Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway

Abstract

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 Ca(2+)-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 Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ 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 Ca(2+)-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 (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, 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 pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.

Caffeine‐evoked, calcium‐sensitive membrane currents in rabbit aortic endothelial cells

“…In the absence of extracellular Ca2+, a decrease in the magnitude of the initial agonist-evoked transient increase of [Ca2+], was observed in bovine aortic endothelial cells (Schilling et al, 1988;Lfickhoff et al, 1988). Furthermore, depletion of the intracellular Ca2+ stores has been reported to be linked to the agonist-induced Ca2" influx from the extracellular space (Hallam et al, 1989;Jacob, 1990;Schilling et al, 1992). The precise mechanism(s) which brings about these changes in cytoplasmic [Ca2"] in native endothelial cells is ill-defined.…”

Na+/Ca2+ Exchange Facilitates Ca2+-dependent Activation of Endothelial Nitric-oxide Synthase

“…Physiological and Pathophysiological Implications-In the present study, Ca 2ϩ -dependent activation of eNOS was primarily investigated in store-depleted endothelial cells. This method is widely accepted as a physiological relevant model of endothelial cell activation, since direct depletion of intracellular Ca 2ϩ stores by inhibitors of endoplasmic Ca 2ϩ -ATPase such as thapsigargin mimics the stimulation of phospholipase C-coupled receptors in terms of Ca 2ϩ /NO signaling due to activation of the same Ca 2ϩ entry pathway (8,30). It is therefore conceivable to conclude that the effects of Na ϩ loading observed in thapsigargin-treated endothelial cells will take place similarly in cells activated by stimulation of phospholipase C-coupled receptors.…”

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

“…To exclude the effect of Ni 2ϩ on other mechanisms of Ca 2ϩ regulation a specific cation channel blocker, 100 M SKF (17,28), was used instead of Ni 2ϩ . Vessels were perfused with TG and SKF for 20 min once a baseline with 1% BSA had been established.…”