Na+/Ca2+ exchange facilitates Ca2+-dependent activation of endothelial nitric-oxide synthase

Recent evidence suggests the expression of a Na+/ Ca2+ exchanger (NCX) in vascular endothelial cells, To elucidate the functional role of endothelial NCX, we studied Ca2+ signaling and Ca2+-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na+ gradients and after loading of endothelial cells with Na+ ions using the ionophore monensin. Monensin-induced Na+ loading markedly reduced Ca2+ entry and, thus, steady-state levels of intracellular free Ca2+ ([Ca2+](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca2+](i), Ca2+-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca2+ concentration response curve in monensin-treated cells. This facilitation of Ca2+-dependent activation of eNOS was strictly dependent on the presence of Na+ ions during treatment of the cells with monensin, Na+-induced facilitation of eNOS activation was not due to a direct effect of Na+ ions on the Ca2+ sensitivity of the enzyme. Moreover, the effect of Na+ was not related to Na+ entry-induced membrane depolarization or suppression of Ca2+ entry, since neither elevation of extracellular K+ nor the Ca2+ entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) mimicked the effects of Na+ loading. The effects of monensin were completely blocked by 3',4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na+/Ca2+ exchange, was ineffective. Consistent with a pivotal role of Na+/Ca2+ exchange in Ca2+-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na+ gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.