Qtaishat Nasser M, Redmond T Michael, Pepperberg David R
Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1435-46. doi: 10.1167/iovs.02-0679.
Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues.
Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol. The mice were maintained in darkness for a defined period ( approximately 1.5, 4.5, 24, or 48 hours) and then anesthetized, exsanguinated, and killed. Retinoids contained in the retina, RPE, serum, and liver were extracted and analyzed for ((3)H) radioactivity and molar level.
The specific activity (SA, in counts per minute per nanomole) of serum all-trans ((3)H)retinol in all mice exhibited a peak at postinjection times of 1.5 or 4.5 hours, and by 48 hours declined to approximately 7% or less of the peak. In Rpe65(+/+) mice, the average SA of RPE ((3)H)retinyl ester similarly exhibited an early peak (4.5 hours) and by 48 hours declined to approximately 6% to 10% of the peak. By contrast, the average SA of RPE ((3)H)retinyl ester in Rpe65(-/-) mice exhibited a peak at 24 or 48 hours. Radioactivity and molar data for serum all-trans retinol and RPE retinyl ester obtained at 4.5 hours were analyzed to infer the molar influx of all-trans retinol from the circulation into the RPE. Levels of all-trans retinol influx derived from this analysis (mean +/- SD: 0.014 +/- 0.004 nmol in 1-month Rpe65(+/+) mice; 0.021 +/- 0.009 nmol in 1-month Rpe65(-/-) mice; 0.016 +/- 0.013 nmol in 3-month Rpe65(+/+) mice; 0.026 +/- 0.018 nmol in 3-month Rpe65(-/-) mice) did not differ significantly from one another (P > 0.169). However, the inferred fractional influx (molar amount of entering all-trans retinol divided by the molar amount of RPE retinyl ester) in Rpe65(+/+) animals (0.34 +/- 0.04 and 0.10 +/- 0.03, respectively, for 1- and 3-month mice) substantially exceeded that for Rpe65(-/-) animals (0.055 +/- 0.023 and 0.015 +/- 0.006, respectively, for 1- and 3-month mice). Significant levels of ((3)H)retinaldehydes were detected in the retinas of Rpe65(+/+) mice, but not in those of Rpe65(-/-) mice, after the longer postinjection periods.
The results indicate preservation of a substantial inward flux of all-trans retinol from the circulation into the RPE of Rpe65(-/-) mice, despite the presence of abnormally high molar levels of RPE retinyl ester. They further imply the occurrence of a robust outward movement of all-trans retinol from the RPE into the circulation in Rpe65(+/+) mice, and substantial impairment of this efflux process in Rpe65(-/-) mice. These findings raise the hypothesis that in normal RPE, 11-cis retinal and/or 11-cis retinol stimulate the efflux of all-trans retinol at the RPE basolateral membrane. In 3-month Rpe65(+/+) mice, the observed relationship between the SAs of retinaldehydes in the retina and of RPE retinyl ester is consistent with a last-in/first-out processing of all-trans retinol to 11-cis retinal within normally functioning RPE.
视网膜色素上皮（RPE）中通常高度表达的一种蛋白质RPE65编码基因发生靶向破坏的小鼠，其RPE中全反式视黄酯异常高水平蓄积，而视网膜中11-顺式视黄醛极少。本研究旨在确定Rpe65基因缺陷小鼠在体循环和眼组织之间是否存在类视黄醇的异常通量。
将约1月龄和3月龄的暗适应Rpe65基因缺陷小鼠（Rpe65(-/-)）和野生型对照小鼠（Rpe65(+/+)）腹腔注射全反式（³H）视黄醇。小鼠在黑暗中饲养一定时间（约1.5、4.5、24或48小时），然后麻醉、放血并处死。提取视网膜、RPE、血清和肝脏中的类视黄醇，分析其³H放射性和摩尔水平。
所有小鼠血清全反式（³H）视黄醇的比活性（SA，每纳摩尔每分钟计数）在注射后1.5或4.5小时出现峰值，到48小时降至峰值的约7%或更低。在Rpe65(+/+)小鼠中，RPE（³H）视黄酯的平均SA同样出现早期峰值（4.5小时），到48小时降至峰值的约6%至10%。相比之下，Rpe65(-/-)小鼠中RPE（³H）视黄酯的平均SA在24或48小时出现峰值。分析4.5小时时获得的血清全反式视黄醇和RPE视黄酯的放射性和摩尔数据，以推断全反式视黄醇从循环进入RPE的摩尔通量。该分析得出的全反式视黄醇流入水平（平均值±标准差：1月龄Rpe65(+/+)小鼠为0.014±0.004 nmol；1月龄Rpe65(-/-)小鼠为0.021±0.009 nmol；3月龄Rpe65(+/+)小鼠为0.016±0.013 nmol；3月龄Rpe65(-/-)小鼠为0.026±0.018 nmol）彼此之间无显著差异（P>0.169）。然而，Rpe65(+/+)动物（1月龄和3月龄小鼠分别为0.34±0.04和0.10±0.03）的推断分数流入量（进入的全反式视黄醇摩尔量除以RPE视黄酯摩尔量）显著超过Rpe65(-/-)动物（1月龄和3月龄小鼠分别为0.055±0.023和0.015±0.006）。注射后较长时间后，在Rpe65(+/+)小鼠的视网膜中检测到显著水平的（³H）视黄醛，但在Rpe65(-/-)小鼠的视网膜中未检测到。
结果表明，尽管RPE视黄酯的摩尔水平异常高，但Rpe65(-/-)小鼠从循环到RPE的全反式视黄醇仍有大量内向通量。它们进一步表明，在Rpe65(+/+)小鼠中，全反式视黄醇从RPE到循环有强劲的外向运动，而在Rpe65(-/-)小鼠中这种流出过程有严重受损。这些发现提出了一个假设，即在正常RPE中，11-顺式视黄醛和/或11-顺式视黄醇刺激全反式视黄醇在RPE基底外侧膜的流出。在3月龄Rpe65(+/+)小鼠中，视网膜中视黄醛和RPE视黄酯的SA之间观察到的关系与正常功能的RPE中全反式视黄醇向11-顺式视黄醛的后进先出处理一致。
