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Differential potentiation of mitogen-stimulated phosphoinositide hydrolysis in protein kinase C-depleted Swiss 3T3 cells.

ABSTRACT: In Swiss 3T3 cells, depletion of protein kinase C (PKC) by prolonged incubation with phorbol esters potentiates the formation of total inositol phosphates in response to bombesin or vasopressin [Blakeley, Corps & Brown (1989) Biochem. J. 258, 177-185]. The characteristics of the accumulation of inositol phosphates in control and PKC-depleted cells stimulated by bombesin, vasopressin or prostaglandin F2 alpha (PGF2 alpha) have now been compared. The potentiation of the PGF2 alpha response was greater than that of the vasopressin response which was, in turn, greater than that of the bombesin response. The time courses of the responses to all three agonists were biphasic, and both phases of the response were amplified in the PKC-depleted cells. These results provide further evidence for the involvement of a PKC-mediated negative-feedback loop regulating phosphoinositide hydrolysis in response to several 3T3 cell mitogens. The differential potentiation of the response to these agonists suggests that PKC might act at multiple sites within the signal transduction pathway.

SUBMITTER: Brown KD

PROVIDER: S-EPMC1131761 | biostudies-other | 1990 Sep

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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Differential potentiation of mitogen-stimulated phosphoinositide hydrolysis in protein kinase C-depleted Swiss 3T3 cells.

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