POSSIBLE RELATIONSHIP BETWEEN THE 23-kDa PHOSPHOPROTEIN AND THE IP3 -INDUCED Ca2+RELEASE IN HUMAN PLATELETS

Abstract

The regulation of Ca2+ concentration in human platelets involves intracellular membranes i.e. dense tubular system (DTS). Agonist-induced platelet activation generates inositol 1,4,5 trisphosphate (IP3) which is responsible for Ca2+ mobilization from DTS. However, its mechanism of action is still unknown. cAMP has been shown to regulate Ca2+ transport by isolated membrane vesicles. This effect was correlated with the phosphorylation of a 23 kDa protein. We investigated whether this phosphorylation could play a role in the mechanism of IP3-induced Ca release.We isolated a membrane fraction enriched in intracellular membranes, which actively sequesters Ca2++. The Ca2+ uptake was mediated by a characterized (Ca2+ + Mg2+)-ATPase of a molecular weight 120 kDa. As well, the characterization of the 23-kDa protein phosphorylation by the catalytic subunit of the cAMP dependent protein kinase (C. Sub.) has been achieved. IP^-induced Ca release was tested on our membrane preparations. The transient effect was maximal at one minute and a dose-response curve was obtained.The cAMP dependent phosphorylation of the 23-kDa protein increased the Ca2+ liberation induced by IP by two fold whatever the IP3 concentration. The addition on the protein kinase inhibitor inhibited the IP3 -induced Ca2+ release.The effect of IP3 on the cAMP-mediated phosphorylation of the 23-kDa protein has been examinated.A dose dependent stimu-ulation of the 23-kDa protein phosphorylation in the presence of C. Sub. was initiated by IP3. The maximal effect was observed after 1-2 min and obtained with an IP3 concentration similar to that producing the maximal calcium release. The stimulation of the phosphorylation by IP3 was detected in the absence of Ca2+ and in the presence of phosphatase inhibitors.Therefore, we suggest a possible correlation between cAMP dependent phosphorylation of the 23-kDa protein and the IP3-induced Ca2+ release in human platelet membrane vesicles.