Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with -threo-β-benzyloxyaspartate

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na⁺-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using -threo-β-benzyloxyaspartate ( -TBOA), a newly developed competitive, non-transported blocker of the EAAT 1–3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus -TBOA (100 μM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of -TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of -TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of -TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na⁺, and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na⁺ uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na⁺-dependent, glutamate transporters, -TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.     

Indomethacin modulates ischemia-evoked release of glutamate and adenosine from the rat cerebral cortex

The effects of indomethacin (10 mg/kg) on the release of the transmitter amino acids, glutamate, aspartate, GABA, and of the purines, adenosine and inosine, from the cerebral cortex was studied in a four-vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, indomethacin significantly attenuated the ischemia-evoked release of glutamate and aspartate, but not of GABA. Adenosine levels in the cortical superfusates were significantly elevated following indomethacin administration. As indomethacin is a potent inhibitor of adenosine uptake, these results suggest that, by blocking adenosine uptake, indomethacin could elevate extracellular adenosine levels and depress glutamate and asparte efflux as a consequence of the activation of adenosine A₁ receptors.     

Effects of the anion channel blocker DIDS on ouabain- and high K(+)-induced release of amino acids from the rat cerebral cortex

Amino acid release from the rat cerebral cortex was analyzed using an in vivo cortical cup perfusion model. Topical applications of ouabain or high extracellular K(+) were used to mimic two dimensions of ischemic conditions which promote cell swelling and amino acid release. Ouabain (30 microM) induced significant releases of taurine, gamma-aminobutyric acid (GABA), aspartate, glutamate and phosphoethanolamine. The anion channel blocker, 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS; 1 mM), inhibited ouabain-induced release of all these amino acids except for glutamate. Exposure to high extracellular K(+) (75 mM) induced a delayed rise in the levels of taurine in the superfusates and an immediate increase in GABA levels. There were no significant releases of other amino acids. The release of taurine and GABA was sensitive to the blocking of anion channels with DIDS. Both ouabain- and high K(+)-induced taurine release is likely to be mediated by DIDS sensitive anion channels. The extracellular accumulation of the other amino acids, where insensitive to DIDS, may be mediated by mechanisms other than swelling-induced anion channels.     

Further studies on the effects of topical lactate on amino acid efflux from the ischemic rat cortex

A rat four-vessel cerebral occlusion model was used to examine the effects of D-lactate and oxamate, a lactate dehydrogenase inhibitor, on cortical window superfusate levels of amino acids, glucose and L-lactate. Superfusate levels of aspartate, glutamate, taurine, GABA and phosphoethanolamine rose during ischemia and then declined during reperfusion. Glycine and alanine levels tended to increase during reperfusion, whereas glutamine levels were lower. Serine levels were not altered. Glucose levels declined rapidly during ischemia and recovered during reperfusion. Lactate levels were sustained during ischemia and increased during reperfusion. Unlike L-lactate, which attenuated ischemia/reperfusion (I/R) evoked amino acid release (J.W. Phillis, D. Song, L.L. Guyot, M.H. O'Regan, Lactate reduces amino acid release and fuels recovery of function in the ischemic brain, Neurosci. Lett. 272 (1999) 195-198), topical application of D-lactate (20 mM), which is not used as an energy substrate, enhanced the I/R release of aspartate, glutamate, GABA and taurine into cortical superfusates, and also elevated L-lactate levels above those in the controls. Glucose levels were not altered. Oxamate (20 mM) application elevated the pre-ischemia levels of alanine, glycine and GABA and those of GABA during ischemia. Levels of all amino acids, with the exception of phosphoethanolamine, were elevated during reperfusion. Oxamate, an inhibitor of lactate dehydrogenases 1 and 5, did not alter the pattern of efflux of glucose and L-lactate. In the presence of oxamate, L-lactate (20 mM) failed to inhibit amino acid release. The failure of D-lactate to attenuate amino acid release confirms the inability of this isomer to act as a metabolic substrate. The oxamate data indicate that inhibition of lactate dehydrogenase is detrimental to the viability of cortical cells during I/R, even though extracellular lactate levels are elevated. The pre-ischemia increases in alanine and glycine are suggestive of elevations in pyruvate as a result of the block of its conversion to lactate, with transamination reactions converting pyruvate to form these amino acids. In summary, the results further substantiate the concept of a role for L-lactate as a cerebral energy substrate.     

Tamoxifen, a chloride channel blocker, reduces glutamate and aspartate release from the ischemic cerebral cortex

The effects of the anti-estrogen, anion channel blocker, tamoxifen on amino acid release from the ischemic rat cerebral cortex was investigated using a cortical cup technique. Tamoxifen (20 μM in artificial cerebrospinal fluid), applied topically, inhibited the ischemia-evoked efflux of aspartate, glutamate, taurine and phosphoethanolamine. Reductions in the ischemia-evoked levels of these amino acids suggest that tamoxifen may attenuate chloride-related osmotic cell swelling and the associated regulatory volume decrease (RVD) release of amino acids.     

Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex

Elevated levels of glutamate and aspartate have been implicated in the pathogenesis of neural injury and death induced by ischemia. The mechanism(s) whereby they escape into the extracellular environment have been a subject of controversy. This study evaluated the contribution of phospholipases and protein kinases to ischemia-evoked glutamate and aspartate release from the ischemic/reperfused rat cerebral cortex. Changes in the extracellular levels of these amino acids during four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked amino acid release was compared in control vs. drug treated animals, in which selective inhibitors of phospholipases and protein kinases were applied topically onto the cerebral cortex. The phospholipase inhibitors tested included 4-bromophenacyl bromide, a non-selective inhibitor; 7,7-dimethyleicosadienoic (DEDA), an inhibitor of secretory type phospholipase A₂ (PLA₂); AACOCF₃, an inhibitor of the Ca²⁺-dependent cytoplasmic form of PLA₂, HELSS, which inhibits a Ca²⁺-independent cytoplasmic PLA₂, and U73122, a selective inhibitor of phospholipase C (PLC). All five phospholipase inhibitors significantly attenuated glutamate and aspartate release into the extracellular milieu, indicating the possibility that several forms of the enzyme are likely to be involved. The protein kinase C (PKC) inhibitor, chelerythrine chloride, also reduced excitatory amino acid efflux, whereas the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced their release. The non-selective kinase inhibitor, staurosporine, and H-89, which selectively inhibits protein kinase A, did not reduce ischemia-evoked amino acid efflux. These results suggest that ischemia-evoked release of the excitatory transmitters amino acids is a result, in part, of the activation of phospholipases A₂ and C, with PKC involvement in the transduction process. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis, may allow these transmitter amino acids to diffuse down their concentration gradients into the extracellular fluid.     

Hyposmotically induced amino acid release from the rat cerebral cortex: role of phospholipases and protein kinases.

In an evaluation of the contribution of swelling-induced amino acid release, through the regulatory volume decrease (RVD) process, to cerebral ischemic injury, studies of the role of phospholipases and protein kinases in the response to hyposmotic stress were undertaken using an in vivo rat cortical cup model. Hyposmotic stress induced significant releases of aspartate, glutamate, glycine, phosphoethanolamine, taurine and GABA from the rat cerebral cortex. Taurine release was most affected, exhibiting a greater than 9-fold increase during the hyposmotic stimulus. The phospholipase A2 (PLA2) inhibitors 4-bromophenacyl bromide (1 microM) and 7,7-dimethyleicosadienoic acid (5 microM) had no significant effects on hyposmotically induced amino acid release. AACOCF3 (50 microM), an inhibitor of cytosolic PLA2 decreased taurine release to 84% of DMSO controls. The release of the other amino acids was not affected. The phospholipase C inhibitor U73122 (5 microM) had no significant effects on amino acid release. The protein kinase C (PKC) inhibitor chelerythrine (5 microM) significantly reduced hyposmotically induced taurine release to 72% of saline controls but had no significant effects on the other amino acids. Stimulation of PKC with phorbol 12-myristate, 13-acetate (10 microM) did not significantly change taurine, glutamate, glycine or phosphethanolamine release. The releases of aspartate and GABA were enhanced 2 to 3 fold. Phorbol 12,13-didecanoate (10 microM), another potent stimulator of PKC, significantly increased taurine release to 122% of DMSO controls. The releases of aspartate, glutamate and glycine were enhanced 2.5 to 3.5 fold. Similarly, stimulation of protein kinase A with forskolin (100 microM) significantly increased taurine, aspartate, and glycine release 1.5- to 2-fold compared to DMSO controls. In summary, phospholipases may play a minor role in volume regulation. These studies also support the hypothesis that protein kinases play a modulatory role in the RVD response. The results show that although RVD may play a role, additional mechanisms, including phospholipase activation, must be involved in the ischemia-evoked release of excitotoxic amino acids.     

Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport

Astrocytes have essential roles for neuron survival and function, so that their demise in neurodegenerative insults, such as ischemia, deserves attention. A major event of the cell death cascade in ischemia is the reversed operation of excitatory amino acid transporters (EAAT), releasing glutamate. Cytotoxicity is conventionally attributed to extracellular glutamate accumulation. We previously reported that mimicking such dysfunction by EAAT substrate inhibitors, whose uptake induces glutamate release by heteroexchange, triggers glutathione (GSH) depletion and oxidative death of differentiated astrocytes in culture. Here we demonstrate that astrocyte death, although correlated with glutamate release, is not resulting from high extracellular glutamate-mediated toxicity. L-glutamate per se was gliotoxic only at concentrations much higher than the maximum reached with the potent EAAT substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and toxicity was lower. Moreover, high glutamate concentrations offered protection against PDC. Protection was also provided by L-aspartate, which is both transported by EAAT and metabolized into glutamate, and by inhibiting glutamine synthetase, which uses transported glutamate to synthesize glutamine. Neither D-aspartate, a metabolically inert EAAT substrate, nor compounds that can provide glutamate intracellularly but are not EAAT substrates offered protection. Interestingly, only the compounds providing protection prevented PDC-induced GSH depletion. These data strongly suggest that reversed uptake-mediated astrocyte death results from the leakage of glutamate from a compartmentalized intracellular metabolic pool specifically fuelled by EAAT, crucial for preserving GSH contents. In addition, we provide evidence for a minor contribution of the cystine-glutamate antiporter x(c) (-) but a major role of the 5-lipoxygenase pathway in this death mechanism.     

Inhibition of Na(+)/H(+) exchange by 5-(N-ethyl-N-isopropyl)-amiloride reduces free fatty acid efflux from the ischemic reperfused rat cerebral cortex

Brain tissue acidosis is considered to be a contributor to ischemic brain injury. The deleterious effects of marked acidosis may be associated with reperfusion and an excessive entry of Na(+) into cerebral neurons and glia as intracellular pH is restored by Na(+)/H(+) exchange. Normalization of pH, with activation of many calcium-dependent and other phospholipases and proteases with pH optima in the neutral or alkaline range, could account for the pronounced elevation in extracellular levels of free fatty acids which occurs during reperfusion following cerebral ischemia. In the present investigation we evaluated the effects of inhibition of Na(+)/H(+) exchange with N-(N-ethyl-N-isopropyl)-amiloride (EIPA; 25 microM) applied topically onto the rat cerebral cortex prior to and during ischemia. Free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high pressure liquid chromatography. EIPA application effectively inhibited the increases in arachidonic and linoleic acid release observed in the control rats during reperfusion, and non-significantly depressed that of palmitic and oleic acids. Superfusate levels of glucose, which decline to near zero levels during ischemia and then rebound during reperfusion, were not affected by EIPA administration. Lactate levels in cortical superfusates from EIPA-treated animals rose more rapidly during reperfusion than did those in the control rats and then significantly declined towards basal levels. The data indicate that inhibition of Na(+)/H(+) exchange prevented the activation of phospholipases that usually occurs during reperfusion following a cerebral ischemic episode. These results are the first demonstration of such an effect and may provide an explanation for the cerebroprotective effects that have been observed in stroked animals following administration of Na(+)/H(+) exchange inhibitors.     

Inhibition of Na(+)/Ca(2+) exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury

Reversal of the Na(+)/Ca(2+) exchanger (NCX) occurs during ischemia-reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 microM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 microM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na(+)/Ca(2+) exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na(+)/Ca(2+) exchange inhibitors in ischemia-reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.     

Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats

The aim of this study was to measure extracellular chemical changes in the cerebral cortex in response to compression contusion trauma in rats. Energy-related metabolites (i.e., lactate, pyruvate, adenosine, inosine, and hypoxanthine) and amino acids were harvested from the extracellular fluid (ECF) using microdialysis and analyzed by high-performance liquid chromatography. The measurements were performed in cortical tissue, where neuronal injury occurs in this model. The severity of the trauma was varied by using different depths of impact: mild trauma, 1.5 mm; severe trauma, 2.5 mm. The trauma induced a dramatic increase in the ECF levels of energy-related metabolites that was conditioned by the severity of the insult. The ECF level of taurine, glutamate, aspartate, and gamma-aminobutyric acid (GABA) also rose markedly, while other amino acids did not change significantly. The results suggest that the trauma induced a transient, profound focal disturbance of energy metabolism in the cortical tissue, probably as a result of mechanically induced disruption of ion homeostasis and reduced blood flow in combination. The data support the potential role of glutamate and aspartate as mediators of traumatic brain injury. However, the concomitantly released adenosine, GABA, and taurine may be protective and ameliorate excitotoxicity. In analogy with the reported cumulative damaging effects of repeated ischemic insults, the observed ECF changes may help explain the vulnerability of traumatized brain tissue to secondary ischemia.     

Depolarization, exocytosis and amino acid release evoked by hyposmolarity from cortical synaptosomes

External osmolarity reduction (20%) led to labelled glutamate, GABA and taurine release from rat brain cortical synaptosomes. A Cl--independent, Na+-dependent, La3+-sensitive and tetrodotoxin (TTX) reduced depolarization of synaptosomes occurred upon hyposmolarity, suggestive of Na+ entry through nonselective cation channels. This depolarization, together with cytosolic Ca2+ ([Ca2+]I) increase, resulted in exocytosis, monitored by FM1-43. The release fraction resulting from these phenomena was estimated, by its decrease, by La3+, EGTA-AM and tetanus toxin (TeTX), as 34-44% for glutamate, 21-29% for GABA and 18-22% for taurine. Protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate (PMA) increased the hyposmolarity-elicited exocytosis and this activation increased glutamate (80%), GABA (51%) and taurine (42%) hyposmotic efflux. Inhibition by chelerythrine reduced glutamate, GABA and taurine efflux by 64%, 50% and 24%, respectively. The Na+-dependence of amino acid release (glutamate 63%, GABA 46% and taurine 29%) may result from both, prevention of the depolarization-exocytosis efflux, and blockade of the carrier reversal operation. Carrier blockade by dl-threo-beta-benzyloxy aspartate (TBOA) and NO-711 resulted in 37% and 28% reduction of glutamate and GABA release, respectively. Contribution of the osmolyte leak pathway to amino acid release, estimated by the influence of Cl- (NPPB) and tyrosine kinase (AG18) blocker, was up to 55% for taurine, but only 10-18% for GABA, with apparently no contribution for glutamate. The predominant osmolyte-type mechanism of taurine release suggest its function in volume control in nerve endings, while glutamate and GABA respond to events concurrent with hyposmolarity by a neurotransmitter-like release mechanism. The hyposmolarity-induced amino acid efflux from nerve endings may have consequences for neuronal excitability during hyponatremia.     

Melittin enhances amino acid and free fatty acid release from the in vivo cerebral cortex

The effects of the phospholipase activator melittin on amino acid and free fatty acid release from the rat cerebral cortex were monitored and compared with those of a secretory PLA(2), using a cortical cup technique with topical application of these agents in artificial cerebrospinal fluid. Melittin (10 microg/ml; 3.5 microM) elicited a rapid increase in the levels of superfusate amino acids; aspartate, glutamate, GABA, glycine, taurine, glutamine, phosphoethanolamine, alanine, serine and the free fatty acids arachidonic, linoleic, palmitic and oleic acid. PLA(2) (25 microg/ml) also enhanced amino acid efflux but its effects were significantly slower to develop than those of melittin. The results confirm previous indications of an ability of phospholipases to augment extracellular levels of several amino acids, including the excitotoxins glutamate and aspartate, and further implicate phospholipase activation as a significant contributor to cerebral ischemic injury. Melittin has the potential to be a useful tool with which to evaluate the role of phospholipases in ischemia injury.     

Glial transporters for glutamate, glycine and GABA I. Glutamate transporters

The termination of chemical neurotransmission in the CNS involves the rapid removal of neurotransmitter from synapses by specific transport systems. Such mechanism operates for the three major amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA) and glycine. To date, five different high-affinity Na(+)-dependent glutamate (Glu) transporters have been cloned: GLT1, GLAST, EAAC1, EAAT4 and EAAT5. The first two are expressed mainly by glial cells, and seem to be the predominant Glu transporters in the brain. A major function of Glu uptake in the nervous system is to prevent extracellular Glu concentrations from raising to neurotoxic levels in which glial transporters seem to play a critical role in protecting neurons from glutamate-induced excitotoxicity. Under particular conditions, glial GluTs have been shown to release Glu by reversal of activity, in a Ca(2+)--and energy-independent fashion. Furthermore, an activity of these transporters as ion channels or transducing units coupled to G-proteins has recently been reported. The localization, stoichiometry, and regulation of glial GluTs are outlined, as well as their possible contributions to nervous system diseases as ALS, AD and ischemic damage.     

Effect of hyperglycemia on extracellular levels of amino acids and free fatty acids in the ischemic/reperfused rat cerebral cortex

This study analyzed the effects of pre-existing hyperglycemia on the extracellular levels of glutamate, other amino acids and free fatty acids, including arachidonic acid, in the ischemic/reperfused rat cerebral cortex, using a cortical cup technique. Forebrain cerebral ischemia (20 min) was induced by four vessel occlusion. Glucose (3.4 g/kg) was administered 30 min prior to ischemia. Glucose administration had no effect on basal levels of superfusate amino acids and reduced basal levels of linoleic and oleic acids. Cerebral ischemia elicited increased superfusate levels of aspartate, glutamate, phosphoethanolamine, taurine, gamma-aminobutyric acid (GABA) and arachidonic acid when compared with basal levels. Reperfusion caused a further increase in phosphoethanolamine and arachidonic acid levels and transient increases in linoleic, oleic and palmitic acids. Hyperglycemia resulted in significantly reduced levels of glutamate, phosphoethanolamine, GABA and arachidonic, myristic, palmitic, linoleic and oleic acids during ischemia/reperfusion in comparison with the saline-injected ischemic controls. The results indicate that ischemia/reperfusion-evoked increases in the extracellular levels of glutamate, certain other amino acids and free fatty acids are attenuated by prior systemic glucose administration.     

Hypothyroidism reduces glutamate-synaptic release by ouabain depolarization in rat CA3-hippocampal region

Thyroid hormones modulate the physiology of the hippocampus in humans, where glutamate plays an important role as neurotransmitter. The aim of this work was to study the effect of hypothyroidism on hippocampal glutamate extracellular levels, release, uptake, and synthesis. The effects of PDC (a glutamate transporter inhibitor) and ouabain (a Na(+) /K(+) -ATPase inhibitor) infusion on microdialysate glutamate and aspartate levels of CA3 hippocampal region were evaluated. Animals were assigned to one of the following groups: hypothyroid group (Hyp), receiving methimazole (anantithyroid drug); replacement group (Hyp + T(4) ), receiving antithyroid treatment plus thyroxine; and euthyroid control group (Eut). Dialysate fractions were collected every 15 min to determine basal glutamate levels for 1 hr. Then, PDC (10 mM) or ouabain (100 μM) was infused for 30 min. Results showed lower glutamate and aspartate basal levels in Hyp than in Eut groups. PDC infusion increased amino acids levels in all groups, whereas ouabain infusion increased glutamate and aspartate levels only in the Eut group. The infusion of tetrodotoxin (TTX; a voltage-gated sodium channel inhibitor) prevented the glutamate increase in euthyroid rats. The Hyp + T(4) group showed glutamate levels similar to those found in the Eut group. Additionally, glutaminase activity in hippocampus was lower in the Hyp group than in the Eut or Hyp + T(4) group. Results suggest that high-affinity glutamate transporters are not altered by hypothyroidism; however, decreased hypotyroidism reduced vesicular glutamate release in the CA3-hippocampal region as a consequence of diminished glutamate synthesis.     

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.     

Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca(2+) and Na(+) in cultured cerebellar neurons

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.     

Taurine uptake by chick embryo retinal neurons and glial cells in purified culture

The properties and cellular distribution of a high-affinity uptake mechanism for taurine have been investigated using separate populations of purified chick embryo neural retina neurons and glia. Purified neuronal monolayers, cultured in serum-free medium, were incubated in radioactive taurine under different conditions and studied autoradiographically and biochemically. Labeling with radioactive taurine was detected in the perikaryon of most of the neurons present in the cultures. Neuronal uptake occurred by means of a high-affinity mechanism which was completely inhibited at low temperatures or in the absence of sodium ions. The uptake was linear for at least 1 hr and, as is the case in vivo, could be inhibited by gamma-aminobutyric acid (GABA) or beta-alanine. Incubation in ouabain, glutamate, or high K+ concentrations failed to cause any increase in the amount of taurine released by neurons preloaded with the radioactive amino acid. The rather wide-spread distribution of high-affinity taurine uptake was confirmed using separate retinal cultures rich in glial cells. Practically 100% of the glial cells appeared labeled after incubation in 10(-7) M [3H] taurine, and this uptake was also inhibited by low-temperature, Na+-free medium, GABA, or beta-alanine. Several pieces of evidence indicate that high-affinity taurine uptake coexists with uptake mechanisms for other amino acids, such as GABA, glutamate, and aspartate, in retinal neurons as well as glial cells. These in vitro populations offer a promising experimental system for the investigation of the effects of taurine on retinal cells.