Characterization of platelet-activating factor-induced elevation of cytosolic free-calcium level in neurohybrid NCB-20 cells

Abstract

The effect of platelet-activating factor on the intracellular cytosolic level of free calcium ([Ca 2+] i) was studied in neurohybrid NCB-20 cells. In fura-2-loaded NCB-20 cells, platelet-activating factor induced an immediate and concentration-dependent increase in [Ca 2+] 1 with a maximum increase of334 ± 27nM above a basal value of147 ± 6nM (n = 40). Platelet-activating factor-induced [Ca 2+] i mobilization was inhibited by the platelet-activating factor antagonists BN 50739, WEB 2086, SRI 63-441 and BN 52021 in a dose-dependent manner with ic 50 values of 12, 38, 897 and 45000 nM, respectively. The calcium-channel blockers nifedipine (10 μM) and diltiazem (10 μM) had no effect on the platelet-activating factor-induced increase in [Ca 2+] i; however, extracellular Ca 2+-depletion caused a63.6 ± 4.7% reduction of platelet-activating factor-induced increase in [Ca 2+] i (n = 5,P < 0.001). The remaining 36% contributed from intracellular sources was completely inhibited by 10 μM of8-(N,N-diethylamine)octyl 3,4,5-trimethoxytenzoate hydrochloride (TMB-8). NCB-20 cells exhibited homologous desensitization to sequential addition of platelet-activating factor, but no heterologous desensitization between platelet-activating factor and bradykinin or ATP was observed. These data suggest that activation of the neuronal platelet-activating factor receptor results in an increase in [Ca 2+] i primarily via a receptor-operated rather than a voltage-dependent calcium-channel and to a lesser extent from intracellular Ca 2+ release. Our findings may contribute to an understanding of the mechanism of platelet-activating factor actions on neuronal cells.