Critical role of serine 465 in isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3

Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) alpha-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isoflurane-increased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKCalpha in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKCalpha mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.     

蛋白激酶C对大鼠缺血海马突触体谷氨酸摄取的调控作用

采用大鼠海马脑片体外缺血模型,观察海马突触体内蛋白激酶Ｃ（ＰＫＣ）活性的变化,以及这种变化对突触体谷氨酸（ＧＬＵ）摄取的影响。结果显示：海马脑片体外“缺血”１０ｍｉｎ,其突触体内ＰＫＣ活性基本不变,而缺血３０ｍｉｎ,突触体内ＰＫＣ活性显著上升（Ｐ＜０．０１,ｎ＝６）;非Ｎ－甲基－Ｄ－天门冬氨酸（ＮＭＤＡ）受体拮抗剂ＤＮＱＸ有效地抑制ＰＫＣ活性的同时,可降低胞外ＧＬＵ的堆积,而ＮＭＤＡ受体阻断剂ＡＰ＿５无作用。进一步实验证明,ＰＫＣ激动剂ＰＤＢ浓度依赖性地抑制突触体对３Ｈ－ＧＬＵ的摄取（ＩＣ５０＝１３１±１０μｍｏｌ／Ｌ）,此抑制作用可由ＰＫＣ抑制剂Ｈ－７（１００μｍｏｌ／Ｌ）抵消。提示脑缺血诱发ＧＬＵ堆积的作用机理可能是：脑缺血引发钙内流导致ＧＬＵ过量释放,ＧＬＵ又通过突触前非ＮＭＤＡ受体激活ＰＫＣ,抑制其自身摄取,正反馈性加重胞外ＧＬＵ的堆积。     

Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl^– currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-Glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential –60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential –17 mV at extracellular Cl^– concentration ([Cl^–]_o) 104 mM with an 60-mV shift per 10-fold change in [Cl^–]_o, properties consistent with Cl^–-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [³H]-L-glutamate. Thus PMA-enhanced Cl^– currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl^– currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl^– currents only at a high concentration (1 µM). Intracellular injection of BAPTA, a Ca²⁺-chelating agent, did not affect the PMA-enhanced Cl^– currents. 4-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl^– currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently. oocytes; protein kinase C     

The release of glutamate and aspartate from rat brain synaptosomes in response to domoic acid (amnesic shellfish toxin) and kainic acid

Kainic acid is known to stimulate the release of glutamate (GLU) and aspartate (ASP) from presynaptic neurons. It has been suggested that the enhanced release of these endogenous EAA's plays a significant role in the excitotoxic effects of KA. Domoic acid (DOM), a shellfish toxin, is structurally similar to KA, and has been shown to be 3–8 times more toxic than KA. In this study, effects of KA and DOM on the release of GLU and ASP from rat brain synaptosomes were investigated. Amino acid analysis was performed by the reversed phase HPLC, following derivatization with 9-fluorenylmethyl chloroformate (FMOC). Potassium chloride (40 mM) was used as a positive control, and stimulated GLU release from rat brain synaptosomes in presence or absence of Ca²⁺. DOM enhanced the release of GLU, whereas KA stimulated the release of both GLU and ASP from synaptosomes in the presence of Ca²⁺. However, their potency to stimulate GLU and ASP release was enhanced in absence of Ca²⁺. These results indicate that diferent mechanisms may be involved in the release of GLU and ASP in response to DOM and KA, and that neurotransmitter release appeared to be highly specific for these agonists. It would appear that DOM and KA may interact with different receptors on the presynaptic nerve terminal, and/or activate different subtypes of voltage-dependent Ca²⁺ channels to promote influx of Ca²⁺ which is targeted for different pools of neurotransmitters.Abbreviations ANOVA analysis of variance - ASP aspartate - DOM domoic acid - DHKA dihydrokainic acid - EAA excitatory amino acid - FMOC 9-fluorenylmethyl chloroformate - GLU glutamate - KA kainic acid     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)₂D₃ formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (I_glu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased I_glu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.     

Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH₂-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC.     

Endogenous excitatory amino acid release from brain slices and astrocyte cultures evoked by trimethyltin and other neurotoxic agents

Trimethyltin (TMT) is a toxic alkyltin compound that is known to produce neuronal necrosis in the CNS. The present study examined the effects of TMT on the release of excitatory amino acids (EAA) from cortical slices prepared from adult and aged (24 months old) rats. The calcium dependence of TMT-induced EAA efflux was evaluated and compared to other neurotoxic agents. The actions of TMT were also evaluated in an astrocyte culture model to assess glial contributions to TMT-induced EAA efflux. TMT (10–1000 M) evoked a dose-related increase in GLU and ASP efflux during a 30 min incubation period and this efflux was sustained or slightly higher during a 15 min recovery period. TMT-stimulated GLU efflux was not altered in aged rats. TMT-induced GLU efflux was significantly reduced by removing extracellular calcium and including 10 M EGTA in the incubation media. Calcium channel blockers (nifedipine, verapamil, flunarizine, amiloride, neomycin) and MK-801 did not significantly attenuate TMT-induced GLU efflux. Diltiazem (25 M) produced modest but inconsistent reductions in TMT-induced GLU efflux from brain slices, and significantly inhibited the leakage of lactate dehydrogenase (LDH) from TMT-treated astrocyte cultures. TMT did not increase GLU efflux from glial cultures during a 30 min incubation period, but did significantly elevate GLU efflux during the 15 min recovery period. TMT evoked the release of EAA by both calcium dependent and independent mechanisms in brain slices. TMT at high concentrations also produced a delayed increase in glial GLU efflux. These studies suggest that excitotoxic mechanisms may contribute to TMT-induced neurotoxicity.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.     

Excitatory amino acid transporters: keeping up with glutamate

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Among the five known human EAAT subtypes, the glial carriers, EAAT1 and EAAT2 have the greatest impact on clearance of glutamate released during neurotransmission. Studies of carriers expressed on neurons, Purkinje cells and photoreceptor cells (EAAT3, EAAT4 and EAAT5, respectively) suggest more subtle roles for these subtypes in regulating excitability and signalling. The data suggest that EAA transporters may influence glutamatergic transmission by regulating the amount of glutamate available to activate pre- and post-synaptic metabotropic receptors and by altering neuronal excitability through a transporter-associated anion conductance that is activated by carrier substrates. Recent studies on structural, mechanistic and physiological aspects of carrier function in a variety of model systems and organisms have led to surprising insights into how excitatory amino acid transporters shape cellular communication in the nervous system.     

Protein Kinase C-Mediated Suppression of the Presynaptic Adenosine A1 Receptor by a Facilitatory Metabotropic Glutamate Receptor

Abstract: KCl-evoked glutamate exocytosis from cerebrocortical synaptosomes can be inhibited by the adenosine A1 receptor agonist cyclohexyladenosine (CHA). Inhibition is associated with a decreased KCl-evoked Ca²⁺ level elevation, and the effect of the agonist is occluded by prior incubation with the Agelenopsis aperta neurotoxin ω-agatoxin-IVA at 250 n M . The inhibition is suppressed in the presence of 3 n M phorbol dibutyrate (PDBu) or by activation of the protein kinase C (PKC)-coupled metabotropic glutamate receptor by 100 µ M (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )ACPD]. A tonic inhibition of release by leaked exogenous adenosine can be reversed by adenosine deaminase or by PDBu addition. The CHA-induced inhibition can be enhanced by the PKC inhibitor Ro 31-8220. The mechanism for the suppression of the adenosine A1 receptor-mediated inhibition is distinct from that previously described for the (1 S ,3 R )ACPD-evoked, PKC-mediated, facilitatory pathway, which enhances phosphorylation of the MARCKS protein, 4-aminopyridine-induced action potentials, and release of glutamate because the latter requires at least 100 n M PDBu [or the combination of (1 S ,3 R )ACPD and arachidonic acid] and is not seen following KCl depolarization. Both PKC-mediated pathways may be involved in the presynaptic events associated with the establishment of synaptic plasticity.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP-43 in Rat Hippocampal Slices and Synaptosomes

Abstract: Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 µM), an N-methyl-d -aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates—arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)—were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 µM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.     

Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium‐dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCα, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCα can be co‐immunoprecipitated with EAAC1. When the glial glutamate transporter GLT‐1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester‐activated subtypes of PKC was examined in C6 glioma transfected with GLT‐1. Of the classical subtypes, only PKCα was detected, and of the non‐classical subtypes, PKCδ and PKCε were detected. In this system, phorbol ester‐dependent internalization of GLT‐1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCδ (rottlerin). PKCα immunoreactivity was found in GLT‐1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCα in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCα may be required for the regulated redistribution of GLT‐1.     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

Glutamate transport is coupled to the co-transport of 3 Na(+) and 1 H(+) followed by the counter-transport of 1 K(+). In addition, glutamate and Na(+) binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [(14)C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.     

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.     

Galanin reduces PDBu-induced protein phosphorylation in rat ventral hippocampus.

The effect of galanin (GAL) on basal and phorbol-12,13-dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2 microM) inhibited PDBu stimulation in a concentration-dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity. Depolarization of miniprisms from ventral hippocampi by 18 mM K+ prevented the effect of GAL on PDBu-induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor-mediated action.