bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing
Authors: Azaryan Anahit V. , Schiller Martin , Mende‐Mueller Liane , Hook Vivian Y. H.
Published: July 2010

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([35S]Met)preproenkephalin. The 70‐kDa CG aspartic proteinase was purified by concanavalin A‐Sepharose, Sephacryl S‐200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val‐d‐Leu‐Pro‐Phe‐Val‐d‐Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys172‐Arg173 pair located at the COOH‐terminus of (Met)enkephalin‐Arg6‐Gly7‐Leu8, as assessed by peptide microsequencing. The importance of full‐length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze 35S‐labeled proenkephalin and proopiomelanocortin and its inability to cleave tri‐ and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.
Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated
Authors: Bharadwaj Mausumi , Bizzozero Oscar A.
Published: July 2010

Abstract: P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153‐containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl‐CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl‐CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second‐order reaction (k2 = 246 ± 6 M−1 min−1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl‐CoA. The activation energy calculated from the Arrhenius plot is ∼2 kcal/mol and much lower than that of enzyme‐catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol‐containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.
hVH‐5: A Protein Tyrosine Phosphatase Abundant in Brain that Inactivates Mitogen‐Activated Protein Kinase
Authors: Martell Karen J. , Seasholtz Audrey F. , Kwak Seung P. , Clemens Kristina K. , Dixon Jack E.
Published: July 2010

Abstract: A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH‐5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen‐activated protein kinase. The catalytic region of hVH‐5 was expressed as a fusion protein and was shown to hydrolyze p‐nitrophenylphosphate and inactivate mitogen‐activated protein kinase, thus proving that hVH‐5 possessed phosphatase activity. A unique proline‐rich region distinguished hVH‐5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH‐5 from related phosphatases was that hVH‐5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH‐5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH‐5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate‐early gene, suggesting a possible role in the signal transduction cascade.
ATP‐Binding Proteins on the External Surface of Synaptic Plasma Membranes: Identification by Photoaffinity Labeling
Authors: Nagy Agnes K. , Shuster Terrence.A.
Published: July 2010

Abstract: 8‐Azidoadenosine triphosphate labeled in the α or γ position with 32P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate‐polyacrylamide gel electrophoretic patterns, UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5–10 µM resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8‐azidoadenosine [α‐32P]triphosphate‐labeled protein complex of ∼45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 mM EDTA, a band at 44 kDa was labeled within this ATP‐binding complex. The labeling pattern of the subbands of this 45/46‐kDa complex is consistent with these bands being extracellular ATP‐binding proteins on the surface of the synaptosome.
Differences in Nuclear Signaling by Leukemia Inhibitory Factor and Interferon‐γ: The Role of STAT Proteins in Regulating Vasoactive Intestinal Peptide Gene Expression
Authors: Symes Aviva J. , Corpus Lisa , Fink J. Stephen
Published: July 2010

Abstract: To investigate the importance of STAT protein activation in leukemia inhibitory factor (LIF)‐mediated induction of neuropeptide gene transcription, we compared signaling to the 180‐bp cytokine response element (CyRE) in the vasoactive intestinal peptide (VIP) promoter by interferon‐γ (IFN‐γ) and LIF. We show that LIF and IFN‐γ activate STAT proteins but only LIF activates VIP gene transcription. Thus STAT activation is not sufficient for VIP transcriptional activation. In a CyRE reporter plasmid, in which the STAT site has been deleted, LIF, but not IFN‐γ, activates transcription, indicating that sequences within the CyRE distinct from the STAT site are important to LIF‐mediated transcriptional activation. The CyRE does not mediate transcriptional activation to LIF in a non‐VIPergic cell line, suggesting that cell‐specific factors exist which are permissive for cytokine‐dependent regulation of gene expression. Human and mouse sequences are highly conserved in the region of the CyRE, consistent with the functional importance of multiple regions of the CyRE. These findings show that regions within the CyRE distinct from the STAT site are important to the LIF‐dependent regulation of VIP gene expression and enable the CyRE to respond in a cell‐specific and cytokine‐specific manner.
The Response of the Tyrosine Hydroxylase Gene to Cyclic AMP Is Mediated by Two Cyclic AMP‐Response Elements
Authors: Best Joseph A. , Chen Yang , Piech Kristen M. , Tank A. William
Published: July 2010

Abstract: Tyrosine hydroxylase (TH) gene transcription rate is stimulated by cyclic AMP in cultured rat pheochromocytoma cells. This effect is at least partially due to the interaction of transcription factors with the canonical cyclic AMP‐response element (CRE) at position −45 to −38 within the TH gene promoter. In this study we test whether a region of the TH gene promoter, which is adjacent to and upstream of the canonical TH CRE, also participates in the response of the promoter to cyclic AMP. Using electrophoretic mobility shift assays, we demonstrate that nuclear proteins from rat pheochromocytoma cell lines bind to the region of the TH gene from −102 to −73. A comparison of promoter sequences indicates that sequences within this region of the TH gene are highly homologous to proenkephalin promoter sequences (between −110 and −80) designated ENKCRE‐1 and ENKCRE‐2. We designated the TH gene sequence homologous to ENKCRE‐1 as TH E1 and the sequence homologous to ENKCRE‐2 as TH E2. Competition displacement binding assays suggest that protein binding to the −102/−73 region of the TH gene is critically dependent on the TH E1 sequence. Transient transfection assays using minimal promoter constructs demonstrate that this region acts as a cyclic AMP‐responsive element. Mutagenesis of the TH E1 sequence within the normal context of the TH gene proximal promoter leads to a 50% decrease in the cyclic AMP inducibility of the promoter. These results support the hypothesis that the full response of the TH gene to cyclic AMP requires both the canonical TH CRE and this newly discovered element, which we term TH CRE2.
Analysis of the Neuronal Promoter of the Rat Aromatic l‐Amino Acid Decarboxylase Gene
Authors: Aguanno Ann , Lee Mavis R. , Marden Chloe M. , Rattray Marcus , Gault Andrea , Albert Vivian R.
Published: July 2010

Abstract: The rat aromatic l‐amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5′ untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non‐AADC‐expressing cells. We demonstrate the use of the neuronal‐specific UTR in individual dopamine‐, norepinephrine‐, and serotonin‐containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68‐bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis‐active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue‐specific expression of the neuronal promoter may not be controlled by cis‐active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.
NMDA Receptor Activation Produces Concurrent Generation of Nitric Oxide and Reactive Oxygen Species: Implications for Cell Death
Authors: Gunasekar P. G. , Kanthasamy A. G. , Borowitz J. L. , Isom G. E.
Published: July 2010

Abstract: The ability of glutamate to stimulate generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant‐sensitive fluorescent dye 2,7‐dichlorofluorescin (DCF). Exposure of cells to glutamate (10 µM) produced a rapid generation of oxidants that was blocked ∼70% by MK‐801 (a noncompetitive NMDA‐receptor antagonist). To determine if nitric oxide (NO) or reactive oxygen species (ROS) contributed to the oxidation of DCF, cells were treated with compounds that altered their generation. NO production was inhibited with NG‐nitro‐l‐arginine methyl ester (l‐NAME) (nitric oxide synthase inhibitor) and reduced hemoglobin (NO scavenger). Alternatively, cells were incubated with superoxide dismutase (SOD) and catalase, which selectively metabolize O2−· andH2O2. Concurrent inhibition of O2−· and NO production nearly abolished intracellular oxidant generation. Pretreatment of cells with either chelerythrine (1 µM, protein kinase C inhibitor) or quinacrine (5 µM, phospholipase A2 inhibitor) before addition of glutamate also blocked oxidation of DCF. Generation of oxidants by glutamate was significantly reduced by incubating the cells in Ca2+‐free buffer. In cytotoxicity studies, a positive correlation was observed between glutamate‐induced death and oxidant generation. Glutamate‐induced cytotoxicity was blocked by MK‐801 and attenuated by treatment with l‐NAME, chelerythrine, SOD, or quinacrine. It is concluded that glutamate induces concurrent generation of NO and ROS by activation of both NMDA receptors and non‐NMDA receptors through a Ca2+‐mediated process. Activation of NO synthase and phospholipaseA2 contribute significantly to this response. It is proposed that simultaneous generation of NO and ROS results in formation of peroxynitrite, which initiates the cellular damage.
Neuroleptics Up‐Regulate Adenosine A2a Receptors in Rat Striatum: Implications for the Mechanism and the Treatment of Tardive Dyskinesia
Authors: Parsons Bruce , Togasaki Daniel M. , Kassir Suham , Przedborski Serge
Published: July 2010

Abstract: Neuroleptics, which are potent dopamine receptor antagonists, are used to treat psychosis. In the striatum, dopamine subtype‐2 (D2) receptors interact with high‐affinity adenosine subtype‐2 (A2a) receptors. To examine the effect of various neuroleptics on the major subtypes of striatal dopamine and adenosine receptors, rats received 28 daily intraperitoneal injections of these drugs. Haloperidol (1.5 mg/kg/day) increased the density of striatal D2 receptors by 24% without changing their affinity for [3H]sulpiride. Haloperidol increased the density of striatal A2a receptors by 33% (control, 522.4 ± 20.7 fmol/mg of protein; haloperidol, 694.6 ± 23.6 fmol/mg of protein; p < 0.001) without changing their affinity for [3H]CGS‐21680 (control, 19.2 ± 2.2 nM; haloperidol, 21.4 ± 2.3 nM). In contrast, haloperidol had no such effect on striatal dopamine subtype‐1 (D1) and adenosine subtype‐1 (A1) receptors. Binding characteristics and the pharmacological displacement profile of the increased [3H]CGS‐21680 binding sites confirmed them as A2a receptors. Comparing different classes of neuroleptics showed that the typical neuroleptics haloperidol and fluphenazine (1.5 mg/kg/day) increased D2 receptor densities, whereas the atypical neuroleptics sulpiride (100 mg/kg/day) and clozapine (20 mg/kg/day) did not (control, 290.3 ± 8.7 fmol/mg of protein; haloperidol, 358.1 ± 6.9 fmol/mg of protein; fluphenazine, 381.3 ± 13.6 fmol/mg of protein; sulpiride, 319.8 ± 18.9 fmol/mg of protein; clozapine, 309.2 ± 13.7 fmol/mg of protein). Similarly, the typical neuroleptics increased A2a receptor densities, whereas the atypical neuroleptics did not (control, 536.9 ± 8.7 fmol/mg of protein; haloperidol, 687.9 ± 28.0 fmol/mg of protein; fluphenazine, 701.1 ± 31.6 fmol/mg of protein; sulpiride, 563.3 ± 27.2 fmol/mg of protein; clozapine, 550.9 ± 40.9 fmol/mg of protein). There were no differences in affinities for [3H]CGS‐21680 or [3H]sulpiride among the various treatment groups. This study demonstrates that typical neuroleptics induce comparable up‐regulation in both striatal D2 and A2a receptors. Thus, A2a receptors might be a pharmacologic target for the development of novel therapeutic strategies to minimize the adverse effects of antipsychotic treatment.
Ca2+‐Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin‐Dependent Kinases
Authors: Uchikawa Tomoyoshi , Kiuchi Yuji , Yura Akihiko , Nakachi Noriyuki , Yamazaki Yukako , Yokomizo Chie , Oguchi Katsuji
Published: July 2010

Abstract: We studied effects of Ca2+ in the incubation medium on [3H]dopamine ([3H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca2+ (0–30 min) and Ca2+ concentration (0–10 mM) in Krebs‐Ringer medium affected [3H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 mM Ca2+ after a 10‐min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in Vmax of the [3H]DA uptake process. On the other hand, [3H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 mM Ca2+. The Ca2+‐dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca2+‐free medium following preincubation with 1 mM Ca2+. Protein kinase C inhibitors did not affect apparently Ca2+‐dependent enhancement of the uptake, whereas 1‐[N,O‐bis(1,5‐isoquinolinesulfonyl)‐N‐methyl‐l‐tyrosyl]‐4‐phenylpiperazine (KN‐62; a Ca2+/calmodulin‐dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN‐62 and wortmannin appeared to be additive. N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide hydrochloride (W‐7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca2+‐dependent enhancement of [3H]DA uptake is mediated by activation of calmodulin‐dependent protein kinases.
Two Distinct ATP Receptors Activate Calcium Entry and Internal Calcium Release in Bovine Chromaffin Cells
Authors: Reichsman F. , Santos S. , Westhead E. W.
Published: July 2010

Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca2+ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP‐induced rise in Ca2+ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca2+ causes secretion; the primary role of Ca2+ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca2+ increases. The agonist potency order for the increase in fluorescence from fura‐2‐loaded chromaffin cells due to release of Ca2+ from internal stores is ATP = UTP > ADP > 2‐methylthio‐ATP, α,β‐methylene ATP, identifying the receptor as a P2U purinoceptor. The potency order for secretion is 2‐methylthio‐ATP > ATP > α,β‐methylene ATP, ADP, UTP, placing the receptor in the P2Y subtype. Thus, two distinct receptors are responsible for Ca2+ release and secretion. Agonists were more effective in the absence of extracellular Mg2+, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage‐dependent Ca2+ current and secretion.
The Tyrosine Kinase Inhibitor Genistein Increases Endogenous Dopamine Release from Normal and Weaver Mutant Mouse Striatal Slices
Authors: Bare D. J. , Ghetti B. , Richter J. A.
Published: July 2010

Abstract: Protein tyrosine kinases that are known to have major roles in the control of cell growth and transformation are abundant and have numerous phosphoprotein substrates in the adult CNS. Although less well characterized than serine/threonine kinases, tyrosine kinases are also concentrated in the synapse. The effect of genistein, a selective inhibitor of tyrosine kinase activity, was examined on the in vitro release of endogenous dopamine (DA) from superfused mouse striatal slices. Fractional release of DA was significantly increased over basal release levels by genistein (100 and 200 µM). The effect was concentration dependent and rapidly reversible on washout of the kinase inhibitor. No significant change from basal release levels was observed with two structural analogues of genistein that do not inhibit tyrosine kinase activity at the same concentration. We have previously described alterations in basal and evoked DA release from the striatum of the weaver (wv/wv) mutant mouse, and genotypic differences in fractional release were also observed with genistein stimulation. The total evoked release was 25–50% greater from the wv/wv striatum. These results suggest a modulatory role for tyrosine kinase activity in neurotransmitter release and perhaps an alteration of kinase‐regulated mechanisms in the DA‐deficient wv/wv striatum.
The F3 Neuronal Glycosylphosphatidylinositol‐Linked Molecule Is Localized to Glycolipid‐Enriched Membrane Subdomains and Interacts with L1 and Fyn Kinase in Cerebellum
Authors: Olive Sylviane , Dubois Catherine , Schachner Melitta , Rougon Geneviève
Published: July 2010

Abstract: The F3 molecule is a member of the immunoglobulin superfamily anchored to plasma membranes by a glycosylphosphatidylinositol group. In adult mouse cerebellum, F3 is predominantly expressed on a subset of axons, the parallel fibers, and at their synapses. In vitro studies established that it is a plurifunctional molecule that, depending on the cellular context and the ligand with which it interacts, either mediates repulsive interactions or promotes neurite outgrowth. In the present study, we report the isolation of two fractions of F3‐containing microdomains from adult cerebellum on the basis of their resistance to solubilization by Triton X‐100 at 4°C. Both fractions were composed of vesicles, ranging from 100 to 200 nm in diameter. Lipid composition analysis indicated that the lighter fraction was enriched in cerebrosides and sulfatides. F3 sensitivity to phosphatidylinositol phospholipase C differed between the two fractions, possibly reflecting structural differences in the lipid anchor of the F3 molecule. Both fractions were highly enriched in other glycosylphosphatidylinositol‐anchored proteins such as NCAM 120 and Thy‐1. It is interesting that these vesicles were devoid of the transmembrane forms (NCAM 180 and NCAM 140), which were recovered in Triton X‐100‐soluble fractions, but contained the L1 transmembrane adhesion molecule that is coexpressed with F3 on parallel fibers and the fyn tyrosine kinase. Immunoprecipitation experiments indicated that F3, but not NCAM 120 or Thy‐1, was physically associated in a complex with both L1 and fyn tyrosine kinase. This strongly suggests that the interaction between L1 and F3, already described to occur with isolated molecules, is present in neural tissue. More important is that our study provides information on the molecular machinery likely to be involved in F3 signaling.
Comparison of the Molecular Forms of the Kex2/Subtilisin‐Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl‐Terminus Truncation and Membrane Association
Authors: Hill R. M. , Ledgerwood E. C. , Brennan S. O. , Pu L.‐P. , Loh Y. P. , Christie D. L. , Birch N. P.
Published: July 2010

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full‐length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full‐length 86‐kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl‐terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane‐associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86‐kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus‐truncated 64‐kDa form.
Differential Oligodendroglial Expression of the Tumor Necrosis Factor Receptors In Vivo and In Vitro
Authors: Tchélingérian Jean‐Léon , Monge Madeleine , Le Saux Françoise , Zalc Bernard , Jacque Claude
Published: July 2010

Abstract: The cytokine tumor necrosis factor‐α (TNFα) has been proposed to play a key role in the degenerative processes observed in demyelinating diseases such as multiple sclerosis (MS). In the immune system the cellular responses to TNF are mediated by two different receptors: TNF‐RI, which is involved in cell death, and TNF‐RII, which has been shown to mediate cell proliferation. We investigated the oligodendroglial expression of TNF‐RI and ‐RII. In vivo, in normal adult rodent brain, oligodendrocytes express TNF‐RII but not TNF‐RI. However, after 3 days in culture, both types of receptors were expressed by mature oligodendrocytes, purified from 4‐week‐old rats, suggesting that expression of TNF‐RI was induced by either the isolation process or the culture conditions. This inducibility of TNF‐RI may explain the differences in oligodendrocyte cell death reported in various experimental conditions and in the pathology of MS lesions.
Molecular Cloning of α1d‐Adrenergic Receptor and Tissue Distribution of Three α1‐Adrenergic Receptor Subtypes in Mouse
Authors: Alonso‐Llamazares Ana , Zamanillo Daniel , Casanova Emilio , Ovalle Sergio , Calvo Pedro , Chinchetru Miguel A.
Published: July 2010

Abstract: A partial cDNA encoding most of the third intracellular loop of the mouse α1d‐adrenergic receptor subtype was amplified from hippocampus by reverse transcription‐polymerase chain reaction (RT‐PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an α1d‐adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0‐kb transcript. Partial cDNA fragments of the α1b‐ and α1a‐adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT‐PCR indicated that the α1‐adrenergic receptors are widely distributed in mouse tissues. The α1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The α1a subtype is also expressed in many tissues, whereas the α1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.
Cloning of the α Component of the Chick Ciliary Neurotrophic Factor Receptor: Developmental Expression and Down‐Regulation in Denervated Skeletal Muscle
Authors: Ip Fancy C. F. , Fu Amy K. Y. , Tsim Karl W. K. , Ip Nancy Y.
Published: July 2010

Abstract: A full‐length cDNA clone encoding for the chick CNTFRα (α component of the ciliary neurotrophic factor receptor) was isolated by screening an embryonic day 13 chick brain cDNA library with a rat CNTFRα probe. The isolated cDNA clone contained a ∼2‐kb insert with an open reading frame of 362 amino acids. The identification of this clone as chick CNTFRα was based on the homology in amino acid sequence (∼70%) with the rat and human CNTFRα. Hydropathy analysis revealed that the chick CNTFRα contains a hydrophobic region at the amino terminus that is typical of secretory signal peptides, as well as a hydrophobic region at the carboxyl terminus that is characteristic of glycosylphosphatidylinositol‐linked proteins. The expression of chick CNTFRα was developmentally regulated and was widely distributed in neural tissues, such as brain and spinal cord. In the periphery, chick CNTFRα transcript was expressed at high levels in the skeletal muscle and was only barely detectable in the liver. Unexpectedly, the expression of chick CNTFRα mRNA in skeletal muscle was decreased by ∼10‐fold at 1.5 days after denervation. This is in sharp contrast to the result previously obtained with CNTFRα in denervated rat muscle.
The Human Aromatic l‐Amino Acid Decarboxylase Gene Can Be Alternatively Spliced To Generate Unique Protein Isoforms
Authors: O'Malley Karen L. , Harmon Steve , Moffat Mark , Uhland‐Smith Ann , Wong Shou
Published: July 2010

Abstract: Aromatic l‐amino acid decarboxylase (AADC) is expressed in a wide variety of tissues, including those where it is known to convert l‐DOPA and 5‐hydroxytryptophan to dopamine and serotonin, respectively. AADC has been cloned from many species and shown to undergo alternative splicing within its 5′ untranslated region. Here, we report that the human AADC gene can undergo additional alternative splicing of exon 3, generating two different protein isoforms (termed AADC480 and AADC442). Both transcripts are widely expressed, with AADC442 predominating in many neuronal and nonneuronal tissues. When homogenates were prepared from COS‐7 cells transfected with expression vectors containing either cDNA, AADC480 catalyzed the decarboxylation of both l‐DOPA and 5‐hydroxytryptophan. AADC442 was inactive in either assay. These findings suggested that AADC442 may have a different function in non‐monoamine‐expressing tissues. Taken together, these results suggest that the human AADC gene undergoes complex processing, leading to the formation of both tissue‐specific transcripts as well as unique protein isoforms.
Tissue‐Specific Processing of the Neuroendocrine Protein VGF
Authors: Trani E. , Ciotti T. , Rinaldi A. M. , Canu N. , Ferri G. L. , Levi A. , Possenti R.
Published: July 2010

Abstract: VGF is a neuroendocrine‐specific gene product that is up‐regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N‐terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C‐terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low‐molecular‐weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.
Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)‐1‐Aminocyclopentane‐trans‐1,3‐Dicarboxylic Acid‐Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells
Authors: Gorman Adrienne M. , Grieve Angus , Griffiths Roger
Published: July 2010

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (trans‐ACPD) was studied in mouse cerebellar granule cells. In Mg2+‐containing buffers, NMDA (50–100 µM), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans‐ACPD. Potentiation of the trans‐ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans‐ACPD‐stimulated phosphoinositide hydrolysis was blocked by d,l‐2‐amino‐5‐phosphonopentanoic acid (APV), a competitive NMDA‐receptor antagonist. Under Mg2+‐free conditions, the accumulation of inositol phosphates in the presence of trans‐ACPD alone was equal to that attained by trans‐ACPD in Mg2+‐containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans‐ACPD potentiated synergistically the NMDA‐evoked increases in cytosolic free‐Ca2+ levels in Mg2+‐containing but not in Mg2+‐free solutions, and moreover did not enhance the AMPA‐evoked increases in cytosolic free‐Ca2+ levels. The calcium ionophore A23187 caused a dose‐dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans‐ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein‐coupled phospholipase C activation and regulated elevation of cytosolic free‐Ca2+ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.
Tyrosine Kinase Activity Is Essential for Interleukin‐1β‐Stimulated Production of Interleukin‐6 in U373 Human Astrocytoma Cells
Authors: Carlson Robert O. , Aschmies Suzan H.
Published: July 2010

Abstract: We have investigated the roles of tyrosine kinase and protein kinase C activity in interleukin‐1β‐induced interleukin‐6 production, using the U373 human astrocytoma cell line as a model system for astrocytes. Compounds known to inhibit tyrosine kinases were tested for effects on interleukin‐6 production in U373 cells stimulated with interleukin‐1β. Complete to nearly complete inhibition of interleukin‐1β‐induced interleukin‐6 production was observed with the flavonoids genistein and quercetin, the bisindole alkaloids staurosporine and K‐252a, or the tyrphostin AG879. Herbimycin A was a potent inhibitor but did not induce complete inhibition at saturating dose. Calphostin C, an inhibitor of protein kinase C, also completely inhibited interleukin‐6 production. The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate induced interleukin‐6 production, and treatment with a combination of this phorbol ester and interleukin‐1 produced synergistic stimulation. Prolonged exposure to phorbol ester eliminated subsequent stimulation by phorbol ester but only partially decreased interleukin‐1‐induced interleukin‐6 and had no effect on the activities of selected inhibitors including calphostin C. We conclude that tyrosine kinase activity is essential for interleukin‐1‐induced interleukin‐6 production in U373 astrocytoma cells and that activity of a phorbol ester‐insensitive, atypical protein kinase C isozyme may also be involved.
Ischemia‐Triggered Translocation and Inactivation of Protein Kinase C Isoforms in the Fetal Brain
Authors: Yavin Ephraim , Inserte‐Igual Javier , Gil Shosh
Published: July 2010

Abstract: The kinetics of protein kinase C (PKC) translocation and down‐regulation in the 20‐day‐old fetal brain following short and long episodes of maternal‐fetal blood flow occlusion were examined. Restriction for up to 15 min increased the specific enzymatic activity in the membrane by 73%, indicative of translocation. After a 30‐min restriction and a 2.5‐h reperfusion the total PKC activity in the cytosol was reduced to ∼50%, consistent with down‐regulation/inactivation. The total membrane PKC activity remained unchanged. Several PKC isoenzymes, including α, β1, β2, ε, and ζ, but not γ, were identified in the fetal brain on western blots using specific antibodies. Compared with postnatal day 15, a greater proportion of the fetal PKC isoforms, particularly α and ε, were membrane bound. α, β2, ε, and ζ, but not β1, were translocated into the membrane compartment after episodes of ischemia alone or ischemia and reperfusion. There were no major identifiable proteolytic fragments in the 50‐kDa region. Major losses in the total enzymatic activity were encountered in both cytosol and membrane fractions after storage of the enzyme for 10 days at 4°C. These losses were less profound in membrane fractions from ischemic than control animals, suggesting a relative sparing of activity in the membrane as a result of the insult. Preincubation of DEAE‐purified PKC for 30 min at 50°C resulted in enzyme inactivation. This was accompanied by a size reduction (∼2–5 kDa) in the gel migration of several isozymes in both cytosol and membrane fractions. At 42°C, although the molecular size was apparently reduced, limited PKC activity was observed, suggesting either that the two processes are not mutually related or that certain PKC isoforms can act after partial modification. The data suggest that ischemic episodes stimulate two apparently adverse processes in the PKC signal transduction cascade: a decline in the cytosol and a sparing of the membrane‐translocated PKC activity. The latter may provide an important regulatory mechanism for PKC long‐term activation in nerve cells.
Cyclooxygenase‐2 Induction in Cerebral Cortex: An Intracellular Response to Synaptic Excitation
Authors: Adams Julian , Collaço‐Moraes Yolanda , De Belleroche Jacqueline
Published: July 2010

Abstract: We have characterised the induction of the mitogen‐inducible form of cyclooxygenase, COX‐2, in the rat cerebral cortex in response to excitotoxin injection into the nucleus basalis. This model is associated with intense stimulation of the ascending pathway to the cerebral cortex, seizure activity, and subsequent ipsilateral cortical induction of various immediate early genes (IEGs), including c‐fos, c‐jun, and zif268, and ornithine decarboxylase enzyme activity and mRNA, all of which processes are sensitive to treatment with the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801. In this study we show that excitotoxin injection also causes a marked induction of COX‐2 mRNA in ipsilateral cortex detectable at 1 h and peaking at 4 h, where COX‐2 mRNA levels were 19 times those in unoperated animals. Levels of COX‐2 mRNA remained significantly elevated at 24 h. The early induction of COX‐2 at 1 h was also seen in sham‐operated animals, but at 4 h the COX‐2 mRNA level was significantly increased (4.4‐fold) in animals injected with excitotoxin compared with sham‐operated animals. The induction at this time point (4 h) was explored pharmacologically and found to be significantly attenuated by treatment with MK‐801 (1.5 mg/kg), lamotrigine (10 mg/kg), which prevents presynaptic glutamate release by blocking voltage‐sensitive Na+ channels, and the glucocorticoid dexamethasone (3 mg/kg), which has an indirect inhibitory effect on phospholipase A2 and COX activity. These results demonstrate that the induction of COX‐2 mRNA occurs by two distinct mechanisms: the rapid and transient response to tissue damage and a second delayed and more substantial response, which is initiated by excitotoxin stimulation and is mediated by presynaptic glutamate release, NMDA receptor activation, and subsequent phospholipase A2 activity. We propose a model to demonstrate the similarities between COX‐2 and IEG mRNA induction and highlight possible mechanistic differences in the nature of the induction by the phospholipase A2 pathway.
Solubilization of High‐Affinity, Guanine Nucleotide‐Sensitive μ‐Opioid Receptors from Rat Brain Membranes
Authors: Weems Henri B. , Chalecka‐Franaszek Elzbieta , Côté Thomas E.
Published: July 2010

Abstract: High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐selective opioid agonist, with high affinity (KD = 1.90 ± 0.93 nM; Bmax = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [3H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 ± 0.39 nM, Bmax = 920 ± 154 fmol/mg of protein). Guanosine 5′‐O‐(3‐thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two‐site than a one‐site model with KDhigh = 2.17 ± 1.5 nM, Bmax = 648 ± 110 fmol/mg of protein and KDlow = 468 ± 63 nM, Bmax = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with KD = 815 ± 33 nM, Bmax = 965 ± 124 fmol/mg of protein.
Differential Effect of NMDA on Extracellular Serotonin in Rat Midbrain Raphe and Forebrain Sites
Authors: Tao Rui , Auerbach Sidney B.
Published: July 2010

Abstract: The contribution of NMDA receptors to regulation of serotonin (5‐HT) release was assessed by in vivo microdialysis in freely behaving rats. During infusion of NMDA (30, 100, and 300 µM) into the dorsal raphe nucleus (DRN), 5‐HT was increased by ∼25, 100, and 280%, respectively. Competitive and noncompetitive NMDA‐receptor antagonists blocked this effect on DRN 5‐HT. Infusion of NMDA (300 µM) into the DRN also produced an 80% increase in extracellular 5‐HT in the nucleus accumbens. During infusion of NMDA (100 and 300 µM) into the median raphe nucleus (MRN), 5‐HT was increased by ∼15 and 80%, respectively. NMDA‐receptor antagonists blocked this effect on MRN 5‐HT. Infusion of NMDA into the MRN also produced a significant increase in hippocampal 5‐HT. In contrast, infusion of NMDA into the nucleus accumbens, frontal cortex, or hippocampus produced small decreases in 5‐HT in these forebrain sites. Taken together, these results suggest that NMDA receptors in the midbrain raphe, but not the forebrain, can have an excitatory influence on 5‐HT neurons and, thus, produce increased 5‐HT release in the forebrain. Furthermore, in comparison with the MRN, DRN 5‐HT neurons were more sensitive to the excitatory effect of NMDA.
Real‐Time Effects of N‐Methyl‐d‐Aspartic Acid on Dopamine Release in Slices of Rat Caudate Putamen: A Study Using Fast Cyclic Voltammetry
Authors: Iravani M. M. , Kruk Z. L.
Published: July 2010

Abstract: The functional role of N‐methyl‐d‐aspartic acid (NMDA) glutamate receptors in the real‐time regulation of single electrical pulse (1 p)‐stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg2+, 20 µM NMDA had a weak effect on background signals but did not affect 1 p‐stimulated dopamine release. Removal of Mg2+ increased the background and doubled 1 p‐stimulated dopamine release. In the absence of Mg2+, 20 µM NMDA caused a transient “release” of dopamine and decreased the background signal. The 1 p‐stimulated dopamine release was subsequently reduced. In the presence of 1 µM (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP), superfusion with 20 µM NMDA did not cause a transient “release” of dopamine, and 1 p‐stimulated dopamine release was not subsequently attenuated. In the presence of 1 µM tetrodotoxin, 1 p‐stimulated dopamine release was abolished, but 20 µM NMDA still caused a transient “release” of dopamine. Removal of Ca2+ from the artificial CSF abolished 1 p‐stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine “release” when 20 µM NMDA was added. The dopamine release‐inducing effect of 20 µM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency‐dependent dopamine release (cold spots). Subsequent 1 p‐stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg2+, NMDA induces release of dopamine by acting at CPP‐sensitive NMDA receptors in a Ca2+‐independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real‐time observations of the effects of NMDA on electrical stimulus‐independent and ‐dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.
Differential Regulation of Histamine‐ and Bradykinin‐Stimulated Phospholipase C in Adrenal Chromaffin Cells: Evidence for Involvement of Different Protein Kinase C Isoforms
Authors: Sena Cristina M. , Rosário Luís M. , Parker Peter J. , Patel Viral , Boarder Michael R.
Published: July 2010

Abstract: In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC‐α, ε, and ζ were identified. To characterize down‐regulation of these enzymes, cells were incubated for 6–48 h with 1 µM phorbol myristate acetate (PMA). PKC‐ε down‐regulated more rapidly than PKC‐α. At 12 h, PMA pretreatment, for example, PKC‐ε was maximally down‐regulated (23 ± 4% of controls), whereas PKC‐α was unchanged. PKC‐α showed partial down‐regulation by 24 h of PMA pretreatment. PKC‐ζ did not down‐regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC‐ε than for PKC‐α. The accumulation of total 3H‐inositol (poly)phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10‐min PMA treatment (1 µM). However, with 12‐h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC‐ε, whereas the restoration of the histamine response corresponds to the loss of PKC‐α. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down‐regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC‐linked receptors in chromafin cells are differentially regulated by PKC isoforms.