Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells
The relative importance of intracellular and extracellular Ca2+ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca2+ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC50 of 4x10-10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca2+. However, in the absence of external Ca2+, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with45Ca, bradykinin increased the45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in45Ca efflux induced by bradykinin of 1.3x10-9 M. The involvement of an intracellular Ca2+ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca2+,Mg2+-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca2+ concentrations showed at least two compartments involved in the uptake of Ca2+. The45Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the45Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP3). The IP3-induced release was not affected by apyrase or exogenous GTP. The EC50 for the release of Ca2+ by IP3 was 1.0 μM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca2+ influx and the release of intracellular Ca2+. The release of calcium from one of the high affinity intracellular Ca2+ stores is mediated by the intracellular second messenger, IP3.