Close correlation between cytoplasmic Ca++ levels and release of an endothelium-derived relaxing factor from cultured endothelial cells.

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 EDRF released was determined on the basis of endothelium-free rabbit aortic ring relaxation and cultured smooth muscle cell cGMP content. 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 EDRF released. BKN increased both [Ca++]i and EDRF release upon initial exposure (10(-8)M), but there were no further changes at higher concentrations. ACh induced no significant changes in either [Ca++]i or EDRF release. There was a close quantitative correlation between agonist-induced changes in [Ca++]i and the amount of EDRF released (relaxation response in aortic rings and cGMP levels.) (p < 0.001) Removal of extracellular Ca++ eliminated continuous elevation in both [Ca++]i and the amount of EDRF induced by ATP (10(-5)M), BKN (10(-8)M) and A23187 (10(-6)M). These findings suggest that intracellular Ca++ levels are directly linked to the amount of EDRF released, and that extracellular Ca++ is essential for EDRF release because its influx is involved in the continuous elevation of [Ca++]i.