Characterization of nitric oxide production following isolation of rat hepatocytes

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) p roduce large amounts of nitrite following isolation. Nitrite production was inhibited by the inducible nitric oxide synthase (iNOS) inhibitor aminogua nidine, as well as the transcription inhibitor actinomycin D. Increases in iNOS mRNA, protein, and activity levels correlated with the formation of ni trite. iNOS mRNA was first detectable 2 h after the onset of hepatocyte inc ubations and peaked at 4 h. These results indicate that nitrite formation i s a result of the large scale production of nitric oxide (NO) by hepatocyte s in response to the time-dependent induction of iNOS. NO production by hep atocytes was attenuated by pretreatment of rats with the Kupffer cell inhib itor, gadolinium chloride. Also, the addition of the endotoxin neutralizing agent, polymyxin B; the protein kinase inhibitor, staurosporine, and antio xidants to perfusion buffers and hepatocyte suspensions also decreased nitr ite formation. Collectively, our results suggest that iNOS is induced in he patocytes in response to the stresses generated during collagenase isolatio n procedures. The response appears to be triggered by a complex interaction between several different factors including Kupffer cell activation, react ive oxygen species generation, and endotoxin contamination of collagenase p reparations.