nNOS Expression Research Articles

Mechanisms of NO-Mediated Protein S-Nitrosylation in the Lens-Induced Myopia

Myopia is a chronic ocular disease, emerging as the most common type of refractive error. This study intends to preliminarily explore the roles of protein S-nitrosylation of nitric oxide (NO) in the regulation of myopia by detecting the expression of neuronal nitric oxide synthase (nNOS) and downstream S-nitrosylation, using the animal model of lens-induced myopia (LIM) in mice. The 3-week-old C57BL/6 J mice were divided into three groups: group I, lens-induced 0-week group (take eyeballs at the age of 3 weeks); group II, self-control eyes of experimental group (take eyeballs at the age of 7 weeks); and group III, lens-induced 4-week group (take eyeballs at the age of 7 weeks). The diopter and axial length of each group were measured by streak retinoscopes and optical coherence tomography (OCT) before and after model establishment. The protein expressions and locations of nNOS and S-nitrosylated proteins (PSNOs) were measured by western blot and immunofluorescence staining. Site-specific proteomic for protein S-nitrolysation was used to detect the existence and location of S-nitrosylation proteins in the retina of myopic and nonmyopic mice. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and motif enrichment analyses were performed. The differential sites were analyzed by GO, KEGG, and motif. Irreversible biotinylation procedure combined with protein purification and western blot was used to detect the protein expression of α-enolase (ENO1), a key player in the hypoxia-related signal pathway. The expressions of nNOS and PSNOs were significantly lower in the retina of experimental eyes than that in self-control eyes and 3-week-old baseline group. A total of 595 S-nitrosylated proteins, 709 S-nitrosylated peptides, and 708 S-nitrosylated sites were identified by site-specific S-nitrolysation proteomics in the retina of myopic and control eyes. A total of 19 differentiation loci were screened, of which 13 sites were downregulated and 6 sites were upregulated in experimental eyes compared with the self-control group. Specifically, the expression of SNO-ENO1 was significantly lower in the retina of experimental eyes than that in self-control eyes and 3-week-old baseline group. LIM induces the decrease of nNOS and PSNO protein levels in the retina of myopic mice. NO-mediated nonclassical protein S-nitrosylation modification may play an important role in the regulation of lens-induced myopia. ENO1 may be a key factor in the regulation of S-nitrosylation modification of myopia.

Low-Load Resistance Exercise with Blood Flow Restriction Increases Hypoxia-Induced Angiogenic Genes Expression.

The aim of the study was to determine whether low-load exercise (LL) with blood flow restriction (LL-BFR) would induce similar changes in expression of genes involved in hypoxia and angiogenesis compared to LL and high-load exercise (HL). Twenty-four males (age: 21.3 ± 1.9 years, body height: 1.74 ± 0.8 m, body mass: 73 ± 1.8 kg) were allocated into three groups: low-load exercise (LL), low-load exercise with blood-flow restriction (LL-BFR), and high-load exercise (HL). For the LL-BFR group a pneumatic cuff was inflated at 80% of the arterial occlusion pressure. All participants performed bilateral knee extension exercise, twice a week, for 8 weeks. LL and LL-BFR groups performed 3-4 sets of 15 reps at 20% 1RM, whilst the HL group performed 3-4 sets of 8-10 reps at 80% 1RM with a 60-s rest interval between sets. The hypoxia-inducible factor-1 alpha (HIF-1α) and beta (HIF-1β), vascular endothelial growth factor (VEGF), neuronal (nNOS), and inducible nitric oxide synthase (iNOS) genes expression were assessed before and after training. HIF-1α and HIF-1β mRNA levels significantly increased in the LL-BFR group and exceeded those elicited by HL and LL groups (p < .0001). VEGF gene expression was increased in both LL-BFR and HL groups, however, LL-BFR elicited a greater increase than LL (p < .0001). nNOS and iNOS genes expression significantly increased in all groups with greatest increases being observed in the LL-BFR group (p < .0001). The findings suggest that LL-BFR induces greater increases in genes expression related to hypoxia and angiogenesis than traditional resistance training.

ODP228 Obesity Induced a Down-Regulation of nNOS in Intrapancreatic Nervous System

Abstract Objective Obesity has become a serious global public health problem. Obesity is frequently preceding and the most important risk factor of type 2 diabetes. Pancreas regulates glucose metabolism and glucose homeostasis, and abnormality of pancreas play an important role in insulin resistance and diabetes. Intrapancreatic nerve innervation modulates insulin release and is tightly linked to the onset and development of diabetes. However, it is not known whether the activity of the intrapancreatic nervous system has been altered before the onset of diabetes. In this study, we examined the intrapancreatic nervous system in rats with obesity looking for the changes induced by obesity. Methods Obese Sprague-Dawley rats were developed with feeding high fat diet for 10 weeks. Using Immunofluorescence method, choline acetyl transferase (ChAT) and nNOS positive neurons in intrapancreatic ganglia were counted. ChAT and nNOS expression in pancreas were also measured with western blot method. Results (1) There was no significant difference in fasting blood glucose between obese rats and normal rats, but oral glucose tolerance test showed that the oral glucose tolerance was impaired in obese rats. (2) In rats fed with normal diet, 80.2%±1.5 neurons in intrapancreatic ganglia were ChAT positive, and in the obese rats, 79.3%±1.8 neurons were ChAT positive. There was no significant difference between the two groups (p&amp;gt;0. 05, n=5). In intrapancreatic ganglia of obese rats, 40%±1.4 neurons were nNOS positive, which was significantly lower than that (60.5%±1.2) of rats fed with normal diet (p&amp;lt;0. 01,n=5). (3) In pancreatic tissue, the relative expression level of ChAT protein was 1. 02±0. 03 in rats fed with normal diet and 0.98±0. 05 in obese rats (p&amp;gt;0. 05, n=5). The relative expression level of nNOS protein was 1.70±0. 08 in normal rats and 1.10±0. 05 in obese rats (p&amp;gt;0. 01, n=5). Conclusion The cholinergic nerves in intrapancreatic nervous system was not altered in obese rats, however, nNOS nerves was down-regulated in obese rats. The down-regulation of nNOS would induce a hyperactivity in intrapancreatic nervous system and this would cause an enhancement of insulin release, which has been observed in obese individuals. Presentation: No date and time listed

The interleukin-enhanced binding factor 3-mediated inhibition of nitric oxide production via phosphoinositide 3-kinase/protein kinase B pathway contributes to central cardiovascular regulation in the rostral ventrolateral medulla in hypertension.

Hypertension is characterized by sympathetic hyperactivity, which is related to the overexcitation of the presympathetic neurons in the rostral ventrolateral medulla (RVLM). Nitric oxide (NO) has been reported to be a vital neuromodulator involved in central cardiovascular regulation. However, the mechanism of interleukin-enhanced binding factor 3 (ILF3) participating in blood pressure (BP) regulation is still unclear. Therefore, this study aims to clarify the role of ILF3 within the rostral ventrolateral medulla (RVLM) in regulating NO in hypertension. It was found that the expression level of ILF3 was significantly increased in the RVLM of spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats through microarray gene expression analysis, Western blot, and immunofluorescence. Overexpression of ILF3 by injecting constructed adenovirus into the RVLM increased the BP and renal sympathetic nerve activity (RSNA) of the WKY rats, significantly decreasing NO production and neuronal nitric oxide synthase (nNOS) expression. Knockdown of ILF3 in the RVLM of SHR significantly reduced BP but increased NO production and the neuronal nitric oxide synthase (nNOS) expression. Furthermore, it was found that the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was activated via Western blotting in the RVLM after overexpression of ILF3, whereas it was attenuated after knockdown of ILF3 in SHR. In addition, inhibition of PI3K by intracisternal infusion of the PI-103 attenuated the increase in Akt phosphorylation and decrease in nNOS expression and NO production caused by overexpressing ILF3, which ultimately blunted high BP induced by overexpressing ILF3. Taken together, this current study suggests that ILF3 participates in high BP via reducing NO production in the RVLM through PI3K/Akt pathway.

Nobiletin protects enteric nerves and ameliorates disordered bowel motility in diet-induced obese mice via increasing Trem2 expression

Nobiletin can regulate lipid metabolism and protect the central nervous system. However, its role in the enteric nervous system (ENS) of obese subjects is still unclear. To investigate the ENS protective effects and mechanism of nobiletin in obese mice, male C57BL/6 mice were fed a chow diet and a high-fat diet (HFD) for 8 weeks. The identified obese and control mice were grouped and administered vehicle, nobiletin 40 mg/kg, 100 mg/kg or 200 mg/kg daily for 4 weeks. The major indexes of obesity, intestinal transit rate, PGP9.5, nNOS, TNF-α, IL-1β, IL-6, IL-10, Bcl2 and Bax were measured. The full-length transcriptome was used to analyze differentially expressed genes (DEGs) in the colon. The results indicated that nobiletin effectively improved major indexes of obesity and bowel motility function, suppressed the expression of TNF-α, IL-1β, IL-6 and Bax, and upregulated the expression of IL-10, Bcl2, PGP9.5 and nNOS. Based on full-length transcriptome sequencing, nobiletin regulated lipid metabolism and inflammation via the PPAR and NOD-like receptor signaling pathways. Trem2 expression was significantly reduced in obese mice. However, Trem2 expression was significantly increased after nobiletin treatment in obese mice. The enrichment analysis showed that Trem2 plays an important role in enteric neuroinflammation. In conclusion, nobiletin regulates lipid metabolism and inflammation in obese mice. Trem2 is a potential target of nobiletin for ENS protection in obese mice.

Age-related changes of neurons from the large intestine containing neuronal nitric oxide synthase

The aim of the study was to identify the localization, percentage and morphometric characteristics of neuronal NO synthase (nNOS)-immunoreactive (IR) neurons in the intramural ganglia of the myenteric (MP) and submucous plexus (SP) of the large intestine of rats of different age groups.&#x0D; Material and methods. The work was performed on Wistar rats aged 1, 10, 20, 30, 60 days and 2 years using immunohistochemical methods.&#x0D; Results. nNOS-IR neurons were found in the large intestine from the moment of birth and during the remaining age periods. In the intramural ganglia of the MP, the largest percentage of nNOS-IR neurons was detected in the newborn rat and decreased in ontogenesis up to 60 days of life, then did not change until senescence. In the SP, nNOS-IR neurons were also detected in the greatest number in newborns, in the next 20 days the percentage decreased significantly, nNOS-IR neurons were not detected in 30-day and two-month-old animals, but again appeared in large numbers in aged rats. The average cross-sectional area of nNOS-IR neurons increased in the MP from birth during the first two months of life. In the SP, the average size of nNOS-IR cells increased in the first 30 days of life and was significantly larger in aged rats compared with other ages.&#x0D; Conclusions. In early postnatal ontogenesis, there is a decrease in the expression of nNOS in neurons of the large intestine and a subsequent increase in aged rats.

Ketamine alleviating depressive-like behaviors is associated with regulation of nNOS-CAPON-Dexras1 complex in chronic unpredictable mild stress rats.

BackgroundA growing number of studies have demonstrated that ketamine induces rapid and sustained antidepressant action. Neuronal nitric oxide synthase (nNOS) signaling has been explored for the treatment of neuropsychiatric disorders for decades. But the effect of ketamine on nNOS signaling is poorly understood. The aim of the present study was to investigate the effect of ketamine on nNOS signaling in a chronic unpredictable mild stress (CUMS) model of depression.MethodsForty-eight rats were randomly divided into four groups: the control group of healthy rats (group C), the healthy rats treated with ketamine 10 mg/kg for 3 days (group CK), the rats model of stress-induced depression group (group D), and the depressed group treated with ketamine 10 mg/kg for 3 days (group DK). The sucrose preference test and open field test were used to assess behavioral changes. Immunohistochemistry, immunofluorescence, and real-time PCR analysis were carried out to measure the expression of nNOS, CAPON, and Dexras1 in the prefrontal cortex (PFC) of the CUMS rats.ResultsCompared with healthy rats, the total distance traveled, the rearing counts, the sucrose preference percentage (SPP), and CAPON and Dexras1 expression in the PFC significantly decreased, while nNOS expression increased in CUMS rats. After treating with ketamine, the total distance traveled, the rearing counts, the SPP, and CAPON and Dexras1 expression significantly increased, while nNOS expression significantly decreased.ConclusionThe results indicated that ketamine improved the depressive behavior of rats, which may be related to the reduced nNOS expression and enhanced CAPON and Dexras1 expression.

Effects of Simo decoction on gastric motility of diabetic rats.

To investigate the effects of simo decoction (SMD) on the gastric motility of diabetic rats. Diabetic rats were gavaged with various doses of SMD (0.15, 1.5, and 3.0ml/kg/d) or saline, and their blood glucose levels and body weight were monitored. Gastric emptying and antral motility were assessed by phenol red retention and contractions of antral strips, respectively. The levels of substance P (SP), vasoactive intestinal peptide (VIP), and neurogenic nitric oxide synthase (nNOS) in the gastric antrum were determined by real-time polymerase chain reaction and Western blot. Gastric emptying was delayed in diabetic rats (p< 0.01 vs. non-diabetic controls) but accelerated after SMD administration (p< 0.01). The contractions of antral strips were reduced in diabetic rats (p< 0.01 vs. non-diabetic controls) but improved after SMD intervention (p< 0.05). The mRNA expressions of SP, VIP, and nNOS in diabetic rats were downregulated compared with non-diabetic controls (all p< 0.01). Simo decoction treatment did not affect the expression of these factors in diabetic rats. The protein levels of SP, VIP, and nNOS in diabetic rats were decreased (p< 0.01), increased (p< 0.01), and comparable (p> 0.05), respectively, in comparison with non-diabetic controls. Simo decoction administration increased SP protein expression (p< 0.01) and decreased the levels of VIP (p< 0.01) and nNOS (p< 0.01) in diabetic rats. Simo decoction improved gastric dysmotility of diabetic rats possibly by upregulating SP and downregulating VIP and nNOS.

Esculentoside A Alleviates Intestinal Dysmotility in Ulcerative Colitis by Regulating H2S/CSE and NO/nNOS Systems.

Background Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that commonly affects the health of many individuals. Esculentoside A (EsA), a saponin extracted from the roots of Phytolacca esculenta, has antioxidative and anti-inflammatory effects against various diseases. Nonetheless, its role in UC is undetermined. Hence, in this study, we examined the therapeutic effects of EsA in UC. Methods Primary intestinal neuronal cells (in vitro) were treated with lipopolysaccharide (LPS) to induce inflammatory injury. An in vivo UC rat model was created by the administration of dextran sulfate sodium (DSS) to rats, which were subsequently treated with different doses of EsA. The effects of EsA on intestinal motility, histological score, inflammatory response, hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) system, NO/neuronal nitric oxide synthase (nNOS) system, and LPS-induced primary intestinal neuronal cell viability loss, proliferation inhibition, and apoptosis were detected. Results In vitro, EsA treatment increased the number of DSS-inhibited bowel movements and body weight, improved the histological score of colitis, and inhibited the inflammatory response by reducing IL-6 and TNF-α levels in rats. More importantly, EsA reduced the NO and H2S levels in serum and CSE, CBS, and nNOS expressions in the colon tissue. In vivo, EsA treatment eased the viability loss, proliferation inhibition, and apoptosis of LPS-stimulated primary intestinal neuronal cells, as well as inhibited the expressions of IL-6, TNF-α, CSE, CBS, and nNOS in cells. Conclusion EsA improved intestinal motility and suppressed inflammatory response in DSS-induced UC, which may be mediated by H2S/CSE and NO/nNOS systems.

Neuronal nitric oxide synthases in the pathogenesis of metabolic syndrome

The study of the molecular mechanisms of metabolic syndrome (MS) and its complications are among the most acute problems of modern endocrinology. Functional changes in the expression, activity, and regulatory properties of neuronal NO synthase (nNOS), which catalyzes the formation of the most important secondary mediator, nitric oxide (NO), and its dependent NO/cGMP signaling pathways in the brain, myocardium, and skeletal muscles, play a key role among the molecular causes of MS. In the brain, nNOS is associated with NMDA receptors, the hyperactivation of which in MS leads to excessive stimulation of nNOS and hyperproduction of NO, which leads to NO-induced damage to neurons and disruption of the central regulation of physiological processes and neurodegeneration. In the myocardium with MS, there are changes in the expression and localization of nNOS, as well as its functional interaction with cytoskeletal proteins, which leads to disorders of myocardial contraction and hypertrophy. In skeletal muscles, nNOS controls their contraction, oxidative metabolism, is involved in the regulation of vascular relaxation, and also participates in the regulation of glucose transport. A decrease in the expression and activity of nNOS, as well as dysregulation of its activity in MS, cause disturbances of these processes and make a significant contribution to the development of insulin resistance and deterioration of glucose homeostasis. Thus, nNOS can be considered an important therapeutic target in the treatment of MS and other metabolic disorders, as well as to prevent their complications from the nervous and cardiovascular systems and the musculoskeletal system.

Behavioral defects and downregulation of hippocampal BDNF and nNOS expression in db/db mice did not improved by chronic TGF-β2 treatment.

Epidemiological evidence suggests that there is a link between diabetes and mood disorders, such as depression and anxiety. Although peripheral or central inflammation may explain this link, the molecular mechanisms are not fully understood and few effective treatments for diabetes or mood disorders are available. In the present study, we aimed to determine whether transforming growth factor (TGF)-β2, an anti-inflammatory substance, might represent a potential therapeutic agent for diabetes-related mood behaviors. TGF-β2 expression in the hippocampus is affected by anxiolytic drugs and stress exposure, it is able to cross the blood-brain barrier, and it is as an exercise-induced physiological adipokine that regulates glucose homeostasis. Therefore, we hypothesized that a chronic TGF-β2 infusion would ameliorate diabetes-related glucose intolerance and mood dysregulation. To determine the effects of the chronic administration of TGF-β2 on diabetes, we implanted osmotic pumps containing TGF-β2 into type 2 diabetic mice (db/db mice), and age-matched non-diabetic control wild type mice and db/db mice were infused with vehicle (PBS), for 12 consecutive days. To assess anxiety-like behaviors and glucose homeostasis, the mice underwent elevated plus maze testing and intraperitoneal glucose tolerance testing. Hippocampal and perigonadal visceral white adipose tissue perigonadal white adipose tissue samples were obtained 12 days later. Contrary to our hypothesis, TGF-β2 infusion had no effect on diabetes-related glucose intolerance or diabetes-related behavioral defects, such as inactivity. In db/db mice, the expression of inflammatory markers was high in pgWAT, but not in the hippocampus, and the former was ameliorated by TGF-β2 infusion. The expression of brain-derived neurotrophic factor and neuronal nitric oxide synthase, important regulators of anxiety-like behaviors, was low in db/db mice, but TGF-β2 infusion did not affect their expression. We conclude that although TGF-β2 reduces the expression of pro-inflammatory markers in the adipose tissue of diabetic mice, it does not ameliorate their obesity or mood dysregulation.

Influence of Acupuncture on Expression of Mitochondrial Fusion and Fission Mediators in Rat Liver

Background: We aimed to elucidate the mechanism of change in mitochondrial fusion- and fission-related mediators for maintaining cell function through the effects of acupuncture treatment and apply findings to a liver disease model. We focused on the optic atrophy-1 (OPA1) and fission protein 1 (Fis1) genes of rat liver cells. Methods: Sprague Dawley rats were divided into a control group (no treatment) and LR2, LR3, LR4 and LR8 groups (acupuncture treatment to those points). Acupuncture was performed on each point for 10 minutes once daily for 4 days. Changes in the mRNA expression of Peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α) and Fis1 were observed via quantitative real-time polymerase chain reaction (qRT-PCR); changes in OPA1, mitofusin-2 (MFN2), mitofusin-1 (MFN1), dynamin-related protein 1 (DRP1) and adenosine monophosphate-activated protein kinase (AMPK) proteins were observed through western blotting and endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) expressions were observed through immunohistochemistry. Result: OPA1 decreased in the LR3 and LR8 groups and Fis1 increased in the LR2 and LR4 groups. AMPK and PGC-1α decreased significantly in all acupuncture groups. eNOS and nNOS expression reduced in all acupuncture groups. Therefore, acupuncture can regulate mitochondrial fusion/fission by influencing the following mediators: AMPK, PGC-1α, OPA1, Fis1, eNOS and nNOS.

Xiongshao Zhitong Recipe Attenuates Nitroglycerin-Induced Migraine-Like Behaviors via the Inhibition of Inflammation Mediated by Nitric Oxide Synthase.

Migraine is a major cause of disability worldwide, particularly in young adults and middle-aged women. Xiongshao Zhitong Recipe (XZR) is a traditional Chinese medicine prescription used for treating migraine, but its bioactive components and therapeutic mechanisms remain unclear. We aimed to confirm the therapeutic effect of XZR on migraine and to determine the possible mechanism and bioactive components of XZR. Here, a sensitive UHPLC-LTQ-Orbitrap MS assay was carried out to analyze the ingredients of XZR, and a total of 62 components were identified, including coumarins, phenolic acids, phthalides, flavonoids, and terpenoids; among them, 15 components were identified in the serum samples after XZR treatment. We established a rat model of migraine via nitroglycerin (NTG) injection. The in vivo experiments demonstrated that XZR attenuated allodynia and photophobia in rats with NTG-induced migraine, and XZR also demonstrated analgesic effects. XZR reversed the abnormal levels of nitric oxide, 5-hydroxytryptamine (5-HT), calcitonin gene-related peptide (CGRP), and substance P (SP) to normal levels. XZR also downregulated inflammatory reactions, including mast cell degranulation and serum IL-1β, IL-6, and TNF-α levels. In terms of mechanism, we revealed that XZR treated NTG-induced migraine through the inhibition of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression in both the trigeminal nucleus caudalis (TNC) and periaqueductal gray matter (PAG), as well as the total NOS enzyme activity, which regulated the NF-κB signaling pathway. Additionally, imperatorin and xanthotoxin, two major ingredients of XZR, showed a high binding affinity to nNOS (Gly468-Leu616). In vitro, XZR, imperatorin, and xanthotoxin inhibited the nNOS expression and the NF-κB signaling pathway in lipopolysaccharide (LPS)-stimulated PC12 cells. In conclusion, we demonstrated the therapeutic effects of XZR and provided evidence that XZR played a critical anti-inflammatory role by suppressing NOS and NF-κB signaling pathway activation. Imperatorin and xanthotoxin were potential bioactive components of XZR. The findings from this study supported that XZR was a candidate herbal drug for migraine therapy.

The N-methyl-D-aspartate receptor antagonist ketamin exerts analgesic effects via modulation of the nitric oxide pathway.

Ketamine, an NMDA receptor antagonist, has been approved to have analgesic effects. It is known that nitric oxide pathway is involved in antinociception but with dual effects. In this study, we investigated the role of nitric oxide in ketamine-induced analgesia. Ketamine was administered to mice acute and chronically with/without nitric oxide synthase (NOS) inhibitors. Experimental models of nociception pain, including hot plate, tail flick, and formalin tests, were performed. Western blot was used to measure levels of nitric oxide synthase enzymes in the brain. Ketamine doses of 0.03 and 0.3 mg/kg had significant analgesic effects (p < 0.01). High-dose chronic ketamine could induce analgesia in later phases of the treatment in tail flick test (p < 0.01). Pretreatment with various NOS inhibitors decreased the analgesic effect. In western blot analysis, the expression of NOS proteins was decreased. Low-dose ketamine is effective in analgesia induction. The expression of nNOS and iNOS proteins is dependent on the inhibition of the NMDA/NO pathway.

Evidence that resistin acts on the mechanical responses of the mouse gastric fundus

Resistin, among its several actions, has been reported to exert central anorexigenic effects in rodents. Some adipokines which centrally modulate food intake have also been reported to affect the activity of gastric smooth muscle, whose motor responses represent a source of peripheral signals implicated in the control of the hunger-satiety cycle through the gut-brain axis. On this basis, in the present experiments, we investigated whether resistin too could affect the mechanical responses in the mouse longitudinal gastric fundal strips. Electrical field stimulation (EFS) elicited tetrodotoxin- and atropine-sensitive contractile responses. Resistin reduced the amplitude of the EFS-induced contractile responses. This effect was no longer detected in the presence of L-NNA, a nitric oxide (NO) synthesis inhibitor. Resistin did not influence the direct muscular response to methacholine. In the presence of carbachol and guanethidine, EFS elicited inhibitory responses whose amplitude was increased by resistin. L-NNA abolished the inhibitory responses evoked by EFS, indicating their nitrergic nature. In the presence of L-NNA, resistin did not have any effect on the EFS-evoked inhibitory responses. Western blot and immunofluorescence analysis revealed a significant increase in neuronal nitric oxide synthase (nNOS) expression in neurons of the myenteric plexus following resistin exposure. In conclusion, the present results offer the first evidence that resistin acts on the gastric fundus, likely through a modulatory action on the nitrergic neurotransmission.

4'-Iodo-α-Pyrrolidinononanophenone Provokes Differentiated SH-SY5Y Cell Apoptosis Through Downregulating Nitric Oxide Production and Bcl-2 Expression.

Abuse of pyrrolidinophenone derivatives (PPs) is known to cause severe damage to the central nervous system due to their high lipophilicity. In this study, we compared sensitivity to toxicity elicited by 4'-iodo-α-pyrrolidinononanophenone (I-α-PNP), one of the most potent cytotoxic derivatives among PPs synthesized previously, between SH-SY5Y cells differentiated by all-trans-retinoic acid (ATRA) and the undifferentiated cells, and found that the differentiated cells are more sensitive to I-α-PNP toxicity than the undifferentiated cells. Treatment with I-α-PNP elicited some apoptotic alterations (Bax expression, loss of mitrochondrial membrane potential, and activation of caspases) in the differentiated cells, whose patterns were similar to those in the undifferentiated cells. I-α-PNP treatment resulted in no significant alteration in Bcl-2 expression in the undifferentiated cells, whereas it considerably downregulated the protein expression in the differentiated cells, suggesting that the high I-α-PNP sensitivity of the differentiated cells is mainly due to downregulation of Bcl-2 expression. I-α-PNP treatment decreased nitric oxide (NO) production and neuronal NOS (nNOS) expression in the differentiated cells, and the patterns of I-α-PNP-evoked alterations in phosphorylation of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression were almost the same as that in nNOS expression. Additionally, the addition of an NO donor restored the I-α-PNP-evoked alterations in expressions of Bcl-2, BDNF, and nNOS in the differentiated cells. These findings suggest that the downregulation of Bcl-2 expression by I-α-PNP in differentiated cells is attributed to the acceleration of two negative feedback loops (nNOS/NO/CREB loop and CREB/BDNF loop) triggered by decreased NO production.

The nNOS and NeuN expression aspects in the cerebral cortex of rats with different cognitive abilities in cerebral hypoperfusion with short-term physical exercise

Introduction. Physical activity is often used in rehabilitation to restore cognitive function after cerebral blood flow impairment and typological features like baseline cognitive ability may alter their effectiveness.The aim was to evaluate the expression of nNOS and NeuN in the large hemisphere cortex in the cerebral hypoperfusion occurrence in rats having different scores in the Morris maze.Materials and methods. Cerebral hypoperfusion was simulated by bilateral ligation of the common carotid arteries. Before surgery, Wistar rats were divided into equal subgroups with high (HCA) and low levels of cognitive ability (LCA) according to the results of Morris maze testing. Animals were removed from the experiment at 8, 21, 35, 60, and 90 days after surgery, a total of 184 rats, 24 formed the control group and 80 formed the cerebral hypoperfusion subgroup with 80 animals undergoing short-term swimming daily starting on day 7 of the experiment.Results. We revealed the earlier changes in HCA animals (8th day of the experiment) induced by cerebral hypoperfusion accompanied by a more intense nNOS-positive neurons density reduction to 47 % of control values, the length of their processes, and an increase in pericarion area. In LCA the decrease of density was only 75 % of control values and pericarion area does not differ from control values. At later periods on the 60 and 90 days of the experiment LCA demonstrates the preservation of the neurons density and greater length of their processes (67 % of the control subgroup values).Conclusion. HCA is a risk factor for damage of nNOS-positive neurons in cerebral hypoperfusion. Physical exercise more effectively decreases nNOS expression in animals with HCA, which may be one of the sanogenetic mechanisms of this factor.

Expression and localization of neuronal nitric oxide synthase in the brain and sensory tissues of the African cichlid fish Astatotilapia burtoni.

Nitric oxide (NO) produced by the enzyme neuronal nitric oxide synthase serves as an important neurotransmitter in the central nervous system that is involved in reproductive regulation, learning, sensory processing, and other forms of neural plasticity. Here, we map the distribution of nnos-expressing cells in the brain and retina of the cichlid fish Astatotilapia burtoni using in situ hybridization. In the brain, nnos-expressing cells are found from the olfactory bulbs to the hindbrain, including within specific nuclei involved in decision-making, sensory processing, neuroendocrine regulation, and the expression of social behaviors. In the retina, nnos-expressing cells are found in the inner nuclear layer, presumably in amacrine cells. We also used quantitative PCR to test for differences in nnos expression within the eye and olfactory bulbs of males and females of different reproductive states and social statuses. In the eye, males express more nnos than females, and socially dominant males express more nnos than subordinate males, but expression did not differ among female reproductive states. In the olfactory bulbs, dominant males had greater nnos expression than subordinate males. These results suggest a status-specific function for NO signaling in the visual and olfactory systems that may be important for sensory perception related to mating or territorial interactions to maintain the social hierarchy. The widespread distribution of nnos-expressing cells throughout the cichlid brain is similar to that in other teleosts, with some conserved localization patterns across vertebrates, suggesting diverse functions for this important neurotransmitter system.

Enhanced Glial Reaction and Altered Neuronal Nitric Oxide Synthase are Implicated in Attention Deficit Hyperactivity Disorder.

Attention deficit hyperactivity disorder (ADHD) has a complex etiology, and its specific causal factors remain to be elucidated. Aberration of nitric oxide synthase (nNOS) and inflammation, together with astrocytic and microglial cells have been continually associated with several neurological disorders, including ADHD. Using spontaneously hypertensive rat (SHR), we investigated the changes in nNOS, inflammatory, microglial and astrocytic markers in the frontal cortex and hippocampus at three different ages: onset of hypertension stage (i.e., 6 weeks after birth of SHR), established hypertension stage (i.e., 12 weeks after birth of SHR) and senescent stage (i.e., 12 months after birth of SHR), and compared with its age-matched normotensive control, Wistar-Kyoto (WKY) rats. A significant upregulation of Iba-1 expression in the senescent stage of SHR was observed. Further, we observed an upregulated nNOS expression in both onset and established stages of SHR, and a downregulated nNOS in the senescent stage. Our study showed an age-related increment of astrogliosis in the cortex and hippocampi of aged SHR. On the basis of our results, alterations in the nNOS and Iba-1 expressions, as well as age-related astrogliosis, may contribute to ADHD pathogenesis.

Dysregulation of the Enteric Nervous System in the Mid Colon of Complement Component 3 Knockout Mice with Constipation Phenotypes.

Complement component 3 (C3) contributes to neurogenesis, neural migration, and synaptic elimination under normal and disease conditions of the brain, even though it has not been studied in the enteric nervous system (ENS). To determine the role of C3 in the regulatory mechanism of ENS during C3 deficiency-induced constipation, the changes in the markers of neuronal and interstitial cells of Cajal (ICCs), the markers for excitatory and inhibitory transmission of ENS, and expression of C3 receptors were analyzed in the mid colon of C3 knockout (KO) mice at 16 weeks of age. Prominent constipation phenotypes, including the decrease in stool parameters, changes in the histological structure, and suppression of mucin secretion, were detected in C3 KO mice compared to wildtype (WT) mice. The expression levels of the neuron specific enolase (NSE), protein gene product 9.5 (PGP9.5), and C-kit markers for myenteric neurons and ICCs were lower in the mid colon of C3 KO mice than WT mice. Excitatory transmission analysis revealed similar suppression of the 5-hydroxytryptamine (5-HT) concentration, expression of 5-HT receptors, acetylcholine (ACh) concentration, ACh esterase (AChE) activity, and expression of muscarinic ACh receptors (mAChRs), despite the mAChRs downstream signaling pathway being activated in the mid colon of C3 KO mice. In inhibitory transmission analysis, C3 KO mice showed an increase in the nitric oxide (NO) concentration and inducible nitric oxide synthase (iNOS) expression, while neuronal NOS (nNOS) expression, cholecystokinin (CCK), and gastrin concentration were decreased in the same mice. Furthermore, the levels of C3a receptor (C3aR) and C3bR expression were enhanced in the mid colon of C3 KO mice compared to the WT mice during C3 deficiency-induced constipation. Overall, these results indicate that a dysregulation of the ENS may play an important role in C3 deficiency-induced constipation in the mid colon of C3 KO mice.