bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture
Authors: Dessi Frédéric , Pollard Hélène , Moreau Joelle , Ben‐Ari Yezekiel , Charriaut‐Marlangue Christiane
Published: July 2010

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration‐dependent fashion with an EC50 of ∼60 µM when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC50 = 1 µM), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC‐treated neurons by two‐dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.
Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors
Authors: Bettendorff L. , Goessens G. , Sluse F. , Wins P. , Bureau M. , Laschet J. , Grisar T.
Published: July 2010

Abstract: When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16‐fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 ± 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high‐affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low‐affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine‐deficient neuroblastoma cells.
Blockade of Neurotensin Receptor by SR 48692 Potentiates the Facilitatory Effect of Haloperidol on the Evoked In Vivo Dopamine Release in the Rat Nucleus Accumbens
Authors: Brun P. , Steinberg R. , Le Fur G. , Soubrié P.
Published: July 2010

Abstract: The present experiments assessed the effects of SR 48692, a selective nonpeptide antagonist of neurotensin receptors, on mesolimbic dopaminergic neurotransmission. Dopamine release evoked by the electrical stimulation of the median forebrain bundle (20 Hz, 10 s) was measured in the nucleus accumbens of urethane‐anesthetized rats using differential pulse amperometry combined with carbon fiber electrodes. SR 48692 (0.1 mg/kg, i.p.) alone did not affect this release, whereas it dose‐dependently (0.03–1 mg/kg, i.p.) enhanced the haloperidol (50 µg/kg, i.p.)‐induced facilitation of the electrically evoked DA release. The increase induced by haloperidol (92 ± 26% above control values 30 min after injection) was potentiated by SR 48692 (264 ± 75% at 0.03 mg/kg, 428 ± 113% at 0.1 mg/kg, and 480 ± 135% at 1 mg/kg). Effects identical to those of SR 48692 were obtained with SR 48527, a chemically related compound with a high affinity for neurotensin receptors, but not with SR 49711, its low‐affinity antipode. The potentiating effects of SR 48692 were positively related to the stimulation frequency (from 6 to 20 Hz) and to the dose of haloperidol (from 12.5 to 50 µg/kg) and were abolished after prior kainic acid lesion (1 µg/1 µl) of the nucleus accumbens. Thus, the effects of SR 48692 required the integrity of postsynaptic elements of the nucleus accumbens and occurred under the combination of two, at least partly, interdependent conditions: strong D2 autoreceptor blockade and high‐intensity stimulation likely to release neurotensin. It is interesting that these potentiating effects of SR 48692 did not appear in the striatum. In conclusion, these findings suggest that endogenous neurotensin may attenuate the facilitation of D2 receptor blockade on mesolimbic but not nigrostriatal dopamine transmission.
Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain
Authors: Cousin M. A. , Nicholls D. G. , Pocock J. M.
Published: July 2010

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage‐sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L‐type Ca2+ channel antagonist nifedipine, the spider toxin Aga‐Gl, and the NMDA receptor antagonist MK‐801. Single‐cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6–10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 mM KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V‐type ATPase inhibitor bafilomycin, and Aga‐Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.
Glutamate‐Induced Loss of Ca2+/Calmodulin‐Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons
Authors: Morioka Motohiro , Fukunaga Kohji , Nagahiro Shinji , Kurino Masahito , Ushio Yukitaka , Miyamoto Eishichi
Published: July 2010

Abstract: The exposure of cultured rat hippocampal neurons to 500 µM glutamate for 20 min induced a 55% decrease in the total Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) activity. The Ca2+‐independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK‐801, an N‐methyl‐d‐aspartate (NMDA)‐type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca2+ ionophore, ionomycin. Immunoblot analysis with the anti‐CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.
Full‐Length and Truncated Alzheimer Amyloid Precursors in Chromaffin Granules: Solubilization of Membrane Amyloid Precursor Is Mediated by an Enzymatic Mechanism
Authors: Vassilacopoulou Dido , Ripellino James A. , Tezapsidis Nikolaos , Hook Vivian Y. H. , Robakis Nikolaos K.
Published: July 2010

Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was indistinguishable from full‐length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full‐length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full‐length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full‐length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full‐length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.
Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro
Authors: Uto A. , Dux E. , Kusumoto M. , Hossmann K.‐A.
Published: July 2010

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 mM) applied for 5 min to 8‐day‐old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK‐801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 mM. These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free‐radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.
Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40
Authors: Goldstein M. , Lee K. Y. , Lew J. Y. , Harada K. , Wu J. , Haycock J. W. , Hokfelt T. , Deutch A. Y.
Published: July 2010

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti‐pTH32–47 antibodies with phospho‐ and dephospho‐Ser40 forms of TH protein and peptide TH32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody‐capture ELISAs, anti‐pTH32–47 was more reactive with the phospho‐TH than with the dephospho‐TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho‐TH forms. In western blots, labeling of the ∼60‐kDa TH band by anti‐pTH32–47 was readily detectable in lanes containing protein kinase A‐phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti‐pTH32–47. Similarly, anti‐pTH32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8‐bromoadenosine 3′,5′‐cyclic monophosphate or forskolin enabled removal of TH activity by anti‐pTH32–47. Lastly, in immunohistochemical studies, anti‐pTH32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion‐fixed rat brain.
N‐Cadherin Is a Major Glycoprotein Component of Isolated Rat Forebrain Postsynaptic Densities
Authors: Beesley Philip W. , Mummery Rosemary , Tibaldi John
Published: July 2010

Abstract: We have previously described a monoclonal antibody, PAC 1, that recognises two postsynaptic density (PSD)‐enriched glycoproteins (pgps) of apparent Mr 130,000 (pgp130) and 117,000 (pgp117). Immunodevelopment of western blots of rat forebrain homogenate, synaptic membrane (SM), and PSD samples with PAC 1 and an N‐cadherin antiserum shows that pgp130 and N‐cadherin are of identical apparent Mr and show identical patterns of enrichment in these fractions. The apparent molecular masses of pgp130 and N‐cadherin are both lowered by 11 kDa following removal of N‐linked carbohydrate with endoglycosidase‐F containing N‐glycopeptidase. The two molecules show an identical pattern of migration when separated by two‐dimensional electrophoresis. A single 130‐kDa band immunoprecipitated from solubilised PSD preparations by the N‐cadherin antiserum is recognised by PAC 1 on western blots. We conclude that pgp130 is N‐cadherin. Development of western blots of two‐dimensional gel separations of SM and PSD glycoproteins shows that N‐cadherin is a major glycoprotein component of PSDs. The immunoprecipitation experiments show that the Mr of N‐cadherin is greater than that of the major pgp, PSD gp116. The PAC 1 antibody recognises two concanavalin A‐binding glycoproteins with apparent molecular masses of 136 and 127 kDa in liver samples. The 136‐kDa band is also recognised by the N‐cadherin antiserum. These observations, together with data showing that the PAC 1 epitope is intracellular, suggest that PAC 1 is a pan‐cadherin antibody and recognises an epitope on the conserved cadherin intracellular carboxyl‐terminal domain.
Nondestructive Measurement of Retinal Glucose Transport and Consumption In Vivo Using NMR Spectroscopy
Authors: Berkowitz Bruce A. , Garner Margaret H. , Wilson Charles A. , Corbett Ronald J. T.
Published: July 2010

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6‐2H2]glucose or 3‐deoxy‐3‐fluoro‐d‐glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, 2H or 19F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.
Increased Expression of Retinal TIMP3 mRNA in Simplex Retinitis Pigmentosa Is Localized to Photoreceptor‐Retaining Regions
Authors: Jomary Catherine , Neal Michael J. , Jones Stephen E.
Published: July 2010

Abstract: The human tissue inhibitor of metalloproteinases‐3 (TIMP3) gene is the most recently characterized member of a family of genes whose products are implicated in extracellular matrix (ECM) remodelling. We previously described an increase in expression of TIMP3 mRNA in retinas affected by the progressive photoreceptor degenerative disease, simplex retinitis pigmentosa (RP). To gain further insight into the association between TIMP3 overexpression and retinal degeneration, we have analyzed the cellular localization of TIMP3 mRNA in control and simplex RP retinas using in situ hybridization. No TIMP3 mRNA expression was detectable in control neural retina. In RP‐affected retinas, overexpression of TIMP3 mRNA was observed in photoreceptor inner segments and in the ganglion cell layer only in those regions retaining relatively nondystrophic retinal architecture. Modulation of TIMP3 expression in these regions, possibly in association with matrix metalloproteinases, may reflect remodelling of the retinal ECM and concomitant reorganization of neuronal connectivity.
Molecular Cloning and Differential Expression Patterns of Avian Glutamate Receptor mRNAs
Authors: Ottiger Hans‐Peter , Gerfin‐Moser Andrea , Del Principe Franco , Dutly Fabrizio , Streit Peter
Published: July 2010

Abstract: We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand‐gated ion channel family. One of them, GluP‐II, encodes a full‐length AMPA‐sensitive glutamate receptor GluR2 (GluR‐B) homologue, whereas the other two partial clones, designated as GluP‐III and ‐IV, are nearly identical to rodent GluR3 (GluR‐C) and GluR4 (GluR‐D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP‐II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR‐B) subunit genes. Immunoreactivity for a monoclonal sequence‐specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.
Nitroprusside and Cyclic GMP Stimulate Na+‐Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes
Authors: Asano Shoichi , Matsuda Toshio , Takuma Kazuhiro , Kim Hye Sun , Sato Tomoaki , Nishikawa Takashige , Baba Akemichi
Published: July 2010

Abstract: The effects of nitric oxide (NO)‐generating agents on 45Ca2+ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na+‐Ca2+ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µM increased monensin‐stimulated Ca2+ uptake in the slices, although it did not affect high K+‐stimulated Ca2+ uptake. Another NO donor, 3‐morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µM), a NO scavenger, and mimicked by 8‐bromo‐cyclic GMP (100 µM). In rat brain synaptosomes, SNP increased monensin‐stimulated Ca2+ uptake, but it did not affect high K+‐stimulated Ca2+ uptake. 8‐Bromocyclic GMP, but not SNP, increased Na+‐dependent Ca2+ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8‐bromo‐cyclic GMP increased Ca2+ uptake in the presence of ouabain and monensin, which were required for the Ca2+ uptake in the cells. These findings suggest that NO stimulates the Na+‐Ca2+ exchanger in neuronal preparations and astrocytes in a cyclic GMP‐dependent mechanism.
Galectin‐3, a β‐Galactoside‐Binding Animal Lectin, Binds to Neural Recognition Molecules
Authors: Probstmeier Rainer , Montag Dirk , Schachner Melitta
Published: July 2010

Abstract: In this study, we have investigated the ability of galectin‐3, a β‐galactoside‐binding animal lectin, to interact in vitro with different neural tissue‐derived glycoproteins involved in cell‐cell and cell‐substrate adhesion. Galectin‐3 interacted to varying degrees with the cell recognition molecules L1, the myelin‐associated glycoprotein, and the neural cell adhesion molecule and the extracellular matrix molecules tenascin‐C and tenascin‐R but not with collagen type I. Binding of galectin‐3 to the different glycoproteins tested was carbohydrate dependent and could be specifically inhibited by the addition of lactose and, to a lesser extent, galactose.
Activation of Muscarinic Receptors Inhibits Apoptosis in PC12M1 Cells
Authors: Lindenboim Liora , Pinkas‐Kramarski Ronit , Sokolovsky Mordechai , Stein Reuven
Published: July 2010

Abstract: Previous studies have shown that PC12 cells depend on growth factors for their survival. When deprived of growth factors, the cells undergo a dying process termed “apoptosis” (programed cell death). We show here that muscarinic agonists inhibited the apoptotic death of growth factor‐deprived PC12M1 cells (PC12 cells stably expressing cloned m1 muscarinic acetylcholine receptors). This protective effect of the muscarinic agonists was observed in both proliferating and neuronal PC12M1 cells, was blocked by the muscarinic antagonist atropine, and was not observed in PC12 cells lacking m1 receptors. Muscarinic receptors therefore mediate inhibition of apoptosis in these cells. In addition to its effect on survival, the muscarinic agonist oxotremorine induced inhibition of DNA synthesis as well as growth arrest of exponentially growing PC12M1 cells at the S and G2/M phases of the cell cycle. Muscarinic receptors in these cells may therefore mediate inhibition of cell cycle progression.
Stimulation of Adenylyl Cyclase Mediated by Phospholipase C‐Linked M3 Muscarinic Receptor in Human Neuroblastoma SK‐N‐BE(2)C Cells
Authors: Suh Byung‐Chang , Kim Kyong‐Tai
Published: July 2010

Abstract: Muscarinic receptor in human neuroblastoma SK‐N‐BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5‐trisphosphate (IP3) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca2+ ([Ca2+]i) with an EC50 value of 35 µM. In addition, carbachol produced a 1.3–3‐fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E2 (PGE2). The M3 antagonist p‐fluorohexahydrosiladifenidol (IC50 = 0.5–0.8 µM) inhibited the increases in [Ca2+]i, IP3, and cAMP more effectively than the M1 antagonist pirenzepine (IC50 = 5–9 µM) and the M2 antagonist methoctramine (IC50 = 20–30 µM). The involvements of [Ca2+]i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol‐induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE2 on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 µM) clearly enhanced PGE2‐induced cAMP production. However, phorbol 12‐myristate 13‐acetate did not enhance PGE2‐stimulated cAMP production. These data suggest that phospholipase C‐linked M3 receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca2+‐dependent system in the neuronal cells.
Pharmacological Characterization of the Voltage‐Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System
Authors: Maubecin Verónica Alvarez , Sanchez Viviana N. , Rosato Siri Marcelo D. , Cherksey Bruce D. , Sugimori Mutzuyuki , Llinás Rodolfo , Uchitel Osvaldo D.
Published: July 2010

Abstract: The voltage‐dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L‐type channels (1,4‐dihydropyridines), N‐type channels (ω‐conotoxin GVIA), and P‐type channels [funnel web toxin (FTX) and ω‐agatoxin IVA]. K+‐induced Ca2+ uptake by chicken synaptosomes was blocked by ω‐conotoxin GVIA (IC50 = 250 nM). This toxin at 5 µM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and ω‐agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω‐agatoxin IVA affected Ca2+ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with ω‐agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω‐conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω‐conotoxin GVIA was never greater than the one observed with ω‐conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by ω‐conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N‐type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P‐type Ca2+ channel blockers, thus indicating that P‐type channels are predominant in this preparation. In contrast, Ca2+ uptake by chicken synaptosomes is sensitive to ω‐conotoxin GVIA, FTX, ω‐agatoxin IVA, and ω‐conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.
Anesthetic Barbiturates Enhance Gsα‐Dependent Cyclic AMP Production in S49 Mouse Lymphoma Cells
Authors: Gonzales Jerry M.
Published: July 2010

Abstract: Cyclic AMP (cAMP) regulates many important physiological processes. Barbiturates influence cAMP regulation, possibly through effects on G proteins. This study used intact S49 mouse lymphoma cells to characterize the role of G proteins in the effect of barbiturates on cAMP regulation. cAMP accumulation was determined in intact S49 WT (wild‐type) and S49 cyc− cells (the Gsα‐deficient mutant) by measuring the conversion of [3H]‐ATP to [3H]cAMP in cells preloaded with [3H]adenine. Pentobarbital enhanced cAMP accumulation in WT cells in the absence (basal) or presence of isoproterenol but had no effect on the EC50 for isoproterenol. This effect was dose dependent with a 50–60% enhancement at 2 mM pentobarbital. Pentobarbital did not affect forskolin‐stimulated cAMP accumulation in WT cells. In cyc− cells, basal and forskolin‐stimulated cAMP accumulation were stimulated only at the highest concentration of pentobarbital used (2 mM). Pentobarbital did not affect the inhibition of cAMP accumulation by somatostatin in WT cells, and pertussis toxin treatment of WT cells did not affect the action of pentobarbital on cAMP accumulation. Pentobarbital did not affect isoproterenol‐stimulated adenylyl cyclase activity in whole‐cell homogenates or membranes prepared from WT cells. The S‐(−)‐isomer of pentobarbital enhanced isoproterenol‐stimulated cAMP accumulation more than the R‐(+)‐isomer. Phenobarbital and barbituric acid did not enhance isoproterenol‐stimulated cAMP accumulation, whereas the anesthetic barbiturates hexobarbital, pentobarbital, and thiopental all enhanced activity. These results suggest that pentobarbital enhances cAMP accumulation in intact WT cells by a mechanism that is dependent on Gsα but independent of Gi. The properties of barbiturates that are responsible for the enhancement of cAMP accumulation may be related to the properties that are responsible for producing sedation and anesthesia.
Opposite Changes in GABAA Receptor Function in the CA1‐3 Area and Fascia Dentata of Kindled Rat Hippocampus
Authors: Titulaer M. N. G. , Ghijsen W. E. J. M. , Kamphuis W. , De Rijk T. C. , Lopes da Silva F. H.
Published: July 2010

Abstract: Muscimol‐stimulated radiotracer 36Cl− uptake in synaptoneurosomes was used to investigate the function of the GABAA receptor complex in the CA1‐3 area and fascia dentata (granular and molecular layers and hilus) of rats kindled by stimulation, twice a day, of the Schaffer collateral fibers. Two kindled groups were studied: (a) 24 h after the last generalized tonic‐clonic seizure [fully kindled (FK) stage] and (b) 28 days after the last generalized seizure (long‐term stage). Synaptoneurosomes were prepared in parallel from subslices of the CA1‐3 area and fascia dentata. In FK animals, the muscimol‐stimulated 36Cl− uptake was significantly reduced by 21% in the CA1‐3 area in comparison with nonstimulated controls, whereas a significant increase of 29% was found in the fascia dentata. Significant changes were no longer present at 4 weeks after the last generalized seizure. The observed changes in muscimol‐stimulated 36Cl− uptake at the FK stage closely parallel the recently observed changes in [3H]muscimol binding in the CA1 area and fascia dentata. These results indicate that kindling causes a transiently decreased GABAA receptor‐mediated function in the CA1‐3, in contrast to an increased GABAA receptor‐mediated function in the fascia dentata.
Carbonyl‐Related Posttranslational Modification of Neurofilament Protein in the Neurofibrillary Pathology of Alzheimer's Disease
Authors: Smith Mark A. , Rudnicka‐Nawrot Maria , Richey Peggy L. , Praprotnik Darja , Mulvihill Paul , Miller Carol A. , Sayre Lawrence M. , Perry George
Published: July 2010

Abstract: We present the first evidence for carbonyl‐related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimer's disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress‐type mechanisms, through the formation of cross‐links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo.
Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108‐15 Neuroblastoma/Glioma Hybrid Cells
Authors: Fried Gabriel , Han Hui Quan , Meister Björn , Hökfelt Tomas , Greengard Paul
Published: July 2010

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108‐15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E1 plus 3‐isobutyl‐1‐methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense‐cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense‐cored vesicles and of an extracellular matrix protein associated with neuronal maturation.
Preincubation with Creatine Enhances Levels of Creatine Phosphate and Prevents Anoxic Damage in Rat Hippocampal Slices
Authors: Carter Adrian J. , Müller R. Enzio , Pschorn Uwe , Stransky Werner
Published: July 2010

Abstract: We have investigated the relationship between energy metabolism, NMDA‐receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [14C]lysine into perchloric acid‐insoluble tissue extracts. The concentrations of energy metabolites were measured by ion‐exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose‐dependently (0.03–3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia‐induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with (R,S)‐3‐guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia‐induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK‐801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.
Choroid Plexus Potassium Cotransport: Modulation by Osmotic Stress and External Potassium
Authors: Keep Richard F. , Xiang Jianming
Published: July 2010

Abstract: The choroid plexuses are involved in CSF secretion and CSF K homeostasis. This study examines the potential role of K cotransport in these two processes using isolated rat lateral ventricle choroid plexuses. Bumetanide‐sensitive 86Rb influx and efflux were measured to assess the response of K cotransport to changes in media osmolality and K concentration. Alterations in osmolality had no effect on K uptake (in the presence or absence of bumetanide). However, the efflux rate constant for K was 0.29 ± 0.02, 0.44 ± 0.04, and 0.84 ± 0.06 min−1 in 240, 300, and 424 mOsm/kg solutions, respectively (p < 0.001). This increase in efflux with osmolality, an opposite effect to that found in many cells, was solely due to enhanced K cotransport. The increased cotransport may be involved in limiting brain shrinkage during hyperosmotic stress if the cotransporter is present on the apical membrane. The rate of bumetanide‐sensitive efflux was unaffected by changes in external [K]. However, the rate of K uptake (measured on return to normal [K] media) was reduced gradually by exposure to low [K]. It was 21 ± 1, 19 ± 3, 13 ± 2, and 6 ± 1 nmol/mg/min after 0, 10, 30, and 60‐min exposure to 1 mM K. Sixty minutes of exposure to 1 mM [K] abolished the bumetanide‐sensitive K uptake present in plexuses exposed continually to normal media. This modulation of K cotransport by external [K] may be important in CSF K homeostasis by limiting K loss from the CSF if CSF [K] is low.
Dopamine D2 Receptors Potentiate Arachidonate Release via Activation of Cytosolic, Arachidonate‐Specific Phospholipase A2
Authors: Vial Daniel , Piomelli Daniele
Published: July 2010

Abstract: Several Gi‐linked neurotransmitter receptors, including dopamine D2 receptors, act synergistically with Ca2+‐mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor‐dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D2‐receptor cDNA, CHO(D2). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA‐specific phospholipase A2 (cPLA2) were synthesized and added to cultures of CHO(D2) cells. Incubation with antisense oligodeoxynucleotides inhibited D2 receptor‐dependent release of AA but had no effect on D2‐receptor binding or D2 inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D2 receptor‐dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane‐bound, non‐AA‐specific PLA2 (such as p‐bromophenacyl bromide). cPLA2 is expressed in brain tissue. The results, showing that cPLA2 participates in receptor‐dependent potentiation of AA release in CHO(D2) cells, suggest that this phospholipase may serve a similar signaling function in brain.
Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP
Authors: Kirk Ian P. , Richardson Peter J.
Published: July 2010

Abstract: The adenosine A2a receptor inhibition of potassium (15 mM)‐evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 mM) reduced the effect of the A2a receptor agonist CGS‐21680 (1 nM). CGS‐21680 inhibited GABA release in the presence of the L‐type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω‐Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS‐21680. Forskolin and 8‐bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS‐21680 without affecting the evoked release. The nonselective protein kinase inhibitor H‐7 inhibited both the evoked release and the inhibition by CGS‐21680, whereas the selective protein kinase A and G inhibitor HA‐1004 had no effect on either evoked release or the action of CGS‐21680. Pretreatment with pertussis toxin did not affect the A2a receptor‐mediated inhibition. Therefore, the effect of A2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N‐type calcium channel and protein kinase C.
Molecular Characterization of a Type II Cyclic GMP‐Dependent Protein Kinase Expressed in the Rat Brain
Authors: El‐Husseini Alla El‐Din , Bladen Christopher , Vincent Steven R.
Published: July 2010

Abstract: We applied reverse transcription‐PCR to examine the gene expression of cyclic GMP (cGMP)‐dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP‐dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.
Identification of New Oligodendrocyte‐ and Myelin‐Specific Genes by a Differential Screening Approach
Authors: Schaeren‐Wiemers N. , Schaefer C. , Valenzuela D. M. , Yancopoulos G. D. , Schwab M. E.
Published: July 2010

Abstract: We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte‐free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide‐synthesizing enzyme UDP‐galactose:ceramide galactosyl‐transferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T‐lymphocyte‐specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl‐CoA‐desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long‐chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the β‐tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.
Ascorbate Transport and Intracellular Concentration in Cerebral Astrocytes
Authors: Siushansian Ramin , Wilson John X.
Published: July 2010

Abstract: Regulation of the initial rate of uptake and steady‐state concentration of ascorbate (reduced vitamin C) was investigated in rat cerebral astrocytes. Although these cells did not synthesize vitamin C, they accumulated millimolar concentrations of ascorbate when incubated with medium containing the vitamin at a level (200 µM) typical of brain extracellular fluid. Initial rate of [14C]‐ascorbate uptake and intracellular ascorbate concentration were dependent on extracellular Na+ and sensitive to the anion transport inhibitor sulfinpyrazone. Comparison of the efflux profiles of ascorbate and 2′,7′‐bis(carboxyethyl)‐5 (or ‐6)‐carboxyfluorescein from astrocytes permeabilized with digitonin localized most intracellular ascorbate to the cytosol. Pretreatment of astrocytes with dibutyryl cyclic AMP (dBcAMP) doubled their initial rate of sulfinpyrazone‐sensitive [14C]ascorbate uptake compared with cells treated with either n‐butyric acid or vehicle. dBcAMP also increased steady‐state intracellular ascorbate concentration by 39%. The relatively small size of the change in astrocytic ascorbate concentration was explained by the finding that dBcAMP increased the rate of efflux of the vitamin from ascorbate‐loaded cells. These results indicate that uptake and efflux pathways are stimulated by cyclic AMP‐dependent mechanisms and that they regulate the cytosolic concentration of ascorbate in astrocytes.
Delayed Excitotoxic Neurodegeneration Induced by Excitatory Amino Acid Agonists in Isolated Retina
Authors: Romano Carmelo , Price Madelon T. , Olney John W.
Published: July 2010

Abstract: Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N‐methyl‐d‐aspartate (NMDA) or non‐NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non‐NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non‐NMDA agonists kainate (KA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non‐NMDA receptors, as a combination of receptor‐selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the retina. It is interesting that brief exposure to the non‐NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delayed excitotoxic neurodegeneration.
Characterization of l‐Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors
Authors: Stuhlmiller David F. E. , Boje Kathleen M. K.
Published: July 2010

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N‐methyl‐d‐aspartate‐mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l‐[14C]arginine and the NOS inhibitor [14C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l‐[14C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors NG‐methyl‐l‐arginine, NG‐amino‐l‐arginine, and N5‐imidoethyl‐l‐ornithine. l‐[14C]Arginine uptake was not inhibited by aminoguanidine or NG‐nitro‐l‐arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l‐arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [14C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l‐[14C]arginine.