Extracellular ATP and UTP activation of phospholipase D is mediated by protein kinase C‐ε in rat renal mesangial cells

Abstract

We have studied whether a nucleotide receptor mediates the effects of extracellular ATP and UTP on phosphatidylcholine metabolism in rat cultured glomerular mesangial cells. 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. 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. 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. Elevation of cytosolic free Ca2+ by the calcium ionophore A23187 (1 μm) 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. 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. 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. 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. 1993 British Pharmacological Society