ACCUMULATION OF PHOSPHATIDYLALCOHOL IN CULTURED-CELLS - USE OF SUBCELLULAR FRACTIONATION TO INVESTIGATE PHOSPHOLIPASE-D ACTIVITY DURING SIGNAL-TRANSDUCTION

Phosphatidylalcohol accumulates as a product of a phospholipase D (PLD )-catalysed transphosphatidylation reaction in cells incubated in the presence of a primary alcohol, In the presence of ethanol the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the accumulat ion of [H-3]phosphatidylethanol (PEth) in HeLa cells prelabelled with [H-3]palmitic acid. Radioactivity associated with PEth increased linea rly during a 30 min incubation, indicating that a sustained activation of PLD is caused by PMA in these cells. This was accompanied by the m embrane association of protein kinase C-alpha (PKC-alpha), the PKC iso form that recent studies indicate is involved in the activation of PLD . In similar experiments, the neuropeptide bradykinin stimulated an ac cumulation of PEth in 3T3 Li cells. The radioactivity associated with PEth increased to a maximal level at 30 s and plateaued after this tim e, suggesting that bradykinin induces only a transient activation of P LD in these cells. This is consistent with the effects of bradykinin o n PKC-alpha, which underwent a rapid and transient association with ce ll membranes. The subcellular localization of PEth was examined using the technique of subcellular fractionation on Percoll density gradient s to isolate organelle-enriched fractions from HeLa and 3T3 Li cells. An accumulation of [H-3]PEth was measured in the plasma-membrane (PM)- enriched fractions of both HeLa and 3T3 Li cells after incubation with PMA and bradykinin respectively. This was accompanied by a time-depen dent accumulation of [H-3]PEth in the combined mitochondrial and endop lasmic reticulum (MER)-enriched fractions of both cell lines. PMA was also found to cause translocation of PKC-alpha to both the PM- and MER -enriched fractions in HeLa cells. However, bradykinin stimulated the translocation of PKC-alpha to the PM-enriched fractions only of 3T3 Li cells. The results show that PLD activation leads to the accumulation of PEth in both the PM and MER fractions. We therefore propose that e ither bradykinin activates a PM-associated PLD and the PLD reaction pr oduct is rapidly translocated to other membrane systems or it activate s an MER-associated PLD by a mechanism that does not involve PKC-alpha .