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

Depletion of agonist-sensitive Ca2+ stores results in activation of capacit ative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded singl e endothelial cells freshly isolated from bovine left circumflex coronary a rteries, we investigated whether a resting period in a Ca2+-free environmen t results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activat ion 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, a ctivity 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)) decr eased 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 Ca 2+ on [Ca2+](b) was reduced while it did not change the time the cells rest ed in Ca2+-free solution. After a 2 min period in low Na+ conditions, ryano dine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re- established the effect of Ca2+ on [Ca2+](b) in low Na+ conditions. Collapsi ng 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 activa tion of CCE is not associated with the agonist-sensitive Ca2+ pools that de plete rapidly in a Ca2+-free environment. Subplasmalemmal ryanodine-sensiti ve Ca2+ stores (RsS) are emptied in Ca2+-free /low Na+ solution and re-sequ ester Ca2+ which enters the cells prior an increase in [Ca2+](b) occurs. Th us, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activa tion of CCE or regulation of subplasmalemmal Ca2+.