sorbinil and tolrestat

Aldose reductase (AR) is widely expressed aldehyde-metabolizing enzyme. The reduction of glucose by the AR-catalyzed polyol pathway has been linked to the development of secondary diabetic complications. Although treatment with AR inhibitors has been shown to prevent tissue injury in animal models of diabetes, the clinical efficacy of these drugs remains to be established. Recent studies suggest that glucose may be an incidental substrate of AR, which appears to be more adept in catalyzing the reduction of a wide range of aldehydes generated from lipid peroxidation. Moreover, inhibition of the enzyme has been shown to increase inflammation-induced vascular oxidative stress and prevent myocardial protection associated with the late phase of ischemic preconditioning. On the basis of these studies, several investigators have ascribed an important antioxidant role to the enzyme. Additionally, ongoing work indicates that AR is a critical component of intracellular signaling, and inhibition of the enzyme prevents high glucose-, cytokine-, or growth factor-induced activation of protein kinase C and nuclear factor-kappa-binding protein. Thus, treatment with AR inhibitors prevents vascular smooth muscle cell growth and endothelial cell apoptosis in culture and inflammation and restenosis in vivo. Additional studies indicate that the antioxidant and signaling roles of AR are interlinked and that AR regulates protein kinase C and nuclear factor-kappaB via redox-sensitive mechanisms. These data underscore the need for reevaluating anti-AR interventions for the treatment of diabetic complications. Potentially, the development of newer drugs that selectively inhibit AR-mediated glucose metabolism and signaling, without affecting aldehyde detoxification, may be useful in preventing inflammation associated with the development of diabetic complications, particularly micro- and macrovascular diseases.

Topics: Aldehyde Reductase; Animals; Cataract; Diabetes Complications; Humans; Imidazolidines; Models, Molecular; Naphthalenes; Oxidation-Reduction; Oxidative Stress; Signal Transduction

Aldose reductase ([EC1.1.1.21]: AR) acts on the first step of the polyol metabolic pathway to catalyze the reduction of glucose to sorbitol with NADPH as a coenzyme. Hyperactivity of the pathway in individuals with high blood glucose level is closely related to the onset or progression of diabetic complications. AR inhibitors have therefore been noted as possible pharmacotherapeutic agents for the treatment of diabetic complications. One AR inhibitor has been on the market in Japan, while some potent inhibitors are in clinical trials. Reviewed are the physiological roles of AR, the chemical structures of AR inhibitors, interactions of AR inhibitors with AR using X-ray studies, and the following potencies of AR inhibitors: in vitro activities for AR, in vitro selectivities between AR and aldehyde reductase, their pharmacological effects in vivo, and their effectiveness in clinical trials. Also discussed are directions for the design of future AR inhibitors.

Topics: Aldehyde Reductase; Binding Sites; Clinical Trials as Topic; Crystallography, X-Ray; Diabetic Neuropathies; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Naphthalenes; Quinazolines

Despite numerous attempts over 16 years, the results of aldose reductase inhibitor (ARI) trials for the treatment of diabetic neuropathy have not proven efficacy. This paper reviews each of the ARI trials, examines confounding factors, and proposes a future course. The confounding factors considered are pharmacokinetics (ARI penetration of human nerve), length of trial (in terms of the natural history of diabetic neuropathy), trial endpoints (reversibility or slowing of progression), reproducibility of clinical measurements (in terms of power calculations), standardization and quality control of endpoints, and clinically meaningful differences in endpoints. We conclude that ARIs are most likely to have a beneficial effect in the management of diabetic distal symmetrical polyneuropathy and autonomic neuropathy but that the clinical role of ARIs is to slow the progression of diabetic neuropathy rather than to reverse it. Future trials should be designed with adequate statistical power, with consideration of the variability of the endpoint measurements for long enough duration, and with rigorous quality control to definitively confirm the utility of ARIs in the treatment of diabetic distal symmetrical polyneuropathy and autonomic neuropathy.

Topics: Aldehyde Reductase; Autonomic Nervous System; Diabetic Neuropathies; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Isoquinolines; Naphthalenes; Phthalazines; Time Factors

Recently, aldose reductase inhibitors (ARIs) have been registered in several countries for the improvement of glycaemic control. However, their efficacy is still controversial. ARIs inhibit the enhanced flux of glucose through the polyol pathway. As such, they can never be more effective than normoglycaemia, and so their potential benefits and limitations should be considered relative to the effects of prolonged euglycaemia. The clinical effects of ARIs can be put into perspective by assessing the effects of improved glycaemic control attained in randomised trials of intensive insulin treatment [such as the Diabetes Control and Complications Trial (DCCT)] and after pancreatic transplantation. Although direct comparison of these 3 interventions is hampered by differences in patient populations, duration and methods of follow-up and in the potency of ARIs, the effects of these 3 metabolic interventions and their course in time appear remarkably similar. For neuropathy, all 3 interventions induce an increase in average motor nerve conduction velocity of approximately 1 m/sec during the first months of treatment. At the same time, improvement of painful symptoms may occur. These changes probably largely represent a metabolic amelioration of the condition of the nerves. Around the second year of treatment with all 3 forms of metabolic improvement, an acceleration of nerve conduction of a similar magnitude occurs, with signs of structural nerve regeneration and some sensory recuperation. Experience with ARIs in nephropathy is still limited, but similar improvements in glomerular filtration rate and, less consistently, in urinary albumin excretion were found during short term normoglycaemia produced by all 3 forms of treatment. Comparison of a small number of studies, however, shows differences between intensive insulin regimens, pancreatic transplantation and ARIs in effects on retinopathy. Retinopathy often temporarily deteriorates in the early phases of improved glycaemic control, but this is not noted with ARIs. New microaneurysm formation was slightly reduced in a single long term study with the ARI sorbinil, but the preventive effects on the overall levels of retinopathy seemed less strong than in normoglycaemia trials of similar duration. However, the pharmacodynamic effects on inhibiting the polyol pathway differ among ARIs, and the half-life of the inhibiting effect of sorbinil may have been too short for a complete reduction of polyol pathway activity.

Topics: Adult; Aged; Aldehyde Reductase; Cross-Sectional Studies; Diabetes Mellitus; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Humans; Imidazoles; Imidazolidines; Insulin; Islets of Langerhans Transplantation; Middle Aged; Naphthalenes; Pancreas Transplantation; Treatment Outcome

To update readers on research being conducted with the aldose reductase inhibitor (ARI) tolrestat in treating complications of diabetes mellitus. The article briefly describes early investigations with other ARIs and reviews the more recent studies of tolrestat. In addition, the article gives readers a simplified overview of the biochemical background pertinent to the use of these agents.. A MEDLINE search was performed to identify articles relating to the clinical use of, and research involving, the following ARIs: sorbinil, alrestatin, ponalrestat, and tolrestat. In addition, pharmaceutical manufacturers were contacted in an attempt to obtain data relating to ongoing investigations.. Review articles and clinical trials of sorbinil, alrestatin, and ponalrestat were included. Articles dealing with clinical trials of tolrestat were selected from the MEDLINE search. As there were only a few trials, all studies identified were included. No additional written data were available from the manufacturers.. ARIs, which when first introduced were proclaimed to be major advances in treating diabetic complications, have never produced the expected results. Problems with efficacy and toxicity relegated most of this class of agents to historical interest. One compound, tolrestat, has continued to be tested and has potential clinical application. To date, the extent of benefit that has been realized in tolrestat-treated patients is small to moderate. Improvements have occurred in paresthesia and neuropathy, but unfortunately, not in pain symptoms. Adverse effects have been minor and are primarily confined to elevations of hepatic alanine aminotransferase. Additional clinical trials are being conducted with this agent.. Tolrestat is the only one of the original ARIs still undergoing clinical trials. Results so far have been encouraging, but by no means definitive, for improvement in some aspects of diabetic neuropathy. Information from ongoing investigations is necessary before the true usefulness of tolrestat therapy can be determined.

Topics: Aldehyde Reductase; Clinical Trials as Topic; Diabetes Complications; Diabetes Mellitus; Humans; Imidazoles; Imidazolidines; Isoquinolines; Naphthalenes; Phthalazines

Topics: Aldehyde Reductase; Animals; Diabetic Neuropathies; Double-Blind Method; Humans; Imidazoles; Imidazolidines; Naphthalenes; Nerve Regeneration; Sural Nerve

Topics: Aldehyde Reductase; Blood Glucose; Diabetes Complications; Diabetes Mellitus; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Humans; Imidazoles; Imidazolidines; L-Iditol 2-Dehydrogenase; Naphthalenes; Phthalazines; Sugar Alcohol Dehydrogenases

Topics: Aldehyde Reductase; Animals; Binding Sites; Blood Glucose; Cataract; Chemical Phenomena; Chemistry; Corneal Diseases; Diabetes Complications; Diabetes Mellitus; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Disease Models, Animal; Fluorenes; Galactose; Humans; Hydantoins; Imidazoles; Imidazolidines; Models, Molecular; Naphthalenes; Phthalazines; Rhodanine; Sorbitol; Structure-Activity Relationship; Substrate Specificity; Sugar Alcohol Dehydrogenases; Thiazolidines; Tissue Distribution

Despite numerous attempts over 16 years, the results of aldose reductase inhibitor (ARI) trials for the treatment of diabetic neuropathy have not proven efficacy. This paper reviews each of the ARI trials, examines confounding factors, and proposes a future course. The confounding factors considered are pharmacokinetics (ARI penetration of human nerve), length of trial (in terms of the natural history of diabetic neuropathy), trial endpoints (reversibility or slowing of progression), reproducibility of clinical measurements (in terms of power calculations), standardization and quality control of endpoints, and clinically meaningful differences in endpoints. We conclude that ARIs are most likely to have a beneficial effect in the management of diabetic distal symmetrical polyneuropathy and autonomic neuropathy but that the clinical role of ARIs is to slow the progression of diabetic neuropathy rather than to reverse it. Future trials should be designed with adequate statistical power, with consideration of the variability of the endpoint measurements for long enough duration, and with rigorous quality control to definitively confirm the utility of ARIs in the treatment of diabetic distal symmetrical polyneuropathy and autonomic neuropathy.

Topics: Aldehyde Reductase; Autonomic Nervous System; Diabetic Neuropathies; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Isoquinolines; Naphthalenes; Phthalazines; Time Factors

Topics: Aldehyde Reductase; Animals; Diabetic Neuropathies; Double-Blind Method; Humans; Imidazoles; Imidazolidines; Naphthalenes; Nerve Regeneration; Sural Nerve

The acetic acid derivatives of [1,2,4]triazino[4,3-a]benzimidazole (TBI) were synthesized and tested in vitro and in vivo as selective aldose reductase (ALR2) inhibitors. Compound PS11 showed highest inhibitory activity (IC(50)) 0.32 microM and was found to be effective in preventing cataract development in severely galactosemic rats when administered as an eyedrop solution. All the compounds investigated were selective for ALR2, since none of them inhibited appreciably aldehyde reductase, sorbitol dehydrogenase, or glutathione reductase.

Topics: Acetic Acid; Aldehyde Reductase; Animals; Benzimidazoles; Cataract; Disease Models, Animal; Enzyme Inhibitors; Galactosemias; Inhibitory Concentration 50; Molecular Structure; Naphthalenes; Ophthalmic Solutions; Rats

Phenolic marine natural product is a kind of new potential aldose reductase inhibitors (ARIs). In order to investigate the binding mode and inhibition mechanism, molecular docking and dynamics studies were performed to explore the interactions of six phenolic inhibitors with human aldose reductase (hALR2). Considering physiological environment, all the neutral and other two ionized states of each phenolic inhibitor were adopted in the simulation. The calculations indicate that all the inhibitors are able to form stable hydrogen bonds with the hALR2 active pocket which is mainly constructed by residues TYR48, HIS110 and TRP111, and they impose the inhibition effect by occupying the active space. In all inhibitors, only La and its two ionized derivatives La_ion1 and La_ion2, in which neither of the ortho-hydrogens of 3-hydroxyl is substituted by Br, bind with hALR2 active residues using the terminal 3-hydroxyl. While, all the other inhibitors, at least one of whose ortho-sites of 3- and 6-hydroxyls are substituted by Br substituent which take much electron-withdrawing effect and steric hindrance, bind with hALR2 through the lactone group. This means that the Br substituent can effectively regulate the binding modes of phenolic inhibitors. Although the lactone bound inhibitors have relatively high RMSD values, our dynamics study shows that both binding modes are of high stability. For each inhibitor molecule, the ionization does not change its original binding mode, but it does gradually increase the binding free energy, which reveals that besides hydrogen bonds, the electrostatic effect is also important to the inhibitor-hALR2 interaction.

Topics: Aldehyde Reductase; Enzyme Inhibitors; Hydrogen Bonding; Imidazolidines; Molecular Dynamics Simulation; Naphthalenes; Protein Structure, Secondary; Rhodanine; Thiazolidines

Atherosclerotic lesion formation is associated with the accumulation of oxidized lipids. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion; however, their contribution to atherosclerotic lesion formation remains unclear.. To test the hypothesis that inhibition of aldehyde removal by aldose reductase (AR), which metabolizes both free and phospholipid aldehydes, exacerbates atherosclerotic lesion formation.. In atherosclerotic lesions of apolipoprotein (apo)E-null mice, AR protein was located in macrophage-rich regions and its abundance increased with lesion progression. Treatment of apoE-null mice with AR inhibitors sorbinil or tolrestat increased early lesion formation but did not affect the formation of advanced lesions. Early lesions of AR(-/-)/apoE(-/-) mice maintained on high-fat diet were significantly larger when compared with age-matched AR(+/+)/apoE(-/-) mice. The increase in lesion area attributable to deletion of the AR gene was seen in both male and female mice. Pharmacological inhibition or genetic ablation of AR also increased the lesion formation in male mice made diabetic by streptozotocin treatment. Lesions in AR(-/-)/apoE(-/-) mice exhibited increased collagen and macrophage content and a decrease in smooth muscle cells. AR(-/-)/apoE(-/-) mice displayed a greater accumulation of the AR substrate 4-hydroxy trans-2-nonenal (HNE) in the plasma and protein-HNE adducts in arterial lesions than AR(+/+)/apoE(-/-) mice.. These observations indicate that AR is upregulated in atherosclerotic lesions and it protects against early stages of atherogenesis by removing toxic aldehydes generated in oxidized lipids.

Topics: Aldehyde Reductase; Aldehydes; Animals; Apolipoproteins E; Atherosclerosis; Diabetes Mellitus, Experimental; Dietary Fats; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Imidazolidines; Macrophages; Male; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Naphthalenes; Oxidation-Reduction; Phospholipids; Up-Regulation

Aldose reductase (AR) catalyzes the reduction of several aldehydes ranging from lipid peroxidation products to glucose. The activity of AR is increased in the ischemic heart due to oxidation of its cysteine residues, but the underlying mechanisms remain unclear. To examine signaling mechanisms regulating AR activation, we studied the role of nitric oxide (NO). Treatment with the NO synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester prevented ischemia-induced AR activation and myocardial sorbitol accumulation in rat hearts subjected to global ischemia ex vivo or coronary ligation in situ, whereas inhibition of inducible NOS and neuronal NOS had no effect. Activation of AR in the ischemic heart was abolished by pretreatment with peroxynitrite scavengers hesperetin or 5, 10, 15, 20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron [III]. Site-directed mutagenesis and electrospray ionization mass spectrometry analyses showed that Cys-298 of AR was readily oxidized to sulfenic acid by peroxynitrite. Treatment with bradykinin and insulin led to a phosphatidylinositol 3-kinase (PI3K)-dependent increase in the phosphorylation of endothelial NOS at Ser-1177 and, even in the absence of ischemia, was sufficient in activating AR. Activation of AR by bradykinin and insulin was reversed upon reduction with dithiothreitol or by inhibiting NOS or PI3K. Treatment with AR inhibitors sorbinil or tolrestat reduced post-ischemic recovery in the rat hearts subjected to global ischemia and increased the infarct size when given before ischemia or upon reperfusion. These results suggest that AR is a cardioprotective protein and that its activation in the ischemic heart is due to peroxynitrite-mediated oxidation of Cys-298 to sulfenic acid via the PI3K/Akt/endothelial NOS pathway.

Topics: Aldehyde Reductase; Animals; Bradykinin; Dithiothreitol; Enzyme Activation; Enzyme Inhibitors; Ferric Compounds; Glucose; Hesperidin; Hypoglycemic Agents; Imidazolidines; Insulin; Lipid Peroxidation; Male; Metalloporphyrins; Myocardial Reperfusion Injury; Myocardium; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sorbitol; Vasodilator Agents

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of how the slight change of a chemically extended side-chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships.

Topics: Acetates; Aldehyde Reductase; Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Imidazolidines; Ligands; Models, Molecular; Naphthalenes; Protein Conformation; Thioamides; Thiocarbamates

Four different types of marine natural compounds isolated from tunicates were found to inhibit human aldose reductase. They all are characterized by a heterocyclic system, and at least two phenolic groups are present in the structure. Two of the compounds tested showed an inhibitory potency 5/6-fold higher than that of the known AR inhibitor sorbinil. One notable structural feature of these active compounds is the lack of either the carboxylic acid or the spiro-hydantoin commonly present in the principal classes of currently used inhibitors.

Topics: Aldehyde Reductase; Animals; Biological Products; Humans; Imidazolidines; Marine Biology; Naphthalenes; Quinazolines; Rhodanine; Thiazolidines; Urochordata

The objective of this study was to determine whether the polyol pathway enzyme aldose reductase mediates diabetes abnormalities in vascular smooth muscle cell (SMC) growth. Aldose reductase inhibitors (tolrestat or sorbinil) or antisense aldose reductase mRNA prevented hyperproliferation of cultured rat aortic SMCs induced by high glucose. Cell cycle progression in the presence of high glucose was blocked by tolrestat, which induced a G0-G1 phase growth arrest. In situ, diabetes increased SMC growth and intimal hyperplasia in balloon-injured carotid arteries of streptozotocin-treated rats, when examined 7 or 14 days after injury. Treatment with tolrestat (15 mg x kg(-1) x day(-1)) diminished intimal hyperplasia and decreased SMC content of the lesion by 25%. Although tolrestat treatment increased immunoreactivity of the lesion with antibodies raised against protein adducts of the lipid peroxidation product 4-hydroxy trans-2-nonenal, no compensatory increase in lesion fibrosis was observed. Collectively, these results suggest that inhibition of aldose reductase prevents glucose-induced stimulation of SMC growth in culture and in situ. Even though inhibition of aldose reductase increases vascular oxidative stress, this approach may be useful in preventing abnormal SMC growth in vessels of diabetic patients.

Topics: Aldehyde Reductase; Animals; Aorta; Cell Cycle; Cell Division; Cells, Cultured; Diabetes Mellitus, Experimental; Enzyme Inhibitors; Glucose; Hyperplasia; Imidazolidines; Male; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley; Reference Values; Tunica Intima

Bacterial infections are one of the major causes of human eye disease. Because the bacterial endotoxin lipopolysaccharide (LPS) is known to cause cytotoxicity through oxidative stress and an earlier study has shown that aldose reductase (AR) mediates oxidative stress signals, the purpose of this study was to investigate the anti-inflammatory effects of AR inhibition on LPS-induced activation of NF-kappaB-dependent signals in human lens epithelial cells (HLECs).. Growth-arrested HLECs were cultured without or with AR inhibitors or transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with LPS (1-10 mug/mL) for 24 hours. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by nucleosomal degradation. Electrophoretic mobility gel shift assays were performed to determine the activation of NF-kappaB and AP1. The levels of nitric oxide, MMP-2, MMP-9, Cox-2, and TNF-alpha were measured by using specific ELISA kits. Western blot analysis was performed to determine the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of PKC and mitogen-activated protein kinase (MAPK).. Bacterial LPS caused apoptosis of HLECs. Inhibition of AR by two structurally unrelated inhibitors, sorbinil and tolrestat, or ablation by AR siRNA prevented the LPS-induced apoptosis, activation of caspase-3 and cleavage of PARP protein. Inhibition of AR in HLECs also prevented the LPS-induced activation of redox-sensitive transcription factors such as NF-kappaB and AP1 and their downstream signals that lead to expression of Cox-2, MMP-2, MMP-9, and TNF-alpha proteins. In addition, inhibition of AR prevented LPS-induced activation of protein kinases upstream to NF-kappaB activation such as PKC and MAPK in HLECs.. The results indicate that AR mediates the bacterial endotoxin signaling that could damage HLECs by regulating the signals that activate the redox-sensitive transcription factor NF-kappaB and cause inflammation. Thus, inhibition of AR could be a therapeutic target for Gram-negative bacterial infection-induced visual complications.

Topics: Aldehyde Reductase; Blotting, Western; Caspase 3; Collagen Type XI; Cyclooxygenase 2; Dinoprostone; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Imidazolidines; Infant; Lens, Crystalline; Lipopolysaccharides; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase Kinases; Naphthalenes; NF-kappa B; Nitric Oxide; Tumor Necrosis Factor-alpha

Aldose reductase (AR; AKR1B1) a member of aldo-keto reductase super family, that we had shown earlier mediates cytotoxic signals induced by high glucose, cytokines and growth factors, also mediates the inflammatory signals induced by Gram-negative bacterial endotoxin, lipopolysaccharide (LPS). Inhibition of AR by three distinct AR inhibitors sorbinil, tolrestat or zopolrestat suppressed the LPS-induced production of inflammatory cytokines such as TNF-alpha, IL-6, IL-1beta, IFN-gamma, and chemokine MCP-1 in murine peritoneal macrophages. Inhibition of AR also prevented the production of nitric oxide, and prostaglandin E2 and expression of iNOS and Cox-2 proteins. The LPS-induced DNA binding activity of NF-kappaB and AP1 were significantly inhibited by AR inhibitors, and this effect was mediated through the inhibition of phosphorylation of IkappaB-alpha, IKK alpha/beta and PKC. These results suggest the therapeutic use of AR inhibitors as anti-inflammatory drugs.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Chemokine CCL2; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; I-kappa B Kinase; Imidazolidines; Inflammation Mediators; Interferon-gamma; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Naphthalenes; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; Phthalazines; Protein Kinase C; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Increased expression of adhesion molecules by the activated endothelium is a critical feature of vascular inflammation associated with several disease states such as atherosclerosis. However, mechanisms regulating the endothelial induction of adhesion molecules are not entirely clear. Herein we report that inhibition of the polyol pathway enzyme aldose reductase (AR) prevents the increase in ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs) and decreases monocyte adhesion to these cells. In TNF-alpha-stimulated HUVECs, treatment with AR inhibitors sorbinil and tolrestat diminished NF-kappaB activity, phosphorylation and degradation of Ikappa-Balpha, and the nuclear translocation of NF-kappaB. Inhibition of AR abrogated TNF-alpha-induced activation and membrane translocation of PKC, and antisense ablation of AR prevented both TNF-alpha-induced PKC and NF-kappaB activation. However, inhibition of AR did not prevent phorbol ester-induced activation of PKC or NF-kappaB, indicating that inhibition of AR does prevents events upstream of PKC activation. These results identify a novel regulator of endothelial activation and suggest that AR is an obligatory mediator of TNF-alpha signaling leading to an increase in the expression of adhesion molecules and increased binding of monocytes to the endothelium.

Topics: Aldehyde Reductase; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Humans; I-kappa B Proteins; Imidazolidines; Inflammation; Intercellular Adhesion Molecule-1; Monocytes; Naphthalenes; NF-kappa B; NF-KappaB Inhibitor alpha; Oligodeoxyribonucleotides, Antisense; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Transcriptional Activation; Transfection; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Cell Adhesion Molecule-1

Chronic hyperglycemia and cytokines such as tumor necrosis factor alpha (TNF-alpha) cause oxidative stress leading to dysregulated cell growth or apoptosis that contributes to the development of inflammation and secondary complications of diabetes. However, the mechanisms regulating hyperglycemic or cytokine injury are not well understood. Herein we report that inhibition of the polyol pathway enzyme aldose reductase (AR) by two structurally unrelated inhibitors--sorbinil and tolrestat--prevents, in the human lens epithelial cell line B-3, the apoptosis and activation of caspase-3 caused by exposure to high glucose levels or TNF-alpha. Inhibition of AR attenuated TNF-alpha and hyperglycemia-induced activation of protein kinase C (PKC), phosphorylation of the inhibitory subunit of nuclear factor-kappaB (NF-kappaB), and stimulation of NF-kappaB, but it did not prevent the activation of NF-kappaB and PKC by phorbol ester. Inhibition of AR also attenuated the increase in p38 mitogen-activated protein kinase and c-Jun N-terminal kinase phosphorylation. These signaling pathways were also inhibited in cells in which the expression of AR was reduced by antisense ablation. Collectively, these results identify a new participant in apoptotic signaling and suggest that AR is an obligatory mediator of the apoptotic events upstream of PKC. These observations could provide new insights into the pathophysiology of diabetes and the role of aberrant glucose metabolism in apoptotic cell death.

Topics: Aldehyde Reductase; Apoptosis; Caspase 3; Caspases; Cell Division; Cell Survival; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Glucose; Humans; I-kappa B Kinase; Imidazoles; Imidazolidines; Lens, Crystalline; Naphthalenes; NF-kappa B; Oxidation-Reduction; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha

Increased glucose utilization by aldose reductase (AR) has been implicated in the development of diabetes complications. However, the mechanisms that regulate AR during diabetes remain unknown. Herein we report that several nitric oxide (NO) donors prevent ex vivo synthesis of sorbitol in erythrocytes obtained from diabetic or nondiabetic rats. Compared with erythrocytes of nondiabetic rats, the AR activity in the erythrocytes of diabetic rats was less sensitive to inhibition by NO donors or by AR inhibitors-sorbinil or tolrestat. Treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, enhanced AR activity and sorbitol accumulation in tissues of nondiabetic rats. Application of transdermal nitroglycerin patches or treatment with L-arginine did not inhibit AR activity or sorbitol accumulation in the tissues of nondiabetic animals. Treatment with L-NAME increased, whereas treatment with L-arginine or nitroglycerine patches decreased AR activity and sorbitol content in tissues of diabetic rats. These observations suggest that NO maintains AR in an inactive state and that this repression is relieved in diabetic tissues. Thus, increasing NO availability may be a useful strategy for inhibiting the polyol pathway and preventing the development of diabetes complications.

Topics: Aldehyde Reductase; Animals; Diabetes Mellitus, Experimental; Erythrocytes; Glucose; Imidazoles; Imidazolidines; Naphthalenes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Sprague-Dawley; Sorbitol

The activation of the polyol pathway through aldose reductase (AR) might be involved in diabetic neuropathy. A considerable structural similarity exists between AR and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) (both belonging to aldo-keto reductase superfamily); 3alpha-HSD forms 5alpha-reduced-3alpha-hydroxylated steroids, possibly possessing neurotrophic functions. Aim of these experiments was to test "in vitro" in rat sciatic nerves, whether glucose concentrations in the diabetic range might affect the capacity of 3alpha-HSD to transform dihydroprogesterone (DHP) into tetrahydroprogesterone (THP), a steroid proved to possess neurotrophic effects. The capability of AR inhibitors, drugs used to avoid diabetic complications, to decrease THP formation was also assessed. 3alpha-HSD activity was evaluated by the conversion of labelled DHP into THP (in a single case dihydrotestosterone was used as substrate, and the corresponding 3alpha-hydroxylated metabolite was evaluated). Freshly prepared rat sciatic nerve homogenates were used as source of the enzyme. Whole brain, liver and prostate served as "control" tissues. The results show that glucose added up to a concentration of 400 mg/dL (well above the euglycemic upper level) does not affect the 3alpha-HSD activity in the sciatic nerve and in the other tissues considered. Similarly, when the enzyme was challenged by two AR inhibitors, tolrestat and sorbinil, added in a concentration about 10 times higher than their IC50 for AR, no significant changes were observed. Analogous results were achieved when DHT was used in presence of glucose (400 mg/dL) and sorbinil. We conclude that hyperglycemia or the administration of the AR inhibitors do not affect 3alpha-HSD activity in peripheral nerves and therefore do not reduce the formation of steroid metabolites possibly endowed with neurotrophic action.

Topics: 20-alpha-Dihydroprogesterone; 3-Hydroxysteroid Dehydrogenases; Aldehyde Reductase; Animals; Diabetic Neuropathies; Dihydrotestosterone; Enzyme Inhibitors; Hyperglycemia; Imidazoles; Imidazolidines; Male; Naphthalenes; Rats; Rats, Sprague-Dawley; Sciatic Nerve

Several (Z)-5-arylidene-2,4-thiazolidinediones were synthesized and tested as aldose reductase inhibitors (ARIs). The most active of the N-unsubstituted derivatives (2) exerted the same inhibitory activity of Sorbinil. The introduction of an acetic side chain on N-3 of the thiazolidinedione moiety led to a marked increase in lending inhibitory activity, conducting to the discovery of a very potent ARI (4c), whose activity level (IC50=0.13 microM) was in the same range of Tolrestat. Moreover, the corresponding methyl esters (3), devoid of any acidic functionality, showed appreciable inhibitory activity similar to that of the N-unsubstituted compounds. It was also found that the substitution pattern on the 5-benzylidene moiety markedly influenced the activity of N-unsubstituted 2,4-thiazolidinediones 2, compounds with substituents at the meta position being generally more effective than the para-substituted ones; however, this SAR was not evidenced in acetates 3 and acids 4.

Topics: Aldehyde Reductase; Animals; Cattle; Enzyme Inhibitors; Hypoglycemic Agents; Imidazoles; Imidazolidines; Inhibitory Concentration 50; Magnetic Resonance Spectroscopy; NADP; Naphthalenes; Structure-Activity Relationship; Thiazoles; Thiazolidinediones

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.

Topics: Aldehyde Reductase; Animals; Carotid Artery Injuries; Cell Division; Cells, Cultured; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Mitogens; Muscle, Smooth, Vascular; Naphthalenes; Rabbits; Rats; RNA, Messenger; Up-Regulation

Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [(3)H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60-65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography-chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25-30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.

Topics: Aldehyde Reductase; Aldehydes; Alkenes; Animals; Aorta; Cells, Cultured; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Glutathione; Imidazoles; Imidazolidines; Lipoproteins, LDL; Male; Mass Spectrometry; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy-trans-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg. kg(-1). d(-1)) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.

Topics: Aldehyde Reductase; Aldehydes; Angioplasty, Balloon; Animals; Aorta; Carotid Artery Injuries; Carotid Stenosis; Cell Division; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Glutathione; Hemostatics; Humans; Imidazoles; Imidazolidines; Lipid Peroxidation; Male; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley; Recurrence; RNA, Messenger; Thrombin; Tritium

Aldose and aldehyde reductases are monomeric NADPH-dependent oxidoreductases that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The overall three-dimensional structures of the enzymes are composed of similar alpha/beta TIM-barrels, and the active site residues Tyr 50, His 113, and Trp 114 interacting with the hydrophilic heads of inhibitors are conserved. We have used molecular modeling and mass spectrometry to characterize the interactions between the enzymes and three aldose reductase inhibitors: tolrestat, sorbinil, and zopolrestat. Unlike the IC(50) values (concentration of inhibitor giving 50% of inhibition in solution), the Vc(50) values measured by mass spectrometry (accelerating voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the two enzymes are similar, and they correlate with the electrostatic and hydrogen-bonding energies calculated between the conserved Tyr 50, His 113, and Trp 114 and the inhibitors. The results of our comparison agree with detailed structural information obtained by X-ray crystallography, suggesting that nonconserved residues from the C-terminal loop account for differences in IC(50) values for the two enzymes. Additionally, they confirm our previous assumption that the Vc(50) values reflect the enzyme-inhibitor electrostatic and hydrogen-bonding interactions and exclude the hydrophobic interactions.

Topics: Aldehyde Reductase; Animals; Benzothiazoles; Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Inhibitory Concentration 50; Models, Molecular; Naphthalenes; Phthalazines; Spectrometry, Mass, Electrospray Ionization; Swine; Thiazoles

The effects of aldose reductase inhibitors on lens protein modifications induced by naphthalene-1,2-dihydrodiol were investigated in vitro to confirm the role of aldose reductase on naphthalene cataract formation. HPLC analysis of naphthalene-1, 2-dihydrodiol incubated with aldose reductase and NAD+indicated the formation of a metabolite peak corresponding to 1,2-naphthoquinone. Soluble proteins from rat lenses prepared by gel filtration of crude lens extracts through Sephadex PD-10, incubated with naphthalene-1, 2-dihydrodiol in the presence of NAD+displayed an absorbance ca 450 nm and their spectra were essentially identical to those of 1, 2-naphthoquinone-protein adducts. Similar spectra were also obtained from proteins isolated from the intact rat lens after in vitro incubation in medium containing naphthalene-1,2-dihydrodiol. The spectra obtained from lens proteins incubated with 1, 2-dihydroxynaphthalene were distinct from those of either naphthalene-1,2-dihydrodiol or 1,2-naphthoquinone. Aldose reductase inhibitors possessing either hydantoin or carboxylic acid groups prevented protein modification induced by naphthalene-1, 2-dihydrodiol but not protein modification induced by 1, 2-dihydroxynaphthalene or 1,2-naphthoquinone. Therefore, the metabolite formed from naphthalene-1,2-dihydrodiol by aldose reductase is 1,2-naphthoquinone. Lens proteins modified by naphthalene-1,2-dihydrodiol appear essentially identical to protein adducts formed with 1,2-naphthoquinone and their formation can be prevented by both hydantoin and carboxylic acid containing aldose reductase inhibitors.

Topics: Aldehyde Reductase; Animals; Cataract; Chromatography, High Pressure Liquid; Crystallins; Enzyme Inhibitors; Fluorenes; Hydantoins; Imidazoles; Imidazolidines; Lens, Crystalline; Naphthalenes; Naphthols; Naphthoquinones; Phthalazines; Rats; Spectrophotometry

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Topics: Aldehyde Reductase; Benzothiazoles; Binding Sites; Computer Simulation; Enzyme Inhibitors; Humans; Imidazoles; Imidazolidines; Kinetics; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; NADP; Naphthalenes; Phthalazines; Recombinant Proteins; Thiazoles

Specific non-covalent interactions between aldose reductase (AR), its NADP+ cofactor and five inhibitors have been characterized by electrospray mass spectrometry (ES-MS). These results indicated that the protein could be desorbed and maintained in the gas phase in a form very close to its native conformation. Collisionally induced dissociation (CID)-MS and CID-MS-MS showed that the adenosine diphosphate part of the cofactor interacts strongly with AR. The relative stability of the ternary AR x NADP+ x inhibitor complexes was established and successfully correlated with the IC50 values. All inhibitors were shown to only bind to AR holoenzyme. These results are important for the field of drug development insofar as ES-MS might provide a rapid and very sensitive method for the screening of potential drugs or for the identification of compounds displaying high binding affinity to a target biomolecule.

Topics: Acetates; Aldehyde Reductase; Animals; Apoenzymes; Enzyme Inhibitors; Furans; Imidazoles; Imidazolidines; Lens, Crystalline; Mass Spectrometry; NADP; Naphthalenes; Rhodanine; Swine; Thiazoles; Thiazolidines

Topics: Aldehyde Reductase; Animals; Enzyme Inhibitors; Eye Proteins; Fluorenes; Hydantoins; Imidazoles; Imidazolidines; Lens, Crystalline; Naphthalenes; Naphthoquinones; Oxidoreductases; Phthalazines; Rats

Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket.. The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase.. The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

Topics: Aldehyde Reductase; Animals; Binding Sites; Computer Simulation; Crystallography; Enzyme Inhibitors; Imidazoles; Imidazolidines; Lens, Crystalline; Models, Molecular; Molecular Conformation; Molecular Sequence Data; NADP; Naphthalenes; Swine

The inhibitory effects of seven agents on aldose reductase were studied by enzymatic kinetics using the purified enzyme from bovine testis, sorbinil, tolrestat, silybin and berberini were found to exhibit a marked enzyme-inhibitory effect with Ki values of 1.08 mumol/L, 0.12 mumol/L, 3.5 micrograms/ml, and 2.7 micrograms/ml respectively. The established assay system described in this paper may be useful in screening aldose reductase inhibitors.

Topics: Aldehyde Reductase; Animals; Cattle; Enzyme Inhibitors; Imidazoles; Imidazolidines; Male; Naphthalenes; Silymarin; Testis

Naphthalene-induced cataract in rat lenses can be completely prevented by AL01576, an aldose reductase inhibitor (ARI). In an attempt to understand the mechanism of this inhibition, several ARIs were examined to compare their efficacies in preventing naphthalene cataract, using both in vitro and in vivo models. Two classes of ARIs were tested: One group including AL01576, AL04114 (a AL01576 analog) and Sorbinil contained the spirohydantoin group, while Tolrestat contained a carboxylic acid group. Furthermore, to clarify if aldose reductase played a role in naphthalene-induced cataractogenesis in addition to its role in sugar cataract formation, a new dual cataract model was established for ARI evaluations. This was achieved by feeding rats simultaneously with high galactose and naphthalene or incubating rat lenses in culture media containing high galactose and naphthalene dihydrodiol. Under these conditions, both cortical cataract and perinuclear cataract developed in the same lens. It was found that at the same dosage of 10 mg/kg/day, both AL01576 and AL04114 completely prevented all morphological and biochemical changes in the lenses of naphthalene-fed rats. Sorbinil was less efficacious, while Tolrestat was inactive. AL01576 showed a dose-response effect in preventing naphthalene cataract and at 10 mg/kg/day, it was also effective as an intervention agent after cataractogenesis had begun. With the dual cataract model, Tolrestat prevented the high galactose-induced cortical cataract but showed no protection against the naphthalene-induced perinuclear cataract. AL01576, on the other hand, prevented both cataract formations. Results for dulcitol and glutathione levels were in good agreement with the morphological findings. AL04114, and ARI as potent as AL01576 but without its property for cytochrome P-450 inhibition, displayed similar efficacy in preventing naphthalene cataract. Based on these results, it was concluded that the prevention of the naphthalene cataract probably results from inhibition of the conversion of naphthalene dihydrodiol to 1,2-dihydroxynaphthalene and that the effect of the ARIs cannot be explained by their inhibition of the dihydrodiol dehydrogenase activity of aldose reductase.

Topics: Aldehyde Reductase; Animals; Cataract; Culture Techniques; Disease Models, Animal; Enzyme Inhibitors; Fluorenes; Galactose; Hydantoins; Imidazoles; Imidazolidines; Male; Naphthalenes; Rats; Spiro Compounds

It has been demonstrated that activation of aldose reductase (AR; EC 1.1.1.21) in diabetic tissues plays an important role in the pathogenesis of diabetic complications. In the present study, the effects of non-enzymatic glycation of recombinant human AR (rhAR) on enzyme activity and affinity for its substrate (glyceraldehyde), co-factor (NADPH) and inhibitors (ARI; Sorbinil, Tolrestat, AL-1576 and Statil) were examined. Although rhAR was successfully non-enzymatically glycated with HPLC-purified [3H]D-glucose, the Michaelis constant (Km) and catalytic efficiency (Kcat/Km) for glyceraldehyde, the Km for NADPH and the inhibitor constant (Ki) for ARI did not change. These results suggest that the mechanism of AR activation and its insensitivity to inhibition observed in diabetic tissues cannot be attributed to its non-enzymatic glycation.

Topics: Aldehyde Reductase; Enzyme Inhibitors; Fluorenes; Glucose; Glycosylation; Humans; Hydantoins; Imidazoles; Imidazolidines; Kinetics; Naphthalenes; Phthalazines; Recombinant Proteins; Substrate Specificity; Transfection

Steady-state kinetic and inhibition properties of human placental aldose reductase carboxymethylated at Cys-298 were investigated. A comparison of the primary deuterium kinetic isotope effect on the reduced and the carboxymethylated enzymes suggests that carboxymethylation did not affect the reaction sequence of substrate binding and release. Values of DV/KD-glyceraldehyde greater than DV suggest that steps in the reaction scheme subsequent to hydride transfer, particularly the release of NADP may be rate limiting. Carboxymethylation of Cys-298 was also found to affect NADPH and aldehyde binding to the enzyme. Carboxymethylation had little effect on the secondary structure of the enzyme, but a comparison of the circular dichroic spectra of the reduced and carboxymethylated enzyme, suggests a weakened interaction between the nicotinamide and 2'-monophosphoadenosine 5'-diphosphoribose of NADPH, and the carboxymethylated enzyme. Interaction between Cys-298 and NADPH appears to determine the rate of isomerization of the E:NADP binary complex and carboxymethylation-induced decrease in kcat may be due to slower isomerization of the E:NADP binary complex. The carboxymethylated enzyme was less sensitive than the reduced enzyme to most aldose reductase inhibitors including sorbinil (d-6-fluoro-spiro[chroman-4,4'-imidazolidine]-2',5'-dione), except tolrestat (N-methyl-N-[(5-trifluromethyl-6-methoxy-1-naphthalenyl)- thiomethyl]glycine) and quercetin. On the basis of these observations it is suggested that Cys-298 may form a part of the 'S'-inhibitor binding site of the enzyme and may be responsible for tight binding of NADPH.

Topics: Aldehyde Reductase; Aldehydes; Circular Dichroism; Cysteine; Deuterium; Female; Humans; Imidazoles; Imidazolidines; Iodoacetates; Iodoacetic Acid; Kinetics; NADP; Naphthalenes; Placenta; Pregnancy; Quercetin; Spectrophotometry, Ultraviolet

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only approximately 7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was approximately 1-5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.

Topics: Aldehyde Reductase; Amino Acid Oxidoreductases; Animals; Arginine; Benzothiazoles; Blood Pressure; Body Weight; Capillary Permeability; Citrulline; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Glycation End Products, Advanced; Guanidines; Imidazoles; Imidazolidines; Inositol; Iodine Radioisotopes; Male; Methylguanidine; Naphthalenes; Nitric Oxide Synthase; omega-N-Methylarginine; Phthalazines; Rats; Rats, Sprague-Dawley; Retina; Sciatic Nerve; Serum Albumin, Bovine; Sorbitol; Thiazoles; Uvea

The role of His42, His188, and Lys263 residues in the catalytic action of human aldose reductase was investigated in association with various inhibitors of this enzyme by site-directed mutagenesis. While mutations at His42- greater than Gln, His42- greater than Tyr, His188- greater than Gln, and His188- greater than Tyr brought small change in the kinetic parameters, Lys263- greater than Glu mutation markedly increased the Km value for the substrate DL-glyceraldehyde by a factor of 60. Lys263- greater than Met substitution resulted in approximately 14 fold elevation of Km for the substrate. By contrast, mutation of Lys263- greater than Arg significantly decreased the Km for the substrate with concomitant reduction in kcat. Moderate increase in Km values for the cofactor NADPH was demonstrated for mutated enzymes. These results are indicative of the possible role of Lys263 in the substrate binding through electrostatic interaction. The inhibitor constants (Ki) for structurally diverse aldose reductase inhibitors against mutated enzymes demonstrated different degree of alteration, indicating binding sites of aldose reductase inhibitors on the enzyme molecule vary from one another, and some of the sites are more closely correlated with the physicochemical property of Lys263.

Topics: Aldehyde Reductase; Amino Acid Sequence; Base Sequence; Binding Sites; Cloning, Molecular; Fluorenes; Humans; Hydantoins; Imidazoles; Imidazolidines; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; NADP; Naphthalenes; Structure-Activity Relationship

A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.

Topics: Administration, Oral; Aldehyde Reductase; Animals; Cattle; Chemical Phenomena; Chemistry; Diabetes Mellitus, Experimental; Diaphragm; Galactitol; Galactose; Glycine; Lens, Crystalline; Male; Molecular Structure; Naphthalenes; Prodrugs; Rats; Rats, Inbred Strains; Sciatic Nerve

The ability of aldose reductase inhibitors to prevent the decline in neural Na+,K(+)-ATPase activity in diabetic rats has not been confirmed by all laboratories. In this study, the efficacy of two structurally different aldose reductase inhibitors was evaluated under different experimental conditions. Na+,K(+)-ATPase activity was measured in sciatic nerves from streptozocin-induced diabetic rats fed normal rodent chow or a chow supplemented with 68% sucrose. Nerve homogenates from chow-fed rats were prepared with a Dounce tissue grinder, whereas homogenates from the sucrose-fed rats were prepared with an Ultra-Turrax disperser. In the chow-fed rats, 4 weeks of untreated diabetes resulted in an increase in neural sorbitol and fructose, a decrease in myoinositol, and a 54% decline in Na+,K(+)-ATPase activity. Sorbinil administration (20 mg/kg/day) completely prevented the rise in sorbitol and fructose and the depletion of myoinositol, but did not prevent the decline in Na+,K(+)-ATPase activity. In diabetic rats fed the sucrose diet for 4, 6, and 8 weeks, the neural sorbitol and fructose levels were elevated, the myoinositol concentration declined, and the Na+,K(+)-ATPase activity was 26 to 28% below the control. Prevention or intervention treatment with sorbinil (20 mg/kg/day) or tolrestat (50 mg/kg/day) for 4 to 6 weeks prevented the alterations in sorbitol, fructose, and myoinositol, and also prevented the decline in Na+,K(+)-ATPase activity. In conclusion, prevention and intervention therapy with aldose reductase inhibitors prevented the decline in Na+,K(+)-ATPase in sciatic nerves of sucrose-fed streptozocin-diabetic rats that were homogenized with an Ultra-Turrax disperser, but not in sciatic nerves from streptozocin-diabetic rats fed normal rodent chow that were homogenized with a Dounce tissue grinder. These findings indicate that the assessment of aldose reductase inhibitor efficacy is dramatically affected by the type of nerve preparation assayed and/or the diet.

Topics: Aldehyde Reductase; Animals; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Dietary Carbohydrates; Fructose; Glucose; Imidazoles; Imidazolidines; Inositol; Male; Naphthalenes; Rats; Rats, Inbred Strains; Sciatic Nerve; Sodium-Potassium-Exchanging ATPase; Sorbitol; Sucrose

The mechanism by which hyperglycaemia leads to diabetic complications has not been fully elucidated. Non-enzymatic glycosylation leads to considerable functional and structural alteration of proteins. Hyperglycaemia also induces changes in intracellular metabolites, particularly in the polyol pathway. Aldose reductase inhibitors, which block the polyol pathway, have been shown to prevent complications in animal models, and this provides the rationale for the large scale trials that are presently being conducted.

Topics: Aldehyde Reductase; Diabetes Complications; Diabetes Mellitus; Glycolysis; Glycosylation; Humans; Hyperglycemia; Imidazoles; Imidazolidines; Naphthalenes; Phthalazines; Rhodanine; Thiazolidines

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.

Topics: Aldehyde Reductase; Animals; Cell Aggregation; Cell Division; Cell Line; Cell Survival; Clone Cells; Glucose; Imidazoles; Imidazolidines; Kidney Medulla; Kinetics; Naphthalenes; Sodium Chloride; Sugar Alcohol Dehydrogenases

Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).

Topics: Alcohol Dehydrogenase; Aldehyde Reductase; Chromatography, High Pressure Liquid; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Female; Glyceraldehyde; Humans; Hydrogen-Ion Concentration; Imidazoles; Imidazolidines; Immunoenzyme Techniques; Kinetics; NADP; Naphthalenes; Phthalazines; Placenta; Pregnancy; Substrate Specificity; Sugar Alcohol Dehydrogenases

Topics: Aldehyde Reductase; Diabetic Retinopathy; Humans; Imidazoles; Imidazolidines; Naphthalenes; Sugar Alcohol Dehydrogenases

Kinetic patterns of inhibition of homogenous human kidney aldose reductase (AR, EC 1.1.1.21) and aldehyde reductase II (AR II, EC 1.1.1.19) by statil, ICI 105552 [1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-yl acetic acid], tolrestat, alrestatin, chromone carboxylic acid (CCA), quercetin, phenobarbital and sorbinil were studied. On the basis of the kinetic nature of inhibition, the inhibitors were classified into four distinct categories. For aldose reductase, sorbinil and phenobarbital were noncompetitive (NC; category I) and CCA and alrestatin were uncompetitive (UC; category II) to both the aldehyde substrate and NADPH. Quercetin and ICI 105552 were NC to the aldehyde and UC to NADPH (category III) and tolrestat and statil were UC to the aldehyde and NC to NADPH (category IV). For AR II, sorbinil and alrestatin were category I inhibitors, ICI 105552 and statil belong to category II, phenobarbital, tolrestat and CCA to category III, and quercetin to category IV. To determine the specificity of inhibition, the ratios of the inhibition constants (Kii) for AR and AR II were calculated. A lower ratio indicates greater specificity. With aldehyde as the varied substrate the specificity ratios were: statil less than ICI 105552 less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital, and with NADPH as the varied substrate, ICI 105552 less than statil less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital. For AR, double-inhibition plots generated for one inhibitor from each kinetic category versus sorbinil showed that AR inhibitors of categories I-III bind to the same site on the protein molecule as sorbinil. However, tolrestat seemed to bind to a site different from the sorbinil binding site. For AR II, inhibitors from all the four categories appeared to bind to the same inhibitor binding site.

Topics: Aldehyde Reductase; Aldehydes; Carbohydrate Dehydrogenases; Chromones; Humans; Imidazoles; Imidazolidines; Isoquinolines; Kidney; Kinetics; Naphthalenes; Phenobarbital; Phthalazines; Quercetin; Quinolones; Substrate Specificity; Sugar Alcohol Dehydrogenases

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.

Topics: Aldehyde Reductase; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Durapatite; Humans; Hydroxyapatites; Imidazoles; Imidazolidines; Isoelectric Focusing; Isoenzymes; Kinetics; Molecular Weight; Muscles; Myocardium; Naphthalenes; Phthalazines

A series of aldose reductase inhibitors was prepared in which structural modifications were made to three positions of the potent, orally active inhibitor tolrestat (1), namely, the 6-methoxy substituent, thioamide sulfur, and the N-methyl moiety. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated rat sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. Bioisosteric replacement of the 6-methoxy group of 1 with a methylthio substituent gave 5, and replacement of the thioamide substituent of 1 with a cyanoamidine gave 7. Both 5 and 7 retained high in vitro potency but were less potent in vivo than 1. Replacement of the tolrestat N-methyl group by a carbomethoxy moiety gave 10 and led to a substantial reduction in activity in each of the three assays employed. However, this same structural modification on oxo-tolrestat (2) led to 11 and resulted in an enhancement of the intrinsic activity and a comparable in vivo potency. The isolated nerve data suggest that some compounds in these series do not readily penetrate into peripheral nerves, and this presumably is a factor in their lack of oral activity.

Topics: Aldehyde Reductase; Animals; Chemical Phenomena; Chemistry; In Vitro Techniques; Male; Naphthalenes; Rats; Rats, Inbred Strains; Sciatic Nerve; Sugar Alcohol Dehydrogenases

Experimental dietary galactosemia is known to result in accumulation of the polyol, galactitol, via the aldose reductase metabolic pathway in a variety of tissues, including renal glomeruli. Because increased polyol accumulation also occurs in insulin-dependent diabetes mellitus (IDDM), in which marked renal glomerular hyperperfusion occurs, we have studied glomerular hemodynamics in rats with experimental galactosemia. Insulin deficiency and its accompanying metabolic disorders are not present in this experimental model. In additional groups of animals, aldose reductase inhibitors, either sorbinil or tolrestat, were added to the galactose diet. In all, five groups of rats were studied: regular diet, 50% galactose diet, regular diet plus sorbinil, 50% galactose diet plus sorbinil, and 50% galactose plus tolrestat. The diets were comparable in protein and salt, and the rats were pair fed. Micropuncture and whole kidney clearance measurements were carried out after 10-14 days on these diets. Compared with rats fed the regular diet, those fed with 50% galactose diet had significantly higher glomerular filtration rates, renal plasma flow, single-nephron glomerular filtration rates, and plasma flow (QA), whereas afferent vascular resistance (RA) was decreased. Addition of sorbinil or tolrestat to the high-galactose diet not only prevented renal hyperperfusion but RA and single-nephron filtration fraction (SNFF) were higher than in normal rats, and QA was lower. In addition, sorbinil administration to rats on the control diet caused significant decreases in single-nephron blood flow and the ultrafiltration coefficient and a rise in SNFF.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aldehyde Reductase; Animals; Galactose; Glomerular Filtration Rate; Imidazoles; Imidazolidines; Kidney Glomerulus; Male; Mathematics; Models, Theoretical; Naphthalenes; Nephrons; Rats; Rats, Inbred Strains; Reference Values; Sugar Alcohol Dehydrogenases

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on 1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and 2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic changes. We hypothesize that 1) increases in blood flow may reflect impaired contractile function of smooth muscle cells in resistance arterioles and 2) increases in vascular 125I-BSA permeation and urinary albumin excretion reflect impaired vascular barrier functional integrity in addition to increased hydraulic conductance secondary to microvascular hypertension associated with decreased vascular resistance.

Topics: Albuminuria; Aldehyde Reductase; Animals; Cardiac Output; Diabetes Mellitus, Experimental; Fluorenes; Glomerular Filtration Rate; Hemodynamics; Hydantoins; Imidazoles; Imidazolidines; Iodine Radioisotopes; Male; Naphthalenes; Rats; Rats, Inbred Strains; Reference Values; Regional Blood Flow; Serum Albumin, Bovine; Sorbitol; Strontium; Sugar Alcohol Dehydrogenases; Vascular Resistance

The distribution of aldose reductase and aldehyde reductase II in the epithelium, cortex and nuclear regions of the bovine lens has been studied. The levels of the two enzymes in different regions of the bovine lens were determined after partial purification by DEAE-cellulose (DE-52) column chromatography. Aldose reductase was present in all the three regions of the lens, whereas aldehyde reductase II was present mainly in the epithelium and cortex. The activity of the enzymes, expressed per mg protein, was 10-15 fold higher in lens epithelium as compared to cortex and when expressed per g tissue wet weight, was approximately 2 fold higher. Substrate specificity of aldose reductase purified from all three regions of the lens was comparable, but the susceptibility to inhibition by various aldose reductase inhibitors was significantly different. As compared to the enzyme of cortex and nucleus, the epithelial aldose reductase was less (30-40%) susceptible to inhibition by aldose reductase inhibitors such as sorbinil, tolrestat, statil and tetramethylene glutaric acid. The substrate specificity and characteristics of inhibition of aldehyde reductase II purified from epithelium and cortex were similar.

Topics: Alcohol Dehydrogenase; Aldehyde Reductase; Animals; Cattle; Chromatography, Ion Exchange; Epithelium; Fluorometry; Glutarates; Imidazoles; Imidazolidines; Kinetics; Lens Cortex, Crystalline; Lens Nucleus, Crystalline; Lens, Crystalline; Naphthalenes; Phenobarbital; Phthalazines; Spectrophotometry; Substrate Specificity

Topics: Aldehyde Reductase; Animals; Diabetes Mellitus, Experimental; Imidazoles; Imidazolidines; Male; Naphthalenes; Phenylephrine; Pupil; Rats; Rats, Inbred Strains; Sugar Alcohol Dehydrogenases

Topics: Aldehyde Reductase; Animals; Capillary Permeability; Diabetes Mellitus, Experimental; Galactose; Horseradish Peroxidase; Imidazoles; Imidazolidines; Iris; Male; Naphthalenes; Rats; Rats, Inbred Strains; Reflex, Pupillary; Sugar Alcohol Dehydrogenases

In view of the similarity of cataracts and neuropathy in galactose-fed and diabetic rats, the present experiments were undertaken to determine whether consumption of galactose-enriched diets (10, 25, or 50% by weight) also increases collagen crosslinking and permeation of vessels by 125I-albumin analogous to that observed in diabetic rats. The observations in these experiments: demonstrate that consumption of galactose-enriched diets for 3 wk selectively increases 125I-albumin permeation of the same vascular beds affected in diabetic rats and by diabetic vascular disease in humans (i.e., the aorta and vessels in the eye, kidney, sciatic nerve, and new tissue formed in the diabetic milieu); demonstrate that the susceptibility of the vasculature to aldose reductase-linked injury (increased permeability) varies greatly in different tissues; indicate that collagen solubility (crosslinking) changes in galactose-fed rats differ sharply from those in diabetic rats; and provide new evidence that consumption of galactose-enriched diets induces a hypogonadal state in male rats.

Topics: Aldehyde Reductase; Animals; Body Weight; Capillary Permeability; Collagen; Galactose; Imidazoles; Imidazolidines; Male; Naphthalenes; Organ Size; Prostate; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Solubility; Testosterone; Tissue Distribution

Carbon-13 nuclear magnetic resonance spectroscopy has been used in the study of glucose metabolism, specifically aldose reductase inhibition, in intact rabbit lenses maintained in organ culture. This technique provides an effective method of screening potential inhibitors of aldose reductase under conditions that more closely approximate in vivo conditions than do earlier methods. The aspirin substitutes acetaminophen and ibuprofen were studied as aldose reductase inhibitors and were found to be effective in reducing sorbitol accumulation in lenses exposed to high glucose stress. Results of this work with various inhibitors of aldose reductase are discussed in terms of lens metabolism and implications regarding diabetic complications such as cataract formation.

Topics: Acetaminophen; Aldehyde Reductase; Animals; Ibuprofen; Imidazoles; Imidazolidines; Lens, Crystalline; Magnetic Resonance Spectroscopy; Naphthalenes; Organ Culture Techniques; Rabbits; Sorbitol; Sugar Alcohol Dehydrogenases

Two structurally unrelated aldose reductase inhibitors (sorbinil and tolrestat) were used for assessing the possible role of aldose reductase in the thickening of retinal capillary basement membranes of rats. Rats were fed either a normal diet, a 50% galactose diet, or a 50% galactose diet with the addition of an aldose reductase inhibitor. Micrographs of capillaries in the outer plexiform layer of the retina were analysed. This showed that the basement membrane thickening and the ultrastructural changes in the retinal capillaries that were induced by the galactose diet were inhibited by the addition of an aldose reductase inhibitor. Thickening of the basement membrane may be related to other tissue lesions in diabetic retinopathy. Therefore, its prevention might have a favourable influence on other aspects of this retinopathy.

Topics: Aldehyde Reductase; Animals; Diabetic Retinopathy; Galactose; Imidazoles; Imidazolidines; Male; Naphthalenes; Rats; Rats, Inbred Strains; Sugar Alcohol Dehydrogenases; Sugar Alcohols

Topics: Aldehyde Reductase; Animals; Cattle; Diabetes Mellitus, Experimental; Galactitol; Isoquinolines; Lens, Crystalline; Naphthalenes; Rats; Sorbitol; Sugar Alcohol Dehydrogenases