Center for Exercise Science, Department of Applied Physiology & Kinesiology, University of Florida, Gainesville, FL, USA.
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.
我们假设内皮型一氧化氮合酶（eNOS）基因的靶向突变会降低骨骼肌细胞中与 Akt 相关的信号事件，与野生型（WT）对照相比。结果表明，与 WT 相比，eNOS（-/-）小鼠比目鱼肌中的慢肌肌球蛋白重链（I 型/β）表达和慢收缩纤维的丰度降低。此外，eNOS（-/-）小鼠原代肌管中 Akt（p-Akt（Ser-473）/总 Akt）和 GSK-3β（GSK-3β（Ser-9）/总 GSK-3β）的基础磷酸化减少了 60-70%。用钙离子载体 A23187（0.4 μM，1 h）处理后，WT 小鼠肌管中 Akt 和 GSK-3β的磷酸化增加了约 2 倍（P<0.05），但对 eNOS（-/-）肌管中这些蛋白的磷酸化没有影响。此外，A23187 处理未能诱导 eNOS（-/-）肌管中转录因子 NFATc1 的核易位。用一氧化氮供体丙脒丙脒 NONOate（PAPA-NO；1 μM，1 h）处理可增加 Akt 和 GSK-3β的磷酸化，并诱导 WT 和 eNOS（-/-）肌管中转录因子 NFATc1 的核易位，并消除了在 NOS 敲除培养物中与 WT 的差异。在 C2C12 肌管中的平行实验发现，NO 或鸟苷酸环化酶激活剂 YC-1 诱导的 Akt 磷酸化被鸟苷酸环化酶或 PI3K 抑制剂（10 μM ODQ 或 25 μM LY2904002）共同处理所阻止。这些数据表明，eNOS 活性对于钙诱导的 Akt 通路的激活是必需的，并且一氧化氮足以提高原代肌管中的 Akt 活性。NO 似乎通过 cGMP、PI3K 依赖性途径影响 Akt 信号转导。
