Intratracheal adenoviral-mediated delivery of iNOS decreases pulmonary vasoconstrictor responses in rats

2004 ◽  
Vol 97 (5) ◽  
pp. 1814-1822 ◽  
Author(s):  
Louis G. Chicoine ◽  
Edith Tzeng ◽  
Rebekah Bryan ◽  
Steven Saenz ◽  
Michael L. Paffett ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Exhaled Nitric Oxide ◽  
Time Dependent ◽  
Inos Mrna ◽  
Alveolar Wall ◽  
Gene Transduction ◽  
Inos Protein ◽  
Protein Levels ◽  
Inos Gene ◽  
Adenoviral Construct

We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS overexpression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter-regulated human iNOS gene (Adeno-iNOS), or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS-transduced rats demonstrated human iNOS mRNA and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS-treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS-transduced rats, the expression of iNOS protein and exhaled nitric oxide concentrations were increased on days 1–4 and 7 but returned to baseline values by day 14. The administration of the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine dihydrochloride (l-NIL) decreased exhaled nitric oxide concentrations to levels found in Adeno-Control-transduced lungs. In a second group of rats, the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days after transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial, and venous vasoconstrictor responses to U-46619 compared with sham, Adeno-Control, and control groups. In a third set of experiments, the response to 400 nM U-46619 in the presence of 10 μM l-NIL was not different in the isolated lungs from Adeno-Control- and Adeno-iNOS-transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression, which attenuates the vascular constrictor responses to the thromboxane mimetic U-46619.

scholarly journals Exendin-4 inhibits interleukin-1β-induced iNOS expression at the protein level, but not at the transcriptional and posttranscriptional levels, in RINm5F β-cells

2009 ◽  
Vol 202 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Jung-Hoon Kang ◽  
Seo-Yoon Chang ◽  
Hyun-Jong Jang ◽  
Dong-Bin Kim ◽  
Gyeong Ryul Ryu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inos Expression ◽  
Interleukin 1Β ◽  
Inos Mrna ◽  
Β Cells ◽  
Β Cell ◽  
Inos Protein ◽  
Inos Gene ◽  
Nitrite Production ◽  
Inos Promoter

Cytokines such as interleukin-1β (IL-1β) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to β-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for β-cell proliferation. However, the protective mechanisms of GLP-1 in β-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1β-induced iNOS gene expression were investigated employing RINm5F β-cells. EX-4 inhibited IL-1β-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1β-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3′-untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1β-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1β-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1β-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.

scholarly journals Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages

2002 ◽  
Vol 173 (2) ◽  
pp. 285-296 ◽  
Author(s):  
C Boiti ◽  
D Zampini ◽  
G Guelfi ◽  
F Paolocci ◽  
M Zerani ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Expression Patterns ◽  
Physiological Role ◽  
Total Activity ◽  
Pcr Analysis ◽  
Inos Mrna ◽  
Corpora Lutea ◽  
Mrna Levels ◽  
Isolated Cells ◽  
Protein Levels

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.

Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells

1995 ◽  
Vol 268 (4) ◽  
pp. F770-F777 ◽  
Author(s):  
M. G. Mohaupt ◽  
J. Schwobel ◽  
J. L. Elzie ◽  
G. S. Kannan ◽  
B. C. Kone
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Cytokines ◽  
Collecting Duct ◽  
Tnf Alpha ◽  
Inos Mrna ◽  
No Production ◽  
Inos Gene ◽  
Medullary Collecting Duct ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.

Time-Dependent Effect of Nitrate-Rich Meals on Exhaled Nitric Oxide in Healthy Subjects

CHEST Journal ◽  
2005 ◽  
Vol 128 (4) ◽  
pp. 2465-2470 ◽  
Author(s):  
Anne-Marie Vints ◽  
Ellie Oostveen ◽  
Guy Eeckhaut ◽  
Mieke Smolders ◽  
Wilfried A. De Backer
Keyword(s):  
Nitric Oxide ◽  
Healthy Subjects ◽  
Exhaled Nitric Oxide ◽  
Time Dependent ◽  
Dependent Effect ◽  
Time Dependent Effect

Dexamethasone suppresses iNOS yet induces GTPCH and CAT-2 mRNA expression in rat lungs

2003 ◽  
Vol 285 (2) ◽  
pp. L484-L491 ◽  
Author(s):  
Jeffrey W. Skimming ◽  
Omer Nasiroglu ◽  
Chun-Jen Huang ◽  
Charles E. Wood ◽  
Bruce R. Stevens ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Hemorrhagic Shock ◽  
Mrna Expression ◽  
Control Group ◽  
Inos Mrna ◽  
Oxide Formation ◽  
Inos Protein ◽  
Arginine Transport ◽  
Nitric Oxide Formation ◽  
Dexamethasone Group

The in vivo mechanisms by which glucocorticoids inhibit nitric oxide expression await detailed investigation. In cell culture experiments, glucocorticoids have been shown to inhibit inducible nitric oxide synthase (iNOS) formation and activity. Glucocorticoids can inhibit iNOS activity in cultured cells by blocking arginine transport and inhibiting tetrahydrobiopterin biosynthesis. We recently reported that changes in intrapulmonary formation of nitric oxide in endotoxemic rats correspond with changes in transcription of the predominant arginine transporter cationic amino acid transporter (CAT)-2. Realizing that hemorrhagic shock induces nitric oxide overproduction in intact animals, we sought to explore whether glucocorticoids attenuate hemorrhagic shock-induced increases in intrapulmonary nitric oxide formation and whether they might do so by inhibiting the formation of tetrahydrobiopterin, iNOS protein, and CAT-2. We randomly assigned 10 male Sprague-Dawley rats to receive dexamethasone or normal saline. Bleeding the animals to a mean systemic blood pressure of between 40 and 45 mmHg created the hemorrhagic shock. Dexamethasone abrogated the increase in exhaled nitric oxide concentrations caused by hemorrhagic shock. At the end of the experiment, plasma nitrate/nitrite values were lower in the dexamethasone group than in the control group. The iNOS protein concentrations were also lower in the dexamethasone group than in the control group. Dexamethasone decreased the intrapulmonary iNOS mRNA concentrations yet increased both guanosine triphosphate cyclohydrolase I mRNA and CAT-2 mRNA. Our results support the idea that dexamethasone inhibits nitric oxide formation in a manner that is independent of tetrahydrobiopterin and arginine transport yet dependent on downregulation of iNOS mRNA expression.

scholarly journals Expression of Endomyocardial Nitric Oxide Synthase and Coronary Endothelial Function in Human Cardiac Allografts

Circulation ◽  
2001 ◽  
Vol 104 (suppl_1) ◽  
Author(s):  
Stephen M. Wildhirt ◽  
Michael Weis ◽  
Costas Schulze ◽  
Nicole Conrad ◽  
Sinan Pehlivanli ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Endothelial Function ◽  
Heart Transplantation ◽  
Mrna Expression ◽  
Inos Mrna ◽  
Inos Protein ◽  
Cardiac Allografts ◽  
Oxide Synthase

Background Inducible nitric oxide synthase (iNOS) is expressed and is functionally active in the presence of transplant arteriosclerosis. However, the early involvement of iNOS in alterations of microvascular endothelial function in the absence of preexisting lesions remains unclear; this information would be of prognostic value. We studied the course of iNOS mRNA expression, transcardiac nitric oxide production, and their potential association with microvascular coronary endothelial dysfunction in human cardiac allografts. Methods and Results A total of 42 patients were studied at 1, 6, and 12 months after heart transplantation. Microvascular coronary flow velocity reserve (CFVR) was tested in an endothelium-dependent (acetylcholine) and -independent manner (adenosine) using a Doppler flow wire. Endomyocardial iNOS expression was determined by reverse transcription polymerase chain reaction. iNOS protein and nitrotyrosine levels were detected by immunohistochemistry. Transcardiac plasma nitrite/nitrate (NOx) levels were measured by the Griess reaction. CFVR was impaired in 26.1% of patients (n=11) at 1 month and in 31% of patients (n=13) at 12 months after heart transplantation. Patients who developed impaired CFVR in the first year showed a significant increase in iNOS gene expression. Patients with impairment of CFVR 1 month after heart transplantation had higher levels of iNOS mRNA than patients with a normal CFVR. Patients with an initial impairment of CFVR who did not improve over time presented with significantly higher iNOS mRNA levels. iNOS protein and nitrotyrosine were expressed in the endomyocardial vessels of patients with impaired CFVR. Transcardiac NOx release was higher in patients with impaired CFVR. Conclusions In human cardiac allografts, microvascular endothelial dysfunction is associated with an enhanced endomyocardial iNOS mRNA expression and higher transcardiac NOx production and is accompanied by the expression of nitrotyrosine protein, suggesting peroxynitrite plays a role in the disease process. The data from the present study suggest an important role for the iNOS/nitric oxide pathway in the regulation of microvascular function in the absence of preexisting atherosclerotic lesions.

scholarly journals miR-129-5p Regulates the Immunomodulatory Functions of Adipose-Derived Stem Cells via Targeting Stat1 Signaling

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
He-Yang Zhang ◽  
Yu-Han Wang ◽  
Yan Wang ◽  
Yan-Nv Qu ◽  
Xiao-Hui Huang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Inflammatory Factors ◽  
Inos Mrna ◽  
No Production ◽  
Adipose Derived Stem Cells ◽  
Protein Levels ◽  
Bowel Diseases

Adipose-derived stem cells (ASCs) have become one of the most promising stem cell populations for cell-based therapies in regenerative medicine and for autoimmune disorders owing to their multilineage differentiation and immunomodulatory capacities, respectively. One advantage of ASC-based therapy lies in their immunosuppressive potential. However, how to get ASCs to provide consistent immunosuppression remains unclear. In the current study, we found that miR-129-5p was induced in ASCs treated with inflammatory factors. ASCs with miR-129-5p knockdown exhibited enhanced immunosuppressive capacity, as evidenced by reduced expression of proinflammatory factors, with concurrent increased expression of inducible nitric oxide synthases (iNOS) and nitric oxide (NO) production. These cells also had an increased capacity to inhibit T cell proliferation in vitro. ASCs with miR-129-5p knockdown alleviated inflammatory bowel diseases and promoted tumor growth in vivo. Consistently, ASCs that overexpressed miR-129-5p exhibited reduced iNOS expression. Furthermore, we show that miR-129-5p knockdown in ASCs results in hyperphosphorylation of signal transducer and activator of transcription 1 (Stat1). When fludarabine, an inhibitor of Stat1 activation, was added to ASCs with miR-129-5p knockdown, iNOS mRNA and protein levels were significantly reduced. Collectively, these results reveal a new role for miR-129-5p in regulating the immunomodulatory activities of ASCs by targeting Stat1 activation. These novel insights into the mechanisms of ASC immunoregulation may lead to the consistent production of ASCs with strong immunosuppressive functions and thus better clinical utility of these cells.

Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin

2001 ◽  
Vol 354 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Riku KORHONEN ◽  
Hannu KANKAANRANTA ◽  
Aleksi LAHTI ◽  
Mari LÄHDE ◽  
Richard G. KNOWLES ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Cell Activation ◽  
Inos Mrna ◽  
No Production ◽  
Ionophore A23187 ◽  
Inos Protein ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+]i), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased [Ca2+]i on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in [Ca2+]i in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor κB (NF-κB) was investigated by electrophoretic mobility-shift assay. TG (100nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100nM) did not affect the NF-κB activity induced by low (1ng/ml) or high (100ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline (‘XTT’) method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1µM) also transiently increased [Ca2+]i and had opposite effects on NO production depending on the LPS concentration. Our results show that increased [Ca2+]i induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in [Ca2+]i might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.

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