Previous studies have demonstrated a strict extracellular Ca2+ dependence for the G(0) to G(1) and G(1) to S transition in growth factor-treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid-derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G(0) to G(1) transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca2+-independent increase in the levels of inositol 1,4,5-trisphosphate (lns[1,4,5]P-3), inositol 1,4-bisphosphate (Ins[1,4]P-2), and inositol 4-monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51 B cells with EGF results in a slower, more prolonged extracellular Ca2+-dependent increase in both [H-3]-glycerol radiolabeled diacylglycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (Ptdlns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated Ptdlns turnover, and that Ptdlns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca2+-dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca2+-dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine-linked phospholipase D. These results suggest that diacylglycerol sources other than Ptdlns's may be important in the extracellular Ca2+-dependent regulation of EGF-mediated cell replication. (C) 1995 Wiley-Liss, Inc.
