Canals Santiago, Casarejos Maria José, de Bernardo Sonsoles, Rodríguez-Martín Eulalia, Mena Maria Angeles
Departamento de Investigación, Servicio de Neurobiología, Hospital Ramón y Cajal, Ctra. de Colmenar, Km. 9, 28034 Madrid, Spain.
J Biol Chem. 2003 Jun 13;278(24):21542-9. doi: 10.1074/jbc.M213174200. Epub 2003 Apr 4.
Glutathione (GSH) depletion is the earliest biochemical alteration shown to date in brains of Parkinson's disease patients. However, data from animal models show that GSH depletion by itself is not sufficient to induce nigral degeneration. We have previously shown that non-toxic inhibition of GSH synthesis with l-buthionine-(S,R)-sulfoximine in primary midbrain cultures transforms a nitric oxide (NO) neurotrophic effect, selective for dopamine neurons, into a toxic effect with participation of guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG) (Canals, S., Casarejos, M. J., de Bernardo, S., Rodríguez-Martín, E., and Mena, M. A. (2001) J. Neurochem. 79, 1183-1195). Here we demonstrate that arachidonic acid (AA) metabolism through the 12-lipoxygenase (12-LOX) pathway is also central for this GSH-NO interaction. LOX inhibitors (nordihydroguaiaretic acid and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) ones, prevent cell death in the culture, even when added 10 h after NO treatment. Furthermore, the addition of AA to GSH-depleted cultures precipitates a cell death process that is indistinguishable from that initiated by NO in its morphology, time course, and 12-LOX, GC, and PKG dependence. The first AA metabolite through the 12-LOX enzyme, 12-hydroperoxyeicosatetraenoic acid, induces cell death in the culture, and its toxicity is greatly enhanced by GSH depletion. In addition we show that if GSH synthesis inhibition persists for up to 4 days without any additional treatment, it will induce a cell death process that also depends on 12-LOX, GC, and PKG activation. In this study, therefore, we show that the signaling pathway AA/12-LOX/12-HPETE/GC/PKG may be important in several pathologies in which GSH decrease has been documented, such as Parkinson's disease. The potentiating effect of NO over such a signaling pathway may be of relevance as part of the cascade of events leading to and sustaining nerve cell death.
谷胱甘肽（GSH）耗竭是迄今为止在帕金森病患者大脑中发现的最早的生化改变。然而，动物模型的数据表明，GSH耗竭本身不足以诱导黑质变性。我们之前已经表明，在原代中脑培养物中用L-丁硫氨酸-（S，R）-亚砜亚胺对GSH合成进行无毒抑制，会将对多巴胺神经元具有选择性的一氧化氮（NO）神经营养作用转变为一种有毒作用，该过程涉及鸟苷酸环化酶（GC）和环磷酸鸟苷依赖性蛋白激酶（PKG）（卡纳尔斯，S.，卡萨雷霍斯，M. J.，德贝尔纳多，S.，罗德里格斯-马丁，E.，和梅纳，M. A.（2001年）《神经化学杂志》79卷，1183 - 1195页）。在这里，我们证明通过12-脂氧合酶（12-LOX）途径进行的花生四烯酸（AA）代谢对于这种GSH - NO相互作用也至关重要。脂氧合酶抑制剂（去甲二氢愈创木酸和黄芩苷），而非环氧合酶（吲哚美辛）或环氧酶（克霉唑）抑制剂，可预防培养物中的细胞死亡，即使在NO处理10小时后添加也有效。此外，向GSH耗竭的培养物中添加AA会引发一个细胞死亡过程，该过程在形态、时间进程以及对12-LOX、GC和PKG的依赖性方面与由NO引发的过程无法区分。通过12-LOX酶产生的首个AA代谢产物12-氢过氧二十碳四烯酸会诱导培养物中的细胞死亡，并且GSH耗竭会极大地增强其毒性。此外，我们表明，如果GSH合成抑制持续长达4天而不进行任何额外处理，它将诱导一个同样依赖于12-LOX、GC和PKG激活的细胞死亡过程。因此，在本研究中，我们表明信号通路AA/12-LOX/12-HPETE/GC/PKG在一些已记录有GSH降低的病症（如帕金森病）中可能很重要。NO对这样一条信号通路的增强作用可能作为导致并维持神经细胞死亡的一系列事件的一部分而具有相关性。
