bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of a Nerve Growth Factor‐Sensitive Kinase that Phosphorylates the DNA‐Binding Domain of the Orphan Nuclear Receptor NGFI‐B
Authors: Hirata Yoko , Whalin Michael , Ginty David D. , Xing Jun , Greenberg Michael E. , Milbrandt Jeffrey , Guroff Gordon
Published: July 2010

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI‐B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA‐binding domain inhibits its binding to the NGFI‐B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI‐B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA‐binding domain with this purified preparation inhibits its binding to the NGFI‐B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg2+ but will use Mn2+. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90rsk. Comparison with a partially purified preparation of cyclic AMP response element‐binding protein kinase, however, indicates that the two are either very similar or identical.
Increase in Chloride‐Dependent l‐Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment
Authors: Koyama Yutaka , Ishibashi Tadashi , Baba Akemichi
Published: July 2010

Abstract: The effect of energy failure on Cl−‐dependent l‐glutamate (l‐Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose‐deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl−‐dependent l‐[3H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l‐[3H]Glu uptake was inhibited by l‐Glu, dl‐2‐amino‐4‐phosphonobutyrate, l‐homocysteic acid, l‐cystine, 4,4′‐diisothiocyano‐2,2′‐disulfonic stilbene, and removal of Cl−. Uptakes of Na+‐dependent l‐[3H]‐Glu, [3H]GABA, and [3H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the Vmax value without affecting the Km value. The increased l‐[3H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose‐containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose‐containing solution. Treatment with veratrine, 2,4‐dinitrophenol, carbonyl cyanide m‐chlorophenylhydrazone, and NaCN in normoxic conditions increased l‐[3H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl−‐dependent l‐Glu transport activity in synaptic membranes increases in ischemia‐ or hypoxia‐induced brain energy failures.
Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord
Authors: Tsuji Koji , Nakamura Yoichi , Ogata Tadanori , Shibata Taihoh , Kataoka Kiyoshi
Published: July 2010

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µM) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca2+‐free medium and was inhibited by an antagonist of NMDA receptors or a voltage‐sensitive Ca2+ channel blocker. Preincubation with W7 also inhibited the glutamate‐evoked cyclic AMP increase. NMDA, aspartate, and high‐K+ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2S,1′S,2′S)‐2‐(carboxycyclopropyl)‐glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca2+ influx via both NMDA receptors and voltage‐sensitive Ca2+ channels followed by an activation of the Ca2+/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.
Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long‐Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD‐GP180
Authors: Au Kinh N. , Gurd James W.
Published: July 2010

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic‐like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine‐phosphorylated proteins and two prominent tyrosine‐phosphorylated glycoproteins of apparent Mr 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)‐associated glycoprotein PSD‐GP180. When hippocampal slices were incubated in oxygenated Krebs‐Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of Mr 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N2 instead of O2 and that did not contain glucose. Tyrosine‐phosphorylated GP110 and PSD‐GP180 could no longer be detected after 10 min of exposure of the slices to ischemic‐like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose‐containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long‐term loss of phosphotyrosine [Tyr(P)]‐PSD‐GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD‐GP180 after ischemia was Ca2+ dependent, the levels of Tyr(P)‐PSD‐GP180 slowly increasing to preischemic values if Ca2+ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca2+‐dependent, transient tyrosine phosphorylation of p42. The major PSD‐associated, tyrosine‐phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.
Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells
Authors: Rosales Fritz Víctor M. , Maccioni Hugo J. F.
Published: July 2010

Abstract: Ganglioside GT3 is the precursor of c‐series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub‐Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10‐day‐old chick embryo were cultured with [3H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C. perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC‐fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA‐treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a‐, b‐, and c‐ series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi‐synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.
Presynaptic Nicotinic Receptors Stimulate Increases in Intraterminal Calcium of Chick Sympathetic Neurons in Culture
Authors: Doležal Vladimír , Schobert Angelika , Hertting Georg
Published: July 2010

Abstract: Stimulation of chick sympathetic neurons in culture by the cholinergic agonists acetylcholine, nicotine, and 1,1‐dimethyl‐4‐phenylpiperazinium (all at 10–1,000 µmol/L) induced concentration‐dependent increases of free calcium levels measured by fura 2 fluorescence in neuronal processes. The response evoked by acetylcholine had both nicotinic and muscarinic components, whereas that induced by 1,1‐dimethyl‐4‐phenylpiperazinium was purely nicotinic. Tetrodotoxin (0.3 µmol/L) blocked completely the increase of intraterminal free calcium level evoked by electrical stimulation. On the other hand, stimulation with 1,1‐dimethyl‐4‐phenylpiperazinium still evoked 20–25% of the control response in the presence of tetrodotoxin. The concentration‐response relationship of 1,1‐dimethyl‐4‐phenylpiperazinium stimulation did not differ in the absence and in the presence of tetrodotoxin. The nicotinic antagonists d‐tubocurarine (10 µmol/L) and mecamylamine (10 µmol/L), but not α‐bungarotoxin (125 nmol/L), prevented the increase of intraterminal free calcium level evoked by 1,1‐dimethyl‐4‐phenylpiperazinium (100 µmol/L) in the presence of tetrodotoxin. These observations indicate the presence of nicotinic receptors on neuronal processes that increase the intraterminal concentration of free calcium and probably modulate transmitter release. Their pharmacological properties are similar to those of nicotinic receptors located on neuronal cell bodies.
A New Cys2/His2 Zinc Finger Gene, rKr2, Is Expressed in Differentiated Rat Oligodendrocytes and Encodes a Protein with a Functional Repressor Domain
Authors: Pott Uwe , Thiesen Hans‐Jürgen , Colello Raymond J. , Schwab Martin E.
Published: July 2010

Abstract: The function of the vertebrate nervous system is dependent on the proper myelination of its fiber tracts. Myelin of the CNS is produced by oligodendrocytes. To identify gene regulatory proteins expressed in this particular glial cell type, we isolated cDNAs coding for Cys2/His2 zinc finger proteins from a rat oligodendrocyte cDNA library. One clone, named rKr2 (rKr for rat Krüppel‐type protein), encodes a protein with 19 carboxy‐terminal zinc finger domains and an amino‐terminal Krüppel‐associated box domain. This amino‐terminal domain of the rKr2 protein behaved as a strong transcriptional repressor module when fused to the DNA binding domain of yeast GAL4 and tested on an appropriate reporter construct. High levels of rKr2 mRNA in adult rat tissues were found only in the CNS and testis; in the CNS, the message was predominantly expressed in differentiated oligodendrocytes. The modular structure of the rKr2 protein (carboxy‐terminal DNA binding domain, amino‐terminal repressor module) and its expression pattern suggest that it acts as a sequence‐specific transcriptional repressor in the myelin‐producing glial cells of the CNS.
Antisense Oligodeoxynucleotides to the Cloned δ Receptor DOR‐1: Uptake, Stability, and Regulation of Gene Expression
Authors: Standifer Kelly M. , Jenab Shirzad , Su Wendy , Chien Chih‐Cheng , Pan Ying‐Xian , Inturrisi Charles E. , Pasternak Gavril W.
Published: July 2010

Abstract: Phosphodiester antisense oligodeoxynucleotides (ODNs) directed against various domains of the cloned mouse δ receptor DOR‐1 reduce δ‐opioid receptor binding in vivo and in vitro. The present study examines the stability of an antisense ODN (275 nM) directed against the δ‐opioid receptor and its effect on DOR‐1 mRNA in cultured neuroblastoma cells and in vivo. When added to NG108‐15 cells, much of the antisense ODN is degraded. However, >1% is intact, associated with cells, and stable for at least 72 h. Northern blot analysis demonstrates that treatment of NG108‐15 cells with the antisense ODN reduces the levels of a species of DOR‐1 mRNA by ∼25%. Similarly, intrathecal administration of the antisense ODN results in the accumulation of intact ODN within the spinal cord, which is stable for at least 72 h, although the levels of accumulation in vivo are lower than in vitro after either 4 or 72 h. Antisense ODN treatment lowers DOR‐1 mRNA levels by ∼25%. The loss of mRNA both in vivo and in vitro corresponds quite well to the decreases in receptor binding previously observed by our laboratory and is consistent with reduction of δ‐opioid receptor protein in vitro as determined by western blot with a monoclonal antibody selective for the δ‐opioid receptor. In conclusion, these studies indicate that a small, but significant, proportion of ODN is taken up by cells and remains intact for up to 72 h. This appears to be sufficient to down‐regulate mRNA levels of δ‐opioid receptors and their expression.
Induction of Gene Expression of the Catecholamine‐Synthesizing Enzymes by Insulin‐Like Growth Factor‐I
Authors: Hwang Onyou , Choi Hyun Jin
Published: July 2010

Abstract: The effects of insulin‐like growth factor‐I (IGF‐I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DBH), and phenylethanolamine N‐methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum‐free medium. The mRNA level of TH was maximally elevated in the presence of IGF‐I by 3.1 ± 0.4‐fold after 48 h, DBH by 5.1 ± 0.3‐fold in 24 h, and PNMT by 2.8 ± 0.5‐fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF‐I‐exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time‐zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF‐I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF‐I‐induced DBH increase and the effects of IGF‐I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF‐I in these cells.
Expression of Somatostatin Receptor Subtypes in Cultured Astrocytes and Gliomas
Authors: Feindt Janka , Becker Inga , Blömer Ulrike , Hugo Heinz‐Hermann , Mehdorn Hubertus M. , Krisch Brigitte , Mentlein Rolf
Published: July 2010

Abstract: Expression of receptors for the neuropeptide somatostatin was investigated in vitro in rat and human astrocytes, glioma cell lines, and solid human glial tumors that were all immunopositive for the astrocytic marker glial fibrillary acidic protein. After affinity labelling with a peptide‐gold conjugate of known biological activity, somatostatin‐binding sites could be visualized at the light‐ and electron‐microscopic level on the surface of glial cells. Glioma cells were generally labeled more strongly than were normal astrocytes and preferentially bound the ligand at their processes and not at their somata as were normal cells. Somatostatin transmembrane receptor (SSTR) subtype expression was probed by reverse transcription‐polymerase chain reaction: In rat and human cortical astrocytes and in one glioma cell line (U 118), a pattern of three subtypes (SSTR‐1, SSTR‐2, and SSTR‐4) was detected, whereas, in all other glioma cell lines and in six solid glial tumors investigated, the SSTR‐2 subtype was relatively stronger, expressed either alone or in combination with SSTR‐1; sometimes SSTR‐3 or SSTR‐4 was demonstrated in clearly reduced amounts. In astrocytes and gliomas, somatostatin reduced the levels of cyclic AMP elicited by the adenylate cyclase activator forskolin indicating that at least one of the receptor subtypes is negatively linked to adenylate cyclase. In contrast to other cell types, somatostatin did not inhibit the basal or the fetal calf serum‐stimulated proliferation of astrocytes, glioma cell lines, or glial tumors in culture. Thus, strong SSTR‐2 subtype expression characterizes glial tumors, but somatostatin is ineffective in inhibiting their growth.
Photoaffinity Labeling with 2‐[2‐(4‐Azido‐3‐[125I]‐Iodophenyl)ethylamino]adenosine and Autoradiography with 2‐[2‐(4‐Amino‐3‐[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain
Authors: Luthin David R. , Lee Kevin S. , Okonkwo David , Zhang Peijun , Linden Joel
Published: July 2010

Abstract: The A2a adenosine receptor agonist 2‐[2‐(4‐amino‐3‐iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125I]APE, discriminates between high‐ and low‐affinity conformations of A2a adenosine receptors. In this study, [125I]APE was used for rapid (24‐h) autoradiography in rat brain sections. The pattern of [125I]APE binding is consistent with that expected of an A2a‐selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125I]APE binding to these brain regions is abolished by 1 µM 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680) but is little affected by 100 nM 8‐cyclopentyl‐1,3‐dipropylxanthine. Conversion of [125I]APE to the corresponding arylazide results in [125I]AzPE. The rank‐order potency of compounds to compete for [125I]AzPE binding in the dark is CGS‐21680 > d‐(R)‐N6‐phenylisopropyladenosine > N6‐cyclopentyladenosine, indicating that it also is an A2a‐selective ligand. Specific photoaffinity labeling by [125I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 ± 4% by 100 µM guanosine 5′‐O‐(3‐thiotriphosphate) and 91 ± 1.3% by 100 nM CGS‐21680. [125I]APE and [125I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP‐binding proteins by autoradiography and photoaffinity labeling.
Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction
Authors: Cho William , Taylor Larry P. , Mansour Alfred , Akil Huda
Published: July 2010

Abstract: Dopamine receptors belong to the seven transmembrane helix‐containing, G protein‐coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate‐114 in helix 3 and two helix 5 serines (194 and 197) of the D2 receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site‐directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D2 receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine‐198 with an alanine selectively disrupted [3H]N‐0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine‐411 or the helix 6 leucine‐387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine‐411 in helix 7 and leucine‐387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D2 receptor.
Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan
Authors: Meyer‐Puttlitz Birgit , Milev Peter , Junker Ernst , Zimmer Irene , Margolis Richard U. , Margolis Renée K.
Published: July 2010

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid‐binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor‐type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N‐terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N‐terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid‐binding domain of link protein. The presence in brain of these N‐terminal fragments is consistent with the developmentally regulated appearance of the C‐terminal half of neurocan, which we described previously. We have also used a slot‐blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan‐KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ∼1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo‐β‐galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.
Chemical and Immunological Characterization of Galactosyl‐β1‐3‐Globoside in Bovine, Human, and Rat Brain
Authors: Dasgupta Somsankar , Hogan Edward L. , Van Halbeek Herman
Published: July 2010

Abstract: Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short‐chain (containing ‐CHO 1–4) and previously uncharacterized long‐chain (−CHO > 4–5) Ngsls. We reported the structural characterization of brain GgOse4Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse4Cer and nLcOse5Cer on a TLC plate employing two different solvent systems. The carbohydrate molar composition indicated the presence of Gal/Glc/GalNAc in a ratio of 2.8:1.0:0.9. Five permethylated alditol acetate peaks were characterized as 2,3,4,6‐OMe4Gal, 2,4,6‐OMe3Gal, 2,3,6‐OMe3Gal, 2,3,6‐OMe3Glc, and 4,6‐OMe2GalNAcMe by gas chromatography‐mass spectrometry. The anomeric carbohydrate sequence has been determined by specific exoglycosidase digestion. Six‐hundred megahertz 1H NMR spectroscopy of the oligosaccharide released by ceramide glycanase hydrolysis confirmed the structure of the Ngsl as Galβ1‐3GalNAcβ1‐3Galα1‐4Galβ1‐4Glc\1‐1Cer or IV3GalGbOse4Cer. Using the immunooverlay technique with anti‐stage‐specific embryonic antigen 3 antibody, it was found in bovine, rat, and normal adult human brain and bovine myelin, but not in human or rat myelin.
Characterization of Na+‐Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons
Authors: Glinn Michele , Ni Binhui , Paul Steven M.
Published: July 2010

Abstract: Our laboratory has recently cloned and expressed a brain‐ and neuron‐specific Na+‐dependent inorganic phosphate (Pi) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na+‐dependent 32Pi cotransport in cultured fetal rat cortical neurons, where >90% of saturable Pi uptake is Na+‐dependent. Saturable, Na+‐dependent 32Pi uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na+‐dependent Pi transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent Km values for Pi and Na+ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (>60%) Na+‐dependent Pi uptake, with a threshold for maximal Pi import of 1–2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na+‐dependent Pi import rates. These data establish the presence of a Na+‐dependent Pi cotransport system in neurons of the CNS, demonstrate the Ca2+‐dependent nature of 32Pi uptake, and suggest that the neuronal Na+‐dependent Pi cotransporter may import Pi required for the production of high‐energy compounds vital to neuronal metabolism.
Peroxynitrite‐Mediated Inhibition of DOPA Synthesis in PC12 Cells
Authors: Ischiropoulos Harry , Duran Daniel , Horwitz Joel
Published: July 2010

Abstract: Experimental evidence has implicated oxidative stress in the development of Parkinson's disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion‐limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant‐induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinson's disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3‐morpholinosydnonimine (SIN‐1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN‐1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN‐1‐mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN‐1 persisted even after removal of SIN‐1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.
Phenotypic and Genotypic Analysis of Rats with Cerebellar GABAA Receptors Composed from Mutant and Wild‐Type α6 Subunits
Authors: Mäkelä Riikka , Wong Garry , Lüddens Hartmut , Korpi Esa R.
Published: July 2010

Abstract: The alcohol‐sensitive (ANT) rat line, developed for high behavioral sensitivity to ethanol, also exhibits enhanced sensitivity to benzodiazepines, such as diazepam. The rat line carries a point mutation in the cerebellum‐specific γ‐aminobutyric acid type A (GABAA) receptor subunit α6, making their diazepam‐insensitive (DIS) receptors sensitive to diazepam. We now report that phenotypes of individual ANT and alcohol‐insensitive rats, classified on diazepam sensitivity of cerebellar [3H]Ro 15‐4513 binding, correlated well with homozygous wild‐type, homozygous mutant, and heterozygous genotypes, although some heterozygotes were biased toward the parental phenotypes. GABA down‐modulated DIS [3H]Ro 15‐4513 binding in mutant homozygotes but tended to up‐modulate it in heterozygotes and wild‐type homozygotes. Slopes for GABA inhibition of cerebellar t‐butylbicyclophosphoro[35S]thionate binding were larger in mutant than in wild‐type homozygotes, with heterozygotes being intermediate. Diazepam displacement of [3H]Ro 15‐4513 binding in heterozygotes revealed three components, with their affinities indistinguishable from those in combined wild‐type and mutant homozygotes. This lack of interaction in DIS binding between wild‐type and mutant α6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the α6 subunit‐containing GABAA receptors in the heterozygotes are formed from individual mutant and wild‐type subunits with their relative expression differing from animal to animal.
Generation of Ketone Bodies from Leucine by Cultured Astroglial Cells
Authors: Bixel Maria Gabriele , Hamprecht Bernd
Published: July 2010

Abstract: To elucidate the significance of branched‐chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia‐rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first‐order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl‐CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, d‐β‐hydroxybutyrate, and α‐ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5‐3H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite α‐ketoisocaproate, to acetoacetate, which can be further reduced to d‐β‐hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.
Pro‐Thyrotropin‐Releasing Hormone Processing by Recombinant PC1
Authors: Nillni Eduardo A. , Friedman Theodore C. , Todd Roberta B. , Birch Nigel P. , Loh Y. Peng , Jackson Ivor M. D.
Published: July 2010

Abstract: Pro‐thyrotropin‐releasing hormone (proTRH) is the precursor to thyrotropin‐releasing hormone (TRH; pGlu‐His‐Pro‐NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln‐His‐Pro‐Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26‐kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine‐labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26‐kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well‐established antibodies, we propose that the initial cleavage gave rise to the formation of a 15‐kDa N‐terminal peptide (preproTRH25–152 or preproTRH25–158) and a 10‐kDa C‐terminal peptide (preproTRH154–255 or preproTRH160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5‐kDa C‐terminal peptide. The 15‐kDa N‐terminal intermediate was further processed to a 6‐kDa peptide (preproTRH25–76 or preproTRH25–82) and a 3.8‐kDa peptide (preproTRH83–108), whereas the 10‐kDa C‐terminal intermediate was processed to a 5.4‐kDa peptide (preproTRH206–255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25–50), one of the proTRH N‐terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.
Neurochemical Effects Following Peripheral Administration of Tetrahydropterin Derivatives to the hph‐1 Mouse
Authors: Brand Michael P. , Hyland Keith , Engle Todd , Smith Isabel , Heales Simon J. R.
Published: July 2010

Abstract: The hph‐1 mouse, which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 µmol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6‐methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N‐benzyl‐1,4‐dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1–4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 µmol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.
Nigral and Striatal Comparative Study of the Neurotoxic Action of 1‐Methyl‐4‐Phenylpyridinium Ion: Involvement of Dopamine Uptake System
Authors: Santiago Marti , Machado Alberto , Cano Josefina
Published: July 2010

Abstract: Microdialysis was used in a comparative study of the neurotoxic action of MPP+ in the absence or presence of nomifensine (20 µM) in the striatum and substantia nigra. Three different concentrations of MPP+ (1, 2.5, and 5 mM) were perfused for 15 min at 24 (day 1) and 48 h (day 2) after surgery. The dopamine basal value in the striatum was ∼17 fmol/min. Nomifensine (20 µM) stimulated dopamine release to ∼170 fmol/min. The increase of dopamine extracellular output in the striatum after MPP+ perfusion on day 1 was independent of the concentration of MPP+ perfused and of the absence or presence of nomifensine (20 µM), being ∼2,500 fmol/min. The dopamine basal value in the substantia nigra was below the detection limit of our HPLC equipment. Nomifensine (20 µM) stimulated dopamine release to ∼6.3 fmol/min. The increase of dopamine extracellular output in the substantia nigra was MPP+ dose‐dependent (1 mM, 75 fmol/min; 2.5 mM, 150 fmol/min; and 5 mM, 250 fmol/min) and independent of the presence or absence of nomifensine. On day 2, the presence of nomifensine on day 1 produced a total protection against MPP+ (1 mM) perfusion in the striatum, which was not observed against MPP+ (5 mM). MPP+ (1 mM) did not produce any neurotoxic action in the substantia in the absence or presence of nomifensine. The MPP+ (2.5 mM) effect on dopamine extracellular output in the absence of nomifensine (20 µM) in the substantia nigra on day 2 was similar to that of MPP+ (1 mM) in the striatum. The presence of nomifensine (20 µM) partially prevented the neurotoxic effect of MPP+ (2.5 mM) on dopaminergic cell bodies/dendrites in the substantia nigra. The MPP+ (5 mM) effect on dopamine extracellular output was similar in both structures studied in the absence or presence of nomifensine on day 2. These results suggest that terminals in the striatum are more sensitive to the neurotoxicity of MPP+ than cell bodies/dendrites in the substantia nigra.
Norepinephrine Is Lateralized Within the Olfactory Bulbs of Male Mice
Authors: Dluzen Dean E. , Kreutzberg Jennifer D.
Published: July 2010

Abstract: Concentrations and in vitro release of norepinephrine were determined from left and right olfactory bulbs of adult male CD‐1 mice. Norepinephrine concentrations from the left olfactory bulbs were significantly greater than those of the right (p < 0.01). No statistically significant differences were obtained for dopamine, nor were there differences in tissue weights between the left and right olfactory bulb tissue samples. To verify further this asymmetry, release rates of norepinephrine were measured from superfused left and right olfactory bulbs. Norepinephrine output from the left olfactory bulb was significantly greater than that from the right (p < 0.05). These results show that norepinephrine is lateralized within the olfactory bulbs of male mice. This asymmetry shows a clear catecholamine specificity with regard to concentrations and may be related to the lateralized olfactory processing that has been reported to occur in humans.
AP1‐Mediated Transcriptional Enhancement of the Rat Tyrosine Hydroxylase Gene by Muscarinic Stimulation
Authors: Chae Hee‐Don , Suh Byung‐Chang , Joh Tong H. , Kim Kyong‐Tai
Published: July 2010

Abstract: We investigated transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by muscarinic stimulation in human neuroblastoma SK‐N‐BE(2)M17 cells. Carbachol treatment increased the levels of intracellular Ca2+ and inositol 1,4,5‐trisphosphate (IP3) and enhanced transcription of the TH gene. The muscarinic receptor antagonist atropine completely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 µM 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid/acetoxymethyl ester (BAPTA/AM) to chelate intracellular Ca2+, carbachol still raised intracellular IP3 level and enhanced TH gene expression. Transient transfection analysis of the 5′ upstream region of TH gene revealed that the AP1 cis‐acting element at −205 to −199 bp was responsible for carbachol stimulation. But carbachol did not enhance TH gene expression in protein kinase C (PKC)‐activated or down‐regulated cells that had been induced by 5‐min or 24‐h exposure to phorbol 12‐myristate 13‐acetate (PMA), respectively. Thus, Ca2+‐independent PKC may play a role in carbachol‐induced TH gene expression. We demonstrated by gel retardation and competition assays that a DNA sequence containing the wild‐type AP1 site formed the specific DNA‐protein complex. However, treatment with carbachol or PMA did not change the amount of the specific DNA‐protein complex. Our results indicate that stimulation of phospholipase C‐linked muscarinic receptors leads to elevated TH gene expression via AP1‐mediated enhancement in a PKC‐dependent pathway.
Potentiation and Inhibition of Ionotropic Neurotransmitter Receptors Expressed in Xenopus Oocyte by Linoleic Acid and Its Hydroperoxide
Authors: Aoshima Hitoshi
Published: July 2010

Abstract: To study the effects of lipid hydroperoxide on ionotropic neurotransmitter receptors, γ‐aminobutyric acid (GABA), N‐methyl‐d‐aspartate (NMDA), and non‐NMDA receptors (GABARs, NMDARs, and non‐NMDARs, respectively) were expressed in Xenopus oocytes that received an injection of mRNA prepared from rat whole brain. Linoleic acid (LA) and its hydroperoxide 13‐l‐hydroperoxylinoleic acid (LOOH) prepared with soybean lipoxygenase inhibited the response of GABARs in the presence of GABA at high concentrations. The inhibition was stronger when the inhibitors were perfused 1 min before a mixture of GABA and the inhibitors than when they were perfused simultaneously with GABA. On the other hand, only LOOH potentiated the response of GABARs in the presence of GABA at low concentrations, possibly increasing the affinity of GABA to the receptors. Both LA and LOOH accelerated the rate of desensitization of GABARs, but LOOH did not affect their equilibrium between the active and desensitized form of the receptors. They also inhibited the response of NMDARs in a noncompetitive manner but barely inhibited the response of non‐NMDARs in the presence of kainate at various concentrations. These results suggest the possibility that production of lipid hydroperoxide modulates the neural transmission in the brain, especially through GABARs.
Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA
Authors: Falk‐Vairant J. , Corrèges P. , Eder‐Colli L. , Salem N. , Meunier F. M. , Lesbats B. , Loctin F. , Synguelakis M. , Israël M. , Dunant Y.
Published: July 2010

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real‐time recording with an embryonic Xenopus myocyte. Glioma C6‐Bu‐1 cells were found to be competent for Ca2+‐dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG‐2 cells. However, acetylcholine release could be restored when N18TG‐2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine‐releasing capacity. The transfected N18TG‐2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.
Characterization of Dystroglycan‐Laminin Interaction in Peripheral Nerve
Authors: Yamada Hiroki , Chiba Atsuro , Endo Tamao , Kobata Akira , Anderson Louise V. B. , Hori Hisae , Fukuta‐Ohi Hiroko , Kanazawa Ichiro , Campbell Kevin P. , Shimizu Teruo , Matsumura Kiichiro
Published: July 2010

Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α‐ and β‐dystroglycan, by posttranslational processing. The 120‐kDa peripheral nerve isoform of α‐dystroglycan binds laminin‐2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan‐laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan‐laminin interaction in peripheral nerve. We demonstrate that (1) α‐dystroglycan is an extracellular peripheral membrane glycoprotein that links β‐dystroglycan in the Schwann cell outer membrane with laminin‐2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α‐dystroglycan in the interaction with laminin.
Involvement of an Intercellular Adhesion Molecule 1‐Dependent Pathway in the Pathogenesis of Secondary Changes After Spinal Cord Injury in Rats
Authors: Hamada Yoshitaka , Ikata Takaaki , Katoh Shinsuke , Nakauchi Kenji , Niwa Mineo , Kawai Yumiko , Fukuzawa Kenji
Published: July 2010

Abstract: The intercellular adhesion molecule 1 (ICAM‐1) plays an important role in immune responses by promoting infiltration of neutrophils into tissues; however, its implication in the secondary destructive pathomechanism after the initial mechanical injury to the spinal cord has not been clarified yet. This study was conducted to examine the role of ICAM‐1 in this process after spinal cord injury (SCI) in rats. The expression of ICAM‐1 mRNA was investigated by the reverse transcription‐PCR method and the effect of monoclonal antibody (mAb) to ICAM‐1 on SCI was evaluated by measuring various parameters. ICAM‐1 mRNA expression correlated with the severity of injury and reached its maximum level 6 h after SCI. Intravenous injection of ICAM‐1 mAb (1 mg/kg) 30 min after SCI reduced motor disturbance and enhanced recovery. Moreover, it significantly suppressed myeloperoxidase activity by 43.0% and spinal cord edema by 1.1% in the injured spinal cord tissue. The posttraumatic drop in spinal cord blood flow was also improved. These results suggest that ICAM‐1 is deeply involved in the secondary self‐destructive process after mechanical injury of the spinal cord and should be an effective target for developing a pharmacological treatment for SCI.
Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder
Authors: Poltorak Maciej , Frye Mark A. , Wright Renee , Hemperly John J. , George Mark S. , Pazzaglia Peggy J. , Jerrels Shari A. , Post Robert M. , Freed William J.
Published: July 2010

Abstract: Neural cell adhesion molecule (N‐CAM) is involved in cell‐cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N‐CAM in the CSF. Soluble human N‐CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N‐CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N‐CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N‐CAM immunoreactive proteins, primarily the 120‐kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N‐CAM concentrations. It is possible that the 120‐kDa N‐CAM band present in the CSF is derived from CNS cells as a secreted soluble N‐CAM isoform. Our results suggest the possibility of latent state‐related disturbances in N‐CAM cellular function, i.e., residue from a previous episode, or abnormal N‐CAM turnover in the CNS of patients with mood disorder.