Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2011 Sep 16;286(37):32491-501. doi: 10.1074/jbc.M111.255877. Epub 2011 Jul 24.
α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.
α-苯基-N-叔丁基硝酮（PBN），一种自由基捕获剂，先前已被证明可保护视网膜免受光诱导的神经退行性变，但保护机制尚不清楚。在这里，我们报告说，PBN 介导的视网膜保护可能是通过抑制 RPE65 活性来减缓视紫红质再生的速度来实现的。当在 2700 勒克斯强度的破坏性光暴露前 0.5-12 小时注射 50mg/kg 的 PBN 时，PBN 可保护白化 Sprague-Dawley 大鼠的视网膜，但在暴露后给药时则没有效果。PBN 注射显著抑制了杆状光感受器的体内恢复和功能性视紫红质光色素的恢复速度。视觉循环酶活性测定表明，PBN 抑制了视觉循环的关键酶之一 RPE65，IC50=0.1mm。发现 RPE65 的抑制类型为非竞争性，K(i)=53μm。PBN 对其他视觉循环酶，即卵磷脂视黄醇酰基转移酶和视黄醇脱氢酶的活性没有影响。有趣的是，一种更具可溶性的 PBN 形式，N-叔丁基-α-（2-磺苯基）硝酮，具有相似的自由基捕获活性，它不能保护视网膜或抑制 RPE65 活性，这为 PBN 的特异性和作用机制提供了一些启示。减缓视觉循环被认为是治疗视网膜疾病的一种策略，如 Stargardt 病和干性年龄相关性黄斑变性，其中视觉循环的有毒副产物在视网膜细胞中积累。因此，PBN 抑制 RPE65 的催化作用可能为这些视网膜疾病提供治疗益处。
