Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes

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-1α (IL-1), tumor necrosis factor-α (TFN), nor interferon-γ (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.