bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Serotonin Receptors Recognized by Anti‐Idiotypic Antibodies
Authors: Tamir Hadassah , Liu Kuo‐peing , Hsiung Shu‐chi , Yu Peiying , Kirchgessner Annette L. , Gershon Michael D. 
Published: October 2006

AbstractAnti‐idiotypic antibodies were generated by immunizing rabbits with affinity‐purified antibodies to serotonin (5‐hydroxytryptamine; 5‐HT). Anti‐5‐HT activity was removed from the resulting antisera by chromatography through a 5‐HT affinity column. The anti‐idiotypic antibodies were demonstrated by enzyme‐linked immunosorbent assay to bind to affinity‐purified whole anti‐5‐HT antibodies and their Fab fragments. Anti‐idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5‐HT were coupled, competed with 5‐HT (covalently bound to protein) for the binding sites on anti‐5‐HT antibodies and serotonin binding protein. The anti‐idiotypic antibodies antagonized the binding of [3H]5‐HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti‐ idiotypic antibodies also blocked the binding of the 5‐HT1B‐ selective ligand (‐)‐[125I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti‐idiotypic antibodies failed to inhibit binding of the 5‐ HT1A‐selective ligand 8‐hydroxy‐2‐(di‐n)‐[3H]propylamino)‐ tetralin ([3H]8‐OH‐DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti‐idiotypic antibodies may recognize some 5‐HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti‐idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5‐ HT1C or 5‐HT2 receptor or with a genomic clone encoding the 5‐HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5‐ HT1C and 5‐HT2 receptors, but not on the surfaces of HeLa cells that expressed 5‐HT1A receptors. Immunostaining of cells by the anti‐idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5‐HT1C receptors was blocked by mesulergine (but not ketanserin, 8‐OH‐DPAT. or spiperone), whereas that of cells expressing 5‐HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8‐OH‐DPAT). The anti‐ idiotypic antibodies failed to inhibit the uptake of [3H]5‐HT by serotonergic neurons. It is concluded that the anti‐idiotypic antibodies generated with anti‐5‐HT serum recognize the 5‐ HT1B, 5‐HTlC, and 5‐HT2 receptor subtypes; however, neither 5‐HT1A receptors nor 5‐HT uptake sites appear to react with these antibodies.
Binding of Botulinum and Tetanus Neurotoxins to Ganglioside GT1b and Derivatives Thereof
Authors: Schengrund Cara‐Lynne , DasGupta Bibhuti R. , Ringler Nancy J. 
Published: October 2006

AbstractThe ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b‐coated plastic wells was investigated and compared with the binding characteristics of the parent ∼ 150‐kDa protein. Although the ∼50‐kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b‐coated wells was exhibited by the ∼ 50‐kDa carboxy‐terminal half of the ∼ 100‐kDa heavy chain of BTxA; the amino‐terminal half of the heavy chain lacked the ability to bind. Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy‐terminal half of its heavy chain. Binding of BTxA and the C‐terminal half of the heavy chain was optimal in buffers of low ionic strength (μ≤ 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at μ≤ 0.10. TTx and the ∼ 50‐kDa C‐terminal half of its ∼ 100‐kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA. Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b. Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by ∼60 and ∼90%, respectively. Determination of the IC50 values for GT1b derivatives (molar concentration needed to reduce toxin binding to GT1b by 50%) indicated that (a) the carboxyl moiety of the sialosyl residues is recognized by BTxA and TTx, (b) the lipid segment of GT1b enhances BTxA and TTx binding, and (c) the N‐acetyl groups and C(8)‐C(9) on the sialosyl residues are not essential components of the binding site for BTxA and TTx.
Depolarization and Agonist‐Stimulated Changes in Inositol 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex
Authors: Challiss R. A. John , Nahorski Stefan R. 
Published: October 2006

AbstractMuscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and myo‐inositol 1,3,4,5‐tetrakis‐phosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values forcarbachol. Omission of buffer Ca2+ ([Ca2+]e 10–20 μM) reduced basal lns (1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K+‐evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L‐type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3‐kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.
Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin‐Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells
Authors: Purkiss John , Murrin Richard A. , Owen P. Jane , Boarder Michael R. 
Published: October 2006

AbstractThe role of lipid‐bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal Chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor‐linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase(DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of Chromaffin cells prelabelled (24 h) with 32P1 leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When Chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [γ‐32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.
Ca2+‐Independent Release of Glutamate During In Vitro Anoxia in Isolated Nerve Terminals
Authors: Rubio Inmaculada , Torres Magdalena , Miras‐Portugal María Teresa , Sánchez‐Prieto Jose 
Published: October 2006

AbstractThe effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extrasynaptosomal concentration of glutamate ([Glu]cxt) under aerobic conditions was 2.3 μM and increased to 4.9 μM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]cxt The accumulation of glutamate in the external medium during anoxia was Ca2+ in dependent and insensitive to a significant reduction of the Ca2+‐dependent release of the amino acid. These results indicate that a Ca2+‐independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.
Solubilized Rat Brain Adenosine Receptors Have Two High‐Affinity Binding Sites for l, 3‐Dipropyl‐8‐Cyclopentylxanthine
Authors: Oliveira Júlia C. , Sebastião A. M. , Ribeiro J. A. 
Published: October 2006

AbstractThe specific binding of L‐N6‐[3H]phenylisopropyladenosine (L‐[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L‐[3H]PIA (0.5–12.0 nA/) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 ± 0.03 and 0.66 ± 0.05 pmol/mg of protein. In the presence of GTP, the binding of L‐[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 ± 0.09 and 0.93 ± 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L‐[3H]PIA binding by 1, 3‐dipropyl‐8‐cydopentylxanthine (DPCPX) (0.1–300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 ± 0.02 and 0.74 ± 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.
Neuronal Survival Factor from Bovine Brain Is Identical to Neuron‐Specific Enolase
Authors: Takei Nobuyuki , Kondo Jun , Nagaike Kazuhiro , Ohsawa Keiko , Kato Kanefusa , Kohsaka Shinichi 
Published: October 2006

AbstractNeuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high‐speed centrif‐ugation was used as the starting material. At the step of DE‐52 ion‐exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti‐F14 antiserum recognized mainly a 30‐kDa protein in F14 and anti‐F23 antiserum recognized mainly a 44‐kDa protein in F23. After sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of F23, the 44‐kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron‐specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti‐NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose‐dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.
Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain
Authors: Raisman‐Vozari Rita , Hirsch Etienne , Javoy‐Agid France , Vassort Cecile , Savasta Marc , Feuerstein Claude , Thibault Jean , Agid Yves 
Published: October 2006

AbstractLevels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S‐labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high‐sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer‐assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S‐labelled antibody, the detection of TH could be performed at the cellular level.
Resolution of Biphasic Binding of the Opioid Antagonist Naltrexone in Brain Membranes
Authors: Remraers Ann E. , Medzihradsky Fedor 
Published: October 2006

AbstractIn synaptosomal membranes from rat brain cortex, in the presence of 150 mM NaC1, the opioid antagonist [3H] naltrexone bound to two populations of receptor sites with affinities of 0.27 and 4.3 nM, respectively. Guanosine‐5′‐(3‐thiotriphosphate) had little modulating effect and did not alter the biphasic nature of ligand binding. On the other hand, receptor‐selective opioids differentially inhibited the two binding components of [3H] naltrexone. As shown by nonlinear least‐squares analysis, the μ opioids Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol or sufentanil abolished high‐affinity [3H] naltrexone binding, whereas the δ‐selective ligands [D‐ Pen2, D‐Pen5] enkephalin, ICI 174, 864, and oxymorphindole inhibited and eventually eliminated the low‐affinity component in a concentration‐dependent manner. These results indicate that, in contrast to the guanine nucleotide‐sensitive biphasic binding of opioid‐alkaloid agonists, the heterogeneity of naltrexone binding in brain membranes reflects ligand interaction with different opioid‐receptor types.
Glutamic Acid Decarboxylase Activity of the Preoptic Area and Hypothalamus Is Influenced by the Serotonergic System
Authors: Munaro N. I. , Morello H. , Taleisnik S. 
Published: October 2006

AbstractThe effect of the serotonergic system on glutamic acid decarboxylase (GAD) activity of the preoptic area and the hypothalamus was studied in female rats on the day of proestrus. A circadian rhythm of GAD activity was observed with higher values in rats killed at 1130 h than in rats killed at 1500 h. In rats bearing lesions of the median raphe nucleus (MRn), a nucleus that sends 5‐hydroxytryptamine nerve terminals to the areas under study decreased GAD activity. On the contrary, electrochemical stimulation of the MRn enhanced GAD activity in intact rats killed at 1500 h, but not in those killed at 1130 h, an effect that was prevented by the injection of the 5‐hydroxytryptamine antagonist, methysergide. Furthermore, the injection of 5‐hydroxytryptamine into the third ventricle, either in intact rats in the afternoon or in MRn‐lesioned rats in the morning, also increased GAD activity. The results of the present study suggest that activation of the serotonergic system increases GAD activity in the preoptic area and hypothalamus.
Vasoactive Intestinal Peptide Stimulates Catecholamine Biosynthesis in Isolated Adrenal Chromaffin Cells: Evidence for a Cyclic AMP‐Dependent Phosphorylation and Activation of Tyrosine Hydroxylase
Authors: Waymire J. C. , Craviso G. L. , Lichteig K. , Johnston J. P. , Baldwin C. , Zigmond R. E. 
Published: October 2006

AbstractVasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel‐filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12‐O‐tetradecanoylphorbol 13‐acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP‐dependent phosphorylation of TH is responsible for these effects.
Changes in Extracellular Concentrations of Glutamate, Aspartate, Glycine, Dopamine, Serotonin, and Dopamine Metabolites After Transient Global Ischemia in the Rabbit Brain
Authors: Baker A. J. , Zornow M. H. , Scheller M. S. , Yaksh T. L. , Skilling S. R. , Smullin D. H. , Larson A. A. , Kuczenski R‐ 
Published: October 2006

AbstractAlthough considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmittiers (e.g., glycine and doparaine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1‐, 5‐, and 13‐fold and by 4‐, 9‐, and 31‐fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700‐fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4‐fold, but remained elevated throughout the 80‐min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann‐Whitney test). That glutamate and aspartate concentrations in the hippocampus co‐vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury. The observation that dopamine concentrations increased independently of ischemic duration indicates a maximal release with only S min of ischemia and suggests that dopamine's role in the incremental injury seen with increasing ischemic duration is limited to prolonged high concentrations rather than increasing peak levels as seen with glutamate and aspartate. The sustained elevation of glycine concentrations after ischemia may explain the ability of excitatory amino acids to produce delayed toxicity in the reperfusion phase, as glycine has been shown to facilitate glutamate's activity at the N‐ methyl‐D‐aspartate receptor.
Stimulation of γ‐[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur‐Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release
Authors: Dunlop John , Grieve Angus , Schousboe Arne , Griffiths Roger 
Published: October 2006

AbstractIn primary cultures of mouse cerebral cortex neurons, sulphur‐containing excitatory amino acids (SAAs; namely, L‐cysteine sulphinate, L‐cysteate, L‐homocysteine sulphinate, L‐homocysteate, S‐sulphocysteine) at concentrations ranging from 0.1 μM to 1 mM evoked a saturable release of ‐γ‐[3H] aminobutyric acid ([3H] GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50), 100–150 nM) in releasing [3H] GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H] tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA‐stimulated release of [3H] GABA was shown to comprise two distinct components, calcium‐dependent and calcium‐independent, which occur after activation of N‐methyl‐B‐aspartate (NMDA) and non‐NMDA receptors. Thus, all SAA‐evoked responses were antagonized by the selective, competitive NMDA‐receptor antagonist, 3‐[(±)‐2‐carboxypiperazin‐4‐yl] propyl‐1‐phosphonic acid (IC50range, >50 μM) and the non‐NMDA‐receptor antagonist, 6, 7‐dinitroquinoxalinedione (IC50 range, 5–50 nM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA‐evoked responses without having any effect on basal levels of [3H] GABA efflux, a result consistent with an involvement of NMDA‐receptor activation. Calcium‐independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 pMexcitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 fiMSKF‐89976‐A, a non‐ transportable inhibitor of theGABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are dearly consistent with a receptor‐mediated, depolarization‐induced reversal of the GABA carrier.