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.

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.

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.

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.

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.

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.

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.

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.

Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography-mass spectrometry with electrospray ionization.

We have developed a liquid chromatographic-mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH(4)Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C(18) column (100 mm x 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5-15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 degrees C. The degradation half-life of NTG was found to be 4.5 +/- 0.4 h and 39.2 +/- 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 +/- 0.1 and 0.2 +/- 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 degrees C was shown to be 26.8 +/- 1.8 min, consistent with previous values obtained using gas chromatography.

Determination of nitric oxide-donor effects on tissue gene expression in vivo using low-density gene arrays.

Gene array technology has been used to examine gene expression changes following drug treatments, including administration of nitric oxide (NO) donors. High-density arrays represent a powerful and popular method to analyze a large number of genes simultaneously. On the other hand, low-density arrays, available commercially at a lower cost, allow for the use of gene-specific primers, which reduces the risk of cross-hybridization among genes with similar sequence. For certain experiments in which the hypothesis is focused on a selected set of genes, use of low-density arrays might be more productive and cost-effective. Here, we describe our experience using low-density arrays to examine the effect of exposure to the NO-donor isobutyl nitrite on the expression of 23 cancer- and angiogenesis-related genes in mouse tissues. Detailed descriptions of data capture procedures, statistical tests, and confirmation studies using real-time quantitative (RTQ) reverse transcription polymerase chain reaction (RT-PCR) are presented. Three simple statistical methods, namely Student's t test, significant analysis of microarrays (SAM), and permutation adjusted t statistics (PATS), were applied on our gene array data, and their utilities were compared. All three methods yielded concordant results for the most significant genes, namely vascular endothelial growth factor (VEGF), VEGF receptor 3, Smad5, and Smad7. RT-PCR confirmed VEGF upregulation as observed via gene arrays. PATS appeared to be more robust than SAM in handling our small gene array data set. This statistical method, therefore, appears more suited for analyzing low-density gene array data. We conclude that low-density gene array is a useful screening method that can be performed with lower cost and less cumbersome data treatment.

Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice.

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (-/-)) and wild-type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg(-1)s.c. 3 x daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (-/-) and (+/+) mice, the EC(50) from NTG concentration-response curve was increased by approximately 3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (-/-) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.

eNOS-dependent vascular interaction between nitric oxide and calcitonin gene-related peptide in mice: gender selectivity and effects on blood aggregation.

The present study was performed to explore a possible vascular interplay between nitric oxide (NO) and calcitonin gene-related peptide (CGRP). We examined factors affecting CGRP release by the NO donor, nitroglycerin (NTG) and the potential involvement of endothelial NO synthase (eNOS) using eNOS knockout (-/-) vs. wild-type (+/+) mice. In the female eNOS (+/+) mice, but not in males, in vitro NTG (0.73 mM) induced significant increases in the release of CGRP-like immunoreactivity (CGRP-LI) from the aorta and the heart but not from the small intestine. In eNOS (-/-) mice, NTG incubation did not induce any CGRP-LI changes in either gender. These results suggest that NTG-induced CGRP release is eNOS-dependent and tissue- and gender-selective. The functional implication of this NO-CGRP interaction was further examined by testing the anti-aggregatory action of acetylcholine (Ach). Ach-induced platelet inhibition was significantly enhanced by the addition of aorta segments of either gender. However, the female aorta segments exhibited a greater platelet inhibitory effect, which could be reversed by the blockade of either CGRP or eNOS. Our study revealed a novel eNOS-dependent interaction between NO and CGRP, and the possible participation of regulatory peptides in affecting platelet function and possibly cardiovascular protection in females.

Liquid chromatographic-mass spectrometric determination of endogenous gamma-hydroxybutyrate concentrations in rat brain regions and plasma.

A new liquid chromatographic-mass spectrometric (LC-MS) method for determining trace concentrations of gamma-hydroxybutyric acid (GHB) in biological samples has been developed. This method utilizes solid-phase extraction for separation, deuterated GHB as an internal standard (IS) and multiple reaction monitoring (MRM) in the negative ion mode to detect the parent and product ions (103 and 57 for GHB, and 109 and 61 for D6-GHB, respectively). The assay produces excellent linearity and reproducibility, with a limit of quantification (LOQ) of about 0.1 microg/ml. The method has been applied for the determination of endogenous GHB in various rat brain regions.

Effects of obesity on the pharmacodynamics of nitroglycerin in conscious rats.

Literature reports have suggested that hemodynamic response toward organic nitrates may be reduced in obese patients, but this effect has not been studied. We compared the mean arterial pressure (MAP) responses toward single doses of nitroglycerin (NTG, 0.5-50 micro g) in conscious Zucker obese (ZOB), Zucker lean (ZL), and Sprague-Dawley (SD) rats. NTG tolerance development in these animal groups was separately examined. Rats received 1 and 10 micro g/min of NTG or vehicle infusion, and the maximal MAP response to an hourly 30 micro g NTG IVchallenge dose (CD) was measured. Steady-state NTG plasma concentrations were measured during 10 micro g/min NTG infusion. The Emax and ED50 values obtained were 33.9 +/- 3.6 and 3.5 +/- 1.7 micro g for SD rats, 33.2 +/- 4.1 and 3.0 +/- 1.4 micro g for ZL rats, and 34.8 +/- 3.9 and 5.3 +/- 2.8 micro g for ZOB rats, respectively. No difference was found in the dose-response curves among these 3 groups (P >.05, 2-way ANOVA). Neither the dynamics of NTG tolerance development, nor the steady-state NTG plasma concentrations, were found to differ among these 3 animal groups. These results showed that ZOB rats are not more resistant to the hemodynamic effects of organic nitrates compared with their lean controls. Thus, the acute and chronic hemodynamic effects induced by NTG are not sensitively affected by the presence of obesity in a conscious animal model of genetic obesity.

cDNA microarray analysis of vascular gene expression after nitric oxide donor infusions in rats: implications for nitrate tolerance mechanisms.

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 microg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-microg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P <.001, analysis of variance [ANOVA]), but not after SNAP (P >.05, ANOVA) or control infusion (P >.05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the down-regulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.