Bombesin and platelet-derived growth factor stimulate phosphatidylcholine breakdown by a common mechanism.

The two Swiss 3T3 cell mitogens, bombesin and plateletderived growth factor (PDGF ) both stimulate the phosphoinositidase C-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptdlns[4,5]P2). Bombesin stimulates rapid generation of inositol 1,4,5-trisphosphate (Ins[ 1,4,5]P,) in these cells [ 11, whereas there is a distinct lag before PDGFstimulated InsP, generation is observed 12, 31. Bombesin is believed to stimulate phosphoinositidase C via an interaction with a receptor-G-protein complex [4]. However, recent evidence suggests that PDGF-stimulated PtdIns(4,5)P2 hydrolysis occurs via tyrosine phosphorylation of PLCy with no G-protein involvement (51. We have recently demonstrated that bombesin stimulates phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 cells [6] which may be important in generating a sustained increase in diacylglycerol (DAG) levels and thus protein kinase C activity. PDGFstimulated PtdCho hydrolysis has been demonstrated in 3T3-Ll cells [7]. Since the two mitogens regulate Ptdlns( 4,5)P2 hydrolysis by different mechanisms, we investigated the interrelationship between PtdIns(4,5)Pz and PtdCho breakdown in response to each agonist. For assay of PtdCho hydrolysis, Swiss 3T3 cells were labelled with 2 pCi of [methyl-’Hlcholine chloride in Dulbecco’s modified Eagle’s medium (DMEM) containing 2% (v/v) calf serum for 48 h. After treatment with the agonists, [ 3H]choline metabolites were separated on Dowex50W-H + cation-exchange columns according to Cook & Wakelam 161. Assay of agonist-stimulated increases in Ins( 1,4,5)P, mass was achieved using the competitive binding assay of Palmer er al. [8]. Both PDGF and c-sis were used and gave similar results. Bombesin (6 17 n M ) stimulated the rapid elevation of Ins( 1,4,5)P,, which was maximal at 5 s and rapidly returned to unstimulated levels by 30 s (typical experiment: basal, 3.15kO.31 pmol; stimulated, 33.13f2.65 pmol at 5 s).This response occurred with a concentration required to give 50”/0 of maximal response (EC,,,) of 5 .88f3.66 nM, which is similar to that for bombesin-stimulated DAG production 191 and DNA synthesis [ 11. Maximum concentrations of PDGF (30 ng/ml) also stimulated the formation of Ins( I ,4,5)P, in Swiss 3T3 fibroblasts. As expected, the onset of the response was slower than that obtained for bombesin, with a lag time of 10 s before any increase in mass Ins( 1,4,5)P, was observed. This lag period is, however, considerably less than previously reported in studies using [ 3H]inositol-labelled cells [2, 31. Furthermore, although the magnitude of the response was smaller than that for bombesin (bombesin, 10-1 2-fold, n = 8; PDGF, 2.5-3.5-fold at 20 s, n = 3), the increased levels were maintained for up to 5 min. We have previously shown that bombesin stimulates PtdCho hydrolysis subsequent to Ins( 1,4,5)P, production. Since PDGF stimulates Ins( 1,4,5)P3 production by a mechanism fundamentally different to bombesin, the effects