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Clinical Science (2010) 118, (657–668) (Printed in Great Britain)
Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms
Alvaro Yogi*, Glaucia E. Callera*, Ulisses V. Hipólito†‡, Catiane R. Silva‡, Rhian M. Touyz* and Carlos R. Tirapelli‡
*Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada, †Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), SP, Brazil, and ‡Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP, Brazil
Key words: aorta, calcium, hydrogen peroxide, prostanoid, reactive oxygen species, superoxide anion. Abbreviations: [Ca2+]i, intracellular Ca2+ concentration; CM-H2DCFDA, 2′,7′ dichlorodihydrofluorescein diacetate; COX, cyclo-oxygenase; DHE, dihydroethidium; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; fura 2/AM, fura 2 acetoxymethyl ester; H2DCFDA, 2′,7′ dichlorodihydrofluorescein diacetate; HBSS, Hanks balanced salt solution; L-NAME, NG-nitro-L-arginine methyl ester; NOS, NO synthase; O2−, superoxide anion; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PGF2α, prostaglandin F2α; PGH2, prostaglandin H2; ROS, reactive oxygen species; SOD, superoxide dismutase; TEA, tetraethylammonium; TXA2, thromboxane A2; VSMC, vascular smooth muscle cell. Correspondence: Professor Carlos R. Tirapelli (email crtirapelli@eerp.usp.br). The present study investigated the role of ROS (reactive oxygen species) and COX (cyclo-oxygenase) in ethanol-induced contraction and elevation of [Ca2+]i (intracellular [Ca2+]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1–800 mmol/l) induced contraction in endothelium-intact (EC50: 306±34 mmol/l) and endothelium -denuded (EC50: 180±40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [NG-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100μmol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100μmol/l], oxyhaemoglobin (NO scavenger, 10μmol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1μmol/l) increased ethanol-induced contraction. Tiron [O2− (superoxide anion) scavenger, 1 mmol/l] and catalase (H2O2 scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10μmol/l), SC560 (selective COX-1 inhibitor, 1μmol/l), AH6809 [PGF2α (prostaglandin F2α)] receptor antagonist, 10μmol/l] or SQ29584 [PGH2(prostaglandin H2)/TXA2 (thromboxane A2) receptor antagonist, 3μmol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O2− and H2O2. Ethanol induced a transient increase in [Ca2+]i, which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca2+ signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear. Received 6 July 2009/12 November 2009; accepted 3 December 2009 Published as Immediate Publication 3 December 2009, doi:10.1042/CS20090352 © The Authors Journal compilation © 2010 Biochemical Society |
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