• Advances in pediatric surgery
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• v.11 no.1
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• pp.9-18
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• 2005

Abnormal distribution of enteric nerves such as adrenergic, cholinergic and non-adrenergic non-cholinergic nerves (NANC) may cause the failure of relaxation at the involved bowel segment in Hirschsprung's disease (HD). Nitric oxide (NO) is a major inhibitory NANC neurotransmitter in the gastrointestinal tract. NO is synthesized by activation of nNOS (neuronal nitric oxide synthase) in the intramural ganglion cells and regulates bowel movement. To assess the distribution of nNOS in HD, immunohistochemical staining to nNOS was utilized on paraffin embedded specimens. Ten control colon specimens were tested for feasibility of staining. Immunohistochemisrty was done on ganglionic colon as well as aganglionic segment of 15 patients with HD. nNOS immunoreactivity was observed in the neuronal cells, small cells and nerve fibers in the muscle layer and submucosal neuronal cells of control specimens. This finding was also observed in the ganglionic segments of HD. But, there was no nNOS immunoreactivity in aganglionic segments of HD. In conclusion nNOS immunohistochemical staining of paraffin embedded specimen is feasible and reliable. And the results suggest that the relaxation failure of the aganglionic bowel in HD is related to the absence of nNOS containing cells and nerve fibers.

• Journal of Acupuncture Research
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• v.23 no.5
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• pp.199-206
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• 2006

Objectives : The aim of this study was to investigate the effect of various electroacupuncture stimulation on neuronal nitric oxide synthase(nNOS) in cerebral cortex, brain stem, cerebellum of spontaneously hypertensive rats. Methods : We evaluated the changes of nNOS-positive neurons using a immunohistochemical method. The staining intensity of nNOS positive neurons was assessed in a quantitative fashion using a microdensitometrical method based on optical density by means of an image analyzer. Results : The average optical density of nNOS-positive neurons of 100 Hz (bipolar square wave 0.2 ms duration and 100 Hz frequency) electroacupuncture treatment group significantly decreased in most cortical areas comparison between the manual acupuncture and 2 Hz (bipolar square wave 0.2 ms duration and 2 Hz frequency) electroacupuncture groups. In the brain stem, the optical density of nNOS-positive neuron at superficial gray layer of the superior colliculus area, dorsolateral periaqueductal gray area and paralemniscal nucleus were same as cerebral cortex. Conclusion : We conclude that the morphological evidence for nNOS-positive neurons may be have regional change in cerebral cortex brain stem and cerebellum according to various electroacupuncture stimulations.

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.215-221
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• 2007

Neuronal cell toxicity induced by decreased nitric oxide (NO) production may be caused by modulation of constitutive neuronal NO synthase (nNOS). We used lead acetate ($Pb^{2+}$) to modulate physiological NO release and the related pathways of protein kinases like PKC, CaM-KII, and PKA in CATH.a cells, a dopaminergic cell line that has constitutive nNOS activity. In the cells treated with $Pb^{2+}$, cell viability and modulation (phosphorylation) levels of nNOS were determined by MTT assay and Western blot analysis, respectively. nNOS reductase activity (cytochrome c) was also assessed to compare the phosphorylation site-specific nNOS activity. nNOS activity was also determined by NADPH consumption rates. $Pb^{2+}$ treatment alone increased the phosphorylation of nNOS with decreased reductase activity. The phosphorylation levels increased markedly with decreased nNOS reductase activity, when $Pb^{2+}$ was combined with inhibitors for two (PKC and CaM-KII) or three (PKA, PKC and CaM-KII) protein kinases. Interestingly, when the cells were exposed to $Pb^{2+}$ plus PKC or CaM-KII inhibitor, the nNOS was phosphorylated strongly with the lowest activity. However, the levels of phosphorylated nNOS following $Pb^{2+}$ treatment decreased significantly after combined treatment with the PKA inhibitor, and $Pb^{2+}$-induced suppression of reductase activity did not occur. These results demonstrate that physiological NO release in the neuronal cells exposed to $Pb^{2+}$ can be decreased by PKA-mediated nNOS phosphorylation that may be caused by interactions with PKC and/or CaM-KII.

• Journal of Korean Neurosurgical Society
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• v.29 no.7
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• pp.877-885
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• 2000

Objective : The NOS inhibitors exhibit antinociceptive activity in rat model of neuropathic pain. NOS activity increases in the dorsal root ganglia(DRG) in neurop-athic pain. However, NOS activity decreases in the dorsal horn of spinal cord in the nerve injury models of neuropathic pain. To investigate whether the mechanism of decrease of NOS expression in the dorsal horn is related to a secondary effect resulting from increased NO production and likewise in the spinal DRG in the spinal nerve ligation model of neuropathic pain. Methods : We conducted behavioral tests for neuropathic pain, and nNOS immunohistochemistry and NADPH-diaphorase histochemistry after tight ligation of the 5th lumbar(L5) and 6th lumbar(L6) spinal nerves and L5 dorsal rhizotomy. Results : Typical neuropathic pain behaviors occurred 7 days after post-ligation in the neuropathic surgery group, but neuropathic pain behaviors in the dorsal rhizotomy group were absent or weak 7 days after post-operation. There was a decrease in the number of nNOS immunoreactive dorsal horn neurons on the both side(especially ipsilateral side) 7 days after post-ligation. The number of nNOS immunoreactive neurons in both side of the dorsal horn was not decreased 7 days after L5 dorsal rhizotomy. Conclusion : These data indicate that the changes in the injured DRG is essential for development and maintenance of neuropathic pain, and mechanism of decrease of nNOS expression in the dorsal horn is a secondary effect against the changes in the DRG including increased NO production in the spinal nerve ligation model of neuropathic pain.

• Journal of Korean Physical Therapy Science
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• v.10 no.2
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• pp.193-198
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• 2003

Acupuncture has been used as a clinical treatment in Oriental medicine for various diseases. In the present study was carried out to investigate the effects of acupuncture and electrical stimulation on the change neuronal nitric oxide synthase(nNOS) immunoreactive cells in the periaqueductal gray(PAG) area of the male SD rats. Enhanced expression of nNOS was detected in the dorsolateral-PAG(DL-PAG) area of rat with stress by fixed body, and acupuncture and needle electrode electrical stimulation groups at Hapgok like acupoint decreased the stress-induced enhancement in the expression of nNOS. The present results demonstrate that acupuncture and needle electrode electrical stimulation is effective in the modulation of expression of nNOS in the DL-PAG area under stress conditions.

• Molecular & Cellular Toxicology
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• v.4 no.1
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• pp.11-15
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• 2008

The expression of neuronal nitric oxide synthase (nNOS) is regulated by various spliced first exons (exon 1a-1i), sharing differentially common exon 2 in diverse human tissues. The highly complex structure and regulation of human nNOS gene gave limitations of information for the precise mechanism of nNOS regulation. In the present study, we report that the repeats of polymorphic dinucleotides $(GT)^nA(TG)^n$ repeats located in just upstream to the exon 1f in human nNOS gene play suppressive role in transcription, as shown in the characteristics of Z-DNA motif in other genes. In neuronal and trophoblast cells transfected transiently with luciferase construct without dinucleotide repeats at the 5'-flanking region of exon 1f in nNOS gene, the luciferase activity was increased markedly. However, the presence of the dinucleotide repeats dramatically suppressed the luciferase activity to the basal level, and which was dependent on the length of $(GT)^n$ and $(TG)^n$ repeats. More importantly, we found the polymorphisms in the length of dinucleotide repeats in human. Furthermore, we show for the first time here that there is a significant association of the lengths of polymorphic dinucleotide $(GT)^n$ and $(TG)^n$ repeats with the risk of schizophrenia.

• Journal of Acupuncture Research
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• v.21 no.5
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• pp.1-12
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• 2004

Objective : This study was to investigate the effect of acupuncture on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats. Methods : The experimental groups were divided into four groups : Normal, Choksamni(ST36), Kokchi(LI11), arbitrary group. Thereafter we evaluated changes in NADPH-diaphorase positive neurons histochemically and changes in nNOS neurons immunohistochemically. Results : 1. The optical densities of NADPH-diaphorase positive neurons of all the Choksamni & Kokchi groups were significantly different in SuG, DLPAG, IP, Pr, Gi areas of brain stem and cerebellum as compared to normal & arbitrary groups. In PPTg only Choksamni group was significantly different as compared to normal and arbitrary groups. 2. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG areas of brain stem as compared to normal group. In IP, Pr only Kokchi group was significantly different as compared to normal group. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG, PPTg, Pr, Gi areas of brain stem as compared to arbitrary group. In IP, Pr only Kokchi group was significantly different as compared to arbitrary group. 3. The optical densities of nNOS-positive neurons of all the Choksamni & Kokchi groups were not significantly different in cerebellum as compared to normal & arbitrary groups. Conclusions : We found out that acupuncture have effects on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.344-351
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• 2005

Nitric oxide (NO) and calcium-binding proteins occur in various types of cells in the central nervous system. They are important signaling and calcium buffering molecules, respectively. In the present study, using immunocytochemistry we examined the distribution and the co-localization pattern of neurons containing neuronal nitric oxide synthase (nNOS) and parvalbumin in the visual cortex of hamster. The overall number of parvalbumin-immunoreactive (IR) neurons was 17 times higher than that of the nNOS-IR neurons in the hamster visual cortex. The highest differences were found in layer V, where parvalbumin-IR neurons were 54.7 times more abundant than nNOS-IR neurons. Many nNOS- and parvalbumin-IR neurons were similar in size, shape, and manner of distribution in the visual cortex. However, two-color immunofluorescence revealed that no neurons in the hamster visual cortex expressed both nNOS and parvalbumin. The present results indicate that there are subtle species differences in the co-localization pattern between nNOS and calcium-binding proteins. The present results also suggest not only the heterogeneity and functional diversity of nNOS-IRneurons in the visual cortex, but also the importance of understanding animal diversity

• Journal of Life Science
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• v.29 no.1
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• pp.18-28
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• 2019

Recent studies reported that obesity upregulated the expression of neuronal nitric oxide synthase (nNOS) and regulated particular behavior patterns in animal models. They also reported that ameliorated the increase in nNOS expression and decreased depression and anxiolytic effects. Thus, exercise seems to be an effective strategy for improving brain function by downregulating nNOS. However, the immune response differs greatly, depending on the exercise intensity. The aim of the present study was to investigate differences in brain nNOS expression in obese C57BL/6 mice that performed exercise of different intensities. Obesity was induced in 6-wks-old mice (n=35) by feeding a 60%-fat diet for 6-wks. A control (CON) group (n=14) was fed a normal diet. At the end of the induction 6-wks period of obesity, seven animals in the CON group and obesity-induced group were sacrificed to confirm obesity induction (preliminary experiments and confirmation of visceral fat accumulation). The remaining animals were then used in an 8-wks exercise intervention. Other than the CON (n=7), the obesity-induced animals were divided into the following groups: high-fat diet (HFD, n=7), HFD-low intensity (HFD-LI, n=7, 12 m/min for 75 min), HFD-moderate intensity (HFD-MI, n=7, 15 m/min for 60 min), and HFD-high intensity (HFD-HI, n=7, 18 m/min for 50 min). The exercise was performed on an animal treadmill. The expression of the nNOS protein in the hippocampus was significantly higher in the HFD group as compared with that in the CON group (p<0.01). However, there was no difference in the hippocampal expression of the nNOS protein in the other exercise groups as compared with that in the CON group. In contrast, nNOS expression in the HFD-HI group was significantly lower than that in the HFD-LI group (p<0.05). The expression of phosphorylated Akt (pAkt) was significantly higher in all the exercise groups as compared with that in the CON and HFD groups. There was no difference in the expression of pAkt in the cerebral cortex among groups, and the expression of pAkt in the cerebellum was significantly higher in the HFD-HI group as compared with that in the CON group (p<0.05). There were also no between-group differences in pAkt expression in the cerebellum among the various exercise groups. In conclusion, nNOS seems to be overexpressed in response to obesity, and it appears to be downregulated by exercise. Relatively high-intensity exercise may be effective in improving brain function by downregulating nNOS.

• Clinical and Experimental Pediatrics
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• v.52 no.5
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• pp.594-602
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• 2009

Purpose : Transforming growth factor (TGF)-${\beta}1$ reportedly increases neuronal survival by inhibiting the induction of inducible nitric oxide synthase (NOS) in astrocytes and protecting neurons after excitotoxic injury. However, the neuroprotective mechanism of $TGF-{\beta}1$ on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether $TGF-{\beta}1$ has neuroprotective effects via a NO-mediated mechanism and N-methyl-D-aspartate (NMDA) receptor modulation on perinatal HI brain injury. Methods : Cortical cells were cultured using 19-day-pregnant Sprague-Dawley (SD) rats treated with $TGF-{\beta}1$ (1, 5, or 10 ng/mL) and incubated in a 1% O2 incubator for hypoxia. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 h of hypoxic exposure (7.5% $O_2$). $TGF-{\beta}1$ (0.5 ng/kg) was administered intracerebrally to the rats 30 min before HI brain injury. The expressions of NOS and NMDA receptors were measured. Results : In the in vitro model, the expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS) increased in the hypoxic group and decreased in the 1 ng/mL $TGF-{\beta}1-treated$ group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the $TGF-{\beta}1$-treated group. The expressions of eNOS and nNOS were reversed compared with the expression of iNOS. The expressions of all NMDA receptor subunits decreased in hypoxia group and increased in the $TGF-{\beta}1$-treated group except NR2C. Conclusion : The administration of $TGF-{\beta}1$ could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.