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

Risa Korenaga, Joji Ando, Akira Ohtsuka, Ichiro Sakuma, Weidong Yang, Teruhiko Toyo-oka, Akira Kamiya
1993 Cell Structure and Function  
BSTRACT. Westudied whether there is a quantitative relationship between free cytosolic Ca44 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
more » ... free rabbit aortic ring relaxation and cultured smooth muscle cell CGMPcontent. Changes in intracellular CaH concentration ([Ca^D 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^L and the amount of EDRFreleased. BKNincreased both [Ca^L and EDRFrelease upon initial exposure (10~8 M), but there were no further changes at higher concentrations. AChinduced no significant changes in either [Ca^L or EDRFrelease. There was a close quantitative correlation between agonist-induced changes in [Ca^L 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^L and the amount of EDRFinduced 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 EDRFreleased, and that extracellular Ca++ is essential for EDRFrelease because its influx is involved in the continuous elevation of [Ca^li. Vascular endothelial cells produce and secrete vasodilator and vasoconstrictor substances whichare closely related to regulation of vascular tonus (2, 8, 12, 32, 36) . Endothelium-derived relaxing factor (EDRF) (1 1), a vasodilator substance produced by endothelium, is believed to be nitric oxide (NO) (27, 28) or an NO-related substance such as S-nitroso-L-cysteine (25). EDRFactivates the intracellular soluble guanylate cyclase of subendothelial vascular smooth muscle and elevates CGMP to induce dephosphorylation of myosin light chain, leading to smooth muscle relaxation (24, 30). EDRFrelease from the endothelium is stimulated by hormones or autacoids in the blood stream, or by neurotransmitters. It is known to be affected not only by such chemical agonists but by physical stimulation such as shear stress caused by blood flow (10, 22, 31). The details of the mechanismof EDRFrelease in response to the chemt To whomreprint requests and correspondence should be addressed.
doi:10.1247/csf.18.95 fatcat:dlyxljxbqjhybdmwbpcxal334u