PHYSIOLOGICAL RESEARCH ISSN 0862-8408 © 2007 Institute of Physiology, v. v. i., Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164 E-mail: physres@biomed.cas.cz http://www.biomed.cas.cz/physiolres Physiol. Res. 56: 427-432, 2007 Effect of Nitric Oxide Release from NOR-3 on Urea Synthesis, Viability and Oxygen Consumption of Rat Hepatocyte Cultures R. CHIMENTI, G. MARTINO, S. MAZZULLA, S. SESTI Department of Cell Biology, University of Calabria, Arcavacata di Rende, Italy Received August 9, 2006 Accepted August 17, 2006 On-line available August 22, 2006 Summary As nitric oxide is considered a mediator of liver oxidative metabolism during sepsis, we studied the effects of exogenous nitric oxide, produced by NO-donor, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), on cell viability, urea biosynthesis and oxygen consumption in rat hepatocyte cultures. Nitric oxide release from NOR-3 was studied using 4,5-diaminofluorescein diacetate. Urea levels were measured by the spectrophotometric method. Cell viability was determined by the MTT test and trypan blue exclusion test, whereas oxygen consumption was measured by a polarographic technique. After 2 h treatment, NOR-3 induced an increase in the levels of nitric oxide. After 2 h of treatment and 24 h after the end of the treatment with NOR-3, both cell viability and urea synthesis were significantly reduced in comparison to the controls for NOR-3 concentrations equal to or greater than 50 μM. A reduction in oxygen consumption was observed in hepatocytes after 40 min treatment with 100 μM NOR-3, even if the cell viability was unchanged. Reduction of oxygen consumption is an early indicator of the metabolic alterations in hepatocytes exposed to nitric oxide. These findings suggest that nitric oxide accumulation acts on hepatocyte cultures inducing cell death and reduction of urea synthesis after 2 hours. Key words 4,5 diamminofluorescein diacetate • Nitric oxide • Nitric oxide donor • Rat hepatocytes Introduction Nitric oxide (NO) is a free radical, present in conditions such as atmospheric pollution and cigarette smoke. In biological systems, NO has a short half-life in the order of seconds. NO has an oxidation intermediate state and is consequently capable both to oxidize and to reduce the chemical compounds with which it comes into contact. The biosynthesis mainly occurs through the transformation of L-arginine amino acid to citrulline. This reaction requires the participation of NO synthase (NOS) enzyme. Recently, mitochondrial NOS (mtNOS) has been detected (Giulivi 2003). The endothelial (eNOS), the neuronal (nNOS) and the mitochondrial types of enzyme are constitutive. The inducible type (iNOS) is not modulated in its enzymatic activity by the intracellular Ca 2+ concentration, as happens in the constitutive type (Taylor et al. 1998). The mechanisms by which NO can elicit changes in the hepatic metabolism can be considered from two aspects: 1) by exerting a