Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.