bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Glucokinase Isoforms Are Expressed in Rat Brain
Authors: Roncero Isabel , Alvarez Elvira , Vázquez Patricia , Blázquez Enrique
Published: March 2003

Abstract: Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6‐phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT‐PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT‐2 in the same hypothalamic neurons suggests a role in glucose sensing.
Increase of Brain‐Derived Neurotrophic Factor Gene Expression in NG108‐15 Cells by the Nuclear Isoforms of Ca2+/Calmodulin‐Dependent Protein Kinase II
Authors: Takeuchi Yusuke , Yamamoto Hideyuki , Miyakawa Taihei , Miyamoto Eishichi
Published: March 2003

Abstract: We have reported that the δ3 isoform of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain‐derived neurotrophic factor (BDNF), we transiently overexpressed the δ3 isoform in NG108‐15 cells. The quantitative RT‐PCR analysis revealed that rat cerebellum and NG108‐15 cells expressed the exon IV‐containing mRNA of BDNF (exon IV‐BDNF mRNA) more than the exon III‐BDNF mRNA. Treatment of NG108‐15 cells with Bay K 8644 increased both exon III‐ and exon IV‐BDNF mRNAs, and overexpression of the δ3 isoform potentiated the expression of the exon IV‐BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the δ1 isoform, a nonnuclear isoform, or the inactive mutant of the δ3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the δ3 isoform increased luciferase gene expression. Double‐immunostaining of NG108‐15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the δ3 isoform or the αB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF.
Azido‐ and Isothiocyanato‐Substituted Aryl Pyrazoles Bind Covalently to the CB1 Cannabinoid Receptor and Impair Signal Transduction
Authors: Howlett Allyn C. , Wilken Gerald H. , Pigg J. Jill , Houston Devin B. , Lan Ruoxi , Liu Qian , Makriyannis Alexandros
Published: March 2003

Abstract: 3‐Azidophenyl‐ and 3‐isothiocyanatophenyl‐ and 2‐(5′‐azidopentyl)‐ and 2‐(5′‐isothiocyanatopentyl)pyrazoles were synthesized to determine whether these compounds could behave as covalently binding ligands for the CB1 cannabinoid receptor in rat brain membranes. Heterologous displacement of [3H]CP55940 indicated that the apparent affinity of these compounds for the CB1 receptor was similar to that of the parent compound, SR141716A, with the exception of the 3‐isothiocyanato derivatives, which showed a 10‐fold loss of affinity. The 3‐azidophenyl and 3‐isothiocyanatophenyl compounds behaved as antagonists against the cannabinoid agonist desacetyllevonantradol in activation of G proteins [guanosine 5′‐O‐(γ‐35S]thio)triphosphate ([35S]GTPγS) binding] and regulation of adenylyl cyclase. The 2‐(5′‐azidopentyl)‐ and 2‐(5′‐isothiocyanatopentyl)pyrazoles were poor antagonists for [35S]GTPγS binding, and both compounds failed to antagonize the cannabinoid regulation of adenylyl cyclase. After incubation with the isothiocyanato analogues or UV irradiation of the azido analogues, the 3‐substituted aryl pyrazoles formed covalent bonds with the CB1 receptor as evidenced by the loss of specific binding of [3H]CP55940. In the case of the isothiocyanato analogues, the log concentration‐response curve for cannabinoid‐stimulated [35S]GTPγS binding was shifted to the right, indicating that loss of receptors compromised signal transduction capability. These irreversibly binding antagonists might be useful tools for the investigation of tolerance and receptor down‐regulation in both in vitro and in vivo studies.
Expression of mRNAs Encoding for Two Different Olfactory Receptors in a Subset of Olfactory Receptor Neurons
Authors: Rawson Nancy E. , Eberwine Jim , Dotson Ryan , Jackson Jennifer , Ulrich Patricia , Restrepo Diego
Published: March 2003

Abstract : Evidence has accumulated to support a model for odorant detection in which individual olfactory receptor neurons (ORNs) express one of a large family of G protein‐coupled receptor proteins that are activated by a small number of closely related volatile chemicals. However, the issue of whether an individual ORN expresses one or multiple types of receptor proteins has yet to be definitively addressed. Physiological data indicate that some individual ORNs can be activated by odorants differing substantially in structure and/or perceived quality, suggesting multiple receptors or one nonspecific receptor per cell. In contrast, molecular biological studies favor a scheme with a single, fairly selective receptor per cell. The present studies directly assessed whether individual rat ORNs can express multiple receptors using single‐cell PCR techniques with degenerate primers designed to amplify a wide variety of receptor sequences. We found that whereas only a single OR sequence was obtained from most ORNs examined, one ORN produced two distinct receptor sequences that represented different receptor gene families. Double‐label in situ hybridization studies indicated that a subset of ORNs co‐express two distinct receptor mRNAs. A laminar segregation analysis of the cell nuclei of ORNs labeled with the two OR mRNA probes showed that for one probe, the histogram of the distribution of the cell nuclei along the depth of the epithelium was bimodal, with one peak overlapping the (unimodal) histogram for the other probe. These results are consistent with co‐expression of two OR mRNAs in a population of single ORNs.
Interleukin‐10 and Interleukin‐13 Inhibit Proinflammatory Cytokine‐Induced Ceramide Production Through the Activation of Phosphatidylinositol 3‐Kinase
Authors: Pahan Kalipada , Khan Mushfiquddin , Singh Inderjit
Published: March 2003

Ceramide produced by hydrolysis of plasma membrane sphingomyelin (SM) in different cells including brain cells in response to proinflammatory cytokines [tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β)] plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The present study underlines the importance of IL‐10 and IL‐13, cytokines with potent antiinflammatory properties, in inhibiting the proinflammatory cytokine (TNF‐α and IL‐1β)‐mediated degradation of SM to ceramide in rat primary astrocytes. Treatment of rat primary astrocytes with TNF‐α or IL‐1β led to rapid degradation of SM to ceramide, whereas IL‐10 and IL‐13 by themselves were unable to induce the degradation of SM to ceramide. Interestingly, both IL‐10 and IL‐13 prevented proinflammatory cytokine‐induced degradation of SM to ceramide. Both IL‐10 and IL‐13 caused rapid activation of phosphatidylinositol (PI) 3‐kinase, and inhibition of that kinase activity by wortmannin and LY294002 potently blocked the inhibitory effect of IL‐10 and IL‐13 on proinflammatory cytokine‐mediated induction of ceramide production. This study suggests that the inhibition of proinflammatory cytokine‐mediated degradation of SM to ceramide by IL‐10 and IL‐13 is mediated through the activation of PI 3‐kinase. As ceramide induces apoptosis and IL‐10 and IL‐13 inhibit the induction of ceramide production, we examined the effect of IL‐10 and IL‐13 on proinflammatory cytokine‐mediated apoptosis. Inhibition of TNF‐α‐induced apoptosis by IL‐10 and IL‐13 suggests that the antiapoptotic nature of IL‐10 and IL‐13 is probably due to the inhibition of ceramide production.
Coupling Efficacy and Selectivity of the Human μ‐Opioid Receptor Expressed as Receptor—Gα Fusion Proteins in Escherichia coli
Authors: Stanasila Laura , Lim William K. , Neubig Richard R. , Pattus Franc
Published: March 2003

Abstract: Two constructs encoding the human μ‐opioid receptor

(hMOR) fused at its C terminus to either one of two Gα subunits,

Gαo1 (hMOR‐Gαo1) and Gαi2

(hMOR‐Gαi2), were expressed in Escherichia coli at

levels suitable for pharmacological studies (0.4‐0.5 pmol/mg). Receptors fused

to Gαo1 or to Gαi2 maintained high‐affinity

binding of the antagonist diprenorphine. Affinities of the μ‐selective

agonists morphine,

[D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin

(DAMGO), and endomorphins as well as their potencies and intrinsic activities

in stimulating guanosine

5′‐O‐(3‐[35S]thiotriphosphate)

([35S]GTPγS) binding were assessed in the presence of added

purified Gβγ subunits. Both fusion proteins displayed high‐affinity

agonist binding and agonist‐stimulated [35S]GTPγS binding. In

the presence of Gβγ dimers, the affinities of DAMGO and

endomorphin‐1 and ‐2 were higher at hMOR‐Gαi2 than at

hMOR‐Gαo1, whereas morphine displayed similar affinities at

the two chimeras. Potencies of the four agonists in stimulating

[35S]GTPγS binding at hMOR‐Gαo1 were

similar, whereas at hMOR‐Gαi2, endomorphin‐1 and morphine

were more potent than DAMGO and endomorphin‐2. The intrinsic activities of the

four agonists at the two fusion constructs were similar. The results confirm

hMOR coupling to Gαo1 and Gαi2 and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.
Induction of Glycerol Phosphate Dehydrogenase Gene Expression During Seizure and Analgesia
Authors: Link Wolfgang A. , Kauselmann Gunther , Mellström Britt , Kuhl Dietmar , Naranjo Jose R.
Published: March 2003

Abstract: Using mRNA differential display, we found that the gene
for NAD+‐dependent glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) is induced in rat brain following seizure activity. Northern blot and in situ hybridization analysis confirmed the differential display results; they also showed, in a separate model of neuronal activation, that after thermal noxious stimulation of the hind‐paws, a similar increase in GPDH mRNA occurs in the areas of somatotopic projection in the lumbar spinal cord. Surprisingly, administration of analgesic doses of morphine or the nonsteroidal antiinflammatory drugs aspirin, metamizol (dipyrone), and indomethacin also increased GPDH mRNA levels in rat spinal cord. The opioid receptor antagonist naloxone completely blocked morphine induction of GPDH but had no effect on GPDH induction by noxious heat stimulation or metamizol treatment, implicating different mechanisms of GPDH induction. Nevertheless, in all cases, induction of the GPDH gene requires adrenal steroids and new protein synthesis, as the induction was blocked in adrenalectomized rats and by cycloheximide treatment, respectively. Our results suggest that the induction of the GPDH gene upon peripheral noxious stimulation is related to the endogenous response to pain as it is mimicked by exogenously applied analgesic drugs.
Identification of a Peptide Sequence in Albumin that Potentiates Superoxide Production by Microglia
Authors: Nakamura Yoichi , Si Qiu‐Sheng , Takaku Takeshi , Kataoka Kiyoshi
Published: March 2003

Abstract: Microglial activation has recently been recognized as acause of damage in various neurodegenerative diseases. A possible mechanismunderlying this damage is the activation of microglia by serum factors leakedthrough a disruption of the blood—brain barrier, which in turn triggermicroglial cell proliferation and the release of various substances toxic toneurons, such as superoxide (O2‐). We recently reportedthat serum albumin enhanced O2‐ producation in culturedrat microglia stimulated by phorbol ester. In the present report, we identifythe active site of this enhancement within the albumin molecule. We purifiedan active subfragment from trypsin‐treated bovine serum albumin that wascomposed of 12‐mer and 33‐mer peptides connected by a disulfide bond. Thechemically synthesized 12‐mer peptide showed activity within a concentrationrange (∼10‐7M) equivalent to that of albumin. Theactivities of a series of synthesized peptides conclusively indicated that theminimum active sequence was Leu‐His‐Thr‐Leu. The present study may shed lighton the mechanism of neuronal cell damage in various neurodegenerativediseases.
Selective Synthesis of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase Isoform 2 and Identification of Specifically Phosphorylated Serine Residues
Authors: O'Neill Ryan C. , Braun Peter E.
Published: March 2003

Abstract: 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) is a protein found abundantly in the cytoplasmic compartments of CNS myelin. Two isoforms of this protein, CNP1 and CNP2, are detectable. They differ by a 20‐amino acid extension exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically in 293T cells and labeled with 32P. Immunoprecipitation of labeled CNP2 and tryptic phosphopeptide mapping analyses identified serines 9 and 22 as the major sites of phosphorylation. Only serine 22 was phosphorylated initially in oligodendrocyte‐enriched cultures of neonatal rat brain glial cells. However, 4β‐phorbol 12, 13‐dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These results suggest that serine 9 is phosphorylated by a PDB‐sensitive kinase, likely protein kinase C, and that serine 22 appears to be constitutively phosphorylated.
Phe208 and Ile199 in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat
Authors: Geha Rani Maurice , Chen Kevin , Shih Jean Chen
Published: March 2003

Abstract:
It has been reported previously that reciprocally switching Phe208 and Ile199 in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A‐F208I showed a sixfold decrease in the specificity constant kcat/Km for both the MAO A‐ and the MAO B‐preferring substrates 5‐hydroxytryptamine and β‐phenylethylamine, respectively. The reciprocal point mutant MAO B‐I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159‐214 in MAO A and 150‐205 in MAO B. Chimerics MAO AB159‐214A and MAO BA150‐205B had small changes in Km and IC50 values when compared with MAO A and B, respectively, but did not exhibit a preference switch. The results suggest that Phe208 in MAO A and amino acid segments 159‐214 and 150‐205 in MAO A and B, respectively, influence the enzyme active site. However, substrate and inhibitor preferences of human MAO A and B are not determined by the respective residues Phe108 and Ile199 as in rat MAO nor by their neighboring regions.
Characterization of 3‐[123I]iodo‐L‐α‐methyl tyrosine transport in astrocytes of neonatal rats
Authors: Kopka Klaus , Riemann Burkhard , Friedrich Matthias , Winters Silke , Halfter Hartmut , Weckesser Matthias , Stögbauer Florian , Ringelstein E. Bernd , Schober Otmar
Published: March 2003

3‐[123I]Iodo‐L‐α‐methyl tyrosine (123I‐IMT) is used for diagnosis and monitoring of brain tumours by means of single‐photon emission tomography. As recently shown, 123I‐IMT is predominantly mediated into rat C6 glioma cells by sodium‐independent system L for large neutral amino acids. Until now, 123I‐IMT transport in non‐neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of 123I‐IMT uptake into astrocytes of neonatal rats. In particular sodium‐independent 123I‐IMT transport into neonatal astrocytes was compared with sodium‐independent 123I‐IMT uptake into neoplastic rat C6 glioma cells.

Competitive inhibition experiments showed that 123I‐IMT is exclusively transported via sodium‐independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium‐independent 123I‐IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants KM = 13.9 ± 0.5 µm and KM = 33.9 ± 4.1 µm, respectively, which are in the same range of KM values as those recently determined for amino acid transport into neoplastic and non‐neoplastic glial cells. Indeed, the KM values in the micromolar range correspond to the expression of the LAT‐1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium‐independent maximum transport velocities (Vmax) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 ± 0.3 and 39.9 ± 3.3 nmol/mg protein/10 min, respectively).
Oxidative post‐translational modifications of α‐synuclein in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinson's disease
Authors: Przedborski Serge , Chen Qiping , Vila Miquel , Giasson Benoit I. , Djaldatti Ruth , Vukosavic Slobodanka , Souza José M. , Jackson‐Lewis Vernice , Lee Virginia M.‐Y. , Ischiropoulos Harry
Published: March 2003

Structural and functional alterations of α‐synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). α‐Synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD‐linked α‐synuclein mutations may be achieved by oxidative post‐translational modifications. Here, we show that wild‐type α‐synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of α‐synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Conversely, β‐synuclein and synaptophysin were not nitrated in MPTP‐intoxicated mice. Our data demonstrate that α‐synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of α‐synuclein in the neurodegeneration associated with MPTP toxicity and with PD.
Molecular consequences of activated microglia in the brain: overactivation induces apoptosis
Authors: Liu Bin , Wang Kevin , Gao Hui‐Ming , Mandavilli Bhaskar , Wang Jia‐Yi , Hong Jau‐Shyong
Published: March 2003

Microglia, the resident immune cells in the brain, play a pivotal role in immune surveillance, host defense, and tissue repair in the CNS. In response to immunological challenges, microglia readily become activated as characterized by morphological changes, expression of surface antigens, and production of immune modulators that impact on neurons to induce neurodegeneration. However, little is known concerning the fate of activated microglia. In the present study, stimulation of cultured rat primary microglia with 1 ng/mL of the inflammagen lipopolysaccharide (LPS) resulted in a maximal activation as measured by the release of tumor necrosis factor alpha (TNFα). However, treatment with higher concentrations of LPS resulted in significantly lower quantities of detectable TNFα. Further analysis revealed that overactivation of microglia with higher concentrations of LPS (> 1 ng/mL) resulted in a time‐ and dose‐dependent apoptotic death of microglia as defined by DNA strand breaks, surface expression of apoptosis‐specific markers (phosphatidylserine), and activation of caspase‐3. In contrast, astrocytes were insensitive to LPS‐induced cytotoxicity. In light of the importance of microglia and the limited replenishment mechanism, depletion of microglia from the brain may severely hamper its capacity for combating inflammatory challenges and tissue repair. Furthermore, overactivation‐induced apoptosis of microglia may be a fundamental self‐regulatory mechanism devised to limit bystander killing of vulnerable neurons.
Mechanisms of inverse agonism of antipsychotic drugs at the D2 dopamine receptor: use of a mutant D2 dopamine receptor that adopts the activated conformation
Authors: Wilson Janet , Lin Hong , Fu Dingyi , Javitch Jonathan A. , Strange Philip G.
Published: March 2003

The antipsychotic drugs have been shown to be inverse agonists at the D2 dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D2 dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D2 dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G‐protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non‐activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.
Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers
Authors: Chang Michael C. J. , Contreras Miguel A. , Rosenberger Thad A. , Rintala Jyrki J. O. , Bell Jane M. , Rapoport Stanley I.
Published: March 2003

Both (Li+) and valproic acid (VPA) are effective in treating bipolar disorder, but the pathway by which either works, and whether it is common to both drugs, is not agreed upon. We recently reported, using an in vivo fatty acid model, that Li+ reduces the turnover rate of the second messenger arachidonic acid (AA) by 80% in brain phospholipids of the awake rat, without changing turnover rates of docosahexaenoic or palmitic acid. Reduced AA turnover was accompanied by down‐regulation of gene expression and protein levels of an AA‐specific cytosolic phospholipase A2 (cPLA2). To see if VPA had the same effect on AA turnover, we used our in vivo fatty acid model in rats chronically administered VPA (200 mg/kg, i.p. for 30 days). Like Li+, VPA treatment significantly decreased AA turnover within brain phospholipids (by 28–33%), although it had no effect on cPLA2 protein levels. Thus, both mood stabilizers, Li+ and VPA have a common action in reducing AA turnover in brain phospholipids, albeit by different mechanisms.
Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system
Authors: Loureiro‐dos‐Santos Nelson E. , Reis Ricardo A. M. , Kubrusly Regina C. C. , De Almeida Olga M. M. S. , Gardino Patricia F. , De Mello Maria C. F. , De Mello Fernando G.
Published: March 2003

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 µm kainate (T1/2 : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide ‐ thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium‐3 sensitive choline uptake and hemicolinium‐3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mmNG‐nitro‐l‐arginine methyl ester (l‐NAME). Veratridine (3μM) also reduced ChAT by a Ca2+ dependent, but glutamate independent mechanism and was prevented by 1μM tetrodotoxin.
Elevated levels of group‐III metabotropic glutamate receptors in the inferior colliculus of genetically epilepsy‐prone rats following intracollicular administration of l‐serine‐O‐phosphate
Authors: Yip Ping K. , Meldrum Brian S. , Rattray Marcus
Published: March 2003

The selective group‐III metabotropic glutamate receptor agonist, l‐serine‐O‐phosphate (l‐SOP), when injected bilaterally into the inferior colliculus of the sound sensitive genetically epilepsy‐prone (GEP) rats produces a short proconvulsant excitation followed by a long phase of protection against sound‐induced seizures lasting for 2–4 days. We have studied this prolonged suppression of audiogenic seizures using pharmacological and molecular biological approaches including semiquantitative RT‐PCR and western blotting. The intracerebroventricular injection of the protein synthesis inhibitor cycloheximide (120 µg) 30 min beforehand significantly reduces the proconvulsant seizure activity and the prolonged anticonvulsant effect of intracollicular l‐SOP (500 nmol/side). The sensitive semiquantitative RT‐PCR revealed a significant up‐regulation in mGlu4 and mGlu7 mRNA levels in the inferior colliculus at 2 days (maximum suppression of audiogenic seizures) after intracollicular l‐SOP injection compared with the non‐injected, 2‐day post‐vehicle treated and 7‐day (return to expressing audiogenic seizures) post‐drug or vehicle‐treated groups. No significant changes were observed in mGlu6 or mGlu8 mRNA expression levels in drug‐treated compared with control groups. Examination of mGlu4a and mGlu7a protein levels using western blotting showed a significant increase in mGlu7a but no significant change in mGlu4a protein levels 2 days after l‐SOP treatment compared with the control groups (non‐injected and 2‐day vehicle‐injected group). These results suggest that up‐regulation of mGlu7 receptors is involved in the prolonged anticonvulsant effect of l‐SOP against sound‐induced seizures in GEP rats. The potential use of mGlu7 agonists as novel anti‐epileptic agents merits investigation.
A novel untranslated ‘exon H’ of the human choline acetyltransferase gene in placenta
Authors: Robert Isabelle , Quirin‐Stricker Christiane
Published: March 2003

To investigate the existence of 5′‐region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5′‐untranslated regions (UTR) in human neuronal and non‐neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG‐3 and neuroblastoma CHP126 and MC‐IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT‐PCR). We used a sense primer located in the 5′‐flanking region, in the previously defined intronic sequence and an anti‐sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG‐3 cells whereas it was absent in spinal cord, CHP126 and MC‐IXC cells. It was designated ‘H‐type’ of ChAT mRNA. Whereas CHP126 produced the R‐ and N‐type of ChAT mRNAs, no transcript of the N‐and R‐type was detected in JEG‐3 and human placenta. In addition, CHP126 and JEG‐3 cells and placenta showed the expression of the M‐type of ChAT mRNA. The idendity of the amplified 567 bp product (H‐type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H‐type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M‐type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.
Mutagenesis of the GABAA receptor α1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine‐site ligands
Authors: Davies Martin , Newell J. Glen , Dunn Susan M. J.
Published: March 2003

We have mutated several amino acids in the region of the GABAA receptor α1 subunit predicted to form a small extracellular loop between transmembrane domains two and three to investigate its possible role in ligand sensitivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mutant α1 subunits were co‐expressed with β2 and γ2 subunits in tsA201 cells or Xenopus oocytes. Binding studies revealed that the only mutation that significantly affected [3H]Ro15–4513 binding was the V279A substitution which reduced the affinity for this ligand. Electrophysiological examination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration‐response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteric modulation by benzodiazepine‐site ligands was examined. V279A reduced the potency of both flunitrazepam and Ro15–4513 but, in each case, their efficacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy without affecting its potency. Additionally, P277A and A280S resulted in Ro15–4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII–TMIII plays a role in determining the sensitivity of GABAA receptors to both GABA and benzodiazepine‐site ligands.
Characterization of a phospholipid antigen reacting with serum antibody in patients with peripheral neuropathies and paraproteinemia
Authors: Suzuki Minoru , Suetake Keiji , Kasama Takeshi , Ariga Toshio , Shiina Meiko , Kusunoki Susumu , Yu Robert K.
Published: March 2003

A phospholipid antigen that reacted with the serum antibody from a patient with peripheral neuropathy and paraproteinemia with both impaired sensory and motor functions, but not with sera from patients with only impaired sensory functions and healthy controls, was purified from bovine cauda equina as a minor component with a concentration of about 0.6 µg per gram wet‐weight tissue. The structure of the phospholipid was characterized as lysophosphatidylinositol by means of thin‐layer chromatography, gas–liquid chromatography, and negative‐ion fast‐atom‐bombardment mass spectrometry. The major fatty acid component of this phospholipid was stearic acid (> 81%). Our data suggest the possible involvement of a lysophospholipid antigen in the immunopathogenesis of peripheral neuropathies with severe motor and sensory dysfunctions. There is an intriguing possibility that the difference in immunoreactivity of serum antibodies may underlie the differential clinical manifestations in patients with peripheral neuropathy and paraproteinemia.
Divergent pathways account for two distinct effects of amyloid β peptides on exocytosis and Ca2+ currents: involvement of ROS and NF‐κB
Authors: Green Kim N. , Peers Chris
Published: March 2003

Amyloid peptides (AβPs) are implicated in neuronal death associated with Alzheimer's disease. Their toxicity involves disruption cellular Ca2+ homeostasis, leading to activation of caspases and cell death. Antioxidants can prevent such cell death and show beneficial clinical effects in Alzheimer's disease patients. Using the model neurosecretory cell line, PC12, we have shown that AβPs cause enhancement of evoked exocytosis via formation of a Cd2+‐resistant Ca2+ influx pathway, and also cause selective, functional up‐regulation of current through L‐type Ca2+ channels. The involvement of reactive oxygen species (ROS) in these effects were investigated by examining the ability of various antioxidants to interfere with these responses. Both melatonin and ascorbic acid fully blocked the enhancement of catecholamine secretion caused by application of AβP(1−40), as monitored in real time amperometrically, but inhibition of the transcriptional regulator NF‐κB with SN‐50 did not affect secretion. Enhanced immunofluorescence, observed in AβP‐treated cells using a monoclonal antibody raised against the N‐terminus of AβP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen also fully prevented augmentation of whole‐cell Ca2+ currents caused by application of AβP(1−40). By contrast, inhibitors of NF‐κB (sulfasalazine and SN‐50) were able to prevent AβP induced Ca2+ channel current enhancement, whilst inhibitors of mitogen‐activated protein kinase and protein kinase C could not. Our results indicate that augmentation or induction by AβPs of two important, distinct factors regulating Ca2+ homeostasis is mediated by increased ROS production, but only one of these (up‐regulation of native Ca2+ channels) requires activation of NF‐κB.
Further characterization of the molecular interaction between PSD‐95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating
Authors: Rutter A. Richard , Freeman Fiona M. , Stephenson F. Anne
Published: March 2003

Coexpression of PSD‐95c‐Myc with NR1‐1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3H]MK801 binding similar to that previously described for l‐glutamate. The inhibition constants (KIs) for the binding of l‐glutamate and glycine to NR1‐1a/NR2A determined by [3H]CGP 39653 and [3H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1‐1a/NR2A receptors coexpressed ± PSD‐95c‐Myc. The increased EC50 for l‐glutamate enhancement of [3H]MK801 binding was also found for NR1‐2a/NR2A and NR1‐4b/NRA receptors thus the altered EC50 is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1‐4b but not the NR1‐1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1‐4b and NR1‐4b/NR2A expression was enhanced by PSD‐95c‐Myc but whole cell enzyme‐linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD‐95c‐Myc has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C‐terminal E(T/S)XV PSD‐95 binding motifs.
Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons
Authors: Holm Pontus C. , Åkerud Peter , Wagner Joseph , Arenas Ernest
Published: March 2003

Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c‐ret, which binds ligands of the glial cell line‐derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)‐positive neurons in the LC express both GFRα1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down‐regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH‐positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6‐hydroxydopamine (6‐OHDA) lesion model, but induced the sprouting of TH‐positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.
Aspirin provides cyclin‐dependent kinase 5‐dependent protection against subsequent hypoxia/reoxygenation damage in culture
Authors: Vartiainen Nina , Keksa‐Goldsteine Velta , Goldsteins Gundars , Koistinaho Jari
Published: March 2003

Aspirin [acetylsalicylic acid (ASA)] is an anti‐inflammatory drug that protects against cellular injury by inhibiting cyclooxygenases (COX), inducible nitric oxide synthase (iNOS) and p44/42 mitogen‐activated protein kinase (p44/42 MAPK), or by preventing translocation of nuclear factor κB (NF‐κB). We studied the effect of ASA pre‐treatment on neuronal survival after hypoxia/reoxygenation damage in rat spinal cord (SC) cultures. In this injury model, COX, iNOS and NF‐κB played no role in the early neuronal death. A 20‐h treatment with 3 mm ASA prior to hypoxia/reoxygenation blocked the hypoxia/reoxygenation‐induced lactate dehydrogenase (LDH) release from neurons. This neuroprotection was associated with increased phosphorylation of neurofilaments, which are substrates of p44/42 MAPK and cyclin‐dependent kinase 5 (Cdk5). PD90859, a p44/42 MAPK inhibitor, had no effect on ASA‐induced tolerance, but olomoucine and roscovitine, Cdk5 inhibitors, reduced ASA neuroprotection. Hypoxia/reoxygenation alone reduced both the protein amount and activity of Cdk5, and this reduction was inhibited by pre‐treatment with ASA. Moreover, the protein amount of a neuronal Cdk5 activator, p35, recovered after reoxygenation only in ASA‐treated samples. The prevention of the loss in Cdk5 activity during reoxygenation was crucial for ASA‐induced protection, because co‐administration of Cdk5 inhibitors at the onset ofreoxygenation abolished the protection. In conclusion, pre‐treatment with ASA induces tolerance against hypoxia/reoxygenation damage in spinal cord cultures by restoring Cdk5 and p35 protein expression.
Tetrahydrobiopterin deficiency increases neuronal vulnerability to hypoxia
Authors: Delgado‐Esteban María , Almeida Angeles , Medina Jose M.
Published: March 2003

Tetrahydrobiopterin (BH4) is an essential co‐factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre‐incubating neurones with 5 mm 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia‐induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4‐deficient neurones. All these effects were counteracted whenneuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia‐induced damage in BH4‐deficient neurones was prevented when Nω‐nitro‐l‐arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability tohypoxia‐induced mitochondrial damage and necrosis.
Long‐term post‐synaptic consequences of methamphetamine on preprotachykinin mRNA expression
Authors: Johnson‐Davis Kamisha L. , Hanson Glen R. , Keefe Kristen A.
Published: March 2003

Exposure to repeated high doses of methamphetamine produces long‐term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by ∼20% in the middle striatum and ∼32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by ∼20% in the middle striatum and ∼14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either timepoint. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.
The human SH‐SY5Y neuroblastoma cell‐line expresses a functional P2X7 purinoceptor that modulates voltage‐dependent Ca2+ channel function
Authors: Per Larsson Kim , Jon Hansen Anker , Dissing Steen
Published: March 2003

Fura‐2 imaging of purinergic stimulation of non‐differentiated neuronal human SH‐SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 µm) was as follows: 2′,3′‐O‐(4‐benzoyl‐benzoyl)‐ATP (BzATP) > ATP4– > ATP; whereas 2‐(methylthio)‐ATP, ADP, UTP and α,β‐methylene‐ATP and β,γ‐methylene‐ATP were ineffective. The response to BzATP was inhibited by pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic‐acid (PPADS, 1 µm), 1‐(N,O‐bis[5‐isoquinolinesulfonyl]‐N‐methyl‐l‐tyrosyl)‐4‐phenylpiperazine (KN‐62, 100 nm) and 8‐(3‐benzamido‐4–4‐methylbenzamido)‐naphthalene‐1,3,5‐trisulfonic‐acid (suramin, 200 µm). The presence of a P2X7 receptor was confirmed by western blot studies using anti‐P2X7. EC50 for BzATP was 212 ± 6 µm. BzATP > 30 µm induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 µm only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 µm) and to a smaller degree by ω‐conotoxin GVIA (1 µm) despite an almost equal presence of L and N‐type Ca2+‐channels. In whole‐cell voltage‐clamp studies at − 90 mV, BzATP (300 µm) produced a fast activating inward current with a similar pharmacology as observed with Fura‐2 imaging. Current clamp studies showed a dose‐dependent depolarization to BzATP and ATP4–. BzATP also triggered transmitter release. Thus, the human neuronal SH‐SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+‐channels.
Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization
Authors: Le Crom Stéphane , Prou Delphine , Vernier Philippe
Published: March 2003

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS‐7 cells, long‐term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS‐15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long‐term desensitization time‐course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre‐treatment of cells with R(–)‐PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.
β‐Amyloid peptide induces formation of actin stress fibers through p38 mitogen‐activated protein kinase
Authors: Song Cheng , Perides George , Wang Dechun , Liu Ya Fang
Published: March 2003

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial β‐amyloid precursor protein APP‐V642I (IAPP) or mutant presenilin‐1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild‐type amyloid precursor protein or presenilin‐1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with β‐amyloid (Aβ) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a γ‐secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Aβ peptide induces actin stress fibers. Because activation of the p38 mitogen‐activated protein kinase (p38MAPK)–mitogen‐associated protein kinase‐associated protein kinase (MAPKAPK)‐2–heat‐shock protein 27 signaling pathway mediates actin polymerization, we explored whether Aβ peptide activates p38MAPK and MAPKAPK‐2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK‐2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Aβ peptide, IAPP or mPS1. Moreover, treatment with a γ‐secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK‐2. In summary, our data suggest that overproduction of Aβ peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.