DEPLETION OF THE INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE INTRACELLULAR CA2+ STORE IN VASCULAR ENDOTHELIAL-CELLS ACTIVATES THE AGONIST-SENSITIVE CA2+-INFLUX PATHWAY

Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca2+-ATPase/pump inhibitor 2.5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ Concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca2+-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both Ca-45(2+) influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca2+-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pH(o)), and was inhibited by extracellular La3+, by depolarization of the membrane. and by thc novel Ca2+-influx blocker 1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both Ca-45(2+) and Ba-133(2=), consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ. thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pH(o) and BHQ stimulated the influx of both Ca-45(2+) and Ba(133)2+ to the same extent. These results demonstrate that the agonist-activated Ca2+-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.