Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca 2+] i levels in neonatal rat cardiomyocytes

Abstract

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca 2+] i) and inositol-1,4,5-trisphosphate (IP 3) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP 3 and [Ca 2+] i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP 3 and [Ca 2+] i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca 2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca 2+] i increase but had no significant effect on IP 3 accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP 3 and [Ca 2+] i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca 2+] i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca 2+] i. The PLA 2 inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca 2+] i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca 2+] i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA 2, may enhance Ca 2+ release from IP 3 sensitive SR Ca 2+ stores via activation of PLC in neonatal rat cardiomyocytes.