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Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

ABSTRACT: Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.

SUBMITTER: Brown KD

PROVIDER: S-EPMC1148179 | biostudies-other | 1987 Aug

REPOSITORIES: biostudies-other

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Rapid desensitization and resensitization of bombesin-stimulated phospholipase D activity in Swiss 3T3 cells.

Project description:The kinetics of bombesin-stimulated phospholipase D (PLD) activity were examined in Swiss 3T3 fibroblasts. The stimulated activity was found to rapidly desensitize, being completely absent after 40 s. This activity then quickly, but incompletely, resensitized, with PLD being detectable after a 4.5 min wash of the desensitized cells and 75-80% of the activity being recovered after 10 min. The desensitization was dose-dependent; however, the half-maximal stimulatory concentration of bombesin was an order of magnitude lower than that required for bombesin-stimulated second messenger generation and the KD for bombesin receptor binding. This suggested that desensitization was stimulated by a 'downstream' effect, but experiments have ruled out changes in protein kinase C activity and Ca2+ concentration. Binding experiments suggested that part of the desensitization is due to receptor internalization, and the requirement for an extracellular agonist for resensitization implies that receptor recycling plays a role. Over an extended time course, cycles of desensitization and resensitization of bombesin-stimulated PLD activity were apparent which may be relevant to mitogenic signalling. These studies add further evidence for a second messenger pathway of PLD activation, and the disparity between the kinetics of diacylglycerol generation and PLD activation supports the possibility that phosphatidic acid may have a messenger role in stimulated cells.

| S-EPMC1137983 | biostudies-other

The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

Project description:The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

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Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein.

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

Project description: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.

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