Cardiovascular characterization of the novel organic mononitrate NDIBP in rats.

Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 µM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 µM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.

Chemical Structure Regulates the Formation of Secondary Organic Aerosol and Brown Carbon in Nitrate Radical Oxidation of Pyrroles and Methylpyrroles.

Nitrogen-containing heterocyclic volatile organic compounds (VOCs) are important components of wildfire emissions that are readily reactive toward nitrate radicals (NO3) during nighttime, but the oxidation mechanism and the potential formation of secondary organic aerosol (SOA) and brown carbon (BrC) are unclear. Here, NO3 oxidation of three nitrogen-containing heterocyclic VOCs, pyrrole, 1-methylyrrole (1-MP), and 2-methylpyrrole (2-MP), was investigated in chamber experiments to determine the effect of precursor structures on SOA and BrC formation. The SOA chemical compositions and the optical properties were analyzed using a suite of online and offline instrumentation. Dinitro- and trinitro-products were found to be the dominant SOA constituents from pyrrole and 2-MP, but not observed from 1-MP. Furthermore, the SOA from 2-MP and pyrrole showed strong light absorption, while that from 1-MP were mostly scattering. From these results, we propose that NO3-initiated hydrogen abstraction from the 1-position in pyrrole and 2-MP followed by radical shift and NO2 addition leads to light-absorbing nitroaromatic products. In the absence of a 1-position hydrogen, NO3 addition likely dominates the 1-MP chemistry. We also estimate that the total SOA mass and light absorption from pyrrole and 2-MP are comparable to those from phenolic VOCs and toluene in biomass burning, underscoring the importance of considering nighttime oxidation of pyrrole and methylpyrroles in air quality and climate models.

S-Nitrosylation of Akt by organic nitrate delays revascularization and the recovery of cardiac function in mice following myocardial infarction.

The effects of long-term nitrate therapy are compromised due to protein S-Nitrosylation, which is mediated by nitric oxide (NO). This study is to determine the role of Akt S-Nitrosylation in the recovery of heart functions after ischaemia. In recombinant Akt protein and in HEK293 cells, NO donor decreased Akt activity and induced Akt S-Nitrosylation, but was abolished if Akt protein was mutated by replacing cysteine 296/344 with alanine (Akt-C296/344A). In endothelial cells, NO induced Akt S-Nitrosylation, reduced Akt activity and damaged multiple cellular functions including proliferation, migration and tube formation. These alterations were ablated if cells expressed Akt-C296/344A mutant. In Apoe-/- mice, nitroglycerine infusion increased both Akt S-Nitrosylation and infarct size, reduced Akt activity and capillary density, and delayed the recovery of cardiac function in ischaemic hearts, compared with mice infused with vehicle. Importantly, these in vivo effects of nitroglycerine in Apoe-/- mice were remarkably prevented by adenovirus-mediated enforced expression of Akt-C296/344A mutant. In conclusion, long-term usage of organic nitrate may inactivate Akt to delay ischaemia-induced revascularization and the recovery of cardiac function through NO-mediated S-Nitrosylation.

Highly Oxygenated Organic Nitrates Formed from NO3 Radical-Initiated Oxidation of ß-Pinene.

The reactions of biogenic volatile organic compounds (BVOC) with the nitrate radicals (NO3) are major night-time sources of organic nitrates and secondary organic aerosols (SOA) in regions influenced by BVOC and anthropogenic emissions. In this study, the formation of gas-phase highly oxygenated organic molecules-organic nitrates (HOM-ON) from NO3-initiated oxidation of a representative monoterpene, ß-pinene, was investigated in the SAPHIR chamber (Simulation of Atmosphere PHotochemistry In a large Reaction chamber). Six monomer (C = 7-10, N = 1-2, O = 6-16) and five accretion product (C = 17-20, N = 2-4, O = 9-22) families were identified and further classified into first- or second-generation products based on their temporal behavior. The time lag observed in the peak concentrations between peroxy radicals containing odd and even number of oxygen atoms, as well as between radicals and their corresponding termination products, provided constraints on the HOM-ON formation mechanism. The HOM-ON formation can be explained by unimolecular or bimolecular reactions of peroxy radicals. A dominant portion of carbonylnitrates in HOM-ON was detected, highlighting the significance of unimolecular termination reactions by intramolecular H-shift for the formation of HOM-ON. A mean molar yield of HOM-ON was estimated to be 4.8% (-2.6%/+5.6%), suggesting significant HOM-ON contributions to the SOA formation.

The new organic nitrate 2-nitrate-1,3-diocthanoxypropan (NDOP) induces nitric oxide production and vasorelaxation via activation of inward-rectifier potassium channels (KIR).

INTRODUCTION: Cardiovascular diseases are coupled to decreased nitric oxide (NO) bioavailability, and there is a constant search for novel and better NO-donors. Here we synthesized and characterized the cardiovascular effects of the new organic nitrate 2-nitrate-1,3-dioctanoxypropan (NDOP). METHODS: A combination of in vitro and in vivo experiments was performed in C57BL/6 mice and Wistar rats. Thus, the ability of NDOP in donating NO in a cell-free system and in vascular smooth muscles cells (VSMC) and its ability to induce vasorelaxation in aortic rings from mice were evaluated. In addition, changes in blood pressure and heart rate to different doses of NDOP were evaluated in conscious rats. Finally, acute pre-clinical toxicity to oral administration of NDOP was assessed in mice. RESULTS: In cell-free system, NDOP increased NO levels, which was dependent on xanthine oxidoreductase (XOR). NDOP also increased NO levels in VSMC, which was not influenced by endothelial NO synthase. Furthermore, incubation with the XOR inhibitor febuxostat blunted the vasorelaxation in aortic ring preparations. In conscious rats, NDOP elicited dose-dependent reduction in blood pressure accompanied with increased heart rate. In vessel preparations, NDOP (10-8-10-3 mol/L) induced endothelium-independent vasorelaxation, which was inhibited by the NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and hydroxocobalamin or by inhibition of soluble guanylyl cyclase using H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one. To investigate if NDOP acts through potassium channels, selective blockers were used. Inhibition of BKCa, Kv or KATP subtypes of potassium channels had no effect, but inhibition of inward-rectifier potassium channels (KIR) significantly reduced NDOP-mediated vasorelaxation. Lastly, NDOP showed low toxicity (LD50 ~5000 mg/kg). CONCLUSION: Bioactivation of NDOP involves functional XOR, and this new organic nitrate elicits vasorelaxation via NO-cGMP-PKG signaling and activation of KIR channels. Future studies should further characterize the underlying mechanism and evaluate the therapeutic benefits of chronic NDOP treatment in relevant cardiovascular disease models.

A chamber study of alkyl nitrate production formed by terpene ozonolysis in the presence of NO and alkanes.

Organic nitrates are relatively long-lived species and have been shown to have a potential impact on atmospheric chemistry on local, regional, and even global scales. However, the significance of these compounds in the indoor environment remains to be seen. This work describes an impinger-based sampling and analysis technique for organic nitrate species, focusing on formation via terpene ozonolysis in the presence of nitric oxide (NO). Experiments were conducted in a Teflon film environmental chamber to measure the formation of alkyl nitrates produced from α-pinene ozonolysis in the presence of NO and alkanes using gas chromatography with an electron capture detector. For the different concentrations of NO and O3 analyzed, the concentration ratio of [O3]/[NO] around 1 was found to produce the highest organic nitrate concentration, with [O3] = 100 ppb & [NO] = 105 ppb resulting in the most organic nitrate formation, roughly 5 ppb. The experiments on α-pinene ozonolysis in the presence of NO suggest that organic nitrates have the potential to form in indoor air between infiltrated ozone/NO and terpenes from household and consumer products.

Novel (pyrazolyl)benzenesulfonamides with a nitric oxide-releasing moiety as selective cyclooxygenase-2 inhibitors.

Inhibition of cyclooxygenase-2 (COX-2) is a promising anti-inflammatory therapeutic strategy, but long-term medication with COX-2-inhibitors (coxibs) may be associated with adverse cardiovascular effects. Functionalization of existing lead structures with nitric oxide (NO)-releasing moieties is an auspicious approach to minimize these effects. In this regard, an organic nitrate (-O-NO2) substituent was introduced at a (pyrazolyl)benzenesulfonamide lead structure. The novel NO-coxibs selectively inhibited COX-2 in a low micromolar range (IC50(COX-2): 0.22-1.27 µM) and are supposed to be promising anti-inflammatory compounds with, in parallel, positive effects on vascular homeostasis.

Efficacy of the long-acting nitro vasodilator pentaerithrityl tetranitrate in patients with chronic stable angina pectoris receiving anti-anginal background therapy with beta-blockers: a 12-week, randomized, double-blind, placebo-controlled trial.

BACKGROUND: The organic nitrate pentaerithrityl tetranitrate (PETN) has been shown to have ancillary properties that prevent the development of tolerance and endothelial dysfunction. This randomized, double-blind, placebo-controlled, multicentre study ('CLEOPATRA' study) was designed to investigate the anti-ischaemic efficacy of PETN 80 mg b.i.d. (morning and mid-day) over placebo in patients with chronic stable angina pectoris. METHODS AND RESULTS: A total of 655 patients were evaluated in the intention-to-treat population, randomized to PETN (80 mg b.i.d., n = 328) or placebo (n = 327) and completed the study. Patients underwent treadmill exercise tests at randomization, after 6 and 12 weeks of treatment. Treatment with PETN over 12 weeks did not modify the primary endpoint total exercise duration (TED, P = 0.423). In a pre-specified sub-analysis of patients with reduced exercise capacity (TED at baseline ≤9 min, n = 257), PETN appeared more effective than placebo treatment (P = 0.054). Superiority of PETN over placebo was evident in patients who were symptomatic at low exercise levels (n = 120; P = 0.017). Pentaerithrityl tetranitrate 80 mg b.i.d. was well tolerated, and the overall safety profile was comparable with placebo. CONCLUSION: Although providing no additional benefit in unselected patients with known coronary artery disease, PETN therapy, administered in addition to modern anti-ischaemic therapy, could increase exercise tolerance in symptomatic patients with reduced exercise capacity.

Chronic therapy with isosorbide-5-mononitrate causes endothelial dysfunction, oxidative stress, and a marked increase in vascular endothelin-1 expression.

AIMS: Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was corrected by vitamin C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. METHODS AND RESULTS: Male Wistar rats (n= 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Isosorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan in vivo/ex vivo treatment and suppressed by apocynin. In addition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed. CONCLUSION: Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-dependent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates.

Organic Nitrate Additive for High-Rate and Large-Capacity Lithium Metal Anode in Carbonate Electrolyte.

Lithium nitrate has been widely used to improve the interfacial stability of Li metal anode in ether electrolyte. However, the low solubility limits its application in carbonate electrolytes for high-voltage Li metal batteries. Herein, nitrated polycaprolactone (PCL-ONO2 ), which is prepared via the acylation of polycaprolactone diol (PCL-diol) followed by the grafting of nitrate group, has been proposed as an electrolyte additive to introduce high-concentration NO3 - into carbonate electrolytes for the first time. The theoretical calculations and X-ray photoelectron spectroscopy depth profiling demonstrate that the PCL-ONO2 additive preferentially reacts with Li metal and in situ constructs a stable dual-layered solid electrolyte interphase film, presenting an inner nitride-rich layer and an outer flexible PCL-based layer on the surface of Li metal anode. As a result, the Li metal anode delivers an impressive long-term cycling performance over 1400 h at an elevated area capacity of 10.0 mAh cm-2 and an ultrahigh current density of 10.0 mA cm-2 in the Li symmetrical cells. Moreover, the PCL-ONO2 additive enables the full cells constructed by coupling high-loading LiFePO4 (20.0 mg cm-2 ) or LiNi0.5 Co0.2 Mn0.3 (16.5 mg cm-2 ) cathode and thin Li metal anode (≈50 µm) to demonstrate greatly improved cycling stability and rate capability.

Measurement report of the change of PM2.5 composition during the COVID-19 lockdown in urban Xi'an: Enhanced secondary formation and oxidation.

Enhanced secondary aerosol formation was observed during the COVID-19 lockdown in Xi'an, especially for polluted episodes. More oxidized­oxygenated organic aerosol (MO-OOA) and sulfate showed the dominant enhancements, especially in large particle-mode. Meanwhile, relative humidity (RH) showed a positive promotion on the formation of sulfate and MO-OOA during the lockdown, but had no obvious correlation with less oxidized­oxygenated organic aerosol (LO-OOA) or nitrate. Organosulfurs (OS) displayed a higher contribution (~58%) than inorganic sulfate to total sulfate enhancement in the polluted episode during the lockdown. Although the total nitrate (TN) decreased during the lockdown ascribing to a larger reduction of inorganic nitrate, organic nitrate (ON) showed an obvious increase from pre-lockdown (0.5 ± 0.6 µg m-3 and 1 ± 2% of TN) to lockdown (5.3 ± 3.1 µg m-3 and 17 ± 9% of TN) in the polluted case (P < 0.05). In addition, RH also displayed a positive promotion on the formation of ON and OS, and the increases of both OS and ON were much efficient in the nighttime than in the daytime. These results suggest that higher RH and stagnant meteorology might facilitate the sulfate and MO-OOA enhancement, especially in the nighttime, which dominated the secondary aerosol enhancement in haze pollution during the lockdown.

Exploring the inorganic and organic nitrate aerosol formation regimes at a suburban site on the North China Plain.

Nitrate-driven aerosol pollution frequently occurs during winter over the North China Plain (NCP). Extensive studies have focused on inorganic nitrate formation, but few have focused on organic nitrates in China, precluding a thorough understanding of the nitrogen cycle and nitrate aerosol formation. Here, the inorganic (NO3,inorg) and organic nitrate (NO3,org) formation regimes under aerosol liquid water (ALW) and aerosol acidity (pH) influences were investigated during winter over the NCP based on data derived from an Aerodyne high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS). The campaign-averaged concentration of the total nitrate was 5.3 µg m-3, with a 13% contribution from NO3,org, which exhibited a significantly decreased contribution with increasing haze episode evolution. The diurnal cycles of NO3,inorg and NO3,org were similar, with high concentrations during the nighttime at a high ALW level, revealing the important role of aqueous-phase processes. However, the correlations between the aerosol pH and NO3,inorg (R2 = 0.13, P < 0.01) and NO3,org (R2 = 0.63, P < 0.01) during polluted periods indicated a contrasting effect of aerosol pH on inorganic and organic nitrate formation. Our results provide a useful reference for smog chamber studies and promote a better understanding of organic nitrate formation via anthropogenic emissions.

3-NOP: ADME studies in rats and ruminating animals.

3-NOP (3-nitrooxypropanol) reduces enteric methane formation in ruminants. A series of ADME studies in rats, lactating goats and beef cattle was performed. 3-NOP was entirely absorbed from the GIT of rats: approximately 75% of the administered 3-NOP was eliminated as carbon dioxide via exhalation and approximately 20% were excreted via urine. 3-NOP is oxidized to 3-nitrooxypropionic acid (NOPA) which is then hydrolyzed to 3-hydroxypropionic acid (HPA) and inorganic nitrate, the major rat plasma metabolites. NOPA is also a plasma metabolite in beef. The metabolism of 3-NOP is fast as indicated by the negligible amounts of 3-NOP found in rat plasma 2 h after dosing. HPA is a naturally occurring metabolite. It is either metabolized into carbon dioxide and acetyl-CoA or into propanoyl-CoA, the latter serves as substrate for gluconeogenesis. Gluconeogenesis is very prominent in lactating ruminants which use propanoyl-CoA as their main carbon source. Thus, the formation of lactose from 3-NOP by lactating goats is not unexpected. Lactose was the major metabolite of 3-NOP in the aqueous phase of milk. The incorporation of 3-NOP into endogenous metabolism makes it difficult to derive a marker residue, however, conservative risk assessment could be based on the measured radioactivity in tissues.

The potential of aldehyde dehydrogenase 2 as a therapeutic target in cardiovascular disease.

INTRODUCTION: Mitochondrial aldehyde dehydrogenase (ALDH-2) plays a major role in the ethanol detoxification pathway by removing acetaldehyde. Therefore, ALDH-2 inhibitors such as disulfiram represent the first therapeutic targeting of ALDH-2 for alcoholism therapy. Areas covered: Recently, ALDH-2 was identified as an essential bioactivating enzyme of the anti-ischemic organic nitrate nitroglycerin, bringing ALDH-2 again into the focus of clinical interest. Mechanistic studies on the nitroglycerin bioactivation process revealed that during bioconversion of nitroglycerin and in the presence of reactive oxygen and nitrogen species the active site thiols of ALDH-2 are oxidized and the enzyme activity is lost. Thus, ALDH-2 activity represents a useful marker for cardiovascular oxidative stress, a concept, which has been meanwhile supported by a number of animal disease models. Mechanistic studies on the protective role of ALDH-2 in different disease processes identified the detoxification of 4-hydroxynonenal by ALDH-2 as a fundamental process of cardiovascular, cerebral and antioxidant protection. Expert opinion: The most recent therapeutic exploitation of ALDH-2 includes activators of the enzyme such as Alda-1 but also cell-based therapies (ALDH-bright cells) that deserve further clinical characterization in the future.

Effects of organic and inorganic nitrate on aortic and carotid haemodynamics in heart failure with preserved ejection fraction.

AIMS: To assess the haemodynamic effects of organic vs. inorganic nitrate administration among patients with heart failure with preserved ejection fraction (HFpEF). METHODS AND RESULTS: We assessed carotid and aortic pressure-flow relations non-invasively before and after the administration of 0.4 mg of sublingual nitroglycerin (n = 26), and in a separate sub-study, in response to 12.9 mmoL of inorganic nitrate (n = 16). Nitroglycerin did not consistently reduce wave reflections arriving at the proximal aorta (change in real part of reflection coefficient, 1st harmonic: -0.09; P = 0.01; 2nd harmonic: -0.045, P = 0.16; 3rd harmonic: +0.087; P = 0.05), but produced profound vasodilatation in the carotid territory, with a significant reduction in systolic blood pressure (133.6 vs. 120.5 mmHg; P = 0.011) and a marked reduction in carotid bed vascular resistance (19 580 vs. 13 078 dynes · s/cm5 ; P = 0.001) and carotid characteristic impedance (3440 vs. 1923 dynes · s/cm5 ; P = 0.002). Inorganic nitrate, in contrast, consistently reduced wave reflections across the first three harmonics (change in real part of reflection coefficient, 1st harmonic: -0.12; P = 0.03; 2nd harmonic: -0.11, P = 0.01; 3rd harmonic: -0.087; P = 0.09) and did not reduce blood pressure, carotid bed vascular resistance, or carotid characteristic impedance (P = NS). CONCLUSIONS: Nitroglycerin produces marked vasodilatation in the carotid circulation, with a pronounced reduction in blood pressure and inconsistent effects on central wave reflections. Inorganic nitrate, in contrast, produces consistent reductions in wave reflections, and unlike nitroglycerin, it does so without significant hypotension or cerebrovascular dilatation. These haemodynamic differences may underlie the different effects on exercise capacity and side effect profile of inorganic vs. organic nitrate in HFpEF.

Cardiorespiratory effects induced by 2-nitrate-1,3-dibuthoxypropan are reduced by nitric oxide scavenger in rats.

The search for new nitric oxide donors is warranted by the limitations of organic nitrates currently used in cardiology. The new organic nitrate 2-nitrate-1,3-dibuthoxypropan (NDBP) exhibited promising cardiovascular activities in previous studies. The aim of this study was to investigate the cardiorespiratory responses evoked by NDBP and to compare them to the clinically used organic nitrate nitroglycerine (NTG). Arterial pressure, heart rate and respiration were recorded in conscious adult male Wistar rats. Bolus i.v. injection of NDBP (1 to 15mg/kg; n=8) and NTG (0.1 to 5mg/kg; n=8) produced hypotension. NDBP induced bradycardia at all doses, while NTG induced tachycardia at three lower doses but bradycardia at higher doses. Hydroxocobalamin (20mg/kg; HDX), a NO scavenger, blunted hypotension induced by NDBP (15mg/kg), and its bradycardic effect (n=6). In addition, HDX blunted both hypotension and bradycardia induced by a single dose of NTG (2.5mg/kg; n=6). Both NDBP and NTG altered respiratory rate, inducing a biphasic effect with a bradypnea followed by a tachypnea; HDX attenuated these responses. Our data indicate that NDBP and NTG induce hypotension, bradycardia and bradypnea, which are mediated by nitric oxide release.

Nitric oxide as a target for the hypotensive and vasorelaxing effects induced by (Z)-ethyl 12-nitrooxy-octadec-9-enoate in rats.

The cardiovascular effects induced by a new organic nitrate were investigated in rats. The (Z)-ethyl 12-nitrooxy-octadec-9-enoate (NCOE) was synthesized from ricinoleic acid, the major compound of the castor oil. NCOE induced significant and dose-dependent hypotension and bradycardia in normotensive rats. In rats pretreated with NCOE (60 mg/kg, i.v., once a day) for 4 consecutive days, hypotension induced by the nitrate was similar to that observed in rats that were not pretreated with the compound. The vasorelaxation induced by the compound was concentration-dependent (10(-10)-10(-3) M) in rat mesenteric artery rings, pre-contracted with phenylephrine (1 µM), with or without endothelium. Pre-incubation with PTIO (300 µM), a free radical form of NO (NO) scavenger, attenuated the NCOE vasorelaxation potency. However, in the presence of L-cysteine (3 mM), a reduced form of NO (NO-) scavenger, NCOE response was potentiated. NCOE effect was not changed in the presence of an inhibitor of cytochrome P450, proadifen (10 µM). On the other hand, the vasodilation was reduced in the presence of mitochondrial aldehyde dehydrogenase inhibitor (mtALDH), cyanamide (1 mM); soluble guanylyl cyclase inhibitor (sGC), ODQ (10 µM); and non-selective K+ channels blocker, TEA (3 mM). In addition the NCOE-induced vasorelaxation was reduced by BKCa (iberiotoxin, 100 nM) and KATP selective (glibenclamide, 10 µM) blockers, however the effect was not modified by a KV blocker (4-aminopyridine, 1 mM). Furthermore, NCOE increased NO levels in rat aortic smooth muscle cultured cells, detected by NO-sensitive probe DAF-2DA, by flow cytometry. These results together suggest that NCOE induces short-lasting hypotension and bradycardia, and promotes vasorelaxation due to NO release through the compound metabolism via mtALDH and consequent sGC, KATP and BKCa activation. Furthermore, the compound was not able to induce tolerance.