shown that the overwhelming
majority (>90%) of receptors for atrial natriuretic factor that are
present in the kidney are biologically silent. That is to say, they do
not mediate any of the known renal effects of the hormone. The
authors speculate that these silent receptors may serve as "peripheral
storage clearance binding sites, acting as a hormonal buffer system to
modulate plasma levels of atrial natriuretic factor". The physiological
significance of silent or spare receptors and their regulation remains
to be explored.
In summary, the present study shows that V2 vasopressin
receptors are differentially expressed in basolateral membrane
preparations from the kidneys of water-replete and water-deprived

53
rats. Scatchard analyses indicate that the difference in binding is
due to a reduction of receptor numbers on the membranes prepared
from the dehydrated animals. These results show that AVP receptor
downregulation can occur in response to physiologically elevated
levels of circulating vasopressin. Such a condition could ultimately
affect the antidiuretic action of the hormone.

CHAPTER 4
REGULATION OF VASOPRESSIN BINDING
IN MINERALOCORTICOID-DEPENDENT HYPERTENSION
InLroducti.on
The
role
of
vasopressin
in the
etiology
of hypertension
has
received
considerable
attention,
much of
it being
reviewed
recently
by
McNeill
(1983) and
Johnston
(1985).
The pressor action
of vaso-
pressin
was
the
first physiological
response
attributed
to
this
posterior
pituitary
substance
(Oliver
and Schafer, 1895)
and
the
vasoconstrictive properties of the hormone are now well-documented
both ¡n vivo (Liard, 1984) and in vitro (Altura and Altura, 1984).
One line of evidence which points to a relationship between
vasopressin and high blood pressure is that circulating levels of
vasopressin are elevated in several models of hypertension. Crofton
et al. (1978) noted higher plasma vasopressin concentrations in the
spontaneously hypertensive rat. Similar observations were noted in
renovascular (one kidney,one clip) (Pulían et al., 1978) and volume-
expanded (DOCA-salt) (Mohring et al., 1977; Crofton et al., 1979)
models of hypertension. While the circulating levels of vasopressin
are significantly increased in all of the above studies, the plasma
concentrations are, nonetheless, below the threshold for pressor
activity. This discrepancy can be resolved, however, by invoking
increased vascular reactivity toward vasopressin. Such an enhanced
pressor sensitivity to AVP has been noted in the SHR (Crofton et al.,
1978) and in DOCA-salt hypertensive rats (Crofton et al., 1980).
54

55
Added support for the involvement of vasopressin in the pathogenesis
of DOCA-salt hypertension comes from the study of Friedman et al.
(1960). These investigators noted that the administration of AVP
accelerated the development of DOCA-salt hypertension. The most
telling piece of evidence however, which implicated vasopressin in
DOCA-salt hypertension was the finding of Crofton et al. (1979) who
reported that Brattleboro rats, which are unable to synthesize AVP,
failed to develop DOCA-salt hypertension.
The development of selective antagonists to the pressor and
antidiuretic effects of vasopressin greatly facilitated our
understanding of the involvement of vasopressin in hypertension.
Crofton et al. (1979) found that the administration of a selective V j
(vascular) antagonist caused an acute fall in blood pressure in DOCA-
salt hypertensive rats. Similarly, Mohring et al. (1977) were able to
lower the blood pressure by the injection of a highly specific
vasopressin antibody. Hofbauer et al. (1984) attenuated the
development of DOCA-salt hypertension by the simultaneous infusion
of Vj antagonists. Collectively, these studies suggest that
vasopressin may contribute to the development of hypertension by
virtue of its vasoconstrictive properties. Conversely, other
investigators
(Rascher et
al., 1983;
Yamamoto et
al.,
1984; Filep et
al., 1987)
have been
unable to
reduce the
high
blood pressure
associated with DOCA-salt hypertension by the administration of V|
blockers.
A renal locus in the pathogenesis of DOCA-salt hypertension has
been largely overlooked until recently. Saito and Yajima (1982)
observed that when Brattleboro rats were treated with DDAVP, a

56
selective
tubular (V2)
agonist, DOCA-salt hypertension
developed. A
similar
phenomenon
was observed by Woods
and
Johnston (1981),
using
renovascular
models of hypertension
in
Brattleboro rats.
Whereas
Hofbauer et
al. (1984) attenuated the
development of DOCA-
salt hypertension with
V1 blockers as noted
above,
the simultaneous
infusion of a combined V1/V2 antagonist completely abolished the rise
in blood pressure. These studies suggest a tubular site of action for
vasopressin in the development of DOCA-salt hypertension.
The role of the kidney in the pathogenesis of hypertension has
been a major focus of Guyton and his colleagues (Guyton et al.,
1972). The action of vasopressin in this context was underscored in
the findings of Manning et al. (1979) and Cowley et al. (1984b) who
reported that subpressor doses of vasopressin can contribute to a rise
in arterial pressure, which is dependent on the fluid-retaining
properties of the hormone. In addition, it has been shown that
vasopressin stimulates sodium reabsorption in the medullary thick
ascending limb of the mouse (Hebert et al., 1981a) and the cortical
collecting tubule of the rat (Reif et al., 1986). The latter authors
also showed that the effects of vasopressin on sodium reabsorption
are augmented in DOCA-treated rats.
Thus, it appears that there is an interaction between vasopressin
and mineralocorticoids at the level of the renal tubule. Increased
activity of the salt and fluid transport processes would lend itself to
a volume-expanded state, with the potential for raising arterial
pressure. The question of how vasopressin activity is increased in
DOCA-treated animals remains unanswered. This study will address
the question at the cellular level. Vasopressin receptors and

57
vasopressin-stimulated adenylate cyclase activity will be measured in
basolateral membranes prepared from renal tubular epithelia in normo-
tensive and DOCA-salt hypertensive rats.
Materials and Methods
Chemicals
^H-argininie-vasopressin (^H-AVP), ^I-arginine-vasopressin
(*^I-AVP) and 3H-3',5'-cyclic adenosine monophosphate (^H-cAMP)
were purchased from New England Nuclear Corporation (Boston).
Peptides, cAMP, protein kinase, desoxycorticosterone acetate (DOCA),
protein kinase, and the regenerating reagents for adenylate cyclase
stimulation (theophylline, creatine phosphokinase, phosphocreatinine,
ATP and GTP) were all obtained from Sigma Chemical Company (St.
Louis). Vasopresssin antibody was purchased from Amersham
(Arlington Heights, IL) and Liquiscint liquid scintillation fluid from
National Diagnostics (New Jersey).
Animals
Thirty male Sprague Dawley rats weighing 175-225 g were
obtained from the Animal Resource facility at the University of
Florida. Rats were housed in a climate-controlled room and caged
individually in 20x18x24 cm wire mesh cages. They were provided a
standard Purina rat chow diet and allowed free access to tap water.
The rats were divided into five groups containing six rats per group.
Under chloral hydrate anesthesia (0.4 g/kg body weight, i.p.), animals
in Group I were implanted subcutaneously with two silastic capsules,
5 cm long, containing DOCA. They were unilaterally nephrectomized
using a retroperitoneal approach and provided with a 1% NaCl
drinking solution in lieu of water. Group II animals received 1% NaCl

58
drinking solution and was unilaterally nephrectomized according to
the procedures described above, but received no DOCA implants.
Animals in Group III received DOCA implants, tap water to drink and
their kidneys remained intact. Those in Group IV were implanted
with DOCA capsules, received 1% NaCl to drink, but their kidneys
were left intact. Animals in Group V received no special treatment
and were provided with tap water to drink. Blood pressures were
recorded by tail cuff plethysmography prior to surgery and once
weekly, thereafter. Values are expressed as mm Hg.
Membrane PrqpargtiQn and Plasma Collection
After four weeks of treatment, animals were sacrificed by
decapitation. Trunk blood was collected in chilled tubes containing
0.3 M EDTA (50 /il/ml blood) for the determination of plasma
vasopressin concentration. Blood samples were processed according to
the procedure described in Chapter 3. Renal basolateral membranes
were prepared by modifying the density gradient centrifugation
method of Boumendil-Podevin and Podevin (1983), according to the
description in Chapter 2. For vasopressin receptor binding assays,
membranes were stored in liquid N2 until use. For adenylate cyclase
measurements, membranes were used immediately.
Vasopressin Receptor Assay
Binding studies were conducted according to the procedure
described in Chapter Two. Briefly, membrane samples were incubated
in triplicate at 20°C for 45 minutes with concentrations of -^H-AVP
ranging from 50 pM to 2,500 pM. Non-specific binding was
determined in the presence of 2.5 /iM unlabelled AVP.

59
Vasopressin Radioimmunoassay
Plasma vasopressin concentrations were determined according to
the method described in Chapter 3.
Adenylate Cvclase Stimulation
Basal and vasopressin-stimulated adenylate cyclase activity in
renal basolateral membranes was performed according to the following
protocol: Basal samples (membranes corresponding to 100 /xg protein)
were suspended in a total volume of 150 /xl of 50 mM Tris buffer, pH
7.4, containing 138 /xg ATP, 39 /xg GTP, 150 /xg MgCl2, 38 /xg EGTA,
150 /xg BSA, and a regenerating system consisting of 228 /xg theo¬
phylline, 60 /xg creatine phosphokinase and 100 /xg phosphocreatine.
Agonist-stimulated samples were prepared as above but were incubated
in the presence of A VP ranging in concentration from 0.1 nM to
10 /xM. Triplicate samples were incubated at 37°C for 10 minutes.
The reaction was stopped by the addition of 300 /xl of 100 mM Tris
buffer containing 5 mM EDTA, pH 7. The samples were boiled for 5
minutes and then centrifuged at 3,000 rpm for 5 minutes. Super¬
natants were removed at stored at -20°C for subsequent determination
of cAMP formation.
cAMP Determination
The cellular cAMP content was determined by the competitive
protein binding assay as modified by Baker et al. (1986). Samples for
cAMP determination were reconstituted in 200 /xl of 25 mM Tris
buffer, pH 7.0. 10 /xl of each sample were incubated at 4°C for 60
minutes in a total volume of 200 /xl Tris buffer containing 25,000 cpm
of ^H-cAMP, 42 /xg BSA, 280 /xg theophylline and the reaction
initiated by the addition of 24 /xg of cAMP-dependent protein kinase.

60
At the end of the incubation, 70 /¿I of a hydroxyapatite suspension
(50% w/v) was added to each tube, vortexed and allowed to sit for
six minutes. The contents of the tube were then filtered under
vacuum through Whatman GF/C filters, then washed with 12 ml of 10
mM Tris buffer at pH 7.0. Filters were removed and transferred to
scintillation vials containing 1 ml of 0.3N HC1. After the hydroxy¬
apatite had dissolved (~10 min), 9 ml of liquid scintillation fluid
(Liquiscint) was added and each vial counted. Non-specific binding
was determined in the presence of 10 cAMP. The amount of
cAMP present in each sample was calculated from a standard curve
determined with known amounts of unlabeled cAMP.
Analysis
Data from saturation studies were analyzed using iterative curve
fitting programs. Statistical significance was assessed by one-way
analysis of variance. The post-hoc analysis between treatment pairs
was performed by Duncan’s method. A "p" value of 0.05 or less was
considered an acceptable level of significance.
Results
The combination of desoxycorticosterone acetate treatment and
increased sodium intake precipitates the development of the well-
characterized syndrome of DOCA-salt hypertension. Figure 4-1
depicts the rapid development of high blood pressure in rats
administered the mineralocorticoid and offered 1% NaCl to drink.
Those animals in group I with a reduced renal mass became
hypertensive most rapidly and attained the highest arterial pressure
(176±5 mm Hg). Even those animals with intact kidneys but provided
a similar regimen (Group IV) became hypertensive (154±8 mm Hg)

61
Figure 4-1. Arterial pressure in control and DOCA-treated rats.
Blood pressures were determined prior to experiment and once per
week thereafter by tail cuff plethysmography. Arrow indicates start
of treatment. Error bars represent S.E.M. (n = 6 per group except V,
where n = 4). *p<.05 vs. V and II. See "Methods" section for key to
group designations.

62
after only 4 weeks of treatment. This contrasts to the animals in
group III who received only DOCA and displayed an arterial pressure
of 121 ±7 mm Hg. This value was not significantly different from the
values observed in animals in Groups V (108±8 mm Hg) and II (110±6
mm Hg), receiving no treatment or 1% NaCl and unilateral
nephrectomy, respectively.
^H-vasopressin binding in renal epithelial basolateral membranes
prepared from the DOCA-salt hypertensive rats (Group I) and their
appropriate controls (Group II) revealed a higher level of binding in
membranes from the hypertensive group (Figure 4-2). As depicted in
Figure 4-3, Scatchard analysis of these data indicate that this higher
level of binding is due to an increased number of binding sites
(Group I = 307±29 fmoles/mg protein vs. Group II = 179±17 fmoles/mg
protein) [p<.05], rather than a change in affinity (Group I = 0.59±.ll
nM vs. Group II = 0.54±.09 nM) [p>.05]. This represents a 71%
increase over control values. Table 4-1 summarizes the binding data
for the other experimental groups. It can been noted that a higher
Bmax *s a*so ev^ent for the hypertensive animals in Group IV
(281 ±33 f moles/mg protein). This too, is significantly higher than the
values for Group II (p<.05). Furthermore, there were no significant
differences between the values in Group II and the untreated animals
in Group V (Bmax = 196±23 fmoles/mg protein; Kj) = 0.67±.12) [p>.05].
Binding (Bmax = 191± 14 fmoles/mg protein; Kj) = 0.63±.04) in
membranes prepared from the animals receiving DOCA only (Group
III) was not significantly different from either of the control groups
(II or V).

63
Figure 4-2. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Details of binding protocol described in "Methods" section of
Chapter 2. Error bars represent S.E.M. of triplicate determinations.
DOCA, desoxycorticosterone acetate; Na, 1% NaCl; IK, one kidney.

64
Bound (fmoles/mg P)
Figure 4-3. Scatchard analyses of 3H-vasopressin binding. Binding
determined in basolateral membranes prepared from kidneys of DOCA-
salt hypertensive rats and normotensive sham-operated control rats.
Analyses of data in Figure 4-2. *p<.05 vs. control. Abbreviations are
as in Figure 4-2.

65
TABLE 4-1.
VASOPRESSIN BINDING IN DOCA-TREATED RATS.
Treatment
Group
Bmax
(fmoles/mg P)
Kp
(nM)
n
I
307 ± 29*
0.59 ± .11
4
II
179 ± 17
0.54 ± .09
4
III
191 ± 14
0.63 ± .04
3
IV
281 ± 33*
0.62 ± .14
2
V
196 ± 23
0.67 ± .12
3
p < .05 vs. Groups II and V

66
Basal and vasopressin-stimulated cyclic AMP formation was
measured in freshly prepared membranes from the various groups.
The values were derived from the standard curve depicted in Figure
4-4. Figure 4-5 represents the production of cAMP in membranes
prepared from animals in the DOCA-salt hypertensive group (I) and
the corresponding control group (II). Whereas basal values did not
differ from each another, membranes from the hypertensive animals
were stimulated to a greater degree than membranes from the control
group. The enhanced sensitivity to vasopressin is evidenced by a
shift
toward the
left
for
the curve depicting
Group
I values.
Subtracting basal
values,
the
calculated EDyg’s for
the
hypertensive
and
control group
are
2x10'
10 M and 8.5xl0‘10
M,
respectively.
Table 4-2 summarizes the basal and vasopressin-stimulated cyclic AMP
production in membranes of the other experimental groups. Basal
values did not differ from one another but a greater level of
stimulation and an enhanced sensitivity toward vasopressin were also
noted in membranes from the hypertensive animals in Group IV.
Finally, plasma vasopressin levels were determined in the
animals of the various groups and those data are presented in Table
4-3. Values for the non-hypertensive animals in Groups II and V
ranged from 1.2±0.2 pg/ml to 1.8±0.2 pg/ml, respectively. Plasma
vasopressin concentrations were significantly elevated among the
hypertensive animals. Measured values in Groups I and IV were
6.H0.8 pg/ml and 8.414.5 pg/ml, respectively. This represents a five¬
fold elevation in plasma vasopressin concentrations between the
hypertensive and normotensive animals.

67
cyclic AMP (pmoles)
Figure 4-4. Standard curve for cyclic AMP. Values for cyclic AMP
determined by competitive binding assay, using 3H-cAMP. Details of
procedure described in "Methods" section. cAMP, adenosine 3'5'-
cyclic monophosphate.

68
O 11 10 9 8 7 6 5
[AVPJ (-log M)
Figure 4-5. Basal and vasopressin-stimulated cyclic AMP formation.
Cyclic AMP production by basolateral membranes prepared from
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Membrane preparation described in "Methods" section of
Chapter 2. Procedures for the stimulation and determination of
cyclic AMP described in "Methods" section. Abbreviations are as in
Figure 4-2 except AVP, arginine vasopressin; cAMP, adenosine 3'5'-
cyclic monophosphate.

69
TABLE 4-2.
VASOPRESSIN STIMULATED cAMP FORMATION IN DOCA SALT
HYPERTENSIVE RATS
Group
Basal
pmoles/10 min./mg P
Maximal Stimulation
pmoles/10 min./mg P
IC50
I
72 + 14
410 ± 20
2xlO~10M
II
61 + 12
355 ± 10
8xlO-10M
hi
64 + 07
360 + 10
7xlO'10M
IV
65 + 10
400 ± 12
2.5xlO_10M
V
58 ± 15
345 + 15
8.5xlO"‘°M

70
TABLE 4-3.
PLASMA A VP LEVELS IN DOCA-SALT
HYPERTENSIVE RATS
Group
[AVP] (pg/ml)
I
6.1 ± 0.8
II
1.8 ± 0.2
III
1.9 ± 0.3
IV
8.4 ± 4.5
V
1.2 ± 0.2

71
Discussion
The role of vasopressin in the development of DOCA-salt
hypertension appears to be unequivocal. Blood pressure will not
increase in this model unless vasopressin is present. This was
demonstrated most elegantly by the use of Brattleboro rats, animals
which are genetically incapable of synthesizing AVP (Crofton et al.,
1979). What is unclear is the mechanism or mechanisms which
underlie the etiology of the blood pressure increase. Various target
tissues have been implicated to account for the hypertensive actions
of vasopressin. A central site of action has been suggested by the
study of Chen et al. (1986). These investigators reported heightened
vasopressinergic activity in DOCA-salt hypertensive rats. This
augmentation in hypothalamic release of vasopressin was exclusive of,
and preceeded the well-known increase in peripheral AVP levels or
elevation of blood pressure. The authors suggested that this
alteration in central vasopressinergic activity may affect blood
pressure by modulation of the cardiovascular centers in the brain.
Studies with vasopressor antagonists suggest a vascular site of action.
Crofton et al. (1979) reported that the acute administration of V]
blockers lowered blood pressure in animals with established DOCA-salt
hypertension. Hofbauer et al. (1984) found that the chronic
administration of V j blockers during the DOCA-salt regimen
attenuated the development and magnitude of hypertension associated
with this model. Other studies however, tend to refute these
findings (Rascher et al., 1983; Yamamoto et al., 1984; Filep et al.,
1987).

72
A tubular site of action for vasopressin in the development of
DOCA-salt hypertension is suggested by the study of Saito and
Yajima (1982). Using Brattleboro rats, these investigators observed
that DOCA-salt hypertension would develop following the chronic
administration of DDAVP, a V2*specific tubular agonist. These
findings are consistent with the hypothesis of Cowley et al. (1984)
that subpressor doses of vasopressin can contribute to a rise in
arterial pressure, by virtue of
the
fluid-retaining properties of
the
hormone.
Indeed, plasma
levels
of
vasopressin tend to
be raised
in
DOCA-salt
hypertension.
If the
tubular sensitivity to
vasopressin
is
concommitantly increased,
as is
the
vascular sensitivity
(Berecek
et
al., 1982),
then these
processes
may lend themselves to
the
development of hypertension.
The
present study
shows
that V2 vasopressin
receptors
in
basolateral membranes prepared from renal tubular epithelia in DOCA-
salt treated rats are significantly elevated relative to tissue from
non-DOCA treated animals. Animals treated with DOCA-salt and
having a reduced renal mass (Group I) displayed a 56% increase in
binding with respect to tissue from untreated animals (Group V) and
a 71% increase compared to uninephrectomized animals receiving 1%
NaCl (Group II). Those DOCA-salt hypertensive rats with intact
kidneys (Group IV) displayed increases of 43% and 56%, respectively.
The present finding that mineralocorticoids modulate the expression
of vasopressin receptors in the kidney is consistent with the study of
Rajerison et al. (1974). These authors observed that adrenalectomy
resulted in a reduction of ^H-lysine vasopressin binding in a renal
medullary membrane preparation. A concomitant lowering of

73
vasopressin-stimulated adenylate cyclase activity was also noted.
Replacement therapy with aldosterone restored the hormone binding
capacity and adenylate cyclase activity to control levels. In the
present study, vasopressin-stimulated cAMP production was
commensurately increased in membranes prepared from the DOCA-salt
hypertensive rats. Furthermore, the effective concentration which
caused half-maximal stimulation (ED50) was reduced. Thus, an
increased sensitivity to vasopressin is suggested on the basis of
adenylate cyclase activity, although no change in the affinity
constant of the receptor was observed. Using microdissected renal
tubules, Pettinger et al. (1986) have observed a similar increase in
the sensitivity of adenylate cyclase in response to vasopressin in
tubules obtained from DOCA-salt treated rats. This response was
specific for vasopressin and occurred only in the segment of the
cortical collecting tubule. The phenomenon was observed as early as
five days post-treatment, a period which preceded the elevation of
blood pressure.
The mechanism of vasopressin receptor up-regulation associated
with DOCA-salt hypertension is not clear. It might have been
expected that vasopressin binding would decrease in the face of
elevated circulating levels of AVP, as was observed in Chapter 3.
However, the magnitude of change in plasma vasopressin levels in the
present study was only half of what occurred with dehydration.
Clearly, factors other than homologous regulation must be invoked.
As a steroid, DOCA probably acts by increasing the transcriptional
rate for the vasopressin receptor protein. Increased expression of
other tubular basolateral membrane proteins is also observed with

74
DOCA treatment. Garg et al. (1981) reported that Na-K-ATPase
activity was markedly elevated in the cortical collecting duct of
rabbits treated with DOCA. Similar results were observed by El
Mernissi et al. (1983) and Mujáis et al. (1985). Mineralocorticoids
can affect the expression of receptors in other tissues as well.
Wilson et al. (1986) observed a greater density of angiotensin II
receptors in brain tissue. Angiotensin II receptors are also increased
in vascular smooth muscle tissue in DOCA-salt hypertensive rats
(Schiffrin et al., 1983). These findings correlate quite nicely with
the increased dipsogenic and pressor responsiveness to Ang II in
DOCA-salt treated animals. Not all receptors are modulated by
mineralcorticoids, however, Alphaj-adrenergic receptors were not
significantly altered in membrane fractions prepared from DOCA-salt
hypertensive and normotensive rats (Fukuda et al., 1983). These
authors did observe differences however, in -’I I-yohimbine binding
between spontaneously hypertensive rats and the their normotensive
counterparts, Wistar-Kyoto rats.
How might an up-regulation of renal tubular vasopressin
receptors contribute to the development of DOCA-salt hypertension?
Vasopressin regulates fluid reabsorption in the distal nephron and also
stimulates sodium
reabsorption
in at
least
two distinct sites:
the
medullary thick
ascending limb
(Hebert et
al., 1981a) and in
the
cortical collecting
tubule (Reif
et al.,
1986).
A relationship between
vasopressin-stimulated cAMP production and Na-K-ATPase activity,
the enzyme involved in active transport of sodium, has been
demonstrated by Tomita et al. (1987). As shown in the present study,
the increased density of AVP receptors appears to render the tubule

75
more sensitive to vasopressin, reflected by the enhanced AVP-
stimulated production of cAMP. Reif et al. (1986) have shown that
chronic DOCA treatment enhances vasopressin-stimulated sodium
reabsorption in the cortical collecting tubule. Treatment with DOCA
also increases Na-K-ATPase activity in distal segments of the nephron
(Garg et al., 1981). Thus, the increased concentration of plasma
vasopressin noted in the present study coupled with an enhanced
sensitivity to AVP in renal tubules may lead to an exaggerated
retention of salt and fluids, resulting in an expanded blood volume.
Extracellular volume expansion has been noted in at least some stage
of the development of DOCA-salt hypertension (Villamil et al., 1982;
Tajima et al., 1983).
Taken together, the present findings and the above studies
suggest that the development of DOCA-salt hypertension may be
caused, in part, by an increased tubular sensitivity to vasopressin.
Increased activity of vasopressin-mediated processes would lend itself
to a volume-expanded state, a condition necessary to prevent hyper¬
natremia. Accordingly, the resultant blood pressure elevation could
be viewed as a compensatory mechanism in response to an increase in
cardiac output, secondary to hypervolemia and aimed at the
restoration of fluid and electrolyte balance.

CHAPTER 5
EFFECTS OF MINERALOCORTICOIDS ON VASOPRESSIN
RECEPTORS AND ADENYLATE CYCLASE-COUPLED RESPONSES
IN LLC-PKj CELLS
Introduction
Although vasopressin can act on the kidney and affect renin
secretion, glomerular filtration and tubular transport, a detailed
understanding of how AVP or other hormones affect these processes
at the cellular level has been difficult to ascertain. In large part,
this is due to the complexity of the kidney and the veritable myriad
of potential target tissues. One attempt to solve this problem has
been to
study
the action of
vasopressin on
isolated
glomeruli,
vascular
tissue
or discretely dissected tubular
segments.
While
somewhat
more
refined than an
isolated whole kidney, this
approach
still ignores the heterogeneity
of cell types
within anatomically
distinct
regions.
In order to
circumvent these
potential
problems,
one technique that has been applied to answer many of the questions
concerning the cellular actions of vasopressin in the kidney is cell
culture. The use of primary cultures and cell lines has been utilized
successfully in a number of studies, ranging from transport physiology
to hormonal regulation. Handler (1986) has recently written an
elegant review on the use of kidney cells in culture and succinctly
summarizes the wide-ranging studies amenable to these techniques.
The primary objective for using cell culture techniques in this
study was to establish an in vitro system in which to evaluate the
effects of mineralocorticoids on vasopressin binding, in order to
76

77
compare these results with those observed in vivo that are associated
with mineralocorticoid-dependent hypertension. To this end,
vasopressin binding was characterized in LLC-PKj cells (ATCC # CL
101), a continuous cell line of renal epithelial origin, established by
Hull et al. (1976).
Materials and Methods
Chemicals
Tritiated 8-L-arginine [phenylalanine - 3,4,5-^I I]-vasopressin (3H-
AVP) and tritiated 3',5'-cyclic adenosine monophosphate (^H-cAMP)
were purchased from New England Nuclear Corporation (Boston).
Desoxycorticosterone acetate (DOCA), arginine and lysine vasopressin
(AVP, LVP), oxytocin (oxy), angiotensin II (ang II), D-ala-met-
enkephalinamide (DAME), cAMP and cAMP-dependent protein kinase
were all purchased from Sigma Chemical Co. (St. Louis). Dulbecco’s
Modified
Eagle’s Medium and
fetal
bovine serum were
obtained
from
GIBCO
(Long Island, NY).
All
other reagents were
obtained
from
Sigma, unless otherwise indicated.
Cell Culture
LLC-PKj cells (ATCC # CL 101) were routinely grown as mono-
layers in plastic 75 cm^ Corning flasks using Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), streptomycin [100 /¿g/ml], penicillin [100 units/ml], and
NaHCC>3 [44 mM], Cell cultures were maintained at 37°C in an
atmosphere of 95% O2 and 5%CC>2. Dome formation typically appeared
after several days of growth. Dome formation in epithelial cell
cultures is acceptable presumptive evidence of the following processes
that are required for its expression: functional plasma membrane

78
polarization, formation of occluding
junctions
and vectorial
trans-
epithelial active ion
transport (Lever,
1985).
At confluence,
cells
were subcultured as
follows: the medium was
removed and the
cells
washed with once
with trypsin-EDTA
solution
(0.05% trypsin,
0.2%
EDTA) for 10-15 minutes. The cells were centrifuged (500 x g, 5
min), resuspended in DMEM + 10% FBS and plated at a density of 2-
2.5 x 104 cells/cm^ in flasks as above for continued passage or in 35
mm plastic Falcon petri dishes and utilized upon achieving confluence.
Vasopressin Receptor Binding Assay
The vasopressin binding assay was carried out directly in the
dish, on the monolayer. Twenty four hours prior to the experiment,
the growth medium was removed and replaced with serum-free DMEM.
At the time of the experiment, the medium was aspirated from the
plates and the monolayer washed once with phosphate-buffered saline
(PBS). PBS was composed of NaCl (8g/L), KC1 (0.2g/L), Na2HP04
(1.15g/L) and KH2PO4 (0.2g/L). Binding was initiated by the addition
of 0.5 ml of the reaction buffer (PBS containing 0.8% bovine serum
albumin, 1 /xg/ml aprotinin, 1 /ig/ml pepstatin A, 1 /xg/ml leupeptin,
pH 7.2) containing ^H-AVP. Non-specific binding was determined in
the presence of a 1000-fold excess of non-radioactive A VP. Studies
were conducted in triplicate using ^H-AVP at a concentration of 2.5
nM, for 30 minutes at 20°C, unless indicated otherwise and repeated
at least once. For dissociation studies, the reaction medium was
removed after 60 minutes and replaced with buffer containing 2.5 fiM
AVP. Saturation studies were performed with concentrations of -^H-
AVP ranging from 0.5-20 nM. Binding was terminated by rapidly
aspirating the reaction mixture from the cells and washing twice with

79
ice-cold PBS.
The cells were
dissolved
by
the addition
of 0.5 ml
0.1N NaOH,
scraped free from
the plate
with a
rubber
policeman,
transferred to
a vial containing
10 ml
of
liquid
scintillation fluid
(Liquiscint), and counted for 5
minutes in
an
LKB
liquid
scintillation
counter.
Stimulation of Adenylate Cvclase
Twenty four hours prior to the experiment, the growth medium
was removed and replaced with serum-free medium. At the time of
the experiment, the medium was aspirated and the cells washed once
with PBS. The "control" buffer consisted of serum-free DMEM
containing 11 /¿g/ml isobutyl-methyl xanthine (IBMX), a phospho-
diesterase
inhibitor and 0.1
mg/ml
aprotinin, a
serine
protease
inhibitor.
The "stimulation"
buffer
consisted of
the
components
described
above, plus AVP in
concentrations ranging
from
1 nM to
1 /xM. One ml of the control or stimulation buffer was added in
triplicate to the plates and incubated at 37°C for 20 minutes. The
reaction was terminated by rapidly aspirating the reaction mixtures,
washing the monolayer once with PBS and adding 1 ml of boiling IN
HC1. The cells were scraped with a rubber policeman and the
contents transferred into a 4 ml polypropylene tube with a Pasteur
pipette. Samples were centrifuged at 3000 rpm for 5 minutes. The
supernatants were removed and blown to dryness under a stream of
air, before being stored at -20°C, for subsequent determination of
cAMP formation. The pellets were saved for protein determination,
according to the method of Lowry et al. (1951).

80
cAMP Determination
The cellular cAMP content was determined by the competitive
protein binding assay as modified by Baker et al. (1986), as described
in detail in Chapter 4.
POCA Treatment of Cell Cultures
Desoxycorticosterone acetate (DOCA) was dissolved in
dimethylsulfoxide (DMSO) at a concentration of 1 mg/ml. This stock
concentration was initially diluted with PBS and subsequent dilutions,
ranging from 1 pg/ml to 10 /xg/ml were made in serum-free DMEM.
The solutions were sterilized by passing them through 0.2 micron
mesh Nalgene filters. Growth medium was aspirated off of the cells
and replaced with DMEM containing DOCA. Cells were treated for 1-
24 hours with DOCA concentrations ranging from 1 pg/ml to 1 /¿g/ml.
Results
Binding of ^H-AVP to monolayers of LLC-PKj cells increased
rapidly over time and attained equilibrium after 45 minutes. At this
time, specific binding, represented ~75% of total -^H-AVP bound.
Upon equilibrium, binding was shown to be reversible by the removal
of the radioligand and the addition of buffer containing unlabeled
vasopressin. As with association, dissociation was rapid and reached
levels equivalent to non-specific binding after 45 minutes (Figure 5-
1). The specificity of the receptor was determined by competition of
^H-AVP binding with a variety of related and unrelated peptides.
Lysine^-vasopressin and arginine^-vasopressin were equally effective
(IC^q = 2.1 nM) in terms of their ability of displacing the radioligand
from the binding sites on the cells. On the other hand, oxytocin,
angiotensin II and D-ala-met-enkephalinamide were ineffective even at

81
Figure 5-1. 3H-vasopressin (3H-AVP) binding in LLC-PK^ cells as a
function of time. Receptor binding protocol described in "Methods"
section. Association: Total binding determined at 2.5 nM [3H-AVP].
Non-specific binding (NSB) determined in the presence of 2.5 nM [3H-
AVP] + 2.5 /¿M unlabeled AVP. Total - NSB = Specific (75%).
Dissociation: At equilibrium binding (1 hr), total 3H-AVP binding is
dissociated from receptor by the addition of 2.5 /¿M unlabeled AVP.

82
the highest concentration (10 ^M) in displacing ^H-AVP (Figure 5-2).
Using increasing concentrations of ^H-AVP, binding sites on the
surface of the cells appear to be saturable (Figure 5-3). Scatchard
analysis of the saturation isotherm data reveals a single class of high
affinity binding sites (ICq = 2.98 nM). This is in good agreement
with the value of 2.1 mM observed for the IC50 for vasopressin. The
maximal binding capacity (Bmax) was calculated to be 155 fmoles/mg
protein (Figure 5-3, insert). Vasopressin stimulated cAMP
accumulation in a dose-dependent manner with LLC-PKi cells (Figure
5-4) which was abolished by pre-treatment with V2 antagonists.
Treatment of the cell cultures with DOCA, either over varying
periods of time or with varying concentrations, failed to alter the
binding of ^H-AVP (Figure 5-5). While binding was significantly
reduced at 1 pg/ml DOCA, this effect can be attributed to the "toxic”
effect of the mineralocorticoid, as cell viability was adversely
affected. Basal and vasopressin-stimulated cyclic AMP formation in
LLC-PK1 cells were not significantly affected by DOCA treatment
(Figure 5-6).
Discussion
The structural and functional heterogeneity of the kidney is not
particularly amenable to the study of hormonal action at the cellular
level. One attempt to resolve this dilemma has been the use of
tissue cultures of specific cellular elements in the kidney. These
techniques have facilitated our understanding of vasopressin action at
targets as diverse as glomerular mesangial cells (Bonventre et al.,
1986) and tubular epithelial cells (Tang and Weinberg, 1986). In the
present study, the interaction of vasopressin with its receptor was

83
[Peptide] (-log M)
Figure 5-2. Specificity of vasopressin receptor in LLC-PKj cells.
Competition of 3H-vasopressin (3H-AVP) binding with vasopressin
analogues and unrelated peptides. Receptor binding protocol
described in "Methods" section. Abbreviations are as in Figure 2-4.

84
Figure 5-3. Saturation isotherm and Scatchard plot (insert) of 3H-
vasopressin (3H-AVP) binding in LLC-PKj cells. Concentration of
3H-AVP ranged from 500 pM to 20,000 pM. Specific binding was
determined using a thousand-fold excess of unlabeled AVP. Details of
the binding procedure are described in the "Methods" section.

85
Figure 5-4. Basal and vasopressin-stimulated cyclic AMP formation in
LLC-PKj cells. Procedures for the stimulation and determination of
cyclic AMP are described in the "Methods" section. AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate; V, blocker,
d(CH2)5D-Ile2-Ile4-AVP.

fmoles 3H-AVP bound/mg
86
[DOCA1
Figure 5-5. Effect of DOCA on 3H-vasopressin (3H-AVP) binding in
LLC-PKj cells. Cells were treated for 1-24 hours with DOCA ranging
in concentration from 1 pg/ml to 1 pg/ml. Binding was assessed at
3.0 nM [3H-AVP], with details provided in the "Methods” section.
DOCA, desoxycorticosterone acetate. *p.05 vs. control.

87
DOCA [pg/ml]
Figure 5-6. Effect of DOCA on basal and vasopressin-stimulated
cAMP production in LLC-PKj cells. Basal cAMP was measured in the
absence of AVP. Stimulated values were assessed in the presence of
10 nM AVP. Details of procedures are found in the "Methods"
section. DOCA, desoxycorticosterone acetate; AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate.

88
examined in a homogeneous population of tubular epithelial cells and
the influence of mineralocorticoids on AVP binding and adenylate
cyclase activity was determined.
Binding of ^H-arginine vasopressin to LLC-PKj cells was rapid,
reversible and specific. The receptor displayed a high affinity (2-3
nM) for arginine vasopressin and the closely related moiety, lysine
vasopressin, but for no other peptide, including oxytocin. This
finding differs somewhat from that reported by Roy and Ausiello
(1981a) who found that the IC50 for AVP was more than an order of
magnitude greater than that for LVP. This may be due to differences
in the radioligands used between the two studies. Roy and Ausiello
utilized a tritiated lysine vasopressin compound with a specific
activity of 8.5 Ci/mmol, whereas the tritiated vasopressin analogue
used in the present study contained arginine in the number 8 position
of the peptide and had a specific activity eight times greater (70
Ci/mmol). Variability in the phenotypic expression of the cells from
passage
to passage
must
also be considered
when
interpreting
any
result.
Scatchard
analysis
performed on the
saturation data in
the
present
study
revealed a
single class of
binding
sites with
an
apparent
B
max
of
155 fmol/mg protein. This
finding
too differs
from
those of
Roy
and
Ausiello
(1981a). They reported
a nonlinear
plot,
indicative of heterogeneity in the population of binding sites.
Comparison of the maximal binding capacities for the high affinity
sites in each of the studies is difficult, as Roy and Ausiello (1981a)
expressed their data on the basis of cell number, rather than per mg
of protein. However, the of 155 fmols/mg protein calculated in
max

89
the present study is in close agreement with the value of 166
fmoles/mg protein, as determined by Jans et al. (1987).
Roy
and
Ausiello (1981b)
found that cells
suspended
in
hypertonic
saline
or treated with
EDTA exhibited
a homogeneous
population
of
binding sites.
They proposed a
hypothesis
for
vasopressin-receptor interactions involving a hormone-induced
alteration in binding affinity or so-called "receptor transition". In
other words, rather than postulating two independent binding sites, as
a nonlinear Scatchard plot might suggest, they believe in an
interconversion of receptor states of different affinity. Why a
similar situation was not observed in the present study is unclear.
The binding sites characterized in the present study apparently
represent vasopressin receptors, as vasopressin was shown to
stimulate cAMP production in a dose-dependent manner. Agonist-
induced stimulation resulted in a sixteen-fold increase over basal
levels. This effect was completely abolished by pretreatment of the
cells with a V2 antagonist. The values for basal and AVP-stimulated
cAMP levels are in good agreement with those reported by Lester et
al. (1985) and Skorecki et al. (1987).
The synthetic mineralocorticoid DOC A, had no demonstrable
effect on either vasopressin binding or cAMP production in LLC-PKj
cells in this study. This contrasts with the observations noted in
vivo, as described in Chapter 4. In that study, a significant increase
was found in the vasopressin-stimulated adenylate cyclase activity and
number of vasopressin binding sites on tubular epithelial membranes
prepared from the kidneys of rats chronically treated with DOC A and
NaCl.

90
It may be appropriate to consider several possibilities to account
for the lack of mineralocorticoid effect noted in the LLC-PKj cells.
The most obvious explanation is that this particular cell line does not
possess receptors for mineralocorticoids, whereas cytosolic and
nuclear binding sites for mineralocorticoids have been reported for A$
cells (Handler et al., 1981) and MDCK cells (Luden et al., 1978). No
such reports have appeared in the literature for LLC-PKj epithelial
cells. This is consistent with the observation that LLC-PKj cells,
unlike the A$ and MDCK epithelial cell lines, are relatively refractory
to the effects of aldosterone, at least with respect to transepithelial
Na+ transport (Meier and Insel, 1985).
Perhaps the most pertinent evidence concerning the lack of
effect of mineralocorticoids in LLC-PKj cells comes from a study by
Roy et al. (1980). These investigators examined the regulation of
vasopressin receptors in LLC-PKj ^ cells, a subline of the parent
LLC-PKj strain. These cells are characterized by possessing only 5%
as many vasopressin receptors as the parent cell line. They reported
that fetal bovine serum or insulin could markedly stimulate the
expression of receptors in these cells. In contrast, thyroid hormone,
dexamethasone and aldosterone were ineffective in evoking a change
in receptor number.
Alternatively, methodological considerations must not be
overlooked. DOCA was only soluble in organic solvents such as
absolute ethanol or DMSO. Dilution of the DOCA-containing solvents
with aqueous buffers often resulted in the precipitation of the
steroid. Thus, it is impossible to say with any degree of certainty
exactly how much DOCA was applied to the cell cultures. On the

91
other hand, at high doses (>1 pg/ml), viability of the cells became a
significant factor. One study reported that ^H-DOCA receptor
binding was inhibited by a broad class of protease inhibitors (Baker
and Fanestil, 1977). Whether mineralocorticoids exert any
physiological effects in LLC-PK] cells remains in question, but it is
entirely possible that the use of protease inhibitors in the present
study may have antagonized the effect of the steroid.
Thus, vasopressin receptors are expressed in vivo on renal
tubular epithelial membranes and in vitro on renal epithelial cells.
Both receptors are coupled to adenylate cyclase. In addition, both
receptors are susceptible to regulation by vasopressin (Chapter 3 and
Lester et al., 1985). It appears however, that the receptors respond
differentially to the effects of mineralocorticoids. The question of
whether mineralocorticoids directly affect the expression of
vasopressin receptors at the cellular level remains unanswered using
LLC-PK] cells as a model. The question may be more suitably
addressed using a primary culture of epithelial cells prepared from rat
collecting tubules.

CHAPTER 6
INTRACELLULAR SECOND MESSENGERS IN LLC-PKj CELLS:
A DUAL TRANSMEMBRANE SIGNALING SYSTEM
Introduction
It is now well-documented that vasopressin receptors are
integral components in the plasma membrane of LLC-PKj cells, a
continous cell line of renal epithelial origin (Ausiello et al., 1987).
Vasopressin interacts with these receptors to initiate the sequence of
intracellular events that ultimately lead to the transepithelial
transport of fluid and solutes, in a manner not unlike that seen in
the distal collecting segments of the nephron. As in the kidney,
vasopressin receptors in LLC-PKj cells display characteristics of the
V2 subtype and are coupled to adenylate cyclase, resulting in a
hormone-specific stimulation of cAMP accumulation (Goldring et al.,
1978; Roy and Ausiello, 1981a). Recently, it was reported that
vasopressin stimulated transient increases in cytosolic calcium in
LLC-PKi cells (Tang and Weinberg, 1986). This action is not unlike
the vasopressin-stimulated calcium fluxes noted in hepatocytes
(Michell et al., 1979) and smooth muscle cells (Aiyar et al., 1986). In
these tissues however, vasopressin exerts its action through V j
receptors, which are coupled to phospholipase C and the hydrolysis of
phosphatidyl inositol (PI). This leads to the generation of inositol
phosphates (IP) and diacylglycerol (DG). Inositol triphosphate (IP3) is
thought to be involved in the mobilization of calcium from
intracellular organelles such as the endoplasmic reticulum whereas DG
92

93
has been shown to be an endogenous activator of protein kinase C
(Berridge, 1987), as illustrated in Figure 6-1.
Roy and Ausiello (1981b) have demonstrated that the vasopressin
receptor-effector system in LLC-PK | cells can be modulated by
altering the osmolality of the medium to which the cells are exposed.
Variation in osmotic conditions is analogous to the situation in the
kidney where segments of the tubule are exposed to a continous
corticomedullary osmotic gradient. Thus, the epithelial cells and the
receptors situated on the plasma membranes of their surfaces are
exposed to conditions ranging from isotonicity to hypertonicity. Roy
and Ausiello (1981b) reported that incubation in hypertonic NaCl will
increase the binding of vasopressin and the coupling between the
receptor and adenylate cyclase, leading to elevated levels of CAMP.
Thus, while it is well known that vasopressin stimulates the
production of cAMP in LLC-PK] cells, the following study was
designed to determine if vasopressin stimulates the hydrolysis of
phosphatidyl inositol, resulting in the formation of inositol
phosphates. Furthermore, the influence of increasing the osmolality
of the incubation medium on vasopressin-stimulated PI hydrolysis was
also examined.
Materials and Methods
Chemicals
Peptides (arginine vasopressin, lysine vasopressin, angiotensin II)
and the cholinergic agonist carbachol were purchased from Sigma (St.
Louis). Vasopressin antagonists were a gift from Dr. M. Manning of
the Medical College of Ohio. ^H-myo-inositol and OCS liquid
scintillation fluid were obtained from Amersham (Arlington Hts., IL).

94
Figure 6-1. Phosphoinositide cycle. Myo-inositol is incorporated in
phosphoinositides (PI, PIP, PIP2). Agonist-occupied receptors
stimulate phospholipase C, which acts to cleave PIP2 and IP3 and DG.
IP3 acts to mobilize intracellular stores of calcium, then IP3 is
metabolically converted to IP2 and IP under the action of the
appropriate phosphatases. DG activates a calcium-phospholipid-
dependent protein kinase C, which can phosphorylate a number of
cellular proteins. ATP, adenosine triphosphate; PI, phosphatidyl
inositol; PIP, phosphatidyl inositol-4-phosphate; PIP2, phosphatidyl
inositol-4,5-phosphate; DG, diacylglycerol; IP3, inositol 1,4,5-
phosphate; IP2, inositol 4-phosphate; IP, inositol 1-phosphate; P¡,
inorganic phosphate.

95
Dowex was obtained from Biorad Laboratories (New York). Liquiscint
liquid scintillation fluid was purchased from National Diagnostics (New
Jersey). Dulbecco’s Modified Eagle’s Medium and fetal bovine serum
were obtained from GIBCO (New York). Other reagents were
obtained from Fisher Scientific (Orlando) unless indicated otherwise.
Culturing and Preparation of LLC-PK| Cells
LLC-PK ] cells (ATCC # CL 101) were routinely grown in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%
fetal bovine serum. Cells were plated at an initial density of 2-2.5 x
10^ cells/cm^ in plastic 75 cm^ flasks and on 35 mm plates. The
cultures were maintained at 37°C in a humidified atmosphere of 95%
O2 and
5% C02.
Cells in
the
35 mm plates were
utilized upon
achieving
confluence
whereas
the
monolayers in the
flasks were
subcultured according to the procedure described in Chapter Five.
The cells to be utilized for the experiments were washed twice with
buffer I, a regular Krebs-Ringer Bicarbonate (KRB) buffer, which
contained 118 mM NaCl, 4.7 mM KC1, 0.75 mM CaCl2, 1.18 mM
KH2PO4, 24.8 mM NaHCC>3 and 10 mM glucose and had an osmolality
of 300 mOsm/kg H2O. The cells from each plate were scraped with a
rubber policeman, transferred to a tube containing KRB buffer and
centrifuged at 500 x g for 5 minutes. The cells were then
resuspended in the appropriate buffer. In some experiments, buffers
of different osmolality than that of buffer I were used. Buffer II
(600 mOsm/kg H2O) was prepared by the addition of urea (300
mOsm/kg H2O) to buffer I (which had NaCl as its major component).
Buffers III (900 mOsm/kg H20) and IV (1200 mOsm/kg H20) were
prepared by the addition of equiosmolar amounts of NaCl and urea to

96
buffer II. In this way, the buffers simulated the osmolality and
composition of the renal medullary interstitium.
IncorpQ ration of 3H.-jnpsitpl intg Membrang PhosEhoinositides and
Release of 3H-inositol Phosphates
The method for measurement of phosphoinositide (PI) hydrolysis
in
LLC-PK] cells
was
similar
to that
described
by Gonzales
and
Crews (1984) for
brain
slices.
LLC-PK]
cells were incubated
with
0.1
/¿M -^H-inositol
(Specific
activity =
16.0
Ci/mmol) in
the
appropriate buffer (I to IV) for 3 hours at 37°C in a shaking water
bath. The cells were then washed with buffer of the same
osmolality (except that it did not contain -’ll-inositol) and divided
into several aliquots. In each aliquot, more of the same buffer but
containing 20 mM LiCl was added. LiCl inhibits dephosphorylation of
inositol phosphates (IP), and has been used to amplify hormone-
dependent PI responses in numerous tissues including LLC-PKj cells
(Berridge, 1987; Kinoshita et al., 1987).
To
determine the
hormone-stimulated release
of
3H-IP, the
reaction
was
started by
adding vasopressin
or other
agents and
incubating
the
samples at
37°C in a shaking
water
bath. During
incubation
the
buffers
were kept saturated
with
02:C02 (95:5)
mixture.
After
one hour,
the reaction was terminated
by
the addition
of chloroform:methanol (1:2 v/v) mixture. The organic and aqueous
layers were separated by centrifugation.
Quantitation of Inositol Phosphates (IP) Released
In order to eliminate the variation due to differences in number
of cells or 3H-inositol incorporation in each sample, values are
expressed as 3H-inositol phosphates released as a percentage of the
total amount of 3H-inositol incorporated in each sample. To do this,

97
radioactivity was determined in the organic layer (which contained
unhydrolyzed ^H-phosphoinositides) and in the aqueous layer (which
contained ^H-inositol phosphates). An aliquot of the organic layer
was placed in a scintillation vial and the chloroform was allowed to
evaporate in a fume hood. The radioactivity in the vial was
determined by the addition of OCS scintillation fluid and counted in a
Beckman (LS 7000) scintillation counter. The aliquots of aqueous
layers were mixed with Dowex (50% v/v, 100-200 mesh, formate form)
and poured into polypropylene columns. After the liquid was drained,
the Dowex was washed with 5 mM (unlabeled) inositol to remove free
^H-inositol. Total ^H-inositol phosphates were eluted with 0.1 M
formic acid/1.0 M ammonium formate. The radioactivity of the eluted
^H-inositol phosphates in each sample was measured by adding
Liquiscint and counted in a Beckman (LS 7000) scintillation counter.
The release of ^H-inositol phosphates (IP) were calculated as follows:
[3H]-IPs RELEASED = dom of IP in aqueous layer
(% Total Incorp.) dpm of IP in aq. layer + dpm of PI in org. layer
Statistical Analysis
The data on the release of IP with different drugs or in
different buffers were analyzed by one-way ANOVA (analysis of
variance). The individual contrasts between treatment pairs were
made by Duncan’s method. When only two groups were used, the
data were analyzed by the Student t-test. Differences with P < 0.05
were considered significantly different from each other.
Results
After incorporation of -^H-inositol into phosphatidyl inositol of
the membranes of LLC-PK^ cells for 3 hours, the vasopressin-

98
stimulated release of inositol phosphates was determined. The data in
Figure 6-2 indicate that vasopressin-stimulated IP release increased
with time at least up to 120 minutes. Sixty minutes was chosen as
the incubation period for measurement of IP release from radiolabeled
PI in LLC-PKj cells.
Stimulation of PI hydrolysis in LLC-PKj cells by vasopressin
proceeded in a dose-dependent manner, as seen in Figure 6-3. At at
-8
concentration of 10 M, there was a > 100% increase in PI
hydrolysis in these cells. The effect was dose-dependent up to 10 ^
M vasopressin in the incubation medium.
The specificity of the agonist-stimulated PI hydrolysis is
illustrated in Figure 6-4. Both arginine vasopressin and lysine
_7
vasopressin in equimolar concentrations (10 M) significantly
stimulated PI hydrolysis when compared to the incubation buffer
(p<.05). Carbachol, a cholinergic agonist that has been reported to
stimulate PI hydrolysis in kidney slices (Garg et al., 1986), exerted no
significant effect in LLC-PKj cells at concentrations up to 1 mM.
Angiotensin II, an octapeptide which is structurally different from
vasopressin but shares some physiological and biochemical properties
(vasoconstrictor, stimulates PI hydrolysis in vascular smooth muscle
cells), failed to stimulate PI hydrolysis in LLC-PKj cells at
concentrations up to 10 /¿M. An attempt was made to deduce the
nature of the vasopressin receptor mediating the PI response seen in
this cell system. As can be seen in Figure 6-5, the Vj antagonist
significantly reduced the amount of vasopressin-stimulated PI
hydrolysis (p<.05). The V2 receptor blocker also significantly lowered
the amount of vasopressin-stimulated release of PI (p<.05). Both the

99
Time (min)
Figure 6-2. Time dependence of 3H-inositol phosphate release in
LLC-PKj cells. Control represents Krebs-Ringers buffer. Vasopressin
(AVP)-stimulated release was conducted at a concentration of 10 pM
AVP. Results are expressed as 3H-inositol incorporated into the
phosphatidyl inositol pool. Details of the procedure are found in the
"Methods” section.

100
co
Q)
(D
cr
i
i
â– D
0)
CO
k_
o
CL
k_
o
o
c
Figure 6-3. Dose-dependent stimulation of 3H-inositol phosphate
release in LLC-PKj cells. Vasopressin (A VP), in concentrations
ranging from 1 nM to 10 /iM, were used to stimulate the release of
3H-inositol phosphate. Results are expressed as in Figure 6-2.
Details of the procedure are found in the "Methods" section.

101
Figure 6-4. Specificity of agonist-stimulated 3H-inositol phosphate
release in LLC-PKj cells. Peptides and the muscarinic agonist
carbachol were used, in the concentrations indicated to stimulate 3H-
inositol phosphate release. Results are expressed as in Figure 6-2.
Details of the procedure are found in the "Methods" section. A VP,
arginine vasopressin; LVP, lysine vasopressin; CARB, carbachol; ANG
II, angiotensin II.

102
12
A B
A) Buffer
B) AVP [10 7M]
C) AVP + V1 antagonist
D) AVP + V2 antagonist
* p<.05 vs A.C.D
C D
Figure 6-5. Antagonism of vasopressin-stimulated 3H-inositol
phosphate release in LLC-PKj cells. Vasopressin at a concentration
of 100 nM, stimulated the release of 3H-inositol phosphate. This
effect was totally abolished by pretreatment with equimolar amounts
of the Vj antagonist and attenuated by the V2 antagonist. Results
are expressed as in Figure 6-2. Details of the procedure are found
in the "Methods" section. Abbreviations for the Vj and V2
antagonists are as in Figure 2-4. AVP, arginine vasopressin.

103
Vj and V2 blockers by themselves caused a small but statistically
non-significant stimulation of PI hydrolysis (data not shown).
The effect of increasing the osmolality of the incubation medium
was assessed on vasopressin-stimulated PI hydrolysis. These results
are presented in Figure 6-6. Raising the osmolality to 600 mosmol/kg
H2O had no significant effect on AVP-evoked PI release. However,
when the osmolality was increased further, to 900 and 1200
mosmol/kg H2O, vasopressin-stimulated PI hydrolysis was significantly
reduced
with respect
to the
isoosmotic
buffer
(p<.05).
Altering the
osmotic
environment
had no
significant
effect
on the
buffer-evoked
release of PI.
Discussion
The vast preponderance of evidence suggests that vasopressin
causes the transepithelial transport of fluid and solutes in mammalian
renal tubules and amphibian urinary bladder by stimulating adenylate
cyclase which leads to the activation of cAMP-dependent protein
kinases. Vasopressin-stimulated cAMP production has also been
demonstrated in LLC-PKj cells, epithelial cells of renal epithelial
origin (Goldring et al., 1978; Chapter 5). The experiments conducted
in the present study represent the first documentation of vasopressin-
stimulated hydrolysis of phosphatidyl inositol in cells of renal
epithelial origin.
The effect of AVP-stimulated PI hydrolysis was time- and dose-
-8
dependent, with the minimum effective dose being 10 M. While this
concentration of vasopressin is generally higher than that required to
stimulate adenylate cyclase activity in these cells (Goldring et al.,
1978; Ausiello et al., 1987), the value is identical to that required to

104
Osmolality (mosmol/L)
Figure 6-6. The effect of osmolality on 3H-inositol phosphate release
in LLC-PKj cells. Osmolality was increased from normal iso-osmotic
conditions by the addition of NaCl and urea. This mimics the
corticomedullary osmotic gradient as seen in the kidney. Control
represents Krebs-Ringers buffer. Vasopressin (AVP), at a
concentration of 1 /xM, was used to stimulate release. Results are
expressed as in Figure 6-2. Details of the procedure are found in
the "Methods" section.

105
elicit an increase in cytosolic calcium in these cells (Tang and
Weinberg, 1986). Why the concentrations of vasopressin necessary to
stimulate these second messenger systems are higher than circulating
levels of vasopressin in vivo is unclear. However, it should be noted
that in certain tissues (kidney, urinary bladder), activation of only a
small fraction of hormone receptors is necessary to produce maximal
physiological responses.
Only arginine- and lysine-vasopressin were effective in
stimulating PI hydrolysis in LLC-PKj cells. The muscarinic agonist
carbachol and angiotensin II were without effect in this cell system.
This differs from the findings of Garg et al. (1986 and unpublished
observations) in which the cholinergic agent and peptide were shown
to stimulate PI hydrolysis in kidney slices. This suggests that the
stimulation of PI hydrolysis in LLC-PKj cells by vasopressin is
hormone-specific.
The nature of the receptors which mediate the vasopressin-
induced release of inositol phosphates from the membranes of LLC-
PKj cells remains to be clarified. Two major subtypes of vasopressin
receptors have been characterized, based on the effector system to
which they are coupled. Vj receptors, which mediate the
glycogenolytic action in hepatocytes and the vasoconstrictor action in
smooth muscle cells, are coupled to the hydrolysis of phosphatidyl
inositol and the generation of inositol phosphates and diacylglycerol.
V2 receptors on the other hand, mediate the antidiuretic action of
the hormone and are linked to adenylate cyclase and the production
of cAMP (Michell et al., 1979). Based on these criteria, it would
appear that the vasopressin-induced release of inositol phosphates in

106
LLC-PKj cells as seen in the present study, is mediated through Vj-
type vasopressin receptors. The use of selective Vj and V2
antagonists proved somewhat equivocal, however. A series of these
compounds have been developed which antagonize the vasoconstrictive
and antidiuretic actions of the hormone, respectively (Sawyer et al.,
1981; Manning and Sawyer, 1986). In the present study, vasopressin-
stimulated PI hydrolysis was completely abolished by pretreatment
with the selective Vj antagonist d(CH2)5-Tyr(Me)-AVP. The finding
that the V2 antagonist d(CH2)-D-Ile^-Ile^-AVP partially blocked the
vasopressin-induced release of inositol phosphates was rather
surprising. Several explanations may be considered when interpreting
these data. This first deals with the relative potency displayed by
d(CH2)-D-Ile^-Ile^-AVP. This compound displays only a forty-fold
greater anti-antidiuretic activity than anti-vasopressor activity,
compared to d(CH2)5-Tyr(Me)-AVP, which is the most potent anti¬
vasopressor available and displays little or no antidiuretic-blocking
activity (Manning and Sawyer, 1986). Alternatively, the vasopressin
receptor present in LLC-PKj may display different physical
characteristics from vasopressin receptors for which these analogues
were designed. Kinter et al. (1988) have noted several instances
where the selectivity of V2 antagonists is greatly reduced.
Another factor that should be considered is that V2 antagonists
also inhibit vasopressin-stimulated cAMP production. This points out
the possible interaction between the two intracellular signaling
systems. Berridge (1987) describes several examples of interactions
between the PI and adenylate cyclase pathways. Friedlander and
Amiel (1987) reported that activators of protein kinase C inhibit

107
vasopressin-stimulated cAMP formation in MDCK cells. This action
may occur by covalent modification of the vasopressin receptor or the
adenylate cyclase enzyme. This latter concept is strengthened by the
finding of Yoshimasa et al. (1987), who reported that treatment of
frog erythrocytes with phorbol esters resulted in phosphorylation of
the catalytic subunit of the adenylate cyclase enzyme coupled to the
/3-adrenergic receptor. Conversely, it is conceivable that in the LLC-
PKj cell line, a cAMP-dependent mechanism may act to potentiate PI
hydrolysis. Therefore, blockade of vasopressin-stimulated cAMP
production by V2 antagonists may lead to a diminution of
vasopressin-induced phosphatidyl inositol hydrolysis. The role of the
V2 receptor-mediated cAMP pathway in modulating vasopressin-
stimulated PI hydrolysis is unclear and remains to be clarified.
Alteration of the osmotic environment to which these cells are
exposed affected the vasopressin-induced release of inositol
phosphates. Increasing the osmolality of the incubation medium above
600 mosmol/kg H2O resulted in a significant reduction in the amount
of inositol phosphates released. It is doubtful that the hypertonicity
adversely affected the viability of the cells. On the contrary, Roy
and Ausiello (1981b) have shown that LLC-PK] cells display an
increased vasopressin receptor density and an enhanced vasopressin-
stimulated
production of
cAMP
when incubated
under
hypertonic
conditions.
The opposite
effects
of hypertonic NaCl on
vasopressin-
stimulated
PI hydrolysis
and
cAMP formation
only
serves to
underscore the apparent interaction between these two second
messenger systems in this cell system and may represent some form
of negative feedback.

108
The physiological significance of vasopressin-induced PI
hydrolysis in LLC-PKj cells and its regulation by osmolality is
uncertain at the present time. These epithelial cells display transport
characteristics not only of the distal nephron (H2O reabsorption), but
also of the proximal tubule (glucose, amino acid transport) and the
medullary thick ascending limb (sodium transport) (Horster and Stopp,
1986). In this regard, it should be noted that an increase in
peritubular osmolality has been shown to modulate the effect of
vasopressin on NaCl transport and cAMP formation in epithelial cells
of the medullary thick ascending limb of Henle (Hebert et al., 1981b;
Torikai and Imai, 1984). While caution should be exercised when
making comparisons between in vitro and in vivo situations, it is
tempting to speculate that vasopressin-stimulated PI hydrolysis may
be involved in NaCl transport or cell volume regulation of renal
epithelial cells and these hormonal effects may be modulated by
peritubular osmolality.
In summary, this study represents the first demonstration that
vasopressin stimulates phosphatidyl inositol hydrolysis in a time- and
dose-dependent manner. Furthermore, the response appears to be
hormone-specific, in
that
none of
the
other agents tested
caused a
release
of inositol
phosphates.
The
vasopressin-mediated
response
could
be modulated
by
altering
the
osmotic conditions
of the
incubation medium; a situation expected to be encountered in the
corticomedullary gradient of peritubular osmolality in the kidney.
Because the vasopressin-stimulated adenylate cyclase system is well-
documented in this cell line, the LLC-PK] system represents an

109
experimental model to study the interaction of the two
messenger systems in epithelial cells of renal origin.
second

CHAPTER 7
SUMMARY
Arginine^-vasopressin, a neurohypophyseal hormone, is
synthesized
as
a prohormone in
the
supraoptic and
paraventricular
nuclei of
the
hypothalamus, transported
and
modified
axonally and
stored in
nerve
terminals within
the
neural
lobe of
the posterior
pituitary gland. An extrahypothalamic distribution of vasopressinergic
fibers also innvervates multiple regions of the central nervous system,
with an especially dense field of terminals found in the brainstem.
The peripheral and central vasopressin systems act, by vastly
different mechanisms, to maintain fluid balance and circulatory
homeostasis. Centrally, vasopressin can affect circulatory control by
modulating the activity of the baroreceptor reflex and more directly,
by affecting sympathetic and parasympathetic outflow. In the
periphery vasopressin is a pressor substance, acting on resistance
vessels and causing vasoconstriction. More importantly, vasopressin
represents the principal hormone of fluid balance, acting at discrete
tubular sites in the kidney to promote the reabsorption of water and
electrolytes, principally sodium. Therefore, it is not surprizing that
the regulation of vasopressin synthesis and release should be
governed by both osmotic stimuli, mediated by centrally-located
osmosensitive cells and non-osmotic stimuli, sensed by volume-
sensitive mechanoreceptors in the heart and peripheral vessels.
Alterations in the synthesis or release of the hormone can have
110

Ill
profound physiological consequences. These range from diabetes
insipidus, a condition characterized by an inability to synthesize
vasopressin and manifested clinically by polydipsia and polyuria, to
the syndrome of inappropriate ADH secretion wherein circulating
levels of vasopressin are dramatically elevated, promoting fluid
retention and often resulting in hyponatremia.
Conversely, the biological activity of vasopressin can be affected
by modifying the sensitivity of the target tissue to the hormone.
AVP initiates a biological response, be it central, renal, vascular or
otherwise, by interacting with plasma membrane-bound receptors
located on the cells of the target tissue. These receptors are
integral proteins which transduce the principal (hormone) signal into
a cellular response
by activation
of
"second messenger"
systems.
Currently, vasopressin
receptors
have
been
classified
into two sub-
types (Michell et al.,
1979).
The
Vi
receptor is
coupled
to the
activation of phosopholipase C, resulting in the hydrolysis of
phosphatidyl inositol and the generation of inositol phosphates (IP)
and diacylglycerol (DG). DG is an endogenous activator of protein
kinase C, an enzyme which causes the phosphorylation of numerous
cells proteins. IP acts to cause the release of calcium from intra¬
cellular stores, which in turn, can affect the activity of calcium-
dependent enzymes (Berridge, 1987). Vj receptors have been
characterized in smooth muscle tissue where they mediate the
vasoconstrictive actions of the hormone. In addition Vj sites have
also been reported on glomerular mesangial cells and in gonadal, liver
and adrenal tissue (Chapter 1). In addition, it now apprears that Vj-
type receptors are also present in epithelial cells of renal origin

112
(Chapter 6). V2 receptors, thought to located exclusively on renal
tubular epithelial cells, are coupled to adenylate cyclase and the
formation of cyclic AMP (Morel et al., 1987). These receptors are
responsible for transducing the antidiuretic action of the hormone.
By means of cAMP-dependent protein kinases, phosphorylation of
integral proteins in the apical membrane of a discrete population of
epithelial cells leads to the formation of aqueous channels (Gluck and
Al-Awqati, 1980) and an enhanced apical permeability to tubular fluid.
Vasopressin-stimulated, cyclic AMP-dependent activation of Na-K-
ATPase, the active transporter of sodium may account for the ability
of certain epithelial
tissues to
promote
reabsorption of
this
electrolyte (Tomita, et
al., 1987).
Clearly,
modification of
these
receptor-mediated processes will have
as dramatic an impact
on
physiological function as did the alteration of the hormone itself.
Pathophysiological conditions associated with receptor defects are
most dramatically depicted in cases of hypercholesterolemia and non¬
insulin-dependent diabetes mellitus, where low density lipoprotein
receptor and insulin receptor numbers are diminished, thereby
reducing the sensitivity
of
the
target tissues to
the respective
hormones. By analogy,
if
the
sensitivity of renal
tubular target
tissues to vasopressin was altered, this could have profound effects
on the transepithelial transport of fluid and electrolytes, with the
potential for influencing fluid homeostasis.
In this dissertation, the effect of alterations in fluid balance on
vasopressin receptors was studied. Vasopressin receptors were
characterized on basolateral membranes prepared from rat kidney
tubular epithelia. Basolateral membranes were isolated by differential

113
and density gradient centrifugation techniques. Activity of Na-K-
ATPase, a marker enzyme for these membranes, was increased ten¬
fold. Binding of a tritiated radioligand was rapid, reversible,
saturable and specific. The binding site displayed a high affinity
(~lnM) and fit a pharmacological profile consistent for a V2-type
vasopressin receptor. Furthermore, AVP-stimulated cAMP formation
was demonstrated using this membrane preparation.
Vasopressin receptors undergo regulation, in vivo and |n vitro.
Homologous downregulation has been demonstrated previously, but
only under the influence of pharmacological doses of AVP (Rajerison
et al, 1977; Lester et al., 1985; Eggena and Ma, 1986). What effect,
if any, do physiological changes in circulating levels of vasopressin
have on renal tubular vasopressin receptors? The present study
showed that V2 vasopressin receptor number is reduced in basolateral
membranes prepared from water-deprived rats. This has important
ramifications with respect to fluid homeostasis, especially in an
animal already compromised by dehydration. One plausible
explanation in this case is that the downregulation may reflect a
diminution of a vasopressin receptor "reserve" (Ariens et al., 1960).
"Spare" receptors have been noted in other adenylate cyclase-linked
tissues as well (Flores et al., 1974). Alternatively, a fraction of the
total number of receptors present in any given tissue may represent
"silent" receptors (Maack et al., 1987). These binding sites are not
coupled to a biological effector system and therefore, elicit no
response. However, they may serve as "peripheral clearance sites",
acting as a buffer system to modulate plasma levels of the hormone.
Thus, downregulation of the inert hormone-receptor complex may

114
represent one avenue for buffering the system against high
circulating levels of the hormone. Evidence is accumulating that
receptor reserve and signal transduction relationships are impacted by
changes in physiological state (Kinter et al., 1988). The physiological
significance of "silent” and "spare” receptors and their regulation
remains to be explored.
Heterologous regulation of vasopressin receptors implies that
other hormones or factors can regulate AVP receptor expression.
Roy et al. (1980) described the regulation of vasopressin receptors by
insulin, in an in vitro preparation. In the present study, the
expression of vasopressin receptors and vasopressin-stimulated
adenylate cyclase activity was dramatically increased in rats treated
with a synthetic steroid hormone, desoxycorticosterone acetate
(DOCA) + 1% NaCl. The upregulation occurred despite an elevated
plasma concentration of vasopressin. This suggests that the increased
density of AVP receptors is due to an increased transcriptional rate
of vasopressin receptor protein synthesis, induced by the steroid
hormone. Desoxycorticosterone acetate (DOCA) has previously been
shown to affect the expression of other basolateral membrane
proteins, including Na-K-ATPase (Garg et al., 1981). Reif et al.
(1986) have demonstrated that vasopressin-stimulated sodium
reabsorption is augmented in the presence of DOCA. Taken together
with the findings of Pettinger et al. (1986) that vasopressin-
stimulated cyclic
AMP
formation
was enhanced in
microdissected
cortical
collecting
tubules
of DOCA
-treated rats, the
above
studies
suggest
that the
tubular
effects of
vasopressin play a
role
in the
etiology of mineralocorticoid-dependent hypertension. The present

115
findings indicate that transporting-epithelial tissue in the kidney
display an increased sensitivity toward vasopressin, as indicated by
the enhanced formation of A VP-stimulated cyclic AMP. The increased
fluid and sodium-retaining properties of the hormone lend themselves
to the development of hypertension, exclusive of its vasopressor
activity. The
role of
the
kidneys
in the pathophysiology
of
hypertension is
supported
by
Guyton
et al. (1972) and
the
observations of Manning et al. (1979) and Cowley et al. (1984).
Regulation of vasopressin receptors in vitro has been reported
by Roy et al. (1980) and Lester et al. (1985). The lack of a
demonstable effect of DOCA in LLC-PK] cells in the present study
may reflect an absence of receptors for mineralocorticoids in this cell
line. This is supported by the findings of Roy et al. (1980) who were
unable to modulate receptor expression by aldosterone in LLC-PK jl
cells, a subline of the parent LLC-PK \ strain. This contrasts with
the ability of mineralocorticoids to exert effects on physiological
processes in A^ and MDCK epithelial cell lines (Meier and Insel,
1985). Methodological considerations should not be overlooked
however, when interpreting the results of DOCA-treated LLC-PK \
cells.
Perhaps the most exciting finding in this series of studies is
that vasopressin is coupled to two distinct receptor subtypes in LLC-
PK i cells. This exciting discovery would redefine the long-held
dogma that VI receptors are present on a variety of tissues, but that
transport-epithelia possess only V2 receptors, linked to adenylate
cyclase. The present study demonstrated that indeed, vasopressin-
stimulated cyclic AMP formation does occur in these cells. By

116
loading ^H-inositol into the cell membrane, it was shown that
vasopressin was also effective in stimulating the hydrolysis of
phosphatidyl inositol which had incorporated the radioligand. This
resulted in the release of -^H-inositol phosphates which, according to
Berridge (1987), act to mobilize intracellular stores of calcium. This
finding complements the study of Tang and Weinberg (1986) who
measured vasopressin-stimulated calcium fluxes in the same cell line.
In addition, release of inositol phosphates in the present study was
modulated by altering the ionic milieu of the incubation buffer. This
effect mimicked the changing osmotic composition found in the
corticomedullary gradient of interstitial fluid in the kidney. Whereas
IP released was diminished with increasing osmotic concentration, Roy
and Ausiello (1981b) reported that vasopressin-stimulated cyclic AMP
production increased with increasing osmotic concentration. This
underscores the potential interaction between the two transmembrane
signaling systems.
Therefore, according to the classification scheme of Michell et
al. (1979), it appears that LLC-PKj cells possess not only V2-type
receptors linked to adenylate cyclase but that V]-type receptors
coupled to PI hydrolysis are also present on the cell surface. The
present finding and those of Roy and Ausiello (1981b) suggest that
vasopressin is capable of regulating multiple effector systems.
Conversely, these signal transduction systems appear to be regulated
differentially by the ionic milieu to which they are exposed. This
may allow for one action of vasopressin to prevail in a particular
segment of the nephron. Only very recently reports have surfaced in
the literature describing the expression of multiple vasopressin

117
receptor subtypes in renal epithelial tissue (Star et al., 1988;
Burnatowska-Hledin and Spielman, 1988). Even more extraordinary, a
single muscarinic receptor subtype (M2) has been shown to be coupled
to both adenylate cyclase and phosphoinositide hydrolysis (Ashkenazi
et al., 1987).
These studies were intended to further our understanding of
vasopressin receptors and receptor-mediated signal transduction
processes in renal epithelial tissue. The series of experiments
demonstrate:
1) The characterization of vasopressin binding sites in a Na+-K+-
ATPase-enriched preparation of renal tubular epithelial basolateral
membranes prepared by density-gradient centrifugation;
2) Dehydration-induced elevation of circulating levels of vasopressin
down-regulates vasopressin receptors;
3) Excess exposure to mineralocorticoids up-regulates vasopressin
receptors
and increases the
tubular
sensitivity of
vasopressin-
stimulated
adenylate cyclase.
Enhanced
sensitivity to
the fluid-
retaining properties of the hormone provides a cellular and systemic
basis for the role of vasopressin in the pathogenesis of DOCA-salt
hypertenion;
4)Vasopressin receptors are coupled to the formation of cAMP,
and also to phosphatidyl inositol turnover in LLC-PKj cells. The two
transduction processes are modulated differentially by the osmotic
strength of the incubation medium. The coupling of vasopressin
receptors to multiple effector systems redefines our concept of signal
transduction in renal tubular epithelia.

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BIOGRAPHICAL SKETCH
Martin Steiner was born March 3, 1952, in New York City,
where he lived for a period of one year. He moved with his family
to Chicago, where he remained for the next 17 years. He attended
the University of Iowa where he received a B.S. degree in zoology in
1974. After five years as a research assistant in the Departments of
Pharmacology and Pyschiatry at The University of Iowa, he began his
graduate studies, receiving a M.S. degree in zoology from Clemson
University in 1982. He proceeded to the University of Florida and
entered the graduate program in physiology.
Under the supervision of Dr. M. Ian Phillips, Martin Steiner was
awarded a Ph.D. in April, 1988. There is no truth to the rumor that
he will pursue other courses of study, thereby ending his quest to
become the longest living "professional student." Therefore, he
expects to a) get married, b) get a job, and c) live happily ever
after.
132

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.
M. Ian Phillips, D.Sc., Chairman
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.
\
Steven R. Childers, Ph.D.
Associate Professor of
Pharmacology and Therapeutics
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.

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.
^—
Colin Sumners, Ph.D.
Associate Professor of Physiology
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 requirements for the degree of Doctor of
Philosophy.
April, 1988
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 8336



21
Lutz-Bucher and Koch (1983). Antoni (1984) reported that the ligand
specificity of pituitary vasopressin receptors was distinct from
previously characterized Vj and V2 receptors although no
measurements of the second messengers were conducted. Gaillard et
al. (1984) however, did report that vasopressin failed to influence
adenylate cyclase activity in pituitary membrane fractions, but did
potentiate CRF-stimulated cAMP accumulation. Balia et al. (1985)
described the binding and signal transduction mechanisms for
vasopressin in membranes prepared from rat adrenal capsules (chiefly
zona glomerulosa). Displacement studies suggested that the receptor
was of the V\ variety and incorporation studies indicated that A VP
32
promoted P uptake into PI. This effect was blocked by V\
antagonists as was AVP-stimulated PI hydrolysis. The net effect of
vasopressin binding in isolated zona glomerulosa cells was the
stimulation of aldosterone secretion. Whereas vasopressin stimulates
steroid biosynthesis in the adrenals, it inhibits testicular Leydig cell
steroidogenesis. Receptors which mediate this effect were described
by Meidan and Hsueh (1985). Relative binding potencies of selective
agonists and antagonists suggest that it is a V\ subtype. Finally, a
number of cellular elements in the bloodstream are reported to
possess vasopressin receptors. Binding sites on platelets were
described by Berrettini et al. (1982). Mononuclear phagocytes were
shown by Block et al. (1981) to possess vasopressin receptors.
Although A VP has been shown to cause platelet aggregation, the
function of vasopressin receptors in monocytes is unknown. Table 1-
1 summarizes the current state of knowledge concerning vasopressin
receptors in biological tissues and their significance.


91
other hand, at high doses (>1 pg/ml), viability of the cells became a
significant factor. One study reported that ^H-DOCA receptor
binding was inhibited by a broad class of protease inhibitors (Baker
and Fanestil, 1977). Whether mineralocorticoids exert any
physiological effects in LLC-PK] cells remains in question, but it is
entirely possible that the use of protease inhibitors in the present
study may have antagonized the effect of the steroid.
Thus, vasopressin receptors are expressed in vivo on renal
tubular epithelial membranes and in vitro on renal epithelial cells.
Both receptors are coupled to adenylate cyclase. In addition, both
receptors are susceptible to regulation by vasopressin (Chapter 3 and
Lester et al., 1985). It appears however, that the receptors respond
differentially to the effects of mineralocorticoids. The question of
whether mineralocorticoids directly affect the expression of
vasopressin receptors at the cellular level remains unanswered using
LLC-PK] cells as a model. The question may be more suitably
addressed using a primary culture of epithelial cells prepared from rat
collecting tubules.


66
Basal and vasopressin-stimulated cyclic AMP formation was
measured in freshly prepared membranes from the various groups.
The values were derived from the standard curve depicted in Figure
4-4. Figure 4-5 represents the production of cAMP in membranes
prepared from animals in the DOCA-salt hypertensive group (I) and
the corresponding control group (II). Whereas basal values did not
differ from each another, membranes from the hypertensive animals
were stimulated to a greater degree than membranes from the control
group. The enhanced sensitivity to vasopressin is evidenced by a
shift
toward the
left
for
the curve depicting
Group
I values.
Subtracting basal
values,
the
calculated EDygs for
the
hypertensive
and
control group
are
2x10'
10 M and 8.5xl010
M,
respectively.
Table 4-2 summarizes the basal and vasopressin-stimulated cyclic AMP
production in membranes of the other experimental groups. Basal
values did not differ from one another but a greater level of
stimulation and an enhanced sensitivity toward vasopressin were also
noted in membranes from the hypertensive animals in Group IV.
Finally, plasma vasopressin levels were determined in the
animals of the various groups and those data are presented in Table
4-3. Values for the non-hypertensive animals in Groups II and V
ranged from 1.20.2 pg/ml to 1.80.2 pg/ml, respectively. Plasma
vasopressin concentrations were significantly elevated among the
hypertensive animals. Measured values in Groups I and IV were
6.H0.8 pg/ml and 8.414.5 pg/ml, respectively. This represents a five
fold elevation in plasma vasopressin concentrations between the
hypertensive and normotensive animals.


122
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20
data which contend that vasopressin acts in the brain as a neuro-
transmitter or neuromodulator. The presence of AVP receptors in the
regions mentioned above parallel the vasopressinergic pathways
involved in central cardiovascular regulation.
The nature of the effector system coupled to the vasopressin
receptor in the brain still remains in question. Competition of ^H-
vasopressin binding with vasopressin analogues suggests that the
receptor resembles the vascular or V \ subtype (Cornett and Dorsa,
1985; Audigier and Barberis, 1985), although no direct measurements
of vasopressin-stimulated PI hydrolysis have been reported. However,
a presynaptic membrane phosphoprotein known as B50 which is a
substrate for protein kinase C, is phosphorylated upon the addition of
AVP to rat hippocampal synaptic membranes (Hinko and Perlmutter,
1987).
These data
tend
to
suggest that
vasopressin acts through Vi
type
receptors in
neuronal
membranes
to cause the formation of
inositol
phosphates
and
diacylglycerol.
DG may activate
protein
kinase
C which in
turn,
phosphorylates
integral membrane
proteins.
Given
the importance
of
calcium in
the process of
synaptic
transmission, the actions of IP cannot be excluded either.
Other Tissues
A burgeoning number of studies have recently reported the
presence of vasopressin receptors in a variety of tissues. ^H-
vasopressin binding and its relation to glycogen phosphorylase activity
was reported by Cantau et al. (1980). Activation of this enzyme and
its resulting glycogenolytic action is mediated by V\ receptors
coupled to PI hydrolysis (Michell et al., 1979). In the anterior
pituitary gland, vasopressin receptors were first characterized by


109
experimental model to study the interaction of the two
messenger systems in epithelial cells of renal origin.
second


124
Hull, R.N., W.R. Cherry and G.W. Weaver. The origin and character
istics of a pig kidney cell strain, LLC-PKIN VITRO 12:670-
677, 1976.
Hwang, B.H., J.Y. Wu and W.B. Severs. Effects of chronic
dehydration on angiotensin II receptor binding in the subfornical
organ, paraventricular hypothalamic nucleus and adrenal medulla
of Long-Evans rats. Neurosci. Lett. 65:35-40, 1986.
Ichikawa, I and B.M. Brenner. Evidence for glomerular actions of
ADH and dibutyryl cyclic AMP in the rat. Am. J. Physiol.
233:F102-F117, 1977.
Imai, Y., P.L. Nolan and C.I. Johnston. Restoration of suppressed
baroreflex sensitivity in rats with hereditary diabetes insipidus
(Brattleboro rats) by arginine vasopressin and DDAVP.
Circ. Res. 53:140-149, 1983.
Ingram, W.R., C. Fisher and S.W. Ranson. Experimental diabetes
insipidus in the monkey. Arch. Intern. Med. 57:1067-1080, 1936.
Jans, D.A., T.J. Resink and B.A. Hemmings. Complementation between
LLC-PK! mutants affected in polypeptide hormone-receptor
function. Eur. J. Biochem. 162:571-576, 1987.
Jard, S., Vasopressin isoreceptors in mammals: relation to cyclic AMP-
dependent and cyclic AMP-independent transduction mechanism.
Curr. Topics Memb. Transport 18:255-285, 1983.
Jewell, P.A., and E.B. Verney. An experimental attempt to determine
the site of the neurohypophysial osmoreceptors in the dog.
Philos. Trans. R. Society 240:197:324, 1957.
Johnston, C.I. Vasopressin in circulatory control and hypertension.
J. Hyperten. 3:557-569, 1985.
Kimura, T., L. Share, B.C. Wang and J.T. Crofton. The role of
central adrenoceptors in the control of vasopressin release and
blood pressure. Endocrinology 108:1829-1836, 1981.
Kinoshita, Y., M. Fukase, T. Yamatani, T. Chiba, M. Nakai, M.
Tsutsumi and K. Fujita. Possible involvement of inositol
phosphates and calmodulin in calmodulin-induced stimulation of
phosphate transport in LLC-PK! cells. Biochem. Biophys. Res.
Comm. 144:741-748, 1987.
Kinter, L.B., W.F. Huffman and F.L. Stassen. Antagonists of the
antidiuretic activity of vasopressin. Am. J. Physiol.
254:F165,F177, 1988.
Kirk, K.L. Antidiuretic hormone (ADH)-mediated water reabsorption
stimulates internalization of basolateral cell membrane in rabbit
cortical collecting tubule (CCT). Federation Proc. 46:1285, 1987
(Abstract).


93
has been shown to be an endogenous activator of protein kinase C
(Berridge, 1987), as illustrated in Figure 6-1.
Roy and Ausiello (1981b) have demonstrated that the vasopressin
receptor-effector system in LLC-PK | cells can be modulated by
altering the osmolality of the medium to which the cells are exposed.
Variation in osmotic conditions is analogous to the situation in the
kidney where segments of the tubule are exposed to a continous
corticomedullary osmotic gradient. Thus, the epithelial cells and the
receptors situated on the plasma membranes of their surfaces are
exposed to conditions ranging from isotonicity to hypertonicity. Roy
and Ausiello (1981b) reported that incubation in hypertonic NaCl will
increase the binding of vasopressin and the coupling between the
receptor and adenylate cyclase, leading to elevated levels of cAMP.
Thus, while it is well known that vasopressin stimulates the
production of cAMP in LLC-PK] cells, the following study was
designed to determine if vasopressin stimulates the hydrolysis of
phosphatidyl inositol, resulting in the formation of inositol
phosphates. Furthermore, the influence of increasing the osmolality
of the incubation medium on vasopressin-stimulated PI hydrolysis was
also examined.
Materials and Methods
Chemicals
Peptides (arginine vasopressin, lysine vasopressin, angiotensin II)
and the cholinergic agonist carbachol were purchased from Sigma (St.
Louis). Vasopressin antagonists were a gift from Dr. M. Manning of
the Medical College of Ohio. ^H-myo-inositol and OCS liquid
scintillation fluid were obtained from Amersham (Arlington Hts., IL).


62
after only 4 weeks of treatment. This contrasts to the animals in
group III who received only DOCA and displayed an arterial pressure
of 121 7 mm Hg. This value was not significantly different from the
values observed in animals in Groups V (1088 mm Hg) and II (1106
mm Hg), receiving no treatment or 1% NaCl and unilateral
nephrectomy, respectively.
^H-vasopressin binding in renal epithelial basolateral membranes
prepared from the DOCA-salt hypertensive rats (Group I) and their
appropriate controls (Group II) revealed a higher level of binding in
membranes from the hypertensive group (Figure 4-2). As depicted in
Figure 4-3, Scatchard analysis of these data indicate that this higher
level of binding is due to an increased number of binding sites
(Group I = 30729 fmoles/mg protein vs. Group II = 17917 fmoles/mg
protein) [p<.05], rather than a change in affinity (Group I = 0.59.ll
nM vs. Group II = 0.54.09 nM) [p>.05]. This represents a 71%
increase over control values. Table 4-1 summarizes the binding data
for the other experimental groups. It can been noted that a higher
Bmax *s a*so ev^ent for the hypertensive animals in Group IV
(281 33 f moles/mg protein). This too, is significantly higher than the
values for Group II (p<.05). Furthermore, there were no significant
differences between the values in Group II and the untreated animals
in Group V (Bmax = 19623 fmoles/mg protein; Kj) = 0.67.12) [p>.05].
Binding (max = 191 14 fmoles/mg protein; Kp = 0.63.04) in
membranes prepared from the animals receiving DOCA only (Group
III) was not significantly different from either of the control groups
(II or V).


REFERENCES
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Ausiello, D.A., J.I. K riesberg. C. Roy and J.J. Karnovsky. Contraction
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J. Clin. Invest. 65:754-760, 1980.
Ausiello, D.A., K.L. Skorecki, A.S. Verkman and J.V. Bonventre.
Vasopressin signaling in kidney cells. Kidney Int. 31:521-529,
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Baddouri, K., D. Butlen, M. Imbert-Teboul, F. Le Bouffant, J.
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1984.
118


CHARACTERIZATION AND REGULATION OF RENAL
VASOPRESSIN RECEPTORS
By
MARTIN STEINER
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
1988


BIOGRAPHICAL SKETCH
Martin Steiner was born March 3, 1952, in New York City,
where he lived for a period of one year. He moved with his family
to Chicago, where he remained for the next 17 years. He attended
the University of Iowa where he received a B.S. degree in zoology in
1974. After five years as a research assistant in the Departments of
Pharmacology and Pyschiatry at The University of Iowa, he began his
graduate studies, receiving a M.S. degree in zoology from Clemson
University in 1982. He proceeded to the University of Florida and
entered the graduate program in physiology.
Under the supervision of Dr. M. Ian Phillips, Martin Steiner was
awarded a Ph.D. in April, 1988. There is no truth to the rumor that
he will pursue other courses of study, thereby ending his quest to
become the longest living "professional student." Therefore, he
expects to a) get married, b) get a job, and c) live happily ever
after.
132


116
loading ^H-inositol into the cell membrane, it was shown that
vasopressin was also effective in stimulating the hydrolysis of
phosphatidyl inositol which had incorporated the radioligand. This
resulted in the release of -^H-inositol phosphates which, according to
Berridge (1987), act to mobilize intracellular stores of calcium. This
finding complements the study of Tang and Weinberg (1986) who
measured vasopressin-stimulated calcium fluxes in the same cell line.
In addition, release of inositol phosphates in the present study was
modulated by altering the ionic milieu of the incubation buffer. This
effect mimicked the changing osmotic composition found in the
corticomedullary gradient of interstitial fluid in the kidney. Whereas
IP released was diminished with increasing osmotic concentration, Roy
and Ausiello (1981b) reported that vasopressin-stimulated cyclic AMP
production increased with increasing osmotic concentration. This
underscores the potential interaction between the two transmembrane
signaling systems.
Therefore, according to the classification scheme of Michell et
al. (1979), it appears that LLC-PKj cells possess not only V2-type
receptors linked to adenylate cyclase but that V]-type receptors
coupled to PI hydrolysis are also present on the cell surface. The
present finding and those of Roy and Ausiello (1981b) suggest that
vasopressin is capable of regulating multiple effector systems.
Conversely, these signal transduction systems appear to be regulated
differentially by the ionic milieu to which they are exposed. This
may allow for one action of vasopressin to prevail in a particular
segment of the nephron. Only very recently reports have surfaced in
the literature describing the expression of multiple vasopressin


38
complement other studies that have examined the vasopressin-sensitive
adenylate cyclase system in the kidney and its coupling to V2-type
receptors (Roy et al., 1975; Rajerison et al., 1977; Butlen et al., 1978).


71
Discussion
The role of vasopressin in the development of DOCA-salt
hypertension appears to be unequivocal. Blood pressure will not
increase in this model unless vasopressin is present. This was
demonstrated most elegantly by the use of Brattleboro rats, animals
which are genetically incapable of synthesizing AVP (Crofton et al.,
1979). What is unclear is the mechanism or mechanisms which
underlie the etiology of the blood pressure increase. Various target
tissues have been implicated to account for the hypertensive actions
of vasopressin. A central site of action has been suggested by the
study of Chen et al. (1986). These investigators reported heightened
vasopressinergic activity in DOCA-salt hypertensive rats. This
augmentation in hypothalamic release of vasopressin was exclusive of,
and preceeded the well-known increase in peripheral AVP levels or
elevation of blood pressure. The authors suggested that this
alteration in central vasopressinergic activity may affect blood
pressure by modulation of the cardiovascular centers in the brain.
Studies with vasopressor antagonists suggest a vascular site of action.
Crofton et al. (1979) reported that the acute administration of V]
blockers lowered blood pressure in animals with established DOCA-salt
hypertension. Hofbauer et al. (1984) found that the chronic
administration of V j blockers during the DOCA-salt regimen
attenuated the development and magnitude of hypertension associated
with this model. Other studies however, tend to refute these
findings (Rascher et al., 1983; Yamamoto et al., 1984; Filep et al.,
1987).


5
and water reabsorption. Vasopressin is a potent inhibitor of renin
secretion. Subpressor doses of AVP decrease plasma renin levels. It
is thought that vasopressin acts directly on the juxtaglomerular cells,
as the effect can be demonstrated in the isolated kidney and occurs
with no demonstrable effect on renal hemodynamics (Vander, 1968).
Arginine^-vasopressin reduces the glomerular ultrafiltration
permeability coefficient (Kf) and may decrease the glomerular
filtration rate (Ichikawa and Brenner, 1977). While the mechanism by
which AVP decreases the Kf is not clear, Ausiello et al. (1980) have
shown that vasopressin stimulates the contraction of glomerular
mesangial cells grown in culture. Therefore, it is possible that
mesangial contraction reduces the surface area available for
glomerular ultrafiltration.
The next demonstrable action
of vasopressin occurs at
the
thick
ascending Limb
of Henle. Morel et al. (1976)
made
the
first
observation of vasopressin-sensitive
adenylate cyclase
activity
in
both
the medullary and
cortical portions
of the thick ascending limb.
The
concentration of
AVP necessary
to stimulate
adenylate
cyclase
activity in the
thick ascending
limb is one or
two
orders of
magnitude higher
than that needed
in more distal
tubular
segments.
The physiological significance of this finding remained in doubt until
Hebert et al. (1981a) found that AVP, in pico-molar concentrations,
stimulated NaCl transport out of the tubular lumen into the
interstitium. This suggests a role for vasopressin in the maintenance
of the corticomedullary interstitial osmotic gradient.
The major target for the action of vasopressin is the distal
segment of the collecting tubule. Here vasopressin, through a cAMP-


85
Figure 5-4. Basal and vasopressin-stimulated cyclic AMP formation in
LLC-PKj cells. Procedures for the stimulation and determination of
cyclic AMP are described in the "Methods" section. AVP, arginine
vasopressin; cAMP, adenosine 3'5'-cyclic monophosphate; V, blocker,
d(CH2)5D-Ile2-Ile4-AVP.


26
sensitive adenylate cyclase system is present on the peritubular side
of tubular epithelia (Grantham and Burg, 1966), the characterization
of vasopressin receptors localized specifically to the basolateral
membrane has not been previously described. This study was
conducted to determine the characteristics of vasopressin binding for
a basolateral membrane preparation of rat renal tubular epithelia.
Materials and Methods
Animals and Chemicals
Male Sprague-Dawley rats weighing 200-250g were obtained from
the Animal Resource Facility at the University of Florida. Rats were
housed individually in 20x18x24 cm wire mesh cages, exposed to a
12:12 hour light:dark cycle, provided a standard Purina rat chow diet
and allowed free access to tap water.
Percoll and synthetic peptides were purchased from Sigma
Chemical Co. (St. Louis). d(Et)2Tyr(Et)D-AVP (Vj) and d(CH2)5D-
Ile^-Ile^-AVP (V2) vasopressin analogues were generous gifts of Dr.
M. Manning of the Medical College of Ohio in Toledo. Tritiated 8-L-
arginine [phenylalanine-3,4,5-^H]-AVP (^H-AVP) used in receptor
binding studies was purchased from New England Nuclear Corp.
(Boston) with a specific activity of 70 Ci/mmol. Other reagents were
obtained from Sigma, unless otherwise indicated.
Membrane Preparation
Animals were sacrificed by decapitation. Renal basolateral
membranes were prepared according to the density gradient
centrifugation method of Boumendil-Podevin and Podevin (1983), with
slight modification. Kidneys were removed, decapsulated and rinsed
in a chilled buffer containing 250 mM sucrose, 2 mM Tris-HEPES, 0.1


IBMX
isobutylmethylxanthine
IP
inositol phosphate
K.IU
kallikrein inhibitory units
KRB
Krebs Ringers buffer
LVP
lysine vasopressin
MDCK
Madine-Darby canine kidney
NTS
nucleus of the tractus solitarius
OXY
oxytocin
PBS
phosphate-buffered saline
PI
phosphatidyl inositol
PMSF
phenylmethylsulfonyl fluoride
PNPP
p-nitrophenyl phosphate
PVN
paraventricular nucleus
RIA
radioimmunoassay
RPM
revolutions per minute
SEM
standard error of the mean
SHR
spontaneously hypertensive rat
SON
supraoptic nucleus
TFA
trifluoroacetic acid
WKY
Wistar-Kyoto
vii


KEY TO ABBREVIATIONS
ACTH
adrenocorticotropic hormone
ADH
antidiuretic hormone
ANF
atrial natriuretic factor
ANG II
angiotensin II
ANOVA
analysis of variance
ATCC
American Tissue Culture Collection
ATP
adenosine triphosphate
AVP
arginine vasopressin
BSA
bovine serum albumin
CAMP
cyclic adenosine 3\5'-monophosphate
cGMP
cyclic guanosine 3',5'-monophosphate
CNS
central nervous system
CPM
counts per minute
DAME
D-ala-met-enkephalinamide
DDAVP
deamino-D-arginine vasopressin
DG
diacylglycerol
DMEM
Dulbeccos modified Eagles medium
DMSO
dimethylsulfoxide
DOCA
desoxycorticosterone acetate
DPM
disintegrations per minute
EDTA
ethylenediaminetetraacetic acid
EGTA
ethyleneglycoltetraacetic acid
FBS
fetal bovine serum
GTP
guanosine triphosphate
vi


81
Figure 5-1. 3H-vasopressin (3H-AVP) binding in LLC-PK^ cells as a
function of time. Receptor binding protocol described in "Methods"
section. Association: Total binding determined at 2.5 nM [3H-AVP].
Non-specific binding (NSB) determined in the presence of 2.5 nM [3H-
AVP] + 2.5 /M unlabeled AVP. Total NSB = Specific (75%).
Dissociation: At equilibrium binding (1 hr), total 3H-AVP binding is
dissociated from receptor by the addition of 2.5 /M unlabeled AVP.


130
Stoeckel, M.E., M.J. Freund-Mercier, J.M. Palacios, Ph. Richard and A.
Porte. Autoradiographic localization of binding sites for
oxytocin and vasopressin in the rat kidney. J. Endocr. 113:179-
182, 1987.
Swanson, L.W., and H.G.J.M. Kuypers. The paraventricular nucleus of
the hypothalamus: Cytoarchitectonic subdivisions and the
organization of projections to the pituitary, dorsal vagal complex
and spinal cord as demonstrated by retrograde fluorescence
double-labeling method. J. Comp. Neurol. 194:555-570, 1980.
Swanson, L.W., and P.E. Sawchenko. Paraventricular nucleus: A site
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Neuroendocrinology 31:410-417, 1980.
Swanson, L.W., and P.E. Sawchenko. Hypothalamic integration:
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Tajima, Y., S. Ichikawa, T. Sakamaki, H. Matsuo, F. Aizawa, M.
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Tang, M.J., and J.M. Weinberg. Vasopressin-induced increases of
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Thrasher, T.N., C.J. Brown, L.C. Keil and D.J. Ramsay. Thirst and
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receptor mechanism? Am. J. Physiol. 238.R333-R339, 1980.
Thrasher, T.N., L.C. Keil and D.J. Ramsay. Lesions of the organum
vasculosum of the lamina terminalis (OVLT) attenuate
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dog. Endocrinology 110:1837-1839, 1982.
Tomita, K., A. Owada, Y. Ino, N. Yoshiyama, T. Shiigai and J.
Takeuchi. The effect of vasopressin on Na-K-ATPase in cortical
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(Abstract).
Torikai, S. and S. Imai. Effects of solute concentration on
vasopressin-stimulated cyclic AMP generation in the rat
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400:306-308, 1984.


61
Figure 4-1. Arterial pressure in control and DOCA-treated rats.
Blood pressures were determined prior to experiment and once per
week thereafter by tail cuff plethysmography. Arrow indicates start
of treatment. Error bars represent S.E.M. (n = 6 per group except V,
where n = 4). *p<.05 vs. V and II. See "Methods" section for key to
group designations.


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.
^
Colin Sumners, Ph.D.
Associate Professor of Physiology
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 requirements for the degree of Doctor of
Philosophy.
April, 1988
Dean, College of Medicine
Dean, Graduate School


47
pg AVP
Figure 3-3. Standard curve for arginine vasopressin (AVP). Values
determined by radioimmunoassay. Details described in "Methods"
section.


125
Lefkowitz, R.J., M.G. Caron and G.L. Stiles. Mechanisms of
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240:E441-446, 1981.


30
Radioactivity was determined in an LKB LS counter. Samples were
corrected for variations in efficiency of counting by an internal
program and expressed as disintegrations per minute (dpm). Based on
the radioactivity and protein in each sample, values are reported as
fmoles bound/mg protein. Specific binding was calculated as the
difference between total binding and non-specific binding.
Analysis
Data from saturation and competition experiments were analyzed
using iterative curve fitting programs. Data are reported as the
mean + S.E.
Results
The activity of Na+K+-ATPase, a marker enzyme for the
basolateral membrane was markedly enriched in the F\ fraction,
which displayed a 3.25-fold increase in activity over that of the P2
pellet and a 10-fold increase when compared to the homogenate
(Table 2-1). A two-fold enrichment of A VP binding per unit protein
was achieved in the BL membrane preparation compared to the P2
pellet. A tritiated ligand was chosen for these experiments since
iodine destroys the biological activity of the vasopressin molecule
(Dorsa et
al.,
1983).
The
binding of ^H-AVP
to renal basolateral
membranes
was
linearly
related to the amount
of protein (i.e.,
membrane)
present in
the
assay (Figure 2-2).
Association and
dissociation
of
3h-avp
is
illustrated in Figure
2-3. Equilibrium
binding of the ligand occurred within 30 minutes and was stable for
up to 2 hours. Upon equilibrium, binding was shown to be reversible
by the addition of 1 /iM AVP. Specific binding, determined in the
presence of an excess amount of AVP was 80-85% of total binding.


128
Rajerison, R., J. Marchetti, C. Roy, J. Bockaert and S. Jard. The
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control of blood pressure. Hypertension 6(Suppl.II):7-15, 1984.
Roth, J., and S.I. Taylor. Receptors for peptide hormones: alterations
in disease of humans. Annu. Rev. Physiol. 44:639-651, 1982.
Roy, C., and D.A. Ausiello. Characterization of (8-lysine)-vasopressin
binding sites on a pig kidney cell line (LLC-PKj). J. Biol.
Chem. 256:3415-3422, 1981a.
Roy, C., and D.A. Ausiello. Regulation of vasopressin binding to
intact cells. Ann. N.Y. Acad. Sci. 372:92-105, 1981b.
Roy, C., T. Barth and S. Jard. Vasopressin-sensitive kidney adenylate
cyclase. Structural requirements for attachment to the receptor
and enzyme activation. Studies with vasopressin analogues. J.
Biol. Chem. 250:3149-3156, 1975.
Roy, C., G. Guillon and S. Jard. Hormone-dependent desensitization
of vasopressin-sensitive adenylate cyclase. Biochem. Biophys.
Res. Comm. 72:1265-1270, 1976.
Roy, C., A.S. Preston and J.S. Handler. Insulin and serum increase
the number of receptors for vasopressin in a kidney-derived line
of cells grown in a defined medium. Proc. Natl. Acad. Sci.
U.S.A. 77:5979-5983, 1980.
Sachs, H., and Y. Takabatake. Evidence for a precursor in
vasopressin biosynthesis. Endocrinology 75:943-948, 1964.
Saito, T., and T. Yajima. Development of DOCA-salt hypertension in
the Brattleboro rat. Ann. N.Y. Acad. Sci. 394:309-318, 1982.
Sawyer, W.H. Evolution of neurohypophyseal hormones and their
receptors. Federation Proc. 36:1842-1847, 1977.
Sawyer, W.H., Z. Gronzka and M. Manning. Neurohypophyseal
peptides: Design of tissue-specific agonists and antagonists. Mol.
Cell. Endocrinol. 22:117-134, 1981.


2
demonstrated that the vasopressin-neurophysin molecule is formed as
part of a common precursor hormone. The precursor molecule is
synthesized in the neuronal cell body by a ribosomal mechanism and
packaged into secretory granules by the Golgi apparatus. The
vesicles are transported by axoplasmic flow toward the nerve terminal
in the neural lobe of the pituitary where they are stored. Gainer et
al. (1977) have shown that the prohormone undergoes post-
translational processing during axonal transport, suggesting that the
granules must contain both the precursor molecule and enzymes
responsible for its modification. The contents of the secretory
granules (hormone, neurophysin, and residual peptide fragments) are
released upon depolarization of the nerve terminal. This exocytotic
process involves a calcium-dependent fusion of the secretory vesicle
with the nerve terminal membrane, followed by an opening of the
vesicle and an emptying of its contents.
Control of Vasopressin Secretion
It is reasonable to expect that the release of vasopressin from
the neural lobe of the pituitary gland should be influenced by factors
pertaining to vasopressins role in fluid homeostasis and circulatory
control.
Furthermore,
regulation
of
vasopressin secretion
would
be
expected
to affect the
activity
of
the paraventricular
(PVN)
and
supraoptic
nuclei (SON).
Indeed,
the
activity of these
structures
is
influenced by both osmotic and non-osmotic stimuli.
The
pioneering study
of
Verney (1947)
was
the first to
demonstrate that
centrally
located structures
which
were osmo-
sensitive,
affected
the secretion
of vasopressin.
These
structures are
so exquisitely sensitive that changes in plasma osmolality as small as


ACKNOWLEDGEMENTS
I wish to express my most sincere gratitude and appreciation to
the chairman of my supervisory committee, Dr. M. Ian Phillips, for
his friendship, guidance and support and the confidence in me that he
exhibited. Sincere thanks are extended to the members of my
committee, Drs. Steven R. Childers, Mohan K. Raizada and Colin
Sumners, for helpful advice and invaluable criticism throughout this
project. Thanks go to the many faculty members in the Departments
of Physiology and Pharmacology, but especially Drs. Melvin J. Fregly,
Bruce R. Stevens, Lai C. Garg and Stephen P. Baker, who offered me
guidance, suggestions or the use of their facilities. Thanks also go
to my friends and colleagues, past and present, Dr. Rod Casto, Elaine
M. Richards and Dr. Klaus Hermann, for continued encouragement.
The expert secretarial skills of Carolyn Gabbard are greatly
appreciated. And a very special note of gratitude is extended to
Birgitta Stenstrom Kimura, for expert technical assistance and, more
importantly, for her kindness the last 4 1/2 years.


This dissertation is dedicated to my parents, Lillian and Murry Steiner.


19
vasopressin binding sites must be present on the smooth muscle cells
of the resistance vasculature. This observation has proven difficult
due to the high degree of cellular heterogeneity of the vascular wall
and technical problems encountered in preparing pure plasma
membrane fractions derived from smooth muscle cells. These
obstacles have apparently been overcome as judged by the report by
Schiffrin and Genest (1983) describing vasopressin binding in a
mesenteric artery membrane preparation. AVP binding has also been
demonstrated in aortic smooth muscle cells in primary culture (Penit
et al., 1983) and in an established smooth muscle cell line (A-10
cells) (Stassen et al., 1987). These receptors, like those in renal
tissue,
exhibit
nanomolar affinity
constants
and
a
single
class of
binding
sites.
Whereas renal
tubular
vasopressin
receptors are
coupled
to
adenylate cyclase activation,
those
in
the
vasculature
stimulate
the
hydrolysis of phosphatidyl inositol
and
the
mobilization
of intracellular stores of calcium, as a means of signal transduction
(Aiyar et al., 1986).
Central Nervous System
Vasopressin receptors in the brain have been characterized using
traditional biochemical techniques and their distribution analyzed by
means of autoradiography (Dorsa et al., 1983). This group found that
the receptor behaves kinetically as those found in the periphery.
Furthermore, they reported that the NTS in the brainstem displayed a
high level of binding. Other investigators have confirmed this
finding and extended their observations to include AVP binding in the
hippocampus and lateral septum (Biegon et al., 1984). These findings
are consistent with the functional, behavioral and immunocytochemical


7
A
Figure 1-1. Model of antidiuretic hormone (ADH)-stimulated
adenylate cyclase cascade in renal tubular epithelial cell. ADH
combines with receptor in basolateral membrane. Interaction of
hormone and receptor stimulates adenylate cyclase, resulting in the
formation of cyclic AMP and the activation of cAMP-dependent
protein kinases. These enzymes act to phosphorylate microtubules,
microfilaments or integral proteins in the apical membrane. While the
mechanism of increased apical permeability to water remains unclear,
the most widely accepted model postulates the formation of aqueous
channels (Gluck and Al-Awqati, 1980).


106
LLC-PKj cells as seen in the present study, is mediated through Vj-
type vasopressin receptors. The use of selective V] and V2
antagonists proved somewhat equivocal, however. A series of these
compounds have been developed which antagonize the vasoconstrictive
and antidiuretic actions of the hormone, respectively (Sawyer et al.,
1981; Manning and Sawyer, 1986). In the present study, vasopressin-
stimulated PI hydrolysis was completely abolished by pretreatment
with the selective Vj antagonist d(CH2)5-Tyr(Me)-AVP. The finding
that the V2 antagonist d(CH2)-D-Ile^-Ile^-AVP partially blocked the
vasopressin-induced release of inositol phosphates was rather
surprising. Several explanations may be considered when interpreting
these data. This first deals with the relative potency displayed by
d(CH2)-D-Ile^-Ile^-AVP. This compound displays only a forty-fold
greater anti-antidiuretic activity than anti-vasopressor activity,
compared to d(CH2)5-Tyr(Me)-AVP, which is the most potent anti
vasopressor available and displays little or no antidiuretic-blocking
activity (Manning and Sawyer, 1986). Alternatively, the vasopressin
receptor present in LLC-PKj may display different physical
characteristics from vasopressin receptors for which these analogues
were designed. Kinter et al. (1988) have noted several instances
where the selectivity of V2 antagonists is greatly reduced.
Another factor that should be considered is that V2 antagonists
also inhibit vasopressin-stimulated cAMP production. This points out
the possible interaction between the two intracellular signaling
systems. Berridge (1987) describes several examples of interactions
between the PI and adenylate cyclase pathways. Friedlander and
Amiel (1987) reported that activators of protein kinase C inhibit


119
Baker, M.E., and D.D. Fanestil. Effect of protease inhibitors and
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cultures of rat brain. J. Neurochem. 47:1318-1326, 1986.
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pressin receptors in the adrenal cortex: properties of binding,
effects of phosphoinositide metabolism and aldosterone secretion.
Endocrinology 117:421-423, 1985
Bartelstone, H.S., and P.A. Nasmyth. Vasopressin potentiation of
catecholamine actions in dog, rat, cat and rat aortic strip. Am.
J. Physiol. 208:754-759, 1965.
Berecek, K.H., R.D. Murray, F. Gross and M.J. Brody. Vasopressin
and vascular reactivity in the development of DOCA
hypertension in rats with hereditary diabetes insipidus.
Hypertension 4:3-12, 1982.
Berecek, K.H., H.R. Olpe, R.S.G. Jones and K.G. Hofbauer.
Microinjection of vasopressin into the locus coeruleus of
conscious rats. Am. J. Physiol. 247:H675-681, 1984.
Berrettini, W.H., R.M. Post, E.K. Worthington and J.B. Casper. Human
platelet vasopressin receptors. Life Sci. 30:425-432, 1982.
Berridge, M.J. Inositol triphosphate and diacylglycerol: two
interacting second messengers. Annu. Rev. Biochem. 56:159-193,
1987.
Biegon, A., M. Terlou, Th.D. Voorhuis and E.R. De Kloet. Arginine
vasopressin binding sites in rat brain: a quantitative
autoradiographic study. Neurosci. Lett. 44:229-234, 1984.
Blessing, W.W., A.F. Sved and D.J. Reis. Destruction of noradrenergic
neurons in rabbit brainstem elevates plasma vasopressin, causing
hypertension. Science 217:661-663, 1982.
Block, L.H., R. Locher, W. Tenschert, W. Siegenthaler, T. Hofmann, R.
Mettler and W. Vetter. 1?-5I-8-L-arginine vasopressin binding
to human mononuclear phagocytes. J. Clin. Invest. 68:374-381,
1981.
Bockaert, J., C. Roy, R. Rajerison and S. Jard. Specific binding of
3H-lysine-vasopressin to pig kidney plasma membranes. J. Biol.
Chem. 248:5922-5931, 1973.
Bonventre, J.V., K.L. Skorecki, J.I. Kreisberg and J.Y. Cheung.
Vasopressin increases cytosolic free calcium concentration in
glomerular mesangial cells. Am. J. Physiol. 25LF94-F102, 1986.


121
Cowley, A.W., D.C. Merrill, J. Osborn and B.J. Barber. Influence of
vasopressin and angiotensin on baroreflexes in the dog. Circ.
Res. 54:163-172, 1984a.
Cowley, A.W., D.C. Merrill, E.W. Quillen and M.M. Skelton. Longterm
hemodynamic and metabolic effects of vasopressin with servo-
controlled fluid in dogs. Am. J. Physiol. 247:R537-R545, 1984b.
Crofton, J.T., L. Share, R.E. Shade, C. Allen and D. Tarnowski.
Vasopressin in the rat with spontaneous hypertension. Am. J.
Physiol. 235:H361-H366, 1978.
Crofton, J.T., L. Share, R.E. Shade. W.J. Lee-Kwon, M. Manning and
W.H. Sawyer. The importance of vasopressin in the development
and maintenance of DOCA-salt hypertension. Hypertension 1:31-
38, 1979.
Crofton, J.T., L. Share, B. Wang and R.E. Share. Pressor
responsiveness to vasopressin in the rat with DOCA-salt
hypertension. Hypertension 2:424-431, 1980.
Darmady, E.M., J. Durant, E.R. Mathews and F. Stranack.
Localization of 131I-pitressin in the kidney by autoradiography.
Clin. Sci. 19:229-241, 1960.
De Wied, D. Central actions of neurohypophyseal hormones. Prog.
Brain Res. 60:155-167, 1983.
Dierickx, K F. Vandesande and J. DeMey. Identification, in the
external region of the rat median eminence, of separate
neurophsyin-vasopressin and neurophysin-oxytocin containing
nerve fibers. Cell Tiss. Res. 168:141-151, 1976.
Di Nicolantonio, R., and F.A.O. Mendelsohn. Plasma renin and angio
tensin in dehydrated and rehydrated rats. Am. J. Phsyiol.
250:R898-R901, 1986.
Dorsa, D.M., L.A. Majumdar, F.M. Petracca, D.G. Baskin and L.E.
Cornett. Characterization and localization of 3H-arginine8
vasopressin binding to rat kidney and brain tissue. Peptides
4:699-706, 1983.
Eggena, P. Vasopressin resistance of toad urinary bladder: in vivo
and in vitro studies. Endocrinology 108:1125-1131, 1981.
Eggena, P., and C.L. Ma. Downregulation of vasopressin receptors in
toad bladder. Am. J. Physiol. 250:C453-459, 1986.
El Mernissi, G., D. Charbardes, A. Doucet, A. Hus-Citharel, M.
Imbert-Teboul, F. Le Bouffant, M. Montegut, S. Siaume and F.
Morel. Changes in tubular basolateral membrane markers after
chronic DOCA treatment. Am. J. Physiol. 245:F100-F109, 1983.


114
represent one avenue for buffering the system against high
circulating levels of the hormone. Evidence is accumulating that
receptor reserve and signal transduction relationships are impacted by
changes in physiological state (Kinter et al., 1988). The physiological
significance of "silent and "spare receptors and their regulation
remains to be explored.
Heterologous regulation of vasopressin receptors implies that
other hormones or factors can regulate AVP receptor expression.
Roy et al. (1980) described the regulation of vasopressin receptors by
insulin, in an in vitro preparation. In the present study, the
expression of vasopressin receptors and vasopressin-stimulated
adenylate cyclase activity was dramatically increased in rats treated
with a synthetic steroid hormone, desoxycorticosterone acetate
(DOCA) + 1% NaCl. The upregulation occurred despite an elevated
plasma concentration of vasopressin. This suggests that the increased
density of AVP receptors is due to an increased transcriptional rate
of vasopressin receptor protein synthesis, induced by the steroid
hormone. Desoxycorticosterone acetate (DOCA) has previously been
shown to affect the expression of other basolateral membrane
proteins, including Na-K-ATPase (Garg et al., 1981). Reif et al.
(1986) have demonstrated that vasopressin-stimulated sodium
reabsorption is augmented in the presence of DOCA. Taken together
with the findings of Pettinger et al. (1986) that vasopressin-
stimulated cyclic
AMP
formation
was enhanced in
microdissected
cortical
collecting
tubules
of DOCA
-treated rats, the
above
studies
suggest
that the
tubular
effects of
vasopressin play a
role
in the
etiology of mineralocorticoid-dependent hypertension. The present


27
mM PMSF, pH 7.4. The tissue was minced and then homogenized
with 5 strokes of a Dounce homogenizer followed by 5 strokes of a
teflon pestle homogenizer at a setting of 800 rpm. The resulting
homogenate was centrifuged at 1,000 x g for 10 minutes at 4C. The
pellet (P]) is discarded and the supernatant (Sj) centrifuged at 22,000
x g for 15 minutes at 4C. The supernatant (Sj) is discarded, leaving
a triple-layered pellet (P2). The upper two layers of the pellet were
removed by gentle vortexing and homogenized in 10 ml of the Tris-
HEPES-sucrose buffer without PMSF. Percoll (1.2 ml) is added and
mixed, then centrifuged at 40,000 x g for 35 minutes at 4C. The F |
band (Basolateral membranes, density = 1.037 g/ml) is aspirated and
diluted in 5 volumes of 2 mM Tris-HEPES, 85 mM KC1, 85 mM
sucrose, pH 7.4. Percoll
was removed
from the
mixture
by
ultracentrifugation at 60,000
x g for
15
minutes at
4C.
The
supernatant is decanted, the
membranes
are
gently washed
away
from
the Percoll pellet, resuspended in Tris-HEPES-sucrose buffer with
PMSF and stored in liquid N2. An outline of this membrane
preparation is depicted in Figure 2-1. Protein content was
determined with the Folin reagent, using bovine serum albumin as the
standard, according to the method of Lowry et al. (1951).
Determination of Na+K+-ATPase Activity
The Na+K+-ATPase activity was determined by measuring the K+-
stimulated, ouabain-sensitive p-nitrophenylphosphate (PNPP) hydrolysis
at pH 7.8, based on the method of Yoshida et al. (1969). Briefly, the
assay buffer was composed of 50 mM Tris-HCl (pH 7.8), containing 5
mM MgCl2, 2.5 mM EDTA, 50 mM KCI and 5 mM PNPP. The reaction
was carried out in the presence and absence of ouabain (1 mg/ml) by


UNIVERSITY OF FLORIDA
3 1262 08554 8336


112
(Chapter 6). V2 receptors, thought to located exclusively on renal
tubular epithelial cells, are coupled to adenylate cyclase and the
formation of cyclic AMP (Morel et al., 1987). These receptors are
responsible for transducing the antidiuretic action of the hormone.
By means of cAMP-dependent protein kinases, phosphorylation of
integral proteins in the apical membrane of a discrete population of
epithelial cells leads to the formation of aqueous channels (Gluck and
Al-Awqati, 1980) and an enhanced apical permeability to tubular fluid.
Vasopressin-stimulated, cyclic AMP-dependent activation of Na-K-
ATPase, the active transporter of sodium may account for the ability
of certain epithelial
tissues to
promote
reabsorption of
this
electrolyte (Tomita, et
al., 1987).
Clearly,
modification of
these
receptor-mediated processes will have
as dramatic an impact
on
physiological function as did the alteration of the hormone itself.
Pathophysiological conditions associated with receptor defects are
most dramatically depicted in cases of hypercholesterolemia and non
insulin-dependent diabetes mellitus, where low density lipoprotein
receptor and insulin receptor numbers are diminished, thereby
reducing the sensitivity
of
the
target tissues to
the respective
hormones. By analogy,
if
the
sensitivity of renal
tubular target
tissues to vasopressin was altered, this could have profound effects
on the transepithelial transport of fluid and electrolytes, with the
potential for influencing fluid homeostasis.
In this dissertation, the effect of alterations in fluid balance on
vasopressin receptors was studied. Vasopressin receptors were
characterized on basolateral membranes prepared from rat kidney
tubular epithelia. Basolateral membranes were isolated by differential


53
rats. Scatchard analyses indicate that the difference in binding is
due to a reduction of receptor numbers on the membranes prepared
from the dehydrated animals. These results show that AVP receptor
downregulation can occur in response to physiologically elevated
levels of circulating vasopressin. Such a condition could ultimately
affect the antidiuretic action of the hormone.


88
examined in a homogeneous population of tubular epithelial cells and
the influence of mineralocorticoids on AVP binding and adenylate
cyclase activity was determined.
Binding of -^H-arginine vasopressin to LLC-PKj cells was rapid,
reversible and specific. The receptor displayed a high affinity (2-3
nM) for arginine vasopressin and the closely related moiety, lysine
vasopressin, but for no other peptide, including oxytocin. This
finding differs somewhat from that reported by Roy and Ausiello
(1981a) who found that the IC50 for AVP was more than an order of
magnitude greater than that for LVP. This may be due to differences
in the radioligands used between the two studies. Roy and Ausiello
utilized a tritiated lysine vasopressin compound with a specific
activity of 8.5 Ci/mmol, whereas the tritiated vasopressin analogue
used in the present study contained arginine in the number 8 position
of the peptide and had a specific activity eight times greater (70
Ci/mmol). Variability in the phenotypic expression of the cells from
passage
to passage
must
also be considered
when
interpreting
any
result.
Scatchard
analysis
performed on the
saturation data in
the
present
study
revealed a
single class of
binding
sites with
an
apparent
B
max
of
155 fmol/mg protein. This
finding
too differs
from
those of
Roy
and
Ausiello
(1981a). They reported
a nonlinear
plot,
indicative of heterogeneity in the population of binding sites.
Comparison of the maximal binding capacities for the high affinity
sites in each of the studies is difficult, as Roy and Ausiello (1981a)
expressed their data on the basis of cell number, rather than per mg
of protein. However, the of 155 fmols/mg protein calculated in
max


127
Muller, J., W.A. Kachadorian and V.A. DiScala. Evidence that ADH-
stimulated intramembrane particle aggregates are transferred
from cytoplasmic to luminal membranes in toad bladder epithelial
cells. J. Cell Biol. 85:83-95, 1980.
Nakahara, T., S. Terada, J. Pincus, G. Flouret and O. Hechter. Neuro
hypophyseal hormone-responsive renal adenylate cyclase. J. Biol.
Chem. 253:3211-3218, 1978.
Nakashima, A., J.P. Angus and C.I. Johnston. Chronotropic effects of
angiotensin II, bradykinin and vasopressin in guinea pig atria.
Eur. J. Pharmacol. 81:479-485, 1982.
Nicholson, H.D., R.W. Swann, G.D. Burford, D.C. Wathes, D.G. Porter
and B.T. Pickering. Identification of oxytocin and vasopressin in
the testis and in adrenal tissue. Reg. Peptides 8:141-146, 1984.
Ogawa, K., M.A. Henry, J. Tange, E.A. Woodcock and C.I. Johnston.
Atrial natriuretic peptide in dehydrated Long-Evans rats and
Brattleboro rats. Kidney Int. 31:760-765, 1987.
Oliver, G., and E.A. Schaefer. On physiological action of extracts of
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Penit, J., M. Faure and S. Jard. Vasopressin and angiotensin II
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^-Adrenergic regulation of insulin and epidermal growth factor
receptors in rat adipocytes. J. Biol. Chem. 258:7386-7394, 1983.
Pettinger, W.A., R. Fallet, Y. Wang, L.T. Tam and W.B. Jeffries.
Enhanced cAMP response to vasopressin in the CCT of DOCA-Na
hypertensive rats. Am. J. Physiol. 25LF1096-F1100, 1986.
Phillips, M.I. Functions of angiotensin in the central nervous system.
Ann. Rev. Physiol. 49:413-435, 1987.
Poste, G. New insights into receptor regulation. J. Appl. Physiol.:
Respirat. Environ. Exercise Physiol. 57:1297-1305, 1984.
Pulan, P.T., C.I. Johnston, W.P. Anderson and P.I. Korner. The role
of vasopressin in blood pressure control and in experimental
hypertension. Clin. Sci. Mol. Med. 55:251s-254s, 1978.
Rajerison, R.M., D. Butlen and S. Jard. Effects of in vivo treatment
with vasopressin and analogues on renal adenylate cyclase
responsiveness to vasopressin stimulation in. vitro.
Endocrinology 101:1-12, 1977.


CHAPTER 4
REGULATION OF VASOPRESSIN BINDING
IN MINERALOCORTICOID-DEPENDENT HYPERTENSION
InLroducti.on
The
role
of
vasopressin
in the
etiology
of hypertension
has
received
considerable
attention,
much of
it being
reviewed
recently
by
McNeill
(1983) and
Johnston
(1985).
The pressor action
of vaso-
pressin
was
the
first physiological
response
attributed
to
this
posterior
pituitary
substance
(Oliver
and Schafer, 1895)
and
the
vasoconstrictive properties of the hormone are now well-documented
both in vivo (Liard, 1984) and in vitro (Altura and Altura, 1984).
One line of evidence which points to a relationship between
vasopressin and high blood pressure is that circulating levels of
vasopressin are elevated in several models of hypertension. Crofton
et al. (1978) noted higher plasma vasopressin concentrations in the
spontaneously hypertensive rat. Similar observations were noted in
renovascular (one kidney,one clip) (Pulan et al., 1978) and volume-
expanded (DOCA-salt) (Mohring et al., 1977; Crofton et al., 1979)
models of hypertension. While the circulating levels of vasopressin
are significantly increased in all of the above studies, the plasma
concentrations are, nonetheless, below the threshold for pressor
activity. This discrepancy can be resolved, however, by invoking
increased vascular reactivity toward vasopressin. Such an enhanced
pressor sensitivity to AVP has been noted in the SHR (Crofton et al.,
1978) and in DOCA-salt hypertensive rats (Crofton et al., 1980).
54


77
compare these results with those observed in vivo that are associated
with mineralocorticoid-dependent hypertension. To this end,
vasopressin binding was characterized in LLC-PKj cells (ATCC # CL
101), a continuous cell line of renal epithelial origin, established by
Hull et al. (1976).
Materials and Methods
Chemicals
Tritiated 8-L-arginine [phenylalanine-3,4,5-^] I]-vasopressin (3H-
AVP) and tritiated 3',5'-cyclic adenosine monophosphate (^H-cAMP)
were purchased from New England Nuclear Corporation (Boston).
Desoxycorticosterone acetate (DOCA), arginine and lysine vasopressin
(AVP, LVP), oxytocin (oxy), angiotensin II (ang II), D-ala-met-
enkephalinamide (DAME), cAMP and cAMP-dependent protein kinase
were all purchased from Sigma Chemical Co. (St. Louis). Dulbeccos
Modified
Eagles Medium and
fetal
bovine serum were
obtained
from
GIBCO
(Long Island, NY).
All
other reagents were
obtained
from
Sigma, unless otherwise indicated.
Cell Culture
LLC-PKj cells (ATCC # CL 101) were routinely grown as mono-
layers in plastic 75 cm^ Corning flasks using Dulbeccos Modified
Eagles Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), streptomycin [100 /xg/ml], penicillin [100 units/ml], and
NaHCC>3 [44 mM], Cell cultures were maintained at 37C in an
atmosphere of 95% O2 and 5%CC>2. Dome formation typically appeared
after several days of growth. Dome formation in epithelial cell
cultures is acceptable presumptive evidence of the following processes
that are required for its expression: functional plasma membrane


31
TABLE 2-1.
K+-STIMULATED, OUABAIN-SENSITIVE Na+-K+ ATPase ACTIVITY
Fraction
Units*/Mg Protein (x 10
Purification
H
0.08 .04
IX
P2
0.26 + .01
3.25X
Fl
0.80 .01
10X
Potassium-stimulated, ouabain-sensitive Na+-K+-ATPase activity.
Activity determined by measuring K.+-stimulated, ouabain-sensitive p-
nitrophenylphosphate (PNPP) hydrolysis at pH 7.8. Values are
expressed as units/mg protein, where 1 unit = 1 pmole PNPP
hydrolyzed per minute. H, homogenate. P2, 224,000g pellet. Fj,
basolateral membrane fraction. *(1 unit = 10~6 moles/min.)


117
receptor subtypes in renal epithelial tissue (Star et al.t 1988;
Burnatowska-Hledin and Spielman, 1988). Even more extraordinary, a
single muscarinic receptor subtype (M2) has been shown to be coupled
to both adenylate cyclase and phosphoinositide hydrolysis (Ashkenazi
et al., 1987).
These studies were intended to further our understanding of
vasopressin receptors and receptor-mediated signal transduction
processes in renal epithelial tissue. The series of experiments
demonstrate:
1) The characterization of vasopressin binding sites in a Na+-K+-
ATPase-enriched preparation of renal tubular epithelial basolateral
membranes prepared by density-gradient centrifugation;
2) Dehydration-induced elevation of circulating levels of vasopressin
down-regulates vasopressin receptors;
3) Excess exposure to mineralocorticoids up-regulates vasopressin
receptors
and increases the
tubular
sensitivity of
vasopressin-
stimulated
adenylate cyclase.
Enhanced
sensitivity to
the fluid-
retaining properties of the hormone provides a cellular and systemic
basis for the role of vasopressin in the pathogenesis of DOCA-salt
hypertenion;
4)Vasopressin receptors are coupled to the formation of cAMP,
and also to phosphatidyl inositol turnover in LLC-PKj cells. The two
transduction processes are modulated differentially by the osmotic
strength of the incubation medium. The coupling of vasopressin
receptors to multiple effector systems redefines our concept of signal
transduction in renal tubular epithelia.


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
CHARACTERIZATION AND REGULATION
OF RENAL VASOPRESSIN RECEPTORS
By
Martin Steiner
April, 1988
Chairman: M. Ian Phillips
Major Department: Physiology
Vasopressin receptors were characterized on basolateral
membranes of renal tubular epithelia and their capacity for regulation
was studied during alterations in fluid balance. Membranes were
obtained from rat kidney and prepared by differential and density
gradient centrifugation techniques. Radioreceptor methodologies were
used to demonstrate that binding was rapid, reversible, specific and
saturable. The binding site displayed a pharmacological profile for
the V2 receptor subtype, based on competition studies with selective
V] and V2 antagonists.
Vasopressin receptors were impacted by the physiological state
of the animal. Water-deprived animals displayed significantly elevated
plasma vasopressin levels. As a consequence vasopressin binding sites
were downregulated. Conversely, the administration of the
viii


78
polarization, formation of occluding
junctions
and vectorial
trans-
epithelial active ion
transport (Lever,
1985).
At confluence,
cells
were subcultured as
follows: the medium was
removed and the
cells
washed with once
with trypsin-EDTA
solution
(0.05% trypsin,
0.2%
EDTA) for 10-15 minutes. The cells were centrifuged (500 x g, 5
min), resuspended in DMEM + 10% FBS and plated at a density of 2-
2.5 x 104 cells/cm^ in flasks as above for continued passage or in 35
mm plastic Falcon petri dishes and utilized upon achieving confluence.
Vasopressin Receptor Binding Assay
The vasopressin binding assay was carried out directly in the
dish, on the monolayer. Twenty four hours prior to the experiment,
the growth medium was removed and replaced with serum-free DMEM.
At the time of the experiment, the medium was aspirated from the
plates and the monolayer washed once with phosphate-buffered saline
(PBS). PBS was composed of NaCl (8g/L), KC1 (0.2g/L), Na2HP04
(1.15g/L) and KH2PO4 (0.2g/L). Binding was initiated by the addition
of 0.5 ml of the reaction buffer (PBS containing 0.8% bovine serum
albumin, 1 /xg/ml aprotinin, 1 /ig/ml pepstatin A, 1 /xg/ml leupeptin,
pH 7.2) containing ^H-AVP. Non-specific binding was determined in
the presence of a 1000-fold excess of non-radioactive A VP. Studies
were conducted in triplicate using ^H-AVP at a concentration of 2.5
nM, for 30 minutes at 20C, unless indicated otherwise and repeated
at least once. For dissociation studies, the reaction medium was
removed after 60 minutes and replaced with buffer containing 2.5 /M
AVP. Saturation studies were performed with concentrations of ^H-
AVP ranging from 0.5-20 nM. Binding was terminated by rapidly
aspirating the reaction mixture from the cells and washing twice with


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24
regulate the receptor of a second hormone. For example, a steroid
hormone may affect the expression of a peptide hormone receptor
through transcriptional events. In any case, regulation of receptors
can affect the cellular sensitivity to hormonal stimulation. For the
antidiuretic hormone, this is of tanamount importance in its ability to
maintain fluid homeostasis.
The studies delineated in this dissertation were conducted to
(a) characterize antidiuretic hormone receptors in the kidney, (b)
study their physiological regulation in vivo, by altering the state of
fluid balance, ranging from a condition of dehydration to a volume-
expanded model of hypertension, and to (c) establish a "parallel"
system n vitro. to examine receptor regulation and signal
transduction mechanisms at the cellular level. Hopefully, this
information will expand the current state of thinking about the
mechanisms of action of arginine vasopressin and its role in fluid and
electrolyte homeostasis and circulatory control.


108
The physiological significance of vasopressin-induced PI
hydrolysis in LLC-PKj cells and its regulation by osmolality is
uncertain at the present time. These epithelial cells display transport
characteristics not only of the distal nephron (H2O reabsorption), but
also of the proximal tubule (glucose, amino acid transport) and the
medullary thick ascending limb (sodium transport) (Horster and Stopp,
1986). In this regard, it should be noted that an increase in
peritubular osmolality has been shown to modulate the effect of
vasopressin on NaCl transport and cAMP formation in epithelial cells
of the medullary thick ascending limb of Henle (Hebert et al., 1981b;
Torikai and Imai, 1984). While caution should be exercised when
making comparisons between in vitro and in vivo situations, it is
tempting to speculate that vasopressin-stimulated PI hydrolysis may
be involved in NaCl transport or cell volume regulation of renal
epithelial cells and these hormonal effects may be modulated by
peritubular osmolality.
In summary, this study represents the first demonstration that
vasopressin stimulates phosphatidyl inositol hydrolysis in a time- and
dose-dependent manner. Furthermore, the response appears to be
hormone-specific, in
that
none of
the
other agents tested
caused a
release
of inositol
phosphates.
The
vasopressin-mediated
response
could
be modulated
by
altering
the
osmotic conditions
of the
incubation medium; a situation expected to be encountered in the
corticomedullary gradient of peritubular osmolality in the kidney.
Because the vasopressin-stimulated adenylate cyclase system is well-
documented in this cell line, the LLC-PK] system represents an


50
two groups (Control Kjj = 0.61+.04 nM vs. Dehydrated Kjj = 0.64+.03
nM).
Discussion
Alterations in fluid balance can affect not only the hormones
themselves which participate in its regulation but also the receptors
which mediate the biological responses of the hormones. Thus,
dehydration causes a reduction in /c-opiate receptor binding in the
neurohypophysis (Brady and Herkenham, 1987) and a decrease in
angiotensin II binding in the adrenal medulla (Hwang et al., 1986).
Alternatively, receptors for atrial natriuretic factor are increased in
the kidney and adrenal gland, in response to reduced circulating
levels of the hormone (Lynch et al., 1986).
These data in the present study show that V2-type vasopressin
receptors located on renal tubular epithelial basolateral membranes
are modulated when fluid homeostasis is compromised by dehydration.
A significant reduction in the number of vasopressin receptors (£*max)
was noted in the renal tubular basolateral membranes prepared from
the water-deprived animals. There was no difference between the
two groups with respect to the affinity (K q) of the receptor for the
hormone. Water deprivation for 72 hours significantly elevated
plasma AVP concentrations 11.5-fold. The magnitude of this increase
is comparable to the response observed by Woods and Johnston (1983)
in rats dehydrated for a similar period of time. Thus, it would
appear that the receptors are sensitive to the circulating levels of
vasopressin. This is supported by the study of Shewey and Dorsa
(1986) who reported finding no difference in the level of ^H-AVP
binding in renal medullary membranes prepared from heterozygous


32
ug Protein
Figure 2-2. 3H-vasopressin binding as a function of
membrane protein concentration. Receptor binding protocol
in "Methods" section. Protein concentration determined
method of Lowry et al. (1951).
basolateral
described
by the


18
isolated renal tubules (Grantham and Burg, 1966). It may be
concluded that these vasopressin binding sites constitute the
receptors which mediate the antidiuretic action of the hormone in the
distal tubular collecting segments of the nephron. Since then,
vasopressin binding sites have been characterized on a variety of
membranes prepared from porcine, bovine, rat and human kidneys
(Jard, 1983). A more convincing arguement for the epithelial
localization of vasopressin binding sites comes from the finding of
Roy and Ausiello (1981a). Using tissue culture techniques, these
investigators were the first to characterize vasopressin receptors in
LLC-PKj cells. This cell line is derived from tubular elements of the
pig kidney (Hull et al., 1976). Both intact cells and renal membranes
all appear to possess a single class of high affinity binding sites (Kj)
= 1-25 nM).
All vasopressin-sensitive renal tissue need not be of epithelial
origin, however. Vasopressin receptors have been characterized on
glomerular mesangial cells (Jard et al., 1987) and appear to mediate
the vasopressin-induced contraction of this tissue (Ausiello et al.,
1980). This action may account for the alteration in glomerular
ultrafiltration observed with vasopressin in the intact glomerulus.
Vasopressin binding sites in intact glomeruli has recently been
reported (Stoeckel et al., 1987). Thus, caution must be employed
when interpreting vasopressin binding studies conducted in the kidney
by autoradiography or when using crude membrane preparations.
Vasculature
In order to effect the cardiovascular actions of vasopressin
mediated through its vasoconstrictive properties, it follows that


104
Osmolality (mosmol/L)
Figure 6-6. The effect of osmolality on 3H-inositol phosphate release
in LLC-PKj cells. Osmolality was increased from normal iso-osmotic
conditions by the addition of NaCl and urea. This mimics the
corticomedullary osmotic gradient as seen in the kidney. Control
represents Krebs-Ringers buffer. Vasopressin (AVP), at a
concentration of 1 /xM, was used to stimulate release. Results are
expressed as in Figure 6-2. Details of the procedure are found in
the "Methods" section.


12
Maraka et al. (1981) have suggested that vasopressin released into
this capillary portal zone may affect the synthesis and release of
ACTH and other adenohypophyseal hormones.
Parvocellular neurons of the PVN, which are morphologically and
topographically distinct from magnocellular neurons, have recently
been shown to project to neural lobe of the pituitary (Swanson and
Kuypers, 1980). Vasopressinergic fibers from the hypothalamus are
reported to innervate the lateral septum in the forebrain (Sofroniew
and Weindl, 1978). More extensive descending projections have been
traced to the intermediolateral column of the spinal cord, the nucleus
tractus
solitarius and
the
dorsal
vagal complex in
the
brainstem
(Buijs,
1978; Swanson
and
Kuypers, 1980) and the
locus
coeruleus
(Sofroniew, 1980).
Despite
the
complex organization
of
the PVN,
Swanson et al. (1980), using retrograde labeling techniques, have
convincingly demonstrated that separate neurons project to each of
the areas described above.
Afferent Projections to the PVN
The development
of histofluorescence
methods lead to
the
demonstration that the
PVN and
the SON
contained one of
the
highest densities of
noradrenergic
terminals
in all of the
brain
(Carlsson et al., 1962). By a variety of anterograde and retrograde
labeling techniques, the origins of these synaptic inputs have been
traced to A\ and Cj cell groups of the ventral medulla, the A2 and
Ci cell groups in the medial NTS and the A$ cell group in the locus
coeruleus. The parvocellular neurons of the PVN are preferentially
innervated by noradrenergic axons from the medulla and locus
coeruleus (A2 and A$) while the A\ area of the ventrolateral medulla


63
Figure 4-2. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Details of binding protocol described in "Methods" section of
Chapter 2. Error bars represent S.E.M. of triplicate determinations.
DOCA, desoxycorticosterone acetate; Na, 1% NaCl; IK, one kidney.


10
Vasopressin is not a hormone unto itself. It is reasonable to
expect that AVP should interact with other regulatory systems to
acheive homeostasis. This is most clear in terms of the cardio
vascular effects of vasopressin. Vasopressin, the renin-angiotensin
system and the autonomic nervous system all interact, to the point of
redundancy in maintaining circulatory control. As noted previously,
vasopressin
can
inhibit the
secretion
of
renin
from
the kidney
(Vander, 1968).
Conversely,
angiotensin
has
been
shown
to stimulate
the release
of
AVP (Phillips,
1987).
Vasopressin
and
the autonomic
nervous system interact extensively within the brain to acheive
circulatory control, as is discussed in a subsequent section. In the
periphery, vasopressin potentiates the vasoconstrictor action of nor
epinephrine and sympathetic nervous stimulation (Bartlestone and
Nasmyth, 1965). Conversely, blockade of the sympathetic nervous
system greatly accentuates the pressor effect of vasopressin (Pulan
et al., 1978). A methodical study by Gavras et al. (1982) provides
experimental evidence which supports the interaction of these three
effector systems in the maintenance of blood pressure.
Other Biological Effects
While the renal and cardiovascular actions of vasopressin are
perhaps the best known, other biological effects have recently been
attributed to
vasopressin.
The
central
effects of vasopressin in
cardiovascular
regulation will
be
discussed in
a subsequent
section.
Acting as a
neuropeptide,
AVP
has
been
ascribed a
role in
thermoregulation, nociception,
memory
and
behavior.
Another
neuroendocrine
action of vasopressin
is to
cause
the release of ACTH
from the anterior pituitary, either by acting directly or by


97
radioactivity was determined in the organic layer (which contained
unhydrolyzed ^H-phosphoinositides) and in the aqueous layer (which
contained ^H-inositol phosphates). An aliquot of the organic layer
was placed in a scintillation vial and the chloroform was allowed to
evaporate in a fume hood. The radioactivity in the vial was
determined by the addition of OCS scintillation fluid and counted in a
Beckman (LS 7000) scintillation counter. The aliquots of aqueous
layers were mixed with Dowex (50% v/v, 100-200 mesh, formate form)
and poured into polypropylene columns. After the liquid was drained,
the Dowex was washed with 5 mM (unlabeled) inositol to remove free
^H-inositol. Total ^H-inositol phosphates were eluted with 0.1 M
formic acid/1.0 M ammonium formate. The radioactivity of the eluted
^H-inositol phosphates in each sample was measured by adding
Liquiscint and counted in a Beckman (LS 7000) scintillation counter.
The release of ^H-inositol phosphates (IP) were calculated as follows:
[3H]-IPs RELEASED = dom of IP in aqueous layer
(% Total Incorp.) dpm of IP in aq. layer + dpm of PI in org. layer
Statistical Analysis
The data on the release of IP with different drugs or in
different buffers were analyzed by one-way ANOVA (analysis of
variance). The individual contrasts between treatment pairs were
made by Duncans method. When only two groups were used, the
data were analyzed by the Student t-test. Differences with P < 0.05
were considered significantly different from each other.
Results
After incorporation of -^H-inositol into phosphatidyl inositol of
the membranes of LLC-PK^ cells for 3 hours, the vasopressin-


13
projects to magnocellular and parvocellular neurons of the PVN and
SON. In addition to rostral projections from the brainstem, the PVN
receives numerous inputs from diencephalic and telencephalic
structures. The efferent connections of the PVN and the complex
organization of this hypothalamic nucleus have been elegantly
reviewed by Swanson and Sawchenko (1983).
The ascending catecholaminergic pathways and descending
vasopressinergic pathways provide an anatomical basis for the
feedback loops which enable vasopressin to influence autonomic and
cardiovascular function and in turn, allow for adrenergic control of
vasopressin
release.
This
latter point
may
account
for the tonic
inhibitory
influence
that
oc-adrenergic
stimuli
have
on vasopressin
secretion (Kimura et al., 1981).
Role of AVP in Central Cardiovascular Regulation
The diversity of afferent and efferent projections of the PVN
has lead Swanson and Sawchenko (1980) to view this vasopressin-
synthesizing site as a visceral effector nucleus, capable of influencing
the secretion of hormones from the anterior and posterior lobes of
the pituitary and neural activity in the sympathetic and
parasympathetic divisions of the autonomic nervous system. The
vasopressinergic innervation of certain brainstem and forebrain nuclei
tends to suggest that AVP plays an important role in the central
control of circulation, exclusive of its well known vasoconstrictive
action in the periphery.
One potential role of AVP in the central control of
cardiovascular regulation involves its ability to modulate neuronal
activity of the NTS, which constitutes the anatomical substrate of the


34
Specificity of the receptor for related A VP analogues and non-
related peptides is illustrated in Figure 2-4. The data show that
lysine vasopressin (LVP), a vasopressin peptide differing from AVP by
only one amino acid in the number 8 position, is some 13 times less
effective in displacing the ligand than AVP (IC50: AVP = 0.74 nM vs.
LVP = 9.7 nM). Furthermore, the synthetic vasopressin analogue
d(CH2)5D-Ile2-Ile4-AVP, a specific tubular (V2) antagonist is some
3,700 times more effective in displacing the ligand than is the
vascular (Vj) antagonist d(Et)2Tyr(Et)D-AVP (IC50: V2 = 0.0039 nM
vs. V\ = 14.7 nM). At concentrations of 1 /xM, oxytocin, angiotensin
II and D-ala-met-enkephalinamide were incapable of displacing even
50% of the ligand. A representative saturation isotherm for ^H-AVP
binding to membranes is depicted in Figure 2-5. Transformation of
the data for Scatchard analysis is also represented in Figure 2-5
(insert). Analysis of the data indicates that the maximal binding
capacity (^max) for vasopressin receptors on the membranes is ~220
f mol/mg protein and the affinity constant for the receptors is 0.61
nM.
Discussion
Several studies have demonstrated the presence of vasopressin
receptors in the kidney (Campbell et al., 1972; Bockaert et al., 1973;
Dorsa et al., 1983; Stoeckel et al., 1987). The present study reports
the preparation of Na+K+-ATPase-enriched renal tubular epithelial
basolateral membranes and the characterization of ^H-AVP binding in
these membranes. This preparation allows one to separate, by
differential and density gradient centrifugation, tubular epithelial
membranes possessing vasopressin receptors linked to adenylate


51
Brattleboro
rats and
their
chronically dehydrated
homozygous
counterparts,
which are
unable
to synthesize AVP.
This would
suggest that
the reduction
in vasopressin receptor density
observed in
the present study is attributable to the elevated plasma levels of
vasopressin, rather than some nonspecific effect of dehydration.
Homologous downregulation of vasopressin receptors has been
reported in a renal epithelial cell line (Lester et al., 1985) and in the
toad urinary bladder (Eggena and Ma, 1986), following treatment with
exogenous vasopressin. The phenomenon of downregulation results
from an increased rate of internalization when the hormone
concentration has been greatly enhanced (Catt et al., 1979). The
"anatomical correlate" of downregulation has been described by Kirk
(1987). He has elegantly shown by morphometric analysis that
retrieval or internalization of the basolateral membrane occurs in the
cells of the rabbit cortical collecting tubule when induced by a
vasopressin-stimulated transcellular water flow. He suggests that
membrane internalization could reduce the sensitivity of the cortical
collecting tubule cells to vasopressin if AVP receptors are present on
the retrieved membrane. While not addressed in the present study,
the issue of adenylate cyclase responsiveness has been examined in
conjunction with the downregulation of AVP receptors. Rajerison et
al. (1977) showed in the rat that vasopressin-sensitive adenylate
cyclase activity in membrane preparations from the rat kidney medulla
is greatly reduced after the infusion of pharmacological doses of AVP
to the animals.
In the present study, it would seem odd that an animal in dire
need of water conservation would lose AVP receptors as the need


131
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95
Dowex was obtained from Biorad Laboratories (New York). Liquiscint
liquid scintillation fluid was purchased from National Diagnostics (New
Jersey). Dulbeccos Modified Eagles Medium and fetal bovine serum
were obtained from GIBCO (New York). Other reagents were
obtained from Fisher Scientific (Orlando) unless indicated otherwise.
Culturing and Preparation of LLC-PK| Cells
LLC-PK ] cells (ATCC # CL 101) were routinely grown in
Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10%
fetal bovine serum. Cells were plated at an initial density of 2-2.5 x
10^ cells/cm^ in plastic 75 cm^ flasks and on 35 mm plates. The
cultures were maintained at 37C in a humidified atmosphere of 95%
O2 and
5% C02.
Cells in
the
35 mm plates were
utilized upon
achieving
confluence
whereas
the
monolayers in the
flasks were
subcultured according to the procedure described in Chapter Five.
The cells to be utilized for the experiments were washed twice with
buffer I, a regular Krebs-Ringer Bicarbonate (KRB) buffer, which
contained 118 mM NaCl, 4.7 mM KC1, 0.75 mM CaCl2, 1.18 mM
KH2PO4, 24.8 mM NaHCC>3 and 10 mM glucose and had an osmolality
of 300 mOsm/kg H2O. The cells from each plate were scraped with a
rubber policeman, transferred to a tube containing KRB buffer and
centrifuged at 500 x g for 5 minutes. The cells were then
resuspended in the appropriate buffer. In some experiments, buffers
of different osmolality than that of buffer I were used. Buffer II
(600 mOsm/kg H2O) was prepared by the addition of urea (300
mOsm/kg H2O) to buffer I (which had NaCl as its major component).
Buffers III (900 mOsm/kg H20) and IV (1200 mOsm/kg H20) were
prepared by the addition of equiosmolar amounts of NaCl and urea to


84
Figure 5-3. Saturation isotherm and Scatchard plot (insert) of 3H-
vasopressin (3H-AVP) binding in LLC-PKj cells. Concentration of
3H-AVP ranged from 500 pM to 20,000 pM. Specific binding was
determined using a thousand-fold excess of unlabeled AVP. Details of
the binding procedure are described in the "Methods" section.


28
Kidney tissue homogenized in 2 mM Tris-HEPES, 250 mM Sucrose buffer,
pH 7.4 containing 0.1 mM PMSF.
Homogenate- IK x g, 10
t
S, 22K x g, 15
1
P2 (upper two layers of "triple pellet)
l
10% Percoll Gradient- 40K x g, ;
F] = Basolateral Membrane (1.037g/ml)
Fj = Brush Border Membrane (1.041g/ml)
F3 = Mitochondrial Membrane (1.067g/ml)
Figure 2-1. Scheme for preparation of basolateral membranes from
renal tubular epithelia. Kidneys from male Sprague Dawley rats
homogenized in Tris-Sucrose buffer containing the protease inhibitor,
PMSF. Membranes prepared by density gradient and differential
centrifugation, as described in "Methods" section.


69
TABLE 4-2.
VASOPRESSIN STIMULATED cAMP FORMATION IN DOCA SALT
HYPERTENSIVE RATS
Group
Basal
pmoles/10 min./mg P
Maximal Stimulation
pmoles/10 min./mg P
IC50
I
72 + 14
410 20
2xlO~10M
II
61 + 12
355 10
8xlO-10M
hi
64 + 07
360 + 10
7xlO'10M
IV
65 + 10
400 12
2.5xlO_10M
V
58 15
345 + 15
8.5xl0"M


36
[3H-AVP] Figure 2-5. Saturation isotherm and Scatchard plot (insert)) of 3H-
vasopressin (3H-AVP) binding in renal tubular basolateral membranes.
Concentration of 3H-AVP ranged from 50 pM to 3,000 pM. Specific
binding determined in the presence of 3 /M unlabeled AVP. Details
described in "Methods" section.


TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
KEY TO ABBREVIATIONS v i
ABSTRACT viii
CHAPTER 1 BACKGROUND 1
Synthesis, Transport and Release of
Vasopressin 1
Control of Vasopressin Secretion 2
Biological Actions of AVP in the
Periphery 4
Vasopressin in the Central Nervous
System 11
Vasopressin Receptors 15
Significance 23
CHAPTER 2 CHARACTERIZATION OF 3H-VASOPRESSIN
BINDING SITES IN RENAL TUBULAR
BASOLATERAL MEMBRANES 25
Introduction 25
Materials and Methods 26
Results 30
Discussion 34
CHAPTER 3 DEHYDRATION-INDUCED DOWNREGULATION OF
RENAL TUBULAR VASOPRESSIN
RECEPTORS 39
Introduction 39
Materials and Methods 41
Results 44
Discussion 50
CHAPTER 4 REGULATION OF VASOPRESSIN BINDING IN
MINERALOCORTICOID-DEPENDENT
HYPERTENSION 54
Introduction 54
Materials and Methods 57
Results 60
Discussion 71
iv


16
Vasopressin Receptors
Current
dogma
holds
that all peptide or protein
hormones
initiate their
actions
by
binding to membrane-bound receptors
located
on the cell
surface
of
the
target tissues. The interaction
of the
hormone with its receptor causes the activation of one of a number
of second messenger systems (cAMP, cGMP, PI). Depending on the
tissue and receptor involved, this can result in the activation of
enzymes, mobilization of intracellular stores of calcium (which, in
turn can affect enzyme activity), phosphorylation of membrane
proteins or the activation of ion channels, leading to the appropriate
biological response.
AVP interacts with at least two subclasses of vasopressin
receptors, depending on the tissue in question. Michell et al. (1979)
formally designated these different subtypes as Vj and V2 vasopressin
receptors on the basis of the effector systems to which these
receptors are coupled. Elucidation of the subtypes has been greatly
facilitated by the synthesis of structurally distinct vasopressin
analogues which bind differentially to the V\ or V2 receptor.
Development of these analogues has resulted in the design of specific
agonists and antagonists for each of the receptor subtypes (Sawyer et
al., 1981).
V1 receptors are coupled to phospholipase C and the hydrolysis
of phosphatidyl inositol (PI) with the subsequent formation of inositol
phosphates (IP) and diacylglycerol (DG). Inositol phosphates have
been shown to increase the mobilization of intracellular stores of
calcium. Diacylglycerol on the other hand, activates the enzyme
protein kinase C. V2 receptors are coupled to adenylate cyclase and


57
vasopressin-stimulated adenylate cyclase activity will be measured in
basolateral membranes prepared from renal tubular epithelia in normo-
tensive and DOCA-salt hypertensive rats.
Materials and Methods
Chemicals
^H-argininie-vasopressin (^H-AVP), ^I-arginine-vasopressin
(^I-AVP) and 3n-3',5'-cyclic adenosine monophosphate (^H-cAMP)
were purchased from New England Nuclear Corporation (Boston).
Peptides, cAMP, protein kinase, desoxycorticosterone acetate (DOCA),
protein kinase, and the regenerating reagents for adenylate cyclase
stimulation (theophylline, creatine phosphokinase, phosphocreatinine,
ATP and GTP) were all obtained from Sigma Chemical Company (St.
Louis). Vasopresssin antibody was purchased from Amersham
(Arlington Heights, IL) and Liquiscint liquid scintillation fluid from
National Diagnostics (New Jersey).
Animals
Thirty male Sprague Dawley rats weighing 175-225 g were
obtained from the Animal Resource facility at the University of
Florida. Rats were housed in a climate-controlled room and caged
individually in 20x18x24 cm wire mesh cages. They were provided a
standard Purina rat chow diet and allowed free access to tap water.
The rats were divided into five groups containing six rats per group.
Under chloral hydrate anesthesia (0.4 g/kg body weight, i.p.), animals
in Group I were implanted subcutaneously with two silastic capsules,
5 cm long, containing DOCA. They were unilaterally nephrectomized
using a retroperitoneal approach and provided with a 1% NaCl
drinking solution in lieu of water. Group II animals received 1% NaCl


98
stimulated release of inositol phosphates was determined. The data in
Figure 6-2 indicate that vasopressin-stimulated IP release increased
with time at least up to 120 minutes. Sixty minutes was chosen as
the incubation period for measurement of IP release from radiolabeled
PI in LLC-PKj cells.
Stimulation of PI hydrolysis in LLC-PKj cells by vasopressin
proceeded in a dose-dependent manner, as seen in Figure 6-3. At at
-8
concentration of 10 M, there was a > 100% increase in PI
hydrolysis in these cells. The effect was dose-dependent up to 10 ^
M vasopressin in the incubation medium.
The specificity of the agonist-stimulated PI hydrolysis is
illustrated in Figure 6-4. Both arginine vasopressin and lysine
_7
vasopressin in equimolar concentrations (10 M) significantly
stimulated PI hydrolysis when compared to the incubation buffer
(p<.05). Carbachol, a cholinergic agonist that has been reported to
stimulate PI hydrolysis in kidney slices (Garg et al., 1986), exerted no
significant effect in LLC-PKj cells at concentrations up to 1 mM.
Angiotensin II, an octapeptide which is structurally different from
vasopressin but shares some physiological and biochemical properties
(vasoconstrictor, stimulates PI hydrolysis in vascular smooth muscle
cells), failed to stimulate PI hydrolysis in LLC-PKj cells at
concentrations up to 10 /M. An attempt was made to deduce the
nature of the vasopressin receptor mediating the PI response seen in
this cell system. As can be seen in Figure 6-5, the Vj antagonist
significantly reduced the amount of vasopressin-stimulated PI
hydrolysis (p<.05). The V2 receptor blocker also significantly lowered
the amount of vasopressin-stimulated release of PI (p<.05). Both the


mineralocorticoid desoxycorticosterone acetate (DOCA) to rats
resulted in the increased expression of tubular vasopressin receptors
and the development of DOCA-salt hypertension. This was associated
with an increase in adenylate cyclase activity in the basolateral
membrane preparations. These data provide a cellular basis for the
increased tubular sensitivity to vasopressin and its fluid-retaining and
sodium-reabsorbing properties that are characteristic of this volume-
dependent model of hypertension. The heightened expression of
vasopressin receptors appears to result from an increased
transcriptional rate of protein synthesis induced by the
mineralocorticoid DOCA.
Desoxycorticosterone acetate (DOCA) was ineffective in
regulating vasopressin receptors or adenylate cyclase activity in LLC-
PKj cells, a cell line of renal epithelial origin. This coincides with a
lack of mineralocorticoid receptors in this particular cell strain.
Signal transduction mechanisms were also evaluated and vasopressin
stimulated cyclic AMP formation and phosphatidyl inositol hydrolysis,
suggesting that these cells possess both Vj and V2 receptor subtypes.
This demonstrates that one hormone can modulate cellular activity by
interacting with multiple effector systems.
These studies demonstrate that
renal
vasopressin
receptors
are
influenced by
the physiological state
of
the animal.
Similarly,
receptor-coupled
signal transduction
mechanisms are
subject
to
modification, in
vitro.
Alterations
in
vasopressin
receptors
or
receptor-mediated
processes
may provide
a cellular
basis
for
pathophysiological
conditions
associated
with
fluid and electrolyte
balance.
IX


6
dependent mechanism, can exert its well known antidiuretic effect, by
altering the permeability of the tubular membrane to water (Grantham
and Burg, 1966). The cellular events beyond cAMP formation include
stimulation of cAMP-dependent protein kinase in the cytosol and
protein kinase A-dependent phosphorylation of the apical membrane,
resulting in an increased permeability of the apical membrane to
water (Figure 1-1). At present, the precise mechanism by which
protein kinase activation, calcium activation and microtubule-
microfilament assembly result in an alteration in membrane
permeability remain unclear, despite the well-established antidiuretic
effect of the hormone. The most favored hypothesis to emerge,
however, is that vasopressin induces, by some conformational change,
the formation of aqueous channels in the apical membrane (Gluck and
Al-Awqati, 1980). This is supported by the appearance of ADH-
induced intramembraneous particle aggregates in the luminal
membrane. The number of aggregates and the cumulative area of
membrane occupied by the particles are directly related to the
magnitude of ADH-induced changes in water permeability (Muller et
al., 1980).
Cardiovascular Effects
Although an elevation of blood pressure was the first
physiological effect noted following the administration of vasopressin,
the cardiovascular actions of vasopressin have generally been
regarded as physiologically unimportant. This is somewhat surprising
as
vasopressin
has been
shown
to be
one of
the
most potent
vasoconstrictive
agents
(Altura and
Altura,
1984).
In
addition, even
the
antidiuretic
action
of
the hormone
contributes
to
the regulation


22
TABLE 1-1.
BIOLOGICAL ACTIONS OF ARGININE8-VASOPRESSIN.
Tissue
Action
Receptor Subtype
Reference
Brain
Neuromodulator:
cardiovascular
control, nociception,
thermoregulation
Vl
DeWied, 1983
Anterior
pituitary
ACTH release
Vlb
Antoni, 1984
Platelets
Aggregation
7
Berrettini et
al 1982
Mononuclear
phagocytes
?
?
Block et al.,
1981
Blood vessels
Vasoconstriction
Vl
Schiffrin &
Genest, 1983
Liver
Glycogenolysis
Vl
Michell et al.,
1979
Adrenal
glomerulosa
cells
Steroidogenesis
Vl
Balia et al.,
1985
Testes
Inhibits androgen
biosynthesis
Vl
Meidan &
Hsueh, 1985
Juxtaglo
merular cells
Inhibits renin
secretion
?
Vander, 1968
Glomerular
mesangial cells
Contraction
Vl
Jard et al.,
1987
Renal tubular
epithelium
Solute, ion
transport
v2
Morel et al.,
1987


79
ice-cold PBS.
The cells were
dissolved
by
the addition
of 0.5 ml
0.1N NaOH,
scraped free from
the plate
with a
rubber
policeman,
transferred to
a vial containing
10 ml
of
liquid
scintillation fluid
(Liquiscint), and counted for 5
minutes in
an
LKB
liquid
scintillation
counter.
Stimulation of Adenylate Cvclase
Twenty four hours prior to the experiment, the growth medium
was removed and replaced with serum-free medium. At the time of
the experiment, the medium was aspirated and the cells washed once
with PBS. The "control" buffer consisted of serum-free DMEM
containing 11 /g/ml isobutyl-methyl xanthine (IBMX), a phospho-
diesterase
inhibitor and 0.1
mg/ml
aprotinin, a
serine
protease
inhibitor.
The "stimulation"
buffer
consisted of
the
components
described
above, plus AVP in
concentrations ranging
from
1 nM to
1 /xM. One ml of the control or stimulation buffer was added in
triplicate to the plates and incubated at 37C for 20 minutes. The
reaction was terminated by rapidly aspirating the reaction mixtures,
washing the monolayer once with PBS and adding 1 ml of boiling IN
HC1. The cells were scraped with a rubber policeman and the
contents transferred into a 4 ml polypropylene tube with a Pasteur
pipette. Samples were centrifuged at 3000 rpm for 5 minutes. The
supernatants were removed and blown to dryness under a stream of
air, before being stored at -20C, for subsequent determination of
cAMP formation. The pellets were saved for protein determination,
according to the method of Lowry et al. (1951).


68
O 11 10 9 8 7 6 5
[AVPJ (-log M)
Figure 4-5. Basal and vasopressin-stimulated cyclic AMP formation.
Cyclic AMP production by basolateral membranes prepared from
DOCA-salt hypertensive rats and normotensive sham-operated control
rats. Membrane preparation described in "Methods" section of
Chapter 2. Procedures for the stimulation and determination of
cyclic AMP described in "Methods" section. Abbreviations are as in
Figure 4-2 except AVP, arginine vasopressin; cAMP, adenosine 3'5'-
cyclic monophosphate.


CHAPTER 2
CHARACTERIZATION OF 3H-VASOPRESSIN BINDING
SITES IN RENAL TUBULAR BASOLATERAL MEMBRANES
Introduction
Arginine^-vasopressin in most species is the mammalian anti
diuretic hormone. It acts upon distal tubular segments of the
nephron to promote fluid reabsorption by altering the permeability of
the tubular luminal membrane to water. Vasopressin-stimulated
activation of the enzyme adenylate cyclase initiates the biochemical
cascade responsible for generating transepithelial net fluxes of ions
and solutes.
The presence of vasopressin binding sites in the kidney was
described as early as 1960 (Darmady et al.) and autoradiographic
reports of vasopressin receptors continue up to the present day
(Stoeckel et al., 1987). Early studies which utilized traditional
biochemical techniques to characterize vasopressin receptors in plasma
membranes prepared from renal medullary tissue did so in somewhat
crude preparations which may have been contaminated with vascular
and non-epithelial elements (Campbell et al., 1972; Bockaert et al.,
1973). These types of studies, therefore, fail to distinguish between
vasopressin-sensitive cells of tubular epithelial origin from cells which
respond to AVP located in the glomerulus and the renal vasculature.
Nor do these studies allow one to localize the binding specifically to
the basolateral as opposed to the apical region of hormone-sensitive
epithelia. Although it is well-established that the vasopressin-
25


55
Added support for the involvement of vasopressin in the pathogenesis
of DOCA-salt hypertension comes from the study of Friedman et al.
(1960). These investigators noted that the administration of AVP
accelerated the development of DOCA-salt hypertension. The most
telling piece of evidence however, which implicated vasopressin in
DOCA-salt hypertension was the finding of Crofton et al. (1979) who
reported that Brattleboro rats, which are unable to synthesize AVP,
failed to develop DOCA-salt hypertension.
The development of selective antagonists to the pressor and
antidiuretic effects of vasopressin greatly facilitated our
understanding of the involvement of vasopressin in hypertension.
Crofton et al. (1979) found that the administration of a selective V j
(vascular) antagonist caused an acute fall in blood pressure in DOCA-
salt hypertensive rats. Similarly, Mohring et al. (1977) were able to
lower the blood pressure by the injection of a highly specific
vasopressin antibody. Hofbauer et al. (1984) attenuated the
development of DOCA-salt hypertension by the simultaneous infusion
of Vj antagonists. Collectively, these studies suggest that
vasopressin may contribute to the development of hypertension by
virtue of its vasoconstrictive properties. Conversely, other
investigators
(Rascher et
al., 1983;
Yamamoto et
al.,
1984; Filep et
al., 1987)
have been
unable to
reduce the
high
blood pressure
associated with DOCA-salt hypertension by the administration of V |
blockers.
A renal locus in the pathogenesis of DOCA-salt hypertension has
been largely overlooked until recently. Saito and Yajima (1982)
observed that when Brattleboro rats were treated with DDAVP, a


59
Vasopressin Radioimmunoassay
Plasma vasopressin concentrations were determined according to
the method described in Chapter 3.
Adenylate Cvclase Stimulation
Basal and vasopressin-stimulated adenylate cyclase activity in
renal basolateral membranes was performed according to the following
protocol: Basal samples (membranes corresponding to 100 /xg protein)
were suspended in a total volume of 150 /xl of 50 mM Tris buffer, pH
7.4, containing 138 /xg ATP, 39 /xg GTP, 150 /xg MgCl2, 38 /xg EGTA,
150 /xg BSA, and a regenerating system consisting of 228 /xg theo
phylline, 60 /xg creatine phosphokinase and 100 /xg phosphocreatine.
Agonist-stimulated samples were prepared as above but were incubated
in the presence of A VP ranging in concentration from 0.1 nM to
10 /xM. Triplicate samples were incubated at 37C for 10 minutes.
The reaction was stopped by the addition of 300 /xl of 100 mM Tris
buffer containing 5 mM EDTA, pH 7. The samples were boiled for 5
minutes and then centrifuged at 3,000 rpm for 5 minutes. Super
natants were removed at stored at -20C for subsequent determination
of cAMP formation.
cAMP Determination
The cellular cAMP content was determined by the competitive
protein binding assay as modified by Baker et al. (1986). Samples for
cAMP determination were reconstituted in 200 /xl of 25 mM Tris
buffer, pH 7.0. 10 /xl of each sample were incubated at 4C for 60
minutes in a total volume of 200 /xl Tris buffer containing 25,000 cpm
of -^H-cAMP, 42 /xg BSA, 280 /xg theophylline and the reaction
initiated by the addition of 24 /xg of cAMP-dependent protein kinase.


45
Figure 3-1. Urine output and urine osmolality in control and
dehydrated rats. A: urine output (Uv). B: urine osmolality (Uosm).
Osmolality determined by vapor pressure osmometry. Error bars
represent S.E.M. *p<.05 vs. control.


41
rats injected with pharmacological doses of vasopressin (Rajerison et
al., 1977). A VP-induced downregulation of vasopressin receptors and
a concomitant desensitization of vasopressin-sensitive adenylate
cyclase activity has been demonstrated in a renal epithelial cell line
(Lester et al., 1985).
Thus, vasopressin can affect the cellular sensitivity of its target
tissue by regulating its own receptor. Because dehydration is a
condition characterized by high circulating levels of AVP, the present
study was conducted in order to determine whether vasopressin
receptors are commensurately regulated under conditions of water
deprivation.
Materials and Methods
Animals
Male Sprague-Dawley rats (n=6 per group) weighing 200-250g
were obtained from the Animal Resource Facility at the University of
Florida.
Rats were
housed individually in 20x18x24
cm
wire mesh
cages, exposed to a
12:12 hour light:dark cycle,
provided
a standard
Purina rat
chow diet
and allowed free access
to tap
water. The
dehydrated
experimental
group was deprived of
water
for
72 hours
prior to sacrifice.
Chemicals
Tritiated 8-L-arginine [phenylalanine-3,4,5-^H]-AVP ([^H]-AVP)
used in
receptor binding studies
was purchased
from
New
England
Nuclear
Corp. (Boston) with a
specific
activity
of
70
Ci/mmol.
lodinated
125
vasopressin ( I-AVP),
specific
activity
of
2200
Ci/mmol,
was also
obtained from New
England
Nuclear
for
use
in the
vasopressin
radioimmunoassay (RIA). AVP antibody
was
purchased


82
the highest concentration (10 ^M) in displacing ^H-AVP (Figure 5-2).
Using increasing concentrations of ^H-AVP, binding sites on the
surface of the cells appear to be saturable (Figure 5-3). Scatchard
analysis of the saturation isotherm data reveals a single class of high
affinity binding sites (K-q = 2.98 nM). This is in good agreement
with the value of 2.1 mM observed for the IC50 for vasopressin. The
maximal binding capacity (Bmax) was calculated to be 155 fmoles/mg
protein (Figure 5-3, insert). Vasopressin stimulated cAMP
accumulation in a dose-dependent manner with LLC-PK-i cells (Figure
5-4) which was abolished by pre-treatment with V2 antagonists.
Treatment of the cell cultures with DOCA, either over varying
periods of time or with varying concentrations, failed to alter the
binding of ^H-AVP (Figure 5-5). While binding was significantly
reduced at 1 pg/ml DOCA, this effect can be attributed to the "toxic
effect of the mineralocorticoid, as cell viability was adversely
affected. Basal and vasopressin-stimulated cyclic AMP formation in
LLC-PK1 cells were not significantly affected by DOCA treatment
(Figure 5-6).
Discussion
The structural and functional heterogeneity of the kidney is not
particularly amenable to the study of hormonal action at the cellular
level. One attempt to resolve this dilemma has been the use of
tissue cultures of specific cellular elements in the kidney. These
techniques have facilitated our understanding of vasopressin action at
targets as diverse as glomerular mesangial cells (Bonventre et al.,
1986) and tubular epithelial cells (Tang and Weinberg, 1986). In the
present study, the interaction of vasopressin with its receptor was


CHAPTER 3
DEHYDRATION-INDUCED DOWNREGULATION OF RENAL
TUBULAR VASOPRESSIN RECEPTORS
Introduction
Several endocrine systems act to maintain fluid and electrolyte
balance. Alterations of fluid homeostasis influence plasma levels of a
number of hormones. This is particularly evident during dehydration.
This condition leads to elevated circulating levels of vasopressin
(Aisenbrey et al.,
1981;
Woods and Johnston,
1983),
angiotensin II
(Di Nicolantonio
and
Mendelsohn, 1986)
and
endogenous opiate
peptides (Mata et
al.,
1977). Plasma levels of
atrial natriuretic
peptide on the other hand tend to be lower, which is consistent with
the physiological role of this hormone (Ogawa et al., 1987). In turn,
these hormones act upon their respective receptors in proportion to
the response required to re-establish volume homeostasis. Paramount
to this task is the action of arginine vasopressin, exerting its well-
known antidiuretic and vasoconstrictive effects, through interactions
with V2 and V] receptors, respectively.
Peptide hormone receptors can be regulated by the concentration
of hormone to which they are exposed. This can often lead to a
condition described as "downregulation." This phenomenon results
from withdrawal of cell surface membrane receptors by an
internalization process. Reduced cellular sensitivity to hormonal
stimulation can also occur by an uncoupling of the existing receptors
from the signal-transducing effector system. The most well-
39


129
Schiffrin, E.L., and J. Genest. 3H-vasopressin binding to the rat
mesenteric artery. Endocrinology 113:409-411, 1983.
Schiffrin, E.L., F.S. Thome and J. Genest. Vascular angiotensin II
receptors in renal and DOCA-salt hypertensive rats.
Hypertension 5 (Suppl. V):V16-V21, 1983.
Schrier, R.W., T. Berl and R.J. Anderson. Osmotic and non-osmotic
control of vasopressin release. Am. J. Physiol. 236:F321-F332,
1979.
Schwartz, J., L.C. Keil, J. Maselli and I.A. Reid. Role of vasopressin
in blood pressure regulation during adrenal insufficiency.
Endocrinology 112:234-238, 1983.
Schwartz, J., and I.A. Reid. Role of vasopressin in blood pressure
regulation in conscious water-deprived dogs. Am. J. Physiol.
245:R74-R77, 1983.
Shewey, L.M., and D.M. Dorsa. Enhanced binding of 3H-arginine
vasopressin in the Brattleboro rat. Peptides 7:701-704, 1986.
Sklar, A.H., and R.W. Schrier. Central nervous system mediators of
vasopressin release. Physiol. Rev. 63:1243-1280, 1983.
Skorecki, K.L., J.M. Conte and D.A. Ausiello. Effects of hypertonicity
on cAMP production in cultured renal epithelial cells (LLC-PK¡).
Mineral Electrolyte Metab. 13:165-172, 1987.
Sladek, C.D. Regulation of vasopressin release by neurotransmitters,
neuropeptides and osmotic stimuli. Prog. Brain Res. 60:71-90,
1983.
Sofroniew, M.V. Projections from vasopressin, oxytocin and
neurophysin neurons to neural targets in rat and human. J.
Histochem. Cytochem. 28:475-478, 1980.
Sofroniew, M.V., and A. Weindl. Projections from parvocellular
vasopressin- and neurophysin-containing neurons of the
suprachiasmatic nucleus. Am. J. Anat. 153:391-430, 1978.
Star, R.A., H. Nonoguchi, R.B. Balaban and M.A. Knepper. Calcium
and cyclic AMP as second messengers in rat inner medullary
collecting duct. Kidney Int. 33:286, 1988 (Abstract).
Starling, E.H., and E.B. Verney. The secretion of urine as studied on
the isolated kidney. Proc. Roy. Soc. 97B:321-363, 1924.
Stassen, F.L., G. Heckman, D. Schmidt, N. Aiyar, P. Nambi and S.T.
Crooke. Identification and characterization of vascular (Vt)
vasopressin receptors of an established smooth muscle cell line.
Mol. Pharmacol. 31:259-266, 1987.


29
the addition of 10 /xl membranes (100 /xg protein) to 0.5 ml of the
assay buffer. The mixtures were incubated for 2-10 minutes at 37C.
The reaction was stopped by the addition of 3.5 ml IN NaOH and
absorbance was read at 410 nm. Net Na+K+-ATPase activity was
determined by subtracting the PNPP hydrolysis activity in the
presence of ouabain from the activity in the absence of ouabain.
Specific activity was calculated based on a molar extinction
coefficient e = 15,400 M_1cm_1, and final values were expressed as
units/mg protein (where 1 unit = 1 /xmol PNPP hydrolyzed per
minute).
Vasopressin Receptor Assay
Membranes, corresponding to 100-200 /xg protein were suspended
in 300 /xl of 100 mM Tris-HCl buffer containing 5 mM MgCl2, 1 mM
EGTA and 0.1% BSA, pH 7.3 at 20C. The incubation buffer
contained the peptidase inhibitors aprotinin (1000 K.I.U./ml), leupeptin
(1 /xg/ml) and pepstatin A (1 /xg/ml), to prevent proteolytic
degradation of the peptides or radioligand. For saturation studies,
triplicate samples were incubated for 45 minutes at 20C with
concentrations of ^H-AVP ranging from 50 pM to 5,000 pM. Non
specific binding was determined in the presence of 5 /xM unlabelled
AVP. Competitive inhibition by various peptides was assessed in the
presence of 1.0 nM ^H-AVP. The incubation was terminated by the
addition of 1 ml ice-cold Tris-buffer and the bound tracer was
rapidly separated from the unbound moiety by centrifugation at 10,000
x g for 2.5 minutes at 4C. The membrane pellet was dissolved in
100 /xl of formic acid and transferred to a mini vial containing 4 ml of
liquid scintillation fluid (Liquiscint; National Diagnostics).


42
from Amersham (Arlington Hts., IL) while AVP standard was obtained
from Peninsula Labs (Belmont, CA). Other reagents were obtained
from Sigma, unless otherwise indicated.
Membrane Preparation and Plasma Collection
Animals were sacrificed by decapitation and trunk blood was
collected. A 100 y\ sample was obtained separately for the
determination of plasma osmolality while the remainder was collected
in chilled tubes containing 0.3 M EDTA (50 y 1/ml) for the
determination of plasma AVP concentration. Blood samples were
centrifuged at 3,000 x g for 10 minutes at 4C, the plasma removed
and stored at -20C. Renal basolateral membranes were prepared by
modifying the density gradient centrifugation method of Boumendil-
Podevin and Podevin (1983), according the description provided in
Chapter 2.
Vasopressin Receptor Assay
Membranes, corresponding to 100-200 /g protein were suspended
in 300 yl of 100 mM Tris-HCl buffer containing 5 mM MgCl2, 1 mM
EGTA and 1
3.1%
BSA, pH
7.3 at
20C.
Triplicate
samples
were
incubated for
45
minutes
at 20C
with
concentrations
of 3H-
-AVP
ranging from
50
pM to
5,000
pM.
Non-specific
binding
was
determined in the presence of 5 tM unlabelled AVP. Further details
of the receptor binding protocol are described in Chapter 2.
R1A for Vasopressin
Plasma samples for the determination of AVP were thawed and
purified using SepPak Cjg cartridges (Waters). Briefly, the
purification process is as follows: Cartridges are pre-wetted with 5 ml
methanol followed by 5 ml 1% trifluroacetic acid (TFA). A 1 ml


48
Figure 3-4. Saturation isotherms of 3H-vasopressin (3H-AVP) binding.
Binding determined in basolateral membranes prepared from kidneys of
water-replete and water-deprived (72 hr) rats. Details of binding
protocol described in "Methods" section of Chapter 2. Error bars
represent S.E.M. of triplicate determinations.


113
and density gradient centrifugation techniques. Activity of Na-K-
ATPase, a marker enzyme for these membranes, was increased ten
fold. Binding of a tritiated radioligand was rapid, reversible,
saturable and specific. The binding site displayed a high affinity
(~lnM) and fit a pharmacological profile consistent for a V2-type
vasopressin receptor. Furthermore, AVP-stimulated cAMP formation
was demonstrated using this membrane preparation.
Vasopressin receptors undergo regulation, in vivo and |n vitro.
Homologous downregulation has been demonstrated previously, but
only under the influence of pharmacological doses of AVP (Rajerison
et al, 1977; Lester et al., 1985; Eggena and Ma, 1986). What effect,
if any, do physiological changes in circulating levels of vasopressin
have on renal tubular vasopressin receptors? The present study
showed that V2 vasopressin receptor number is reduced in basolateral
membranes prepared from water-deprived rats. This has important
ramifications with respect to fluid homeostasis, especially in an
animal already compromised by dehydration. One plausible
explanation in this case is that the downregulation may reflect a
diminution of a vasopressin receptor "reserve" (Ariens et al., 1960).
"Spare" receptors have been noted in other adenylate cyclase-linked
tissues as well (Flores et al., 1974). Alternatively, a fraction of the
total number of receptors present in any given tissue may represent
"silent" receptors (Maack et al., 1987). These binding sites are not
coupled to a biological effector system and therefore, elicit no
response. However, they may serve as "peripheral clearance sites",
acting as a buffer system to modulate plasma levels of the hormone.
Thus, downregulation of the inert hormone-receptor complex may


14
baroreceptor reflex. The NTS receives information from afferent
baroreceptor fibers originating in the carotid sinus and aortic arch.
The NTS influences cardiovascular function in three ways: (a) it
relays baroreceptor activity to other brainstem areas to modulate
spinal preganglionic neuronal activity; (b) it relays baroreceptor and
chemoreceptor activity to the hypothalamus, which serves to regulate
and integrate autonomic and cardiovascular responses; and (c) it
participates in the regulation of fluid and electrolyte homeostasis by
influencing the release of A VP (Reis et al., 1984). Experimental
evidence tends to support the hypothesis that bloodborne AVP
enhances the sensitivity of the baroreceptor efferent pathway.
Courtice et al. (1984) have electrophysiological data which show that
AVP enhances the baroreflex by increasing parasympathetic vagal tone
and depressing barosympathetic outflow. In addition, Brattleboro rats,
which are deficient in vasopressin, have a depressed baroreflex, which
can be corrected by intravenous infusions of vasopressin (Imai et al.,
1983). Though somewhat paradoxical, centrally administered AVP
acts in an opposite manner to peripherally administered AVP.
Intracerebroventricular administration of AVP elcits a pressor
response, tachycardia, increased sympathetic outflow and increased
levels of plasma catecholamines (Unger et al., 1984). Matsuguchi et
al. (1982) reported that the microinjection of AVP directly into the
NTS caused increases in arterial pressure and heart rate. Berecek et
al. (1984) reported similar findings following microinjections of AVP
into the locus coeruleus.
In addition to the reciprocal neuroanatomical pathways between
the PVN and NTS that are described above, functional interactions


52
became greater. An explanation for this may be that the kidney
possesses "spare" receptors (Ariens et al., 1960). Baddouri et al.
(1984) noted a downregulation of vasopressin receptors in the kidney
of the water deprived jerboa, Jaculus orientalis. Water-deprived
animals in that study displayed a five-fold elevation in plasma AVP
levels and a 36% reduction in ^H-lysine vasopressin binding to renal
medullary membranes. Despite reduced vasopressin binding, the
kidneys of some of these desert rodents still retained the capacity to
significantly concentrate urine under these conditions. The latter
finding suggests that "spare" receptors may have been involved in the
regulatory phenomenon. A receptor reserve has also been noted in
other adenylate cyclase-linked tissues as well (Flores et ah, 1974).
Alternatively, reduced binding without a decrement in biological
responsiveness may occur if those receptors which were
downregulated are "silent", or not coupled to an effector system.
Maack et ah (1987) have recently shown that the overwhelming
majority (>90%) of receptors for atrial natriuretic factor that are
present in the kidney are biologically silent. That is to say, they do
not mediate any of the known renal effects of the hormone. The
authors speculate that these silent receptors may serve as "peripheral
storage clearance binding sites, acting as a hormonal buffer system to
modulate plasma levels of atrial natriuretic factor". The physiological
significance of silent or spare receptors and their regulation remains
to be explored.
In summary, the present study shows that V2 vasopressin
receptors are differentially expressed in basolateral membrane
preparations from the kidneys of water-replete and water-deprived


72
A tubular site of action for vasopressin in the development of
DOCA-salt hypertension is suggested by the study of Saito and
Yajima (1982). Using Brattleboro rats, these investigators observed
that DOCA-salt hypertension would develop following the chronic
administration of DDAVP, a V2*specific tubular agonist. These
findings are consistent with the hypothesis of Cowley et al. (1984)
that subpressor doses of vasopressin can contribute to a rise in
arterial pressure, by virtue of
the
fluid-retaining properties of
the
hormone.
Indeed, plasma
levels
of
vasopressin tend to
be raised
in
DOCA-salt
hypertension.
If the
tubular sensitivity to
vasopressin
is
concommitantly increased,
as is
the
vascular sensitivity
(Berecek
et
al., 1982),
then these
processes
may lend themselves to
the
development of hypertension.
The
present study
shows
that V2 vasopressin
receptors
in
basolateral membranes prepared from renal tubular epithelia in DOCA-
salt treated rats are significantly elevated relative to tissue from
non-DOCA treated animals. Animals treated with DOCA-salt and
having a reduced renal mass (Group I) displayed a 56% increase in
binding with respect to tissue from untreated animals (Group V) and
a 71% increase compared to uninephrectomized animals receiving 1%
NaCl (Group II). Those DOCA-salt hypertensive rats with intact
kidneys (Group IV) displayed increases of 43% and 56%, respectively.
The present finding that mineralocorticoids modulate the expression
of vasopressin receptors in the kidney is consistent with the study of
Rajerison et al. (1974). These authors observed that adrenalectomy
resulted in a reduction of ^H-lysine vasopressin binding in a renal
medullary membrane preparation. A concomitant lowering of


9
the baroreceptor reflex. Furthermore, vasopressin actually tends to
enhance the baroreflex by increasing vagal tone and inhibiting
sympathetic outflow following i.v. administration, apparently through
an interaction with the cardiovascular centers in the brainstem
(Cowley et al., 1984a). The role of vasopressin in the central control
of the circulation is addressed later in this chapter.
Because the pressor effects of vasopressin are normally obscured
by the baroreflex, the physiological importance of AVP in
cardiovascular function has often been ignored. Nevertheless,
vasopressin plays an important role in the maintenance of blood
pressure under conditions of dehydration (Schwartz and Reid, 1983),
when circulatory volume is compromised as in the case of hemorrhage
(Zerbe et al., 1982), in a situation of adrenal insufficiency (Schwartz
et al., 1983) or when the vasoregulatory reflex mechanisms are
interrupted. In each of these cases, plasma levels of vasopressin rise
in excess of the concentration necessary for maximal antidiuresis.
That vasopressin is essential for maintaining circulatory homeostasis
in even a moderately compromised condition is evident in the findings
of Aisenbrey et al. (1981). In that study, rats were dehydrated for a
period of 72 hours and then challenged with a vascular vasopressin
blocker. Whereas the blocker had no intrinsic cardiovascular activity
nor did it affect blood pressure in normally hydrated animals, blood
pressure fell precipitously in the dehydrated rats. In a related study,
Brattleboro rats, which are unable to synthesize vasopressin, were
unable to maintain their blood pressure when subjected to dehydration
(Woods and Johnston, 1983). Collectivelly, these data lend support
for a physiological role for vasopressin in circulatory control.


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.
M. Ian Phillips, D.Sc., Chairman
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.
\
Steven R. Childers, Ph.D.
Associate Professor of
Pharmacology and Therapeutics
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.


89
the present study is in close agreement with the value of 166
fmoles/mg protein, as determined by Jans et al. (1987).
Roy
and
Ausiello (1981b)
found that cells
suspended
in
hypertonic
saline
or treated with
EDTA exhibited
a homogeneous
population
of
binding sites.
They proposed a
hypothesis
for
vasopressin-receptor interactions involving a hormone-induced
alteration in binding affinity or so-called "receptor transition". In
other words, rather than postulating two independent binding sites, as
a nonlinear Scatchard plot might suggest, they believe in an
interconversion of receptor states of different affinity. Why a
similar situation was not observed in the present study is unclear.
The binding sites characterized in the present study apparently
represent vasopressin receptors, as vasopressin was shown to
stimulate cAMP production in a dose-dependent manner. Agonist-
induced stimulation resulted in a sixteen-fold increase over basal
levels. This effect was completely abolished by pretreatment of the
cells with a V2 antagonist. The values for basal and AVP-stimulated
cAMP levels are in good agreement with those reported by Lester et
al. (1985) and Skorecki et al. (1987).
The synthetic mineralocorticoid DOC A, had no demonstrable
effect on either vasopressin binding or cAMP production in LLC-PKj
cells in this study. This contrasts with the observations noted in
vivo, as described in Chapter 4. In that study, a significant increase
was found in the vasopressin-stimulated adenylate cyclase activity and
number of vasopressin binding sites on tubular epithelial membranes
prepared from the kidneys of rats chronically treated with DOC A and
NaCl.


75
more sensitive to vasopressin, reflected by the enhanced AVP-
stimulated production of cAMP. Reif et al. (1986) have shown that
chronic DOCA treatment enhances vasopressin-stimulated sodium
reabsorption in the cortical collecting tubule. Treatment with DOCA
also increases Na-K-ATPase activity in distal segments of the nephron
(Garg et al., 1981). Thus, the increased concentration of plasma
vasopressin noted in the present study coupled with an enhanced
sensitivity to AVP in renal tubules may lead to an exaggerated
retention of salt and fluids, resulting in an expanded blood volume.
Extracellular volume expansion has been noted in at least some stage
of the development of DOCA-salt hypertension (Villamil et al., 1982;
Tajima et al., 1983).
Taken together, the present findings and the above studies
suggest that the development of DOCA-salt hypertension may be
caused, in part, by an increased tubular sensitivity to vasopressin.
Increased activity of vasopressin-mediated processes would lend itself
to a volume-expanded state, a condition necessary to prevent hyper
natremia. Accordingly, the resultant blood pressure elevation could
be viewed as a compensatory mechanism in response to an increase in
cardiac output, secondary to hypervolemia and aimed at the
restoration of fluid and electrolyte balance.


60
At the end of the incubation, 70 /I of a hydroxyapatite suspension
(50% w/v) was added to each tube, vortexed and allowed to sit for
six minutes. The contents of the tube were then filtered under
vacuum through Whatman GF/C filters, then washed with 12 ml of 10
mM Tris buffer at pH 7.0. Filters were removed and transferred to
scintillation vials containing 1 ml of 0.3N HC1. After the hydroxy
apatite had dissolved (~10 min), 9 ml of liquid scintillation fluid
(Liquiscint) was added and each vial counted. Non-specific binding
was determined in the presence of 10 cAMP. The amount of
cAMP present in each sample was calculated from a standard curve
determined with known amounts of unlabeled cAMP.
Analysis
Data from saturation studies were analyzed using iterative curve
fitting programs. Statistical significance was assessed by one-way
analysis of variance. The post-hoc analysis between treatment pairs
was performed by Duncans method. A "p" value of 0.05 or less was
considered an acceptable level of significance.
Results
The combination of desoxycorticosterone acetate treatment and
increased sodium intake precipitates the development of the well-
characterized syndrome of DOCA-salt hypertension. Figure 4-1
depicts the rapid development of high blood pressure in rats
administered the mineralocorticoid and offered 1% NaCl to drink.
Those animals in group I with a reduced renal mass became
hypertensive most rapidly and attained the highest arterial pressure
(1765 mm Hg). Even those animals with intact kidneys but provided
a similar regimen (Group IV) became hypertensive (1548 mm Hg)


67
cyclic AMP (pmoles)
Figure 4-4. Standard curve for cyclic AMP. Values for cyclic AMP
determined by competitive binding assay, using 3H-cAMP. Details of
procedure described in "Methods" section. cAMP, adenosine 3'5'-
cyclic monophosphate.


74
DOCA treatment. Garg et al. (1981) reported that Na-K-ATPase
activity was markedly elevated in the cortical collecting duct of
rabbits treated with DOCA. Similar results were observed by El
Mernissi et al. (1983) and Mujis et al. (1985). Mineralocorticoids
can affect the expression of receptors in other tissues as well.
Wilson et al. (1986) observed a greater density of angiotensin II
receptors in brain tissue. Angiotensin II receptors are also increased
in vascular smooth muscle tissue in DOCA-salt hypertensive rats
(Schiffrin et al., 1983). These findings correlate quite nicely with
the increased dipsogenic and pressor responsiveness to Ang II in
DOCA-salt treated animals. Not all receptors are modulated by
mineralcorticoids, however, Alphaj-adrenergic receptors were not
significantly altered in membrane fractions prepared from DOCA-salt
hypertensive and normotensive rats (Fukuda et al., 1983). These
authors did observe differences however, in -I I-yohimbine binding
between spontaneously hypertensive rats and the their normotensive
counterparts, Wistar-Kyoto rats.
How might an up-regulation of renal tubular vasopressin
receptors contribute to the development of DOCA-salt hypertension?
Vasopressin regulates fluid reabsorption in the distal nephron and also
stimulates sodium
reabsorption
in at
least
two distinct sites:
the
medullary thick
ascending limb
(Hebert et
al., 1981a) and in
the
cortical collecting
tubule (Reif
et al.,
1986).
A relationship between
vasopressin-stimulated cAMP production and Na-K-ATPase activity,
the enzyme involved in active transport of sodium, has been
demonstrated by Tomita et al. (1987). As shown in the present study,
the increased density of AVP receptors appears to render the tubule


107
vasopressin-stimulated cAMP formation in MDCK cells. This action
may occur by covalent modification of the vasopressin receptor or the
adenylate cyclase enzyme. This latter concept is strengthened by the
finding of Yoshimasa et al. (1987), who reported that treatment of
frog erythrocytes with phorbol esters resulted in phosphorylation of
the catalytic subunit of the adenylate cyclase enzyme coupled to the
/3-adrenergic receptor. Conversely, it is conceivable that in the LLC-
PKj cell line, a cAMP-dependent mechanism may act to potentiate PI
hydrolysis. Therefore, blockade of vasopressin-stimulated cAMP
production by V2 antagonists may lead to a diminution of
vasopressin-induced phosphatidyl inositol hydrolysis. The role of the
V2 receptor-mediated cAMP pathway in modulating vasopressin-
stimulated PI hydrolysis is unclear and remains to be clarified.
Alteration of the osmotic environment to which these cells are
exposed affected the vasopressin-induced release of inositol
phosphates. Increasing the osmolality of the incubation medium above
600 mosmol/kg H2O resulted in a significant reduction in the amount
of inositol phosphates released. It is doubtful that the hypertonicity
adversely affected the viability of the cells. On the contrary, Roy
and Ausiello (1981b) have shown that LLC-PK] cells display an
increased vasopressin receptor density and an enhanced vasopressin-
stimulated
production of
cAMP
when incubated
under
hypertonic
conditions.
The opposite
effects
of hypertonic NaCl on
vasopressin-
stimulated
PI hydrolysis
and
cAMP formation
only
serves to
underscore the apparent interaction between these two second
messenger systems in this cell system and may represent some form
of negative feedback.


94
Figure 6-1. Phosphoinositide cycle. Myo-inositol is incorporated in
phosphoinositides (PI, PIP, PIP2). Agonist-occupied receptors
stimulate phospholipase C, which acts to cleave PIP2 and IP3 and DG.
IP3 acts to mobilize intracellular stores of calcium, then IP3 is
metabolically converted to IP2 and IP under the action of the
appropriate phosphatases. DG activates a calcium-phospholipid-
dependent protein kinase C, which can phosphorylate a number of
cellular proteins. ATP, adenosine triphosphate; PI, phosphatidyl
inositol; PIP, phosphatidyl inositol-4-phosphate; PIP2, phosphatidyl
inositol-4,5-phosphate; DG, diacylglycerol; IP3, inositol 1,4,5-
phosphate; IP2, inositol 4-phosphate; IP, inositol 1-phosphate; P¡,
inorganic phosphate.


70
TABLE 4-3.
PLASMA A VP LEVELS IN DOCA-SALT
HYPERTENSIVE RATS
Group
[AVP] (pg/ml)
I
6.1 0.8
II
1.8 0.2
III
1.9 0.3
IV
8.4 4.5
V
1.2 0.2


fmoles 3H-AVP bound/mg
86
[DOCA1
Figure 5-5. Effect of DOCA on 3H-vasopressin (3H-AVP) binding in
LLC-PKj cells. Cells were treated for 1-24 hours with DOCA ranging
in concentration from 1 pg/ml to 1 pg/ml. Binding was assessed at
3.0 nM [3H-AVP], with details provided in the "Methods section.
DOCA, desoxycorticosterone acetate. *p.05 vs. control.


56
selective
tubular (V2)
agonist, DOCA-salt hypertension
developed. A
similar
phenomenon
was observed by Woods
and
Johnston (1981),
using
renovascular
models of hypertension
in
Brattleboro rats.
Whereas
Hofbauer et
al. (1984) attenuated the
development of DOCA-
salt hypertension with
V1 blockers as noted
above,
the simultaneous
infusion of a combined Vj/V2 antagonist completely abolished the rise
in blood pressure. These studies suggest a tubular site of action for
vasopressin in the development of DOCA-salt hypertension.
The role of the kidney in the pathogenesis of hypertension has
been a major focus of Guyton and his colleagues (Guyton et al.,
1972). The action of vasopressin in this context was underscored in
the findings of Manning et al. (1979) and Cowley et al. (1984b) who
reported that subpressor doses of vasopressin can contribute to a rise
in arterial pressure, which is dependent on the fluid-retaining
properties of the hormone. In addition, it has been shown that
vasopressin stimulates sodium reabsorption in the medullary thick
ascending limb of the mouse (Hebert et al., 1981a) and the cortical
collecting tubule of the rat (Reif et al., 1986). The latter authors
also showed that the effects of vasopressin on sodium reabsorption
are augmented in DOCA-treated rats.
Thus, it appears that there is an interaction between vasopressin
and mineralocorticoids at the level of the renal tubule. Increased
activity of the salt and fluid transport processes would lend itself to
a volume-expanded state, with the potential for raising arterial
pressure. The question of how vasopressin activity is increased in
DOCA-treated animals remains unanswered. This study will address
the question at the cellular level. Vasopressin receptors and


40
documented examples of these types of regulation come from the
elegant studies by Lefkowitz et al. (1984) involving /9-adrenergic
receptors. His group has demonstrated examples of "homologous"
regulation by receptor-specific agonists such as isoproterenol and
"heterologous" regulation, where stimulation by a drug or hormone
leads to a change in sensitivity to another unrelated drug or
hormone. They found that thyroid status could have a profound
effect on both the number and coupling of oc-adrenergic and p-
adrenergic receptors. Presumably, this control of adrenergic
receptors was exerted at the level of gene transcription. Adrenergic
receptors are by no means unique in terms of their susceptibility to
regulation. Insulin receptors too, undergo homologous regulation by
insulin (Gavin et al., 1974). Insulin receptors also undergo
heterologous regulation, under the action of ^-adrenergic agents
(Pessin et al., 1983). Additional examples and more insight into the
phenomena of receptor regulation may be gained from the recent
reviews by Roth and Taylor (1982) and Poste (1984).
The cellular sensitivity to vasopressin can be drastically reduced
following chronic stimulation by the hormone. When the isolated
toad bladder, an epithelial model for the mammalian renal tubule, is
stimulated repetitively with high concentrations of the neuro
hypophyseal hormone, the osmotic water flow across the bladder
diminishes progressively (Eggena, 1981). Renal medullary plasma
membranes incubated with high concentrations of vasopressin display
reduced hormone-sensitive adenylate cyclase activity (Roy et al.,
1976). Furthermore, vasopressin-elicited desensitization of adenylate
cyclase activity has been shown in renal membranes prepared from


17
the formation of cAMP. Both V] and V2 receptors are functionally
linked to their respective effector systems through guanine nucleotide
regulatory proteins (G proteins). Thus, the antidiuretic action of
vasopressin is mediated through V'2-type receptors while the
vasoconstrictive, glycogenolytic and steroidogenic actions of AVP are
mediated by Vj-type receptors, based on structure-activity
relationship studies with vasopressin analogues or by directly
determining the second messenger. A novel vasopressin receptor
(Vib) which displays a pharmacological profile that does not clearly
fall into one category or the other has been reported to mediate the
effects of AVP in the anterior pituitary (Antoni, 1984). Furthermore,
recent evidence suggests that at least one tissue may possess both V]
and V2 receptor subtypes (Chapter 6).
Kidney
Evidence for the binding of vasopressin to hormone-responsive
cells in the kidney was first suggested by the autoradiographic
studies of Darmady et al. (1960). Specific binding of ^H-AVP to
plasma membranes of the renal medulla was subsequently reported by
two independent laboratories (Campbell, et al., 1972; Bockaert et al.,
1973). Neither study succeeded in localizing the binding to plasma
membranes of renal tubular origin nor were they able to distinguish
between basolateral and apical membranes. However, Bockaert et al.
(1973) and others (Nakahara et al., 1978) were able to demonstrate
the correlation between receptor occupancy and the activation of
adenylate cyclase. These studies were consistent with the finding
that cAMP and vasopressin-induced changes in membrane permeability
were only noted following application to the peritubular side of