bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of Quinolinic Acid by 3‐Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro
Authors: Foster Alan C. , White Robert J. , Schwarcz Robert 
Published: July 2010

AbstractIn mammalian peripheral organs, 3‐hydroxy‐anthranilic acid oxygenase (3HAO), catalyzing the conversion of 3‐hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HA0 in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy‐14C]3‐hydroxy‐anthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chro‐matographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed aKmof 3.6 ± 0.5 μMfor 3‐hydroxyanthranilic acid and aVmaxof 13.1± 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3‐hydroxyanthranilic acid caused <25% inhibition of activity at 500 μM. Both the Fe2+dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10‐fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at — 80°C. Taken together, these data indicate that measurements of brain 3HA0 may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.
Effects of Purine Nucleotides on the Binding of [3H]Cyclopentyladenosine to Adenosine A‐l Receptors in Rat Brain Membranes
Authors: Williams Michael , Braunwalder Albert 
Published: July 2010

AbstractAdenine nucleotides displace the binding of the selective adenosine A‐l receptor ligand [3H]cyclopentyl‐adenosine (CPA) to rat brain membranes in a concentration‐dependent manner, with the rank order of activity being ATP > ADP > AMP. Binding was also displaced by GTP, ITP, adenylylimidodiphosphate (AppNHp), 2‐meth‐ylthioATP, and the β‐γ‐methylene isostere of ATP, but was unaffected by the α‐β‐methylene isosteres of ADP and ATP, and UTP. At ATP concentrations >100 μM, the inhibitory effects on CPA binding were reversed, until at 2 mMATP, specific binding of CPA was identical to that seen in controls. Concentrations of ATP >10 mMtotally inhibited specific binding. Inclusion of the catabolic enzyme adenosine deaminase in the incubation medium abolished the inhibitory effects of ATP, indicating that these were due to adenosine formation, presumably due to ectonucleotidase activity. The inhibitory effects were also attenuated by the α‐β‐methylene isostere of ATP, an ectonucleotidase inhibitor. Adenosine deaminase, α‐β‐methylene ATP (100 μM), and β‐γ‐methylene ATP (100 μM) had no effect on the “stimulatory” phase of binding, although GTP (100 μM) slightly attenuated it. Comparison of the binding of [3H]CPA in the absence and presence of 2 mMATP by saturation analysis showed that theKDand apparent Bmaxvalues were identical. Examination of the pharmacology of the control and “ATP‐dependent” CPA binding sites showed slight changes in binding of adenosine agonists and antagonists. The responses observed with high concentrations of ATP were not observed with GTP, AppNHp, the chelating agents EDTA and EGTA, or inorganic phosphate. The divalent cations Mg2+and Ca2+at 10 mMattenuated the stimulatory actions of high (2 mM) concentrations of ATP, whereas EGTA and EDTA (10 mM) enhanced the “stimulatory” actions of ATP. EDTA (10 mM) abolished the inhibitory effects of ATP, indicating a specific dependence on Mg2+for the inhibitory response. The effects of ATP on [3H]CPA binding were reversible for antagonists but not agonists. The mechanism by which ATP reverses its own inhibitory action on adenosine A‐l radioligand binding is unclear, and from the observed actions of the divalent cations and chelating agents probably does not involve a phosphorylation‐dependent process.
Adenosine Inhibition of Calmodulin‐Sensitive Adenylate Cyclase from Bovine Cerebral Cortex
Authors: Yeager Robert E. , Nelson Richard , Storm Daniel R. 
Published: July 2010

AbstractCalmodulin (CaM)‐sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM‐sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3‐[3‐(cholamido‐propyl)‐dimethylammonio]‐propanesulfonate and Mn2+retained sensitivity to adenosine inhibition. TheKifor adenosine inhibition of the CaM‐sensitive adenylate cyclase was approximately 2.6 × 10‐4M. 5′‐Guanylylimi‐dodiphosphate and CaM did not affect theKiof 3′‐deoxy‐adenosine for the enzyme, but the presence of Ca2+in the millimolar range raised theKiby a factor of 5. These results show that the CaM‐sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine‐specific (“P”) site located on the catalytic subunit of the enzyme.
Posttranslational Modification of Calmodulin in Rat Brain and Pituitary
Authors: Murtaugh Timothy J. , Wright Lynda S. , Siegel Frank L. 
Published: July 2010

AbstractThe posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N‐methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl‐3H]‐S‐adenosyl‐l‐methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobaltstimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N‐methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.
Production of Monoclonal Antibody to 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase from Bovine Cerebral White Matter
Authors: Fujishiro Masatoshi , Kohsaka Shinichi , Nagaike Kazuhiro , Tsukada Yasuzo 
Published: July 2010

AbstractLewis rats were immunized with partially purified 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) from bovine cerebral white matter and the spleen cells were fused with cell of a mouse myeloma cell line (SP‐2). The production of monoclonal antibody was detected by (1) enzyme‐linked immunoadsorbent assay, (2) immunohistochemical staining of bovine cerebrum, (3) Western blotting analysis, and (4) CNPase binding assay. Monoclonal antibody that specifically binds CNPase molecules was obtained. However, the antibody did not suppress the enzyme activity. Western blotting analysis demonstrated that the monoclonal antibody binds both CNa (Wla) and CNb (Wlb). The monoclonal antibody was identified as being of the IgG2c subclass. Immunohistochemical examination revealed that the myelin sheath in the CNS was heavily stained with the monoclonal antibody in several species (bovine, mouse, rat, and human). In contrast, peripheral nervous system myelin was not stained even in bovine tissue. These results suggest that the monoclonal antibody obtained in the present study specifically recognizes the CNPase molecules in the CNS.
Identification of the D2‐‐Dopamine Receptor Binding Subunit in Several Mammalian Tissues and Species by Photoaffinity Labeling
Authors: Amlaiky Nourdine , Caron Marc G. 
Published: July 2010

AbstractPhotoaffinity labeling of the D2‐‐dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2‐‐dopaminergic antagonist probe [125I]N‐(p‐azidophenethyl)spiperone ([125I]N3‐‐NAPS). In tissues containing D2‐‐receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr= 94,000. Specificity of the labeling is typically D2‐‐dopaminergic in character. The covalent labeling is blocked by (+)‐butaclamol but not by the inactive (‐) isomer. Agonists block incorporation with the order of potency:N‐n‐propylnorapomorphine > apomorphine > dopamine. The D2‐‐selective antagonist spiperrone blocks labeling of the Mr= 94,000 peptide whereas the D1‐‐selective antagonist SCH‐23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2‐‐dopamine receptor resides on a Mr= 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr(32–38,000) are labeled at the expense of the Mr= 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mrreceptor fragments might be the result of a metal‐dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2‐‐receptors. [125I]N3‐‐NAPS specifically labels a major peptide of Mr± 120,000 inadditionto the Mr= 94,000 peptide. This peptide may represent an unprocessed or differently processed form of the receptor or a pharmacologically similar but biochemically distinct form of the receptor in this tissue. Alternatively, the Mr= 94,000 peptide labeled in all the other tissues, even under the conditions used, may already represent a fragment of this high‐Mrpeptide.
Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5‐Hydroxytryptamine in the Rat Hypothalamus
Authors: Lookingland K. J. , Shannon N. J. , Chapin D. S. , Moore K. E. 
Published: July 2010

AbstractThe effects of tryptophan administration on neurochemical estimates of synthesis [5‐hydroxytryptophan (5‐HTP) accumulation following administration of a decarboxylase inhibitor], storage [5‐hydroxytryptamine (5‐HT) concentrations], and metabolism [5‐hydroxyin‐doleacetic acid (5‐HIAA) concentrations] of 5‐HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30–300 mg/kg i.p.) produced a dose‐dependent increase in the rate of 5‐HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4‐dihydroxyphen‐ylalanine. Peak 5‐HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose‐dependent increases in 5‐HT and 5‐HIAA concentrations in these same regions without changes in the 5‐HIAA/5‐HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5‐HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5‐HT uptake with chlorimipramine or fluoxetine produced modest (10–40%) reductions in 5‐HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5‐HIAA is derived from released and recaptured 5‐HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5‐HT. In both chlorimipramine‐ and fluoxetine‐treated rats, 5‐HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5‐HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5‐HT.
Characterizaton and Biosynthesis of the Plasma Membrane Proteolipid Protein in Neural Tissue
Authors: Fischer Itzhak , Sapirstein Victor S. 
Published: July 2010

AbstractIn this study we have characterized, in brain, the expression of a plasma membrane proteolipid protein (PM‐PLP) complex that can form cation‐selective channels in lipid bilayers. We isolated PLP fractions from synaptic plasma membrane and glial microsomes and found a high degree of similarity in both size and amino acid composition to the complex we had previously isolated from kidney. Antibodies specific to the kidney PM‐PLP were prepared, and, on the basis of immunoblot and immunoprecipitation studies, the PM‐PLP complex isolated from neural membranes was shown to be immuno‐logically related to the kidney PM‐PLP. These proteolipid proteins exhibited a molecular weight of approximately 14K and contained a high percentage of hydrophobic amino acids with an apparent absence of cysteine. The biogenesis of PM‐PLP in brain was studied by in vitro translation of free and bound polysomes and total RNA in a rabbit reticulocyte lysate followed by immunoprecipitation of the translation products. From these studies it is concluded that the PM‐PLP complex is synthesized on the rough endoplasmic reticulum. On the basis of the identical electrophoretic mobility of material isolated from plasma membranes and material immunoprecipitated after translation of bound polysomes and isolated RNA, it appears that the PM‐PLP does not undergo detectable posttranslational processing between its site of synthesis and its incorporation into the plasma membrane.
Regulation of the Actin‐Activated Mg‐ATPase of Brain Myosin via Phosphorylation by the Brain Ca2+, Calmodulin‐Dependent Protein Kinases
Authors: Tanaka Etsuro , Fukunaga Kohji , Yamamoto Hideyuki , Iwasa Takafumi , Miyamoto Eishichi 
Published: July 2010

AbstractWe have previously isolated two Ca2+, calmodulin‐dependent protein kinases with molecular weights of 120,000 (120K enzyme) and 640,000 (640K enzyme), respectively, by gel filtration analysis from rat brain. Chicken gizzard myosin light‐chain kinase and the 120K enzyme phosphorylated two light chains of brain myosin, whereas the 640K enzyme phosphorylated both the two light chains and the heavy chain. The phosphopeptides of the light chains digested by Staphylococcus aureus V8 protease were similar among chicken gizzard myosin light‐chain kinase, the 120K enzyme, and the 640K enzyme. Only the seryl residue in the light chains and the heavy chain was phosphorylated by the enzymes. The phosphorylation of brain myosin by any of these enzymes led to an increase in actin‐activated Mg‐ATPase activity. The results suggest that brain myosin is regulated by brain Ca2+, calmodulin‐dependent protein kinases in a similar but distinct mechanism in comparison with that of smooth muscle myosin.
Pharmacological Characterization of Cholinergic Receptors in a Human Neuroblastoma Cell Line
Authors: Clementi F. , Cabrini D. , Gotti C. , Sher E. 
Published: July 2010

AbstractA human neuroblastoma cell line, IMR32, has been characterized as far as morphology, membrane receptors for neurotransmitters, and uptake and release of [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine). These cells expressed at their surface both nicotinic and muscarinic cholinergic receptors, revealed by [125I]α‐bungarotoxin and [3H]quinuclidinylbenzilate ([3H]QNB) binding, respectively. [125I]α‐Bungarotoxin binding was efficiently inhibited by α‐bungarotoxin, nicotine, carbachol, andd‐tubocurarine. [3H]QNB binding was competitively inhibited by atropine, pirenzepine, and carbachol. Hexamethonium did not affect the binding of either ligand. In competition experiments with [3H]QNB, pirenzepine recognized only one binding site with “low affinity,” and carbachol recognized two sites with different affinities, β‐adrenergic receptors were present in a very low amount, whereas α‐adrenergic and dopaminergic receptors were not detectable. IMR32 cells had an imipramine‐sensitive [3H]dopamine uptake, but carbachol, high levels of K+, the calcium ionophore A23187, and α‐latrotoxin were not able to induce release of [3H]dopamine that had been taken up. The ultrastructural analysis showed that IMR32 cells contained very few dense‐core vesicles, suggesting a low storage capacity for neurotransmitter. These cells could be an useful in vitro model for studying neurotransmitter receptors of the human CNS.
Regional Distribution of Immunoreactive‐Thyrotrophin‐Releasing Hormone and Substance P, and Indoleamines in Human Spinal Cord
Authors: Bennett G. W. , Nathan P. A. , Wong K. K. , Marsden C. A. 
Published: July 2010

Abstract:The regional distributions of thyrotrophin‐releasing hormone (TRH) and substance P in postmortem human spinal cord were determined by radioimmunoassay in fresh tissue taken from 22 patients who died without known neurological disease. Dorsal, ventral, and intermediolateral spinal cord regions were obtained from different segmental levels (lumbar L1, 2, 3, and 4; thoracic groups T1–3, T4–6, T7–9, and T10–12) together with selective regions of grey matter of lumbar spinal cord. The effects on peptide levels of (a) the age of the patient, (b) the postmortem time interval, and (c) freezing the tissue samples prior to assay were assessed. Levels of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) were determined in regional lumbar and thoracic tissue using HPLC with electrochemical detection. Substance P was found in the highest concentration in the dorsal spinal cord, with no significant segmental differences. In contrast, TRH was present in higher levels in the ventral rather than the dorsal spinal cord, with segmental differences. There was a significant difference in the 5‐HT/5‐HIAA ratio between dorsal and ventral spinal cord, with the highest ratio in the ventral spinal cord. There were no significant differences in substance P, TRH, or 5‐HT levels in spinal cords between 5 and 20 h postmortem or from patients aged between 65 and 90 years. Freezing the tissue (– 80°C for 24 h) prior to assay significantly reduced TRH and substance P levels compared to samples assayed immediately without prior freezing. The levels and distribution of peptides and indoleamines in human spinal cord are compared with those previously reported in the rat and other species and the possible roles of these substances in the control of motor function and autonomic control are discussed.
Measurement of Endogenous Noradrenaline Release in the Rat Cerebral Cortex In Vivo by Transcortical Dialysis: Effects of Drugs Affecting Noradrenergic Transmission
Authors: L'Heureux Rémy , Dennis Trevor , Curet Olivier , Scatton Bernard 
Published: July 2010

Abstract:The release of endogenous noradrenaline was measured in the cerebral cortex of the halothane‐anesthetized rat by using the technique of brain dialysis coupled to a radioenzymatic assay. A thin dialysis tube was inserted transversally in the cerebral cortex (transcortical dialysis) and perfused with Ringer medium (2 μl min−1). Under basal conditions, the cortical output of noradrenaline was stable over a period of at least 6 h and amounted to 8.7 pg/20 min (not corrected for recovery). Histological control of the perfused area revealed very little damage and normal morphology in the vicinity of the dialysis tube. Omission of calcium from the perfusion medium caused a marked drop in cortical noradrenaline output. Bilateral electrical stimulation (for 10 min) of the ascending noradrenergic pathways in the medial forebrain bundle caused a frequency‐dependent increase in cortical noradrenaline output over the range 5–20 Hz. Stimulation at a higher frequency (50 Hz) resulted in a levelling off of the increase in cortical noradrenaline release. Systemic administration of the dopamine‐β‐hydroxylase inhibitor bis‐(4‐methyl‐1‐homopiperazinylthiocarbonyl) disulfide (FLA 63) (25 mg/kg i.p.) markedly reduced, whereas injection of the monoamine oxidase inhibitor pargyline (75 mg/kg i.p.) resulted in a progressive increase in, cortical noradrenaline output.d‐Amphetamine (2 mg/kg i.p.) provoked a sharp increase in cortical noradrenaline release (+ 450% over basal values within 40 min). Desmethylimipramine (10 mg/kg i.p.) produced a twofold increase of cortical noradrenaline release. Finally, idazoxan (20 mg/kg i.p.) and clonidine (0.3 mg/kg i.p.), respectively, increased and decreased the release of noradrenaline from the cerebral cortex. It is concluded that the technique of transcortical dialysis is a suitable means to investigate the effect of centrally acting drugs on the release of endogenous noradrenaline in the cerebral cortex of the rat.
Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit
Authors: Jackisch Rolf , Geppert Martin , Illes Peter 
Published: July 2010

Abstract:Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 μMcocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential K‐agonists ethylketocyclazocine, dynorphin A1‐13, dynorphin A,trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]‐benzeneacetamide (U‐50,488), and (–)‐5,9‐dimethyl‐2′‐OH‐2‐tetrahydrofurfuryl‐6,7‐benzomorphan [(–)‐MR 2034], whereas (+)‐MR 2035 [the (+)‐enantiomer of (–)‐MR 2034] was ineffective. In contrast, the preferential δ‐agonists Leu‐enkephalin, Met‐enkephalin, and D‐Ala2,‐D‐Leu5‐enkephalin (DADLE) as well as the μ‐agonists morphine, normorphine, D‐Ala2Gly‐ol5‐enkephalin (DAGO), and β‐casomorphin1‐4amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 μM) was much more potent than ethylketocyclazocine (1 μM) or DADLE (1 μM). (–)‐N‐(3‐furylmethyl)‐α‐noretazocine [(–)‐MR 2266], 1 μM, a preferential K‐antagonist, antagonized the effect of ethylketocyclazocine more potently than (–)‐naloxone or (+)‐MR 2267 [the (+)‐enantiomer of (–)‐MR 2266]. Given alone, (–)‐MR 2266 slightly and (+)‐MR 2267 (1 μMeach) greatly enhanced NA release, apparently due to α2‐adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 μM). The effects of DADLE (1 μM) and DAGO (1 μM) were also antagonized by (–)MR 2266 (0.1 μM) but not by the δ‐antagonistN,N‐diallyl‐Tyr‐Aib‐Phe‐Leu‐OH (ICI 174864), 0.3 μM. It is concluded that NA release in the rabbit hippocampus is inhibited via K‐receptors; our results do not support the presence of modulatory μ‐ and δ‐receptors in this tissue. However, in the rat hippocampus μ‐receptors may modulate NA release.