Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.
Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.
Font
J
J
Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Basque Country, Bilbao, Spain.
Azula
F J
FJ
Marino
A
A
Nieva
N
N
Trueba
M
M
Macarulla
J M
JM
D000470:Alkaloids / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004357:Drug Synergism; D006801:Humans; D008563:Membrane Lipids / Q000302:isolation & purification / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000096:biosynthesis*
D000470:Alkaloids; D008563:Membrane Lipids; D010743:Phospholipids; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D013928:Thromboxane A2; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium