bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoprecipitation of High‐Affinity, Guanine Nucleotide‐Sensitive, Solubilized μ‐Opioid Receptors from Rat Brain
Authors: Chalecka‐Franaszek Elzbieta , Weems Henri B. , Crowder A. Tamara , Cox Brian M. , Côté Thomas E.
Published: March 2003

Abstract:Antibodies directed against the C‐terminal and the N‐terminal regions of the μ‐opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the μ receptor. Two fusion proteins were constructed: One contained the 50 C‐terminal amino acids of the μ receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating ∼70% of solubilized rat brain μ receptors as determined by [3H][D‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) had an EC50of 0.4 nMin diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to μ receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity‐purified antibody was screened for the presence of G protein α subunits, it was determined that Gαo, Gαi1, Gαi3, and to a lesser extent Gαi2, but not Gαsor Gαq/11, were coimmunoprecipitated with the μ receptor. Inclusion of GTPγS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.
Spatiotemporal Interaction of α2 Autoreceptors and Noradrenaline Transporters in the Rat Locus Coeruleus
Authors: Callado Luis F. , Stamford Jonathan A
Published: March 2003

Abstract: We investigated the roles of α2 autoreceptors and noradrenaline (NA) transporters on NA efflux and uptake in the rat locus coeruleus after electrical stimulation. NA efflux was evoked by various trains (50 pulses, 10‐500 Hz) and measured by fast cyclic voltammetry. NA efflux and uptake half‐time (t1/2) were stimulus‐dependent, ranging from 43 ± 3 nM and 2.45 ± 0.21 s, respectively, with 500‐Hz stimuli to 127 ± 11 nM and 4.41 ± 0.34 s, respectively, with 100‐Hz trains. Based on these data, we calculate that each transporter removes 0.19 NA molecules from the extracellular space every second, a velocity compatible more with transporter‐than channel‐mode conduction. Dexmedetomidine (10 nM) decreased NA efflux by ∼30% on stimulations of ≤1 s in duration. BRL 44408 (1 μM) increased NA efflux on stimuli of ≥2 s (by up to 92 ± 16%). Desipramine (50 nM) increased NA efflux on stimuli of ≥1 s (by 113 ± 24%) but slowed NA uptake on all stimuli. When given together, the effects of desipramine and BRL 44408 were additive at stimuli of ≥1 s but showed potentiation on shorter trains. There was a significant time delay for the elevation of NA efflux by blockade of uptake (0.79 s) or autoreceptors (1.14 s), suggesting that both are located extrasynaptically and that NA must diffuse through the extracellular space to these structures. We suggest that released NA may interact with α2 autoreceptors and NA transporters as far as 10 μm from the release sites, an action compatible with a volume transmission role of NA in the locus coeruleus.
Identification of the Isoforms of Ca2+/Calmodulin‐Dependent Protein Kinase II in Rat Astrocytes and Their Subcellular Localization
Authors: Takeuchi Yusuke , Yamamoto Hideyuki , Fukunaga Kohji , Miyakawa Taihei , Miyamoto Eishichi
Published: March 2003

Abstract: Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT‐PCR followed by the “nested” PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was δ2. Immunostaining suggested that the δ2 isoform was localized predominantly at the Golgi apparatus. The localization of the δ2 isoform at the Golgi apparatus was also observed in NG108‐15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108‐15 cells. In contrast to the αB and δ3 isoforms that entered the nucleus, as reported, the γA isoform was excluded from the nucleus in the transfected NG108‐15 cells. These results suggest that the 15‐amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the γA isoform.
Glutamic Acid Decarboxylase‐Expressing Astrocytes Exhibit Enhanced Energetic Metabolism and Increase PC12 Cell Survival Under Glucose Deprivation
Authors: Bellier J.P. , Sacchettoni S. , Prod'hon C. , Perret‐Liaudet A. , Belin M.F. , Jacquemont B.
Published: March 2003

Abstract : Astrocytes play a key role by catabolizing glutamate from extracellular space into glutamine and tricarboxylic acid components. We previously produced an astrocytic cell line that constitutively expressed glutamic acid decarboxylase (GAD67), which converts glutamate into GABA to increase the capacity of astrocytes to metabolize glutamate. In this study, GAD‐expressing astrocytes in the presence of glutamate were shown to have increased energy metabolism, as determined by a moderate increase of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, by an increased ATP level, and by enhanced lactate release. These changes were due to GAD transgene expression because transient expression of a GAD antisense plasmid resulted in partial suppression of the ATP level increase. These astrocytes had an increased survival in response to glucose deprivation in the presence of glutamate compared with the parental astrocytes, and they were also able to enhance survival of a neuronal‐like cell line (PC12) under glucose deprivation. This protection may be partially due to the increased lactate release by GAD‐expressing astrocytes because PC12 cell survival was enhanced by lactate and pyruvate under glucose deprivation. These results suggest that the establishment of GAD expression in astrocytes enhancing glutamate catabolism could be an interesting strategy to increase neuronal survival under hypoglycemia conditions.
Nuclear Factor‐κB Mediates the Cell Survival‐Promoting Action of Activity‐Dependent Neurotrophic Factor Peptide‐9
Authors: Glazner Gordon W. , Camandola Simonetta , Mattson Mark P.
Published: March 2003

Abstract : Activity‐dependent neurotrophic factor (ADNF) is produced

by astrocytes in response to neuronal depolarization and, in turn, promotes

neuronal survival. A nineamino acid ADNF peptide (ADNF9) exhibits full

neurotrophic activity and potently protects cultured embryonic rat hippocampal

neurons from oxidative injury and apoptosis. Picomolar concentrations of ADNF9

induced an increase in nuclear factor‐κB (NF‐κB) DNA‐binding

activity within 1 h of exposure, with a maximum increase of ~10‐fold by 6 h.

Activation of NF‐κB was correlated with increased resistance of neurons

to apoptosis induced by exposure to Fe2+. The antiapoptotic action of ADNF9 was abolished when NF‐κB activation was specifically blocked with κB decoy DNA. Oxidative stress was attenuated in neurons pretreated with ADNF9, and this effect of ADNF9 was blocked by κB decoy DNA, suggesting that ADNF9 suppresses apoptosis by reducing oxidative stress. ADNF9 also prevented neuronal apoptosis following trophic factor withdrawal via an NF‐κB‐mediated mechanism. Thus, NF‐κB mediates the neuron survival‐promoting effects of ADNF9 in experimental models relevant to developmental neuronal death and neurodegenerative disorders.
Site‐Specific Phosphorylation of Neurofilament‐L Is Mediated by Calcium/Calmodulin‐Dependent Protein Kinase II in the Apical Dendrites During Long‐Term Potentiation
Authors: Hashimoto Ryota , Nakamura Yu , Komai Shoji , Kashiwagi Yujiro , Tamura Keiko , Goto Takahiro , Aimoto Saburo , Kaibuchi Kozo , Shiosaka Sadao , Takeda Masatoshi
Published: March 2003

Abstract : Neurofilament‐L (NF‐L), one subunit of the neuronal

intermediate filaments, is a major element of neuronal cytoskeletons. The

dynamics of NF‐L are regulated by phosphorylation of its head domain. The

phosphorylation sites of the NF‐L head domain by protein kinase A, protein

kinase C, and Rho‐associated kinase have been previously identified, and those

by calcium/calmodulin‐dependent protein kinase II (CaMKII) were identified in

this study. A series of site‐ and phosphorylation state‐specific antibodies

against NF‐L was prepared to investigate NF‐L phosphorylation in neuronal

systems. Long‐term potentiation (LTP) is a cellular model of neuronal

plasticity that is thought to involve the phosphorylation of various proteins.

NF‐L is considered a possible substrate for phosphorylation. During LTP

stimulation of mouse hippocampal slices, the series of antibodies demonstrated

the increase in the phosphorylation level of Ser57 in NF‐L and the

visualization of the localized distribution of Ser57

phosphorylation in a subpopulation of apical dendrites of the pyramidal

neurons. Furthermore, Ser57 phosphorylation during LTP is suggested

to be mediated by CaMKII. Here we show that NF‐L is phosphorylated by CaMKII

in a subpopulation of apical dendrites during LTP, indicating that

Ser57 is a novel phosphorylation site of NF‐L in vivo related to the neuronal signal transduction.
Lactate Prevents the Alterations in Tissue Amino Acids, Decline in ATP, and Cell Damage Due to Aglycemia in Retina
Authors: Zeevalk Gail D. , Nicklas William J.
Published: March 2003

Abstract: Under conditions of energy impairment, CNS tissue can

utilize substrates other than glucose to maintain energy metabolism. Retinas

produce large amounts of lactate, although it has not been shown that lactate

can be utilized by retina to prevent the cell damage associated with

hypoglycemia. To investigate this, intact, isolated retinas were subjected to

aglycemic conditions in the presence or absence of 20 mM lactate.

Retinas incubated in the absence of glucose for 60 min showed a threefold

elevation in tissue aspartate and 60% decreases in tissue glutamate and

glutamine, demonstrating a mobilization of carbon from glutamine and glutamate

to the tricarboxylic acid cycle. Lactate prevented these changes in tissue

amino acids, indicating metabolism of lactate with sparing of tissue glutamate

and glutamine. Tissue ATP was 20 and 66% of control values with zero glucose

or zero glucose plus lactate, respectively. Consistent with previous findings,

incubation of retinas in the absence of glucose caused acute swelling of

retinal neurons and release of GABA into the medium at 60 min. These acute

toxic affects caused by the absence of glucose were completely prevented by

the presence of lactate. At 24 h of recovery following 60 min of zero glucose,

many pyknotic profiles were observed and lactate dehydrogenase (LDH) release

into the medium was elevated sevenfold, indicating the extent of cell death.

In contrast, no elevation in LDH was found and histology appeared normal in

retinas exposed to zero glucose in the presence of lactate.

α‐Cyano‐4‐hydroxy cinnamate (4‐CIN; 0.5 mM), an inhibitor of

the monocarboxylic acid transporter and mitochondrial pyruvate carrier,

blocked the ability of lactate to maintain ATP and protect retinas from

aglycemia but had no effect on ATP or toxicity per se. Derangements in tissue

aspartate, glutamate, and glutamine, which were prevented by lactate during

zero glucose incubation, were again observed with lactate plus zero glucose in

the presence of 4‐CIN. However, 0.5 mM 4‐CIN alone in the presence of

glucose produced similar increases in aspartate and decreases in glutamate and

glutamine as observed with zero glucose while having only modest inhibitory

effects on [U‐14C]lactate uptake, suggesting the mitochondrial pyruvate carrier as the main site of action. The above findings show that lactate is readily utilized by the chick retina during glucose deprivation to prevent derangements in tissue amino acids and ATP and retinal neuronal cell death.
The Potent, Selective mGlu2/3 Receptor Agonist LY379268 Increases Extracellular Levels of Dopamine, 3,4‐Dihydroxyphenylacetic Acid, Homovanillic Acid, and 5‐Hydroxyindole‐3‐Acetic Acid in the Medial Prefrontal Cortex of the Freely Moving Rat
Authors: Cartmell Jayne , Perry Kenneth W. , Salhoff Craig R. , Monn James A. , Schoepp Darryle D.
Published: March 2003

Abstract: Previous work has shown that the potent, selective

metabotropic glutamate mGlu2/3 receptor agonist LY379268 acts like the

atypical antipsychotic clozapine in behavioral assays. To investigate further

the potential antipsychotic actions of this agent, we examined the effects of

LY379268 using microdialysis in awake, freely moving rats, on extracellular

levels of dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid

(HVA), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) in rat medial prefrontal

cortex. Systemic LY379268 increased extracellular levels of dopamine, DOPAC,

HVA, and 5‐HIAA in a dose‐dependent, somewhat delayed manner. LY379268 (3

mg/kg s.c.) increased levels of dopamine, DOPAC, HVA, and 5‐HIAA to 168, 170,

169, and 151% of basal, respectively. Clozapine (10 mg/kg) also increased

dopamine, DOPAC, and HVA levels, with increases of 255, 262, and 173%,

respectively, but was without effect on extracellular 5‐HIAA levels by 3 mg/kg

LY379268 were reversed by the selective mGlu2/3 receptor antagonist LY341495

(1 mg/kg). Furthermore, LY379268 (3 mg/kg)‐evoked increases in DOPAC and HVA

were partially blocked and the increase in 5‐HIAA was completely blocked by

local application of 3 μM tetrodotoxin. Therefore, we have demonstrated that mGlu2/3 receptor agonists activate dopaminergic and serotonergic brain pathways previously associated with the action of atypical antipsychotics such as clozapine and other psychiatric agents.
Regional and Developmental Expression of the Npc1 mRNA in the Mouse Brain
Authors: Prasad A. , Fischer W. A. , Maue R. A. , Henderson L. P.
Published: March 2003

Abstract: Niemann‐Pick type C (NP‐C) disease is a fatal, autosomal

recessive disorder of cholesterol metabolism that results in progressive

central nervous system deterioration and premature death. Recently, a gene

mutated in NP‐C disease (NPC1) was identified in both human patients

and in the npcnih mouse model. Although the function of

the NPC1 gene is at present unknown, determining the pattern of its

expression in the brain may facilitate identification of the mechanisms

underlying the neuropathology of this disease and in identifying relevant

targets for any potential therapeutic intervention. We have used in situ

hybridization techniques to characterize the pattern of Npc1 mRNA

expression in both the wild‐type and the npcnih mutant

mouse brain. In adult animals of both genotypes, the Npc1 mRNA was

detected in the majority of neurons in nearly all regions, but at

significantly higher levels in the cerebellum and in specific pontine nuclei.

Analysis of Npc1 mRNA levels during development in the wild‐type

mouse indicated that this transcript was expressed in neurons as early as

embryonic day 15 and that a significant region‐specific pattern of expression

was established by postnatal day 7. Our data suggest that whereas the

NPC1 gene is widely expressed in neurons of the brain, the higher levels of expression in the cerebellum and pontine structures established by early postnatal ages may make these regions more susceptible to neuronal dysfunction in NP‐C disease.
Thrombin‐Induced Activation of Cultured Rodent Microglia
Authors: Möller Thomas , Hanisch Uwe‐Karsten , Ransom Bruce R.
Published: March 2003

Abstract: Microglia are the resident immune cells of the CNS. Upon
brain damage, these cells are rapidly activated and function as tissue
macrophages. The first steps in this activation still remain unclear, but it
is widely believed that substances released from damaged brain tissue trigger
this process. In this article, we describe the effects of the blood
coagulation factor thrombin on cultured rodent microglial cells. Thrombin
induced a transient Ca2+ increase in microglial cells, which
persisted in Ca2+‐free media. It was blocked by thapsigargin,
indicating that thrombin caused a Ca2+ release from internal
stores. Preincubation with pertussis toxin did not alter the thrombin‐induced
[Ca2+]i signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor‐α, interleukin‐6, interleukin‐12, the chemokine KC, and the soluble tumor necrosis factor‐α receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.
Mice with Markedly Reduced PACAP (PAC1) Receptor Expression by Targeted Deletion of the Signal Peptide
Authors: Hashimoto Hitoshi , Shintani Norihito , Nishino Akiko , Okabe Masaru , Ikawa Masahito , Matsuyama Shogo , Itoh Kyoko , Yamamoto Kyohei , Tomimoto Shuhei , Fujita Takashi , Hagihara Nami , Mori Wakaba , Koyama Yutaka , Matsuda Toshio , Nagata Shigekazu , Baba Akemichi
Published: March 2003

Abstract: In an attempt to study the pituitary adenylate
cyclase‐activating polypeptide (PACAP) type 1 (PAC1) receptor
(PAC1R) function in vivo and to produce a mouse model with altered
expression of PAC1R, we have used gene targeting in embryonic stem
cells to disrupt exon 2 of the PAC1R gene, which contains the ATG
translation start site and the signal peptide. Un‐expectedly, active
transcription of PAC1R mRNA was detected in the mutant mice;
however, exon 1 was spliced to exon 3 (skipping exon 2), and
125I‐PACAP27 binding in brain was greatly reduced. PAC1R
exon 2‐/‐ mice were viable, fertile, and morphologically and
histologically indistinguishable from their wild‐type counterparts. We next
examined the ligand binding and cell surface expression of the mutant receptor
lacking the signal peptide in transfected COS‐7 cells. 125I‐PACAP27
binding of the mutant receptor was approximately one‐tenth of that in the
wild‐type receptor. Although the wild‐type receptor was expressed abundantly
in both the plasma membrane and the cytoplasm around the nucleus, the mutant
receptor was expressed in the plasma membrane with a markedly reduced level.
Digestion of the membranes with endoglycosidase F greatly reduced the size of
the wild‐type receptor but only slightly reduced that of the mutant receptor.
These results demonstrate that the signal peptide is required for efficient
cell surface expression and N‐linked glycosylation of the PAC1R.
However, the mutant receptors still functionally coupled to adenylate cyclase
in COS‐7 cells, suggesting the presence of sufficient spare receptors such
that the mutant receptors are capable of activating the second messenger
system. We suggest that the mutant mice with markedly reduced PAC1R
expression can serve as a useful animal model or cell culture system for
further studies in PAC1R function.
Induction of Adrenomedullin During Hypoxia in Cultured Human Glioblastoma Cells
Authors: Kitamuro Tomomi , Takahashi Kazuhiro , Nakayama Masaharu , Murakami Osamu , Hida Wataru , Shirato Kunio , Shibahara Shigeki
Published: March 2003

Abstract: Adrenomedullin is a potent vasodilator peptide originally isolated from pheochromocytoma. Adrenomedullin is produced by various types of cells including neurons and astrocytes. To explore possible pathophysiological roles of adrenomedullin in hypoxic brain, we studied the effects of hypoxia on the expression of adrenomedullin in T98G human glioblastoma cells by radioimmunoassay and northern blot analysis. Expression levels of adrenomedullin mRNA and immunoreactive adrenomedullin levels in the culture medium were increased by hypoxia about six‐ and about threefold, respectively. Treatment with cobalt chloride increased expression levels of adrenomedullin mRNA about threefold and immunoreactive adrenomedullin levels in the culture medium about threefold in T98G cells. Using actinomycin D, we showed that hypoxia did not cause the stabilization of the adrenomedullin mRNA, suggesting that the increased adrenomedullin mRNA levels in response to hypoxia are caused mainly by increased transcription. Treatment with cycloheximide caused increases in adrenomedullin mRNA levels in both normoxic and hypoxic states, raising the possibility that some protein(s) may act as a suppressor of adrenomedullin gene expression in T98G cells. These findings indicate that adrenomedullin is highly induced during hypoxia in T98G glioblastoma cells and suggest that increased expression of adrenomedullin during hypoxia may be important in the defense against hypoxia or ischemia in the brain.
Cyclic AMP‐Induced Neuronal Differentiation via Activation of p38 Mitogen‐Activated Protein Kinase
Authors: Hansen Thomas v. O. , Rehfeld Jens F. , Nielsen Finn C.
Published: March 2003

Abstract: The p38 mitogen‐activated protein kinase (MAPK) pathway mediates cellular responses to inflammatory cytokines and environmental stress, but recent studies have indicated that p38 MAPK may be involved in a more widespread set of cellular functions. Here we show that activation of the cyclic AMP (cAMP) pathway induces a rapid, dose‐dependent phosphorylation and activation of p38 MAPK and that combined stimulation with forskolin and growth factors results in additive stimulation of p38 MAPK. Forskolin‐stimulated neurite out‐growth in rat pheochromocytoma PC12 cells was inhibited by the p38 MAPK inhibitor SB203580. With the combination of forskolin and nerve growth factor, neurite outgrowth was additively increased, and this effect was also inhibited by SB203580. Finally, transfection of p38AGF, which exhibits a mutated activation loop, inhibited cAMP‐mediated neuronal differentiation. The results indicate that p38 MAPK is a downstream target of the cAMP signaling pathway and that p38 MAPK plays a key role in neuronal differentiation induced by cAMP and growth factors by integration of signals from both pathways.
Tyrosine Phosphorylation of the α Subunit of Transducin and Its Association with Src in Photoreceptor Rod Outer Segments
Authors: Bell Michael W. , Desai Nirav , Guo Xiao X. , Ghalayini Abboud J.
Published: March 2003

Abstract: Recent evidence indicates that tyrosine phosphorylation
may play important roles in retinal photoreceptor rod outer segments (ROS). We
investigated the tyrosine phosphorylation of endogenous proteins in isolated
bovine ROS. Several proteins with apparent molecular masses of 31, 39, 60, 83,
90, 97, 120, 140, and 180 kDa were tyrosine‐phosphorylated in ROS incubated
with Mg2+, ATP, and orthovanadate. Several tyrosine kinase
inhibitors significantly inhibited tyrosine phosphorylation of these proteins
in ROS. The 39‐ and 60‐kDa tyrosine‐phosphorylated proteins were identified as
the α subunit of the G protein transducin (Tα) and the tyrosine
kinase Src, respectively. The presence of Src and tyrosine kinase activity in
bovine ROS was confirmed by their cofractionation with rhodopsin and Tα
on continuous sucrose gradients. Several tyrosine‐phosphorylated proteins,
including Src, coimmunoprecipitated with Tα. The association of Src with
Tα was detected in the absence of tyrosine phosphorylation, but was
enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine
kinase activity also associated with Tα was sevenfold higher under
tyrosine‐phosphorylating conditions. The recovery of transducin by hypotonic
GTP extraction from tyrosine‐phosphorylated ROS was significantly less than
that from nonphosphorylated ROS. We localized the site on Tα
phosphorylated by Src to the amino‐terminal half by limited tryptic digests,
and further mapped it by ion trap mass spectrometry to Tyr142 in the helical domain of Tα. Tα was also tyrosine‐phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.
Rat α‐Synuclein Interacts with Tat Binding Protein 1, a Component of the 26S Proteasomal Complex
Authors: Ghee Medeva , Fournier Alain , Mallet Jacques
Published: March 2003

Abstract:The α‐synuclein gene, which encodes a brain presynaptic nerve terminal protein of unknown function, is linked to familial early‐onset Parkinson's disease (PD). The finding that α‐synuclein forms the major fibrillary component of Lewy bodies in brains of PD patients suggests that the two point mutations in α‐synuclein (Ala53Thr, Ala30Pro) may promote the aggregation of α‐synuclein into filaments. To address the role of α‐synuclein in neurodegenerative diseases, we performed a yeast two‐hybrid screen of a rat adult brain cDNA library using rat α‐synuclein 2 (αSYN2). Here we report that αSYN2 interacts specifically with Tat binding protein 1, a subunit of the 700‐kDa proteasome activator (PA700), the regulatory complex of the 26S proteasome and of the modulator complex, which enhances PA700 activation of the proteasome.
Coexpression of Postsynaptic Density‐95 Protein with NMDA Receptors Results in Enhanced Receptor Expression Together with a Decreased Sensitivity to L‐Glutamate
Authors: Rutter A. Richard , Stephenson F. Anne
Published: March 2003

Abstract: Coexpression in human embryonic kidney (HEK) 293 cells ofthe postsynaptic density‐95 protein (PSD‐95) with NMDA receptor NR2A or NR2Bsingle subunits or NR1‐1a/NR2A and NR1‐1a/NR2B subunit combinations induced anapproximately threefold increase in NR2A and NR2B subunit expression. Deletionof the NR2 C‐terminal ESDV motifs resulted in the loss of this increasefollowing coexpression of NR1‐1a/NR2ATrunc andNR1‐1a/NR2BTrunc with PSD‐95. Characterisation of the radioligandbinding properties of [3H]MK‐801 to NR1‐1a/NR2A receptors with orwithout PSD‐95 showed that PSD‐95 induced a threefold increase inBmax values and an apparent approximately fivefolddecrease in affinity in the presence of 10 μM L‐glutamate. In thepresence of 1 mM L‐glutamate, the Ki for MK‐801binding to NR1‐1a/NR2A with PSD‐95 was not significantly different from thatfor NR1‐1a/NR2A without PSD‐95. The EC50 value for the enhancementof [3H]MK‐801 binding by L‐glutamate to NR1‐1a/NR2A was 1.8± 0.4 (n = 4) and 8.9 (mean of n = 2) μM in the absence andpresence of PSD‐95, respectively. Thus, coexpression of PSD‐95 withNR1‐1a/NR2A results in a decreased sensitivity to L‐glutamate and an enhancedexpression of NR2A and NR2B subunits. Deletion studies show that this effectis mediated via interaction of the C‐terminal ESDV motif of the NR2 subunitwith PSD‐95.
An Allelic Variation in the Human Prodynorphin Gene Promoter Alters Stimulus‐Induced Expression
Authors: Zimprich Alexander , Kraus Jürgen , Wöltje Michael , Mayer Peter , Rauch Elisabeth , Höllt Volker
Published: March 2003

Abstract: Prodynorphin, the precursor of the dynorphin opioid peptides, has been shown to play an important role in several aspects of human diseases and complex traits, e.g., drug abuse, epilepsy, and mood disorders. The objective of this study was to identify polymorphisms in the 5′ control region of the human prodynorphin gene and to relate these polymorphisms to prodynorphin gene expression. Within the core promoter region, a 68‐bp sequence was found to occur as a polymorphic element, either singular or as tandemly repeated element two, three, or four times. This 68‐bp repeat element contains an AP‐1 transcription factor binding site as demonstrated by electrophoretic mobility shift assay. Reporter gene assays were performed and provided evidence for allele dependent different promoter activity. Dynorphin was found to be involved in many pathophysiological processes so that the described prodynorphin alleles may correlate with the occurrence of several diseases, for example, drug addiction. However, prodynorphin allelic distributions were not significantly different in heroin addicts and control subjects.
Synergistic Induction of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Gene Expression by Nerve Growth Factor and PACAP in PC12 Cells
Authors: Hashimoto Hitoshi , Hagihara Nami , Koga Kazumi , Yamamoto Kyohei , Shintani Norihito , Tomimoto Shuhei , Mori Wakaba , Koyama Yutaka , Matsuda Toshio , Baba Akemichi
Published: March 2003

Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter‐luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT‐PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12‐myristate 13‐acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin‐induced PACAP mRNA expression. Down‐regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen‐activated protein kinase extracellular signal‐regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF‐induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.
Synaptotagmin IV Is Present at the Golgi and Distal Parts of Neurites
Authors: Ibata Keiji , Fukuda Mitsunori , Hamada Tomohiro , Kabayama Hiroyuki , Mikoshiba Katsuhiko
Published: March 2003

Abstract: Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor‐differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform‐specific antibody (anti‐SytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A‐dependent gene up‐regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity.
Functional Interplay Between Nuclear Factor‐κB and c‐Jun Integrated by Coactivator p300 Determines the Survival of Nerve Growth Factor‐Dependent PC12 Cells
Authors: Maggirwar Sanjay B. , Ramirez Servio , Tong Ning , Gelbard Harris A. , Dewhurst Stephen
Published: March 2003

Abstract: Nerve growth factor (NGF) activates the transcription factors nuclear factor κB (NF‐κB) and activator protein‐1 (AP‐1) in sympathetic neurons. Whereas NGF‐inducible NF‐κB is required for the survival of neurons, c‐Jun has the ability to promote neuronal death. In this report, we have examined the effect of NGF withdrawal on c‐Jun and NF‐κB transcription factors in PC12 cells differentiated to a neuronal phenotype. We show that the withdrawal of NGF from these cultures results in de novo synthesis of c‐Jun, increase in AP‐1 activity, and down‐regulation of NF‐κB activity. To investigate how the signal transduction pathways activating c‐Jun and NF‐κB are differentially regulated by NGF, we performed transcriptional analyses. Expression of RelA (NF‐κB) suppressed the c‐Jun‐dependent transcription of c‐jun, and this effect was reversed by overexpression of the coactivator p300. RelA’s effects on c‐Jun transcription were mediated by competitive binding of the carboxy‐terminal region of RelA to the CH1 domain of p300, which also binds to c‐Jun; deletion of this region abrogated the ability of RelA to inhibit c‐Jun activity. Furthermore, the inhibition of endogenous NF‐κB in NGF‐maintained neuronal PC12 cells led to the induction of c‐Jun synthesis and a marked increase in cell death. Together, these studies demonstrate a functional interaction between NF‐κB and c‐Jun and suggest a novel mechanism of NF‐κB‐mediated neuroprotection.
Correlating Physiology with Gene Expression in Striatal Cholinergic Neurones
Authors: Richardson Peter J. , Dixon Alistair K. , Lee Kevin , Bell Matt I. , Cox Peter J. , Williams Richard , Pinnock Robert D. , Freeman Tom C.
Published: March 2003

Abstract: The expression of 34 transmitter‐related genes has been examined in the cholinergic neurones of rat striatal brain slices, with the aim of correlating gene expression with functional activity. The mRNAs encoding types I, II/IIA, and III α subunits of the voltage‐sensitive sodium channels were detected, suggesting the presence of these three types of sodium channel. Similarly, mRNAs encoding all four α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA)‐type glutamate receptor subunits and the NR1 and NR2A, 2B, and 2D subunits of the NMDA‐type glutamate receptors were detected, suggesting that various combinations of these subunits mediate the cellular response to synaptically released glutamate. Other mRNAs detected included the NK1 and NK3 tachykinin receptors, all four known adenosine receptors, and the GABA‐synthesising enzyme glutamate decarboxylase. Subpopulations of these cholinergic neurones have been identified on the basis of the expression of the NK3 tachykinin receptor in 5% and the trkC neurotrophin receptor in 12% of the cells investigated.
Insect Nicotinic Acetylcholine Receptor: Conserved Neonicotinoid Specificity of [3H]Imidacloprid Binding Site
Authors: Zhang Aiguo , Kaiser Hartmut , Maienfisch Peter , Casida John E.
Published: March 2003

Abstract:
The insect nicotinic acetylcholine receptor (nAChR) is a major target for insecticide action. The rapidly expanding use of neonicotinoid insecticides of varied structures makes it increasingly important to define similarities and differences in their action, particularly for the first‐generation chloropyridinyl compounds versus the second‐generation chlorothiazolyl derivatives. We have shown with Musca domestica that a convenient and relevant determination of the neonicotinoid insecticide target is a binding site assay with [3H]imidacloprid ([3H]IMI). This study uses membranes from the aphids Myzus persicae and Aphis craccivora and from heads of the flies Drosophila melanogaster and Musca domestica to characterize the [3H]IMI binding sites relative to their number and possible species variation in structure‐activity relationships. With emphasis on commercial neonicotinoids, six potent chloropyridinyl compounds are compared with the corresponding six chlorothiazolyl analogues (syntheses are given for chemicals prepared differently than previously described). The preference for chloropyridinyl versus chlorothiazolyl is not dependent on the insect species examined but instead on other structural features of the molecule. The chlorothiazolyl substituent generally confers higher potency in the clothianidin and desmethylthiamethoxam series and the chloropyridinyl moiety in the imidacloprid, thiacloprid, acetamiprid, and nitenpyram series. Two chlorothiazolyl compounds compete directly with the chloropyridinyl [3H]IMI for the same binding sites in Myzus and Drosophila membranes. This study shows conserved neonicotinoid specificity of the [3H]IMI binding site in each of the four insect species examined.
ortho‐Substituted PCB95 alters intracellular calcium signaling and causes cellular acidification in PC12 cells by an immunophilin‐dependent mechanism
Authors: Wong Patty W. , Garcia Eric F. , Pessah Isaac N.
Published: March 2003

ortho‐Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low µM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre‐incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR‐sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre‐incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho‐substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.
Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine
Authors: Hens Jurgen , Vanderwinden Jean‐Marie , De Laet Marc‐Henri , Scheuermann Dietrich W. , Timmermans Jean‐Pierre
Published: March 2003

Data on the axonal projections of enteric neurones in the human intestine are still scarce. The present study aimed to identify the morphology and neurochemical coding of enteric neurones in the human small intestine, which are involved in the innervation of the mucosa. The lipophilic neuronal tracer DiI was applied to one mucosal villus of small intestinal resection specimens. The tissue was kept in organotypic culture and subsequently processed for immunohistochemistry. Neurones labelled from the mucosa were located in all ganglionated nerve networks, including the myenteric plexus. In all plexuses, at least five neurochemical types of neurones could be observed, i.e. SOM‐IR neurones, SP‐IR neurones, SOM/SP‐IR neurones, VIP‐IR neurones and neurones lacking immunoreactivity for any of these markers. Most of the DiI‐labelled neurones were multidendritic; a minority of neurones could be identified as Dogiel type II cells, suggesting the existence of a subgroup of primary afferent neurones in the DiI‐filled cell population. The ratio of labelled multidendritic neurones (assumed to be secretomotor) to labelled Dogiel type II neurones (assumed to be primary afferent) in the myenteric plexus is higher in large mammals (pig and human) than in small mammals (guinea pig). This might point to the existence of a different topographical distribution of subsets of primary afferent neurones and/or topographically distinct intrinsic mucosal reflex circuits in large mammals, including humans.
Expression of splice variants of the NR1 subunit of the N‐methyl‐d‐aspartate receptor in the normal and injured rat spinal cord
Authors: Prybylowski K. L. , Grossman S. D. , Wrathall J. R. , Wolfe B. B.
Published: March 2003

Quantitative western blot analysis in laminectomy control spinal cords of adult rats was used to provide the first report of the normal expression patterns of the N1, C1, C2 and C2′ cassettes in the cervical, thoracic and lumbar regions of the spinal cord as a percent of total NR1 subunit protein. In all regions studied, the C1 and C2 cassettes were usually contained in less than 10% of total NR1 protein. In contrast, approximately 90% of total NR1 protein contained the C2′ cassette. A significant proportion of total NR1 protein (approximately 30%) also contained the N1 cassette. These data are consistent with expression of NR1000 (NR1–4a) and NR1100 (NR1–4b) as the dominant splice forms in the spinal cord. Splice variant expression was also studied following incomplete, contusive spinal cord injury (SCI) to the thoracic level 8 (T8) region. This injury did not change expression of the C1 or C2 cassette in any region of the spinal cord acutely at 24 h or chronically at 1 month. There was an increase in expression of the N1 cassette in the lumbar regions 1 month after injury (p < 0.05). These data indicate that SCI induces distal changes in NR1 splice variant expression, which may play a role in the adaptive response of neurons in the chronically injured spinal cord.
Antiapoptotic role of NF‐κB in the auto‐oxidized dopamine‐induced apoptosis of PC12 cells
Authors: Lee Heon‐Jin , Kim Sun‐Hee , Kim Kwang‐Woon , Um Jee‐Hyun , Lee Hye‐Won , Chung Byung‐Seon , Kang Chi‐Dug
Published: March 2003

Current concepts of the pathogenesis of Parkinson's disease (PD) center on the formation of reactive oxygen species (ROS), and dopamine has been considered to be a major source of ROS. Recently, it has been shown in a postmortem study that nuclear translocation of nuclear factor‐kappa B (NF‐κB) was observed in dopaminergic neurons of patient with PD. However, its role is not known. The present study examined the possible role of NF‐κB in ODA (auto‐oxidized dopamine)‐induced apoptosis to understand the process of PD. Using the electrophoretic mobility shift assay, it was found that ODA activated the DNA binding activity of NF‐κB. Suppression of the transcriptional activity of NF‐κB in PC12 cells by overexpression of a wild‐type and a dominant negative mutant form (S32A/S36A) of inhibitor kappa B (IκB)‐α led to increase of apoptotic cell death induced by treatment of ODA. In addition, overexpression of NF‐κB in PC12 cells blocked ODA‐induced cell death. However, JNK/SAPK activities, which mediate various stress signals, were similar among the parental, NF‐κB‐ or dominant negative mutant IκBα‐transfected cells. Therefore, these results suggest that activation of NF‐κB during ODA‐induced apoptosis may have a counteracting activity against the signals mediating apoptotic cell death and thereby delay the process of Parkinson's disease.
Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis
Authors: Torrecilla Amparo , Marques Agostinho F. P. , Buscalioni Rafael D. , Oliveira Jorge M. A. , Teixeira Natercia A. , Atencia Eva Ana , Günther Sillero María A. , Sillero Antonio
Published: March 2003

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the Pigenerated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) theVmaxandKmvalues of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nm–µmconcentrations of ATP on the metabolism of AMP.
Dexamethasone up‐regulates type II IL‐1 receptor in mouse primary activated astrocytes
Authors: Pousset F. , Cremona S. , Dantzer R. , Kelley K. W. , Parnet P.
Published: March 2003

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL‐1 receptors including the type I IL‐1 receptor (IL‐1RI) that transduces IL‐1 signals in cooperation with the IL‐1 receptor accessory protein (IL‐1RAcP) and the type II IL‐1 receptor (IL‐1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL‐1 receptors expression. IL‐1β‐activated mouse primary astrocytes were treated with 10−6 M dexamethasone, and IL‐1 receptors were studied at the mRNA and protein levels. Using RT‐PCR, IL‐1RI and IL‐1RII but not IL‐1RAcP mRNAs were found to be up‐regulated by dexamethasone in a time‐dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL‐1RI but strongly increased IL‐1RII mRNA expression. Binding studies revealed an increase in the number of IL‐1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL‐1.
A single β subunit M2 domain residue controls the picrotoxin sensitivity of αβ heteromeric glycine receptor chloride channels
Authors: Shan Qiang , Haddrill Justine L. , Lynch Joseph W.
Published: March 2003

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the αβ heteromeric glycine receptor relative to the α homomeric receptor. By analogy with structurally related receptors, the β subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the α subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the α homomeric receptor. Furthermore, the converse F282T mutation in the β subunit increased the picrotoxin sensitivity of the αβ heteromeric receptor. The P278G mutation in the β subunit did not affect the picrotoxin sensitivity of the αβ heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to α subunit T258 is specifically required for picrotoxin sensitivity. Mutations to α subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting ‘competitive’ antagonist) binds to this residue.