bluuebunny/crossref_metadata_2025 huggingface dataset:
Release of Endogenous and Accumulated Exogenous Amino Acids from Slices of Normal and Climbing Fibre‐Deprived Rat Cerebellar Slices
Authors: Toggenburger Gerhard , Wiklund Leif , Henke Hermann , Cuénod Michel
Published: October 2006

Abstract: Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for γ‐aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low‐Ca2+ (0.1 mM), high‐Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+‐induced efflux was similar under normal and low‐Ca2+ concentrations. The K+‐induced efflux of exogenously accumulated l‐[3H]aspartate, d‐[3H]aspartate, and l‐[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low‐Ca2+, high‐Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3‐acetylpyridine, release of endogenous aspartate and exogenous l‐[3H]glutamate and d‐[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, l‐[3H]glutamate and l‐[3H]aspartate differ in several aspects: (1) l‐[3H]glutamate uptake was 4 times higher than that of l‐[3H]aspartate; (2) fractional rate constant of K+‐evoked release of l‐[3H]aspartate was 7%× 2.5 min−1. and of l‐[3H]glutamate 36%× 2.5 min−1; and (3) specific activity of l‐[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for l‐[3H]aspartate, specific activities in the eluate and tissue were similar.
Developmental Changes in the Oligosaccharide Composition of Synaptic Junctional Glycoproteins
Authors: Fu Sheung Chun , Gurd James W.
Published: October 2006

Abstract: Synaptic junctions (SJs) were isolated from the forebrains of rats ranging in postnatal age from 10 days to greater than 1 year. SJ glycoproteins that react with Concanavalin A (Con A) were isolated by chromatography on Con A‐agarose and separated by gel electrophoresis. The concentrations of the major SJ Con A binding (Con A+) glycoproteins (apparent Mr 180,000, 130,000, and 110,000) increased between 10 and 28 days, with GP180 and GP110 showing greater relative increases than GP130. Con A binding oligosaccharides associated with 10‐day SJs were sensitive to digestion with endoglycosidase C11 and α‐mannosidase, indicating that they were of the high‐mannose type, as previously shown for 28‐day SJs. Con A+ oligosaccharides from rats of increasing postnatal age were analyzed by chromatography on Biogel P‐4. Two major oligosaccharides, containing five and eight mannose residues, were present in SJs of all ages examined. During development the ratio of man5 to man8 oligosaccharides increased, so that man5 constituted the predominant species in 28‐day and adult SJs. Peptide mapping experiments showed that GP180, GP130, and GP110 were each associated with a unique polypeptide composition. Little or no change in peptide composition of the major SJ glycoproteins occurred during development.
Peripheral Nervous System Myelin Assembly In Vitro: Perturbation by the Ionophore Monensin
Authors: Linington C. , Waehneldt T. V.
Published: October 2006

Abstract: Sciatic nerves from 13‐day‐old rats were incubated in vitro with [35S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′‐cyclic nucleotide phospho‐diesterase (myelin), 5′‐nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorography, and bands corresponding to the P0 and myelin basic proteins were identified by co‐migration with unlabelled myelin marker proteins on both one‐dimensional SDS‐PAGE and two‐dimensional nonequilibrium isoelectric focussing/SDS‐PAGE systems. Following a 90‐min incubation with [35S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P0 protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P0 and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P0: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P0 were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P0 glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.
Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium‐Independent Efflux by Oleic and Arachidonic Acids
Authors: Rhoads D. E. , Osburn L. D. , Peterson N. A. , Raghupathy E.
Published: October 2006

Abstract: The release of preloaded [14C]neuroactive amino acids (glutamic acid, proline, γ‐aminobutyric acid) from rat brain synaptosomes can occur via a time‐dependent, Ca2+ ‐independent process. This Ca2+‐independent efflux is increased by compounds that activate Na+ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [14C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on 22Na+ permeability is observed. Since Na+ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na+ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na+ gradient.
Effects of Thyroxine on Methionine Adenosyltransferase Activity in Rat Cerebral Cortex and Cerebellum During Postnatal Development
Authors: Di Giorgio Rosa M. , Fodale Vincenzo , Macaione Salvatore , De Luca Grazia C.
Published: October 2006

Abstract: Methionine adenosyltransferase (MAT) activity was evaluated in cerebral cortex and cerebellum in controls and in rats treated with thyroxine. In controls the enzyme showed a different pattern in cerebral cortex and cerebellum during neonatal and late suckling periods. Hyperthyroid rats showed a significant increase of the enzyme in cerebral cortex only at the 2nd day of the neonatal period; in cerebellum the developmental pattern of MAT in neonatal period was anticipated temporally by 2–4 days. During the late suckling period thyroxine treatment produced in cerebellum a significant decrease in MAT activity at the 15th day after birth. From these data, we propose that hyperthyroidism may cause precocious induction of MAT both in cerebral cortex and in cerebellum and that the increased availability of S‐adenosyll‐methionine during the neonatal period could be related to its utilization also in polyamine biosynthesis.
Evidence for Membrane‐Associated Choline Kinase Activity in Rat Striatum
Authors: Reinhardt Rick R. , Wecker Lynn
Published: October 2006

Abstract: The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X‐100, was 5.90 μmol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X‐100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 μmol/g initial wet weight/h, respectively. Enzyme activity in the paniculate fraction was not detected in the absence of Triton X‐100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane‐associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane‐solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the paniculate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane‐associated enzyme has not yet been determined.
Human Platelet Phenol Sulfotransferase: Partial Purification and Detection of Two Forms of the Enzyme
Authors: Butler Paula R. , Anderson Robert J. , Venton Duane L.
Published: October 2006

Abstract: To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36–120‐fold. Activity toward 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), dopamine, 5‐hydroxytryptamine (5‐HT), and phenol eluted in the same Sephadex G‐100 and Affi‐Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5‐HT, and phenol as substrates were1198, 1068,401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 μM, 21 μM, 19 μM, and 500 μM for MHPG, dopamine, phenol, and 5‐HT, respectively. Apparent Km values for 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 μM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5‐HT. An additional pH optimum at 8.6 was present for 5‐HT. A thermolabile form of the enzyme measured with dopamine and 5‐HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10−5M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.
Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes
Authors: Crosland Richard D. , Martin Joseph V. , McClure William O.
Published: October 2006

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [3H]acetylcholine ([3H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [3H]choline were incubated with liposomes containing Mg2+, Ca2+, Mn2+, Co2+, Sr2+, or Ba2+. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [3H]ACh and [3H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg2+, Mn2+, or Co2+ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K+/Na+. Synaptosomes incubated with liposomes containing Ca2+, Sr2+, or Ba2+, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [3H]ACh. Liposomes containing either Ca2+ or Sr2+ were equally effective in promoting the release of [3H]ACh from synaptosomes, whereas liposomes containing Ba2+ were 2.5 times more effective in promoting the release of [3H]ACh than were liposomes containing either Ca2+ or Sr2+. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [3H]ACh caused by liposomes containing Ca2+, Sr2+, or Ba2+ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.
Identification and Determination of 1‐Methylimidazole‐4‐Acetic Acid in Human Cerebrospinal Fluid by Gas Chromatography‐Mass Spectrometry
Authors: Swahn Carl‐Gunnar , Sedvall Göran
Published: October 2006

Abstract: A method for the quantification of 1‐methylimidazole‐4‐acetic acid in human CSF was developed. Methylimidazole‐acetic acid was identified and quantitated in CSF. The method involves concentration of the compound on a cation exchanger, extraction of the methyl ester with ethyl acetate, and preparation of a heptafluorobutyryl derivate of the methyl ester, which is finally purified by chromatography on silica gel and quantitated by gas chromatography‐mass spectrometry with the deuterated analogue as internal standard. The coefficient of variation at 1 ng/ml was 13%. The limit of sensitivity was about 0.2 ng/ml. The concentration of methylimidazole‐acetic acid in lumbar CSF from healthy volunteers was below 1 ng/ml. Ventricular CSF contained higher concentrations than lumbar fluid. The existence of a rostrocaudal concentration gradient was established. There was a correlation between the concentration of methylimidazole‐acetic acid and tele‐methylhistamine in CSF. The concentration of methylimidazole‐acetic acid in lumbar CSF from schizophrenic patients, patients with subarachnoidal haemorrhage, or patients with rheumatic disease was in the range of that in healthy volunteers.
Posttranslational Modifications of Nuclear Proteins in Rat Cerebral Hemispheres During Postnatal Development
Authors: Serra I. , Kamiyama M. , Hashim G. A. , Ragonese P. , Giuffrida A. M.
Published: October 2006

Abstract: The processes of acetylation, phosphorylation, and methylation of nuclear proteins in cerebral hemispheres of 10‐ and 30‐day‐old rats were investigated. The experiments were carried out in vitro by measuring the incorporation of labeled precursors into histones and nonhistone chromosomal proteins (NHP) extracted from nuclei and separated by polyacrylamide gel electrophoresis. The results obtained indicate that there are age‐specific differences in the processes of phosphorylation and methylation of chromosomal proteins, whereas the acetylation process did not change significantly between 10 and 30 days of age. Electrophoretic analysis of histones indicated that the histone H3 was labeled to a greater degree than the other fractions and showed major changes in the processes of phosphorylation and methylation during postnatal development. The electrophoretic analysis of NHP showed considerable changes between 10 and 30 days of age. Certain components of NHP became increasingly evident as the brain developed. The methylation of an as yet unidentified protein with a molecular weight of approximately 118,000 daltons occurred at both ages.
Calmodulin and Ca2+‐ and Calmodulin‐Dependent Protein Kinase in Rat Anterior Pituitary Gland
Authors: Hatada Yasuyuki , Munemura Masahide , Fukunaga Kohji , Yamamoto Hideyuki , Maeyama Masao , Miyamoto Eishichi
Published: October 2006

Calmodulin and Ca2+‐ and calmodulin‐dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 ± 0.11 μg/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co‐migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 μ.M in the presence of 0.30μ.M calmodulin. Trifluoperazine and chlorpromazine, calmodulin‐interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 x 10‐5M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+‐ and calmodulin‐dependent manner. In light of these results, the possibility that calmodulin and the calmodulin‐activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.
Regulation of Rat Pineal Hydroxyindole‐O‐Methyltransferase in Neonatal and Adult Rats
Authors: Sugden David , Klein David C.
Published: October 2006

The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole‐O‐methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine‐treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the Km of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.
The Nerve Growth Factor Receptor on PC 12 Cells: Interconversion Between Two Forms with Different Binding Properties
Authors: Block Timothy , Bothwell Mark
Published: October 2006

PC 12 cells possess two classes of nerve growth factor (NGF) receptors on their surfaces which can be distinguished by kinetic criteria. The majority class binds and releases 125I‐NGF at a relatively rapid rate and has been called fast. The second class of receptors has been called slow because of relatively slower rates of binding and release of 125I‐NGF, and also may be distinguished from fast receptors by their cytoskeletal association and resistance to trypsin. PC 12 cell plasma membranes were prepared and shown to have only the fast class of receptors. These membranes were fused to receptorless 3T3 cells with polyethylene glycol. The resultant fused cells were shown to possess NGF receptors, essentially all of which behave like slow receptors. Immunofluorescence microscopy was used to monitor the introduction of PC 12 cell membrane and NGF receptorsinto 3T3 cells. Results obtained with C10‐2, a monoclonal antibody specific for a major PC12 cell‐surface antigen, show that up to 90% of 3T3 cells receive PC 12 membrane and that the PC 12 membrane becomes integrally incorporated into the 3T3 cell plasma membrane. It is suggested that an association of receptors with cytoskeleton may be involved in the conversion of fast to slow receptor behavior, and that the differing proportion of fast and slow NGF receptors in PC 12 and 3T3 cells reflects the differing cytoskeletal organization of these cells.
The Effect of the Shiverer Mutation on Myelin Basic Protein Expression in Homozygous and Heterozygous Mouse Brain
Authors: Barbarese E. , Nielson M. L. , Carson J. H.
Published: October 2006

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co‐dominantly with the wild‐type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wildtype amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild‐type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +shi animals. Third, the small amount of myelin basic protein‐related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild‐type brain. The pattern of myelin basic protein‐related polypeptides in +lshi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein‐related polypeptides qualitatively similar to that encoded by wild‐type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein‐related translation products are spared. The pattern of myelin basic protein‐related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis‐acting regulatory element controlling that gene (or genes).
Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate‐Binding Protein
Authors: Michaelis Elias K. , Michaelis Mary L. , Stormann Thomas M. , Chittenden W. Leroy , Grubbs Robert D.
Published: October 2006

A glutamate‐binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine its low isoelectric point (4.82). The protein exhibits either a single, high‐affinity class of sites for L‐[3H]glutamate binding (KD= 0.13 μM) when binding is measured at low protein concentrations, or two classes of sites with high (KD= 0.17 μM) and low affinities (KD= 0.8 μM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L‐glutamate to a self‐associating form of the protein. The displacement of protein‐bound L‐[3H]glutamic acid by other neuroac‐tive amino acids has characteristics similar to those observed for displacement of L‐glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.
Different Rates of Synthesis and Turnover of Ribosomal RNA in Rat Brain and Liver
Authors: Stoykova A. S. , Dudov K. P. , Dabeva M. D. , Hadjiolov A. A.
Published: October 2006

Abstract: The kinetics of in vivo labeling of cellular free UMP and nucleolar, nucleoplasmic, and cytoplasmic rRNA with [14C]orotate in rat brain and liver were investigated. Evaluation of the experimental data shows: (a) The rate of nucleolar precursors of ribosomal RNA (pre‐rRNA) synthesis and the deduced rate of ribosome formation in brain is about fivefold lower than in liver and corresponds to 220–260 ribosomes/min/nucleus. (b) The lower rate of in vivo pre‐rRNA synthesis is correlated with a lower activity of RNA polymerase I in isolated brain nuclei, (c) The half‐lives of nucleolar rRNA in brain and liver are 210 and 60 min, respectively, thus showing a slower rate of processing of pre‐rRNA in brain nucleoli. (d) The nucleo‐cytoplasmic transport of ribosomes in brain is also markedly slower than in liver and reflects the lower rates of synthesis and processing of pre‐rRNA. (e) Cytoplasmic ribosomes in brain and liver turn over with half‐lives of about 6 and 4 days, respectively. It is concluded that the markedly lower rate of ribosome biogenesis in brain is specified mainly at the level of transcription of rRNA genes.
Postmortem Changes in Binding to the Muscarinic Receptor from Human Cerebral Cortex
Authors: Undén A. , Meyerson B. , Winblad B. , Sachs C. , Bartfai T.
Published: October 2006

Abstract: The effects of storage at 4°C on the antagonist and agonist binding properties of the muscarinic acetyl‐choline receptor from fresh surgical and frozen autopsy samples from human cerebral cortex were studied. The number of 1‐[3H]3‐quinuclidinyl benzilate binding sites and their affinities were stable up to 51 h, both when stored as pieces of intact nonfrozen tissue and as a homogenate. The agonist binding properties as measured by the ability of the muscarinic agonist carbachol to compete with l‐[3H]3‐quinuclidinyl benzilate were also stable up to 51 h when the tissue was stored in the form of pieces. The affinity for carbachol decreased when the tissue was stored as a homogenate. The frozen autopsy samples showed no significant differences in binding properties in comparison with fresh neurosurgical tissue.
Decreased Number of Benzodiazepine Receptors in Frontal Cortex of Rat Brain Following Long‐Term Lithium Treatment
Authors: Hetmar Ole , Nielsen Mogens , Braestrup Claus
Published: October 2006

Abstract: Chronic administration of lithium led to a decreased number of benzodiazepine receptors (ca. 20%) in frontal cortex of rat brain, whereas no change was observed in the binding characteristics in the remaining part of the cortex and in the hippocampus and the cerebellum. Long‐term lithium treatment did not change the binding of [3H]lysergic acid diethylamide and [3H]quinuclidinyl benzilate to membranes of various brain regions in the rat. We concluded that the effect of lithium on the benzodiazepine receptor is brain region specific and cannot be explained as a consequence of a reduced γ‐aminobutyric acid‐ergic stimulation of benzodiazepine receptor, as the change in receptor binding was due to a change in the number of receptors rather than in the affinity constant.
125I‐Antibody Autoradiography and Peptide Fragments of Albumin in Cerebral Edema
Authors: Bodsch Wolfram , Hossmann Konstantin‐Alexander
Published: October 2006

Abstract: The regional distribution of albumin in serum extravasations of cerebral edema was visualized on intact brain slices by autoradiography of 125I‐labeled antibodies directed against albumin. Following autoradiographic imaging of edema protein spread, concentrations of total serum proteins were determined by radioimmunoassay in tissue micro samples taken from various regions of the brain. Peptide fragments of albumin—produced upon proteolytic breakdown of the native protein in vivo— were separated by affinity chromatography and HPLC. The combination of techniques for imaging, direct quantification, and analysis of molecular structure of serum proteins was proved to be valid in three different types of experimental cerebral edema in the rat: cortical cryogenic lesion, brain tumors, and stroke‐prone spontaneous hypertension. The results indicate differences in the reactivity of edematous tissue with respect to proteolytic activity, depending on the susceptibility of serum proteins to in vivo fragmentation.
Ethanolamine Glycerophospholipid Formation by Decarboxylation of Serine Glycerophospholipids in Myelinating Organ Cultures of Cerebellum
Authors: Bradbury Keith
Published: October 2006

Abstract: Serine decarboxylation as a source of glycer‐ophospholipid ethanolamine is known to occur in mammals. However, early investigators failed to demonstrate the pathway in brain. In the present study serine is shown to be decarboxylated to glycerophospholipid ethanolamine in myelinating organ cultures of rat cerebellum up to 32 days in vitro. The pattern of incorporation of l‐[3‐14C]serine into culture phospholipids strongly suggests a precursor‐product relationship between serine glycero‐phospholipids (SGP) and ethanolamine glycerophospho‐lipids (EGP), with serine label appearing in the ethanolamine moiety of EGP. The time course of labelling was similar for both acid‐stable and acid‐labile EGP In contrast DL‐[l‐14C]serine failed to label EGP significantly due to the loss of serine carbon C1 on decarboxylation. Through the systematic hydrolysis of phospholipids from cerebellar cultures incubated with l‐[3‐14C], it was clear that in SGP, acid‐stable EGP, and acid‐labile EGP >70% of radiolabel resides in the base moiety of each of these molecular species. It is proposed that serine decarboxylation as a source of EGP ethanolamine may be important in the early stages of brain development.
Evidence for the Presence of Lectins with Mannose Specificity in the Rat Cerebellum
Authors: Zanetta J. P. , Reeber A. , Dontenwill M. , Vincendon G.
Published: October 2006

Abstract: Two different methods were set up to detect the possible presence of lectin‐like molecules with a specificity for mannose‐rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11‐day‐old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine‐labeled proteins, kept in solution, with insolubilized [14C]glucosamine‐labeled glyco proteins. The results of both methods are compatible with the presence of lectin‐like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.
Tryptic Peptide Mapping Studies on the Regulatory Subunits of Type II Protein Kinases from Cerebral Cortex and Heart. Evidence for Overall Structural Divergence and Differences in the Autophosphorylation and cAMP‐binding Domains
Authors: Stein Jill C. , Sarkar Dwijen , Rubin Charles S.
Published: October 2006

Abstract: Regulatory subunits of type II cAMP‐dependent protein kinases (RII) (EC 2.7.1.37) from bovine brain and heart exhibit similar physicochemical and functional properties in vitro. However, the two forms of RII are markedly different in their (a) antigenic determinants, (b) cell and tissue distribution, and (c) subcellular localization. This suggests that each of these cAMP‐binding proteins may possess some unique structural features. To assess the degree of overall divergence between the primary structures of brain RII and heart RII, tryptic peptides derived from the two proteins were mapped by reverse phase HPLC on a C18 column. When the column effluent was monitored at 280 nm, 15 peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. More complex HPLC profiles were observed by following peptide absorbance at 210 nm, but a similar level of diversity was apparent: 13 brain‐RII‐specific and 15 heart‐RII‐specific tryptic peptides were identified and resolved with a gradient (0–50%) of acetonitrile in 0.1% trifluoroacetic acid. In complementary experiments, classical two‐dimensional mapping analyses revealed that several 32P‐labeled tryptic fragments derived from autophosphorylated and photoaffinity‐labeled brain RII were separate and distinct from the 32P‐peptides isolated from similarly treated heart RII. The HPLC mapping data document a structural basis for the immunological disparity between brain RII and heart RII and suggest that the two cAMP‐binding proteins are different proteins rather than interconvertible forms of a single protein. The two‐dimensional maps further indicate that significant structural dissimilarities between brain RII and heart RII also occur within the functionally conserved autophosphorylation and cAMP‐binding domains.
Photochemical Damage in the Albino Rat Retina: Oral Taurine Has No Effect on DNA and Protein Loss from Severely Damaged Photoreceptor Cells
Authors: Voaden M. J. , Hussain A. A. , Lalji K.
Published: October 2006

Abstract: Albino Wistar rats were exposed continuously to fluorescent light, causing photoreceptor destruction over a period of 4 days. The rate of DNA and protein loss from the cells was not affected by administration of 5% taurine in the drinking water.
Partial Purification and Properties of the Inhibitors of Na,K‐ATPase and Ouabain‐Binding in Bovine Central Nervous System
Authors: Akagawa Kimio , Hara Naoyuki , Tsukada Yasuzo
Published: October 2006

Abstract: Endogenous inhibitors of Na,K‐ATPase and ouabain‐binding were partially purified from bovine central nervous system, and some of their properties were studied. They were eluted as low‐molecular‐weight fractions by gel filtration. They could be adsorbed by both Amberlite IR 120 and Amberlite IRA 400 at acidic and basic pH, respectively, indicating that they could act as both anions and cations at different pH. These inhibitors of ouabain‐binding appeared to affect specific binding of ouabin, and Scatchard plot analysis showed that the in hibition was competitive, suggesting that they could bind to the same site as ouabain, presumably to Na,K‐ATPase itself. The inhibitory activities were heat stable, but charring inactivated them completely.
Kinetic Effects of Terbium Ions on Muscarinic Acetylcholine Receptors of Murine Neuroblastoma Cells
Authors: El‐Fakahany Esam E. , Pfenning Michael , Richelson Elliott
Published: October 2006

Abstract: Preincubation of murine neuroblastoma cells (clone NIE‐115) with terbium chloride resulted in a significant potentiation of carbachol‐mediated increase in cyclic GMP formation. This effect was accompanied by a shift of the peak response from 30 s to 120 s and a 6‐fold decrease in carbachol concentration producing half‐maximal responses, in addition to a significant increase in the Hill coefficient. Terbium ions also caused a significant decrease in the affinity and an increase in the maximum binding of [3H]quinuclidinyl benzilate to muscarinic receptors, the change in affinity being mainly due to a decrease in the association rate. Preincubation of cells with 1 mM carbachol for 4 h (the desensitized state of the muscarinic receptor) resulted in a decrease in the ability of terbium to alter [3H]quinuclidinyl benzilate binding. The effects of terbium reported here might be due to its affecting muscarinic receptor‐effector coupling, which is considered to be lost upon receptor desensitization.
Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes
Authors: Law Che‐Leung , Wong Patrick C. L. , Fong Wang‐Fun
Published: October 2006

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω‐diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC50= 0.22 mM), less so by spermidine (IC50= 4.0 mM), but not by putrescine (IC50 > 100 mM). At 10 mM, 1,3‐diaminopropane, cadaverine, and 1,8‐diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC50 values of 2.7 and 2.2 mM, respectively. Putrescine was only slightly inhibitory (IC50= 17.3 mM) and the other diamines were inactive. The uptake of γ‐aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 mM. With the exception of inhibition of glycine uptake by 1,8‐diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 mM, affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.
Developmental Changes in the Neutral α‐Amino Acid Transport Systems of Rat Brain Over the First Three Weeks After Birth
Authors: Riggs Thomas R. , Pote Kenneth G. , Im Hae‐Sook , Huff Daniel W.
Published: October 2006

Abstract: Transport of seven different amino acids into brain slices increased as donor rats aged from 1 to 6 days. Uptakes of 2‐aminoisobutyric acid, 2‐(methyl‐amino)isobutyric acid, and L‐alanine then decreased by day 14, while uptakes of other amino acids continued to increase or remained fairly constant. Neutral α‐amino acid transport systems were characterized by measuring inhibition of uptakes and kinetics for representative amino acids at different ages. Results indicate that 2‐ami‐noisobutyrate and 2‐(methylamino)isobutyrate used only one (and the same) system in brain slices from 6‐day‐old rats, with characteristics of system A (the major sodium‐dependent system in most mammalian cells). They used at least two systems at ages 1, 14, and 23 days, but, of these, only at 1 day did they use the same systems in the same proportions. Alanine and leucine used more than one system at all four ages, and somewhat different combinations than used by each other or by 2‐aminoisobu‐tyrate or 2‐(methylamino)isobutyrate. Their transport characteristics showed they used mostly system ASC (a sodium‐dependent system distinguished from A) and/or system L (sodium‐independent). We conclude that system A increases as the brain ages from 1 to 6 days and declines thereafter. System L probably increases with aging from 1 to 23 days.
A Possible Interrelationship Between Extracellular Taurine and Phosphoethanolamine in the Hippocampus
Authors: Lehmann Anders , Hamberger Anders
Published: October 2006

Abstract: The effect of guanidinoethane sulfonic acid (GES), an inhibitor of taurine uptake, was examined with respect to endogenous amino acids in the hippocampus of the freely moving rabbit. GES increased the extracellular levels of both taurine and phosphoethanolamine (PEA), other amino acids being unaffected. However, long‐term oral administration of GES selectively reduced endogenous taurine levels. The effect of GES on PEA appeared to be a consequence of the elevated extracellular taurine as exogenously administered taurine per se increased PEA levels in the extracellular space. The findings are discussed in conjunction with the proposed membrane‐stabilizing effects of taurine.
Ontogeny of Calcium Antagonist Binding Sites in Chick Brain and Heart
Authors: Marangos P. J. , Sperelakis †N. , Patel J.
Published: October 2006

Abstract: The ontogeny of chick brain and heart ventricle calcium antagonist binding sites was determined, using [3H]nitrendipine ([3H]NDP), as the ligand. The binding of [3H]NDP to adult heart and brain was kinetically very similar, with the former displaying a KD of 0.28 ± 0.02 nM and a Bmax of 138 ± 17 fmol/mg protein, and the latter a KD of 0.30 ± 0.02 nM and a Bmax of 160 ± 12 fmol/mg protein. The binding site in both brain and heart was highly specific for dihydropyridine calcium antagonists, such as nifedipine, nimodipine, and nisoldipine, since these drugs were several orders of magnitude more potent as inhibitors of [3H]NDP binding than verapamil, methoxyverapamil, or diltiazem. The developmental appearance of [3H]NDP binding sites in brain was rather gradual, with adult levels being attained just prior to birth. This was in contrast to the profile in heart ventricle which showed essentially adult levels at seven days gestation. The acquisition of [3H]NDP binding sites in chick brain roughly paralleled the onset of neuronal maturation and functional activity. In both chick brain and heart, verapamil and methoxyverapamil were weak inhibitors of [3H]NDP binding. However, the inhibition of binding in both tissues was markedly biphasic, with only 50% of the binding sites being susceptible to inhibition by each agent, suggesting that multiple calcium antagonist binding sites may exist in both tissues.
Study of the Mechanism of Release of [3H]GABA from a Teleost Retina In Vitro
Authors: Jaffé E. H. , Hernández N. , Holder L. G.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca2+ dependency of the process of release of [3H]GABA from the retina of the teleost Eugenes plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration‐dependent release of [3H]GABA. The veratridine effect was inhibited in Na+‐free solution, but was not affected by 1 μM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine‐and potassium‐stimulated release of [3H] GABA occurred in Ca2+‐free medium. Inhibitors of the Ca2+ channel, such as Mg2+(20 mM), La3+ (0.1 mM), and methoxy‐verapamil (4 μM‐0.4 mM), inhibited the veratridine‐and K+‐stimulated release. However, Co2+ and Cd2+ caused a potentiation and no change of the K+‐and veratridine‐stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [3H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradio‐graphic studies with [3H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Muller cells. β‐Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [3H]GABA was found with β‐alanine, and no inhibition of the subsequent veratridine‐stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [3H]GABA and lacked the capacity to induce the veratridine‐stimulated release of [3H]GABA. These results suggest a voltage‐and Ca2+‐dependent neuronal release of [3H]GABA from retina.
Muscarinic Acetylcholine Receptors in Neuroblastoma Cells: Lack of Effect of Veratrum Alkaloids on Receptor Number
Authors: Milligan Graeme , Strange Philip G.
Published: October 2006

Abstract: The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable (“control” cells) and excitable (“differentiated” cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6‐h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor‐active component of veratrine (not veratridine) occluded by the cells and released into the binding assays ùpon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.
5‐Hydroxytryptamine Measurements in Molluscan Ganglia and Neurons Using a Modified Radioenzymatic Assay
Authors: McCaman M. W. , Ono J. K. , McCaman R. E.
Published: October 2006

Abstract: A radioenzymatic procedure for the determination of sub‐picomole amounts of 5‐hydroxytryptamine (5‐HT) is described. It is a modification of the method originally described by Saavedra et al. (1973), in which 5‐HT was measured as the radiolabelled product [3H]5‐ methoxy‐N‐acetyltryptamine, after incubation with [3H]S‐adenosylmethionine, acetyl‐CoA, and the enzymes hydroxyindole‐O‐methyltransferase (EC 2.1.1.4) and N‐ acetyltransferase (EC 2.3.1.5). Ganglia from various gastropod molluscs (Aplysia californica, Tritonia diomedia, Lymnaea stagnalis, and Helisoma trivolvis), as well as individual neuronal somata isolated from these ganglia, were assayed for 5‐HT. Among the homologous giant cerebral cells in these animals, the 5‐HT concentrations were similar. Statistical analysis of the 5‐HT values in paired 5‐HT‐containing neurons demonstrated that the variability was considerably greater in samples obtained from different animals than in those obtained from the same animal. This suggests that experiments aimed at manipulating amine levels in individual neurons may benefit by using a paired‐cell paradigm. The effects of incubating Aplysia ganglia with 5‐HT or with the 5‐HT precursors tryptophan and 5‐hydroxytryptophan (5‐HTP) were studied. High concentrations of 5‐HTP and 5‐HT (100 μM) increased the levels of 5‐HT in ganglia, but only incubation in high concentrations of 5‐HTP resulted in an increase of 5‐HT in the isolated somata of 5‐HT‐containing neurons C1 and P5.
In Vivo Effects of β‐Bungarotoxin on the Acetylcholine System in Different Brain Areas of the Rat
Authors: Gulya K. , Budai D. , Kása P. , Rakonczay Z.
Published: October 2006

Abstract: The in vivo effects of β‐bungarotoxin (β‐BT) on the acetylcholine (ACh) system were studied in the whole cerebrum and in different brain regions. The effect of β‐BT on cerebral ACh and choline (Ch) contents was time‐dependent. The results show that a singe intrace‐ rebroventricular injection of 1 μg toxin increased both the ACh and Ch contents in the cortex, hippocampus, and cerebellum, while in the striatum the ACh level was decreased. Ten nanograms of toxin injected into the lateral ventricle twice, on the first and third days, led to a reduced ACh level 2 days after the last treatment. In animals treated with the same dose three times, on the first, third, and fifth days, and sacrificed 2 days after the last injection, the choline acetyltransferase and acetylcholinesterase activities were reduced and the number of muscarinic acetylcholine receptors was decreased. A biphasic effect of the toxin was therefore demonstrated. It is suggested that in the first phase of the toxin effect the increased levels of ACh and Ch may be due to the inhibition of neuronal transmission, while in the second phase, when the elements of the ACh system are reduced, the neuronal degenerating effect of β‐BT plays a significant role.
Similarities and Differences Between High‐Affinity Binding Sites for Cocaine and Imipramine in Mouse Cerebral Cortex
Authors: Reith Maarten E. A. , Allen Donald L. , Sershen Henry , Lajtha Abel
Published: October 2006

Abstract: Previously we found close similarities between high‐affinity binding sites for [3H]cocaine and those for [3H]imipramine in the mouse cerebral cortex in regard to their association with neuronal uptake of serotonin. In the present study we investigated whether the two ligands bind to the same site. The two ligands had the following high‐affinity binding properties in common: localization in both synaptosomal and microsomal fractions; vulnerability to treatment with N‐ethylmaleimide, trypsin, and phospholipase A2; and resistance to exposure to dithiothreitol. In contrast, cocaine binding in the cerebral cortex was more sensitive to heat inactivation than imipraminebinding. In addition, the mechanism by which cocaine inhibited [3H]imipramine binding differed from that by which imipramine inhibited [3H]cocaine binding. These data suggest that the high‐affinity binding sites for [3H]cocaine and [3H]imipramine in the cerebral cortex are distinct entities.