目的:观察内源性一氧化氮合酶(NOS)抑制物非对称性二甲基精氨酸(ADMA)及其信号通路在4周运动大鼠NO水平及骨骼肌收缩功能与线粒体生物合成中的调节作用。方法:建立4周运动大鼠模型,检测离体比目鱼肌对电刺激的单次、强直和疲劳收缩的最大张力;并检测骨骼肌中ATP和线粒体DNA含量以及过氧化物酶增殖体受体γ辅激活因子1α(PGC-1α)、核呼吸因子(NRF)mRNA的表达以反映线粒体生物合成及功能;用高效液相色谱测定血清ADMA浓度;用Western blot法检测骨骼肌中内源性ADMA生成酶PRMT1和ADMA代谢酶DDAH2种亚型以及NOS 3种亚型蛋白的表达;用比色法测定NOS活性及一氧化氮(NO)含量等。结果:与正常对照组相比,运动组大鼠比目鱼肌对电刺激诱导的各种收缩张力均明显增强,比目鱼肌ATP含量、线粒体DNA含量和PGC-1α、NRF mRNA增加显著(P〈0.01)。运动组大鼠比目鱼肌中构成型NOS(cNOS)的蛋白表达及其NOS活性明显上调(P〈0.01),而NO含量仅小幅增加(P〈0.05);同时,4周运动增加大鼠血清ADMA浓度,并伴有骨骼肌DDAH2表达下调。结论:短期耐力运动增强比目鱼肌单次收缩、强直收缩和抗疲劳收缩肌功能,其机制可能与过度增加的cNOS促使ADMA水平反馈性升高,从而维持骨骼肌NO低幅度增加,促进线粒体生物合成有关。
AIM: To observe the effect of endogenous nitric oxide synthase( NOS) inhibitor asymmetric dimethylarginine( ADMA) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats.METHODS: The 4 weeks running rat model was established. The twitch tension,tetanic tension and the fatigue test of soleus muscle induced by electrical stimulation ex vivo were detected. The ATP content,mitochondrial DNA levels and the mRNA expression of peroxisome proliferator-activated receptor γ coactivator-1α( PGC-1α),nuclear respiratory factor( NRF)were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle. Serum ADMA concentration was detected by high performance liquid chromatography. The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA metabolism enzyme DDAH,3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot. NOS activity and nitric oxide( NO) content were analyzed by colorimetric method. RESULTS: Compared with normal control group,the twitch tension,tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced,ATP content,mitochondrial DNA content and the mRNA expression of PGC-1α and NRF were significantly increased( P〈 0. 01). In addition,the protein expression of constitute type NOS( cNOS) and NOS activity were significantly increased( P〈 0. 01),but the increase in NO content was relatively smaller in soleus muscle in exercise group( P〈 0. 05).Moreover,serum ADMA concentration in running group was increased,while the DDAH2 expression in skeletal muscle was decreased. CONCLUSION: Short-term endurance exercise enhances the twitch tension,tetanic tension and fatigue resistance of soleus muscle. The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration,thus maintaining the increase in NO synthesis at a relatively low level,and resulting in promoting skeletal muscle mitochondria biosynthes