Asymmetric Dimethylarginine Content Research Articles

Asymmetrical dimethylarginine induces dysfunction of insulin signal transduction via endoplasmic reticulum stress in the liver of diabetic rats

AimsEndoplasmic reticulum stress (ERS) as an emerging factor is involved in insulin resistance (IR), which is the pathological basis of diabetes mellitus. Accumulation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase is associated with IR, but the underlying mechanisms have not been elucidated. This study was to reveal the important role of ADMA in IR and determine whether endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats and hepatocytes. Materials and methodsDiabetic rat model was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg). Phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) was detected to evaluate IR. The protein kinase PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, x-box binding protein-1 (XBP-1) splicing, glucose-regulated protein 78 (GRP78) and C/EBP homologues protein (CHOP) expressions were measured to assess ERS. Key findingsEndogenous ADMA content was significantly increased and positively correlated with either IR as evidenced by increased IRS1 at serine and reduced Akt phosphorylation or ERS as indicated by upregulations of PERK and eIF2α phosphorylation, XBP-1 splicing, GRP78 and CHOP expressions in the liver of diabetic rats compared with control rats. Exogenous ADMA directly caused IR and ERS in dose- and time-dependent manners in primary mouse hepatocytes. Pretreatment with ERS inhibitor 4-phenylbutyrate or ADMA antagonist L-arginine not only improved ADMA-associated or -induced hepatic IR but also attenuated ADMA-associated or -induced ERS in diabetic rats or hepatocytes. SignificanceThese findings indicate that endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats.

Endogenous asymmetric dimethylarginine accumulation contributes to the suppression of myocardial mitochondrial biogenesis in type 2 diabetic rats

BackgroundSuppressed mitochondrial biosynthesis has been reported to be the early signal of mitochondrial dysfunction which contributes to diabetic cardiomyopathy, but the mechanism of mitochondrial biosynthesis suppression is unclear. Nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is closely related to diabetic cardiovascular complications. This study was to determine whether endogenous ADMA accumulation was involved in the suppression of myocardial mitochondrial biogenesis in diabetic rats and to elucidate the potential mechanism in rat cardiomyocytes.MethodsType 2 diabetic rat model was induced by high-fat feeding plus single intraperitoneal injection of small dose streptozotocin (35 mg/kg). The copy number ratio of mitochondrial gene to nuclear gene was measured to reflect mitochondrial biogenesis. The promoter activity of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its post-translational modifications were detected by dual-luciferase reporter assay and immunoprecipitation.ResultsMyocardial ADMA content was enhanced and associated with suppressions of myocardial mitochondrial biogenesis and cardiac function in parallel with PGC-1α downregulation and uncoupling protein 2 (UCP2) upregulation in the myocardium of diabetic rats compared with control rats. Similarly, ADMA and its homolog could inhibit myocardial mitochondrial biogenesis and PGC-1α expression, increase UCP2 expression and oxidative stress in vitro and in vivo. Moreover, ADMA also suppressed the promoter activity and PGC-1α expression but boosting its protein acetylation and phosphorylation in rat cardiomyocytes.ConclusionsThese results indicate that endogenous ADMA accumulation contributes to suppression of myocardial mitochondrial biogenesis in type 2 diabetic rats. The underlying mechanisms may be associated with reducing PGC-1α promoter activity and expression but boosting its protein acetylation and phosphorylation.

458-P: Resveratrol Attenuates Diabetic Cardiomyopathy by Inhibiting Asymmetric Dimethylarginine-Induced PGC-1a Acetylation

The cardiac protection of resveratrol may due to improvement of mitochondrial function through SIRT1 activation and PGC-1α deacetylation. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, is associated with diabetic cardiovascular complications and reported to have cross-talk with lysine acetylation in cells. This study was to determine the role of ADMA in the pathogenesis of diabetic cardiomyopathy, whether resveratrol could revise ADMA-induced pathological changes and elucidate the underlying mechanisms in rat model of type 2 diabetes mellitus (T2DM). Resveratrol was orally administered (50mg/kg/d) to T2DM rats for 16w. Echocardiography was performed to detect cardiac function. Contents of myocardial ATP and mitochondrial DNA were measured to evaluate mitochondrial function and biogenesis. Elevated endogenous ADMA content and its signal disorders were associated with cardiac and mitochondrial dysfunction in the myocardium of T2DM rats compared with control rats. Treatment with resveratrol significantly attenuated endogenous ADMA accumulation and cardiac dysfunction, increased myocardial mitochondrial DNA and ATP contents, as well as upregulated PGC-1α expression in the myocardium of resveratrol-treated diabetic rats compared with untreated diabetic rats. A positive feedback was observed in vitro that elevated ADMA return promoting its synthetic enzyme PRMT1 expression, reducing its metabolic enzymes DDAH2 expression, reproducing myocardial hypertrophy and mitochondrial dysfunction. Resveratrol treatment could reverse these pathological changes. More importantly, ADMA could enhance PGC-1α acetylation and reduced SIRT1 expressions, all of which were antagonized by resveratrol in cardiomyocytes. These results suggest that ADMA-induced PGC-1α hyperacetylation may serve as potential therapeutic targets for resveratrol to improve diabetic cardiomyopathy.

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells.

Trichophyton rubrum (T. rubrum) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum, investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells.

Iron Overload Damages the Endothelial Mitochondria via the ROS/ADMA/DDAHII/eNOS/NO Pathway.

It has been recognized that iron overload may harm the body's health. Vascular endothelial cells (VECs) are one of the main targets of iron overload injury, and the mechanism involved was thought to be related to the excessive generation of reactive oxygen species (ROS). However, the subcellular and temporal characteristics of ROS generation, potential downstream mechanisms, and target organelles in VECs injured by iron overload have not been expounded yet. In this study, we elucidated the abovementioned issues through both in vivo and in vitro experiments. Mice were fed pellet diets that were supplemented with iron for 4 consecutive months. Results showed that the thoracic aortic strips' endothelium-dependent dilation was significantly impaired and associated with inflammatory changes, noticeable under brown TUNEL-positive staining in microscopy analysis. In addition, the serum content of asymmetric dimethylarginine (ADMA) increased, whereas nitric oxide (NO) levels decreased. Furthermore, the dimethylarginine dimethylaminohydrolase II (DDAHII) expression and activity, as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) in aortic tissue, were inhibited. Human umbilical vein endothelial cells were treated with 50 μM iron dextran for 48 hours, after which the cell viability, NO content, DDAHII expression and activity, and phosphorylation of eNOS decreased and lactate dehydrogenase and caspase-3 activity, ADMA content, and apoptotic cells significantly increased. After the addition of L-arginine (L-Arg) or pAD/DDAHII, the abovementioned changes were reversed. By dynamically detecting the changes of ROS generation in the cytoplasm and mitochondria and interfering with different aspects of signaling pathways, we have confirmed for the first time that excessive ROS originates from the cytoplasm and activates the ROS-induced ROS release (RIRR) mechanism, leading to mitochondrial dysfunction. Together, our data suggested that excessive free iron ions produced excess ROS in the cytoplasm. Thus, excess ROS create one vicious circle by activating the ADMA/eNOS/DDAHII/NO pathway and another vicious circle by activation of the RIRR mechanism, which, when combined, induce a ROS burst, resulting in mitochondrial dysfunction and damaged VECs.

Dimethylarginine dimethylaminohydrolase 1 deficiency aggravates monocrotaline-induced pulmonary oxidative stress, pulmonary arterial hypertension and right heart failure in rats

Patients with pulmonary arterial hypertension (PAH) and right ventricular (RV) failure have a poor clinical outcome, but the mechanisms of PAH and RV failure development are not totally clear. PAH is associated with reduced NO bioavailability and increased endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase-1 (DDAH1) plays a critical role in ADMA degradation. Here we generated a novel DDAH1 deficiency rat strain using the CRISPR-Cas9 technique, and studied the effect of DDAH1 dysfunction on monocrotaline-induced PAH, lung vascular remodeling and RV hypertrophy. DDAH1 knockout resulted in abolished DDAH1 expression in various tissues, and significant increases of plasma and lung ADMA content. DDAH1 knockout has no detectable effect on cardiac and lung structure, and LV function under control conditions in rats. However, DDAH1 knockout significantly aggravated monocrotaline-induced lung and RV oxidative stress, lung vascular remodeling and fibrosis, pulmonary hypertension and RV hypertrophy in rats. DDAH1 KO resulted in significantly greater increases of plasma and lung ADMA content under control conditions. In the wild type rats monocrotaline resulted in significant increases of plasma and lung ADMA contents and reduction of lung eNOS protein content and these changes were more marked in DDAH1 KO rats. Together, our results demonstrated that DDAH1 plays an important role in attenuating monocrotaline-induced lung oxidative stress, pulmonary hypertension and RV hypertrophy in rats.

Xin-Ji-Er-Kang Alleviates Myocardial Infarction-Induced Cardiovascular Remodeling in Rats by Inhibiting Endothelial Dysfunction.

The present study was designed to elucidate the beneficial effects of XJEK on myocardial infarction (MI) in rats, especially through the amelioration of endothelial dysfunction (ED). 136 Sprague-Dawley rats were randomized into 13 groups: control group for 0wk (n = 8); sham groups for 2, 4, and 6 weeks (wk); MI groups for 2, 4, and 6 wk; MI+XJEK groups for 2, 4, and 6w k; MI+Fosinopril groups for 2, 4, and 6 wk (n = 8~10). In addition, 8 rats were treated for Evans blue staining and Tetrazolium chloride (TTC) staining to determine the infarct size. Cardiac function, ECG, and cardiac morphological changes were examined. Colorimetric analysis was employed to detect nitric oxide (NO), and enzyme-linked immunosorbent assay (ELISA) was applied to determine N-terminal probrain natriuretic peptide (NT-ProBNP), endothelin-1 (ET-1), angiotensin II (Ang II), asymmetric dimethylarginine (ADMA), tetrahydrobiopterin (BH4), and endothelial NO synthase (eNOS) content. The total eNOS and eNOS dimer/(dimer+monomer) ratios in cardiac tissues were detected by Western blot. We found that administration of XJEK markedly ameliorated cardiovascular remodeling (CR), which was manifested by decreased HW/BW ratio, CSA, and less collagen deposition after MI. XJEK administration also improved cardiac function by significant inhibition of the increased hemodynamic parameters in the early stage and by suppression of the decreased hemodynamic parameters later on. XJEK also continuously suppressed the increased NT-ProBNP content in the serum of MI rats. XJEK improved ED with stimulated eNOS activities, as well as upregulated NO levels, BH4 content, and eNOS dimer/(dimer+monomer) ratio in the cardiac tissues. XJEK downregulated ET-1, Ang II, and ADMA content obviously compared to sham group. In conclusion, XJEK may exert the protective effects on MI rats and could continuously ameliorate ED and reverse CR with the progression of MI over time.

411-P: PRMT1 Upregulation and ADMA Accumulation Contribute to the High Sensitivity of Zucker Diabetic Fatty Rats to Myocardial Ischemia/Reperfusion Injury

Diabetic patients show a high sensitivity to myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism has remained obscure. Increasing evidence indicates that protein arginine methyltransferase1 (PRMT1) is implicated in modulation of cellular processes including gene transcription, energy metabolism, apoptosis and generation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS). ADMA as an intermediate of protein arginine methylation catalyzed by PRMT1 is associated with diabetic cardiovascular complications. This study was to examine whether PRMT1-ADMA disorder is involved in the high sensitivity to myocardial I/R injury in Zucker diabetic fatty (ZDF) rats. Myocardial I/R injury was induced by 30min of ligation in left anterior descending coronary artery followed by 2∼24h reperfusion. Cardiac function was measured by M-echocardiography, and ADMA signaling pathway was evaluated by expressions, activities or contents of signal molecules. ZDF rats showed severer cardiac dysfunction and larger area of infarction after myocardial I/R injury compared with Zucker-control rats. Expression and activity of PRMT1 and ADMA content were higher while expression and activity of NOS and nitric oxide (NO) content were lower in ZDF rats. Treatment with PRMT1 inhibitor TC-E or knockdown of PRMT1 with shRNA-PRMT1-LV reduced myocardial ADMA accumulation and protected cardiac function of ZDF rats from I/R injury. Exogenous ADMA perfusion of isolated heart from control rats could induce similar cardiac dysfunction to that of I/R injury. Treatment with TC-E or NO donor protected the isolated heart from I/R injury or ADMA-induced impairment. These results indicate that PRMT1 upregulation and ADMA accumulation contribute to the high sensitivity of ZDF rats to myocardial I/R injury and reveal the novel target for prevention and treatment of myocardial I/R injury in diabetes mellitus. Disclosure Y. Xiong: None. Y. Lin: None. Y. Lei: None. Z. He: None. X. Zhou: None. Funding National Natural Science Foundation of China (81570751); Guangdong Province (2016A030311050)

Protective effect of Xin-Ji-Er-Kang on cardiovascular remodeling in high-salt induced hypertensive mice: Role ofoxidative stress and endothelial dysfunction

BackgroundXin-Ji-Er-Kang (XJEK) is a Chinese herbal formula that has beenreported to exert effective protection against cardiovascular diseases, such as hypertension and myocarditis. ObjectiveThe aim of the present study was to investigate the effect of XJEK on high-salt-induced hypertensive mice and its possible mechanism. MethodsThe model of hypertension was established through a high-salt diet. Sixty male Kunming mice were randomized into six groups, namely the Control, Model, Low-dose XJEK, Middle-dose XJEK, High-dose XJEK and Fosinopril groups (n=10 per group). Different steady interventions were given to each group: 0.9% Sodium chloride was added to the diet of the Control group and 8% sodium chloride to the diet of the other five groups from the very beginning. An additional 4, 8 and 12 g/kg/day XJEK were intragastrically administered to the Low-dose, Middle-dose and High-dose XJEK groups, respectively, and 2 mg/kg/day fosinopril to the fosinopril group, from the start of week 5. Systolic blood pressure (SBP) was measured weekly from weeks 1 to 8 using the tail-cuff method. At the end of week 8, left ventricular (LV) systolic pressure, LV end-diastolic pressure and rate of rise of LV pressure were examined using a TransonicScisense catheter (Transonic Systems Inc,Ithaca, NY,USA). Endothelium-dependent relaxations induced by acetylcholine were observed in an isolated thoracic aorta ring. Serum and heartsweresampled for the measurement of the following indexes:Serum nitric oxide (NO), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content (determined by colorimetricanalysis); serum angiotensin II(Ang II), endothelin-1, endothelial NO synthase (eNOS), asymmetric dimethylarginine (ADMA), tetrahydrobiopterin (BH4) concentration and l-arginine (determined by enzyme-linked immunosorbent assay); heart to body weight (HW/BW) ratio; myocardial morphological change (determined by HE and VG staining); myocardial eNOS expression (determined by immunofluorescence), and myocardial endothelin receptor A (ETA) expression (determined by western blotting).Results:Statistical data showed that the HW/BW ratio was significantly decreased in the drug treatment group. XJEK treatment could improve the heart systolic and diastolic function and ameliorate hemodynamic parameters and vascular remodeling indexes. Colorimetric results showed that, compared with the model group, XJEK increased serum SOD, NOlevels, and decreased those of serum MDA and Ang II. XJEK reverted changes in cardiac pathology, decreased the myocardial cross-sectional area, collagen volume fraction and perivascular collagen area and improved endothelial dysfunction (ED) by promoting eNOS activity, enhancing NO bioavailability, increasing the expression of BH4 and decreasing ETA content. In addition, treatment with XJEK decreased ADMA content in the myocardium.Conclusion:In conclusion, XJEK mitigates cardiac remodeling in high-salt-induced hypertensive mice. The potential mechanism involves improved oxidative stress and endothelial dysfunction, independently of ameliorating BP.

Clinical value assessment of chemiluminescence for urine asymmetric dimethylarginine

Objective To explore the clinical application value of chemiluminescence detection of urine asymmetric dimethylarginine (ADMA) in pregnancy-induced hypertension (PIH). Methods Collected the 24 h urine from 60 normal pregnancy women and 72 PIH pregnancy women who were admitted to Huzhou Maternal and Child Health Hospital from May 2014 to April 2015 by the case-control study, Determination of urine ADMA content by chemiluminescence (CLIA) and high performance liquid chromatography (HPLC), the results between two assays were analyzed by the Rank sum test, receiver operating characteristic (ROC) curve and pearson correlation analysis. Results Compared with the normal control group, the urine ADMA concentration in the PIH group was significantly increased by HPLC and CLIA, and the concentration of ADMA by CLIA in the PIH group was 68.18(57.25, 81.55)μmol/L higher than that of the normal control group 30.11(22.69, 42.97)μmol/L(Z=-8.139, P<0.001), and the concentration of ADMA by HPLC in the PIH group by HPLC was 71.11(57.65, 82.89)μmol/L higher than that of the normal control group 28.11(21.06, 42.99)μmol/L(Z=-8.356, P<0.001). The difference was statistically significant.The two methods of urine ADMA concentration were highly positively correlated with PIH blood pressure. The correlation coefficient r values were 0.746 and 0.763, respectively, the P values were 0.007 and 0.008 respectively. Conclusions CLIA can better detect the ADMA concentration in urine of pregnant women with PIH, and has a good clinical diagnosis ability. The ADMA concentration in urine is related to the blood pressure level of PIH.(Chin J Lab Med, 2018, 41: 948-951) Key words: Arginine; Urinalysis; Luminescence; Chromatography, high pressure liquid; Hypertension, pregnancy-induced

Accumulation of Endogenous Asymmetric Dimethylarginine Contributes to Diabetic Cardiomyopathy via Endoplasmic Reticulum Stress in Diabetic Rats

Diabetic cardiomyopathy is a major cause of morbidity and mortality among diabetic patients. Endoplasmic reticulum (ER) stress has been involved in diabetic cardiomyopathy. Accumulation of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) is associated with diabetic cardiovascular complications. This study was to determine whether ADMA accumulation contributes to diabetic cardiomyopathy and whether its mechanism is related to ER stress in streptozotocin-induced diabetic rats and cultured rat cardiomyocytes. Cardiac function was measured by M-echocardiography. ER stress was reflected by transcription of glucose-regulated protein78, phosphorylation of eukaryotic initiation factor 2α kinase, splicing of X box-binding protein-1 mRNA and expression of C/EBP homologues protein. Changes of ADMA signal pathway were evaluated by ADMA and nitric oxide contents, NOS and dimethylarginine dimethylaminohydrolase activities, as well as their protein expression. The levels of ADMA in serum and myocardium were significantly elevated in diabetic rats compared to control rats. This elevation was associated with left ventricular diastolic and systolic dysfunction of diabetic rats in comparison with control rats. Furthermore, ADMA accumulation was not only coupled with the disorder of ADMA signal pathway, but also tied to ER stress and oxidative stress in myocardium of diabetic rats. Treatment of diabetic rats with antioxidant N-acetylcysteine could reduce oxidative stress and ER stress, but also ameliorate cardiac dysfunction of diabetic rats. Similar results to diabetic rats were observed in the exogenous NOS inhibitor-treated normal rats and ADMA-incubated cardiomyocytes. These results indicate that endogenous ADMA accumulation plays a pivotal role in the development of diabetic cardiomyopathy, and the underlying mechanism is close related to ER stress and oxidative stress. Disclosure Y. Xiong: None. Y. He: None. W. Fang: None. Y. Lei: None. Y. Lin: None. Z. He: None.

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.

Role of nitric oxide synthase inhibitor in the skeletal muscle contractile dysfunction of type 2 diabetic rats

Objective To investigate the role of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in the contractile dysfunction of skeletal muscle in diabetic rats and on which the therapeutic effects of L-Arginine. Methods Type 2 diabetic rats were induced by high fat diet and a single intraperitoneal injection of streptozotocin (STZ, 30 mg/kg), followed by high fat diet for 8 weeks. Specific twicth tension and specific tetanic tension of soleus (SOL) and extensor digitorum longus (EDL) isolated from control and diabetic rats were detected by electric stimulation to reflect contractile function of skeletal muscle. ADMA content of skeletal muscle was analyzed by enzyme linked immunosorbent assay (ELISA), and activities of dimethylarginie dimethylaminohydrolase (DDAH) and NOS, NO content were measured by colorimetry. The protein expression of ADMA synthetase protein arginine methyl transferase 1 (PRMT1) and ADMA hydrolase DDAH and NOS were determined detected by Western blott-ing. Oral glucose tolerance test and protein expressions of phosphorylated insulin receptor substrate 1(p-IRS-1) and protein kinase B (p-Akt) as well as the membrane transportation of glucose transporter 4 (Glut4) were measured to reflect insulin resistance. Results In comparison with control rats, specific twicth tension and tetanic tension of SOL and EDL in diabetic rats were significantly decreased (P<0.01), indicating the contractile dysfunction. Increased ADMA content (P<0.05), decreased DDAH and NOS activities as well as NO content (P<0.01) in comparison with up-regulated protein expression of PRMT1 and down-regulated protein expression of DDAH, endothelial NOS (eNOS) and neuronal NOS (nNOS)(P<0.05) were observed in the skeletal muscle of diabetic rats compared to control rats, indicating that the pathway of PRMT1/ADMA/DDAH/ NOS/NO was disordered in the skeletal muscle of diabetic rats. Furthermore, the glucose tolerance, both IRS-1 and Akt protein phosphorylation as well as the membrane translocation of Glut4 were decreased (P<0.05), indicating the insulin resistance in diabetic rats. Treatment with L-Arginine for 8 weeks not only significantly improved the contractile dysfunction but also reversed the disorder of ADMA signaling pathway and insulin resistance in skeletal muscle of diabetic rats compared to untreated diabetic rats. Conclusions The accumulation of endogenous NOS inhibitor ADMA contributes to the contractile dysfunction of skeletal muscle in diabetic rats, the underlying mechanism may be related to insulin resistance. Key words: Asymmetric dimethylarginine; Type 2 diabetes mellitus; Contractile dysfunction; Insulin resistance; L-arginine

Cardiomyocyte dimethylarginine dimethylaminohydrolase-1 (DDAH1) plays an important role in attenuating ventricular hypertrophy and dysfunction.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases that limits nitric oxide bioavailability. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) exerts a critical role for ADMA degradation and plays an important role in NO signaling. In the heart, DDAH1 is observed in endothelial cells and in the sarcolemma of cardiomyocytes. While NO signaling is important for cardiac adaptation to stress, DDAH1 impact on cardiomyocyte homeostasis is not clear. Here we used the MerCreMer-LoxP model to specifically disrupt cardiomyocyte DDAH1 expression in adult mice to determine the physiological impact of cardiomyocyte DDAH1 under basal conditions and during hypertrophic stress imposed by transverse aortic constriction (TAC). Under control conditions, cardiomyocyte-specific DDAH1 knockout (cDDAH KO) had no detectable effect on plasma ADMA and left ventricular (LV) hypertrophy or function in adult or aging mice. In response to TAC, DDAH1 levels were elevated 2.5-fold in WT mice, which exhibited no change in LV or plasma ADMA content and moderate LV hypertrophy and LV dysfunction. In contrast, cDDAH1 KO mice exposed to TAC showed no increase in LV DDAH1 expression, slightly increased LV tissue ADMA levels, no increase in plasma ADMA, but significantly exacerbated LV hypertrophy, fibrosis, nitrotyrosine production, and LV dysfunction. These findings indicate cardiomyocyte DDAH1 activity is dispensable for cardiac function under basal conditions, but plays an important role in attenuating cardiac hypertrophy and ventricular remodeling under stress conditions, possibly through locally confined regulation of subcellular ADMA and NO signaling.

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.

OS 32-09 THE BENEFICIAL EFFECT OF LOSARTAN ON PLATELET AGGREGATION RESULTS FROM THE REDUCTION OF ENDOGENOUS ASYMMETRIC DIMETHYL ARGININE IN SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Previous studies show that platelet aggregation and asymmetric dimethyl arginine (ADMA) are increased in hypertensive patients and animals. However, the relationship between endogenous ADMA and platelet aggregation in spontaneous hypertensive rats (SHR) remains unclear. Since Losartan can increase the nitric oxide (NO) release and ameliorate the state of thrombosis, we aimed to test whether the beneficial effect of losartan on platelet aggregation results from the reduction of ADMA generation. Design and Method: With male Wistar Kyoto rats (WKY) as normal control, we randomly divided male SHR into three groups: SHR control group, L-arginine-treated (1.0 g/kg) and losartan-treated (30 mg/kg) group. For each group, we measured systolic blood pressure (SBP), platelet aggregation rate (pt-Ag), NO level, NOS activity and ADMA content in both plasma and platelets. Results: Compared with WKY rats, the ADMA level in both plasma and in platelets and platelet aggregation rate in SHR were higher, while the NO level and NOS activity were lower. L-arginine (1.0 g/kg) and losartan (30 mg/kg) treatment decreased platelet aggregation rate concomitantly with the decreased level of ADMA and the increased level of NO and the activity of NOS. Incubating platelets from SD rats with thoracic aortic rings for 5 min, the platelet aggregation rate in SHR was higher than that of WKY rats. L-arginine (1.0 g/kg) and losartan (30 mg/kg) significantly reduced the platelet aggregation rate. Conclusions: Our results suggest that endogenous ADMA plays a key role in platelet aggregation, and the beneficial effect of losartan is related to the reduced ADMA level.

Phosphorylation of Nonmuscle Myosin Light Chain Promotes Endothelial Injury in Hyperlipidemic Rats Through a Mechanism Involving Downregulation of Dimethylarginine Dimethylaminohydrolase 2.

Suppression of dimethylarginine dimethylaminohydrolase (DDAH) activation is related to endothelial dysfunction in hyperlipidemia, and nonmuscle myosin regulatory light chain (nmMLC20) has been show to exert transcriptional function in regulation of gene expression. This study aims to explore whether the suppression of DDAH activation promotes endothelial injury under the condition of hyperlipidemia and whether nmMLC20 can regulate DDAH expression in a phosphorylation-dependent manner. The rats were fed with high-fat diet for 8 weeks to establish a hyperlipidemic model, which showed an increase in plasma lipids and endothelial injury, accompanied by an elevation in myosin light chain kinase (MLCK) activity, phosphorylated nmMLC20 (p-nmMLC20) level, and asymmetric dimethylarginine (ADMA) content as well as a reduction in DDAH2 expression, DDAH activity, and nitric oxide (NO) content. Next, human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL; 100 μg/mL) for 24 hours to establish a cellular injury model in vitro. Consistent with the finding in vivo, ox-LDL induced HUVECs injury (apoptosis and necrosis) concomitant with an increase in MLCK activity, p-nmMLC20 level (in total or nuclear proteins), and ADMA content as well as a reduction in DDAH2 expression, DDAH activity, and NO content; these phenomena were attenuated by MLCK inhibitor. Either in hyperlipidemic rats or in ox-LDL-treated HUVECs, there was not significant change in DDAH1 expression. Based on these observations, we conclude that the suppression of DDAH2 expression might account for, at least partially, the vascular endothelial dysfunction in hyperlipidemia, and nmMLC20 plays a role in suppression of DDAH2 expression in a phosphorylation-dependent manner.

Assessment of ADMA, estradiol, and progesterone in severe preeclampsia

ABSTRACTBackground: Decreased nitrous oxide (NO) levels are crucial factors in severe preeclampsia (sPE), and asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthetase. Steroid hormones are closely related to the vascular endothelium. This study determined the levels of and correlations between ADMA, estradiol (E2), and progesterone (Pg) in sPE to investigate the roles of these factors in this disease. Methods: Sixty-two sPE patients (sPE group) were divided into the sPE1 subgroup (28+1–32+0 weeks of pregnancy), the sPE2 subgroup (32+1–36+0 weeks), and the sPE3 subgroup (36+1–40+0 weeks) and 75 normal pregnant women (NC group) were divided into the NC1 subgroup (28+1–32+0 weeks of gestation), the NC2 subgroup (32+1–36+0 weeks), and the NC3 subgroup (36+1–40+0 weeks). Serum and placental ADMA levels were measured by enzyme-linked immunosorbent assay (ELISA), and serum E2 and Pg concentrations were determined by the chemilumineseent immunoassay (CLIA). Results: ADMA concentrations in both the placenta and the maternal serum were significantly higher in the sPE group (p < 0.05). Higher ADMA contents were observed in the placenta relative to the maternal serum (p < 0.05). Serum E2 levels were significantly lower in the sPE group (p < 0.05). For Pg, the only significant difference was observed between the sPE1 and NC1 subgroups (p < 0.05). The Pg/E2 ratios in the sPE groups were significantly higher, with a significant high positive correlation between Pg/E2 ratios and serum ADMA levels. Conclusion: Increased serum levels of ADMA in sPE may result from increased secretion from the placenta, and the increased Pg/E2 ratio may play a role in the development of sPE by aggravating ADMA.

Abstract 453: Transgenic Overexpression of Alanine-glyoxylate Aminotransferase 2 in Mice Lowers Asymmetric Dimethylarginine and Improves Vasomotor Function

Background: Elevation of the endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been shown to be associated with increased risk of cardiovascular diseases. There are two major pathways of ADMA catabolism: 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 poorly characterized. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of systemic 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. There were no developmental or phenotypic changes in the TG animals. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels were decreased by 15% (p&lt;0.05) in the TG mice, whereas ADGV plasma levels were 6 times higher in comparison with wild-types littermates (p&lt;0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p&lt;0.05). TG mice demonstrated improved endothelium-dependent vasodilation (in response to acetylcholine) in aortic rings. The endothelium-independent relaxation (in response to sodium nitroprusside) was unchanged. There was no difference in mean arterial blood pressure measured by telemetry between the wild type and AGXT2 TG mice. 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. 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. 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.