RESUMO
In the present study, we investigated the role of angiotensin type I (AT1) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.
Assuntos
Artérias/metabolismo , Etanol/administração & dosagem , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Ativação Enzimática , Masculino , Fosforilação , Ratos , Ratos WistarRESUMO
We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with l-NAME (non selective NOS inhibitor, 100 µmol/L), 7-nitroindazole (selective nNOS inhibitor, 100 µmol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, 1 µmol/L), glibenclamide (selective blocker of ATP-sensitive K(+) channels, 3 µmol/L) and 4-aminopyridine (selective blocker of voltage-dependent K(+) channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O(2)(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H(2)O(2)) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 µmol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 µmol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by l-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca(2+) concentration ([Ca(2+)]c), O(2)(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca(2+)]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Etanol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Oxirredução , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Chronic ethanol consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks' treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (Emax) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the Emax values for phenylephrine were observed between groups in the presence of indometacin. L-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the Emax for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Etanol/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Endotélio Vascular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipertensão/induzido quimicamente , Masculino , Artérias Mesentéricas/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilefrina/farmacologia , Prostaglandinas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Vasoconstrição/efeitos dos fármacosRESUMO
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
Endothelins (ETs) and sarafotoxins (SRTXs) are active isopeptides that have very similar structures and functions. All isoforms interact with two specific G-protein-coupled receptors, ET(A) and ET(B). To characterize functional vascular ET receptors in the poisonous snake, Bothrops jararaca, cumulative concentration-response curves to ETs and SRTXs were performed in isolated aortic rings, in the absence and presence of selective ET receptor antagonists. Vascular expression of ET receptor messenger RNA (mRNA) was evaluated by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis, and a fragment of the ET(A) receptor was cloned and sequenced. In vivo, ET-1 induced a dose-dependent biphasic response on anesthetized B. jararaca snakes. In vitro, ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 induced concentration-dependent vasoconstriction, with a potency order suggesting the presence of typical ET(A) receptors. BQ-123, a selective ET(A) antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, K(B), (pK(B)) values for mixed ET(A)/ET(B) receptor populations. The nonselective ET(A)/ET(B) receptors antagonist, PD-142893, produced similar inhibition. The ET(B) antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) plus indomethacin. Processing of the B. jararaca aortic first-strand complementary DNA, by RT-PCR with primers designed from the Gallus gallus ET(A) receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ET(A) receptor showed a very high sequence similarity with ET(A) receptor sequences from chicken, rat, human, and Xenopus. In conclusion, vascular responses to SRTXs/ETs in the B. jararaca aorta are mediated predominantly, but not exclusively, by typical ET(A) receptors.
Assuntos
Bothrops , Venenos de Crotalídeos/farmacologia , Receptor de Endotelina A/química , Receptor de Endotelina A/metabolismo , Sequência de Aminoácidos , Animais , Aorta Torácica/efeitos dos fármacos , Sequência de Bases , Pressão Sanguínea/efeitos dos fármacos , Sequência Conservada , DNA Complementar/genética , Relação Dose-Resposta a Droga , Antagonistas do Receptor de Endotelina A , Feminino , Infusões Intravenosas , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Perfusão , RNA Mensageiro/análise , Receptor de Endotelina A/agonistas , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
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.
Assuntos
Artérias Carótidas/metabolismo , Receptor de Endotelina A/fisiologia , Receptor de Endotelina B/fisiologia , Animais , Western Blotting , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Endotelina-1/farmacologia , Endotelinas/farmacologia , Endotélio Vascular/fisiologia , Fatores Relaxantes Dependentes do Endotélio/fisiologia , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Óxido Nítrico/fisiologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Canais de Potássio/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
In the search for new antimalarial agents, nine Brazilian plant species were selected, from the Annonaceae (6), Menispermaceae (2) and Siparunaceae (1) families naturally occurring at the cerrado and Atlantic rainforest regions, in order to investigate their in vitro antiplasmodial activity. The ethanol and the alkaloid extracts were tested against K1, chloroquine-resistant, and Palo Alto, chloroquine-sensitive, strains of Plasmodium falciparum. The majority of the alkaloid extracts were more active than the ethanol ones, with IC(50) ranging 0.3-8.2 microg/mL. The crude Guatteria australis alkaloids were the most active against K1 with an IC(50) = 0.3 microg/mL. The most promising total alkaloid fractions for further bioguided isolation are those with the IC(50) < or = 5 microg/mL: G. australis, Cissampelos ovalifolia and Duguetia lanceolata.
Assuntos
Alcaloides/farmacologia , Antimaláricos/farmacologia , Isoquinolinas/farmacologia , Magnoliopsida/química , Plasmodium falciparum/efeitos dos fármacos , Animais , Brasil , Concentração Inibidora 50 , Extratos Vegetais/farmacologia , Folhas de Planta/químicaRESUMO
In the search for new antimalarial agents, nine Brazilian plant species were selected, from the Annonaceae (6), Menispermaceae (2) and Siparunaceae (1) families naturally occurring at the cerrado and Atlantic rainforest regions, in order to investigate their in vitro antiplasmodial activity. The ethanol and the alkaloid extracts were tested against K1, chloroquine-resistant, and Palo Alto, chloroquine-sensitive, strains of Plasmodium falciparum The majority of the alkaloid extracts were more active than the ethanol ones, with IC50 ranging 0.3¨C8.2 ¦Ìg/mL The crude Guatteria australis alkaloids were the most active against K1 with an IC50 = 0.3 ¦Ìg/mL. The most promising total alkaloid fractions for further bioguided isolation are those with the IC50 ¡Ü 5 ¦Ìg/mL: G. australis, Cissampelos ovalifolia and Duguetia lanceol.