bluuebunny/crossref_metadata_2025 huggingface dataset:
Ceramide Protects Hippocampal Neurons Against Excitotoxic and Oxidative Insults, and Amyloid β‐Peptide Toxicity
Authors: Goodman Yadong , Mattson Mark P.
Published: July 2010

Abstract: The transcription factor NFκB is activated by various signals associated with brain injury, including tumor necrosis factor (TNF), oxidative insults, and amyloid β‐peptide (Aβ). We recently reported that TNFs activate NFκB in neurons and protect them against excitotoxic and oxidative insults, including Aβ toxicity. We now report that C2‐ceramide (C2), a membrane‐permeant activator of NFκB, protects cultured rat hippocampal neurons against death induced by glutamate, FeSO4, and Aβ. Protection was concentration dependent, specific (a ceramide analogue known not to activate NFκB was ineffective), required pretreatment, and was blocked by inhibitors of RNA and protein synthesis. Lipid peroxidation and accumulation of cellular peroxides induced by glutamate, FeSO4, and Aβ were significantly attenuated in neurons pretreated with C2. The data indicate that C2 induces antioxidant pathways in neurons and suggest novel approaches for reducing neuronal injury in both acute and chronic neurodegenerative conditions.
The Sec1 Family: A Novel Family of Proteins Involved in Synaptic Transmission and General Secretion
Authors: Halachmi Naomi , Lev Zeev
Published: July 2010

Abstract: The Sec1 family, a novel family of proteins involved in synaptic transmission and general secretion, is described. To date, 14 members of this family have been identified: four yeast proteins, Sec1, Sly1, Slp1/Vps33, and Vps45/Stt10; three nematode proteins, Unc‐18 and the homologues of Sly1 and Slp1; the Drosophila Rop; and six mammalian proteins, the rat Munc‐18/n‐Sec1/rbSec1A and rbSec1B, the mouse Munc‐18b/muSec1 and Munc‐18c, and the bovine Munc‐18 and mSec1. The mammalian proteins share 44–63% sequence identity with the nematode Unc‐18 and Drosophila Rop proteins and 20–29% with the yeast proteins and their nematode homologues. The Sec1 proteins are mostly hydrophilic and lack a transmembrane domain. Nevertheless, Sec1 proteins are found as membrane‐bound proteins. Some of them are also found as soluble, cytoplasmic proteins. Binding of the rat brain Sec1 to the presynaptic membrane may be due to strong interaction with syntaxin, an integral component of this membrane. The rat brain Sec1 is also bound to Cdk5, a neural cyclin‐dependent kinase. The Sec1 proteins play a positive role in exocytosis. Loss of function mutations in SEC1, SLY1, or SLP1 result in blocking of protein transport between distinct yeast subcellular compartments. Inactivation of unc‐18 and rop results in inhibition of neurotransmitter release and, in the case of rop, inhibition of general secretion as well. In addition, studies of Rop and n‐Sec1 indicate that they also play a negative role in synaptic transmission, mediated by their interaction with syntaxin. A working model addressing the dual regulative role of the Sec1 proteins in secretion is presented.
Modulation of the Stress‐Induced Synthesis of hsp27 and αB‐Crystallin by Cyclic AMP in C6 Rat Glioma Cells
Authors: Kato Kanefusa , Ito Hidenori , Hasegawa Kaori , Inaguma Yutaka , Kozawa Osamu , Asano Tomiko
Published: July 2010

Abstract: The possible participation of cyclic AMP in the stress‐induced synthesis of two small stress proteins, hsp27 and αB‐crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB‐crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β‐adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite‐ or heat‐induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB‐crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB‐crystallin showed that the modulation of the stress‐induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB‐crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB‐crystallin is suppressed.
An Early Loss in Membrane Protein Kinase C Activity Precedes the Excitatory Amino Acid‐Induced Death of Primary Cortical Neurons
Authors: Durkin J. P. , Tremblay R. , Buchan A. , Blosser J. , Chakravarthy B. , Mealing G. , Morley P. , Song D.
Published: July 2010

Abstract: Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15‐min exposure of day 14–18 cortical neurons to 100 µM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30–50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC‐selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral ischemia in vivo.
Suramin Induces Phosphorylation of the High‐Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons
Authors: Gill Jagjit S. , Connolly Denise C. , McManus Michael J. , Maihle Nita J. , Windebank Anthony J.
Published: July 2010

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet‐derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin‐like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose‐dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin‐induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine‐phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high‐affinity NGF (TrkA) receptor. Activation of other members of the signal‐transduction cascade (Shc, p21ras, Raf‐1, ERK‐1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.
Exposure of Astrocytes to Hypoxia/Reoxygenation Enhances Expression of Glucose‐Regulated Protein 78 Facilitating Astrocyte Release of the Neuroprotective Cytokine Interleukin 6
Authors: Hori Osamu , Matsumoto Masayasu , Kuwabara Keisuke , Maeda Yusuke , Ueda Hirokazu , Ohtsuki Toshiho , Kinoshita Taroh , Ogawa Satoshi , Stern David M. , Kamada Takenobu
Published: July 2010

Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78‐kDa polypeptide that demonstrated sequence identity with glucose‐regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high‐molecular‐weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti‐sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R‐induced astrocyte release of IL‐6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL‐6 under the stress of H/R.
Immunological Characterization and Transmembrane Topology of 5‐Hydroxytryptamine3 Receptors by Functional Epitope Tagging
Authors: Mukerji Jishnu , Haghighi Ali , Séguéla Philippe
Published: July 2010

Abstract: 5‐Hydroxytryptamine3 (5‐HT3) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABAA, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5‐HT3 receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5‐HT3 receptors in transfected COS‐7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti‐fusion protein antibodies directed against a wild‐type receptor intracellular domain. The extracellular location of the distal C‐terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5‐HT3 serotonin‐gated channels. In this region, the significant homology between members of this class of neurotransmitter‐gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate‐gated and ATP‐gated channels.
Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion
Authors: Tandon A. , Bachoo M. , Weldon P. , Polosa C. , Collier B.
Published: July 2010

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine‐treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2‐Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2‐Hz stimulation of colchicine‐treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20‐Hz stimulation was depressed. The amount of ACh released during 5‐Hz stimulation in the presence of vesamicol by the terminals of colchicine‐treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine‐sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half‐life of ChAT in sympathetic preganglionic terminals is ∼4 days. (c) One consequence of colchicine‐induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.
Gαq/11 Mediates Cholecystokinin Activation of the Cationic Conductance in Rat Substantia Nigra Dopaminergic Neurons
Authors: Wu Tony , Wang Hung‐Li
Published: July 2010

Abstract: Using acutely isolated rat substantia nigra neurons, our previous studies indicated that sulfated cholecystokinin octapeptide (CCK‐8) excites substantia nigra dopaminergic neurons by increasing the cationic conductance and that pertussis toxin‐insensitive G proteins mediate CCK‐8 induction of cationic currents. Gαq and Gα11 are expressed in various tissues, including the brain, and likely to mediate pertussis toxin‐insensitive neural signal transductions. In the present study, two different experiments were performed to test the hypothesis that Gαq/11 mediates CCK‐8 enhancement of the cationic conductance. First, we investigated the expression of Gαq and Gα11 mRNAs in CCK‐8‐responsive substantia nigra dopaminergic neurons by combining whole‐cell patch‐clamp recordings with a single‐cell reverse transcriptase‐polymerase chain reaction assay. After CCK‐8‐evoked cationic currents were recorded, cellular RNA was harvested from single neurons and used as a template for the subsequent reverse transcriptase‐polymerase chain reaction analysis. Gαq and Gα11 mRNAs were present in all substantia nigra dopaminergic neurons that responded to CCK‐8. Substantia nigra dopaminergic neurons were also internally perfused with the antibody raised against the common C‐terminus of Gαq and Gα11 during whole‐cell recordings. CCK‐8 failed to induce cationic currents after dopaminergic neurons were dialyzed with the anti‐Gαq/11 antibody. Our studies suggest that CCK‐8 activation of the cationic conductance in substantia nigra dopaminergic neurons is transduced by Gαq and/or Gα11.
Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen‐Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells
Authors: Cox Michael E. , Ely Constance M. , Catling Andrew D. , Weber Michael J. , Parsons Sarah J.
Published: July 2010

Abstract: Nicotine‐induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen‐activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 mM KCI, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down‐stream of Ca2+ influx and upstream of MEK activation. This Ca2+‐activated tyrosine kinase activity could not be accounted for entirely by c‐Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca2+‐activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.
Binding Interactions of Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor with the Different Subunits of Their High Affinity Receptors
Authors: Robledo Olivier , Auguste Patrick , Coupey Lionel , Praloran Vincent , Chevalier Sylvie , Pouplard Annick , Gascan Hugues
Published: July 2010

Abstract: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin‐6‐receptor transducer as well as gp190/low‐affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high‐affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high‐affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high‐affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high‐affinity‐type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10−10M, whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.
Cytokine‐Regulated Expression of Platelet‐Derived Growth Factor Gene and Protein in Cultured Human Astrocytes
Authors: Silberstein Francesca Ceccherini , De Simone Roberta , Levi Giulio , Aloisi Francesca
Published: July 2010

Abstract: To elucidate mechanisms regulating the production of platelet‐derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte‐enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum‐free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor‐β1 (TGF‐β1) or tumor necrosis factor‐α (TNF‐α); the largest increase was detected after combined treatment with the two cytokines. Interleukin‐1β (IL‐1β) by itself had little or no effect but synergized with TGF‐β1 in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A‐ and PDGF B‐chain mRNAs were detected in untreated astrocytes. PDGF B‐chain mRNA levels were increased by TGF‐β1, TNF‐α, TNF‐α/TGF‐β1, or IL‐1β/TGF‐β1, whereas PDGF A‐chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF‐β1, TNF‐α, and IL‐1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.
Mesenteric Organ Production, Hepatic Metabolism, and Renal Elimination of Norepinephrine and Its Metabolites in Humans
Authors: Eisenhofer Graeme , Åneman Anders , Hooper Douglas , Rundqvist Bengt , Friberg Peter
Published: July 2010

Abstract: This study used regional differences in plasma concentrations of norepinephrine and its metabolites to examine how production of the transmitter by sympathetic nerves, in particular, those innervating mesenteric organs, is integrated with metabolism by the liver and elimination by the kidneys. Higher concentrations of norepinephrine, its glycol metabolites 3,4‐dihydroxyphenylglycol and 3‐methoxy‐4‐hydroxyphenylglycol and their sulfate conjugates in portal venous than arterial plasma indicate substantial production of norepinephrine by mesenteric organs (15.5 nmol/min). Much lower concentrations of norepinephrine and its glycol metabolites in plasma leaving than entering the liver indicate their efficient hepatic removal (20 nmol/min). Higher concentrations of vanillylmandelic acid in the hepatic outflow than inflow indicate that this metabolic end product is produced largely from the norepinephrine and glycol metabolites removed by the liver. Renal elimination of vanillylmandelic acid (18–20 nmol/min), produced mainly by the liver (17 nmol/min), and of 3‐methoxy‐4‐hydroxyphenylglycol sulfate (7–9 nmol/min), produced largely by mesenteric organs (7 nmol/min), comprised 86–91% of the total renal elimination of norepinephrine metabolites. The results show that mesenteric organs produce about one‐half of the norepinephrine formed in the body. The liver removes substantial amounts of circulating norepinephrine and its glycol metabolites and converts these compounds to vanillylmandelic acid, which is then eliminated from the body by the kidneys. The sulfate conjugates are also metabolic end products eliminated by the kidneys. However, these metabolites are produced by extrahepatic tissues, in particular, mesenteric organs, which represent a significant source of sulfate‐conjugated norepinephrine and 3,4‐dihydroxyphenylglycol, and the main source of sulfate‐conjugated 3‐methoxy‐4‐hydroxyphenylglycol.
The Rate of Valine Incorporation into Proteins with Correction for Valine Recycling, Measured in Two Brain Tumor Models and the Cortex
Authors: Jevric‐Causevic A. , Diksic M.
Published: July 2010

Abstract: The tissue dilution factor (λ) for the incorporation of valine into proteins in the rat cortex and in two different tumors, AA ascites and C6 glioma, was determined from measurements of specific activities in the tissue acid‐soluble and aminoacyl‐tRNA pools and in the plasma. A constant plasma specific activity was achieved by a constant infusion rate of [3H]valine. The data showed that the λ for valine was the same in the cortex as in the tumors, and the recycling was ∼36%. There was no difference in the λ calculated on the basis of the specific activities in the tissue acid‐soluble or aminoacyl‐tRNA pools. The average dilution factor was found to be 0.64 ± 0.05. The rate of valine incorporation into proteins was on average 3.2 ± 0.4 and 4.9 ± 0.4 nmol/g/min in the cortex for the groups of rats used in the AA ascites and C6 glioma experiments, respectively. In the AA ascites tumor the rate was ∼41 and 29 nmol/g/min 4 and 7 days after tumor implantation, respectively, whereas in the C6 glioma the rate was ∼41 and 72 nmol/g/min 6 and 13 days after inoculation, respectively. The tumors had, in comparison with the cortex, a significantly greater volume of distribution of valine. The amounts of valyl‐tRNA were significantly greater in the tumors as compared with the normal cortex, with the exception of the glioma 6 days after implantation where the concentration was the same as in the cortex.
A Novel Rat Tyrosine Hydroxylase mRNA Species Generated by Alternative Splicing
Authors: Lanièce Philippe , Le Hir Hervé , Bodeau‐Péan Sylvie , Charon Yves , Valentin Luc , Thermes Claude , Mallet Jacques , Dumas Sylvie
Published: July 2010

Abstract: Tyrosine hydroxylase (TH) catalyzes the first and rate‐limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase‐PCR experiments were performed with RNA from PC12 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N‐terminal regulatory domain. A recombinant protein was produced in E. coli. Its in vitro specific activity was similar to that of the previously identified TH protein.
Noradrenaline Increases the Expression of the Proenkephalin Gene in Cultured Astroglial Cells by Acting on β1‐ and α1‐Adrenoceptors
Authors: Hildebrand B. , Olenik C. , Theodoridu A. , Boeckh C. , Uhl A. , Meyer D. K.
Published: July 2010

Abstract: In primary culture, type I astroglial cells from neocortex of newborn rats express the proenkephalin gene. The glial cells are not homogeneous but differ in their morphology; i.e., polygonal and process‐bearing cells are found. Transcription of the proenkephalin gene is increased via protein kinase A upon stimulation with cyclic AMP (cAMP) analogues. In the present study, how noradrenaline affected the expression of the proenkephalin gene in both cell types was investigated. Noradrenaline enhanced the levels of proenkephalin mRNA in a concentration‐dependent manner. Experiments with subtype‐selective antagonists suggested that β1‐adrenoceptors were involved. In situ hybridization showed that proenkephalin mRNA was induced only in polygonal cells. Noradrenaline also increased the levels of cAMP. However, concentrations of noradrenaline that produced a maximal increase in cAMP caused only submaximal elevations of proenkephalin mRNA. This discrepancy was explained by the finding that noradrenaline increased the expression of the proenkephalin gene also via α1‐adrenoceptors. It is concluded that β1‐ and α1‐adrenoceptors can act in a synergistic manner on the expression of the proenkephalin gene in astroglial cells.
Interferon‐γ and Nitric Oxide Down‐Regulate Lipopolysaccharide‐Induced Prostanoid Production in Cultured Rat Microglial Cells by Inhibiting Cyclooxygenase‐2 Expression
Authors: Minghetti Luisa , Polazzi Elisabetta , Nicolini Alessia , Créminon Christophe , Levi Giulio
Published: July 2010

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down‐regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l‐arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor NG‐monomethyl‐l‐arginine, led to a remarkable increase in lipopolysaccharide‐induced prostanoid release. Moreover, the nitric oxide‐generating compound 3‐morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon‐γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide‐evoked production of prostaglandin E2, prostaglandin D2, and thromboxane. It is interesting that the inhibitory effect of interferon‐γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon‐γ and nitric oxide act mainly by down‐regulating the lipopolysaccharide‐induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase‐2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon‐γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.
(1S,3R)‐1‐Aminocyclopentane‐1,3‐Dicarboxylic Acid‐Induced Increases in Cyclic AMP Formation in the Neonatal Rat Hippocampus Are Mediated by a Synergistic Interaction Between Phosphoinositide‐ and Inhibitory Cyclic AMP‐Coupled mGluRs
Authors: Schoepp Darryle D. , Johnson Bryan G. , Monn James A.
Published: July 2010

Abstract: A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1S,3R)‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), which is an agonist for phosphoinositide‐coupled and inhibitory‐coupled cAMP‐linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five‐ to 10‐fold). However, the agonists 3,5‐dihydroxyphenylglycine (DHPG) and (2R,4R)‐4‐aminopyrrolidine‐2,4‐dicarboxylate (2R,4R‐APDC), which selectively act on phosphoinositide‐coupled and inhibitory cAMP‐coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype‐selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1S,3R‐ACPD, were found. Stimulations of cAMP content evoked by 1S,3R‐ACPD and combined additions of DHPG plus 2R,4R‐APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8‐p‐sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)‐α‐methyl‐4‐carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1S,3R‐ACPD‐induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).
Activation of Mitogen‐Activated Protein Kinases in Oligodendrocytes
Authors: Bhat Narayan R. , Zhang Peisheng
Published: July 2010

Abstract: The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen‐activated protein (MAP) kinase [also called extracellular signal‐regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet‐derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an in‐gel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti‐MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C‐activating tumor promoter, phorbol 12‐myristate 13‐acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti‐phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42‐kDa phosphoprotein band cross‐reacting with anti‐MAP kinase antibodies. The phosphorylation of p42mapk in PDGF‐treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti‐pan‐ERK antibodies indicated the presence of ERK‐immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan‐ERK‐immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis.
Inhibition of Prolyl Oligopeptidase by Fmoc‐Aminoacylpyrrolidine‐2‐Nitriles
Authors: Li Jingrong , Wilk Elizabeth , Wilk Sherwin
Published: July 2010

Abstract: Prolyl oligopeptidase (EC 3.4.21.26), a widely distributed cytosolic enzyme, cleaves peptidylprolyl peptide and peptidylprolyl amino acid bonds in many neuropeptide substrates. Its action on vasopressin has been proposed as the underlying mechanism accounting for the ability of inhibitors of prolyl oligopeptidase to reverse scopolamine‐induced amnesia in rats. Future behavioral studies would be facilitated by the availability of potent inhibitors readily synthesized from common intermediates. A series of Fmoc‐aminoacylpyrrolidine‐2‐nitriles prepared by a simple two‐step synthesis were found to be potent noncompetitive inhibitors of the rabbit brain enzyme. The most potent inhibitors, Fmoc‐prolyl‐pyrrolidine‐2‐nitrile and Fmoc‐alanyl‐pyrrolidine‐2‐nitrile, each have a Ki of 5 nM. The compounds are cell permeable and stable. They do not inhibit the related enzyme dipeptidyl peptidase IV (EC 3.4.14.5). When administered intraperitoneally to mice, Fmoc‐alanyl‐pyrrolidine‐2‐nitrile crosses the blood‐brain barrier to inhibit brain prolyl oligopeptidase. The ease of synthesis, potency, efficacy in vivo, stability, and specificity of Fmoc‐aminoacylpyrrolidine‐2‐nitriles may make them inhibitors of choice in studies probing the physiological significance of prolyl oligopeptidase.
Modulation of GABAA Receptor tert‐[35S]Butylbicyclophosphorothionate Binding by Antagonists: Relationship to Patterns of Subunit Expression
Authors: Korpi E. R. , Seeburg P. H. , Lüddens H.
Published: July 2010

Abstract: The multisubunit γ‐aminobutyric acid type A (GABAA) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABAA‐receptor ionophore ligand tert‐[35S]butylbicyclophosphorothionate ([35S]TBPS) and its modulation by GABA and the GABAA antagonists bicuculline and 2′‐(3′‐carboxy‐2′,3′‐propyl)‐3‐amino‐6‐p‐methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABAA subunits in brain as reported previously. In some brain regions SR 95531 reduced [35S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline‐ to SR 95531‐modulated [35S]TBPS binding. This ratio correlated positively with α2‐subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit‐containing GABAA receptors. The data aid in developing GABAA‐receptor subtype‐specific antagonists and in establishing receptor domains critical for the actions of GABAA antagonists.
Dopamine D3 (Auto)receptors Inhibit Dopamine Release in the Frontal Cortex of Freely Moving Rats In Vivo
Authors: Gobert A. , Lejeune F. , Rivet J.‐M. , Cistarelli L. , Millan M. J.
Published: July 2010

Abstract: In freely moving rats, the novel, selective dopamine (DA) D3 receptor agonist PD 128,907 dose‐dependently [effective dose (ED25) = 0.07 mg/kg, s.c.] reduced dialysate levels of DA in the frontal cortex, a structure innervated by the ventral tegmental area (VTA). This action of PD 128,907 (0.16 mg/kg, s.c.) was abolished by a selective DA D3 receptor antagonist S 14297 (1.25 mg/kg, s.c.), which alone did not modify levels of DA. In contrast to S 14297, its inactive distomer, S 17777, did not modify the actions of PD 128,907. In addition, PD 128,907 dose‐dependently and potently inhibited the firing rate of VTA‐localized neurons in anesthetized rats (ED50 = 0.001 mg/kg, i.v.). S 14297, but not S 17777, completely reversed the actions of PD 128,907 (0.005 mg/kg, i.v.) with a 50% inhibitory dose of 0.03 mg/kg, i.v. and did not itself significantly modify the firing rate. In conclusion, these data provide the first direct evidence that DA D3 (auto)receptors modulate (inhibit) the release of DA in the frontal cortex.
Molecular Cloning, Expression, and Chromosomal Localization of a Human Brain‐Specific Na+‐Dependent Inorganic Phosphate Cotransporter
Authors: Ni Binhui , Du Yansheng , Wu Xin , DeHoff Bradley S. , Rosteck Paul R. , Paul Steven M.
Published: July 2010

Abstract: We describe the molecular cloning of a cDNA encoding a human brain Na+‐dependent inorganic phosphate (Pi) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na+‐dependent inorganic Pi cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS‐1 cells results in Na+‐dependent Pi uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron‐enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia‐enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late‐onset familial Alzheimer's disease locus.
A τ Promoter Region Without Neuronal Specificity
Authors: Andreadis Athena , Wagner Bridget K. , Broderick Jennifer A. , Kosik Kenneth S.
Published: July 2010

Abstract: The microtubule‐associated protein τ is produced from a 6‐kb mRNA expressed primarily in neurons. A 2‐kb τ mRNA has also been characterized, which produces a τ isoform that localizes to the nucleus, and an 8‐kb mRNA is expressed in the PNS. Mapping and sequencing of the human τ gene start showed that it has an unusually GC‐rich 5′‐untranslated region coded by a single untranslated exon (designated −1). Primer extensions and expression assays indicated that upstream of exon −1 is a promoter that is not neuron specific. This region contains consensus binding sites for transcription factors AP2, Sp1, and GCF.
Induction of Proenkephalin Gene Expression in Cultured Bovine Chromaffin Cells Is Dependent on Protein Synthesis of AP‐1 Proteins
Authors: Bacher Bruno , Wang Xiaomin , Schulz Stefan , Höllt Volker
Published: July 2010

Abstract: In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c‐fos, c‐jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE‐2 element of the proenkephalin promoter was sufficient for basal and second messenger‐induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE‐2 and AP‐1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP‐1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c‐fos, but not of c‐jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP‐1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells.
Transduction Mechanisms Involved in Thrombin Receptor‐Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes
Authors: Debeir Thomas , Gueugnon Josseline , Vigé Xavier , Benavides Jesús
Published: July 2010

Abstract: In astrocytes, thrombin and thrombin receptor‐activating activating peptide (TRAP‐14), a 14‐amino‐acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [3H]thymidine incorporation into DNA (EC50 = 1 nM and +650% at 100 nM for thrombin; EC50 = 3 µM and +600% at 100 µM for TRAP‐14) and nerve growth factor (NGF) secretion (approximately twofold at 100 nM thrombin or 100 µM TRAP‐14). The [3H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down‐regulation of this enzyme by chronic exposure of astrocytes to phorbol 12‐myristate 13‐acetate (PMA). Thrombin‐induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19‐fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin‐induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin‐ and TRAP‐14‐induced DNA synthesis. PART activation caused a decrease in forskolin‐stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX‐sensitive G protein, such as Gi or Go, is involved in thrombin‐induced cell division. In contrast, thrombin‐induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin‐ and TRAP‐14‐induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)‐triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.
A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue‐Specific Expression and Functional Analysis
Authors: Takeda Yasuo , Asou Hiroaki , Murakami Yoshinori , Miura Masayuki , Kobayashi Masaaki , Uyemura Keiichi
Published: July 2010

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full‐length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full‐length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12‐bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs‐transfected cells (L4‐2) were similar to those of full‐length L1‐transfected cells (L3‐1), but the cell migratory activity of L4‐2 itself was clearly lower than that of L3‐1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full‐length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.
A Prototypic Intracellular Calcium Antagonist, TMB‐8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium‐Dependent Component of Glutamate Neurotoxicity
Authors: Malcolm Craig S. , Ritchie Lyndsay , Grieve Angus , Griffiths Roger
Published: July 2010

Abstract: The effect(s) of a prototypic intracellular Ca2+ antagonist, 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), on glutamate‐induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free‐Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate‐induced toxicity, quantified by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na+/Cl−‐dependent component observed within minutes of glutamate exposure, and a “delayed” Ca2+‐dependent component (ED50∼50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB‐8 (at >100 µM) protects granule cells from the Ca2+‐dependent, but not the Na+/Cl−‐dependent, component of glutamate‐induced neurotoxic damage, and furthermore, where TMB‐8 inhibits glutamate‐evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB‐8‐sensitive intracellular store may be a necessary step in the onset of glutamate‐induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB‐8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate‐evoked increases in [Ca2+]i and subsequently effect a pronounced time‐dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate‐evoked increases in [Ca2+]i and glutamate‐induced neurotoxicity.