Isomerization and oxidation of vitamin a in cone-dominant retinas: a novel pathway for visual-pigment regeneration in daylight

The first step toward light perception is 11-cis to all-trans photoisomerization of the retinaldehyde chromophore in a rod or cone opsin-pigment molecule. Light sensitivity of the opsin pigment is restored through a multistep pathway called the visual cycle, which effects all-trans to 11-cis re-isomerization of the retinoid chromophore. The maximum throughput of the known visual cycle, however, is too slow to explain sustained photosensitivity in bright light. Here, we demonstrate three novel enzymatic activities in cone-dominant ground-squirrel and chicken retinas: an all-trans-retinol isomerase, an 11-cis-retinyl-ester synthase, and an 11-cis-retinol dehydrogenase. Together these activities comprise a novel pathway that regenerates opsin photopigments at a rate 20-fold faster than the known visual cycle. We suggest that this pathway is responsible for sustained daylight vision in vertebrates.     

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

Evolution and the origin of the visual retinoid cycle in vertebrates

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.     

Retinal pigment epithelium-retinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells

Visual perception begins with the absorption of a photon by an opsin pigment, inducing isomerization of its 11-cis-retinaldehyde chromophore. After a brief period of activation, the resulting all-trans-retinaldehyde dissociates from the opsin apoprotein rendering it insensitive to light. Restoring light sensitivity to apo-opsin requires thermal re-isomerization of all-trans-retinaldehyde to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle in retinal pigment epithelial (RPE) cells. Vertebrates can see over a 10(8)-fold range of background illumination. This implies that the visual cycle can regenerate a visual chromophore over a similarly broad range. However, nothing is known about how the visual cycle is regulated. Here we show that RPE cells, functionally or physically separated from photoreceptors, respond to light by mobilizing all-trans-retinyl esters. These retinyl esters are substrates for the retinoid isomerase and hence critical for regenerating visual chromophore. We show in knock-out mice and by RNA interference in human RPE cells that this mobilization is mediated by a protein called "RPE-retinal G protein receptor" (RGR) opsin. These data establish that RPE cells are intrinsically sensitive to light. Finally, we show that in the dark, RGR-opsin inhibits lecithin:retinol acyltransferase and all-trans-retinyl ester hydrolase in vitro and that this inhibition is released upon exposure to light. The results of this study suggest that RGR-opsin mediates light-dependent translocation of all-trans-retinyl esters from a storage pool in lipid droplets to an "isomerase pool" in membranes of the endoplasmic reticulum. This translocation permits insoluble all-trans-retinyl esters to be utilized as substrate for the synthesis of a new visual chromophore.     

DISCO! Dissociation of cone opsins: the fast and noisy life of cones explained

Vertebrate retinas contain two types of photoreceptors. Rods are for vision in dim light, while cones provide high-speed color vision in bright light. In this issue of Neuron, Kefalov et al. present data to explain the reduced sensitivity and faster response kinetics of cones. They show that the chromophore dissociates from cone but not rod visual pigment, yielding apo-opsin. This apo-opsin activates the signaling cascade to desensitize cones and speed the photoresponse.     

An Alternative Pathway Mediates the Mouse and Human Cone Visual Cycle

One of the fundamental mysteries of the human visual system is the continuous function of cone photoreceptors in bright daylight. As visual pigment is destroyed, or bleached, by light [1], cones require its rapid regeneration, which in turn involves rapid recycling of the pigment's chromophore. The canonical visual cycle for rod and cone pigments involves recycling of their chromophore from all-trans retinol to 11-cis retinal in the pigment epithelium, adjacent to photoreceptors [2]. However, shortcomings of this pathway indicate the function of a second, cone-specific, mechanism for chromophore recycling [3]. Indeed, biochemical [3], [4], [5], [6] and [7] and physiological [8] studies on lower species have described a cone-specific visual cycle in addition to the long-known pigment epithelium pathway. Two important questions remain, however: what is the role of this pathway in the function of mammalian cones, and is it present in higher mammals, including humans? Here, we show that mouse, primate, and human neural retinas promote pigment regeneration and dark adaptation selectively in cones, but not in rods. This pathway supports rapid dark adaptation of mammalian cones and extends their dynamic range in background light independently of the pigment epithelium. This pigment-regeneration mechanism is essential for our daytime vision and appears to be evolutionarily conserved.     

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.     

Molecular Evidence that Only Two Opsin Subfamilies,the Blue Light- (SWS2) and Green Light-Sensitive (RH2), Drive Color Vision in Atlantic Cod (Gadus morhua)

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.     

Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium

The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.     

Cone visual pigments

Cone visual pigments are visual opsins that are present in vertebrate cone photoreceptor cells and act as photoreceptor molecules responsible for photopic vision. Like the rod visual pigment rhodopsin, which is responsible for scotopic vision, cone visual pigments contain the chromophore 11-cis-retinal, which undergoes cis–trans isomerization resulting in the induction of conformational changes of the protein moiety to form a G protein-activating state. There are multiple types of cone visual pigments with different absorption maxima, which are the molecular basis of color discrimination in animals. Cone visual pigments form a phylogenetic sister group with non-visual opsin groups such as pinopsin, VA opsin, parapinopsin and parietopsin groups. Cone visual pigments diverged into four groups with different absorption maxima, and the rhodopsin group diverged from one of the four groups of cone visual pigments. The photochemical behavior of cone visual pigments is similar to that of pinopsin but considerably different from those of other non-visual opsins. G protein activation efficiency of cone visual pigments is also comparable to that of pinopsin but higher than that of the other non-visual opsins. Recent measurements with sufficient time-resolution demonstrated that G protein activation efficiency of cone visual pigments is lower than that of rhodopsin, which is one of the molecular bases for the lower amplification of cones compared to rods. In this review, the uniqueness of cone visual pigments is shown by comparison of their molecular properties with those of non-visual opsins and rhodopsin. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Mutation of cGMP phosphodiesterase 6alpha'-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.     

The Action of 11-cis-Retinol on Cone Opsins and Intact Cone Photoreceptors

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-Retinol is the precursor to 11-cis-retinal, the 11-cis-aldehyde form of vitamin A and the chromophore that combines covalently with rod and cone opsin proteins to form visual pigments. 11-cis-Retinal is consumed during visual signaling, and its continual synthesis is required. Photon absorption by the visual pigments causes the isomerization of its chromophore to the all-trans configuration. This initiates two processes critical for vision: activation of the photoreceptor cell and the eventual recovery of the original photosensitivity of the cells, requiring regeneration of the visual pigments. As cones are used for bright light vision, these two processes must work more rapidly in cones than in rods and thus cones have a higher requirement of 11-cis-retinoids as suggested by Rushton (1, 2).Photoreceptor activation begins with photoisomerization of the chromophore within the visual pigment. This results in a subsequent conformational change of the protein part of the visual pigment that is able to activate its G protein transducin, which in turn activates a PDE that lowers the concentration of cGMP and closes cGMP-gated ion channels. These steps comprise the visual signal transduction cascade (see Ref. 3 for review).The visual cycle involves regeneration of the visual pigment, which ultimately deactivates the protein and accomplishes the recovery of the photosensitivity of the photoreceptor cell. Classically, this process involves both the photoreceptor cell and the retinal pigment epithelium (RPE).⁴ After photoisomerization of the chromophore and formation of the active visual pigment, all-trans-retinal is released from the opsin and reduced to all-trans-retinol, which is then transported to the RPE where it is isomerized to 11-cis-retinol through a number of steps. In the RPE, 11-cis-retinol is oxidized to the aldehyde form, which is transported back to the photoreceptor cell and can be directly used by all of the opsins to regenerate an inactive pigment ready for photoactivation. The details of this model have been extensively reviewed (4, 5). Alternatively, recent work suggests that cones have an additional source of 11-cis-retinoids from Müller cells (6–8). Like the RPE cells, Müller cells have been shown to be able to convert all-trans-retinol to 11-cis-retinol (6). Unlike in the RPE cells, 11-cis-retinol is not oxidized to 11-cis-retinal in Müller cells.Jones et al. (9) demonstrated that administration of 11-cis-retinol to bleached salamander red cones could restore photosensitivity. A logical conclusion was that red cones were able to oxidize 11-cis-retinol to the aldehyde and regenerate visual pigments although noncovalent binding of 11-cis-retinol to red cone opsins generating a light-sensitive complex could not be excluded. On the other hand, 11-cis-retinol does not restore photosensitivity to bleached salamander rod cells but appears to directly activate the cells (9, 10). The data suggested that the rods were not able to oxidize 11-cis-retinol, but that the retinol itself could activate the signal transduction cascade, and indeed we recently demonstrated that 11-cis-retinol acts as an agonist to expressed bovine rod opsin (11). Our aim here was to study the action of 11-cis-retinol on cone opsins and cone photoreceptor cells to determine the efficacy of an alternate visual cycle for cones.The photoreceptor cells used in this study are from tiger salamander, and the expressed opsins used for biochemical experiments are those from salamander and human. Photoreceptor cells are generally identified by cell morphology and the type of opsin it contains that can be further complicated by the findings that some cone cells have multiple opsins (12, 13). Recently genetic analysis has determined that opsins fall into five classes (reviewed in Refs. 14 and 15). We have studied opsins falling into four of these classes and use common color-derived names for the opsins and photoreceptor cells. The classic rod cells used for scotopic vision contain rhodopsin, the visual pigment for the rod opsin (RH1 opsin) and appeared red and thus have been designated as red rods. Some species such as salamanders have an additional rod cell whose photosensitivity is blue-shifted from that of the red rod and thus designated as green rods. In the tiger salamander, the green rods contain the identical opsin (SWS2 opsin) found in blue cones (16). The human blue cones contain an opsin from a different class (SWS1 opsin), which is homologous to the salamander UV cone opsin. The human red and green and salamander red cone opsins all belong to the same class of opsins (M/LWS opsins). Absorption properties of visual pigments are further modulated in some animals including the tiger salamander by use of 11-cis-retinal with an additional double bond (3,4-dehydro or A2 11-cis-retinal) resulting in red-shifted absorbance from pigments containing 11-cis-retinal (A1 11-cis-retinal).We show here that 11-cis-retinol is not an agonist to cone opsins and does not itself generate a light-sensitive opsin. We further show using microspectrophotometry that both red and blue salamander cone cells regenerate visual pigments from 11-cis-retinol, whereas pigments could not be regenerated with 11-cis-retinol in bleached salamander red and green rods even though the latter contains the same opsin as the salamander blue cone. Thus, rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type, and these mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.     

Aberrant metabolites in mouse models of congenital blinding diseases: formation and storage of retinyl esters

Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. All-trans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cis-retinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

Most vertebrate retinas contain two types of photoreceptor cells, rods and cones, which show different photoresponses to mediate scotopic and photopic vision, respectively. These cells contain different types of visual pigments, rhodopsin and cone visual pigments, respectively, but little is known about the molecular properties of cone visual pigments under physiological conditions, making it difficult to link the molecular properties of rhodopsin and cone visual pigments with the differences in photoresponse between rods and cones. Here we prepared bovine and mouse rhodopsin (bvRh and mRh) and chicken and mouse green-sensitive cone visual pigments (cG and mG) embedded in nanodiscs and applied time-resolved fluorescence spectroscopy to compare their G_t activation efficiencies. Rhodopsin exhibited greater G_t activation efficiencies than cone visual pigments. Especially, the G_t activation efficiency of mRh was about 2.5-fold greater than that of mG at 37 °C, which is consistent with our previous electrophysiological data of knock-in mice. Although the active state (Meta-II) was in equilibrium with inactive states (Meta-I and Meta-III), quantitative determination of Meta-II in the equilibrium showed that the G_t activation efficiency per Meta-II of bvRh was also greater than those of cG and mG. These results indicated that efficient G_t activation by rhodopsin, resulting from an optimized active state of rhodopsin, is one of the causes of the high amplification efficiency of rods.     

Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day-night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.     

Biochemical characterization of the retinoid isomerase system of the eye

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.     

A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.