nNOS Levels Research Articles

Effect of Dp71 deficiency on the oxytocin hypothalamic axis in osmoregulation function in mice

Dp71 is the major form of dystrophins (Dp) in the supraoptic nucleus (SON) and in the neural lobe of hypophysis (NL/HP). Dp71-null mice exhibit a hypo-osmolar status attributed to an altered osmosensitivity of the SON and to a perturbed vasopressinergic axis. Because oxytocin (OT) is implicated in osmoregulation via natriuresis, this study explored the oxytocinergic axis in Dp71-null mice after salt-loading (SL).Under normosmolar conditions, OT-mRNA expression was higher in the Dp71-null SON compared to wild-type (wt) and the OT peptide level has not changed. Dp-immunostaining was localized in astrocytes end-feet surrounding vessels in wt SON. This distribution changed in Dp71-null SON, Dp being detected in OT-soma of MCNs. nNOS and NADPH-diaphorase levels increased in the OT area of the Dp71-null SON compared to wt. In the NL/HP, OT level reduced in Dp71-null mice and Dp localization changed from pituicytes end-feet in wt SON to OT terminals in Dp71-null SON.Salt-Loading resulted in an increase of OT-mRNA and peptide levels in wt SON but had no effect in Dp71-null SON. In the NL/HP, OT content was reduced after SL. For Dp71-null mice, OT level, already low in control, was not modified by SL. Dp level was not affected by SL in the SON nor in the NL/HP.Our data confirmed the importance of Dp71 for the SON functionality in osmoregulation. The localization of Dp71 at the glial-vascular interface could be associated with SON osmosensitivity, leading to an adequate OT synthesis in the SON and release from the NL/HP upon plasmatic hyperosmolality.

A novel large animal model of recurrent migraine established by repeated administration of inflammatory soup into the dura mater of the rhesus monkey.

Several animal models of migraine have been established, and those based on trigeminovascular system activation are widely accepted. However, most of these models have been established on lower animals, such as rodents, and involve only a single administration of a noxious stimulus. In this study, an inflammatory soup (10 μL), consisting of prostaglandin E2 (0.2 mM), serotonin (2 mM), bradykinin (2 mM) and histamine (2 mM), was injected into the dura mater of conscious rhesus monkeys through an indwelling catheter. The infusion started on day 8 and was repeated every 3 days, for a total of six administrations, to induce neurogenic inflammation. We performed behavioral assessments and measured the expression of the oncogene c-fos, neuronal nitric oxide synthase (nNOS) and calcitonin gene related peptide (CGRP) in the trigeminal system and in multiple brain regions involved in pain processing by immunohistochemical staining. Compared with monkeys in the control group, three of the four animals in the inflammatory soup group displayed decreased motor behaviors, and two showed increased ipsilateral nose and mouth secretions during the stimulus period. Higher expression levels of c-fos, nNOS and CGRP were found in various brain areas of experimental animals compared with controls, including the trigeminal nucleus caudalis, thalamus, hypothalamus, midbrain, pons and other areas involved in pain perception. These results suggest that repeated inflammatory soup stimulation of the dura activates the trigeminovascular system and produces migraine-like pathological changes and abnormal behaviors in conscious rhesus monkeys.

1019: Progesterone attenuates brain inflammatory response and protects the brain from inflammation-induced immature myeloid cells increase

Pregnancy is a challenge for the maternal immune system since it has to defend against pathogens while tolerating alloantigenic fetal tissue. Little is known about maternal brain immune system during pregnancy. During pregnancy there is a marked increase in endogenous progesterone (PRO) and PRO supplementation is used for prevention of preterm birth. We sought to determine the maternal brain immune response to inflammation in pregnant (P) and non-pregnant (NP) mice and whether PRO supplementation attenuates this response. NP & P mice were pretreated with vaginal PRO or carrier from E13-E16. Lipopolysaccharide (LPS 30μg) or vehicle were administered intraperitoneally at E15 & E16. Serum was analyzed for IL-6 levels by ELISA. Brain was analyzed for nNOS, NFkB, IL-6 & TNF-a levels by western blot. Brain immature myeloid cells (IMC; CD45high, CD11b+, Gr1+) as % of CD45high hematopoietic brain cells and microglial (CD45low cells) activity as medial CD45 intensity was analyzed by FACS. LPS significantly increased brain nNOS, NFkB, IL-6 & TNF-a protein brain levels in NP & P mice. PRO pretreat ment significantly reduced nNOS and NFkB brain levels following LPS treatment in both groups. In NP mice, LPS treatment significant increased brain IMC population, and PRO pretreatment reduced the increase. In P mice, neither LPS nor LPS & PRO altered the brain IMC population. LPS led to a significant increase in microglial activity both groups. PRO pretreatment led to significant decrease in microglial activity in P mice. In NP mice, there was a trend but it did not reach statistical significance. PRO had no effect on serum IL-6 levels in both groups. PRO attenuates the inflammatory response in the brain in NP & P mice but had no effect on systemic (IL-6) inflammation. LPS increased brain IMC population in NP mice but not in P mice and PRO pretreatment reduced the IMC increase. These results suggest that endogenous PRO during pregnancy may protect the brain from LPS-induced central inflammation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Age- and sex-related changes in cortical and striatal nitric oxide synthase in the Q175 mouse model of Huntington's disease

In Huntington's disease (HD), corticostriatal and striatopallidal projection neurons preferentially degenerate as a result of mutant huntingtin expression. Pathological deficits in nitric oxide (NO) signaling have also been reported in corticostriatal circuits in HD, however, the impact of age and sex on nitrergic transmission is not well characterized. Thus, we utilized NADPH-diaphorase (NADPH-d) histochemistry and qPCR assays to assess neuronal NO synthase (nNOS) activity/expression in aged male and female Q175 heterozygous mice. Compared to age-matched controls, male Q175 mice exhibited reductions in NADPH-d staining in the motor cortex at 21, but not, 16 months of age. Comparisons across genotypes showed that striatal NADPH-d staining was significantly decreased at both 16 and 21 months of age. Comparisons within sexes in 21 month old mice revealed a decrease in striatal NADPH-d staining in males, but no changes were detected in females. Significant correlations between cortical and striatal NADPH-d staining deficits were also observed in males and females at both ages. To directly assess the role of constitutively active NOS isoforms in these changes, nNOS and endothelial NOS (eNOS) mRNA expression levels were examined in R6/2 (3 month old) and Q175 (11.5 month old) mice using qPCR assays. nNOS transcript expression was decreased in the cortex (40%) and striatum (54%) in R6/2 mice. nNOS mRNA down-regulation in striatum of Q175 animals was more modest (19%), and no changes were detected in cortex. eNOS expression was not changed in the cortex or striatum of Q175 mice. The current findings point to age-dependent deficits in nNOS activity in the HD cortex and striatum which appear first in the striatum and are more pronounced in males. Together, these observations and previous studies indicate that decreases in nitrergic transmission progress with age and are likely to contribute to corticostriatal circuit pathophysiology particularly in male patients with HD.

Neuronal Nitric Oxide Synthase Expression in the Anterior Vaginal Wall of Patients with Stress Urinary Incontinence.

Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common medical problems, particularlyamong older women. In this study, we aim to explore the relationship between the neurotransmitter nNOS in the vaginal epithelium, and the occurrence of SUI and changes of nNOS levels according to menopausal status.Matherials and Methods: Fourty women were enrolled. The patients were divided into four groups according to menstruaiton status and SUI. The vagina specimens were taken during transobturator tape application. The specimens were examined pathologically in terms of n-NOS expression. nNOS expression was compared between SUI and control groups. The results were evaluated statistically. Epithelial total nNOS score in group 1 and group 3 were 2.4 ± 0.5 and 1.4 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 2.2 ± 0.4 in group 1 and 1.3 ± 0.5 in group 3 (P = .001). Epithelial total nNOS score in group 2 and group 4 were 4.4 ± 0.5 and 3.5 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 4.4 ± 0.5 in group 2 and 3.6 ± 0.5 in group 4 ( P = .006). Our results show that expression of nNOs in the anterior vaginal epithelium decreased significantly in the SUI group. Altough our findings indicate important results, well designed further studies are needed to comprehend the role of NOS pathways better in SUI pathophysiology.

Epalrestat, an Aldose Reductase Inhibitor, Restores Erectile Function in Streptozocin-induced Diabetic Rats

Epalrestat, an aldose reductase inhibitor (ARI), was adopted to improve the function of peripheral nerves in diabetic patients. The aim of this study was to investigate whether epalrestat could restore the erectile function of diabetic erectile dysfunction using a rat model. From June 2016, 24 rats were given streptozocin (STZ) to induce the diabetic rat model, and epalrestat was administered to ten diabetic erectile dysfunction (DED) rats. Intracavernous pressure (ICP) and mean systemic arterial pressure (MAP), levels of aldose reductase (AR), nerve growth factor (NGF), neuronal nitric oxide synthase (nNOS), α-smooth muscle antigen (α-SMA), and von Willebrand factor (vWF) in the corpus cavernosum were analyzed. We discovered that epalrestat acted on cavernous tissue and partly restored erectile function. NGF and nNOS levels in the corpora were increased after treatment with epalrestat. We also found that the content of α-SMA-positive smooth muscle cells and vWF-positive endothelial cells in the corpora cavernosum were declined. Accordingly, epalrestat might improve erectile function by increasing the upregulation of NGF and nNOS to restore the function of the dorsal nerve of the penis.

Preventive Effect of Lactobacillus Plantarum CQPC10 on Activated Carbon Induced Constipation in Institute of Cancer Research (ICR) Mice

Chinese Paocai is a traditional fermented food containing an abundance of beneficial microorganisms. In this study, the microorganisms in Szechwan Paocai were isolated and identified, and a strain of lactic acid bacteria (Lactobacillus plantarum CQPC10, LP-CQPC10) was found to exert an inhibitory effect on constipation. Microorganisms were isolated and identified via 16S rDNA. Activated carbon was used to induce constipation in a mouse model and the inhibitory effect of LP-CQPC10 on this induced constipation was investigated via both pathological sections and qPCR (quantitative polymerase chain reaction). A strain of Lactobacillus plantarum was identified and named LP-CQPC10. The obtained results showed that, as compared to the control group, LP-CQPC10 significantly inhibited the amount, weight, and water content of faeces. The defecation time of the first tarry stool was significantly shorter in LP-CQPC10 groups than in the control group. The activated carbon progradation rate was significantly higher when compared to the control group and the effectiveness was improved. LP-CQPC10 increased the serum levels of MTL (motilin), Gas (gastrin), ET (endothelin), AchE (acetylcholinesterase), SP (substance P), and VIP (vasoactive intestinal peptide), while decreasing the SS (somatostatin) level. Furthermore, it improved the GSH (glutathione) level and decreased the MPO (myeloperoxidase), MDA (malondialdehyde), and NO (nitric oxide) levels. The results of qPCR indicated that LP-CQPC10 significantly up-regulated the mRNA expression levels of c-Kit, SCF (stem cell factor), GDNF (glial cell-derived neurotrophic factor), eNOS (endothelial nitric oxide synthase), nNOS (neuronal nitric oxide synthase), and AQP3 (aquaporin-3), while down-regulating the expression levels of TRPV1 (transient receptor potential cation channel subfamily V member 1), iNOS (inducible nitric oxide synthase), and AQP9 (aquaporin-9). LP-CQPC10 showed a good inhibitory effect on experimentally induced constipation, and the obtained effectiveness is superior to that of Lactobacillus bulgaricus, indicating the better probiotic potential of this strain.

Short-term 2.1 GHz radiofrequency radiation treatment induces significant changes on the auditory evoked potentials in adult rats

Purpose: There is a growing interest in the usage of radiofrequency radiation (RF) as a noninvasive brain stimulation method. Previously reported data demonstrated that RF exposure caused a change in brain oscillations. Therefore, we aimed to investigate effects of RF on brain oscillation by measuring the auditory response of different brain regions in rats.Materials and methods: Rats were randomly divided into three groups (n = 12 per each group): Cage control (C), sham rats (Sh), and rats exposed to 2.1 GHz RF for 2 h/day for 7 days. At the end of the exposure, auditory evoked potentials (AEPs) were recorded at different locations in rats. Latencies and amplitudes of AEPs, evoked power, inter-trial phase synchronization, and auditory evoked gamma responses were obtained in response to an auditory stimulus. Furthermore, TBARS levels and 4-HNE, GFAP, iNOS, and nNOS expressions were evaluated in all groups.Results: Peak-to-peak amplitudes of AEPs were significantly higher in the RF group compared with the Sh group. There is no significant difference in peak latencies of AEPs between groups. Beside, evoked power, inter-trial phase synchronization, and auditory evoked gamma responses were significantly higher in the RF group compared with the Sh group. In addition, the RF group had significantly lower TBARS and 4-HNE levels than the Sh group. There were no significant differences between groups for GFAP, nNOS, and iNOS levels, and between the C and RF groups for all parameters.Conclusions: Our present findings suggest that short-term RF treatment under chosen experimental conditions have statistically significant effect on neuronal networks of rats by probably reducing oxidative damage. However, this effect must be further studied for possible noninvasive brain stimulation.

S-nitrosylation of endothelial nitric oxide synthase impacts erectile function

Neuronal and endothelial nitric oxide synthases (nNOS and eNOS respectively) play major roles in generating the nitric oxide bioactivity necessary for erectile function. S-nitrosylation has been shown to regulate NOS activity. The presence of S-nitrosylated NOS in the penis and the impact of NOS S-nitrosylation/denitrosylation on erectile function were examined. S-nitrosylated forms of NOS were identified by biotin-switch assay followed by western blot analysis. Erectile function in S-nitrosoglutathione reductase deficient (GSNO+/-) and null (GSNO-/-) mice were assessed by continuous cavernous nerve electrical stimulation (CCNES). Glutathione ethyl ester (GSHee) was used to manipulate S-nitrosylated NOS levels. Immunohistological and immunofluorescence analyses were used to identify the location of eNOS and GSNO-R in corporal tissue. eNOS and nNOS were S-nitrosylated in unstimulated penises of the mice. CCNES resulted in a time-dependent increase in eNOS S-nitrosylation with peak eNOS S-nitrosylation observed during detumescence. S-nitrosylated nNOS levels were unchanged. Intracorporal injection of GSHee reduced S-nitrosylated eNOS levels, enhancing time to maximum intracorporal pressure (ICP). eNOS and GSNO-R co-localize to the endothelium of the corpus cavernosum in the mouse and the human. ICP measurements obtained during CCNES demonstrate GSNO-R+/- and GSNO-R-/- animals cannot maintain an elevated ICP. Results suggest eNOS S-nitrosylation/denitrosylation is an important mechanism regulating eNOS activity during erectile function. GSNO-R is a key enzyme involved in the eNOS denitrosylation. The increase in eNOS S-nitrosylation (inactivation) observed with tumescence may begin a cycle leading to detumescence. Clinically this may indicate that alterations in the balance of S-nitrosylation/denitrosylation either directly or indirectly contribute to erectile dysfunction.

Decreased Number and Expression of nNOS-Positive Interneurons in Basolateral Amygdala in Two Mouse Models of Autism.

The basolateral amygdala (BLA) controls socio-emotional behaviors and is involved in the etiology of autism. We have recently shown that virtually every neuronal nitric oxide synthase (nNOS) positive cell is a GABAergic inhibitory interneuron in the mouse BLA. Here, stereology was used to quantify the number of nNOS-expressing interneurons in valproic acid (VPA)-exposed C57BL/6J (B6) and BTBR T+Itpr3tf/J (BTBR) mice models of autism. Additionally, the protein and mRNA levels of nNOS in the BLA were quantitatively assessed by western blot and qRT-PCR analysis, respectively. Our results showed the decreased number of nNOS interneurons in the BLA of animal models relative to autism. Consistently, nNOS was significantly reduced in the VPA-exposed and BTBR mice at both protein and mRNA levels. Together, these preliminary findings suggest that down-regulation of nNOS may be an attractive target for the pharmacological intervention in autism.

Restorative effect of resveratrol on expression of endothelial and neuronal nitric oxide synthase in cavernous tissues of chronic unpredictable mild stress-exposed rats: an impact of inflammation.

We investigated the effect of resveratrol on endothelial and neuronal nitric oxide synthase (eNOS and nNOS) expression in the corpus cavernosum from chronic unpredictable mild stress (CUMS)-exposed rats in order to examine possible role of proinflammatory cytokines, which might play a role on erectile dysfunction (ED). Rats were randomly and equally divided into fourgroups such as control,control+resveratrol, CUMS and CUMS + resveratrol (20 mg/kg/day, i.p/8 weeks). Sucrose intake and forced swimming tests were used to evaluate depressive-like behaviors. nNOS, eNOS expressions, inflammatory markers, corticosterone and testosterone levels were analyzed either in blood samples and/or penile tissues. CUMS-exposed rats displayed depressive-like behaviors, reduced penile nNOS and eNOS expressions, and serum testosterone levels and enhanced serum and penile tissue levels of proinflammatory markers compared to controls. Resveratrol reversed depressive-like behaviors and suppressed serum and penile levels of proinflammatory markers, increased nNOS and eNOS expressions and testosterone levels in CUMS-exposed rats. Resveratrol exerted antidepressant-like effects and protected the development of CUMS-induced impairment of cavernosal eNOS and nNOS expressions associated with ED, which might be related to its anti-inflammatory action.

Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration.

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1β and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD.

Clinical antibacterial effectiveness and biocompatibility of gaseous ozone after incomplete caries removal.

To evaluate local effect of gaseous ozone on bacteria in deep carious lesions after incomplete caries removal, using chlorhexidine as control, and to investigate its effect on pulp vascular endothelial growth factor (VEGF), neuronal nitric oxide synthase (nNOS), and superoxide dismutase (SOD). Antibacterial effect was evaluated in 48 teeth with diagnosed deep carious lesion. After incomplete caries removal, teeth were randomly allocated into two groups regarding the cavity disinfectant used: ozone (open system) or 2% chlorhexidine. Dentin samples were analyzed for the presence of total bacteria and Lactobacillus spp. by real-time quantitative polymerase chain reaction. For evaluation of ozone effect on dental pulp, 38 intact permanent teeth indicated for pulp removal/tooth extraction were included. After cavity preparation, teeth were randomly allocated into two groups: ozone group and control group. VEGF/nNOS level and SOD activity in dental pulp were determined by enzyme-linked immunosorbent assay and spectrophotometric method, respectively. Ozone application decreased number of total bacteria (p = 0.001) and Lactobacillus spp. (p < 0.001), similarly to chlorhexidine. The VEGF (p < 0.001) and nNOS (p = 0.012) levels in dental pulp after ozone application were higher, while SOD activity was lower (p = 0.001) comparing to those in control pulp. Antibacterial effect of ozone on residual bacteria after incomplete caries removal was similar to that of 2% chlorhexidine. Effect of ozone on pulp VEGF, nNOS, and SOD indicated its biocompatibility. Ozone appears as effective and biocompatible cavity disinfectant in treatment of deep carious lesions by incomplete caries removal technique.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion

IntroductionLung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. Materials and methodsA total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. ResultsmRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (−63.7%), LTC4S (−35.9%) and iNOS (−60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). ConclusionLazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Pathology of nNOS-Expressing GABAergic Neurons in Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia that is often accompanied by mood and emotional disturbances and seizures. There is growing body of evidence that neurons expressing γ-aminobutyric acid (GABA) play an important role in regulation of cognition, mood, and emotion as well as seizure susceptibility, but participation of GABAergic neuronal pathology in Alzheimer’s disease (AD) is not understood well at present. Here, we report that transgenic mice expressing human amyloid precursor protein Swedish–Dutch–Iowa mutant (APPSweDI) exhibit early loss of neurons expressing GAD67, a GABA-synthesizing enzyme, in advance of the loss of pyramidal neurons in hippocampal CA1 region. The loss of GAD67+ neurons in APPSweDI mice accompanied with decreased spatial cognition as well as increased anxiety-like behaviors and kainic acid-induced seizure susceptibility at early phase. In the hippocampal CA1 region, GAD67+ neurons expressed high basal levels of neuronal nitric oxide synthase (nNOS) and nitrosative stress (nitrotyrosine). Similarly, GAD67+ neurons in primary cortical and hippocampal neuron cultures also expressed high basal levels of nNOS and degenerated in response to lower Aβ concentrations due to their high basal levels of nitrosative stress. Given the role of GABAergic neurons in cognitive and neuropsychiatric functions, this study reports the role of nNOS-mediated nitrosative stress in dysfunction of GABAergic neurons and its potential participation in early development of cognitive and neuropsychiatric symptoms in AD.

Endurance exercise training protects against the upregulation of nitric oxide in the striatum of MPTP/probenecid mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder, caused by the gradual loss of cells in substantia nigra. Nitric oxide (NO) plays an important role in a variety of signal transduction pathways that are crucial for maintaining the physiologic functions of nervous system. The aims of this study are: 1) To investigate the expression of the inducible form of NO (iNOS), and compare it to neuronal nitric oxide (nNOS) in the brain of a chronic mouse model of PD and 2) To study the effect of endurance exercise training on the expression of these markers.METHODMouse models of PD were obtained using 10 doses of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) (25 mg/kg) and probenecid (250 mg/kg) over 5 weeks. Forty C57BL/6 albino mice were randomly divided into four groups: sedentary control (SC, N = 10), exercise control (EC, N = 10), sedentary PD (SPD, N = 10), exercise PD (EPD, N = 10). At the end of training program, nNOS and iNOS were evaluated in the striatum in all animal groups using immunohistochemistry.RESULTSnNOS showed significant increases in striatum (ST) of SPD mice compared to SC mice (P &gt; 0.03). There was also decreased expression of nNOS in EC group compared to SC mice, but this decrease was not significant (P &gt; 0.8). Exercise training significantly decreased the level of nNOS in the EPD compared to SPD, (P &gt; 0.04). Although, iNOS expression followed almost the same trend as nNOS, but exercise training did not significantly decrease the expression of iNOS in both EC and EPD groups, P &gt; 0.2 and 0.3 respectively.DISCUSSIONThe data from this study suggests that 4 weeks of treadmill exercise has a positive impact on the expression of nNOS and iNOS in the striatum of a PD model. This might clear in part the pathogenicity of the diseases and the positive impact of training on PD.Support or Funding InformationThis work was funded from the Deanship of Scientific Research at Jordan University of Science and TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.

Early hyperbaric oxygen effects on neuropathic pain and nitric oxide synthase isoforms in CCI rats.

Neuropathic pain is pain caused by injury or dysfunction in the central and/or peripheral nervous system. Neuropathic pain has a high incidence with a complex mechanism, but effective treatment remains elusive. Hyperbaric oxygen (HBO) therapy has been widely used in the treatment of a variety of neurological diseases. The current study used a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. We observed the effects of early use of 2.5 absolute atmosphere (ATA) HBO on neuropathic pain-related behaviors and the expression of nitric oxide synthase (NOS) isoforms in the spinal dorsal horn. In the CCI group, mechanical withdrawal threshold (MWT) was decreased, Thermal withdrawal latency (TWL) was shortened, and mRNA and protein levels of iNOS and nNOS were significantly increased compared to the sham group. MWT was increased, TWL was enhanced, and iNOS and nNOS levels were significantly decreased in the HBO group compared to the CCI group. There was no change in eNOS levels across all groups. HBO treatment at early stages can improve hyperalgesia.

Increase of n-NOS and i-NOS in Rat Colon After Sacral Neuromodulation

Sacral neuromodulation (SNM) is proposed to treat different anorectal dysfunctions but its mechanism of action is not yet known. Our previous study demonstrated how SNM can significantly increase neuronal nitric oxide synthase NOS (n-NOS) and inducible NOS (i-NOS) expression in the anus and rectum of rats. There are no reports regarding the relation between SNM and NOS in colonic cells: our aim was to assess NOS expression in colonic rat model after SNM. Twenty-six female Sprangue-Dawley rats were considered: group I, normal control rats; group II, sham treatment rats, in whom electrodes for electrical stimulation were placed in S1 foramen bilaterally and left in place, without performing neuromodulation; group III, rats in whom SNM was performed. After 14 days, the rats were sacrificed and we evaluated n-NOS and i-NOS in colonic specimens by immunohistochemistry and Western Blot analysis. Western Blot analysis showed that levels of n-NOS and i-NOS were higher in colon of the III group rats respect to the others; in particular, immunohistochemistry revealed that, after neuromodulation, n-NOS expression in the muscle cells and i-NOS expression in glandular epithelium and nervous cells were highly represented (p < 0.05). Our study showed that in colon, SNM is able to influence NO synthesis, activating n-NOS expression in muscle cells and i-NOS expression in glandular epithelium and nervous cells. Our study showed a complex colonic response to SNM. This experimental model could be applied to better understand the mechanism of action of SNM in bowel dysfunction.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.