PHOSPHATIDYLINOSITOL-DEPENDENT AND PHOSPHATIDYLCHOLINE-DEPENDENT PHOSPHOLIPASES-C ARE INVOLVED IN THE MECHANISM OF ACTION OF ATRIAL-NATRIURETIC-FACTOR IN CULTURED RAT AORTIC SMOOTH-MUSCLE CELLS

We have investigated the involvement of specific phospholipase systems and their possible mutual relationship with the mechanism by which at rial natriuretic factor (ANF) increases phosphatidate (PA) and diacylg lycerol (DAC) in rat aortic smooth muscle cells (RASMC), one of the ma jor targets of this hormone. Our results indicate that ANF initially s timulates a phosphatidylinositol-dependent phospholipase C (PI-PLC) wi th a significant increase of DAC, enriched in arachi donate, and inosi tol trisphosphate (IP3) and then a phosphatidylcholine-dependent phosp holipase C (PC-PLO with formation of DAG, enriched in myristate, and p hosphocholine (Pcho). Moreover, ANF stimulates PA formation at an inte rmediate stage between early and late DAG formation. The transphosphat idylation reaction. as well as its labeling ratio, demonstrate that ph osphatidylcholine-dependent phospholipase D IPC-PLD) is not involved. Our experiments with R59022, a DAC kinase (DAGK) inhibitor, indicate t hat such an increase may be due to the phosphorylation of DAG derived from phosphatidylinositol (PI) hydrolysis. Our results show that phorb ol 12-myristate 13 acetate (PMA) plays a significant role in late DAG formation and that Pcho is released concomitantly, suggesting there is a relationship between the two phospholipase Cs (PLCs) that occurs th rough a protein kinase C (PKC) translocation from cytosol to the plasm a membrane. These findings are confirmed by the use of PKC inhibitors calphostin, H7, and staurosporine. The involvement of membrane phospho lipid hydrolysis and the ensuing production of second messengers might explain the vasorelaxant effect of ANF. (C) 1997 Wiley-Liss, Inc.