Surface carbonic anhydrase activity on astrocytes and neurons facilitates lactate transport

A number of studies have provided physiological evidence for extracellular carbonic anhydrase (CA) in brain. Association of extracellular CA with glia has been limited to functional studies of gliotic slices and retinal Muller cells. While astrocytes contain intracellular CA, there has been no direct evidence for surface CA on these cells. In fact, some morphological studies suggest that the extracellular CA in brain parenchyma resides on neurons, not glia. There has been no functional demonstration of extracellular CA activity on CNS neurons, however. Here we capitalized on the H(+) dependence of inward lactate transport to reveal functional extracellular CA activity on cultured astrocytes and acutely isolated hippocampal pyramidal neurons. Exposure to 20 mM L-lactate produced a rapid acidification of astrocytes that was reversibly blocked by 10 microM benzolamide. The lactate-induced acidification (LIA) was also blocked by a dextran-conjugated CA inhibitor. In CO(2)/HCO(3) (-)-free, HEPES-buffered media, the LIA was largely unaffected. Acutely dissociated hippocampal pyramidal neurons underwent a similar LIA that was reversibly blocked by benzolamide. Surface CA is likely to facilitate lactate transport by enabling rapid replenishment (i.e., buffering) of surface H(+) required for inward lactate-H(+) cotransport. These results demonstrate functional surface CA for the first time on individual mammalian astrocytes and neurons and suggest that this enzyme may play a role in the utilization of monocarboxylate substrates such as lactate and pyruvate by the brain.     

Activity-dependent pH shifts in hippocampal slices from normal and carbonic anhydrase II-deficient mice

The type II isoform of carbonic anhydrase is abundant in astrocytes and oligodendroglia. To explore whether the expression of the type II isoform is required for interstitial carbonic anhydrase activity, we studied extracellular pH transients in hippocampal slices from mutant mice devoid of carbonic anhydrase type II and from wild-type littermates. Stimulation of the Schaffer collateral afferents evoked similar extracellular pH transients in the CA1 stratum pyramidale, consisting of a predominant alkaline shift and little or no subsequent acidosis. After 5-s stimulus trains at 10 Hz, alkaline shifts were not significantly different in carbonic anhydrase II-deficient and wild-type preparations, averaging 0.09 +/- 0.04 and 0.08 +/- 0.04 unit pH, respectively. Addition of 1.5 microM benzolamide amplified the alkaline shifts by 385 +/- 146 and 345 +/- 75% in the mutant and wild-type preparations, respectively. Dose response studies with benzolamide displayed similar sensitivity to this carbonic anhydrase inhibitor over a concentration range of 0. 03-10 microM. These data indicate that interstitial carbonic anhydrase activity is effectively unaltered in brains devoid of carbonic anhydrase type II. The results are consistent with the interpretation that a distinct extracellular isoform of carbonic anhydrase exists in brain.     

The Molecular Mechanisms of Neural Flow Coupling: A New Concept

The phenomenon known as neural flow coupling (NFC) occurs at the capillary level where there are no known pressure controlling structures. Recent developments in advanced magnetic resonance imaging technologies have made possible in vivo direct investigations of water physiology that have shed new insight on the water dynamics of the cortical pericapillary space and their complex functionality in relation to NFC. Neural activities initiate a chain of events that ultimately affect NFC. First, neural activities generate extracellular acidification. Extracellular acidosis in turn produces inhibition of aquaporin‐4 (AQP‐4) located at the end feet of pericapillary astrocytes, the water channel which regulates water influx into the pericapillary space and, hence, interstitial flow. Reduction of pericapillary water pressure results in a negative balance between pericapillary and intraluminal capillary pressure, allowing for capillary caliber expansion. Proton permeability through the tight junctions of the blood brain barrier is significantly high owing to the Grotthuss proton “tunneling” mechanism and, therefore, carbonic anhydrase (CA) type IV (CA‐IV) anchored to the luminal surface of brain capillaries functions as scavenger of extracellular protons. CA‐IV inhibition by acetazolamide or carbon dioxide results in the accumulation of extracellular protons, causing AQP‐4 inhibition and a secondary increase in rCBF.     

Rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Death of astrocytes requires hours to days in injury models that use hypoxia, acidosis, or calcium paradox protocols. These methods do not incorporate the shifts in extracellular K(+), Na(+), Cl(-), and Ca(2+) that accompany acute brain insults. We studied astrocyte survival after exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), with respective [Ca(2+)], [K(+)], [Na(+)], [Cl(-)], and [HCO(-)(3)] of 0.13, 65, 51, 75, and 13 mM (15% CO(2)/85% N(2), pH 6.6). Intracellular pH (pH(i)) was monitored with the fluorescent dye BCECF. Cell death was indicated by a steep fall in the pH-insensitive, 440-nm-induced fluorescence (F440) and was confirmed by propidium iodide staining. After 15-40-min HAIR exposure, reperfusion with standard Ringer caused death of most cultured (and acutely dissociated) astrocytes within 20 min. Cell death was not prevented if low Ca(2+) was maintained during reperfusion. Survival fell with increased HAIR duration, elevated temperature, or absence of external glucose. Comparable durations of hypoxia, acidosis, or ion shifts alone did not lead to acute cell death, while modest loss was noted when acidosis was paired with either hypoxia or ion shifts. Severe cell loss required the triad of hypoxia, acidosis, and ion shifts. Intracellular pH was significantly higher in HAIR media, compared with solutions of low pH alone or with low pH plus hypoxia. These results indicate that astrocytes can be killed rapidly by changes in the extracellular microenvironment that occur in settings of traumatic and ischemic brain injury.     

Acute feedback control of astrocytic glycolysis by lactate

Neuronal activity is accompanied by a rapid increase in interstitial lactate, which is hypothesized to serve as a fuel for neurons and a signal for local vasodilation. Using FRET microscopy, we report here that the rate of glycolysis in cultured mice astrocytes can be acutely modulated by physiological changes in extracellular lactate. Glycolytic inhibition by lactate was not accompanied by detectable variations in intracellular pH or intracellular ATP and was not dependent of mitochondrial function. Pyruvate was also inhibitory, suggesting that the effect of lactate is not mediated by the NADH/NAD(+) ratio. We propose that lactate serves as a fast negative feedback signal limiting its own production by astrocytes and therefore the amplitude of the lactate surge. The inhibition of glucose usage by lactate was much stronger in resting astrocytes than in K(+)-stimulated astrocytes, which suggests that lactate may also help diverting glucose from resting to active zones.     

Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca(2+) and Zn(2+) in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0-6.8 and the pH(50) of 6.1, the reversal potential close to the Na(+) equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC(50)=19.6+/-2.1 microM). Either low [Ca(2+)](o) or high [Zn(2+)](o) increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a+2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn(2+)](o) (>10(-4) M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca(2+)](o) and high [Zn(2+)](o) on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn(2+) elevation and/or Ca(2+) reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention.     

Localization of desmin in cultured astrocytes and its distribution in relation to glial fibrillary acidic (GFA) protein

In dissociated cultures from newborn rat brain, it is shown that astrocytes which are positive with GFA antisera also stain with desmin antisera. However, the localization of these two proteins within the same cell seems to be different as revealed by double immunofluorescence employing both these antisera.     

Carbonic anhydrase inhibitor sulthiame reduces intracellular pH and epileptiform activity of hippocampal CA3 neurons

PURPOSE: Sulthiame is a carbonic anhydrase (CA) inhibitor with an anticonvulsant effect in the treatment of benign and symptomatic focal epilepsy in children. The aim of the study was to elucidate the mode of action of sulthiame with respect to possible changes of intracellular pH (pHi) that might develop along with sulthiame's anticonvulsant properties. METHODS: The effects of sulthiame (a) on pHi of 2',7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester (BCECF-AM) loaded CA3 neurones as well as (b) on epileptiform activity (induced by 50 microM 4-aminopyridine) were compared with those of the CA inhibitors acetazolamide and benzolamide. RESULTS: In the majority of neurons, sulthiame (1.0-1.5 mM; n = 8) as well as the membrane permeant acetazolamide (0.5-1.0 mM; n = 6) reversibly decreased pHi by 0.18 +/- 0.05 (SD) and 0.17 +/- 0.10 (SD) pH units, respectively, within 10 min. The poor membrane permeant benzolamide (1.0-2.0 mM) had no influence on pHi (n = 8). Sulthiame (1.0-2.5 mM) and acetazolamide (1.0-2.0 mM) reversibly reduced the frequency of action potentials and epileptiform bursts after 10-15 min (n = 9, n = 7), whereas benzolamide (1.0-2.0 mM) had no effect (n = 6). CONCLUSIONS: The results suggest that sulthiame acts as a membrane-permeant CA inhibitor whose beneficial effect on epileptiform activity results at least in part from a modest intracellular acidosis of central neurons.     

Extracellular matrix modulates the proliferation of rat astrocytes in serum-free culture

The mechanism of glial proliferation in the developing nervous system, as well as in response to injury, inflammation, and tumor invasion, is unknown. Several growth factors and extracellular matrices have been shown to stimulate the proliferation of cultured cells of various origin, including astrocytes. We investigated the effect of extracellular matrix components, including fibronectin (FN), laminin (LN), and collagen types I and IV, on the growth of astrocytes during stimulation by various growth factors. When astrocytes were grown on FN- and LN-coated wells in a serum-free, chemically defined medium, their increase in number significantly exceeded that of cells grown on plastic wells. The addition of platelet-derived or basic fibroblast growth factor to cells cultured on FN - or LN -coated wells significantly potentiated astrocyte proliferation. The collagen preparations had no such effect. These observations indicate that FN and LN have a fundamental part in converting the quiescent astrocyte into the proliferating phenotype, which may be required for remodeling damaged brain tissues in vivo. © 1993 Wiley-Liss, Inc.     

Neurofibromatosis 1 (NF1) heterozygosity results in a cell-autonomous growth advantage for astrocytes

Individuals with neurofibromatosis 1 (NF1) develop low-grade astrocytomas at an increased frequency. To gain insight into the function of the Nf1 gene product as a growth regulator for astrocytes, we examined mice heterozygous for a targeted Nf1 mutation. In our previous studies, we demonstrated increased numbers of proliferating astrocytes in Nf1 heterozygote (Nf1+/-) mice in vivo. We now show that cultured Nf1+/- astrocytes exhibit a cell-autonomous growth advantage in vitro associated with increased p21-ras pathway activation. Furthermore, we demonstrate that Nf1+/-;wild-type N-ras mice have a similar astrocyte growth advantage in vitro and in vivo as either oncogenic N-ras or Nf1+/-; oncogenic N-ras mice. Lastly, mice heterozygous for targeted defects in both Nf1 and p53 as well as Nf1 and Rb exhibit 3- and 2.5-fold increases in astrocyte proliferation in vivo, respectively, suggesting that abnormalities in Nf1- and p53/Rb-regulated pathways cooperate in the heterozygous state to confer a growth advantage for brain astrocytes. Collectively, these results provide evidence for a cell-autonomous growth advantage in Nf1+/- astrocytes and suggest that some of the brain pathology in individuals with NF1 might result from reduced, but not absent, NF1 gene function.     

A chamber for electrophysiological recording from cultured neurones allowing perfusion and temperature control

With the increasing use of cell culture in electrophysiology there has arisen a need for more rigorous control of the extracellular fluid composition and temperature. We describe here an environmental chamber designed to permit perfusion and temperature control of the extracellular medium during electrophysiological studies of cultured or acutely dissociated neurones in 35 mm Petri dishes. The chamber consists of a Peltier driven heat exchange, which allows temperature control above and below ambient. The Petri dish temperature is controlled in 3 ways: (1) through direct conduction from the controlled surface; (2) by flow of gas around the dish; and (3) by flow of extracellular medium to the Petri dish. We demonstrate the temperature control achieved with this equipment and the ability to exchange the extracellular medium in the whole dish. Using the whole-cell-patch recording technique to record from hippocampal neurones, Q10 parameters were estimated for the membrane time constant, input resistance and action potential halfwidth, over a range of 15-36 degrees C. The mean Q10 values (+/- S.E.M.) were 0.71 +/- 0.05, 0.58 +/- 0.03 and 0.35 +/- 0.1, respectively. Because of rapid diffusion and mixing of drugs when using puffer pipettes, quantitative pharmacological studies or manipulations of the extracellular recording medium are not possible. By perfusing the whole recording chamber the final concentration can be accurately known. Using this method we obtained an estimate of the relative potency of the non-specific excitatory amino acid antagonist, kynurenate, for synaptically activated N-methyl-d-aspartate (NMDA) receptors of 1:0.62.     

The role of integrin receptors in aspects of glioma invasion in vitro.

Integrins are heterodimers consisting of non-covalently associated alpha and beta subunits. They mediate adherence of normal and tumour cells to the extracellular matrix, a property which is essential for migration of neoplastic astrocytes as they invade into the normal brain parenchyma. Flow cytometry and immunocytochemical analysis of cultured cells derived from 10 gliomas (1 pilocytic astrocytoma, 1 astrocytoma, 1 oligoastrocytoma, 1 anaplastic oligoastrocytoma, 4 anaplastic astrocytomas and 2 glioblastoma multiforme) revealed that the beta1 integrin subunit was generally expressed more strongly than alpha4 or alpha(v) integrin subunits. Subsequent studies with function-blocking antibodies against the beta1 subunit inhibited adhesion, motility and invasion of the gliomas in vitro, to varying degrees, on all extracellular matrix substrates investigated (laminin, collagen type IV, fibronectin and vitronectin), the inhibition by beta1 subunit was greatest on collagen type IV. These studies therefore substantiate the case for a role of the beta1 integrin subunit in neoplastic glial cell invasion of the brain.     

GABA-activated Cl- channels in astrocytes of hippocampal slices

We used kainic acid-lesioned hippocampal slices to examine glial responses to the inhibitory neurotransmitter GABA in a neuron-free environment. Slices were prepared from rats which received intracerebroventricular injections of kainic acid 1 month prior to experiments. Astrocytes (membrane potential averaged 81.4 +/- 5.5 mV; n = 46; mean +/- SD) were impaled in the CA3 region of the slice, which was completely depleted of neurons. GABA (1 mM) application by bath perfusion depolarized membrane potential from 1 to 5 mV. The GABA-induced depolarization was not affected by a tetrodotoxin (1 microM)/high-Mg2+/low-Ca2+ solution. Changing the Cl- equilibrium potential by reducing extracellular Cl- greatly increased the GABA-induced depolarization. Muscimol mimicked the GABA response, while picrotoxin (0.1 mM), an antagonist of the GABA-activated Cl- channel, resulted in a 60% blockade. The barbiturate, pentobarbital (0.1 mM), and the benzodiazepine agonist, flunitrazepam (1 mM), enhanced the depolarization by 60 and 40%, respectively. A blocker of glial GABA uptake, beta-alanine (1 mM), did not affect the GABA-induced membrane depolarization, indicating that the depolarization is not caused by electrogenic uptake of the amino acid. The pharmacological properties of the GABA response of astrocytes from the hippocampal slice is similar to that previously described for cultured astrocytes from rat cerebral hemispheres. Our data suggest that GABA receptors, which are coupled to Cl- channels, are also expressed by astrocytes in an intact tissue.     

Simvastatin and atorvastatin facilitates amyloid β‐protein degradation in extracellular spaces by increasing neprilysin secretion from astrocytes through activation of MAPK/Erk1/2 pathways

One of the major neuropathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid β‐protein (Aβ) in the brain. Aβ accumulation seems to arise from an imbalance between Aβ production and clearance. Neprilysin (NEP) and insulin‐degrading enzyme (IDE) are the important Aβ‐degrading enzymes in the brain, and deficits in their expression may promote Aβ deposition in patients with sporadic late‐onset AD. Statins, which are used clinically for reducing cholesterol levels, can exert beneficial effects on AD. Therefore, we examined whether various statins are associated with Aβ degradation by inducing NEP and IDE expression, and then evaluating the relation between activation of intracellular signaling transduction, inhibition of cholesterol production, and morphological changes to astrocytes. Treating cultured rat astrocytes with simvastatin and atorvastatin significantly decreased the expression of NEP but not IDE in a concentration‐ and time‐dependent manner. The decrease in NEP expression was a result of activation of extracellular signal‐regulated kinase (ERK) but not the reduction of cholesterol synthesis pathway. This NEP reduction was achieved by the release to the extracellular space of cultured astrocytes. Furthermore, the cultured medium prepared from simvastatin‐ and atorvastatin‐treated astrocytes significantly induced the degradation of exogenous Aβ. These results suggest that simvastatin and atorvastatin induce the increase of Aβ degradation of NEP on the extracellular of astrocytes by inducing ERK‐mediated pathway activity and that these reagents regulate the differential mechanisms between the secretion of NEP, the induction of cholesterol reduction, and the morphological changes in the cultured astrocytes. GLIA 2016;64:952–962     

Importance of magnesium ions in development of tolerance to ethanol: studies on cultured cerebral vascular smooth muscle cells, type-2 astrocytes and intact rat brain

This study was designed to examine the roles of intracellular free magnesium ion concentration ([Mg(2+)](i)) in ethanol-induced intoxication and development of tolerance in cultured canine cerebral vascular smooth muscle cells and astrocytes as well as intact rat brain. The basal, resting level of [Mg(2+)](i) in cerebrovascular cells was 732.5 +/- 82.4 microM. Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10 and 25 mM) for 24 h reduced the concentrations of [Mg(2+)](i) to 521.1 +/- 59.6 microM, and 308.2 +/- 37.8 microM, respectively. However, exposure of these cultured vascular cells to the same concentrations of ethanol, after initial pretreatment with ethanol for 24 h, failed to interfere with the levels of [Mg(2+)](i). Measurement of [Mg(2+)](i) at 48 h and 72 h indicated that the decreased levels of [Mg(2+)](i) induced by ethanol at 24 h treatment returned toward baseline. Similar experiments were performed in cultured type-2 astrocytes isolated from neonatal rat brain. The basal level of [Mg(2+)](i) in type-2 astrocytes was about 125 microM. Incubation of these cells with 10 mM ethanol for 10 min resulted in a 27% reduction in the level of [Mg(2+)](i), whereas incubation with 25 mM ethanol resulted in almost a 50% reduction in [Mg(2+)](i). The decreased levels of [Mg(2+)](i) lasted around 30 min, until the measurement finished. Continuous incubation of these cultured astrocytes, with ethanol (either 10 mM or 25 mM), for more than 24 h, indicated that the concentrations of [Mg(2+)](i) in type-2 astrocytes were equivalent to those at basal, resting levels. In vivo 31P-NMR spectroscopy, performed on intact rat brains, indicated that an initial administration of 4 mg/kg ethanol ( approximately 20-25 mM blood alcohol level) resulted (after 20-40 min of exposure) in severe deficits in whole brain [Mg(2+)](i) (550 +/- 33 microM to 358 +/- 24 microM). Repeated injections of ethanol (4 mg/kg) over the next 24-72 h resulted in progressively diminishing effects on brain [Mg(2+)](i). These experimental data indicate that chronic ethanol treatment can induce a tolerance to depletion of [Mg(2+)](i) in cerebrovascular smooth muscle cells, type-2 astrocytes as well as intact rat brain. The results suggest that [Mg(2+)](i) might play a major role in alcohol-induced tolerance in the brain.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Glycogen phosphorylase isozyme pattern in mammalian retinal Müller (glial) cells and in astrocytes of retina and optic nerve

Müller cells, the radially oriented dominant macroglial cells of the retina, are known to contain abundant glycogen as well as the key enzyme for its degradation, glycogen phosphorylase (GP), but the expressed isozyme pattern is unknown. To elucidate the isoform expression pattern, specific antisera directed against the brain (BB) and muscle (MM) isoforms of GP were applied to retinal sections, isolated Müller cells, and sections of the optic nerve. We show that Müller cells of rat, rabbit, guinea pig, and mouse retina exclusively express the BB isoform. Astrocytes of rat and rabbit optic nerve, as well as retina express only the BB isoform. In contrast, astrocytes in the brain and spinal cord as well as the epithelial cells of the pars caeca and of the ciliary body express both the BB and MM isoform. This result may indicate some differences in the role of glycogen in retinal macroglia and brain astrocytes, reflecting a local specialization of macroglia in the retina proper.     

Methylmercury-mediated inhibition of 3H-D-aspartate transport in cultured astrocytes is reversed by the antioxidant catalase

Astrocytes are essential for removal of glutamate from the extracellular space in the central nervous system. The neurotoxic heavy metal methylmercury potently and specifically inhibits the transport of glutamate in cultured astrocytes by an unknown mechanism. Glutamate transport in astrocytes is also inhibited by reactive oxygen species. A glutamate-induced transporter current is inhibited both by reactive oxygen species and thiol oxidizing agents. These observations suggest that oxidation of the transporter might mediate methylmercury-induced inhibition of glutamate transport. In the present study, we examined the ability of thiol reducing or oxidizing agents to inhibit transport of 3H-D-aspartate, a glutamate analog, in primary cultures of neonatal rat astrocytes. To assess if methylmercury-mediated inhibition of 3H-aspartate transport was due to overproduction of reactive oxygen species, we tested the ability of Trolox, alpha-phenyl-tert-butyl nitrone (PBN), or catalase to attenuate the methylmercury-induced inhibition of aspartate uptake. Neither the thiol reducing agent dithiothreitol (DTT), nor the thiol oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) had any effect on 3H-aspartate transport suggesting that the thiol redox state does not alter transporter function. In contrast, the antioxidant catalase (1000 U/ml) significantly attenuated methylmercury-induced inhibition of 3H-aspartate uptake, suggesting that excess reactive oxygen species, specifically H2O2, inhibit the function of an astrocytic excitatory amino acid transporter (EAAT1). Prolonged exposure (6 h) to inhibitors of glutamate transport significantly decreased EAAT1 mRNA levels suggesting that transporter expression is related to function. This study suggests that methylmercury-induced overproduction of H2O2 is a mechanism for inhibition of glutamate transport and transporter expression in cultured astrocytes.