Conducted signals within arteriolar networks initiated by bioactive amino acids.
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Our purpose was to determine the specificity of L-arginine (L-Arg)-induced conducted signals for intra- vs. extracellular actions of L-Arg. Diameter and red blood cell velocities were measured for arterioles [18 +/- 1.6 (SE) micrometer] in the cremaster muscle of pentobarbital sodium-anesthetized (Nembutal, 70 mg/kg) hamsters (n = 53). Remote (conducted) responses were viewed approximately 1,000 micrometer upstream from the local (micropipette) application. Six amino acids were tested: L-arginine, L-cystine, L-leucine, L-lysine, L-histidine, and L-aspartate (100 microM each). Only L-Arg induced a remote dilation; L-lysine and L-aspartate had no effect, and the others each induced a significant remote constriction. There is a second conducted signal initiated by L-arginine that preconditions the arteriolar network and upregulates a direct response of L-arginine to dilate the remote site. This was blocked by inhibition of L-arginine uptake at the local (preconditioning) site (100 microM L-histidine or 1 mM phenformin). Arginine-glycine-aspartate (100 microM)-induced remote dilations (+3. 2 +/- 0.3 micrometer) were not mimicked by a peptide control and were prevented by anti- integrin alphav monoclonal antibody. Remote dilations were greater in animals with a higher wall shear stress for arginine-glycine-aspartate (r2 = 0.92) but not for L-arginine (r2 = 0.12). Thus L-arginine initiates separate conducted signals related to system y+ transport, integrins, and baseline flow. (+info)
Asymmetric dimethylarginine plasma concentrations differ in patients with end-stage renal disease: relationship to treatment method and atherosclerotic disease.
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Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide (NO) synthase. Its concentration is elevated in patients with end-stage renal disease (ESRD), in part because it is excreted via the kidneys. In this study, the plasma concentrations of ADMA, symmetric dimethylarginine, and L-arginine were determined in relation to plasma nitrate levels (as an index of NO formation) for a group of 80 patients with ESRD. The effects of two treatment methods, i.e., hemodialysis (HD) and peritoneal dialysis (PD), and the role of the presence of atherosclerotic disease were evaluated. Forty-three patients receiving HD and 37 patients receiving PD were compared with healthy control subjects. Plasma L-arginine and dimethylarginine levels were determined by HPLC, using precolumn derivatization with o-phthaldialdehyde. Plasma nitrate levels were determined by gas chromatography-mass spectrometry. Predialysis ADMA concentrations in HD-treated patients were approximately sixfold higher than those in the control group (6.0+/-0.5 versus 1.0+/-0.1 micromol/L; P < 0.05). Plasma nitrate concentrations were significantly lower in HD-treated patients, which suggests that ADMA may inhibit NO synthase. In contrast, plasma ADMA levels and nitrate concentrations in PD-treated patients were similar to those in control subjects. Plasma L-arginine concentrations were not significantly decreased in patients with ESRD. ADMA concentrations were significantly decreased 5 h after HD, compared with baseline values. ADMA levels were significantly higher in HD-treated patients with manifest atherosclerotic disease than in HD-treated patients without atherosclerotic disease (7.31+/-0.70 versus 3.95+/-0.52 micromol/L; P < 0.05). This study confirms that ADMA is accumulated in ESRD. PD-treated patients exhibit significantly lower ADMA levels than do HD-treated patients. Accumulation of ADMA may be a risk factor for the development of endothelial dysfunction and cardiovascular disease in patients with ESRD. (+info)
Continuous perivascular L-arginine delivery increases total vessel area and reduces neointimal thickening after experimental balloon dilatation.
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The aim of this study was to evaluate whether vascular remodeling and neointimal thickening occur after balloon dilatation of the nonatherosclerotic rabbit carotid artery, and whether both processes are influenced by continuous perivascular delivery of L-arginine or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In the first experiment, histological and morphometric evaluation of arteries was performed at different time points after balloon dilatation: 10 minutes (n=7), and 1 (n=7), 2 (n=9), 3 (n=20), or 10 (n=5) weeks. Neointimal thickening progressively contributed to luminal narrowing for at least 10 weeks after angioplasty. During the first 2 weeks after dilatation, a significant decrease of the total vessel area was measured. Ten weeks after dilatation, both the neointimal and total vessel area were increased without further changing of the luminal area. In the second experiment, endothelial injured rabbits were randomly assigned to receive 2 weeks of continuous local perivascular physiological salt solution (n=6), L-arginine (n=8), or L-NAME (n=7), starting immediately after balloon dilatation (ie, local drug delivery during the first phase of the biphasic vascular remodeling process). Perivascular L-arginine delivery significantly reduced the neointimal area, despite an increased number of neointimal Ki-67-positive smooth muscle cells. Both the luminal area and total vessel area were significantly increased. Serum L-arginine levels remained unchanged. L-NAME administration had no effect on the neointimal area, nor on the luminal and total vessel area. Neointimal formation and biphasic vascular remodeling occur after experimental balloon dilatation of the nonatherosclerotic rabbit carotid artery, and can be influenced by continuous local perivascular delivery of L-arginine. (+info)
Paraoxonase 192 Gln/Arg gene polymorphism, coronary artery disease, and myocardial infarction in type 2 diabetes.
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Paraoxonase is an HDL-associated enzyme implicated in the pathogenesis of atherosclerosis by protecting lipoproteins against peroxidation. Its biallelic gene polymorphism at codon 192 (glutamine/arginine) has been associated with coronary artery disease (CAD). To further evaluate the role of this paraoxonase gene polymorphism for CAD in type 2 diabetes, we determined the paraoxonase genotype in 288 type 2 diabetic patients (170 with and 118 without angiographically documented CAD). The paraoxonase 192 Gln/Arg genotype was assessed using polymerase chain reaction followed by AlwI digestion. The frequency of the Gln allele was 0.656 in the CAD patients and 0.746 in the controls (chi2 = 5.36, P = 0.02). Compared with the Gln/Gln genotypes, the age-adjusted odds ratio for CAD was 1.78 (95% CI 1.08-2.96, P = 0.02) in subjects carrying at least one Arg allele. In the multivariate analysis, this association was even stronger after correction for the possible confounders age, sex, smoking history, and hypertension. Among current and former smokers, the odds ratio (OR) for having CAD among patients with at least one Arg allele was 3.58 (1.45-9.53, P < 0.01). The paraoxonase Arg allele was not associated with the history of myocardial infarction (OR 1.20 [0.73-1.99, NS]), but was with the extent of CAD (OR for three-vessel disease 1.92 [1.15-3.27, P = 0.01]). Our data indicate that the 192 Arg allele of the human paraoxonase gene is a risk factor for CAD but not myocardial infarction in type 2 diabetic patients, a risk factor further modified by cigarette smoking. This risk could possibly be explained by a reduced ability of the paraoxonase Arg isoform to protect lipoproteins against peroxidation. (+info)
Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling.
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OBJECTIVES: The purpose of this study was to assess the effects of L-arginine and N(G)-nitro-L-arginine methyl ester (L-NAME) on neointimal hyperplasia and vascular remodeling after balloon angioplasty in the hypercholesterolemic rabbit. BACKGROUND: Restenosis after balloon angioplasty is a consequence of both neointimal hyperplasia and vessel remodeling. Nitric oxide inhibits neointimal hyperplasia, but its effect on vessel remodeling is unknown. METHODS: Six weeks after induction of bilateral iliac atherosclerosis, 48 rabbits underwent successful angioplasty in 75 vessels. Eight rabbits (acute group) were sacrificed immediately after angioplasty. The remaining animals received either placebo (chronic control group), or a diet supplemented with either L-arginine (1.5 g/kg/day), or L-NAME (15 mg/kg/day) for 4 weeks after angioplasty. RESULTS: The intimal area was significantly greater in the chronic control group compared to the acute group (2.60+/-1.03 mm2 vs. 1.35+/-0.62 mm2). This increase in intimal area was lower in the L-arginine group (1.79+/-0.61 mm2), and greater in the L-NAME group (3.23+/-0.92 mm2). The area circumscribed by the internal elastic lamina (IEL) increased significantly in the control group compared to the acute group (from 2.52+/-0.66 to 3.33+/-0.85 mm2); a more marked increase occurred in the L-NAME group (3.90+/-0.85 mm2). By contrast, IEL area was unchanged in the L-arginine group (2.41+/-0.62 mm2). As a result, there was no significant difference in lumen area after 4 weeks in the chronic groups (control: 0.74+/-0.38 mm2; L-arginine: 0.50+/-0.43 mm2; L-NAME: 0.48+/-0.42 mm2). CONCLUSIONS: Our results demonstrate that L-arginine inhibits whereas L-NAME stimulates neointimal hyperplasia after experimental balloon angioplasty in the hypercholesterolemic rabbit. However, the lack of vessel enlargement in the L-arginine group resulted in a similar final lumen size in the L-NAME and L-arginine groups. (+info)
Aromatic L-amino acid decarboxylase: conformational change in the flexible region around Arg334 is required during the transaldimination process.
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Aromatic L-amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C-terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L-dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine. For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327-Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L-dopa as substrate, but its k(cat) value was decreased 57-fold, and the Km value was increased 9-fold compared with those of the wild-type AADC. The absorption spectra of the fragmentary AADC in the presence of L-dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328-Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L-dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process. (+info)
Anion-coordinating residues at binding site 1 are essential for the biological activity of the diphtheria toxin repressor.
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The homodimeric diphtheria toxin repressor (DtxR) uses Fe2+ as a corepressor, binds to iron-regulated promoters, and negatively regulates the syntheses of diphtheria toxin, corynebacterial siderophore, and several other Corynebacterium diphtheriae products. The crystal structure of DtxR shows that the second domain of each monomer has two binding sites for Fe2+ or certain other divalent metal ions. In addition, site 1 binds a sulfate or phosphate anion, suggesting that phosphate may function intracellularly as a co-corepressor. The effects of alanine substitutions for selected residues in sites 1 and 2 were determined by measuring the beta-galactosidase activities of a tox operator/promoter-lacZ reporter construct in Escherichia coli strains expressing each DtxR variant. Our studies demonstrated that single alanine substitutions for the anion-binding residues in site 1 (R80A, S126A, or N130A) caused severely decreased DtxR activity, similar to the effects of alanine substitutions for metal-binding residues in site 2 (C102A, E105A, or H106A) and greater than the effects of alanine substitutions for metal-binding residues in site 1 (H79A, E83A, or H98A) reported previously by other investigators. Various combinations of alanine substitutions for site 1 and site 2 residues were also analyzed to further elucidate the roles of these cation- and anion-binding ligands in DtxR activity. Furthermore, the interaction between residue E20 in the DNA binding domain and R80 in anion/cation binding site 1 was analyzed, and the E20A variant of DtxR was shown to have a phenotype indistinguishable from that of the R80A variant. Our data demonstrated for the first time that the anion-binding residues R80, S126, and N130 at site 1 are essential for DtxR activity. The data also showed that the interaction of E20 in domain 1 with R80 in domain 2, first revealed by X-ray crystallography in apo-DtxR and holo-DtxR, is a structural feature of DtxR that is important for its repressor activity. (+info)
Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases. (+info)