Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

Pharmac. Ther.Vol. 54, pp. 129-149,1992 Printed in GreatBritain.All rightsreserved

0163-7258/92$15.00 © 1992PergamonPressLid

Associate Editor: P. K. CHIANG

ENDOTHELINS A N D SARAFOTOXINS: PHYSIOLOGICAL REGULATION, RECEPTOR SUBTYPES A N D T R A N S M E M B R A N E SIGNALING MORDECHAI SOKOLOVSKY Laboratory of Neurobiochemistry, Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Abstract--The endothelins and sarafotoxins are two structurally related families of potent vasoactive peptides. Although the physiological functions of these peptides are not entirely clear, the endothelins are probably involved in pathophysiological conditions such as hypertension and heart failure. This review summarizes the state of the art in some areas of this intensively studied subject, including: (1) structure-function relationships of ET/SRTX, (2) ET concentrations in plasma, (3) ET/SRTX receptor subtypes and (4) signaling events mediated by the activation of ET/SRTX receptors. CONTENTS 1. Introduction 2. Structural Aspects 3. Plasma Concentrations of Endothelins in Humans 4. Receptor Subtypes 5. Kinetics of Binding 6. Covalent Labeling of ET/SRTX Receptors 7. Cloning and Expression of ET Receptors 8. Tissue Distribution of the Receptor mRNA 9. Endothelin/Sarafotoxin Signal Transduction Acknowledgements References

129 130 133 134 137 137 139 140 141 143 143

1. I N T R O D U C T I O N The endothelins (ET) and sarafotoxins (SRTX) (Fig. 1) are two structurally related families of potent vasoactive peptides (for reviews see Yanagisawa and Masaki, 1989; Kloog and Sokolovsky, 1989; Lovenberg and Miller, 1990; Simonson and Dunn, 1990a; Sokolovsky, 1991). Endothelin-1 (ET-1) was first isolated from the supernatant of cultured porcine aortic endothelial cells (Yanagisawa et al., 1988), while the existence of ET-2 and ET-3 was predicted following the isolation of genes related to ET-I (Yanagisawa and Masaki, 1989). Another member of the endothelins, known as vasoactive intestinal contractor (VIC), has also been identified (Saida et ai., 1989). Recent studies have demonstrated the presence of endothelins in a variety of tissues, such as glial cells (MacCumber et al., 1990), pituitary (Matsumoto et al., 1989; Yoshizawa et al., 1990) and spinal cord (Giaid et al., 1989). These findings suggest that ET synthesis may not be confined to the endothelium. Four sarafotoxins, SRTX-a, -b, -c and -d, all derived from the native Israeli snake Atractaspis engaddensis, have been characterized so far (Bdolah et al., 1989). The identification of these peptides and the subsequent characterization of their binding sites has generated Abbreviations--DAG, diacylglycerol; DSP, dithiobis (succinimidyl propionate); DSS, disuccinimidyl suberate; ET, endothelin; ET-R, endothelin receptor; IP3, inositol-l,4,5-trisphosphate;LT, leukotriene; LX, lipoxin; PA, phosphatidic acid; PG, prostaglandin; PKC, protein kinase C; PLA2, phospholipaseA2; PLC, phosphoinositide-speciflcphospholipase C; PLD, phospholipase D; SRTX, sarafotoxin; TX, thromboxane; VIC, vasoactive intestinal contractor. 129

M . SOKOLOVSKY

130

ENDOTHELINS

Variable region 1

5

i0

15

20

IASP LYS GLU CYS VAL TYRITYRICYS HIS LEU ASP ILE ILE TRP ASP LYS GLU CYS VAL TYR PHE CYS HIS LEU ASP ILE ILE TRP ASP LYS GLU CYS VAL TYR PHE CYS M S LEU ASP ILE ILE TRP ASP LYS GLU CYS VAL TYR PHE CYS HIS LEU ASP l i e II2 TRP

ET-3

I

ET-I ET-3 VIC

(ET-4)

SARAFOTOXINS

=

CYS SER C Y S ~ A S P

LYS GLU CYS LEU ASN PHE CYS HIS GLN ASP VAL lie TRP ~SRTX-a

FIG. 1. Structure of endothelins (Yanagisawa and Masaki, 1989; Saida et al., 1989) and sarafotoxins (Bdolah et aL, 1989). The shadowed area marks the variable regions of the peptides. intense and widespread study of the physiology and pathophysiology of the endothelin system. Of particular interest are the findings that ET production is not restricted to endothelial cells, that ETs are present in the plasma and that plasma ET concentrations are increased in various pathological states, indicating its potential involvement in diseases. It should be pointed out that the sarafotoxins while homologous with ETs are not endogenous in mammals. Like other peptide hormones and neurotransmitters, ET isopeptides arise through proteolytic processing of isopeptide-specific prohormones. Their precursor, preproendothelin, is a polypeptide consisting of about 200 amino acids that undergo proteolytic cleavage to form a 38 to 39 amino acid peptide designated as 'big endothelin' (Fig. 2) (Inoue et al., 1989). A protease termed ET-converting enzyme then cleaves Trp21-Valn in big ET to form the mature endothelin. Such processing of big ET is essential for the full expression of biological activity (Kimura et al., 1989; Yanagisawa et al., 1989). Ultimately, scientists hope to be able to regulate ET levels in the body. One logical approach is to manipulate the receptors, for example by the use of specific antagonist(s). This appears to be technically difficult, although two recent reports describe the synthesis of such antagonists (Ihara et al., 1991; Spinella et al., 1991). Alternatively, or in addition, inhibition of the enzymes that cleave the precursor seems to be a promising approach. The purpose of this review is to describe recent advances in the biochemical characterization of the ET/SRTX system. We will focus on (1) structure-function relationships of ET/SRTX, (2) ET concentrations in plasma, (3) ET/SRTX receptor subtypes and (4) signaling events mediated by the activation of ET/SRTX receptors.

2. STRUCTURAL ASPECTS Eight naturally occurring peptides of the ET/SRTX family are now known. Although from diverse sources, they exhibit a high degree of sequence homology (Fig. 1). Each one possesses four cysteinyl residues, and about 60-70% of their 21 amino acid residues are identical. Common to

Endothelins, sarafotoxins and receptor subtypes

NH

131

COOH

2 1

20

53

74

92

203 SPECIFIC ENDOPEPTIDASE(S)

NH

COOH

2

53

74

BIG ENDOTHELIN

92 ENDOTHELIN CONVERTING ENZYME

NH 2 ~

53

COOH

ENDOTHELIN

74

FIG. 2. Proposed proteolytic processing pathway for conversion of preproendothelin to endothelin (Yanagisawa et al., 1988, Itoh et al., 1988; Yanagisawa and Masaki, 1989). all of them are: (1) two disulfide bonds, Cysl-Cys ]5 and Cys3-Cysll; (2) a hydrophobic C-terminus (residues 16-21); and (3) three polar charged side chains (residues 8-10). The role of specific amino acids in the functioning of ETs and SRTXs is demonstrated, for example, by the fact that removal of Trp 2~ displaces the EDs0 for contraction by nearly three orders of magnitude (Kimura et al., 1988). The important role of Trp 2~ as the C-terminal residue is also evident from the study of Nishikori et al. (1991), which showed that elongation of the C-terminal by one amino acid results in a derivative with considerably reduced binding affinity and vasoconstrictive potency. Nakajima et al. (1989) concluded that the terminal amino acid and carboxyl groups of Asp 8 and Glu t° and the aromatic moiety of Phe '4 are important for the binding of ET-1 to its receptor. Recently, N'.9-azidobenzoyl-ET-1 was synthesized and purified as a monoreactive affinity-labeling reagent (Kundu and Misono, 1991). The binding properties of this reagent were similar to those of ET-1, indicating that attachment of the azidobenzoyl at the E-amino group of Lys9 does not interfere with the binding of the peptide to the receptor. This is consistent with the earlier report that the substitution of Leu for Lys9 did not affect biological activity (Nakajima et al., 1989). The sequence of the carboxy-terminal tail is conserved and the amino-terminal residue is always Cys, which in SRTX-a, SRTX-b, ET-1 and ET-2 is followed by Ser and in SRTX-c, SRTX-d and ET-3 by Thr. Since these two last amino acids are structurally related, it might be assumed that the presence of one rather than the other does not affect function. It was recently demonstrated (Yellen et al., 1991), however, that in a voltage-gated potassium channel such a change alters ionic selectivity, indicating that even a conservative change might be important in modifying the functions of peptides and proteins, as was indeed suggested by Bdolah et al. (1989) in connection with the SRTX family. The most important differences between the various peptides of the ET/SRTX family are to be found within the sequences of the inner loop Cys3-Cys H. All of these peptides possess Glu ~°, and except for SRTX-c all have AspS-Lys9. The sequences at positions 4-7 therefore represent the variable region (Kloog and Sokolovsky, 1989) of the ET/SRTX peptide family (Fig. 1). Thus, unlike the "constant" C-terminal tail of these peptides, their N-terminal sequences are variable. At least one interesting difference between the various peptides, involving their net charges, appears to derive from variations in the loop created by their disulfide bonds. Thus, SRTX-a,

132

M. SOKOLOVSKY

SRTX-b and SRTX-d (each having two positive and three negative charges) and ET-1 and ET-2 (each with one positive and two negative charges) would all have a net charge of - 1 within the loop, SRTX-c (with four negative charges) would have a net charge of - 4 , and ET-3 (with two positive and two negative charges) would have a net charge of zero. As SRTX-b and ET-1 have similar vasoconstrictive and cardiotoxic effects, which are different from those of SRTX-c and ET-3, it seems that an overall net charge of - 1 within the Cys3-Cys ~ loop is required for the expression of these biological activities. The absence of this single net negative charge in SRTX-c and ET-3 is therefore another common feature of these two peptides (aside from their common Cys~-ThrE-Cys 3) that may contribute to the vasodilatory activity observed at low ET concentrations (Warner et al., 1989; Faraci, 1989). However, the marked difference between the intraloop charges of SRTX-c ( - 4 ) and ET-3 (0) may explain the differences in potencies and/or in mechanisms of binding and second messenger systems between the two peptides, as well as between them and ET-1 or SRTX-b. Kimura et aL 0988) showed that reduction of the four cysteinyl residues lowers the contractile potency of porcine coronary artery strips. Hirata et al. (1989a) suggested that the double cyclic structure of ET-1, formed by Cysl-Cys 15 and Cys3-Cys 11, is critical for the induction of vasoconstriction by receptor binding. Kloog et al. (1988) demonstrated that the reduction of disulfides in SRTX resulted in the loss of binding properties in rat atrium and cerebellum. Hirata et al. (1989b, c) showed that [Cys j-l~, Cys3-tS]SRTX, in which the Cys bonds are "wrongly" connected, is inactive in increasing [Ca2+ ]i while [Cys HS, Cys3-H]SRTX was effective in binding to ET receptors as well as in increasing [Ca2+]~. Kitazumi et al. (1990), in a study of vasoconstriction of rat thoracic aorta for example, showed that reduction of the two sets of intrachain disulfide linkages or the presence of the two non-natural disulfide bonds [Cys 1-11,Cys3-15] in SRTX-b caused disappearance of constrictive activity. These data suggest that the proper intramolecular loop structures are essential in order for the constrictive activity to be expressed. On the other hand, the vasoconstrictive effects of ET-1, and of three analogs in which alanyl residues were substituted for the cysteinyl residues, indicate that the presence of the two disulfide bonds is not essential for ET-1 activity (Randall et al., 1989; Topouzis et al., 1989). Thus, chemical modifications of ET and SRTX, such as reduction of one or both disulfide bridges, cleavage of the intramolecular loop by lysyl endopeptidase or removal/modification of the C-terminal Trp, all lower their vasoconstrictive activity and in several cases also alter their binding characteristics. Two other studies have direct relevance in this connection. In the first, the C-terminal fragment (16-21) of ETs was shown to act as an agonist in the isolated guinea pig bronchus, while it was inactive in the isolated rat aorta (Maggi et al., 1989). In the second, the C-terminal hexapeptide that corresponds to the one in SRTX-b and in SRTX-c, and is very similar to that of endothelins (Fig. 1), was surprisingly devoid of biological activity in the guinea pig bronchus (Rovero et al., 1990). This unexpected observation might be related to the existence of more than one ET/SRTX receptor in this tissue (see below). Proteolytic cleavage of ETs by neutral endopeptidase (Sokolovsky et al., 1990) at AsptS-Ile ~9, resulting in the release of the C-terminal peptide Ile'9-IIC°-Trp2~, renders them biologically inactive as indicated by four assays: binding to the receptor, induction of phosphoinositide hydrolysis, immunogenicity and lethality. This proteolytic cleavage is a two-step process in which the C-terminal tripeptide is cleaved off only after the formation of a nick between positions 5 and 6, suggesting that the ETs assume a compact structure in which the tail is hidden and is unavailable for enzymatic cleavage. This suggestion is supported by NMR studies (Saudek et aL, 1989, 1991; Krystek et al., 1991; Bortmann et al., 1991), all of which describe a well-structured conformation within residues 1-16 and a helical segment between residues 9 and 15. Other NMR studies did not, however, provide evidence of the presumed interaction between the C-terminus of ET-l or SRTX-b and the peptide's N-terminal portion (Mills et al., 1991; Munro et al., 1991; Tamaoki et al., 1991). It should be noted that the proposed conformation (described above) is based on measurements recorded at very high concentrations and often in media containing organic solvents, clearly non-physiological experimental conditions. A further complication arises from the recent observation that in aqueous solutions ET-I, when present at concentrations higher than about


133

0.05 mg/ml, forms aggregates through the formation of micelles (Bennes et al., 1990). As these authors noted, such aggregation highlights the importance of the experimental conditions and may partly account for the conflicting interpretations that exist. Two new compounds, each acting as an antagonist to ET-1, were recently described in two laboratories. Ihara et aL (1991), while screening natural products in an attempt to find a selective ET-antagonist, isolated a novel cyclic pentapeptide from the fermentation products of S t r e p t o m y c e s misakiensis. This peptide, cyclo(-D-Glu-L-Ala-allo-D-Ile-L-IIe-D-Trp), binds to some ET receptors but not to others, i.e. to ETA-R but not to ETB-R (see below). It also antagonizes ET-l-induced vasoconstriction in rabbit iliac artery and pressor action in rats. Spinella et al. (1991) designed and synthesized an ET-1 analog by replacing the Cys~-Cys ~5bond with an amide linkage. As discussed above, several studies have suggested that the Cys~-Cys ~5 bond is more important than the Cys3-Cys II bond for the maintenance of ET activity. In their synthetic peptide, Spinella et al. (1991) incorporated diaminopropionic acid into ET-1 at position 1; this was bound to Asp ~5, which was incorporated into the peptide chain, replacing Cys 15. The resulting analog displayed potent and specific antagonism to ET-1. The specificity of this antagonist is strikingly illustrated by its failure to affect ET-3-induced vasoconstriction of guinea pig lung. Its selectivity might be explained if one assumes the presence of distinct receptor subtypes, as discussed below.

3. PLASMA C O N C E N T R A T I O N S OF E N D O T H E L I N S IN H U M A N S In an attempt to determine whether ET levels are altered in pathophysiological situations, their concentrations were measured in the circulating blood of healthy individuals and of patients with various diseases. This was achieved by the use of a specific radioimmunoassay (RIA) for ET-1, ET-3 and big ET (Ando et al., 1989, Davenport et al., 1990; Togashi et al., 1991; Miyauchi et al., 1991). The values recorded in Table 1 indicate that endothelin is present in normal human plasma at a basal level of around 1.5-2 pg/ml, and at concentrations several times higher in various pathological states. It should however be emphasized that in each case the measured concentration of ET may be an underestimation, for several reasons. (1) Endothelins bind very tightly to their receptors and their dissociation rate is very slow. Thus, the amount of circulating ET may not be TABLE1. Plasma Levels o f Endothelin-1 in Healthy and Diseased States

Normal

Patients

Increase (X-fold) in the diseased state

0.76 + 0.38 (22)

4.95 + 0.8 (22)

6.5

Stewart et al. (1991a)

i.5 + 0.3 (11)

3.7 _ 0.9 (6)

2.5

Miyauchi et al. (1989)

1.5 _ 0.07 (40)

3.3 (12)

2.16

Toyo-oka et al. (1991)

0.26 _ 0.23 (14)

3.65+ 1.14 (6)

--

Cernacek and Stewart (1989)

1.5 ___0.3 (14) < 1.8 (10) < 3.5 (19)

1.88 _ 0.32 (22) 10.9 + 3.4 (24) 12.3 _+ 5.5

1.3 6 3.5

Yamane et al. (1991) Totsune et al. (1989) Koyama et al. (1989)

1.5 ___0.5 (16)

9.1 + 3.2 (12)

6

Masaoka et al. (1989)

2.4

Stewart et al. (1991b)

4.4 1.86

Ziv et al. (1992) Taylor et al. (1990); Schiff et al. (1991)

Levels of ET-I (pg/ml) (n*) Disease Myocardial infarction Myocardial infarction Vasospastic angina pectoris Cardiogenic shock Raynaud's phenomenon Hemodialysis Uremia Subarachnoid hemorrhage Pulmonary hypertension Acute ischemic cerebral stroke Preeclampsia

1.45 _ 0.45 2.6+0.1 (13) 6. I + 0.7 (10)

*n = number of patients.

3.5 _ 2.5 11.5-1-1.7(16) 1i.4 _ 0.9 (10)

Reference

134

M. SOKOLOVSKY

an accurate reflection of the biological activity of the system. (2) Increases in local concentrations of ET in specific target organs may be greater than in the plasma. (3) Big ET-1, which is functionally a much less active but metabolically more stable form, is cosecreted and can be converted into ET-1. (4) The extensive local removal of endothelins clearly limits the amount of relevant peptides that might escape local degradation, and may thus give rise to false negative results. Attempts to correlate plasma concentrations with data obtained from in vitro studies have so far met with difficulties; for example, the highest plasma level measured to date, i.e. 12-15 pg/ml, is below the threshold estimated to induce vasoconstriction in vitro (Costello et al., 1990). The question then arises: does interference with the action of endothelins protect against the increase in level of ET observed in various pathological states, as recorded in Table 1? Obviously, specific antagonists would be helpful in addressing this question. Furthermore, it will be difficult to distinguish the role of the circulating peptide from that of the locally formed one. Moreover, there is no evidence associating increased ET concentrations (Table 1) in humans with biological activity; obviously, experimental investigation of such a correlation is subject to limitations of an ethical nature in human subjects. The functional significance of elevated plasma ET in patients can therefore at present only be speculated on. However, an important step was recently taken by Vierhapper et al. (1990), who investigated the effects of exogenous application of ET- 1 in humans. A rise in serum ET concentrations and a concomitant rise in blood pressure and serum potassium concentrations were observed, clearly indicating that an increase in circulating endothelins might be associated with biological activity. It should be noted that the doses used in this human study (carried out with six healthy men) were well below those used in experiments with animals. In an important recent study Lerman et al. (1991) investigated the biological action of endothelin on dogs through administration of exogenous ET in amounts causing a two-fold increase in plasma ET, i.e. equivalent to the reported increase in various pathological conditions (Table 1). They observed a decrease in cardiac rate and output, in association with increased renal and systemic vascular resistance and antinatriuresis. These findings are consistent with a functional role for endogenous ET as a potentially pathogenic vasoconstrictor peptide hormone in the regulation of cardiovascular, renal and endocrine function (Lerman et al., 1991).

4. RECEPTOR SUBTYPES Since the discovery of ET/SRTX receptors, numerous radioligand binding studies have been undertaken in an attempt to characterize them. A diversity of molecular, pharmacological and functional data were obtained, suggesting the existence of several ET/SRTX receptor subtypes. To date, as discussed in detail below, two molecular species of ET/SRTX receptors have been cloned and expressed (Arai et al., 1990; Sakurai et al., 1990; Lin et al., 1991; Ogawa et al., 1991; Sakamoto et al., 1991; Nakamuta et al., 1991; Hosoda et al., 1991). It is, however, difficult to assign a well-defined pharmacological profile to each of the molecular species, since the binding properties of ET/SRTX receptor subtypes are similar and there is as yet no known drug that can be used to identify a specific subtype. Traditionally, receptors are classified on the basis of their differing affinities for antagonists. However, unlike other receptor systems (e.g. muscarinic, see Sokolovsky (1989) for review) the ET/SRTX system lacks a selective group of antagonists and, as discussed above, the first two specific antagonists were only recently described. As a result, identification of these receptor subtypes has had to depend on the use of agonists. In addition, the binding properties of receptors are probably sensitive to their environment, so that the binding properties of a cloned receptor transfected into a cell line may be different from those observed in vivo or in vitro (see for example Pinkas-Kramarski et al., 1988). Since every methodological approach, including in vivo experimentation in animals, has its own advantages and limitations arising from the biological complexity of the specific experimental model used, one would expect a consistent picture to emerge from each one. In the following we discuss some of the technical problems associated with these experiments and describe the pharmacological, functional and molecular activities that indicate heterogeneity among the ET/SRTX receptors.


135

In most of the studies reported so far, a single class of high-affinity binding sites for [J2~I]-ET-1 was identified. In several cases, however, two sites were observed. For example, the binding isotherm obtained from [t25I]-ET-1 binding to membranes prepared from rat aorta was best fitted by a two-site model with Kd values of 0.47 + 0.35 nM and 93 + 80 nM and a relative abundance of 33% and 66%, respectively (Jones et al., 1991). Emori et al. (1990) recently reported the presence in cultured bovine endothelial cells of two distinct subclasses of ET-3 receptors with high (7 pM) and low (250 pM) affinities. Also in the rabbit uterus two populations of binding sites for ET-1 were noted (Maggi et al., 1991), one of them (1 pmol/mg protein, Kd = 100 pM) having a rank order of potency ET-I = ET-3 = SRTXs, and the other (10 pmol/mg protein, Kd = 400 pM) having the rank order ET-1 > SRTXb --- ET-3. In contrast, in the rat uterus [125I]-ET-1 was bound to a single site of 170 + 50 fmol/mg protein and Kd = 0.12 + 0.03 nM (Bousso-Mittler et al., 1989; Hagiwara et al., 1990). Significant differences in the dissociation constants of [125I]-E-1 were reported for example in rat cerebellar and cardiac preparations, with values ranging from 2-1900 pM (Williams et al., 1991; Hiley et al., 1990; Jones et al., 1991; Bousso-Mittler et al., 1991) and from 1-1900 pM (Williams et al., 1991; Gu et al., 1989; Bolger et al., 1990). These variations in binding characteristics might result from one or more differences in experimental protocol. For example: 1. Differences in tissue preparation, e.g. subcellular fractions vs cell cultures. Differences might also be detected within similar preparations; as an example, Kanba et al. (1990) demonstrated that agonist-induced down-regulation of muscarinic receptors in cell suspensions differs significantly from that in monolayer cultures of the same cells. 2. Differences in purity of the synaptosomal preparations used. Binding studies employing preparations of high purity are likely to yield high Bmaxvalues expressed in mol/mg protein. 3. Variations in receptor-concentration as well as in the ratio of receptor to ligand. 4. Variations in ligand concentration. It should be noted that when high concentrations of ligand are used, the non-specific binding is likely to increase beyond acceptable values. As an example, if the Kd is above 50 riM, it will be necessary to use ligand concentrations higher than 10 -7 M, an experimental condition in which the non-specific binding will be unacceptably high for practical purposes. 5. Degradation. Both the ligands and the receptors might be susceptible to the action of various endogenous proteases, necessitating the use of a cocktail consisting of several protease inhibitors. Different cocktails are used by different laboratories. Other factors may induce variation, e.g. proteolysis is more pronounced at 37°C than at lower temperatures, and the process might be more active in some tissue (e.g. rat atrium) than in others (e.g. rat cortex). 6. Possible creation of artifacts as a result of trapping ligand within the cell or tissue slices. This might explain the observation of Devesly et al. (1990) that there is a 20-fold difference between the affinities of ET-1 and ET-2 for Swiss 3T3 cells at 4°C and at 37°C. Hirata et al. (1988) suggested that ET might be trapped within the intramembranal lipid environment. Trapping of the ligand, together with the extremely slow dissociation of ET from the receptor (Bolger et al., 1990; Galron et al., 1991; Devesly et al., 1990), may explain, at least in part, why the kinetic profile of ET appears to be complex and irreversible. It may also explain its prolonged mode of action in several isolated tissues and in vivo. 7. Variations in buffer composition and pH, the presence or absence of BSA, chelators, Mg 2÷, Ca 2÷ and other cations (Bolger et al., 1990; Maggi et al., 1991). The pH-profile of [~25I]-ET-I binding indicated that the optimal pH for binding was between 6 and 7 in rat ventricular membranes (Bolger et al., 1990) and between 4 and 6 in cardiac tissue (Liu et al., 1990). In the former preparation, the decrease in binding on the alkaline side of the optimum, i.e. at pH 7-10, was not as marked as it was on the acidic side, i.e. at pH 4-6. In the second preparation there was a dramatic decrease in binding on both sides of the optimum pH. Thus, the use of neutral pH seems to be adequately suited to binding studies, at least under the conditions employed in these two studies. Binding studies using [~25I]-labeled ETs and SRTX-b identified specific saturable high-affinity binding sites in a variety of tissues (e.g. brain, heart, lung, kidney, liver, intestine, from humans and other mammals, i.e. rats, pigs, dogs. etc.) (Kloog and Sokolovsky, 1989; Galron et al., 1989; Nayler, 1990; Marsault et al., 1990; MacCumber et al., 1990; Hemsen et al., 1990; Henry et al.,

136

M. SOKOLOVSKY

1990; Loftier and Lohrer, 1991; Jones et al., 1991). Membranes, isolated cells, or whole tissue slices were used in these studies. Localization of ET-1 by binding studies as well as by in vitro a u t o r a d i o g r a p h y showed that in h u m a n brain the highest concentration appears to be in the cerebellum and h i p p o c a m p u s (Jones et al., 1989). In the rat the pattern of binding-site distribution (highest to lowest) was: cerebellum - liver - h y p o t h a l a m u s > h i p p o c a m p u s ~_ lung > heart ~ kidney (Kloog and Sokolovsky, 1989; Jones et al., 1989, 1991; Neuser et al., 1991; K o h z u k i et al., 1991). similarly, in the dog the pattern was cerebellum ~ lung -~ spleen > liver > kidney > heart (Loftier and Lohrer, 1991). In spite o f the marked structural similarities between ETs and SRTXs, some differences were noted between the binding o f the various peptides to their receptors in different tissues (see Table 2). This led to the suggestion that multiple receptor subtypes exist for these peptides (Kloog et aL, 1989b; M a g g i e t al., 1989; Badr et al., 1989; Fu et al., 1989; R o v e r o et al., 1990; Jones et al., 1991; Loftier and Lohrer, 1991). Our initial binding experiments, performed with two ETs (l and 3) and two S R T X s (b and c) and using membranal preparations o f rat atrium, aorta, uterus, cerebellum and caudate putamen, indicated heterogeneity o f E T / S R T X receptors. The evidence pointed to the existence o f at least three subtypes: a high-affinity subtype for ET-1 and S R T X - b (c¢), a high-affinity subtype for S R T X - c (fl), and a less selective type that binds all o f these peptides with high affinity (7). Other studies (Williams et al., 1991; Schvartz et al., 1991) confirmed and extended these findings. Loftier and Lohrer (1991) suggested the existence o f at least four subtypes.

TABLE2. Rank Order o f Potency o f E T / S R T X Peptides Determined Either From Direct Binding o f [12sI]-ET - I or [12sI]-SRTX-b or by Competitive Binding o f These Ligands to Their Receptors in Various Tissues

Receptor subtype

Tissue

Rank order

Astroglia (mice) (rat)

ET-l ~-ET-3 ET-1 -~ ET-2 _> ET-3

Aorta (rat)

ET-1 - SRTX-b > ET-3 > SRTX-c ET-1 - SRTX-b > ET-3 ET-1 > ET-3

ET A ETA(?) ETA

Kloog et al. (1989b) Williams et al. (1991) Jones et al. (1991)

Atrium (rat)

ET-I _~ SRTX-b > ET-3 > SRTX-c ET-1 _~ SRTX-b > ET-3 > SRTX-c

ET A

Kloog et al. (1989b); Galron et al. (1989) Williams et al. (1991); Hemsen et al. (1990)

ETB

Kloog et al. (1989b)

(human)

ETa

ET-I --, ET-2 ~ SRTX-b > ET-3

Caudate putamen (rat) ET-1 _~ SRTX-b -~ SRTX-c ~ ET-3 Cerebellum (rat)

SRTX-c = SRTX-b > ET-1 = ET-3 ET-3 ~- SRTX-b > ET-1 -~ ET-3 ET-1 ~ ET-3 ~- SRTX-c

Reference MacCumber et al. (1990); Ehrenreich et aL (1991)

ET x and ET A Hiley et al. (1990) ET x Williams et al. (1991) Kloog et al. (1989b); ET 8 Kloog and Sokolovsky (1989)

Hippocampus (rat)

ET-1 - SRTX-b = ET-3

Ileum (guinea pig)

ET-1 ~- SRTX-b -~ ET-3 - VIC ~- SRTX-c

ET B

Wolberg et aL (1991)

Kidney (rat)

ET-1 ~_ ET-3

ET a

Jeng et al. (1990)

Liver (rat)

ET-1 _~ ET-3

ETa

Jeng et al. (1990); Serradeil le Gal et al. (1991)

Lung (human) (rat) (guinea pig)

ET-1 --, ET-2 -~ SRTX-b > ET-3 ET-1 > ET-3 ET-l -~ ET-2 -~ ET-3

ET A ET A ETa

Hemsen et al. (1990) Jeng et aL (1990) Pons et al. (1991)

Uterus (rat)

SRTX-b - ET-l > ET-3 > SRTX-c

ETA

ET-1 - SRTX-b -~ ET-3 ET-1 > SRTX-b > ET-3

ETa ETA

Bousso-Mittler et al. (1989); Hagiwara et al. (1990); Maggi et al. (1991); Maggi et al. (1991)

(rabbit)

ET a

Kloog and Sokolovsky (1989) Williams et al. (1991);

ETA-selective subtype, ETs-non-selective subtype, ETx-subtypes other than ET A and ETB.


137

The findings of Eta and Triggle (1991) suggest that ET and SRTX activate different cell-signaling systems to different extents in rat aorta and anococcygeus, indicating that the receptors mediating the responses to ET and SRTX in these two tissues are not identical. An interesting observation, consistent with the above, was that of Rovero et al. (1990), who showed that the hexapeptide (16--21) corresponding to the C-terminal portion of SRTX, unlike the C-terminal portion of ET, was devoid of biological activity in the guinea pig bronchus--again indicating the possible existence of more than one receptor for the ET/SRTX family. We have designated different subtypes by the Greek letters, ~, fl and 7, a nomenclature used for several other enzymatic and receptor systems. Maggi et al. (1989) have suggested the use of ET A (A for aorta) and ET B (B for bronchus). According to the IUPHAR recommendation, receptors showing rank orders of affinity of ET-1 - ET-2 > ET-3 are called ETA-R, while those sharing similar affinities (i.e. less selective ones) are called ETB-R (Table 2). It should be pointed out that in addition to differences in ligand-receptor interaction, other differences--possibly arising in the signaling pathways--also exist between these receptors. As an example, Galron et al. (1990) showed that different pathways of ET-1 and SRTX-b stimulate phosphoinositide hydrolysis in rat heart myocytes, while Henry et al. (1990) demonstrated the possible existence of an interspecies difference in the receptor-effector system for ET-1.

5. KINETICS OF BINDING One of the characteristics of ET/SRTX binding in the various tissues investigated is the extremely slow rate of dissociation of receptor-ligand complexes. In rat vascular smooth muscle cells, for example, binding was almost irreversible (Hirata et al., 1988). The half-life of circulating endothelins for example in rat kidney is about 2 min, in marked contrast to the long-lasting functional effect. This apparent discrepancy may be at least partly due to the extremely slow rate of dissociation. Kinetic studies of ET-1 and ET-2 receptor interaction in Swiss 3T3 fibroblasts revealed different rates and extents of dissociation for the two isoforms (Devesly et al., 1990). The dissociation of bound [125I]-labeled ET-1, ET-3 and SRTX-b from their preformed receptor-ligand complexes was recently studied in two membranal preparations, rat cerebellum and guinea pig ileum (Galron et al., 1991). The dissociation rates for all three ligands were significantly lower in the cerebellar than in the ileum preparations. In addition, in both preparations the rates of dissociation of ET-3 from the receptor were significantly slower than those of ET-1 or SRTX-b. The tl/2 values in the ileum are about 18 min for ET-1 and 7min for SRTX-b, in contrast to more than 2 hr for ET-3; in the cerebellum the t,/2 values are more than 2-3 hr for SRTX-b and ET-1, while for ET-3 the rate of dissociation from the receptor is negligible. The fact that for each of the three ligands the dissociation rate in the cerebellar preparation was significantly different from that in the ileum supports the suggested existence of different receptor subtypes in these two regions. Alternatively, these findings might be explained in terms of interactions of the same receptor with different membrane components in the different tissues. Also worth noting are the differences in dissociative behavior between ET-3 vs ET-1 and SRTX-b in both the cerebellum and the ileum. These differences could stem from dissimilarities in the nature of the receptor-ligand complexes (i.e. different modes of binding) (Galron et al., 1989).

6. COVALENT LABELING OF ET/SRTX RECEPTORS Affinity labeling with bifunctional cross-linking reagents has proved to be an extremely useful procedure in the biochemical and pharmacological analyses of many receptors, especially when the questions to be addressed concern multiple receptor subtypes. As shown in Table 3, different laboratories report specific labeling of proteins with different molecular masses. The labeled proteins can be grouped into several classes, of molecular mass 70, 50, 42-44 and 32-34 kDa. There are several possible reasons which, alone or in combination, might account for differences in labeling patterns, among them: (1) proteolysis (2) the use of different cross-linking reagents, e.g. disuccinimidyl suberate (DSS) or dithiobis (succinimidyl propionate) (DSP). In DSP a S-S bond

138

M. SOKOLOVSKY

TABLE3. C r o s s - L i n k i n g o f Endothelins to Their Putatioe Receptor(s) Molecular mass of the [12sI]-labeled proteins (kDa) Preparation Chick heart Rat lung Rat mesangial cells Human placenta Rat cerebellum, cortex, caudate putamen Rat A10 VSMC* C6 glial cells Osteoblastic cells (rat calvanae) Guinea pig ileum Rat atrium Bovine aorta Rat brain Bovine atrium

ET- 1 50 44 58, 34 32 50

ET-2 44

73, 60 70 70 70 43 52, 30 50

ET-3 34, 46 32

References

50, 38

Watanabe et al. (1989) Masuda et al. (1989) Sugiura et al. (1989) Nakajo et al. (1989) Ambar et al. (1990)

60

Martin et al. (1990)

70

Takuwa et al. (1990) 70 70 50

Galron et al. (1991) Glaron et al. (1991) Bender et al. (1991) Schvartz et al. (1990) Schvartz et al. (1991)

*Vascular smooth muscle cells.

replaces the CH2-CH 2 bond present in DSS. The resulting small difference in molecular size, together with the structural differences between e.g. ET-I and ET-3, might give rise to differences between the two ligands in the proximity of their reactive components, thus in turn yielding different patterns of labeling. (3) The use of different membrane preparations; as an example, Ambar e t al. (1990) used homogenates, Watanabe e t al. (1989) used cardiac membranes from newborn chicks prepared by sucrose density gradient centrifugation, while Sugiura e t al. (1989) used a microsomal fraction prepared from cultured rat mesangial cells. (4) The use of different tissues, e.g. rat brain as compared to peripheral tissues such as chick cardiac preparations or rat mesangial cells. (5) Inter-species differences (compare rat atrium with bovine atrium, Table 3). It should be noted that these labeled proteins (Table 3) were the major species detected. In addition, multiple minor bands have frequently been observed. The nature and origin of these minor labeled bands are not known, but can probably be explained in terms of proteolysis, tissue differences and/or other technical variations. Several authors have mentioned the possibility that the low molecular weight peptide, i.e. ca. 30 kDa, is a proteolytic degradation product. For example, comparison of the peptide maps of the 52 and 30 kDa endothelins from rat brain revealed that the latter is a proteolytic product of the former (Schvartz e t al., 1990). Purification of bovine lung receptor by affinity chromatography followed by microsequencing of tryptic fragments (Kozuka e t al., 1991) revealed the presence of ETB receptors (see below). Purification of the receptor in the presence of low (1 mM) and high (50 mM) concentrations of EDTA yielded a 34 kDa and a 52 kDa species, respectively, as a major form, indicating that the former polypeptide is a proteolytic product of the latter. Recently, affinity labeling of ET-1 receptors in bovine and rat lung membranes by W'9-azidobenzyol-[~zsI]-ET-I yielded a radiolabeled protein band with an apparent Mr of 34 kDa in both preparations (Kundu and Misono, 1991). These authors argue that under their experimental conditions (0.05% bacitracin, longer incubation time, etc.) receptor proteolysis seems unlikely. However, in view of the absence of EDTA from the medium as well as the cloning data (see next section, Table 4), it appears that proteolysis should probably not be discounted. As shown in Table 3 in rat cerebellum, cortex and caudate putamen, ET-I specifically labeled a major component with a molecular mass of around 50 kDa. In the same tissues ET-3 specifically labeled, in addition to the 50 kDa, a 38 kDa band. This result indicates that in the rat brain the endothelin binding site resides within a polypeptide with an apparent Mr of 50 kDa and that the 38 kDa polypeptide is labeled exclusively by ET-3. Since all the experiments were conducted under similar experimental conditions (Ambar e t al., 1990) it seems less likely that the 38 kDa polypeptide


139

TABLE4. Molecular Cloning o f Endothelin Receptor Subtypes (ET-R) cDNA library Rat lung Bovine lung Rat A-10 VSMC* Human liver Human placenta Human placenta Human jejunum

Amino acid composition 415 427 426 442 442 427 416

Rank order of potency ET- 1 "-~ET-2 -'- ET-3 ET-1 > ET-2 > SRTX-b > ET-3 ET-1 = ET-2 > ET-3 ET-1 = ET-2-,~ ET-3 ET-1 = ET-2-,~ ET-3 ET-1 > ET-2 > ET-3 ET-1 ~ ET-2 = ET-3

Type of receptor ET B ETA ETA ET B ET B ETA ETB

References Sakurai et al. (1990) Arai et al. (1990) Linet al. (1991) Nakamuta et al. (1991) Ogawa et al. (1991) Hosoda et al. (1991) Sakamoto et al. (1991)

*Vascular smooth muscle cells. represents product of proteolysis, although one cannot completely discount the possibility for example that binding of ET-3 to the receptor sensitizes it to proteolysis more than e.g. ET-1. As reported by Watanabe et al. (1989) for chick cardiac membranes, [~25I]-ET-3 labels two major bands and one minor band with Mr values of 34, 46 and 50 kDa, respectively. Thus, in contrast to labeling with ET-1, the results obtained with ET-3 for chick cardiac membranes differ from those obtained for rat brain tissues. Recently, Schvartz et al. (1991) labeled bovine atrial membrane preparation with [125I]-ET-1 and [~25I]-ET-3, and this resulted in the labeling of a single 50 kDa band. The cross-linked receptors yielded different peptide maps. These results, together with previous binding data (Kloog et al., 1989b) indicating distinct binding properties exhibited by ET-1, ET-3 and SRTXs in the atrium (which was also confirmed by Schvartz et al., 1991), can be explained in terms of the existence of multiple receptor subtypes.

7. C L O N I N G A N D EXPRESSION OF ET RECEPTORS Several laboratories have recently succeeded in cloning, sequencing and functionally expressing receptors for ETs (see Table 4 for refs). The expression was carried out in COS-7 cells in all of these studies except that of Lin et al. (1991), who expressed the receptors in CHO-KI cells, and Hosoda et al. (1991), who in addition expressed the receptor in Xenopus oocytes. These findings clearly lend further support to the existence of several endothelin receptor subtypes. Sequence analysis of the various cDNA clones predicted that the encoded receptor polypeptides are composed of roughly the same number of amino acid residues (Table 4), ranging from 415 to 442, and of a molecular mass ranging from 46,900 to 49,629, which is consistent with the values of around 50 kDa obtained by affinity cross-linking (Table 3). To date, two subtypes have been identified; however, as discussed above, binding studies predict the presence of at least three or four. Indeed, Sakamoto et al. (1991) have postulated the existence of a third endothelin receptor gene and predicted that its sequence will have a low degree of similarity to those of the two known receptor genes. Hydropathicity analysis of the amino acid sequences of the cloned receptors indicates that all of them (Table 4) contain seven hydrophobic clusters of 22-26 residues each, separated by stretches of hydrophilic residues. It thus appears that the cDNA encodes a protein with seven membrane domains, an extracellular tail and a cytoplasmic C-tail, consistent with the superfamily of G-protein-receptor complexes. As noted in all the reports on the subject, there are several potential sites for post-translational modifications: (1) Consensus sites for N-glycosylation (Asn-X Ser/Thr); two such sites, Asn 29 and Asn 62, exist in rat and bovine lung receptors but only one, Asn 5°, in the human receptor. In other non-endothelin receptors the number of potential glycosylation sites ranges from two to five. (2) Six cysteinyl residues in the C-terminal cytoplasmic domain (including a common sequence of Cys-Leu-Cys-Cys-Cys-Cys), one of which may be palmitoylated as in the case of the ~-adrenergic receptor (O'Dowd et al., 1989), and several Ser residues in the third cytoplasmic loop and the C-terminal tail, which may potentially serve as substrate sites for Ser/Thr protein kinase (Kemp and Pearson, 1990). The N-terminal segment exhibits some interesting features. One of these is the presence of a putative N-terminal signal sequence (Sakurai et aL, 1990; Lin et al., 1991). As noted by Saudou

140

M, SOKOLOVSKY

et al. (1990), this feature has thus far been associated only with receptors containing a long extracellular tail. All the sequences of the receptor indicate the presence of a relatively long and proline-rich putative N-terminal region. From a comparison of the seven amino acid sequences it is clear that there are major differences in the N-terminal segments, especially between ETA-R and ETa-R; in the former, irrespective of tissue source (rat, human, etc.) the N-terminal portion is composed of 80 residues, while in the latter the corresponding segment comprises 101 residues, and there is little homology between the two. It was suggested for fl-adrenergic receptors (Dohlman et al., 1990) and for peptide hormone receptors (Moyle et al., 1991) that in addition to the well-documented involvement of the membrane-spanning domains of receptors in ligand binding, an important role is played by the hydrophilic extracellular domains in forming the ligand-binding site. Thus, it is possible that the N-terminal region of the ET-R may be involved in ligand binding and therefore confers ligand selectivity. Nearly all ET receptors contain the conserved sequence Asp-Arg-Tyr which, as pointed out by Lin et al. (1991), is present in 66 of 80 nonendothelin receptors and has been shown to be important for coupling of receptors to G-proteins (Franke et al., 1990). Moreover, inspection of the published sequences indicates that the position of this tripeptide in the sequence is typical of the specific subtype irrespective of its source. Thus, in ETA-R its position is at residues 182-184, while in ETB-R it is found at 198-200 (in rat lung it is at 197-199). It is interesting to note that modification of the rat cerebellar ET receptor with tetranitromethane implicated a tyrosyl residue(s) in the binding site (M. Sokolovsky, unpublished results). The location of this residue(s) is unknown, though it is tempting to speculate that the nitration reaction is facilitated by interaction of the aromatic ring of the tyrosyl residue with other residues, i.e. through hydrogen bonding with an aspartic or glutamic acid residue. Obviously, such interaction between Tyr and Asp could occur between residues in the same or in different transmembrane domains, and may play a role in ligand binding. Previous results of nitration with tetranitromethane pointed to the possible role of a tyrosyl residue(s) in the binding of ligands to muscarinic receptors (Gurwitz and Sokolovsky, 1985) and to ~2-adrenoceptors (Nakata et al., 1986). In an attempt to determine whether the ET/SRTX receptors are glycoproteins, we examined the ability of neuraminidase (EC.3.2.1.18), an exoenzyme that serially cleaves glycosidically bound sialic acid residues, to induce an enzymatic deglycosylation process in the receptor (Bousso-Mittler et al., 1991). Incubation of rat cerebellar membranes with the enzyme was accompanied by a reduced ability of the receptors to bind ET-1 and SRTX-b. In contrast, no effect on binding or on molecular mass was observed when rat caudate putamen was used. Thus, the receptors of rat cerebellum appear to be glycoprotein, and the presence of the sugar moiety is a prerequisite for binding of the ligands to their receptors. The finding that the cerebellar receptor corresponding to the ETB-receptor appears to be a glycosylated protein agrees with the observation that the cloned receptors possess potential N-glycosylation sites, and is also consistent with their tissue distribution. The observed rank order of potency of the ET/SRTX receptors characterized them as belonging to the ETB-R type. However, in spite of their similar binding profiles, the data indicate that the susceptibility of ET/SRTX receptors to deglycosylation in the cerebellum differs from that in the caudate putamen, suggesting that the ETB-R might represent heterogeneous rather than a homogeneous family of receptor subtypes. One should, however, note the possibility that the receptors of e.g. the caudate putamen are not sialoglycosylated proteins but are glycosylated by other carbohydrates, and/or that they are organized in the membrane in such a way that their sugar moieties are inaccessible to neuroaminidase. These possibilities preclude definite conclusions with respect to heterogeneity among the same ET/SRTX receptor subtypes.

8. TISSUE DISTRIBUTION OF THE RECEPTOR mRNA Two major forms of human ETB-R mRNA (4.3 Kb and 1.7 Kb) were expressed in a wide variety of human tissues (Ogawa et al., 1991). The highest levels were detected in the brain, i.e. in the cerebral cortex and cerebellum. Moderate levels were found in placenta, lung, kidney, adrenal gland, colon and duodenum. Northern blot analysis of human ETA-R mRNA (4.3 Kb) (Hosoda et al., 1991) showed its highest abundance in the aorta, a high level in the lung, atrium colon and

141


placenta, and moderate levels in cerebral cortex, cerebellum, heart ventricle, kidney, adrenal gland and duodenum. In the rat, Northern blot analysis of ETA-R mRNA demonstrated its highest levels in the lung and the heart with lower amounts in brain, muscle and kidney (Lin et al., 1991), indicating that the tissue distribution of ETA-R was similar in the two species. As pointed out by Hosoda et al. (1991), their cloned human ETA-R is the major ET receptor subtype expressed in vascular smooth muscle cells, and they therefore suggest that ET-1 released from the endothelial cells (in which there is no hybridization signal) binds to the receptor in vascular smooth muscle cells and acts as a local regulator in cardiovascular homeostasis.

9. ENDOTHELIN/SARAFOTOXIN SIGNAL TRANSDUCTION The diversity of action of the endothelins may be explained in terms of (1) the existence of several-receptor subtypes and/or (2) the activation of different signal pathways, such as those involving PLC, PLA2 and PLD (Fig. 3, Table 5). Other possible pathways are via Na+/H ÷ exchange (Simonson et al., 1989; Kramer et al., 1990; Vigne et al., 1991; Battistini et al., 1991). In addition, ET-induced cAMP formation (Abdel-Latif and Zhang, 1991) was observed in one specific system, namely, the iris sphincter of various species including rabbit, dog, cat, cow, monkey and human. The latter is so far the only positive report with regard to the involvement of cAMP formation; all other studies dealing with signal transduction has indicated negative results. It should be pointed out that the accumulation of cAMP might have resulted from the stimulation of adenylate cyclase not directly but rather indirectly via the activation of PLC (Felder et al., 1989; Tomita et al., 1990), or like in the rat Glomerular mesangial cells in which ET-I amplified fl-adrenergic mediated cAMP accumulation by a PGE2-dependent mechanism (Simonson and Dunn, 1990b). A large body of accumulated evidence indicates that endothelins and sarafotoxins act as neuropeptides which, via their receptor, activate the phosphatidylinositol 4,5-bisphosphate (PIP2)specific phospholipase C (PLC) (Auguet et al., 1988; Ambar et al., 1988, 1989; Resink et al., 1988; Van Renterghem et al., 1988; Bousso-Mittler et al., 1989; Galron et al., 1989; Kai et al., 1989; Marsden et al., 1989; Vigne et al., 1989). Activation of PLC by hormone or neurotransmitter leads to the cleavage of membrane-bound phosphoinositides into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), each of which subsequently activates separate signal transduction pathways.

MEMBRANE ou

I

r

RECEPTOR

IN

~_~ ARACHIDONICACID alP3 Cyclooxygonaee Products

Lipoxygenase Products

(PGTX)

(LTLX) j ~i

/

I

~ DAGq

1! N./H" PORTER

T

PA

P!C

[PHOSPHORYLATIONEVENTSl

FIG. 3. Schematic representation of signal transduction pathways following activation of the ET/SRTX receptor.

JPT 54/2--B

M. SOKOLOVSKY

142

TABLE5. Signaling Events I n d u c e d by Activation o f the E T / S R T X Receptors cAMP Na+/H ÷ Tissue PLC PLA2 PLD formation exchange Reference Filep et al. (1990); Airways (guinea pig) + + Pons et al. (1991); Battistini et al. (1991) Anterior pituitary cells (rat)

Stojikovic et al. (1991); H. Lewy, R. Galron, A. Bdolah, M. Sokolovsky and Z. Naor, submitted

+

+

Kai et al. (1989); Van Renterghem et al. (1989); Konishi et al. (1991)

Aortic SMC* (bovine and rat)

+

+

Aortic VSMCt (rabbit)

+

Resink et al. (1988, 1989); Marsden et al. (1989); Reynolds and Mok (1990)

Atrium (rat)

+

Kloog et al. (1988, 1989a,b); Ambar et al. (1989); Vigne et al. (1989) Kloog et al. (1988, 1989a,b); Kloog and Sokolovsky (1989)

Brain (rat) + (various regions, e.g. putamen, hypothalamus etc.) +

Brain capillary endothelial cells Cardiocytes (rat)

Hirata et al. (1989a); Galron et al. (1989)

+ +

Eye (rabbit) Iris sphincter (rabbit)

+

+

Mesangial cells (rat)

+

+

Granstam et al. (1991) +

+

Abdel-Latif and Zhang, (1991) Abdel-Latif et al. (1991) +

+

Fibroblasts (rat-l) Uterus (rat)

Vigne et al. (1991)

Simonson et al. (1989); Simonson and Dun (1990b) MacNulty et al. (1990); Bousso-Mittler et al. (1989)

*SMC--smooth muscle cells. tVSMC--vascular smooth muscle cells.

Diacylglycerol activates protein kinase C (PKC), while IP3 triggers the rapid mobilization of intracellular Ca 2÷ (as reviewed by Nishizuka, 1988). Increases in intracellular Ca 2÷ can directly activate Ca2+-dependent protein kinases as well as Ca2+-sensitive PLC and phospholipase A2 (PLA2), leading to the release of arachidonic acid. Recent investigations of the signaling cascade in mesangial and vascular smooth muscle cells showed that following IP 3 formation, which results in mobilization of [Ca 2÷ ]~, activation of chloride channels occurs (Iijima et al., 1991). The ensuing C1- efflux cause membrane depolarization and, in turn, activation of voltage-dependent Ca 2÷ channels, resulting in sustained elevation of [Ca 2÷ ]i. It should be noted that in several of the reports cited in Table 5, stimulation of PLC activity by endothelins and/or sarafotoxins occurred at higher concentrations than the Kd values for their binding. One possible explanation for this inconsistency might be that only a fraction of the receptors are coupled to PLC. In addition, or alternatively, the reaction may involve a specific


143

receptor subtype, or the activation of more than one receptor might be necessary in order to obtain the response. Endothelin has also been reported to activate phospholipase A2 in several tissues, leading to the release of arachidonic acid. This is then metabolized to prostaglandins, thromboxanes and leukotrienes (Resink et al., 1988; Marsden et al., 1989; Reynolds and Mok, 1990; Pons et al., 1991), suggesting the possibility that eicosanoid metabolites of arachidonic acid may play an important role as second messengers in mediating some of the biological activities of endothelins. It should be noted that two mechanisms might be responsible for the induced generation of arachidonic acid by endothelins in various cells: (1) activation of PLA2 and direct formation of arachidonic acid; and (2) an indirect pathway via PLC. The former mechanism elevates [Ca2÷ ]i, thereby activating Ca2+-sensitive PLA 2, while the latter stimulates production of e.g. DAG, which might be a substrate for lipases producing arachidonic acid (for review see Burgoyne and Morgan, 1990; Naor, 1991). Phospholipase D (PLD) hydrolyz~s phospholipids such as phosphatidylcholine and phosphatidylethanolamine, producing phosphatidic acid and releasing the free polar head-group, e.g. choline and ethanolamine. Recent evidence suggests that the activation of PLD by neurotransmitters, hormones and growth factors may represent a novel and ubiquitous signal transduction pathway in mammalian cells, mediated by accumulation of phosphatidic acid and/or diacylglycerol (reviewed by Dennis et al., 1991; Thompson et al., 1991). Stimulation by ET-I of choline generation from phosphatidylcholine was recently discovered in Rat-1 fibroblasts (MacNulty et al., 1990), leading these authors to suggest that endothelin activates PLD by both PKC-dependent and PKC-independent mechanisms. In line with this notion, Konishi et al. (1991) demonstrated that in aortic vascular smooth muscle cells, activation of PLD by endothelin may be closely linked to a signal transduction event. In rat mesangial cells, ET-1 activated not only PLC but also Na+/H + exchange. Vigne et al. (1991), using brain capillary endothelial cells, demonstrated that high-affinity E T r R and ET3-R (i.e. ETB-R) control Na+/H ÷ exchange activity via a PKC-independent mechanism. In the same preparations, receptors of the ET A type control ET-I action via activation of PLC. In view of the diverse signal-transduction pathways that can be activated by ET-R, the question arises whether a specific pathway can be associated with a given receptor subtype. Examination of the data published so far does not allow the assignment of a particular receptor subtype to a specific signal event. Future research may lead to the discovery of more subtypes, and should establish whether such a correlation exists. The biochemical characterization of the family of endothelins, sarafotoxins and their receptors has advanced at a remarkable rate. Future research can be expected to focus, for example, on: (1) Identification and synthesis of ET-selective antagonists, as well as the search for the possible existence of multiple (high and low) affinity states for agonist binding and their pharmacological relevance; (2) Enzymatic processing of ET precursors; (3) Construction of chimeric receptor genes and site-directed mutagenesis; (4) Interaction of signal transduction pathways associated with each subtype; (5) Study of physiological implications of the distribution of ET receptor subtypes; (6) Production of receptor-specific antibodies. All of these topics are of interest and importance in the attempt to further understand the physiological function of ET as well as its role in various clinical disorders, among them hypertension, cerebral/coronary vasospasm, asthma, and renal failure. Acknowledgements--I thank Ms Shirley Smith for excellent editorial assistance.

REFERENCES

ABDEL-LATIF,A. A. and ZHANG,Y. (1991) Species differences in effects of endothelin-I on myo-inositol triphosphate accumulation, cyclic AMP formation and contraction of isolated iris sphincter of rabbit and other mammalian species. Invest Ophthal. Vis. Sci. 32: 2432-2438.

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M. SOKOLOVSKY

ABDEL-LATIF,A. A., ZHANG, Y. and YOUSUFZAI,S. Y. K. (1991) Endothelin-I stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle: activation of phospholipase A 2. Curt. Eye Res. 10: 259-265. AMBAR, I, KLOOG, Y., KOCHVA, E., WOLEBERG,Z., BDOLAH,A., ORON, U. and SOKOLOVSKY,M. (1988) Characterization and localization of a novel neuroreceptor for the peptide sarafotoxin. Biochem. biophys. Res. Commun. 157:1104-1110. AMBAR,I., KLOOG,Y., SCHVARTZ,I., HAZUM,E. and SOKOLOVSKY,M. (1989) Competitive interaction between endothelin and sarafotoxin: Binding and phosphoinositides hydrolysis in rat atria and brain. Biochem. biophys. Res. Commun. 158: 195-201. AMBAR,I., KLOOG,Y. and SOKOLOVSKY,N. (1990) Cross-linking of endothelin 1 and endothelin 3 to rat brain membranes: Identification of the putative receptor(s). Biochemistry 29: 6415-6418. ANDO, K., HIRATA,Y., SHICHIRI,M., EMORI,T. and MARUMO,F. 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Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

GM-CSF: receptor structure and transmembrane signaling.

LKB1 in transmembrane receptor signaling.

Kinetic and cross-linking studies indicate different receptors for endothelins and sarafotoxins in the ileum and cerebellum.

Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus.

Receptors and transmembrane signaling.

Conformational study of endothelins and sarafotoxins with the cystine-stabilized helical motif by means of CD spectra.

Regulation, Signaling, and Physiological Functions of G-Proteins.

Endothelins.

Regulation of adrenal atrial natriuretic hormone receptor subtypes.

Regulation of cell differentiation by Eph receptor and ephrin signaling.

The growth hormone secretagogue receptor: its intracellular signaling and regulation.

B lymphocyte antigen receptor signaling: initiation, amplification, and regulation.

T-cell development and transmembrane signaling: changing biological responses through an unchanging receptor.

Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: stimulation of phosphoinositide turnover and inhibition of prolactin release.

Redox regulation of T-cell receptor signaling.

Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor.

Structure-activity relationships of sarafotoxins: chemical syntheses of chimera peptides of sarafotoxins S6b and S6c.

Juxtamembrane contribution to transmembrane signaling.

Angiotensin-II receptor subtypes and coupling to signaling systems in cultured fetal fibroblasts.

Differential coupling of subtypes of the muscarinic receptor to signaling mechanisms in brain and peripheral tissues.

Transmembrane signaling changes with aging.

Endothelins.

Glucose concentrations in brain and blood: regulation by dopamine receptor subtypes.