Vasopressin: Mechanisms of Receptor Activation

The Neurohypophysis: Structure. Function and Control, Progress in Bruin Reseurch, Vol. 60, edited by B.A. Cross and G . Leng 01983 Elsevier Science Publishers B.V.

Vasopressin : Mechanisms of Receptor Activation S . JARD Centre CNRS-INSERM de Pharinacologie-Endoc.rinologie, rue de l r Cardonille, B . P . 5055, 34033 Montpellier Cedcx (France)

INTRODUCTION Besides its well known roles in the regulation of body fluid osmolality and blood pressure, vasopressin exerts a large variety of biological effects. Among these are: increased glycogenolysis and neoglucogenesis by liver cells (Hems and Whitton, 1973), increased corticotropin release by the adenohypophysis (Gillies et al., 1978), platelet aggregation (Haslam and Rosson, 1972), mitogenic effects on several cell types (Hunt et al., 1977 ; Miller et al., 1977; Whitfield et al., 1970 ;Rozengurt et al. , 1979), increased firing rate of several neuronal groups in the brain (Muhlethaler et al., 1982), and several effects on animal behaviour (De Wied and Bohus, 1978 ;De Wied and Versteeg, 1979). It is clearly established (Sawyer et al., 1974) that structural modifications of the vasopressin molecule affect its biological activities in a differential manner depending on the target tissue considered. In addition, vasopressin acts either through cyclic AMP-dependent (antidiuretic effect) or cyclic AMP-independent, calcium-dependent (vasopressor and glycogenolytic effects, among others) mechanisms (for review see Jard and Bockaert, 1975; De Wulf et al., 1980). Therefore, it can be concluded that, in mammals, there are at least two types of vasopressin receptors (vasopressin isoreceptors). The subdivision of vasopressin receptors into two classes (V, and V, vasopressin receptors) was formally introduced by Michell et al. (1979) on the basis of the different nature of the effectors to which these receptors are coupled : adenylate cyclase (V2-receptors)or the effector responsible for an increase in calcium entry into the cell and(or) mobilization of cellular calcium stores. The purpose of the present article is to review the available pharmacological and biochemical data on vasopressin receptors in mammals. The discussion will be restricted to those receptors for which binding data are presently available. KINETICS OF VASOPRESSIN BINDING TO VASOPRESSIN RECEPTORS Specific vasopressin binding sites which could be identified to vasopressin receptors involved in vasopressin-induced adenylate cyclase activation have been characterized on membranes prepared from porcine (Bockaert et al., 1973; Roy et al., 1975a, b), bovine (Hechter et al., 1978), rat (Rajerison et al., 1974; Butlen et al., 1978) and human kidneys (Guillon et al., 1982). In all cases [3H]vasopressinbinding was found to be time-dependent, reversible and saturable. Scatchard plots derived from the determination of the dose-depen-

384 dency for hormone binding at equilibrium did not show any clear deviation from linearity. This indicates that there is no cooperativity in hormonal binding. This conclusion was confirmed by the similarity of the dissociation constants as evaluated independently from equilibrium saturation and kinetic data. The apparent homogeneity in the population of vasopressin binding sites present in membrane preparations derived from the medullopapillary portion of the kidney suggests that vasopressin receptors from collecting ducts and ascending limbs of the loops of HenlC, the two segments of the nephron containing a vasopressin-sensitive adenylate cyclase (Imbert et al., 1975a, b), have similar properties with respect to the kinetics of hormonal binding. As indicated in Table I, the determined dissociation constants (K,) for vasopressin binding to renal vasopressin receptors are somewhat different depending on the mammalian species considered, ranging from 0.4 nM in the rat to 10-20 nM in the pig. In contrast to the data obtained on membrane preparations, binding studies on intact LLC-PK 1 cells * showed a marked heterogeneity in the population of vasopressin binding sites present on these cells as revealed by curvilinear Scatchard plots of the equilibrium dose-binding relationship (Roy and Ausiello, 1981). A precise analysis of the kinetics of vasopressin binding to LLC-PK1 cells, including determination of the rate of formation and dissociation of hormone-receptor complexes, led Roy and Ausiello to conclude that neither negative cooperativity nor binding to two or more independent populations of binding sites could adequately account for the kinetics of hormonal binding to LLC-PK 1 cells. The experimental data could be fitted with a model involving a hormone-induced change in receptor affinity. The authors (Roy et al., 1981) provided convincing evidence suggesting that this change in receptor affinity reflects a rapid desensitization of vasopressin-sensitive adenylate cyclase activity. Thus, receptor transition was not apparent in purified membranes from LLC-PKI cells or EDTA-treated cells, two preparations in which rapid desensitization of vasopressin-sensitive adenylate cyclase activity did not occur. For all the above-mentioned biological preparations numerous correlations between hormone binding and adenylate cyclase activation could be demonstrated (for review see Jard et al., 1975). These correlations clearly demonstrate that the specific vasopressin binding sites detected on kidney membrane fractions and LLC-PK 1 cells are the hormonal receptors involved in adenylate cyclase activation.

TABLE I KINETICS OF VASOPRESSIN BINDING TO RENAL MEDULLOPAPILLARY MEMBRANES Spec.ie.7

Equilihriicm dhsociation constmi ( n M )

Binding capaciiy pmollmg protein

Refirence ~~~

ox Pig Rat Human a b

I .44" 10-20h 0.4f0.1 a 4.08

1.34 1 .O 0.22 i0.02 0.5-0.8

Hechter et al. (1978) Bockaert et al. (1973) Rajerison et al. (1974) Guillun et al. (1982)

Value for arginine-vasopressin. Value for lysine-vasopressin.

* LLC-PK 1 cells are an established pig kidney cell line which maintains characteristics of polar epithelial cells and responds to vasopressin by an increased CAMP production and subsequent activation of CAMP-dependent protein kinase (Mills et al., 1979; Ausiello et al., 1980).

385 TABLE I1 KINETICS OF VASOPRESSIN BINDING T O LIVER AND VASCULAR VASOPRESSIN RECEPTORS All values refer to arginine-vasopressin.

Isolated rat hepatocytes Rat liver membranes Rat aortic inyocytes in culture

8

3 12

320 0.8

40

Cantau et al. (1980) Cantau et al. (1980) Penit et al. (1982)

Extrarenal vasopressin receptors have been characterized on isolated rat hepatocytes and purified liver membranes (Cantau et al., 1980), and on rat aortic smooth muscle cells in primary culture (Penit et al., 1983). On isolated hepatocytes and purified liver membranes vasopressin binds to an apparently homogeneous population of sites. The apparent dissociation constants for [ 3 H ] v a ~ o p r e ~binding ~ i n to isolated cells and purified membranes are different : 8 and 3 nM, respectively. As will be discussed later, this difference probably reflects the effects of “agonist-specific” modulators of receptor function operating in intact cells. The dissociation constant of 3 nM determined on purified rat liver membranes is about ten times higher than that determined on kidney membranes from the same species (compare data from Tables I and 11). The maximal vasopressin binding capacity of isolated hepatocytes was 320 fmoli lo6 cells, a figure about three times higher than those found on LLC-PK1 cells. The vasopressin binding sites detected on isolated hepatocytes were identified to the receptors involved in phosphorylase activation on the following grounds : (1) [3H]vasopressin binding was inhibited by vasopressin structural analogues which were shown to inhibit vasopressin-induced phosphorylase activation. A close correspondence was found between the K, values for the binding of these analogues to hepatocytes and the corresponding inhibition constants (K,); and (2) the same order of potency was found when the activities of a series of vasopressin analogues were measured by their abilities either to promote phosphorylase activation or to inhibit [3HJvasopressin binding. However, the apparent dissociation constants (K,) were found to be 5-SO times higher than the corresponding phosphorylase activation constants (K(J. The specific vasopressin binding sites detected on rat aortic myocytes maintained in primary culture had a similar affinity for vasopressin to that of vasopressin receptors on isolated hepatocytes (Table 11). The maximal binding capacity of myocytes (40 fmoU106 cells) was about eight times less than that of hepatocytes. For a series of five antivasopressor peptides, the determinedpKd* values for binding to aortic myocytes were almost identical to the corresponding PA, * values for inhibition of the vasopressor response to vasopressin in vivo. For a series of 15 vasoactive vasopressin analogues a clear correlation could be demonstrated between their respective vasopressor activities and the corresponding K,, values for binding to aortic myocytes. Together these data suggest that the vasopressin binding sites on aortic myocytes in primary culture belong to the main class of vasopressin receptors involved in the vasopressor response to vasopressin.

* pK, is the negative logarithm of the binding dissociation constant (K,) ; PA, is the negative logarithm of the molar concentration of antagonist that reduces the response to 2 x units of agonist to equal the response to 1 x unit in the absence of antagonist.

386

MODULATORS OF VASOPRESSIN RECEPTOR FUNCTION : COMPARATIVE STUDIES WITH RENAL AND EXTRARENAL VASOPRESSIN RECEPTORS The kinetics of vasopressin binding to renal and extrarenal vasopressin receptors is affected by magnesium ions and triphosphonucleotides. As shown in Fig. 1 , binding of ['H]vasopressin to LLC-PK 1 cells and to purified liver membranes exhibits an absolute requirement for the presence of magnesium ions in the incubation medium. The effect of reducing the magnesium concentration is to reduce receptor affinity. On liver membranes it could be shown that the magnesium effect is agonist-specific. Receptor affinity for vasopressin and several analogues active in promoting phosphorylase activation is reduced when magnesium concentration is decreased while, under the same conditions, receptor affinity for antagonists of the glycogenolytic response is unchanged. The corresponding data for renal vasopressin receptors have

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(

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Fig. 1. Effect of Mg2+ on [3H]vasopressin binding to LLC-PK 1 cells and rat liver membranes. LLC-PK I cells (0) and rat liver membranes ( 0 ) were incubated in the presence of the indicated amounts of magnesium together with 3.3nM (0)or 0.SnM ( 0 ) [3H]vasopressin. For both LLC-PKI cells and liver membranes the [3H]vasopressin concentrations used represented about one-third of the correspondingKdvalues. The observed changes in the amount of hormone bound reflect changes in receptor affinity. Data on LLC-PKI cells are taken from Roy and Ausiello (1981). Details on the vasopressin binding assay on liver membranes are given in Cantau et al. (1980).

387

not been collected. The dose-dependencies determined on liver membranes and LLC-PK I cells are similar, with an apparent K , value for magnesium of about 0.5 mM. These data suggest that magnesium ions act at the external border of the membrane. Kidney and liver vasopressin receptors are sensitive to triphosphonucleotides. The nucleotide effect results in an increased dissociation rate of hormone-receptor complexes and a corresponding increase in the equilibrium dissociation constant. The nucleotide effects on kidney and hepatic vasopressin receptors could be clearly distinguished on the basis of their dose-dependencies and specificities (Fig.2). ATP and GTP are equally active on liver membranes, with an apparentK, value of 0.5 mM. It is likely that hydrolysis of ATP or GTP is involved in their effects. Thus, 5’-guanylylimidodiphosphate (Gpp(NH)p) and 5’-adenylylimidodiphosphate are inactive. In contrast, Gpp(NH)p is almost as active as GTP on renal vasopressin receptors with an apparentK,n value in the micromolar range. ATP is far less active than GTP or Gpp(NH)p. Furthermore, in the experiments shown in Fig. 2 , one can hardly exclude that the ATP effect observed in the millimolar range is not due to small amounts of GTP contaminating the ATP preparation used. On both liver and kidney membranes the nucleotide effect was found to be agonist-specific. The agonist-specific character of the magnesium and nucleotide effects on renal and extrarenal vasopressin receptors could indicate that these agents are involved in the transduction mechanisms triggered by these receptors. Alternatively, magnesium and triphosphonucleotides could be involved in the desensitization of vasopressin receptors. Indeed, it has been clearly established that desensitization of membrane receptors for hormones and neurotransmitters is induced by the binding of agonists but not by the binding of antagonists. Anyway, the above-described data indicate clearly that renal and hepatic vasopressin receptors differ with respect to their sensitivities to triphosphonucleotides. L i v e r membranes

Kidney membranes

100-

8060-

F

60-

20-

a

01 -1

-6

5

I

I

-5

-4

-3

Nuckotide

(log M I

-7

t

I

I

I

-6

-5

-4

-3

Nucleotide

((09 MI

Fig. 2. Effects of triphosphonucleotides on [‘H]vasopressin binding to rat kidney and rat liver membranes. Rat kidney membranes (left panel) and rat liver membranes (right panel) were incubated in the presence of [3H]vasopressin and the indicated amounts of ATP (A ), GTP ( A ) o r 5’-guanylylimidodiphosphate( 0 ) .The [SH]vasopressin concentrations used were adjusted to equal about one-third of the K d value. The observed changes in the amount of bound hormone mainly reflect changes in receptor affinity. Data on rat kidney membranes are taken from Rajerison (1979). For experimental details on the vasopressin binding assays see Rajerison (1979) and Cantau et al. (1980).

388

PHYSICOCHEMICAL PROPERTIES AND RECOGNITION PATTERNS OF RENAL AND EXTRARENAL VASOPRESSIN RECEPTORS Vasopressin receptors from kidney and liver membranes can be solubilized in an active form under the influence of non-ionic detergents (Roy et al., 197%; Guillon et al., 1980). Determination of the hydrodynamic properties of rat kidney and rat liver receptors indicated (Guillon et a]., 1981) that these two receptors are hydrophobic and highly asymmetrical proteins of similar sizes (Table 111). Kidney and liver vasopressin receptors could not be TABLE 111 MOLECULAR PARAMETERS OF SOLUBILIZED VASOPRESSIN RECEPTORS Experimental data are taken from Guillon et al. ( 1980) Liver receptor

Stokes radius (nmf Apparent sedimentation coefficient in H,O gradient, Sapp (S) D,O gradient, Sapp (S) Standard sedimentation coefficient, S,OW (S) Partial specific volume (ml/g) Frictional ratio, FIF, Apparent molecular weight. M, Detergent bound (nigimg protcin) Molecular weight of the protein moiety, M,

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+

3.5 0.2 (6) 3 . 0 + 0 . I (6) 3.7 0.78 0.02 (14) I.77 101,000

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+

1.81

92,000 0.09 83,000

0.22

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-

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d PVDAVP,/ dlCH215VDAVP

10

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d DAVP v) a l

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0VAVP

AVP

DAVP

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D L

E

9.0 d PVDAVP

x c rn

0

x

= D

60d

/

OAVP

,d V D A V P

60

7.0 pKd

LVP

a

8.0

80

V D,"V P

7.0

6.0

Hepatocytes

pKd

80

Hepatocytes

Fig. 3. Recognition patterns of vasopressin receptors from rat kidney membrancs, isolated hepatocytes and aortic myocytcs. The figure was constructed from published data by Butlen et al. ( 1978). Cantau et al. ( I 980) and Penit et al. ( 1982). Abbreviations: AVP, arginine-vasopressin: LVP, lysine-vasopressin; DAVP, [8-~-arginine]vasopressin; VAVP, [4-valine]arginine-vasopressin; VDAVP, [4-valine, 8-o-argininelvasopressin;dC6AVP, deamino-h-carba-arginine-vasopressin ; dC60VP, deamino-6-carba-8-ornithine-vasopressin; dVDAVP, deamino-[4-valine, 8-D-arginine]vasopressin ; d(CH2)5-VDAVP, [ 1 -(p-mercapto-p, P-cyclopentamcthylenepropionic acid),4-valine,8-o-arginine]vasoprcssin; dPTOT, 1-deminopenicillaniine-[4-threonine]oxytocin:dPVDAVP. I-deaniinopenicillaminc-[4-

valine,8-~-arginine]vasopressin.

389

distinguished on the basis of any of the hydrodynamic parameters which can be determined by classical biochemical methods. Pharmacological studies using a large series of vasopressin structural analogues revealed (Fig. 3) the existence of striking similarities in the respective recognition patterns of vasopressin receptors from rat hepatocytes and rat aortic myocytes. Marked differences could be demonstrated between the recognition patterns of hepatic and aortic receptors on the one hand, and renal receptors on the other hand. As recognized from pharmacological studies in vivo (for review see Manning and Sawyer, 1977), substitution of D-arginine for L-arginine in position 8 and(or) substitution of valine for asparagine in position 4, affect the affinity of the vasopressin molecule for renal and extrarenal receptors in a clearly differential manner. TRANSDUCTION MECHANISMS TRIGGERED BY VASOPRESSIN RECEPTORS The molecular mechanisms involved in renal adenylate cyclase activation by vasopressin are probably similar to those which operate in the most extensively studied systems such as the glucagon-sensitive adenylate cyclase from liver or the p-adrenergic receptor-coupled enzyme from turkey erythrocytes (for review see Levitsky, 1982). Hormone-sensitive adenylate cyclases are composed of three distinct molecular entities: a receptor unit (R), a catalytic unit (C), and a coupling unit frequently denominated nucleotide regulatory unit (N). According to current views (see Levitski, 1982), both hormone binding to R and GTP binding to N are required to induce activation of C. The active state of the system decays concomitantly with the hydrolysis of GTP to GDP and P, at the N regulatory site. Replenishment of N with GTP and the continued presence of hormone at the receptor ensures the ability of the system to reacquire its active CAMP producing state. This “on-off” cycle accounts for most of the properties of hormone-dependent adenylate cyclases. In the case of vasopressin-sensitive adenylate cyclase, it could be demonstrated that: (1) vasopressin receptors and adenylate cyclase are distinct molecular entities which can be physically separated by various biochemical methods (Guillon et al., 198 1) ; (2) the guanylnucleotide binding component involved in adenylate cyclase activation by sodium fluoride and guanylnucleotides has been indirectly identified in renal membranes (Guillon et al., 198 1) ; and (3) GTP and the non-hydrolysable GTP analogue 5’-guanylylimidodiphate, markedly increase the sensitivity of rat (Rajerison, 1979) and human (Guillon et al., 1982) kidney adenylate cyclase to stimulation by vasopressin. Several models of receptors to adenylate coupling have been proposed. The more frequently discussed models are the so-called “collision coupling” model and the shuttle-ternary activation model. According to the first model (Tolkovsky and Levitski, 1978), the hormone-receptor complex makes a transient encounter with the adenylate cyclase complex viewed as a fairly stable complex formed between the catalytic unit and the N component ( I ) . The intermediary HR.N,,,C complex does not accumulate.

+

HR N,,C +HRN,,,C 4 HR N G T p C ’ 7NC GDP Pi

+

+

+ NGTPC’

in which C‘ and C represent the activated and non-activated forms of the catalytic unit, respectively. The collision coupling model contains the implicit assumption that one hormone-receptor complex can activate more than one adenylate cyclase molecule, as suggested by Jard et al. (1 975) in the case of vasopressin-sensitive adenylate cyclase. In the shuttle ternary activation

390 model it is assumed that the hormone-receptor complex can form a fairly stable complex with the N regulatory component according to the following scheme:

+

-

HR NGTp-+HRN,,, NfCTP+ C NfGTPC'

-

R

+ NIGTP+ H

This model offers the advantage of accounting for the existence of two affinity states of the receptor and the role of guanylnucleotides in the interconversion between these two states. A precise analysis of the kinetics of adenylate cyclase activation by vasopressin allowing to select the most adequate model has not been performed yet. Anyway, the abovementioned models do not predict that for all hormonal concentrations the fractional enzyme activation is identical to the fractional receptor occupancy. They can account for the observation that renal adenylate cyclase activation is a saturable function of receptor occupancy. Half-maximal adenylate cyclase activation is obtained for a fractional receptor occupancy less than 0.5. The non-linearity in the coupling of hormone binding to response, as estimated by the ratio of vasopressin concentrations leading to half-maximal binding and half-maximal adenylate cyclase activation, varies somewhat depedeqding on the mammalian species considered: 40,5, 5 and 1.2, for porcine (Jard et al., 1975), bovine (Hechter et al., 1978), rat (Butlen et al., 1978) and human (Guillon et al., 1982) renal adenylate cyclases, respectively. These differences might reflect species differences in the relative number of interacting R, N, and C units. Although marked amplification of the hormonal signal occurs at the cyclic AMP production step, it is clear that vasopressin concentrations eliciting half-maximal adenylate cyclase activation are much higher that hormonal concentrations needed to elicit half-maximal increase in water permeability of isolated collecting ducts (Grantham and Burg, 1966) or half-maximal antidiuretic response in vivo. These observations suggest that an additional amplification of the hormonal signal also occurs at steps beyond the cyclic AMP production step. This conclusion is supported by the observation that vasopressin structural analogues which behave as partial agonists of low intrinsic activity at the adenylate cyclase activation step are able to induce a full antidiuretic response in vivo (Butlen et al., 1978). The primary involvement of changes in cell calcium fluxes in the glycogenolytic response to vasopressin stimulation of isolated hepatocytes has been convincingly established (for review see De Wulf et al., 1980). There is also much evidence that a rise in cytosolic calcium concentration is also the initiator of the contraction of vascular smooth muscle in response to vasopressin stimulation. Conversely it has been established that vasopressin receptors in liver and vascular smooth muscle cells are neither positively nor negatively coupled to adenylate cyclase. In rat hepatocytes (Kirk et al., 1977) and rat aorta (Takhar and Kirk, 1981) vasopressin increases the incorporation of 32Pinto phosphatidylinositol. Stimulated phosphatidylinositol breakdown followed by compensatory resynthesis is a response of a wide variety of cells to many hormones and neurotransmitters acting through cyclic-AMP independent mechanisms. Michell and collaborators (Michell et al., 1979; Billah and Michell, 1979) have developed the view that phosphatidylinositol breakdown might be a reaction intrinsic to the same unitary mechanism whereby these hormones bring about calcium mobilization in their target cells. Several arguments support the proposal by Michell and collaborators that a causal relationship between hormone-induced increase in phosphatidylinositol breakdown and calcium mobilization are as follows : (1) the phosphatidylinositol response appears to occur independently of hormone-induced changes in cytosolic calcium concentration (Billah and Michell, 1979). Thus, phosphatidylinositol breakdown and labelling are resistant at least partially to

391 calcium elimination from the incubation medium, a situation in which phosphorylase activation by vasopressin is abolished. Furthermore, admission of calcium into hepatocytes with the ionophore A23 187 does not elicit the phosphatidylinositol response (Billah and Michell, 1979);(2) stimulation of phosphatidylinositol breakdown by vasopressin is rapid. The effect is clearly detectable within 1-2 min after addition of vasopressin (Kirk et al., 1977; Tolbert et al., 1980); (3) studies by Kirk et al. (1981), using vasopressin structural analogues, indicate that dose-response curves for phosphorylase activation and for enhanced phosphatidylinositol metabolism are parallel suggesting that the same receptor population is responsible for triggering both responses ; (4) comparison of the dose-dependencies for the four different detectable effects of vasopressin on isolated hepatocytes, namely binding of the hormone to the receptor (Cantau et al., 1980), increase in phosphatidylinositol turnover (Kirk et al., 19Sl), increase in calcium fluxes and the final glycogenolytic response, indicate that the A,, value for the phosphatidylinositol effect is almost identical to the dissociation constant for vasopressin binding to hepatocytes. On the other hand, vasopressin-induced increase in 45Cauptake can be detected upon exposure of hepatocytes to 0.1 nM vasopressin; phosphorylase activation is detectable for about ten times less than concentration. These results are compatible with the supposed sequence of events : hormone binding to the receptor, increase in phosphatidylinosito1 turnover, rise in cytosolic calcium concentration with subsequent triggering of the calciumdependent mechanisms of phosphorylase activation. They also suggest that a marked amplification exists between the primary signal in hormone action and the final biological response, and that the larger part of this amplification occurs between the phosphatidylinositol breakdown step and calcium mobilization. Parallel determinations ofK, values for the binding of vasopressin structural analogues to rat hepatocytes and of K, values for phosphorylase activation, showed (Cantau et al., 1980) that the ratios K,IK,, an estimate of the amplification of the hormonal signal, were not identical for all analogues tested. Furthermore, the additive character of several amino acid substitutions was clearly apparent. Thus (Table IV), substitution of D-arginine for L-arginine led to a 2.5-fold decrease in the K,IK, ratio as compared to that observed for arginine-vasopressin. Deamination of [8-D-arginine]vasopressin led to a further 5-fold decrease in the KJK, ratio. The same structural modifications introduced in [Cvalinelarginine-vasopressin led to deamino-[Cvaline, 8-D-arginine]vasopressin,an antagonist of vasopressin-induced phosphorylase activation for which the K,IK, ratio was equal to unity. TABLE fV EFFECTS OF STRUCTURAL MODIFICATIONS OF THE VASOPRESSIN MOLECULE ON THE RELATION OF BINDING TO PHOSPHOKYLASE ACTIVATION IN RAT HEPATOCYTES TheKcI/Kuvalue is an estimate of the amplification of the hormonal signal. It is maximal for arfiinine-vasopress~n.It equals unity for the competitive antagonist deamino-[4-valine,8-D-arginine]vasopressin.Intermediate value5 observed for several analogues might indicate that these analogues are partial agonists at an early step of the hormonal action. Experimental data are from Cantau et al. (1980). ~~

Pepride

KdK

Magnitude of’the phosphorylusr rpsponse (% of rrr~inine-vnsopressin-inducrdresponse) -

Arginine-vasopressin

[8-D-Arginine]vasopressin Deamino-[ 8-~-arfiinine]vasopressin [4-Valine]arginine-vasopressin [4-Valine, 8-o-arginineIvasopressin Deamino-[4-valine, 8-D-arginine]vasopressin

50 20 4 50 2 1

100 I00

100 I00 100

0

302

These data suggest that [8-D-arginine]vasopressin,deamino-[8-~-arginine]vasopressin and [4-valine, 8-~-arginine]vasopressinbehave like partial agonists at the primary step of vasopressin action on isolated hepatocytes. In line with Michell’s hypothesis, it would be of great interest to compare the magnitude of the maximal effect of these analogues on phosphatidylinositol breakdown with that of vasopressin. The nature of the effector molecule to which hepatic and vascular vasopressin receptors are coupled is not known. By analogy with hormone-responsive adenylate cyclases, it is possible that several receptors triggering calcium-dependent responses in the same cell are coupled to the same pool of effector molecules. In line with such a view is the recent demonstration by Breant et al. ( 1 98 1) of a heterologous desensitization of the glycogenolytic response in rat liver cells induced by occupation by an agonist of one of three independent receptors which activate the calcium-dependent pathway for phosphorylase activation namely, vasopressin, angiotensin and a-adrenergic receptors.

SUMMARY Vasopressin receptors have been characterized on renal membranes from several mammalian species and on two extrarenal vasopressin-sensitive cell types in the rat, namely isolated hepatocytes and aortic smooth muscle cells in primary culture. On all biological preparations so far tested vasopressin binds to a single class of sites. The dissociation constant for vasopressin binding to renal receptors varies depending on the mammalian species considered, from 0.4 nM in the rat to 10-20 nM in the pig. In the rat, the affinities of hepatic and vascular vasopressin receptors for vasopressin are similar and about ten times lower than the affinity of kidney receptors. Renal and extrarenal vasopressin receptors have similar hydrodynamic properties. They can be distinguished on the following grounds. ( I ) Renal vasopressin receptors are coupled to adenylate cyclase. Hepatic and vascular receptors have no direct functional relationship with adenylate cyclase. A primary involvement of a rise of cytosolic calcium in the biological responses triggered by these receptors has been established. In isolated hepatocytes and aorta, vasopressin stimulates phosphatidylinositol breakdown. Available experimental data are compatible with the existence of a causal relationship between vasopressin-induced phosphatidylinositol breakdown and the increase in cytosolic calcium. ( 2 ) Kidney and liver vasopressin receptors are affected by triphosphonucleotides. The nucleotide effect results in a reduction in receptor affinity for agonists but not for antagonists. On kidney receptors, the nucleotide effect is specific for guanylnucleotides ; it is observable in a micromolar range and does not involve GTP hydrolysis. On liver receptors ATP and GTP are equally active in a millimolar range. Non-hydrolysable ATP and GTP analogues are inactive. (3) Hepatic and aortic vasopressin receptors in the rat have almost identical recognition patterns. This recognition pattern is clearly different from that of kidney receptors. In all vasopressin-sensitive cells so far tested the dissociation constant for vasopressin binding to the receptors is much higher than the hormonal concentration eliciting half-maximal biological response. This suggests that a marked amplification occurs at steps beyond adenylate cyclase activation (renal receptors) or activation of the primary effector involved in calcium mobilization (hepatic and vascular receptors). REFERENCES Ausiello, D.A., Hall, D.H. and Daycr, J.M. (1980) Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK 1 cclls. R i o c h ~ mJ . , 186: 773-780.

393 Billah. M.M. and Michell, R.H. ( 1979) Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, adrenaline, ionophore A23 187 and calcium-ion deprivation. Biochern. J . . 182: 661-668. Bockaert, J., Roy, C., Rajerison, R. and Jard, S. (1973) Specific binding of (’H)-lysine-vasopressin to pig kidney plasma membranes. J . hid. Chem., 249: 5922-5931. Breant, B., Keppens, S. and De Wulf, H. (1981) Heterologous desensitization of the cyclic-AMP-independent glycogenolytic response in rat liver cells. Biochem. J . . 200: 509-5 14. Butlen, D., Guillon, G., Rajerison, R.M., Jard, S., Sawyer, W.H. andManning. M. (1978) Structural requirements for activation of vasopressin-sensitive adenylate cyclase. hormone binding, and antidiuretic action : effects of highly potent analogues and competitive inhibitors. Molec,. Phurmacol.. 14: 1 0 0 ~ 1 0 1 7 . Cantau, B.. Keppens, S . , De Wulf, H. and Jard, S. 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