Inhibitory effect of ethanol on hepatocyte growth factor-induced DNA synthesis and Ca2+ mobilization in rat hepatocytes

Abstract

Hepatocyte growth factor (HGF) is the most potent mitogen identified for hepatocytes and is thought to be an important growth factor in the regulation of liver regeneration. Its effects are mediated through a tyrosine kinase receptor, the product of c-met proto-oncogene. One of the downstream signaling processes activated by HGF is phospholipase C-gamma. HGF stimulation of liver cells causes formation of inositol 1,4,5-triphosphate, which releases Ca2+ from intracellular Ca2+ ([Ca2+]i) stores, and causes elevation of cytosolic Ca2+ levels. It is known that liver regeneration is inhibited by both acute and chronic ethanol (EtOH) treatment. We investigated the effect of EtOH on HGF-induced DNA synthesis and mobilization of [Ca2+]i in rat hepatocytes in primary culture. DNA synthesis was monitored by [3H]thymidine incorporation in primary cultures of hepatocytes 42 hr after stimulation with HGF. HGF concentration required for maximum DNA synthesis was 0.3 to 1 ng/ml, and DNA synthesis was inhibited by 100 mM EtOH at HGF concentrations in the range of 0.1 to 5 ng/ml. This inhibition was strongest (45 to 47% inhibition) at a low concentration of HGF (0.1 to 0.3 ng/ml) and decreased at an HGF concentration > 1 ng/ml. HGF-induced changes in [Ca2+]i were measured in single fura 2-loaded hepatocytes by fluorescence imaging techniques. The Ca2+ response induced by HGF (0.3 to 5 ng/ml) was inhibited by EtOH, with an EC50 of approximately 50 mM. Analysis of Ca2+ response patterns in individual cells indicated that EtOH suppressed the number of responsive cells and made Ca2+ responses more transient, but did not affect peak [Ca2+]i elevation; thus suggesting an inhibition at the level of phospholipase C-gamma-activation. These data indicate that inhibition by EtOH of the response of liver cells to HGF may contribute to the inhibitory effect of EtOH on liver regeneration.