PHYSIOLOGY OF RESPIRATORY MUSCLE CELLS

我们对呼吸肌生理学有着长期的兴趣， 自由基对肌肉功能的影响。 当前项目 研究了细胞内信号传导机制，其中自由基可能 作为重要的组成部分。 肿瘤坏死因子-α（TNF-α） α）介导各种骨骼肌萎缩和无力， 慢性疾病;然而，很少有人知道的直接影响， TNF α对肌肉或相关信号机制的影响。 在非肌肉组织中 细胞类型，TNF α刺激活性氧的产生 (ROS)其可激活核因子-kB（NF-kB）。 这种TNF α/NF- 然而，kB途径并非普遍存在。 受体后信号传导 TNF α的机制是细胞类型特异性的， 研究肌肉。 目前的项目通过以下方式解决这一问题： 测试TNF α对膈肌和其他骨骼的直接影响， 通过评估TNF-α/NF-kB通路作为一种假定的 信号机制 实验将在纤维束上进行 分离自小鼠隔膜和培养的肌肉细胞，以解决 四个具体目标：目标1。为了确定TNF-α是否直接 刺激肌肉中的蛋白质流失。 其他调查人员得出结论， TNF α并不直接刺激肌肉紧张症。 我们将 通过将培养的肌肉细胞暴露于TNF α来测试这一断言， 24-96小时;将通过测量肌细胞大小、总 蛋白质含量和肌球蛋白水平。 目标2.评估NF-kB激活 作为肌肉对TNF α的早期反应。 我们将评估NF-kB 通过测试TNF-α刺激的肌肉中的 I-kB（一种NF-κ B抑制蛋白）与NF-κ B的核转位。 目标3。确定ROS是否介导肌肉中的NF-κ B活化。 我们 将测试TNF α刺激ROS产生的能力， 外源性ROS激活NF-κ B，抗氧化酶抑制NF-κ B， TNF α激活NF-kB。 目标4。 测试泛素的重要性 在TNF α激活NF-κ B中。在其他细胞类型中，TNF α 刺激I-kB与泛素结合;这导致I-kB 通过26 S蛋白酶体复合物进行蛋白水解，从而激活NF-κ B。 我们将在TNF-α刺激的肌肉中评估这种机制， 测试增加的泛素结合，并通过测试能力 蛋白酶体抑制剂来阻断NF-κ B的激活。

We have a longstanding interest in respiratory muscle physiology and the effects of free radicals on muscle function. The current project examines an intracellular signaling mechanism in which free radicals may function as essential components. Tumor necrosis factor-alpha (TNF- alpha) mediates skeletal muscle atrophy and weakness in a variety of chronic diseases; however, little is known about the direct effects of TNFalpha on muscle or the signaling mechanism(s) involved. In nonmuscle cell types, TNFalpha stimulates production of reactive oxygen species (ROS) which can activate nuclear factor-kB (NF-kB). This TNF alpha/NF- kB pathway is not ubiquitous, however. Post-receptor signaling mechanisms for TNFalpha are cell type-specific and have not been investigated in muscle. The current project addresses this issue by testing the direct effects of TNFalpha on diaphragm and other skeletal muscle cells and by evaluating the TNF-alpha/NF-kB pathway as a putative signaling mechanism. Experiments will be conducted on fiber bundles isolated from mouse diaphragm and on cultured muscle cells to address four specific aims: Aim 1. To determine whether TNF-alpha directly stimulates protein loss in muscle. Other investigators have concluded that TNFalpha does not stimulate muscle catabolism directly. We will test this assertion by exposing cultured muscle cells to TNFalpha for 24-96 hrs; catabolism will be assessed by measuring myocyte size, total protein content, and myosin levels. Aim 2. Evaluate NF-kB activation as an early response of muscle to TNFalpha. We will evaluate NF-kB activation in TNF-alpha-stimulated muscle by testing for depletion of I-kB (a NF-kB inhibitory protein) and nuclear translocation of NF-kB. Aim 3. Determine whether ROS mediate NF-kB activation in muscle. We will test the capacities of TNFalpha to stimulate ROS production, of exogenous ROS to activate NF-kB, and of antioxidant enzymes to inhibit NF-kB activation by TNFalpha. Aim 4. Test the importance of ubiquitin in NF-kB activation by TNFalpha. In other cell types, TNFalpha stimulates I-kB conjugation with ubiquitin; this leads to I-kB proteolysis via the 26S proteasome complex thereby activating NF-kB. We will evaluate this mechanism in TNF-alpha-stimulated muscle by testing for increased ubiquitin conjugation and by testing the capacity of proteasome inhibitors to block NF-kB activation.