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Yiannis Koutalos - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein removes all-trans-retinol and Retinal from rod outer segments, preventing lipofuscin precursor formation.
Journal of Biological Chemistry, 2017Co-Authors: Chunhe Chen, Leopold Adler, Patrice Goletz, Debra A. Thompson, Federico Gonzalez-fernandez, Yiannis KoutalosAbstract:: Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of Retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-Retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-Retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-Retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the Retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-Retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 μm-1 IRBP also removed all-trans-Retinal, but with much less efficacy, indicating that the reduction of Retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-Retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-Retinal.
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Formation of all-trans retinol after visual pigment bleaching in mouse photoreceptors.
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Chunhe Chen, Lorie R. Blakeley, Yiannis KoutalosAbstract:Vision is initiated by the absorption of light by the visual pigment present in the outer segments of the rod and cone photoreceptor cells in the retina. In both cell types, the first step in the detection of light is the photoisomerization of the retinyl chromophore of the visual pigment from 11-cis to all-trans.1,2 This isomerization of the chromophore bleaches the pigment, necessitating its regeneration with fresh 11-cis Retinal. The production of 11-cis Retinal includes the recycling of the all-trans chromophore of the bleached pigment through a series of reactions called the visual cycle.3–5 These reactions begin in the outer segment with the release of all-trans Retinal from the photoactivated pigment and its reduction to all-trans retinol by retinol dehydrogenase in a reaction using NADPH.6 The all-trans retinol is then transferred from outer segments to the adjacent Retinal pigment epithelial cells,7 where it is esterified to form retinyl ester.8,9 The ester is converted to 11-cis retinol,10–13 which is then oxidized to 11-cis Retinal.14 Studies of cone-dominant ground squirrel and chicken retinas have provided evidence of the presence of an additional visual cycle used by cones.15–17 Continuous vision depends on the regeneration of the visual pigment and defects in the processing of the chromophore through the reactions of the visual cycle are responsible for a wide range of visual defects.3,18 Extensive studies using whole eyes have argued for the presence of two slow steps in the operation of the mouse visual cycle: the formation of all-trans retinol and the isomerization of all-trans retinyl ester.19–21 Although the formation of all-trans retinol has been characterized in detail in amphibian photoreceptors,22–24 these results cannot provide a quantitative insight into the operation of the mouse visual cycle because of critical species differences, such as body temperature. Therefore, we undertook the measurement of the kinetics of all-trans retinol formation in mouse photoreceptors by HPLC of retinoid extracts and fluorescence imaging. In our results, all-trans retinol formation was not the slowest step in the mouse visual cycle, which supports the notion that the isomerization of retinyl esters is the slowest step.20,21 Throughout the text, unqualified Retinal and retinol refer to the all-trans isomers.
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Formation Of All-Trans Retinol In Mouse Rod Photoreceptors
Biophysical Journal, 2009Co-Authors: Lorie R. Blakeley, Chunhe Chen, Yiannis KoutalosAbstract:Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans Retinal and its reduction to all-trans retinol. All-trans retinol is then transported to the neighboring Retinal pigment epithelial cells where it is used to remake 11-cis Retinal. The reduction of all-trans Retinal to all-trans retinol is catalyzed by retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans retinol formation proceeds with a rate of ∼0.06 min−1, which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis Retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans Retinal, as formation of all-trans retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans retinol formation on the level of metabolic activity.
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interphotoreceptor retinoid binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments
Biochemistry, 2007Co-Authors: Qingqing Wu, Lorie R. Blakeley, Barbara N Wiggert, Rosalie K. Crouch, Carter M Cornwall, Yiannis KoutalosAbstract:The vertebrate cells responsible for vision are the rod and cone photoreceptors of the retina that convert incoming light to an electrical signal. This conversion takes place in the photoreceptor outer segments, which are full of membrane disks containing the visual pigment, and, in a physiologically important arrangement, are enveloped by the Retinal pigment epithelium (RPE). The visual pigment is composed of a chromophore, 11-cis Retinal, attached to an integral membrane protein, opsin. The detection of light begins with the absorption of incoming photons by the visual pigment. An absorbed photon isomerizes the chromophore moiety from 11-cis to all-trans bringing about a conformational change that initiates a cascade of reactions culminating in membrane potential change. The recovery of the cell from light involves the deactivation of the intermediates activated by light, and the reestablishment of membrane potential (1, 2). However, the isomerized chromophore, all-trans Retinal, remains. For vision to be possible, it is essential that the visual pigment regenerate: that is, the all-trans Retinal has to be removed, and fresh 11-cis Retinal has to be provided to combine with opsin and reform the visual pigment. The reactions regenerating the pigment are known as the Visual Cycle (3–5). In the case of the rod photoreceptors, the cells responsible for vision at low light intensities, the Visual Cycle encompasses reactions in the outer segment and in the adjacent RPE cells. The first step in the Cycle is the release of all-trans Retinal from photoactivated rhodopsin after hydrolysis of the Schiff base bond linking the chromophore to opsin. All-trans Retinal is then reduced to all-trans retinol in a reaction catalyzed by retinol dehydrogenase (6, 7), requiring NADPH, and taking place on the cytoplasmic side of the outer segment disks. It is possible that all-trans Retinal ends up inside the disks, bound via a Schiff base to phosphatidylethanolamine, in which case the phosphatidylethanolamine-all-trans-Retinal compound is transported to the cytoplasmic side by the ABCR transporter (8–10) making all-trans Retinal available for reduction. The all-trans retinol formed in the rod outer segment is transported to the RPE, in a process that can be facilitated by the interphotoreceptor retinoid binding protein (IRBP; (11–13)). In the RPE, retinol is converted by lecithin-retinol acyltransferase to retinyl ester (LRAT; (14, 15)), which is isomerized to 11-cis retinol (16, 17) by the RPE65 protein (18–21). 11-cis retinol is then oxidized to 11-cis Retinal, and transported back to photoreceptor outer segments where it associates with opsin to reform rhodopsin. Previous work (22–25) has established that all-trans retinol can be monitored in the outer segments of living isolated rod and cone photoreceptors from its distinctive fluorescence. Here, we have taken advantage of two properties of frog rod photoreceptors to actually measure the amounts of all-trans retinol produced with quantitative biochemical and physiological methods. One, in contrast to larval salamander photoreceptors that contain two types of chromophore (based on vitamins A1 and A2) and in widely varying ratios (24), frog rods contain a single, vitamin A1-based chromophore. Two, the metabolic supply of NADPH is not limiting for the formation of all-trans retinol in the case of frog rods (23), allowing a simplified analysis and direct comparisons between biochemical and physiological data. This has further allowed us to properly characterize the removal of all-trans retinol by different lipophilic carriers. In experiments with purified rod outer segment membranes, whole retinas, and living isolated rods, we have separately measured the different steps involved in all-trans retinol formation and removal. On the basis of these measurements, we have calculated the predicted kinetics of retinol formation and removal in rod outer segments, and found that they are in close agreement with those measured directly from isolated rod photoreceptors. We also characterized the effect of different concentrations of lipophilic carriers on the removal of all-trans retinol, and established that for the physiological concentrations of carriers the rate of all-trans retinol removal is determined by the IRBP concentration. Our results strongly support a specific interaction mediating the removal of all-trans retinol by IRBP, perhaps through a receptor on the rod outer segment plasma membrane. Throughout the text, when not specifically designated as the 11-cis isomers, unqualified Retinal and retinol refer to the all-trans forms.
Alejandro Catala - One of the best experts on this subject based on the ideXlab platform.
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The effect of alpha tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation of rod outer segments.
Molecular and cellular biochemistry, 1999Co-Authors: Mario Guajardo, A Terrasa, Alejandro CatalaAbstract:The effect of a tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation induced by ascorbate-Fe2+ of rod outer segment membranes isolated from bovine retina was examined. The inhibition of light emission (maximal induced chemiluminescence) by alpha tocopherol, all-trans retinol and retinyl palmitate was concentration dependent. All trans retinol showed a substantial degree of inhibition against ascorbate-Fe2+ induced lipid peroxidation in rod outer segment membranes that was 10 times higher than the observed in the presence of either at tocopherol or retinyl palmitate. Inhibition of lipid peroxidation of rod outer segment membranes by alpha tocopherol and retinyl palmitate was almost linear for up to 0,5 micromol vitamin/mg membrane protein, whereas all-trans retinol showed linearity up to 0,1 micromol vitamin/mg membrane protein. Incubation of rod outer segments with increasing amounts of low molecular weight cytosolic proteins carrying I-[14C] linoleic acid, [3H] retinyl palmitate or [3H] all-trans retinol during the lipid peroxidation process produced a net transfer of ligand from soluble protein to membranes. Linoleic acid was 4 times more effectively transferred to rod outer segment membranes than all-trans retinol or retinyl palmitate. Incubation of rod outer segments with delipidated low molecular weight cytosolic proteins produced inhibition of lipid peroxidation. The inhibitory effect was increased when the soluble Retinal protein fraction containing alpha tocopherol was used. These data provide strong support for the role of all-trans retinol as the major Retinal antioxidant and open the way for many fruitful studies on the interaction and precise roles of low molecular weight cytosolic Retinal proteins involved in the binding of antioxidant hydrophobic compounds with rod outer segments.
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The effect of α tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation of rod outer segments
Molecular and Cellular Biochemistry, 1999Co-Authors: Mario Guajardo, A Terrasa, Alejandro CatalaAbstract:The effect of α tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation induced by ascorbate-Fe2+ of rod outer segment membranes isolated from bovine retina was examined. The inhibition of light emission (maximal induced chemiluminescence) by α tocopherol, all-trans retinol and retinyl palmitate was concentration dependent. All trans retinol showed a substantial degree of inhibition against ascorbate-Fe2+ induced lipid peroxidation in rod outer segment membranes that was 10 times higher than the observed in the presence of either α tocopherol or retinyl palmitate. Inhibition of lipid peroxidation of rod outer segment membranes by α tocopherol and retinyl palmitate was almost linear for up to 0,5 μmol vitamin/mg membrane protein, whereas all-trans retinol showed linearity up to 0,1 μmol vitamin/mg membrane protein. Incubation of rod outer segments with increasing amounts of low molecular weight cytosolic proteins carrying 1-[14C] linoleic acid, [3H] retinyl palmitate or [3H] all-trans retinol during the lipid peroxidation process produced a net transfer of ligand from soluble protein to membranes. Linoleic acid was 4 times more effectively transferred to rod outer segment membranes than all-trans retinol or retinyl palmitate. Incubation of rod outer segments with delipidated low molecular weight cytosolic proteins produced inhibition of lipid peroxidation. The inhibitory effect was increased when the soluble Retinal protein fraction containing a tocopherol was used. These data provide strong support for the role of all-trans retinol as the major Retinal antioxidant and open the way for many fruitful studies on the interaction and precise roles of low molecular weight cytosolic Retinal proteins involved in the binding of antioxidant hydrophobic compounds with rod outer segments.
George Wolf - One of the best experts on this subject based on the ideXlab platform.
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The visual cycle of the cone photoreceptors of the retina.
Nutrition Reviews, 2004Co-Authors: George WolfAbstract:The photoreceptors of the eye's retina consist of rods and cones; rods serve vision in dim light, whereas cones serve high-resolution color vision in daylight. The first event in vision is the light-initiated isomerization of 11-cis-Retinal, which is attached to rod or cone opsin, to all-trans-Retinal. The regeneration of 11-cis-Retinal comprises the well-known visual cycle in rods. By using cone-dominant retinas from chickens and ground squirrels, a visual cycle has been discovered in cones that differs radically from that in rods, mainly in the mechanism of isomerization of all-trans-retinol to 11-cis-retinol, and the latter's oxidation to 11-cis-Retinal.
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A newly discovered visual cycle necessary for vision during continuous illumination.
Nutrition Reviews, 2002Co-Authors: George WolfAbstract:Rhodopsin, the light-sensitive visual pigment of the retina, is activated through photoisomerization of its prosthetic group, 11-cis-Retinal, to all-trans-Retinal. A protein found in the Retinal pigment epithelium named Retinal G protein- coupled receptor (RGR) reacts to light in an opposite but parallel way: its prosthetic group, all-trans-Retinal, is photoisomerized to 11-cis-Retinal. The latter is reduced to 11-cis-retinol by a cis-retinol dehydrogenase that co-purifies with the RGR. The resulting 11-cis-retinol feeds into the visual cycle to be oxidized to 11-cis-Retinal, thus replenishing the 11-cis-Retinal of the rhodopsin. During continuous intense illumination, RGR can supplement the 11-cis-Retinal required to regenerate rhodopsin.
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a case of human vitamin a deficiency caused by an inherited defect in retinol binding protein without clinical symptoms except night blindness
Nutrition Reviews, 1999Co-Authors: George WolfAbstract:Two German siblings were found to suffer from night blindness and mild Retinal dystrophy but no other clinical symptoms of vitamin A deficiency. Even though they had no detectable plasma Retinal-binding protein (RBP) and their plasma retinol was exceedingly low, they showed normal physiologic functions and growth. Their RBP gene was found to harbor two point mutations. Their post-prandial plasma levels of retinyl esters were normal, and it is likely that they derived their tissue retinol from retinyl esters.
Barbara Wiggert - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination
Experimental Eye Research, 2005Co-Authors: Nasser M. Qtaishat, Barbara Wiggert, David R. PepperbergAbstract:Abstract All- trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the Retinal pigment epithelium (RPE), where it is enzymatically converted to 11- cis Retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all- trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all- trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad ( Bufo marinus ) eyes were bleached and then incubated in darkness for defined periods (5–180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed ‘IRBP I’) at defined concentrations (2–90 μ m ). Retinoids present in the retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with ≥10 μ m IRBP I showed a pronounced release of all- trans retinol with increasing period of incubation. As determined with 25 μ m IRBP I, the increase of all- trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all- trans Retinal and all- trans retinol contained in the retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90 μ m bovine serum albumin. However, incubation with ‘IRBP II’, a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all- trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all- trans retinol from the rods during operation of the visual cycle.
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Retinoid processing in Retinal pigment epithelium of toad (Bufo marinus).
The Journal of biological chemistry, 1994Co-Authors: Ting Ing L Okajima, Gerald J. Chader, Barbara Wiggert, David R. PepperbergAbstract:Abstract The formation of 11-cis-[3H]Retinal in the Retinal pigment epithelium (RPE) and its release to extracellular medium containing interphotoreceptor retinoid-binding protein (IRBP) were studied in the RPE eyecup of the toad (Bufo marinus). The RPE was labeled with all-trans-[3H]retinol during an initial 1-h incubation. In phase 2 of the incubation (0-2 h), the extracellular medium contained initially ligand-free IRBP (0-26 microM). Retinoids subsequently extracted from the extracellular medium and RPE were analyzed by high performance liquid chromatography and scintillation counting. IRBP increased both the molar amount and specific radioactivity of 11-cis-Retinal released by the RPE during phase 2. The molar amount of 11-cis-Retinal in the RPE was small relative to that of Retinal released with high IRBP. With 21 microM IRBP and phase 2 incubations of > or = 10 min, the specific radioactivity of released 11-cis-Retinal exceeded that of all-trans-retinyl ester in the RPE. The specific radioactivity of 11-cis-retinyl ester was less than that of all-trans-ester, independent of IRBP concentration. The results indicate that IRBP promotes the formation (from all-trans-precursor) as well as the release of 11-cis-Retinal and suggest the preferred utilization of recently incorporated and esterified all-trans-retinol in 11-cis-Retinal synthesis in a "last in/first out" manner.
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increase in retinyl palmitate concentration in eyes and livers and the concentration of interphotoreceptor retinoid binding protein in eyes of vitiligo mutant mice
Biochemical Journal, 1994Co-Authors: Sylvia B. Smith, Todd Duncan, Geetha Kutty, R K Kutty, Barbara WiggertAbstract:Retinyl esters play an important role in the visual cycle because they are involved in regeneration of 11-cis-Retinal for use in rhodopsin formation. In the present study, retinyl ester concentrations were significantly elevated in eyes and livers of mice homozygous for the vitiligo mutation (mivit/mivit). Vitiligo mice demonstrate a slowly progressing Retinal degeneration characterized by gradual loss of photoreceptor cells and rhodopsin as well as uneven pigmentation of the Retinal pigment epithelium (RPE). Analysis of retinoids by h.p.l.c. indicated that the retinyl palmitate level was increased fivefold in eyes of affected mice at 10 weeks postnatally and was threefold higher at 22 weeks of age. Accumulation of retinyl palmitate occurred in the RPE rather than the neural retina. Furthermore, the concentration of all-trans-retinol was elevated in the RPE of vitiligo mice. Levels of interphotoreceptor retinoid binding protein (IRBP) were increased in vitiligo mice between ages 4 and 14 weeks, but returned to normal by 16 weeks. Increased IRBP levels were not due to increased protein synthesis because IRBP mRNA levels did not differ significantly between control and affected animals. To examine possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Mean hepatic total vitamin A levels in affected mice were approximately 1.7 times higher than controls. Analysis of esterified and non-esterified retinoids in liver showed that the concentration of retinyl palmitate was elevated. Plasma retinol levels were normal. This study provides the first evidence of altered systemic retinoid metabolism in vitiligo mice, which occurs, significantly, under normal dietary conditions.
Chunhe Chen - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein removes all-trans-retinol and Retinal from rod outer segments, preventing lipofuscin precursor formation.
Journal of Biological Chemistry, 2017Co-Authors: Chunhe Chen, Leopold Adler, Patrice Goletz, Debra A. Thompson, Federico Gonzalez-fernandez, Yiannis KoutalosAbstract:: Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of Retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-Retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-Retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-Retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the Retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-Retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 μm-1 IRBP also removed all-trans-Retinal, but with much less efficacy, indicating that the reduction of Retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-Retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-Retinal.
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Formation of all-trans retinol after visual pigment bleaching in mouse photoreceptors.
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Chunhe Chen, Lorie R. Blakeley, Yiannis KoutalosAbstract:Vision is initiated by the absorption of light by the visual pigment present in the outer segments of the rod and cone photoreceptor cells in the retina. In both cell types, the first step in the detection of light is the photoisomerization of the retinyl chromophore of the visual pigment from 11-cis to all-trans.1,2 This isomerization of the chromophore bleaches the pigment, necessitating its regeneration with fresh 11-cis Retinal. The production of 11-cis Retinal includes the recycling of the all-trans chromophore of the bleached pigment through a series of reactions called the visual cycle.3–5 These reactions begin in the outer segment with the release of all-trans Retinal from the photoactivated pigment and its reduction to all-trans retinol by retinol dehydrogenase in a reaction using NADPH.6 The all-trans retinol is then transferred from outer segments to the adjacent Retinal pigment epithelial cells,7 where it is esterified to form retinyl ester.8,9 The ester is converted to 11-cis retinol,10–13 which is then oxidized to 11-cis Retinal.14 Studies of cone-dominant ground squirrel and chicken retinas have provided evidence of the presence of an additional visual cycle used by cones.15–17 Continuous vision depends on the regeneration of the visual pigment and defects in the processing of the chromophore through the reactions of the visual cycle are responsible for a wide range of visual defects.3,18 Extensive studies using whole eyes have argued for the presence of two slow steps in the operation of the mouse visual cycle: the formation of all-trans retinol and the isomerization of all-trans retinyl ester.19–21 Although the formation of all-trans retinol has been characterized in detail in amphibian photoreceptors,22–24 these results cannot provide a quantitative insight into the operation of the mouse visual cycle because of critical species differences, such as body temperature. Therefore, we undertook the measurement of the kinetics of all-trans retinol formation in mouse photoreceptors by HPLC of retinoid extracts and fluorescence imaging. In our results, all-trans retinol formation was not the slowest step in the mouse visual cycle, which supports the notion that the isomerization of retinyl esters is the slowest step.20,21 Throughout the text, unqualified Retinal and retinol refer to the all-trans isomers.
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Formation Of All-Trans Retinol In Mouse Rod Photoreceptors
Biophysical Journal, 2009Co-Authors: Lorie R. Blakeley, Chunhe Chen, Yiannis KoutalosAbstract:Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans Retinal and its reduction to all-trans retinol. All-trans retinol is then transported to the neighboring Retinal pigment epithelial cells where it is used to remake 11-cis Retinal. The reduction of all-trans Retinal to all-trans retinol is catalyzed by retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans retinol formation proceeds with a rate of ∼0.06 min−1, which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis Retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans Retinal, as formation of all-trans retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans retinol formation on the level of metabolic activity.
