Department of Pharmacy-DIFAR, Biochemistry and Physiology Lab, University of Genoa, Genova, Italy.
Biol Cell. 2013 Aug;105(8):345-58. doi: 10.1111/boc.201300003. Epub 2013 Jun 18.
The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks.
OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F₁F₀-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O₂) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections.
Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O₂. The ability of OS to manipulate O₂ may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.
杆状细胞的外节（OS）是光转化发生的特化细胞器。我们之前对纯化的杆状细胞盘进行的蛋白质组学和生化分析表明，呼吸链复合物 I-IV 和 F1 Fo-ATP 合酶在 OS 盘中具有功能性表达，以及活跃的可溶性三羧酸循环酶。在这里，我们将研究重点放在整个 OS 上，它包含细胞质和质膜以及盘状结构，这些盘状结构呈天然扁平的小泡状，而不是球形的渗透完整盘状结构。
从牛视网膜中纯化 OS 并对其纯度进行了表征。进行了耗氧量测定、ATP 合成和细胞色素 c 氧化酶（COX）测定。通过半定量 Western 印迹、免疫荧光或共焦激光扫描显微镜对整个牛视网膜和牛视网膜切片以及通过免疫金透射电子显微镜（TEM）对纯化的 OS 或牛视网膜切片进行 COX 和 F₁F₀-ATP 合酶（ATP 合酶）的存在评估。在 OS 中，ATP 合酶和 COX 均具有催化活性。在存在丙酮酸和苹果酸的情况下，它们能够消耗氧气（O₂）。CLSM 分析显示，视紫红质自发荧光和 MitoTracker Deep Red 633 荧光在杆状细胞 OS 上共定位。通过免疫荧光和 TEM 在牛视网膜切片中对 Rh 与 ATP 合酶在杆状细胞 OS 中的共定位研究，证实了数据的可靠性。
我们的数据证实 COX 和 ATP 合酶在 OS 中的表达和活性，表明杆状细胞 OS 中存在额外的线粒体氧化磷酸化，旨在为视觉转导提供 ATP。在这方面，富含膜的 OS 环境旨在吸收光和 O₂。OS 对 O₂ 的操纵能力可能为许多归因于氧化应激的视网膜退行性疾病的发病机制以及目前用作支持治疗的饮食补充剂的治疗效果提供新的认识。
