bluuebunny/crossref_metadata_2025 huggingface dataset:
α2‐Macroglobulin Complexes with and Mediates the Endocytosis of β‐Amyloid Peptide via Cell Surface Low‐Density Lipoprotein Receptor‐Related Protein
Authors: Narita Masaaki , Holtzman David M. , Schwartz Alan L. , Bu Guojun
Published: July 2010

Abstract: A primary histopathological feature of Alzheimer's disease is the accumulation of β‐amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α2‐macroglobulin (α2M⋆), a physiological ligand for the low‐density lipoprotein receptor‐related protein (LRP) that is abundantly expressed in the CNS. These α2M⋆/125I‐Aβ complexes are immunoreactive with both anti‐Aβ and anti‐α2M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α2M⋆/125I‐Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor‐associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free 125I‐Aβ is not inhibited by RAP and thus must be mediated via an LRP‐independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.
Sensitization of G Protein‐Coupled Benzodiazepine Receptors in the Striatum of 6‐Hydroxydopamine‐Lesioned Rats
Authors: Tenn C. C. , Niles L. P.
Published: July 2010

Abstract: The nonselective benzodiazepine (BZ) agonist diazepam is a potent inhibitor of adenylyl cyclase (AC) activity in the rat striatum. To examine this inhibitory action of diazepam further, its effects were examined in 6‐hydroxydopamine‐lesioned animals, which reportedly exhibit sensitization of the striatal AC pathway. As previously observed, inhibition of AC activity by diazepam was biphasic, with the first phase being receptor‐mediated, whereas the second phase involves a direct action on the enzyme itself. In the presence of NaCl (120 mM), a marked sensitization to the receptor‐mediated inhibitory effect of diazepam on AC activity was observed in striatal membranes of lesioned animals. EC50 values were 10.4 ± 1.1 and 4.8 ± 0.9 nM (p < 0.05) for intact and lesioned striata, respectively. An examination of [3H]diazepam binding revealed a significant increase in the density of binding sites in denervated striata, with no change in affinity. A time‐dependent increase in [α‐32P]GTP labeling of two distinct striatal proteins with apparent molecular masses of 40 and 45 kDa, suggestive of the α subunits of Gi and Gs, respectively, was observed. There was a significant increase in basal [α‐32P]GTP binding to both proteins in lesioned striata. In addition, diazepam stimulated [α‐32P]GTP binding to the 40‐kDa protein, especially in lesioned striata. These data indicate that the sensitization of the receptor‐mediated inhibitory effect of diazepam on AC activity in denervated striata may involve up‐regulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins.
Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle
Authors: Cabezas‐Herrera Juan , Moral‐Naranjo María Teresa , Campoy F. Javier , Vidal Cecilio J.
Published: July 2010

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X‐100. Asymmetric (A12) and globular hydrophilic and amphiphilic (GH4, GA4, GA2, and GA1) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of GH4 and GA4 decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent‐released A12 AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA‐I). Conversely, the collagenase‐detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of GA4 AChE in DMV and a few in NMV bound to RCA. Most of the RCA‐unreactive GA4 forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.
Purification and Characterization of Naturally Soluble Neuropathy Target Esterase from Chicken Sciatic Nerve by HPLC and Western Blot
Authors: Escudero M. A. , Vilanova E.
Published: July 2010

Abstract: Neuropathy target esterase (NTE) activity is defined operatively as the paraoxon‐resistant mipafox‐sensitive phenyl valerate esterase activity. A preparation containing a soluble isoform (S‐NTE2) has been obtained from sciatic nerve. It was inhibited by the biotinylated organophosphorous ester S9B [1‐(saligenin cyclic phospho)‐9‐biotinyldiaminononane] in a progressive manner showing a second‐order rate constant of (3.50 ± 0.26) × 106M−1· min−1 with an I50 for 30 min of 6.6 ± 0.4 nM. S‐NTE2 was enriched 218‐fold by gel filtration followed by strong and weak anion‐exchange chromatographies in HPLC. In western blots, this enriched sample showed two bands of endogenous biotinylated polypeptides after treating the blots with streptavidin‐alkaline phosphatase complex. When the sample was treated with S9B, another biotinylated band was observed with a molecular mass of ∼56 kDa, which was not seen when the sample had been pretreated with mipafox before the S9B labeling. It was deduced that this band represents a polypeptide (identified as the S‐NTE2 protein) that is bound by both mipafox and S9B and that should be responsible for the progressive S9B inhibition. It is possible that S‐NTE2 is the target for attack by compounds that promote delayed neuropathy.
Transient Global Ischemia Alters NMDA Receptor Expression in Rat Hippocampus: Correlation with Decreased Immunoreactive Protein Levels of the NR2A/2B Subunits, and an Altered NMDA Receptor Functionality
Authors: Zhang Liang , Hsu Jee Ching , Takagi Norio , Gurd James W. , Wallace M. Christopher , Eubanks James H.
Published: July 2010

Abstract: We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N‐methyl‐d‐aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham‐operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single‐channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18–24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only ∼10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.
Effect of Sorbinil and Ascorbic Acid on myo‐Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose
Authors: Karihaloo Anil K. , Joshi Kusum , Chopra Jagjit S.
Published: July 2010

Abstract: The effect of long‐term (2 weeks) exposure to 0–50 mM glucose and 0–1 mM sorbitol on myo‐inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo‐inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo‐Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 mM glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose‐dependent fashion. Significant prevention was achieved with 0.5 and 1 mM sorbinil. Ascorbic acid also prevented the reduction in myo‐inositol uptake due to excess extracellular glucose, at 3 and 30 µM concentrations, but not at 300 µM. Neither sorbinil nor ascorbic acid could prevent the alterations in myo‐inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose‐induced alteration of myo‐inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo‐inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.
Degradation of Tau by Lysosomal Enzyme Cathepsin D: Implication for Alzheimer Neurofibrillary Degeneration
Authors: Kenessey Agnes , Nacharaju Parimala , Ko Li‐wen , Yen Shu‐Hui
Published: July 2010

Abstract: The degradation of different isoforms of human recombinant tau (R‐tau; T39, T40, and T44) and fetal tau (F‐tau) by cathepsin D (CD) was investigated. Gel electrophoresis and Coomassie Blue staining of different R‐tau species digested at pH 3.5 showed very little differences in CD susceptibility. Immunoblotting analyses revealed that amino and carboxy termini of tau were cleaved before other regions. F‐tau was most vulnerable to proteolysis at both termini. Digestion of R‐tau with 0.01 unit of CD/ml at pH 3.5 resulted in cleavage between Phe8‐Glu9, Met419‐Val420, Thr427‐Leu428‐Ala429, and Leu436‐Ala437 as determined by amino acid sequencing and mass spectroscopy (numbering of amino acids was based on T40). With higher concentrations of CD (1 unit/ml), additional sites of digestion were detected between amino acids 34–161, 200–257, and 267–358. The cleavage sites at amino acids 34–161 and 267–358 were observed at pH 3.5, whereas that at amino acids 200–257 was detected at pH 7.0. Our results suggest that CD cleavage of tau could generate tau fragments with intact microtubule binding domains, which could have a role in the pathogenesis of paired helical filaments (PHFs) in Alzheimer's disease. Such proteolysis might also contribute to the changes of PHF phenotype observed in intracellular and extracellular tangles.
Serotonin Acts as a Radical Scavenger and Is Oxidized to a Dimer During the Respiratory Burst of Activated Microglia
Authors: Huether Gerald , Fettkötter Inga , Keilhoff Gerburg , Wolf Gerald
Published: July 2010

Abstract: Serotonin (5‐HT) is known to be readily oxidized and to act as a scavenger of reactive oxygen species produced, e.g., in the presence of peroxidase and H2O2 or during the respiratory burst of phagocytes. One major oxidation product formed under these conditions, the 5‐HT dimer 5,5′‐dihydroxy‐4,4′‐bitryptamine (DHBT), was suggested to have neurotoxic properties and to contribute to neuronal damage in neurodegenerative disorders. It is shown in the present study that the luminol‐enhanced chemiluminescence signal measured after stimulation of the respiratory burst activity of cultivated rat microglial cells by the addition of phorbol 12‐myristate 13‐acetate is suppressed by 5‐HT in a dose‐dependent manner. During this process, 5‐HT is oxidized to DHBT. Neither the intraventricular injection of DHBT nor the addition of DHBT to cultured astrocytes, neurons, or PC‐12 cells was found to cause measurable cytotoxic effects. It is concluded that extracellular 5‐HT locally released from platelets and 5‐HT nerve endings at sites of brain damage or inflammation, through its suppressant effect on the release of reactive oxygen species during the respiratory burst of activated microglia, may contribute to attenuate secondary tissue damage in the CNS.
High‐Density Lipoprotein Aggregated by Oxidation Induces Degeneration of Neuronal Cells
Authors: Kivatinitz Silvia C. , Pelsman María A. , Del C. Alonso Alejandra , Bagatolli Luis , Quiroga Santiago
Published: July 2010

Abstract: We have previously reported that high‐density lipoprotein (HDL) exhibits antineuritogenic effects on chicken cerebral cells in culture. In the present study, we show the effects of HDLs, oxidized by UV irradiation or heating, on chicken cerebral neurons in culture. Both treatments produced several physical and chemical changes in the HDLs, i.e., formation of lipid peroxides, enlargement of HDL diameters, an increased exposure of the tryptophan groups of the apolipoprotein A‐I to a more hydrophilic environment, formation of bityrosines, and cross‐linking of apolipoprotein A‐I. When these treatments were performed in the absence of EDTA, most of the modifications described above were more intense and HDLs formed a macroaggregate that displays a rosette‐like structure. The aggregated HDLs produced neurodegeneration and death when added to both undifferentiated and differentiated cerebral neurons in culture. This process was accompanied by the disorganization of the cellular microtubular cytoskeleton and hyperphosphorylation of the microtubule‐associated protein tau. Native HDL or HDLs treated in the presence of EDTA inhibited the neuritogenesis of undifferentiated neurons but did not show any significant effect on the differentiated neurons in culture. The effects on the cellular cytoskeleton and morphology of aggregated HDLs recall those of the fibrillar β‐amyloid peptide. The present results suggest that aggregated HDLs could participate in neurodegeneration associated with oxidative stress in the CNS.
Ethanol Specifically Inhibits NMDA Receptors with Affinity for Ifenprodil in the Low Micromolar Range in Cultured Cerebellar Granule Cells
Authors: Engblom A. Christine , Courtney Michael J. , Kukkonen Jyrki P. , Åkerman Karl E. O.
Published: July 2010

Abstract: The effect of ethanol on the intracellular Ca2+ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCI concentrations has been studied using image analysis. The cells grown in low KCI displayed high sensitivity for glycine. The subtype‐selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high‐potency component in these cultures. In cells grown in high KCI the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.
Development and Characterization of Antibodies Against the N Terminus of the Human Dopamine D4 Receptor
Authors: Lanau Fabienne , Brockhaus Manfred , Pink J. Richard L. , Franchet Christelle , Wildt‐Perinic Dunja , Goepfert Corine , Probst Alphonse , Hartman Deborah S.
Published: July 2010

Abstract: The human dopamine D4 receptor (hD4R), which has been implicated in human diseases such as schizophrenia and in a personality trait called “novelty seeking,” has not yet been characterized at the protein level. Following epitope scanning of the hD4R, we have produced a highly specific monoclonal antibody named DFR1 raised against an amino‐terminal peptide in a predicted extracellular region of the receptor. DFR1 decorated recombinant hD4Rs on the surface of intact Chinese hamster ovary (CHO) cells by flow cytometry and fluorescence microscopy and also recognized recombinant hD4.2, hD4.4, and hD4.7 receptor isoforms by western blot analysis. When expressed stably in CHO cells, all three hD4R isoforms contained N‐linked glycosylation and showed apparent molecular masses of 48, 55, and 67 kDa for hD4.2, hD4.4, and hD4.7, respectively. DFR1 immunoreactivity representing hD4R protein or dopamine D4 receptor‐like antigens was observed in crude membrane extracts of postmortem human brain tissue by immunoblotting. The DFR1 antibody provides a new immunological tool with the potential to further our understanding of the human dopamine D4 receptor protein.
Cardiotrophin‐1 Induces the Same Neuropeptides in Sympathetic Neurons as Do Neuropoietic Cytokines
Authors: Cheng Jr‐Gang , Pennica Diane , Patterson Paul H.
Published: July 2010

Abstract: Cardiotrophin‐1 (CT‐1) was cloned from mouse embryoid body for its ability to induce growth of heart cells. Predictions of its secondary structure indicate that CT‐1 belongs to a family of cytokines with a four‐helical bundle structure, and sequence comparisons reveal a weak homology to leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). Using a reverse transcriptase‐polymerase chain reaction assay with rat sympathetic neuron cultures, we find that CT‐1 induces and suppresses the expression of the same set of neuropeptide and neurotransmitter synthetic enzyme mRNAs as do LIF and CNTF. In addition, the effects of CT‐1 and LIF are not additive, and CT‐1 does not require a GPI‐linked component to mediate its actions. Our functional data confirm that CT‐1 is a member of the neuropoietic cytokine family and suggest that the CT‐1 receptor complex contains the gp130 signal transducing component.
Lithium Increases Transcription Factor Binding to AP‐1 and Cyclic AMP‐Responsive Element in Cultured Neurons and Rat Brain
Authors: Ozaki Naoko , Chuang De‐Maw
Published: July 2010

Abstract: We have investigated whether lithium has effects on transcription factor binding to consensus DNA sequences of AP‐1 and cyclic AMP‐responsive element (CRE) in cultured rat neurons and in vivo. Treatment of rat cerebellar granule cells (CGC) with lithium chloride induced a concentration‐dependent increase in AP‐1 and CRE binding activities with maximal effects at therapeutically relevant concentrations of 0.5 and 1.0 mM. Time‐course studies show that lithium's effects on AP‐1 and CRE binding were biphasic within the first 24 h of treatment in immature CGC in culture and persistent in mature CGC, lasting as long as 7 days. These actions were concurrent with an increase in the mRNA levels of c‐fos and c‐jun, as well as the protein levels of c‐Fos, c‐Jun, and phosphorylated CRE binding protein (p‐CREB). Gel supershift assays using transcription factor‐specific antibodies revealed that p‐CREB, Jun D, and a Fos family protein(s) are components of the AP‐1 binding complex in untreated and lithium‐treated CGC. Chronic dietary treatment of rats with lithium carbonate for 4 weeks also significantly increased AP‐1 and CRE binding activity in the frontal cortex, hippocampus, amygdala, and cerebellum. Similar to the results obtained in CGC, p‐CREB, Jun D, and Fos family proteins are present in the AP‐1 binding sites in the frontal cortex and hippocampus of untreated and lithium‐treated rats. Lithium‐induced activation of transcription factor binding to AP‐1 and CRE sites in vivo and in vitro provides a new avenue to study the mechanisms of action of lithium in the treatment of manic depressive illness.
α3, β2, and β4 Form Heterotrimeric Neuronal Nicotinic Acetylcholine Receptors in Xenopus Oocytes
Authors: Colquhoun Lorna M. , Patrick James W.
Published: July 2010

Abstract: One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different β subunits assemble with an α subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for α3, β2, and β4 subunits. β2 and β4, in pairwise combination with α3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). α3β4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine‐evoked currents through α3β2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for α3, β2, and β4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both β2 and β4 subunits in functional receptors.
Comparison of Serotonin and Dopamine Release in Substantia Nigra and Ventral Tegmental Area: Region and Species Differences
Authors: Cragg S. J. , Hawkey C. R. , Greenfield S. A.
Published: July 2010

Abstract: In this study, we compare the electrically evoked, somatodendritic release of dopamine (DA) with axonal release of serotonin (5‐HT) in the substantia nigra (SN) and ventral tegmental area (VTA) in vitro by using fast‐scan cyclic voltammetry with carbon‐fibre microelectrodes. Furthermore, we have examined transmitter release in these regions in guinea‐pig compared with rat. Somatodendritic DA was released, as shown previously, in guinea‐pig VTA, SN pars compacta (SNc), and occasionally in SN pars reticulata (SNr). 5‐HT was rarely released, except in SNr, where nonetheless it only contributed to <30% of amine signals. In rat midbrain, somatodendritic DA release was evoked to a similar extent as in guinea‐pig. However, a clear species difference was apparent; i.e., 5‐HT and DA were detected equally in rat SNc, whereas in rat SNr, 5‐HT was the predominant transmitter detected. Nevertheless, electrically evoked extracellular concentrations of 5‐HT in SNc and SNr were, respectively, seven‐ and fourfold less than DA in SNc. 5‐HT release was low in all regions in neonatal rat slices before the maturation of 5‐HT terminals. Hence, axonal 5‐HT transmission in midbrain exhibits both species and site selectivity. Moreover, whereas somatodendritic DA release is conventionally regarded as modest compared with axon terminal release in striatum, somatodendritic DA release can result in significantly greater extracellular levels than a transmitter released from axon terminals in the same locality.
Agonist‐Induced Phosphorylation of the κ‐Opioid Receptor
Authors: Appleyard Suzanne M. , Patterson Terrell A. , Jin Wenzhen , Chavkin Charles
Published: July 2010

Abstract: Antipeptide antibodies against the κ‐opioid receptor were used to test whether acute or chronic exposure to κ agonists altered the phosphorylation state of the κ‐opioid receptor. Immunoprecipitation of the κ receptor from guinea pig hippocampal slices preincubated in [32P]orthophosphoric acid revealed a basal phosphorylation of the κ‐opioid receptor. The amount of 32P incorporation into the receptor was increased following a 75‐min treatment with the κ agonist U50,488H. This effect was blocked by the selective κ receptor antagonist norbinaltorphimine. The time course of this change in the phosphorylation state of the receptor correlated with a desensitization of the electrophysiological response to κ agonists measured in the dentate gyrus of hippocampal slices. The phosphorylation state of the κ‐opioid receptor was also elevated in brain slices from guinea pigs made tolerant to U50,488H by 5 days of continuous exposure and then maintained in κ agonist to avoid acute opiate withdrawal. The results of this study show that the κ‐opioid receptor was phosphorylated in an agonist‐dependent manner in brain slices taken from untreated and U50,488H‐tolerant animals.
Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides
Authors: Chritton S. L. , Chinnow S. L. , Grabau C. , Dousa M. K. , Lucas D. , Roddy D. , Yaksh T. L. , Tyce G. M.
Published: July 2010

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4‐dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4‐dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4‐dihydroxyphenylacetic acid > 3‐methoxy‐4‐hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4‐dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µM carbachol in a calcium‐dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca2+‐dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.
Uptake of Agmatine into Rat Brain Synaptosomes: Possible Role of Cation Channels
Authors: Sastre Magdalena , Regunathan Soundararajan , Reis Donald J.
Published: July 2010

Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy‐ and temperature‐dependent and saturable with a Km of 18.83 ± 3.31 mM and a Vmax of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na+,K+‐ATPase inhibitor) or removal of extracellular Na+ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion‐channel agents on agmatine uptake, only Ca2+‐channel blockers inhibited the uptake, whereas a reduction in extracellular Ca2+ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na+‐independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.
Cellular Expression and Proteolytic Processing of Presenilin Proteins Is Developmentally Regulated During Neuronal Differentiation
Authors: Capell Anja , Saffrich Rainer , Olivo Jean‐Christophe , Meyn Liane , Walter Jochen , Grünberg Jürgen , Mathews Paul , Nixon Ralph , Dotti Carlos , Haass Christian
Published: July 2010

Abstract: We have determined the expression of the Alzheimer's disease‐associated proteins presenilin‐1 and presenilin‐2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin‐1 and presenilin‐2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin‐1 and presenilin‐2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin‐1 and presenilin‐2 differs slightly, with more presenilin‐2 expressed in axons compared with presenilin‐1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full‐length presenilin‐1 or presenilin‐2 could be detected within cell lysates. At early developmental stages the expected ∼34‐kDa N‐terminal proteolytic fragment of presenilin‐1 and the ∼38‐kDa fragment of presenilin‐2 were detected. Later during differentiation we predominantly detected a ∼38‐kDa fragment for presenilin‐1 and a ∼42‐kDa fragment for presenilin‐2. By epitope mapping, we show that these slower migrating peptides represent N‐terminal proteolytic fragments, cleaved C‐terminal to the conventional site of processing. It is noteworthy that both presenilin‐1 and presenilin‐2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.
Perlecan Binds to the β‐Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability
Authors: Castillo Gerardo M. , Ngo Catherine , Cummings Joel , Wight Thomas N. , Snow Alan D.
Published: July 2010

Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β‐amyloid (Aβ) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth‐Holm‐Swarm tumor was used to define perlecan's interactions with Aβ and its effects on Aβ fibril formation. Using a solid‐phase binding immunoassay, freshly solubilized full‐length Aβ peptides bound immobilized perlecan at two sites, representing both high‐affinity [KD = ∼5.8 × 10−11M for Aβ (1–40); KD = ∼6.5 × 10−12M for Aβ (1–42)] and lower‐affinity [KD = 3.5 × 10−8M for Aβ (1–40); KD = 4.3 × 10−8M for Aβ (1–42)] interactions. An increase in the binding capacity of Aβ (1–40) to perlecan correlated with an increase in Aβ amyloid fibril formation during a 1‐week incubation period. The high‐capacity binding of Aβ (1–40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin‐4‐sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of Aβ (1–40) amyloid fibril formation, causing a significant increase in Aβ fibril assembly over a 2‐week incubation period at 1 h (2.8‐fold increase), 1 day (3.6‐fold increase), and 3 days (2.8‐fold increase) in comparison with Aβ (1–40) alone. Perlecan also initially accelerated the formation of Aβ (1–42) fibrils within 1 h and maintained significantly higher levels of Aβ (1–42) thioflavin T fluorescence throughout a 2‐week experimental period in comparison with Aβ (1–42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on Aβ (1–40) fibril formation and maintenance of Aβ (1–42) fibril stability occurred in a dose‐dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of Aβ fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, α1‐antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of Aβ, but also accelerates Aβ fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of Aβ amyloidosis in Alzheimer's disease.
Enhanced Depolarization‐Evoked Calcium Signal and Reduced [ATP]/[ADP] Ratio Are Unrelated Events Induced by Oxidative Stress in Synaptosomes
Authors: Tretter Laszlo , Chinopoulos Christos , Adam‐Vizi Vera
Published: July 2010

Abstract: Oxidative insult elicited by hydrogen peroxide (H2O2) was previously shown to increase the basal intracellular Ca2+ concentration in synaptosomes. In the present study, the effect of H2O2 on the depolarization‐evoked [Ca2+] signal was investigated. Pretreatment of synaptosomes with H2O2 (0.1–1 mM) augmented the [Ca2+] rise elicited by high K+ depolarization with essentially two alterations, the sudden sharp rise of [Ca2+]i due to K+ depolarization is enhanced and, instead of a decrease to a stable plateau, a slow, steady rise of [Ca2+]i follows the peak [Ca2+]i. H2O2 in the same concentration range lowered the ATP level and the [ATP]/[ADP] ratio. When carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP) (1 µM) or rotenone (2 µM)/oligomycin (10 µM) was applied initially to block mitochondrial ATP production, the lowered [ATP]/[ADP] ratio was further reduced by subsequent addition of 0.5 mM H2O2. The decline of the [ATP]/[ADP] ratio was parallel with but could not explain the enhanced K+‐evoked [Ca2+]i signal, indicated by experiments in which the [ATP]/[ADP] ratio was decreased by FCCP (0.1 µM) or rotenone (2 µM) to a similar value as by H2O2 without causing any alteration in the [Ca2+]i signal. These results indicate that H2O2‐evoked oxidative stress, in its early phase, gives rise to a complex dysfunction in the Ca2+ homeostasis and, parallel with it, to an impaired energy status.
Temporal Analysis of Changes in Neuronal c‐fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels
Authors: Gissel Cornelia , Doutheil Jens , Paschen Wulf
Published: July 2010

Abstract: Activation of immediate early gene expression is a key event in stress‐induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+‐ATPase. Tg‐induced rise in [Ca2+]i and the effect of loading neurons with the cell‐permeable calcium chelator BAPTA‐AM on this increase in [Ca2+]i were measured in fura‐2‐loaded cells by fluorescence microscopy. Changes in c‐fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c‐fos mRNA levels (∼10‐fold more than DMSO) which peaked at 1 h after exposure. The Tg‐induced rise in c‐fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c‐fos. Thus, stress‐induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress‐induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.
Regulation of L‐Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors
Authors: Zapata Rafael , Navarro Angels , Canela Enric I. , Franco Rafael , Lluis Carmen , Mallol Josefa
Published: July 2010

Abstract: Identification of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A1Rs in the regulation of calcium conductance was studied in these cells. The agonist N6‐(R)‐(2‐phenylisopropyl)adenosine (R‐PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 mM KCl depolarization. The antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine led to the opening of voltage‐dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µM) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R‐PIA, thus suggesting that A1R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L‐type voltage‐dependent calcium channels.
Agrin‐Deficient Myotube Retains Its Acetylcholine Receptor Aggregation Ability when Challenged with Agrin
Authors: Pun San , Ng Yu P. , Yang Jie F. , Ip Nancy Y. , Tsim Karl W. K.
Published: July 2010

Abstract: Agrin is a synapse‐organizing molecule that mediates the nerve‐induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron‐derived agrin is the AChR‐aggregating factor, but the possible roles of muscle‐derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin‐deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin‐deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin‐deficient myotubes could also respond to neuron‐induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin‐deficient myotubes retain their ability to exhibit the agrin‐ or neuron‐induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron‐derived agrin but not the agrin from muscle.
Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol
Authors: Barbosa J. , Clarizia A. D. , Gomez M. V. , Romano‐Silva M. A. , Prado V. F. , Prado M. A. M.
Published: July 2010

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3H]‐acetylcholine ([3H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3H]ACh release. The effect of PMA was dose‐dependent, was sensitive to calphostin C, a PKC‐selective inhibitor, and could not be mimicked by α‐PMA, an inactive phorbol ester. PMA did not alter the release of [3H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32P‐labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.
Characterization of a Dopamine D1 Receptor from Apis mellifera: Cloning, Functional Expression, Pharmacology, and mRNA Localization in the Brain
Authors: Blenau Wolfgang , Erber Joachim , Baumann Arnd
Published: July 2010

Abstract: The neurotransmitter dopamine is an important regulator of physiological and behavioral functions in both vertebrates and invertebrates. We have isolated a homologue of the vertebrate dopamine D1 receptor subfamily from the honeybee Apis mellifera. [3H]Lysergic acid diethylamide specifically binds to the heterologously expressed receptor with KD∼5 nM. Dopaminergic receptor ligands compete for this high‐affinity binding, with the following order of potency: R(+)‐lisuride > chlorpromazine = cis(Z)‐flupentixol > dopamine > S(+)‐butaclamol > R(+)‐SCH 23390 > haloperidol. Activation of the heterologously expressed receptor of Apis mellifera leads to cyclic AMP production. Receptor mRNA is expressed in perikarya of different brain neuropils, including those of mushroom body intrinsic neurons. These results suggest that this dopamine receptor is involved in signal processing of visual and olfactory information in the honeybee.
Regulation of α2A‐Adrenergic Receptor Expression by Epinephrine in Cultured Astroglia from Rat Brain
Authors: Reutter Michael A. , Richards Elaine M. , Sumners Colin
Published: July 2010

Abstract: Epinephrine (Epi) mediates various physiological effects via α2A‐adrenergic receptors (α2A‐ARs). Studies in mice with a point mutation in the gene for α2A‐AR have shown that these receptors are responsible for the centrally mediated depressor effects of α2‐AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α2A‐ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague‐Dawley rats as a model system in which to study factors that regulate α2A‐AR expression. These cells contain α2‐ARs, which are predominately of the α2A‐AR subtype. Our studies have shown that Epi causes a dose‐ and time‐dependent decrease in steady‐state levels of α2A‐AR mRNA and number of α2A‐ARs, effects that are mediated via α1‐ and β‐adrenergic receptors (α1‐ARs and β‐ARs). These effects of Epi on α2A‐AR mRNA and α2A‐AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α1‐ARs and β‐ARs, respectively. Taken together, these results indicate that expression of α2A‐ARs is regulated in a heterologous manner by Epi, via α1‐AR‐ and β‐AR‐mediated intracellular pathways.
Antibody to Epitope Tag Induces Internalization of Human Muscarinic Subtype 1 Receptor
Authors: Tolbert Lara M. , Lameh Jelveh
Published: July 2010

Abstract: We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody‐induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody‐mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody‐induced internalization, similar to the agonist‐induced process, is mediated by clathrin‐coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein‐coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.
Potassium‐Induced Release of Endogenous Morphine from Rat Brain Slices
Authors: Guarna M. , Neri C. , Petrioli F. , Bianchi E.
Published: July 2010

Abstract: Endogenous morphine has been clearly demonstrated by gas chromatography/mass spectrometry in the brain, spinal fluid, adrenal glands, and liver of mammals. To clarify the role of endogenous morphine, its release from rat brain slices was studied in vitro in the presence of high potassium concentrations, with and without calcium in the medium. The perfusate was hydrolyzed, solid phase‐extracted, and then analyzed by gas chromatography/mass spectrometry. Depolarization due to high potassium concentrations increased the release of the alkaloid manyfold with respect to the basal value, and the release was dependent on the presence of calcium in the medium. These results suggest that endogenous morphine might act as a neurotransmitter or neuromodulator in the rat CNS.
Effects of 2‐Deoxy‐d‐Glucose on Methamphetamine‐Induced Dopamine and Serotonin Neurotoxicity
Authors: Callahan Brian , Yuan Jie , Stover Gretchen , Hatzidimitriou George , Ricaurte George
Published: July 2010

Abstract: The mechanisms underlying the neurotoxic actions of methamphetamine (METH) and related substituted amphetamines are unknown. Previous studies with 2‐deoxyglucose (2‐DG) have suggested that METH‐induced neurotoxicity may involve exhaustion of intracellular energy stores. However, because 2‐DG also produces hypothermic effects, and because METH's neurotoxic actions are highly susceptible to thermoregulatory influence, previous findings with 2‐DG are difficult to interpret. The present studies were undertaken to further examine the influence of 2‐DG's glucoprivic and thermic effects in the context of METH‐induced dopamine (DA) and serotonin (5‐HT) neurotoxicity. 2‐DG protected against METH‐induced DA neurotoxicity in both rats and mice. In both species, 2‐DG, alone or in combination with METH, produced hypothermic effects. METH's toxic effects on brain 5‐HT neurons were either unaffected or exacerbated by 2‐DG, depending on species, brain region, and dose of METH tested. These results indicate that different mechanisms may underlie METH‐induced DA and 5‐HT neurotoxicity, and suggest that, as compared with 5‐HT neurons, DA neurons are more susceptible to temperature influence, whereas 5‐HT neurons are more vulnerable than DA neurons to metabolic compromise. Additional studies are needed to further assess the role of energy stores in the neurotoxic effects of METH and related drugs.