Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Dissection of mitochondrial Ca2+ uptake and release fluxes in situ after depolarization-evoked [Ca2+](i) elevations in sympathetic neurons

We studied how mitochondrial Ca2+ transport influences [Ca2+](i) dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+(i); the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+](i) recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+](i), while the net mitochondrial flux (J(mito)) is biphasic, indicative of Ca+) accumulation during the initial phase of recovery when [Ca2+](i) is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. J(mito) was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na(+)/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of J(mito) increases steeply with [Ca2+](i), as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na(+) concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na(+)/Ca2+ exchanger. Above approximately 400 nM [Ca2+](i), net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+](i) is approximately 200-300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca(2+)](i) to levels below approximately 500 nM.     

Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase.

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.     

Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca²⁺ responses to ATP, including a transition of Ca²⁺ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10⁻⁴ and 10⁻² M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP₃) mediated Ca²⁺ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca²⁺ responses due to the biphasic nature of IP₃Rs and the interaction of SERCA pumps with IP₃Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP₃Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca²⁺ response signatures, which are important in mediating bone mechanoadaptation.     

The effect of anions on the human P2X7 receptor

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

2',3'-Dialdehyde ATP analog labels the Ca(2+)-ATPase of sarcoplasmic reticulum via the catalytic adenosine-nucleotide-binding site.

The 2',3'-dialdehyde ATP analog (oATP) was synthesized and its ability to activate the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum via the adenosine-nucleotide-binding site was investigated. After reduction by sodium borohydride, oATP binds covalently to the catalytic adenosine-nucleotide-binding site of the enzyme, resulting in 85% loss of acetyl-phosphate-driven Ca2+ uptake and ATP-hydrolysing ability. In the absence of a reducing agent, oATP serves as a substrate for the Ca(2+)-ATPase, as indicated by Pi formation (hydrolysis) and Ca(2+)-uptake ability. oATP binding to the intact light sarcoplasmic reticulum is observed in the absence and presence of the competitive adenosine nucleotide inhibitor, fluorescein isothiocyanate with apparent affinity constants of 1.2 mM and 2.2 mM, respectively. Autoradiography of tryptic fragments from partially purified Ca(2+)-ATPase labeled with [alpha-32P]oATP or [gamma-32P]oATP locates the covalent binding site to the A1 fragment, even in the fluorescein-isothiocyanate-labeled pump protein. With high probability, a lysine residue in the tryptic A1 fragment is labeled by the ribose-modified ATP analog close to the phosphorylation site at Asp351.     

Methyl-beta-cyclodextrin but not retinoic acid reduces EAAT3-mediated glutamate uptake and increases GTRAP3-18 expression

The Na+-dependent glutamate transporter EAAT3 facilitates glutamate uptake into neurons as well as many other cell types. GTRAP3-18 (JWA, Arl6ip5) is a novel protein that interacts with EAAT3 and negatively modulates EAAT3-mediated glutamate uptake. Previous studies suggest that retinoic acid (RA) decreases Na+-dependent glutamate uptake and increases GTRAP3-18 protein expression. However, the RA used in those studies was complexed with methyl-beta-cyclodextrin (MebetaCD). In the present study we found that MebetaCD, but not RA, significantly reduced Na+-dependent EAAT3-mediated [3H]glutamate uptake in human embryonic kidney 293 (HEK293) cells. MebetaCD also significantly increased GTRAP3-18 protein expression in HEK293 cells as well as in rat hypothalamic neuron cultures. Intracerebroventricular administration of MebetaCD to the mouse brain resulted in a significant increase in GTRAP3-18 immunoreactivity in the hippocampus and cerebral cortex. In conclusion, we have shown that MebetaCD reduces EAAT3-mediated glutamate uptake and induces the expression of GTRAP3-18 protein.     

Ethanol Alters Glutamate but Not Adenosine Uptake in Rat Astrocytes: Evidence for Protein Kinase C Involvement

Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [³H]glutamate uptake and inhibits [³H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [³H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [³H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [³H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [³H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [³H]glutamate uptake but not [³H]adenosine uptake by affecting PKC modulation of transporter activity.     

Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina

Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.     

Affinity labelling of rat liver acetyl-CoA carboxylase by a 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (kmod) were 0.11 and 0.26 min-1 respectively. oATP completely inhibited the [14C]ADP in equilibrium ATP exchange and did not effect the [14C]acetyl-CoA in equilibrium malonyl-CoA exchange. Incorporation of approximately 1 equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to 3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA carboxylase by oATP. It was shown that the reagent apparently interacted selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.