High Sensitivity of Glutamate Uptake to Extracellular Free Arachidonic Acid Levels in Rat Cortical Synaptosomes and Astrocytes

By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.     

Glucocorticoids Inhibit Glucose Transport and Glutamate Uptake in Hippocampal Astrocytes: Implications for Glucocorticoid Neurotoxicity

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15-30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).     

Chondroitin Sulfate Proteoglycan Tenascin-R Regulates Glutamate Uptake by Adult Brain Astrocytes

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.     

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 n M LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca²⁺]_i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca²⁺]_i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca²⁺]_i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca²⁺]_i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca²⁺]_i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.     

Manganese Decreases Glutamate Uptake in Cultured Astrocytes

Recent data have shown an accumulation of manganese in the basal ganglia in patients with chronic hepatic encephalopathy (HE). Astrocytes and ammonia are critically involved in the pathogenesis of HE, and we have recently demonstrated that ammonia decreases glutamate uptake in cultured astrocytes. Since failure by astrocytes to take up glutamate may represent an important pathogenetic mechanism in HE, we, therefore, examined the effect of manganese on glutamate transport in these cells. Treatment of cultured astrocytes with 100 M manganese for 2 days resulted in a 54% decrease in the uptake of D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed a 28% decline in V_max, with no change in the K_m. Treatment of cultures with 5 mM NH₄Cl inhibited D-aspartate uptake by 21%, and a combination of 5 mM NH₄Cl with 100 M manganese produced an additive effect on uptake inhibition. These results suggest a pathogenetic role for manganese in HE, possibly involving glutamate transport.     

Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes

The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na(+)-dependent and a Na(+)-independent system. The Na(+)-dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X-AG. The Na(+)-independent system was inhibited by cystine and was consistent with system x-c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na(+)-independent glutamate uptake. In C-6 cells, the glutamate uptake via systems X-AG and x-c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X-AG was very potent, whereas the uptake via system xc- was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system xc- was necessary for the uptake of cystine, another substrate of system xc-. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.     

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Abstract

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D‐glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.     

Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

High Glutamate Decreases S100B Secretion by a Mechanism Dependent on the Glutamate Transporter

Several molecules have been shown to be involved in glial-neuronal communication, including S100B, an astrocyte-derived neurotrophic cytokine. Extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by an unknown mechanism. Here, we investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. DCG-IV, an agonist of group II metabotropic glutamate receptors, caused a smaller decrease in S100B secretion when compared to 1 mM glutamate. d-aspartate partially reverted the glutamate effect on S100B release and two other inhibitors, PDC and DIDS, reverted it completely. These findings suggest that S100B secretion is inversely coupled to glutamate uptake. Decrease in S100B secretion may be considered as direct excitotoxic damage, but a beneficial mechanism effect cannot be ruled out, because S100B elevation could cause an additional cell death.The authors Francine Tramontina and Marina C. Leite are equally contributed to this work.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.     

Expression of Lysophosphatidic Acid Receptor in Rat Astrocytes: Mitogenic Effect and Expression of Neurotrophic Genes

Lysophosphatidic acid (LPA) is a phospholipid mediator with a variety of biological activities. It remains unknown, however, which cells in the brain express the LPA receptor. The present study was undertaken to identify cells in the rat brain expressing functional LPA receptors, and to explore biological roles of LPA in these cells. We found that the LPA receptor was most dominantly expressed in rat astrocytes, determined by LPA-induced Ca²⁺ imaging, and by Northern blot analyses. LPA induced a mitogenic response and expression of immediate early genes in astrocytes, through pertussis-toxin sensitive G-protein(s). LPA also stimulated the expression of various cytokine genes, including nerve growth factor, interleukin (IL)-1, IL-3 and IL-6. Thus, astrocytes are the major target of LPA in the brain. We propose that LPA may play important roles in neuronal development, gliosis and wound-healing process in the brain.     

Synaptic Vesicular Glutamate Uptake: Modulation by a Synaptosomal Cytosolic Factor

We have demonstrated previously that L-glutamate is taken up into isolated synaptic vesicles in an ATP-dependent manner, supporting the neurotransmitter role of this acidic amino acid. We now report that a nerve terminal cytosolic factor inhibits the ATP-dependent vesicular uptake of glutamate in a dose-dependent manner. This factor appears to be a protein with a molecular weight greater than 100,000, as estimated by size exclusion chromatography, and is precipitated by ammonium sulfate (40% saturation). The inhibitory factor is inactivated by heating to 100 degrees C. Proteolytic digestion of the ammonium sulfate fraction by trypsin or chymotrypsin did not reduce, but rather increased slightly, the inhibition of glutamate uptake. Unlike the native factor, the digest retained inhibitory activity after heating, suggesting that proteolytic digestion may generate active fragments. The inhibition of ATP-dependent vesicular glutamate uptake is not species-specific, as the factor obtained from both rat and bovine brains produced an equal degree of inhibition of glutamate uptake into vesicles of each species. These observations raise the possibility that vesicular uptake of glutamate may be regulated by an endogenous factor in vivo.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Ascorbic Acid Uptake by a High-Affinity Sodium-Dependent Mechanism in Cultured Rat Astrocytes

Ascorbic acid (vitamin C) is synthesized in rodent liver, circulates in the blood, and is concentrated in the brain. Experiments were performed to characterize the mechanism of ascorbate uptake by rat cerebral astrocytes in primary culture. Astroglial uptake of L-[14C]ascorbate was observed to be both saturable and stereoselective. In addition, uptake was dependent on both the incubation temperature and the concentration of Na+ because it was largely inhibited by cooling to 4 degrees C, by treatment with ouabain to increase intracellular Na+, and by the substitution of K+, Li+, or N-methyl-D-glucamine for extracellular Na+. The affinity for ascorbate was relatively high in cells incubated with a physiological concentration of extracellular Na+, because the apparent Km was 32 microM in 138 mM Na+. However, the affinity for ascorbate was significantly decreased when the extracellular Na+ concentration was lowered. Treatment of astrocytes with dibutyryl cyclic AMP induced stellation and increased the maximum rate of ascorbate uptake by 53%. We conclude that astrocytes possess a stereoselective, high-affinity, and Na+-dependent uptake system for ascorbate. This system may regulate the cerebral ascorbate concentration and consequently modulate neuronal function.     

Glutamate Uptake into Synaptic Vesicles: Competitive Inhibition by Bromocriptine

The ATP-dependent uptake of L-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the peptide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested: the IC50 was determined to be 22 microM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate translocator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.     

Glucose Transport in Astrocytes: Regulation by Thyroid Hormone

Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate-saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid-hormone-free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these "hypothyroid" cells markedly increased the net rate of 2-deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.     

Glutamate Uptake Is Stimulated by Extracellular S100B in Hippocampal Astrocytes

Summary  

Specificity and Reversibility of the Inhibition by HgCl2 of Glutamate Transport in Astrocyte Cultures

Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2-deoxyglucose uptake. Uptake of 2-deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+-ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated X-AG. Acute exposure to 0.5 microM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2-deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 microM as chlorides). The inhibitory action of 0.5 microM HgCl2 on glutamate transport was rapidly reversible. The action of 1-2 microM HgCl2 was progressive when exposures were extended to 1-3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.