Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor.
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ABSTRACT:
SUBMITTER: Cattaneo MG
PROVIDER: S-EPMC1133319 | biostudies-other | 1989 Sep
REPOSITORIES: biostudies-other
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Project description:Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.
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Project description:In previous studies, activators of protein kinase C, sphingosine, ATP and various oncogenes were each found to enhance phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here I examined possible stimulation of PtdEtn hydrolysis by various growth-stimulatory agents, including serum, bombesin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. Treatment of NIH 3T3 fibroblasts, prelabelled with [14C]Etn or [32P]PtdEtn, with PDGF-BB resulted in enhanced formation of [14C]Etn or [32P]phosphatidic acid from the respective labelled cellular pools of PtdEtn. A maximal effect (approximately 3-fold stimulation) on PtdEtn hydrolysis was obtained with 50 ng of PDGF/ml after 5 min of treatment. Phosphatidylcholine (PtdCho) was also hydrolysed, although less extensively than PtdEtn, in PDGF-stimulated cells. PDGF-stimulate hydrolysis of both PtdEtn and PtdCho was prevented by prolonged (30 h) treatment of cells with 400 nM-phorbol 12-myristate 13-acetate (PMA). Similar to PDGF, fetal calf serum (1-10%) also stimulated PtdEtn hydrolysis. However, in contrast to PDGF, the effect of serum on PtdEtn hydrolysis (i) was not diminished by pretreatment with PMA, and (ii) was synergistic with that of PMA after a 1 h incubation. Compared with PDGF and serum, bombesin had less effect on PtdEtn hydrolysis, while FGF and insulin had no effects at all. In contrast to PDGF or serum, bombesin inhibited the effect of PMA on PtdEtn hydrolysis.
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