Mei J M, Chi W M, Trump B F, Eccles C U
Department of Pharmaceutical Sciences, University of Maryland at Baltimore 21201, USA.
Mol Chem Neuropathol. 1996 Feb;27(2):155-66. doi: 10.1007/BF02815091.
Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca2+]i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca2+]i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca2+]i, which returned to near basal levels on restoration of O2-containing medium. Prolonged hypoxic conditions (> 60 min) caused irreversible elevation of [Ca2+]i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake ("point of no return"). Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca2+]i. The hypoxia-induced elevation of [Ca2+]i was also greatly attenuated if L-NAME (100 microM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca2+]i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the "point of no return," as characterized above, greatly potentiated [Ca2+]i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca2+]i and subsequent cell death, was not blocked by NOS inhibitor, L-NAME.
一氧化氮（NO）已被提出作为缺氧/缺血性细胞损伤中的一种神经信使分子（诺维茨基等人，1991年；特里菲利蒂，1992年）。我们使用培养的大鼠小脑颗粒细胞在葡萄糖-氧剥夺联合模型中进行了研究。实验旨在检验以下假设：胞质钙（[Ca2+]i）的持续升高和NO生成共同作用，在缺氧损伤后触发神经元损伤。通过用预先用氩气平衡的培养基灌注细胞来实现缺氧状态。使用数字成像荧光显微镜监测加载了fura-2 AM的细胞中的[Ca2+]i。在短期缺氧条件下，细胞显示[Ca2+]i逐渐且持续适度增加，在恢复含O2培养基后恢复到接近基础水平。长时间缺氧条件（>60分钟）导致[Ca2+]i不可逆升高，随后细胞膜完整性破坏，表现为严重肿胀、正常细胞形状和突起丧失、fura-2染料泄漏以及碘化丙啶摄取（“不可逆转点”）。用特异性NO合酶抑制剂NG-硝基-L-精氨酸甲酯（L-NAME，100 microM）预处理显著延迟了[Ca2+]i升高强度的起始时间。如果在细胞显示不可逆损伤迹象之前将L-NAME（100 microM）添加到氩气灌注培养基中，缺氧诱导的[Ca2+]i升高也会大大减弱。然而，长时间或反复缺氧条件会导致[Ca2+]i迅速而强烈增加，这不能被NO合酶（NOS）抑制所阻断。此外，如上述“不可逆转点”后的复氧极大地增强了[Ca2+]i过载并促进了细胞损伤过程。NOS抑制剂L-NAME不能阻断如[Ca2+]i迅速大量增加及随后细胞死亡所证明的细胞损伤的增强和促进作用。
