title: Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca(2+)-free environment.
abstract: Depletion of agonist-sensitive Ca2+ stores results in activation of capacitative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca(2+)-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca(2+)-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca(2+)-free solution). This effect was prevented by inhibition of the Na(+)-Ca2+ exchange but not by a blockade of ryanodine-sensitive Ca2+ release (RsCR). In contrast to BsS, mitochondrial Ca2+ content remained unchanged in the Ca(2+)-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca(2+)-free environment. In contrast to Mn2+ influx, the effect of re-addition of Ca2+ to elevate bulk Ca2+ concentration ([Ca2+]b) decreased with the time the cells rested in Ca(2+)-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+ conditions the effect of Ca2+ on [Ca2+]b was reduced while it did not change the time the cells rested in Ca(2+)-free solution. After a 2 min period in low Na+ conditions, ryanodine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+ on [Ca2+]b in low Na+ conditions. Collapsing subplasmalemmal Ca2+ stores with nocodazole, increased the effect of Ca2+ on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+ on [Ca2+]b was not reduced in Ca(2+)-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca(2+)-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca(2+)-free/low Na+ solution and re-sequester Ca2+ which enters the cells prior an increase in [Ca2+]b occurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.
authors.affiliation: Department of Medical Biochemistry, Karl-Franzens University of Graz, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Paltauf-Doburzynska
authors.forename: M
authors.initials: M
authors.lastname: Frieden
authors.forename: W F
authors.initials: WF
authors.lastname: Graier
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D019831:Sodium-Calcium Exchanger; D012433:Ryanodine; D008274:Magnesium; D001920:Bradykinin; D015739:Nocodazole; D002118:Calcium