Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca2+-free environment

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 Ca2+-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca2+-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca2+-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 Ca2+-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca2+-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca2+-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+.