Åstrand Laboratory of Work Physiology, Swedish School of Sport and Health Sciences, GIH, Stockholm, Sweden.
Department of Physiology and Pharmacology, Biomedicum C5, Karolinska Institutet, Solna, Sweden.
Am J Physiol Endocrinol Metab. 2021 Apr 1;320(4):E691-E701. doi: 10.1152/ajpendo.00506.2020. Epub 2021 Feb 8.
Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of the redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to HO had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half-maximal inhibition at ∼200 µM ONOO), which was fully reversed by the presence of an ONOO scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ∼60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant -acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions. Here we show that exogenous peroxynitrite results in nitration of phosphorylase as well as inhibition of glycogenolysis in isolated intact mouse skeletal muscle during short-term repeated contractions. However, repeated contractions in the absence of exogenous peroxynitrite do not result in nitration of phosphorylase or affect glycogenolysis, nor does the addition of antioxidants alter glycogenolysis during repeated contractions. Thus phosphorylase is not subject to redox control during repeated contractions.
磷酸化酶是历史上研究最深入的蛋白质之一，但对其在剧烈肌肉收缩期间的调节知之甚少。骨骼肌磷酸化酶的酪氨酸硝化导致酶失活，抗氧化剂可防止这种情况发生。在收缩肌肉中，氧化还原状态的改变是否会影响磷酸化酶活性和糖原分解尚不清楚。在这里，我们研究了氧化还原状态在控制分离的小鼠快肌（伸趾长肌，EDL）和慢肌（比目鱼肌）肌肉制剂中磷酸化酶和糖原分解中的作用在重复收缩期间。HO 暴露于粗肌肉提取物对磷酸化酶活性几乎没有影响。然而，暴露于过氧亚硝酸盐（ONOO），一种硝化/氧化试剂，导致磷酸化酶完全失活（半最大抑制在约 200μM ONOO），这可被 ONOO 清除剂二硫苏糖醇（DTT）完全逆转。ONOO 孵育分离的肌肉会导致磷酸化酶硝化，并在重复收缩期间显着抑制糖原分解。ONOO 还导致休息状态和重复收缩后高能磷酸化合物（ATP 和磷酸肌酸）大量减少。这些代谢变化与重复收缩时的力产生减少有关（约为对照的 60%）。相比之下，重复收缩不会导致磷酸化酶硝化，DTT 或一般抗氧化剂乙酰半胱氨酸也不会改变重复收缩期间的糖原分解。这些发现表明，ONOO 在极端条件下抑制活肌肉中的磷酸化酶和糖原分解。然而，硝化在重复收缩期间对磷酸化酶和糖原分解的控制没有起到重要作用。在这里，我们表明外源性过氧亚硝酸盐会导致分离的完整小鼠骨骼肌在短期重复收缩期间磷酸化酶硝化以及糖原分解抑制。然而，在没有外源性过氧亚硝酸盐的重复收缩不会导致磷酸化酶硝化或影响糖原分解，并且抗氧化剂的添加也不会改变重复收缩期间的糖原分解。因此，磷酸化酶在重复收缩期间不受氧化还原控制。
