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Biochemistry Oct 1993Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids...
Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. The interactions of IRBP with all-trans-retinol have been studied by three fluorescence-based methods and by measurements of binding of 3[H]-labeled all-trans-retinol to this protein. It was found that IRBP contains two sites with similar but not identical affinities for all-trans-retinol. The dissociation constant of the all-trans-retinol-IRBP complex at the first site was 0.1 microM, which is about 10-fold lower than previously reported values. The second site had about 2.5-fold lower affinity for all-trans-retinol as compared to the first site. Long-chain fatty acids were found in this study to displace all-trans-retinol from the stronger retinol-binding site on IRBP. Displacement of all-trans-retinol was used to study the interactions of fatty acids with this protein. It was found that docosahexaenoic acid (DHA C22:6n-3), an essential fatty acid which plays an important role in vision, had the highest apparent affinity for the site probed on IRBP of all the fatty acids studied.
Topics: Animals; Binding Sites; Binding, Competitive; Cattle; Docosahexaenoic Acids; Fatty Acids, Nonesterified; Isomerism; Kinetics; Mathematics; Photoreceptor Cells; Retina; Retinol-Binding Proteins; Spectrometry, Fluorescence; Tritium; Vitamin A
PubMed: 8218196
DOI: 10.1021/bi00093a007 -
Drug Metabolism Reviews 1995Retinol (vitamin A) is an essential nutrient which has many physiological effects throughout the body. Our studies have demonstrated that retinol modulation of immune... (Review)
Review
Retinol (vitamin A) is an essential nutrient which has many physiological effects throughout the body. Our studies have demonstrated that retinol modulation of immune response, through alteration of macrophage and neutrophil function, can have dramatic effects on the toxicity of some compounds. Based on these studies, our current hypothesis for retinol potentiation of chemical-induced liver injury is that retinol administered to rats prior to the hepatotoxicant (CCl4 and AA in rats; and AA, APAP, and GalN in mice) primes the Kupffer cells to a more active state. This may occur in part as a result of increases in chemical mediators such as TNF from these Kupffer cells. Following hepatocyte damage by a toxicant, Kupffer cells are activated to release reactive oxygen species, immune mediators, and chemotactic factors which all serve to enhance the inflammatory response. This increased inflammatory response then results in increased injury to the already toxicant-damaged hepatocytes. In addition, retinol modulation of toxicant activation and detoxification may also make important contributions to the potentiation of some toxicants such as AA. Retinol protection of CCl4 hepatotoxicity in mice is more difficult to explain at this time but is possibly related to alterations in CCl4 metabolism in this species. Differences in response between pulmonary and liver macrophages (Kupffer cells) may explain the retinol protection from 1-NN pulmonary toxicity. Retinol may decrease the inflammatory response through downregulation of pulmonary macrophage function, thus resulting in decreased pulmonary injury. Finally, since retinol protection of cadmium toxicity in the liver and testis requires 7 days of retinol pretreatment, we suspect that retinol is inducing protective protein(s) in these organs. Aside from its normal biological role in rhe body, clinical medicine has found new uses for retinol in the treatment and prevention of some cancers, and in the treatment of certain dermatologic conditions. Since these patients are frequently administered or exposed to other potentially toxic compounds, it is obviously prudent and necessary to continue research into the effects of retinol on immune modulation and interaction with other compounds. More importantly, these studies demonstrate the modulation of immune function is one mechanism by which one chemical can influence the toxicity of another.
Topics: Animals; Cadmium; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Drug Synergism; Ethanol; Liver; Liver Diseases; Mice; Rats; Vitamin A
PubMed: 7641581
DOI: 10.3109/03602539509029828 -
Biophysical Journal Dec 2006The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption....
The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.
Topics: Animals; Biological Transport; Cell Membrane; Diffusion; Fluorescence; Fluorescence Recovery After Photobleaching; In Vitro Techniques; Rana pipiens; Rod Cell Outer Segment; Tretinoin; Vitamin A
PubMed: 17012326
DOI: 10.1529/biophysj.106.086728 -
Biochemical Pharmacology Oct 1995Catalysis of the oxidation of all-trans-retinol (vitamin A1) or of all-trans-retinal to all-trans-retinoic acid (all-trans-RA) by rat conceptal enzymes was investigated... (Comparative Study)
Comparative Study
Catalysis of the oxidation of all-trans-retinol (vitamin A1) or of all-trans-retinal to all-trans-retinoic acid (all-trans-RA) by rat conceptal enzymes was investigated during organogenesis. Products of the reaction were identified and quantified with HPLC by comparing their elution times with those of authentic standard retinoids. Under the incubation and assay conditions utilized, all-trans-retinol and all-trans-retinal were converted to readily detectable quantities of all-trans-RA. Rat conceptal homogenates from gestational days 10.5, 11.5 and 12.5 each exhibited enzymatic activity for oxidation of all-trans-retinol and all-trans-retinal to all-trans-RA. Enzymatic catalysis was verified by showing that: (1) both reactions were coenzyme dependent; (2) the rates of reactions increased as concentrations of conceptal protein increased; (3) both reactions were abolished by heating the tissue homogenates (100 degrees, 5 min); and (4) both reactions exhibited substrate saturation. Under the same experimental conditions, formation of all-trans-RA from all-trans-retinol was much slower than from all-trans-retinal, suggesting that oxidation of all-trans-retinol to all-trans-retinal was the rate-limiting step for biotransformation of all-trans-retinol to all-trans-RA in embryonic tissues. When NAD or NADP were replaced by NADH or NADPH, the rate of oxidation of all-trans-retinol was reduced markedly, indicating that the reaction was catalyzed primarily by an NAD/NADP-dependent dehydrogenase(s). Carbon monoxide (CO:O2 = 90:10) did not inhibit the reaction. NAD appeared to be a more effective cofactor than NADP in catalyzing oxidation of all-trans-retinal to all-trans-RA. When NAD was omitted, formation of all-trans-RA from all-trans-retinal was reduced by approximately 55%. Replacing NAD by NADH or NADPH also reduced the conversion of all-trans-retinal to all-trans-RA by about 60%. These observations suggested at least two pathways for the generation of all-trans-RA from all-trans-retinal in embryos: oxidation catalyzed by an NAD/NADP-dependent dehydrogenase(s) and oxidation catalyzed by an oxidase(s) that did not require NAD, NADH, NADP or NADPH. Conversion of all-trans-retinol to all-trans-RA was inhibited strongly by low concentrations of citral, but not by high concentrations of sodium azide, 4-methylpyrazole, or metyrapone. Similarly, oxidation of all-trans-retinal was inhibited strongly by citral but not by metyrapone.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Acyclic Monoterpenes; Animals; Biotransformation; Embryo, Mammalian; Embryonic and Fetal Development; Enzyme Inhibitors; Gestational Age; In Vitro Techniques; Metyrapone; Monoterpenes; NAD; NADP; Rats; Rats, Sprague-Dawley; Rats, Wistar; Retinaldehyde; Terpenes; Tretinoin; Vitamin A
PubMed: 7488242
DOI: 10.1016/0006-2952(95)02005-w -
Biomedicine & Pharmacotherapy =... Aug 2023Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid....
Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid. Retinol and all-trans-retinoic acid (atRA) penetrate the blood-brain barrier and are reported to be neuroprotective in several animal models. We characterised the impact of retinol and its metabolites, all-trans-retinal (atRAL) and atRA, on ferroptosis-a programmed cell death caused by iron-dependent phospholipid peroxidation. Ferroptosis was induced by erastin, buthionine sulfoximine or RSL3 in neuronal and non-neuronal cell lines. We found that retinol, atRAL and atRA inhibited ferroptosis with a potency superior to α-tocopherol, the canonical anti-ferroptotic vitamin. In contrast, we found that antagonism of endogenous retinol with anhydroretinol sensitises ferroptosis induced in neuronal and non-neuronal cell lines. Retinol and its metabolites atRAL and atRA directly interdict lipid radicals in ferroptosis since these compounds displayed radical trapping properties in a cell-free assay. Vitamin A, therefore, complements other anti-ferroptotic vitamins, E and K; metabolites of vitamin A, or agents that alter their levels, may be potential therapeutics for diseases where ferroptosis is implicated.
Topics: Animals; Vitamin A; Ferroptosis; Lipid Peroxidation; Tretinoin; Vitamins; Retinaldehyde; Lipids
PubMed: 37236031
DOI: 10.1016/j.biopha.2023.114930 -
Investigative Ophthalmology & Visual... Aug 2009To test whether the formation of all-trans retinol limits the regeneration of the visual pigment. all-trans retinol is formed after visual pigment bleaching through the...
PURPOSE
To test whether the formation of all-trans retinol limits the regeneration of the visual pigment. all-trans retinol is formed after visual pigment bleaching through the reduction of all-trans retinal in a reaction involving NADPH. This reduction begins the recycling of the chromophore for the regeneration of the visual pigment.
METHODS
Experiments were performed with dark-adapted, isolated retinas and isolated photoreceptor cells from wild-type and Nrl(-/-) mice. The photoreceptors of Nrl(-/-) mice are conelike and contain only cone pigments. The formation of all-trans retinol after pigment bleaching was measured by quantitative HPLC of retinoids extracted from isolated retinas and by imaging the fluorescence of retinol in photoreceptor outer segments. Experiments were performed at 37 degrees C.
RESULTS
In rods, the formation of all-trans retinol proceeded with first-order kinetics, with a rate constant of 0.06 +/- 0.02 minute(-1), significantly faster than the reported rate constant for rhodopsin regeneration. In Nrl(-/-) photoreceptors, the formation of all-trans retinol occurred at least 100 times faster than in rods. For both cell types, the fraction of all-trans retinal converted to all-trans retinol at equilibrium is approximately 0.8, indicating the presence of a similar fraction of reduced NADPH.
CONCLUSIONS
Formation of all-trans retinol does not limit the regeneration of bleached visual pigment. Formation of all-trans retinol in the cone-like Nrl(-/-) photoreceptors is much faster than in rods, consistent with a faster regeneration of the visual pigment after bleaching. Different types of photoreceptors contain a comparable fraction of reduced NADPH to drive the reduction of all-trans retinal.
Topics: Animals; Basic-Leucine Zipper Transcription Factors; Chromatography, High Pressure Liquid; Dark Adaptation; Eye Proteins; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; NADP; Photoreceptor Cells, Vertebrate; Retinal Pigments; Vitamin A
PubMed: 19264891
DOI: 10.1167/iovs.08-3336 -
Biochemistry Sep 2000In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that... (Comparative Study)
Comparative Study
In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that ultimately results in phototransduction and visual sensation. The production of 11-cis-retinal in adjacent retinal pigment epithelial (RPE) cells is a fundamental process that allows regeneration of the vertebrate visual system. Here, we present evidence that all-trans-retinol is unstable in the presence of H(+) and rearranges to anhydroretinol through a carbocation intermediate, which can be trapped by alcohols to form retro-retinyl ethers. This ability of all-trans-retinol to form a carbocation could be relevant for isomerization. The calculated activation energy of isomerization of all-trans-retinyl carbocation to the 11-cis-isomer was only approximately 18 kcal/mol, as compared to approximately 36 kcal/mol for all-trans-retinol. This activation energy is similar to approximately 17 kcal/mol obtained experimentally for the isomerization reaction in RPE microsomes. Mass spectrometric (MS) analysis of isotopically labeled retinoids showed that isomerization proceeds via alkyl cleavage mechanism, but the product of isomerization depended on the specificity of the retinoid-binding protein(s) as evidenced by the production of 13-cis-retinol in the presence of cellular retinoid-binding protein (CRBP). To test the influence of an electron-withdrawing group on the polyene chain, which would inhibit carbocation formation, 11-fluoro-all-trans-retinol was used in the isomerization assay and was shown to be inactive. Together, these results strengthen the idea that the isomerization reaction is driven by mass action and may occur via carbocation intermediate.
Topics: Animals; Cattle; Diterpenes; Humans; Hydrochloric Acid; Isomerism; Mass Spectrometry; Mathematical Computing; Microsomes; Photochemistry; Pigment Epithelium of Eye; Retinaldehyde; Retinoids; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Retinyl Esters; Sodium Hydroxide; Vitamin A; cis-trans-Isomerases
PubMed: 10985782
DOI: 10.1021/bi001061c -
Investigative Ophthalmology & Visual... Jan 2017Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown...
PURPOSE
Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol.
METHODS
Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics.
RESULTS
Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds.
CONCLUSIONS
This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.
Topics: Animals; Dark Adaptation; Female; Macaca fascicularis; Male; Models, Animal; Ophthalmoscopy; Optical Imaging; Retinal Pigments; Retinal Rod Photoreceptor Cells; Retinol-Binding Proteins; Rhodopsin; Vitamin A
PubMed: 28129424
DOI: 10.1167/iovs.16-20061 -
Biophysical Journal Mar 2005The 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... (Comparative Study)
Comparative Study
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.
Topics: Animals; Cells, Cultured; Female; Fluorescence Recovery After Photobleaching; Isomerism; Kinetics; Light; Male; Metabolic Clearance Rate; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; NADP; Oxidation-Reduction; Photoreceptor Cells; Rana pipiens; Retinaldehyde; Rod Cell Outer Segment; Species Specificity; Vitamin A
PubMed: 15626704
DOI: 10.1529/biophysj.104.054254 -
Sub-cellular Biochemistry 2016Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and... (Review)
Review
Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and cone photoreceptor cells in vertebrate retina. In all known visual systems, the chromophore is 11-cis-retinal complexed with a protein, called opsin, and photoisomerization produces all-trans-retinal. In mammals, regeneration of 11-cis-retinal following photoisomerization occurs by a thermally driven isomerization reaction. Additional reactions are required during regeneration to protect cells from the toxicity of aldehyde forms of vitamin A that are essential to the visual process. Photochemical and phototransduction reactions in rods and cones are identical; however, reactions of the rod and cone visual pigment regeneration cycles differ, and perplexingly, rod and cone regeneration cycles appear to use different mechanisms to overcome the energy barrier involved in converting all-trans- to 11-cis-retinoid. Abnormal processing of all-trans-retinal in the rod regeneration cycle leads to retinal degeneration, suggesting that excessive amounts of the retinoid itself or its derivatives are toxic. This line of reasoning led to the development of various approaches to modifying the activity of the rod visual cycle as a possible therapeutic approach to delay or prevent retinal degeneration in inherited retinal diseases and perhaps in the dry form of macular degeneration (geographic atrophy). In spite of great progress in understanding the functioning of rod and cone regeneration cycles at a molecular level, resolution of a number of remaining puzzling issues will offer insight into the amelioration of several blinding retinal diseases.
Topics: Animals; Carrier Proteins; Darkness; Forecasting; Geographic Atrophy; Humans; Isomerism; Light; Molecular Structure; Photochemistry; Photons; Pregabalin; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Pigments; Retinal Rod Photoreceptor Cells; Retinaldehyde; Schiff Bases; Vertebrates; Vision, Ocular; Vitamin A; cis-trans-Isomerases
PubMed: 27830507
DOI: 10.1007/978-94-024-0945-1_9