bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Neuropeptide‐Processing Enzymes by Nitric Oxide in Cultured Astrocytes
Authors: Devi Lakshmi , Petanceska Suzana , Liu Roy , Arbabha Babak , Bansinath Mylarao , Garg Uttam
Published: July 2010

Abstract: Nitric oxide (NO), a recently discovered neuro‐transmitter, has been shown to have a cytostatic effect on cultured glia. A NO‐generating agent, S‐nitroso‐N‐acetyl‐penicillamine (SNAP), was used to treat C6 glioma and primary cortical astrocytes. The levels of a monobasic peptide‐processing enzyme activity and carboxypeptidase E activity were examined. The cellular levels of these two enzymes are specifically reduced in response to treatment with SNAP. A decrease of ˜30–50% in these two’enzyme activities was seen in both primary astrocytes and C6 glioma cells. This decrease in cellular enzyme activities is not due to increased secretion because the secreted activity is also reduced in response to SNAP treatment in both the glioma cells and the primary astrocytes. Removal of SNAP treatment causes the carboxypeptidase enzyme activity to return to control levels within 3 days. Northern and western blot analyses indicate that the reduced cellular level of carboxypeptidase E is not due to reduced expression of the messenger RNA or protein, suggesting that the SNAP treatment is affecting factors that influence carboxypeptidase E activity. Taken together, these results imply that NO is involved in the regulation of peptide biosynthetic enzymes and this could lead to the antimitogenic action of SNAP on glia.
Large Dense‐Core Vesicles in Rat Adrenal After Reserpine: Levels of mRNAs of Soluble and Membrane‐Bound: Constituents in Chromaffin and Ganglion Cells Indicate a Biosynthesis of Vesicles with Higher Secretory Quanta
Authors: Laslop Andrea , Mahata Sushil K. , Wolkersdorfer Martin , Mahata Manjula , Srivastava Meera , Seidah Nabil G. , Fischer‐Colbrie Reiner , Winkler Hans
Published: July 2010

Abstract:Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense‐core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense‐core vesicles (chromogranins A and B., secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215–857% after 1–3 days of drug treatment. For partly membrane‐bound components (dopamine β‐hydroxylase, prohormone convertase 2, carboxypeptidase H., and peptidylglycine α‐amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochromeb661and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y., secretogranin II, carboxypeptidase H., and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long‐lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.Key Words: ChromograninSecretogranin II—Monoamine transporter—Prohormone convertase 2—Carboxypeptidase H—Cytochromeb661‐Clusterin.
Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells
Authors: Wahl Philip , Elster Lisbeth , Schousboe Arne
Published: July 2010

Abstract:Poly(A)+mRNA was isolated from cultured mouse cerebellar granule cells and injected intoXenopusoocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current‐responses were also obtained after application of β‐alanine and taurine, but these were very low relative to that of glycine (maximal β‐alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K+‐evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloadedd‐[3H]aspartate evoked by 40 mMK+was dose dependently inhibited by glycine, and the concentration producing half‐maximal inhibition was 50 μM.Taurine, β‐alanine, and the specific GABAAreceptor agonist isoguvacine also inhibited K+‐evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC50value was 200 μMfor taurine, 70 μMfor β‐alanine, and 4 μMfor isoguvacine. Bicuculline (150 μM) antagonized the inhibitory effect of isoguvacine (150 μM) but not that of glycine (1 mM). In contrast, strychnine (20 μM) antagonized the inhibitory effect of glycine (1 mM) but not that of isoguvacine (150 μM). The pharmacology of the responses to β‐alanine and taurine showed that these agonists activate both glycine and GABAAreceptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.
Calcium‐Mediated Degeneration of the Axonal Cytoskeleton in the Ola Mouse
Authors: Glass Jonathan D. , Schryer Brenda L. , Griffin John W.
Published: July 2010

Abstract: The C57BL/Ola (Ola) mouse is a mutant substrain in which transected axons undergo very slow Wallerian degeneration. Because axonal degradation during Wallerian degeneration is calcium dependent, we tested whether Ola axons are susceptible to calcium‐mediated axonal degeneration by comparing neuro‐filament degradation between Ola and C57BL/6 mice in sciatic nerve explants. Using immunoblot analysis of neurofilament degradation and electron microscopy we found that as in normal axons, axonal degeneration in the Ola is calcium dependent. However, when compared with normal animals, higher levels of calcium were required for complete degradation of neurofilaments in Ola nerve, suggesting a relative insensitivity to calcium‐mediated degeneration in the Ola. We conclude that calcium‐activated proteases are present and active in Ola axons but that higher levels of calcium are required to accomplish complete axonal degradation. These results suggest a possible mechanism for prolonged survival of transected Ola axons and provide potential insight into the pathophysiology of axonal degeneration in injury and disease.
Stable Expression of the Cloned Rat Brain Neurotensin Receptor into Fibroblasts: Binding Properties, Photoaffinity Labeling, Transduction Mechanisms, and Internalization
Authors: Chabry Joëlle , Labbé‐Jullié Catherine , Gully Danielle , Kitabgi Patrick , Vincent Jean‐Pierre , Mazella Jean
Published: July 2010

Abstract: The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration‐dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly undissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature‐dependent mechanism. Indeed, the sequestration of the neurotensin‐receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.
Organization, Sequence, and Expression of a Gene Encoding Goldfish Neurofilament Medium Protein
Authors: Glasgow Eric , Hall Christina M. , Schechter Nisson
Published: July 2010

Abstract: The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF‐M) protein. The tissue‐specific expression of goldfish NF‐M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF‐M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF‐M protein is similar to higher vertebrate NF‐M proteins. In addition, the organization of the goldfish NF‐M gene is identical to the NF‐M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF‐M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF‐M genes.
Characterization of the Functional Activity of Dopamine Ligands at Human Recombinant Dopamine D4 Receptors
Authors: Chabert Christian , Cavegn Catherine , Bernard Alain , Mills Ann
Published: July 2010

Abstract: The human D4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [35S]GTPγS binding assay. By measuring D4 receptor stimulation of [35S]‐GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D4 receptor, dopamine, quinpirole, and dp‐2‐aminodihydroxy‐1,2,3,4‐tetrahydronaphthalene were all full agonists, whereas (−)‐apomorphine appeared to be a partial agonist. No increase in [35S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole‐stimulated [35S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [3H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein‐coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.
Melatonin Receptor‐Mediated Inhibition of Cyclic AMP Accumulation in Chick Retinal Cell Cultures
Authors: Iuvone P. Michael , Gan Jiwei
Published: July 2010

Abstract: Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high‐affinity 2‐[125I]iodomelatonin binding sites (KD = 41.6 pM), similar to those in intact retina. The effects of melatonin and related indoles on cyclic AMP accumulation were examined. Melatonin (10−7M) had no effect on basal or K+‐stimulated cyclic AMP accumulation, but inhibited forskolin‐stimulated cyclic AMP accumulation by approximately 50%. Melatonin inhibited forskolin‐stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation. Half‐maximal inhibition was observed at 5.9 × 10−10M melatonin. The relative order of potency among melatonin analogues was 2‐iodomelatonin > melatonin ≈ 6‐chloromelatonin ≥ 6‐hydroxymelatonin > N‐acetylserotonin ≈ 5‐methoxytryptophol > serotonin. The EC50 value for inhibition of cyclic AMP accumulation by 2‐iodomelatonin (36.7 pM) was comparable to the KD value for binding of the radioligand, suggesting that the binding sites represent functional receptors. The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N‐acetyltryptamine, and N‐(2,4‐dinitrophenyl)‐5‐methoxytryptamine, with estimated KB values of 0.12, 0.17, and 1 µM, respectively. At a concentration of 10 µM, N‐(2,4‐dinitrophenyl)‐5‐methoxytryptamine significantly inhibited forskolin‐stimulated cyclic AMP accumulation when added alone; at 30 µM, luzindole and N‐acetyltryptamine also had significant inhibitory effects. The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin. The results of this study indicate that melatonin receptors on retinal cells are coupled via inhibitory G proteins to cyclic AMP accumulation. Thus, some of the effects of melatonin on retinal physiology may be related to regulation of cyclic nucleotide metabolism.
Different Modulation of Protein Kinase C Isozymes in Dibutyryl Cyclic AMP‐Differentiated PC12h Cells
Authors: Uehara‐Kunugi Yukiko , Shimohama Shun , Kobayakawa Hitoshi , Tamura Hitomi , Taniguchi Takashi
Published: July 2010

Abstract: PC12h cells can be differentiated into sympathetic neuron‐like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP‐treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.
4‐Bromohomoibotenic Acid Selectively Activates a 1‐Aminocyclopentane‐1S,3R‐Dicarboxylic Acid‐Insensitive Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis in Rat Cortical Slices
Authors: Chung Dorothy S. , Winder Danny G. , Conn P. Jeffrey
Published: July 2010

Abstract: Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (1S,3R‐ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue (R,S)‐4‐bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration‐dependent manner (EC50 = 190 µM). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1S,3R‐ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to l‐2‐amino‐3‐phosphonopropionic acid (l‐AP3), whereas the response to 1S,3R‐ACPD is partially blocked by l‐AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis‐linked mGluR that is insensitive to 1S,3R‐ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.
Detection of Superoxide Production by Activated Microglia Using a Sensitive and Specific Chemiluminescence Assay and Microglia‐Mediated PC12h Cell Death
Authors: Tanaka M. , Sotomatsu A. , Yoshida T. , Hirai S. , Nishida A.
Published: July 2010

Abstract: Superoxide production by cultured microglia derived from neonatal rat brains and the cytotoxicity of these cells were evaluated. The chemiluminescence (photon counts) detected in the presence of MCLA, a new chemiluminescence probe, was strongly correlated with the microglial cell count. Chemiluminescence observed in this system was confirmed to originate specifically from superoxide produced by activated microglia. Phorbol myristate acetate‐stimulated microglia caused a pronounced reduction of PC12h cell numbers in coculture. The addition of superoxide dismutase with catalase or the addition of deferoxamine mesylate inhibited PC12h cell death, suggesting that active oxygen species derived from superoxide generated by the microglia or iron‐oxygen complex formation were responsible for the cytotoxicity. These results imply that activated microglia may participate in the progression of the pathologic process in some neurodegenerative disorders.
Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6‐Hydroxydopamine
Authors: Unnerstall James R. , Ladner Allison
Published: July 2010

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6‐hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 mM 6‐methyl‐5,6,7,8‐tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 mM 6‐methyl‐5,6,7,8‐tetrahydropterin), tyrosine hydroxylase activity in 2‐ and 12‐month‐old lesioned animals was twice that measured in vehicle‐treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age‐matched vehicle‐treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two‐dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2‐month‐old lesioned animals was greater than that observed in vehicle‐treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24‐month‐old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24‐month‐old Fischer 344 rats due to a deficit in stimulus‐evoked enzyme phosphorylation.
Early Effects of β,β′‐Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon
Authors: Tashiro Tomoko , Imai Reiko , Komiya Yoshiaki
Published: July 2010

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β′‐iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l‐[35S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold‐insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold‐soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200‐kDa neurofilament protein was detected in the ventral root using phosphorylation‐dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.
The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum
Authors: Khan Zafar U. , Gutierrez Antonia , De Blas Angel L.
Published: July 2010

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABAA receptors that show diazepam (or clonazepam)‐insensitive [3H]Ro 15‐4513 binding has been determined by immunoprecipitation with subunit‐specific antibodies. These receptors have α6, α1, γ2S, γ2L, and β2 or β3 subunits colocalizing in the same receptor complex.
Preferential Stimulation of Extracellular Release of Dopamine in Rat Frontal Cortex to Striatum Following Competitive Inhibition of the N‐Methyl‐d‐Aspartate Receptor
Authors: Nishijima Koichi , Kashiwa Atsushi , Nishikawa Toru
Published: July 2010

Abstract: Using a brain microdialysis technique, we have shown in the rat that local infusion of a selective and competitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist, cis‐4‐phosphonomethyl‐2‐piperidine carboxylic acid (CGS‐19755), into the medial frontal cortex via dialysis tubing caused a concentration‐related increase in the extracellular release of dopamine, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid in the cortical region. Coinfusion of a sodium channel blocker, tetrodotoxin, completely inhibited the ability of the NMDA antagonist to augment frontal dopamine metabolism. These findings suggest that dopamine neurons projecting to the frontal cortex might be under a tonic transsynaptic inhibition exerted by excitatory amino acid neurotransmission via the NMDA receptor at the level of dopamine terminal fields.
Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide mRNAs by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons
Authors: Lewis Susan E. , Rao Mahendra S. , Symes Aviva J. , Dauer William T. , Fink J. Stephen , Landis Story C. , Hyman Steven E.
Published: July 2010

Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine‐mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.
Mutations in the unc‐41 Gene Cause Elevation of Acetylcholine Levels
Authors: Harada S. , Hori I. , Yamamoto H. , Hosono R.
Published: July 2010

Abstract: Mutations in the Caenorhabditis elegans unc‐41 gene result in an allele‐dependent elevation of acetylcholine content. Eight recessive alleles (cn252, e268, e399, e650, e1175, e1199, e1294, and e870) lead to phenotypes including uncoordinated locomotion, slow growth, a small mature body, and resistance to the acetylcholinesterase inhibitors as well as the elevation of acetylcholine content. The remaining two alleles, e554 and e1162, exhibit normal acetylcholine levels but display the short‐body phenotype in a semidominant way. To determine the localization of the elevated acetylcholine content, a method for the isolation of synaptic vesicles from C. elegans was established. The elevation of acetylcholine content in the unc‐41 mutants is accompanied by the accumulation of synaptic vesicles. We propose that at least one function of the unc‐41 gene relates to the release of neurotransmitters.
Expression of Non‐NMDA Glutamate Receptor Channel Genes by Clonal Human Neurons
Authors: Hardy Mattie , Younkin Donald , Tang Cha‐Min , Pleasure Jeanette , Shi Qing‐Yao , Williams Margaret , Pleasure David
Published: July 2010

Abstract: Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N‐methyl‐d‐aspartate (NMDA) and non‐NMDA glutamate receptor channels. We now show by reverse transcriptase‐polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non‐NMDA glutamate receptor genes (GluR1‐7, Ka‐1, and Ka‐2) and that four of these genes (GluR2, GluR6, GluR7, and Ka‐1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non‐NMDA glutamate receptor channels are readily isolated from NT2‐derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2‐derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate. We expect that the NT2‐derived human neuronal culture system will facilitate studies of human neuronal non‐NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.
Synergistic Activation of DNA Synthesis in Astrocytes by Fibroblast Growth Factors and Extracellular ATP
Authors: Neary J. T. , Whittemore S. R. , Zhu Q. , Norenberg M. D.
Published: July 2010

Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet‐derived growth factor or epidermal growth factor. ATP appears to act via P2 purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis‐resistant P2 agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.
Oligodendroglial Signal Transduction Systems Are Developmentally Regulated
Authors: He Minghua , McCarthy Ken D.
Published: July 2010

Abstract: Studies from several laboratories indicate that oligodendroglia exhibit signal transduction systems that can be activated by classical neurotransmitters. Previous studies from this laboratory indicate that oligodendroglia express neuroligand receptors linked to the regulation of Ca2+i. Experiments presented in this article were designed to determine if developmental processes that influence the ability of oligodendroglia to respond to neuroligands with an increase in Ca2+i proceed either in vitro or in vivo. Findings support the view that developmental processes markedly affected the sensitivity of these cells to both purinergic and cholinergic receptor agonists, whereas their responsiveness to either histamine or bradykinin appeared relatively stable over time. Approximately 90 and 75% of oligodendroglia responded to ATP or carbachol, respectively, after 4 days in vitro, whereas <10% of these cells responded to either of these neuroligands after 8 days in vitro. The decrease in the percentage of oligodendroglia responding to ATP, but not carbachol, could be prevented by including dibutyryl cyclic AMP in the culture medium during the final 4 days in vitro. However, once the loss in responsiveness to ATP had occurred, it could not be reversed by exposure to dibutyryl cyclic AMP. Developmental changes in the ATP sensitivity of oligodendroglia occurred in cells expressing galactocerebroside and myelin basic protein. The neuroligand sensitivity of oligodendroglia isolated from either neonatal, 2‐, 3‐, or 5‐week‐old spinal cord was examined to determine if developmental changes in oligodendroglial Ca2+ regulation occurred in vivo. The results of these experiments indicate that the percentage of oligodendroglia responding to either ATP or carbachol markedly decreased as a function of the age of the animal used to prepare the cultures; this was not the case for the stimulation of Ca2+i by histamine. The decreased sensitivity of oligodendroglia isolated from older animals could not be reversed through the addition of dibutyryl cyclic AMP. Overall, the results of these experiments indicate that developmental processes selectively influence the sensitivity of oligodendroglia to specific neuroligands and suggest that oligodendroglial processes unrelated to myelin formation may be regulated by neuroligands in vivo.
Expression of Neurotrophins and Their Receptors in Primary Astroglial Cultures: Induction by Cyclic AMP‐Elevating Agents
Authors: Condorelli D. F. , Dell'Albani P. , Mudò G. , Timmusk T. , Belluardo N.
Published: July 2010

Abstract: By northern blot analysis and ribonuclease protection assay, we observed the presence of a high level of trkB mRNA in primary brain cultures devoid of neuronal cells and highly enriched in glial fibrillary acidic protein‐positive astroglial cells prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum. In primary astroglial cultures, the more abundant trkB transcripts code for the truncated receptor without tyrosine kinase activity; probes specific for the full‐length trkB mRNA did not detect any signal in northern blot analysis. By the sensitive ribonuclease protection assay, we could show the presence of trkC mRNA in cultured astrocytes, whereas no trkA mRNA was detected. We confirmed the presence of relatively high levels of nerve growth factor and neurotrophin‐3 mRNA, and very low basal level of brain‐derived neurotrophic factor mRNA. Moreover, we demonstrated that another member of the neurotrophin family, neurotrophin‐4, is also expressed in cultured astroglial cells. In view of the fact that many functional receptors for conventional neurotransmitters or neuropeptides present on astroglial cells may act via the adenylate cyclase system, we studied also the effect of agents able to increase the intracellular cyclic AMP concentration. A sharp increase in the trkB mRNA level was observed after treatment of primary astroglial cultures with dibutyryl cyclic AMP, 8‐bromo‐cyclic AMP, or the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine. On the contrary, trkC mRNA levels were unaffected by treatment with cyclic AMP‐elevating agents. All the neurotrophin mRNAs examined, except neurotrophin‐4, were increased by 3‐isobutyl‐1‐methylxanthine treatment. Therefore, in cultured astroglial cells, gene expression of neurotrophins and trkB is regulated by activation of the cyclic AMP‐second messenger system. This process may take part in the neuronal‐glial interactions during the normal neuronal activity or after injury events.
Selective Potentiation of DNA Binding Activities of Both Activator Protein 1 and Cyclic AMP Response Element Binding Protein Through In Vivo Activation of N‐Methyl‐d‐Aspartate Receptor Complex in Mouse Brain
Authors: Ogita Kiyokazu , Yoneda Yukio
Published: July 2010

Abstract: DNA binding activities of several transcription factors were evaluated in nuclear extracts from brains of mice that were injected intracerebroventricularly with N‐methyl‐d‐aspartic acid (NMDA) using gel‐shift assays. An injection of saline transiently increased binding of both probes for activator protein 1 (AP1) and cyclic AMP response element binding protein (CREB) 30 min after the injection, and NMDA was effective in inducing a more potent increment of binding of both probes 1–5 h after the injection than did saline. However, no significant alterations were found in binding of probes for other transcription factors tested up to 4 h following the injection of NMDA. The potentiation by NMDA was prevented in a dose‐dependent manner by administration of the non‐competitive NMDA antagonist (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine, the NMDA antagonist d,l‐(E)‐2‐amino‐4‐propyl‐5‐phosphono‐3‐pentenoic acid, and the glycine antagonist 5,7‐dichlorokynurenic acid, whereas administration of the proposed polyamine antagonist ifenprodil was rather ineffective in protecting against the potentiation by NMDA. These results support the proposal that an intracerebroventricular injection of NMDA may selectively potentiate DNA binding activities of both AP1 and CREB through activation of the NMDA receptor complex in mouse brain.
Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes
Authors: Dauphin François , Linville Donald G. , Hamel Edith
Published: July 2010

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration‐dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP3) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10−3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10−5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol‐induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP3 accumulation induced by 10−7M (endothelium‐dependent submaximal effect) and 10−4M (endothelium‐independent near‐maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP3 production by 10−4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in ‐log IC50): 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP; 8.65) > pirenzepine (8.28) > 6‐chloro‐5,10‐dihydro‐5‐[(1‐methyl‐4‐piperidinyl)acetyl]‐11H‐dibenzo[b,e][1,4]diazepine‐11‐one (UH‐AH 371; 7.87) > 11‐[[2‐[(diethylamino)methyl]‐1‐piperidinyl]acetyl]‐5,‐11‐dihydro‐6H‐pyridol[2,3‐b][1,4]benzodiazepine‐6‐one (AF‐DX 116; 6.62), a pharmacological profile compatible with an M1 receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10−7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in ‐log IC50): 4‐DAMP (8.38) > pirenzepine (7.25) > UH‐AH 371 (6.25) > AF‐DX 116 (5.72), which was reminiscent of an M3 pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M1 and M3 subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M3 dilatatory receptors within the vessel wall.
Characterization of Nicotine‐Induced Desensitization of Evoked Dopamine Release from Rat Striatal Synaptosomes
Authors: Rowell Peter P. , Hillebrand Julie A.
Published: July 2010

Abstract: Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [3H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of l‐nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [3H]dopamine release. The IC50 for nicotine‐induced desensitization was found to be 12 nM with a maximum inhibition of >90% at 300 nM. Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 mM K+. The onset of nicotine‐induced desensitization occurred with a t1/2 of 43 s at 30 nM nicotine. The temperature dependence of onset yielded a Q10 of 1.2.Recovery from desensitization was slower (t1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first‐order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine‐induced neurotransmitter release.
In Vitro and In Vivo Expression of Opioid and σ Receptors in Rat C6 Glioma and Mouse N18TG2 Neuroblastoma Cells
Authors: Barg Jacob , Thomas Gail E. , Bem Wojciech T. , Parnes Michael D. , Ho Andrew M. , Belcheva Mariana M. , McHale Robert J. , McLachlan Julie A. , Tolman Kym C. , Johnson Frank E. , Coscia Carmine J.
Published: July 2010

Abstract: Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for δ opioid binding using [3H][d‐Ala2,d‐Leu5]enkephalin and for σ1 and σ2 binding with 1,3‐[3H]di‐o‐tolylguanidine in the presence and absence of 1 µM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and σBmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/σ ligands were similar in vitro and in vivo. With successive passages in the murine host, δ opioid and σ1 binding of the neuroblastoma‐derived solid tumors became undetectable. In contrast, σ2 receptor Bmax values were unchanged with successive passages of the neuroblastoma‐derived tumors and doubled in the nude mouse‐borne gliomas. When neuroblastoma‐derived solid tumors that were devoid of δ opioid binding were returned to culture, opioid receptors appeared to be up‐regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for σ binding was more complex, with the σ2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of σ sites in transformed human meninges, kidney, and colon tissue.
Enhanced Aspartate Release Related to Epilepsy in (EL) Mice
Authors: Flavin Helen J. , Seyfried Thomas N.
Published: July 2010

Abstract: Previous studies have shown that potassium‐evoked, calcium‐dependent, endogenous aspartate release is greater from hippocampal slices of adult epileptic (EL) mice than from nonepileptic control C57BL/6J (B6) mice. To examine further the association between epilepsy and enhanced aspartate release in EL mice, endogenous neurotransmitter release from hippocampal slices was studied in young, seizure‐free EL mice and in two nonseizure control mouse strains, DDY and B6. DDY is the parental strain from which EL arose, and it has a genetic background similar to EL. Released amino acid neurotransmitters were quantitated by HPLC with fluorescent detection and were expressed as picomoles of amino acid released per minute of incubation per slice ± SEM. Aspartate release was significantly higher in EL mice (15.8 ± 0.8) than in either the control B6 or DDY mice (8.5 ± 1.4 and 8.4 ± 1.7, respectively). No significant differences were found among the B6, DDY, and EL mice for the release of glutamate (23.0 ± 2.0, 32.3 ± 5.8, and 25.9 ± 2.6, respectively) or GABA (23.5 ± 0.7, 19.5 ± 3.2, and 21.8 ± 3.2, respectively). Thus, enhanced aspartate release precedes the onset of EL seizures and may be related to the cause rather than to the effects of seizure activity.
Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes
Authors: Mokuno Kenji , Ohtani Kunihiko , Suzumura Akio , Kiyosawa Kazuhiro , Hirose Yoshikiyo , Kawai Kuniyuki , Kato Kanefusa
Published: July 2010

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 102 to 104 U/ml tumor necrosis factor‐α for 3 days increased the levels of Mn SOD in a dose‐ and time‐dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3‐day exposure to interleukin‐1α at concentrations of 102 or 103 U/ml. However, these stimuli had no effect on levels of copper‐zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon‐γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.
Agonist Binding to α2‐Adrenoceptors Is Elevated in the Locus Coeruleus from Victims of Suicide
Authors: Ordway Gregory A. , Widdowson Peter S. , Smith Karen Streator , Halaris Angelos
Published: July 2010

Abstract: The binding of an agonist, p‐[125I]iodoclonidine, and an antagonist, [3H]yohimbine, to α2‐adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant‐free victims of suicide and age‐matched controls. Agonist binding to α2‐adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α2‐adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α2‐adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high‐affinity state of autoinhibitory α2‐adrenoceptors with suicide.
Effects of 1‐Methyl‐4‐Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion
Authors: Bates T. E. , Heales S. J. R. , Davies S. E. C. , Boakye P. , Clark J. B.
Published: July 2010

Abstract: The effects of 1‐methyl‐4‐phenylpyridinium (MPP+) on the oxygen consumption, ATP production, H2O2 production, and mitochondrial NADH‐CoQ1 reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µM MPP+ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP+ produced a concentration‐dependent decrease in H2O2 production. Incubation of mitochondria with ADP for 60 min after loading with 100 µM MPP+ caused no loss of complex I activity after washing of MPP+ from the mitochondrial membranes. These data are consistent with MPP+ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H2O2 by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H2O2 in the presence of Cu2+ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP+ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.
Studies on the Characterization of the Inhibitory Mechanism of 4′‐Alkylated 1‐Methyl‐4‐Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles
Authors: Gluck Martin R. , Youngster Stephen K. , Ramsay Rona R. , Singer Thomas P. , Nicklas William J.
Published: July 2010

Abstract: 1‐Methyl‐4‐phenylpyridinium (MPP+), the toxic agent in MPTP‐induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′‐alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4′‐alkylated phenylpyridine derivatives to inhibit NADH‐linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1‐octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ∼600‐fold, versus only a 36‐fold, increase in the IC50 of MPP+ versus 4′‐pentyl‐MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′‐decyl‐MPP+ was nearly 80‐fold more potent than phenylpyridine although the latter compound partitions twice as much into 1‐octanol. In addition, the lipophilic anion TPB− was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.