NO Signaling Pathway Research Articles

The apoptotic inducible effects of salicylic acid on hepatoma cell line: relationship with nitric oxide signaling.

Clinical and experimental data suggest that salicylic acid (SA) is tumor preventive and NO has a multitude of effects on tumor biology. Therefore, firstly, the aim of our study is to explore the important role of SA in apoptotic induction of liver cancer cells. Secondly, we investigate whether SA mediates the anti-tumor effects by NO signaling pathway. The liver cancer cell line was treated with different concentrations of SA. Cell proliferation was tested using MTS assay and cell apoptosis was assessed by flow cytometry. NO content and NOS activities were measured by biochemical assay. The anti- or pro-apoptotic regulator gene expressions were analyzed by real-time PCR. Our data illustrated that high concentration of SA significantly inhibited liver cancer cell proliferation accompanied by apoptosis induction. In addition, SA led to the release of NO and the increase of NOS activities in above process. Importantly, SA up-regulated a series of apoptosis-related gene expression and reduced the mRNA level of HMGB1. Meanwhile, we also found that NOS inhibitor L-NAME and NO scavenger cPTIO attenuated the above SA-induced effects. Thus, we provided the evidence that SA exerted anti-tumor effects in liver cancer cell in part mediated by the NO pathway.

The Endophytic Fungus Phomopsis liquidambari Increases Nodulation and N2 Fixation in Arachis hypogaea by Enhancing Hydrogen Peroxide and Nitric Oxide Signalling

The continuous cropping obstacles in monoculture fields are a major production constraint for peanuts. Application of the endophytic fungus Phomopsis liquidambari has increased peanut yields, and nodulation and N2 fixation increases have been considered as important factors for P. liquidambari infection-improved peanut yield. However, the mechanisms involved in this process remain unknown. This work showed that compared with only Bradyrhizobium inoculation, co-inoculation with P. liquidambari significantly elevated endogenous H2O2 and NO levels in peanut roots. Pre-treatment of seedlings with specific scavengers of H2O2 (CAT) and NO (cPTIO) blocked P. liquidambari-induced nodulation and N2 fixation. CAT not only suppressed the P. liquidambari-induced nodulation and N2 fixation, but also suppressed the enhanced H2O2 and NO generation. Nevertheless, the cPTIO did not significantly inhibit the induced H2O2 biosynthesis, implying that H2O2 acted upstream of NO production. These results were confirmed by observations that exogenous H2O2 and sodium nitroprusside (SNP) reversed the inhibition of P. liquidambari-increased nodulation and N2 fixation by the specific scavengers. The transcriptional activities of the symbiosis-related genes SymRK and CCaMK of peanut-Bradyrhizobium interactions also increased significantly in response to P. liquidambari, H2O2 and SNP treatments. The pot experiment further confirmed that the P. liquidambari infection-enhanced H2O2 and NO signalling pathways were significantly related to the increase in peanut nodulation and N2 fixation. This is the first report that endophytic fungus P. liquidambari can increase peanut-Bradyrhizobium interactions via enhanced H2O2/NO-dependent signalling crosstalk, which is conducive to the alleviation of continuous cropping obstacles via an increase in nodulation and N2 fixation.

Abstract TP277: Adropin is Profoundly Neuroprotective in Experimental Ischemic Stroke

Adropin has been shown to improve peripheral endothelial dysfunction, reduce insulin resistance, and increase glucose utilization in obesity and diabetes. Although we showed that adropin reduces paracellular permeability of brain endothelial cells exposed to ischemia-like conditions, little is known of the effects of adropin in the brain. We hypothesized that adropin exerts neuroprotection through activation of endothelial nitric oxide synthase (eNOS)/NO signaling pathway and reduction of blood-brain barrier (BBB) damage. Male mice were subjected to permanent middle cerebral artery occlusion (MCAO) and treated intravenously with either vehicle or synthetic adropin (90, 900 and 2700 nmol/kg) at the onset of ischemia and sacrificed to determine infarct size at 48 h. Cellular localization of adropin was determined using immunofluorescence. Brain tissue was collected at 24 h after stroke to determine BBB damage, matrix metalloproteinase (MMP)-9 activity, and levels of tight junction proteins, eNOS, and gp91 phox -containing NADPH oxidase. We determined the effects of a 3-h delayed administration on stroke outcomes to evaluate the translational value of adropin therapy. In the brain, adropin was expressed on endothelial cells and at a low level on astrocytes after stroke. Adropin given at the onset of MCAO dose-dependently reduced infarct size. More importantly, delaying adropin administration to 3 h post-MCAO profoundly reduced infarct size compared to vehicle-treated mice. Ischemia slightly increased eNOS phosphorylation at Ser1176, significantly increased gp91 phox (a major source of reactive oxygen species), and reduced brain adropin levels. Adropin treatment induced a dramatic increase in eNOS phosphorylation, significantly reduced gp91 phox levels, and restored endogenous adropin levels compared to the vehicle group. Additionally, adropin protected against a stroke-induced increase in MMP-9 activity, loss of zona occludens-1 and occludin, and BBB disruption as assessed by extravasated IgG and albumin. Collectively, these findings indicate that adropin-mediated neuroprotection in ischemic stroke may be through an increase in eNOS-derived NO bioavailability, reduction of gp91 phox , and decreased MMP-9 activity and BBB damage.

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction.

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients' miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states.

344 - The Blood Pressure-Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor

Inorganic nitrate and nitrite from dietary and endogenous sources are metabolized to nitric oxide (NO) and other bioactive nitrogen oxides that affect blood pressure. The mechanisms for nitrite bioactivation are unclear but recent studies in rodents suggest that gastric acidity may influence the systemic effects of this anion. In a randomized double-blind placebo-controlled crossover study we tested the effects of a proton pump inhibitor on the acute cardiovascular effects of nitrite. 15 healthy non-smoking, normotensive subjects, age 19-39 were pretreated with placebo or esomeprazole (3 x 40 mg) prior to ingesting sodium nitrite (0.3 mg·kg −1 ), followed by blood pressure monitoring. Nitrite reduced systolic blood pressure by a maximum of 6 ± 1.3 mmHg when taken after placebo while pretreatment with esomeprazole blunted this effect. Peak plasma nitrite, nitrate and RXNO levels after nitrite ingestion were similar in both interventions. In 8 healthy volunteers we then infused increasing doses of sodium nitrite (1, 10, and 30 nmol · kg −1 · min −1 ) intravenously. Interestingly, while plasma nitrite peaked at similar levels as with orally ingested nitrite (~ 1.8 µM), no changes in blood pressure were observed. In rodents, esomeprazole did not affect the blood pressure response to the NO-donor DEANONOate, or vascular relaxation to nitroprusside and acetylcholine, demonstrating an intact downstream NO-signaling pathway. We conclude that the acute blood pressure-lowering effect of nitrite requires an acidic gastric environment. Future studies will reveal if the cardiovascular complications associated with the use of proton pump inhibitors are linked to interference with the nitrate-nitrite-NO pathway.

Blood Pressure-Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor.

Inorganic nitrate and nitrite from dietary and endogenous sources are metabolized to NO and other bioactive nitrogen oxides that affect blood pressure. The mechanisms for nitrite bioactivation are unclear, but recent studies in rodents suggest that gastric acidity may influence the systemic effects of this anion. In a randomized, double-blind, placebo-controlled crossover study, we tested the effects of a proton pump inhibitor on the acute cardiovascular effects of nitrite. Fifteen healthy nonsmoking, normotensive subjects, aged 19 to 39 years, were pretreated with placebo or esomeprazole (3×40 mg) before ingesting sodium nitrite (0.3 mg kg-1), followed by blood pressure monitoring. Nitrite reduced systolic blood pressure by a maximum of 6±1.3 mm Hg when taken after placebo, whereas pretreatment with esomeprazole blunted this effect. Peak plasma nitrite, nitrate, and nitroso species levels after nitrite ingestion were similar in both interventions. In 8 healthy volunteers, we then infused increasing doses of sodium nitrite (1, 10, and 30 nmol kg-1 min-1) intravenously. Interestingly, although plasma nitrite peaked at similar levels as with orally ingested nitrite (≈1.8 µmol/L), no changes in blood pressure were observed. In rodents, esomeprazole did not affect the blood pressure response to the NO donor, DEA NONOate, or vascular relaxation to nitroprusside and acetylcholine, demonstrating an intact downstream NO-signaling pathway. We conclude that the acute blood pressure-lowering effect of nitrite requires an acidic gastric environment. Future studies will reveal if the cardiovascular complications associated with the use of proton pump inhibitors are linked to interference with the nitrate-nitrite-NO pathway.

Adenosine A2A receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies.

We aim to investigate whether A2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A2A agonist, 24h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC50 10-9.2M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (∼1.5-fold) and pre-eclamptic pregnancies (∼1.2-fold), an effect blocked by the A2A antagonist, ZM-241385 (10-5M) and the inhibitor of NO synthase, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A2A. CGS-21680 also increased (∼1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A2A decrease (∼30%) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.

Effects of chronic l-DOPA administration on neurogenic and endothelium-dependent relaxation responses in rabbit corpus cavernosum

Dopamine is a crucial central neurotransmitter that plays a fundamental role in the autonomic and somatic components of penile reflexes in animals and humans. Similar to the erectile responses of dopamine, systemic administration of l-DOPA induces yawning and penile erection in some species. The possible effects of l-DOPA on nitric oxide (NO)-dependent and -independent non-adrenergic non-cholinergic (NANC) relaxation responses mediated by electrical field stimulation (EFS) and endothelium-dependent relaxation were investigated in this study. Thirty-two adult albino male rabbits, in two- and four-week-treatment groups, were divided into three subgroups: control group (saline-injected) (n=4), 3mg/kg/day (low dose) l-DOPA-injected groups (n=6) and 12mg/kg/day (high dose) l-DOPA-injected groups (n=6). After the intraperitoneal injection treatments, the corpus cavernosum tissues were placed in organ bath chambers. The EFS-mediated responses, and the concentration-response curve to carbachol, sodium nitroprusside (SNP), sildenafil were assessed. The two-week treatment with high-dose l-DOPA decreased the NO-dependent NANC relaxation responses, while there was no change in the low-dose two- and four-week treatment groups. The NO-independent NANC relaxation responses in the two-week groups decreased, and the responses in the four-week groups were unchanged when compared to the controls. The relaxation responses to carbachol showed no differences among all groups except for the high-dose four-week l-DOPA group. The relaxation responses of SNP and sildenafil were increased in all of the treatment groups when compared to the controls. The observed increases in SNP- and sildenafil-induced responses, along with the decreased EFS-mediated responses, suggest increased sensitivity in the NO-signalling pathway following l-DOPA administration.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries.Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively.SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC.In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

H2S- and NO-Signaling Pathways in Alzheimer's Amyloid Vasculopathy: Synergism or Antagonism?

Alzheimer's type of neurodegeneration dramatically affects H2S and NO synthesis and interactions in the brain, which results in dysregulated vasomotor function, brain tissue hypoperfusion and hypoxia, development of perivascular inflammation, promotion of Aβ deposition, and impairment of neurogenesis/angiogenesis. H2S- and NO-signaling pathways have been described to offer protection against Alzheimer's amyloid vasculopathy and neurodegeneration. This review describes recent developments of the increasing relevance of H2S and NO in Alzheimer's disease (AD). More studies are however needed to fully determine their potential use as therapeutic targets in Alzheimer's and other forms of vascular dementia.

Thrombospondin-1 Gene Polymorphism Is Associated with Estimated Pulmonary Artery Pressure in Patients with Sickle Cell Anemia

Thrombospondin-1 (TSP1) is a multifunctional glycoprotein present in platelet α-granules that contains domains for adhesive proteins, enzymes, and cell receptors. It is released by activated platelets and is increased in the plasma of patients with sickle cell anemia (SCA). TSP1 is known to mediate adherence of sickle reticulocytes to endothelial cells. Furthermore, TSP1 also inhibits the NO signaling pathway through its binding to the CD47 cell receptor on endothelial cells and platelets. Thus, we hypothesized that genetic variants in TSP1 may contribute to the marked phenotypic variation seen in SCA.We tested whether genetic variants of the TSP1 gene are associated with biomarkers of SCA severity and specific complications in a cohort of 452 patients with homozygous HbSS disease from the multicenter walk-PHaSST (Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy) trial. Genetic data from 406 patients were included in the analysis. Patients ranged in age from 12 to 70 years, and 51% were females. 307 patients were enrolled at various sites in the United States, while 99 patients were enrolled at Hammersmith Hospital in the UK. A total of 10 SNPs were selected, including haplotype tagged SNPs, coding variants, and SNPs potentially regulating gene expression and alternative splicing. DNA isolated from peripheral blood was used for genotyping.We found that the TSP1 SNPs rs1478604 and rs1478605 were significantly positively correlated with tricuspid regurgitation velocity (TRV) >2.5 m/sec, an echocardiographic marker of pulmonary hypertension associated with increased mortality, under a recessive model (r=0.10, p=0.0288 and r=0.13, p=0.0047 respectively). TRV was also analyzed as a categorical variable using regression analysis, and subjects were grouped based on the following clinically relevant cut-off points, 2.5 m/sec, 2.7 m/sec, and 3 m/sec. Univariate regression analyses showed TRV>2.5 m/sec was independently associated with the recessive genotypes of rs1478604 and rs1478605 (p=0.069 and 0.017 respectively). As previously published, we confirmed the association of TRV with age, gender, NT-proBNP and creatinine, and we adjusted for these variables in multivariate regression analyses for the association of TRV and each SNP. The results showed that the two SNPs were significantly associated with TRV>2.5 m/sec (rs1478604, OR 2.54, 95% CI (1, 6.44), p<0.05 and rs1478605, OR 4.20, 95% CI (1.41, 12.49), p<0.01) even after controlling for these variables.The results suggest that genetic variations in the TSP1 gene are associated with variations in estimated pulmonary artery pressure in patients with SCA. Both rs1478604 and rs1478605 are localized to the 5' untranslated region. Bioinformatic analysis of these SNPs for putative transcription factor binding sites demonstrated that the two SNPs are associated with both gain and loss of transcription factor binding compared to the wild type. Therefore, both SNPs are potentially functional in TSP1 regulation. These data also suggest possible translational implications in light of earlier pre-clinical reports that showed that mice lacking TSP1 were protected from hypoxia-mediate pulmonary and right ventricular remodeling and showed less alterations in PA pressure.In populations without SCA, these alleles have been independently linked to risk of malaria, corneal immune function, and gastric cancer. TSP1 activity has also been linked to fibrotic renal disease in a rat model and to IRI-mediated AKI in a murine model. The mechanism implicated in some of these cases involves disordered TSP1 regulation of transforming growth factor beta (TGF-beta), whose superfamily of receptors is dysregulated in the pathogenesis of pulmonary hypertension and kidney disease.Our findings support a hypothetical model of TSP1 involvement in the pathophysiology of SCA. Efforts to seek validation of the genetic findings from this study in an independent SCA cohort are currently underway. DisclosuresIsenberg:RCTI, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313; Vasculox, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313.

The involvement of neuronal nitric oxide synthase in the anti-epileptic action of curcumin on pentylenetetrazol-kindled rats

In this study, it was investigated whether a NO signaling pathway is involved in the anti-epileptic effect of curcumin on pentylenetetrazol (PTZ)-kindled rats. PTZ-kindled rats received different doses of curcumin that were administered intraperitoneally for 24 days. Either a non-selective inhibitor of nitric oxide synthase (NOS) (N-nitro-L-arginine methyl ester (L-NAME)), a selective inhibitor of neuronal NOS (7-Nitroindazole (7-NI)), a selective inhibitor of inducible NOS (aminoguanidine (AG)), or a NO precursor (L-arginine (L-ARG)) was administered chronically to evaluate the role of NO in curcumin's anti-seizure effect. A chronic administration of curcumin (200 mg/kg) was most effective for decreasing the mean frequency of epileptiform discharge. Furthermore, a pretreatment with L-NAME or 7-NI augmented the anti-epileptic effect of curcumin. In contrast, AG failed to significantly alter the anti-epileptic effect of curcumin. A pretreatment with L-ARG temporally reversed the anti-epileptic effect of curcumin in the early stage, but in the late stage, it potentiated curcumin's anti-epileptic effect. These findings suggest that the L-arginine-nitric oxide pathway may be involved in the anti-epileptic properties of curcumin, and that the role of nNOS (and not iNOS) is prominent in this neuroprotective feature.

Synergistic apoptosis-inducing effect of aspirin and isosorbide mononitrate on human colon cancer cells.

Aspirin and isosorbide mononitrate (ISMN) are two commonly used drugs, which are clinically applied for the treatment of inflammatory and cardiovascular diseases, respectively. Recently, aspirin has attracted interest due to its potential application for the treatment of cancer, particularly colon cancer. NO-aspirin, an aspirin derivative containing a covalently bound NO-donating moiety, has been proven to be an effective anti‑tumor agent with apoptosis-inducing ability. In the present study, ISMN was used as an NO donor and its synergic effect with aspirin was assessed in human colon cancer cells. In vitro, an MTT assay demonstrated that ISMN had a synergistic effect on the growth inhibitory effects of aspirin on HCT116 and SW620 colon cancer cells, while the growth of EA.hy926 normal endothelial cells was unaffected. This synergistic anti‑tumor effect was further validated in vivo using nude mouse HCT116 cell xenograft model. Observation of nuclear morphology, Annexin V-fluorescein isothiocyanate/propidium iodide double staining and a caspase-3 activity assay suggested that the combination of the two drugs induced apoptosis in HCT116 cells. Furthermore, the molecular mechanisms of the apoptotic effect of the drugs was assessed using an NO release assay, reverse transcription quantitative polymerase chain reaction analysis, western blot analysis and a luciferase reporter assay. It was certified that the increase in the amount of NO release, the decrease in the luciferase promoter activity and the expression of cyclin D1 and c-myc in HCT116 cells were affected by aspirin and ISMN in a synergistic manner. In conclusion, the present study was the first, to the best of our knowledge, to report on the synergistic apoptosis-inducing effects of aspirin and ISMN in human colon cancer cells, which were mediated via Wnt and NO signaling pathways. The results of the present study will facilitate the development of future therapeutic strategies.

PPARγ–PI3K/AKT–NO signal pathway is involved in cardiomyocyte hypertrophy induced by high glucose and insulin

ObjectiveTo investigate the role of the PPARγ–PI3K/AKT–NO signaling pathway in cardiac hypertrophy induced by high glucose and insulin. MethodsCardiomyocyte hypertrophy induced by high glucose (25.5mmol/L) and insulin (0.1μmol/L) (HGI) was characterized in rat primary cardiomyocytes by measuring the cell surface area, protein content, and atrial natriuretic factor mRNA expression level. Protein and mRNA expressions were measured by Western blotting and real time RT-PCR, respectively. A spectrophotometric assay was used to measure the enzymatic concentration of endothelial NO synthase (eNOS), and the Griess assay measured the NO concentration. ResultsHGI induced significant cardiomyocyte hypertrophy and decreased the expression of PPARγ, AKT and eNOS at the mRNA and protein levels, which occurred in parallel with declining eNOS activity and NO concentration (P<0.05). The effects of HGI were inhibited by rosiglitazone (0.1μmol/L), a selective PPARγ agonist (P<0.05). However, GW9662, a selective PPARγ antagonist, abolished the effects of rosiglitazone (P<0.05). LY294002, a PI3K/AKT inhibitor, and NG-nitro-l-arginine-methyl ester, an NOS inhibitor, partially blocked the effects of rosiglitazone (P<0.05). ConclusionThese results suggest that the PPARγ–PI3K/AKT–NO signal transduction pathway might be involved in HGI-induced cardiomyocyte hypertrophy.

Sepiapterin Attenuates Increased Mitochondrial Fission in Human Cardiomyocytes Exposed to High Glucose

Anesthetic cardioprotection by volatile anesthetics is shown to reduce the infarct size after a myocardial ischemia/reperfusion injury. This protective effect is abolished under diabetic conditions, but the causal mechanisms have not been completely elucidated. Previously, we have demonstrated that high glucose increased reactive oxygen species (ROS) generation and mitochondrial fission. Sepiapterin, a BH4 analog, could decrease ROS by increasing nitric oxide synthase. In this study, we examined the relationship between mitochondrial fission and NO signaling pathways. We hypothesized that modulation of BH4/NO signaling with sepiapterin can restore the attenuated anesthetic cardioprotection conferred by high glucose through decreasing mitochondrial fission.Human induced pluripotent stem cell derived cardiomyocytes (CMs) were exposed to 5 or 25 mM glucose for 3 days with or without sepiapterin. CMs were exposed to isoflurane for 30 minutes followed by 6 hours of H2O2 exposure. Cell viability was measured using a lactate dehydrogenase assay or propidium iodide staining. Mitochondria were loaded with Mitotracker Green and imaged using confocal microscopy. The images were analyzed using the ImageJ software to assess the form factor and aspect ratio.CMs in 5 and 11 mM glucose were protected against oxidative stress as demonstrated by increased cell viability and decreased mitochondrial fission in isoflurane‐treated CMs. However, CMs exposed to 25 mM glucose had an attenuation of cardioprotection and increased mitochondrial fission. Addition of sepiapterin reduced mitochondrial fission and increased cell viability in CMs cultured in 25 mM glucose.

The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture.

Angiotensin-converting enzyme 2 (ACE2), its product angiotensin-(1-7), and its receptor Mas have been shown to moderate the adverse effects of the ACE-angiotensin II-AT1 axis in many diseases. The aim of this study was to determine whether the ACE2-Ang-(1-7)-Mas axis could have similar effects in a cell culture model of pancreatic damage. AR42J cells were stimulated with 10 nmol/L cerulein to simulate acute pancreatitis. ACE2, Ang-(1-7), Mas receptor, and PI3K/AKT pathway were measured by quantitative real-time polymerase chain reaction and Western blot analysis. ACE2 and Mas receptor protein levels in AR42J cells were significantly increased (P < 0.05) between 30 minutes and 6 hours postdisease induction compared with the control group. Mas receptor gene expression was significantly increased (P < 0.05) at 2 hours postdisease induction, and Ang-(1-7) was increased at 6 hours. Treatment with Ang-(1-7) in AR42J cells increased IL-10, decreased IL-6 and IL-8, and reduced the damage to pancreatic cells. Levels of IL-6 and IL-8 in AR42J cell culture were increased significantly after treatment with A779. Moreover, Ang-(1-7) increased the concentration of PI3K/AKT pathway and eNOSin AR42J cells. ACE2-angiotensin-(1-7)-Mas axis significantly inhibits pancreatitis in response to decreased inflammatory factors by the activation of endothelial nitric oxide synthase and NO signaling pathways.

S-Nitrosylation Positively Regulates Ascorbate Peroxidase Activity during Plant Stress Responses

Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways.

Age-associated changes of nitric oxide concentration dynamics in the central nervous system of Fisher 344 rats.

The increase in life expectancy is accompanied by an increased risk of developing neurodegenerative disorders and age is the most relevant risk factor for the appearance of cognitive decline. While decreased neuronal count has been proposed to be a major contributing factor to the appearance of age-associated cognitive decline, it appears to be insufficient to fully account for the decay in mental function in aged individuals. Nitric oxide ((•)NO) is a ubiquitous signaling molecule in the mammalian central nervous system. Closely linked to the activation of glutamatergic transmission in several structures of the brain, neuron-derived (•)NO can act as a neuromodulator in synaptic plasticity but has also been linked to neuronal toxicity and degenerative processes. Many studies have proposed that changes in the glutamate-(•)NO signaling pathway may be implicated in age-dependent cognitive decline and that the exact effect of such changes may be region specific. Due to its peculiar physical-chemical properties, namely hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of (•)NO concentration changes are critical determinants for the understanding of its bioactivity in the brain. Here we show a detailed study of how (•)NO concentration dynamics change in the different regions of the brain of Fisher 344 rats (F344) during aging. Using microelectrodes inserted into the living brain of anesthetized F344 rats, we show here that glutamate-induced (•)NO concentration dynamics decrease in the hippocampus, striatum, and cerebral cortex as animals age. performance in behavior testing of short-term and spatial memory, suggesting that the impairment in the glutamate:nNOS pathway represents a functional critical event in cognitive decline during aging.

Deficiency of endothelial nitric oxide signaling pathway exacerbates peritoneal fibrosis in mice

Long-term peritoneal dialysis (PD) causes peritoneal dysfunction and structural alterations, eventually leading to peritoneal fibrosis. The endothelial nitric oxide synthase (eNOS)-NO signaling pathway contributes to the progression of organ fibrosis. However, it remains unknown whether NO signaling is involved in the process of peritoneal fibrosis. We evaluated the role of the eNOS-NO signaling pathway in the development of peritoneal fibrosis and whether stimulation of soluble guanylate cyclase (sGC), a downstream effector of NO, could attenuate peritoneal fibrosis. We used wild-type (WT) and eNOS-deficient mice (eNOSKO). The mice underwent mechanical peritoneal stripping-induced peritoneal fibrosis at day 0. At 3, 7, 14, and 28 days after peritoneal stripping, the mice were killed. In some eNOSKO mice, the sGC stimulator Bay 41-2272 was administered by intraperitoneal injection. In WT mice, granulomatous tissue formation was observed in the submesothelial area at days 3 and 7. After day 7, the peritoneal membrane thickness gradually decreased and peritoneal tissue was repaired with leaving only slight fibrosis at day 28. However, eNOSKO mice demonstrated more progression of peritoneal fibrosis than WT mice at 28 days after peritoneal stripping. Expression of vimentin in the thickened peritoneum was prolonged after day 7 in eNOSKO mice. Treatment with Bay 41-2272 significantly attenuated peritoneal vimentin expression and fibrosis in the eNOSKO mice. Disruption of the eNOS-NO signaling pathway exacerbates peritoneal fibrosis by delaying wound healing. sGC stimulation may be a useful therapy for prevention of peritoneal fibrosis.

Evolving possible link between PI3K and NO pathways in neuroprotective mechanism of ischemic postconditioning in mice.

The present study was conducted to pharmacologically investigate the influence of NO signaling pathway in PI3K mediated neuroprotective mechanism of ischemic postconditioning (iPoCo). Bilateral carotid artery occlusion (BCAO) of 12min followed by reperfusion for 24h was employed to produce ischemia/reperfusion-induced cerebral injury in male Swiss mice. Memory was assessed using Morris water maze test. Degree of motor incoordination was evaluated using inclined beam walk test, rotarod test, and lateral push test. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Brain acetylcholinesterase activity, thiobarbituric acid reactive species (TBARS), nitrite/nitrate and reduced glutathione (GSH) levels were also estimated. BCAO followed by reperfusion produced significant rise in cerebral infarct size, acetylcholinesterase activity, and TBARS levels along with fall in nitrite/nitrate and GSH levels. A significant impairment of memory and motor coordination was also noted. iPoCo consisting of three episodes of 10s carotid artery occlusion and reperfusion significantly attenuated infarct size, memory impairment, motor incoordination, and altered biochemicals. iPoCo-induced neuroprotective effects were significantly abolished by wortmannin (a selective PI3K inhibitor). However, administration of L-Arginine (a NO precursor) in the presence of wortmannin restored the protective effect of ischemic postconditioning. It may be concluded that neuroprotective mechanism of iPoCo involves PI3K-mediated pathway with NO signaling as an important downstream step.