bluuebunny/crossref_metadata_2025 huggingface dataset:
Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia
Authors: Yamashita Kimihiro , Niwa Masami , Kataoka Yasufumi , Shigematsu Kazuto , Himeno Akihiko , Tsutsumi Keisuke , Nakano‐Nakashima Mihoko , Sakurai‐Yamashita Yasuko , Shibata Shigeki , Taniyama Kohtaro
Published: July 2010

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke‐prone spontaneously hypertensive rats subjected to a 10‐min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of 125I‐ET‐1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ETB receptor. The de novo 125I‐ET‐1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET‐1‐ and ET‐3‐like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ETB receptor are activated to participate in the pathophysiology of ischemia‐related neural tissue damage by astrocytic ET‐1 and ET‐3 produced in response to transient forebrain ischemia would have to be considered.
The [3H]Tryptamine Receptor in Human Brain: Kinetics, Distribution, and Pharmacologic Profile
Authors: Mousseau Darrell D. , Butterworth Roger F.
Published: July 2010

Abstract: The kinetics and distribution of [3H]tryptamine binding sites in human brain were investigated. Specific [3H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single‐site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [3H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na+, K+, Ca2+, Mg2+, Cu2+) indicate stereospecific displacement as well as a rank‐order potency profile that is different from that reported for the rat [3H]tryptamine binding site. This suggests the presence of distinct species‐dependent [3H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine‐mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [3H]‐tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [3H]‐tryptamine in human brain.
Subcellular Distribution of Calpain and Calpastatin Immunoreactivity and Fodrin Proteolysis in Rabbit Hippocampus After Hypoxia and Glucocorticoid Treatment
Authors: Ostwald Klas , Hayashi Masami , Nakamura Megumi , Kawashima Seiichi
Published: July 2010

Abstract: Rabbits were subjected to hypoxia (5% O2) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for μ‐calpain, m‐calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short‐term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta‐methasone, 0.4 mg × kg−1× day−1) for 7 days produced a decrease in the ratio of activated μ‐calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short‐term recovery, indicating impairment of calpain activation.
Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury
Authors: Renuka Prasad M. , Ramaiah C. , McIntosh T. K. , Dempsey R. J. , Hipkens S. , Yurek D.
Published: July 2010

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury (p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site (p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury (p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury (p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.
Binding of γ‐Aminobutyric AcidA Receptors to Tubulin
Authors: Item Chike , Sieghart Werner
Published: July 2010

Abstract: Tubulin cofractionated with γ‐aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7‐1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate‐gel electrophoresis of affinity‐purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.
Fenfluramine Releases Serotonin from Human Brain Nerve Endings by a Dual Mechanism
Authors: Bonanno Giambattista , Fassio Anna , Severi Paolo , Ruelle Antonio , Raiteri Maurizio
Published: July 2010

Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d‐fenfluramine has been shown to act as a potent releaser of brain serotonin [5‐hydroxytryptamine (5‐HT)]. Here we have investigated the effects of d‐fenfluramine on the release of [3H]5‐HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [3H]5‐HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d‐fenfluramine used. At 5 µM, the release of [3H]5‐HT was blocked by the 5‐HT transporter inhibitor fluoxetine and was Ca2+ independent and insensitive to the human autoreceptor 5‐HT1D agonist sumatriptan. The release of [3H]5‐HT elicited by 0.5 µMd‐fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca2+ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d‐fenfluramine largely diffuses into serotonergic terminals and causes release of 5‐HT through the 5‐HT carrier working in the inside‐outside direction; at relatively low concentrations d‐fenfluramine enters the terminals through the 5‐HT transporter but elicits release of 5‐HT by an exocytotic‐like mechanism.
Small N‐Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function
Authors: Korpi E. R. , Kuner T. , Kristo P. , Köhler M. , Herb A. , Lüddens H. , Seeburg P. H.
Published: July 2010

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ‐aminobutyric acid type A (GABAA) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)‐amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single‐stranded phage plaques hybridized specifically to the short (α6S) and long (wild‐type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30‐nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABAA receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15‐4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA‐induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30‐nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit‐containing receptors.
N‐Acetylaspartylglutamate Selectively Inhibits Neuronal Responses to N‐Methyl‐d‐Aspartic Acid In Vitro
Authors: Burlina Alessandro P. , Skaper Stephen D. , Mazza Maria Rosaria , Ferrari Vanni , Leon Alberta , Burlina Alberto B.
Published: July 2010

Abstract: Canavan's disease is an autosomal recessive disorder characterized by a deficiency of aspartoacylase and accumulation of N‐acetylaspartic acid (NAA), leading to a severe leukodystrophy and spongy degeneration of the brain. N‐Acetylaspartylglutamate (NAAG), the presumed product of NAA, also accumulates in this disease. The endogenous dipeptide NAAG has been suggested to have low potency at NMDA receptors. Here we have tested the actions of NAAG and NAA on NMDA‐evoked responses in cultured cerebellar granule cells. In differentiating granule cells grown in low‐K+ medium, NAAG negated the survival‐promoting effects of NMDA but not K+ depolarization. Neither NAAG nor NAA alone promoted cell survival in low‐K+ medium. The modest trophic action of 50 µM kainic acid in low‐K+ medium was reinforced by the NMDA receptor antagonist dizocilpine maleate and by NAAG. In K+‐differentiated granule cells, NAAG raised the threshold of NMDA neurotoxicity but not that of kainate. The observed activities of NAAG were overcome by excess NMDA and were not mimicked by NAA. These data raise the possibility that disruption of NMDA receptor processes by NAAG may be of pathophysiological relevance.
Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus
Authors: Rocha Elizabete , Rodnight Richard
Published: July 2010

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li+ in serum reached 0.6–1.2 mM, a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [32P]‐phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium‐treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more 32P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.
Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells
Authors: Ng Gordon Y. K. , O'Dowd Brian F. , Caron Mirelle , Dennis Michael , Brann Mark R. , George Susan R.
Published: July 2010

Abstract: We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor‐specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin‐solubilized preparation of cells expressing D2L receptor prelabeled with 32Pi or [3H]‐palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.
Agonist and Cyclic AMP‐Mediated Regulation of β1‐Adrenergic Receptor mRNA and Gene Transcription in Rat C6 Glioma Cells
Authors: Hosoda Kohkichi , Feussner Gretchen K. , Rydelek‐Fitzgerald Laura , Fishman Peter H. , Duman Ronald S.
Published: July 2010

Abstract: Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down‐regulation of β1‐adrenergic receptors (β1AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of β1AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered β1AR mRNA levels in a biphasic manner; i.e., short‐term exposure (30–60 min) increased by 50%, whereas longer exposure (2–6 h) decreased by 50% the levels of β1AR mRNA. The extent of both the up‐ and down‐regulation was dependent on agonist concentration. Similar regulation of β1AR mRNA was observed in forskolin‐treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol‐ and forskolin‐mediated down‐regulation of β1AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6‐h time course. The mechanisms underlying β1AR mRNA down‐regulation were examined. The half‐life of β1AR mRNA was slightly increased (from 61 to 77 min) after a 2‐h exposure of the cells to either isoproterenol or forskolin. Nuclear run‐on analysis demonstrated that the rate of β1AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90–240 min, consistent with the time course for up‐ and down‐regulation of β1AR mRNA. Isoproterenol treatment (120 min) also decreased the level of β1AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4‐thiouridine‐pulsed cells. The results demonstrate that β1AR mRNA has a relatively short half‐life and that agonist regulation of β1AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of β1AR mRNA occurs at the level of β1AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that β1AR mRNA down‐regulation may be mediated by the induction of a repressor of the β1AR gene.
The Calcium Content of Mitochondria from Brain Subregions Following Short‐Term Forebrain Ischemia and Recirculation in the Rat
Authors: Zaidan Emad , Sims Neil R.
Published: July 2010

Abstract: A procedure was established for determining the calcium content of mitochondria isolated from rat brain subregions based on changes in fura‐2 fluorescence after disruption of the organelles with Triton X‐100 and sodium dodecyl sulfate. Mitochondria isolated from the forebrain of normal rats contained 2.5 ± 0.9 nmol of calcium/mg of protein. A 30‐min ischemic period produced an approximately twofold increase in the calcium content of mitochondria isolated from the dorsolateral striatum, a region in which most neurons die within 24 h after this period of ischemia. The calcium content of mitochondria from the paramedian cortex, a region in which there are few ischemia‐susceptible neurons, tended to be similarly increased, although this difference was not statistically significant. Larger increases (to approximately five times control values) were seen in mitochondria isolated from both regions after 10 min of recirculation. By 1 h of recirculation, mitochondrial calcium had returned close to preischemic control values in both regions. Longer recirculation periods produced no further changes in the calcium content of mitochondria from the paramedian cortex. However, mitochondrial calcium was again increased in the dorsolateral striatum after 6 h (6.5 nmol of calcium/mg of protein) and 24 h (8.7 nmol of calcium/mg of protein) of recirculation. This regionally selective increase in calcium in the dorsolateral striatum preceded the period during which the majority of neurons in this region exhibit advanced degenerative changes. Thus, this increase may be an essential step, albeit a late one, in the development of neuronal loss.
Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes
Authors: Hayashi Teruo , Kagaya Ariyuki , Motohashi Nobutaka , Yamawaki Shigeto
Published: July 2010

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose‐dependent Ca2+ influx, which was reduced to 14% by the absence of external Ca2+. Dantrolene inhibited the C48/80‐induced increase in Ca2+ permeability of plasma membranes in a concentration‐dependent manner (0.33–10 µM, IC50 value was 5 µM). We next examined C48/80‐induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h0/h−1 was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration‐dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µM) itself did not change the membrane fluidity, but it significantly reduced the C48/80‐induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80‐induced increase in fluidity was dependent on extracellular Ca2+. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80‐induced membrane perturbation, which causes Ca2+ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.
Ethanol‐Induced Oxygen Radical Formation and Lipid Peroxidation in Rat Brain: Effect of Chronic Alcohol Consumption
Authors: Montoliu Carmina , Vallés Soraya , Renau‐Piqueras Jaime , Guerri Consuelo
Published: July 2010

Abstract: The effect of chronic and in vitro ethanol exposure on brain oxygen radical formation and lipid peroxidation was analyzed. Ethanol induces a dose‐dependent increase in lipid peroxidation in brain homogenates. The peroxidative effects of alcohol seem to be related to both cytochrome P450 and the ethanol‐inducible form of cytochrome P450 (CYP2E1), because preincubation with metyrapone (an inhibitor of cytochrome P450) or with an antibody against CYP2E1 abolished the ethanol‐increased lipid peroxidation. Using the formation of dichlorofluorescein, we also demonstrated that both in vitro and chronic alcohol exposure significantly enhanced the formation of oxygen radical species in synaptosomes. Chronic alcohol treatment also leads to an induction of cytochrome P450 (230%), NADPH cytochrome c reductase (180%), NADPH oxidation (184%), and CYP2E1 in brain microsomes. In addition, this treatment produced a decrease in the GSH/GSSG ratio in brain and significantly enhanced the levels of superoxide dismutase and catalase activities. This mechanism could be involved in the toxic effects of ethanol on brain and membrane alterations occurring after chronic ethanol intake.
Membrane Clustering and Bungarotoxin Binding by the Nicotinic Acetylcholine Receptor: Role of the β Subunit
Authors: Wheeler Susan V. , Jane Steven D. , Cross Kathryn M. L. , Chad John E. , Foreman Richard C.
Published: July 2010

Abstract: Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the postsynaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43‐kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43‐kDa protein and to compare them with hybrid muscle nAChRs containing a β subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit α1β1γδ and 43‐kDa protein transcripts produced discrete receptor aggregates with a diameter of 1–5 µm whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the β1 subunit by the neuronal β2 protein produced a functioning receptor that did not aggregate in the presence of 43‐kDa protein and was substantially blocked by the same concentration of NBT. Hybrid α1β4γδ receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43‐kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing β2. These results suggest that the β subunit is an important determinant in receptor localisation and sensitivity to NBT.
Glial Fibrillary Acidic Protein Transcriptional Regulation Is Independent of a TFIID‐Binding Downstream Initiator Sequence
Authors: Hatch Nan , Sarid Jacob
Published: July 2010

Abstract: Previous studies have shown that the promoter of the glial fibrillary acidic protein (GFAP) gene contains a transcriptional initiator located downstream from the transcription initiation (cap) site. This initiator was shown to be essential for efficient in vitro TATA box binding and function of TATA box‐binding factor (TFIID); its deletion significantly reduced in vitro template transcription by the GFAP promoter and inhibited almost completely expression of a reporter gene under control of human GFAP promoter and upstream sequences in glial cells. However, although activity of initiator‐containing GFAP promoter in human and murine GFAP‐reporter constructs in transfected cells was shown to increase sharply when upstream cis‐acting elements are added, initiator‐lacking murine constructs have shown a small GFAP promoter region to have high activity in glioma cells, with no increase in activity when regulatory sequences are extended further upstream. These findings suggested a possible difference between the in vitro and in vivo effects of the downstream initiator on the promoter region itself, as well as an in vivo interplay between the initiator and regulatory sequences upstream from the promoter region. Here we have analyzed the effect of the downstream initiator on gene expression in cells using matched (pairs of otherwise identical initiator‐containing and initiator‐deleted plasmids) constructs extending to different upstream positions. We show that matched initiator‐containing and initiator‐deleted counterparts direct similar expression levels, and that, with or without the downstream initiator, expression levels increase sharply when upstream sequences are added to the GFAP promoter. Our results show that the downstream initiator is not required for GFAP transcriptional activity in cultured cells. Thus, it is possible that the initiator sequences are required in vitro to compensate for an absence or lack of function of GFAP transcriptional machinery component(s) that are fully active in vivo.
A Carboxyl Terminal Leucine Zipper Is Required for Tyrosine Hydroxylase Tetramer Formation
Authors: Vrana Kent E. , Walker Stephen J. , Rucker Paul , Liu Xuan
Published: July 2010

Abstract: Tyrosine hydroxylase catalyzes the rate‐limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C‐terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C‐terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu → Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.
Tryptamine Induces Phosphoinositide Turnover and Modulates Adrenergic and Muscarinic Cholinergic Receptor Function in Cultured Cerebellar Granule Cells
Authors: Ishitani Ryoichi , Kimura Mitsutoshi , Takeichi Masami , Chuang De‐Maw
Published: July 2010

Abstract: Tryptamine dose‐dependently increased phosphoinositide (PI) hydrolysis by approximately fourfold in primary cultures of rat cerebellar granule cells (EC50 = 56 µM). The PI response stimulated by tryptamine was dependent on the presence of extracellular Ca2+ and Na+. Tryptamine‐induced PI breakdown could be partially inhibited by pretreatment with 4β‐phorbol 12‐myristate 13‐acetate but not pertussis toxin. The presence of tryptamine markedly attenuated PI responses induced by norepinephrine (NE) and carbachol, with no apparent effect on the responses to 5‐hydroxytryptamine and glutamate. The inhibition of NE‐ and carbachol‐induced PI turnover by tryptamine was dose dependent with IC50 values of ∼0.4 and ∼2.5 mM, respectively. Pretreatment of cells with tryptamine (0.5 mM) also attenuated NE‐ and carbachol‐induced PI turnover, but failed to affect 5‐hydroxytryptamine‐ and glutamate‐induced responses. Furthermore, ketanserin, atropine, and prazosin did not have any effect on inositol phosphate formation induced by tryptamine. These observations indicate that tryptamine markedly increased Ca2+‐ and Na+‐dependent PI turnover in cerebellar neurons and selectively inhibited NE‐ and carbachol‐induced PI hydrolysis.
Agonist‐Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK‐N‐MCIXC Neuroepithelioma Cells
Authors: Palmer R. Kyle , Yule David I. , McEwen Edward L. , Williams John A. , Fisher Stephen K.
Published: July 2010

Abstract: Fura‐2 digital imaging microfluorimetry was used to evaluate the Ca2+ signals generated in single clonal human neuroepithelioma cells (SK‐N‐MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin‐1 (ET‐1), ATP, oxotremorine‐M (Oxo‐M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca2+i) but of different magnitudes (ET‐1 = ATP> NE). The Ca2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca2+ influx. The Ca2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca2+ signal, which was characteristic of the response to Oxo‐M, was observed in <40% of cells stimulated with ET‐1 and absent from Ca2+ signals elicited after NE addition. Removal of extracellular Ca2+ enhanced the rate of decay of Ca2+ signals generated by ATP, ET‐1, or Oxo‐M and, when evident, abolished the sustained phase of Ca2+ influx. In the absence of extracellular Ca2+, NE elicited asynchronous multiple Ca2+ transients. In either the absence or presence of extracellular Ca2+,>94% of cells responded to ET‐1 or ATP, whereas corresponding values for Oxo‐M and NE were ∼74 and ∼48%. Sequential addition of agonists to cells maintained in a Ca2+‐free buffer indicated that each ligand mobilized Ca2+ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four‐ to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET‐1 or ATP resulted in larger increases in the net formation of inositol 1,4,5‐trisphosphate than did either Oxo‐M or NE. These results indicate that, in SK‐N‐MCIXC cells, the characteristics of both Ca2+ signaling and inositol phosphate production are agonist specific.
Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+‐ATPase
Authors: Kosenko Elena , Kaminsky Yuri , Grau Eugenio , Miñana María‐Dolores , Marcaida Goizane , Grisolía Santiago , Felipo Vicente
Published: July 2010

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium‐induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK‐801, an antagonist of the NMDA receptor, prevented ammonia‐induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na+,K+‐ATPase activity by 76%. This increase was also prevented by previous injection of MK‐801. The molecular mechanism leading to activation of the ATPase was further studied. Na+,K+‐ATPase activity in samples from ammonia‐injected rats was normalized by “in vitro” incubation with phorbol 12‐myristate 13‐acetate, an activator of protein kinase C. The results obtained suggest that ammonia‐induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C‐mediated phosphorylation of Na+,K+‐ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.
Effects of Organotins on Rat Brain Astrocytes in Culture
Authors: Richter‐Landsberg Christiane , Besser Andreas
Published: July 2010

Abstract: The interaction of triethyltin (TET) and trimethyltin (TMT) with rat brain astrocytes in vitro was investigated. Both compounds are highly neurotoxic after in vivo application, cause neurobehavioral changes, and elicit neuronal and glial responses in the CNS. In this study, 5‐week‐old cultures were exposed to TMT or TET (0.1–2.5 µM) for 24 h. A concentration‐dependent cytotoxicity was observed for both agents by vital dye uptake assay using neutral red (NR). The order of potency for half‐maximal cytotoxicity (NR‐50) was TET (0.7 µM)> TMT (2.5 µM), in agreement with results found after in vivo administration. TET and TMT caused similar morphological changes: large holes extending through the plasma membrane appeared initially in the flattened cell bodies, cytoplasmic extensions were retracted, and long cellular processes formed. Later, the cell bodies rounded up and had only a few extremely long and thin processes. Indirect immunofluorescence staining using anti‐vimentin and anti‐glial fibrillary acidic protein (GFAP) antibodies revealed that the orderly array of the intermediate filament system was severely disturbed. At lower concentrations, an increased bundling was observed, and at higher concentrations the disassembly of the intracellular framework was seen, and cellular staining appeared rather diffuse. Western blot analysis of cellular extracts was carried out to determine the protein levels of GFAP and vimentin. In this culture system, TET and TMT caused an almost two‐fold increase in the levels of GFAP at concentrations around and below NR‐50, indicating that astrocytes react to organotics independently of neuronal signals. Concomitantly, TET (0.7 µM) and TMT (2.5 µM) led to a 63% decrease in the activity of ecto‐5′‐nucleotidase, which in addition to its enzymatic function, represents a transmembrane cell surface protein and has been implicated in cellular adhesion and cell communication processes. Thus, triorganotins directly affect astrocytes in culture and alter their functional stage even in the absence of neurons.
Evidence Against a Role for the Kunitz Domain in Amyloidogenic and Secretory Processing of the Amyloid Precursor Protein
Authors: Ladror Uri S. , Kohnken Russell E. , Wang Gary T. , Manelli Arlene M. , Frail Donald E. , Klein William L. , Holzman Thomas F. , Krafft Grant A.
Published: July 2010

Abstract: The effect of the Kunitz proteinase inhibitor (KPI) on potential β‐amyloid precursor protein (βPP)‐processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in βPP. In addition, the level of secretion of KPI‐containing βPP751 and KPI‐lacking βPP695 from transfected cells was examined to assess the effect of the KPI on βPP secretion. βPP751 and βPP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations βPP751, but not βPP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of βPP or in the amyloidogenic cleavage of βPP. The amounts of βPP695 and βPP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in βPP751 did not inhibit the secretory cleavage in transfected cells.
τ Regulation of Microtubule‐Microtubule Spacing and Bundling
Authors: Frappier Thierry F. , Georgieff Irene S. , Brown Kristy , Shelanski Michael L.
Published: July 2010

Abstract: τ proteins are microtubule‐associated proteins that promote microtubule polymerization in vitro and in vivo. They are a family of neuronal proteins with apparent molecular weights in the range 50,000–68,000 determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned. It has an apparent molecular weight of 116,000 and has been called high‐molecular‐weight τ (HMW τ). All the τ proteins are encoded by a single gene, which undergoes complex alternative splicing. In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW τ as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the τ microtubule‐binding domain. HMW τ‐recombinant‐virus‐infected Sf9 cells overexpressed HMW τ, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low‐molecular‐weight τ (LMW τ) overexpression. Process cross sections revealed a larger spacing (≈35 nm) between microtubules when induced by HMW τ than when induced by LMW τ (≈20 nm). The truncated construct also induces processes, where microtubules were packed far more closely together (≈10 nm). Although branching did not occur in processes induced by intact τs, 10% of the processes induced by the truncated τ protein branched.
Some Common Properties Between a Brain Protein that Is Modified by Posttranslational Arginylation and the Microtubule‐Associated STOP Protein
Authors: Bongiovanni Guillermina , Barra Héctor S. , Hallak Marta E.
Published: July 2010

Abstract: Properties so far studied of the 125‐kDa 14C‐arginylated protein from rat brain show remarkable similarities with those of the STOP (stable tubule only polypeptide) protein. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis the 125‐kDa 14C‐arginylated protein moves to the same position as the STOP protein. The 125‐kDa 14C‐arginylated protein was immunoprecipitated by the monoclonal Mab 296 antibody specific for neuronal STOP protein. The 125‐kDa 14C‐arginylated protein was retained by a calmodulin column like STOP protein. As occurs with the STOP protein, the 125‐kDa 14C‐arginylated protein is found in higher proportion in cold‐stable than in cold‐labile microtubules. However, the modified protein associates with microtubules in a lower proportion than the STOP protein. We conclude that the STOP protein incorporates arginine by a posttranslational reaction but that only a small fraction of the STOP protein shows acceptor capacity in vitro.
Diversity of High‐Molecular‐Weight τ Proteins in Different Regions of the Nervous System
Authors: Mavilia Carmelo , Couchie Dominique , Nunez Jacques
Published: July 2010

Abstract: We show in this work that high‐molecular‐weight (HMW) τ transcripts are present in all the regions of the CNS and PNS studied, i.e., the dorsal root ganglia (DRG), spinal cord, cerebellum, and forebrain. However, the relative amount of HMW and low‐molecular‐weight (LMW) τ variants and their sequence vary depending on the region. Two HMW τ variants that contain either both exons 4A and 6 or only exon 6 have been identified in the adult spinal cord by PCR amplification and sequenced. In contrast, a single HMW τ variant that contains exon 4A but not exon 6 was detected in the adult rat DRG. This means that at least part of the HMW τ expressed in the spinal cord is produced locally and not transported within this structure by fibers arising in the DRG. The expression of the HMW τ isoforms is developmentally regulated both quantitatively and qualitatively in the spinal cord. At immature stages very low levels of the HMW τ transcript containing both exons 4A and 6 are expressed, whereas the τ species containing only exon 6 is absent. Rat forebrain, rat cerebellum, and human forebrain express much lower levels of HMW τ transcripts compared with the spinal cord and the DRG. Their sequence contains both exons 4A and 6, i.e., is identical to that of the major HMW τ transcript detected in the adult rat spinal cord. The minor spinal cord species that contains only exon 6 was not identified in the rat forebrain and rat cerebellum but was present among the human brain PCR fragments. These data demonstrate that the family of HMW τ proteins is heterogeneous and that it includes transcripts containing exon 4A, 6, or both. Six of 15 exons of the τ gene are alternatively spliced, and the pattern of splicing varies quantitatively and/or qualitatively depending on the region of the nervous system and on the developmental stage.
Differential Regulation of Two Classes of Neuronal Intermediate Filament Proteins During Optic Nerve Regeneration
Authors: Hall Christina M.
Published: July 2010

Abstract: The regulation of expression of two different types of neuronal intermediate filament proteins, ON1/ON2 and plasticin, was studied during optic nerve regeneration in the goldfish. During regenerative growth of optic axons, there is a rapid and dramatically increased expression of plasticin, a recently cloned, novel type III intermediate filament protein, in the retinal ganglion cells. At the time when the growing axons reinnervate the optic tectum, expression of plasticin declines and there is an increased expression of ON1 and ON2. This time course suggests that the target tissue participates in the regulation of these proteins. The aim of this study was to characterize the regulatory role played by the optic tectum. To address this issue, a repeated‐crush paradigm was used whereby growing axons were hindered from reaching their target. It was found that in absence of tectal contact, the increased expression of ON1 and ON2 normally seen during regeneration was not induced. In contrast, expression of plasticin increased both in the presence and in the absence of tectal contact.
Differential Expression of MARCKS and Other Calmodulin‐Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells
Authors: Rosé Sergio D. , Cook Harold W. , Palmer Frederick B. St. C. , Ridgway Neale D. , Byers David M.
Published: July 2010

Abstract: Expression of the protein kinase C substrate MARCKS and other heat‐stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB‐11 (SK‐N‐SH) neuroblastoma cells than in HTB‐10 (SK‐N‐MC) or mouse N1E‐115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H‐myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50‐kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E‐115 and HTB‐10) with less MARCKS, whereas neuromodulin (GAP‐43) was detected in N1E‐115 and HTB‐11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate‐labeled MARCKS and may be due to a conformational change affecting the C‐terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β‐12‐O‐tetradecanoylphorbol 13‐acetate resulted in three‐ to fourfold increased phosphorylation of MARCKS in HTB‐11 cells, with little increase noted in HTB‐10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin‐binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.
Bafilomycin A1 Inhibits the Action of Tetanus Toxin in Spinal Cord Neurons in Cell Culture
Authors: Williamson Lura C. , Neale Elaine A.
Published: July 2010

Abstract: Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium‐evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar‐type H+‐ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose‐dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 nM baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1‐sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.
Analysis by Polymerase Chain Reaction of α1 and α6 GABAA Receptor Subunit mRNAs in Individual Cerebellar Neurons After Whole‐Cell Recordings
Authors: Santi Maria Rita , Vicini Stefano , Eldadah Basil , Neale Joseph H.
Published: July 2010

Abstract: With the use of the single‐cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole‐cell patch‐clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to α1 and α6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme‐mediated cleavage of the amplification products. In all Purkinje neurons assayed, α1 subunit mRNA but not α6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the α1 and α6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA‐mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of α6 subunit mRNA.
Chronic Sodium Valproate Selectively Decreases Protein Kinase C α and ε In Vitro
Authors: Chen Guang , Manji Husseini K. , Hawver David B. , Wright Clinton B. , Potter William Z.
Published: July 2010

Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first‐line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to “therapeutic” concentrations (0.6 mM) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme‐selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or 3H‐phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross‐talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.