bluuebunny/crossref_metadata_2025 huggingface dataset:
Methylmercury‐Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F‐NMR Study
Authors: Denny Michael F. , Atchison William D.
Published: July 2010

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108‐15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108‐15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg‐induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108‐15 cells. The pattern of alterations in fura‐2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F‐NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5F‐BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 µM EGTA to prevent the Ca2+e‐dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 ± 0.20 nM; following a 40‐min exposure to MeHg‐free buffer [Zn2+]i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn2+]i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn2+]i to 3.99 ± 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell‐permeant heavy metal chelator N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca2+]i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40‐min exposure to MeHg‐free buffer. [Ca2+]i was 498 ± 28 or 492 ± 53 nM during a 40‐min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca2+]i differed significantly from either pretreatment levels or buffer‐treated controls.
Levels of Benzodiazepine Receptor Subtypes and GABAA Receptor α‐Subunit mRNA Do Not Correlate During Development
Authors: Williamson Robert E. , Pritchett Dolan B.
Published: July 2010

Abstract: Developmental changes in the pharmacological properties of the GABAA receptor have been suggested to result from changes in the subunit composition of the receptor complex. The nicotinic acetylcholine receptor is structurally related to the GABAA receptor and undergoes a developmental subunit switch at the neuromuscular synapse. To examine the mechanistic similarities between these systems we sought to find whether the changes in GABAA receptor subunits are controlled by changes in messenger RNA levels, as they are for the nicotinic acetylcholine receptor. We found a 10‐fold increase in the level of α1‐subunit mRNA, and a small increase in levels of GABAA/benzodiazepine receptors from day 1 to day 24 of rat cerebellar development. We also found that the levels of α1‐subunit mRNA were higher than the levels of mRNA encoding other α subunits at all developmental time points. The low levels of messenger RNA for α2, α3, and α5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these α subunits have been shown to form GABAA receptors with type II benzodiazepine binding. These findings are inconsistent with simple models that would explain the developmental differences in GABAA receptor pharmacology simply as a result of changes in α‐subunit gene expression.
Transcriptional and Posttranscriptional Mechanisms Involved in the Interleukin‐1, Steroid, and Protein Kinase C Regulation of Nerve Growth Factor in Cortical Astrocytes
Authors: Pshenichkin Sergey P. , Szekely Anna Maria , Wise Bradley C.
Published: July 2010

Abstract: Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF). Treatment of astrocytes with interleukin‐1β (IL‐1) or the protein kinase C (PKC) activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) increased NGF mRNA content by six‐ to 10‐fold, followed in time by increases in cell content and cell secretion of NGF. Dexamethasone potently inhibited the effects of IL‐1 and TPA on astroglial cell NGF expression. The action of IL‐1 was not mediated by PKC because treatment of cells with maximal concentrations of both IL‐1 and TPA gave an additive increase in NGF mRNA content and NGF secretion, and because down‐regulating PKC activity failed to inhibit the stimulatory effects of IL‐1. Moreover, both agents increased NGF gene transcription in nuclear run‐on assays, but only IL‐1 significantly stabilized the NGF mRNA. An analysis of the effects of IL‐1 and TPA on immediate early gene expression indicated that IL‐1 preferentially induced c‐jun gene expression, whereas TPA greatly increased c‐fos and zif/268 gene expression. These results suggest that IL‐1 activates c‐jun and NGF gene expression, and NGF mRNA stabilization in astrocytes by a distinct PKC‐independent signaling pathway.
Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2
Authors: Theveniau Magali A. , Yasuda Kazuki , Bell Graeme I. , Reisine Terry
Published: July 2010

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide‐directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93‐kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2‐specific ligand 125I‐MK‐678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93‐kDa [35S]methionine‐labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148‐kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for 125I‐MK‐678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93‐kDa protein was detected specifically in the rat brain. The 148‐kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with 125I‐MK‐678. As in rat brain, 2e3 interacted specifically with a single 148‐kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for 125I‐MK‐678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post‐translational processing of SSTR2 because 2e3 immunoprecipitated 41‐kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41‐kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide‐directed antibodies suggests the existence of multiple forms of SSTR2.
Cloning and Expression of a 5‐Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase
Authors: Tsou Ann‐ping , Kosaka Alan , Bach Chinh , Zuppan Patti , Yee Calvin , Tom Leonard , Alvarez Robert , Ramsey Scott , Bonhaus Douglas W. , Stefanich Eric , Jakeman Lyn , Eglen Richard M. , Chan Hardy W.
Published: July 2010

Abstract: A cDNA clone (designated as GP2‐7) encoding a novel 5‐hydroxytryptamine (5‐HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5‐HT receptor subtypes (34–48%). The sequence of GP2‐7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5‐HT7. mRNA for GP2‐7 was detected in cortical and limbic brain regions. Transiently expressed GP2‐7 showed high‐affinity binding to [3H]5‐HT (pKi = 9.0) with the following rank order of affinities: 5‐carboxyamidotryptamine (5‐CT) > 5‐HT = 5‐methoxytryptamine (5‐MeOT) > methiothepin > 8‐hydroxy‐2‐(dipropylamino)tetralin (8‐OH‐DPAT) > spiperone ≫ sumatriptan. Adenylyl cyclase activity in CHO‐K1 cells transiently transfected with GP2‐7 was stimulated by several analogues of 5‐HT with the following order of potency: 5‐CT > 5‐HT = 5‐MeOT > dipropyl‐5‐CT > 8‐OH‐DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2‐7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5‐CT.
Primary Structure of the Human Platelet Serotonin 5‐HT2A Receptor: Identity with Frontal Cortex Serotonin 5‐HT2A Receptor
Authors: Cook Edwin H. , Fletcher Kathlyn E. , Wainwright Mark , Marks Nancy , Yan Shu‐ya , Leventhal Bennett L.
Published: July 2010

Abstract: Previous radioligand binding studies have demonstrated human platelet serotonin2A (5‐HT2A) receptor binding sites. Pharmacological similarities between platelet and frontal cortex 5‐HT2A receptor binding parameters have been demonstrated. However, it is not clear whether the platelet 5‐HT2A receptor primary structure is identical to that of the brain receptor. Three overlapping cDNAs were obtained to span completely the coding region of the 5‐HT2A receptor. These clones were sequenced with external and internal primers. The nucleotide sequence of human platelet 5‐HT2A cDNA was identical to that reported for the human frontal cortex 5‐HT2A receptor, except for nucleotide 102 (T → C), which has been reported to represent a normal DNA polymorphism that does not alter the amino acid sequence. This finding may have implications in the study of neuropsychiatric disorders for which altered platelet 5‐HT2A receptor binding has been demonstrated.
Involvement of Calcium Influx in Muscarinic Cholinergic Regulation of Phospholipase C in Cerebellar Granule Cells
Authors: Del Río E. , Nicholls D. G. , Downes C. P.
Published: July 2010

Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca2+ concentration ([Ca2+]c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca2+]c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca2+]c and agonist‐stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol‐stimulated inositol phosphate accumulation was achieved using 20 mM KCl, which increased [Ca2+]c from ∼20 to ∼75 nM, indicating the involvement of relatively low threshold Ca2+ channels and the high sensitivity of the relevant PLC to small changes in [Ca2+]c. By contrast, increases in [Ca2+]c induced by the Ca2+ ionophore ionomycin were associated with more modest and less potent effects on agonist‐stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein‐regulated PLC and voltage‐stimulated elevations of [Ca2+]c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.
Schwann Cells Exhibit P2Y Purinergic Receptors that Regulate Intracellular Calcium and Are Up‐Regulated by Cyclic AMP Analogues
Authors: Lyons Susan A. , Morell Pierre , McCarthy Ken D.
Published: July 2010

Abstract: Schwann cells establish close contact with axons during development, and this is maintained throughout life. Signaling by neurotransmitters may play an important role in Schwann cell‐axon interaction. Schwann cells were examined for the presence of neuroligand receptors that are linked to increases in levels of cytoplasmic calcium. Schwann cell cultures were prepared from neonatal rat sciatic nerve and, after 0.25, 1, 4, 7, and 14 days in vitro (DIV), loaded with the calcium indicator dye fura 2‐AM. The influence of neuroligands on the cytosolic free calcium concentration ([Ca2+]i) was then examined at each time point using a video‐based imaging system. Approximately 80–95% of all freshly isolated Schwann cells responded to 10 µM ATP with a three‐fold rise in [Ca2+]i. Bradykinin, glutamate, and histamine had no or only partial and inconsistent responses. The ATP‐induced calcium response disappeared within 4 DIV. Culturing cells in the presence of cyclic AMP (cAMP) analogues (which induce proliferation and differentiation in vitro) restored the ability of Schwann cells to respond to ATP with increased [Ca2+]i. In the presence of cAMP analogues the extent of recovery of ATP responsiveness was dependent on serum concentration. Fifty to ninety percent of cells regained calcium responsiveness to ATP when grown in medium containing cAMP analogues and 1% serum. These cells also exhibited immunoreactivity to P0 antibody, characteristic of the myelinating lineage. In contrast, only 15–30% of the Schwann cells regained calcium responsiveness when grown in medium containing cAMP analogues and 10% serum. Under these conditions all Schwann cells exhibited immunoreactivity to antibodies against nerve growth factor receptor, characteristic of the nonmyelinating lineage, although some also contained galactocerebroside immunoreactivity. The correlation between the recovery of the ATP response and the recovery of stage‐specific markers suggests that Schwann cell ATP receptor expression may be a developmental process, preferentially associated with Schwann cells moving toward the myelinating lineage.
Stress‐Induced Sensitization of Dopamine and Norepinephrine Efflux in Medial Prefrontal Cortex of the Rat
Authors: Gresch Paul J. , Sved Alan F. , Zigmond Michael J. , Finlay Janet M.
Published: July 2010

Abstract: We examined whether prior exposure to chronic cold (17–28 days, 5°C) alters basal or stress‐evoked (30‐min tail shock) catecholamine release in medial prefrontal cortex, nucleus accumbens, and striatum, using in vivo microdialysis. Basal norepinephrine (NE) concentrations in medial prefrontal cortex did not differ between chronically cold‐exposed rats and naive control rats (2.7 ± 0.3 vs. 2.5 ± 0.2 pg/20 µl, respectively). Basal dopamine (DA) efflux in any of the brain regions was not significantly different between chronically cold‐exposed rats and naive rats. However, a trend for lower basal DA efflux in the cold‐exposed relative to naive rats was observed in medial prefrontal cortex (1.5 ± 0.2 vs. 2.2 ± 0.3 pg/20 µl, respectively), nucleus accumbens (3.7 ± 0.8 vs. 5.4 ± 0.9 pg/20 µl, respectively), and striatum (4.4 ± 0.5 vs. 7.2 ± 1.5 pg/20 µl, respectively). In medial prefrontal cortex of rats previously exposed to cold, tail shock elicited a greater increase from baseline in both DA and NE efflux relative to that measured in naive rats (DA, 2.3 ± 0.3 vs. 1.2 ± 0.1 pg, respectively; NE, 3.8 ± 0.4 vs. 1.4 ± 0.2 pg, respectively). However, in nucleus accumbens or striatum of rats previously exposed to cold, the stress‐induced increase in DA efflux was not significantly different from that of naive rats (nucleus accumbens, 1.8 ± 0.7 vs. 1.5 ± 0.3 pg, respectively; striatum, 1.9 ± 0.4 vs. 2.6 ± 0.7 pg, respectively). Thus, both cortical NE projections and cortically projecting DA neurons sensitize after chronic exposure to cold. In contrast, subcortical DA projections do not sensitize under these conditions.
Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas
Authors: Iversen Lars , Mulvihill Eileen , Haldeman Betty , Diemer Nils Henrik , Kaiser Frank , Sheardown Malcolm , Kristensen Peter
Published: July 2010

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin‐stimulated cyclic AMP formation. The mGluR4 subtypes may represent l‐2‐amino‐4‐phosphonobutyrate‐sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre‐ and postsynaptically located metabotropic glutamate receptors.
Chemical Modifications of Melatonin Receptors in Chicken Brain
Authors: Kosař E. , Teisinger J. , Vyskočil F. , Vaněček J.
Published: July 2010

Abstract: The membrane‐bound or solubilized melatonin receptors were treated with protein‐modifying agents under specific conditions and then assayed for 125I‐melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N‐ethylmaleimide and p‐chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid and 1‐fluoro‐2,4‐dinitrobenzene) inhibited 125I‐melatonin binding in a dose‐dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I‐melatonin binding to guanine nucleotides after N‐ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole, N‐acetylimidazole, and p‐nitrobenzenesulfonyl fluoride also inhibited 125I‐melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross‐reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I‐melatonin binding in a dose‐dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.
Cytochrome P450s of the 4A Subfamily in the Brain
Authors: Strömstedt Maria , Warner Margaret , Gustafsson Jan‐Åke
Published: July 2010

Abstract: Members of the P450 4A subfamily are key enzymes in the synthesis and degradation of metabolites of arachidonic acid, which are of physiological importance in the brain. In the rat, four members of this subfamily, 4A1, 4A2, 4A3, and 4A8, have been described. In this study, the expression of members of the 4A subfamily in the rat brain has been examined by PCR amplification, by western and northern blotting, and by protein N‐terminal sequencing. With PCR all four members of the subfamily were detectable in the liver and kidney. P450 4A1 was found exclusively in the liver and kidney, whereas P450 4A2 was detectable in all the tissues tested, including the lung, seminal vesicles, prostate, cerebral cortex, hypothalamic preoptic area, cerebellum, and brainstem. The tissue distribution of P450 4A3 was similar to that of 4A2 except that it was not detectable in seminal vesicles. A P450 4A8‐specific fragment was amplified from the kidney, liver, and prostate and weakly from the cerebral cortex but not from other brain regions. Despite the evidence of their presence by PCR, no members of the 4A family were detectable on northern blots with mRNA from the brain. On western blots a P450 4A‐specific antiserum recognized a band in P450 fractions prepared from the brain. The intensity of the signal with 30 pmol of P450 from the brain was similar to that with 10 pmol of liver microsomal P450. The brain P450 was extracted from 1 g of brain, whereas the 10 pmol of liver P450 is the equivalent of 1 mg of liver. This suggests a brain content of 4A P450 that is 0.1% of that in the liver. N‐terminal sequencing of the protein bands in the brain P450 fraction revealed the presence of both P450 4A8 and 4A3. These data show the presence in the brain of forms of P450 whose level of mRNA is too low to be detected on northern blots. The specificity of tissue distribution shows that this is not just a nonspecific background level of expression and suggests a role of brain P450 in the synthesis and degradation of arachidonic acid metabolites.
Neuronal Glucoprivation Enhances Hypothalamic Histamine Turnover in Rats
Authors: Oohara Akihiko , Yoshimatsu Hironobu , Kurokawa Mamoru , Oishi Ryozo , Saeki Kiyomi , Sakata Toshiie
Published: July 2010

Abstract: Histamine (HA) turnover in the rat hypothalamus following insufficient energy supply due to glucoprivation was examined after administration of insulin or 2‐deoxy‐d‐glucose (2‐DG). HA turnover was assessed by accumulation of tele‐methylhistamine (t‐MH), a major metabolite of brain HA, following administration of pargyline. Intraperitoneal injection of 1, 2, and 4 U/kg of insulin, which had no influence on steady‐state levels of HA and t‐MH, increased pargyline‐induced accumulation of t‐MH. Accumulation of t‐MH due to pargyline was inversely related to the concomitant plasma glucose concentration after different doses of insulin. The level of t‐MH accumulated by pargyline did not change compared with that of controls, when a euglycemic condition was maintained or insulin at a dose of 6 mU per rat was infused into the third cerebroventricle. Intracerebroventricular infusion of 24 µmol per rat of 2‐DG, which had no influence on steady‐state levels of HA and t‐MH, increased the level of t‐MH enhanced by pargyline. The results indicate that an increase in hypothalamic HA turnover in response to glucoprivation may be involved in homeostatic regulation of energy metabolism in the brain.
Phorbol Ester Administration Transiently Increases Aromatic l‐Amino Acid Decarboxylase Activity of the Mouse Striatum and Midbrain
Authors: Young E. A. , Neff N. H. , Hadjiconstantinou M.
Published: July 2010

Abstract: Aromatic l‐amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12‐myristate 13‐acetate administered intracerebroventricularly transiently increased AAAD activity by 30–50% over control values within ∼30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of Vmax with little change of Km for either l‐3,4‐dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester‐induced increase of AAAD. Moreover, okadaic acid, a serine/threonine‐selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C‐mediated pathways modulate AAAD activity in vivo.
Iron, Transferrin, and Ferritin in the Rat Brain During Development and Aging
Authors: Roskams A. Jane I. , Connor James R.
Published: July 2010

Abstract: Iron is a universal cofactor for mitochondrial energy generation and supports the growth and differentiation of all cell types. In the CNS, iron is a key component of systems responsible for myelination and the synthesis of several neurotransmitters. In this study the spatial and temporal pattern of iron and its regulatory proteins transferrin and ferritin are quantitatively examined in the rat CNS during the first 3 weeks of postnatal life and in adults and aged animals. The midbrain, the cerebral cortex, and the cerebellum‐pons are examined independently. Iron, transferrin, and ferritin concentrations are highest in all three brain regions at birth and decrease in each region to minimum levels during the third postnatal week. The decrease in levels of iron, transferrin, and ferritin is most pronounced in the cerebellum‐pons and cortex and least in the midbrain. From postnatal day 17, iron (total iron content) and ferritin levels increase throughout the lifetime of the rat. In contrast, transferrin levels remain fairly constant in each brain region after postnatal day 24. The midbrain region, which includes the iron‐rich regions such as the globus pallidus, substantia nigra, and red nucleus, has the least change in iron with development, has the highest level of ferritin during development, and consistently has the highest level of transferrin at all ages. These observations are consistent with reports that iron is important for normal motor function. Transferrin did not increase after postnatal day 24 in the three brain regions examined despite increasing amounts of iron, which implies a decrease in iron mobility in the aged rats, a finding that is consistent with observations of human brain tissue. The data reported in this study demonstrate that iron acquisition and mobilization systems in the CNS are established early in development and that the overall pattern of acquisition among brain regions is similar. These data offer support and insight into established concepts that a sufficient iron supply is critical for normal neurological development.
Purification and Initial Characterization of Rat B49 Glial Cell Line‐Derived Neurotrophic Factor
Authors: Lin Leu‐Fen H. , Zhang Tie Jin , Collins Frank , Armes Lyman G.
Published: July 2010

Abstract: The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin‐binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and on molecular sieve chromatography with an apparent mass of ∼33–45 kDa. The factor behaves like a disulfide‐bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS‐PAGE or reversed‐phase (RP)‐HPLC. The apparent mass of the monomer is ∼16 kDa after deglycosylation with N‐Glycanase. This factor has been purified 34,000‐fold to apparent homogeneity by a combination of heparin‐affinity chromatography, molecular sieving chromatography, SDS‐PAGE, and RP‐HPLC. The purified rat protein promotes the survival, morphological differentiation, and high‐affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high‐affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at ∼40 pg/ml, or 1 pM, and has unique amino‐terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line‐derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.
Enhanced Phosphodiestric Breakdown of Phosphatidylinositol Bisphosphate After Experimental Brain Injury
Authors: Prasad M. Renuka , Dhillon H. S. , Carbary T. , Dempsey R. J. , Scheff S. W.
Published: July 2010

Abstract: Regional levels of lactate and inositol 1,4,5‐trisphosphate (IP3), a cellular second messenger of the excitatory neurotransmitter system, were measured after lateral fluid percussion (FP) brain injury in rats. At 5 min postinjury, tissue lactate concentrations were significantly elevated in the cortices and hippocampi of both the ipsilateral and contralateral hemispheres. By 20 min postinjury, lactate concentrations were elevated only in the cortices and hippocampus of the ipsilateral hemisphere. Whereas the IP3 concentrations were elevated in the hippocampi of the ipsilateral and contralateral hemisphere and in the cortex of ipsilateral hemisphere at 5 min postinjury, no elevation in these sites was found at 20 min postinjury. Histologic analysis revealed neuronal damage in the cortex and CA3 regions of hippocampus ipsilateral to the injury at 24 h postinjury. The present results suggest activation of the phosphoinositide signal transduction pathway at the onset of injury and of a possible requirement of early persistent metabolic dysfunction (>20 min) such as the lactate accumulation in the delayed neuronal damage.
Glutamatergic Control of Dopamine Release During Stress in the Rat Prefrontal Cortex
Authors: Jedema Hank Peter , Moghaddam Bita
Published: July 2010

Abstract: In vivo microdialysis was used to assess the hypothesis that the stress‐induced increase in dopamine release in the prefrontal cortex is mediated by stress‐activated glutamate neurotransmission in this region. Local perfusion of an α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, blocked the stress‐induced increase in dopamine levels, whereas an NMDA receptor antagonist, 2‐amino‐5‐phosphonopentanoic acid, at the dose tested, was not able to alter this response significantly. These data indicate that the effect of stress on dopamine release in the prefrontal cortex is mediated locally by activation of AMPA/kainate receptors, which modulate the release of dopamine in this region.
Altered Brain Metabolism of Iron as a Cause of Neurodegenerative Diseases?
Authors: Gerlach M. , Ben‐Shachar D. , Riederer P. , Youdim M. B. H.
Published: July 2010

Abstract: Iron is the most abundant metal in the human body (Pollitt and Leibel, 1982; Youdim, 1988), and the brain, like the liver, contains a substantially higher concentration of iron than of any other metal (Yehuda and Youdim, 1988). Within the brain, iron shows an uneven distribution, with high levels in the basal ganglia (substantia nigra, putamen, caudate nucleus, and globus pallidus), red nucleus, and dentate nucleus (Spatz, 1922; Hallgren and Sourander, 1958; Hill and Switzer, 1984; Riederer et al., 1989). Iron deposition in the brain is mainly in organic storage forms such as ferritin but not hemosiderin (Hallgren and Sourander, 1958; Octave et al., 1983), with relatively little in a free and reactive form. Although the function of a regionally high brain iron content is unknown, the homeostasis of brain iron is thought to be necessary for normal brain function, especially in learning and memory (Youdim et al., 1989; Yehuda and Youdim, 1989; Pollit and Metallinos‐Katsaras, 1990; Youdim, 1990). Thus, a high content of brain iron may be essential, particularly during development, but its presence means that injury to brain cells may release iron ions that can lead to oxidative stress via formation of oxygen free radicals. Such radicals are thought to be involved in lipid peroxidation of the cell membrane, leading to increased membrane fluidity, disturbance of calcium homeostasis, and finally cell death (Youdim et al., 1989; Halliwell, 1992). Iron is an essential participant in many metabolic processes, including (a) DNA, RNA, and protein synthesis, (b) as a cofactor of many heme and nonheme enzymes, (c) the formation of myelin, and (d) the development of the neuronal dendritric tree (Ben‐Shachar et al., 1986; Youdim et al., 1991b). A deficiency of iron metabolism would therefore be expected to alter some or all of these processes (Jacobs and Worwood, 1980; Youdim, 1985, 1988). Studies of iron distribution in the human brain have demonstrated that the degree of iron deposition, primarily in the basal ganglia (a predominantly dopamine structure), increases with age (Hallgren and Sourander, 1958) and in certain disorders, most notably the basal ganglia disorders (Seitelberger, 1964). This review will present some of the experimental evidence indicating a role of disturbed iron metabolism as a cause of the neurodegenerative disorder Parkinson's disease and possibly other neurodegenerative disorders such as Alzheimer's disease. In addition, some of the neurochemical and histochemical findings obtained at autopsy from analyses of the brain from patients with neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and progressive supranuclear palsy (Steele‐Richardson‐Olszewski's disease) will be discussed. Special attention will be paid to clarifying the possible implication of the observed changes in the etiology of neurodegenerative disorders.
Increase in Rat Adrenal Phenylethanolamine N‐Methyltransferase mRNA Level Caused by Immobilization Stress Depends on Intact Pituitary ‐ Adrenocortical Axis
Authors: Viskupic Emil , Kvetnansky Richard , Sabban Esther L. , Fukuhara Koki , Weise Virginia K. , Kopin Irwin J. , Schwartz Joan P.
Published: July 2010

Abstract: The effects of a single and of repeated immobilization stress on the expression of the final enzyme involved in epinephrine biosynthesis, phenylethanolamine N‐methyltransferase (PNMT), are described. A single immobilization (whether lasting 5 or 120 min) caused a severalfold increase of the adrenal PNMT mRNA level as measured 2 h after the beginning of the procedure. This elevation was of a transient nature, peaked 3–6 h after the 2‐h immobilization, and returned to control values by 12 h after the stress. When the animals were immobilized for 2 h/day for seven consecutive days, an increase in content of PNMT mRNA of a similar magnitude was observed, which persisted for at least 2 days after the seventh immobilization. The immobilization‐induced increase was completely abolished in hypophysectomized animals, whereas adrenal denervation failed to prevent it. These data suggest that the immobilization‐induced increase in adrenal PNMT mRNA level depends primarily on pituitary‐adrenocortical regulation.
A Combination of PLP and DM20 Transgenes Promotes Partial Myelination in the Jimpy Mouse
Authors: Nadon Nancy L. , Arnheiter Heinz , Hudson Lynn D.
Published: July 2010

Abstract: Mutations in the myelin proteolipid protein (PLP) gene, such as that found in the jimpy mouse, result in an abnormal structure of the myelin, severe dysmyelination, and a reduction in the number of mature oligodendrocytes. To examine the functions of the two alternatively spliced isoforms of proteolipid protein, transgenic mice were generated that express either PLP or DM20 cDNAs placed under control of the PLP upstream regulatory region. The transgenes were bred into jimpy mice, and the effect of the transgenes on the dysmyelinating phenotype was analyzed. Neither the PLP transgene nor the DM20 transgene alone had an effect on myelination in the jimpy mice. Combining the two transgenes substantially increased the number of myelinated axons, suggesting that the two alternatively spliced products of the PLP locus perform distinct functions in oligodendrocytes. The enhanced myelination was not sufficient, however, for completely correcting the dysmyelinating phenotype of the jimpy mice, nor was it accompanied by the restoration of normal levels of myelin gene expression. The inability to rescue the jimpy phenotype is most likely attributable to a dominant negative action of the abnormal proteolipid proteins present in jimpy mice. These results demonstrate the complexity of proteolipid protein function in myelination.
Comparison of the Potency of Competitive NMDA Antagonists Against the Neurotoxicity of Glutamate and NMDA
Authors: Speliotes Elizabeth K. , Hartnett Karen A. , Blitzblau Rachel C. , Aizenman Elias , Rosenberg Paul A.
Published: July 2010

Abstract: The object of this investigation was to determine whether glutamate uptake affects the apparent potency of the competitive antagonists dl‐2‐amino‐5‐phosphonovalerate and CGS‐19755 in blocking NMDA receptor‐mediated neurotoxicity. In astrocyte‐rich rat cortical cultures we observed that dl‐2‐amino‐5‐phosphonovalerate and CGS‐19755 were 24 and 16 times more potent against NMDA than against glutamate‐induced toxicity. In contrast, dl‐2‐amino‐5‐phosphonovalerate was equipotent against the two agonists in astrocyte‐poor cultures, in which dendrites are directly exposed to the extracellular medium. With the noncompetitive NMDA antagonist MK‐801, similar potencies were observed against glutamate (212 ± 16 nM) and against NMDA (155 ± 9 nM) neurotoxicity. These results may be explained if we assume that the neuronal cell body is less susceptible than the dendrites to NMDA receptor‐mediated toxicity, and that the action of glutamate in astrocyte‐rich cultures is confined to the cell body. In this case, one would expect that higher concentrations of glutamate would be needed to produce toxicity in astrocyte‐rich cultures, and that higher concentrations of competitive antagonists would be needed to overcome this toxicity. Our observations help explain the pharmacology of the competitive NMDA antagonists against NMDA receptor‐mediated neurotoxicity but also suggest the possibility that, because the cell body and dendrites may be distinct sites for neurotoxicity, they might also involve different mechanisms of toxicity.
Effects of Extracellular ATP on Fe2+‐Induced Cytotoxicity in PC‐12 Cells
Authors: Cheng Y. , Wixom P. , James‐Kracke M. R. , Sun A. Y.
Published: July 2010

Abstract: Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca2+ influx via the activation of a P2y purinoceptor. Rat pheochromocytoma (PC‐12) cells are known to resemble rat sensory neurons and to possess a P2y purinoceptor. In this study, we demonstrated that extracellular ATP dose‐dependently increased PC‐12 cell death in the presence of ferrous ions. Voltage‐sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC‐12 cells from Fe2+/ATP‐induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe2+/ATP‐induced neuronal cell death.
Nitric Oxide‐Mediated Inhibition of the Mitochondrial Respiratory Chain in Cultured Astrocytes
Authors: Bolaños J. P. , Peuchen S. , Heales S. J. R. , Land J. M. , Clark J. B.
Published: July 2010

Abstract: The Ca2+‐independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon‐γ (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH‐ubiquinone‐1 reductase (complex I) and succinate‐cytochrome c reductase (complex II–III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate‐cytochrome c reductase activity, but NADH‐ubiquinone‐1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG‐monomethyl‐l‐arginine. The lipopolysaccharide/interferon‐γ treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide‐mediated neurotoxicity.
In Vivo Dynamics of Norepinephrine Release‐Reuptake in Multiple Terminal Field Regions of Rat Brain
Authors: Mitchell Kim , Oke Arvin F. , Adams R. N.
Published: July 2010

Abstract: Electrical stimulation of the ascending dorsal tegmental bundle of the locus ceruleus was used to elicit controlled release of norepinephrine. Real‐time in vivo monitoring in the brains of urethane‐anesthetized rats was observed with high speed chronocoulometry at rapidly responding carbon fiber electrodes. Using modeling similar to that developed for dopamine release, the electrochemical signals were characterized as the balance between norepinephrine release per electrical stimulation pulse and apparent Michaelis‐Menten reuptake parameters. Stimulation produced simultaneous overflow release at all terminal fields examined. The release and reuptake characteristics varied considerably in different regions. If the parameters are normalized to endogenous concentration in the terminal fields, release but not reuptake correlates with innervation density in several regions. Stimulated release results in norepinephrine overflow and transport in most brain regions with half‐lives of 1–3 s and overflow distances of 25–50 µm at most. A surprising exception occurs in the upper layers of cortex (cingulate and sensory) where half‐lives may be in the 10s of seconds and spatial reach may be up to 100 µm. The uptake in the outer cortical layers appears to be minimal and comparable with only nonspecific reuptake.
3‐Methoxytyramine Is the Major Metabolite of Released Dopamine in the Rat Frontal Cortex: Reassessment of the Effects of Antipsychotics on the Dynamics of Dopamine Release and Metabolism in the Frontal Cortex, Nucleus Accumbens, and Striatum by a Simple Two Pool Model
Authors: Karoum Farouk , Chrapusta Stanislaw J. , Egan Michael F.
Published: July 2010

Abstract: 3‐Methoxytyramine (3‐MT) and 3,4‐dihydroxyphenylacetic acid (DOPAC) rates of formation were used, respectively, to assess the dynamics of dopamine (DA) release and turnover in the rat frontal cortex, nucleus accumbens, and striatum. Assuming total (re)uptake and metabolism of released DA are relatively uniform among the three brain regions, a simplified two pool model was used to assess the metabolic fate of released DA. Under basal conditions, 3‐MT formation was found to comprise >60% of total DA turnover (sum of 3‐MT plus DOPAC rates of formation) in the frontal cortex, and not more than 15% in the nucleus accumbens and striatum. Haloperidol increased the 3‐MT rate of formation to a greater extent in the frontal cortex than in the two other regions. Clozapine increased the 3‐MT rate of formation in the frontal cortex and decreased it in the striatum. Both drugs increased DOPAC rate of formation in the frontal cortex and nucleus accumbens. It was elevated by haloperidol but not clozapine in the striatum. It is concluded that (1) O‐methylation is a prominent step in the catabolism of DA in the frontal cortex under both physiological conditions and after acute treatment with antipsychotics, (2) 3‐MT is the major metabolite of released DA in the frontal cortex and possibly also in the nucleus accumbens and striatum, (3) in contrast to the frontal cortex, most of the DOPAC in the nucleus accumbens and striatum appear to originate from intraneuronal deamination of DA that has not been released, (4) because presynaptic uptake and metabolism of DA give rise to DOPAC, whereas postsynaptic uptake and metabolism produced both DOPAC and 3‐MT, the ratio of 3‐MT to DOPAC rates of formation can be a useful index of reuptake inhibition.
Possible Role of Nitric Oxide in Catecholamine Secretion by Chromaffin Cells in the Presence and Absence of Cultured Endothelial Cells
Authors: Torres Magdalena , Ceballos Guillermo , Rubio Rafael
Published: July 2010

Abstract: We studied the effect of cultured endothelial cells on the secretion of catecholamines by cultured bovine chromaffin cells. Chromaffin cell catecholamine secretion was stimulated by either boluses of potassium (K+) or the nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP). Endothelial cells inhibited the catecholamine release and stimulatory effects of K+ and DMPP. This inhibition increased with time, and in 25 min the initial stimulated secretory response (100%) to 30 mM K+ or 25 μM DMPP dropped to 45 ± 3% and 53.5 ± 2.3%, respectively. This endothelial cell‐induced inhibition was blocked by the nitric oxide synthase inhibitors N‐nitro‐l‐arginine methyl ester (l‐NAME) and N‐monoethyl‐l‐arginine (l‐NMMA), and by the guanylate cyclase inhibitor methylene blue, indicating that the l‐arginine/nitric oxide/ cyclic GMP pathway is involved in this endothelial cell‐chromaffin cell interaction. In the absence of endothelial cells, incubation of chromaffin cells with l‐NAME, l‐NMMA, or methylene blue also augmented the secretagogue‐induced catecholamine secretion, indicating that nitric oxide from chromaffin cells could be implicated in an autoinhibitory process of catecholamine release. These results provide indirect evidence for the presence of nitric oxide synthase in bovine adrenomedullary chromaffin cells. Our results show that there is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that an additional level of inhibition is observed when cultured vascular endothelial cells are present. These two inhibitory processes may have different origins, but they appear to converge into a common pathway, the l‐arginine/nitric oxide synthase/guanylate cyclase pathway.
Extracellular Glutamate During Focal Cerebral Ischaemia in Rats: Time Course and Calcium Dependency
Authors: Wahl Florence , Obrenovitch Tihomir P. , Hardy Aidan M. , Plotkine Michel , Boulu Roger , Symon Lindsay
Published: July 2010

Abstract: The time course of changes in extracellular glutamic acid levels and their Ca2+ dependency were studied in the rat striatum during focal cerebral ischaemia, using microdialysis. Ischaemia‐induced changes were compared with those produced by high K+‐evoked local depolarization. To optimize time resolution, glutamate was analysed continuously as the dialysate emerged from the microdialysis probe by either enzyme fluorimetry or biosensor. The Ca2+ dependency of glutamate changes was examined by perfusing the probe with Ca2+‐free medium. With normal artificial CSF, ischaemia produced a biphasic increase in extracellular glutamate, which started from the onset of ischaemia. During the first phase lasting ∼10 min, dialysate glutamate level increased from 5.8 ± 0.9 µM· min−1 to 35.8 ± 6.2 µM where it stabilized for ∼3 min. During the second phase dialysate glutamate increased progressively to its maximum (82 ± 8 µM), reached after 55 min of ischaemia, where it remained for as long as it was recorded (3 h). The overall changes in extracellular glutamate were similar when Ca2+ was omitted from the perfusion medium, except that the first phase was no longer detectable and, early in ischaemia, extracellular glutamate increased at a significantly slower rate than in the control group (2.2 ± 1 µM· min−1; p < 0.05). On the basis of these data, we propose that most of the glutamate released in the extracellular space in severe ischaemia is of metabolic origin, probably originating from both neurons and glia, and caused by altered glutamate uptake mechanisms. Comparison with high K+‐induced glutamate release did not suggest that glutamate “exocytosis,” early after middle cerebral artery occlusion, was markedly limited by deficient ATP levels.