Density-dependent proliferation of adult rat hepatocytes in primary culture induced by epidermal growth factor is potentiated by cAMP-elevating agents.
We investigated whether or not epidermal growth factor (EGF) and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early (4 h after adding EGF) and late phases (21 h after adding EGF) of primary cultures. Adult rat hepatocytes did not significantly proliferate after culture with 20 ng/ml EGF for 4 h at a density of 1 X 10(5) cells/cm2. In contrast, when the density was decreased by about one-third to 3.3 X 10(4) cells/cm2, the number of nuclei increased about 1.2-fold after culture with 10-20 ng/ml EGF for 4 h. Under these culture conditions, DNA synthesis began within 2-4 h of exposure to 20 ng/ml of EGF, although at the high cell density, DNA was not synthesized during this period. The beta-adrenoceptor agonists, metaproterenol and isoproterenol, and other cAMP-elevating agents, such as glucagon, forskolin, and dibutyryl cAMP, potentiated both hepatocyte DNA synthesis and proliferation about 1.4-fold when cultured in combination with 20 ng/ml EGF. The stimulatory effects of metaproterenol and other cAMP-elevating agents were specifically blocked by the cAMP-dependent protein kinase inhibitor, H-89 (10(-7) M). The effect of EGF was almost completely suppressed by genistein (5 X 10(-6) M) and rapamycin (10 ng/ml), but it was unaffected by wortmannin (10(-7) M). These results demonstrate that mature rat hepatocytes can proliferate very rapidly in low-density cultures with EGF, the effects of which were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. In addition, the activation of receptor tyrosine kinase and p70 ribosomal protein S6 kinase may be involved in EGF-induced hepatocyte DNA synthesis and proliferation.
Kimura
M
M
Biochemical Pharmacology Group, Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama, Japan.
Ogihara
M
M
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002452:Cell Count / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008297:Male; D009921:Metaproterenol / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013449:Sulfonamides
D000318:Adrenergic beta-Agonists; D007529:Isoflavones; D007546:Isoquinolines; D013449:Sulfonamides; D009921:Metaproterenol; D004815:Epidermal Growth Factor; D003994:Bucladesine; D004247:DNA; D019833:Genistein; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide