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 ε-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine skeletal muscle and C2C12 cells but differ in their expressional regulation.