Cybulsky A V, Cyr M D
Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Am J Physiol. 1993 Oct;265(4 Pt 2):F551-60. doi: 10.1152/ajprenal.1993.265.4.F551.
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.
在大鼠膜性肾病中，补体C5b - 9可诱导肾小球上皮细胞（GEC）损伤和蛋白尿。在培养的大鼠GEC中，C5b - 9刺激磷脂酰肌醇定向磷脂酶（PL）C，且PLC的产物可下调C5b - 9介导的GEC损伤。我们现在报告，C5b - 9诱导的磷脂酰胆碱（PC）水解提供了1,2 - 二酰基甘油（DAG）的额外来源。通过与1 - O - [烷基 - 3H]2 - 溶血磷脂酰胆碱短暂孵育，完整的GEC中的PC被标记。C5b - 9的组装刺激了PC衍生的[3H]DAG增加（为对照的173±18%），在用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）长时间预孵育使蛋白激酶C（PKC）耗竭的GEC中，这种增加减少。与C5b - 9类似，在用钙离子载体A23187加PMA短暂孵育GEC后，[3H]DAG从PC中释放。C5b - 9和A23187加PMA诱导的[3H]DAG增加与DAG质量的增加平行。C5b - 9还增加了[3H]磷脂酸（PA；为对照的182±37%），但DAG和PA之间没有明显的相互转化。因此，DAG可能起源于PLC。还通过从外源性1 - 棕榈酰 - 2 - [花生四烯酰 - 14C]PC释放[14C]DAG，在GEC匀浆中研究了PC定向的PLC活性。在生理钙离子浓度（200 - 1200 nM）下存在PLC活性，并且PMA刺激细胞匀浆中的PLC活性（在ATP存在下）。这些结果直接证明PMA刺激DAG从PC中释放，并且与PMA在[3H]溶血磷脂酰胆碱标记的GEC中的作用一致。因此，GEC含有一种PC定向的PLC，其活性受到生理调节且在纳摩尔钙离子浓度下存在。C5b - 9刺激PC定向的PLC，导致DAG产生。这种DAG可能触发一种在补体攻击期间限制损伤的机制。
