Kanzaki Keita, Watanabe Daiki, Aibara Chihiro, Kawakami Yuki, Yamada Takashi, Takahashi Yoshitaka, Wada Masanobu
Department of Clinical Nutrition, Faculty of Health Science and Technology, Kawasaki University of Medical Welfare, Okayama, Japan.
Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan.
Physiol Rep. 2018 Jan;6(2). doi: 10.14814/phy2.13582.
It has been shown that calpains are involved in the proteolysis of muscle proteins that occurs with eccentric contraction (ECC) and that exogenously applied nitric oxide decreases the calpain-mediated proteolysis. The aim of this study was to examine the effects of ingestion of l-arginine (ARG), a nitric oxide precursor, on ECC-related calpain activation. In the first and second experiments, male Wistar rats were given ARG in water for 7 days starting from 3 days before the ECC protocol (average ingestion, ~600 mg kg-body wt day ). Tibialis anterior muscles underwent 200 repeated ECCs and, subsequently, were excised 3 days later. Whole muscle analyses (the first experiment) revealed that ARG attenuated ECC-induced force deficit and autolysis of calpain-1, and increased the amounts of S-nitrosylated calpain-1. Regarding ryanodine receptor (RyR) and dihydropyridine receptor (DHPR), ECC-induced proteolysis was completely inhibited by ARG, whereas the inhibition was partial for junctophilin-1 (JP1). Skinned fiber analyses (the second experiment) showed that ARG also inhibited ECC-elicited reductions in the ratio of depolarization-induced to maximum Ca -activated force. In the third experiment, homogenates of rested muscles were treated with S-nitrosylating agent, S-nitrosoglutathione (GSNO), and/or high Ca concentration ([Ca ]). Treatment with high [Ca ] and without GSNO produced proteolysis of RyR, DHPR, and JP1. On the other hand, treatment with high [Ca ] and GSNO caused complete inhibition of RyR and DHPR proteolysis and partial inhibition of JP1 proteolysis. These results indicate that ARG ingestion can attenuate ECC-induced proteolysis of Ca regulatory proteins and force deficit by decreasing calpain activation via S-nitrosylation.
研究表明，钙蛋白酶参与了离心收缩（ECC）时发生的肌肉蛋白水解过程，并且外源性一氧化氮可减少钙蛋白酶介导的蛋白水解。本研究的目的是检测一氧化氮前体L-精氨酸（ARG）的摄入对ECC相关钙蛋白酶激活的影响。在第一个和第二个实验中，从ECC实验方案前3天开始，雄性Wistar大鼠饮用含ARG的水7天（平均摄入量约为600毫克/千克体重/天）。胫前肌进行200次重复的ECC，随后在3天后切除。全肌肉分析（第一个实验）显示，ARG减轻了ECC诱导的力量缺失和钙蛋白酶-1的自溶，并增加了S-亚硝基化钙蛋白酶-1的量。关于兰尼碱受体（RyR）和二氢吡啶受体（DHPR），ARG完全抑制了ECC诱导的蛋白水解，而对连接蛋白-1（JP1）的抑制是部分性的。去表皮肌纤维分析（第二个实验）表明，ARG也抑制了ECC引起的去极化诱导力与最大钙激活力比值的降低。在第三个实验中，对静息肌肉的匀浆用S-亚硝基化剂S-亚硝基谷胱甘肽（GSNO）和/或高钙浓度（[Ca]）进行处理。高[Ca]且无GSNO处理导致RyR、DHPR和JP1的蛋白水解。另一方面，高[Ca]和GSNO处理导致RyR和DHPR蛋白水解完全被抑制，JP1蛋白水解部分被抑制。这些结果表明，摄入ARG可通过S-亚硝基化减少钙蛋白酶激活，从而减轻ECC诱导的钙调节蛋白的蛋白水解和力量缺失。
