tretinoin and 9-cis-retinal

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LL

Topics: Aldehydes; Animals; Cattle; Cell Line; Cells, Cultured; Diterpenes; Flow Cytometry; Humans; Lipofuscin; Liposomes; Microscopy, Confocal; Phagocytosis; Phosphatidylcholines; Phosphatidylethanolamines; Retinal Pigment Epithelium; Retinaldehyde; Retinoids; Rod Cell Outer Segment; Tretinoin

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

Topics: Algorithms; Alitretinoin; Animals; Binding, Competitive; Diterpenes; Fluorometry; Humans; Kinetics; Protein Binding; Retinaldehyde; Retinoid X Receptors; Retinol-Binding Proteins, Cellular; Tretinoin; Vitamin A

All-trans and 9-cis retinoic acids function as ligands for retinoic acid receptors (RARs and RXRs), which are ligand-dependent transcription factors and play important roles in development and cellular differentiation. Several retinal dehydrogenases are likely to contribute to the production of all-trans and 9-cis RAs in vivo, but their respective roles in different tissues are still poorly characterized. We have previously characterized and cloned from kidney tissues the rat retinal dehydrogenase type 1 (RALDH1), which oxidizes all-trans and 9-cis retinal with high efficiency but is inactive with 13-cis retinal. Here we have characterized the retinal-oxidizing activity in monkey JTC12 cells, which are derived from kidney proximal tubules. In vitro assay of cell lysates revealed the presence of a NAD+-dependent dehydrogenase that catalyzed the oxidation of all-trans, 9-cis, and 13-cis retinal. Northern blot analysis of JTC12 RNAs and cloning by reverse transcription-polymerase chain reaction demonstrated expression of a monkey homolog of RALDH1. Bacterially expressed JTC12 RALDH1 catalyzed conversion of all three retinal isomers, with a higher catalytic efficiency for 9-cis retinal than for all-trans and 13-cis retinal. Accordingly, live JTC12 produced 9-cis retinoic acid more efficiently than all-trans retinoic acid from their respective retinal precursors. Only metabolites corresponding to the same steric conformation were formed from 9-cis or all-trans retinal, indicating a lack of detectable isomerizing activity in JTC12 cells.

Topics: Aldehyde Oxidoreductases; Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; Diterpenes; Haplorhini; Humans; Hydrogen-Ion Concentration; Isomerism; Isotretinoin; Kidney Tubules, Proximal; Molecular Sequence Data; Rats; Retinal Dehydrogenase; Retinaldehyde; Sequence Alignment; Substrate Specificity; Tretinoin

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37 degrees C compared to 25 degrees C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The K(m) for all-trans and 13-cis retinal were similar (0.66 and 0.62 microM, respectively). However, the K(m) of RALDH2 for 9-cis retinal substrate (2.25 microM) was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl(2) activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low K(m) and high catalytic efficiency for all-trans retinal, it may likely be involved in the production of all-trans RA in vivo.

Topics: Aldehyde Oxidoreductases; Alitretinoin; Animals; Catalysis; Cloning, Molecular; Diterpenes; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Isotretinoin; Kinetics; Mice; Recombinant Proteins; Retinal Dehydrogenase; Retinaldehyde; Temperature; Tretinoin; Vitamin A

Retinoic acids have important pleiotropic biological effects and thus the potential for human cytochrome P-450s (CYPs) to mediate retinoic acid synthesis was investigated. We examined the retinoic acid synthetic activity of human cDNA-expressed CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A4+ cytochrome b(5) (b(5)), 3A5, and 4A11, expressed individually in insect cells together with NADPH-P-450 reductase. Only CYP1A1, 1A2, 1B1, and 3A4+b(5) converted all-trans-retinal (20 microM) to all-trans-retinoic acid with turnover numbers of 0.53, 0.18, 0.20, and 0.41 nmol/min/nmol P-450, respectively. With 9-cis-retinal as substrate, CYP1A2 exhibited a turnover number of 1.58 nmol/min/nmol P-450 whereas CYP1A1, 2C19, and 3A4+b(5) had turnover numbers of 0.40, 0.27, and 0.41 nmol/min/nmol P-450, respectively. For CYP3A4 activities with both retinals, b(5) was required. Kinetic analyses revealed that CYP1A1, 1A2, and 3A4+b(5) with all-trans-retinal had apparent K(m) values of 55, 356, and 255 microM, and V(max) values of 2.0, 8.3, and 6.3 nmol/min/nmol P-450, respectively, and with 9-cis-retinal had K(m) values of 77, 91, and 368 microM, and V(max) values of 2.7, 9.7, and 7.6 nmol/min/nmol P-450, respectively. The 9-cis retinoic acid synthetic activity of a group of 12 human liver microsomes correlated only with the CYP1A2 activity (r = 0.96), implicating CYP1A2 in human liver microsomal metabolism of 9-cis- retinal to 9-cis-retinoic acid. These studies have indicated that human CYPs are capable of catalyzing retinal to retinoic acid metabolism, but the physiological relevance of this metabolism is still unclear.

Topics: Animals; Benzoflavones; Cytochrome P-450 Enzyme System; Diterpenes; Humans; Isoenzymes; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Rats; Recombinant Proteins; Retinaldehyde; Tretinoin

Topics: Acetaldehyde; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Cell Differentiation; Disulfiram; Diterpenes; Ethanol; Humans; Isoenzymes; Neurons; Retinal Dehydrogenase; Retinaldehyde; Retinoids; Sheep; Tretinoin; Tumor Cells, Cultured

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.

Topics: Animals; Biotransformation; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Diterpenes; In Vitro Techniques; Intestine, Small; Isomerism; Kinetics; Microsomes; NADP; Rats; Retinaldehyde; Tretinoin

The underlying nature of the defect of CVID is not understood, and the treatment at present is life-long infusion of replacement immunoglobulin. Attempts have been made to use other therapeutic agents, such as IL-2 and retinoic acid (RA), with mixed results. RA is a morphogenetic signalling molecule related to vitamin A and involved in vertebrate development. We report here our in vitro evaluation of the effects of three vitamin A analogues, 9-cis retinal, 13-cis RA and all-trans RA, on antibody production of PBMC from normal donors and patients with CVID. At 10(-5) M, 9-cis retinal strongly augmented IgM production of lymphocytes from normal individuals and to a much lesser extent, mild, non-granulomatous (group C) CVID patients, but IgG production was not affected. In the presence of anti-human IgM and IL-2, 9-cis retinal at 10(-5) M elevated IgM and IgG production by normal PBMC, but the effect on PBMC of mild CVID was minimal. The effect of 9-cis retinal was significantly reduced at 10(-7) and 10(-9) M. Only minimal effects were found using 13-cis RA and all-trans RA under these conditions. No detectable antibody production was found in severe, granulomatous (group A) CVID patients under any conditions tested. Taking all data into account, 9-cis retinal is the most potent stimulator for antibody production compared with 13-cis RA and all-trans RA as tested in this in vitro study.

Topics: Blood Donors; Cells, Cultured; Common Variable Immunodeficiency; Diterpenes; Humans; Immunoglobulins; Isotretinoin; Leukocytes, Mononuclear; Retinaldehyde; Tretinoin; Vitamin A

The pleiotropic effects of retinoids are mediated by two families of nuclear receptors: RAR (retinoic acid receptors) and RXR (retinoid X receptors). 9-cis-Retinoic acid is a specific ligand for RXR receptors, whereas either 9-cis- or all-trans-retinoic acid activates the RAR receptor family. The existence of RXRs suggests a new role for isomerization in the biology of retinoic acid. We report here the identification of an aldehyde dehydrogenase in the rat kidney that catalysed the oxidation of 9-cis- and all-trans-retinal to corresponding retinoic acids with high efficiency, 9-cis-retinal being 2-fold more active than all-trans-retinal. Based on several criteria, such as amino acid sequence, pH optimum, and inhibition by chloral hydrate, this enzyme was found to be a novel isoenzyme of aldehyde dehydrogenase. 9-cis-Retinol, the precursor for the biosynthesis of 9-cis-retinal was identified in the rat kidney. The occurrence of endogenous 9-cis-retinol and the existence of specific dehydrogenase which participates in the catalysis of 9-cis-retinal suggest that all-trans-retinoi(d) isomerization to 9-cis-retinoi(d) occurs at the retinol level, analogous to all-trans-retinol isomerization to 11-cis-retinol in the visual cycle.

Topics: Aldehyde Dehydrogenase; Amino Acid Sequence; Animals; Diterpenes; Isoenzymes; Isomerism; Kidney; Molecular Sequence Data; Peptide Fragments; Rats; Rats, Sprague-Dawley; Retinaldehyde; Sequence Analysis; Sequence Homology, Amino Acid; Substrate Specificity; Tretinoin