myo-Inositol 1,4,5-trisphosphate stimulates protein phosphorylation in saponin-permeabilized human platelets.
In an attempt to establish a system with physiological substrates and phospholipid surfaces to investigate Ca2+- and 1,2-diacylglycerol-dependent protein kinase C activation, saponized platelets were used. Saponin, through interaction with plasma membrane cholesterol, makes cells permeable without major disruption of organelles. Washed platelets, prelabeled with 32P, were treated with 1-50 micrograms of saponin per ml. Permeabilization was evident at a concentration of 10 micrograms of saponin per ml, as indicated by the action of extracellular Ca2+ on the phosphorylation of the 20,000- and 40,000-Da proteins. These proteins are, respectively, the substrates for myosin light chain kinase and protein kinase C. Activation of these enzymes occurred when the estimated free [Ca2+] was changed from approximately equal to 80 nM to 300 nM. The effect of Ca2+ on kinase C-induced phosphorylation was potentiated by 1,2-didecanoylglycerol (1 microM). myo-Inositol 1,4,5-trisphosphate (5-20 microM) increased phosphorylation of the 20,000- and 40,000-Da proteins. This action was time and concentration dependent. The effect of myo-inositol 1,4,5-trisphosphate on the activation of kinase C was additive with 1,2-didecanoylglycerol. The action of myo-inositol 1,4,5-trisphosphate could be due to mobilization of Ca2+ from platelet organelles and/or to a direct effect on protein kinases.
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D012503:Saponins / Q000494:pharmacology*; D013403:Sugar Phosphates / Q000494:pharmacology*
D001798:Blood Proteins; D007295:Inositol Phosphates; D012503:Saponins; D013403:Sugar Phosphates; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium