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LECTURE

LECTURE

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

P hototransduction 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 of neurotransmitter by photoreceptor cells. The visual cycle re- stores 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 published 24 ; however, molecular infor- mation 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, 57 and conversion of rhodopsin to photorhodopsin requires only 200 fsec. 8 How- ever, 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 rods 5 and cones 9 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.

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 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 Ophthalmol- ogy, 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

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 explana- tion for this log–linear relationship is not well understood, and the 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 desensitizing intermediate(s) remains a matter of active investigation, 10,1216 it is clear that visual cycle reactions are important in determin- ing 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. 1,1719 The reader is referred to Marmor and Martin 20 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 ani- mal, as will be discussed later. Fifty years later, Wald 21 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 of- fering more resolution verified the fundamental observa- tion. 23,24 Bernstein et al. 25 and Rando 26 provided a molecular explanation 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 transcellular migration of the retinoids during bleaching and regeneration is all the more remarkable, considering the anatomy of the jour- ney (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 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-

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338 Saari IOVS, February 2000, Vol. 41, No. 2 F IGURE 1. The chromophore of visual

FIGURE 1. The chromophore of visual pigments alternates between 11-cis and all-trans configurations. 11-cis-Retinal or a closely related derivative is the chromophore for all known visual pigments. Its bind- ing to opsin freezes the receptor in a stable, inactive form ensuring a low rate of thermal isomerization in the dark. Photoisomerization converts 11-cis-retinal to all-trans-retinal, activates the receptor, and initiates phototransduction. The 11-cis configuration is produced by a sequence of reactions that are thermally driven (i.e., can occur in the dark).

ments has not been completely determined. 27 The enzymes are depicted as part of one continuous internal membrane com- partment for simplicity. Absorption of light by rhodopsin in the disc membrane converts 11-cis-retinal to all-trans-retinal and generates the active photoproduct, metarhodopsin II (Rho*). The G-protein–stimulating activity of Rho* is quenched by phosphorylation and by the binding of arrestin (not shown in the figure). The Schiff base linking all-trans-retinal and opsin is hydrolyzed to release free all-trans-retinal. Recent reports sug- gest 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, 28 perhaps as an adduct with phosphatidyl ethanolamine. 29 all- trans-Retinol dehydrogenase (RDH) catalyzes the reduction of all-trans-retinal to all-trans-retinol by reduced nicotinamide adenine dinucleotide phosphate (NADPH). all-trans-Retinol leaves the photoreceptor cell, traverses the interphotoreceptor matrix space (the subretinal space) where it encounters inter- photoreceptor retinoid-binding protein (IRBP), and enters the RPE where it is esterified by lecithin-retinol acyltransferase (LRAT). all-trans-Retinyl ester is converted to 11-cis-retinol and free fatty acid by an isomerohydrolase. 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). 11- cis-Retinal diffuses into the photoreceptor cell where it asso- ciates with opsin to regenerate the visual pigment. all-trans-

with opsin to regenerate the visual pigment. all- trans - F IGURE 3. Electron photomicrograph of

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

Retinol can be taken up from the blood and esterified by LRAT in RPE cells (not shown). 11-cis-Retinyl esters can be hydro- lyzed and used for visual pigment regeneration by 11-cis-retinyl ester hydrolase (11-REH). Cellular retinaldehyde-binding pro- tein (CRALBP), a water soluble retinoid-binding protein, is shown (Fig. 4) with its high-affinity ligands, 11-cis-retinal or 11-cis-retinol. Cellular retinol-binding protein (CRBP) is shown associated with all-trans-retinol, although the protein also binds 11-cis-retinol in vitro. IRBP is present in the photorecep- tor matrix. Its role in retinoid transport is uncertain (discussion to follow). Opsin is shown on the lower side of the disc only for reasons of symmetry. Evidence leading to this working hypothesis of the rod visual cycle has been presented in several recent re- views. 14,26,3034 Each of the enzymatic reactions shown in the RPE or rod outer segments (ROSs) has been demonstrated in RPE microsome or ROS preparations, respectively. The water- soluble retinoid-binding proteins (CRALBP, CRBP, and IRBP) have been localized to the compartments in which they are shown by immunocytochemistry. 3537 CRBP and CRALBP are also found in Mu¨ller cells. Mammalian rods cannot use exoge- nous 11-cis-retinol for regeneration of visual pigments. 38,39

-retinol for regeneration of visual pigments. 3 8 , 3 9 F IGURE 2. The general

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

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2000, Vol. 41, No. 2 The Friedenwald Lecture 339 F IGURE 4. A schematic illustrating a

FIGURE 4. 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 abbreviations used are: ABCR, ATP-binding cassette, retina; CRBP, cellular retinol-binding protein; CRALBP, cellular retinaldehyde-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.

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

ANALYSIS OF THE FLOW OF RETINOIDS IN THE MOUSE VISUAL CYCLE

Dowling 22 determined the flow of retinoids in and out of the RPE 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 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 Mu¨ller cells play essential roles in the visual cycle? We addressed these questions by analyzing the composition of visual cycle retinoids during re- covery from a flash or from steady illumination. We chose mice as the experimental animals because of the increasing availabil- ity of animals with targeted disruption of genes encoding putative visual cycle components.

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 approxi- mately 40% of their visual pigment. Retinoids were extracted and analyzed before bleaching (dark adapted) and during the recovery period in the dark. The high-performance liquid chro- matography traces from an experiment using flash illumination are shown in Figure 5, and the results are summarized in Figure 6. 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-retinal were rapid, including intercellular transport, esterification, isomerization, oxidation, and conjugation with opsin. 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. These results refer to the experimental situation in which approximately 40% of the mouse visual pigment was bleached. It is possible that intermediates other than all-trans-retinal would appear if the visual system were challenged with larger

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340 Saari IOVS, February 2000, Vol. 41, No. 2 F IGURE 5. HPLC [high-performance liquid chromatography]

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-retinal oxime; 7, all-trans-retinol; 8, anti all-trans-retinal oxime. Reprinted, with permission, from Van Hooser JP, Garwin GG, Saari JC. Analysis of the visual cycle in transgenic mice. Methods Enzymol. 2000, Academic Press. In press.

fractional bleaches. At the limit of 100% bleach, Dowling 22 and Zimmerman 24 observed in rats the progressive appearance and disappearance of all the visual cycle intermediates that could be resolved. In addition, Perlman et al. 41 observed that the time constant 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 and transport processes involved, it is possible that another step could become rate limiting as flux through the pathway is increased. Flash illumination of animals and humans has been used with great success in a number of experimental situations. However, it could be argued that physiological conditions are more closely approximated with steady illumination. Thus, 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. Dark-adapted mice were subjected to illumination from two 60-W fluores- cent bulbs (50 foot-candles). Retinoids were extracted and analyzed at various times after onset of the lights and during the recovery period in the dark. Again, all-trans-retinal was the only retinoid that accumulated in substantial amounts during bleaching and recovery. Figure 7 depicts the amount of all- trans-retinal accumulated during steady state bleaching and

trans -retinal accumulated during steady state bleaching and F IGURE 6. Composition of polar retinoids in

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; ( ) 11-cis-retinol. Arrow: a flash. Retinyl esters did not change significantly with this amount of bleaching (not shown). Modified, with permission, from Palczewski K, Van Hooser JP, Garwin GG, Saari JC. Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotore- ceptor retinoid-binding protein or arrestin. Biochemistry. 1999;39:

12012–12019. American Chemical Society.

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

same amount of visual pigment was bleached in each case. The F IGURE 7. Recovery from

FIGURE 7. Recovery from steady state bleaching. The bar at the top of the figure illustrates the lighting regimen used (filled bar, dark; open bar, light). In the dark (0 on the abscissa) 5% of the retinals are in the all-trans configuration. The onset of light increased the amount of all-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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2000, Vol. 41, No. 2 The Friedenwald Lecture 341 F IGURE 8. Recovery from flash and

FIGURE 8. Recovery from flash and steady state bleaching. Mice were subjected to either a flash or to constant light that bleached 40% of their visual pigment, and placed in the dark. At intervals retinoids were extracted and analyzed by high-performance liquid chromatography. Gray bars, recovery after a flash; open bars, recovery after steady illumination. 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-retinal limits regeneration of visual pigment in mice. Vision Res. 1998;38:

1325–1333. Elsevier Science.

results, shown in Figure 8, illustrate that the recovery from steady illumination is approximately 3.5 times more rapid (half- life [t 1/2 ], 5 minutes with constant illumination; t 1/2 , 17 min- utes with flash illumination). Similar results were reported in a study of phosphorylation of rhodopsin. 42 What can account for the difference in the rates of decay of 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 from a difference in rate of reduction of all-trans-retinal. It is possi- ble that the reaction is subject to control at the level of the enzyme, all-trans-retinol dehydrogenase. Alternatively, it is possible that flash illumination fails to fully activate the pentose phosphate pathway, which is largely responsible for genera- tion of NADPH for retinal reduction. Modulation of the rate of all-trans-retinal reduction will be discussed further in subse- quent sections.

The Block in the Visual Cycle in Excised Mouse Eyes

Dissected frog eyes, which contain preformed 11-cis-retinyl ester in their RPE, 23 regenerate their visual pigment after bleaching. 1,1720 However, mouse eyes, with little if any pre- formed 11-cis-retinyl ester, 40 do not regenerate their visual pigment once removed from the animal. 43,44 What step in the visual cycle is blocked in excised mouse eyes? The visual cycle, as currently postulated, does not include any obvious step that requires metabolic energy except for the ABCR transporter reaction 29 (see Fig. 4). Formation of the 11-cis configuration is an endergonic process, 45,46 but the energy required for forma-

tion of the hindered 11-cis configuration has been postulated to come from the hydrolysis of the ester bond of all-trans-retinyl ester. 47 Thus, it would not be anticipated that depriving an eye of 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 mouse eyes. 44 Eyes were removed from dark-adapted mice and sub- jected to constant illumination (Fig. 9A). 11-cis-Retinal steadily disappeared during the illumination period. all-trans-Retinal transiently appeared, and ultimately all-trans-retinol and all- trans-retinyl ester accumulated. No 11-cis-retinoids were formed. Based on current ideas about the visual cycle (Fig. 4), this suggests that the isomerization reaction is not functional in excised mouse eyes. This block in the cycle could result from several causes. Perhaps the isomerization reaction requires metabolic energy (e.g., ATP), contrary to what has been sug- gested, and the ATP stores in these excised eyes are rapidly depleted. In support of this are reports that the electrical responses of rabbit eyes rapidly decay in the absence of oxygen and glucose 48 and that excised mouse eyes do not regenerate their visual pigment unless they are perfused with oxygen and glucose. 43 However, it is also possible that changes in cellular pH, 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 of excised 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- olized 44 (Fig. 9B). No reduction was evident, nor did any other metabolites appear. This striking result indicates that the reac- tion 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. Why does reduction of all-trans-retinal not occur, and is this related to the differences we have observed in the rates of regenera- tion after flash or constant illumination? Several possibilities will be discussed in the subsequent sections. Reduction of all-trans-retinal requires a source of reducing power, and numerous studies have demonstrated that in the visual cycle this source must be NADPH. 49,50 The pentose phosphate 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 for superoxide production, in adipose tissue where NADPH is used for fatty acid biosynthesis, and in dividing cells that require ribose-P for DNA synthesis. Activation of glucose 6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate pathway, 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. 52,53 Studies of glucose metabolism emphasized that ROSs were capable of producing NADPH through the pentose phosphate pathway in amounts sufficient to account for reduc- tion of all-trans-retinal. 54 However, in dark-adapted rabbit and monkey retina, the ratio of NADPH to nicotinamide adenine dinucleotide phosphate (NADP) was reported to be 0.3, 55 indicating that the pathway would have to be activated for

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342 Saari IOVS, February 2000, Vol. 41, No. 2 F IGURE 9. Effect of bleaching on

FIGURE 9. Effect of bleaching on dark-adapted excised mouse eyes or eyecups. (A) Excised mouse eyes were subjected to constant illumination. Note the progressive loss of 11-cis-retinal and the accumulation of 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 in buffer were exposed to constant illumination. Note the accumulation of all-trans-retinol. Retinoids were extracted and analyzed at intervals as shown for all three treatments. Open circles, 11-cis-retinal; filled circles, 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. 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. Biochemistry. 1999;39:12012–12019. American Chemical Society.

reduction to occur. In vivo [ 13 C] nuclear magnetic resonance studies of rabbit retina have demonstrated an activation of the pentose phosphate pathway in constant light. 56 Perhaps flash illumination does not fully activate the pentose phosphate pathway in normal mouse eyes and not at all in excised mouse eyes. The Schiff base linking all-trans-retinal and opsin must be hydrolyzed before the retinoid can be reduced to all-trans- retinol. Differences in the rates of reduction in vivo or the absence of reduction in excised eyes could be due to differ- ences in the rates of hydrolysis of the Schiff bases formed with intermediates generated by a flash or constant illumination. Flash illumination is known to result in phosphorylation pri- marily of ser334 of opsin, whereas constant illumination results in phosphorylation primarily of ser338. 42 Complex formation of these differently phosphorylated opsins with arrestin could further affect the rates of hydrolysis of the Schiff base, although our studies of arrestin knockout mice indicate otherwise 44 (see following discussion). It is also possible that NADPH and all-trans-retinal are generated in separate compartments, and an active process must occur to unite them. The rim protein of ROSs 57 has recently been identified as a member of the ABC-transporter family (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 for the transporter. The investigators propose that the function of the protein is to pump all-trans-retinal from inside the disc, where it is generated, to the cytosolic side, where it can be reduced by NADPH. 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. 29 Thus, depletion of ATP in excised mouse eyes could prevent the two substrates of the reaction from uniting. However, this explanation seems unlikely to account for the absence of reduction of all-trans- retinal that we have observed in flashed, excised mouse eyes because reduction occurs in the same experimental system with constant illumination (Fig. 9A). In addition, a normal rate of visual pigment regeneration was observed in ABCR / animals, indicating that the visual cycle is not dependent on the transporter. Finally, it is possible that the enzyme is directly regulated by unknown mechanisms. The lack of structural information about rod RDH considerably hinders further progress in this area.

Blocked Transport of Retinoids in the Visual Cycle

Constant illumination of mouse eyecups immersed in buffer revealed a third pattern of metabolism. 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. 9C). No retinyl esters were produced. This result suggests that the transport of all-trans-retinol to the RPE, where LRAT is localized 60,61 did not occur. Examination of the eyecups at the end of the experiment provides the explanation 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 in detecting abnormal visual cycle function.

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2000, Vol. 41, No. 2 The Friedenwald Lecture 343 F IGURE 10. Recovery of 11- cis

FIGURE 10. Recovery of 11-cis-retinal in the dark after a flash. (F) Normal mice; (E) IRBP / mice; ( ) arrestin / mice.

In summary, 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. Continued study will lead to a solu- tion to this problem and very likely to enhanced understanding of the control of the visual cycle.

ANALYSIS OF THE FLOW OF RETINOIDS IN KNOCKOUT MICE

The Visual Cycle in Arrestin / Mice

Photoactivated Rho is quenched in a two-step process involv- ing opsin phosphorylation by rhodopsin kinase 62 and binding of 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 ROS membranes results in a 40% inhibition of the rate of reduction of all-trans-retinal, 64 whereas we observed inhibition of RDH in whole ROS preparations (containing rhodopsin kinase and arrestin) when ATP and guanosine triphosphate were added. 65 We attribute the ATP effect to phosphorylation of Rho* by rhodopsin kinase, formation of a complex with arrestin, and a reduction of the accessibility of all-trans-retinal to RDH. 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. To address these possibilities, we examined the composi- tion of retinoids during bleaching and regeneration in arres- tin / mice. These animals were born and raised in the dark and subjected to flash bleaching followed by a recovery period in the dark. The distribution of visual cycle retinoids observed before and after a flash is very similar to that seen with normal mice (not shown). The recovery of 11-cis-retinal in the dark was slower in arrestin / mice than in normal mice (0.6 and 1.1% per minute, respectively; Fig. 10). However, the rates of rhodopsin 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 arrestin / mice is surprising 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 within the rod outer segment. 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. Perhaps an examination of the kinetics of visual pigment regeneration in rhodopsin kinase knockout mice will resolve this issue.

The Visual Cycle in IRBP / Mice

There is much circumstantial evidence to support a role for IRBP in the diffusion of retinoids between RPE and photore- ceptor cells: 1) IRBP possesses two or more high-affinity bind- ing sites for retinoids. 6670 (2) all-trans-Retinol or 11-cis-retinal has been found to be associated with IRBP purified from dark- or light-adapted retina, respectively. 71 3) IRBP occurs precisely in the extracellular compartment separating photoreceptor and RPE cells. 36,72,73 4) Photoreceptor cells die in mice with a targeted 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 in retinoid transport: 1) IRBP is a large, cigar-shaped protein (axial ratio 7:1), an unlikely shape for a transport protein. 69,78,79 2) Diffusion of all-trans-retinol between populations of vesicles is relatively rapid and inhibited in the presence of IRBP. 80 We examined the kinetics of recovery of 11-cis-retinal and of rhodopsin after a flash with IRBP / mice. 44 IRBP / mice were dark adapted and exposed to a flash that bleached approximately 35% of their visual pigment. Animals were killed before the flash (dark adapted) and at various intervals in the dark after the flash. Retinoids were extracted from the poste- rior poles of the eyes and analyzed by high-performance liquid chromatography. 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 product all-trans-retinal. 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. Small, transient increases of all-trans-retinol and retinyl ester were observed during the return to the dark-adapted state. The overall pattern strongly resembled that observed with wild-type mice. The recovery kinetics resulting from four experiments with IRBP / mice are shown in Figure 10, along with the recovery curve ob- tained with wild-type mice. IRBP / mice regenerated their 11-cis-retinal at a rate of 0.8% per minute, compared with a value of 1.1% per minute for a mixed population of wild-type mice. 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. However, we have ex- amined only visual cycle retinoids after a modest bleaching of visual 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 visual physiology because photoreceptor cells die in its absence. 74 IRBP may act as a buffer in the subretinal space, limiting the concentration of free retinoid and preventing oxidative degra- dation. 80,81 Perhaps the results obtained here will direct re-

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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 the available evidence regarding the regeneration of rod visual pigments. However, this model must be regarded as a working hypothesis for several reasons. First, several “orphan” retinoid- binding components have been described, and some are likely to be involved in the visual cycle. For instance, RPE65 must play a major role in retinoid metabolism, based on the inability of RPE65 / mice to make 11-cis-retinoids, 82 but its function is not understood at this time. Other proteins such as perop- sin 83 and RGR opsin 84 have amino acid sequences related to that of opsin. What are their functions? Peropsin has been localized to the apical plasma membrane of RPE. 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, 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 phenotype associated with knockout mice may provide an answer. Sec- ond, the cone visual cycle differs from the rod visual cycle in several quantitative aspects (e.g., faster turnover) and perhaps qualitative aspects as well. Mu¨ller cells are known to contain two retinoid-binding proteins (CRALBP and CRBP) 3537 and to perform several transformations of retinoids in vitro. 86 Do Mu¨ller cells contribute to the regeneration of visual pigments? The answers to these and other fascinating questions await the results of further experimentation. However, the questions emphasize 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 bleached visual pigments. Invertebrates rely on the establishment of photoequilibrium, in which the first photon absorbed converts 11-cis-retinal to all-trans-retinal (Fig. 11). A second photon can then convert all-trans-retinal back to 11-cis-retinal. At constant levels of illumination a steady state level of bleached visual pigment is generated, which is a factor in determining the sensitivity of the visual system, as has been discussed. Thus, 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, in vertebrates there is a complicated system involving dissociation of the chromophore and regeneration of the 11-cis configuration in a neighboring nurse cell. What are the advantages and disadvantages of the two systems? The invertebrate system is inherently more sim- ple and elegant in design. However, the bleaching rate and the regeneration rate are tied to the photon flux, a feature perhaps disadvantageous 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-

regeneration rate is independent of the photon flux, allow- F IGURE 11. Regeneration of visual pigments

FIGURE 11. Regeneration of visual pigments in invertebrates is achieved 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 the receptor. Vertebrates have a more complex system in which the 11-cis configuration is regenerated in a light-independent reaction in an adjacent cell.

ing relatively rapid restoration of visual sensitivity even in the dark.

Is Night Blindness Caused by Defects in the Visual Cycle?

Night blindness is a common hallmark of many inherited reti- nal diseases and nutritional disorders. In general, 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. The former mechanism results in relatively mild elevations of the scotopic threshold. For instance, a 25% reduction in rhodopsin level increases the scotopic threshold by 1.5. However, active species produced by mutation, photobleaching or vitamin A deficiency, result in very large elevations in scotopic threshold. For example, 25% bleaching of rhodopsin increases the scotopic threshold by 10 4 . The night blindness reported in many retinitis pigmentosa cases results simply from the re- duced photon catch associated with decreased amounts of rhodopsin in the affected retina. 87,88 However, several muta- tions in the opsin gene result in constitutively active species that further desensitize the retina. 89,87,90 In addition, mutations in other genes that produce constitutive activation of photo- transduction 91 or that affect photointermediate quenching pathways 92,93 result in delayed dark adaptation.

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. The roles of ABCR in Stargardt disease 94 and of RPE65 in Leber’s congenital amaurosis 95,96 and in other retinal diseases have been discussed in detail elsewhere and will not be covered here. Recently, missense mutations in the gene encoding 11-cis-retinol dehydrogenase have been found in pa- tients with fundus albipunctatus. 97 This form of congenital stationary night blindness results in delayed dark adaptation and delayed regeneration of visual pigments. 98100

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CRALBP has several distinguishing characteristics that strongly suggest its participation in the visual process. First, the protein has a high-affinity binding site for either 11-cis-retinal (K d , 10 nM) or 11-cis-retinol (K d , 60 nm). 101 Second, CRABLP purifies from RPE saturated with 11-cis-retinal and from neural retinal saturated with 11-cis-retinal and 11-cis-retinol, 102 retinoids of the visual cycle. Third, in retina, CRALBP is found in Mu¨ller and RPE cells. 36 The RPE, of course, is the site of intense retinoid metab- olism related to the visual cycle. Fourth, CRALBP affects the enzyme activity of four enzymes of the visual cycle in vitro. The binding protein reduces LRAT-mediated esterification of 11-cis- retinol by 90% and modestly stimulates oxidation of 11-cis-retinol by 11-RDH. 103 Apo-CRALBP is required for retinol isomerase (isomerohydrolase) activity 104,105 and for release of 11-cis-retinol from endogenous 11-cis-retinyl esters by 11-REH. 104 The develop- ment of CRALBP knockout mice should resolve the question of the role of CRALBP in visual physiology. 106 Mutations in the gene encoding CRALBP have been asso- ciated with several forms of retinal degeneration. Affected siblings 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) did not bind 11-cis-retinal in vitro, 107 stressing the importance of the retinoid-binding site. Four mutations in the gene encoding CRALBP were found in three unrelated patients with reces- sively inherited retinitis punctata albescens 108. Twenty pa- tients 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. 109 These results suggest that CRALBP plays an important role in visual physiology.

Mu¨ller 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 Mu¨ller cells may be involved. Two retinoid-binding proteins, CRALBP 36 and CRBP, 5,37 are found in Mu¨ller and RPE cells in retinas from several species. The presence of CRALBP is par- ticularly intriguing, because the binding protein, as mentioned above, has been demonstrated to purify from neural retina as a mixture of complexes with 11-cis-retinol or 11-cis-retinal. 102 The all-trans- and 9-cis-retinoids modulate many biologic pro- cesses 110 but 11-cis-retinoids are only known to be involved in the visual process or in the absorption of light (pineal). Cul- tured chick Mu¨ller cells take up exogenous all-trans-retinol and convert 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 amphibian cone cells resensitize with exogenous 11-cis-retinol, whereas rods require 11-cis-retinal. 111 Goldstein noted that the ampli- tude of the cone but not the rod early receptor potential reaches a steady state in illuminated, isolated frog retina and recovers with a t 1/2 of 5 to 6 minutes in the dark. 112114 Thus, it appears that cones can regenerate their visual pigment in frog retina in the absence of RPE. However, the interpretation of these provocative studies hinges on the validity of the demonstration 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 Mu¨ller 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 past few years the number of published studies related to visual pigment regeneration has increased dramatically. Some well- established players have been characterized at the molecular level (for instance, cone RDH, 11-RDH, and LRAT), and several other proteins have been shown to play very important roles in the visual cycle (RPE65 and ABCR). 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. We can anticipate the elabo- ration of further complexities and further medical relevance as investigators obtain the tools and molecular information nec- essary for precise dissection of individual and integrated steps of the visual cycle.

Acknowledgments

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

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