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Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes
G Williams1, T Brown, L Becker
1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235.
Abstract:
Nitric oxide (NO) is an important mediator of diverse physiological and pathological responses. To determine whether NO production can be induced in skeletal muscle, we stimulated C2C12 mouse skeletal muscle myocytes with putative inducers of nitric oxide synthase (NOS). Neither lipopolysaccharide (LPS), interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), nor interferon-gamma (IFN) was able to stimulate nitrite production by C2C12 cells when administered alone. However, combinations of IFN with either TNF or IL-1 resulted in significant nitrite production; simultaneous stimulation of cells with all three cytokines resulted in significantly increased nitrite production compared with any combination of two cytokines. Northern analysis of RNA obtained from stimulated C2C12 cells revealed induction of a single mRNA band that precisely coincided with the mRNA band of mouse macrophage-inducible NOS (iNOS). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis followed by sequencing of the 5' 765 bases of the skeletal muscle iNOS cDNA demonstrated exact homology with mouse macrophage iNOS. These findings indicate that combinations of cytokines stimulate NO production in skeletal muscle cells via induction of the macrophage-type iNOS gene.
Insights
Cytokine combinations, including interferon-gamma (IFN) with tumor necrosis factor-alpha (TNF) or interleukin-1 (IL-1), induce nitric oxide (NO) production in skeletal muscle cells. This occurs through the induction of the inducible nitric oxide synthase (iNOS) gene, similar to macrophages.
Area of Science:
- Cellular Biology
- Immunology
- Physiology
Background:
- Nitric oxide (NO) plays a crucial role in various physiological and pathological processes.
- Understanding NO production in skeletal muscle is important for comprehending its functions.
- The induction of nitric oxide synthase (NOS) is a key regulatory step in NO synthesis.
Purpose of the Study:
- To investigate whether nitric oxide (NO) production can be induced in skeletal muscle cells.
- To identify specific stimuli that trigger NO production in C2C12 mouse skeletal muscle myocytes.
- To determine the molecular mechanism underlying NO induction in skeletal muscle.
Main Methods:
- Stimulation of C2C12 mouse skeletal muscle myocytes with various cytokines: lipopolysaccharide (LPS), interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), and interferon-gamma (IFN).
- Measurement of nitrite production as an indicator of NO synthesis.
- Northern blot analysis to detect mRNA expression of inducible nitric oxide synthase (iNOS).
- Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing to confirm iNOS gene homology.
Main Results:
- Individual cytokines (LPS, IL-1, TNF, IFN) did not induce significant nitrite production alone.
- Combinations of IFN with TNF or IL-1 significantly stimulated nitrite production.
- Simultaneous stimulation with all three cytokines (IFN, TNF, IL-1) resulted in the highest nitrite production.
- Northern analysis revealed the induction of an mRNA band identical to macrophage-inducible NOS (iNOS).
- RT-PCR and sequencing confirmed the skeletal muscle iNOS cDNA homology with the macrophage-type iNOS gene.
Conclusions:
- Combinations of cytokines are required to induce nitric oxide (NO) production in skeletal muscle cells.
- The macrophage-type inducible nitric oxide synthase (iNOS) gene is induced in skeletal muscle by these cytokine combinations.
- These findings highlight a conserved mechanism for NO regulation in different cell types, including skeletal muscle.