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 4 x 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 with 45Ca, bradykinin increased the 45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in 45Ca efflux induced by bradykinin of 1.3 x 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+. The 45Ca 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 the 45Ca 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 microM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid).(ABSTRACT TRUNCATED AT 250 WORDS)
Freay
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A
Department of Pharmacology, University of Miami School of Medicine, FL 33101.
Johns
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A
Adams
D J
DJ
Ryan
U S
US
Van Breemen
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002132:Calcium Radioisotopes; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D011343:Procaine / Q000494:pharmacology; D011651:Pulmonary Artery; D011817:Rabbits
D002132:Calcium Radioisotopes; D009569:Nitric Oxide; D011343:Procaine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium