Biotransformation of organic nitrates by glutathione S-transferases and other enzymes: An appraisal of the pioneering work by William B. Jakoby.

Organic nitrates (R-ONO2; R, organic residue) such as nitroglycerin are used as drugs in part for more than a century. Their pharmacological use is associated with clinically relevant tolerance which is reportedly known since 1888. The underlying mechanisms of both, the mechanisms of action and the main pharmacological effect, which is vasodilatation and reduction of blood pressure, and the development of tolerance, which means increasing need of drug amount in sustained long-term therapy, are still incompletely understood. William B. Jakoby and associates were the first to report the biotransformation of organic nitrates, notably including nitroglycerin (i.e., glycerol trinitrate; GTN), by glutathione S-transferase (GST)-catalyzed conjugation of glutathione (GSH) to the nitrogen atom of one of the three nitrate groups of GTN to generate glutathione sulfenyl nitrite (glutathione thionitrate, S-nitroglutathione; GSNO2). Jakoby's group was also the first to suggest that GSNO2 reacts with a second GSH molecule to produce inorganic nitrite (ONO-) and glutathione disulfide (GSSG) without the catalytic involvement of GST. This mechanism has been adopted by others to the biotransformation of GTN by mitochondrial aldehyde dehydrogenase (mtALDH-(CysSH)2) which does not require GSH as a substrate. The main difference between these reactions is that mtALDH forms an internal thionitrate (mtALDH-(CysSH)-CysSNO2) which releases inorganic nitrite upon intra-molecular reaction to form mtALDH disulfide (mtALDH-(CysS)2). Subsequently, ONO- and GSNO2 are reduced by several proteins and enzymes to nitric oxide (NO) which is a very potent activator of soluble guanylyl cyclase to finally relax the smooth muscles thus dilating the vasculature. GSNO2 is considered to rearrange to GSONO which undergoes further reactions including GSNO and GSSG formation. The present article is an appraisal of the pioneering work of William B. Jakoby in the area of the biotransformation of organic nitrates by GST. The two above mentioned enzymatic reactions are discussed in the context of tolerance development to organic nitrates, still a clinically relevant pharmacological concern.

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase.

BACKGROUND: Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. METHODS: Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. RESULTS: In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. CONCLUSIONS: Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

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.

Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction: targeting nitric oxide and endothelin.

Hemoglobin-based oxygen carrier (HBOC)-201 is a cell-free modified hemoglobin solution potentially facilitating oxygen uptake and delivery in cardiovascular disorders and hemorrhagic shock. Clinical use has been hampered by vasoconstriction in the systemic and pulmonary beds. Therefore, we aimed to 1) determine the possibility of counteracting HBOC-201-induced pressor effects with either adenosine (ADO) or nitroglycerin (NTG); 2) assess the potential roles of nitric oxide (NO) scavenging, reactive oxygen species (ROS), and endothelin (ET) in mediating the observed vasoconstriction; and 3) compare these effects in resting and exercising swine. Chronically instrumented swine were studied at rest and during exercise after administration of HBOC-201 alone or in combination with ADO. The role of NO was assessed by supplementation with NTG or administration of the eNOS inhibitor Nω-nitro-l-arginine. Alternative vasoactive pathways were investigated via intravenous administration of the ETA/ETB receptor blocker tezosentan or a mixture of ROS scavengers. The systemic and to a lesser extent the pulmonary pressor effects of HBOC-201 could be counteracted by ADO; however, dosage titration was very important to avoid systemic hypotension. Similarly, supplementation of NO with NTG negated the pressor effects but also required titration of the dose. The pressor response to HBOC-201 was reduced after eNOS inhibition and abolished by simultaneous ETA/ETB receptor blockade, while ROS scavenging had no effect. In conclusion, the pressor response to HBOC-201 is mediated by vasoconstriction due to NO scavenging and production of ET. Further research should explore the effect of longer-acting ET receptor blockers to counteract the side effect of hemoglobin-based oxygen carriers.NEW & NOTEWORTHY Hemoglobin-based oxygen carrier (HBOC)-201 can disrupt hemodynamic homeostasis, mimicking some aspects of endothelial dysfunction, resulting in elevated systemic and pulmonary blood pressures. HBOC-201-induced vasoconstriction is mediated by scavenging nitric oxide (NO) and by upregulating endothelin (ET) production. Pressor effects can be prevented by adjuvant treatment with NO donors or direct vasodilators, such as nitroglycerin or adenosine, but dosages must be carefully monitored to avoid hypotension. However, hemodynamic normalization is more easily achieved via administration of an ET receptor blocker.

Tumor-Targeting Salmonella typhimurium A1-R: An Overview.

The present chapter reviews the development of the tumor-targeting amino-acid auxotrophic strain S. typhimurium A1 and the in vivo selection and characterization of the high-tumor-targeting strain S. typhimurium A1-R. Efficacy of S. typhimurium A1-R in nude-mouse models of prostate, breast, pancreatic, and ovarian cancer, as well as sarcoma and glioma in orthotopic mouse models is described. Also reviewed is efficacy of S. typhimurium A1-R targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma, breast-cancer brain metastasis, and experimental breast-cancer bone metastasis in orthotopic mouse models. The efficacy of S. typhimurium A1-R on pancreatic cancer stem cells, on pancreatic cancer in combination with anti-angiogenic agents, as well as on cervical cancer, soft-tissue sarcoma, and pancreatic cancer patient-derived orthotopic xenograft (PDOX) mouse models, is also described.

Enhancement of Tumor-Targeted Delivery of Bacteria with Nitroglycerin Involving Augmentation of the EPR Effect.

The use of bacteria, about 1 µm in size, is now becoming an attractive strategy for cancer treatment. Solid tumors exhibit the enhanced permeability and retention (EPR) effect for biocompatible macromolecules such as polymer-conjugated anticancer agents, liposomes, and micelles. This phenomenon permits tumor-selective delivery of such macromolecules. We report here that bacteria injected intravenously evidenced a property similar to that can of these macromolecules. Bacteria that can accumulate selectively in tumors may therefore be used in cancer treatment.Facultative or anaerobic bacteria will grow even under the hypoxic conditions present in solid tumors. We found earlier that nitric oxide (NO) was among the most important factors that facilitated the EPR effect via vasodilatation, opening of endothelial cell junction gaps, and increasing the blood flow of hypovascular tumors. Here, we describe the augmentation of the EPR effect by means of nitroglycerin (NG), a commonly used NO donor, using various macromolecular agents in different tumor models. More importantly, we report that NG significantly enhanced the delivery of Lactobacillus casei to tumors after intravenous injection of the bacteria, more than a tenfold increase in bacterial accumulation in tumors after NG treatment. This finding suggests that NG has a potential advantage to enhance bacterial therapy of cancer, and further investigations of this possibility are warranted.

Relation of mitochondrial oxygen consumption in peripheral blood mononuclear cells to vascular function in type 2 diabetes mellitus.

Recent studies have shown mitochondrial dysfunction and increased production of reactive oxygen species in peripheral blood mononuclear cells (PBMCs) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to adenosine triphosphate (ATP) production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMCs that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMCs and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192 ± 47 vs 161 ± 44 pmoles/min, p = 0.01), uncoupled (64 ± 16 vs 53 ± 13 pmoles/min, p = 0.007), and maximal (795 ± 87 vs 715 ± 128 pmoles/min, p=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1 ± 6.6 vs 15.8 ± 4.8%, p = 0.03) and correlated with maximal oxygen consumption (r = -0.64, p=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for the assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Biodegradation of nitroglycerin and ethylene glycol dinitrate by free and immobilized mixed cultures.

Aerobic biodegradation of nitroglycerin (NG) and ethylene glycol dinitrate (EGDN), both as individual substrates and in their mixture, was tested using batch or fed-batch cultivation with free suspended cells enriched from a soil sample subjected to a long-term contamination with explosives. EGDN was degraded only in the presence of glycerol as a co-substrate whereas NG could serve as a sole carbon, energy and nitrogen source for growth, its degradation being only slightly boosted by either glycerol or pyruvate. NG was not sufficient as a co-substrate for microbial growth on EGDN; furthermore, the presence of EGDN inhibited the NG degradation. The growth inhibition by both NG and EGDN was alleviated by the addition of glycerol. At an optimum nitroglycerin concentration of 30 mg/L, a maximum specific degradation rate of 60.9 ± 1.8 mg/gdw/h was observed. The biodegradation of both pollutants occurred with a release of nitrite. A method was developed for growing substantial amounts of NG-degrading biomass in the presence of glycerol for its immobilization on expanded slate in a pot-scale packed-bed reactor. Preliminary reactor tests were conducted in a continuous operation mode yielding a 70-90% NG biodegradation up to a load of 20 mg/L/h, with a removal rate up to 16 mg/L/h.

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity.

Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K(m) 0.84 mM; V(max) 36 µM/min) and the embryonic eye (K(m) 0.20 mM; V(max) 30 µM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.

Direct intramyocardial mesenchymal stromal cell injections in patients with severe refractory angina: one-year follow-up.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6-8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up (p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 (p < 0.001), angina attacks per week from 13.8 to 3.2 (p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week (p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life (p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Key enzymes enabling the growth of Arthrobacter sp. strain JBH1 with nitroglycerin as the sole source of carbon and nitrogen.

Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG.

Vascular bioactivation of nitroglycerin is catalyzed by cytosolic aldehyde dehydrogenase-2.

RATIONALE: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. OBJECTIVE: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. METHODS AND RESULTS: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. CONCLUSIONS: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.

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.

Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

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.

Evaluation of anti-migraine potential of Areca catechu to prevent nitroglycerin-induced delayed inflammation in rat meninges: possible involvement of NOS inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Areca catechu nut extract is a popular folk remedy for the treatment of migraine in Kerala and Tamil Nadu states of India. AIM OF THE STUDY: In order to prove the claimed utilization of plant, the effect of hydroalcoholic extract of Areca catechu nut (ANE) was investigated in nitroglycerine induced inflammation in rat meninges. In these models infusion of nitric oxide donor glyceryl trinitrate (GTN) produces augmented plasma protein extravasation (PPE) in dura mater, provides an important substrate for the development of migraine in rats. MATERIALS AND METHODS: The effect on plasma protein extravasation was assessed in both the models of intravenous and topical GTN application following oral administration of ANE (250 mg/kg and 500 mg/kg) in both curative and preventive treatment and compared with that of control positive. The l-NAME (15 mg/kg, i.v.) was used as reference standard. Plasma protein extravasation was measured using fluorescein as marker and was measured using a Perkin-Elmer LS-30 luminescence spectrometer. RESULTS: Expression of iNOS in the spleen after intravenous injection produced PPE into the dura mater in control positive group was significantly (P<0.01) reduced to 1.553±0.02499 and 1.398±0.01887 by preventive treatment with ANE at the dose of 250 and 500 mg/kg, orally, respectively. The extravasation produced by topical GTN due to expression of iNOS in dural macrophages was also reduced to 1.555±0.03384 and 1.425±0.01204 by preventive treatment with ANE at the dose of 250 and 500 mg/kg, orally, respectively. While ANE do not showed any significant results in curative treatment in both the models of i.v. and topical GTN application. CONCLUSION: These findings collectively indicate that the extract exhibited significant inhibition of iNOS, which may be the probable mechanism for its anti-migraine activity, providing evidence, at least in part, for its folkloric use.

Nitric oxide synthase-dependent responses of the basilar artery during acute infusion of nicotine.

INTRODUCTION: Our goals were to determine whether acute exposure to nicotine alters nitric oxide synthase (NOS)-dependent responses of the basilar artery and to identify a potential role for activation of NAD(P)H oxidase in nicotine-induced impairment in NOS-dependent responses of the basilar artery. METHODS: We measured in vivo diameter of the basilar artery in response to NOS-dependent (acetylcholine) and NOS-independent (nitroglycerin) agonists before and during an acute infusion of nicotine (2 microg/kg/min intravenously for 30 min followed by a maintenance dose of 0.35 microg/kg/min). In addition, we measured superoxide anion production (lucigenin chemiluminescence) by the basilar artery in response to nicotine in the absence or presence of apocynin. RESULTS: We found that NOS-dependent, but not NOS-independent, vasodilation was impaired during infusion of nicotine. In addition, treatment of the basilar artery with apocynin (100 microM, 30 min prior to infusion of nicotine) prevented nicotine-induced impairment in NOS-dependent vasodilation. Further, the production of superoxide anion was increased in the basilar artery by nicotine, and this increase could be inhibited by apocynin. DISCUSSION: Our findings suggest that acute exposure to nicotine impairs NOS-dependent dilation of the basilar artery by a mechanism that appears to be related to the release of superoxide anion. A possible source of superoxide may be via the activation of NAD(P)H oxidase.

Implantation of paclitaxel-eluting stent impairs the vascular compliance of arteries in porcine coronary stenting model.

BACKGROUND: The impaired compliance of large and medium-sized muscular arteries has been shown to correlate with the risk of adverse cardiovascular events. We assessed coronary artery distensibility using simultaneous intracoronary ultrasound and pressure wire measurements in porcine coronary arteries after implantation of paclitaxel-eluting (PES) and bare metal stents (BMS) and compared this with the histopathology of the arterial wall injury. METHODS: PES and BMS were implanted into porcine left coronary arteries under general anesthesia. At 1-month follow-up (FUP) the endothelium-dependent and endothelium-independent vascular compliances were measured after intracoronary infusion of 10(-6)M acetylcholine for 2.5min, and intracoronary bolus of 100microg nitroglycerine, respectively. The arterial stiffness index, distensibility and reflexion index were calculated in stented arteries (n=25 PES and n=25 BMS), and correlated with histopathologic and histomorphometric changes of the vessel wall. RESULTS: In spite of smaller neointimal area, the fibrin deposition, medial thickening, vascular wall inflammation scores and arterial remodeling index were elevated and endothelialization was impaired in arteries with PES. Arteries with PES exhibited significantly worse endothelium-dependent vascular compliance: the stiffness (p<0.001) and reflexion index (p<0.001) were significantly higher and the distensibility index (p<0.001) lower as compared with the arteries with BMS. The endothelium-independent vascular reaction was similarly impaired in arteries with PES, as the stiffness index (p<0.001) and the distensibility index (p<0.001) differed significantly between the PES and BMS groups. Incomplete endothelialization (r=0.617, p<0.001) was significantly associated with the endothelium-dependent increased vascular stiffness. The increased fibrin score (r=0.646, p<0.001), vessel wall inflammation (r=0.657, p<0.001) and medial thickening (r=0.672, p<0.001) correlated significantly with the endothelium-independent stiffness index. CONCLUSIONS: Implantation of PES impairs the coronary artery wall structure and the endothelium-dependent and independent vessel wall dynamics more than does the implantation of BMS.

Partially irreversible inactivation of mitochondrial aldehyde dehydrogenase by nitroglycerin.

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.

Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway.

AIM: To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. METHODS: Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free alpha-minimum essential media plus 10% fetal bovine serum (dextran-coated charcoal stripped) supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L beta-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 micromol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. RESULTS: The treatment of HBMSC with NTG (0.1-10 micromol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evidenced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 micromol/L) could not be abolished by either N(G ) -nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity. CONCLUSION: NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.

Dynamics of nitroglycerin-induced exhaled nitric oxide after lung transplantation: evidence of pulmonary microvascular injury?

BACKGROUND: In search of real-time molecular correlates to ischemia-reperfusion-induced lung injury, we explored the hypothesis that liberation of nitric oxide (NO) into exhaled breath after pulmonary microvascular bioconversion of nitroglycerin (GTN) is attenuated in clinical lung transplantation. METHODS: Exhaled NO was measured under basal conditions and after intravenous administration of GTN in patients undergoing lung transplantation. Patients undergoing routine cardiac surgery served as controls. Basal and GTN-induced exhaled NO was also measured in donors before retrieval and after implantation in recipients. RESULTS: The characteristic GTN-induced exhaled NO response observed in cardiac surgical patients before cardiopulmonary bypass and in lung transplant and multiple-organ donors was nearly totally abolished in lung transplant recipients. This response was also attenuated to a lesser degree in the routine cardiac surgery patients after cardiopulmonary bypass. CONCLUSIONS: These results suggest a graded influence of time-factored complete and partial ischemia on GTN-induced evolution of NO into exhaled breath, providing biochemical evidence for a degree of microvascular injury, which can be monitored non-invasively at the bedside.