ABSTRACT
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ABSTRACT
Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein ß (C/EBPß), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPß variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.
Subject(s)
Adipocytes/drug effects , Adipogenesis , CCAAT-Enhancer-Binding Protein-beta/metabolism , Nitric Oxide Donors/pharmacology , S-Nitrosoglutathione/pharmacology , 1-Methyl-3-isobutylxanthine/pharmacology , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/metabolism , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , Dexamethasone/pharmacology , Insulin/pharmacology , Leptin/genetics , Leptin/metabolism , Mice , PPAR gamma/genetics , PPAR gamma/metabolismABSTRACT
Nitric oxide synthase (NOS) catalyzes the transformation of l-arginine, molecular oxygen (O2), and NADPH-derived electrons to nitric oxide (NO) and l-citrulline. Under some conditions, however, NOS catalyzes the reduction of O2 to superoxide (O2-) instead, a phenomenon that is generally referred to as uncoupling. In principle, both the heme in the oxygenase domain and the flavins in the reductase domain could catalyze O2- formation. In the former case the oxyferrous (Fe(II)O2) complex that is formed as an intermediate during catalysis would dissociate to heme and O2-; in the latter case the reduced flavins would reduce O2 to O2-. The NOS cofactor tetrahydrobiopterin (BH4) is indispensable for coupled catalysis. In the case of uncoupling at the heme this is explained by the essential role of BH4 as an electron donor to the oxyferrous complex; in the case of uncoupling at the flavins it is assumed that the absence of BH4 results in NOS monomerization, with the monomers incapable to sustain NO synthesis but still able to support uncoupled catalysis. In spite of little supporting evidence, uncoupling at the reductase after NOS monomerization appears to be the predominant hypothesis at present. To set the record straight we extended prior studies by determining under which conditions uncoupling of the neuronal and endothelial isoforms (nNOS and eNOS) occurred and if a correlation exists between uncoupling and the monomer/dimer equilibrium. We determined the rates of coupled/uncoupled catalysis by measuring NADPH oxidation spectrophotometrically at 340â¯nm and citrulline synthesis as the formation of [3H]-citrulline from [3H]-Arg. The monomer/dimer equilibrium was determined by FPLC and, for comparison, by low-temperature polyacrylamide gel electrophoresis. Uncoupling occurred in the absence of Arg and/or BH4, but not in the absence of Ca2+ or calmodulin (CaM). Since omission of Ca2+/CaM will completely block heme reduction while still allowing substantial FMN reduction, this argues against uncoupling by the reductase domain. In the presence of heme-directed NOS inhibitors uncoupling occurred to the extent that these compound allowed heme reduction, again arguing in favor of uncoupling at the heme. The monomer/dimer equilibrium showed no correlation with uncoupling. We conclude that uncoupling by BH4 deficiency takes place exclusively at the heme, with virtually no contribution from the flavins and no role for NOS monomerization.
Subject(s)
Nitric Oxide Synthase/chemistry , Biopterins/analogs & derivatives , Biopterins/chemistry , Citrulline/chemistry , Enzyme Inhibitors/chemistry , Heme/chemistry , Humans , Imidazoles/chemistry , NADP/chemistry , Nitric Oxide Synthase/antagonists & inhibitors , Nitroarginine/chemistry , Oxygen/chemistry , Pichia/genetics , Protein MultimerizationABSTRACT
Soluble guanylyl cyclase (sGC, EC 4.6.1.2) is a key enzyme in the regulation of vascular tone. In view of the therapeutic interest of the NO/cGMP pathway, drugs were developed that either increase the NO sensitivity of the enzyme or activate heme-free apo-sGC. However, modulation of sGC activity by endogenous agents is poorly understood. In the present study we show that the maximal activity of NO-stimulated purified sGC is significantly increased by cytosolic preparations of porcine coronary arteries. Purification of the active principle by several chromatographic steps resulted in a protein mixture consisting of 100, 70, and 40â¯kDa bands on SDS polyacrylamide gel electrophoresis. The respective proteins were identified by LC-MS/MS as gelsolin, annexin A6, and actin, respectively. Further purification resulted in loss of activity, indicating an interaction of sGC with a protein complex rather than a single protein. The partially purified preparation had no effect on basal sGC activity or enzyme activation by the heme mimetic BAY 60-2770, suggesting a specific effect on the conformation of the NO-bound heterodimeric holoenzyme. Since the three proteins identified are all related to contractile elements of smooth muscle, our data suggest that regulation of vascular tone involves a modulatory interaction of sGC with the cytoskeleton.
Subject(s)
Cytoskeletal Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Nitric Oxide/pharmacology , Soluble Guanylyl Cyclase/metabolism , Animals , Coronary Vessels , Cytoskeletal Proteins/genetics , Gene Expression Regulation, Enzymologic/drug effects , Soluble Guanylyl Cyclase/genetics , SwineABSTRACT
According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.
Subject(s)
Aldehyde Dehydrogenase, Mitochondrial/metabolism , Drug Tolerance/physiology , Muscle, Smooth, Vascular/metabolism , Nitrates/metabolism , Nitric Oxide/metabolism , Nitroglycerin/metabolism , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Cell Line, Transformed , Cell Line, Tumor , Dithiothreitol/pharmacology , Female , Humans , Male , Mice , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Nitrates/pharmacology , Organ Culture Techniques , Rats , Rats, Sprague-DawleyABSTRACT
Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of â¼0.2 µM and maximal cGMP formation at 10 µM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one-oxidized (ferric) sGC was moderate, reaching â¼10%-15% of maximal activity. Dilution experiments of cinaciguat/Tween 20-preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide-sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.
Subject(s)
Benzoates/pharmacology , Enzyme Inhibitors/pharmacology , Molecular Mimicry , Protoporphyrins/metabolism , Soluble Guanylyl Cyclase/antagonists & inhibitors , Vasodilation/drug effects , Animals , Aorta, Thoracic/physiology , Cattle , Coronary Vessels/metabolism , Cyclic GMP/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Activation , Enzyme Stability , Lung/drug effects , Lung/enzymology , Protoporphyrins/chemistry , Rats, Sprague-Dawley , Soluble Guanylyl Cyclase/metabolism , Swine , Vasodilator Agents/pharmacologyABSTRACT
Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of â¼2.4·10(-5) and â¼7.9·10(-5) m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to â¼1.5·10(-5) m at pH 6.0 and increased to â¼8.3·10(-5) m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by â¼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of â¼1.9·10(-5) m in the presence of Arg and BH4 but exhibited much higher IC50 values (â¼1.0-6.1·10(-4) m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 â¼ 1.3-2.0·10(-5) m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.
Subject(s)
Enzyme Inhibitors/pharmacology , Hydrogen Sulfide/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide/pharmacology , Animals , Citrulline/biosynthesis , Drug Interactions , Escherichia coli/cytology , Escherichia coli/drug effects , Escherichia coli/metabolism , Glutathione/pharmacology , Humans , Mice , NADP/metabolism , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidation-Reduction/drug effects , Sulfhydryl Compounds/pharmacologyABSTRACT
Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9 ± 0.4 and 0.55 ± 0.06 M(-1)s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From this observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) seems to involve formation of a Mg(2+)â¢glutathione (GSH) complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.
Subject(s)
Glutathione/chemistry , Magnesium/pharmacology , Nitric Oxide/pharmacology , Nitrosation , Nitroso Compounds/chemistry , Oxygen/metabolism , Sulfhydryl Compounds/chemistry , Electrochemistry , Kinetics , Luminescent Measurements , Superoxide Dismutase/metabolismABSTRACT
(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) availability regulates nitric oxide and superoxide formation by endothelial nitric oxide synthase (eNOS). At low BH4 or low BH4 to 7,8-dihydrobiopterin (BH2) ratios the enzyme becomes uncoupled and generates superoxide at the expense of NO. We studied the effects of exogenously added BH2 on intracellular BH4/BH2 ratios and eNOS activity in different types of endothelial cells. Incubation of porcine aortic endothelial cells with BH2 increased BH4/BH2 ratios from 8.4 (controls) and 0.5 (BH4-depleted cells) up to ~20, demonstrating efficient reduction of BH2. Uncoupled eNOS activity observed in BH4-depleted cells was prevented by preincubation with BH2. Recycling of BH4 was much less efficient in human endothelial cells isolated from umbilical veins or derived from dermal microvessels (HMEC-1 cells), which exhibited eNOS uncoupling and low BH4/BH2 ratios under basal conditions and responded to exogenous BH2 with only moderate increases in BH4/BH2 ratios. The kinetics of dihydrofolate reductase-catalyzed BH4 recycling in endothelial cytosols showed that the apparent BH2 affinity of the enzyme was 50- to 300-fold higher in porcine than in human cell preparations. Thus, the differential regulation of eNOS uncoupling in different types of endothelial cells may be explained by striking differences in the apparent BH2 affinity of dihydrofolate reductase.
Subject(s)
Biopterins/analogs & derivatives , Biopterins/metabolism , Endothelium, Vascular/metabolism , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide/antagonists & inhibitors , Tetrahydrofolate Dehydrogenase/metabolism , Animals , Aorta/cytology , Aorta/metabolism , Cell Line , Cells, Cultured , Cyclic GMP/metabolism , Dermis/blood supply , Dermis/cytology , Dermis/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/enzymology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Microvessels/cytology , Microvessels/enzymology , Microvessels/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Superoxides/metabolism , Sus scrofaABSTRACT
Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10(-7)-10(-6) M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10(-7) M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 µM BH4 (2 min with 1 µM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.
Subject(s)
Biopterins/analogs & derivatives , Nitric Oxide Synthase Type I/metabolism , Animals , Binding Sites , Biopterins/metabolism , Biopterins/pharmacology , Enzyme Stability , Kinetics , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type I/chemistry , Oxygen/metabolism , Protein Binding/drug effects , Protein Multimerization , Protein Structure, Quaternary , Rats , Sf9 Cells , SpodopteraABSTRACT
Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.
Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Mitochondrial Proteins/antagonists & inhibitors , Nitroglycerin/metabolism , Onium Compounds/pharmacology , Vasodilator Agents/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase, Mitochondrial , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Catalytic Domain , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Female , Humans , In Vitro Techniques , Male , Molecular Docking Simulation , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Protein Binding , Rats , Rats, Sprague-Dawley , Swine , Vasodilation/drug effectsABSTRACT
Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg(2+). These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.
Subject(s)
Glutathione/metabolism , Nitric Oxide/metabolism , Oxygen/metabolism , Aerobiosis , Humans , Nitric Oxide Donors/metabolism , Nitrosation , Nitroso Compounds/metabolism , S-Nitrosoglutathione/metabolism , Sulfhydryl Compounds/metabolismABSTRACT
Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Blood Vessels/metabolism , Mutant Proteins/metabolism , Nitroglycerin/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase, Mitochondrial , Amino Acid Substitution , Biocatalysis/drug effects , Catalytic Domain , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Humans , Isoflavones/chemistry , Isoflavones/pharmacology , Mass Spectrometry , Models, Molecular , Molecular Structure , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation , Nitrates/chemistry , Nitrates/metabolism , Nitroglycerin/chemistry , Protein Binding , Protein Structure, Tertiary , Sulfur Compounds/chemistry , Sulfur Compounds/metabolismABSTRACT
Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.
Subject(s)
Biopterins/analogs & derivatives , Guanylate Cyclase/metabolism , Oxidative Stress/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Aorta/drug effects , Aorta/metabolism , Biopterins/pharmacology , Cardiovascular Diseases/metabolism , Cells, Cultured , Cyclic GMP/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Heme/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitroglycerin/pharmacology , Oxidation-Reduction/drug effects , Peroxynitrous Acid/metabolism , Pterins/pharmacology , Soluble Guanylyl Cyclase , Superoxides/metabolism , SwineABSTRACT
To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.
Subject(s)
Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Mutagenesis, Site-Directed/methods , Nitroglycerin/metabolism , Signal Transduction/genetics , Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase, Mitochondrial , Animals , Biotransformation/genetics , Cattle , Gene Silencing , Humans , Nitroglycerin/chemistry , NitrosationABSTRACT
Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 µM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 µM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Mitochondrial Proteins/metabolism , Pentaerythritol Tetranitrate/analogs & derivatives , Aldehyde Dehydrogenase, Mitochondrial , Animals , Aorta/drug effects , Aorta/enzymology , Aorta/metabolism , Dose-Response Relationship, Drug , Guanylate Cyclase/metabolism , Male , Mice , Mice, Inbred C57BL , Nitric Oxide/biosynthesis , Nitroglycerin/metabolism , Nitroglycerin/pharmacology , Pentaerythritol Tetranitrate/metabolism , Pentaerythritol Tetranitrate/pharmacology , Rats , Rats, Sprague-Dawley , Vasodilation/drug effectsABSTRACT
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Benzamides/pharmacology , Benzodioxoles/pharmacology , Genetic Variation , Mitochondrial Proteins/metabolism , Nitroglycerin/pharmacology , Vasodilator Agents/pharmacology , Acetaldehyde/metabolism , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase, Mitochondrial , Animals , Benzamides/metabolism , Benzodioxoles/metabolism , Cattle , Enzyme Activation/drug effects , Enzyme Activation/genetics , Asia, Eastern , Guanylate Cyclase/metabolism , Humans , Mitochondrial Proteins/genetics , NAD/metabolism , Nitric Oxide/biosynthesis , Nitroglycerin/metabolism , Oxidation-Reduction/drug effects , Vasodilator Agents/metabolismABSTRACT
Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited approximately 50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.
Subject(s)
Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Nitroglycerin/metabolism , Superoxides/metabolism , Aldehyde Dehydrogenase/isolation & purification , Aldehyde Dehydrogenase, Mitochondrial , Animals , Cattle , Escherichia coli/genetics , Guanylate Cyclase/isolation & purification , Guanylate Cyclase/metabolism , Humans , Lung/chemistry , NAD/metabolism , Nitric Oxide/metabolism , Nitroglycerin/analogs & derivatives , Point Mutation , Receptors, Cytoplasmic and Nuclear/isolation & purification , Receptors, Cytoplasmic and Nuclear/metabolism , Soluble Guanylyl Cyclase , Superoxide Dismutase/metabolismABSTRACT
Mitochondrial aldehyde dehydrogenase (ALDH2) may be involved in the biotransformation of glyceryl trinitrate (GTN), and the inactivation of ALDH2 by GTN may contribute to the phenomenon of nitrate tolerance. We studied the GTN-induced inactivation of ALDH2 by UV/visible absorption spectroscopy. Dehydrogenation of acetaldehyde and hydrolysis of p-nitrophenylacetate (p-NPA) were both inhibited by GTN. The rate of inhibition increased with the GTN concentration and decreased with the substrate concentration, indicative of competition between GTN and the substrates. Inactivation of p-NPA hydrolysis was greatly enhanced in the presence of NAD(+), and, to a lesser extent, in the presence of NADH. In the presence of dithiothreitol (DTT) inactivation of ALDH2 was much slower. Dihydrolipoic acid (LPA-H(2)) was less effective than DTT, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. When DTT was added after complete inactivation, dehydrogenase reactivation was quite modest (< or =16%). The restored dehydrogenase activity correlated inversely with the GTN concentration but was hardly affected by the concentrations of acetaldehyde or DTT. Partial reactivation of dehydrogenation was also accomplished by LPA-H(2) but not by GSH. We conclude that, in addition to the previously documented reversible inhibition by GTN that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to ALDH inactivation. Importantly, ALDH2-catalyzed GTN reduction was partly inactivated by preincubation with GTN, suggesting that the inactivation of GTN reduction is also partly irreversible. These observations are consistent with a significant role for irreversible inactivation of ALDH2 in the development of nitrate tolerance.
Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase/chemistry , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/chemistry , Nitroglycerin/chemistry , Acetaldehyde/chemistry , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase, Mitochondrial , Ascorbic Acid/chemistry , Cysteine/chemistry , Dithiothreitol/chemistry , Drug Tolerance , Enzyme Activation/drug effects , Glutathione/chemistry , Humans , Mitochondrial Proteins/metabolism , NAD/chemistry , Nitroglycerin/metabolism , Nitroglycerin/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Vasodilator Agents/chemistry , Vasodilator Agents/metabolism , Vasodilator Agents/pharmacologyABSTRACT
Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.