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Journal of the American Heart... Aug 2020Background Basal release of nitric oxide (NO) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular... (Randomized Controlled Trial)
Randomized Controlled Trial
Background Basal release of nitric oxide (NO) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular arteries could be particularly important during exercise when shear stress may stimulate increased NO synthesis. Methods and Results We investigated acute effects of NO synthase inhibition on exercise hemodynamics using N-monomethyl-l-arginine (l-NMMA), a nonselective NO synthase -inhibitor. Healthy volunteers (n=10, 5 female, 19-33 years) participated in a 2-phase randomized crossover study, receiving l-NMMA (6 mg/kg, iv over 5 minutes) or placebo before bicycle exercise (25-150 W for 12 minutes). Blood pressure, cardiac output (measured by dilution of soluble and inert tracers) and femoral artery diameter were measured before, during, and after exercise. At rest, l-NMMA reduced heart rate (by 16.2±4.3 bpm relative to placebo, <0.01), increased peripheral vascular resistance (by 7.0±1.4 mmHg per L/min, <0.001), mean arterial blood pressure (by 8.9±3.5 mmHg, <0.05), and blunted an increase in femoral artery diameter that occurred immediately before exercise (change in diameter: 0.14±0.04 versus 0.32±0.06 mm after l-NMMA and placebo, <0.01). During/after exercise l-NMMA had no significant effect on peripheral resistance, cardiac output, or on femoral artery diameter. Conclusions These results suggest that NO plays little role in modulating muscular artery function during exercise but that it may mediate changes in muscular artery tone immediately before exercise.
Topics: Adult; Arterial Pressure; Arteries; Cardiac Output; Cross-Over Studies; Enzyme Inhibitors; Exercise; Exercise Test; Female; Femoral Artery; Humans; Male; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Placebos; Pulse Wave Analysis; Vascular Resistance; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 32781940
DOI: 10.1161/JAHA.119.013849 -
Amino Acids Dec 2015Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and...
Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS(3)), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS(3) and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS(3) and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.
Topics: Arginine; Gene Deletion; Heat-Shock Response; Hot Temperature; Mass Spectrometry; Methylation; Protein-Arginine N-Methyltransferases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; omega-N-Methylarginine
PubMed: 26189025
DOI: 10.1007/s00726-015-2047-5 -
Immunology Letters Oct 2017Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli by expressing a variety of surface and intracellular receptors...
Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli by expressing a variety of surface and intracellular receptors and triggering multiple signal transduction pathways. The onset of microbial infection is primarily determined by the initial contacts made by the microbes with the host macrophages. Although there prevail a relationship between the chemokine receptor and Toll like receptors during disease, particularly TLR-2 and CCR-2 signaling interdependence on each other has not been yet investigated during acute staphylococcal infection. Thus, the present study was aimed to trace possible interaction between CCR-2 and TLR-2 in peritoneal macrophages during acute Staphylococcus aureus infection. We found that neutralization of CCR-2 attenuates TLR-2 expression and restricts S. aureus burden but TLR-2 neutralization augments CCR-2 expression in macrophages, along with compromised host-derived reactive oxygen species production. S. aureus infection to CCR-2 intact but TLR-2 neutralized macrophages triggered production of IL-1β, TNF-α, IL-6, IFN-γ, MCP-1 and expression of iNOS, TNFR-1 and GPx with concomitant decrease in IL-10 production. Further, study with NG-monomethyl-l-arginine (L-NMMA) [iNOS blocker] and buthionine sulfoximine (BSO) [GPx blocker] revealed that S. aureus infection enhanced TLR-2 expression in CCR-2 intact and TLR-2 neutralized macrophages possibly via iNOS and TNFR-1 up regulation and GPx down regulation. Overall, our data indicate that targeting CCR-2 with neutralizing antibody in the early phase of S. aureus infection could restrict excessive inflammation with less compromised bacterial killing. It certainly would be a therapeutic strategy in S. aureus induced inflammatory and infective diseases.
Topics: Animals; Antibodies, Neutralizing; Bacteriolysis; Buthionine Sulfoximine; Cells, Cultured; Cytokines; Host-Pathogen Interactions; Macrophages, Peritoneal; Mice; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Receptors, CCR2; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; omega-N-Methylarginine
PubMed: 28736159
DOI: 10.1016/j.imlet.2017.07.011 -
Hypertension (Dallas, Tex. : 1979) Oct 2014Obesity is an important risk factor for the development of insulin resistance. Initial compensatory mechanisms include an increase in insulin levels, which are thought... (Randomized Controlled Trial)
Randomized Controlled Trial
Obesity is an important risk factor for the development of insulin resistance. Initial compensatory mechanisms include an increase in insulin levels, which are thought to induce sympathetic activation in an attempt to restore energy balance. We have previously shown, however, that sympathetic activity has no beneficial effect on resting energy expenditure in obesity. On the contrary, we hypothesize that sympathetic activation contributes to insulin resistance. To test this hypothesis, we determined insulin sensitivity using a standard hyperinsulinemic euglycemic clamp protocol in obese subjects randomly assigned in a crossover design 1 month apart to receive saline (intact day) or trimetaphan (4 mg/min IV, autonomic blocked day). Whole-body glucose uptake (MBW in mg/kg per minute) was used as index of maximal muscle glucose use. During autonomic blockade, we clamped blood pressure with a concomitant titrated intravenous infusion of the nitric oxide synthase inhibitor N-monomethyl-L-arginine. Of the 21 obese subjects (43±2 years; 35±2 kg/m(2) body mass index) studied, 14 were insulin resistant; they were more obese, had higher plasma glucose and insulin, and had higher muscle sympathetic nerve activity (23.3±1.5 versus 17.2±2.1 burst/min; P=0.03) when compared with insulin-sensitive subjects. Glucose use improved during autonomic blockade in insulin-resistant subjects (MBW 3.8±0.3 blocked versus 3.1±0.3 mg/kg per minute intact; P=0.025), with no effect in the insulin-sensitive group. These findings support the concept that sympathetic activation contributes to insulin resistance in obesity and may result in a feedback loop whereby the compensatory increase in insulin levels contributes to greater sympathetic activation.
Topics: Adult; Autonomic Nervous System; Blood Glucose; Blood Pressure; Cross-Over Studies; Enzyme Inhibitors; Female; Ganglionic Blockers; Glucose Clamp Technique; Humans; Insulin; Insulin Resistance; Male; Middle Aged; Muscles; Nitric Oxide Synthase; Obesity; Trimethaphan; omega-N-Methylarginine
PubMed: 25001269
DOI: 10.1161/HYPERTENSIONAHA.114.03738 -
PloS One 2015Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood...
Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250-300 g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.
Topics: Animals; Arterioles; Blood Flow Velocity; Endothelium, Vascular; Female; Fluoresceins; Glycocalyx; Hemodynamics; Intravital Microscopy; Microvessels; Nitric Oxide; Nitric Oxide Synthase Type III; Polysaccharide-Lyases; Rats; Rats, Sprague-Dawley; omega-N-Methylarginine
PubMed: 25575016
DOI: 10.1371/journal.pone.0117133 -
Parasites & Vectors Jul 2015Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal...
BACKGROUND
Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis.
METHODS
T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining.
RESULTS
We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions.
CONCLUSION
The findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.
Topics: Adaptation, Physiological; Animals; Base Sequence; Benzimidazoles; Carbocyanines; Cell Survival; Enzyme Inhibitors; High-Throughput Nucleotide Sequencing; Humans; Iron Deficiencies; Leupeptins; Models, Biological; Nitric Oxide; Nitric Oxide Synthase; Protozoan Proteins; Reactive Oxygen Species; Sequence Analysis, RNA; Transcriptome; Trichomonas Infections; Trichomonas vaginalis; omega-N-Methylarginine
PubMed: 26205151
DOI: 10.1186/s13071-015-1000-5 -
Cell Death & Disease Jun 2015The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic...
The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.
Topics: AMP-Activated Protein Kinases; Aminoimidazole Carboxamide; Animals; Apoptosis; Calcium; Cycloheximide; Dactinomycin; Diabetes Mellitus; Endoplasmic Reticulum; Enzyme Activation; Fura-2; Humans; Insulin-Secreting Cells; Insulinoma; Interleukin-1beta; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; RNA, Messenger; Rats; Rats, Wistar; Ribonucleotides; S-Nitroso-N-Acetylpenicillamine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; omega-N-Methylarginine
PubMed: 26086963
DOI: 10.1038/cddis.2015.154 -
Diabetes Jun 2017Insulin resistance is a major health risk, and although exercise clearly improves skeletal muscle insulin sensitivity, the mechanisms are unclear. Here we show that...
Insulin resistance is a major health risk, and although exercise clearly improves skeletal muscle insulin sensitivity, the mechanisms are unclear. Here we show that initiation of a euglycemic-hyperinsulinemic clamp 4 h after single-legged exercise in humans increased microvascular perfusion (determined by contrast-enhanced ultrasound) by 65% in the exercised leg and 25% in the rested leg ( < 0.05) and that leg glucose uptake increased 50% more ( < 0.05) in the exercised leg than in the rested leg. Importantly, infusion of the nitric oxide synthase inhibitor l--monomethyl-l-arginine acetate (l-NMMA) into both femoral arteries reversed the insulin-stimulated increase in microvascular perfusion in both legs and abrogated the greater glucose uptake in the exercised compared with the rested leg. Skeletal muscle phosphorylation of TBC1D4 Ser and Ser and glycogen synthase activity were greater in the exercised leg before insulin and increased similarly in both legs during the clamp, and l-NMMA had no effect on these insulin-stimulated signaling pathways. Therefore, acute exercise increases insulin sensitivity of muscle by a coordinated increase in insulin-stimulated microvascular perfusion and molecular signaling at the level of TBC1D4 and glycogen synthase in muscle. This secures improved glucose delivery on the one hand and increased ability to take up and dispose of the delivered glucose on the other hand.
Topics: Adult; Contrast Media; Enzyme Inhibitors; Exercise; Femoral Artery; GTPase-Activating Proteins; Glucose; Glucose Clamp Technique; Glycogen Synthase; Healthy Volunteers; Humans; Hypoglycemic Agents; Insulin; Insulin Resistance; Leg; Male; Microvessels; Muscle, Skeletal; Nitric Oxide Synthase; Phosphorylation; Signal Transduction; Ultrasonography; Young Adult; omega-N-Methylarginine
PubMed: 28292969
DOI: 10.2337/db16-1327 -
Arteriosclerosis, Thrombosis, and... Jun 2015The insulinotropic gut-derived hormone glucagon-like peptide-1 (GLP-1) increases capillary perfusion via a nitric oxide-dependent mechanism in rodents. This improves...
OBJECTIVE
The insulinotropic gut-derived hormone glucagon-like peptide-1 (GLP-1) increases capillary perfusion via a nitric oxide-dependent mechanism in rodents. This improves skeletal muscle glucose use and cardiac function. In humans, the effect of clinically used GLP-1 receptor agonists (GLP-1RAs) on capillary density is unknown. We aimed to assess the effects of the GLP-1RA exenatide on capillary density as well as the involvement of nitric oxide in humans.
APPROACH AND RESULTS
We included 10 healthy overweight men (age, 20-27 years; body mass index, 26-31 kg/m(2)). Measurements were performed during intravenous infusion of placebo (saline 0.9%), exenatide, and a combination of exenatide and the nonselective nitric oxide-synthase inhibitor L-N(G)-monomethyl arginine. Capillary videomicroscopy was performed, and baseline and postocclusive (peak) capillary densities were counted. Compared with placebo, exenatide increased baseline and peak capillary density by 20.1% and 8.3%, respectively (both P=0.016). Concomitant L-N(G)-monomethyl arginine infusion did not alter the effects of exenatide. Vasomotion was assessed using laser Doppler fluxmetry. Exenatide nonsignificantly reduced the neurogenic domain of vasomotion measurements (R=-5.6%; P=0.092), which was strongly and inversely associated with capillary perfusion (R=-0.928; P=0.036). Glucose levels were reduced during exenatide infusion, whereas levels of insulin were unchanged.
CONCLUSIONS
Acute exenatide infusion increases capillary perfusion via nitric oxide-independent pathways in healthy overweight men, suggesting direct actions of this GLP-1RA on microvascular perfusion or interaction with vasoactive factors.
Topics: Adult; Blood Glucose; Blood Pressure; Capillaries; Cross-Over Studies; Exenatide; Glucagon-Like Peptide 1; Humans; Insulin; Male; Microcirculation; Microscopy, Video; Nitric Oxide; Overweight; Peptides; Skin; Venoms; Young Adult; omega-N-Methylarginine
PubMed: 25908765
DOI: 10.1161/ATVBAHA.115.305447 -
Journal of Molecular Endocrinology May 2017Diabetic foot ulceration (DFU) represents a common vascular complication of diabetes mellitus (DM) with high morbidity and disability resulting from amputation. Netrin-1...
Diabetic foot ulceration (DFU) represents a common vascular complication of diabetes mellitus (DM) with high morbidity and disability resulting from amputation. Netrin-1 level was decreased in type 2 DM patients and has been identified as a protective regulator against diabetes-triggered myocardial infarction and nephropathy. Unfortunately, its role and molecular mechanism in DFU remain poorly elucidated. Here, netrin-1 levels were reduced in DM and DFU patients relative to healthy controls, with netrin-1 levels being the lowest in DFU patients. Moreover, exposure to high glucose (HG) also suppressed netrin-1 expression in human umbilical vein endothelial cells (HUVECs). Elevated netrin-1 expression by infection with Ad-netrin-1 adenovirus vector protected against HUVEC injury in response to HG by ameliorating the inhibitory effects on cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, cell apoptotic rate and caspase-3 activity. Importantly, HG-impaired angiogenesis was improved after netrin-1 overexpression by elevating cell migration, capillary-like tube formation and VEGF production. Mechanism assay substantiated that netrin-1 elevation increased the phosphorylation levels of AKT and eNOS, and NO production, which was notably suppressed by HG, indicating that netrin-1 overexpression restored HG-triggered impairment of the PI3K/AKT-eNOS pathway. More intriguingly, preconditioning with LY294002 (PI3K/AKT antagonist) or -monomethyl-l-arginine (eNOS inhibitor) antagonized netrin-1-induced activation of the PI3K/AKT-eNOS pathway. Concomitantly, treatment with these antagonists also attenuated the protective role of netrin-1 in endothelial dysfunction upon HG stimulation. These results suggest that elevation of netrin-1 may restore HG-triggered impairment of HUVEC and angiogenesis by activating the PI3K/AKT-eNOS pathway, indicating a potential agent for wound healing in DFU patients.
Topics: Adenoviridae; Case-Control Studies; Caspase 3; Cell Survival; Cells, Cultured; Chromones; Diabetes Mellitus, Type 2; Diabetic Foot; Female; Gene Expression Regulation; Genetic Vectors; Glucose; Human Umbilical Vein Endothelial Cells; Humans; L-Lactate Dehydrogenase; Male; Malondialdehyde; Middle Aged; Morpholines; Neovascularization, Physiologic; Netrin-1; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; omega-N-Methylarginine
PubMed: 28250059
DOI: 10.1530/JME-16-0239