RESUMO
The purpose of the present work was to investigate whether conversion of exogenous applied big-endothelin-1 (Big-ET-1) as well as the basal release and mRNA levels of endothelin-1 (ET-1) is altered by ethanol consumption in the rat carotid. The measurement of the contraction induced by Big-ET-1 served as an indicative of functional endothelin (ET)-converting enzyme (ECE) activity. Cumulative application of exogenous Big-ET-1 elicited a concentration-related contraction with the concentration-response curve shifted to the right when compared to ET-1. In endothelium-intact rings, phosphoramidon (1 mmol/l), a nonselective ECE/neutral endopeptidase (NEP) inhibitor, produced a rightward displacement of the concentration-response curves and reduced the maximal contractile response to Big-ET-1. However, in endothelium-denuded rings phosphoramidon reduced the maximum contraction for Big-ET-1 but did not alter the potency when compared to the curves obtained in the absence of the inhibitor. Ethanol consumption for 2, 6, or 10 weeks reduced the contractile effect elicited by Big-ET-1 in carotid rings with intact endothelium when compared to control or isocaloric rings. However, no differences on Big-ET-1-induced contraction were observed after endothelial denudation. On the other hand, ethanol consumption increased ET-1-induced contraction. Finally, chronic ethanol consumption did not alter either the mRNA levels for pre-pro-ET-1 nor the basal release of ET-1. The present findings show that chronic ethanol consumption does not alter the mRNA levels for ET-1 or its basal release in the rat carotid. Moreover, ethanol intake reduces the contraction induced by exogenously applied Big-ET-1 in carotid rings with intact endothelium, a fact that might be the result of a reduced conversion of this peptide by ECE on its mature active peptide ET-1.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Ácido Aspártico Endopeptidases/metabolismo , Artérias Carótidas/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Endotelina-1/metabolismo , Etanol/farmacologia , Metaloendopeptidases/metabolismo , Vasoconstrição/efeitos dos fármacos , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Artérias Carótidas/metabolismo , Artérias Carótidas/fisiopatologia , Depressores do Sistema Nervoso Central/administração & dosagem , Relação Dose-Resposta a Droga , Endotelina-1/genética , Endotelina-1/farmacologia , Enzimas Conversoras de Endotelina , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Etanol/administração & dosagem , Glicopeptídeos/farmacologia , Masculino , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
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 [succinyl-(Glu(9),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-l-arginine 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.
Assuntos
Artérias Carótidas/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Endotelina-1/farmacologia , Etanol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Glicemia/metabolismo , Western Blotting , Peso Corporal/efeitos dos fármacos , Depressores do Sistema Nervoso Central/sangue , Relação Dose-Resposta a Droga , Endotelinas/farmacologia , Etanol/sangue , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Fenilefrina/farmacologia , Piperidinas/farmacologia , Ratos , Ratos Wistar , Receptor de Endotelina A/biossíntese , Receptor de Endotelina B/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium-denuded vessels, levels of ETB receptor mRNA were reduced. 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 ETA and ETB receptors, respectively, produced concentration-dependent rightward displacements of the ET-1 concentration-response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620-induced contraction was augmented after endothelium removal. 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. 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. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage-dependent K+ channels.