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Ca2+ liberation by IP3: Mechanism of regulation
8
THROMBOSIS RESEARCH
Suppl. VI, 1986
15 Ca2+ LIBERATION BY IP3: MECHANISM OF REGULATION. J. Enouf, R. Bredoux, N. Bourdeau, *F. Giraud, *M. Claret, S. Levy-Toledano. U INSERM 150, H6pital Lariboisisre, Paris, France. * Universit6 Paris Sud, Orsay. The rapid turnover of the polyphosphoinositides is responsible for inositol trisphosphate (IP3) formation. 1~3 is synthetized in platelets and mobilizes Ca2+ from a Ca2+-accumulating membrane fraction enriched in intracellular membranes i.e. dense tubular system. Its mechanism of action was however unknown. The catalytic subunit of the kinase CAMP dependent (C sub) phosphorylates a 23 Kd protein (P 23) involved in Ca2+ fluxes. This phosphorylation increases the Ca2+ liberation induced by IP3 by two fold whatever the IP3 concentration. The increased Ca2+ liberation was not due to an increase in the IP3releasable calcium fraction. The maximal stimulation was obtained with C sub 5 ug/ml. The addition of the protein-kinase inhibitor inhibits the IP3induced Ca2+ stimulation by C sub. Stimulation of the IPg-induced Ca2+ liberation is correlated with an effect on the P 23 induced phosphorylation. This observation is based on the following results : the maximal effect was obtained after 1-2 minutes and reaches 150% with an IP3 concentration similar to the dose producing the maximal calcium release. The phosphorylation stimulation results neither from IP3 kinase activation, nor from a Ca2+-dependent kinase; phosphatase inhibition is neither involved. Therefore we suggest that the CAMP dependent phosphorylation of the P 23 is implicated in the mechanism of Ca2+ liberation by IP3.
16 EARLY BIOCHEMICAL EVENTS IN THROMBIN-INDUCED ACTIVATION OF NORMAL AND PATHOLOGICAL PLATELETS. F. Rendu, M. Lebret, J. Maclouf, S. Levy-Toledano and J.P. Caen. U 150 Inserm, H6pital Lariboisizre, Paris, France. Thrombin stimulation of washed human platelets prelabeled with 32P Pi and isolated on metrizamide gradient results in a transient decrease (30 %) in 32~ phosphatidyl 4,5 bisphosphate (P1P2), accompanied by synthesis of 32P phosphatidate (PA) and phosphorylations of myosin light chain (P 20) and of 43K protein (P 43). These events occur during pseudopod formation and granule Similar studies were centralization, i.e. prior to release and aggregation. Thrombasthenic performed on pathological platelets from inherited disorders. platelets undergo normal biochemical responses in the absence of aggregation. In Gray syndrome, characterized by a lack ofol-granules and a decreased release of dense body constituents , platelet morphological changes and granule centralization are delayed; no PIP2 breakdown is detectable although PA formation is normal, and the reduced calcium mobilization and thromboxane formation could account for the markedly decreased P 20 and P 43 phosphorylations in the first 30 seconds. Platelets from Hermansky-Pudlak syndrome, lacking dense bodies, undergo inositol lipid metabolism, granule however the release of q centralization and protein phosphorylations: -granules and lysosomes remains lower than normal. From these results and those obtained in normal platelets in presence of different inhibitors we conclude that the cascade of events is amplified by calcium and other end-products such as thromboxanes.
THROMBOSIS RESEARCH
Suppl. VI, 1986
15 Ca2+ LIBERATION BY IP3: MECHANISM OF REGULATION. J. Enouf, R. Bredoux, N. Bourdeau, *F. Giraud, *M. Claret, S. Levy-Toledano. U INSERM 150, H6pital Lariboisisre, Paris, France. * Universit6 Paris Sud, Orsay. The rapid turnover of the polyphosphoinositides is responsible for inositol trisphosphate (IP3) formation. 1~3 is synthetized in platelets and mobilizes Ca2+ from a Ca2+-accumulating membrane fraction enriched in intracellular membranes i.e. dense tubular system. Its mechanism of action was however unknown. The catalytic subunit of the kinase CAMP dependent (C sub) phosphorylates a 23 Kd protein (P 23) involved in Ca2+ fluxes. This phosphorylation increases the Ca2+ liberation induced by IP3 by two fold whatever the IP3 concentration. The increased Ca2+ liberation was not due to an increase in the IP3releasable calcium fraction. The maximal stimulation was obtained with C sub 5 ug/ml. The addition of the protein-kinase inhibitor inhibits the IP3induced Ca2+ stimulation by C sub. Stimulation of the IPg-induced Ca2+ liberation is correlated with an effect on the P 23 induced phosphorylation. This observation is based on the following results : the maximal effect was obtained after 1-2 minutes and reaches 150% with an IP3 concentration similar to the dose producing the maximal calcium release. The phosphorylation stimulation results neither from IP3 kinase activation, nor from a Ca2+-dependent kinase; phosphatase inhibition is neither involved. Therefore we suggest that the CAMP dependent phosphorylation of the P 23 is implicated in the mechanism of Ca2+ liberation by IP3.
16 EARLY BIOCHEMICAL EVENTS IN THROMBIN-INDUCED ACTIVATION OF NORMAL AND PATHOLOGICAL PLATELETS. F. Rendu, M. Lebret, J. Maclouf, S. Levy-Toledano and J.P. Caen. U 150 Inserm, H6pital Lariboisizre, Paris, France. Thrombin stimulation of washed human platelets prelabeled with 32P Pi and isolated on metrizamide gradient results in a transient decrease (30 %) in 32~ phosphatidyl 4,5 bisphosphate (P1P2), accompanied by synthesis of 32P phosphatidate (PA) and phosphorylations of myosin light chain (P 20) and of 43K protein (P 43). These events occur during pseudopod formation and granule Similar studies were centralization, i.e. prior to release and aggregation. Thrombasthenic performed on pathological platelets from inherited disorders. platelets undergo normal biochemical responses in the absence of aggregation. In Gray syndrome, characterized by a lack ofol-granules and a decreased release of dense body constituents , platelet morphological changes and granule centralization are delayed; no PIP2 breakdown is detectable although PA formation is normal, and the reduced calcium mobilization and thromboxane formation could account for the markedly decreased P 20 and P 43 phosphorylations in the first 30 seconds. Platelets from Hermansky-Pudlak syndrome, lacking dense bodies, undergo inositol lipid metabolism, granule however the release of q centralization and protein phosphorylations: -granules and lysosomes remains lower than normal. From these results and those obtained in normal platelets in presence of different inhibitors we conclude that the cascade of events is amplified by calcium and other end-products such as thromboxanes.