Staurosporine induces hydrolysis of phosphatidyl inositol 4,5‐bisphosphate in human platelets

Abstract

Staurosporine in the micromolar range raised inositol trisphosphate in intact human platelets to levels comparable to that mediated by thrombin. This response was inhibited by neomycin, a phospholipase C antagonist. Staurosporine alone induced a weak, transient rise in cytosolic free calcium levels ([Ca2']J from release of internal Ca*+ stores but potentiated the effect induced by thrombin. Therefore, it is unlikely that this alkaloid suppressed inositol trisphosphate mobilization of Ca2'. Additional studies show that staurosporine, OS-5 ,uM, stimulated GTPase activity in platelet membranes while 2 PM K252a and 20 FM H7 were inactive. Present results suggest that staurosporine may activate platelet phospholipase C at the level of G proteins or receptors. Key wor& Staurosporine;Platelet; Phospholipa~ C; Calcium; Inositol phosphate _ _ lntrodwtionStaurosporine, a microbial alkaloid from Streptomyces sp. is a potent but nonspecific inhibitor of protein kinase C (PI(C) (IC,, of 3 nM) [l]. This reagent has been used in numerous studies to inhibit PKC-dependent responses in platelets [3-61. Recently we showed that staurosporine in the micromolar range causes a rapid and sustained elevation of cytosolic free Ca" concentration ([Ca"+]J in neutrophils via an indirect mechanism [7f. Subsequently, Himpens and coworkers [S] reported that staurosporine mobilizes intra~ll~ar Ca" stores and induces Ca2' influx in cultured DDTlMF-2 smooth muscle cells. In both studies, the effect of staurosporine on [Ca2']i is not correlated with PKC inhibition or phospholipase C (PLC) activation. The present study was undertaken to investigateand compare the effect of staurosporine on [Ca2']i in human platelets. We report here that micromolar staurosporine induced significant hydrolysis of phosphatidyl inositol4,5-bisphosphate (PIP2) but weak transient elevations of [Ca2'J,. Materials and methods Materials['H]Myo-inositol, (W-120 Ciimmol) and a ligand binding assay kit for Ins(1,4,5)P, were from Amersham Canada Ltd. (Oakville, Qnt.); human thrombin, apyrase, neomycin sulfate, and prostacyclin were from Sigma Chemical Co. (St. Louis, MO); staurosporine was obtained from Sigma and Boehringer Mannheim (Montreal, P.Q.); Fura-AM and ionomycin were from Calbiochem Corp. (San Diego, CA); Stock solutions of staurosporine and ionomycin were dissolved in dimethyl sulfoxide (MesSO) and stored at -80°C. separation of platelet ~pe~~nBlood was drawn by ve~p~cture from drug-free volunteers into 116 volume acid citrate dextrose. Platelet-rich plasma was obtained by centrifuging whole blood at 300 x g at room temperature for 15 min. After careful removal, the plasma was further centrifuged at 2500 x g at room temperature for 10 min to pellet the platelets. Platelets were resuspended in Tyrode-HEPES buffer (134 mM NaCl, 12 mM NaHCO,, 2.9 mM KCl, 0.36 mM NaH,PQ4, 1 mM MgCl,, 5 mM HEPES,J mM glucose; pH 7.2) containing apyrase (0.6 ADPase U/ml) and 0.35% bovine serum albumin. For labeling platelets with [3H]myoinositol, the buffer also contained 1 mM EG...

Staurosporine Mobilizes Ca2+ from Secretory Granules by Inhibiting Protein Kinase C in Rat Submandibular Acinar Cells

“…However, staurosporine is not specific, and it has also been shown to increase [Ca 2+ ] c by mechanisms that are independent of protein kinase C (Wong et al, 1992;Turkson et al, 1994;Tojyo et al, 1995). Therefore, in this study, we used various protein kinase C inhibitors-such as K252a and chelerythrine, which inhibit the catalytic domain of protein kinase C, and H-7 and calphostin C, which inhibit the regulatory domain-to examine whether the staurosporine-induced [Ca 2+ ] c increase resulted from the inhibition of protein kinase C. All of the inhibitors induced qualitatively similar changes in [Ca 2+ ] c , although the magnitudes and the time courses of [Ca 2+ ] c increases were not identical.…”

The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF‐2 cells

“…. However, several studies, including one on DDT, MF-2 cells (Himpens et al, 1993), have described effects of staurosporine on [Ca2+], that were independent of PKC activity (Nigam et al, 1992;Wong et al, 1992;Turkson et al, 1994). Since GF109203X-induced inhibition of Ca2+ release elicited by thapsigargin was also observed after prolonged exposure of cells to PMA, a treatment known to downregulate PKC, the effect of GF109203X The presence of the PKC isozymes a, e and C was shown in DDT, MF-2 cells (Assender et al, 1994).…”

Cell Death and Transcriptional Responses Induced in Larvae of the Nematode Haemonchus contortus by Toxins/Toxicants with Broad Phylogenetic Efficacy

“…The most significantly enriched KEGG pathway among the genes upregulated by staurosporine treatment ( Table 3 ) was “signaling pathways regulating pluripotency of stem cells”, a mechanism directly supported by previous research showing that staurosporine regulates lineage choice in pluripotent cells by acting as a broad-spectrum inducer of molecular gastrulation [ 31 ]. The other pathways only enriched among genes upregulated by staurosporine treatment include “Phosphatidylinositol signaling system” and “Inositol phosphate metabolism”, pathways which were shown to be affected by staurosporine in human platelets through the hydrolysis of phosphatidyl inositol 4,5-bisphosphate [ 32 ], as well as “mTOR signaling pathway”, which was previously shown to be affected by staurosporine through the inhibition of phosphorylation of mTOR transcriptional regulators [ 33 ]. Interestingly, “synaptic vesicle cycle” was upregulated by staurosporine, which contains the “exocytosis” target pathway for which staurosporine was originally prioritized as a NIT [ 3 ].…”