bluuebunny/crossref_metadata_2025 huggingface dataset:
The N‐Methyl‐d‐Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C‐γ
Authors: Gurd James W. , Bissoon N.
Published: July 2010

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C‐γ (PLC‐γ). A glutathione S‐transferase (GST) fusion protein containing both the N‐ and the C‐proximal SH2 domains of PLC‐γ was bound to glutathione‐agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine‐phosphorylated PSD‐GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2‐agarose beads in a phosphorylation‐dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2‐agarose. No binding occurred to GST‐agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC‐γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine‐phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.
Normal Binding and Reactivity of Copper in Mutant Superoxide Dismutase Isolated from Amyotrophic Lateral Sclerosis Patients
Authors: Marklund Stefan L. , Andersen Peter M. , Forsgren Lars , Nilsson Peter , Ohlsson Per‐Ingvar , Wikander Göran , Öberg Agneta
Published: July 2010

Abstract: In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn‐superoxide dismutase. The mutant CuZn‐superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active‐site Cu ions. Asp90Ala mutant CuZn‐superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp90Ala mutant CuZn‐superoxide dismutase was 45 Da lower than that of the wild‐type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn‐superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild‐type enzyme in enzymic activity, in content and affinity for active‐site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn‐superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active‐site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.
Domoic Acid Neurotoxicity in Cultured Cerebellar Granule Neurons Is Mediated Predominantly by NMDA Receptors that Are Activated as a Consequence of Excitatory Amino Acid Release
Authors: Berman Frederick W. , Murray Thomas F.
Published: July 2010

Abstract: The participation of NMDA and non‐NMDA receptors in domoic acid‐induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 µMl‐glutamate or 10 µM domoate for 2 h in physiologic buffer at 22°C followed by a 22‐h incubation in 37°C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24‐h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist d(−)‐2‐amino‐5‐phosphonopentanoic acid and the non‐NMDA receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate‐induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65–77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate‐mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate‐stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas glutamate‐induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non‐NMDA receptors. Domoate‐induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptor‐mediated stimulation of EAA efflux.
Changes in the Activity and mRNA Levels of Phospholipase D During Ceramide‐Induced Apoptosis in Rat C6 Glial Cells
Authors: Yoshimura Shin‐ichi , Sakai Hideki , Ohguchi Kenji , Nakashima Shigeru , Banno Yoshiko , Nishimura Yasuaki , Sakai Noboru , Nozawa Yoshinori
Published: July 2010

Abstract: N‐Acetylsphingosine (C2‐ceramide), a membrane‐permeable analogue, induced apoptosis in C6 glial cells. Phase‐contrast micrographs showed that the round cells appeared 3 h after exposure to 25 µM C2‐ceramide and the number of floating cells increased time‐dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C2‐ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPγS‐dependent PLD activity was down‐regulated after ceramide treatment in a time‐dependent manner. In the in vitro PLD assay, membrane‐associated PLD activation in response to recombinant ADP‐ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down‐regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription‐polymerase chain reaction. Decreases in GTPγS‐dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.
Phosphorylation of Neurofilament Heavy‐Chain Side‐Arm Fragments by Cyclin‐Dependent Kinase‐5 and Glycogen Synthase Kinase‐3α in Transfected Cells
Authors: Bajaj Narinder P. S. , Miller Christopher C. J.
Published: July 2010

Abstract: The side‐arm domain of neurofilament heavy‐chain (NF‐H) is heavily phosphorylated in axons. Much of this phosphate is located within a multiphosphorylation repeat (MPR) domain situated toward the carboxy terminus of the molecule. The MPR domain contains the repeat motif KSP of which there are two broad categories, KSPXX and KSPXK. In mouse NF‐H, the KSPXK repeats are situated toward the latter part of the MPR domain. We have expressed in mammalian cells fragments of mouse NF‐H side‐arm containing all of the MPR domain, the latter part of the MPR domain containing the KSPXK repeats, and the complementary amino‐terminal part of the MPR domain, which contains the KSPXX repeats. By cotransfecting these fragments with the neurofilament kinases cyclin‐dependent kinase‐5 (cdk‐5)/p35 and glycogen synthase kinase‐3α (GSK‐3α), we show that cdk‐5 induces cellular phosphorylation of the KSPXK‐containing fragment of NF‐H. Using the transfected fragments, we also map the epitopes for several commonly utilised NF‐H monoclonal antibodies and describe the effects that phosphorylation by cdk‐5 and GSK‐3α have on their reactivities.
Characterization of Glutamate‐Induced Formation of N‐Acylphosphatidylethanolamine and N‐Acylethanolamine in Cultured Neocortical Neurons
Authors: Hansen Harald S. , Lauritzen Lotte , Strand Anne Mette , Vinggaard Anne Marie , Frandsen Aase , Schousboe Arne
Published: July 2010

Abstract: Glutamate‐induced formation of N‐acylethanolamine (NAE) and N‐acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [14C]ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate‐induced formation was seen in 2‐day‐old cultures, whereas glutamate induced a pronounced formation in 6‐day‐old cultures. The calcium ionophore A23187 (2 µM) stimulated, within 2 h, formation of NAPE in 2‐day‐old cultures (fourfold) as well as in 6‐day‐old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA‐selective receptor antagonists d‐(−)‐2‐amino‐5‐phosphonovalerate and N‐(1‐(2‐thienyl)‐cyclohexyl)piperidine and the lack of effect of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate‐receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). In 6‐day‐old cultures, exposure to NMDA (100 µM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40–50% neuronal death, as measured by a decrease in cellular formazan formation [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death. Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the use of different pharmacological agents. Exposure to 5 mM NaN3 for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25–30% of total labeling). [14C]Anandamide was also formed in [14C]arachidonic acid‐labeled neurons exposed to NaN3. No NAPE formation was detected in A23187‐stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate‐induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.
Biochemical Analysis of Myelin Proteins in a Novel Neurological Mutant: The Taiep Rat
Authors: Möller J. R. , Durr P. G. , Quarles R. H. , Duncan I. D.
Published: July 2010

Abstract: Hemispheres, spinal cords, and sciatic nerves were taken from taiep, carrier, and control rats at ages ranging from 1 day to 16 months. Absolute myelin yields from CNS taiep tissues peaked at ∼2 months and then decreased until they reached a low but stable level. Myelin yield from the affected hemispheres expressed as a percentage of age‐matched controls decreased continuously from 2 weeks until it reached a stable level of ∼10–15%. The same was true for the spinal cords, but here the myelin yield reached a plateau at a slightly higher percentage of 20–25%. In comparison with control rats, isolated CNS myelin fractions from the affected rats had a greater content of high molecular weight proteins. Western blot analyses of CNS homogenates revealed that myelin basic protein (MBP), proteolipid protein, and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase were all present but decreased to levels generally consistent with the deficiencies of myelin. However myelin‐associated glycoprotein (MAG) levels always were reduced much more than those of the other three myelin proteins, and at younger ages the apparent molecular weight for MAG was increased in the mutants. Western blot analyses of sciatic nerve homogenates showed that the levels of MBP, MAG, and P0 were not significantly different in control and mutant animals. These results suggested an early hypomyelination of the CNS, with peak levels of myelin at 2 months, followed by a prolonged period of myelin loss, until a very low but stable myelin level was reached. The consistently greater loss of MAG, in comparison with other CNS myelin proteins, is different from most other hypomyelinating mutants in which MAG is relatively preserved in comparison with the proteins of compact myelin. This might be due to microtubular abnormalities in the taiep mutant interfering with transport of myelin proteins and having the greatest effect on MAG because of its most distal location in the periaxonal oligodendroglial membranes.
Functional Mapping of Dorsal and Median Raphe 5‐Hydroxytryptamine Pathways in Forebrain of the Rat Using Microdialysis
Authors: McQuade R. , Sharp T.
Published: July 2010

Abstract: Recent neurochemical studies of the properties of 5‐hydroxytryptamine (5‐HT) pathways arising from the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) have measured extracellular 5‐HT in brain regions with reported preferential DRN or MRN 5‐HT inputs. Here, we have tested whether electrical stimulation of the DRN and MRN releases 5‐HT in rat forebrain regions in a pattern that fits the reported distribution of DRN/MRN pathways. The effect on extracellular 5‐HT of electrical stimulation (5 Hz, 300 µA, 20 min) of the DRN, and then MRN, was determined in six regions of the anaesthetised rat. Stimulation of the DRN evoked a short‐lasting but clear‐cut release of 5‐HT (+70–100%) in regions (frontal cortex, dorsal striatum, globus pallidus, and ventral hippocampus) reported to receive a 5‐HT projection from the DRN. Regions receiving an MRN innervation (dorsal hippocampus, medial septum, and ventral hippocampus) released 5‐HT (+70–100%) in response to MRN stimulation. Regions reported to receive a preferential DRN innervation (frontal cortex, dorsal striatum, and globus pallidus) did not respond to MRN stimulation. Of two regions (dorsal hippocampus and medial septum) reported to receive a preferential MRN innervation, one did not respond to DRN stimulation (dorsal hippocampus) although the other (medial septum) did. In summary, electrical stimulation of the DRN and MRN released 5‐HT in a regionally specific pattern. With the exception of one region (medial septum), this pattern of release bears a strong relationship to the distribution of 5‐HT projections from the DRN and MRN reported by anatomical studies. The combination of raphe stimulation with microdialysis may be a useful way to study the in vivo neurochemistry of DRN/MRN 5‐HT pathways.
Effects of Milnacipran and Pindolol on Extracellular Noradrenaline and Serotonin Levels in Guinea Pig Hypothalamus
Authors: Moret Chantal , Briley Mike
Published: July 2010

Abstract: Milnacipran, a dual noradrenaline (NA) and serotonin (5‐hydroxytryptamine, 5‐HT) uptake inhibitor, increased extracellular levels of NA and 5‐HT in hypothalamus of freely moving guinea pigs as measured by microdialysis. The basal levels of both monoamines, which were tetrodotoxin sensitive, were increased in a dose‐dependent manner and to a similar extent after the intraperitoneal administration of milnacipran (10 and 40 mg/kg i.p.). Levels of the NA metabolite 4‐hydroxy‐3‐methoxyphenylglycol (MHPG) were decreased by milnacipran at 10 and 40 mg/kg i.p., whereas those of the 5‐HT metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) showed no effect. Subcutaneous injection of 5‐HT1A and β‐adrenergic receptor antagonist (−)‐pindolol alone, at 10 mg/kg, had no effect on the extracellular levels of NA or 5‐HT. The concomitant administration of (−)‐pindolol (10 mg/kg s.c.) with milnacipran (10 mg/kg i.p.) increased severalfold the effect of milnacipran on the extracellular levels of NA and 5‐HT. These results indicate that milnacipran, by blocking the uptake of NA and 5‐HT, increases virtually equipotently the extracellular levels of NA and 5‐HT, confirming previous in vitro studies. In addition, the antagonism of 5‐HT1A autoreceptors by (−)‐pindolol potentiates the action of milnacipran on both NA and 5‐HT systems, without modifying the ratio of these activities.
Use of Dipeptides for the Synthesis of Glutathione by Astroglia‐Rich Primary Cultures
Authors: Dringen Ralf , Kranich Oliver , Löschmann Peter‐Andreas , Hamprecht Bernd
Published: July 2010

Abstract: The intracellular content of glutathione in astroglia‐rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cells to use dipeptides for glutathione synthesis. For restoration of the glutathione level, after a 24‐h starvation period in the absence of glucose and amino acids, glucose, glutamate, cysteine, and glycine have to be present in the incubation buffer. The dipeptides CysGly and γGluCys were able to substitute for cysteine plus glycine and glutamate plus cysteine, respectively. Half‐maximal contents of glutathione were found at 20 µM CysGly and 3 mMγGluCys. In addition, the oxidized forms of the dipeptides CysGly and GlyCys could replace cysteine plus glycine for glutathione restoration, and the glycine‐containing dipeptides GlyGly, GlyLeu, GlyGlu, GlyGln, and γGluGly could partially substitute for the glycine necessary for the replenishment of glutathione. The glutathione resynthesis in the presence of CysGly plus glutamate was totally inhibited in the presence of buthionine sulfoximine, an inhibitor of γ‐glutamylcysteine synthetase. In contrast, glutathione restoration from γGluCys at a concentration of 10 mM in the presence of glycine was not influenced by the inhibitor. The use of CysGly or γGluCys was not affected by the presence of the dipeptidase inhibitors cilastatin or bestatin. In addition, carnosine and several other dipeptides applied in a 50‐fold excess only slightly prevented the use of CysGly, hinting at the existence in astroglial cells of a transport system specific for CysGly. The results demonstrate that astroglial cells can use dipeptides for intracellular glutathione synthesis and that the dipeptides most likely are taken up as intact molecules into astroglial cells before intracellular hydrolysis occurs.
Differential Vulnerability of the CA1 and CA3 Subfields of the Hippocampus to Superoxide and Hydroxyl Radicals In Vitro
Authors: Wilde G. J. C. , Pringle A. K. , Wright P. , Iannotti F.
Published: July 2010

Abstract: The relative roles of the superoxide and hydroxyl radicals in oxidative stress‐induced neuronal damage were investigated using organotypic hippocampal slice cultures. Cultures exposed to 100 µM duroquinone, a superoxide‐generating compound, for 3 h developed CA1‐selective lesions over a period of 24 h. The damage accounted for ∼64% of the CA1 subfield, whereas CA3 showed just 6% damage, a pattern of damage comparable to that observed following hypoxia/ischaemia. Duroquinone‐induced damage was attenuated by a spin‐trap agent. In contrast, hydroxyl radical‐mediated damage, generated by exposure to 30 µM ferrous sulphate for 1 h, resulted in a CA3‐dominant lesion. The damage developed over 24 h, similar to that observed with duroquinone, but with ∼45% damage in CA3 compared with only 7% in CA1. These data demonstrate a selective vulnerability of the CA1 pyramidal neurones to superoxide‐induced damage and suggest that of the free radicals generated following hypoxia/ischaemia, superoxide, rather than hydroxyl radical, is instrumental in producing neuronal damage.
Phospholipase A2 and Its Role in Brain Tissue
Authors: Farooqui Akhlaq A. , Yang Hsiu‐Chiung , Rosenberger Thad A. , Horrocks Lloyd A.
Published: July 2010

Abstract: Phospholipase A2 (PLA2) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn‐2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2‐catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet‐activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA2+‐dependent and Ca2+‐independent, based on their catalytic dependence on Ca2+. Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.
Evidence for Nicotinic Receptors Potentially Modulating Nociceptive Transmission at the Level of the Primary Sensory Neuron: Studies with F11 Cells
Authors: Puttfarcken Pamela S. , Manelli Arlene M. , Arneric Stephen P. , Donnelly‐Roberts Diana L.
Published: July 2010

Abstract: F11 cells are a dorsal root ganglion (DRG) cell line used to model the function of authentic type C, peptidergic, nociceptive neurons. The cellular events underlying the antinociceptive effects of (±)‐epibatidine, a nicotinic acetylcholine receptor (nAChR) ligand that is 200‐fold more potent than morphine, is unknown. The present study investigated the ability of cholinergic channel activators (ChCAs) to effect nAChR‐gated ion flux and modulate the release of substance P (SP), a neuropeptide identified to play a critical role in nociception. The prototypical agonists (−)‐nicotine and (−)‐cytisine, the ganglionic stimulant 1,1‐dimethyl‐4‐phenylpiperazinium, the novel ChCA ABT‐418 [(S)‐3‐methyl‐5‐(‐1‐methyl‐2‐pyrrolidinyl)isoxazole], and (±)‐epibatidine evoked a concentration‐dependent stimulation of rubidium (86Rb+) efflux with EC50 values of 14.2 ± 1.6, 63.4 ± 24, 3.8 ± 2.0, 29.8 ± 2.6, and 0.019 ± 0.001 µM as well as maximal intrinsic activities of 100, 97, 69, 75, and 102%, respectively. The noncompetitive nAChR antagonist mecamylamine potently antagonized (−)‐nicotine‐evoked ion flux, whereas the competitive antagonist dihydro‐β‐erythroidine was a weak antagonist, giving support to an α3β4 nAChR subtype. In addition, concentrations of (±)‐epibatidine, similar to those necessary to induce maximal 86Rb+ efflux, evoked spontaneous release of SP from these cells, which was blocked by mecamylamine. Furthermore, prolonged exposure to (±)‐epibatidine desensitized the functional response of the nAChR in this cell line (IC50 = 12 ± 9 nM). These findings in F11 cells provide a model to investigate the role nAChRs play in modulating DRG cell function, and may lead to insights into the role these receptors have in modulating nociceptive transmission.
Cholinergic Control of Nerve Growth Factor in Adult Rats: Evidence from Cortical Cholinergic Deafferentation and Chronic Drug Treatment
Authors: Roßner Steffen , Wörtwein Gitta , Gu Zezong , Yu Juan , Schliebs Reinhard , Bigl Volker , Perez‐Polo J. Regino
Published: July 2010

Abstract: It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG‐saporin and chronic pharmacological treatment of sham‐treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham‐treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham‐treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion‐induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.
Rubidium Uptake and Accumulation in Peripheral Myelinated Internodal Axons and Schwann Cells
Authors: Lehning Ellen J. , Gaughan Christopher L. , Eichberg Joseph , LoPachin Richard M.
Published: July 2010

Abstract: To study mechanisms of K+ transport in peripheral nerve, uptake of rubidium (Rb+), a K+ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero‐K+ Ringer's solutions containing Rb+ (1–20 mM) and x‐ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb+ (Rb+o) and exchanging it for internal K+. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1–10 mM Rb+o concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb+ concentrations, which suggests involvement of a nonenzymatic process. At 20 mM Rb+o, a differential stimulatory response was observed; i.e., axoplasmic Rb+ uptake velocities increased more than fivefold relative to the 10 mM rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb+o uptake rate into axons and glia was completely inhibited by ouabain (2–4 mM) exposure or incubation at 4°C. These results suggest that Rb+ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na+,K+‐ATPase activity and implicate the presence of axonal‐ and glial‐specific Na+ pump isozymes.
Transient Association of the Phosphotyrosine Phosphatase SHP‐2 with TrkA Is Induced by Nerve Growth Factor
Authors: Goldsmith Bruce A. , Koizumi Shinichi
Published: July 2010

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP‐2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP‐2 with TrkA was accompanied by the tyrosine phosphorylation of SHP‐2 and an association of SHP‐2 with multiple tyrosine‐phosphorylated proteins. In addition, the PTPase activity in SHP‐2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP‐2 with TrkA is induced by NGF and that this association leads to SHP‐2 activation and tyrosine phosphorylation. We conclude that SHP‐2 plays a significant role in early biochemical events in TrkA‐mediated signal transduction.
Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I
Authors: Xie Han‐qing , Johnson Gail V. W.
Published: July 2010

Abstract: Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule‐associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated PC12 cells with the cell‐permeable ceramide derivative N‐acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N‐hexanoylsphingosine (C6). In addition, C2 treatment increased the levels of a calpain‐derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, α‐actin and α‐tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium‐activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of PC12 cells with C2 enhanced calcium‐stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane‐permeable calpain‐selective fluorescence probe N‐succinyl‐l‐leucyl‐l‐leucyl‐l‐valyl‐l‐tyrosine‐7‐amido‐4‐methylcoumarin. This activity increase was not due to a direct effect of C2 on calpains, because C2 did not alter the activities of purified calpain I or II. In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain‐specific inhibitor N‐carbobenzoxy‐l‐leucyl‐l‐leucyl‐l‐tyrosine diazomethyl ketone blocked the C2‐induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramide‐mediated changes in normal and pathological neuronal processes.
Mianserin‐Induced Down‐Regulation of Human 5‐Hydroxytryptamine2A and 5‐Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH‐SY5Y
Authors: Newton R. A. , Elliott J. M.
Published: July 2010

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5‐hydroxytryptamine2A (5‐HT2A) and 5‐HT2C receptors stably expressed in the human neuroblastoma cell line SH‐SY5Y. Incubation of cells expressing the 5‐HT2A receptor with mianserin (100 nM) for 24 h caused a significant decrease (48%) in the binding capacity of [3H]ketanserin. This receptor down‐regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5‐HT but not by carbachol. Exposure of cells expressing the 5‐HT2C receptor to mianserin (100 nM) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [3H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5‐HT at the 5‐HT2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down‐regulation is evident at human 5‐HT2A and 5‐HT2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.
Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool
Authors: Pan Chien‐Yuan , Kao Lung‐Sen
Published: July 2010

Abstract: The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high‐K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin‐sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.
Activating Mutations of the Serotonin 5‐HT2C Receptor
Authors: Herrick‐Davis Katharine , Egan Christina , Teitler Milt
Published: July 2010

Abstract: Site‐directed mutagenesis was performed to create a mutant serotonin 5‐HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS‐7 cells, the binding affinity of 5‐HT for [3H]‐mesulergine‐labeled 5‐HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5‐HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five‐fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5‐HT and [3H]‐mesulergine labeled the same number of S312K mutant receptors and 5′‐guanylylimidodiphosphate had no effect on [3H]5‐HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high‐affinity state of the 5‐HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.
Glutamate Uptake Impairment and Neuronal Damage in Young and Aged Rats In Vivo
Authors: Massieu Lourdes , Tapia Ricardo
Published: July 2010

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3‐month‐old and 22–24‐month‐old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.
Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species
Authors: Berman Sarah B. , Hastings Teresa G.
Published: July 2010

Abstract: Dopamine can form reactive oxygen species and other reactive metabolites that can modify proteins and other cellular constituents. In this study, we tested the effect of dopamine oxidation products, other generators of reactive oxygen species, and a sulfhydryl modifier on the function of glutamate transporter proteins. We also compared any effects with those on the dopamine transporter, a protein whose function we had previously shown to be inhibited by dopamine oxidation. Preincubation with the generators of reactive oxygen species, ascorbate (0.85 mM) or xanthine (500 µM) plus xanthine oxidase (25 mU/ml), inhibited the uptake of [3H]glutamate (10 µM) into rat striatal synaptosomes (−54 and −74%, respectively). The sulfhydryl‐modifying agent N‐ethylmaleimide (50–500 µM) also led to a dose‐dependent inhibition of [3H]glutamate uptake. Preincubation with dopamine (100 µM) under oxidizing conditions inhibited [3H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amounts of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase (2–50 U/ml) further inhibited [3H]glutamate uptake, an effect prevented by the addition of glutathione. The effects of free radical generators and dopamine oxidation on [3H]glutamate uptake were similar to the effects on [3H]dopamine uptake (250 nM). Our findings suggest that reactive oxygen species and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine‐induced toxicity, and Parkinson's disease.
Expression of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Receptors and PACAP in Human Fetal Retina
Authors: Olianas M. C. , Ingianni A. , Sogos V. , Onali P.
Published: July 2010

Abstract: Specific receptors for pituitary adenylate cyclase‐activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12–18‐week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 125I‐PACAP 27 with IC50 values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine‐isoleucine, which are homologous to PACAP, displayed lower affinities for the 125I‐PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription‐polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation.
Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry
Authors: Abbruscato Thomas J. , Thomas Sarah A. , Hruby Victor J. , Davis Thomas P.
Published: July 2010

Abstract: Biphalin [(Tyr‐d‐Ala‐Gly‐Phe‐NH)2] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood‐brain and blood‐cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [125I‐Tyr1]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [125I‐Tyr1]‐biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ‐ and μ‐opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [125I‐Tyr1]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [125I‐Tyr1]biphalin was decreased with naloxone, d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ‐ and μ‐opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [125I‐Tyr1]biphalin, which could be described by Michaelis‐Menten kinetics with a Km of 2.6 ± 4.8 µM, Vmax of 14.6 ± 2.89 pmol−1·min−1·g−1, and Kd of 0.568 ± 0.157 µl·min−1·g−1. Brain entry of [125I‐Tyr1]biphalin was sensitive to 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid and l‐phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.
Opposite Regulation of Adenylyl Cyclase by Protein Kinase C in Astrocyte and Microglia Cultures
Authors: Patrizio Mario , Slepko Natalia , Levi Giulio
Published: July 2010

Abstract: We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10‐min treatment with the phorbol esters phorbol 12‐myristate 13‐acetate (PMA) and 4β‐phorbol 12,13‐didecanoate (4β‐PDD) (but not with 4α‐PDD) or with diacylglycerol, which activate PKC, dose‐dependently enhanced cyclic AMP accumulation induced by the β‐adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin‐C and by down‐regulation of PKC and was not related to activation of membrane receptors or Gs proteins or to inhibition of Gi proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA‐treated astrocytes. In microglia, a 10‐min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4β‐PDD (but not 4α‐PDD) inhibited the cyclic AMP response in a time‐ and dose‐dependent manner. Such inhibition was mimicked by staurosporine and calphostin‐C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.
In Vivo Evidence for the Link Between l‐ and d‐Serine Metabolism in Rat Cerebral Cortex
Authors: Takahashi Katsunobu , Hayashi Fumihiko , Nishikawa Toru
Published: July 2010

Abstract: To obtain an insight into the metabolic pathways of endogenous d‐serine in mammalian brains, we have investigated in the infant rat the effects of systemic administration of l‐serine, d‐serine, and related amino acids, including glycine and threonine, on the amino acid contents in the cerebral cortex. Intraperitoneal injection of l‐serine induced a rapid and transient elevation of the levels of l‐serine itself in the neocortex, with its peak at 3 h post injection, and a delayed and prolonged increase in d‐serine contents from 1.5 h to at least 24 h thereafter. Similarly, a significant augmentation in cerebral d‐serine contents was observed 6 h after intraperitoneal administration of glycine, which also elevated the cortical l‐serine levels. In contrast, l‐threonine injection affected the concentrations of neither d‐ nor l‐serine in the cortex of the pups. d‐Serine given systemically, in turn, increased the neocortical contents of l‐serine as well as d‐serine itself, but failed to alter those of glycine and l‐threonine. These in vivo data suggest the possible link between metabolic pathways of d‐ and l‐serine in the cerebral cortex of the rat.
Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors
Authors: Moody Eric J. , Knauer Christopher , Granja Ricardo , Strakhova Marina , Skolnick Phil
Published: July 2010

Abstract: Most general anesthetics produce two distinct actions at GABAA receptors. Thus, these drugs augment GABA‐gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a β subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABAA receptors, site‐directed mutagenesis of the β3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABAA receptor composed of α1β3γ2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate‐induced chloride currents and its ability to stimulate [3H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA‐gated chloride currents and GABA‐enhanced [3H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on β3(Asn290) indicates that the dual actions of this intravenous anesthetic at GABAA receptors are mediated via distinct loci.