nNOS Nitric Oxide Research Articles

Investigation of the effect of Sugammadex on glutamate-induced neurotoxicity in C6 cell line and the roles played by nitric oxide and oxidative stress pathways.

This experiment was intended to evaluate the effect of sugammadex on the cytotoxicity induced by glutamate, involving the nitric oxide and oxidative stress pathways. C6 glioma cells were used in the study. Glutamate was given to cells in the glutamate group for 24 h. Sugammadex at different concentrations was given to cells in the sugammadex group for 24 h. Cells in the sugammadex + glutamate group were pre-treated with sugammadex at various concentrations for 1 h and then exposed to glutamate for 24 h. XTT assay was used to assess the cell viability. The levels of nitric oxide (NO), neuronal nitric oxide synthase (nNOS), total antioxidant (TAS), and total oxidant (TOS) in the cells were calculated using commercial kits. Apoptosis was detected by TUNEL assay. Sugammadex at concentrations of 50 and 100 μg/mL significantly enhanced the cell viability in C6 cells after the cytotoxicity induced by glutamate (p < 0.001). Moreover, sugammadex considerably decreased the levels of nNOS NO and TOS and the number of apoptotic cells and increased the level of TAS (p < 0.001). Sugammadex has protective and antioxidant properties on cytotoxicity and could be an effective supplement for neurodegenerative diseases such as Alzheimer and Parkinson if further research in vivo supports this claim.

Involvement of NMDAR/PSD-95/nNOS–NO–cGMP pathway in embryonic exposure to BPA induced learning and memory dysfunction of rats

Bisphenol A (BPA), can lead to learning and memory impairment, but the underlying mechanism is poorly understood. Researchers have indicated that the N-methyl-D-aspartate receptor (NMDAR)/postsynaptic density protein 95 (PSD-95)/neuronal nitric oxide synthase (nNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway greatly contributes to learning and memory process. Pregnant rats were exposed to 0, 0.05, 0.5, 5 and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19. Morris water maze, transmission electron microscope, western blot, real time PCR, biochemical analysis and ELISA were used to analyze the changes in behavior, synaptic ultrastructure, protein and gene expression of NMDAR, PSD-95, nNOS, together with nNOS activity, NO (Nitrate reductase method) and cGMP levels of the rat pups at different growth stages. Results of this research displayed that exposure to 0.5 mg/kg/day BPA could damage the spatial learning ability of rats at postnatal day (PND) 56. However, spatial memory ability could be affected by exposure to BPA at doses up to 5 mg/kg/day. Moreover, the thickness of the postsynaptic density decreased after exposure to BPA at doses of 5 and 50 mg/kg/day. Levels of NR1, NR2A, PSD-95 protein and mRNA were downregulated to some extent after exposure to BPA, whereas the expression of NR2B increased at GD 20 but decreased at PND 21 and 56. Contrarily, the nNOS expression along with the enzyme activity were promoted after exposure to BPA. Meanwhile, the NO and cGMP levels were suppressed at GD 20 but promoted at PND 21 and 56. In conclusion, these results demonstrated that NMDAR/PSD-95/nNOS–NO–cGMP pathway could be affected by embryonic exposure to BPA, which may involve in the spatial learning and memory dysfunction of rats in later life.

Angiotensin (1–7) through modulation of the NMDAR–nNOS–NO pathway and serotonergic metabolism exerts an anxiolytic-like effect in rats

Although recent studies have shown that angiotensin (1–7) (Ang [1–7]) exerts anti-stress and anxiolytic-like effects, the underlying mechanisms remain elusive. The ventral hippocampus (VH) is proposed to be a critical brain region for mood and stress management through the N-methyl-d-aspartate receptor (NMDAR) signaling pathway. However, the role of VH NMDAR signaling in the effects of Ang (1–7) remains unclear. In the present study, Ang (1–7) was injected into the bilateral VH of stressed rats, or in combination with a Fyn kinase inhibitor, NMDAR antagonist, neuronal nitric oxide synthase (nNOS) inhibitor, or nitric oxide (NO) scavenger. Anxiety-like behaviors were assessed using the open field test and elevated plus maze test, while alterations in NMDAR–nNOS–NO signaling and serotonergic metabolism were examined in the VH. After 21 days of chronic restraint stress, anxiety-like behaviors were evident. Levels of phosphorylated NR2B (a key NMDAR subunit), its upstream kinase Fyn, as well as activity of nNOS and monoamine oxidase (MAO) were markedly reduced. In contrast, levels of serotonin were increased. Bilateral VH infusion of Ang (1–7) recovered NMDAR–nNOS–NO signaling and MAO-mediated serotonin metabolism, as well as reducing anxiety-like behaviors in stressed rats. These effects were diminished by blockade of MasR (Ang [1–7]-specific receptor), Fyn kinase, NMDAR, nNOS, or NO production. Altogether, these findings indicate that Ang (1–7) exerts anxiolytic effects through modulation of the NMDAR–nNOS–NO pathway and serotonergic metabolism. Future translational research should focus on the relationship between Ang (1–7), glutamatergic neurotransmission, and serotonergic neurotransmission in the VH.

Activity-Induced SUMOylation of Neuronal Nitric Oxide Synthase Is Associated with Plasticity of Synaptic Transmission and Extracellular Signal-Regulated Kinase 1/2 Signaling.

Aims: Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) signaling have been implicated in learning, memory, and underlying long-lasting synaptic plasticity. In this study, we aimed at detecting whether nNOS is a target protein of SUMOylation in the hippocampus and its contributions to hippocampal long-term potentiation (LTP) of synaptic transmission. Results: We showed that N-methyl-d-aspartate receptor-dependent neuronal activity enhancement induced the attachment of small ubiquitin-like modifier 1 (SUMO1) to nNOS. Protein inhibitor of activated STAT3 (PIAS3) promoted SUMO1 conjugation at K725 and K739 on nNOS, which upregulated NO production and nNOS S1412 phosphorylation (activation). In addition, the N-terminus (amino acids 43-86) of PIAS3 bound nNOS directly. Tat-tagged PIAS3 segment representing amino acids 43-86, a cell-permeable peptide containing PIAS3 residues 43-86, suppressed activity-induced nNOS SUMOylation by disrupting PIAS3-nNOS association. It also decreased LTP-related expression of Arc and brain-derived neurotrophic factor and blocked signaling via extracellular signal-regulated kinase (ERK) 1/2 and Elk-1 in the hippocampus. More importantly, PIAS3-mediated nNOS SUMOylation was required for activity-regulated ERK1/2 activation in nNOS-positive neurons and hippocampal LTP induction. Innovation and Conclusion: These findings indicated that network activity-regulated nNOS SUMOylation underlies excitatory synaptic LTP by facilitating nNOS-NO-ERK1/2 signal cascades.

Sepiapterin alleviates impaired gastric nNOS function in spontaneous diabetic female rodents through NRF2 mRNA turnover and miRNA biogenesis pathway.

An impaired nitrergic system and altered redox signaling contribute to gastric dysmotility in diabetics. Our earlier studies show that NF-E2-related factor 2 (NRF2) and phase II antioxidant enzymes play a vital role in gastric neuronal nitric oxide synthase (nNOS) function. This study aims to investigate whether supplementation of sepiapterin (SEP), a precursor for tetrahydrobiopterin (BH4) (a cofactor of NOS) via the salvage pathway, restores altered nitrergic systems and redox balance in spontaneous diabetic (DB) female rats. Twelve-week spontaneous DB and age-matched, non-DB rats, with and without dietary SEP (daily 20 mg/kg body wt for 10 days) treatment, were used in this study. Gastric antrum muscular tissues were excised to investigate the effects of SEP in nitrergic relaxation and the nNOS-nitric oxide (NO)-NRF2 pathway(s). Dietary SEP supplementation significantly ( P < 0.05) reverted diabetes-induced changes in nNOS dimerization and function; nitric oxide (NO) downstream signaling molecules; HSP-90, a key regulator of nNOSα activity and dimerization; miRNA-28 that targets NRF2 messenger RNA (mRNA), and levels of microRNA (miRNA) biogenesis pathway components, such as DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and TRBP (HIV1-1 transactivating response RNA-binding protein). These findings emphasize the importance of the BH4 pathway in regulating gastric motility functions in DB animals by modulating nNOSα dimerization in association with changes in enteric NRF2 and NO downstream signaling. Our results also identify a new pathway, wherein SEP regulates NRF2 mRNA turnover by suppressing elevated miRNA-28, which could be related to alterations in miRNA biogenesis pathway components. NEW & NOTEWORTHY This study is the first to show a causal link between NF-E2-related factor 2 (NRF2) and neuronal nitric oxide synthase (nNOS) in gastric motility function. Our data demonstrate that critical regulators of the miRNA biosynthetic pathway are upregulated in the diabetic (DB) setting; these regulators were rescued by sepiapterin (SEP) treatment. Finally, we show that low dihydrofolate reductase expression may lead to impaired nNOS dimerization/function-reduced nitric oxide downstream signaling and elevate oxidative stress by suppressing the NRF2/phase II pathway through miRNA; SEP treatment restored all of the above in DB gastric muscular tissue. We suggest that tetrahydrobiopterin supplementation may be a useful therapy for patients with diabetes, as well as women with idiopathic gastroparesis.

CD70 Modulates the Role of eNOS In Endothelial Cells

CD70 has traditionally been identified on antigen presenting cells and interacts with CD27 on T cells, promoting proliferation and memory cell formation. We have recently shown that CD70 is important in the formation of effector memory T cells in hypertension. However, the precise cell types that express CD70 and the role of CD70 on non‐immune cells has not been investigated. Here we describe a new role of CD70 in endothelial function. We found that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by &gt; 30 fold by laminar (15 dynes/cm2) compared to oscillatory shear. Studies of mesenteric vascular reactivity showed that CD70−/− mice have markedly impaired endothelium‐dependent vasodilation to acetylcholine compared to WT mice (45± 5 vs 25 ± 4 %) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. Pre‐incubating vessels with the endothelial nitric oxide synthase (eNOS) inhibitor L‐NAME blocked endothelium‐dependent vasodilatation to acetylcholine in WT mice nearly 75%; in contrast, L‐NAME had no effect on endothelial vessel relaxation in CD70−/− mice. This suggests that vessels of CD70−/− mice lack the capacity to produce nitric oxide (NO). Western blot for the endothelial nitric oxide synthase (eNOS) showed a marked reduction of this enzyme in the aortas of CD70−/− as compared to wild type mice. We found that CD70 and eNOS interact by co‐immunoprecipitation, however this association is abolished by siRNA against CD70. In additional experiments, aortas from CD70−/− did demonstrate expression of both neuronal NOS (nNOS) and inducible NOS (iNOS). In order to assess functional effects of this expression, nNOS NO production was blocked with LVNIO, resulting in further impairment in vasodilation of CD70−/− mesenteric vessels (−logEC50 of 5.4 after treatment, compared to 6.2 before treatment); however blockade of iNOS with 1400 W resulted in apparent recovery of acetylcholine‐induced vasodilation. These data indicate that loss of CD70−/− leads to destabilization of the eNOS protein in endothelial cells resulting in endothelial dysfunction. In the absence of eNOS in CD70−/− vessels, iNOS contributes to endothelial dysfunction, while nNOS has a compensatory role. These studies have identified a new role of the immune co‐stimulatory protein CD70 in modulating endothelial eNOS protein levels and vascular function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.