Effect of ATP and platelet-activating factor on intracellular calcium concentrations of cultured oviductal cells from cows

Abstract

Oviductal endosalpingeal cells were mechanically isolated from heifers at different reproductive stages (cyclic: days 8-14, at oestrus: day 0 and pregnant: day 7) and cultured until 100% confluent. The cells were loaded with the Ca2+-sensitive fluorochrome fura-2/AM, and cytosolic free calcium ([Ca2+](i)) was monitored spectrofluorimetrically and by use of a microscope image analysis system. ATP (400 μmol l-1) evoked a prompt increase in [Ca2+](i) in all cell preparations in both the presence or absence of extracellular Ca2+ when measured with the cuvette method. Single cell measurements using oviductal cells from cyclic heifers revealed a heterogeneous [Ca2+](i) pattern in response to ATP, with some cells either failing to respond or reacting very slowly. Platelet-activating factor (PAF, 30 nmol l-1) induced a rapid increase in [Ca2+](i) especially in cells derived from cyclic and pregnant animals, but the effect was significantly less in cells from heifers at oestrus. The increase in [Ca2+](i) in bovine cells induced by PAF was reduced when extracellular calcium was depleted, indicating that the effect of PAF on [Ca2+](i) involves an influx from the extracellular space. Voltage-sensitive calcium channels do not appear to be involved in the influx of extracellular Ca2+ since verapamil had no effect on the PAF-induced increase in [Ca2+](i). The PAF receptor antagonist WEB 2086 inhibited the PAF-mediated effects on [Ca2+](i).