Platelet-activating factor induced calcium mobilization and phosphoinositide metabolism in cultured bovine cerebral microvascular endothelial cells

Abstract

Platelet-activating factor (PAF) is a powerful lipid autacoid with a variety of biological activities. More and more evidence suggests that PAF might play an important role in modulation of cerebrovascular system function, particularly during ischemia-induced cerebrovascular damage. However, the mechanisms involved in PAF actions on cerebrovascular or other brain cells are virtually unknown. Therefore, this study was designed to investigate PAF receptor-mediated cellular signal transduction in bovine cerebral microvascular endothelial (CME) cells with the aid of a potent PAF antagonist, WEB 2086. PAF induced an immediate and concentration-dependent increase in [Ca 2+] i with an EC 50 of 4.75 nM. PAF-induced [Ca 2+] i mobilization was inhibited by PAF antagonist WEB 2086, in a dose-dependent manner (IC 50 = 15.53 nM). The calcium channel blockers diltiazem (10 μM) and verapamil (10 μM) had no effect on the PAF-induced increase in [Ca 2+] i, but depletion of Ca 2+ from the incubation buffer caused a 45.26% reduction of PAF-induced [Ca 2+] i elevation. PAF stimulated phosphoinositide metabolism in a dose-dependent manner with an EC 50 of 12.4 nM for IP 3 formation, which was also inhibited by the PAF antagonist WEB 2086 in a dose-dependent manner with IC 50 value of 16.97 nM for IP 3 production. These data indicate that bovine CME cells respond to biologically relevant concentrations of PAF and this response involves activation of phospholipase C and increase in [Ca 2+] i via specific PAF receptors. Our results may contribute to further understanding of the mechanism behind PAF actions on cerebrovascular cells.