Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Buenos Aires, Argentina.
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):1171-80. doi: 10.1167/iovs.09-3909. Epub 2009 Sep 24.
Oxidative stress is involved in inducing apoptosis of photoreceptors in many retinal neurodegenerative diseases. It has been shown that oxidative stress increases in photoreceptors the synthesis of ceramide, a sphingolipid precursor that then activates apoptosis. In several cell types, ceramide is converted by ceramidases to sphingosine (Sph), another apoptosis mediator; hence, this study was undertaken to determine whether Sph participates in triggering photoreceptor apoptosis.
Rat retina neurons were incubated with [(3)H]palmitic acid and treated with the oxidant paraquat (PQ) to evaluate Sph synthesis. Sph was added to cultures with or without docosahexaenoic acid (DHA), the major retina polyunsaturated fatty acid and a photoreceptor survival factor, to evaluate apoptosis. Synthesis of Sph and sphingosine-1-phosphate (S1P), a prosurvival signal, were inhibited with alkaline ceramidase or sphingosine kinase inhibitors, respectively, before adding PQ, C(2)-ceramide, or Sph. Apoptosis, mitochondrial membrane polarization, cytochrome c localization, and reactive oxygen species (ROS) production were determined.
PQ increased [(3)H]Sph synthesis in photoreceptors and blocking this synthesis by inhibiting alkaline ceramidase decreased PQ-induced apoptosis. Addition of Sph induced photoreceptor apoptosis, increased ROS production, and promoted cytochrome c release from mitochondria. Although DHA prevented this apoptosis, inhibiting Sph conversion to S1P blocked DHA protection.
These results suggest that oxidative stress enhances formation of ceramide and its subsequent breakdown to Sph; ceramide and/or Sph would then trigger photoreceptor apoptosis. Preventing Sph synthesis or promoting its phosphorylation to S1P rescued photoreceptors, suggesting that Sph is a mediator of their apoptosis and modulation of Sph metabolism may be crucial for promoting photoreceptor survival.
氧化应激参与了许多视网膜神经退行性疾病中光感受器细胞凋亡的诱导。研究表明，氧化应激会增加光感受器细胞中神经酰胺的合成，神经酰胺是一种鞘脂前体，可激活细胞凋亡。在几种细胞类型中，神经酰胺可被神经酰胺酶转化为鞘氨醇（Sph），这是另一种凋亡介质；因此，本研究旨在确定 Sph 是否参与引发光感受器细胞凋亡。
用 [(3)H]棕榈酸孵育大鼠视网膜神经元，并使用氧化剂百草枯（PQ）处理以评估 Sph 的合成。在有或没有二十二碳六烯酸（DHA）的培养物中添加 Sph，DHA 是主要的视网膜多不饱和脂肪酸和光感受器存活因子，以评估细胞凋亡。在添加 PQ、C(2)-神经酰胺或 Sph 之前，用碱性神经酰胺酶或鞘氨醇激酶抑制剂分别抑制 Sph 和鞘氨醇-1-磷酸（S1P）的合成，S1P 是一种促进存活的信号。然后测定细胞凋亡、线粒体膜极化、细胞色素 c 定位和活性氧（ROS）的产生。
PQ 增加了光感受器中的 [(3)H]Sph 合成，通过抑制碱性神经酰胺酶抑制 Sph 的合成可减少 PQ 诱导的细胞凋亡。添加 Sph 诱导光感受器细胞凋亡，增加 ROS 产生，并促进细胞色素 c 从线粒体释放。尽管 DHA 可预防这种凋亡，但抑制 Sph 转化为 S1P 则会阻断 DHA 的保护作用。
这些结果表明，氧化应激增强了神经酰胺的形成及其随后向 Sph 的分解；然后，神经酰胺和/或 Sph 将引发光感受器细胞凋亡。阻止 Sph 的合成或促进其磷酸化为 S1P 可挽救光感受器，表明 Sph 是其凋亡的介质，调节 Sph 代谢对于促进光感受器存活可能至关重要。
