Hormonal regulation of phospholipase D activity in Ca2+ transporting cellsof rabbit connecting tubule and cortical collecting duct

Phospholipase D (PLD is distributed widely in mammalian tissues where it is believed to play an important role in the regulation of cell functions and cell fate by a variety of extracellular signals. In this study, we used pr imary cultures of rabbit connecting tubule (CNT) and cortical collecting du ct (CCD) cells, sown to confluence on a permeable support, to investigate t he possible involvement of PLD in the mechanism of action of hormones that regulate Ca2+ reabsorption. RT-PCR revealed the presence of transcripts of PLD1b and PLD2, but not PLD1a, in these cultures. Moreover, the expression of substantial amounts of PLD1 protein was demonstrated by Western blotting . To measure PLD activity, cells were labelled with [H-3]myristic acid afte r which the PLD-catalysed formation of radiolabelled phosphatidylethanol ([ H-3]PtdEth) was measured in the presence of 1% (v/v) ethanol. Deamino-Cys.D -Arg(8)-vasopressin (dDAVP) and N-6-cyclopentyladenosine (CPA), two potent stimulators of Ca2+ transport across these monolayers, stimulated PLD activ ity as was indicated by a marked increase in [H-3]PtdEth. Similarly, ATP, a potent inhibitor of dDAVP- and CPA-stimulated Ca2+ transport, increased th e formation of [H-3]PtdEth. PLD activity was furthermore increased by 8Br-c AMP and following acute (30 min) stimulation of protein kinase C (PKC) with a phorbol ester (PMA). Chronic PMA treatment (120 hi to downregulate phorb ol ester-sensitive PKC isoforms did not affect PLD activation by dDAVP, CPA and 8Br-cAMP, while markedly decreasing the effect of ATP and abolishing t he affect of PMA. The PKC inhibitor chelerythrine significantly reduced PLD activation by dDAVP, CPA and 8Br-cAMP, without changing the effect of ATP. The inhibitor only partially reduced the effect of PMA. This study shows t hat Ca2+ transporting cells of CNT and CCB contain a regulated PLD activity . The physiological relevance of this activity, which is not involved in Ca 2+ reabsorption, remains to be established (C) 2001 Elsevier Science B.V. A ll rights reserved.