BACKGROUND Eosinophilic esophagitis(EoE)is an eosinophilic-predominant inflammation of the esophagus diagnosed by upper endoscopy and biopsies.A non-invasive and cost-effective alternative for management of EoE is bei...BACKGROUND Eosinophilic esophagitis(EoE)is an eosinophilic-predominant inflammation of the esophagus diagnosed by upper endoscopy and biopsies.A non-invasive and cost-effective alternative for management of EoE is being researched.Previous studies assessing utility of fractional exhaled nitric oxide(FeNO)in EoE were low powered.None investigated the contribution of eosinophilic inflammation of the stomach and duodenum to FeNO.AIM To assess the utility of FeNO as a non-invasive biomarker of esophageal eosinophilic inflammation for monitoring disease activity.METHODS Patients aged 6-21 years undergoing scheduled upper endoscopy with biopsy for suspected EoE were recruited in our observational study.Patients on steroids and with persistent asthma requiring daily controller medication were excluded.FeNO measurements were obtained in duplicate using a chemiluminescence nitric oxide analyzer(NIOX MINO,Aerocrine,Inc.;Stockholm,Sweden)prior to endoscopy.Based on the esophageal peak eosinophil count(PEC)/high power field on biopsy,patients were classified as EoE(PEC≥15)or control(PEC≤14).Mean FeNO levels were correlated with presence or absence of EoE,eosinophil counts on esophageal biopsy,and abnormal downstream eosinophilia in the stomach(PEC≥10)and duodenum(PEC≥20).Wilcoxon rank-sum test,Spearman correlation,and logistic regression were used for analysis.P value<0.05 was considered significant.RESULTS We recruited a total of 134 patients,of which 45 were diagnosed with EoE by histopathology.The median interquartile range FeNO level was 17 parts per billion(11-37,range:7-81)in the EoE group and 12 parts per billion(8-19,range:5-71)in the control group.After adjusting for atopic diseases,EoE patients had significantly higher FeNO levels as compared to patients without EoE(Z=3.33,P<0.001).A weak yet statistically significant positive association was found between the number of esophageal eosinophils and FeNO levels(r=0.30,P<0.005).On subgroup analysis within the EoE cohort,higher FeNO levels were noted in patients with abnormal gastric(n=23,18 vs 15)and duodenal eosinophilia(n=28,21 vs 14);however,the difference was not statistically significant.CONCLUSION After ruling out atopy as possible confounder,we found significantly higher FeNO levels in the EoE cohort than in the control group.展开更多
Bronchial asthma is a common chronic airway inflammatory disease. Asthma is associated with high mortality, especially in the elderly patients. Repeated exacerbations cause disease progression. Therefore, identifying ...Bronchial asthma is a common chronic airway inflammatory disease. Asthma is associated with high mortality, especially in the elderly patients. Repeated exacerbations cause disease progression. Therefore, identifying the onset of acute elderly asthma as soon as possible and giving the effective treatment is crucial to improve the prognosis. This study was to investigate the significance of fractional exhaled nitric oxide (FeNO) combined with serum procalcitonin (PCT) and C-reactive protein (CRP) in the evaluation of elderly asthma. A total of 120 elderly patients with an acute attack of asthma from July, 2010 to May, 2012 were studied. On presentation, FeNO, serum PCT and CRP concentrations were measured and sputum culture was also performed. The elderly patients were re-evaluated when they had returned to their stable clinical state. The elderly patients were classified into two groups: positive bac- terial culture group (A) and negative bacterial culture group (B). The results showed that: (1) In patients with an acute exacerbation of asthma, 48 (40%) patients had positive sputum bacterial culture and 72 (60%) had negative sputum bacterial culture. (2) The levels of FeNO in patients with acute exacerbation of asthma were significantly higher than in those with no acute exacerbation state (63.8±24.6 vs. 19±6.5 ppb, P〈0.05). There was no significant difference in FeNO between group A and group B (P〉0.05). (3) The levels of PCT and CRP in group A patients with an acute exacerbation of asthma were significantly higher (P〈0.05) than in group B (for PCT: 27.46±9.32 vs. 7.85±3.52 ng/mL; for CRP: 51.25±11.46 vs. 17.11±5.87 mg/L, respectively). When they had returned to stable clinical state, the levels of PCT and CRP in group A were decreased significantly (P〈0.05), and those in group B had no significant change (P〉0.05) when compared with the exacerbation group. There were no significant differences in the levels of PCT and CRP between the two groups in non-acute exacerbation state (/9〉0.05). These results suggest that the increase in FeNO indicates the acute exacerbation of asthma, and the elevation of serum PCT and CRP levels may be associated with bacterial infection.展开更多
Background: Some patients present clinical features of both asthma and chronic obstructive pulmonary disease (COPD), which has led to the recent proposal of asthma-COPD overlap (ACO) as a diagnosis. Fractional exhaled...Background: Some patients present clinical features of both asthma and chronic obstructive pulmonary disease (COPD), which has led to the recent proposal of asthma-COPD overlap (ACO) as a diagnosis. Fractional exhaled nitric oxide (FeNO) is a candidate biomarker to diagnose ACO. We assessed the effect of an add-on treatment with budesonide/formoterol (BUD/FM) combination in patients with ACO, which was diagnosed by FeNO. Methods: This was a prospective, single-arm, open-label, before and after comparison study. Subjects included 83 patients with COPD who attended outpatient clinics for routine checkups at Shizuoka General Hospital between June and November 2016. All patients fulfilled the GOLD definition of COPD and were receiving long-acting muscarinic antagonist (LAMA) or LAMA/long-acting β2 agonist (LABA) combinations. After an 8-week run-in period, BUD/FM was added to the patients with FeNO levels of ≥35 ppb, defined as having ACO. For patients receiving LAMA/LABA, BUD/FM was added after the discontinuation of LABA. The modified British Medical Research Council (mMRC) score, COPD assessment test (CAT) score, spirometric indices, forced oscillation parameters, and FeNO were assessed before and after 8 weeks of BUD/ FM add-on treatment. Results: Twenty-four patients (28.9%) had FeNO levels ≥ 35 ppb, and 17 patients completed the study (mean age: 73 years and GOLD I/II/III/IV, 5/10/1/1). The mean CAT scores significantly improved (9.2 to 5.4, p = 0.015) and 10 patients (58.8%) showed ≥2 points improvement, a minimal clinically important difference. The mean FeNO levels significantly decreased from 63.0 to 34.3 ppb (p Conclusions: FeNO-guided treatment with BUD/FM improves symptoms in patients with ACO.展开更多
Aim: The goal of this study was to compare asthma treatment guidance based on bronchial hyper-responsiveness to mannitol, spirometry or exhaled nitric oxide (FeNO) in stable asthmatic children. Methods: 60 stable alle...Aim: The goal of this study was to compare asthma treatment guidance based on bronchial hyper-responsiveness to mannitol, spirometry or exhaled nitric oxide (FeNO) in stable asthmatic children. Methods: 60 stable allergic asthmatic children aged 7 to 16 years on a low to medium dose treatment with inhaled corticosteroids (ICS) were recruited to a double blind randomised controlled trial. At study entry (visit 1), the following was assessed: FeNO, spirometry, bronchial hyper-responsiveness to mannitol (MDP-?test), quality of life (paediatric asthma quality-of-life questionnaire;PAQLQ) and asthma control (asthma control test;ACT). Subjects were randomly assigned to one of three groups and treatment was modified by a blinded respiratory physician according to the test results of visit 1: ICS dose was doubled when FeNO was >22 ppb (group 1), in case of a positive MDP-test (group 2) or when FEV1 was <80% of a predicted one (group 3), respectively, or remained unchanged for the remaining subjects. After 3 months (visit 2), the subjects were reassessed and all tests were repeated. Results: 48 children successfully completed the study. At the first visit, 8 out of 16 (50%) children in group 1 showed a FeNO > 22 ppb, 8 children out of 16 (50%) in group 2 showed a positive MDP-test and 3 children out of 16 (18.7%) in group 3 had a FEV1 < 80% of that predicted and had their ICS-dose doubled. In group 1, FeNO decreased significantly after the intervention (p = 0.005), whereas the self-administered and the interviewer-administered PAQLQ (p = 0.02 resp. p = 0.033) as well as the ACT (p = 0.031) increased. Neither the number of children with a positive mannitol challenge nor spirometric results changed significantly. In group 2 and group 3, there were no significant changes in none of the assessed parameters. Conclusion: In this small pragmatic double blind randomised controlled study, we showed that ICS dose modification based on FeNO led to increased quality of life and enhanced asthma control, and to a reduction in airway inflammation and was superior to treatment modifications based on bronchial hyper-responsiveness to mannitol or on FEV1.展开更多
Objective: Highland natives adapt well to the hypoxic environment at high altitude(HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial nitric oxide synthas...Objective: Highland natives adapt well to the hypoxic environment at high altitude(HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial nitric oxide synthase(ENOS) G894 T polymorphism contributed to the physiology and pathophysiology of humans at HA by regulating the production of NO. In this meta-analysis, we evaluate the association between the ENOS G894 T polymorphism and HA adaptation through analyzing the published data. Methods: We searched all relevant literature about the ENOS G894 T polymorphism and HA adaptation in Pub Med, Medline, and Embase before Step 2015. A random-effects model was applied(Revman 5.0), and study quality was assessed in duplicate. Six studies with 634 HA native cases and 621 low-altitude controls were included in this meta-analysis. Results: From the results, we observed that the wild-type allele G was significantly overrepresented in the HA groups(OR=1.85; 95% CI, 1.47–2.33; P<0.0001). In addition, the GG genotype was significantly associated with HA adaptation(OR=1.99; 95% CI, 1.54–2.57; P<0.0001). Conclusion: Our results showed that in 894 G allele carriers, the GG genotype might be a beneficial factor for HA adaptation through enhancing the level of NO. However, more studies were needed to confirm our findings due to the limited sample size.展开更多
Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopte...Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclobydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4:BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.展开更多
The aim of the present study was to measure intrarenal spatial and temporal localization of all three nitric oxide synthase (NOS) isoforms in the developing ovine kidney. Reverse transcriptase-polymerase chain reactio...The aim of the present study was to measure intrarenal spatial and temporal localization of all three nitric oxide synthase (NOS) isoforms in the developing ovine kidney. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western Blot analyses, and in situ hybridization techniques were performed for NOS I -III isoforms in renal tissue obtained from sheep aged ~24 h, one, three, six, and 12 weeks post natally (N = 3). RT-PCR performed on cortical and medullary kidney tissue revealed the presence of all three NOS isoforms from day one to 12 weeks postnatally. NOS I and NOS II mRNA levels were greater in cortex compared to medulla during the first three weeks whereas NOS III mRNA levels were predominantly transcribed within the medulla. In all NOS isoforms, there was a decrease in cortical mRNA levels after three to six weeks. Protein levels confirmed the presence of all three NOS isoforms over the first three months of postnatal life. By demonstrating NOS isoform transcripts to be more abundant in the early post natal period, these findings may provide insight into the age dependent role of NO in modulating kidney function during ontogeny.展开更多
Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiologic...Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.展开更多
文摘BACKGROUND Eosinophilic esophagitis(EoE)is an eosinophilic-predominant inflammation of the esophagus diagnosed by upper endoscopy and biopsies.A non-invasive and cost-effective alternative for management of EoE is being researched.Previous studies assessing utility of fractional exhaled nitric oxide(FeNO)in EoE were low powered.None investigated the contribution of eosinophilic inflammation of the stomach and duodenum to FeNO.AIM To assess the utility of FeNO as a non-invasive biomarker of esophageal eosinophilic inflammation for monitoring disease activity.METHODS Patients aged 6-21 years undergoing scheduled upper endoscopy with biopsy for suspected EoE were recruited in our observational study.Patients on steroids and with persistent asthma requiring daily controller medication were excluded.FeNO measurements were obtained in duplicate using a chemiluminescence nitric oxide analyzer(NIOX MINO,Aerocrine,Inc.;Stockholm,Sweden)prior to endoscopy.Based on the esophageal peak eosinophil count(PEC)/high power field on biopsy,patients were classified as EoE(PEC≥15)or control(PEC≤14).Mean FeNO levels were correlated with presence or absence of EoE,eosinophil counts on esophageal biopsy,and abnormal downstream eosinophilia in the stomach(PEC≥10)and duodenum(PEC≥20).Wilcoxon rank-sum test,Spearman correlation,and logistic regression were used for analysis.P value<0.05 was considered significant.RESULTS We recruited a total of 134 patients,of which 45 were diagnosed with EoE by histopathology.The median interquartile range FeNO level was 17 parts per billion(11-37,range:7-81)in the EoE group and 12 parts per billion(8-19,range:5-71)in the control group.After adjusting for atopic diseases,EoE patients had significantly higher FeNO levels as compared to patients without EoE(Z=3.33,P<0.001).A weak yet statistically significant positive association was found between the number of esophageal eosinophils and FeNO levels(r=0.30,P<0.005).On subgroup analysis within the EoE cohort,higher FeNO levels were noted in patients with abnormal gastric(n=23,18 vs 15)and duodenal eosinophilia(n=28,21 vs 14);however,the difference was not statistically significant.CONCLUSION After ruling out atopy as possible confounder,we found significantly higher FeNO levels in the EoE cohort than in the control group.
基金supported by Fundation of Henan Provincial Medical Science and Technology Research(No.201003098)
文摘Bronchial asthma is a common chronic airway inflammatory disease. Asthma is associated with high mortality, especially in the elderly patients. Repeated exacerbations cause disease progression. Therefore, identifying the onset of acute elderly asthma as soon as possible and giving the effective treatment is crucial to improve the prognosis. This study was to investigate the significance of fractional exhaled nitric oxide (FeNO) combined with serum procalcitonin (PCT) and C-reactive protein (CRP) in the evaluation of elderly asthma. A total of 120 elderly patients with an acute attack of asthma from July, 2010 to May, 2012 were studied. On presentation, FeNO, serum PCT and CRP concentrations were measured and sputum culture was also performed. The elderly patients were re-evaluated when they had returned to their stable clinical state. The elderly patients were classified into two groups: positive bac- terial culture group (A) and negative bacterial culture group (B). The results showed that: (1) In patients with an acute exacerbation of asthma, 48 (40%) patients had positive sputum bacterial culture and 72 (60%) had negative sputum bacterial culture. (2) The levels of FeNO in patients with acute exacerbation of asthma were significantly higher than in those with no acute exacerbation state (63.8±24.6 vs. 19±6.5 ppb, P〈0.05). There was no significant difference in FeNO between group A and group B (P〉0.05). (3) The levels of PCT and CRP in group A patients with an acute exacerbation of asthma were significantly higher (P〈0.05) than in group B (for PCT: 27.46±9.32 vs. 7.85±3.52 ng/mL; for CRP: 51.25±11.46 vs. 17.11±5.87 mg/L, respectively). When they had returned to stable clinical state, the levels of PCT and CRP in group A were decreased significantly (P〈0.05), and those in group B had no significant change (P〉0.05) when compared with the exacerbation group. There were no significant differences in the levels of PCT and CRP between the two groups in non-acute exacerbation state (/9〉0.05). These results suggest that the increase in FeNO indicates the acute exacerbation of asthma, and the elevation of serum PCT and CRP levels may be associated with bacterial infection.
文摘Background: Some patients present clinical features of both asthma and chronic obstructive pulmonary disease (COPD), which has led to the recent proposal of asthma-COPD overlap (ACO) as a diagnosis. Fractional exhaled nitric oxide (FeNO) is a candidate biomarker to diagnose ACO. We assessed the effect of an add-on treatment with budesonide/formoterol (BUD/FM) combination in patients with ACO, which was diagnosed by FeNO. Methods: This was a prospective, single-arm, open-label, before and after comparison study. Subjects included 83 patients with COPD who attended outpatient clinics for routine checkups at Shizuoka General Hospital between June and November 2016. All patients fulfilled the GOLD definition of COPD and were receiving long-acting muscarinic antagonist (LAMA) or LAMA/long-acting β2 agonist (LABA) combinations. After an 8-week run-in period, BUD/FM was added to the patients with FeNO levels of ≥35 ppb, defined as having ACO. For patients receiving LAMA/LABA, BUD/FM was added after the discontinuation of LABA. The modified British Medical Research Council (mMRC) score, COPD assessment test (CAT) score, spirometric indices, forced oscillation parameters, and FeNO were assessed before and after 8 weeks of BUD/ FM add-on treatment. Results: Twenty-four patients (28.9%) had FeNO levels ≥ 35 ppb, and 17 patients completed the study (mean age: 73 years and GOLD I/II/III/IV, 5/10/1/1). The mean CAT scores significantly improved (9.2 to 5.4, p = 0.015) and 10 patients (58.8%) showed ≥2 points improvement, a minimal clinically important difference. The mean FeNO levels significantly decreased from 63.0 to 34.3 ppb (p Conclusions: FeNO-guided treatment with BUD/FM improves symptoms in patients with ACO.
文摘Aim: The goal of this study was to compare asthma treatment guidance based on bronchial hyper-responsiveness to mannitol, spirometry or exhaled nitric oxide (FeNO) in stable asthmatic children. Methods: 60 stable allergic asthmatic children aged 7 to 16 years on a low to medium dose treatment with inhaled corticosteroids (ICS) were recruited to a double blind randomised controlled trial. At study entry (visit 1), the following was assessed: FeNO, spirometry, bronchial hyper-responsiveness to mannitol (MDP-?test), quality of life (paediatric asthma quality-of-life questionnaire;PAQLQ) and asthma control (asthma control test;ACT). Subjects were randomly assigned to one of three groups and treatment was modified by a blinded respiratory physician according to the test results of visit 1: ICS dose was doubled when FeNO was >22 ppb (group 1), in case of a positive MDP-test (group 2) or when FEV1 was <80% of a predicted one (group 3), respectively, or remained unchanged for the remaining subjects. After 3 months (visit 2), the subjects were reassessed and all tests were repeated. Results: 48 children successfully completed the study. At the first visit, 8 out of 16 (50%) children in group 1 showed a FeNO > 22 ppb, 8 children out of 16 (50%) in group 2 showed a positive MDP-test and 3 children out of 16 (18.7%) in group 3 had a FEV1 < 80% of that predicted and had their ICS-dose doubled. In group 1, FeNO decreased significantly after the intervention (p = 0.005), whereas the self-administered and the interviewer-administered PAQLQ (p = 0.02 resp. p = 0.033) as well as the ACT (p = 0.031) increased. Neither the number of children with a positive mannitol challenge nor spirometric results changed significantly. In group 2 and group 3, there were no significant changes in none of the assessed parameters. Conclusion: In this small pragmatic double blind randomised controlled study, we showed that ICS dose modification based on FeNO led to increased quality of life and enhanced asthma control, and to a reduction in airway inflammation and was superior to treatment modifications based on bronchial hyper-responsiveness to mannitol or on FEV1.
基金supported by the National Natural Science Foundation of China(81372125)
文摘Objective: Highland natives adapt well to the hypoxic environment at high altitude(HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial nitric oxide synthase(ENOS) G894 T polymorphism contributed to the physiology and pathophysiology of humans at HA by regulating the production of NO. In this meta-analysis, we evaluate the association between the ENOS G894 T polymorphism and HA adaptation through analyzing the published data. Methods: We searched all relevant literature about the ENOS G894 T polymorphism and HA adaptation in Pub Med, Medline, and Embase before Step 2015. A random-effects model was applied(Revman 5.0), and study quality was assessed in duplicate. Six studies with 634 HA native cases and 621 low-altitude controls were included in this meta-analysis. Results: From the results, we observed that the wild-type allele G was significantly overrepresented in the HA groups(OR=1.85; 95% CI, 1.47–2.33; P<0.0001). In addition, the GG genotype was significantly associated with HA adaptation(OR=1.99; 95% CI, 1.54–2.57; P<0.0001). Conclusion: Our results showed that in 894 G allele carriers, the GG genotype might be a beneficial factor for HA adaptation through enhancing the level of NO. However, more studies were needed to confirm our findings due to the limited sample size.
文摘Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclobydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4:BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.
文摘The aim of the present study was to measure intrarenal spatial and temporal localization of all three nitric oxide synthase (NOS) isoforms in the developing ovine kidney. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western Blot analyses, and in situ hybridization techniques were performed for NOS I -III isoforms in renal tissue obtained from sheep aged ~24 h, one, three, six, and 12 weeks post natally (N = 3). RT-PCR performed on cortical and medullary kidney tissue revealed the presence of all three NOS isoforms from day one to 12 weeks postnatally. NOS I and NOS II mRNA levels were greater in cortex compared to medulla during the first three weeks whereas NOS III mRNA levels were predominantly transcribed within the medulla. In all NOS isoforms, there was a decrease in cortical mRNA levels after three to six weeks. Protein levels confirmed the presence of all three NOS isoforms over the first three months of postnatal life. By demonstrating NOS isoform transcripts to be more abundant in the early post natal period, these findings may provide insight into the age dependent role of NO in modulating kidney function during ontogeny.
文摘Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.