1 We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ET A and ET B receptors, evaluated by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ET B receptors are expressed in the endothelium and smooth muscle cells, while ET A receptors are expressed only in the smooth muscle cells. In endothelium-denuded vessels, levels of ET B receptor mRNA were reduced. 2 Vascular reactivity experiments, using standard muscle bath procedures, showed that ET-1 induces contraction in endothelium-intact and -denuded carotid rings in a concentration-dependent manner. Endothelial removal enhanced ET-1-induced contraction. BQ123 and BQ788, selective antagonists for ET A and ET B receptors, respectively, produced concentration-dependent rightward displacements of the ET-1 concentration-response curves. 3 IRL1620, a selective agonist for ET B receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620-induced contraction was augmented after endothelium removal. 4 ET-1 concentration dependently relaxed phenylephrine-precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. 5 Preincubation of intact rings with N G -nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620-induced relaxation. The combination of L-NAME, indomethacin and TEA completely abolished IRL1620-induced relaxation while sulfaphenazole did not affect this response. 4-aminopyridine (4-AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620-induced relaxation. 6 The major finding of this work is that it firstly demonstrated functionally the existence of both ET A and ET B vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ET B receptors that mediated vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage-dependent K þ channels.

We investigated the mechanisms involved in the enhancement of endothelin (ET)-1 vascular reactivity induced by ethanol consumption. Ethanol intake for 2, 6, and 10 weeks enhanced the ET-1-induced contractile response of endothelium-intact but not endothelium-denuded rat carotid rings independently of the treatment duration. Conversely, phenylephrine-induced contraction was not affected by ethanol intake. The contraction induced by IRL1620 ,Ala 11,15 )-ET-1-(8 -21)], a selective ET B agonist, was increased after treatment with ethanol in endothelium-intact but not in endothelium-denuded carotid rings. Moreover, ET-1-and IRL1620-induced relaxation was reduced in endothelium-intact phenylephrine-precontracted rings from ethanol-treated rats. Acetylcholine-induced relaxation was not affected by ethanol treatment. N G -Nitro-Larginine methyl ester, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin, and tetraethylammonium reduced the relaxation induced by IRL1620 in carotid glands from control but not ethanol-treated rats. The mRNA levels for ET A and ET B receptors were not altered by ethanol consumption. However, ethanol treatment reduced the protein expression of ET B receptors. Furthermore, immunohistochemical assays showed reduced immunostaining for endothelial ET B receptors after treatment with ethanol. We conclude that ethanol consumption enhances ET-1-induced contraction in the rat carotid and that this response is not different among the three periods of treatment used in this study. Finally, the potentiation of ET-1-induced vascular reactivity is probably caused by reduced expression of relaxing endothelial ET B receptors.

Background and purpose: The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. Experimental approach: The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg À1 , i.v.) or to the selective ET B agonist, IRL1620 (0.001-1.0 nmol kg À1 , i.v.), with or without ET A or ET B antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg À1 and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-g-methylleucyl1-D-1methoxycarbonyltryptophanyl-Dnorleucine) at 0.25 mg kg À1 , respectively) were tested in anaesthetized rats, after 2 weeks' chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. Key results: The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg À1 reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg À1 ) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg À1 were enhanced following ET B receptor blockade. Finally, we observed higher levels of ET A receptor in the heart and mesenteric artery and a reduction of ET B receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. Conclusions and implications: Increased vascular reactivity to ET-1 and altered protein levels of ET A and ET B receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.

1 We investigated whether blood vessels contribute to the production of ET-1(1-31) from exogenous big endothelin-1 (BigET-1) in the rabbit and assessed which enzymes are involved in this process. 2 Vascular reactivity experiments, using standard muscle bath procedures, showed that BigET-1 induces contraction in endothelium-intact rabbit aortic rings. Preincubation of the rings with phosphoramidon, CGS35066 or thiorphan reduced BigET-1-induced contraction. Conversely, chymostatin did not affect BigET-1-induced contraction. 3 Thiorphan and phosphoramidon, but not CGS35066 or chymostatin, reduced ET-1(1-31)-induced contraction. None of the enzymatic inhibitors affected the contraction afforded by ET-1. 4 BQ123-, but not BQ788-, selective antagonists for ET A and ET B receptors, respectively, produced concentration-dependent rightward displacements of the ET-1(1-31) and ET-1 concentrationresponse curves. 5 By the use of enzymatic assays, we found that the aorta, as well as the heart, lung, kidney and liver, possess a chymase-like activity. 6 Enzyme immunoassays detected significant levels of Ir-ET-1(1-31) in bathing medium of aortas after the addition of BigET-1 (30 nM). Neither thiorphan nor chymostatin altered the levels of Ir-ET-1(1-31). Conversely, the levels of Ir-ET-1(1-31) were increased in the presence of phosphoramidon. This marked increase of the 31-amino-acid peptide was abolished when phosphoramidon and chymostatin were added simultaneously. 7 The major new finding of the present work is that the rabbit aorta generates ET-1(1-31) from exogenously administered BigET-1. Additionally, by measuring the production of ET-1(1-31), we showed that a chymase-like enzyme is involved in this process when ECE and NEP are inhibited by phosphoramidon. Our results also suggest that ET-1(1-31) is an alternate intermediate in the production of ET-1 following BigET-1 administration. Finally, we showed that NEP is the predominant enzymatic pathway involved in the cleavage of ET-1(1-31) to a bioactive metabolite that will act on ET A receptors to induce contraction in the rabbit aorta.

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