Characterization and regulation of renal vasopressin receptors

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Characterization and regulation of renal vasopressin receptors
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Steiner, Martin, 1952-
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Receptors, Cell Surface -- physiology   ( mesh )
Vasopressins -- physiology   ( mesh )
Binding Sites -- drug effects   ( mesh )
Hypertension, Renal -- chemically induced   ( mesh )
Muridae   ( mesh )
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bibliography   ( marcgt )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 118-131).
Statement of Responsibility:
by Martin Steiner.
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Typescript.
General Note:
Vita.

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University of Florida
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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.




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INGEST IEID EQC2VGGR8_IHH1R5 INGEST_TIME 2012-12-07T22:00:43Z PACKAGE AA00012907_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES