Search

Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Ne-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.

Background Sevoflurane induces preconditioning (SevoPC). The effect of aprotinin and the involvement of endothelial nitric-oxide synthase (NOS) on SevoPC are unknown. We investigated (1) whether SevoPC is strengthened by multiple preconditioning cycles, (2) whether SevoPC is blocked by aprotinin, and (3) whether endothelial NOS plays a crucial role in SevoPC. Methods Anesthetized male Wistar rats were randomized to 15 groups (each n = 6) and underwent 25-min regional myocardial ischemia and 2-h reperfusion. Controls were not treated further. Preconditioning groups inhaled 1 minimum alveolar concentration of sevoflurane for 5 min (SEVO-I), twice for 5 min each (SEVO-II), three times for 5 min each (SEVO-III), or six times for 5 min each (SEVO-VI). Aprotinin was administered with and without sevoflurane. Involvement of endothelial NOS was determined with the nonspecific NOS blocker N-nitro-l-arginine-methyl-ester, the specific neuronal NOS blocker 7-nitroindazole, and the specific inducible NOS blocker aminoguanidine. Results SevoPC reduced infarct size in all protocols (SEVO-I, 42 ± 6%; SEVO-II, 33 ± 4%; SEVO-III, 11 ± 5%; SEVO-VI, 16 ± 4%; all P < 0.001 vs. control, 67 ± 3%) and was least after three and six cycles of sevoflurane (P < 0.001 vs. SEVO-II and -I, respectively). Aprotinin alone had no effect on infarct size but blocked SevoPC. N-nitro-l-arginine-methyl-ester abolished SevoPC (67 ± 4%; P < 0.05 vs. SEVO-III). Aminoguanidine and 7-nitroindazole blocked SevoPC only partially (25 ± 6 and 31 ± 6%, respectively; P < 0.05 vs. SEVO-III and control). SevoPC induced endothelial NOS phosphorylation, which was abrogated by aprotinin. Conclusion SevoPC is strengthened by multiple preconditioning cycles, and phosphorylation of endothelial NOS is a crucial step in mediating SevoPC. These effects are abolished by aprotinin.

Background Nitric oxide (NO) is produced by resident and inflammatory cells in the respiratory tract by the enzyme NO synthase (NOS), which exists in three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS. NO production is increased in patients with COPD, and the production of NO under oxidative stress conditions generates reactive nitrogen species that may amplify the inflammatory response in COPD. Methods To examine the role of increased NO in COPD, we administered a relatively selective iNOS inhibitor, aminoguanidine, by nebulization in a double-blind, placebo-controlled study in COPD patients, healthy smokers, and healthy nonsmoking subjects. We investigated whether aminoguanidine had any effect on exhaled NO produced in the central lung (flux of NO from the airways [J no ] and peripheral lungs (concentration of NO in peripheral lung [C alv ], on NO metabolites (nitrite [NO 2 − ]/nitrate [NO 3 − ], peroxinitrite [ONOO − ], nitrotyrosine), and on a marker of oxidative stress (8-isoprostane) in exhaled breath condensate (EBC) and in sputum. Results Aminoguanidine administration resulted in a significant reduction in J no compared with administration of the saline solution control in healthy subjects, smokers, and COPD patients. C alv in smokers and in COPD patients was not completely inhibited 1 h after aminoguanidine inhalation, in marked contrast to previous results in asthma. Moreover, ONOO − and NO 2 − /NO 3 − levels were also increased in EBC and in sputum of smokers and COPD and were not completely inhibited following aminoguanidine inhalation. 8-Isoprostane levels were also increased in smokers and in COPD patients but were not reduced after aminoguanidine inhalation. Conclusions These results suggest that the constitutive NOS isoform as well as iNOS might be involved in NO release and contribute to the high C alv and ONOO − production in patients with COPD. Trial registration: Clinicaltrials.gov Identifier: NCT00180635.

Background The aim of this study was to observe the time-course changes of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) induction in aorta during the development of hypertension, as well as the relationship of HO-1/carbon monoxide (CO) system and iNOS/nitric oxide (NO) system in spontaneously hypertensive rats (SHR). Methods The systolic blood pressure (SBP) was determined in conscious rats by the tail-cuff method. The tissue HO-1 and iNOS mRNA and protein levels were estimated with reverse transcription polymerase chain reaction and Western blot method. Results The expression of HO-1 and iNOS in aorta increased with the SBP elevation during the development of SHR and was attenuated when the hypertension was lowered with the vasodilator hydralazine. At 8 weeks, only HO-1 was induced, whereas at 12 and 16 weeks, both HO-1 and iNOS were observed. The level of plasma nitrite/nitrate was associated with the change in iNOS expression in SHR. In addition, the SBP of 8-week-old SHR was significantly increased after pretreatment with zinc protoporphyrin IX for 7 consecutive days. Chronic blockade of iNOS activity by aminoguanidine resulted in significant up-regulation of HO-1, but the pressor effect was blunt. Conclusions These results suggest that the up-regulation of HO-1 and iNOS in aorta is a compensatory mechanism for the elevation of SBP during the development of hypertension in SHR. The expression of HO-1 is earlier than that of iNOS. Our data suggest that the HO-1/CO system takes over and acts as a major modulator for the regulation of SBP when the iNOS/NO system is suppressed.

Objective: We tested the hypothesis that occlusion of the superior mesenteric artery induces late preconditioning against myocardial infarction and examined the effects of pharmacological modifiers of inducible nitric oxide synthase activity on the late preconditioning in anesthetized rats. Methods: Rats underwent an intestinal ischemia preconditioning protocol (30 min occlusion of the superior mesenteric artery) or were sham-operated. They were subjected to a sustained 30 min of coronary occlusion and 180 min of reperfusion 24 h later. Results: In rats receiving no pharmacological intervention, the percentage of myocardial infarct within the area at risk and left ventricle was 72±4% and 31±2%, respectively, in sham-operated rats, and these were significantly reduced to 44±4% and 23±2% ( P

Background: Ischemia/reperfusion (I/R) leads to the induction of inducible nitric oxide synthase. The present study investigated the effects of selective and continuous inhibition of iNOS on myocardial performance, infarct size and histomorphological changes after I/R in rabbits. Methods and Results: Ischemia/reperfusion (I/R) was induced by occlusion of the circumflex coronary artery for 30 min followed by 48 h of reperfusion. Sham animals (group A) served as control. Three groups were subjected to I/R: (B) placebo; (C) aminoguanidine (AMG; 10 mg/kg bolus) given prior to and 48 h after I/R to test its acute effects; (D) AMG (300 mg/kg/day sc) to test effects of continuous treatment. Hemodynamics, myocardial blood flow, infarct size, iNOS activity, cGMP levels, immunohistochemical analysis of iNOS expression and AMG tissue levels were determined.Continuous AMG treatment improved myocardial performance (hemodynamics and blood flow) compared to placebo group. iNOS was highest in placebo-treated animals. AMG tissue levels were highest in tissues affected by I/R. Infarct size (% of the circumflex region) was significantly smaller in group D when compared to group B. Conclusions: This is the first study showing that activation of myocardial iNOS isozyme during 48 h of reperfusion contributes to a late phase of I/R-induced injury in rabbits. Selective and continuous modulation of iNOS by AMG over this time period exerts protective effects with respect to myocardial performance, coronary blood flow, cellular infiltration and reduction of infarct size; this may be a novel therapeutic approach in the clinical situation to limit irreversible myocardial injury associated with ischemia and reperfusion.

The objectives of this study were to determine the effect of diet restriction (DR) and the crosslinking inhibitor, aminoguanidine (AG), on PMA-induced respiratory burst, concentrations of uric acid, and the rate of pentosidine accumulation in the skin (Ps) of naturally hyperglycemic broiler breeder hens. Female chicks (n = 450) were randomly assigned to four groups from 8 to 92 weeks after hatch: ad libitum (AL), diet restricted (DR), AL and DR groups supplemented with 400 ppm AG each (AL + AG and DR + AG). No consistent effects of treatments were observed on plasma concentrations of glucose. The accumulation of Ps in AL birds increased linearly with age (p < .001) and was significantly retarded in all treatment groups (p < .001). Ps in the AL + AG group was comparable to that in the DR or DR + AG groups. PMA-induced respiratory bursts in blood leukocytes were significantly retarded in DR or AG-supplemented (p < .0001) groups. Although there was a marginal increase in overall mean concentrations of plasma uric acid for the DR group, no consistent differences were observed on individual time points. It is concluded that the glycosylation process may not be the primary cause of glucose-derived crosslinks and that the accumulation of Ps can be retarded by DR and AG in broiler breeder hens.

This study explored the role of inducible nitric oxide (NO) synthase (iNOS) in an infant rat model of group B streptococcal meningitis. Brain iNOS activity increased during meningitis (P < .001), and iNOS was detected by immunocytochemistry in the walls of meningeal vessels and cells of the cerebrospinal fluid (CSF) inflammation. Animals treated with iNOS inhibitor aminoguanidine (AG; 130 mg/kg every 8 h) had reduced NO production (P < .05), higher CSF bacterial titers (P < .05), and increased incidence of seizures (P < .01) compared with untreated infected animals. AG also increased areas of severe hypoperfusion in the cortex (31% +/- 14% in controls vs. 56% +/- 16% in AG; P < .01) and the extent of cortical neuronal injury, both when administered at the time of infection (P < .05) and in established meningitis (P < .02). Thus, NO produced by iNOS may be beneficial in this model of experimental meningitis by reducing cerebral ischemia.

Subclinical lymphocytic choriomeningitis virus infection primes mice expressing a V beta 8.1D beta 2J beta 2.3C beta 2 T cell receptor as a transgene for induction of fatal hematogenous shock after administration of a dose of staphylococcal enterotoxin B (SEB) that is tolerated by uninfected controls. The lethal effect is greatly diminished by prior depletion of the virus-primed CD4+ T cells. Evidence of transient tumor necrosis factor (TNF) secretion is detected in serum within 1 h of SEB administration, and massive amounts of interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) are present within 4-6 h. Mice are partly protected by treatment with dimeric soluble TNF receptor-Fc fusion protein or the nitric oxide synthase inhibitor, aminoguanidine, neither of which blocks SEB-induced IFN-gamma or IL-6 production. Administration of a monoclonal antibody to IFN-gamma concomitant with SEB effectively neutralizes this cytokine but has no effect on survival.

Current treatment of the nephropathy complication of diabetes mellitus is suboptimal in halting the progression of the complex disease. Among the irreversible effects of sustained hyperglycemia is the heightened formation of advanced glycation end products (AGEs). The role of AGEs in diabetic nephropathy has been established by years of basic research. This article reports progression through human studies of the few AGE inhibitors that have reached clinical development, including pimagedine, pyridoxamine, and alagebrium.

Aminoguanidine (AG) is a prototype therapeutic agent for the prevention of formation of advanced glycation endproducts. It reacts rapidly with alpha,beta-dicarbonyl compounds such as methylglyoxal, glyoxal, and 3-deoxyglucosone to prevent the formation of advanced glycation endproducts (AGEs). The adducts formed are substituted 3-amino-1,2,4-triazine derivatives. Inhibition of disease mechanisms, particularly vascular complications in experimental diabetes, by AG has provided evidence that accumulation of AGEs is a risk factor for disease progression. AG has other pharmacological activities, inhibition of nitric oxide synthase and semicarbazide-sensitive amine oxidase (SSAO), at pharmacological concentrations achieved in vivo for which controls are required in anti-glycation studies. AG is a highly reactive nucleophilic reagent that reacts with many biological molecules (pyridoxal phosphate, pyruvate, glucose, malondialdehyde, and others). Use of high concentrations of AG in vitro brings these reactions and related effects into play. It is unadvisable to use concentrations of AG in excess of 500 microM if selective prevention of AGE formation is desired. The peak plasma concentration of AG in clinical therapy was ca. 50 microM. Clinical trial of AG to prevent progression of diabetic nephropathy was terminated early due to safety concerns and apparent lack of efficacy. Pharmacological scavenging of alpha-oxoaldehydes or stimulation of host alpha-oxoaldehyde detoxification remains a worthy therapeutic strategy to prevent diabetic complications and other AGE-related disorders.

Throughout the industrialized (well-fed) world, diabetes mellitus is the most prevalent cause of end-stage renal disease (ESRD). Diabetic nephropathy is as likely to develop in long-duration non-insulin-dependent diabetes (type 2) as in insulin-dependent diabetes mellitus (type 1). Nephropathy in diabetes follows a well outlined course, starting with microalbuminuria through proteinuria, azotaemia and culminating in ESRD. Renal functional decline in diabetic nephropathy is slowed by establishment of euglycaemia and normalization of hypertensive blood pressure. Diabetic ESRD patients, compared with other causes of ESRD, sustain greater mortality and morbidity due to concomitant systemic disorders, especially coronary artery and cerebrovascular disease. A central role for glucose toxicity, especially the adverse impact of accumulated advanced glycosylated end-products (AGEs), appears likely from experimental data generated both in induced diabetic rodents and diabetic individuals. Treatment with aminoguanidine raises the possibility of blocking end-organ damage in diabetes without the necessity for correcting hyperglycaemia.