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.     

Glutamate Induces Glutathione Efflux Mediated by Glutamate/Aspartate Transporter in Retinal Cell Cultures

This study was undertaken in order to characterize the role of the glutamate/aspartate transporter (GLAST) in the glutathione (GSH) efflux induced by glutamate. Our results demonstrated that retinal cell cultures exhibit two mechanisms of GSH release, one Na⁺-independent and other Na⁺-dependent. Glutamate and aspartate induced GSH efflux only in presence of Na⁺. Treatment with PCD (L-trans-Pyrrolidine-2,4-dicarboxylate), a transportable glutamate uptake blocker, increased GSH release indicating that GSH can be carried by glutamate transporters in retinal cell cultures. Added to this, treatment with zinc ion cultures, a recognized inhibitor of GLAST blocked GSH efflux evoked by glutamate. Treatment with NMDA antagonist (MK-801) did not have any effect on the GSH release induced by glutamate. These results suggest that glutamate induces GLAST-mediated release of GSH from retinal cell cultures and this could represent an important mechanism of cellular protection against glutamate toxicity in the CNS.     

Muscimol Potentiation of Acidic Amino Acid Release from Cerebellar Synaptosomes Is Chloride Dependent

In previous studies we have shown that the depolarization-induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by γ-aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K⁺-evoked release of d-[³H]aspartate. Our studies show that: Ca²⁺, Na⁺, and Mg²⁺ are not required for muscimol to exert its effect; a depolarizing concentration of K⁺ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl^- concentration (50–70 mM) is also required. A possible model based on these findings is proposed.     

GLUTAMINE UPTAKE AND METABOLISM BY THE ISOLATED TOAD BRAIN: EVIDENCE PERTAINING TO ITS PROPOSED ROLE AS A TRANSMITTER PRECURSOR

Abstract— Hemisections of toad brains, when incubated in a physiological medium containing no glutamine. released considerable amounts of this amino acid into the medium. When glutamine was included in the medium at a concentration of 0.2 mm the net efflux from the tissue was reduced but not totally prevented. Although there was no net uptake of glutamine, the tissue did accumulate [U-¹⁴C]glu-tamine and some of this labelled glutamine was rapidly metabolized to glutamate, GABA and aspartate. The precursor-product relationship for the metabolism of glutamine to glutamate differed from the classic single compartment model in that the specific radioactivity of glutamate rose very quickly to approx one-tenth that of glutamine, but increased slowly thereafter. These data suggest that the [¹⁴C]glutamine was taken up into two metabolically distinct compartments and/or that some of the [¹⁴C]glutamine was converted to [¹⁴C]glutamate during the uptake process. The uptake of [¹⁴C]glutamine was diminished when the tissue was incubated in a non-oxygenated medium or when Na⁺ was omitted (substituted with sucrose) and K⁺ was concomitantly elevated. However, on a relative basis, the incorporation of radioactivity into glutamate and GABA was increased by these incubation conditions. The metabolism of glutamine to aspartate was greatly depressed when the tissue was not oxygenated. The glutamate formed from [U-¹⁴C]glutamine taken up by the tissue was converted to GABA at a faster rate than was glutamate derived from [U-¹⁴C]glucose. [U-¹⁴C]gly-cerol or exogenous [U-¹⁴C]glutamate. This suggests that glutamine was metabolized to GABA selectively; i.e. on a relative basis, glutamine served as a better source of carbon for the synthesis of GABA than did glucose, glycerol or exogenous glutamate. When the brain hemisections were incubated in the normal physiological medium with or without glutamine. there was very little efflux of glutamate, GABA or aspartate from the tissue. However when NaCl was omitted from the medium (substituted with sucrose) and K⁺ was elevated to 29 miu. a marked efflux of these three amino acids into the medium did occur, and over a period of 160min, the content of each amino acid in the tissue was depleted considerably. When glutamine (0.2 mm ) was included in the Na⁺ deficient-high K.⁺ medium, the average amount of glutamate, GABA and aspartate in the tissue plus the medium was greater than when glutamine was not included in the medium. Such data indicate that CNS tissues can utilize glutamine for a net synthesis of glutamate, GABA and aspartate. The results of this study provide further evidence in support of the concept that the functional (transmitter) pools of glutamate and GABA are maintained and regulated in part via biosynthesis from glutamine. One specific mechanism instrumental in regulating the content of glutamate in nerve terminals may be a process of glutamine uptake coupled to deamidation.     

Sodium ion transport in isolated intestinal epithelial cells. The effect of actively transported sugars on sodium ion efflux

A technique to measure Na⁺ efflux from isolated intestinal epithelial cells has permitted us to examine the mechanisms responsible for Na⁺ transport in absorptive cells without contamination by other cell types. We examined the effect of actively transported sugars on Na⁺ efflux from isolated rat jejunal epithelial cells to evaluate the mechanism by which actively transported non-electrolytes stimulate Na⁺ absorption. Glucose, galactose and 3-O-methylglucose, sugars known to be actively transported by the small intestine, stimulate total Na⁺ efflux from isolated epithelial cells. This stimulation results from an increase of active Na⁺ transport, since it is inhibited by ouabain. Glucose stimulation is significantly greater than that produced by galactose or 3-O-methylglucose, 2-Deoxyglucose, a sugar that is not actively transported, has no effect on total Na⁺ efflux from isolated cells. Phloridzin, which has no effect on Na⁺ efflux in a sugar-free medium, completely abolishes the effect of galactose. These findings (a) support the hypothesis that the increase in intestinal absorption of Na⁺ in the presence of actively transported non-electrolytes occurs by a transcellular route; and (b) are consistent with the ion-gradient model. The results are not compatible with the direct energy-coupling model.     

Modulation of Aspartate Release by Ascorbic Acid and Endobain E,an Endogenous Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na⁺, K⁺-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[³H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[³H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na⁺, K⁺-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[³H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na⁺, K⁺-ATPase, may well modulate neurotransmitter release at synapses.     

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.     

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.     

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.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

COUPLED TRANSPORT OF GLUTAMATE AND SODIUM IN A CEREBELLAR NERVE CELL LINE

The cerebellar nerve cell line ε¹ has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na⁺ and furthermore, ²²Na⁺ uptake is stimulated by glutamate, indicating that glutamate uptake and Na⁺ uptake are coupled. Two molecules of Na ⁺ are transported for each molecule of glutamate. The K_m for glutamate is found to be 5 × 10^?5M in both the glutamate uptake assay and the ²²Na⁺ uptake assay, providing additional evidence for glutamate-Na⁺ coupling. Pre-incubation with ouabain, which inhibits the Na⁺-K⁺ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na⁺ gradient. Tetrodotoxin, however, has no effect on glutamate-induced ²²Na⁺ uptake, showing that this Na⁺ flux does not occur via voltage-dependent Na⁺ channels. Studies on the specificity of the ε¹ glutamate transport system show that it is distinct from systems that transport alanine and glycine. l -Glutamate, d -aspartate, l -cysteate, and l -cysteine sulfinate are able to utilize the transport system efficiently. d -Glutamate, l -homocysteate, N-methyl-d , l -aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate ²²Na⁺ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, ε¹ does not have an excitatory glutamate receptor.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Cation flux in the ehrlich ascites tumor cell evidence for Na-for-Na and K-for-K exchange diffusion

In a previous study, evidence was presented for an external Na⁺-dependent, ouabain-insensitive component of Na⁺ efflux and an external K⁺-dependent component of K⁺ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na⁺ and K⁺ they represent Na⁺-for-Na⁺ and K-⁺for-K⁺ exchange mechanisms. Using ⁸⁶Rb to monitor K⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb⁺ influx and a component of Rb⁺ efflux, both representing approx. 30% of the total fux. Inhibition of Rb⁺ efflux is greatly reduced by removal of extracellular K⁺. Furosemide does not alter steady-state levels of intracellular K⁺ and it does not prevent cells depleted of K⁺ by incubation in the cold from regaining K⁺ upon warming. Using ²²Na to monitor Na⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na⁺ efflux which represents approx. 22% of total Na⁺ efflux. Furosemide does not alter steady-state levels of intracellular Na⁺ and does not prevent removal of intracellular Na⁺ upon warming from cells loaded with Na⁺ by preincubation in the cold. The ability of furosemide to affect unidirectional Na⁺ and K⁺ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na⁺-dependent amino acid transport system A.     

Voltage-Sensitive Ca2+ Channels Involved in Nicotinic Receptor-Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [³H]dopamine release from striatal synaptosomes, and this action is both Na⁺ and Ca²⁺ dependent and is blocked by Cd²⁺. This suggests that stimulation of presynaptic nicotinic receptors results in Na⁺ influx and local depolarisation that activates voltage-sensitive Ca²⁺ channels, which in turn provide the Ca²⁺ for exocytosis. Here we have investigated the subtypes of Ca²⁺ channels implicated in this mechanism. [³H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a ⁸⁶Rb⁺ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [³H]dopamine release by 60% but had no significant effect on ⁸⁶Rb⁺ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ～30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [³H]dopamine release by activation of N-type Ca²⁺ channels. In contrast, KCl-evoked [³H]dopamine release appears to involve both N-type and P-type channels.     

GABA efflux from synaptosomes: Effects of membrane potential,and external GABA and cations

Summary Presynaptic GABAergic nerve terminals accumulate -aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na⁺ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from³H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent evoked release), external GABA promotes GABA efflux when the medium contains Na⁺, butinhibits GABA efflux in the absence of Na⁺. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K⁺). These data, and published data on the internal Na⁺ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na⁺. The stimulatory effect of depolarization is consistent with efflux of Na⁺ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na⁺+GABA. The inhibitory effect of GABA in Na⁺-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na⁺+1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.     

Presynaptic k-Opioid and Muscarinic Receptors Inhibit the Calcium-Dependent Component of Evoked Glutamate Release from Striatal Synaptosomes

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.     

Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt_Ph, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt_Ph as an aspartate transporter driven by Na⁺ or a glutamate transporter driven by either Na⁺ or H⁺. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt_Ph transport. We confirm that Glt_Ph is a Na⁺-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na⁺ ions. In contrast to the EAATs, transport via Glt_Ph is independent of H⁺ and K⁺. We propose a kinetic model of transport in which at least two Na⁺ ions are coupled to the cotransport of each aspartate molecule by Glt_Ph, and where an ion- and substrate-free transporter reorients to complete the transport cycle.     

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.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na⁺ efflux goes through the Na⁺ pump. We found that (i) internal K⁺ had a strong inhibitory effect on Na⁺ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na⁺, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K⁺, but reduced its effectiveness compared with other monovalent cations, as an activator of Na⁺ efflux, and (iii) the relative effectiveness of different K⁺ congeners as external activator of the Na⁺ efflux, though affected by the ATP concentration, was not affected by the Na⁺/⁺ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na⁺ + K⁺)-ATPase can be reached by interaction with external K⁺ after phosphorylation and with internal K⁺ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.