CHARACTERIZATION OF CYTOSOLIC PHOSPHOLIPASES-C FROM PORCINE AORTIC ENDOTHELIAL-CELLS

Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells ar e involved in myriad normal and pathophysiologic functions. Although i t is known that agonists activate PLCs in endothelial cells, second me ssengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosoli c PLCs from porcine aortic endothelial cells were partially purified b y ammonium sulfate fractionation and column chromatography on DEAE-Sep harose CL-6B and heparin-agarose. Three PLC isozymes immunologically s imilar to bovine brain PLC-beta, PLC-gamma, and PLC-delta were identif ied. The relative levels of PLC activities in the cytosol were: PLC-be ta, 50%; PLC-gamma, 44%; PLC-delta, 6%. The level of PLC-beta activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may p lay a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P- 2) as substrate were similar to those with phosphatidylinositol (PtdIn s) as substrate, indicating that cytosolic PLCs hydrolyze both PtdIns and PtdIns(4,5)P-2 and no PtdIns(4,5)P-2-specific PLC was present in t he cytosol. The catalytic properties of the partially purified PLC iso zymes from porcine endothelial cells were similar to their counterpart s from bovine brain. These include the dependence of hydrolysis of Ptd Ins on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptd Ins and PtdIns(4,5)P-2, the pH optima, and the stimulatory effects of deoxycholate.