Abstract
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.
Highlights
The endothelial isoform of nitric-oxide synthase1 is constitutively expressed in endothelial cells and cardiac myocytes and dynamically regulated by Ca2ϩ/calmodulin
To test for a contribution of Naϩ/Ca2ϩ exchanger (NCX) in Ca2ϩ homeostasis, endothelial cells were loaded with Naϩ by incubation with the Naϩ ionophore monensin, which is known to promote Ca2ϩ entry via reversed mode Naϩ/Ca2ϩ exchange [26, 36]
To test whether the monensin-induced facilitation of endothelial isoform of nitric-oxide synthase (eNOS) activation was due to a change in cellular Naϩ gradients or rather a general phenomenon associated with membrane depolarization and/or inhibition of Ca2ϩ entry, we investigated the effects of Kϩ-induced membrane depolarization and inhibition of Ca2ϩ entry by SK&F 96365
Summary
The endothelial isoform of nitric-oxide synthase (eNOS)1 is constitutively expressed in endothelial cells and cardiac myocytes and dynamically regulated by Ca2ϩ/calmodulin. Determination of eNOS Activity in Resting and Bradykinin-stimulated Endothelial Cells—Endothelial cells grown in 6-well plates were washed with 50 mM HEPES buffer, pH 7.4, containing 2.5 mM CaCl2, 5 mM KCl, 1 mM MgCl2, and 100 mM NaCl or choline chloride and preincubated for 15 min at 37 °C in the absence or presence of 1 M monensin.
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