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Clinical and Experimental Immunology Jul 2014Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These...
Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.
Topics: Adult; Cells, Cultured; Chronic Disease; Cystic Fibrosis; Humans; Lung; Male; Middle Aged; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Oxygen Consumption; Pseudomonas Infections; Pseudomonas aeruginosa; Respiratory Mucosa; Sputum; Young Adult; omega-N-Methylarginine
PubMed: 24611476
DOI: 10.1111/cei.12318 -
PloS One 2015Glucagon levels are often moderately elevated in diabetes. It is known that glucagon leads to a decrease in hepatic glutathione (GSH) synthesis that in turn is...
Glucagon levels are often moderately elevated in diabetes. It is known that glucagon leads to a decrease in hepatic glutathione (GSH) synthesis that in turn is associated with decreased postprandial insulin sensitivity. Given that cAMP pathway controls GSH levels we tested whether insulin sensitivity decreases after intraportal (ipv) administration of a cAMP analog (DBcAMP), and investigated whether glucagon promotes insulin resistance through decreasing hepatic GSH levels.Insulin sensitivity was determined in fed male Sprague-Dawley rats using a modified euglycemic hyperinsulinemic clamp in the postprandial state upon ipv administration of DBcAMP as well as glucagon infusion. Glucagon effects on insulin sensitivity was assessed in the presence or absence of postprandial insulin sensitivity inhibition by administration of L-NMMA. Hepatic GSH and NO content and plasma levels of NO were measured after acute ipv glucagon infusion. Insulin sensitivity was assessed in the fed state and after ipv glucagon infusion in the presence of GSH-E. We founf that DBcAMP and glucagon produce a decrease of insulin sensitivity, in a dose-dependent manner. Glucagon-induced decrease of postprandial insulin sensitivity correlated with decreased hepatic GSH content and was restored by administration of GSH-E. Furthermore, inhibition of postprandial decrease of insulin sensitivity L-NMMA was not overcome by glucagon, but glucagon did not affect hepatic and plasma levels of NO. These results show that glucagon decreases postprandial insulin sensitivity through reducing hepatic GSH levels, an effect that is mimicked by increasing cAMP hepatic levels and requires physiological NO levels. These observations support the hypothesis that glucagon acts via adenylate cyclase to decrease hepatic GSH levels and induce insulin resistance. We suggest that the glucagon-cAMP-GSH axis is a potential therapeutic target to address insulin resistance in pathological conditions.
Topics: Adenylyl Cyclases; Animals; Blood Glucose; Bucladesine; Cyclic AMP; Dose-Response Relationship, Drug; Glucagon; Glucose Clamp Technique; Glutathione; Injections, Intravenous; Insulin; Insulin Resistance; Liver; Male; Nitric Oxide; Portal Vein; Postprandial Period; Rats; Rats, Sprague-Dawley; omega-N-Methylarginine
PubMed: 25961284
DOI: 10.1371/journal.pone.0127221 -
Journal of Applied Physiology... Feb 2018Nitric oxide (NO)-mediated vasodilation contributes to the rapid rise in muscle blood flow at exercise onset. This occurs via increased cyclic guanosine monophosphate...
Nitric oxide (NO)-mediated vasodilation contributes to the rapid rise in muscle blood flow at exercise onset. This occurs via increased cyclic guanosine monophosphate (cGMP), which is catabolized by phosphodiesterase-5 (PDE-5). Whether PDE-5 limits exercise vasodilation onset kinetics is unknown. We hypothesized the time course of exercise vasodilation would be 1) accelerated during PDE-5 inhibition (sildenafil citrate, SDF) and 2) decelerated during NO synthase inhibition ( N-monomethyl-l-arginine, l-NMMA), and 3) the effect of SDF on vasodilation onset kinetics would be attenuated with concurrent l-NMMA. Data from 29 healthy adults were analyzed. Individuals completed 5 min of moderate-intensity forearm exercise under control conditions and during 1) oral SDF ( n = 8), 2) intra-arterial l-NMMA ( n = 15), or 3) combined SDF + l-NMMA ( n = 6). Forearm blood flow (FBF; Doppler ultrasound of the brachial artery) and mean brachial artery blood pressure (MAP) were measured continuously. Forearm vascular conductance (FVC, FBF ÷ MAP) was curve-fit with a monoexponential model, and vasodilation onset kinetics were assessed by mean response time (MRT, time to achieve 63% of steady state). SDF had no effect on MRT ( P = 0.90). NOS inhibition increased MRT ( P = 0.01). MRT during SDF+l-NMMA was not different from control exercise ( P = 0.76). PDE-5 inhibition alone has no effect on rapid-onset vasodilation. Whereas NOS inhibition decelerates vasodilator kinetics, when combined with SDF, vasodilator kinetics do not differ from control. These data suggest NO-independent activation of cGMP occurs at exercise onset; thus PDE-5 inhibition may improve vasodilation in pathologies where NO bioavailability is impaired. NEW & NOTEWORTHY We show that when NO bioavailability is reduced, PDE-5 inhibition can restore vasodilation onset kinetics of exercise-mediated vasodilation via NO-independent cGMP pathways. These data suggest PDE-5 inhibition may improve exercise vasodilation onset kinetics in pathologies where NO bioavailability is impaired.
Topics: Adult; Cyclic GMP; Exercise; Female; Humans; Male; Nitric Oxide Synthase; Phosphodiesterase 5 Inhibitors; Sildenafil Citrate; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 28982942
DOI: 10.1152/japplphysiol.00483.2017 -
Nitric Oxide : Biology and Chemistry Nov 2020Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the...
Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the window of effect for L-NMMA on human vascular function is unknown, which is critical for designing and interpreting human-based studies. This study utilized the passive leg movement (PLM) assessment of vascular function, which is predominantly NO-mediated, in 7 young male subjects under control conditions, immediately following intra-arterial L-NMMA infusion (0.24 mg⋅dl⋅min), and at 45-60 and 90-105 min post L-NMMA infusion. The leg blood flow (LBF) and leg vascular conductance (LVC) responses to PLM, measured with Doppler ultrasound and expressed as the change from baseline to peak (ΔLBF and ΔLVC) and area under the curve (LBF and LVC), were assessed. PLM-induced robust control ΔLBF (1135 ± 324 ml⋅min) and ΔLVC (10.7 ± 3.6 ml⋅min⋅mmHg) responses that were significantly attenuated (704 ± 196 ml⋅min and 6.7 ± 2 ml⋅min⋅mmHg) immediately following L-NMMA infusion. Likewise, control condition PLM ΔLBF (455 ± 202 ml) and ΔLVC (4.0 ± 1.4 ml⋅mmHg) were significantly attenuated (141 ± 130 ml and 1.3 ± 1.2 ml⋅mmHg) immediately following L-NMMA infusion. However, by 45-60 min post L-NMMA infusion all PLM variables were not significantly different from control, and this was still the case at 90-105 min post L-NMMA infusion. These findings reveal that the potent reduction in NO bioavailability afforded by NOS inhibition with L-NMMA has a window of effect of less than 45-60 min in the human vasculature. These data are particularly important for the commonly employed approach of pharmacologically inhibiting NOS with L-NMMA in the human vasculature.
Topics: Adult; Enzyme Inhibitors; Femoral Artery; Hemodynamics; Humans; Leg; Male; Nitric Oxide; Nitric Oxide Synthase; Regional Blood Flow; Time Factors; Young Adult; omega-N-Methylarginine
PubMed: 32979497
DOI: 10.1016/j.niox.2020.09.001 -
Diabetes, Obesity & Metabolism Feb 2019The associations between arginine-based metabolites and incident type 2 diabetes (T2D) are unknown. We employed a case-cohort design, nested within the PREDIMED trial,... (Observational Study)
Observational Study
The associations between arginine-based metabolites and incident type 2 diabetes (T2D) are unknown. We employed a case-cohort design, nested within the PREDIMED trial, to examine six plasma metabolites (arginine, citrulline, ornithine, asymmetric dimethylarginine [ADMA], symmetric dimethylarginine [SDMA] and N-monomethyl-l-arginine [NMMA]) among 892 individuals (251 cases) for associations with incident T2D and insulin resistance. Weighted Cox models with robust variance were used. The 1-year changes in arginine (adjusted hazard ratio [HR] per SD 0.68, 95% confidence interval [CI] 0.49, 0.95; Q4 vs. Q1 0.46, 95% CI 0.21, 1.04; P trend = 0.02) and arginine/ADMA ratio (adjusted HR per SD 0.73, 95% CI 0.51, 1.04; Q4 vs. Q1 0.52, 95% CI 0.22, 1.25; P trend = 0.04) were associated with a lower risk of T2D. Positive changes of citrulline and ornithine, and negative changes in SDMA and arginine/(ornithine + citrulline) were associated with concurrent 1-year changes in homeostatic model assessment of insulin resistance. Individuals in the low-fat-diet group had a higher risk of T2D for 1-year changes in NMMA than individuals in Mediterranean-diet groups (P interaction = 0.02). We conclude that arginine bioavailability is important in T2D pathophysiology.
Topics: Aged; Aged, 80 and over; Arginine; Case-Control Studies; Citrulline; Cohort Studies; Diabetes Mellitus, Type 2; Diet, Fat-Restricted; Diet, Mediterranean; Female; Humans; Incidence; Insulin Resistance; Male; Middle Aged; Ornithine; Risk Factors; omega-N-Methylarginine
PubMed: 30146690
DOI: 10.1111/dom.13514 -
The Journal of Toxicological Sciences 2020Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of...
Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells.
Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.
Topics: Animals; Benzhydryl Compounds; Brain; Depression, Chemical; Hippocampus; Male; Neuronal Outgrowth; Neurotoxins; Nitric Oxide; Oxidation-Reduction; PC12 Cells; Phenols; Protein Disulfide-Isomerases; Rats; Rats, Sprague-Dawley; Ribonucleases; Rotenone; omega-N-Methylarginine
PubMed: 33268678
DOI: 10.2131/jts.45.783 -
Redox Biology Sep 2019L-N-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS)....
L-N-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature.
Topics: Animals; Arginine; Enzyme Inhibitors; Female; Mesenteric Arteries; Mice; Myography; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroglycerin; RAW 264.7 Cells; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sheep; Stereoisomerism; Vasodilation; Vasodilator Agents; omega-N-Methylarginine
PubMed: 31200239
DOI: 10.1016/j.redox.2019.101238 -
Microcirculation (New York, N.Y. : 1994) Apr 2020The aim of this study was to investigate whether the effects on local blood flow and metabolic changes observed in the skin after an endogenous systemic increase in... (Clinical Trial)
Clinical Trial
OBJECTIVE
The aim of this study was to investigate whether the effects on local blood flow and metabolic changes observed in the skin after an endogenous systemic increase in insulin are mediated by the endothelial nitric oxide pathway, by administering the nitric oxide synthase inhibitor N -monomethyl l-arginine using microdialysis.
METHODS
Microdialysis catheters, perfused with N -monomethyl l-arginine and with a control solution, were inserted intracutaneously in 12 human subjects, who received an oral glucose load to induce a systemic hyperinsulinemia. During microdialysis, the local blood flow was measured by urea clearance and by laser speckle contrast imaging, and glucose metabolites were measured.
RESULTS
After oral glucose intake, microvascular blood flow and glucose metabolism were both significantly suppressed in the N -monomethyl l-arginine catheter compared to the control catheter (urea clearance: P < .006, glucose dialysate concentration: P < .035). No significant effect of N -monomethyl l-arginine on microvascular blood flow was observed with laser speckle contrast imaging (P = .81).
CONCLUSION
Local delivery of N -monomethyl l-arginine to the skin by microdialysis reduces microvascular blood flow and glucose delivery in the skin after oral glucose intake, presumably by decreasing local insulin-mediated vasodilation.
Topics: Adult; Blood Flow Velocity; Blood Glucose; Female; Glucose Tolerance Test; Humans; Male; Microcirculation; Microdialysis; Regional Blood Flow; omega-N-Methylarginine
PubMed: 31628700
DOI: 10.1111/micc.12597 -
Clinical Cancer Research : An Official... Mar 2018Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress...
Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC. TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay. L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9. iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. .
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Docetaxel; Drug Synergism; Female; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 5; MAP Kinase Signaling System; Mice; Mice, SCID; Nitric Oxide Synthase Type II; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays; omega-N-Methylarginine
PubMed: 29301832
DOI: 10.1158/1078-0432.CCR-17-1437 -
Nitric oxide synthase inhibition restores orthostatic tolerance in young vasovagal syncope patients.Heart (British Cardiac Society) Nov 2017Syncope is sudden transient loss of consciousness and postural tone with spontaneous recovery; the most common form is vasovagal syncope (VVS). We previously...
OBJECTIVE
Syncope is sudden transient loss of consciousness and postural tone with spontaneous recovery; the most common form is vasovagal syncope (VVS). We previously demonstrated impaired post-synaptic adrenergic responsiveness in young VVS patients was reversed by blocking nitric oxide synthase (NOS). We hypothesised that nitric oxide may account for reduced orthostatic tolerance in young recurrent VVS patients.
METHODS
We recorded haemodynamics in supine VVS and healthy volunteers (aged 15-27 years), challenged with graded lower body negative pressure (LBNP) (-15, -30, -45 mm Hg each for 5 min, then -60 mm Hg for a maximum of 50 min) with and without NOS inhibitor N-monomethyl-L-arginine acetate (L-NMMA). Saline plus phenylephrine (Saline+PE) was used as volume and pressor control for L-NMMA.
RESULTS
Controls endured 25.9±4.0 min of LBNP during Saline+PE compared with 11.6±1.4 min for fainters (p<0.001). After L-NMMA, control subjects endured 24.8±3.2 min compared with 22.6±1.6 min for fainters. Mean arterial pressure decreased more in VVS patients during LBNP with Saline+PE (p<0.001) which was reversed by L-NMMA; cardiac output decreased similarly in controls and VVS patients and was unaffected by L-NMMA. Total peripheral resistance increased for controls but decreased for VVS during Saline+PE (p<0.001) but was similar following L-NMMA. Splanchnic vascular resistance increased during LBNP in controls, but decreased in VVS patients following Saline+PE which L-NMMA restored.
CONCLUSIONS
We conclude that arterial vasoconstriction is impaired in young VVS patients, which is corrected by NOS inhibition. The data suggest that both pre- and post-synaptic arterial vasoconstriction may be affected by nitric oxide.
Topics: Administration, Intravenous; Adolescent; Adult; Age Factors; Arterial Pressure; Arteries; Cardiac Output; Enzyme Inhibitors; Female; Hemodynamics; Humans; Lower Body Negative Pressure; Male; Nitric Oxide Synthase; Syncope, Vasovagal; Time Factors; Treatment Outcome; Vascular Resistance; Vasoconstriction; Young Adult; omega-N-Methylarginine
PubMed: 28501796
DOI: 10.1136/heartjnl-2017-311161