Vicinal Thiols Research Articles

Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild-type TP53, and vicinal thiol/disulfide status

Nonresponse to remission-induction chemotherapy, which remains a major problem in acute myeloblastic leukemia (AML), has been linked to cellular resistance to apoptosis. Because the apoptosis induced by chemotherapeutic drugs is mediated by loss of mitochondrial transmembrane potential (MTP), it was postulated that sensitivity to mitochondrial membrane depolarization might be heterogeneous in AML. Using the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (mClCCP), the mitochondrial membrane sensitivity to depolarization (mClCCP concentrations that inhibit 50% of the transmembrane potential [IC(50)]) in AML blasts was measured and demonstrated marked interclonal heterogeneity, with the existence of comparatively sensitive (median mClCCP IC(50), 4 microM) and resistant (median mClCCP IC(50), 10 microM) clones. Furthermore, the mClCCP IC(50) was inversely associated with spontaneous in vitro apoptosis (P =.001). It was high in cases with mutant TP53 and correlated with the total cellular level of the multidrug resistance-associated protein (P =.019) but not of bcl-2, bax, or bcl-x. It was also found that the dithiol oxidant diamide, in contrast to the monovalent thiol oxidant diethyl maleate, increased the sensitivity of mitochondrial membranes to mClCCP. To confirm that TP53 directly affects MTP in leukemic cells and to establish the role of vicinal thiol oxidation in the TP53-dependent pathway, CEM 4G5 leukemia cells with forced, temperature-dependent expression of TP53 were studied. Monobromobimane, which inhibits mitochondrial membrane depolarization by preventing dithiol cross-linking, inhibited depolarization and apoptosis in 4G5 cells. It was concluded that in leukemia, TP53 and vicinal thiol/disulfide status are determinants of mitochondrial membrane sensitivity to depolarization, which is in turn associated with spontaneous apoptosis.

Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca 2+ release

Mitochondria contribute to the maintenance of the intracellular Ca 2+ homeostasis by taking up and releasing the cation via separate and specific pathways. The molecular details of the release pathway are elusive but its stimulation by the cross-linking of some vicinal thiols and consequently NAD + hydrolysis are known. Thiol cross-linking and NAD + hydrolysis can be achieved by addition of peroxynitrite (ONOO −), the product of the reaction between superoxide (O 2 −) and nitric oxide (nitrogen monoxide, NO ) to mitochondria. Mitochondria contain an NO synthase (mtNOS), which is stimulated by Ca 2+, and are a copious source of O 2 −. We show here that intramitochondrially formed ONOO − stimulates the specific, NAD +-linked Ca 2+ release from mitochondria. Our findings that upon Ca 2+ uptake mtNOS is stimulated, that ONOO − is formed, and that Ca 2+ is subsequently released from intact mitochondria suggest the existence of a feedback loop, which prevents overloading of mitochondria with Ca 2+.

G6PD Deficient Cells and the Bioreduction of Disulfides: Effects of DHEA, GSH Depletion and Phenylarsine Oxide

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.

Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine.

Peroxynitrite-dependent formation of nitrotyrosine has been associated with inactivation of various enzymes and proteins possessing functionally important tyrosines. We have previously reported an enzymatic activity modifying the nitrotyrosine residues in nitrated proteins. Here we are describing a nonenzymatic reduction of nitrotyrosine to aminotyrosine, which depends on heme and thiols. Various heme-containing proteins can mediate the reaction, although the reaction also is catalyzed by heme. The reaction is most effective when vicinal thiols are used as reducing agents, although ascorbic acid also can replace thiols with lesser efficiency. The reaction could be inhibited by (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, but not other tested NO donors. HPLC with electrochemical detection analysis of the reaction identified aminotyrosine as the only reaction product. The reduction of nitrotyrosine was most effective at a pH close to physiological and was markedly decreased in acidic conditions. Various nitrophenol compounds also were modified in this reaction. Understanding the mechanism of this reaction could help define the enzymatic modification of nitrotyrosine-containing proteins. Furthermore, this also could assist in understanding the role of nitrotyrosine formation and reversal in the regulation of various proteins containing nitrotyrosine. It also could help define the role of nitric oxide and other reactive species in various disease states.

Modulation of Tau Phosphorylation Within Its Microtubule‐Binding Domain by Cellular Thiols

Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Dithiothreitol Enhances Arsenic Trioxide-Induced Apoptosis in NB4 Cells

Recently, arsenic trioxide (As2O3) was reported to induce clinical remission in patients with acute promyelocytic leukemia. Modulation of protein phosphorylation by binding to the vicinal thiols has been suggested as a possible mechanism. We found that phenylarsine oxide, a strong vicinal thiol-binding agent, neither induced nuclear fragmentation or DNA laddering nor increased caspase activity in NB4 cells; however, As2O3 and a weak thiol-binding agent, dimethylarsinic acid, did increase activity. Dithiothreitol (DTT) effectively suppressed the phenylarsine oxide-inhibited cellular reductive capacity, but unexpectedly, enhanced As2O3-induced apoptosis in NB4 cells. As2O3-induced and As2O3-plus-DTT-induced apoptosis in NB4 cells was modulated by oxidant modifiers, but not by nitric oxide synthase inhibitors. These results demonstrate that DTT, a dithiol agent and known antidote for trivalent inorganic arsenic, enhances the toxicity of As2O3, thereby opening a new research direction for the mechanisms of arsenic toxicity and perhaps also helping in the development of new therapeutic strategies for treating leukemias.

Caspase Involvement in the Induction of Apoptosis by the Environmental Toxicants Tributyltin and Triphenyltin

Organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) can kill target cells by triggering apoptosis. The mechanism by which these environmental toxicants activate the apoptotic program is currently unclear. We have studied the effect of TBT and TPT in the human Hut-78 and Jurkat T-lymphocyte cell lines. Within 1 h there was a 30-fold increase in caspase activity, as measured by the cleavage of the fluorescent peptide DEVD-AMC. Morphological changes characteristic of apoptosis, such as membrane blebbing and nuclear fragmentation, were readily detectable. Blocking caspase activity with the peptide inhibitor z-VAD-fmk prevented all subsequent apoptotic changes. The optimal concentration range for induction of apoptosis was 0.5 to 5 μM TBT. TPT was also able to trigger caspase activity in the lymphocyte cell lines, but it took over 2 h to detect and occurred at a lower concentration range of 0.01 to 1 μM. Higher concentrations of TBT and TPT caused cell necrosis, and we showed that these concentrations were able to inhibit caspase activity in apoptotic cells. TBT and TPT were able interact with a vicinal thiol compound, similar to the known caspase inhibitor phenylarsine oxide, providing a potential mechanism for caspase inhibition. We propose that vicinal thiol proteins may be a general biological target of these organotin compounds, leading to the induction of caspase activity and apoptosis at low concentrations, and more extensive cell damage and necrotic cell death at higher concentrations.

Inhibition of tumor necrosis factor-alpha- and interleukin-1-induced endothelial E-selectin expression by thiol-modifying agents.

The expression of endothelial-leukocyte adhesion molecules has been postulated to be regulated by redox-sensitive events. Tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1 (IL-1)-induced E-selectin expression was analyzed after pretreating human umbilical vein endothelial cells with different thiol-modifying agents, ie, diamide, phenylarsine oxide, N-ethylmaleimide, and diethyl maleate. E-selectin protein expression was quantified by indirect immunofluorescence. All compounds suppressed the cytokine-induced E-selectin expression in a concentration-dependent manner, whereas the antioxidant N-acetylcysteine showed no effect. The inhibitory effect of diamide (100 micromol/L, 1 hour) was reversible within 6 hours when the cells were allowed to recover before application of cytokines. Reversibility was strongly delayed when cells were deprived of glutathione by buthionine sulfoximine pretreatment. Glutathione depletion alone did not influence cytokine-induced E-selectin expression. Analysis of cellular glutathione status showed a 3-fold increase in oxidized glutathione after diamide treatment. Monochlorobimane labeling also revealed a decrease in total cellular thiols. During recovery, the glutathione status was restored within 1 hour, whereas total thiol content and E-selectin expression needed at least 6 hours to return to baseline. Complete inhibition of E-selectin expression by the vicinal thiol blocker phenylarsine oxide (0.5 micromol/L) was reversed by dithiols like dithiothreitol or dimercaptopropanol, but not by the monothiol 2-mercaptoethanol. These data suggest that proteins with essential thiols, most probably vicinal thiols. are involved in the IL-1- and TNF-alpha-mediated induction of E-selectin. These thiols must be in the reduced state; oxidation or other modification thereof attenuates or abolishes the cells' response to the cytokines.

Nitric oxide and its congeners in mitochondria: implications for apoptosis.

Apoptosis is an evolutionarily conserved form of physiologic cell death important for tissue development and homeostasis. The causes and execution mechanisms of apoptosis are not completely understood. Nitric oxide (NO) and its congeners, oxidative stress, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. Recent findings strongly suggest that mitochondria contain a factor or factors that upon release from the destabilized organelles, induce apoptosis. We have found that oxidative stress-induced release of Ca2+ from mitochondria followed by Ca2+ reuptake (Ca2+ cycling) causes destabilization of mitochondria and apoptosis. The protein product of the protooncogene bcl-2 protects mitochondria and thereby prevents apoptosis. We have also found that NO and its congeners can induce Ca2+ release from mitochondria. Thus, nitrogen monoxide (.NO) binds to cytochrome oxidase, blocks respiration, and thereby causes mitochondrial deenergization and Ca2+ release. Peroxynitrite (ONOO-), on the other hand, causes Ca2+ release from mitochondria by stimulating a specific Ca2+ release pathway. This pathway requires oxidized nicotinamide adenine dinucleotide (NAD+) hydrolysis to adenosine diphosphate ribose and nicotinamide. NAD+ hydrolysis is only possible when some vicinal thiols are cross-linked. ONOO- is able to oxidize them. Our findings suggest that NO and its congeners can induce apoptosis by destabilizing mitochondria via deenergization and/or by inducing a specific Ca2+ release followed by Ca2+ cycling.

Thiol Chelation of Cu 2 By Dihydrolipoic Acid Prevents Human Low Density Lipoprotein Peroxidation

Mono-thiols can act either as pro- or anti-oxidants during metal-catalyzed low density lipoprotein (LDL) peroxidation, however investigation of the role of vicinal thiols has been neglected. Therefore dihydrolipoic acid (DHLA), a vicinal dithiol, and lipoic acid, its oxidized form, were used to investigate Cu 2+-mediated LDL peroxidation. We demonstrate here that DHLA inhibited Cu 2+-dependent LDL peroxidation by chelating copper. DHLA (0–20 μM) increased lag-times of conjugated diene formation in LDL (100 μg/ml) oxidized with 5 μM Cu 2+ in a concentration dependent manner, and this effect was saturated after 5 μM DHLA; enough to chelate all of the added Cu 2+. In a similar fashion DHLA prevented LDL-mediated reduction of Cu 2+ to Cu +. Lipoic acid had no effect in these systems. DHLA alone also reduced Cu 2+, however this was inhibited when DHLA was in excess of the copper concentration. Hence there is complex formation between the two species. Copper:DHLA complex formation was further investigated and found to be dependent upon pH and the presence of oxygen. At low pH (<6), or in the absence of oxygen, the complex is stable, presumably due to vicinal thiol chelation. As the pH is increased, the carboxylate group also participates in copper chelation, this results in a less stable complex which is susceptible to oxidation, and copper is eventually released. Electron spin resonance studies demonstrate the formation of hydroxyl, but not superoxide, radicals during Cu 2+-catalyzed DHLA oxidation. Thus in our LDL experiments at physiological pH, DHLA is able to either reductively inactivate Cu 2+ when Cu 2+ is in excess, or effectively chelate Cu 2+ when DHLA is in excess. The Cu 2+:DHLA complex eventually undergoes copper-catalyzed oxidation, copper is released and LDL peroxidation proceeds. DHLA, thus, has both pro- and antioxidant properties depending upon the ratio of Cu 2+:DHLA and the pH. These results provide an additional mechanism of thiol-mediated formation of radicals and metal chelation.

Purification and characterization of N-acetylglucosamine kinase from rat liver--comparison with UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

N-Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase (EC 2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39-kDa subunits. About 25% of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (EC 5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver. The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N-ethylmaleimide, iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol-modifying chemical reagents, sodium meta-periodate, sodium meta-arsenite/2,3-dimercaptopropanol and diazenedicarboxylic acid bis-N,N'-dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDP-GlcNAc 2-epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N-[3H]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.

Vicinal Thiols Are Involved in Inositol 1,2,3,5,6-Pentakisphosphate 5-Phosphatase Activity from Fetal Calf Thymus

Inositol 1,2,3,5,6-pentakisphosphate (Ins(1,2,3,5,6)P5) 5-phosphatase present in fetal calf thymus has been partially purified. This enzyme was inhibited dose-dependently by different thiol modifiers likeN-ethylmaleimide (NEM),p-chloromercuribenzene sulfonate (PCMBS), diamide, and phenylarsine oxide (PAO). The inhibition by PCMBS and diamide was protected by preincubation with dithiothreitol (DTT) and the phosphatase substrate, Ins(1,2,3,5,6)P5. Diamide, a compound that specifically modifies vicinal thiol groups, also blocked the 5-phosphatase dose-dependently. Specificity of this blockade was proven by using dimercaptopropanol (DMP), a compound known to protect vicinal thiol groups. DMP prevented the enzyme from inhibition by diamide. These data suggest that vicinal thiols are involved in Ins(1,2,3,5,6)P55-phosphatase activity.

General Method to Identify and Enrich Vicinal Thiol Proteins Present in Intact Cells in the Oxidized, Disulfide State

Some 5% of the soluble proteins of L1210 murine leukemia lymphoblasts contain surface vicinal thiols (Kalef, E., Walfish, P. G., and Gitler, C. (1993)Anal. Biochem.212, 325–334). Redox dithiol to intraprotein disulfide conversion could regulate the cellular function of these proteins. A general method is presented to identify and enrich vicinal thiol proteins existing in cells in their oxidized, disulfide state. The method is based on thein situblockage by cell permeableN-ethylmaleimide (NEM) of readily accessible cellular protein sulfhydryls. Following removal of the excess NEM, disulfide-containing proteins were identified by reduction with DTT and specific labeling withN-iodoacetyl-[125I]-3-iodotyrosine. The vicinal thiol proteins formed could also be enriched, prior to labeling with [125I]IAIT, by their selective binding to Sepharose–aminohexanoyl-4-aminophenylarsine oxide. Exponentially growing L1210 lymphoblasts contain more than 20 proteins with thiols in the oxidized, disulfide state. The majority derive from vicinal thiol proteins. The fraction oxidized, in some proteins, represents almost the totality of the protein present in the cell. Exposure of lymphoblasts to diamide increases the number and concentration of proteins with intraprotein disulfides. This method allows sensitive direct identification of vicinal thiol proteins that participate in redox regulation and those that are targets to oxidative stress conditions.

Neutrophil-associated inflammatory responses in rats are inhibited by phenylarsine oxide

NADPH oxidase is a phagocyte-specific enzyme which produces O 2 − and so initiates a cascade of reactive oxygen species formation. Inflammatory diseases involve overproduction of reactive oxygen species which induce tissue damage. Phenylarsine oxide has been described previously as a complete and direct inhibitor of NADPH oxidase in vitro that acts by covalently binding to vicinal thiol groups of a membrane-associated component of the enzyme. In the present work, the potential anti-inflammatory effect of phenylarsine oxide was tested on two experimental models in rats, carrageenan-induced paw oedema and lipopolysaccharide-mediated lung inflammation. Intraperitoneal injection of phenylarsine oxide reduced (i) reactive oxygen species production by rat phagocytes, (ii) neutrophil infiltration into the lung after inhalation of lipopolysaccharide and (iii) neutrophil-dependent oedema induced by carrageenan in hindpaws. We conclude that phenylarsine oxide has anti-inflammatory properties which are probably exerted by its ability to inhibit neutrophil NADPH oxidase-dependent reactive oxygen species production. The present work provides the basis for the development of new anti-inflammatory, arsenic-free agents reacting at the phenylarsine oxide site, which seems to be the Achilles' heel of NADPH oxidase. © 1997 Elsevier Science B.V. All rights reserved.

Reactive oxygen and nitrogen species regulate mitochondrial Ca2+ homeostasis and respiration.

The reduction of molecular oxygen to water provides most of the biologically useful energy. However, oxygen reduction is a mixed blessing because incompletely reduced oxygen species such as superoxide or peroxides are quite reactive and can, when out of control, cause damage. In mitochondria, where most of the oxygen utilized by eukaryotic cells is reduced, the dichotomy of oxygen shows itself best. Thus, reactive oxygen is a threat to them, as is evident from oxidative damage to mitochondrial lipids, proteins, and nucleic acids. Reactive oxygen, in the form of peroxides, also serves useful functions in mitochondria. This is exemplified by the control of mitochondrial and cellular calcium homeostasis, whose understanding has improved greatly during the last few years. An exciting new aspect is the discovery that nitric oxide and congeners have an enormous impact on mitochondria. Physiological concentrations of nitrogen monoxide (NO) at physiological cellular oxygen pressure inhibit cytochrome oxidase and thereby respiration. A transient inhibition of cytochrome oxidase by NO appears to be used in at least some forms of cell signalling. Peroxynitrite, the product of the reaction between superoxide and NO, can stimulate the specific calcium release pathway from mitochondria by oxidizing some vicinal thiols in mitochondria. There is evidence mounting that mitochondrial calcium handling and its modulation by reactive oxygen and nitrogen species is important for necrotic and apoptotic cell death.

Regulation of the thyroid NADPH-dependent H2O2 generator by Ca2+: studies with phenylarsine oxide in thyroid plasma membrane.

Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct action of Ca2+ on the PAO-enzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH: ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2.3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.

Inhibition of the pyridine nucleotide-linked mitochondrial Ca(2+) release by 4-hydroxynonenal: the role of thiolate-disulfide conversion.

Rat liver mitochondria contain a specific Ca(2+) release pathway which operates when intramitochondrial NAD(+) is hydrolyzed to ADPribose and nicotinamide. The molecular details of this pathway are incompletely understood. It has been reported that NAD(+) hydrolysis and therefore Ca(2+) release stimulated by t-butylhydroperoxide is prevented by 4-hydroxynonenal (HNE). The reason underlying inhibition by HNE, however, remained unclear. It has also been reported that NAD(+) hydrolysis and Ca(2+) release are stimulated when some vicinal thiols are cross-linked, as shown with phenylarsine oxide or gliotoxin (GT). We now show that HNE also prevents the GT-induced Ca(2+) release, but only when given before GT. Conversely, GT stimulates Ca(2+) release only when given before HNE. Inhibition of Ca(2+) release by HNE is reduced by its preincubation with thiol compounds, the effectiveness of which increases with decreasing pKa of their sulfhydryl group. Preincubation of HNE with glutathione at high, but not at low, pH similarly reduces inhibition of Ca(2+) release by HNE. These findings provide evidence that HNE inhibition of Ca(2+) release is due to a modification of mitochondrial thiolates in a way that their cross-linking is prevented, and give further insight into the regulation of Ca(2+) release from intact mitochondria.

Stimulation of Ca 2+ release from rat liver mitochondria by the dithiol reagent α-lipoic acid

Rat liver mitochondria contain a Ca 2+-specific release pathway stimulated by Ca 2+-dependent hydrolysis of oxidized intramitochondrial pyridine nucleotides to ADP ribose and nicotinamide. We have previously shown that NAD + hydrolysis and subsequent Ca 2+ release are inhibited by cyclosporine A and that they are only possible when some critical thiols are cross-linked or oxidized, e.g. by phenylarsine oxide, gliotoxin, or peroxynitrite. We now report that the antioxidant α-lipoic acid stimulates Ca 2+ release from intact mitochondria, i.e. with preservation of the mitochondrial membrane potential and without large-amplitude swelling. The release stimulated by α-lipoic acid is inhibited by cyclosporine A and is more effective when the pyridine nucleotides are oxidized. The results strongly suggest that α-lipoic acid stimulates the Ca 2+-specific release pathway from intact mitochondria by oxidizing some vicinal thiols, thereby stimulating hydrolysis of oxidized pyridine nucleotides. These observations further corroborate that intact rat liver mitochondria contain a specific Ca 2+ release pathway stimulated by modification of vicinal thiols. Prolonged stimulation of Ca 2+ release by lipoic acid followed by its re-uptake (Ca 2+ “cycling”) may contribute to the detrimental, prooxidant-like effects seen with higher concentrations of lipoic acid.

Focal Adhesion and Stress Fiber Formation Is Regulated by Tyrosine Phosphatase Activity

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.