EFFECTS OF GLUTAMATE TRANSPORT SUBSTRATES AND GLUTAMATE RECEPTOR LIGANDS ON THE ACTIVITY OF Na+/K+-ATPase IN BRAIN TISSUE IN VITRO (original) (raw)
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Glutamate transporter coupling to Na, K-ATPase
2009
Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The ␣1-␣3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [ 3 H]D-aspartate, [ 3 H]L-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 M ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [ 3 H]D-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.
Journal of Molecular Neuroscience, 2003
Two glutamate receptor agonists, NMDA (N-methyl-D-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na + /K +-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na + /K +-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na + /K +-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.
Substrate-induced up-regulation of Na+-dependent glutamate transport activity
Neurochemistry International, 2000
Sodium-dependent transporters regulate extracellular glutamate in the CNS. Recent studies suggest that the activity of several dierent neurotransmitter transporters can be rapidly regulated by a variety of mechanisms. In the present study, we report that pre-incubation of primary`astrocyte-poor' neuronal cultures with glutamate (100 mM) for 30 min nearly doubled the V max for Na + -dependent accumulation of L-[ 3 H]-glutamate, but had no eect on Na + -dependent [ 3 H]-glycine transport. Pre-incubation with glutamate also increased the net uptake of non-radioactive glutamate, providing evidence that the increase in accumulation of L-[ 3 H]-glutamate was not related to an increase in intracellular glutamate and a subsequent increase in exchange of intracellular non-radioactive glutamate for extracellular radioactive glutamate. The glutamate receptor agonists, a-amino-3hydroxy-5-methylisoxazole-4-propionate, quisqualate, and (1 S, 3R )-1-aminocyclopentane-1,3-dicarboxylic acid did not mimic the eect of pre-incubation with glutamate and the glutamate-induced increase was not blocked by receptor antagonists. However, compounds known to interact with the transporters, including L-aspartate, D-aspartate, L-(-)-threo-3-hydroxyaspartate (L-THA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), caused variable increases in transport activity and attenuated the increase induced by glutamate, suggesting that the increase is related to the interaction of glutamate with the transporters. Several studies were attempted to de®ne the mechanism of this regulation. We found no evidence for increases in transporter synthesis or cell surface expression. Inhibitors of signaling molecules known to regulate other neurotransmitter transporters had no eect on this stimulation. Using a variety of cultures, evidence is provided to suggest that this substrate-induced upregulation of glutamate transport is speci®c for the GLT-1 and GLAST subtypes and does not in¯uence transport mediated by EAAC1. These studies suggest that the interaction of glutamate with some of the subtypes of glutamate transporters causes an increase in transport activity. Conceivably, this phenomenon provides an endogenous mechanism to increase the clearance of glutamate during periods of prolonged elevations in extracellular glutamate. 7
Neurochemistry International, 2000
Sodium-dependent binding of [ 3 H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not ®xed) rat brain. After the incubation with [ 3 H]L-aspartate, the sections were exposed against a 3 H-sensitive ®lm and the resulting autoradiograms were evaluated by quantitative densitometry. Eects of several inhibitors were examined and their potency expressed as IC 50 and n H. Together with previously published data, the present study supports the view that [ 3 H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [ 3 H]L-aspartate binding were detected. It is not certain whether these dierences re¯ect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to dierentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly dierentiate between the binding of [ 3 H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: Land D-CCG I, L-CCG III and L-CCG IV) identi®ed at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [ 3 H]L-aspartate or other transporter-speci®c ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ.
Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na + /K + ATPase
European Journal of Neuroscience, 1995
In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na+/K+ ATPase to maintain ionic gradients. Considering that impairment of Na+/K+ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na+/K+ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current-and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1-3 pM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 pM) or strophanthidin (30 pM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1-1 0 yM) and strophanthidin (1-1 0 pM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na+/Kt ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na+/K+ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na+/K+ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.
Neurosci Lett, 1996
Quantitative autoradiography of [3H]L-aspartate binding in thaw-mounted sections of rat brain has shown that L-trans-pyrrolidine-2,4dicarboxylate and D-threo-3-hydroxyaspartate but not DL-2-aminoadipate strongly interacted with the binding sites while dihydrokainate, kainate and/~-arninoadipate produced only weak effects. The potency of inhibitors did not vary from one region to another in the telencephalon (neocortex, hippocampus and neostriatum) but, n-threo-3-hydroxyaspartate, L-trans-pyrrolidine-2,4-dicarboxylate, kainate and dihydrokainate inhibited [3H]L-aspartate binding in the cerebellar cortex less potently than that in the forebrain. Characteristics of the known excitatory amino acid transporters can, in part, explain the present results but contributions from additional transporter molecules to the heterogeneity of [3H]L-aspartate binding sites cannot be ruled out.
Neuroscience Letters, 1996
Quantitative autoradiography of [3H]L-aspartate binding in thaw-mounted sections of rat brain has shown that L-trans-pyrrolidine-2,4dicarboxylate and D-threo-3-hydroxyaspartate but not DL-2-aminoadipate strongly interacted with the binding sites while dihydrokainate, kainate and/~-arninoadipate produced only weak effects. The potency of inhibitors did not vary from one region to another in the telencephalon (neocortex, hippocampus and neostriatum) but, n-threo-3-hydroxyaspartate, L-trans-pyrrolidine-2,4-dicarboxylate, kainate and dihydrokainate inhibited [3H]L-aspartate binding in the cerebellar cortex less potently than that in the forebrain. Characteristics of the known excitatory amino acid transporters can, in part, explain the present results but contributions from additional transporter molecules to the heterogeneity of [3H]L-aspartate binding sites cannot be ruled out.
Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes
Neurochemical Research, 2003
The regulation of the Na ϩ -dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with L-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca 2ϩ / diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [ 3 H]-D-aspartate uptake. This reduction was dose-and time-dependent and sensitive to PKC inhibitors. Furthermore, the L-glutamate-dependent [ 3 H]-D-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the L-glutamate effect. In sharp contrast, in cortical astrocytes, both L-glutamate and TPA pre-exposure result in an augmentation of the [ 3 H]-D-aspartate uptake. These findings suggest that the Na ϩ -dependent glutamate uptake GLAST undergoes a region-specific regulation.
The Journal of physiology, 2016
Management of glutamate and K(+) in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na(+) /K(+) -ATPase isoform combination is activated by the K(+) transients occurring during neuronal activity. In the present study, we report that glutamate transporter-mediated astrocytic Na(+) transients stimulate the Na(+) /K(+) -ATPase and thus the clearance of extracellular K(+) . Specifically, the astrocytic α2β1 Na(+) /K(+) -ATPase subunit combination displays an apparent Na(+) affinity primed to react to physiological changes in intracellular Na(+) . Accordingly, we demonstrate a distinct physiological role in K(+) management for each of the two astrocytic Na(+) /K(+) -ATPase β-subunits. Neuronal activity is associated with transient [K(+) ]o increases. The excess K(+) is cleared by surrounding astrocytes, partly by the Na(+) /K(+) -ATPase of which several subunit isoform combinations exist. The astrocytic Na(+) /K(+) -ATPase α2β2 isoform conste...
Regulation of the Na + -Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astr
Neurochem Res, 2003
The regulation of the Na ϩ -dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with L-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca 2ϩ / diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [ 3 H]-D-aspartate uptake. This reduction was dose-and time-dependent and sensitive to PKC inhibitors. Furthermore, the L-glutamate-dependent [ 3 H]-D-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the L-glutamate effect. In sharp contrast, in cortical astrocytes, both L-glutamate and TPA pre-exposure result in an augmentation of the [ 3 H]-D-aspartate uptake. These findings suggest that the Na ϩ -dependent glutamate uptake GLAST undergoes a region-specific regulation.