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The direct nitric oxide donor molsidomine is commonly used to relieve symptoms in chronic angina thanks to its vasodilatory properties that induce both a reduction in myocardial oxygen demand and an increase in coronary blood flow. The objective of this study was to compare the short-term effect of molsidomine 16 mg once daily (Coruno(®), Therabel Pharmaceuticals Ltd, Dublin, Ireland) in patients with stable angina previously on molsidomine 8 mg twice daily, in care of general practitioners (GPs) versus cardiologists.A total of 53 and 80 patients treated by GPs and cardiologists, respectively, took part in this multicenter, open-label clinical trial. Frequency of anginal attacks, short-acting nitroderivative tablets consumption, and subjective clinical status were evaluated, as was tolerability to molsidomine through the reporting of adverse events (AEs), the recording of vital parameters-resting blood pressure, heart rate, and electrocardiogram-and routine blood analyses.Although demographic and clinical characteristics were significantly different in GPs' compared with cardiologists' patients, the effect of either the 8 mg or the 16 mg molsidomine formulation on anginal crises and nitroderivatives consumption was similar in both patient cohorts, with a trend for the 16 mg formulation to be more efficient on symptoms in elderly patients. Subjective assessment of the beneficial effect of molsidomine 16 versus 8 mg was comparable in GPs' and cardiologists' patients independently of age, "no change" being the most often reported item. Self-evaluation of functional capacity in elderly showed on the whole no difference between the two patient cohorts, only magnitude of pace and total score on molsidomine 16 mg being higher in cardiologists' compared with GPs' patients, and total score in cardiologists' patients higher on molsidomine 16 mg compared with 8 mg. Incidences of AEs and drug-related AEs, as well as proportions of patients reporting such AEs, were similar between GPs' and cardiologists' patient cohorts as between molsidomine 8 and 16 mg formulations. Molsidomine 16 mg once daily had no clinically significant effect on blood pressure, heart rate, electrocardiogram or blood parameters, and compliance with treatment was excellent in whatever patient cohort.Despite significant demographic and clinical differences between patients in care of GPs and cardiologists, molsidomine was equally efficient in these two patient cohorts, with a trend towards less anginal attacks in the elderly when treated with the 16 mg compared with the 8 mg formulation. Compliance and tolerability to the drug were excellent in both patient cohorts.

Background and Aims Despite its beneficial effects, cisplatin has considerable nephrotoxic, ototoxic, neurotoxic and hepatotoxic side effects. It has been documented that reactive oxygen radical species are involved with the pathophysiology of cisplatin-induced hepatotoxicity. Molsidomine (MOL) can exert antioxidant and anti-inflammatory effects. Therefore, the current study was planned to determine the effects of cisplatin on the liver oxidant/antioxidant system and the possible protective effects of (MOL) on liver toxicity. Methods Animals were divided into four groups as follows: (1) control; (2) MOL; (3) cisplatin and (4) MOL plus cisplatin group. Biochemical and histopathological evaluations were performed on the extracted liver tissue. Also, serum levels of serum aspartate transaminase (AST) and serum alanine transaminase (ALT) were determined. Results Our results clearly indicated that liver antioxidant enzyme activities and ALT levels were significantly decreased, whereas lipid peroxidation and neutrophil accumulation were increased in the cisplatin-treated animals (5 mg/kg single dose, i.p.) compared to the control rats. MOL treatment (4 mg/kg/day, i.p.) for 3 consecutive days provided a significant protection against cisplatin-induced hazardous changes in the liver tissue. Our histopathological findings including caspase-3 activity were also in accordance with the biochemical results. Conclusions We propose that MOL acts in the liver as a potent scavenger of free radicals, anti-inflammatory and anti-apoptotic effects to prevent the toxic effects of cisplatin, both at the biochemical and histopathological levels.

A series of unmodified and organically modified mesoporous silica materials was prepared. The unmodified mesoporous silica was synthesized via sol–gel synthesis in the presence of d -glucose as pore-forming agent. The functionalized by phenyl, aminopropyl and mercaptopropyl groups silica materials were prepared via grafting. The fabricated adsorbent materials were characterized by Fourier transform infrared spectroscopy (FTIR) analysis, N 2 adsorption/desorption and elemental analysis methods. Then their adsorption properties for mesoionic dug molsidomine were investigated at 290–313 K and physiological pH value. Thermodynamic parameters of molsidomine adsorption on the synthesized materials have been calculated. The obtained results showed that the adsorption process of molsidomine on the phenyl modified silica is the most quantitatively and energetically favorable. The unmodified and mercaptopropyl modified silica materials exhibit significantly higher adsorption capacities and energies for molsidomine than the aminopropyl modified sample. The effects are discussed from the viewpoint of nature of specific interactions responsible for the adsorption.

Sauchinone is a phytochemical known as a nitric oxide (NO) inhibitor. NO is a kind of neurotransmitter and involved in psychotic effect of abuse drug. In present, we carried out a study on the effect of sauchinone on methamphetamine-induced alteration of behavior in mice. Locomotory activity and conditioned place preference (CPP) were used to evaluate behavioral changes. As a result, sauchinone inhibited the methamphetamine-induced hyperlocomotion in dose-dependent manner, whereas sauchinone had no effect on normal locomotory activity. The inhibitory effect of sauchinone on methamphetamine-induced hyperlocomotion was reversed by treatment of molsidomine, a NO donor. Sauchinone also significantly blocked the acquisition and expression of CPP induced by methamphetamine in mouse. However, it did not produce place preference or place aversion, when it was treated alone in animals. Taken together, sauchinone blocked drug reward-related behavior as well as acute hyperlocomotion induced by methamphetamine treatment.

Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), a peroxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole-cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate independent, whereas in the ventricle APD, prolongation was rate dependent. In addition to prolongation of the action potential, beat-to-beat variability of repolarization was significantly increased in ventricular but not in atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1-induced increase in ventricular beat-to-beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.

Reactive oxygen species (ROS)-induced pancreatic β cell death affects insulin secretion and is important in the pathogenesis of diabetes. Lagerstroemia speciosa, a traditional folk medicine, has been used for t he prevention and treatment of diabetes. However, whether Lagerstroemia speciosa has a cytoprotective effect on pancreatic β cells remains to be elucidated. The present study aimed to investigate the cytoprotective effects of hot water extracts from Lagerstroemia speciosa leaves (LWE) on 3-morpholinosydnonimine (SIN-1)-induced oxidative damage in Syrian hamster pancreatic insulinoma HIT-T15 cells. The HIT-T15 cells were first treated with SIN-1 (50 µM) for 24 h and then co-incubated with LWE for 48 h. SIN-1 significantly decreased HIT-T15 cell viability (P

Increasing of peroxynitrite (ONOO − ) production during ischemia in the immature brain was considered to be associated with impaired cognitive function. GABAergic synapses played an important role in memory formation including the induction of long-term potentiation (LTP) and long-term depression (LTD) in hippocampus. In the present study, we examined the effects of acute exposure of the ONOO − donor, SIN-1 on GABAergic synaptic transmission in immature rat hippocampal slices with whole-cell patch-clamp recordings. The results showed that SIN-1 increased the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) and decreased paired pulse ratio via the formation of ONOO − . In addition, it also increased the frequency of spontaneous (but not miniature) IPSCs in a dose-dependent manner without altering amplitudes or rise and decay times of both (sIPSCs and mIPSCs). It further demonstrated that the presynaptic action of SIN-1 was external calcium dependent and was not related to the changes of interneuron excitability. This study provides electrophysiological evidences from developing hippocampal slices to support that SIN-1 enhances action potential-dependent GABA release. It suggests that the potentiation effect of ONOO − may contribute to hyperexcitability and seizures and may underlie one of the mechanisms by which ischemia increases seizure susceptibility in the immature brain.

Purpose: To explore the protective and curative effects of molsidomine (MOL) on doxorubicin (DOX)-induced cardiac damage in the in vivo rat heart. Methods: Forty rats were randomized into five groups (n = 8): (1) the control group; (2) the MOL group (10 mg/kg for 21 days); (3) the DOX group (a single dose of 20 mg/kg); (4) the DOX + MOL group (3 days after the single dose of DOX, 10 mg/kg MOL continued for 21 days), and (5) the MOL + DOX group (24 h after a 21-day regimen of 10 mg/kg MOL, a single dose of DOX). The rats were monitored for mean arterial blood pressure, heart rate, O2 saturation, and electrocardiography. Heart tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and nitric oxide (NO) were determined. Results: Blood pressure and O2 saturation values indicated a significant decrease in the DOX group compared with the control group. T negativity was observed in 4 of 8 rats in the DOX group, in 1 of 8 rats in the DOX + MOL group, and in 4 of 8 rats in the MOL + DOX group. MDA levels were significantly higher in the DOX group. SOD, GSH, and NO levels were significantly lower in the DOX group compared with the other groups. There was no statistically significant difference in the CAT levels in any of the study groups compared with controls. DOX treatment induced morphological alterations, such as disorganization of cardiomyocytes, loss of myofibrils, and cytoplasmic vacuolization in the heart. On the other hand, histological damage was significantly reduced in the DOX + MOL and MOL + DOX groups. Conclusion: This study implies that there are cardioprotective effects of MOL on DOX-induced cardiotoxicity.

Inflammation plays a crucial role in muscle remodeling and repair after acute and chronic damage, in particular in muscular dystrophies, a heterogeneous group of genetic diseases leading to muscular degeneration. Defect of nitric oxide (NO) generation is a key pathogenic event in muscular dystrophies, thus NO donors have been explored as new therapeutics for this disease. We have investigated the immune-modulating effect of one of such drugs, molsidomine, able to slow the progression of muscular dystrophy in the α-Sarcoglican-null mice, a model for the limb girdle muscular dystrophy 2D, sharing several hallmarks of muscle degeneration with other muscular dystrophies. α-Sarcoglican-null mice were treated with molsidomine and drug effects on the inflammatory infiltrates and on muscle repair were assessed at selected time points. We found that molsidomine treatment modulates effectively the characteristics of the inflammatory infiltrate within dystrophic muscles, enhancing its healing function. Initially molsidomine amplified macrophage recruitment, promoting a more efficient clearance of cell debris and effective tissue regeneration. At a later stage molsidomine decreased significantly the extent of the inflammatory infiltrate, whose persistence exacerbates muscle damage: most of the remaining macrophages displayed characteristics of the transitional population, associated with reduced fibrosis and increased preservation of the muscle tissue. The dual action of molsidomine, the already known NO donation and the immunomodulatory function we now identified, suggests that it has a unique potential in tissue healing during chronic muscle damage. This, alongside its already approved use in human, makes molsidomine a drug with a significant therapeutic potential in muscular dystrophies.

The work is devoted to synthesis and study of warfarin composites with unmodified, methyl and phenyl modified silica in order to develop controlled release formulation of the anticoagulant. The composites were prepared by two routes, adsorption and sol-gel, and characterized with FTIR spectroscopy, dynamic light scattering and DSC methods. The drug release behavior from the composites in media with pH 1.6, 6.8 and 7.4 was analyzed in vitro. The release kinetics of the warfarin – silica composites prepared by the two routes was compared among each other and with analogous silica composites with water soluble drug molsidomine. The comparative analysis showed that in general the kinetic regularities and mechanisms of release for both drugs are similar and determined by nonuniform distribution of the drugs over the silica matrixes and stability of the matrixes in the studied media for the adsorbed composites and uniformly distributed drug and more brittle structure for the sol-gel composites. The sol-gel composite of warfarin - phenyl modified silica is perspective for further development of novel warfarin formulation with controlled release because it releases warfarin according to zero-order kinetic law with approximately equal rate in the media imitating different segments of gastrointestinal tract.

Peroxynitrite and heme peroxidases (or heme)-H

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS® system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility. Abbreviations: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO−: peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation ANOVA: analysis of variance Fil: Cabrillana, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad del Aconcagua; Argentina Fil: Uribe, Pamela. Universidad de La Frontera; Chile Fil: Villegas, Juana V.. Universidad de La Frontera; Chile Fil: Álvarez, Juan. Harvard Medical School; Estados Unidos Fil: Sánchez, Raúl. Universidad de La Frontera; Chile Fil: Fornes, Miguel Walter. Universidad del Aconcagua; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Authentic nitric oxide (NO; 0.1 - 10 micromoles) caused transient, dose-dependent relaxation of phenylephrine-induced tone without changing membrane potential in mesenteric arteries. Larger doses, above 10 micromoles, did not evoke more relaxation (maximal relaxation to 150 micromoles NO in denuded arteries, 69+/-7%, n=8) but stimulated muscle hyperpolarization (maximum 19+/-3 mV, n=5). The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), abolished relaxation to low doses of NO (n=4), but did not modify hyperpolarization with higher doses of NO (n=4). The potassium channel blocker charybdotoxin (ChTX; 50 nM) abolished hyperpolarization to high doses of NO and significantly reduced the maximal relaxation (to 43+/-6%, n=4; P

Recent studies suggest that nitric oxide donors capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of biofilms. Encased in extracellular polymeric substances, human and plant pathogens within biofilms are significantly more resistant to sanitizers. This is particularly a problem in refrigerated environments where food is processed. In an exercise aimed to study the potential of nitric oxide donors as biofilm dispersal in refrigerated conditions, we compared the ability of different nitric oxide donors (SNAP, NO-aspirin and Noc-5) to dislodge biofilms formed by foodborne, human and plant pathogens treated at 4 °C. The donors SNAP and Noc-5 were efficient in dispersing biofilms formed by Salmonella enterica, pathogenic Escherichia coli and Listeria innocua. The biomasses were decreased up to 30 % when compared with the untreated controls. When the plant pathogens Pectobacterium sp. and Xanthomonas sp. were tested the dispersion was mainly limited to Pectobacterium carotovorum biofilms, decreasing up to 15 % after exposure to molsidomine. Finally, the association of selected nitric oxide donors with sanitizers (DiQuat, H2O2, peracetic acid and PhenoTek II) was effective in dispersing biofilms. The best dispersal was achieved by pre-treating P. carotovorum with molsidomine and then peracetic acid. The synergistic effect was estimated up to ~35 % in dispersal when compared with peracetic acid alone. The association of nitric oxide donors with sanitizers could provide a foundation for an improved sanitization procedure for cleaning refrigerate environments. Electronic supplementary material The online version of this article (doi:10.1186/s13568-016-0220-1) contains supplementary material, which is available to authorized users.

Advanced paternal age is linked to complications in pregnancy and genetic diseases in offspring. Aging results in excess reactive oxygen species (ROS) and DNA damage in spermatozoa; this damage can be transmitted to progeny with detrimental consequences. Although there is a loss of antioxidants with aging, the impact on aging male germ cells of the complete absence of either catalase (CAT) or superoxide dismutase 1 (SOD1) has not been investigated. We used CAT-null (Cat(-/-)) and SOD1-null (Sod(-/-)) mice to determine whether loss of these antioxidants increases germ cell susceptibility to redox dysfunction with aging. Aging reduced fertility and the numbers of Sertoli and germ cells in all mice. Aged Sod(-/-) mice displayed an increased loss of fertility compared to aged wild-type mice. Treatment with the pro-oxidant SIN-10 increased ROS in spermatocytes of aged wild-type and Sod(-/-) mice, while aged Cat(-/-) mice were able to neutralize this ROS. The antioxidant peroxiredoxin 1 (PRDX1) increased with age in wild-type and Cat(-/-) mice but was consistently low in young and aged Sod(-/-) mice. DNA damage and repair markers (γ-H2AX and 53BP1) were reduced with aging and lower in young Sod(-/-) and Cat(-/-) mice. Colocalization of γ-H2AX and 53BP1 suggested active repair in young wild-type mice but reduced in young Cat(-/-) and in Sod(-/-) mice and with age. Oxidative DNA damage (8-oxodG) increased in young Sod(-/-) mice and with age in all mice. These studies show that aged Sod(-/-) mice display severe redox dysfunction, while wild-type and Cat(-/-) mice have compensatory mechanisms to partially alleviate oxidative stress and reduce age-related DNA damage in spermatozoa. Thus, SOD1 but not CAT is critical to the maintenance of germ cell quality with aging.

Purpose: The purpose of this study was to investigate the therapeutic and protective effects of molsidomine (MLS) against doxorubicin (DOX)-induced renal damage in rats. Methods: Forty rats were randomly divided into five groups (control, MLS, DOX, DOX+MLS and MLS+DOX groups). Thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), nitric oxide (NO) and glutathione peroxidase (GPx) levels were determined from kidney tissues and blood urea nitrogen (BUN), creatinine (Cr) and albumin (Alb) levels also determined. Results: DOX treatment caused a significant increase in TBARS levels and a significant decrease in the GSH and CAT levels compared with the control group. In comparison, MLS administration before DOX injection caused a significant decrease in TBARS levels and also increases in GSH and CAT levels, whereas treatment of MLS after DOX injection did not show any beneficial effect on these parameters. All groups showed a significant increase in NO levels compared to the control group. There were no significant differences among the all groups for BUN and Cr levels. Serum level of Alb decreased in the DOX-treated groups when compared with control and MLS groups. The histopathological findings were in accordance with the biochemical results. MLS treatment reversed the DOX-induced kidney damage in group 4. MLS treatment before DOX injection exerted a protective effect against DOX-induced kidney damage. Conclusions: MLS shows promise as a possible therapeutic intervention for the prevention of kidney injury associated with DOX treatment. Additional studies are warranted.

Embryonic VE-Cadherin-expressing progenitors (eVE-Cad+), including hemogenic endothelium, have been shown to generate hematopoietic stem cells and a variety of other progenitors, including mesoangioblasts, or MABs. MABs are vessel-associated progenitors with multilineage mesodermal differentiation potential that can physiologically contribute to skeletal muscle development and regeneration, and have been used in an ex vivo cell therapy setting for the treatment of muscular dystrophy. There is currently a therapeutic need for molecules that could improve the efficacy of cell therapy protocols; one such good candidate is nitric oxide. Several studies in animal models of muscle dystrophy have demonstrated that nitric oxide donors provide several beneficial effects, including modulation of the activity of endogenous cell populations involved in muscle repair and the delay of muscle degeneration. Here we used a genetic lineage tracing approach to investigate whether the therapeutic effect of nitric oxide in muscle repair could derive from an improvement in the myogenic differentiation of eVE-Cad+ progenitors during embryogenesis. We show that early in vivo treatment with the nitric oxide donor molsidomine enhances eVE-Cad+ contribution to embryonic and fetal myogenesis, and that this effect could originate from a modulation of the properties of yolk sac hemogenic endothelium.

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.

Introduction Long‐term daily administration of phosphodiesterase type 5 (PDE5) inhibitors in the rat prevents or reverses corporal veno‐occlusive dysfunction (CVOD) and smooth muscle cell (CSMC) loss and fibrosis, in both aging and bilateral cavernosal nerve resection (BCNR) models for erectile dysfunction. In the aging rat model, corporal implantation of skeletal muscle‐derived stem cells (MDSC) reverses CVOD. Nitric oxide (NO) and cyclic guanosine monophosphate can modulate stem cell lineage. Aim To investigate in the BCNR model the effects of sildenafil at lower doses, alone or in combination with MDSC or the NO donor molsidomine, on CVOD and the underlying corporal histopathology. Main Outcomes Measures CVOD, histological, and biochemical markers in rat corporal tissue. Methods Rats subjected to BCNR were maintained for 45 days either untreated, or received sildenafil in the water or retrolingually at 10, 2.5, and 1.25 mg/kg/day (medium, low, and very low doses), or intraperitoneal molsidomine, or MDSC implantation into the corpora cavernosa separately or in combination. Cavernosometry evaluated CVOD. Histopathology was assessed on penile sections by Masson trichrome, immunohistochemistry for α‐smooth muscle actin (ASMA), or immunofluorescence for neuronal nitric oxide synthase (nNOS)/neurofilament 70, and in fresh tissue by Western blot for various markers and picrosirius red for collagen. Results All treatments normalized erectile function (drop rate), and most increased the CSMC/collagen ratio and ASMA expression in corporal tissue sections, and reduced collagen content in the penile shaft. MDSC also increased nNOS and brain‐derived neurotrophic factor. The combination treatment was not superior to MDSC or sildenafil given alone, and upregulated PDE5. Conclusions. Lowering the dose of a continuous long‐term sildenafil administration still maintained the prevention of CVOD in the BCNR rat previously observed, but it was less effective on the underlying histopathology. As in the aging rat model, MDSC also counteracted CVOD, but supplementation with very low‐dose sildenafil did not improve the outcome. Kovanecz I, Rivera S, Nolazco G, Vernet D, Segura D, Gharib S, Rajfer J, and Gonzalez‐Cadavid NF. Separate or combined treatments with daily sildenafil, molsidomine, or muscle‐derived stem cells, prevent erectile dysfunction in a rat model of cavernosal nerve damage. J Sex Med 2012;9:2814–2826.

Heme proteins share the ability to detoxify reactive nitrogen intermediates (NO and peroxynitrite). But, to date, no heme-containing enzymatic defense against toxic reactive nitrogen intermediates has been discovered in Leishmania species. We have cloned, expressed, and characterized a pseudoperoxidase from Leishmania major (LmPP) that is capable of detoxifying peroxynitrite (ONOO(-)). Optical, EPR, and resonance Raman spectral studies demonstrate that ONOO(-) can rapidly convert the six-coordinate ferric low-spin to a ferric high-spin form at neutral pH. Western blotting and immunofluorescence studies with anti-LmPP antibody show that the mature enzyme is located at the plasma membrane of amastigotes and is expressed eightfold higher in amastigotes compared to promastigotes. Moreover, to further investigate its exact physiological role in Leishmania, we have created LmPP-knockout mutants by gene replacement in L. major strains. IC(50) values for exogenously added H(2)O(2) or 3-morpholinosydnonimine (SIN1) show that deletion of LmPP in L. major renders the cell more susceptible to SIN1. The null mutant cells exhibit a marked decrease in virulence on infection with activated macrophages as well as inoculation into BALB/c mice. Collectively, these data provide strong evidence that LmPP plays an important role in the enzymatic defense against ONOO(-) within macrophages.

Molsidomine is one of the sydnonimine class of antianginal drugs that due to its structure exhibits both dipolar nature and aromatic properties. To select efficient carrier for the drug, unmodified and modified mesoporous silica materials were synthesized using phenyltriethoxysilane and 3-aminopropyltriethoxysilane via cocondensation and grafting routes. Synthesis of the mesoporous silica materials via cocondensation was carried out in the presence of D-glucose as pore-forming agent. Equilibrium isotherms for the adsorption of mesoionic compound molsidomine on the mesoporous silica materials were analyzed by the Langmuir, Freundlich, Redlich-Peterson and Langmuir–Freundlich (Sips) models. Langmuir model is found to be the best to explain the equilibrium data. Comparative study of the adsorption properties of the unmodified and modified mesoporous silica materials demonstrated that the phenyl-modified silica materials are the most efficient adsorbents for molsidomine. They exhibit the highest adsorption capacity and affinity in relation to the mesoionic compound.

The mineralocorticoid receptor (MR) plays an important role in salt and water homeostasis and pathological tissue modifications, such as cardiovascular and renal fibrosis. Importantly, MR activation by aldosterone per se is not sufficient for the deleterious effects but requires the additional presence of a certain pathological milieu. Phenomenologically, this milieu could be generated by enhanced nitrosative stress. However, little is known regarding the modulation of MR transcriptional activity in a pathological milieu. The glucocorticoid receptor (GR), the closest relative of the MR, binds to the same hormone-response element but elicits protective effects on the cardiovascular system. To investigate the possible modulation of MR and GR by nitrosative stress under controlled conditions we used human embryonic kidney (HEK) cells and measured MR and GR transactivation after stimulation with the nitric oxide (NO)-donor SNAP and the peroxynitrite-donor Sin-1. In the presence of corticosteroids NO led to a general reduced corticosteroid receptor activity by repression of corticosteroid receptor–DNA interaction. The NO-induced diminished transcriptional MR activity was most pronounced during stimulation with physiological aldosterone concentrations, suggesting that NO treatment prevented its pathophysiological overactivation. In contrast, single peroxynitrite administration specifically induced the MR transactivation activity whereas genomic GR activity remained unchanged. Mechanistically, peroxynitrite permitted nuclear MR translocation whereas the cytosolic GR distribution was unaffected. Consequently, peroxynitrite represents a MR-specific aldosterone mimetic. In summary, our data indicate that the genomic function of corticosteroid receptors can be modulated by nitrosative stress which may induce the shift from physiological toward pathophysiological MR effects.

As a part of an ongoing search for novel antioxidants from the salt marsh plants, bioactivity-isolation and structure determination of constituents from Salicornia herbacea were performed. One new triterpenoid saponin ( 4 ), along with three known saponins ( 1 – 3 ), has been isolated from n -BuOH fraction of S. herbacea . On the basis of the spectroscopic methods, the structure of the new saponin 4 was elucidated as 3β-hydroxy-23-oxo-30-noroleana-12, 20(29)-diene-28-oic acid 3- O -β- d -glucuronopyranosyl-28- O -β- d -glucopyranoside. Scavenging effects of saponins 1 - 4 were examined on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and peroxynitrite. Particularly, saponin 3 exerted significant antioxidant activity on both authentic peroxynitrite and peroxynitrite generated from morpholinosydnonimine (SIN-1).

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA-NONOate, a pure NO slow generator, and of SIN-1 (3-morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non-enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA-NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn-SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN-1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn-SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO-resistant AP and mitochondrial APX may be important components of the H(2) O(2) -signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn-SOD, against NO and ONOO(-) stress in plant mitochondria.

To study oxidative stress in biological systems, chemical compounds capable of producing free radicals have been widely used. Here, we compared two free-radical generators, 3-morpholinosydnonimine (SIN-1) and 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH), by measuring the thiol oxidation kinetics of various thiols. We found that SIN-1 is 30 times potent in causing thiol oxidation than AAPH. Kinetic simulations revealed that in the SIN-1 system (0.1 mM), superoxide, nitrogen dioxide and carbonate radicals are the major reactive species which, in combination, induce ∼50% of thiol molecules to undergo one-electron oxidation, thereby forming the thiyl radical which propagates further thiol oxidation by direct coupling with thiolates. Similarly, the alkyl peroxyl radical derived from AAPH (3 mM) initiates comparable extent of one-electron oxidation and formation of the thiyl radical. In conclusion, our study provides experimental and theoretical evidence that SIN-1 is mainly an one-electron oxidizing agent that can be functionally mimicked by AAPH.

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.

The PET imaging agent CuII(atsm) improves motor and cognitive function in Parkinson’s disease., Parkinson’s disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified CuII(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that CuII(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.

The role of taurine in regulating glucose-induced nitrosative stress has been examined in human Schwann cells, a model for understanding the pathogenesis of diabetic neuropathy. Exposure to high glucose increased nitrated proteins (1.56 fold p

Satellite cells are myogenic precursors that proliferate, activate, and differentiate on muscle injury to sustain the regenerative capacity of adult skeletal muscle; in this process, they self-renew through the return to quiescence of the cycling progeny. This mechanism, while efficient in physiological conditions does not prevent exhaustion of satellite cells in pathologies such as muscular dystrophy where numerous rounds of damage occur. Here, we describe a key role of nitric oxide, an important signaling molecule in adult skeletal muscle, on satellite cells maintenance, studied ex vivo on isolated myofibers and in vivo using the α-sarcoglycan null mouse model of dystrophy and a cardiotoxin-induced model of repetitive damage. Nitric oxide stimulated satellite cells proliferation in a pathway dependent on cGMP generation. Furthermore, it increased the number of Pax7+/Myf5− cells in a cGMP-independent pathway requiring enhanced expression of Vangl2, a member of the planar cell polarity pathway involved in the Wnt noncanonical pathway. The enhanced self-renewal ability of satellite cells induced by nitric oxide is sufficient to delay the reduction of the satellite cell pool during repetitive acute and chronic damages, favoring muscle regeneration; in the α-sarcoglycan null dystrophic mouse, it also slowed disease progression persistently. These results identify nitric oxide as a key messenger in satellite cells maintenance, expand the significance of the Vangl2-dependent Wnt noncanonical pathway in myogenesis, and indicate novel strategies to optimize nitric oxide-based therapies for muscular dystrophy. Disclosure of potential conflicts of interest is found at the end of this article.

3-Morpholinosydnonimine (SIN-1) is used as a donor of peroxynitrite (ONOO−) in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s) in the medium, rather thanONOO−, exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1.

Treatment of PC12 cells with fungus-derived alkaloid neoechinulin A for more than 12 h renders the cells resistant to subsequent superoxide (O₂⁻)/nitric oxide (NO) insults derived from 3-morpholinosydnonimine (SIN-1). However, the underlying mechanism(s) remains largely unclear. To elucidate the mechanism(s), we assessed the specificity of the cytoprotection afforded by neoechinulin A treatment using other cytocidal stressors and also clarified the resulting cellular alterations, focusing on the antioxidant and metabolic enzymes systems. Neoechinulin A treatment for more than 12 h endowed PC12 cells with significant resistance to transient NO toxicity, but not persistent NO toxicity, bolus H₂O₂ toxicity, or oxidative insult from the redox cycling quinone menadione. Cellular antioxidant system profiling revealed no substantial potentiation of the activity of any antioxidant enzyme in lysate from the neoechinulin A-treated cells excluding glutathione (GSH) content, which was significantly decreased (>50%), resulting in a proportional compromise in the thiol-reducing activity of the intact cells. In addition, no differences were observed in the activity for any nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H)-generating enzyme, steady-state NAD(P)H/nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD(P)⁺) ratios, or the levels of total NAD(P)H. Nevertheless, the neoechinulin A-treated intact cells exhibited increased NAD(P)H redox turnover when driven by extracellular tetrazolium. The structurally inactive analog preechinulin failed to protect cells against NO toxicity or induce these alterations, suggesting their link with the cytoprotective mechanism. These results suggest that neoechinulin A, despite disabling the GSH defense system, confers cytoprotection against nitrosative stresses by elevating the cellular reserve capacity for NAD(P)H generation, which could offset crippling of energy-supplying systems due to nitrosative stress.

Endothelial dysfunction is associated with the formation of peroxynitrite, described to be toxic. Recent data also suggests that peroxynitrite is able to activate the protective Nrf2 pathway and/or the unfolded protein response (UPR). The aim of our work was to study the response of human endothelial cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, and to highlight the possible protective roles of Nrf2 or the UPR pathway in this response. Immortal and primary human umbilical vein endothelial cells were exposed to SIN-1. SIN-1 incubation led to Nrf2 activation and to the overexpression of Nrf2-regulated genes, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1. We also demonstrated that this defensive response protected cells against cell death induced by serum starvation, by reducing apoptosis (monitored by caspase-3 activity and DNA fragmentation) and favoring autophagosome formation, as evidenced by LC3-II accumulation. Interestingly, we observed an activation of the UPR, with a rapid and significant overexpression of CHOP in serum starved cells stimulated with SIN-1. While siRNA mediated knockdown of CHOP had no effect on DNA fragmentation, the invalidation of Nrf2 or HO-1 by siRNA strongly increased DNA fragmentation, but also reinforced the SIN-1-induced LC3-II accumulation. This study shows that peroxynitrite, at least at sublethal concentrations and within a narrow concentration range, could exert protective effects on endothelial cells by modulating the balance between autophagy and apoptosis, through Nrf2-dependent pathways.

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50-100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 μM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)β(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, μM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)β(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.

Peripheral nerve injury often results in neuropathic pain that is manifested as hyperalgesia, and allodynia. Several studies suggest a functional role for neuronal nitric oxide synthase (nNOS) in the development or maintenance of neuropathic pain, but such a contribution remains unclear. In our current study, we found that intraplantar injection of the NOS substrate L-arginine or NO donor 3-morpholino-synonimine (SIN-1) produced mechanical hypersensitivity that lasted more than 24 h. Following L5 spinal nerve ligation (L5 SNL), immunoreactivity for nNOS in the ipsilateral L5 but not L4 dorsal root ganglion (DRG) was dramatically increased in mainly small- and medium-sized neurons and non-neuronal cells. L5 SNL caused increased nNOS immunoreactivity in the ipsilateral sciatic nerve, mainly in Schwann cells and the ipsilateral glabrous hind paw skin, mainly on the basement membrane. Furthermore, total nNOS protein and mRNA in the ipsilateral sciatic nerve and hind paw skin were markedly upregulated following nerve injury. Intraplantar injection of the NOS inhibitor 7-nitroindazole (7-NI) or the non-specific NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME) effectively suppressed SNL-induced mechanical allodynia. Collectively, these data suggest that in the periphery nNOS upregulation induced by peripheral nerve injury contributes to mechanical hypersensitivity during the maintenance phase of neuropathic pain. Blocking nNOS signaling in the periphery may thus be a novel therapeutic strategy for the treatment of neuropathic pain.

The Na+/Ca2+exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H2O2), on the Na+/Ca2+exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na+/Ca2+exchanger by impairing the intracellular Ca2+(Cai2+)-regulatory sites, leaving unharmed the intracellular Na+− and Ca2+−transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect Hi+and (Hi++ Nai+) inhibition of Cai2+-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na+/Ca2+exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca2+concentration that seems to be the main factor responsible for cell damage.

Peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of myricetin is associated with inhibition of peroxynitrite-mediated DNA damage, a critical event leading to peroxynitrite elicited cytotoxicity. We observed that peroxynitrite can cause DNA damage in ϕX-174 plasmid DNA and rat primary astrocytes. The presence of myricetin at physiological concentration was found to significantly inhibit DNA strand breakage induced by both peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1). Moreover, the consumption of oxygen caused by SIN-1 was found to be decreased in the presence of myricetin, indicating that myricetin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that myricetin inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that myricetin can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical formation.

The IOP lowering effects of NCX 139, a new chemical entity comprising latanoprost amide and a NO-donating moiety, were compared to those of the respective des-nitro analog in in vitro assays and in rabbit and dog models of ocular hypertension. The NO donor, molsidomine as well as the prostamide bimatoprost (Lumigan(®)) and the prostaglandin agonist, latanoprost (Xalatan(®)) were also investigated for comparison. NCX 139 but not its des-nitro analog resulted in NO-mediated vascular relaxant effect in pre-contracted rabbit aortic rings (EC(50)=0.70±0.06 μM; E(max)=80.6±2.9%). Like bimatoprost (IC(50)=3.07±1.3 μM) or latanoprost (IC(50)=0.48±0.15 μM), NCX 139 displaced (3)H-PGF2α binding on recombinant human prostaglandin-F (FP) receptors with an estimated potency of 0.77±0.13 μM. In transient ocular hypertensive rabbits, bimatoprost and latanoprost were not effective while molsidomine elicited a dose-dependent reduction of IOP confirming the responsiveness of rabbits to NO but not to FP receptor agonists. NCX 139 tested at a therapeutically relevant dose, significantly lowered IOP while the des-nitro analog was not effective (0.03% NCX 139, Δ(max)=-12.8±2.0 mmHg). In glaucomatous dogs, 0.03% NCX 139 decreased IOP to a greater extent compared to an equimolar dose of the respective des-nitro derivative (Δ(max)=-4.6±1.0 and -2.7±1.3 mmHg, respectively for NCX 139 and its des-nitro analog). Albeit with low potency, NCX 139 also resulted effective in normotensive dogs while it did not reduce IOP in normotensive rabbits. NCX 139, a compound targeting two different and important mechanisms, is endowed with ocular hypotensive effects more evident in hypertensive conditions which may be of interest in the search of more effective treatments for hypertensive glaucoma.

Study Type – Aetiology (case control) Level of Evidence 1b What's known on the subject? and What does the study add? The development of penile fibrosis in diabetes is associated with an increase in oxidative stress and the key pro-fibrotic factor. TGFβ1 within the corpora. As a consequence, a putative compensatory expression of inducible nitric oxide synthase (iNOS) cause a steady output of nitric oxide and cGMP which act as endogenous antifibrotic agents by quenching oxidative stress and inhibiting collagen synthesis and myofibroblast formation. This study adds to the growing body of evidence that the use of antioxidant or antifibrotic therapies may be effective in preventing and possibly ameliorating penile corporal fibrosis and therefore improving erectile function in diabetes, by targeting different pathways involved in the chronic histological damage that underlies erectile dysfunction. OBJECTIVE • To investigate whether sustained long-term separate treatments of diabetic inducible nitric oxide synthase knockout (iNOSKo) mice with allopurinol, an antioxidant inhibiting xanthine oxidoreductase, decorin, a transforming growth factor-β1 (TGFβ1) -binding antagonist, and molsidomine, a long-life nitric oxide donor, prevent the processes of diabetes-induced cavernosal fibrosis. MATERIALS AND METHODS • Eight week old male iNOS knock out (iNOSKo) mice were made diabetic by injecting 150 mg/kg B.W Streptozotocin (1P) with were either left untreated or treated with the oral antioxidant allopurinol (40 mg/kg/day), or decoin (50 mg, 1P, twice), as an anti-TGFβ1 agent (n = 8/group). • Glycemia and oxidative stress markers were determined in blood and urine. • Paraffin-embedded tissue sections from the penile shaft were subjected to Masson trichrome staining for the smooth muscle (smc)/collagen ratio, and imunostaining for smc content, profibrotic factors, oxidative stress, cell replication and cell death markers followed by quantitative image analysis. RESULTS • Eight-week treatment with either allopurinol or decorin counteracted the decrease in smooth muscle cells and the increase in apoptosis and local oxidative stress within the corpora tissue. • Decorin but not allopurinol increased the smooth muscle cell/collagen ratio, whereas allopurinol but not decorin inhibited systemic oxidative stress. • Molsidomine was effective in reducing both local and systemic oxidative stress, but did not prevent corporal fibrosis. CONCLUSION • Both allopurinol and decorin appear as promising approaches either as a single or a combined pharmacological modality for protecting the diabetic corpora from undergoing apoptosis and fibrosis although their functional effects still need to be defined.

Drug-induced vascular injury (DIVI) is observed in rat mesenteric arterioles in response to treatment with phosphodiesterase-4 inhibitors (PDE4i). However, the mechanisms responsible for causing the characteristic vascular lesions are unclear. Nitrotyrosine (NT) adducts, markers of local nitric oxide (NO) production, have been observed in close proximity to the arterial lesions and in the inflammatory cells associated with DIVI. To determine if NO has a direct role in DIVI, rats were treated with the PDE4i CI-1044 at 10, 20, or 40 mg/kg alone or in combination with the nitric oxide synthase inhibitor L-NAME (60 mg/kg) or the nitric oxide donor SIN-1 (30 mg/kg). Mesenteries were collected and processed for microscopic evaluation. NT formation was evaluated in situ via immunohistochemical staining. Serum nitrite (SN), a marker of in vivo NO production, was measured. Compared with vehicle controls, treatment with CI-1044 alone resulted in dose-related increases in the frequency and severity of vascular injury, SN levels, and NT residues. SIN-1 coadministration caused vascular injury to occur at lower doses of CI-1044, compared with CI-1044 alone, with the overall incidence and severity of injury being greater across all CI-1044-dose groups. Following administration of 20 or 40 mg/kg CI-1044, there were also increases in NT immunoreactivity when SIN-1 was coadministered and significant increases in SN. Conversely, coadministration of L-NAME resulted in marked reduction of injury, NT, and SN when compared with CI-1044 alone. The present study suggests that NO production is closely linked to PDE4i-induced vascular injury.

The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT(1A) autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT(1A) receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT(1A) receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F(7,63) = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT(1A) receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

AIMS Rapid atrial pacing for 1 week leads to decreased expression of endocardial nitric oxide (NO)-synthase and decreased NO concentrations. We hypothesized that increasing NO bioavailability may reduce electrical remodelling induced by atrial tachycardia. METHODS AND RESULTS We examined the effect of molsidomine, a NO donor, and N(ω)-nitro-l-arginine methylester (l-NAME), a NO-synthase inhibitor, on electrical remodelling occurring during 4 h of rapid atrial pacing in sheep. Haemodynamic and electrophysiological parameters were measured at baseline, 1 h after the start of the infusion and before the start of pacing, and 2 and 4 h after pacing. We measured the effect of molsidomine on atrial monophasic action potentials (MAPs) in non-instrumented sheep and on l-type Ca(2+) currents and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients in right atrial cells, isolated from control sheep. In control sheep, rapid atrial pacing shortened the atrial effective refractory period (AERP) by 12 ± 0.18% after 4 h, an effect that was unaffected by l-NAME. Infusion of molsidomine increased AERP at baseline (+13.4 ± 1.04%) and transiently attenuated pacing-induced AERP shortening (13.6 ± 0.1% at 2 h). Molsidomine tended to increase MAP duration by 20.7 ± 13.4 ms. Incubation of isolated atrial myocytes with NO donor 3-morpholino-sydnonimine (SIN-1) increased significantly l-type Ca(2+) current and [Ca(2+)](i) transients. CONCLUSION Infusion of molsidomine, a NO donor, delayed shortening of the action potential during short-term rapid atrial pacing, by increasing [Ca(2+)](i). Whereas the former could be protective against repetitive short episodes of atrial fibrillation, the latter might be detrimental in the long term.

Inflammatory conditions are characterized by continuous overproduction of nitric oxide (NO) that can contribute to cell survival but also to cell demise by affecting apoptosis. These facts are important in regulation of hepatic fibrogenesis during exposure to inflammatory stress, since elevated NO may pose the risk of cells with a pro-fibrogenic phenotype giving rise to a sustained proliferation leading to chronic fibrosis. Since nitration of tyrosine residues occurs in a range of diseases involving inflammation, we tested the hypothesis that nitration of specific proteins could result in apoptosis of hepatic stellate cells (HSC), the primary cellular source of matrix components in liver diseases. We found the peroxynitrite generator SIN-1 to promote apoptosis in human and rat HSC, based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax protein. We also showed that SIN-1-induced apoptosis of HSC was due to protein nitration. Among the tyrosine-nitrated proteins, tyrosine kinase Lyn was identified. SIN-1 triggered a signaling pathway through Src kinase Lyn activation that resulted in increased activity of the tyrosine kinase Syk. The involvement of these signaling molecules in the apoptotic process induced by SIN-1 as well as the mechanism by which they are activated was confirmed by using specific inhibitors. In summary, NO, via protein-nitration, could play an important role in controlling liver fibrosis resolution by regulation of HSC apoptosis.

Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT1A or 5-HT2A receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT1A receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT1A receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid d , l -homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT1A autoreceptors are under tonic inhibitory influence by 5-HT.

WOS: 000280190900020, PubMed ID: 20697210, Objectives: Nitric oxide (NO) is a potent nonadrenergic, noncholinergic mediator of gastrointestinal smooth muscle. We aimed to investigate the effects of new NO/cyclic guanosine monophosphate (cGMP) pathway-affecting agents at the sheep sphincter of Oddi (SO) in vitro. Methods: Sheep SO rings were mounted in organ baths and tested for isometric tension and cGMP levels in response to 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; 3-morpholinosydnonimine hydrochloride (SIN-1); and BAY 41-2272 in the presence and absence of 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). Results: 3,3-bis(Aminoethyl)-1-hydroxy-2-oxo-1-triazene; SIN-1; and BAY 41-2272 relaxed SO rings in a concentration-dependent manner. These relaxations were significantly decreased in the presence of ODQ (P < 0.05). All agents significantly increased the cGMP levels compared with the control group (P < 0.05). The increased cGMP levels in the 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; and BAY 41-2272-treated groups were significantly different from both control and carbachol groups (P < 0.05), whereas the increase in the SIN-1 group was significantly different from all groups (P < 0.05). The cGMP levels were significantly lower in the presence of ODQ compared with its absence (P < 0.05). Conclusions: The relaxation of SO rings by these agents may be via increasing the levels of cGMP. The additional increase produced by SIN-1 may be the combined effects of NO generation and activation of guanylyl cyclase.

Dimethyl sulfoxide (DMSO) is frequently used as a solvent for many water-insoluble drugs in biological studies at concentrations often up to 1%. However, little is known about its effects on oxidatively generated DNA damage at very low concentrations (0.005–0.5%). This study was undertaken to investigate the effects of DMSO on peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA, with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of DNA strand breaks in a concentration- and time-dependent manner. The presence of DMSO at concentrations of 0.005–0.5% was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. However, DMSO at the above concentrations showed no affect on SIN-1-mediated oxygen consumption, indicating that DMSO did not affect the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that incubation of the plasmid DNA with authentic peroxynitrite resulted in significant formation of DNA strand breaks, which could also be dramatically inhibited by the presence of DMSO at 0.005–0.5%. Electron paramagnetic resonance spectroscopy, using 5,5-dimethylpyrroline- N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct from the SIN-1 and authentic peroxynitrite. DMSO at the concentrations ranging from 0.01% to 0.5% significantly inhibited the adduct signal. Taken together, these studies demonstrate, for the first time, that DMSO at extremely low concentrations (0.005–0.5%) can potently inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. The results of this study suggest that, where DMSO is applied as a solvent, caution should be observed when evaluating the actions of drugs in experiments involving DNA damage.

The phosphatase vs. kinase equilibrium plays a critical role in the regulation of myocardial contractility. Previous studies have demonstrated that peroxynitrite exerts a biphasic effect on cardiomyocyte contraction, such that high peroxynitrite reduced beta-adrenergic-stimulated myocyte contraction by inducing the dephosphorylation of phospholamban (PLB) via phosphatase activation. Conversely, low peroxynitrite increased basal and beta-adrenergic-stimulated contraction also through a PLB-dependent mechanism. However, previous studies have not elucidated the mechanism underlying the positive effects of low peroxynitrite on myocyte contraction. In the current study, we examined the phosphatase vs. kinase equilibrium as a potential mechanism underlying the positive effects of peroxynitrite. SIN-1 (peroxynitrite donor, 10 mumol/L) increased myocyte Ca(2+) transient and shortening amplitude, accelerated myocyte relaxation, and enhanced PLB phosphorylation. Specific inhibition of PP1/PP2a with okadaic acid failed to inhibit this positive effect. However, inhibition of PKA with KT5720 completely abolished the effects of SIN-1 on myocyte contraction. Additionally, SIN-1 induced a significant increase in PKA activity in cardiac homogenates, which was inhibited with FeTPPS (peroxynitrite decomposition catalyst). Surprisingly, SIN-1 also increased activity in purified preparations (i.e., in the absence of cAMP) of PKA. Therefore, our data suggest that peroxynitrite directly activates PKA (independent from cAMP), resulting in the enhancement of myocyte contraction and relaxation through the phosphorylation of PLB.

Supercritical carbon dioxide (scCO 2 ) processing was performed with mixtures of CO 2 -soluble peracetylated- β -cyclodextrin (PAc- β -CD) heptakis(2,3,6-tri-O-acetyl)- β -cyclodextrin, and highly water-soluble drug molsidomine (MOL) to prepare inclusion complexes of MOL and PAc- β -CD. The MOL/PAc- β -CD inclusion complex was confirmed by differential scanning calorimetry, X-ray diffractometry, and 1 H NMR analyses. The complexes were further investigated for their potential use in controlled drug delivery applications. The in-vitro release of MOL from the peanut oil suspensions into aqueous phase was found to be significantly retarded by the complexation with PAc- β -CD, mainly due to the hydrophobic properties associated with the PAc- β -CD.