Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods.
Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods.
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DOI：
10.1085/jgp.200910267
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发表时间：
2009-09
期刊：
影响因子：
--
通讯作者：
Sampath AP
中科院分区：
文献类型：
--
作者：
Miyagishima KJ;Cornwall MC;Sampath AP
光传导级联中活性中间体的关闭和视觉色素的重建在强光照射后视杆和视锥光感受器的敏感性恢复中起着关键作用。从漂白的蝾螈杆获得的生理证据表明，与尖端相比，这种敏感性的恢复在外节基部发生得更快。对类似漂白的蝾螈杆的微荧光测量表明，全反式视网膜到全反式视黄醇的还原在外节基部也比在尖端发生得更快。本文报道的实验旨在验证这两种现象之间存在联系的假设，例如，由于全反式视网膜还原的减少和全反式视网膜还原的还原剂细胞质烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的缺乏，外节尖端的灵敏度恢复速度减慢。在适应黑暗和漂白的条件下，对分离的蝾螈杆进行细胞外膜电流和灵敏度的测量，而细胞内NADPH浓度通过附着在内段的微管透析来改变。在漂白前和漂白后分别评估外节的基部和尖端的敏感性。暴露在光激活50%视觉色素的光下后，杆状体在近10分钟内完全不敏感，之后基底恢复灵敏度和响应性，时间常数为~ 200秒，但尖端灵敏度恢复得更慢，时间常数为~ 680秒。将5 mM NADPH透析到棒中，促进了早期恢复，消除了先前观察到的尖端/碱基差异。1.66 mM的NADPH透析未能消除尖端/碱基恢复差异，表明杆细胞的稳态NADPH浓度为~ 1 mM。这些结果表明，色素漂白后，内段是还原等量物的主要来源，全反式视网膜还原为全反式视黄醇在敏感性恢复中起着关键作用。
The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of ∼200 seconds, but tip sensitivity recovered more slowly with a time constant of ∼680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is ∼1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.
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