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Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD + as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S -nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO - ) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO - (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO - can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.

Peripheral neurotoxicity is a dose-limiting adverse reaction of primary frontline chemotherapeutic agents, including vincristine. Neuropathy can be so disabling that patients drop out of potentially curative therapy, negatively impacting cancer prognosis. The hallmark of vincristine neurotoxicity is axonopathy, yet its underpinning mechanisms remain uncertain. We developed a comprehensive drug discovery platform to identify neuroprotective agents against vincristine-induced neurotoxicity. Among the hits identified, SIN-1-an active metabolite of molsidomine-prevents vincristine-induced axonopathy in both motor and sensory neurons without compromising vincristine anticancer efficacy. Mechanistically, we found that SIN-1's neuroprotective effect is mediated by activating soluble guanylyl cyclase. We modeled vincristine-induced peripheral neurotoxicity in rats to determine molsidomine therapeutic potential in vivo. Vincristine administration induced severe nerve damage and mechanical hypersensitivity that were attenuated by concomitant treatment with molsidomine. This study provides evidence of the neuroprotective properties of molsidomine and warrants further investigations of this drug as a therapy for vincristine-induced peripheral neurotoxicity., (© 2024. The Author(s).)

The cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by nitric oxide (NO). During the early developmental period, rat hearts exhibit higher resistance to ischemia-reperfusion (I/R) injury, contain higher levels of serum nitrates, and their resistance cannot be further increased by IPC or IPoC. NOS blocker (L-NAME) lowers their high resistance. Wistar rat hearts (postnatal Days 1 and 10) were perfused according to Langendorff and exposed to 40 min of global ischemia followed by reperfusion with or without IPoC. NO and reactive oxygen species donors (DEA-NONO, SIN-1) and L-NAME were administered. Tolerance to ischemia decreased between Days 1 and 10. DEA-NONO (low concentrations) significantly increased tolerance to I/R injury on both Days 1 and 10. SIN-1 increased tolerance to I/R injury on Day 10, but not on Day 1. L-NAME significantly reduced resistance to I/R injury on Day 1, but actually increased resistance to I/R injury on Day 10. Cardioprotection by IPoC on Day 10 was not affected by either NO donors or L-NAME. It can be concluded that resistance of the neonatal heart to I/R injury is NO dependent, but unlike in adult hearts, cardioprotective interventions, such as IPoC, are most likely NO independent., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Numerous research projects focused on the management of acute pulmonary hypertension as Coronavirus Disease 2019 (COVID-19) might lead to hypoxia-induced pulmonary vasoconstriction related to acute respiratory distress syndrome. For that reason, inhalative therapeutic options have been the subject of several clinical trials. In this experimental study, we aimed to examine the hemodynamic impact of the inhalation of the SIN-1A formulation (N-nitroso-N-morpholino-amino-acetonitrile, the unstable active metabolite of molsidomine, stabilized by a cyclodextrin derivative) in a porcine model of acute pulmonary hypertension. Landrace pigs were divided into the following experimental groups: iNO (inhaled nitric oxide, n = 3), SIN-1A-5 (5 mg, n = 3), and SIN-1A-10 (10 mg, n = 3). Parallel insertion of a PiCCO system and a pulmonary artery catheter (Swan-Ganz) was performed for continuous hemodynamic monitoring. The impact of iNO (15 min) and SIN-1A inhalation (30 min) was investigated under physiologic conditions and U46619-induced acute pulmonary hypertension. Mean pulmonary arterial pressure (PAP) was reduced transiently by both substances. SIN-1A-10 had a comparable impact compared to iNO after U46619-induced pulmonary hypertension. PAP and PVR decreased significantly (changes in PAP: -30.1% iNO, -22.1% SIN-1A-5, -31.2% SIN-1A-10). While iNO therapy did not alter the mean arterial pressure (MAP) and systemic vascular resistance (SVR), SIN-1A administration resulted in decreased MAP and SVR values. Consequently, the PVR/SVR ratio was markedly reduced in the iNO group, while SIN-1A did not alter this parameter. The pulmonary vasodilatory impact of inhaled SIN-1A was shown to be dose-dependent. A larger dose of SIN-1A (10 mg) resulted in decreased PAP and PVR in a similar manner to the gold standard iNO therapy. Inhalation of the nebulized solution of the new SIN-1A formulation (stabilized by a cyclodextrin derivative) might be a valuable, effective option where iNO therapy is not available due to dosing difficulties or availability.

Background: The study's objective is to evaluate if Molsidomine (MOL), an anti-oxidant, anti-inflammatory, and anti-apoptotic drug, is effective in treating hyperoxic lung injury (HLI)., Methods: The study consisted of four groups of neonatal rats characterized as the Control, Control+MOL, HLI, HLI + MOL groups. Near the end of the study, the lung tissue of the rats were evaluated with respect to apoptosis, histopathological damage, anti-oxidant and oxidant capacity as well as degree of inflammation., Results: Compared to the HLI group, malondialdehyde and total oxidant status levels in lung tissue were notably reduced in the HLI + MOL group. Furthermore, mean superoxide dismutase, glutathione peroxidase, and glutathione activities/levels in lung tissue were significantly higher in the HLI + MOL group as compared to the HLI group. Tumor necrosis factor-α and interleukin-1β elevations associated with hyperoxia were significantly reduced following MOL treatment. Median histopathological damage and mean alveolar macrophage numbers were found to be higher in the HLI and HLI + MOL groups when compared to the Control and Control+MOL groups. Both values were increased in the HLI group when compared to the HLI + MOL group., Conclusions: Our research is the first to demonstrate that bronchopulmonary dysplasia may be prevented through the protective characteristics of MOL, an anti-inflammatory, anti-oxidant, and anti-apoptotic drug., Impact: Molsidomine prophylaxis significantly decreased the level of oxidative stress markers. Molsidomine administration restored the activities of antioxidant enzymes. Molsidomine prophylaxis significantly reduced the levels of inflammatory cytokines. Molsidomine may provide a new and promising therapy for BPD in the future. Molsidomine prophylaxis decreased lung damage and macrophage infiltration in the tissue., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Ulcerative colitis (UC) is a subcategory of intestinal inflammatory bowel disease characterized by up-regulation of proinflammatory cytokines and oxidative stress. The current study was designed to assess the probable protective effect of the nitric oxide (NO) donor, molsidomine, in experimental colitis model in rats. Rats were haphazardly classified into four groups: control, acetic acid, acetic acid + molsidomine (1 mg/kg) and acetic acid + molsidomine (2 mg/kg). Molsidomine (1 and 2 mg/kg/day) was administered by intra-peritoneal injection for 7 days prior to induction of UC. On the 8th day, colitis was induced by intra-rectal instillation of 2 ml of (4% v/v) acetic acid in normal saline using a pediatric plastic catheter. The rats were sacrificed 1 day following colitis induction, blood samples were obtained; colons and livers were isolated then underwent macroscopic, biochemical, histopathological and immunohistochemical examination. Pretreatment with molsidomine significantly reduced disease activity index, colon mass index, colonic macroscopic and histological damage. Besides, molsidomine significantly reduced the serum levels of alanine transaminase (ALT) (58.7 ± 8.9 & 59.7 ± 8 vs 288.75 ± 31.4 in AA group) and aspartate transaminase (AST) (196.2 ± 37.4 & 204 ± 30 vs 392.7 ± 35.6 in AA group). Moreover, molsidomine effectively decreased malondialdehyde (MDA) and total nitrate/nitrite (NOx) contents, and up regulated the enzymatic activity of superoxide dismutase (SOD) and glutathione level (GSH) in colonic and hepatic tissues. With regard to anti-inflammatory mechanisms, molsidomine suppressed tumor necrosis factor-alpha (TNF-α) (792.5 ± 16.7 & 448 ± 12.1 vs 1352.5 ± 45.8 in AA group) in colonic tissues and (701 ± 19 & 442.5 ± 22.5 vs 1501 ± 26 in AA group) in hepatic tissues as well as nuclear transcription factor kappa B (NF-kB/p65) levels (416.2 ± 4.1 & 185.5 ± 14.2 vs 659.2 ± 11.5 in AA group) in colonic tissues and (358 ± 6.2 & 163.5 ± 9.6 vs 732.5 ± 5.5 in AA group) in hepatic tissues. In addition, molsidomine significantly decreased inducible nitric oxide synthase (iNOS) levels (8.1 ± 0.1 & 4.9 ± 0.1 vs 16 ± 0.1 in AA group) in colonic tissues and (8.6 ± 0.3 & 6.1 ± 0.1 vs 17.8 ± 0.1 in AA group) in hepatic tissues, and myeloperoxidase (MPO) contents (10.5 ± 0.4 & 6.6 ± 0.3 vs 20.9 ± 0.6 in AA group) in colonic tissues and (13.1 ± 0.2 & 6.3 ± 0.06 vs 23.9 ± 1.4 in AA group) in hepatic tissues at p > 0.05. Furthermore, it suppressed apoptosis by reducing expression of Caspase 3 and Bax in colonic and hepatic tissues. Therefore, molsidomine might be a promising candidate for the treatment of UC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Diabetic neuropathy is a disorder that affects various regions of the nervous system and there is no specific treatment available for it. This study evaluated the protective effect of molsidomine in diabetic neuropathy in rats. Diabetes was induced in male Wistar rats by administrating streptozotocin (52 mg/kg ip). Diabetic rats were treated with molsidomine 5 mg/kg po and 10 mg/kg po. After 8 weeks of treatment, motor coordination, mechanical allodynia, mechanical hyperalgesia, nerve conduction velocity, and glycosylated hemoglobin were assessed. Thereafter, animals were killed and the sciatic nerve was isolated for measurement of reduced glutathione and lipid peroxidation, and histopathological analysis. Treatment with molsidomine significantly improved motor coordination, paw withdrawal threshold, mechanical threshold, and nerve conduction velocity. Furthermore, molsidomine treatment also reduced malondialdehyde levels and prevented depletion of reduced glutathione in the sciatic nerve homogenate. Histopathology revealed that molsidomine treatment maintained normal architecture of the sciatic nerve. The results of our study strengthen the alternative use of molsidomine in diabetic neuropathy., Competing Interests: There is no conflict of interest., (© 2021 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency. NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Background: Previous studies suggested that Cg1 area of the cingulate cortex of rats controls glutamate-mediated fear-induced defensive behaviour and antinociception organised at the posterior hypothalamus. In turn, microinjection of the nitric oxide donor SIN-1 into the anterior hypothalamus of mice produced defensive behaviours and fear-induced antinociception. However, it remains unknown whether Cg1 also modulates the latter mechanisms in mice., Aims: The present study examined the influence of Cg1 on SIN1-evoked fear-induced defensive behaviour and antinociception organised at the anterior hypothalamus of mice., Methods: The fear-like behavioural and antinociceptive responses to the microinjection of SIN-1 (300 nmol) into the anterior hypothalamus were evaluated after the microinjection of either N-methyl-D-aspartic acid receptor agonist (0.1, 1 and 10 nmol) or physiological saline into the cingulate cortex of C57BL/6 male mice. In addition, neurotracing and immunohistochemistry were used to characterise Cg1-anterior hypothalamus glutamatergic pathways., Results: The data showed that activation of Cg1 N-methyl-D-aspartic acid receptors increased escape while reducing freezing and antinociceptive responses to SIN-1 microinjections into the anterior hypothalamus. Anterograde neural tract tracer co-localised with VGLUT2-labelled fibres suggests these responses are mediated by glutamatergic synapses at the anterior hypothalamus., Conclusions: In contrast with previous studies showing that Cg1 facilitates both escape and antinociception to chemical stimulation of the posterior hypothalamus in rats, the present data suggest that Cg1 facilitates escape while inhibiting defensive antinociception produced by the microinjection of SIN-1 in the anterior hypothalamus of mice. Accordingly, Cg1 may have opposite effects on antinociceptive responses organised in the anterior and posterior hypothalamus of mice and rats, respectively.

Molsidomine is currently used as a vasodilator drug for the treatment of myocardial ischemic syndrome and congestive heart failure, although still presenting some mitochondrial-targeted side effects in many human cells. As a model of molsidomine mitotoxicity, the reaction of cytochrome c with phosphatidylserine (PS)- and cardiolipin (CL)-containing liposomes was investigated in oxidative/nitrosative conditions imposed by SIN-1 decomposition, which renders peroxynitrite (ONOO - ) as a main reactive product. In these conditions, the production of thiobarbituric acid-reactive substance (TBARs) and LOOH was affected by the lipid composition and the oxidative/nitrative conditions used. The oxidative/nitrative conditions were the exposure of lipids to SIN-1 decomposition, native cytochrome c after previous exposure to SIN-1, concomitantly to SIN-1 and native cytochrome c, native cytochrome c, and cytochrome c modified by SIN-1 that presents a less-rhombic heme iron (L-R cytc). TBARs and LOOH production by lipids and cytochrome c exposed concomitantly to SIN-1 differed from that obtained using L-R cytc and featured similar effects of SIN-1 alone. This result suggests that lipids rather than cytochrome c are the main targets for oxidation and nitration during SIN-1 decomposition. PS- and CL-containing liposomes challenged by SIN-1 were analyzed by Fourier transform infrared spectroscopy that revealed oxidation, trans-isomerization, and nitration. These products are consistent with reaction routes involving lipids and NOx formed via peroxynitrite or direct reaction of NO • with molecular oxygen that attacks LOOH and leads to the formation of substances that are not reactive with thiobarbituric acid.

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu 2+ ), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH ( P < 0.05; IC 50 PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18  μ g/ml) and peroxynitrite scavenging assays. PIT at 75  μ g/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu 2+ , SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST ( P < 0.05; IC 50 PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67  μ g/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Kittipot Sirichaiwetchakoon et al.)

Aim: Brain nitric oxide (NO) have been reported in regulation of the sympatho-adrenomedullary system, which can affect voiding and storage functions. Therefore, we investigated effects of intracerebroventricularly (icv) administered 3-(4-morpholinyl)sydnonimine, hydrochloride (SIN-1) (NO donor) on the micturition reflex, focusing on their dependence on the sympatho-adrenomedullary system and on brain N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats., Methods: Plasma noradrenaline and adrenaline were measured just before and 5 minutes after SIN-1 administration. Evaluation of urodynamic parameters was started 1 hour before SIN-1 administration or intracerebroventricular pretreatment with other drugs., Results: SIN-1 (100 and 250 µg/animal) elevated plasma adrenaline and reduced intercontraction interval ([ICI] values; 110.5% [SIN-1, 0 µg] and 54.9% [SIN-1, 250 µg] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.59]) without affecting plasma noradrenaline or maximal voiding pressure. SIN-1 (250 µg/animal) reduced single-voided volume and bladder capacity without affecting post-voiding residual volume. The SIN-1 (250 µg/animal)-induced adrenaline elevation and ICI reduction were attenuated by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) (NO scavenger, icv) (ICI values; 44.7% [vehicle + SIN-1] and 77.5% [carboxy-PTIO + SIN-1] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.51]). Acute bilateral adrenalectomy abolished SIN-1-induced adrenaline elevation, while showed no effect on the SIN-1-induced ICI reduction. The ICI reduction was attenuated by MK-801 (NMDA receptor antagonist, icv) (ICI values; 47.0% [vehicle + SIN-1] and 87.6% [MK-801 + SIN-1] during 15 minutes after SIN-1 administration [P < .05; η 2  = 0.61]), but not by DNQX (AMPA receptor antagonist, icv)., Conclusion: Brain NO is involved in facilitation of the rat micturition reflex through brain NMDA receptors, independently of the sympatho-adrenomedullary outflow modulation., (© 2020 Wiley Periodicals LLC.)

We recently showed that red blood cells (RBCs) from patients with type 2 diabetes mellitus (T2DM-RBCs) induce endothelial dysfunction through a mechanism involving arginase I and reactive oxygen species. Peroxynitrite is known to activate arginase in endothelial cells. Whether peroxynitrite regulates arginase activity in RBCs, and whether it is involved in the cross-talk between RBCs and the vasculature in T2DM, is unclear and elusive. The present study was designed to test the hypothesis that endothelial dysfunction induced by T2DM-RBCs is driven by peroxynitrite and upregulation of arginase. RBCs were isolated from patients with T2DM and healthy age matched controls. RBCs were co-incubated with aortae isolated from wild type rats for 18 h in the absence and presence of peroxynitrite scavenger FeTTPS. Evaluation of endothelial function in organ chambers by cumulative addition of acetylcholine as well as measurement of RBC and vessel arginase activity was performed. In another set of experiments, RBCs isolated from healthy subjects (Healthy RBCs) were incubated with the peroxynitrite donor SIN-1 with subsequent evaluation of endothelial function and arginase activity. T2DM-RBCs, but not Healthy RBCs, induced impairment in endothelial function, which was fully reversed by scavenging of RBC but not vascular peroxynitrite with FeTPPS. Arginase activity was up-regulated by the peroxynitrite donor SIN-1 in Healthy RBCs, an effect that was inhibited by FeTTPS. Healthy RBCs co-incubated with aortae in the presence of SIN-1 caused impairment of endothelial function, which was inhibited by FeTTPS or the arginase inhibitor ABH. T2DM-RBCs induced up-regulation of vascular arginase, an effect that was fully inhibited by FeTTPS. Collectively, our data indicate that RBCs impair endothelial function in T2DM via an effect that is driven by a peroxynitrite-mediated increase in arginase activity. This mechanism may be targeted in patients with T2DM for improvement in endothelial function.

Background: Nitric oxide (NO) donors have been used to control biofilm formation. Nitric oxide can be delivered in situ using organic carriers and acts as a signaling molecule. Cells exposed to NO shift from biofilm to the planktonic state and are better exposed to the action of disinfectants. In this study, we investigate the capability of the NO donors molsidomine, MAHAMA NONOate, NO-aspirin and diethylamine NONOate to act as anti-adhesion agents on ready-to-eat vegetables, as well as dispersants for a number of pathogenic biofilms on plastic., Results: Our results showed that 10 pM molsidomine reduced the attachment of Salmonella enterica sv Typhimurium 14 028 to pea shoots and coriander leaves of about 0.5 Log(CFU/leaf) when compared with untreated control. The association of 10 pmol L -1 molsidomine with 0.006% H 2 O 2 showed a synergistic effect, leading to a significant reduction in cell collection on the surface of the vegetable of about 1 Log(CFU/leaf). Similar results were obtained for MAHMA NONOate. We also showed that the association of diethylamine NONOate at 10 mmol L -1 and 10 pmol L -1 with the quaternary ammonium compound diquat bromide improved the effectiveness of biofilm dispersal by 50% when compared with the donor alone., Conclusions: Our findings reveal a dual role of NO compounds in biofilm control. Molsidomine, MAHMA NONOate, and diethylamine NONOate are good candidates for either preventing biofilm formation or dispersing biofilm, especially when used in conjunction with disinfectants. Nitric oxide compounds have the potential to be developed into a toolkit for pro-active practices for good agricultural practices (GAPs), hazard analysis and critical control points (HACCP), and cleaning-in-place (CIP) protocols in industrial settings where washing is routinely applied. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate-glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine-cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate-glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO - ) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO - donor (SIN-1) or directly with ONOO - , which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO - treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate ( g app ) in combination with a conservation of the force redevelopment rate (k Tr ) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

Nitric oxide (NO) plays a leading role in learning and memory processes. Previously, we showed its ability to modify the deleterious effect of immunotoxin 192 IgG-saporin (192-IgG-SAP) in the cholinergic system. The aim of this study was to analyze the potential of a NO donor (molsidomine, MOLS) to prevent the recognition memory deficits resulting from the septal cholinergic denervation by 192 IgG-SAP in rats. Quantification of neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively) expression was evaluated in striatum, prefrontal cortex, and hippocampus. In addition, a choline acetyltransferase immunohistochemical analysis was performed in medial septum and assessed the effect of MOLS treatment on the spatial working memory of rats through a recognition memory test. Results showed that 192-IgG-SAP reduced the immunoreactivity of cholinergic septal neurons (41%), compared with PBS-receiving control rats (p < 0.05). Treatment with MOLS alone failed to antagonize the septal neuron population loss but prevented the progressive abnormal morphological changes of neurons. Those animals exposed to 192-IgG-SAP immunotoxin exhibited a reduction of cortical nNOS expression against the control group, whereas expression was enhanced in the 192-IgG-SAP + MOLS group. The most relevant finding was the recovering of the discrimination index exhibited by the 192-IgG-SAP + MOLS group. When compared with the rats exposed to the 192-IgG-SAP immunotoxin, they reached values similar to those observed in the PBS group. Our results show that although MOLS failed to block the cholinergic neurons loss induced by 192-IgG-SAP, it avoided the neuronal damage progression., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE -/- Fbn1 C1039G+/- ) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE -/- Fbn1 C1039G+/- mouse model. Female ApoE -/- Fbn1 C1039G+/ - mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 ± 1.2 vs. 8.1 ± 0.7 μm) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 ± 2% vs. 27 ± 2%) combined with a decreased end diastolic (3.1 ± 0.2 vs. 3.9 ± 0.2 mm) and end systolic diameter (1.9 ± 0.1 vs. 2.9 ± 0.2 mm). Furthermore, perivascular fibrosis (23 ± 2 vs. 30 ± 2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 ± 0.04 vs. 2.91 ± 0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

What We Already Know About This Topic: Some general anesthetics have been shown to have adverse effects on neuronal development that affect neural function and cognitive behavior.Clinically relevant concentrations of inhalational anesthetics inhibit the postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domain-mediated protein-protein interaction between PSD-95 or PSD-93 and N-methyl-D-aspartate receptors or neuronal NO synthase., What This Article Tells Us That Is New: Neonatal PSD-95 PDZ2WT peptide treatment mimics the effects of isoflurane (~1 minimum alveolar concentration) by altering dendritic spine morphology, neural plasticity, and memory without inducing detectable increases in apoptosis or changes in synaptic density.These results indicate that a single dose of isoflurane (~1 minimum alveolar concentration) or PSD-95 PDZ2WT peptide alters dendritic spine architecture and functions important for cognition in the developing brain. This impairment can be prevented by administration of the NO donor molsidomine., Background: In humans, multiple early exposures to procedures requiring anesthesia constitute a significant risk factor for development of learning disabilities and disorders of attention. In animal studies, newborns exposed to anesthetics develop long-term deficits in cognition. Previously, our laboratory showed that postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domains may serve as a molecular target for inhaled anesthetics. This study investigated a role for PDZ interactions in spine development, plasticity, and memory as a potential mechanism for early anesthetic exposure-produced cognitive impairment., Methods: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 PDZ2WT peptide. Apoptosis, hippocampal dendritic spine changes, synapse density, long-term potentiation, and cognition functions were evaluated (n = 4 to 18)., Results: Exposure of postnatal day 7 mice to isoflurane or PSD-95 PDZ2WT peptide causes a reduction in long thin spines (median, interquartile range [IQR]: wild type control [0.54, 0.52 to 0.86] vs. wild type isoflurane [0.31, 0.16 to 0.38], P = 0.034 and PDZ2MUT [0.86, 0.67 to 1.0] vs. PDZ2WT [0.55, 0.53 to 0.59], P = 0.028), impairment in long-term potentiation (median, IQR: wild type control [123, 119 to 147] and wild type isoflurane [101, 96 to 118], P = 0.049 and PDZ2MUT [125, 119 to 131] and PDZ2WT [104, 97 to 107], P = 0.029), and deficits in acute object recognition (median, IQR: wild type control [79, 72 to 88] vs. wild type isoflurane [63, 55 to 72], P = 0.044 and PDZ2MUT [81, 69 to 84] vs. PDZ2WT [67, 57 to 77], P = 0.039) at postnatal day 21 without inducing detectable differences in apoptosis or changes in synaptic density. Impairments in recognition memory and long-term potentiation were preventable by introduction of a NO donor., Conclusions: Early disruption of PDZ domain-mediated protein-protein interactions alters spine morphology, synaptic function, and memory. These results support a role for PDZ interactions in early anesthetic exposure-produced cognitive impairment. Prevention of recognition memory and long-term potentiation deficits with a NO donor supports a role for the N-methyl-D-aspartate receptor/PSD-95/neuronal NO synthase pathway in mediating these aspects of isoflurane-induced cognitive impairment.

Several studies have shown that peroxynitrite (ONOO-), formed upon the reaction of •NO and O2-, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO--induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO- donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1-200 μM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO- donor with FeTMPyP (1 μM), an ONOO- decomposition catalyst, inhibited the effects induced by ONOO-. Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO- (SIN-1 200 μM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO- induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO-. Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO- in cardiac tissue.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Introduction and Aim: Methotrexate (Mtx) is an antineoplastic and immunosuppressive drug that may cause hepatotoxicity, whereas molsidomine (Mol) is a vasodilating and antioxidant agent. This study aimed to investigate the potential protective effects of Mol in Mtx-induced liver toxicity in rats., Materials and Methods: Forty Wistar albino rats were equally divided into five groups: control, Mol, Mtx, Mol-Mtx, and Mtx-Mol. Following treatment, the animals were sacrificed, and liver tissue samples were histopathologically evaluated using Roening grading and Bcl-2 antibody staining. Tissue oxidants, antioxidants, and serum transaminases were measured and statistically compared across all groups., Results: No hepatic fibrosis or steatosis was observed in any of the groups. In the Mtx group, grade 2 liver injury and score 2 Bcl-2 antibody staining were observed; however, in the Mol-Mtx group, these were lower (grade 1, score 1). There were no statistically significant differences in serum transaminase levels among groups. Malondialdehyde levels were higher in all rats that received Mtx, but no differences in myeloperoxidase levels were observed among the groups. Levels of tissue antioxidants, including superoxide dismutase, glutathione (GSH) peroxidase (GSH-Px), and reduced GSH, were significantly higher in the Mol-treated and Mol pre-treated groups. Catalase (CAT) levels were elevated in all Mol-treated groups, but only in that group were CAT levels statistically significantly higher than in the control group., Conclusion: Our results suggest that some oxidant levels could increase following Mtx administration in the liver, possibly contributing to liver damage, whereas Mol could mitigate the histopathological and biochemical effects of hepatotoxicity. However, molecular studies are required to understand the exact mechanisms of these alterations., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The ability to detect and image secreted peroxynitrite (ONOO - ) along the extracellular surface of a single cell is biologically significant, as ONOO - generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO - , precise determination of the ONOO - level at the cell surface remains a challenging task. In this paper, the use of a membrane-anchored streptavidin-biotin-controlled binding probe (CBP), ONOO-CBP, to determine quantitatively the ONOO - level at the cell surface and to investigate the effect of different stimulants on the production of ONOO - along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO - secretion, achieving more than a 25-fold increase in ONOO - relative to untreated cells. After 1 h of phorbol-12-myristate-13-acetate (PMA) stimulation, the amount of ONOO - secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3-morpholinosydnonimine hydrochloride (SIN-1), which was estimated to release about 20 μm of ONOO - into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We show that 3-morpholinosydnonimine (SIN-1)-induced nitric oxide (NO) triggers the formation of SGs. Whereas the composition of NO-induced SGs is initially similar to sodium arsenite (SA)-induced type I (cytoprotective) SGs, the progressive loss of eIF3 over time converts them into pro-death (type II) SGs. NO-induced SG assembly requires the phosphorylation of eIF2α, but the transition to type II SGs is temporally linked to the mTOR-regulated displacement of eIF4F complexes from the m 7 guanine cap. Whereas SA does not affect mitochondrial morphology or function, NO alters mitochondrial integrity and function, resulting in increased ROS production, decreased cytoplasmic ATP, and plasma membrane permeabilization, all of which are supported by type II SG assembly. Thus, cellular energy balance is linked to the composition and function of NO-induced SGs in ways that determine whether cells live or die.

Chondrocyte is targeted for disruption in Osteoarthritis (OA) and Kashin-Beck Disease (KBD), and chondrocyte death in cartilage may contribute to the progression of OA and KBD. Oxidative stress leads to increased risk for OA. Previous work in our laboratory implicates oxidative stress as a potential mediator in children with KBD. While these studies suggest a role for oxidative stress in the modulation of OA and KBD, the direct effects of reactive oxygen species/reactive nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, decreased the cell viability in hypertrophic chondrocytes in a dose- and time- dependent manner. SIN-1 induced necrosis in hypertrophic chondrocytes, whereas triggered apoptosis in non-hypertrophic cells of non-differentiated ATDC5 cells and C28/I2 cells. Ultrastructural analysis of hypertrophic chondrocyte treated with SIN-1 revealed morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and organelles, even to disappearance. Moreover, SIN-1 induced chondronecrosis in the deep zone of engineered cartilage tissue, such as cell-free vacancy and "red ghost" cells. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenous ROS/RNS, leads to necrosis in hypertrophic chondrocytes. Oxidative stress-mediated necrotic cell death contributes to chondronecrosis in the deep zone of cartilage in both OA and KBD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Objective: The aim of this study was to investigate FGF8 and FGFR3 expression in clinical samples of Kashin-Beck disease (KBD), an endemic osteochondropathy found in China, as well as in pre-clinical models of this disease., Method: Cartilage was collected from the hand phalanges of five patients with KBD and from five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for four weeks prior to exposure to the T-2 toxin. ATDC5 cells were differentiated into hypertrophic chondrocytes for twenty-one days, and then treated with 3-morpholinosydnonimine (SIN-1) (0, 1, 3, or 5 mM) for 24 h. FGF8 and FGFR3 were visualized using immunohistochemistry; protein levels were assessed by western blotting, and mRNA levels were determined by real-time RT-PCR., Results: Increased staining of FGF8 and FGFR3 was observed in the cartilage of children with KBD compared to normal children. Both increased FGF8 and FGFR3 staining, as well as protein levels, were also observed in the cartilage of rats fed normal or Se-deficient diets plus T-2 toxin exposure, compared to those in rats fed with normal or Se-deficient diets alone. SIN-1 treatment of hypertrophic chondrocytes (ATCD5 cells) increased FGF8 and FGFR3 protein and mRNA levels in a dose-dependent manner., Conclusion: Our data indicate that SIN-1 induces FGF8 and FGFR3 overexpression and this is involved in the abnormal terminal differentiation and degradation of the ECM in cartilage. FGF8 and FGFR3 may therefore play an important role in the onset of deep zone necrosis and pathogenesis in KBD in adolescent children., (Copyright © 2018. Published by Elsevier Inc.)

Background: An excitatory imbalance in the hypothalamus of rodents caused by local chemical stimulation elicits fear-related defensive reactions such as escape and freezing. In addition, these panic attack-like defensive reactions induced by hypothalamic neurons may cause antinociception. However, there is a shortage of studies showing the participation of the anterior hypothalamic nucleus in these adaptive defensive mechanisms. Nitric oxide (NO) donors have been shown to evoke fear-related defensive responses when microinjected into paralimbic and limbic structures, and this excitatory neuromodulation can recruit the glutamatergic system., Aims: The aim of this work was to investigate the influence of the glutamatergic system in the nitrergic effects on fear-related defensive responses organised by anterior hypothalamic neurons., Methods: The present study evaluates the effects of the molsidomine active metabolite SIN-1 NO donor administered into the anterior hypothalamus (AH) of mice at different concentrations (75, 150 and 300 nmol/0.1 μL). Then, we investigated the effects of pre-treatment of the AH with AP-7 (an N-methyl-d-aspartate (NMDA) receptor-selective antagonist; 0.02, 0.2 and 2 nmol/0.1 μL) on the behavioural and antinociceptive effects provoked by AH chemical stimulation with SIN-1 microinjections., Results: The 300 nmol dose of SIN-1 was the most effective at causing panic-like defensive behaviours followed by a significant antinociceptive response. In addition, both of these effects were attenuated or inhibited by AH pre-treatment with AP-7., Conclusions: These findings suggest that the panicogenic and antinociceptive effects evoked by intra-AH microinjections of SIN-1 depend on NMDA receptor activation.

The aim of this study was to assess whether there are differences between the results of determining oxidative stress markers obtained from different origin cell lines after exposure to chemicals generating free radicals. The studies considered two markers of oxidative stress: the level of thiobarbituric acid reactive substances (TBARS) and superoxide dismutase activity. The evaluation was performed in five cell lines: Chinese hamster ovary (CHO-9) cells, lung adenocarcinoma A549, macrophages RAW264.7, skin carcinoma cells A431, and keratinocytes HaCaT. Three compounds generating free radicals were used as a source of reactive oxygen/nitrogen: 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), sodium persulfate (SP), and 3-morpholinosydnonimine hydrochloride (SIN-1). The most appropriate cell line to assess the level of TBARS proved to be the murine macrophage cell line RAW 264.7. Equally, good performance was observed in the lung cancer cell line A549, but only when tested with AAPH and SP. In the case of measuring superoxide dismutase activity, it appeared that the most suitable cell line was also the RAW 264.7 line, although dispersion increased significantly at the highest concentrations of AAPH and SP measurements. When choosing a cell line to determine oxidative stress, the specificity of the stress-inducing compound and the parameter determined should be taken into consideration.

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.

Background: Well known risk factors for diabetic erectile dysfunction include impaired nitric oxide synthesis and endothelial dysfunction. We proposed to evaluate the efficacy of nitric oxide donor, molsidomine in rat model of diabetic erectile dysfunction., Methods: Streptozotocin (52mg/kg, ip) induced diabetic male rats were treated with molsidomine (5 and 10mg/kg, po) for 8 weeks. The sexual behaviour of male rat in presence of the female rat in oestrous phase was observed at the end of study. The effect of treatment on serum testosterone level, sperm parameters and penile tissue histopathology was also evaluated. Further anti-inflammatory activity and antioxidant potential of molsidomine was evaluated by in vitro method. In silico docking study was carried out to appreciate binding conformation of the molsidomine to its plausible target, phosphodiesterase enzyme., Results: Molsidomine significantly and dose dependently increased sexual behaviour, sperm count and serum testosterone level in diabetic rats. Further, the protective effect of molsidomine was also substantiated by pathological changes in the architect of the penile tissue. Molsidomine showed good membrane stability accounting for its significant anti-inflammatory action and also significantly scavenged DPPH radical activity showing its antioxidant action. Molsidomine was found to settle well in the active site of PDE-5 enzyme with less binding affinity than the standard drug sildenafil., Conclusion: The results highlight the rationale behind the repositioning of molsidomine therapy for the management of diabetic erectile dysfunction., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

The axon initial segment (AIS), the domain responsible for action potential initiation and maintenance of neuronal polarity, is targeted for disruption in a variety of central nervous system pathological insults. Previous work in our laboratory implicates oxidative stress as a potential mediator of structural AIS alterations in two separate mouse models of central nervous system inflammation, as these effects were attenuated following reactive oxygen species scavenging and NADPH oxidase-2 ablation. While these studies suggest a role for oxidative stress in modulation of the AIS, the direct effects of reactive oxygen and nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, drives a reversible loss of AIS protein clustering in primary cortical neurons in vitro. Pharmacological inhibition of both voltage-dependent and intracellular calcium (Ca 2+ ) channels suggests that this mechanism of AIS disruption involves Ca 2+ entry specifically through L-type voltage-dependent Ca 2+ channels and its release from IP 3 -gated intracellular stores. Furthermore, ROS/RNS-induced AIS disruption is dependent upon activation of calpain, a Ca 2+ -activated protease previously shown to drive AIS modulation. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenously applied ROS/RNS, is capable of driving structural alterations in the AIS complex.

Aim: Multiple interacting pathways contribute to progression of renal and cardiac damage in chronic kidney disease followed by chronic heart failure (renocardiac syndrome). We hypothesized that simultaneous pharmacological modulation of critical pathways implicated in renocardiac syndrome would effectively reduce fibrosis in and preserve function of heart and kidney., Methods: Rats were subjected to subtotal nephrectomy followed 9 weeks later by coronary artery ligation. From week 11 until week 16, rats received vehicle or losartan, or a combination of the NF-kB inhibitor PDTC, the NO donor molsidomine and superoxide dismutase mimetic tempol, or a combination of all four of these plus metoprolol together. At week 16, renal and cardiac structure, function and gene expression were assessed., Results: Individual and combined treatments were similarly effective in limiting cardiac fibrosis and further decline in systolic function. Combined treatment with all five drugs reduced renal fibrosis and CTGF gene expression more effectively than other strategies. Combining all five drugs reduced heart rate, inotropy and mean arterial pressure (MAP)., Conclusion: Thus, in our model of chronic renocardiac syndrome, combined treatments similarly decreased cardiac fibrosis and stabilized systolic function as losartan alone, perhaps suggesting a dominant role for a single factor such as angiotensin II type 1 (AT1) receptor activation or inflammation in the network of aberrant systems in the heart. However, tubulointerstitial fibrosis was most effectively reduced by a five-drug regimen, pointing to additive effects of multiple pathophysiological pathways in the kidney., (© 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Purpose: To determine the role of Molsidomine in preventing radiation-induced retinopathy after head and neck region irradiation of rats with a single radiation dose of 15 Gy., Materials and Methods: Male Wistar albino rats were randomly grouped into five as follows: (1) control group rats, which were applied through an intraperitoneal (i.p.) vehicle without radiotherapy (RT); (2) RT group rats received a single dose of 15 Gy irradiation and after daily 0.1 ml vehicle i.p. for 5 consecutive days; (3) molsidomine (MOL) group rats were treated for 5 consecutive days by i.p. with 4 mg/kg/day MOL; (4) irradiation plus MOL group (RT+MOL) rats received irradiation and after 10 days single daily i.p. dose of MOL for 5 consecutive days; and (5) MOL+RT group rats were treated for 5 consecutive days by i.p. with MOL before RT. At the end of the work the rats were sacrificed under high-dose anesthesia on the 16 th day and then eye tissues were taken for histopathological, immunohistochemical (caspase-3), and biochemical analyses (superoxide dismutase [SOD], glutathione peroxidase [GSH], and malondialdehyde [MDA])., Results: RT significantly decreased both the content of GSH and the activity of SOD, and significantly increased the production of MDA level in the rat eyes. MOL treatment significantly increased the SOD and GSH levels and significantly decreased the MDA production (p < 0.0001). In addition, RT significantly increased the number of ganglion cells (GCs; p = 0.001), whereas especially pretreatment with MOL improved (p = 0.013). RT led to significant retinopathy formation, and MOL therapy protected the retina from radiation-induced retinopathy (p < 0.0001)., Conclusions: We suggest that MOL is a powerful antioxidant and free radical scavenger that prevents the rat eyes from radiation-induced retinopathy and oxidative stress.

Molsidomine is a well-known vasodilatating, antianginal drug. Despite earlier studies with its metabolites (3-morpholino-syndnonimine (SIN-1) and N-nitroso-N-morpholino-amino-acetonitrile (SIN-1A)), which indicated a potential favorable cardioprotective activity, a lot of controversy remains. The aim of our research was to compare molsidomine, SIN-1, SIN-1A, and lidocaine influence on arrhythmias and hemodynamic parameters in 2 experimental models in rats. In the Langendorff heart study, SIN-1A markedly elevated left ventricular systolic pressure, maximum rise and fall of the first pressure derivative, coronary flow, and myocardial oxygen consumption. In addition, SIN-1A more so than SIN-1 significantly lowered creatine kinase release. The antiarrhythmic action of SIN-1 was observed, while lidocaine significantly diminished ventricular arrhythmias duration in comparison with the control. In the ischemia-reperfusion-induced arrhythmias model, hypotensive action of molsidomine was observed as well as the reduction in pressure rate product. Molsidomine also prolonged ventricular tachycardia duration. On the other hand, no significant effects on hemodynamic parameters as well as on ventricular arrhythmias were found in any of the SIN-1 and SIN-1A groups. In conclusion, our research suggests a possible direct, cardioprotective action of SIN-1A. It seems worthwhile to further investigate molsidomine derivatives, especially SIN-1A, because of its potential use in invasive cardiology procedures such as percutaneous transluminal coronary angioplasty.

Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100 μM SIN-1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production., (© 2016 Blackwell Verlag GmbH.)

Chronic anthracycline (ANT) cardiotoxicity is a serious complication of cancer chemotherapy. Molsidomine, a NO-releasing drug, has been found cardioprotective in different models of I/R injury and recently in acute high-dose ANT cardiotoxicity. Hence, we examined whether its cardioprotective effects are translatable to chronic ANT cardiotoxicity settings without induction of nitrosative stress and interference with antiproliferative action of ANTs. The effects of molsidomine (0.025 and 0.5mg/kg, i.v.) were studied on the well-established model of chronic ANT cardiotoxicity in rabbits (daunorubicin/DAU/3mg/kg/week for 10 weeks). Molsidomine was unable to significantly attenuate mortality, development of heart failure and morphological damage induced by DAU. Molsidomine did not alter DAU-induced myocardial lipoperoxidation, MnSOD down-regulation, up-regulation of HO-1, IL-6, and molecular markers of cardiac remodeling. Although molsidomine increased 3-nitrotyrosine in the myocardium, this event had no impact on cardiotoxicity development. Using H9c2 myoblasts and isolated cardiomyocytes, it was found that SIN-1 (an active metabolite of molsidomine) induces significant protection against ANT toxicity, but only at high concentrations. In leukemic HL-60 cells, SIN-1 initially augmented ANT cytotoxicity (in low and clinically achievable concentrations), but it protected these cells against ANT in the high concentrations. UHPLC-MS/MS investigation demonstrated that the loss of ANT cytotoxicity after co-incubation of the cells in vitro with high concentrations of SIN-1 is caused by unexpected chemical depletion of DAU molecule. The present study demonstrates that cardioprotective effects of molsidomine are not translatable to clinically relevant chronic form of ANT cardiotoxicity. The augmentation of antineoplastic effects of ANT in low (nM) concentrations may deserve further study., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

This study investigated the effects and associated underlying mechanisms of molsidomine, a nitric oxide (NO) donor, on cardiac electrical remodeling and ventricular tachycardias (VTs) induced by chronic isoprenaline (ISO) stimulation in rats. The rats were randomly divided into groups that were treated with saline (control group), ISO (ISO group), ISO + molsidomine (ISO + M group), and ISO + molsidomine + the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, ISO + M + O group) for 14 days. An electrophysiological study was performed to assess cardiac repolarization, action potential duration restitution, and the induction of action potential duration alternans and VTs in vitro. The properties of the Ca transients, Ca handling-related proteins, and NO/guanosine 3'5'-cyclic monophosphate (cGMP)/protein kinase G (PKG) pathway were examined. Compared with the control group, chronic ISO stimulation prolonged the cardiac repolarization, decreased the Ca transient alternans and action potential duration alternans thresholds, and increased the maximum slope (Smax) of the action potential duration restitution curve and incidence of VTs in vitro. All these effects were attenuated by molsidomine treatment (P < 0.05). Moreover, molsidomine activated cGMP/PKG signaling and stabilized the expression of calcium handling-related proteins compared with the ISO group. However, the protective effects of molsidomine were partially inhibited by ODQ. Our results suggest that molsidomine stabilizes calcium handling and attenuates cardiac electrical remodeling and arrhythmogenesis in rats with chronic β-adrenergic receptor activation. These effects are at least partially mediated by the activation of NO/cGMP/PKG pathway.

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., Competing Interests: The authors have declared that no competing interests exist.

Objective and Design: To evaluate the beneficial effects of exogenous NO and an inhibitor of the COX2, and their action levels in a model of SIRS/bacterial translocation (BT) induced by Zymosan A(®)., Material and Methods: Ninety Wistar rats were submitted to different treatments, and after 12h and 24h they were anaesthetized in order to collect blood, mesenteric lymph nodes, and kidney for subsequent biochemical analyses and microbiological examinations., Treatments: A nitric oxide donor, Molsidomine(®), was compared with a COX2 inhibitor, Celecoxib(®)., Methods: Zymosan A(®) was administered to Wistar rats. The animals were divided into 6 groups: one group for survival study, Group (1) No manipulation (BASAL); Group (2) vehicle of Zymosan A(®) given intraperitoneally (SHAM); Group I (control), with Zymosan A(®) (0.6g/kg) intraperitoneally; Group II (Molsidomine), with Molsidomine(®) (4mg/kg) through the penis dorsal vein, 30min prior to administration of the Zy(®) (0.6g/kg); Group III (Celecoxib), with Celecoxib(®) (400mg/kg) orally through a stomach tube, 6h prior to administration of the Zy (0.6g/kg)., Determinations: The parameters survival, bacterial translocation, renal function, neutrophil accumulation, oxygen free radicals (OFR), detoxifying enzymes, and cytokines were measured at different times after Zymosan administration., Results: The model established induced a mortality rate of 100% and generated BT and systemic inflammatory response syndrome (SIRS) in all samples. It also significantly increased all variables, with p<.001 for MPO and all pro-inflammatory cytokines, and p<.01 for all OFR. Treatment with Molsidomine reduced mortality to 0%, decreased BT, MPO, pro-inflammatory cytokines and OFR (p<.001) significantly and increased IL-10 and IL-6 production. Moreover, the Celecoxib(®) showed a lower capacity for SIRS regulation., Conclusions: The exogenous administration of NO prevented BT and controlled SIRS. Therefore these results suggest that Molsidomine could be used as a therapeutic strategy to protect against BT., (Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

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.

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., (© 2016 by the Society for the Study of Reproduction, Inc.)

Nitrous oxide (N2O)-induced antinociception in mice is dependent on the neuromodulator nitric oxide (NO). In contrast to C57BL/6J (B6) mice, DBA/2J (D2) mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of D2 mice to respond to N2O might result from a deficit of NO function. Therefore, it was of interest to determine whether increasing the availability of NO might increase sensitivity of D2 mice to N2O. Male D2 mice were pretreated with sub-antinociceptive intracerebroventricular doses of the NO donor 3-morpholinosydnoimine or the NO precursor l-arginine then assessed for responsiveness to N2O-induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of D2 mice to N2O. These results indicate that the NOS enzyme in D2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O-induced antinociception may lie in availability or utilization of l-arginine., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Experimental evidence indicates the implication of the nitric oxide (NO) in anxiety. Contradictory results were reported however, concerning the effects of NO donors in animal models of anxiety disorders. The present study investigated the effects of the NO donor molsidomine on anxiety-like behaviour and compared them with the anxiolytic diazepam in rats. For this purpose, the light/dark and the open field tests were used. The effects of molsidomine on motility were also assessed. Intraperitoneal (i.p.) administration of molsidomine (1 and 4mg/kg) did not influence rats' performance either in the light/dark or in the open field test. Administration of 2mg/kg molsidomine significantly prolonged the time spent in the light chamber in the rats compared with the vehicle-treated animals, did not affect the first latency to enter the dark chamber and did not influence the number of transitions between the light and dark compartments of the apparatus. In the open field test, rats that received 2mg/kg molsidomine spent more time in the central zone of the apparatus and exhibited an increment of rearing episodes compared with control and to molsidomine 1 and 4mg/kg-treated rats. Nevertheless, molsidomine, at any dose tested, did not alter locomotor activity compared with vehicle-treated rats in a motility test. The present results indicate that the 2mg/kg molsidomine induced anxiolytic-like effects in the light/dark and open field tests in the rat cannot be attributed to changes in locomotor activity. The magnitude of the molsidomine (2mg/kg)-induced anxiolytic-like effects was not different to that produced by the benzodiazepine anxiolytic diazepam (1mg/kg)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Cisplatin, an effective chemotherapeutic agent, is used for the treatment of several types of cancers. However, cisplatin has some severe side effects such as nephrotoxicity. On the other hand, molsidomine, a NO donor, has anti-oxidative and vasodilator effects., Objectives: The aim of this study was to estimate the protective effects of molsidomine on cisplatin-induced nephrotoxicity., Material and Methods: Thirty-two rats were randomly divided into 4 groups as follows: (1) control; (2) received a single-dose intraperitoneal (i.p.) injection of 5 mg/kg cisplatin; (3) received single i.p. dose of molsidomine (4 mg/kg/day) for 3 consecutive days before cisplatin treatment; (4) received single i.p. dose of molsidomine (4 mg/kg/day) for 3 consecutive days. The specific biochemical markers, including antioxidants, and the histopathological alterations were evaluated., Results: Cisplatin significantly increased malondialdehyde (MDA) and myeloperoxidase (MPO) levels and decreased glutathione peroxidase (GPX) level. Molsidomine significantly decreased MPO level nearly to control level; however, its ameliorating effects on MDA, SOD, CAT and GPX did not reach to significant levels. Cisplatin-induced elevation of blood-urea-nitrogen and serum-creatinine were diminished after molsidomine administration. Cisplatin also induced severe tubular degeneration, nuclear condensation, apoptosis and scattered patchy inflammation in the histological examination. Molsidomine improved all of these histological damages., Conclusions: In this study, the beneficial effect of molsidomine against cisplatin nephrotoxicity has been evaluated for the first time.

Hydrogen sulfide (H₂S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H₂S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H₂S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H₂S in inflammation., (Copyright © 2015 the American Physiological Society.)

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Excessive intracellular accumulation of zinc (Zn(2+)) is neurotoxic and contributes to a number of neuropathological conditions. Here, we investigated the protective effect of 3-morpholinosydnonimine (SIN-1) against Zn(2+)-induced neuronal cell death in differentiated PC12 cells. We found that Zn(2+)-induced PC12 cell death was reduced in a concentration-dependent manner by pretreatment with SIN-1. The intracellular accumulation of Zn(2+) was not affected by pretreatment with SIN-1, indicating that SIN-1-induced neuroprotection was not attributable to reduced influx of Zn(2+) into cells. SIN-1C, the stable decomposition product of SIN-1, failed to prevent Zn(2+)-induced cell death. Furthermore, the protective effect of SIN-1 against Zn(2+)-induced PC12 cell death was almost completely abolished by uric acid, a free radical scavenger, suggesting that reactive oxygen and nitrogen species generated by SIN-1 may contribute to the protective effect. SIN-1 prevented the inactivation of glutathione reductase (GR) and the increase in the ratio of oxidized glutathione/total glutathione (GSSG/total GSH) induced by Zn(2+). Addition of membrane permeable GSH ethyl ester (GSH-EE) to PC12 cells prior to Zn(2+) treatment significantly increased cell viability. We therefore conclude that SIN-1 may exert neuroprotective effect against Zn(2+)-induced cell death in differentiated PC12 cells by preventing inhibition of GR and increase in GSSG/total GSH ratio., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chondrocyte senescence is associated with the aging and degeneration of cartilage, and eventually leads to joint destruction. The aim of this study was to elucidate the mechanisms responsible for the cytoprotective effects of heme oxygenase‑1 (HO‑1) on chondrocytes in cartilage. Chondrocyte senescence was induced using cilostazol and measured using a specific senescence‑associated β‑galactosidase (SA‑β‑gal) staining assay. Cilostazol altered the expression of type Ⅱ collagen and β‑catenin, which are phenotypic markers of the differentiation and dedifferentiation of chondrocytes. Cilostazol also significantly induced HO‑1 expression, and the induction of HO‑1 expression was affected by a significant increase in reactive oxygen species (ROS) production caused by cilostazol treatment. Of note, pre‑treatment with 3‑morpholinosydnonimine hydrochloride (SIN‑1), an inducer of HO‑1 expression, markedly attenuated cilostazol‑induced chondrocyte senescence, and thus, we examined whether HO‑1 directly modulates chondrocyte senescence induced by cilostazol. The upregulation of HO‑1 was found to suppress cilostazol‑induced cellular senescence. In addition, the inhibition of HO‑1 activity with the iron chelator, desferrioxamine (DFO), or HO‑1 siRNA increased cilostazol‑induced chondrocyte senescence. Based on these results, it can be concluded that HO‑1 is associated with the suppression of chondrocyte senescence, and that the enforced overexpression of HO‑1 protects chondrocytes against stress‑induced senescence.