bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis
Authors: Herrera‐Marschitz M. , You Z.‐B. , Goiny M. , Meana J. J. , Silveira R. , Godukhin O. V. , Chen Y. , Espinoza S. , Pettersson E. , Loidl C. F. , Lubec G. , Andersson K. , Nylander I. , Terenius L. , Ungerstedt U.
Published: July 2010

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ‐aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K+ depolarization, (b) Na+ channel blockade, (c) removal of extracellular Ca2+, and (d) depletion of presynaptic vesicles by local administration of α‐latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K+ depolarization and were increased by perfusing with tetrodotoxin or with Ca2+‐free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K+‐depolarizing conditions by α‐latrotoxin. Because the effect of K+ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K+ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.
In Situ Analysis of Na,K‐ATPase α1‐ and α3‐Isoform mRNAs in Aging Rat Hippocampus
Authors: Chauhan N. B. , Siegel G. J.
Published: July 2010

Abstract: Age‐related changes in the expression of Na,K‐ATPase α1‐ and α3‐isoform mRNAs were analyzed by in situ hybridization in the Fischer‐344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24‐month‐old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold (p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age‐related, cell‐ and isoform‐specific alterations in pretranslational regulation of Na,K‐ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age‐related alterations in hippocampal function, before cell loss.
Overexpression of MARCKS, but Not Protein Kinase C‐α, Increases Phorbol Ester‐Stimulated Synthesis of Phosphatidylcholine in Human SK‐N‐MC Neuroblastoma Cells
Authors: Rosé S. D. , Morash S. C. , Ridgway N. D. , Byers D. M. , Cook H. W.
Published: July 2010

Abstract: To investigate the regulation of phorbol ester‐stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine‐rich protein kinase C substrate (MARCKS) and the α‐isoform of protein kinase C (PKC‐α) were overexpressed in a human neuroblastoma (SK‐N‐MC) cell line that does not increase PtdCho synthesis in response to 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl‐3H]choline was stimulated 1.88–2.34‐fold in the presence of 100–200 nM TPA. In clones overexpressing PKC‐α (30–40‐fold increased level of protein) or in mock‐transfected vector controls, TPA had much less of a stimulatory effect (1.04–1.43‐fold) on PtdCho synthesis. TPA caused translocation of PKC‐α and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK‐N‐MC cells, MARCKS is required for TPA‐stimulated synthesis of PtdCho, and PKC‐α alone is insufficient for supporting enhanced synthesis.
Suppression of p140trkA Does Not Abolish Nerve Growth Factor‐Mediated Rescue of Serum‐Free PC12 Cells
Authors: Taglialatela Giulio , Hibbert Chris J. , Hutton Leslie A. , Werrbach‐Perez Karin , Perez‐Polo J. Regino
Published: July 2010

Abstract: Programmed cell death, the intrinsic form of apoptosis, plays an integral role in those neurodegenerative events associated with age‐related neuropathology. Neurotrophins (NTs), such as nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and NT‐3, are required for survival of certain neurons, and thus their clinical use to counteract age‐ and pathology‐associated neurodegeneration has been suggested, although mechanistic descriptions for NT cell rescue from apoptosis are not definitive. Here we attempted to isolate the individual actions of high‐affinity tyrosine kinase (Trk) receptors and p75NGFR, the common low‐affinity NT receptor, in NT rescue of apoptotic PC12 cells. Our results showed that whereas inhibiting Trk receptor phosphorylation abolishes NGF rescue of serum‐deprived PC12 cells from apoptosis, TrkA suppression with antisense oligonucleotides did not. Also, although BDNF did not rescue naive serumless PC12 cells, which lack the BDNF‐specific TrkB receptor, it significantly increased survival of TrkA‐suppressed serum‐starved PC12 cells. These data confirm the hypothesis that binding of any NT to Trk‐free p75NGFR‐bearing cells blocks apoptosis but also suggest that if Trk receptors are expressed, prohibiting Trk phosphorylation also blocks NT‐mediated rescue from apoptosis.
Glutathione Efflux from Cultured Astrocytes
Authors: Sagara Jun‐ichi , Makino Nobuo , Bannai Shiro
Published: July 2010

Abstract: The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25°C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis‐Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p‐Chloromercuribenzenesulfonic acid, a thiol‐reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.
Adenosine A2a Receptor‐Mediated Modulation of Striatal Acetylcholine Release In Vivo
Authors: Kurokawa Masako , Koga Kumiko , Kase Hiroshi , Nakamura Joji , Kuwana Yoshihisa
Published: July 2010

Abstract: To determine the functions of striatal adenosine A2a receptors in vivo, the effects of a selective agonist, 2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680), and an antagonist, (E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine (KF17837), on acetylcholine release were investigated in the striatum of awake freely moving rats using microdialysis. Intracerebroventricular injection of CGS 21680 (10 µg) increased acetylcholine release in striatum and KF17837 (30 mg/kg p.o.) antagonized the CGS 21680‐induced acetylcholine elevation. To investigate the contribution of dopaminergic and GABAergic neurons on A2a receptor‐mediated acetylcholine release, the effects of CGS 21680 were studied by using dopamine‐depleted rats in the presence or absence of GABA antagonists. In the dopamine‐depleted striatum, the intrastriatal application of CGS 21680 (0.3–30 µM) increased extracellular acetylcholine, which was significantly greater than that in normal striatum. The CGS 21680‐induced elevation of acetylcholine release was still observed in the presence of GABA antagonists bicuculline (30 µM) and 2‐hydroxysaclofen (100 µM) and was similar in both normal and dopamine‐depleted striatum. These results suggest that A2a agonist stimulates acetylcholine release in vivo, and this effect of A2a agonist is modulated by dopaminergic and GABAergic neurotransmission.
Protein Kinases Are Involved in Prolonged Acetylcholine Release from Rat Hippocampus Induced by Thyrotropin‐Releasing Hormone Analogue NS‐3
Authors: Oka Michiko , Itoh Yoshinori , Ukai Yojiro , Yoshikuni Yoshiaki , Kimura Kiyoshi
Published: July 2010

Abstract: The effects of various protein kinase inhibitors on acetylcholine release from the rat hippocampus induced by the local application of NS‐3 (montirelin hydrate, CG‐3703), a thyrotropin‐releasing hormone analogue, into the medial septum‐diagonal band were examined using in vivo microdialysis. Perfusion of NS‐3 (1 µM) into the medial septum‐diagonal band for 20 min produced a pronounced and prolonged increase in the hippocampal acetylcholine efflux. Pretreatment of the medial septum‐diagonal band with either K‐252a, a nonselective protein kinase inhibitor, or selective protein kinase A inhibitor H‐89 almost completely blocked the acetylcholine efflux evoked by NS‐3, and selective protein kinase C inhibitor calphostin C inhibited the action of NS‐3. On the other hand, NS‐3 (0.1–10 µM) or TRH (1–100 µM) increased the cyclic AMP efflux from the medial septum‐diagonal band in a concentration‐dependent manner, as measured by microdialysis. These findings suggest that protein kinases A and C in the neurons of the medial septum‐diagonal band are involved in the mechanism of the prolonged stimulation of acetylcholine release from the hippocampus induced by thyrotropin‐releasing hormone and its analogue, NS‐3.
Diminished Noradrenergic Stimulation Reduces the Activity of Rolipram‐Sensitive, High‐Affinity Cyclic AMP Phosphodiesterase in Rat Cerebral Cortex
Authors: Ye Ying , O'Donnell James M.
Published: July 2010

Abstract: The present study examined the in vivo regulation of rolipram‐sensitive, high‐affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of β‐adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4‐selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6‐hydroxydopamine (6‐OHDA; 200 µg) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half‐life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6‐OHDA infusion, respectively. This was followed by the development of supersensitivity of β‐adrenergic receptor‐linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6‐OHDA (300 µg) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6‐OHDA treatment, continuous blockade of β‐adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of β‐adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor‐effector system in the brain.
Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors
Authors: Trimmer Barry A. , Qazi Sanjive
Published: July 2010

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta. Cyclic AMP levels were increased by treatment with 3‐isobutyl‐1‐methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine‐M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine‐M and by oxotremorine‐M in the presence of 3‐isobutyl‐1‐methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine‐M. The primary inositol metabolite generated during a 5‐min exposure to oxotremorine‐M co‐eluted from ion‐exchange columns with inositol‐1‐monophosphate, although other more polar metabolites were also detected. This agonist‐evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell‐free preparation of nerve cord membranes labeled with [3H]inositol. Incubation with oxotremorine‐M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell‐free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.
Vesicular Quantal Size Measured by Amperometry at Chromaffin, Mast, Pheochromocytoma, and Pancreatic β‐Cells
Authors: Finnegan Jennifer M. , Pihel Karin , Cahill Paula S. , Huang Lan , Zerby Susan E. , Ewing Andrew G. , Kennedy Robert T. , Wightman R. Mark
Published: July 2010

Abstract: Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic β‐cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for <6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine‐containing vesicles at later days in culture is responsible for this alteration.
Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation
Authors: Issa Amalia M. , Gauthier Serge , Collier Brian
Published: July 2010

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 nM) and okadaic acid (50 nM) reduced significantly (p < 0.01) the synthesis of [3H]ACh from [3H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration‐dependent manner (p < 0.01). This depletion was not the result of a drug‐induced increase of spontaneous ACh release, which was not changed significantly (p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration‐dependent manner (p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly (p > 0.4) in the presence of calyculin A or okadaic acid. Both high‐affinity and low‐affinity [3H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration‐dependent manner in the presence of calyculin A or okadaic acid; these agents reduced Vmax values for high‐ and low‐affinity choline uptake (p < 0.01) with no significant change in Km values (p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.
Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S‐Nitrosylation
Authors: Wolosker Herman , Reis Marcelo , Assreuy Jamil , De Meis Leopoldo
Published: July 2010

Abstract: Nitric oxide (NO; including NO•, NO+, and NO−) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S‐nitrosogluthathione and S‐nitroso‐N‐acetylpenicillamine, were used. The primary target of NO is the H+‐ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µM) and, to a much lesser extent, methemoglobin protected the vacuolar H+‐ATPase from inhibition. Inhibition of H+ pumping by NO was reversed by addition of 0.5 mM dithiothreitol. The results indicate that the vacuolar H+‐ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S‐nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.
β25–35 Alters Calcium Homeostasis and Induces Neurotoxicity in Cerebellar Granule Cells
Authors: Scorziello Antonella , Meucci Olimpia , Florio Tullio , Fattore Morena , Forloni Gianluigi , Salmona Mario , Schettini Gennaro
Published: July 2010

Abstract: We studied the neurotoxic effects of β25–35 amyloid fragment (β25–35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 mM KCl but slightly modified the survival of 25 mM KCl‐cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3‐DIV‐treated cultures; whereas in β25–35‐pretreated cells, a significant glutamate toxicity was observed. Treatment of 6‐DIV cells with β25–35, performed with 25 mM KCl, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate‐related neurotoxicity, and this effect was enhanced by β25–35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of β25–35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and β25–35‐treated cells. β25–35 did not affect basal [Ca2+]i but modified glutamate‐induced [Ca2+]i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that β25–35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.
Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain
Authors: Conn Kelly J. , Pietropaolo Michael , Ju Shyr‐Te , Abraham Carmela R.
Published: July 2010

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β‐precursor protein. In our search for proteases that can generate the N‐terminus of amyloid β protein (β‐secretases), we discovered a thiol‐dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine‐aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N‐terminus of amyloid β protein, and generates amyloidogenic fragments from full‐length recombinant amyloid β‐precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not “ghost” tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.
Glutamine Synthetase‐Induced Enhancement of β‐Amyloid Peptide Aβ(1–40) Neurotoxicity Accompanied by Abrogation of Fibril Formation and Aβ Fragmentation
Authors: Aksenov M. Y. , Aksenova M. V. , Butterfield D. A. , Hensley K. , Vigo‐Pelfrey C. , Carney J. M.
Published: July 2010

Abstract: β‐Amyloid peptide (Aβ) is the main constituent in both senile plaques and diffuse deposits in Alzheimer's diseased brains. It was previously shown that synthetic Aβs were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of Aβ(1–40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of Aβ toxicity to hippocampal cell cultures. In the present study the enhancement of Aβ toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of Aβ(1–40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of Aβ(1–40). The fragmentation of Aβ was inhibited by immunoprecipitation of GS.
Calcium Binds Dynamin I and Inhibits Its GTPase Activity
Authors: Liu Jun‐Ping , Zhang Qun‐Xing , Baldwin Graham , Robinson Phillip J.
Published: July 2010

Abstract: Synaptic vesicle recycling is a neuronal specialization of endocytosis that requires the GTPase activity of dynamin I and is triggered by membrane depolarization and Ca2+ entry. To establish the relationship between dynamin I GTPase activity and Ca2+, we used purified dynamin I and analyzed its interaction with Ca2+ in vitro. We report that Ca2+ bound to dynamin I and this was abolished by deletion of dynamin's C‐terminal tail. Phosphorylation of dynamin I by protein kinase C promoted formation of a dynamin I tetramer and increased Ca2+ binding to the protein. Moreover, Ca2+ inhibited dynamin I GTPase activity after stimulation by phosphorylation or by phospholipids but not after stimulation with a GST‐SH3 fusion protein containing the SH3 domain of phosphoinositide 3‐kinase. These results suggest that in resting nerve terminals, phosphorylation of dynamin I by protein kinase C converts it to a tetramer that functions as a Ca2+‐sensing protein. By binding to Ca2+, dynamin I GTPase activity is specifically decreased, possibly to regulate synaptic vesicle recycling.
Changes of Neuropeptide Y Messenger RNA and Peptide by Drugs Influencing Endogenous Norepinephrine Content in Cerebrocortex of the Rat
Authors: Niu Chiang‐Shan , Chang Tzen Kwan , Cheng Juei‐Tang
Published: July 2010

Abstract: Cotransmission of classic transmitters at the synapse has been mentioned for both the CNS and the PNS. Neuropeptide Y (NPY) is a cotransmitter in noradrenergic neurotransmission. In an attempt to understand the heteroregulation of norepinephrine (NE) and NPY biosynthesis, the present study was performed using radioimmunoassay of NPY and northern blotting of cDNA probes for characterization of NPY mRNA. Values of NPY‐like immunoreactivity (NPY‐ir) were elevated in the cerebrocortex from rats that received treatment with fusaric acid, an inhibitor of dopamine‐β‐hydroxylase, with a parallel decrease in NE. Similar results were also observed in rats treated with DSP‐4, an alkylator of vesicles in noradrenergic nerve terminals. Moreover, cerebrocortical NPY‐ir was reduced in rats receiving treatment with pargyline, an inhibitor of monoamine oxidase, with an elevation of catecholamine in parallel. Activity of NPY mRNA was modified by these drugs in a similar way. However, values of NPY‐ir and NE in the cerebrocortex were not influenced by treatment with sodium nitroprusside or guanethidine at a dose producing hypotensive effect. Mediation of hypotensive reflex can thus be ruled out. The data obtained suggest that in vivo decrease of NE by drugs increases biosynthesis of NPY in the cerebrocortex of rats.
Amino Acid Composition, Immunoreactivity, Sequence Analysis, and Function of Bovine Hippocampal Metallothionein Isoforms
Authors: Ebadi M. , Perini F. , Mountjoy K. , Garvey J. S.
Published: July 2010

Abstract: The high concentration of zinc in the hippocampal mossy fiber axon boutons is localized in the vesicles and is mobilized by exocytosis of the zinc‐laden vesicles. Because “free” zinc in excess is a neurotoxic substance inhibiting an extensive number of sulfhydryl‐containing enzymes and receptor sites, we hypothesized that low‐molecular‐weight zinc binding proteins must exist in the hippocampus to regulate the steady‐state concentration of zinc. In this communication, we report that the bovine hippocampus synthesizes metallothionein (MT) isoforms that are similar, but not identical, to those of the rat brain MT isoforms and cross‐react poorly with antibodies formed against the hepatic MT isoforms, suggesting that the immunologically dominant regions of hippocampal MT (residues 1–29) are not conserved. A comparative sequence analysis of bovine hippocampal MTs and bovine hepatic MT isoforms I and II revealed a 90% sequence identity, being mostly different in residues 1–29. The results of these studies suggest that the hippocampal MT isoforms, which are synthesized on a continuous basis, may play a role in regulating the transport, accumulation, and compartmentation of zinc in the hippocampus.
Docosahexaenoic Acid Is a Major n‐3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels
Authors: Lecomte Marc , Paget Clarisse , Ruggiero Daniel , Wiernsperger Nicolas , Lagarde Michel
Published: July 2010

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100‐µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µM) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.
Characterization of a Novel Platelet‐Derived Growth Factor‐Associated Protein
Authors: Fischer W. H. , Schubert D.
Published: July 2010

Abstract: A novel mitogen‐associated protein was isolated from a rat neural retina cell line. The protein copurified with platelet‐derived growth factor (PDGF)‐A and was therefore termed PDGF‐associated protein (PAP). cDNAs corresponding to the protein were characterized from both rat and human cDNA libraries. PAP binds to PDGF with low affinity and enhances the mitogenic effect of PDGF‐A but lowers the mitogenic activity of PDGF‐B. PAP mRNA is abundant in the brain of newborn rats and is found in several other tissues.
Brain‐Derived Neurotrophic Factor Stimulates AP‐1 and Cyclic AMP‐Responsive Element Dependent Transcriptional Activity in Central Nervous System Neurons
Authors: Gaiddon C. , Loeffler J. P. , Larmet Y.
Published: July 2010

Abstract: Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high‐affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1‐dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c‐fos and c‐jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis‐acting elements by which BDNF stimulates c‐fos transcription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum‐responsive element, Fos AP1‐like element, and cyclic AMP (cAMP)‐responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c‐fos transcription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c‐fos promoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1‐ and CRE‐dependent transcription through a mechanism that is distinct from the cAMP‐ and Ca2+‐dependent pathways in CNS neurons.
Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress
Authors: Ben‐Yoseph Oded , Boxer Peter A. , Ross Brian D.
Published: July 2010

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6‐13C2,6,6‐2H2]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration‐dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2‐induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.
Extracellular Striatal Dopamine and Glutamate After Decortication and Kainate Receptor Stimulation, as Measured by Microdialysis
Authors: Smolders I. , Sarre S. , Vanhaesendonck C. , Ebinger G. , Michotte Y.
Published: July 2010

Abstract: Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 µM concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so‐called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study.
Phorbol Ester‐Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C‐α in Human SH‐SY5Y Neuroblastoma Cells
Authors: Turner Neil A. , Walker John H. , Ball Stephen G. , Vaughan Peter F. T.
Published: July 2010

Abstract: The effect of inhibition and down‐regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH‐SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31‐7549 inhibited carbachol‐evoked NA release (IC50 0.6 µM) but not 100 mM K+‐evoked release. In addition, Ro 31‐7549 inhibited the enhancement of carbachol‐ and K+‐evoked release after pretreatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 100 nM) for 8 min, with IC50 values of 0.7 and 2.4 µM, respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH‐SY5Y cells to phorbol 12,13‐dibutyrate (PDBu) or bryostatin‐1 caused down‐regulation of PKC‐α and PKC‐ε but not PKC‐ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA‐enhanced secretion was also apparent after only 2‐h exposure to either PDBu or bryostatin‐1, conditions that caused down‐regulation of PKC‐α, but not PKC‐ε or ζ. The PKC inhibitor Gö‐6976 (2 µM), which has been shown to inhibit selectively PKC‐α and β in vitro, also inhibited the TPA enhancement of carbachol‐ and K+‐evoked NA release by >50%. These data suggest that in SH‐SY5Y cells, the ability of TPA to enhance carbachol‐ and K+‐evoked NA secretion is due to activation of PKC‐\ga.
Coupling of Astroglial α2‐Adrenoreceptors to Second Messenger Pathways
Authors: Enkvist M. O. Kristian , Hämäläinen Heli , Jansson Christian C. , Kukkonen Jyrki P. , Hautala Rasmus , Courtney Michael J. , Åkerman Karl E. O.
Published: July 2010

Abstract: We have investigated which α2‐receptor subtypes are expressed in cultured cortical astroglia, and their coupling to second messengers. Binding assays using [3H]rauwolscine showed a very low number of α2 receptors in the astrocytic cultures. Treatment of cultures with dibutyryl cyclic AMP (dBcAMP) increased significantly the number of receptors. The RNase protection assay was used to investigate which receptor subtype the cells express. The α2B message was expressed at a low level in both treated and untreated cells, the levels of mRNA for the α2A/D subtype were up‐regulated significantly in cells treated with dBcAMP and no expression of mRNA for the α2C subtype was detected. The α2 agonist dexmedetomidine inhibited forskolin‐induced increases in cyclic AMP both in treated and untreated cultures in a pertussis toxin‐dependent manner. This effect was abolished by the α2‐receptor antagonist rauwolscine. Selective α2‐receptor agonists dexmedetomidine, clonidine, and UK14,304 all increased intracellular calcium only in dBcAMP‐treated cells. The antagonist rauwolscine abolished this effect. Ca2+ responses were also seen in the absence of extracellular Ca2+ and they were inhibited by the phospholipase C inhibitor U‐73122, suggesting that astroglial α2 receptors are coupled to the inositol phospholipid pathway. We therefore also tested the effect of dexmedetomidine directly on inositol 1,4,5‐trisphosphate accumulation. A significant increase was seen that was blocked by the antagonist rauwolscine and, as expected, by U‐73122. In short, the results demonstrate that the α2 receptors in astroglia are coupled to multiple second messenger pathways. They are up‐regulated in cells treated with dBcAMP, which simultaneously assume a process‐bearing morphology. If this morphological change reflects some in vivo process such as reactive gliosis, the up‐regulation of α2‐receptor expression could mean an adaptive change in astrocytic responses to a common neurotransmitter, noradrenaline.
gp130, a Shared Signal Transducing Receptor Component for Hematopoietic and Neuropoietic Cytokines
Authors: Taga Tetsuya
Published: July 2010

Abstract: Most of the receptors for soluble factors functioning in the hematopoietic system belong to the class I cytokine receptor family. These receptors often share common signal transducing receptor components in the same family, which explains the functional redundancy of cytokines. One typical example is a group of receptor systems for interleukin‐6 (IL‐6) and related cytokines that share gp130 as a signal transducer. This subset of cytokines, i.e., IL‐6, IL‐11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin‐1, are all pleiotropic, exhibiting overlapping biological activities, and are known to function also in the neuronal system. In their receptor complexes, gp130 and ligand‐specific chains possess no intrinsic tyrosine kinase domain but are associated with members of the Jak family of cytoplasmic tyrosine kinases. The Jak kinases become activated after ligand‐induced homo‐ or heterodimerization of gp130. This activation appears to link the cell surface receptors to the nuclear genes through a series of biochemical changes, including tyrosine phosphorylation and activation of a latent cytoplasmic transcription factor called signal transducer and activator of transcription 3 (STAT3).
Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells
Authors: Guo Xi , Przywara Dennis A. , Wakade Taruna D. , Wakade Arun R.
Published: July 2010

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+‐free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo‐1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine‐ but not muscarine‐stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine‐evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine‐evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ∼20‐s refractory period was found between repeated caffeine‐evoked exocytotic bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.
Endogenous GABA Modulates Histamine Release from the Anterior Hypothalamus of the Rat
Authors: Okakura‐Mochizuki Kaori , Mochizuki Takatoshi , Yamamoto Yumiko , Horii Arata , Yamatodani Atsushi
Published: July 2010

Abstract: Using a microdialysis method, we investigated the effects of the nipecotic acid‐induced increase in content of endogenous GABA on in vivo release of histamine from the anterior hypothalamus (AHy) of urethane‐anesthetized rats. Nipecotic acid (0.5 mM), an inhibitor of GABA uptake, decreased histamine release to ∼60% of the basal level. This effect was partially antagonized by picrotoxin (0.1 mM), an antagonist of GABAA receptors, or phaclofen (0.1 mM), an antagonist of GABAB receptors. These results suggest that histamine release is modulated by endogenous GABA through both GABAA and GABAB receptors. When the tuberomammillary nucleus, where the cell bodies of the histaminergic neurons are localized, was stimulated electrically, the evoked release of histamine from the nerve terminals in the AHy was significantly enhanced by phaclofen, suggesting that GABAB receptors may be located on the histaminergic nerve terminals and modulate histamine release presynaptically. On the other hand, picrotoxin caused an increase in histamine release to ∼170% of the basal level, and this increase was diminished by coinfusion with d(−)‐2‐amino‐5‐phosphonopentanoic acid (0.1 mM), an antagonist of NMDA receptors. Previously, we demonstrated tonic control of histamine release by glutamate mediated through NMDA receptors located on the histaminergic terminals in the AHy. These results suggest the possible localization of GABAA receptors on glutamatergic nerve terminals and that the receptors may regulate the basal release of histamine indirectly.
Release of Adenosine from Rat Hippocampal Slices by Nitric Oxide Donors
Authors: Fallahi Nazanin , Broad R. Michael , Jin Shaoyu , Fredholm Bertil B.
Published: July 2010

Abstract: In the present study we have used perfused hippocampal slices to examine the hypothesis that nitric oxide (NO) can evoke the release of adenosine from nervous tissue. ATP stores were labeled by incubation with [3H]adenine. Electrical field stimulation at 5 Hz for 5 min evoked the release of 3H‐purines, and this was enhanced by the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP). Stimulation at 10 Hz for 15 min evoked a larger release of 3H‐purines, which was enhanced in a concentration‐dependent manner by both SNAP and sodium nitroprusside (SNP), with SNP being 100‐fold less potent than SNAP. N‐Acetylpenicillamine (N‐AP) was without effect. SNAP had a markedly reduced, although significant, effect when given in the absence of field stimulation. Using HPLC it was found that SNAP enhanced the release of both endogenous and labeled adenosine in a concentration‐dependent manner. At the highest concentration used (1 mM), SNAP increased electrically evoked release of endogenous adenosine 100‐fold and unstimulated adenosine release eightfold. The ability of SNAP to enhance adenosine release was eliminated in the added presence of hemoglobin (10 µM), further supporting the proposal that the effects of SNAP were due to the liberation of NO. These data provide direct evidence that NO evokes a concentration‐dependent release of adenosine from both stimulated and unstimulated nerves of the hippocampus. It is suggested that such NO‐stimulated adenosine release may contribute to some of the reported effects of NO donors in the nervous system.