Ceruti S, Giammarioli A M, Camurri A, Falzano L, Rufini S, Frank C, Fiorentini C, Malorni W, Abbracchio M P
Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133, Milan, Italy.
Neuromuscul Disord. 2000 Aug;10(6):436-46. doi: 10.1016/s0960-8966(00)00118-8.
We recently suggested that, in muscular dystrophies, the excessive accumulation of adenosine as a result of an altered purine metabolism may contribute to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which can be differentiated in vitro into multinucleated cells (myotubes). Exposure of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptotic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive transporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-chloro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2-chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which blocks intracellular methylation-dependent reactions. Both nucleosides markedly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morphological alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via completely different metabolic pathways, and are consistent with the hypothesis that adenosine accumulation in dystrophic muscles may represent a novel pathogenetic pathway in muscle diseases.
我们最近提出，在肌肉营养不良症中，嘌呤代谢改变导致的腺苷过度积累可能会导致功能逐渐恶化和肌肉细胞死亡。为了验证这一假设，我们利用了C2C12成肌细胞，这种细胞可以在体外分化为多核细胞（肌管）。将增殖的成肌细胞和分化的肌管暴露于腺苷或其代谢稳定的类似物2-氯腺苷中，会导致凋亡性细胞死亡和肌管破坏。两种核苷的细胞毒性不依赖于细胞外腺苷受体，而是至少部分地通过膜硝基苄基硫代肌苷敏感转运体进入细胞。腺苷激酶抑制剂5-碘结核菌素可预防2-氯腺苷诱导的（但不是腺苷诱导的）效应，这表明细胞内磷酸化/激活反应在2-氯腺苷介导的细胞毒性中起关键作用。相反，同型半胱氨酸的添加加剧了腺苷的细胞毒性，这表明腺苷的作用可能是由于S-腺苷同型半胱氨酸的积累，它会阻断细胞内甲基化依赖性反应。两种核苷均通过对肌动蛋白细胞骨架的作用显著破坏了肌管结构；然而，对于肌管来说，这两种核苷诱导的形态学改变也存在明显差异。这些结果表明，腺苷和2-氯腺苷通过完全不同的代谢途径诱导成肌细胞凋亡，这与营养不良肌肉中腺苷积累可能代表肌肉疾病中一种新的致病途径的假设一致。
