bluuebunny/crossref_metadata_2025 huggingface dataset:
t‐[35S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues
Authors: Olsen Richard W. , Szamraj Olga , Miller Thomas
Published: October 2006

Abstract Specific high affinity binding of the cage convulsant t‐[35S] butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S] TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22°C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4°C temperature and 150‐min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD= 50 nM, Bmax= 250 fmol/mg protein in housefly; KD= 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1=8 × 106M‐1 min‐1), but dissociation was described better by two rate constants (k‐1, = 0.28 min‐1 and 0.042 min‐1; calculated Kd values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50= 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1‐2 μM). Stereospecific inhibition by cypermethrin isomers agreed partially with biological activity, suggesting the possible role of this site in some toxicological actions of pyrethroids, although these compounds have more potent effects on voltage‐regulated sodium channels. [35S]TBPS binding sites in crayfish muscle appear identical to γ‐aminobutyric acid (GABA) receptor‐chloride channels labeled by radiolabeled picrotoxin in this tissue. The sites in both invertebrate tissues examined are similar to those in mammalian brain and appear to be the target of important drugs.
Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay
Authors: Gillon J. Y. , Labatut R. , Renaud B. , Pujol J. F.
Published: October 2006

Abstract: The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (< 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.
Effect of Ouabain Applied by Intrastriatal Microdialysis on the In Vivo Release of Dopamine, Acetylcholine, and Amino Acids in the Brain of Conscious Rats
Authors: Westerink B. H. C. , Damsma G. , de Vries J. B.
Published: October 2006

Abstract: In the present study we have applied a brain microdialysis technique to investigate the effects of ouabain infusion on the release of dopamine, acetylcholine, and amino acids from striatal neurons in freely moving rats. Ouabain caused an increase in the dialysate levels of dopamine; its metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC); and the amino acids glutamate, aspartate, taurine, glycine, alanine, serine, asparagine, and threonine. The ouabain‐induced increase in dopamine was dose dependent and explosive (100‐ fold at an infusion concentration of 1 mmol/L) and contrasted strongly with the small effect of the glycoside on the output of DOPAC. We investigated the nature of ouabain‐induced transmitter release by determining its sensitivity to coinfusion with tetrodotoxin or the calcium antagonist Mg2+.In the case of dopamine two mechanisms of ouabain‐induced release could be established. At lower infusion concentrations ouabain induced an exocytotic type of release whereas at higher concentrations the release was probably camer mediated. In the case of amino acids we noticed a calcium‐independent release which was nerve impulse flow dependent in the case of glutamate and aspartate and impulse flow independent in the case of alanine, serine, glycine, threonine, and asparagine. Ouabain induced a decrease in the release of acetylcholine and glutamine.
Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol
Authors: Stephens L. R. , Logan S. D.
Published: October 2006

Abstract: Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion‐exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4‐trisphosphate, [3H]inositol 1,4,5‐trisphosphate, and [3H]inositol 1,3,4,5‐tetrakisphos‐phate. The levels of [3H]inositol 1,4,5‐trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5‐trisphosphate was incubated with hippocampal formation homogenates in an ATP‐containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4‐bisphosphate, [3H]inositol 1,3,4‐trisphosphate, and [3H]inositol 1,3,4,5‐tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5‐tetrakisphosphate is metabolised to [3H]inositol 1,3,4‐trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4‐bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5‐bisphosphate selective phospholipase C.
Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain
Authors: Hara Masayuki , Matsuda Yoshihiro , Okumura Nobuaki , Hirai Keisuke , Nakagawa Hachiro
Published: October 2006

Abstract: The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2‐deoxy‐d‐glucose (2‐DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2‐DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.
Cloning of a cDNA Encoding MAP1B in Rat Brain: Regulation of mRNA Levels During Development
Authors: Safaei Roohangiz , Fischer Itzhak
Published: October 2006

Abstract: This article describes the isolation of a microtubule‐associated protein 1B (MAP1B) cDNA clone from a rat brain λ gt11 library and the study of MAP1B mRNA expression during brain development. On Northern blots, the cDNA hybridized with an mRNA of > 10 kilobases which was present only in the brain. The identity of the cDNA was confirmed by the characterization of the antiserum against the fusion protein, and also by comparing both the original antibody and the anti‐fusion protein antiserum with a panel of well‐studied monoclonal antibodies against different forms of MAP1 and MAP2. The regulation of MAP1B mRNA during development was studied in whole brain, cerebral cortex, hypothalamus, brainstem, and olfactory bulbs. The steady‐state levels of MAP1B mRNA in all tissues examined were relatively low in the adult compared to developing brains. This decrease varied in different brain regions, and its time course appeared to coincide with the pattern of postnatal developmental and morphological events. The developmental patterns of the MAP1B mRNA and protein in the brain were similar, suggesting that expression of this protein is under transcriptional control. The RNA blots were also probed with β‐actin and β‐tubulin to compare the levels of MAP1B mRNA with other cytoskeletal elements and as controls for the quality of the RNA.
Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains
Authors: Wood W. Gibson , Gorka Christine , Schroeder Friedhelm
Published: October 2006

Abstract: Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol‐treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol‐treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.
Mouse Brain c‐fos mRNA Distribution Following a Single Electroconvulsive Shock
Authors: Daval Jean‐Luc , Nakajima Takashi , Gleiter Christoph H. , Post Robert M. , Marangos Paul J.
Published: October 2006

Abstract: The regional distribution of c‐fos mRNA in the mouse brain has been investigated by in situ hybridization autoradiography after seizures induced by an acute electroconvulsive shock (ECS). ECS led to a widespread induction of the proto‐oncogene c‐fos in the brain, with highest concentrations in discrete areas within the limbic system and also in the hypothalamus and cerebellum. The mild stress of sham treatment in earclipped animals induced a weaker and qualitatively different pattern of c‐fos mRNA expression involving the cortex, hippocampus, and cerebellum. These data suggest the usefulness of c‐fos in situ hybridization as a marker of neuronal stimulation and in mapping a range of effects from a mild stress to the robust changes of an electroconvulsive seizure.
Observation of Cerebral Metabolites in an Animal Model of Acute Liver Failure In Vivo: A 1H and 31P Nuclear Magnetic Resonance Study
Authors: Bates Timothy E. , Williams Stephen R. , Kauppinen Risto A. , Gadian David G.
Published: October 2006

Abstract: Acute liver failure was induced in rats by a single intragastric dose of carbon tetrachloride. This causes hepatic centrilobular necrosis, as indicated by histological examinations, and produces a large increase in the activity of serum alanine aminotransferase. The plasma NH4+ level (mean ±SEM) was 123 ± 10 μM the control group and 564 ± 41 μM in animals with acute liver failure (each n = 5). 31P nuclear magnetic resonance (NMR) was used to monitor brain cortical high‐energy phosphate compounds, Pi, and intracellular pH. 1H NMR spectroscopy was utilised to detect additional metabolites, including glutamate, glutamine, and lactate. The results show that the forebrain is capable of maintaining normal phosphorus energy metabolite ratios and intracellular pH despite the metabolic challenge by an elevated blood NH4+level. There was a significant increase in the brain glutamine level and a concomitant decrease in the glutamate level during hyperammonaemia. The brain lactate level increased twofold in rats with acute liver failure. The results indicate that 1H NMR can be used to detect cerebral metabolic changes in this model of hyperammonaemia, and our observations are discussed in relation to compartmentation of NH4+ metabolism.
Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue
Authors: Elliott J. Martin , Kent Amanda
Published: October 2006

Abstract: The human platelet contains a functional 5‐hy‐droxytryptamine (5‐HT) receptor that appears to resemble the 5‐HT2 subtype. In this study, we have used the iodinated derivative [I25I]iodolysergic acid diethylamide ([I25I]iodoLSD) in an attempt to label 5‐HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high‐affinity site (KD= 0.35 ± 0.02 nM). Displacement of specific [l25I]iodoLSD binding indicated a typical 5‐HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r= 0.97, p > 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax= 136 ± 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax= 258 ± 19 fmol/mg of protein) (p > 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD= 0.37 ± 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r.= 0.96, p > 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5‐HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.
Regulation of UDP‐Galactose:Ceramide Galactosyltransferase and UDP‐Glucose:Ceramide Glucosyltransferase After Crush and Transection Nerve Injury
Authors: Costantino‐Ceccarini Elvira , Poduslo Joseph F.
Published: October 2006

Abstract: The enzyme activities of ceramide galactosyltransferase and ceramide glucosyltransferase were assayed as a function of time (0, 1, 2, 4, 7, 14, 21, 28, and 35 days) after crush injury or permanent transection of the adult rat sciatic nerve. These experimental models of neuropathy are characterized by the presence and absence of axonal regeneration and subsequent myelin assembly. Within the first 4 days after both injuries, a 50% reduction of ceramide galactosyltrans‐ferase‐specific activity was observed compared to values found in the normal adult nerve. This activity remained unchanged at 7 days after injury; however, by 14 days the ceramide galactosyltransferase activity diverged in the two models. The activity increased in the crushed nerve and reached control values by 21 days, whereas a further decrease was observed in the transected nerve such that the activity was nearly immeasurable by 35 days. In contrast, the ceramide glucosyltransferase activity showed a rapid increase between 1 and 4 days, followed by a plateau that was 3.4‐fold greater than that in the normal adult nerve, which persisted throughout the observation period in both the crush and transection models. [3H]Galactose precursor incorporation studies at 7, 14, 21, and 35 days after injury confirmed the previously observed shift in biosynthesis from the galactocerebrosides during myelin assembly in the crush model to the glucocere‐brosides and oligohexosylceramide homologues in the absence of myelin assembly in the transection model. The transected nerves were characterized by a peak of biosynthesis of the glucocerebrosides at 14 days. Of particular interest is the biosynthesis of the glucocerebrosides and the oligohexosyl‐ceramides at 7 and 14 days after crush injury. This biosynthesis was dramatically reduced by 21 days after crush, even though the ceramide glucosyltransferase activity remained elevated. The present data indicate that this shift in myelina‐tion‐dependent glycolipid biosynthesis is controlled at least partially at the level of these sugar transferases.
α2‐Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison
Authors: Richards Elaine M. , Sumners Colin , Chou Yun‐Chia , Raizada Mohan K. , Phillips M. Ian
Published: October 2006

Abstract: Membranes prepared from either neuronal or glial cultures contain α2‐adrenergic receptors as determined by the characteristics of [3H]yohimbine ([3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 ± 1.35 nM (n = 10) for neuronal cultures and 18.42 ± 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a 5max of 1.6 ± 0.33 pmol/mg protein (n = 10) compared with 0.143 ± 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for α2‐adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the α2‐adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynan‐thine, or propranolol. The agonists showed the same pattern with the α2‐selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog, 5′‐guanylyl‐imidodiphos‐phate, were able to lower the affinity of the α2‐receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of α2‐adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain α‐adrenoceptors.
In Vivo Measurement Using Microdialysis of the Release and Metabolism of 5‐Hydroxytryptamine in Raphe Neurones Grafted to the Rat Hippocampus
Authors: Sharp T. , Foster G. A.
Published: October 2006

Abstract: The overflow and metabolism of serotonin (5‐hydroxy‐tryptamine; 5‐HT) from transplants of embryonic medullary and mesencephalic raphe neurones in the previously 5‐HT‐denervated hippocampus have been analyzed in vivo using intracerebral dialysis. The average density of 5‐HT‐immunoreactive fibres in the grafted hippocampus was less than in nonlesioned hippocampus. Nonetheless, both basal and potassium‐stimulated levels of 5‐HT in the dialysates were restored to approximately normal after transplantation of medullary raphe cells, whereas mesencephalic implants resulted in over twice the 5‐HT output observed in control hippocampus. However, 5‐hydroxyindoleacetic acid (5‐HIAA) overflow was increased only after grafting of mesencephalic raphe and then only to normal levels; medullary implants, by contrast, failed to enhance 5‐HIAA output above that from lesion‐only hippocampus. The evidence of a relative hyperactivity of the grafted neurones may explain the disproportionate improvements in various lesion‐induced behavioural deficits after grafting of nervous tissue. In addition, differences in the presynaptic regulation of 5‐HT release and metabolism are also apparent in the transplants; these variations are dependent on the precise origin of the serotoninergic cells.
Changes in Amino Acid Contents in the Spinal Cord and Brainstem of Rats with Experimental Autoimmune Encephalomyelitis
Authors: Honegger Conrad G. , Krenger Werner , Langemann Helen
Published: October 2006

Abstract: The effects of experimental autoimmune encephalomyelitis (attack and recovery) on levels of six amino acids have been investigated in nine regions of the Lewis rat spinal cord between segments C3 and Col and in the brainstem. Amino acids were analyzed by separation of their 4′‐dimeth‐ylaminoazobenzene‐4‐sulfonyl chloride derivatives on a re‐versed‐phase column using a ternary gradient. Glutamate and γ‐aminobutyric acid were reduced by 10‐30% in all segments during the attack, whereas taurine, lysine, glutami|ne, and glycine were all greatly increased (up to 300%). Most values except those of taurine, as well as glutamate in certain segments, returned to normal on recovery. Because some of these compounds have neurotransmitter function, these changes may contribute to the neurological symptoms of experimental autoimmune encephalomyelitis.
Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine
Authors: Yudkoff Marc , Zaleska Malgorzata M. , Nissim Itzak , Nelson David , ErecinAska Maria
Published: October 2006

Abstract: Gas chromatography‐mass spectrometry was used to evaluate the metabolism of [I5N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25‐35 nmol/mg protein at an initial rate >9 nmol/min/mg of protein. The metabolism of [15NJglutamine generated l5N‐labelled glutamate, aspartate, and γ‐aminobutyric acid (GABA). An efflux of both [15N]gIutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of ,5N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2‐oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70‐80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.
α1Adrenoreceptpr‐Mediated Increase in Acetylcholine Release in Braip Slices During Morphine Tolerance
Authors: Tanganelli S. , Antonelli T. , Simonato M. , Spalluto G. , Tomasini C. , Bianchi C. , Beani L.
Published: October 2006

Abstract: Norepinephrine, clonidine, and phenylephrine increased the electrically evoked release of endogenous acetylcholine in cortical slices taken from morphine‐tolerant guinea pigs. This effect was α1adrenoreceptor mediated and was opposite to the α2‐adrenoreceptor‐mediated inhibition of acetylcholine release, normally elicited by norepinephrine and clonidine. In the presence of prazosin, clonidine recovered its normal inhibitory properties, suggesting that morphine tolerance induced the appearance of an α1adrenoreceptor‐mediated response that overshadowed, but did not cancel, the still present α2‐adrenoreceptor inhibitory control. The attempt to prove the presence of α‐adrenoreceptors on the nerve endings by testing the effect of norepinephrine in synaptosomal preparations (preloaded with [3H]choline and de‐polarized with KCL and veratridine) was unsuccessful. Therefore the problem of the exact location of this excitatory input remains to be solved. These results confirm previous findings reporting the increase in cortical acetylcholine release induced by the α‐adrenoreceptor agonists in morphine‐tolerant, freely moving guinea pigs and demonstrate that opiate tolerance inverts the direction of the noradrenergic modulation even in the isolated intracortical cholinergic structures.
Potassium‐Stimulated Release of [3HJTaurine from Cultured GABAergic and Glutamatergic Neurons
Authors: Schousboe Arne , Pasantes‐Morales Herminia
Published: October 2006

Abstract: The effect of depolarizing concentrations of potassium (56 mM) on the release of [3H]taurine was examined in two types of cultured neurons from mouse brain: cerebral cortex neurons, which are largely GABAergic, and cerebellar neurons, which after treatment with kainate consist almost entirely of glutamatergic granule cells. The release of [3H]taurine was compared to that of γ‐[3H]aminobutyric acid ([3H]GABA) in cortical neurons and to that of D‐[3HJaspartate in granule cells. Cortical neurons responded to potassium stimulation (1 min or continuously) by an immediate increase in [3H]GABA efflux of more than six times over the basal efflux, followed by a sharp decline despite the persistence of the stimulatory agent. The potassium‐induced release of [3H]GABA was largely calcium‐dependent. The release of [3H]taurine was considerably less in magnitude, only doubling after the stimulus, with a time course delayed in both onset and decline. The release of [3H]taurine was partially calcium‐dependent and was also decreased in low‐chloride solutions. In cerebellar granule cells, exposure to potassium resulted in a large (sixfold) and prompt release of D‐[3H]aspartate, largely calcium‐dependent. A totally different pattern was observed for the release of [3H]taurine. A stimulatory effect occurred only when cells were exposed continuously to potassium. Taurine efflux was very delayed, with a broad stimulus plateau reached after 15‐20 min of stimulation. Taurine release was unaffected by omission of calcium, but it was abolished in a low‐chloride medium. These results suggest that taurine is released from cells handling other neuroactive amino acids as neurotransmitters. A co‐release of GABA/taurine or glu‐tamate/taurine is unlikely due to the marked differences observed’ in their efflux patterns, clearly indicating different mechanisms for release. Instead, taurine release may result from a cell response different from the stimulus‐secretion coupling of neurotransmitters.
Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane‐Bound and Solubilized Specific Sites
Authors: Morishima Yoshiyuki , Nakata Yoshihiro , Segawa Tomio
Published: October 2006

Abstract: Mg2+ increased but Na+ and GTP decreased [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3‐[(3‐cholamidopropyl)dimethyl‐ammonio]‐l‐propane sulfonate (CHAPS)‐solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least‐squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two‐site model, corresponding to a “high‐affinity (H)” and a “low‐affinity (L)” state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new “super‐low affinity (SL)” state were observed. The SP/ [3HJSP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet‐activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgC12, NaCl, and GTP were greater in the CHAPS‐solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3HJSP binding by unlabeled SP in the presence of MgCl2 in the CHAPS‐solubilized fraction were best fitted to a one‐site model. The KD value was relatively close to that of the low‐affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCI2concentration to 1 mM, although the inhibition curves consistently fit the one‐site model, the KD values changed only slightly.
Changes in [3H]Nitrendipine Binding and γ‐Aminobutyric Acid Release in Rat Hippocampus Following Repeated Morphine Administration
Authors: Ohnishi Tetsuo , Saito Kihachi , Matsumoto Ken , Sakuda Masayoshi , Ishii Kenji , Inoki Reizo
Published: October 2006

Abstract: An antagonistic effect of calcium on the action of morphine was studied in rat hippocampal slices. The effect of repeated administration of morphine on γ‐aminobutyric acid (GABA) release and binding of [3H]nitrendipine, a cal‐cium antagonist, was also examined. (1) In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons, disinhibiting them through basket cells. When the calcium concentration was elevated, potentiation of the field potentials by morphine was reduced. Decrease of the calcium concentration, on the other hand, enhanced the potentiating effect of morphine. Following repeated administration of morphine, its enhancing effect on the field potentials in slices was not observed. (2) In hippocampal membrane fractions obtained from rats repeatedly treated with morphine, enhancement of [3H]nitrendipine binding was observed. (3) In hippocampal slice preparations from rats receiving morphine repeatedly, K+ (45 mM)‐stim‐ulated [3H]GABA efflux was enhanced. The above results indicate that morphine antagonizes calcium, thereby reducing the release of transmitters. Furthermore, increase in calcium channels following repeated treatment of rats with morphine may explain the mechanism underlying development of tolerance.
Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP‐Dependent Protein Kinase in Rat Frontal Cortex
Authors: Nestler Eric J. , Terwilliger Rose Z. , Duman Ronald S.
Published: October 2006

Abstract: The influence of chronic administration of antidepressants on cyclic AMP‐dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP‐dependent protein kinase activity in soluble fractions by ∼25%, whereas enzyme activity was increased in the particulate fractions by ∼20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant jdrugs, because representatives of several other classes of psychotropic drugs—namely, haloperidol, morphine, and diazepam—failed to alter either soluble or particulate levels of cyclic AMP‐dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP‐dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP‐dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long‐term adaptive changes in brain thought to be essential for the blinical actions of these treatments.
Actin Involvement in Exocytosis from PC12 Cells: Studies on the Influence of Botulinum C2 Toxin on Stimulated Noradrenaline Release
Authors: Matter Karin , Dreyer Florian , Aktories Klaus
Published: October 2006

Abstract: Botulinum C2 toxin is known to ADP‐ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five‐ to 15‐fold by carbachol (100 μM) or K+ (50 mM) and 10–30‐fold by the ionophore A23187 (5 μM). Pretreatment of PC12 cells with botulinum C2 toxin for 4–8 h at 20°C, increased carbachol‐, K+‐, and A23187‐induced, but not basal, [3H]noradrenaline release maximally 1.5‐ to threefold, whereas ≥75% of the cellular actin pool was ADP‐ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37°C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for > 1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+ but not by A23187. Concomitantly with toxin‐induced stimulation of secretion, 20–50% of the cellular actin was ADP‐ribosylated, whereas >60% of actin was modified when exocytosis was attenuated. The data indicate that ADP‐ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.
Basal Lipid Peroxidation in Substantia Nigra Is Increased in Parkinson's Disease
Authors: Dexter D. T. , Carter C. J. , Wells F. R. , Javoy‐Agid F. , Agid Y. , Lees A. , Jenner P. , Marsden C. D.
Published: October 2006

Abstract: Polyunsaturated fatty acid (PUFA) levels (an index of the amount of substrate available for lipid peroxidation) were measured in several brain regions from patients who died with Parkinson's disease and age‐matched control human postmortem brains. PUFA levels were reduced in parkinsonian substantia nigra compared to other brain regions and to control tissue. However, basal malondialdehyde (MDA; an intermediate in the lipid peroxidation process) levels were increased in parkinsonian nigra compared with other parkinsonian brain regions and control tissue. Expressing basal MDA levels in terms of PUFA content, the difference between parkinsonian and control substantia nigra was even more pronounced. Stimulating MDA production by incubating tissue with FeSO4 plus ascorbic acid, FeSO4 plus H2O2, or air alone produced lower MDA levels in the parkinsonian substantia nigra, probably reflecting the lower PUFA content. These results may indicate that an increased level of lipid peroxidation continues to occur in the parkinsonian nigra up to the time of death, perhaps because of continued exposure to excess free radicals derived from some endogenous or exogenous neurotoxic species.
Mechanism of Agonist‐Induced Down‐Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line
Authors: Ray Prabhati , Middleton Wilbert , Berman Jonathan D.
Published: October 2006

Abstract: The mechanisms of carbachol‐induced muscarinic acetylcholine receptor (mAChR) down‐regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma X glioma hybrid NG108‐15 cell line by specific ligand binding assays. N‐[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by ∼80%. Bacitracin (1–4 mg/ml) and methyl‐amine (1–15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist‐induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1–10 μM) and colchicine (1–10 μM) prevented carbachol‐induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist‐induced mAChR down‐regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 μM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.
Multiple Factors Influencing the In Vitro Release of [Met5]‐Enkephalin from Rat Hypothalamic Slices
Authors: Nikolarakis K. E. , Almeida O. F. X. , Herz A.
Published: October 2006

Abstract: This study examined several in vivo and in vitro factors which influence the release of [Met5]‐enkephalin (Met‐ENK) from male rat hypothalamic slices superfused in vitro. Met‐ENK release was significantly stimulated by corticotropin‐releasing hormone (CRH; 10−12–10−8M), an effect which was abolished in the presence of the CRH‐receptor antagonist, α‐helical CRF9–41 (10−6M). The amount of Met‐ENK release diminished with time in experiments in which the slices were continuously exposed to CRH. The opioid receptor antagonist naloxone (10−6M) stimulated Met‐ENK release, even in the presence of the Na+‐channel blocker tetrodotoxin (10−6M), a result indicating presynaptic opioid feedback inhibition of Met‐ENK release. The role of gonadal steroids in the control of Met‐ENK release in vitro was also examined. It was found that the basal and CRH‐induced release of Met‐ENK was not changed 1 week after castration. However, a significant increase in the basal release of this peptide was observed 4 weeks after gonadectomy, and the Met‐ENK–releasing efficacy of CRH was found to be reduced. The Met‐ENK content of hypothalami from 1‐week castrates was not significantly changed from control levels, but was significantly reduced in those from 4‐week castrates. These long‐term effects of castration could be overcome by the subcutaneous implantation of testosterone‐containing capsules at the time of castration.
Transition Metals, Ferritin, Glutathione, and Ascorbic Acid in Parkinsonian Brains
Authors: Riederer Peter , Sofic Emin , Rausch Wolf‐Dieter , Schmidt Bruno , Reynolds Gavin P. , Jellinger Kurt , Youdim Moussa B. H.
Published: October 2006

Abstract: The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinson's disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)‐binding protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.
Electroconvulsive Shock (ECS) and the Adenosine Neuromodulatory System: Effect of Single and Repeated ECS on the Adenosine A1 and A2 Receptors, Adenylate Cyclase, and the Adenosine Uptake Site
Authors: Gleiter Christoph H. , Deckert Jurgen , Nutt David J. , Marangos Paul J.
Published: October 2006

Abstract: The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once‐daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6‐[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5′N‐[3H]ethylcarboxyamidoadenosine ([3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well‐known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once‐daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.
Human Brain‐Type Glycogen Phosphorylase: Quantitative Localization in Human Tissues Determined with an Immunoassay System
Authors: Kato Kanefusa , Shimizu Atsuko , Kurobe Naomi , Takashi Munehisa , Koshikawa Takashi
Published: October 2006

Abstract: Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity‐purified with a column of brain phosphorylase‐coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase‐coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich‐type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross‐reactivity of the assay with muscle phosphorylase was <1%. Tissue concentrations of immunoreactive brain‐type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.
Differential Effect of Stress on In Vivo Dopamine Release in Striatum, Nucleus Accumbens, and Medial Frontal Cortex
Authors: Abercrombie Elizabeth D. , Keefe Kristen A. , DiFrischia Daniel S. , Zigmond Michael J.
Published: October 2006

Abstract: Microdialysis was used to assess extracellular dopamine in striatum, nucleus accumbens, and medial frontal cortex of unanesthetized rats both under resting conditions and in response to intermittent tail‐shock stress. The dopamine metabolites 3,4‐dihydroxy‐phenylacetic acid and homovanillic acid also were measured. The resting extracellular concentration of dopamine was estimated to be approximately 10 nM in striatum, 11 nM in nucleus accumbens, and 3 nM in medial frontal cortex. In contrast, the resting extracellular levels of 3,4‐dihydroxyphenylacetic acid and homovanillic acid were in the low micromolar range. Intermittent tail‐shock stress increased extracellular dopamine relative to baseline by 25% in striatum, 39% in nucleus accumbens, and 95% in medial frontal cortex. 3,4‐Dihydroxyphenylacetic acid and homovanillic acid also were generally increased by stress, although there was a great deal of variability in these responses. These data provide direct in vivo evidence for the global activation of dopaminergic systems by stress and support the concept that there exist regional variations in the regulation of dopamine release.
Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix
Authors: Probstmeier Rainer , Kühn Klaus , Schachner MeLitta
Published: October 2006

Abstract: A soluble form of the neural cell adhesion molecule (N‐CAM) was obtained from 100,000‐g supernatants of crude brain membrane fractions by incubation for 2 h at 37°C. The isolated N‐CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N‐CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N‐CAM 110 bound to different types of collagen (collagen types I‐VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fi‐bronectin). (b) Binding of N‐CAM 110 to heat‐denatured collagens was absent or substantially reduced, (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3‐2.0 ± 10−9M, depending on ihe collagen type and buffer conditions). Binding of N‐CAM 110 to collagens could be prevented in a concentration‐dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N‐CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell‐cell adhesion, N‐CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo
Atrial Natriuretic Peptide Modulates Amiloride‐Sensitive Na+ Transport Across the Blood‐Brain Barrier
Authors: Ibaragi Masa‐aki , Niwa Masami , Ozaki Masayori
Published: October 2006

Abstract: We obtained evidence that amiloride specifically potentiates 125I‐labeled α‐rat atrial natriuretic peptide (1‐28) [atrial natriuretic peptide (ANP)‐(99‐126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/ mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10−4M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10−7M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCI in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride‐sensitive Na+ transport. Thus, the possibility that ANPs control amiloride‐sensitive Na4+ transport at the blood‐brain barrier by interacting with specific receptors has to be considered