Endotoxic lipid A induces intracellular Ca2+ increase in human platelets.
The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (tmax). Maximum [Ca2+]i levels, observed at 30 microM lipid A, were 432 +/- 60 nM. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of tmax, but shortening the time for 50% recovery (t50) from greater than 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1 microM], prolonging the tmax. to 54.7 +/- 5.1 s, but decreasing the t50 to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0.15 microM). These results suggest that platelet activation by lipid A involves an interaction between [Ca2+]i elevation and protein kinase C activation.
Romano
M
M
Istituto di Patologia Medica e Medicina Mediterranea, Università di Messina, Italy.
Molino
M
M
Cerletti
C
C
D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood; D007295:Inositol Phosphates / Q000097:blood; D008050:Lipid A / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D011493:Protein Kinase C / Q000097:blood
D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D008050:Lipid A; D008345:Manganese; D004533:Egtazic Acid; D009532:Nickel; D015544:Inositol 1,4,5-Trisphosphate; C058097:inositol 1,3,4-trisphosphate; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2