Vol. 282, Issue 3, 1146-1154, 1997

Proliferation of Adult Rat Hepatocytes by Hepatocyte Growth Factor Is Potentiated by Both Phenylephrine and Metaproterenol

Mitsutoshi Kimura and Masahiko Ogihara

Biochemical Pharmacology Group, Faculty of Pharmaceutical Sciences, Josai University. 1-1, Keyakidai, Sakado, Saitama 350-02 Japan

We investigated whether or not beta and alpha adrenergic agonists could affect proliferation of adult rat hepatocytes induced by hepatocyte growth factor (HGF) during the early and late phases of primary culture. Adult rat hepatocytes underwent significant DNA synthesis after culture with 5 ng/ml HGF for 3 h at a low cell density (3.3 × 10⁴ cells/cm²). Under these culture conditions, the number of nuclei increased significantly during a subsequent 4-h culture period. Hepatocyte DNA synthesis and proliferation induced by 5 ng/ml HGF was reduced at high cell densities near confluence. A beta adrenergic agonist, metaproterenol (10⁷ M), and dibutyryl cAMP significantly potentiated hepatocyte DNA synthesis and proliferation at a concentration as low as 10⁷ M when cultured in combination with 5 ng/ml HGF. Similarly, an alpha-1 adrenergic agonist, phenylephrine (10⁶-10⁴ M) markedly potentiated HGF-induced hepatocyte DNA synthesis and proliferation. The phenylephrine effect was mimicked by a phorbol ester (10⁶ M), but not by ionomycin (10⁶ M). The mitogenic effects of HGF were almost completely blocked by simultaneous treatment of hepatocytes with genistein (5 × 10⁶ M), U-73122 (10⁶ M), wortmannin (10⁷ M), sphingosine (3 × 10⁶ M) and rapamycin (10 ng/ml). These results demonstrate that HGF can rapidly induce proliferation of adult rat hepatocytes in primary culture. However, this effect is dependent on the initial plating density. The co-mitogenic effects of metaproterenol and phenylephrine may involve both protein kinase A and protein kinase C activation, respectively. The results also suggest that following stimulation with HGF, activation of tyrosine kinase, phosphatidylinositol 3-kinase, phospholipase C and p70 ribosomal protein S6 kinase is essential for hepatocyte proliferation.