Post-isolation inducible nitric oxide synthase gene expression due to collagenase buffer perfusion and characterization of the gene regulation in primary cultured murine hepatocytes

The traditional two-step EGTA/collagenase method is widely used in studying nitric oxide (NO) production in hepatocytes, The present study first revealed that hepatocytes isolated by this method spontaneously express an iNOS mRNA. Thereafter, based on this novel finding, we characterized the expression and regulation of the gene in primary cultured hepatocytes, Using Northern blot analysis, the iNOS mRNA was observed 4 h after isolation, reached peak at 8 h, and declined to an undetectable level after 24 h. iNOS gene expression was shown to be serum-independent and not due to lipopolysaccharide contamination. Time-course analysis of the effects of actinomycin D demonstrated that the increase in iNOS transcripts is the result of an accompanying great increase in iNOS gene transcription and lower iNOS mRNA stability; also blockage by cycloheximide suggests that it is dependent on de novo protein synthesis. Inhibition by pyrrolidine dithiocarbamate, a NF-kappa B/c-rel inhibitor, further implies the involvement of NF-kappa B/c-rel. To clarify reason(s) for the induction, hepatocytes were isolated with the collagenase buffer perfusion step omitted. As a consequence, iNOS mRNA was undetectable in the hepatocytes. These findings show that the traditional hepatocyte-isolation culture does indeed transiently express a serum-independent but de novo protein synthesis-dependent iNOS mRNA due to collagenase (type IV) buffer perfusion.