EXTRACELLULAR ATP AND UTP ACTIVATION OF PHOSPHOLIPASE-D IS MEDIATED BY PROTEIN-KINASE C-EPSILON IN RAT RENAL MESANGIAL CELLS

1 We have studied whether a nucleotide receptor mediates the effects of extracellular ATP and UTP on phosphatidylcholine metabolism in rat cultured glomerular mesangial cells. 2 ATP and UTP stimulated a biphasic 1,2-diacylglycerol (DAG) formation in [H-3]-arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [H-3]-myristic acid, a tracer that preferentially marks phosphatidylcholine, both nucleotides induced a delayed monophasic production of DAG with a concomitant increase in phosphatidic acid and choline formation. 3 4 A phospholipase D-mediated phosphatidylcholine hydrolysis was further suggested by the observation that ATP and UTP stimulate the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells. 4 The rank order of potency of a series of nucleotide analogues for stimulation of phosphatidylethanol formation was UTP = ATP > ITP > ATPgammaS > betagamma-imido-ATP = ADP > 2-methylthio-ATP = betagamma-methylene-ATP = ADPbetaS, while AMP, adenosine, CTP and GTP were inactive, indicating the presence of a nucleotide receptor. 5 Elevation of cytosolic free Ca2+ by the calcium ionophore A23187 (1 muM) or the Ca2+-ATPase inhibitor, thapsigargin (200 nM) slightly increased phosphatidylethanol formation. However, chelation of cytosolic Ca2+ with high concentrations of Quin 2 did not attenuate ATP- and UTP-induced phosphatidylethanol production, thus suggesting that Ca2+ is not crucially involved in agonist-stimulated phospholipase D activation. 6 The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but not the biologically inactive 4alpha-phorbol 12,13-didecanoate, increased phospholipase D activity in mesangial cells, suggesting that PKC may mediate nucleotide-induced phosphatidylcholine hydrolysis. 7 Down-regulation of PKC-alpha and -beta isoenzymes by 8 h PMA treatment still resulted in full phospholipase D activation. In contrast, a 24 h treatment of mesangial cells with PMA, a regimen that also causes depletion of PKC-epsilon, markedly attenuated nucleotide-evoked phosphatidylethanol formation. In addition, the selective PKC inhibitor, calphostin C attenuated ATP- and UTP-induced phosphatidylethanol production. 8 In summary, these data suggest that extracellular ATP and UTP use a common nucleotide receptor to activate phospholipase D-mediated phosphatidylcholine hydrolysis. Stimulation of phospholipase D appears to involve the PKC-epsilon isoenzyme, activated by DAG derived from phosphoinositide hydrolysis by phospholipase C.