Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca 2+-sensitive fluorochrome, fura-2 / acetoxymethylester, and cytosolic free calcium ([Ca2+](i)) was monitored by spectrofluorimetry. Platelet-activatin g factor, at a concentration of 30 nmol l(-1), induced an intracellular Ca2 + increase in cultured bovine oviductal cells, mainly via influx from the e xtracellular space. In fura-2-loaded oviductal cells, different Ca2+ channe l blockers were investigated to characterize the pathways responsible for t he Ca2+ influx. The negative effects of Ni2+-, La3+- and Ca2+-activated Kchannel blockers, such as apamin and charybdotoxin, and Ca2+ channel blocke rs, such as dotarizine, on the platelet-activating factor-induced [Ca2+](i) increase indicate the minor participation of the voltage-gated Ca2+ channe ls. TMB-8 and flufenamic acid blocked the platelet-activating factor-induce d Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentr ations of 1.25 mu mol l(-1) and 2.5 mu mol l(-1), significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mu mol l(-1 ) significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferat ion was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mu mol l(-1) and 95.29 mu mol l(-1), respectively. The depolarization was significantly inhibited at 50 mu mol l(-1) for both TMB-8 and flufenam ic acid. The results of the present study may contribute to further underst anding of the mechanism behind the actions of platelet-activating factor on oviductal cells.