Biomedical subjects

E Aizenman

Publications and source records attributed to E Aizenman.

At least 37 records · Page 2Linked to original sources

PubMed1998

Dihydrokainate-sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate.

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l-serine-O-sulphate, but not by l-alpha-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20-24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 +/- 9.8% in neuronal cultures (P < 0.001; n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05; n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 +/- 4.9 micrometer, compared to 1.2 +/- 0.3 micrometer in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 micrometer MK-801, 10 micrometer tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm) inhibited the 45calcium uptake stimulated by 30 micrometer N-methyl-d-aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced approximately 25% inhibition of 30 micrometer NMDA-induced current in cortical neurons. Dihydrokainate (1 mm) alone generated a small current (17% of the current produced by 30 micrometer NMDA) that was blocked by 30 micrometer 5,7-dichlorokynurenate and only weakly by 10 micrometer cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.

Animals↗

PubMed1998

Reverse Na+/Ca2+ exchange contributes to glutamate-induced intracellular Ca2+ concentration increases in cultured rat forebrain neurons.

Activation of ionotropic glutamate receptors causes increases in intracellular Ca2+ concentration ([Ca2+]i) and intracellular Na+ concentration in neurons. It has been suggested that reversal of the plasma membrane Na+/Ca2+ exchanger (NCE) may account in part for the rise in [Ca2+]i. Recently, KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) was reported to selectively inhibit the reverse mode of the NCE in non-neuronal cells. We investigated the effects of KB-R7943 on glutamate-stimulated increases in [Ca2+]i. In cultured rat forebrain neurons loaded with indo-1 acetoxymethyl ester, KB-R7943 inhibited the reverse mode of NCE (IC50 = 0.7 microM). When tested against kainate- (100 microM), N-methyl-D-aspartate- (30 microM), glutamate- (3 microM), or KCl- (50 mM) induced [Ca2+]i transients (15 sec, in the presence of Na+ and Ca2+), KB-R7943 inhibited these transients with IC50 values of 6. 6, 8.2, 5.2, and 2.9 microM, respectively. [Ca2+]i increases caused by a higher concentration of glutamate (100 microM) also were inhibited by KB-R7943 (10 microM). However, KB-R7943 had no effect on peak [Ca2+]i changes caused by prolonged application of glutamate and did not inhibit glutamate-induced neuronal injury. KB-R7943 did not inhibit N-methyl-D-aspartate- or kainate-induced whole-cell currents, nor did it substantially inhibit voltage-sensitive Ca2+ currents, excluding a direct inhibition of these ion channels. These results suggest that reverse NCE contributes to the immediate rise in [Ca2+]i resulting from glutamate receptor activation. However, reverse NCE becomes less important as the stimulus time is increased, and Ca2+ entry by this route is not critical for the expression of excitotoxic injury.

Animals↗

PubMed1998

An NR2B point mutation affecting haloperidol and CP101,606 sensitivity of single recombinant N-methyl-D-aspartate receptors.

Haloperidol and ifenprodil are N-methyl-D-aspartate (NMDA) receptor (NR) antagonists with preference for the NR1/NR2B subunit combination. Previous investigations utilizing 125I-MK801 binding assays with recombinant receptors distinguished certain structural determinants on the NR2B subunit for these two drugs, with glutamate 201 being critical for haloperidol sensitivity and arginine 337 being important for ifenprodil block. Other studies, however, suggested that these two sites pharmacologically overlap. In an attempt to resolve these discrepancies, we have characterized the actions of haloperidol and CP101,606, an ifenprodil analog, on the single-channel properties of NR1/NR2B(E201R) receptors transiently expressed in Chinese hamster ovary cells, because receptors formed by NR1/NR2B(R337K) appear to be nonfunctional. Haloperidol (10 microM) inhibited wild-type NR1/NR2B channels by decreasing the frequency of channel opening, whereas CP101,606 (0.5 microM) antagonized NR1/NR2B channel activity by decreasing both the open dwell time and the frequency of channel opening. The inhibitory actions of both drugs were virtually absent in the mutant NR1/NR2B(E201R) receptors. These results suggest that glutamate 201 is critical for both haloperidol and CP101,606 inhibition, thus demonstrating common features in the action of these two antagonists.

Animals↗

PubMed1997

Functional consequences of NR2 subunit composition in single recombinant N-methyl-D-aspartate receptors.

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-D-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5, 5'-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

Animals↗

PubMed1997

Effects of pyrroloquinoline quinone on glutamate-induced production of reactive oxygen species in neurons.

Pyrroloquinoline quinone may act as a free radical scavenger and also as a modulator of the NMDA receptor associated redox modulatory site. Using the oxidation sensitive dye dihydroethidium, we examined the effects of pyrroloquinoline quinone on free radical production in cultured forebrain neurons following glutamate receptor activation. Both glutamate (100 microM) and hydrogen peroxide (30 mM) produced a rapid increase in dihydroethidium fluorescence indicating dye oxidation. Pyrroloquinoline quinone (5-200 microM) effectively inhibited dihydroethidium fluorescence induced by glutamate but not by hydrogen peroxide. Glutamate-induced dihydroethidium fluorescence was inhibited by the thiol oxidant 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). Pyrroloquinoline quinone (50 microM) inhibited glutamate responses in control and in dithiothreitol treated neurons. However, pyrroloquinoline quinone did not further decrease the response to glutamate in DTNB treated neurons. These results suggest that pyrroloquinoline quinone inhibits the free radical-generating response to glutamate by oxidizing the NMDA receptor redox site and not by scavenging reactive oxygen species.

Animals↗

PubMed1997

Intrinsic redox properties of N-methyl-D-aspartate receptor can determine the developmental expression of excitotoxicity in rat cortical neurons in vitro.

The sensitivity of central neurons in culture to N-methyl-D-aspartate (NMDA) receptor-mediated cell death increases with development. In this study, we show that this phenomenon in vitro may be due, at least in part, to changes in the redox properties of the NMDA receptor itself. With increasing days in culture, NMDA-induced electrical responses in rat cortical neurons are less sensitive to dithiothreitol-induced potentiation and spontaneously oxidize less readily than in younger cells. These results imply that at earlier developmental ages NMDA receptors prefer a more oxidized state. Hence, in the presence of a reducing agent, NMDA-induced neurotoxicity was produced in normally resistant younger neurons. The observed changes in NMDA receptor properties with development could not be attributed to long-range diffusible redox endogenous factors. An oxidized NMDA receptor thus confers maturing neurons a protective mechanism against glutamate toxicity during development.

Animals↗

PubMed1997

NMDA receptor-mediated neurotoxicity: a paradoxical requirement for extracellular Mg2+ in Na+/Ca2+-free solutions in rat cortical neurons in vitro.

Accumulation of intracellular Ca2+ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2+ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2+ content independently of Ca2+ to investigate the potential role of Mg2+ in excitotoxicity in rat cortical neurons in vitro. In Ca2+-free solutions in which the Na+ was replaced by N-methyl-D-glucamine or Tris (but not choline), which also contained 9 mM Mg2+, exposure to 100 microM glutamate or 200 microM NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2+ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2+-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg2+ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.

Animals↗

PubMed1997

Trapping channel block of NMDA-activated responses by amantadine and memantine.

We investigated the mechanisms by which the antiparkinsonian and neuroprotective agents amantadine and memantine inhibit responses to N-methyl-D-aspartic acid (NMDA). Whole cell recordings were performed using cultured rat cortical neurons or Chinese hamster ovary (CHO) cells expressing NMDA receptors. Both amantadine and memantine blocked NMDA-activated channels by binding to a site at which they could be trapped after channel closure and agonist unbinding. For neuronal receptors, the IC50s of amantadine and memantine at -67 mV were 39 and 1.4 microM, respectively. When memantine and agonists were washed off after steady-state block, one-sixth of the blocked channels released rather than trapped the blocker; memantine exhibited "partial trapping." Thus memantine appears to have a lesser tendency to be trapped than do phencyclidine or (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[1,d]cyclihepten-5,1 0-imine (MK-801). We next investigated mechanisms that might underlie partial trapping. Memantine blocked and could be trapped by recombinant NMDA receptors composed of NR1 and either NR2A or NR2B subunits. In these receptors, as in the native receptors, the drug was released from one-sixth of blocked channels rather than being trapped in all of them. The partial trapping we observed therefore was not due to variability in the action of memantine on a heterogeneous population of NMDA receptors in cultured cortical neurons. Amantadine and memantine each noncompetitively inhibited NMDA-activated responses by binding at a second site with roughly 100-fold lower affinity, but this form of inhibition had little effect on the extent to which memantine was trapped. A simple kinetic model of blocker action was used to demonstrate that partial trapping can result if the presence of memantine in the channel affects the gating transitions or agonist affinity of the NMDA receptor. Partial trapping guarantees that during synaptic communication in the presence of blocker, some channels will release the blocker between synaptic responses. The extent to which amantadine and memantine become trapped after channel block thus may influence their therapeutic effects and their modulation of NMDA-receptor-mediated excitatory postsynaptic potentials.

Amantadine↗

PubMed1996

An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.

Bacterial Proteins↗

PubMed1996

Pharmacological properties of acquired excitotoxicity in Chinese hamster ovary cells transfected with N-methyl-D-aspartate receptor subunits.

The cytotoxicity induced by the transient expression of functional N-methyl-D-aspartate (NMDA) receptors has been examined with the use of a luciferase reporter assay in Chinese hamster ovary cells. Various NMDA receptor antagonists, in a dose-dependent manner, prevented a loss of luciferase activity 24 to 48 hr post-transfection of either the NR1/NR2A or NR1/ NR2B subunit receptor configurations, likely correlating to the time required to express functionally these receptors. Both glutamate and NMDA were potently cytotoxic to transfected cells previously protected by antagonists. The novel ifenprodil analog (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP101,606-27) protected cells expressing NR1/NR2B, but not those cells expressing either NR1/NR2A or, putatively, NR1/NR2A/NR2B. Decreased cytotoxicity was observed when a mutated NR1 subunit (N616R) with reduced Ca++ permeability was used in coexpression studies with NR2A or NR2B. In contrast to our results with NR1/NR2A or NR1/NR2B, cells expressing NR1/NR2C did not perish. Our studies suggest that expression of functional NMDA receptors in non-neuronal cells leads to a form of excitotoxicity similar to that observed in mammalian neurons in vitro.

Animals↗

PubMed1995

Modulation of N-methyl-D-aspartate receptors by hydroxyl radicals in rat cortical neurons in vitro.

Oxygen-derived reactive species generated by xanthine/xanthine oxidase (X/XO) can modulate the N-methyl-D-aspartate (NMDA) receptor via its redox-sensitive site. Here it is shown that hydroxyl radicals are the agents responsible for NMDA receptor oxidation by X/XO. Spectrophotometric assays revealed that the amounts of superoxide anion and H2O2 produced by X/XO were not decreased by the hydroxyl radical-specific scavenger mannitol. This sugar, however, could prevent most of the oxidizing actions of NMDA receptors by X/XO, but not by the thiol oxidizing agent 5,5 -dithio-bis-nitrobenzoic acid. Finally, a non-enzymatic source of hydroxyl radicals was also effective in oxidizing the receptor's redox site. Hydroxyl radicals may thus represent the final common pathway for the modulation of NMDA receptor function by oxygen-derived free radical generating systems.

Animals↗

PubMed1995

Iron-mediated oxidation of 3,4-dihydroxyphenylalanine to an excitotoxin.

2,4,5-Trihydroxyphenylalanine (TOPA) oxidizes in solution to form a quinone derivative that is a non-NMDA glutamatergic agonist and neurotoxin. DOPA can autoxidize in physiological solutions to form small amounts of both TOPA and TOPA quinone. We report here that this conversion can be dramatically enhanced by iron (II) alone, but more so by iron (II) in the presence of hydrogen peroxide. This conversion is of sufficient magnitude that the resulting product can elicit non-NMDA, glutamate receptor-mediated electrical responses in cultured cortical neurons isolated from rat. This finding suggests that TOPA quinone may play a role in pathological processes involving abnormal iron metabolism in catecholaminergic neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1995

TOPA quinone, a kainate-like agonist and excitotoxin is generated by a catecholaminergic cell line.

The quinone derivative of 2,4,5-trihydroxyphenylalanine (TOPA) is a selective non-NMDA agonist and excitotoxin. While 3,4-dihydroxyphenylalanine (DOPA)-containing physiological solutions have been shown to generate TOPA and TOPA quinone (TOPA compounds), there have been no previous reports demonstrating the formation of this toxin in biological preparations. Here, using a pheochromocytoma catecholaminergic clonal cell line (PC12), we have identified TOPA compounds as by-products of catecholamine synthesis. PC12 cells incubated for 45 min with 30 microM tyrosine as a catecholamine precursor produced 1.0 +/- 0.2 pmol/10(6) cells of total TOPA compounds. The formation of these compounds could be enhanced nearly twofold when the cells were stimulated with 56 mM KCl. Moreover, the addition of a DOPA decarboxylase inhibitor (30 microM NSD-1015) increased the formation of TOPA compounds in both the unstimulated and stimulated conditions to a maximum of 5.5 +/- 0.7 pmol/10(6) cells after a 45 min incubation. A time-course analysis revealed that DOPA production above baseline levels coincided with the detectable generation of TOPA compounds. Finally, we observed an inhibition of TOPA compounds formation by 100 microM reduced glutathione, suggesting that these catecholamine products are formed from the extracellular autoxidation of DOPA. We propose that TOPA quinone may be an underestimated component of catecholamine toxicity that could be partly responsible for the demise of neurons in several neurodegenerative disorders, including Parkinson's and Huntington's disease. In addition, TOPA quinone may represent the first identified selective non-NMDA agonist that may be synthesized in the brain.

Adrenal Gland Neoplasms↗

PubMed1994

Stable transfection of the NR1 subunit in Chinese hamster ovary cells fails to produce a functional N-methyl-D-aspartate receptor.

The NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor cDNA was stably transfected into Chinese hamster ovary (CHO) cells. Northern analysis revealed 3 clonal cell lines expressing high levels of NMDAR1 mRNA (BA1, BA2 and BA3). NMDAR1 protein was readily detected by Western analysis in only one of the clonal populations, BA1. Whole-cell patch clamp analysis revealed a lack of functional NMDA receptors in all of the cell lines, including BA1. NMDA-mediated responses were obtained only after a second subunit, NMDAR2A, was transiently expressed in BA1 cells. Therefore, the NR1 subunit alone may not be sufficient to form functional NMDA receptors in CHO cells.

Animals↗

PubMed1994

Further evidence that pyrroloquinoline quinone interacts with the N-methyl-D-aspartate receptor redox site in rat cortical neurons in vitro.

In this study, we show that the essential nutrient pyrroloquinoline quinone (PQQ; 50 microM) regulates N-methyl-D-aspartate (NMDA; 10 microM) receptor activity primarily by reversing the increase in the frequency of openings of the receptor-associated ion channel after chemical reduction with dithiothreitol (DTT; 1 mM). Similar to other redox-active agents, PQQ (50-200 microM) had no effect on the single-channel conductance or arithmetic mean open time of NMDA-activated events. In other experiments, we observed that inhibitory effects of PQQ (50 microM) on NMDA (30 microM)-induced whole-cell responses could be abolished by prior N-ethylmaleimide (500 microM) alkylation of the putative thiol residues that likely comprise the redox site of the receptor. These results demonstrate that PQQ modulates the NMDA receptor by directly oxidizing its redox modulatory site.

Animals↗

PubMed1994

The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic brain injury.

Pyrroloquinoline quinone is a ubiquitous redox cofactor and putative essential nutrient in mammals. Pyrroloquinoline quinone has recently been demonstrated to depress N-methyl-D-asparate induced electrical responses and is neuroprotective in vitro. In addition, pyrroloquinoline quinone has been demonstrated to act as a free radical scavenger in mammalian tissues. In this study, we demonstrate a neuroprotective effect of pyrroloquinoline quinone in an in vivo cerebral hypoxia/ischemia model in the rodent. Significant reduction in infarct size resulted from pyrroloquinoline quinone pretreatment and also when pyrroloquinoline quinone was administered following induction of hypoxia/ischemia. The neuroprotective effect was not dependent on change in core or cranial temperatures, as there was no difference between temperature measurements in pyrroloquinoline quinone-treated and vehicle-treated controls. No changes in electroencephalographic activity were observed at neuroprotective doses. These findings suggest that pyrroloquinoline quinone may represent a novel class of quinoid reagents of potential use in the treatment of neurological disorders that involve excitotoxicity. This study demonstrates a protective effect of the novel essential nutrient pyrroloquinoline quinone on brain injury in a rodent model of cerebral hypoxia/ischemia. Pyrroloquinoline quinone was neuroprotective when administered before and even after the insult, and did not appear to have significant neurobehavioral side effects. Pyrroloquinoline quinone represents a new class of agents with potential use in the therapy of stroke.

Animals↗

PubMed1994

Comparison of the potency of competitive NMDA antagonists against the neurotoxicity of glutamate and NMDA.

The object of this investigation was to determine whether glutamate uptake affects the apparent potency of the competitive antagonists DL-2-amino-5-phosphonovalerate and CGS-19755 in blocking NMDA receptor-mediated neurotoxicity. In astrocyte-rich rat cortical cultures we observed that DL-2-amino-5-phosphonovalerate and CGS-19755 were 24 and 16 times more potent against NMDA than against glutamate-induced toxicity. In contrast, DL-2-amino-5-phosphonovalerate was equipotent against the two agonists in astrocyte-poor cultures, in which dendrites are directly exposed to the extracellular medium. With the noncompetitive NMDA antagonist MK-801, similar potencies were observed against glutamate (212 +/- 16 nM) and against NMDA (155 +/- 9 nM) neurotoxicity. These results may be explained if we assume that the neuronal cell body is less susceptible than the dendrites to NMDA receptor-mediated toxicity, and that the action of glutamate in astrocyte-rich cultures is confined to the cell body. In this case, one would expect that higher concentrations of glutamate would be needed to produce toxicity in astrocyte-rich cultures, and that higher concentrations of competitive antagonists would be needed to overcome this toxicity. Our observations help explain the pharmacology of the competitive NMDA antagonists against NMDA receptor-mediated neurotoxicity but also suggest the possibility that, because the cell body and dendrites may be distinct sites for neurotoxicity, they might also involve different mechanisms of toxicity.

2-Amino-5-phosphonovalerate↗

PubMed1993

Studies on the effects of several pentamidine analogues on the NMDA receptor.

We have investigated the interactions of six analogues of pentamidine with the N-methyl-D-aspartate (NMDA) receptor complex. All six compounds were effective NMDA receptor antagonists based upon their ability to inhibit [3H]dizocilpine binding to rat brain membranes. IC50 values ranged from 2 to 18 microM, and all compounds had Hill coefficients in excess of 1 suggesting a non-competitive interaction with [3H]dizocilpine. All compounds also inhibited NMDA- and glycine-induced intracellular Ca2+ changes measured in cultured rat forebrain neurons using the fluorescent indicator, fura-2. IC50 values in this assay ranged from 0.4 to 4.7 microM. Whereas pentamidine is directly toxic to cultured neurons, this was not a consistent finding with the pentamidine analogues tested, indicating that the toxic effects are not related to NMDA receptor antagonism. Finally, all of the agents tested were also effective in protecting neurons from NMDA-induced neurotoxicity. These data emphasize the possible utility of pentamidine-like drugs as neuroprotective agents and suggest that it is possible to generate compounds with a wider margin of safety than pentamidine itself.

Animals↗