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 alpha-bungarotoxin) and nNOS (stained with a specific antibody) in murine C(2)C(12) myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C(2)C(12) myotubes. An antibody to the alpha(1)-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the alpha(1)-subunit of the nAChR. Treatment of mice with bacterial LPS downregulated the expression of nNOS in skeletal muscle, and treatment of C(2)C(12) cells with bacterial LPS and interferon-gamma markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (alpha-, gamma-, and epsilon-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine skeletal muscle and C(2)C(12) cells but differ in their expressional regulation.
Ebert
Jutta G
JG
Department of Pharmacology, Johannes Gutenberg University, D-55101 Mainz, Germany.
Zelenka
Marek
M
Gath
Ingolf
I
Gödtel-Armbrust
Ute
U
Förstermann
Ulrich
U
D000818:Animals; D002460:Cell Line; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
D008070:Lipopolysaccharides; D020033:Protein Isoforms; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse