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PubMed1992

Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.

This study investigates whether the interphotoreceptor retinoid-binding protein (IRBP) is necessary for the release of 11-cis-retinaldehyde (RAL) or if the retinoid is constitutively released from the retinal pigment epithelium (RPE) following synthesis. The strategic location of IRBP in the interphotoreceptor matrix (IPM) and its retinoid-binding ability make it a candidate for a role in 11-cis-RAL release. Fetal bovine RPE cells were grown in permeable chambers, and their apical surfaces were incubated with medium containing either apo-IRBP, the apo form of cellular retinaldehyde-binding protein (CRALBP), the apo form of serum retinol-binding protein (RBP), or bovine serum albumin (BSA) or with medium devoid of binding proteins. [3H]-all-trans-Retinol (ROL) was delivered to the basal surface of the cells by RBP. High-performance liquid chromatography demonstrated that [3H]-11-cis-RAL was optimally released into the apical medium when apo-IRBP was present. The most surprising result was the diminished level of [3H]-11-cis-RAL when apo-CRALBP was in the apical medium. Circular dichroism demonstrated that CRALBP had not been denatured by the photobleaching required for endogenous ligand removal. Therefore, apo-CRALBP should have been able to bind [3H]-11-cis-RAL if it was constitutively released into the apical medium. In addition, when proteins other than apo-IRBP were present, or if the cells were incubated with medium alone, the observed decrease in apical [3H]-11-cis-RAL was concomitant with a buildup of intracellular [3H]-all-trans-retinyl palmitate and [3H]-all-trans-ROL in the basal culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Blue and ultraviolet light-absorbing opsin from the retinal pigment epithelium.

The retinal pigment epithelium (RPE) contains an abundant opsin that is distinct from rhodopsin and cone visual pigments and is able to bind the retinaldehyde chromophore. The putative retinal G protein-coupled receptor (RGR) was isolated in digitonin solution from bovine RPE microsomes and copurified consistently with a minor 34-kDa protein. The absorption spectrum of RGR revealed endogenous pH-sensitive absorbance in the blue and near-ultraviolet regions of light. Membrane-bound RGR was incubated with exogenously added all-trans-retinal and formed two long-lived pH-dependent photopigments with absorption maxima of 469 +/- 2.4 and 370 +/- 7.3 nm. The effects of hydrogen ion concentration suggest that the blue and near-UV photopigments are tautomeric forms of RGR, in which an all-trans-retinal Schiff base is protonated or unprotonated, respectively. The RPE pigment was also demonstrable by its reactivity to hydroxylamine in the dark. The retinaldehyde-RGR conjugate at neutral pH favors the near-UV pigment and is a novel light-absorbing opsin in the vertebrate eye.

Animals↗
PubMed1992

Muller cells of chicken retina synthesize 11-cis-retinol.

The amounts of endogenous retinyl palmitate, retinol and retinaldehyde were measured in the neural retina and retinal pigment epithelium (RPE) of predominantly cone (chicken), rod (rat) and more mixed (cat, human) retinae. The ratio of 11-cis to all-trans isomers of retinyl palmitate and retinol in the neural retina and the RPE increases progressively with the increase in diurnality of the species from rat to chicken. The membrane fractions of both chicken and bovine RPE enzymically isomerize all-trans retinol to 11-cis-retinol. Chicken neural retina membranes enzymically form 11-cis-retinol and all-trans-retinyl palmitate from all-trans-retinol. Light and electron microscopy revealed no contamination of chicken neural retina by RPE. Muller cells from chicken retina were isolated, cultured and characterized by immunocytochemical localization of cellular retinaldehyde-binding protein. Cultured chicken Muller cells form all-trans-retinyl palmitate, 11-cis-retinol and 11-cis-retinyl palmitate from all-trans-retinol and release most of the 11-cis-retinol into the medium. The results indicate that chicken neural retina and Muller cells in particular synthesize 11-cis-retinoids from all-trans-retinol.

Animals↗
PubMed1998

Expression, characterization and engineered specificity of rat epididymal retinoic acid-binding protein.

Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.

Animals↗
PubMed2003

Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

PURPOSE: Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues. METHODS: Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol. The mice were maintained in darkness for a defined period ( approximately 1.5, 4.5, 24, or 48 hours) and then anesthetized, exsanguinated, and killed. Retinoids contained in the retina, RPE, serum, and liver were extracted and analyzed for ((3)H) radioactivity and molar level. RESULTS: The specific activity (SA, in counts per minute per nanomole) of serum all-trans ((3)H)retinol in all mice exhibited a peak at postinjection times of 1.5 or 4.5 hours, and by 48 hours declined to approximately 7% or less of the peak. In Rpe65(+/+) mice, the average SA of RPE ((3)H)retinyl ester similarly exhibited an early peak (4.5 hours) and by 48 hours declined to approximately 6% to 10% of the peak. By contrast, the average SA of RPE ((3)H)retinyl ester in Rpe65(-/-) mice exhibited a peak at 24 or 48 hours. Radioactivity and molar data for serum all-trans retinol and RPE retinyl ester obtained at 4.5 hours were analyzed to infer the molar influx of all-trans retinol from the circulation into the RPE. Levels of all-trans retinol influx derived from this analysis (mean +/- SD: 0.014 +/- 0.004 nmol in 1-month Rpe65(+/+) mice; 0.021 +/- 0.009 nmol in 1-month Rpe65(-/-) mice; 0.016 +/- 0.013 nmol in 3-month Rpe65(+/+) mice; 0.026 +/- 0.018 nmol in 3-month Rpe65(-/-) mice) did not differ significantly from one another (P > 0.169). However, the inferred fractional influx (molar amount of entering all-trans retinol divided by the molar amount of RPE retinyl ester) in Rpe65(+/+) animals (0.34 +/- 0.04 and 0.10 +/- 0.03, respectively, for 1- and 3-month mice) substantially exceeded that for Rpe65(-/-) animals (0.055 +/- 0.023 and 0.015 +/- 0.006, respectively, for 1- and 3-month mice). Significant levels of ((3)H)retinaldehydes were detected in the retinas of Rpe65(+/+) mice, but not in those of Rpe65(-/-) mice, after the longer postinjection periods. CONCLUSIONS: The results indicate preservation of a substantial inward flux of all-trans retinol from the circulation into the RPE of Rpe65(-/-) mice, despite the presence of abnormally high molar levels of RPE retinyl ester. They further imply the occurrence of a robust outward movement of all-trans retinol from the RPE into the circulation in Rpe65(+/+) mice, and substantial impairment of this efflux process in Rpe65(-/-) mice. These findings raise the hypothesis that in normal RPE, 11-cis retinal and/or 11-cis retinol stimulate the efflux of all-trans retinol at the RPE basolateral membrane. In 3-month Rpe65(+/+) mice, the observed relationship between the SAs of retinaldehydes in the retina and of RPE retinyl ester is consistent with a last-in/first-out processing of all-trans retinol to 11-cis retinal within normally functioning RPE.

Animals↗
PubMed2001

Ocular phenotype of bothnia dystrophy, an autosomal recessive retinitis pigmentosa associated with an R234W mutation in the RLBP1 gene.

OBJECTIVE: To describe the phenotype of Bothnia dystrophy, an autosomal recessive retinal dystrophy with an R234W mutation in the RLBP1 gene encoding cellular retinaldehyde-binding protein. DESIGN: Medical records were reviewed retrospectively. Ophthalmologic examination, including kinetic perimetry and, in selected cases, adaptometry, color vision tests, fluorescein angiography, and electrophysiologic studies, was performed. The study included 24 individuals, all homozygous for an R234W mutation in the RLBP1 gene. RESULTS: Patients typically show night blindness from early childhood. In young adults, retinitis punctata albescens was observed, followed by macular degeneration and a decrease in visual acuity that led to legal blindness in early adulthood. Dark adaptometry and electrophysiologic testing showed an initial loss of rod function followed by a progressive reduction of the cone responses in older ages. CONCLUSIONS: Bothnia dystrophy is a unique retinal dystrophy belonging to the rod-cone dystrophies and has a high prevalence in northern Sweden. Fifty-seven cases of Bothnia dystrophy have been diagnosed, indicating a prevalence as high as 1 per 4500 population in the geographic area studied. A defect ability of mutated cellular retinaldehyde-binding protein to bind retinoid probably explains the defect rod function followed by central and peripheral degeneration. CLINICAL RELEVANCE: Retinal dystrophies associated with other mutations of the RLBP1 gene, including retinitis pigmentosa of Bothnia type, might account for a considerable number of cases of autosomal recessive retinitis pigmentosa in other geographic areas as well.

Adolescent↗
PubMed2001

Retinoic acid biosynthesis by normal human breast epithelium is via aldehyde dehydrogenase 6, absent in MCF-7 cells.

Retinoic acid (RA) is the form of vitamin A that controls differentiation and proliferation of epithelia. Our previous work established that normal breast epithelia synthesize RA from retinol, an ability retained by three immortalized but nontumorigenic cell lines but lost in five of six breast cell lines. In this work, we characterize the cause of this defect in one of the lines, the MCF-7 line. We have determined that the immortalized but nontumorigenic cell line, MTSV1.7, capable of synthesizing RA from both retinol and retinal, contains a retinaldehyde dehydrogenase activity for the second step in RA biosynthesis. We have identified it, after isolation, as a previously described enzyme, aldehyde dehydrogenase 6 (ALDH6). Immunohistochemical analysis of normal human breast with antibodies to ALDH6 showed expression of this enzyme in the glandular epithelia colocalized with cellular RA-binding protein type II, a possible marker for certain cells able to synthesize RA. ALDH6 was not present in MCF-7 cells, and these cells were unable to oxidize retinal to RA in culture. When MCF-7 cells were then transfected with ALDH6, they (re)gained the ability to oxidize retinal to RA as well as some ability to synthesize RA when provided with retinol. This suggests that loss of ALDH6 expression is the defect in RA biosynthesis in these cells. Identification of ALDH6 as the retinaldehyde dehydrogenase present in normal human breast epithelia provides the first tool necessary for studying the loss of RA synthetic ability in cancer cells and the relationship of this process to malignant transformation.

Aldehyde Dehydrogenase↗
PubMed2004

Isotretinoin treatment inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration.

Recessive Stargardt's macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardt's discase is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium (RPE). This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardt's patients. Here, we tested a novel therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardt's disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde (11cRAL) and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase (11cRDH) in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde (atRAL), which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr-/- knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr-/- mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardt's patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

Animals↗
PubMedDate not supplied

Absence of isomerization of retinyl palmitate, retinol, and retinal in chlorinated and nonchlorinated solvents under gold light.

Purified solutions of all-trans retinyl palmitate, retinol, and retinaldehyde in chloroform, methylene chloride, or hexane were exposed to white light or gold fluorescent light or were kept in the dark, and the resulting isomer distributions were determined by LC (liquid chromatography). No significant isomerization of any of the retinoids occurred either in the dark or on exposure to gold light in any of the solvents tested. However, a large amount of the 9-cis isomer and only much smaller amounts of other cis isomers were produced when retinol or retinyl palmitate in chloroform or methylene chloride solution was exposed to white light. The isomerization pattern of retinyl palmitate in chloroform was not altered by the addition of free-radical scavengers, addition of an organic base, or substitution of deuterochloroform for chloroform as solvent. Use of other polar solvents such as tetrahydrofuran, acetone, or methanol produced isomer distributions similar to those obtained in chloroform solution. Retinol and retinyl palmitate in hexane solution, on exposure to white light, were isomerized much less extensively than in chloroform or methylene chloride and produced a significant amount of the 13-cis, as well as the 9-cis, isomer. Isomerization of retinaldehyde in chloroform or in methylene chloride solution under white light yielded 13-cis, 11-cis, 9-cis, and 7-cis isomers, in order of decreasing amount, whereas in hexane solution, only the 13-cis and 9-cis isomers were produced in significant quantity.

Chemical Phenomena↗
PubMed1982

Determination of vitamin D and retinoid activity in eggs by HPLC.

Reverse phase high performance liquid chromatography (HLPC) was used to estimate the vitamin D3 content of battery and free range eggs. At the same time another HPLC technique was used to estimate the retinol activity in the battery eggs derived from the separate determination of all-trans retinol, 13-cis retinol and retinaldehyde concentrations. There was some variation in both the vitamin D3 and retinol activity in eggs over the laying year but no significance could be drawn from this. Vitamin D3 levels in 12 monthly samples each of 120 battery eggs averaged 1.2 micrograms/100 g edible portion, while the levels in each of four quarterly samples of 36 free range eggs from the London and Newcastle areas averaged 1.4 and 0.8 micrograms/100 g respectively. These differences between battery and free range eggs were not statistically significant. The average retinol activity in four samples of 120 battery eggs was calculated to be 190 micrograms/100 g, and the components of this activity were identified as 132 micrograms all-trans retinol and 21 micrograms of retinaldehyde, as well as 53 micrograms 13-cis retinol which has not previously been reported in hens' eggs.

Animals↗
PubMed1996

Structures important in NAD(P)(H) specificity for mammalian retinol and 11-Cis-retinol dehydrogenases.

Mammalian retinol and 11-cis retinol dehydrogenases catalyze the formation of retinaldehyde from retinol and 11-cis retinaldehyde from 11-cis retinol, respectively. Although their amino acid sequences are 54% identical, these enzymes have different cofactor specificities: rat retinol dehydrogenase uses NADP+, while cow 11-cis retinol dehydrogenase uses NAD+. We used their close sequence similarity and the availability of 3D structures of their homologs to construct a 3D model of the two retinol dehydrogenases to investigate the determinants for cofactor specificity. The structure of rat retinol dehydrogenase shows that lysine-64 is important in stabilizing binding of 2'-phosphate on NADP+ in two ways: lysine's positively charged side chain has a coulombic attraction to the 2'-phosphate and partially compensates for the negative charge of aspartic acid-38. Cow 11-cis retinol dehydrogenase has threonine-61 at the position homologous to lysine-64. Threonine-61 does not have a stabilizing coulombic interaction with NADP+, nor can threonine-61 counteract the repulsive interaction between NADP+ and aspartic acid-37 in 11-cis retinol dehydrogenase. This suggests that aspartic acid-37 and threonine-61 are important in the specificity of 11-cis retinol dehydrogenase for NAD+.

Alcohol Oxidoreductases↗
PubMed1999

Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes.

The normal growth and differentiation of the epidermis require an adequate supply of vitamin A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/nuclear receptor complexes at specific DNA sequences in their control regions. The message conveyed by RA is likely modulated by the concentration of the ligand available for binding to the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydrogenase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound to cellular retinol-binding protein (holo-CRBP). However, little is known about these enzymes and their genes in the epidermal cells. Our work describes the first member of the RDH family found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NAD+ as a preferred cofactor, utilizes free and CRBP-bound all-trans-retinol and steroids as substrates.

Alcohol Oxidoreductases↗
PubMed1997

Sex differences in endogenous retinoid release in the post-embryonic spinal cord of the western mosquitofish, Gambusia affinis affinis.

In this study we have shown sex differences in endogenous retinoic acid synthesis and retinaldehyde dehydrogenase activity in the post-embryonic spinal cords of immature female and male G. a. affinis. The F9 reporter cell assay and the zymography bioassay showed that the endogenous retinoic acid levels correlated with the levels of the endogenous enzyme(s) responsible for retinoic acid synthesis. These data also showed that both the endogenous retinoic acid levels and the enzyme(s) were higher in spinal cord segments 8-16 of immature males than in immature females. We have also shown that exogenous treatment of 17 alpha-methyltestosterone results in the masculinization of the immature female's anal fin and its appendicular support elements as well as the endogenous synthesis of retinoic acid and retinaldehyde dehydrogenase activity. The F9 reporter cell assay showed that the endogenous retinoic acid levels were relatively unchanged in spinal cord segments 8-16 of immature males treated with 17 alpha-methyltestosterone. However, the F9 reporter cell assay showed that the endogenous retinoic acid levels in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone were markedly higher than levels observed in the immature males. The data also showed that the activity of the enzyme(s) responsible for the synthesis of endogenous retinoic acid was higher in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone than in immature males treated with 17 alpha-methyltestosterone. Currently under investigation is the question of what role the endogenous enzyme(s) responsible for the synthesis of retinoic acid plays either alone or in concert with androgen in organizing hormone-dependent sexually dimorphic areas in the teleost body plan.

Aging↗
PubMed1992

Synthesis of retinoic acid from retinol by cultured rabbit Müller cells.

Previous observations have shown that Müller glial cells of the vertebrate retina contain cellular retinoid-binding proteins, that the retina contains retinoic acid, and that cellular retinoic acid-binding protein is present in amacrine neurons (and, in some species, Müller cells) within the retina. These findings led to the suggestion that Müller cells may synthesize retinoic acid and release it for use by other retinal cells. To test this possibility, we cultured Müller cells from adult rabbit retinas, incubated the cultures with radioactive retinol, and identified and quantified the resultant radioactive retinoids by HPLC. Retinaldehyde was rapidly synthesized from retinol, reaching a plateau of 1-2 pmol mg-1 cell protein by 30 min. Retinoic acid initially accumulated more slowly, but by 30 min constituted most of the synthesized retinoid. While the retinaldehyde remained within the cells, retinoic acid was rapidly released into the medium; extracellular retinoic acid exceeded the intracellular amount after 30 min of incubation. Smaller amounts of retinyl esters were also synthesized and retained by the cells. These results are consistent with the suggestion that Müller glia are a source of retinoic acid in the retina. The synthesis of retinoic acid by these cells, and the presence of retinal neurons that contain cellular retinoic acid-binding protein, raise the possibility that retinoic acid plays a role in the retina, although this role is not presently known. Furthermore, these results may have implications for other parts of the adult nervous system. Adult brain contains retinol- and retinoic acid-binding proteins, and, therefore, may also be a site of retinoic acid metabolism. Because of the relatively simple cellular organization of the retina and its demonstrated capacity to synthesize retinoic acid, the retina may be a system of choice for further studies of the synthesis and function of retinoic acid in adult neural tissue.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Acyl CoA:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium.

Visual perception is mediated by a family of G protein-coupled receptors called the opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde upon absorption of a photon. Restoration of light sensitivity to the photobleached opsin requires chemical re-isomerization of the chromophore. This is carried out by an enzymatic pathway called the visual cycle in retinal pigment epithelial cells. The isomerase in this pathway uses fatty-acyl esters of all-trans-retinol as substrate. A retinyl-ester synthase that produces these esters, called lecithin:retinol acyltransferase (LRAT), has been extensively characterized. Based on prior biochemical studies and the phenotype in lrat(-/-) knockout mice, it has been assumed that LRAT is the sole or dominant retinyl-ester synthase in the retinal pigment epithelium. Here we demonstrate the presence of a second ester synthase activity in these cells called acyl CoA:retinol acyltransferase (ARAT). We show that this activity uses palmitoyl coenzyme A as an acyl donor, unlike LRAT which uses phosphatidylcholine. Similar to LRAT, ARAT esterifies both all-trans-retinol and 11-cis-retinol. LRAT and ARAT are both potently inhibited by the retinyl-ester analog, all-trans-retinylbromoacetate, but only ARAT is inhibited by progesterone. Unexpectedly, the maximum turnover rate (V(max)) of ARAT was similar to that of LRAT. However, the Michaelis constant (K(M)) of ARAT was 10-fold higher than the K(M) of LRAT for all-trans-retinol. These observations suggest that ARAT may complement LRAT to provide additional retinyl-ester synthase activity under conditions of high all-trans-retinol. These conditions occur in the retina following exposure to bright light.

Acyltransferases↗
PubMed1999

Antioxidant enzymes in the macular retinal pigment epithelium of eyes with neovascular age-related macular degeneration.

PURPOSE: To test the hypothesis that neovascular age-related macular degeneration is related to oxidative stress involving the macular retinal pigment epithelium. This study investigated, as a function of age, levels of enzymes that defend tissues against oxidative stress in the macular retinal pigment epithelium of human eyes with this disease. METHODS: Surgical specimens of macular choroidal neovascular membranes from eyes with age-related macular degeneration and the macular regions of whole donor eyes with neovascular age-related macular degeneration or without evident ocular disease were studied by quantitative electron microscopic immunocytochemistry with colloidal gold-labeled second antibodies. Relative levels in retinal pigment epithelium cell cytoplasm and lysosomes were determined of five enzymes believed to protect cells from oxidative stress, as well as levels of the retinal pigment epithelium marker cytoplasmic retinaldehyde-binding protein, for comparison with the enzymes. RESULTS: Copper, zinc superoxide dismutase immunoreactivity increased and catalase immunoreactivity decreased with age in cytoplasm and lysosomes from macular retinal pigment epithelium cells of normal eyes and eyes with age-related macular degeneration. Cytoplasmic retinaldehyde-binding protein immunoreactivity showed no significant relationship to age or the presence of neovascular age-related macular degeneration. Glutathione peroxidase immunoreactivity was absent from human retinal pigment epithelium cells. Both heme oxygenase-1 and heme oxygenase-2 had highly significantly greater immunoreactivity in retinal pigment epithelium cell lysosomes than in cytoplasm, differing from the much greater cytoplasmic immunoreactivity of the other proteins studied. This immunoreactivity decreased with age, particularly in the lysosomes of retinal pigment epithelium cells from eyes with neovascular age-related macular degeneration. These decreases were of borderline significance (P = .067 for heme oxygenase-1; P = .12 for heme oxygenase-2) when eyes with age-related macular degeneration were compared with normal eyes by multivariable logistic regression. CONCLUSIONS: The high heme oxygenase-1 and heme oxygenase-2 lysosomal antigen levels in macular retinal pigment epithelium cells of eyes with neovascular age-related macular degeneration suggest that oxidative stress causes a pathologic upregulation of these enzymes. Increased lysosomal disposal may indicate that the reparative functions of these enzymes are accompanied by deleterious effects, necessitating their rapid removal from the cell. The much higher heme oxygenase-1 and heme oxygenase-2 antigen levels in macular retinal pigment epithelium cells from younger individuals suggest that protective mechanisms against oxidation and, hence, presumably to the development of age-related macular degeneration, decrease with age.

Adult↗
PubMed1995

Histopathology of bone spicule pigmentation in retinitis pigmentosa.

PURPOSE: To evaluate bone spicule pigmentation, a fundus feature in retinitis pigmentosa (RP) formed by migration of pigment-containing cells to perivascular sites in the inner retina. METHODS: The authors performed light and electron microscopy, including immunocytochemistry, on the retinas from ten patients with RP and five normal donors. RESULTS: The pigment-containing cells in regions of bone spicule pigmentation were derived from the retinal pigment epithelium (RPE). The translocated cells were remarkably polarized with a number of specializations characteristic of RPE cells in situ, but they did not contain lipofuscin granules and were not immunoreactive for cellular retinaldehyde-binding protein. The cells were linked by junctional complexes and formed epithelial layers around retinal vessels and next to the inner limiting membrane. Adjacent Müller cell processes contained glial fibrillary acidic protein-positive filaments and formed microvilli and intermediate junctions, resembling those in the external limiting membrane. Vascular endothelial cells adjacent to the translocated RPE cells were thin and fenestrated, resembling the choriocapillaris, and were separated from the pigmented cells by a layer of extracellular matrix similar in organization to Bruch membrane. Thickening of the matrix layer caused narrowing and occlusion of the vascular lumina. CONCLUSIONS: The lack of lipofuscin granules and cellular retinaldehyde-binding protein immunoreactivity in the translocated RPE cells is probably related to the loss of photoreceptors. The development of fenestrations in the endothelial cells correlates with the leakiness of retinal vessels to fluorescein observed in some cases of RP. Narrowing and occlusion of vascular lumina by thickening of the surrounding layer of extracellular matrix may contribute to the loss of inner retinal neurons found in RP. These changes in the RPE, blood vessels, glia, and inner neurons warrant consideration in designing therapies to restore vision to degenerate retinas.

Adult↗