Winkler B S, Dang L, Malinoski C, Easter S S
Eye Research Institute, Oakland University, Rochester, Michigan 48309, USA.
Invest Ophthalmol Vis Sci. 1997 Jul;38(8):1569-77.
To report results of functional, biochemical and structural studies of photoreceptor mitochondria in isolated rat retinas under conditions of mitochondrial inhibition.
Dark-adapted rat retinas were incubated in a modified Ringer's bicarbonate medium under aerobic and anaerobic conditions. Several different procedures were used to inhibit mitochondrial function; N2, 0.01 mM antimycin A, and 1 and 10 mM potassium cyanide (KCN). Measurements were made of lactic acid production, retinal adenosine triphosphate (ATP) content, and receptor potentials. Morphology of the inner segment mitochondria was examined by electron microscopy.
In the presence of N2, 0.01 mM antimycin, or 1 mM KCN, lactic acid production was linear throughout the 60- minute period; and the rate was similar for each condition. Retinal ATP content and the amplitude of the receptor potential were also maintained at high levels after short-term incubations with either N2, antimycin A, or 1 mM KCN. In contrast, use of 10 mM KCN produced an entirely different set of results. These effects were studied both at the alkaline pH (8.9) found when this concentration of KCN was simply added to bicarbonate-buffered media and at the normal pH (after readjustment) of 7.4. With 10 mM KCN (pH 8.9), retinal lactate production was severely depressed, retinal ATP content was nearly depleted within 5 to 10 minutes, and the amplitude of the receptor potential rapidly declined to a low level. The deleterious effects of 10 mM KCN on these parameters were lessened to varying degrees when pH was readjusted to 7.4. Electron microscopic observations of rat rod inner segments indicated generally excellent survival of these organelles after incubation with either N2, antimycin A, or 1 mM KCN in comparison with their appearance under oxygenated conditions. However, the inner segments were significantly disrupted after incubation of retinas with 10 mM KCN.
Findings suggest that the loss of the receptor potential and depletion of ATP observed with minutes after exposing isolated rat retinas to media containing 10 mM KCN results from the inhibition of both respiration and glycolysis by this high concentration of KCN. In contrast, when conditions are chosen so that only respiration is impaired (as with N2, antimycin A, or 1 mM KCN) photoreceptor cells are resistant to short-term episodes of mitochondrial inhibition, principally because the upregulation of glycolysis generates sufficient ATP to compensate reasonably well for the loss in mitochondrially produced ATP.
报告在离体大鼠视网膜中线粒体抑制条件下光感受器线粒体的功能、生化及结构研究结果。
将暗适应的大鼠视网膜在改良的林格氏碳酸氢盐培养基中于需氧和厌氧条件下孵育。采用几种不同方法抑制线粒体功能；分别为氮气、0.01 mM抗霉素A以及1和10 mM氰化钾（KCN）。测量乳酸生成量、视网膜三磷酸腺苷（ATP）含量及感受器电位。通过电子显微镜检查内节线粒体的形态。
在氮气、0.01 mM抗霉素或1 mM KCN存在的情况下，整个60分钟期间乳酸生成呈线性；且每种条件下速率相似。在用氮气、抗霉素A或1 mM KCN短期孵育后，视网膜ATP含量及感受器电位幅度也维持在较高水平。相比之下，使用10 mM KCN产生了完全不同的一组结果。在将该浓度KCN简单添加到碳酸氢盐缓冲培养基时发现的碱性pH（8.9）以及重新调整后的正常pH（7.4）条件下均对这些效应进行了研究。在10 mM KCN（pH 8.9）条件下，视网膜乳酸生成严重受抑，视网膜ATP含量在5至10分钟内几乎耗尽，感受器电位幅度迅速降至低水平。当pH重新调整至7.4时，10 mM KCN对这些参数的有害影响在不同程度上有所减轻。大鼠视杆细胞内节的电子显微镜观察表明，与在有氧条件下的外观相比，在用氮气、抗霉素A或1 mM KCN孵育后这些细胞器总体上存活良好。然而，在用10 mM KCN孵育视网膜后内节明显受损。
研究结果表明，将离体大鼠视网膜暴露于含10 mM KCN的培养基后数分钟内观察到的感受器电位丧失及ATP耗竭是由于该高浓度KCN对呼吸和糖酵解的抑制所致。相比之下，当选择仅损害呼吸的条件时（如氮气、抗霉素A或1 mM KCN），光感受器细胞对短期线粒体抑制事件具有抗性，主要是因为糖酵解的上调产生了足够的ATP以相当好地补偿线粒体产生的ATP的损失。
