Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes

In mammalian skeletal muscle, neuronal-type nitric oxide synthase (nNOS) is found to be enriched at neuromuscular endplates. Here we demonstrate the colocalization of the nicotinic acetylcholine receptor (nAChR, stained with α-bungarotoxin) and nNOS (stained with a specific antibody) in murine C2C12 myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C2C12myotubes. An antibody to the α1-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the α1-subunit of the nAChR. Treatment of mice with bacterial LPS downregulated the expression of nNOS in skeletal muscle, and treatment of C2C12 cells with bacterial LPS and interferon-γ markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (α-, γ-, and e-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine s...