In vivo and in vitro action of endothelin-1 on goat cerebrovascular bed

This study concerned the effects and mechanisms of action of endothelin-1 on the cerebral circulation. Cerebral blood flow was electromagnetically measured in awake goats. Endothelin-1 (0.01-0.3 nmol) produced dose-dependent decreases in this flow (maximal reduction = 34%) and increases in cerebrovascular resistance (maximal increase = 74%) (P < 0.01). IRL 1620 (Suc-[Glu9, Ala11,15]endothelin-1-(8-21), agonist for endothelin ET(B) receptors, 0.01-0.3 nmol) slightly decreased cerebral blood flow. The effects of endothelin-1, but not those of IRL 1620, on cerebral blood flow were diminished by 50% during infusion of the antagonist for endothelin ET(A) receptors, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-Trp), 2 nmol min(-1)), but not affected during infusion of the antagonist for endothelin ET(B) receptors, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-Leucyl-1-(met hoxycarbonyl)-D-tryptophyl]-Dnorleucine monosodium), 2 nmol min(-1)). Intravenous administration of NW-nitro-L-arginine methyl ester (L-NAME, 47 mg kg(-1)) or NW-nitro-L-arginine (L-NNA, 47 mg kg(-1)) reduced basal cerebral blood flow by 39 and 33%, increased cerebrovascular resistance by 108 and 98% and mean arterial pressure by 23 and 17%, and decreased heart rate by 27 and 25%, respectively (all at least P < 0.05). The increases in cerebrovascular resistance (as absolute values) induced by endothelin-1 were not affected during either L-NAME or L-NNA (as absolute values and percentages). Intravenous administration of meclofenamate (5 mg kg(-1)) did not change the cerebrovascular effects of endothelin-1 and IRL 1620. In isolated goat cerebral arteries under control, resting conditions, endothelin-1 (10(-11)-10(-7) M) induced concentration-dependent contractions (EC50 = 4.78 X 10(-9) M; maximal contraction = 3177+/-129 mg), whereas IRL 1620 (10(-11)-10(-7) M) produced no effect. This contraction produced by endothelin-1 was competitively blocked by BQ-123 (10(-7)-3 X 10(-6) M), and was not affected by BQ-788 (10(-6) and 10(-5) M). L-NAME (10(-4) M), meclofenamate (10(-5) M), indomethacin (10(-5) M), L-NAME (10(-4) M) plus meclofenamate (10(-5) M) and phosphoramidon (10(-4) M) did not affect the contraction in response to endothelin-1. Endothelium removal increased the response to endothelin-1, as well as the BQ-123 antagonism against endothelin-1 (pA2 values, 7.62 vs. 6.88; P < 0.01). In both intact and de-endothelized arteries precontracted with prostaglandin F2alpha endothelin-1 induced a further contraction, and IRL 1620 caused no effect. These results suggest that: (1) endothelin-1 produces cerebral vasoconstriction by activating endothelin ET(A) receptors probably located in smooth muscle; (2) endothelin ET(B) receptors, nitric oxide and prostanoids might be not involved in the cerebrovascular action of endothelin-1, and (3) endothelium removal may increase cerebrovascular reactivity by increasing sensitivity of endothelin ET(A) receptors to endothelin-1.     

Endothelin-1 induces vasoconstriction and nitric oxide release via endothelin ET(B) receptors in isolated perfused rat liver

Endothelin-1 (0.1, 1 and 10 nM) induced a significant increase in portal pressure and nitric oxide (NO) release in the isolated rat liver. The endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala(11,15)]endothelin-1-(8-21)) (0.1, 1 and 10 nM) also elicited a marked increase in portal pressure and NO release. The potency of endothelin-1 was higher than that of IRL 1620. The endothelin ET(A) receptor antagonist, BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu)) (1 and 10 microM), had no effect on the endothelin-1-induced change in portal pressure and NO current. In contrast, the endothelin ET(B) receptor antagonist, BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (1 and 10 nM), attenuated the endothelin-1-induced change in portal pressure and NO current. Administration of N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, completely abolished the endothelin-1- or IRL 1620-induced NO release. L-NMMA enhanced the increase in portal pressure and decrease in O2 consumption caused by endothelin-1. These results indicated that endothelin ET(B) receptors mediate both vasoconstriction and NO release and that NO plays a significant role in stabilizing microcirculation in isolated perfused rat liver.     

Teratocarcinosarcoma of the paranasal sinuses: a clinicopathologic and immunohistochemical study

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 > sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

High levels of cytokine-producing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome

Activation of endothelin receptors on the vasculature can produce a variety of responses from potent vasoconstriction to mild vasodilation, depending on the receptor complement within the tissue. To elucidate the potential role of endothelin analogues as tumour blood flow modifiers, we have evaluated the effect of the ET(B) receptor agonist, IRL 1620 ([Suc-(Glu9, Ala(11,15))-ET-1(8-21)]) in CBH/CBi rats bearing an HSN fibrosarcoma. Tissue blood flow and vascular resistance were determined, 20 min following administration of IRL 1620 (bolus intravenous), using the uptake of radiolabelled iodoantipyrine (125I-IAP). Blood flow was unchanged in most tissues. However, at doses > or = 1.0 nmol kg(-1) IRL 1620, blood flow in the brain and heart was increased, whereas in the small intestine it was reduced. Blood flow in the skeletal muscle was reduced at 1.0 nmol kg(-1) only. Tumour blood flow was significantly reduced at 3.0 and 5.0 nmol kg(-1). Vascular resistance was unchanged in most tissues although it was increased in the skeletal muscle at 1.0 nmol kg(-1), in the kidney at 1.0 and 3.0 nmol kg(-1) and in the brain and heart, it was reduced at 5.0 nmol kg(-1) IRL 1620. Vascular resistance was significantly increased in the tumour and the small intestine at doses > or = 1 nmol kg(-1) IRL 1620. Pretreatment of rats with BQ-788, an ET(B) receptor antagonist, selectively attenuated the tumour vascular response to 3 nmol kg(-1) IRL 1620 with no changes observed in the normal tissue responses. Our results demonstrate that the HSN tumour vasculature is selectively responsive to IRL 1620 at doses > 1 nmol kg(-1) compared with the majority of normal tissues with the exception of the small intestine, and that only the tumour response is highly sensitive to BQ-788 antagonism, under the experimental dosing regime investigated. These differences may be exploitable for therapeutic benefit.     

Diuresis and natriuresis produced by long term administration of a selective Angiotensin-(1-7) antagonist in normotensive and hypertensive rats

Intrarenal arterial infusion of endothelin-1 (1, 3 and 10 ng/kg per min) reduced renal blood flow, urine flow rate and urinary Na+ excretion without affecting fractional Na+ excretion in anesthetized rabbits. An endothelin ET(A) receptor antagonist (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-me thyl-pentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl]amino-3-(2-pyr idyl)propionic acid (FR139317, 1 microg/kg per min) attenuated the endothelin-1 (1 ng/kg per min)-induced renal responses. An endothelin ET(B) receptor antagonist N-cis 2,6-dimetylpiperidinocarbonyl-L-gamma-metylleucyl-D-1-met hoxycarbonyltryptophanyl-D-norleucine (BQ-788, 1 microg/kg per min) potentiated the endothelin-1-induced changes in renal blood flow, urine flow rate and urinary Na+ excretion. A nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 50 microg/kg per min) also potentiated the endothelin-1-induced reductions in urine flow rate and urinary Na+ excretion but not the reduction in renal blood flow. Endothelin-1 reduced fractional Na+ excretion in the presence of BQ-788 or L-NAME. A spontaneous NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (30 ng/kg per min) slightly attenuated the antinatriuresis but not the vasoconstriction induced by endothelin-1. These results suggest that in the rabbit kidney in vivo endothelin ET(A) receptors mediate endothelin-1-evoked vasoconstriction and tubular Na+ reabsorption, that the concomitant stimulation of endothelin ET(B) receptors by endothelin-1 counteracts both the ET(A) receptor-mediated vascular and tubular actions, and that the tubular action, but not the vascular action, of endothelin-1 is also susceptible to changes in renal NO level.     

Atypical receptors mediate the response to endothelin-1 and sarafotoxin S6b in the human umbilical artery

The receptors mediating smooth muscle response to endothelin-1 and sarafotoxin S6b in the human umbilical artery were investigated in vitro. Both agonists induced contractions that were unaffected by the endothelin ET(B) receptor antagonist BQ 788 (10(-9), 10(-8), 10(-7) M). The non-selective endothelin ET(A/B) receptor antagonist PD 142893 (10(-7) M) decreased the contraction induced by endothelin-1. PD 142893 (10(-9) M) enhanced the contraction induced by sarafotoxin S6b whereas higher concentrations had no effect. Removing the endothelium did not affect the antagonising action of PD 142893 on endothelin-1-induced contractions while the enhancement of the sarafotoxin S6b-induced contraction was abolished. Sarafotoxin S6b induced relaxation in segments precontracted by 5-hydroxytryptamine and exposed to the endothelin ET(A) receptor antagonist BQ 123 (10(-7) M) and PD 142893 (10(-9) M) abolished this relaxation. These endothelial receptors seem neither to be classical endothelin ET(A) nor endothelin ET(B) receptors and they are not activated by endothelin-1.     

Effect of endothelin-1 on corticosteroid secretion by the frog adrenal gland is mediated by an endothelinA receptor

We have previously reported that endothelin-1 (ET-1) stimulates the in vitro secretion of corticosterone and aldosterone from the adrenal gland of the frog Rana ridibunda. The aim of the present study was to investigate the pharmacological profile of the endothelin receptor subtype involved in the corticotropic effect of ET-1. The mixed ET(A)/ET(B) receptor antagonist Ro 47-0203 (10(-5) M) totally blocked the stimulatory effect of ET-1 (5 x 10(-9) M) on corticosterone and aldosterone secretion. The action of ET-1 was also inhibited by the selective ET(A) receptor antagonist BQ-485 (10(-7) M). In contrast, the selective ET(B) receptor antagonist IRL 1038 (10(-6) M) did not affect the response of the frog adrenal gland to ET-1. In addition, the selective ET(B) receptor agonist IRL 1620 (10(-6) M) did not mimic the stimulatory effect of ET-1. The high affinity ET(C) receptor agonist endothelin-3 (ET-3) stimulated corticosteroid secretion, but was 400 times less potent than ET-1. Moreover, the action of ET-3 was also blocked by BQ-485 (10(-7) M). These data indicate that the stimulatory effects of ET-1 and ET-3 on corticosteroid secretion by the frog adrenal gland are mediated by an ET(A) receptor subtype.     

ET(B) receptor and nitric oxide synthase blockade induce BQ-123-sensitive pressor effects in the rabbit

Endothelin-1 (0.25 nmol/kg, injected into the left cardiac ventricle) induces a protracted increase of mean arterial pressure that is significantly reduced by the selective ET(A) receptor antagonist BQ-123 (1 and 10 mg/kg) in the anesthetized rabbit. The sole administration of the selective ET(B) antagonist BQ-788 (0.25 mg/kg) induces a pressor response abolished by BQ-123 (1 mg/kg). Concomitant to the increase in mean arterial pressure, BQ-788 induces a significant increase in plasma levels of endothelin-1 and its precursor big endothelin-1. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) also increases arterial blood pressure, and the response is reduced dose-dependently by BQ-123 (1 and 10 mg/kg). In addition, the administration of BQ-788 in the presence of L-NAME induced a further increase in arterial blood pressure. The duration of the pressor response to L-NAME is also significantly reduced by an endothelin-converting enzyme inhibitor, phosphoramidon (10 mg/kg). Finally, L-NAME induces an increase in plasma levels of big endothelin-1 but not endothelin-1. Our results illustrate that blockade of either nitric oxide synthase or ET(B) receptors triggers a raise in plasma levels of endothelin-1 or its precursor. These later moieties are suggested to be significantly involved, through the activation of ET(A) receptors, in the pressor effects of L-NAME and BQ-788 in the anesthetized rabbit.     

Endothelin-1-(1-31), a novel vasoactive peptide, increases [Ca2+]i in human coronary artery smooth muscle cells

We have previously found that human chymase cleaves big endothelins at the Tyr31-Gly32 bond and produces 31-amino acid long endothelins-(1-31), without any further degradation products. In this study, we investigated the effect of synthetic endothelin-1-(1-31) on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Endothelin-1-(1-31) increased [Ca2+]i in a concentration-dependent manner (10(-14) to 10(-10) M). This endothelin-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon (N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-L-Leucyl-L-Tryptoph an), an inhibitor of endothelin-converting enzyme. It was, however, inhibited by 10(-10) M BQ123 (Cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu-)), an endothelin ET(A) receptor antagonist, but not by 10(-10) M BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-yMeLeu-D-Trp(COOM e)-D-Nle-ONa), an endothelin ET(B) receptor antagonist. These results suggest that endothelin-1-(1-31) by itself exhibits vasoactive properties probably through endothelin ET(A) receptors. Since human chymase has been reported to play a role in atherosclerosis, endothelin-1-(1-31) may be one of the candidate substances for its cause.     

Endothelin ET(B) receptors counteract venoconstrictor effects of endothelin-1 in anesthetized rats

The inhibitory effects of BQ 788 (3 mg/kg, i.v., ET(B)-receptor antagonist) on endothelin-1 (ET-1)- or IRL 1620 (ET(B)-receptor agonist)-induced changes in mean arterial pressure (MAP), mean circulatory filling pressure (MCFP, driving force of venous return), arterial resistance (RA), venous resistance (RV) and cardiac output (CO) were characterized in 6 groups of pentobarbital-anesthetized rats. ET-1 or IRL 1620 (0.5, 1 and 2 nmol/kg, i.v.) dose-dependently increased MAP, RA, RV and MCFP and decreased CO. Maximum changes in RA, RV and CO elicited by ET-1 were greater than those by IRL 1620. Equimolar doses of ET-1 and IRL 1620 also caused similar initial transient decreases in MAP. BQ 788 alone slightly elevated MCFP, but did not alter other variables. The ET(B)-blocker abolished all changes elicited by IRL 1620, but only partially inhibited its responses on MCFP, showing the presence of BQ 788-insensitive receptors. BQ 788 also abolished ET-1's depressor response, partially inhibited its effect on MCFP, and markedly augmented its effects on RA, RV and CO. Thus, ET(B)-receptors counteract the sustained constrictor effects of ET-1 on arterial and venous resistance vessels Our results indicate a substantial arterial and venous dilator role for ET(B) receptors.     

Role of endothelium in the endothelin-1-mediated potentiation of the norepinephrine response in the aorta of hypertensive rats

OBJECTIVE: To investigate the role of the endothelium in the functional interaction between endothelin-1 and norepinephrine in the contractile response of aortas from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Thoracic aorta rings with and without endothelium from SHR and from WKY rats were suspended in an organ bath to record the isometric tension. After an equilibration period of 120 min, the preparations with and without endothelin-1 were subjected to single and cumulative additions of norepinephrine in different experiments. To characterize the mechanisms involved in the interaction between endothelin-1 and norepinephrine, the aortic rings were pretreated with a cyclooxygenase pathway inhibitor (piroxicam, SO29548), an inhibitor of NO synthase [NG-nitro-L-arginine (NLA)], or selective endothelin receptor blockers (BQ-123 or BQ-788). In some experiments we examined the contractile responses to norepinephrine in aortas pretreated either with angiotensin II (AII) or with U46619, an agonist of prostaglandin H2-thromboxane A2 receptors. Finally, we examined the effect of the combination of calcium-entry blockade by administration of nifedipine and treatment with either endothelin-1 or U46619 on the norepinephrine reactivity. RESULTS: Administration of 3 x 10(-10) mol/l endothelin-1 potentiated the contractile response to norepinephrine in SHR aortas with endothelium, irrespective of whether they had been treated with NLA. No endothelin-1-mediated enhancement of the response to norepinephrine was observed in SHR denuded rings and in untreated and NLA-treated WKY rat aortas. All did not affect the response to norepinephrine in SHR rings with endothelium. The amplification by endothelin-1 of the response to (1-100) x 10(-9) mol/l norepinephrine was abolished by blockade of the cyclooxygenase pathway with piroxicam or SO29548. In WKY rat and SHR denuded aortas, 10(-8) mol/l U46619 potentiated the contractile responses to norepinephrine. Administration of 3 x 10(-6) mol/l BQ-123 abolished the increase in reactivity to norepinephrine evoked by endothelin-1 in intact SHR aorta, whereas 3 x 10(-6) mol/l BQ-788 failed to modify this potentiating effect. Administration of 10(-8) mol/l nifedipine inhibited the potentiation of the norepinephrine-induced contractions evoked both by endothelin-1 in SHR aortic rings with endothelium and by U46619 in SHR denuded rings. CONCLUSION: Our results show that a low concentration of endothelin-1 induced potentiation of the contractile response to norepinephrine in SHR aortas but not in WKY rat aortas. This response was endothelium-dependent. Furthermore, our study affords functional arguments that both endothelial and smooth muscle pathways are involved in the potentiating interaction. We propose that endothelin-1 stimulates the production of endothelium- and cyclooxygenase-generated vasoconstrictor factors, which in turn may serve directly as priming stimuli at the vascular smooth muscle level, to activate the Ca(2+)-signal pathway and consequently to increase locally the vascular sensitivity to norepinephrine.     

Mechanism of prostaglandin E2-, F2alpha- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E2-, prostaglandin F2alpha- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F2alpha)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E2 caused maximum relaxation of endothelin-1 precontracted vessels (EC50: 1.8 x 10(-8) M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor, L-NAME (N(G)-Nitro-L-arginine methylester). CGRP-(8-37) (calcitonin gene-related peptide fragment (8-37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK1 receptor blocker, GR 82334 ([D-Pro9[Spiro-gamma-Lactam]Leu10,Trp11]physalaemin (1-11)), markedly attenuated the response. Both prostaglandin F2alpha and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1alpha(Z),2beta,3beta,5alpha]]-(+)-7-[5-([1,1'-b iphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-he ptenoic acid), a thromboxane receptor antagonist (EC50: for prostaglandin F2alpha 7.9 x 10(-9) M, and for latanoprost acid 4.9 x 10(-9) M). L-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8-37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E2 could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Characterization of endothelin receptors in the human umbilical artery and vein

The aim of the present study was to characterize pharmacologically endothelin receptors that are present in human umbilical vessels. 2. Endothelin-1 (ET-1) and endothelin-2 (ET-2) are potent stimulants of both the human umbilical artery (pEC50 7.9 and 7.5) and vein (pEC50 8.1 and 8.0). Endothelin-3 (ET-3) is inactive on the artery but contracts the vein (pEC50 7.6). IRL1620 is inactive in both vessels. The order of potency of agonists is suggestive of a typical ET(A) receptor in the artery (ET-1 = ET-2 > > ET-3) and a mixture of ET(A) and ET(B) receptors in the vein (ET-1 = ET-2 > or = ET-3). 3. The selective ET(A) receptor antagonist, BQ123, competitively inhibits the effect of ET-1 in the human umbilical artery (pA2 6.9), while in the vein, only a mixture of BQ123 and BQ788 (a selective ET(B) antagonist) weakly displaces to the right of the cumulative concentration-response curve to ET-1. Contractions induced by ET-3 in the vein are inhibited by BQ788 (pA2 7.6), but not by BQ123. 4. Inhibition of Ca2+ channels by nifedipine (0.1 microM) is accompanied by a significant decrease of the maximal response to ET-1 by 40% in the artery and by 30% in the vein. The response of the vein to ET-3 is almost abolished by nifedipine. 5. The results indicate that: (i) endothelins contract the human isolated umbilical artery via stimulation of an ET(A) receptor type; (ii) the contraction induced by ET-1 in the vein is mediated by both ET(A) and ET(B) receptors, while ET-3 stimulates the ET(B) receptor; (iii) the contribution of Ca2+ channels to the contraction mediated by the ET(B) receptor appears to be more important than to that mediated by the ET(A) receptor.     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists. 2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (-log EC50) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18). 3. FR139317 (1-3 microM) and BQ-123 (1 microM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 microM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 microM) had no additional effect. RES-701-1 (0.1-1 microM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 microM). 4. Modulation of the Ca2+ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 microM) abolished rhythmic motility induced by ET-1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 microM) completely inhibited ET-1-induced contractions. 5. We conclude that there are two types of ET-receptors, excitatory ET(A)- and ET(B)-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca2+ channels (ROCs) and partly by opening T-type Ca2+ channels, and mediate rhythmic motility by opening L-type Ca2+ channels.     

ETA and ETB receptors on vascular smooth muscle cells from mesenteric vessels of spontaneously hypertensive rats

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.     

Mechanisms of endothelin-induced macromolecular leakage in microvascular beds of rat mesentery

Microvascular responses to endothelin-3 were investigated in the rat mesentery under fluorescence microscopy. Endothelin-3 in a range of 0.1-100 pM induced arteriolar constriction in a dose-dependent manner, and stimulated Ca2+ mobilization, demonstrated by fura-2-associated fluorography, in both arterioles and venules. Cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-) (BQ123), and endothelin ETA receptor antagonist, at a concentration of 10 microM inhibited the endothelin-3 (100 pM)-induced arteriolar constriction and Ca2+ mobilization in arterioles but not in venules. In venules, an early onset leakage of FITC (fluorescein isothiocyanate)-labeled albumin and subsequent reduction of red blood cell velocity without arteriolar constriction were observed after the superfusion of endothelin-3 with BQ123, suggesting that a non-endothelin ETA receptor mediates macromolecular leakage followed by a decrease in blood flow. Endothelin-3 with BQ123 neither stimulated leukocyte adhesion nor activated luminol-dependent chemiluminescence in venules, showing that endothelin-3-increased permeability may be induced by leukocyte-independent and oxyradical-independent mechanisms. These microvascular alterations of permeability and red blood cell velocity were significantly attenuated by the addition of phalloidin, an F-actin stabilizer, suggesting the involvement of endothelial cell contraction. Nicardipine (1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]methyl-2- [methyl(phenylmethyl)amino]-3,5-pyridinedicarboxylic acid ethyl ester), a dihydropyridine-type Ca2+ channel antagonist, eliminated endothelin-3-induced arteriolar constriction; however, it did not affect albumin leakage promoted by endothelin-3 with BQ123, suggesting that a non-voltage-dependent Ca2+ channel(s) is involved in non-endothelin ETA receptor-mediated Ca2+ mobilization and contraction of venular endothelial cells. Overall, it is conceivable that endothelin ETA receptor and voltage-dependent Ca2+ channel are involved in endothelin-3-induced arteriolar constriction. In addition, the present results suggest that Ca2+ mobilization in venular endothelium, which is mediated by a non-endothelin ETA receptor, possibly endothelin ETB receptor and regulated by non-voltage-dependent Ca2+ channel(s), may cause endothelial cell contraction and subsequently increase macromolecular permeability in microvascular beds treated with endothelin-3.     

Regulation of the immune response--lessons from transgenic models

Volatile anaesthetics induce hypotension by both indirect and direct effects; they have been reported to inhibit vasoconstriction produced by a variety of agonists. These studies were performed to see if halothane, enflurane and isoflurane attenuate endothelin-1-evoked contraction and if they interact with endothelium-dependent or -independent vasoactive substances. Rat aortic rings were suspended in aerated Krebs' solution (37 degrees C) and contracted by incremental doses of endothelin-1 5 x 10(-10) to 5 x 10(-8) mol litre-1. Volatile anaesthetics at 1 and 2 MAC were tested on endothelium intact and denuded rings. They were tested also on L-NAME incubated endothelium intact and indomethacin-incubated endothelium intact and denuded rings. Responses to endothelin-1 were compared in the presence and absence of volatile anaesthetics. Isoflurane at 1 and 2 MAC concentration, and enflurane at 2 MAC, induced a rightward shift of the dose-response curve obtained with endothelin-1 in both endothelium denuded and intact rings, associated with a decrease in maximal tension generated in the latter rings. In L-NAME-incubated endothelium intact rings and in indomethacin endothelium denuded rings, the anaesthetics induced a rightward shift of the dose-response curve without modification of maximal tension. In indomethacin-incubated endothelium intact rings there was significant attenuation of endothelin-1 contraction in control rings which was not enhanced by volatile anaesthetics in treated rings. The present study indicates that isoflurane at 1 and 2 MAC, and enflurane at 2 MAC, significantly decreased endothelin-1-induced contraction of isolated rat aorta. This inhibition was observed in both intact and denuded rings and probably involves mechanisms within the smooth muscle. Nevertheless, our results suggest that part of the anaesthetic-induced inhibition of endothelin-1-evoked vasocontraction involves an "indomethacin-like" effect on an endothelial-derived vasoconstricting cyclo-oxygenase product.     

Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin

This study shows the effects of a selective endothelin ET(B) receptor agonist, IRL 1720 {Ac-[Ala11,15]endothelin-1-(8-21)}, on cardiovascular responses in anesthetized spontaneously hypertensive rats and Wistar-Kyoto rats. Single intravenous bolus injection of IRL 1720 caused a dose-related short-lasting fall in blood pressure, left ventricular pressure and myocardial contractility. However, repeated intravenous bolus injection of 10(-5) mol/kg IRL 1720 produced a biphasic response consisting of an initial short-lasting decrease followed by a sustained increase in these parameters. The initial decrease was reduced, whereas the following increase was enhanced with the repeated injections of IRL 1720. The cardiovascular pressor response was not inhibited by the endothelin ET(A) receptor antagonist, FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl] amino-4-+methylpentanoyl] amino-3-[3-(1-methyl-1H-indolyl)]propionyl]amino -3- (2-pyridyl)propionic acid). The effects of IRL 1720 were qualitatively similar but more potent in spontaneously hypertensive rats than in Wistar-Kyoto rats. These results suggest the existence of two types of endothelin ET(B) receptor for IRL 1720: a tachyphylactic endothelin ET(B) receptor that mediates cardiovascular depressor responses and a less tachyphylactic endothelin ET(B) receptor that mediates pressor responses in the rat.