Inhibition Of Endogenous Nitric Oxide Research Articles

Molecular Dynamics-Ensemble Docking and Biophysical Studies for Structure-Based Identification of Non-Amino Acidic Ligands of DDAH-1.

Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) accounts for the catabolism of the endogenous inhibitors of nitric oxide (NO) synthases, namely, ADMA (Nω,Nω-dimethyl-l-arginine) and NMMA (Nω-monomethyl-l-arginine). Inhibition of DDAH-1 may prove a therapeutic benefit in diseases associated with elevated nitric oxide (NO) levels by providing a tissue-specific increase of ADMA and NMMA. In this work, we have used molecular dynamics to generate a pool of DDAH-1 conformations in the apo and holo forms. Ensemble docking has been instrumental in screening an in-house fragment-based library of 824 compounds. Resulting virtual hits have been validated for their binding activity to recombinant human DDAH-1 using microscale thermophoresis (MST). As a key result, three non-amino acidic ligands of DDAH-1 (VIS212, VIS268, VIS726) are identified with higher binding efficiency index than ADMA. Amid these compounds, purpurogallin (VIS726) proves a potent ligand of DDAH-1, showing a mixed behavior of enzymatic inhibition in a biochemical assay. This finding widens the panel of known molecular targets of purpurogallin and provides clues into the molecular mechanisms of its cellular NO inhibition activity as well as its anti-inflammatory and neuroprotective effects.

The effect of exenatide (a GLP-1 analogue) and sitagliptin (a DPP-4 inhibitor) on asymmetric dimethylarginine (ADMA) metabolism and selected biomarkers of cardiac fibrosis in rats with fructose-induced metabolic syndrome

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, is a risk factor for endothelial dysfunction, a common pathophysiological denominator for both atherogenesis and cardiac fibrosis.We aimed to investigate whether the cardioprotective and antifibrotic effects of incretin drugs, exenatide and sitagliptin, may be associated with their ability to affect circulating and cardiac ADMA metabolism.Normal and fructose-fed rats were treated with sitagliptin (5.0/10 mg/kg) or exenatide (5/10 µg/kg) for 4 weeks. The following methods were used: LC-MS/MS, ELISA, Real-Time-PCR, colorimetry, IHC and H&E staining, PCA and OPLS-DA projections.Eight-week fructose feeding resulted in an increase in plasma ADMA and a decrease in NO concentration. Exenatide administration into fructose-fed rats reduced the plasma ADMA level and increased NO level. In the heart of these animals exenatide administration increased NO and PRMT1 level, reduced TGF-ß1, α-SMA levels and COL1A1 expression. In the exenatide treated rats renal DDAH activity positively correlated with plasma NO level and negatively with plasma ADMA level and cardiac α-SMA concentration. Sitagliptin treatment of fructose-fed rats increased plasma NO concentration, reduced circulating SDMA level, increased renal DDAH activity and reduced myocardial DDAH activity. Both drugs attenuated the myocardial immunoexpression of Smad2/3/P and perivascular fibrosis.In the metabolic syndrome condition both sitagliptin and exenatide positively modulated cardiac fibrotic remodeling and circulating level of endogenous NOS inhibitors but had no effects on ADMA levels in the myocardium.

Nitric Oxide Precursors and Dimethylarginines as Risk Markers for Accelerated Measured GFR Decline in the General Population

Nitric oxide (NO) deficiency is associated with endothelial dysfunction, hypertension, atherosclerosis, and chronic kidney disease (CKD). Reduced NO bioavailability is hypothesized to play a vital role in kidney function impairment and CKD. We investigated the association of serum levels of endogenous inhibitors of NO, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), and precursors of NO, arginine, citrulline, and ornithine, with a decline in glomerular filtration rate (GFR) and new-onset CKD. In a prospective cohort study of 1407 healthy, middle-aged participants of Northern European origin in the Renal Iohexol Clearance Survey (RENIS), GFR was measured repeatedly with iohexol clearance during a median follow-up time of 11 years. GFR decline rates were analyzed using a linear mixed model, new-onset CKD (GFR< 60 ml/min per 1.73 m2) was analyzed with interval-censored Cox regression, and accelerated GFR decline (the 10% with the steepest GFR decline) was analyzed with logistic regression. Higher SDMA was associated with slower annual GFR decline. Higher levels of citrulline and ornithine were associated with accelerated GFR decline (odds ratio [OR], 1.43; 95% confidence interval [CI] 1.16-1.76 per SD higher citrulline and OR 1.23; 95% CI 1.01 to 1.49 per SD higher ornithine). Higher citrulline was associated with new-onset CKD, with a hazard ratio of 1.33 (95% CI 1.07-1.66) per SD higher citrulline. Associations between NO precursors and the outcomes suggest that NO metabolism plays a significant role in the pathogenesis of age-related GFR decline and the development of CKD in middle-aged people.

Acute effects of online hemodiafiltration versus high-flux hemodialysis on serum levels of asymmetric dimethylarginine and tumor necrosis factor-alpha

Background Chronic inflammation as a major determinant of ‘dialysis syndrome’ is considered as the main factor of morbidity and mortality in dialysis patients. Tumor necrosis factor-alpha (TNF-α) may play important roles in the development of T helper (Th) imbalance, cardiovascular disease, and wasting in the uremic milieu. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide that may be an independent risk factor for endothelial dysfunction and cardiovascular disease. In hemodialysis (HD) patients, plasma ADMA is a strong and independent predictor of overall mortality and cardiovascular outcome. The aim of this study to evaluate the acute effects of hemodiafiltration (HDF) compared with conventional HD on blood levels of ADMA and TNF-α. Patients and methods A cross-sectional study was conducted on 20 patients with end-stage renal disease receiving dialysis in the dialysis unit of Ain Shams Specialized Hospital receiving twice weekly HD session with high-flux (HF) dialyzer and once weekly HDF session. Blood samples were collected from all participants before and after HD session and from the same participants before and after HDF session. ADMA and TNF-α levels were assessed by enzyme-linked immunosorbent assay techniques. Results The decrease in TNF-α was higher after HDF session versus HF HD session (79.47 ± 14.16 vs. 50.43 ± 31.05), with P value of 0.001. Moreover, the decrease in ADMA was higher after HDF session versus HF HD session (75.01 ± 12.55 vs. 41.79 ± 24.73), with P value of 0.001. Conclusion The use of online HDF technique showed a significant reduction of ADMA and TNF-α in adults with end-stage renal disease.

Asymmetric Dimethylarginine in an NMU-induced Rat Mammary Tumor Model.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) production and a newly discovered risk factor involved in endothelial dysfunction and adverse cardiovascular events. Recently, both NO and ADMA have also emerged as molecules of interest in carcinogenesis and tumor growth progression. Our earlier studies have confirmed elevated plasma ADMA levels in patients with hematological malignancies. However, the cause of elevated ADMA was unclear. The aim of this study was to assess the concentrations of ADMA, symmetric dimethylarginine (SDMA) and L-arginine in rats exposed to N-nitroso-N-methylurea (NMU) for the induction of mammary tumors. A total of 95 female rats of the Sprague-Dawley strain were used in the study. Plasma concentrations of ADMA, SDMA and L-arginine were quantified and statistically analyzed. Mean ADMA levels were higher in the tumor-bearing group compared to the control group. Mean plasma levels of SDMA and L-Arginine were not significantly different between the groups. The L-ARG/ADMA ratio was lower in rats with tumors compared to controls. Histological assessment confirmed expression of ADMA within the tumor cells, which strongly suggests that these tumor cells were the source of ADMA. Other studies are warranted to further explain the role of ADMA in neoplastic diseases.

IDF21-0159 Asymmetric Dimethylarginine and 20-Hydroxyeicosatetraenoic Acid – A lethal combo in diabetic kidney disease

Background: 20-Hydroxyeicosatetraenoic Acid (20-HETE), an important metabolite of arachidonic acid, is known to be involved in causing renal cell injury. Cytochrome P450 is the enzyme responsible for the synthesis of 20-HETE. Cytochrome P450 is negatively related to nitric oxide (NO) levels. We previously reported that Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide, is significantly elevated in diabetes patients with albuminuria. Hence, we hypothesize that the inhibition of NO by ADMA in turn may augment the synthesis of 20-HETE. Further, we hypothesis that 20-HETE may also play a vital role in the ADMA induced kidney fibrosis. Hence, in this study we plan to measure the HETE levels in patients with Type 2 DM and study its association with ADMA and NO levels. Aim: Our aim is to study the urinary level of 20-HETE in patients with type 2 DM with and without nephropathy and to analyse the association of 20-HETE with ADMA, NO and other biochemical parameters. We also aimed to evaluate the efficiency of 20-HETE as a potential non-invasive biomarker for predicting diabetic nephropathy. Method: We recruited 120 patients from a centre for diabetes South India. Patients were divided into four groups namely normal glucose tolerance (NGT) (n=30), type 2 DM without nephropathy (T2DM) (n=30), type 2DM with microalbuminuria (MIC) (n=30) and type 2 DM with macroalbuminuria (MAC) (n=30). The urinary 20-HETE levels and circulatory ADMA levels were assessed using ELISA kit. Plasma total nitrite was quantified as a measure of nitric oxide formation by using Griess method as per the manufacturer's instructions. Urinary albumin was measured in fasting urine sample using immunoturbiometric assay and serum creatinine measured using Jaffe's method. Statistical analysis was performed using SPSS 20. Results: Urinary 20-HETE levels and the circulatory ADMA levels were significantly increased in concomitant with the significant decrease in nitric oxide levels in MIC and MAC groups when compared with NGT and T2DM groups. 20-HETE showed a significant positive correlation with the ADMA (r=0.362, p<0.001). As expected, 20-HETE showed a significant negative correlation with total nitric oxide (r=-0.252, p<0.05). Also with FBS (r=0.537, p<0.001), HbA1c (r=0.502, p<0.001) and urinary albumin to creatinine ratio (r=0.576, p<0.001) 20-HETE showed significant positive correlation confirming its association with diabetes and kidney damage. We found that the urinary 20-HETE level of 252.79 pg/ml was significant to detect the diabetic nephropathy in T2DM. The predicted cut-off value of 20-HETE had a specificity of 75% and sensitivity of 81%. Discussion: From this clinical study, we found that the exaggerated synthesis of ADMA under diabetic conditions contributes to the synthesis of 20-HETE. This is due to the inhibitory action of ADMA on the nitric oxide synthesis and the reduction in NO level promotes the synthesis of 20-HETE via CYP4A enzyme. Thus under hyperglycemic conditions, ADMA and 20-HETE together contribute to the diabetic kidney injury. Further, we suggest that 20-HETE can be used as a non-invasive biomarker for detecting diabetic kidney disease at an early stage.

Effect of profilin-1 on the asymmetric dimethylarginine-induced vascular lesion associated hypertension

Abstract Funding Acknowledgements Type of funding sources: None. OnBehalf Guohua Ni Objective Previous studies have demonstrated that the levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, are strongly associated with hypertension, diabetes, and cardiovascular diseases. Profilin-1, an actin-binding protein, has been documented to be involved in endothelial injury and in the proliferation of vascular smooth muscle cells (VSMCs) resulting from hypertension. However, the role of profilin-1 in ADMA-induced vascular injury in hypertension remains largely unknown. Methods Forty healthy subjects and forty-two matched patients with essential hypertension were enrolled, and the related indexes of vascular injury in plasma were detected. Rat aortic smooth muscle cells (RASMCs) were treated with different concentrations of ADMA for different periods of time and transfected with profilin-1 shRNA to interrupt the expression of profilin-1. To determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway, RASMCs were pretreated with AG490 or rapamycin. The expression of profilin-1 was tested using real-time PCR and western blot analysis. Cell proliferation was measured by flow cytometry and MTT assays. Results Compared with healthy subjects, the levels of ADMA and profilin-1 were markedly elevated in hypertensive individuals, while the levels of NO were significantly decreased(P &amp;lt; 0.05). In vitro, studies showed ADMA induced profilin-1 expression in a concentration- and time-dependent manner in RASMCs (P &amp;lt; 0.05), concomitantly with promoting the proliferation of RASMCs. Furthermore, ADMA-mediated proliferation of RASMCs and up-regulation expression of profilin-1 were inhibited by blockade of the JAK2/STAT3 pathway or knockdown of profilin-1. Conclusions Profilin-1 implicated in the ADMA-mediated vascular lesions in hypertension. Abstract Figure 12 Abstract Figure 3

Effect of profilin-1 on the asymmetric dimethylarginine-induced vascular lesion-associated hypertension.

Previous studies have demonstrated that the levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, are strongly associated with hypertension, diabetes, and cardiovascular diseases. Profilin-1, an actin-binding protein, has been documented to be involved in endothelial injury and in the proliferation of vascular smooth muscle cells resulting from hypertension. However, the role of profilin-1 in ADMA-induced vascular injury in hypertension remains largely unknown. Forty healthy subjects and forty-two matched patients with essential hypertension were enrolled, and the related indexes of vascular injury in plasma were detected. Rat aortic smooth muscle cells (RASMCs) were treated with different concentrations of ADMA for different periods of time and transfected with profilin-1 small hairpin RNA to interrupt the expression of profilin-1. To determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway, RASMCs were pretreated with AG490 or rapamycin. The expression of profilin-1 was tested using real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation was measured by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide assays. Compared with healthy subjects, the levels of ADMA and profilin-1 were markedly elevated in hypertensive individuals, while the levels of NO were significantly decreased (p < 0.05). In vitro, studies showed ADMA-induced profilin-1 expression in a concentration- and time-dependent manner in RASMCs (p < 0.05), concomitantly with promoting the proliferation of RASMCs. Furthermore, ADMA-mediated proliferation of RASMCs and upregulation expression of profilin-1 were inhibited by blockade of the JAK2/STAT3 pathway or knockdown of profilin-1. Profilin-1 implicated in the ADMA-mediated vascular lesions in hypertension.

Upregulation of DDAH2 Limits Pulmonary Hypertension and Right Ventricular Hypertrophy During Chronic Hypoxia in Ddah1 Knockout Mice.

Objective: Chronic hypoxia causes pulmonary vasoconstriction leading to pulmonary hypertension and right ventricular hypertrophy. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis; its level increases in hypoxia (HX) concomitantly with reduced activity of dimethylarginine dimethylaminohydrolases (DDAH-1 and DDAH-2), enzymes metabolizing ADMA. Ddah1 knockout (KO) mice may therefore help to understand the pathophysiological roles of this enzyme and its substrate, ADMA, in the development of hypoxia-associated pulmonary hypertension.Methods: Ddah1 KO mice and their wild-type (WT) littermates were subjected to normoxia (NX) or for 21 days. We measured ADMA concentration in plasma and lungs, DDAH1 and DDAH2 mRNA and protein expression in the lungs, right ventricular systolic pressure (RVSP), right ventricular hypertrophy by the Fulton index, and cardiomyocyte hypertrophy by dystrophin staining of the heart.Results: Ddah1 KO mice had higher ADMA concentrations in plasma and in lung tissue than WT in NX (p < 0.05). ADMA significantly increased in WT-HX in plasma and lungs, while there were no significant differences in WT-HX vs. KO-HX. This finding was paralleled by a 38 ± 13% reduction in Ddah1 but not Ddah2 mRNA expression, and reduced DDAH1 protein expression but stable DDAH2 protein levels in WT mice. Ddah1 KO mice showed significant elevation of DDAH2 protein but not mRNA levels, which further increased in HX. HX led to increased RVSP and right ventricular hypertrophy in both, WT and KO mice, with no significant differences between both genotypes.Conclusions: Chronic hypoxia causes an elevation of ADMA, which may impair NO production and lead to endothelial dysfunction and vasoconstriction. Downregulation of DDAH1 expression and activity may be involved in this; however, knockout of the Ddah1 gene does not modify the hypoxia-induced pathophysiological changes of pulmonary blood pressure and right ventricular hypertrophy, possibly due to compensatory upregulation of DDAH2 protein.

Prognostic Value of Asymmetric Dimethylarginine in Patients with Heart Failure: A Systematic Review and Meta-analysis.

Objective Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, is reported to be a risk factor for cardiovascular disease. The purpose of the present study is to investigate whether ADMA is an independent predictor for future mortality and adverse clinical events among patients with heart failure (HF). Methods Electronic literature databases (Central, MEDLINE, and Embase) were searched for relevant observational studies on the prognostic value of ADMA in HF patients published before January 2019. Pooled hazard ratios (HRs) or odds ratio and the corresponding 95% confidence interval (CI) were calculated for risk evaluation. Results 10 studies with 2195 participants were identified and analyzed. The pooled HR of composite clinical events for the highest vs. lowest quartiles from categorical variable results was 1.34 (95% CI: 1.15-1.57, P < 0.001, I2 = 0%), which is 1.31 (95% CI: 1.10-1.55, P < 0.005, I2 = 0%) in the subgroup of acute decompensated HF. The pooled HR of composite clinical events from continuous variable results was 1.41 (95% CI: 1.21-1.63, P < 0.001, I2 = 21.9%), with 0.1 μM increment accounting for the increasing 25% risk for composite adverse clinical events. The pooled HR for all-cause mortality was 2.38 (95% CI: 1.48-3.82, P < 0.001, I2 = 0%) after sensitivity analysis. Two studies reporting the HR of inhospital mortality in HF patients regarded it as a prognostic indicator, with categorical variable HR as 1.26 (95% CI: 1.07-1.84, P < 0.05) and continuous variable OR as 2.15 (95% CI: 1.17–4.29, P < 0.05). Conclusions ADMA is an independent predictor for composite clinical outcomes among HF patients with both short-term and long-term prognostic value.

Protective Effect of Epigallocatechin Gallate on Endothelial Disorders in Atherosclerosis.

Healthy vascular endothelial cells regulate vascular tone and permeability, prevent vessel wall inflammation, enhance thromboresistance, and contribute to general vascular health. Furthermore, they perform important functions including the production of vasoactive substances such as nitric oxide (NO) and endothelium-derived hyperpolarizing factors, as well as the regulation of smooth muscle cell functions. Conversely, vascular endothelial dysfunction leads to atherosclerosis, thereby enhancing the risk of stroke, myocardial infarction, and other cardiovascular diseases (CVDs). Observational studies and randomized trials showed that green tea intake was inversely related to CVD risk. Furthermore, evidence indicates that epigallocatechin gallate (EGCG) found in green tea might exert a preventive effect against CVDs. EGCG acts as an antioxidant, inducing NO release and reducing endothelin-1 production in endothelial cells. EGCG enhances the bioavailability of normal NO by reducing levels of the endogenous NO inhibitor asymmetric dimethylarginine. Furthermore, it inhibits the enhanced expression of adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and attenuates monocyte adhesion. In addition, EGCG prevents enhanced oxidative stress through the Nrf2/HO-1 pathway. These effects indicate that it might prevent the production of reactive oxygen species, inhibit inflammation, and reduce endothelial cell apoptosis during the initial stages of atherosclerosis. The current review summarizes recent research in this area and discusses novel findings regarding the protective effect of EGCG on endothelial dysfunction and CVDs in general.

Asymmetric Dimethylarginine (ADMA) and Symmetric Dimethylarginine (SDMA) Concentrations in Patients with Obesity and the Risk of Obstructive Sleep Apnea (OSA).

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthesis, and play a critical role in the process of endothelial dysfunction, and are considered markers of oxidative stress. The aim of the present study was to explore relationships between ADMA and/or SDMA and the occurrence of OSA in obese patients as well as the effect of the endothelial nitric oxide synthase (eNOS) gene polymorphism, which may modify the influence of ADMA or SDMA on NO production. A total of 518 unrelated obese subjects were included in this study. Body weight, height and blood pressure were measured and data on self-reported smoking status were collected. Obstructive sleep apnea (OSA) was assessed by the apnea hypopnea index (AHI). Blood samples were collected to measure serum concentrations of glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, creatinine, HbA1c (%), folic acid, vitamin B12, C-reactive protein (CRP), aspartate aminotransferase (ASP), alanine aminotransferase (ALT) and IL-6 by routine methods. The NOS3 gene G894T and 4a/4b polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. ADMA, SDMA and arginine concentrations were assessed simultaneously using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method. Adjusted multivariate logistic regression analysis showed a significant association between the occurrence of OSA and high serum ADMA levels, BMI above 40, age > 43 years, hypertension and male sex. Heterozygotes for the G894T eNOS polymorphism have the lowest serum concentrations of ADMA and SDMA, while no effect of the 4a/4b variants was observed. The results indicate that OSA in obese individuals can coexist with high ADMA levels, which appear as a potential OSA predictor.

Genetic regulation of dimethylarginines and endothelial dysfunction in rheumatoid arthritis.

Rheumatoid Arthritis (RA) confers an increased cardiovascular disease (CVD) risk which accounts for much of the premature morbidity and mortality observed in this population. Alterations in vascular function and morphology leading to increased atherosclerotic burden are considered the main drivers of CVD in RA individuals with systemic inflammation playing a key role in the dysregulation of endothelial homeostasis and initiation of vascular injury. Dimethylarginines are endogenous inhibitors of nitric oxide (NO) synthase and have emerged as novel, independent biomarkers of CVD in a wide range of conditions associated with vascular pathology. In RA several reports have demonstrated abnormal dimethylarginine metabolism attributable to various factors such as systemic inflammation, decreased degradation or upregulated synthesis. Although a causal relationship between dimethylarginines and vascular damage in RA has not been established, the tight interrelations between inflammation, dimethylarginines and endothelial dysfunction suggest that determination of dimethylarginine regulators may shed more light in the pathophysiology of the atherosclerotic process in RA and may also provide new therapeutic targets. The Alanine-Glyoxylate Aminotransferase 2 (AGTX2)-dependent pathway is a relatively recently discovered alternative pathway of dimethylarginine catabolism and its role on RA-related atherosclerotic disease is yet to be established. As factors affecting dimethylarginine concentrations linked to CVD risk and endothelial dysfunction are of prominent clinical relevance in RA, we present preliminary evidence that gene variants of AGTX-2 may influence dimethylarginine levels in RA patients and provide the rationale for larger studies in this field.

Plasma asymmetric dimethylarginine concentrations are not related to differences in maximal oxygen uptake in endurance trained and untrained men.

What is the central question of this study? Is there an association of plasma concentration of asymmetric dimethylarginine, which is related to exercise capacity in patients with cardiovascular diseases, with oxygen delivery and subsequently exercise capacity in healthy subjects in the absence of the potentially confounding influence of inflammation and oxidative stress? What is the main finding and its importance? Plasma asymmetric dimethylarginine concentrations are not related to exercise capacity in healthy subjects, while O2 delivery in the working skeletal muscle during the maximal graded-exercise test is not associated with any of the l-arginine analogues. ADMA alone does not play a crucial role in local muscle perfusion and in maintaining exercise capacity. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis that could limit oxygen (O2 ) delivery in the working skeletal muscles by altering endothelium-dependent vasodilatation. Exercise capacity is associated with plasma ADMA concentrations in patients with cardiovascular diseases, but this issue has still not been investigated in healthy subjects. We aimed to determine whether plasma ADMA concentrations were negatively associated with exercise capacity in young healthy male subjects. Ten men with maximal oxygen uptake ( )> 65mLkg-1 min-1 were included in the high exercise capacity group (HI-FIT), and 10 men with < 45mLkg-1 min-1 were included in the low exercise capacity group (LO-FIT). Plasma ADMA and other l-arginine analogue concentrations were measured before and after a maximal graded-exercise test by liquid chromatography-tandem mass spectrometry. Microvascular O2 delivery during exercise was estimated through the pattern from the sigmoid model of muscle deoxygenation in the vastus lateralis measured by near infrared spectroscopy. was 60% higher in the HI-FIT group (median: 70.2mLkg-1 min-1 ; IQR: 68.0-71.9mLkg-1 min-1 ) than in the LO-FIT group (median: 43.8mLkg-1 min-1 ; IQR: 34.8-45.3mLkg-1 min-1 ). Plasma ADMA concentrations did not differ between the LO-FIT and HI-FIT groups before (0.50± 0.06 vs. 0.54± 0.07μmolL-1 , respectively) and after the maximal incremental exercise test (0.49± 0.08 vs. 0.55± 0.03μmolL-1 , respectively). There was no significant association of plasma ADMA concentrations with the pattern of local muscle deoxygenation and exercise capacity. Exercise capacity and microvascular O2 delivery are not related to plasma ADMA concentrations in young healthy male subjects. Our findings show that ADMA does not play a crucial role in local muscle perfusion and in maintaining exercise capacity without pathological conditions.

Pharmacological activation of dimethylarginine dimethylaminohydrolase (DDAH) activity by inorganic nitrate and DDAH inhibition by NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline: results and overview.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of nitric oxide (NO) synthase. SDMA is excreted in the urine without major metabolization. About 10% of daily produced ADMA are excreted unchanged in the urine. The major elimination route of ADMA (about 90%) involves its hydrolysis to dimethylamine (DMA) and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and excretion of DMA in the urine. High circulating and low excretory concentrations of ADMA are considered risk factors. Experimentally, DDAH activity can be inhibited by SH-specific agents such as inorganic and organic mercury compounds, and by S-nitrosothiols which block the SH group of a particular cysteine moiety of DDAH that is essential for its hydrolytic activity. Alternatively, DDAH activity can be inhibited by organic compounds that compete with the substrate ADMA for DDAH. Arginine analogs that contain substituents on guanidine nitrogen atom(s) (NG) represent a class of DDAH inhibitors. In the present study, we investigated the effects of physiological and natural amino acid derivatives of L-arginine and L-citrulline as well as of nitrate and nitrite, the major circulating and excretory metabolites of NO and NO donating drugs. Here, we report for the first time that the physiological NG-hydroxy-L-arginine, an isolable intermediate in NO synthesis, inhibits recombinant DDAH-1 activity (IC50 ≈ 100µM). Two plant L-citrulline derivatives, i.e., Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline (connatin), were found to inhibit almost completely hepatic DDAH activity in vitro in rat homogenate at a concentration of 100µM each. At pharmacological concentrations (i.e., 1mM), inorganic nitrate, but not inorganic nitrite, was found to increase rat liver DDAH activity. In urine of 18 patients with Becker's muscular dystrophy, nitrate was found to correlate closely with DMA (Spearman, r = 0.73, p = 0.002). In summary, NG-hydroxy-L-arginine, Nω,Nω-dimethyl-L-citrulline and Nω,Nω-dimethyl-Nδ-hydroxy-L-citrulline are novel inhibitors of DDAH activity. This article provides an overview of amino acid-based DDAH inhibitors and discusses potential underlying inhibition mechanisms.

Metabolomic assessment of exposure to near-highway ultrafine particles.

Exposure to traffic-related air pollutants has been associated with increased risk of adverse cardiopulmonary outcomes and mortality; however, the biochemical pathways linking exposure to disease are not known. To delineate biological response mechanisms associated with exposure to near-highway ultrafine particles (UFP), we used untargeted high-resolution metabolomics to profile plasma from 59 participants enrolled in the Community Assessment of Freeway Exposure and Health (CAFEH) study. Metabolic variations associated with UFP exposure were assessed 3 using a cross-sectional study design based upon low (mean 16,000 particles/cm3) and high (mean 24,000 particles/cm3) annual average UFP exposures. In comparing quantified metabolites, we identified five metabolites that were differentially expressed between low and high exposures, including arginine, aspartic acid, glutamine, cystine and methionine sulfoxide. Analysis of the metabolome identified 316 m/z features associated with UFP, which were consistent with increased lipid peroxidation, endogenous inhibitors of nitric oxide and vehicle exhaust exposure biomarkers. Network correlation analysis and metabolic pathway enrichment identified 38 pathways and included variations related to inflammation, endothelial function and mitochondrial bioenergetics. Taken together, these results suggest UFP exposure is associated with a complex series of metabolic variations related to antioxidant pathways, in vivo generation of reactive oxygen species and processes critical to endothelial function.

ASSOCIATION OF DIETARY SODIUM INTAKE AND 24H URINE SODIUM EXCRETION WITH ENDOTHELIAL DYSFUNCTION AND URINARY ALBUMIN EXCRETION IN EARLY-STAGE HYPERTENSIVE INDIVIDUALS

Objective: Monitoring of dietary sodium intake is essential in hypertension and efforts have been made to find the least troublesome method to accurately estimate sodium consumption. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide, which is implicated in abnormal pressure-natriuresis. Increased urinary albumin excretion (UAE) has been associated with sodium intake, possibly through alteration of intrarenal hemodynamics. We investigated which method of measuring sodium intake, including dietary and urinary assessment, correlates better with endothelial dysfunction and UAE and may be a better indicator of vascular damage in newly-diagnosed, never-treated hypertensive patients. Design and method: Untreated individuals free from cardiovascular comorbidities were eligible to participate, whose 24 h systolic/diastolic blood pressure exceeded 130 and/or 80 mmHg based on their ambulatory blood pressure recording (MobilOGraph). Asymmetric dimethylarginine (ADMA) was measured in serum samples using commercially available competitive enzyme-linked immunosorbent assay (ELISA) kit. UAE was measured in 24 h urine samples. Dietary sodium intake was estimated with a) a 24hour diet recall that corresponded to the same day as the 24hour urine collection, b) a salt score questionnaire based on a 0–10 scale, with higher values indicating higher salt intake, and c) urinary sodium excretion in 24hour urine sample. Results: A total of 50 hypertensive patients, 28 males and 22 females, with a mean age of 47.0 ± 10.9 years and mean 24hour ambulatory systolic/diastolic blood pressure 135.2 ± 10.0/88.5.0 ± 9.1 mmHg, were included. Urinary albumin excretion was 6.1 (4.3–10.5) mg/24 h and ADMA levels were 0.99 ± 0.42 μmol/l. Urinary sodium excretion was 83.3 (48.4–146.5) mmol/24 h, estimated dietary sodium intake was 79.2 (36.7–130.4) mmol/24 h, and salt score was scaled at 4.9 ± 1.5. Urinary sodium excretion was the only sodium indicator that positively and strongly correlated with both urinary albumin excretion (r = 0.347, p = 0.043) and ADMA levels (r = 0.389, p = 0.025). Conclusions: In a population of untreated, relatively young, early-stage hypertensive individuals, urinary sodium excretion, yet not other dietary measures of sodium, may reflect microvascular damage. These results support the widely perceived premise that 24hour urine collection is more accurate, although more cumbersome to perform, than other dietary sodium assessments, in terms of pathophysiology and clinical significance.

Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S-nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust

Objective: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair.Materials/methods: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH1 transgenic) mice and measured wound repair using the electric cell impedance sensing system.Results: Wound closure in epithelial cells from WT mice occurred within 24 h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH1 transgenic mice, control wounds were repaired up to 8 h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24 h. Likewise, cells from DDAH1 transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC.Discussion/conclusion: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.

Inverse correlation between maternal plasma asymmetric dimethylarginine (ADMA) and birthweight percentile in women with impaired placental perfusion: circulating ADMA as an NO-independent indicator of fetal growth restriction?

L-Arginine (Arg) is the enzymatic precursor of nitric oxide (NO) which has multiple biological functions. Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO. We hypothesized that the ADMA and SDMA have additional biological functions in pregnancy, beyond NO synthesis, and may play a role in the regulation of birthweight (BW). To investigate this issue, we measured the plasma concentration of ADMA, SDMA, Arg and the NO metabolites nitrite and nitrate, at 23-25weeks of gestation in women with normal placental function (Group 1) and in women with impaired placental perfusion; 19 of these women had normal outcome (Group 2), 14 had a fetus that was growth restricted (Group 3), and 10 women eventually developed preeclampsia (Group 4). BW percentile was found to inversely correlate with maternal plasma ADMA concentration in Group 3 (r=-0.872, P<0.001) and in Group 4 (r=-0.800, P<0.05). But, BW percentile did not correlate with the maternal plasma concentration of Arg, SDMA, nitrate or nitrite. Our results suggest that maternal plasma ADMA concentration is an important indicator of fetal growth restriction in women with impaired placental perfusion independent of NO.