Fatokun Amos A, Smith Robert A, Stone Trevor W
Institute of Biomedical and Life Sciences, University of Glasgow, West Medical Building, Glasgow G12 8QQ, UK.
Brain Res. 2008 Jun 18;1215:200-7. doi: 10.1016/j.brainres.2008.04.013. Epub 2008 Apr 16.
During cerebral hypoxia or ischaemia, mitochondrial dysfunction is induced which can lead to free radical production and cell death. This phenomenon is mimicked by the acute administration of mitochondrial poisons such as 3-nitropropionic acid (3-NPA) and potassium cyanide (KCN), with the production of reactive molecular species secondary to the activation of glutamate receptors. Also during ischaemia, the kynurenine pathway of tryptophan metabolism is activated, leading to the production of quinolinic acid and kynurenic acid which can modulate N-methyl-D-aspartate (NMDA) receptors as agonist and antagonist respectively. Since kynurenic acid is known to be neuroprotective, we have now examined its ability to prevent the neurotoxic effects of mitochondrial dysfunction in primary cultures of postnatal rat cerebellar granule neurons. Viability was quantified using the Alamar Blue (AB) assay and by direct morphological examination. Both 3-NPA and KCN (10 microM-1 mM) reduced neuronal viability in a concentration-dependent manner. The NMDA receptor antagonists 2-amino-5-phosphonopentanoic acid (D-AP5) at a concentration of 50 microM, and a 10 microM dose of (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801) prevented cell death, although the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at a concentration of 10 microM did not. The antioxidant enzymes catalase and superoxide dismutase, and the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) afforded partial protection. Kynurenic acid, a glutamate antagonist with preference for the glycine site of the NMDA receptors, had no protective effect at all against 3-NPA or KCN toxicity at concentrations up to 1 mM. Although these data confirm a major role for NMDA receptors and oxidative stress in the neurotoxic effects of mitochondrial inhibitors, they reveal a resistance to kynurenic acid which suggests a non-classical activation of NMDA receptors by mitochondrial inhibitors that is independent of the glycine site or which occurs distal to the site of action of kynurenic acid.
在脑缺氧或缺血期间，会诱导线粒体功能障碍，进而导致自由基产生和细胞死亡。这种现象可通过急性给予线粒体毒物如3 - 硝基丙酸（3 - NPA）和氰化钾（KCN）来模拟，继谷氨酸受体激活后会产生活性分子。同样在缺血期间，色氨酸代谢的犬尿氨酸途径被激活，导致喹啉酸和犬尿喹啉酸的产生，它们可分别作为激动剂和拮抗剂调节N - 甲基 - D - 天冬氨酸（NMDA）受体。由于已知犬尿喹啉酸具有神经保护作用，我们现在研究了其在新生大鼠小脑颗粒神经元原代培养物中预防线粒体功能障碍神经毒性作用的能力。使用alamar蓝（AB）检测法并通过直接形态学检查对细胞活力进行定量。3 - NPA和KCN（10微摩尔 - 1毫摩尔）均以浓度依赖性方式降低神经元活力。浓度为50微摩尔的NMDA受体拮抗剂2 - 氨基 - 5 - 膦酰基戊酸（D - AP5）和10微摩尔剂量的（+） - 5 - 甲基 - 10,11 - 二氢 - 5H - 二苯并（a，d）环庚烯 - 5,10 - 亚胺马来酸氢盐（MK - 801）可防止细胞死亡，尽管浓度为10微摩尔的非NMDA受体阻滞剂6 - 氰基 - 7 - 硝基喹喔啉 - 2,3 - 二酮（CNQX）不能。抗氧化酶过氧化氢酶和超氧化物歧化酶以及一氧化氮合酶抑制剂盐酸Nω - 硝基 - L - 精氨酸甲酯（L - NAME）提供了部分保护作用。犬尿喹啉酸是一种优先作用于NMDA受体甘氨酸位点的谷氨酸拮抗剂，在浓度高达1毫摩尔时对3 - NPA或KCN毒性完全没有保护作用。尽管这些数据证实了NMDA受体和氧化应激在线粒体抑制剂神经毒性作用中的主要作用，但它们揭示了对犬尿喹啉酸的抗性，这表明线粒体抑制剂对NMDA受体的非经典激活独立于甘氨酸位点，或者发生在犬尿喹啉酸作用位点的远端。
