Angiotensin Receptors in Vascular Tissue

MARIE-A.

DEVYNCK,

Ph.D.

PHILIPPE MEYER, M.D. Paris, France

From the Vascular and Renal Pharmacology and Physiology Unit (INSERM U7), Mpital Necker, 750 15 Paris, France. Requests for reprints should be addressed to Dr. Marie-A. Devynck, Vascular and Renal Pharmacology and Physiology Unit (INSERM U7), H6pital Necker, 161 rue de S&we% 75015 Paris, France.

756

November 1976

The biologic effect of angiotensin II is triggered by its interaction with components of target organs, which specifically recognize the hormone. These receptors have been studied with the use of radioactive angiotensin and, as for other peptidic hormones, have been localized in the plasma membrane of target cells. Such angiotensin receptors have been characterized in three target organs: vascular tissue, uterus and adrenal cortex. The binding characteristics differ in contractile tissue and in adrenal glands, the N and C terminal ends of angiotensin being involved in the former, whereas the N terminus does not appear to have the same importance in the latter. Numerous factors, including ionic composition, seem to be able to modify angiotensin-receptor interaction in vascular smooth muscle. However, the molecular mechanisms responsible for angiotensin binding and for the transmission of the signal determined by receptor-angiotensin interaction are not yet understood. As observed with other peptidic hormones, the number of angiotensin receptors seems to be susceptible to variation under certain conditions. in uterine smooth muscle, it was shown that the number of receptors increased after nephrectomy, a phenomenon which was prevented by the prolonged infusion of angiotensin. The significance of such a variation remains unknown, but it may be partially responsible for the inverse relationship that exists between the endogenous angiotensin level and the pressor effect of exogenous angiotensin. In the near future, investigation of the angiotensin-receptor mechanism will probably answer whether the variation in angiotensin receptors is similar in all target tissues and whether an angiotensin-receptor mechanism is involved in the pathogenesis of certain varieties of hypertension. In addition, a precise understanding of the angiotensin-receptor interaction will help the development of new angiotensin antagonists. A hormone receptor is a particular cellular component possessing the dual function of recognition and stimulation. Recognition is specific for certain structural elements of a given hormone which interact with complementary structures on the binding site of the receptor. Stimulation results from the interaction between active groups in the hormone and the receptor, and initiates the series of cellular events leading to the biologic response. The mechanism of receptor activation is not well understood, but it may be related to variations in receptor conformation or charge distribution. Before the interaction of a radioactive hormone with a cellular binding site can be considered to correspond to a receptor interaction, it should fulfil the criteria of a hormonal response, exhibiting specificity, high affinity, reversibility and saturability. In addition, the kinetic characteristics of the binding should correlate with those of the hor-

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ANGIOTENSIN RECEPTORS IN VASCULAR TISSUE-DEVYNCK,

monal effect or, ideally, with those of the first biochemical event in the series culminating in the biologic response. Employing these precise characteristics, it is possible to distinguish between the binding of a hormone to “receptor” sites and to “acceptor” sites [ 1,2]. The function of these latter binding sites remains uncertain. Since they usually exhibit some specificity for the hormone, they may represent enzymes involved in its metabolism, or receptors responsible for as yet unidentified biologic effects, or they may correspond to immature receptors not yet coupled to the biologic response. For numerous technical reasons, it is difficult to prove that the binding sites in subcellular fractions are the actual receptors. It is possible that the target tissue is altered during its preparation or that the hormone is inactivated by degradative enzymes released from damaged subcellular material. Many of these difficulties have been overcome, and numerous hormone receptors have been characterized [3]. The study of the interaction of angiotensin with its receptors is associated with such problems that their detailed analysis seems justified. In this review we will summarize the present state of the studies on the binding of angiotensin II to vascular smooth muscle and compare them with the binding of angiotensin II to receptors in other target organs. SPECIFIC PROBLEMS ENCOUNTERED IN THE CHARACTERIZATION OF ANGIOTENSIN II RECEPTORS

The physiologic concentration of angiotensin II in the plasma of the many species studied is in the order of ?O-” M. In order to study the binding of the hormone at these low concentrations, it is necessary to use a radioactive hormone of high specific activity. This is usually achieved by introducing atoms of radioactive iodine into the hormone molecule. However, the major drawback resulting from the iodination of small peptide hormones is a loss of biologic activity. This has been established for oxytocin [4] as well as for angiotensin II, at least as far as the contractile response is concerned [5-71 (Table I). In the case of angiotensin II, the halogenation is mainly made on the tyrosine (the fourth amino acid) in the CYposition of the hydroxyl group. This results both in a lowering of the pK of the hydroxyl group, which is necessary for biologic activity [8-l 11, and a steric hindrance, which is appreciable by a comparison of the dimensions of iodine and the benzene ring of tyrosine (about 2.1 and 2.7 A, respectively).

This modification is particularly important since the integrity of the tyrosine is necessary for the initiation of contraction [ 12,131. In addition, halogenation may be performed on the histidine (the sixth amino acid). The substitution of hydrogen by tritium or carbon by 14C does not provoke major structural changes and

TABLE I

MEYER

Biologic Activities of lodinated Angiotensin (expressed as per cent of the activity of angiotensin I I considered as 100 per cent) Di-

Monolodinated Angiotensin II

lodinated Angiotensin

33

13

f51

Rat uterus

-

Rat colon

25

Rabbit

70 -

2 23 24 24 21 10

[51 [Sl 151 161 [71 161

Effect Pressor response ContractlIe effect on

aorta

II

References

does not perturb the biologic activity of the hormone [ 141. However, the specific activities obtained are much lower than those of the iodinated hormone. Since 3H bears 29 Ci/mAtom the maximum specific activity of an angiotensin II possessing 2 to 3 atoms of tritium per molecule is 58 to 87 Ci/mmol, whereas that for a monoiodinated ( lz51 or 13’1) angiotensin II can reach 2,000 CVmmol. It is clear that one must either work with physiologic concentrations of an iodinated hormone, which interacts badly with its receptor, or with higher pharmacologic concentrations of a tritiated hormone, which interact normally with the receptor. We preferred this latter solution in our own studies and chose the rabbit aorta as a model. This target organ has the advantage of being a vascular segment whose contractile responses to angiotensin have been extensively studied [ 11,15-181. In addition, the rabbit aorta has a sympathetic innervation which is so weak [ 191 that the eventuality that tritiated (3H) angiotensin II could bind to nervous receptors (reasonable in view of the effects of the hormone on sympathetic nerve terminals) is unlikely. There are obvious problems in generalizing the results obtained on the rabbit aorta to all vascular tissue, and in particular to those which respond physiologically to angiotensin II [20]. Nonetheless, the angiotensin II receptors in the rabbit aorta exhibit the major characteristics of angiotensin receptors in other arteries inasmuch as can be judged from their contractile responses. The second difficulty in the study of the angiotensin II receptor arises from a lack of knowledge concerning the first intracellular biochemical event provoked by the hormone-receptor interaction. The action of angiotensin II on smooth muscle adenylcyclase is uncertain [21,22], and its effect on the binding of membrane calcium has not been explained satisfactorily [23,24]. Thus, in the case of this hormone, one is reduced to correlating its specific binding with a global biologic effect, contraction measured in vitro, without being absolutely certain of the existence of a

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linear relationship between the occupation of receptors and the contraction, given the large number of intermediate steps. In this respect, the choice of the rabbit aorta has a great advantage in that pharmacologic studies have demonstrated a direct proportionality between the number of angiotensin II receptors and the contractile response [lo]. This proportionality implies firstly, that all the receptors are coupled to the response, i.e., that a contractile response is obligatorily provoked by the occupation of receptors and that there are no spare receptors; secondly, that the receptors are comprised of homogeneous molecular entities which function independently (noncooperatively); and thirdly, that the mean effective dose (EDso) corresponds to the affinity constant of the hormone (apparent dissociation constant, KJ 1251. LOCALIZATION

OF ANGIOTENSIN

II RECEPTORS

It seemed very likely that the angiotensin II receptors would be localized on the plasma membrane since the receptors for most other polypeptide hormones are situated at this level. Furthermore, the contractile response of angiotensin II is extremely rapid in onset, in contrast to the slow onset of action of hormones which act inside the cell, such as steroids [26]. The contractile response of angiotensin II is closely associated with measurable membrane phenomena. During the first seconds after exposure to the hormone, there is a depolarization due to changes in the membrane permeability to various ions [27,28]. An essentially membranous effect of the hormone is suggested more directly by the fact that the contractile effect of polymerized angiotensin II (N-poly-O-acetyl-l-angiotensin II) and angiotensin II bound covalently by its N terminal to a macromolecule (gamma globulin, cytochrome C or horseradish peroxidase [29,30]) is quantitatively reduced but not delayed. These results are not conclusive since the angiotensin II molecules could eventually, under the influence of tissue hydrolases, be dissociated from their macromolecular support and have free access to smooth muscle cells. The problem was complicated by the demonstration of a possible direct action of angiotensin II on isolated mitochondria [31] and by autoradiographic results showing a nuclear localization in myocardial and arterial cells short time intervals after the intravenous injection of tritiated angiotensin II [32]. In reality, the autoradiographic results cannot be considered conclusive because the specificity of the nuclear localization was not demonstrated and could be due to radioactive fragments of the hormone. Other investigators have been unable to confirm these autoradiographic findings in myocardial cells [33] and kidney [34]. Furthermore, the stimulation of nucleic acid synthesis apparently induced by angiotensin II in myo-

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cardial cells does not appear to correlate with the much more rapid induction of contraction. Localization of the angiotensin II receptors on the plasma membrane is directly demonstrable biochemically. By ultracentrifugation of a tissue homogenate in a density gradient, it is possible to obtain membranous fractions enriched in plasma membranes which are recognized by their high concentration of marker enzymes, such as adenylate cyclase and 5’nucleotidase. The enrichment of this membrane fraction in angiotensin receptors has been shown to be as great as that of the marker enzymes in vascular smooth muscle (rabbit aorta) [35], nonvascular smooth muscle (rat uterus) [36] and in bovine adrenal cortex [37]. This result is a strong argument supporting localization of angiotensin II receptors at the level of the plasma membrane.

The American Journal of Medicine

CHARACTERIZATION

OF AORTIC RECEPTORS

Tritiated angiotensin II binding sites exhibiting characteristics expected for receptors were demonstrated initially in the intact rabbit aorta [38] and subsequently in membrane fractions [35]. Table II shows the association and dissociation constants measured on microsomes and establishes the reversibility of the binding reaction. Study of the binding as a function of angiotensin II concentration demonstrated two classes of sites. The first high affinity sites are saturable at a ligand concentration of 2 to 3 X 1Om8M, whereas the second class of sites exhibits low affinity and is not saturable under our experimental conditions (up to 5 X 10e7 M). Identification of the first class of sites as angiotensin II receptors is strongly supported by the similarity between the apparent dissociation constant and the EDso (Table II). Nevertheless, positive identification of these sites as the angiotensin II receptors was hampered in our initial studies by the absence of a correlation between the biologic activity and the binding of some analogues and fragments of angiotensin [35,39]. In these experiments, the affinity of these components for the angiotensin II receptor was studied by measuring the decrease in 3H angiotensin II binding (2.5-5 X 1Om8 M) in the presence of a twentyfold excess of the nonradioactive angiotensin II derivative. Recently, we realized that in using such high concentrations we would, in fact, principally be measuring the affinity of these structural analogues for the second, low affinity class of angiotensin II binding sites. The competition experiments, repeated subsequently employing lower concentrations of both 3H angiotensin El and the nonradioactive derivatives, showed that their affinity for the first class of sites correlated well with their biologic activity (Figure 1). This observation is interesting for a number of reasons. Firstly, it permits definite identification of the first class of angiotensin II binding sites as

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ANGIOTENSIN

TABLE

II

Binding

Parameters

for Angiotensin

RECEPTORS

IN VASCULAR

TISSUE-OEVYNCK,

MEYER

II Receptors

Rabbit Aorta Membranes (29°C)

Parameter Association (lO’M_’ Dissociation (10m4 Equilibrium

rate constant X set-‘) rate constant X set-‘) constant

From

k f

From

Scatchard

Rat Uterus ____ Membranes (29°C)

DOC-Solubilized Material (29°C)

k, 1.3

1.6

2.5

k_, 8

41

13

20

20

Kd

1 (IO+ Contractile (1O-9 NOTE:

MI response

plots 6.2 ED,,

7.8 8.5

MI DOC

[I31 1531

10 [Sl

= deoxycholate.

the angiotensin II receptors. Secondly, it poses the problem of the significance of the second class of low affinity sites. According to the competition experiments, these sites have a marked specificity for the C terminal of angiotensin II, whereas both the N and the C terminal are necessary for the correct interaction with the first class of sites. The existence of a membranous class of sites, with a lower affinity than that of the hormone receptor, has been demonstrated for numerous other polypeptide hormones, but their significance remains obscure [ 11. One of the more commonly advanced hypotheses is that these sites, which are hormonal acceptors, could correspond to the enzymes implicated in the inactivation of the hormone. This, however, is unlikely since we have been unable to detect significant degradation of angiotensin II in contact with aortic microsomal membranes under our experimental conditions. As has been achieved with membrane receptors for many other polypeptide hormones, it has been possible

to separate the angiotensin II receptors from their membranous environment by treating them with detergents [38] (sodium deoxycholate in the case of angiotensin). Biochemical studies performed so far cannot explain the phenomena of desensitization or tachyphylaxis. Desensitization could perhaps be related to dissociation of the hormone-receptor complex, or to a conformational change which shifts the receptor from an active to an inactive form. Similarly, tachyphylaxis has been interpreted in many different ways [20,40]. It may result from events occurring at the receptor level, such as an occupation of all the receptors by the added hormone, or from a slowly reversible conformational change in the receptor which makes it incapable of conveying the hormonal response. Since many factors are able to modify the contractile

+ Figure 1. Specificity of angiotensin binding on rabbit aorta microsomal membranes. Displacement of =H angiotensin II by unlabelledangiotensin derivatives. The ordinate shows the percentage of specific binding after competition with unlabelled analogues added simultaneously. In these experiments =H angiotensin II concentration was lOma M, correspondingto the high affinity bindingsites. The incubation was performed at 29% for 8 minutes. The relative affinity of these compounds,determinedfrom their contractileeffects on rabbit aorta strips are: 100 for Vap-angiotensin II, 5.6 for the heptapeptide 2-8 and 5.1 for Asp’ -angiotensin I, < 0.1 for the pentapeptide4-8 and 0 for the heptapeptidel-7 [ 131. The Phe4-angiotensin II analog was reported to have 10 to 20 per cent of the angiotensin II pressor response on the nephrectomized rat [I 1] and a relative affinity of 0.54 on rabbit aorta strip [ 101. Sar’ ,Ile8-angiotensin II is a competitive antagonist with a pAp of 9.33 on rabbit aorta [ 121.

November 1976

4-8 PENTAPEPTIDE

-8

-1 LOG CONCENTRATION

-6 OF ANGIOTENSIN

The American Journal of Medicine

-5 PEPTIDES (Ml

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TABLE III

TISSUE-DEVYNCK,

MEYER

Effect of Various Compounds on 3 H-Angiotensin II Specific Binding (% of control)* Specific Binding

Concentration IOW

Sodium Potassium Magnesium Calcium EDTA -SH and -S-S-Reagents N-ethylmaleimide Dimercaprol Glutathione “High energy” compounds Adenosine triphosphate Guanosine triphosphate Phosphoenol pyruvate kinase 1 Pvruvate Enzymatic treatment Trypsine (E.C.3.4.4.4) Pepsine (E.C.3.4.4.1) Phospholipase A (E.C.3.1.1.4) Phospholipase C (E.C.3.1.4.3) Phospholipase C + 5.10’ M Caf+ Neuraminidase (E.C.3.2.1.18) NOTE: -SH * Each value

5 fig 20 /Jg 10 pg 50 /Jg 50 pg 12Obg

ml-’ ml-’ ml-’ ml-’ ml-’ ml-’

-

100 63 39 50 109

M M

IO4 M 1O-3 M lO-3 M

20 100

IO- M 1O-3 M

55 22

5.10+

5 fig ml-’

M M

20 40 30 30 30 30 15

61

M

min min min min min min min

100

37°C 37°C 37°C 37°C 37°C 37’C 37°C

__~__~

200 70 180 121 109 105 60

= sulfhydryl; -S=S= disulfide. IS the mean of four to six determinations

effect of angiotensin II, it seems reasonable that certain of them may act by modifying the binding of angiotensin to its vascular receptors. Although the available evidence is still fragmentary and derives principally from our own laboratory, we have summarized it in Table III. (1) Increasing the concentration of divalent cations (calcium and magnesium) strongly inhibits the specific binding of angiotensin II. Transmembrane movements of calcium occur during variations in mechanical activity of vascular smooth muscle and could thus be able to subtly modulate the hormone-receptor interaction at the same time. Such a phenomenon has been described with adrenocorticotropic hormone at the level of the adrenal cortical cell [41]. The inhibitory effect of magnesium on the contractile effect of angiotensin II [42] is often interpreted as being secondary to a competition with extracellular calcium. Binding studies suggest that magnesium could also act directly on the formation of the hormone-receptor complex. Potassium has a weak inhibitory effect on the specific binding of angiotensin II, and sodium has no effect. This is in agreement with the absence of an effect on angiotensin contractile response as a result of changes in sodium concentration in vitro [43]. The lack of a direct sodium effect on angiotensin-elicited contraction must be clearly distinguished from the variations resulting from modifications of the sodium balance which will be discussed subsequently. (2) Sulfhydryl (-SH) groups have been implicated in contraction and relaxation of vascular smooth muscle [44,45]. N-ethylmaleimide, which binds covalently to sulfhydryl groups, nonspecifically

762

200-IO-” 80 - IO-” 90-IO-‘M 6 - IO+ 1 - 1O-3

decreases the reactivity of the rabbit aorta, whereas dithiothreitol, which reduces disulfide bridges, specifically abolishes the response to angiotensin II [45]. During recent experiments (unpublished), we showed that 10m4 M N-ethylmaleimide decreased the specific binding of 5 X 10m8 M angiotensin II by 40 per cent and that 10m3 M dimercaprol decreased it by 80 per cent. These results do not, however, provide conclusive proof that -SH groups are part of the receptor structure; it is possible that modification of sulfhydryl groups in close proximity to the receptor on the membrane could indirectly modify the hormone-receptor interaction. (3) The same problems are associated with the analysis of the studies on the alteration of the membranes provoked by different enzymatic treatments, the results of which are presented in Table III. The increase in the binding capacity of the membranes following hydrolysis of phospholipids or proteins does not suggest that the receptor does not contain either phospholipid or protein, but rather that a disorganization of the membrane structure increases the accessibility of the receptor. However, prolonged treatment with either trypsin or phospholipase C redticed the binding of 3H-angiotensin II. The physiologic significance of the angiotensin II binding sites exposed by hydrolysis is unknown. An analogous phenomenon was observed with insulin receptors [46]. The results obtained after treatment with neuraminidase are more easy to interpret since the decrease in 3H angiotensin II specific binding was found both in the membrane preparations as well as on the solubilized material. The resistance of angiotensin

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ANGIOTENSIN RECEPTORS IN VASCULAR TISSUE-DEVYNCK,

binding sites to moderate proteolysis suggests that receptors are integral proteins in the sense proposed by Singer and Nicolson [47]. The effects of neuraminidase suggest that the receptor is a glycoprotein, the sialic acid of which is directly implicated in the formation of the angiotensin II-receptor complex. (4) Nucleotides appear to be able to modify the binding of diverse polypeptide hormones [48,49]. The results obtained with angiotensin II on vascular smooth muscle cell membranes (Table Ill) show that at high concentrations, adenosine triphosphate and to a lesser extent guanosine triphosphate, inhibit the specific binding of angiotensin II to aortic microsomal membranes. This decrease in binding is correlated with phosphorylation since the presence of the energy generating system, phosphoenol pyruvate-pyruvate kinase, prevents specific binding to these membranes. It is conceivable that changes in phosphorylation could be implicated in the receptor activation-inactivation process. (5) The specific binding of angiotensin II is not modified by I-noradrenaline [35], but it is increased by the prostaglandins of the E series. The interpretation of this phenomenon is more difficult in view of the variability of the experimental results of studies aimed at demonstrating an interference by endogenous prostaglandins in the angiotensin II contractile response [ 10,50,5 l] and merits further investigation. COMPARISON OF THE VASCULAR RECEPTORS WITH RECEPTORS IN OTHER ANGIOTENSIN II TARGET ORGANS The arigiotensin receptors of contractile tissues have been studied essentially with the use of classic pharmacologic methods. According to Schild [52], it is possible to demonstrate a difference in a hormone receptor in various target organs if the pharmacologic studies take into account the following parameters: (1) apparent affinity and intrinsic activity of the agonists. In the case of a linear coupling, the apparent affinity corresponds to the apparent dissociation constant and is indicated by the EDs0 value. (2) Apparent affinity of competitive antagonists or PAZ* values. (3) Measurement of contractile effects of analogues in organs rendered tachyphylactic with angiotensin II. In the case of smooth muscle, pharmacologic results suggest that differences may exist among angiotensin receptors in the rabbit aorta, the rat uterus and the rat colon [53]. However, other studies did not demonstrate any difference between vascular and intestinal smooth muscles of two different species [53]. These investigations should be completed by direct studies on the binding of angiotensin II, which offer obvious advantages in comparison to pharmacologic studies. Results obtained so far in our laboratory have not demonstrated any difpAp = negative logarithm of the concentration of antagonist that reduces the effect of a double dose of agonist to that of a single

MEYER

ference between vascular smooth muscle receptors (rabbit aorta) and nonvascular smooth muscle receptors (rat uterus) [ 361. However, great differences exist between the receptors of vascular smooth muscle and those of the adrenal gland. These differences are particularly marked in the adrenal cortex. (1) Although the N terminal of the hormone is indispensable for full contractile activity, it is not necessary for the steroidogenic action: the 2-8 heptapeptide, sometimes referred to as angiotensin Ill, stimulates the secretion of aldosterone as well as [55-571 or better [58] than angiotensin II even though it only has 10 to 50 per cent of its contractile activity. It is worth noting here that the competitive antagonists Sar’,Ala*-angiotensin II and Sar’,Ile*angiotensin II, which are relatively resistant to aminopeptidase degradation, inhibit the myotropic response more than the steroidogenic response [58,59]. These pharmacologic observations suggest that receptor occupancy in the adrenal cortex is maximally insured by a sever? amino acid hormone. However, binding experiments on the adrenal cortex produced conflicting results: on the one hand, they have confirmed that the 2-8 heptapeptide angiotensin II binds nearly as well as the intact angiotensin II [37,60], and yet, on the other hand, these studies have shown that Sari-angiotensin derivatives are also capable of binding as efficiently to receptor sites. Further studies are obviously needed to determine whether angiotensin II receptors are identical to those of angiotensin III. (2) In contrast to vascular smooth muscle the binding of angiotensin II to the adrenal cortex is unaffected by calcium [37], variably affected by sodium [61,62] and greatly inhibited by guanosine triphosphate [49]. As discussed previously, these differences may stem from variations occurring at the level of the receptor or from changes occurring in the environment of the membrane receptor. In the adrenal medulla, the possible differences in the receptor can only be discussed in terms of pharmacologic studies. Interestingly, the C terminal of angiotensin II can be modified without greatly affecting its activity in the adrenal medulla [63]: for example, the decapeptide angiotensin II, without apparently being converted to the octapeptide, and the Ala*-angiotensin derivative stimulate catecholamine release. Binding studies would seem to be indispensable to better characterize this phenomenon and to demonstrate eventually that the angiotensin-receptor interaction in the adrenal medulla does not involve the C terminal of angiotensin, contrary to other target tissues. HYPOTHESES CONCERNING THE REGULATION OF THE VASCULAR RESPONSE TO ANGIOTENSIN II MEDIATED BY CHANGES AT ANGIOTENSIN II RECEPTORS

THE

LEVEL

OF THE

l

dose.

The pressor effect of angiotensin II is modified by numerous factors. Two of them, the plasma concentration

1976

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of endogenous angiotensin II and the sodium balance (acting perhaps via endogenous angiotensin II), are specific for this hormone in the sense that they do not affect (or very little) the pressor action of other vasoconstrictor agents [64-701. The specificity of these factors implies that their action cannot be attributed to a modification of the intracellular machinery responsible for the mechanical activity of vascular smooth muscle. Their action is probably related either to the events occurring at the level of the angiotensin II receptor and concerning the formation of angiotensin II receptor complexes, or to modifications affecting the coupling of the receptors to intracellular events ultimately responsible for contraction. The results of variations in sodium balance on the pressor effect of angiotensin II are well established. Vascular reactivity to exogenous angiotensin II increases in positive sodium balance and decreases in negative sodium balance. It is interesting to note that these effects of variations in sodium balance are the opposite in the adrenal cortex in which the steroidogenie effect of angiotensin II is potentiated by a negative sodium balance. The variations in sodium balance, via their influence on renin secretion, modify the plasma concentration of angiotensin II which increases and decreases with a deficiency or an excess of sodium, respectively. The fluctuations in endogenous angiotensin II are obviously involved in the development of specific hypersensitivity to angiotensin which occurs after bilateral nephrectomy. When plasma angiotensin II has been lowered by nephrectomy, the pressor effect of an injection of exogenous angiotensin is markedly enhanced. Several hypotheses may be proposed to explain the inverse relationship between the contractile effect and the endogenous hormone level. (1) A first hypothesis could be that the variations in pressor activity of exogenous angiotensin II are inversely proportional to the occupation of these vascular receptors by the endogenous angiotensin II. During negative sodium balance, circulating angiotensin II is increased and the proportion of free receptors accessible to the exogenous hormone is decreased, resulting in a reduction in the pressor response. During positive sodium balance and after nephrectomy, the inverse results are obtained and are related to an increase in the proportion of free vascular receptors. This reasoning has often been proposed to explain the variations in the pressor response to angiotensin II as an inverse function of the circulating level of angiotensin II as in the test of Kaplan and Silah [67]. This interpretation has been applied to the variations in angiotensin II liberated in the vascular wall under the action of iso-renin [71]. (2) A second hypothesis could be that the changes in endogenous angiotensin II, produced by variations in sodium balance or by nephrectomy, modify the “avidity” of vascular

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receptors for the hormone [72j. The nature of the events involved in this phenomenon is not clear. (3) The third hypothesis is that the variation in the pressor effect of exogenous angiotensin II is explained by a variation in the number of total angiotensin receptors in vascular tissues. Assuming an unmodified coupling between receptor occupancy and contraction and the law of mass action, an increase in the total number of receptors would, of course, if the hormone concentration is able to saturate binding sites, increase the angiotensin response. The difficulties of conducting studies of receptors in arterial wall due to scarcity of material led us to study this question on receptors in the uterine membrane. Our results [73,74] have established that the total number of uterine angiotensin II receptors increases by 20 per cent and 80 per cent, respectively, 19 hours and 22 hours after nephrectomy. This increase in the number of sites is not associated with a significant variation in their affinity for the hormone. The increase in uterine receptors after nephrectomy is real and does not correspond to freeing of sites secondary to the disappearance of endogenous angiotensin II following nephrectomy. In fact, the appearance of the increased number of receptors following nephrectomy is not apparent until after 18 hours and is much slower than the disappearance of renin and endogenous angiotensin II [ 7 1,75,76], and also much slower than the dissociation of angiotensin II from its membrane receptors. In addition, the increased number of receptors is not suppressed by the injection of a dose of hormone capable of occupying all the receptors immediately before sacrifice. The mechanism of the increase in the total number of receptors after suppression of the endogenous hormone is not yet understood. However, it provides a satisfactory explanation for the supersensitivity observed in vitro in the uterus of a rat previously nephrectomized. Under normal experimental conditions, this supersensitivity can no longer be explained by a freeing of receptors [77]. Although direct binding studies have not yet been conducted with vascular receptors, one may conceive that the same variation occurs at this level and causes the supersensitivity observed in vivo when endogenous angiotensin is low (in case of positive sodium balance and after nephrectomy). Comparable observations have been made with other polypeptide hormones and transmitters, such as prostaglandins and catecholamines [78--841. In most of these studies the effect provoked by a given hormone is specifically reduced by the prior exposure of a target tissue to this same hormone; conversely, there appears to be a specific supersensitivity after suppression of the endogenous hormone. This effect has been attributed to a variation in the total number of receptors without modification of their affinity with many substances such

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ANGIOTENSIN RECEPTORS IN VASCULAR TISSUE-DEVYNCK,

as insulin [85-901 and the catecholamines [91]. This effect could also concern steroid hormones: the number of estrogen-induced cytosolic receptors of progesterone in the uterus progressively decreases when plasma progesterone increases in the dioestrus phase [92]. In the case of insulin it has been clearly shown that the variation of receptor number develops directly as an inverse function of the concentration of endogenous hormone. This inverse relation between the number of re&eptors and the concentration of circulating hormone has been verified in the case of angiotensin and its uterine receptors, when long-term infusions of suppressor doses of angiotensin II were capable of inhibiting the increase in the maximum binding capacity. Gavin et al. [86] have suggested that the inverse relationship between the total number of receptors (and hence the hormonal effect) and the level of circulating hormone is a regulatory phenomenon occurring in target tissues and counteracting the variations in secretion of the hormone. It is conceivable that an abnormality in this regulatory phenomenon could be the origin of certain pathologic conditions. Before admitting such a modulatory process at the receptor level as a general phenomenon, one must demonstrate that it occurs in all target tissues of a given hormone. In the case of insulin the phenomenon seems still to be controversial in some tissues [93]. As pre-

MEYER

viously emphasized, in the case of angiotensin II the vascular receptor has not been investigated. In addition, since the effects of changes in sodium balance on the steroidogenic effect of angiotensin are opposite to those of its myotropic action, one should expect a different receptor mechanism in the adrenal cortex. Only after completion of these physiologic studies may the angiotensin receptor mechanism in hypertension be understood. The search for an abnormality at the receptor level in pathologic states is legitimized by the discovery of a decreased number of receptors in various pathologic conditions such as genetic and experimental diabetes [85,87-901 and myasthenia gravis [ 941. ACKNOWLEDGMENT We wish to acknowledge the help of V. Koreve and M. G. Pernollet for their contribution in the unpublished experiments presented in this paper, and to K. O’Dea and S. Hamon for their help with the manuscript. ADDENDUM Since completion of this article, Le Morvan 951 have reported results concerning 54C-angiotensin II on guinea pig aorta, previous results on rabbit aorta from our

and Palaic binding of confirming laboratory

[351.

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Birnbaumer L, Pohl SL, Kaumann AJ: Receptors and acceptors: a necessary distinction in hormone binding studies. Advances in Cyclic Nucleotide Research. IV (Greengard P, Robison GA, eds), New York, Raven Press, 1974, p 239. Cuatrecasas P, Hollenberg MD: Binding of insulin and other hormones to non-receptor material. Saturability, specificity and apparent negative cooperativity. Biochem Biophys Res Commun 62: 31, 1975. Cuatrecasas P: Membrane receptors. Ann Rev Biochem 43: 169, 1974. Thomson EE, Freychet P, Roth J: Monoiodoocytocin: demonstration of its biological activity and specific binding to isolated fat cells. Endocrinology 91: 1199, 1972. Lin SY. Ellis l-l. Weisblum B. Goodfriend TL: Preoaration and properties of iodinated angiotensins. Biochem Pharmacol 19: 651, 1970. Papadimitriou A, Worcel M: Dose-response curves for angiotensin II and synthetic analogues in three types of smooth muscle: existence of different forms of receptor sites for angiotensin II. Br J Pharmacol 50: 291, 1974. Kurcbart H, Coli A, Vancheri L, et al.: Chemical and biological effects associated with the iodination of angiotensin II. Biochim Biophys Acta 230: 160, 1971. Schrbder E: Synthese von Lys6Va15, von Tyr3Va14, von Tyr(Me)4Va15 und von TyreVa15 angiotensin II-Asp’-P-amid. Justus Liebigs Ann Chem 664: 243, 1965. Park WK, Choi C, Rioux F, et al.: Synthesis of peptides with the solid-phase method. II. Octapeptide analogues of angiotensin II. Can J Biochem 52: 113, 1974. Rioux F, Park WK, Regoli D: Application of drug-receptor theories to angiotensin. Can J Physiol Pharmacol 51: 665, 1973.

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