bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Photoaffinity 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

The 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

In 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

We 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

A 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.
Dihydrotetrabenazine Binding and Monoamine Uptake in Mouse Brain Regions
Authors: Scherman Daniel
Published: July 2010

Abstract:Binding of [2‐3H]dihydrotetrabenazine and uptake of 5‐hydroxytryptamine (serotonin) were studied in mouse brain cerebellum, pons‐medulla, frontal cortex, hypothalamus, hippocampus, and striatum. Binding of [2‐3H]dihydrotetrabenazine to homogenates of these brain areas is stable for several hours and occurs at a homogeneous class of binding sites (KD= 2.4 nM). Subcellular fractionation and regional distribution of [2‐3H]dihydrotetrabenazine binding and serotonin uptake showed that the ligand binds to synaptic vesicles. Dihydrotetrabenazine inhibited serotonin uptake with the same inhibitory constant (IC50= 2.6 nM) for synaptic vesicles from brain regions containing 3,4‐dihydroxy‐phenylethylamine (dopamine) or serotonin and noradrenaline in different proportions. This constant is similar to theKDof [2‐3H]dihydrotetrabenazine, which suggests that the latter ligand labels specifically and with the same affinity the monoamine transporter from various mono aminergic synaptic vesicles. Therefore the regional differences in central monoamine depletion induced in vivo by tetrabenazine are not due to regional differences in inhibition of vesicular monoamine uptake. Moreover, vesicular monoamine transporters from the central and peripheral nervous systems of various mammals and from bovine adrenal glands have comparable affinity for substrate and inhibitor (Kmvalues for serotonin and IC50for dihydrotetrabenazine are about 0.8 μMand 3 nM, respectively) and comparable turnover number (10–35 molecules transported per transporter per minute), which suggests the involvement of a common transporter molecule in the process of monoamine uptake by the various monoaminergic storage vesicles.
A Bioluminescence Method for the Measurement of l‐Glutamate: Applications to the Study of Changes in the Release of l‐Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats
Authors: Fosse Viggo M. , Kolstad Jens , Fonnum Frode
Published: July 2010

Abstract: We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of l‐glutamate (l‐Glu). Oxidation of l‐Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from < 0.5 to 500 pmol of l‐Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long‐term stability of the reagent mixture (> 8 h) were established. Bioluminescence analysis of L‐Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl‐evoked release of endogenous L‐Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.
Effects of S‐100 Proteins on Assembly of Brain Microtubule Proteins: Correlation Between Kinetic and Ultrastructural Data
Authors: Donato Rosario , Battaglia Francesco , Cocchia Domenico
Published: July 2010

Abstract: Microtubules formed in vitro in the presence of S‐100 proteins and micromolar Ca2+ concentrations are fewer in number and longer than those formed in the presence of Ca2+ alone. Moreover, microtubules growing after addition of microtubule fragments to a microtubule protein solution in the presence of S‐100 are shorter than those growing in its absence. These data lend support to previous results of kinetic studies indicating that S‐100 interferes with both the nucleation and the elongation of microtubules in vitro.
Rate of Protein Glycosylation in Rat Cerebral Cortex
Authors: Alperin Daniel Mario , Idoyaga‐Vargas Victor P. , Carminatti Hector
Published: July 2010

Abstract: Quantitative aspects of the pathway leading to the formation of asparagine‐linked oligosaccharides were investigated in rat cerebral cortex. Steady‐state labeling conditions were achieved with [2‐3H]mannose by developing a micromethod of incubation of cerebral cortex particles in the presence of physiological concentrations of glucose (1 g/L). The rate of [2‐3H]mannose uptake and incorporation into protein was markedly affected when the concentration of glucose was lowered to 0.05 g/L. It was found that in the presence of glucose (1 g/L), a minor fraction of the utilized [2‐3H]mannose is used in glyco‐protein formation and the remaining labeled sugar enters the other major metabolic pathways, generating tritiated water which is rapidly exchanged with that of the medium. Under these conditions, the intracellular isotopic dilution of [2‐3H]mannose‐labeled precursors was calculated to be about 11.5‐fold. These data allow determination of the rate of the net transfer of mannose into proteins. Comparison of the rate of glycosylation between 5‐ and 30‐day‐old cerebral cortex revealed a striking difference: 2.1 and 0.3 ng of mannose/mg protein/h, respectively.
Mutual Inhibition Kinetic Analysis of γ‐Aminobutyric Acid, Taurine, and β‐Alanine High‐Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β‐Alanine Carriers in Both Cell Types
Authors: Larsson O. M. , Griffiths R. , Allen I. C. , Schousboe A.
Published: July 2010

Abstract: The transport kinetics of γ‐aminobutyric acid (GABA), taurine, and β‐alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high‐affinity uptake system for each amino acid was demonstrated both in neurons (KmGABA= 24.9 ± 1.7 μM; KmTau= 20.0 ± 3.3 μM; Kmβ‐Ala= 73.0 ± 3.6 μM) and astrocytes (KmGABA= 31.4 ± 2.9 μM, KmTau= 24.7 ± 1.3 μM; Kmβ‐Ala= 70.8 ± 3.6 μM). The maximal uptake rates (Vmax) determined were such that, in neurons, VmaxGABA> Vmaxβ‐Ala=VmaxTau, whereas in astrocytes, Vmaxβ‐Ala > VmaxTau=VmaxGABA. Taurine was found to inhibit β‐alanine uptake into neurons and astro cytes in a competitive manner, with Ki values of 217 μM in neurons and 24 μM in astrocytes. β‐Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 μM in neurons and 71 μM in astrocytes. However, β‐alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of β‐alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons. Taken together, the similarity between the results obtained for β‐alanine and taurine uptake in addition to their competitive mode of mutual inhibition strongly suggest that these compounds share a common carrier in neurons and astrocytes and that neither amino acid in either cell type is transported appreciably, if at all, by the GABA carrier, and vice versa.
Characteristics of the Large Neutral Amino Acid Transport System of Bovine Brain Microvessel Endothelial Cell Monolayers
Authors: Audus Kenneth L. , Borchardt Ronald T.
Published: July 2010

Abstract: The large neutral amino acid (LNAA) transport system, or L (leucine) system, in primary cultures of bovine brain microvessel endothelial cell monolayers has been characterized. The transendothelial transport of leucine in this in vitro blood‐brain barrier (BBB) model was determined to be bidirectional and time, temperature, and concentration dependent. Leucine transport was saturable, and the apparent Km and Vmax were determined to be 0.18 mM and 6.3 nmol/mg/min, respectively. Transendothelial transport of leucine was resistant to inhibition by ouabain and sodium azide. Other LNAAs, including the centrally acting drugs α‐methyl‐3,4‐dihydroxyphenylalanine (α‐methyldopa), l‐3,4‐dihydroxyphenyl alanine (l‐DOPA), α‐methyltyrosine, and baclofen, inhibited leucine transport. The leucine carrier system was also found to be stereospecific. Sucrose, used as a diffusion marker, moved across the monolayers slowly, and its concentration was not significant for at least 30 min.
Ionizing Radiation Alters the Properties of Sodium Channels in Rat Brain Synaptosomes
Authors: Mullin Michael J. , Hunt Walter A. , Harris R. Adron
Published: July 2010

Abstract:The effect of ionizing radiation on neuronal membrane function was assessed by measurement of neurotoxin‐stimulated22Na+uptake by rat brain synaptosomes. High‐energy electrons and γ photons were equally effective in reducing the maximal uptake of22Na+with no significant change in the affinity of veratridine for its binding site in the channel. Ionizing radiation reduced the veratridine‐stimulated uptake at the earliest times measured (3 and 5 s), when the rate of uptake was greatest. Batrachotoxin‐stimulated22Na+uptake was less sensitive to inhibition by radiation. The binding of [3H]saxitoxin to its receptor in the sodium channel was unaffected by exposure to ionizing radiation. The effect of ionizing radiation on the lipid order of rat brain synaptic plasma membranes was measured by the fluorescence polarization of the molecular probes 1,6‐diphenyl‐1,3,5‐hexatriene and l‐[4‐(trimethylammonium)phenyl]‐6‐phenyl‐l,3,5‐hexatriene. A dose of radiation that reduced the veratridine‐stimulated uptake of22Na+had no effect on the fluorescence polarization of either probe. These results demonstrate an inhibitory effect of ionizing radiation on the voltage‐sensitive sodium channels in rat brain synaptosomes. This effect of radiation is not dependent on changes in the order of membrane lipids.
Expression of Classical Mitochondrial Respiratory Responses in Homogenates of Rat Forebrain
Authors: Sims Neil R. , Blass John P.
Published: July 2010

Abstract: Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected sub fractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by “free” mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m‐chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs‐Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol‐rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.
Intractable Unphysiologically Low Adenylate Energy Charge Values in Synaptosome Fractions: An Explanatory Hypothesis Based on the Fraction's Heterogeneity
Authors: Kyriazi Harold T. , Basford R. E.
Published: July 2010

Abstract: Synaptosomes prepared and incubated in a variety of ways from rat cerebra exhibited intractable, unphysiologically low adenylate energy charge values (∼0.37–0.60), low total adenine nucleotide contents (∼8–10 nmol/mg protein), and much higher adenylate kinase apparent Kcq values (∼3–8) as compared to intact brain tissue (values of ∼0.90, 25 nmol/mg, and 0.74. respectively). Synaptosomes prepared from mouse, dog, and chicken cerebra had values essentially identical to those from rat. When incubated under oxygen in a physiological salt solution containing glucose, synaptosomes metabolized more glucose to lactic acid than to CO2, and the addition of 100 μM veratridine caused a two‐ to threefold stimulation of O2 uptake, lactate accumulation, and CO2 output. It is known that synaptosome fractions contain a substantial number (at least 30–45% by volume) of cytoplasm‐containing particles devoid of mitochondria (henceforth termed “cytosolic particles”), and that ∼80% of brain hexokinase is bound to the outer mitochondrial membrane. For the cytosolic particles, lacking oxidative phosphorylation, to maintain their “in vivo” ATP turnover would require about a 19‐fold increase in the glycolytic rate, which is not possible due to limiting amounts of hexokinase, and thus these particles are postulated to be responsible for the high level of aerobic lactate accumulation and the intractable low energy charge values found in synaptosome fractions. The mitochondria‐containing particles are postulated to have a normal energy charge, a submaximal glycolytic rate, and minimal lactate production, on the basis of the capacity of veratridine to stimulate synaptosomal O2 uptake and CO2 and lactate output. Calculations based on this “two populations of particles” hypothesis indicate that for synaptosome fractions in general, (1) the cytosolic particles contain ∼35–64% of the total adenine nucleotides and maintain an energy charge of ∼0.12; (2) the cytosolic particles and mitochondria‐containing particles have adenylate kinase apparent Keq values of ∼0.21–1.66 and 0.74, respectively, revealing that the higher apparent Kcq values of the synaptosome fractions probably are not real departures from equilibrium; and (3) ∼31–45% of synaptosome fraction protein is contained in debris, which, when taken into account, yields total adenine nucleotide contents in the cytosolic particles and mitochondria‐containing particles of ∼15–24 and ∼11 −19 nmol/mg of particle protein, respectively.
Accurate Evaluation of 1,2‐Diacylglycerol in Gerbil Forebrain Using HPLC and In Situ Freezing Technique
Authors: Abe Koji , Kogure Kyuya
Published: July 2010

Abstract: Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2‐diacylglycerols were first measured quantitatively by HPLC. Although 1,2‐diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2‐diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one‐fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2‐diacylglycerols in gerbil forebrain was first reported and the control 1,2‐diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2‐diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2‐diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2‐diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.
Changes in Nine Enzyme Markers for Neurons, Glia, and Endothelial Cells in Agonal State and Huntington's Disease Caudate Nucleus
Authors: Butterworth John
Published: July 2010

Abstract: Enzymes considered to be markers for neurons (angiotensin converting enzyme, thermolysin‐like metal‐loendopeptidase, alanine aminopeptidase, and glutamate‐oxaloacetate transaminase), glia (glutamine synthetase, pyruvate carboxylase, and β‐glucuronidase), and endothelial cells (alkaline phosphatase and plasminogen activator) were measured in caudate nucleus from 10 sudden death controls, eight agonal state controls, and 16 Huntington's disease patients. Glutamate‐oxaloacetate transaminase was slightly reduced by agonal state. The four enzymes with a neuronal distribution were all correlatively reduced in Huntington's disease caudate nucleus. Glutamine synthetase activity was reduced and β‐glucuronidase mean activity increased over twofold in Huntington's disease caudate nucleus, with the two enzyme activities being inversely related. Pyruvate carboxylase was markedly affected by agonal state and was very variable in Huntington's disease caudate nucleus. The two endothelial enzymes were unaltered in Huntington's disease caudate nucleus. The findings are indicative of neuronal loss, an increased proportion of altered glia, and also of maintained vasculature in Huntington's disease caudate nucleus. Measurement of enzyme activities can help to delineate the types of cell altered in Huntington's disease.
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

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

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

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.
Acetylation and Phosphorylation of Histones and Nonhistone Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Developing Rat Brain
Authors: Serra I. , Avola R. , Condorelli D. F. , Surrentino S. , Renis M. , Kamiyama M. , Hashim G. A. , Giuffrida A. M.
Published: July 2010

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl‐CoA and of [γ‐32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low‐molecular‐weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000–90,000 daltons)—absent at 1 day, appearing at 10 days, and more evident at 30 days of age—was observed in both neuronal and glial nuclei.
Putative Cholinergic‐Specific Gangliosides in Guinea Pig Forebrain
Authors: Ferretti Patrizia , Borroni Edilio
Published: July 2010

The nature of the cholinergic‐specific antigen Chol‐1 recognized by an antiserum raised against Torpedo cholinergic electromotor synaptosomal plasma membranes was investigated in guinea pig forebrain to establish whether it has a gangliosidic nature in guinea pig as in Torpedo. Gangliosides extracted from guinea pig forebrain and extensively purified to eliminate peptide contaminants were effective in inhibiting the selective lysis of the cholinergic subpopulation of cortical synaptosomes induced by the antiserum. Neuraminidase, protease, alkali, and heat treatment did not impair the inhibitory activity of gangliosides. Whereas the antiserum recognized many gangliosides from Torpedo electric organ, the immunostaining of guinea pig forebrain gangliosides separated on TLC showed only two immunopositive bands migrating close to GT1b and GQ. After affinity purification on Torpedo electric organ gangliosides the activity of the antiserum in inducing complement‐mediated lysis was increased and it still recognized the two ganglioside bands on TLC. These results strongly suggest the existence of two polysialogangliosides bearing antigenic determinants specific for the cholinergic neurons.
Inhibition of Aromatic L‐Amino Acid Decarboxylase and Tyrosine Aminotransferase by the Monoamine Oxidase Inhibitor Phenelzine
Authors: Dyck Lillian E. , Dewar Karen M.
Published: July 2010

The concentration ofp‐tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect onp‐tyramine levels in the first 1–2 h following injection. The high dose of phenelzine increased thep‐tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatalp‐tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine amino‐transferase activity of rat liver homogenates; the IC50was 50 μM. Phenelzine also inhibited the aromatic L‐amino acid decarboxylase activity of rat brain homogenate with an IC50of 25 μM.Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L‐amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatalp‐tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect onp‐tyramine levels followed later by very large increases inp‐tyramine levels.
Regional Cerebral Glucose Utilization During Insulin‐Induced Hypoglycemia in Unanesthetized Rats
Authors: Bryan Robert M. , Keefer Kerry A. , MacNeill Colin
Published: July 2010

Regional cerebral glucose utilization (rCMRgl) was studied during insulin‐induced hypoglycemia in unanesthetized rats. Rats were surgically prepared using halothane and nitrous oxide anesthesia and allowed 5 h to recover from the anesthesia before rCMRgl was measured. The rCMRgl was measured using [6‐14C]glucose in a normoglycemic control group and two hypoglycemic groups, A (30 min after insulin injection) and B (2 h after insulin injection). The mean plasma glucose level was 7.03 μmol/ml in the normoglycemic group, 1.96 μmol/ml in hypoglycemic group A, and 1.40 μmol/ml in hypoglycemic group B. The rCMRgl in hypoglycemic group A decreased 8–18% in 17 brain regions measured; five changes were statistically significant. The rCMRgl in hypoglycemic group B decreased significantly in all but one of the brain regions measured; the decrease ranged from 15% in the pyramidal tract to 36% in the motor and auditory cortices. The rCMRgl in every brain region decreased when the plasma glucose level fell below 1.5–2.5 μmol/ml. No brain region could maintain rCMRgl at plasma glucose concentrations lower than predicted by regional glucose influx described in previous studies. Glucose utilization in all brain regions appears to be limited by the influx of glucose.
Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase
Authors: Rothstein Jeffrey D. , Tabakoff Boris
Published: July 2010

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+‐dependent, K+‐stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium‐dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.
Characterization of Curaremimetic Neurotoxin Binding Sites on Membrane Fractions Derived from the Human Medulloblastoma Clonal Line, TE671
Authors: Lukas Ronald J.
Published: July 2010

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High‐affinity binding sites (KD= 2 nMfor 1‐h incubation at 20°C) and low‐affinity binding sites (KD= 40 nM) for125I‐labeled α‐bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high‐affinity binding of toxin is 0.56 nM(k1= 6.3 · 10−1min−1nM−1;k‐1= 3.5 · 10−3min−1) at 20°C. Nicotine,d‐tubocurarine, and acetylcholine are among the most effective inhibitors of high‐affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulthydryl residues. High‐affinity toxin binding sites that have been subjected to reaction with the sulthydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High‐affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.
Distribution of Free Methylarginines in Rat Tissues and in the Bovine Brain
Authors: Ueno Shu‐ichi , Sano Akira , Kotani Kazuhiko , Kondoh Keiji , Kakimoto Yasuo
Published: July 2010

Abstract:A sensitive and specific method for determining three forms of methylarginine, i.e.,NG‐monomethylarginine,NG,NG‐dimethylarginine, andNG,NG‐dimethylarginine, in mammalian tissues was developed. After partial purification by ion‐exchange chromatography, the methyl‐arginines were derivatized to phenylthiocarbamyl compounds and quantitatively determined using HPLC with a reverse‐phase C18column. In rat organs, the highest concentrations of methylarginines were observed in the spleen. In rat brain, cerebellum and olfactory bulb contained large amounts ofNG‐monomethylarginine andNG,NG‐dimethylarginine. A detailed study of the distribution of methylarginines in the bovine brain was also made, and the concentration ofNG,NG‐dimethylarginine was almost the same in all regions. The cerebellar gray matter, hippocampus, and hypothalamus contained large amounts of methylarginines. The distribution of methylarginines seems to parallel the distribution of nitric oxide synthase, which is known to be inhibited byNG‐monomethylarginine. This may indicate that methylarginines play some role in controlling nitric oxide synthase activity.
Tetrahydrobiopterin Turnover in Cultured Rat Sympathetic Neurons: Developmental Profile, Pharmacologic Sensitivity, and Relationship to Norepinephrine Synthesis
Authors: Kapatos Gregory , Hirayama Kei , Hasegawa Hiroyuki
Published: July 2010

Abstract:We have examined the turnover of 5,6,7,8‐tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE‐containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half‐life of ∼2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition byN‐acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90–95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24‐h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels. In contrast, treatment of cultures for 24 h with the direct‐acting inhibitor of TH, α‐methyl‐p‐tyrosine, produced an 84% decline in NE content that was maintained over the 4‐day treatment period, indicating that the slow decline in NE content following inhibition of BH4 synthesis was not the result of the slow turnover rate of NE. These results demonstrate that despite an almost complete loss of BH4, sympathetic neurons were able to maintain neurotransmitter content, albeit at reduced levels, by retaining a level of TH activity above the value that might have been predicted based on the reduced level of BH4.
Relationship Between Platelet Monoamine Oxidase B Activity and Alleles at the MAOB Locus
Authors: Girmen A. Sule , Baenziger John , Hotamisligil Gokhan S. , Konradi Christine , Shalish Christo , Sullivan Jack L. , Breakefield Xandra O.
Published: July 2010

Abstract: Genetic variations in monoamine oxidase (MAO)‐B activity have been proposed to have a contributory role in several neurologic and psychiatric diseases. Variations in activity could affect rates of degradation of exogenous amines, including toxins, precursors of toxins (like 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine), or false transmitters, and of endogenous amines, such as neurotransmitters. In this study a highly polymorphic (GT)n repeat element was used to mark alleles at the MAOB locus. The MAOB allele status and levels of platelet MAO‐B activity were determined for 41 control males. No correlation was noted between specific alleles and levels of MAO‐B activity in this sample set. This suggests that the structural gene for MAOB is not usually the primary determinant of activity levels in platelets.
Stimulation of Cultured Cerebellar Granule Cells via Glutamate Receptors Induces TRE‐ and CRE‐Binding Activities Mediated by Common DNA‐Binding Complexes
Authors: Sakurai Hiroaki , Kurusu Rie , Sano Kuniaki , Tsuchiya Tomofusa , Tsuda Masaaki
Published: July 2010

Abstract:By use of nuclear mini‐extracts prepared from cultured cerebellar granule cells in a gel‐mobility assay, exogenousN‐methyl‐D‐aspartate (NMDA) or kainate was shown to increase both 12‐O‐tetradecanoylphorbol 13‐acetate‐responsive element (TRE)‐ and cyclic AMP‐responsive element (CRE)‐binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L‐2‐amino‐5‐phosphonovalerate and the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, respectively. The increase of TRE‐binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE‐binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA‐binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE‐binding activity could be mediated by the TRE‐binding activity. Thus, the TRE‐binding activity cross‐binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c‐Fos or Fos‐related proteins in the TRE‐ and CRE‐binding complexes was shown by a supershift gel‐mobility assay using anti‐c‐Fos antiserum.
Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions
Authors: Russi P. , Alesiani M. , Lombardi G. , Davolio P. , Pellicciari R. , Moroni F.
Published: July 2010

Abstract: Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N‐methyl‐D‐aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringer's solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 ± 0.9 to 48 ± 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200–400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound‐induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D‐serine (100 μg intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.