Role of diacylglycerol (DAG) in hormonal induction of S phase in hepatocytes: The DAG-dependent protein kinase C pathway is not activated by epidermal growth factor (EGF), but is involved in mediating the enhancement of responsiveness to EGF by vasopressin, angiotensin II, and norepinephrine

The role of diacylglycerol (DAC) in hormonal induction of S phase was inves tigated in primary cultures of rat hepatocytes. In this model, several agon ists that bind to G protein-coupled receptors act as comitogens when added to the cells soon after plating (i.e., in G(O)/early G(I) phase), while the cells are most responsive to the mitogenic effect of epidermal growth fact or (EGF) at 24-48 h of culturing (i.e., mid/late G(I)). It was found that t he cellular concentration of DAC rose markedly and progressively during the first 24 h of culturing. Exposure of the hepatocytes at 3 h to alpha(I)-ad renergic stimulation (norepinephrine with timolol), vasopressin, or angiote nsin II further increased this rise, producing a sustained increase in the DAG level. Norepinephrine, which was the most efficient comitogen, produced the most prolonged DAC elevation. In contrast, no significant increase of DAC was found in response to EGF, neither at 3 nor at 24 h, using concentra tions that markedly stimulated the ERK subgroup of the mitogen-activated pr otein kinases (MAPK) and DNA synthesis. Addition of Bacillus cereus phospha tidylcholine-specific phospholipase C (PC-PLC) strongly elevated DAG, while Streptomyces phospholipase D (PLD) increased phosphatidic acid (PA) but no t DAG. B. cereus PC-PLC ana-the protein kinase C (PKC) activator tetradecan oyl phorbol-acetate (TPA), like norepinephrine, vasopressin, and angiotensi n II, stimulated MAPK and enhanced the stimulatory effect of EGF on DNA syn thesis. The PKC inhibitor GF109203X did not diminish the effect of EGF on M APK or DNA synthesis, but strongly inhibited the effects of norepinephrine, vasopressin, angiotensin II, TPA and B. cereus PC-PLC on MAPK and almost a bolished the enhancement by these agents of EGF-stimulated DNA synthesis. T hese results suggest that although generation of DAG is not a direct downst ream response mediating the effects of the EGF receptor in hepatocytes, a s ustained elevation of DAG with activation of PKC markedly increases the res ponsiveness to EGF. Mechanisms involving DAG and PKC seem to play a role in the comitogenic effects of various agents that bind to C protein-coupled r eceptors and activate the cells early in G(I), such as norepinephrine, angi otensin II, and vasopressin. (C)1999 Wiley-Liss, Inc.