Dimethylarginine Dimethylaminohydrolase Activity Research Articles

Asymmetric dimethylarginine in angiotensin II-induced hypertension

Recent studies have shown that asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, is increased in hypertension and chronic kidney disease. However, little is known about the effects of hypertension per se on ADMA metabolism. The purpose of this study was to test the hypothesis that ANG II-induced hypertension, in the absence of renal injury, is associated with increased oxidative stress and plasma and renal cortex ADMA levels in rats. Male Sprague-Dawley rats were treated with ANG II at 200 ng.kg(-1).min(-1) sc (by minipump) for 1 or 3 wk or at 400 ng.kg(-1).min(-1) for 6 wk. Mean arterial pressure was increased after 3 and 6 wk of ANG II; however, renal injury (proteinuria, glomerular sclerosis, and interstitial fibrosis) was only evident after 6 wk of treatment. Plasma thiobarbituric acid reactive substances concentration and renal cortex p22(phox) protein abundance were increased early (1 and 3 wk), but urinary excretion of isoprostane and H(2)O(2) was only increased after 6 wk of ANG II. An increased in plasma ADMA after 6 wk of ANG II was associated with increased lung protein arginine methyltransferase-1 abundance and decreased renal cortex dimethylarginine dimethylaminohydrolase activity. No changes in renal cortex ADMA were observed. ANG II hypertension in the absence of renal injury is not associated with increased ADMA; however, when the severity and duration of the treatment were increased, plasma ADMA increased. These data suggest that elevated blood pressure alone, for up to 3 wk, in the absence of renal injury does not play an important role in the regulation of ADMA. However, the presence of renal injury and sustained hypertension for 6 wk increases ADMA levels and contributes to nitric oxide deficiency and cardiovascular disease.

Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: Role of asymmetric dimethylarginine

Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to l-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NO x ) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.

Regulation of the ADMA-DDAH system in endothelial cells: a novel mechanism for the sterol response element binding proteins, SREBP1c and -2

Asymmetric dimethylarginine (ADMA) has been implicated in the progression of cardiovascular disease as an endogenous inhibitor of nitric oxide synthase. The regulation of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme responsible for metabolizing ADMA, is poorly understood. The transcription factor sterol response element binding protein (SREBP) is activated by statins via a reduction of membrane cholesterol content. Because the promoters of both DDAH1 and DDAH2 isoforms contain sterol response elements, we tested the hypothesis that simvastatin regulates DDAH1 and DDAH2 transcription via SREBP. In cultured endothelial cells, simvastatin increased DDAH1 mRNA expression compared with vehicle. In an ADMA loading experiment, simvastatin treatment resulted in a decrease in ADMA content, an indication of increased DDAH activity. The knockdown of SREBP1c protein led to an increase in DDAH1 mRNA expression and activity, whereas the knockdown of SREBP2 led to a decrease in DDAH1 mRNA expression. The role of SREBP2 in the activation of the DDAH1 was supported by chromatin immunoprecipitation studies demonstrating increased binding of SREBP2 to the DDAH1 promoter upon simvastatin stimulation. These data indicate that SREBP1c might act as a repressor and SREBP2 as an activator of DDAH transcription and activity. This study describes a novel mechanism of reciprocal regulation by the SREBP family members of the DDAH-ADMA system, which represents a potential link between cellular cholesterol content and endothelial dysfunction observed in cardiovascular disease.

Red wine decreases asymmetric dimethylarginine via SIRT1 induction in human endothelial cells

To investigate the effect of three red wines (RWs) from different growing areas and made from different grapes on asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, in young and senescent human endothelial cells (ECs). All RWs decreased ADMA levels, but 2-fold concentration of German RW was necessary to reach the same effect on ADMA compared to Italian RW and French RW without affecting the cell viability and morphology. The ADMA-lowering effect of RW was increased in senescent compared to young cells, accompanied by enhanced activity of the metabolizing enzyme: dimethylarginine dimethylaminohydrolase (DDAH) II, whereas the same amount in the upregulated protein expression of DDAH II and the downregulated protein expression of the synthesizing enzyme: protein arginine methyltransferase 1 was revealed. These effects were associated with decreased 8-iso-prostaglandin F 2α and peroxynitrite formation, enhanced protein expression of NAD +-dependent class III histone deacetylase sirtuin (SIRT) 1, and downregulated protein expression of histone senescence factor p53. Blockade of SIRT1 activity abolished the effect of red wine on ADMA. These data are the first demonstration that RW by activating SIRT1 impairs synthesis and increases metabolism of ADMA. This effect of RW is accentuated in senescent cells probably due to enhanced DDAH activity.

Dimethylarginine Dimethylaminohydrolase-1 Transgenic Mice Are Not Protected from Ischemic Stroke

BackgroundMethylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).Methodology/Principal FindingsWe investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.Conclusion/SignificanceOur study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.

Ex vivo gene transferring of human dimethylarginine dimethylaminohydrolase-2 improved endothelial dysfunction in diabetic rat aortas and high glucose-treated endothelial cells

ObjectivesElevated level of asymmetric dimethylarginine (ADMA) is an independent risk factor for endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme responsible for the degradation of endogenous ADMA. The purposes of this study were to determine whether suppressed DDAH2 expression would implicate in endothelial dysfunction associated with diabetes mellitus and further to investigate whether adenovirus-mediated DDAH2 gene overexpression could improve the hyperglycemia-induced endothelial dysfunction. MethodsDiabetic model was induced by intraperitoneal injection of streptozotocin to male Sprague–Dawley rats. Recombinant adenoviral vector encoding human DDAH2 gene driven by a cytomegalovirus promoter was constructed to overexpress hDDAH2 gene in isolated rat aortas and endothelial cells. Changes in DDHA/ADMA/nitric oxide (NO) pathway in diabetic rats and high glucose-treated endothelial cells were examined. ResultsDDAH2 expression was distinctly suppressed, which was accompanied by inhibited DDAH activity and impaired endothelium-dependent relaxation in aortas, and elevated ADMA concentrations in serum of diabetic rats compared to control rats. Suppressions of DDAH2 expression and DDAH activity, accumulation of ADMA, and inhibition of NO synthesis were observed in high glucose-treated endothelial cells. DDAH2 overexpression not only improved endothelial dysfunction in diabetic aortas but also attenuated hyperglycemia-induced changes in DDAH/ADMA//NO pathway in endothelial cells. ConclusionThese results indicate that suppression of DDAH2 expression contributes to hyperglycemia-induced endothelial dysfunction, which can be improved by DDAH2 overexpression. This study suggests that targeted modulation of DDAH2 gene in vascular endothelium may be a novel approach for the treatment of endothelial dysfunction in diabetes mellitus.

Effect of 3,4,5,6-tetrahydroxyxanthone on erythrocyte deformability in apolipoprotein E-deficient mice

Previous investigations have indicated that reduced erythrocyte deformability may be an important factor contributing to the development of atherosclerosis, and endogenous asymmetric dimethylarginine (ADMA) might be an important contributor to reduction of erythrocyte deformability in atherosclerosis. In this study, the effect of 3,4,5,6-tetrahydroxyxanthone (1), a kind of polyphenolic compound, on erythrocyte deformability in apolipoprotein E-deficient (apoE − / − ) mice was evaluated. After treatment with compound 1 (10 or 20 mg/kg per day) for 4 weeks, erythrocyte deformability, antioxidant enzymes activity, erythrocyte dimethylarginine dimethylaminohydrolase (DDAH) activity, the plasma level of ADMA and malondialdehyde (MDA) level were determined. Treatment with compound 1 (10 or 20 mg/kg) increased erythrocyte deformability, antioxidant enzymes activity concomitantly, a decrease in the plasma levels of MDA and ADMA, and an increase in erythrocyte DDAH activity. The present result suggests that the beneficial effect of 1 on the erythrocyte deformability, besides inhibiting lipid peroxidation, may be related to reduction of ADMA concentration via an increase in DDAH activity.

Regulation of myometrial contractivity during pregnancy in the rat: potential role for DDAH

There has been little information demonstrating the roles of dimethylarginine dimethylaminohydrolase (DDAH), which is the hydrolyzing enzyme of endogenous nitric oxide synthase (NOS) inhibitors and, in turn, modulates the intracellular concentrations of NOS inhibitors, in the myometrium during the course of pregnancy. Therefore, the present experiments were designed to investigate whether or not DDAH activity, protein and mRNA expression levels are altered during gestation of the rat and, if altered, those changes reflect on the levels of endogenous inhibitors and endothelin-1 (ET-1), and NO-dependent cyclic GMP generation in the myometrium. The up-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression at mid-gestation were accompanied by the reduced monomethylarginine and asymmetric dimethylarginine as NOS inhibitors, and ET-1 levels, and by the enhanced NO-dependent cyclic GMP production. At term gestation, on the other hand, down-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression were accompanied by the increased NOS inhibitors and ET-1 levels, and decreased NO-dependent cyclic GMP generation. These results suggest that alterations in DDAH/NOS inhibitors/NO-dependent cyclic GMP/ET-1 pathway are possibly involved in maintaining myometrial quiescence during gestation and controlling delivery at term.

Hepatoprotective potential of crocin and curcumin against iron overload-induced biochemical alterations in rat.

The present study was undertaken to evaluate the possible ameliorating effect of crocin and curcumin on certain biochemical alterations associated with iron overload-induced liver injury in rats. 5 groups of rats were used, a normal control group received daily i.p. injections of saline and 4 groups received daily i.p. injections of ferric nitrilotriacetate (FeNTA) for 8 successive days, the dose of iron was increased during the experimental period (from 6 to 15 mg Fe/kg). The first iron overloaded group kept without further treatment and served as a positive control group. The second iron overloaded group received daily i.p injections of crocin (200 mg/kg ) in saline. The 3rd and the 4th iron overloaded groups received orally either 0.5% carboxy methyl cellulose (CMC) or curcumin (100 mg/kg) in CMC respectively. Treatment started 3 days before and concurrently with iron administration for 8 days. Results revealed that iron-induced liver injury was reflected by significant changes in the liver function indices, hyperammonemia and reduced serum urea level. A significant deposition of iron in liver was associated with enhanced oxidative and nitrosative stress status. Moreover, iron overloaded rats exhibited significant alterations in liver energy metabolism together withdiminished ureogenesis and a decline in dimethylarginine dimethylaminohydrolaseactivity. Supplementation with either crocin or curcumin ameliorated most of the biochemical changes induced by iron overload in rat liver. A function that may be beneficial for populations at risk for iron overload. Key words: Iron overload, liver, rat, oxidative stress, energy metabolism, ureogenesis, crocin, curcumin.

Dimethylarginine dimethylaminohydrolase regulation: a novel therapeutic target in cardiovascular disease

Asymmetric dimethylarginine (ADMA), an endogenous methylated form of the amino acid l-arginine, inhibits the activity of the enzyme endothelial nitric oxide synthase, with consequent reduced synthesis of nitric oxide. ADMA is metabolised to l-citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The modulation of DDAH activity and expression plays a pivotal role in regulating intracellular ADMA concentrations, with important effects on vascular homeostasis. For example, impairment in DDAH activity, resulting in elevated ADMA concentrations and reduced nitric oxide synthesis, can promote the onset and progression of atherosclerosis in experimental models. This review discusses the current role of ADMA and DDAH in vascular health and disease, the techniques used to assess DDAH activity and expression, and the results of recent studies on pharmacological and biological agents modulating DDAH activity and expression. Suggestions for future basic and clinical research directions are also discussed.

All‐trans retinoic acid inhibits cobalt chloride‐induced apoptosis in PC12 cells: Role of the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine pathway

Previous studies have shown that the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its specific hydrolase dimethylarginine dimethylaminohydrolase (DDAH) are involved in the regulation of apoptosis in different cell types. In the present study, we investigated the role of the DDAH/ADMA pathway in cobalt chloride (CoCl(2))-induced apoptosis and the antiapoptotic effect of all-trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl(2) (125 microM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase-3 activity. CoCl(2) treatment also decreased the activity of DDAH and the expression of DDAH2 (mRNA and protein), resulting in an increased level of ADMA. All these alterations induced by CoCl(2) were attenuated by atRA (0.1, 1, or 10 microM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl(2). We also found that treatment of exogenous ADMA (3, 10, or 30 microM) induced the apoptosis of PC12 cells in a concentration- and time-dependent manner, which was inhibited by the antioxidant or the caspase-3 inhibitor. These findings suggest that the modulation of the DDAH/ADMA/ROS pathway plays an important role in CoCl(2)-induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells.

Asymmetric Dimethylarginine Impairs Glucose Utilization via ROS/TLR4 Pathway in Adipocytes: an Effect Prevented by Vitamin E

Background: Asymmetric dimethylarginine (ADMA), the inhibitor of nitric oxide synthase (NOS), has been reported to be associated with glucose metabolism, but its mechanisms remain unknown. Methods: In 3T3-L1 adipocytes, we measured the effects of ADMA on glucose transport process under basal or insulin-induced condition, and examined the production of nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α), and the expression of toll-like receptor 4 (TLR4). Results: ADMA significantly impaired basal or insulin-stimulated 2-deoxy- [³H] glucose uptake, and decreased the expression of insulin receptor substrate-1 (IRS-1) and glucose transporter-4 (GLUT4). Phosphorylated protein of IRS-1 and translocation of GLUT4 with insulin-stimulation were also inhibited by ADMA. NO decreased, while production of ROS and TNF-α, and expression of TLR4 increased after ADMA treatment. Vitamin E reduced the effects of ADMA on glucose transport system, and on NO, ROS and TLR4. Moreover, vitamin E decreased ADMA contents by up-regulating dimethylarginine dimethylaminohydrolase (DDAH) activity in adipocytes. Though L-arginine also increased NO level, but failed to reduce the effects of ADMA. Conclusion: ADMA significantly impairs both basal and insulin-stimulated glucose transport in adipocytes, which may relate to activation of the ROS/TLR4 pathway.

Involvement of asymmetric dimethylarginine (ADMA) in tubulointerstitial ischaemia in the early phase of diabetic nephropathy

Decreased peritubular capillary (PTC) flow due to impaired endothelial function elicits tubulointerstitial ischaemia, thereby enhancing renal damage in chronic kidney disease, including diabetic nephropathy. Since nitric oxide (NO) is a vasodilator and known to play an important role in the maintenance of PTC flow, it is conceivable that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, may cause tubulointerstitial ischaemia, thus being involved in the progression of diabetic nephropathy. In this study, we investigated whether overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that degrades ADMA, could improve tubulointerstitial ischaemia in streptozotocin (STZ)-induced diabetic rats. Recombinant adenovirus vector encoding DDAH-I (Adv-DDAH) or control vector expressing bacterial beta-galactosidase (Adv-LZ) was intravenously administrated to diabetic rats. Three days after the treatment, effects of DDAH overexpression on plasma or urinary levels of ADMA or NO metabolites (NOx), tubulointerstitial ischaemia and renal expression of transforming growth factor-beta (TGF-beta) were evaluated. Renal DDAH expression and activity were reduced in diabetic rats. Urinary levels of ADMA and TGF-beta were increased, while NOx levels were decreased in diabetic rats. Compared with control rats, pimonidazole-detected hypoxic areas were larger in the kidney of diabetic rats, although the number of capillaries in tubulointerstitial regions was not different between the two groups. In addition, renal expression levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and TGF-beta were also increased in diabetic rats. DDAH overexpression significantly inhibited the increase of ADMA and the decrease of NOx and subsequently decreased urinary albumin excretion levels and ameliorated tubulointerstitial hypoxia and HIF-1alpha as well as TGF-beta expression in diabetic rats. The present study demonstrated for the first time that the suppression of ADMA by DDAH overexpression could improve tubulointerstitial ischaemia and subsequent renal damage in experimental diabetic nephropathy. Substitution of DDAH protein or enhancement of its activity may become a novel therapeutic strategy for the treatment of early diabetic nephropathy.

Structure–activity relationship of novel and known inhibitors of human dimethylarginine dimethylaminohydrolase-1: Alkenyl-amidines as new leads

Recent studies demonstrated that inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity could be a new strategy to indirectly affect nitric oxide (NO) formation by elevating N ω-methylated l-arginine (NMMA, ADMA) levels. This approach is an alternate strategy for the treatment of diseases associated with increased NO-concentrations. To date, three classes of potent inhibitors are known: (1) pentafluorophenyl sulfonates (IC 50 = 16–58 μM, PaDDAH), which are also inhibitors for the arginine deiminase; (2) the most potent inhibitors are based on indolylthiobarbituric acid (IC 50 = 2–17 μM, PaDDAH), which were identified by virtual modelling; and (3) l-arginine analogs, whose best representative is N ω-(2-methoxyethyl)- l-arginine (IC 50 = 22 μM, rat DDAH). Based on these known structures, we aimed to develop inhibitors for the human DDAH-1 with improved potency and better relative selectivity for DDAH-1 over NOS. Particularly, the binding pocket of the guanidine-moiety was investigated by screening differently substituted guanidines, amidines and isothioureas in order to collect information on possible binding modes in the active site. All substances were tested in a plate-reader format and HPLC assay and several potent inhibitors were identified with K i-values varying from 2 to 36 μM, with N 5-(1-iminobut-3-enyl)- l-ornithine ( l-VNIO) being the most potent inhibitor of the human DDAH-1 so far described. Besides these potent inhibitors alternate substrates for hDDAH-1 were identified as well.

The l-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation: Biological and analytical ramifications

The l-arginine/nitric oxide ( l-Arg/NO) pathway is altered in liver and kidney diseases. However, the status of the l-Arg/NO pathway during and after orthotopic transplantation is insufficiently investigated and findings are uncertain because of analytical shortcomings. Also, most human studies have focused on individual members of the l-Arg/NO pathway such as nitrate or asymmetric dimethylarginine (ADMA). In the present article we report on a pilot study investigating extensively the status of the l-Arg/NO pathway before and during orthotopic liver transplantation (OLT). By using fully validated, highly sensitive and specific GC–MS and GC–MS/MS methods nitrite, nitrate, ADMA and its hydrolysis product dimethylamine (DMA), l-arginine and l-ornithine were measured in blood and urine. Our study gives strong evidence of the exceptional importance of hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity for the elimination of systemic ADMA. In end-stage liver disease the synthesis of NO and ADMA as well as the DDAH activity are elevated. However, increase in DDAH activity is insufficient to efficiently eliminate overproduced ADMA. The transplanted liver graft is capable of clearing ADMA in a rapid and sufficient manner. In contrast to studies from other groups, our study shows that in OLT as well as in living donor kidney transplantation, the second study reported here, reperfusion of the graft does not cause drastic alterations to the l-Arg/NO pathway with regard to NO synthesis. In the OLT study the concentration of circulating l-arginine fell temporally dramatically, while l-ornithine levels increased diametrically, most likely due to elevation of arginase activity. However, the relatively long-lasting decrease in plasmatic l-arginine in OLT seems not to have affected NO synthesis after reperfusion. Our OLT study suggests that liver reperfusion is associated with greatly elevated activity of proteolytic and hydrolytic enzymes including DDAH and arginase. Suppression of proteolytic and hydrolytic activity in transplantation could be a useful measure to improve outcome and remains to be investigated in further studies on larger patient collectives. The importance of analytical chemistry in this area of research is also discussed in this article.

Reduction of asymmetric dimethylarginine in the protective effects of rutaecarpine on gastric mucosal injury

Our recent study has shown that asymmetric dimethylarginine (ADMA) plays an important role in facilitating gastric mucosal injury by multiple factors. To explore whether the protection of rutaecarpine against gastric mucosal injury is related to reduction of ADMA content, a model of ethanol-induced gastric mucosal injury in rats was selected for this study. The ulcer index, the content of ADMA and NO, and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in gastric tissues were measured in vivo after pretreatment with rutaecarpine. The in vitro effect of rutaecarpine on the release of calcitonin gene-related peptide (CGRP) and NO from isolated gastric tissues was also determined. The results showed that ethanol significantly increased the ulcer index, decreased the DDAH activity and the NO level, and elevated the ADMA level, which was attenuated by pretreatment with rutaecarpine (0.6 mg/kg or 1.2 mg/kg). In the isolated gastric tissues, rutaecarpine significantly increased the release of both CGRP and NO; the release of NO, but not CGRP, was abolished in the presence of l-NAME (10(-4) mol/L). The present results suggest that rutaecarpine protects the gastric mucosa against injury induced by ethanol and that the gastroprotection of rutaecarpine is related to reduction of ADMA levels through stimulating the release of CGRP.

Nitric Oxide Upregulates Dimethylarginine Dimethylaminohydrolase-2 via Cyclic GMP Induction in Endothelial Cells

Dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme that metabolizes asymmetrical N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-monomethyl-L-arginine (MMA), which are competitive endogenous inhibitors of NO synthase. However, it remains unknown whether NO itself influences DDAH activity and/or ADMA/MMA contents to regulate NO generation via a biofeedback mechanism. The present study was designed to examine the effects of NO on intracellular ADMA and MMA contents and DDAH gene expression levels and enzymatic activities in cultured rat aortic endothelial cells. The NO donors SNAP and NOR3 did not influence DDAH-1 expression but increased DDAH-2 mRNA and protein levels in concentration-dependent manners. SNAP upregulated DDAH enzymatic activity and reduced the MMA and ADMA contents but did not affect the symmetrical N(G),N'(G)-dimethyl-L-arginine and L-arginine levels, thereby negating a mediatory role for system y(+) in ADMA/MMA downregulation. The cGMP agonists 8-bromo-cGMP and C-type natriuretic peptide also stimulated DDAH-2 gene and protein expression levels and DDAH activity and increased the amount of nitrite/nitrate released into the culture supernatants. SNAP-induced DDAH-2 gene expression and DDAH activity were significantly inhibited by a protein kinase G inhibitor, KT5823, and a soluble guanylate cyclase inhibitor, ODQ, suggesting a mediatory role for cGMP in NO-induced DDAH-2 expression. Suppression of DDAH-2 mRNA using small interfering RNA technology abrogated NO-induced DDAH-2 expression. These data demonstrate that NO acts on endothelial cells to induce DDAH-2 expression via a cGMP-mediated process to reduce ADMA/MMA. Thus, the DDAH-2-ADMA/MMA-endothelial NO synthase regulatory pathway and NO-induced cGMP constitute a positive feedback loop that ultimately serves to maintain NO levels in the endothelial environment.

Dual molecular regulations of nitric oxide at the peripheral and central levels in experimental African sleeping sickness

The two-stage trypanosomiasis exists in rats infected with Trypanosoma brucei brucei, the neurological phase appearing after 9 to 12 days of infection. NO was determined in rats before and after infection (15 and 21 days) using a specific voltammetric sensor in the central nervous system (CNS) and peripheral (blood) compartments. Opposite changes were observed in the two compartments: a decrease in the periphery, an increase in the CNS (13 days after infection). This duality was analyzed in both healthy and infected Wistar rats using a molecular approach of the metabolic pathways of L-arginine, as it is the substrate of NOS, arginase and protein synthesis. In the peripheral compartment of infected animals, the decrease in inducible iNOS activity is counterbalanced by the rise in arginase activity resulting in an overproduction of related aminoacids (arginine, proline, glutamine, glutamate). Contrarily in the CNS, iNOS activity increased while that of arginase did not vary (no variation in related aminoacid concentrations). Concomitantly, there was an increased activity of dimethylarginine dimethyl aminohydrolase (DDAH), a regulatory enzyme metabolizing asymmetric dimethylarginine (ADMA), an end product of proteolysis inhibiting iNOS activity. In conclusion, the duality of NO behavior in the CNS and periphery is backed by different molecular regulations. In the peripheral compartment of rats, mice and humans (a decrease in blood NO was also observed in patients), the reduced NO production reflects the strategy deployed by the trypanosome to impair the immune processes The increased brain NO certainly accounts for blood-brain barrier and CNS pathophysiological alterations. from Infectious diseases of the nervous system: pathogenesis and worldwide impact Paris, France. 10–13 September 2008

ADMA induces monocyte adhesion via activation of chemokine receptors in cultured THP-1 cells

Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is also an important inflammatory factor contributing to the development of atherosclerosis (AS). The present study was to test the effect of ADMA on angiotensin (Ang) II-induced monocytic adhesion. Human monocytoid cells (THP-1) or isolated peripheral blood monocyte cells (PBMCs) were incubated with Ang II (10−6M) or exogenous ADMA (30μM) for 4 or 24h in the absence or presence of losartan or antioxidant PDTC. In cultured THP-1 cells, Ang II (10−6M) for 24h elevated the level of ADMA in the medium, upregulated the protein expression of protein arginine methyltransferase (PRMT) and decreased the activity of dimethylarginine dimethylaminohydrolase (DDAH). Both of Ang II and ADMA increased monocytic adhesion to human umbilical vein endothelial cells (HUVECs), elevated the levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and tumor necrosis factor (TNF)-α and upregulated CCR2 and CXCR2 mRNA expression, concomitantly with increase in reactive oxygen species (ROS) generation and activation of nuclear factor (NF)-κB. Pretreatment with losartan (10μM) or PDTC (10μM) abolished the effects mediated by Ang II or ADMA. In isolated PBMCs from healthy individuals, ADMA upregulated the expression of CXCR2 mRNA, which was attenuated by losartan (10μM), however, ADMA had no effect on surface protein expression of CCR2. The present results suggest that ADMA may be involved in monocytic adhesion induced by Ang II via activation of chemokine receptors by ROS/NF-κB pathway.

Regulation of Dimethylarginine Dimethylaminohydrolase (DDAH) in Rat Preglomerular Vascular Smooth Muscle Cells (PGVSMCs) by Reactive Oxygen Species (ROS)

DDAH degrades asymmetric dimethylarginine (ADMA) which is an endogenous NOS inhibitor and risk factor for cardiovascular disorders. We tested the hypothesis that ROS regulate the transcription, translation or activity of DDAH. We created a stable rat PGVSMC line overexprissing p22phox (S‐p22phox) using a p22phox‐pcDNA4/HisMax construct (a generous gift from Kathy Griendling). Quantitative real time PCR and Western blotting confirmed a marked increase in p22phox mRNA and protein of 4.7±0.6 and 2.3±0.5 fold, (p<0.01), compared to wild type cells (WT). S‐p22phox cells had elevated superoxide and hydrogen peroxid generation (2.3±0.09 fold and 1.8±0.1 fold p<0.01). The mRNAs for DDAH ‐1 and ‐2 were increased by 2.8 ± 0.2 and 1.8 ± 0.1 fold in S‐p22phox cells p< 0.01). DDAH‐2 promoter activity, determined by transient transfection and luciferase assay, was 4.1 fold increased in S‐p22phox cells (p < 0.01). Interestingly, both DDAH‐1 and ‐2 proteins were significantly lower in S‐p22phox cells (0.9±0.2 and 0.9±0.1 fold; p<0.01), yet β‐actin was not different. DDAH activity measured by a colorimetric assay (Kidney Int. 72:886–9, 2007) was reduced by 62±2% in S‐p22phox(p<0.01). The results reveal for the first time an important effect but complex of p22phox – dependent ROS generation on DDAH. Thus, ROS upregulate the expression of DDAH‐1 and ‐2 genes via activating a promoter, at least for DDAH‐2, yet downregulate both DDAH‐1 and ‐2 proteins and their enzyme activity. This is a specific effect since ROS do not change the expression of cell structure proteins. These findings in PGVSMCs provide a novel regulatory pathway whereby ROS may modulate renal hemodynamics, and thereby BP and salt homeostasis.