Nitric Oxide Signaling Research Articles

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia–reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia–reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia–reperfusion injury through the P2Y13 receptor and the NO–sGC–PKG pathway.

Quantitative aspects of nitric oxide production in the heart.

Nitric oxide (NO) has essential roles in heart physiology, including the regulation of myocardial contractility and coronary blood flow, and in heart pathophysiology, particularly in the ischemic heart. NO is produced by both NO synthase (NOS)-dependent and NOS-independent pathways in the heart. This review summarizes quantitative aspects of NO production in the heart; the contribution of cardiomyocytes, endothelial cells (ECs), red blood cells (RBCs), and neurons are also discussed. Based on the available data, under normal conditions, the human heart produces about 50-70 µmol NO per day, primarily attributed to eNOS activity; ECs produce the highest amount of NO compared to other cell types in the heart. On the other hand, during ischemic conditions, NOS-independent NO production participates a significant role in the heart NO production that can exceed NOS-dependent NO generation. These data are relevant as most cardiovascular disorders are associated with NO dysfunction, and increasing NO bioavailability and signaling is a potential therapeutic approach for cardiovascular diseases.

Haptoglobin treatment contributes to regulating nitric oxide signal and reduces oxidative stress in the penis: A preventive treatment for priapism in sickle cell disease.

Background: In sickle cell disease (SCD), reduced bioavailability of endothelial NO and cGMP results in reduced expression of phosphodiesterase type 5 (PDE5), thus impairing the penile erection control mechanism and resulting in prolonged penile erection (priapism). In SCD, reduced NO bioavailability is associated with excess plasma hemoglobin due to intravascular hemolysis and increased oxidative stress. Haptoglobin is the plasma protein responsible for reducing plasma hemoglobin levels, but in SCD, haptoglobin levels are reduced, which favors the accumulation of hemoglobin in plasma. Therefore, we aimed to evaluate the effects of haptoglobin treatment on functional and molecular alterations of erectile function, focusing on the contractile and relaxant mechanisms of corpus cavernosum (CC), as well as oxidative stress. Methods: SCD mice were treated with haptoglobin (400 mg/kg, subcutaneous) or vehicle of Monday, Wednesday and Friday for a period of 1 month. Corpus cavernosum strips were dissected free and placed in organ baths. Cumulative concentration-response curves to the acetylcholine, sodium nitroprusside, phenylephrine and KCL, as well as to electrical field stimulation (EFS), were obtained in CC. Protein expressions of eNOS, phosphorylation of eNOS at Ser-1177, nNOS, PDE5, ROCK1, ROCK2, gp91phox, 3-nitrotyrosine, and 4-HNE were measured by western blot in CC. Results: Increased CC relaxant responses to acetylcholine, sodium nitroprusside and electrical-field stimulation were reduced by haptoglobin in SCD mice. Reduced CC contractile responses to phenylephrine and KCl were increased by haptoglobin in SCD mice. Haptoglobin prevented downregulated eNOS, p-eNOS (Ser-1177), PDE5, and ROCK2 protein expressions and reduced protein expressions of reactive oxygen species markers, NADPH oxidase subunit gp91phox, 3-nitrotyrosine and 4-HNE in penises from SCD mice. Haptoglobin treatment did not affect ROCK1 and nNOS protein expressions in penises from SCD mice. Basal cGMP production was lower in the SCD group, which was normalized by haptoglobin treatment. Conclusion: Treatment with haptoglobin improved erectile function due to up-regulation of eNOS-PDE5 expression and down-regulation of the gp91phox subunit of NADPH oxidase and oxidative/nitrosative stress in the penises of SCD mice. Treatment with haptoglobin also increased contractile activity due to up-regulation of ROCK2. Therefore, haptoglobin treatment may be an additional strategy to prevent priapism in SCD.

Dibutyl phthalate promotes angiogenesis in EA.hy926 cells through estrogen receptor-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways

Dibutyl phthalate (DBP) is an endocrine disruptor that has been widely used in various products of human use. DBP exposure has been associated with reproductive and cardiovascular diseases and metabolic disorders. Although dysfunction of the vascular endothelium is responsible for many cardiovascular and metabolic diseases, little is known about the effects of DBP on human endothelium. In this study, we investigated the effect of three concentrations of DBP (10−6, 10−5, and 10−4 M) on angiogenesis in human endothelial cell (EC) line EA.hy926 after acute exposure. Tube formation assay was used to investigate in vitro angiogenesis, whereas qRT-PCR was employed to measure mRNA expression. The effect of DBP on extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS) activation was examined using Western blotting, whereas the Griess method was used to assess NO production. Results show that the 24-h-long exposure to 10−4 M DBP increased endothelial tube formation, which was prevented by addition of U0126 (ERK1/2 inhibitor), wortmannin (PI3K-Akt inhibitor), and l-NAME (NOS inhibitor). Short exposure to 10−4 M DBP (from 15 to 120 min) phosphorylated ERK1/2, Akt, and eNOS in different time points and increased NO production after 24 and 48 h of exposure. Application of nuclear estrogen receptor (ER) and G protein-coupled ER (GPER) inhibitors ICI 182,780 and G-15, respectively, abolished the DBP-mediated ERK1/2, Akt, and eNOS phosphorylation and increase in NO production. In this study, we report for the first time that DBP exerts a pro-angiogenic effect on human vascular ECs and describe the molecular mechanism involving ER- and GPER-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

The Glycolysis Inhibitor 2-Deoxy-D-Glucose Exerts Different Neuronal Effects at Circuit and Cellular Levels, Partially Reverses Behavioral Alterations and does not Prevent NADPH Diaphorase Activity Reduction in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy is the most drug-resistant type with the highest incidence among the other focal epilepsies. Metabolic manipulations are of great interest among others, glycolysis inhibitors like 2-deoxy d-glucose (2-DG) being the most promising intervention. Here, we sought to investigate the effects of 2-DG treatment on cellular and circuit level electrophysiological properties using patch-clamp and local field potentials recordings and behavioral alterations such as depression and anxiety behaviors, and changes in nitric oxide signaling in the intrahippocampal kainic acid model. We found that epileptic animals were less anxious, more depressed, with more locomotion activity. Interestingly, by masking the effect of increased locomotor activity on the parameters of the zero-maze test, no altered anxiety behavior was noted in epileptic animals. However, 2-DG could partially reverse the behavioral changes induced by kainic acid. The findings also showed that 2-DG treatment partially suppresses cellular level alterations while failing to reverse circuit-level changes resulting from kainic acid injection. Analysis of NADPH-diaphorase positive neurons in the CA1 area of the hippocampus revealed that the number of positive neurons was significantly reduced in dorsal CA1 of the epileptic animals and 2-DG treatment did not affect the diminishing effect of kainic acid on NADPH-d+ neurons in the CA1 area. In the control group receiving 2-DG, however, an augmented NADPH-d+ cell number was noted. These data suggest that 2-DG cannot suppress epileptiform activity at the circuit-level in this model of epilepsy and therefore, may fail to control the seizures in temporal lobe epilepsy cases.

Vanillic Acid Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension by Enhancing NO Signaling Pathways

Pulmonary arterial hypertension (PAH) is a severe progressive disease characterized by elevated mean pulmonary arterial pressure, right ventricular hypertrophy, and eventual progression to right heart failure and death. This study aimed to examine the effect of the natural product vanillic acid (VA) on monocrotaline (MCT)-induced PAH in rats. The arginase inhibitory activity and enzyme kinetic reaction of VA were also investigated. The results showed that VA could improve pulmonary arterial pressure, pulmonary artery vascular remodeling, and right ventricular remodeling induced by MCT in rats and reduce the degree of pulmonary tissue fibrosis. Moreover, VA downregulated the gene and protein expression levels of Hif-2α, Hif-1β, and Arg2 and increased the P-eNOS/eNOS levels, thus increasing nitric oxide (NO) levels in PAH rats. Furthermore, VA was determined to be a mixed competitive arginase inhibitor with an IC50 of 26.1 μM. In conclusion, the arginase inhibitor VA exerted protective effects on MCT-induced PAH and pulmonary vascular remodeling by enhancing NO signaling pathways.

Abstract P002: Mechanism Of Mesenteric Artery Vasodilation And Blood Pressure Regulation By Statins

Purpose: Statins are the world’s most prescribed drugs with a wide variety of cholesterol-dependent and -independent cardiovascular effects. Pre-clinical and clinical data suggest that statins lower blood pressure (BP) in some patients but have no effect in others. However, the underlying mechanism for such differences remain unclear. By studying the direct vascular effects of therapeutically relevant concentrations of statins in resistance mesenteric arteries from normotensive Sprague Dawley (SD) rats and Spontaneously Hypertensive (SHR) rats, we sought to determine the mechanism of differential BP regulation by statins. Methods: Pressurized arterial myography, phosphodiesterase (PDE) activity assay, ELISA-based cGMP measurement, blood pressure measurement with tail-cuff. Results: Our pressure myography data revealed that statins induce rapid vasodilation in resistance mesenteric arteries. Endothelium denudation, inhibition of endothelial nitric oxide (NO) synthase (eNOS) with L-NNA, smooth muscle cell (SMC) guanylyl cyclase (sGC) inhibition with ODQ, or the inhibition of protein kinase G (PKG) with KT5823 each abolished statin-evoked vasodilation, demonstrating a critical role of endothelium and NO signaling in this process. However, contrary to previous reports, mevalonate application had no effect on vasodilation. Statins selectively inhibited PDE1A in SMCs, elevated [cGMP] i , which could potentiate NO-GC-cGMP-PKG signaling, leading to vasodilation. Statin-evoked vasodilation is attenuated in SHR rat mesenteric arteries that have reduced eNOS expression and impaired NO signaling. Consistent with the vasodilatory action, acute statin treatment lowered systolic and diastolic BP in healthy SD rats. In contrast, acute statin treatment failed to lower systemic BP in SHR rats with reduced eNOS expression. Conclusion: Statins are endothelium-dependent vasodilator, and selective inhibitor of PDE1A in resistance mesenteric arteries. Intact endothelial function and NO production are essential for statin-induced vasodilation and the reduction of BP. Our study could provide explanation to the anti-hypertensive and BP neutral effects of statins.

Synthesis and Chromatographic Determination of S-Nitrosopantetheine: Exploring Reactivity and Stability in Different Aqueous Solutions

S-nitrosothiols (RSNOs) are a group of sulfur-containing compounds biologically involved in nitric oxide (NO˙) release and signalling pathway. NO˙ plays important physiological and pharmacological activities, particularly in vasodilation and in inducing muscle relaxation. Several RSNO compounds have been detected in biological systems, and many of them have been chemically synthesized in the laboratory. To date, no works describing the synthesis of the S-nitrosopantetheine (SNOPANT) are reported in the literature. Taking into account that pantetheine is a biological thiol with a crucial function in metabolism, its nitrosylation in vivo could play a role in various metabolic signalling pathways. In this paper, the synthesis and the chromatographic determination of SNOPANT is reported for the first time, as well as a brief investigation of its reactivity in aqueous solutions in the presence of factors known to affect its stability.

Sickle cell disease in the new era: advances in drug treatment

Sickle cell disease is an inherited blood disorder afflicting an estimated 100,000 individuals in the United States and over 20 million people worldwide. The disease is heralded as the first molecular disease. However, despite its genetic simplicity, the pathophysiologic processes leading to its clinical sequelae are complex, heterogeneous and interrelated, making drug development to treat the disease challenging. For over two decades only one drug, hydroxyurea, had been used as disease-modifying therapy. New pharmacologic agents are rapidly evolving with three new drugs, with different mechanisms of action, approved by the United States Food and Drug Administration in recent years (L-glutamine, crizanlizumab and voxelotor). Several therapeutic approaches targeting different pathways in the disease pathophysiology are being investigated. These include inhibition of hemoglobin S polymerization such as by fetal hemoglobin induction or by increasing hemoglobin oxygen affinity, as well as intervention of downstream pathways including inhibiting cellular adhesion, reducing inflammation and oxidant stress, modulating platelet activation and coagulation abnormalities, and targeting nitric oxide signaling. This review will provide an overview of these therapeutic strategies, discuss the four currently approved drugs in detail, and summarize ongoing clinical trials of new drugs or drug indications for the treatment of sickle cell disease in different phases of development excluding those related to cellular therapies.

Biomimetic niche of vascular intima with biophysical orientation and biochemical stimulation for rapid endothelialization and long-term patency

Clinical replacement of small-diameter vascular grafts (SDVG) for vessel lesion often encounters highly occluded risk due to acute platelet activation in a short run and poor endothelialization. Recent strategies built with either biochemical modification of heparinization or biophysical manipulation of hemodynamics have attempted to address the issue of unqualified long-term patency rate, but the major challenges on suppressing thrombosis and elevating fast endothelialization are yet untangled. Herein, we designed a biomimetic SDVG made of biochemical regulation of dopamine-mediated copper ion (DA-CuII) and biophysical regulation of intimal oriented nanotopography. The DA-CuII system can significantly modulate the cell fates of vascular cells containing platelets, endothelial cells, and smooth muscle cells via catalytically producing nitric oxide (NO) in situ and the intimal oriented nanotopography can effectively promote the oriented growth and elongation of endothelial cells to achieve luminal remodeling and maintain an instructive long-term patency. Consequently, the current work integrates biochemical cue of NO signaling molecule with biophysical stimulation of intimal oriented nanotopography to create an off-the-shelf SDVG with specific anti-thrombosis and rapid endothelialization.

Exogenously Applied Sodium Nitroprusside Mitigates Lead Toxicity in Rice by Regulating Antioxidants and Metal Stress-Related Transcripts.

Sustainable agriculture is increasingly being put in danger by environmental contamination with dangerous heavy metals (HMs), especially lead (Pb). Plants have developed a sophisticated mechanism for nitric oxide (NO) production and signaling to regulate hazardous effects of abiotic factors, including HMs. In the current study, we investigated the role of exogenously applied sodium nitroprusside (SNP, a nitric oxide (NO) donor) in ameliorating the toxic effects of lead (Pb) on rice. For this purpose, plants were subjected to 1.2 mM Pb alone and in combination with 100 µM SNP. We found that under 1.2 mM Pb stress conditions, the accumulation of oxidative stress markers, including hydrogen peroxide (H2O2) (37%), superoxide anion (O2−) (28%), malondialdehyde (MDA) (33%), and electrolyte leakage (EL) (34%), was significantly reduced via the application of 100 µM SNP. On the other hand, under the said stress of Pb, the activity of the reactive oxygen species (ROS) scavengers such as polyphenol oxidase (PPO) (60%), peroxidase (POD) (28%), catalase (CAT) (26%), superoxide dismutase (SOD) (42%), and ascorbate peroxidase (APX) (58%) was significantly increased via the application of 100 µM SNP. In addition, the application of 100 µM SNP rescued agronomic traits such as plant height (24%), number of tillers per plant (40%), and visible green pigments (44%) when the plants were exposed to 1.2 mM Pb stress. Furthermore, after exposure to 1.2 mM Pb stress, the expression of the heavy-metal stress-related genes OsPCS1 (44%), OsPCS2 (74%), OsMTP1 (83%), OsMTP5 (53%), OsMT-I-1a (31%), and OsMT-I-1b (24%) was significantly enhanced via the application of 100 µM SNP. Overall, our research evaluates that exogenously applied 100 mM SNP protects rice plants from the oxidative damage brought on by 1.2 mM Pb stress by lowering oxidative stress markers, enhancing the antioxidant system and the transcript accumulation of HMs stress-related genes.

Nitric oxide metabolites: associations with cardiovascular biomarkers and clinical parameters in patients with HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is one of the most rapidly growing cardiovascular health burden worldwide, but there is still a lack of understanding about the HFpEF pathophysiology. The nitric oxide (NO) signalling pathway has been identified as a potential key element. The aim of our study was to investigate markers of NO metabolism [l-arginine (l-Arg), homoarginine (hArg), and asymmetric and symmetric dimethylarginine (ADMA and SDMA)], additional biomarkers [N-terminal pro-B-type natriuretic peptide (NT-proBNP), endothelin-1 (ET-1), mid-regional pro-adrenomedullin (MR-proADM), copeptin, and high-sensitivity C-reactive protein (hsCRP)], and the endothelial function in an integrated approach focusing on associations with clinical characteristics in patients with HFpEF. Seventy-three patients, prospectively enrolled in the 'German HFpEF Registry', were analysed. Inclusion criteria were left ventricular ejection fraction (LVEF)≥50%; New York Heart Association functional class≥II; elevated levels of NT-proBNP>125pg/mL; and at least one additional criterion for structural heart disease or diastolic dysfunction. All patients underwent transthoracic echocardiography, cardiopulmonary exercise testing, and pulse amplitude tonometry (EndoPAT™). Patients were categorized in two groups based on their retrospectively calculated HFA-PEFF score. Serum concentrations of l-Arg, hArg, ADMA, SDMA, NT-proBNP, ET-1, MR-proADM, copeptin, and hsCRP were determined. Patients had a median age of 74years, 47% were female, and median LVEF was 57%. Fifty-two patients (71%) had an HFA-PEFF score≥5 (definitive HFpEF), and 21 patients (29%) a score of 3 to 4 (risk for HFpEF). Overall biomarker concentrations were 126±32μmol/L for l-Arg, 1.67±0.55μmol/L for hArg, 0.74 (0.60;0.85) μmol/L for SDMA, and 0.61±0.10μmol/L for ADMA. The median reactive hyperaemia index (RHI) was 1.55 (1.38;1.87). SDMA correlated with NT-proBNP (r=0.291; P=0.013), ET-1 (r=0.233; P=0.047), and copeptin (r=0.381; P=0.001). ADMA correlated with ET-1 (r=0.250; P=0.033) and hsCRP (r=0.303; P=0.009). SDMA was associated with the left atrial volume index (β=0.332; P=0.004), also after adjustment for age, sex, and comorbidities. Biomarkers were non-associated with the RHI. A principal component analysis revealed two contrary clusters of biomarkers. Our findings suggest an impaired NO metabolism as one possible key pathogenic determinant in at least a subgroup of patients with HFpEF. We argue for further evaluation of NO-based therapies. Upcoming studies should clarify whether subgroups of HFpEF patients can take more benefit from therapies that are targeting NO metabolism and pathway.

Combined effects of nitric oxide synthase 3 genetic variant and childhood emotional abuse on earlier onset of suicidal behaviours

Marked heterogeneity in suicide attempters has been observed, with earlier onset being linked to stronger heritability, more childhood maltreatment. Nitric oxide signalling system might be implicated in this relationship through its role in the stress response/adaptation. This study examined how NOS genetic variants and childhood maltreatment were associated with age at first suicide attempt (SA). Adult patients with SA history (N = 414) filled in the Childhood Trauma Questionnaire, and six functionally relevant NOS2 and NOS3 polymorphisms were genotyped. Analyses included χ2, Mann-Whitney U tests, Kendall's regression, multivariate linear and Cox survival regressions, and a moderation analysis. The NOS3 promotor 27-bp variable number tandem repeat (VNTR) bb homozygous state and childhood emotional abuse were independently associated with earlier age at first SA, which was robust after controlling for confounders [regression coefficient − 3.975, 95% CI −6.980 – (−0.970), p = 0.010, and − 1.088, 95% CI −2.172 – (−0.004), p = 0.049]. No interaction was observed. In the Cox proportional hazards model for age at first SA, the hazard ratio for patients with childhood emotional abuse and NOS3 27-bp VNTR bb was 0.533 (95% CI 0.394–0.720, p < 0.001) compared to patients without. Intermediate scores were observed with either only the risk genotype or only childhood emotional abuse. A graded relationship was also observed for repeated SA, family history of SA, and severe SA history. These results are preliminary due to a low statistical power and call for replication and further characterization of the role of nitric oxide system in the susceptibility to early-onset SB.

Effects of fumarate on renal vascular reactivity and the modulation of blood pressure in normotensive rats: Possible contribution of the nitric oxide synthase-nitric oxide system

Introduction: Fumarate, the tricarboxylic acid (TCA) cycle intermediary, has been linked to nitric oxide (NO) produc­tion. NO plays a prominent role in the physiological regulation of blood pressure and renal hemodynamics. This study is aimed to investigate any contribution of fumarate to blood pressure and renal hemodynamics in normotensive rats with a possible link to the nitrergic system.&#x0D; Materials and methods: Fumarate (1, 3 and 10 μmol) was injected into isolated perfused kidneys, pre-constricted with epinephrine (30 μM). The fumarase inhibitor, pyromellitic acid (PMA) (1, 3 and 10 μM), was used to perfuse the iso­lated kidney and perfusate was collected for nitric oxide and fumarate assays. An acute blood pressure study involved the injection of bolus doses of fumarate (0.1, 0.3 and 1 μg/kg, iv) or PMA (1, 3 and 10 μg/kg, iv) to normotensive rats in the presence of N(ω)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg, iv) or PMA (1, 3 and 10 μg/kg).&#x0D; Results and discussion: Fumarate reduced perfusion pressure and elicited a peak reduction at the highest dose. Per­fusing the kidney with PMA caused a paradoxical increase in perfusion pressure (70%, p&lt;0.05), compared to baseline. Bolus doses of fumarate reduced blood pressure (-29.3±6.2 mmHg, p&lt;0.05), cortical blood flow (CBF) and increased medullary blood flow (MBF). L-NAME did not abolish the vasodilatory effect of fumarate, but reduced the magnitude of response (50%, p&lt;0.05). PMA did not significantly affect the vasodilatory effect of fumarate (p&gt;0.05).&#x0D; Conclusion: These data suggest that fumarate exerts a vasodilatory effect on renal and systemic hemodynamics that may partly involve the nitric oxide signaling.&#x0D; Grapgical abstract:

Vasoactive Effects of Chronic Treatment with Fructose and Slow-Releasing H2S Donor GYY-4137 in Spontaneously Hypertensive Rats: The Role of Nitroso and Sulfide Signalization.

Increased fructose consumption induces metabolic-syndrome-like pathologies and modulates vasoactivity and the participation of nitric oxide (NO) and hydrogen sulfide (H2S). We investigated whether a slow-releasing H2S donor, GYY-4137, could exert beneficial activity in these conditions. We examined the effect of eight weeks of fructose intake on the blood pressure, biometric parameters, vasoactive responses, and NO and H2S pathways in fructose-fed spontaneously hypertensive rats with or without three weeks of GYY-4137 i.p. application. GYY-4137 reduced triacylglycerol levels and blood pressure, but not adiposity, and all were increased by fructose intake. Fructose intake generally enhanced endothelium-dependent vasorelaxation, decreased adrenergic contraction, and increased protein expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and concentration of conjugated dienes in the left ventricle (LV). Although GYY-4137 administration did not affect vasorelaxant responses, it restored disturbed contractility, LV oxidative damage and decreased protein expression of TNFα in fructose-fed rats. While the participation of endogenous H2S in vasoactive responses was not affected by fructose treatment, the expression of H2S-producing enzyme cystathionine β-synthase in the LV was increased, and the stimulation of the NO signaling pathway improved endothelial function in the mesenteric artery. On the other hand, chronic treatment with GYY-4137 increased the expression of H2S-producing enzyme cystathionine γ-lyase in the LV and stimulated the beneficial pro-relaxant and anti-contractile activity of endogenous H2S in thoracic aorta. Our results suggest that sulfide and nitroso signaling pathways could trigger compensatory vasoactive responses in hypertensive rats with metabolic disorder. A slow H2S-releasing donor could partially amend metabolic-related changes and trigger beneficial activity of endogenous H2S.

NNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation–contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

Synthesis of new imidazole-triazole-glycoside hybrids as anti-breast cancer candidates

One of the leading causes of death worldwide among women is breast cancer. Thus, discovering new multi-target anti-breast cancer drugs is mandatory to stimulate nitric oxide (NO) and cyclooxygenase-2 (COX-2) as a cross-talking strategy for killing breast cancerous cells. 1-(3-(4-bromophenyl)-5-methyl-2-thioxo-2,3-dihydro-1H -imidazol-4-yl)ethan-1-one 1 was prepared and allowed to react with various alkyl halide propargyl bromide; 1,2-dibromoethane; and 1,3-dichloropropane to afford the corresponding alkyl imidazolo derivatives 2, and 14a,b respectively; Compound 2 allowed to react undergoes click reaction condition afforded the corresponding 1,2,3-triazole-N-acetylated glycosides 4 and 5 which reacted with methanolic ammonia solution to obtain free hydroxyl 1,2,3-triazole N-glycosides 6 and 7; respectively. Also, Compounds 14a,b reacted with sodium azide to give the azido derivatives 15,16; which reacted under click reaction condition to produce 1,2,3-triazole-C-glycosides compounds 18-21, respectively. Meanwhile, the previous compounds can be deacetylated to afford the free hydroxyl thioglycosides 22-25. Moreover, the thioglycoside derivatives of compound 1 were synthesized and underwent glycosylation conditions utilizing acetylated glycosyl bromides to give S-glycoside analogs 8-10 which proceeded to undergo deacetylated condition to obtain free hydroxyl imidazolo- S-glycosides 11-13; respectively. All newly synthesized compounds were tested against human breast MCF-7 and MDA-MB-231 cancerous cell lines. Cytotoxicity using MTT assay was performed as well as IC50 was measured for all candidates. Then, nitric oxide (NO) levels and cyclooxygenase-2 (COX-2) enzyme activity were investigated in the proposed cancerous cells. We resulted that the prepared compounds were more cytotoxic against MDA-MB-231 cells than MCF-7 cells. MCF-7 breast cancerous cells were resistant to doxorubicin in addition to the applied compounds, while MDA-MB-231 cells were sensitive. It was reported that compounds 22, 12, and 6 recorded the highest cytotoxic effects against both cancer cell lines. The signaling crosstalk between NO and COX-2 in cancer cells achieved that compounds 12 and 22 induced the significant elevation of NO levels in MCF-7 cells which in turn inhibited the COX-2 enzyme activity. In conclusion, the newly prepared compounds containing an imidazole nucleus have anti-breast cancer potential through modulation of NO and COX-2 signaling track.

Nitric oxide generated by Piriformospora indica-induced nitrate reductase promotes tobacco growth by regulating root architecture and ammonium and nitrate transporter gene expression

ABSTRACT Nitric oxide (NO) is involved not only in the regulation of plant growth, development, and stress responses but also in the regulation of plant-microbe interactions. Here, we demonstrate that Piriformospora indica can induce tobacco nitrate reductase to produce a NO signal in roots which enhances nitrogen uptake capacity by inducing the expression of ammonium and nitrate transporter genes and the development of lateral root and root hair, thereby promoting tobacco growth. In addition, the NO signal induced by P. indica is significantly different from that induced by the pathogen Phytophthora nicotianae. Inoculation with P. indica did not produce H2O2 and maintained high expression of Phytoglobin 1 in roots, resulting in a significantly lower NO level than in the roots inoculated with P. nicotianae. These findings suggest that an appropriate NO level is the likely basis of plant-P. indica symbiosis, which promotes the growth of host plants.

Inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse vulnerability in rats.

Glutamate signalling through the N‐methyl‐d‐aspartate receptor (NMDAR) activates the enzyme neuronal nitric oxide synthase (nNOS) to produce the signalling molecule nitric oxide (NO). We hypothesized that disruption of the protein–protein interaction between nNOS and the scaffolding protein postsynaptic density 95 kDa (PSD95) would block NMDAR‐dependent NO signalling and represent a viable therapeutic route to decrease opioid reward and relapse‐like behaviour without the unwanted side effects of NMDAR antagonists. We used a conditioned place preference (CPP) paradigm to evaluate the impact of two small‐molecule PSD95‐nNOS inhibitors, IC87201 and ZL006, on the rewarding effects of morphine. Both IC87201 and ZL006 blocked morphine‐induced CPP at doses that lacked intrinsic rewarding or aversive properties. Furthermore, in vivo fast‐scan cyclic voltammetry (FSCV) was used to ascertain the impact of ZL006 on morphine‐induced increases in dopamine (DA) efflux in the nucleus accumbens shell (NAc shell) evoked by electrical stimulation of the medial forebrain bundle (MFB). ZL006 attenuated morphine‐induced increases in DA efflux at a dose that did not have intrinsic effects on DA transmission. We also employed multiple intravenous drug self‐administration approaches to examine the impact of ZL006 on the reinforcing effects of morphine. Interestingly, ZL006 did not alter acquisition or maintenance of morphine self‐administration, but reduced lever pressing in a morphine relapse test after forced abstinence. Our results provide behavioural and neurochemical support for the hypothesis that inhibition of PSD95‐nNOS protein–protein interactions decreases morphine reward and relapse‐like behaviour, highlighting a previously unreported application for these novel therapeutics in the treatment of opioid addiction.

High-performance amperometric determination of nitric oxide released by endothelial cells using flow injection analysis

An amperometric flow injection analysis (FIA) method based on an optimized carbon paste electrode (CPE) for the determination of nitric oxide (NO) production from endothelial cells (ECs) was developed. The CPE was modified by Nafion, gold nanoparticles (AuNPs) and graphene nanoribbons (GNRs) via drop casting method. The proposed sensor exhibited excellent sensitivity and selectivity to the oxidation of NO with a linear correlation of 0.02 - 22.4 μM and a detection limit of 6 nM. The developed method is applied for real-time measurements of NO released from endothelial cells by stimulation of endothelial NO synthase (eNOS). Induction of NO synthesis by eNOS was successfully initiated by the calcium ionophore A23187, acetylcholine (ACh), or L-arginine. Furthermore, a reduction of eNOS activity could be observed after the addition of a NOS inhibitor, resulting in a decrease in NO signaling. Targeting eNOS could be of considerable value with regard to preventing or treating the development of cardiovascular diseases. The sensor’s ease of fabrication and applicability for cell measurements enables the device to be used in pharmacological research and drug discovery.