ENDOTHELIN STIMULATES PHOSPHATIDYLCHOLINE HYDROLYSIS THROUGH BOTH PLCAND PLD PATHWAYS IN MESANGIAL CELLS

Endothelin (ET) is a recently characterized vasoconstrictor hormone th at has potent effects on glomerular function. Many vasoconstrictors, l ike ET, that stimulate phospholipase C (PLC) hydrolysis of polyphospho inositides also stimulate phosphatidylcholine (PtdCho) hydrolysis via both PLC and phospholipase D (PLD) pathways. We have previously report ed that ET stimulates a protein kinase C (PKC)regulated, intracellular calcium-insensitive PLD activity that forms phosphatidic acid (PA) in rat mesangial cells (MC). We now ask whether ET-induced diglyceride ( DG) production is also, in part, a result of either PLC- or PLD-induce d hydrolysis of PtdCho. ET induced both a time- and dose-dependent sti mulation in DG as measured by radioflux and mass assays. ET-stimulated DG production was still elevated even at time points where inositol p olyphosphates had returned to basal levels. In addition, using [H-3]ch oline-labeled cells, ET stimulated [H-3]phosphocholine accumulation, s uggesting a PLC-mediated hydrolysis of PtdCho. Stimulation of DG was u naffected by the presence of ethanol or propranolol, suggesting that E T-stimulated DG were not a result of a sequential PLD/PA phosphohydrol ase activity. We further dissociated PtdCho-dependent PLC and PLD acti vities because, in contrast to ET-induced stimulation of PLD, the effe ct of ET on DG formation was mimicked with ionomycin and was inhibited with ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid but not ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. ET stimulation of DG could not be mimicked by phorbol myristate acetat e and was not blocked by PKC inhibition or depletion. Together, these data suggest that ET stimulates multiple signaling pathways in MC that hydrolyze PtdCho via separate PLC and PLD mechanisms. The potential s ignificance of ET-stimulated PtdCho hydrolysis includes the generation of diverse species of DG and PA, lipid second messengers that may aug ment an activated mesangial cell phenotype.