Anderson Robert E, Maude Maureen B, McClellan Mark, Matthes Michael T, Yasumura Douglas, LaVail Matthew M
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Mol Vis. 2002 Sep 23;8:351-8.
Previous studies have shown that the level of docosahexaenoic acid (22:6n-3, DHA) is lower in the rod outer segment (ROS) membranes of dogs and mice with inherited retinal degeneration than in ROS from appropriate controls. In the present study, we analyzed the ROS fatty composition of several lines of transgenic rats with P23H and S334ter rhodopsin mutations. Lines were chosen that have different rates of retinal degeneration.
At 21-22 days of age, animals were perfused and eyes fixed and sectioned for morphologic examination. Others were killed and retinas isolated for preparation of ROS by sucrose step-gradient centrifugation. Fatty acid composition of ROS phospholipids was determined by gas-liquid chromatography. Membrane purity was assessed by polyacrylamide gel electrophoresis.
Retinas of the slow degenerating lines were indistinguishable from controls, whereas there was a 15-20% and 50-60% loss of photoreceptor cell nuclei in intermediate and fast degenerating lines, respectively. Except for the slow P23H line, all mutant lines had lower levels of 22:6n-3 and total n-3 fatty acids in ROS phospholipids, compared to wild-type controls, and the level of 22:6n-3 was lowest in those lines with the fastest rate of degeneration. The relative levels of the other fatty acid families (saturated, monoenoic, and n-6) increased proportionately. The n-6/n-3 ratio increased in the more rapidly degenerating lines, but the phospholipid/protein ratios did not change. The low levels of 22:6n-3 in the ROS membranes were not compensated for by an increase in 22:5n-6, which always occurs in the retina of animals where 22:6n-3 levels are reduced by dietary manipulation.
Rats that express mutant rhodopsins have lower levels of 22:6n-3 in their ROS phospholipids than wild-type animals. We propose that photoreceptor-specific mutations provoke a metabolic stress in rod photoreceptor cells that generates an oxidant stress in these cells. The retina responds to this stress by reducing the level of substrate for lipid peroxidation (22:6n-3).
先前的研究表明，患有遗传性视网膜变性的狗和小鼠的视杆细胞外段（ROS）膜中二十二碳六烯酸（22:6n-3，DHA）的水平低于相应对照组的ROS中的水平。在本研究中，我们分析了几株具有P23H和S334ter视紫红质突变的转基因大鼠品系的ROS脂肪酸组成。选择了具有不同视网膜变性速率的品系。
在21-22日龄时，对动物进行灌注，固定眼睛并切片进行形态学检查。处死其他动物，分离视网膜，通过蔗糖梯度离心制备ROS。通过气液色谱法测定ROS磷脂的脂肪酸组成。通过聚丙烯酰胺凝胶电泳评估膜纯度。
缓慢退化品系的视网膜与对照组无明显差异，而在中度和快速退化品系中，光感受器细胞核分别损失15%-20%和50%-60%。除了缓慢退化的P23H品系外，与野生型对照组相比，所有突变品系的ROS磷脂中22:6n-3和总n-3脂肪酸水平较低，并且在退化速率最快的那些品系中22:6n-3水平最低。其他脂肪酸家族（饱和、单不饱和和n-6）的相对水平成比例增加。在退化较快的品系中n-6/n-3比值增加，但磷脂/蛋白质比值没有变化。ROS膜中22:6n-3的低水平并未因22:5n-6的增加而得到补偿，22:5n-6的增加总是发生在通过饮食控制降低22:6n-3水平的动物视网膜中。
表达突变视紫红质的大鼠其ROS磷脂中22:6n-3的水平低于野生型动物。我们提出光感受器特异性突变在视杆光感受器细胞中引发代谢应激，进而在这些细胞中产生氧化应激。视网膜通过降低脂质过氧化底物（22:6n-3）的水平来应对这种应激。
