A Novel Role of Irbesartan in Gastroprotection against Indomethacin-Induced Gastric Injury in Rats: Targeting DDAH/ADMA and EGFR/ERK Signaling.
ABSTRACT: The advent of angiotensin II type 1 receptor blockers (ARBs) as intriguing gastroprotective candidates and the superior pharmacokinetics and pharmacodynamics displayed by irbesartan compared to many other ARBs raised the interest to investigate its gastroprotective potential in a rat model of gastric injury. Irbesartan (50?mg/Kg) was orally administered to male Wistar rats once daily for 14 days; thereafter gastric injury was induced by indomethacin (60?mg/Kg, p.o). Irbesartan reduced gastric ulcer index, gastric acidity, and ameliorated indomethacin-induced gastric mucosal apoptotic and inflammatory aberrations, as demonstrated by hampering caspase-3, prostaglandin E2 and tumor necrosis factor-alpha levels and cyclooxygenase-2 mRNA expression. This ARB increased mucosal dimethylarginine dimethylaminohydrolase-1 (DDAH-1) gene expression and decreased elevated levels of matrix metalloproteinase-9, asymmetric dimethylarginine (ADMA), epidermal growth factor receptor (EGFR) mRNA and phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Histopathological evaluation corroborated biochemical findings. Overall efficacy of irbesartan was comparable to ranitidine, the widely used H2 receptor blocker. In conclusion, irbesartan exerts significant gastroprotection against indomethacin-induced mucosal damage via acid-inhibitory, anti-inflammatory, anti-apoptotic and extracellular matrix remodeling mechanisms that are probably mediated, at least partly, by down-regulating DDAH/ADMA and EGFR/ERK1/2 signaling.
Project description:BACKGROUND AND PURPOSE: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.
Project description:Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.
Project description:Asymmetric dimethylarginine (ADMA) has been shown to be an independent predictor of cardiovascular diseases. Dimethylarginine dimethylaminohydrolase 2 (DDAH 2) promotes the metabolism of ADMA and plays a key role in the regulation of acute inflammatory response. With the present study, we investigated the relationship between DDAH 2 polymorphisms and risk of coronary artery disease (CAD) and its association to plasma ADMA concentrations. We used the haplotype-tagging SNP approach to identify tag SNPs in DDAH 2. The SNPs were genotyped by PCR and sequenced in 385 CAD patients and 353 healthy controls. Plasma concentrations of ADMA were determined using enzyme-linked immunosorbent assay (ELISA). A promoter polymorphism -449C/G (rs805305) in DDAH 2 was identified. Compared with the ADMA concentrations in CC genotype (0.328?±?0.077??mol/l), ADMA concentrations in CG?+?GG genotype were significantly increased (0.517?±?0.090??mol/l, P?<?0.001). No significant associations between the -449C/G and risk of CAD were detected in the genetic models. The results of this study suggest that Genetic -499C/G polymorphism in DDAH 2 gene may affect the plasma ADMA concentrations in patients with CAD. However, it does not indicate a novel genetic risk marker for CAD.
Project description:Nitric oxide prevents hypertension yet enhances proximal tubule Na+ reabsorption. Nitric oxide synthase is inhibited by asymmetric dimethylarginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is expressed abundantly in the proximal tubule (PT). We hypothesize that ADMA metabolized by DDAH-1 inhibits fluid reabsorbtion (Jv) by the proximal tubule. S2 segments of the PT were microperfused between blocks in vivo to assess Jv in anesthetized rats. Compared with vehicle, microperfusion of ADMA or N?-nitro-l-arginine methyl ester (l-NAME) in the proximal tubule reduced Jv dose dependently. At 10-4 mol/l both reduced Jv by ~40% (vehicle: 3.2?±?0.7 vs. ADMA: 2.1?±?0.5, P < 0.01 vs. l-NAME: 1.9?±?0.4 nl·min-1·mm-1, P < 0.01; n = 10). Selective inhibition of DDAH-1 in rats with intravenous L-257 (60 mg/kg) given 2 h before and L-257 (10-5 mol/l) perfused in the proximal tubule for 5 min reduced Jv by 32?±?4% (vehicle: 3.2?±?0.5 vs. L-257: 2.2?±?0.5 nl·min-1·mm-1; P < 0.01) and increased plasma ADMA by ?50% (vehicle: 0.46?±?0.03 vs. L-257: 0.67?±?0.03 µmol/l, P < 0.0001) without changing plasma symmetric dimethylarginine. Compared with nontargeted control small-interference RNA, knock down of DDAH-1 in mice by 60% with targeted small-interference RNAs (siRNA) reduced Jv by 29?±?5% (nontargeted siRNA: 2.8?±?0.20 vs. DDAH-1 knockdown: 1.9?±?0.31 nl·min-1·mm-1, P < 0.05). In conclusion, fluid reabsorption in the proximal tubule is reduced by tubular ADMA or by blocking its metabolism by DDAH-1. L-257 is a novel regulator of proximal tubule fluid reabsorption.
Project description:Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with the severity of portal hypertension. Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through farnesoid X receptor (FXR) agonism and DDAH-1 gene therapy.DDAH-1 immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with the FXR agonist obeticholic acid (OA, 5 mg/kg) or vehicle for 5 days. Further, animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control, which resulted in the following groups: sham+saline, BDL+saline, BDL+DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by COBAS INTEGRA, DDAH-1 expression by qPCR and Western blot, eNOS activity by radiometric assay.Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL+OA vs. BDL+vehicle (8±1 vs. 13.5±0.6 mmHg; p<0.01) with no change in the mean arterial pressure (MAP). Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL+DDAH-1 vs. BDL+saline (p<0.01).This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies, targeting DDAH-1 in cirrhosis and portal hypertension.
Project description:Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.
Project description:We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.
Project description:Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase (eNOS). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of ADMA lead to reduction in nitric oxide (NO) production, which is linked to endothelial dysfunction and atherosclerosis. Piper sarmentosum is an herb that has shown stimulation on endothelial NO production by increasing both expression and activity of eNOS. Thus, this study determined whether the positive effect of P. sarmentosum on NO production is related to its modulation on the DDAH-ADMA pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to tumor necrosis factor-? (TNF-?). HUVEC were divided into four groups: control, treatment with 250 µg/ml of aqueous extract of P. sarmentosum leaves (AEPS), treatment with 30 ng/ml of TNF-?, and concomitant treatment with AEPS and TNF-? for 24 h. After treatments, HUVEC were collected to measure DDAH1 messenger RNA (mRNA) expression using quantitative real-time polymerase chain reaction. DDAH1 protein level was measured using enzyme-linked immunosorbent assay (ELISA), and DDAH enzyme activity was measured using colorimetric assay. ADMA concentration was measured using ELISA, and NO level was measured using Griess assay. Compared to control, TNF-?-treated HUVEC showed reduction in DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05). Treatment with AEPS successfully increased DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05) in TNF-?-treated HUVEC. Treatment with TNF-? caused an increase in ADMA level (P < 0.01) and a decrease in endothelial NO production (P < 0.001). Whereas treatment with AEPS was able to reduce ADMA level (P < 0.01) and restore NO (P < 0.001) in TNF-?-treated HUVEC. The results suggested that AEPS promotes endothelial NO production by stimulating DDAH activity and thus reducing ADMA level in TNF-?-treated HUVEC.
Project description:<h4>Aims</h4>Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.<h4>Methods and results</h4>In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.<h4>Conclusion</h4>Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.
Project description:BACKGROUND:Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the synthesis of nitric oxide (NO) through the metabolism of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA). Pilot studies have associated the rs805305 SNP of DDAH2 with ADMA concentrations in sepsis. This study explored the impact of the rs805305 polymorphism on DDAH activity and outcome in septic shock. METHODS:We undertook a secondary analysis of data and samples collected during the Vasopressin versus noradrenaline as initial therapy in septic shock (VANISH) trial. Plasma and DNA samples isolated from 286 patients recruited into the VANISH trial were analysed. Concentrations of L-Arginine and the methylarginines ADMA and symmetric dimethylarginine (SDMA) were determined from plasma samples. Whole blood and buffy-coat samples were genotyped for polymorphisms of DDAH2. Clinical data collected during the study were used to explore the relationship between circulating methylarginines, genotype and outcome. RESULTS:Peak ADMA concentration over the study period was associated with a hazard ratio for death at 28 days of 3.3 (95% CI 2.0-5.4), p < 0.001. Reduced DDAH activity measured by an elevated ADMA:SDMA ratio was associated with a reduced risk of death in septic shock (p = 0.03). The rs805305 polymorphism of DDAH2 was associated with reduced DDAH activity (p = 0.004) and 28-day mortality (p = 0.02). Mean SOFA score and shock duration were also reduced in the less common G:G genotype compared to heterozygotes and C:C genotype patients (p = 0.04 and p = 0.02, respectively). CONCLUSIONS:Plasma ADMA is a biomarker of outcome in septic shock, and reduced DDAH activity is associated with a protective effect. The polymorphism rs805305 SNP is associated with reduced mortality, which is potentially mediated by reduced DDAH2 activity. TRIAL REGISTRATION:ISRCTN Registry, ISRCTN20769191 . Registered on 20 September 2012.