CHARACTERIZATION OF PLATELET-ACTIVATING FACTOR-INDUCED ELEVATION OF CYTOSOLIC FREE-CALCIUM LEVEL IN NEUROHYBRID NCB-20 CELLS

The effect of platelet-activating factor on the intracellular cytosolic level of free calcium ([Ca2+]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 [Ca2+]i with a maximum increase of 334 +/- 27 nM above a basal value of 147 +/- 6 nM (n = 40). Platelet-activating factor-induced [Ca2+]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 IC50 values of 12, 38, 897 and 45000 nM, respectively. The calcium-channel blockers nifedipine (10-mu-M) and diltiazem (10-mu-M) had no effect on the platelet-activating factor-induced increase in [Ca2+]i; however, extracellular Ca2+-depletion caused a 63.6 +/- 4.7% reduction of platelet-activating factor-induced increase in [Ca2+]i (n = 5, P < 0.001). The remaining 36% contributed from intracellular sources was completely inhibited by 10-mu-M of 8-(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 [Ca2+]i primarily via a receptor-operated rather than a voltage-dependent calcium-channel and to a lesser extent from intracellular Ca2+ release. Our findings may contribute to an understanding of the mechanism of platelet-activating factor actions on neuronal cells.