Neuronal Nitric Oxide Synthase Inhibitor Research Articles

Nitrosative Stress in Autism: Supportive Evidence and Implications for Mitochondrial Dysfunction.

A recent study by the Amal team published in this journal in May 2023 proved for the first time the link of nitric oxide (NO) with autism spectrum disorder (ASD), thereby opening new venues for the potential use of neuronal nitric oxide synthase (nNOS) inhibitors as therapeutics for improving the neurological and behavioral symptoms of ASD. The authors conclude that their findings demonstrate that NO plays a significant role in ASD. Indeed, earlier studies support elevated NO and its metabolites, nitrite, and peroxynitrite, in individuals diagnosed with ASD. Dysregulated NOS activity may underlie the well-documented mitochondrial dysfunction in a subset of individuals with ASD. Strategies for treating ASD shall also consider NO effects on mitochondrial respiration in modulating NOS activity. Further experimental evidence and controlled clinical trials with NOS modifiers are required for assessing their therapeutic potential for individuals with ASD.

The anterior cingulate cortex and its interface with the dorsal periaqueductal grey regulating nitric oxide-mediated panic-like behaviour and defensive antinociception

The anterior cingulate cortex (ACC) Cg1 (24b) area modulates glutamate-mediated unconditioned fear and antinociception organised by hypothalamus. However, it remains unknown whether 24b area also modulates these latter defensive responses through connections with dorsal periaqueductal grey matter (dPAG), a midbrain structure implicated in the genesis of innate fear-induced defence. The aim of this work is to examine the correlation between the behavioural effects of intra-ACC microinjections of vehicle, NMDA (1 nmol) or lidocaine (2%) with Fos protein expression and nitrergic activity in the dPAG of male C57BL/6 mice that were threatened by snakes. In addition, the 24b area-dPAG pathways were also characterised by neural tract tracing procedures. Finally, the effect of dPAG pretreatment with the neuronal nitric oxide synthase inhibitor N(omega)-propyl-l-arginine (NPLA; 0.2, 0.4 or 0.8 nmol) 10 min before 24b area treatment with NMDA on behavioural and nociceptive responses of threatened mice was studied. The activation of 24b area N-methyl-d-aspartic acid receptors facilitated escape and freezing rather than risk assessment, and enhanced Fos expression and nitrite levels in dPAG, while lidocaine decreased escape and risk assessment as well as Fos and nitrergic activity in dPAG. In addition, dPAG pretreatment with NPLA suppressed intra-24b NMDA-facilitated panicogenic effects while increased nociception. Infusions of an antegrade neurotracer into 24b area showed axonal fibres surrounding both dorsomedial and dorsolateral PAG perikarya. Neurons were identified in 24b area after deposits of a retrograde neurotracer into dPAG. Our findings suggest that the ACC/24b area modulates innate defensive responses through the recruitment of dPAG nitrergic neurons.

Indazole derivatives as novel inhibitors of monoamine oxidase and D-amino acid oxidase

The monoamine oxidase (MAO) enzymes metabolize neurotransmitter amines in the peripheral and central tissues, and inhibitors of these enzymes find application in the treatment of neuropsychiatric and neurodegenerative disorders. Based on reports that the neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole, inhibits the MAO-B isoform, the present study investigated the MAO inhibition potencies of a synthetic series of fifteen C5- and C6-substituted indazole derivatives. While only one derivative (5c) was a submicromolar inhibitor of human MAO-A (IC50 = 0.745 µM), all compounds inhibited human MAO-B with submicromolar IC50 values. Substitution on C5 of indazole yielded particularly potent MAO-B inhibition with IC50 values that ranged from 0.0025–0.024 µM. Further investigation of a selected indazole derivative showed a competitive mode of MAO inhibition. To further explore the pharmacological properties of the indazole derivatives, they were also evaluated as potential inhibitors of porcine D-amino acid oxidase (DAAO). None of the synthetic compounds were noteworthy DAAO inhibitors, however, 1H-indazol-5-ol, a synthetic precursor, was found to be a good potency inhibitor with an IC50 value of 2.03 µM.

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.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.

Facilitating the drug repurposing with iC/E strategy: A practice on novel nNOS inhibitor discovery.

Over the past decades, many existing drugs and clinical/preclinical compounds have been repositioned as new therapeutic indication from which they were originally intended and to treat off-target diseases by targeting their noncognate protein receptors, such as Sildenafil and Paxlovid, termed drug repurposing (DRP). Despite its significant attraction in the current medicinal community, the DRP is usually considered as a matter of accidents that cannot be fulfilled reliably by traditional drug discovery protocol. In this study, we proposed an integrated computational/experimental (iC/E) strategy to facilitate the DRP within a framework of rational drug design, which was practiced on the identification of new neuronal nitric oxide synthase (nNOS) inhibitors from a structurally diverse, functionally distinct drug pool. We demonstrated that the iC/E strategy is very efficient and readily feasible, which confirmed that the phosphodiesterase inhibitor DB06237 showed a high inhibitory potency against nNOS synthase domain, while other two general drugs, i.e. DB02302 and DB08258, can also inhibit the synthase at nanomolar level. Structural bioinformatics analysis revealed diverse noncovalent interactions such as hydrogen bonds, hydrophobic forces and van der Waals contacts across the complex interface of nNOS active site with these identified drugs, conferring both stability and specificity for the complex recognition and association.

Influence of NO Synthase Inhibitors on Serotonin Release in the Medial Prefrontal Cortex during the Formation and Generalization of the Conditioned Fear Response in Rats

Serotonergic and nitrergic systems of the medial prefrontal cortex (mPFC) are involved in the control of fear generalization, but their local interaction during this function has been little studied. The aim of the work was to study the effect of blockade of endogenous nitrergic signals on serotonin release in the mPFC during the acquisition of a conditioned fear response (CFR – a fear model) and on the dynamics of its generalization. In male Sprague-Dawley rats using intracranial microdialysis in vivo and high-performance liquid chromatography with electrochemical detection, we found that the intra-mPFC infusion through the dialysis probe of the NO synthase inhibitor N-ω-nitro-L-arginine (NA, 0.5 mM) and selective neuronal NO synthase inhibitor N-ω-propyl-L-arginine (NPLA, 2mM) decreased the basal level of extracellular serotonin in the mPFC and reduced its rise, caused by the CFR acquisition (a paired presentation of a conditioned cue (CS+) and inescapable footshock). The intra-mPFC infusion of NA and NPLA increased animals’ freezing to a differential cue (CS–) not associated with footshock, during the first test, carried out 70 minutes after the CFR acquisition, but reduced it during repeated testing a day after the infusion, without changing freezing of the same animals to the potentially dangerous CS+. The data obtained indicate the involvement of endogenous NO in the activation of serotonin release in the mPFC, caused by the CFR acquisition. In addition, they show that the blockade of endogenous nitrergic signals of the mPFC, which enhances the initial generalization of the fear reaction, contributes to the extinction of the generalized fear, possibly due to inhibition of the serotonin release in the mPFC.

Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.

ARL-17477 is a dual inhibitor of NOS1 and the autophagic-lysosomal system that prevents tumor growth in vitro and in vivo

ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic.

The NO Answer for Autism Spectrum Disorder.

Autism spectrum disorders (ASDs) include a wide range of neurodevelopmental disorders. Several reports showed that mutations in different high-risk ASD genes lead to ASD. However, the underlying molecular mechanisms have not been deciphered. Recently, they reported a dramatic increase in nitric oxide (NO) levels in ASD mouse models. Here, they conducted a multidisciplinary study to investigate the role of NO in ASD. High levels of nitrosative stress biomarkers are found in both the Shank3 and Cntnap2 ASD mouse models. Pharmacological intervention with a neuronal NO synthase (nNOS) inhibitor in both models led to a reversal of the molecular, synaptic, and behavioral ASD-associated phenotypes. Importantly, treating iPSC-derived cortical neurons from patients with SHANK3 mutation with the nNOS inhibitor showed similar therapeutic effects. Clinically, they found a significant increase in nitrosative stress biomarkers in the plasma of low-functioning ASD patients. Bioinformatics of the SNO-proteome revealed that the complement system is enriched in ASD. This novel work reveals, for the first time, that NO plays a significant role in ASD. Their important findings will open novel directions to examine NO in diverse mutations on the spectrum as well as in other neurodevelopmental disorders. Finally, it suggests a novel strategy for effectively treating ASD.

Improved synthesis and anticancer activity of a potent neuronal nitric oxide synthase inhibitor

An improved synthesis of 4-methyl-7-(3-((methylamino)methyl)phenethyl)quinolin-2-amine (1) is reported. A scalable, rapid, and efficient methodology was developed to access this compound with an overall yield of 35%, which is 5.9-fold higher than the previous report. The key differences in the improved synthesis are a high yielding quinoline synthesis by a Knorr reaction, a copper-mediated Sonogashira coupling to the internal alkyne in excellent yield, and a crucial deprotection of the N-acetyl and N-Boc groups achieved under acidic conditions in a single step rather than a poor yielding quinoline N-oxide strategy, basic deprotection conditions, and low yielding copper-free conditions that were reported in the previous report. Compound 1, which previously was shown to inhibit IFN-γ-induced tumor growth in a human melanoma xenograft mouse model, was found to inhibit the growth of metastatic melanoma, glioblastoma, and hepatocellular carcinoma in vitro.

Contextual fear expression engages a complex set of interactions between ventromedial prefrontal cortex cholinergic, glutamatergic, nitrergic and cannabinergic signaling

Rats re-exposed to an environment previously associated with the onset of shocks evoke a set of conditioned defensive responses in preparation to an eventual flight or fight reaction. Ventromedial prefrontal cortex (vmPFC) is mutually important for controlling the behavioral/physiological consequences of stress exposure and the one's ability to satisfactorily undergo spatial navigation. While cholinergic, cannabinergic and glutamatergic/nitrergic neurotransmissions within the vmPFC are shown as important for modulating both behavioral and autonomic defensive responses, there is a gap on how these systems would interact to ultimately coordinate such conditioned reactions. Then, males Wistar rats had guide cannulas bilaterally implanted to allow drugs to be administered in vmPFC 10 min before their re-exposure to the conditioning chamber where three shocks were delivered at the intensity of 0.85 mA for 2 s two days ago. A femoral catheter was implanted for cardiovascular recordings the day before fear retrieval test. It was found that the increment of freezing behavior and autonomic responses induced by vmPFC infusion of neostigmine (acetylcholinesterase inhibitor) were prevented by prior infusion of a transient receptor potential vanilloid type 1 (TRPV1) antagonist, N-methyl-d-aspartate receptor antagonist, neuronal nitric oxide synthase inhibitor, nitric oxide scavenger and soluble guanylate cyclase inhibitor. A type 3 muscarinic receptor antagonist was unable to prevent the boosting in conditioned responses triggered by a TRPV1 agonist and a cannabinoid receptors type 1 antagonist. Altogether, our results suggest that expression of contextual conditioned responses involves a complex set of signaling steps comprising different but complementary neurotransmitter pathways.

A Comparative Study of the Impact of NO-Related Agents on MK-801- or Scopolamine-Induced Cognitive Impairments in the Morris Water Maze

Learning and memory deficits accompany numerous brain dysfunctions, including schizophrenia and Alzheimer's disease (AD), and many studies point to the role of nitric oxide (NO) in these processes. The present investigations constitute the follow-up of our previous research, in which we investigated the activity of NO releasers and a selective inhibitor of neuronal NO synthase (nNOS) to prevent short-term memory deficits in novel object recognition and T-maze. Here, the ability of the compounds to prevent the induction of long-term memory deficits by MK-801 or scopolamine administration was investigated. The Morris Water Maze test, a reliable and valid test of spatial learning and memory, was used, in which escape latency in the acquisition phase and nine different parameters in the retention phase were measured. A fast NO releaser (spermine NONOate), a slow NO releaser (DETA NONOate), and a nNOS inhibitor, N(ω)-propyl-L-arginine (NPLA), were used. The compounds were administered i.p. at a dose range of 0.05-0.5 mg/kg. All compounds prevented learning deficits in the acquisition phase and reversed reference memory deficits in the retention phase of the scopolamine-treated mice. Spermine NONOate was the least effective. In contrast, the drugs poorly antagonised MK-801-induced deficits, and only the administration of DETA NONOate induced some improvements in the retention trial.

Role of nitric oxide on defensive behavior and long-term aversive learning induced by chemical stimulation of the dorsolateral periaqueductal gray matter

The midbrain periaqueductal gray matter, especially the dorsolateral portion (dlPAG), coordinates immediate defensive responses (DR) to threats, but also ascends forebrain information for aversive learning. The synaptic dynamics in the dlPAG regulate the intensity and type of behavioral expression, as well as long-term processes such as memory acquisition, consolidation, and retrieval. Among several neurotransmitters and neural modulators, nitric oxide seems to play an important regulatory role in the immediate expression of DR, but it remains unclear if this gaseous on-demand neuromodulator contributes to aversive learning. Therefore, the role of nitric oxide in the dlPAG was investigated, during conditioning in an olfactory aversive task. The behavioral analysis consisted of freezing and crouch-sniffing in the conditioning day after glutamatergic NMDA agonist injection into the dlPAG. Two days later, rats were re-exposed to the odor cue and avoidance was measured. 7NI, a selective neuronal nitric oxide synthase inhibitor (40 and 100 nmol), injected before NMDA (50 pmol) impaired immediate DR and consequent aversive learning. The scavenging of extrasynaptic nitric oxide by C-PTIO (1 and 2 nmol) induced similar results. Moreover, spermine NONOate, a nitric oxide donor (5, 10, 20, 40, and 80 nmol), produced DR by itself, but only the low dose also promoted learning. The following experiments utilized a fluorescent probe, DAF-FM diacetate (5 µM), directly into the dlPAG, to quantify nitric oxide in the three previous experimental situations. Nitric oxide levels were increased after NMDA stimulation, decreased after 7NI, and increased after spermine NONOate, in line with alterations in defensive expression. Altogether, the results indicate that nitric oxide plays a modulatory and decisive role in the dlPAG regarding immediate DR and aversive learning.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy.

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity, which triggers cell death in various neuropathological diseases, including epilepsy. Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold, that is, has an anticonvulsant effect. However, the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear. In this study, we performed RNA sequencing, functional enrichment analysis, and weighted gene coexpression network analysis of the hippocampus of tremor rats, a rat model of genetic epilepsy. We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity. In addition, we used a pilocarpine-induced N2a cell model to mimic epileptic injury. After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole, changes in malondialdehyde, lactate dehydrogenase and superoxide dismutase, which are associated with oxidative stress, were reversed, and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine. Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells. Furthermore, 7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3, gasdermin-D, interleukin-1β and interleukin-18. This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death. Taken together, our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells, and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Role of NO/cGMP/kATP pathway on diosgenin induced antinociceptive activity by formalin and hotplate induced model in rats

Diosgenin is a natural steroid sourced from plants and exhibits good analgesic and anti-inflammatory activity. However, mechanism of action associated with the analgesic activity of diosgenin is not yet delineated. Hence, in the present study, we explored the mechanism of analgesic activity of diosgenin on the nitric oxide/cyclic guanosine monophosphate/adenosine monophosphate sensitive potassium channels (NO/cGMP/kATP). The role of (NO/cGMP/kATP) in the Wistar rat was studied using formalin-induced models and hot plate models with various antagonists, such as N-nitro-L-arginine methyl ester hydrochloride (L NAME), glibenclamide, 1H-(1,2,4) oxadiazole (4,3-A) quinoxaline-1-one (ODQ) and 7-nitroindazole. Two doses of diosgenin were used in the study (25 and 50 mg/kg). Diosgenin reached its maximal effect (P ˂0.001) at (50 mg/kg po) in formalin as well as a hot plate induced nociception. This study has demonstrated that prior administration of the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole (0.1-1 mg/kg i.p.), glibenclamide, an ATP K+ channel inhibitor, L-NAME (10 mg/kg i.p.) and ODQ (2 mg/kg i.p.) significantly prevents the antinociceptive effect of diosgenin in formalin and hot plate induced nociception (50 mg/kg i.p administered 10 min after). It suggests that NO/cGMP/kATP has a significant role in the antinociceptive activity of diosgenin.

Pharmacological correction of the sequelae of acute alcohol-induced myocardial damage with new derivatives of neuroactive amino acids coupled with the blockade of the neuronal NO synthase isoform

Introduction: Acute alcohol intoxication (AAI) induces a number of myocardial disorders, which result in mitochon­drial dysfunction in cardiomyocytes, oxidative stress, and decreased cardiac contractility. Nitric oxide produced by the nNOS is one of the major modulators of cardiac activity. New derivatives of GABA (RSPU-260 compound) and glu­tamate (glufimet) can be potentially regarded as such agents as the interaction between the NO system and the GABA and glutamatergic systems has been proved.&#x0D; Materials and methods: All the studies were performed on female white Wistar rats, aged 10 months, whose weight was 280–320g AAI intoxication was modeled of 32% ethanol (gavage, 4g/kg).&#x0D; Results and discussion: Glufimet and the RSPU-260 compound caused a significant improvement in myocardial contractility, increased oxygen consumption in the V3 state according to Chance, raised the respiratory control ratio and decreased the intensity of LPO intensity. Their effectiveness exceeded that of mildronate, their comparator. nNOS inhibition resulted in a pronounced aggravation of oxidative stress implicated in MDA accumulation in cardiac mito­chondria and decreased activity of SOD; myocardial contractility and mitochondrial function indicators did not show a significant difference from the control group. The compounds under study coupled with nNOS inhibition had a car­dioprotective effect.&#x0D; Conclusion: Glufimet and the RSPU-260 compound, derivatives of neuroactive amino acids, have a pronounced cardioprotective effect, restrict LPO processes, enhance SOD activity, improve the mitochondrial respiratory function after acute alcohol intoxication when coupled with neuronal NO-synthase inhibition, the expression of which persists after AAI.&#x0D; Graphical abstract:

Neuronal nitric oxide synthase inhibition accelerated the removal of fluoxetine’s anxiogenic activity in an animal model of PTSD

While SSRIs are the current first-line pharmacotherapies against post-traumatic stress disorder (PTSD), they suffer from delayed onset of efficacy and low remission rates. One solution is to combine SSRIs with other treatments. Neuronal nitric oxide synthase (nNOS) has been shown to play a role in serotonergic signaling, and there is evidence of synergism between nNOS modulation and SSRIs in models of other psychiatric conditions. Therefore, in this study, we combined subchronic fluoxetine (Flx) with 7-nitroindazole (NI), a selective nNOS inhibitor, and evaluated their efficacy against anxiety-related behavior in an animal model of PTSD.We used the underwater trauma model to induce PTSD in rats. Animals underwent the open field (OFT) and elevated plus maze tests on days 14 (baseline) and 21 (post-treatment) after PTSD induction to assess anxiety-related behaviors. Between the two tests, the rats received daily intraperitoneal injections of 10 mg/kg Flx or saline, and were injected intraperitoneally before the second test with either 15 mg/kg NI or saline. The change in behaviors between the two tests was compared between treatment groups.Individual treatment with both Flx and NI had anxiogenic effects in the OFT. These effects were associated with modest increases in cFOS expression in the hippocampus. Combination therapy with Flx + NI did not show any anxiogenic effects, while causing even higher expression levels of cFOS.In conclusion, addition of NI treatment to subchronic Flx therapy accelerated the abrogation of Flx’s anxiogenic properties. Furthermore, hippocampal activity, as evidenced by cFOS expression, was biphasically related to anxiety-related behavior.

Nitric Oxide Linked to mGluR5 Upregulates BDNF Synthesis by Activating MMP2 in the Caudate and Putamen after Challenge Exposure to Nicotine in Rats.

Nitric oxide (NO) linked to glutamate receptors in the caudate and putamen (CPu) regulates neuroadaptation after drug exposure. Matrix-metalloproteinase (MMP), a Ca2+-dependent zinc-containing endopeptidase, increases mature brain-derived neurotrophic factor (BDNF) synthesis after drug exposure in the brain. The present study determined that NO synthesis linked to metabotropic glutamate receptor subtype 5 (mGluR5) stimulation after challenge exposure to nicotine activates MMP, which upregulates BDNF synthesis in the CPu. Subcutaneous injection of challenge nicotine (1.0 mg/kg) after repeated injections of nicotine (1.0 mg/kg/day) for 14 days and 7 days of nicotine withdrawal increased MMP2 activity and BDNF expression in the CPu of rats. These increases were prevented by the bilateral intra-CPu infusion of the mGluR5 antagonist, MPEP (0.1 nmol/side), the IP3 receptor antagonist, xestospongin C (0.004 nmol/side) or the neuronal nitric oxide synthase (nNOS) and NO inhibitor, Nω-propyl (0.1 nmol/side) prior to the challenge nicotine. Furthermore, bilateral intra-CPu infusion of the MMP2 inhibitor, OA-Hy (1 nmol/side) prevented the challenge nicotine-induced increase in the expression of BDNF. These findings suggest that elevation of NO synthesis linked to mGluR5 potentiates BDNF synthesis via activation of MMP2 after challenge exposure to nicotine in the CPu of rats.

The role of the L-arginine-NO-cGMP-dependent pathway in the development of sensitization to mephedrone effects on the locomotor activity in mice

Mephedrone, a popular psychostimulating substance widely used illegally in recreational purposes, exerts in rodents that regularly and intermittently were exposed to it a sensitized response to the drug. Behavioral sensitization is one of experimental models of drug dependency/abuse liability. In the present study we evaluated a potential involvement of the L-arginine-NO-cGMP pathway in the development of sensitization to the mephedrone-induced hyperlocomotion. Locomotor activity was measured automatically and experiments were performed on male Albino Swiss mice. We demonstrated that a 5-day administration of 7-nitroindazole (10 or 20 mg/kg/day) and L-NAME (50 mg/kg/day) suppressed the development of sensitization to the mephedrone-induced hyperlocomotion. As for L-arginine (125 or 250 mg/kg/day) and methylene blue (5 or 10 mg/kg/day) the obtained outcomes are inconclusive. Furthermore, the lower dose of L-NAME (25 mg/kg/day) surprisingly potentiated the development of sensitization to the mephedrone-induced effects on the spontaneous locomotor activity in mice. In conclusion, our data demonstrated that modulators of the L-arginine-NO-cGMP pathway may differently affect the development of sensitization to the locomotor stimulant effects of mephedrone. Inhibition of neuronal nitric oxide synthase (NOS) seems to prevent this process quite profoundly, non-selective inhibition of NOS may have a dual effect, whereas inhibition of soluble guanylate cyclase may only partially suppress the development of sensitization to the mephedrone-induced effects.