Retinaldehyde Research Articles

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line.

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.

Tolerance Profile of Retinol, Retinaldehyde and Retinoic Acid under Maximized and Long-Term Clinical Conditions

Background: Topical retinoic acid (RA) causes irritation of the skin. To prevent this side effect, natural precursors of RA have been proposed. The aim of the present study was to compare the local tolerance profiles of retinol (ROL), retinaldehyde (RAL) and RA. Methods: ROL, RAL and RA were studied using repeated insult patch tests for 14 days (n = 6). Similarly, RAL and RA were assessed in long-term clinical use for 44 weeks (n = 355). Clinical scoring on irritation, measurement of transepidermal water loss (barrier function) and laser Doppler blood flow perfusion units (irritation) were performed. Results: Under maximized conditions, an equally low irritation potential for ROL and RAL and a more pronounced irritant effect with RA could be demonstrated clinically (p < 0.05 in the intergroup analysis). Furthermore, RAL and RA induced more scaling than ROL (p < 0.05), and ROL and RA tended to induce more burning/pruritus than RAL (nonsignificant). The TEWL values were low with ROL and high with RAL and RA (nonsignificant, intergroup analysis). The laser Doppler measurements confirmed pro-irritating effects of RA and the nonirritating effects of ROL and RAL (p = 0.001, intergroup analysis). The long-term clinical study showed that the study population developed a high frequency of erythema (44% of the population), scaling (35%) and burning/pruritus (29%) with RA in the first 4 weeks of treatment, whereas these 3 parameters were significantly less frequent with RAL (p < 0.0001 in the intergroup analysis). Conclusion: The natural retinoids ROL and RAL do have a good tolerance profile, in contrast with the irritating potential of RA.

Biological Activities of Topical Retinaldehyde

Background: We had hypothesised that retinaldehyde (RAL) should be an interesting precursor for topical use. Aim: We review our observations about its biological activities. Methods: We performed pilot studies to explore its biological effects and tolerability in human skin and compared the effects of topical RAL to that of all-trans-retinoic acid (RA) in the mouse tail test. Results: The biological activities of RAL were found to be qualitatively identical to that of RA: (i) induction of cellular RA-binding protein type 2 mRNA and protein, (ii) increase in epidermal proliferation (increase in DNA synthesis, epidermal thickness, induction of 50-kD keratin mRNA and reduction in 70-kD keratin mRNA), and (iii) metaplastic effects (induction of orthokeratosis, reduction of 65-kD keratin mRNA, increase in filaggrin and loricrin mRNAs). When associated with RAL, citral (known for its capacity to inhibit the oxidation of retinol to RA in epidermis) counteracted the effects induced by RAL indicating that RAL exerts biological activities through transformation to RA. Hypothesising that keratinocytes would metabolize 9-cis-RAL to 9-cis-RA, we compared the biological effects induced by topical 9-cis-RAL and found that hyperplastic and metaplastic responses were lower than those induced by all-trans-RAL or all-trans-RA at similar concentrations. This suggests that 9-cis-RAL has no advantage over all-trans-RAL for specific delivery of natural retinoids into the skin. As in clinical studies conducted in human skin, we also found topical RAL less irritant than RA. Conclusion: These studies indicate that topical RAL has biological activity and is well tolerated.

Lack of effect of RPE65 removal on the enzymatic processing of all- trans-retinol into 11- cis-retinol in vitro

RPE65 is a major membrane associated protein found in the vertebrate retinal pigment epithelium (RPE). Various studies have shown this protein to be essential for visual function, possibly at the level of the processing of retinoids. The pigment epithelium is the anatomical site in which the visual chromophore 11- cis retinal is generated. The two critical RPE enzymes in the isomerization pathway are lecithin retinol acyl transferase (LRAT) and isomerohydrolase, which processes all- trans-retinyl esters into 11- cis-retinol. Both enzymes are membrane bound. It is shown here that RPE65 can be largely extracted (90–95%) from RPE membranes by 1 M KCl by itself, or with added detergent CHAPS. The almost quantitative extraction of RPE65 from RPE membranes has little or no effect on in vitro LRAT and isomerohydrolase activities in quantitative enzymatic assays using RPE membranes, suggesting that RPE65 may not have an important role to play in the enzymatic processing of all- trans-retinol into 11- cis-retinol in vitro.

The metabolic pathway of visual pigment chromophore formation in Drosophila melanogaster--all-trans (3S)-3-hydroxyretinal is formed from all-trans retinal via (3R)-3-hydroxyretinal in the dark.

Carotenoid-depleted fruit flies, Drosophila melanogaster, were reared on yeast/glucose medium containing lipid-depleted white corn grits and cholesterol. After rearing for more than a year, the yield of flies remained constant and the content of 3-hydroxyretinal in a head was three logarithmic units less than that of normal flies reared on medium containing yellow corn grits. When all-trans retinal was supplied as the sole source of retinoids, the flies formed and accumulated all-trans 3-hydroxyretinal in the dark. To examine the metabolic pathway to produce (3S)-3-hydroxyretinal in Drosophila, all-trans retinal was supplemented for two hours to carotenoid-depleted flies in the dark, and the subsequent changes in the composition of 3-hydroxyretinal enantiomers were analyzed using a chiral column on HPLC. The results indicated initial formation of (3R)-3-hydroxyretinal followed by isomerization into the 3S enantiomer. In another set of experiments, the membrane fraction was obtained from the head homogenate of retinoid-depleted flies and an in vitro assay of 3-hydroxyretinal formation from retinal was performed. The 3-hydroxyretinal produced was the 3R enantiomer, supporting the result obtained from the in vivo experiment whereby (3S)-3-hydroxyretinal is produced from retinal via (3R)-3-hydroxyretinal. Addition of NADPH enhanced 3-hydroxyretinal formation and the presence of carbon monoxide inhibited it, suggesting that hydroxylation at the C3 position of retinal occurred via the monooxygenase activity of cytochrome P-450.

Distribution of 11- cis LRAT, 11- cis RD and 11- cis REH in bovine retinal pigment epithelium membranes

Our recent finding of the co-localization of 11- cis retinyl esters and 11- cis retinyl ester hydrolase (11- cis REH) activity in bovine retinal pigment epithelium (RPE) plasma membrane (PM) has led us to explore the possibility that the PM may provide 11- cis retinal for rhodopsin regeneration. In the RPE, visual chromophore is synthesized via a membrane associated 11- cis retinol dehydrogenase (11- cis RD). Accordingly, bovine RPE membranes enriched with either endoplasmic reticulum (ER) or plasma membrane (PM) enzyme markers were prepared and assayed for visual cycle enzyme activities. Pronounced 11- cis RD activity was associated with both ER- and PM-enriched membrane fractions. In contrast, 11- cis REH activity was mostly recovered in PM-enriched fractions while LRAT activity was found only in ER-enriched membranes. The finding that both 11- cis retinol and 11- cis retinal can be produced at the PM of the bovine RPE strongly suggests that 11- cis retinyl esters at this subcellular locale serve as a precursor of visual chromophore for pigment regeneration.

Alternate Sources and Dynamics of Vitamin A and its Incorporation Into the Eyes During the Early Endotrophic and Exotrophic Larval Stages of Atlantic Halibut ( Hippoglossus hippoglossus L.)

The content of various forms of vitamin A were determined in developing eggs and larval stages of Atlantic halibut ( Hippoglossus hippoglossus) both prior to and after the onset of first feeding. Almost all (95%) of the vitamin A present in newly fertilized eggs was in the all- trans retin al form, the rest being all- trans retin ol and 13- cis retin ol. No other major forms or precursors of vitamin A (carotenoids) were found in the eggs. During the ensuing yolk-dependent development, until the onset of first feeding (day 33 posthatch [PH]), the content of all- trans retin ol increased steadily with a concurrent decline observed in all- trans retin al. Half of the increase in all- trans retin ol occurred during the last 12 days before the onset of first feeding. Specific dissection and analyses of the eyes showed that at hatching, neither retin al nor retin ol was present. However, with continued development, primarily all- trans retin al, with smaller amounts of all- trans retin ol and 13- cis retin ol, were gradually incorporated into the eyes. Approximately 75% of the observed increase occurred between days 21 to 33 PH, which coincided with the period when the eyes became pigmented. Following day 26 PH, when the liver anlage had become the major axis of yolk absorption, greater amounts of particularly all- trans retin ol began to be incorporated into the eyes, while both all- trans retin al and 13 cis retin ol continued their incorporation, albeit at a slower rate. After the onset of first feeding on natural zooplankton (>95% the calanoid copepod Temora longicornis,) the trend established at day 26 PH continued throughout the studied exotrophic stages. The planktonic prey was found to contain high levels of carotenoids, but only trace amounts of all isomers of retin ol and retin al. We argue that prior to the onset of first feeding, vitamin A is derived from all- trans retin al stored in the yolk. This form may be more suited for storage in marine fish eggs and may also be readily incorporated in the developing retina. After first feeding is established, vitamin A is derived from precursors present in the planktonic prey, mainly carotenoids. We also propose that the increased content of all- trans retin ol in the eyes may form a labile source of vitamin A that can be readily be converted to the functional aldehyde form, thereby allowing increased photo-sensitivity control of retin al pigments during reduced light conditions.

Differential rhodopsin regeneration in photoreceptor membranes is correlated with variations in membrane properties.

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less "fluid", lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.

Z-scan measurements of the nonlinear refraction in retinal derivatives

The nonlinear refractive index, n 2, of 13-cis retinal aldehyde, 13-cis retinoic acid and all-trans retinoic acid were determined. For 13-cis retinal aldehyde solutions which were left to age for a few days, an increase of over one order of magnitude for n 2 was observed. With basis on the comparison of infrared spectra of freshly prepared and aged solutions, we suggest that the enhancement of the nonlinear response is associated to a spontaneous cis to trans isomerization of the retinal molecule. The second order hyperpolarizabities of all the retinal derivatives were also inferred from the measurements.

Photoinitiated electron transfer interaction of all-trans retinal with electron donors and acceptors

Formation of radical ions of all-trans retinal (ATR) has been demonstrated using 1H CIDNP on its photoinitiated interaction with electron donor (stilbene) and acceptors (2,5-dichlorobenzoquinone, chloranil). CIDNP data show qualitative agreement with calculations of spin density distribution in ATR radical ions conducted by means of the INDO/AM1 method. It has been found that CIDNP effects are formed in the reaction of reversible electron transfer between ATR and either electron donors or acceptors. Retinal isomers arising under irradiation also give rise to nonequilibrium population of spin states in NMR spectra. The correlation between CIDNP effects and the processes of cis-trans isomerization has been discussed.

An ultrafast one-photon and two-photon transient absorption study of the solvent-dependent photophysics in all-trans retinal

Ultrafast transient absorption has been measured for all-trans retinal in hexane and ethanol with 150 fs time resolution. Excitation by one-photon absorption at 400 nm or two-photon absorption at 800 nm allows selection of the initially populated state. One-photon excitation leads to decays in 300 fs to 2 ps in hexane and about 2 ps in ethanol. On a longer time scale, intersystem crossing is observed in about 30 ps in both solvents even though the order of the two lowest excited singlet states is reversed. A model of the photophysics of all-trans retinal in both solvents is presented.

Towards understanding the ultra-fast dynamics of rhodopsin

Abstract

Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes

During the aging process the retinal pigment epithelium (RPE) accumulates autofluorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis that at least one of the fluorescent components of RPE lipofuscin is formed by reaction of vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All- trans retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two different lipid compositions. One type of liposome consisted of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a primary amine. The other liposome type was identical in composition except that some of the PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After incubation of the samples, aliquots were examined with fluorescence microscopy to assess whether any lipofuscin-like fluorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chromatography. Photoreceptor outer segments incubated with retinaldehyde developed an intense golden yellow fluorescent emission when illuminated with 395–440 nm light. Similar fluorescence developed in the liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did not develop any detectable fluorescence. The development of fluorescence in the samples in situ correlated with the appearance of an orange colored component in the lipid extracts that displayed a weak red emission upon ultraviolet light illumination. Incorporation of this component into liposomes resulted in the appearance of a golden yellow fluorescent emission. The results of these experiments suggest that retinal, generated during visual pigment bleaching, can react with PE in the photoreceptor outer segments to form a fluorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE lipofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably derived from the reaction product of outer segment PE and retinal.

The exchange polarization model of photoisomerization: A rationale for profound solvent effects on photoisomerization of trans-stilbene and all-trans retinal

The exchange model of photoisomerization proposed recently by us for S 1 trans-stilbene is further examined and confirmed with a set of new data on trans-stilbene-d 10. The model is then generalized (termed the exchange polarization model) in order to account for the known photoisomerization behaviors of all-trans retinal in polar and non-polar solvents. This new model gives a clear picture of how the isomerization reaction proceeds in the excited electronic state. It leads to a working hypothesis for controlling the photoisomerization pathways of polyenes.

Light-mediated retinoic acid production.

Retinoids serve two main functions in biology: retinaldehyde forms the chromophore bound to opsins, and retinoic acid (RA) is the activating ligand of transcription factors. These two functions are linked in the vertebrate eye: we describe here that illumination of the retina results in an increase in RA synthesis, as detected with a RA bioassay and by HPLC. The synthesis is mediated by retinaldehyde dehydrogenases which convert some of the chromophore all-trans retinaldehyde, released from bleached rhodopsin, into RA. As the eye contains high levels of retinaldehyde dehydrogenases, and as the oxidation of retinaldehyde is an irreversible reaction, RA production has to be considered an unavoidable by-product of light. Through RA synthesis, light can thus directly influence gene transcription in the eye, which provides a plausible mechanism for light effects that cannot be explained by electric activity. Whereas the function of retinaldehyde as chromophore is conserved from bacteria to mammals, RA-mediated transcription is fully evolved only in vertebrates. Invertebrates differ from vertebrates in the mechanism of chromophore regeneration: while in the invertebrate visual cycle the chromophore remains bound, it is released as free all-trans retinaldehyde from illuminated vertebrate rhodopsin. RA synthesis occurring as corollary of dark regeneration in the vertebrate visual cycle may have given rise to the expansion of RA-mediated transcriptional regulation.

Plasma retinoids after topical use of retinaldehyde on human skin.

Retinaldehyde (RAL), a natural metabolite of beta-carotene and retinol (ROL), is tolerated by human skin after topical application. To see if topical application of a large quantity of RAL on human skin is associated with a detectable alteration of constitutive levels of plasma retinoids resulting from metabolism of RAL in the skin. Plasma retinoids [ROL, all-trans-retinoic acid (all-trans-RA), RAL, retinyl palmitate/oleate, 13-cis-RA and 4-oxo-13-cis-RA] were analyzed by high-pressure liquid chromatography. Determinations were done in 10 healthy male volunteers kept on a vitamin-A-poor diet before, during and after daily topical application of 7 mg of RAL to 40% of the body surface for 14 days. The introduction of a restricted vitamin A diet before RAL application resulted in a decrease in the plasma levels of ROL, all-trans-RA and retinyl palmitate/oleate. Topical application of RAL did not induce an alteration of the plasma levels of retinoid metabolites. No RAL was detectable in any of the plasma samples. The skin metabolism of topically applied RAL does not result in detectable alterations of constitutive levels of plasma retinoids in humans.

Ab initio molecular dynamics of retinals

A Car-Parrinello ab initio molecular dynamics calculation is presented of all-trans and 11-cis retinals. The minimum energy configurations of the two isomers have been determined by a simulated annealing procedure. The backbone conjugation is properly described within the local density approximation. The vibrational frequencies have been determined from the molecular dynamics trajectories. The theoretical results show an excellent agreement with experiment and provide grounds for an analysis of the retinal vibrations in terms of localized modes.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-CIS retinol dehydrogenase.

Identification of a 32-kd protein in the bovine retinal pigment epithelium.A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated.The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity.Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Photoactivated state of rhodopsin and how it can form

A variety of spectroscopic and biochemical studies of the photoreceptor rhodopsin have revealed conformational changes which occur upon its photoactivation. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogs or recombinant opsins regenerated with 11- cis retinal. We propose a model for the photoactivation mechanism which defines ‘off’ and ‘on’ states for individual molecular groups. These groups have been identified to undergo structural alterations during photoactivation. Analysis of mutant pigments in which specific groups are locked into their respective ‘on’ or ‘off’ states provides a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions to switch to this conformation. The simple model proposed for the active-state of rhodopsin can be compared to structural models of its ground-state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.

Erratum

The FASEB JournalVolume 9, Issue 8 p. 691-691 ErratumFree Access Erratum This article corrects the following: Enzymatic formation of 9-cis, 13-cis, and all-trans retinals from isomers of β-carotene Akihiko Nagao, James Allen Olson, Volume 8Issue 12The FASEB Journal pages: 968-973 First Published online: September 1, 1994 First published: 01 May 1995 https://doi.org/10.1096/j.1530-6860.1995.tb93391.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume9, Issue8May 1995Pages 691-691 RelatedInformation