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E Aizenman

Publications and source records attributed to E Aizenman.

At least 55 records · Page 3Linked to original sources
PubMed1993

Allosteric modulation of the NMDA receptor by dihydrolipoic and lipoic acid in rat cortical neurons in vitro.

The mitochondrial cofactor dihydrolipoic acid (DHLA) was observed to potentiate N-methyl-D-aspartate (NMDA), but not non-NMDA, receptor-mediated whole-cell responses in cultured neurons. This potentiation was readily reversed by the oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB). DHLA was unable to increase NMDA responses previously potentiated by dithiothreitol, nor did it have an effect on NMDA receptors alkylated with N-ethylmaleimide. Single-channel recordings revealed that DHLA produced an increase in NMDA channel open frequency, with no change in single-channel conductance or open time. In contrast, lipoic acid reversed the potentiation of NMDA-evoked responses produced by dithiothreitol and had no effect on NMDA receptors previously oxidized by DTNB. DHLA and lipoic acid are pervasively found substances that readily permeate cellular membranes and thus may influence NMDA receptor activity in vivo by modifying its redox site.

Animals↗
PubMed1993

Nonenzymatic conversion of 3,4-dihydroxyphenylalanine to 2,4,5-trihydroxyphenylalanine and 2,4,5-trihydroxyphenylalanine quinone in physiological solutions.

2,4,5-Trihydroxyphenylalanine (TOPA) oxidizes in solution to form a quinone derivative that is a non-N-methyl-D-aspartate agonist and neurotoxin. Although pathways have been postulated for the formation of both TOPA and TOPA quinone from closely related catecholamines, the generation of these compounds has not been conclusively demonstrated by analytical techniques. Reverse-phase HPLC with a dual electrode coulometric detector was used to analyze TOPA containing solutions in an effort to rigorously characterize the behavior of this substance under physiological conditions. Electrode potential, buffer system, and methanol concentration were varied to obtain optimal conditions to selectively detect and quantify TOPA and TOPA quinone from closely related catecholamines. TOPA was shown to rapidly autoxidize to TOPA quinone by a process dependent on pH. TOPA was the dominant species at acidic pHs (below 5-6), whereas TOPA quinone was dominant at physiological pHs. This conversion was reversible upon acidification. In addition, we found that 3,4-dihydroxyphenylalanine can autoxidize to form both TOPA and TOPA quinone under physiological conditions. This partial conversion (0.5%) is time dependent and can be substantially decreased (0.2%) in acidic conditions (pH < or = 3). These results suggest that some of the excitatory and excitotoxic properties that some investigators have attributed to DOPA may actually be due to its conversion to TOPA and TOPA quinone.

Buffers↗
PubMed1993

The modulation of N-methyl-D-aspartate receptors by redox and alkylating reagents in rat cortical neurones in vitro.

1. The properties of sulfhydryl redox modulation of the N-methyl-D-aspartate (NMDA) receptor have been examined in rat cortical neurones in culture. Electrophysiological measurements were performed with the whole-cell and outside-out patch variants of the patch-clamp technique. 2. The disulphide reducing agent dithiothreitol (DTT; 0.1-10 mM) potentiated 10 microM NMDA-mediated whole-cell currents when applied slowly alone via the superfusate. The initial rate of reduction, as well as the degree of potentiation, was dependent on the concentration of DTT although the process was complicated by the fact that a second, large component appeared at a concentration of 10 mM of this agent. 3. DTT (0.1-10 mM) was also rapidly applied together with the agonist from a perfusion pipette. With this method, the second component was not readily apparent, and the concentration of DTT producing a half-maximal potentiation of the NMDA response was 1.9 +/- 0.3 mM. Two other disulphide reducing agents, ethylene glycol bisthioglycolate and meso-bis(N,N-dimethyl)adipamide-2,5-dithiol, also potentiated NMDA responses, but were not as effective as DTT. 4. Following a 4 mM DTT treatment, we observed that the NMDA receptor underwent spontaneous oxidation with a half-time of 1.9 min. In contrast, the sulfhydryl oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB; 500 microM) produced a more rapid reversal of the effects of DTT (t1/2 = 0.6 min). The spontaneously oxidized receptor could be further oxidized with DTNB and fully reduced with DTT. 5. After receptor oxidation with 500 microM DTNB, NMDA produced whole-cell responses with an EC50 of 68.4 +/- 9.4 microM, whereas after reduction with 4 mM DTT the EC50 for NMDA was 32.5 +/- 3.4 microM. In addition, the maximum response after reduction with DTT was substantially increased over that observed after oxidation. 6. Single channel measurements performed on outside-out patches revealed that reduction produced a dramatic increase in the number of NMDA-induced channel openings. We observed a 2.1 +/- 0.2-fold increase in the frequency of openings during reduction with 500 microM DTT when compared to patches which had been exposed to 500 microM DTNB. Small but significant differences were observed in the single channel conductance for the oxidized (34.6 +/- 1.1 pS) and reduced (37.6 +/- 1.5 pS) states of the receptor. In contrast, no significant changes were seen in the arithmetic mean channel open time between the two redox conditions (5.4 +/- 0.3 ms after oxidation, 6.0 +/- 0.7 ms after reduction).(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed1993

Long-lasting modification of the N-methyl-D-aspartate receptor channel by a voltage-dependent sulfhydryl redox process.

Ionic currents through the N-methyl-D-aspartate (NMDA) receptor channel are modulated by sulfhydryl redox reagents. We report here a novel form of redox modulation that alters NMDA channel kinetics in a voltage-dependent manner. The effects of the thiol reductant dithiothreitol (DTT) and the oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) on NMDA-activated whole-cell currents were examined at various transmembrane voltages in cultured rat cortical neurons. DTT produced a similar level of potentiation of NMDA-induced currents at both -60 mV and +30 mV. However, the reversal of this potentiation by a sulfhydryl-oxidizing agent was dependent on the holding potential, because DTNB decreased the DTT-potentiated NMDA responses more effectively at negative voltages. Interestingly, the NMDA peak current-voltage relationship became substantially outwardly rectifying when sequential DTT/DTNB treatments took place at a positive holding potential, but not under any other circumstances. Single-channel recordings from outside-out patches revealed that this phenomenon was likely produced by a significant and long-lasting 2.3-fold prolongation of the mean open time of NMDA channels at a positive holding potential. Thus, a voltage-dependent chemical alteration in NMDA receptor structure modified the kinetic properties of the associated ion channel.

Animals↗
PubMed1992

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons.

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.

Animals↗
PubMed1992

Glutathione prevents 2,4,5-trihydroxyphenylalanine excitotoxicity by maintaining it in a reduced, non-active form.

2,4,5-Trihydroxyphenylalanine (TOPA) in aqueous solution has been shown to form an non-N-methyl-D-aspartate (non-NMDA) agonist and neurotoxin, TOPA quinone. We examined whether the endogenous chemical reductant glutathione (GSH) could abolish the agonist properties of TOPA and block its excitotoxicity in rat cortical neurons in culture by preventing the formation of TOPA quinone. The oxidative formation of TOPA quinone from TOPA (30-500 microM) at pH 7.2 was measured spectrophotometrically. Using glutathione (0.05-3 mM) as the reducing agent, we found that the optimal [GSH]:[TOPA] ratio which significantly retarded TOPA quinone formation was 10:1. Thus, 3 mM GSH prevented whole-cell currents induced by a solution of 300 microM TOPA but did not affect currents elicited by 300 microM kainate. In addition, 2 mM GSH protected neurons from the toxic effects of 200 microM TOPA, but was not effective against 200 microM NMDA. These results suggest that the presence of endogenous reductants may limit the toxicity of TOPA.

Animals↗
PubMed1992

The action of CGS-19755 on the redox enhancement of NMDA toxicity in rat cortical neurons in vitro.

The effects of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) were studied in cultures of rat cerebral cortex under normal and altered redox conditions. CGS-19755 was effective in preventing delayed neuronal death produced by an acute exposure to either glutamate (500 microM) or NMDA (200 microM), but was ineffective in protecting neurons against the toxicity induced by a prolonged exposure to kainate (500 microM). We observed that the reducing agent dithiothreitol (DTT, 500 microM), could dramatically enhance toxicity and electrophysiological responses produced by 50 microM NMDA. CGS-19755 (100 microM) could effectively block both of these effects of DTT. Any toxicity produced by DTT alone was also antagonized by CGS-19755. In contrast, oxidized DTT did not enhance NMDA toxicity nor was it toxic when added alone. These results indicate that CGS-19755 is an effective and specific neuroprotectant acting at the NMDA receptor in vitro, and that the enhancement in NMDA toxicity induced by DTT is mediated by an increase in activity at this receptor complex.

Animals↗
PubMed1992

Interaction of the putative essential nutrient pyrroloquinoline quinone with the N-methyl-D-aspartate receptor redox modulatory site.

The putative essential nutrient pyrroloquinoline quinone (PQQ) can efficiently mediate reduction and oxidation reactions in a variety of systems. Therefore, we investigated whether this compound could alter the function of the NMDA receptor via a recently described redox modulatory site. In rat cortical neurons in vitro, 50 microM PQQ could reverse the enhancement of 30 microM NMDA-induced whole-cell ionic currents produced by the reducing agent dithiothreitol (DTT; 2-4 mM). PQQ also depressed native responses in a DTT-reversible fashion. In addition, 50-200 microM PQQ produced a significant degree of neuroprotection in an acute model of NMDA-mediated neurotoxicity in astrocyte-rich cultures of rat cerebral cortex. Under certain conditions, PQQ can lead to the formation of oxygen-derived free radicals, and we have previously observed that these reactive species can oxidize the NMDA receptor. Nevertheless, the enzymatic free radical scavengers superoxide dismutase and catalase (10 micrograms/ml each) did not abolish the actions of PQQ. This observation held true even in astrocyte-poor cortical cultures, where neuronal processes are directly exposed to the extracellular milieu. Therefore, under in vitro conditions in which PQQ is presented without an exogenous electron donor, it appears as if the entire neuroprotective effect of PQQ is attributable to a direct oxidation of the NMDA receptor redox site. These results suggest the possibility of a novel role for PQQ, PQQ-like substances, and quinone-containing proteins in the brain, and may represent a novel therapeutic approach for the amelioration of NMDA receptor-mediated neurotoxic injury.

Animals↗
PubMed1992

Pentamidine is an N-methyl-D-aspartate receptor antagonist and is neuroprotective in vitro.

Acquired immunodeficiency syndrome (AIDS) is frequently associated with dementia. The wide spectrum of neurological abnormalities associated with this dementia may involve a neurotoxin that activates the NMDA subtype of glutamate receptor in neurons. We have found that the antimicrobial agent pentamidine, which is prescribed for AIDS patients for the prophylaxis and treatment of Pneumocystis carinii pneumonia, is an effective NMDA receptor antagonist. Pentamidine inhibited 3H-dizocilpine binding to the NMDA receptor in rat brain membranes at a site separate from glutamate, glycine, and spermidine, with an affinity near 2 microM. Similar concentrations of pentamidine block NMDA-induced increases in intracellular Ca2+ and NMDA-induced currents in cultured forebrain and cortical neurons, apparently without use dependence or voltage dependence, suggesting that pentamidine may represent a novel chemical class of NMDA receptor antagonist. Finally, pentamidine protects neurons from the lethal effects of acute NMDA exposure in vitro. AS pentamidine may accumulate in the brain at relevant concentrations following repeated high-dose parenteral administration, these findings suggest that the drug may be neuroprotective in vivo.

Animals↗
PubMed1991

Oxidized glutathione modulates N-methyl-D-aspartate- and depolarization-induced increases in intracellular Ca2+ in cultured rat forebrain neurons.

We have investigated the interaction of reduced and oxidized glutathione (GSSG) with intracellular Ca2+ increases produced by N-methyl-D-aspartate (NMDA), kainate and KCl in primary cultures of forebrain neurons derived from fetal rats. Responses to NMDA, applied with glycine, were inhibited by GSSG (10 mM), but were unaffected by reduced glutathione and L-cysteine. Inhibition by GSSG was still apparent after cells were oxidized by 5,5'-dithio-bis-2-nitrobenzoic acid, and this effect showed spontaneous but only partial reversal. This suggests that modulation of the redox site on the NMDA receptor could not account for all of the effects produced by GSSG. However, the observation that complete recovery from GSSG treatment required exposure of cells to dithiothreitol suggests that oxidation of the redox site contributes to the action of GSSG. GSSG also inhibited responses produced by 50 mM KCl but not those produced by 50 microM kainate. The effects of GSSG on KCl responses were fully and rapidly reversible. These results suggest that high concentrations of GSSG may modulate NMDA receptors, and that some of the actions of GSSG may be mediated by the redox site on the receptor complex.

Amino Acids↗
PubMed1991

Effects of nicotinic agonists on the NMDA receptor.

The cholinergic nicotinic agonists (-)nicotine and lobeline were observed to partially inhibit whole-cell N-methyl-D-aspartate (NMDA)-induced responses in rat cortical neurons in culture. In addition we found that (-)nicotine, (+)nicotine, and lobeline, but not the nicotine metabolite (-)cotinine nor acetylcholine, were able to displace [3H]dizocilpine ([3H]MK 801) binding in well-washed membranes obtained from rat brain. These results show that certain nicotinic agonists can interact with the NMDA receptor and block its function.

Animals↗
PubMed1991

2,4,5-trihydroxyphenylalanine in solution forms a non-N-methyl-D-aspartate glutamatergic agonist and neurotoxin.

We have investigated the pharmacologic and neurotoxic properties of 2,4,5-trihydroxyphenylalanine [topa; the 6-hydroxylated derivative of 3,4-dihydroxyphenylalanine (dopa)] in central neurons. Application of solutions of topa to the chicken eyecup preparation results in glutamatergic responses mediated predominantly by non-N-methyl-D-aspartate receptors. Pharmacological activity depends upon oxidation in solution to a new compound. This compound is tentatively identified as topa quinone. Solutions of topa are toxic to cortical neurons in culture, and this toxicity is blocked by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. These results suggest that production or accumulation of topa or its oxidation products might be involved in excitotoxicity, especially in dopaminergic neurons and their projection targets.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

Overexpression of the relA gene in Escherichia coli.

Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.

Base Sequence↗
PubMed1991

N-methyl-D-aspartate antagonists prevent kainate neurotoxicity in rat retinal ganglion cells in vitro.

Under defined culture conditions, exogenous glutamate (Glu), NMDA, or an endogenous Glu-related toxin is lethal to rat retinal ganglion cells; these detrimental effects are NMDA receptor mediated because specific NMDA antagonists can prevent cellular injury. In the presence of an endogenous Glu-like toxin, 125 microM kainate (KA) increases the proportion of retinal ganglion cells that die, but the toxicity (due to both KA and the endogenous toxin) is totally prevented by 2-amino-5-phosphonovalerate (APV), a specific NMDA receptor antagonist. These findings indicate that the KA-induced portion of retinal ganglion cell death also appears to be mediated via NMDA receptors. There are at least 2 possible mechanisms for this lethal effect. In addition to KA receptors, KA could directly stimulate NMDA receptors. Alternatively, KA might activate its own specific receptor, which in turn leads to a net increase in the release of an endogenous Glu-related toxin; this endogenous substance would then activate NMDA receptors. Patch-clamp electrophysiology experiments have helped to distinguish between these possibilities. Concentrations of APV that completely block the current elicited by maximal nondesensitizing doses of NMDA exert no detectable inhibition of KA-evoked currents. Hence, at the concentrations used, it appears unlikely that KA directly activates NMDA receptors in this preparation. Furthermore, the fraction of toxicity attributed to the addition of KA can be blocked by the relatively specific non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This finding is consistent with the hypothesis that KA adds an increment of toxicity in this system by directly interacting with KA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1991

Autoradiographic localization of putative nicotinic receptors in the rat brain using 125I-neuronal bungarotoxin.

Neuronal bungarotoxin (NBT), a snake venom neurotoxin, selectively blocks nicotinic receptors in many peripheral and central neuronal preparations. alpha-Bungarotoxin (alpha BT), on the other hand, a second toxin isolated from the venom of the same snake, is an ineffective nicotinic antagonist in most vertebrate neuronal preparations studied thus far. To examine central nicotinic receptors recognized by NBT, we have characterized the binding of 125I-labeled NBT (125I-NBT) to rat brain membranes and have mapped the distribution of 125I-NBT binding in brain sections using quantitative light microscopic autoradiography. The binding of 125I-NBT was found to be saturable, of high affinity, and heterogeneously distributed in the brain. Pharmacological studies suggested that more than one population of sites is labeled by 125I-NBT. For example, one component of 125I-NBT binding was also recognized by alpha BT, while a second component, not recognized by alpha BT, was recognized by the nicotinic agonist nicotine. The highest densities of these alpha BT-insensitive, nicotine-sensitive sites were found in the fasciculus retroflexus, the lateral geniculate nucleus, the medial terminal nucleus of the accessory optic tract, and the olivary pretectal nucleus. alpha BT-sensitive NBT binding sites were found in highest density in the lateral geniculate nucleus, the subthalamic nucleus, the dorsal tegmental nucleus, and the medial mammillary nucleus (lateral part). The number of brain regions with a high density of 125I-NBT binding sites, blocked either by alpha BT or by nicotine, is low when compared with results obtained using other approaches to studying the central distribution of nicotinic receptors, such as labeling with 3H-nicotine or labeling with cDNA probes to mRNAs coding for putative receptor subunits. It is proposed that 125I-NBT labels a subpopulation of nicotinic receptors in the rat brain, and that, given its ability to block nicotinic receptors in a variety of neuronal preparations, NBT will be a useful probe for studying the functional properties of these sites.

Animals↗
PubMed1990

A 3,4-dihydroxyphenylalanine oxidation product is a non-N-methyl-D-aspartate glutamatergic agonist in rat cortical neurons.

Applications of solutions of 2,4,5-trihydroxyphenylalanine (TOPA or 6-hydroxyDOPA) to rat cortical neurons in culture monitored under whole-cell voltage clamp with patch electrodes resulted in currents which could be nearly completely blocked by the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only weakly antagonized by the NMDA antagonist D.L-2-amino-5-phosphonovalerate (APV). Thus, TOPA can generate glutamatergic responses by interacting preferentially with non-NMDA receptors in cortical neurons. As these results show that a product closely related to the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has glutamatergic agonist properties, it is conceivable that catecholamine-containing brain areas may be at special risk for excitotoxic damage under certain conditions.

2-Amino-5-phosphonovalerate↗
PubMed1990

Blockade of nicotinic responses in rat retinal ganglion cells by neuronal bungarotoxin.

The effects of neuronal bungarotoxin (NBT) on nicotinic acetylcholine responses recorded from rat retinal ganglion cells in culture were studied with patch electrodes. We observed that the concentration of this toxin needed to induce a total blockade of nicotinic currents varied according to the method of toxin application utilized. Rapid addition of 20 microM NBT by pressure ejection from micropipettes produced total blockade of 50-microM acetylcholine-induced currents. In contrast, when added slowly via the physiological solution which continuously superfused the cells, NBT was able to produce a complete blockade of the response at 200 nM. The IC50 determined for NBT by the superfusion method was 55 nM. Recovery from block was slow and incomplete with both drug application methods, although some differences were found. The results are discussed with reference to a scheme in which NBT binds with both low and high affinity to functional nicotinic receptors in rat retinal ganglion cells in culture.

Acetylcholine↗