Phosphatidic acid increases intracellular free Ca2+ and cardiac contractile force

Although phosphatidic acid (PA) is mainly formed due to the hydrolysis of phosphatidylcholine by myocardial phospholipase D, its functional significance in the heart is not fully understood. The present study was designed to determine the effects of PA on intracellular free Ca 2+ level ([Ca2 + ] i ) in freshly isolated adult rat cardiomyocytes by using fura 2-acextoxmethylester and free fura 2 technique. Addition of PA at concentrations of 1-200 μM produced a concentration-dependent increase in [Ca 2+ ] i from the basal level of 117 ± 8 nM; maximal increase in [Ca 2+ ] i was 233 ± 50 nM, whereas median effective concentration (EC 50 ) for PAwas 45 ± 1.2 μM. This increase in [Ca 2+ ] i was abolished by the removal of extracellular Ca 2+ with ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and was partially attenuated by Ca 2+ channel blockers, verapamil or diltiazem. Preincubation of cardiomyocytes with cyclopiazonic acid and thapsigargin or with ryanodine [to deplete sarcoplasmic reticulum (SR) Ca 2+ ] attenuated the PA-induced increase in [Ca 2+ ] i by 66, 37, and 43%, respectively. Furthermore, the response of [Ca 2+ ] i to PA was blunted by 2-nitro-4 carboxyphenylcarbonate, an inhibitor of phospholipase C, but was unaffected by staurosporine, a protein kinase C inhibitor. PA was also observed to induce Ca 2+ efflux from the myocytes. In addition, an injection of PA (0.34 μg/100 g body wt iv) in rats produced a significant increase of the left ventricular developed pressure as well as the maximum rates of cardiac contraction and relaxation within 5 min. These data suggest that the PA-induced increase in [Ca 2+ ] i in cardiomyocytes is a consequence of both Ca 2+ influx from the extracellular source and Ca 2+ release from the intracellular SR stores. Furthermore, these in vitro data suggest the possibility that PA may regulate [Ca 2+ ] i and contractile parameters in the heart.