Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle.
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.
Drenning
Jason A
JA
Center for Exercise Science, Department of Applied Physiology & Kinesiology, University of Florida, Gainesville, FL, USA.
Lira
Vitor A
VA
Soltow
Quinlyn A
QA
Canon
Claire N
CN
Valera
Lauren M
LM
Brown
Dana L
DL
Criswell
David S
DS
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D018485:Muscle Fibers, Skeletal / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D009154:Mutation; D018995:Myosin Heavy Chains / Q000096:biosynthesis; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction
D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D018995:Myosin Heavy Chains; D006152:Cyclic GMP; D002118:Calcium