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Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography. Bombesin and TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells by a common mechanism.

ABSTRACT: A method for the rapid and quantitative separation of glycerophosphocholine, choline phosphate and choline upon ion-exchange columns is described. The method has been utilized to examine the stimulation of phosphatidylcholine breakdown in quiescent Swiss 3T3 cells in response to bombesin and 12-O-tetradecanoylphorbol 13-acetate (TPA). The stimulated generation of choline is shown to precede that of choline phosphate, with no effect upon glycerophosphocholine levels; but was attenuated in cells in which protein kinase C activity was down-regulated. The results thus suggest that stimulation of the cells with either bombesin or TPA activates phospholipase D-catalysed phosphatidylcholine breakdown by a common mechanism involving the activation of protein kinase C.

SUBMITTER: Cook SJ

PROVIDER: S-EPMC1133466 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Project description:Treatment of Swiss-mouse 3T3 fibroblasts with a tumour-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), increases the absolute amount of 1,2-diacylglycerol (1,2-DG) in a time- and dose-dependent manner. Within 1 min of TPA (0.1 microM) addition to confluent monolayer cells, cellular 1,2-DG content increases significantly, and reaches a value nearly 4-fold above control at 10 min. The content of 1,2-DG then falls, but still remains at twice the basal value for up to 60 min. The effect of TPA on 1,2-DG content is dose-dependent, with a half-maximal effect obtained with 6 nM. Studies with a series of tumour promoters have revealed that stimulating effect on 1,2-DG formation is closely related to their relative potency as a tumour promoter, as well as their mitogenic effect on 3T3 cells. TPA does not have any stimulatory effect on phosphoinositide turnover, which is a major mechanism of 1,2-DG formation by natural agonists, including a peptide mitogen, bombesin, but significantly inhibits bombesin-induced phosphoinositide turnover. Nevertheless, the content of 1,2-DG is even higher in cells treated with both bombesin and TPA than in cells treated with bombesin alone. In addition, a diacylglycerol kinase inhibitor, R59022, increases 1,2-DG content synergistically with bombesin, but not with TPA. Measurements of the absolute amounts of various phospholipids show that TPA does not increase phosphatidic acid, despite a remarkable increase in 1,2-DG. Further studies on phospholipid metabolism reveal that TPA stimulates metabolic turnover of phosphatidylcholine (PC), without changing the mass of PC. TPA stimulates the incorporation of both [3H]choline and [32P]Pi into PC, and the release of [3H]choline metabolites from prelabelled cells. These findings suggest that TPA increases cellular 1,2-DG content mainly via increased PC turnover and diacylglycerol kinase inhibition. The present study provides evidence for a novel effect of TPA on the metabolism of 1,2-DG in Swiss-mouse 3T3 cells.

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Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography. Bombesin and TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells by a common mechanism.

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