Eye (1990) 4, 326-332 Processing and Transport of Retinoids by the Retinal Pigment Epithelium DE AN BOK Los Angeles Summary Recent developments regarding our understanding of retinoid processing and trans­ port during the visual cycle and related events are reviewed. Retinoids are bound and protected by a cohort of retinoid binding proteins, each of whiCh is unique. The production of retinol (Vitamin A) derivatives is accomplished by a group of mem­ brane-bound enzymes, some of which appear to be coupled in their actions. Historical Perspectives on a series of biochemical reactions involving The photopigments of all organisms studied the coordinated activity of the retinal pigment to date consist of a chromophore derived from epithelium (RPE) and the photoreceptors.3 Vitamin A (Retinol)! covalently bound to a These events begin with the photobleaching protein by a protonated aldimine bond of rhodopsin to form opsin and all-trans·ret· (Schiff's base).2 The chromophore or pros­ inal, the production of various retinol deriva­ thetic group is, in all cases, retinaldehyde (ret­ tives, the regeneration of ll-cis-retinal and inal) the oxidised form of retinol. Retinol and ultimately, the regeneration of the photopig­ its derivatives are collectively referred to as ment itself. This complex series of interac­ retinoids and the proteins that covalently bind tions is known as the visual cycle. In retinal are called opsins. Both the retinoids Dowling's original observations, light was and the opsins are hydrophobic molecules, found to cause a cis to trans isomerisation of retinol being a fat-soluble vitamin, and the opsin-bound retinal following which retinal opsins belonging to a class of membrane com­ dissociated from the protein and was reduced ponents collectively known as intrinsic pro­ to form all-trans-retinol. The all-trans-retinol teins. These are amphipathic molecules was then observed to diffuse out of the photo· (meaning to tolerate both) whose fat-loving receptors, into the subretinal (retinal ventri· moieties are dissolved in the lipid bilayer of cular) space and into the retinal pigment cell membranes and whose water-loving epithelium (RPE) where it was converted to domains are exposed to the cytoplasm or cell retinyl esters. Subsequently, in the dark, ret· exterior. inyl esters were hydrolysed and the photo­ The photopigments of the vertebrate retina pigments were regenerated. utilise either the ll-cis-retinal stereoisomer or Initial studies of the visual cycle were not the ll-cis-dehydroretinal form. Humans util­ able to determine precisely the cellular sites in ise 11-cis-retinal exclusively as far as we know. which all of the requisite steps take place, In 1960, Dowling published his classic work although it was assumed that many occurred From: Department of Anatomy and Cell Biology and the Jules Stein Eye Institute, C�nter for the Health Sciences, University of California, Los Angeles, CA 90024. Some of the work cited in this publication was supported by USPHS grants EYOO444 and EYOO331 and by a Center Grant from Retinitis Pigmentosa Fighting Blindness Inc. DB is Dolly Green Professor of Ophthalmology. Correspondence to: Dean Bok, PhD, Jules Stein Eye Institute, University of California, Los Angeles,CA 90024. PROCESSING AND TRANSPORT OF RETINOIDS BY THE RETINAL PIGMENT EPITHELIUM 327 in the RPE. It was clear that all-trans-retinol protection of retinoids because of their was formed in the photoreceptors and that extreme hydrophobicity and susceptibility to retinyl esters were formed in the RPE. oxidation. The solubility of retinol in water is Beyond that, specific details were lacking. about one nanomolar, yet its concentration in Clearly there had to be a site of reisomer­ the blood is approximately one micromolar, a isation of the retinoids at some point and for one thousand-fold difference. In 1976 Heller the oxidation of retinol to retinal. In spite of and Bok showed that RBP delivers all-trans­ an early, unconfirmed report that isomerisa­ retinol to the RPE by interacting with mem­ tion was an event that occurs in the RPE,4 brane receptors on its basolateral plasma very little progress was made toward a further membrane (the side facing Bruch's mem­ understanding of the multiple steps in the brane).9 It arrives at this location by penetrat­ visual cycle for many years, although Krinsky ing the endothelium of the choriocapillaris demonstrated the presence of a retinyl ester through its fenestrations. Once RBP delivers synthetase in the RPE5 and Lion et al. were its cargo of retinol to the RPE, it loses its high able to demonstrate that mammalian RPE, affinity for TTR and the two diffuse back into but not neurosensory retina, contains a the choriocapillaris. At the time that these stereospecific oxidoreductase capable of con­ phenomena were discovered, Chader and verting ll-cis-retinol to ll-cis-retinal.6 Pep­ associates were discovering other, intracellu­ perberg and Masland showed that lar retinoid binding proteins in the retinaiO ll-cis-retinal oxidation could take place in the and shortly thereafter, Saari and coworkers outer segments of amphibians.7 began the purification and characterisation of One of the major reasons why events in the these proteins.ll,12 They and others, including visual cycle were so difficult to study was the our laboratory, began the systematic local­ extreme hydrophobicity of the retinoids and isation by immunocytochemistry of each of the difficultythat this property causes in aque­ these binding proteins within retinal cell ous systems. Furthermore, the retinoids are populations. A protein that binds all-trans­ highly labile to oxidation and artifactual iso­ retinol intracellularly, named cellular retinol merisation and are difficult to separate. It was binding protein (CRBP) by Bashor et al. in not until the advent of high performance their study of non-ocular tissues,13 was local­ liquid chromatography that these compounds ised to RPE and Muller cells.14 Another bind­ could be resolved rapidly from complex mix­ ing protein, unique to the retina, was tures in tissue homogenates. The lack of a discovered by Futterman et al. to bind the IS wide variety of detergents for the solubilisa­ ll-cis stereoisomers of retinol and retinal. tion of membrane-bound enzymes was an Since this protein was found to bind ll-cis­ important impediment to our understanding retinal initially, it was named cellular retinal of visual cycle enzymes. Lacking also was an binding protein (CRalBP). Like CRBP, it was appreciation of the important role for retinoid found to exist in RPE and Muller cells. binding proteins in the solubilisation of ret­ Finally, Adler et al.16 discovered another inoids. The door to the world of these binding large, extracellular protein in the subretinal proteins was opened in the late 1960s with the space which was shown to have retinoid bind­ discovery of the first retinoid binding protein. ing properties by the laboratories of Chaderl7 and Bridges. 18 This is a glycoprotein with low Retinoid Binding Proteins and Their Role in specificity for individual retinoids. It is syn­ Retinoid Protection and Transport thesised and secreted by the photoreceptorsl9 Kanai et al. reported the discovery of plasma and, by virtue of its location, is called inter­ retinol binding protein (RBP) in 1968.8 This photoreceptor or interstitial retinoid binding protein is secreted by the liver as a complex protein (lRBP). All of these binding proteins with a larger protein named transthyretin have been sequenced by molecular cloning (TTR) because of its binding of thyroxin and methods and some by conventional protein its indirect binding of retinol through its chemistry as well. CRBP belongs to a gene association with RBP. Retinoid binding pro­ family of proteins that bind retinal, retinoic teins are required for the solubilisation and acid and fatty acids (the latter are called fatty 328 DEAN BOK acid binding proteins).20 CRalBP has a unique gave negative results. Likewise, since enzyme amino acid sequence and is found in no tissues activity could not be studied there was con­ outside the eye.21 IRBP is found in small siderable uncertainty as to the energy require­ quantities in the brain and is abundant in the ments and natural substrate for the enzyme. pineal gland, a structure with primitive photo­ These problems were recently resolved in a receptive function. 19 Interestingly, it has some series of publications from Robert Rando's sequence homology with a cellular retinal laboratory. First, it was shown by Bernstein binding protein in cephalopods.22 and Rand024 that isomerisation occurs at the The discovery of retinoid binding proteins alcohol-oxidation state. Subsequently, in an and their localisations to specific cell types interesting series of experiments, Bernstein et within the retina have helped significantly in al. discovered that the 30-year quest for iso­ our understanding of the division of labour merising activity had been thwarted by the that exists within the retina with respect to ret­ organic solvents in which the substrates had inoids. Much remains to be learned, however been delivered to tissue homogenates.25 about the function of these proteins. Aside When the all-trans-retinol was solubilised by from serving as protective depots for ret­ bovine serum albumin rather than ethanol, inoids, more sophisticated functions remain the traditional solvent, isomerising activity to be elucidated. As yet, with the exception of was readily detected and virtually all of this RBP, none have been implicated in intra or activity was found in the microsomal fraction extracellular receptor-mediated processes, of the RPE-choroid complex. Virtually none although Saari and Bredberg have shown that was found in the neurosensory retina. CRalBP can serve in vitro as a substrate car­ Improved cell culture techniques have also rier for the oxidation of 11-cis-retinol to 11- supported the in vitro evidence that retinoid cis-retinal.23 More will be said later about isomerase is an RPE enzyme.