Still NAAG'ing After All These Years: The Continuing Pursuit of GCPII Inhibitors.

Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca(2+)-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) (Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy.

The central nervous system effects, pharmacokinetics and safety of the NAALADase-inhibitor GPI 5693.

AIM: The aim was to assess the central nervous system (CNS) effects, pharmacokinetics and safety of GPI 5693, an inhibitor of a novel CNS-drug target, NAALADase which is being evaluated for the treatment of neuropathic pain. METHODS: This was a double-blind, placebo-controlled, exploratory study in healthy subjects receiving oral GPI 5693 single ascending doses of 100, 300, 750, 1125 mg with a placebo treatment randomly interspersed. An open-label, parallel extension examined the effects of food and sex on the pharmacokinetics of 750, 1125 and 1500 mg doses. Blood samples were collected for pharmacokinetic and biochemical/haematological safety analysis, vital signs, ECG and adverse event checks were performed regularly up to 48 h postdose. Postdose CNS effects were assessed using eye movements, adaptive tracking, electroencephalography (EEG), body sway and Visual Analogue Scales (VAS). RESULTS: CNS effects were mainly observed after the 1125 mg dose, showing a significant decrease of adaptive tracking performance, VAS alertness and VAS mood, and an increase of EEG occipital alpha and theta power. Gastro-intestinal (GI) adverse effects were frequent at higher doses. No clinically significant changes in vital signs or ECG were noted during any of the treatments. The therapeutically relevant concentration range (950-11 100 ng ml(-1)) as determined from animal experiments was already reached after the 300 mg dose. C(max) after the 300 mg and 750 mg dose was 2868 and 9266 ng ml(-1) with a t(1/2) of 2.54 and 4.78 h, respectively. Concomitant food intake (with the 750 mg and 1125 mg doses) reduced C(max) by approximately 66% and AUC by approximately 40%. With concomitant food intake, the dose-normalized C(max) also decreased significantly by -5.6 (CI: -2.6 to -8.7) ng ml(-1) mg(-1). The pharmacokinetic variability was largest after the 300 mg and 750 mg dose, resulting in a SD of approximately 50% of the C(max). CONCLUSION: NAALADase inhibition with GPI 5693 was safe and tolerable in healthy subjects. Plasma concentrations that were effective in the reversal of hyperalgesia in the chronic constrictive injury animal model of neuropathic pain were obtained at doses of 300, 750 and 1125 mg in the fasted state. Comcomitant food intake reduced C(max) and AUC. CNS effects and GI AEs increased in incidence over placebo only at the 1125 mg dose.

Pharmacokinetics and pharmacodynamics of the new propofol prodrug GPI 15715 in rats.

BACKGROUND AND OBJECTIVE: We studied the pharmacokinetics and pharmacodynamics of GPI 15715 (Aquavan injection), a new water-soluble prodrug metabolized to propofol by hydrolysis. METHODS: Nine adult male Sprague-Dawley rats (398 +/- 31 g) received a bolus dose of 40 mg GPI 15715. The plasma concentrations of GPI 15715 and propofol were determined from arterial blood samples, and the pharmacokinetics of both compounds were investigated using compartment models whereby the elimination from the central compartment of GPI 15715 was used as drug input for the central compartment of propofol. Pharmacodynamics were assessed using the median frequency of the EEG power spectrum. RESULTS: A maximum propofol concentration of 7.1 +/- 1.7 microg mL(-1) was reached 3.7 +/- 0.2 min after bolus administration. Pharmacokinetics were best described by two-compartment models. GPI 15715 showed a short half-life (2.9 +/- 0.2 and 23.9 +/- 9.9 min), an elimination rate constant of 0.18 +/- 0.01 min(-1) and a central volume of distribution of 0.25 +/- 0.02 L kg(-1). For propofol, the half-life was 1.9 +/- 0.1 and 45 +/- 7 min, the elimination rate constant was 0.15 +/- 0.02 min(-1) and the central volume of distribution was 2.3 +/- 0.6 L kg(-1). The maximum effect on the electroencephalogram (EEG)--EEG suppression for >4 s--occurred 6.5 +/- 1.2 min after bolus administration and baseline values of the EEG median frequency were regained 30 min later. The EEG effect could be described by a sigmoid Emax model including an effect compartment (E0 = 16.9 +/- 7.9 Hz, EC50 = 2.6 +/- 0.8 microg mL(-1), ke0 = 0.35 +/- 0.04 min(-1)). CONCLUSIONS: Compared with known propofol formulations, propofol from GPI 15715 showed a longer half-life, an increased volume of distribution, a delayed onset, a sustained duration of action and a greater potency with respect to concentration.

N-Acetylated alpha-linked acidic dipeptidase converts N-acetylaspartylglutamate from a neuroprotectant to a neurotoxin.

We previously reported that inhibition of the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase; glutamate carboxypeptidase II) robustly protects cortical neurons from ischemic injury. Since NAALADase hydrolyzes N-acetylaspartylglutamate (NAAG) to glutamate we hypothesized that inhibiting NAALADase would both decrease glutamate and increase NAAG. Increasing NAAG is potentially important because NAAG is a metabotropic glutamate receptor agonist and an N-methyl-D-aspartate (NMDA) partial antagonist, both of which have previously been shown to be neuroprotective. To understand the likely effects of endogenous NAAG in the central nervous system, we have now investigated the activity of NAAG in primary cortical cultures while manipulating NAALADase activity. Under hydrolyzing conditions, when NAALADase was active, NAAG had toxic effects that were blocked by NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists and by NAALADase inhibition. NAAG's toxic effects were presumably due to the liberation of glutamate. Under nonhydrolyzing conditions, when NAALADase was inhibited, NAAG demonstrated neuroprotective effects against both NMDA toxicity and metabolic inhibition. In the case of NMDA-induced toxicity, NAAG provided neuroprotection through its partial antagonist activity at the NMDA receptor. In the case of metabolic inhibition, NAAG had an additional neuroprotective effect mediated through its agonist properties at the type II metabotropic glutamate receptor. These results indicate that NAAG might play an important role in the central nervous system, under certain pathological conditions, as a neurotoxin or as a neuroprotectant, depending on the activity of NAALADase.

Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

Toxicity induced by a polyglutamated folate analog is attenuated by NAALADase inhibition.

Folates have been shown to be neurotoxic and convulsive. Endogenously, folates exist in the brain in a polyglutamated form with 1-7 terminal glutamates (approx. 1 microM). The brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase) has been shown to remove sequentially the gamma-linked glutamates from folic acid polyglutamates. We report that, at high concentrations (300 microM-30 mM), a folic acid hexaglutamate analog is dose-dependently toxic to dissociated rat cortical cultures and that this toxicity is reversed by 2-PMPA, a potent and selective NAALADase inhibitor. These data suggest a new mechanism for folic acid toxicity.

Inhibition of virus-induced neuronal apoptosis by Bax.

The Bax protein is widely known as a pro-apoptotic Bcl-2 family member that when overexpressed can trigger apoptosis in multiple cell types and is important for the developmental cell death of neurons. However, Bax was found here to be a potent inhibitor of neuronal cell death in mice infected with Sindbis virus. Newborn mice, which are highly susceptible to a fatal infection with neurotropic Sindbis virus, were significantly protected from neuronal apoptosis and fatal disease when infected with a recombinant Sindbis virus encoding Bax. Deletion of the N terminus of Bax, which mimics cleaved Bax, converted Bax into a pro-apoptotic factor in vivo. As mice mature during the first week after birth, they acquire resistance to a fatal Sindbis virus infection. However, Bax-deficient mice remained very sensitive to fatal disease compared with their control littermates, indicating that endogenous Bax functions as a survival factor and contributes to age-dependent resistance to Sindbis virus-induced mortality. The protective effects of Bax were reproduced in cultured hippocampal neurons but not in cultured dorsal root ganglia neurons. These findings indicate that cell-specific factors determine the anti-apoptotic versus pro-apoptotic function of Bax.

Blockade of NAALADase: a novel neuroprotective strategy based on limiting glutamate and elevating NAAG.

Excessive glutamate receptor activation is thought to be involved in the neuronal injury caused by stroke. Based on the hypothesis that N-acetyl-aspartyl-glutamate (NAAG) is a modulatory neurotransmitter or storage form of glutamate, we have pursued a novel strategy of therapeutic intervention, blockade of N-acetylated alpha-linked acidic dipeptidase (NAALADase), the enzyme that hydrolyzes NAAG to liberate glutamate. Using the suture model of transient middle cerebral artery occlusion (MCAO) in rats, the prototype NAALADase inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) dramatically reduced extracellular glutamate accumulation measured by microdialysis both during a 2-hour occlusion and during reperfusion, consistent with an effect on glutamate supply. During reperfusion, the decrease in glutamate was accompanied by an equimolar, reciprocal rise in extracellular NAAG. NAALADase inhibition may prove to be a well tolerated therapy for cerebral ischemia. In addition, NAALADase inhibitors should prove to be important tools in understanding the physiological role of NAAG in the brain.

Ion channels and exchangers that mediate ischemic neuronal injury.

Preventing the loss of ion homeostasis or promoting its recovery are two primary targets of neuroprotective strategies. The contribution of excitatory neurotransmitter receptor linked ion channels is now well established. Sodium and calcium may also enter neurons and glia through voltage sensitive channels and exchangers, contributing directly and indirectly to injury.

Regional vulnerability to endogenous and exogenous oxidative stress in organotypic hippocampal culture.

Organotypic cultures of the brain provide a unique opportunity to directly examine the regional vulnerability of specific brain regions like the hippocampus. Two well-characterized models of oxidative stress were used to examine the regional vulnerability of the hippocampus. Endogenous oxidative stress was induced by blocking synthesis of the endogenous antioxidant, glutathione with buthionine sulfoximine (BSO). Exogenous oxidative stress was induced with paraquat, an intracellular generator of superoxide. Injury was measured by quantitative fluorescence microscopy using the vital dye propidium iodide. BSO caused dose- and time-dependent injury that took at more than 24 h to develop. Injury began in discrete patches in the culture. In any given culture, each patch increased in size and intensity as incubation continued. The pattern was not clearly correlated with neuronal anatomy and may demonstrate glial vulnerability. Injury caused by BSO could be prevented with the antioxidants trolox or the 21-aminosteroid U-83836E, both of which are vitamin E derivatives. Paraquat also caused dose- and time-dependent injury, but the CA1 region of the hippocampus was most vulnerable. The same pattern of selective CA1 injury was caused by brief exposures to high concentrations and by prolonged exposures to much lower concentrations. Under some conditions, paraquat injury was prevented by iron chelation with deferoxamine or by blockade of either NMDA or AMPA/ kainate glutamate receptors. During paraquat exposure, glutathione concentration in the cultures was reduced prior to onset of propidium staining. The observation that the hippocampus has a similar selective regional pattern of vulnerability to paraquat and ischemia suggests that their mechanisms of injury may be related.

Cerebellar top-of-the-basilar syndrome.

We present a patient with unusual cerebrovasculature due to the absence of anastomosis between anterior and posterior circulations and bilateral fetal posterior cerebral arteries. This resulted in an atypical 'cerebellar' top-of-the-basilar syndrome, with bilateral superior cerebellar artery infarctions. We review the clinical presentation and radiologic findings and explain the embryologic origin of this vascular anatomy.

Protective effects of extracellular acidosis and blockade of sodium/hydrogen ion exchange during recovery from metabolic inhibition in neuronal tissue culture.

Acidosis is a universal response of tissue to ischemia. In the brain, severe acidosis has been linked to worsening of cerebral infarction. However, milder acidosis can have protective effects. As part of our investigations of the therapeutic window in our neuronal tissue culture model of ischemia, we investigated the effects of acidosis during recovery from brief simulated ischemia. Ischemic conditions were simulated in dissociated cortical cultures by metabolic inhibition with potassium cyanide to block oxidative metabolism and 2-deoxyglucose to block glycolysis. Lowering the extracellular pH (pH0) to 6.2 during metabolic inhibition had no effect on injury, as measured by lactate dehydrogenase release from cultures after 24 h of recovery. Lowering the pH0 during the first hour of recovery, in contrast, had profound protective effects. When the duration of metabolic inhibition was lengthened to 30 min, most of the protective effects of the NMDA receptor antagonist MK-801 were lost. However, the protective effects of acidosis were unchanged. This suggested that the protective effects of extracellular acidosis could be due to more than blockade of NMDA receptors. Intracellular acidosis might be responsible. To test this, recovery of intracellular pH (pH1) was slowed by incubation with blockers of Na+/H+ exchangers at normal pH0. The two compounds tested, dimethylamiloride and harmaline, had protective effects when present during recovery from metabolic inhibition. Measurements of pH1 confirmed that the blockers slowed recovery from intracellular acidosis; more rapid pH1 recovery was correlated with injury. The protective effects of acidosis could be reversed by brief incubation with the protonophore monensin, which rapidly normalized pH1. These results are the first demonstration of the protective effects of blocking Na+/H+ exchange in a model of cerebral ischemia. The protective effects of acidosis appear to arise either from suppressing pH-sensitive mechanisms of injury or from blocking sodium entry due to Na+/H+ exchange.

Toxic NMDA-receptor activation occurs during recovery in a tissue culture model of ischemia.

In some animal models of reversible ischemia, there is a therapeutic window during early recovery when glutamate receptor antagonists can rescue neurons from injury. We have previously reported that organotypic cultures of the hippocampus can be protected by NMDA-receptor antagonists during recovery from a brief period of simulated ischemia. To model ischemia, we have used potassium cyanide to inhibit oxidative metabolism and 2-deoxyglucose to inhibit glycolysis. To study the time course and mechanisms of delayed NMDA-receptor toxicity in more detail, we have extended these studies to dissociated cortical cultures. Injury was assessed by release of lactate dehydrogenase into the culture medium. Metabolic inhibition for 15 min caused dose-dependent injury. Morphologic signs of neuronal toxicity were delayed until the recovery period. MK-801 reduced injury significantly when present throughout the experiment. Surprisingly, MK-801 provided the same protection when administration was delayed until after the end of the metabolic inhibition, blocking NMDA receptors only during recovery. To examine NMDA toxicity during metabolic inhibition, the competitive NMDA-receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid was added during exposure. The protective effect of NMDA-receptor blockade was completely lost if the antagonist was removed during 1 min of continuing selective inhibition of oxidative metabolism. The toxic potency and effectiveness of glutamate were enhanced during metabolic inhibition, showing that receptors were not inactivated by simulated ischemia. These results are consistent with the specific hypothesis that return of oxidative metabolism triggers a critical period of toxic NMDA-receptor activation.

Neurotoxicity of acute glutamate transport blockade depends on coactivation of both NMDA and AMPA/Kainate receptors in organotypic hippocampal cultures.

Excessive activation of glutamate receptors is neurotoxic, contributing to brain injury caused by cerebral ischemia. The pharmacology of glutamate neurotoxicity is difficult to study in animals because it is efficiently cleared from the extracellular space by a family of glutamate transporters. We have investigated the receptor specificity of endogenous glutamate's toxic effects in organotypic cultures of the hippocampus by acute blockade of these transporters. The organotypic cultures used in these transporters. The organotypic cultures used in these experiments preserve the intrinsic connections and regional differentiation of the hippocampus in long term culture and may more closely reproduce the pharmacology of the mature brain region. Membrane injury was measured with digital fluorescence imaging of the vital dye, propidium iodide, 24 h after a 30-min exposure to glutamate receptor agonists or to antagonists of glutamate transport. Confirming our previous results, bath-applied, exogenous glutamate caused dose-dependent neuronal injury. Glutamate was less potent than the selective agonists NMDA, AMPA, and quisqualate. Blockade of glutamate transport with the selective antagonists threo-hydroxy-aspartate and pyrrolidine-dicarboxylic acid also caused dose-dependent neuronal injury. Endogenous or exogenous glutamate toxicity was caused by a coactivation of both NMDA and AMPA/kainate receptors; blockade of either was sufficient to substantially prevent neuronal injury. Protective effects of combined application of antagonists were generally less than additive. We conclude that AMPA/kainate receptors play a more prominent role in glutamate neurotoxicity in organotypic cultures than in dissociated cortical or hippocampal cultures, acting together with NMDA receptors to cause neuronal injury.

Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischemia.

BACKGROUND AND PURPOSE: The hippocampus demonstrates a regional pattern of vulnerability to ischemic injury that depends on its characteristic differentiation and intrinsic connections. We now describe a model of ischemic injury using organotypic hippocampal culture, which preserves the anatomic differentiation of the hippocampus in long-term tissue culture. METHODS: Ischemic conditions were modeled by metabolic inhibition. Cultures were briefly exposed to potassium cyanide to block oxidative phosphorylation and 2-deoxyglucose to block glycolysis. The fluorescent dye propidium iodide was used to observe membrane damage in living cultures during recovery. RESULTS: 2-Deoxyglucose/potassium cyanide incubation resulted in dose-dependent, regionally selective neuronal injury in CA1 and the dentate hilus, which began slowly after 2 to 6 hours of recovery. Subsequent histological examination of cultures after 1 to 7 days of recovery demonstrated neuronal pyknosis that was correlated with the early, direct observation of membrane damage with propidium. Both propidium staining and histological degeneration were prevented by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 when administered 30 minutes after the end of the exposure to 2-deoxyglucose and potassium cyanide. Tetrodotoxin, which blocks voltage-dependent sodium channels, had protective effects that were greatest during the period of 2-deoxyglucose and potassium cyanide incubation but also produced protection against the mildest conditions of metabolic inhibition when administered after 30 minutes of recovery. CONCLUSIONS: This in vitro model reproduced elements of the time course, regional vulnerability, and pharmacologic sensitivities of in vivo ischemic hippocampal injury. Inhibition of metabolism in organotypic culture provides a rapid, easily controlled injury and reproduces the in vitro pattern of hippocampal regional vulnerability to ischemia. It is the first in vitro model of ischemia to exhibit complete protection by delayed administration of an NMDA receptor antagonist during recovery from a brief insult. The protective effects of tetrodotoxin suggest that an early period of sodium entry into cells during and after ATP depletion may be responsible for the more prolonged period of toxic NMDA receptor activation.

The regional vulnerability to hypoglycemia-induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801.

Profound hypoglycemia selectively damages CA1 and the dentate gyrus of the hippocampus. We have examined the time course of hippocampal neuronal injury in organotypic cultures following in vitro "hypoglycemia," using the fluorescent vital dye propidium iodide to observe directly the regional distribution of early neuronal membrane injury in living cultures. The in vivo hippocampal pattern of hypoglycemic injury was reproduced by a 2 hr exposure to glucose-free media, which resulted in simultaneous, selective propidium staining of CA1 and the dentate gyrus starting by 4 hr after exposure. After 24 hr of recovery, CA3 remained spared. A similar pattern of propidium staining was produced by incubation of cultures for briefer periods in glucose-free medium containing 5 mM 2-deoxyglucose (2-DG) to inhibit glycolysis. This "hypoglycemic" pattern and time course of neuronal injury was mimicked by 300 microM aspartate but not by glutamate. The NMDA receptor antagonists MK-801 and CPP, but not the relatively selective non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, prevented the development of propidium staining. MK-801 protected against injury even if added to the recovery media 30 min after the insult, while TTX (10 microM) protected only if added by the end of the exposure. The appearance of propidium staining after 4-6 hr of recovery was well correlated with histological observation of pyknotic neuronal nuclei in the injured regions. The characteristic hippocampal regional vulnerability of CA1 and the dentate gyrus to injury following profound hypoglycemia can be reproduced in organotypic hippocampal culture and appears to be mediated both by an early TTX-sensitive component and by a more prolonged period of toxic NMDA receptor activation, extending for at least 30 min into the recovery period.

Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.

Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.

Enhancement of NMDA receptor-mediated neurotoxicity in the hippocampal slice by depolarization and ischemia.

Evidence from animal stroke models suggests that the proximate cause of neuronal degeneration after ischemia is massive release of glutamate and activation of NMDA receptors. However, in the physiologic presence of oxygen and glucose in the rat hippocampal slice preparation, the neurotoxicity of glutamate, as measured by inhibition of protein synthesis, requires high concentrations and is not prevented by glutamate receptor antagonists. Thus, the NMDA receptor-mediated neurotoxic effects of extracellular glutamate accumulation during ischemia might depend on additional factors, such as neuronal depolarization. In the experiments reported here, slices were exposed to glutamate in a medium intended to mimic the ionic conditions found during ischemia, high potassium (128 mM) and low sodium (26 mM). This depolarizing medium itself inhibited protein synthesis in a manner which was partially mediated by NMDA receptor activation, since it was significantly reversed by the noncompetitive NMDA antagonist, MK-801. Furthermore, the effect of glutamate under depolarizing conditions was also significantly decreased by MK-801, suggesting that glutamate was acting at NMDA receptors. Thus, depolarization appears to enhance the sensitivity of neurons to toxic NMDA receptor activation by glutamate. Under conditions that mimic ischemia, hypoxia plus hypoglycemia, a similar protective effect of NMDA receptor antagonists was observed. Depolarization and ischemia both appeared to attenuate the neurotoxicity of non-NMDA receptor agonists. It appears that under conditions of normal glucose and oxygen, high concentrations of bath applied glutamate inhibit protein synthesis at sites other than the NMDA receptor. However, when the Na+ gradient is decreased, as occurs during ischemia, glutamate's NMDA effects predominate. These findings suggest that ionic shifts may play a central role in permitting NMDA receptor-mediated ischemic neuronal damage.