Effects of bradykinin on prostaglandin I2 synthesis in human vascular endothelial cells

The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca2+ kinetics, cytosolic phospholipase A(2) (cPLA(2)) activity, and mRNA expression of cPLA(2) and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release, Bradykinin increased both the influx of extracellular Ca2+ and Ca2+ release from the intracellular Ca2+ storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA(2) to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA(2), PGHS-1. and PGHS-2 was 87, 562, and 47 amol/mu g RNA, respectively. With the stimulation of bradykinin, cPLA(2) mRNA increased to 746 amol/mu g RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/mu g RNA, and PGHS-2 mRNA increased to 22 400 amol/mu g RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA(2) and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA(2) and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA(2) activity caused by the increase of intracellular Ca2+ concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA(2) are different from those regulating PGHS isoforms in endothelial cells.