Richard J. Bridges

Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversal

Na+‐dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. lthreo‐β‐hydroxyaspartate (l‐TBHA), dlthreo‐β‐benzyloxyaspartate (dl‐TBOA), ltrans‐pyrrolidine‐2,4‐dicarboxylic acid (ltrans‐2,4‐PDC) (+/–)‐cis‐4‐methy‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (cis‐4‐methy‐trans‐2,4‐PDC) and lantiendo‐3,4‐methanopyrrolidine‐2,4‐dicarboxylic acid (lantiendo‐3,4‐MPDC) inhibited l‐[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 µm (dl‐TBOA and l‐TBHA) – 43 µm (cis‐4‐methy‐trans‐2,4‐PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While l‐TBHA, ltrans‐2,4‐PDC, cis‐4‐methy‐trans‐2,4‐PDC and lantiendo‐3,4‐MPDC displayed significant transporter substrate activities in neurons and astrocytes, dl‐TBOA was a substrate only in astrocytes. This effect of dl‐TBOA was concentration‐dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal dl‐TBOA uptake velocity (≤ 10 µm), dl‐TBOA blocked uptake reversal in ATP‐depleted astrocytes; this blockade was negated at concentrations that drove substantial dl‐TBOA uptake (> 10 µm). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions.

Autoradiographic characterization of putative excitatory amino acid transport sites

Removal of excitatory amino acids from the extracellular space is now postulated to occur through at least two distinct transport systems that are distinguished by their ionic dependency. Thus, both sodium-dependent and chloride-dependent systems have been described in the mammalian central nervous system. In this report we attempt to characterize these sites by autoradiography, using D-[3H]aspartate and L-[3H]glutamate as ligands. Previous studies have shown that sequestration of radioligand into membrane vesicles can be a potential artifact when examining transport sites. We have found that sequestration can be alleviated by incubation of tissue sections in xylenes prior to incubation with radioligand. Using in vitro autoradiography we have characterized the two binding sites with respect to their distribution, kinetics and pharmacology. Both appeared to have a single, saturable binding site with Kds in the low micromolar range. Sodium-dependent D-aspartate binding predominated, having a Bmax that was five times greater than chloride-dependent L-glutamate binding in whole brain. The levels of binding to the two sites varied between brain regions. Sodium-dependent D-aspartate binding was highest in the cerebellar molecular layer greater than dentate gyrus molecular layer greater than entorhinal cortex. Chloride-dependent L-glutamate binding was highest in the outer layers of cerebral cortex greater than dentate gyrus molecular layer greater than entorhinal cortex greater than striatum. Pharmacological characterization of these sites also showed major differences. Sodium-dependent D-aspartate binding was most potently inhibited by L-aspartate greater than threo-beta-hydroxyaspartate greater than L-cysteine sulfinic acid greater than L-cysteic acid. Chloride-dependent glutamate binding was most potently inhibited by L-glutamate greater than L-alpha-amino adipic acid greater than quisqualate greater than L-serine-o-sulfate. The differences in distribution, ligand binding properties and pharmacology of these sites suggest that a significant variable in excitatory amino acid circuitry may include heterogeneity in transporters associated with excitatory pathways.

Conformationally restricted inhibitors of the high affinity L-glutamate transporter

Abstract A series of acidic amino acids has been prepared and evaluated in an effort to identify the structural features required for binding to and inhibiting the high affinity uptake system that clears L-glutamate from the synaptic cleft during excitatory amino acid-mediated neurotransmission in the mammalian CNS.

A Novel Chloride-Dependent L-[3H]Glutamate Binding Site in Astrocyte Membranes

Abstract: Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L‐[3H]glu‐tamate that is Cl−‐dependent and Na+‐independent. The binding site is a single saturable site with a KD of about 0.5 μM, is inhibited by L‐aspartate, L‐cysteate, and quisqualate, and is insensitive to kainate, N‐methyl‐D‐aspartate, α‐ami‐no‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate, and 2‐ami‐no‐4‐phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl−‐dependent high‐affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamates excitatory action by high‐affinity uptake, and the excitotoxic action of acidic amino acids in membranes of a single cell type.

N-methyl-D-aspartate receptors and Alzheimer's disease

The results of several studies now suggest that the density of N-methyl-D-aspartate (NMDA) receptors is maintained in many Alzheimers disease (AD) cases, although loss of these receptors can occur in specific regions as a consequence of severe neuronal loss. Recent findings demonstrate that there are at least two subtypes of the NMDA receptor which are allosterically regulated. To determine the status of the NMDA receptor in AD, studies are required which will examine the activation state of the NMDA receptor and the properties of subtypes in relation to neuronal density and structure.

Glutathione levels in olfactory and non-olfactory neural structures of rats

Olfactory receptor neurons are a CNS entry point for a wide variety of airborne substances. Therefore, it is probable that detoxification mechanisms are present in these neurons to neutralize such agents. Glutathione (GSH) is an essential component of several detoxification schemes, and in this study we examined the distribution and levels of GSH in the olfactory epithelium, olfactory bulb, cortex, hippocampus and cerebellum in neonatal, weanling, adult and aged rats. We report that GSH is primarily localized to the olfactory receptor neurons and their oxons within the olfactory epithelium. It is also localized within the glomerular neuropil and granule cells of the olfactory bulb. Levels of GSH in the olfactory epithelium and hippocampus do not change as a function of age, although GSH levels decrease in several brain regions, including the olfactory bulb, cerebellum and cortex.

A comparison of 2-amino-4-phosphonobutyric acid (AP4) receptors and [3H]AP4 binding sites in the rat brain

The glutamate analogue 2-amino-4-phosphonobutyric acid (AP4) is a potent antagonist at several synapses where an excitatory amino acid appears to be the neurotransmitter. Previous studies identified a Cl-/Ca2+ dependent [3H]glutamate binding site in synaptic plasma membrane (SPM) preparations that was also labeled by [3H]AP4 and exhibited a pharmacology similar to the AP4 receptor. This report examines the pharmacological specificity in both biochemical and electrophysiological preparations in greater detail. Several compounds are identified which readily interact with the apparent binding site in membranes, but neither mimic nor inhibit the action of AP4 in electrophysiological studies. The rate of dissociation of [3H]AP4 from SPMs is shown to increase in the presence of added AP4 and increasing the osmolarity in the SPM binding assay decreases the level of observed [3H]AP4 binding. These findings indicate both a heterogeneous population of binding sites and the occurrence of transport. It is concluded that much of the AP4 binding observed in SPM preparations is to a site other than the AP4 receptor. The results provide a further pharmacological description of AP4 receptors which should facilitate the identification of the receptor in biochemical preparations.

Increased density of excitatory amino acid transport sites in the hippocampal formation following an entorhinal lesion.

High affinity transport of excitatory amino acids such as L-glutamate into astrocytes is necessary for the termination of its excitatory signal and the prevention of its excitotoxic effects. The removal of glutamate from the synaptic cleft is carried out by both sodium- and chloride-dependent systems. Both sodium-dependent D-[3H]aspartate and chloride-dependent L-[3H]glutamate binding were found to increase in the dentate gyrus molecular layer of rats following an entorhinal lesion. The increased binding reached a maximum at 5 and 7 days postlesion and returned to normal by 12 days postlesion. No changes in binding were observed at long time points postlesion. This increased ability to transport glutamate may be a compensatory response to protect the remaining neurons from the excitotoxic conditions that accompany neuronal degeneration.

Gliotoxic properties of theLathyrus excitotoxinβ-N-oxalyl-l-α,β-diaminopropionic acid (β-l-ODAP)

Abstract β-N-Oxalyl- l -α,β-diaminopropionic acid (β- l -ODAP) is an excitatory amino acid agonist found in the seeds ofLathyrus sativus that is believed to be the major causative agent in the pathology of human lathyrism. We have found that in addition to its previously recognized neurotoxic properties, β- l -ODAP is also gliotoxic. When added to cultures of neonatal rat astrocytes, β- l -ODAP induced a series of morphological changes (e.g., extensive vacuole formation, pale and swollen nuclei with obvious nucleoli, and cellular swelling) that led to the eventual lysis of the glial cells. If the β- l -ODAP was removed prior to the lysis of the astrocytes, many of the early morphological changes appeared to be reversible. When quantitated by a loss of the lactate dehydrogenase activity, β- l -ODAP lysed the astrocytes with an LD50 of2.1 ± 0.2mM following 48 h of exposure. Lower concentrations of β- l -ODAP were found to be more toxic if the duration of the exposure was increased. The results suggest that the overall impact of the toxin on the CNS may represent the cumulative action of β- l -ODAP at a number of distinct points on both neurons and astrocytes. The potential that these multiple sites of action may affect the normal regulation of extracellular glutamate and, consequently, disturb the balance between its normal and pathological roles is discussed.

A l-[3H]glutamate binding site on glia: an autoradiographic study on implanted astrocytes

In the present study cultured astrocytes were implanted into the inferior colliculus of rats to create an astrocyte-enriched field that could be examined autoradiographically. The presence of the astrocytes was confirmed with anti-glial fibrillary acidic protein (GFA) immunocytochemistry. We report the presence of a chloride-dependent glutamate binding site on the implanted astrocytes. In the presence of chloride, the specific glutamate binding detected in the implant area was 5-fold greater than that found in a corresponding contralateral region. When the chloride was replaced with acetate, glutamate binding to the astrocytes decreased by more than 80%. The chloride-dependent binding to the astrocytes was insensitive to inhibition by kainic acid (KA) and N-methyl-D-aspartate (NMDA) and sensitive to quisqualate, L-aspartate, L-2-amino-4-phosphonobutyrate, and L-alpha-aminoadipate. The pharmacology of the binding was very similar to that of the in vitro glutamate binding to membranes from cultured astrocytes and to that of a chloride-dependent transport system identified in a glioma cell line. We conclude that the interaction of glutamate with astrocytes is an important component of the total glutamate binding observed in brain slices.