Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Ferricytochrome (c) directly oxidizes aminoacetone to methylglyoxal, a catabolite accumulated in carbonyl stress.

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH4(+) ion, and H2O2 coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O2(•-) and HO (•) radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E0 values = -0.51 and -1.0 V) to ferricytochrome c (E0 = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH4(+) ion. In the presence of oxygen, aminoacetone enoyl and O2(•-) radicals propagate aminoacetone oxidation to methylglyoxal and H2O2. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.

Acetilação radicalar de amino ácidos, peptídeos e nucleobases pelos sistemas biacetilo/peroxinitrito e metilglioxal/peroxinitrito / Radical acetylation of aminoacids, peptides, and nucleobases by the biacetyl or methylglyoxal/peroxynitrite systems

Biacetilo (2,3-butanediona) é um contaminante de comida e cigarro, também implicado na hepatoxicidade do álcool e em doenças pulmonares. O metilglioxal (MG), um α-oxoaldeído reativo frequentemente associado ao diabetes e envelhecimento, é produto da fragmentação oxidativa de trioses fosfato, acetona e aminoacetona. Por sua vez, peroxinitrito - um potente oxidante, agente nitrante e nucleófilo formado in vivo pela reação controlada por difusão do ânion radical superóxido com o radical óxido nítrico (k ~1010 M-1s-1) é capaz de se adicionar a CO2 e compostos carbonílicos, gerando produtos potencialmente tóxicos ou sinalizadores celulares. Aminoácidos, peptídeos e nucleobases podem ser acetilados nos grupos amina e na porção desoxiribose. Relativamente ao tratamento com peroxinitrito isolado, níveis superiores de 3-nitrotirosina foram detectados quando tirosina foi tratada com peroxinitrito/biacetilo ou metilglioxal. Ambos os grupos amina de lisina (Lys) ou um deles de derivados de lisina bloqueados e um deles (Ac-Lys-OMe, Z-Lys-OMe) foram acetilados pelo sistema biacetilo ou metilglioxal/peroxinitrito. Em tetrapeptídeos sintéticos contendo lisina como aminoácido amino-terminal (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), a lisina foi acetilada pelo sistemas dicarbonilico/peroxinitrito no grupo α-amina (em maior extensão) e/ou no ε-amina (em menor extensão). No conjunto, estes resultados podem ser interpretados à luz do mecanismo proposto para a reação de compostos α-dicarbonílicos com peroxinitrito, o qual envolve sequencialmente: (i) adição nucleofílica de peroxinitrito à carbonila; (ii) homólise do aduto peroxinitroso formado, liberando •NO2 e um radical oxila do reagente carbonílico; (iii) β-clivagem do radical oxila a um ácido carboxílico (ácido acético no caso de biacetilo e ácido fórmico, a partir de metilglioxal) e radical acetila; (iv) captação do radical acetila pelo oxigênio molecular dissolvido dando acetato, ou por aminoácido ou nucleobase...

Diacetyl (2,3-butanedione) is a food and cigarette contaminant recently implicated in alcohol hepatotoxicity and lung disease. In turn, methylglyoxal (MG) is an α-oxoaldehyde frequently associated with diabetes and aging that is putatively formed by the oxidative fragmentation of trioses phosphate, acetone and aminoacetone. Peroxynitrite - a potent oxidant, nitrating agent and nucleophile formed in vivo by the diffusion-controlled reaction of superoxide radical with nitric oxide (k ~1010 M-1s-1) - is able to form adducts with carbon dioxide and carbonyl compounds. When initially present in the reaction mixtures before addition of ONOO-, amino acids, peptides and nucleobases undergo acetylation at the amino group and purine moieties in the presence of biacetyl or methylglyoxal. Higher levels of 3-nitrotyrosine nitration were measured when peroxynitrite/biacetyl or metilglioxal was added to tyrosine, in comparison with peroxynitrite alone. Both amino groups of L-lysine or one of the amino groups of L-lysine derivatives (Z-Lys-OH and Ac-Lys-OH) were acetylated by biacetyl and methylglyoxal/peroxynitrite system. Using tetrapeptides containing lysine at the terminal amino acid (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), the lysine residue was acetylated at both or either α-amino (major adduct) and ε-amino group (minor adduct). Altogether these data can be interpreted by the mechanism proposed to describe the reaction of α-dicarbonyls with peroxynitrite as follows: (i) nucleophilic addition of peroxynitrite to the carbonyl group of the reagent; (ii) homolysis of the formed peroxynitroso carbonyl adduct to •NO2 and a carbonyloxyl radical; (iii) β-cleavage of the oxyl radical to acetyl radical plus acetic acid (from diacetyl) or formic acid (from methylglyoxal); (iv) competitive scavenging of the acetyl radical by dissolved molecular oxygen and by added amino acid, peptide or nucleobase, ultimately yielding acetate or acetylated biomolecule. If occurring in vivo...

Generation of singlet oxygen by the glyoxal-peroxynitrite system.

Diacetyl, methylglyoxal, and glyoxal are α-dicarbonyl catabolites prone to nucleophilic additions of amino groups of proteins and nucleobases, thereby triggering adverse biological responses. Because of their electrophilicity, in aqueous medium, they exist in a phosphate-catalyzed dynamic equilibrium with their hydrate forms. Diacetyl and methylglyoxal can be attacked by peroxynitrite (k(2) ≈ 1.0 × 10(4) M(-1) s(-1) and k(2) ≈ 1.0 × 10(5) M(-1) s(-1), respectively), a potent biological nucleophile and oxidant, yielding the acetyl radical from the homolysis of peroxynitrosocarbonyl adducts, and acetate or formate ions, respectively. We report here that glyoxal also reacts with peroxynitrite, yielding formate ion at rates at least 1 order of magnitude greater than does methylglyoxal. A triplet EPR signal (1:2:1; a(H) = 0.78 mT) attributable to hydrated formyl radical was detected by direct flow experiments. In the presence of the spin trap 2-methyl-2-nitrosopropane, the EPR spectrum displays the di-tert-butyl nitroxide signal, another signal assignable to the spin trapping adduct with hydrogen radical (a(N) = a(H) = 1.44 mT), probably formed from formyl radical decarbonylation, and a third EPR signal assignable to the formyl radical adduct of the spin trap (a(N) = 0.71 mT and a(H) = 0.14 mT). The novelty here is the detection of singlet oxygen ((1)Δ(g)) monomol light emission at 1270 nm during the reaction, probably formed by subsequent dioxygen addition to formyl radical and a Russell reaction of nascent formylperoxyl radicals. Accordingly, the near-infrared emission increases upon raising the peroxynitrite concentration in D(2)O buffer and is suppressed upon addition of O(2) ((1)Δ(g)) quenchers (NaN(3), l-His, H(2)O). Unequivocal evidence of O(2) ((1)Δ(g)) generation was also obtained by chemical trapping of (18)O(2) ((1)Δ(g)) with anthracene-9,10-divinylsulfonate, using HPLC/MS/MS for detection of the corresponding 9,10-endoperoxide derivative. Our studies add insights into the molecular events underlying nitrosative, oxidative, and carbonyl stress in inflammatory processes and aging-associated maladies.

Acetyl radical production by the methylglyoxal-peroxynitrite system: a possible route for L-lysine acetylation.

Methylglyoxal is an α-oxoaldehyde putatively produced in excess from triose phosphates, aminoacetone, and acetone in some disorders, particularly in diabetes. Here, we investigate the nucleophilic addition of ONOO(-), known as a potent oxidant and nucleophile, to methylglyoxal, yielding an acetyl radical intermediate and ultimately formate and acetate ions. The rate of ONOO(-) decay in the presence of methylglyoxal [k(2,app) = (1.0 ± 0.1) × 10(3) M(-1) s(-1); k(2) ≈ 1.0 × 10(5) M(-1) s(-1)] at pH 7.2 and 25 °C was found to be faster than that reported with monocarbonyl substrates (k(2) < 10(3) M(-1) s(-1)), diacetyl (k(2) = 1.0 × 10(4) M(-1) s(-1)), or CO(2) (k(2) = 3-6 × 10(4) M(-1) s(-1)). The pH profile of the methylglyoxal-peroxynitrite reaction describes an ascendant curve with an inflection around pH 7.2, which roughly coincides with the pK(a) values of both ONOOH and H(2)PO(4)(-) ion. Electron paramagnetic resonance spin trapping experiments with 2-methyl-2-nitrosopropane revealed concentration-dependent formation of an adduct that can be attributed to 2-methyl-2-nitrosopropane-CH(3)CO(•) (a(N) = 0.83 mT). Spin trapping with 3,5-dibromo-4-nitrosobenzene sulfonate gave a signal that could be assigned to a methyl radical adduct [a(N) = 1.41 mT; a(H) = 1.35 mT; a(H(m)) = 0.08 mT]. The 2-methyl-2-nitrosopropane-CH(3)CO(•) adduct could also be observed by replacement of ONOO(-) with H(2)O(2), although at much lower yields. Acetyl radicals could be also trapped by added L-lysine as indicated by the presence of (ε)N-acetyl-L-lysine in the spent reaction mixture. This raises the hypothesis that ONOO(-)/H(2)O(2) in the presence of methylglyoxal is endowed with the potential to acetylate proteins in post-translational processes.

Radical acetylation of 2'-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium.

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant ( k 2 = 1.0 x 10 (4) M (-1) s (-1)) when compared with monocarbonyl substrates ( k 2 < 10 (3) M (-1) s (-1)). Phosphate ions (100-500 mM) do not affect the rate of spontaneous peroxynitrite decay, but the H 2PO 4 (-) anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum ( a N = a H = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either l-histidine or 2'-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS ( n ). These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.