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.

Aerobic co-oxidation of hemoglobin and aminoacetone, a putative source of methylglyoxal.

Aminoacetone (1-aminopropan-2-one), a putative minor biological source of methylglyoxal, reacts like other α-aminoketones such as 6-aminolevulinic acid (first heme precursor) and 1,4-diaminobutanone (a microbicide) yielding electrophilic α-oxoaldehydes, ammonium ion and reactive oxygen species by metal- and hemeprotein-catalyzed aerobic oxidation. A plethora of recent reports implicates triose phosphate-generated methylglyoxal in protein crosslinking and DNA addition, leading to age-related disorders, including diabetes. Importantly, methylglyoxal-treated hemoglobin adds four water-exposed arginine residues, which may compromise its physiological role and potentially serve as biomarkers for diabetes. This paper reports on the co-oxidation of aminoacetone and oxyhemoglobin in normally aerated phosphate buffer, leading to structural changes in hemoglobin, which can be attributed to the addition of aminoacetone-produced methylglyoxal to the protein. Hydroxyl radical-promoted chemical damage to hemoglobin may also occur in parallel, which is suggested by EPR-spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide and ethanol. Concomitantly, oxyhemoglobin is oxidized to methemoglobin, as indicated by characteristic CD spectral changes in the Soret and visible regions. Overall, these findings may contribute to elucidate the molecular mechanisms underlying human diseases associated with hemoglobin dysfunctions and with aminoacetone in metabolic alterations related to excess glycine and threonine.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells.

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4(+) ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O2(*-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 microM) and Fe(3+) ion (100 microM) addition to the cell cultures had no effect, whereas Cu(2+) (5.0-100 microM) significantly increased cell death. Supplementation of the AA- and Cu(2+)-containing culture medium with antioxidants, such as catalase (5.0 microM), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu(2+) ions (10-50 microM) relative to control cultures. This may imply higher activity of antioxidant enzymes in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) plus Cu(2+) (100 microM) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

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.

The dual face of endogenous alpha-aminoketones: pro-oxidizing metabolic weapons.

Amino metabolites with potential prooxidant properties, particularly alpha-aminocarbonyls, are the focus of this review. Among them we emphasize 5-aminolevulinic acid (a heme precursor formed from succinyl-CoA and glycine), aminoacetone (a threonine and glycine metabolite), and hexosamines and hexosimines, formed by Schiff condensation of hexoses with basic amino acid residues of proteins. All these metabolites were shown, in vitro, to undergo enolization and subsequent aerobic oxidation, yielding oxyradicals and highly cyto- and genotoxic alpha-oxoaldehydes. Their metabolic roles in health and disease are examined here and compared in humans and experimental animals, including rats, quail, and octopus. In the past two decades, we have concentrated on two endogenous alpha-aminoketones: (i) 5-aminolevulinic acid (ALA), accumulated in acquired (e.g., lead poisoning) and inborn (e.g., intermittent acute porphyria) porphyric disorders, and (ii) aminoacetone (AA), putatively overproduced in diabetes mellitus and cri-du-chat syndrome. ALA and AA have been implicated as contributing sources of oxyradicals and oxidative stress in these diseases. The end product of ALA oxidation, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties, promote protein cross-linking, and damage GABAergic receptors of rat brain synaptosome preparations. In turn, methylglyoxal (MG), the end product of AA oxidation, is also highly cytotoxic and able to release iron from ferritin and copper from ceruloplasmin, and to aggregate proteins. This review covers chemical and biochemical aspects of these alpha-aminoketones and their putative roles in the oxidative stress associated with porphyrias, tyrosinosis, diabetes, and cri-du-chat. In addition, we comment briefly on a side prooxidant behaviour of hexosamines, that are known to constitute building blocks of several glycoproteins and to be involved in Schiff base-mediated enzymatic reactions.

Ischemic preconditioning inhibits mitochondrial respiration, increases H2O2 release, and enhances K+ transport.

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+ channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease ( approximately 25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2 release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+ transport, indicative of higher mitoKATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.

Toxicidade de aminoacetona e células produtoras de insulina / Cytotoxity of aminoacetone on insulin-producing cells

Danos induzidos por hiperglicemia em tecidos no diabetes são caracterizados por quatro mecanismos conectados: aumento do fluxo metabólico através da via do poliol, ativação da proteína quinase C (PKC), aumento da atividade da via das hexosaminas e aumento da produção intracelular dos precursores dos produtos finais de glicação avançada (AGEs). Entre eles, os derivados de metilglioxal, um potente agente de modificação de proteínas e DNA, têm sido associados a complicações microvasculares no diabetes: nefropatia, retinopatia e neuropatia. O metilglioxal é produzido a partir das trioses fosfato, acetona e aminoacetona, um catabólito de treonina e glicina, gerado na matriz mitocondrial. A aminoacetona sofre oxidação enzimática, catalisada por aminoxidase sensível a semicarbazida (SSAO), ou química, catalisada por íons de cobre e ferro, produzindo metilglioxal, H2O2 e NH4 +. Sabendo que metilglioxal e H2O2 são capazes de induzir apoptose e/ou necrose em células produtoras de insulina (RINm5f) propomos uma possível atividade pró-oxidante da aminoacetona sobre células beta do pâncreas. O tratamento destas linhagens com aminoacetona/Cu(II) aumentou a morte celular, fluxo de Ca2+ intracelular, produção de NO, fragmentação do DNA, depleção dos níveis de glutationa reduzida (GSH), expressão gênica da proteína apoptótica Bax, enzimas antioxidantes - glutationa peroxidase (GPx), glutationa redutase (GRd), catalase e isoformas de superóxido dismutases (CuZnSOD e MnSOD) - e óxido nítrico sintase induzida (iNOS). Embora as concentrações normais e patológicas da aminoacetona, provavelmente seja muito menores que as usadas nos experimentos, sugerimos que, em tecidos de diabéticos, um acúmulo da aminoacetona em longo prazo pode conduzir a danos oxidativos e eventualmente morte das células beta do pâncreas.

Tissue damages induced by hyperglycemia in diabetics are characterized by four linked mechanisms: increased flux through the polyol pathway, protein kinase C (PKC) activation, increased hexosamine pathway activity and intracellular production of advanced glycation end product (AGE) precursors. The production of AGEs by modifying proteins and DNA agent, such as methylglyoxal, has been implicated in microvascular complications in diabetes: nephropathy, retinopathy and neuropathy. Methylglyoxal is putatively produced in vivo from trioses phosphate, acetone and aminoacetone, a catabolite of threonine and glycine synthesized in the mitochondrial matrix. Aminoacetone has been reported to undergo semicarbazide sensitive amine oxidase- catalyzed and copper- and iron-catalyzed oxidations by molecular oxygen to methylglyoxal, NH4 + ion and H2O2. Considering that methylglyoxal and H2O2 have been found to promote apoptosis/necrosis to insulin-producing cells (RINm5f), we propose a possible pro-oxidant role of aminoacetone in pancreatic beta-cells. Treatment of RINm5f cells with aminoacetone plus Cu(II) ion promotes an increase of non-viable cells, influx of Ca2+ ions, NO production, DNA fragmentation, depletion of reduced glutathione (GSH) levels, and increased mRNA expression of pro-apoptotic protein (Bax), antioxidant enzymes - glutathione peroxidase (GPx), glutathione reductase (GRd), MnSOD, CuZnSOD and catalase - and inducible nitric oxide synthase (iNOS). Although both normal and pathological concentrations of aminoacetone are probably much lower than those used here, it is tempting to propose that excess aminoacetone in diabetic patients, at long term, may drive oxidative damage and eventually death of pancreatic beta-cells.