Biochemistry of Visual Pigment RegenerationThe Friedenwald Lecture

John C. Saari

Rods bleached in vivo therefore do not regenerate theirvisual purple from material within themselves or in thetissues proximal to them, but from the distally placedpigment epithelium: the pigment epithelium functions inthe regeneration of visual purple; it exerts a regenerativeaction on the rods.1

Phototransduction and the visual cycle play complemen-tary roles in vertebrate vision. Phototransduction is ini-tiated by the photoisomerization of 11-cis-retinal bound

to opsin and ultimately results in a change in the release ofneurotransmitter by photoreceptor cells. The visual cycle re-stores the product of photoisomerization, all-trans-retinal, tothe 11-cis configuration and allows the regeneration ofbleached visual pigments (Fig. 1). The biochemical mechanismof phototransduction has been extensively studied during thepast 2 decades, and as a result, the process serves as theparadigm for understanding G-protein–coupled receptors ingeneral. In contrast, molecular understanding of the visualcycle is poorly developed, and many fundamental questionsregarding reactions, enzymes, and control mechanisms remainunanswered. Sequences of cDNAs encoding three visual cycleenzymes have been published2–4; however, molecular infor-mation is unavailable for the other three presumed enzymes ofthe cycle, including retinol isomerase (isomerohydrolase).

It has been reported that regeneration of visual pigmentsis a slow process and that photoisomerization of 11-cis-retinalin rhodopsin is very rapid. In fact, complete dark adaptation inhumans requires approximately 40 minutes,5–7 and conversionof rhodopsin to photorhodopsin requires only 200 fsec.8 How-ever, this is not a fair comparison, because it is clear that thephotolysis and regeneration rates in the living eye must beequal and opposite in sign at ambient levels of illumination.Any other situation would not be compatible with vision,because visual pigment would dissipate rapidly. Alpern hasdemonstrated for human rods5 and cones9 that a steady statelevel of bleached visual pigments, in which the bleach andregeneration rates are equal and of opposite sign, is present atdifferent levels of physiologic illumination.

In humans the progress curves for the regain of visualthreshold and for the regeneration of visual pigment coincidewhen displayed on a semi–log plot.6,7 The molecular explana-tion for this log–linear relationship is not well understood, andthe relationship may be fortuitous, but most agree that a pho-toproduct is responsible for desensitization of the visual sys-tem.6,10,11 Although the molecular identity of the desensitizingintermediate(s) remains a matter of active investigation,10,12–16

it is clear that visual cycle reactions are important in determin-ing the steady state level of bleached visual pigment and thusthe sensitivity of the retina.

The critical role of the retinal pigment epithelium (RPE) invisual pigment regeneration is apparent from the studies of19th century investigators who demonstrated that dissectedfrog retina could regenerate its bleached visual pigment onlywhen in contact with the RPE.1,17–19 The reader is referred toMarmor and Martin20 for a depiction of some of their insightfulexperiments. It is fortunate that the early physiologists usedfrog eyes for their experiments because rodent eyes do notregenerate their visual pigments when removed from the ani-mal, as will be discussed later. Fifty years later, Wald21 usedextraction techniques to show that vitamin A was involved inthe visual process and formulated the first modern version ofthe visual cycle including the participation of the RPE (Fig. 2).

The role of the RPE in the visual cycle became more clearafter the classic study by Dowling, 22 which demonstratedmovement of retinoid out of the neural retina and into RPEduring extensive bleaching and return during recovery in thedark. Later studies by other investigators with techniques of-fering more resolution verified the fundamental observa-tion.23,24 Bernstein et al.25 and Rando26 provided a molecularexplanation for the necessity of the RPE with the demonstra-tion that the critical enzymatic regeneration of the 11-cis con-figuration occurred within this tissue.25,26 The transcellularmigration of the retinoids during bleaching and regeneration isall the more remarkable, considering the anatomy of the jour-ney (Fig. 3). The relatively insoluble retinoid must leave thedisc membranes, diffuse through a cytosolic compartment toreach the plasma membrane of the rod outer segment, traversethe plasma membrane, diffuse across the subretinal space toreach the plasma membrane of the RPE cell, enter into thereactions of the visual cycle in this cell, and make the returnjourney!

A current working hypothesis for the reactions of thevertebrate rod visual cycle is shown in schematic form inFigure 4. The figure depicts internal and plasma membranes ofthe RPE and rod photoreceptor cells and the interphotorecep-tor matrix space (subretinal space) separating these two cells.The visual cycle enzymes in RPE are all associated with mem-branes; however, their localization to subcellular compart-

From the Departments of Ophthalmology and Biochemistry, Uni-versity of Washington School of Medicine, Seattle, Washington.

Supported in part by National Institutes of Health Grants RO1EY02317, EY01730, and EY09339 and by unrestricted awards fromResearch to Prevent Blindness. JCS is a Senior Scientific Investigator ofResearch to Prevent Blindness.

Submitted for publication August 6, 1999; accepted August 30,1999.

Commercial relationships policy: N.Corresponding author: John C. Saari, Department of Ophthalmol-

ogy, Box 356485, University of Washington, Seattle, WA 98195-6485.jsaari@u.washington.edu

Investigative Ophthalmology & Visual Science, February 2000, Vol. 41, No. 2Copyright © Association for Research in Vision and Ophthalmology 337

L E C T U R E

ments has not been completely determined.27 The enzymes aredepicted as part of one continuous internal membrane com-partment for simplicity. Absorption of light by rhodopsin in thedisc membrane converts 11-cis-retinal to all-trans-retinal andgenerates the active photoproduct, metarhodopsin II (Rho*).The G-protein–stimulating activity of Rho* is quenched byphosphorylation and by the binding of arrestin (not shown inthe figure). The Schiff base linking all-trans-retinal and opsin ishydrolyzed to release free all-trans-retinal. Recent reports sug-gest that an adenosine triphosphate (ATP)–binding cassettetransporter (ABCR) is involved in moving all-trans-retinal fromthe intradiscal to the cytosolic aspect of the disc membrane,28

perhaps as an adduct with phosphatidyl ethanolamine.29 all-trans-Retinol dehydrogenase (RDH) catalyzes the reduction ofall-trans-retinal to all-trans-retinol by reduced nicotinamideadenine dinucleotide phosphate (NADPH). all-trans-Retinolleaves the photoreceptor cell, traverses the interphotoreceptormatrix space (the subretinal space) where it encounters inter-photoreceptor retinoid-binding protein (IRBP), and enters theRPE where it is esterified by lecithin-retinol acyltransferase(LRAT). all-trans-Retinyl ester is converted to 11-cis-retinol andfree fatty acid by an isomerohydrolase. 11-cis-Retinol can beesterified by LRAT and stored (reaction not shown) or oxidizedto 11-cis-retinal by 11-cis-retinol dehydrogenase (11-RDH). 11-cis-Retinal diffuses into the photoreceptor cell where it asso-ciates with opsin to regenerate the visual pigment. all-trans-

Retinol can be taken up from the blood and esterified by LRATin RPE cells (not shown). 11-cis-Retinyl esters can be hydro-lyzed and used for visual pigment regeneration by 11-cis-retinylester hydrolase (11-REH). Cellular retinaldehyde-binding pro-tein (CRALBP), a water soluble retinoid-binding protein, isshown (Fig. 4) with its high-affinity ligands, 11-cis-retinal or11-cis-retinol. Cellular retinol-binding protein (CRBP) is shownassociated with all-trans-retinol, although the protein alsobinds 11-cis-retinol in vitro. IRBP is present in the photorecep-tor matrix. Its role in retinoid transport is uncertain (discussionto follow). Opsin is shown on the lower side of the disc onlyfor reasons of symmetry.

Evidence leading to this working hypothesis of the rodvisual cycle has been presented in several recent re-views.14,26,30–34 Each of the enzymatic reactions shown in theRPE or rod outer segments (ROSs) has been demonstrated inRPE microsome or ROS preparations, respectively. The water-soluble retinoid-binding proteins (CRALBP, CRBP, and IRBP)have been localized to the compartments in which they areshown by immunocytochemistry.35–37 CRBP and CRALBP arealso found in Muller cells. Mammalian rods cannot use exoge-nous 11-cis-retinol for regeneration of visual pigments.38,39

FIGURE 1. The chromophore of visual pigments alternates between11-cis and all-trans configurations. 11-cis-Retinal or a closely relatedderivative is the chromophore for all known visual pigments. Its bind-ing to opsin freezes the receptor in a stable, inactive form ensuring alow rate of thermal isomerization in the dark. Photoisomerizationconverts 11-cis-retinal to all-trans-retinal, activates the receptor, andinitiates phototransduction. The 11-cis configuration is produced by asequence of reactions that are thermally driven (i.e., can occur in thedark).

FIGURE 2. The general features ofthe regeneration cycle were alreadyapparent in this early depiction. Re-printed, with permission, from WaldG. Carotenoids and the vitamin A cy-cle in vision. Nature. 1934;134:65.Macmillan Magazines, Ltd.

FIGURE 3. Electron photomicrograph of the tip of a primate rod outersegment (ROS) embedded in the apical processes of a retinal pigmentepithelial (RPE) cell. Photoisomerization occurs within the disc mem-brane system of the ROS whereas the isomerase reaction occurs withinthe RPE cell. Intercellular diffusion of retinoids couples the processesin the two cells into a visual cycle. Reprinted, in modified form, withpermission, from Hogan MJ, Alvarado JA, Weddell JE. Histology of theHuman Eye. Philadelphia: WB Saunders; 1971:412.

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Thus, 11-cis-retinal is the presumed product of RPE retinoidmetabolism for the mammalian rod visual cycle.

ANALYSIS OF THE FLOW OF RETINOIDS IN THE

MOUSE VISUAL CYCLE

Dowling22 determined the flow of retinoids in and out of theRPE by analysis of their temporal appearance during pro-longed, total bleaching and recovery in the dark. Several fun-damental questions remained regarding the flux of the cycle.What step of the cycle determines the rate of visual pigmentregeneration? Is there biochemical evidence to suggest that thecycle is regulated? Do the several retinoid-binding proteins thathave been characterized in RPE and Muller cells play essentialroles in the visual cycle? We addressed these questions byanalyzing the composition of visual cycle retinoids during re-covery from a flash or from steady illumination. We chose miceas the experimental animals because of the increasing availabil-ity of animals with targeted disruption of genes encodingputative visual cycle components.

The Rate-Limiting Step in the Mouse Visual Cycle

Lightly pigmented mice were dark adapted and subjected toeither a flash or to steady illumination that bleached approxi-mately 40% of their visual pigment. Retinoids were extractedand analyzed before bleaching (dark adapted) and during therecovery period in the dark. The high-performance liquid chro-matography traces from an experiment using flash illuminationare shown in Figure 5, and the results are summarized in Figure6. Surprisingly, the only retinoid that accumulated in substan-tial amounts during the recovery period was all-trans-retinal.40

In other words, all processes after reduction of all-trans-retinalwere rapid, including intercellular transport, esterification,isomerization, oxidation, and conjugation with opsin. Thisfinding led to the conclusion that reduction of all-trans-retinalby NADPH determines the rate of entry of retinoid into thevisual cycle and emphasizes the importance of this reaction.

These results refer to the experimental situation in whichapproximately 40% of the mouse visual pigment was bleached.It is possible that intermediates other than all-trans-retinalwould appear if the visual system were challenged with larger

FIGURE 4. A schematic illustrating a working hypothesis for the mammalian rod visual cycle. The reactions and compartments are discussed inmore detail in the text. The abbreviations used are: ABCR, ATP-binding cassette, retina; CRBP, cellular retinol-binding protein; CRALBP, cellularretinaldehyde-binding protein; IMH, isomerohydrolase; IRBP, interphotoreceptor retinoid-binding protein: LRAT, lecithin:retinol acyltransferase;RDH, all-trans-retinol dehydrogenase; 11-RDH, 11-cis-retinol dehydrogenase; Rho, rhodopsin; Rho*, activated rhodopsin; 11-Ral, 11-cis-retinal;11-Rol, 11-cis-retinol; at-Ral, all-trans-retinal; at-Rol, all-trans-retinol; at-RE, all-trans-retinyl ester.

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fractional bleaches. At the limit of 100% bleach, Dowling22 andZimmerman24 observed in rats the progressive appearance anddisappearance of all the visual cycle intermediates that couldbe resolved. In addition, Perlman et al.41 observed that the timeconstant for the regeneration of visual pigment in rats is in-versely proportional to the amount of visual pigment bleached.Because of the complex nature of the chemical reactions andtransport processes involved, it is possible that another stepcould become rate limiting as flux through the pathway isincreased.

Flash illumination of animals and humans has been usedwith great success in a number of experimental situations.However, it could be argued that physiological conditions aremore closely approximated with steady illumination. Thus, wethought it important to verify that our observation of theaccumulation of all-trans-retinal during recovery from a flashwas not an artifact of the illumination conditions. Dark-adaptedmice were subjected to illumination from two 60-W fluores-cent bulbs (50 foot-candles). Retinoids were extracted andanalyzed at various times after onset of the lights and duringthe recovery period in the dark. Again, all-trans-retinal was theonly retinoid that accumulated in substantial amounts duringbleaching and recovery. Figure 7 depicts the amount of all-trans-retinal accumulated during steady state bleaching and

during recovery in the dark. The constant light resulted in asteady state with approximately 35% of the visual pigmentbleached. When the light was turned off, the all-trans-retinalrapidly decayed to the original dark-adapted value. We com-pared the rate of decay of all-trans-retinal produced by steadyillumination with that produced by a flash. Approximately thesame amount of visual pigment was bleached in each case. The

FIGURE 5. HPLC [high-performance liquid chromatography] separa-tion of visual cycle retinoids. (A) dark adapted mice; (B) mice imme-diately following a flash; (C) mice 60 minutes in the dark after a flash.The numbers indicate the elution positions of 1, retinyl palmitate; 2,all-trans-retinyl acetate (internal standard); 3, syn 11-cis-retinal oxime;4, syn all-trans-retinal oxime; 5, 11-cis-retinol; 6, anti 11-cis-retinaloxime; 7, all-trans-retinol; 8, anti all-trans-retinal oxime. Reprinted,with permission, from Van Hooser JP, Garwin GG, Saari JC. Analysis ofthe visual cycle in transgenic mice. Methods Enzymol. 2000, AcademicPress. In press.

FIGURE 6. Composition of polar retinoids in dark-adapted mice recov-ering from a flash. (E) 11-cis-Retinal; (F) all-trans-retinal; (f) all-trans-retinol; (M) 11-cis-retinol. Arrow: a flash. Retinyl esters did not changesignificantly with this amount of bleaching (not shown). Modified,with permission, from Palczewski K, Van Hooser JP, Garwin GG, SaariJC. Kinetics of visual pigment regeneration in excised mouse eyes andin mice with a targeted disruption of the gene encoding interphotore-ceptor retinoid-binding protein or arrestin. Biochemistry. 1999;39:12012–12019. American Chemical Society.

FIGURE 7. Recovery from steady state bleaching. The bar at the top ofthe figure illustrates the lighting regimen used (filled bar, dark; openbar, light). In the dark (0 on the abscissa) ;5% of the retinals are in theall-trans configuration. The onset of light increased the amount ofall-trans-retinal to ;40%. When the light was turned off (90 minutes)all-trans-retinal rapidly decreased to the dark-adapted value.

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results, shown in Figure 8, illustrate that the recovery fromsteady illumination is approximately 3.5 times more rapid (half-life [t1/2], 5 minutes with constant illumination; t1/2, 17 min-utes with flash illumination). Similar results were reported in astudy of phosphorylation of rhodopsin.42

What can account for the difference in the rates of decayof all-trans-retinal (and rate of 11-cis-retinal formation) gener-ated by the two different bleaching regimens? Because all-trans-retinal was the only visual cycle intermediate that accu-mulated in both cases, the difference in rates must result froma difference in rate of reduction of all-trans-retinal. It is possi-ble that the reaction is subject to control at the level of theenzyme, all-trans-retinol dehydrogenase. Alternatively, it ispossible that flash illumination fails to fully activate the pentosephosphate pathway, which is largely responsible for genera-tion of NADPH for retinal reduction. Modulation of the rate ofall-trans-retinal reduction will be discussed further in subse-quent sections.

The Block in the Visual Cycle in ExcisedMouse Eyes

Dissected frog eyes, which contain preformed 11-cis-retinylester in their RPE,23 regenerate their visual pigment afterbleaching.1,17–20 However, mouse eyes, with little if any pre-formed 11-cis-retinyl ester,40 do not regenerate their visualpigment once removed from the animal.43,44 What step in thevisual cycle is blocked in excised mouse eyes? The visual cycle,as currently postulated, does not include any obvious step thatrequires metabolic energy except for the ABCR transporterreaction29 (see Fig. 4). Formation of the 11-cis configuration isan endergonic process,45,46but the energy required for forma-

tion of the hindered 11-cis configuration has been postulated tocome from the hydrolysis of the ester bond of all-trans-retinylester.47 Thus, it would not be anticipated that depriving an eyeof its source of blood would prevent the formation of 11-cis-retinoids. We addressed this question by analyzing the compo-sition of visual cycle retinoids in bleached, excised mouseeyes.44 Eyes were removed from dark-adapted mice and sub-jected to constant illumination (Fig. 9A). 11-cis-Retinal steadilydisappeared during the illumination period. all-trans-Retinaltransiently appeared, and ultimately all-trans-retinol and all-trans-retinyl ester accumulated. No 11-cis-retinoids wereformed. Based on current ideas about the visual cycle (Fig. 4),this suggests that the isomerization reaction is not functional inexcised mouse eyes. This block in the cycle could result fromseveral causes. Perhaps the isomerization reaction requiresmetabolic energy (e.g., ATP), contrary to what has been sug-gested, and the ATP stores in these excised eyes are rapidlydepleted. In support of this are reports that the electricalresponses of rabbit eyes rapidly decay in the absence of oxygenand glucose48 and that excised mouse eyes do not regeneratetheir visual pigment unless they are perfused with oxygen andglucose.43 However, it is also possible that changes in cellularpH, ionic concentrations, or oxidative stress results in inhibi-tion of the isomerase.

A Block of the Visual Cycle at the RDH Reaction

Based on the results obtained with constant illumination ofexcised mouse eyes, it could be predicted that flash illumina-tion would produce a similar metabolic pattern—namely, ac-cumulation of all-trans-retinyl ester. However, the actual ex-perimental result we obtained was completely unanticipated.all-trans-Retinal generated by the flash was not further metab-olized44 (Fig. 9B). No reduction was evident, nor did any othermetabolites appear. This striking result indicates that the reac-tion catalyzed by RDH is more complicated than had beenanticipated and that simply supplying one of the substrates(all-trans-retinal) is not sufficient to activate the reaction. Whydoes reduction of all-trans-retinal not occur, and is this relatedto the differences we have observed in the rates of regenera-tion after flash or constant illumination? Several possibilitieswill be discussed in the subsequent sections.

Reduction of all-trans-retinal requires a source of reducingpower, and numerous studies have demonstrated that in thevisual cycle this source must be NADPH.49,50 The pentosephosphate pathway (also known as the hexose monophos-phate shunt) supplies most of the NADPH in most tissues.51

This pathway is considered to be constitutive in most quies-cent cells except in neutrophils where NADPH is required forsuperoxide production, in adipose tissue where NADPH isused for fatty acid biosynthesis, and in dividing cells thatrequire ribose-P for DNA synthesis. Activation of glucose6-phosphate dehydrogenase (G6PD), the first enzyme of thepentose phosphate pathway, by epidermal growth factor hasbeen studied in detail in growing cells where it appears toinvolve release of the enzyme from structural elements withinthe cell.52,53 Studies of glucose metabolism emphasized thatROSs were capable of producing NADPH through the pentosephosphate pathway in amounts sufficient to account for reduc-tion of all-trans-retinal.54 However, in dark-adapted rabbit andmonkey retina, the ratio of NADPH to nicotinamide adeninedinucleotide phosphate (NADP) was reported to be 0.3,55

indicating that the pathway would have to be activated for

FIGURE 8. Recovery from flash and steady state bleaching. Mice weresubjected to either a flash or to constant light that bleached ;40% oftheir visual pigment, and placed in the dark. At intervals retinoids wereextracted and analyzed by high-performance liquid chromatography.Gray bars, recovery after a flash; open bars, recovery after steadyillumination. The rate of recovery from steady state bleaching was;3.5 times more rapid. Modified, with permission, from Saari JC,Garwin GG, Van Hooser JP, Palczewski K. Reduction of all-trans-retinallimits regeneration of visual pigment in mice. Vision Res. 1998;38:1325–1333. Elsevier Science.

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reduction to occur. In vivo [13C] nuclear magnetic resonancestudies of rabbit retina have demonstrated an activation of thepentose phosphate pathway in constant light.56 Perhaps flashillumination does not fully activate the pentose phosphatepathway in normal mouse eyes and not at all in excised mouseeyes.

The Schiff base linking all-trans-retinal and opsin must behydrolyzed before the retinoid can be reduced to all-trans-retinol. Differences in the rates of reduction in vivo or theabsence of reduction in excised eyes could be due to differ-ences in the rates of hydrolysis of the Schiff bases formed withintermediates generated by a flash or constant illumination.Flash illumination is known to result in phosphorylation pri-marily of ser334 of opsin, whereas constant illumination resultsin phosphorylation primarily of ser338.42 Complex formationof these differently phosphorylated opsins with arrestin couldfurther affect the rates of hydrolysis of the Schiff base, althoughour studies of arrestin knockout mice indicate otherwise44 (seefollowing discussion).

It is also possible that NADPH and all-trans-retinal aregenerated in separate compartments, and an active processmust occur to unite them. The rim protein of ROSs57 hasrecently been identified as a member of the ABC-transporterfamily (ABCR).58,59 The rate of hydrolysis of ATP by this pro-tein is stimulated by addition of 11-cis- or all-trans-retinal,28

suggesting that one or both of these retinoids are substrates forthe transporter. The investigators propose that the function ofthe protein is to pump all-trans-retinal from inside the disc,where it is generated, to the cytosolic side, where it can bereduced by NADPH. Recent reports of the accumulation ofcondensation products of phosphatidylethanolamine and reti-

nal in the ABCR knockout mouse suggests that these may bethe actual substrates for the transporter.29 Thus, depletion ofATP in excised mouse eyes could prevent the two substrates ofthe reaction from uniting. However, this explanation seemsunlikely to account for the absence of reduction of all-trans-retinal that we have observed in flashed, excised mouse eyesbecause reduction occurs in the same experimental systemwith constant illumination (Fig. 9A). In addition, a normal rateof visual pigment regeneration was observed in ABCR2/2animals, indicating that the visual cycle is not dependent onthe transporter.

Finally, it is possible that the enzyme is directly regulatedby unknown mechanisms. The lack of structural informationabout rod RDH considerably hinders further progress in thisarea.

Blocked Transport of Retinoids in theVisual Cycle

Constant illumination of mouse eyecups immersed in bufferrevealed a third pattern of metabolism. 11-cis-Retinal steadilydisappeared concomitant with a transient increase in theamount of all-trans-retinal and an eventual accumulation ofall-trans-retinol (Fig. 9C). No retinyl esters were produced.This result suggests that the transport of all-trans-retinol to theRPE, where LRAT is localized60,61 did not occur. Examinationof the eyecups at the end of the experiment provides theexplanation for this result. The retinas became detached dur-ing the incubation. This somewhat trivial explanation nonethe-less provides information relative to retinoid transport in de-tached retinas and illustrates the power of retinoid analysis indetecting abnormal visual cycle function.

FIGURE 9. Effect of bleaching on dark-adapted excised mouse eyes or eyecups. (A) Excised mouse eyeswere subjected to constant illumination. Note the progressive loss of 11-cis-retinal and the accumulationof all-trans-retinal, -retinol and retinyl esters. (B) Excised mouse eyes were subjected to a flash (arrow).Note the accumulation of all-trans-retinal and the lack of regeneration. (C) Excised eyecups immersed inbuffer were exposed to constant illumination. Note the accumulation of all-trans-retinol. Retinoids wereextracted and analyzed at intervals as shown for all three treatments. Open circles, 11-cis-retinal; filledcircles, all-trans-retinal; filled squares, all-trans-retinol; filled triangles, retinyl esters; open squares,11-cis-retinol. Modified, with permission, from Palczewski K, Van Hooser JP, Garwin GG, Saari JC. Kineticsof visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the geneencoding interphotoreceptor retinoid-binding protein or arrestin. Biochemistry. 1999;39:12012–12019.American Chemical Society.

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In summary, the metabolic inertness of all-trans-retinal inexcised mouse eyes generated by a flash and the differences inthe rates of regeneration after flash or steady bleaching pointout how poorly we understand the processes by which thevisual cycle is controlled. Continued study will lead to a solu-tion to this problem and very likely to enhanced understandingof the control of the visual cycle.

ANALYSIS OF THE FLOW OF RETINOIDS IN

KNOCKOUT MICE

The Visual Cycle in Arrestin2/2 Mice

Photoactivated Rho is quenched in a two-step process involv-ing opsin phosphorylation by rhodopsin kinase62 and bindingof arrestin to phosphorylated opsin.63 Two studies have sug-gested that arrestin affects the activity of RDH in ROS prepa-rations. Direct addition of arrestin to washed, flashed ROSmembranes results in a 40% inhibition of the rate of reductionof all-trans-retinal,64 whereas we observed inhibition of RDHin whole ROS preparations (containing rhodopsin kinase andarrestin) when ATP and guanosine triphosphate were added.65

We attribute the ATP effect to phosphorylation of Rho* byrhodopsin kinase, formation of a complex with arrestin, and areduction of the accessibility of all-trans-retinal to RDH. Theseresults led to the prediction that reduction of all-trans-retinaland regeneration of visual pigments would be more rapid in amouse without functional arrestin than in normal subjects.

To address these possibilities, we examined the composi-tion of retinoids during bleaching and regeneration in arres-tin2/2 mice. These animals were born and raised in the darkand subjected to flash bleaching followed by a recovery periodin the dark. The distribution of visual cycle retinoids observedbefore and after a flash is very similar to that seen with normalmice (not shown). The recovery of 11-cis-retinal in the darkwas slower in arrestin2/2 mice than in normal mice (0.6 and1.1% per minute, respectively; Fig. 10). However, the rates ofrhodopsin regeneration were similar (0.8% per minute and 1%per minute, respectively).

The failure to detect major changes in the rates of rho-dopsin and 11-cis-retinal regeneration in arrestin2/2 mice issurprising in view of the in vitro results mentioned earlier.Perhaps the experimental situation in which we observed the

ATP effect poorly approximates the conditions found withinthe rod outer segment. An alternative possibility is that ourinterpretation of the in vitro results was incorrect and thatphosphorylation of opsin alone was sufficient to alter the rateof release of all-trans-retinal. Perhaps an examination of thekinetics of visual pigment regeneration in rhodopsin kinaseknockout mice will resolve this issue.

The Visual Cycle in IRBP2/2 Mice

There is much circumstantial evidence to support a role forIRBP in the diffusion of retinoids between RPE and photore-ceptor cells: 1) IRBP possesses two or more high-affinity bind-ing sites for retinoids.66–70 (2) all-trans-Retinol or 11-cis-retinalhas been found to be associated with IRBP purified from dark-or light-adapted retina, respectively.71 3) IRBP occurs preciselyin the extracellular compartment separating photoreceptorand RPE cells.36,72,73 4) Photoreceptor cells die in mice with atargeted disruption of the IRBP gene.74 5) Studies with cul-tured RPE cells suggest that IRBP causes the release of 11-cis-retinal, whereas other binding proteins with affinity for 11-cis-retinal are ineffective.75,76 6) IRBP efficiently delivers 11-cis-retinal to toad photoreceptors for rhodopsin regeneration.77

Other evidence argues against an active role for IRBP inretinoid transport: 1) IRBP is a large, cigar-shaped protein (axialratio .7:1), an unlikely shape for a transport protein.69,78,79 2)Diffusion of all-trans-retinol between populations of vesicles isrelatively rapid and inhibited in the presence of IRBP.80

We examined the kinetics of recovery of 11-cis-retinal andof rhodopsin after a flash with IRBP2/2 mice.44 IRBP2/2mice were dark adapted and exposed to a flash that bleachedapproximately 35% of their visual pigment. Animals were killedbefore the flash (dark adapted) and at various intervals in thedark after the flash. Retinoids were extracted from the poste-rior poles of the eyes and analyzed by high-performance liquidchromatography. The flash produced an immediate 35% de-crease in the amount of 11-cis-retinal and a concomitant, pro-portional increase in the amount of the photolysis productall-trans-retinal. Recovery in the dark resulted in an increase inthe amount of 11-cis-retinal and a corresponding decrease inthe amount of all-trans-retinal. Small, transient increases ofall-trans-retinol and retinyl ester were observed during thereturn to the dark-adapted state. The overall pattern stronglyresembled that observed with wild-type mice. The recoverykinetics resulting from four experiments with IRBP2/2 miceare shown in Figure 10, along with the recovery curve ob-tained with wild-type mice. IRBP2/2 mice regenerated their11-cis-retinal at a rate of 0.8% per minute, compared with avalue of 1.1% per minute for a mixed population of wild-typemice. The relatively modest difference in rates of recoverysuggests that IRBP does not influence the rate of visual pigmentregeneration.

The normal turnover rates for visual pigments and retinoidcomposition in IRBP2/2 mice are surprising in view of thewealth of evidence mentioned earlier. However, we have ex-amined only visual cycle retinoids after a modest bleaching ofvisual pigments. Perhaps abnormal patterns of retinoid metab-olism would become apparent with other bleaching regimens.It is apparent that IRBP plays an important role in visualphysiology because photoreceptor cells die in its absence.74

IRBP may act as a buffer in the subretinal space, limiting theconcentration of free retinoid and preventing oxidative degra-dation.80,81 Perhaps the results obtained here will direct re-

FIGURE 10. Recovery of 11-cis-retinal in the dark after a flash. (F)Normal mice; (E) IRBP2/2 mice; (M) arrestin2/2 mice.

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search toward other potential roles for IRBP in visual physiol-ogy.

PERSPECTIVE

Outstanding Problems

The visual cycle shown in Figure 4 is consistent with theavailable evidence regarding the regeneration of rod visualpigments. However, this model must be regarded as a workinghypothesis for several reasons. First, several “orphan” retinoid-binding components have been described, and some are likelyto be involved in the visual cycle. For instance, RPE65 mustplay a major role in retinoid metabolism, based on the inabilityof RPE652/2 mice to make 11-cis-retinoids,82 but its functionis not understood at this time. Other proteins such as perop-sin83 and RGR opsin84 have amino acid sequences related tothat of opsin. What are their functions? Peropsin has beenlocalized to the apical plasma membrane of RPE.83 Could it beinvolved in the export of 11-cis-retinal from RPE or in theimport of all-trans-retinol from the interphotoreceptor matrix?Eicosanoids such as prostaglandins are secreted from cellsthrough a transmembrane protein called the prostaglandintransporter, which facilitates secretion and uptake of the hy-drophobic signaling molecules.85 There is insufficient informa-tion to draw any conclusions at this time, but the phenotypeassociated with knockout mice may provide an answer. Sec-ond, the cone visual cycle differs from the rod visual cycle inseveral quantitative aspects (e.g., faster turnover) and perhapsqualitative aspects as well. Muller cells are known to containtwo retinoid-binding proteins (CRALBP and CRBP)35–37 and toperform several transformations of retinoids in vitro.86 DoMuller cells contribute to the regeneration of visual pigments?The answers to these and other fascinating questions await theresults of further experimentation. However, the questionsemphasize our rudimentary understanding of the complex pro-cess of visual pigment regeneration.

Why Is the Visual Cycle So Complex?

Two general systems are used for regeneration of bleachedvisual pigments. Invertebrates rely on the establishment ofphotoequilibrium, in which the first photon absorbed converts11-cis-retinal to all-trans-retinal (Fig. 11). A second photon canthen convert all-trans-retinal back to 11-cis-retinal. At constantlevels of illumination a steady state level of bleached visualpigment is generated, which is a factor in determining thesensitivity of the visual system, as has been discussed. Thus, thechromophore does not dissociate from the opsin to which it iscovalently bound and the meta II species absorbs in the visiblerange of the spectrum. In contrast, in vertebrates there is acomplicated system involving dissociation of the chromophoreand regeneration of the 11-cis configuration in a neighboringnurse cell. What are the advantages and disadvantages of thetwo systems? The invertebrate system is inherently more sim-ple and elegant in design. However, the bleaching rate and theregeneration rate are tied to the photon flux, a feature perhapsdisadvantageous in rapidly changing light conditions. In con-trast, the vertebrate regeneration system is more complex,because it involves the participation of two different cell types,several enzymes, and intercellular flow of retinoids. However,the regeneration rate is independent of the photon flux, allow-

ing relatively rapid restoration of visual sensitivity even in thedark.

Is Night Blindness Caused by Defects in theVisual Cycle?

Night blindness is a common hallmark of many inherited reti-nal diseases and nutritional disorders. In general, the conditionappears to result from two phenomena: decreased photoncatch resulting from diminished rhodopsin content and/or thegeneration of a species that actively desensitizes the retina. Theformer mechanism results in relatively mild elevations of thescotopic threshold. For instance, a 25% reduction in rhodopsinlevel increases the scotopic threshold by 1.5. However, activespecies produced by mutation, photobleaching or vitamin Adeficiency, result in very large elevations in scotopic threshold.For example, 25% bleaching of rhodopsin increases thescotopic threshold by 104. The night blindness reported inmany retinitis pigmentosa cases results simply from the re-duced photon catch associated with decreased amounts ofrhodopsin in the affected retina.87,88 However, several muta-tions in the opsin gene result in constitutively active speciesthat further desensitize the retina.89,87,90 In addition, mutationsin other genes that produce constitutive activation of photo-transduction91 or that affect photointermediate quenchingpathways92,93 result in delayed dark adaptation.

Visual Cycle Defects and Inherited RetinalConditions

Mutations in several genes encoding presumptive visual cyclecomponents have recently been implicated in several inheritedretinal diseases. The roles of ABCR in Stargardt disease94 and ofRPE65 in Leber’s congenital amaurosis95,96 and in other retinaldiseases have been discussed in detail elsewhere and will notbe covered here. Recently, missense mutations in the geneencoding 11-cis-retinol dehydrogenase have been found in pa-tients with fundus albipunctatus.97 This form of congenitalstationary night blindness results in delayed dark adaptationand delayed regeneration of visual pigments.98–100

FIGURE 11. Regeneration of visual pigments in invertebrates isachieved by photoisomerization of a complex of opsin and all-trans-retinal absorbing in the visible range. Thus, photoequilibrium is estab-lished in the light, and the chromophore does not dissociate from thereceptor. Vertebrates have a more complex system in which the 11-cisconfiguration is regenerated in a light-independent reaction in anadjacent cell.

344 Saari IOVS, February 2000, Vol. 41, No. 2

CRALBP has several distinguishing characteristics thatstrongly suggest its participation in the visual process. First, theprotein has a high-affinity binding site for either 11-cis-retinal (Kd,10 nM) or 11-cis-retinol (Kd, 60 nm).101 Second, CRABLP purifiesfrom RPE saturated with 11-cis-retinal and from neural retinalsaturated with 11-cis-retinal and 11-cis-retinol,102 retinoids of thevisual cycle. Third, in retina, CRALBP is found in Muller and RPEcells.36 The RPE, of course, is the site of intense retinoid metab-olism related to the visual cycle. Fourth, CRALBP affects theenzyme activity of four enzymes of the visual cycle in vitro. Thebinding protein reduces LRAT-mediated esterification of 11-cis-retinol by 90% and modestly stimulates oxidation of 11-cis-retinolby 11-RDH.103 Apo-CRALBP is required for retinol isomerase(isomerohydrolase) activity104,105 and for release of 11-cis-retinolfrom endogenous 11-cis-retinyl esters by 11-REH.104 The develop-ment of CRALBP knockout mice should resolve the question ofthe role of CRALBP in visual physiology.106

Mutations in the gene encoding CRALBP have been asso-ciated with several forms of retinal degeneration. Affectedsiblings in a consanguineous pedigree segregating for nonsyn-dromic autosomal recessive retinitis pigmentosa were homozy-gous for a G4763A nucleotide substitution in the CRALBP gene.Recombinant CRALBP bearing this substitution (R150Q) didnot bind 11-cis-retinal in vitro,107 stressing the importance ofthe retinoid-binding site. Four mutations in the gene encodingCRALBP were found in three unrelated patients with reces-sively inherited retinitis punctata albescens108. Twenty pa-tients from seven families with features of retinitis punctataalbescens and macular degeneration (Bothnia dystrophy) werehomozygous for a missense mutation (R234W) in the CRALBPgene.109 These results suggest that CRALBP plays an importantrole in visual physiology.

Muller Cells and Cone Visual PigmentRegeneration

The literature contains many intriguing suggestions that thevisual cycle in cones differs from that in rods and that Mullercells may be involved. Two retinoid-binding proteins,CRALBP36 and CRBP,5,37 are found in Muller and RPE cells inretinas from several species. The presence of CRALBP is par-ticularly intriguing, because the binding protein, as mentionedabove, has been demonstrated to purify from neural retina as amixture of complexes with 11-cis-retinol or 11-cis-retinal.102

The all-trans- and 9-cis-retinoids modulate many biologic pro-cesses110 but 11-cis-retinoids are only known to be involved inthe visual process or in the absorption of light (pineal). Cul-tured chick Muller cells take up exogenous all-trans-retinol andconvert it to all-trans- and 11-cis-retinyl palmitate and 11-cis-retinol. The latter retinoid has been found in the culture me-dium.86 Although no oxidation to 11-cis-retinal has been ob-served, other investigators have noted that isolated amphibiancone cells resensitize with exogenous 11-cis-retinol, whereasrods require 11-cis-retinal.111 Goldstein noted that the ampli-tude of the cone but not the rod early receptor potentialreaches a steady state in illuminated, isolated frog retina andrecovers with a t1/2 of 5 to 6 minutes in the dark.112–114 Thus,it appears that cones can regenerate their visual pigment infrog retina in the absence of RPE. However, the interpretationof these provocative studies hinges on the validity of thedemonstration that early receptor potential amplitude is di-rectly proportional to the amount of unbleached visual pig-ment.115,116 Thus, it seems clear that some interesting retinoid

metabolism occurs in Muller cells, and the presence of 11-cis-retinoids strongly suggests that it is related to the visual cycle.

CONCLUSION

This is an exciting time in visual cycle research. During the pastfew years the number of published studies related to visualpigment regeneration has increased dramatically. Some well-established players have been characterized at the molecularlevel (for instance, cone RDH, 11-RDH, and LRAT), and severalother proteins have been shown to play very important roles inthe visual cycle (RPE65 and ABCR). Further indication of thesophistication of the cycle has been shown by studies thatpoint out the complexity of a seemingly simple step such asthe reduction of all-trans-retinal. We can anticipate the elabo-ration of further complexities and further medical relevance asinvestigators obtain the tools and molecular information nec-essary for precise dissection of individual and integrated stepsof the visual cycle.

Acknowledgments

The author thanks, in particular, Lucille Bredberg, John Crabb, JingHuang, Greg Garwin, Breandan Kennedy, Ann Milam, Maria Nawrot,Kris Palczewski, and Dan Possin, among the many colleagues whocontributed to these studies.

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