Differential induction of constitutive and inducible nitric oxide synthases by distinct inflammatory stimuli in bovine aortic endothelial cells
| Autor: | Yumi Kaku, Hiroki Nanri, Masaharu Ikeda, Kuniaki Ejima, Akio Kuroiwa, Tomoyo Sakimura |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Lipopolysaccharides
Gene isoform Transcription Genetic Lipopolysaccharide Tumor necrosis factor medicine.medical_treatment Molecular Sequence Data (Bovine aorta) Polymerase Chain Reaction Nitric oxide chemistry.chemical_compound Endothelial cell Interferon medicine Animals Amino Acid Sequence RNA Messenger Inducible nitric oxide synthase Molecular Biology Cells Cultured biology Cell Biology Molecular biology Isoenzymes Nitric oxide synthase Endothelial stem cell Constitutive nitric oxide synthase Cytokine chemistry Enzyme Induction biology.protein Cytokines Calcium Cattle lipids (amino acids peptides and proteins) Tumor necrosis factor alpha Endothelium Vascular Nitric Oxide Synthase Sequence Alignment Subcellular Fractions medicine.drug |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1356:43-52 |
| ISSN: | 0167-4889 |
| Popis: | Exposure to various combinations of cytokines and lipopolysaccharide (LPS) has been reported to increase NO production in vascular endothelial cells. The molecular entity of the newly expressed nitric oxide synthase (NOS) in endothelial cells, however, has not yet been examined in detail. In this report, we carried out biochemical characterizations and molecular identification of NOS isoform(s) expressed in cytokine/LPS-treated bovine aortic endothelial cells (BAEC). The increased NOS activity in tumor necrosis factor-alpha (TNF-alpha)/LPS-treated BAEC was localized mainly in the cytosolic fraction and Ca2+-independent, whereas that in interferon-alpha,beta(IFN-alpha,beta)/LPS-treated BAEC was preferentially in the membrane fraction and Ca2+-dependent, suggesting that TNF-alpha/LPS increased an inducible NOS (iNOS)-like activity, and IFN-alpha,beta/LPS increased an endothelial constitutive NOS (ecNOS)-like activity. Correspondingly, the different responses to the cytokine/LPS pretreatment were demonstrated in semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for iNOS or ecNOS, that is, TNF-alpha/LPS elicited the expression of iNOS mRNA whereas IFN-alpha,beta/LPS increased that of ecNOS mRNA. A nuclear run-on transcription assay and an inhibition experiment by actinomycin D indicated that the apparent increase of ecNOS in the IFN-alpha,beta/LPS-treated BAEC was at least in part ascribed to the transcriptional activation. The nucleotide sequences of the amplified PCR products in TNF-alpha/LPS- and IFN-alpha,beta/LPS-treated BAEC were 93% and 99% identical to the corresponding regions of human hepatocyte iNOS and bovine ecNOS, respectively. These findings indicated that, in cytokine/LPS-treated BAEC, two NOS isoforms whose molecular natures were closely homologous to the conventional isoforms of iNOS and ecNOS were differently induced in response to distinct inflammatory stimuli. |
| Databáze: | OpenAIRE |
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