The Nitric Oxide (NO) Donor Molsidomine Attenuates Memory Impairments Induced by the D1/D2 Dopaminergic Receptor Agonist Apomorphine in the Rat.

Several lines of evidence suggest that scarcity of the gaseous molecule nitric oxide (NO) is associated with the pathogenesis of schizophrenia. Therefore, compounds, such as NO donors, that can normalize NO levels might be of utility for the treatment of this pathology. It has been previously shown that the NO donor molsidomine attenuated schizophrenia-like behavioral deficits caused by glutamate hypofunction in rats. The aim of the current study was to investigate the efficacy of molsidomine and that of the joint administration of this NO donor with sub-effective doses of the non-typical antipsychotics clozapine and risperidone to counteract memory deficits associated with dysregulation of the brain dopaminergic system in rats. Molsidomine (2 and 4 mg/kg) attenuated spatial recognition and emotional memory deficits induced by the mixed dopamine (DA) D1/D2 receptor agonist apomorphine (0.5 mg/kg). Further, the joint administration of sub-effective doses of molsidomine (1 mg/kg) with those of clozapine (0.1 mg/kg) or risperidone (0.03 mg/kg) counteracted non-spatial recognition memory impairments caused by apomorphine. The present findings propose that molsidomine is sensitive to DA dysregulation since it attenuates memory deficits induced by apomorphine. Further, the current findings reinforce the potential of molsidomine as a complementary molecule for the treatment of schizophrenia.

Molsidomine decreases hyperoxia-induced lung injury in neonatal rats.

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-1ß 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.

The Nitric Oxide (NO) Donor Molsidomine Counteract Social Withdrawal and Cognition Deficits Induced by Blockade of the NMDA Receptor in the Rat.

The deficiency of the gaseous molecule nitric oxide (NO) seems to be critically involved in the pathogenesis of schizophrenia. Thus, molecules that can normalize NO levels, as are NO donors, might be of utility for the medication of this psychiatric disease. The aim of the present study was to detect the ability of the NO donor molsidomine to reduce schizophrenia-like impairments produced by the blockade of the N-methyl-D-aspartate (NMDA) receptor in rats. Molsidomine's ability to attenuate social withdrawal and spatial recognition memory deficits induced by the NMDA receptor antagonist ketamine were assessed using the social interaction and the object location test, respectively. Further, the efficacy of the combination of sub-effective doses of molsidomine with sub-effective doses of the atypical antipsychotic clozapine in alleviating non-spatial recognition memory deficits was evaluated utilizing the object recognition task. Molsidomine (2 and 4 mg/kg) attenuated social withdrawal and spatial recognition memory deficits induced by ketamine. Co-administration of inactive doses of molsidomine (1 mg/kg) and clozapine (0.1 mg/kg) counteracted delay-dependent and ketamine-induced non-spatial recognition memory deficits. The current findings suggest that molsidomine is sensitive to glutamate hypofunction since it attenuated behavioral impairments in animal models mimicking the negative symptoms and cognitive deficits of schizophrenia. Additionally, the present results support the potential of molsidomine as an adjunctive drug for the therapy of schizophrenia.

Impact of endothelial nitric oxide synthase activation on accelerated liver regeneration in a rat ALPPS model.

BACKGROUND: Although the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) induces more rapid liver regeneration than portal vein embolization, the mechanism remains unclear. AIM: To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase (eNOS) activation on liver regeneration in ALPPS. METHODS: The future liver remnant/body weight (FLR/BW) ratio, hepatocyte proliferation, inflammatory cytokine expression, and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation (PVL) models. Hepatocyte proliferation was assessed based on Ki-67 expression, which was confirmed using immunohistochemistry. The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays. The Akt-eNOS pathway was assessed using western blotting. To explore the role of inflammatory cytokines and NO, Kupffer cell inhibitor gadolinium chloride (GdCl3), NOS inhibitor N-nitro-arginine methyl ester (L-NAME), and NO enhancer molsidomine were administered intraperitoneally. RESULTS: The ALPPS group showed significant FLR regeneration (FLR/BW: 1.60% ± 0.08%, P < 0.05) compared with that observed in the PVL group (1.33% ± 0.11%) 48 h after surgery. In the ALPPS group, serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group. However, the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3 (1.72% ± 0.19%, P < 0.05; 22.25% ± 1.30%, P < 0.05) than in the PVL group (1.33% ± 0.11% and 12.78% ± 1.55%, respectively). Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group. There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index. In the PVL group treated with molsidomine, the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group. CONCLUSION: Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS, whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.

Dynamics of Irreversible NO Release from Photoexcited Molsidomine.

Molsidomine (SIN-10), an orally administered NO-delivery drug for vasodilation, cannot be used to alleviate hypertensive crisis because it releases NO at a slow rate. SIN-10 may be used to treat sudden cardiac abnormalities if the rapid and immediate release of NO is achieved via photoactivation. The photodissociation dynamics associated with the NO release process from SIN-10 in CHCl3 was investigated using time-resolved infrared spectroscopy. Approximately 41% of photoexcited SIN-10 at 360 nm decomposed into CO2, CH2CH3 radical, and the remaining radical fragment [SIN-1A(-H)] with a time constant of 43 ps. All SIN-1A(-H) released NO spontaneously with a time constant of 68 ns, becoming N-morpholino-aminoacetonitrile, resulting in 41% for the quantum yield of immediate NO release from SIN-10. The results obtained can be used to realize the quantitative control of the NO administration at a specific time, and SIN-10 can be potentially used to address the phenomenon of hypertensive crisis.

Prevalence of nitrosamine contaminants in drug samples: Has the crisis been overcome?

Various drug samples (N = 249; drug substances, tablets, capsules, solutions, crèmes, and more) from the European pharmaceutical market were collected since 2019 and analyzed for 16 nitrosamines (NAs). In 2.0% of the cases, NAs were detected. These findings included four active pharmaceutical ingredients already known for potential NA contamination: losartan (N-nitrosodimethylamine [NDMA] and N-nitrosodiethylamine, simultaneously), valsartan (NDMA), metformin (NDMA) and ranitidine (NDMA). The fifth new finding, which has not been reported yet, discovered contamination of a molsidomine tablet sample with N-nitrosomorpholine (NMor). The tablet contained 144% of the toxicological allowable intake for NMor. NMor was included in our screening from the beginning and is currently the focus of regulatory authorities, but was added to the guidelines only last year. Thus, it may not have been the focus of regulatory investigations for too long. Our results indicate that the majority of drug products in the market are nonhazardous in terms of patient safety and drug purity. Unfortunately, the list of individual affected products keeps growing constantly and new NA cases, such as molsidomine or nitrosated drug substances (nitrosamine drug substance-related impurities [NDSRI]), continue to emerge. We therefore expect nitrosamine screenings to remain a high priority.

Effects of low doses of different nitric oxide (NO) donors in rat models of obsessive-compulsive disorder (OCD) and posttraumatic stress disorder (PTSD).

Several lines of evidence suggest that the intra- and inter-cellular messenger nitric oxide (NO) is critically involved in anxiety. Contrasting findings are reported, however, regarding the effects of NO donors in preclinical models of anxiety. Previous research has shown that challenge with a low dose range of the NO donors sodium nitroprusside (SNP) and molsidomine induce anti-anxiety-like effects in rodents. There is poor information concerning the effects of these NO donors on preclinical models mimicking the obsessive-compulsive disorder (OCD) and the post-traumatic stress disorder (PTSD). The present research was designed to investigate this issue in the rat. To this end, the mCPP-induced excessive self-grooming and the contextual fear conditioning (CFC) test which are behavioural paradigms resembling OCD and PTSD respectively in rodents were used. Acute administration of SNP (1 mg/kg) and molsidomine (4 mg/kg) attenuated excessive self-grooming induced by the 5-HT2C receptor agonist mCPP (0.6 mg/kg). Further, at the same dosage, both these NO donors reduced freezing behaviour evidenced in the CFC test. The present results suggest that NO donors are efficacious in attenuating abnormal behaviours revealed in animal models of OCD and PTSD which are among the most severe pathologies of anxiety.

Molsidomine ameliorates diabetic peripheral neuropathy complications in Wistar rats.

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.

Molsidomine alleviates acetic acid-induced colitis in rats by reducing oxidative stress, inflammation and apoptosis.

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.

Nitric oxide-mediated defensive and antinociceptive responses organised at the anterior hypothalamus of mice are modulated by glutamatergic inputs from area 24b of the cingulate cortex.

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.

Peroxynitrite decomposition catalyst enhances respiratory function in isolated brain mitochondria.

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.

The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis).

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 (Cu2+), 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; IC50PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18 µg/ml) and peroxynitrite scavenging assays. PIT at 75 µg/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, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0.05; IC50PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67 µg/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.

Red Blood Cell Peroxynitrite Causes Endothelial Dysfunction in Type 2 Diabetes Mellitus via Arginase.

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.

Oxidative/Nitrative Mechanism of Molsidomine Mitotoxicity Assayed by the Cytochrome c Reaction with SIN-1 in Models of Biological Membranes.

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.

Brain nitric oxide induces facilitation of the micturition reflex through brain glutamatergic receptors in rats.

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; Î·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; Î·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; Î·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.

Deletion of P2X7 Receptor Decreases Basal Glutathione Level by Changing Glutamate-Glutamine Cycle and Neutral Amino Acid Transporters.

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.

Exploitation of nitric oxide donors to control bacterial adhesion on ready-to-eat vegetables and dispersal of pathogenic biofilm from polypropylene.

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% H2 O2 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.

Nitric Oxide-Based Treatment of Poor-Grade Patients After Severe Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Patients with aneurysmal subarachnoid hemorrhage (aSAH) require close treatment in neuro intensive care units (NICUs). The treatments available to counteract secondary deterioration and delayed ischemic events remain restricted; moreover, available neuro-monitoring of comatose patients is undependable. In comatose patients, clinical signs are hidden, and timing interventions to prevent the evolution of a perfusion disorder in response to fixed ischemic brain damage remain a challenge for NICU teams. Consequently, comatose patients often suffer secondary brain infarctions. The outcomes for long-term intubated patients w/wo pupil dilatation are the worst, with only 10% surviving. We previously added two nitroxide (NO) donors to the standard treatment: continuous intravenous administration of Molsidomine in patients with mild-to-moderate aSAH and, if required as a supplement, intraventricular boluses of sodium nitroprusside (SNP) in high-risk patients to overcome the so-called NO-sink effect, which leads to vasospasm and perfusion disorders. NO boluses were guided by clinical status and promptly reversed recurrent episodes of delayed ischemic neurological deficit. In this study, we tried to translate this concept, the initiation of intraventricular NO application on top of continuous Molsidomine infusion, from awake to comatose patients who lack neurological-clinical monitoring but are primarily monitored using frequently applied transcranial Doppler (TCD). METHODS: In this observational, retrospective, nonrandomized feasibility study, 18 consecutive aSAH comatose/intubated patients (Hunt and Hess IV/V with/without pupil dilatation) whose poor clinical status precluded clinical monitoring received standard neuro-intensive care, frequent TCD monitoring, continuous intravenous Molsidomine plus intraventricular SNP boluses after TCD-confirmed macrospasm during the daytime and on a fixed nighttime schedule. RESULTS: Very likely associated with the application of SNP, which is a matter of further investigation, vasospasm-related TCD findings promptly and reliably reversed or substantially weakened (p < 0.0001) afterward. Delayed cerebral ischemia (DCI) occurred only during loose, low-dose or interrupted treatment (17% vs. an estimated 65% with secondary infarctions) in 17 responders. However, despite their worse initial condition, 29.4% of the responders survived (expected 10%) and four achieved Glasgow Outcome Scale Extended (GOSE) 8-6, modified Rankin Scale (mRS) 0-1 or National Institutes of Health Stroke Scale (NIHSS) 0-2. CONCLUSIONS: Even in comatose/intubated patients, TCD-guided dual-compartment administration of NO donors probably could reverse macrospasm and seems to be feasible. The number of DCI was much lower than expected in this specific subgroup, indicating that this treatment possibly provides a positive impact on outcomes. A randomized trial should verify or falsify our results.

Force generated by myosin cross-bridges is reduced in myofibrils exposed to ROS/RNS.

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 (gapp) in combination with a conservation of the force redevelopment rate (kTr) 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 donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice.

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-/-Fbn1C1039G+/-) 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-/-Fbn1C1039G+/- mouse model. Female ApoE-/-Fbn1C1039G+/- 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 ±â€¯1.2 vs. 8.1 ±â€¯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 ±â€¯2% vs. 27 ±â€¯2%) combined with a decreased end diastolic (3.1 ±â€¯0.2 vs. 3.9 ±â€¯0.2 mm) and end systolic diameter (1.9 ±â€¯0.1 vs. 2.9 ±â€¯0.2 mm). Furthermore, perivascular fibrosis (23 ±â€¯2 vs. 30 ±â€¯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 ±â€¯0.04 vs. 2.91 ±â€¯0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability.