bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake, Release, and Metabolism of Citrate in Neurons and Astrocytes in Primary Cultures
Authors: Westergaard Niels , Sonnewald Ursula , Unsgård Germund , Peng Liang , Hertz Leif , Schousboe Arne
Published: July 2010

Abstract: Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (∼70 nmol × h−1× mg of protein−1). The rate of release of citrate from neurons was <5% of this value. Using [14C]citrate it could be shown that citrate was oxidatively metabolized to 14CO2 at a modest rate (∼1 nmol × n−1× mg−1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca2+ and Mg2+ by chelation, thereby modulating neuronal excitability.
Hippocampal Serotonin 5‐HT1A Receptor Enhances Acetylcholine Release in Conscious Rats
Authors: Izumi Junkichi , Washizuka Masataka , Miura Naoyoshi , Hiraga Yoshihiro , Ikeda Yugo
Published: July 2010

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5‐HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; 20 μM), a 5‐HT1A agonist, increased ACh levels, whereas 7‐trifluoromethyl‐4‐(4‐methyl‐1 ‐piperazinyl)‐pymoto[1,2‐a]quinoxaline (CGS‐12066B; 100 μM), a 5‐HT1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5‐HT, had no effect on ACh levels. Following perfusion of 1‐(2‐methoxyphenyl)‐4‐[4‐ (2‐phthalimido)butyl]piperazine (NAN‐190; 10 μM), which is a selective 5‐HT1A antagonist, the effect of 8‐OH‐DPAT was totally abolished, whereas CGS‐12066B decreased extracellular ACh levels. 5‐HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN‐190. These results indicate that the 5‐HT1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5‐HT1A receptor and the suppressive one via the 5‐HT1B receptor in the dorsal hippocampus of rats.
Characterization of Low pH‐induced Catecholamine Secretion in the Rat Adrenal Medulla
Authors: Fujiwara Naoshi , Warashina Akira , Shimoji Koki
Published: July 2010

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES‐buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O2/5% CO2. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ∼6.4. The low pH‐induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca2+ antagonist, 3,4,5‐trimethoxybenzoic acid 8‐(N,N‐diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage‐gated Ca2+ channel, but were completely inhibited in Ca2+‐free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH4CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH‐induced CA secretion may be mediated by dihydropyridine‐insensitive Ca2+ influx. Furthermore, the initial transient phase of the low pH‐induced CA secretion might be caused by a Ca2+ release from intracellular stores, which is also induced by the Ca2+ influx.
Glutamine Transaminase K and ω‐Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching
Authors: Makar Tapas K. , Nedergaard Maiken , Preuss Axel , Hertz Leif , Cooper Arthur J. L.
Published: July 2010

Abstract: Glutamine transaminase K and ω‐amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of gluta‐mine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (< 15‐fold). Cerebral ω‐amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and ω‐amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of ω‐amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S‐conjugate β‐lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.
Diurnal Variations in Cyclic AMP and Melatonin Content of Golden Hamster Retina
Authors: Faillace María P. , Sarmiento María I. Keller , Siri Leonardo Nicola , Rosenstein Ruth E.
Published: July 2010

Abstract: The diurnal variations and photic regulation of cyclic AMP and melatonin content in golden hamster retina were studied. Both parameters showed significant diurnal variations with maximal values at night. Light exposure during the night inhibited retinal cyclic AMP and melatonin levels, whereas exposure to darkness during the day significantly increased cyclic AMP and melatonin content. Incubation with melatonin of retinas excised at different intervals indicated that the methoxyindole inhibited cyclic AMP accumulation in a time‐dependent manner. The inhibitory effect of melatonin at 2400 h and at noon showed a threshold concentration of 1 and 10 pM, respectively. At 0400 h melatonin did not affect cyclic AMP accumulation. The results indicate a diurnal variability of retinal cyclic AMP and melatonin content in hamsters, mainly influenced by a photic stimulus. Cyclic AMP could be a putative second messenger for melatonin action in golden hamster retina.
Cross Talk Among Cyclic AMP, Cyclic GMP, and Ca2+ ‐Dependent Intracellular Signalling Mechanisms in Brain Capillary Endothelial Cells
Authors: Vigne Paul , Lund Lise , Frelin Christian
Published: July 2010

Abstract: C‐type natriuretic peptide and sodium nitroprusside, a nitric oxide donor molecule, induced large increases in cyclic GMP formation in cultured rat brain capillary endothelial cells. Isoproterenol, a potent agonist of adenylate cyclase, potentiated the actions of C‐type natriuretic peptide and of sodium nitroprusside. These actions were not observed in the presence of isobutylmethylxanthine and were mimicked by forskolin. Endothelin‐1 had no action on basal cyclic GMP levels. It reduced cyclic GMP formation induced by C‐type natriuretic peptide and sodium nitroprusside by about 50%. These actions involved an ETA receptor subtype and a Ca2+‐dependent and protein kinase C‐independent mechanism. Finally, increasing cyclic GMP slightly prolonged intracellular Ca2+ transients induced by endothelin‐1. The results suggest the presence of extensive cross talk among cyclic AMP, cyclic GMP, and Ca2+‐dependent mechanisms in endothelial cells of brain microvessels. The relevance of the results to the regulation of the blood‐brain barrier permeability is discussed.
Direct Measurement of Extracellular Lactate in the Human Hippocampus During Spontaneous Seizures
Authors: During Matthew J. , Fried Itzhak , Leone Paola , Katz Amiram , Spencer Dennis D.
Published: July 2010

Abstract: The effect of clinical, spontaneous‐onset seizures on extracellular fluid lactate was investigated by the method of lactography, the in vivo on‐line measurement of lactate levels using microdialysis. Studies of experimental animals have suggested that generation of extracellular lactate as measured by microdialysis is an index of local glucose utilization and is dependent on the activity of neurons under physiological conditions. Patients with medically refractory complex partial epilepsy underwent stereo‐tactic implantation of combination depth electrode/micro‐dialysis probes into both hippocampi for 7–16 days. During spontaneous complex partial seizures with secondary generalization, extracellular lactate levels rose by 91 β 32%. Moreover, this increase persisted for 60–90 min. During a unilateral hippocampal seizure that did not propagate to the contralateral hippocampus, the increase in lactate content was restricted to the side of seizure activity. Between seizures, extracellular lactate levels correlated with the frequency of interictal spikes. In summary, these data suggest that brief clinical seizures increase nonoxidative glucose metabolism significantly as measured by the generation of extracellular lactate. Furthermore, the increase in extracellular lactate level is limited to the site of seizure activity. Lactate is transported extracellularly via a lactate/proton cotransporter; therefore, the rise in extracellular lactate level may mediate the drop in pHo associated with seizure activity. As acidification of the extracellular compartment has an inhibitory effect on neuronal excitability, the rise in extracellular lactate content may be a mechanism of seizure arrest and postictal refractoriness. Moreover, extracellular lactate may also mediate the decreased seizure susceptibility associated with frequent interictal spikes.
Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells
Authors: Sampath Deepa , Jackson George R. , Werrbach‐Perez Karin , Perez‐Polo J. Regino
Published: July 2010

Abstract: Nerve growth factor (NGF) is a member of the neuro‐ trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per‐ oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time‐ and dose‐dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.
(S)‐Carboxy‐3‐Hydroxyphenylglycine, an Antagonist of Metabotropic Glutamate Receptor (mGluR)1a and an Agonist of mGluR2, Protects Against Audiogenic Seizures in DBA/2 Mice
Authors: Thomsen Christian , Klitgaard Henrik , Sheardown Malcolm , Jackson Helen C. , Eskesen Karen , Jacobsen Palle , Treppendahl Svend , Suzdak Peter D.
Published: July 2010

Abstract: The in vivo anticonvulsant effects and in vitro metabo‐tropic glutamate receptor selectivity of (S)‐4‐carboxy‐3‐hydroxy‐phenylglycine [(S)‐4C3HPG] were examined. Intracerebroventricular injection of (S)‐4C3HPG dose‐dependently antagonized audiogenic‐induced clonic and tonic convulsions in DBA/2 mice with ED60 values of 76 and 110‐nmol per mouse, respectively. (S)‐4C3HPG dose‐dependently inhibited the spontaneously evoked epileptic spikes in a cingulate cortex‐corpus callosum slice preparation. (SJ‐4C3HPG displaced the binding of [3H]glutamate in membranes prepared from baby hamster kidney (BHK) cells expressing the metabotropic glutamate receptor mGluR1a with an EC50 of 5 β 1 uM. (S)‐4C3HPG dose‐dependently antagonized glutamate‐stimulated phosphoinositide hydrolysis in BHK cells expressing mGluR 1a with an IC50 of 15 β 3 μM. (S)‐4C3HPG was, however, an agonist at mGluR2 with an EC60 of 21 β 4 μM for inhibition of forskolin‐stimulated cyclic AMP formation in BHK cells expressing the mGluR2. (S)‐4C3HPG had no effects at mGluR4a. These data suggest that the anticonvulsant action of (S)‐4C3HPG is mediated by combined antagonism of mGluRIa and agonism of mGluR2. These results suggest the importance of mGluR1a and/or mGluR2 in the control of epileptic activity.
Carbohydrate‐Binding Proteins in the Leech: I. Isolation and Characterization of Lactose‐Binding Proteins
Authors: Cole Robert N. , Zipser Birgit
Published: July 2010

Abstract: Three lactose‐binding proteins with apparent molecular masses of 16, 35, and 63 kDa [leech lectin 16, 35, and 63 (LL16, LL35, and LL63, respectively)] were isolated from leech membranes. Polyclonal antibodies raised against LL35 cross‐reacted with LL16 and LL63, indicating that all three lectins were immunologically related. These leech lectins, however, can be subdivided into two groups based on their tissue distributions and binding affinities for galactose derivatives. LL16 and LL35 are endogenous to the leech's CNS, whereas LL63 is only present in peripheral organs. LL16 and LL35, found in the CNS, bind both the α and β anomers of methylgalactose, whereas the peripheral lectin LL63 binds only the β form. LL35 and LL63 also differ in their binding affinities for galactosamine and N‐acetylgalactosamine. The binding activity of LL35 was calcium independent and active over a wide pH range. Triton X‐100 and 2‐mercaptoethanol were necessary to recover LL35 binding activity during extraction. These characteristics strongly suggest that LL35 is another member of the calcium‐independent galactose/lactose‐specific lectins previously described in vertebrates and recently demonstrated in sponges and nematodes. Because a single leech expresses up to 100 µg of LL35, this leech lectin is readily amenable to structural and functional analysis.
Serotonin Binding Protein: Synthesis, Secretion, and Recycling
Authors: Tamir Hadassah , Liu Kuo‐peing , Hsiung Shu‐chi , Adlersberg Mella , Gershon Michael D.
Published: July 2010

Abstract: Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5‐HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5‐HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56‐ and 45‐kDa forms of SBP are produced by posttranslational cleavage of a 68‐kDa precursor molecule; and (b) that 45‐kDa SBP is a constituent of serotonergic secretory vesicles. Pulse‐chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5‐HT and all three forms of SBP. Following pulse labeling for 20 min with l‐[35S]methionine, the cells were incubated in the presence of an excess of unlabeled l‐methionine for 0, 30, 60, or 90 min at 37°C. Alternatively, the chase was performed under conditions (20°C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S‐labeled 68‐, 56‐, and 45‐kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20°C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post‐Golgi compartment. When secretion was induced, 45‐kDa SBP, but not 56‐ or 68‐kDa SBP, was released to the medium. When antibodies to 45‐kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45‐kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces after 20 min, at which time antibodies to SBP were found inside the cells. It is suggested that 45‐kDa SBP is packaged with 5‐HT in secretory vesicles. Some 45‐kDa SBP is lost during secretion as a result of exocytosis; however, a fraction of the 45‐kDa SBP remains bound to the luminal surface of the membrane of secretory vesicles. This protein is exposed to the ambient medium as a consequence of exocytosis, but is reinternalized when the vesicular membrane is recaptured during vesicle recycling.
Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes
Authors: Giambalvo C. T. , Wagner R. L.
Published: July 2010

Abstract: The effects of D1 and D2 dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D1 agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D2 agonists [LY 171555, (+)‐3‐(3‐hydroxyphenyl)‐N‐n‐propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)‐3‐(3‐Hydroxyphenyl)‐N‐n‐propylpiperidine had a D2 antagonist profile. Apomorphine showed a biphasic dose‐response change; i.e., it decreased particulate PKC activity at the D2 receptor at low concentrations (0.1 µM) and increased it at the D1 receptor at higher concentrations (10 µM). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D2 agonists, but it reversed the changes in PKC activity induced by the D1 agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D1 and D2 dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D1 drugs in the presence of calcium is mediated indirectly via a transneuronal effect.
Muscarinic Receptor‐Mediated Inositol 1,4,5‐Trisphosphate Formation in SH‐SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization
Authors: Wojcikiewicz Richard J. H. , Tobin Andrew B. , Nahorski Stefan R.
Published: July 2010

Abstract: Stimulation of muscarinic receptors expressed in SH‐SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5‐trisphosphate (InsP3) accumulation, with a dramatic increase (six‐ to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ∼60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ channels with Ni2+ showed that the plateau phase was dependent upon Ca2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca2+ from intracellular stores revealed that Ca2+ from this source was capable of supporting the peak phase of the InsP3 response. Carbachol‐stimulated phosphoinositidase C activity in permeabilized SH‐SY5Y cells was also shown to be highly dependent on free Ca2+ concentration (20–100 nM) and suggests that under normal conditions, InsP3 formation is enhanced by increases in cytosolic free Ca2+ concentration that accompany muscarinic receptor activation. Measurement of carbachol‐stimulated total inositol phosphate accumulation in the presence of Li+ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor‐mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP3 accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca2+ concentration amplify InsP3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP3 accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca2+ or protein kinase C activity.
Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus
Authors: Cunha Rodrigo A. , Milusheva E. , Vizi E. S. , Ribeiro J. A. , Sebastião A. M.
Published: July 2010

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [3H]acetylcholine ([3H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A1/A2 agonist 2‐chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration‐dependent manner, the release of [3H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [3H]ACh release was prevented by the A1 antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A2A agonist CGS‐21680 (30 nM) produced a greater increase of the evoked release of [3H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS‐21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7‐dimethyl‐1‐propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [3H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [3H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A1 inhibitory receptors modulate ACh release, whereas in the CA3 area, both A2A excitatory and A1 inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A1 inhibitory and A2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.
Cyclothiazide Selectively Potentiates AMPA‐ and Kainate‐Induced [3H]Norepinephrine Release from Rat Hippocampal Slices
Authors: Desai Manisha A. , Burnett J. Paul , Schoepp Darryle D.
Published: July 2010

Abstract: Activation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA‐ and kainate (KA)‐induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA‐induced [3H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA‐ and KA‐induced [3H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N‐methyl‐d‐aspartate‐induced [3H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4‐aminopyridine. These results demonstrate that AMPA receptor‐mediated modulation of [3H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.
ADP‐Ribosylation of Human Myelin Basic Protein
Authors: Boulias Chris , Moscarello Mario A.
Published: July 2010

Abstract: When isolated myelin membranes were ADP‐ribosylated by [32P]NAD+ either in the absence of toxin (by the membrane ADP‐ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP‐ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP‐ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline‐urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP‐ribosylated. Component 1 (C‐1), the most cationic charge isomer, incorporated 1.79 mol of ADP‐ribose/mol of protein. C‐2 and C‐3 (which differ from C‐1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP‐ribose/mol of protein, respectively, whereas C‐8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP‐ribosyl bond was shown to have a half‐life of about 80 min, suggesting an N‐glycosidic linkage between ADP‐ribose and an arginyl residue of the protein. As MBP contains several components that are ADP‐ribosylated to different specific activities, the use of MBP, ADP‐ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP‐ribosylated, an endoproteinase Lys‐C digest of C‐1 ADP‐ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed‐phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP‐ribose/nmol of peptide, respectively). The ADP‐ribosylated residues were identified as Arg9 and Arg54 by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.
Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS
Authors: Glasgow Eric , Druger Robert K. , Fuchs Chana , Levine Edward M. , Giordano Suzanne , Schechter Nisson
Published: July 2010

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two‐dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.
Isolated Cerebral and Cerebellar Mitochondria Produce Free Radicals when Exposed to Elevated Ca2+ and Na+: Implications for Neurodegeneration
Authors: Dykens James A.
Published: July 2010

Abstract: The evidence is compelling that free radicals, plus increases in free cytosolic Ca2+ and Na+, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca2+ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (•OH) radicals, plus ascorbyl and other carbon‐centered radicals when exposed to 2.5 µM Ca2+, 14 mM Na+, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin‐induced neurodegeneration. In a feed‐forward cycle, exposure of isolated mitochondria to •OH significantly increases subsequent radical production five‐ to 16‐fold (average = 8.8 ± 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH‐CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after •OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.
Effects of Ischaemic Conditions on Uptake of Glutamate, Aspartate, and Noradrenaline by Cell Lines Derived from the Human Nervous System
Authors: O'Neill Clare M. , Ball Stephen G. , Vaughan Peter F. T.
Published: July 2010

Abstract: The effect of hypoglycaemic, hypoxic, and ischaemic conditions on high‐affinity neurotransmitter transport was studied in the human astrocytoma clone D384 and the human neuroblastoma clone SH‐SY5Y. Both cell lines expressed a sodium‐dependent glutamate/aspartate transporter. Km values for d‐[3H]aspartate uptake were 6.1 ± 0.9 µM for D384 cells and 5.3 ± 0.3 µM for SH‐SY5Y cells (mean ± SEM of three experiments). In addition, SH‐SY5Y, but not D384, expressed a sodium‐dependent noradrenaline transporter with Km = 0.6 ± 0.1 µM (mean ± SEM of three experiments). Up to 3 h of hypoglycaemic conditions had no effect on neurotransmitter uptake or on ATP levels of each cell line. In sharp contrast, during hypoxic conditions, the uptake of d‐[3H]aspartate and [3H]noradrenaline declined by 43–56% within 5 min. These reduced rates of neurotransmitter uptake were maintained over 30 min of hypoxic conditions. Five minutes of ischaemic conditions caused similar reductions in neurotransmitter uptake rates. A correlation between reductions in rates of neurotransmitter uptake and in ATP levels was observed for each cell line. Results are discussed in relation to other brain preparations, which are used as models of the nervous system to study the effects of ischaemic conditions on neurotransmitter and energy metabolism.
Increased Retinal Synthesis of Heparan Sulfate Proteoglycan and HNK‐1 Glycoproteins Following Photoreceptor Degeneration
Authors: Landers Robert A. , Rayborn Mary E. , Myers Kathleen M. , Hollyfield Joe G.
Published: July 2010

Abstract: In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of 35S‐amino acid utilization reported here confirm that the 35SO42− differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of 35SO42−‐labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na235SO4 isolated retinal cultures from the following: (a) mice with light‐induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light‐induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan‐enriched fractions were prepared from the incubation media and guanidine HCI/detergent extracts of the retinas by ion‐exchange chromatography. The 35SO42−‐proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of 35SO42−‐HSPG found in control incubations. The RCS and light‐damaged rats also showed increased synthesis of 35SO42−‐chondroitin sulfate proteoglycan relative to the control, though the increase was of lesser magnitude than the HSPG effect. 35SO42−‐protein in degenerate and light‐damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N‐linked oligosaccharides, including several recognized by the HNK‐1 antibody. These data suggest that a sustained increase in HSPG and HNK‐1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.
Secreted Forms of β‐Amyloid Precursor Protein Protect Against Ischemic Brain Injury
Authors: Smith‐Swintosky Virginia L. , Pettigrew L. Creed , Craddock Susan D. , Culwell Alan R. , Rydel Russell E. , Mattson Mark P.
Published: July 2010

Abstract: The β‐amyloid precursor protein (βAPP) is the source of the amyloid β‐peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β‐peptide sequence that liberates secreted forms of βAPP (APPSs) into the extracellular milieu. We now report that postischemic administration of these APPSs intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APPS695 or APPS751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APPSs in vivo.
A New Look at the Promoter of the Human Monoamine Oxidase A Gene: Mapping Transcription Initiation Sites and Capacity to Drive Luciferase Expression
Authors: Denney Richard M. , Sharma Abha , Dave Sanat K. , Waguespack Ann
Published: July 2010

Abstract: Monoamine oxidase (MAO) A (EC 1.4.3.4) oxidizes norepinephrine and serotonin and is expressed in a cell type‐specific manner. Recent evidence that MAO A‐deficient males in a large Dutch kindred suffer from mild mental retardation and occasional episodes of impulsive aggressive behavior makes it important to understand how the human MAO A promoter is regulated. Conventional primer extension analyses of MAO A mRNA in earlier studies predicted incorrect transcription initiation sites for the human MAO A promoter. Reverse transcription and polymerase chain reaction (PCR) readily detected MAO A mRNA initiated 5′ to −135 bp but not 5′ to −226 bp (5′ to the ATG initiation codon). PCR‐assisted primer extension and RNase protection assays reveal that most MAO A mRNA is initiated between −30 and −40, which resembles a eukaryotic initiator element. Depending on the tissue source, a minor, variable proportion of MAO A mRNAs is initiated more distally at approximately −95 and −136, within the more proximal of two 90‐bp GC‐rich tandem repeats. Genomic DNA segments spanning −4 to −200 and −465 or −935, but not −4 to −82, drive robust luciferase expression in mammalian cells. We conclude that (a) the primary transcription initiation site occurs at a putative initiator (Inr) element located between −30 and −40, with a minor, tissue‐specific proportion of additional initiation near −95 and −136; and (b) MAO A‐luciferase reporter constructs that contained all the known transcription initiation sites exhibited no evidence for inhibitory cis elements between −200 and at least −935. The apparent inhibitory activity previously reported for sequences 5′ to the most proximal PvuII site may have resulted from the use of partial promoter constructs that omitted the putative Inr element.
Microtubule‐Associated Protein 2 as an Early Indicator of Ischemia‐Induced Neurodegeneration in the Gerbil Forebrain
Authors: Matesic Diane F. , Lin Rick C. S.
Published: July 2010

Abstract: Microtubule‐associated protein 2 (MAP‐2) was studied in the gerbil hippocampus and striatum after transient ischemia. Western immunoblot analysis shows that there is a significant decrease of MAP‐2 in the dorsolateral sector of the striatum and a slight decrease of MAP‐2 in the CA1 region of the hippocampus 6–12 h after ischemia in the gerbil forebrain. The immunohistochemical staining pattern of MAP‐2 in these two regions also shows a loss of immunostaining of MAP‐2. In particular, a beaded MAP‐2 immunostaining pattern at the apical dendritic region of the CA1 neurons of the hippocampus was found within 12 h after ischemia compared with the smooth dendritic immunostaining of MAP‐2 in normal CA1 neurons. In vitro assays of MAP‐2 degradation suggest that dendritic loss of immunoreactivity after ischemia seen on western blots may be due to calpain I degradation of MAP‐2. Loss of MAP‐2 in both the striatum and hippocampus was found to occur earlier than spectrin degradation by western blot analysis. These results suggest that loss of MAP‐2 may participate in the initial phase of neuronal dysfunction and that dendritic breakdown may be a first sign of neurodegeneration.
Accurate Differentiation of Neuronopathic and Nonneuronopathic Forms of Niemann‐Pick Disease by Evaluation of the Effective Residual Lysosomal Sphingomyelinase Activity in Intact Cells
Authors: Graber Denis , Salvayre Robert , Levade Thierry
Published: July 2010

Abstract: Niemann‐Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein‐Barr virus‐transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low‐density lipoproteins. This lipoprotein‐associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short‐term pulse‐chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half‐time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann‐Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann‐Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann‐Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.
Characterization of gp93, a Novel, Highly Heterogeneous Glycoprotein Present in Growth Cone Membranes
Authors: Quiroga Santiago , Pfenninger Karl H.
Published: July 2010

Abstract: gp93 was first described in growth cones from fetal rat brain as a 90–97‐kDa glycoprotein family that binds wheat‐germ agglutinin and consists of at least 12 different isoelectric variants (pl range ∼4.9–6.4). Of particular interest is that different sets of gp93 variants are expressed in growth cones isolated from different brain regions. The preparation of a polyclonal antibody to gp93 allowed further characterization of this glycoprotein. The carbohydrate groups of gp93 were partially characterized by digestion with different glycosidases. The results indicate that most or all oligosaccharide units are N‐linked (asparagine‐linked) and contain sialic acid. Two‐dimensional polyacrylamide gel electrophoresis and western blot with anti‐gp93 show that deglycosylated gp93 is an only slightly heterogeneous polypeptide of 66 kDa, indicating that gp93 heterogeneity is due, primarily or exclusively, to differential glycosylation. Analysis of the tissue distribution in fetal rat showed gp93 to be highly enriched in the brain. Immunoblots and immunostaining of cross sections of developing cerebellum revealed that gp93 is developmentally regulated in this tissue, associated primarily with growing parallel fibers and Purkinje dendrites. Immunostaining of neurons in culture shows significant amounts of gp93 in elongating neurites and growth cones. Our results indicate that gp93 is a developmentally regulated glycoprotein of the brain that is most prominent in growth cones and growing neurites and that appears to be glycosylated differentially by different neurons.
In Vitro Hypoxia Induces Expression of the NR2C Subunit of the NMDA Receptor in Rat Cortex and Hippocampus
Authors: Perez‐Velazquez Jose L. , Zhang Liang
Published: July 2010

Abstract: In mammalian brain, ischemic injury could be mediated by delayed glutamate neurotoxicity. We have studied the possibility of altered genetic expression of quiescent NMDA receptor subunits in an in vitro model of hypoxia‐hypoglycemia, using the reverse transcription‐polymerase chain reaction technique. It was found that mRNA corresponding to the NR2C subunit was present 1 h after a brief hypoxic‐hypoglycemic episode in adult rat hippocampus and cortex but absent in control tissue. These findings indicate that the phenotypic characteristics of certain brain cells after an ischemic insult are altered by the expression of genes that are quiescent in those cells under normal physiological conditions.
Are Striatal Dopamine D4 Receptors Increased in Schizophrenia?
Authors: Reynolds Gavin P. , Mason Sarah L.
Published: July 2010

Abstract: The density of dopamine D2‐like receptors was determined using [3H]emonapride binding in putamen tissue taken postmortem from schizophrenic subjects and matched controls. A 72% increase in number of these receptors was identified in the schizophrenics, although three patients not receiving antipsychotic drug treatment before death exhibited receptor densities in the control range. Displacement of 1 nM [3H]emonapride binding by raclopride was used to define the contribution of the D4 subtype of dopamine receptors to total [3H]emonapride binding. No evidence was obtained for the presence of D4 receptors in putamen tissue from either control or schizophrenic subjects, indicating that the increase in D2‐like receptor density in schizophrenia is due not to an increase in number of D4 sites in the disease, but to an up‐regulation of D2 or D3 receptors probably induced by chronic treatment with antipsychotic drugs.
RNA Editing of the Glutamate Receptor Subunits GluR2 and GluR6 in Human Brain Tissue
Authors: Paschen Wulf , Hedreen John C. , Ross Christopher A.
Published: July 2010

Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ∼10% edited compared with ∼90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue‐specific regulation.