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

摘要

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