Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells.

Abstract

In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca2+]i) causes activation of nitric oxide synthase (NOS) and release of nitric oxide (NO). The goal of the study was to characterize the interplay between [Ca2+]i and NO production in this cell type. Simultaneous measurements of [Ca2+]i and intracellular NO concentration ([NO]i) in cultured bovine vascular endothelial cells (CPAE cell line) with the fluorescent indicators fura-2 and DAF-2, respectively, revealed that Ca2+ influx following agonist-induced intracellular Ca2+ store depletion (capacitative Ca2+ entry, CCE) represents the preferential Ca2+ source for the activation of the Ca2+-calmodulin-dependent endothelial NOS (eNOS). Exposure to the NO donor sodium nitroprusside (SNP) showed that high NO levels suppressed CCE and had an inhibitory effect on Ca2+ extrusion by the plasmalemmal Ca2+-ATPase. This inhibitory effect on CCE was mimicked by the membrane-permeant cGMP analogue 8-bromo-cGMP, but was reversed by the NO scavenger haemoglobin and prevented by the inhibitor of the NO-sensitive guanylate cyclase ODQ. Brief exposure to SNP reduced the peak of ATP-induced Ca2+ release from the endoplasmic reticulum (ER) and accelerated Ca2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca2+ loading of the ER, as revealed by direct measurements of store content with the ER-entrapped low-affinity Ca2+ indicator mag-fura-2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO-dependent [Ca2+]i regulation. Via cGMP-dependent inhibition of CCE and acceleration of Ca2+ sequestration into the ER, NO can lower [Ca2+]i and therefore exert an autoregulatory negative feedback on its own Ca2+-dependent synthesis.