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

• Published in: The Journal of physiology 2002-02, Vol.539 (1), p.77-91
• Main Authors: Dedkova, Elena N., Blatter, Lothar A.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Calcium - metabolism; Calcium-Transporting ATPases - antagonists & inhibitors; Cattle; Cells, Cultured; Cyclic GMP - physiology; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme Inhibitors - pharmacology; Intracellular Membranes - metabolism; Nitric Oxide - metabolism; Nitric Oxide - pharmacology; Nitroprusside - pharmacology; Original; Osmolar Concentration; Thapsigargin - pharmacology
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2001.013258

In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca 2+ ] 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 [Ca 2+ ] i and NO production in this cell type. Simultaneous measurements of [Ca 2+ ] 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 Ca 2+ influx following agonist-induced intracellular Ca 2+ store depletion (capacitative Ca 2+ entry, CCE) represents the preferential Ca 2+ source for the activation of the Ca 2+ -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 Ca 2+ extrusion by the plasmalemmal Ca 2+ -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 Ca 2+ release from the endoplasmic reticulum (ER) and accelerated Ca 2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca 2+ loading of the ER, as revealed by direct measurements of store content with the ER-entrapped low-affinity Ca 2+ indicator mag-fura-2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO-dependent [Ca 2+ ] i regulation. Via cGMP-dependent inhibition of CCE and acceleration of Ca 2+ sequestration into the ER, NO can lower [Ca 2+ ] i and therefore exert an autoregulatory negative feedback on its own Ca 2+ -dependent synthesis.