PROTEIN-KINASE C-ALPHA MEDIATES PHOSPHOLIPASE-D ACTIVATION BY NUCLEOTIDES AND PHORBOL ESTER IN MADIN-DARBY CANINE KIDNEY-CELLS - STIMULATION OF PHOSPHOLIPASE-D IS INDEPENDENT OF ACTIVATION OF POLYPHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C AND PHOSPHOLIPASE A(2)

Protein kinase C (PKC) has been implicated in the activation of phosph olipase D (PLD) in a number of systems. By antisense technology, we ha ve ''knocked out'' alpha and beta isoforms of PKC to study the role of these isoforms in PLD activation in Madin-Darby canine kidney (MDCK) cells. To this end, we have studied PLD activation by phorbol 12-myris tate 13 acetate (PMA), ATP, UTP, and 2-methylthio-ATP in cells labeled with [H-3]palmitic acid. [H-3]Phosphatidylethanol (PEt) production ca talyzed by PLD in the presence of ethanol was time- and concentration- dependent in PMA- and nucleotide stimulated cells. In Ca2+-free medium , [H-3]PEt accumulation was diminished for all stimuli assayed. Treatm ent of cells with chelerythrine, an inhibitor of PKC, and phorbol este r down-regulation of PKC inhibited [H-3]PEt production by both PMA and nucleotides. In cells transfected with antisense PKCalpha or both PKC alpha and PKCbeta, PLD activation was inhibited by both PMA and nucleo tides, whereas in cells transfected with antisense PKCbeta, PLD activa tion was similar to that of control cells. Moreover, inhibition of pol yphosphoinositide-specific PLC (by neomycin) or of release of arachido nic acid and arachidonic acid metabolites (by nordihydroguaiaretic aci d or by indomethacin) failed to decrease [H-3]PEt accumulation in PMA- and nucleotide-stimulated MDCK-D1 cells. From these data, we conclude that in MDCK-D1 cells PMA and nucleotide receptors utilize PKCalpha t o regulate PLD activity and that PLD activation is independent of the activation of polyphosphoinositide-specific PLC and phospholipase A(2) -mediated release of arachidonic acid or arachidonic acid metabolites.