bluuebunny/crossref_metadata_2025 huggingface dataset:
Rate of Glutamate Synthesis from Leucine in Rat Brain Measured In Vivo by 15N NMR
Authors: Kanamori Keiko , Ross Brian D. , Kondrat Richard W.
Published: July 2010

Abstract: The rate of glutamate synthesis from leucine by the branched‐chain aminotransferase was measured in rat brain in vivo at steady state. The rats were fed exclusively by intravenous infusion of a nutrient solution containing [15N]leucine. The rate of glutamate synthesis from leucine, determined from the rate of increase of brain [15N]glutamate measured by 15N NMR and the 15N enrichments of brain and blood leucine analyzed by gas chromatography‐mass spectrometry, was 0.7–1.8 µmol/g/h at a steady‐state brain leucine concentration of 0.25 µmol/g. A comparison of the observed fractional 15N enrichments of brain leucine (0.42 ± 0.03) and glutamate (0.21 ± 0.015) showed that leucine provides ∼50% of glutamate nitrogen under our experimental condition. From the observed rate (0.7–1.8 µmol/g) and the known Km of the branched‐chain aminotransferase for leucine (1.2 mM), the rate of glutamate synthesis from leucine at physiological brain leucine concentration (0.11 µmol/g) was estimated to be 0.35–0.9 µmol/g/h, with leucine providing ∼25% of glutamate nitrogen. The results strongly suggest that plasma leucine from dietary source, transported into the brain, is an important external source of nitrogen for replenishment of brain glutamate in vivo. Implications of the results for treatment of maple‐syrup urine disease patients with leucine‐restricted diet are discussed.
Veratridine‐Induced Depolarization Reduces the Palmitoylation of Brain and Myelin Glycerolipids
Authors: Sanchez Paul , Tetzloff Sabine U. , Bizzozero Oscar A.
Published: July 2010

Abstract: In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60‐day‐old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 µM) reduced the incorporation of [3H]palmitate into all brain glycerolipids by 40–50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [3H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 µM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage‐gated sodium channels and involves the stimulation of the Na+/K+ pump. Decreased levels of both ATP, due to activation of the Na+,K+‐ATPase, and the precursor palmitoyl‐CoA were found in the veratridine‐treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage‐dependent Na+ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K+]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.
Extracellular Calcium Deprivation in Astrocytes: Regulation of mRNA Expression and Apoptosis
Authors: Chiesa Roberto , Angeretti Nadia , Del Bo Roberto , Lucca Elisa , Munna Elettra , Forloni Gianluigi
Published: July 2010

Abstract: Cell viability and gene expression were studied in primary astroglial cells cultured in a nominally calcium‐free medium. Ca2+ deprivation reduced progressively the astrocytes' viability, starting from 12 h; the restoration of a normal Ca2+ concentration (1.8 mM) in the medium after 12‐h deprivation reversed the degenerative effect within 24 h. Biochemical and morphological examinations indicated that cell death induced by Ca2+ deprivation was mediated by apoptosis. This was associated with the expression of c‐fos, c‐jun, and c‐myc, which, with different time courses, were induced in astrocytes after Ca2+ deprivation. Furthermore, shifting to a Ca2+‐free medium modified the expression of Ich‐1S transcript and rapidly increased intracellular cyclic AMP, which has been implicated in the transcriptional activation of immediate‐early genes. The absence of Ca2+ in the medium reduced the expression of constitutive proteins such as α‐actin, clusterin, glial fibrillary acidic protein, amyloid precursor protein, and glucose‐6‐phosphate dehydrogenase. The expression of these mRNAs was reduced >50% after 8 h of Ca2+ deprivation, when the effect on cell viability was negligible. When Ca2+ deprivation was prolonged for 24 h the expression of mRNA dropped completely, and restoration of the Ca2+ ions in the medium for 48 h did not reverse this effect. In contrast with general assumption, the apoptotic machinery in astrocytes is activated similarly not only by increased Ca2+ influx but also with the extracellular Ca2+ deprivation.
Effect of Immobilization Stress on Transcriptional Activity of Inducible Immediate‐Early Genes, Corticotropin‐Releasing Factor, Its Type 1 Receptor, and Enkephalin in the Hypothalamus of Borderline Hypertensive Rats
Authors: Mansi James A. , Rivest Serge , Drolet Guy
Published: July 2010

Abstract: The effects of immobilization on the expression of immediate‐early gene c‐fos and nerve growth factor‐inducible (NGFI)‐B mRNAs, corticotropin‐releasing factor (CRF) mRNA, CRF heteronuclear RNA (hnRNA), CRF receptor types 1 and 2α mRNA, and enkephalin hn/mRNA were investigated in the hypothalamic paraventricular nucleus of Wistar‐Kyoto (WKY) rats and borderline hypertensive rats (BHRs). Rats were deeply anesthetized 0, 30, 60, and 180 min after the beginning of the immobilization session (60 min maximum). BHR paraventricular nuclei displayed slight differences in their resting levels of NGFI‐B mRNA and CRF hnRNA, both being significantly elevated compared with those of WKY rats. Conversely, basal levels of enkephalin primary transcript were significantly lower in BHRs. Immobilization, however, induced transient variations in the hybridization signals for all evaluated genes within the paraventricular nucleus (except for CRF 2α receptor). Immediate‐early gene mRNA levels were higher and more prolonged in BHRs than in WKY rats. This heightened neuronal activation in the BHRs was associated with a more rapid increase in CRF mRNA expression (30 min) compared with that in WKY rats (60 min). It is interesting that a transient rise in CRF hnRNA levels was detected in stressed WKY rats, whereas the BHR group displayed a progressive decline in this transcript, being significantly below resting levels 180 min after the immobilization session. The stress‐induced expression of CRF type 1 receptor mRNA was similar in both strains. Moreover, no significant differences were observed for enkephalin hn/mRNA in either strain during the immobilization session. Therefore, the hypothalamic paraventricular nucleus appears to be involved in the functional hyperreactivity of the hypothalamic‐pituitary‐adrenal and autonomic axes to stress observed in BHRs, which may lead ultimately to a stress‐induced hypertensive state.
Analysis of Local Structure in the D2/S1–S2 Region of the Rat Skeletal Muscle Type 1 Sodium Channel Using Insertional Mutagenesis
Authors: Kraner Susan D. , Filatov Gregory N. , Sun Weijing , Bannerman Peter , Lindstrom Jon , Barchi Robert L.
Published: July 2010

Abstract: A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full‐length membrane‐associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two‐electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1–S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1–S2 loop predicted by current models of channel tertiary structure.
Pituitary Adenylate Cyclase‐Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells
Authors: Tanaka Keiko , Shibuya Izumi , Uezono Yasuhito , Ueta Yoichi , Toyohira Yumiko , Yanagihara Nobuyuki , Izumi Futoshi , Kanno Tomio , Yamashita Hiroshi
Published: July 2010

Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP‐induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT‐PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP‐induced Ca2+ release was unaffected by Rp‐adenosine 3′,5′‐cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U‐73122, Ca2+ release in response to the peptides was unaffected by U‐73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.
Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways
Authors: Ding Wei‐Qun , Larsson Christer , Alling Christer
Published: July 2010

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH‐SY5Y cells. Stimulation with carbachol induced expression of c‐fos, fosB, c‐jun, junB, and junD. This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca2+ concentration with a Ca2+ ionophore. The Ca2+ effect was inhibited by KN‐62, suggesting an induction through Ca2+/calmodulin‐dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol‐stimulated increase in mRNA levels of c‐fos, fosB, and junB by ∼70% but had only minor effects on the expression of c‐jun and junD. On the other hand, preincubation with KN‐62 attenuated the carbachol‐induced increase in c‐jun and junD expression by 70% but had no effect on c‐fos, fosB, and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca2+/calmodulin‐dependent kinase II completely abolished the carbachol‐stimulated expression of c‐jun and junD, but c‐fos, fosB, and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor‐stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor‐induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca2+/calmodulin‐dependent kinase II.
Biotransformation of l‐DOPA to Dopamine in the Substantia Nigra of Freely Moving Rats: Effect of Dopamine Receptor Agonists and Antagonists
Authors: Sarre Sophie , Vandeneede Diederik , Ebinger Guy , Michotte Yvette
Published: July 2010

Abstract: We investigated the effects of continuous intranigral perfusion of dopamine D1 and D2 receptor agonists and antagonists on the biotransformation of locally applied l‐DOPA to dopamine in the substantia nigra of freely moving rats by means of in vivo microdialysis. The “dual‐probe” mode was used to monitor simultaneously changes in extracellular dopamine levels in the substantia nigra and the ipsilateral striatum. Intranigral perfusion of 10 µMl‐DOPA for 20 min induced a significant 180‐fold increase in extracellular nigral dopamine level. No effect of the intranigral l‐DOPA administration was observed on dopamine levels in the ipsilateral striatum, suggesting a tight control of extracellular dopamine in the striatum after enhanced nigral dopamine levels. Continuous nigral infusion with the D1 receptor agonist CY 208243 (10 µM) and with the D2 receptor agonist quinpirole at 10 µM (a nonselective concentration) attenuated the l‐DOPA‐induced increase in dopamine in the substantia nigra by 85 and 75%, respectively. However, perfusion of the substantia nigra with a lower concentration of quinpirole (1 µM) and the D1 antagonist SCH 23390 (10 µM) did not affect the nigral l‐DOPA biotransformation. The D2 antagonist (−)‐sulpiride (10 µM) also attenuated the l‐DOPA‐induced dopamine release in the substantia nigra to ∼10% of that of the control experiments. We confirm that there is an important biotransformation of l‐DOPA to dopamine in the substantia nigra. The high concentrations of dopamine formed after l‐DOPA administration may be the cause of dyskinesias or further oxidative stress in Parkinson's disease. Simultaneous administration of D1 receptor agonists with l‐DOPA attenuates the biotransformation of l‐DOPA to dopamine in the substantia nigra. The observed effects could occur via changes in nigral GABA release that in turn influence the firing rate of the nigral dopaminergic neurons.
Neurotrophins Rescue Cerebellar Granule Neurons from Oxidative Stress‐Mediated Apoptotic Death: Selective Involvement of Phosphatidylinositol 3‐Kinase and the Mitogen‐Activated Protein Kinase Pathway
Authors: Skaper Stephen D. , Floreani Maura , Negro Alessandro , Facci Laura , Giusti Pietro
Published: July 2010

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum‐free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation‐induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation‐induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high‐K+ to low‐K+/serum‐free medium decreased viability by >50% when measured after 30 h. Treatment of low‐K+ granule neurons at DIV 4 with nerve growth factor, brain‐derived neurotrophic factor (BDNF), neurotrophin‐3, or neurotrophin‐4/5 (NT‐4/5) demonstrated concentration‐dependent (1–100 ng/ml) protective effects only for BDNF and NT‐4/5. Between DIV 10 and 20, K+‐deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT‐4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin‐3. BDNF and NT‐4/5 also circumvented glutamate‐induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate‐triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin‐protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3‐kinase or the mitogen‐activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival‐promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.
Ultrastructural Studies of Carboxyl‐Terminal Truncation Mutants of the Neuronal Intermediate Filament Protein Peripherin
Authors: Ho C.‐L. , Chien C.‐L. , Brown K. , Liem R. K. H.
Published: July 2010

Abstract: We have prepared carboxyl‐terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per‐C424) self‐assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per‐C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin‐positive cells are transfected with Per‐C424 or Per‐C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles and clumps formed by Per‐C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per‐C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per‐C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal ribosomal entry site, we prepared a construct that expresses Per‐C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per‐C356 by fluorescence‐activated cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.
Enhanced [3H]DOPA and [3H]Dopamine Turnover in Striatum and Frontal Cortex In Vivo Linked to Glutamate Receptor Antagonism
Authors: Reith Jakob , Cumming Paul , Gjedde Albert
Published: July 2010

Abstract: We tested the hypothesis that blockade of NMDA glutamate receptors in brain enhances dopamine turnover. We blocked this class of glutamate receptors in the rat brain in vivo with dizocilpine (MK‐801) and measured the accumulation of radiolabeled DOPA and its metabolites as functions of time after intravenous bolus injection. Using the time courses of the accumulated metabolites, we calculated the turnover constants of enzymes mediating dopamine synthesis and catabolism. Dizocilpine treatment for 8 days enhanced the rates of DOPA decarboxylation and dopamine oxidation (monoamine oxidation) 4‐ and 16‐fold, respectively, in neostriatum and 10‐ and 3‐fold, respectively, in frontal cortex. The findings are not inconsistent with the hypothesis that the psychotomimetic properties of dizocilpine may be the manifestation of denervation hypersensitivity linked to activation of key enzymes of dopamine turnover in striatum.
3‐Hydroxybutyrate Aids the Recovery of the Energy State from Aglycaemic Hypoxia of Adult but Not Neonatal Rat Brain Slices
Authors: Brooks Keith J. , Clark John B. , Bates Timothy E.
Published: July 2010

Abstract: The level of phosphocreatine (PCr) and the intracellular pH (pHi) of superfused cortical brain slices from adult or 10‐day‐old rats were monitored using 31P NMR. When the glucose in the superfusing medium was replaced by 3‐hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pHi was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pHi returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.
Involvement of Calcineurin in Ca2+ Paradox‐Like Injury of Cultured Rat Astrocytes
Authors: Matsuda Toshio , Takuma Kazuhiro , Asano Shoichi , Kishida Yoko , Nakamura Hiroaki , Mori Koichi , Maeda Sadaaki , Baba Akemichi
Published: July 2010

Abstract: The Ca2+/calmodulin‐dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+‐containing medium after exposure to Ca2+‐free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+‐mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge‐induced injury in a dose‐dependent manner (10−10–10−8M). Cyclosporin A at 1 µM mimicked the effect of FK506. Rapamycin (1 µM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge‐induced increase in cytosolic Ca2+ level nor Na+‐Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.
Hydrogen Peroxide Enhances Signal‐Responsive Arachidonic Acid Release from Neurons: Role of Mitogen‐Activated Protein Kinase
Authors: Samanta Sujon , Perkinton Michael S. , Morgan Michael , Williams Robert J.
Published: July 2010

Abstract: Hydrogen peroxide (H2O2) is a potent stimulator of signal‐responsive phospholipase A2 (PLA2) in vascular smooth muscle and cultured endothelial cells. We investigated whether H2O2 plays a similar regulatory role in neurons. H2O2 did not stimulate a release of arachidonic acid from cultured neurons when applied alone but strongly enhanced the liberation of arachidonic acid evoked by maximally effective concentrations of either glutamate, the glutamate receptor agonist N‐methyl‐d‐aspartate (NMDA), the muscarinic receptor agonist carbachol, the Na+‐channel opener veratridine, or the Ca2+‐ionophore ionomycin. The potentiating effects of H2O2 were strongly inhibited in the presence of the PLA2 inhibitor mepacrine, suggesting that the site of action was within the signal responsive arachidonic acid cascade. The enhancing effect of H2O2 was not reversed by protein kinase C inhibitors (chelerythrine chloride or GF 109203X) nor was it mimicked by phorbol ester treatment. H2O2 alone strongly enhanced the levels of immunodetectable activated mitogen‐activated protein kinase (activated MAP kinases ERK1 and ERK2) in a Ca2+‐dependent manner and this effect was additive with increases in the levels of activated MAP kinase evoked by glutamate. The enhanced release of arachidonic acid, however, was not clearly reversed by the MAP kinase kinase (MEK) inhibitor PD 98059, although this treatment effectively abolished H2O2 activation of MAP kinase. Thus, MAP kinase activation and Ca2+‐dependent arachidonic acid release are regulated by oxidative stress in cultured striatal neurons.
Involvement of Pituitary Adenylate Cyclase‐Activating Polypeptide II Vasoactive Intestinal Peptide 2 Receptor in Mouse Neocortical Astrocytogenesis
Authors: Zupan Véronique , Hill Joanna M. , Brenneman Douglas E. , Gozes Illana , Fridkin Mati , Robberecht Patrick , Evrard Philippe , Gressens Pierre
Published: July 2010

Abstract: At the end of neuronal migration, the neopallial germinative zone produces glial cells destined to colonize the upper layers of neocortex. High densities of binding sites for vasoactive intestinal peptide (VIP) have been found in the rodent germinative zone just after completion of neuronal migration, suggesting a possible role of VIP in neocortical astrocytogenesis. In the present study, administration of a VIP antagonist at embryonic days 17 and 18 to pregnant mice was followed by a dramatic depletion of astrocytes in the upper cortical layer of the offspring. The depletion of astrocytes was dose‐dependent, with a 42% reduction in the density of astrocytes observed with 50 µg of antagonist. The antagonist effect was reversed by cotreatment with VIP or pituitary adenylate cyclase‐activating polypeptide (PACAP), suggesting the involvement of a receptor common to these two neuropeptides. VIP antagonist‐induced inhibition of astrocytogenesis was also blocked by Ro 25‐1553, a long‐acting cyclic VIP analogue selective for the PACAP II VIP2 receptor subclass. Our results demonstrate that VIP and/or PACAP play a crucial physiological role in neocortical astrocytogenesis, possibly through interaction with PACAP II VIP2 receptors.
Differential Coupling of μ‐, δ‐, and κ‐Opioid Receptors to Gα16‐Mediated Stimulation of Phospholipase C
Authors: Lee Jonathan W. M. , Joshi Sushma , Chan Joy S. C. , Wong Yung H.
Published: July 2010

Abstract: The μ‐opioid receptor has recently been shown to stimulate phosphoinositide‐specific phospholipase C via the pertussis toxin‐sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both δ‐ and κ‐opioid receptors are likely to activate G16. Interactions of δ‐ and κ‐opioid receptors with G16 were examined by coexpressing the opioid receptors and Gα16 in COS‐7 cells. The δ‐selective agonist [d‐Pen2,d‐Pen5]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ‐opioid receptor and Gα16. The δ‐opioid receptor‐mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators.

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Med Phys
Regulators of G Protein Signaling: Rapid Changes in mRNA Abundance in Response to Amphetamine
Authors: Burchett Scott A. , Volk Michael L. , Bannon Michael J. , Granneman James G.
Published: July 2010

Abstract: This study examined mRNAs encoding regulators of G protein signaling (RGSs) expressed within the striatum and determined whether their expression in the caudate putamen was altered by amphetamine. RT‐PCR techniques were used to clone cDNA probes of RGSs expressed within the rat striatum. Northern blot analysis of caudate putamen and nucleus accumbens RNA determined the relative abundance of RGS mRNA expressed within the caudate putamen and adjacent nucleus accumbens to be RGS 2 > RGS 5 > RGS 16 > RGS 4 = RGS 9 > RGS 8 = RGS 3. A single injection of amphetamine rapidly and transiently induced RGS 2 mRNA. The temporal pattern of induction of RGS 2 strongly resembled that of the immediate early gene c‐fos. Levels of mRNAs of RGS 3 and 5 steadily increased over a 4‐h interval, as did that of the 6.6‐kb transcript of RGS 8. The level of RGS 9 mRNA, which shows strong striatal‐specific expression, steadily decreased over a 4‐h interval, whereas RGS 4 and 16 and the 3.9‐kb transcript of RGS 8 were not significantly affected at any point examined. The ability of amphetamine to alter RGS mRNA expression within the caudate putamen suggests these proteins may play an important role in adaptive processes to psychostimulant exposure.
The Cholinergic Gene Locus
Authors: Eiden Lee E.
Published: July 2010

Abstract: Messenger RNAs and the cognate gene(s) encoding choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) have been cloned from mammals and several other animal classes in the last decade. These have provided molecular tools for investigating acetylcholine synthesis and packaging into synaptic vesicles, the genesis of cholinergic vesicles, and the development and senescence of the cholinergic nervous system. VAChT and ChAT have been found to share a common gene locus and regulatory elements for gene transcription. The cholinergic gene locus represents a previously undiscovered type of neuronal transcriptional unit controlling chemically coded neurotransmission. In vitro assays for the transport function of VAChT have shed light on the bioenergetics of amine accumulation in secretory vesicles. Manipulation of VAChT expression in vivo has demonstrated unequivocally the primacy of vesicular exocytosis as the mode of transmitting quanta of acetylcholine at the neuromuscular junction, as in vivo manipulation of acetylcholinesterase levels has demonstrated the importance of acetylcholine metabolism in the regulation of complex functions such as cognition. Light and electron microscopic visualization of VAChT, complementing previous ChAT immunohistochemistry, has improved understanding of the genesis and function of the cholinergic vesicle, neuron, and synapse. These advances should accelerate the development of “cholinergic” pharmacological and gene therapeutic approaches to treatment of human diseases that are associated with cholinergic surfeit and insufficiency.
Glucocorticoid‐Dependent Action of Neural Crest Factor AP‐2: Stimulation of Phenylethanolamine N‐Methyltransferase Gene Expression
Authors: Ebert Steven N. , Ficklin Mary Beth , Her Song , Siddall Brenda J. , Bell Rose Ann , Ganguly Karunesh , Morita Kyoji , Wong Dona L.
Published: July 2010

Abstract: AP‐2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP‐2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N‐methyltransferase (PNMT). However, stimulation of the PNMT gene by AP‐2 requires glucocorticoids and appears to be mediated through the interaction of AP‐2 with activated type II glucocorticoid receptors. Mutation of AP‐2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP‐2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP‐2 stimulates gene expression through a novel glucocorticoid‐dependent mechanism.
Possible Involvement of Phosphatidylinositol 3‐Kinase in Regulated Exocytosis: Studies in Chromaffin Cells with Inhibitor LY294002
Authors: Chasserot‐Golaz Sylvette , Hubert Pierre , Thiersé Danièle , Dirrig Sylvie , Vlahos Chris J. , Aunis Dominique , Bader Marie‐France
Published: July 2010

Abstract: Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3‐kinase (PI 3‐kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3‐kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine‐stimulated chromaffin cells. Attempts to immunoprecipitate PI 3‐kinase with anti‐phosphotyrosine antibodies failed, suggesting that the activity of PI 3‐kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2‐containing proteins in stimulated chromaffin cells. LY294002 [2‐(4‐morpholinyl)‐8‐phenyl‐4H‐1‐benzopyran‐4‐one], a potent inhibitor of PI 3‐kinase, produced a dose‐dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine‐labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3‐kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.
Prostaglandin E2 and 4‐Aminopyridine Prevent the Lipopolysaccharide‐Induced Outwardly Rectifying Potassium Current and Interleukin‐1β Production in Cultured Rat Microglia
Authors: Caggiano Anthony O. , Kraig Richard P.
Published: July 2010

Abstract: Brain inflammation includes microglial activation and enhanced production of diffusible chemical mediators, including prostaglandin E2. Prostaglandin E2 is generally considered a proinflammatory molecule, but it also promotes neuronal survival and down‐regulates some aspects of microglial activation. It remains unknown, however, if and how prostaglandin E2 prevents microglial activation. In primary culture, microglial activation is predicted by a characteristic pattern of whole‐cell potassium currents and interleukin‐1β production. We investigated if prostaglandin E2 could alter these currents and, if so, whether these currents are necessary for microglial activation. Microglia were isolated from mixed cell cultures prepared from neonatal rat brains and exposed to 0–10 µM prostaglandin E2 and lipopolysaccharide for 24 h. Currents were elicited by using standard patch‐clamp technique, and interleukin‐1β production was measured by ELISA. Peak outward current densities in microglia treated with lipopolysaccharide plus prostaglandin E2 (10 nM) were reduced significantly from those of cells treated with lipopolysaccharide alone. Prostaglandin E2 and 4‐aminopyridine (a blocker of outward potassium currents) also significantly reduced interleukin‐1β production. Thus, although prostaglandin E2 is classified generally as a proinflammatory chemical, it has complex roles in brain inflammation that include preventing microglial activation, perhaps by reducing the outward potassium current.
Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells
Authors: Crick Dean C. , Andres Douglas A. , Danesi Romano , Macchia Marco , Waechter Charles J.
Published: July 2010

Abstract: It is well documented that 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate‐derived product. Recently, it has been shown that free geranylgeraniol (GG‐OH) and farnesol (F‐OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG‐OH and F‐OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post‐translational modification of key regulatory proteins. In this study we report that GG‐OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F‐OH was added. Under these conditions [3H]mevalonate and [3H]GG‐OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP‐binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG‐OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG‐OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG‐OH was added to lovastatin‐treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.
High‐ and Low‐Affinity α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid ([3H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits
Authors: Standley Steve , Tocco Georges , Wagle Naveed , Baudry Michel
Published: July 2010

Abstract: Quantitative α‐[3H]amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid ([3H]AMPA) binding autoradiography was performed on frozen‐thawed sections from rat brain after preincubation at 0 or 35°C for 1 h. Preincubation at 35°C instead of 0°C resulted in a selective decrease of [3H]AMPA binding assayed at a low concentration of [3H]‐AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [3H]AMPA binding after preincubation at 35°C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [3H]AMPA(KD∼14 nM), whereas heavier organelles exhibited lower affinity for AMPA (KD∼190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti‐GluR2/3, C‐terminal antibodies). Experiments using two deglycosylating enzymes, N‐glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5‐kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high‐mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N‐terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high‐affinity [3H]AMPA binding sites represent nonsynaptic, intracellular membrane‐bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [3H]AMPA binding/GluR sites.
Multiple Coupling of Human D5 Dopamine Receptors to Guanine Nucleotide Binding Proteins GS and Gz
Authors: Sidhu Anita , Kimura Kazuhiro , Uh Misook , White Beatrix H. , Patel Shutish
Published: July 2010

Abstract: We have demonstrated previously that D1 dopamine receptors are coupled to both Gsα and Goα. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high‐affinity binding sites, indicating D5 receptors couple to both Gsα and cholera toxin‐insensitive G proteins. The interaction between D5 receptors and Gsα was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin‐sensitive G‐protein coupling to Goα or Giα. D5 receptors were also not coupled to Gqα and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF−4‐sensitive, N‐ethylmaleimide‐resistant G protein. Anti‐Gzα caused immunoprecipitation of 24.2 ± 5.2% of G protein‐associated D5 receptors, indicating coupling between D5 and Gzα. The coupling to Gzα was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gzα.
Ontogeny and Cellular Localization of the Pyruvate Recycling System in Rat Brain
Authors: Cruz Fátima , Scott Steve R. , Barroso Isabel , Santisteban Pilar , Cerdán Sebastián
Published: July 2010

Abstract: The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of 13C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2‐13C2]acetate as a substrate, was detected by 13C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole‐brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.
The Cell‐Specific Silencer Region of the Human Dopamine β‐Hydroxylase Gene Contains Several Negative Regulatory Elements
Authors: Kim Hee‐Sun , Yang Chunying , Kim Kwang‐Soo
Published: July 2010

Abstract: Dopamine β‐hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons in the nervous system. Transient transfection assays have indicated that cell‐specific transcription of the human DBH gene may require a cell‐specific silencer region residing at −486 to −263 bp upstream of the transcription start site. This region includes a putative DBH negative regulatory element (DNRE) with sequence homology to the restrictive element‐1 (RE1)/neuron‐restrictive silencer element identified in many other neural‐specific genes. However, DNRE exerted negative regulation in both neuronal and nonneuronal cells alike, and site‐directed mutation of this element did not significantly diminish the repressive activity of the DBH silencer region. Furthermore, expression of RE1‐silencing transcription factor/neuron‐restrictive silencer factor repressed neither DBH nor tyrosine hydroxylase promoter activity. We now report identification of three protein binding sites in the silencer region of the human DBH gene: SI at −271 to −250 bp, SII at −316 to −295 bp, and SIII at −348 to −324 bp. In vitro binding studies showed that SI and SIII, but not SII, interact with nuclear proteins from DBH‐negative cells in a cell‐specific manner. Furthermore, SI and SIII preferentially repressed the heterologous thymidine kinase and homologous DBH proximal promoter activities in nonneuronal cells. Taken together, the cell‐specific silencer function of the upstream DBH region appears to involve several cis‐regulatory elements, including two cell‐specific repressor elements, SI and SIII, and a general negative regulatory element, DNRE. Based on these data, we propose that a highly restricted pattern of DBH gene expression in (nor)adrenergic cells of the nervous system may be controlled by multiple negative regulatory elements/silencers.
Induction of Nitric Oxide Synthase and Nitric Oxide‐Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor‐α/Lipopolysaccharide
Authors: Heneka Michael T. , Löschmann Peter‐A. , Gleichmann Marc , Weller Michael , Schulz Jörg B. , Wüllner Ullrich , Klockgether Thomas
Published: July 2010

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor‐α (TNF‐α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N‐monomethylarginine, aminoguanidine, and 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine HCl prevented TNF‐α/LPS‐induced cell death and DNA fragmentation, suggesting that the TNF‐α/LPS‐induced cell death is mediated by iNOS‐derived NO. This hypothesis is supported by the finding that addition of l‐arginine, which serves as a precursor and limiting factor of enzyme‐derived NO production, potentiated TNF‐α/LPS‐induced loss of viability.
Association of the Src Family Tyrosine Kinase Fyn with TrkB
Authors: Iwasaki Yasuno , Gay Brigitte , Wada Kazuyo , Koizumi Shinichi
Published: July 2010

Abstract: Fyn tyrosine kinase, a member of the Src family, was recently reported to be present in neurons and glia cells. We investigated whether Fyn is involved in the Trk‐dependent signal transduction pathways of neurotrophin. The Fyn‐Src homology domain 2 (SH2) was observed to associate in vitro with the intracellular domain of TrkB (ICD‐TrkB). This association was dependent on the autophosphorylation of ICD‐TrkB. The Fyn‐SH2 domains bound to phosphorylated ICD‐TrkB (plCD‐TrkB) with an affinity similar to the binding of phospholipase Cγ (PLCγ)‐SH2 domains to its autophosphorylation site in TrkB. The Src‐SH2 domains showed substantially lower affinity with plCD‐TrkB, suggesting that the association between Fyn‐SH2 and plCD‐TrkB is not due to non‐specific interactions of SH2 domains with phosphorylated tyrosine residues. This is further supported by the observation that Fyn‐SH2 was able to trap phosphorylated TrkB in cell lysate prepared from primary rat cortical neurons stimulated with brain‐derived neurotrophic factor (BDNF). In contrast, endogenous Fyn was coprecipitated with TrkB from cortical neurons without BDNF stimulation. This basal association showed a threefold increase on BDNF stimulation, probably due to the SH2/phosphotyrosine interaction that was observed in the cell‐free system. All these data suggest the involvement of Fyn in the neurotrophin signal transduction pathways downstream of TrkB.