Glutamate Uptake In Synaptosomes Research Articles

Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake.

(1) In this study, we examined the effects of crude venom from the spider Parawixia bistriata on glutamate and GABA uptake into synaptosomes prepared from rat cerebral cortex. Addition of venom to cortical synaptosomes stimulated glutamate uptake and inhibited GABA uptake in a concentration-dependent manner. (2) The venom was fractionated using reverse-phase high-performance liquid chromatography on a preparative column. The fraction that retained glutamate uptake-stimulating activity was further purified on a reverse-phase analytical column followed by ion-exchange chromatography. (3) The active fraction, referred to as PbTx1.2.3, stimulated glutamate uptake in synaptosomes without changing the K(M) value, and did not affect GABA uptake. Additional experiments showed that the enhancement of glutamate uptake by PbTx1.2.3 occurs when ionotropic glutamate receptors or voltage-gated sodium and calcium channels are completely inhibited or when GABA receptors and potassium channels are activated, indicating that the compound may have a direct action on the transporters. (4) In an experimental model for glaucoma in which rat retinas are subjected to ischemia followed by reperfusion, PbTx1.2.3 protected neurons from excitotoxic death in both outer and inner nuclear layers, and ganglion cell layers. (5) This active spider venom component may serve as a basis for designing therapeutic drugs that increase glutamate clearance and limit neurodegeneration.

Morphine Withdrawal Increases Glutamate Uptake and Surface Expression of Glutamate Transporter GLT1 at Hippocampal Synapses

Opiate abuse causes adaptive changes in several processes of synaptic transmission in which the glutamatergic system appears a critical element involved in opiate tolerance and dependence, but the underlying mechanisms remain unclear. In the present study, we found that glutamate uptake in hippocampal synaptosomes was significantly increased (by 70% in chronic morphine-treated rats) during the morphine withdrawal period, likely attributable to an increase in the number of functional glutamate transporters. Immunoblot analysis showed that expression of GLT1 (glutamate transporter subtype 1) was identified to be upregulated in synaptosomes but not in total tissues, suggesting a redistribution of glutamate transporter expression. Moreover, the increase in glutamate uptake was reproduced in cultured neurons during morphine withdrawal, and the increase of uptake in neurons could be blocked by dihydrokainate, a specific inhibitor of GLT1. Cell surface biotinylation and immunoblot analysis showed that morphine withdrawal produced an increase in GLT1 expression rather than EAAC1 (excitatory amino acids carrier 1), a neuronal subtype, at the cultured neuronal cell surface, whereas no significant change was observed in that of cultured astrocytes. Electron microscopy also revealed that GLT1 expression was markedly increased in the nerve terminals of hippocampus and associated with the plasma membrane in vivo. These results suggest that GLT1 in hippocampal neurons can be induced to translocate to the nerve terminals and express on the cell surface during morphine withdrawal. The translocation of GLT1 at synapses during morphine withdrawal provides a neuronal mechanism for modulation of excitatory neurotransmission during opiate abuse.

Impaired Spinal Cord Glutamate Transport Capacity and Reduced Sensitivity to Riluzole in a Transgenic Superoxide Dismutase Mutant Rat Model of Amyotrophic Lateral Sclerosis

We characterized synaptosomal glutamate transport activity in a recently developed transgenic rat model of amyotrophic lateral sclerosis (ALS) overexpressing the G93A Cu(2+)/Zn(2+) superoxide dismutase (SOD1) mutation. Using spinal cord synaptosomes, a significant reduction (43%) in the maximal velocity for high-affinity, Na(+)-dependent glutamate uptake was observed at disease end stage in G93A rats compared with age-matched controls. Similarly, a 27% reduction in maximum velocity (V(max)) was measured at disease onset, but no difference in spinal cord V(max) values were observed with presymptomatic animals compared with controls. In comparison, we observed no differences in the V(max) for glutamate clearance at disease end stage with synaptosomes from cortex, hippocampus, striatum, cerebellum, and brainstem, indicating a specific deficit in the spinal cord. The pharmacological sensitivity of spinal cord uptake to dihydrokainate suggests that the GLT-1 (glutamate transporter-1) subtype primarily mediates the transport activity. Expression analysis revealed a loss of GLT-1 as well as qualitative changes in GLAST (glutamate/aspartate transporter) but no measurable changes in EAAC1 (excitatory amino acid carrier 1) in spinal cord of end-stage G93A rats, indicating that deficits in glutamate transporters in this rat model may be glial specific. Riluzole, a neuroprotective agent used clinically to slow the progression of ALS, produced an enhancement of spinal cord synaptosomal glutamate uptake in control animals and early-stage disease G93A rats, but this effect was lost in end-stage animals. Altered expression of astroglial glutamate transporters accompanied by reduced capacity for spinal cord clearance of extracellular glutamate in the G93A SOD1 transgenic rat may account for a dampened effect of riluzole to enhance glutamate uptake at end-stage disease.

Synaptosomal glutamate transport studies in a transgenic rat model of amyotrophic lateral sclerosis

Transgenic rats over‐expressing the G93A Co/Zn superoxide dismutase (SOD1) mutation have recently been generated (Howland et al. PNAS99, 1604–1609) and found to replicate an amyotrophic lateral sclerosis (ALS) – like motor neuron disease previously described in transgenic mice harboring the same mutation. Impairments in synaptosomal glutamate transport capacity have been documented both in the transgenic mouse model and with tissue obtained from human ALS patients. We have measured synaptosomal glutamate transport in the G93A SOD1 rat to further characterize these animals and to provide a comparison with the data described above. We observed a 36% decrease in the maximum velocity of glutamate uptake in synaptosomes prepared from spinal cord of G93A SOD1 rats compared with age matched control animals (Vmax values for glutamate transport were 152 and 98 pmol/min/mg, respectively, in control and transgenic animals) while the affinity for glutamate (Km 3–5 μm) was unchanged. In contrast, no differences in Vmax values were observed when synaptosomes were prepared from cortex, hippocampus, striatum, brainstem or cerebellum. Pharmacological sensitivity to the selective GLT‐1 transport inhibitor dihydrokainate (IC50 74 μm) demonstrates that glutamate uptake in spinal cord synaptosomes is predominantly mediated by this subtype. Incubation of spinal cord synaptosomes with riluzole (100 μm) resulted in a 20–30% increase in glutamate uptake in the control animals while in the G93A SOD1 rats this effect was less pronounced. Our results indicate both a reduction in spinal cord glutamate transport capacity in the SOD1 rats and an altered functional response to riluzole.

Ibogaine alters synaptosomal and glial glutamate release and uptake.

Ibogaine has aroused expectations as a potentially innovative medication for drug addiction. It has been proposed that antagonism of the NMDA receptor by ibogaine may be one of the mechanisms underlying its antiaddictive properties; glutamate has also been implicated in ibogaine-induced neurotoxicity. We here report the effects of ibogaine on [3H]glutamate release and uptake in cortical and cerebellar synaptosomes, as well as in cortical astrocyte cultures, from mice and rats. Ibogaine (2-1000 microM) had no effects on glutamate uptake or release by rat synaptosomes. However, ibogaine (500-1000 microM) significantly inhibited the glutamate uptake and stimulated the release of glutamate by cortical (but not cerebellar) synaptosomes of mice. In addition, ibogaine (1000 microM) nearly abolished glutamate uptake by cortical astrocyte cultures from rats and mice. The data provide direct evidence of glutamate involvement in ibogaine-induced neurotoxicity.

Effects of linalool on glutamate release and uptake in mouse cortical synaptosomes.

Linalool, a monoterpene compound prevalent in essential oil of plant species traditionally used as sedatives, has been characterized as anticonvulsant in several experimental models. Linalool inhibits the binding of [3H]glutamate and [3H]dizocilpine to brain cortical membranes, indicating a participation of the glutamatergic transmission its mechanism of action. In this study, we investigated the effects of linalool on [3H]glutamate release (basal and potassium-stimulated) and [3H]glutamate uptake in mice cortical synaptosomes. Linalool significantly reduced potassium-stimulated glutamate release as well as glutamate uptake, not interfering with basal glutamate release. The data indicates that linalool may interfere with several relevant elements of the glutamatergic transmission, including detriment of the K+-stimulated glutamate release.

Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes

The purpose of this study was to examine the effect of the anti-convulsant agent, riluzole, on high-affinity glutamate uptake as measured in rat spinal cord synaptosomes. The rate of glutamate uptake was significantly increased in the presence of 0.1 μM and 1.0 μM riluzole, but not at the higher concentrations examined. Kinetics analysis demonstrated that riluzole (0.1 μM) decreased the apparent K m by 21% and increased the V max by 31%. Glutamate uptake also was significantly increased in spinal cord synaptosomes obtained from rats treated with 8 mg/kg (i.p.) of riluzole and sacrificed 4 h later. The increase in glutamate uptake in vitro was not affected by pretreatment either with H7, an inhibitor of PKA and PKC, or with the PKC activating phorbol ester, 12- O-tetradecanoylphorbol 13-acetate. Previous studies have shown that some of the actions of riluzole are mediated by G proteins sensitive to pertussis toxin. Surprisingly, treatment of synaptosomes with pertussis toxin alone increased the rate of glutamate uptake, while having no effect on uptake in the presence of riluzole. However, pretreatment with cholera toxin was found to completely block the effects of riluzole on glutamate uptake. These results reveal an additional mechanism by which riluzole can affect glutamatergic neurotransmission, and provides further support that riluzole may prove beneficial in the treatment of traumatic central nervous system injuries involving the excitotoxic actions of glutamate.

Inhibition of glutamate uptake by Tx3-4 is dependent on the redox state of cysteine residues.

Glutamate transporters are essential for the homeostasis of glutamate and normal function of glutamatergic synapses. Their function was shown to be regulated by redox agents and dimerizations that involves redox changes of cysteine residues. Peptide neurotoxins are also known to be rich in cysteine residues that contribute to their activity and stability. Among them is the toxin Tx3-4, from the spider Phoneutria nigriventer, which is able to inhibit glutamate uptake in rat hippocampal synaptosomes. Based on results obtained with manipulation of the redox state of cysteine residues in synaptosomes and in Tx3-4, we suggest that the effect of this toxin on glutamate uptake is due to interactions that involve cysteines both in the toxin and in the transporters.

Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex.

Long-term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium-independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2+ NO3- (NOx-) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx- accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Effects of the crude venom of the social wasp Agelaia vicina on γ‐aminobutyric acid and glutamate uptake in synaptosomes from rat cerebral cortex

Glutamate (L-glu) is the most important excitatory neurotransmitter in the mammalian central nervous system. Its action is terminated by transporters located in the plasma membrane of neurons and glial cells, which have a critical role in preventing glutamate excitotoxicity under normal conditions. The neurotransmitter γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Venoms of solitary wasps and orb-spiders are composed of large proteins, medium-size peptides, polyamine amides (PAs), and other neuroactive components that are highly selective to nervous tissues. The abnormal operation of uptake systems is involved in several failures. Several studies indicate alterations in extracellular GABA and glutamate concentrations in epilepsy conditions that may relate to transporter functions. The effects of the crude and boiled venom of the social wasp Agelaia vicina, “cassununga,” on GABA and L-glu uptake in rat cerebral cortex synaptosomes are related. The venom uncompetitively inhibited high- and low-affinity GABA uptake by 91.2% and by 76%, respectively. This kind of inhibition was also found to affect high- (99.6%) and low-affinity (90%) uptake of L-glu. These results suggest that the effects observed in these experiments indicate the venom of A. vicina to be a useful tool to further characterize GABA- and L-glu-uptake systems. © 2000 John Wiley & Sons, Inc. J Biochem Toxicol 14: 88–94, 2000

Effects of the crude venom of the social wasp Agelaia vicina on gamma-aminobutyric acid and glutamate uptake in synaptosomes from rat cerebral cortex.

Glutamate (L-glu) is the most important excitatory neurotransmitter in the mammalian central nervous system. Its action is terminated by transporters located in the plasma membrane of neurons and glial cells, which have a critical role in preventing glutamate excitotoxicity under normal conditions. The neurotransmitter gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Venoms of solitary wasps and orb-spiders are composed of large proteins, medium-size peptides, polyamine amides (PAs), and other neuroactive components that are highly selective to nervous tissues. The abnormal operation of uptake systems is involved in several failures. Several studies indicate alterations in extracellular GABA and glutamate concentrations in epilepsy conditions that may relate to transporter functions. The effects of the crude and boiled venom of the social wasp Agelaia vicina, "cassununga," on GABA and L-glu uptake in rat cerebral cortex synaptosomes are related. The venom uncompetitively inhibited high- and low-affinity GABA uptake by 91.2% and by 76%, respectively. This kind of inhibition was also found to affect high- (99.6%) and low-affinity (90%) uptake of L-glu. These results suggest that the effects observed in these experiments indicate the venom of A. vicina to be a useful tool to further characterize GABA- and L-glu-uptake systems.

Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters.

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.

Nontransportable inhibitors attenuate reversal of glutamate uptake in synaptosomes following a metabolic insult.

Na+-dependent, high-affinity glutamate transporters in the central nervous system are generally credited with regulating extracellular levels of L-glutamate and maintaining concentrations below those that would induce excitotoxic injury. Under pathological conditions, however, it has been suggested that these same transporters may contribute to excitotoxic injury by serving as sites of efflux for cellular L-glutamate. In this study, we examine the efflux of [3H]D-aspartate from synaptosomes in response to both alternative substrates (i.e., heteroexchange), such as L-glutamate, and a metabolic insult (5 mM potassium cyanide and 1 mM iodoacetate). Exposure of synaptosomes containing [3H]D-aspartate to either L-glutamate or metabolic inhibitors increased the efflux of the radiolabeled substrate to over 200% of control values. Two previously identified competitive transport inhibitors (L-trans-2, 3-pyrrolidine dicarboxylate and dihydrokainate) failed to stimulate [3H]D-aspartate efflux but did inhibit glutamate-mediated heteroexchange, consistent with the action of nontransportable inhibitors. These compounds also attenuated the efflux of [3H]D-aspartate from synaptosomes exposed to the metabolic inhibitors. These results add further strength to the model of central nervous system injury-induced efflux of L-glutamate through its high-affinity transporters and identify a novel strategy to attenuate this process.

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [ 3 H ]- l-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC 50 value obtained was 2.35±0.9 μg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3±0.69%, n=3).

The Vigilance Promoting Drug Modafinil Increases Extracellular Glutamate Levels in the Medial Preoptic Area and the Posterior Hypothalamus of the Conscious Rat: Prevention by Local GABAA Receptor Blockade

The effects of modafinil on glutamatergic and GABAergic transmission in the rat medial preoptic area (MPA) and posterior hypothalamus (PH), are analysed. Modafinil (30–300 mg/kg) increased glutamate and decreased GABA levels in the MPA and PH. Local perfusion with the GABAA agonist muscimol (10 μM), reduced, while the GABAA antagonist bicuculline (1 μM and 10 μM) increased glutamate levels. The modafinil (100 mg/kg)-induced increase of glutamate levels was antagonized by local perfusion with bicuculline (1 μM). When glutamate levels were increased by the local perfusion with the glutamate uptake inhibitor L-trans-PDC (0.5 mM), modafinil produced an additional enhancement of glutamate levels. Modafinil (1–33 μM) failed to affect [3H]glutamate uptake in hypothalamic synaptosomes and slices. These findings show that modafinil increases glutamate and decreases GABA levels in MPA and PH. The evidence that bicuculline counteracts the modafinil-induced increase of glutamate levels strengthens the evidence for an inhibitory GABA/glutamate interaction in the above regions controlling the sleep-wakefulness cycle.

Uptake of l-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups

The specificity of the vesicular l-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular l-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the ic 50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with ic 50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their ic 50 values for the vesicular uptake of γ-aminobutyric acid (GABA) were all higher than 20 μM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with ic 50 values for the vesicular uptake of glutamate ranging between 1 and 30 μM and for γ-aminobutyric acid higher than 50 μM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on γ-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.

Lithium acutely inhibits and chronically up-regulates and stabilizes glutamate uptake by presynaptic nerve endings in mouse cerebral cortex.

We previously reported that lithium stimulated extracellular glutamate accumulation in monkey and mouse cerebrocortical slices. We report here that this is caused by lithium-induced inhibition of glutamate uptake into the slice. Glutamate release was amplified 5-fold over inhibition of uptake. When the effects of lithium and the specific glutamate transporter inhibitors, L-trans-pyrrolidine-2, 4-dicarboxylic acid and dihydrokainic acid, were plotted as glutamate accumulation vs. inhibition of glutamate uptake, the plots were superimposable. This finding strongly indicates that lithium-induced glutamate accumulation is caused entirely by inhibition of uptake. With cerebrocortical synaptosomes, inhibition of glutamate uptake was greater than in slices, suggesting that presynaptic nerve endings are the primary site of inhibition of uptake by lithium. Inhibition of uptake was caused by a progressive lowering of Vmax, as the lithium concentration was increased, whereas the Km remained constant, indicating that lithium inhibited the capacity of the transporter but not its affinity. Chronic treatment of mice with lithium, achieving a blood level of 0.7 mM, which is on the low side of therapeutic, up-regulated synaptosomal uptake of glutamate. This would be expected to exert an antimanic effect. Lithium is a mood stabilizer, dampening both the manic and depressive phases of bipolar disorder. Interestingly, although the uptake of glutamate varied widely in individual control mice, uptake in lithium-treated mice was stabilized over a narrow range (variance in controls, 0.423; in lithium treated, 0.184).

Aging is associated with a decrease in synaptosomal glutamate uptake and an increase in the susceptibility of synaptosomal vitamin E to oxidative stress.

We examined the influence of aging upon the uptake of glutamate by synaptosomes, and the oxidation of synaptosomal vitamin E. Synaptosomes isolated from the cerebral hemispheres of Fischer 344 rats, 4 and 24 months old, were suspended at 37 degrees C in buffer (pH 7.4) simulating extracellular fluid containing 10 mM glucose. The Km for the high affinity uptake of tritium labeled glutamate was approximately 10 microM. The uptake of glutamate was lower in synaptosomes from older animals than those from younger animals for periods of up to 20 minutes. Upon incubation with a mixture of ferrous iron and ascorbate, more of the alpha tocopherol in synaptosomes derived from older rats was oxidized than in those derived from younger ones. Older animals may be more susceptible to excitotoxicity because: a) synaptosomal reuptake of glutamate is less efficient and b) oxidative stress induced by various agents including glutamate may be higher in synaptosomes from the older animal.

ARACHIDONIC ACID INHIBITS3H-GLUTAMATE UPTAKE WITH DIFFERENT POTENCIES IN RODENT CENTRAL NERVOUS SYSTEM REGIONS EXPRESSING DIFFERENT TRANSPORTER SUBTYPES

High-affinity glutamate reuptake in neurons and glial cells, a mechanism involved in the maintenance of physiological excitatory amino acid neurotransmission, can be inhibited by arachidonic acid (AA). We studied the effect of different doses (from 10 to 500gmM) of AA onL-[3H]glutamate uptake in synaptosomes from rat cortex, rat cerebellum and mouse spinal cord. We found that AA inhibition was dose-dependent, but theIC50in the cortex differed significantly from those in the cerebellum and spinal cord (170±7.9gmMvs42.5±5.4gmMand 34.7±2.2gmMrespectively). We therefore suggest that arachidonic acid modulates uptake differently in relation to the regional expression of the glutamate transporter subtypes.

Possible regulation of high-affinity glutamate uptake in synaptosomes of normal and epileptic mice.

Glutamate (Glu) uptake is the primary mechanism for its removal from the synapse. In genetic audiogenic seizures (AGS), Glu uptake is elevated prior to the appearance of seizures. Increased Glu uptake is also observed in synaptosomes from normal mice preincubated with lithium or nitroarginine, an NO synthase inhibitor. Pertussis and cholera toxins cause a marked reduction in Glu uptake. In contrast, neither lithium nor nitroarginine affected Glu uptake by synaptosomes from genetic epileptic mice. Arachidonic acid inhibits Glu uptake, whereas synaptosomes from epileptic mouse brain appear to be more sensitive to arachidonic acid as indicated by a shift of the inhibition curve to the left. These observations are indicative of the possible regulation of Glu uptake by second messengers and its alteration in genetic epilepsy.