Cellular interactions between L-arginine and asymmetric dimethylarginine: Transport and metabolism.

This study was aimed to examine the effect of L-arginine (ARG) exposure on the disposition of asymmetric dimethylarginine (ADMA) in human endothelial cells. Although the role of ADMA as an inhibitor of endothelial nitric oxide synthase (eNOS) is well-recognized, cellular interactions between ARG and ADMA are not well-characterized. EA.hy926 human vascular endothelial cells were exposed to 15N4-ARG, and the concentrations of 15N4-ARG and ADMA in the cell lysate and incubation medium were determined by a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) assay. Nitric oxide (NO) production was estimated by utilizing cumulative nitrite concentration via a fluorometric assay. Cells incubated with 15N4-ARG exhibited enhanced nitrite production as well as 15N4-ARG cellular uptake. These changes were accompanied by a decrease in cellular ADMA level and increase in extracellular ADMA level, indicating an efflux of endogenous ADMA from the cell. The time courses of ADMA efflux as well as nitrite accumulation in parallel with 15N4-ARG uptake were characterized. Following preincubation with 15N4-ARG and D7-ADMA, the efflux of cellular 15N4-ARG and D7-ADMA was significantly stimulated by high concentrations of ARG or ADMA in the incubation medium, demonstrating trans-stimulated cellular transport of these two amino acids. D7-ADMA metabolism was inhibited in the presence of added ARG. These results demonstrated that in addition to an interaction at the level of eNOS, ARG and ADMA may mutually influence their cellular availability via transport and metabolic interactions.

Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of 15N3 -citrulline in biological samples.

RATIONALE: We showed that the metabolite peaks of (15)N(3) -citrulline ((15)N(3) -CIT) and (15)N(3) -arginine ((15)N(3) -ARG) could be detected when (15) N(4) -ARG was metabolized by nitric oxide synthase (NOS) in endothelial cells. The usefulness of these metabolites as potential surrogate indices of nitric oxide (NO) generation is evaluated. METHODS: A hydrophilic-interaction liquid chromatography/electrospray tandem mass spectrometric assay (LC/MS/MS) was utilized for the simultaneous analysis of (15)N(4) -ARG, ARG, CIT, (15)N(3) -CIT and (15)N(3) -ARG. (15)N(3) -CIT and (15)N(3) -ARG from impurities of (15)N(4) -ARG were determined and corrected for the calculation of their concentration. (15)N(4) -ARG-derived NO, i.e., (15)NO formation was determined by analyzing (15)N-nitrite accumulation by another LC/MS/MS assay. RESULTS: After EA.hy926 human endothelial cells were challenged with (15)N(4) -ARG for 2 hours, the peak intensities of (15)N(3) -CIT and (15)N(3) -ARG significantly increased with (15)N(4) -ARG concentration and positively correlated with (15)N-nitrite production. The estimated Km values were independent of the metabolite (i.e., (15)N(3) -CIT, (15)N(3) -CIT+(15)N(3) -ARG or (15) N-nitrite) used for calculation. However, after correction for its presence as a chemical contaminant of (15)N(4) -ARG, (15)N(3) -ARG was only a marginal contributor for the estimation of NOS activity. CONCLUSIONS: These data suggest that the formation of (15)N(3) -CIT can be used as an indicator of NOS activity when (15)N(4) -ARG is used as a substrate. This approach may be superior to the radioactive (14)C-CIT method which can be contaminated by (14)C-urea, and to the (14)N-nitrite method which lacks sensitivity.

In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1.

Organic nitrates (ORNs) are commonly used anti-ischemic and anti-anginal agents, which serve as an exogenous source of the potent vasodilator nitric oxide (NO). Recently, both mitochondrial aldehyde dehydrogenase-2 (ALDH2) and cytosolic aldehyde dehydrogenase-1a1 (ALDH1A1) have been shown to exhibit the ability to selectively bioactivate various ORNs in vitro. The objective of the present research was to examine the potential role of ALDH3A1, another major cytosolic isoform of ALDH, in the in vitro bioactivation of various ORNs, and to estimate the enzyme kinetic parameters toward ORNs through mechanistic modeling. The extent of bioactivation was assayed by exposing recombinant ALDH3A1 to various concentrations of ORNs, and measuring the concentration-time profiles of released NO via a NO-specific electrode. Metabolite formation kinetics was monitored for nitroglycerin (NTG) using LC/MS/MS. Our results showed that ALDH3A1 mRNA and protein were highly expressed in C57BL/6 mouse aortic, cardiac, and hepatic tissues, and it was able to release NO from several ORNs, including NTG, isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN), and nicorandil with similar Vmax (0.175-0.503nmol/min/mg of ALDH3A1), and Km values of 4.01, 46.5, 818 and 5.75×10(3)µM, respectively. However, activation of isosorbide-5-mononitrate (IS-5-MN) by ALDH3A1 was undetectable in vitro. ALDH3A1 was also shown to denitrate NTG, producing primarily glyceryl 1,2-dinitrate (1,2-GDN) in preference to glyceryl 1,3-dinitrate (1,3-GDN). Therefore, ALDH3A1 may contribute to the bioactivation of ORNs in vivo.

Organic nitrate metabolism and action: toward a unifying hypothesis and the future-a dedication to Professor Leslie Z. Benet.

This review summarizes the major advances that had been reported since the outstanding contributions that Professor Benet and his group had made in the 1980s and 1990s concerning the metabolism and pharmacologic action of organic nitrates (ORNs). Several pivotal studies have now enhanced our understanding of the metabolism and the bioactivation of ORNs, resulting in the identification of a host of cysteine-containing enzymes that can carry out this function. Three isoforms of aldehyde dehydrogenase, all of which with active catalytic cysteine sites, are now known to metabolize, somewhat selectively, various members of the ORN family. The existence of a long-proposed but unstable thionitrate intermediate from ORN metabolism has now been experimentally observed. ORN-induced thiol oxidation in multiple proteins, called the "thionitrate oxidation hypothesis," can be used not only to explain the phenomenon of nitrate tolerance, but also the various consequences of chronic nitrate therapy, namely, rebound vasoconstriction, and increased morbidity and mortality. Thus, a unifying biochemical hypothesis can account for the myriad of pharmacological events resulting from nitrate therapy. Optimization of the future uses of ORN in cardiology and other diseases could benefit from further elaboration of this unifying hypothesis.

Mechanism of cellular oxidation stress induced by asymmetric dimethylarginine.

The mechanism by which asymmetric dimethylarginine (ADMA) induces vascular oxidative stress is not well understood. In this study, we utilized human umbilical vein endothelial cells (HUVEC) to examine the roles of ADMA cellular transport and the uncoupling of endothelial nitric oxide synthase (eNOS) in contributing to this phenomenon. Dihydroethidium (DHE) fluorescence was used as an index of oxidative stress. Whole cells and their isolated membrane fractions exhibited measureable increased DHE fluorescence at ADMA concentrations greater than 10 µM. ADMA-induced DHE fluorescence was inhibited by co-incubation with L-lysine, tetrahydrobiopterin (BH(4)), or L-nitroarginine methyl ester (L-NAME). Oxidative stress induced in these cells by angiotensin II (Ang II) were unaffected by the same concentrations of L-lysine, L-NAME and BH(4). ADMA-induced reduction in cellular nitrite or nitrite/nitrate production was reversed in the presence of increasing concentrations of BH(4). These results suggest that ADMA-induced DHE fluorescence involves the participation of both the cationic transport system in the cellular membrane and eNOS instead of the Ang II-NADPH oxidase pathway.

Continuous exposure to L-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells.

The therapeutic benefits of L-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells. Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on endothelial nitric oxide synthase (eNOS) expression and function, as well as glucose and superoxide (O2(·-) ) accumulation. Short-term (2 h) exposure to at least 50 µM ARG moderately increased eNOS activity and intracellular glucose (p < 0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O2(·-) accumulation. Co-incubation with 100 µM ascorbic acid, 300 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 100 µM L-lysine or 30 µM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido)-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.

Intracellular L-arginine concentration does not determine NO production in endothelial cells: implications on the "L-arginine paradox".

We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of (15)N(4)-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, (15)N(4)-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by(15)N-nitrite or estimated (15)N(3)-citrulline concentrations when (15)N(4)-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced (15)N(4)-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by (15)N-nitrite, total nitrite and (15)N(3)-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the "L-arginine paradox" should not consider intracellular ARG concentration as a reference point.

Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action.

The utility of an LC-MS/MS assay for nitrite determination in studying L-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0-500 µM of (15)N(4)-ARG for 2 h. (14)N-nitrite and (15)N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form (14)N- and (15)N-naphthotriazole (i.e., (14)N-NAT and (15)N-NAT). Peak responses of (14)N-NAT and (15)N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized (14)N-NAT and (15)N-NAT from (14)N-nitrite and (15)N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2% in quality control samples. Following incubation of EA.hy926 cells with (15)N(4)-ARG, saturable increases of (15)N-nitrite accumulation with increasing (15)N(4)-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in (14)N-nitrite level observed. When cellular fractions were exposed to (15)N(4)-ARG, (15)N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC-MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC-MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.

Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites.

Organic nitrate vasodilators (ORN) exert their pharmacologic effects through the metabolic release of nitric oxide (NO). Mitochondrial aldehyde dehydrogenase (ALDH2) is the principal enzyme responsible for NO liberation from nitroglycerin (NTG), but lacks activity towards other ORN. Cytosolic aldehyde dehydrogenase (ALDH1a1) can produce NO from NTG, but its activity towards other ORN is unknown. Using purified enzymes, we showed that both isoforms could liberate NO from NTG, isosorbide dinitrate (ISDN), and nicrorandil, while only ALDH1a1 metabolized isosorbide-2-mononitrate and isosorbide-5-mononitrate (IS-5-MN). Following a 10-min incubation with purified enzyme, 0.1 mM NTG and 1 mM ISDN potently inactivated ALDH1a1 (to 21.9% ± 11.1% and 0.44% ± 1.04% of control activity, respectively) and ALDH2 (no activity remaining and 4.57% ± 7.92% of control activity, respectively), while 1 mM IS-5-MN exerted only modest inactivation of ALDH1a1 (reduced to 89% ± 4.3% of control). Cytosolic ALDH in hepatic homogenates incubated at the vascular EC(50) concentrations of ORN was inactivated by NTG (to 45.1% ± 8.1% of control activity) while mitochondrial ALDH was inactivated by NTG and nicorandil (to 68.2% ± 10.0% and 78.7% ± 19.8% of control, respectively). Via site-directed mutagenesis, the active sites of ORN metabolism of ALDH2 (Cys-319) and ALDH1a1 (Cys-303) were found to be identical to those responsible for their dehydrogenase activity. Cysteine-302 of ALDH1a1 and glutamate-504 of ALDH2 were found to modulate the rate of ORN metabolism. These studies provide further characterization of the substrate selectivity, inactivation, and active sites of ALDH2 and ALDH1a1 toward ORN.

Identification of nitroglycerin-induced cysteine modifications of pro-matrix metalloproteinase-9.

Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase-9 (MMP-9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro-form of MMP-9 (proMMP-9), has not been investigated and was examined here using nano-flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision-induced dissociation (CID) and electron-transfer dissociation (ETD), were employed for the identification of various modifications. A high-resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP-9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP-9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr-262, in close proximity to an oxidized Cys-256 of proMMP-9. The intramolecular interaction between these two residues toward NTG-induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG.

Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS.

PURPOSE: L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹5N4-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples. METHODS: Concentrations of unlabeled ARG, ¹5N4-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C6-ARG, D4-CIT and D7-ADMA were used as internal standards for ARG and ¹5N4-ARG, CIT, and dimethylarginines, respectively. RESULTS: The calibration curves of ARG, ¹5N4-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹5N4-ARG, which allowed the observation of generation of ¹5N3-CIT and ¹5N3-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats. CONCLUSION: An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.

Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats.

Several studies suggested that long-term nitrate therapy may produce negative outcomes in patient mortality and morbidity. A possible mechanism may involve nitrate-mediated activation of various extracellular matrix (ECM) proteases, particularly matrix metalloproteinase-9 (MMP-9), and adhesion molecules in human macrophages, leading to the destabilization of atherosclerotic plaques. We examined the gene and protein regulating effects on THP-1 human macrophages by repeated exposure to therapeutically relevant concentrations of nitroglycerin (NTG) and possible involvement of nuclear factor (NF)-κB signaling mechanism in mediating some of these observed effects. THP-1 human macrophages repeatedly exposed to NTG (at 10 nM, added on days 1, 4 and 7) exhibited extensive alterations in the expression of multiple genes encoding ECM proteases and adhesion molecules. These effects were dissimilar to those produced by a direct nitric oxide donor, diethylenetriamine NONOate. NTG exposure significantly up-regulated NF-κB DNA nuclear binding activity and MMP-9 protein expression, and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression; these effects were abrogated in the presence of the NF-κB inhibitor parthenolide (a chemical inhibitor derived from the feverfew plant). Further, we examined whether our in vitro findings (an elevated MMP-9/TIMP-1 ratio and gelatinase activity) can be translated to in vivo effects, in a rat model. Sprague-Dawley rats exposed continuously to NTG subcutaneously for 8 days via mini-osmotic pumps showed significant induction of plasma MMP-9 dimer concentrations and the expression of a complex of MMP-9 with lipocalin-2 or neutrophil gelatinase associated lipocalin (NGAL). Plasma gelatinase activity was significantly increased by NTG over the entire study period, attaining peak elevation at day 6. Plasma TIMP-1 protein was down-regulated significantly by day 2 and days 4-7 in the NTG-treated rats. Pharmacokinetic monitoring of NTG and its dinitrate metabolites indicated that concentrations were well within therapeutic levels observed in humans. Our studies indicate that clinically relevant concentrations of NTG not only altered ECM matrix by changing the expression of multiple genes that govern cellular integrity, affecting cellular MMP-9/TIMP-1 balance in THP-1 human macrophages possibly via NF-κB activation, but also led to systemic changes in MMP-9/TIMP-1 expression and gelatinase activity in rats. These effects may contribute to extracellular matrix degradation and possible atherosclerotic plaque destabilization.

Broad regulation of matrix and adhesion molecules in THP-1 human macrophages by nitroglycerin.

Although nitroglycerin (NTG) is effective for the acute relief in coronary ischemic diseases, its long-term benefits in mortality and morbidity have been questioned. The possibility has been raised that NTG may increase the activity of matrix metalloproteinases (MMP), which could lead to disruption and dislodging of atherosclerotic plaques. This study examined the broad effects of acute NTG exposure on the expression and activity of genes encoding MMP-9, as well as an array of ECM and adhesion molecules in THP-1 human macrophages. Gene array studies identified that while NTG exposure (100microM, 48h) did not significantly increase MMP-9 gene expression, genes encoding testican-1, integrin alpha-1, thrombospondin-3, fibronectin-1 and MMP-26 were significantly down-regulated. On the other hand, genes encoding catenin beta-1 and vascular cell-adhesion molecule-1 were up-regulated. Real-time PCR studies confirmed significant down-regulation of testican-1 gene expression, but its protein expression was not significantly altered. NTG exposure, caused a significant increase in total MMP-9 protein expression (1.96-fold) and active MMP-9 (3.7-fold) concentrations. Recombinant MMP-9 was significantly activated by NTG and its dinitrate metabolites, indicating post-translation modification of this protein by organic nitrates. These results indicate that NTG exposure could broadly affect the gene expression and activity of proteases that govern the ECM cascade, thereby potentially altering atherosclerotic plaque stability.

Role of glutaredoxin-mediated protein S-glutathionylation in cellular nitroglycerin tolerance.

We hypothesize that nitroglycerin (NTG) causes direct oxidation of multiple cellular sulfhydryl (SH) proteins and that manipulation of SH redox status affects NTG tolerance. In LLC-PK1 cells, we found that nitrate tolerance, as indicated by cGMP accumulation toward NTG, was accompanied by increased protein [(35)S]cysteine incorporation, significant S-glutathionylation of multiple proteins, and decreased metabolic activity of several SH-sensitive enzymes, including creatine kinase, xanthine oxidoreductase, and glutaredoxin (GRX). Cells overexpressing GRX exhibited reduced cellular protein S-glutathionylation (PSSG) and absence of NTG tolerance, whereas those with silenced GRX showed increased extent of NTG-induced tolerance. Incubation of LLC-PK1 cells with oxidized glutathione led to several major observations associated with nitrate tolerance, namely, reduced cGMP accumulation, PSSG formation, superoxide accumulation, and the attenuation of these events by vitamin C. Aortic S-glutathionylated proteins increased approximately 3-fold in rats made tolerant in vivo to NTG and showed significant negative correlation with vascular responsiveness ex vivo. NTG incubation in EA.hy926 endothelial cells and LLC-PK1 cells led to increased S-glutathionylation and activity of p21(ras), a known mediator of cellular signaling. These results indicate that the hallmark events of NTG tolerance, such as reduced bioactivation and redox signaling, are associated with GRX-dependent protein deglutathionylation.

Dissociation between superoxide accumulation and nitroglycerin-induced tolerance.

We hypothesize that superoxide (O(2)(*-)) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O(2)(*-) accumulation and reduced cGMP response to NTG versus vehicle control. O(2)(*-) stimulated by NTG was reduced by oxypurinol (100 microM), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O(2)(*-) but did not reduce cGMP response. The O(2)(*-) scavenger tiron reduced Ang II-induced O(2)(*-) production but did not increase NTG-stimulated cGMP production. Using p47(phox-/-) and gp91(phox-/-) mice versus their respective wild-type controls (WT), we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg s.c., t.i.d. for 3 days), as indicated by their ex vivo pEC(50) and cGMP accumulation upon NTG challenge. In vitro aorta O(2)(*-) production was enhanced by NTG incubation in both p47(phox) null and WT mice. Pre-exposure of isolated mice aorta to 100 microM NTG for 1 h resulted in vascular tolerance toward NTG and increased O(2)(*-) accumulation. Oxypurinol (1 mM) reduced O(2)(*-) but did not attenuate vascular tolerance. These results suggest that O(2)(*-) does not initiate either in vitro and in vivo NTG tolerance, and that the p47(phox) and gp91(phox) subunits of NADPH oxidase are not critically required. Increased O(2)(*-) accumulation may be an effect, rather than an initiating cause, of NTG tolerance.

Pharmacokinetics of 1,4-butanediol in rats: bioactivation to gamma-hydroxybutyric acid, interaction with ethanol, and oral bioavailability.

1,4-Butanediol (BD), a substance of abuse, is bioactivated to gamma-hydroxybutyrate (GHB), but its fundamental pharmacokinetics (PK) have not been characterized. Because this bioactivation is partly mediated by alcohol dehydrogenase, we hypothesized that there may also be a metabolic interaction between ethanol (ETOH) and BD. We therefore studied, in rats, the plasma PK of GHB, BD and ETOH each at two intravenous (IV) doses, when each substance was given alone, and when GHB or BD was co-administered with ETOH. Results showed that bioconversion of intravenously administered BD to GHB was complete, and that both GHB and BD exhibited nonlinear PK. Various population PK models were analyzed using NONMEM VI, and the best disposition model was found to include two PK compartments each for BD, an (unmeasured) putative semialdehyde intermediate (ALD), GHB and ETOH, the presence of nonlinear (Michaelis-Menten) elimination for each compound, and several mutual inhibition processes. The most prominent mutual metabolic inhibition was found between ETOH and BD, while that between GHB and ETOH was not significant. In vitro studies using liver homogenates confirmed mutual metabolic inhibitions between GHB and BD. Oral absorption of BD was best described by a first-order process with lag-time and pre-systemic metabolism from BD to ALD. Oral absorption of BD (as BD plus ALD) was rapid and complete. The fraction of the absorbed dose entering the central compartment as BD was 30% for the 1.58 mmol/kg dose and 55% for the 6.34 mmol/kg dose. At 6.34 mmol/kg IV, the onset of loss of righting reflex (LRR) for BD was significantly delayed vs. that produced by GHB (72.0 +/- 9.1 min vs. 6.7 +/- 0.6 min, respectively, p < 0.001), and the total duration of LRR was prolonged for BD vs. GHB (192 +/- 28 min vs. 117 +/- 2 min, respectively, p < 0.05). Relative to IV dosing, oral BD produced similar but more variable LRR effects. These results may provide a quantitative PK framework for the understanding of the toxicokinetics and toxicodynamics of both BD and GHB.

Inactivation of hepatic enzymes by inhalant nitrite--in vivo and in vitro studies.

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P < .05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P < .05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2-) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.