We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH2 (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 mu M, 0.1 U/ml, and 1 mu M, respectively. Ionomycin elevated [Ca2+](i) in a concentration-dependent manner. However, AYPGKF-NH2 and thrombin induced no [Ca2+](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca2+](i) elevation induced by 1 mu M ionomycin, while it had no significant effect on the AYPGKF-NH2-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH2-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca2+ signal and also that G(i/o) is involved in such NO production in vascular endothelial cells. (c) 2006 Elsevier Inc. All rights reserved.