Neuronal Nitric Oxide Synthase Research Articles

Modulatory effect of olanzapine on neuronal nitric oxide synthase (nNOS) expression in the rat striatum.

Nitric oxide (NO) has been thought to be a novel factor involved in the mechanisms of mental disorders pathogenesis for quite some time. However, little is known about potential crosstalk between neuronal NO signaling and neuroleptics action. The present work was, therefore, focused on gene expression of neuronal NO synthase (nNOS) in the brains of rats chronically treated with olanzapine, an atypical antipsychotic drug. Studies were carried out on adult, male Sprague-Dawley rats that were divided into 2 groups: control and experimental animals treated with olanzapine (28-day-long intraperitoneal injection, at dose 5mg/kg daily). All individuals were killed under anesthesia and the whole brains excised. Immunohistochemical procedure was used for histological assessment of the whole brain, and for both descriptive and quantitative analysis of nNOS protein distribution in selected brain structures. Long-term treatment with olanzapine is reflected in different changes in the number of enzyme-expressing cells in the rat brain. Olanzapine decreased the number of nNOS-expressing cells and possibly reduced NO synthesis in the rat striatum. Olanzapine can be taken into account as a potential inhibitor of NO synthesis in the rat striatum.

Gene therapy delivered micro-dystrophins co-localize with transgenic utrophin in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (μDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (μDysH3) or AAV6-CK8e-μDys5 (μDys5). We used immunofluorescence to qualitatively assess the localization of μDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. μDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with μDys5, but not with transgenic utrophin expression or μDysH3. Transgenic utrophin expression and μDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of β-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.

Pharmacokinetics, biodistribution, and in vivo toxicity of 7-nitroindazole loaded in pegylated and non-pegylated nanoemulsions in rats

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. The development of sepsis is associated with excessive nitric oxide (NO) production, which plays an important role in controlling vascular homeostasis. 7-nitroindazole (7-NI) is a selective inhibitor of neuronal nitric oxide synthase (NOS-1) with potential application for treating NO imbalance conditions. However, 7-NI exhibits a low aqueous solubility and a short plasma half-life. To circumvent these biopharmaceutical limitations, pegylated (NEPEG7NI) and non-pegylated nanoemulsions (NENPEG7NI) containing 7-NI were developed. This study evaluates the pharmacokinetic profiles and toxicological properties of 7-NI loaded into the nanoemulsions. After a single intravenous administration of the free drug and the nanoemulsions at a dose of 10 mg.kg−1 in Wistar rats, 7-NI was widely distributed in the organs. The pharmacokinetic parameters of Cmax, t1/2, and AUC0-t were significantly increased after administration of the NEPEG7NI, compared to both free 7-NI and NENPEG7NI (p < 0.05). No observable adverse effects were observed after administering the free 7-NI, NEPEG7NI, or NENPEG7NI in the animals after a single dose of up to 3.0 mg.kg−1. The results indicated that 7-NI-loaded nanoemulsions are safe, constituting a promising approach to treating sepsis.

Evaluation of serum nitric oxide synthase levels in patients with coronary slow flow based on corrected TIMI frame count.

The coronary slow flow phenomenon (CSFP) finding in angiography is characterized by the delayed filling of the terminal vessels without significant epicardial coronary disease. The endothelium performs a vital role in cardiovascular homeostasis by releasing vasoactive substances. Endothelial cells produce nitric oxide (NO) as one of these essential compounds. Three isoforms of nitric oxide synthase (NOS) are endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and induced nitric oxide synthase (iNOS). We aimed to determine the role of NOS in the development of CSFP as the first human study. A total of 129 patients who met the inclusion criteria were enrolled in the study. The patients were classified into five groups based on the results of coronary angiography: Group 1 without coronary artery disease (CAD) and without CSF, group 2 without CAD and with CSF, group 3 with CAD (< 50%) and without CSF, group 4 with CAD (50-90%) and without CSF, and group 5 with CAD and CSF. The serum level of NOS was determined in the participants. Coronary flow was quantified in patients with CSFP using the corrected TIMI frame count (CTFC) method, and the correlation between the levels of this biomarker and CTFC was investigated. In this study, the NOS serum levels were not significantly correlated with the mean CTFC. Since the total amount of NOS was measured as a result of 3 isoforms of this enzyme, the lack of correlation could be related to increased iNOS level and decreased eNOS concentration. These results should be confirmed by more human studies.

Zingerone attenuates sciatic nerve damage caused by sodium arsenite by inhibiting NF-κB, caspase-3, and ATF-6/CHOP pathways and activating the Akt2/FOXO1 pathway.

In the present study, the potential protective effects of zingerone (ZNG) against sciatic nerve damage caused by sodium arsenite (SA), a common environmental pollutant, were evaluated by various biochemical, molecular, and histological methods. In the study, SA and ZNG were given to 35 male Sprague Dawley rats for 14 days. At the end of the period, the sciatic nerve tissues were taken and the markers involved in oxidative stress, endoplasmic reticulum stress, inflammation, and apoptosis were analyzed. The data obtained showed that SA decreased glutathione (GSH) levels and increased malondialdehyde (MDA) levels in the sciatic nerve tissue. However, it was determined that these markers approached the control group levels due to the anti-oxidant properties of ZNG. While SA triggered endoplasmic reticulum stress and apoptosis pathways, ZNG suppressed them. Moreover, SA up-regulated inflammatory markers such as nuclear factor kappa-B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), and neuronal nitric oxide synthases (nNOS) in the sciatic nerves and caused neuro-inflammation and inhibited cell survival by suppressing serine/threonine-protein kinase 2 (Akt2) and forkhead box protein O1 (FOXO1) genes. It has also been shown histopathologically that SA causes degeneration in the sciatic nerves. In contrast, ZNG suppressed neuro-inflammation, activated Akt2/FOXO1 signaling, and repaired histological irregularities. In general, SA caused oxidative stress, inflammation, ER stress, and apoptosis in the sciatic nerves of rats, causing damage to the tissues, however, ZNG suppressed these pathways and protected the sciatic nerves from the destructive effect of SA.

Impact of intravascular hemolysis on functional and molecular alterations in the urinary bladder: implications for an overactive bladder in sickle cell disease.

Patients with sickle cell disease (SCD) display an overactive bladder (OAB). Intravascular hemolysis in SCD is associated with various severe SCD complications. However, no experimental studies have evaluated the effect of intravascular hemolysis on bladder function. This study aimed to assess the effects of intravascular hemolysis on the micturition process and the contractile mechanisms of the detrusor smooth muscle (DSM) in a mouse model with phenylhydrazine (PHZ)-induced hemolysis; furthermore, it aimed to investigate the role of intravascular hemolysis in the dysfunction of nitric oxide (NO) signaling and in increasing oxidative stress in the bladder. Mice underwent a void spot assay, and DSM contractions were evaluated in organ baths. The PHZ group exhibited increased urinary frequency and increased void volumes. DSM contractile responses to carbachol, KCl, α-β-methylene-ATP, and EFS were increased in the PHZ group. Protein expression of phosphorylated endothelial NO synthase (eNOS) (Ser-1177), phosphorylated neuronal NO synthase (nNOS) (Ser-1417), and phosphorylated vasodilator-stimulated phosphoprotein (VASP) (Ser-239) decreased in the bladder of the PHZ group. Protein expression of oxidative stress markers, NOX-2, 3-NT, and 4-HNE, increased in the bladder of the PHZ group. Our study shows that intravascular hemolysis promotes voiding dysfunction correlated with alterations in the NO signaling pathway in the bladder, as evidenced by reduced levels of p-eNOS (Ser-1177), nNOS (Ser-1417), and p-VASP (Ser-239). The study also showed that intravascular hemolysis increases oxidative stress in the bladder. Our study indicates that intravascular hemolysis promotes an OAB phenotype similar to those observed in patients and mice with SCD.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites β-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

Investigation of the Effect of Pumpkin (Cucurbita pepo L.) Seed Oil on Pentylenetetrazole-induced Neuronal Damage in HT-22 Cell Line

Recent studies have shown the positive effects of Cucurbita pepo L. (pumpkin) seed oil (PSO) in different disease models. However, the effect of PSO on neurological diseases has not been clarified yet. Therefore, this study aims to elucidate the effects of BBS on pentylenetetrazole (PTZ)-induced neuronal damage and the possible roles of oxidative and nitrosative stress in this effect in vitro. The HT-22 hippocampal neuronal cell line was used in the study. Cell survival after PTZ-induced neuronal damage was evaluated with the XTT test in the groups. While the effects of BBS on total antioxidant status (TAS) and total oxidant status (TOS) after PTZ were measured with colorimetric commercial kits, its effects on neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) levels were also determined by ELISA kits. In light of the data obtained, it was found that pre-treatment with PSO prevented the decrease in cell survival after exposure to PTZ. In addition, it has been found that PSO normalizes the increase in TOS, nNOS, and NO in neuronal cells after PTZ. As a result, it was determined that the treatment of neuronal cells with PSO prevented neuronal damage caused by PTZ and showed neuroprotective properties. It is thought that PSO may achieve these effects through oxidative and nitrosative systems. Enrichment of a daily diet with PSO might be beneficial in reducing the risks of neurological diseases.

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 the 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.

"NO" Time in Fear Response: Possible Implication of Nitric-Oxide-Related Mechanisms in PTSD.

Post-traumatic stress disorder (PTSD) is a psychiatric condition characterized by persistent fear responses and altered neurotransmitter functioning due to traumatic experiences. Stress predominantly affects glutamate, a neurotransmitter crucial for synaptic plasticity and memory formation. Activation of the N-Methyl-D-Aspartate glutamate receptors (NMDAR) can trigger the formation of a complex comprising postsynaptic density protein-95 (PSD95), the neuronal nitric oxide synthase (nNOS), and its adaptor protein (NOS1AP). This complex is pivotal in activating nNOS and nitric oxide (NO) production, which, in turn, activates downstream pathways that modulate neuronal signaling, including synaptic plasticity/transmission, inflammation, and cell death. The involvement of nNOS and NOS1AP in the susceptibility of PTSD and its comorbidities has been widely shown. Therefore, understanding the interplay between stress, fear, and NO is essential for comprehending the maintenance and progression of PTSD, since NO is involved in fear acquisition and extinction processes. Moreover, NO induces post-translational modifications (PTMs), including S-nitrosylation and nitration, which alter protein function and structure for intracellular signaling. Although evidence suggests that NO influences synaptic plasticity and memory processing, the specific role of PTMs in the pathophysiology of PTSD remains unclear. This review highlights pathways modulated by NO that could be relevant to stress and PTSD.

Dual Strategy with Adipose-Derived Stem Cells and l-arginine Recovered Cavernosal Functions in a Rat Model of Radical Prostatectomy.

As standard therapy for prostate cancer, radical prostatectomy causes cavernous nerve (CN) injury and increases fibrosis and hypoxia-induced penile structural alterations. This study aimed to determine the potential beneficial effects of adipose-derived stem cells (ADSCs) and l-arginine alone or in combination on the penile erection in a rat model of erectile dysfunction caused by bilateral cavernous nerve transection (CNT). Male rats (n = 35) were randomized into five groups: Sham-operated; CNT (4-weeks); CNT plus ADSCs (1 × 106 cells by intracavernosal injection); CNT plus l-arginine (4 weeks, 10 mg/kg/day, oral); and ADSCs combined with l-arginine in CNT. In vivo erectile responses and in vitro relaxant responses were measured. Western blot and immunohistochemistry analyses were used to determine the expression and localization of endothelial nitric oxide synthase, neuronal nitric oxide synthase, transforming growth factor-beta 1, hypoxia-inducible factor-1 alpha (HIF-1α), and apoptosis markers (Bax and Bcl-2). The ratio of smooth muscle to collagen and nerve regeneration were calculated using Masson's trichrome and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. The combined treatment restored diminished erectile responses, endothelium-dependent acetylcholine, and electrical field stimulation-induced relaxation of the corpus cavernosum in rats with CNT, whereas either monotherapy produced only partial improvements. All treatment regimens restored increases in the protein expression of HIF-1 and Bax in rats with CNT. The decrease in smooth muscle mass and NADPH-diaphorase-positive nerve fibers was partially ameliorated by monotherapy, whereas combined therapy led to recovery. These findings indicate that combined treatment with ADSCs and l-arginine may restore erectile function in rats with CNT by inhibiting hypoxia-induced neurotoxicity and preserving endothelium function and smooth muscle content.

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.

Modification-Specific Proteomic Analysis Reveals Cysteine S-Nitrosylation Mediated the Effect of Preslaughter Transport Stress on Pork Quality Development.

This study aimed to explore the effects of preslaughter transport stress on protein S-nitrosylation levels and S-nitrosylated proteome in post-mortem pork longissimus thoracis (LT) muscle. Pigs (N= 16) were randomly divided into 3 h transport (high-stress group, HS) and 3 h transport followed by 3 h resting treatments (low-stress control group, LS). Results demonstrated that high transport stress levels induced nitric oxide (NO) overproduction by promoting NO synthase (NOS) activity and neuronal NOS (nNOS) expression, which thereby notably increased protein S-nitrosylation levels in post-mortem muscle (p < 0.05). Proteomic analysis indicated that 133 S-nitrosylation-modified cysteines belonging to 85 proteins were significantly differential, of which 101 cysteines of 63 proteins were higher in the HS group (p < 0.05). Differential proteins including cytoskeletal and calcium-handling proteins, glycolytic enzymes, and oxidoreductase were mainly involved in the regulation of muscle contraction and energy metabolism that might together mediate meat quality development. Overall, this study provided direct evidence for changes in S-nitrosylation levels and proteome in post-mortem muscle in response to preslaughter transport stress and revealed the potential impact of S-nitrosylated proteins on meat quality.

The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens

6-Nitrodopamine (6-ND) is released from rat and human vas deferens and is considered a major mediator of both tissues contractility. The contractions induced by 6-ND are selectively blocked by both tricyclic antidepressants and α1-adrenoceptor antagonists. Endothelial nitric oxide synthase (eNOS) is the major isoform responsible for 6-ND release in mouse isolated heart, however the origin of 6-ND in the vas deferens is unknown. Here it was investigated by LC-MS/MS the basal release of 6-ND from isolated vas deferens obtained from control, eNOS−/−, nNOS−/−, and iNOS−/− mice. In addition, it was evaluated in vitro vas deferens contractility following electric field stimulation (EFS).Basal release of 6-ND was significantly reduced in nNOS−/− mice compared to control mice, but not decreased when the vas deferens were obtained from either eNOS−/− or iNOS−/− mice. Pre-incubation of the vas deferens with tetrodotoxin (1 μM) significantly reduced the basal release of 6-ND from control, eNOS−/−, and iNOS−/− mice but had no effect on the basal release of 6-ND from nNOS−/− mice. EFS-induced frequency-dependent contractions of the vas deferens, which were significantly reduced when the tissues obtained from control, eNOS−/− and iNOS−/− mice, were pre-incubated with l-NAME, but unaltered when the vas deferens was obtained from nNOS−/− mice. In addition, the EFS-induced contractions were significantly smaller when the vas deferens were obtained from nNOS−/− mice.The results clearly demonstrate that nNOS is the main NO isoform responsible for 6-ND release in mouse vas deferens and reinforces the concept of 6-ND as a major modulator of vas deferens contractility.

Inhibition of nitric oxide synthase modulates learned helplessness response in female rats

Learned helplessness is the condition of humans or animals that have learned to behave helplessly, failing to respond even though there are opportunities to help themselves by avoiding unpleasant circumstances. It was investigated the participation of nitric oxide (NO) in female Wistar rats in learned helplessness tests. The animals received acute treatment with N–nitro-l-arginine methyl ester (L-NAME, 10 mg/kg or 100 mg/kg), an inhibitor of NO synthase, 30 minutes before random inescapable shock sessions lasting 25 minutes. On the test day, the animals that received L-NAME of 10 mg/kg or 100 mg/kg 24 hours beforehand, demonstrated reduced difference between stress and non-stress groups compared with control groups. The activity of the neuronal nitric oxide synthase was lower in animals pretreated with L-NAME, especially at a dose of 100 mg/kg. These results indicate that acute inhibition of NO synthase improved learned helplessness response, suggesting that NO is involved with stress and depression.

The neuronal nitric oxide synthase expression increases during satellite cell-derived primary myoblasts differentiation.

Theneuronal nitric oxide synthase (nNOS; encoded byNOS1)-derivednitric oxide (NO) plays an important role in maintaining skeletal muscle mass. In adult skeletal muscle, nNOS localizes to the cell membrane, cytosol, and nucleus, and regulates muscle hypertrophy and atrophy in various subcellular fractions.However, its role in muscle stem cells (also known as muscle satellite cells), which provide myonuclei for postnatal muscle growth, maintenance, and regeneration, remains unclear. The present study aimed to determinenNOS expression in muscle satellite cell-derived primary myoblasts during differentiation and its DNA methylation levels, an epigenetic modification that controls gene expression. Undifferentiated and differentiated satellite cell-derived primary myoblasts were found to express nNOS. Immunohistochemical analysis revealed that nNOS colocalized with Pax7 (satellite cell marker) only in the undifferentiated myoblasts. Furthermore, nNOS immunoreactivity spread to the cytosol of Pax7-negative differentiated myotube-like cells. Thelevel of Nos1µmRNA, the main isoform of skeletal muscle nNOS, was increased in differentiated satellite cell-derived primary myoblasts compared to that in the undifferentiated cells. However,Nos1methylation levelsremained unchangedduring differentiation.These findings suggest that nNOS induction and the appropriate transition of its subcellular localization may contribute to muscle differentiation.

γ-Oryzanol Ameliorates Depressive Behavior in Ovariectomized Mice by Regulating Hippocampal Nitric Oxide Synthase: A Potential Therapy for Menopausal Depression.

Depression is a severe mental condition, common among menopausal women. γ-Oryzanol (ORY) has various biological properties; however, the effect of ORY on menopausal depression and its underlying mechanisms have not been investigated. ORY is orally administered to ovariectomized (OVX) mice for 20 weeks. ORY administration results in lower immobility time in the tail suspension and forced swim test and increases locomotor activity in the open field test. In the primary hippocampal neurons and hippocampi of OVX mice, ORY treatment increases nitric oxide (NO) production and neuronal NO synthase (nNOS) expression. Further, the phosphorylation of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), and tropomyosin receptor kinase B, along with the expression of brain-derived neurotrophic factior (BDNF), is upregulated. These stimulatory effects of ORY are diminished by treatment with estrogen receptor β (ERβ) antagonist. ORY similarly interacts with ERβ in the molecular docking analysis. Moreover,intracerebroventricular injection of 7-nitroindazole, a nNOS inhibitor, abolishes the antidepressant effects of ORY. The results indicate that ORY attenuates depressive behavior in OVX mice by upregulating ERβ-mediated hippocampal nNOS expression and activating the ERK-CREB-BDNF signaling networks. The findings suggest that ORY is a potential therapeutic agent for attenuating menopausal depression.

Circadian disturbance induces erectile dysfunction by impairing endothelial function.

In order to explore the impact of circadian disturbance on erectile function, we randomly divided 24 adult male rats into groups of control (light on at 8:00 a.m. and off at 8:00 p.m.), dark/dark (DD; constant dark), light/light (LL; constant light), and shift dark/light (DL; light off at 8:00 a.m. and on at 8:00 p.m.). Four weeks later, erectile function was measured and corpora cavernosa were harvested for analysis. The maximum intracavernous pressure (mICP) and mICP/mean arterial pressure (MAP) ratio in the DD, LL, and DL groups were significantly lower than that in the control group. The LL and DL groups showed significantly attenuated endothelial nitric oxide synthase (eNOS), while DD, LL, and DL showed reduced neuronal nitric oxide synthase (nNOS) at both mRNA and protein levels. The production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was inhibited by altered light/dark cycles to varying degrees. Circadian disturbance impaired endothelial function and contributed to erectile dysfunction. For the core circadian elements, mRNA expression of circadian locomotor output cycles kaput ( Clock ) and brain/muscle aryl-hydrocarbon receptor nuclear translocator-like protein 1 ( Bmal1 ) was elevated in the DL group, but their protein expression was not significantly changed. DD, LL, and DL increased period 1 ( Per1 ) and Per3 levels, while LL and DL increased PER1 levels. No significant difference was found for Per2 levels, and PER2 and PER3 concentrations were not significantly changed. Moreover, LL and DL significantly increased cryptochrome-1 (CRY1) and CRY2 at both mRNA and protein levels. The altered light/dark rat model showed that circadian disturbance contributed to erectile dysfunction probably by impairing endothelial function. Meanwhile, the core circadian elements were detected in the corpora cavernosa, but these were disrupted. However, which circadian element regulates erectile function and how it works need further analysis.

PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase

BackgroundProtection against ischemic stroke may be most effective when multiple components of the neurovascular unit are protected, yet current treatments target mainly neurons. Here we explored whether the PSD-95 inhibitor Tat-NR2B9c (NA-1) can protect not only neurons but also the blood-brain barrier. MethodsAdult male Sprague-Dawley rats were randomly divided into three groups, which were subjected to either sham surgery or transient cerebral ischemia-reperfusion, after which some animals were treated with Tat-NR2B9c. The therapeutic efficacy of Tat-NR2B9c was assessed in terms of the degree of neurological deficit and cerebral infarction, integrity of the blood-brain barrier, cerebral water content, as well as expression of PSD-95, nitric oxide synthase, and matrix metalloprotease-9. ResultsTat-NR2B9c (NA-1) ameliorated neurofunctional deficit, reduced cerebral infarction, mitigated blood-brain barrier injury and improved its integrity following ischemia-reperfusion, leading to less cerebral edema. These improvements were associated with upregulation of tight junction proteins in the blood-brain barrier. At the same time, Tat-NR2B9c (NA-1) downregulated neuronal nitric oxide synthase and matrix metalloprotease-9, while reversing the ischemia-induced downregulation of endothelial nitric oxide synthase in brain. We report here the first evidence that PSD-95 is expressed in vascular endothelial cells in the brain. ConclusionOur experiments in a rat model of transient occlusion of the middle cerebral artery suggest that Tat-NR2B9c (NA-1) can mitigate ischemic injury to the blood-brain barrier, and that it may do so by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19–22% and 13–17%, respectively, P < 0.001–0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62–85%), whereas 18–38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33–36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.