Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+](i) levels in neonatal rat cardiomyocytes

Phosphatidic acid (PA), which can be synthesized de novo, or as a product o f phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycer ol (DAG), mediates diverse cellular functions in various cell types, includ ing cardiomyocytes. We set out to characterize the effect of PA on intracel lular free calcium ([Ca2+](i)) and inositol-1,4,5-trisphosphate (IP3) level s in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP3 and [Ca2+](i ) levels in adherent cells. There was strong correlation in the concentrati on-response relationships between IP3 and [Ca2+](i) increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclo piazonic acid (CPA), significantly attenuated the PA evoked [Ca2+](i) incre ase but had no significant effect on IP3 accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP3 and [Ca2+](i) elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibito r, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca2 +](i). Depletion of extracellular calcium attenuated both basal and PA evok ed increases in [Ca2+](i). The PLA(2) inhibitors, bromophenylacyl-bromide ( BPB) and CDP-choline, had no effect on PA evoked [Ca2+](i) responses. Neith er the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949 , affected PA evoked changes in [Ca2+](i). Taken together, these data indic ate that PA, in a manner independent of PKC, DAG, or PLA(2), may enhance Ca 2+ release from IP3 sensitive SR Ca2+ stores via activation of PLC in neona tal rat cardiomyocytes. (C) 1999 Elsevier Science B.V. All rights reserved.