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1From the Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois; and the 2National Eye Institute, Bethesda, Maryland.
PURPOSE. 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.
METHODS. 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 (3H)retinol. The mice were maintained in darkness for a defined period (
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 (3H) radioactivity and molar level.
RESULTS. The specific activity (SA, in counts per minute per nanomole) of serum all-trans (3H)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 (3H)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 (3H)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 (3H)retinaldehydes were detected in the retinas of Rpe65+/+ mice, but not in those of Rpe65-/- mice, after the longer postinjection periods.
CONCLUSIONS. 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.
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