Induction of Normal Cardiovascular Development in the Vitamin A-Deprived Quail Embryo by Natural Retinoids

The biological activity of various natural retinoids and the time "window" when vitamin A activity is required for normal cardiovascular development were examined in vitamin A-deprived Japanese quail embryos. The administration of 1 μg of retinol at the beginning of incubation resuited in normal cardiovascular development in 97% of embryos; retinoic acid was toxic at this dose level. Treatment of embryos with 0.1 μg of all-trans-retinol or 13-cis-retinoic acid at the beginning of incubation resulted in normal cardiovascular development in 47 and 12% of embryos, respectively; administration of these retinoids at other time points attenuated the percentage of embryos with normal cardiovascular development. Didehydroretinol, 0.1 μg, and 9-cis-retinoic acid, 0.1 μg, were inactive at all time points examined; 9-cis-retinoic acid did not enhance the biological activity of all-trans-retinoic acid. All-trans-retinoic acid, 0.1 μg, administered during 22-28 hr of incubation induced normal cardiovascular development in 20-34% of embryos; biological activity was optimal when it was administered at 24 hr. All retinoids tested were inactive in establishing normal cardiovascular development when administered at 36 hr of incubation or later. The studies suggest that all-trans-retinoic acid is the biologically active form of vitamin A required for normal cardiovascular development in the avian embryo. There is a critical time point within the first 22-28 hr of quail embryogenesis when all-trans-retinoic acid initiates events that lead to normal cardiovascular development.     

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

Background

Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.

Methods

We have determined apparent dissociation constants, K′_d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.

Results

CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′_d, 11 nM and 68 nM) and 9-cis-retinal (K′_d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.

Conclusions

CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.

General significance

These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

Site of conversion of endogenous all-trans-retinoids to 11-cis-retinoids in the bovine eye

By use of a new high-resolution high-pressure liquid chromatographic method for the separation of isomeric forms of retinol, retinal, retinyl ester and retinal oxime, various retinoids were analyzed in separated retinal pigment epithelial tissue or neural retinal tissue from fresh bleached bovine eyes after incubation in the dark at either 30 or 4°C for 90 min. 11-cis-Retinoids significantly increased during incubation at 30°C, relative to those at 4°C, in the retinal pigment epithelium, but not in the retina. The major forms of vitamin A in incubated retinal pigment epithelium and neural retina were retinyl esters (70%) and all-trans-retinol (69%), respectively. Thus, in keeping with observations on the isomerization of radioactive retinol in homogenates of eye tissues, the retinal pigment epithelium seems to be the primary site of 11-cis-retinoid formation from endogenous all-trans-retinoids in the bovine eye.     

FATP1 Inhibits 11-cis Retinol Formation via Interaction with the Visual Cycle Retinoid Isomerase RPE65 and Lecithin:Retinol Acyltransferase

The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.     

Molecular Framework of Steroid/Retinoid Discrimination in 17β-Hydroxysteroid Dehydrogenase Type 1 and Photoreceptor-associated Retinol Dehydrogenase

Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17β-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17β-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17β-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17β-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17β-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17β-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17β-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17β-HSD1 developed for cancer treatment.     

Isocratic reversed-phase liquid chromatographic of all-trans-retinoic acid and its major metabolites in new potential supplementary test systems for developmental toxicology

An isocratic reversed-phase high-performance liquid chromatographic procedure for the determination of all-trans-retinoic acid (all-trans-RA) and its metabolites, all-trans-4-oxo-RA, 5,6-epoxy-RA, 9-cis-RA and13-cis-RA, in mouse plasma and embryo and in new in vitro potential test systems for development toxicology has been developed. These compounds, their biological precursor retinol (vitamin A) and the internal standard were resolved on a Spherisorb ODS-2 (5 μm) column (250×4.6 mm I.D.) with acetonitrile-water-methanol-n-butyl alcohol (56:37:4:3, v/v) containing 100 mM ammonium acetate and 70 mM acetic acid as the elution system with a total run time of 23 min. The assay was linear over a wide range, with a lower limit of quantitation of 50 ng/ml or 10 ng/ml of protein for all-trans-RA, 13-cis-RA and 9-cis-RA and of 25 ng/ml or 5 ng/ml protein for the 4-oxo- and 5,6-epoxy-metabolites. At these concentrations, intra-assay coefficients of variation (C.V.) of the retinoids were 3–9%. Mean intra-assay C.V. averaged 5–7% in the tissues studied. Its use is discussed for RA measurements in some of the new test systems — Drosophila melanogaster, sea urchin embryos and cultured human keratinocytes — that have to be evaluated in toxicological testing, supplementary to standard assays in mammals.     

The Retinol Dehydrogenase Rdh10 Localizes to Lipid Droplets during Acyl Ester Biosynthesis

Rdh10 catalyzes the first step of all-trans-retinoic acid biogenesis physiologically, conversion of retinol into retinal. We show that Rdh10 associates predominantly with mitochondria/mitochondrial-associated membrane (MAM) in the absence of lipid droplet biosynthesis, but also locates with lipid droplets during acyl ester biosynthesis. Targeting to lipid droplets requires the 32 N-terminal residues, which include a hydrophobic region followed by a net positive charge. Targeting to mitochondria/MAM and/or the stability of Rdh10 require both the N-terminal and the 48 C-terminal hydrophobic residues. Rdh10 behaves similarly to cellular retinol-binding protein, type 1, which also localizes to mitochondria/MAM before lipid droplet synthesis, and associates with lipid droplets during acyl ester synthesis (Jiang, W., and Napoli, J. L. (2012) Biochem. Biophys. Acta 1820, 859–8692). LRAT, an ER protein, also associates with lipid droplets upon acyl ester biosynthesis. Colocalization of Rdh10, Crbp1, and LRAT on lipid droplets suggests a metabolon that mediates retinol homeostasis.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Biosynthesis of 9-cis-Retinoic Acid from 9-cis-β-Carotene in Human Intestinal Mucosa in Vitro

The role of 9-cis-β-carotene (9-cis-β-C) as a potential precursor of 9-cis-retinoic acid (9-cis-RA) has been examined in human intestinal microcosa in vitro. By using HPLC, uv spectra, and chemical derivatization analysis, both 9-cis-RA and all-trans-retinoic acid (all-trans-RA) have been identified in the postnuclear fraction of human intestinal microcosa after incubation with 9-cis-β-C at 37°C. The biosynthesis of both 9-cis-RA and all-trans-RA from 9-cis-β-C was linear with increasing concentrations of 9-cis-β-C (2-30 μM) and was linear with respect to tissue protein concentration up to 0.75 mg/ml. Retinoic acid was not detected when a boiled incubation mixture was incubated in the presence of 9-cis-β-C. The rate of synthesis of 9-cis- and all-trans-RA from 4 μM 9-cis-β-C were 16 ± 1 and 18 ± 2 pmol/hr/mg of protein, respectively. However, when 2 μM all-trans-β-C was added to the 4 μM 9-cis-β-C, the rate of all-trans-RA synthesis was increased to 38 ± 6 pmol/hr/mg of protein, whereas the rate of 9-cis-RA synthesis remained the same. These results suggest that 9-cis-RA is produced directly from 9-cis-β-C. Furthermore, incubations of either 0.1 μM 9-cis- or all-trans-retinal under the same incubation conditions showed that 9-cis-RA could also arise through oxidative conversion of 9-cis-retinal. Although only 9-cis-RA was detected when 9-cis-RA was used as the substrate, the isomerization of the all-trans-RA to 9-cis-RA cannot be ruled out, since both all-trans-RA and trace amounts of 9-cis-RA were detected when all-trans-retinal was incubated as the substrate. These data indicate that 9-cis-β-C can be a source of 9-cis-RA in the human. This conversion may have a significance in the anticarcinogenic action of β-C.     

An Analysis of the Effects of Retinoic Acid and Other Retinoids on the Development of Adrenergic Cells from the Avian Neural Crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-transRA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-transRA and 13-cisRA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-transRA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-α, produced an increase in adrenergic cells similar to that seen with all-transRA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-transRA is mediated by RAR-α and possibly RAR-β. To further define the actions of all-transRA on the neural crest we incubated cultures with 5-bromo-2′-deoxyuridine (BrdU) to determine whether all-transRA could affect the rate of proliferation. The results show that while all-transRA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-transRA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.     

Retinoids regulate tight junctional resistance of cultured human cervical cells

The objective ofthe study was to determine the effect of retinoids on paracellularresistance across the cervical epithelium and the mechanisms involved.The experimental model was cultures of human CaSki cells on filters,which retain phenotypic characteristics of the endocervical epithelium.End points for paracellular resistance were measurements oftransepithelial electrical resistance and fluxes of pyranine (atrisulfonic acid that traverses the epithelium via the intercellularspace). Paracellular resistance was significantly increased in cellsgrown in retinoid-free medium; the effect could be blocked and reversedwith all-trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol.The effect of tRA was dose dependent and saturable, with a 50%effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D(which decreases tight junctional resistance) and additive to those ofionomycin (which decreases the resistance of the lateral intercellularspace), suggesting that tRA modulates tight junctional resistance. Itis concluded that vitamin A determines the degree of paracellularresistance across cervical cells by a mechanism that involvesmodulation of tight junctional resistance.

     

Metabolism and biological activity of all-trans 4,4-difluororetinyl acetate

All-trans [11-³H]4,4-difluororetinyl acetate was synthesized by treating methyl all-trans [11-³H]4-oxoretinoate with diethylaminosulfurtrifluoride, followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol, difluororetinyl palmitate and related esters, 4-oxoretinol, 4-oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and / or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by ¹⁹F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 ± 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8–3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD⁺ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium

Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1 ^-/- mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1 ^-/- mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE’s) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1 ^-/- mice. Further, total retinyl-ester synthase activity was reduced in both dgat1 ^-/- retina and RPE.     

Morphogenetic effects of 9-cis-retinoic acid on the regenerating limbs of the axolotl

9-cis-retinoic acid has recently been found to be a high affinity ligand for the retinoic X receptor (RXR). RXRs are believed to be involved in metabolic activities rather than in morphogenetic ones. Interestingly, RXR has been found to form heterodimers involving other receptors from the steroid family, such as the thyroid hormone receptor, vitamin D receptor or retinoic acid receptors (RARs). In this paper we examined whether or not 9-cis-retinoic acid had any morphogenetic properties on the regenerating axolotl limb. It is shown that 9-cis-retinoic acid proximalized regenerating limbs and was somewhat more potent in this action than all-trans-retinoic acid. Based on these observations, the possible roles of other receptors during pattern formation is discussed. Correspondence to: P.A. Tsonis     

Effects of visual pigments on the organization of phospholipid multibilayer model membranes--a spin probe study

A cholestane spin probe was used to study the effects of the all-trans isomers of retinal and retinol, and the 13-cis- isomer of retinal on the degree of organization of oriented phospholipid multibilayer model membranes in the dark. Concentration-dependent effects were observed indicative of changes in the degree of organization of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, sphingomyelin, and brain lipids. In general the all-trans compounds improved the degree of order of the lipid films at low concentrations, and decreased it at high concentrations. The 13-cis retinal served only to decrease the degree of order. The magnitudes of these effects depend on lipid composition and the nature of the pigment polar residue. Chemical as well as nonbonding interactions are considered to be responsible for these various effects.