Solessio Eduardo, Umino Yumiko, Cameron David A, Loew Ellis, Engbretson Gustav A, Knox Barry E, Barlow Robert B
Department of Ophthalmology, SUNY Upstate Medical University, Center for Vision Research, Syracuse, New York 13210, USA.
Invest Ophthalmol Vis Sci. 2009 Sep;50(9):4477-86. doi: 10.1167/iovs.08-3186. Epub 2009 Apr 30.
Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration.
The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry.
The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)).
Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.
视紫红质突变或视觉循环功能不足导致的游离视蛋白积累可引发视网膜变性。游离视蛋白激活光转导；然而，持续性激活与视网膜变性之间的联系尚不清楚。在本研究中，检测了因维生素A缺乏而呈现持续性激活的非洲爪蟾视杆细胞的光反应。与哺乳动物的视杆细胞不同，非洲爪蟾的视杆细胞不会退化。将维生素A缺乏的非洲爪蟾视杆细胞的光转导与持续性激活的哺乳动物视杆细胞的光转导进行对比，可能会为导致视网膜变性的机制提供新的认识。
用吸力电极记录法测量非洲爪蟾蝌蚪视杆细胞的光电流，并用异丁基甲基黄嘌呤（IBMX）跃升技术测量鸟苷酸环化酶活性。通过显微分光光度法测定视杆细胞中视紫红质的含量。
维生素A缺乏的视杆细胞外段长度和直径为年龄匹配动物视杆细胞的60%至70%。其视蛋白含量约90%为游离或未结合形式。类似于漂白适应，光反应脱敏（10至20倍）且更快。与漂白适应不同的是，维生素A缺乏的视杆细胞通过异常高的cGMP合成速率维持接近正常的饱和（暗）电流密度。它们在黑暗中的cGMP合成速率（15秒-1）比对照视杆细胞可达到的最大水平（约7秒-1）高两倍。
维持循环电流密度和反应范围似乎是视杆细胞稳态的一个重要目标。然而，非洲爪蟾视杆细胞中与维生素A缺乏相关的代偿性变化是以基础ATP消耗增加15倍的高代谢成本为代价的。
