Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.     

Cyanide interaction with redox modulatory sites enhances NMDA receptor responses.

Activation of NMDA receptors plays an important role in cyanide neurotoxicity. Cyanide indirectly activates the receptor by inducing neuronal release of glutamate and also enhances receptor-mediated responses by a direct interaction with the receptor complex. This study investigated the mechanism in cerebellar granule cells by which cyanide enhances NMDA-induced Ca2+ influx. Cyanide (50 microM) increased the influx of Ca2+ over the NMDA concentration range of 0.5-500 microM. Experiments showed that cyanide does not interact with the receptor's glycine or PKC mediated phosphorylation regulatory sites. N-ethylmaleimide, a thiol alkylating agent which inactivates the redox regulatory sites of the receptor, blocked the enhancing effect of cyanide. Pretreatment of cells with 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), a compound that oxidizes the receptor redox sites, had no effect on the response to cyanide. On the other hand, the nonpermeant reducing agents, dithiothreitol or cysteine, further increased the cyanide effect. These observations can be explained by cyanide interacting with redox sensitive disulfide groups that are not accessible to the non-permeant reducing agents. It is proposed that cyanide interacts with a redox site(s) located either on the intracellular receptor domain or in the transmembrane hydrophobic domain. Furthermore the enhancement by cyanide of the excitotoxic actions of NMDA involves receptor sites that are sensitive to oxidation/reduction and this interaction contributes to the neurotoxic action of cyanide.     

6,7-Dinitroquinoxaline-2,3-Dione Blocks the Cytotoxicity of N-Methyl-D-Aspartate and Kainate, but Not Quisqualate, in Cortical Cultures

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.     

Transient oxidation as a mechanistic strategy in enzymatic catalysis

Enzymes that employ a transient oxidation mechanism catalyze transformations that are overall redox neutral, but involve intermediates that have a higher oxidation state than the substrates or products. An oxidation/reduction sequence may be used directly to promote isomerization reactions or indirectly to permit the formation of stabilized intermediates such as carbanions. This review will focus on three recent examples of nicotinamide-dependent enzymes that have been found to employ transient oxidation during catalysis: ADP-L-glycero-D-manno-heptose 6-epimerase, GDP-mannose 3,5-epimerase, and the 6-phosphoglucosidases from family 4. These enzymes are remarkable in their ability to catalyze either nonstereospecific hydride transfers or multiple chemical steps within a single active site.     

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site

A novel modulatory site on the N-methyl-D-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.     

Interactions of Glutamate Receptor Agonists Coupled to Changes in Intracellular Ca2+ in Rat Cerebellar Granule Cells in Primary Culture

Changes in cytosolic free Ca2+ concentrations in response to glutamate receptor agonists and their interactions were studied in rat cerebellar granule cells grown on coverslips. The intracellular Ca2+ as measured with fura-2 increased by applying kainate (KA), quisqualate (QU), and N-methyl-D-aspartate (NMDA). The effect of KA could not be blocked by the NMDA receptor blocker 2-amino-5-phosphonovaleric acid (AP5). The KA- and QU-induced increase in intracellular free Ca2+ was also observed in a Na(+)-free medium, indicating that this response is not secondarily due to the depolarization. The effect of 10 microM QU on the KA-induced changes in cytosolic free Ca2+ was additive only at low KA concentrations, but QU at 0.1 mM totally blocked the response to KA. In the presence of 10 microM KA, the dose-response curve of QU became biphasic, whereas with 50 microM KA, a reduction of the response was seen around 1-100 microM QU. The effect of NMDA on the QU-induced response was additive only at low QU concentrations. It is proposed that rat cerebellar granule cells in primary culture express separate receptor-channel complexes for NMDA, QU, and KA, but interactions between agonists for these receptor sites exist. Thus, QU when present at intermediate concentrations seems to interact with the KA type of receptor, causing its desensitization. At high QU concentrations, an interaction of QU with the NMDA receptor site is apparent.     

Simple shifts in redox/thiol balance that perturb blood coagulation.

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.     

Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the membrane-bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a protonmotive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron-sulfur clusters transferring electrons between the two active sites. Here, we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc1 complex. No evidence for the 'reductant-induced oxidation' of ubiquinol could be detected; therefore no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.     

A tetrahydrobiopterin radical forms and then becomes reduced during Nomega-hydroxyarginine oxidation by nitric-oxide synthase

Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (>0.5 per heme) formed during reactions catalyzed by iNOSoxy that contained either H4B or 5-methyl-H4B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a heme-NO product complex. The biopterin radical then decayed within a 200-300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H4B (or 5MeH4B). These results provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H4B first provides an electron to a heme-dioxy intermediate, and then the H4B radical receives an electron from a downstream reaction intermediate to regenerate H4B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology.     

Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.     

Reduction-reoxidation cycles contribute to catalysis of disulfide isomerization by protein-disulfide isomerase

Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.     

Subunit-specific redox modulation of nmda receptors expressed in xenopus oocytes

Abstract

We have examined the effects of oxidizing and reducing agents on a number of subtypes of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA for the ε1 subunit from mouse to express homomeric receptors or with ε1 in combination with either ε1, ε2, ε3 or ε4 subunits to express heteromeric receptors. All heteromeric combinations resulted in receptors that were affected by the redox reagents, dithiothreitol (DTT) and 5-5-dithio-bis-2-nitrobenzoic acid (DTNB). However, the effects on the small currents from homomeric receptors were quite variable. The ε1/ε3 combination showed a greater enhancement by DTT than any of the other combinations. All four receptors expressed showed both a component of persistent potentiation and a slowly reversible component. The reversible component was largest for ε1/ε3. Additional experiments were done with S-nitrosocysteine (SNOC), a nitric oxide donor that may affect NMDA receptors by oxidation. SNOC had transient effects on the four heteromeric subunit combinations. The different sensitivities of particular subunit combinations may have pharmacological and clinical significance.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

A concerted,alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc(1) complex

A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron-sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c(1). A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol-cytochrome c reductase kinetics of the bc(1) complex include both a pH dependence and a dependence on Rieske iron-sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc(1) complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc(1) activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.     

Evidence for a concerted mechanism of ubiquinol oxidation by the cytochrome bc1 complex

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b(L) within the cytochrome bc(1) complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc(1) complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c(1) reduction by approximately 10-fold but had no effect upon the rate of cytochrome c(1) reduction in bc(1) complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c(1), reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b(H) and b(L), exert negative control on the rate of reduction of cytochrome c(1) and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b(H) and b(L) to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.     

Pharmacological evidence for N-methyl-D-aspartate receptors on nigrostriatal dopaminergic nerve terminals.

The efflux of tritium from rat striatal synaptosomes labelled with [3H]dopamine was utilized as an index of dopamine (DA) release for the purpose of characterizing the receptors underlying the effects of L-glutamate. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA), and kainate each induced DA release in the absence of Mg2+, through NMDA was much more efficacious and only the NMDA response was inhibited by Mg2+. The response to L-glutamate was potentiated in a concentration-dependent manner by glycine. Further, it was completely inhibited by the competitive NMDA antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and by the NMDA channel blocker phencyclidine. Finally, the response to L-glutamate was unaffected by either tetrodotoxin or the kainate-AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. These data demonstrate the presence of NMDA receptors on dopaminergic nerve terminals that mediate the ability of L-glutamate to release DA and suggest an additional mechanism by which information from the nigrostriatal and corticostriatal pathways may be integrated.     

Considerations on the mechanism of photosynthetic water oxidation – dual role of oxo-bridges between Mn ions in (i) redox-potential maintenance and (ii) proton abstraction from substrate water

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.     

N-Methyl-D-Aspartate Receptors and Ethanol: Inhibition of Calcium Flux and Cyclic GMP Production

Measurements of calcium uptake and cyclic GMP production by cerebellar granule cells grown in primary culture demonstrated that ethanol preferentially inhibited N-methyl-D-aspartate (NMDA) receptor-gated cation channel function. Concentrations of ethanol as low as 10 mM inhibited NMDA-stimulated Ca2+ uptake by greater than 30%, and ethanol also inhibited NMDA-stimulated (Ca2+-dependent) cyclic GMP accumulation in a similar, dose-dependent manner. Responses to kainate were significantly less sensitive to ethanol. Studies using various concentrations of NMDA, as well as phencyclidine (PCP) and glycine, suggested that ethanol affected the "coagonist" binding site of the NMDA receptor-channel complex, rather than the PCP recognition site.     

Redox regulation of neuronal migration in a Down Syndrome model

Down Syndrome (DS), one of the major genetic causes of mental retardation, is characterized by disrupted corticogenesis produced, in part, by an abnormal layering of neurons in cortical laminas II and III. Because defects in the normal migration of neurons during corticogenesis can result in delayed cortical radial expansion and abnormalities in cortical layering, we have examined neuronal migration in murine trisomy 16 (Ts16), a mouse model for DS. Using an in vitro assay for chemotaxis, our data demonstrate that the number of acutely dissociated Ts16 cortical neurons migrating in response to glutamate or N-methyl-D-aspartate (NMDA), known chemotactic factors, was decreased compared to normal littermates, suggesting a defect in NMDA receptor- (NMDAR-) mediated events. Ts16 neurons did not lack NMDAR since expression of mRNA and protein for NMDAR subunits was observed in Ts16 cells. However, the number of cells that generated an observable current in response to NMDA was decreased compared to normal littermates. Similar to DS, Ts16 CNS demonstrated an inherent oxidative stress likely caused by the triplication of genes such as SOD1. To determine if the abnormal redox state was a factor in the failure of NMDAR-mediated migration in Ts16, we treated Ts16 neurons with either n-acetyl cysteine (NAC) or dithiothrietol (DTT), known antioxidants. The reduction in NMDAR-mediated migration observed in Ts16 neurons was returned to normal littermate values by NAC or DTT. Our data indicate that oxidative stress may play a key role in the abnormal glutamate-mediated responses during cortical development in the Ts16 mouse and may have an impact on neuronal migration at critical stages.