bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain Regional Correspondence Between Alzheimer's Disease Histopathology and Biomarkers of Protein Oxidation
Authors: Hensley Kenneth , Hall Nathan , Subramaniam Ramachandran , Cole Pamela , Harris Marni , Aksenov Michael , Aksenova Marina , Gabbita S. Prasad , Wu Jun F. , Carney John M. , Lovell Mark , Markesbery William R. , Butterfield D. Allan
Published: July 2010

Abstract: Four biomarkers of neuronal protein oxidation [W/S ratio of MAL‐6 spin‐labeled synaptosomes, phenylhydrazine‐reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)‐demented and age‐matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation‐sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque‐dense regions of the AD brain may represent environments of elevated oxidative stress.
Gangliosides Inhibit Platelet‐Derived Growth Factor‐Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH‐SY5Y Cells
Authors: Hynds DiAnna L. , Summers Monica , Van Brocklyn James , O'Dorisio M. Sue , Yates Allan J.
Published: July 2010

Abstract: SH‐SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK‐N‐SH. It grows well in serum‐containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet‐derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF‐BB, we found that SH‐SY5Y cells specifically bind PDGF with a KD = 0.14 ± 0.06 nM and Bmax = 7.3 ± 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]‐thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF‐BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24‐h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170‐kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti‐phosphotyrosine antibody. Immunoprecipitation with anti‐PDGF β‐receptor antibody and visualization on a western blot with an anti‐phosphotyrosine antibody also revealed a 170‐kDa protein. Maximum phosphorylation of the 170‐kDa protein occurred after 5‐min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH‐SY5Y cells have specific receptors for PDGF‐BB that are functional, and can be modulated by gangliosides.
The Mechanism of Muscarinic Receptor‐Stimulated Phosphatidylinositol Resynthesis in 1321N1 Astrocytoma Cells and Its Inhibition by Li+
Authors: Batty Ian H. , Downes C. Peter
Published: July 2010

Abstract: The coupling of muscarinic receptor‐stimulated phosphatidylinositol 4,5‐bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li+ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium ± inositol (40 µM). In inositol‐replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0–30 min) ∼≥20‐fold activation of phospholipase C, whereas prolonged (>60 min) stimulation turned over Ptdlns equal to the cellular total mass, involving ∼80% of the cellular Ptdlns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC50∼ 20 µM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol‐replete or depleted cells basal concentrations of inositol and CMP‐phosphatidate were respectively ∼20 mM or ≤100–500 µM and ∼0.1 or ∼≥1–10 pmol/mg of protein. Comparison of the effects of agonist ± Li+ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP‐phosphatidate or inositol in inositol‐replete or depleted cells, respectively. Thus, the preferential capacity of Li+ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.
Distribution of Branch Point Prenyltransferases in Regions of Bovine Brain
Authors: Runquist M. , Parmryd I. , Thelin A. , Chojnacki T. , Dallner G.
Published: July 2010

Abstract: Bovine brains contain large amounts of isoprenoid compounds and the enzymes involved in their biosynthesis were investigated. Ten different regions were dissected from fresh bovine brains and, in addition, fractions from cerebellum, spinal cord, and hypophysis were obtained. The cholesterol concentration was found to be ∼8 mg/g in the cortex regions and three times higher in the pons, medulla oblongata, and white matter. Dolichol concentration varied between 8 and 40 µg/g in the different tissues, and ubiquinone was found at a lower level, which varied between 3 and 25 µg/g. Farnesyl‐pyrophosphate synthase activity in cytosolic fractions from various regions exhibited only a twofold variation, whereas geranylgeranyl pyrophosphate synthase displayed larger differences, being particularly rich in the pons, medulla oblongata, white matter, and spinal cord. Squalene synthase activity was lowest in the thalamus and threefold higher in the pons. Determination of specific activity based on cholesterol content revealed that enzyme activities in various regions are not related to the actual lipid amount present. Both cis‐ and trans‐prenyltransferases exhibited similarities in their regional distribution showing up to 20‐fold differences in activity. Thus, it appears that the mevalonate pathway lipids and the various branch point enzymes involved in their syntheses vary greatly in different brain regions and are subjected to separate regulation.
Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study
Authors: Steinberg R. , Rodier D. , Souilhac J. , Bougault I. , Emonds‐Alt X. , Soubrié P. , Le Fur G.
Published: July 2010

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane‐anesthetized rats by using microdialysis. Substance P (0.01–1 µM), [Sar9,Met(O2)11]substance P (1–10 µM), septide (0.1–3 µM), neurokinin (NK) A (0.1–10 µM), and senktide (0.1–10 µM) produced concentration‐dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK1 antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK2 receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK1 receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK2 receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P‐induced ACh release suggested that the effect of this peptide involves the stimulation of both NK1 and NK2 receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D1 agonist (+)‐SKF‐38393 (3 µM) may be mediated indirectly through local release of NKA or substance P acting at NK2 receptors.
Suppression of Mitochondrial Succinate Dehydrogenase, a Primary Target of β‐Amyloid, and Its Derivative Racemized at Ser Residue
Authors: Kaneko Isao , Yamada Noriko , Sakuraba Yuko , Kamenosono Masahiro , Tutumi Shinya
Published: July 2010

Abstract: β‐Amyloid cores contain considerable amounts of d‐Ser and d‐Asp residues in Alzheimer's disease. We investigated the cytotoxic effects of various synthetic β‐amyloids, including d‐Ser‐substituted derivatives, on primary cultured neurons and nonneuronal HeLa cells. β25–35, its d‐Ser26‐substituted derivative, and β1–40 in 10–100 nM specifically suppressed mitochondrial succinate dehydrogenase activity [MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] reduction] in HeLa cells, which are dependent on ATP production mainly from glycolysis, but did not exert detectable cytotoxicity, assessed by dye exclusion test, NADH levels, and uptake of [3H]Leu and [3H]Tdr. The β‐amyloids, on the other hand, did exert neurodegenerative effects on rat hippocampal cultured neurons in which ATP is mostly synthesized by the mitochondrion. The activities of β25–35 and [d‐Ser26]β25–35 are dependent on their having β‐structures and not random forms. Although β25–35 was degraded rapidly by proteinase(s) in brain extract or leucine aminopeptidase, [d‐Ser26]β25–35 is fairly resistant. These results indicate that one of the primary targets of β‐amyloids is suppression of mitochondrial succinate dehydrogenase, and the vulnerability of the brain to β‐amyloids can be explained by its large dependence on mitochondrial energy production. Moreover, racemization of serine residues of β‐amyloids may be involved in neurodegeneration and formation of senile plaques through escaping from the degradation process by brain proteinases.
Uptake of Dopamine Released by Impulse Flow in the Rat Mesolimbic and Striatal Systems In Vivo
Authors: Suaud‐Chagny M. F. , Dugast C. , Chergui K. , Msghina M. , Gonon F.
Published: July 2010

Abstract: The release of dopamine in the striatum, nucleus accumbens, and olfactory tubercle of anesthetized rats was evoked by electrical stimulation of the mesolimbic dopaminergic pathway (four pulses at 15 Hz or four pulses at 200 Hz). Carbon fiber electrodes were implanted in these regions to monitor evoked dopamine overflow by continuous amperometry. The kinetics of dopamine elimination were estimated by measuring the time to 50% decay of the dopamine oxidation current after stimulation ceased. This time ranged from 64 ms in the striatum to 113 ms in the nucleus accumbens. Inhibition of dopamine uptake by nomifensine (2–20 mg/kg), GBR 12909 (20 mg/kg), cocaine (20 mg/kg), mazindol (10 mg/kg), or bupropion (25 mg/kg) enhanced this decay time by up to +602%. Uptake inhibition also produced an increase in the maximal amplitude of dopamine overflow evoked by four pulses at 15 Hz. This latter effect was larger in the striatum (+420%) than in mesolimbic areas (+140%). These results show in vivo that these uptake inhibitors actually slow the clearance of dopamine released by action potentials and suggest that dopaminergic transmission is both prolonged and potentiated strongly by these drugs, in particular in the striatum.
Ornithine Decarboxylase Activity and Edema Formation in Cerebral Ischemia of Conscious Gerbils
Authors: Rao A. Muralikrishna , Başkaya Mustafa K. , Maley Mary E. , Prasad M. Renuka , Dempsey Robert J.
Published: July 2010

Abstract: General anesthetic agents often affect the biochemical and physiologic changes triggered by cerebral ischemia. This study examined the regional activities of ornithine decarboxylase (ODC) in gerbils subjected to 5 min of bilateral carotid occlusion without anesthesia. At 2, 4, and 6 h of reperfusion, significant ODC activity was observed in both the cortex and the hippocampus. Pretreatment with α‐difluoromethylornithine (DFMO) significantly blocked the ODC activity at 2, 4, and 6 h. Significant edema formation was found at 2, 4, and 6 h. At 2 h, edema formation was unaffected by administration of DFMO. However, DFMO treatment reduced later edema formation at 4 and 6 h. These results demonstrate that ODC activity and edema formation are delayed in gerbils after the induction of transient ischemia even with the removal of anesthetic agents and their potentially protective effects. These findings suggest that ODC activity and its induction of delayed cerebral edema are specific to cerebral ischemia and not to an anesthetic effect. DFMO treatment reduced both the ODC activity and edema formation, indicating a role for polyamines in postischemic edema formation.
Incremental Changes in Extracellular Noradrenaline Availability in the Frontal Cortex Induced by Naturalistic Environmental Stimuli: A Microdialysis Study in the Freely Moving Rat
Authors: Dalley Jeffrey W. , Stanford S. Clare
Published: July 2010

Abstract: Changes in levels of extracellular noradrenaline (NA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) in the frontal cortex, induced by exposure of unrestrained, conscious rats to novel environments, were compared using in vivo microdialysis. NA efflux increased when rats were transferred to a novel cage, but this was not significant when compared with either basal efflux or with changes after equivalent handling in their home cage. Increasing the intensity of illumination of the novel cage by fivefold significantly increased NA efflux with respect to basal efflux but not handled controls. However, a sustained and significant increase in NA efflux (cf. basal efflux or handled controls) was found when an unfamiliar conspecific was also present in the novel cage. In all cases, basal efflux was restored within 1 h of returning rats to their home cage. Neither handling nor environmental stimuli described above affected DOPAC efflux. 5‐HIAA efflux was increased (cf. basal) in the presence of an unfamiliar conspecific, but this increase was no greater than that in handled rats. It is concluded that different naturalistic stimuli cause incremental changes in the levels of extracellular NA in the frontal cortex; these changes affect both phasic and tonic components of the response.
Biosynthetic Processing of Preprovasoactive Intestinal Polypeptide in Parasympathetic Neurons of the Sphenopalatine Ganglion
Authors: Nilsson Christer , Fahrenkrug Jan
Published: July 2010

Abstract: The precursor for rat vasoactive intestinal polypeptide (preproVIP) is processed by proteolytic cleavage into a signal peptide and five further functional domains: preproVIP 22–79, peptide histidine isoleucine (PHI), preproVIP 111–122, VIP, and preproVIP 156–170. To investigate the biosynthetic processing of preproVIP in peripheral parasympathetic neurons, the sphenopalatine ganglion and one of its projection areas, the nasal mucosa, were used. By immunohistochemistry it was shown that in the sphenopalatine ganglion, preproVIP‐derived peptides are localized mainly in neuronal cell bodies, whereas in the nasal mucosa immunoreactivity was found only in nerve fibers and terminals. The peptides were quantified and characterized by radioimmunoassay, HPLC, and gel chromatography using antisera specific for the different precursor products. In the rat sphenopalatine ganglion, the different peptides were found in approximately equimolar amounts, with the exception of PHI and its C‐terminally extended variant, PHV, which were present at considerably lower concentrations. However, in the nasal mucosa there was a preferential accumulation of VIP to at least three times the concentration of any of the other peptides. Our results suggest that all preproVIP‐derived peptides are present and processed in the sphenopalatine ganglion but that there is a selective accumulation of VIP in the nerve terminals. This indicates that VIP is physiologically the most important transmitter among the preproVIP‐derived peptides in parasympathetic nerves originating in the sphenopalatine ganglion.
Activation of Type II Adenylyl Cyclase by the Cloned μ‐Opioid Receptor: Coupling to Multiple G Proteins
Authors: Chan Joy S. C. , Chiu Tillie T. , Wong Yung H.
Published: July 2010

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ‐opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ‐opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ‐opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ‐selective agonist, [d‐Ala2, N‐Me‐Phe4,Gly5‐ol]enkephalin, to stimulate cyclic AMP accumulation in a dose‐dependent manner. The opioid‐induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin‐sensitive Gi proteins, because it was abolished completely by the toxin. Possible coupling between the μ‐opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid‐induced response became pertussis toxin‐insensitive and was enhanced significantly upon co‐expression with the α subunit of Gz, whereas those of Gq, G12, or G13 inhibited the opioid response. When pertussis toxin‐sensitive G protein α subunits were tested under similar conditions, all three forms of αi and both forms of αo were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co‐expression of α subunits reflects a functional coupling between α subunits and the μ‐opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ‐opioid receptor can couple to Gi1–3, Go1–2, and Gz, but not to Gs, Gq, G12, G13, or Gt.
Differential Effects of D1 and D2 Dopamine Receptor Antagonists on Acute Amphetamine‐ or Methamphetamine‐Induced Up‐Regulation of zif/268 mRNA Expression in Rat Forebrain
Authors: Wang John Q. , McGinty Jacqueline F.
Published: July 2010

Abstract: This study investigated the hypothesis that D1 and D2 dopamine receptors interact to regulate the expression of zif/268 mRNA in rat forebrain after an acute injection of amphetamine or methamphetamine. Forty‐five minutes and 3 h after a single injection of amphetamine (4 mg/kg i.p.) or methamphetamine (4 mg/kg i.p.), the mRNA expression of zif/268 in dorsal striatum and sensorimotor cortex was increased, as revealed by quantitative in situ hybridization histochemistry. Induction was more intense in striatal patches at 45 min than at 3 h, when a more homogeneous pattern of zif/268 mRNA induction was observed. SCH 23390, a selective D1 receptor antagonist, suppressed, and eticlopride, a D2 receptor antagonist, elevated, constitutive zif/268 mRNA levels in the striatum, but neither antagonist had a significant effect on the constitutive expression of zif/268 in sensorimotor cortex. Pretreatment with SCH 23390 completely blocked the stimulant‐induced zif/268 expression in striatum and partially blocked the stimulant‐induced zif/268 expression in cortex. Pretreatment with eticlopride augmented zif/268 mRNA expression in patch and matrix compartments of dorsal and ventral striatum 45 min after amphetamine or methamphetamine injection. However, at 3 h, eticlopride completely blocked amphetamine‐ and methamphetamine‐stimulated zif/268 mRNA in dorsomedial, but not dorsolateral, striatum. In addition, eticlopride partially blocked cortical zif/268 induction by both amphetamines. Both antagonists prevented stimulant‐induced hyperlocomotion and stereotypies. These results demonstrate that D1 and D2 receptors in mesolimbic/mesostriatal pathways both regulate amphetamine‐and methamphetamine‐stimulated behaviors and zif/268 mRNA expression. Furthermore, the effect of D2 receptor blockade on zif/268 expression was found to be contingent on the time interval investigated after psychostimulant administration.
Tumor Necrosis Factor‐α and Basic Fibroblast Growth Factor Decrease Glial Fibrillary Acidic Protein and Its Encoding mRNA in Astrocyte Cultures and Glioblastoma Cells
Authors: Murphy Greer M. , Lee Yuen LIng , Jia Xiao‐Chi , Yu Albert C. H. , Majewska Anna , Song Yaoli , Schmidt Karen , Eng Lawrence F.
Published: July 2010

Abstract: Tumor necrosis factor‐α is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor‐α. Treatment with tumor necrosis factor‐α for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor‐α treatment increased the expression of the cytokine interleukin‐6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor‐α, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor‐α induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor‐α. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor‐α treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.
Roles of Prostaglandins D2 and E2 in Interleukin‐1‐Induced Activation of Norepinephrine Turnover in the Brain and Peripheral Organs of Rats
Authors: Terao Akira , Kitamura Hiroshi , Asano Atsushi , Kobayashi Mari , Saito Masayuki
Published: July 2010

Abstract: Possible roles of prostaglandins (PGs) in interleukin‐1 (IL‐1)‐induced activation of noradrenergic neurons were examined by assessing norepinephrine (NE) turnover in the brain and peripheral organs of rats. An intraperitoneal injection of human recombinant IL‐1β accelerated NE turnover in the hypothalamus, spleen, lung, diaphragm, and pancreas. A similar increase in NE turnover was also observed after intracerebroventricular injection of corticotropin‐releasing hormone (CRH). Pretreatment with indomethacin (cyclooxygenase inhibitor) abolished the IL‐1‐induced, but not the CRH‐induced, increase in hypothalamic and splenic NE turnover. To elucidate which eicosanoid‐cyclooxygenase product(s) is responsible for accelerating NE turnover, PGD2, PGE2, PGF2α, U‐46619 (stable thromboxane A2 analogue), or carbacyclin (stable prostacyclin analogue) was administered intracerebroventricularly. Among them, PGE2 was the only eicosanoid effective in increasing NE turnover in spleen, whereas PGD2 was effective in the hypothalamus. The stimulative effect of PGD2 was abolished by pretreatment with intracerebroventricular injection of a CRH antiserum. These results suggest that the action of IL‐1 is mediated through PGD2 production to activate the noradrenergic neurons in the hypothalamus, and through PGE2 production to increase sympathetic nerve activity in spleen.
Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag‐Fura‐2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP
Authors: García‐Martín E. , Martín‐Romero F. J. , Gutiérrez‐Merino C.
Published: July 2010

Abstract: Synaptosomes can be loaded with mag‐fura‐2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag‐fura‐2 acetoxymethyl ester in Hanks'‐HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′‐(βγ‐imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto‐ATPase or an ecto‐protein kinase.
The Effect of pH on Glycolysis and Phosphofructokinase Activity in Cultured Cells and Synaptosomes
Authors: Erecińska M. , Deas J. , Silver I. A.
Published: July 2010

Abstract: The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK‐21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [Pi] ([ATP]i, [ADP]i, and [Pi]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [Pi], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] and lower [glucose‐6‐phosphate] and [fructose‐6‐phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [Pi] rose under all pHe conditions. Simultaneously [glucose‐6‐phosphate] and [fructose‐6‐phosphate] decreased whereas [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4. Alterations in concentrations of cations other than H+ were small and unlikely to contribute to the regulation of glycolysis. It is concluded that (a) under intracellular conditions, in the presence of a high [ATP] and a low [ADP] and [AMP], a fall in [H+] powerfully inhibits phosphofructokinase; lactate production correlates with the levels of glycolytic intermediates. (b) A small decline in cellular energy state is sufficient to release phosphofructokinase inhibition by protons. (c) Once activated by the decrease in energy level, the steady‐state glycolytic rate is dependent on other factors, such as glycolytic intermediates and enzymes.
Regional Metabolism of Met‐Enkephalin and Cholecystokinin on Intact Rat Brain Slices: Characterization of Specific Peptidases
Authors: Konkoy Christopher S. , Davis Thomas P.
Published: July 2010

Abstract: The metabolism of Met‐enkephalin and cholecystokinin (CCK) 8‐(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met‐enkephalin (400 µM) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met‐enkephalin was high in caudate‐putamen (5,160 ± 120) and lower in nucleus accumbens (3,630 ± 110) and frontal cortex (3,180 ± 120). Inhibition of aminopeptidases decreased Met‐enkephalin degradation (50–97% vs. control) in frontal cortex but was less effective in caudate‐putamen (20–34%). Tyr‐Gly‐Gly and Phe‐Met were recovered in caudate‐putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met‐enkephalin degradation in caudate‐putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met‐enkephalin from degradation (recovery >95%) in caudate‐putamen. CCK 8‐(sulfated) degradation on slices from caudate‐putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13–30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.
Covalent Affinity Labeling of Brain Catecholamine‐Absorbing Proteins Using a High‐Specific‐Activity Substituted Tetrahydronaphthalene
Authors: Ross Gregory M. , McCarry Brian E. , Mishra Ram K.
Published: July 2010

Abstract: The recently described catecholamine‐absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high‐specific‐activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high‐specific‐activity substituted tetrahydronaphthalene derivative (6‐hydroxy‐[125I]iodo‐[N‐(N‐2′,4′‐dinitrophenyl)aminopropyl]‐2‐amino ‐ 1,2,3,4‐tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.
Regional Deafferentiation Down‐Regulates Subtypes of Glutamate Transporter Proteins
Authors: Ginsberg Stephen D. , Martin Lee J. , Rothstein Jeffrey D.
Published: July 2010

Abstract: Low extracellular glutamate content is maintained primarily by high‐affinity sodium‐dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT‐1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria‐fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT‐1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d‐[3H]aspartate binding. GLT‐1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d‐[3H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d‐[3H]aspartate binding returned to control levels. This study demonstrates the down‐regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.
Rapid Down‐Regulation of GABAA Receptors in the Gerbil Hippocampus Following Transient Cerebral Ischemia
Authors: Alicke Bruno , Schwartz‐Bloom Rochelle D.
Published: July 2010

Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABAA/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down‐regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5‐min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABAA receptor antagonist [3H]SR‐95531 and the hydrophobic benzodiazepine agonist [3H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [3H]SR‐95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the Bmax as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in KD). As expected, there were no changes in the binding of [3H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABAA receptors to high concentrations of synaptic GABA in vivo causes receptor down‐regulation, perhaps via receptor internalization.
Alternate Promoters in the Rat Aromatic l‐Amino Acid Decarboxylase Gene for Neuronal and Nonneuronal Expression: An In Situ Hybridization Study
Authors: Jahng Jeong Won , Wessel Thomas C. , Houpt Thomas A. , Son Jin H. , Joh Tong H.
Published: July 2010

Abstract: Aromatic l‐amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue‐specific expression of the rat AADC gene.
Glial Cell Line‐Derived Neurotrophic Factor Exerts Neurotrophic Effects on Dopaminergic Neurons In Vitro and Promotes Their Survival and Regrowth After Damage by 1‐Methyl‐4‐Phenylpyridinium
Authors: Hou Jyh‐Gong Gabriel , Lin Leu‐Fen H. , Mytilineou Catherine
Published: July 2010

Abstract: The effect of glial cell line‐derived neurotrophic factor (GDNF) on the growth of mesencephalic dopaminergic neurons and on their survival following exposure to the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP+) was examined in vitro. In cultures developing under normal conditions, GDNF at 1 ng/ml optimally improved the survival and stimulated the growth of dopaminergic neurons without affecting glial growth. In cultures treated with MPP+, GDNF could not prevent toxicity to dopaminergic neurons. The uptake of [3H]dopamine and the number of tyrosine hydroxylase‐positive neurons were similarly reduced by MPP+ in the presence or absence of GDNF. However, after removal of MPP+, GDNF protected dopaminergic neurons from the continuous cell death and stimulated the regrowth of dopaminergic fibers damaged by MPP+. We conclude that GDNF supports the growth of normally developing dopaminergic neurons and stimulates their survival and recovery after damage. These findings suggest that GDNF could be useful in the development of therapeutic approaches to Parkinson's disease, which is characterized by dopaminergic cell loss.
Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP‐Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells
Authors: Huang Chun‐Ming , Kao Lung‐Sen
Published: July 2010

Abstract: To study how growth factors affect stimulus‐secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP‐induced [Ca2+]i rise and dopamine secretion in PC12 cells. After a 4‐day incubation of cells, all three factors increased ATP‐induced dopamine secretion significantly. We then examined which step of ATP‐induced secretion was affected by the growth factors. Cellular levels of dopamine‐β‐hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP‐induced [Ca2+]i rise was also enhanced after growth factor treatment. The EC50 of ATP for inducing [Ca2+]i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca2+]i of dopamine secretion was increased significantly only in NGF‐treated cells. Our results suggest that for EGF‐ and insulin‐treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca2+]i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca2+ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca2+]i.
Potassium‐Induced Stimulation of Glutamate Uptake in Mouse Cerebral Astrocytes: The Role of Intracellular pH
Authors: Judd Michael G. , Nagaraja Tavarekere N. , Brookes Neville
Published: July 2010

Abstract: The Na+‐glutamate cotransporters are believed to countertransport OH− and K+. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO3− dependent. Also, it was reported recently that raising extracellular [K+] alkalinizes astrocyte cytoplasm by an HCO3−‐dependent mechanism. Here, we have compared glutamate uptake in HCO3−‐buffered and HCO3−‐depleted solutions at varying [K+]. We observed a pronounced stimulation of glutamate uptake by extracellular K+ (3–24 mM) that was substantially HCO3− dependent and affected preferentially the uptake of high concentrations (>25 µM) of glutamate. Stimulation of uptake by low extracellular [K+] (1.5–3 mM) was less dependent on HCO3−. Potassium‐induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba2+ and amiloride on glutamate uptake, as well as the HCO3−‐dependent stimulatory effects of K+ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH−. A “bimodal” scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.
Calcium Ion Impedes Translation Initiation at the Synapse
Authors: Weiler Ivan Jeanne , Childers William S. , Greenough William T.
Published: July 2010

Abstract: Stimulation of synaptoneurosome suspensions by the neurotransmitter glutamate gives rise to rapid loading of ribosomes onto mRNA and increased incorporation of amino acids into trichloroacetic acid‐precipitable polypeptides. Metabotropic glutamate receptors (mGluRs) are responsible for this effect. Although simultaneous Ca2+ entry and mGluR stimulation do not change the response, entry of Ca2+ 30 s or 3 min before mGluR stimulation markedly depresses the polyribosomal loading. Either NMDA or ionophore (A23187) produces the depression. A calmodulin antagonist, W7, alleviates the effect, suggesting that inactivation of phospholipase A2 by calcium‐calmodulin‐dependent kinase II is partially responsible for the phenomenon. Thus, interaction between different classes of glutamate receptors affects the control of protein translation at the synapse. This effect may partially explain recent observations of negative interactions between receptor classes in induction of long‐term potentiation.
Serotonin Enhances Striatal Dopamine Outflow In Vivo Through Dopamine Uptake Sites
Authors: De Deurwaerdère Philippe , Bonhomme Norbert , Lucas Guillaume , Le Moal Michel , Spampinato Umberto
Published: July 2010

Abstract: Serotonin (5‐HT) administered at 1, 3, and 10 µM into the striatum of halothane‐anesthetized rats by in vivo microdialysis increased extracellular dopamine (DA) in a concentration‐dependent manner (approximately 65, 190, and 440%, respectively). These effects were reduced by 50% in the presence of 1 µM tetrodotoxin (TTX) or in the absence of Ca2+ ions. The DA uptake blocker nomifensine (0.1 µM) significantly lowered (by 50%) the enhancement of DA outflow induced by 3 µM 5‐HT. Nomifensine (1 µM) coperfused with 1 µM TTX abolished the 1 and 3 µM 5‐HT‐induced DA outflow, whereas the effect of 10 µM 5‐HT was significantly reduced by 1 (−55%) and 10 µM (−70%) nomifensine. These data demonstrate that, in vivo, striatal DA uptake sites are partially involved in the DA‐releasing action of 5‐HT.
Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108‐15 Cells Studied by Concurrent 31P/19F and 23Na Double‐Quantum Filtered NMR Spectroscopy
Authors: Tauskela Joseph S. , Shoubridge Eric A.
Published: July 2010

Abstract: The role of voltage‐sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108‐15 cells, a neuronal cell line that does not express glutamate‐sensitive receptor‐mediated Ca2+ channels. Concurrent 31P/19F and 23Na double‐quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2‐bis‐(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization‐induced increase in [Ca2+]i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage‐gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia.
Inhibition of Excitatory Amino Acid‐Induced Phosphoinositide Hydrolysis as a Possible Mechanism of Nitroprusside Neurotoxicity
Authors: Yu Onnfoh , Chuang De‐Maw
Published: July 2010

Abstract: Inclusion of sodium nitroprusside {Na2[Fe2+‐(CN)5NO]} into the culture medium is toxic to cultured rat cerebellar granule neurons. A possible underlying mechanism may be the inhibition of phosphoinositide (PI) response to excitatory amino acids (EAAs) because activation of glutamate receptors can be neuroprotective and neurotrophic in differentiating neurons. Sodium nitroprusside selectively inhibited the PI response to EAAs (NMDA > glutamate = quisqualate > kainate) without affecting that to carbachol or KCI. In contrast, S‐nitroso‐N‐acetylpenicillamine (SNAP), another nitric oxide (NO) donor, potentiated NMDA‐induced PI hydrolysis. Hemoglobin reversed the effects of nitroprusside and SNAP. However, NO may not be involved because NO solution was without effect and N‐acetylpenicillamine, a SNAP analogue that does not contain a NO moiety, also potentiated NMDA‐induced PI hydrolysis in a hemoglobin‐sensitive manner. Furthermore, the metabolites of NO (nitrate and nitrite), l‐arginine, reduced glutathione, 8‐bromo‐cyclic guanosine 3′:5′‐cyclic monophosphate (8‐Br‐cGMP), and atrial natriuretic peptide, which accelerates the production of cGMP independent of NO, were ineffective as modulators. However, potassium ferrocyanide {K4[Fe2+(CN)6]}, but not potassium ferricyanide {K3[Fe3+(CN)6]}, inhibited NMDA‐induced PI hydrolysis as effectively as nitroprusside, but this inhibition was not reversed by hemoglobin. Cyanide, a product from the disintegration of nitroprusside, potentiated rather than inhibited NMDA‐induced PI hydrolysis. Taken together, these results suggest that the parent molecule itself, nitroprusside, contributes primarily in inhibiting EAA‐induced PI hydrolysis. Inhibition of EAA‐induced PI hydrolysis may in part mediate the mechanisms of nitroprusside toxicity in primary cultures of differentiating cerebellar granule neurons.