Pharmacological modulation of oscillations in the blowfly salivary gland

Abstract

lation counting and expressed as d.p.m./culture well. (ii) Alternatively, the various inositol phosphates within the sample were separated by h.p.1.c. on a Partisil Sax column eluted with a 0-3 M-ammonium formate/O.l M-formic acid gradient at a flow rate of 1.25 ml/min. Fractions (0.5 min) were collected and radioactivity in them determined as above expressed as d.p.m./fraction. Treatment of 3T3 cultures with FGF stimulates total inositol phosphate formation in cells. Total [3HH]inotisol phosphates in control 3T3 cultures was 12.5 1 f 36 d.p.m./culture well; in cultures exposed to basic FGF (50 ng/ml) for 2 h it was 2 126 f 77 d.p.m./culture well. Table 1 shows different inositol phosphates produced during this response analysed by h.p.1.c. By 10 s of stimulation, inositol trisphosphate levels are raised, as are levels of inositol tetrakisand pentakisphosphate. After 10 min, the inositol trisand tetrakis-phosphate levels have fallen to control levels while the raised inositol pentakisphosphate levels are maintained. By 30 min of stimulation, although still higher than controls, the inositol pentakisphosphate levels are falling towards control values. This observation of a rapid and transient inositol trisphosphate signal accompanied by a much more persistent inositol pentakisphosphate signal suggests a multiple signalling pathway coupled by phospholipase C and activated by FGF. What mechanisms the two intracellular signals are targeting remain to be established, but it might be that the more highly phosphorylated inositol derivatives are involved in mediating the long-term regulation of cell growth. The responses to FGF in vitro can be divided into very early events, which include ionic fluxes, protein kinase activation and jntracellular pH changes, and later events such as gene transcription and protein synthesis (Gospodarowicz, 1087). It is interesting to speculate that the transient and persistent inositol phosphate signals observed here may be linked t o early and late responses to FGF. Overall these data establish that FGF does stimulate phosphoinositide hydrolysis, at least in Balb/c 3T3 fibroblast cells. However, the relative importance of this signalling pathway for mitogenesis has still to be defined.