Correspondence to G. Balfagón (Tel.: +34 91 4975450; Fax. +34 91 4975478; e-mail: gloria.balfagon@uam.es). Chronic ouabain treatment increases the contribution of nitric oxide to endothelium- dependent relaxation R. Aras-López 1 , J. Blanco-Rivero 1 , R. Hernanz 2 , A.M. Briones 2 , L.V. Rossoni 3 , M. Ferrer 1 , M. Salaices 2 and G. Balfagón 1 Departamentos de 1 Fisiología, 2 Farmacología y Terapéutica, Facultad de Medicina, Uni- versidad Autónoma de Madrid, 28029 Madrid, Spain. 3 Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil (Received on November, 2007) R. ARAS-LÓPEZ, J. BLANCO-RIVERO, R. HERNANZ, A.M. BRIONES, L. V. ROSSONI, M. FERRER, M. SALAICES and G. BALFAGÓN. Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relax- ation. J Physiol Biochem, 64 (2), 115-126, 2008. The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega- nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were high- er in segments from ouabain-treated rats. Preincubation with the prostacyclin syn- thesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca 2+ -dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompa- nied by increased endothelial nitric oxide participation and impaired endothelium- dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta. Keywords: Nitric oxide, Endothelial-dependent hyperpolarizing factor, Prostacyclin, Acetyl- choline. J Physiol Biochem, 64 (2), 115-126, 2008