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1
Rao, Y. Manjula, Arun Chaudhury, and Raj K. Goyal. "Active and inactive pools of nNOS in the nerve terminals in mouse gut: implications for nitrergic neurotransmission." American Journal of Physiology-Gastrointestinal and Liver Physiology 294, no. 3 (March 2008): G627—G634. http://dx.doi.org/10.1152/ajpgi.00519.2007.
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Nitric oxide (NO) is responsible for nitrergic neurotransmission in the gut, and its release is dependent on its de novo synthesis by neuronal nitric oxide synthase (nNOS). The magnitude of NO synthesis and release during neurotransmission may be related to the fraction of catalytically active nNOS out of a larger pool of inactive nNOS in the nerve terminals. The purpose of the present study was to identify catalytically active and inactive pools of nNOS in the varicosities from mouse gut. Enteric varicosities were confirmed as nitrergic by colocalization of nNOS with the nerve varicosity marker synaptophysin. Low-temperature SDS-PAGE of these varicosity extracts showed 320-, 250-, and 155-kDa bands when blotted with anti-nNOS1422–1433 and 320- and 155-kDa bands when blotted with anti-nNOS1–20 antibodies, respectively. The 320- and 155-kDa bands represent dimers and monomers of nNOSα; the 250- and 135-kDa bands represent dimers and monomers of nNOSβ. Immunoprecipitation with calmodulin (CaM) showed that a portion of nNOSα dimer was bound with CaM. On the other hand, a portion of nNOSα dimer, nNOSβ dimer, and all monomers lacked CaM binding. The CaM-lacking nNOS fractions reacted with anti-serine 847-phospho-nNOS. In vitro assays of NO production revealed that only the CaM-bound dimeric nNOSα was catalytically active; all other forms were inactive. We suggest that the amount of catalytically active nNOSα dimers may be regulated by serine 847 phosphorylation and equilibrium between dimers and monomers of nNOSα.
2
Saur, Dieter, Winfried L. Neuhuber, Bernd Gengenbach, Andrea Huber, Volker Schusdziarra, and Hans-Dieter Allescher. "Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract." American Journal of Physiology-Gastrointestinal and Liver Physiology 282, no. 2 (February 1, 2002): G349—G358. http://dx.doi.org/10.1152/ajpgi.00226.2001.
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5′ mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOSα) or alternative splicing encoding NH2-terminally truncated proteins (nNOSβ/γ) that lack the PDZ/GLGF domain for protein-protein interaction of nNOSα. We studied the expression of 5′ nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH2-terminal nNOS antibodies. 5′ nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOSα-a, -b, -c; nNOSβ) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOSα-a and nNOSβ mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOSα and nNOSβ cDNAs revealed proteins of ∼155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH2-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5′ nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOSα knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.
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Huber, Andrea, Dieter Saur, Manfred Kurjak, Volker Schusdziarra, and Hans-Dieter Allescher. "Characterization and splice variants of neuronal nitric oxide synthase in rat small intestine." American Journal of Physiology-Gastrointestinal and Liver Physiology 275, no. 5 (November 1, 1998): G1146—G1156. http://dx.doi.org/10.1152/ajpgi.1998.275.5.g1146.
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The aim of this study was to characterize neuronal nitric oxide synthase (nNOS) activity and 5′-end splice variants in rat small intestine. nNOS was predominantly expressed in the longitudinal muscle layer, with attached myenteric plexus (LM-MP). The biochemical properties of NOS activity in enriched nerve terminals resemble those of nNOS isolated from the brain. Western blot analysis of purified NOS protein with an nNOS antibody showed a single band in the particulate fraction and three bands in the soluble fraction. Rapid amplification of 5′ cDNA ends-PCR revealed the presence of three different 5′-end splice variants of nNOS. Two variants encode for nNOSα, which has a specific domain for membrane association. The third variant encodes for nNOSβ, which lacks the domain for membrane association and should therefore be soluble. nNOS is predominantly expressed in LM-MP and can be enriched in enteric nerve terminals. We present the first evidence that three 5′-end splice variants of nNOS encoding two different proteins are expressed in rat small intestine. These two nNOS enzymes exhibit different subcellular locations and might be implicated in different biological functions.
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Chaudhury, Arun, Y. Manjula Rao, and Raj K. Goyal. "PIN/LC8 is associated with cytosolic but not membrane-bound nNOS in the nitrergic varicosities of mice gut: implications for nitrergic neurotransmission." American Journal of Physiology-Gastrointestinal and Liver Physiology 295, no. 3 (September 2008): G442—G451. http://dx.doi.org/10.1152/ajpgi.90280.2008.
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This investigation demonstrates the presence and binding of the protein LC8 (described as “protein inhibitor of nNOS” or PIN in some reports) to different components of neuronal nitric oxide synthase (nNOS) in nitrergic varicosities of mice gut. Whole varicosity extracts showed three (320-, 250-, and 155-kDa) nNOS bands with anti-nNOS1422–1433 antibody and a 10-kDa band with anti-LC8 antibody. The LC8 immunoprecipitate (IP) showed three nNOS bands, suggesting that LC8 was bound with all three forms of nNOS but dissociated from them during SDS-PAGE. Studies using LC8 IP and supernatant and probed with anti-CaM showed that LC8 was not associated with CaM-bound 320-kDa nNOS but was present in the CaM-lacking fraction. Probing these fractions with anti-serine847-P-nNOS showed that 320-kDa serine847-phosphorylated-nNOS consisted of LC8-bound and LC8-lacking components. Subsequent studies with varicosity membrane and cytosolic fractions separately showed that membrane contained CaM-bound and CaM-lacking, serine847-phosphorylated 320-kDa nNOS; both these fractions lacked LC8. On the other hand, the cytosolic fraction contained CaM-lacking, serine847-phosphorylated 320-kDa, 250-kDa, and 155-kDa nNOS bands that were all associated with LC8. These studies, along with in vitro nitric oxide assays, show that in gut nitrergic nerve varicosities 1) all cytosolic serine847-phosphorylated nNOS was catalytically inactive and bound with LC8, and 2) membrane-associated nNOS consisted of catalytically active, CaM-bound and catalytically inactive, CaM-lacking, serine847-phosphorylated nNOSα dimers, both of which lacked LC8. These results suggest that LC8 may dissociate from the 320-kDa nNOSα dimer upon binding to membrane, thus supporting the view that LC8 may transport nNOSα dimer to the varicosity membrane for participation in nitrergic neurotransmission.
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Smith, Cheryl A., Beth Santymire, Aaron Erdely, Vasuki Venkat, György Losonczy, and Chris Baylis. "Renal nitric oxide production in rat pregnancy: role of constitutive nitric oxide synthases." American Journal of Physiology-Renal Physiology 299, no. 4 (October 2010): F830—F836. http://dx.doi.org/10.1152/ajprenal.00300.2010.
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Functional studies show that increased renal nitric oxide (NO) mediates the renal vasodilation and increased glomerular filtration rate that occur during normal pregnancy. We investigated whether changes in the constitutive NO synthases (NOS), endothelial (eNOS) and neuronal (nNOS), were associated with the increased renal NO production in normal midterm pregnancy in the rat. In kidneys from midterm pregnant (MP: 11–13 days gestation), late-term pregnant (LP: 18–20 days gestation), and similarly aged virgin (V) rats, transcript and protein abundance for eNOS and the nNOSα and nNOSβ splice variants, as well as the rate of l-arginine-to-l-citrulline conversion, were determined as a measure of NOS activity. At MP, renal cortical abundance of the total eNOS protein and phosphorylated (Ser1177) eNOS was reduced, and l-arginine-to-l-citrulline conversion in the cortical membrane fraction was decreased; these declines were also seen in LP. There were no changes in the eNOS transcript. In contrast, l-arginine-to-l-citrulline conversion in the soluble fraction of renal cortex increased at MP and then declined at LP. This MP increase was ablated by S-methylthiocitrulline, a nNOS inhibitor. Using Western blotting, we did not detect a change in the protein abundance or transcript of the 160-kDa nNOSα, but protein abundance and transcript of the nNOSβ were increased at MP in cortex. Collectively, these studies suggest that the soluble nNOSβ is responsible for the increased renal cortical NO production during pregnancy.
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Tu, Angyan, Jun Ye, and Bing Wang. "Neutrosophic Number Optimization Models and Their Application in the Practical Production Process." Journal of Mathematics 2021 (April 19, 2021): 1–8. http://dx.doi.org/10.1155/2021/6668711.
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In order to simplify the complex calculation and solve the difficult solution problems of neutrosophic number optimization models (NNOMs) in the practical production process, this paper presents two methods to solve NNOMs, where Matlab built-in function “fmincon()” and neutrosophic number operations (NNOs) are used in indeterminate environments. Next, the two methods are applied to linear and nonlinear programming problems with neutrosophic number information to obtain the optimal solution of the maximum/minimum objective function under the constrained conditions of practical productions by neutrosophic number optimization programming (NNOP) examples. Finally, under indeterminate environments, the fit optimal solutions of the examples can also be achieved by using some specified indeterminate scales to fulfill some specified actual requirements. The NNOP methods can obtain the feasible and flexible optimal solutions and indicate the advantage of simple calculations in practical applications.
7
Sampath, Chethan, Abhinav V. Raju, Michael L. Freeman, Shanthi Srinivasan, and Pandu R. Gangula. "Nrf2 attenuates hyperglycemia-induced nNOS impairment in adult mouse primary enteric neuronal crest cells and normalizes stomach function." American Journal of Physiology-Gastrointestinal and Liver Physiology 322, no. 3 (March 1, 2022): G368—G382. http://dx.doi.org/10.1152/ajpgi.00323.2021.
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Primary neuronal cell crust (pENCs) in the intestine habitats nNOS and Nrf2, which was suppressed in diabetic gastroparesis. Activation of Nrf2 restored nNOS by suppressing inflammatory markers in pENCs cells. Inhibition of Nrf2 reveals a negative feedback mechanism for the activation of GSK-3. Activation of Nrf2 alleviates STZ-induced delayed gastric emptying and nitrergic relaxation in female mice. Activation of Nrf2 restored impaired gastric BH4 biosynthesis enzyme GCH-1, nNOSα expression thus regulating nitric oxide levels.
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Ihara, Hideshi, Atsushi Kitamura, Shingo Kasamatsu, Tomoaki Ida, Yuki Kakihana, Hiroyasu Tsutsuki, Tomohiro Sawa, Yasuo Watanabe, and Takaaki Akaike. "Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation." Biochemical Journal 474, no. 7 (March 15, 2017): 1149–62. http://dx.doi.org/10.1042/bcj20160999.
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We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3′,5′-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.
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Zhao, Tianxue, Qian Li, Qianyun Mao, Kaida Mu, and Chen Wang. "Hepatic nNOS impaired hepatic insulin sensitivity through the activation of p38 MAPK." Journal of Endocrinology 248, no. 3 (March 2021): 265–75. http://dx.doi.org/10.1530/joe-20-0322.
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Neuronal nitric oxide synthase (nNOS) interacts with its adaptor protein NOS1AP through its PZD domain in the neurons. Previously, we had reported that NOS1AP enhanced hepatic insulin sensitivity through its PZD-binding domain, which suggested that nNOS might mediate the effect of NOS1AP. This study aimed to examine the role and underlying mechanisms of nNOS in regulating hepatic insulin sensitivity. nNOS co-localized with NOS1AP in mouse liver. The overexpression of NOS1AP in mouse liver decreased the level of phosphorylated nNOS (p-nNOS (Ser1417)), the active form of nNOS. Conversely, the liver-specific deletion of NOS1AP increased the level of p-nNOS (Ser1417). The overexpression of nNOS in the liver of high-fat diet-induced obese mice exacerbated glucose intolerance, enhanced intrahepatic lipid accumulation, decreased glycogen storage, and blunted insulin-induced phosphorylation of IRbeta and Akt in the liver. Similarly, nNOS overexpression increased triglyceride production, decreased glucose utilization, and downregulated insulin-induced expression of p-IRbeta, p-Akt, and p-GSK3beta in the HepG2 cells. In contrast, treatment with Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, improved glucose tolerance and upregulated insulin-induced phosphorylation of IRbeta and Akt in the liver of ob/ob mice. Furthermore, overexpression of nNOS increased p38MAPK phosphorylation in the HepG2 cells. In contrast, inhibition of p38MAPK with SB203580 significantly reversed the nNOS-induced inhibition of insulin-signaling activity (all P < 0.05). This indicated that hepatic nNOS inhibited the insulin-signaling pathway through the activation of p38MAPK. These findings suggest that nNOS is involved in the development of hepatic insulin resistance and that nNOS might be a potential therapeutic target for diabetes.
10
Choate, J. K., E. J. F. Danson, J. F. Morris, and D. J. Paterson. "Peripheral vagal control of heart rate is impaired in neuronal NOS knockout mice." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 6 (December 1, 2001): H2310—H2317. http://dx.doi.org/10.1152/ajpheart.2001.281.6.h2310.
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The role of nitric oxide (NO) in the vagal control of heart rate (HR) is controversial. We investigated the cholinergic regulation of HR in isolated atrial preparations with an intact right vagus nerve from wild-type (nNOS+/+, n = 81) and neuronal NO synthase (nNOS) knockout (nNOS−/−, n = 43) mice. nNOS was immunofluorescently colocalized within choline-acetyltransferase-positive neurons in nNOS+/+ atria. The rate of decline in HR during vagal nerve stimulation (VNS, 3 and 5 Hz) was slower in nNOS−/− compared with nNOS+/+ atria in vitro ( P < 0.01). There was no difference between the HR responses to carbamylcholine in nNOS+/+ and nNOS−/− atria. Selective nNOS inhibitors, vinyl-l-niohydrochloride or 1–2-trifluoromethylphenyl imidazole, or the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one significantly ( P < 0.05) attenuated the decrease in HR with VNS at 3 Hz in nNOS+/+ atria. NOS inhibition had no effect in nNOS−/− atria during VNS. In all atria, the NO donor sodium nitroprusside significantly enhanced the magnitude of the vagal-induced bradycardia, showing the downstream intracellular pathways activated by NO were intact. These results suggest that neuronal NO facilitates vagally induced bradycardia via a presynaptic modulation of neurotransmission.
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Lu, Deyin, Yiling Fu, Arnaldo Lopez-Ruiz, Rui Zhang, Ramiro Juncos, Haifeng Liu, R. Davis Manning, Luis A. Juncos, and Ruisheng Liu. "Salt-sensitive splice variant of nNOS expressed in the macula densa cells." American Journal of Physiology-Renal Physiology 298, no. 6 (June 2010): F1465—F1471. http://dx.doi.org/10.1152/ajprenal.00650.2009.
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Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-β is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-α, nNOS-β, and nNOS-γ were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, α-, β-, and γ-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-α decreased markedly, whereas nNOS-β increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 ± 3% in normal-salt rats and 52 ± 6% ( P < 0.05) in the high-salt group. We found 1) macula densa cells express nNOS-α, nNOS-β, and nNOS-γ, 2) a high-salt diet enhances nNOS-β, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-β. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were α-, β-, and γ-isoforms and propose that enhanced level of nNOS-β during high-salt intake may contribute to macula densa NO production and help attenuate TGF.
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SONG, Yao, Jay L. ZWEIER, and Yong XIA. "Heat-shock protein 90 augments neuronal nitric oxide synthase activity by enhancing Ca2+/calmodulin binding." Biochemical Journal 355, no. 2 (April 6, 2001): 357–60. http://dx.doi.org/10.1042/bj3550357.
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Heat-shock protein 90 (hsp90) has been shown to facilitate neuronal NO synthase (nNOS, type 1) activity in vivo. But the direct effect of hsp90 on purified nNOS has not been determined yet. Moreover, the mechanism underlying the action of hsp90 is not known. nNOS activity is primarily initiated and regulated by the binding of Ca2+/calmodulin (CaM). Therefore, we explored whether hsp90 modulates nNOS activity by affecting CaM binding. Recombinant rat nNOS was purified from the stably transfected cells by affinity chromatography. hsp90 increased nNOS activity in a dose-dependent manner with an EC50 of 24.1±6.4nM. In the presence of hsp90, the CaM-nNOS dose-response curve was shifted markedly to the left and the maximal activity was also elevated. Further in vitro protein-binding experiments confirmed that hsp90 increased the binding of CaM to nNOS. Taken together, these data indicate that hsp90 directly augments nNOS catalytic function and that this effect is, at least partially, mediated by CaM-binding enhancement.
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YONEYAMA, Hirohito, Akira YAMAMOTO, and Hiroaki KOSAKA. "Neuronal nitric oxide synthase generates superoxide from the oxygenase domain." Biochemical Journal 360, no. 1 (November 8, 2001): 247–53. http://dx.doi.org/10.1042/bj3600247.
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When l-arginine is depleted, neuronal nitric oxide synthase (nNOS) has been reported to generate superoxide. A flavoprotein module construct of nNOS has been demonstrated to be sufficient for superoxide production. In contrast, nNOS was reported not to be involved in superoxide formation, because such formation occurred with a mixture of the boiled enzyme and redox-active cofactors. We aimed to resolve these controversial issues by examining superoxide generation, without the addition of redox-active cofactors, by recombinant wild-type nNOS and by C415A-nNOS, which has a mutation in the haem proximal site. In a superoxide-sensitive adrenochrome assay, the initial lag period of C415A-nNOS was increased 2-fold compared with that of native nNOS. With ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, prominent signals of the superoxide adduct were obtained with wild-type nNOS, whereas an enzyme preparation boiled for 5min did not produce superoxide. Higher concentrations of NaCN (10mM) decreased superoxide formation by 63%. Although the activity of the reductase domain was intact, superoxide generation from C415A-nNOS was decreased markedly, to only 10% of that of the wild-type enzyme. These results demonstrate that nNOS truly catalyses superoxide formation, that this involves the oxygenase domain, and that full-length nNOS hinders the reductase domain from producing superoxide.
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Wang, Xianhong, Ming Lu, Yongzong Gao, Andreas Papapetropoulos, William C. Sessa, and Wenhui Wang. "Neuronal nitric oxide synthase is expressed in principal cell of collecting duct." American Journal of Physiology-Renal Physiology 275, no. 3 (September 1, 1998): F395—F399. http://dx.doi.org/10.1152/ajprenal.1998.275.3.f395.
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We used the RT-PCR technique and immunocytochemical methods to determine the expression of endothelial nitric oxide synthase (eNOS) or neuronal nitric oxide synthase (nNOS) in the cortical collecting duct (CCD) in rats on high-K+ diet. The microdissected CCDs of the rat kidney were lysed, and RT-PCR was carried out using rat nNOS and eNOS gene-specific primers. Southern analysis showed the presence of mRNA of nNOS but not eNOS in the CCD. The presence of nNOS in the CCD was further confirmed by light microscopy. We used the polyclonal nNOS antibody in immunocytochemical studies of the isolated CCD. We found that immunoreactivity to nNOS was present in the CCD and heterogeneous with positive and negative immunostaining. We performed the immunocytochemical studies in the split-open CCD and found that the immunoreactivity to nNOS was detected only in principal cells but not in intercalated cells. We conclude that nNOS is expressed in the rat CCD in rats on high-K+ diet. The presence of nNOS in the CCD is heterogeneous and mainly located in principal cells.
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Gangula, Pandu R., Kishore B. Challagundla, Kalpana Ravella, Sutapa Mukhopadhyay, Vijayakumar Chinnathambi, Mukul K. Mittal, K. Raja Sekhar, and Chethan Sampath. "Sepiapterin alleviates impaired gastric nNOS function in spontaneous diabetic female rodents through NRF2 mRNA turnover and miRNA biogenesis pathway." American Journal of Physiology-Gastrointestinal and Liver Physiology 315, no. 6 (December 1, 2018): G980—G990. http://dx.doi.org/10.1152/ajpgi.00152.2018.
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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.
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Choate, J. K., S. M. Murphy, R. Feldman, and C. R. Anderson. "Sympathetic control of heart rate in nNOS knockout mice." American Journal of Physiology-Heart and Circulatory Physiology 294, no. 1 (January 2008): H354—H361. http://dx.doi.org/10.1152/ajpheart.00898.2007.
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Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS−/−) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1–10 Hz) and bath-applied norepinephrine (NE; 10−8–10−4 mol/l) on HR were examined in atria from nNOS−/− and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS−/− atria showed significantly reduced HR responses to sympathetic nerve activation and NE ( P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10−7–10−5 mol/l) was attenuated in nNOS−/− atria ( P < 0.05). Constitutive NOS inhibition with l-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS−/− and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.
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Han, Jeong Pil, Jeong Hyeon Lee, Geon Seong Lee, Ok Jae Koo, and Su Cheong Yeom. "Positive Correlation between nNOS and Stress-Activated Bowel Motility Is Confirmed by In Vivo HiBiT System." Cells 10, no. 5 (April 27, 2021): 1028. http://dx.doi.org/10.3390/cells10051028.
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Neuronal nitric oxide synthase (nNOS) has various roles as a neurotransmitter. However, studies to date have produced insufficient data to fully support the correlation between nNOS and bowel motility. This study aimed to investigate the correlation between nNOS expression and gastrointestinal (GI) tract motility using a stress-induced neonatal maternal separation (NMS) mouse model. In this study, we generated a genetically modified mouse with the HiBiT sequence knock-in into the nNOS gene using CRISPR/Cas9 for analyzing accurate nNOS expression. nNOS expression was measured in the stomach, small intestine, large intestine, adrenal gland, and hypothalamus tissues after establishing the NMS model. The NMS model exhibited a significant increase in nNOS expression in large intestine, adrenal gland, and hypothalamus. Moreover, a significant positive correlation was observed between whole gastrointestinal transit time and the expression level of nNOS. We reasoned that NMS induced chronic stress and consequent nNOS activation in the hypothalamic-pituitary-adrenal (HPA) axis, and led to an excessive increase in intestinal motility in the lower GI tract. These results demonstrated that HiBiT is a sensitive and valuable tool for analyzing in vivo gene activation, and nNOS could be a biomarker of the HPA axis-linked lower intestinal tract dysfunction.
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Nagl, Florian, Katrin Schönhofer, Barbara Seidler, Jörg Mages, Hans-Dieter Allescher, Roland M. Schmid, Günter Schneider, and Dieter Saur. "Retinoic acid-induced nNOS expression depends on a novel PI3K/Akt/DAX1 pathway in human TGW-nu-I neuroblastoma cells." American Journal of Physiology-Cell Physiology 297, no. 5 (November 2009): C1146—C1156. http://dx.doi.org/10.1152/ajpcell.00034.2009.
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Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a–1l, transcribed from distinct promoters, leading to nNOS variants with different 5′-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.
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VALLON, VOLKER, TIMOTHY TRAYNOR, LUCIANO BARAJAS, YUNING G. HUANG, JOSIE P. BRIGGS, and JÜRGEN SCHNERMANN. "Feedback Control of Glomerular Vascular Tone in Neuronal Nitric Oxide Synthase Knockout Mice." Journal of the American Society of Nephrology 12, no. 8 (August 2001): 1599–606. http://dx.doi.org/10.1681/asn.v1281599.
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Abstract. For further elucidation of the role of neuronal nitric oxide synthase (nNOS) in macula densa (MD) cells, experiments were performed in anesthetized nNOS knockout mice (nNOS -/-). At comparable levels of arterial BP, renal blood flow was not significantly different between nNOS +/+ and nNOS -/- (1.7 ± 0.2 versus 1.4 ± 0.1 ml/min), and autoregulation of renal blood flow was maintained to a pressure level of approximately 85 mmHg in both groups of mice (n = 6 in each group). The fall in proximal tubular stop-flow pressure in response to an increase in loop of Henle perfusion rate from 0 to 30 nl/min was comparable in nNOS +/+ and -/- mice (40.7 ± 1.6 to 32 ± 2 mmHg versus 40.6 ± 1.6 to 31.6 ± 2 mmHg; not significant; n = 13 versus 18 nephrons). Luminal application of the nonselective NOS inhibitor nitro-L-arginine (10-3 and 10-2 M) enhanced the perfusion-dependent fall in stop-flow pressure in nNOS +/+ (7 ± 1 to 13 ± 2 mmHg; P < 0.05) but not in nNOS -/- (7 ± 1 to 8 ± 1 mmHg; not significant) mice. nNOS -/- mice exhibited a lower nephron filtration rate, compared with nNOS +/+, during free-flow collections from early distal tubules (influence of MD intact, 7 ± 0.7 versus 10.9 ± 1 nl/min; P = 0.002) but not from late proximal tubule (influence of MD minimized, 10.1 ± 1 versus 11.7 ± 1 nl/min; not significant; n = 16 nephrons). Distal Cl concentration and fractional absorption of fluid or chloride up to the early distal tubule was not different between nNOS -/- and +/+ mice. The data indicate that nNOS in MD tonically attenuates the GFR-lowering influence of ambient luminal NaCl, which may serve to increase the fluid and electrolyte load to the distal tubule, consistent with a role of MD nNOS in tubuloglomerular feedback resetting.
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ROCZNIAK, AGNES, JAMES N. FRYER, DAVID Z. LEVINE, and KEVIN D. BURNS. "Downregulation of Neuronal Nitric Oxide Synthase in the Rat Remnant Kidney." Journal of the American Society of Nephrology 10, no. 4 (April 1999): 704–13. http://dx.doi.org/10.1681/asn.v104704.
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Abstract. Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42 ± 15.60 versus 5/6 Nx: 47.55 ± 12.78 arbitrary units, P < 0.05, n = 4) and inner medulla (sham: 147.70 ± 26.96 versus 5/6 Nx: 36.95 ± 17.24 arbitrary units, P < 0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66 ± 0.04-fold of sham values, P < 0.05, n = 6) and inner medulla (0.74 ± 0.12-fold of sham values, P < 0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.
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Kumar, Gaurav, Pankaj Paliwal, Nishant Patnaik, and Ranjana Patnaik. "Withania somniferaphytochemicals confer neuroprotection by selective inhibition of nNos: Anin silicostudy to search potent and selective inhibitors for human nNOS." Journal of Theoretical and Computational Chemistry 16, no. 05 (August 2017): 1750042. http://dx.doi.org/10.1142/s0219633617500420.
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Neuronal nitric oxide synthase (nNOS or NOS1) is an important therapeutic target for the treatment of various neurological diseases. A major challenge faced in the design of nNOS inhibitors emphasizes on potency in humans and selectivity over other NOS isoforms — eNOS and iNOS. The present structural-based in silico study was carried out to search potent and selective inhibitor for human nNOS from a set of 40 Withania somnifera phytochemicals structure. Ten phytochemicals appear as dual-selective inhibitors of nNOS over both iNOS and eNOS. Here we report five potent and selective human nNOS inhibitors, namely, Chlorogenic Acid, Withanolide B, Withacnistin Pelletierine, and Calystegine B2 based on their selectivity, binding energy and nNOS active site residues interaction profile. These phytochemicals have nNOS selectivity higher than 4-methyl-6-(2-(5-(3-(methylamino)propyl)pyridin-3-yl)-ethyl)pyridin-2-amine inhibitor and have potential as an oral neurotherapeutic agent to combat neurological disorders mediated by nNOS activation.
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Ebert, Jutta G., Marek Zelenka, Ingolf Gath, Ute Gödtel-Armbrust, and Ulrich Förstermann. "Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes." American Journal of Physiology-Cell Physiology 284, no. 4 (April 1, 2003): C1065—C1072. http://dx.doi.org/10.1152/ajpcell.00476.2002.
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In mammalian skeletal muscle, neuronal-type nitric oxide synthase (nNOS) is found to be enriched at neuromuscular endplates. Here we demonstrate the colocalization of the nicotinic acetylcholine receptor (nAChR, stained with α-bungarotoxin) and nNOS (stained with a specific antibody) in murine C2C12 myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C2C12myotubes. An antibody to the α1-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the α1-subunit of the nAChR. Treatment of mice with bacterial LPS downregulated the expression of nNOS in skeletal muscle, and treatment of C2C12 cells with bacterial LPS and interferon-γ markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (α-, γ-, and ε-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine skeletal muscle and C2C12 cells but differ in their expressional regulation.
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Stegbauer, Johannes, Yvonne Kuczka, Oliver Vonend, Ivo Quack, Lorenz Sellin, Andreas Patzak, Andreas Steege, Kristina Langnaese, and Lars Christian Rump. "Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 294, no. 2 (February 2008): R421—R428. http://dx.doi.org/10.1152/ajpregu.00481.2007.
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Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(−/−)], and nNOS-deficient [nNOS(−/−)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC50 and maximal pressor responses to angiotensin II were greater in eNOS(−/−) than in nNOS(−/−) and smaller in wild-type mice. The nonselective NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(−/−) and wild-type mice but not in eNOS(−/−) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. l-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(−/−) and wild-type mice but not in eNOS(−/−) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3′, 5′-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.
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Paliege, Alex, Diane Mizel, Carmen Medina, Anita Pasumarthy, Yuning G. Huang, Sebastian Bachmann, Josephine P. Briggs, Jurgen B. Schnermann, and Tianxin Yang. "Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E2." American Journal of Physiology-Renal Physiology 287, no. 1 (July 2004): F152—F159. http://dx.doi.org/10.1152/ajprenal.00287.2003.
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It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS −/− mice on a mixed genetic background and in COX-2 −/− mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of PGE2 on nNOS expression. In a cultured macula densa cell line, PGE2 significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 −/− mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day ( PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 −/− mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE2 on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation.
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Bedenbaugh, Michelle N., Richard B. McCosh, Justin A. Lopez, John M. Connors, Robert L. Goodman, and Stanley M. Hileman. "Neuroanatomical Relationship of Neuronal Nitric Oxide Synthase to Gonadotropin-Releasing Hormone and Kisspeptin Neurons in Adult Female Sheep and Primates." Neuroendocrinology 107, no. 3 (2018): 218–27. http://dx.doi.org/10.1159/000491393.
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Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity. Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships. Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates. Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep.
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Lawler, John M., Mary Kunst, Jeff M. Hord, Yang Lee, Kumar Joshi, Rachel E. Botchlett, Angelo Ramirez, and Daniel A. Martinez. "EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 306, no. 7 (April 1, 2014): R470—R482. http://dx.doi.org/10.1152/ajpregu.00371.2013.
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Reduced mechanical loading during bedrest, spaceflight, and casting, causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma to the cytosol. We used EUK-134, a cell-permeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOSμ translocation and muscle fiber atrophy as a result of short-term (54 h) hindlimb unloading. Fischer-344 rats were divided into ambulatory control, hindlimb-unloaded (HU), and hindlimb-unloaded + EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOSμ immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOSμ protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr-32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOSμ translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus, redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers.
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Sharma, Neeru M., Tamra L. Llewellyn, Hong Zheng, and Kaushik P. Patel. "Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN." American Journal of Physiology-Heart and Circulatory Physiology 305, no. 6 (September 15, 2013): H843—H855. http://dx.doi.org/10.1152/ajpheart.00170.2013.
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An increased sympathetic drive is an adverse characteristic in chronic heart failure (CHF). The protein expression of neuronal nitric oxide synthase (nNOS)- and hence nitric oxide (NO)-mediated sympathoinhibition is reduced in the paraventricular nucleus (PVN) of rats with CHF. However, the molecular mechanism(s) of nNOS downregulation remain(s) unclear. The aim of the study was to reveal the underlying molecular mechanism for the downregulation of nNOS in the PVN of CHF rats. Sprague-Dawley rats with CHF (6–8 wk after coronary artery ligation) demonstrated decreased nNOS dimer/monomer ratio (42%), with a concomitant increase in the expression of PIN (a protein inhibitor of nNOS known to dissociate nNOS dimers into monomers) by 47% in the PVN. Similarly, PIN expression is increased in a neuronal cell line (NG108) treated with angiotensin II (ANG II). Furthermore, there is an increased accumulation of high-molecular-weight nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of CHF rats (29%). ANG II treatment in NG108 cells in the presence of a proteasome inhibitor, lactacystin, also leads to a 69% increase in accumulation of nNOS-Ub conjugates immunoprecipitated by an antiubiquitin antibody. There is an ANG II-driven, PIN-mediated decrease in the dimeric catalytically active nNOS in the PVN, due to ubiquitin-dependent proteolytic degradation in CHF. Our results show a novel intermediary mechanism that leads to decreased levels of active nNOS in the PVN, involved in subsequent reduction in sympathoinhibition during CHF, offering a new target for the treatment of CHF and other cardiovascular diseases.
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van’t Hof, Robert J., Jeny MacPhee, Helene Libouban, Miep H. Helfrich, and Stuart H. Ralston. "Regulation of Bone Mass and Bone Turnover by Neuronal Nitric Oxide Synthase." Endocrinology 145, no. 11 (November 1, 2004): 5068–74. http://dx.doi.org/10.1210/en.2004-0205.
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Abstract Nitric oxide (NO) is produced by NO synthase (NOS) and plays an important role in the regulation of bone cell function. The endothelial NOS isoform is essential for normal osteoblast function, whereas the inducible NOS isoform acts as a mediator of cytokine effects in bone. The role of the neuronal isoform of NOS (nNOS) in bone has been studied little thus far. Therefore, we investigated the role of nNOS in bone metabolism by studying mice with targeted inactivation of the nNOS gene. Bone mineral density (BMD) was significantly higher in nNOS knockout (KO) mice compared with wild-type controls, particularly the trabecular BMD (P < 0.01). The difference in BMD between nNOS KO and control mice was confirmed by histomorphometric analysis, which showed a 67% increase in trabecular bone volume in nNOS KO mice when compared with controls (P < 0.001). This was accompanied by reduced bone remodeling, with a significant reduction in osteoblast numbers and bone formation surfaces and a reduction in osteoclast numbers and bone resorption surfaces. Osteoblasts from nNOS KO mice, however, showed increased levels of alkaline phosphatase and no defects in proliferation or bone nodule formation in vitro, whereas osteoclastogenesis was increased in nNOS KO bone marrow cultures. These studies indicate that nNOS plays a hitherto unrecognized but important physiological role as a stimulator of bone turnover. The low level of nNOS expression in bone and the in vitro behavior of nNOS KO bone cells indicate that these actions are indirect and possibly mediated by a neurogenic relay.
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González-Martínez, Jorge A., Gabriel Möddel, Zhong Ying, Richard A. Prayson, William E. Bingaman, and Imad M. Najm. "Neuronal nitric oxide synthase expression in resected epileptic dysplastic neocortex." Journal of Neurosurgery 110, no. 2 (February 2009): 343–49. http://dx.doi.org/10.3171/2008.6.17608.
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Object Nitric oxide has been associated with epileptogenesis. Previous studies have shown increased expression of N-methyl-d-aspartate (NMDA) subunit NR2B receptors in epileptic dysplastic human neocortex. The expression of neuronal nitric oxide synthase (nNOS), and its relation to this subunit NR2B in epileptic dysplastic tissue has never been addressed. Methods Ten patients with medically intractable epilepsy caused by focal cortical dysplasia (CD), and 2 patients with mesial temporal sclerosis (control group) underwent pre- and/or intraoperative invasive monitoring evaluations. Cortical samples from epileptogenic and nonepileptogenic areas were collected from each patient intraoperatively. Samples were processed for cresyl violet staining, immunocytochemical tests with nNOS, NeuN, and NR2B, and immunofluorescence analyses to evaluate colocalized immunoreactivity between nNOS and NR2B. Results . All samples obtained in the patients with epilepsy revealed CD in various degrees. In the nonepileptic sample group, cresyl violet staining revealed normal cortical architecture in 9 samples, but a mild degree of CD in 3. The density and intensity of nNOS-stained neurons was remarkably increased in the epileptic tissue compared with nonepileptic samples (p < 0.05). Two types of nNOS-stained neurons were identified: Type I, expressing strong nNOS immunoreactivity in larger neurons; and Type II, expressing weak nNOS immunoreactivity in slightly smaller neurons. Different from Type I neurons, Type II nNOS-stained neurons revealed immunoreactivity colocalized with NR2B antibody. Conclusions The overexpression of nNOS in the epileptic samples and the immunoreactivity colocalization between nNOS and NR2B may suggest a possible role of nNOS and NO in the pathophysiological mechanisms related to in situ epileptogenicity.
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Bandyopadhyay, A., S. Chakder, and S. Rattan. "Regulation of inducible and neuronal nitric oxide synthase gene expression by interferon-gamma and VIP." American Journal of Physiology-Cell Physiology 272, no. 6 (June 1, 1997): C1790—C1797. http://dx.doi.org/10.1152/ajpcell.1997.272.6.c1790.
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The studies examined the regulation of inducible and neuronal nitric oxide synthases (iNOS and nNOS, respectively) in the rat brain, stomach, rectum, and spleen. Relative expression of iNOS and nNOS mRNAs was quantified by the sensitive method of polymerase amplification reactions. The NOS proteins were determined by Western blot, using specific antibodies. Highest levels of nNOS and iNOS mRNAs were expressed in the rat brain and spleen, respectively. Furthermore, both nNOS and iNOS were expressed in the stomach and rectum. Interestingly, treatment of tissues with lipopolysaccharides or cytokine interferon-gamma (IFN-gamma) induced the expression of iNOS and decreased that of nNOS, representing a shift from one isoform to the other. When the tissues were treated with IFN-gamma followed by vasoactive intestinal polypeptide (VIP), the induction of iNOS was reduced by VIP. The changes in iNOS and nNOS expression at the transcriptional level corresponded to those at the translational level. The data suggest a regulatory role of IFN-gamma and VIP in the expression iNOS and nNOS and a counterregulation of iNOS and nNOS in rat tissues.
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Song, Yao, Jay L. Zweier, and Yong Xia. "Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy." American Journal of Physiology-Cell Physiology 281, no. 6 (December 1, 2001): C1819—C1824. http://dx.doi.org/10.1152/ajpcell.2001.281.6.c1819.
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Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.
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CAO, LUXIANG, and WILLIAM D. ELDRED. "Subcellular localization of neuronal nitric oxide synthase in turtle retina: Electron immunocytochemistry." Visual Neuroscience 18, no. 6 (November 2001): 949–60. http://dx.doi.org/10.1017/s0952523801186128.
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Recent studies imaging nitric oxide (NO) production in the retina have indicated a much wider distribution of NO production than would be suggested by previous light-microscopic localizations of neuronal nitric oxide synthase (nNOS). To help resolve this discrepancy, the present study analyzed the ultrastructural localization of nNOS-like immunoreactivity (-LI) in all layers of the retina. In the ellipsoids of rod photoreceptors and the accessory elements of double cones, nNOS-LI was associated with some atypical mitochondria. In the outer plexiform layer, nNOS-LI was in some postsynaptic horizontal and bipolar cell processes at photoreceptor ribbon synapses. In some amacrine and ganglion cell somata, nNOS-LI was diffusely localized in the cytoplasm and associated with the endoplasmic reticulum. In the inner plexiform layer, nNOS-LI diffusely filled some amacrine cell processes, while in other amacrine cells nNOS-LI was selectively localized at the presynaptic specializations of conventional synapses. Neuronal NOS-LI was also found at membrane specializations in bipolar cell terminals that were distinct from their normal ribbon synapses. Finally, some nNOS-LI was found in mitochondria in Müller cells. The diverse subcellular localizations of nNOS-LI indicates that NO may play distinct functional roles in many retinal cells, which correlates well with the widespread NO production found in previous NO imaging studies.
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Cao, Shaonan, Emily E. Greenberger, Michael W. Epperly, Anthony J. Kanai, and Joel S. Greenberger. "Absence of nNOS Increases Longevity of Long Term Bone Marrow Cultures and Radiation Resistance." Blood 106, no. 11 (November 16, 2005): 4197. http://dx.doi.org/10.1182/blood.v106.11.4197.4197.
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Abstract Neuronal nitric oxide synthase (nNOS) has been shown to be localized to the mitochondrial membrane. The mitochondria play an important role in irradiation-induced apoptosis. Following irradiation, there is increased production of superoxide as well as an induction of nitric oxide in mitochondria. The combination of superoxide and nitric oxide results in production of peroxynitrite, a very strong oxidant that produces lipid peroxidation. Previous data have demonstrated that lack of nNOS protects the bladder from ionizing irradiation damage. To determine the role of nNOS in hematopoiesis, we established long term bone marrow cultures (LTBMCs) from nNOS deletion recombinant negative (knockout) mice as well as nNOS+/+ littermate mice. LTBMCs from nNOS knockout mice compared to +/+ control demonstrated increased total cumulative cell production (32.2 x 106 and 15.9 x 106 non-adherent cells, respectively), cobblestone island formation (5073 and 1106, respectively), and increased cumulative generation of day 14 CFU-GEMM colony forming cells per 105 plated (325 ± 30.4 and 9 ± 2.5 colonies, respectively) over 20 weeks in culture. IL-3 dependent non-adherent cell lines derived from the nNOS-/- and the nNOS+/+ cultures were tested for radiosensitivity. Cells from the nNOS−/− cell line demonstrated decreased radiation apoptosis 24 hours following irradiation, 5.89 ± 0.71% apoptotic cells compared to 10.42 ± 1.19% for the control littermates (p=0.041). Cell cycle analysis of littermate cells at 24 hours after 10 Gy demonstrated a G0/G1 and a G2/M block while there was no change in the cell cycle distribution of the nNOS−/− cells. The data demonstrate that absence of nNOS increases the longevity of hematopoiesis in LTBMCs and increases the radiation resistance of hematopoietic cell lines derived from such cultures.
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Ba, M., W. Ding, L. Guan, Y. Lv, and M. Kong. "S-nitrosylation of Src by NR2B-nNOS signal causes Src activation and NR2B tyrosine phosphorylation in levodopa-induced dyskinetic rat model." Human & Experimental Toxicology 38, no. 3 (October 23, 2018): 303–10. http://dx.doi.org/10.1177/0960327118806633.
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Abnormality in Src PSD-95 NR2B signaling complex assemble occurs in levodopa-induced dyskinesia (LID). N-methyl-D-aspartate receptor (NMDAR) subunit NR2B tyrosine phosphorylation mediated by Src family protein tyrosine kinases is closely associated with dyskinesia. Src autophosphorylation (p-Src) is an important part of Src-catalyzed phosphorylation of NR2B. In addition, the neuronal nitric oxide synthase (nNOS)-derived NO (nNOS/NO) signal which was also involved in dyskinesia recently was proved to participate in the regulation of Src function. Yet, the detailed signal mechanism about the interactions of NR2B, nNOS, and Src is still unknown. In the present study, we investigated the influences of nNOS on Src activation and NR2B tyrosine phosphorylation in dyskinetic rat model by immunoblotting and immunoprecipitation. The results demonstrated that chronic levodopa treatment resulted in downregulation of p-nNOS-S847, one marker of nNOS overactivation. Coinstantaneously, the S-nitrosylation (SNO-Src) and autophosphorylation (p-Src) of Src and NR2B tyrosine phosphorylation were upregulated in dyskinetic rat model. Conversely, administration of 7-NI, one nNOS inhibitor, reversed all these effects of levodopa treatment. Besides, NR2B-containing NMDAR (NR2B/NMDAR) antagonist CP-101,606 could upregulate p-nNOS-S847 and thus attenuate nNOS activation and simultaneously reduce the SNO-Src, p-Src, and NR2B tyrosine phosphorylation. Taken together, the S-nitrosylation of Src is caused by nNOS/NO signal, which is overactivated via Ca2+ influx dependent on NR2B/NMDAR, and subsequently facilitates Src auto-tyrosine phosphorylation and further phosphorylates NR2B. The “NR2B/NMDAR–nNOS/NO–SNO-Src–p-Src–NR2B/NMDAR” signaling cycle may be the molecular basis of NR2B tyrosine phosphorylation upward positive feedback, which demonstrates the possibility as one latent target for dyskinesia therapy.
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Poon, Chi H., Ka C. Tsui, Sze C. Chau, Pit S. Chong, Sylvia W. Y. Lui, Luca Aquili, Kah H. Wong, and Lee W. Lim. "Functional Roles of Neuronal Nitric Oxide Synthase in Neurodegenerative Diseases and Mood Disorders." Current Alzheimer Research 18, no. 10 (September 2021): 831–40. http://dx.doi.org/10.2174/1567205018666211022164025.
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Nitric oxide synthase (NOS) is well known for its involvement in the regulation of the nervous, cardiovascular, and immune systems. Neuronal NOS (nNOS) is the most characterized NOS among all the isoforms. It accounts for most of the production of nitric oxide (NO) in the nervous system required for synaptic transmission and neuroplasticity. Previous studies have described the localization of nNOS in specific brain regions of interest. There is substantial evidence in the literature suggesting that nNOS signaling has significant involvement in several disease pathologies. However, the association between brain nNOS expression profiles and disease remains largely unknown. In this review, we attempt to delineate the contribution of nNOS signaling in memory and mood disorders in order to achieve a better understanding of nNOS in disease modulation.
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Kourosh-Arami, Masoumeh, Nasrin Hosseini, Monireh Mohsenzadegan, Alireza Komaki, and Mohammad Taghi Joghataei. "Neurophysiologic implications of neuronal nitric oxide synthase." Reviews in the Neurosciences 31, no. 6 (August 27, 2020): 617–36. http://dx.doi.org/10.1515/revneuro-2019-0111.
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AbstractThe molecular and chemical properties of neuronal nitric oxide synthase (nNOS) have made it a key mediator in many physiological functions and signaling transduction. The NOS monomer is inactive, but the dimer form is active. There are three forms of NOS, which are neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthase. nNOS regulates nitric oxide (NO) synthesis which is the mechanism used mostly by neurons to produce NO. nNOS expression and activation is regulated by some important signaling proteins, such as cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), calmodulin (CaM), heat shock protein 90 (HSP90)/HSP70. nNOS-derived NO has been implicated in modulating many physiological functions, such as synaptic plasticity, learning, memory, neurogenesis, etc. In this review, we have summarized recent studies that have characterized structural features, subcellular localization, and factors that regulate nNOS function. Finally, we have discussed the role of nNOS in the developing brain under a wide range of physiological conditions, especially long-term potentiation and depression.
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Guerra, Damian D., Rachael Bok, Vibhuti Vyas, David J. Orlicky, Ramón A. Lorca, and K. Joseph Hurt. "Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum." Proceedings of the National Academy of Sciences 116, no. 35 (August 12, 2019): 17541–46. http://dx.doi.org/10.1073/pnas.1905902116.
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Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca2+/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca2+ requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOSS1412A knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOSS1412A ileum. At higher frequencies, nNOS S1412 had no effect. nNOSS1412A ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOSS1412A ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.
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Guerra, Damian D., Rachael Bok, Evelyn Llerena Cari, Cari Nicholas, David J. Orlicky, Joshua Johnson, and K. Joseph Hurt. "Effect of neuronal nitric oxide synthase serine-1412 phosphorylation on hypothalamic–pituitary–ovarian function and leptin response." Biology of Reproduction 102, no. 6 (February 26, 2020): 1281–89. http://dx.doi.org/10.1093/biolre/ioaa025.
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Abstract Hypothalamic neuronal nitric oxide synthase (nNOS) potentiates adult female fertility in rodents by stimulating gonadotropin releasing hormone (GnRH) secretion, which in turn promotes luteinizing hormone (LH) release and ovulation. The mechanism of hypothalamic nNOS activation is not clear but could be via nNOS serine1412 (S1412) phosphorylation, which increases nNOS activity and physiologic NO effects in other organ systems. In female rodents, hypothalamic nNOS S1412 phosphorylation reportedly increases during proestrus or upon acute leptin exposure during diestrus. To determine if nNOS S1412 regulates female reproduction in mice, we compared the reproductive anatomy, estrous cycle duration and phase proportion, and fecundity of wild-type and nNOS serine1412➔alanine (nNOSS1412A) knock-in female mice. We also measured hypothalamic GnRH and serum LH, follicle stimulating hormone (FSH), estradiol, and progesterone in diestrus mice after intraperitoneal leptin injection. Organ weights and histology were not different by genotype. Ovarian primordial follicles, antral follicles, and corpora lutea were similar for wild-type and nNOSS1412A mice. Likewise, estrous cycle duration and phase length were not different, and fecundity was unremarkable. There were no differences among genotypes for LH, FSH, estradiol, or progesterone. In contrast to prior studies, our work suggests that nNOS S1412 phosphorylation is dispensable for normal hypothalamic–pituitary–ovarian function and regular estrous cycling. These findings have important implications for current models of fertility regulation by nNOS phosphorylation.
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Baum, Oliver, Felicitas A. M. Huber-Abel, and Martin Flück. "nNOS Increases Fiber Type-Specific Angiogenesis in Skeletal Muscle of Mice in Response to Endurance Exercise." International Journal of Molecular Sciences 24, no. 11 (May 26, 2023): 9341. http://dx.doi.org/10.3390/ijms24119341.
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We studied the relationship between neuronal NO synthase (nNOS) expression and capillarity in the tibialis anterior (TA) muscle of mice subjected to treadmill training. The mRNA (+131%) and protein (+63%) levels of nNOS were higher (p ≤ 0.05) in the TA muscle of C57BL/6 mice undergoing treadmill training for 28 days than in those of littermates remaining sedentary, indicating an up-regulation of nNOS by endurance exercise. Both TA muscles of 16 C57BL/6 mice were subjected to gene electroporation with either the pIRES2-ZsGreen1 plasmid (control plasmid) or the pIRES2-ZsGreen1-nNOS gene-inserted plasmid (nNOS plasmid). Subsequently, one group of mice (n = 8) underwent treadmill training for seven days, while the second group of mice (n = 8) remained sedentary. At study end, 12–18% of TA muscle fibers expressed the fluorescent reporter gene ZsGreen1. Immunofluorescence for nNOS was 23% higher (p ≤ 0.05) in ZsGreen1-positive fibers than ZsGreen1-negative fibers from the nNOS-transfected TA muscle of mice subjected to treadmill training. Capillary contacts around myosin heavy-chain (MHC)-IIb immunoreactive fibers (14.2%; p ≤ 0.05) were only higher in ZsGreen1-positive fibers than ZsGreen1-negative fibers in the nNOS-plasmid-transfected TA muscles of trained mice. Our observations are in line with an angiogenic effect of quantitative increases in nNOS expression, specifically in type-IIb muscle fibers after treadmill training.
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Patzak, Andreas, Andreas Steege, En Yin Lai, Jan Ole Brinkmann, Eckehardt Kupsch, Nadine Spielmann, Adrian Gericke, et al. "Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform of nitric oxide synthase." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 294, no. 2 (February 2008): R429—R437. http://dx.doi.org/10.1152/ajpregu.00482.2007.
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The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(−/−)], or nNOS gene [nNOS(−/−)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at 10−8 and 10−6 mol/l reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). NG-nitro-l-arginine methyl ester (l-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at 10−8 and 10−6 mol/l. The response to angiotensin II was enhanced in nNOS(−/−) mice (41% and 25% at 10−8 and 10−6 mol/l) and even more enhanced in eNOS(−/−) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). l-NAME led to complete constriction of Af in these groups. Media-to-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types 1A and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation.
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Church, Jarrod E., Stefan M. Gehrig, Annabel Chee, Timur Naim, Jennifer Trieu, Glenn K. McConell, and Gordon S. Lynch. "Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 301, no. 5 (November 2011): R1358—R1366. http://dx.doi.org/10.1152/ajpregu.00096.2011.
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Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscle's ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS−/− mice. We found that nNOS−/− mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS−/− mice were more resistant to fatigue than those of wild-type littermates ( P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS−/− mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS−/− mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.
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Kuznetsova, L. A., and N. E. Basova. "The role of the neural NO synthase adapter protein in the pathogenesis of metabolic syndrome and type 2 diabetes mellitus." Сибирский научный медицинский журнал 43, no. 5 (October 27, 2023): 34–49. http://dx.doi.org/10.18699/ssmj20230504.
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The pathogenesis of metabolic syndrome (MS) is characterized by obesity, hypertension, dyslipidemia and insulin resistance. MS increases the risk of developing type 2 diabetes mellitus (DM2). The neuronal isoform of nitric oxide synthase (nNOS) is defined by complex protein-protein interactions, since nNOS, unlike other isoforms of NOS,contains a C-terminal PDZ domain, which allows it to conjugate with other proteins and, first of all, to interact with an adapter of neuronal, or type 1, nitric oxide synthase (NOS1AP), also denoted CAPON in our work. Changes in the interaction between nNOS and NOS1AP lead to metabolic disorders in brain, heart, liver and skeletal muscles, which plays a key role in the development of MS and T2DM. NOS1AP, interacting with the PDZ domain of nNOS, competes with the postsynaptic density protein (PSD95) and regulates the stability of subcellular localization of nNOS and enzyme expression during synapse formation. NOS1AP promotes nNOS binding to targets such as small GTPase (Dexras1), synapsines, regulating the formation of dendritic roots, mediates activation of the nNOS-p38MAP kinase pathway during excitotoxicity. It has been shown that single-nucleotide polymorphism of the NOS1AP gene and its overexpression in the myocardium leads to the manifestation of long QT syndrome, which is most clearly manifested in elderly patients with DM2. It was found that the genetic polymorphism of NOS1AP affects insulin secretion when using calcium blockers, and can promote the development of DM2. The functional role of NOS1AP in stabilizing the functions of skeletal muscle nNOS in the cytoskeletal complex associated with dystrophin/utrophin was discovered. The purpose of the review is to provide updated information on the role of NOS1AP and the nNOS/NOS1AP complex in the pathogenesis of MS and DM2. The potential molecular mechanisms of the interaction of NOS1AP with nNOS and with other proteins, which leads to change in nNOS activity, localization and content, are discussed.
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Terauchi, Akiko, Daisuke Kobayashi, and Hiroshi Mashimo. "Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 2 (August 2005): G291—G299. http://dx.doi.org/10.1152/ajpgi.00005.2005.
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Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient (nNOS−/−), eNOS-deficient (eNOS−/−), and W/Wv mice (lacking certain c-Kit-expressing ICC) with or without l-arginine or Nω-nitro-l-arginine methyl ester (l-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of eNOS−/− was higher and W/Wv was lower than wild-type and nNOS−/− mice. l-Arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, nNOS−/−, and W/Wv mice. However, neither l-arginine nor l-NAME affected basal tone in eNOS−/− mice. In vitro, l-arginine decreased basal tone in wild-type and nNOS−/− IAS but not in eNOS−/− or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, eNOS−/−, and W/Wv mice but absent in all nNOS−/− mice. EFS-induced IAS relaxation was also reduced in nNOS−/− IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR.
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Osuka, Koji, Yasuo Watanabe, Nobuteru Usuda, Ayami Nakazawa, Kohji Fukunaga, Eishichi Miyamoto, Masakazu Takayasu, Masaaki Tokuda, and Jun Yoshida. "Phosphorylation of Neuronal Nitric Oxide Synthase at Ser847 by CaM-KII in the Hippocampus of Rat Brain after Transient Forebrain Ischemia." Journal of Cerebral Blood Flow & Metabolism 22, no. 9 (September 2002): 1098–106. http://dx.doi.org/10.1097/00004647-200209000-00007.
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The authors previously demonstrated that Ca 2+/calmodulin (CaM)-dependent protein kinase IIα (CaM-KIIα) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study, they established that forebrain ischemia causes an increase in the phosphorylation of nNOS at Ser847 in the hippocampus. This nNOS phosphorylation appeared to be catalyzed by CaM-KII: (1) it correlated with the autophosphorylation of CaM-KIIα; (2) it was blocked by the CaM-KII inhibitor, KN-93; and (3) nNOS and CaM-KIIα were found to coexist in the hippocampus. Examination of the spatial relation between nNOS and CaM-KIIα in the brain revealed coexistence in the hippocampus but not in the cortex during reperfusion, with a concomitant increase in autophosphorylation of CaM-KIIα. The phosphorylation of nNOS at Ser847 probably takes place in nonpyramidal hippocampal neurons, which increased after 30 minutes of reperfusion in the hippocampus, whereas no significant increase was detected in the cortex. An intraventricular injection of KN-93 significantly decreased the phosphorylation of nNOS in the hippocampus. These results point to CaM-KII as a protein kinase, which by its colocalization may attenuate the activity of nNOS through its Ser847 phosphorylation, and may thus contribute to promotion of tolerance to postischemic damage in hippocampal neurons.
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Bayram, Zübeyde, Mevlüt Asar, Sevil çayli, and Ramazan Demir. "Immunocytochemical Detection of Neuronal Nitric Oxide Synthase (nNOS)-IR in Embryonic Rat Stomach Between Days 13 and 21 of Gestation." Journal of Histochemistry & Cytochemistry 50, no. 5 (May 2002): 671–79. http://dx.doi.org/10.1177/002215540205000508.
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In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 13–21 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.
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Zhang, Lin, Hong-Jin Yuan, Bo Cao, Cheng-Cheng Kong, Fang Yuan, Jun Li, Huan-Yu Ni, et al. "MGE-derived nNOS + interneurons promote fear acquisition in nNOS −/− mice." Biochemical and Biophysical Research Communications 493, no. 4 (December 2017): 1560–66. http://dx.doi.org/10.1016/j.bbrc.2017.09.158.
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Roczniak, Agnes, Joseph Zimpelmann, and Kevin D. Burns. "Effect of dietary salt on neuronal nitric oxide synthase in the inner medullary collecting duct." American Journal of Physiology-Renal Physiology 275, no. 1 (July 1, 1998): F46—F54. http://dx.doi.org/10.1152/ajprenal.1998.275.1.f46.
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Nitric oxide (NO) derived from neuronal NO synthase (nNOS) in the kidney inner medulla has been implicated in the regulation of arterial blood pressure. The purpose of the present study was to determine the effect of high dietary NaCl on the expression of nNOS in the rat inner medullary collecting duct (IMCD). After 3 days or 3 wk of high (4.0%)-NaCl diet in rats, urinary[Formula: see text]/[Formula: see text]excretion significantly increased. In freshly microdissected IMCD, nNOS was readily detected by immunofluorescence with polyclonal antibody, an effect that was completely blocked by neutralization of antibody with immunizing antigen. In rats fed a 4.0% NaCl diet for 3 days, IMCD nNOS mRNA, detected by RT-PCR, did not change from control values (0.3% NaCl, 19.84 ± 1.57 × 103, vs. 4.0% NaCl, 20.44 ± 3.14 × 103 cpm; P = not significant, n = 3). By Western blotting however, nNOS protein expression significantly increased (0.3% NaCl, 0.51 ± 0.12, vs. 4.0% NaCl, 0.92 ± 0.14 arbitrary units; P < 0.05, n = 5). After 3 wk of 4.0% dietary NaCl, expression of nNOS mRNA and protein in IMCD did not differ significantly from control values. In contrast to these data, renal cortical expression of nNOS mRNA and protein was significantly decreased after 4.0% NaCl diet for 3 days. High dietary NaCl had no significant effect on expression of mRNA for inducible NO synthase (iNOS) in IMCD after either 3 days or 3 wk. In summary, our data indicate that nNOS mRNA and protein are expressed in IMCD and that high dietary NaCl differentially regulates nNOS expression in IMCD and cortex. The early increase in nNOS protein in IMCD may contribute to enhanced local production of NO and thereby represent an adaptive response to salt intake.
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Hori, N., M. C. Lee, K. Sasaguri, H. Ishii, M. Kamei, K. Kimoto, M. Toyoda, and S. Sato. "Suppression of Stress-induced nNOS Expression in the Rat Hypothalamus by Biting." Journal of Dental Research 84, no. 7 (July 2005): 624–28. http://dx.doi.org/10.1177/154405910508400708.
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Nitric oxide (•NO) modulates the activity of the endocrine system in the behavioral response to stress. The purpose of this study was to investigate the effect of restraining the body of an animal on expression of neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) of the hypothalamus, and the inhibitory effect of para-masticatory activity on restraint-induced nNOS expression. We observed an increase in nNOS mRNA expression and nNOS-positive neurons in the rat hypothalamus after 30 or 60 min of restraint. Biting on a wooden stick during bodily restraint decreased nNOS mRNA expression in the hypothalamus. In addition, the number of nNOS-positive neurons was significantly reduced in the PVN of the hypothalamus. These observations clearly suggest a possible anti-stress effect of the masticatory activity of biting, and this mechanism might be unconsciously in operation during exposure to psychological stressors.
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Chen, Lichao, Deborah Duricka, Scott Nelson, Sanjib Mukherjee, Stewart G. Bohnet, Ping Taishi, Jeannine A. Majde, and James M. Krueger. "Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases." Journal of Applied Physiology 97, no. 1 (July 2004): 17–28. http://dx.doi.org/10.1152/japplphysiol.01355.2003.
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Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1β and TNF-α, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2′,5′-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1β mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.
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Hahn, F. Ekkehardt, Christoph Jocher, and Henning Schröder. "A Novel Methylene Dithioether as a Ligand: Synthesis and Molecular Structure of a Zinc(II) Complex with N4S2 Coordination Environment." Zeitschrift für Naturforschung B 58, no. 10 (October 1, 2003): 1027–29. http://dx.doi.org/10.1515/znb-2003-1016.
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The octadentate ligand [N(CH2CH2NH2)(CH2CH2CH2OH)(CH2CH2S)]2CH2, (NNOS-232)2CH2, was synthesized accidentally by the reaction of the unsymmetrically substituted tripod [N(CH2CH2NH2)(CH2CH2CH2OH)(CH2CH2SH)], NNOS-232, with dichloromethane in the presence of aluminum hydroxide. Ligand (NNOS-232)2CH2 was reacted with zinc bis(perchlorate) hexahydrate to yield the complex [Zn((NNOS-232)2CH2)](ClO4)2 1 exhibiting a distorted octahedrally coordinated zinc atom in an N4S2 coordination environment, as shown by an X-ray diffraction study.