Biochemistry of Visual Pigment Regeneration The Friedenwald Lecture
John C. Saari
Rods bleached in vivo therefore do not regenerate their visual purple from material within themselves or in the tissues proximal to them, but from the distally placed pigment epithelium: the pigment epithelium functions in the regeneration of visual purple; it exerts a regenerative action on the rods.1 hototransduction and the visual cycle play complementary roles in vertebrate vision. Phototransduction is initiated by the photoisomerization of 11-cis-retinal bound to opsin and ultimately results in a change in the release of neurotransmitter by photoreceptor cells. The visual cycle restores the product of photoisomerization, all-trans-retinal, to the 11-cis configuration and allows the regeneration of bleached visual pigments (Fig. 1). The biochemical mechanism of phototransduction has been extensively studied during the past 2 decades, and as a result, the process serves as the paradigm for understanding G-protein– coupled receptors in general. In contrast, molecular understanding of the visual cycle is poorly developed, and many fundamental questions regarding reactions, enzymes, and control mechanisms remain unanswered. Sequences of cDNAs encoding three visual cycle enzymes have been published2– 4; however, molecular information is unavailable for the other three presumed enzymes of the cycle, including retinol isomerase (isomerohydrolase). It has been reported that regeneration of visual pigments is a slow process and that photoisomerization of 11-cis-retinal in rhodopsin is very rapid. In fact, complete dark adaptation in humans requires approximately 40 minutes,5–7 and conversion of rhodopsin to photorhodopsin requires only 200 fsec.8 However, this is not a fair comparison, because it is clear that the photolysis and regeneration rates in the living eye must be equal 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 has demonstrated for human rods5 and cones9 that a steady state level of bleached visual pigments, in which the bleach and regeneration rates are equal and of opposite sign, is present at different levels of physiologic illumination. In humans the progress curves for the regain of visual threshold and for the regeneration of visual pigment coincide when displayed on a semi–log plot.6,7 The molecular explanation for this log–linear relationship is not well understood, and the relationship may be fortuitous, but most agree that a photoproduct is responsible for desensitization of the visual system.6,10,11 Although the molecular identity of the desensitizing intermediate(s) remains a matter of active investigation,10,12–16 it is clear that visual cycle reactions are important in determining the steady state level of bleached visual pigment and thus the sensitivity of the retina. The critical role of the retinal pigment epithelium (RPE) in visual pigment regeneration is apparent from the studies of 19th century investigators who demonstrated that dissected frog retina could regenerate its bleached visual pigment only when in contact with the RPE.1,17–19 The reader is referred to Marmor and Martin20 for a depiction of some of their insightful experiments. It is fortunate that the early physiologists used frog eyes for their experiments because rodent eyes do not regenerate their visual pigments when removed from the animal, as will be discussed later. Fifty years later, Wald21 used extraction techniques to show that vitamin A was involved in the visual process and formulated the first modern version of the visual cycle including the participation of the RPE (Fig. 2). The role of the RPE in the visual cycle became more clear after the classic study by Dowling, 22 which demonstrated movement of retinoid out of the neural retina and into RPE during extensive bleaching and return during recovery in the dark. Later studies by other investigators with techniques offering more resolution verified the fundamental observation.23,24 Bernstein et al.25 and Rando26 provided a molecular explanation for the necessity of the RPE with the demonstration that the critical enzymatic regeneration of the 11-cis configuration occurred within this tissue.25,26 The transcellular migration of the retinoids during bleaching and regeneration is all the more remarkable, considering the anatomy of the journey (Fig. 3). The relatively insoluble retinoid must leave the disc membranes, diffuse through a cytosolic compartment to reach the plasma membrane of the rod outer segment, traverse the plasma membrane, diffuse across the subretinal space to reach the plasma membrane of the RPE cell, enter into the reactions of the visual cycle in this cell, and make the return journey! A current working hypothesis for the reactions of the vertebrate rod visual cycle is shown in schematic form in Figure 4. The figure depicts internal and plasma membranes of the RPE and rod photoreceptor cells and the interphotoreceptor matrix space (subretinal space) separating these two cells. The visual cycle enzymes in RPE are all associated with membranes; however, their localization to subcellular compart337


From the Departments of Ophthalmology and Biochemistry, University of Washington School of Medicine, Seattle, Washington. Supported in part by National Institutes of Health Grants RO1 EY02317, EY01730, and EY09339 and by unrestricted awards from Research to Prevent Blindness. JCS is a Senior Scientific Investigator of Research to Prevent Blindness. Submitted for publication August 6, 1999; accepted August 30, 1999. Commercial relationships policy: N. Corresponding author: John C. Saari, Department of Ophthalmology, Box 356485, University of Washington, Seattle, WA 98195-6485. jsaari@u.washington.edu
Investigative Ophthalmology & Visual Science, February 2000, Vol. 41, No. 2 Copyright © Association for Research in Vision and Ophthalmology

The Schiff base linking all-trans-retinal and opsin is hydrolyzed to release free all-trans-retinal. from Wald G. The chromophore of visual pigments alternates between 11-cis and all-trans configurations. a water soluble retinoid-binding protein. and initiates phototransduction. with permission. 1934. Retinol can be taken up from the blood and esterified by LRAT in RPE cells (not shown). Recent reports suggest that an adenosine triphosphate (ATP)– binding cassette transporter (ABCR) is involved in moving all-trans-retinal from the intradiscal to the cytosolic aspect of the disc membrane. in modified form.338 Saari IOVS. The G-protein–stimulating activity of Rho* is quenched by phosphorylation and by the binding of arrestin (not shown in the figure). with permission. all-trans-Retinyl ester is converted to 11-cis-retinol and free fatty acid by an isomerohydrolase. Philadelphia: WB Saunders. Macmillan Magazines. Reprinted.e. and IRBP) have been localized to the compartments in which they are shown by immunocytochemistry.134:65. Reprinted. Mammalian rods cannot use exoge¨ nous 11-cis-retinol for regeneration of visual pigments. Vol. IRBP is present in the photoreceptor matrix. Photoisomerization converts 11-cis-retinal to all-trans-retinal. Opsin is shown on the lower side of the disc only for reasons of symmetry. 4) with its high-affinity ligands.29 alltrans-Retinol dehydrogenase (RDH) catalyzes the reduction of all-trans-retinal to all-trans-retinol by reduced nicotinamide adenine dinucleotide phosphate (NADPH).39 FIGURE 2. 11-cis-Retinyl esters can be hydrolyzed and used for visual pigment regeneration by 11-cis-retinyl ester hydrolase (11-REH). Intercellular diffusion of retinoids couples the processes in the two cells into a visual cycle.38. The general features of the regeneration cycle were already apparent in this early depiction. . activates the receptor. is shown (Fig. Electron photomicrograph of the tip of a primate rod outer segment (ROS) embedded in the apical processes of a retinal pigment epithelial (RPE) cell. Alvarado JA. 11-cis-Retinol can be esterified by LRAT and stored (reaction not shown) or oxidized to 11-cis-retinal by 11-cis-retinol dehydrogenase (11-RDH). all-trans-Retinol leaves the photoreceptor cell. Its binding to opsin freezes the receptor in a stable. and enters the RPE where it is esterified by lecithin-retinol acyltransferase (LRAT).28 perhaps as an adduct with phosphatidyl ethanolamine. No. Ltd. February 2000. although the protein also binds 11-cis-retinol in vitro.26. traverses the interphotoreceptor matrix space (the subretinal space) where it encounters interphotoreceptor retinoid-binding protein (IRBP). Evidence leading to this working hypothesis of the rod visual cycle has been presented in several recent reviews.35–37 CRBP and CRALBP are also found in Muller cells.14. The watersoluble retinoid-binding proteins (CRALBP. Nature. Cellular retinol-binding protein (CRBP) is shown associated with all-trans-retinol. Absorption of light by rhodopsin in the disc membrane converts 11-cis-retinal to all-trans-retinal and generates the active photoproduct. CRBP. 1971:412. 2 FIGURE 1. 11cis-Retinal diffuses into the photoreceptor cell where it associates with opsin to regenerate the visual pigment. Cellular retinaldehyde-binding protein (CRALBP). Photoisomerization occurs within the disc membrane system of the ROS whereas the isomerase reaction occurs within the RPE cell. Weddell JE. metarhodopsin II (Rho*). 41.30 –34 Each of the enzymatic reactions shown in the RPE or rod outer segments (ROSs) has been demonstrated in RPE microsome or ROS preparations. The 11-cis configuration is produced by a sequence of reactions that are thermally driven (i. all-trans- FIGURE 3. inactive form ensuring a low rate of thermal isomerization in the dark. 11-cis-retinal or 11-cis-retinol. respectively. 11-cis-Retinal or a closely related derivative is the chromophore for all known visual pigments.. Histology of the Human Eye. can occur in the dark). ments has not been completely determined. Carotenoids and the vitamin A cycle in vision. from Hogan MJ.27 The enzymes are depicted as part of one continuous internal membrane compartment for simplicity. Its role in retinoid transport is uncertain (discussion to follow).

2 The Friedenwald Lecture 339 FIGURE 4. 11-Ral. all-trans-retinyl ester. RDH. oxidation. retina. the only retinoid that accumulated in substantial amounts during the recovery period was all-trans-retinal. isomerohydrolase. isomerization. including intercellular transport. . activated rhodopsin. cellular retinol-binding protein. What step of the cycle determines the rate of visual pigment regeneration? Is there biochemical evidence to suggest that the cycle is regulated? Do the several retinoid-binding proteins that have been characterized in RPE and Muller cells play essential ¨ roles in the visual cycle? We addressed these questions by analyzing the composition of visual cycle retinoids during recovery from a flash or from steady illumination. Rho. It is possible that intermediates other than all-trans-retinal would appear if the visual system were challenged with larger ANALYSIS OF THE FLOW MOUSE VISUAL CYCLE OF RETINOIDS IN THE Dowling22 determined the flow of retinoids in and out of the RPE by analysis of their temporal appearance during prolonged. and the results are summarized in Figure 6. This finding led to the conclusion that reduction of all-trans-retinal by NADPH determines the rate of entry of retinoid into the visual cycle and emphasizes the importance of this reaction. We chose mice as the experimental animals because of the increasing availability of animals with targeted disruption of genes encoding putative visual cycle components. 11-cis-retinol dehydrogenase. and conjugation with opsin. all-trans-retinol. cellular retinaldehyde-binding protein. IRBP. The Rate-Limiting Step in the Mouse Visual Cycle Lightly pigmented mice were dark adapted and subjected to either a flash or to steady illumination that bleached approximately 40% of their visual pigment. Rho*. Vol. These results refer to the experimental situation in which approximately 40% of the mouse visual pigment was bleached.40 In other words. 11-Rol. at-RE. CRALBP.IOVS. Surprisingly. all-trans-retinol dehydrogenase. at-Ral. 11-cis-retinol. Several fundamental questions remained regarding the flux of the cycle. all-trans-retinal. lecithin:retinol acyltransferase. Retinoids were extracted and analyzed before bleaching (dark adapted) and during the recovery period in the dark. CRBP. 11-cis-retinal. interphotoreceptor retinoid-binding protein: LRAT. esterification. The abbreviations used are: ABCR. all processes after reduction of all-trans-retinal were rapid. rhodopsin. A schematic illustrating a working hypothesis for the mammalian rod visual cycle. The reactions and compartments are discussed in more detail in the text. The high-performance liquid chromatography traces from an experiment using flash illumination are shown in Figure 5. No. Thus. February 2000. 11-cis-retinal is the presumed product of RPE retinoid metabolism for the mammalian rod visual cycle. 41. ATP-binding cassette. total bleaching and recovery in the dark. 11-RDH. IMH. at-Rol.

February 2000. Perlman et al. ( ) 11-cis-retinol. Methods Enzymol. No. we thought it important to verify that our observation of the accumulation of all-trans-retinal during recovery from a flash was not an artifact of the illumination conditions. Modified. Again. 7. Academic Press. 8. The FIGURE 7. . all-trans-retinyl acetate (internal standard). 4. 6. Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin. FIGURE 6. 5. In the dark (0 on the abscissa) 5% of the retinals are in the all-trans configuration. Van Hooser JP. Reprinted. The bar at the top of the figure illustrates the lighting regimen used (filled bar. 2 FIGURE 5. Vol. Recovery from steady state bleaching. light). Saari JC. HPLC [high-performance liquid chromatography] separation of visual cycle retinoids. dark. anti 11-cis-retinal oxime. 41. from Palczewski K. Analysis of the visual cycle in transgenic mice. (f) all-transretinol. However.41 observed that the time constant for the regeneration of visual pigment in rats is inversely proportional to the amount of visual pigment bleached. The constant light resulted in a steady state with approximately 35% of the visual pigment bleached. open bar. 2000. Because of the complex nature of the chemical reactions and transport processes involved. The numbers indicate the elution positions of 1. (B) mice immediately following a flash. When the light was turned off. Thus. the all-trans-retinal rapidly decayed to the original dark-adapted value. Saari JC. When the light was turned off (90 minutes) all-trans-retinal rapidly decreased to the dark-adapted value. Retinyl esters did not change significantly with this amount of bleaching (not shown). (F) all-trans-retinal. (A) dark adapted mice. fractional bleaches.340 Saari IOVS. syn 11-cis-retinal oxime. The onset of light increased the amount of all-trans-retinal to 40%. (E) 11-cis-Retinal. In press. it is possible that another step could become rate limiting as flux through the pathway is increased. 1999. 2. with permission.39: 12012–12019. In addition. Approximately the same amount of visual pigment was bleached in each case. Garwin GG. At the limit of 100% bleach. Figure 7 depicts the amount of alltrans-retinal accumulated during steady state bleaching and during recovery in the dark. 11-cis-retinol. Arrow: a flash. Flash illumination of animals and humans has been used with great success in a number of experimental situations. retinyl palmitate. American Chemical Society. from Van Hooser JP. all-trans-retinol. Garwin GG. it could be argued that physiological conditions are more closely approximated with steady illumination. syn all-trans-retinal oxime. Retinoids were extracted and analyzed at various times after onset of the lights and during the recovery period in the dark. Dark-adapted mice were subjected to illumination from two 60-W fluorescent bulbs (50 foot-candles). with permission. 3. Dowling22 and Zimmerman24 observed in rats the progressive appearance and disappearance of all the visual cycle intermediates that could be resolved. We compared the rate of decay of all-trans-retinal produced by steady illumination with that produced by a flash. anti all-trans-retinal oxime. all-trans-retinal was the only retinoid that accumulated in substantial amounts during bleaching and recovery. Composition of polar retinoids in dark-adapted mice recovering from a flash. Biochemistry. (C) mice 60 minutes in the dark after a flash.

55 indicating that the pathway would have to be activated for results. February 2000. which is largely responsible for generation of NADPH for retinal reduction. it could be predicted that flash illumination would produce a similar metabolic pattern—namely.50 The pentose phosphate pathway (also known as the hexose monophosphate shunt) supplies most of the NADPH in most tissues.IOVS. tion of the hindered 11-cis configuration has been postulated to come from the hydrolysis of the ester bond of all-trans-retinyl ester. Mice were subjected to either a flash or to constant light that bleached 40% of their visual pigment.54 However. Recovery from flash and steady state bleaching. A Block of the Visual Cycle at the RDH Reaction Based on the results obtained with constant illumination of excised mouse eyes. does not include any obvious step that requires metabolic energy except for the ABCR transporter reaction29 (see Fig.5 times more rapid. all-trans-retinol dehydrogenase. Garwin GG.23 regenerate their visual pigment after bleaching.44 Eyes were removed from dark-adapted mice and subjected to constant illumination (Fig. in dark-adapted rabbit and monkey retina. excised mouse eyes. and in dividing cells that require ribose-P for DNA synthesis. At intervals retinoids were extracted and analyzed by high-performance liquid chromatography. Van Hooser JP. Similar results were reported in a study of phosphorylation of rhodopsin. The Block in the Visual Cycle in Excised Mouse Eyes Dissected frog eyes.45. 1998.1. the first enzyme of the pentose phosphate pathway. all-trans-Retinal generated by the flash was not further metabolized44 (Fig.5 times more rapid (halflife [t1/2]. mouse eyes.44 What step in the visual cycle is blocked in excised mouse eyes? The visual cycle.52.47 Thus. 9B). the actual experimental result we obtained was completely unanticipated. it is also possible that changes in cellular pH. all-trans-Retinal transiently appeared. illustrate that the recovery from steady illumination is approximately 3.3. shown in Figure 8. Palczewski K. 4). and placed in the dark. Reduction of all-trans-retinal limits regeneration of visual pigment in mice. 2 The Friedenwald Lecture 341 FIGURE 8. Alternatively. 17 minutes with flash illumination).42 What can account for the difference in the rates of decay of all-trans-retinal (and rate of 11-cis-retinal formation) generated by the two different bleaching regimens? Because alltrans-retinal was the only visual cycle intermediate that accumulated in both cases. contrary to what has been suggested.53 Studies of glucose metabolism emphasized that ROSs were capable of producing NADPH through the pentose phosphate pathway in amounts sufficient to account for reduction of all-trans-retinal. it would not be anticipated that depriving an eye of its source of blood would prevent the formation of 11-cisretinoids. or oxidative stress results in inhibition of the isomerase. Reduction of all-trans-retinal requires a source of reducing power. No. Vol. ATP). and numerous studies have demonstrated that in the visual cycle this source must be NADPH. 4). Elsevier Science. t1/2. Perhaps the isomerization reaction requires metabolic energy (e. Activation of glucose 6-phosphate dehydrogenase (G6PD). Formation of the 11-cis configuration is an endergonic process. No 11-cis-retinoids were formed.g. as currently postulated. ionic concentrations.43 However. Why does reduction of all-trans-retinal not occur.17–20 However. with little if any preformed 11-cis-retinyl ester. No reduction was evident. by epidermal growth factor has been studied in detail in growing cells where it appears to involve release of the enzyme from structural elements within the cell. Modulation of the rate of all-trans-retinal reduction will be discussed further in subsequent sections. Gray bars. this suggests that the isomerization reaction is not functional in excised mouse eyes. the ratio of NADPH to nicotinamide adenine dinucleotide phosphate (NADP) was reported to be 0. the difference in rates must result from a difference in rate of reduction of all-trans-retinal. Modified. recovery after steady illumination. In support of this are reports that the electrical responses of rabbit eyes rapidly decay in the absence of oxygen and glucose48 and that excised mouse eyes do not regenerate their visual pigment unless they are perfused with oxygen and glucose..40 do not regenerate their visual pigment once removed from the animal. from Saari JC. We addressed this question by analyzing the composition of visual cycle retinoids in bleached. with permission.51 This pathway is considered to be constitutive in most quiescent cells except in neutrophils where NADPH is required for superoxide production. nor did any other metabolites appear. and ultimately all-trans-retinol and alltrans-retinyl ester accumulated.49. This striking result indicates that the reaction catalyzed by RDH is more complicated than had been anticipated and that simply supplying one of the substrates (all-trans-retinal) is not sufficient to activate the reaction. However. which contain preformed 11-cis-retinyl ester in their RPE. The rate of recovery from steady state bleaching was 3.43. and is this related to the differences we have observed in the rates of regeneration after flash or constant illumination? Several possibilities will be discussed in the subsequent sections. This block in the cycle could result from several causes. accumulation of all-trans-retinyl ester. Vision Res. 41. It is possible that the reaction is subject to control at the level of the enzyme. in adipose tissue where NADPH is used for fatty acid biosynthesis.46but the energy required for forma- . open bars.38: 1325–1333. Based on current ideas about the visual cycle (Fig. recovery after a flash. 9A). 11-cis-Retinal steadily disappeared during the illumination period. it is possible that flash illumination fails to fully activate the pentose phosphate pathway. 5 minutes with constant illumination. and the ATP stores in these excised eyes are rapidly depleted.

Flash illumination is known to result in phosphorylation primarily of ser334 of opsin. whereas constant illumination results in phosphorylation primarily of ser338. Biochemistry. where LRAT is localized60. filled circles. (C) Excised eyecups immersed in buffer were exposed to constant illumination. In vivo [13C] nuclear magnetic resonance studies of rabbit retina have demonstrated an activation of the pentose phosphate pathway in constant light.39:12012–12019. 11-cis-Retinal steadily disappeared concomitant with a transient increase in the amount of all-trans-retinal and an eventual accumulation of all-trans-retinol (Fig.28 suggesting that one or both of these retinoids are substrates for the transporter. Vol. The investigators propose that the function of the protein is to pump all-trans-retinal from inside the disc. This somewhat trivial explanation nonetheless provides information relative to retinoid transport in detached retinas and illustrates the power of retinoid analysis in detecting abnormal visual cycle function. No. all-trans-retinol. (A) Excised mouse eyes were subjected to constant illumination.29 Thus.342 Saari IOVS. where it is generated. retinyl esters.58. The Schiff base linking all-trans-retinal and opsin must be hydrolyzed before the retinoid can be reduced to all-transretinol. Effect of bleaching on dark-adapted excised mouse eyes or eyecups. and an active process must occur to unite them. indicating that the visual cycle is not dependent on the transporter. (B) Excised mouse eyes were subjected to a flash (arrow). this explanation seems unlikely to account for the absence of reduction of all-transretinal that we have observed in flashed.59 The rate of hydrolysis of ATP by this protein is stimulated by addition of 11-cis. Note the progressive loss of 11-cis-retinal and the accumulation of all-trans-retinal. February 2000.42 Complex formation of these differently phosphorylated opsins with arrestin could further affect the rates of hydrolysis of the Schiff base. filled triangles. 41. 1999. Garwin GG. Retinoids were extracted and analyzed at intervals as shown for all three treatments. Examination of the eyecups at the end of the experiment provides the explanation for this result. depletion of ATP in excised mouse eyes could prevent the two substrates of the reaction from uniting. although our studies of arrestin knockout mice indicate otherwise44 (see following discussion). American Chemical Society. Differences in the rates of reduction in vivo or the absence of reduction in excised eyes could be due to differences in the rates of hydrolysis of the Schiff bases formed with intermediates generated by a flash or constant illumination. 11-cis-retinal. No retinyl esters were produced. from Palczewski K. This result suggests that the transport of all-trans-retinol to the RPE. -retinol and retinyl esters. The retinas became detached during the incubation.61 did not occur. Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin. The rim protein of ROSs57 has recently been identified as a member of the ABC-transporter family (ABCR). a normal rate of visual pigment regeneration was observed in ABCR / animals. where it can be reduced by NADPH. with permission. 9A). However. Saari JC. Note the accumulation of all-trans-retinol. It is also possible that NADPH and all-trans-retinal are generated in separate compartments. Van Hooser JP. to the cytosolic side. Finally. excised mouse eyes because reduction occurs in the same experimental system with constant illumination (Fig. In addition. it is possible that the enzyme is directly regulated by unknown mechanisms. . reduction to occur. filled squares. Open circles. The lack of structural information about rod RDH considerably hinders further progress in this area. Note the accumulation of all-trans-retinal and the lack of regeneration.56 Perhaps flash illumination does not fully activate the pentose phosphate pathway in normal mouse eyes and not at all in excised mouse eyes.or all-trans-retinal. open squares. Modified. all-trans-retinal. 11-cis-retinol. Recent reports of the accumulation of condensation products of phosphatidylethanolamine and reti- nal in the ABCR knockout mouse suggests that these may be the actual substrates for the transporter. 9C). Blocked Transport of Retinoids in the Visual Cycle Constant illumination of mouse eyecups immersed in buffer revealed a third pattern of metabolism. 2 FIGURE 9.

proportional increase in the amount of the photolysis product all-trans-retinal. Perhaps the experimental situation in which we observed the There is much circumstantial evidence to support a role for IRBP in the diffusion of retinoids between RPE and photoreceptor cells: 1) IRBP possesses two or more high-affinity binding sites for retinoids. Recovery of 11-cis-retinal in the dark after a flash. (E) IRBP / mice. respectively. However. However. formation of a complex with arrestin.78. whereas other binding proteins with affinity for 11-cisretinal are ineffective.72.79 2) Diffusion of all-trans-retinol between populations of vesicles is relatively rapid and inhibited in the presence of IRBP. 10). cigar-shaped protein (axial ratio 7:1).66 –70 (2) all-trans-Retinol or 11-cis-retinal has been found to be associated with IRBP purified from darkor light-adapted retina. Vol. It is apparent that IRBP plays an important role in visual physiology because photoreceptor cells die in its absence. In summary. These animals were born and raised in the dark and subjected to flash bleaching followed by a recovery period in the dark. These results led to the prediction that reduction of all-trans-retinal and regeneration of visual pigments would be more rapid in a mouse without functional arrestin than in normal subjects. Small.6 and 1.81 Perhaps the results obtained here will direct re- .36. Recovery in the dark resulted in an increase in the amount of 11-cis-retinal and a corresponding decrease in the amount of all-trans-retinal.77 Other evidence argues against an active role for IRBP in retinoid transport: 1) IRBP is a large. an unlikely shape for a transport protein.74 IRBP may act as a buffer in the subretinal space.69. To address these possibilities.1% per minute. the metabolic inertness of all-trans-retinal in excised mouse eyes generated by a flash and the differences in the rates of regeneration after flash or steady bleaching point out how poorly we understand the processes by which the visual cycle is controlled. Perhaps abnormal patterns of retinoid metabolism would become apparent with other bleaching regimens. respectively.71 3) IRBP occurs precisely in the extracellular compartment separating photoreceptor and RPE cells. flashed ROS membranes results in a 40% inhibition of the rate of reduction of all-trans-retinal. The relatively modest difference in rates of recovery suggests that IRBP does not influence the rate of visual pigment regeneration. The normal turnover rates for visual pigments and retinoid composition in IRBP / mice are surprising in view of the wealth of evidence mentioned earlier. Direct addition of arrestin to washed. The recovery kinetics resulting from four experiments with IRBP / mice are shown in Figure 10.76 6) IRBP efficiently delivers 11-cisretinal to toad photoreceptors for rhodopsin regeneration. ANALYSIS OF THE FLOW KNOCKOUT MICE OF RETINOIDS Mice IN The Visual Cycle in Arrestin / Photoactivated Rho is quenched in a two-step process involving opsin phosphorylation by rhodopsin kinase62 and binding of arrestin to phosphorylated opsin. we examined the composition of retinoids during bleaching and regeneration in arrestin / mice.74 5) Studies with cultured RPE cells suggest that IRBP causes the release of 11-cisretinal.IOVS. along with the recovery curve obtained with wild-type mice.64 whereas we observed inhibition of RDH in whole ROS preparations (containing rhodopsin kinase and arrestin) when ATP and guanosine triphosphate were added. The distribution of visual cycle retinoids observed before and after a flash is very similar to that seen with normal mice (not shown). respectively). 41. No. and a reduction of the accessibility of all-trans-retinal to RDH.44 IRBP / mice were dark adapted and exposed to a flash that bleached approximately 35% of their visual pigment.8% per minute and 1% per minute. The flash produced an immediate 35% decrease in the amount of 11-cis-retinal and a concomitant.63 Two studies have suggested that arrestin affects the activity of RDH in ROS preparations.8% per minute. Retinoids were extracted from the posterior poles of the eyes and analyzed by high-performance liquid chromatography. Animals were killed before the flash (dark adapted) and at various intervals in the dark after the flash.80. limiting the concentration of free retinoid and preventing oxidative degradation.80 We examined the kinetics of recovery of 11-cis-retinal and of rhodopsin after a flash with IRBP / mice.73 4) Photoreceptor cells die in mice with a targeted disruption of the IRBP gene.1% per minute for a mixed population of wild-type mice. February 2000. Perhaps an examination of the kinetics of visual pigment regeneration in rhodopsin kinase knockout mice will resolve this issue.65 We attribute the ATP effect to phosphorylation of Rho* by rhodopsin kinase. ( ) arrestin / mice. Fig. An alternative possibility is that our interpretation of the in vitro results was incorrect and that phosphorylation of opsin alone was sufficient to alter the rate of release of all-trans-retinal.75. The Visual Cycle in IRBP / Mice FIGURE 10. transient increases of all-trans-retinol and retinyl ester were observed during the return to the dark-adapted state. The recovery of 11-cis-retinal in the dark was slower in arrestin / mice than in normal mice (0. 2 The Friedenwald Lecture 343 ATP effect poorly approximates the conditions found within the rod outer segment. compared with a value of 1. IRBP / mice regenerated their 11-cis-retinal at a rate of 0. (F) Normal mice. The failure to detect major changes in the rates of rhodopsin and 11-cis-retinal regeneration in arrestin / mice is surprising in view of the in vitro results mentioned earlier. we have examined only visual cycle retinoids after a modest bleaching of visual pigments. the rates of rhodopsin regeneration were similar (0. Continued study will lead to a solution to this problem and very likely to enhanced understanding of the control of the visual cycle. The overall pattern strongly resembled that observed with wild-type mice.

The former mechanism results in relatively mild elevations of the scotopic threshold. Thus. For instance.96 and in other retinal diseases have been discussed in detail elsewhere and will not be covered here. a feature perhaps disadvantageous in rapidly changing light conditions. At constant levels of illumination a steady state level of bleached visual pigment is generated. February 2000. the chromophore does not dissociate from the opsin to which it is covalently bound and the meta II species absorbs in the visible range of the spectrum. In contrast. result in very large elevations in scotopic threshold. Regeneration of visual pigments in invertebrates is achieved by photoisomerization of a complex of opsin and all-transretinal absorbing in the visible range. the questions emphasize our rudimentary understanding of the complex process of visual pigment regeneration. Vol. and the chromophore does not dissociate from the receptor. In contrast. Recently.98 –100 .83 Could it be involved in the export of 11-cis-retinal from RPE or in the import of all-trans-retinol from the interphotoreceptor matrix? Eicosanoids such as prostaglandins are secreted from cells through a transmembrane protein called the prostaglandin transporter. the bleaching rate and the regeneration rate are tied to the photon flux. However. 11). the cone visual cycle differs from the rod visual cycle in several quantitative aspects (e. the regeneration rate is independent of the photon flux. What are their functions? Peropsin has been localized to the apical plasma membrane of RPE.85 There is insufficient information to draw any conclusions at this time. Vertebrates have a more complex system in which the 11-cis configuration is regenerated in a light-independent reaction in an adjacent cell. photoequilibrium is established in the light. based on the inability of RPE65 / mice to make 11-cis-retinoids. Other proteins such as peropsin83 and RGR opsin84 have amino acid sequences related to that of opsin. However. faster turnover) and perhaps qualitative aspects as well. FIGURE 11. in which the first photon absorbed converts 11-cis-retinal to all-trans-retinal (Fig.90 In addition. and some are likely to be involved in the visual cycle. the vertebrate regeneration system is more complex.89. Why Is the Visual Cycle So Complex? Two general systems are used for regeneration of bleached visual pigments. active species produced by mutation. several mutations in the opsin gene result in constitutively active species that further desensitize the retina. in vertebrates there is a complicated system involving dissociation of the chromophore and regeneration of the 11-cis configuration in a neighboring nurse cell.86 Do Muller cells contribute to the regeneration of visual pigments? ¨ The answers to these and other fascinating questions await the results of further experimentation. Muller cells are known to contain ¨ two retinoid-binding proteins (CRALBP and CRBP)35–37 and to perform several transformations of retinoids in vitro. a 25% reduction in rhodopsin level increases the scotopic threshold by 1.88 However. For instance. Is Night Blindness Caused by Defects in the Visual Cycle? Night blindness is a common hallmark of many inherited retinal diseases and nutritional disorders. Thus. mutations in other genes that produce constitutive activation of phototransduction91 or that affect photointermediate quenching pathways92. several enzymes.5. For example.344 Saari IOVS. 41.93 result in delayed dark adaptation. because it involves the participation of two different cell types. as has been discussed. A second photon can then convert all-trans-retinal back to 11-cis-retinal. However. The night blindness reported in many retinitis pigmentosa cases results simply from the reduced photon catch associated with decreased amounts of rhodopsin in the affected retina.82 but its function is not understood at this time. What are the advantages and disadvantages of the two systems? The invertebrate system is inherently more simple and elegant in design. PERSPECTIVE Outstanding Problems The visual cycle shown in Figure 4 is consistent with the available evidence regarding the regeneration of rod visual pigments.97 This form of congenital stationary night blindness results in delayed dark adaptation and delayed regeneration of visual pigments. First. but the phenotype associated with knockout mice may provide an answer. which facilitates secretion and uptake of the hydrophobic signaling molecules.87. and intercellular flow of retinoids. missense mutations in the gene encoding 11-cis-retinol dehydrogenase have been found in patients with fundus albipunctatus.87. The roles of ABCR in Stargardt disease94 and of RPE65 in Leber’s congenital amaurosis95. the condition appears to result from two phenomena: decreased photon catch resulting from diminished rhodopsin content and/or the generation of a species that actively desensitizes the retina. However.. No. photobleaching or vitamin A deficiency. allow- Visual Cycle Defects and Inherited Retinal Conditions Mutations in several genes encoding presumptive visual cycle components have recently been implicated in several inherited retinal diseases. RPE65 must play a major role in retinoid metabolism. ing relatively rapid restoration of visual sensitivity even in the dark.g. which is a factor in determining the sensitivity of the visual system. 25% bleaching of rhodopsin increases the scotopic threshold by 104. this model must be regarded as a working hypothesis for several reasons. 2 search toward other potential roles for IRBP in visual physiology. However. Second. In general. several “orphan” retinoidbinding components have been described. Invertebrates rely on the establishment of photoequilibrium.

1961. Matthews HR. Bok D.57:335–340. Rhodopsin kinetics in the human eye. Lamb TD. 41. Ruiz A. During the past few years the number of published studies related to visual pigment regeneration has increased dramatically.106 Mutations in the gene encoding CRALBP have been associated with several forms of retinal degeneration. Rushton WAH.19:502–507. CRALBP36 and CRBP. Vision Res. Basic. 3. Huang J. and several other proteins have been shown to play very important roles in the visual cycle (RPE65 and ABCR). J Biol Chem. 8. 14. The binding protein reduces LRAT-mediated esterification of 11-cisretinol by 90% and modestly stimulates oxidation of 11-cis-retinol by 11-RDH. Further indication of the sophistication of the cycle has been shown by studies that point out the complexity of a seemingly simple step such as the reduction of all-trans-retinal. Ohba N. Lisman JE. 12.5.IOVS. J Biol Chem. Reuter R. Schoenlein RW. Lamb TD.103 Apo-CRALBP is required for retinol isomerase (isomerohydrolase) activity104.105 and for release of 11-cis-retinol from endogenous 11-cis-retinyl esters by 11-REH. 1879:235–342.104 The development of CRALBP knockout mice should resolve the question of the role of CRALBP in visual physiology. Cultured chick Muller cells take up exogenous all-trans-retinol and ¨ convert it to all-trans. is the site of intense retinoid metabolism related to the visual cycle.36 The RPE. 1998. 10. Maria Nawrot. Dark-adaptation and the regeneration of rhodopsin. 1995. Leibrock CS.17:1269 –1316. Dark adaptation: a re-examination. ¨ ¨ ed. Leipzig: Vogel. has been demonstrated to purify from neural retina as a mixture of complexes with 11-cis-retinol or 11-cis-retinal. Lim Y-H.271:20621–20630. Nordby K. 5.102 The all-trans. Twenty patients from seven families with features of retinitis punctata albescens and macular degeneration (Bothnia dystrophy) were homozygous for a missense mutation (R234W) in the CRALBP gene.and 9-cis-retinoids modulate many biologic processes110 but 11-cis-retinoids are only known to be involved in the visual process or in the absorption of light (pineal). Gilbert BA. Palczewski K.107 stressing the importance of the retinoid-binding site. Saari JC.37 are found in Muller and RPE cells in ¨ retinas from several species. Trends Neurosci. Handbuch der Physiologie. 1996. CONCLUSION This is an exciting time in visual cycle research. CRALBP affects the enzyme activity of four enzymes of the visual cycle in vitro. Fain GL. J Biol Chem. The latter retinoid has been found in the culture medium. Rando RR. Proc R Soc Lond B Biol Sci. 1977. Masseidvaag F.162:20 – 46. and Dan Possin. First. J Physiol. Wernstedt C. 4. John Crabb. Muller Cells and Cone Visual Pigment ¨ Regeneration The literature contains many intriguing suggestions that the visual cycle in cones differs from that in rods and that Muller ¨ cells may be involved. Rushton WAH. Lucille Bredberg. The retinal pigment epithelial-specific 11-cis-retinol dehydrogenase belongs to the family of short chain alcohol dehydrogenases. other investigators have noted that isolated amphibian cone cells resensitize with exogenous 11-cis-retinol.102 retinoids of the visual cycle. Clinical and Applied Aspects. In: Hermann L. 1993. in particular. J Biol Chem. Vol. 13.109 These results suggest that CRALBP plays an important role in visual physiology.11:539 –549.and 11-cis-retinyl palmitate and 11-cisretinol. Hellman U. 1998. Crouch RK. 7. Simon A. cone RDH. Mechanisms of opsin activation.115. 1991. Chemische vorgange in der netzhaut.273:21790 – 21799. J Physiol.86 Although no oxidation to 11-cis-retinal has been observed. We can anticipate the elaboration of further complexities and further medical relevance as investigators obtain the tools and molecular information necessary for precise dissection of individual and integrated steps of the visual cycle. The kinetics of cone visual pigments in man.112–114 Thus. The Ferrier Lecture. Palczewski K. Visual adaptation. Kris Palczewski. Greg Garwin. Some wellestablished players have been characterized at the molecular level (for instance. Exp Eye Res. The presence of CRALBP is particularly intriguing. Molecular and biochemical characterization of lecithin retinol acyltransferase. Breandan Kennedy. Recombinant CRALBP bearing this substitution (R150Q) did not bind 11-cis-retinal in vitro. Ann Milam. 1965. in retina. Acknowledgments The author thanks. it seems clear that some interesting retinoid . and the presence of 11-cis¨ retinoids strongly suggests that it is related to the visual cycle. References 1. 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