Differential effects of resveratrol on the dilator responses of femoral arteries, ex vivo Publication date: 1 November 2019 Source: Nitric Oxide, Volume 92 Author(s): Miguel Diaz, Vijay Parikh, Saira Ismail, Raja Maxamed, Emily Tye, Clare Austin, Tristan Dew, Brigitte A. Graf, Luc Vanhees, Hans Degens, May Azzawi Abstract Resveratrol is a plant-derived phytoalexin with antioxidant, anti-inflammatory and cardio-protective properties and may be a promising therapeutic intervention strategy in cardiovascular disease. Here, we investigated the acute direct effects of trans-resveratrol (RV), on acetylcholine (ACh)-induced and flow-mediated dilation (FMD) of isolated pressurized femoral arteries of young (4-month-old) and old (26-month-old) mice. Vessel exposure to RV enhanced ACh (0.01–1.0 mM)-induced dilation (p < 0.05), but not FMD (@ 5–10 μL⋅min−1) (p < 0.05) in both young and old mice. After RV incubation, acute nitric oxide (NO) production by cultured endothelial cells was increased in response to 0.01 mM ACh, but reduced by flow (5–10 μL⋅min−1; p < 0.05). In isolated femoral arteries from endothelial nitric oxide synthase knockout (eNOS−/-) mice, RV had no overall effect on FMD, but potentiated ACh induced dilation, that was completely abolished by potassium channel blockers, Apamin and Tram 34 (p < 0.01). We demonstrate that the non-metabolised form of RV stimulates ACh-induced dilation via the NO and EDHF pathways, but not FMD by interaction with the cyclo-oxygenase pathway. Our findings have important implications in the use of RV (for both young and aged) under ‘normal’ non-diseased physiological states.

Hydrogen sulfide stimulates activation of hepatic stellate cells through increased cellular bio-energetics Publication date: 1 November 2019 Source: Nitric Oxide, Volume 92 Author(s): Turtushikh Damba, Mengfan Zhang, Manon Buist-Homan, Harry van Goor, Klaas Nico Faber, Han Moshage Abstract Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extracellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

S-Nitrosoglutathione exhibits greater stability than S-nitroso-N-acetylpenicillamine under common laboratory conditions: A comparative stability study Publication date: 1 November 2019 Source: Nitric Oxide, Volume 92 Author(s): Alyssa C. Melvin, W. Matthew Jones, Alec Lutzke, Christopher L. Allison, Melissa M. Reynolds Abstract S-Nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) are susceptible to decomposition by stimuli including heat, light, and trace metal ions. Using stepwise isothermal thermogravimetric analysis (TGA), we observed that NO-forming homolytic cleavage of the S–N bond occurs at 134.7 ± 0.8 °C in GSNO and 132.8 ± 0.9 °C in SNAP, contrasting with the value of 150 °C that has been previously reported for both RSNOs. Using mass spectrometry (MS), nuclear magnetic resonance (NMR), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), we analyzed the decomposition products from TGA experiments. The organic product of GSNO decomposition was glutathione disulfide, while SNAP decomposed to form N-acetylpenicillamine disulfide as well as other products, including tri- and tetrasulfides. In addition, we assessed the relative solution stabilities of GSNO and SNAP under common laboratory conditions, which include variable temperature, pH, and light exposure with rigorous exclusion of trace metal ions by chelation. GSNO exhibited greater stability than SNAP over a 7-day period except in one instance. Both RSNOs demonstrated an inverse relationship between solution stability and temperature, with refrigeration considerably extending shelf life. A decrease in pH from 7.4 to 5.0 also enhanced the stability of both RSNOs. A further decrease in pH from 5.0 to 3.0 resulted in decreased stability for both RSNOs, and is notably the only occasion in which SNAP proved more stable than GSNO. After 1 h of exposure to overhead fluorescent lighting, both RSNOs displayed high susceptibility to light-induced decomposition. After 7 h, GSNO and SNAP decomposed 19.3 ± 0.5% and 30 ± 2%, respectively.

DDAH1 and DDAH2 polymorphisms associate with asymmetrical dimethylarginine plasma levels in erectile dysfunction patients but not in healthy controls Publication date: 1 November 2019 Source: Nitric Oxide, Volume 92 Author(s): Guilhermo Brites-Anselmi, Ana Maria Milanez Azevedo, Anderson Heiji Lima Miyazaki, Lucas Cezar Pinheiro, Fernanda Borchers Coeli-Lacchini, Murilo Ferreira de Andrade, Carlos Augusto Fernandes Molina, Silvio Tucci, Emilio Hirsch, Jose Eduardo Tanus-Santos, Riccardo Lacchini Abstract Erectile Dysfunction (ED) is one of the main complaints of aging male. A reduced production of Nitric Oxide (NO) may be involved in ED pathogenesis. NO is synthesized from l-Arginine, and asymmetrical dimethylarginine inhibits all NO synthases. DDAH1 and DDAH2 are genes that encode enzymes responsible for metabolizing ADMA. We aimed to assess whether: 1) ADMA and nitrite levels associated with ED risk and with symptoms intensity; and whether 2) DDAH1 and DDAH2 gene polymorphisms associate with changes in biochemical data, and with ED risk and symptoms intensity. In this study were included 98 healthy controls and 130 ED patients. ADMA levels were measured by ELISA and nitrite levels by Chemiluminescence. DDAH1 and DDAH2polymorphisms were assessed by Taqman assays. We found that ED had increased nitrite levels and lower ADMA levels than Control group (P < 0.05). We found a significant correlation of ADMA with Nitrite levels only in ED (B = -0.57, P < 0.001). Genotypes and haplotypes of DDAH1 were associated with ADMA levels in ED (P < 0.05), while haplotypes of DDAH2 were associated with levels of nitrite in ED (P < 0.05). Erectile dysfunction patients show an association between DDAH1 and DDAH2 polymorphisms with ADMA levels, which in turn are negatively correlated with nitrite levels. This is not evident on healthy controls.

Visible light-controlled NO generation for photoreceptor-mediated plant root growth regulation Publication date: 1 November 2019 Source: Nitric Oxide, Volume 92 Author(s): Suprakash Biswas, Neha Upadhyay, Debojyoti Kar, Sourav Datta, Apurba Lal Koner Abstract Nitric oxide (NO) is an essential redox-signaling molecule free radical, contributes a significant role in a diverse range of physiological processes. Photo-triggered NO donors have significant potential compared to other NO donors because it releases NO in the presence of light. Hence, an efficient visible light-triggered NO donor is designed and synthesized by coupling 2,6-dimethyl nitrobenzene moiety at the peri-position of 1, 8-naphthalimide. The NO-releasing ability is validated using various spectroscopic techniques, the photoproduct is characterized, and finally, the NO generation quantum yield is also determined. Furthermore, the photo-generated NO has been employed to Arabidopsis thaliana as a model plant to examine the effect of photoreceptor-mediated NO uptake on plant root growth regulation molecule. Graphical abstract

Upregulation of the nitrosylome in bipolar disorder type 1 (BP1) and major depression, but not BP2: Increased IgM antibodies to nitrosylated conjugates are associated with indicants of leaky gut Publication date: 1 October 2019 Source: Nitric Oxide, Volume 91 Author(s): Michael Maes, Denitsa Simeonova, Drozdstoy Stoyanov, Jean–Claude Leunis Abstract Objective Major depression (MDD) and a lifetime history of MDD are characterized by increased nitrosylation, while bipolar disorder type 1 (BP1), but not BP2, is accompanied by highly increased levels of oxidative stress and nitric oxide (NO) production. Nevertheless, it is unknown whether nitrosylation is involved in BP and whether there are differences in nitrosylation between BP1 and BP2. Methods Serum IgM antibodies directed against nitroso (NO)-adducts were examined in MDD, BP1, BP2 and healthy controls, namely IgM responses to NO-cysteine, NO-tryptophan (NOW), NO-arginine and NO-albumin (SBA) in association with IgA/IgM responses to LPS of Gram-negative bacteria, IgG responses to oxidized low-density lipoprotein (ox-LDL) and serum peroxides. Results Serum IgM levels against NO adducts were significantly higher in BP1 and MDD as compared with healthy controls, whereas BP2 patients occupied an intermediate position. IgM responses to NO-albumin were significantly higher in BP1 and MDD than in BP2 patients. There were highly significant associations between the IgM responses to NO-adducts and IgG responses to ox-LDL and IgA/IgM responses to Gram-negative bacteria. Conclusions BP1 and MDD are characterized by an upregulation of the nitrosylome (the proteome of nitrosylated proteins) and increased IgM responses to nitrosylated conjugates. Increased nitrosylation may be driven by increased bacterial translocation and is associated with lipid peroxidation processes. Innate-like (B1 and marginal zone) B cells and increased nitrosylation may play a key role in the major affective disorders through activation of immune-inflammatory and oxidative pathways, cardiovascular comorbidity and impairments in antioxidant defenses, neuro-glial interactions, synaptic plasticity, neuroprotection, neurogenesis.

Amyloid-β induced neuropathological actions are suppressed by Padina gymnospora (Phaeophyceae) and its active constituent α-bisabolol in Neuro2a cells and transgenic Caenorhabditis elegans Alzheimer's model Publication date: 1 October 2019 Source: Nitric Oxide, Volume 91 Author(s): Balakrishnan Shanmuganathan, Sethuraman Sathya, Boopathi Balasubramaniam, Krishnaswamy Balamurugan, Kasi Pandima Devi Abstract The inhibition of Aβ peptide development and aggregation is a hopeful curative approach for the discovery of disease modifying drugs for Alzheimer's disease (AD) treatment. Recent research mainly focuses on the discovery of drugs from marine setting due to their immense therapeutic potential. The present study aims to evaluate the brown macroalga Padina gymnospora and its active constituent α-bisabolol against Aβ25-35 induced neurotoxicity in Neuro2a cells and transgenic Caenorhabditis elegans (CL2006 and CL4176). The results of the in vitro study revealed that the acetone extract of P. gymnospora (ACTPG) and its active constituent α-bisabolol restores the Aβ25-35 induced alteration in the oxidation of intracellular protein and lipids. In addition, ACTPG and α-bisabolol inhibited cholinesterase and β-secretase activity in Neuro2a cells. Moreover, the intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) production was reduced by ACTPG and α-bisabolol in Neuro2a cells. The decrease in the expression level of apoptotic proteins such as Bax and caspase-3 in ACTPG and α-bisabolol treated group indicates that the seaweed and its bioactive compound have anti-apoptotic property. Further, the in vivo study revealed that the ACTPG and α-bisabolol exerts neuroprotective effect against Aβ induced proteotoxicity in transgenic C. elegans strains of AD. Moreover it altered the Aβ mediated pathways, lifespan, macromolecular damage and down regulated the AD related gene expression of ace-1, hsp-4 and Aβ, thereby preventing Aβ synthesis. Overall, the outcome of the study signifies the neuroprotective effect of ACTPG and α-bisabolol against Aβ mediated AD pathology. Graphical abstract

Structure effect of water-soluble iron porphyrins on catalyzing protein tyrosine nitration in the presence of nitrite and hydrogen peroxide Publication date: 1 October 2019 Source: Nitric Oxide, Volume 91 Author(s): Jiayu Li, Zhen Yang, Hailing Li, Zhonghong Gao Abstract Water-soluble iron porphyrins, such as FeTPPS (5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III)), FeTMPyP (5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphyrinato iron (III) chloride) and FeTBAP (5,10,15,20-tetrakis (4-benzoic acid) porphyrinato iron (III)), are highly active catalysts for peroxynitrite decomposition and thereby have been suggested as therapeutic agent for inflammatory diseases that implicate the involvement of nitrotyrosine formation. Here, we systemically investigated catalytic properties of FeTPPS, FeTMPyP and FeTBAP on protein nitration in the presence of hydrogen peroxide and nitrite. We showed that FeTPPS, FeTBAP and FeTMPyP all exhibited higher peroxidase activity in compared with hemin. As to protein nitration, the catalytic effect of FeTPPS and FeTBAP are effective in the presence of hydrogen peroxide and nitrite, while negligible BSA nitration was observed in the case of FeTMPyP. Moreover, the underlying mechanism of the oxidation of FeTPPS, FeTBAP and FeTMPyP was further studied. Collectively, our results suggest that, compound I and II species are involved in as the key intermediates in FeTMPyP/H2O2 system as similar as those in FeTPPS/H2O2 and FeTBAP/H2O2 system. As compared to weak antioxidants, TPPS and TBAP, however, TMPyP scavenges oxo-Fe (IV) intermediates of FeTMPyP at a faster rate by significant self-degradation; results in the shortest lifetimes of OFeIV-TMPyP and the lowest catalytic activity on oxidizing tyrosine and nitrite; and therefore, attributes to inactivation of FeTMPyP in protein nitration. In addition, association of FeTMPyP to BSA was found weak, while strong binding of FeTPPS and FeTBAP were observed. The weak binding keeps away of target residue of BSA from the center of FeTMPyP where the RNS is generated, which might be attributed as additional factors to the inactivation of FeTMPyP in protein nitration. Graphical abstract The modification group of water soluble iron porphyrin plays an important role in determining its catalytic effect on catalyzing protein tyrosine nitration in the presence of H2O2 and NO2−.

Electroacupuncture ameliorates neuronal injury by Pink1/Parkin-mediated mitophagy clearance in cerebral ischemia-reperfusion Publication date: 1 October 2019 Source: Nitric Oxide, Volume 91 Author(s): Huanyuan Wang, Suhui Chen, Yamin Zhang, Hong Xu, Hua Sun Abstract The accumulation of dysfunctional mitochondria induced by the impairment of the autophagy-lysosome pathway (ALP), especially mitophagy is an important cause of cerebral ischemia-reperfusion (I/R) injury. Electroacupuncture (EA) exerts remarkable effects in treating ischemic stroke; however, the detailed mechanism remains unclear. In this study, rats were treated with mitochondrial permeability transition pore (mPTP) opening inhibitor, peroxynitrite (ONOO−) scavenger, or selective inhibitor of mitophagy activation during 2-h middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion in combination with EA treatment. RNA-Seq analysis showed that EA treatment in cerebral I/R was linked to the autophagosome, the PI3K/Akt signaling pathway and metabolic pathways. We found that I/R resulted in significantly mitochondrial function impairments including decreased mitochondrial membrane potential (MMP) and ATP levels, aggregation of damaged mitochondria, excessive nitro/oxidative stress, PI3K/Akt/mTOR-mediated ALP dysfunction and deficiency of Pink1/Parkin-mediated mitophagy clearance. The treatment with EA, cyclosporine-A (CsA, a potent inhibitor of mPTP opening) or FeTMPyP (a type of ONOO− scavenger) could significantly increase MMP and/or ATP levels, improve mitochondrial function and decrease neuronal injury. At the same time, EA also improved ALP dysfunction and the deficiency of mitophagy clearance; however, mitochondrial division inhibitor-1 (Mdivi-1, a selective inhibitor of mitophagy activation) blocked mitophagy clearance and aggravated neuronal injury. Taken together, EA ameliorates nitro/oxidative stress-induced mitochondrial functional damage and decreases the accumulation of damaged mitochondria via Pink1/Parkin-mediated mitophagy clearance to protect cells against neuronal injury in cerebral I/R. Graphical abstract

Validity and reliability of test strips for the measurement of salivary nitrite concentration with and without the use of mouthwash in healthy adults Publication date: 1 October 2019 Source: Nitric Oxide, Volume 91 Author(s): Abrar M. Babateen, Oliver M. Shannon, John C. Mathers, Mario Siervo Abstract The nitrate (NO3−)-nitrite (NO2−)-nitric oxide (NO) pathway has received considerable interest in recent years as a potential target for nutritional interventions designed to increase NO production, and elicit therapeutic effects in humans. In particular, studies have evaluated the effects of supplemental dietary NO3−, which serves as a ‘substrate’ for this pathway, on numerous different health outcomes. One challenge has been to evaluate compliance with the NO3− interventions. A recent advance in this field has been the development of a non-invasive, simple and rapid method to measure nitrite concentrations in saliva using small test salivary strips. In the present study, ten healthy adults were recruited to a randomised, crossover study and received an acute dose of NO3−-rich beetroot juice (BJ) after rinsing their mouth with either water or commercially available antibacterial mouthwash. Salivary NO3− and NO2− concentrations were measured at baseline and up to 5 h after BJ consumption using the gold-standard chemiluminescence and a colorimetric Griess assay. In addition, two salivary test strips (Berkeley Test strips, CA, USA) were used to measure NO2−concentrations at the same time points. Five observers read the strips and inter- and intra-observer reliability was measured. The Bland-Altman method was used to provide a visual representation of the agreement between the methods used to evaluate salivary NO3−/NO2−concentration. Sialin concentrations were measured at baseline and up to 5 h after BJ consumption. BJ elevated salivary NO3− and NO2− concentrations when the mouth was rinsed with water (both P < 0.01), as assessed via both chemiluminescence and Griess methods. Rinsing the mouth with antibacterial mouthwash attenuated markedly the increase in NO2− (P < 0.001), while NO3−concentrations were unaffected (P > 0.05). The Intra-Class Coefficients of Correlation (ICC) showed a high inter- and intra-observer reliability (r > 0.8). A significant positive correlation was found between absolute salivary NO2−concentrations measured by strips and Griess and chemiluminescence methods (rho = 0.83 and 0.77, respectively) and also when expressed as changes in salivary NO2− concentrations (rho = 0.80 and 0.79, respectively). Bland Altman analysis indicated a poor agreement for absolute NO2− concentrations between salivary strips and the chemiluminescence and Griess methods. A small significant negative correlation was found between changes in salivary sialin and salivary NO2ˉ concentrations (r = −0.20, P = 0.04). A non-significant positive correlation was observed between the change in salivary sialin and salivary NO3ˉ concentrations (r = 0.18, P = 0.06). This study suggests that commercially available salivary NO2− test strips provide a reasonable surrogate marker for monitoring changes in salivary NO2− concentrations in humans. However, the strips do not provide accurate estimates of absolute NO2−concentrations.