Close Correlation between Cytoplasmic Ca++ Levels and Release of an Endothelium-Derived Relaxing Factor from Cultured Endothelial Cells.

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Abstract
We studied whether there is a quantitative relationship between free cytosolic Ca++ levels and the release of an endothelium-derived relaxing factor (EDRF) from cultured fetal bovine aortic endothelial cells (EC). EC pretreated with indomethacin were stimulated by the agonists adenosine triphosphate (ATP), bradykinin (BKN), acetylcholine (ACh) and calcium ionophore (A23187) in various concentrations (10-8-10-5 M), and the amount of EDRFreleased was determined on the basis of endothelium-free rabbit aortic ring relaxation and cultured smooth muscle cell CGMPcontent. Changes in intracellular Ca++ concentration ([Ca++]i in response to the same stimuli were determined by photometric fluorescence microscopy using the fluorescent calcium indicator Fura-2. EC stimulation by ATP and A23187 induced dose-dependent increases in both [Ca++]i and the amount of EDRFreleased. BKNincreased both [Ca++]i and EDRFrelease upon initial exposure (10-8M), but there were no further changes at higher concentrations. AChinduced no significant changes in either [Ca++]i or EDRFrelease. There was a close quantitative correlation between agonist-induced changes in [Ca++]i and the amount of EDRFreleased (relaxation response in aortic rings and CGMPlevels.) (p < 0.001) Removal of extracellular Ca++ eliminated continuous elevation in both [Ca++]i and the amount of EDRFinduced by ATP (10-5 M), BKN(10-8M) and A23187 (10-6M). These findings suggest that intracellular Ca++ levels are directly linked to the amount of EDRFreleased, and that extracellular Ca++ is essential for EDRFrelease because its influx is involved in the continuous elevation of [Ca++]i.

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