bluuebunny/crossref_metadata_2025 huggingface dataset:
Depolarization of Cerebellar Granule Cells Increases Phosphorylation of Rabphilin‐3A
Authors: Fykse Else Marie
Published: July 2010

Abstract: Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin‐3A is present in cerebellar granule cells as a phosphoprotein, by using 32P‐labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 ± 13%) (mean ± SEM) on depolarization of the cells with K+ (56 mM) in the presence of external Ca2+ (1 mM). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13‐dibutyrate) also enhanced the phosphorylation of rabphilin‐3A (217 ± 21%). Inhibitors of Ca2+/calmodulin‐stimulated protein kinases or protein kinase C reduced the depolarization‐enhanced phosphorylation of rabphilin‐3A, indicating that rabphilin‐3A is one of the targets for Ca2+‐activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13‐dibutyrate and K+ depolarization produced an increased level of phosphorylation of rabphilin‐3A compared with either stimulus alone (287 ± 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin‐3A is phosphorylated in vivo and undergoes synaptic activity‐dependent phosphorylation during Ca2+‐activated K+ depolarization.
Functional Differentiation of Multiple Dopamine D1‐Like Receptors by NNC 01‐0012
Authors: Sugamori Kim S. , Hamadanizadeh Soheila A. , Scheideler Mark A. , Hohlweg Rolf , Vernier Philippe , Niznik Hyman B.
Published: July 2010

Abstract: Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype‐specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1‐like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS‐7 cells. Of all the compounds tested, only NNC 01‐0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH‐23390 binding with an estimated affinity (∼0.6 nM) 20‐fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01‐0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 µM)‐stimulated adenylyl cyclase activity. In contrast, NNC 01‐0012 (10 µM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by ∼33% relative to the full agonist dopamine (10 µM), an effect that was blocked by the selective D1 receptor antagonist NNC 22‐0010. These data clearly suggest that the benzazepine NNC 01‐0012, despite lacking the N‐methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01‐0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.
The Family of Na+/Cl− Neurotransmitter Transporters
Authors: Nelson Nathan
Published: July 2010

Abstract: The termination of neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific high‐affinity neurotransmitter transporters. Most of these transporters are encoded by a family of genes (Na+/Cl− transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed five distinct subfamilies: γ‐aminobutyric acid transporters, monoamine transporters, amino acid transporters, “orphan” transporters, and the recently discovered bacterial transporters. The bacterial transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the three‐dimensional structure of these membrane proteins. Some of the neurotransmitter transporters have been implicated as important sites for drug action. Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of neurotransmitter transport, the structure of the transporters, and the interaction of each transporter in complex neurological activities.
Molecular Cloning of a Rat Brain cDNA, with Homology to a Tyrosine Kinase Substrate, that Induces Galactosylceramide Expression in COS‐7 Cells
Authors: Ogura Kiyoshi , Kohno Kenji , Tai Tadashi
Published: July 2010

Abstract: A rat brain cDNA clone has been isolated, using a eukaryotic cell transient expression system in conjunction with an anti‐galactosylceramide (anti‐GalCer) monoclonal antibody that induces GalCer expression in COS‐7 cells. The protein was designated as GalCer expression factor‐1 (GEF‐1). A good correlation between GalCer expression and the level of the enzyme activity of UDP‐galactose:ceramide galactosyltransferase (CGT) was demonstrated. The cDNA insert encoded a polypeptide of 771 amino acids with a calculated molecular mass of 85,787 Da. The cDNA hybridized to a single mRNA of 3.1 kb in all rat organs examined, including brain, testis, and skeletal muscle. The cDNA product was determined to be a tyrosine‐phosphorylated protein with a molecular mass of 110 kDa in transfected COS‐7 cells and adult rat brain. COS‐7 cells transfected with the cDNA clone showed dramatic morphological changes: The transfected cells appeared to be fibroblast‐like cells, whereas the parent COS‐7 cells were typical epithelial‐like cells. The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor‐regulated tyrosine kinase substrate but no homology to CGT. Taking these results together, it is suggested that GEF‐1 may play an important role in regulating GalCer expression in the brain.
Differential Release of Dopamine by Nitric Oxide in Subregions of Rat Caudate Putamen Slices
Authors: Iravani Mahmoud M. , Millar Julian , Kruk Zygmunt L.
Published: July 2010

Abstract: Fast cyclic voltammetry was used to measure NO and dopamine (DA) simultaneously in rat caudate putamen (CPu) slices. Analysis of electrochemical signals obtained from mixtures of DA and NO showed that subtraction of either the DA or the NO component revealed the contribution of the other component. Application of such data manipulation to signals obtained in CPu slices indicated that DA and NO components contributed to electrochemical signals. Following electrical stimulation (>1 s), site‐dependent NO‐like signals were observed. Pressure ejection of NMDA yielded a biphasic electrochemical signal. The first phase (lasting 10–20 s) had electrochemical characteristics of DA and was observed only during the first application of NMDA. The second phase developed more slowly, was of longer duration (1–3 min), and had electrochemical characteristics of a mixture of DA and NO. Generation of the NO‐like signal by NMDA was antagonised by l‐N‐monomethylarginine. Pressure ejection of NO into CPu slices caused dose‐ and site‐dependent DA release. More DA was released in the dorsolateral than in the dorsomedial CPu. Pressure ejection of DA did not generate NO. It is concluded that electrically stimulated DA release is not mediated by prior release of NO.
Calcium Dependence of Synaptic Vesicle Recycling Before and After Synaptogenesis
Authors: Coco Silvia , Verderio Claudia , De Camilli Pietro , Matteoli Michela
Published: July 2010

Abstract: Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium‐ and depolarization‐dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low‐affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.
NMDA and Non‐NMDA Ionotropic Glutamate Receptors Modulate Striatal Acetylcholine Release via Pre‐ and Postsynaptic Mechanisms
Authors: Morari M. , Sbrenna S. , Marti M. , Caliari F. , Bianchi C. , Beani L.
Published: July 2010

Abstract: The effects of NMDA and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1–300 µM) facilitated acetylcholine release from slices in a dose‐dependent manner. NMDA (100–300 µM) and AMPA (30–300 µM), however, subsequently inhibited acetylcholine release. NMDA (100 µM)‐induced facilitation was antagonized by 3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP) and dizocilpine (both 1–10 µM), whereas the 10 µM AMPA effect was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 1–30 µM). NMDA (100 µM)‐induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor l‐nitroarginine (1–100 µM). Tetrodotoxin (0.5 µM) prevented the facilitatory effect of 3 µM NMDA and AMPA, but left unchanged that of 30 µM NMDA and 100 µM AMPA. Acetylcholine release from synaptosomes was stimulated by KCI (7.5–100 mM) in a dose‐dependent manner. NMDA and AMPA maximally potentiated the 20 mM KCl effect at 1 µM and 0.01 µM, but were ineffective at 100 µM and 10 µM, respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 µM NMDA and 0.01 µM AMPA were antagonized by CPP (0.0001–1 µM) or dizocilpine (0.0001–10 µM) and by CNQX (0.001–1 µM), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre‐ and post‐synaptic NMDA and non‐NMDA ionotropic receptors.
Examination of the Role of ADP‐Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells
Authors: Glenn Daphne E. , Thomas Geraint M. H. , O'Sullivan Antony J. , Burgoyne Robert D.
Published: July 2010

Abstract: The possible role of ADP‐ribosylation factor (ARF)‐activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin‐permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF‐stimulated PLD activity in these cell types. Exocytosis from cytosol‐depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol‐depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA‐sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.
Role of Endogenous Glutathione in the Oxidation of Dopamine
Authors: Rabinovic Ariel D. , Hastings Teresa G.
Published: July 2010

Abstract: Intrastriatal injection of dopamine causes the selective degeneration of tyrosine hydroxylase‐containing terminals and an increase in content of cysteinylcatechols, an index of dopamine oxidation. Both of these effects can be attenuated by coadministration of antioxidants such as glutathione. Therefore, we investigated the effects of decreased endogenous glutathione on the neurotoxic potential of dopamine. We observed that pretreatment with either l‐buthionine sulfoximine, a specific inhibitor of glutathione synthesis, or diethyl maleate, which forms adducts with glutathione, caused significant decreases in endogenous glutathione levels at the time of dopamine injection. Pretreatment with l‐buthionine sulfoximine potentiated the formation of protein cysteinyl‐dopamine after intrastriatal injection of 1.0 µmol of dopamine. We also observed that intrastriatal injection of 1.0 µmol of dopamine decreased striatal glutathione content in all pretreatment conditions. However, injection of a low dose (0.05 µmol of dopamine) caused a decrease in striatal glutathione levels only in the l‐buthionine sulfoximine‐pretreated rats. Diethyl maleate pretreatment was not effective in potentiating either cysteinyl‐catechol formation or glutathione loss after dopamine injection. We conclude that dopamine contributes to cellular oxidative stress and that this can be exacerbated, or at least unmasked, if glutathione synthesis is compromised.
Extracellular Glutamate Levels Are Chronically Elevated in the Brains of LP‐BM5‐Infected Mice: A Mechanism of Retrovirus‐Induced Encephalopathy
Authors: Espey Michael Graham , Kustova Yelena , Sei Yoshitatsu , Basile Anthony S.
Published: July 2010

Abstract: Mice infected with the LP‐BM5 leukemia retrovirus mixture develop a progressive immunodeficiency with associated behavioral, histological, and neurochemical alterations consistent with glutamatergic hyperactivation. To gain insight into the contribution of excitatory amino acids to the neurodegeneration observed in these mice, their concentrations were measured in the CSF and striatal microdialysates. Glutamate concentrations were significantly elevated in CSF but not plasma as early as 4 weeks postinoculation. Steady‐state glutamate levels in striatal microdialysates were increased threefold and could be reduced 40% by application of l‐α‐aminoadipate, an inhibitor of microglial glutamate transport. Stimulation of infected mice with KCl/l‐trans‐2,4‐pyrrolidine dicarboxylate further increased glutamate levels 170–270% above those evoked in control mice. Tetrodotoxin suppressed the depolarization‐evoked increase in glutamate by 88% in control mice, but it had only negligible effects in 40% of infected mice. Analysis of glutamate transport and catabolism suggests that abnormal astrocytic function does not contribute to the increase in basal extracellular glutamate levels. These findings are the first direct evidence that infection with an immunodeficiency‐inducing retrovirus leads to a chronic elevation of extracellular free glutamate levels in the brain, which contributes to the neurodegenerative and cognitive deficits observed in these mice.
Monitoring of Brain Spiking Activity and Autoantibodies to N‐Terminus Domain of GluR1 Subunit of AMPA Receptors in Blood Serum of Rats with Cobalt‐Induced Epilepsy
Authors: Dambinova Svetlana A. , Granstrem Oleg K. , Tourov Alexandre , Salluzzo Roberto , Castello Filippa , Izykenova Galina A.
Published: July 2010

Abstract: We have monitored EEG spontaneous spiking activity and analyzed serum from rats with cobalt‐induced epilepsy for the presence of autoreactive antibodies to α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) glutamate receptor subunits. The presence and the level of autoantibodies were assessed using immunoblot and ELISA with synthetic peptide specific to the N‐terminus domain of the GluR1 subunit of the AMPA receptor. Rats with cobalt‐induced epilepsy exhibited strong GluR1 immunoreactivity at the end of the first week after surgery compared with vehicle‐treated rats. We showed that GluR1 autoantibodies in blood serum of rats with cobalt‐induced epilepsy preceded the spiking activity maximum in the EEG. Levels of autoantibodies to GluR1 detected in blood of these rats remained elevated when EEG spiking activity was significantly reduced and seizures disappeared. The EEG monitoring of spiking activity showed a correlation with accumulation of GluR1 autoantibodies in blood serum of rats with cobalt‐induced epilepsy.
Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells
Authors: Fukuda Kazuhiko , Shoda Takehiro , Morikawa Hitoshi , Kato Shigehisa , Mima Hiroyuki , Mori Kenjiro
Published: July 2010

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time‐ and dose‐dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti‐Ca2+‐dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time‐ and dose‐dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin‐induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX‐sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen‐activated protein kinases.
Pertussis Toxin‐Insensitive Signaling of the ORL1 Receptor: Coupling to Gz and G16 Proteins
Authors: Chan Joy S. C. , Yung Lisa Y. , Lee Jonathan W. M. , Wu Ya‐Lan , Pei Gang , Wong Yung H.
Published: July 2010

Abstract: Nociceptin/OFQ is the endogenous ligand for the G protein‐coupled opioid receptor‐like (ORL1) receptor. To elucidate the cellular functions of the ORL1 receptor, we examined its ability to interact with Gz and G16, two pertussis toxin (PTX)‐insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL1 and dopamine D1 receptors, nociceptin/OFQ dose‐dependently inhibited dopamine‐stimulated cyclic AMP (cAMP) accumulation in a PTX‐sensitive manner. However, PTX failed to block the nociceptin/OFQ‐induced inhibition of dopamine‐stimulated cAMP accumulation in HEK 293 cells co‐expressing the α‐subunit of Gz. This result indicates functional interaction between the ORL1 receptor and Gz. A similar result was obtained with retinoic acid‐differentiated SH‐SY5Y cells, which endogenously express both the ORL1 receptor and Gz. When the ORL1 receptor was transiently co‐expressed in COS‐7 cells with the α‐subunit of G16, nociceptin/OFQ dose‐dependently stimulated the formation of inositol phosphates. Nociceptin‐induced stimulation of phospholipase C was absolutely dependent on the co‐expression of α16 and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX‐insensitive G proteins, the ORL1 receptor behaves very much like the opioid receptors.
Brain β‐Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine‐347 as a Heparin‐Sensitive Protein Kinase Phosphorylation Site
Authors: Sihag Ram K.
Published: July 2010

Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β‐spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO4/mol of β‐spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO4/mol of spectrin heterodimer, respectively. The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P]orthophosphate showed phosphorylation of only β‐spectrin in vivo. Two‐dimensional phosphopeptide map analyses showed that most of the in vivo sites on β‐spectrin were phosphorylated by either a heparin‐sensitive endogenous cytoskeleton‐associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β‐spectrin showed that [32P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin‐sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α‐chymotryptic 32P‐labeled peptide phosphorylated by the heparin‐sensitive cytoskeleton‐associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β‐spectrinG (βSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of β‐spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.
Differential Membrane Targeting and Pharmacological Characterization of Chimeras of Rat Serotonin 5‐HT1A and 5‐HT1B Receptors Expressed in Epithelial LLC‐PK1 Cells
Authors: Darmon Michèle , Langlois Xavier , Suffisseau Laurent , Fattaccini Claude‐Michelle , Hamon Michel
Published: July 2010

Abstract: The serotonin 5‐HT1A and 5‐HT1B receptors are two structurally related but pharmacologically distinguishable 5‐HT receptor types. In brain, the 5‐HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5‐HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC‐PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5‐HT1A and 5‐HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5‐HT1B receptor was responsible for its Golgi‐like localization in transfected LLC‐PK1 cells. In contrast, the third intracellular domain of the 5‐HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C‐terminal domain of the 5‐HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5‐HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).
Osmotic Swelling‐Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes
Authors: Morales‐Mulia Sandra , Vaca Luis , Hernandez‐Cruz Arturo , Pasantes‐Morales Herminia
Published: July 2010

Abstract: Hyposmotic swelling‐induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1–1 mM). Verapamil did not affect either the swelling‐evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+‐free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5‐di‐tert‐butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA‐acetoxymethyl ester (20 µM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.
Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina
Authors: Zeevalk G. D. , Davis N. , Hyndman A. G. , Nicklas W. J.
Published: July 2010

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time‐dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [3H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing 3H‐labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [3H]acetate plus [U‐14C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [3H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5‐ and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [3H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, 14C‐labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of 14C‐labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans‐pyrrolidine‐2,4‐dicarboxylate (t‐PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na+‐dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [3H]glutamate, constancy of the specific activity of extracellular [3H]glutamate, decrease in the specific activity of extracellular [14C]glutamate, and attenuation of release by prior loading with t‐PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.
Role of Acetyl‐l‐Carnitine in Rat Brain Lipogenesis: Implications for Polyunsaturated Fatty Acid Biosynthesis
Authors: Ricciolini Rita , Scalibastri Maurizio , Kelleher Joanne K. , Carminati Paolo , Calvani Menotti , Arduini Arduino
Published: July 2010

Abstract: This study was undertaken to explore the metabolic fate of acetyl‐l‐carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1‐14C]acetyl‐l‐carnitine into the lateral brain ventricle of conscious rats. After injection of [1‐14C]acetyl‐l‐carnitine, the majority of radioactivity was recovered as 14CO2 expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl‐l‐carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl‐CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl‐l‐carnitine following intracerebroventricular injection. In summary, these data implicate acetyl‐l‐carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.
Expression and Localization of Brain Ankyrin Isoforms and Related Proteins During Early Developmental Stages of Rat Nervous System
Authors: Kunimoto Manabu , Adachi Tatsumi , Ishido Masami
Published: July 2010

Abstract: Expression and localization of two isoforms of brain ankyrin, 440‐ and 220‐kDa ankyrinB, were studied in the developing nervous system of the rat fetus. The 440‐kDa ankyrinB appeared on as early as embryonic day 13, and its level increased progressively toward the day of birth, which was similar to the expression pattern of growth‐associated protein (GAP)‐43, a well‐established axonal protein. On the other hand, 220‐kDa ankyrinB was expressed at a low level but constitutively throughout the latter prenatal period and was a major isoform even before embryonic day 14. Whereas the localization of 440‐kDa ankyrinB was essentially confined to the axons, judging from the similarity with that of GAP‐43, 220‐kDa ankyrinB showed a rather general distribution in neural tissue. The localization of L1, known as an ankyrinB‐binding protein, was similar to that of 440‐kDa ankyrinB in the brain tissue, whereas it was similar to that of 220‐kDa ankyrinB in cultured neurons, suggesting that the interaction of L1 with brain ankyrins in neurons is affected by their environment.
Expression and Activity of Cyclin‐Dependent Kinase 5/p35 in Adult Rat Peripheral Nervous System
Authors: Terada Masahiko , Yasuda Hitoshi , Kogawa Syuro , Maeda Kengo , Haneda Masakazu , Hidaka Hideki , Kashiwagi Atsunori , Kikkawa Ryuichi
Published: July 2010

Abstract: In spite of the clarification in the temporal and spatial expression pattern of a cyclin‐dependent kinase (Cdk) 5 and its neuron‐specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co‐immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.
Functional Expression of the Serotonin Transporter in Immortalized Rat Brain Microvessel Endothelial Cells
Authors: Brust Peter , Friedrich Anne , Krizbai Istvan A. , Bergmann Ralf , Roux Françoise , Ganapathy Vadivel , Johannsen Bernd
Published: July 2010

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5‐hydroxytryptamine (5‐HT)]. This neurotransmitter is expected to be involved in the regulation of the blood‐brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor‐mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5‐HT molecules. To realize this, a similar mechanism of 5‐HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT‐PCR that 5‐HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5‐HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5‐HT by an active saturable process with a Km value of 397 ± 64 nmol/L and a transport capacity of 51.7 ±

3.5 pmol · g‐1· min‐1. The 5‐HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5‐HT uptake was sensitive to specific 5‐HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5‐HT, which supports the concept of direct serotoninergic regulation of the BBB function.
Molecular mechanism underlying neuroprotection by rasagiline
Authors: Naoi M. , Maruyama W. , Youdium M. B. H.
Published: July 2010

1‐Amino‐N‐propargylaminoindan (rasagiline) is now under phase 3 clinical trials for the treatment of Parkinson's disease (PD). In vivo and in vitro experiments using animal and cellular PD models proved that rasagiline protects neurons from cytotoxicity of various insults. Rasagiline prevents apoptosis induced by a dopaminergic neurotoxin, N‐methyl(R)salsolinol in dopaminergic SH‐SY5Y cells. It suppressed the decline in mitochondrial membrane potential, and following activation of apoptotic cascade. The stabilization of mitochondria membrane potential was confirmed also in isolated mitochondria. To examine the effect of rasagiline on antiapoptotic bcl‐2 gene expression, the levels of mRNA and protein of Bcl‐2 protein family were examined in SH‐SY5Y cells treated with rasagiline. The levels of antiapoptotic bcl‐2 and bcl‐xL increased significantly, but those of pro‐apoptotic bax and bad were not. The study on the structure‐activity relationship among propargylamines revealed the chemical structure required for the stabilization of PT and the induction of bcl‐2 genes, respectively. In addition, rasagiline increased the level of glial cell line‐derived nutrition factor (GDNF). The results suggest that rasagiline protects neurons by increased expression of cyto‐protective genes, and may rescue declining neurons in PD and other neurodegenerative disorders.
Regulation of human UDP‐galactose: ceramide β‐galactosyltransferase (CGT) gene expression
Authors: Yu R. K. , Tencomnao T.
Published: July 2010

Myelination is a highly coordinated event during nervous system development, which involves the synthesis of many myelin specific components. The most abundant glycolipid in myelin is galactocerebroside (GC), whose synthesis is catalysed by CGT. To elucidate the transcriptional control of the hCGT gene, we sequenced 2.3 kb of the 5′‐flanking region of this gene. Transient transfection of the human oligodendroglioma (HOG; GC+) and neuroblastoma (LAN‐5; GC–) cell lines using a series of luciferase reporter plasmids containing deletion fragments of the hCGT 5′‐flanking region showed that this promoter functions in a cell‐specific manner. Three positive regulatory regions were identified: a region at −292/−256 containing an Sp1 site, a region at −747/−688 containing sites for TGGCA‐BP, CRE, and ER half‐site, a distal region at −1325/−1279 containing a nitrogen regulatory site. A negative regulatory domain (−1594/−1326) was also identified. Using mutation analyses and cotransfection experiments in HOG cells, we confirmed that the Sp1 motif (−267/−259) and the CRE motif (−697/−690) were critical for hCGT expression. Gel shift assays showed that these motifs are bound by nuclear extracts from both cell lines and mouse brains. Using antibodies to Sp1, Sp3, and CREB, these proteins were shown to be components of the complexes. However, the CRE complexes were the only notable differences between HOG and LAN‐5 cells, which may be responsible for the differential transcription of this gene. Thus, the hCGT promoter contains both positive and negative regulatory regions for the cell‐specific expression, and these regions function cooperatively.

Acknowledgements:  Supported by NIH NS11853‐27.
Workshop 4: Compartmentation of Neuronal Metabolism. Compartmentation of energy metabolism and amino acid synthesis in synaptosomes
Authors: McKenna M. C. , Hopkins I. B. , O'Brien J. A. , Carey A.
Published: July 2010

There is strong evidence that the brain can use multiple substrates for energy including glucose, lactate, ketone bodies, glutamate and glutamine. Competition studies show that certain substrates are preferentially used for energy by synaptic terminals even when other substrates are available. It has recently been shown that synaptosomes can use both glutamine and glutamate for energy and synthesis of amino acids; however, these substrates yield very different patterns of 13C‐labelling of end products. These findings provide evidence of differential compartmentalisation of the metabolism of glutamate taken up from the extracellular milieu as compared to the glutamate produced from glutamine within synaptic terminals. This compartmentalisation is related to the specific role(s) of glutamate vs. glutamine in synaptic terminals as well as the metabolism of these amino acids in either partial or complete TCA cycles for energy. The presence of glucose, which provides a source of acetyl‐CoA, can greatly modulate both the metabolic fate of other substrates and the pool size of amino acids such as glutamate and GABA. The differential localization of the enzymes glutamate dehydrogenase and aspartate aminotransferase contribute to this compartmentalisation as does the necessity that synaptic terminals balance their energy needs with the requirement to synthesize neurotransmitters.
Compartmentalized neurotransmitter and amino acid synthesis in vivo studied by high field MRS
Authors: Gruetter R. , Henry P. G. , Lei H. , Choi I.‐Y.
Published: July 2010

Glutamatergic action occurs by the glial uptake of extracellular glutamate and conversion into glutamine, which is exported to neurons. Recently, a 1 : 1 stoichiometry between total oxidative brain glucose consumption, CMRglc(ox), and glutamatergic action was reported. This relationship required, that all of the glucose phosphorylated in glia was exported to neurons in the form of lactate. We recently reaffirmed in the awake human brain that the majority of ATP produced in glia is oxidative (Gruetter et al. 2001). In that study we also observed that the exchange rate between TCA cycle intermediates and amino acids, Vx, is on the order of the flux through pyruvate dehydrogenase, VPDH, which implied that this exchange rate is influenced by the malate–aspartate shuttle (MAS). Recently we have extended this line of investigation to the intact rat brain. TCA cycle flux and glutamate/glutamine metabolism was assessed in vivo at two different anesthetic levels (pentobarbital and α‐chloralose anesthesia) using 13C NMR in conjunction with 13C glucose infusions. Several metabolic rates were extracted from the rate of label incorporation using a mathematical model (Gruetter et al. 2001). The apparent rate of glutamatergic neurotransmission, VNT, was low, even though glucose metabolism was still substantial, suggesting that the stoichiometry between glucose metabolism and glutamatergic neurotransmission is far from 1 : 1. The rate of pyruvate carboxylation was ∼25% of glutamine synthase flux. Vx was found to be equal to VPDH, indicating that Vx is coupled to CMRglc(ox), consistent with MAS being the major mechanism that facilitates label exchange across the inner mitochondrial membrane.

Reference Gruetter et al. (2001) Am. J. Physiol.281, 100E.
The role of NAAG and NAALADase in axon‐to‐glia signalling
Authors: Grossfeld R. M. , Urazaev A. K. , Lieberman E. M.
Published: July 2010

Recent investigations from our laboratories have identified N‐acetylaspartylglutamate (NAAG) as the probable primary axon‐to‐glia signalling agent and source of signalling glutamate (GLU) at nonsynaptic regions of crayfish nerve fibers. NAAG is released from giant axons and mimics the action of the natural signalling agent in hyperpolarizing the glia. GLU formed extracellularly during inactivation of NAAG by NAALADase has a similar, but smaller, effect on the glia. A consequence of these actions of NAAG and GLU appears to be an activation of a signal transduction cascade leading to modulation of water and K+ homeostasis of the neuronal microenvironment. GLU or a related substance also appears to be a neuron‐glia signalling agent at nonsynaptic and synaptic regions of vertebrate nervous system. Therefore, conserved mechanisms of neuron‐glia signalling, mediated at least in part by NAAG and/or GLU, may regulate signal transmission and processing at axons and synapses under physiological and pathological conditions in invertebrates and vertebrates.

Acknowledgements:  Supported by NIH grant NS34799 and Guilford Pharmaceuticals (Baltimore, MD).
NAALADase (GCP II) inhibition as a novel therapeutic target for neuropathic pain, diabetic neuropathy, and ALS
Authors: Slusher B. S. , Rojas C. , Sima A. , Majer P. , Tsukamoto T. , Jackson P. J. , Dal Canto M. , Lu X. M. , Vomov J. J. , Burak E. , Rhodes C. , Limsakun T. , Potter B. , Wozniak K.
Published: July 2010

NAALADase (N‐acetylated‐α‐linked‐acidic dipeptidase; also termed glutamate carboxypeptidase II) is an enzyme that catalyzes hydrolysis of the abundant brain peptide N‐acetyl‐aspartyl‐glutamate (NAAG) to N acetyl‐aspartate (NAA) and glutamate. We have synthesized many potent and selective NAALADase inhibitors from distinct chemical classes. In rodents, selective inhibitors have been shown to increase NAAG and decrease glutamate extracellularly and provide therapeutic benefits in animal models of neurological diseases where excess glutamate neurotransmission has been implicated. Specifically, NAALADase inhibitors exhibit neuroprotection in rats following transient middle cerebral artery occlusion, attenuate neuropathic pain following chronic constrictive injury, prevent nerve conduction velocity deficits and sural nerve degeneration following chronic diabetes in STZ and BB/W rats, and delay clinical symptoms and mortality in the mouse SOD transgenic model of ALS. These biological effects are likely mediated by increases in extracellular NAAG and decreases in extracellular glutamate as a consequence of NAALADase inhibition. NAALADase inhibition may be useful in neurological disorders in which excessive excitatory amino acid transmission is pathogenic. Also, NAALADase inhibition may represent a novel glutamate regulating strategy devoid of the side‐effects that have hampered development of postsynaptic glutamate receptor antagonists. A lead NAALADase inhibitor termed GPI 5693 is currently completing Phase I clinical testing.
Mechanisms of the system a neuronal glutamine transporter SAT1
Authors: Mackenzie B. , Hediger M. A. , Varoqui H.
Published: July 2010

Glutamine, the preferred precursor for neurotransmitter glutamate, is likely to be the principal substrate for the neuronal System A transporter SAT1 in vivo. By measuring currents associated with SAT1 expression in Xenopus oocytes, we found that SAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine, alanine and the System A‐specific analogue 2‐(methylamino) isobutyrate, each with K0.5 of 0.3–0.5 mm. Amino acid transport is driven by the Na+ electrochemical gradient. Kinetic data indicates that Na+/cotransport comprises the ordered binding first of Na+ (a voltage‐dependent step), then alanine, then simultaneous translocation. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SAT1 mediates a cation leak with selectivity Na+, Li+, H+, K+. The temperature‐dependence of the leak current (Ea = 17 ± 3 kcal/mol) is consistent with carrier‐mediated Na+ uniport activity (cf 13 ± 2 kcal/mol for Na+/alanine cotransport) but the leak does not saturate at physiological [Na+], suggesting channel activity. Despite a Na+ Hill coefficient of 1, we obtained Na+/amino acid coupling coefficients greater than 1 from simultaneous measurement of charge and [3H]alanine or [3H]glutamine uptake. Interpretation of these data is model‐dependent and consistent with either (1) an all‐carrier model in which Na+/amino acid cotransport is thermodynamically coupled 2 : 1, cotransport is preferred over Na+ uniport, and in which there is little cooperativity between Na+ binding events, or (2) 1 : 1 coupling in parallel with an always‐on Na+ channel activity. In either scenario, the presence of SAT1 at the plasma membrane and resultant Na+ fluxes will place a significant energy burden on the cell.
Redefining chemical anatomy of glutamatergic neurotransmission
Authors: Schafer M. K. , Weihe E.
Published: July 2010

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region‐ and pathway‐specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.

Our results suggest pathway‐specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.