Endogenous NO Research Articles

Oxygen-Evolving Radiotherapy-Radiodynamic Therapy Synergized with NO Gas Therapy by Cerium-Based Rare-Earth Metal-Porphyrin Framework.

The efficacy of traditional radiotherapy (RT) has been severely limited by its significant side effects, as well as tumor hypoxia. Here, the nanoscale cerium (Ce)-based metaloxo clusters (Ce(IV)6)-porphyrin (meso-tetra (4-carboxyphenyl) porphyrin, TCPP) framework loaded with L-arginine (LA) (denoted as LA@Ce(IV)6-TCPP) is developed to serve as a multifarious radio enhancer to heighten X-ray absorption and energy transfer accompanied by O2/NO generation for hypoxia-improved RT-radiodynamic therapy (RDT) and gas therapy. Within tumor cells, LA@Ce(IV)6-TCPP will first react with endogenous H2O2 and inducible NO synthase (iNOS) to produce O2 and NO to respectively increase the oxygen supply and reduce oxygen consumption, thus alleviating tumor hypoxia. Then upon X-ray irradiation, LA@Ce(IV)6-TCPP can significantly enhance hydroxyl radical (•OH) generation from Ce(IV)6 metaloxo clusters for RT and synchronously facilitate singlet oxygen (1O2) generation from adjacently-coordinated TCPP for RDT. Moreover, both the •OH and 1O2 can further react with NO to generate more toxic peroxynitrite anions (ONOO-) to inhibit tumor growth for gas therapy. Benefitting from the alleviation of tumor hypoxia and intensified RT-RDT synergized with gas therapy, LA@Ce(IV)6-TCPP elicited superior anticancer outcomes. This work provides an effective RT strategy by using low doses of X-rays to intensify tumor suppression yet reduce systemic toxicity.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels

SignificanceNitric oxide (NO) is involved in the regulation of physiological and pathological mechanisms of the cardiovascular, nervous and immune systems; and plays an important role in cancer being involved in tumor growth and suppressing processes depending on its concentration. ApproachThe development of a near-infrared excitable nanoprobe, consisting of gold nanoparticles functionalized with a two-photon excitable NO probe, for the detection of intracellular NO is reported. ResultsThe nanoprobe showed good selectivity towards NO over cellular interferences and excellent stability in aqueous-medium over time. The nanoprobe was able to selectively detect endogenous and exogenous NO in different cell lines and it accumulated in the acidic organelles showing negligible toxicity. Importantly, the nanoprobe showed potential to quantify intracellular NO concentrations in breast cancer cells. ConclusionsThe novel gold-based two-photon nanoprobe showed an excellent performance and versatility and could potentially be applied for the spatiotemporal monitoring of in vivo NO levels.

Effects of training flights of combat jet pilots on parameters of airway function, diffusing capacity and systemic oxidative stress, and their association with flight parameters

BackgroundFighter aircraft pilots are regularly exposed to physiological challenges from high acceleration (Gz) forces, as well as increased breathing pressure and oxygen supply in the support systems. We studied whether effects on the lung and systemic oxidative stress were detectable after real training flights comprising of a wide variety of exposure conditions, and their combinations.MethodsThirty-five pilots of the German Air Force performed 145 flights with the Eurofighter Typhoon. Prior to and after flight lung diffusing capacity for carbon monoxide (DLCO) and nitric oxide (DLNO), alveolar volume (VA), and diffusing capacities per volume (KCO, KNO) were assessed. In addition, the fractional concentration of exhaled nitric oxide (FeNO) was determined, and urine samples for the analysis of molecular species related to 8-hydroxy-2’-deoxyguanosine (8-OHdG) were taken. For statistical analysis, mixed ANOVA models were used.ResultsDLNO, DLCO, KNO, KCO and VA were reduced (p < 0.001) after flights, mean ± SD changes being 2.9 ± 5.0, 3.2 ± 5.2, 1.5 ± 3.7, 1.9 ± 3.7 and 1.4 ± 3.1%, respectively, while FeNO decreased by 11.1% and the ratio of 8-OHdG to creatinine increased by 15.7 ± 37.8%. The reductions of DLNO (DLCO) were smaller (p < 0.001) than those of KNO (KCO). In repeated flights on different days, baseline values were restored. Amongst various flight parameters comprising Gz-forces and/or being indicative of positive pressure breathing and oxygenation support, the combination of long flight duration and high altitude appeared to be linked to greater changes in DLNO and DLCO.ConclusionsThe pattern of reductions in diffusing capacities suggests effects arising from atelectasis and increased diffusion barrier, without changes in capillary blood volume. The decrease in exhaled endogenous NO suggests bronchial mucosal irritation and/or local oxidative stress, and the increase in urinary oxidized guanosine species suggests systemic oxidative stress. Although changes were small and not clinically relevant, their presence demonstrated physiological effects of real training flights in a modern 4th generation fighter jet.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Use of thiacalix[4]arene C-1193 for a directed influence on the functional activity of mitochondria and simulation of this process using a Petri nets.

In molecular biological studies, considerable attention is paid to macrocyclic nanoscale compounds known as calix[4]arenes. An imperative concern in biochemical membranology and molecular biotechnology is the exploration of effectors capable of modifying the intensity of redox reactions within the inner mitochondrial membrane and influencing the activity of its Ca2+ transport systems. The simulation model development is relevant to formalize and generalize the experimental data and assess the conformity of experimental results with theoretical predictions. Experiments were carried out on a suspension of isolated rat myometrial mitochondria. The synthesized thiacalix[4]arene C-1193, containing four sulfur atoms, was employed. Demonstrations of time-dependent and concentration-dependent (0.01-10 μM) inhibition of Ca2+ accumulation and reactive oxygen species (ROS) formation by mitochondria in the presence of C-1193 were observed. While C-1193 inhibited the oxidation of NADH and FADH2, it did not induce mitochondrial swelling. The thiacalix[4]arene also inhibited the synthesis of nitric oxide, with a Ki of 5.5 ± 1.7 nM, positioning it as a high-affinity blocker of endogenous NO generation in mitochondria. These results are the basis for the possible application of the synthesized thiacalix[4]arene as a tool in researching biochemical processes in mitochondria. A simulation model employing functional hybrid Petri nets was developed, reproducing the functional activity of mitochondria, including simultaneous NADH oxidation, ROS formation, NO synthesis, and Ca2+ accumulation. The derived equations formalize and describe the time dependencies of the listed processes in the medium under the influence of thiacalix[4]arene C-1193.

GABA and nitric oxide reduce AsV toxicity in tomato and brinjal seedlings by regulating sulphur assimilation and proline metabolism

Arsenate (AsV) is one of the most hazardous elements, and its increasing accumulation in plants is the major area of concern in present days. Therefore, an environmentally reliable approach is required to reduce the load of AsV into vegetables. In the current study, the potential role of gamma amino butyric acid (GABA; 10 µM) and sodium nitroprusside a donor of NO (SNP; 10 µM) in 21 days old seedlings of tomato and brinjal grown in Hoagland medium containing 25 µM of AsV was analysed by estimating physiological and biochemical attributes. AsV exposure reduced root and shoot length and dry mass by 18, 9 % and 15 % in tomato, and 23 and 10 %, and 27 % in brinjal seedlings, respectively. Additionally, AsV decreased leaf gas exchange parameters i.e. stomatal conductance, internal CO2 concentration, transpiration rate and subsequently photosynthetic rate. Although AsV stimulated sulphur assimilation and proline metabolism, but it raised the oxidative stress as evident by increased biomarkers; superoxide radicals (SOR), hydrogen peroxide (H2O2), lipid peroxidation (MDA), and protein oxidation in both the seedlings. The application of GABA and SNP significantly mitigated negative effects of AsV on both the vegetables. The results indicate that GABA was unable to reduce AsV stress in the presence of L-NAME (an endogenous NO biosynthetic inhibitor), but addition of SNP rescued the effect of L-NAME. Overall, the findings suggest that GABA and SNP effectively mitigate AsV stress however, the involvement of NO appears to be necessary for GABA to achieve this task efficiently in the studied vegetables. These results will help to design strategies for mitigating arsenate stress in vegetable crops.

Tetrazine-Based Ratiometric Nitric Oxide Sensor Identifies Endogenous Nitric Oxide in Atherosclerosis Plaques by Riding Macrophages as a Smart Vehicle.

Atherosclerosis (AS) is the formation of plaques in blood vessels, which leads to serious cardiovascular diseases. Current research has disclosed that the formation of AS plaques is highly related to the foaming of macrophages. However, there is a lack of detailed molecular biological mechanisms. We proposed a "live sensor" by grafting a tetrazine-based ratiometric NO probe within macrophages through metabolic and bio-orthogonal labeling. This "live sensor" was proved to target the AS plaques with a diameter of only tens of micrometers specifically and visualized endogenous NO at two lesion stages in the AS mouse model. The ratiometric signals from the probe confirmed the participation of NO during AS and indicated that the generation of endogenous NO increased significantly as the lesion progressed. Our proposal of this "live sensor" provided a native and smart strategy to target and deliver small molecular probes to the AS plaques at the in vivo level, which can be used as universal platforms for the detection of reactive molecules or microenvironmental factors in AS.

Rational engineering of novel rhodamines with stable far red to NIR emission and high brightness for in vivo imaging

Rhodamine fluorophore has attracted increasing attention due to its good stability and high fluorescence quantum yield. However, it is still a challenge to design long-wavelength rhodamine with high performance. In this work, by the reasonable design, we have constructed a novel rhodamine fluorophore YL602-F. YL602-F not only displays long absorption/emission (602/638 nm), but also possesses excellent chemical stability, high brightness (φ × ε/M-1 cm-1 = 26,450), environmental insensitive emission and easy synthesis, thus displaying a good potential for bioimaging application. As a proof-of-concept, based on dye YL602-F, we then designed and developed a nitric oxide-activated fluorescent probe YL602-F-NO. Experiment results showed that probe YL602-F-NO could respond to NO with a 35-fold fluorescence enhancement together with a high sensitivity and the detection limit was calculated to be 83.1 nM. Notably, YL602-F-NO has also been successfully used to sensitively recognize endogenous NO in cells and living mice, especially for real-time monitoring of NO levels in a mouse model of DSS-induced enteritis. We expect the YL602-F fluorophore to be a promising molecule that will help researchers develop activated fluorescent probes with even better performance.

Exogenous hydrogen sulphide alleviates senescence of postharvest pakchoi through regulating antioxidant system, endogenous hydrogen sulphide and nitric oxide metabolism

Previous studies showed that hydrogen sulphide (H2S) fumigation retarded the yellowing of detached pakchoi leaves. However, whether H2S regulated specific metabolic pathways and gene expression levels in this process remained unclear. In this study, the effect of H2S, DL-propargylglycine (PAG, H2S synthetic inhibitor) on antioxidant capacity, endogenous H2S synthesis, and endogenous NO synthesis in pakchoi was investigated. After H2S fumigation (40 L L−1, 6 h), the pakchoi stored at 20 °C ± 1 °C with 90–95% relative humidity exhibited the best quality, with the highest chlorophyll content and the lowest respiration rate and malondialdehyde content. Contrarily, PAG accelerated pakchoi senescence. H2S treatment reduced superoxide anion and hydrogen peroxide by increasing the activities of superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, partially up-regulating the expression of enzyme genes. The increased expression of BcLCD, BcDCD and BcCAS C1 activated L- and D-cysteine desulfhydrase activities so that more endogenous H2S were retained in the pakchoi. Meanwhile, H2S treatment increased cysteine content in pakchoi via up-regulating BcSAT5 expression and enhancing the activities of sulfite reductase and serine acetyltransferase. Moreover, H2S increased endogenous NO primarily by preserving the activities of nitrate reductase and nitrite reductase instead of NO synthase (NOS)-like activity and decreased nitrate and nitrite as well. Overall, exogenous H2S alleviated the senescence of postharvest pakchoi via positively regulating antioxidant system, endogenous H2S and NO metabolism. Moreover, this work described a putative regulatory mechanism and provided some relevant information for future research into the interaction of H2S and NO in this process.

Role of nitric oxide and calcium ions in the effects of hydrogen sulfide on contractile activity of rat jejunum

This study was performed to explore the role of nitric oxide, intracellular and extracellular calcium in the effects of hydrogen sulfide on spontaneous and carbachol-induced contractions of a rat jejunum preparation during a isometric contraction. Application of H2S donor, sodium hydrosulfide, led to a decrease in tonic tension, the amplitude and frequency of spontaneous contractions, as well as in the amplitude induced by carbachol, a nonspecific acetylcholine receptor agonist. Inhibiting the production of endogenous NO synthesis by with L-NAME, the effect of H2S donor remained unchanged, while in the presence of SNAP, a NO donor, the effects of NaHS on the amplitude of spontaneous and carbachol-induced contractions were less pronounced. Dantrolene, a ryanodine receptor inhibitor was used to stop a decrease in tonic tension in the presence of NaHS. The calcium-free solution reduced the inhibitory effect of NaHS on carbachol-induced contractions. This suggests that the inhibitory effect of H2S is associated with the dynamics of the intracellular concentration of calcium ions, and the interaction between NO and H2S occurs at the level of common targets of two gases.

M1 macrophage-derived exosome for reprograming M2 macrophages and combining endogenous NO gas therapy with enhanced photodynamic synergistic therapy in colorectal cancer

Reprogramming immunosuppressive M2 macrophages into M1 macrophages in tumor site provides a new strategy for the immunotherapy of colorectal cancer. In this study, M1 macrophage-derived exosome nanoprobe (M1UC) with Ce6-loaded upconversion material is designed to enhance the photodynamic performance of Ce6 while reprogramming M2 macrophages at tumor site and producing NO gas for three-mode synergistic therapy. Under the excitation of near-infrared light at 808 nm, the probe can generate 660 nm up-conversion fluorescence, which enables the photosensitizer Ce6 to produce ROS efficiently. In addition, the probe leads the production of NO by nitric oxide synthase on exosomes. Confocal laser and flow cytometry results show that M1UC probe reprograms M2 macrophages into M1 macrophages with an efficiency of 95.12%. The cell experiments show that the apoptosis rate of the three-mode synergistic therapy group is 78.8%, and the therapeutic effect is significantly higher than those of the other single treatment groups. In vivo experiments results show that M1UC probes maximally gather at the tumor site after 12 h of intravenous injection in orthotopic colorectal cancer mice. After 808 nm laser irradiation, the survival rate of mice is 100% and the recurrence rate was 0 within 60 d, and the therapeutic effect is significantly higher than those of other single treatment groups, which is also confirmed by immunohistochemistry. This M1 macrophage-derived exosome nanoplatform which is based on the three modes of immunotherapy, gas therapy and photodynamic therapy, provides a new design idea for the diagnosis and treatment of deep tumors.

The effects of inorganic nitrate supplementation on exercise economy and endurance capacity across the menstrual cycle.

Oral inorganic nitrate (NO3-) supplementation has been shown to increase bioavailable NO and provide potential ergogenic benefits in males; however, data in females is scarce. Estrogen is known to increase endogenous NO bioavailability and to fluctuate throughout the menstrual cycle (MC), being lowest in the early follicular (EF) phase and highest during the late follicular (LF) phase. This study examined the effects of oral NO3- supplementation on exercise economy, endurance capacity, and vascular health in young females across the MC. Ten normally menstruating females' MCs were tested in a double-blinded, randomized design during both the EF and LF phases of the MC. Participants consumed ∼13 mmol NO3-, in the form of 140 mL beetroot juice (BRJ) or an identical NO3--depleted placebo (PL) for ∼3 days before lab visits and 2 h before testing on lab visits. Plasma nitrate, nitrite, and estradiol were assessed, as was blood pressure and pulse wave velocity. Moderate-intensity exercise economy and severe intensity time to exhaustion (TTE) were tested on a cycle ergometer. As expected, plasma estradiol was elevated in the LF phase, and plasma nitrite and nitrate were elevated in the BRJ condition. Exercise economy was unaltered by BRJ or the MC, however TTE was significantly worsened by 48 s (∼10%) after BRJ supplementation (P = 0.04), but was not different across the MC with no interaction effects. In conclusion, NO3- supplementation did not affect exercise economy or vascular health and worsened aerobic endurance capacity (TTE), suggesting healthy females should proceed with caution when considering supplementation with BRJ.NEW & NOTEWORTHY Although inorganic nitrate (NO3-) supplementation has increased in popularity as a means of improving exercise performance, data in females at different phases of the menstrual cycle are lacking despite known interactions of estrogen with NO. This study revealed neither NO3- supplementation nor the menstrual cycle influenced exercise economy or vascular health in healthy young naturally menstruating females, while NO3- supplementation significantly worsened endurance capacity (10%) independent of the menstrual cycle phase.

Influence of NO Synthase Inhibitors on Serotonin Release in the Medial Prefrontal Cortex during the Formation and Generalization of the Conditioned Fear Response in Rats

Serotonergic and nitrergic systems of the medial prefrontal cortex (mPFC) are involved in the control of fear generalization, but their local interaction during this function has been little studied. The aim of the work was to study the effect of blockade of endogenous nitrergic signals on serotonin release in the mPFC during the acquisition of a conditioned fear response (CFR – a fear model) and on the dynamics of its generalization. In male Sprague-Dawley rats using intracranial microdialysis in vivo and high-performance liquid chromatography with electrochemical detection, we found that the intra-mPFC infusion through the dialysis probe of the NO synthase inhibitor N-ω-nitro-L-arginine (NA, 0.5 mM) and selective neuronal NO synthase inhibitor N-ω-propyl-L-arginine (NPLA, 2mM) decreased the basal level of extracellular serotonin in the mPFC and reduced its rise, caused by the CFR acquisition (a paired presentation of a conditioned cue (CS+) and inescapable footshock). The intra-mPFC infusion of NA and NPLA increased animals’ freezing to a differential cue (CS–) not associated with footshock, during the first test, carried out 70 minutes after the CFR acquisition, but reduced it during repeated testing a day after the infusion, without changing freezing of the same animals to the potentially dangerous CS+. The data obtained indicate the involvement of endogenous NO in the activation of serotonin release in the mPFC, caused by the CFR acquisition. In addition, they show that the blockade of endogenous nitrergic signals of the mPFC, which enhances the initial generalization of the fear reaction, contributes to the extinction of the generalized fear, possibly due to inhibition of the serotonin release in the mPFC.

Gas station in blood vessels: An endothelium mimicking, self-sustainable nitric oxide fueling stent coating for prevention of thrombosis and restenosis

Stenting is the primary treatment for vascular obstruction-related cardiovascular diseases, but it inevitably causes endothelial injury which may lead to severe thrombosis and restenosis. Maintaining nitric oxide (NO, a vasoactive mediator) production and grafting endothelial glycocalyx such as heparin (Hep) onto the surface of cardiovascular stents could effectively reconstruct the damaged endothelium. However, insufficient endogenous NO donors may impede NO catalytic generation and fail to sustain cardiovascular homeostasis. Here, a dopamine-copper (DA-Cu) network-based coating armed with NO precursor L-arginine (Arg) and Hep (DA-Cu-Arg-Hep) is prepared using an organic solvent-free dipping technique to form a nanometer-thin coating onto the cardiovascular stents. The DA-Cu network adheres tightly to the surface of stents and confers excellent NO catalytic activity in the presence of endogenous NO donors. The immobilized Arg functions as a NO fuel to generate NO via endothelial nitric oxide synthase (eNOS), while Hep works as eNOS booster to increase the level of eNOS to decompose Arg into NO, ensuring a sufficient supply of NO even when endogenous donors are insufficient. The synergistic interaction between Cu and Arg is analogous to a gas station to fuel NO production to compensate for the insufficient endogenous NO donor in vivo. Consequently, it promotes the reconstruction of natural endothelium, inhibits smooth muscle cell (SMC) migration, and suppresses cascading platelet adhesion, preventing stent thrombosis and restenosis. We anticipate that our DA-Cu-Arg-Hep coating will improve the quality of life of cardiovascular patients through improved surgical follow-up, increased safety, and decreased medication, as well as revitalize the stenting industry through durable designs.

New biofunctional effects of oleanane-type triterpene saponins.

In the current review, we describe the novel biofunctional effects of oleanane-type triterpene saponins, including elatosides, momordins, senegasaponins, camelliasaponins, and escins, obtained from Aralia elata (bark, root cortex, young shoot), Kochia scoparia (fruit), Polygala senega var. latifolia (roots), Camellia japonica (seeds), and Aesculus hippocastanum (seeds), considering the following biofunctional activities: (1) inhibitory effects on elevated levels of blood alcohol and glucose in alcohol and glucose-loaded rats, respectively, (2) inhibitory effects on gastric emptying in rats and mice, (3) accelerative effects on gastrointestinal transit in mice, and (4) protective effects against gastric mucosal lesions in rats. In addition, we describe (5) suppressive effects of the extract and chakasaponins from Camellia sinensis (flower buds) on obesity based on inhibition of food intake in mice. The active saponins were classified into the following three types: (1) olean-12-en-28-oic acid 3-O-monodesmoside, (2) olean-12-ene 3,28-O-acylated bisdesmoside, and (3) acylated polyhydroxyolean-12-ene 3-O-monodesmoside. Furthermore, common modes of action, such as involvements of capsaicin-sensitive nerves, endogenous NO and PGs, and possibly sympathetic nerves, as well as common structural requirements, were observed. Based on our findings, a common mechanism of action might mediate the pharmacological effects of active saponins. It should be noted that the gastrointestinal tract is an important action site of saponins, and the role of the saponins in the gastrointestinal tract should be carefully considered.

Metallacage-based enhanced PDT strategy for bacterial elimination via inhibiting endogenous NO production

Antibiotics are among the most used weapons in fighting microbial infections and have greatly improved the quality of human life. However, bacteria can eventually evolve to exhibit antibiotic resistance to almost all prescribed antibiotic drugs. Photodynamic therapy (PDT) develops little antibiotic resistance and has become a promising strategy in fighting bacterial infection. To augment the killing effect of PDT, the conventional strategy is introducing excess ROS in various ways, such as applying high light doses, high photosensitizer concentrations, and exogenous oxygen. In this study, we report a metallacage-based PDT strategy that minimizes the use of ROS by jointly using gallium-metal organic framework rods to inhibit the production of bacterial endogenous NO, amplify ROS stress, and enhance the killing effect. The augmented bactericidal effect was demonstrated both in vitro and in vivo. This proposed enhanced PDT strategy will provide a new option for bacterial ablation.

The importance of the endothelial nitric oxide synthase on the release of 6-nitrodopamine from mouse isolated atria and ventricles and their role on chronotropism

6-nitrodopamine (6-ND) is released from rat isolated atria, where it acts as a potent positive chronotropic agent. The release of 6-ND from rat isolated atria and ventricles is significantly reduced when pre-incubated with l-NAME, and the release was not affected by tetrodotoxin pre-treatment, indicating that in the heart, the origin of 6-ND is not neurogenic. Since l-NAME inhibits all three isoforms of NO synthase, it was investigated the basal release of 6-ND from isolated atria and ventricles from nNOS−/−, iNOS−/− and eNOS−/− mice of either sex. The release of 6-ND was measured by LC-MS/MS.There were no significant differences in the 6-ND basal release from isolated atria and ventricles from male control mice, as compared to female control mice. The 6-ND release from atria obtained from eNOS−/− mice was significantly reduced when compared to atria obtained from control mice. The 6-ND release in nNOS−/− mice was not significantly different compared to control animals whereas the 6-ND release from atria obtained from iNOS−/− mice was significantly higher when compared to control group. Incubation of the isolated atria with l-NAME caused a significant decrease in the basal atrial rate of control, nNOS−/−, and iNOS−/− mice, but not in eNOS−/− mice.The results clearly indicate that eNOS is the isoform responsible for the synthesis of 6-ND in the mice isolated atria and ventricles and supports the concept that 6-ND is the major mechanism by which endogenous NO modulates heart rate.

STUDY OF C-786T POLYMORPHISM IN THE NOS3 GENE IN THE DEVELOPMENT OF PREECLAMPSIA

Objective: Given the importance of endogenous NO in maintaining normal endothelial function, it is possible that polymorphisms in the gene encoding the enzyme that synthesizes NO may contribute to the development of preeclampsia. The purpose of the work was to determine the role of the C-786T polymorphism in the NOS3 gene in the development of preeclampsia. Design and method: We conducted a study of 90 pregnant women in the II-III trimester, which were divided into two groups: The main group of 53 pregnant women with severe preeclampsia. Control group - 47 pregnant women with a physiological course of the gestational period. The age of the examined women ranged from 19 to 41 years. Samples of pregnant women served as the material for the study. We have identified gene polymorphism - C-786T in the NOS3 gene. Isolation of DNA from blood and PCR analysis were carried out using kits of reagents and test systems from Ampli Prime Ribo-prep. Statistical processing of the results was carried out using statistical programs EpiCalc 2000. Results: There was a trend towards the prevalence of the heterozygous C/T genotype in the group of patients with severe preeclampsia, where it was detected with a frequency of 39.5% (x2-2.23; p-0.15; RR-1.46; 95% CI: 0.54-3.91; OR -1.76.; 95% CI: 0.84-3.69), relative to 27.1% in the control group. The T/T genotype was slightly more common – in 11.6% of cases it was detected among patients with severe preeclampsia (x2-1.08; p-0.30; RR-1.78; 95%CI:0.43-7.32; OR-1.88; 95%CI:0.57- 6.19), while in the control group - with a frequency of 6.5%. The C/C genotype was found in 59.8% of patients with severe preeclampsia (x2-1.04; p-0.31; RR-0.81; 95% CI: 0.37-1.74; OR-0.62) The T allele among patients with severe preeclampsia was detected in 31.4% of cases, which was statistically significantly higher (x2-4.38; p-0.04; RR-1.17; 95%CI:0.79-1.73; OR-1.82; 95%CI:1.04- 3.19) than in the control group. Conclusions: Thus, the results of our study allow us to conclude that the adverse genotypic variants of the C-786T polymorphism of the NOS3 gene (associated with vascular endothelial dysfunction) contribute significantly to the mechanism of preeclampsia.

Effects of Nitric Oxide on the Activity of P2X and TRPV1 Receptors in Rat Meningeal Afferents of the Trigeminal Nerve.

Nitric oxide is one of the endogenous molecules that play a key role in migraine. However, the interaction between NO and the main players in the nociceptive activity of the meningeal trigeminal afferents-TRPV1 and P2X3 receptors-remains unstudied. In the current project, the effects of acute and chronic NO administration on the activity of TRPV1 and P2X3 receptors in the peripheral afferents were studied using electrophysiological recording of action potentials of the trigeminal nerve in the rat hemiskull preparations. The data obtained indicate that exogenous and endogenous NO increased the activity of the trigeminal nerve independent on the inhibition of the TRPV1 and P2X3 receptors. The activity of the trigeminal nerve triggered by ATP changed neither in acute incubation in the NO donor-sodium nitroprusside (SNP) nor in the chronic nitroglycerine (NG)-induced migraine model. Moreover, the chronic NG administration did not increase in the number of degranulated mast cells in the rat meninges. At the same time, the capsaicin-induced activity of the trigeminal nerve was higher with chronic NO administration or after acute NO application, and these effects were prevented by N-ethylmaleimide. In conclusion, we suggested that NO positively modulates the activity of TRPV1 receptors by S-nitrosylation, which may contribute to the pro-nociceptive action of NO and underlie the sensitization of meningeal afferents in chronic migraine.

Nitric oxide treatment delays quality deterioration and enzymatic browning of Agaricus bisporus via reactive oxygen metabolism regulation

AbstractNitric oxide (NO) is a highly reactive gaseous small molecule that regulates plant growth. Sodium nitroprusside (SNP) was used as an NO donor to study the effects of NO treatment on browning and antioxidant activity of button mushrooms (Agaricus bisporus). The findings revealed that exogenous NO treatment alleviated the browning, decreased hardness, and increased weight loss of mushrooms stored at 4°C for 15 days, and reduced the accumulation of reactive oxygen species and membrane lipid peroxidation. Moreover, exogenous NO treatment dramatically increased the anabolism of endogenous NO, maintained the content of antioxidant substances including flavonoids and total phenols, and kept catalase and superoxide dismutase activities at a high level. Further studies revealed that exogenous NO treatment inhibited the activities of enzymes that are involved in browning, which including polyphenol oxidase (PPO) and tyrosinase (TYR), and increased phenylalanine ammonia‐lyase (PAL) activity, with SNP treatment at 0.05 mM being the most effective. Furthermore, SNP treatment at 0.05 mM also inhibited PPO and TYR gene expressions while increasing PAL gene expression. Therefore, our findings suggest that exogenous NO has the potential for development in delaying postharvest browning and maintaining the quality of mushrooms.