Components of the Retinoid Cycle and Retinoid Analysis in the Bovine Ciliary Epithelium

Abstract

Abstract: : Purpose: The iris–ciliary epithelium shares embryological relationship with the retinal pigment epithelium and the sensory layers of the retina. Recent observations have documented the expression of components of rod–phototransduction in the iris–ciliary epithelium. The purpose of this work was to determine what components of the retinoid cycle (visual cycle) are expressed and what retinoids are associated in the ciliary epithelium. Methods: RT–PCR was used to verify the expression of components of the retinoid cycle including IRBP, RDH, LRAT, ABCR, CRALBP, and RPE65. DNA products from PCR amplification were gel purified and sequenced. Antibodies to CRALBP, IRBP and RPE65 were used to determine their cellular distribution along the ciliary epithelium by indirect immunofluorescence, and their pattern of protein expression by Western blot. Retinoids were extracted from pooled ciliary processes dissected, under dim red illumination, from eyes of one–week–old calves, and from eyes of 6–12 week old cows. Retinoids were extracted with hexane and analyzed in a normal–phase HPLC column, and the spectra of the main peaks obtained. Results: We found expression of CRBP, IRBP, RDH, LRAT, ABCR and CRALBP mRNA in ciliary processes. CRALBP antibodies labeled the bovine PE cell layer of the ciliary epithelium, whereas IRBP labeled the NPE cell layer. Anti–CRALBP sera cross–reacted with a 36–kDa protein, the anti–IRBP sera recognized a 145–kDa protein and the anti–RPE65 sera cross–reacted with a 61–kDa protein respectively. The main retinoids identified consisted of retinyl esters [7.40±3.53 pmol/mg of protein (n=7)], all–trans–retinol [14.86±1.13 pmol/mg of protein (n=7)] and vitamin A in the form of beta–carotene. We could not detected 11–cis retinol or 11–cis–retinal. Conclusions: The ciliary epithelium expresses many of the known genes encoding proteins involved in the retinoid cycle. The differential expression of CRALBP and IRBP along the ciliary epithelium suggests a potential role of these carrier proteins in retinoid transport and/or metabolism. However, the lack of detection of chromophore, contrasted with the detection of the substrate and expression of enzymes involved in the isomerization process. The putative role of the components of the retinoid cycle in the CE remains to be demonstrated.

Keywords: ciliary body • retinoids/retinoid binding proteins • carotenoids/carotenoid binding proteins