bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance
Authors: Fitzpatrick Susan M. , Hetherington Hoby P. , Behar Kevin L. , Shulman Robert G.
Published: October 2006

Abstract: The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (∼ 500 μM, arterial blood) did not cause significant changes in arterial Pco2, Po2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed ∼ 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate. The results reported here support earlier findings that an increased blood ammonia concentration has a pronounced effect on the brain concentrations of two important amino acids, glutamate and glutamine. They also provide in vivo evidence for the absence of a sustained alteration in either brain intracellular pH or in the concentration of high‐energy phosphate compounds during a period of acute hyperammonemia. The technique of in vivo NMR spectroscopy permits multiple, simultaneous measurements of important intermediary and energy metabolites in a single animal, in real time, prior to and during the systemic perturbation.
Thermodynamics of Agonist and Antagonist Interaction with the Strychnine‐Sensitive Glycine Receptor
Authors: Ruiz‐Gómez Ana , García‐Calvo Margarita , Vázquez Jesús , Marvizón Juan Carlos G. , Valdivieso Fernando , Mayor Federico
Published: October 2006

Abstract: The thermodynamic parameters associated with the interactions of agonists and antagonists with glycine receptors in rat spinal cord membranes were determined. The binding of the antagonist [3H]strychnine and the inhibition of strychnine binding by 11 different glycinergic ligands were examined at temperatures between 0.5 and 37°C The density of receptors was not affected by the temperature at which the incubation was performed, but the ability of glycine receptor agonists and antagonists to compete with [3H]strychnine binding varied markedly. The affinity of the receptor for the antagonists strychnine, 2‐aminostrychnine, RU‐5135,5,6,7,8‐tetrahydro‐4H‐Msoxazolo[5,4‐c]azepin‐3‐ol, and the ligands bicuculline, norharmane, and PK‐8165 decreased at higher temperatures. The binding of these ligands was enthalpydriven. In contrast, the affinity of the agonists glycine, β‐alanine, and taurine and of the antihelmintic ivermectin increased at higher temperatures, and their binding was characterized by substantial increases in entropy. In addition, temperature affected the allosteric interaction between the glycine and strychnine sites of the receptor, as indicated by changes in the Hill number of the competition curves for glycine. Our results clearly indicate that the binding of agonists and antagonists to the glycine receptor is differentially affected by temperature, probably as a consequence of the different changes induced in the receptor conformation.
Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes
Authors: Clark Mike , Dar M. Saeed
Published: October 2006

Abstract: The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC–fluorescence detection. Both basal and KCl‐stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 ± 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 ± 83 pmol/mg protein/5 s. Ethanol caused a dose‐dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol‐stimulated release does not involve the dilazep‐sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol‐induced motor disturbances in the rat.
Sequence Analysis of a Proteolyzed Site in Drosophila Choline Acetyltransferase
Authors: Slemmon J. Randall
Published: October 2006

Abstract: The amino terminal sequence of the 13‐kilodalton (kD) polypeptide present in purified Drosophila acetyl‐CoA: choline‐O‐acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse‐phase HPLC. The 13‐, 54‐, and 67‐kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13‐kD fragment yielded a sequence. The 67‐ and 54‐kD polypeptides appeared completely blocked to gas‐phase Edman sequencing. The location of the amino terminal sequence from the 13‐kD polypeptide in the cDNA‐deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67‐kD enzyme, with the 54‐kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13‐kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.
Calcitonin Gene‐Related Peptide‐Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization
Authors: Sano Yoshihisa , Hiroshima Osamu , Yuzuriha Teruaki , Yamato Chiyuki , Saito Akira , Kimura Sadao , Hirabayashi Tamio , Goto Katsutoshi
Published: October 2006

Abstract: Calcitonin gene‐related peptide (CGRP)‐binding sites were solubilized, using digitonin, from the porcine spinal cord, atria, and coronary arteries. The specific binding of 125I‐human α‐CGRP to the solubilized binding sites was inhibited by human α‐ and β‐CGRP and by rat α‐CGRP, but not by angiotensin II or human calcitonin. Scatchard plot analysis of saturation gave the same KD value for CGRP in the crude membrane fractions of the tissues examined. The affinity of CGRP to the binding sites was decreased by solubilization in the atria and coronary arteries, but not in the spinal cord. Affinity labeling followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed distinct molecular sizes of the specific binding sites among the tissues; 70K for the spinal cord, 70K and 90K for the coronary arteries, and 70K and 120K for the atria. These results indicate that the molecular characteristics of the specific binding sites of CGRP in the cardiovascular system are distinct from those in the central nervous system.
Solubilisation and Characterisation of a Putative Quisqualate‐Type Glutamate Receptor from Chick Brain
Authors: Henley Jeremy M. , Barnard Eric A.
Published: October 2006

Abstract: The brains of 1‐day‐old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate‐type glutamate receptor. α‐[3H]Amino‐3‐hydroxy‐5‐methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0°C in the presence of the chaotrope potassium thiocyanate, of 55 nMand 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane‐bound [3H]AMPA binding sites were efficiently solubilised by N‐octyl‐β‐D‐glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate ∼ AMPA > 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione > L‐glutamate > kainate and was identical for the membrane‐bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min−1 and 0.015 min−1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 × 106M−1 min−1 and 6.55 × 106M−1 min−1 The kinetically derived KD values were 80.7 nM and 2.3 nM The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high‐affinity sites. Solubilised [3H]AMPA binding sites were specifically retained on a wheat germ lectin column, but did not migrate as a single peak in sucrose density gradients or elute from a cation‐exchange column at a specific NaCl concentration. The results demonstrate the solubilisation of a stable [3H]AMPA binding protein and suggest that chick brain, and specifically chick telencephalon, will provide a useful system for further characterisation and purification of the quisqualate‐type glutamate receptor.
[3H]Diprenorphine Binding to k‐Sites in Guinea‐Pig and Rat Brain: Evidence for Apparent Heterogeneity
Authors: Wood M. S. , Traynor J. R.
Published: October 2006

Abstract: The binding of the unselective opioid antagonist [3H]diprenorphine to homogenates prepared from rat brain and from guinea‐pig brain and cerebellum has been studied in HEPES buffer containing 10 mM Mg2+ ions. Sequential displacement of bound [3H]diprenorphine by ligands with selectivity for μ‐, 8‐ δ, and k‐opioid receptors uncovers the multiple components of binding. In the presence of cold ligands that occupy all μ‐, δ‐, and k‐sites, opioid binding still remains. This binding represents 20% of total specific sites and is displaced by naloxone. The nature of these undefined opioid binding sites is discussed.
Characterization of the Effect of Monensin on γ‐Amino‐n‐Butyric Acid Release from Isolated Nerve Terminals
Authors: Sitges María
Published: October 2006

Abstract: The action of the polyether antibiotic monensin on the release of γ‐[3H]amino‐n‐butyric acid ([3H]GABA) from mouse brain synaptosomes is characterized. Monensin enhances the release of this amino acid transmitter in a dose‐dependent manner and does not modify the efflux of the nontransmitter amino acid α‐[3H]aminoisobMtyrate. The absence of external Ca2+ fails to prevent the stimulatory effect of monensin on [3H]GABA release. Furthermore, monensin is less effective in stimulating [3H]GABA release in the presence of Ca2+. The releasing response to monensin is absolutely dependent on external Na+. The blockade of voltage‐sensitive Na+ or Ca2+ channels does not modify monensin‐induced release of the transmitter. Also, the blockade of the GABA uptake pathway fails to prevent the stimulatory effect of monensin on [3H]GABA release. Although monensin markedly increases Na+ permeability in synaptosomes, these data indicate that the Ca2+‐independent monensin‐stimulated transmitter release is not mediated by the Na+‐dependent uptake pathway. It is concluded that the entrance of Na+through monensin molecules inserted in the presynaptic membrane might be sufficient to initiate the intraterminal molecular events underlying transmitter release.
Methylmercury‐Induced Movement and Postural Disorders in Developing Rat: High‐Affinity Uptake of Choline, Glutamate, and γ‐Aminobutyric Acid in the Cerebral Cortex and Caudate‐Putamen
Authors: O'Kusky John R. , McGeer Edith G.
Published: October 2006

Abstract: Subcutaneous administration of methylmercuric chloride to neonatal rats resulted in movement and postural disorders during the fourth postnatal week. Sodium‐dependent high‐affinity uptake of radiolabeled choline, glutamate, and γ‐aminobutyric acid (GABA) was measured in homog‐enates of cerebral cortex and caudate‐putamen. There was a significant decrease in the uptake of [3H]choline in the cerebral cortex, but not in the caudate‐putamen, at the onset of neurological impairment (73‐75%) and at one subclinical stage of toxicity (58‐64%). No significant differences in [3H]glutamate uptake were detected in either region. The uptake of [3H]GABA in the presence of 1 mMβ‐alanine, which was employed to inhibit the glial uptake process, was reduced significantly in both the cerebral cortex and caudate‐putamen at the onset of neurological impairment (50‐62%) and at one subclinical stage (40‐51%). This decrease in [3H]GABA up‐take is consistent with the results of previous studies using this animal model, which demonstrated a preferential degeneration of GABAergic neurons in the cerebral cortex and caudate‐putamen of methylmercury‐treated animals. Because the high‐affinity uptake of choline is the rate‐limiting step for acetylcholine synthesis by cholinergic neurons, the decrease in [3H]choline uptake may reflect an abnormal development of cholinergic innervation of the cerebral cortex.
Uptake and Utilization of Double‐Labeled High‐Density Lipoprotein Sphingomyelin in Isolated Brain Capillaries of Adult Rats
Authors: Homayoun P. , Bentejac M. , Lecerf Jean , Bourre J. M.
Published: October 2006

Abstract: Isolated rat brain capillaries were incubated in the presence of high‐density lipoprotein (HDL) containing [stearic acid‐14C, (methyI‐3H)choline]sphmgomyelim. This double‐labeled sphingomyelin was taken up in a concentration‐dependent manner. Cerebral capillary‐associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hy‐drolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of in‐corporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of “pulse‐chase” experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidylcholine of capillaries.
Characterization of Receptor‐Coupled Phosphoinositide Hydrolysis in the Rat Hippocampus After Intradentate Colchicine
Authors: Tandon Pushpa , Ali Syed F. , Bonner Meta , Tilson Hugh A.
Published: October 2006

Abstract: Lesions produced by intradentate hippocampal administration of colchicine have been reported to produce several time‐dependent behavioral and neurochemical changes, including a possible change in the signal transduction process for the cholinergic muscarinic receptor. To characterize further the effects of colchicine on receptor‐coupled hydrolysis of phosphoinositides, colchicine was injected ste‐reotaxically into the dentate gyrus of rats at a dose of 2.5 μg/ site. The animals were killed 1, 3, or 12 weeks after injection and the hippocampi removed and sliced. [3H]Inositol was incorporated into slices, and various receptor agonists known to stimulate inositol phosphate (IP) metabolism were studied. Colchicine administration altered agonist‐stimulated turnover in the hippocampus in a time‐dependent manner. This hy‐perstimulation was receptor‐mediated, because it was blocked by pirenzepine. The hyperstimulation of turnover was observed also with norepinephrine and serotonin. Colchicine had no effect on IP turnover in vitro. The effect of the colchicine lesion was observed only in the hippocampus, because no change in cholinergic muscarinic receptor‐stimulated phosphatidylinositol turnover was observed in the cortex. These studies indicate that intradentate administration of colchicine produces a compensatory change in the signal transduction process in the hippocampus detectable 12 weeks after the lesion.
Protein Carboxylmethyltransferase Activity in Intact, Differentiated Neuroblastoma Cells: Quantitation by S[3H]Adlenosylmethionine Prelabeling
Authors: Barten Donna M. , O'Dea Robert F.
Published: October 2006

Abstract: Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid‐precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [3H]methionine. These unexpected findings led to the development of a modified assay using S‐[3H]‐adenosylmethionine as the radiolabeled precursor for quan‐titating carboxyl methylation in intact cells. Data obtained from either acid‐precipitated proteins or those separated on an electrophoresis gel following S‐[3H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [3H]methionine incubation. Using each of the three methods, an approxi‐mately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 mMhexa‐methylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.
Allosteric Activation of Brain Mitochondrial Ca2+ Uptake by Spermine and ty Ca2+: Brain Regional Differences
Authors: Jensen John R. , Lynch Gary , Baudry Michel
Published: October 2006

Abstract: Analysis of the initial rates of 45Ca21 uptake by rat brain mitochondria in Ca2+‐1,2‐bis(o‐aminophenoxy)ethane‐N, N, N′,N′‐tetraacetic acid buffers indicated t[hat nontelen‐cephalic mitochondria exhibited both a mnch less pronounced stimulatory effect of spermine and signjificantly more hyperbolic kinetics of Ca2+ uptake than telencjephalic mitochondria. Nontelencephalic mitochondria were also markedly less susceptible to a Ca2+‐induced hysteretic allosteric activation of the Ca2+ uniporter. A new Ca2+ loading procedure, which strikingly illustrates differences in mitochondrial Ca2+buffering characteristics, is also described. In thjis procedure, low concentrations of Ca2+ (1, 2, or 5 μM) were repetitively added to mitochondria every 30 s while changek in free Ca2+concentration were recorded. Spermine induced a marked attenuation of the rise in free Ca2+ level underj these conditions. Steady‐state rates of Ca2+ uptake were determined by a quantitative analysis of the buffering of repetitive Ca2+ additions, and, again, brain regional differences Were qualitatively similar to those observed in the initial rate kinetics; Ca2+ uptake by nontelencephalic mitochondria in the steady state was markedly less responsive to stimulation by spermine and appeared to have a more hyperbolic dependence on Ca2in the absence of spermine. These results also suggested that there is a lag time in the activation of the uniporter by Ca2+, in addition to the hysteresis that has previously been observed in the deactivation of the uniporter. In summary, the present results indicate that the Ca2+ uniporter of nontelencephalic mitochondria is markedly less responsive to allosteric activation by both Ca2+ and spermine and that this is related to. but not entirely explained by, the more hyperbolic dependence of uptake on Ca2+ in nontelencephalic mitochondria. It is proposed that mitochondrial buffering of intracellular Ca2+ and the modulation of this process by spermine differ in telencephalic and nontelencephalic regions of brain.
Calcium/Diacylglycerol‐Dependent Protein Kinase and Its Major 87‐Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia
Authors: Walaas S. Ivar , Wang James K. T. , Albert Katherine A. , Greengard Paul
Published: October 2006

Abstract: When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol‐depenpent protein kinase (protein kinase C) and an 87‐kiIodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chen|iical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87‐kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal gaijglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87‐kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87‐kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.
13C Nuclear Magnetic Resonance Evidence for γ‐Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0
Authors: Brainard James R. , Kyner Eric , Rosenberg Gary A.
Published: October 2006

Abstract: The compartmentation of amino acid metabolism is an active and important area of brain research. I3C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of γ‐aminobutyric acid (GABA) and glutamate in rat brain. [l‐l3C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the l3C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This l3C labeling pattern occurs when [1‐l3Cjglucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (>80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.
[3H]GBR 12935 Binding to Dopamine Uptake Sites in the Human Brain
Authors: Keyser Jacques De , Backer Jean‐Paul De , Ebinger Guy , Vauquelin Georges
Published: October 2006

Abstract: Binding of l‐[2‐(diphenylmethoxy)ethyl]‐4‐(3‐phenylpropyl)piperazine ([3H]GBR 12935) was studied in membrane preparations of several human brain regions. In putamen, the substituted piperazine derivates cis‐ and trans‐flupenthixol displaced 90% of the total [3H]GBR 12935 binding. Computer‐assisted analysis of the competition curves revealed a high‐affinity site (30%; KiH= 54 μM) and a low‐affinity site (60%; KiL= 4.5 μM). The dopamine uptake blockers mazindol and nomifensine only displaced 30% of the total [3H]GBR 12935 binding in a monophasic way. Binding of [3H]GBR 12935 to the dopamine uptake sites, i.e., that displaced by dopamine uptake blockers, corresponded to part of the binding having low affinity for flupen‐thixol and was only detected in putamen, nucleus caudatus, nucleus accumbens, and substantia nigra. Even after masking the high‐affinity binding site for flupenthixol by including 1 μM cis‐flupenthixol in the binding assays, no dopamine uptake sites could be detected in globus pallidus, amygdala, thalamus, hippocampus, and cerebral cortex. Binding of [3H]GBR 12935 to dopamine uptake sites was lost in the nucleus caudatus ipsilateral to ventral midbrain infarctions, confirming their location on nigrostriatal nerve endings. Gross unilateral lesions of the striato‐ and pallidonigral pathways did not affect the number of dopamine uptake sites in the ipsilateral substantia nigra, suggesting that they may reside on the soma or dendrites of nigral neurons
Depolarization Increases Chloride‐Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex
Authors: Iwata Heitaroh , Koyama Yutaka , Baba Akemichi
Published: October 2006

Abstract: To assess the functions of Cl−‐dependent glutamate “binding” (Cl−‐dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 γg/ml), 10 μM aconitine, 56 mM K+, and 50 μM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60–85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2‐. The bindings of γ‐[3H]aminobutyric acid and 5‐[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without afecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl−‐dependent glutamate uptake sites. Na+‐dependent glutamate binding (Na+‐dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl−‐dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.
Source of Methyl Groups in Brain and Nerve Tissue in the Rat
Authors: Long Mary , Weir Donald , Scott John
Published: October 2006

Abstract: Previous studies that demonstrated that mouse brain accumulated significantly more radioactivity from subcutaneously administered 5‐methyltetrahydrofolate labelled in the methyl group compared to the label in the folate moiety are open to two interpretations. The methyl group could have been transferred to another compound (probably methionine) prior to its transport into the brain. Alternatively, if plasma 5‐methyltetrahydrofolate per se is significantly involved in the provision of methyl groups to brain and nerve tissue it would be expected that the folate moiety would be returned to the plasma to complete the cycle and thus would appear not to have been taken up. In this article, using competition experiments that exploit the differences in the mechanism of transport of methionine and 5‐methyltetrahydrofolate into brain and nerve, evidence is presented that in the rat the methyl group of 5‐methyltetrahydrofolate is transported after its conversion to methionine.
Modulation of Retinal Aromatic l‐Amino Acid Decarboxylase via α2 Adrenoceptors
Authors: Rossetti Zvani , Krajnc Dimitrij , Neff Norton H. , Hadjiconstantinou Maria
Published: October 2006

Abstract: Aromatic l‐amino acid decarboxylase (AAAD) activity of the rat retina increases when animals are placed in a lighted environment from the dark. The rise of activity can be inhibited by administering α2 adrenoceptor agonists. In the dark, the enzyme activity can be made to increase by administering α2 adrenoceptor antagonist drugs. Kinetic analysis indicates that the maximum velocity of the enzyme increases with little change of the Km for the substrate l‐3,4‐dihydroxyphenylalanine or the cofactor pyridoxal‐5′‐phosphate. The rise of activity in the light and in the dark after α2 antagonists can be blocked by administering cycloheximide, suggesting that protein synthesis is needed for the response. We speculate that epinephrine released in the dark from a subpopulation of retinal amacrine cells onto α2 receptors suppresses AAAD activity that is associated with dopaminergic amacrines.
Calcium Uptake and Protein Phosphorylation in Myenteric Neurons, Like the Release of Vasoactive Intestinal Polypeptide and Acetylcholine, Are Frequency Dependent
Authors: Agoston Denes V. , Lisziewicz Julianna
Published: October 2006

Abstract: The mechanism of the electrical‐to‐chemical decoding involved in the preferential release of the transmitters acetylcholine and vasoactive intestinal polypeptide (VIP) by electrical field stimulation at low (5 Hz) and high (50 Hz) frequencies was studied in superfused myenteric neurons. The stimulation‐induced uptake of 45Ca2+ accompanying high frequency stimulation was markedly reduced by 10 μM nifedipine, a specific blocker of l‐type voltage‐sensitive Ca2+ channels (VSCCs), as was also the preferential high‐frequency release of VIP. By contrast, the 45Ca2+ uptake during low‐frequency stimulation was somewhat lower per pulse, and neither this uptake nor the preferential release of acetylcholine occurring at this frequency was significantly reduced by nifedipine. These findings suggest that the release of acetylcholine and VIP involve different VSCCs. The pattern of in vitro protein thiophosphorylation in tissue extracts of differentially stimulated myenteric neurons involved polypeptides of 205, 173, 86, 73, 57, 54, 46, 32, 28, and 24 kDa and was also markedly stimulus and nifedipine dependent. This suggests that different phosphoproteins are involved during the frequency‐dependent activation of the different Ca2+ channels and exocytotic mechanisms.
Use of Inhibitors of γ‐Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid
Authors: Löscher Wolfgang , Hönack Dagmar , Gramer Martina
Published: October 2006

Abstract: The technique of estimating γ‐aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA‐T (4‐aminobutyrate:2‐oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA‐T inhibition in different brain regions after systemic injection of GABA‐T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA‐T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA‐T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA‐T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA‐synthesizing enzyme GAD (L‐glutamate‐1‐decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats. but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/ kg i.p., 5–30 min prior to AOAA, reduced the AOAA‐induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA‐T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation
Intracerebroventricular Administration of l‐Buthionine Sulfoximine: A Method for Depleting Brain Glutathione
Authors: Pileblad Erik , Magnusson Tor
Published: October 2006

Abstract: Sprague‐Dawley rats (200‐260 g) were anesthetized with chJoral hydrate (400 mg/kg) and polyethylene cannulae were permanently implanted into the lateral ventricles. One or two days later, l‐buthionine‐[S,R]‐sulfoximine (L‐BSO), an apparently selective inhibitor of γ‐glutamylcysteine synthetase, was administered intracerebroventricularly through the cannulae. The brain content of glutathione (GSH) was determined by HPLC with electrochemical detection (gold/ mercury electrode) using N‐acetylcysteine as internal standard. A time‐course study of the changes in the striatum following a single dose of l‐BSO (3.2 mg) revealed a maximal depletion of GSH (‐60%) approximately 48 h after the administration. The effects of various doses of l‐BSO on GSH in the striatum, in the limbic region, and in the cortex were assessed at 24 h and 48 h after the administration. l‐BSO (0.02‐3.2 mg) produced dose‐dependent reductions of GSH in all brain regions studied at both time intervals. In a long‐term experiment l‐BSO (3.2 mg) was administered every second day. After 4 days, i.e., after two injections, striatal GSH was reduced by approximately 70%. No further depletion of GSH was obtained by additional injections of l‐BSO, but GSH was maintained at this low level for the 12 days studied. These results suggest that l‐BSO, administered intracerebroventricularly, would serve as a useful tool for evaluation of the biological role of GSH in the CNS.
Evidence for a Single Protein Kinase C‐Mediated Phosphorylation Site in Rat Brain Protein B‐50
Authors: Coggins Philip J. , Zwiers Henk
Published: October 2006

Abstract: The neuronal protein B‐50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B‐50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid‐dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B‐50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B‐50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabeled [32P]B‐50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B‐60 that have their N‐termini at, or C‐terminal to, the Ser41 residue and are the major products of specific B‐50 proteolysis. Only those forms of B‐60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B‐50 phosphorylation by PKC and the proposed structure of the B‐50 calmodulin binding domain.
Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type‐2 Astrocyte Cell Lineage
Authors: Gallo Vittorio , Giovannini Claudio , Suergiu Rossana , Levi Giulio
Published: October 2006

Abstract: We have used postnatal rat cerebellar astrocyte‐enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type‐2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ‐[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N‐Methyl‐d‐aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l‐Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l‐glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d‐[3H]aspartate was taken up by both type‐1 and type‐2 astrocytes, but only type‐2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type‐2 astrocytes were also expressed by the immature, bipotential progenitors of type‐2 astrocytes and oligodendrocytes.
Rat Brain Protein Kinase C: Purification, Antibody Production, and Quantification in Discrete Regions of Hippocampus
Authors: Roth Bryan L. , Mehegan John P. , Jacobowitz David M. , Robey Frank , Iadarola Michael J.
Published: October 2006

Abstract: Protein kinase C (PKC), a calcium‐ and phospholipid‐dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three‐column procedure of diethylaminoethyl‐cellulose, phenyl‐Sepharose, and protamine‐agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol‐12,13‐dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a β‐type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78‐kilodalton form of PKC. Micropunches (300 μm in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS–10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I‐protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 ± 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 ± 0.02 and 0.085 ± 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.
Evidence for the Presence of Serotonin Receptors Negatively Coupled to Adenylate Cyclase in the Rabbit Iris‐Ciliary Body
Authors: Tobin A. B. , Osborne N. N.
Published: October 2006

Serotonin has no obvious effect on basal cyclic AMP levels but reduces the forskolin‐, isoproterenol‐, and vasoactive intestinal peptide‐induced stimulation of cyclic AMP levels in a dose‐dependent manner. Serotonergic, cholinergic, muscarinic, α‐adrenergic, and dopaminergic antagonists have no effect on the serotonin response. Topical Application of a serotonin/pargyline solution to the living eye causes desen‐sitisation of the serotonin response in the iris‐ciliary body, an observation confirming the presence of specific serotonergic receptors linked to adenylate cyclase. The 5‐HT1A[5‐hy‐droxytryptamine (serotonin) type 1A] receptor agonists 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin and bujspirone mimic the serotonin response in reducing the forskolin‐stimulated cyclic AMP levels, as do the indole derivatives 5‐methoxy‐ tryptamine, 5‐hydroxtryptophan, and tryptamine. However, the ineffectiveness of the 5‐HT1Aagonist ipsapirone and the inability of spiroxatrine to block the serotonin response show that classical 5‐HT1Areceptors are not involved. The serotonin response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor theophylline, which indicates the involvement of an inhibitory guanine regulatory protein in the coupling of the serotonin receptor to the adenylate cyclase catalytic unit.
Developmental Changes in the Calcium Dependency of γ‐Aminobutyric Acid Release from Isolated Growth Cones: Correlation with Growth Cone Morphology
Authors: Taylor Joanne , Gordon‐Weeks Phillip R.
Published: October 2006

We have investigated the development of Ca2+‐dependent ‐γ‐[3H]aminobutyric acid ([3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+‐evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, wfiich increased with age, such that by 10 days approximately 50% of the K+‐evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle‐containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+‐dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.
Effect of Halide Ions on t‐[35S]Butylbicyclophosphorothionate Binding
Authors: Garrett Kennon M. , Blume Arthur J. , Abel Marc S.
Published: October 2006

The binding of t‐[35S]butylbicyclophosphoro‐thionate ([35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I‐, followed by Br‐< CI‐< F‐. A similar rank order was observed for the concentration of ion required to produce half‐maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I‐ than in Cl‐. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.
Clinical Concentrations of Volatile Anesthetics Reduce Depolarization‐Evoked Release of [3H]Norepinephrine, but Not [3H]Acetylcholine, from Rat Cerebral Cortex
Authors: Bazil Carl W. , Minneman Kenneth P.
Published: October 2006

We compared the potencies of halothane, enflurane, and methoxyflurane in producing unconsciousness in vivo and in inhibiting the release of [3H]norepinephrine and [3H]acetylcholine in vitro. Rats were anesthetized with various concentrations of each anesthetic, and responsiveness was determined by a hemostat tail pinch. Slices of cerebral cortex were equilibrated with similar concentrations of each agent in vitro, and potassium‐evoked release of [3H]norepinephrine and [3H]acetylcholine was determined. For both studies, brain concentrations of the anesthetics were determined by heptane extraction and gas chromatography. Using this method, we found that brain concentrations of all three agents which caused unconsciousness in vivo also reduced depolarization‐evoked release of [3H]norepinephrine by approximately 30% in vitro. The release of [3H]acetylcholine was unaffected b/similar concentrations of these anesthetics. Such selective interference with stimulus‐secretion coupling in central noradrenergic, and possibly other, neurons might contribute to the depressant actions of volatile anesthetics. The differential effects on norepinephrine and acetylcholine release also suggest differences in the mechanisms by which these two transmitters are released.
Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia
Authors: Nowak Thaddeus S. , Bond Ursula , Schlesinger Milton J.
Published: October 2006

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70‐kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of ± the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several‐fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70‐related cDNA probes. Transient ischemia resulted in 5‐ to 10‐fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia. These studies emphasize the importance of using probes selective for stress‐inducible hsp70 sequences in evaluation of the heat shock response, particularly in tissues such as brain in which there is significant constitutive expression of hsp70‐related proteins.