bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Types of Apoptotic Cell Death of Rat Central Nervous System‐Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation
Authors: Honma Naoyuki , Uchida Atsuko , Hirose Hiroya , Srsen Vlastimil , Kishimoto Takeo , Hisanaga Shin‐ichi
Published: July 2010

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS‐derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 mM DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.
Changes of Free Long‐Chain Bases in Neuronal Cells During Differentiation and Aging in Culture
Authors: Valsecchi Manuela , Chigorno Vanna , Nicolini Marco , Sonnino Sandro
Published: July 2010

Abstract: Changes in the free long‐chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o‐phthalaldehyde and fractionated by reversed‐phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: C18‐sphingosine, C18‐sphinganine, C20‐sphingosine, and C20‐sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18‐sphingosine> C20‐sphinganine and varying from 0.02 ± 0.015 pmol/mg of cell protein for C20‐sphinganine at day 0 to 223 ± 22 pmol/mg of cell protein for C18‐sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20‐sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20‐sphinganine progressively increased from day 0 to 22 of culture; that of C18‐sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18‐ and C20‐sphingosine levels during cell culture were qualitatively similar to those of C18‐ and C20‐sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.
Tissue‐Specific Expression of the Vasoactive Intestinal Peptide Gene Requires Both an Upstream Tissue Specifier Element and the 5′ Proximal Cyclic AMP‐Responsive Element
Authors: Hahm Sung Ho , Eiden Lee E.
Published: July 2010

Abstract: An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is important for cell type‐specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 100 bases of the VIP promoter [the VIP cyclic AMP‐responsive element (VIP‐CRE)] confers cyclic AMP and phorbol ester responsiveness to heterologous promoters. The possibility that these two regions of the VIP gene function cooperatively to determine tissue‐specific and second messenger‐dependent expression of the VIP gene was addressed by assaying transcription from a VIP‐luciferase reporter gene with progressive deletions from the 5′ flanking sequence of the gene, with or without inactivation of the proximal VIP‐CRE. Basal expression of the reporter gene in both SH‐EP and SK‐N‐SH human neuroblastoma cells, which express endogenous VIP mRNA, was absolutely dependent on the presence of the upstream TSE. Full constitutive expression was also dependent on the intact VIP‐CRE. Forskolin‐mediated induction of the reporter gene in SH‐EP and SK‐N‐SH cells was completely abolished by mutations in the VIP‐CRE but not by deletion of the upstream sequence, indicating that the VIP‐CRE alone determines cyclic AMP responsiveness. In contrast to reports that the VIP‐CRE imparts 12‐O‐tetradecanoylphorbol 13‐acetate (phorbol 12‐myristate 13‐acetate; PMA) responsiveness to heterologous promoters, PMA stimulation in SK‐N‐SH cells was independent of an intact VIP‐CRE but dependent on a region between −2.5 kb and the VIP‐CRE. Sequencing of the entire 5.2‐kb VIP 5′ flank revealed a consensus PMA‐responsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the VIP gene.
Differential Inhibition of Secretagogue‐Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors
Authors: Dahmer Mary K. , Senogles Susan E.
Published: July 2010

Abstract: Recent studies have demonstrated that D1‐selective and D2‐selective dopamine receptor agonists inhibit catecholamine secretion and Ca2+ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1‐like dopamine receptor subfamily, and D4, a member of the D2‐like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na+, which could explain inhibition of secretion and Ca2+ uptake by dopamine agonists. D1‐selective agonists preferentially inhibited both dimethylphenylpiperazinium‐ (DMPP) and veratridine‐stimulated 22Na+ influx into chromaffin cells. The D1‐selective agonists chloro‐APB hydrobromide (CI‐APB; 100 µM) and SKF‐38393 (100 µM) inhibited DMPP‐stimulated Na+ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2‐selective agonist bromocriptine (100 µM) inhibited Na+ uptake by only 22.9 ± 5.0%. Veratridine‐stimulated Na+ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µM CI‐APB or bromocriptine, respectively. The effect of CI‐APB was concentration dependent. A similar IC50 (∼18 µM) for inhibition of both DMPP‐ and veratridine‐stimulated Na+ uptake was obtained. The addition of 8‐bromo‐cyclic AMP (1 mM) had no effect on either DMPP‐ or veratridine‐stimulated Na+ uptake. These observations suggest that D1‐selective agonists are inhibiting secretagogue‐stimulated Na+ uptake in a cyclic AMP‐independent manner.
Effects of Subchronic Clozapine and Haloperidol on Striatal Glutamatergic Synapses
Authors: Meshul Charles K. , Bunker Gillian L. , Mason John N. , Allen Cynthia , Janowsky Aaron
Published: July 2010

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [3H]MK‐801 to the NMDA receptor‐linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [3H]MK‐801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63‐base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.
Aggregation of Neurofilaments in NF‐L Transfected Neuronal Cells: Regeneration of the Filamentous Network by a Protein Kinase C Inhibitor
Authors: Carter J. E. , Gallo J.‐M. , Anderson V. E. R. , Anderton B. H. , Robertson J.
Published: July 2010

Abstract: Cytoplasmic inclusion bodies that are accumulations of neurofilaments are the pathological hallmark of many neurodegenerative diseases and have been produced in transgenic mice by overexpression of mouse (NF‐L and NF‐M; light and medium chains, respectively) and human (NF‐M and NF‐H; medium and heavy chains, respectively) neurofilament subunits. This report describes a neuronal culture model in which human NF‐L was overexpressed to produce cytoplasmic accumulations of neurofilaments within cell bodies concomitant with the collapse of the endogenous neurofilament network. Electron microscopy showed that, within accumulations, neurofilaments retained a filamentous structure. The culture model thus provides a novel system in which the effect on neurofilament accumulations of manipulating protein phosphorylation can be studied. Treatment of cells containing neurofilament accumulations with bisindolylmaleimide, a specific protein kinase C inhibitor, resulted in regeneration of the filamentous network; this effect was not due to a change in the level of transfected NF‐L expression. These findings lend support to the suggestion that an impairment in the regulation of protein phosphorylation may lead to the accumulation of neurofilaments seen in neurodegenerative disease.
Patients with Amyotrophic Lateral Sclerosis and Other Neurodegenerative Diseases Have Increased Levels of Neurofilament Protein in CSF
Authors: Rosengren Lars E. , Karlsson Jan‐Erik , Karlsson Jan‐Olof , Persson Lennart I. , Wikkelsø Carsten
Published: July 2010

Abstract: In the present study we describe an ELISA to quantify the light subunit of the neurofilament triplet protein (NFL) in CSF. The method was validated by measuring CSF NFL concentrations in healthy individuals and in two well‐characterized groups of patients with amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). The levels were increased in ALS (1,743 ± 1,661 ng/L; mean ± SD) and AD (346 ± 176 ng/L) compared with controls (138 ± 31 ng/L; p < 0.0001 for both). Within the ALS group, patients with lower motor neuron signs only had lower NFL levels (360 ± 237 ng/L) than those with signs of upper motor neuron disease (2,435 ± 1,633 ng/L) (p < 0.05). In a second study patients with miscellaneous neurodegenerative diseases were investigated (vascular dementia, olivopontocerebellar atrophy, normal pressure hydrocephalus, cerebral infarctions, and multiple sclerosis), and the CSF NFL level was found to be increased (665 ± 385 ng/L; p < 0.0001). NFL is a main structural protein of axons, and we suggest that CSF NFL can be used to monitor neurodegeneration in general, but particularly in ALS with involvement of the pyramidal tract.
Amyloid β Toxicity Consists of a Ca2+‐Independent Early Phase and a Ca2+‐Dependent Late Phase
Authors: Abe Kazuho , Kimura Hideo
Published: July 2010

Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca2+ channel blockers or in Ca2+‐free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca2+ channel blockers such as Co2+, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long‐lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca2+‐independent early phase and a Ca2+‐dependent late phase—and that the early phase may be required to induce the late phase.
Neurotransmitter‐Mediated Regulation of Brain Aromatase: Protein Kinase C‐ and G‐Dependent Induction
Authors: Abe‐Dohmae Sumiko , Takagi Yasuyuki , Harada Nobuhiro
Published: July 2010

Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α1‐adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α1‐agonist phenylephrine than in control neurons, whereas prazosin, an α1‐antagonist, suppressed this increase, and ligands for α2‐ or β‐adrenergic receptors did not exert any influence. The profile of α1‐adrenergic receptor subtypes during actual development in vivo suggested that the α1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12‐myristate 13‐acetate and dibutyryl‐cyclic GMP, whereas forskolin and dibutyryl‐cyclic AMP caused a decrease. These data indicate that stimulation via α1 (possibly α1B)‐adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β‐Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.
Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)
Authors: Bourguet Denis , Raymond Michel , Fournier Didier , Malcolm Colin A. , Toutant Jean‐Pierre , Arpagaus Martine
Published: July 2010

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens. The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol‐specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide‐susceptible strain S‐LAB, AChE1 is inhibited by 5 × 10−4M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide‐resistant strain, MSE, there is no mortality after exposure to 5 × 10−4M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10−4M propoxur. The possibility that AChE1 and AChE2 are products of tissue‐specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.
Rate of Retrograde Transport of Cholera Toxin from the Plasma Membrane to the Golgi Apparatus and Endoplasmic Reticulum Decreases During Neuronal Development
Authors: Sofer Anat , Futerman Anthony H.
Published: July 2010

Abstract: Various glycolipid‐binding toxins are internalized from the cell surface to the Golgi apparatus. Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity. We now demonstrate that rhodamine (Rh)‐CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG‐2 cells and that the A subunit is essential for retrograde transport to the ER. In addition, the rate of internalization of Rh‐CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature. The Golgi apparatus was labeled in almost all 1‐day‐old neurons after <1 h of incubation with Rh‐CT but was labeled in <10% of 14‐day‐old neurons after 1 h. During the first 14 days in culture, there was a 15‐fold increase in the number of 125I‐CT‐binding sites per cell, indicating that the decrease in the rate of internalization of Rh‐CT is not due to reduced levels of cell surface GM1 in older neurons. These results imply that the rate of retrograde transport of CT from the plasma membrane to the Golgi apparatus and ER is regulated during neuronal development and differentiation.
Effect of Buthionine Sulfoximine, a Synthesis Inhibitor of the Antioxidant Glutathione, on the Murine Nigrostriatal Neurons
Authors: Andersen J. K. , Mo J. Q. , Hom D. G. , Lee F. Y. , Harnish P. , Hamill R. W. , McNeill T. H.
Published: July 2010

Abstract: This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose‐response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age‐related oxidative stress.
Scavenging Effects of Dopamine Agonists on Nitric Oxide Radicals
Authors: Nishibayashi Sakiko , Asanuma Masato , Kohno Masahiro , Gómez‐Vargas Marvin , Ogawa Norio
Published: July 2010

Abstract: It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson's disease (PD), and the level of nitric oxide radical (•NO), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for •NO in an in vitro •NO‐generating system using 3‐(2‐hydroxy‐1‐methylethyl‐2‐nitrosohydrazino)‐N‐methyl‐1‐propanamine as an •NO donor and 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (carboxy‐PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of •NO generated. •NO appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be ∼23 and 200 µM, respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6‐hydroxydopamine‐injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges •NO radicals and hence protects against •NO‐related cytotoxicity. This ESR spectrometry method using carboxy‐PTIO may be useful for screening other drugs that can quench •NO.
Expression of Native GABAA Receptors in Xenopus Oocytes Injected With Rat Brain Synaptosomes
Authors: Sanna Enrico , Motzo Costantino , Murgia Antonella , Amato Francesco , Deserra Tiziana , Biggio Giovanni
Published: July 2010

Abstract: Oocytes from the frog Xenopus laevis were shown recently to express native nicotinic acetylcholine receptors after injection with purified Torpedo electroplaque membrane vesicles. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes has now been shown to result in the expression of γ‐aminobutyric acid type A (GABAA) receptor‐mediated Cl− currents. Electrophysiological characterization of the responses of these receptors to GABA and other agents revealed that they were incorporated into the oocyte membrane and that they retained their original pharmacological properties, such as sensitivity to Cl− channel blockers, benzodiazepines, and general anesthetics. These results suggest that this approach to the expression of heterologous proteins in Xenopus oocytes may facilitate the study of native synaptic proteins derived from brain tissue.
Intracerebral NMDA Injection Stimulates Production of Interleukin‐1β in Perinatal Rat Brain
Authors: Hagan P. , Poole S. , Bristow A. F. , Tilders F. , Silverstein F. S.
Published: July 2010

Abstract: Susceptibility to NMDA neurotoxicity peaks in the early postnatal period in rats. Although indirect evidence suggests that interleukin‐1β is a mediator of NMDA neurotoxicity in perinatal rats, direct confirmation of NMDA‐induced interleukin‐1β production in the brain has not been reported previously. The primary goal of this study was to determine if intracerebral injection of a neurotoxic dose of NMDA stimulates interleukin‐1β production acutely. We used a rat‐specific interleukin‐1β ELISA to quantify brain tissue homogenate interleukin‐1β content, and an immunocytochemical assay with a monoclonal anti‐rat interleukin‐1β antibody to visualize its distribution. NMDA (10 nmol) was injected stereotaxically into 7‐day‐old rats, using coordinates that targeted the striatum and overlying dorsal hippocampus. Interleukin‐1β concentrations were measured in samples from the injected and contralateral cerebral hemispheres 0–12 h later; in addition, the impact of treatment with the noncompetitive NMDA antagonist MK‐801 on interleukin‐1β production was assessed. We found marked increases in tissue content of interleukin‐1β in the lesioned hemisphere; values peaked at 6 h post injection. Treatment with MK‐801 (1 mg/kg) blocked NMDA‐induced increases in interleukin‐1β. Preliminary immunocytochemical analysis demonstrated high concentrations of interleukin‐1β‐immunoreactive cells in the lesioned hippocampus, and concurrent increases in interleukin‐1β immunoreactivity diffusely in the ependyma at 6 h after NMDA administration. Our data provide the first direct evidence that NMDA‐induced excitotoxic injury stimulates interleukin‐1β production in vivo.
2′‐Deoxyadenosine Induces Apoptosis in Rat Chromaffin Cells
Authors: Wakade Arun R. , Guo Xi , Palmer Kenneth C. , Kulkarni Jayant S. , Przywara Dennis A. , Wakade Taruna D.
Published: July 2010

Abstract: We show here that 2′‐deoxyadenosine (2′‐dAdo) but not adenosine was toxic to chromaffin cells of 3–4‐week‐old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3‐day period in the presence of 100 µM 2′‐dAdo plus 3 µM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase‐mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis‐like cell death by 2′‐dAdo. Lethal effects of 2′‐dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2′‐dAdo‐prompted cell death was not prevented by inhibitors of nucleoside transporter (3 µM dilazep or 1 µM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 µM uridine or 100 µM 2′‐deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2′‐dAdo (100 and 300 µM) showed a three‐ and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2′‐dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2′‐dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.
Differential Effects of δ‐ and μ‐Opioid Receptor Antagonists on the Amphetamine‐Induced Increase in Extracellular Dopamine in Striatum and Nucleus Accumbens
Authors: Schad Christina A. , Justice Joseph B. , Holtzman Stephen G.
Published: July 2010

Abstract: The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine‐induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective δ‐opioid receptor antagonist, but not by the irreversible μ‐opioid receptor antagonist β‐funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 µg) or vehicle was given 15 min before and β‐funaltrexamine (10 µg) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10‐µl volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneous d‐amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30‐min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose‐dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 µg) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 µg of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, β‐funaltrexamine (10 µg) had no effect in the striatum but significantly attenuated the amphetamine‐induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that δ‐opioid receptors play a relatively larger role than μ‐opioid receptors in mediating the amphetamine‐induced increase in extracellular dopamine content in the striatum, whereas μ‐opioid receptors play a larger role in mediating these effects in the nucleus accumbens.
Lysophosphatidic Acid Decreases Glutamate and Glucose Uptake by Astrocytes
Authors: Keller Jeffrey N. , Steiner Marion R. , Mattson Mark P. , Steiner Sheldon M.
Published: July 2010

Abstract: The brain is a rich source of the lipid biomediator lysophosphatidic acid, and lysophosphatidic acid levels can significantly increase following brain trauma. Responses of primary rat brain astrocytes to this novel lipid are defined in the current study. Treatment of cells with lysophosphatidic acid resulted in a time‐ and dose‐dependent inhibition of glutamate uptake. Inhibition of glutamate uptake was specific because the related phospholipids, phosphatidic acid, lysophosphatidylcholine, and lysophosphatidylglycerol, did not inhibit this uptake under comparable conditions, i.e., treatment with 10 µM lipid for 30 min. Lysophosphatidic acid treatment of cells resulted in an increase in lipid peroxidation, as measured by the thiobarbituric acid assay. This increase in content of thiobarbituric acid‐reactive substances was largely inhibited by treatment with dithiothreitol or propyl gallate; however, such treatment did not affect the lysophosphatidic acid‐induced inhibition of glutamate uptake. Lysophosphatidic acid also inhibited glucose uptake with a dose‐response curve that paralleled the inhibition of glutamate uptake. By impairing uptake of glutamate by astrocytes, lysophosphatidic acid may exacerbate excitotoxic processes in various neurodegenerative conditions.
Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells
Authors: Menezes Ana , Zeman Richard , Sabban Esther
Published: July 2010

Abstract: The relationship between elevations in intracellular free Ca2+ concentration ([Ca2+]i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K+ concentration triggered rapid and sustained increases in [Ca2+]i from a basal level of ∼50 to 110–150 nM and three‐ to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5‐trisphosphate (IP3). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP3 or inhibition of Ca2+‐ATPase, rapidly elevated [Ca2+]i to >200 nM and increased TH gene expression (three‐to fivefold). Confocal imaging showed that the elevations in [Ca2+]i in each case occurred throughout the cyto‐ and nucleoplasm. The initial rise in [Ca2+]i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid or 3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)‐octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca2+]i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca2+]i, there are important differences among them in terms of the magnitude of elevated [Ca2+]i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca2+]i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.
Dantrolene Is Cytoprotective in Two Models of Neuronal Cell Death
Authors: Wei Huafeng , Perry David C.
Published: July 2010

Abstract: The neuroprotective effects of dantrolene, an inhibitor of calcium release from intracellular stores, were investigated in a model of cell death induced by calcium release from endoplasmic reticulum in vitro. Thapsigargin (50 nM), a selective inhibitor of endoplasmic reticular Ca2+‐ATPase, significantly increased the cytosolic Ca2+ concentration to 230% over basal levels, induced DNA fragmentation, and reduced cell viability from 94% in control cells to 41% after a 24‐h treatment in GT1‐7 hypothalamic neurosecretory cells. Pretreatment with dantrolene for 30 min significantly inhibited elevation of cytosolic Ca2+ levels, DNA fragmentation, and GT1‐7 cell death induced by thapsigargin in a dose‐dependent manner. To determine if dantrolene would also be protective in an in vivo model of neurodegeneration, it was administered intravenously immediately following a 5‐min global cerebral ischemia in gerbils, and the number of intact hippocampal CA1 pyramidal neurons was counted 7 days later. The effects of dantrolene on brain and rectal temperature were monitored in a separate experiment. Dantrolene significantly increased the number of intact CA1 pyramidal neurons from 40% (untreated ischemic animals) to 67 (10 mg/kg), 78 (25 mg/kg), or 83% (50 mg/kg) of values in sham controls (all p < 0.001). No significant changes in brain or rectal temperature were detected for 4 h following 50 mg/kg dantrolene. These results suggest that abnormal Ca2+ release from intracellular stores can induce neuronal death and that such a mechanism may contribute to delayed hippocampal neuronal death after cerebral ischemia. Dantrolene may be a potentially useful drug for neuroprotection after cerebral ischemia.
Human Brain γ‐Aminobutyric Acid Levels and Seizure Control Following Initiation of Vigabatrin Therapy
Authors: Petroff Ognen A. C. , Behar Kevin L. , Mattson Richard H. , Rothman Douglas L.
Published: July 2010

Abstract: Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain γ‐aminobutyric acid (GABA) concentrations. Seizure control is not improved significantly when the daily dose is increased beyond 50 mg/kg. Serial, in vivo measurements of GABA levels in human occipital lobe were made using 1H NMR spectroscopy before and after the start of vigabatrin treatment. We used a 2.1‐T magnetic resonance imagerspectrometer and an 8‐cm surface coil to examine serially a 14‐cm3 volume in the occipital lobe of 26 patients with complex partial seizures. Brain GABA content increased following the start of vigabatrin treatment up to a daily dose of 60 mg/kg. Additional increases in dose failed to increase brain GABA content further. GABA synthesis may decrease with sustained elevations of human brain GABA levels. Starting vigabatrin treatment reduced seizure frequency by >50%, from six to seven per month to three. Improved seizure control was not associated with further increases of vigabatrin dose. Increased brain GABA concentration was associated with improved seizure control. Starting vigabatrin treatment improved seizure control twofold when GABA levels increased above 1.8 mmol/kg. Further increases in brain GABA content above 2.5 mmol/kg provided less protection. Measuring occipital lobe GABA concentrations may predict improved seizure control when using antiepileptic drugs designed to increase brain GABA levels.
The δ‐Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK‐N‐BE
Authors: Allouche Stéphane , Polastron Jocelyne , Jauzac Philippe
Published: July 2010

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca2+ concentration ([Ca2+]i) either by regulating plasma membrane Ca2+‐channel activity or by mobilizing intracellular Ca2+ stores. The present report documents the [Ca2+]i increase induced by opioid agonists in a human neuroblastoma cell line, SK‐N‐BE, expressing δ‐opioid receptors. In the presence, as well as in the absence, of extracellular Ca2+, opioid agonists enhanced significantly [Ca2+]i, whereas carbachol, known to mobilize specifically inositol 1,4,5‐trisphosphate‐sensitive intracellular Ca2+ stores, acted only in the presence of extracellular Ca2+. The opioid‐induced increase in [Ca2+]i was not affected by treatments modifying the trimeric Gi, Go, and Gs protein transduction mechanisms or the activity of adenylyl cyclase. The Ca2+‐ATPase pump‐inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid‐induced [Ca2+]i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK‐N‐BE cells, δ‐opioid receptors mobilize Ca2+ from intracellular ryanodine‐sensitive stores and the mechanism involved is independent of Gi/Go and Gs proteins and protein kinase A activation.
Association of the Tetraspan Protein CD9 with Integrins on the Surface of S‐16 Schwann Cells
Authors: Hadjiargyrou Michael , Kaprielian Zaven , Kato Nobuo , Patterson Paul H.
Published: July 2010

Abstract: The cell surface glycoprotein CD9 is a member of a group of proteins known as the “tetraspan” or “transmembrane 4 superfamily.” Previous work with non‐neural cells has shown that CD9 associates in cis with integrins and small GTP‐binding proteins on the cell surface. To extend our recent findings showing that perturbation of CD9 alters Schwann cell adhesion, proliferation, and migration, as well as neurite outgrowth in sympathetic neurons, we have searched for CD9‐associated proteins in S‐16 Schwann cells. We demonstrate here that CD9 is specifically coprecipitated from S‐16 cell extracts by antibodies against integrins α3, α6, and β1. In addition, double immunofluorescence labeling and co‐capping experiments indicate that CD9 is specifically colocalized with these integrins on the cell membrane of S‐16 Schwann cells.
Long‐Term Effect of Forskolin on the Activation of Adenylyl Cyclase in Astrocytes
Authors: El Jamali Amina , Rachdaoui Nadia , Jacquemin Claude , Corrèze Claude
Published: July 2010

Abstract: Long‐term (48‐h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive‐intestinal peptide, guanyl 5′‐(βγ‐imido)diphosphate, guanosine 5′‐O‐(3‐thiotriphosphate) [GTP(S)], and AIF4− in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5‐fold. These two opposite effects of forskolin were mediated by a cyclic AMP‐dependent mechanism. No changes in Gsα, Giα, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin‐treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc− cells, lacking Gsα protein. Gsα proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted Gsα protein toward the light‐density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via Gsα, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long‐term treatment by forskolin.
Tetrahydrobiopterin Biosynthesis Enhanced by Lipopolysaccharide Stimulation in Murine Neuroblastoma Cell Line N1E‐115
Authors: Ota Akira , Yoshida Satoshi , Nomura Takahide , Matsui Shigeru , Hagino Yasumichi , Umezawa Kazuo , Katoh Setsuko , Nagatsu Toshiharu
Published: July 2010

Abstract: We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6R‐l‐erythro‐1′,2′‐dihydroxypropyl)‐2‐amino‐4‐hydroxy‐5,6,7,8‐tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E‐115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E‐115 cells with 1 µg/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity (Vmax) of GTP cyclohydrolase I, a rate‐limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP‐binding proteins into serum‐free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E‐115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopolysaccharide signal to tetrahydrobiopterin production, and (b) there may be a cross‐talk between GTP‐binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E‐115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.
Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State
Authors: Kotzyba‐Hibert F. , Kessler P. , Zerbib V. , Bogen C. , Snetkov V. , Takeda K. , Goeldner M. , Hirth C.
Published: July 2010

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4‐diazocyclohexa‐2,5‐dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch‐clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.
Cellular Distribution of 6‐Phosphofructo‐1‐Kinase Isoenzymes in Rat Brain
Authors: Zeitschel U. , Bigl M. , Eschrich K. , Bigl V.
Published: July 2010

Abstract: In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6‐phosphofructo‐1‐kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo‐ and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk‐isolated cells of rat brain by means of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M‐type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L‐type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C‐type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.
Radiolabeled Ligands Specific for the G Protein‐Coupled State of Neurotensin Receptors
Authors: Gaudriault Georges , Zsürger Nicole , Vincent Jean‐Pierre
Published: July 2010

Abstract: Radiolabeled analogues of neuromedin N have been prepared by acylation of the α, ε1, and ε2 amino groups of [Lys2]neuromedin N (Lys‐Lys‐Pro‐Tyr‐Ile‐Leu) either with the 125I‐labeled Bolton‐Hunter reagent or with N‐succinimidyl[2,3‐3H]propionate. The binding properties of the purified analogues toward newborn mouse brain homogenate or toward membranes of cells transitorily (COS) or permanently (AA1) transfected with the cloned rat brain neurotensin receptor cDNA were evaluated and compared with those of radiolabeled neurotensin. The α‐modified analogue of [Lys2]neuromedin N behaves exactly like neurotensin in these binding experiments, whereas the ε1‐ and ε2‐modified analogues selectively recognize the fraction of neurotensin binding sites that is sensitive to GTPγS. The proportion of neurotensin receptors coupled to GTP binding proteins is ∼50% in membranes of newborn mouse brain or of AA1 cells that respond to neurotensin by an increase of the intracellular inositol trisphosphate concentration. By contrast, membranes of transitorily transfected COS cells that do not respond to neurotensin exhibit very low levels of GTP‐sensitive receptors labeled with the ε1‐ or ε2‐modified analogues. These radiolabeled peptides offer new tools to selectively detect active neurotensin receptors.
Pharmacology and Regional Distribution of the Binding of 6‐[3H]Nitro‐7‐Sulphamoylbenzo[f]‐Quinoxaline‐2,3‐Dione to Rat Brain
Authors: Dev Kumlesh K. , Petersen Vibeke , Honoré Tage , Henley Jeremy M.
Published: July 2010

Abstract: 6‐Nitro‐7‐sulphamoylbenzo[f]quinoxaline‐2,3‐dione (NBQX) is a competitive antagonist selective for α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [3H]NBQX binding to rat brain. The association rate of [3H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off‐rate was also rapid, with near‐complete dissociation of the radioligand within 5 min of addition of 1 mM unlabelled l‐glutamate. [3H]NBQX bound to a single class of sites with KD and Bmax values of 47 nM and 2.6 pmol mg−1 of protein, respectively. The rank order of inhibition of [3H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) ≥ (S)‐5‐fluorowillardiine ≥ AMPA ≫l‐glutamate. The chaotrope KSCN had no effect on the IC50 value of unlabelled NBQX displacement of [3H]NBQX binding. The kainate receptor‐selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [3H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [3H]NBQX to coronal sections showed a distribution compatible with that of [3H]AMPA binding. These data indicate that [3H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.