bluuebunny/crossref_metadata_2025 huggingface dataset:
Agonist‐Independent Internalization and Activity of a C‐Terminally Truncated Somatostatin Receptor Subtype 2(δ349)
Authors: Schwartkop Claus‐Peter , Kreienkamp Hans‐Jürgen , Richter Dietmar
Published: March 2003

Abstract : The rat somatostatin receptor subtype 2 (SSTR2) is rapidly internalized and phosphorylated in the presence of somatostatin 14 (SST14). Several C‐terminal deletion constructs of SSTR2 have been investigated for their ability to undergo agonist‐dependent internalization by using biochemical ligand binding assays and confocal microscopic analysis. Whereas mutant receptors lacking either 10 (δ359), 30 (δ339), or 44 (δ325) amino acid residues at the C terminus required SST14 for internalization, a construct lacking the last 20 amino acids (δ349) was detected mostly intracellularly and independently of the presence of the agonist. When internalization was blocked by sucrose, the δ349 receptor remained at the cell surface, strongly indicating that this mutant is internalized in an agonist‐independent fashion. An increased affinity for agonists as measured in membrane binding assays and a reduced level of forskolin‐stimulated cyclic AMP accumulation in human embryonic kidney cells expressing δ349 are properties that are characteristic of agonist‐independent receptor activity. δ349 is not phosphorylated detectably in the absence of agonist, demonstrating that phosphorylation per se is not a prerequisite for internalization of SSTR2. This observation is in line with data obtained for the δ325 mutant, which was internalized in an agonist‐dependent manner, but not phosphorylated in either the presence or absence of SST14. We conclude that truncation of the SSTR2 C terminus at position 349 leads to agonist‐independent, constitutive activity and internalization.
Hydrophobic Interactions Mediate Binding of the Glycine Receptor β‐Subunit to Gephyrin
Authors: Kneussel Matthias , Hermann Achim , Kirsch Joachim , Betz Heinrich
Published: March 2003

Abstract : Glycine receptors (GlyRs) are ligand‐gated chloride channel proteins composed of α‐ and β‐subunits. GlyRs are located to and anchored at postsynaptic sites by the receptorassociated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR β‐subunit. Here, we localized amino acid residues implicated in gephyrin binding by site‐directed mutagenesis. In a novel transfection assay, a green fluorescent protein‐gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for β‐subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.
Low‐Level Lead Exposure in the Early Postnatal Period Results in Persisting Neuroplastic Deficits Associated with Memory Consolidation
Authors: Murphy Keith J. , Regan Ciaran M.
Published: March 2003

Abstract: Prospective studies in humans and experimental investigations in animals have correlated elevated perinatal blood lead levels with enduring behavioural and cognitive perturbations. Although deficits in neuroplastic events necessary for long‐term memory consolidation have been observed during the postnatal period, there is little evidence that these persist into adulthood in the absence of continued lead exposure. To address this issue, we exposed Wistar rat pups to 400 mg of PbCl2/L via their dams’ drinking water from postnatal day 1 to 30. At postnatal day 80, the animals were trained in a onetrial, step‐through, light‐dark passive avoidance paradigm. Prior postnatal lead exposure resulted in a significant decline in recall latency on posttraining day 5, an effect that was specific to the learned response as no obvious behavioural alterations were apparent in open‐field studies. As recall was unaffected in the immediate 48‐h posttraining period, this suggested an enduring impairment in events associated with long‐term memory storage. To investigate this further, we determined the influence of prior lead exposure on the transient modulations of hippocampal neural cell adhesion molecule polysialylation state that occur in the 10–12‐h posttraining period, a neuroplastic event associated with memory consolidation. Direct quantification of polysialylated dentate neurons revealed prior lead exposure to have no effect on basal number but to significantly delay and blunt the transient increase observed in control animals at the 12‐h posttraining time. These findings confirm that lead exposure in the postnatal period results in enduring neuroplastic deficits most likely associated with reordering of connections in pathways subservient to memory consolidation.
Sequential Cleavage of Poly(ADP‐Ribose)polymerase and Appearance of a Small Bax‐Immunoreactive Protein Are Blocked by Bcl‐XL and Caspase Inhibitors During Staurosporine‐Induced Dopaminergic Neuronal Apoptosis
Authors: Kim Ji‐Eun , Oh Jae H. , Choi Won‐Seok , Chang In I. , Sohn Seonghyang , Krajewski Stanislaw , Reed John C. , O'Malley Karen L. , Oh Young J.
Published: March 2003

Abstract: To assess the role of Bcl‐XL and its splice
derivative, Bcl‐Xs, in staurosporine‐induced cell death, we used a
dopaminergic cell line, MN9D, transfected with bcl‐xL
(MN9D/Bcl‐XL), bcl‐xs (MN9D/Bcl‐Xs),
or control vector (MN9D/Neo). Only 8.6% of MN9D/Neo cells survived after 24 h
of 1 μM staurosporine treatment. Caspase activity was implicated
because a caspase inhibitor,
N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone (Z‐VAD‐fmk),
attenuated staurosporine‐induced cell death. Bcl‐XL rescued MN9D
cells from death (89.4% viable cells), whereas Bcl‐Xs had little or
no effect. Bcl‐XL prevented morphologically apoptotic changes as
well as cleavage of poly(ADP‐ribose)polymerase (PARP) induced by
staurosporine. It is interesting that a small Bax‐immunoreactive protein
appeared 4‐8 h after PARP cleavage in MN9D/Neo cells. The appearance of the
small Bax‐immunoreactive protein, however, may be cell type‐specific as it was
not observed in PC12 cells after staurosporine treatment. The sequential
cleavage of PARP and the appearance of the small Bax‐immunoreactive protein in
MN9D cells were blocked either by Z‐VAD‐fmk or by Bcl‐XL. Thus, our
present study suggests that Bcl‐XL but not Bcl‐Xs prevents staurosporine‐induced apoptosis by inhibiting the caspase activation that may be directly or indirectly responsible for the appearance of the small Bax‐immunoreactive protein in some types of neurons.
Regulation of Tyrosine Hydroxylase Gene Expression by Muscarinic Agonists in Rat Adrenal Medulla
Authors: Piech‐Dumas Kristen M. , Sterling Carol R. , Tank A. William
Published: March 2003

Abstract: Tyrosine hydroxylase (TH) gene expression in the adrenal medulla is regulated by numerous stimuli via transsynaptic mechanisms. The adrenal chromaffin cell receptors that mediate this transsynaptic response remain unidentified. In this report we demonstrate that the muscarinic acetylcholine receptor agonist bethanechol stimulates the TH gene transcription rate in both innervated and denervated adrenal glands. Hence, this muscarinic response is not dependent on transsynaptic influences, suggesting that agonist occupation of adrenal chromaffin cell muscarinic receptors is sufficient to activate intracellular signaling pathways that stimulate the TH gene. When bethanechol is administered repeatedly over a 3‐h interval (four injections spaced 1 h apart), TH mRNA levels are increased two‐ to threefold at 6 and 12 h after the initial injection of drug. It is surprising that this induction of TH mRNA does not lead to increases in TH activity or TH protein level. These results are consistent with the hypothesis that both transcriptional and posttranscriptional mechanisms must be regulated to induce TH protein and that muscarinic agonists activate only a subset of these mechanisms.
Temporal Patterns of Poly(ADP‐Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat
Authors: LaPlaca Michelle C. , Raghupathi Ramesh , Verma Ajay , Pieper Andrew A. , Saatman Kathryn E. , Snyder Solomon H. , McIntosh Tracy K.
Published: March 2003

Abstract: The activation of poly(ADP‐ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site‐specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP‐ribose) polymerase activity and cleavage. Male Sprague‐Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5‐2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP‐ribose) polymerase. Endogenous poly(ADP‐ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP‐ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP‐ribose) polymerase‐mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP‐ribose) polymerase activation does not occur, possibly owing to delayed apoptosis‐associated proteolysis, which may impair the repair of damaged DNA.
The “Orphan” Na+/Cl‐‐Dependent Transporter, Rxt1, Is Primarily Localized Within Nerve Endings of Cortical Origin in the Rat Striatum
Authors: Kachidian P , Masson J , Aïdouni Z , Gaspar P , Hamon M , Mestikawy S. El , Goff L. Kerkerian‐Le
Published: March 2003

Abstract : Previous studies have shown that the striatum expresses

very low levels of Na+/Cl‐‐dependent “orphan” transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6‐hydroxydopamine‐induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin‐dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one‐third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1‐positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.
Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases
Authors: Hilfiker Sabine , Pieribone Vincent A. , Nordstedt Christer , Greengard Paul , Czernik Andrew J.
Published: March 2003

Abstract : Synaptotagmin I has been suggested to function as a low‐affinity calcium sensor for calcium‐triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin‐dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagl revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas CaskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin‐coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+‐evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.
hFE65L Influences Amyloid Precursor Protein Maturation and Secretion
Authors: Guénette Suzanne Y , Chen Jing , Ferland Amber , Haass Christian , Capell Anja , Tanzi Rudolph E.
Published: March 2003

Abstract : The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective α‐secretase‐derived secreted APP (APPsα) and the Alzheimer’s disease‐associated β‐amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C‐terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin‐coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a threefold increase in the amount of APPsα recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPsα secreted is due neither to a simple increase in the steady‐state levels of APP nor to activation of the protein kinase C‐regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.
Prion Protein Peptide Neurotoxicity Can Be Mediated by Astrocytes
Authors: Brown David R
Published: March 2003

Abstract : A peptide based on amino acids 106‐126 of the sequence of human prion protein (PrP106‐126) is neurotoxic in culture. A role for astrocytes mediating PrP106‐126 toxicity was investigated. The toxicity of PrP106‐126 to cerebellar cell cultures was reduced by aminoadipate, a gliotoxin. Normally, PrP106‐126 is not toxic to cultures containing neurones deficient in the cellular isoform of prion protein (PrPc). However, PrP106‐126 was toxic to cerebellar cells derived from Prnp0/0 mice (deficient in PrPc expression) when those cerebellar cells were cocultured with astrocytes. This toxicity was found to occur only in the presence of PrPc‐positive astrocytes and to be mediated by glutamate. Furthermore, PrPc‐positive astrocytes were shown to protect Prnp0/0 cerebellar cells from glutamate toxicity. This effect could be inhibited by PrP106‐126. PrP106‐126 did not enhance the toxicity of glutamate to neurones directly. When cerebellar cells were cocultured with astrocytes, the neurones became dependent on astrocytes for protection from glutamate toxicity and expressed an increased sensitivity to glutamate. In such a system, the protective effects of astrocytes against glutamate toxicity to neurones were inhibited by PrP106‐126, resulting in a greater reduction in neuronal survival than would have been caused by PrP106‐126 when astrocytes were not present. This new model provides a possible mechanism by which the gliosis in prion disease may accelerate the neurodegeneration seen in the later stages of the disease.
Calbindin D28K Gene Transfer via Herpes Simplex Virus Amplicon Vector Decreases Hippocampal Damage In Vivo Following Neurotoxic Insults
Authors: Phillips Russell G. , Meier Timothy J. , Giuli Lisa C. , McLaughlin John R. , Ho Dora Y. , Spolsky Robert M.
Published: March 2003

Abstract : Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) can lead to neuron death. Preventing a rise in [Ca2+]i by removing Ca2+ from the extracellular space or by adding Ca2+ chelators to the cytosol of target cells ameliorates the neurotoxicity associated with [Ca2+]i increases. Another potential route of decreasing the neurotoxic impact of Ca2+ is to overexpress one of the large number of constitutive calcium‐binding proteins. Previous studies in this laboratory demonstrated that overexpression of the gene for the calcium‐binding protein calbindin D28K, via herpes simplex virus (HSV) amplicon vector, increases the survival of hippocampal neurons in vitro following energetic or excitotoxic insults but not following application of sodium cyanide. We now report that in vivo hippocampal infection with the calbindin D28K HSV vector increases neuronal survival in the dentate gyrus after application of the antimetabolite 3‐acetylpyridine and increases transsynaptic neuronal survival in area CA3 following kainic acid neurotoxicity. The protective effects of infection with the calbindin D28K vector in an intact brain may prove to be beneficial during changes in Ca2+ homeostasis caused by neurological trauma associated with aging and certain neurological diseases.
Blood‐Brain Barrier Produces Significant Efflux of L‐Aspartic Acid but Not D‐Aspartic Acid
Authors: Hosoya Ken‐ichi , Sugawara Michiko , Asaba Hiroshi , Terasaki Tetsuya
Published: March 2003

Abstract : The brain efflux index method has been used to clarify the mechanism of efflux transport of acidic amino acids such as L‐aspartic acid (L‐Asp), L‐glutamic acid (L‐Glu), and D‐aspartic acid (D‐Asp) across the blood‐brain barrier (BBB). About 85% of L‐[3H]Asp and 40% of L‐[3H]Glu was eliminated from the ipsilateral cerebrum within, respectively, 10 and 20 min of microinjection into the brain. The efflux rate constant of L‐[3H]Asp and L‐[3H]Glu was 0.207 and 0.0346 min‐1, respectively. However, D‐[3H]Asp was not eliminated from brain over a 20‐min period. The efflux of L‐[3H]Asp and L‐[3H]Glu was inhibited in the presence of excess unlabeled L‐Asp and L‐Glu, whereas D‐Asp did not inhibit either form of efflux transport. Aspartic acid efflux across the BBB appears to be stereospecific. Using a combination of TLC and the bioimaging analysis, attempts were made to detect the metabolities of L‐[3H]Asp and L‐[3H]Glu in the ipsilateral cerebrum and jugular vein plasma following a microinjection into parietal cortex, area 2. Significant amounts of intact L‐[3H]Asp and L‐[3H]Glu were found in all samples examined, including jugular vein plasma, providing direct evidence that at least a part of the L‐Asp and L‐Glu in the brain interstitial fluid is transported across the BBB in the intact form. To compare the transport of acidic amino acids using brain parenchymal cells, brain slice uptake studies were performed. Although the slice‐to‐medium ratio of D‐[3H]Asp was the highest, followed by L‐[3H]Glu and L‐[3H]Asp, the initial uptake rate did not differ for both L‐[3H]Asp and D‐[3H]Asp, suggesting that the uptake of aspartic acid in brain parenchymal cells is not stereospecific. These results provide evidence that the BBB may act as an efflux pump for L‐Asp and L‐Glu to reduce the brain interstitial fluid concentration and act as a static wall for D‐Asp.
Bradykinin Induces Interleukin‐6 Expression in Astrocytes Through Activation of Nuclear Factor‐κB
Authors: Schwaninger Markus , Sallmann Svea , Petersen Nicole , Schneider Armin , Prinz Simone , Libermann Towia A. , Spranger Matthias
Published: March 2003

Abstract : Bradykinin, a mediator of inflammation, is produced in

the brain during trauma and stroke. It is thought to open the blood‐brain

barrier, although the mechanism is unclear. We have investigated, therefore,

the effect of bradykinin on the expression of interleukin‐6 (IL‐6), a putative

modulator of the blood‐brain barrier, in astrocytes. IL‐6 gene transcription

was evaluated by transient transfection of the human IL‐6 promoter linked to

the luciferase gene. In murine astrocytes, bradykinin stimulated IL‐6

secretion and gene transcription. The effect of bradykinin was blocked by

KN‐93, an inhibitor of Ca2+/calmodulin‐dependent protein kinases, and by bisindolyl‐maleimide I, an inhibitor of protein kinase C, suggesting the involvement of these protein kinases. Mutations in the multiple response element and the binding site for nuclear factor‐κB (NF‐κB), but not in other known elements of the IL‐6 promoter, interfered with induction of IL‐6 transcription. The involvement of NF‐κB was supported further by the binding that overexpression of nmIκBα, a stable inhibitor of NF‐κB, inhibited the induction of IL‐6 by bradykinin. Bradykinin activated NF‐κB in primary astrocytes as shown by increased DNA binding of NF‐κB. These data demonstrate that bradykinin stimulates IL‐6 expression through activation of NF‐κB, which may explain several inflammatory effects of bradykinin.
The Hydrophobic Core Sequence Modulates the Neurotoxic and Secondary Structure Properties of the Prion Peptide 106‐126
Authors: Jobling Michael F. , Stewart Leanne R. , White Anthony R. , McLean Catriona , Friedhuber Anna , Maher Fran , Beyreuther Konrad , Masters Colin L. , Barrow Colin J. , Collins Steven J. , Cappai Roberto
Published: March 2003

Abstract : The neurodegeneration seen in spongiform encephalopathies is believed to be mediated by protease‐resistant forms of the prion protein (PrP). A peptide encompassing residues 106‐126 of human PrP has been shown to be neurotoxic in vitro. The neurotoxicity of PrP 106‐126 appears to be dependent upon its adoption of an aggregated fibril structure. To examine the role of the hydrophobic core, AGAAAAGA, on PrP106‐126 toxicity, we performed structure‐activity analyses by substituting two or more hydrophobic residues for the hydrophilic serine residue to decrease its hydrophobicity. A peptide with a deleted alanine was also synthesized. We found all the peptides except the deletion mutant were no longer toxic on mouse cerebellar neuronal cultures. Circular dichroism analysis showed that the nontoxic PrP peptides had a marked decrease in β‐sheet structure. In addition, the mutants had alterations in aggregability as measured by turbidity, Congo red binding, and fibril staining using electron microscopy. These data show that the hydrophobic core sequence is important for PrP106‐126 toxicity probably by influencing its assembly into a neurotoxic structure. The hydrophobic sequence may similarly affect aggregation and toxicity observed in prion diseases.
Neuronal Nicotinic Receptors in Dementia with Lewy Bodies and Schizophrenia
Authors: Court Jennifer , Spurden Dean , Lloyd Stephen , McKeith Ian , Ballard Clive , Cairns Nigel , Kerwin Robert , Perry Robert , Perry Elaine
Published: March 2003

Abstract : Neuronal nicotinic receptors have been implicated in schizophrenia on the basis of the high incidence of tobacco smoking in patients, abnormalities in cytisine and α‐bungarotoxin (αBGT) binding in the hippocampus, and linkage between auditory P50 deficits and the region of chromosome 15 coding the α7 subunit. In another disease associated with psychosis, dementia with Lewy bodies (DLB), in which visual hallucinations predominate, reductions in nicotine binding have been identified in various cortical and subcortical regions. We investigated both αBGT and nicotine binding autoradiographically in different thalamic nuclei in autopsy brain tissue from patients with schizophrenia and DLB. αBGT binding in the reticular nucleus was moderately reduced (25%) in schizophrenia and more extensively reduced (50%) in DLB. There were no significant alterations in nicotine binding in schizophrenia, and in DLB, a trend towards moderate reductions in most nuclei reached significance in the lateral dorsal nucleus. It is concluded that widespread abnormalities of thalamic nicotine are not implicated in schizophrenia or DLB, but that reticular αBGT binding may be involved to a lesser and greater extent in the pathophysiology or psychopathology of both disorders.
Striatal Dopamine Metabolism in Monoamine Oxidase B‐Deficient Mice : A Brain Dialysis Study
Authors: Fornai Francesco , Chen Kevin , Giorgi Filippo Sean , Gesi Marco , Alessandri Maria Grazia , Shih Jean Chen
Published: March 2003

Abstract : We have studied striatal dopamine (DA) metabolism in

monoamine oxidase (MAO) B‐deficient mice using brain microdialysis. Baseline

DA levels were similar in wild‐type and knock‐out (KO) mice. Administration of

a selective MAO A inhibitor, clorgyline (2 mg/kg), increased DA levels and

decreased levels of its metabolites in all mice, but a selective MAO B

inhibitor, l‐deprenyl (1 mg/kg), had no effect. Administration of 10

and 50 mg/kg l‐DOPA, the precursor of DA, increased the levels of

DA similarly in wild‐type and KO mice. The highest dose of l‐DOPA

(100 mg/kg) produced a larger increase in DA in KO than wild‐type mice. This

difference was abolished by pretreating wild‐type mice with

l‐deprenyl. These results suggest that in mice, DA is only metabolized by MAO A under basal conditions and by both MAO A and B at high concentrations. This is in contrast to the rat, where DA is always metabolized by MAO A regardless of concentration.
Vesicular Monoamine Transporter 1 Is Responsible for Storage of 5‐Hydroxytryptamine in Rat Pinealocytes
Authors: Hayashi Mitsuko , Haga Megumi , Yatsushiro Shouki , Yamamoto Akitsugu , Moriyama Yoshinori
Published: March 2003

Abstract : Vesicular monoamine transporters (VMATs) are involved in

chemical transduction in monoaminergic neurons and various endocrine cells

through the storage of monoamines in secretory vesicles. Mammalian

pinealocytes contain more 5‐hydroxytryptamine (5‐HT) than any other cells and

are expected on contain VMAT, although no information is available so far.

Upon the addition of ATP, radiolabeled 5‐HT was taken up by a particulate

fraction prepared from cultured rat pinealocytes. The 5‐HT uptake was

inhibited significantly by bafilomycin A1 (an inhibitor of vacuolar

H+‐ATPase),

3,5‐di‐tert‐butyl‐4‐hydroxybenzyli‐denemalononitrile (a proton conductor), or reserpine (an inhibitor of VMAT). RT‐PCR analysis suggested that VMAT type 1 (VMAT1), but not type 2, is expressed. Antibodies against VMAT1 recognized a single polypeptide with an apparent molecular mass of ~55 kDa, and specifically immunostained pinealocytes. VMAT1 immunoreactivity was high in the vesicular structures in the varicosities of long branching processes and was associated with 5‐HT, but not with synaptophysin, a marker protein for microvesicles. The 5‐HT immunoreactivity in the long branching processes disappeared upon incubation with reserpine. These results indicate that 5‐HT, at least in part, is stored in vesicles other than microvesicles in pinealocytes through a mechanism similar to that of various secretory vesicles.
Serotonin‐Mediated Increases in the Extracellular Levels of β‐Endorphin in the Arcuate Nucleus and Nucleus Accumbens : A Microdialysis Study
Authors: Zangen Abraham , Nakash Rachel , Yadid Gal
Published: March 2003

Abstract : Although the involvement of both endogenous opioid and

serotonergic systems in modulation of pain and emotion was suggested, the

neurochemical interaction between these systems in the brain has not

previously been studied directly. Herein, the effects of the local application

of serotonin (5‐HT) and fluoxetine (a 5‐HT reuptake inhibitor) on

extracellular levels of β‐endorphin in the arcuate nucleus and nucleus

accumbens were assessed in freely moving rats using in vivo microdialysis. The

mean basal concentrations of β‐endorphin in dialysates obtained from the

arcuate nucleus and nucleus accumbens were 259.9 and 143.3 pM,

respectively. Specific lesion of the serotonergic system by

5,7‐dihydroxytryptamine (5,7‐DHT) caused a significant decrease in these

dialysate β‐endorphin levels. When 5‐HT (0.25‐5 μM) was added

to the perfusion solution, the levels of β‐endorphin in the dialysate

from the arcuate nucleus increased (186‐296% of baseline), in a

concentration‐dependent manner. In the nucleus accumbens, 0.5 and 2

μM 5‐HT in the perfusion fluid did not affect the levels of

β‐endorphin in the dialysate, whereas 5 and 10 μM 5‐HT caused

an increase of ~190% of baseline. When fluoxetine (250 μM) was present in the perfusing solution, the levels of β‐endorphin in the dialysates from the arcuate nucleus and nucleus accumbens increased two‐ to three‐fold. This effect was not obtained in the 5,7‐DHT‐lesioned rats. Thus, 5‐HT, either endogenously or exogenously delivered, appears to facilitate the release of β‐endorphin in the arcuate nucleus and nucleus accumbens. This indication of an interaction between serotonergic and endorphinic systems may be relevant for assessing pain and mood disorder circuits and the mode of action of antidepressant drugs.
Differentiated Human NT2‐N Neurons Possess a High Intracellular Content of myo‐Inositol
Authors: Novak James E. , Turner R. Scott , Agranoff Bernard W. , Fisher Stephen K.
Published: March 2003

Abstract:
myo‐Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2‐N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2‐N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2‐N cells, respectively. The high concentrations of inositol in NT2‐N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2‐N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2‐N neurons vs 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.
Phoneutria nigriventer Toxin Tx3‐1 Blocks A‐Type K+ Currents Controlling Ca2+ Oscillation Frequency in GH3 Cells
Authors: Kushmerick C. , Kalapothakis E. , Beirão P. S. L. , Penaforte C. L. , Prado V. F. , Cruz J. S. , Diniz C. R. , Cordeiro M. N. , Gomez M. V. , Romano‐Silva M. A. , Prado M. A. M.
Published: March 2003

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.
Tumor Necrosis Factor‐α and Phorbol 12‐Myristate 13‐Acetate Differentially Modulate Cytotoxic Effect of Nitric Oxide Generated by Serum Deprivation in Neuronal PC12 Cells
Authors: Chung Kwang Chul , Park Jae Hyun , Kim Chul Hoon , Ahn Young Soo
Published: March 2003

Abstract:Nitric oxide (NO) is a signaling molecule that mediates several physiological processes in a range of cell and tissue types. Here we investigated the effect of serum deprivation in the absence or presence of phorbol 12‐myristate 13‐acetate (PMA) or tumor necrosis factor‐α (TNFα) on cell viability, NO formation, inducible NO synthase (iNOS) induction, and activation of mitogen‐activated protein kinase in neuronal PC12 cells. Within 24 h of serum deprivation, apoptosis occurred in up to 65‐70% of the cells, and significant levels of NO were generated. When PMA was added in serum‐free medium, NO formation and cell death were decreased. In contrast, addition of TNFα in serum‐free medium increased the levels of NO formation and apoptosis compared with those in serum‐deprived cells. We have demonstrated that differential generation of NO levels by PMA or TNFα under conditions of serum deprivation is mediated by the same pattern of iNOS induction. NO formation via iNOS induction resulted in the activation of c‐JUN N‐terminal kinase (JNK) but not extracellular signal‐regulated kinase. From this study it is suggested that the differential formation of cytotoxic NO by serum deprivation plus PMA or TNFα is primarily mediated by the induction of iNOS enzymes in neuronal PC12 cells and that its action is mediated by the activation of JNK.
Developmental Neurotoxicity of Phenytoin on Granule Cells and Purkinje Cells in Mouse Cerebellum
Authors: Ohmori Hiromitsu , Ogura Hiroo , Yasuda Mineo , Nakamura Shigenobu , Hatta Tatsuo , Kawano Kano , Michikawa Takayuki , Yamashita Keisuke , Mikoshiba Katsuhiko
Published: March 2003

Abstract: Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2‐4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5‐bromo‐2′‐deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5‐trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.
Anoxia‐Induced Dopamine Release from Rat Striatal Slices
Authors: Büyükuysal R. Levent , Mete Birsen
Published: March 2003

Abstract: Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4‐dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia‐induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia‐induced DA depletion, aglycemia‐and ischemia‐induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK‐801 and also by comparing the glutamate‐induced DA release with that during anoxia, aglycemia, or ischemia. MK‐801 decreased the anoxia‐induced DA depletion in a dose‐dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia‐induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK‐801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.
Induction of Glucose‐6‐Phosphate Dehydrogenase by Lipopolysaccharide Contributes to Preventing Nitric Oxide‐Mediated Glutathione Depletion in Cultured Rat Astrocytes
Authors: García‐Nogales Paula , Almeida Angeles , Fernández Emilio , Medina José M. , Bolaños Juan P.
Published: March 2003

Abstract: Treatment of cultured rat astrocytes with lipopolysaccharide (LPS; 1 μg/ml) increased mRNA expression of glucose‐6‐phosphate dehydrogenase (G6PD), the rate‐limiting step in the pentose phosphate pathway (PPP), in a time‐dependent fashion (0‐24 h). This effect was accompanied by an increase in G6PD activity (1.74‐fold) and in the rate of glucose oxidation through the PPP (6.32‐fold). Inhibition of inducible nitric oxide synthase (iNOS) activity by 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine (AMT; 50 μM) did not alter the LPS‐mediated enhancement of G6PD mRNA expression or PPP activity. Blockade of nuclear factor κB (NF‐κB) activation by N‐benzyloxycarbonyl‐Ile‐Glu‐(O‐tert‐butyl)‐Ala‐leucinal (1 μM) prevented the expression of both iNOS mRNA and G6PD mRNA, suggesting that iNOS and G6PD are co‐induced by LPS through a common transcriptional pathway involving NF‐κB activation. Incubation of cells with LPS for 24 h increased intracellular NADPH concentrations (1.63‐fold) as compared with untreated cells, but GSH concentrations were not modified by LPS treatment up to 60 h of incubation. However, inhibition of G6PD activity by dehydroepiandrosterone (DHEA; 100 μM), which prevented LPS‐mediated enhancements in PPP activity and NADPH concentrations, caused a 50% decrease in the GSH/GSSG ratio after 24‐36 h and in GSH concentrations after 60 h of incubation. Furthermore, the changes in glutathione concentrations caused by DHEA were abolished by AMT, suggesting that nitric oxide and/or its reactive derivatives would be involved in this process. From these results, we conclude that LPS‐mediated G6PD expression prevents GSH depletion due to nitric oxide and suggest that this phenomenon may be a contributing factor in the defense mechanisms that protect astrocytes against nitric oxide‐mediated cell injury.
Calcium‐Dependent Interaction Between the Large Myelin‐Associated Glycoprotein and S100β
Authors: Kursula Petri , Tikkanen Gitte , Lehto Veli‐Pekka , Nishikimi Morimitsu , Heape Anthony
Published: March 2003

Abstract : The myelin‐associated glycoprotein is a transmembrane

cell adhesion molecule expressed by myelinating glial cells of the nervous

system. So far, only protein kinases have been reported to interact with the

cytoplasmic domains of the two isoforms of the myelinassociated glycoprotein.

We report here the identification of the first nonkinase intracellular ligand

for the large isoform of the myelin‐associated glycoprotein as the S100β

protein. The interaction is dependent on the presence of calcium. We have also

localized the S100β‐binding site in the cytoplasmic domain specific to

the large myelin‐associated glycoprotein isoform to a putative basic

amphipathic α‐helix. A synthetic peptide corresponding to this region

bound to S100β in a calcium‐dependent manner with a stoichiometric ratio

of 1:1 (KD≈ 7 μM). We suggest that the observed interaction may play a role in the regulation of the myelinating glial cell cytoskeleton and the divalent cation‐dependent signal transduction events during myelin formation and maintenance.
Apoptosis and the Nervous System
Authors: Sastry P. S. , Rao Kalluri Subba
Published: March 2003

Abstract:Apoptosis is now recognized as a normal feature in the development of the nervous system and may also play a role in neurodegenerative diseases and aging. This phenomenon has been investigated intensively during the last 6‐7 years, and the progress made in this field is reviewed here. Besides a few in vivo studies, a variety of neuronal preparations from various parts of the brain, the majority of which were primary cultures, and some cell lines have been investigated. Several apoptosis‐inducing agents have been identified, and these include lack of neurotrophic support, neurotransmitters, neurotoxicants, modulators of protein phosphorylation and calcium homeostasis, DNA‐damaging agents, oxidative stress, nitric oxide, and ceramides. The precise signaling cascade is not well established, and there are lacunae in many suggested pathways. However, it appears certain that the Bcl family of proteins is involved in the apoptotic pathway, and these proteins in turn affect the processing of interleukin‐1β converting enzyme (ICE)/caspases. The available evidence suggests that there may be several apoptotic pathways that may depend on the cell type and the inducing agent, and most of the pathways may converge at the ICE/caspases step.
Neurite Outgrowth Induced by Cyclic AMP Can Be Modulated by the α Subunit of Go
Authors: Ghil Sung‐Ho , Kim Bum‐Jun , Lee Young‐Don , Suh‐Kim Haeyoung
Published: March 2003

Abstract: Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the α subunit of Go (αo) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP‐responsive element binding protein (CREB). Specific inhibition of cAMP‐dependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal‐regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of αo (αowt) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, αoQ205L, which lost GTPase activity and thus could not bind to Gβγ, gave similar results, suggesting that the effect of αo is not mediated through Gβγ. Expression of αowt or αoQ205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and αo may cause changes in NOG by inhibiting CREB activation.
Decreased Protein Levels of Nicotinic Receptor Subunits in the Hippocampus and Temporal Cortex of Patients with Alzheimer’s Disease
Authors: Guan Zhi‐Zhong , Zhang Xiao , Ravid Rivka , Nordberg Agneta
Published: March 2003

Abstract: Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer’s disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR α3, α4, α7, and β2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age‐matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti‐α4, anti‐α7, and anti‐β2 antibodies, respectively. When anti‐α3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age‐matched controls, the α4 subunit was reduced significantly by ∼35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the α3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the α7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the β2 subunit between AD patients and controls. These results reveal brain region‐specific changes in the protein levels of the nAChR, α3, α4, and α7 subunits in AD.
Major Phosphorylation Site (Ser55) of Neurofilament L by Cyclic AMP‐Dependent Protein Kinase in Rat Primary Neuronal Culture
Authors: Nakamura Yu , Hashimoto Ryota , Kashiwagi Yujiro , Aimoto Saburo , Fukusho Eriko , Matsumoto Naohiko , Kudo Takashi , Takeda Masatoshi
Published: March 2003

Abstract: Ser55 of neurofilament L (NF‐L) is reported to
be partly phosphorylated in neurons and to be phosphorylated by cyclic
AMP‐dependent protein kinase (PKA). Bovine NF‐L was phosphorylated by PKA in a
low concentration of MgCl2 (0.3 mM) and digested by
trypsin. Trypsin‐digested fragments were assigned by MALDI/TOF
(matrix‐assisted laser desorption and ionization/time‐of‐flight) mass
spectrometry. Phosphorylation sites were found at Ser41,
Ser55, and Ser62 in the head region, with
Ser55 considered the preferred site. A site‐specific
phosphorylation‐dependent antibody against Ser55 rendered NF‐L
phosphorylated at Ser55 detectable in primary cultured rat neurons.
One‐hour treatment with 20 nM okadaic acid increased the
phosphorylation level of Ser55, and co‐treatment with 10
μM forskolin enhanced it. However, forskolin alone did not elevate
the phosphorylation level. As a consequence, NF‐L may be phosphorylated at
Ser55 by PKA or by a PKA‐like kinase in vivo; however, the
phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.
Turnover and Down‐Regulation of GABAA Receptor α1, β2S, and γ1 Subunit mRNAs by Neurons in Culture
Authors: Lyons Helen R. , Gibbs Terrell T. , Farb David H.
Published: March 2003

Abstract: Benzodiazepines (BZDs), barbiturates, ethanol, and general
anesthetics potentiate the action of γ‐aminobutyric acid (GABA) at the
type A GABA receptor (GABAAR) and have profound effects on mood,
arousal, and susceptibility to seizures. GABAAR number and subunit
mRNA levels change in animal models of epilepsy and anxiety and following
exposure to GABAAR agonists and positive modulators, but the
mechanism of receptor down‐regulation remains unknown. Persistent exposure (48
h) of brain neurons in primary culture to GABA results in a 30% decrease in
the levels of mRNA encoding the α1, β2S, and γ1
GABAAR subunit isoforms, which form a receptor enhanced by
nonselective BZDs. Down‐regulation of α1 mRNA (t1/2
= 8 h) precedes down‐regulation of receptor number (t1/2 =
25 h), suggesting that GABA‐induced GABAAR down‐regulation is a
consequence of decreased mRNA levels. The apparent half‐life of the α1
mRNA in the presence of α‐amanitin (9 h) is consistent with the time
course of α1 mRNA down‐regulation. Moreover, the stability of the
α1, β2S, and γ1 subunit mRNAs is not altered by chronic GABA
exposure. The results demonstrate that GABAAR subunit mRNA
down‐regulation is not a consequence of accelerated mRNA degradation and argue
that GABA‐induced GABAAR down‐regulation is due to inhibition of transcription.