nNOS Inhibitor Research Articles

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.

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.

Abstract P3133: Role Of Myofilament-associatedNeuronal NOSFor Ca 2+ Desensitization Via Troponin I Phosphorylation In The Right Ventricular Cardiomyocytes Of Rats

This study aimed to compare the role of neuronal nitric oxide synthase (nNOS, NOS1) in the excitation-contraction coupling and myofilament Ca 2+ -sensitivity between left ventricular cardiomyocytes (LVCMs) and right ventricular cardiomyocytes (RVCMs). Previous research had only investigated the effects of nNOS on LVCMs, but our study focused on RVCMs which have slightly larger diastolic sarcomere length (SL D ) and smaller sarcomere shortening (ΔSL) than LVCMs. Immunoblot study showed that the total expression and phosphorylated Ser 1417 ratio of nNOS were not different between RVCMs and LVCMs. However, treating RVCMs with the nNOS-specific inhibitor, S-methyl-L-thiocitrulline (SMTC), resulted in more prominent augmentation of ΔSL and shortened SL D in RVCMs, along with a paradoxical decrease of the amplitude of Ca 2+ transient (Δ[Ca 2+ ] i ) due to a more significant inhibition of L-type Ca 2+ current in RVCMs. The Ca 2+ -contraction loop showed a more leftward shift in the RVCMs by nNOS inhibition, indicating Ca 2+ desensitization of the myofilaments by the intrinsic nNOS. Previous studies offered troponin I (cTnI) phosphorylation as the regulatory target of myofilament Ca 2+ -sensitivity through the sGC-cGMP-PKG pathway downstream to NO. The SMTC treatment decreased cTnI phosphorylation in the RVCMs, while not in the LVCMs, suggesting that nNOS plays a more prominent role in myofilament Ca 2+ desensitization in RVCMs by targeting cTnI. Additionally, the higher phosphorylation of nNOSβ, the isoform of nNOS expressed in the myofilaments, was detected in the myofilament of RVCMs than LVCMs. This study is the first to demonstrate the more significant role of nNOS in RVCMs in regulating myofilament Ca 2+ desensitization through cTnI.

IC87201, a PSD-95/nNOS Inhibitor, Ameliorates Heart Rate Variability in the Rat Model of Middle Cerebral Artery Occlusion

Objective: Assessment of heart rate variability (HRV) is a non-invasive and reliable method to evaluate autonomic disorders after cerebral ischemia. The present study was conducted to investigate the therapeutic potential of IC87201 in reducing post-stroke cardiac dysfunction. Materials and methods: Cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) method in 15 anesthetized adult male rats in three MCAO, MCAO+ DXM, and MCAO+ IC87201 groups, for one hour. Electrocardiogram was recorded before, and 48 hours after ischemia and drug administration, and HRV parameters were calculated from R-R intervals. In the treatment groups, IC87201 and Dextromethorphan hydrobromide monohydrate (DXM) were injected after an ischemic period. Results: After brain ischemia, the R-R interval decreased and consequently heart rate increased. The R-R intervals were used to extract the HRV frequency and time domains, including normalized low frequency (LF), high frequency (HF), LF/HF ratio, and standard deviation of R-R interval (SDRR). Normalized LF and LF/HF ratio enhanced 48 hours after ischemia, while normalized HF and SDRR significantly reduced compared to the pre-ischemic state. All HRV parameters had returned to their pre-ischemic level 48 hours after IC87201 and DXM administration, except SDRR, which recovered only in the IC87201 administered group. Conclusion: Based on our findings, it can be concluded that cerebral ischemia significantly worsens HRV parameters as a result of sympathetic overactivity. These changes were reversed by administering DXM and IC87201, but IC87201 has generally been more effective in lowering lesions. As a result, IC87201 can be introduced as an effective substance for the treatment of post-ischemic cardiac side effects.

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.

Butyrate inhibits LPC-induced endothelial dysfunction by regulating nNOS-produced NO and ROS production

Lipids oxidation is a key risk factor for cardiovascular diseases. Lysophosphatidylcholine (LPC), the major component of oxidized LDL, is an important triggering agent for endothelial dysfunction and atherogenesis. Sodium butyrate, a short-chain fatty acid, has demonstrated atheroprotective properties. So, we evaluate the role of butyrate in LPC-induced endothelial dysfunction. Vascular response to phenylephrine (Phe) and acetylcholine (Ach) was performed in aortic rings from male mice (C57BL/6J). The aortic rings were incubated with LPC (10 μM) and butyrate (0.01 or 0.1 Mm), with or without TRIM (an nNOS inhibitor). Endothelial cells (EA.hy296) were incubated with LPC and butyrate to evaluate nitric oxide (NO) and reactive oxygen species (ROS) production, calcium influx, and the expression of total and phosphorylated nNOS and ERK½. We found that butyrate inhibited LPC-induced endothelial dysfunction by improving nNOS activity in aortic rings. In endothelial cells, butyrate reduced ROS production and increased nNOS-related NO release, by improving nNOS activation (phosphorylation at Ser1412). Additionally, butyrate prevented the increase in cytosolic calcium and inhibited ERk½ activation by LPC. In conclusion, butyrate inhibited LPC-induced vascular dysfunction by increasing nNOS-derived NO and reducing ROS production. Butyrate restored nNOS activation, which was associated with calcium handling normalization and reduction of ERK½ activation.

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.

Acute and Sub-chronic Anticonvulsant Effects of Edaravone on Seizure Induced by Pentylenetetrazole or Electroshock in Mice, Nitric Oxide Involvement.

Edaravone is an anti-stroke medication that may have nitric oxide (NO) modulating properties. This study evaluated the role of NO in the acute and sub-chronic anticonvulsant effects of edaravone in murine models of seizures induced by intraperitoneal (IP) or intravenous (IV) injections of pentylenetetrazole (PTZ) or electroshock (maximal electroshock seizure [MES]). 132 male albino mice were randomly divided into 22 groups (n=6) and given IP injections of vehicle or edaravone either acutely or for eight days (sub-chronically). The seizure was induced by electroshock or PTZ (IP or IV). The following edaravone doses were used: 7.5, 10, 12.5 (acute); 5, 7.5, 10 (sub-chronic) in IP PTZ model; 5, 7.5, 10 in IV PTZ model; and 5, 10 mg/Kg in the MES. To evaluate NO involvement, 216 mice were randomly divided into 36 groups (n=6) and pretreated with vehicle, edaravone, a non-specific nitric oxide synthase (NOS) inhibitor: N(ω)-nitro-L-arginine methyl ester (L-NAME) (5 mg/Kg), a specific nNOS inhibitor: 7-nitroindazole (7-NI) (60 mg/Kg), or a combination of edaravone plus L-NAME or 7-NI, either acutely or for eight days before seizure induction. Doses of edaravone were as follows: in IP PTZ model: 12.5 (acute) and 10 (sub-chronic); in IV PTZ model: 10; and in the MES: 5 mg/Kg. Data were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS 18). P≤0.05 was considered statistically significant. In the IP PTZ model, edaravone increased time latencies to seizures (P<0.001), prevented tonic seizures, and death. Edaravone increased the seizure threshold (P<0.001) in the IV PTZ model and shortened the duration of tonic hind-limb extension (THE) in the MES model (P<0.001). In comparison to mice treated with edaravone alone, adding L-NAME or 7-NI reduced seizure time latencies (P<0.001), reduced seizure threshold (P<0.001), and increased THE duration (P<0.001). Edaravone (acute or sub-chronic) could prevent seizures by modulating NO signaling 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.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Adrenomedullin in paraventricular nucleus attenuates adipose afferent reflex and sympathoexcitation via receptors mediated nitric oxide-gamma-aminobutyric acid A type receptor pathway in rats with obesity-related hypertension.

Hypothalamic paraventricular nucleus (PVN) is an important central site for the control of the adipose afferent reflex (AAR) that increases sympathetic outflow and blood pressure in obesity-related hypertension (OH). In this study, we investigated the effects of nitric oxide (NO) and cardiovascular bioactive polypeptide adrenomedullin (ADM) in the PVN on AAR and sympathetic nerve activity (SNA) in OH rats induced by a high-fat diet. The results showed that ADM, total neuronal NO synthase (nNOS) and phosphorylated-nNOS protein expression levels in the PVN of the OH rats were down-regulated compared to the control rats. The enhanced AAR in OH rats was attenuated by PVN acute application of NO donor sodium nitroprusside (SNP), but was strengthened by the nNOS inhibitor nNOS-I, guanylyl cyclase inhibitor (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) and gamma-aminobutyric acid A type receptor (GABAA) antagonist Bicuculline. Moreover, PVN ADM microinjection not only decreased basal SNA but also attenuated the enhanced AAR in OH rats, which were effectively inhibited by ADM receptor antagonist ADM22-52, nNOS-I, ODQ or Bicuculline pretreatment. Bilateral PVN acute microinjection of ADM also caused greater increases in NO and cyclic guanosine monophosphate (cGMP) levels, and nNOS phosphorylation. Adeno-associated virus vectors encoding ADM (AAV-ADM) transfection in the PVN of OH rats not only decreased the elevated AAR, basal SNA and blood pressure (BP), but also increased the expression and activation of nNOS. Furthermore, AAV-ADM transfection improved vascular remodeling in OH rats. Taken together, our data highlight the roles of ADM in improving sympathetic overactivation, enhanced AAR and hypertension, and its related mechanisms associated with receptors mediated NO-cGMP-GABAA pathway in OH condition.

Inhibition of nitric oxide synthase or cystathionine gamma-lyase abolishes leptin-induced fever in male rats

Leptin is an adipokine that regulates energy balance and immune function. Peripheral leptin administration elicits prostaglandin E₂-dependent fever in rats. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H₂S) are also involved in lipopolysaccharide (LPS)-induced fever response. However, there is no data in the literature indicating if these gasotransmitters have a role in leptin-induced fever response. Here, we investigate the inhibition of NO and H₂S enzymes neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and cystathionine γ-lyase (CSE) in leptin-induced fever response, respectively. Selective nNOS inhibitor 7-nitroindazole (7-NI), selective iNOS inhibitor aminoguanidine (AG), and CSE inhibitor dl-propargylglycine (PAG) were administered intraperitoneally (ip). Body temperature (Tb), food intake, and body mass were recorded in fasted male rats. Leptin (0.5 mg/kg ip) induced a significant increase in Tb, whereas AG (50 mg/kg ip), 7-NI (10 mg/kg ip), or PAG (50 mg/kg ip) caused no changes in Tb. AG, 7-NI, or PAG abolished leptin increase in Tb. Our results highlight the potential involvement of iNOS, nNOS, and CSE in leptin-induced febrile response without affecting anorexic response to leptin in fasted male rats 24 h after leptin injection. Interestingly, all the inhibitors alone had the same anorexic effect induced by leptin. These findings have implications for understanding the role of NO and H₂S in leptin-induced febrile response.

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.

Neuronal Nitric Oxide Synthase Regulates Depression-like Behaviors in Shortening-Induced Obese Mice.

Shortening is mainly derived from the partial hydrogenation of palm oil and widely used in fast food. Food processed with shortening contains high levels of industrial trans fatty acids. Studies have shown that there is a correlation between industrial trans fatty acids, obesity, and depression. However, the regulatory effect of neuronal nitric oxide synthase (nNOS) on depression in obese patients is still unknown. The purpose of this study was to explore mood changes in obese mice fed a high shortening diet, and to determine the regulatory effect of nNOS on depressive-like behaviors in obese mice. We used a high shortening diet-induced obesity mouse model to systematically assess the metabolic response, behavioral changes, prefrontal and hippocampal nNOS protein levels, and the effect of nNOS inhibitors (7-nitroindole) on depression-like behavior in obese mice. Interestingly, obese mice on a 9-week high-shortening diet developed short-term spatial working memory impairment and anxiety-like behavior, and obesity may be a risk factor for cognitive impairment and mood disorders. In animals fed a high shortening diet for 12 weeks, obese mice developed depression-like behavior and had significantly elevated levels of nNOS protein expression in the hippocampus and prefrontal lobe. Administration of the nNOS inhibitor 7-nitroindole could improve depression-like behaviors in obese mice, further suggesting that inhibition of nNOS is helpful for depression associated with obesity.

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.

N-isopropylbenzylamine, a methamphetamine mimics, produces toxicity via increasing nitric oxide in vitro

N-isopropylbenzylamine, an isomer of methamphetamine, has been used to adulterate methamphetamine, and distributed as fake “Ice” methamphetamine by illicit manufacturers, leading to a world problem of N-isopropylbenzylamine exposure. Though it is unclear whether N-isopropylbenzylamine has addictive potential like methamphetamine, N-isopropylbenzylamine users reported side effects such as headaches and confusion. However, the pharmacological targets and cytotoxicity of this chemical remained unknown. In this study, in vitro toxicity of N-isopropylbenzylamine and its toxicity-related targets were investigated in SN4741, SH-SY5Y or PC12 cell lines that model neurons. The cell viability was analyzed by using MTT assay after incubation with N-isopropylbenzylamine for 24 h in cells. N-isopropylbenzylamine caused cell death with IC50 values at around 1–3 mM in these cell lines. N-isopropylbenzylamine time- and concentration-dependently facilitated the expression of neuronal nitric oxide synthase (nNOS), and increased intracellular nitric oxide (NO) in SN4741 cells. Furthermore, 7-nitroindazole, a specific inhibitor of nNOS, significantly prevented N-isopropylbenzylamine-induced toxicity in vitro. These results suggested that N-isopropylbenzylamine-induced toxicity is at least partially related to the increased intracellular NO levels and the activated nNOS. Considering the circumstances that N-isopropylbenzylamine was used to adulterate and mimic methamphetamine, and the side effects associated with N-isopropylbenzylamine in abusers, our findings sounded an alarm for abuser and warn the dangerousness of N-isopropylbenzylamine for public health.

Pharmacological nNOS inhibition modified small-conductance Ca2+-activated K+ channel without altering Ca2+ dynamics.

Atrial fibrillation (AF) is associated with electrical remodeling processes that promote a substrate for the maintenance of AF. Although the small-conductance Ca2+-activated K+ (SK) channel is a key factor in atrial electrical remodeling, the mechanism of its activation remains unclear. Regional nitric oxide (NO) production by neuronal nitric oxide synthase (nNOS) is involved in atrial electrical remodeling. In this study, atrial tachyarrhythmia (ATA) induction and optical mapping were performed on perfused rat hearts. nNOS is pharmacologically inhibited by S-methylthiocitrulline (SMTC). The influence of the SK channel was examined using a specific channel inhibitor, apamin (APA). Parameters such as action potential duration (APD), conduction velocity, and calcium transient (CaT) were evaluated using voltage and calcium optical mapping. The dominant frequency was examined in the analysis of AF dynamics. SMTC (100 nM) increased the inducibility of ATA and apamin (100 nM) mitigated nNOS inhibition-induced arrhythmogenicity. SMTC caused abbreviations and enhanced the spatial dispersion of APD, which was reversed by apamin. By contrast, conduction velocity and other parameters associated with CaT were not affected by SMTC or apamin administration. Apamin reduced the frequency of SMTC-induced ATA. In summary, nNOS inhibition abbreviates APD by modifying the SK channels. A specific SK channel blocker, apamin, mitigated APD abbreviation without alteration of CaT, implying an underlying mechanism of posttranslational modification of SK channels.NEW & NOTEWORTHY We demonstrated that pharmacological nNOS inhibition increased the atrial arrhythmia inducibility and a specific small-conductance Ca2+-activated K+ channel blocker, apamin, reversed the enhanced atrial arrhythmia inducibility. Apamin mitigated APD abbreviation without alteration of Ca2+ transient, implying an underlying mechanism of posttranslational modification of SK channels.

Targeting neuronal nitric oxide synthase and the nitrergic system in post-traumatic stress disorder

Current pharmacological approaches to treatment of post-traumatic stress disorder (PTSD) lack adequate effectiveness. As a result, identifying new molecular targets for drug development is necessary. Furthermore, fear learning and memory in PTSD can undergo different phases, such as fear acquisition, consolidation, and extinction. Each phase may involve different cellular pathways and brain regions. As a result, effective management of PTSD requires mindfulness of the timing of drug administration. One of the molecular targets currently under intense investigation is the N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR). However, despite the therapeutic efficacy of drugs targeting NMDAR, their translation into clinical use has been challenging due to their various side effects. One possible solution to this problem is to target signaling proteins downstream to NMDAR to improve targeting specificity. One of these proteins is the neuronal nitric oxide synthase (nNOS), which is activated following calcium influx through the NMDAR. In this paper, we review the literature on the pharmacological modulation of nNOS in animal models of PTSD to evaluate its therapeutic potential. Furthermore, we attempt to decipher the inconsistencies observed between the findings of these studies based on the specific phase of fear learning which they had targeted. Inhibition of nNOS may inhibit fear acquisition and recall, while not having a significant effect on fear consolidation and extinction. However, it may improve extinction consolidation or reconsolidation blockade. Modulation of nNOS has therapeutic potential against PTSD and warrants further development for use in the clinical setting.

Effects of mangiferin and its combination with nNOS inhibitor 7-nitro-indazole (7-NI) in 6-hydroxydopamine (6-OHDA) lesioned Parkinson's disease rats.

Nitric oxide (NO), generated by nitric oxide synthase enzymes (NOS), has an important role in maintaining synapse plasticity, neuro-modulation, and other physiological functions in the brain. NO thus generated also has a key role in formation of reactive oxygen and reactive nitrite species and subsequent neuronal damage due to sustained oxidative stress. Due to its property of ROS and RNOS generation, NO plays a significant role in Parkinson's disease (PD) pathogenesis. Therefore, we evaluated the effect of mangiferin alone and in combination with nNOS inhibitor 7-nitro-indazole (7-NI) in 6-OHDA lesioned rats. Male Wistar rats weighing 200-250 g (n = 8/group) were used in the study. Stereotactic surgeries of rats were done to induce 6-OHDA lesioning in rats. Then, treatment with mangiferin alone and in combination with 7-NI 10mg/kg was done from days 14 to 42 for 28 days. On day 42, rats were subjected to behavioral studies, and their brains were taken out after euthanasia to perform biochemical and molecular studies. Treatment with mangiferin and 7-NI significantly increases locomotor parameters in 6-OHDA lesioned rats. Treatment with mangiferin 45 μg and 7-NI 10mg/kg alone and in combination significantly reduces oxidative stress along with decrease in concentration of pro-inflammatory cytokines, cyclooxygenase 2, total nitrite (NOx) and FOS B concentration. Results of this study suggest that treatment with 7-NI 10mg/kg further enhances anti-inflammatory and anti-parkinsonism activity of mangiferin owing to its property of inhibiting nNOS mediated FOS B signaling and thereby inhibiting mRNA formation of TNF-α and IL-6.