Modulation of Ca2+ fluxes in isolated platelet vesicles: effects of cAMP-dependent protein kinase and protein kinase inhibitor on Ca2+ sequestration and release.
In the present study, we investigated the role of cAMP-dependent protein kinase in the process of Ca2+ uptake and release from platelet-derived membrane vesicles enriched in the dense tubular system. It was found that these membrane vesicles contain endogenous cAMP-dependent protein kinase and that stimulation of protein kinase by cAMP resulted in the phosphorylation of a single protein band (22 kDa). Addition of cAMP-dependent protein kinase produced effects on vesicle Ca2+ accumulation which were dependent on the Ca2+ concentration in the incubation medium. Specifically, at low extravesicular Ca2+ concentrations, cAMP-dependent protein kinase (10-100 micrograms/ml) produced a dose-dependent stimulation of Ca2+ uptake, however, a similar stimulation was not observed at high extravesicular Ca2+ concentrations. When endogenous protein kinase was blocked by the addition of protein kinase inhibitor, (2-160 nM) there was a dose-dependent inhibition of Ca2+ uptake at both low and high concentrations of extravesicular Ca2+. Furthermore, the addition of protein kinase inhibitor at steady state caused a rapid and dose-dependent release of vesicle-accumulated Ca2+. Studies on the phosphorylation profile of vesicle protein indicated that protein kinase inhibitor (80 and 160 nM) was capable of inhibiting the phosphorylation of the 22-kDa protein within 15 s. Finally, the ability of thromboxane A2 to cause Ca2+ release was inhibited by the addition of cAMP-dependent protein kinase (1 mg/ml). These findings suggest that cAMP-dependent protein kinase is not only a major determinant in the accumulation of Ca2+ by the dense tubular system, but may play an important role in the process of intraplatelet Ca2+ release by physiologic agents such as thromboxane A2.
Hettasch
J M
JM
Department of Pharmacology, University of Illinois College of Medicine, Chicago.
Le Breton
G C
GC
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000097:blood*; D013928:Thromboxane A2 / Q000097:blood; D013929:Thromboxane B2 / Q000097:blood
D001095:Arachidonic Acids; D008081:Liposomes; D047428:Protein Kinase Inhibitors; D016718:Arachidonic Acid; D013929:Thromboxane B2; D013928:Thromboxane A2; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium