Role of endogenous nitric oxide in liver-specific functions and survival of cultured rat hepatocytes.

1. The role of endogenous nitric oxide in rat hepatocyte functionality and survival in cell culture was examined. Towards this aim, cytochrome P450 activities (CYP1A1/2, 2B1, 2A1, 2C11, 2D1, 2E1 and 3A1), liver-specific metabolic functions and cell survival were comparatively evaluated in hepatocytes isolated from the male Sprague-Dawley rat and/or cultured in control conditions or in the presence of N-omega-nitro-L-arginine methyl ester (NAME), an inhibitor of nitric oxide synthesis. 2. Suppression of nitric oxide production by NAME paralleled a substantial preservation of hepatocyte phenotype in culture. The presence of NAME was particularly important during isolation and/or the 6-24h culture. By 24h, beneficial effects were evident in parameters particularly unstable in culture (glycogen content, P450), whereas no changes were produced in well-preserved functions (glucose, urea and albumin synthesis, glutathione, drug-conjugating enzymes). 3. Long-term treatment of hepatocytes with NAME also produced a reduction in caspase 3 activation and in the percentage of spontaneous apoptotic cells, and an increase in cell survival and transcriptional activity as shown by attached cellular protein content and the protein-DNA ratio respectively. 4. In conclusion, inhibition of early endogenous nitric oxide formation is an efficient procedure for obtaining hepatocyte cultures with stable expression of differentiated functions, high cell survival and few signs of cell senescence.