Endothelin - a new family of endothelium-derived peptides with widespread biological properties

Life Sciences, Vol. 45, pp. 1499-1513 Printed in the U.S.A.

Pergamon Press

MINIREVIEW ENDOTHnLN

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A NE% FAMILYOF FNDOTHEL.WM-DERIVED PEPJXDESWI'I‘+I WIIXSPREADBIOLXICAL PROPERTIES

Anne-Charlotte Le Yonnier de Gouville,1,3 Homrd L. Lippton,2v3 Icilio Cavero,l Warren R. Sunn~r,~ and Albert L. !+IIEUJ.~

de Vitry, 13 quai Jules Guesde. 94400 Vitry Sur Seine France 2&partment of Pulmonary Medicine, Louisiana State Medical Center. New Orleans, TA 70112 3tipartment of Surgery, Tulane Medical School, New Orleans, LA 70112

$&one

Poulenc-Sante, Centre de Recherche

(Received in final form August 8, 1989)

Smry Endothelin (ET) is a novel Eamilyof three isopeptides(ET-l,m-2, ET-3) each containingtwenty-oneaminoacids and two disulfide of culturedporcine bonds. Initiallyisolatedfrom the supernatant aorticendothelial cetls,l?Tis storedas a preproformand released throughan unusualproteolytic cleavage. In general, ET-l,ET-2, ET-3 differ quantitatively but not qualitatively in theirbiologic activity. Tl? have potentcontractileactivityin a varietyof isolatedtissuesincludingarteriesveins,trachea,duodenumurinary and bladder and uterus. In vivo, ET possesses potentvasodilator vasoconstrictor propertles. Althoughthe mechanismsmediatingthe hemodynamiceEfects of ET are not entirely clarified,recent relaxant factor evidence indicatesa role for endothelium-derived Moreover, ET kinase C and extracellular calcium. @RF), protein appears to produce inflammation and bronchoconstriction through the formation of arachidonic acid metabolites via the cyclooxygenase The presence oE ET binding sites in blood vessels and in pathway. several organ systems suggests EL may have important regulatory fact ions, which remain to be determined.

a newly discovered peptidecontaining21 amino-acids, Endothelin (ET), and two disulfidebonds MS initiallyisolatedfrom su matants of cultured porcineaorticendothelislcellsby Yanagisawaet al. $1). The presence in vascular endotheliumof mRNA encoding for the preproformof ET indicates that ET. is generated-in situ through unique proteolyticcleavage by an "endothelin converting enzyme." The production of ET is regulatedat the level OE mRNA transcriptionwhich ?laybe induced by thrombin (2 U/ml), epinephriine (1 uM), angiotensin11 (LO nM) arginine-vasopressin (0.1ti), (LO to 300 pMj, the proteinkLnaseC activator, transforming growthEactorB TPA (1 ti) and calcium inophores,A 23187 (1 UM) and ionomycine(1 ti) thathumanand porcineET are structurally (1,2,3). Itoh et al. demonstrated identical (4) and differfrom rat ET by six aminoacid substitutions. More recently,the genesencoding three isopeptides for ET have been foundto coexistin the humangenone: ET-L which is the firstisohted ET, ET-2 in whichLeu6-Met7of ET-L are replacedby ~rp6~eu7, and m-3 which is the rat Eorm of ET (5,6). These three peptides(ET-L,ET-2,ET-3)are structurally similarto the snake(Atractapsisengaddensis) venom toxin, sarafotoxin 6b (SRTGb), which may add evolutionarysignificance to the ET molecule (7,8) (Fig. 1).

0024-3205189 $3.00 + .OO Copyright (c) 1989 Pergamon Press plc

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STRUCTURAL ET-l,

ET-2,

DIFFERENCES ET-3

and

ET-

I

(human

Ser

ET-

2

(human)

ET-

3

(rat) 6 b (snake)

NUMBER

5

6

7

12

14

17

19

Ser

Ser

Leu

Met

Val

Phe

Leu

Ile

Gln

Val

Differences

Thr

CZL’

4

porcine)

&

SRT-

2

BETWEEN

SARAFOTOXIN-6b

AMINOA

PEPTIDE

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from

Tw

Leu

Phe

Thr

Tw

LYS

LYS

ASP

Met

Thr

ET-l

Tyr Leu

FIG. 1 Structure of endothelin-1(m-1) and position of amino acid substitutionson ET-1 for ET-2, ET-3 and sarafotoxin-6b. ET-1 has been reported to be a very potent vasoconstrictor -in vitro (1) and similarities to endothelial-derived-constricting-factor (EDCF) many described earlier by O'Brien et al. (9). In addition, this peptide nay be one of the "endogenous regulators" of EDRF described by Furchgott and Zawadski (10). Thus, vascular endotheliummay function as an organ system and play an essential role in the maintenanceof vascular homeostasis.

h&S

The discovery and biochemical characterization of ET-1 have been described in detail in a recent review article by Yanigasawa and Masaki (11). This paper reviews the published results dealing with the biologic effects of ETs on blood vessels and on nonvasculartissues. The ability of ET to release endogenous mediators, its possible mechanisms of .action,and its involvementin cardiovasculardiseases are discussed. In Vitro Studies a) Binding Studies Primary binding studies were carried-outin cultured vascular smooth muscle cells obtained from Wistar rat aorta. Yirata et al., (12) showed that binding of 125I-ET-1is a saturable recess, and characterizedby a sin le ? s. class of sites with an apparent iZdof 8 .4 n?land a Bmax of 13,000 sites/ccl

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This suggests that smooth muscle cells are endowed with a large number of high-aEfinitybinding sites. Power et al. (13) loalized binding sites for ET-1 on the media and adventitia of both human and porcine coronary arteries. In addition, binding sites were seen on nerve trunks and in the adventitiaof small arteries and veins. The presence of binding sites at OK near neural elements provides an ideal location for ET to act as a modulator of autonomic neurotransmissionin the vascular wall. The removal of endotheliumfailed to affect the binding of ET-1 (13). In rat kidneys, a very high density of ET-1 binding sites was found overlying glomeruli in the cortex and over the inner medulla. ET-1 is highly fixed in vasa recta bundles and to a lesser extent in the inter-bundlezone oE the inner stripe of the outer medulla. 4 moderate density of sites was found in the proximal tubules (14). In all these studies the measured aEfinities are of the same order as those reportedby Uirata et al. (12). 1251-ET-1 was not displaced by either angiotensin I, angiotensin arginine-vasopressin, neuropeptide Y, norepinephrine, serotonin, II, histamine, atria1 natriuretic factor (ANF), peptide neurotoxins (apamine, w-conotoxins) or by the Ca2+ -channel blockers nifedipine, nicardipine, diltiazem and verapamil (12). Moreover, bound radioactively-labelled calcium antagonistsW 200/110, D888, and diltiazem were not displacedby ET (15). Autoradiographicstudies show that after i.v. administrationin rats ET was largely localized in lungs, kidneys and liver and to a lesser extent in spleen, heart, stomach, skin, intestineand pancreas. It is uncertain if the high extraction of ET by lung tissue is the result of a catabolic process, or an uptake mechanism or a nonspecificbinding phenomenon. Similar patterns of distributionof radioactivity were found for both ET-1 and ET-3 (X,17). Injected systemiwlly, ET-1 does not appear to cross the blood-brainbarrier, however, under -in vitro conditionsET-l binds specificallyto hypothalamicand thalamic areas, lateral ventricularand subfornicalregions, globus pallidus and caudate putamen. These results imply that ET may participatein a variety of organ functionsand also act as a neuropeptide(18). Following bolus injection of radiolabelledET-1 or ET-3 into the left ventricle, over 60% of the label was removed, whereas the remaining radioactivitywas eliminatedwith a half-life of 10 to 15 min (17). Using a radioimmunoassay technique the mean concentrationof ET in the plasm of sixteen human subjects ~1s 0.6 fm,l/ml. Since the antibody in these experiments crossreacted equally with ET-1 and 'ZI-3, the relative concentrationsof ET-l, ET-2 and ET-3 could not be assessed (19). 'lheauthors of this study concluldedthat ET may be a circulatinghormone in man but the conditions rjhichmay regulate its plasma concentrationare mcertain. b) Isolated Tissues Blood Vessels and ;Ieart ET has potent contractileactivity on a variety of blood vessels from a number of species including rat (20,21), rabbit (22,23,24)! dog (25), and h-n (13). ET-1 elicits contractileeffects which are slow in onset, long in duration, and resistant to wash (1,22). The EC50 for ET-1 on arterial vessels is approximately1 nM and thus it is a more tent vasoactive substancethan epinephrine,angiotensin 11 or vasopressin (1!? . Limited data have suggested that ET-1 has a greater contractile activity on veins rather than on arteries (24,26). Furthermore, the response to ET-1 on isolated renal arteries from spontaneouslyhypertensiverats (SHR) is greater than that from lJistarKyoto rats (WKY), implying that ET-l may play a role in the alteration of renal circulationassociated with certain types of hypertension(20). Since the contractileactivity of ET-1 on porcine coronary artery is not

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indomethacin or altered by phentolamine, diphenhydramine, methysergide, nordihydroguiaret ic acid, this effect is not due to act ivat ion of alpha adrenergic, histaminergicl, and serotonergic receptors as well as formation of cyclooxygenase and lipoxygenase products of arachidonic acid metabolism. The contractile response to ET-1 is completely reversed by isoproterenol or These data suggest that despite tight binding on arterial nitroglycerin. vessels, ET does not appreciably interfere with relaxation of conductance blood vessels mediated by c&iP or cGME’,respectively (1). Further su g;t fob such a conclusion comes from a similar study by Sugiura et al. P_ demonstrated that 8-bromo-cGME’, a lipophilic cGMP analogue, Causes Imsrked relaxation of ET-1-preconstricted rabbit aorta. Conflicting data have accumulated on the dependence of the contractile and lXlt iazem, nifedipine response to ET-1 on extraceLlular calcium. representative agents of three distinct classes of metbxyverapamil (D-600), effect on the contractile calcium channel blockers, appear to have little The concentrations of calcium channel blockers response to ET-l on rat aorta. inhibited the contractile response to bay K used in these experiments In canine felnoral veins, the contractile effects oE ET-1 were 8644 (21). prevented in Ca* free solution but unchanged by the Ca* antagonists verapamil, nimodipine or diltiazem (27). kom these results, the authors concluded that ET-1 does not act as an activator of potential-activated Ca* In contrast, nicardipine inhibits the contractile response to ET-I channels. on porcine coronary artery and large cerebral arteries from the cat but not on rabbit mesenteric artery and vein and rabbit jugular vein. The latter experiments used blood vessels without endothelium (1,28,29). The contractile response to ET on rabbit aorta and on Eeline cerebral arteries is prevented to a large degree by using Ca”-free media containing or not lZTA respectively and can be restored by the subsequent addition of Ca* (1,21,22,29). The contractile response to ET was initially demonstrated using arterial segments with a damaged endothelial cell layer (1). Thus, the modulatory role of endothelial-derived relaxant substances on the eEfects of ET couldn’t be The contractile response to ET-1 in rat aorta, dog femoral vein determined. and cat cerebral artery is significantly enhanced aEter removal of the Further support for the involvement OE a erdothelial cell layer (27,29,30). endot helial origin comes from work by Warner relaxant substance of The vasoconstrictor response to ET-1 and m-3 in the isolated et al. (31). perfused rat mesentery with little baseline tone is enhanced following the addition of inhibitors of soluble guanylate cyclase such as methylene blue and oxyhemoglobin or following the destruction OE the endothelial cell Layer with When the isolated perfused rat mesentery is sodium deoxycholate (28). precontracted with methoxamine, norepinephrine or JJ46619, ET-1 and ET-3 produce vasodilation which is inhibited by methylene blue, oxyhemoglobin OK Since the nonsanguinous perfusate used in sodium deoxycholate treatment. the vascular response to ET in the these experiments contained indomethacin, isolated rat mesentery was not influenced by formation of cyclooxygenase products including thromboxane A2 (TXA2) and prostacyclin (PGI2) (32). It has been proposed that ET promotes the formation and release of EDRF endothelial-related oropert ies. which accounts Eor its vasodilator and rabbit aorta suggest ET-1 and ET-3 are Experiments in the luminally perfused aorta in equiact ive in releasing FJIRFas measured by bioassay with rabbit EDRF was characterized by both the rapid media containing hyoscine. down the bioassay o?scade and by the disappearance of its activity In contrast, prolongation of its survival by superoxide dismutase (32). unlike other substances which relax conductance vessels through the reLease of EDRF, ET-1 produces contraction in precoatractedvessels providingevidence

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against a transient role for EDRFin mediating ET’s actions on blood vessels. ET-1 has also been shown to release TXA and lXI2 from isolated perfused guinea pig and rat lung (24); prostagla I&in E2, PGI2 and TX42 from isolated perfused rabbit spleen; and prostaglandin E2 and PSI2 from rabbit kidney. Moreover, the vasoconstrictor response to ET-1 in rabbit isolated spleen or kidney is enhanced by indunethacin administration. These results support the hypothesis that ET-1 can promote the release of cyclooxygenase products with vasodilator activity (33). The vasodilator and vasoconstrictor effects of ET-1 in the isolated rat mesentery appears to be “tone-dependent .‘I ET-1 produces contractile responses on blood vessels (conductance and resistance) hich are not precontracted and dilates resistance vessels with an experimentally increased tone (32). In ET-1 has not been reported to relax precontracted isolated contrast, Taken together, these observations suggest ET may conductance blood vessels. exert differential effects on conductance, resistance or capacitance vessels of various organs. ET-1 increases the sinoatrial nodal rate of isolated right atria of guinea pigs but has no effect on the frequency of spontaneous contractions of The positive chronotropic activity to ET-1 isolated rat atria (34,35). undergoes tachyphylaxis, and, the mechanism for such a phenomenon is unclear. Altbugh the chronotropic effect of El’ msy be species-dependent, the positive inotropic activity of ET appears miversal. At nanomolar concentrations, ET-1 increases contractile force of the isolated guinea-pi (36), rat and timan This effect is atria (35,37) with ~~11 effects on human ventricle t 37). bunazosin, atenolol, reduced by nicardipine but unchanged by phentolamine, propranolol, atropine, (beta-2 adrenoceptor antagonist), ICI 118,551 diphenhydramine, cimetidine and methysergide (35). These data suggest ET-1 produces an incrmse in atria1 inotropic activity through a direct myocardial ef feet involving L-type voltage-sensitive calcium channels. In contrast, ET-3 has been shown to exert divergent inotropic effects on the isolated langendoff rat-heart preparation. At 0.1 nM (4) or 0.4 n?l (29), ET-3 produced a mild positive inotropic response. However, at 4-43 ti it decrased cardiac performance in a dose-related Eashion. The cardiodepression ms accompanied vasoconstriction cJhich was not altered by phentolamine, by coronary saralasin, L-660,771 (,X-571), a leukotriene D4 receptor meclofenamste, diltiazem, verapamil, nifedipine and hydralazine, but was antagonist (38)) These results suggest prevented by H-7 (protein kinase C inhibitor) (39). that the coronary vasoconstrictor response to ET-3, which likely mediates the of reduction in myocardial performance, occurs independently of the activation II and leukotriene D4 receptors, opening of alpha-adrenergic , angiotensin OK format ion of cyclooxygenase products. However, L-type calcium channels, in the an important role kinase C activation protein might PLY cardiodepressant and coronary vasoconstrictor response to ET-3 in isolated hearts. Non-Cardiovascular

Tissues

The contractile effect of ET is not limited to vascular srrroothmuscles. ET-1 contracts duodenum, stomach (24) bronchi and trachea (40,41). Similarly to isolated rat and rabbit blood vessels, tracheal strips from guinea-pigs respond to ET-1 with a contraction which is slow in onset and long in ‘lhe EC50 for this effect is over 100 fold smaller than those of duration. neurokinin A or leukotriene D4. Although the three substances studied have different potency, their efficacy is of the same order (40). The contractile response to ET-1 on isolated guinea-pig trachea and bronchi is partly inhibited by indometbcin and not by NiC12, w-conotoxin or tetrodotoxin (41).

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These data suggest ET-$;E;i;acts large airways inclependentlys~~i~;and N-type channel voltage-sensitive channels (42.43) or act&ion (44,45). The sn~ll effect of indomethacinindicatescyclooxygenase products play a minor role in the contractileresponse to ET-1 (41).

Conflictingdata on the obligatoryrole of extracellularcalcium in the contractile response to ET-1 have been reported. Nicardipineblocks the contractileresponse to ET-1 on guinea-pi trachea, whereas nifedipinewhich Although the potenciesoE these produces only a partial inhibition(40,41B . dihydropyridinecalcium entry blockers in many preparationsare different, the results of these studies imply that L-type voltage-sensitive calcium channels participateeither directly or indirectlyin the contractionof large airways produced by ET-l. When the epithelialcells of isolatedguinea-pig bronchi were removed, ET-1 produced a greater contractile response suggestingthat ET-1 can release an epithelial substancewith myorelaxantactivity or that ET decreased catabolism due to the absence of endothelial ;$Z::s (41). In isolated rat uterine horns, ET-1 enhances the rhythmicity and the magnitude of contractions; the latter being predominant at higher concentrations(46). These two effects are abolishedwhen Ca* is removed fran the medium. Furthermore,verapamil,nifedipine and diltiazemantagonize the enhanced rate of contractions evoked by ET-1 or oxytocin, whereas the sustainedcontractileeffects to ET-1 remain mchanged. Thus, these two types of effects are mediated by different cellular mechanisms utilizing Caf+. In human urinary bladder, ET induced dose-dependent contractileeffects which were not -modifiedby atropine or nifedipine (48). These data suggest that the contractileeffects of ET may be mediated diEferently in different portions of the genitourinarysystem. In Vivo Studies a) Cardiovasculareffects The available results on the actions of ET in viva, indicate that the integrated cardiovascular response to this peptlde is complex. The hemodynamicresponse to ET in the intact animal depends upon the vascular bed under study, the dose, route and rate of administrationof the peptide as well as the initial degree of vasomotor tone. In the initial study by Yanagisawa ET-1 produces a potent systemic pressor response in the et al. (l), anesthetizedrat pretreatedwith bunazosin,propranololand atropine. In this preparationas in pithed rats, i.v. bolus injectionsoE ET-1 produced a biphasic response, consisting of an initial, short-lasting decrease in systemic arterial pressure followed by pronounced and prolonged systemic hypertension(1,30). In intact SHR rats, the hypotensive eEfects are of larger magnitude than in normotensiverats (30,49). When taken together these studies in the rat suggest that the extent oE systemic hypotension in response to bolus i.v. administrations of ET-1 directly rlependson the degree of initial systemic vasomotor tone. This conclusionwas confirmed in pithed rats in which the systemic hyypotensive response to ET-1 was enhanced when systemic arterial pressure was raised to nonnotensive levels by an infusion of vasopressin(50). Since the systemic hypotensive responses to ET-1 in anesthetized dogs (51) and rats (30,50,52) and in conscious dogs and rats (53,49) are similar, the ability of ET to decrease systemicarterial pressure does not appear to depend on the animal's species and preparation (anesthetizedvs. conscious). In contrast, the hypertensiveresponse to ET-1 is inverselyrelated to the degree of initial systemic vasomotor tone.

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The decrease in systemicarterial pressurein responseto ET-1 is the resultoE the systemic vasodilator properties of this peptide sincecardiac output in rats, cats and dogs (30,54,55)is not decreased or slightly increased. This myocardial effect be due ’ part to baroreceptor-mediated phenomenonand in per?o a directmyo%dial effecti," as much as ET has positiveinotropic and chronotropic activity on isolated cardiactissue. 4lthoug.h all speciesstudiedappearto have a qualitatively uniformsystemichemodynamic responseto ET, the pig is an exception in that it does not respondto ET-1 with systemicvasodilation(56). Ihe reasonfor this differenceis mclear. {Jnlike the initialsystemic hypotensive responsewhich is short-lived, the secondarysystemichypertensive responseto ET-1 in the rat may Last up to sixty minutesdependingon the dose. This long durationdistinguishes ET-1 from othervasoconstrictorsubstances such as angiotensinII. The pressor effectsof bolus i.v.administrationof m-1 are accompaniedby eitherno change or a decrease in cardiac outputindicatingthis peptidepossesses potentsystemicvasoconstrictor activity(30). The rate of administrationappears to play a pivotal role in the hemodynamic resoonse to m-1. Intravenous infusions of ET-l, or conscious dog do (LO, 30 or 50 ng/minfor 60 min), into the anesthetized not provokeinitialhypotension.Rather,systemicarterialpressureincreased and cardiacoutput decreased(51,53). The sole systemic vasoconstrictor response to intravenousinfusionoE ET-1 is not uniqueto the dog since similar data have been reportedin rat (57). Recent data in the cat derrr>nstrate that as the rate of intravenous infusionof ET-1 is increased, the systemicresponseto the infusionbeginsto resemblebolus administration by producingsystemicvasodilation. Thesedata implythe systemicvasodilator responseto ET-1 requiresa relatively higo concentration of the peptideover a slmrtperiodof time. When injectedas an intraarterial holus,ET-1 has a qualitatively similar hemodynamiceEfectin the hindquarters, mesenteric, celiacand renalvascuLar beds. In cats underconditionsof constantregionalblood flow,low doses of ,i.a)typicallyproducevasodilation whereasas the dose ET-l ( 0.4-13pmol/kg is increased, a biphasic response occurs with predominantsecondary vasoconstriction (54). However,the hindquarter vascularbed appearsto be exquisitelysensitiveto the vasodilator propertiesof ET-1 which dilatesthis vascular bed at dosesas low as 400 fmole/kgwith higher doses having considerableefficacy (up to 60% reduction). Although the vasodilator activityof ET-1 in the renal,mesentericand celiacvascularbeds in vivo is small, ET-1 ( 13-130pmoles/kg.i.a)has markedly greater vasoznsmtor activityin thesevascularbeds when comparedto the hindquarter region. ET-1 appearsto have the greatest vasoconstrictor efficacy in the mesenteric vascularbed (30). In the pig, ET-1 (2-100pmol/kgi.v.) evokedan increase in coronary,femoral, bronchial,and renalvascularresistance. The latter regionalcirculationin the pig being the most sensitiveto ET-l (58). These data are consistentwith thoseoE Lipptonet al. (54) in the cat. In the pulmonaryvascularbed underconditionsof constantblood flowand Left atrialpressure,ET-1 elicits a snmll increasein Lobar arterialblood pressurewhich appearswithinone minuteafter Lobarbolus injectionand Lasts activityin the lung 2 to 8 minutes(59). ET-1 also has mild vasoconstrictor oE juvenilepigs in vivo (56). Ihesedata suggestthat at doseswhich have relativelylittlezfw in the pulmonaryvascular bed, ET-1 has a profound inELuenceon systemichemodyn3mics.

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Intra-coronaryadministrationof ET-l, SO p(r/kgto 200 pg/kg, into dogs induced a fall in coronary blood flow and subse uent increase in coronary In another study, ET s 30 to 300 &kg), vascular resistance (60). induced a fall in left circumflex artery blood Elow associated with dose-related falls mean arterial blood pressure, in cardiac output, left ventricular systolic All of tiich were only mildLy pressure and left ventricular contractility. In both studies, the highest doses of GT studied affected by diltiazem (61). induced ventricular fibrillation. Administered intra-cisternally in dogs, ET-L (10 to 1000 pool) induced in the basilar artery diameter. dose-related decreases Three days after administration of 10 pmol, the basilar artery still exhibited prominent vasoconstriction. in systemic blood pressure or heart rate were observed with this dEe’!??E peptide (62). These Eindings and the presence of binding sites for ET in the brain suggest ET may play a role in the development of cerebral vasospasm. bolus administrations of the sa.me dose of ET-1 After re eated i.v. (0.3 or 1.0 ugP into the cat, the decrease in systemic arterial blood pressure is markedly attenuated while the hypertensive phase is not affected, indicating that ET-L develops tachyphylaxis to its systemic hypotensive Furthermore, the Latter phenomenon appears to depend on the total effects. dose administered over the 4 to 6 hours of experimentation. Under the same ET-3 and sarafotoxin 6b Lndergo cross tachyphylaxis (personal conditions, Ihe development of cross-tachyphylaxis suggests that, similar to the data). myocardium (63), systemic blood vessels may have diRerent ET receptors that mediate vasodilation but use a common transduction psthway to elicit their Emct ional response. Alternatively, cross-tachyphylaxis to the systemic vasodilator response to ET may suggest that systemic blood vessels, possess a common ET receptor as described by Hirata et al. (12). It has been reported by De Nucci et al. (24) and Anggard et al. (27) that when ET-1 is infused in isolated guinea-pig Lungs, 60% is removed during the Since Lippton et al. (59) demonstrated that the first psss in the lung. systemic vasodepressor response to right atria1 and to left atria1 injections of ET-l in cat and rabbit are simiLar, Q-1 does not appear to be subjected to significant first-pass pulmonary metabolism. These data also suggest the to ET-L is not due to the release of a systemic hypotensive response vasodiLator substance by the lung. Similar results have been obtained in normotensive rats using intravenous and left ventricular bolus injections of ET-3 (personal data). In in viva microcirculatory preparations, such as the raboit dorsal skin (64F5)smster cheek pouch, and rat mesentery (66)) ET-1 behaves as a very potent vasoconstrictor substance. &I the rabbit skin, a-1 is as potent II, 3rd inhibits the vasodilation induced by as vasopressin and angiotensin calcitonin gene-related peptide (CGKP) (64). In hamster cheek pouch, ET-L is more effective in arterioles than venules and its eEEects are long lasting. No initial vasodilator response to ET-1 was observed in microcirculatory vessels, in contrast to what mis observed in systemic and regional vascular beds cin viva. De Nucci et al. (24) reported that ET-L releases TXA2 and FGI2 frown the Moreover, guinea-pig and rat lung. the secondary systemic vasopressor response to i.v. bolus injections of m-1 into the rat is enhanced by indomethacin and piroxicam. From these results the authors concluded that XI2 is liberated to limit the pressor activity of ?SI’. ‘ke systemic hypertensive response to ET-1 in the pithed rat is unchanged when animals are

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phentolamine, methysergide, enalapril, SK&F 100273 inhibitor), 755~ (Lipoxygenase and cyclooxygenase or desipramine,but is reduced by diltiazem,verapamil,~nitrendipineand cromakalim. These resultssuggestthat the systemichypertensive responseto ET-1 is not mediated by alphaadrenergic,serotonergicor vasopressin-1 receptors and does not depend on formationof angiotensinII as well as cyclooxygenase productsof arachidonic acid metabolism(30). In addition,the fall in systemic arterial pressureobserved in anesthetizedrats is not antagonized by atropine,methysergide, SCH 23390 (a DA-1 dopamineantagonist), (S)-sulpiride,promethazine, idazoxan, propranolol, RP 59227 (PAPreceptor antagonist) and glibenclamide. Thus the hypotensive effectsof ET-1 are not mediated by muscarinic,serotonergic,dopaminergic, histaminergic,PAF, alpha-2 or beta adrenergic receptors, or opening of vascular glibenclamidesensitiverC+ channels. Similarly,the systemicvasodilator responseto i.v.bolusadministration of l?T-Lin cat and rabbit is unchanged by indomethacin, atropine,ICI 118,551,RN 52021 (PAPreceptorantagonist) and naloxone(57). antagonist), BW Pvasopressin

Sincethe initialsystemicvasodepressor responseto ET-1 is not altered by cyclooxygenase blockade,De Nucci et al. (24) suggestedthat the systemic vasodilator response to ET-1 is not mediated by PC12 but is mainly due to releaseof EDRF. However,directevidenceof the lattermechanismis not yet available. b) Otheractivities Under conditions of continuousmechanicalventilation?m-1 has been reported to increase peak inspiratory pressure in the guinea-pigwith an efficacysimilarto that of histamine and plateletactivating factor(PAP). The bronchoconstrictor responseto an aerosolof ET-1 (68) is not associated with changeson systemicarterialpressurewhereasi.v.administered U-1 (69) producessystemicLypertension.The conflicting influenceof MT-1 on systemic arterialpressure in these studiessuggests the interstitial-alveolar wall barriercan preventthe leakage of Locally releasedET-1 to the general responseto aerosolized ET-L, L-LOug/ml circulation. The bronchoconstrictor over 1 ininor 0.05-lnmol/kg i.v. bolus, is not altered by mepyramine, propranolol, nifedipine and verapamilbut is reduced by indomethacinand BY-52021. Thus, PAP and cyclooxygenaseproducts of arachidonic acid metabolism appear to mediate the bronchoconstrictor response to ET-1 in vivo (70). AlthoughET-l constrictspulmonarybloodvessels,this effectE not alteredby indomethacin (personaldata). lhus, ET-1 contractstwo types of smooth muscle(vascular and bronchial) in the lung by two different mechanisms. In perfused hydronephrotic rat kidneys,EX reduced afferentarteriolar diameterin a dose-dependentmanner. This vasoconstriction was associated with oscilhtory vasomotion and reversed by nifedipine. The efferent arteriolardiameterwas only slightlyaffectedby ET. TheseET contractile effectsLead to a dramatic decreasein glomerularfiltration rate, in normal rat kidneys perfusedunder the ssme conditions. Nifedipinealso returned glanerularfiltrationrate to controllevel(47). In rats,ET-1 administered intra-arterially into the left gastricartery inducedgastricdamagesacrossthe wholemucosalsurfaceas indicated by the presence0E areas of vasocongestion,hemorrhage and necrosis. Atropine, phentolamine,propanolol, cimetidine,indanethacin or l3W A4C (5-lipoxygenase inhibitor) failedto antagonizetheseeffects(70).

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ET-1 seems to be involved in inflammatoryprocesses since injected into the rat paw, it induces hyperalgesia,which in contrast to carrageenan-induced blocked by hyperalgesia, is not indomethacin or guanethidine. When administered i.p. to mice, ET-1 evokes abdominal contractionssimilar to those induced by acetic .acid which are antagonizedby indomethacin. ET-1 wokes transient incapacitationwhen administered into a dog knee joint. Moreover, in the same preparationit potentiateda s&effective dose of prostaglandinE2 given 2 hours aEter D-1 suggesting a EacilitatoryeEfect of ET-l. In human forearm, an intradermalinjection of ET-L causes intense and long Lasting pruritus associated with the development oE local erythema and heat. In addition, ET-3 may be released from neural tissue. This can add further relevance to the possible role of isoforms OE the ET peptides in the modulationof nociceptors(71). Influence of Endothelinon the Release oE EndogenousSubstances In cultured rat atria1 myocytes and in rat atria, ET-1 releases ANF (72,73). conscious and anesthetized dogs, ET-1 infusion (10,30, or 50 ng/$ min, over 1 hour) increasesANF Levels. However, these increases in ANF levels in vivo may be due to a bare-release eEfect since atria1 pressure was elevatz by ET-1 infusion. In isolated rat glomeruli and juxtaglomerularcells, ET-1 inhibits renin release (74,75). It is suggested this eEfect is mediated by an increase in intracellular C&*, because it does not occur in Ca* free medium (74). In contrast, plasma renin activity is unchanged 5 min sEter an i.v. bolus injectionof ET-1 (0.25 nmoL/kg) in pithed rats (30). However, in conscious and anesthetized dogs, an hour infusion of ET-1 (30 and 50 ng/kg/min), respectively,evokes a late rise in plasma renin activity which is likely due to renal arterial vasoconstriction(51,53). The effects of ET-1 have been studied in nerve-induced contractile responsesand on the stimulation-evokedrelease oE [3H] norepinephrine (NE) in the guinea pig femoral artery. In these experimentsET-1 is extremely potent in reducing release of NE and nerve-inducedcontraction. These data suggest ET-1 may act presynapticallyto influenceadrenerqic transmitter release and thus mod&&e autonomic nervous system activity (76). These Eindings are supportedby studies in vivo where one hour infusion of ET-1 (lo ng/kg/min) in -7the dog induces a fall in NE plaslnaconcentration. In contrast, a higher inEusion rate of m-1 (30 ng/kg/min) increases cpinephrine and NE plasma content, probably through stimulationof the adrenal medulla (53). Since ET-1 affects the release of ANF, renin and adrenergic transmitter,it may play a role in a variety of cardiovasculardiseases. In platelet rich plasma from humans, dogs and rabbits as well as in washed human platelets,ET-1 has no direct aggregatoryeffect, nor does it influenceaggregatoryresponses to a variety of substancesincluding thrombin, arachidonicacid, U-46619, collagen, GDP and prostaglandinH2 (personaldata). In contrast, the aggregatoryresponse to ADP is markedly depressed in platelet rich plasma taken from anesthetized rabbits pretreated with nmoL/kg, i.a.) (77). This effect is prtially reversed by indoE;kc!i pretreatmentsuggesting that m-1 promotes --in vivo the release 0E PSI2 to lwels which interfere with platelet function. IntracellularYechanism of Action Data have accumulatedconfirning the requirementof calcium for ET-1 to in vitro conditions. produce a contractileresponse in blood vessels under --

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However,it is now clearlydemonstrated thatET-L is not an endogenousagonist of dihydroyridine sensitiveCa* channels(15,21,28) as it was initially ET-1 increases intraceLLuLar calcium in fura- Loaded rat proposed(1. P aorticsmoothmusclecells (12,23)or Labelledwith the fluorescentindo-lin labelled culturedrat aorticsmoothmuscle(78). This effectwas dependent, in part,on extracellular Ca*, howeverother studiesdemonstrate an elevation in intracellular calciumin calcium-free medium. Calcium-dependent effectsof ET-1 have also been characterizedby Miasiro et al. (79) and Marsden et al. (23). ET-L stimulates phospholipase C and phosphoinositide turnover(22,23,80,81)which activates a non-selective cationic channel This depoLarizationbringsthe membraneto potential permeableto Ca*. Levels which activate T,-typeCa++ channels(80). Moreover, Sigiura et al. (22) showedthat the proteinkinase C inhibitor, H-7, reversesET-1 inducedcontractile responses on isolated rabbitaortic strip. Thesedata suggestET-L promotescontraction of vascularsmoothmuscleat Least,in part, throughan increasein IP3 turnoverfollowedby activationof proteinkinase C. Recent studiesby Ohlstein et al. indicate that ET-L does not alter cyclicAMP and cyclic OivmLevels in rabbitaorta(82). These investigators suggest4 thatET-inducedcontractionis not mediatedvia an interaction with voltage-dependent, calciumchannelsand involvesutilizationof superficially bound calciumas well as releaseof calciumfrom intracellular stores. Since staurosporine inhibitedthe contractileresponseto ET-L on rabbit aorta,it was also suggestedET-1 acts through stimulationof phosphatidylinositol turnoversrxlactivationof proteinkinaseC (82). StructureActivityRelationship Endothelinsare 21 amino acid polypeptidespossessingtwo intrachain disulfidebridges. AlthoughET-3 and ET-1 differin amino acid sequenceat positions 2, 4, 5, 6 , 7 and 14 (Fig.L), both have qualitativelysimilar contractile effectsin vitro and vasodilator activit in vivo. ET-2 diEfers from ET-1 in two Gizids at positions"6 and 7K , and its biological activityresemblesthat of ET-1 (personalobservation).ET-L appearsto have, in general, a greater potency than ET-3 in all preparationsstudied so far (5,31),althoughLow doseshad Less vasodilator activitythan ET-3 in the isohted rat mesentery(31). Althoughthese isopeptides (ET-l,m-2, U-3) are homologousat the six aminoacids of the C-terminal tail,the commonhexapeptide has no vascular activity in vitro or in vivo (83). When Trp in position21 is removed from ET-l the EowaL aEi* is reduced by three ordersof magnitude. The progressive removalof amino acid residues fromthe terminal moietyfurther decreasesthe activityand the l-16 residueoE ET is inactive. The reduction of the two disulfidebonds Leading to and openingof the intramolecular Loop reducesthe activityby two ordersof magnitudewhen comparedto nativeET's. The substitution of the cysteinsin position3 and 11 by alanineresidues decrease the potency of ET to contract isolated rat aortic rings by a factor12. However,in contrastto ET, [ALa3'LL]-ET activitywas not affected by removalof endothelium (84). Sarafotoxins (SRT6a, 6b, 6c) are a groupof 21 aminoacid polypeptidesisohted from the venom of snake Atractasis engaddensis (7,8,85,86). These snakepeptidespossessstrikingslml *-=i---+ arlty m structureto EXs and may be consideredas naturalanalogsof ET or "reptilian ETs". Up to now, the predominant biologicactivityfor SRT was reportedto be cardiotoxicity (84,85,86). It is of interestto note that SRT-6bactivates pt~pl-oinositolhydrolysis in brain independently of extraceLLuLar calcium(88,89,93),and similarly to ET-l, binds to rat atria and

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brain (88,89), particularly in cerebellum, thalamus and hypo@$amuu regions (90). Moreover, sarafotoxinSb displaces specificallybound in rat heart membranes (91). Synthetic or purified sarafotoxin 6b h&s approximately50-80% greater vasodilatoractivity than ET-1 in the feline systemic and hindquartervascular bed -in vivo (personaldata). 41though ET-1 constricts the feline mesenteric and pulmonaryvascular bed in vivo, similar doses of sarafotoxin6b dilate both of these vascular beds (esoxdata). The relative rank potency for the systemicvasodilatoreffect oE these four polypeptidesis as follows: SRTGb> ET-l=ET-2>ET-3. Possible Involvementof ET in CardiovascularDisease A pathological state that may involve ET is hypertension. This suggestionis in agreement with the fact that renal arteries from SHR are more sensitive to ET than that of normotensiverats (20) although SHR exhibit increasedvascular reactivityto many constrictor substances. A role of EC in cell proliferationand possibly atherosclerosis is suggested by the study oE Kumuro et al. (78) who demonstratedthat in rat vascular smooth muscle cells incubated with ET-l, c-fos and c-myc mRNA, the incorporation of [methyl-3H]-thymidine,and intracellular I&2+] increased. Moreover, Nakaki et al. (71) demonstratedthat ET stimulatesg: synthesis in vascular smooth muscle (92). In light of ET's effects on blood vessel reactivity smooth muscle cell proliferation,release of ANF, renin and NE, any abnormalityon ET production or its receptor function may lead to cardiovasculardiseases. In conclusion,although the role for ET in health and disease is not yet clarified, future studies should permit us to understand the relevanceof this unique family of endogenousmolecules as endocrine,paracrine, autocrine and intracrineregulatory peptides.

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