Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle

Nitric Oxide. 2009 Nov-Dec;21(3-4):192-200. doi: 10.1016/j.niox.2009.08.001. Epub 2009 Aug 12.


We hypothesized that targeted mutation of the endothelial nitric oxide synthase (eNOS) gene would reduce Akt-related signaling events in skeletal muscle cells, compared to wild type (WT) controls. Results show that slow myosin heavy chain (type I/beta) expression and the abundance of slow-twitch fibers are reduced in plantaris muscle of eNOS(-/-) mice, compared to WT. Further, basal phosphorylation of Akt (p-Akt (Ser-473)/total Akt) and GSK-3beta (GSK-3beta (Ser-9)/total GSK-3beta) are reduced 60-70% in primary myotubes from eNOS(-/-) mice. Treatment with the calcium ionophore, A23187 (0.4 microM, 1 h), increased phosphorylation of Akt and GSK-3beta by approximately 2-fold (P<0.05) in myotubes from WT mice, but had no effect on phosphorylation of these proteins in eNOS(-/-) myotubes. Additionally, A23187 treatment failed to induce nuclear translocation of the transcription factor, NFATc1, in eNOS(-/-) myotubes. Treatment with the nitric oxide donor, propylamine propylamine NONOate (PAPA-NO; 1 microM for 1 h) increased Akt and GSK-3beta phosphorylation, and induced NFATc1 nuclear translocation in WT and eNOS(-/-) myotubes, and eliminated differences from WT in the NOS knockout cultures. Parallel experiments in C2C12 myotubes found that Akt phosphorylation induced by NO or the guanylate cyclase activator, YC-1, is prevented by co-treatment with either a guanylate cyclase or PI3K inhibitor (10 microM ODQ or 25 microM LY2904002, respectively). These data suggest that eNOS activity is necessary for calcium-induced activation of the Akt pathway, and that nitric oxide is sufficient to elevate Akt activity in primary myotubes. NO appears to influence Akt signaling through a cGMP, PI3K-dependent pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Cells, Cultured
  • Cyclic GMP / metabolism
  • Male
  • Mice
  • Mice, Mutant Strains
  • Muscle Fibers, Skeletal / enzymology
  • Muscle, Skeletal / enzymology*
  • Mutation
  • Myosin Heavy Chains / biosynthesis
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism*
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Signal Transduction


  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse
  • Proto-Oncogene Proteins c-akt
  • Myosin Heavy Chains
  • Cyclic GMP
  • Calcium