Vasoactive peptides and characterization of their receptors

Regulatory Peptides, 45 (1993) 323-340

323

© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01378 Review

Vasoactive peptides and characterization of their receptors D. Regoli, P. D ' O r l r a n s - J u s t e , N . R o u i s s i a n d N . E . R h a l e b Department of Pharmacology, Medical School, Universit~de Sherbrooke, Sherbrooke (Canada)

(Received 23 January 1993; revised version received 7 February 1993; accepted 9 February 1993) Key words: Vasoactive peptide; Receptor; Characterization; Pharmacology; Review

Contents I.

Introduction

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

II. Receptors for angiotensin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

326

III. Receptors for kinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

329

IV. Receptors for neurokinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

331

V.

333

Receptors for endothelins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction A variety of peptides take part in the modulation o f peripheral vascular tone, which is primarily controlled by the sympathetic nervous system. Table I gives an overview o f the 15eptides and their receptors. Noteworthy is the fact that each vasoactive endogenous agent has at least two binding and one or more

Correspondence to: D. Regoli, Department of Pharmacology, Medical School, Universit6 de Sherbrooke, Sherbrooke, Qurbec, J1H 5N4, Canada.

336

functional sites. Such a complexity is quite c o m m o n in physiology. The content of the present review will be limited to analysis of receptors for angiotensins, bradykinin and related kinins, substance P and related neurokinins and endothelins. For all these peptides (see Table II), data obtained in pharmacological studies have been confirmed by the identification (with molecular biology techniques) of receptor genes and messenger m R N A s , as well as by the discovery of specific and selective antagonists of peptide or nonpeptide nature. Receptors for vasoactive peptides are found in dif-

324 TABLE I Vasoactive peptides and their receptors Angiotensin Vasopressin Endothelin Neuropeptide Y

ATt, AT 2 VI, V 2

ETA, ETB, ETc? NPY1, NPY2

bradykinin neurokinins VIP ANP

BI, B 2 NK-1, NK-2, NK-3 VIPt, VIP 2 ANP 1, ANP 2

VIP, vasoactive intestinal peptide. ANP, atrial natriuretic peptide.

ferent organs and extravascular tissues (e.g., intestine, urinary bladder, uterus, trachea, bronchi). Several of these organs, isolated and suspended in vitro, have been utilized for the functional characterization of peptide receptors [1-6], together with isolated arteries and veins [7-10] and, in some cases, isolated peripheral vascular beds [ 11]. Over the years, it has become apparent that isolated arteries and veins provide some of the most sensitive and reliable pharmacologic preparations for the characterization

and classification of receptors, at least for some vasoactive peptides [4,7-9]. The preparation of choice for each receptor system is indicated in Table II, together with the naturally-occurring and/or the selective agonist. In pharmacological studies, receptor characterization and classification has been based on the classical criteria proposed by Schild in 1973 [ 12], namely the order of potency of agonists (possibly naturallyoccurring agonists and selective agonists) (Table II)

TABLE II Receptor characterization with agonists Peptide

Receptor

Preparation

Agonist (selective agonist)

pD 2

Agonists order of potency

Angiotensin

AT l AT 2

AT n (pNPhe6-ATii)

8.86 (- )

ATII > AT m > AT I AT m = AT n > AT I

Bradykinin and related kinins

B1 B2

rabbit aorta (RbA) rat brain plasma membranes rabbit aorta rabbit jugular vein (RbJV)

NK- 1

rabbit vena cava (RbVC)

NK-2

rabbit pulmonary artery (RbPA)

NK-3

rat portal vein (RPV)

ET A ET B

rat aorta rat vas deferens rabbit pulmonary artery bovine aorta endothelial cells

7.29 8.48 8.56 8.83 8.86 8.22 8.60 7.68 8.30 8.35 7.45 9.43 100 nM*

desArg9BK > BK BK > desArg9BK

Neurokinins

desArg9BK BK ([Hyp3,Tyr(Me)S ]BK) SP ([Sar9Met(O2) xl ]SP NKA ([flAIaS]NKA(4-10) NKB ([MePhe 7]NKB) endothelin 1 (ET1) endothelin-3 (ETa)

Endothelins

ETc?

endothelin-3 (ET3)

pD2, - l o g of the molar concentration of agonist required to produce 50~o of the maximal effect. * Effective concentration.

SP > N K A > NKB N K A > NKB > SP NKB > NKA > SP ET 1> ET 2 > ET a ET l = ET 2 = ET 3 ET~ = ET2,~ ET 3

325 TABLE III Receptor characterization with antagonists: receptor roles Receptor

Antagonist

Preparation

pA2*

Functional role(s)

AT I

RbA

8.48 8.60

RbA RbJV RbJV RbVC

ET A

DuP 753 [ Sar l,Ala8]ATn PD 127123 [ Leu~]desArg9BK DArg[Hyp3,DPhe7,Leus ]BK Hoe 140 CP-96,345 RP 67580 SR 48968 R-486 BQ - 123

RbPA RPV RbJV

7.29 8.86 9.17 9.50 7.23 9.60 7.45 7.20

ET B

IRL- 1038

RbPA

n.d.

vasoconstriction, aldosterone secretion, hypertension, heart failure smooth muscle proliferation? shock? cell proliferation? inflammation? inflammation, pain, asthma, arthritis, hypotension, plasma extravasation neurogenic inflammation, pain, plasma extravasation asthma, visceral hypermotility intestinal motility, analgesia? tissue ischemia, vasoconstriction, hypertension, prostanoid release hypotension, contraction of venous and pulmonary arterial blood vessels

AT2 BI B2 NK- 1 NK-2 NK-3

pA2, - log of the concentration of antagonists that reduces the effect of a double dose of agonist to that of a single dose [ 13]. p A 2 values have been measured in some of the preparations indicated in Table II. n.d., not determined.

and the a p p a r e n t affinities o f competitive antagonists (Table III), evaluated by the p A z m e t h o d [13] or by the Schild plot [14]. The two classification criteria are p r e s e n t e d for all peptides in T a b l e II (the order o f p o t e n c y o f agonists) and Table I I I (the p A 2 o f the antagonists). M o d e r n technologies have been applied to investigating the peptide receptors and binding assays have been extensively p e r f o r m e d to m e a s u r e actual affinities. It is therefore possible to verify to which extent ICso values, m e a s u r e d by the binding, c o r r e s p o n d to p D 2 or p A 2 values evaluated with biological assays. A p p l i c a t i o n o f molecular biology has lead to coding o f r e c e p t o r genes and to expression o f these receptors [ 15-17,27,59,101,102,122] in specialized cells (e.g., Xenopus oocyte, the m o n k e y kidney C O S - 7 cell) [ 16] such that, in m a n y cases, it is p o s sible to c o m p a r e a p p a r e n t affinity ( p h a r m a c o l o g i c ) and real affinity (biochemical) o f the naturallyoccurring r e c e p t o r with the affinity o f the receptor expressed on the surface o f the Xenopus oocytes or other cells. W h e n available, such d a t a will be analyzed for each peptide category; one such c o m p a r i -

Rabbit vena

cava

lO0

pD 2 for SP 8.63

g,. ro

.,P tO 1o

, _~.-.-'~. 10.~

10 4

, 10.7

, 10-s

10-~

Peptide (M) COS-7 cells

~

80

~

4o

~

2o

• SP

IC5ofor SP 3.0.10 .9 M 8.52 (-log M)

IO-N

iOto

10-9

Peplide

IO s

10 7

10 6

lO s

(M)

Fig. 1. Comparison of biological activities of neurokinins in the rabbit vena cava [ 19] and binding affinities to COS-7 cells [ 17].

326 son is shown in Fig. 1 for the NK- 1 receptor of substance P. From recent data, it is evident that the majority of receptors for vasoactive peptides belong to the rhodopsin family and are constituted of seven hydrophobic transmembraneous domains connected by extracellular and intracellular loops, as illustrated in Fig. 2 by the NK-2 receptor, identified in 1987 by Masu et al. [16]. Peptide receptors are linked to G-proteins and to IP3-Ca 2 + -calmodulin systems that utilize calcium as second messenger and mediate the various biological actions (contraction, secretion, etc.) of the peptides (e.g., the neurokinins [18]). Peptide receptors have also been characterized by the use of antagonists. Major progress has been accomplished with the discovery of potent, selective and specific antagonists of peptide and non-peptide nature. The structures of the most important compounds are presented in Fig. 3.

C00H Fig. 2. Structureof the NK-2 receptor from bovinebrain. Triangles indicatepotentialglycosylationand asterisks phosphorylation sites. Hydrophobictransmembraneresidues are in squares. Constructed from data reproducedfrom Ref. 16.

II. Receptors for angiotensin Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-ProPhe) is the biologically active component of the renin-angiotensin system, whose major role is to sustain blood pressure. The effects of angiotensin are mediated by a single receptor type (ATt), while the second binding site that is found in peripheral organs and in the brain (AT2) is apparently without function. Basic biological data obtained in pharmacological, biochemical and molecular biological studies are presented in Table IV in order to compare the results of different techniques and approaches. Bioassays on AT~ receptors have been performed in the rabbit aorta, a preparation initially described by Furchgott and Bhadrakom [22] and extensively used thereafter by several authors [23-25]. Binding assays have been performed in rat aorta and rabbit uterus plasma membranes, using [ 3H]angiotensin II as a ligand. The same ligand has been used to characterize the vascular (rat aorta) and adrenal (bovine adrenal cortical cells) receptors that have been recently cloned [27,28] and have been expressed in COS-7 cells. Noteworthy is the similarity of data obtained with the three approaches. Firstly, the order of potency of the angiotensins is the same, the most active agent being ATn, followed by ATnI and AT I. Secondly, the relative affinities of the antagonists follow the same order, the peptide analogue saralasin being more active that the non-peptide antagonist, DuP 753. PD 123319 is inactive in the rabbit aorta and on the cloned receptors, while showing some activity on the rabbit uterus, which is a multiple receptor system containing both AT~ and AT 2 sites. Thus, the early suggestion presented by one of us [26] some years ago, that vascular and adrenal receptors for angiotensin are the same entity, is now validated by the cloning of the two receptor sites. Positive and highly significant correlations between biochemical and pharmacological parameters are thus demonstrated by the data presented in Table IV. Such positive correlations have also been re-

327

Receptor

Antagonist

Structure

B~

[LeuS]desArggBK

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu

B,

DArg[Hyp3,DPheT,LeuSIBK

DArg-Arg-Pro-Hyp-Gly-Phe-Ser-DPhe-Leu-Arg

Hoe 140

DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg. N

AT,

CL

DuP 753 K

AT,

NN

PD 127123 NH2

NK-I

CP-96,345

RP 67580

0

(.1

M,

0

NK-2

SR 48968

NK-3

R-486

[TrpT,13AIa~]NKA(4- 10)

ETA

BQ- 123

cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp]

ET.

IRL 103~

[Cysn-CysJSl-ET-l(t 1-21)

Fig. 3. Structures of vasoactive peptide antagonists.

ported with antagonists, as exemplified in Table V, by the data of the DuPont group, who have compared the pA2 (biological) and IDso (biochemical) values of a series of AT t antagonists with their efficacy as antihypertensive agents in non-anesthetized renal hypertensive rats (Table V).

The second site (AT2) is up to now a binding entity, devoid of biological function. It has therefore been characterized only with binding assays [25,29]. The order of potency of agonists in this site, indicated in Table II, is ATnI = AT u > AT I and a selective ligand has been identified. A non-peptidic com-

328 TABLE IV Basic data on angiotensin receptors Agent

AT u AT m AT l [pNPhe6]ATH [ Sar l,Ala s ]AT u DuP 753 PD123177

Pharmacology

Biochemistry

Molecular Biology

rabbit aorta

rat aorta

rabbit uterus

rat aorta ~

pD 2

- log IC50 (nM)

- log of Ki (nM)

- log of K i (nM)

bovine adrenal zona glomerulosa b - log of IC5o (nM)

8.86 7.61 7.58 0 8.6 8.4 Inact.

9.17 8.8 7.0 . 9.3 7.3

8.60 8.77 6.94

8.80 8.27 7.13

~ 8.0 7.52 ~ 5.30

9.37 8.20 >5

8.30 6.52 >5

.

. 8.79 7.44> 5 7.67

.

* Values for DuP 753 were obtained after fit to a two-site model (modified from Dudley et al. [29], with permission). a Displacement of [125I]Sar~,IleS-ATn binding from membranes prepared from pBa23 i401-transfected COS-7 cell. b Displacement of 12SI-labelled ATlI binding from membranes prepared from pAR 1.8-transfected COS-7 cell.

pound, which has been assumed to be an antagonist,

[29]. Recently, investigators working at Parke-Davis

P D 1 2 3 3 1 9 , h a s s h o w n v e r y h i g h affinity f o r t h e A T 2

have reported [30] that PD 123319 promotes

site ( r a b b i t u t e r u s ; I C 5 0 : 2 1 . 2 n M ) w h i l e b e i n g i n a c -

e x c r e t i o n in t h e d o g a n d h a v e s u g g e s t e d t h a t A T 2

t i v e o n t h e A T t site ( r a t liver; IC50: > 10,000 n M )

m a y b e i n v o l v e d in w a t e r h a n d l i n g b y t h e k i d n e y .

water

TABLE V Activities of non-peptide angiotensin II receptor antagonists a

$8307 $8308 EXP6155 EXP6803 EXP7711 EXP9654 EXP9020 EXP9270 DuP753

IC50 (M) b

pA2c

ED30 (mg/kg- 1 i.v.)d

4.0.10 1.3.10 1.6.10 1.4-103.0-103.3-105.5.10 8.0" 10 1.9.10-

5.49 5.74 6.54 7.20 6.90 7.32 7.65 7.93 8.48

30 30 10 11 3.7 1.5 2.0 1.0 0.78

5 5 6 v 7 7 7 s 8

Taken from Timmermans et al. [25]. b Inhibition of specific binding of [3H]angiotensin II (2 nM) to rat isolated adrenal cortical microsomes. c Antagonism of angiotensin II-induced constriction of rabbit isolated aorta. d Antihypertensive potency (i.v.) in conscious renal hypertensive rats. ED3o, dose to decrease mean arterial pressure by 30 mmHg. a

329 Arg9-BK, while B K a n d L y s - B K show residual activity [2] b e c a u s e they are t r a n s f o r m e d by the intramural c a r b o x y p e p t i d a s e (N) into their metabolites. Indeed, t r e a t m e n t o f a o r t a strips with mergetpa, an inhibitor o f c a r b o x y p e p t i d a s e N, prevents completely the effects o f b r a d y k i n i n a n d kallidin [34]. B 1 receptors are contractile a n d are p r e s u m a b l y linked with a G - p r o t e i n and with the IP3-calcium-calmodulin system, as shown recently by Burch et al. [35] a n d others [36]. B 1 receptors are specifically and selectively a n t a g o n i z e d by peptide analogues o f d e s A r g 9B K , for instance [ L e u 8 ] d e s A r g 9 - B K [2], an antag-

Other reports suggest that A T 2 could be involved in the differentiation a n d d e v e l o p m e n t o f nervous structures m o d u l a t e d by angiotensin II [ 31 ]; this r e m a i n s however to be confirmed. Various reports [32,33] point to the inhibitory effect o f D u P 753 on cell multiplication o f vascular s m o o t h muscles i n d u c e d by angiotensin.

III. Receptors for kinins The p h a r m a c o l o g y o f kinin B 1 r e c e p t o r s has been studied on the rabbit aorta, the s a m e p r e p a r a t i o n used for angiotensin (Table II). The m o s t active naturally-occurring kinins on this r e c e p t o r system are the C-terminal d e s A r g metabolites o f k a l l i d i n a n d b r a d y k i n i n (BK), n a m e l y L y s , d e s A r g 9 - B K and des-

onist that shows p A 2 values (7.27) very close to the p D 2 values (7.29) o f desArg9-BK; the antagonist is competitive (Table VI). B 1 receptors mediate c o n t r a c t i o n o f s o m e isolated arteries and relaxation o f others (e.g., the dog renal

TABLE VI Basic data on kinin receptors Agent

Biological activities

Binding*

Molecular biologya

[ 125I-Tyr8]BK dog carotid artery pD 2

epithelium

muscle

pD 2

rabbit jugular vein pD 2

[ 1251]HOE 140 epithelium

- log IC50

- log IC5o

- log Ki (nM)

- log ICs0 (M)

Bradykinin (BK) Lys-BK desArg9BK Lys,desArg9BK [Hyp3,Tyr(Me)S]BK

6.22 7.27 7.29 8.60 In. pA2

8.48 8.63 In. In. 8.56 pA2

8.64 8.44 In. In. 9.07 pA2

9.06 8.28 4.63 In. 8.41

8.66 8.50 In. In. 8.20

9.09 8.30 In. In. n.d.

8.90 9.05 In. 10.20

[LeuS]desArg9BK [ThiS's,DPhe7]BK

7.27 6.23

In. 7.35

In. 6.56

5.76 In.

In. 6.55 p.Ag. 7.29 9.42

In. 7.27

DArg[Hyp3,DPheV,Leu8]BK DArg[Hyp3,ThiS,DTic7,OicS]BK(Hoe 140)

In. 6.71 p.Ag. 8.86 9.17

7.49 9.00

7.44 9.00

7.30 9.09

In. 6.55 p.Ag. 9.35

rabbit aorta

human lung fibroblast

* Taken from Tousignant et al. [66,67]; p.Ag., partial agonist. a Displacement of [3H]BK binding from membranes prepared from CCD-16Lu cells transfected with human B2 receptor cDNA. Recalculated from Ref. 59.

330

artery [37]). The vascular smooth muscle inhibitory effect is indirect and appears to be mediated by prostanoids. Therefore, B 1 receptors might be linked to different G-proteins and transduction systems, as is the case for B 2 receptors (Ref. 35 and other quotations below). B 1receptors have also been implicated, some years ago by Goldstein and Wall [38], in the multiplication of human foetal fibroblasts in vitro and the secretion of collagen by these cells. Such a promitogenic effect has recently been confirmed by Burch et al. in human macrophages [35,39,40]. Still questionable is the hypothetical role of B 1 receptors in the pathogenesis of septic shock in the rat [41,42]. Noteworthy is the fact that B1 receptor number and function in the rabbit are markedly increased a few hours after intravenous administration of a lipopolysaccaride (LPS) [43] and have been recently linked with the interleukins [35,44]. A few reports point to the possible role of interleukins in modulating B 1 receptors number or function [45]; B 1 receptors could be considered to be mediators of biological effects of interleukins [45 ]. The pharmacology of B 1 receptors has been recently reviewed [46] and the reader is referred to this paper for further details. Pharmacological studies on kinin B 2 receptors have been performed in a variety of smooth muscles, the guinea pig ileum being the most widely used [2,47-49]. Indeed, the slow contraction (compared to that of histamine) of this preparation in response to BK is at the origin of the peptide name [50]. Another preparation sometimes used is the rat duodenum which responds to bradykinin with relaxation [51]. However, because of the limitations of these preparations (see below), isolated vessels have been preferentially used to evaluate both the inhibitory and the excitatory effects of kinins (Table II). Thus, the inhibition of smooth muscle tone by BK, which appears to be mediated by nitric oxide released from the endothelium, has been extensively investigated in the dog carotid or renal arteries [ 2,37 ], while the direct contractile effect on the venous smooth muscle has been studied in the rabbit jugular vein [52] (Table VI). The order of potency of

kinins in these preparations is very similar (Table VI) and BK, as well as Lys-BK, are much more potent (by several orders of magnitude) than the C-terminal metabolites. Recently, some selective peptidic agonists for this receptor have been identified, namely [Hyp3,Tyr(Me)8]BK [53] and found to be very active on both isolated vessels (Table VI). B 2 receptors have been further characterized with antagonists, initially with the compounds proposed by Vavrek and Stewart in 1985 [47] and lately by pure competitive antagonists, such as o-Arg[Hyp3,D-PheV,Leu8]BK [54] and long-acting, non-equilibrium antagonists, such as Hoe 140 (DArg[Hyp3,ThiS,D-TicV,OicS]BK [55-57]. Several of the compounds described by Stewart and Vavrek maintain strong agonistic activities in some tissues [54,58] as well as in vivo [54,57] and should be considered as partial agonists, while compounds containing Leu in position 8 and Hoe 140 have no or very little residual agonistic activity and are as potent or even more than the Stewart compounds [54] (Table VI). This has been recently confirmed in CHO transfected cells, expressing the human B 2 receptor, by measuring the accumulation of intracellular Ca: + [ 59]. According to Eggerickx et al. [ 59], 'the agonistic and antagonistic properties of BK analogues do not match strictly the pharmacological profiles described for the rat or guinea pig B 2 receptor subtypes or the putative B 3 subtype' (proposed by Farmer et al. [60]), while they match quite well the profile described in the rabbit jugular vein and other vessels (Ref. 54 and see Table VI). Several of these compounds, but not Hoe 140, are converted into C-terminal desArg metabolites which exert potent antagonistic effects on B 1 receptors and therefore are non-selective for the B 2. Such conversion is prevented by the insertion of unnatural amino acids in positions 7 and 8, as in Hoe 140, which is selective for the B 2 and inactive on the B 1 preparations [55] (Table VI). B 2 receptors mediate a variety of biological effects attributed to kinins, namely hypotension, plasma extravasation, stimulation of sensory fibers that activate cardiovascular reflex or mediate pain

331 and a variety of secretions of endogenous mediators, such as histamine, prostanoids, catecholamines, among others [2,37,54]. Recently, B 2 receptors have been implicated in the inhibitory effects of kinins on cell multiplication of vascular smooth muscle cells [61]. B2 receptors may not be a single entity. Various reports have suggested other receptor types, for instance B 3 [60], B 3 [62], B 4, B 5 [63]. All this work appears to be preliminary and questionable [35,39] and has been recently reassessed by Regoli et al. [ 64]. Consistent evidence has been obtained in favour of the existence of at least two B 2 receptor subtypes, B2A and B2B, which have been recently characterized pharmacologically, both with agonists and antagonists [53,64,65].

IV. Receptors for neurokinins Substance P and related neurokinins have been shown to exert their various biological effects by activating at least three different receptor types. These receptors were named NK-1, NK-2 and NK-3, at the international meeting on Substance P and Neurokinins, held in Montr6al in 1986 [68]. Similar to the B 2 receptor for the kinins, the NK-1 functional site for substance P is inhibitory on the arterial smooth muscle and excitatory on the venous. It has therefore been extensively studied in the same preparations as the B 2 receptor, namely the dog carotid artery (for the inhibitory effect) and the rabbit jugular vein (for the contractile) in an early study [69], and the rabbit vena cava, more recently [ 19]. NK-1 receptors show high sensitivity for substance P and less for the other neurokinins and are very sensitive to selective agonists ([Sar9,Met(O2)ll]SP or acetyl[Arg6,Sar9,Met(O2) 11]SP(6-11). NK-1 receptors are antagonized by peptide antagonists, such as spantide, octa or hexa peptides containing D-Trp residues in their sequences, as well as by newly identified tri and dipeptides [70,71] and even by nonpeptide compounds, such as CP-96,345 and

RP 67580 [72-74]. Non-peptide compounds have been found to be extremely selective for the NK-1 receptor and, in some species, CP-96,345 shows very high pA2 values [70,72]. The antagonism exerted by CP-96,345 and RP 67580 appears to be of the competitive type, as indicated by the linearity of the slope of the Schild plots, which correspond to 1.09 for CP-96,345 and to 0.95 for RP 67580 in the rabbit jugular vein (Regoli, D. and Rouissi, N., unpublished data). A complete pharmacological analysis of the NK-1 receptor features is presented in Table VII. The order of potency of agonists is as follows: SP > N K A > NKB and the antagonistic activities of prototypes of the three generations of antagonists [80] indicate that the non-peptide compounds are the most active, selective and specific. Results of binding assays correlate fairly well with those of biological studies and worthy of notice is the similarity of affinities obtained in biological assays and in COS-7 cells which have been made to express the NK-1 functional site from rat brain (Ref. 17, see Fig. 1). Recently, the human NK-1 receptor has been identified [81] and shown to be homologous to the NK- 1 site of the rat. NK-1 receptors have been implicated in the mediation of a variety of effects of neurokinins in vivo, particularly hypotension, salivary secretion, intestinal and bladder motility, neurogenic inflammation and, most important, in the transmission of pain at the spinal cord level [75-77]. Recent reports with CP-96,345 appear to validate the physiopathological role of NK-1 receptors in hyperalgesia, since the non-peptidic antagonists has been shown to reduce pain in the mouse (hot plate test) [78] and inflammatory pain in the rat [79]. NK-2 receptors have been characterized in the rabbit pulmonary artery [82] and shown to have high sensitivity to NKA and to [/~AlaS]NKA(4-10), the selective agonist described by Rovero et al. [83]. Recently, a non-peptidic antagonist (SR 48968) (Table VII) has been shown to be extremely active and selective for NK-2 receptors, showing even higher

332

o o

o "O

z

o

o

I I ~oo

~

~

0

~o

,.6o<

o~

~H~6

fi

o o

g

o g q~ o

~

o~ ._=

_

~-~

~

o

;> u~ o

?

333 activity for the NK-2A than for the NK-2B subtype [84,851. Because of the implication of NK-2 receptors in bronchoconstriction and in visceral hypermotility in animals and especially in man, which possess almost exclusively NK-2 receptors in the bronchi, the intestine, the urinary bladder and other viscera [86,87], this antagonist (SR 48968) appears to be a promising compound. Indeed, NK-2 functional sites may play important roles in some pathological states, such as asthma, intestinal and bladder hypermotilities [77] and possibly hyperalgesia [ 88]. NK-2 functional sites that are excited by [//Ala 8 ]NKA(4-10) (or by NKA) have been demonstrated in the central nervous system and shown to be involved in the regulation of cardiovascular functions by centrally-acting neurokinins [89], as well as in spinal hyperalgesia [88]. TschOpe etal. [89] have recently described the hypertension and tachycardia induced by intracerebroventricular administration of NKA and the selective blockade of these NKA-induced phenomenons by NK-2 antagonists, administered by the same route. At the spinal cord level, intrathecal administration of NKA has been shown to produce sustained hyperalgesia, which is antagonized by the same type of NK-2 antagonists [90]. Basic pharmacologic data to characterize NK-2 receptors are presented in Table VII. Order of potency of agonists in this functional site is N K A > N K B > SP and is the same as that found by Masu et al. [ 16] on a bovine NK-2 receptor expressed in the xenopus oocyte (Fig. 2). The most active antagonist on the NK-2A receptor of the rabbit pulmonary artery is SR 48968:NK-2 receptor subtypes, NK-2A and NK-2B, have been proposed by Maggi et al. [91], but more recent data are less discriminative and raise questions on the early suggestion [77,91]. NK-3 receptors have been studied on the rat portal vein, a sensitive neurokinin-monoreceptor system which responds with contraction to NKB and to selective NK-3 agonists, such as [MePheT]NKB, while being much less sensitive to N K A and espe-

cially to SP. NK-3 receptors are blocked by a peptidic antagonist, R-496 (Asp-Ser-Phe-Trp-flAla-LeuMet-NH2) discovered in our laboratory (Drapeau et al. [92]). This compound appears to be selective for the NK-3 site but maintains some agonistic activity on NK-1 receptor systems [92]. NK-3 receptors have been characterized with various approaches and the results, summarized in Table VII, indicate that the order of potency of agonists in this site is N K B > N K A > SP; this order of potency is the same as that found by Shigemoto et al. [93] with the cloned receptor expressed in COS-7 cells. [MePheT]NKB [97] and senktide [94] are the selective agonists and R-486 (Fig. 3) is a fairly potent peptide antagonist. NK-3 functional sites have been identified in peripheral tissues and in the central nervous system. They appear to be involved in intestinal motility, by activating the release of acetylcholine from the myenteric plexus [94] and in the bradycardic effect observed with NK-3 agonists that activate the parasympathetic nerves of the heart [95]. At the spinal cord level, NK-3 selective agonists and NKB have been shown to reduce SP-induced hyperalgesia probably by promoting the release of opioids, since the effects of NK-3 receptor agonists are blocked by naloxone [96,97].

V. Receptors for endothelins Of the four peptide groups described in the present review, the endothelin family is the most recently discovered [ 98,105 ]. The three members of the family consist of 21 amino acids and are cyclic peptides with two sulfur bridges between Cysl-Cys 15 and Cys3-Cys 11. Endothelin-1 (ET-1) is generated from endothelial cells under the action of various agents (e.g., angiotensin II or Arg-vasopressin [124] and may also be induced by cytokines and various growth factors (for recent review, Ref. 123). ET-1 is the most potent vasoconstrictive substance yet reported. When administered intravenously in anesthetized

334 rats, ET-1 induces a bi-phasic effect, namely an initial depressor response of short duration followed by a prolonged hypertensive effect, which lasts more than 50 min in that animal model [98,99]. In addition to its potent hemodynamic effects, ET-1 and the two other isomers, ET-2 and ET-3, exert a wide variety of pharmacological effects on vascular and non-vascular tissues [98,99,105]. In addition, ET-1 has been reported to be a potent enhancer of vascular smooth muscle cell proliferation (Refs. 100, 103, 123 (for review)). The majority of the pharmacological effects of endothelins are mediated via the activation of at least three receptor types, ET A, ET a and ET c . Of these three receptors, the first two have been biochemically characterized and genetically expressed in COS-7 (ETA) and COP-5 (ETa) cells, respectively [101,102]. Various studies have also reported the existence of at least three distinct binding sites with radioactivelabelled ET-1 in tissue homogenates and vascular smooth muscle (for review; Ref. 103). In addition, ET A and ET a binding sites have been recently characterized in vascular and non-vascular tissues by the use of selective analogues, such as BQ-123 (ET A antagonist), BQ 3020, I R L 1620 (ET a agonists) and I R L 1038 (ET a antagonist) [104,106,114,121]. The activation of ET A receptors has been initially associated with the vasoconstrictive and pressor effects of endothelins in vivo and, on this later system, ET-1 has been found to be much more potent than ET-3. In contrast, the ET a receptor activation has been associated with the vasodilator properties of the endothelins. Recent studies have shown that utilisation of selective ET a receptor agonists only induce vasodilation in isolated vasculatures in which the natural isomer, ET-3, is equipotent to ET-1 as a releaser of E D R F , yet a much weaker vasoconstrictor than the later peptide [107]. It is of interest to point out that, although ET a receptor activation has been associated with the overall release of E D R F by these peptides in vivo and in vitro [ 108,112,114,121], endothelins also promote the release of vasodilatory

prostanoids from the pulmonary and renal vasculature via the activation of ET A receptors [109,110,120]. One also has to take into account ETa-mediated constrictions of some veins [ 113 ] and of the rabbit pulmonary artery [ 114]. Finally, ET c receptors have been identified in bovine aortic endothelial ceils in culture on which tissue ET-3 was found to be far more active than ET-1 as a generator of E D R F [ 107]. This third receptor is yet to be fully characterized both biochemically and by molecular biology. Also worthy of consideration is the fact that a recent publication has suggested a possible role for ET c receptors in the control of the bloodflow in the hepatic circulation of the anesthetized rat [ 111 ]. Basic pharmacologic data obtained with naturally-occurring peptides and selective analogues in various preparations are presented in Table VIII. As for the other peptides, data derived from pharmacological, biochemical and molecular biological studies are very similar and show good correlations. The data of Table VIII indicate the existence of two receptor types, both by the relative potencies of agonists and the affinities of the two antagonists that are available. In pharmacological assays, the rabbit jugular vein has a mixture of ET A and ET a, the rat aorta has the ET A, while the rabbit pulmonary artery has ET a receptors. ET A are present in porcine aortic muscle and rat aorta (biochemical data) while ET a are found in the guinea pig ileum and porcine cerebellum. BQ-123, the ET A antagonist, is more active on the porcine aortic muscle than in porcine cerebellum by 3 log units, while I R L 1038, the new ET a antagonist, is more active on the guinea pig ileum than on the rat aorta. The relative activities of the three endothelins in cloned receptors, expressed in COS-7 cells (ETA) and COP-5 cells (ETB) follow the same order as in the pharmacological assays. The recent development of selective ET A receptor antagonists is now allowing investigators to further validate functional roles for the different receptors of endothelins in vivo and in vitro (Fig. 3). The most

335 T A B L E VIII Basic d a t a on endothelin receptors Agonistsl

Antagonists 2

ET-1

ET-2

ET-3

ETB*

Pharmacology R a b b i t jugular vein R a b b i t p u l m o n a r y artery R a t aorta

9.69 9.67 8.35

9.22 -

8.48 10.09 6.43

8.656 9.24 > 6

Rat vas deferens

7.43

7.40

7.26

-

BQ-123

7.2 6.90 > 4.77

I R L 1038

6.007 -

Biochemistry 3 G u i n e a pig ileum Porcine aortic vascular s m o o t h muscle Rat aorta Porcine cerebellum M o l e c u l a r biology Bovine lung 4 [ 101 ] Rat lung 5 [102]

11.30

-

11.09

-

-

10.01 10.01 10.15

-

7.15 7.92 10.15

6.02

8.13

9.7

4.80

9.03 8.72

8.14 -

8.04

6.15

6.04 8.7

i pD2. 2 p A z against ET-1. 3 4 5 6 7 *

_ log of IC5o or K i (nM) ( d i s p l a c e m e n t of 125I-ET-1). _ log of K~ expressed in n m o l a r ( d i s p l a c e m e n t of ~251-1abelled from crude m e m b r a n e s of C O S cells transfected with ET A receptor c D N A . * D i s p l a c e m e n t (approx. - l o g of IC50 in n M ) of 125I-labelled ET-1 binding of p r E T R - 7 transfected C O P - 5 cells. Partial agonist. _ log (M) of the minimal effective concentration necessary to block the e n d o t h e l i u m - d e p e n d e n t vasodilation induced by ET-3 [ 125]. E T B selective agonist (BQ 3020: N - a c e t y l - L e u - M e t - A s p - L y s - G l u - A l a - V a l - T y r - P h e - A l a - H i s - L e u - A s p - I l e - I l e - T r p ) [ 114].

interesting prototype, which is now currently used, is the BQ-123 [ 104]. This antagonist interferes with the pressor effects of endothelin-1 in anesthetized rats but is inactive on the initial hypotensive effect induced by this peptide. In addition, in perfused organs, the ETA-selective antagonist interferes in a reversible fashion with the pressor and prostanoidreleasing properties of ET- 1 in the rat perfused lung and in the rabbit kidney [ 116,117]. In addition to the now available ETA-selective antagonist (BQ-123), Urade et al. [ 106] have very recently reported a new antagonist selective for ET B receptors, IRL 1038; its structure is indicated in Fig. 3. ET-1 is generated from a larger intermediate precursor named big-endothelin-1 (1-39 porcine; 1-38

human [98,105]). Big-ET-1 is processed to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme which has been identified in vivo in the rat and guinea pig and in vitro in the rat vas deferens and in various perfused organs [ 110,116-119]. It is of interest to note that BQ-123 not only interferes with the activity of ET-1, but also with those of big-ET-1 in perfused models, such as the rat perfused lung [120]. This observation illustrates that big-ET-1 activates ET A receptors to release prostanoids in this perfused pulmonary system following conversion via the phosphoramidon-sensitive ECE [ 115,119]. Hence, the use of selective antagonists will allow not only to understand the functional roles of the receptors for endothelins, but may also be used

336 to c o n f i r m the c o n v e r s i o n o f p r e c u r s o r s to their active m e t a b o l i t e s p r i o r to the a c t i v a t i o n o f the different r e c e p t o r types by t h e e n d o t h e l i n s .

Note added in proof In a r e c e n t r e v i e w ( B r a d y k i n i n a n d i n f l a m m a t o r y pain, T r e n d s N e u r o s c i . , 16 (1993) 9 9 - 1 0 4 )

A. D r a y

a n d M. P e r k i n s h a v e suggested t h a t B~ r e c e p t o r s m a y p l a y an i m p o r t a n t role in the m a i n t e n a n c e o f h y p e r a l g e s i a since, in p r o l o n g e d i n f l a m m a t o r y states (rat h i n d p a w e x p o s e d to ultraviolet irradiation, rat k n e e j o i n t i n j e c t e d w i t h F r e u n d ' s a d j u v a n t ) , B 1 rec e p t o r a n t a g o n i s t s (but n o t B 2 a n t a g o n i s t s ) r e v e r s e hyperalgesia. A non-peptide endothelin-receptor antagonist, Ro 46-2005, t h a t b i n d s to b o t h E T A a n d E T B r e c e p t o r s has b e e n d e s c r i b e d by Breu, V. et al. ( P r o c e e d i n g s E u r o p e a n P e p t i d e S y m p o s i u m , I n t e r l a k e n , 1992 (to be published)). M o r e recently, a s u l f o n a m i d e , N - [ 5 (2,6

dimethoxybenzyl)-6-(2-hydroxyethoxy)-4-pyri-

midinyl]-4-vinylphenylsulphoramide

been

re-

p o r t e d ( E P - 5 1 0 5 2 6 - A ) by H o f f m a n n - L a R o c h e ,

has

as

another endothelin-receptor antagonist.

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