Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

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 ( 3 H)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 ( 3 H) radioactivity and molar level. RESULTS. 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. 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 alltrans 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. (Invest Ophthalmol Vis Sci. 2003;44:1435‐1446) DOI:10.1167/iovs.020679