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Principles in redox signaling: from chemistry to functional significance.
Full text linkReactive oxygen and nitrogen species are currently considered not only harmful byproducts of aerobic respiration
but also critical mediators of redox signaling. The molecules and the chemical principles sustaining the
network of cellular redox regulated processes are described. Special emphasis is placed on hydrogen peroxide
(H2O2), now considered as acting as a second messenger, and on sulfhydryl groups, which are the direct targets
of the oxidant signal. Cysteine residues of some proteins, therefore, act as sensors of redox conditions and are
oxidized in a reversible reaction. In particular, the formation of sulfenic acid and disulfide, the initial steps of
thiol oxidation, are described in detail. The many cell pathways involved in reactive oxygen species formation
are reported. Central to redox signaling processes are the glutathione and thioredoxin systems controlling H2O2
levels and, hence, the thiol/disulfide balance. Lastly, some of the most important redox-regulated processes
involving specific enzymes and organelles are described. The redox signaling area of research is rapidly expanding,
and future work will examine new pathways and clarify their importance in cellular pathophysiology.
Antioxid. Redox Signal. 18, 1557–159
Effect of pyruvate on rat heart thiol status during ischemia and hypoxia followed by reperfusion.
No full textIschemia or hypoxia followed by reperfusion determine a large release of glutathione from isolated and perfused rat heart. The effects of glucose and/or pyruvate administered during ischemia/reperfusion or hypoxia/reperfusion on the release of cytosolic and mitochondrial glutathione are compared. During ischemia, mitochondrial glutathione is released from the mitochondrion to the cytosol forming a unique pool that leaks out to the interstitial space. Reperfusion causes a large release of total glutathione, particularly from cytosol. Total sulfhydryl groups do not undergo modifications after ischemia, while they appear to decrease upon reperfusion. Pyruvate, which protects the heart by inducing a large recovery of the contractile activity after ischemia, markedly prevents the loss of glutathione. Also total sulfhydryl groups of mitochondria do not undergo significant variations upon ischemia and reperfusion in the presence of pyruvate. During hypoxia, in the absence of glucose, glutathione is mainly lost from the cytosol, while the mitochondrial pool appears to be preserved; in hypoxia, at variance with the ischemic conditions, pyruvate does not show any beneficial effect. The action of pyruvate appears to be multifactorial and its effects are discussed by considering its action on the hydrogen peroxide breakdown, protection of pyruvate dehydrogenase, anaerobic production of ATP and diminution of the intracellular concentration of inorganic phosphate
REDUCTION OF ADRENOCHROME BY RAT-LIVER AND BRAIN DT-DIAPHORASE
No full textLiver and brain exhibit DT-diaphorase activity with adrenochrome as a substrate; the latter is an o-quinone derived from the autoxidation of adrenaline exhibiting neurotoxic and cardiotoxic properties. The reaction is strongly inhibited by dicoumarol, a classical inhibitor of DT-diaphorase. DT-diaphorase-reduced adrenochrome undergoes autoxidation as shown by the oxygen uptake occurring during the reaction. It is proposed that, physiologically, DT-diaphorase might exert a protective role by maintaining adrenochrome in its reduced, non-toxic for
Inhibitory effect of pyruvate on release of glutathione and swelling of rat heart mitochondria.
No full textPyruvate prevents the permeability transition of rat heart mitochondria induced by the system calcium ions + phosphate or by the dithiol reagent phenylarsenoxide and measured as swelling. Since swelling induced by the latter is relieved by the dithiol 2,3-dimercaptopropanol (BAL), it is inferred that the effect of pyruvate might be mediated by the reduction of lipoic acid. In isolated mitochondria, pyruvate also exerts a protective effect when calcium + phosphate-induced swelling is exacerbated by hypoxic conditions. These results agree with our previous observations that pyruvate markedly prevents the loss of cytosolic and mitochondrial glutathione after ischemia or ischemia followed by reperfusion
Inhibition of rat liver mitochondrial permeability transition by respiratory substrates.
Full text linkThe mitochondrial inner membrane can undergo a permeability increase known as ''permeability transition'' elicited by Ca2+ and several other inducing agents. In general, the condition of oxidative stress acts as an inducer, at variance with antioxidants and reducing agents that inhibit the permeability transition. The action of mitochondrial respiratory substrates in preventing the permeability transition induced by Ca2+ and phosphate was examined; pyruvate, isocitrate, and glutamate proved to be particularly effective. The effect of substrates was evident also in the presence of an uncoupler, and, in addition, they were able to counteract the swelling stimulated by acetoacetate and tert-butylhydroperoxide. In the presence of various pyridine nucleotide-dependent substrates, mitochondria are able to reduce the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to an extent far larger than that calculated from the theoretical amount of total mitochondrial thiol groups, indicating the occurrence of a catalytic system. Similarly, the enzymes of the mitochondrial matrix in the presence of either NADH or NADPH are able to reduce DTNB. The results are discussed considering the existence of a close redox communication between pyridine nucleotides and membrane thiol groups, possibly mediated by dithiols such as thioredoxin and lipoic acid. (C
The role of adrenochrome in stimulating the oxidation of catecholamines
No full textAdrenochrome, a stable oxidation product formed after oxidation of adrenaline, strongly stimulates oxygen uptake occurring during the autoxidation of adrenaline, other catecholamines and ascorbate. Oxygen consumed is converted to hydrogen peroxide suggesting the occurrence of a redox cycling process. The reduction of adrenochrome operated by adrenaline is accelerated by the exclusion of oxygen indicating that the oxidation of adrenaline occurs directly and superoxide anion does not necessarily mediate it. Oxygen consumption, observed in the catecholamine/adrenochrome and ascorbate/adrenochrome systems, is due to the autoxidation of leucoadrenochrome that, at variance with adrenaline, easily autoxidizes also at physiological pH. Therefore, in these systems, leucoadrenochrome appears to be the major determinant of the production of superoxide anion
TOXICITY OF AMINOCHROMES
No full textThe first part of the present review deals with the chemical and enzymatic synthesis of adrenochrome and other aminochromes from the corresponding catecholamines. A description of the most significant pathways of formation and the reactivity of the aminochromes is presented. In the second part of the toxicity of aminochromes, mainly at the cardiac and CNS level, is described and some of the molecular mechanisms of the toxic action are outlined. The toxicity of the aminochromes appears to depend mainly on the production of reduced oxygen species through redox cycling. The interaction of aminochromes with sulfhydryl groups and the induced depletion of oxygen, ascorbate and glutathione are additional mechanisms resulting in noxious effects at a cellular level
Mitochondrial thioredoxin reductase and thiol status
No full textRAT-LIVER MITOCHONDRIA; PERMEABILITY TRANSITION; MAMMALIAN THIOREDOXIN; MEMBRANE-PERMEABILITY; SULFHYDRYL-GROUPS; REDOX REGULATION; 5,5'-DITHIOBIS-(2-NITROBENZOIC ACID); MOLECULAR-CLONING; CDNA CLONING; PURIFICATIO
Personal computer control of electrochemical detectors utilized for mitochondrial studies
No full textIn the present communication a personal computer control of electrodes particularly suited for mitochondrial research such as the oxygen electrode, the pH electrode and ion-selective electrodes is described. A personal computer equipped with a data acquisition board, a color monitor, a graphical programming software and a numerical analysis/graphics software provides complete instrumental control, data storage, processing and presentation of experimental data. The major objective of this work is the analysis and utilization of a virtual instrumentation software for data acquisition and control of electrochemical detectors; this may greatly improve the performance and flexibility of the system compared to traditional approaches such as the potentiometric recorders
Mitochondrial thioredoxin reductase: purification, inhibitor studies, and role in cell signaling
No full textMitochondrial thioredoxin reductase (TrxR2) maintains thioredoxin (Trx2) in a reduced state and plays a critical role in mitochondrial and cellular functions. TrxR2 has been identified in many different tissues and can be purified to homogeneity from whole organs and isolated mitochondria. Here we describe the detailed steps required to purify this enzyme. A different initial procedure is needed, according to whether purification starts from whole organs or from isolated and purified mitochondria. In the first case, acid precipitation is a critical preliminary step to separate mitochondrial thioredoxin reductase from the cytosolic isoform. Preparation involves ammonium sulfate fractionation, heating, and freeze/thaw cycles, followed by chromatographic passages involving DEAE-Sephacel, 2',5'-ADP-Sepharose 4B affinity, and omega-Aminohexyl-Sepharose 4B columns. The 2',5'-ADP-Sepharose 4B affinity step can be repeated to remove any contaminating glutathione reductase completely. Although several methods are available to detect the activity of this enzyme, reduction of DTNB is an easy and inexpensive test that can be applied not only to the highly purified enzyme but also to lysed mitochondria, provided non-TrxR2-dependent reaction rates are subtracted. TrxR2, like TrxR1, can be inhibited by several different and chemically unrelated substances, usually acting on the C-terminal containing the cysteine-selenocysteine active site. Many of these inhibitors react preferentially with the reduced form of the C-terminal tail. This condition can be evaluated by estimating enzyme activity after removal of the inhibitor by gel filtration of the enzyme preincubated in oxidizing or reducing conditions. Inhibition of thioredoxin reductase has important consequences for cell viability and can lead to apoptosis. Inhibition of TrxR2 causes large production of hydrogen peroxide, which diffuses from the mitochondrion to the cytosol and is responsible for most of the signaling events observed. Methods to measure hydrogen peroxide in isolated mitochondria or cultured cells are described
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