Nitric oxide: inhibitory effects on endothelial cell calcium signaling, prostaglandin I2 production and nitric oxide synthase expression.
Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase exerts many harmful effects such as stimulation of inflammation and induction of apoptosis. The effects of excessive NO production on functions of endothelial cells, the major physiological source of NO, are not completely known. The aim of this study was to investigate the role of NO on the regulation of endothelial cell Ca2+ signaling and endothelial cell function. Primary porcine aortic endothelial cells (PAECs) were used for all these studies. Intracellular Ca2+ concentrations ([Ca2+]i) were measured using fura-2/AM. Production of prostaglandin I2 (PGI2) and cyclic GMP were assessed using enzyme immunoassays, and endothelial NO synthase protein expression was evaluated by Western blotting. Bradykinin (BK, 10 nM) and thapsigargin (TG, 1 microM) provoked large increases in [Ca2+]i. The NO donor NOC12 reduced these responses, respectively, by 21% and 31% at 100 microM, 60% and 55% at 300 microM, and 74% and 78% at 500 microM. These effects were also observed with other NO donors including spermine NONOate and NOC18, and were completely prevented by carboxy-PTIO (200 microM), an NO scavenger. 8-Bromo-cGMP, however, had no effects on BK- and TG-stimulated Ca2+ responses. A 30-fold increase in PGI2 production was observed in cells stimulated with BK. NOC12 again reduced this response by 12%, 54%, 83% and 95% at 10, 100, 300 and 500 microM, respectively. Endothelial NO synthase protein level was reduced by 2%, 15%, 36 and 47% after 2, 6, 12 and 24 h, respectively, of incubation with NOC18, a NO donor with long half-life. NO, when produced in large amounts, can inhibit agonist-induced Ca2+ responses independently of cyclic GMP, reduce the production of endothelium-derived relaxing factors (EDRFs) and interfere with endothelial NO synthase protein expression.