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 [3H]-arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [3H]-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. 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 > ATP gamma S > beta gamma-imido-ATP = ADP > 2-methylthio-ATP = beta gamma-methylene-ATP = ADP beta S, 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 microM) or the Ca(2+)-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 4 alpha-phorbol 12,13-didecanoate, increased phospholipase D activity in mesangial cells, suggesting that PKC may mediate nucleotide-induced phosphatidylcholine hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)