Post-Isolation Inducible Nitric Oxide Synthase Gene Expression Due to Collagenase Buffer Perfusion and Characterization of the Gene Regulation in Primary Cultured Murine Hepatocytes

Abstract

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-kappaB/c-rel inhibitor, further implies the involvement of NF-kappaB/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.

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

“…Our data showed that NO synthesis reached the highest level within the first 24 h after cell plating and decreased to a lower but relatively stable level thereafter as described in the companion article [Xu et al, 2003]. Significant NO synthase expression and an increase in NO synthesis were observed 4-5 h after cell isolation [Wang et al, 1998;NichollsGrzemski et al, 1999]. However, the present study showed that albumin secretion within the first 24 h was not as low as expected.…”

The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

“…HEK293T cells were transfected at a confluence of 70%. Primary murine hepatocytes were isolated by liver perfusion as previously described (73). About 4 ϫ 10 6 primary murine hepatocytes were added to a collagencoated 10-cm dish (collagen I-coated plates; Nunc, Langenselbold, Germany); after 4 h, adhesion medium (Williams E medium [Sigma-Aldrich, Munich, Germany], 10% FCS, 1% penicillin-streptomycin, 2 mM glutamine, insulin [0.01 mg/ml], 100 nM dexamethasone) was exchanged with the culture medium (Williams E medium [Sigma-Aldrich], 10% FCS, 1% penicillin-streptomycin, 2 mM glutamine).…”

Sporozoite-Mediated Hepatocyte Wounding Limits Plasmodium Parasite Development via MyD88-Mediated NF-κB Activation and Inducible NO Synthase Expression

“…In brief, liver lobules were perfused for 10 min with Ca 2ϩ -free HEPES buffer (pH 7.6) at 37°C followed by perfusion with type IV collagenase (Sigma-Aldrich) solution (Ca 2ϩ -free HEPES buffer (pH 7.6) containing 0.04% type IV collagenase and 0.075% CaCl 2 f 2H 2 O) for another 5 min at 37°C. Resulting cells were purified over a 60% percoll gradient and washed twice by centrifugation at 800 rpm for 30 s. Because collagenase isolation of hepatocytes leads to NF-B activation and iNOS mRNA expression, the cells were first cultured in standard medium (DMEM containing 10% FCS, 1% HEPES, 2 mM L-glutamine, 500 M 2-ME, 100 U/ml penicillin, 100 g/ml streptomycin) containing of 2 M dexamethasone, a known suppressor of NF-B and iNOS (26,27). Cells were seeded on 1% gelatin coated plates.…”