Phorbol esters stimulate the rapid release of choline from prelabelled cells

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Abstract
Evidence implicating the cell surface membrane as the primary site of action of phorbol ester tumor promoters has led us to characterize early changes in phospholipid metabolism which occur in response to these compounds. When C3H10T½ mouse embryo fibroblasts were incubated with [3H]choline for 24 h, ˜78% of the cell associated material was found in phosphatidyl choline and the remainder in sphingomyelin and the acid soluble pool. Within 5 min of exposure of these prelabelled cells to the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (PPD) the release of water soluble [3H] metabolites from cells into the medium was enhanced 2-fold and by 60–120 min the release was 2–5 times that found in control cultures. The released material was identified by chromatography as choline and phosphoryl choline. Evidence was obtained that this material was not derived exclusively from the acid soluble pool or from the degradation of labelled sphingomyelin. Choline metabolite release was concentration dependent between 10−8 and 10−7 M TPA and was not associated with cell toxicity. Phorbol-12,13-didecanoate (PDD) was also active but 4αPDD, which is not a tumor promoter, was inactive. Neither cyclohexlmide (4–40 μg/ml) nor cordycepln (4–40 μg/ml) blocked the TPA induced release. The release was, however, temperature sensitive and did not occur at 4°C. TPA did not induce the release of [3H]inositol from prelabelled phosphatidyl inositol. Although, the calcium ionophore A23187 induced the release of arachidonic acid from prelabelled cells it did not induce choline release. In addition, 5,8,11,14-eicosatetraynoic acid, which inhibits both lipoxygenase and cyclooxygenase activity, did not inhibit TPA stimulation of choline release. We propose that the binding of TPA to cell surface receptors leads to a rapid activation of membrane associated phospholipase(s) that specifically degrade phosphatidyl choline. This effect may be analogous to the ability of other agonists to activate degradation of phosphatidyl inositol.

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