bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium Regulation of Cyclic Nucleotide Signaling in Lobster Olfactory Receptor Neurons
Authors: Reich Gerhard , Boekhoff Ingrid , Breer Heinz , Ache Barry W.
Published: March 2003

Abstract: An elevated free Ca2+ concentration reduces odor‐stimulated production of cyclic AMP (cAMP) in the outer dendritic membranes of lobster olfactory receptor neurons in vitro. This effect can occur within 50 ms of odor stimulation. The effect is concentration‐dependent at submicromolar concentrations of free Ca2+. An elevated free Ca2+ concentration also reduces basal and forskolin‐stimulated cAMP levels in a concentration‐dependent manner, suggesting that Ca2+ is not targeting the activation of the odor receptor/G protein complex. The degradation of synthetic cAMP by phosphodiesterases is not enhanced by an increased free Ca2+ concentration, suggesting that Ca2+ acts by down‐regulating the olfactory adenylyl cyclase. Western blot analysis of the lobster olfactory sensilla that contain the outer dendrites reveals a protein in the transduction zone with a molecular mass of ∼ 138 kDa that is immunoreactive to an antiserum against adenylyl cyclase type III. Given earlier evidence that Ca2+ potentially enters the receptor cell through odor‐activated inositol 1,4,5‐trisphosphate‐gated channels, our results suggest a possible route for cross talk between the cyclic nucleotide and the inositol phospholipid signaling pathways in lobster olfactory receptor neurons.
Alzheimer’s Disease
Authors: Christie G. , Markwell R. E. , Gray C. W. , Smith L. , Godfrey F. , Mansfield F. , Wadsworth H. , King R. , McLaughlin M. , Cooper D. G. , Ward R. V. , Howlett D. R. , Hartmann T. , Lichtenthaler S. F. , Beyreuther K. , Underwood J. , Gribble S. K. , Cappai R. , Masters C. L. , Tamaoka A. , Gardner R. L. , Rivett A. J. , Karran E. H. , Allsop D.
Published: March 2003

Abstract: Peptide aldehyde inhibitors of the chymotrypsin‐like activity of the proteasome (CLIP) such as N‐acetyl‐Leu‐Leu‐Nle‐H (or ALLN) have been shown previously to inhibit the secretion of β‐amyloid peptide (Aβ) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on Aβ formation of a much wider range of peptide‐based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide‐based inhibitors of β‐amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress Aβ formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in Aβ formation.
Molecular Determinants of the Interaction Between the C‐Terminal Domain of Alzheimer's β‐Amyloid Peptide and Apolipoprotein E α‐Helices
Authors: Lins Laurence , Thomas‐Soumarmon Annick , Pillot Thierry , Vandekerckhove Joël , Rosseneu Maryvonne , Brasseur Robert
Published: March 2003

Abstract : In a previous work, we predicted and demonstrated that the 29‐42‐residue fragment of β‐amyloid peptide (Aβ peptide) has in vitro capacities close to those of the titled fragment of viral fusion proteins. We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of Aβ(29‐42) via a direct interaction. Therefore, we suggested that this fragment is implicated in the neurotoxicity of Aβ and in the protective effects of apolipoprotein E in Alzheimer's disease. Because structurally related apolipoproteins do not interact with the Aβ C‐terminal domain but inhibit viral fusion, we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion. In this study, we simulated interactions of all amphipathic helices of apolipoproteins E and A‐I with Aβ and simian immunodeficiency virus (SIV) fusogenic fragments by molecular modeling. We further calculated cross‐interactions that do not inhibit fusion in vitro. The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of Aβ(29‐42) and SIV peptides. Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C‐terminal amphipathic helices.
Depletion of Systemic Macrophages by Liposome‐Encapsulated Clodronate Attenuates Increases in Brain Quinolinic Acid During CNS‐Localized and Systemic Immune Activation
Authors: Koennecke Lutz A. , Zito Mark A. , Proescholdt Margit G. , Van Rooijen Nico , Heyes Melvyn P.
Published: March 2003

Abstract : Quinolinic acid is a neurotoxic tryptophan metabolite produced locally during immune activation. The present study tested the hypothesis that macrophages are an important source. In normal gerbils, the macrophage toxin liposome‐encapsulated clodronate depleted blood monocytes and decreased quinolinic acid levels in liver (85%), duodenum (33%), and spleen (51%) but not serum or brain. In a model of CNS inflammation (an intrastriatal injection of 5 μg of lipopolysaccharide), striatal quinolinic acid levels were markedly elevated on day 4 after lipopolysaccharide in conjunction with infiltration with macrophages (lectin stain). Liposome‐encapsulated clodronate given 1 day before intrastriatal lipopolysaccharide markedly reduced parenchymal macrophage invasion in response to lipopolysaccharide infusion and attenuated the increases in brain quinolinic acid (by 60%). A systemic injection of lipopolysaccharide (450 μg/kg) increased blood (by 38‐fold), lung (34‐fold), liver (23‐fold), spleen (8‐fold), and striatum (25‐fold) quinolinic acid concentrations after 1 day. Liposome‐encapsulated clodronate given 4 days before systemic lipopolysaccharide significantly attenuated the increases in quinolinic acid levels in blood (by 80%), liver (87%), spleen (80%), and striatum (68%) but had no effect on the increases in quinolinic acid levels in lung. These results are consistent with the hypothesis that macrophages are an important local source of quinolinic acid in brain and systemic tissues during immune activation.
Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein‐ and Epitope‐Fused Human M1 Muscarinic Receptors
Authors: Weill Claire , Galzi Jean‐Luc , Chasserot‐Golaz Sylvette , Goeldner Maurice , Ilien Brigitte
Published: March 2003

Abstract: Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild‐type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N‐terminal domain of the fusion proteins. Their functional properties were examined through McN‐A‐343‐evoked Ca2+release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50values) to mediate the Ca2+response, although reduced amplitudes (Emaxvalues) were obtained for the i3‐shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP‐fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP‐ and epitope‐fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.
Characterization of the cDNA Coding for Rat Brain Cysteine Sulfinate Decarboxylase
Authors: Tappaz Marcel , Bitoun Marc , Reymond Isabelle , Sergeant Alain
Published: March 2003

Abstract : Cysteine sulfinate decarboxylase (CSD) is considered as the rate‐limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT‐PCR and RACE‐PCR products of rat brain mRNAs, a 2,396‐bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, ~2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5′ untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR‐amplified products of genomic DNA suggests that the brain form results from the use of a 3′ alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT‐PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue‐specific regulation of CSD genomic expression.
Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits
Authors: Jacomy Hélène , Zhu Qinzang , Couillard‐Després Sébastien , Beaulieu Jean‐Martin , Julien Jean‐Pierre
Published: March 2003

Abstract : To clarify the role of the neurofilament (NF) medium (NF‐M) and heavy (NF‐H) subunits, we generated mice with targeted disruption of both NF‐M and NF‐H genes. The absence of the NF‐M subunit resulted in a two‐ to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF‐H subunits had little effect on the radial growth of motor axons. In NF‐M‐/‐ mice, the velocity of axonal transport of NF light (NF‐L) and NF‐H proteins was increased by about twofold, whereas the steady‐state levels of assembled NF‐L were reduced. Although the NF‐M or NF‐H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF‐M ; NF‐H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF‐L and α‐internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF‐M‐/‐ ; NF‐H‐/‐ mice revealed enhanced NF‐L staining in the perikaryon of motor neurons but a weak NF‐L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF‐L proteins in the sciatic nerve of NF‐M‐/‐ ; NF‐H‐/‐ mice. The combined results demonstrate a requirement of the high‐molecular‐weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF‐L proteins into the axonal compartment.
Fluoxetine Increases Extracellular Dopamine in the Prefrontal Cortex by a Mechanism Not Dependent on Serotonin
Authors: Pozzi Laura , Invernizzi Roberto , Garavaglia Claudio , Samanin Rosario
Published: March 2003

Abstract : Fluoxetine at 10 and 25 mg/kg increased (167 and 205%, respectively) the extracellular dopamine concentration in the prefrontal cortex, whereas 25 (but not 10) mg/kg citalopram raised (216%) dialysate dopamine. No compound modified dialysate dopamine in the nucleus accumbens. The effect of 25 mg/kg of both compounds on cortical extracellular dopamine was not significantly affected by 300 mg/kg p‐chlorophenylalanine (PCPA) (fluoxetine, saline, 235% ; PCPA, 230% ; citalopram, saline, 179% ; PCPA, 181%). PCPA depleted tissue and dialysate serotonin by ~90 and 50%, respectively, and prevented the effect of fluoxetine and citalopram on dialysate serotonin (fluoxetine, saline, 246% ; PCPA, 110% ; citalopram, saline, 155% ; PCPA, 96%). Citalopram significantly raised extracellular serotonin from 0.1 to 100 μM (251‐520%), whereas only 10 and 100 μM increased dialysate dopamine (143‐231%). Fluoxetine similarly increased extracellular serotonin (98‐336%) and dopamine (117‐318%). PCPA significnatly reduced basal serotonin and the effects of 100 μM fluoxetine (saline, 272% ; PCPA, 203%) and citalopram (saline, 345% ; PCPA 258%) on dialysate serotonin but did not modify their effect on dopamine (fluoxetine, saline, 220% ; PCPA, 202% ; citalopram, saline, 191% ; PCPA, 211%). The results clearly show that the effects of fluoxetine and of high concentrations of citalopram on extracellular dopamine do not depend on their effects on serotonin.
Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter
Authors: Malo M. , Diebler M.‐F. , De Carvalho L. Prado , Meunier F.‐M. , Dunant Y. , Bloc A. , Stinnakre J. , Tomasi M. , Tchélingérian J. , Couraud P. O. , Israël M.
Published: March 2003

Abstract : Immortalized rat brain endothelial RBE4 cells do not

express choline acetyltransferase (ChAT), but they do express an endogenous

machinery that enables them to release specifically acetylcholine (ACh) on

calcium entry when they have been passively loaded with the neurotransmitter.

Indeed, we have previously reported that these cells do not release glutamate

or GABA after loading with these transmitters. The present study was set up to

engineer stable cell lines producing ACh by transfecting them with an

expression vector construct containing the rat ChAT. ChAT transfectants

expressed a high level of ChAT activity and accumulated endogenous ACh. We

examined evoked ACh release from RBE4 cells using two parallel approaches.

First, Ca2+‐dependent ACh release induced by a calcium ionophore

was followed with a chemiluminescent procedure. We showed that

ChAT‐transfected cells released the transmitter they had synthesized and

accumulated in the presence of an esterase inhibitor. Second, ACh released on

an electrical depolarization was detected in real time by a whole‐cell

voltageclamped Xenopus myocyte in contact with the cell. Whether

cells synthesized ACh or whether they were passively loaded with ACh

electrical stimulation elicited the release of ACh quanta detected as inward

synaptic‐like currents in the myocyte. Repetitive stimulation elicited a

continuous train of responses of decreasing amplitudes, with rare failures.

Amplitude analysis showed that the currents peaked at preferential levels, as

if they were multiples of an elementary component. Furthermore, we selected an

RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce

the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.
Relationship Between Seizure‐Induced Transcription of the DNA Damage‐Inducible Gene GADD45, DNA Fragmentation, and Neuronal Death in Focally Evoked Limbic Epilepsy
Authors: Henshall David , Sinclair Jennifer , Simon Roger
Published: March 2003

Abstract : We investigated the temporal and spatial profile of mRNA transcription for the growth arrest and DNA damage‐inducible gene GADD45, DNA fragmentation, and neuronal death in rat brain following focally evoked limbic seizures. GADD45 mRNA was detected by in situ hybridization, whereas fragmented DNA was detected using in situ nick end‐labeling by the large (Klenow) fragment of DNA polymerase l. Kainic acid (0.1 μg) was injected into the right amygdala of rats to induce seizures for 45 min, after which diazepam (30 mg/kg) was administered. GADD45 mRNA, DNA fragmentation, and cell death were quantified bilaterally within six limbic brain regions 0‐96 h following seizure cessation. All animals underwent seizures of equivalent severity and duration as determined electrographically. In situ hybridization detected bilateral up‐regulation of GADD45 mRNA throughout the CA1, CA3, and dentate gyrus of the hippocampus, the piriform and retrosplenial cortices, and the thalamus within 1 h of seizure termination. GADD45 mRNA levels remained elevated for up to 6 h, declining to baseline within all structures by 16 h. Klenow‐positive cells were only found within the CA3 pyramidal layer of the ipsilateral hippocampus and appeared 16‐72 h following seizure cessation. Morphologic cell death was also restricted to the CA3 subfield. These data demonstrate that focally evoked limbic seizures trigger early bihemispheric GADD45 mRNA transcription within connected limbic structures, whereas subsequent DNA fragmentation and cell death are restricted to selectively vulnerable brain regions.
D1 Dopamine Receptor‐Induced Cyclic AMP‐Dependent Protein Kinase Phosphorylation and Potentiation of Striatal Glutamate Receptors
Authors: Price Christopher J. , Kim Peter , Raymond Lynn A.
Published: March 2003

Abstract : Dopamine receptor activation regulates cyclic AMP levels

and is critically involved in modulating neurotransmission in the striatum.

Others have shown that α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate

(AMPA)‐type glutamate receptor‐mediated current is potentiated by cyclic

AMP‐dependent protein kinase (PKA) activation. We made whole‐cell patch clamp

recordings from cultured striatal neurons and tested whether D1‐type dopamine

receptor activation affected AMPA receptor‐mediated currents. After a 5‐min

exposure to the D1 agonist SKF 81297 (1 μM), kainate‐evoked

current amplitude was enhanced in ~75% of cells to 121 ± 2.5% of that

recorded prior to addition of drug. This response was inhibited by the D1

antagonist SCH 23390 and mimicked by activators of PKA. Moreover, by western

blot analysis using an antibody specific for the phosphorylated PKA site

Ser845 of GluR1, we observed a marked increase in phosphorylated

GluR1 following a 10‐min exposure of striatal neurons to 1 μM SKF 81297. Our data demonstrate that activation of D1‐type dopamine receptors on striatal neurons promotes phosphorylation of AMPA receptors by PKA as well as potentiation of current amplitude. These results elucidate one mechanism by which dopamine can modulate neurotransmission in the striatum.
Immunocytochemical Localization and Biological Activity of Hydroxysteroid Sulfotransferase in the Frog Brain
Authors: Beaujean Delphine , Mensah‐Nyagan Ayikoe G. , Do‐Rego Jean‐Luc , Luu‐The Van , Pelletier Georges , Vaudry Hubert
Published: March 2003

Abstract : Biosynthesis of the neuroactive steroids pregnenolone
sulfate (▵5PS) and dehydroepiandrosterone sulfate (DHEAS) is
catalyzed by the enzyme hydroxysteroid sulfotransferase (HST), which transfers
the sulfonate moiety from 3′‐phosphoadenosine 5′ ‐phosphosulfate
(PAPS) on thye 3‐hydroxy site of steroids. Although high concentrations of
▵5PS and DHEAS have been detected in the rat brain, the
anatomical localization of HST in the CNS has never been determined. Using an
antiserum against rat liver HST, we have investigated the distribution of
HST‐like Immunoreactivity in the CNS of the frog Rana ridibunda. Two
populations of HST‐immunoreactive neurons were observed in the hypothalamus,
and several bundless of positive nerves fibers were visualized in the
telencephalon and diencephalon. lncubation of frog brain homogenates with
[35S]PAPS and [3H] pregnenolone yielded the formation of
several 3H, 35S‐labeled compounds, including
▵5PS and testosterone sulfate. When [3]
dehydroepiandrosterone and [35S]PAPS were used as precursors, one
of the 3H, 35S‐labeled metabolities coeluted with DHEAS.
Neosynthesis of [3H]▵5PS and [3H]DHEAS
was reduced significantly by 2,4‐dichloro‐6‐nitrophenol, a specific inhibitor
of sulfotransferases. The present study provides the first immunocytochemical
mapping of HSt in the brain. Our data also demonstrate for the first time that
biopsynthesis of the highly poten neuroactive steroids ▵5PS and DHEAS occurs in the CNS of nonmammalian vertebrates.
Group I Metabotropic Glutamate Receptors Mediate Phospholipase D Stimulation in Rat Cultured Astrocytes
Authors: Servitja Joan‐Marc , Masgrau Roser , Sarri Elisabet , Picatoste Fernando
Published: March 2003

Abstract: We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD‐catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi‐prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR‐mediated phosphoinositide phospholipase activation [quisqualate > (S)‐3,5‐dihydroxyphenylglycine > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid]. The response to (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid was inhibited by the mGluR antagonist (S‐α‐methyl‐4‐carboxyphenylglycine and, less potently, by 1‐aminoindan‐1,5‐dicarboxylic acid and 4‐carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5‐selective agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.
Increase in the Number, G Protein Coupling, and Efficiency of Facilitatory Adenosine A2A Receptors in the Limbic Cortex, but not Striatum, of Aged Rats
Authors: Lopes Luísa V. , Cunha Rodrigo A. , Ribeiro J. A.
Published: March 2003

Abstract : Adenosine's effects result from a balanced activation of

inhibitory A1 and facilitatory A2A receptors. Because in

aged animals there is an increased number of A2A receptors, we now

compared the efficiency of A2A receptors in cortical and striatal

preparations of young adult (6‐week‐old) and aged (2‐year‐old) rats. In

cortical, in contrast to striatal, membranes from aged rats, A2A

receptors were more tightly coupled to G proteins, because

5′‐guanylylimidodiphosphate (100 μM) increased by 321% the

Ki of the A2A agonist CGS21680 as a displacer

of binding of the A2A antagonist [3H]ZM241385 (1

nM), compared with a 112% increase in young rats. In cortical slices,

CGS21680 (30‐1,000 nM) was virtually devoid of effect on cyclic AMP

accumulation in young rats but increased cyclic AMP accumulation with an

EC50 of 153 nM in aged rats, whereas the efficiency of

CGS21680 was similar in striatal slices of young and aged rats. CGS21680 (30

nM) was virtually devoid of effect on acetylcholine release from

hippocampal CA1 slices of young rats but caused a 55% facilitation in aged

rats. These results show that the number of A2A receptors, their coupling to G proteins, and their efficiency are enhanced in the limbic cortex of aged rats, suggesting a greater involvement of facilitation in adenosine responses.
Altered Interaction Between PSD‐95 and the NMDA Receptor Following Transient Global Ischemia
Authors: Takagi Norio , Logan Richard , Teves Lucy , Wallace M. Christopher , Gurd James W.
Published: March 2003

Abstract: The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD‐95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD‐95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD‐95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD‐95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD‐95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham‐operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD‐95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD‐95 and the NMDA receptor are modified by an ischemic challenge.
Phosphorylation of HMG‐I by Protein Kinase C Attenuates Its Binding Affinity to the Promoter Regions of Protein Kinase C γ and Neurogranin/RC3 Genes
Authors: Xiao Dian‐Mo , Pak Jhang Ho , Wang Xiaoming , Sato Takayuki , Huang Freesia L. , Chen Hao‐Chia , Huang Kuo‐Ping
Published: March 2003

Abstract: A 20‐kDa DNA‐binding protein that binds the AT‐rich sequences within the promoters of the brain‐specific protein kinase C (PKC) γ and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high‐mobility‐group protein (HMG)‐I. This protein is a substrate of PKC α, β, γ, and δ but is poorly phosphorylated by PKC ε and ζ. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG‐I by PKC resulted in a reduction of DNA‐binding affinity by 28‐fold as compared with 12‐fold caused by the phosphorylation with cdc2 kinase. HMG‐I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a > 100‐fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG‐I are adjacent to the N terminus of two of the three predicted DNA‐binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA‐binding domain, whereas Ser44 is located within the spanning region between the first and second DNA‐binding domains. The current results suggest that phosphorylation of the mammalian HMG‐I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG‐I function.
Enhancement of Dopaminergic Neurotoxicity by the Mercapturate of Dopamine
Authors: Zhang Jing , Kravtsov Vladimir , Amarnath Venkataraman , Picklo Matthew J. , Graham Doyle G. , Montine Thomas J.
Published: March 2003

Abstract: The mechanisms that underlie dopaminergic
neurodegeneration in Parkinson's disease (PD) are not known but have been
proposed to involve oxidation of dopamine and related catechols. In other
organ systems, cytotoxicity from catechol oxidation is profoundly influenced
by mercapturate metabolism. Here we have tested the hypothesis that catechol
thioethers produced in the mercapturic acid pathway may act as dopaminergic
neurotoxins. A rat mesencephalic/neuroblastoma hybrid (MES) cell line was
exposed to dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), or eight
different catechol thioethers for up to 24 h, and the extent of apoptosis was
quantified by a microculture kinetic assay. Apoptosis also was confirmed
morphologically with Giemsa‐stained cultures and by demonstration of
internucleosomal DNA fragmentation. The results showed that dopamine at 5‐50
μM produced concentration‐dependent increases in the percentage of
apoptotic MES cells. At 25 and 50 μM dopamine, the maximal
proportions of apoptotic cells were detected at ∼ 19 (20.7 ± 2.0%)
and 14 h (30.3 ± 3.5%), respectively. None of the catechol thioethers
(up to 5 μM) alone induced significant apoptosis in MES cells.
However, when MES cells were incubated with dopamine (25 μM) and
catechol thioethers (5 μM) to mimic pathological conditions,
5‐S‐N‐acetylcysteinyldopamine,
5‐S‐homocysteinyldopamine, and 5‐S‐homocysteinyl‐DOPAC significantly increased the percentage of apoptotic cells compared with dopamine alone. These results suggest that mercapturate metabolism of endogenous catechols may yield products that facilitate dopaminergic neurodegeneration.
Fatty Acid‐Activated K+ Channels in Autonomic Neurons
Authors: Casavant Reema H. , Xu Zhaohui , Dryer Stuart E.
Published: March 2003

Abstract: Application of arachidonic acid evoked robust activation
of large‐conductance K+ channels in cell‐attached and excised
inside‐out patches from acutely isolated chick ciliary ganglion neurons. A
similar effect was produced by 5,8,11,14‐eicosatetraynoic acid, a
nonmetabolizable analogue of arachidonic acid. The unitary conductance of
fatty acid‐activated channels was 35‐40 pS at +20 mV with physiological
gradients of K+ and 165 pS at +20 mV with an extracellular
K+ concentration of 37.5 mM and an intracellular
K+ concentration of 150 mM. Gating of these channels in
cell‐attached patches was potentiated by membrane stretch. Channel gating
evoked by both lipids was concentration‐dependent, with detectable activation
apparent at 4 μM in the majority of patches and maximal activation
occurring between 32 and 64 μM. Gating was relatively
voltage‐independent. Large‐conductance K+ channels were also
activated in inside‐out patches by the monounsaturated fatty acid
11‐cis‐eicosenoic acid but not by the fully saturated fatty acid
arachidic acid. Application of 100 μM H2O2,
an agent that activates cytosolic phospholipase A2, also caused
activation of large‐conductance K+ channels in intact neurons. The
stimulatory effects of H2O2 were blocked by pretreatment
with 20 μM 4‐bromophenacyl bromide, an irreversible inhibitor of
phospholipase A2. Therefore, mobilization of endogenous fatty acids
can cause activation of large‐conductance K+ channels in autonomic neurons.
Regulation of Adenylyl Cyclase, ERK1/2, and CREB by Gz Following Acute and Chronic Activation of the δ‐Opioid Receptor
Authors: Tso Prudence H. , Yung Lisa Y. , Wong Yung H.
Published: March 2003

Abstract: Opioid tolerance and physical dependence in mammals can be
rapidly induced by chronic exposure to opioid agonists. Recently, opioid
receptors have been shown to interact with the pertussis toxin
(PTX)‐insensitive Gz (a member of the Gi subfamily),
which inhibits adenylyl cyclase and stimulates mitogen‐activated protein
kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell
lines expressing δ‐opioid receptors with or without Gz to
examine the role of Gz in opioid receptor‐regulated signaling
systems. Each cell line was acutely or chronically treated with
[D‐Pen2,D‐Pen5]enkephalin (DPDPE), a δ‐selective
agonist, in the absence or presence of PTX. Subsequently, the activities of
adenylyl cyclase, cyclic AMP (cAMP)‐dependent response element‐binding
proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and
phosphorylation of CREBs and the extracellular signal‐regulated protein
kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited
forskolin‐stimulated cAMP accumulation in a PTX‐insensitive manner, but
Gz could not replace Gi to mediate adenylyl cyclase
supersensitization upon chronic opioid treatment. DPDPE‐induced adenylyl
cyclase supersensitization was not associated with an increase in the
phosphorylation of CREBs. Both Gi and Gz mediated
DPDPE‐induced activation of ERK1/2, but these responses were abolished by
chronic opioid treatment. Collectively, our results show that although
Gz mediated opioid‐induced inhibition of adenylyl cyclase and
activation of ERK1/2, Gz alone was insufficient to mediate opioid‐induced adenylyl cyclase supersensitization.
Ligand Recognition of Serine—Cysteine Amino Acid Exchanges in Transmembrane Domain 5 of α2‐Adrenergic Receptors by UK 14,304
Authors: Cockcroft V. , Frang H. , Pihlavisto M. , Marjamäki A. , Scheinin M.
Published: March 2003

Abstract: Ligand binding of UK 14,304 reveals notable species (i.e.,
human—rodent) and receptor‐subtype differences of
α2‐adrenergic receptors (α2‐ARs). To study
the molecular basis of the selectivity of UK 14,304, we compared a series of
conservative serine—cysteine exchange mutants at ligand‐accessible
positions in transmembrane domain 5 of the human and mouse
α2A‐ARs. UK 14,304 bound with ∼200‐fold higher affinity
to the human α2A‐AR wild‐type receptor compared with the
human α2A‐ARSer201 mutant, but only an
approximately fivefold difference was seen with the corresponding mouse
α2A‐AR variant. These effects of cysteine—serine
exchanges only involved the agonist low‐affinity forms of the receptors, as
the affinity of [3H]UK 14,304 for the agonist high‐affinity
receptor populations was not influenced. The apparent affinities of a set of
eight structurally diverse α2‐AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine—serine exchanges (except for oxymetazoline and yohimbine, with up to nine‐ and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.
Nerve Growth Factor‐Induced Phosphorylation of SNAP‐25 in PC12 Cells
Authors: Kataoka Masakazu , Kuwahara Reiko , Iwasaki Satoshi , Shoji‐Kasai Yoko , Takahashi Masami
Published: March 2003

Abstract: Synaptosomal‐associated protein of 25 kDa (SNAP‐25), a t‐SNARE protein essential for neurotransmitter release, is phosphorylated at Ser187 following activation of cellular protein kinase C by treatment with phorbol 12‐myristate 13‐acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP‐25. Here we studied the phosphorylation of SNAP‐25 in PC12 cells using a specific antibody for SNAP‐25 phosphorylated at Ser187. A small fraction of SNAP‐25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen‐activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP‐25; however, phosphorylation was up‐regulated after a prolonged incubation with NGF. Up‐regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 h after the addition of NGF. Immunofluorescent microscopy showed that SNAP‐25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP‐25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser187 to Ala abolished the NGF‐dependent relocalization. A population of SNAP‐25 in the plasma membrane was not increased by a point mutation at Ser187 to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP‐25 phosphorylation is essential, but not sufficient, for the NGF‐induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up‐regulation of SNAP‐25 phosphorylation and its relocation, and NGF‐induced differentiation of PC12 cells.
Involvement of Phosphatidylcholine‐Specific Phospholipase C in Thromboxane A2‐Induced Activation of Mitogen‐Activated Protein Kinase in Astrocytoma Cells
Authors: Kobayashi Hiroshi , Honma Shigeyoshi , Nakahata Norimichi , Ohizumi Yasushi
Published: March 2003

Abstract: Thromboxane A2 (TXA2) receptor‐mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11‐Epithio‐11,12‐methano‐TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration‐dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol‐specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3′,5′‐cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor‐mediated phosphorylation of mitogen‐activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor‐mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D‐609, an inhibitor of phosphatidylcholine‐specific phospholipase C, potently attenuated the activation in a concentration‐dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D‐609. These results suggest that phosphatidylcholine‐specific phospholipase C and protein kinase C, but not phosphatidylinositol‐specific phospholipase C, are involved in TXA2 receptor‐mediated MAPK activation in 1321N1 human astrocytoma cells.
Kainic Acid‐Induced Neurotrophic Activities in Developing Cortical Neurons
Authors: Lee Yi‐Hsuan , Fang Kwang‐Ming , Yang Chuen‐Mao , Hwang Hwa‐Min , Chiu Chi‐Tso , Tsai Wuhong
Published: March 2003

Abstract: Using primary cultured cortical neurons from embryonic rat brains, we elucidated an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/kainic acid (KA) receptor‐mediated neuroprotective mechanism through actions of nerve growth factor (NGF) in developing neurons. Neurotoxicity of KA in early days in vitro neurons was quite low compared with the mature neurons. However, pretreatment with anti‐NGF antibody or TrkA inhibitor AG‐879 profoundly raised KA toxicity. Furthermore, KA stimulation resulted in an increase of TrkA expression and phosphorylation, which was blocked not only by the AMPA/KA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and AG‐879, but also by the phospholipase C inhibitor U73122 and the intracellular calcium chelator BAPTA. A study of polyphosphoinositide turnover showed that KA‐stimulated phospholipase C (PLC) activity was directly triggered by the AMPA/KA receptor activity, but not by the activity of TrkA or other excitatory amino acid receptor subtypes. Sources of KA‐increased intracellular calcium levels were contributed by both extracellular calcium influx and intracellular calcium release and were partially sensitive to guanosine 5′‐O‐(2‐thiodiphosphate). These results indicate that in developing cortical neurons, activation of AMPA/KA receptors by KA may induce expression, followed by activation of TrkA via PLC signaling and intracellular calcium elevation and hence increase reception of NGF on KA‐challenged neurons. A G protein‐coupled AMPA/KA receptor may be involved in these metabotropic events for neuronal protection.
Regulation of Tyrosine Hydroxylase Activity and Phosphorylation at Ser19 and Ser40 via Activation of Glutamate NMDA Receptors in Rat Striatum
Authors: Lindgren Niklas , Xu Zhi‐Qing David , Lindskog Maria , Herrera‐Marschitz Mario , Goiny Michel , Haycock John , Goldstein Menek , Hökfelt Tomas , Fisone Gilberto
Published: March 2003

Abstract: The activity of tyrosine hydroxylase, the rate‐limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time‐and concentration‐dependent increase in the levels of phospho‐Ser19‐tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser40 and caused a significant increase in DOPA accumulation. NMDA reduced forskolin‐mediated increases in both Ser40 phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho‐Ser40‐tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8‐bromo‐cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser40 via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.
Taurine Blocks Spontaneous Cone Contraction but Not Horizontal Cell Dark Suppression in Isolated Goldfish Retina
Authors: Baldridge William H. , McLure Paul , Pow David V.
Published: March 2003

Abstract:The objective of this study was to investigate the effects of taurine on cone retinomotor movements and the responses of cone‐driven horizontal cells in dark‐adapted teleost retina. In isolated goldfish retina preparations maintained in the dark, cones spontaneously contracted, and the responses of horizontal cells were suppressed. Addition of 5 mMtaurine to the physiological solution blocked the spontaneous contraction of cones in the dark but did not block the dark‐suppression of horizontal cell responses. These results indicate that the mechanism that leads to horizontal cell dark suppression is not sensitive to taurine. Although both cone retinomotor position and horizontal cell responsiveness are known to be modulated by dopamine, the present results do not support the hypothesis that taurine inhibits dopamine release in the dark because only spontaneous cone contraction was affected by taurine. These results also indicate that spontaneous cone contraction in the dark is not the cause of horizontal cell dark suppression because, in the presence of taurine, cones were elongated yet horizontal cell responses were still suppressed. Consequently, these results make it clear that horizontal cell dark suppression is not an artifact produced by incubating isolated teleost retina preparations in taurine‐free physiological solution.
A2‐Pancortins (Pancortin‐3 and ‐4) Are the Dominant Pancortins During Neocortical Development
Authors: Nagano Takashi , Nakamura Akira , Konno Daijiro , Kurata Morito , Yagi Hideshi , Sato Makoto
Published: March 2003

We have identified a novel mouse gene named pancortin that is expressed dominantly in the mature cerebral cortex. This gene produces four different species of proteins, Pancortin‐1‐4, sharing a common region in the middle of their structure with two variations at the N‐terminal (A1 or A2 part) and C‐terminal (C1 or C2 part) sides, respectively. In the present study, we showed that expression of mRNAs for A2‐Pancortins (Pancortin species that contain the A2 part, i.e., Pancortin‐3 and ‐4) is more dominant than that of mRNAs for A1‐Pancortins (Pancortin species that contain the A1 part, i.e., Pancortin‐1 and ‐2) in the prenatal mouse cerebral neocortex. Using western blot analysis, we found that substantial amounts of both A2‐Pancortins were present in the prenatal cerebral neocortex and P19 cells after inducing neuronal differentiation. A2‐Pancortins were still present in the cerebral neocortex of the adult, although their mRNAs were hardly detected. In contrast, the amount of A1‐Pancortins did not increase after the third postnatal week in spite of their intense gene expression. Furthermore, we showed that recombinant Pancortin‐3, one of the A2‐Pancortins, was a secreted protein, in contrast to Pancortin‐1 (one of the A1‐Pancortins). These results suggest that A2‐Pancortins are extracellular proteins essential for neuronal differentiation and that their molecular behavior is distinct from that of A1‐Pancortins.
Alternative Splicing of the Neurotrophin‐3 Gene Gives Rise to Different Transcripts in a Number of Human and Rat Tissues
Authors: Kendall Sharon , Yeo Margaret , Henttu Pirkko , Tomlinson David R.
Published: March 2003

Abstract : The mouse neurotrophin‐3 (NT‐3) gene has been shown to contain two exons (exon 1A and exon 1B) upstream of the single coding exon (exon 2). These upstream exons are alternatively spliced to the coding exon, generating two different NT‐3 transcripts. We investigated whether alternative splicing of two upstream exons also occurs in the human and rat NT‐3 gene. It was found that the human and rat NT‐3 gene also contains two exons upstream of the main coding exon and that alternative splicing of these upstream exons generates two different NT‐3 transcripts : transcript 1A and transcript 1B (TR1B). These two transcripts were widely expressed in several human and rat tissues. Also, a third transcript, transcript 1A1C, derived from splicing of exon 1A to exon 1B before splicing to the coding exon was seen in a small number of rat tissues. Previous quantification of neurotrophin‐3 mRNA has not been transcript‐specific. Here we describe a transcript‐specific semiquantitative RT‐PCR method allowing the quantification of TR1B in human tissues. We show that this is the major NT‐3 transcript and that expression of this transcript was much higher in the adult when compared with the corresponding fetal tissues.