Extracellular ATP and UTP activation of phospholipase D is mediated by protein kinase C‐ε 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 [ 3 H]‐arachidonic acid‐labelled mesangial cells. In contrast, in cells labelled with [ 3 H]‐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 > ATPγS > βγ‐imido‐ATP = ADP > 2‐methylthio‐ATP = βγ‐methylene‐ATP = ADPβS, while AMP, adenosine, CTP and GTP were inactive, indicating the presence of a nucleotide receptor. 5 Elevation of cytosolic free Ca 2+ by the calcium ionophore A23187 (1 μ m ) or the Ca 2+ ‐ATPase inhibitor, thapsigargin (200 n m ) slightly increased phosphatidylethanol formation. However, chelation of cytosolic Ca 2+ with high concentrations of Quin 2 did not attenuate ATP‐ and UTP‐induced phosphatidylethanol production, thus suggesting that Ca 2+ 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 4α‐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‐α and ‐δ 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‐ε, 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‐ε isoenzyme, activated by DAG derived from phosphoinositide hydrolysis by phospholipase C.