Asymmetric dimethylarginine is not involved in ovariectomy-induced osteopenia in rats

Previous studies have indicated that the plasma concentration of nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), was increased in postmenopausal women. In the study reported here, we tested the relationship between the decrease of bone mineral density (BMD) and ADMA concentration in ovariectomized (OVX) rats. Ovariectomized rats at 8 months of age were treated with 17beta-estradiol (10 or 30 microg/kg of body weight/day, s.c.) or L-arginine (300 mg/kg/day, i.p.) for 12 weeks (n = 10 for each group). Pre- and posttreatment total BMD, posttreatment plasma nitrite/nitrate and ADMA concentrations, and posttreatment BMD in the lumbar part of the spine (L4-L6), femurs, and tibias were examined. Ovariectomy caused a significant decrease in several BMD indexes, which was reversed by estrogen treatment (P < 0.05). Plasma nitrite/nitrate concentration was significantly decreased in OVX rats, but was restored by estrogen treatment (P < 0.05). There were no differences in the plasma concentration of ADMA in OVX or estrogen-treated rats. L-Arginine had no effect on plasma nitrite/nitrate concentration and BMD in OVX rats. These results suggest that ovariectomy does not influence the plasma concentration of ADMA, and that ADMA is not involved in ovariectomy-induced osteopenia in rats.     

Relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors

1,3,5,6-tetrahydroxyxanthone was synthesized. The relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors was investigated. Endothelial cells were treated with ox-LDL (100 microg/mL) for 48 h. Adhesion of monocytes to endothelial cells and release of lactate dehydrogenase (LDH) was determined. Levels of tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), nitric oxide (NO) and asymmetric dimethylarginine (ADMA, an endogenous inhibitor of nitric oxide synthase) in conditioned medium and activity of dimethylarginine dimethylaminohydrolase (DDAH) in endothelial cells were measured. Incubation of endothelial cells with ox-LDL (100 microg/mL) for 48 h markedly enhanced the adhesion of monocytes to endothelial cells, increased the release of LDH, the levels of TNF-alpha, MCP-1 and ADMA, and decreased the content of NO and the activity of DDAH. Xanthone (1,3,5,6-tetrahydroxyxanthone) (1, 3 or 10 micromol/L) significantly inhibited the increased adhesion of monocytes to endothelial cells and attenuated the increased levels of LDH, MCP-1 and ADMA induced by ox-LDL. Xanthone (1,3,5,6-tetrahydroxyxanthone) (3 or 10 micromol/L) significantly attenuated the increased level of TNF-alpha and decreased level of NO and activity of DDAH by ox-LDL. The present results suggest that xanthone (1,3,5,6-tetrahydroxyxanthone) preserves endothelial cells and inhibits the increased adhesion of monocytes to endothelial cells induced by ox-LDL, and that the protective effect of xanthone (1,3,5,6-tetrahydroxyxanthone) on endothelial cells is related to reduction of ADMA concentration via increase of DDAH activity.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

The role of asymmetric dimethylarginine in the regulation of nitric oxide level in rats with acute renal injury

Nitric oxide (NO) is synthesized from arginine (ARG) by NO synthase (NOS). Asymmetric dimethylarginine (ADMA), a competitive inhibitor of NOS, participates in the endogenous regulation of NO synthesis. The main amount of ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH) widely expressed in renal tissue. The aim of our study was to compare the changes in DDAH activity and ARG synthesis in kidneys, ADMA and ARG concentration in plasma and their urinary excretion under physiological conditions and in acute renal injury (ARI) induced by glycerol in rats. Urinary nitrite/nitrate excretion (NOx) was estimated as an indicator of whole-body NO synthesis. DDAH activity was decreased, ADMA excretion was increased and plasma ADMA did not change in ARI. Plasma ARG concentration, renal ARG synthesis and urinary NOx excretion were decreased. In conclusion, the diminished enzymatic hydrolysis of the NOS inhibitor ADMA and the reduced synthesis of the NOS substrate ARG might affect NO production in ARI.     

Asymmetric Dimethylarginine (ADMA) and other Endogenous Nitric Oxide Synthase (NOS) Inhibitors as an Important Cause of Vascular Insulin Resistance

Asymmetric dimethylarginine (ADMA) and NG-monomethyl- L-arginine ( L-NMMA) are important endogenous endothelial nitric oxide synthase (eNOS) inhibitors. Studies have shown that patients with insulin resistance have elevated plasma levels of ADMA. Moreover, ADMA levels have a prognostic value on long-term outcome of patients with coronary artery disease. Insulin resistance, a disorder associated to inadequate biological responsiveness to the actions of exogenous or endogenous insulin, is a metabolic condition, which exists in patients with cardiovascular diseases. This disorder affects the functional balance of vascular endothelium via changes of nitric oxide (NO) metabolism. Nitric oxide is produced in endothelial cells from the substrate L-arginine via eNOS. Elevated ADMA levels cause eNOS uncoupling, a mechanism which leads to decreased NO bioavailability and increased production of hydrogen peroxide. According to clinical studies, the administration of L-arginine to patients with high ADMA levels improves NO synthesis by antagonizing the deleterious effect of ADMA on eNOS function, although in specific populations such as diabetes mellitus, this might even been harmful. More studies are required in order to certify the role of NOS inhibitors in insulin resistance and endothelial dysfunction. It is still difficult to say whether increased ADMA levels in certain populations is only a reason or the result of the molecular alterations, which take place in vascular disease states.     

The Effect of Chronic Long-Term Intermittent Hypobaric Hypoxia on Bone Mineral Density in Rats: Role of Nitric Oxide

Intermittent hypoxia is the most common pattern of hypoxic exposure in humans. The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on bone metabolism is not investigated. We examined the effect of CLTIHH on bone metabolism and the role of nitric oxide (NO) in this process. The rats were divided into three groups in this study. The animals in groups I and II have been exposed to CLTIHH. The animals in group II were also treated with nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mm Hg; 5 h/day, 5 days/week, 5 weeks). The group III (control) rats breathed room air in the same environment. At the begining of the experiments, bone mineral density (BMD) of the animals were measured, and blood samples were collected from the tail vein. After the 5-week CLTIHH period, the same measurements were repeated. Parathyroid hormone, calcium, phosphate, bone alkaline phosphatase (b-ALP), NO, interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha levels were determined. The cytokines, NO levels, and BMD in CLTIHH-induced rats were higher compared with baseline and control values. The cytokines, b-ALP, and BMD increased while NO levels decreased in the group II compared with baseline values. BMD values of group II were lower than group I but higher than control group. Our results suggested that CLTIHH has positive effects on bone density. Intermittent hypoxia protocols may be developed for treatment and prevention of osteopenia and osteoporosis.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

Effects of oral L-arginine supplementation on blood pressure and asymmetric dimethylarginine in stress-induced preeclamptic rats

This study was carried out to elucidate the role of asymmetric dimethylarginine (ADMA) and nitric oxide (NO) in preeclampsia development, and to investigate the effect of L-arginine supplementation in rats. Preeclampsia was induced in pregnant rats using a stress model. L-arginine was administered orally and ADMA, urinary nitrate, and protein levels were measured on the 20th day of pregnancy. Compared with the group of rats that are normally pregnant, the levels of blood pressure (BP), protein excretion, and ADMA were significantly increased in preeclampsia which returned to normal levels following the supplementation of L-arginine. Both group of rats had similar urine nitrate levels. Arginine-ADMA-NO pathway is affected in preeclampsia. L-arginine supplementation decreased hypertension (HT), proteinuria, and ADMA levels indicating that taking L-arginine may be beneficial in preeclampsia treatment.     

Increased plasma level of asymmetric dimethylarginine in hypertensive rats facilitates platelet aggregation: role of plasma tissue factor

This study was designed to explore the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in platelet aggregation in hypertension and its possible mechanisms. Spontaneously hypertensive rats (SHR) and L-NAME-induced hypertensive rats were orally administered with L-arginine (1 g/(kg·day) for 14 days. Systolic blood pressure, platelet aggregation, and plasma tissue factor (TF) level and activity were measured. The plasma concentration of ADMA in SHR was determined. In vitro, platelet-rich plasma isolated from Wistar rats was prepared in order to observe the effect of exogenous ADMA on platelet aggregation and TF level and (or) activity in platelet-rich plasma. In both types of hypertensive rats, systolic blood pressure, platelet aggregation, and the level and activity of plasma TF were elevated compared with corresponding control animals. Plasma ADMA level was also increased in SHR. Treatment with L-arginine, a competitor of ADMA, lowered blood pressure and inhibited platelet aggregation concomitantly with a decrease in plasma TF level and activity in both types of hypertensive rats. We also found that exogenous ADMA promoted platelet aggregation and increased TF level and (or) activity in platelet-rich plasma, an effect that was inhibited by pretreatment with L-arginine. Importantly, the enhanced platelet aggregation induced by exogenous ADMA was reduced by pretreatment with anti-TF antibody. The results suggest that endogenous ADMA may be involved in platelet hyperaggregation status in hypertension, and the facilitation of platelet aggregation by ADMA is related to upregulation of the level and activity of plasma TF.     

ADMA-- 一个新的心血管疾病危险因子

非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)为内源性一氧化氮合酶(endogenous Nitric-Oxide Synthase,eNOS)竞争性抑制剂,可抑制NOS的活性,减少一氧化氮(nitric oxide,NO)的合成,致使内皮功能障碍。近年来研究认为ADMA是冠心病的独立预测因子,与心功能不全及心血管疾病死亡率明显相关。本文将针对ADMA与心血管疾病的相关性进行综述。     

Accumulated endogenous NOS inhibitors,decreased NOS activity,and impaired cavernosal relaxation with ischemia

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca(2+)-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excess L-arginine partially recovered both of the impaired relaxations. The contents of N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) but not L-arginine and symmetric N(G),N'(G)-dimethyl-L-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic L-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess L-arginine abolished the inhibition with L-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of L-NMMA and ADMA.     

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.     

Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway

Both NADPH oxidase-derived reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) are increased in hypertension. Apocynin, an NADPH oxidase inhibitor, could inhibit ROS, thus we tested whether apocynin can block NADPH oxidase and prevent increases of ADMA and blood pressure (BP) in spontaneously hypertensive rats (SHRs). SHRs and Wistar Kyoto (WKY) rats, aged 4 weeks, were assigned to four groups: untreated SHRs and WKY rats, SHRs and WKY rats that received 2.5 mM apocynin for 8 weeks. BP was significantly higher in SHRs compared to WKY rats, which was attenuated by apocynin. Apocynin prevented p47phox translocation in SHR kidneys, but not the increase of superoxide and H(2)O(2). Additionally, apocynin did not protect SHRs against increased ADMA. Apocynin blocks NADPH oxidase to attenuate hypertension, but has little effect on the ADMA/nitric oxide (NO) pathway in young SHRs. The reduction of ROS and the preservation of NO simultaneously might be a better approach to restoring ROS-NO balance to prevent hypertension.     

Protective effect of HDL on endothelial NO production: the role of DDAH/ADMA pathway

Accumulating studies have demonstrated that the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine (DDAH/ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of high density lipoprotein (HDL) on endothelial NO production was related to its effect on DDAH/ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were prior exposed to HDL (10, 50, or 100 μg/ml) for 1 h, and then incubated with oxidized low density lipoprotein (ox-LDL) (100 μg/ml) for 24 h. The cultured medium was collected for measuring the concentration of NO and ADMA. The cells were collected for measuring the mRNA and protein expression of DDAH-II as well as DDAH activity. HUVECs treated with ox-LDL (100 μg/ml) for 24 h significantly decreased the concentration of NO, the mRNA and protein expression of DDAH-II as well as DDAH activity and increased the level of ADMA. Pretreatment with HDL (10, 50, or 100 μg/ml) could counteract these changes induced by ox-LDL (100 μg/ml). HDL significantly increased the attenuated endothelial cell NO production induced by ox-LDL, which was attributed to its effect on DDAH/ADMA pathway.     

Aspirin reduces endothelial cell senescence

We report here the effect of aspirin on the onset of replicative senescence. Endothelial cells that were cultured until cumulative population doublings 40 showed clear signs of aging. Incubation with aspirin inhibited senescence-associated beta-galactosidase activity and increased telomerase activity. Along with the delayed onset of senescence, aspirin decreased reactive oxygen species and increased nitric oxide (NO) and cGMP levels. Furthermore, aspirin reduced the elaboration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, and up-regulated the activity of dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA. These effects were specific in that other nonsteroidal anti-inflammatory drugs, such as ibuprofen or acetaminophen, did not prevent the onset of endothelial senescence. The NO synthase inhibitor l-NAME, but not its inactive d-enantiomer, led to complete inhibition of aspirin-delayed senescence. These findings demonstrate that aspirin delays the onset of endothelial senescence by preventing a decrease in NO formation/generation. This might provide a therapeutic strategy aimed at blocking aging-induced NO inhibition.     

Increased Asymmetric Dimethylarginine in Severe Falciparum Malaria: Association with Impaired Nitric Oxide Bioavailability and Fatal Outcome

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 µM; 95% CI 0.74–0.96) compared to those with MSM (0.54 µM; 95%CI 0.5–0.56) and HCs (0.64 µM; 95%CI 0.58–0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0–181; p = 0.01). ADMA was independently associated with decreased exhaled NO (r_s = −0.31) and endothelial function (r_s = −0.32) in all malaria patients, and with reduced exhaled NO (r_s = −0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.     

Enhanced concentrations of relevant markers of nitric oxide formation after exercise training in patients with metabolic syndrome

Metabolic syndrome (MetS) denotes a clustering of risk factors that may affect nitric oxide (NO) bioavailability and predispose to cardiovascular diseases, which are delayed by exercise training. However, no previous study has examined how MetS affects markers of NO formation, and whether exercise training increases NO formation in MetS patients. Here, we tested these two hypotheses. We studied 48 sedentary individuals: 20 healthy controls and 28 MetS patients. Eighteen MetS patients were subjected to a 3-month exercise training (E + group), while the remaining 10 MetS patients remained sedentary (E−group). The plasma concentrations of nitrite, cGMP, and ADMA (asymmetrical dimethylarginine; an endogenous nitric oxide synthase inhibitor), and the whole blood nitrite concentrations were determined at baseline and after exercise training using an ozone-based chemiluminescence assay, and commercial enzyme immunoassays. Thiobarbituric acid reactive species (TBA-RS) were measured in the plasma to assess oxidative stress using a fluorometric method. We found that, compared with healthy subjects, patients with MetS have lower concentrations of markers of NO formation, including whole blood nitrite, plasma nitrite, and plasma cGMP, and increased oxidative stress (all P < 0.05). Exercise training increased the concentrations of whole blood nitrite and cGMP, and decreased both oxidative stress and the circulating concentrations of ADMA (both P < 0.05). These findings show clinical evidence for lower endogenous NO formation in patients with MetS, and for improvements in NO formation associated with exercise training in MetS patients.