Nitric Oxide Synthase Inhibition Prevents Cell Proliferation in Glioblastoma.

Glioblastoma multiforme (GBM) is a prevalent and aggressive primary brain tumor, presenting substantial treatment challenges and high relapse rates. GBM is characterized by alterations in molecular signaling and enzyme expression within malignant cells. This tumor exhibits elevated nitric oxide (NO.) levels. NO. is a crucial signaling molecule involved in the regulation of neuronal functions, synaptic transmission, and cell proliferation. It is primarily synthesized from L-arginine by nitric oxide synthase (NOS) enzymes. The increased levels of NO. in GBM stem from dysregulated activity and expression of clinically relevant NOS isoforms, particularly inducible NOS (iNOS) and neuronal NOS (nNOS). Based on this knowledge, we hypothesize that targeted pharmacological intervention with N6-(1-iminoethyl)-L-lysine (L-NIL), an iNOS inhibitor, and 7-Nitroindazole (7-NI), an nNOS inhibitor, may suggest a promising therapeutic strategy for the treatment of GBM. To test our hypothesis, we utilized the U87-MG cell line as an in vitro model of GBM. Our results showed that treatment with L-NIL and 7-NI led to a reduction in NO. levels, NOS activity, and clonogenic proliferation in U87-MG cells. These findings suggest that NO. and NOS enzymes might be prospective therapeutic targets for GBM.

Effects of extended-release 7-nitroindazole gel formulation treatment on the behavior of Shank3 mouse model of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by behavioral deficits such as abnormalities in communication, social interaction, anxiety, and repetitive behavior. We have recently shown that the Shank3 mutation in mice representing a model of ASD causes excessive nitric oxide (NO) levels and aberrant protein S-nitrosylation. Further, 10-day daily injections of 7-NI, a neuronal nitric oxide synthase inhibitor, into Shank3Δ4-22 and Cntnap2(-/-) mutant mice (models of ASD) at a dose of 80 mg/kg reversed the manifestations of ASD phenotype. In this study, we proposed an extended release of 7-NI using a novel drug system. Importantly, unlike the intraperitoneal injections, our new preparation of poly (sebacic acid-co-ricinoleic acid) (PSARA) gel containing 7-NI was injected subcutaneously into the mutant mice only once. The animals underwent behavioral testing starting from day 3 post-injection. It should be noted that the developed PSARA gel formulation allowed a slow release of 7-NI maintaining the plasma level of the drug at ∼45 µg/ml/day. Further, we observed improved memory and social interaction and reduced anxiety-like behavior in Shank3 mutant mice. This was accompanied by a reduction in 3-nitrotyrosine levels (an indicator of nitrative/nitrosative stress) in plasma. Overall, we suggest that our single-dose formulation of PSARA gel is very efficient in rendering a therapeutic effect of 7-NI for at least 10 days. This approach may provide in the future a rational design of an effective ASD treatment using 7-NI and its clinical translation.

7-Nitroindazole reduces L-DOPA-induced dyskinesias in non-human Parkinsonian primate.

Nitric oxide (NO) plays a pivotal role in integrating dopamine transmission in the basal ganglia and has been implicated in the pathogenesis of Parkinson disease (PD). The objective of this study was to ascertain whether the NO synthase inhibitor, 7-nitroindazole (7-NI), is able to reduce L-DOPA-induced dyskinesias (LIDs) in a non-human primate model of PD chronically intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Six Parkinsonian macaques were treated daily with L-DOPA for 3-4 months until they developed LIDs. Three animals were then co-treated with a single dose of 7-NI administered 45 min before each L-DOPA treatment. Dyskinetic MPTP-treated monkeys showed a significant decrease in LIDs compared with their scores without 7-NI treatment (p < 0.05). The anti-Parkinsonian effect of L-DOPA was similar in all three monkeys with and without 7-NI co-treatment. This improvement was significant with respect to the intensity and duration of LIDs while the beneficial effect of L-DOPA treatment was maintained and could represent a promising therapy to improve the quality of life of PD patients.

Design and synthesis of benzo[d]thiazol-2-yl-methyl-4-(substituted)-piperazine-1-carbothioamide as novel neuronal nitric oxide inhibitors and evaluation of their neuroprotecting effect in 6-OHDA-induced unilateral lesioned rat model of Parkinson's disease.

Neuronal nitric oxide synthase (nNOS) is an enzyme constitutively expressed in the mammalian brain and skeletal muscles. The excessive activation of nNOS in the neurons results in oxidative and nitrosative stress associated with neuronal loss in various neurological disorders. Several nNOS inhibitors have been reported to limit the excessive activation of nNOS. In the present work, we have designed and carried the synthesis of benzo[d]thiazol-2-yl-methyl-4-(substituted)-piperazine-1-carbothioamide as novel neuronal nitric oxide inhibitors (5-28, twenty-four compounds). Stably transfected HEK 293 cells expressing NOS isoforms treated with the compounds (5-28) showed that the eight compounds exhibited > 95% cell survival in the MTT assay. nNOS inhibition assay of the eight compounds illustrated that the compound 18 was most selective for nNOS (nNOS=66.73 ± 1.51; eNOS=28.70 ± 1.39; iNOS =13.26 ± 1.01) in HEK 293 cells expressing NOS isoforms. 6-OHDA-induced unilaterally lesioned rats treated with the compound 18 showed the improvement in motor and non-motor functions. Furthermore, the compound 18 showed the increased levels of dopamine and decreased levels of glutamate and nitrite ions in the isolated rat brain. In the docking analysis, the compound 18 showed the significant binding affinity with the nNOS binding site (the ∆G value = - 9.0 kcal/mol). Overall results demonstrated that the N-(benzo[d]thiazol-2-ylmethyl)-4-(4-nitrophenyl) piperazine-1-carbothioamide (the compound 18) possessed significant nNOS inhibiting activity and neuroprotecting potential in 6-OHDA-induced unilaterally lesioned rat model of PD and more work will be required to establish the role of the compound 18 in the therapy of PD and other neurodegenerative disorders.

Echinodorus macrophyllus fraction with a high level of flavonoid inhibits peripheral and central mechanisms of nociception.

BACKGROUND AND AIM: Echinodorus macrophyllus (Kunth.) Micheli is popularly used for acute and chronic inflammatory conditions. The anti-inflammatory activity was previously demonstrated for its flavonoid-enriched fractions. The aim of this work assessed the antinociceptive properties of both aqueous extract and its fractions. EXPERIMENTAL PROCEDURE: The antinociceptive activity was determined by acetic acid-induced writhing, formalin test, tail immersion test, hot-plate test, xylene-induced ear edema methods, and the evaluation of its mechanism was performed in the writhing model. The aqueous extract of Echinodorus macrophyllus (AEEm) was fractionated, yielding Fr20, and Fr40. Fr40 composition was determined by HPLC-DAD-ESI-MS. RESULTS AND CONCLUSION: Fr20 (all doses) and Fr40 (100 mg/kg) reduced the nociception in the tail-flick model. Both fractions increased the percentage of maximum possible effect with 25 mg/kg, in the hot-plate assay, at 60 min, while AEEm reduced pain only with 50 and 100 mg/kg. There was a reduction in xylene-edema index, with Fr40 (25 mg/kg), AEEm (50 mg/kg) and Fr20 (50 mg/kg). All doses of AEEm, Fr20, and Fr40 reduced both phases of the formalin model. In the abdominal contortion model, Fr40 presented the highest activity, reducing 96% of contortions and its antinociceptive mechanism was evaluated. The results indicated the involvement of NO and adrenergic activation pathways. The main components of Fr40 are swertisin, swertiajaponin, isoorientin 7,3'-dimethyl ether, swertisin-O-rhamnoside, isoorientin, isovitexin, isovitexin-Orhamnoside, and isovitexin-7-O-glucoside. The aqueous extract of E. macrophyllus leaves and its fractions exhibited significant analgesic effect, mediated through both peripheral and central mechanisms being considered a potentially antinociceptive drug.

Cavitation Resistance of WC-10Co4Cr and WC-20CrC-7Ni HVAF Coatings.

Machines operating in aqueous environments may be subjected to cavitation damage during operation. This study aims to evaluate the cavitation resistance of WC-10Co4Cr and WC-20CrC-7Ni coatings under cavitation erosion conditions with additional electrochemical effects. The coatings were deposited on AISI 1040 steel substrates using a high velocity air fuel thermal spray process. The microstructure of the coatings was observed by a scanning electron microscope, while their phase composition was analyzed using an energy-dispersive microanalysis system. In addition, the microhardness of the coatings and substrate was measured, and the surface topography of the eroded surface layers was observed using a 3D optical profilometer. The results revealed that the cavitation resistance of the WC-20CrC-7Ni coatings was better than that of the WC-10Co4Cr coatings. The observation of the structure and surface topography made it possible to identity the reasons for the differences between the cavitation resistance of both coatings: The WC-20CrC-7Ni coatings had a finer grain structure, lower pore density, and lower as-sprayed surface roughness. These differences, along with the presence of a high Cr and Ni content in the feedstock powder, that increased the coating corrosion resistance, contributed to improving the cavitation resistance and reducing the material loss of the WC-20CrC-7Ni coatings.

Challenging the Durability of Intermetallic Mo-Ni Compounds in the Hydrogen Evolution Reaction.

Molybdenum-nickel materials are catalysts of industrial interest for the hydrogen evolution reaction (HER). Well-characterized surfaces of the single-phase intermetallic compounds Ni7Mo7, Ni3Mo, and Ni4Mo were subjected to accelerated durability tests (ADTs) and thorough characterization to unravel whether crystallographic ordering affects the activity. Their intrinsic instability leads to molybdenum leaching, resulting in higher specific surface areas and nickel-enriched surfaces. These are more prone to form Ni(OH)2 layers, which leads to deactivation of the Mo-Ni materials. The crystal structure of the intermetallic compounds has, due to the intrinsic instability of the materials in alkaline media, no effect on the activity. Ni7Mo7, identified earlier as durable, proves to be highly unstable in the applied ADTs. The results show that the enhanced activity of unsupported bulk Mo-Ni electrodes can solely be ascribed to increased specific surface areas.

Effects of onopordia, a novel isolated compound from Onopordon acanthium, on pentylenetetrazole-induced seizures in mice: Possible involvement of nitric oxide pathway.

Epilepsy is identified as a brain disorder and characterized by unpredictable disruption of normal brain function. Due to adverse side effect associated with antiepileptic drugs and also resistance profile, improvement of antiepileptic medications with more beneficial anticonvulsant activity is essential. Natural products have demonstrated their therapeutic properties such as anxiolytic, antidepressant and anticonvulsant activities and a source for identification of novel lead compounds. Therefore, the purpose of this study was to evaluate the effects of Onopordon acanthium secondary metabolite, onopordia, on pentylenetetrazole (PTZ)-induced seizure in male mice and investigate the possible role of nitric oxide pathway. Different doses of onopordia (0.1, 1 and 10 mg/kg) and phenobarbital (20 mg/kg) were administered intraperitoneally (i.p., 30, 60 and 120 min) prior to induction of epileptic seizure and compared to control groups. Onopordia demonstrated anticonvulsant effects when administrated at dose of 10 mg/kg, i.p. and optimum time 60 min prior to induction of seizure. Anticonvulsant effect of onopordia was blocked by applying a single dose of a non-selective nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; 10 mg/kg, i.p.), and also a single dose of a selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; 30 mg/kg, i.p.). Administration of ketamine as a N-Methyl-d-aspartic acid (NMDA) receptor antagonist (0.5 mg/kg; i.p.) with onopordia did not change the anticonvulsant effect of onopordia. The results of the present study demonstrated the anticonvulsant effect of onopordia as a new lead compound and also contribution of NO/nNOS pathway on PTZ-induced seizure in mice.

Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats.

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Thalidomide protects against acute pentylenetetrazol and pilocarpine-induced seizures in mice.

Thalidomide was originally developed to treat primary neurological and psychiatric diseases. There are reports of anticonvulsant effects of thalidomide in rats and antiepileptic effects in patients. Hence, thalidomide (100, 200 and 400 mg/kg) was herein administered to mice to evaluate possible protection against seizures induced by the systemic administration of neurotoxins: 10 mg/kg of 4-aminopyridine (4-AP), 90 mg/kg of pentylenetetrazol (PTZ), or 380 mg/kg of pilocarpine. The effect of an NO and COX inhibitor (7-NI and ibuprofen, respectively) was also examined. The results show that thalidomide (1) induces the typical sedative effects, (2) has no anticonvulsant effect in mice treated with 4-AP, and (3) has anticonvulsant effect (400 mg/kg) in mice treated with PTZ and pilocarpine. It was found that 7-NI has an anticonvulsant effect in the pilocarpine model and that thalidomide's effect is not enhanced by its presence. However, thalidomide (200 mg/kg) plus 7-NI or ibuprofen tend to have a toxic effect in PTZ model. On the other hand, the combination of thalidomide and 7-NI or ibuprofen protects against pilocarpine-induced seizures. In conclusion, thalidomide did not exert an anticonvulsant effect for clonic-tonic type convulsions (4-AP), but it did so for seizures induced by PTZ and pilocarpine (representing absence seizures and status epilepticus, respectively). NO and prostaglandins were involved in the convulsive process elicited by pilocarpine.

The role of striatum and prefrontal cortex in the prevention of amphetamine-induced schizophrenia-like effects mediated by nitric oxide compounds.

Pharmacological manipulation of nitric oxide (NO) has been suggested as a promising treatment for schizophrenia symptoms. A single infusion of sodium nitroprusside, a NO donor with short half-life, was found to improve schizophrenia symptoms. However, an increasing number of preclinical studies have demonstrated the potential beneficial effects of both NO donors and inhibitors. We investigated the potential synergistic effect of sub-effective doses of the NO donor sodium nitroprusside or the NO inhibitor 7-Nitroindazole (7NI) combined with clozapine, a standard atypical antipsychotic, on counteracting amphetamine or MK-801-induced psychosis-like behaviors. The impact of sodium nitroprusside and 7NI on cAMP regulation in the prefrontal cortex and striatum was also evaluated. Confirming previous results, we found that both NO donors and inhibitors prevented amphetamine-induced effects (prepulse inhibition [PPI] disruption and hyperlocomotion). In addition, we observed a synergistic effect of sodium nitroprusside and clozapine on antagonizing the disruptive effects of amphetamine, but not MK-801, in the PPI test. The sub-effective dose of 7NI tested did not prevent amphetamine or MK-induced PPI effects when combined with clozapine. Interestingly, cAMP levels were significantly decreased in the prefrontal cortex after treatment with sodium nitroprusside. In the striatum, both sodium nitroprusside and 7NI blocked the amphetamine-induced increase of cAMP. Our data corroborate previous findings on the dopaminergic mechanisms involved in the action of sodium nitroprusside. It is likely that the differential effects of sodium nitroprusside are related to its ability to modify cAMP levels in the prefrontal cortex.

7-nitroindazol-loaded nanoemulsions: Preparation, characterization and its improved inhibitory effect on nitric oxide synthase-1.

Nitric oxide (NO) participates in several physiological processes such as maintenance of blood pressure, host defense, neurotransmission, inhibition of platelet aggregation and learning and memory. NO is also involved in several diseases or dysfunctions in the cardiovascular, central nervous and musculoskeletal systems. NO also has been shown to be a major player in sepsis. NOS-1-derived NO has been shown to be a relevant species in physiology but also is an important element in pathology. There exist some NOS-1 inhibitors and among of them, 7-nitroindazole has been used for its in vivo selectivity. However, 7-NI has a very short half-life (∼2 h) and a poor water solubility. In this study, we describe the preparation and characterization of 7-NI-loaded nanoemulsions (NE7-NI). The chemical stability of 7-NI was greatly increased and the drug release rate could be controlled after nanoemulsification. NE7-NI reduced NO production in a long-lasting manner in vascular smooth muscle cells and skeletal muscle, without cytotoxicity. Our results evidenced that nanoemulsification approach increases the effective action time of 7-NI, rendering a suitable dosage form, which may be an interesting tool to study the role of NOS-1 in physiology and disease.

Experimental studies on possible regulatory role of nitric oxide on the differential effects of chronic predictable and unpredictable stress on adaptive immune responses.

The present study was designed to investigate the effects of chronic predictable stress (CPS) and chronic unpredictable stress (CUS) on immunological responses in KLH-sensitized rats and involvement of NOergic signaling pathways mediating such responses. Male Wistar rats (200-250g) were exposed to either CPS or CUS for 14days and IgG antibody levels and delayed type hypersensitivity (DTH) response was determined to assess changes in adaptive immunity. To evaluate the role of nitric oxide during such immunomodulation, biochemical estimation of stable metabolite of nitric oxide (NOx) and 3-nitrotyrosine (3-NT, a marker of peroxynitrite formation) were done in both blood and brain. Chronic stress exposure resulted in suppression of IgG and DTH response and elevated NOx and 3-NT levels, with a difference in magnitude of response in CPS vs CUS. Pretreatment with aminoguanidine (iNOS inhibitor) caused further reduction of adaptive immune responses and attenuated the increased NOx and 3-NT levels in CPS or CUS exposed rats. On the other hand 7-NI (nNOS inhibitor) did not significantly affect these estimated parameters. The results suggest involvement of iNOS and lesser/no role of nNOS during modulation of adaptive immunity to stress. Thus, the result showed that predictability of stressors results in differential degree of modulation of immune responses and complex NO-mediated signaling mechanisms may be involved during responses.

Lipopolysaccharide-induced memory impairment in rats is preventable using 7-nitroindazole / O déficit de memória induzido por lipossacarídeos em ratos é prevenido por nitroindazol

Inflammation and oxidative stress have important roles in memory impairment. The effect of 7-nitroindazole (7NI) on lipopolysaccharide (LPS)-induced memory impairment was investigated. Rats were used, divided into four groups that were treated as follows: (1) control (saline); (2) LPS; (3) 7NI-LPS; and (4) 7NI before passive avoidance (PA). In the LPS group, the latency for entering the dark compartment was shorter than in the controls (p < 0.01 and p < 0.001); while in the 7NI-LPS group, it was longer than in the LPS group (p < 0.01 and p < 0.001). Malondialdehyde (MDA) and nitric oxide (NO) metabolite concentrations in the brain tissues of the LPS group were higher than in the controls (p < 0.001 and p < 0.05); while in the 7NI-LPS group, they were lower than in the LPS group (p < 0.001 and p < 0.05, respectively). The thiol content in the brain of the LPS group was lower than in the controls (p < 0.001); while in the 7NI-LPS group, it was higher than in the LPS group (p < 0.001). It is suggested that brain tissue oxidative damage and NO elevation have a role in the deleterious effects of LPS on memory retention that are preventable using 7NI.

Inflamação e estresse oxidativo tem importante papel no déficit de memória. O efeito do 7-nitroindazol (7NI) no déficit de memória induzido por lipossacarídeos (LPS) foi investigado. Foram utilizados ratos que foram divididos em quatro grupos e tratados da seguinte maneira: (1) controles (solução salina); (2) LPS; (3) 7NI-LPS; e (4) 7NI antes da esquiva passiva (PA). No grupo LPS, a latência para entrar no compartimento escuro foi mais curta que nos controles (p < 0,01 e p < 0,001); enquanto no grupo 7NI-LPS, a latência foi maior que aquela do grupo LPS (p < 0,01 e p < 0,001). Concentrações de malondialdeído (MDA) e metabólitos do ácido nítrico (NO) no tecido cerebral do grupo LPS foram maiores que aquelas dos controles (p < 0,001 e p < 0,05); enquanto no grupo 7NI-LPS, as concentrações foram menores do que no grupo LPS (p < 0,001 e p < 0,05, respectivamente). O conteúdo cerebral de tiol no grupo LPS foi menos do que nos controles (p < 0,001); enquanto no grupo 7NI-LPS, este conteúdo foi maior que no grupo LPS (p < 0,001). Sugere-se que o dano oxidativo cerebral e o aumento de NO tenham um papel nos efeitos deteriorativos dos LPS na memória de retenção, e que isto possa ser prevenido com o uso de 7NI.

Fetal tubuloglomerular feedback in an ovine model of mild maternal renal disease.

Fetuses of pregnant ewes, which were subtotally nephrectomized prior to mating, were studied to assess whether mild maternal renal impairment would affect fetal tubuloglomerular feedback (TGF) under control conditions and after the inhibition of macula densa-derived nitric oxide (NO). Based on previous observations we hypothesized that, the TGF curve of fetuses of subtotally nephrectomized (STNx) ewes would resemble that of a volume expanded fetus with a high production rate of NO and that inhibition of neuronal nitric oxide synthase (nNOS) would increase the sensitivity of the TGF system in these fetuses. Renal function studies were performed on anaesthetized fetal sheep (133-140 days gestation; term ~150 days; Isoflurane 2-4% in oxygen). Fetuses were removed from the uterus and placed in a water bath (39.5°C) while maintaining umbilical blood flow. Glomerular filtration rate (GFR) and urine flow rate were markedly increased in fetuses of STNx ewes compared to fetuses of untreated ewes. Interestingly, and contrary to our hypothesis, the fetuses of STNx ewes exhibited no difference in TGF sensitivity in the presence or absence of 7-nitroindazole (7NI; nNOS inhibitor), compared to fetuses of untreated ewes, although sensitivity and reactivity increased in both groups after 7NI. There was however, a decrease in the stop flow pressure and net filtration pressure with an increase in the filtration coefficient (Kf). These factors suggest that maternal renal impairment drives the glomerular hypertrophy which has previously been found to be present in the neonatal period. Thus, we conclude that at ~138 days gestation, the fetal kidney has matured functionally and fetuses of STNx ewes are able to maintain fluid and electrolyte homeostasis even in the presence of increased transplacental flux.

7-NI and ODQ Disturbs Memory in the Elevated Plus Maze, Morris Water Maze, and Radial Arm Maze Tests in Mice.

Nitric oxide (NO) is an atypical neurotransmitter that causes changes in cognition. Nitric oxide synthase (NOS) and guanylate cyclase (GC) inhibitors have been shown to exert some effects on cognition in previous studies; however, the findings have been controversial. This study was aimed at understanding the effects of an NOS inhibitor, 7-nitroindazole (7-NI), and a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), on spatial memory in modified elevated plus maze (mEPM), Morris water maze (MWM), and radial arm maze (RAM) tests. Male Balb-c mice were treated via intraperitoneal injections with 7-NI (15 mg/kg), ODQ (3, 10 mg/kg), L-arginine (100 mg/kg) + 7-NI (15 mg/kg), or physiological saline. ODQ (3 mg/kg) and 7-NI (15 mg/kg) significantly increased the second-day latency in the mEPM test. 7-NI (15 mg/kg) and ODQ (10 mg/kg) significantly increased the escape latency in second, third, and fourth sessions, decreased the time spent in the escape platform's quadrant, and increased the mean distance to the platform in the probe trial of the MWM test. ODQ (3, 10 mg/kg) and 7-NI (15 mg/kg) significantly increased the number of errors, whereas only 7-NI increased the latency in the RAM test. The administration of L-arginine (100 mg/kg) prior to 7-NI inverted the effects of 7-NI, which supports the role of NO on cognition. Our study shows that the NO/cGMP/GS pathway can regulate spatial memory in mice.

The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats.

Wnt-5a increases NO and modulates NMDA receptor in rat hippocampal neurons.

Wnt signaling has a crucial role in synaptic function at the central nervous system. Here we evaluate whether Wnts affect nitric oxide (NO) generation in hippocampal neurons. We found that non-canonical Wnt-5a triggers NO production; however, Wnt-3a a canonical ligand did not exert the same effect. Co-administration of Wnt-5a with the soluble Frizzled related protein-2 (sFRP-2) a Wnt antagonist blocked the NO production. Wnt-5a activates the non-canonical Wnt/Ca(2+) signaling through a mechanism that depends on Ca(2+) release from Ryanodine-sensitive internal stores. The increase in NO levels evoked by Wnt-5a promotes the insertion of the GluN2B subunit of the NMDA receptor (NMDAR) into the neuronal cell surface. To the best of our knowledge, this is the first time that Wnt-5a signaling is related to NO production, which in turn increases NMDARs trafficking to the cell surface.

Depressive-like behavior induced by tumor necrosis factor-α is abolished by agmatine administration.

Agmatine, an endogenous cationic amine, has been shown to exert antidepressant-like effects. This study investigated the ability of agmatine administered orally to abolish the depressive-like behavior induced by the administration of the pro-inflammatory cytokine, tumor necrosis factor (TNF-α) in mice. In control animals, agmatine (0.001, 0.01, 0.1, and 1 mg/kg) reduced the immobility time in the tail suspension test (TST). Acute administration of TNF-α (0.001 fg/mouse, i.c.v.) increased immobility time in the TST, indicative of a depressive-like behavior, and agmatine (0.0001, 0.1, and 1 mg/kg) prevented this effect. Additionally, we examined the effects of the combined administration of sub-effective doses of agmatine with antidepressants, the NMDA receptor antagonist MK-801 and the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) in mice exposed to either TNF-α or saline. In control mice, administration of a sub-effective dose of agmatine (0.0001 mg/kg) combined with sub-effective doses of either fluoxetine (5 mg/kg, p.o.), imipramine (0.1 mg/kg, p.o.), bupropion (1 mg/kg, p.o.), MK-801 (0.001 mg/kg, p.o.) or 7-NI (25 mg/kg, i.p.) produced a synergistic antidepressant-like effect in the TST. All these administrations prevented the increased immobility time induced by TNF-α. The effect of agmatine in the TNF-α model of depression appears to be associated, at least partially, with an activation of the monoaminergic systems and inhibition of NMDA receptors and nitric oxide synthesis, although converging signal transduction pathways that may underlie the effect of agmatine should be further investigated. This set of results indicates that agmatine may constitute a new therapeutic alternative for the treatment of depression associated with inflammation.

Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats.

Nitric oxide (NO) has been reported to be involved in the mechanisms of pain generation throughout the nervous system. We examined the effects of intrathecally (i.t.) administered nitric oxide synthase (NOS) inhibitors on the antinociceptive effects of morphine and endomorphin-1 during acute pain and in chronic constriction injury (CCI)-exposed rats. We used N(G)-nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor; 7-nitroindazole (7-NI) or 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM), selective inhibitors of neuronal NOS (NOS1); and 1400W dihydrochloride, a selective inhibitor of inducible NOS (NOS2). Morphine (0.5-2.5 µg) and endomorphin-1 (2.5-20 µg) in acute pain and morphine (10-40 µg) and endomorphin-1 (5-20 µg) after CCI-injury were combined with NOS inhibitors. For acute pain, the ED50 for endomorphin-1 (7.1 µg) was higher than that of morphine (1.3 µg) in the tail-flick test. For neuropathic pain, the ED50 value for morphine was much higher (43.2 µg) than that of endomorphin-1 (9.2 µg) in von Frey test. NOS inhibitors slightly influenced pain thresholds in both pain models. Moreover, in neuropathic pain, the effects of morphine were more potentiated by L-NAME, TRIM, 7-NI and 1400W (12×, 8.6×, 4.1× and 5.3×, respectively) than were the effects of endomorphin-1 (2.7×, 4.3×, 3.4× and 2.1×, respectively) in the von Frey test. Minocycline which is known to enhance the efficiency of morphine in neuropathic pain, decreased the mRNA expression of NOS1 in the DRG and NOS2 and C1q in the spinal cord after CCI. Both NOS2 and IBA-1 protein levels in the spinal cord and NOS1, NOS2 and IBA1 protein levels in DRG decreased after minocycline administration. In conclusion, our results provide evidence that both neuronal and non-neuronal NOS/NO pathways contribute to the behavioural pain responses evoked by nerve injury. The NOS inhibitors regardless of the type of pain enhanced morphine antinociception and, to a lesser extent, altered the effects of endomorphin-1, an opioid ligand with a peptidergic structure.