Search

Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8-kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open-reading frame of 3441 bases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that the protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulin-binding site, consisting of a strongly hydrophobic region surrounded by basic amino acids, is present. The full-length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin-dependent nitric-oxide synthase. A structural comparison suggested that the liver nitric-oxide synthase is the same as the macrophage enzyme. Northern-blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P. acnes and lipopolysaccharide.

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.

Treatment of mouse maerophage-like RAW 264.7 cells with certain protein synthesis inhibitors is followed by accumulation of the mRNA for the inducible isoiorm of nitric oxide synthase (i-NOS). The activity of these compounds on the i-NOS gene in RAW 264.7 cells was analyzed here in detail. Results show that both cycloheximide and anisomycin can efficiently induce i-NOS mRNA, even when used at concentrations so low ( 0.25μg ml ) to have only negligible effects on protein synthesis; puromycin, on the other hand, shows only a limited effect on i-NOS mRNA expression, detectable only when cells are treated with higher concentrations of inhibitor ( 25μg ml ). In RAW 264.7 cells, low concentrations of cycloheximide trigger an immediate-early gene response, as indicated by induction of c-fos and JE mRNAs, and can efficiently activate transcription of transiently transfeeted recombinant reporter genes including either the i-NOS or the c-fos gene promoters.

Production of nitric oxide (NO) by macrophages is enhanced upon activation by bacterial endotoxins and cytokines mainly via an increase of the intracellular content of the inducible isoform of nitric oxide synthase (i-NOS). We have studied in detail the effect of several modulators of macrophage activity on steady state levels of i-NOS mRNA in the mouse macrophage-like cell line RAW 264.7. Bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) were found to be effective inducers of i-NOS mRNA, in accordance with their known ability to stimulate both i-NOS activity and NO production in macrophages from different sources, while TNF-alpha, IL-1, or IL-6 was ineffective in this regard. Accumulation of i-NOS mRNA in response to either LPS or IFN-gamma stimulation was accompanied by increased i-NOS gene transcription, as detected both by using a nuclear "run-on" transcription assay and by transient transfection of the cloned gene promoter in RAW 264.7 cells. Co-stimulation of the cells with both inducers resulted in higher steady state levels of i-NOS mRNA in the absence, however, of a corresponding potentiation of the rate of gene transcription. This was due primarily to a considerable effect of LPS on i-NOS mRNA stability, with prolongation of its half-life from 1-1.5 h, in the presence of IFN-gamma alone, to 4-6 h in the presence of both LPS and IFN-gamma.

Ca2+/calmodulin-dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of Ca2+/calmodulin-dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.