bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of Hippocampal Neurotrophins During Aging in Rats
Authors: Narisawa‐Saito Mako , Nawa Hiroyuki
Published: July 2010

Abstract: Neurotrophins are a family of neurotrophic factors with considerable structural homology. We used sensitive and specific two‐site enzyme immunoassays to assess age‐associated changes in levels of three neurotrophins—nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin‐3 (NT‐3)—in the hippocampus of Fischer 344 rats. Expressions of these proteins and their mRNAs were compared in the same animals. More than 200 ng of BDNF per gram of tissue was detected in the hippocampus of 2‐month‐old rats. This amount was two and 100 times greater than that of NT‐3 and NGF, respectively. The levels of BDNF and NT‐3 increased further 2–6 months after birth, whereas NGF content declined during this period, and the altered protein levels of all three neurotrophins were maintained 6–18 months postnatally. In contrast to the patterns of protein expression, BDNF mRNA levels increased during both of these periods, and the NT‐3 mRNA levels appeared to decline. Changes in the expression of BDNF mRNA and NGF protein were opposite to those reported to occur in Alzheimer's disease. These results suggest that, during normal aging in rats, neurotrophin expression is regulated independently at both the mRNA and posttranslational levels. Any deficiency in their regulation might contribute to neurodegenerative disorders.
Detection and Quantitation of Perlecan mRNA Levels in Alzheimer's Disease and Normal Aged Hippocampus by Competitive Reverse Transcription‐Polymerase Chain Reaction
Authors: Maresh Grace A. , Erezyilmaz Deniz , Murry Charles E. , Nochlin David , Snow Alan D.
Published: July 2010

Abstract: Our previous studies have implicated perlecan, a specific heparan sulfate proteoglycan, in the pathogenesis of fibrillar β‐amyloid protein (Aβ) accumulation and persistence in Alzheimer's disease (AD) brain. In the present investigation, we determined if perlecan mRNA was present in rodent and human brain tissue and whether perlecan persistence in Aβ amyloid deposits in AD hippocampus may be partly due to increased perlecan expression and/or decreased perlecan degradation. To detect and to quantify low‐abundance perlecan mRNA in rodent and postmortem human brain tissue, regions of perlecan domain I (503 and 366 bp) containing the unique heparan sulfate glycosaminoglycan attachment sites were analyzed by reverse transcription (RT) and polymerase chain reaction (PCR). Perlecan mRNA was detected in rodent brain, kidney, and liver and in human AD and normal aged frontal cortex. Different‐size transcripts of perlecan domain I were found, suggesting the existence of alternatively spliced variants of perlecan or closely related gene products. Quantitative competitive RT‐PCR using a mutant perlecan domain I internal standard was used to determine perlecan mRNA levels in total RNA isolated from the hippocampus of 10 AD (mean ± SEM duration of illness, 11.3 ± 1.4 years) and 10 normal aged controls. No significant difference in perlecan mRNA levels from the hippocampus of AD (1.12 ± 0.29 amol/500 ng of total RNA) versus normal aged controls (1.09 ± 0.30 amol/500 ng of total RNA) was found, indicating that perlecan expression remained at steady‐state levels. These results therefore suggest that perlecan persistence in Aβ‐amyloid deposits in late‐stage AD may be primarily due to decreased perlecan degradation and removal.
Receptor‐Mediated Activation of G Proteins Is Increased in Postmortem Brains of Bipolar Affective Disorder Subjects
Authors: Friedman Eitan , Wang Hoau‐Yan
Published: July 2010

Abstract: Guanine nucleotide binding proteins (G proteins) have been implicated in the pathophysiology of bipolar affective disorder. In the present investigation receptor‐mediated G protein activation and changes in G protein trimeric state were examined in frontal cortical membranes obtained from postmortem brains of bipolar affective disorder subjects and from age‐, sex‐, and postmortem interval‐matched controls. Stimulation of cortical membranes with serotonin, isoproterenol, or carbachol increased guanosine 5′‐O‐(3‐[35S]thiophosphate) ([35S]GTPγS) binding to specific Gα proteins in a receptor‐selective manner. The abilities of these receptor agonists to stimulate the binding of [35S]GTPγS to the Gα proteins was enhanced in membranes from bipolar brains. Immunoblot analyses showed increases in the levels of membrane 45‐ and 52‐kDa Gαs proteins but no changes in the amounts of Gαi, Gαo, Gαz, Gαq/11, or Gβ proteins in membrane or cytosol fractions of bipolar brain homogenates. Pertussis toxin (PTX)‐activated ADP‐ribosylations of Gαi and Gαo were enhanced by ∼80% in membranes from bipolar compared with control brains, suggesting an increase in the levels of the trimeric state of these G proteins in bipolar disorder. Serotonin‐induced, magnesium‐dependent reduction in PTX‐mediated ADP‐ribosylation of Gαi/Gαo in cortical membranes from bipolar brains was greater than that observed in controls, providing further evidence for enhanced receptor‐G protein coupling in bipolar brain membranes. In addition, the amounts of Gβ proteins that coimmunoprecipitated with the Gα proteins were also elevated in bipolar brains. The data show that in bipolar brain membrane there is enhanced receptor‐G protein coupling and an increase in the trimeric state of the G proteins. These changes may contribute to produce exaggerated transmembrane signaling and to the alterations in affect that characterize bipolar affective disorder.
Structural Motifs Encoded by Individual Exons of the Human Neurokinin‐1 Receptor Gene Interact Differentially with Selective Agonists and Antagonists
Authors: Tian Ye , Wu Lan‐Hsin , Pu Yuming , Huang Che C. , Chung Fu‐Zon
Published: July 2010

Abstract: Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N‐terminal sequences of these chimeric receptors are encoded by exon 1, exon 1–2, or exon 1–3 of the NK1 receptor gene, whereas the remaining C‐terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9,Met(O2)11]substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon‐exchanging technique can be a useful tool for studying structure‐function relationships of receptors in which exon‐intron junctions are fully conserved among receptor subtypes.
Fimbria‐Fornix Transections Selectively Down‐Regulate Subtypes of Glutamate Transporter and Glutamate Receptor Proteins in Septum and Hippocampus
Authors: Ginsberg Stephen D. , Rothstein Jeffrey D. , Price Donald L. , Martin Lee J.
Published: July 2010

Abstract: The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria‐fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT‐1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by d‐[3H]aspartate binding. GLT‐1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. d‐[3H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down‐regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down‐regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype‐specific down‐regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.
Differential Regulation of Phenylethanolamine N‐Methyltransferase Expression in Two Distinct Subpopulations of Bovine Chromaffin Cells
Authors: Cahill Anne L. , Eertmoed Alison Leston , Mangoura Dimitra , Perlman Robert L.
Published: July 2010

Abstract: Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N‐methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E‐rich cell population. Cells in the less dense fraction, the norepinephrine (NE)‐rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE‐rich cells had only 3% as much PNMT activity as did the E‐rich cells, 20% of the NE‐rich cells were PNMT immunoreactive. This suggested that the PNMT‐positive cells in the NE‐rich cell cultures contained less PNMT per cell than did E‐rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E‐rich and NE‐rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE‐rich cells were ∼20% of those in E‐rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E‐rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE‐rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE‐rich cells but had little effect in E‐rich cells. Angiotensin II, forskolin, and phorbol 12,13‐dibutyrate elicited large increases in PNMT mRNA levels in E‐rich cells but had no effect in NE‐rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.
Further Identification of Neurokinin Receptor Types and Mechanisms of Calcium Signaling Evoked by Neurokinins in the Murine Neuroblastoma C1300 Cell Line
Authors: Fukuhara Shigetomo , Mukai Hidehito , Kako Koichiro , Nakayama Kazuhisa , Munekata Eisuke
Published: July 2010

Abstract: It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 µM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 µM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, an NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 µM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 µM neurokinin A in the presence of 1.0 µM SR 142801 or 1.0 µM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor‐mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and ω‐conotoxin, L‐ and N‐type voltage‐dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor‐mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage‐independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.
Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury
Authors: Ip Fanny C. F. , Fu Amy K. Y. , Tsim Karl W. K. , Ip Nancy Y.
Published: July 2010

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity‐conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10‐fold after nerve transection; this finding is in sharp contrast to the dramatic up‐regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.
Role of P‐Glycoprotein in the Blood‐Brain Transport of Colchicine and Vinblastine
Authors: Drion N. , Lemaire M. , Lefauconnier J.‐M. , Scherrmann J.‐M.
Published: July 2010

Abstract: Classically, drug penetration through the blood‐brain barrier depends on the lipid solubility of the substance, except for some highly lipophilic drugs, like colchicine and vinblastine, both substrates of P‐glycoprotein, a drug efflux pump present at the luminal surface of the brain capillary endothelial cells. Colchicine and vinblastine uptake into the brain was studied in the rat using the in situ brain perfusion technique and two inhibitors of P‐glycoprotein, verapamil and SDZ PSC‐833. When rats were pretreated with PSC‐833 (10 mg/kg, intravenous bolus), colchicine and vinblastine uptake was enhanced 8.42‐ and 9.08‐fold, respectively, in all the gray areas of the rat brain studied. The mean colchicine distribution volume was increased from 0.67 ± 0.41 to 5.64 ± 0.70 µl/g and vinblastine distribution volume from 2.74 ± 1.15 to 24.88 ± 4.03 µl/g. When rats were pretreated with verapamil (1 mg/kg, intravenous bolus), colchicine distribution volume was increased 3.70‐fold. The increase in colchicine and vinblastine did not differ between the eight brain gray areas. PSC‐833 and verapamil pretreatment had no influence on the distribution volume of either drug in the choroid plexus. Nevertheless, distribution volumes remained small, considering the highly lipophilic nature of the substances. We suggest that P‐glycoprotein is either only partially inhibited (difficulty of fully saturating P‐glycoprotein, especially under in vivo conditions) or not the only barrier to these two drugs.
Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor‐Associated Pores in Dibutyryl Cyclic AMP‐Differentiated NG108‐15 Cells
Authors: Song Shu‐Ling , Chueh Sheau‐Huei
Published: July 2010

Abstract: The effect of replacement of extracellular Na+ with N‐methyl‐d‐glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP‐differentiated NG108‐15 cells. Benzoylbenzoic ATP (BzATP) dose‐dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 µM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4−, replacement of Na+ with NMG had no effect on the ATP‐induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4− was >10 µM. The calculated EC50 value for ATP4− stimulation of the [Ca2+]i increase was 23 µM in NG108‐15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP‐induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP4−‐promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP‐differentiated NG108‐15 cells.
Identification of Two Molecular Species of Rat Brain Phosphatidylcholine that Rapidly Incorporate and Turn Over Arachidonic Acid In Vivo
Authors: Shetty H. Umesha , Smith Quentin R. , Washizaki Kazushige , Rapoport Stanley I. , Purdon A. David
Published: July 2010

Abstract: In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [3H]Arachidonic acid was infused intravenously in pentobarbital‐anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2–10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed‐phase HPLC. Most [3H]arachidonate (>87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn‐2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn‐1 position. However, 10–15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn‐2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn‐1 position. Analysis demonstrated that tracer was present in both the 16:1–20:4 and 18:2–20:4 PtdCho species at specific activities 10–40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half‐lives were calculated for arachidonate of <10 min in 16:1–20:4 and 18:2–20:4 PtdCho and 1–3 h in 16:0–20:4, 18:0–20:4, and 18:1–20:4 PtdCho and PtdIns. The very short half‐lives for arachidonate in the 16:1–20:4 and 18:2–20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.
Portacaval Anastomosis Results in Altered Neuron‐Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C‐NMR Studies
Authors: Sonnewald Ursula , Therrien Guy , Butterworth Roger F.
Published: July 2010

Abstract: 13C‐NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1‐13C]glucose, whereas [2‐13C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1‐13C]glucose as precursor. Brain [1‐13C]glucose metabolism in portacaval‐shunted rats was similar to that in sham‐operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The 13C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2‐13C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval‐shunted rats.
Role of Na+‐Ca2+ Exchanger in Agonist‐Induced Ca2+ Signaling in Cultured Rat Astrocytes
Authors: Takuma Kazuhiro , Matsuda Toshio , Hashimoto Hitoshi , Kitanaka Junichi , Asano Shoichi , Kishida Yoko , Baba Akemichi
Published: July 2010

Abstract: We have previously demonstrated that activation of the Na+‐Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na+‐Ca2+ exchanger is involved in agonist‐induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by l‐glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin‐induced increase in [Ca2+]i. The Na‐induced Ca2+ signal was also attenuated by S‐nitroso‐l‐cysteine and 8‐bromo cyclic GMP, whereas it was enhanced by 3,4‐dichlorobenzamil, an inhibitor of the Na+‐Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na+‐Ca2+ exchanger enhanced the ionomycin‐induced increase in [Ca2+]i and blocked the effects of SNP and 8‐bromo cyclic GMP in reducing the NA‐induced Ca2+ signal. Furthermore, the ionomycin‐induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na+‐Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.
Cytosolic Proteolysis of τ by Cathepsin D in Hippocampus Following Suppression of Cathepsins B and L
Authors: Bednarski Eric , Lynch Gary
Published: July 2010

Abstract: Incubation of cultured hippocampal slices with an inhibitor [N‐CBZ‐l‐phenylalanyl‐l‐alanine‐diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of τ protein and the production of a 29‐kDa τ fragment (τ29). A τ antibody that is sensitive to the phosphorylated state of its epitopes did not recognize τ proteins or the τ29 fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the τ fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. τ29 exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal τ‐tubulin interactions. Three lines of evidence indicated that ZPAD‐induced τ proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co‐incubation of cathepsin D and τ at neutral pH resulted in a loss of intact τ proteins and production of a 29‐kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD‐induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD‐induced τ proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The τ fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age‐related pathological features.
Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor‐Adenylyl Cyclase System in Cerebellar Granule Cells
Authors: Hettinger‐Smith Barbara D. , Leid Mark , Murray Thomas F.
Published: July 2010

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up‐regulation of A1 adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A1 adenosine receptor agonists and antagonists. Exposure to the A1 adenosine receptor agonist N6‐cyclopentyladenosine resulted in (1) a time‐ and concentration‐dependent reduction in the density of receptors labeled by 1,3‐[3H]dipropyl‐8‐cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A1 adenosine receptor sites displaying high affinity for 2‐chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8‐p‐sulfophenyltheophylline produced alterations in A1 adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A1 adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A1 adenosine receptor expression.
Effects of (−)‐Sulpiride on Dopamine Release in Striatum of Developing Rats: Degree of Depolarization Influences Responsiveness
Authors: Andersen Susan L. , Gazzara Russell A.
Published: July 2010

Abstract: The purpose of this study was to determine the effects of localized delivery of the D2 antagonist (−)‐sulpiride (via microdialysis) on spontaneous and evoked dopamine release in the neostriatum of urethane‐anesthetized rats 5, 10, 15, 21, and 70 days of age. Sulpiride increased spontaneous dopamine release approximately threefold relative to baseline measures, and this effect decreased with maturation. The relationship between sulpiride‐ and potassium‐evoked release was complex; sulpiride increased evoked dopamine outflow at 5, 10, and 15 days of age. At 21 and 70 days of age, however, the effects of sulpiride were inversely related to the degree of stimulation with potassium. Furthermore, the D2 agonist quinpirole (100 µM) reversed the effects of sulpiride (10 µM), suggesting receptor mediation. These experiments demonstrate that the maturational decline in the efficacy and potency of D2 antagonism appears to be related to the degree of stimulation at the nerve terminal.
Lack of a Precursor‐Product Relationship Between Histamine and Its Metabolites in Brain After Histidine Loading
Authors: Prell George D. , Hough Lindsay B. , Khandelwal Jai , Green Jack Peter
Published: July 2010

Abstract: Levels of histamine and its major metabolites in brain, tele‐methylhistamine (t‐MH) and tele‐methylimidazoleacetic acid (t‐MIAA), were measured in rat brains up to 12 h after intraperitoneal administration of l‐histidine (His), the precursor of histamine. Compared with saline‐treated controls, mean levels of histamine were elevated at 1 h (+ 102%) after a 500 mg/kg dose; levels of t‐MH did not increase. Following a 1,000 mg/kg dose; levels mean histamine levels were increased for up to 7 h, peaked at 3 h, and returned to control levels within 12 h. In contrast, levels of t‐MH showed a small increase only after 3 h; levels of t‐MIAA remained unchanged after either dose. Failure of most newly formed histamine to undergo methylation, its major route of metabolism in brain, suggested that histamine was metabolized by another mechanism possibly following nonspecific decarboxylation. To test this hypothesis, other rats were injected with α‐fluoromethylhistidine (α‐FMHis; 75 mg/kg, i.p.), an irreversible inhibitor of specific histidine decarboxylase. Six hours after rats received α‐FMHis, the mean brain histamine level was reduced 30% compared with saline‐treated controls. Rats given His (1,000 mg/kg) 3 h after α‐FMHis (75 mg/kg) and examined 3 h later had a higher (+112%) mean level of histamine than rats given α‐FMHis, followed by saline. Levels of t‐MH and t‐MIAA did not increase. These results imply that high doses of His distort the simple precursor‐product relationship between histamine and its methylated metabolites in brain. The possibility that some His may undergo nonspecific decarboxylation in brain after His loading is discussed. These findings, and other actions of His independent of histamine, raise questions about the validity of using His loading as a specific probe of brain histaminergic function.
Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation
Authors: DeGracia Donald J. , Neumar Robert W. , White Blaine C. , Krause Gary S.
Published: July 2010

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF‐4E, eIF‐4G, and eIF‐2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run‐off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro‐initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF‐4E. However, we observed in all 90‐min‐reperfused samples eIF‐4G fragments that also bound eIF‐4E. The amount of eIF‐2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine‐phosphorylated eIF‐2α in normal samples or in those obtained after ischemia without reperfusion. However, serine‐phosphorylated eIF‐2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF‐2α in these samples. The serine phosphorylation of eIF‐2α and partial fragmentation of eIF‐4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.
Changes in Brain Protease Activity in Aging
Authors: Benuck Myron , Banay‐Schwartz Miriam , DeGuzman Teresita , Lajtha Abel
Published: July 2010

Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age‐related changes in calpain specificity and substrate susceptibility were measured. Several age‐related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).
Metabolite Changes in the Cerebral Cortex of Treated and Untreated Infant Hydrocephalic Rats Studied Using In Vitro 31P‐NMR Spectroscopy
Authors: Harris N. G. , Plant H. D. , Briggs R. W. , Jones H. C.
Published: July 2010

Abstract: The effect of hydrocephalus on cerebral energy metabolites and on intermediates of membrane phospholipid metabolism has been studied in H‐Tx rats with inherited infantile hydrocephalus. Hydrocephalic rats and rats with shunts placed at 4–5 days or at 10 days after birth were subjected to magnetic resonance imaging in vivo before 21 days of age to determine the dimensions of the ventricles and cortex. At 21 days, the brains from the three groups of rats, together with age‐matched control littermates, were frozen in situ, and chloroform/methanol extracts of cerebral cortex were prepared for high‐resolution 31P‐NMR spectroscopy. Hydrocephalus resulted in modest decreases in most metabolites quantified. Levels of phosphocreatine, ATP, and diphosphodiesters plus NAD were significantly reduced by 23–32%, and inorganic phosphate content was reduced but not significantly. Levels of the membrane phospholipid intermediates phosphorylethanolamine, glycerophosphorylethanolamine, and glycerophosphorylcholine were also significantly reduced by 30–33%, indicating changes in membrane metabolism. These general decreases are consistent with a loss of cell contents, possibly due to changes in dendrite structure in hydrocephalus. Rats shunt‐treated at 4–5 days were similar to control rats for all energy metabolites, but those treated later at 10 days had reduced phosphocreatine and ATP levels. Shunt‐treated rats also had reductions in levels of membrane phospholipids, some of which occurred in sham‐operated rats. It is concluded that hydrocephalus leads to reductions in levels of energy metabolites and in levels of membrane phospholipids and that the changes in energy metabolites can be reversed by early, but not by later, shunt treatment.
Interleukin‐1β and Tumor Necrosis Factor‐α Induce Expression of α1‐Antichymotrypsin in Human Astrocytoma Cells by Activation of Nuclear Factor‐κB
Authors: Lieb Klaus , Fiebich Bernd L. , Schaller Helen , Berger Mathias , Bauer Joachim
Published: July 2010

Abstract: The protease inhibitor α1‐antichymotrypsin (ACT) has been suggested to be involved in the etiology of Alzheimer's disease (AD). Increased levels of ACT have been found in serum and brains of AD patients, and ACT has been proposed to regulate β‐amyloid fibril formation in vitro. To gain insight into the regulation of ACT in the brain, we investigated the signal transduction pathways involved in ACT gene expression and protein synthesis in the human astrocytoma cell line U373. This cell line has previously been shown to respond with strong ACT synthesis on stimulation with interleukin‐1β (IL‐1β) or tumor necrosis factor‐α (TNFα). Here, we describe that both IL‐1β and TNFα activate the transcription factor nuclear factor‐κB (NF‐κB) via production of reactive oxygen intermediates resulting in ACT expression. In addition, we show that neither protein kinase C nor protein kinase A is involved in IL‐1β‐ or TNFα‐induced ACT expression. These results suggest that activation of NF‐κB may be one possible cause of increased ACT levels in AD and provide a basis for the development of drugs used for the modulation of inflammatory processes occurring in AD.
Two Amino Acid Differences in the Sixth Transmembrane Domain Are Partially Responsible for the Pharmacological Differences Between the 5‐HT1Dβ and 5‐HT1E 5‐Hydroxytryptamine Receptors
Authors: Parker Eric M. , Izzarelli Darcy G. , Lewis‐Higgins Lore , Palmer Dawn , Shapiro Robert A.
Published: July 2010

Abstract: 5‐Hydroxytryptamine elicits its physiological effects by interacting with a diverse group of receptors. Two of these receptors, the 5‐HT1Dβ and the 5‐HT1E receptors, are ∼60% identical in the transmembrane domains that presumably form the ligand binding site yet have very different pharmacological properties. Analysis of the pharmacological properties of a series of chimeric 5‐HT1Dβ/5‐HT1E receptors indicates that sequences in the sixth and seventh transmembrane domains are responsible for the differential affinity of 5‐carboxamidotryptamine for these two receptors. More detailed analysis shows that two amino acid differences in the sixth transmembrane domain (Ile333 and Ser334 in the 5‐HT1Dβ receptor, corresponding to Lys310 and Glu311 in the 5‐HT1E receptor) are largely responsible for the differential affinities of some, but not all, ligands for the 5‐HT1Dβ and 5‐HT1E receptors. It is likely that these two amino acids subtly determine the overall three‐dimensional structure of the receptor rather than interact directly with individual ligands.
Neural Thrombin and Protease Nexin I Kinetics After Murine Peripheral Nerve Injury
Authors: Smirnova Irina V. , Ma Jianxin Y. , Citron Bruce A. , Ratzlaff Keith T. , Gregory Eugene J. , Akaaboune Mohammed , Festoff Barry W.
Published: July 2010

Abstract: We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2–4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I‐labeled target protease and found peak activity occurring later, 6–9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush‐induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti‐PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.
Cloning of CDP‐Diacylglycerol Synthase from a Human Neuronal Cell Line
Authors: Heacock Anne M. , Uhler Michael D. , Agranoff Bernard W.
Published: July 2010

Abstract: A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP‐diacylglycerol, catalyzed by CDP‐diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP‐diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP‐diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP‐diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.
Isolation, Cloning, and Characterisation of a trp Homologue from Squid (Loligo forbesi) Photoreceptor Membranes
Authors: Monk Phillip D. , Carne Alan , Liu Shu‐Hua , Ford John W. , Keen Jeffrey N. , Findlay John B. C.
Published: July 2010

Abstract: The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92‐kDa component isolated from a detergent‐insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full‐length cDNA, encoding the 92‐kDa component, using both cDNA library screening and 5′‐rapid amplification of cDNA ends (5′‐RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl‐terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl‐terminal domain has been shown to be in the regulation of several ligand‐gated channels. The carboxyl‐terminal domain has been expressed and shown to interact with calmodulin in a calcium‐dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.
Inhibition of Muscarinic Receptor‐Stimulated Glial Cell Proliferation by Ethanol
Authors: Guizzetti Marina , Costa Lucio G.
Published: July 2010

Abstract: Acetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3 cholinergic receptors. Ethanol was a potent inhibitor of carbachol‐stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50 of 10 mM. On the other hand, basal and serum‐stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50 = 200–250 mM). Concentration‐response experiments with carbachol, in the presence of 10 mM ethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4‐methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5‐bromodeoxyuridine‐Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 µg/ml insulin and 100 ng/ml platelet‐derived growth factor BB; on the other hand, the mitogenic effect of 1 mM histamine, 100 U/ml interleukin‐1, and 100 ng/ml 12‐O‐tetradecanoylphorbol 13‐acetate were inhibited by ∼50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of the fetal alcohol syndrome.
Expression of a Novel Form of the p56lck Protooncogene in Rat Cerebellar Granular Neurons
Authors: Van Tan Huynh , Allée Guillaume , Benes Cyril , Barnier Jean Vianney , Vincent Jean Didier , Fagard Remi
Published: July 2010

Abstract: The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti‐lck antibody, an lck‐like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56‐kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56‐kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription‐PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5′ end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120 to neurons.
Regulation of GABA Level in Rat Brain Synaptosomes: Fluxes Through Enzymes of the GABA Shunt and Effects of Glutamate, Calcium, and Ketone Bodies
Authors: Erecińska Maria , Nelson David , Daikhin Yevgeny , Yudkoff Marc
Published: July 2010

Abstract: Stable isotopes were used to measure both the rate of GABA formation by glutamic acid decarboxylase (GAD) and the rate of utilization by GABA‐transaminase (GABA‐T). The initial rate of GABA accumulation, determined with either [2‐15N]glutamine or [2H5]glutamine as precursor, was 0.3–0.4 nmol/min/mg of protein. Addition of the calcium ionophore A23187 enhanced GAD activity, whereas changes in levels of inorganic phosphate and H+ were without influence. Flux through GABA‐T (GABA → glutamate), measured with [15N]GABA as precursor, was 0.82 nmol/min/mg of protein, whereas the reamination of succinic acid semialdehyde (reverse flux through GABA‐T) was almost sixfold faster, 4.8 nmol/min/mg of protein. The rate of GABA metabolism via the tricarboxylic acid cycle was very slow, with the upper limit on flux being 0.03 nmol/min/mg of protein. Addition of either acetoacetate or β‐hydroxybutyrate raised the internal content of glutamate and reduced that of aspartate; the GABA concentration and the rate of its formation increased. It is concluded that in synaptosomes (a) GABA‐T is a primary factor in regulating the turnover of GABA, (b) a major regulator of GAD activity is the concentration of internal calcium, (c) GAD in nerve endings may not be saturated with its substrate, glutamate, and the concentration of the latter is a determinant of flux through this pathway, and (d) levels of ketone bodies increase, and maintain at a higher value, the synaptosomal content of GABA, a phenomenon that may contribute to the beneficial effect of a ketogenic diet in the treatment of epilepsy.