Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes

1994 ◽  
Vol 267 (4) ◽  
pp. R1020-R1025 ◽  
Author(s):  
G. Williams ◽  
T. Brown ◽  
L. Becker ◽  
M. Prager ◽  
B. P. Giroir
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Nitric Oxide Synthase ◽  
C2c12 Cells ◽  
Northern Analysis ◽  
Pcr Analysis ◽  
No Production ◽  
Mouse Macrophage ◽  
Nitrite Production ◽  
Oxide Synthase

Nitric oxide (NO) is an important mediator of diverse physiological and pathological responses. To determine whether NO production can be induced in skeletal muscle, we stimulated C2C12 mouse skeletal muscle myocytes with putative inducers of nitric oxide synthase (NOS). Neither lipopolysaccharide (LPS), interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), nor interferon-gamma (IFN) was able to stimulate nitrite production by C2C12 cells when administered alone. However, combinations of IFN with either TNF or IL-1 resulted in significant nitrite production; simultaneous stimulation of cells with all three cytokines resulted in significantly increased nitrite production compared with any combination of two cytokines. Northern analysis of RNA obtained from stimulated C2C12 cells revealed induction of a single mRNA band that precisely coincided with the mRNA band of mouse macrophage-inducible NOS (iNOS). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis followed by sequencing of the 5' 765 bases of the skeletal muscle iNOS cDNA demonstrated exact homology with mouse macrophage iNOS. These findings indicate that combinations of cytokines stimulate NO production in skeletal muscle cells via induction of the macrophage-type iNOS gene.

Inducible nitric oxide synthase in the epithelial epididymal cells of the rat

1997 ◽  
Vol 9 (8) ◽  
pp. 789 ◽  
Author(s):  
Barbara Wiszniewska ◽  
Rafal Kurzawa ◽  
Andrzej Ciechanowicz ◽  
Boguslaw Machalinski
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Epithelial Cells ◽  
Inducible Nitric Oxide Synthase ◽  
Cultured Cells ◽  
No Production ◽  
Nitrite Production ◽  
Oxide Synthase ◽  
Inducible Nos ◽  
Inducible Nitric Oxide

The expression of mRNA for inducible nitric oxide synthase (iNOS) in rat epithelial cells of epididymis was investigated with reverse transcription followed by polymerase chain reaction. Immunocytochemical reaction for iNOS was performed to confirm the enzyme’ s localization in the epididymal epithelium. Additionally, an indirect spectrophotometric method for nitric oxide (NO) determination was applied for measurement of nitrite production by cultured epididymal epithelial cells. Inducible NOS mRNA was detected in freshly isolated epithelial cells, in cultured cells without stimulation as well as in cultured cells after stimulation by lipopolysaccharide and interferon-gamma. Inducible NOS immunoreactivity was observed in the apical part of epithelial cells of epididymal sections and in the cytoplasm of cells in culture. Release of nitrite was observedin vitro in both the unstimulated and stimulated cells of caput (1·44 ± 0·94 v. 4·37 ± 2·42 µM) and cauda (0·69 ± 1·21 v. 5·21 ± 2·76 µM) epididymis (P < 0·001). To the best of our knowledge, this is the first study to demonstrate iNOS in the epididymal epithelial cells of the rat. Nitric oxide released by epididymal epithelial cells may act on cells and tissues located nearby. The results may help explain epididymal function: sperm storage, passage and maturation. Excessive epididymal NO production may also play a role in the inflammatory infertility of the male. Extra keyword: iNOS

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

2002 ◽  
Vol 282 (4) ◽  
pp. C762-C767 ◽  
Author(s):  
Tatsuya Takizawa ◽  
Hiroshi Yoshikawa ◽  
Miho Yamada ◽  
Hidetoshi Morita
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Pcr Analysis ◽  
No Production ◽  
Epr Spectrum ◽  
Pregnant Rats ◽  
Paramagnetic Resonance ◽  
Triplet Signal ◽  
Oxide Synthase ◽  
Nos Isoforms

Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe- N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure ( g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor l-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate α-NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal ( g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term ( day 21.5) placenta was significantly increased compared with that in preterm ( day 19.5) placenta ( P < 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.

Resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves AMPK but not SIRT1

2011 ◽  
Vol 301 (5) ◽  
pp. E922-E930 ◽  
Author(s):  
Carolina Centeno-Baez ◽  
Patrice Dallaire ◽  
André Marette
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Compound C ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide ◽  
L6 Myocytes ◽  
Inos Induction

The plant-derived polyphenol resveratrol (RSV) modulates life span and metabolism, and it is thought that these effects are largely mediated by activating the deacetylase enzyme SIRT1. However, RSV also activates the cell energy sensor AMP-activated protein kinase (AMPK). We have previously reported that AMPK activators inhibit inducible nitric oxide synthase (iNOS), a key proinflammatory mediator of insulin resistance in endotoxemia and obesity. The aim of this study was to evaluate whether RSV inhibits iNOS induction in insulin target tissues and to determine the role of SIRT1 and AMPK activation in this effect. We found that RSV (40 mg/kg ip) treatment decreased iNOS induction and NO production in skeletal muscle and white adipose tissue, but not in liver, of endotoxin (LPS)-challenged mice. This effect of the polyphenol was recapitulated in vitro, where RSV (10–80 μM) robustly inhibited iNOS protein induction and NO production in cytokine/LPS-treated L6 myocytes and 3T3-L1 adipocytes. However, no effect of RSV was observed on iNOS induction in FAO hepatocytes. Further studies using inhibitors of SIRT1 revealed that the deacetylase enzyme is not involved in RSV action on iNOS. In marked contrast, RSV activates AMPK in L6 myocytes, and blunting its activation using Compound C or RNA interference partly blocked the inhibitory effect of RSV on NO production. These results show that RSV specifically inhibits iNOS induction in muscle through a mechanism involving AMPK but not SIRT1 activation. This anti-inflammatory action of RSV likely contributes to the therapeutic effect of this plant polyphenol.

Regulation of endothelial nitric oxide synthase by PGD2 in the developing choroid

2000 ◽  
Vol 278 (1) ◽  
pp. H60-H66 ◽  
Author(s):  
Isabelle Dumont ◽  
Pierre Hardy ◽  
Krishna G. Peri ◽  
Xin Hou ◽  
Stéphane Molotchnikoff ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Choroidal Blood Flow ◽  
Nitrite Production ◽  
Enos Mrna ◽  
Oxide Synthase

We investigated if prostaglandins might regulate the increased choroidal endothelial (e) nitric oxide synthase (NOS) expression in the perinate. Prostaglandins, eNOS mRNA, immunoreactive protein and activity, and nitrite [stable metabolite of nitric oxide (NO)] production were markedly higher in newborn (1 day old) than juvenile (6–8 wk old) pig choroid. Treatment of isolated newborn choroids with the prostaglandin synthase inhibitor ibuprofen for 24 h reduced eNOS mRNA and nitrite production to values in juveniles. This effect was equally observed with the PGD2 receptor (DP) blocker BW A868C and was prevented by cotreatment with PGD2 but not other prostaglandins; similar observations were made on NOS activity in vivo. PGD2 also increased eNOS expression on choroids of juveniles, and this effect was blocked by BW A868C. The manifestation of this upregulation of eNOS by PGD2 on the control of choroidal vasomotor response was tested by using NO-dependent vasorelaxants, ACh, bradykinin (Bk), and substance P (SP). ACh-, Bk-, and SP-elicited choroidal vasorelaxation was greater in saline-treated newborn than juvenile pigs. Ibuprofen (24 h) decreased ACh-, Bk-, and SP-evoked vasorelaxation in newborns, whereas PGD2 increased that in juveniles and prevented the ibuprofen-induced attenuated relaxation in newborns; infusion of N ω-monomethyl-l-arginine in choroids of those animals treated with PGD2 reversed the augmented vasorelaxation to ACh, Bk, and SP. Finally, PGD2-induced upregulation of NOS in the perinate was also reflected by curtailed choroidal blood flow autoregulatory response to increased perfusion pressure. In conclusion, PGD2 exhibits a major role in upregulating eNOS expression and activity in the choroid, which in turn results in greater NO-mediated vasorelaxation; a new mechanism for eNOS regulation via DP is hereby disclosed. The relationship between PGD2 and eNOS in the developing subject provides an explanation for the interactive role of these two factors in the absent choroidal blood flow autoregulation in the perinate.

Dietary salt increases endothelial nitric oxide synthase and TGF-β1 in rat aortic endothelium

1999 ◽  
Vol 277 (4) ◽  
pp. H1293-H1298 ◽  
Author(s):  
Wei-Zhong Ying ◽  
Paul W. Sanders
Keyword(s):  
Nitric Oxide ◽  
Steady State ◽  
Nitric Oxide Synthase ◽  
Arterial Wall ◽  
Transforming Growth Factor ◽  
Northern Analysis ◽  
No Production ◽  
Dietary Salt ◽  
Oxide Synthase ◽  
Tgf Β1

The amount of NaCl in the diet plays an important role in modulating nitric oxide (NO) synthesis in vivo. In the glomerulus, dietary NaCl also regulates transforming growth factor-β1 (TGF-β1) production. We hypothesized that dietary NaCl intake regulated expression of the endothelial isoform of nitric oxide synthase (NOS3) and TGF-β1 in the aorta. Administration of 8.0% NaCl diet to rats for 7 days did not affect blood pressure but increased steady-state mRNA and protein levels of NOS3 in the arterial wall compared with animals on 0.3% NaCl diet. Northern analysis demonstrated increased steady-state amounts of mRNA of TGF-β1 in aortas of rats on 8.0% NaCl diet. By ELISA, both total and active TGF-β1 were increased in these vessel segments. Endothelial denudation of aortic rings reduced active TGF-β1 secretion to undetectable levels. Addition of a neutralizing antibody to TGF-β to aortic ring segments attenuated NO production but not to that observed in animals on the 0.3% NaCl diet. The data showed that dietary NaCl intake modulated NOS3 and TGF-β1 expression in the arterial wall; NOS3 expression was at least partially regulated by endothelial cell production of TGF-β1.

scholarly journals Neuronal nitric oxide synthase expression is lower in areas of the nucleus tractus solitarius excited by skeletal muscle reflexes in hypertensive rats

2013 ◽  
Vol 304 (11) ◽  
pp. H1547-H1557 ◽  
Author(s):  
Megan N. Murphy ◽  
Masaki Mizuno ◽  
Ryan M. Downey ◽  
John J. Squiers ◽  
Kathryn E. Squiers ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Nitric Oxide Synthase ◽  
Protein Expression ◽  
Nucleus Tractus Solitarius ◽  
Neuronal Nitric Oxide Synthase ◽  
No Production ◽  
Hypertensive Rats ◽  
Oxide Synthase ◽  
Muscle Reflexes

The functions of the skeletal muscle exercise pressor reflex (EPR) and its mechanically sensitive component are augmented in hypertension producing exaggerated increases in blood pressure during exercise. Afferent information from the EPR is processed in the nucleus tractus solitarius (NTS). Within the NT, nitric oxide (NO), produced via l-arginine oxidation by neuronal nitric oxide synthase (nNOS), buffers the pressor response to EPR activation. Therefore, EPR overactivity may manifest as a decrease in NO production due to reductions in nNOS. We hypothesized that nNOS protein expression is lower in the NTS of spontaneously hypertensive (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. Further, we examined whether nNOS is expressed with FOS, a marker of neuronal excitation induced by EPR activation. The EPR and mechanoreflex were intermittently activated for 1 h via hindlimb static contraction or stretch, respectively. These maneuvers produced significantly greater pressor responses in SHR during the first 25 min of stimulation. Within the NTS, nNOS expression was lower from −14.9 to −13.4 bregma in SHR compared with WKY. For example, at −14.5 bregma the number of NTS nNOS-positive cells in SHR (13 ± 1) was significantly less than WKY (23 ± 2). However, the number of FOS-positive cells after muscle contraction in this area was not different (WKY = 82 ± 18; SHR = 75 ± 8). In both groups, FOS-expressing neurons were located within the same areas of the NTS as neurons containing nNOS. These findings demonstrate that nNOS protein expression is lower within NTS areas excited by skeletal muscle reflexes in hypertensive rats.

scholarly journals Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase

1997 ◽  
Vol 325 (2) ◽  
pp. 487-493 ◽  
Author(s):  
Sylvain BÉDARD ◽  
Bruno MARCOTTE ◽  
André MARETTE
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Glucose Transport ◽  
Muscle Cells ◽  
Inos Expression ◽  
Skeletal Muscle Cells ◽  
Pcr Analysis ◽  
No Production ◽  
Transporter Protein ◽  
Nitrite Production

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-α, interferon-γ or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. L-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.

Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action

Diabetes ◽  
1997 ◽  
Vol 46 (11) ◽  
pp. 1691-1700 ◽  
Author(s):  
S. Kapur ◽  
S. Bedard ◽  
B. Marcotte ◽  
C. H. Cote ◽  
A. Marette
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Nitric Oxide Synthase ◽  
Insulin Action ◽  
Oxide Synthase

scholarly journals Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

2008 ◽  
Vol 294 (3) ◽  
pp. L582-L591 ◽  
Author(s):  
Neetu Sud ◽  
Stephen Wedgwood ◽  
Stephen M. Black
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Promoter Activity ◽  
Dominant Negative ◽  
No Production ◽  
Enos Expression ◽  
Akt Activation ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression.

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