Asymmetric Dimethylarginine Metabolism Research Articles

Asymmetric Dimethylarginine (ADMA) in Cardiovascular Disease, Cardiac Ischemia/reperfusion Injury, and Ischemic Non-obstructive Coronary Artery Disease: Biochemical and Pharmacological Implications

: Several studies have shown that high plasma concentrations of asymmetric dimethylarginine (ADMA), a known endogenous competitive inhibitor of endothelial nitric oxide synthase (eNOS), correlate with the severity of coronary artery disease (CAD), with worsening of cardiac ischemia/reperfusion (I/R) injury and coronary atherosclerosis. It is believed that it may be an important risk factor for myocardial infarction. ADMA, when in high concentrations, can determine a significant decrease in the synthesis and bioavailability of NO (Nitric oxide) and therefore alter the mechanisms of regulation of coronary vasodilation and vasomotor function of epicardial coronary arteries. Higher serum ADMA concentration is associated with worsening of post-ischemic remodeling since coronary angiogenesis, vasculogenesis, and collateral coronary growth are seriously impaired. In addition, there are reasons to believe that elevated plasma ADMA levels are related to the development of diseases affecting coronary microcirculation, such as ischemic non-obstructive coronary artery disease (INOCA). With the aim of providing the pharmacologist engaged in the design and discovery of new ADMA-lowering drugs with a complete examination of the subject, in this review, we discuss the most important studies related to the correlations between serum ADMA levels and cardiovascular diseases mentioned above. In addition, we critically discuss the main aspects of enzymology, synthesis, and metabolism of ADMA as a prerequisite for understanding the molecular mechanisms through which high concentrations of ADMA could contribute to promoting cardiovascular diseases. ADMA represents a new target for pharmacological modulation of cardiovascular endothelial function and therefore, there is a possibility of using selective pharmacological ADMA lowering drugs in cardiovascular disease with endothelial dysfunction and high plasma ADMA levels.

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Dysregulation of the Nitric Oxide/Dimethylarginine Pathway in Hypoxic Pulmonary Vasoconstriction—Molecular Mechanisms and Clinical Significance

The pulmonary circulation responds to hypoxia with vasoconstriction, a mechanism that helps to adapt to short-lived hypoxic episodes. When sustained, hypoxic pulmonary vasoconstriction (HPV) may become deleterious, causing right ventricular hypertrophy and failure, and contributing to morbidity and mortality in the late stages of several chronic pulmonary diseases. Nitric oxide (NO) is an important endothelial vasodilator. Its release is regulated, amongst other mechanisms, by the presence of endogenous inhibitors like asymmetric dimethylarginine (ADMA). Evidence has accumulated in recent years that elevated ADMA may be implicated in the pathogenesis of HPV and in its clinical sequelae, like pulmonary arterial hypertension (PAH). PAH is one phenotypic trait in experimental models with disrupted ADMA metabolism. In high altitude, elevation of ADMA occurs during long-term exposure to chronic or chronic intermittent hypobaric hypoxia; ADMA is significantly associated with high altitude pulmonary hypertension. High ADMA concentration was also reported in patients with chronic obstructive lung disease, obstructive sleep apnoea syndrome, and overlap syndrome, suggesting a pathophysiological role for ADMA-mediated impairment of endothelium-dependent, NO-mediated pulmonary vasodilation in these clinically relevant conditions. Improved understanding of the molecular (dys-)regulation of pathways controlling ADMA concentration may help to dissect the pathophysiology and find novel therapeutic options for these diseases.

Abstract 13928: Asymmetric Dimethylarginine (ADMA) Mediates the Effect of miRNA-762 on All-Cause Mortality in Patients With Coronary Artery Disease

Introduction: Plasma concentrations of the endogenous L-arginine homologue asymmetric dimethylarginine (ADMA) independently predict cardiovascular and all-cause mortality. We sought to identify miRNAs that mediate the effects of ADMA on mortality or influence mortality through regulation of ADMA concentrations. Methods: In 993 participants of the Ludwigshafen Risk and Cardiovascular Health study (age 63.0±10.5 years, 31.4% females and 74.7% with coronary artery disease (CAD)) we measured ADMA and miRNAs concentrations in citrate plasma and investigated associations between them and with all-cause mortality. Results: miRNA-762 was positively and significantly correlated with ADMA concentrations (p=0.0013) and with all-cause mortality after adjusting for age and sex (HR (95%CI: 1.51(1.26-1.81); p=7.37E-06). The association remained significant after additionally adjusting for body mass index, CAD, arterial hypertension, diabetes, smoking, lipids, C-reactive protein, fibrinogen, renal function, homocysteine and medication (p=0.04). Mediation analyses showed that ADMA mediated 67% of the effect of miRNA-762 on mortality while miRNA-762 only mediated 8% of the effect of ADMA. Conclusions: miRNA-762 is an independent predictor of all-cause mortality in patients with CAD. The effects of miRNA-762 on mortality might be at least partially mediated by ADMA. miRNA-762 has been recently shown to regulate apoptosis in cardiomyocytes in myocardial infarction. We are currently investigating the effects of miRNA-762 on ADMA metabolism and stress responses in cultured cardiomyocytes and endothelial cells.

Nutritional and metabolic regulation of the metabolite dimethylguanidino valeric acid: an early marker of cardiometabolic disease.

Dimethylguanidino valeric acid (DMGV) is a marker of fatty liver disease, incident coronary artery disease, cardiovascular mortality, and incident diabetes. Recently, it was reported that circulating DMGV levels correlated positively with consumption of sugary beverages and negatively with intake of fruits and vegetables in three Swedish community-based cohorts. Here, we validate these results in the Framingham Heart Study Third Generation Cohort. Furthermore, in mice, diets rich in sucrose or fat significantly increased plasma DMGV concentrations. DMGV is the product of metabolism of asymmetric dimethylarginine (ADMA) by the hepatic enzyme AGXT2. ADMA can also be metabolized to citrulline by the cytoplasmic enzyme DDAH1. We report that a high-sucrose diet induced conversion of ADMA exclusively into DMGV (supporting the relationship with sugary beverage intake in humans), while a high-fat diet promoted conversion of ADMA to both DMGV and citrulline. On the contrary, replacing dietary native starch with high-fiber-resistant starch increased ADMA concentrations and induced its conversion to citrulline, without altering DMGV concentrations. In a cohort of obese nondiabetic adults, circulating DMGV concentrations increased and ADMA levels decreased in those with either liver or muscle insulin resistance. This was similar to changes in DMGV and ADMA concentrations found in mice fed a high-sucrose diet. Sucrose is a disaccharide of glucose and fructose. Compared with glucose, incubation of hepatocytes with fructose significantly increased DMGV production. Overall, we provide a comprehensive picture of the dietary determinants of DMGV levels and association with insulin resistance.

Metabolism of asymmetric dimethylarginine in hypoxia: from bench to bedside.

Acute hypoxia and chronic hypoxia induce pulmonary vasoconstriction. While hypoxic pulmonary vasoconstriction is a physiological response if parts of the lung are affected, global exposure to hypoxic conditions may lead to clinical conditions like high-altitude pulmonary hypertension. Nitric oxide is the major vasodilator released from the vascular endothelium. Nitric oxide-dependent vasodilation is impaired in hypoxic conditions. Inhibition of nitric oxide synthesis is the most rapid and easily reversible molecular mechanism to regulate nitric oxide-dependent vascular function in response to physiological and pathophysiological stimuli. Asymmetric dimethylarginine is an endogenous, competitive inhibitor of nitric oxide synthase and a risk marker for major cardiovascular events and mortality. Elevated asymmetric dimethylarginine has been observed in animal models of hypoxia as well as in human cohorts under chronic and chronic intermittent hypoxia at high altitude. In lowlanders, asymmetric dimethylarginine is high in patients with pulmonary hypertension. We have recently shown that high asymmetric dimethylarginine at sea level is a predictor for high-altitude pulmonary hypertension. Asymmetric dimethylarginine is a highly regulated molecule, both by its biosynthesis and metabolism. Methylation of L-arginine by protein arginine methyltransferases was shown to be increased in hypoxia. Furthermore, the metabolism of asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) is decreased in animal models of hypoxia. Whether these changes are caused by transcriptional or posttranslational modifications remains to be elucidated. Current data suggest a major role of asymmetric dimethylarginine in regulating pulmonary arterial nitric oxide production in hypoxia. Further studies are needed to decipher the molecular mechanisms regulating asymmetric dimethylarginine in hypoxia and to understand their clinical significance.

Activated L-Arginine/Nitric Oxide Pathway in Pediatric Cystic Fibrosis and Its Association with Pancreatic Insufficiency, Liver Involvement and Nourishment: An Overview and New Results.

Cystic fibrosis (CF; OMIM 219700) is a rare genetic disorder caused by a chloride channel defect, resulting in lung disease, pancreas insufficiency and liver impairment. Altered L-arginine (Arg)/nitric oxide (NO) metabolism has been observed in CF patients’ lungs and in connection with malnutrition. The aim of the present study was to investigate markers of the Arg/NO pathway in the plasma and urine of CF patients and to identify possible risk factors, especially associated with malnutrition. We measured the major NO metabolites nitrite and nitrate, Arg, a semi-essential amino acid and NO precursor, the NO synthesis inhibitor asymmetric dimethylarginine (ADMA) and its major urinary metabolite dimethylamine (DMA) in plasma and urine samples of 70 pediatric CF patients and 78 age-matched healthy controls. Biomarkers were determined by gas chromatography–mass spectrometry and high-performance liquid chromatography. We observed higher plasma Arg (90.3 vs. 75.6 µM, p < 0.0001), ADMA (0.62 vs. 0.57 µM, p = 0.03), Arg/ADMA ratio (148 vs. 135, p = 0.01), nitrite (2.07 vs. 1.95 µM, p = 0.03) and nitrate (43.3 vs. 33.1 µM, p < 0.001) concentrations, as well as higher urinary DMA (57.9 vs. 40.7 µM/mM creatinine, p < 0.001) and nitrate (159 vs. 115 µM/mM creatinine, p = 0.001) excretion rates in the CF patients compared to healthy controls. CF patients with pancreatic sufficiency showed plasma concentrations of the biomarkers comparable to those of healthy controls. Malnourished CF patients had lower Arg/ADMA ratios (p = 0.02), indicating a higher NO synthesis capacity in sufficiently nourished CF patients. We conclude that NO production, protein-arginine dimethylation, and ADMA metabolism is increased in pediatric CF patients. Pancreas and liver function influence Arg/NO metabolism. Good nutritional status is associated with higher NO synthesis capacity and lower protein-arginine dimethylation.

An Ethnic Comparison of Arginine Dimethylation and Cardiometabolic Factors in Healthy Black and White Youth: The ASOS and African-PREDICT Studies.

Proteinic arginine dimethylation (PADiMe) is a major post-translational modification. Proteolysis of asymmetric and symmetric PADiMe products releases asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), respectively, two endogenous atherogenic substances. SDMA, ADMA, and its major metabolite dimethylamine (DMA) are eliminated by the kidney. The urinary concentrations of DMA+ADMA, SDMA, and DMA+ADMA+SDMA are useful measures of the whole-body asymmetric and symmetric PADiMe, respectively. Urinary (DMA+ADMA)/SDMA is an index of the asymmetric to symmetric PADiMe balance. In two bi-ethnic studies, the ASOS (39 black boys, 41 white boys) and the African-PREDICT (292 black young men, 281 white young men) studies, we investigated whether ethnicity is a major determinant of PADiMe, and whether PADiMe is associated with blood pressure and ethnicity-dependent growth and inflammatory factors, including HDL. DMA, ADMA, and SDMA were measured in spot urine samples by gas chromatography–mass spectrometry, and their excretion was corrected for creatinine excretion. In black boys, creatinine-corrected DMA, DMA+ADMA, and DMA+ADMA+SDMA concentrations were lower by 11.7%, 9.5%, and 7.6% (all p < 0.05), respectively, compared to the white boys, and 3.4%, 2.0%, and 1.8% lower (all p < 0.05), respectively, in black compared to white men. (DMA+ADMA)/SDMA did not differ between black boys and black men, but was higher in white boys compared to white men. ADMA did not differ between black and white boys, or between black and white men. Creatinine-corrected SDMA excretion was lower in black boys compared to white boys (by 8%) and to white men (by 3.1%). None of the PADiMe indices were associated with blood pressure in either study. IGF-binding protein 3 correlated inversely with all PADiMe indices in the black men only. Our study showed that asymmetric proteinic arginine dimethylation is higher in white boys than in black boys, and that this difference disappears in adulthood. ADMA metabolism and SDMA excretion were lower in the black subjects compared to the white subjects, suggesting ethnicity-dependent hepatic and renal elimination of ADMA and SDMA in the childhood. The results of our study may have clinical relevance beyond atherosclerosis, such as in growth and inflammation, which have not been sufficiently addressed thus far.

DDAH1 siRNA Knockdown in a Human Pulmonary Vascular Co‐Culture Cell Model

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients, contributing to morbidity and mortality. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA and DDAH1 is the most important for ADMA metabolism in vivo. Translational studies from our group have identified DDAH1 single nucleotide polymorphisms that are differentially expressed in BPD‐PH patients compared to BPD patients. We hypothesized that inhibiting DDAH1 in human pulmonary microvascular endothelial cells (hPMVECs) will lead to decreased apoptosis and increased proliferation of human pulmonary artery smooth muscle cells (hPASMCs) via effects in hPMVECs. We utilized a co‐culture model where hPMVECs were transfected with DDAH1 siRNA or scramble for 24h and then incubated in co‐culture with hPASMCs for 24h. Analysis included 1) trypan blue exclusion for viable cell numbers and 2) western blot analysis for cleaved caspase‐3 and 8, total caspase‐3 and 8, PCNA, and β‐actin. After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found greater hPASMC viable cell numbers compared to scramble hPASMC controls (N=3, p&lt;0.05). After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found lower hPASMC levels of cleaved caspase‐3 protein (N=3, p&lt;0.05) and cleaved caspase‐8 (N=3, p&lt;0.05), with greater hPASMC protein levels of PCNA (N=3, p&lt;0.05) compared to scramble. DDAH1 siRNA knockdown in hPMVEC results in greater hPASMC proliferation and less cleaved caspase‐3 mediated apoptosis. We speculate that DDAH1 is a potential therapeutic target for BPD‐PH. We speculate that these findings support the concept that DDAH1 regulates vascular remodeling by minimizing ADMA‐inhibition of eNOS, resulting in maintained NO production which would support angiogenesis and prevent/attenuate vascular remodeling. Future studies would include in vivo experiments in transgenic neonatal mice exposed to a BPD‐like lung injury.Support or Funding InformationThis work was funded by NIH‐K08 HL129080This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The renal transport protein OATP4C1 mediates uptake of the uremic toxin asymmetric dimethylarginine (ADMA) and efflux of cardioprotective L-homoarginine

Elevated plasma concentrations of the uremic toxin asymmetrical dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with cardiovascular events and total mortality. Enzymes degrading ADMA [dimethylaminohydrolase 1 (DDAH1)] and synthesizing L-homoarginine [L-arginine:glycine amidinotransferase (AGAT)] are expressed in human proximal tubule cells. So far, it is not known which transport protein in the basolateral membrane of proximal tubule cells is mediating the uptake of ADMA into the cells for subsequent degradation or the export of intracellularly synthesized L-homoarginine. One study suggested that the uptake transporter OATP4C1 (gene symbol SLCO4C1) may be involved in the transport of ADMA and other uremic toxins. OATP4C1 is a member of the SLCO/SLC21 family of solute carriers, localized in the basolateral membrane of human proximal tubule cells. By using stably-transfected HEK cells overexpressing human OATP4C1, we demonstrate that ADMA and L-homoarginine are substrates of OATP4C1 with Km values of 232.1 μM and 49.9 μM, respectively. ADMA and the structurally related uremic toxin SDMA (100 μM) inhibited OATP4C1-mediated L-homoarginine uptake (P < 0.01), whereas other tested uremic toxins such as urea and p-cresyl sulfate have no effect on OATP4C1-mediated transport. Preloading experiments (300 μM for 60 min) with subsequent efflux studies revealed that OATP4C1 also facilitates efflux e.g. of L-homoarginine. Both ADMA and L-homoarginine are substrates of human OATP4C1. Because proximal tubule cells are one site of ADMA metabolism and L-homoarginine synthesis, we postulate a protective role of OATP4C1 by mediating uptake of ADMA from and export of L-homoarginine into the systemic circulation.

Effect of renal function on homeostasis of asymmetric dimethylarginine (ADMA): studies in donors and recipients of renal transplants.

Asymmetric dimethylarginine (ADMA) is a methylated form of arginine and an endogenous nitric oxide synthase inhibitor. Renal function decline is associated with increase of plasma ADMA in chronic kidney disease populations. It is yet unknown how isolated renal function impairment affects ADMA homeostasis in healthy humans. Here, we measured plasma concentrations and urinary excretion of ADMA using GC-MS/MS in 130 living kidney donors before and at 1.6 (1.6-1.9) months after donation. We additionally analyzed 201 stable renal transplant recipients (RTR) that were included > 1year after transplantation, as a model for kidney disease in the context of single kidney state. We measured true glomerular filtration rate (mGFR) using 125I-iothalamate. To study enzymatic metabolism of ADMA, we also measured L-citrulline as primary metabolite. Mean age was 52 ± 10years in donors and 54 ± 12years in RTR. Renal function was significantly reduced from pre- to post-donation (mGFR: 104 ± 17 vs. 66 ± 10ml/min per 1.73m2 BSA, - 36 ± 7%, P < 0.001). Urinary ADMA excretion strongly and significantly decreased from pre- to post-donation (60.6 ± 16.0 vs. 40.5 ± 11.5µmol/24h, - 31.5 ± 21.5%, P < 0.001), while plasma ADMA increased only slightly (0.53 ± 0.08 vs. 0.58 ± 0.09µM, 11.1 ± 20.1%, P < 0.001). Compared to donors post-donation, RTR had significantly worse renal function (mGFR: 49 ± 18ml/min/1.73m2, - 25 ± 2%, P < 0.001) and lower urinary ADMA excretion (30.9 ± 12.4µmol/24h, - 23.9 ± 3.4%, P < 0.001). Plasma ADMA in RTR (0.60 ± 0.11µM) did not significantly differ from donors post-donation (2.9 ± 1.9%, P = 0.13). Plasma citrulline was inversely associated with mGFR (st. β: - 0.23, P < 0.001), consistent with increased ADMA metabolism to citrulline with lower GFR. In both groups, the response of urinary ADMA excretion to renal function loss was much larger than that of plasma ADMA. As citrulline was associated with GFR, our data indicate that with renal function impairment, a decrease in urinary ADMA excretion does not lead to a corresponding increase in plasma ADMA, likely due to enhanced metabolism, thus allowing for lower renal excretion of ADMA.

The role of asymmetric dimethylarginine in the development of arterial&#x0D; hypertension.

The results of numerous studies of recent decades confirm the crucial role of vascular endothelium in regulating vascular homeostasis. A plethora of recent studies have shed light on the clinical significance of endothelial dysfunction in essential hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. At present, it is considered as a generally recognized marker of endothelial dysfunction by most researchers. In vitro experiments showed that ADMA inhibits endothelium-dependent arterial relaxation, increases the level of indicators characterizing the degree of oxidative stress in endothelial cells, enhances the synthesis of the superoxide anion radical by endothelial cells. The molecular mechanisms described above, activated with an increase in the concentration of ADMA, cause various disturbances in the function of the cardiovascular system, which gave grounds to consider the level of ADMA as a criterion and risk factor for the development of cardiovascular diseases. Thus, ADMA plays a key role in the development and progression of CVD associated with a spectrum of diseases and pathological conditions characterized by a disturbance in NO production. Despite clinical and experimental confirmation of the relationship between the increase in ADMA in plasma and the development of cardiovascular events, the unambiguous etiopathogenetic role of ADMA in CVD requires further research. In order to accurately answer the question of whether ADMA is an etiological factor or a biological marker of CVD, additional analysis is needed to study the biochemical, genetic and pharmacological aspects of ADMA metabolism, the results of which are presented in this article.

The farnesoid X receptor agonist obeticholic acid upregulates biliary excretion of asymmetric dimethylarginine via MATE-1 during hepatic ischemia/reperfusion injury.

BackgroundWe previously showed that increased asymmetric dimethylarginine (ADMA) biliary excretion occurs during hepatic ischemia/reperfusion (I/R), prompting us to study the effects of the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) on bile, serum and tissue levels of ADMA after I/R.Material and methodsMale Wistar rats were orally administered 10mg/kg/day of OCA or vehicle for 5 days and were subjected to 60 min partial hepatic ischemia or sham-operated. After a 60 min reperfusion, serum, tissue and bile ADMA levels, liver mRNA and protein expression of ADMA transporters (CAT-1, CAT-2A, CAT-2B, OCT-1, MATE-1), and enzymes involved in ADMA synthesis (protein-arginine-N-methyltransferase-1, PRMT-1) and metabolism (dimethylarginine-dimethylaminohydrolase-1, DDAH-1) were measured.ResultsOCA administration induced a further increase in biliary ADMA levels both in sham and I/R groups, with no significant changes in hepatic ADMA content. A reduction in CAT-1, CAT-2A or CAT-2B transcripts was found in OCA-treated sham-operated rats compared with vehicle. Conversely, OCA administration did not change CAT-1, CAT-2A or CAT-2B expression, already reduced by I/R. However, a marked decrease in OCT-1 and increase in MATE-1 expression was observed. A similar trend occurred with protein expression.ConclusionThe reduced mRNA expression of hepatic CAT transporters suggests that the increase in serum ADMA levels is probably due to decreased liver uptake of ADMA from the systemic circulation. Conversely, the mechanism involved in further increasing biliary ADMA levels in sham and I/R groups treated with OCA appears to be MATE-1-dependent.

Ammonia Reduces Intracellular Asymmetric Dimethylarginine in Cultured Astrocytes Stimulating Its y⁺LAT2 Carrier-Mediated Loss.

Previously we had shown that ammonia stimulates nitric oxide (NO) synthesis in astrocytes by increasing the uptake of the precursor amino acid, arginine via the heteromeric arginine/glutamine transporter y+LAT2. Ammonia also increases the concentration in the brain of the endogenous inhibitor of nitric oxide synthases (NOS), asymmetric dimethylarginine (ADMA), but distribution of ADMA surplus between the intraastrocytic and extracellular compartments of the brain has not been studied. Here we tested the hypothesis that ammonia modulates the distribution of ADMA and its analog symmetric dimethylarginine (SDMA) between the two compartments of the brain by competition with arginine for the y+LAT2 transporter. In extension of the hypothesis we analyzed the ADMA/Arg interaction in endothelial cells forming the blood-brain barrier. We measured by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) technique the concentration of arginine, ADMA and SDMA in cultured cortical astrocytes and in a rat brain endothelial cell line (RBE-4) treated with ammonia and the effect of silencing the expression of a gene coding y+LAT2. We also tested the expression of ADMA metabolism enzymes: protein arginine methyltransferase (PRMT) and dimethylarginine dimethyl aminohydrolase (DDAH) and arginine uptake to astrocytes. Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell types, and the effect in astrocytes was substantially attenuated by silencing of the Slc7a6 gene. Moreover, the y+LAT2-dependent component of ammonia-evoked arginine uptake in astrocytes was reduced in the presence of ADMA in the medium. Our results suggest that increased ADMA efflux mediated by upregulated y+LAT2 may be a mechanism by which ammonia interferes with intra-astrocytic (and possibly intra-endothelial cell) ADMA content and subsequently, NO synthesis in both cell types.

Vitamin D and methylarginines in chronic kidney disease (CKD).

BackgroundVitamin D associates with the plasma concentration of the endogenous inhibitor of the nitric oxide system asymmetric dimethyl arginine (ADMA) and cross-sectional studies in CKD patients treated with the vitamin D receptor activator paricalcitol show that plasma ADMA is substantially less than in those not receiving this drug.MethodsIn the frame of a randomized, double-blind, placebo controlled trial, the Paracalcitol and ENdothelial fuNction in chronic kidneY disease (PENNY), we investigated whether vitamin D receptor activation by paricalcitol (2 μg/day x 12 weeks) affects the plasma concentration of ADMA and symmetric dimethyl arginine (SDMA) in 88 patients with stage 3 to 4 CKD.ResultsParicalcitol produced the expected small rise in serum calcium and phosphate and a marked PTH suppression. However, ADMA [Paricalcitol: baseline 0.75 μMol/L (95%CI: 0.70–0.81), 12 week 0.72 μMol/L (95%CI: 0.66–0.78); Placebo: baseline 0.75 μMol/L (95%CI: 0.70–0.90) 12 weeks 0.70 μMol/L (95%CI: 0.66–0.74)] and SDMA [Paricalcitol: baseline 0.91 μMol/L (95%CI: 0.82–1.00), 12 week 0.94 μMol/L (95%CI: 0.82–0.1.06); Placebo: baseline 0.91 μMol/L (95%CI: 0.82–1.06) 12 weeks 0.99 μMol/L (95%CI: 0.88–1.10)] remained unchanged during the trial and 2 weeks after stopping these treatments.ConclusionsParicalcitol does not modify plasma ADMA and SDMA in patients with stage 3–4 CKD. The apparent beneficial effects of paricalcitol on ADMA registered in cross-sectional studies is likely attributable to confounding by indication rather than to a true effect of this drug on ADMA metabolism.

Abstract 415: Transgenic Expression of Dimethylarginine Dimethylaminohydrolase Attenuates Exercise-induced Fatigue in Duchenne Muscular Dystrophy Carrier Mice

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in dystrophin and characterized by muscle degeneration, cardiomyopathy, and impaired muscle nitric oxide (NO) production that disrupts muscle blood flow regulation and leads to excessive post-exercise fatigue. Interestingly, circulating levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in dystrophin-deficient subjects. Therefore, we hypothesized that excessive circulating ADMA impairs muscle NO production and thus negatively impacts exercise tolerance in DMD. The objectives of this study were to determine whether increased circulating ADMA is itself sufficient to affect exercise performance, and whether transgenic modulation of ADMA metabolism could improve exercise-induced fatigue in the mdx mouse model of DMD. Although infusion of exogenous ADMA did impair exercise performance in healthy, wild-type mice, transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), the enzyme that degrades ADMA, did not affect exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. This improvement in exercise tolerance was associated with reduced heart weight, improved cardiac contractile function, and improved chronotropic responsiveness to beta-adrenergic stimulation in DDAH-transgenic female mdx carriers. In contrast, DDAH transgene expression did not significantly affect skeletal muscle pathology in female mdx carriers. We conclude that DDAH transgene expression improves exercise tolerance in dystrophin-heterozygous females, possibly by limiting pathological cardiac remodeling and helping to maintain heart performance. These findings emphasize the importance of methylated arginines to the development of cardiomyopathy in female DMD carriers, and have interesting implications for current genetic therapies that may only partially restore dystrophin expression in the hearts of male DMD patients.

Metabolism and distribution of pharmacological homoarginine in plasma and main organs of the anesthetized rat.

L-Homoarginine (hArg) and guanidinoacetate (GAA) are produced from L-arginine (Arg) by the catalytic action of arginine:glycine amidinotransferase. Guanidinoacetate methyltransferase methylates GAA on its non-guanidine N atom to produce creatine. Arg and hArg are converted by nitric oxide synthase (NOS) to nitric oxide (NO). NO is oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Asymmetric dimethylarginine (ADMA), an NOS inhibitor, is widely accepted to be exclusively produced after asymmetric N G-methylation of Arg residues in proteins and their regular proteolysis. Low circulating and urinary hArg concentrations and high circulating concentrations of ADMA emerged as risk markers in the human renal and cardiovascular systems. While ADMA's distribution and metabolism are thoroughly investigated, such studies on hArg are sparse. The aim of the present pilot study was to investigate the distribution of exogenous hArg in plasma, liver, kidney, lung, and heart in a rat model of takotsubo cardiomyopathy (TTC). hArg hydrochloride solutions in physiological saline were injected intra-peritoneally at potentially pharmacological, non-toxic doses of 20, 220, or 440mg/kg body weight. Vehicle (saline) served as control. As hArg has been reported to be a pro-oxidant, plasma and tissue malondialdehyde (MDA) was measured as a biomarker of lipid peroxidation. hArg administration resulted in dose-dependent maximum plasma hArg concentrations and distribution in all investigated organs. hArg disappeared from plasma with an elimination half-life ranging between 20 and 40min. hArg administration resulted in relatively small changes in the plasma and tissue content of Arg, GAA, ADMA, creatinine, and of the NO metabolites nitrite and nitrate. Remarkable changes were observed for tissue GAA, notably in the kidney. Plasma and tissue MDA concentration did not change upon hArg administration, suggesting that even high-dosed hArg is not an oxidant. The lowest hArg dose of 20mg/kg bodyweight increased 25-fold the mean hArg maximum plasma concentration. This hArg dose seems to be useful as the upper limit in forthcoming studies on the putative cardioprotective effects of hArg in our rat model of TTC.

Association of circulatory asymmetric dimethylarginine (ADMA) with diabetic nephropathy in Asian Indians and its causative role in renal cell injury

AimAsymmetric dimethylarginine (ADMA) is involved in the regulation of nitric oxide synthesis and in the maintenance of vascular tone and structure. But the role and status of ADMA in diabetes induced kidney injury is not clear. Hence this study is investigating the role of ADMA in the progression of kidney injury and its circulatory status in Asian Indians with and without diabetic nephropathy. MethodsRecruited study subjects were divided into normal glucose tolerance (NGT), type 2 diabetes mellitus (T2DM) and T2DM with micro or macroalbuminuria. Albuminuria was calculated using urinary albumin and creatinine ratio (UACR). ADMA was measured using ELISA. Kidney cell damage in terms of fibrotic markers and ADMA metabolism in terms of DDAH activity were investigated in kidney fibroblasts and mesangial cells. ResultsThere was a significant elevation in plasma ADMA levels in micro and macroalbuminuric diabetic patients. We found a significant positive correlation between ADMA and UACR, serum creatinine, HbA1C and fasting plasma glucose. A cut-off value of ADMA, 0.666μM/l had a sensitivity and specificity of 70.0% and 65.6%, respectively for detecting diabetic nephropathy. DDAH activity was significantly decreased and fibrotic markers such as fibronectin and α-SMA were significantly increased upon high glucose and ADMA treatment. ConclusionWe are suggesting a causative role of ADMA in the development of kidney injury in terms of renal fibrosis and also a cut point of 0.666μM/l of plasma ADMA level appears to be a predictive risk threshold for diabetic nephropathy in south Asian Indian population.

Abstract 185: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function

Background: It has been demonstrated in various studies that ADMA (asymmetric dimethylarginine), an inhibitor of nitric oxide synthase, is associated with the increased risk of cardiovascular diseases. There are two known pathways of ADMA metabolism: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolases (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The second pathway is still poorly understood. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and improvement of vasomotor function. Methods and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. qPCR and Western Blot were used to confirm the ubiquitous expression of the transgene. HPLC-MS/MS was used to generate biochemical data. Systemic ADMA levels were decreased by 15% (p&lt;0.05) in the TG mice, whereas ADGV plasma levels were six times higher in comparison with wild type animals (p&lt;0.001). Heart and lung of TG animals exhibited 2 times lower tissue ADMA content in comparison with wild type littermates (p&lt;0.05). In further experiments, we crossed the AGXT2 TG mice with DDAH1 KO mice and showed that upregulation of AGXT2 protects DDAH1 KO mice from elevation of plasma ADMA levels and restores endothelium-dependent vasodilation in aortic rings. In the current experiments we are assessing whether AGXT2 overexpression also protects DDAH1 KO mice from hypertension. Conclusion: In the current study we demonstrated that upregulation of AGXT2 leads to lowering of ADMA levels and improvement of endothelium-dependent relaxation in vivo in the settings of DDAH1 deficiency . AGXT2 thereby may be a potential drug target for long-term reduction of systemic ADMA levels in cardiovascular pathologies. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far.

ToxicDimethylarginines:Asymmetric Dimethylarginine(ADMA)andSymmetric Dimethylarginine(SDMA).

Asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) are toxic, non-proteinogenic amino acids formed by post-translational modification and are uremic toxins that inhibit nitric oxide (NO) production and play multifunctional roles in many human diseases. Both ADMA and SDMA have emerged as strong predictors of cardiovascular events and death in a range of illnesses. Major progress has been made in research on ADMA-lowering therapies in animal studies; however, further studies are required to fill the translational gap between animal models and clinical trials in order to treat human diseases related to elevated ADMA/SDMA levels. Here, we review the reported impacts of ADMA and SDMA on human health and disease, focusing on the synthesis and metabolism of ADMA and SDMA; the pathophysiological roles of these dimethylarginines; clinical conditions and animal models associated with elevated ADMA and SDMA levels; and potential therapies against ADMA and SDMA. There is currently no specific pharmacological therapy for lowering the levels and counteracting the deleterious effects of ADMA and SDMA. A better understanding of the mechanisms underlying the impact of ADMA and SDMA on a wide range of human diseases is essential to the development of specific therapies against diseases related to ADMA and SDMA.