bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Estrogen and Progesterone on Glutamic Acid Decarboxylase Activity in Discrete Regions of Rat Brain
Authors: Wallis Cleatus J. , Luttge William G.
Published: October 2006

Abstract: Female Charles River rats were ovariectomized and treated for three days with 17/8‐estradiol benzoate (E) (1.0 /μg/day), progesterone (P) (500 μg/day), vehicle, or a combined treatment (2 days E, one day P). Animals were killed on day 3 and the brains were dissected by the micropunch technique. Glutamic acid decarboxylase (GAD) activity was measured by collection of 14CO2. Estradiol benzoate and progesterone were potent inhibitors of GAD activity in regions such as the arcuate nucleus, ventromedial hypothalamus and corticomedial amygdala. Estrogen reduced the Vmax of GAD for glutamate as a substrate without changing the Km. Estrogen also failed to change the Km for pyridoxal phosphate. Combined treatment with estrogen and progesterone did not reduce GAD from ovariectomized levels except in the septum, indicating an interaction of the two hormones at the level of GAD. The suggestion is made that under conditions that inhibit LH secretion GAD activity is low, but when LH secretion is stimulated GAD activity may be comparatively high.
Release of Acetylcholine from Rat Brain Synaptosomes Stimulated with Leptinotarsin, A New Neurotoxin
Authors: Yoshino Jun E. , Baxter Daniel E. , Hsiao Ting H. , McClure William O.
Published: October 2006

Abstract: Leptinotarsin is a neurotoxic protein found in the hemolymph of potato beetles of the genus Leptinotarsa. In order to study the action of leptinotarsin from two species, L. haldemani and L. decemlineata, synaptosomes were prelabeled with [3H]choline in order to synthesize [3H] acetylcholine (ACh). These synaptosomes were then immobilized on Millipore filters and used for assay. Toxins from both species induce the release of radioactivity in this system. Fractionation of the released radioactivity indicated that ACh was released in preference to choline. The toxin that caused release was heat‐labile and was partially dependent on Ca2+ in the perfusing medium. Release followed apparent first order kinetics when stimulation was effected with leptinotarsin from L. haldemani (leptinotarsin‐h), but was more complex when using leptinotarsin from L. decemlineata (leptinotarsin‐d). Increasing the concentration of toxin increased the rate of release, but the shapes of the dose‐release curves elicited by the leptinotarsins from the two species were different. While leptinotarsin‐h exhibited a simple, saturating dose‐release curve, leptinotarsin‐d was characterized by a sigmoid function, which was well described, with a Hill coefficient of 1.8. Antibodies directed toward black widow spider venom glands had no effect upon the releasing activity of leptinotarsin‐h but could partially neutralize that of leptinotarsin‐d. Toxins from both species have been partially purified and do not appear to be identical. The purified toxins should be useful tools with which to study the release of acetylcholine.
Reduction of Vanadate by Ascorbic Acid and Noradrenaline in Synaptosomes
Authors: Ádám‐Vizi Vera , Váradi G. , Simon P.
Published: October 2006

Abstract: The effect of ascorbic acid and noradrenaline on the inhibition of synaptosomal membrane ATPase by vanadate has been studied. Ascorbic acid (2 × 10‐3 M) and noradrenaline (10‐4 M) partly reversed the inhibition by vanadate (10‐6 M); however, when both were administered together the inhibition was completely eliminated. Using electron spin resonance (ESR) spectroscopy, we detected that ascorbic acid (10‐3 M) caused a 42% of reduction of vanadate (10‐4 M). Noradrenaline (10‐4 M) alone also reduced vanadate (10‐4 M) partially. When ascorbic acid and noradrenaline were present together all the vanadate was reduced to vanadyl. The concentration of ascorbic acid present in the brain under physiological conditions is identical to that found effective in our experiments. We suggest that ascorbic acid may protect the ATPase, at least in part, from inhibition by vanadate as a consequence of reducing vanadate to vanadyl. In those tissues where noradrenaline is also present a complete reduction of endogenous vanadium can be presumed.
Alteration of Synaptosomal Plasma Membrane Cholesterol Content: Membrane Physical Properties and Cation Transport Proteins
Authors: Shapiro H. K. , Barchi R. L.
Published: October 2006

Abstract: The level of nerve membrane cholesterol was altered by in vitro incubation of rat brain synaptosomal plasma membrane with liposomes having varying cholesterol contents. The normal plasma membrane cholesterol/phospholipid ratio of 0.3–0.4 (mol/mol) could be decreased by about one‐half or increased more than 100%. Fluorescence polarization measurements were made using the probe 1,6‐diphenyl‐1,3,5‐hexatriene. At temperatures below 35°C, lowering membrane cholesterol led to increased apparent microviscosity, while raising cholesterol content produced little change. However, at 45°C a continuous direct relationship existed between experimental membrane cholesterol/phospholipid ratio (ranging from 0.18 to 0.73) and apparent microviscosity. Under standard liposome‐synaptosomal plasma membrane exchange conditions, 80% of the initial specific [3H]saxitoxin binding activity to the voltage‐dependent sodium channel and at least 95% of the (Mg2+,K+)‐p‐nitrophenylphosphatae activity were preserved. Our results indicate that neither the characteristics of toxin binding nor the kinetics of this enzyme activity is dependent upon membrane cholesterol content.
Analysis of Histones Associated with Neuronal and Glial Nuclei Exhibiting Divergent DNA Repeat Lengths
Authors: Greenwood Paul D. , Silver Julie C. , Brown Ian R.
Published: October 2006

Abstract:Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea‐protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS‐polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.
Regional and Subcellular Localization in Rat Brain of the Enzymes That Can Synthesize γ‐Hydroxybutyric Acid
Authors: Rumigny J. F , Maitre M , Cash C , Mandel P
Published: October 2006

Abstract: Rat brain contains two major NADPH‐linked aldehyde reductases that can reduce succinate semialdehyde to 4‐hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched‐chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.
Alterations of Inositol Lipid Metabolism of Rat Sciatic Nerve in Streptozotocin‐Induced Diabetes
Authors: Natarajan V. , Dyck Peter J. , Schmid Harald H. O.
Published: October 2006

Abstract: The composition and metabolism of rat sciatic nerve phospholipids were studied 20 weeks after induction of chronic diabetes by intraperitoneal injection of streptozotocin (50 mg/kg). On a wet weight basis the nerves from the diabetic animals showed a 7% decrease in total phospholipid from that of controls and a relative decrease in phosphatidylinositol. Incubations of isolated sciatic nerves of diabetic rats in a medium containing [33P]orthophosphate gave decreased labeling of phosphatidylinositol and substantial changes in the labeling pattern of phosphatidylinositol phosphate and 4,5‐bisphosphate from that of controls. The ratio of label in these polyphosphoinositides decreased from 2.5 for normal nerve to about 1.0 for diabetic nerve within a 2‐h incubation period. These metabolic alterations were not observed in acutely diabetic animals 5 days after streptozotocin (100 mg/kg) administration. Because polyphosphoinositides may be involved in the control of membrane permeability during axonal conduction, alterations in their relative amounts or turnover rates could be related to the physiological changes of early diabetic neuropathy.
Biosynthesis of N‐Acetyldopamine and N‐Acetyloctopamine by Schistocerca gregaria Nervous Tissue
Authors: Mir Anis K. , Vaughan Peter F. T.
Published: October 2006

Abstract: N‐Acetyltyramine, N‐acetyldopamine and N‐acetyloctopamine were the major products when either L‐[3H]tyrosine or [3H]tyramine were incubated with thoracic ganglia of the desert locust, Schistocerca gregaria. No label was incorporated into L‐DOPA under these conditions, although 2–3% of the radioactivity could be recovered in dopamine and octopamine. Addition of the aromatic amino acid decarboxylase inhibitor, 3‐hydroxybenzylhydrazine (NSD 1015), prevented the formation of N‐acetylcompounds from L‐[3H]tyrosine, without resulting in an accumulation of label in L‐DOPA. In contrast, incubation of samples of haemolymph with L‐[3H]tyrosine resulted in the recovery of 7% of label in L‐DOPA, which was increased to 17% in the presence of NSD 1015. These results provide evidence that the initial step in the synthesis of dopamine and octopamine by S. gregaria nervous tissue is the conversion of L‐tyrosine to tyramine, which is subsequently metabolised to N‐acetyltyramine, N‐acetyldopamine or N‐acetyloctopamine.
Effect of Carbachol and 56 mM‐Potassium Chloride on the Cyclic AMP‐Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture
Authors: Tank A.W. , Weiner N.
Published: October 2006

Abstract: Tyrosine hydroxylase (TH) activity is increased two‐ to threefold in neuroblastoma cell line NBP2 maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP‐phosphodiesterase (PDE), 4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (RO 20‐ 1724), or the activator of adenylate cyclase, prostaglandin E1 (PGE1). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48‐h treatment with RO 20‐1724 and PGE1, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20‐1724 or PGE1. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20‐1724 or PGE1 is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20‐1724 or PGE1 alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48‐h delay in the induction of TH elicited by either RO 20‐1724 or PGE1. In contrast, the induction elicited by 8‐bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24‐h delay in the induction of TH elicited by RO 20‐1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM‐KCl inhibits the induction of TH elicited by 8‐bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.
Cholesterol Esters of the Human Brain During Fetal and Early Postnatal Development: Content and Fatty Acid Composition
Authors: Yusuf H. K. M. , Dickerson J. W. T. , Hey E. N. , Waterlow J. C.
Published: October 2006

Abstract: The content and fatty acid composition of cholesterol esters of the human brain during development from 13 weeks' gestation up to 26 months of age was studied. The three major brain areas, the forebrain, cerebellum, and the brain stem, were studied separately. The concentration of the esters in each brain region was the highest at the earliest fetal age of 13 weeks and fell during growth. However, transient rises in the concentration were observed, at about birth in the forebrain and at 4–5 months after birth in the cerebellum The peak concentration during the transient period (125–150 μg/g fresh tissue of forebrain and 100–125 μg/g of cerebellum) was similar to the concentrations observed in the two parts respectively during early fetal ages. The brain stem also showed similar transient peak at about a few weeks before birth, but only when the esters were expressed as amount per cell. In absolute terms, a clear transient period was evident in the forebrain between birth and 9 months, while in the cerebellum or the brain stem, the total amount of the esters increased up to about 1 year of age and then remained almost unchanged. The major fatty acids of the esters were palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acid. Most of these fatty acids showed certain changes in relative proportions during development. Thus, in the forebrain, palmitic and oleic acid decreased from about 32% and 40% (weight percentages) at 13–15 weeks of gestation to about 20% and 25% respectively at 26 months of age. During this period, linoleic and arachidonic acid increased from about 3% and S% to about 10% and 24%, respectively. Most of these changes occurred after birth. The cerebellum and the brain stem differed only slightly from the forebrain in either the fatty acid composition or the pattern of the developmental changes in the composition.
Newly Synthesized Cholecystokinin in Subcellular Fractions of the Rat Brain
Authors: Goltermann N. R. , Stengaard‐Pedersen K. , Rehfeld J. F. , Christensen N. J.
Published: October 2006

Abstract: The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [35S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial‐synaptosomal fraction, P2, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH‐terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, Kav, of 1.1 (corresponding to the COOH‐terminal octapeptide) and of 1.40 (a component which may correspond to the COOH‐terminal tet‐rapeptide amide, CCK‐4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.
A Possible Neurochemical Basis of the Central Stimulatory Effects of pp′DDT
Authors: Matin M. A. , Jaffery F. N. , Siddiqui R. A.
Published: October 2006

Abstract: The striatal neurochemical changes induced by pp′DDT (600 mg/kg) in mice were: an increase in the concentration of free ammonia, a decrease in the level of GABA and a reduction in the level of acetylcholine. These changes were maximal 5 h after treatment with pp′DDT, when the animals developed ‘severe’ convulsions. The convulsions and striatal neurochemical changes were modified to different degrees by barbiturates. Phenobarbitone protected all the animals from pp′DDT‐induced convulsions. The levels of striatal acetylcholine and GABA in these animals were within normal limits. Prominal reduced the severity of convulsions in pp′DDT‐treated animals. The levels of striatal acetylcholine and GABA were significantly lower than control values in these animals. Primidone neither modified the convulsions nor the striatal neurochemical changes in pp′DDT‐treated animals. The increase in the concentration of free ammonia, in pp′DDT‐treated animals, was not modified by barbiturates. Aminooxyacetic acid raised the GABA level above normal and abolished the convulsions in pp′DDT‐treated animals; the level of acetylcholine was within normal limits in these animals. Hydroxylamine produced a similar but less marked effect. Pyridoxine had no effect on convulsions or striatal neurochemical changes induced by pp′DDT. The increase in the concentration of free ammonia in pp′DDT‐treated animals was not modified by these agents. It is likely that pp′DDT produced stimulatory effects by increasing the concentration of free ammonia which may be involved in reducing the level of GABA, while changes in the level of acetylcholine may be an effect of pp′DDT‐induced convulsions.
Competitive Inhibition of γ‐Aminobutyric Acid Receptor Binding by N‐2‐Hydroxyethylpiperazine‐N′‐2‐ Ethanesulfonic Acid and Related Buffers
Authors: Tunnicliff Godfrey , Smith Julie A.
Published: October 2006

Abstract: Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high‐affinity and low‐affinity [3H]γ‐aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high‐ and low‐affinity binding sites, respectively. HEPES had Ki, values of 2.25 MM and 115 μM. The buffers had no appreciable effect on sodium‐dependent GABA binding or on γ‐aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high‐ or low‐affinity [3H]muscimol binding. Indeed, they were of the order of 105 times less effective in this case than against [3h]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.
Characterization by [3H]Dihydroergocryptine Binding of Alpha‐Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells
Authors: Haga Tatsuya , Haga Kazuko
Published: October 2006

Abstract: [3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma × glioma hybrid cells (NG 108‐15) that had the characteristics expected of α‐adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of α2 receptors than to that of α1. No specific binding of [3H]WB‐4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the α2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists, including [3H]DHE.
Effect of Nucleotides and Related Compounds on Glutamine Synthetase Activity in Chick Embryo Retina: A Biochemical and Immunohistochemical Study
Authors: Dutt Kamla , Norenberg Michael D. , Reif‐Lehrer Liane
Published: October 2006

Abstract: The effect of a number of nucleotides and related compounds on glutamine synthetase (GS) induction in retina from 12‐day chick embryo was studied with both biochemical and immunohistochemical techniques. A number of these compounds gave rise to GS activity comparable to that induced by treatment with cortisol, which is known to give rise to precocious induction of the enzyme in this system. Of the cyclic nucleotides examined, cAMP (0.5‐1.2 × 10−3 M) gave essentially no increase in GS activity. In contrast, dibutyryl cAMP (0.8 × 10−3 M) had a more significant effect on GS activity, as did 8‐bromo‐cAMP and cGMP at the same concentration. The activity elicited by these nucleotides was generally half that obtained by treatment with 2.8 × 10−7 M‐cortisol for the same length of time, 8‐Bromo‐cGMP (0.8 × 10−3 M) had an effect comparable to the aforementioned concentration of cortisol. Since phosphodiesterase activity is minimal in the chick retina at 12 days of development, addition of isobutylmethylxanthine (1 × 10−5 M) to this system had, as would be expected, little effect on GS activity. Of the noncyclic compounds, 8‐bromoguanosine often gave rise to GS activity comparable to that obtained with cortisol. The other compounds (8‐bromo‐5′‐GMP, guanosine, adenosine, and 5′‐AMP) generally had less of an effect on GS. In general, the degree of staining in the immunohistochemical localization of GS corresponded well with the biochemical results and showed the enzyme to be present in regions consistent with the distribution of Muller cells and their processes. Thin‐layer chromatography and radioimmunoassay of the nucleotides did not show any steroid impurity in any of the compounds used in the study, even when determinations were carried out at five times the concentration of nucleotide used in the experiments.
Rat Brain 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase: Subcellular Distribution and Cold Lability
Authors: Patel Tarun B. , Clark John B.
Published: October 2006

Abstract: Data are provided indicating that the rat brain 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half‐life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3‐hydroxy‐3‐methylglutaryl‐CoA reductase in rat brain is discussed.
Developmental Patterns of Galactosyltransferase Activity in Various Regions of Rat Brain
Authors: Braulke Thomas , Biesold Dietmar
Published: October 2006

Abstract: The developmental pattern of glycoprotein‐galactosyltransferase activity was determined in the microsomal fractions of three regions of the embryonic rat brain and in parts of the visual system and the cerebellum postnatally. It could be shown that the enzyme activity was highest in the embryonic brain, where regional differences were apparent, and decreased progressively after birth. The enzyme profile in the cerebellum showed no marked postnatal changes.
Ibotenic Acid Analogues as Inhibitors of [3H]Glutamic Acid Binding to Cerebellar Membranes
Authors: Honoré Tage , Lauridsen Jørn , Krogsgaard‐Larsen Povl
Published: October 2006

Abstract: The L‐[3H]glutamic acid binding capability of rat cerebellar membranes prepared with or without preincubation at 37°C followed by washing was investigated. The two preparations (KD= 820 nM, Bmax= 54.5 pmol/mg protein; KD= 509 nM, Bmax=13.0 pmol/mg protein) showed no difference in specificity of the binding of the ibotenic acid analogues, consistent with the removal of an endogenous inhibitor by the preincubation at 37°C followed by washing. The order of potency of the ibotenic acid analogues as inhibitors of L‐[3H]glutamic acid binding is different from the order of potency in vivo, suggesting that the binding sites found are different from the physiological glutamic acid receptor.
Chronic Hyperphenylalaninemia Produces Cerebral Hyperglycinemia in Immature Rats
Authors: Dienel Gerald A.
Published: October 2006

The amino acid content of three tissues was measured in 10‐day‐old rats made hyperphenylalaninemic from age 3 to 10 days by daily injections of phenylalanine plus α‐methylphenylalanine to inhibit phenylalanine hydroxylase (PAH). At 12 h after the last injection, the concentrations of alanine, valine, methionine, isoleucine, and leucine in the cerebral hemispheres were depressed by 25–50%, whereas that of glycine was elevated 2.3‐fold. In the spinal cord, the levels of phosphoserine, methionine, and leucine were decreased by 40–50%, and those of serine and threonine increased by 50%. Tyrosine and phenylalanine concentrations were high in all tissues, 2–3 and 15–30 times normal, respectively; of the amino acids investigated, they were the only ones changed in the liver. Cerebral hyperglycinemia was also produced by chronic treatment with phenylalanine plus p‐chlorophenylalanine to inhibit PAH, but not by acute (12 h) hyperphenylalaninemia. An increase in cerebral phosphoserine phosphatase activity was greater in rats treated with phenylalanine plus PAH inhibitor than with inhibitor alone. The content of brain glycine normally declines with age from birth to 15 days; this decrease was prevented by chronic hyperphenylalaninemia. Attempts to reduce the cerebral glycine content of the hyperphenylalaninemic rats were unsuccessful. However, one of the therapeutic protocols, methionine loading, may be useful because it increased the methionine and decreased the phenylalanine contents in the brain.
Regional Distribution and Production Rate of 3‐Methoxy‐4‐Hydroxyphenylethyleneglycol Sulphate (MHPG‐SO4) in Rat Brain
Authors: Kohno Yasuko , Tanaka Masatoshi , Nakagawa Ryoichi , Toshima Nobuo , Nagasaki Nobuyuki
Published: October 2006

The levels of noradrenaline (NA) and 3‐methoxy‐4‐hydroxyphen‐ylethyleneglycol sulphate (MHPG‐SO4) in 15 brain regions showed a parallel distribution in male Wistar rats. The differences in regional distribution of MHPG‐SO4 were similar to those in the rate of NA turnover reported by other investigators. The accumulation rates of MHPG‐SO4 during 45 and 90 min after probenecid injection significantly correlated to the steady state levels of MHPG‐SO4 in nine regions studied. With the results, the regional levels of MHPG‐SO, either in untreated or in probenecid‐treated rats, are considered to be a useful index of NA turnover.
Experimental Galactose Toxicity: Effects on Synaptosomal Phosphatidylinositol Metabolism
Authors: Berry Gerard , Yandrasitz John R. , Segal Stanton
Published: October 2006

Abstract Experimental galactose toxicity was induced by weaning rats onto an isocaloric 40% galactose diet. Synaptosomes were prepared from cerebra of rats at 2‐9 weeks post‐weaning and incubated with [33P]Pi and myo‐[2‐3H]inositol in the presence or absence of 0.2 mM‐acetylcholine. The acetylcholine‐stimulated [33P]Pi labeling of phosphatidylinositol and the changes in amounts of phosphatidylinositol were similar in the normal and galactose‐toxic rats; however, acetylcholine‐stimulated myo‐[2‐3H]inositol labeling of phosphatidylinositol was markedly decreased in the galactose‐toxic rats. The impairment of acetylcholine‐stimulated myo‐[2‐3H]inositol incorporation into phosphatidylinositol observed after 2 weeks on the diet did not vary after more prolonged exposure to galactose.
Recovery of Acetylcholinesterase in the Diaphragm, Brain, and Plasma of the Rat After Irreversible Inhibition by Soman: A Study of Cytochemical Localization and Molecular Forms of the Enzyme in the Motor End Plate
Authors: Grubić Z. , Sketelj J. , Klinar B. , Brzin M.
Published: October 2006

Abstract Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in the end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, to be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.
[3H] Diazepam Displacing Activity in Human Cerebrospinal Fluid
Authors: Kuhn Wilfried , Neuser Dieter , Przuntek Horst
Published: October 2006

Pooled human cerebrospinal fluid was separated by Sephadex G‐50 chromatography. The presence of three peaks, A, B and C, was demonstrated by monitoring absorbance at 254 and 280 nm. All peaks showed [3H]diazepam displacing activity in the membrane receptor test. Peak B was further separated on Bio‐Gel P‐4. At least two major fractions free of salt and GABA in the molecular weight range of approximately 700‐3600 were shown to displace [3H]diazepam in the receptor test. This activity was enhanced by a factor of 3 in the presence of 10 μm‐GABA.
Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity
Authors: Siman Robert G. , Klein William L.
Published: October 2006

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron‐like NG108‐15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [3H]quinuclidinyl benzilate ([3H]QNB), decreased 50‐75% following 24‐h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [3H]deoxy‐d‐glucose ([3H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [3H]valine and [3H]uridine uptake; (4) RNA biosynthesis, measured by [3H]uridine incorporation; (5) protein biosynthesis, measured by [3H]valine and [35S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor‐mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 mm for 24 h) also caused a decrease in [3H]QNB binding levels, but chronic stimulation of α‐adrenergic, opiate, prostaglandin E1, and prostaglandin F2α receptors found on NG108‐15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.
Use of [3H] Spiperone for Labelling Dopaminergic and Serotonergic Receptors in Bovine Caudate Nucleus
Authors: Withy Raymond M. , Mayer R. John , Strange Philip G.
Published: October 2006

Abstract— [3H]Spiperone binding has been used to study neurotransmitter receptors in bovine caudate nucleus in displacement and saturation binding experiments. Displacement curves for several antagonists are biphasic and can be analysed into contributions from dopaminergic and serotonergic sites. Antagonist binding at each class of sites follows the simple mass action equations for binding at a homogeneous set of sites (slope factors close to unity). Agonist displacement curves also indicate complex behaviour, but agonist binding to the dopaminergic sites alone exhibits heterogeneous properties (slope factors less than unity). Saturation binding experiments have been conducted on each class of site, defining dopaminergic binding of [3H]spiperone as that binding displaced by 0.1 mm‐dopamine and serotonergic binding as that displaced by 0.3 μm‐mianserin. In each case, a single class of binding sites was detected: the binding parameters derived in this way have been used to calculate the proportions of the two classes of binding site observed in displacement experiments. Good agreement was obtained between calculated and observed values.
Influence on Turnover and Level of Hypothalamic Noradrenaline by a New Antihypertensive Agent (GYKI 11679)
Authors: Huszti Z. , Szilágyi G. , Mátyus P. , Kasztreiner E.
Published: October 2006

Abstract— In an attempt to delineate the possible importance of the concentration of noradrenaline at hypothalamic noradrenergic receptor sites in a hypotensive response to a drug, the action of a new antihypertensive agent, 1‐(6‐morpholino‐3‐pyridazynyi)‐2‐(1‐[tert‐butoxycarbonyi]‐2‐propylidene)‐diazane (GYKI 11679), on the turnover rate and the endogenous level of noradrenaline (NA) in rat hypothalamus was examined. An effective, antihypertensive i.p. dose of the compound (10 mg/kg) produced a significant but relatively short‐lasting reduction in the hypothalamic noradrenaline content, whereas no change was observed in the cardiac catecholamine level. The NA turnover determinations, carried out in GYKI 11679‐pretreated rats by measuring the disappearance of labeled NA at 1, 2, 3, and 5 h after the injection of the radioactive amine, showed that a 10 mg/kg i.p. dose of the compound, given 1 h prior to the i.c.v. administration of the labeled NA, increased the turnover rate of noradrenaline to a great extent. The estimated half‐lives of NA in the hypothalamus of the treated and of the non‐treated animals were calculated as 1.72 and 3.62 h, respectively. In vitro studies showed that the spontaneous outflow of noradrenaline from hypothalamic slices was accelerated by GYKI 11679 in a dose‐dependent manner in a concentration range of 10−5 to 10−7m. In a 10‐fold higher range, GYKI 11679 produced inhibition of both the hypothalamic and the adrenal tyrosine hydroxylase activity but did not alter DOPA‐decarboxylase, dopamine‐β‐hydroxylase, or monoamine oxidase activities. Direct in vivo measurements of catecholamine synthesis by determining the 3H‐catecholamines (CA) formed from [3H]tyrosine in the hypothalamus after an i.c.v. administration of the labeled precursor showed a moderate increase in [3H]CA formation following a 10 mg/kg dose of the compound. When GYKI 11679 was administered in a 75 mg/kg i.p. dose to rats, the transformation was reduced by –50%. Adenylate cyclase activity measurements did not show stimulatory or inhibitory actions of the drug on the NA‐stimulated adenylate cyclase of the rat hypothalamus, in accordance with previous results. This suggests that the increased NA turnover (utilization) caused by an effective, antihypertensive dose of GYKI 11679 is the direct consequence of an increased outflow, which occurs primarily in the hypothalamus. The increased activity of the noradrenergic neurons in this brain region might lead to a reduced sympathetic activity in the periphery and thus to a significant decrease in blood pressure.
Possible Role of Cyclic AMP in the Receptor‐Mediated Regulation of Glycosyltransferase Activities in Neurotumor Cell Lines
Authors: McLawhon Ronald W. , Schoon Gwendolyn S. , Dawson Glyn
Published: October 2006

Abstract: Exposure of mouse neuroblastoma cell line N4TGI to opiates or [d‐Ala2,d‐Leu5] enkephalin produced a naloxone‐reversible inhibition of cyclic AMP synthesis and prevented, in a concentration‐dependent manner, the formation of both ganglioside GM2 (GalNAc‐[NeuNAc]‐Gal‐Glc‐ceramide) from GM3 (NeuNAc‐Gal‐Glc‐ceramide) and ganglioside GM1 (Gal‐GalNAc‐[NeuNAc]‐Gal‐Glc‐ceramide) from GM2 in cell‐free extracts. In contrast, the receptor‐mediated elevation of intracellular cyclic AMP levels by agents such as prostaglandin E1 (in the presence of isobutylmethylxanthine) or the addition of the cyclic AMP derivatives (dibutyryl cyclic AMP) markedly stimulated the activities of UDP‐GalNAc:GM3,N‐acetylgalactosaminyltransferase and UDP‐Gal:GM2,galactosyltransferase. An overall increase in the synthesis of gangliosides more complex than GM3 was also observed in the mouse neuroblastoma x hamster brain explant hybrid cell line NCB‐20 following elevation of cyclic AMP levels by treatment with serotonin and pargyline. The data presented support the hypothesis that cyclic AMP may have a role in the regulation of sialoglycosphingolipid biosynthesis.
Cerebral Synaptic Transmission During Anoxia Is Protected by Creatine
Authors: Whittingham Tim S. , Lipton Peter
Published: October 2006

Abstract: Synaptic transmission in cerebral tissue fails very rapidly in the absence of oxygen; the metabolic basis for this is not known. We report here that the transmission failure in the guinea pig hippocampal slice can be delayed threefold by exposing the tissue to extracellular creatine (Cr) for 3 h. The improved survival is associated with an increase of tissue phosphocreatine (PCr) concentration. These data argue that the metabolic basis for synaptic transmission failure is a fall in tissue ATP concentrations. They also indicate a way to protect brain tissue against anoxic damage.
Subcellular and Perisynaptic Distribution of Rat Brain Aldehyde Dehydrogenase Activity
Authors: Koivula Timo , Turner Anthony J. , Huttunen Matti , Koivusalo Martti
Published: October 2006

Abstract: The nuclear mitochondrial and synaptosomal fractions of rat brain were each found to contain some 25–30% of the total aldehyde dehydrogenase activity. The cytoplasmic fraction had a very low total aldehyde dehydrogenase activity. There were differences in the distribution of the activity when different aldehydes were used as substrates, suggesting the presence of isoenzymes in the various subcellular compartments. When rats were treated intra‐cisternally with 6‐hydroxydopamine there was no change in brain aldehyde dehydrogenase activity, although the noradrenaline content and the activities of tyrosine hydroxylase and dopamine‐β‐hydroxylase were markedly decreased. Treatment with 6‐hydroxydopamine also had no significant effect on the aldehyde dehydrogenase activity in retinal homogenates. The results suggest that the aldehyde dehydrogenase activity in rat brain is predominantly outside the catecholaminergic nerve terminals.
Developmental Regulation of the Carbohydrate Composition of Glycoproteins Associated with Central Nervous System Myelin
Authors: Poduslo Joseph F.
Published: October 2006

Abstract: Glycoproteins from central nervous system myelin were evaluated for developmental alterations in their carbohydrate composition by autoradiographic analysis of radioiodinated lectin binding after separation by high‐resolution sodium dodecyl sulfate‐pore gradient slab gel electrophoresis (SDS‐PGE). Sixteen lectin‐binding components were assessed in highly purified myelin preparations from 15‐day, 18‐day, and adult rat brains, using the lectins Triticum vulgaris (wheat germ agglutinin) and Ulex europeus (gorse agglutinin I). Developmental changes in lectin binding for individual glycoproteins were evaluated semiquantitatively by comparing densitometric scans of the auto radiographs. Both increases and decreases in lectin binding for individual components were observed as a consequence of development, as well as the appearance and disappearance of lectin binding to three low‐molecular‐weight components. No changes in electrophoretic mobility and hence glycoprotein molecular weight were observed in any components when using these lectins. These developmental changes in lectin binding suggest that increases in glycoprotein (receptor) density occur, as well as an elaboration of oligosaccharide branching for individual glycoproteins. In addition, the appearance of a new glycoprotein in the adult myelin membrane could imply a new functional role not present in the immature membrane. These observations suggest that dynamic alterations of myelin‐associated glycoproteins occur during development. Such developmental regulation of membrane glycoproteins increases the significance of their potential role in myelination and myelin maintenance.
Synaptosomes from Rat Brain: Morphology, Compartmentation, and Transmembrane pH and Electrical Gradients
Authors: Deutsch C. , Drown C. , Rafalowska U. , Silver I. A.
Published: October 2006

Abstract: Morphological studies of synaptosomes isolated from rat brains show that approximately 68% of the synaptosomes in these preparations contain synaptic vesicles (range, 62–72.5%). Approximately 30% of the synaptosomes contain mitochondria, and only less than 20% of the total mitochondria in good preparations are free and not enclosed in synaptic structures. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5% for synaptosomes isolated from anesthetized animals and 11% for synaptosomes isolated from unanesthetized animals. These numbers bracket the value of 8.7% obtained from electron micrographs. The volume percent of intrasynaptic vesicles is 1.5% as calculated from electron micrographs. The pH gradient between the extracellular pH and the mean intracellular pH is ‐0.45, as measured by equilibrium distributions of methylamine and dimethylamine, and ‐0.05, as determined by equilibrium distributions of 5,5‐dimethyloxazolidine‐2,4‐dione and trimethylacetic acid. Analysis of these data shows that there cannot be a large pH gradient (alkaline inside) across the mitochondria, nor can the synaptic vesicle compartment be very large (<1.85%). Equilibrium distribution of [3H]triphenylmethyl‐phosphonium ion in synaptosomal preparations gives a calculated apparent potential of ‐85 mV, in agreement with our previous value. Analysis of these data using the measured volumes of mitochondrial and intrasynaptic vesicular compartments (8.7 and 1.5%, respectively) gives a maximum possible trans mitochondrial membrane potential of ‐59 mV.
Bioluminescent Assay of Total and Brain‐Specific Creatine Kinase Activity in Cerebrospinal Fluid
Authors: Halonen Tuija , Näntö Veikko , Frey Harry , Lövgren Timo
Published: October 2006

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.
Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells
Authors: Reiser G. , Scholz F. , Hamprecht B.
Published: October 2006

Abstract: Interaction of Li+ with the voltage‐dependent Na+ channel has been analyzed in neuroblastoma × glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage‐dependent Na+ channels. Veratridine and acionitine increased the uptake of Li+ to the same degree (EC50 30 μM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine‐stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 μg/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage‐dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.
Glycerol Phosphate Dehydrogenase in Developing Chick Retina and Brain
Authors: Adler Alice J. , Klucznik Katherine M.
Published: October 2006

Abstract: The development of cytoplasmic glycerol phosphate dehydrogenase (GPDH) activity in chick neural retina is compared with that in brain. GPDH converts dihydroxyacetone phosphate to glycerol 3‐phosphate, an intermediate in phospholipid synthesis. The enzyme is known to be under corticosteroid control in rat brain and spinal cord (but not muscle or liver) and in primary oligodendrocyte cultures. It has not been previously studied in the eye. In chick brain the GDPH specific activity rises fivefold from the early embryo to the adult, with nearly all the increase occurring between embryonic day 14 and hatching. This time course correlates well with the known maturation of chick adrenal cortex (which produces corticosteroids). On the other hand, in chick retina the GPDH specific activity remains at a low basal level throughout development. Furthermore, adult rat and beef retinas show much lower enzyme activity than do the corresponding brain tissues. GPDH can be induced precociously by hydrocortisone in embryonic chick brain from days 12 through 16, both in the intact embryo and in tissue culture; however, GPDH is not at all inducible in chick retina. The developmental increase in chick brain GPDH can be correlated qualitatively with myelin formation, as shown by luxol fast blue staining, whereas no myelin is seen in retina at any age. Our results are consistent with recent immunocytochemical studies demonstrating that GPDH in rat brain is associated with myelin‐producing oligodendroglial cells, absent in retina. In comparison, another glial enzyme, glutamine synthetase (GS), known to be inducible in both chick brain and retina, is localized in brain astrocytes and retinal Müller cells.
Calcium‐Dependent Binding of Cytosolic Proteins by Chromaffin Granules from Adrenal Medulla
Authors: Geisow Michael J. , Burgoyne Robert D.
Published: October 2006

Abstract: Purified chromaffin granules from bovine adrenal medulla bound a small group of medullary cell cytosol proteins at micromolar levels of Ca2+ and physiological levels of K+, Mg2+, and Mg‐ATP. The bound proteins had molecular weights of 33,000‐37,000 and 70,000‐71,000 on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and did not correspond with any previously reported cytosolic components of chromaffin cells. The new proteins were eluted from intact granules or resealed granule membranes at 0.1 μM Ca2+; binding was half‐maximal at 2.6 μM. Adsorption and elution in this manner resulted in a high degree of purification of the new proteins that were minor components of the original cytosol. Partially purified fractions enriched in the 33,000‐37,000 and 70,000‐71,000 proteins bound 45Ca2+ at submicromolar levels in the presence of millimolar Mg2+. Calmodulin was also bound by the granule membranes and was present in trace amounts in cytosol eluates from granules, but it did not bind to the new proteins in the presence of calcium ions. The possible significance of the new proteins to calcium‐mediated secretion from chromaffin cells is discussed.