Glutamate release inhibitors: A critical assessment of their action mechanism

Summary A number of important experimental data do not support the widespread hypothesis that Na⁺-channels block is cerebroprotective, essentially because it reduces presynaptic glutamate release: (i) the inhibition of exocytosis by these compounds is not specific to glutamate; (ii) aspartate efflux produced by various stimuli was also reduced, but aspartate cannot be released by exocytosis because it is not concentrated within presynaptic vesicles; and (iii) glutamate accumulated extracellularly during ischaemic or traumatic insult to the CNS is mainly of cytosolic origin. As an alternative, we propose that use-dependent Na⁺-channel blockers enhance the resistance of nerve cells to insults, primarily by decreasing their energy demand, and that reduced efflux of glutamate and other compounds is aconsequence of attenuated cellular stress.     

Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes

The regulation of the Na⁺-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 Ca²⁺/ diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [³H]-d-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the l-glutamate–dependent [³H]-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 [³H]-d-aspartate uptake. These findings suggest that the Na⁺-dependent glutamate uptake GLAST undergoes a region-specific regulation.     

Rottlerin Inhibits (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)-ATPase Activity in Brain Tissue and Alters <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na⁺, K⁺)-dependent ATPase (Na⁺, K⁺-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na⁺, K⁺-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na⁺, K⁺-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na⁺, K⁺-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.     

Thrombin potentiates d‐aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption

Thrombin levels increase in brain during ischemia and hemorrhagic episodes, and may contribute to excitotoxic neural damage. This study examined the effect of thrombin on glutamate efflux from rat cortical cultured astrocytes using ³H‐d ‐aspartate as radiotracer. The glutamate efflux was initiated by addition of 100 mM K⁺ plus 1 mM ouabain (K/O) to replicate extracellular and intracellular ionic changes that occur during cerebral ischemia. Upon exposure to K/O, astrocytes swelled slowly and progressively with no evidence of volume regulation. The K/O‐induced swelling was inhibited by 65% with bumetanide and 25% with BaCl₂, suggesting contribution of Na⁺/K⁺/Cl^? co‐transporter and Kir channels. K/O‐elicited ³H‐d ‐aspartate that consisted of two phases. The first transient component of the release corresponded to 13.5% of total ³H‐d ‐aspartate loaded. It was markedly reduced (61%) by the glutamate transporter blocker DL‐threo‐b‐Benzyloxyaspartic acid and weakly inhibited (21%) by the volume‐sensitive anion channel blocker 4‐[(2‐Butyl‐6,7dichloro‐2‐cyclopentyl‐2,3‐dihidro‐1oxo‐1H‐inden‐5‐yl)oxy] butanoic acid (DCPIB). During the second sustained phase of release, cells lost 45% of loaded of ³H‐d ‐aspartate via a mechanism that was insensitive to DL‐threo‐b‐Benzyloxyaspartic acid but nearly completely suppressed by DCPIB. Thrombin (5 U/mL) had only marginal effects on the first phase but strongly potentiated (more than two‐fold) ³H‐d ‐aspartate efflux in the second phase. The effect of thrombin effect was proportional to cell swelling and completely suppressed by DCPIB. Overall our data showed that under K/O swelling conditions, thrombin potently enhance glutamate release via volume‐sensitive anion channel. Similar mechanisms may contribute to brain damage in neural pathologies which are associated with cell swelling, glutamate efflux and increased thrombin levels.     

Characterization of endogenous amino acid efflux from hippocampal slices during chemically-induced ischemia

Using sodium (NaN₃)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia, we have characterized the endogenous release of excitatory and inhibitory amino acids from superfused hippocampal slices. Chemical ischemia produced an azide (1–30 mM) dose-dependent increase in the efflux of glutamate, aspartate and GABA. These increases were attenuated to varying degrees by removal of Ca²⁺, or the addition of the voltage dependent Na⁺-channel blocker tetrodotoxin (TTX), the selective Ca²⁺ channel blockers conotoxin MVIIA, MVIIC, and nifedipine, the NMDA antagonist MK801, the AMPA antagonist GYKI-52466. Similarly, addition of the GLT-1 glutamate transport inhibitor dihydrokainate (DHK) and the anti-estrogen/anion channel blocker tamoxifen also attenuated the efflux of glutamate and GABA. It would therefore appear that the increases in amino acid efflux induced by chemical ischemia originates from both the neuronal pool, via conventional exocytotic release, and glial sources via reversal of the GLT-1 transporter and anion channel regulated cell swelling.     

Glutamate Transporters and Mitochondria: Signaling,Co-compartmentalization,Functional Coupling,and Future Directions

In addition to being an amino acid that is incorporated into proteins, glutamate is the most abundant neurotransmitter in the mammalian CNS, the precursor for the inhibitory neurotransmitter γ-aminobutyric acid, and one metabolic step from the tricarboxylic acid cycle intermediate α-ketoglutarate. Extracellular glutamate is cleared by a family of Na⁺-dependent transporters. These transporters are variably expressed by all cell types in the nervous system, but the bulk of clearance is into astrocytes. GLT-1 and GLAST (also called EAAT2 and EAAT1) mediate this activity and are extremely abundant proteins with their expression enriched in fine astrocyte processes. In this review, we will focus on three topics related to these astrocytic glutamate transporters. First, these transporters co-transport three Na⁺ ions and a H⁺ with each molecule of glutamate and counter-transport one K⁺; they are also coupled to a Cl^? conductance. The movement of Na⁺ is sufficient to cause profound astrocytic depolarization, and the movement of H⁺ is linked to astrocytic acidification. In addition, the movement of Na⁺ can trigger the activation of Na⁺ co-transporters (e.g. Na⁺–Ca²⁺ exchangers). We will describe the ways in which these ionic movements have been linked as signals to brain function and/or metabolism. Second, these transporters co-compartmentalize with mitochondria, potentially providing a mechanism to supply glutamate to mitochondria as a source of fuel for the brain. We will provide an overview of the proteins involved, discuss the evidence that glutamate is oxidized, and then highlight some of the un-resolved issues related to glutamate oxidation. Finally, we will review evidence that ischemic insults (stroke or oxygen/glucose deprivation) cause changes in these astrocytic mitochondria and discuss the ways in which these changes have been linked to glutamate transport, glutamate transport-dependent signaling, and altered glutamate metabolism. We conclude with a broader summary of some of the unresolved issues.

     

Regulation of the Mouse Na<Superscript>+</Superscript>-Dependent Glutamate/Aspartate Transporter GLAST: Putative Role of an AP-1 DNA Binding Site

Appropriate removal of l-glutamate from the synaptic cleft is important for prevention of the excitotoxic effects of this neurotransmitter. The Na⁺-dependent glutamate/aspartate transporter GLAST is regulated in the short term, by a transporter-dependent decrease in uptake activity while in the long term, a receptor’s-dependent decrease in GLAST protein levels leads to a severe reduction in glutamate uptake. The promoter region of the mouse glast gene harbors an Activator Protein-1 site (AP-1). To gain insight into the molecular mechanisms triggered by Glu-receptors activation involved in GLAST regulation, we took advantage of the neonatal mouse cerebellar prisms model. We characterized the glutamate uptake activity; the glutamate-dependent effect on GLAST protein levels and over the interaction of nuclear proteins with a mouse glast promoter AP-1 probe. A time and dose dependent decrease in transporter activity matching with a decrease in GLAST levels was recorded upon glutamate treatment. Moreover, a significant increase in glast AP-1 DNA binding was found. Pharmacological experiments established that both effects are mediated through α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, favoring the notion of the critical involvement of glutamate in the regulation of its binding partners: receptors and transporters.     

Rottlerin,an inhibitor of protein kinase Cdelta (PKCdelta), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCdelta-independent

Protein kinase C (PKC) regulates the activity and/or cell surface expression of several different neurotransmitter transporters, including subtypes of glutamate transporters. In the present study, the effects of pharmacological inhibitors of PKC were studied in primary astrocyte cultures that express the glutamate aspartate transporter (GLAST) subtype of glutamate transporter. We found that general inhibitors of PKC, bisindolylmaleimide I (Bis I), bisindolylmaleimide II (Bis II), staurosporine and an inhibitor of classical PKCs, Gö6976, had no effect on Na⁺‐dependent glutamate transport activity. However, rottlerin, a putative specific inhibitor of PKCδ, decreased transport activity with an IC₅₀ value (less than 10 µm ) that is comparable to that reported for inhibition of PKCδ. The effect of rottlerin was very rapid (maximal effect within 5 min) and was due to a decrease in the capacity (V_max) for transport. Rottlerin also caused a drastic loss of GLAST immunoreactivity within 5 min, suggesting that rottlerin accelerates GLAST degradation/proteolysis. Rottlerin had no effect on cell surface or total expression of the transferrin receptor, providing evidence that the effect on GLAST cannot be attributed to a non‐specific internalization/degradation of plasma membrane proteins. Down‐regulation of PKCδ with chronic phorbol ester treatment did not block rottlerin‐mediated inhibition of transport activity. These results suggest a novel mechanism for regulation of the GLAST subtype of glutamate transporter and indicate that there is a rottlerin target that is capable of controlling the levels of GLAST by controlling the rate of degradation or limited proteolysis. It appears that the target for rottlerin may not be PKCδ.     

CHARACTERIZATION OF PUTATIVE AMINO ACID TRANSMITTER RELEASE FROM SLICES OF RAT DENTATE GYRUS

Abstract— Superfused slices of the rat dentate gyrus were employed to study the release of GABA, glutamate and aspartate, which are considered strong neurotransmitter candidates in this region. The introduction of Ca²⁺ to a Ca²⁺-free superfusion medium containing a depolarizing agent augmented the efflux of all three amino acids. The response to application of Ca²⁺ nearly always occurred within 30 s, the shortest interval tested in these studies. The efflux rate reached a peak within 90 s and then declined to a level slightly greater than the prestimulation baseline. The failure to maintain the maximal rate with continued exposure to Ca²⁺ and depolarizing influences appeared not to result from a reduction in Ca²⁺ permeability caused by continuous depolarization. Ca²⁺ also stimulated the efflux of exogenously loaded radiolabeled GABA, glutamate and aspartate, but not proline. Exogenously loaded GABA was more readily released than endogenous GABA. Otherwise the effects of various treatments on their efflux rates were qualitatively similar. Mg²⁺ inhibited Ca²⁺-dependent efflux. Ba²⁺, but not Mg²⁺, stimulated amino acid efflux in the absence of Ca²⁺. Extracellular Na⁺ was not required to support Ca²⁺-dependent efflux. Addition of Ca²⁺ to a Ca²⁺-free medium in the absence of a depolarizing agent released GABA from the slices, but not glutamate or aspartate. K⁺-enriched medium and the depolarizing alkaloid, veratridine, stimulated both Ca²⁺-dependent and Ca²⁺-independent release processes. Na⁺-free medium enhanced the Ca²⁺-independent releasing action of elevated K⁺. Ca²⁺-independent release was inhibited by raising the Mg²⁺ concentration by 15 or 30 mM and appeared to be inhibited by Ca²⁺ as well. Amino acid output in the absence of Ca²⁺ is probably not directly related to transmission and is considered to result partially from a general increase in membrane permeability induced by depolarization in a Ca²⁺-free medium and partially from stimulation of carrier-mediated amino acid efflux. These results support previously suggested transmitter roles for GABA, glutamate and aspartate in the rat dentate gyrus.     

The Glutamate Aspartate Transporter (GLAST) Mediates l-Glutamate-Stimulated Ascorbate-Release Via Swelling-Activated Anion Channels in Cultured Neonatal Rodent Astrocytes

Vitamin C (ascorbate) plays important neuroprotective and neuromodulatory roles in the mammalian brain. Astrocytes are crucially involved in brain ascorbate homeostasis and may assist in regenerating extracellular ascorbate from its oxidised forms. Ascorbate accumulated by astrocytes can be released rapidly by a process that is stimulated by the excitatory amino acid, l-glutamate. This process is thought to be neuroprotective against excitotoxicity. Although of potential clinical interest, the mechanism of this stimulated ascorbate-release remains unknown. Here, we report that primary cultures of mouse and rat astrocytes release ascorbate following initial uptake of dehydroascorbate and accumulation of intracellular ascorbate. Ascorbate-release was not due to cellular lysis, as assessed by cellular release of the cytosolic enzyme lactate dehydrogenase, and was stimulated by l-glutamate and l-aspartate, but not the non-excitatory amino acid l-glutamine. This stimulation was due to glutamate-induced cellular swelling, as it was both attenuated by hypertonic and emulated by hypotonic media. Glutamate-stimulated ascorbate-release was also sensitive to inhibitors of volume-sensitive anion channels, suggesting that the latter may provide the conduit for ascorbate efflux. Glutamate-stimulated ascorbate-release was not recapitulated by selective agonists of either ionotropic or group I metabotropic glutamate receptors, but was completely blocked by either of two compounds, TFB-TBOA and UCPH-101, which non-selectively and selectively inhibit the glial Na⁺-dependent excitatory amino acid transporter, GLAST, respectively. These results suggest that an impairment of astrocytic ascorbate-release may exacerbate neuronal dysfunction in neurodegenerative disorders and acute brain injury in which excitotoxicity and/or GLAST deregulation have been implicated.     

Cardiac Glycosides Ouabain and Digoxin Interfere with the Regulation of Glutamate Transporter GLAST in Astrocytes Cultured from Neonatal Rat Brain

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na⁺, K⁺)-dependent ATPase (Na⁺/K⁺-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na⁺/K⁺-ATPase. We now report that Na⁺/K⁺-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na⁺/K⁺-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.     

Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters,Glutamine Synthetase Expression and Activity in Diabetic Retina

This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague–Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as ³H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells’ glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.     

Impairment of Neuronal Glutamate Uptake and Modulation of the Glutamate Transporter GLT-1 Induced by Retinal Ischemia

Excitotoxicity has been implicated in the retinal neuronal loss in several ocular pathologies including glaucoma. Dysfunction of Excitatory Amino Acid Transporters is often a key component of the cascade leading to excitotoxic cell death. In the retina, glutamate transport is mainly operated by the glial glutamate transporter GLAST and the neuronal transporter GLT-1. In this study we evaluated the expression of GLAST and GLT-1 in a rat model of acute glaucoma based on the transient increase of intraocular pressure (IOP) and characterized by high glutamate levels during the reperfusion that follows the ischemic event associated with raised IOP. No changes were reported in GLAST expression while, at neuronal level, a reduction of glutamate uptake and of transporter reversal-mediated glutamate release was observed in isolated retinal synaptosomes. This was accompanied by modulation of GLT-1 expression leading to the reduction of the canonical 65 kDa form and upregulation of a GLT-1-related 38 kDa protein. These results support a role for neuronal transporters in glutamate accumulation observed in the retina following an ischemic event and suggest the presence of a GLT-1 neuronal new alternative splice variant, induced in response to the detrimental stimulus.     

Effects of metabolic inhibitors on the release of glutamate from the retina

—The superfused, isolated retina of the chicken was used to investigate the mechanisms responsible for the increase in retinal transparency and the release of glutamate associated with stimuli known to elicit spreading depression (SD). We sought to distinguish between (1) mechanisms involving glutamate-induced increase in Na⁺ permeability and consequent uptake of extracellular material into the intracellular compartment and (2) mechanisms involving interference with operation of the Na⁺ pump that would result in a similar uptake of extracellular materials. Tetrodotoxin (which inhibits inward movements of Na⁺) depressed the transparency increase caused by stimulation with glutamate but not that elicited by application of KCl. Ouabain (which inhibits the Na⁺ pump) caused a marked increase in tissue transparency. The application of inhibitors of the aerobic metabolism, such as DNP or cyanide, or deprivation of O₂ had no effect on the retinal transparency; results suggesting that the energy for the Na⁺ pump could be supplied by glycolysis. Indeed iodoacetate (which inhibits glycolysis) caused a marked change in transparency. Furthermore we found evidence for a compound in the superfusion fluid supplemented with iodoacetate that may be a reaction product of glutamate and iodoacetate. In some preparations superfusion with glucose-free solutions caused a slowly developing increase in transparency and release of glutamate; in others the increase in transparency was more sudden and there was a larger release of glutamate. Seemingly, interference with the tissue metabolism can cause an uptake of extracellular material either by arrest of the Na⁺ pump or by the release of glutamate, depending on the conditions of the experiment.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

l-Aspartate but Not the d Form Is Secreted Through Microvesicle-Mediated Exocytosis and Is Sequestered Through Na+-Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of l -glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca²⁺ channel blockers. Bay K 8644, an agonist for the L-type Ca²⁺ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ～30% of the total free aspartate. Radiolabeled l -aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺-dependent manner. The Na⁺-dependent uptake of l -aspartate was strongly inhibited by l -cysteine sulfinate, β-hydroxyaspartate, and l -serine-O-sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of l -aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l -aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na⁺-dependent transporter at the plasma membrane. The possible role of l -aspartate as an intercellular chemical transmitter in the pineal gland is discussed.     

K+-stimulated amino acid release from cultured cerebellar neurons: Comparison of static and dynamic stimulation paradigms

The release of several endogenous amino acids and adenosine from rat cerebellar neuronal cultures following elevated K⁺ exposure in the presence and absence of added Ca²⁺ was studied. The amino acids aspartate (ASP), glutamate (GLU) and GABA were released from the cultures in a dose- and Ca²⁺-dependent manner. Taurine (TAU) and the nucleoside adenosine (ADN) efflux rates were dose-dependent but Ca²⁺-independent, and basal levels increased in the absence of Ca²⁺. The K⁺ depolarization induced release of serine (SER), alanine (ALA) and proline (PRO), was not dose-dependent and in the absence of extracellular Ca²⁺ (with added Mg²⁺) higher basal release of SER and ALA, but not PRO, was noted. These findings demonstrate that in addition to known cerebellar neurotransmitters, other neuroactive and neutral amino acids are released from cultured cerebellar neurons in response to K⁺ depolarization. Their observed efflux suggests they may have as yet unidentified roles in neuronal function with different classes of efflux corresponding to: neurotransmitter-type release (ASP, GLU, GABA), and osmoregulatory, possibly neuromodulatory-type release (TAU), a Ca²⁺-insensitive, possibly neuromodulatory-type release (ADN), and a depolarization-sensitive release (SER, ALA, PRO) of which SER and ALA are partially Ca²⁺-sensitive.     

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium‐dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium‐dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so‐called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time‐dependent Na⁺‐dependent glutamate/aspartate transporter/EAAT1‐induced System N‐mediated glutamine release could be demonstrated. Furthermore, D‐aspartate, a specific glutamate transporter ligand, was capable of enhancing the co‐immunoprecipitation of Na⁺‐dependent glutamate/aspartate transporter and Na⁺‐dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron‐derived glutamate through their contribution to the neurotransmitter turnover.