Asymmetric dimethyl-L-arginine Research Articles

Asymmetric Dimethyl l-Arginine (ADMA) is a critical regulator of myocardial reperfusion injury

Endothelial dysfunction by the loss of nitric oxide (NO) is a critical event during reperfusion of ischemic myocardium. Reduced NO availability signals important pathophysiological changes leading to myocardial reperfusion injury. We have recently shown that NO biosynthesis can be disturbed by the endogenous NO synthase (NOS) inhibitor ADMA and that these changes are mediated by an impairment of its metabolism by dimethylarginine dimethylaminohydrolase (DDAH). We therefore analyzed the role of ADMA and its metabolism in the setting of myocardial ischemia and reperfusion. C57-bl6 mice underwent myocardial ischemia for exactly 30 min followed by 2, 4, 8, 12, 24, and 72 h of reperfusion achieved by occlusion and re-opening of the left coronary artery. The reperfused left ventricle was subsequently homogenized for measurements of determinants of the NO synthase pathway. Furthermore, the effects and its mechanisms of ADMA on reperfusion injury were analyzed in a genetic mouse model. A significant accumulation of ADMA was found in myocardial tissue when mice were subjected to 30 min of ischemia followed by reperfusion in our in vivo model. The maximum increase of tissue ADMA at 4 h of reperfusion coincided with reductions of NO tissue concentrations and DDAH activity; protein expression of NOS isoforms, however, was not changed. Furthermore, DDAH overexpression in a genetic mouse model as well as treatment with oral L-arginine markedly reduced reperfusion injury by 40-50% at 4 h of reperfusion. The effects of ADMA on reperfusion injury were shown to be mediated by reduced eNOS activity and phosphorylation, expression of adhesion molecules, and leukocyte activity. Accumulation of tissue ADMA by impairment of DDAH was found to be a significant determinant of reperfusion injury. Our results indicate that ADMA could be a potential new target for the treatment of myocardial ischemia/reperfusion injury.

Plasma Adhesion and Inflammation Markers: Asymmetrical Dimethyl-L-Arginine and Secretory Phospholipase A2 Concentrations before and after Laparoscopic Gastric Banding in Morbidly Obese Patients

The aim of this study was to examine the relationship between subclinical inflammation and weight loss by laparoscopic adjustable gastric banding (LAGB). Plasma concentrations of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), sensitive C-Reactive Protein (sCRP), asymmetrical dimethyl-L-arginine (ADMA), Secretory Phospholipase A2 (sPLA2), and metabolic markers, such as homeostatic model assessment insulin resistance (HOMA-IR) and body mass index (BMI) were determined in morbidly obese patients (n=18, BMI 48.6 +/- 1.7 kg/m2) at baseline and 1 month after operations. Baseline levels in patients were also compared with age-matched controls (n=20, BMI 21.3 +/- 1.8 kg/m2). Plasma ICAM-1, VCAM, sCRP and ADMA, and sPLA2 concentrations were determined by enzyme-linked immunoassay methods and colorimetric method, respectively. Plasma sCRP, ICAM-1, ADMA and sPLA2 concentrations and HOMA-IR were significantly higher in morbidly obese patients than in controls (for each, P<0.01). Plasma VCAM-1 concentration was not changed in obese patients. HOMA-IR was significantly correlated with ICAM-1, ADMA and sPLA2 in the obese group at baseline (for each, P<0.01). There was a significant correlation between plasma sCRP and plasma glucose, VCAM-1, ICAM-1, ADMA and sPLA2 concentrations (for each, P<0.01). 1 month after LAGB, mean body weight loss was 13.2 +/- 6.3 kg, and plasma sCRP and ADMA concentrations and HOMA-IR and BMI were significantly decreased (for each, P<0.01). However, these levels cannot be decreased to the levels of the controls. Obesity and insulin resistance appear to be associated with low-grade inflammation and endothelial dysfunction. Insulin resistance and endothelial dysfunction were improved by weight loss after LAGB.

Nontraditional atherosclerotic risk factors and extent of coronary atherosclerosis in patients with combined impaired fasting glucose and impaired glucose tolerance

Partially inconsistent data exist on mutual relations between nontraditional atherosclerotic risk factors, including the magnitude of insulin resistance (IR), as well as on their relevance for atherogenesis in the metabolic syndrome. Subjects exhibiting combined impaired fasting glucose and impaired glucose tolerance (IFG/IGT) are exposed to an exceptionally high risk for atherogenesis and development of type 2 diabetes mellitus. Because of islet Β-cell dysfunction, the usefulness of commonly used indices of IR is limited in IFG/IGT. Our aim was to assess the relationship between extent of angiographic coronary artery disease (CAD) and nontraditional atherosclerotic risk factors (including IR by a clamp-based golden standard method) in IFG/IGT. Fifty-three subjects (32 men, 21 women; mean age, 55 ± 11 years) with stable angina, preserved left ventricular systolic function, and IFG/IGT were divided into 3 groups: group A (no coronary stenoses >50%, n = 22), group B (1-vessel CAD, n = 15), and group C (2/3-vessel CAD, n = 16). Insulin sensitivity was quantified by a hyperinsulinemic euglycemic clamp technique and expressed as M. M value, plasma homocysteine (Hcy) level, and asymmetric dimethyl- l-arginine (ADMA)/ l-arginine ratio were independent determinants of CAD extent as shown by forward stepwise discriminant function analysis. Compared with group A ( M = 32.7 ± 9.3 μmol/kg fat-free mass [FFM] per minute; Hcy, 8.1 ± 1.4 μmol/L), lower M and higher Hcy levels were found in group B ( M = 16.9 ± 8.2 μmol/kg FFM per minute, P < .001; Hcy, 11.2 ± 2.9 μmol/L, P = .003) and C ( M = 16.4 ± 7.8 μmol/kg FFM per minute, P < .001; Hcy, 12.8 ± 3.9 μmol/L, P < .001). The ADMA/ l-arginine ratio was increased in group C (0.0078 ± 0.0011) compared with group A (0.0063 ± 0.0013, P = .03) and B (0.0058 ± 0.0012, P = .01). Multivariate correlates ( P < .05) of plasma Hcy concentrations were M ( β = −.34 ± .12, P = .008), creatinine clearance ( β = −.23 ± .10, P = .03) and fasting insulin ( β = .25 ± .12, P = .04). This indicates an additive contribution of IR, plasma Hcy, and elevated ADMA/ l-arginine ratio to the extent of angiographic CAD in combined IFG/IGT.

Roads to Dysfunction

See related article, pages 951–960 Oxidized low-density lipoprotein (oxLDL) is considered to be the strongest proatherogenic lipoprotein. OxLDL is rapidly taken up by endothelial cells and macrophages and via the steps of foam cell formation and cell death, oxLDL eventually accumulates in the lipid core of atherosclerotic plaques.1 The interaction of oxLDL with cells is mediated by several receptors.2 Members of the scavenger receptor family predominate in macrophages,3 whereas endothelial cells mainly take up oxLDL by the lectin-like ox-LDL receptor-1 (LOX-1).4 On binding to LOX-1, oxLDL activates a multitude of signaling cascades involving MAP kinases,5,6 protein kinase C (PKC),7,8 and protein kinase B9 in endothelial cells. The consequence of this cellular activation, is apoptosis and superoxide anion production, the latter originating from the NADPH oxidase and uncoupling of the endothelial NO synthase (eNOS). OxLDL therefore is a strong inducer of endothelial dysfunction, a state in which the endothelium, instead of generating NO, produces superoxide anions. OxLDL also modulates the specific activity of NOS. It increases caveolin I expression and the interaction of NOS with this protein impairs NO production.10 Furthermore, oxLDL modulates PKC activity and expression and this family of kinases has been shown to acutely attenuate eNOS-dependent NO production and to promote eNOS uncoupling during prolonged exposure times.11 Several observations suggest that the supply of eNOS with the substrate l-arginine is impaired in the presence of oxLDL: In hypercholesterolemia, supplementation of l-arginine improves endothelium-dependent relaxation and NO production;12,13 l-arginine also attenuates the development of atherosclerosis,14 and normalizes leukocyte adhesion …

High Glucose-Mediated Imbalance of Nitric Oxide Synthase and Dimethylarginine Dimethylaminohydrolase Expression in Endothelial Cells

The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucose-mediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of D-glucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.

Asymmetric dimethyl-L-arginine (ADMA): a possible cause of endothelial dysfunction in the bile-duct excised cirrhotic rat

Asymmetric dimethyl-L-arginine (ADMA): a possible cause of endothelial dysfunction in the bile-duct excised cirrhotic rat

In vitro and in vivo effects of probucol on hydrolysis of asymmetric dimethyl l-arginine and vasospasm in primates

Increased cerebrospinal fluid (CSF) levels of asymmetric dimethyl L-arginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), are associated with delayed vasospasm after subarachnoid hemorrhage (SAH); however, the source, cellular mechanisms, and pharmacological inhibition of ADMA production following SAH are unknown. In an in vitro experiment involving human umbilical vein endothelial cells (HUVECs), the authors examined mechanisms potentially responsible for increased ADMA levels during vasospasm and investigated whether this increase can be inhibited pharmacologically. In a second study, an in vivo experiment, the authors used probucol, which effectively inhibited ADMA increase in HUVEC cultures in vitro, in a randomized double-blind placebo-controlled experiment in a primate model of delayed cerebral vasospasm after SAH. Oxidized low-density lipids (OxLDLs; positive control; p < 0.02) and bilirubin oxidation products (BOXes; p < 0.01), but not oxyhemoglobin (p = 0.74), increased ADMA levels in HUVECs. Probucol inhibited changes in ADMA levels evoked by either OxLDLs (p < 0.001) or BOXes (p < 0.01). Comparable changes were observed in cell lysates. In vivo probucol (100 mg/kg by mouth daily) did not alter serum ADMA levels on Days 7, 14, and 21 after SAH compared with levels before SAH, and these levels were not different from those observed in the placebo group (p = 0.3). Despite achieving therapeutic levels in plasma and measurable levels in CSF, probucol neither prevented increased CSF ADMA levels nor the development of vasospasm after SAH. Increased CSF ADMA and decreased nitrite levels in both groups were strongly associated with the degree of delayed vasospasm after SAH (correlation coefficient [CC] 0.5, 95% confidence interval [CI] 0.19-0.72, p < 0.002 and CC -0.43, 95% CI -0.7 to -0.05, p < 0.03, respectively). Bilirubin oxidation products, but not oxyhemoglobin, increased ADMA levels in the HUVEC. Despite its in vitro ability to lower ADMA levels, probucol failed to inhibit increased CSF ADMA and decreased nitrite levels, and it did not prevent delayed vasospasm in a primate SAH model.

Asymmetrical Dimethylarginine Plasma Concentrations Are Related to Basal Nitric Oxide Release but Not Endothelium-Dependent Vasodilation of Resistance Arteries in Peritoneal Dialysis Patients

Vascular dysfunction in chronic renal failure may be linked to reduced nitric oxide (NO) bioactivity and increased circulating concentrations of the endogenous NO synthase inhibitor asymmetrical dimethyl L-arginine (ADMA). The association between ADMA and basal endothelial NO release and endothelium-dependent vasodilation in resistance arteries of chronic renal failure patients is unknown. Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine, the endothelium-independent vasodilator nitroglycerine, and the endothelium-dependent vasoconstrictor N(G)-monomethyl-L-arginine (L-NMMA) were assessed in 37 peritoneal dialysis patients. L-arginine and ADMA plasma concentrations were measured by HPLC. ADMA (mean +/- SEM: 0.68 +/- 0.02 micromol/L) was associated with basal forearm blood flow (r = -0.33; P < 0.05) and L-NMMA induced vasoconstriction (r = -0.55; P < 0.0005), but not with dilator effects of acetylcholine or nitroglycerine. L-arginine (68 +/- 3 micromol/L) tended to correlate with acetylcholine-induced vasodilation (r = 0.32; P = 0.05) but was not associated with other parameters. ADMA is related to basal but not to acetylcholine-stimulated NO bioactivity in patients on peritoneal dialysis. Impaired endothelium-dependent vasodilation found in chronic renal failure is not explained by elevated circulating NO synthase inhibitors in renal failure.

Renal dysfunction as a novel risk factor: Microalbuminuria and cardiovascular risk

only for elevated glucose or BMI values; the correlation extends well into the range of normal values. Apparently, the metabolic syndrome and microalbuminuria are tightly correlated with each other. This relation is of considerable interest because in patients with renal disease insulin resistance is found even in the very early stages [10], and even when GFR is still within the normal range (unpublished observations). Conversely, in a population sample it was found that insulin resistance (according to the HOMA index) was associated with a higher risk of chronic kidney disease [11]. The information obtained from microalbuminuria cannot be replaced by estimating the glomerular filtration rate. Although the reports in the literature are not entirely consistent, the study in Groningen showed that with increasing quantiles of urinary albumin excretion, the creatinine clearance tended to be higher and was lower only in the uppermost quantiles [12]. Although the creatinine clearance is not a reliable index of glomerular filtration, this observation would be plausible in view of a similar relation between GFR and albuminuria noted previously in diabetic patients [13]. There has been much discussion whether microalbuminuria reflects primarily a generalized endothelial cell dysfunction, as suggested by the Steno hypothesis [14], or whether it reflects a podocyte defect, as proposed more recently [15]. The observation that the rate of albumin escape from the plasma space is increased in patients with microalbuminuria [16] argues for the former possibility. The observation that the risk of being microalbuminuric was significantly higher for carriers of the Q-genotype of the 229 polymorphism of the podocin gene at any given level of body mass index or other correlates of microalbuminuria is more in favor of a podocyte problem [15].

Weight Loss Reduces Circulating Asymmetrical Dimethylarginine Concentrations in Morbidly Obese Women

The endogenous nitric oxide-synthase inhibitor asymmetrical dimethyl-L-arginine (ADMA) is elevated in patients with increased risk for arteriosclerosis. Obesity is a risk factor for cardiovascular disease. We measured plasma ADMA concentrations in morbidly obese women before and after weight loss following gastroplastic surgery. ADMA and symmetrical dimethyl-L-arginine concentrations were analyzed by HPLC from 34 female patients (age 41 +/- 7 yr) with a body mass index (BMI) of 49 +/- 1 kg/m2 before and 14 months after vertical ring gastroplasty. Age-matched healthy women (BMI < 25 kg/m2; n = 24) were studied as controls. After gastroplastic surgery, BMI decreased to 34 +/- 1 kg/m2 in obese women (P < 0.00001), and ADMA concentrations were reduced from 1.06 +/- 0.06 micromol/liter at baseline to 0.81 +/- 0.04 micromol/liter after weight loss (P < 0.00001). Symmetrical dimethyl-L-arginine plasma levels were not affected. ADMA correlated with high-sensitivity C-reactive protein at baseline (r = 0.42; P < 0.05) and after weight loss (r = 0.56; P < 0.005). No association with blood pressure or plasma lipids could be observed. ADMA concentrations were lower in controls (0.68 +/- 0.04 micromol/liter; P < 0.05) compared with obese patients before or after weight reduction. The decrease of highly elevated ADMA concentrations in morbidly obese patients is paralleled by improvement of parameters associated with the metabolic syndrome after weight loss.

Association between cerebrospinal fluid levels of asymmetric dimethyl-L-arginine, an endogenous inhibitor of endothelial nitric oxide synthase, and cerebral vasospasm in a primate model of subarachnoid hemorrhage

Decreased availability of nitric oxide (NO) has been proposed to evoke delayed cerebral vasospasm after sub-arachnoid hemorrhage (SAH). Asymmetric dimethyl-L-arginine (ADMA) inhibits endothelial NO synthase (eNOS) and, therefore, may be responsible for decreased NO availability in cases of cerebral vasospasm. The goal of this study was to determine whether ADMA levels are associated with cerebral vasospasm in a primate model of SAH. Twenty-two cynomolgus monkeys (six control animals and 16 with SAH) were used in this study. The levels of ADMA, L-arginine, L-citrulline, nitrites, and nitrates in cerebrospinal fluid (CSF) and serum were determined on Days 0, 7, 14, and 21 following onset of SAH. Cerebral arteriography was performed to assess the degree of vasospasm. Western blot analyses of the right and left middle cerebral arteries (MCAs) were performed to assess the expression of eNOS, type I protein-arginine methyl transferase (PRMT1) and dimethylarginine dimethylaminohydrolase (DDAH2). Cerebrospinal fluid levels of ADMA remained unchanged in the control group (six animals) and in animals with SAH that did not have vasospasm (five animals; p = 0.17), but the levels increased in animals with vasospasm (11 animals) on Day 7 post-SAH (p < 0.01) and decreased on Days 14 through 21 (p < 0.05). Cerebrospinal fluid levels of ADMA correlated directly with the degree of vasospasm (correlation coefficient = 0.7, p = 0.0001; 95% confidence interval: 0.43-0.83). Levels of nitrite and nitrate as well as those of L-citrulline in CSF were decreased in animals with vasospasm. Furthermore, DDAH2 expression was attenuated in the right spastic MCA on Day 7 post-SAH, whereas eNOS and PRMT1 expression remained unchanged. Changes in the CSF levels of ADMA are associated with the development and resolution of vasospasm found on arteriograms after SAH. The results indicate that endogenous inhibition of eNOS by ADMA may be involved in the development of delayed cerebral vasospasm. Inhibition of ADMA production may provide a new therapeutic approach for cerebral vasospasm after SAH.

Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension

Background Essential hypertension is associated with enhanced LDL oxidation and impaired endothelium-dependent vasodilation. The antioxidant status is linked to the nitric oxide (NO) pathway. Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors inhibit oxidative stress and atherogenesis in experimental models; therefore we tested whether this beneficial antioxidant activity could be also clinically relevant in patients with essential hypertension. Methods Plasma LDL oxidizability was investigated initially in untreated normocholesterolemic patients with moderate essential hypertension without clinically evident target organ damage (n = 96) and in control normotensive subjects (n = 46). Patients were then randomly assigned into two age- and sex-matched groups to receive the new sulfhydryl ACE inhibitor zofenopril (15 to 30 mg/d; n = 48) or enalapril (20 mg/d, n = 48). LDL oxidizability was evaluated (generation of malondialdehyde, MDA) and systemic oxidative stress was evaluated by isoprostanes (8-isoPGF 2α). Asymmetrical dimethyl- l-arginine (ADMA), a competitive inhibitor of endothelial NO synthase, and plasma nitrite and nitrates (NOx) were also measured. Results LDL from hypertensive subjects had enhanced susceptibility to oxidation in vitro compared with that in control subjects ( P < .05). Similarly, isoprostanes were significantly increased ( P < .01) in hypertensive subjects versus control subjects. After 12-week treatment, MDA levels were significantly reduced by zofenopril ( P < .05) but not enalapril treatment ( P = not significant). Isoprostanes were normalized after zofenopril treatment ( P < .03), whereas enalapril was ineffective. After treatment with both ACE inhibitors, plasma NOx concentrations were significantly reduced ( P < .05). Similarly, hypertension increased ADMA concentration compared with the normotensive state, whereas ACE inihibition elicited a significant decrease. However, the reduction of ADMA concentration was significantly higher in patients receiving sulfhydryl ACE inhibition ( P < .05 vs enalapril). Conclusions The sulfhydryl ACE inhibitor zofenopril reduces oxidative stress and improves the NO pathway in patients with essential hypertension. If confirmed in a large multicenter clinical trial, our data suggest a possible vasculoprotective effect of the compound in retarding vascular dysfunction and atherogenesis that often develops rapidly in hypertensive patients.

Plasma concentrations of nitric oxide and asymmetric dimethylarginine in human alcoholic cirrhosis

Background/Aims The liver plays a prominent role in the metabolism of asymmetric dimethyl- l-arginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase. This study was designed to determine whether plasma levels of ADMA and NO production are altered in patients with compensated and decompensated alcoholic cirrhosis. Methods Plasma levels of l-arginine, ADMA, symmetric dimethylarginine (SDMA) and NO (nitrite plus nitrate, NOx) were measured in nine patients with compensated alcoholic cirrhosis (Child-Pugh A) and 11 patients with advanced cirrhosis (Child-Pugh B-C). Seven healthy volunteers served as controls. Results ADMA and NOx concentrations in decompensated cirrhosis were higher than in the compensated group and control group (ADMA: 1.12±0.08 vs. 0.58±0.05 and 0.58±0.07 μmol/l, respectively; P<0.05; NOx 97.90±10.27 vs. 37.42±3.91 and 40.43±5.30 μmol/l, respectively; P<0.05). There was a positive correlation between the clinical score of the patients and concentrations of ADMA ( r 2=0.547, P<0.01) and NOx ( r 2=0.689, P<0.01). SDMA and l-arginine levels were not significantly different between the three groups. Conclusions The results suggest that hepatocellular damage is a main determinant of elevated ADMA concentration in advanced alcoholic cirrhosis. By inhibiting NO release from vascular endothelium, ADMA might oppose the peripheral vasodilation caused by excessive NO production in severe cirrhosis.

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism—an electron paramagnetic resonance investigation

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, l-arginine and S-nitroso- N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N G-nitro- l-arginine-methyl-ester ( l-NAME) and asymmetric dimethyl- l-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.

Circulating concentrations of asymmetrical dimethyl-L-arginine are increased in women with previous gestational diabetes.

The concentration of asymmetrical dimethyl- L-arginine (ADMA), an endogenous inhibitor of the nitric oxide synthase, is increased in patients at risk or with cardiovascular disease. We have investigated ADMA concentrations in women with a history of gestational diabetes (GDM), who could develop endothelial dysfunction and Type II (non-insulin-dependent) diabetes mellitus after delivery, and in healthy control subjects. Previous GDM patients were grouped according to their BMI as obese (> or =25 kg/m(2), n=46) or non-adipose (<25 kg/m(2), n=31). Serum samples were taken 14 to 16 weeks after delivery and after 1 year. The control group comprised 17 healthy women (BMI<25 kg/m(2)). ADMA concentrations were analysed by high performance liquid chromatography. ADMA concentrations were comparable between obese and non-adipose GDM patients (0.58+/-0.02 and 0.57+/-0.02 micro mol/l, respectively), and higher than in the control group (0.47+/-0.03 micro mol/l; p<0.006). Insulin resistance as estimated by the insulin sensitivity index was more frequent among the obese than the non-adipose GDM women (p<0.05) and control subjects (p<0.05, both). No change in ADMA concentrations was found after 1 year in women with GDM. There was only a slight correlation between ADMA and BMI (r=0.26, p<0.02), triglycerides (r=0.29, p<0.004), or fasting plasma glucose (r=0.21, p<0.05), and not with the insulin sensitivity index or other parameters. In a multiple regression analysis ADMA serum concentrations were only associated with triglycerides. Circulating ADMA concentrations are increased in normoglycaemic women with previous GDM. This increase is independent from other risk factors or surrogate markers for diabetes or cardiovascular events.

Blunted acetylcholine relaxation and nitric oxide release in arteries from renal hypertensive rats.

Investigation of the effect of hypertension on endothelium-dependent relaxation and release of nitric oxide (NO) in normotensive and renal hypertensive rats. Sprague-Dawley rats were randomly allocated into two groups: uninephrectomized controls and one-kidney one-clip (Goldblatt hypertension) hypertensive rats, a non-renin dependent model of hypertension. After 10 weeks and in the presence of the cyclooxygenase inhibitor indomethacin, simultaneous measurements of the NO concentration, measured with a NO-specific microelectrode and endothelium-dependent relaxation were performed in isolated rat superior mesenteric arteries. Addition of the NO scavenger, oxyhaemoglobin, showed that basal NO concentration was unaltered in arterial segments from hypertensive rats. In norepinephrine-contracted arteries, acetylcholine increased the NO concentration and caused relaxations, and both parameters were significantly reduced in renal hypertensive arteries. Relaxations induced by the NO donor, S-nitroso-N-acetylpenicillamine were reduced. The superoxide scavenger, superoxide dismutase, and the NO synthase substrate, l-arginine, did not change the increase in NO concentration or acetylcholine relaxation in arteries from normotensive or renal hypertensive animals. In contrast, the NO synthase inhibitor, asymmetric dimethyl l-arginine, reduced the NO concentration and acetylcholine relaxation, while these responses were abolished in the presence of oxyhaemoglobin. This study provides direct evidence that reduced endothelium-dependent relaxations in the superior mesenteric artery from renal hypertensive rats is due, at least in part, to diminished NO release. The reduced NO release and relaxation persist in the presence of excess of substrate for NO synthase.

Synergistic role of progesterone and nitric oxide in the regulation of membrane fluidity of erythrocytes in humans: an electron paramagnetic resonance investigation

Background It has been shown that progesterone may actively participate in the regulation of blood pressure and other cardiovascular regulations. However, the precise mechanism underlying its effects is unclear. Methods In the present study, we examined the effects of progesterone on membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method. Results In an in vitro study, progesterone significantly decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes. The finding indicates that progesterone might increase the membrane fluidity and improve the membrane microviscosity of erythrocytes. The effect of progesterone was significantly potentiated by the nitric oxide (NO) donor, S-nitroso- N-acetylpenicillamine (SNAP) and a cyclic guanosine monophosphate (cGMP) analogue, 8-bromo-cGMP. In contrast, the change in the membrane fluidity evoked by progesterone was attenuated in the presence of the NO synthase inhibitors, N G-nitro- l-arginine-methyl-ester (L-NAME) and asymmetric dimethyl- l-arginine (ADMA). Conclusions The results of the present study showed that progesterone increased the membrane fluidity of erythrocytes and ameliorated the rigidity of cell membranes, at least in part, by an NO-dependent mechanism. Furthermore, the data strongly suggest that progesterone might be involved in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in humans.

Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure

Reduced activity of the nitric oxide (NO) pathway has been implicated in the endothelial dysfunction that occurs in patients with renal failure. NO is generated from L-arginine by NO synthase, and certain uremic toxins including asymmetrical dimethyl-L-arginine (ADMA), inhibit NO synthase and might contribute to endothelial dysfunction. We hypothesized that exogenous L-arginine might improve endothelial function in patients with renal failure by overcoming the effects of uremic toxins. Endothelial function of the forearm resistance vasculature was assessed using plethysmography to measure the dilator response to intra-arterial acetylcholine (25 to 100 nmol/min). Endothelial function of radial and brachial arteries was assessed using vascular ultrasound to measure the dilator response to flow during reactive hyperemia (flow-mediated dilation; FMD). Studies were performed before and after administration of L-arginine by intra-arterial infusion (50 micromol/min) in 8 pre-dialysis patients or by intravenous infusion (10 g) in 18 hemodialysis patients. Local L-arginine did not improve the dilator response of forearm resistance vessels (AUC 23.1 +/- 6.4 pre, 23.1 +/- 5.1 post; P = 0.9) or FMD of the radial artery (6.5 +/- 1.2% pre, 6.3 +/- 0.8% post; P = 0.8). Systemic L-arginine did not improve FMD of the brachial artery (4.1 +/- 1.1% pre, 3.0 +/- 1.1% post; P = 0.07). These data demonstrate that acute local or systemic administration of L-arginine did not improve endothelial function in resistance or conduit arteries of patients with chronic renal failure. The results suggest that competitive inhibition of nitric oxide synthase (NOS) by circulating inhibitors is not the principal explanation for impaired endothelial dilator function in chronic renal failure.

Electron paramagnetic resonance investigation on modulatory effect of 17beta-estradiol on membrane fluidity of erythrocytes in postmenopausal women.

Many studies have shown that estrogen may exert cardioprotective effects and reduce the risk of hypertension and coronary events. On the other hand, it has been proposed that cell membrane abnormalities play a role in the pathophysiology of hypertension, although it is not clear whether estrogen would influence membrane function in essential hypertension. The present study was performed to investigate the effects of 17beta-estradiol (E(2)) on membrane fluidity of erythrocytes in normotensive and hypertensive postmenopausal women. We determined the membrane fluidity of erythrocytes by means of an electron paramagnetic resonance and spin-labeling method. In an in vitro study, E(2) significantly decreased the order parameter for 5-nitroxide stearate and the peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive postmenopausal women. The finding indicates that E(2) might increase the membrane fluidity of erythrocytes. The effect of E(2) was significantly potentiated by the NO donor, S-nitroso-N-acetylpenicillamine, and a cGMP analogue, 8-bromo-cGMP. In contrast, the change in the membrane fluidity evoked by E(2) was attenuated in the presence of the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and asymmetric dimethyl-L-arginine. In hypertensive postmenopausal women, the membrane fluidity of erythrocytes was significantly lower than that in normotensive postmenopausal women. The effect of E(2) on membrane fluidity was significantly more pronounced in the erythrocytes of hypertensive postmenopausal women than in the erythrocytes of normotensive postmenopausal women. The results of the present study showed that E(2) significantly increased the membrane fluidity and improved the microviscosity of erythrocyte membranes, partially mediated by an NO- and cGMP-dependent pathway. Furthermore, the greater action of E(2) in hypertension might be consistent with the hypothesis that E(2) could have a beneficial effect in regulating rheological behavior of erythrocytes and could have a crucial role in the improvement of the microcirculation in hypertension.

Plasma levels of asymmetrical dimethyl-L-arginine in patients with congenital heart disease and pulmonary hypertension.

Asymmetrical dimethyl-L-arginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. We hypothesized that plasma levels of ADMA could be increased in patients with congenital heart disease and pulmonary hypertension. Cardiac catheterization was performed in 20 children and young adults with congenital heart disease with a median age of 10 years (range, 4 months to 33 years). The patients were assigned to group I (high flow, low pressure; n = 14) when Qp/Qs was 1.5 or greater and the mean PAP was less than 25 mm Hg or to group II (high pressure, high resistance; n = 6) when the mean PAP was greater than 25 mm Hg and Rp/Rs was greater than 0.3. Blood samples were taken from pulmonary vein or left ventricle. ADMA was measured by high-performance liquid chromatography. In addition, levels of ADMA were measured in peripheral venous blood obtained from eight control patients. Levels of ADMA in control patients (median, 0.21 microM/l; range, 0.08-0.27 microM/l) did not differ from levels obtained in group I (median, 0.30 microM/l; range, 0.06-0.49) microM/l). Patients in group II showed increased plasma levels of ADMA (median, 0.55; range, 0.25-0.79) (p < 0.01). Inhibition of nitric oxide synthase by increased levels of ADMA might contribute to pulmonary hypertension in patients with congenital heart disease.