The relationship between cytosolic Ca2+, <i>sn</i>-1,2-diacylglycerol and inositol 1,4,5-trisphosphate elevation in platelet-activating-factor-stimulated rabbit platelets. Influence of protein kinase C on production of signal molecules

Murphy, Christine; Elmore, M; Kellie, Stuart; Westwick, John

doi:10.1042/bj2780255

Cited by 36 publications

(37 citation statements)

References 57 publications

(34 reference statements)

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“…platelet responses such as aggregation and degranulation [2-51. We note that Murphy et al [4] reported no effect for 1 PM staurosporine on Ins(1,4,5)P, in rabbit platelets. Species differences may account for the difference in results.…”

Section: Discussionmentioning

confidence: 97%

“…Secondly, initial increases in [Ca2+]i triggered by platelet agonists are amplified by phospholipase A2 activation and TXA, formation [20-221. It is possible that staurosporine failed to activate the amplification pathway or that it blocks amplification at an early step. Direct staurosporine inhibition of TXA, synthesis can be ruled out since 1 PM staurosporine has been shown to enhance thromboxane formation in platelets stimulated by platelet activating factor [4]. The mechanism by which staurosporine activated PLC in platelets remains to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

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

Turkson

Wong

1994

FEBS Letters

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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...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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

Turkson

Wong

1994

FEBS Letters

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“…damage produced by ischemia/reperfusion (Lobner and Lipton, 1993;Zaidan and Sims, 1994). PAF receptor stimulation is known to increase intracellular Ca 2~, both via its release from intracellular stores and by its influx through the cell membrane (Kornecki and Ehrlich, 1991;Murphy et a!., 1991;Bito et a!., 1992;Kamata et al, 1993). We therefore examined the role of Ca2~in PAF-mediated ischemia/ reincubation-induced cell damage.…”

Section: Role Of Calcium In Ischemia/reincubation-induced Cell Deathmentioning

confidence: 99%

Involvement of Platelet‐Activating Factor in Cell Death Induced Under Ischemia/Postischemia‐Like Conditions in an Immortalized Hippocampal Cell Line

Shi

Wang

Friedman

1998

Journal of Neurochemistry

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Abstract:The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemialike conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N 2-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 ±2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protective effect. Maximal protection (85.7 ±5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 p~M BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1 -O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis-an effect that was blocked by BN 50739. Pretreatment of HN33.1 1 cells with the Ca 2chelator 1,2-b/s (2-aminophenoxy)ethane-N , N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/ postischemia-like conditions in HN33.1 1 cells.

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“…To terminate TxB, generation, sub-samples of platelets (400 pl) were withdrawn 3 min after PAF addition and added to 40 pl icecold 100 mM EDTA and 30 pM indomethacin. The samples were centrifuged at 8000 X g for 2 min, the supernatant removed and the TxB, content determined by radioimmunoassay (Johnston et al, 1984) as we have previously described (Murphy et al, 1991). The sensitivity of the assay was CO.1 pmol TxB,/108 platelets.…”

Section: Measurement Of Txb Generationmentioning

confidence: 99%

“…The samples were centrifuged at 8000X g for 2 min, then 2 X 100 p1 samples of the supernatant were removed and the amount of 5-H['4C]T determined by liquid scintillation spectroscopy. The 5-H['4C]T release was determined as previously described (Holmsen and Dangelmaier, 1989 ;Murphy et al, 1991).…”

Section: Measurement Of the Percentage 5-hydro~y['~c]tryptamine Releasementioning

confidence: 99%

Tyrosine‐kinase activity in rabbit platelets stimulated with platelet‐activating factor

Murphy

Kellie

Westwick

1993

European Journal of Biochemistry

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The temporal relationship of tyrosine phosphorylation of proteins in platelet-activating-factor-(PAF)-stimulated rabbit platelets was characterised by Western blotting using a monoclonal antiphosphotyrosine antibody, demonstrated to be specific for detecting only tyrosine phosphorylated proteins. In addition, the protein tyrosine kinase (PTKase) inhibitor genistein, was used to investigate the role of endogenously activated PTKase(s) in the regulation of receptor-stimulated changes in both signal molecule production and in platelet functional responses. Several tyrosine phosphorylated protein bands (52-62 kDa) were observed in unstimulated platelets, however, within 5 s of PAF stimulation, two further groups of tyrosine phosphorylated protein bands were observed (35 -45 kDa and 66-90 kDa) and within 30 s of PAF stimulation a further group was detected (90-150 kDa). Under conditions where intracellular Ca2+ was chelated with acetoxymethyl 1,2-bis(Oaminophen0xy)ethane-N,N,N',N'-tetraacetate (BAPTA-AM) and extracellular Ca2+ was chelated with EGTA, the number of tyrosine-phosphorylated bands was greatly reduced. Tyrosine phosphorylation of the proteins induced by PAF stimulation were differentially inhibited by treatment with genistein. Genistein inhibited PAF-induced elevation of the signal molecule inositol 1,4,5-trisphosphate and also inhibited both mobilization of Ca2+ and the influx of Ca2+ through the plasma membrane. These results suggest a role for endogenously activated PTKase(s) in the early stages of signal transduction in PAF-stimulated platelets. Moreover, inhibition of genistein-sensitive PTKase(s) also caused an inhibition of PAF-induced thromboxane B, generation, dense-granule release and platelet aggregation, indicating a role for PTKase(s) in the regulation of platelet functional responses. Platelets stimulated with a-thrombin, ionomycin and 12-0-tetradecanylphorbol 13-acetate gave a similar pattern of phosphorylated proteins to PAF-stimulated platelets, however, whereas genistein inhibited protein phosphorylation, it had no significant effect on functional responses in platelets stimulated with these agents, suggesting that an alternative signalling pathway exists.Many phosphorylation events observed in platelets have been attributed to the role of protein kinase C (PKC) and myosin light chain kinase, both of which are serinelthreonine specific kinases (for review, see Siess, 1989). However, it is possible that the relatively recently discovered protein tyrosine kinases (PTKases) in platelets may also have a role in signal transduction.

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The relationship between cytosolic Ca2+, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate elevation in platelet-activating-factor-stimulated rabbit platelets. Influence of protein kinase C on production of signal molecules

Cited by 36 publications

References 57 publications

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

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

Involvement of Platelet‐Activating Factor in Cell Death Induced Under Ischemia/Postischemia‐Like Conditions in an Immortalized Hippocampal Cell Line

Tyrosine‐kinase activity in rabbit platelets stimulated with platelet‐activating factor

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