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

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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.