Effects of bradykinin on prostaglandin I-2 synthesis in human vascular endothelial cells

The effects of bradykinin on the regulatory mechanisms of prostacyclin synt hesis in endothelial cells were investigated in association with intracellu lar Ca2+ kinetics, cytosolic phospholipase A(2) (cPLA(2)) activity, and mRN A expression of cPLA(2) and prostaglandin H synthase (PGHS) isoforms. Brady kinin enhanced prostacyclin release from endothelial cells time-dependently , but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced pr ostacyclin release, Bradykinin increased both the influx of extracellular C a2+ and Ca2+ release from the intracellular Ca2+ storage sites. These react ions occurred within 5 minutes after bradykinin stimulation. Within 15 minu tes, bradykinin activated cPLA(2) to 1.3-fold the control level. The consti tutive 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 t o 10 608 amol/mu g RNA, and PGHS-2 mRNA increased to 22 400 amol/mu g RNA i n 180 minutes. Pretreatment with cycloheximide superinduced cPLA(2) and PGH S-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment wi th 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 tha t the elevation of cPLA(2) activity caused by the increase of intracellular Ca2+ concentration is important in the early phase of bradykinin-induced p rostacyclin synthesis and that the mechanisms regulating cPLA(2) are differ ent from those regulating PGHS isoforms in endothelial cells.