Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9

Abstract

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.