bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+/Calmodulin‐Mediated Regulation of the Desensitizing Process in Gq Protein‐Coupled Histamine H1 Receptor‐Mediated Ca2+ Responses in Human U373 MG Astrocytoma Cells
Authors: Hishinuma Shigeru , Ogura Kazumi
Published: March 2003

We investigated Ca2+/calmodulin (CaM)‐mediated regulation of the

desensitizing process of the histamine H1 receptor‐mediated

increase in intracellular Ca2+ concentration in human U373 MG

astrocytoma cells. The desensitizing process was evaluated by measuring the

histamine‐induced Ca2+ responses in cells pretreated with histamine

for 15 s‐30 min under various conditions. Under normal physiological

conditions, desensitization developed with three successive phases : a fast

desensitization within 15 s, a transient resensitization at 45 s, and a prompt

and sustained redesensitization from 1 to 30 min. Similar processes of

desensitization/resensitization occurred even under hypertonic conditions,

where histamine‐mediated internalization of the histamine H1

receptor is inhibited. The transient resensitization phase was selectively

prevented by deprivation of extracellular Ca2+ and, even more

strikingly, by the presence of W‐7 (a CaM antagonist). FK506 and cyclosporin

A, Ca2+/CaM‐dependent protein phosphatase (PP2B) inhibitors,

mimicked such effects. In the presence of KN‐62, a

Ca2+/CaM‐dependent protein kinase II (CaM kinase II) inhibitor, the

early development of desensitization disappeared, allowing a slow and simple

development of desensitization. The early processes of desensitization and

resensitization were unaffected by W‐5, okadaic acid, and KN‐04 (less potent

inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X

and chelerythrine (protein kinase C inhibitors). The high‐affinity site for

histamine was converted to a lower‐affinity site by histamine treatment, which

also showed a transient restoration phase at 45 s in a manner sensitive to

KN‐62 and FK506. These results provide the first evidence that

Ca2+/CaM plays a crucial role in determining the early phase of the

desensitizing process via activation of CaM kinase II and PP2B, by regulating

agonist affinity for histamine H1 receptors.
Increased Exon 5 Expression Alters Extrasynaptic NMDA Receptors in Cerebellar Neurons
Authors: Prybylowski Kate , Rumbaugh Gavin , Wolfe Barry B. , Vicini Stefano
Published: March 2003

Abstract: We investigated the ontogenic changes in expression of alternatively spliced forms of NR1 protein that contain the N1 cassette (exon 5) in comparison with the total population of NR1 within the rat cerebellum. The N1 cassette is strongly developmentally regulated in the cerebellum, with >80% of total NR1 protein in the adult rat containing the N1 cassette. In contrast, early in development, <20% of NR1 protein contained this cassette. Rat cortices from the same ages did not show an increase in the percent of NR1 protein expressing the N1 cassette, indicating that the developmental changes in the cerebellum are tissue‐specific. As the N1 cassette is known to determine NMDA receptor properties, including spermine sensitivity and decay kinetics of glutamate‐induced currents, changes in the characteristics of glutamate‐activated currents in granule cells from cerebellar slices were compared at postnatal day 7 versus 14. Glutamate responses exhibited fast deactivation kinetics and reduced potentiation by spermine at day 14 while maintaining sensitivity to an NR2B‐selective antagonist. These data are consistent with the possibility that N1 cassette expression may be a factor in the developmental changes in properties of extrasynaptic NMDA receptors in the cerebellum.
Nitric Oxide Can Differentially Modulate Striatal Neurotransmitter Concentrations via Soluble Guanylate Cyclase and Peroxynitrite Formation
Authors: Trabace L. , Kendrick K. M.
Published: March 2003

Abstract:
in vivo microdialysis was used to investigate whether
nitric oxide (NO) modulates striatal neurotransmitter release in the rat
through inducing cyclic GMP formation via soluble guanylate cyclase or
formation of peroxynitrite (ONOO‐). When NO donors,
S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP; 1 mM) or
(Z)‐1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate
(NOC‐18; 1 mM), were retrodialysed for 15 min, acetylcholine (ACh),
serotonin (5‐HT), glutamate (Glu), γ‐aminobutyric acid (GABA), and
taurine levels were significantly increased, whereas those of dopamine (DA),
dihydroxyphenylacetic acid (DOPAC), and 5‐hydroxyindoleacetic acid (5‐HIAA)
were decreased. Only effects on ACh, 5‐HT, and GABA showed calcium dependency.
Inhibition of soluble guanylate cyclase by
1H‐[1,2,4]oxadiazolo[4,3‐α]quinoxalin‐1‐one (ODQ; 100 and 200
μM) dose‐dependently reduced NO donor‐evoked increases in ACh,
5‐HT, Glu, and GABA levels. Coperfusion of SNAP or NOC‐18 with an
ONOO‐ scavenger, L‐cysteine (10 mM) resulted in enhanced
concentrations of Glu and GABA. On the other hand, DA concentrations increased
rather than decreased, and no reductions in DOPAC and 5‐HIAA occurred. This
increase in DA and the potentiation of Glu and GABA were calcium‐dependent and
prevented by ODQ. Similar to NO, infusions of ONOO‐ (10 or 100
μM) decreased DA, DOPAC, and 5‐HIAA. Overall, these results
demonstrate that NO increases ACh, 5‐HT, Glu, and GABA levels primarily
through a cyclic GMP‐dependent mechanism. For DA, DOPAC, and 5‐HIAA, effects
are determined by levels of ONOO‐ stimulated by NO donors. When these are high, they effectively reduce extracellular concentrations through oxidation. When they are low, DA concentrations are increased in a cyclic GMP‐dependent manner and may act to facilitate Glu and GABA release further. Thus, changes in brain levels of antioxidants, and the altered ability of NO to stimulate cyclic GMP formation during ageing, or neurodegenerative pathologies, may particularly impact on the functional consequences of NO on striatal dopaminergic and glutamatergic function.
Quantification of Ethanol Concentrations in the Extracellular Fluid of the Rat Brain
Authors: Robinson Donita L. , Lara Jennifer A. , Brunner Lane J. , Gonzales Rueben A.
Published: March 2003

Abstract: Traditional microdialysis techniques provide qualitative
data, although quantitative data are often required for pharmacodynamic
analyses. This study evaluated a potentially usefulin vivo delivery technique
to calibrate microdialysis probes for ethanol. We measuredin vivo delivery
extraction fractions within subjects across 2 days and found no change over
time. We tested the effect of diffusion direction on extraction fraction and
found that it was higher for ethanol diffusion out of the probe than for
diffusion into the probe, bothin vitro andin vivo. Thein vivo extraction
fraction ratio of diffusionIN versus diffusionOUT was
0.65 ± 0.03. Finally, we predicted extracellular brain ethanol
concentrations after 1 g/kg ethanol administration usingin vivo delivery,
“no net flux” dialysis, orin vivo delivery corrected for
diffusion direction with thein vivo extraction fraction ratio. Bothin vivo
delivery and “no net flux” dialysis predicted brain concentrations
that were approximately one‐third lower than blood concentrations, whereas the
correctedin vivo delivery predicted extracellular concentrations very similar to blood concentrations. We conclude that microdialysis calibration methods for ethanol require a measure of extraction fraction for diffusion into the probe. Further studies are needed to establish whether this effect is common to other alcohols.
Induction of Neuronal Apoptosis by Thiol Oxidation
Authors: Aizenman Elias , Stout Amy K. , Hartnett Karen A. , Dineley Kirk E. , McLaughlin BethAnn , Reynolds Ian J.
Published: March 2003

Abstract: The membrane‐permeant oxidizing agent
2,2′‐dithiodipyridine (DTDP) can induce Zn2+ release from
metalloproteins in cell‐free systems. Here, we report that brief exposure to
DTDP triggers apoptotic cell death in cultured neurons, detected by the
presence of both DNA laddering and asymmetric chromatin formation. Neuronal
death was blocked by increased extracellular potassium levels, by
tetraethylammonium, and by the broad‐spectrum cysteine protease inhibitor
butoxy‐carbonyl‐aspartate‐fluoromethylketone.
N,N,N′,N′‐Tetrakis‐(2‐pyridylmethyl)ethylenediamine
(TPEN) and other cell‐permeant metal chelators also effectively blocked
DTDP‐induced toxicity in neurons. Cell death, however, was not abolished by
the NMDA receptor blocker MK‐801, by the intracellular calcium release
antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated
increases in fluorescence signals in cultured neurons loaded with the
zinc‐selective dye Newport Green. The fluorescence signals following DTDP
treatment also increased in fura‐2‐ and magfura‐2‐loaded neurons. These
responses were completely reversed by TPEN, consistent with a DTDP‐mediated
increase in intracellular free Zn2+ concentrations. Our studies
suggest that under conditions of oxidative stress, Zn2+ released from intracellular stores may contribute to the initiation of neuronal apoptosis.
Signaling Within a Caveolae‐Like Membrane Microdomain in Human Neuroblastoma Cells in Response to Fibroblast Growth Factor
Authors: Davy Alice , Feuerstein Claude , Robbins Stephen M.
Published: March 2003

Abstract: It is now clear that the plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these microcompartments are now recognized to be sites of localized signal transduction for several extracellular stimuli. At least two different types of microdomains can be identified, largely based on the presence or absence of the caveolin proteins. The generic name of caveolae‐like domains is commonly used to refer to both domains indistinguishably. Although caveolin proteins were long thought to be absent from the brain, we have shown that the human neuroblastoma cell line LAN‐1 expresses both caveolin‐1 and caveolin‐2. Basic fibroblast growth factor (FGF)‐2 induced a specific signaling response within the caveolae‐like domain of LAN‐1 cells, characterized by the tyrosine phosphorylation of a 75‐80‐kDa protein. This protein present in the caveolae‐like domains has properties suggesting that it is a member of the SNT family of adapter proteins. The signaling event originating in the caveolae‐like domains in response to FGF‐2 appeared to require the activation of at least Fyn and Lyn, two members of the Src family of tyrosine kinases. This work suggests that compartmentalized signaling within caveolae‐like domains may create a level of specificity for certain growth factors such as FGF.
Survival activity of troglitazone in rat motoneurones
Authors: Nishijima Chiaki , Kimoto Kosuke , Arakawa Yoshihiro
Published: March 2003

Troglitazone (TGZ), an antidiabetic drug that improves insulin‐resistance in the peripheral tissues, was tested for neurotrophic activity in motoneurones and other neurones in culture. In rat motoneurones, TGZ had a remarkable effect on survival, which was comparable or superior to that of brain‐derived neurotrophic factor, a known potent neurotrophic factor for rat motoneurones. However, TGZ did not promote the survival of sensory, sympathetic, septal or hippocampal neurones. The effect of TGZ on motoneurones was additive to that of insulin‐like growth factor‐I and both activities were inhibited by phosphatidylinositol 3‐kinase (PI3‐kinase) inhibitors, wortmannin and LY294002, suggesting the involvement of the activation of PI3‐kinase in the activity of TGZ. Pioglitazone, another antidiabetic drug structurally similar to TGZ, did not show any activity, indicating that the agonistic activity of TGZ for peroxisome proliferator‐activated receptor‐γ is not involved in the survival activity. Chromanol, an antioxidant moiety of TGZ, showed little or no survival activity. These results indicate specific neurotrophic activity of TGZ for motoneurones through the activation of PI3‐kinase and support the applicability of TGZ for the treatment of motor neurone diseases such as amyotrophic lateral sclerosis.
Characterization of the blood–brain barrier choline transporter using the in situ rat brain perfusion technique
Authors: Allen David D. , Smith Quentin R.
Published: March 2003

Choline enters brain by saturable transport at the blood–brain barrier (BBB). In separate studies, both sodium‐dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re‐examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (Vmax= 2.4–3.1 nmol/min/g; Km = 39–42 µm) with an apparent affinity (1/Km) for choline five to ten‐fold greater than previously reported in vivo, but less than neuronal ‘high‐affinity’ brain choline transport (Km = 1–5 µm). BBB choline uptake from a sodium‐free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium‐3 inhibited brain choline uptake with a Ki (57 ± 11 µm) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The Vmax of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.
Neurotoxic Aβ peptides increase oxidative stress in vivo through NMDA‐receptor and nitric‐oxide‐synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro
Authors: Parks Janice K. , Smith Trisha S. , Trimmer Patricia A. , Bennett James P. , Parker W. Davis
Published: March 2003

Beta amyloid (Aβ) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Aβ to increase oxygen radical production in vivo. The 1–40 Aβ fragment increased 2,3‐dehydroxybenzoic acid efflux more than the 1–28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Aβ peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Aβ(25–35) and Aβ(35–25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N‐ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Aβ aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Aβ(25–35) but not by Aβ(35–25). Neurotoxic Aβ peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Aβ levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed‐forward hypothesis of Aβ neurotoxicity.
Possible involvement of Fyn kinase in ethanol‐stimulated Cas tyrosine phosphorylation in rat cerebellum and cerebral cortex
Authors: Nishio Hajime , Matsui Kiyoshi , Tsuji Hiroko , Tamura Akiyoshi ,   Koichi Suzuki
Published: March 2003

In the present study, we have investigated the effect of intraperitoneal injection of ethanol (3.5 g/kg) on tyrosine phosphorylation in rat brain. Immunoblot analysis using an antiphosphotyrosine antibody revealed that a 130‐kDa protein band was detected in the brain extract in response to ethanol administration. This ethanol‐stimulated tyrosine phosphorylation of the 130‐kDa protein was found in the brain but not in the heart, liver or thymus. The 130‐kDa phosphotyrosine‐containing protein was identified by immunoprecipitation to be Cas, a crk‐associated src substrate. This ethanol‐stimulated tyrosine phosphorylation of Cas was observed most prominently in the cerebellum and the cerebral cortex. We further examined the possible involvement of Fyn kinase in ethanol‐stimulated Cas tyrosine phosphorylation. Immunecomplex kinase assay showed that Fyn was activated in the cerebellum and cerebral cortex of ethanol‐administered rats. Immunoprecipitation experiments also showed that Fyn was co‐immunoprecipitated with an anti‐Cas antibody in these regions from ethanol‐administered rats. Furthermore, exogenous Fyn was shown to phosphorylate Cas from cerebellum and cerebral cortex in vitro. These findings indicate that ethanol stimulates tyrosine phosphorylation of Cas in rat cerebellum and cerebral cortex, and that Fyn may be involved in the process.
Pitx3 activates mouse tyrosine hydroxylase promoter via a high‐affinity binding site
Authors: Lebel Mélanie , Gauthier Yves , Moreau Alain , Drouin Jacques
Published: March 2003

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme of dopamine and (nor)adrenaline biosynthesis. Regulation of its gene expression is complex and different regulatory mechanisms appear to be operative in various neuronal lineages. Pitx3, a homeodomain‐containing transcription factor, has been cloned from neuronal tissues and, in the CNS, mouse Pitx3 is exclusively expressed in midbrain dopaminergic (MesDA) neurons from embryonic day 11 (E11). TH appears in these neurons at E11.5, consistent with a putative role of Pitx3 in TH transcription. We show that Pitx3 activates the TH promoter through direct interaction with a single high‐affinity binding site within the promoter and that this site is sufficient for Pitx3 responsiveness. In contrast, we did not observe an effect of Nurr1, an orphan nuclear receptor essential for normal development of MesDA neurons, on TH promoter activity. Pitx3 activation of TH promoter activity appears to be cell‐dependent suggesting that Pitx3 action may be modulated by other(s) regulatory mechanism(s) and factor(s).
Expression of KIF3C kinesin during neural development and in vitro neuronal differentiation
Authors: Navone Francesca , Consalez G. Giacomo , Sardella Milena , Caspani Elisabetta , Pozzoli Ombretta , Frassoni Carolina , Morlacchi Elena , Sitia Roberto , Sprocati Teresa , Cabibbo Andrea
Published: March 2003

KIF3A, KIF3B and KIF3C are kinesin‐related motor subunits of the KIF3 family that associate to form the kinesin‐II motor complex in which KIF3C and KIF3B are alternative partners of KIF3A. We have analysed the expression of Kif3 mRNAs during prenatal murine development. Kif3c transcripts are detectable from embryonic day 12.5 and persist throughout development both in the CNS and in some peripheral ganglia. Comparison of the expression patterns of the Kif3 genes revealed that Kif3c and Kif3a mRNAs colocalize in the CNS, while only Kif3a is also present outside the CNS. In contrast, Kif3b is detectable in several non‐neural tissues. We have also performed immunocytochemical analyses of the developing rat brain and have found the presence of the KIF3C protein in selected brain regions and in several fibre systems. Using neuroblastoma cells as an in vitro model for neuronal differentiation, we found that retinoic acid stimulated the expression of the three Kif3 and the kinesin‐associated protein genes, although with different time courses. The selective expression of Kif3c in the nervous system during embryonic development and its up‐regulation during neuroblastoma differentiation suggest a role for this motor during maturation of neuronal cells.
Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes
Authors: Hudson Tracie Y. , Corbett John A. , Howlett Allyn C. , Klein Claudette
Published: March 2003

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague–Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin‐stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin‐stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin‐stimulated activity of the type‐5 and ‐6 isoform family of enzymes is inhibited by NO. To monitor primarily the type‐5 isoform, we examined the ability of NO donors to attenuate D1‐agonist‐stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator‐stimulated cAMP signaling in the striatum.
Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library
Authors: Bracci Luisa , Pini Alessandro , Lozzi Luisa , Lelli Barbara , Battestin Paola , Spreafico Adriano , Bernini Andrea , Niccolai Neri , Neri Paolo
Published: March 2003

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the α‐bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using α‐bungarotoxin‐coated magnetic beads. Toxin‐bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti‐α‐bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of α‐bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C‐terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti‐α‐bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti‐ligand antibody phages can allow the production of receptor‐mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.
Tyrosine phosphorylation of the N‐methyl‐d‐aspartate receptor by exogenous and postsynaptic density‐associated Src‐family kinases
Authors: Cheung Herman H. , Gurd James W.
Published: March 2003

Phosphorylation of the NMDA receptor by Src‐family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2‐domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine‐phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C‐terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine‐phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation‐site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src‐family‐specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.
Inorganic lead stimulates DNA synthesis in human astrocytoma cells: role of protein kinase Cα
Authors: Lu Hailing , Guizzetti Marina , Costa Lucio G.
Published: March 2003

As lead has been shown to activate protein kinase C (PKC), and gliomas are reported to be highly dependent on PKC for their proliferation, this study was undertaken to investigate whether lead may act as a mitogen in human astrocytoma cells, and to determine the role of PKC in this effect. Lead acetate (from 100 nm to 100 µm) induced a concentration‐ and time‐dependent increase in DNA synthesis, as measured by incorporation of [methyl‐3H]thymidine into cell DNA, without causing any cytotoxicity. Flow cytometric analysis showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase. Western blot analyses showed that lead induced translocation of PKCα, but not of PKCε or PKCζ, from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead caused down‐regulation of PKCα, but not of PKCε. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF 109203X and staurosporine, as well as down‐regulation of PKC through prolonged treatment with 12‐O‐tetradecanoylphorbol 13‐acetate, blocked lead‐induced DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs and selective down‐regulation of specific PKC isoforms indicated that the effect of lead on DNA synthesis was mediated by PKCα. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCα.
The chemokine interleukin‐8 acutely reduces Ca2+ currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs
Authors: Puma Carole , Danik Marc , Quirion Rémi , Ramon Fabienne , Williams Sylvain
Published: March 2003

The chemokine IL‐8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL‐8 participates in rapid neuronal signaling by using a combination of whole‐cell recording and single‐cell RT‐PCR on dissociated rat septal neurons. We show that IL‐8 can acutely reduce Ca2+ currents in septal neurons, an effect that was concentration‐dependent, involved the closure of L‐ and N‐type Ca2+ channels, and the activation of Giα1 and/or Giα2 subtype(s) of G‐proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL‐8, expressed mRNAs for either one or both IL‐8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G‐proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL‐8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels.
Relationship between dopamine‐stimulated phospholipid methylation and the single‐carbon folate pathway
Authors: Zhao Ren , Chen Yuen , Tan Wenbo , Waly Mostafa , Sharma Alok , Stover Patrick , Rosowsky Andre , Malewicz Barbara , Deth Richard C.
Published: March 2003

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single‐carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R‐mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA‐stimulated incorporation of [14C]‐labeled formate into phospholipids in cultured SH‐SY5Y neuroblastoma cells. These interventions included: (i) Overexpression of methenyltetrahydrofolate synthetase (MTHFS). (ii) Treatment with 5‐formylTHF. (iii) Treatment with the MTHFS inhibitor 5‐formyltetrahydrohomofolic acid (5‐formylTHHF). (iv) Growth in nucleoside‐free media. 31P‐NMR was also used to follow DA‐induced changes in cell phospholipid composition. MTHFS overexpression and 5‐formylTHHF treatment, both of which lower 5‐methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5‐formylTHF, which increases 5‐methylTHF, caused a dose‐dependent increase in both basal and DA‐stimulated PLM. Growth in nucleoside‐free media caused time‐dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA‐stimulated PLM was initially increased followed by a later decrease. Together, these findings indicate a close functional relationship between single‐carbon folate metabolism and DA‐stimulated PLM, consistent with a role for 5‐methylTHF as the methyl donor for the D4R‐mediated process.
The neuroprotective agent ebselen modifies NMDA receptor function via the redox modulatory site
Authors: Herin Greta Ann , Du Shen , Aizenman Elias
Published: March 2003

Ebselen is a seleno‐organic compound currently in clinical trials for the treatment of ischemic stroke and subarachnoid hemorrhage. Its putative mode of action as a neuroprotectant is via cyclical reduction and oxidation reactions, in a manner akin to glutathione peroxidase. For this reason, we have investigated the effects of ebselen on the redox‐sensitive NMDA receptor. We have found that ebselen readily reversed dithiothreitol (DTT) potentiation of NMDA‐mediated currents in cultured neurons and in Chinese hamster ovary (CHO) cells expressing wild‐type NMDA NR1/NR2B receptors. In contrast, ebselen was unable to modulate NMDA‐induced currents in neurons previously exposed to the thiol oxidant 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB), or in CHO cells expressing a mutant receptor lacking the NR1 redox modulatory site, suggesting that ebselen oxidizes the NMDA receptor via this site. In addition, ebselen was substantially less effective in modifying NMDA responses in neurons exposed to alkylating agent N‐ethylmaleimide (NEM) following DTT treatment. Ebselen also reversed DTT block of carbachol‐mediated currents in Cos‐7 cells expressing the α2βδε subunits of the acetylcholine receptor, an additional redox‐sensitive ion channel. Ebselen was observed to significantly increase cell viability following a 30‐min NMDA exposure in cultured neurons. In contrast, other more typical antioxidant compounds did not afford neuroprotection in a similar paradigm. We conclude that ebselen may be neuroprotective in part due to its actions as a modulator of the NMDA receptor redox modulatory site.
Multiple pathways of Pb2+ permeation in rat cerebellar granule neurones
Authors: Mazzolini Monica , Traverso Sonia , Marchetti Carla
Published: March 2003

The pathways of lead (Pb2+) uptake were studied in fura‐2‐loaded cerebellar granule cells from 8‐day‐old rats. In a nominal Ca‐free external bath, Pb2+ (5–50 µm) determined an increase of the fluorescence emission ratio (R = E340/E380) even in the absence of any specific stimulus. This rise was dose‐dependent, was not significantly affected by mm Mg2+ or Ca2+, but it was readily reversed by the membrane‐permeant heavy metal chelator tetrakis(2‐pyridylmethyl) ethylene‐diamine (TPEN, 100 µm), indicating that it was due to Pb2+ influx. The rate of rise, dR/dt, was increased up to a factor of 5 by depolarizing high‐KCl solution, indicating a sizeable permeation through voltage‐dependent channels. This effect was neither antagonized by nimodipine, nor enhanced by BayK8644, but it was slackened by ω‐agatoxin IVA (200 nm), suggesting an involvement of non‐L‐type calcium channels. Pb2+ influx was also stimulated by glutamic acid or NMDA in the presence of 10–30 µm glycine, but only in Mg‐free solution, suggesting that glutamate channels of the NMDA type are an additional pathway of Pb2+ uptake. Pb2+ caused a time‐, dose‐ and stimulus‐dependent saturation of the dye, whose intracellular concentration is ∼ 10 µm, indicating that intracellular Pb2+ can readily reach a concentration in the micromolar range. These results indicate that the particular vulnerability of neurones to Pb2+ poisoning is linked to the presence of specific transport systems, which mediate the rapid uptake of Pb2+ into the neurone.
Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus
Authors: Silva Ana P. , Carvalho Arsélio P. , Carvalho Caetana M. , Malva João O.
Published: March 2003

In the present work, we investigated the role of pre‐ and post‐synaptic neuropeptide Y1 (NPY1) and Y2 receptors on the calcium responses and on glutamate release in the rat hippocampus. In cultured hippocampal neurones, we observed that only NPY1 receptors are involved in the modulation of intracellular free calcium concentration ([Ca2+]i). In 88% of the neurones analysed, the increase in the [Ca2+]i, in response to depolarization with 50 mm KCl, was inhibited by 1 µm[Leu31,Pro34]NPY, whereas 300 nm NPY13–36 was without effect. However, studies with hippocampal synaptosomes showed that both NPY1 and Y2 receptors can modulate the [Ca2+]i and glutamate release. The pharmacological characterization of the NPY‐induced inhibition of glutamate release indicated that Y2 receptors play a predominant role, both in the modulation of Ca2+‐dependent and ‐independent glutamate release. However, we could distinguish between Y1 and Y2 receptors by using [Leu31,Pro34]NPY and NPY13–36. Active pre‐synaptic Y1 receptors are present in the dentate gyrus (DG) as well as in the CA3 subregion, but its activity was not revealed by using the endogenous agonist, NPY. Concerning the Y2 receptors, they are present in the three subregions (CA1, CA3 and DG) and were activated by either NPY13–36 or NPY. The present data support a predominant role for NPY2 receptors in mediating NPY‐induced inhibition of glutamate release in the hippocampus, but the physiological relevance of the presently described DG and CA3 pre‐synaptic NPY1 receptors remains to be clarified.
Measurement of β‐hydroxybutyrate in acute hyperketonemia in human brain
Authors: Pan J. W. , Telang F. W. , Lee J. H. , De Graaf R. A. , Rothman D. L. , Stein D. T. , Hetherington H. P.
Published: March 2003

We report the measurement of d‐β‐hydroxybutyrate (BHB) in the brains of six normal adult subjects during acute infusions of BHB. We used high field in vivo1H magnetic resonance (MR) spectroscopy in the occipital lobe in conjunction with an acute infusion protocol to elevate plasma BHB levels from overnight fasted levels (0.20 ± 0.10 mm) to a steady state value of 2.12 ± 0.30 mm. At this level of hyperketonemia, we determined a tissue BHB level of 0.24 ± 0.04 mm. No increases in brain lactate levels were seen in these data. The concentrations of BHB and lactate were both considerably lower in comparison with previous data acquired in fasted adult subjects. This suggests that up‐regulation of the monocarboxylic acid transporter occurs with fasting.
Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the β‐amino acid agonist, taurine
Authors: Han Nian‐Lin R. , Haddrill Justine L. , Lynch Joseph W.
Published: March 2003

The β‐amino acid, taurine, is a full agonist of the human glycine receptor α1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed inXenopusoocytes. Several residues in the Ala101–Thr112 domain have previously been identified as determinants of β‐amino acid binding and gating mechanisms inXenopusoocyte‐expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine‐specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist‐ and antagonist‐bound conformations of β‐amino acids. The effects of mutations from Lys104–Thr112 indicate that the mechanism by which this domain controls β‐amino acid‐specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells orXenopusoocytes. Thr112 is the only domain element in mammalian cell‐expressed GlyRs which was demonstrated to discriminate between glycine and taurine.
Nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase and CREB in PC12h cells
Authors: Nakayama Hitoshi , Numakawa Tadahiro , Ikeuchi Toshihiko , Hatanaka Hiroshi
Published: March 2003

We have investigated mechanisms of nicotine‐induced phosphorylation of extracellular signal‐regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 µm. The maximal level of nicotine‐induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)‐induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) α7 subunit‐selective inhibitors had no significant effect on nicotine‐induced ERK phosphorylation. l‐Type voltage‐sensitive calcium channel antagonists inhibited nicotine‐induced ERK phosphorylation. Calcium imaging experiments showed that α7‐containing nAChR subtypes were functional at 1 µm of nicotine in the nicotine‐induced calcium influx, and non‐α7 nAChRs were prominent in the Ca2+ influx at 50 µm of nicotine. An expression of dominant inhibitory Ras inhibited nicotine‐induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine‐induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non‐α7 nAChRs are involved in nicotine‐induced ERK phosphorylation through CaM kinase and the Ras‐MAP kinase cascade and most of the nicotine‐induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.
Brain region‐specific up‐regulation of mouse apolipoprotein E by pharmacological estrogen treatments
Authors: Levin‐Allerhand Justine , McEwen Bruce S. , Lominska Chris E. , Lubahn Dennis B. , Korach Kenneth S. , Smith Jonathan D.
Published: March 2003

Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17β‐estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17β‐Estradiol up‐regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up‐regulated. Treatment of estrogen receptor (ER) α knockout mice with 17β‐estradiol or treatment of C57BL/6 mice with 17α‐estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17β‐estradiol effects on apoE are either directly or indirectly mediated by ERα in the diencephalon, while the effects in the cortex may be mediated by a non‐classical mechanism or by ERβ. Measurement of mRNA levels in estrogen versus placebo‐treated wild‐type mice indicated that the effect of 17β‐estradiol on apoE was not associated with changes in apoE mRNA levels.
Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C
Authors: Peng, Hu , Myers, Jason , Fang, Xiaohong , Stachowiak,* Ewa K. , Maher, Pamela A. , Martins, Gabriel G. , Popescu Gabriela , Berezney‡ Ronald , Stachowiak Michal K.
Published: March 2003

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF‐2) and FGFR1, association of FGFR1 with splicing factor‐rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up‐regulation of endogenous TH protein or a transfected TH promoter‐luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK‐mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF‐2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co‐transfected HMW FGF‐2 (which is exclusively nuclear) but not by cytoplasmic FGF‐1 or extracellular FGFs. Promoter transactivation by HMWFGF‐2 was accompanied by an up‐regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK‐) but not by IP6 or suramin. The TH promoter was also transactivated by co‐transfected wild‐type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP‐/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF‐2 was mediated through the cAMP‐responsive element (CRE) and was associated with induction of CREB‐ and CBP/P‐300‐containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.
D1 dopamine receptor stimulation increases GluR1 phosphorylation in postnatal nucleus accumbens cultures
Authors: Chao Steven Z. , Lu Wenxiao , Lee Hey‐Kyoung , Huganir Richard L. , Wolf Marina E.
Published: March 2003

Postsynaptic interactions between dopamine and glutamate receptors in the nucleus accumbens are critical for acute responses to drugs of abuse and for neuroadaptations resulting from their chronic administration. We tested the hypothesis that D1 dopamine receptor stimulation increases phosphorylation of the AMPA receptor subunit GluR1 at the protein kinase A phosphorylation site (Ser845). Nucleus accumbens cell cultures were prepared from postnatal day 1 rats. After 14 days in culture, GluR1 phosphorylation was measured by western blotting using phosphorylation site‐specific antibodies. The D1 receptor agonist SKF 81297 increased Ser845 phosphorylation in a concentration‐ dependent manner, with marked increases occurring within 5 min. This was prevented by the D1 receptor antagonist SCH 23390 and the protein kinase A inhibitor H89, and reproduced by forskolin. The D2 receptor agonist quinpirole attenuated the response to D1 receptor stimulation. Neither D1 nor D2 receptor agonists altered GluR1 phosphorylation at Ser831, the site phosphorylated by protein kinase C and calcium/calmodulin‐dependent protein kinase II. In other systems, phosphorylation of GluR1 at Ser845 is associated with enhancement of AMPA receptor currents. Thus, the present results suggest that AMPA receptor transmission in the nucleus accumbens may be augmented by concurrent D1 receptor stimulation.
PKC and Raf‐1 inhibition‐related apoptotic signalling in N2a cells
Authors: Bronisz Agnieszka , Gajkowska Barbara , Doma Krystyna , Ska‐Janik ń
Published: March 2003

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS10) or by inhibition PI3‐K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf‐1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10‐ or WM‐induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL‐2‐dependent mode of cell death after both treatments. Furthermore, in typical ‘gain of function’ experiments, cells with overexpression of permanently active Raf‐1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf‐1/BAD112, as well as PI3‐K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross‐talk between these two pathways at a level upstream from Raf‐1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3‐K is able to induce apoptosis.
Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus
Authors: Sperlágh Beáta , Köfalvi Attila , Deuchars Jim , Atkinson Lucy , J. Milligan Carol , Buckley Noel J. , Vizi E. Sylvester
Published: March 2003

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7R) are largely unknown. Here we studied the effect of P2X7R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1–30 mm) and other ATP analogues elicited concentration‐dependent [3H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non‐selective P2‐receptor antagonist (3–30 µm), Brilliant blue G (1–100 nm) the P2X7‐selective antagonist and Zn2+ (0.1–30 µm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3–30 µm) and BzATP (0.6–6 µm) elicited concentration‐dependent [14C]glutamate efflux, and blockade of the kainate receptor‐mediated transmission by CNQX (10–100 µm) and gadolinium (100 µm), decreased ATP evoked [3H]GABA efflux. The Na+ channel blocker TTX (1 µm), low temperature (12°C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP‐induced [3H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation‐induced [3H]GABA efflux. In conclusion, P2X7Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.