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We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11-cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.

Stargardt disease is a juvenile onset retinal degeneration, associated with elevated levels of lipofuscin and its bis-retinoid components, such as N-retinylidene-N-retinylethanolamine (A2E). However, the pathogenesis of Stargardt is still poorly understood and targeted treatments are not available. Utilizing high spatial and high mass resolution matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS), we determined alterations of lipid profiles specifically localized to the retinal pigment epithelium (RPE) in Abca4 -/- Stargardt model mice compared to their relevant background strain. Extensive analysis by LC-MS/MS in both positive and negative ion mode was required to accurately confirm the identity of one highly expressed lipid class, bis(monoacylgylercoro)phosphate (BMP) lipids, and to distinguish them from isobaric species. The same BMP lipids were also detected in the RPE of healthy human retina. BMP lipids have been previously associated with the endosomal/lysosomal storage diseases Niemann-Pick and neuronal ceroid lipofuscinosis and have been reported to regulate cholesterol levels in endosomes. These results suggest that perturbations in lipid metabolism associated with late endosomal/lysosomal dysfunction may play a role in the pathogenesis of Stargardt disease and is evidenced in human retinas.

In this issue of Chemistry & Biology, Srinivasan and colleagues describe their study of ligand-protein interactions in visual pigments. Comparing the more stable isomeric ligand 9-cis retinal to the physiologically occurring 11-cis retinal, they report differences in ligand specificity and opsin conformational stability not previously described for bleached rhodopsin., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE., Methods: Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections., Results: Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels., Conclusions: This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.

RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.

As cone photoreceptors mediate vision in bright light, their photopigments are bleached at a rapid rate and require substantial recycling of the chromophore 11-cis-retinal (RAL) for continued function. The retinal pigment epithelium (RPE) supplies 11-cis-RAL to both rod and cone photoreceptors; however, stringent demands imposed by the function of cones in bright light exceed the output from this source. Recent evidence has suggested that cones may be able to satisfy this demand through privileged access to an additional source of chromophore located within the inner retina. In this study, we demonstrate that the protein RPE65, previously identified in RPE as the isomerohydrolase of the RPE-retinal visual cycle, is found within cones of the rod-dominant mouse retina, and the level of RPE65 in cones is inversely related to the level in the RPE. The light sensitivity of cone ERGs of BALB/c mice, which had an undetectable level of cone RPE65, was enhanced by approximately threefold with administration of exogenous chromophore, indicating that the cones of these animals are chromophore deficient. This enhancement with chromophore administration was not observed in C57BL/6 mice, whose cones contain RPE65. These results demonstrate that RPE65 within cones may be essential for the efficient regeneration of cone photopigments under bright-light conditions.

Purpose: Lipofuscin, a fluorescent lysosomal pigment made of lipophilic molecules, is associated with age-related pathophysiological processes in the retinal pigment epithelium (RPE). The best-characterized components of lipofuscin are A2E and its oxides, but a direct spatial correlation with lipofuscin has not previously been possible., Methods: Lipofuscin fluorescence was mapped across the RPE of Abca4(-/-) and Sv129 (background strain control) mice. In the same tissues, they determined the spatial distribution of A2E and its oxides by using the high molecular specificity of matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS). The fluorescence and tandem mass spectra taken directly from the tissue were compared with those of synthetic A2E standard., Results: In 2-month-old mice, A2E was found in the center of the retinal pigment epithelial tissue; with age, A2E increased across the tissue. With high levels of A2E, there was a marked correlation between A2E and lipofuscin, but with low levels this correlation diminished. The distributions of the oxidized forms of A2E were also determined. The amount of oxidation on A2E remained constant over 6 months, implying that A2E does not become increasingly oxidized with age in this time frame., Conclusions: This report is the first description of the spatial imaging of a specific retinoid from fresh tissue and the first description of a direct correlation of A2E with lipofuscin. The molecule-specific imaging of lipofuscin components from the RPE suggests wide applicability to other small molecules and pharmaceuticals for the molecular characterization and treatment of age-related macular degeneration.

Purpose: The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal., Methods: Experiments were performed in isolated retinas and single living rods derived from 129/sv wild-type mice and Abca4-, arrestin-, and rhodopsin kinase-deficient mice and in genetically modified mice containing unpalmitylated rhodopsin. Formation of all-trans retinol was measured by imaging its fluorescence and by HPLC of retina extracts. The release of all-trans retinal from photoactivated rhodopsin was measured in purified rod outer segment membranes according to the increase in tryptophan fluorescence. All experiments were performed at 37°C., Results: The kinetics of all-trans retinol formation in the different types of genetically modified mice are in reasonable agreement with those in wild-type animals. The kinetics of all-trans retinol formation in 129/sv mice are similar to those in C57BL/6, although the latter are known to regenerate rhodopsin much more slowly. The release of all-trans retinal from rhodopsin in purified membranes is significantly faster than the formation of all-trans retinol in intact cells and is independent of the presence of the palmitate groups., Conclusions: The regeneration of rhodopsin and the recycling of its chromophore are not strongly coupled. Neither the activities of Abca4, rhodopsin kinase, and arrestin, nor the palmitylation of rhodopsin affects the formation of all-trans retinol.

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.

Purpose: To determine the effect of light/dark cycles on the cones of 11-cis retinal-treated RPE65/rhodopsin double knockout (Rpe65(-/-)Rho(-/-)) mice. Studies have shown that cones degenerate in chromophore-deficient mouse models for Leber Congenital Amaurosis (LCA), but exogenous supplementation of the native 11-cis retinal chromophore can inhibit this degeneration, suggesting that 11-cis retinal could be used as a therapeutic agent for preserving functional cones in patients with LCA. However, these treated mice were maintained in the dark., Methods: 11-cis Retinal was introduced into Rpe65(-/-)Rho(-/-) mice at postnatal day 10 as a single subcutaneous injection mixed with a basement membrane matrix. The mice were maintained in either normal light/dark cycles or constant dark conditions. Fluorescence microscopy was used to assess retinal morphology. Cone cell survival was determined by counting cone opsin-containing cells on flat-mounted P30 retinas. Cross-sections of P21 mouse retina were used to assess cone cell integrity by visualizing opsin localization. Cone function was determined by electroretinography (ERG)., Results: Previous studies have shown that 11-cis retinal-treated mice lacking RPE65 and raised in constant dark have higher cone photoreceptor cell number, improved cone opsin localization, and enhanced cone ERG signals when compared with untreated mice. However, in this study the authors show that 11-cis retinal-treated Rpe65(-/-)Rho(-/-) mice raised in cyclic light did not show the improvements seen with the dark-reared mice., Conclusions: Thus, 11-cis retinal by itself, as well as other agents that form photosensitive pigments, will not be good therapeutic candidates for preserving cones in LCA.

Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones use the photosensitive molecule 11-cis-retinal to detect light, and in constant illumination, a continuous supply of 11-cis-retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis-retinal to cones by using 11-cis-retinol generated in Müller cells. In the cycle, 11-cis-retinol is transported from Müller cells to cone inner segments, where it is oxidized to 11-cis-retinal. This oxidation step is only performed in cones, thus rendering the cycle cone-specific. Interphotoreceptor retinoid-binding protein (IRBP) is a retinoid-binding protein in the subretinal space that binds 11-cis-retinol endogenously. Cones in Irbp(-/-) mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis-retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis-retinol to cones by preserving the isomeric state of 11-cis-retinol in light. With electrophysiology, we show that the cone-like photoreceptors of Nrl(-/-) mice use the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolated Nrl(-/-)Rpe65(-/-) retinas, we show that IRBP delivers 11-cis-retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis-retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis-retinol to cones and can facilitate cone function in the presence of light.

Purpose: Delivery of hydrophobic compounds to the retina/RPE has been challenging. The purpose of this study was to develop an effective method for the sustained delivery of retinoids to rod and cone photoreceptors of young mice lacking a normal supply of 11-cis retinal., Methods: Solubilized basement membrane matrix (Matrigel; BD Biosciences, San Jose, CA) loaded with 9-cis retinal was administered subcutaneously into Rpe65(-/-) mouse pups for assessment of delivery to rods and cones and to Rpe65(-/-)Rho(-/-) mouse pups for assessment of delivery to cones. Intraperitoneal injections of 9-cis retinal were used for comparison. Cone density and opsin localization were evaluated with immunohistochemistry. Cone opsin protein levels were assayed with immunoblots, and cone function was analyzed by electroretinography (ERG) recordings. Retinoid content was determined by high-performance liquid chromatography analysis of retinal extracts. Pigment levels were quantified in homogenized retinas by absorption spectroscopy before and after light exposure., Results: Single administration of Matrigel loaded with 9-cis retinal to Rpe65(-/-) mice increased cone densities in all analyzed regions of the retina compared with mice treated using intraperitoneal delivery. Cone opsin levels increased to near wild-type levels. Similar treatment in Rpe65(-/-)Rho(-/-) mice increased b-wave ERG amplitudes significantly, indicating the maintenance of cone function. Matrigel was shown to continuously release 9-cis retinal for periods up to 1 week., Conclusions: As a method for sustained drug delivery, subcutaneous administration using Matrigel proved more efficacious than intraperitoneal injection for in vivo delivery of retinoids to cone photoreceptors. These experiments are the first to show a sustained delivery of retinoids in mice and suggest a strategy for potential clinical therapeutic development.

Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization.

S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.

Purpose: Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined., Methods: Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined., Results: A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner., Conclusions: The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site.

Purpose: The final step in the retinoid visual cycle is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs) that oxidize 11-cis-retinol (11cROL) to 11-cis-retinaldehyde (11cRAL). Genetic studies in mice indicate that the full repertoire of 11-cis-RDH enzymes remains to be identified. This study was conducted to characterize the 11-cis-RDH activity of RDH10 in vitro and specifically to determine whether RDH10 can functionally and physically interact with visual cycle proteins., Methods: Human RDH10 was expressed in COS1 cells to measure its 11-cis-RDH activity in the presence or absence of purified recombinant cellular retinaldehyde-binding protein (CRALBP). The RPE visual cycle was reconstituted in HEK-293A cells by co-expressing RDH10, CRALBP, RPE-specific 65-kDa protein (RPE65) and lecithin retinol acyltransferase (LRAT). The cells were subsequently treated with all-trans-retinol (atROL), and retinoid profiles were quantified by HPLC. Immunocytochemical and co-immunoprecipitation analyses were performed to determine whether RDH10 physically interacts with other visual cycle proteins., Results: RDH10 oxidized 11cROL to generate 11cRAL in vitro in the presence of CRALBP. RDH10 can use both NAD(+) and NADP(+) as cofactors for 11-cis-RDH activity, although NAD(+) cofactor confers more robust activity. In a cell culture model co-expressing RDH10 with RPE65, LRAT and CRALBP, the visual chromophore 11cRAL was generated from atROL. Immunohistochemistry showed that RDH10 co-localizes with RPE65 and CRALBP in vivo in primary bovine RPE cells. Immunoprecipitation analysis demonstrated that RDH10 physically interacts with CRALBP and RPE65., Conclusions: RDH10 may function in the RPE retinoid visual cycle as an 11-cis-RDH, and thereby partially compensate for the loss of RDH5 function in patients with fundus albipunctatus.

Purpose: RPE65, a major retinal pigment epithelium protein, is essential in generating 11-cis retinal, the chromophore for all opsins. Without chromophore, cone opsins are mislocalized and cones degenerate rapidly (e.g., Rpe65(-/-) mouse). Function, survival, and correct targeting of opsins is increased in Rpe65(-/-) cones on supplying 11-cis retinal. Here, we determine the consequences of 11-cis retinal withdrawal and supplementation on cone development in the all-cone Nrl(-/-) retina., Methods: Rpe65(-/-) Nrl(-/-), Nrl(-/-), and wild-type mice were examined. Cone structure was analyzed by using TUNEL assay, electron microscopy, and cone-specific antibodies. Cone function was assessed with light-adapted single-flash ERGs., Results: Rpe65(-/-)Nrl(-/-) mice had an increased number of TUNEL-positive photoreceptors during programmed cell death compared with Nrl(-/-) mice, in addition to accelerated age-related degeneration. Cone function in Rpe65(-/-)Nrl(-/-) mice was minimal, and opsins were mislocalized. Treatment with 11-cis retinal restored cone function, promoted outer segment formation, and enabled opsin trafficking to outer segments. Eliminating Rpe65 prevented rosette formation in Nrl(-/-) retinas; supplementation of Rpe65(-/-)Nrl(-/-) mice with 11-cis retinal resulted in their reoccurrence., Conclusions: Taken together, function and opsin trafficking in Nrl(-/-) and wild-type cones are comparable, confirming and extending our findings that cone maturation and outer segment development are dependent on the presence of chromophore. The data on age-related cone death in Rpe65(-/-)Nrl(-/-) mice and the reintroduction of rosettes after 11-cis retinal injections confirm that outer segments, which for steric reasons appear to introduce rosettes in an all-cone retina, are essential for cell survival. These results are important for understanding and treating chromophore-related cone dystrophies.

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore-opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

11-cis-retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5'-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.

11-cis-retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis-retinal through a series of enzymatic steps known as the visual cycle. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp(-/-) mice have a normal visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. We analyzed the cones in Irbp(-/-) mice to determine whether IRBP has a cone-specific visual cycle function. Cone electroretinogram (ERG) responses were reduced in Irbp(-/-) mice, but similar responses from Irbp(-/-) mice at all ages suggest that degeneration does not underlie cone dysfunction. Furthermore, cone densities and opsin levels in Irbp(-/-) mice were similar to C57BL/6 (wild-type) mice, and both cone opsins were properly localized to the cone outer segments. To test for retinoid deficiency in Irbp(-/-) mice, ERGs were analyzed before and after intraperitoneal injections of 9-cis-retinal. Treatment with 9-cis-retinal produced a significant recovery of the cone response in Irbp(-/-) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.

Purpose: The Rpe65-/- mouse, used as a model for Leber congenital amaurosis, has slow rod degeneration and rapid cone loss, presumably because of the mistrafficking of cone opsins. This animal does not generate 11-cis retinal, and both cone loss and rod response are restored by 11-cis retinal administration. Similarly, the Lrat-/- mouse does not produce 11-cis retinal. The authors sought to determine whether the same effects on rod and cone opsins in the Rpe65-/- mouse are also present in the Lrat-/- mouse, thereby establishing that these changes can be attributed to the lack of 11-cis retinal rather than to some unknown function of RPE65., Methods: Rod and cone opsins were localized by immunohistochemical methods. Functional opsin levels were determined by regeneration with 11-cis retinal. Isorhodopsin levels were determined from pigment extraction. Opsin phosphorylation was determined by mass spectrometry., Results: Rods in both models degenerated slowly. Regenerable rod opsin levels were similar over the 6-month time course investigated, rod opsin was phosphorylated at a low level (approximately 10%), and minimal 9-cis retinal was generated by a nonphotic process, giving a trace light response. In both models, S-opsin and M/L-opsin failed to traffic to the cone outer segments appropriately, and rapid cone degeneration occurred. Cone opsin mistrafficking in both models was arrested on 11-cis retinal administration., Conclusions: These data show that the Lrat-/- and Rpe65-/- mice are comparable models for studies of Leber congenital amaurosis and that the destructive cone opsin mistrafficking is caused by the lack of 11-cis retinal.

Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat(-/-) and Rpe65(-/-) mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat(-/-) and Rpe65(-/-) retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T alpha-subunit, cone phosphodiesterase 6alpha' (PDE6alpha'), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat(-/-) and Rpe65(-/-) cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T alpha and cone PDE6alpha' were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65(-/-)Rho(-/-) double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.

Dark noise, light-induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11-cis-3,4-dehydroretinal (A2) chromophore, and regenerating with the 11-cis-retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue-shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light-like noise in the rod. Complete recordings were obtained in five cells (21 degrees C). Based on the wavelength of maximum absorbance, lambda max,A1 = 502 nm and lambda max,A2 = 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was approximately 0.74 : 0.26 in the native state and approximately 0.09 : 0.91 in the final state. In the native (A2) state, the single-quantum response (SQR) had an amplitude of 0.41 +/- 0.03 pA and an integration time of 3.16 +/- 0.15 s (mean +/- s.e.m.). The low-frequency branch of the dark noise power spectrum was consistent with discrete SQR-like events occurring at a rate of 0.238 +/- 0.026 rod(-1) s(-1). The corresponding values in the final state were 0.57 +/- 0.07 pA (SQR amplitude), 3.47 +/- 0.26 s (SQR integration time), and 0.030 +/- 0.006 rod(-1) s(-1) (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8-fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final state. The power spectrum of the remaining 'continuous' noise component did not differ between the two states. The smaller and faster SQR in the native (A2) state is consistent with the idea that the rod behaves as if light-adapted by dark events that occur at a rate of nearly one per integration time. Both the decreased level of dark noise and the increased SQR amplitude must significantly improve the reliability of photon detection in dim light in the presence of the A1 chromophore compared to the native (A2) state in salamander rods.

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Palmitylation is a widespread modification in G-protein-coupled receptors and often a dynamic process. In rhodopsins, palmitylation is static on C322/C323. Red/green (M/LWS) cone opsins have no cysteines at corresponding positions and no palmitylation. Blue (SWS2) cone opsins have a single corresponding cysteine and mass spectrometric analysis showed partial palmitylation of salamander SWS2 cone opsin. Ultraviolet (SWS1) cone opsins have one corresponding cysteine, but only unpalmitylated opsin was observed for mouse and salamander. The results show that the static palmitylation found on rhodopsin is not found on cone opsins and suggest the possibility of an unidentified role for opsin palmitylation in cones.

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Purpose: Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens., Methods: AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections., Results: The proteomics approach used to identify affinity-purified proteins revealed the lens-specific intermediate filament proteins filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that filensin and AQP0 were present in the same membrane compartments., Conclusions: These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization.

The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

The choroid in the eye provides vascular support for the retinal pigment epithelium (RPE) and the photoreceptors. Vascular endothelial growth factor (VEGF) derived from the RPE has been implicated in the physiological regulation of the choroidal vasculature, and overexpression of VEGF in this epithelium has been considered an important factor in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Here, we demonstrate that RPE-derived VEGF is essential for choriocapillaris development. Conditional inactivation of VEGF expression in the RPE (in VEGFrpe-/- mice) results in the absence of choriocapillaris, occurrence of microphthalmia, and the loss of visual function. Severe abnormalities of RPE cells are already observed when VEGF expression in the RPE is only reduced (in VEGFrpe+/- mice), despite the formation of choroidal vessels at these VEGF levels. Finally, using Hif1arpe-/- mice we demonstrate that these roles of VEGF are not dependent on hypoxia-inducible factor-1alpha-mediated transcriptional regulation of VEGF expression in the RPE. Thus, hypoxia-inducible factor-1alpha-independent expression of VEGF is essential for choroid development.

Rpe65 knockout mice (Rpe65-/-) are unable to synthesize the visual pigment chromophore 11-cis retinal; however, if these animals are reared in complete darkness, the rod photoreceptors accumulate a small amount of 9-cis retinal and its corresponding visual pigment isorhodopsin. Suction-electrode recording of single rods from dark-reared Rpe65-/- mice showed that the rods were about 400 times less sensitive than wild-type control rods and that the maximum responses were much smaller in amplitude. Spectral sensitivity measurements indicated that Rpe65-/- rod responses were generated by isorhodopsin rather than rhodopsin. Sensitivity and pigment concentration were compared in the same mice by measuring light responses from rods of one eye and pigment concentration from the retina of the other eye. Retinas had 11-35% of the normal pigment level, but the rods were of the order of 20-30 times less sensitive than could be accounted for by the loss in quantum catch. This extra desensitization must be caused by opsin-dependent activation of the visual cascade, which leads to a state equivalent to light adaptation in the dark-adapted rod. By comparing the sensitivity of dark-reared Rpe65-/- rods to that produced in normal rods by background light, we estimate that Rpe65-/- opsin is of the order of 2.5x10(-5) as efficient in activating transduction as photoactivated rhodopsin (Rh*) in WT mice. Dark-reared Rpe65-/- rods are less desensitized than rods from cyclic light-reared Rpe65-/- mice, have about 50% more photocurrent and degenerate at a slower rate. Retinas sectioned after 9 months in darkness show a larger number of photoreceptor nuclei in dark-reared animals than in cyclic light-reared animals, though both have fewer nuclei than in cyclic light-reared wild-type retinas. Both also have shorter outer segments and a lower free-Ca2+ concentration. These experiments provide the first quantitative measurement of opsin activation in physiologically responding mammalian rods.

Purpose: In retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65(-/-) mouse, a model for Leber's congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the retinal pigment epithelium is disrupted, and 11-cis retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-cis retinal., Methods: Rpe65(-/-)::Rho(-/-) mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-cis retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR., Results: At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11-cis retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally., Conclusions: The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-cis retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies.

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.

Purpose: RPE65 is essential for the generation of 11-cis retinal. Rod photoreceptors in the RPE65-knockout (Rpe65(-/-)) mouse are known to degenerate slowly with age. This study was designed to examine cone photoreceptors and the expression of cone-specific genes in the Rpe65(-/-) mouse., Methods: Gene expression changes were identified by microarray and confirmed by real-time RT-PCR. Cone photoreceptors were stained by peanut agglutinin (PNA) lectin in the flatmounted retina. The 9- or 11-cis retinal was supplied by intraperitoneal injections., Results: The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin alpha-subunit mRNA decreased at both 4 and 8 weeks of age, whereas again the rod transducin alpha-subunit did not show any significant change. Rpe65(-/-) mice showed significant cone loss in both the central and ventral retina between 2 and 3 weeks of age. Administration of 9- or 11-cis retinal to Rpe65(-/-) mice 2 weeks of age increased cone density by twofold in these areas., Conclusions: In the Rpe65(-/-) mouse, the expression of cone-specific genes is downregulated and is accompanied by cone degeneration at early ages. Early administration of 9- or 11-cis retinal can partially prevent cone loss, suggesting that the absence of 11-cis chromophore may be responsible for the early cone degeneration.

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.

Purpose: Membrane protein 20 (MP20) is the second most abundant integral membrane protein in the lens, yet little is known about its function and post-translational modifications. The purpose of this work was the determination of the primary protein structure of MP20 and the types and sites of in vivo modifications., Methods: Bovine MP20 was isolated by anion exchange chromatography or SDS-PAGE followed by digestion with cyanogen bromide (CNBr) or trypsin. The total membrane protein fraction was also digested with trypsin in solution. The CNBr and trypsin peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry., Results: Using these approaches to integral membrane protein analysis, complete sequence coverage of MP20 was obtained. The in vivo sites of phosphorylation were identified as Ser-170 and Thr-171, the latter being previously unreported. The most abundant form of MP20 is monophosphorylated at Ser-170, whereas unphosphorylated and diphosphorylated forms were in lower abundance. In addition, two sites of a rare type of glycosylation (C-mannosylation) were identified at tryptophan residues 43 and 61., Conclusions: The functional significance of phosphorylated MP20, the predominant form, remains unknown. Glycosylation of tryptophan residues represents a new lens protein modification that can explain galectin-3 interaction and suggests a topology for MP20 in which these peptides are located in an extracellular domain.

Purpose: To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells., Methods: The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry. Expression of RDH10 in rMC-1, a cell line derived from rat Muller cells, was determined by RT-PCR and Western blot analysis. All-trans retinol dehydrogenase activity assays were performed using lysates from rMC-1 cells. The generation of all-trans retinal from tritiated all-trans retinol was analyzed by HPLC., Results: RDH10, retinal G protein-coupled receptor (RGR), and RPE65 all had higher expression levels in the eyecups of BALB/c than in C57Bl/6 mice. In addition to the RPE, RDH10 was also detected at lower levels in the retina and liver. Immunohistochemistry showed that RDH10 was localized in Muller cells in retinal sections. RDH10 was detected in rMC-1 cells, at both the RNA and protein levels. The rat RDH10 cDNA containing the full-length coding region was cloned from rMC-1 cells. The rat RDH10 cDNA encodes a protein of 341 amino acids and shares 99% sequence identity with human, bovine, and mouse RDH10 at the amino acid level. In rMC-1 cells, all-trans retinol dehydrogenase activity was detected in the microsomal fraction. NADP was shown to be the preferred cofactor, which is identical with the cofactor preference of the recombinant RDH10., Conclusions: RDH10 was expressed in retinal Muller cells, in addition to the RPE. RDH10 generates all-trans retinal, which is the substrate for the photoisomerase RGR in Muller cells.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

In HEK293S cells expressing opsin, rhodopsin regenerates on addition of all-trans retinol. This study was to determine if key proteins in the retinal pigment epithelium (RPE) are present in these cells. Cellular retinoid binding protein, cellular retinoic-acid binding protein, RPE65, caveolin-1-alpha- and -beta-isoforms, interphotoreceptor retinoid binding protein, and 11-cis retinol dehydrogenase, but not lecithin:retinol acyltransferase (LRAT), were identified by Western blot analysis. LRAT transcripts were found by RT-PCR and Southern blot analysis. Small interference RNA specific to LRAT reduced ester formation, confirming that the enzyme is present. Therefore, HEK293S cells contain the functional components of the retinoid cycle found in the RPE.

The chromophore of visual pigments is 11-cis-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (Rpe65-/-) mouse, where synthesis of 11-cis-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for cis-retinoid generation in the absence of RPE65. Cyclic-light-reared, 2-month-old Rpe65-/- mice were placed in complete darkness. No exogenous retinoids were administered. After 4 weeks, enhanced a- and b-wave amplitudes were obtained, increasing >10-fold for the a-wave and >3-fold for the b-wave as compared with cyclic-light-reared Rpe65-/- mice. Visual-pigment levels increased to approximately 10 pmol per retina, compared with no measurable pigment for cyclic-light-reared Rpe65-/- mice. The lambdamax of the isolated pigment was 487 nm, characteristic for isorhodopsin. Retinoid extractions confirmed the presence of 9-cis-retinal and the absence of 11-cis-retinal. Once the Rpe65-/- mice were returned to cyclic light, within 48 h the electroretinogram function returned to levels found in Rpe65-/- mice maintained in cyclic light. This dark-mediated pathway is also operational in older animals, because 13-month-old Rpe65-/- mice kept in prolonged darkness (12 weeks) had increased isorhodopsin levels and electroretinogram a- and b-wave amplitudes. These studies demonstrate that a pathway exists in the eye for the generation of 9-cis-retinal that is independent of RPE65 and light.

Purpose: To first assess the distribution of posttranslationally truncated products of aquaporin 0 (AQP0) in dissected sections of a normal human lens and to determine the effect of backbone cleavage on the water permeability of AQP0., Methods: A 27-year-old lens was concentrically dissected into six sections. Membrane protein was isolated from each section and cleaved with cyanogen bromide, and the peptides were separated and analyzed by reverse-phase (RP)-HPLC-mass spectrometry (MS). The sites of posttranslational AQP0 C-terminal truncation were determined by mass spectrometry. Truncated forms of AQP0 were expressed in a Xenopus laevis oocyte system, and the effect of truncation on AQP0 water permeability was assessed in an oocyte osmotic swelling assay., Results: The extent of truncation at many sites within the C terminus increased with fiber cell age, and the effects of truncations after residues 234, 238, and 243 on AQP0 water permeability were examined. Truncation after residue 243 resulted in an approximate 15% decrease in permeability compared with the full-length protein, AQP0 1-263. However, rather than a direct effect on water transport, analysis of surface protein expression indicated that the decrease in permeability was a result of less efficient protein trafficking to the oocyte surface and that the permeabilities of full-length and 1-243 AQP0 were indistinguishable. Further, C-terminal truncation of AQP0 to 1-234 and 1-238, completely impaired trafficking into the plasma membrane, precluding the measurement of permeability., Conclusions: These data provide evidence that loss of 20 amino acids from the C terminus may not directly affect the ability of AQP0 to transport water.