Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.
Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-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 Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-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 Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e 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(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-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.
Teubl
M
M
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Groschner
K
K
Kohlwein
S D
SD
Mayer
B
B
Schmidt
K
K
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D051242:Caveolin 1; D022461:Caveolins; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D004730:Endothelium, Vascular; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D016257:Fura-2 / Q000737:chemistry; D007093:Imidazoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013552:Swine
D002121:Calcium Channel Blockers; D051242:Caveolin 1; D022461:Caveolins; D007093:Imidazoles; D008565:Membrane Proteins; D019831:Sodium-Calcium Exchanger; D002956:Citrulline; D000584:Amiloride; D008985:Monensin; D012964:Sodium; D019001:Nitric Oxide Synthase; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2