Organic nitrate reductase: reassessment of its subcellular localization and tissue distribution and its relationship to the glutathione transferases

1. Using a specific and sensitive GLC method for the determination of glyceryl trinitrate (GTN), its subcellular and tissue distribution were reassessed. Liver was the most active tissue, but activity was also detected in the heart, kidney and gut. In all tissues activity was localized in the soluble fraction. The activity of soluble glutathione S-transferase followed the same pattern, liver exhibiting the highest and the heart the lowest activity. 2. Pretreatment with phenobarbitone and 3-methylcholanthrene stimulated both the glutathione S-transferase and organic nitrate reductase activities. 3. Glutathione S-transferase activity was competitively inhibited by GTN. 4. A comparison of the plasma and hepatic metabolism of GTN revealed higher drug affinity for the hepatic enzyme.     

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).     

Cytochrome P-450 mediated biotransformation of organic nitrates

The vascular biotransformation of organic nitrates appears to be a prerequisite for their action as vasodilators. In the current study, we assessed the involvement of cytochrome P-450 in the denitration of glyceryl trinitrate and the enantiomers of isoidide dinitrate. Denitration of organic nitrates by the microsomal fraction of rat liver was NADPH dependent and followed apparent first-order kinetics. Under aerobic conditions, the t1/2 of D-isoidide dinitrate was significantly shorter than that of L-isoidide dinitrate (11.9 vs. 14.1 min, p less than or equal to 0.05), which is consistent with the greater potency of the D-enantiomer for vasodilation. Under anaerobic conditions, the denitration of glyceryl trinitrate was very rapid (t1/2 approximately 30 s). Organic nitrate biotransformation was inhibited by carbon monoxide, SKF 525A, and dioxygen. This suggests that the biotransformation of organic nitrates can occur through the direct interaction with the heme moiety of cytochrome P-450. The biotransformation of glyceryl trinitrate was catalyzed preferentially by those isoenzymes induced by phenobarbital. The biotransformation of glyceryl trinitrate was regioselective for 1,3-glyceryl dinitrate formation except in phenobarbital-induced microsomes under aerobic conditions, in which preferential formation of 1,2-glyceryl dinitrate occurred. These data suggest that cytochrome P-450 is involved in the biotransformation of organic nitrates and raises the possibility that vascular cytochrome P-450 may play a role in the mechanism-based biotransformation of organic nitrates, the result of which is vascular smooth muscle relaxation.     

A comparative study of glyceryl trinitrate biotransformation and glyceryl trinitrate induced relaxation in bovine pulmonary artery and vein

Recent evidence supports the hypothesis that the mechanism by which glyceryl trinitrate induces relaxation of vascular smooth muscle involves the biotransformation of glyceryl trinitrate. This study was conducted to determine if there was a direct correlation between the capacity of vascular smooth muscle preparations to biotransform glyceryl trinitrate and their sensitivity to the relaxant effect of this organic nitrate. Isolated bovine pulmonary arteries and veins were contracted submaximally and cumulative dose-response relationships to glyceryl trinitrate were obtained; the vein was approximately 10 times more sensitive than the artery to glyceryl trinitrate induced relaxation. In a separate series of experiments, these vascular tissues were contracted submaximally and incubated with 0.5 microM [14C]glyceryl trinitrate for 2 min, during which glyceryl trinitrate induced relaxation was monitored. At 2 min, tissue samples were taken for determination of glyceryl trinitrate and glyceryl-1,2- and 1,3-dinitrate content by thin-layer chromatography and liquid scintillation spectrometry. Biotransformation of glyceryl trinitrate to glyceryl dinitrate occurred concomitantly with relaxation of these blood vessels. The concentration of glyceryl dinitrate in the vein was significantly less than that in the artery (p less than or equal to 0.05), even though significantly greater relaxation of the vein than the artery was observed (p less than or equal to 0.05). From these data, a simple linear relationship between glyceryl trinitrate biotransformation and relaxation is not apparent.     

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation.

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.     

Regulation of human mitochondrial aldehyde dehydrogenase (ALDH-2) activity by electrophiles in vitro

Recently, mitochondrial aldehyde dehydrogenase (ALDH-2) was reported to reduce ischemic damage in an experimental myocardial infarction model. ALDH-2 activity is redox-sensitive. Therefore, we here compared effects of various electrophiles (organic nitrates, reactive fatty acid metabolites, or oxidants) on the activity of ALDH-2 with special emphasis on organic nitrate-induced inactivation of the enzyme, the biochemical correlate of nitrate tolerance. Recombinant human ALDH-2 was overexpressed in Escherichia coli; activity was determined with an HPLC-based assay, and reactive oxygen and nitrogen species formation was determined by chemiluminescence, fluorescence, protein tyrosine nitration, and diaminonaphthalene nitrosation. The organic nitrate glyceryl trinitrate caused a severe concentration-dependent decrease in enzyme activity, whereas incubation with pentaerythritol tetranitrate had only minor effects. 4-Hydroxynonenal, an oxidized prostaglandin J(2), and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg(2+) and Ca(2+). Hydrogen peroxide and NO generation caused only minor inhibition of ALDH-2 activity, whereas peroxynitrite generation or bolus additions lead to severe impairment of the enzymatic activity, which was prevented by the thioredoxin/thioredoxin reductase (Trx/TrxR) system. In the presence of glyceryl trinitrate and to a lesser extent pentaerythritol tetranitrate, ALDH-2 may be switched to a peroxynitrite synthase. Electrophiles of different nature potently regulate the enzymatic activity of ALDH-2 and thereby may influence the resistance to ischemic damage in response to myocardial infarction. The Trx/TrxR system may play an important role in this process because it not only prevents inhibition of ALDH-2 but is also inhibited by the ALDH-2 substrate 4-hydroxynonenal.     

Biotransformation of glyceryl trinitrate by rat brain homogenate.

Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biotransformation of the organic nitrate vasodilator drug to a mixture of glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN). Heating of the RBH at 100 degrees C for 5 min and (or) pretreatment with 5 mM N-ethylmaleimide at 37 degrees C for 10 min demonstrated that about two-thirds of the GTN biotransformation activity was due to a sulfhydryl-dependent enzymatic process resulting in the predominant formation of 1,2-GDN, and that the remaining biotransformation activity was due to a sulfhydryl-dependent nonenzymatic process resulting in the selective formation of 1,3-GDN. In a preliminary experiment, nitric oxide formation was observed during the incubation of GTN with RBH under anaerobic conditions. These data support the idea that some of the therapeutic and adverse effects of GTN are mediated through its action in the central nervous system.     

Computational identification of significantly regulated metabolic reactions by integration of data on enzyme activity and gene expression

The concentrations and catalytic activities of enzymes control metabolic rates. Previous studies have focused on enzyme concentrations because there are no genome-wide techniques used for the measurement of enzyme activity. We propose a method for evaluating the significance of enzyme activity by integrating metabolic network topologies and genome-wide microarray gene expression profiles. We quantified the enzymatic activity of reactions and report the 388 significant reactions in five perturbation datasets. For the 388 enzymatic reactions, we identified 70 that were significantly regulated (P-value < 0.001). Thirty-one of these reactions were part of anaerobic metabolism, 23 were part of low-pH aerobic metabolism, 8 were part of high-pH anaerobic metabolism, 3 were part of low-pH aerobic reactions, and 5 were part of high-pH anaerobic metabolism.     

Interaction between cigarettes and propranolol in treatment of angina pectoris.

To determine whether cigarette smoking interferes with the medical management of angina pectoris, 10 patients with angina pectoris who smoked at least 10 cigarettes a day were studied before, during, and after a standardised maximal exercise test. This was done at the end of four randomly allocated one-week treatment periods during which the patients took glyceryl trinitrate while not smoking, took glyceryl trinitrate while smoking, took glycerly trinitrate and propranolol (380 mg/day) while not smoking, and took glyceryl trinitrate and propranolol while smoking. Carboxyhaemoglobin was measured to ensure compliance. Smoking was associated with a significantly higher heart rate, blood pressure, number of positions with ST-segment depression, and total ST-segment depression after exercise than non-smoking (p < 0.01) whether or not the patients were taking propranolol. These results suggest that smoking aggravates the simple haemodynamic variables used to assess myocardial oxygen requirements and the exercise-induced precordial electrocardiographic signs of myocardial ischaemia. These effects were still evident after treatment with propranolol and represent a hindrance to the effective medical treatment of angina pectoris.     

Buccaling under the pressure: influence of secondary care establishments on the prescribing of glyceryl trinitrate buccal tablets in primary care.

OBJECTIVE: To determine which characteristics were the best predictors of high rates of prescribing of glyceryl trinitrate buccal tablets. DESIGN: Practice and patient characteristics from 197 practices were examined, and a multiple regression analysis was performed to examine which variables were important in predicting this prescribing. SETTING: Former family health services authority (197 practices). MAIN OUTCOME MEASURE: Volume of prescribing of glyceryl trinitrate buccal tablets. RESULTS: Four variables contributed significantly to a multiple regression model: the catchment area of the secondary care establishment; the number of partners in a practice; the level of practice deprivation; and whether the practice served an urban or a rural area. The model suggests that the most important variable was the catchment area of the secondary care establishment in which the practice was located. CONCLUSION: Although only the prescribing of short acting glyceryl trinitrate buccal tablets was studied, an impact of this size on primary care prescribing may have extensive implications for all drug expenditure in primary care.     

Determination of glyceryl trinitrate in human plasma by gas chromatography—negative ion chemical ionization—selected ion monitoring

A specific, sensitive and accurate quantitation method for glyceryl trinitrate was developed using gas chromatography—negative ion chemical ionization—selected ion monitoring with dichloromethane as a reagent gas. [¹⁵N₃] and [²H₅, ¹⁵N₃] variants were synthesized from non-labelled or [²H₈]glycerol and [¹⁵N]nitric acid. The former variant was used for preventing adsorption of glyceryl trinitrate onto active sites on column materials and the latter was used as an internal standard for quantitation of glyceryl trinitrate in biological fluids by selected ion monitoring. The quantitation limit of this method is 0.1 ng/ml of human plasma. When glyceryl trinitrate was administered intravenously in the dose of 4 μg/kg to patients receiving hypotensive anesthesia for surgical operation, the plasma levels exhibited a biexponential decay. The mean and standard deviation of half-lives of the α and β phases were found to be about 0.41 ± 0.13 and 5.34 ± 1.60 min, respectively.     

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay

An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O-pyrenedecyl-sn-glycerol and high-performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on the presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine, and cerebellum. Activity was not uniformly distributed in brain: it was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is found not only in liver and the gastrointestinal tract but also in brain and other organs of the rat and provide an additional goal for tetrahydrobiopterin biosynthesis in these organs.     

Practolol in Treatment of Angina Pectoris. A Double-blind Trial

Twenty-four patients with angina pectoris entered a double-blind trial of the cardioselective beta-adrenergic blocking agent practolol. Seventeen experienced less angina and consumed fewer glyceryl trinitrate tablets when on the active preparation. There was also a decrease in the mean number of attacks suffered by patients while on practolol and a reduction in the number of glyceryl trinitrate tablets taken. These results are of statistical significance at, at least, the 5% level.     

Enzymatic activities in European flat oyster, Ostrea edulis, and pacific oyster, Crassostrea gigas, hemolymph

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.     

Differential biotransformation of glyceryl trinitrate by red blood cell-supernatant fraction and pulmonary vein homogenate

We have demonstrated previously that glyceryl trinitrate (GTN) undergoes biotransformation to two glyceryl dinitrate (GDN) metabolites in the human red blood cell-supernatant fraction (RBC-SF) by hemoglobin-mediated and sulfhydryl-dependent enzymatic mechanisms. In the present study, we have shown that biotransformation of GTN in rabbit RBC-SF yields a glyceryl-1,2-dinitrate (1,2-GDN)/glyceryl-1,3-dinitrate (1,3-GDN) ratio of 5.3. Following inhibition of hemoglobin-mediated biotransformation of GTN by carbon monoxide (CO), the 1,2-GDN/1,3-GDN ratio was 2.1. Following inhibition of sulfhydryl-dependent biotransformation by N-ethylmaleimide (NEM), the 1,2-GDN/1,3-GDN ratio was 30.0. We have demonstrated previously that for GTN-induced vasodilation of isolated bovine pulmonary vein (BPV), the 1,2-GDN/1,3-GDN ratio was 7.1, which indicated that a hemoprotein-dependent process was involved in GTN biotransformation. To determine if this was the case, the biotransformation of GTN (0.51 microM) was studied in BPV homogenates; 31.1 pmol GDN/mg BPV protein was formed in 20 min. The 1,2-GDN/1,3-GDN ratio was 1.1, which indicated that hemoprotein-mediated biotransformation did not occur. This conclusion was supported by the fact that CO did not inhibit GTN biotransformation. GTN biotransformation by BPV homogenate was inhibited 62% by NEM, 89% by boiling of the homogenate, and almost completely by boiling plus NEM. These results indicated that biotransformation of GTN by the BPV homogenate involved in a combination of enzymatic and nonenzymatic processes that were mostly sulfhydryl dependent. It is concluded that the mechanism for GTN biotransformation in isolated intact BPV, which yielded preferential formation of 1,2-GDN, was rendered nonfunctional upon tissue homogenization.     

Formation of glyceryl 2-mononitrate by regioselective bioconversion of glyceryl trinitrate: efficiency of the filamentous fungus Phanerochaete chrysosporium

Various microorganisms have been evaluated for their ability to hydrolyze glyceryl trinitrate (GTN) to glyceryl dinitrates and mononitrates. Provided that the GTN extracellular concentration was under the lethal dose, metabolite formation and regioselectivity depend on the nature of the strain used. In particular, Phanerochaete chrysosporium at a sublethal dose (3 mM) converts GTN into 1,2-glyceryl dinitrate and 2-glyceryl mononitrate (2-GMN) with a 80% regioselectivity in both steps. This bioconversion, when carried out in fermentors at 28 degrees C, allowed formation of 2-GMN at a rate of 12 mumol/h/g of dried mycelium. Successive batches of 3 mM GTN could be converted into 2-GMN as long as consecutive additions of glycerol or glucose were effected to ensure cell survival and the efficiency of the enzymatic system involved.     

Light-limitation on predator-prey interactions: consequences for metabolism and locomotion of deep-sea cephalopods

The present study attempts to correlate the metabolism and locomotory behavior of 25 species of midwater Cephalopoda from California and Hawaii with the maximal activities of key metabolic enzymes in various locomotory muscle tissues. Citrate synthase (CS) and octopine dehydrogenase (ODH) activities were used as indicators of aerobic and anaerobic metabolic potential respectively. CS activity in mantle muscle is highly correlated with whole-animal rates of oxygen consumption, whereas ODH activity in mantle muscle is significantly correlated with a species' ability to buffer the acidic end-products of anaerobic metabolism. Both CS and ODH activities in mantle muscle declined strongly with a species' habitat depth. For example, CS and ODH activities ranged respectively from 0.04 units g(-1) and 0.03 units g(-1) in the deep-living squid Joubiniteuthis portieri, to 8.13 units g(-1) and 420 units g(-1) in the epipelagic squid Sthenoteuthis oualaniensis. The relationships between enzymatic activities and depth are consistent with similar patterns observed for whole-animal oxygen consumption. This pattern is believed to result from a relaxation, among deep-living species, in the need for strong locomotory abilities for visual predator/prey interactions; the relaxation is due to light-limitation in the deep sea. Intraspecific scaling patterns for ODH activities may, for species that migrate ontogenetically to great depths, reflect the counteracting effects of body size and light on predator-prey detection distances. When scaled allometrically, enzymatic activities for the giant squid, Architeuthis sp., suggest a fairly active aerobic metabolism but little burst swimming capacity. Interspecific differences in the relative distributions of enzymatic activities in fin, mantle, and arm tissue suggest an increased reliance on fin and arm muscle for locomotion among deep-living species. We suggest that, where high-speed locomotion is not required, more efficient means of locomotion, such as fin swimming or medusoid arm propulsion, are more prevalent.     

Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the Yeast Saccharomyces bulderi

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.     

Acute and chronic arterial and venous effects of captopril in congestive cardiac failure.

OBJECTIVE--To determine whether captopril alters peripheral venous tone in patients with congestive cardiac failure. DESIGN--Open study of patients at start of captopril treatment and three months later. SETTING--A hospital gamma camera laboratory. PATIENTS--16 Men with congestive cardiac failure in New York Heart Association class II or III, aged 57-73. INTERVENTIONS--Patients were initially given 500 micrograms sublingual glyceryl trinitrate followed by 25 mg oral captopril. The study was then repeated after three months'' captopril treatment. MAIN OUTCOME MEASURES--Previously validated non-invasive radionuclide techniques were used to measure changes in central haemodynamic variables and peripheral venous volumes in the calf. RESULTS--After 25 mg captopril there were falls in blood pressure and relative systemic vascular resistance and increases in cardiac index and left ventricular ejection fraction. This was accompanied by a 16% increase in peripheral venous volume (95% confidence interval 13.4% to 18.4%, p less than 0.01), which compared with an 11% increase after 500 micrograms glyceryl trinitrate (10% to 12%, p less than 0.01). Eleven patients were restudied after three months'' continuous treatment with captopril. The resting venous volume was higher than it had been initially, by about 10%, and increased by a further 8.4% after 25 mg captopril (5.4% to 11.4%, p less than 0.05). CONCLUSIONS--Captopril is an important venodilator. Venous and arterial dilatation are produced short term and during long term treatment.     

Activities of oxidative enzymes in mycoplasmas.

The activities of several oxidoreductases were measured in three fermentative and two nonfermentative Mycoplasma species that were grown under aerobic or anaerobic conditions. Acholeplasma laidlawii MG, Mycoplasma hyorhinis GDL, and Mycoplasma pneumoniae FH had very high apparent activities of pyruvate dehydrogenase and pyruvate dehydrogenase complex compared with the activities of mammalian fibroblasts or human platelet-enriched preparations, while Mycoplasma salivarium VV and Mycoplasma arthritidis 07 had very low apparent activities of these two enzymes. Strictly anaerobic growth diminished both enzymatic activities. The activity of alpha-ketoglutarate dehydrogenase complex was minimal in all five mycoplasmas that were grown under aerobic conditions, anaerobic conditions, or both. All the mycoplasmas that were examined exhibited lactate dehydrogenase and NADH-dichlorophenol indophenol oxidoreductase activities. The properties of mycoplasmal pyruvate dehydrogenase complex suggest that it differs from the mammalian enzyme.