Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

Abstract

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 4×10⁻¹⁰ 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 1.3×10⁻⁹ 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.