bluuebunny/crossref_metadata_2025 huggingface dataset:
PC12 Cells Overexpressing Tissue Plasminogen Activator Regenerate Neurites to a Greater Extent and Migrate Faster than Control Cells in Complex Extracellular Matrix
Authors: Pittman Randall N. , DiBenedetto Angela J.
Published: July 2010

Abstract: PC12 cells were stably transfected with expression vectors containing rat tissue plasminogen activator (tPA) under control of either a cytomegalovirus or rous sarcoma virus promoter. Cell lines were characterized using protease assays, ELISAs, immunoblots, northern blots, and Southern blots. Control PC12 cells or cells containing vectors alone released about 1 pg tPA/cell/24 h, whereas cells stably transfected with a tPA cDNA released 2–5 pg tPA/cell/24 h. A strong correlation existed between the amount of tPA released and the ability of cells to degrade extracellular matrix. Experiments with protease inhibitors and antibodies against tPA and plasminogen indicated that degradation of matrix involved tPA‐generated plasmin and that the amount of matrix degraded was dependent on the amount of tPA released. Cells expressing high levels of tPA migrated on a three‐dimensional matrix about twice as fast as control cells and regenerated neurites within three‐dimensional gels of Matrigel to a greater extent than control cells. Antibodies that inhibited tPA and plasminogen decreased migration and neurite regeneration, indicating that tPA was involved in both events. PC12 cells overexpressing tPA should provide a useful model system for investigating neural functions of tPA including its role in migration and regeneration.
NMDA Receptors in Rat Cerebellum and Forebrain: Subtle Differences in Pharmacology and Modulation
Authors: Widdowson P. S. , Trainor A. , Lock E. A.
Published: July 2010

Abstract: Binding of [3H]glutamate, [3H]glycine, and the glutamate antagonist [3H]CGS‐19755 to NMDA‐type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [3H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [3H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [3H]glycine bound to a second site with a 10‐fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [3H]Glutamate binding was not affected by glycine agonists or antagonists, nor was [3H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS‐19755 and 3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid, glutamate antagonists, reduced [3H]glycine binding in cerebellum, whereas only CGS‐19755 was effective in forebrain. Glycine agonists and antagonists modulated [3H]CGS‐19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.
Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase
Authors: Chen Jiang , Rasenick Mark M.
Published: July 2010

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of Gs‐mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down‐regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl‐5′‐imidodiphosphate [Gpp(NH)p]‐stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21‐day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant‐induced β‐receptor desensitization occurred earlier than the enhancement of Gpp(NH)p‐activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p‐stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p‐stimulated adenylyl cyclase activity is not correlated with desensitization of β‐adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially Gs) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (αs, αi, αo, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between Gs and adenylyl cyclase.
Specific Delay of Degradation of Mitochondrial ATP Synthase Subunit c in Late Infantile Neuronal Ceroid Lipofuscinosis (Batten Disease)
Authors: Ezaki Junji , Wolfe Leonhard S. , Higuti Tomihiko , Ishidoh Kazumi , Kominami Eiki
Published: July 2010

Abstract: Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse‐chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.
Neurotoxic Effect of Intranigral Injection of 1‐Methyl‐4‐Phenylpyridinium on GABA‐Containing Neurons and Its Relation to Circling Behavior
Authors: Jasso‐López Diana , Tapia Ricardo
Published: July 2010

Abstract: The ionic species 1‐methyl‐4‐phenylpyridinium (MPP+) seems to be the metabolite responsible for the damage to dopaminergic neurons occurring after administration of the parkinsonian drug 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. In the present study we show that the unilateral stereotaxic microinjection of MPP+ into the substantia nigra pars reticulata in rats produces immediately intense and long‐lasting (up to 96 h) contralateral turning behavior in a dose‐dependent manner. This behavioral effect was correlated with a dose‐ and time‐dependent decrease (up to 90%) of glutamate decarboxylase activity and with a notable loss of neurons in the injected nigra reticulata. GABA levels in the injected nigra were also decreased, whereas the dopamine concentration in the ipsilateral striatum was not affected at 24 h, when maximal behavioral effects were observed. The circling behavior was prevented by the dopamine carrier blocker nomifensine only during the first 2 h, whereas the dopamine receptor antagonist haloperidol was ineffective. The results indicate that MPP+ is toxic for inhibitory GABAergic neurons in the nigra pars reticulata and, furthermore, suggest that disruption of the function of these GABAergic neurons may be involved in the abnormal motor behavior produced by the injection of MPP+ in the substantia nigra.
Effect of Nerve Growth Factor and Forskolin on Glycosyltransferase Activities and Expression of a Globo‐Series Glycosphingolipid in PC12D Pheochromocytoma Cells
Authors: Kanda Takashi , Ariga Toshio , Yamawaki Masanaga , Pal Shubhro , Katoh‐Semba Ritsuko , Yu Robert K.
Published: July 2010

Abstract: The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK‐dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo‐series neutral GSL, Galα1‐3Galα1‐4Galβ1‐4Glcβ1‐1′Cer (GalGb3), and the activity of GalGb3‐synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)‐, GM3‐, GD1a‐, and GM2‐synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti‐GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3‐synthase activity may be closely related to the cellular differentiation process in this cell line.
Differential Correlation Between Translocation and Down‐Regulation of Conventional and New Protein Kinase C Isozymes in C6 Glioma Cells
Authors: Chen Ching Chow , Cheng Chin San , Chang Jacque , Huang Huei Chen
Published: July 2010

Abstract: Correlation between translocation and down‐regulation of conventional protein kinase Cα (cPKCα) and new PKCδ (nPKCδ) induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) at different time courses (5 min, 30 min, 1 h, 3 h, 6 h, 10 h, 17 h, and 24 h) was studied in C6 glioma cells. From the dose‐dependent translocations of these two isoforms by 10‐min treatment with TPA (1, 3, 10, 30, 100, 300, and 1,000 nM), we found that cPKCα was translocated by 3–1,000 nM and nPKCδ was translocated by 10–1,000 nM TPA. Both isoforms were maximally translocated by 100–1,000 nM TPA, whereas 1 nM did not translocate these two isoforms. When the cells were treated with 1,000 nM TPA for 5 min to 17 h, the translocation of these two isoforms occurred rapidly after 5‐min treatment and could be sustained for 1 h, whereas down‐regulation occurred after 3‐h treatment and almost complete down‐regulation was observed after 17‐h treatment. However, the extent of down‐regulation of nPKCδ was greater than that of cPKCα at 3‐, 6‐, and 10‐h treatment. Further studies by using different doses of TPA (100, 10, 3, and 1 nM) and extending the time to 24 h showed that cPKCα was more resistant to down‐regulation. This conventional isoform was maintained at a translocation state even after long‐term treatment with 3–100 nM TPA, and complete down‐regulation was only shown after 1,000 nM treatment. On the other hand, nPKCδ was almost completely down‐regulated by long‐term treatment with a translocation dose of 10–1,000 nM TPA despite higher membrane content of this new isoform. Therefore, the differential translocation and down‐regulation of cPKCα and nPKCδ was demonstrated in C6 glioma cells and this will be useful for exploring cPKCα‐ or nPKCδ‐specific functional roles in cellular functions and different signal transduction pathways in these cells.
l‐DOPA Cytotoxicity to PC12 Cells in Culture Is via Its Autoxidation
Authors: Basma Alie N. , Morris Erick J. , Nicklas William J. , Geller Herbert M.
Published: July 2010

Abstract: The mechanism of cytotoxicity of l‐DOPA was studied in the rat pheochromocytoma PC12 cell line. The cytotoxicity of l‐DOPA to PC12 cells was time and concentration dependent. Carbidopa, which inhibited the conversion of l‐DOPA to dopamine, did not protect against l‐DOPA cytotoxicity in PC12 cells. Furthermore, clorgyline, a selective inhibitor of monoamine oxidase type A, and pargyline, an inhibitor of both monoamine oxidase types A and B, both did not have an effect on l‐DOPA toxicity. These findings suggest that cytotoxicity was not due to dopamine formed from l‐DOPA. Catalase or superoxide dismutase each partially protected against l‐DOPA toxicity in PC12 cells. In combination, the effects were synergistic and provided almost total protection against cytotoxicity. 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist of non‐NMDA receptors, did not protect against l‐DOPA toxicity. These data suggest that toxicity of l‐DOPA is most likely due to the action of free radicals formed as a result of its autoxidation. Furthermore, these findings suggest that patients on long‐term l‐DOPA therapy are potentially at risk from the toxic intermediates formed as a result of its autoxidation.
Purification and Properties of High Molecular Weight Calpastatin from Bovine Brain
Authors: Mohan Panaiyur S. , Nixon Ralph A.
Published: July 2010

Abstract: Calcium‐activated neutral proteases (calpains) are regulated by specific endogenous protein inhibitors, the calpastatins, which are widely distributed in mammalian tissues. Calpastatins from different species or in various tissues from the same species exhibit considerable size heterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both transcriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was purified 2,463‐fold from a cytosolic fraction of fresh bovine cerebral cortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA44, phenyl‐Sepharose, concanavalin A‐Sepharose, and Q‐Sepharose. The major calpastatin displayed a native molecular mass of 250–300 kDa by gel filtration and was composed of 125‐kDa polypeptide chains by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). Small amounts of a 68‐kDa calpastatin fragment were detected particularly in molecules exhibiting smaller native molecular mass (250 kDa). When electroeluted from SDS gels, the 125‐ and 68‐kDa polypeptides each inhibited calpain. The purified protein was strongly immunoreactive toward antibodies raised against a synthetic peptide, CEKLGEKEETIPPDYR, shown to be a conserved repetitive motif in the calpastatin gene or a recombinant polypeptide corresponding to domains L and 1 of human calpastatin. Calpastatins purified from bovine and human erythrocytes exhibited molecular masses of 78 and 68 kDa, respectively, by SDS‐PAGE. Both erythrocyte calpastatins reacted strongly with antibodies against the conserved sequence but not with antibodies raised against domains L and 1 of human calpastatin, indicating that the erythrocyte inhibitors lack these two domains. The heat‐stable inhibitor was effective toward calpain I and calpain II and showed no inhibitory activity toward other proteases, including trypsin, chymotrypsin, cathepsin D, bromelain, and papain. Brain calpastatin therefore shares molecular properties with calpastatins from other tissues but is distinct from the erythrocyte form.
Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells
Authors: Lee Ken , Ito Akihiro , Koshimura Kunio , Ohue Tetsuya , Takagi Yasutaka , Miwa Soichi
Published: July 2010

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l‐[3H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and 22Na+ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the KD under hypoxic conditions was twice as large as that under control conditions, whereas the Bmax was unchanged. When the specific [3H]nicotine binding was kinetically analyzed, the association constant (k1) but not the dissociation constant (k−1) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine‐evoked 22Na+ flux into the cells was suppressed by hypoxia. In contrast, specific [3H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.
Tumor Necrosis Factor‐α Regulates Nicotinic Responses in Mixed Cultures of Sympathetic Neurons and Nonneuronal Cells
Authors: Soliven Betty , Wang Ning
Published: July 2010

Abstract: It is recognized that tumor necrosis factor‐α (TNF‐α), a pleiotropic cytokine, influences hormone secretion and transmitter release from central neurons. To examine the role of TNF‐α as a modulator of autonomic function of the PNS, we measured [3H]norepinephrine ([3H]NE) secretion evoked by 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP), a nicotinic agonist, in cultures from neonatal rat superior cervical ganglia (SCG). We found that (1) DMPP‐evoked [3H]NE secretion was enhanced in SCG mixed cultures treated for 48 h with recombinant human TNF‐α (rhTNF‐α) plus rat interferon‐γ (IFN‐γ) but not in cultures treated with either cytokine alone; (2) an increase in [3H]NE secretion was also observed in mixed cultures treated with recombinant murine TNF‐α (rmTNF‐α) alone; and (3) the presence of nonneuronal cells or soluble factors released by them was required for the effect of these cytokines on secretion. Electrophysiologic experiments revealed an increase in nicotinic receptor current density in neurons from mixed cultures treated with rhTNF‐α plus IFN‐γ or with rmTNF‐α when compared with control cultures. We conclude that prolonged exposure to rhTNF‐α plus IFN‐γ or rmTNF‐α regulates nicotinic responses in SCG cultures via a soluble factor or factors secreted by nonneuronal cells.
Decrease of Brain Phospholipid Synthesis in Free‐Moving n‐3 Fatty Acid Deficient Rats
Authors: Gazzah Noureddine , Gharib Abdallah , Croset Martine , Bobillier Pierre , Lagarde Michel , Sarda Nicole
Published: July 2010

Abstract: The autoradiographic method with [14C]‐docosahexaenoic acid ([14C]22:6 n‐3) was used to determine whether a diet deficient in n‐3 fatty acids, inducing a decrease in 22:6 n‐3 circulating level, was associated with changes in local rates of phospholipid synthesis in the rat brain. As compared with rats fed a normal diet (peanut plus rapeseed oil), a n‐3 fatty acid deficiency [peanut oil group (P group)] induced a generalized decrease (−35 to −76%) of 22:6 n‐3 incorporation rates into phospholipids in all the regions examined. This effect was confirmed by using [3H]22:6 n‐3 infusion by biochemical analysis and quantifications corrected for the contribution of docosahexaenoate derived from lipid store recycling to the unesterified pool, taken as the precursor pool for phospholipid synthesis in the whole brain. In normal or n‐3 fatty acid‐deficient rats, the values of the brain‐to‐plasma 22:6 n‐3 specific activity ratio (Ψ) were similar (0.03), indicating that a considerable endogenous source of 22:6 n‐3 (97%), likely derived from phospholipid degradation, dilutes the specific activity of the tracer coming from plasma. Using the specific activity of 22:6 n‐3 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis. The results also demonstrate that the pattern of 14C or 3H distribution in brain lipids was not modified by the n‐3 fatty acid‐deficient diet. The major lipids labeled were phospholipids, particularly phosphatidylethanolamine. Nevertheless, the unesterified 22:6 n‐3 concentrations in plasma and brain were significantly reduced (eight‐ and threefold, respectively) in the P group. In addition, the proportion of 22:6 n‐3 in the brain total lipid fraction, total phospholipids, and phosphatidylcholine, ‐ethanolamine, and ‐serine was significantly decreased in n‐3 fatty acid‐deficient rats. This was partially compensated for by an increase in the 22:5 n‐6 level. These results are discussed in relation to the limitation of 22:6 n‐3 use to quantify, by the quantitative autoradiographic method, changes in local rates of phospholipid synthesis in rat brain.
The Simian Virus 40 Large T Antigen Does Not Inhibit Translation of the 14‐kDa Myelin Basic Protein mRNA in Reticulocyte Lysates or in Transfected Cells
Authors: Ueno S. , Foster L. , Hifumi G. T. , Tennekoon G. I. , Campagnoni A. T.
Published: July 2010

Abstract: Viral T antigens are transcription factors that have been suspected of inhibiting expression of the myelin basic protein (MBP) mRNA at the translational level in vitro and in vivo. The effect of simian virus 40 (SV40) large T antigen (T‐ag) was examined on the translation of the 14‐kDa MBP mRNA in reticulocyte lysates and on MBP expression after transfection into cells that express SV40 T‐ag. SV40 T‐ag did not inhibit translation of 14‐kDa MBP cRNAs in cell‐free translations even at 30 µM (∼600 µg/ml) T‐ag. Permanent transfection of COS‐1 cells (which endogenously express SV40 T‐ag) with the 14‐kDa MBP cDNA resulted in the expression of the 14‐kDa MBP as determined by western blot analysis. Permanent transfection of N20.1 cells, an oligodendrocyte line immortalized with a temperature‐sensitive SV40 T‐ag, with the 14‐kDa MBP cDNA construct also resulted in the expression of the 14‐kDa MBP under conditions in which the cells expressed functional SV40 T‐ag. These results indicate that SV40 T‐ag does not prevent expression of the MBP gene at the translational level and that in those immortalized oligodendrocyte lines that express MBP mRNA, but not MBP protein, some factor other than the SV40 large T‐ag is responsible for the posttranscriptional regulation.
Increased Concentrations of the Neurotoxin 3‐Hydroxykynurenine in the Frontal Cortex of HIV‐1‐Positive Patients
Authors: Sardar A. M. , Bell J. E. , Reynolds G. P.
Published: July 2010

Abstract: Human immunodeficiency virus (HIV)‐1‐associated dementia is a frequent consequence of HIV infection and is associated with neuronal deficits. Increased concentrations of the kynurenine pathway metabolites 3‐hydroxykynurenine (3‐HK) and quinolinic acid (QA) may contribute to this neuronal damage. We measured 3‐HK concentrations and the activity of its catabolising enzyme, 3‐hydroxykynureninase, in postmortem brain tissue from eight controls and 32 HIV‐positive patients, including a group that exhibited dementia. 3‐HK concentrations were significantly increased (over threefold) in the HIV‐positive group when compared with controls. This increase was greater in those patients with dementia, but it was still apparent in the nondemented cases. 3‐Hydroxykynureninase activity was significantly increased in the HIV‐infected group compared with the control values. The effect was apparent in both nondementia and dementia cases, although the latter showed a slightly greater increase. The 3‐HK content increase is thus unrelated to a reduction in activity of this enzyme and is likely to reflect an overall increase in the kynurenic metabolic pathway. Elevated levels of the neurotoxin 3‐HK may contribute to the neuronal deficits underlying HIV‐associated dementia.
A Possible Role of Glutathione as an Endogenous Agonist at the N‐Methyl‐d‐Aspartate Recognition Domain in Rat Brain
Authors: Ogita Kiyokazu , Enomoto Riyo , Nakahara Fukiko , Ishitsubo Naoya , Yoneda Yukio
Published: July 2010

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l‐[3H]‐glutamic acid (l‐[3H]Glu) and dl‐(E)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentenoic acid ([3H]CGP‐39653) in rat brain synaptic membranes, with less potent displacement of binding of dl‐α‐amino‐3‐hydroxy‐5‐[3H]‐methylisoxazole‐4‐propionic and [3H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l‐Glu by <1%. Both GSH and GSSG potentiated (+)‐5‐[3H]methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ([3H]MK‐801) binding in a manner similar to that found with l‐Glu. Pre‐treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration‐response curve for l‐Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [3H]MK‐801 binding to a somewhat smaller extent than that found in the presence of l‐Glu at a maximally effective concentration. The potentiation of [3H]MK‐801 binding by GSH was invariably attenuated by addition of CGP‐39653, d‐2‐amino‐5‐phosphonovaleric acid (d‐AP5), and 5,7‐dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP‐39653, d‐AP5, DCKA, and 6,7‐dichloroquinoxaline‐2,3‐dione to inhibit [3H]MK‐801 binding when determined in the presence of both l‐Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N‐methyl‐d‐aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.
Facilitation of Hippocampal Potentials by Suramin
Authors: Wieraszko A.
Published: July 2010

Abstract: The influence of suramin, a suggested purino‐receptor antagonist, on the evoked synaptic potentials recorded from hippocampal slices was evaluated. The suramin induced a nondecremental, concentration‐dependent amplification of the slope of excitatory postsynaptic potential and magnitude of the population spike (long‐term potentiation, LTP). The effect of suramin was completely abolished by adenylimidodiphosphate, a nonhydrolyzable analogue of ATP, and markedly reduced by NMDA‐receptor antagonists dl‐2‐amino‐5‐phosphonovaleric acid and MK‐801. These results indicate that, in addition to acting as an antagonist of P2 receptors, suramin is also able to facilitate hippocampal potentials in a way that involves mechanisms participating in induction of LTP.
Muscarinic Cholinoceptor‐Stimulated Synthesis and Degradation of Inositol 1,4,5‐Trisphosphate in the Rat Cerebellar Granule Cell
Authors: Gray David W. , Whitham Emma M. , Challiss R. A. John , Nahorski Stefan R.
Published: July 2010

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [3H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), as well as inositol mono‐ to tetrakisphosphate, fractions was observed. Addition of carbachol (1 mM) caused an immediate increase in level of Ins(1,4,5)P3 (peak increase two‐fold over basal by 60 s), which was well‐maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP5 and InsP6 was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P3 and inositol 1,3,4,5‐tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P3 metabolism occurs predominantly via the inositol polyphosphate 5‐phosphatase route, with metabolism via the Ins(1,4,5)P3 3‐kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide‐specific phospholipase C hydrolysis of phosphatidylinositol 4,5‐bisphosphate, although some hydrolysis of phosphatidylinositol 4‐phosphate cannot be excluded.
Monoamine Interactions Measured by Microdialysis in the Ventral Tegmental Area of Rats Treated Systemically with (±)‐8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin
Authors: Chen Nian‐Hang , Reith Maarten E. A.
Published: July 2010

Abstract: The effect of (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), a selective serotonin 5‐HT1A agonist, on levels of extracellular norepinephrine (NE), dopamine (DA), and 5‐HT (measured simultaneously) was investigated by microdialysis in the ventral tegmental area (VTA) of freely moving rats, and their behavioral activity was monitored. At 50 µg/kg s.c., 8‐OH‐DPAT reduced 5‐HT levels but enhanced NE and DA levels in VTA dialysate. These effects were not altered by pretreatment with systemic idazoxan (5 mg/kg i.p.), a selective α2 antagonist, or local sulpiride (10 µM), a selective D2/D3 antagonist. At 500 µg/kg s.c., 8‐OH‐DPAT further enhanced or more persistently reduced dialysate NE or 5‐HT content but had little effect on dialysate DA content. Its DA level‐increasing effect could be seen dramatically with local infusion of cocaine (30 µM) and, to a lesser extent, sulpiride (10 µM). Depletion of endogenous 5‐HT with p‐chlorophenylalanine attenuated both the 5‐HT level‐reducing and DA level‐enhancing effects of 8‐OH‐DPAT without affecting its maximal NE effect and the locomotor‐stimulatory effect. Partial depletion of endogenous NE with N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine failed to change the monoamine response but diminished the locomotion induced by 8‐OH‐DPAT. These results suggested that (a) the low dose of 8‐OH‐DPAT may act at presynaptic 5‐HT1A receptors to modulate 5‐HT and DA release, while acting at postsynaptic 5‐HT1A receptors to modulate NE release; (b) the high dose of 8‐OH‐DPAT may activate D2 receptors to offset its DA level‐increasing effect; and (c) the locomotor‐stimulatory effect of 8‐OH‐DPAT might be mediated primarily by postsynaptic 5‐HT1A receptors and the NE system.
Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport‐Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum
Authors: Bloc Alain , Samuel Denise , Forni Claude , Dusticier Nicole , Kerkerian‐Le Goff Lydia
Published: July 2010

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High‐affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), a non‐NMDA receptor antagonist, or dl‐2‐amino‐5‐phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose‐dependent increase in glutamate uptake rate, related to an increase in the Vmax of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10−5M) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (1 mM). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non‐NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.
Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2
Authors: Izenwasser Sari , Côté Thomas E.
Published: July 2010

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D2 receptors. [3H]‐Sulpiride binding to 7315c cell homogenates was specific and saturable, and Ki values for compounds to compete for these sites were highly correlated with values for the same compounds at D2 receptors in brain. Dopamine maximally inhibited ∼65% of forskolin‐stimulated cyclase activity in cell membranes. Some D2 agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that Gi1, Gi2, Gi3, Go, Gq, and Gs are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C‐terminal regions of Gαi1 and Gαi2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C‐terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D2 receptor regulates cyclase inhibition predominantly via Gi1 and/or Gi2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D2 receptors in the absence of other dopamine receptor subtypes.
Microdialysis with High NaCl Causes Central Release of Amino Acids and Dopamine
Authors: Horn Thomas , Bauce Lorenzo , Landgraf Rainer , Pittman Quentin J.
Published: July 2010

Abstract: Recent studies have shown that the neuropeptides arginine‐8‐vasopressin (AVP) and oxytocin (OXT) are released within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to microdialysis of these nuclei with high‐NaCl perfusion media. These results suggest an inherent osmosensitivity of SON and PVN neurons. To investigate whether the observed release of AVP/OXT is a unique phenomenon to these neuropeptides, several brain regions were examined for the release of amino acids or dopamine in response to high‐ or low‐NaCl stimulation. Urethane‐anesthetized male Sprague‐Dawley rats were perfused with five‐ion solution using U‐shaped microdialysis probes. Samples were collected at 30‐min intervals and analyzed for amino acids and dopamine by HPLC. In the dialysates of all perfusion areas, including the SON, PVN, hippocampus, and striatum, concentrations of Asp, Glu, Ser, Gln, Gly, taurine (Tau), and γ‐aminobutyric acid (GABA) were significantly increased during perfusion with high‐NaCl medium. This release was found to be dose dependent when tested in the hippocampus and striatum with perfusion medium containing 0.5 or 1.0 M NaCl. However, only the release of Glu and Ser was found to be Ca2+ dependent. In contrast, the use of mannitol, a nonionic osmolyte, for perfusions in the striatum in concentrations of 0.5 and 1 M resulted in reduced levels of amino acids in the dialysates (Glu, Ser, Gln, and Tau). Low‐NaCl perfusion medium (0.01 M) resulted in significantly increased Glu, Tau, Gly, and GABA levels in the striatum. In addition, dopamine levels in striatal dialysates were significantly increased during stimulation with 1 M NaCl. These results indicate that stimulation with high NaCl concentrations affects the release of several neurotransmitters and is not specific for AVP and OXT. The described phenomenon of the release of amino acids in response to this stimulation seems to be a response to the changed ionic concentration rather than to the osmolality. In light of these findings shown for amino acids and dopamine as well as those previously reported for AVP, OXT, and angiotensin, it would appear that sensitivity to tonicity changes brought about by microdialysis may be a feature of many transmitter systems.
A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: I. Protein Purification and Characterization
Authors: Huang Chang‐Jen , Chen Chien‐Chang , Chen Hsiu‐Jane , Huang Fore‐Lien , Chang Geen‐Dong
Published: July 2010

Abstract: Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate‐polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian α1‐antitrypsin in many aspects, it is likely a fish equivalent of α1‐antitrypsin.
Enkephalin Metabolism by Microglia Aminopeptidase N (CD13)
Authors: Lucius Ralph , Sievers Jobst , Mentlein Rolf
Published: July 2010

Abstract: Rat microglia in culture showed a high capacity to degrade neuropeptides compared with other glial cells. Leu‐enkephalin was readily hydrolyzed to free tyrosine and Gly‐Gly‐Phe‐Leu. Inhibition experiments and immunostaining revealed that aminopeptidase N (CD13) on the surface of microglia was responsible for enkephalin cleavage. Endopeptidase‐24.11 (“enkephalinase”), angiotensin‐converting enzyme, or carboxypeptidases could not be detected on microglia. Aminopeptidase N activity in microglia was considerably higher than in rat peripheral monocytes and macrophages, which both also exhibited low endopeptidase 24.11 activities. Activity of aminopeptidase N was upregulated by culture of microglia on astrocytes and downregulated by exposure of microglia to lipopolysaccharide. The occurrence of aminopeptidase N on microglia is in line with the view that they originate from the monocytic lineage.
Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex
Authors: Thomaidou D. , Dori I. , Patsavoudi E.
Published: July 2010

Abstract: The monoclonal antibody 4C5 recognizes a neuron‐specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age‐dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8‐day‐old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8‐day‐old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron‐neuron interactions.
Role of Cyclic GMP in the Regulation of Neuronal Calcium and Survival by Secreted Forms of β‐Amyloid Precursor
Authors: Barger Steven W. , Fiscus Ronald R. , Ruth Peter , Hofmann Franz , Mattson Mark P.
Published: July 2010

Abstract: The Alzheimer's disease (AD) β‐amyloid precursor proteins (βAPPs) are large membrane‐spanning proteins that give rise to the βA4 peptide deposited in AD amyloid plaques. βAPPs can also yield soluble forms (APPss) that are potently neuroprotective against glucose deprivation and glutamate toxicity, perhaps through their ability to lower the intraneuronal calcium concentration ([Ca2+]i). We have investigated the mechanism through which APPss exert these effects on cultured hippocampal neurons. The ability of APPss to lower rapidly [Ca2+]i was mimicked by membrane‐permeable analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as agents that elevate endogenous levels of these cyclic nucleotides. However, only cGMP content was increased by APPs treatment, and specific inhibition of cGMP‐dependent protein kinase (but not cAMP‐dependent kinase) blocked the activity of APPss. A membrane‐permeable analogue of cGMP (8‐bromo‐cGMP) also mimicked the ability of APPss to attenuate the elevation of [Ca2+]i by glutamate, apparently through inhibition of NMDA receptor activity. In addition, 8‐bromo‐cGMP afforded protection against glucose deprivation and glutamate toxicity, and the protection by APPss against glucose deprivation was blocked by an inhibitor of cGMP‐dependent kinase. Together, these data suggest that APPss mediate their [Ca2+]i‐lowering and excitoprotective effects on target neurons through increases in cGMP levels.
Lipid Metabolism and Membrane Composition Are Altered in the Brains of Type II Diabetic Mice
Authors: Makar Tapas K. , Hungund Basaligappa L. , Cook George A. , Kashfi Khosrow , Cooper Arthur J. L.
Published: July 2010

Abstract: CBL/57 strain db/db mice exhibit type II (non‐insulin‐dependent) diabetes. The affected mice are markedly hyperinsulinemic, hyperglycemic, and hypercholesterolemic, and their serum K+ levels are decreased. The brains of the diabetic mice are significantly smaller than those of their lean, control littermates, but the protein concentration is normal. The low brain weight is accompanied by a loss of major fatty acid components within the whole brain, nerve endings, and mitochondrial membranes. Cholesterol levels are low in whole brain but are not significantly different from normal in the synaptosomal membranes. The phospholipid concentration is significantly decreased in whole brain homogenates, crude synaptosomal membranes, and crude mitochondrial membranes of the diabetic mice. In addition, the specific activities of membrane‐bound synaptosomal acetylcholinesterase, Na+,K+‐ATPase, and Mg2+‐ATPase are decreased in crude synaptosomal membranes of the diabetic mice. The specific activities of carnitine palmitoyltransferase I and carnitine acetyltransferase are significantly increased in the crude mitochondrial fraction isolated from the brains of the type II diabetic mice, whereas the specific activity of pyruvate dehydrogenase complex is decreased. The specific activities of two other mitochondrial enzymes—monoamine oxidase B and citrate synthase—and a cytosolic enzyme—lactate dehydrogenase—are unaltered. The ability to synthesize cyclic AMP is markedly decreased in the brains of the diabetic mice. The concentrations of carnitine and of the amino acids, glutamate, aspartate, glutamine, and serine are unaltered, whereas glycine levels are significantly elevated in the brains of the db/db mice. The data suggest that in vivo the brains of the diabetic mice exhibit a decreased capacity for glucose oxidation and increased capacity for fatty acid oxidation. This hypothesis is supported by the finding that cerebral mitochondria isolated from the db/db mice oxidize [1‐14C]palmitate to 14CO2 at a rate almost twice that of control mitochondria. The present findings emphasize the potentially serious alteration of brain metabolism in uncontrolled type II diabetes.
Oxypurinol‐Enhanced Postischemic Recovery of the Rat Brain Involves Preservation of Adenine Nucleotides
Authors: Phillis J. W. , Perkins L. M. , Smith‐Barbour M. , O'Regan M. H.
Published: July 2010

Abstract: The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20‐min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four‐vessel occlusion with arterial hypotension to 45–50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol‐pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol‐treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol‐treated animals. These beneficial effects of oxypurinol may have been a result of its purine‐sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high‐energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.