Accumulation of phosphatidylalcohol in cultured cells: use of subcellular fractionation to investigate phospholipase D activity during signal transduction

Abstract

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 accumulation of [3H]phosphatidylethanol (PEth) in HeLa cells prelabelled with [3H]palmitic acid. Radioactivity associated with PEth increased linearly during a 30 min incubation, indicating that a sustained activation of PLD is caused by PMA in these cells. This was accompanied by the membrane association of protein kinase C-alpha (PKC-alpha), the PKC isoform that recent studies indicate is involved in the activation of PLD. In similar experiments, the neuropeptide bradykinin stimulated an accumulation of PEth in 3T3 Li cells. The radioactivity associated with PEth increased to a maximal level at 30 s and plateaued after this time, suggesting that bradykinin induces only a transient activation of PLD in these cells. This is consistent with the effects of bradykinin on PKC-alpha, which underwent a rapid and transient association with cell membranes. The subcellular localization of PEth was examined using the technique of subcellular fractionation on Percoll density gradients to isolate organelle-enriched fractions from HeLa and 3T3 Li cells. An accumulation of [3H]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-dependent accumulation of [3H]PEth in the combined mitochondrial and endoplasmic 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 either bradykinin activates a PM-associated PLD and the PLD reaction product is rapidly translocated to other membrane systems or it activates an MER-associated PLD by a mechanism that does not involve PKC-alpha.