bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulphur‐Containing Amino Acids Modulate Noradrenaline Release from Hippocampal Slices
Authors: Selema G. , Cristòfol R. M. , Gassó S. , Griffiths R. , Rodríguez‐Farré E.
Published: July 2010

Abstract: The l‐ and d‐enantiomers of the sulphur‐containing amino acids (SAAs)—homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S‐sulphocysteine—stimulated [3H]noradrenaline release from rat hippocampal slices in a concentration‐dependent manner. The relative potencies of the l‐isomers (EC50 values of 1.05–1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10‐fold lower than that of NMDA (0.15 mM), whereas the d‐isomers exhibited a wider range of potencies (0.75 to >5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage‐sensitive Na+ channel blocker tetrodotoxin (55–71%) and completely blocked by addition of Mg2+ or Co2+ to the incubation medium. All SAA‐evoked responses were concentration‐dependently antagonized by the selective NMDA receptor antagonist d‐(−)‐2‐amino‐5‐phosphonopentanoic acid (IC50 values of 3.2–49.5 µM). 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a non‐NMDA receptor antagonist, at 100 µM inhibited the [3H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65–85%), whereas 10 µM CNQX only inhibited the effects of S‐sulpho‐l‐cysteine and l‐ and d‐homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2,3‐dione (100 µM), which did not antagonize the [3H]noradrenaline release induced by glutamate and NMDA, reduced only the S‐sulpho‐l‐cysteine‐evoked response (25%). Thus, the stimulation of Ca2+‐dependent[3H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.
Occurrence of Two Types of Secretory Vesicles in the Human Neuroblastoma SH‐SY5Y
Authors: Goodall Anna R. , Danks Kirsty , Walker John H. , Ball Stephen G. , Vaughan Peter F. T.
Published: July 2010

Abstract: Western blot analysis showed that the human neuroblastoma SH‐SY5Y expresses the proteins synaptotagmin I, synaptobrevin, synapsin I, rab3a, syntaxin, SNAP‐25, NSF, α‐SNAP, and munc‐18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin I, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine‐β‐hydroxylase on sucrose‐D2O gradient fractions indicates the presence of a population of noradrenaline‐containing large dense‐cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48‐kDa synaptophysin antigen absent from the large dense‐cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin I contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH‐SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization‐evoked release of noradrenaline from SH‐SY5Y occurs by LDCV exocytosis. This enhances the value of SH‐SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.
Sublytic Terminal Complement Attack on Myotubes Decreases the Expression of mRNAs Encoding Muscle‐Specific Proteins
Authors: Lang Thomas J. , Badea Tudor C. , Wade Robert , Shin Moon L.
Published: July 2010

Abstract: Activation of inflammatory and cytotoxic complement effectors that include the C5b‐9 complex plays an important pathogenic role in myasthenia gravis, an inflammatory autoimmune disease of the muscle. Altered muscle‐specific gene expression has been observed in experimental myasthenic rats. In this study, we have examined the effect of sublytic C5b‐9 on myotubes differentiated from C2C12 myoblasts, by generating C5b‐9 with C7‐deficient serum with or without C7. Within 2 h, C7‐deficient serum plus C7, compared with C7‐deficient serum alone, induced markedly decreased levels of mRNAs encoding α‐actin, troponin I slow twitch isoform, acetylcholine receptor α, and muscle aldolase A, whereas the heat shock protein 83 mRNA level remained constant, by northern analysis. Because the half‐life of the acetylcholine receptor α was estimated to be >8 h, the C5b‐9 effect was, in part, due to enhanced mRNA decay. Because C5b‐9 also induced c‐jun mRNA and reduced the myoD mRNA level, a possible inhibition of muscle gene transcription by C5b‐9 was examined in myotubes transfected with troponin promoter‐luciferase gene constructs. Luciferase activity was reduced to 50% in response to C5b‐9 at 2 h. Thus, C5b‐9 appears to inhibit the muscle‐specific gene expression by stimulating mRNA decay and by decreasing the transcription process. The data also indicate a possible pathogenic role of C5b‐9 in immune‐mediated inflammatory muscle disorders in which complement activation has been implicated.
Functional Analysis of the Mouse Myelin/Oligodendrocyte Glycoprotein Gene Promoter in the Oligodendroglial CG4 Cell Line
Authors: Solly S. K. , Daubas P. , Monge M. , Dautigny A. , Zalc B.
Published: July 2010

Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a late phylogenetic acquisition among vertebrates that is found only in mammals. MOG is a minor component of myelin protein, representing ∼0.01–0.05% of the total. Regulatory elements in the MOG gene were identified by transfecting the oligodendroglial CG4 cell line with chimeric MOG‐luciferase genes. Only a few hundred base pairs upstream of the coding sequence were necessary for high‐level activity of the mouse MOG promoter. More distal recognition sites may exist, because silencing activity, indicative of negative regulatory elements, was detected upstream of base pair 657. Transcriptional activity of chimeric MOG‐ and myelin basic protein‐luciferase genes was greater in CG4 cells than in 3T3 fibroblasts or C6 glioblastoma, demonstrating their superiority for functional analysis of myelin gene regulatory elements.
Stress‐Activated Protein Kinase/c‐Jun N‐Terminal Kinase Phosphorylates τ Protein
Authors: Reynolds C. Hugh , Utton Michelle A. , Gibb Graham M. , Yates Alexandra , Anderton Brian H.
Published: July 2010

Abstract: A proportion of the neuronal microtubule‐associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress‐activated protein kinase/c‐Jun N‐terminal kinase, a proline‐directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation‐dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase‐3, the c‐Jun N‐terminal kinase readily phosphorylates Thr205 and Ser422, which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase‐3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress‐activated/c‐Jun N‐terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline‐directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.
An HSV‐1 Vector Containing the Rat Tyrosine Hydroxylase Promoter Enhances Both Long‐Term and Cell Type‐Specific Expression in the Midbrain
Authors: Song Song , Wang Yaming , Bak Sun‐Yung , Lang Phung , Ullrey Donna , Neve Rachael L. , O'Malley Karen L. , Geller Alfred I.
Published: July 2010

Abstract: A defective herpes simplex virus type one (HSV‐1) vector that contains a 6.8‐kb fragment of the rat tyrosine hydroxylase promoter (pTHlac‐7kb) was examined for its capability to target catecholaminergic cell type‐specific expression in the CNS. Cell type‐specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV‐1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac‐7kb supported a seven‐ to 20‐fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac‐7kb supported a 10‐fold targeting of β‐galactosidase expression to tyrosine hydroxylase‐expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac‐7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long‐term expression from pTHlac‐7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased ∼30‐fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV‐1 vector system, the tyrosine hydroxylase promoter enhanced cell type‐specific expression and contributed to stable, long‐term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.
Metabotropic Glutamate Receptors Increase Amyloid Precursor Protein Processing in Astrocytes: Inhibition by Cyclic AMP
Authors: Lee Robert K. K. , Wurtman Richard J.
Published: July 2010

Abstract: Neurotransmitter receptors that increase phosphatidylinositol hydrolysis generate second messengers that activate protein kinase C. Here, we used metabotropic glutamate receptor agonists to increase both phosphatidylinositol hydrolysis and secretion of the soluble extracellular fragment of amyloid precursor protein (APPs) from cortical astrocyte cultures. The increase in APPs secretion was mimicked by direct activation of protein kinase C with phorbol ester and was suppressed by the metabotropic glutamate receptor antagonist l‐(+)‐2‐amino‐3‐phosphonopropionic acid or by the protein kinase C inhibitor GF109203X. Ionotropic glutamate agonists did not increase APPs secretion. Forskolin or dibutyryl cyclic AMP inhibited the increase in APPs secretion caused by metabotropic glutamate receptor agonists or by phorbol ester treatment but did not affect basal APPs levels. Therefore, glutamatergic agonists that increase protein kinase C activation or decrease cyclic AMP formation may enhance the conversion of full‐length APP to nonamyloidogenic APPs in Alzheimer's disease.
Alterations in Phosphatidylcholine Metabolism of Stretch‐Injured Cultured Rat Astrocytes
Authors: Lamb Robert G. , Harper Courtney C. , McKinney Jerry S. , Rzigalinski Beverly A. , Ellis Earl F.
Published: July 2010

Abstract: The primary objective of this study was to determine the influence of stretch‐induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic‐bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50‐ms pulse of compressed air. Stretch‐induced cell injury increased the incorporation of [3H]choline into PC in an incubation time‐ and stretch magnitude‐dependent manner. PC biosynthesis was increased three‐ to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch‐induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch‐induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch‐dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n‐acetylcysteine). Arachidonate‐enriched cells were more susceptible to stretch‐induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non‐arachidonate‐enriched cells. In summary, the data suggest that stretch‐induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron‐catalyzed Haber‐Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch‐induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch‐induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.
Dose‐Response Relationship for Nicotine‐Induced Up‐Regulation of Rat Brain Nicotinic Receptors
Authors: Rowell Peter P. , Li Maureen
Published: July 2010

Abstract: The chronic administration of nicotine to animals has been shown to result in an increase in brain nicotinic acetylcholine receptor (nAChR) density. It has been suggested that this agonist‐induced receptor up‐regulation is a consequence of long‐term nAChR desensitization in vivo. In this study, the effects of different nicotine doses and administration schedules as well as the resulting blood and brain nicotine levels were determined to assess the effect of in vivo nicotine concentration on nAChR density in the brain. Rats with indwelling subcutaneous cannulas were infused for 10 days with 0.6–4.8 mg/kg/day nicotine either 2×, 4×, or 8×/day or by constant infusion. The nAChR density in cortical, striatal, and hippocampal tissue measured by [3H]cytisine binding as well as the corresponding plasma and brain nicotine levels measured by GC analysis were determined. The results showed a dose‐dependent increase in nAChR density with significant increases achieved at 2.4 mg/kg/day in all three brain areas. It is surprising that at this dose there was little difference between the constant infusion of nicotine and twice‐daily administration, whereas more frequent periodic injections were actually less effective at up‐regulating nAChRs. An analysis of the blood and brain levels of nicotine compared with the concentrations that produce nAChR desensitization suggests that in vivo desensitization alone is not sufficient for nAChR up‐regulation to occur.
Time Course and Regional Basis of Pb‐Induced Changes in MK‐801 Binding: Reversal by Chronic Treatment with the Dopamine Agonist Apomorphine but Not the D1 Agonist SKF‐82958
Authors: Cory‐Slechta Deborah A. , McCoy Lance , Richfield Eric K.
Published: July 2010

Abstract: In the present study we attempted to further define the time course and regional specificity of lead (Pb)‐induced changes in the NMDA receptor complex and the influence of dopaminergic system modulations on these changes. Autoradiographic measurements of alterations in MK‐801 binding, as evaluated under four different activation conditions (none, spermidine, glycine, or maximal activation), were performed in medial frontal cortex, dorsal striatum, and nucleus accumbens of male rats after 2 weeks or 8 months of chronic postweaning (from 21 days of age on) exposure to 0, 50, or 150 ppm Pb acetate in drinking water. The 8‐month groups also received chronic intermittent intraperitoneal injections of saline, or of the dopamine (DA) agonist apomorphine or the D1 agonist SKF‐82958 2–3 times per week beginning at 60 days of age. Two weeks of 50 ppm Pb exposure resulted in small but significant increases in MK‐801 binding under conditions of glycine or spermidine activation, whereas decreases were observed in response to 150 ppm under conditions of no or maximal activation in all regions. After 8 months of Pb, concentration‐dependent decreases in MK‐801 binding were observed across regions under all activation conditions. These effects were noted at blood Pb concentrations averaging as low as 16 µg/dl. Pb‐induced decreases in MK‐801 binding were either partially or fully reversed by chronic intermittent treatment with the DA agonist apomorphine but not by the D1 agonist SKF‐82958, implicating D2‐based mechanisms in this reversal. Combined findings from this and previous studies based on this exposure protocol indicate a Pb‐induced pattern of widespread hypoglutamatergic function accompanied by increased DA function in mesolimbic systems, a pattern of changes reminiscent of those proposed to underlie schizophrenia. Such findings suggest that Pb exposure, even at current environmental levels, could be a risk factor for behavioral and/or neurological disturbances arising from imbalances of glutamate/dopamine function in mesocorticolimbic systems.
Amyloid β‐Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes
Authors: Avdulov Nicolai A. , Chochina Svetlana V. , Igbavboa Urule , O'Hare Eugene O. , Schroeder Friedhelm , Cleary James P. , Wood W. Gibson
Published: July 2010

Abstract: Amyloid β‐peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ1–40 and Aβ25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer‐eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p≤ 0.02) by Aβ1–40. Similar effects on fluidity were observed for Aβ25–35 (p≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased (p≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water‐soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ‐induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.
Changes in Cerebral Acyl‐CoA Concentrations Following Ischemia‐Reperfusion in Awake Gerbils
Authors: Rabin Olivier , Deutsch Joseph , Grange Eric , Pettigrew Karen D. , Chang Michael C. J. , Rapoport Stanley I. , Purdon A. David
Published: July 2010

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl‐CoAs via acyl‐CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia‐reperfusion on brain fatty acid and acyl‐CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia‐reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4‐fold in the ischemic‐reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl‐CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl‐ and arachidonoyl‐CoA and a marked decrease in palmitoyl‐ and docosahexaenoyl‐CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl‐CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.
Host Cell‐Specific Folding and Assembly of the Neuronal Nicotinic Acetylcholine Receptor α7 Subunit
Authors: Cooper Sandra T. , Millar Neil S.
Published: July 2010

Abstract: Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α‐bungarotoxin binding could be detected in only two of the nine cell lines. Both of these “α7‐permissive” cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH‐SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope‐tagged α7 subunit, it has been possible to show that the elevation in surface α‐bungarotoxin binding in these two cell lines is due to expression of cDNA‐encoded α7. The cell‐specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero‐oligomeric muscle nAChR or the homo‐oligomeric serotonin receptor 5‐HT3 subunit. Our data also indicate that the cell‐specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter‐gated ion channels.
Isolation and Characterization of Ovine Tyrosine Hydroxylase mRNA
Authors: Tillet Yves , Krieger Monique , Thibault Jean
Published: July 2010

Abstract: Tyrosine hydroxylase (TH) cDNA has been characterized in rodents and primates, but only a few studies have been developed in ungulates, except in cows. Because sheep is a species used for many physiological studies, it was of interest to clone TH cDNA in this species. Ovine TH cDNA was purified from a library of sheep adrenal glands. The entire cDNA was 1,721 bp long. It presented a higher percentage of similarity with bovine TH cDNA (93%) than with rodent cDNAs (75%). The deduced amino acid sequence was 490 amino acids long and had 96% similarity with the bovine amino acid sequence. The entire cDNA and different fragments obtained with endonuclease restriction enzymes were cloned in plasmid pUC 18 and were labeled with 35S‐dATP to detect TH mRNA by in situ hybridization. Strong labelings were observed on adrenal medulla and on noradrenergic and dopaminergic neurons in the sheep but also in the cow and pig. This labeling matched completely TH immunohistochemical staining obtained on the same sections with anti‐TH antibodies. Ovine TH cDNA is a useful tool to study the variations of TH mRNA levels in sheep catecholaminergic neurons.
Chromatographic Discrimination of Soluble Neuropathy Target Esterase Isoenzymes and Related Phenyl Valerate Esterases from Chicken Brain, Spinal Cord, and Sciatic Nerve
Authors: Escudero M. A. , Céspedes M. V. , Vilanova E.
Published: July 2010

Abstract: Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 µM paraoxon but sensitive to 250 µM mipafox. Gel filtration chromatography with Sephacryl S‐300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S‐NTE1 and S‐NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1–250 µM range, in the presence of 40 µM paraoxon. The data revealed that S‐NTE1 and S‐NTE2 have different sensitivities to mipafox with I50 (30 min) values of 1.7 and 19 µM, respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components (Vo peak, or S‐NTE1; and 100‐K peak, or S‐NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high‐molecular‐mass (>700‐kDa) peak or S‐NTE1 was obtained. It showed an I50 of 5.2 µM in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the Vo peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the Vo peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S‐NTE2 should be a different entity than S‐NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.
A2A Adenosine Receptors Inhibit ATP‐Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A
Authors: Park Tae‐Ju , Song Sook‐Keun , Kim Kyong‐Tai
Published: July 2010

Abstract: The regulatory role of A2A adenosine receptors in P2 purinoceptor‐mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2‐p‐(2‐carboxyethyl)‐phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680), a specific agonist of the A2A adenosine receptor, the extracellular ATP‐evoked rise in cytosolic free Ca2+ concentration ([Ca2+]i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5‐trisphosphate production evoked by ATP were not affected by CGS‐21680 treatment. However, ATP‐evoked Ca2+ influx was inhibited following CGS‐21680 stimulation. The CGS‐21680‐mediated inhibition occurred independently of nifedipine‐induced inhibition of the [Ca2+]i rise. The CGS‐21680‐induced inhibition was completely blocked by reactive blue 2. The CGS‐21680 effect was mimicked by forskolin and dibutyryl‐cyclic AMP and blocked by Rp‐adenosine 3′,5′‐cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A2A adenosine receptors leads to inhibition of P2 purinoceptor‐mediated Ca2+ influx through ATP‐gated cation channels and involves protein kinase A.
A Naturally Occurring Amino Acid Substitution of the Human Serotonin 5‐HT2A Receptor Influences Amplitude and Timing of Intracellular Calcium Mobilization
Authors: Ozaki Norio , Manji Husseini , Lubierman Victoria , Lu Sha Jia , Lappalainen Jaakko , Rosenthal Norman E. , Goldman David
Published: July 2010

Abstract: Recently, two naturally occurring amino acid substitutions were identified in the C‐terminal region of the serotonin 5‐HT2A receptor. One of these, His 452Tyr, has a rarer allele Tyr frequency of 9%. If 452Tyr alters 5‐HT2A function, it would thus be a candidate allele for human neurobehavioral variation. The present study was designed to evaluate the potential influence of the 452His and 452Tyr alleles on cellular 5‐HT2A functions. Platelet 5‐HT2A binding and 5‐HT‐induced Ca2+ response were compared in eight 452His/452His homozygous and eight 452His/452Tyr heterozygous individuals matched for sex, age, and diagnosis (all were patients with seasonal affective disorder). There was no difference in 5‐HT2A binding measured using 125I‐lysergic acid diethylamide. Nor were levels of G‐protein subunits or PKC α, δ, ε, or ζ significantly altered. However, when Ca2+ response was stimulated by 2, 5, 10, or 25 µM 5‐HT, significant differences were found. In 452His/452Tyr heterozygotes, 452Tyr was associated with both smaller peak amplitude in Ca2+ mobilization and a different time course of response, with slower peak latency and longer half‐time in 452His/452Tyr heterozygotes compared with 452His/452His homozygotes. The overall difference in the response of the 5‐HT2A receptor in individuals with 452Tyr was a blunting of the shape of the Ca2+ mobilization peak. The data reported here suggest that the primary sequence of this intracellular domain is important in function of the receptor and that the 452His and 452Tyr 5‐HT2A alleles should be carefully evaluated for effects on human neurobehavioral variation.
Endothelin Evokes Efflux of Glutamate in Cultures of Rat Astrocytes
Authors: Sasaki Yukio , Takimoto Misato , Oda Kyoko , Früh Thomas , Takai Michihiro , Okada Toshikazu , Hori Seiji
Published: July 2010

Abstract: Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with l‐[3H]‐glutamate. Glutamate efflux was induced by either 60 mM KCl or Na+‐free medium, suggesting that the efflux is due to the reversed operation of a Na+‐ and K+‐coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ETB‐R) subtype was expressed two to three times more densely than the ET type A receptor (ETA‐R) in astrocytes. The ETB‐R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 nM ET‐1 in a concentration‐dependent manner, causing a maximal inhibition of 60% at 1 µM. However, the ETA‐R antagonist BQ‐123 did not cause significant inhibition even at 10 µM. Combination of both antagonists completely inhibited the ET‐1‐induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ETB‐R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.
1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine (MPTP) Decreases Glutamate Uptake in Cultured Astrocytes
Authors: Hazell Alan S. , Itzhak Yossef , Liu Huaping , Norenberg Michael D.
Published: July 2010

Abstract: The deleterious effect of the parkinsonian neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) on dopaminergic neurons of the substantia nigra is well established. In addition, increased glutamatergic drive to basal ganglia output nuclei is considered a likely contributor to the pathogenesis of Parkinson's disease. One possibility for the increased excitatory tone may be related to an impairment in glutamate uptake. As astrocytes possess efficient transport mechanisms for both MPTP and glutamate, we have examined the effect of this agent on d‐aspartate uptake into these cells. Treatment of cultures with 50 µM MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in Vmax with no change in the Km. Treatment with deprenyl, a monoamine oxidase B inhibitor, produced a complete reversal of MPTP‐induced uptake inhibition, but was ineffective following exposure of cells to the MPTP metabolite, 1‐methyl‐4‐phenylpyridinium (MPP+). Removal of MPTP from cultures resulted in a complete restoration of glutamate uptake after 24 h. These results show that MPTP reversibly compromises glutamate uptake in cultured astrocytes, which is dependent on the conversion of MPTP to MPP+. Such findings suggest that the glutamate transporter in astrocytes plays an important role in MPTP‐induced neurotoxicity and possibly in parkinsonism.
Phosphorylation and Activation of Brain Tryptophan Hydroxylase: Identification of Serine‐58 as a Substrate Site for Protein Kinase A
Authors: Kuhn Donald M. , Arthur Robert , States J. Christopher
Published: July 2010

Abstract: Tryptophan hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin‐dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation‐dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S‐transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full‐length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1–98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99–444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine‐58 to arginine resulted in the expression of a full‐length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine‐58 within the regulatory domain of the enzyme.
Nitric Oxide‐Mediated Mitochondrial Damage in the Brain: Mechanisms and Implications for Neurodegenerative Diseases
Authors: Bolaños Juan P. , Almeida Angeles , Stewart Victoria , Peuchen Stephan , Land John M. , Clark John B. , Heales Simon J. R.
Published: July 2010

Abstract: Within the CNS and under normal conditions, nitric oxide (•NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that •NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances •NO synthesis may be excessive and •NO may become neurotoxic. Excessive glutamate‐receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both •NO and superoxide (O2•−) and hence peroxynitrite (ONOO−) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of •NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of •NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO− interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy‐consuming pathway involving poly(ADP‐ribose) synthetase. Possible mechanisms whereby ONOO− impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO− production. It is concluded that neurotoxicity elicited by excessive •NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.
Distinct Patterns of Expression and Physiological Effects of sst1 and sst2 Receptor Subtypes in Mouse Hypothalamic Neurons and Astrocytes in Culture
Authors: Viollet Cécile , Lanneau Christophe , Faivre‐Bauman Annie , Zhang Ji , Djordjijevic Dragan , Loudes Catherine , Gardette Robert , Kordon Claude , Epelbaum Jacques
Published: July 2010

Abstract: Somatostatin (SRIF) receptor subtypes (sst) were characterized in hypothalamic neurons and astrocytes by quantitative reverse transcription‐polymerase chain reaction and radioreceptor assays using [125I‐Tyr0,d‐Trp8]SRIF‐14 as a ligand in ionic conditions discriminating between SRIF‐1 (sst2, ‐3, and ‐5 receptors) and SRIF‐2 (sst1 and ‐4 receptors) binding sites. In neurons, sst1 mRNA levels were twofold higher than those of sst2, and sst3–5 expression was only minor. Astrocytes expressed 10‐fold less sst mRNAs than neurons, which corresponded mostly (80%) to sst2. SRIF‐1 binding site radioautography indicated that 10% of hypothalamic neurons were labelled on both cell bodies and neuritic processes, as were 35% of astrocytes. On neuronal and glial membranes, SRIF‐14 and octreotide, an sst2/sst3/sst5‐selective analogue, completely displaced SRIF‐1 binding, whereas des‐AA1,2,5[d‐Trp8,IAmp9]SRIF (CH‐275), an sst1‐selective analogue, was ineffective. Using SRIF‐2 conditions, only SRIF‐14 and CH‐275 displaced the binding on neurons. No SRIF‐2 binding was observed on glia. SRIF‐14 and octreotide inhibited forskolin‐stimulated adenylyl cyclase activity in neurons and glia, whereas CH‐275 was effective in neurons only. In patch‐clamp experiments, SRIF‐14 modulated the glutamate sensitivity of hypothalamic neurons with either synergistic or antagonistic effects; CH‐275 was only stimulatory and octreotide inhibitory. It is concluded that hypothalamic neurons express primarily sst1 and sst2, sst2 predominates in astrocytes, and both receptors induce distinct biological effects.
Effects of Transforming Growth Factor‐β and Platelet‐Derived Growth Factor on Oligodendrocyte Precursors: Insights Gained from a Neuronal Cell Line
Authors: Asakura Kunihiko , Hunter Samuel F. , Rodriguez Moses
Published: July 2010

Abstract: Conditioned medium derived from a rat central nervous system neuronal cell line B104 (B104 CM) was shown previously to contain uncharacterized potent mitogen(s) for oligodendrocyte/type‐2 astrocyte (O‐2A) progenitor cells. In this study, we demonstrated that B104 cells produce and secrete platelet‐derived growth factor (PDGF)‐AA homodimer, but not PDGF‐B chain. B104 cells did not express other known potent mitogens for O‐2A progenitor cells, including fibroblast growth factor‐2 and neurotrophin‐3. Unexpectedly, B104 cells also expressed transcripts of transforming growth factor‐β1 (TGF‐β1) and ‐β2 (TGF‐β2), which are known to regulate O‐2A progenitor cell differentiation and proliferation, and secreted exclusively the 25‐kDa active forms of TGF‐β1 and TGF‐β2. Neutralization of B104 CM with anti‐PDGF‐AA antibody decreased proliferation of O‐2A progenitor cells, whereas neutralization with anti‐TGF‐β antibodies had no effect. The combination of PDGF and TGF‐β on proliferation was not equivalent to the effect of B104 CM, indicating the possibility of an unidentified growth factor. B104 CM maintained a high expression of PDGF‐α receptor in oligodendrocytes. The observation that both a stimulatory factor (PDGF‐AA) and a regulatory factor (TGF‐β) for O‐2A progenitor cell proliferation and differentiation are produced from a single neuronal cell line emphasizes the potential critical interaction between neurons and O‐2A progenitor cells in myelination and possibly in remyelination.
Characterization of CPP32‐Like Protease Activity Following Apoptotic Challenge in SH‐SY5Y Neuroblastoma Cells
Authors: Posmantur Rand , McGinnis Kim , Nadimpalli Ravi , Gilbertsen Richard B. , Wang Kevin K. W.
Published: July 2010

Abstract: We characterized the activation of interleukin‐1β‐converting enzyme (ICE)‐like proteases (caspases) in human neuroblastoma cells (SH‐SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti‐poly(ADP‐ribose) polymerase (anti‐PARP) and anti‐CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl‐DEVD‐7‐amido‐4‐methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32‐like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH‐SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH‐SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.
Select Alterations in Protein Kinases and Phosphatases During Apoptosis of Differentiated PC12 Cells
Authors: Davis Penny K. , Dudek Serena M. , Johnson Gail V. W.
Published: July 2010

Abstract: The involvement of cell cycle‐regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13suc1 precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin‐dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin‐dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle‐regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.
Responses of Bergmann Glia and Granule Neurons In Situ to N‐Methyl‐d‐Aspartate, Norepinephrine, and High Potassium
Authors: Shao Yanping , McCarthy Ken D.
Published: July 2010

Abstract: To gain insight into neuronal‐glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N‐methyl‐d‐aspartate (NMDA) with a rise in [Ca2+]i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non‐NMDA glutamate receptor blockers 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and α‐methyl‐4‐carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K+, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca2+, indicating a direct activation of α1‐adrenergic receptors that mediated release of Ca2+ from intracellular stores. The KCI‐induced response in both neurons and glia was dependent on external Ca2+ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.
Monoamine Transporter Gene Expression in the Central Nervous System in Diabetes Mellitus
Authors: Petrišić Melita Šalković , Augood Sarah J. , Bicknell R. John
Published: July 2010

Abstract: Diabetic animals exhibit altered neurotransmission in brain monoaminergic systems. By means of in situ hybridization, we have investigated the expression of dopamine, noradrenaline, and serotonin transporter (DA‐T, NA‐T, and 5‐HT‐T, respectively) mRNAs in the brains of alloxan‐ and streptozotocin‐diabetic rats. The expression of DA‐T mRNA is decreased in 1‐ (−11%) and 4‐(−17%) week alloxan‐diabetic and 4‐ (−9%) and 8‐(−20%) week streptozotocin‐diabetic rats in the ventral medial bundle. The expression of NA‐T mRNA is decreased in the locus coeruleus of 8‐ (−26%) week streptozotocin‐diabetic rats, in the noradrenergic A1 cell group of 4‐(−27%) week alloxan‐ and 8‐ (−25%) week streptozotocin‐diabetic rats, and in the noradrenergic A2 cell group of 1‐ (−21%) and 4‐ (−28%) week alloxan‐diabetic and 4‐ (−27%) and 8‐ (−25%) week streptozotocin‐diabetic animals. The expression of 5‐HT‐T mRNA in the dorsal raphe nucleus is increased in 1‐ (+14%) and 4‐ (+44%) week alloxan‐ and 4‐ (+28%) and 8‐(+44%) week streptozotocin‐diabetic rats. The expression of each of the three monoamine transporter genes may be differentially regulated in diabetes and dependent on the duration of diabetes. Altered monoamine transporter gene expression may possibly contribute to the observed dysfunctions in brain monoamine transmission in chronic diabetes.
Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation‐Induced Damage in Cultured Hippocampal Neurons
Authors: Geng Mei‐Yu , Saito Hiroshi , Nishiyama Nobuyoshi
Published: July 2010

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µM) inhibited this LDH release in a concentration‐dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP‐dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation‐induced damage by enhancing the formation of energy‐yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.