bluuebunny/crossref_metadata_2025 huggingface dataset:
Axonal Transport and Metabolism of Glycoproteins in Rat Sciatic Nerve
Authors: Toews Arrel D. , Saunders Barry F. , Morell Pierre
Published: December 2006

Abstract: The axonal transport and metabolism of glycoproteins in sciatic nerve sensory axons were examined from 2 h to 7 weeks following injection of [3H]fucose into the 5th lumbar dorsal root ganglion of adult rats. Incorporation of fucose into glycoproteins was prolonged; only half of the 3H label in the ganglion was acid‐insoluble after 4 h, and maximal labeling did not occur until approximately 24 h after injection. [3H]Glycoproteins were transported distally at a rate of approximately 310 mm per day after a synthesis and/or processing lag of approximately 40 min. Gel electrophoresis demonstrated that many glycoproteins were transported, including prominent labeled species having apparent M.W. of approximately 49,000, 90,000, 118,000, and 132,000. With increasing time after injection, a peak with an apparent M.W. of 49,000 accounted for an increasing proportion of the total label in the nerve. The accumulation of this glycoprotein (possibly a subunit of Na+,K+‐ATPase, an enzyme known to be present in axolemma) was due in part to its preferential deposition in the axons, while other glycoproteins passed through the axons directly to the nerve terminals. Radioactivity in another labeled glycoprotein, with an apparent M.W. of 30,000, also increased preferentially in the nerve relative to other labeled glycoproteins. This was shown to be the myelin P0 protein. This protein was not transported from the ganglion; rather, its increased prominence with time in the nerve was due both to a decrease in amounts of other labeled species and to an absolute increase in labeling of the P0 protein, possibly due to reutilization by the Schwann cells of fucose released during turnover of labeled glycoproteins delivered to the axons by axonal transport.
Noncorrelation of Choline Kinase or ATP Citrate Lyase with Cholinergic Activity in Rat Brain
Authors: Harvey S. A. K. , Leong S. F. , Clark J. B.
Published: December 2006

Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.
Actions of Ptychodiscus brevis Red Tide Toxin on Metabolic and Transmitter‐Releasing Properties of Synaptosomes
Authors: Risk M. , Norris P. J. , Coutinho‐Netto J. , Bradford H. F.
Published: December 2006

Abstract: A pure toxin isolated from Ptychodiscus brevis stimulated differential release of amino acid neurotransmitters from mammalian cortical syn‐aptosomes together with loss of K+ content and respiratory stimulation at 40 ng/ml. The effect was blocked by tetrodotoxin and by verapamil, implicating Na+ channel activation and possibly Ca2+ influx as necessary for the response, although the response did not change upon omission of Ca2+. Verapamil was therefore likely to be acting as a Na+ channel blocker in this instance. The toxin at 40 ng/ml caused acetylcholine release from guinea pig ileum, which is consistent with the proposed depolarising action for the toxin.
Chronic Manganese Treatment of Rats Alters Synaptosomal Uptake of Dopamine and the Behavioural Response to Amphetamine Administration
Authors: Leung Thomas K. C. , Lai James C. K. , Tricklebank Mark , Davison Alan N. , Lim Louis
Published: December 2006

Abstract: Chronic manganese treatment from conception onward resulted in increased striatal synaptosomal uptake of dopamine, but not of a variety of other neurotransmitters/precursors in 80‐day‐old rats. The open‐field behaviour of these manganese‐treated 80‐day‐old rats was no different from that of untreated age‐matched rats. However, amphetamine administration (1 mg/kg body weight) increased activity to a significantly lower extent in manganese‐treated rats. These observations indicate that chronic manganese treatment results in marked alterations of activities associated with the dopaminergic system.
On the Phospholipid Metabolism of Glial Cell Primary Cultures.
Authors: Witter Brigitte , Gunawan Johannes , Debuch Hildegard
Published: December 2006

Abstract: Primary cultures were prepared from newborn rat brain. After 16‐18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1‐[1‐3H]alkyl‐sn‐glycero‐3‐phosphoethanolamine (1‐alkyl‐GPE), for 1–20 h. Five main products were formed: 1‐alkyl‐2‐acyl‐GPE; 1‐alkyl‐2‐acyksn‐glycero‐3‐phosphocholine (1‐alkyl‐2‐acyl‐GPC); 1‐alkenyl‐2‐acyl‐GPE (ethanolamine plasmalogen); 1‐alkenyl‐2‐acyl‐GPC (choline plasmalogen); and 1‐alkyl‐glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl‐, 27.5% alkyl‐acyl‐, and 46.0% alkenyl‐acyl‐ compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1‐alkyl‐glycerol was a minor reaction.
The Occurrence of γ‐Aminobutyrylcholine in Mammalian Brain—Fact or Artefact?
Authors: Barrett D. A. , Davies W. E.
Published: December 2006

Abstract: γ‐Aminobutyrylcholine (GABACh) has been reported to exist in mammalian brain tissue, but not, as yet, given a specific physiological role in the CNS. In order to investigate further its occurrence and function in the CNS, two new methods have been developed for its isolation and determination at the picomole level. Its isolation has been achieved by ammonium Reineckate precipitation or by cation‐exchange followed by HPLC determination of the dansyl and O‐phthaldialdehyde derivatives. Using these methods, no free endogenous GABACh (<80 pmol/g) was found in rat, guinea pig, cat, pig, marmoset, or human brain tissue. No evidence was obtained, either in vitro or in vivo, for the incorporation of [14C]choline or [14C]γ‐aminobutyric acid into GABACh. GABACh was hydrolysed at a low rate (maximum of 45 nmol/h/g of brain tissue) after incubation with rat, guinea pig, or cat brain minces and homogenates. These results fail to confirm the data of other investigators, and the possible reasons for this are discussed.
Effect of Chronic Hypernatremic Dehydration and Rapid Rehydration on Brain Carbohydrate, Energy, and Amino Acid Metabolism in Weanling Mice
Authors: Thurston Jean Holowach , Hauhart Richard E. , Schulz Demoy W.
Published: December 2006

Abstract: This is a study of the effects of chronic hypernatremic dehydration and rehydration on carbohydrate, energy, and amino acid metabolism in the brains of weanling mice. Chronic hypernatremic dehydration induced by 4 days of water deprivation and salt loading was associated with severe weight loss (no other observed clinical effects), increased brain Na+ levels, and a decreased brain water content. Changes in the concentrations of brain glucose, glycolytic and citric acid cycle metabolic intermediates, and phosphocreatine were compatible with reduced cerebral metabolic rate. In adaptation to chronic hypernatremia, there was a significant increase in the content of the measured brain amino acids. Rapid rehydration over a 4‐h period with 2.5% dextrose in water returned plasma Na+ levels and brain Na+ and water contents to normal. After rehydration, metabolites were altered in a manner consistent with increased fluxes through the glycolytic pathway and citric acid cycle; the brain glycogen content almost tripled. Brain taurine and glutamine levels were not lowered by rehydration, and the total content of the measured amino acids in brain was still significantly higher than in controls. We speculate that these metabolic perturbations may relate to the development of cerebral edema and seizures or coma following rapid rehydration of humans with chronic hypernatremic dehydration.
Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases
Authors: Law P. Y. , Griffin M. T. , Koehler J. E. , Loh H. H.
Published: December 2006

Abstract: The role of membrane phospholipids in enkephalin receptor‐mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma × glioma NG108‐15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108‐15 cells were treated with phospholipase C from Clostridium welchii at 37°C, an enzyme concentration‐dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)‐stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF‐5′‐guanylyl‐imidodiphosphate (Gpp(NH)p)‐sensitive adenylate cyclase activities. Further, Leu5‐enkephalin inhibition of basal or PGE1‐stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]d‐Ala2‐Met5‐enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N‐component. Further, treatment of NG108‐15 hybrid cell membrane with phospholipase C at 24°C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108‐15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108‐15 cells was not altered by treating the cells with phospholipase A2 or phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.
Kinetics of [3H]Ro 15–1788 Binding to Membrane‐Bound Rat Brain Benzodiazepine Receptors
Authors: Brown Chloë L. , Martin Ian L.
Published: December 2006

Abstract: Ro 15–1788 is thought to interact specifically with the benzodiazepine receptor population in the mammalian CNS as an antagonist. We have compared the kinetics of interaction of this ligand with the benzodiazepine agonist flunitrazepam in the rat cerebellum. The association of [3H]Ro 15–1788 with the benzodiazepine receptor in this brain region is monoexponential, and the dissociation, initiated either by dilution or by displacement with a number of different ligands, also obeys simple monoexponential kinetics. There is no evidence of cooperative interactions with this ligand, and its dissociation is unaffected by the presence of 100 μMγ‐aminobutyric acid and/or 150 mM sodium chloride. In contrast, the dissociation of the agonist [3H]flunitrazepam is biphasic, and the possible interpretation of this data in terms of agonist‐induced conformational change is discussed. Evidence is also presented that the mechanism of interaction of Ro 15–1788 with the benzodiazepine receptor population in hippocampal membranes is distinct from that found in the cerebellum.
Turnover Rates of the Molecular Species of Ethanolamine Plasmalogen of Rat Brain
Authors: Masuzawa Yasuo , Sugiura Takayuki , Ishima Yoshio , Waku Keizo
Published: December 2006

Abstract: 1,2‐Diradyl‐3‐acetylglycerols prepared from 1‐O‐alk‐l'‐enyl‐2‐acylglycero‐3‐phosphoethanolamine (alkenylacyl‐GPE, ethanolamine plasmalogen) and 1‐alkyl‐2‐acylglycero‐3‐phosphoethanolamine (alkylacyl‐GPE) of rat brain at 18 days of age were subfractionated into six species by AgNO3impregnated TLC. The percent compositions of subfractions were compared with that of 1,2‐diacylglycero‐3‐phosphoethanolamine (diacyl‐GPE). The incorporation rate of [1‐3H]glycerol into each molecular species was also estimated to examine the turnover rate and selective synthesis of molecular species of ethanolamine phosphoglycerides (EPG). Among the molecular species of EPG, a major proportion contained polyun‐saturated fatty chains, and the sum of tetraene‐, pentaene‐, and hexaene‐containing species was >65% in common with three classes of EPG. It was possible to calculate the turnover time, synthesis rate, and synthesis rate constant of ethanolamine plasmalogen in myelinating rat brain by the equation of Zilversmit et al. since the time‐dependent change of specific activity and the distribution of molecular species indicated that each molecular species of alkenylacyl‐GPE is synthesized from the corresponding species of alkylacyl‐GPE. The observed turnover lime of ethanolamine plasmalogen was about 5 h. The observed turnover times of the various molecular species were of the order: tetraene hexaene > pentaene monoene diene. The synthesis rate constants of each molecular species, in the formation of alkenylacyl‐GPE from alkylacyl‐GPE, were of the order: hexaene > tetraene > pentaene > diene monoene. This result indicated that the hexaene species is preferentially synthesized from alkylacyl‐GPE among molecular species of ethanoiamine plasmalogen in the desaturation of alkylacyl‐GPE.
5′‐Nucleotidase from Bovine Caudate Nucleus Synaptic Plasma Membranes: Specificity for Substrates and Cations; Study of the Carbohydrate Moiety by Glycosidases
Authors: Meflah Khaled , Harb Jean , Duflos Yves , Bernard Serge
Published: December 2006

Abstract: We studied 5:‐nucleotidase in preparations of synaptic plasma membranes from bovine caudate nucleus. The best substrates for this membrane‐bound enzyme were purine nucleotides, particularly 5’AMP. Effects of metal cations and chelating agents suggest that 5′‐nucleotidase is a metalloprotein. Optimal conditions for solubilization of the 5′‐nucleotidase were found by using a low concentration of the zwitterionic detergent sulfobetaine 14. In contrast, another membrane‐bound enzyme, acetylcholinesterase, was not solubilized under these conditions, but only in the presence of Triton X‐100. The effects of lectins (concanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, and Limulus polyphemus agglutinin) showed that both enzymes are glycoproteins. Sequential hydrolysis with specific glycosi‐dases produced modifications of the effect of lectins on these enzymes. The results suggest the presence of a complex‐type glycosylation, with a fucose residue on the internal /V‐acetyl‐D‐glucosamine of the pentasaccharide core.
Magnesium Transport by Brain Mitochondria: Energy Requirement and Dependence on Ca2+ Fluxes
Authors: Rugolo Michela , Zoccarato Franco
Published: December 2006

Abstract: The association of Mg2+ ions with mitochondria isolated from guinea pig cerebral cortex is investigated and resolved into two components, that bound to the surface of both the outer and the inner membranes and that transported into the mitochondrial matrix. When rotenone‐treated mitochondria are preincubated in a Mg2+ ‐containing medium, Mg2+ binding can be measured and actual Mg2+ transport determined after the addition of succinate. Mg2+ uptake as well as retention within mitochondria is an energy‐dependent process linked to substrate oxidation. EGTA completely prevents Mg2+ uptake, while the Ca2+ uniporter inhibitor Ruthenium Red, along with prevention of Mg2+ uptake, induces a slow efflux of accumulated Mg2+ ions. These findings suggest that both inward and outward Mg2+ movements follow Ca2+ fluxes across the mitochondrial membrane. Modulation of Mg2+ movements by mitochondria is therefore suggested to occur within nerve terminals.
Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I‐Labeled Secretin Binding and Association with Adenylate Cyclase
Authors: Roth Bryan L. , Beinfeld Margery C. , Howlett Allyn C.
Published: December 2006

Abstract: Secretin, a gut‐brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin > vasoactive intestinal peptide > peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I‐labeled secretin to the neuroblastoma plasma membranes was saturable, time‐dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5′‐(β,γ‐imino)‐triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.
Postmortem and Regional Changes of Serotonin, 5‐Hydroxyindoleacetic Acid, and Tryptophan in Brain
Authors: McIntyre Iain M. , Stanley Michael
Published: December 2006

Abstract: Using a specific and sensitive high pressure liquid chromatographic technique for the measurement of serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), and tryptophan (TRP), we found that there were no changes in 5‐HT or 5‐HIAA in the rat cortex when left in situ for 6 h at room temperature or 24 h at 4°C. Only a minimal 14% increase in 5‐HT was observed after 24 h at 4°C in the striatum of the same animals. Concentrations of TRP, however, were increased significantly in both brain regions by these postmortem delay procedures. A second study revealed that there were significant regional 5‐HT and 5‐HIAA concentration differences within the cerebral cortex. The frontal cortex was shown to have the highest concentrations of 5‐HT and 5‐HIAA. Further, within the frontal cortex, 5‐HIAA levels varied, showing apparent progressive rostral to caudal increases. 5‐HT concentrations, however, remained constant within the frontal cortex. These results are discussed in reference to the conflicting reports of the previous human suicide and postmortem studies.
Regulation of Calmodulin‐ and Dopamine‐Stimulated Adenylate Cyclase Activities by Light in Bovine Retina
Authors: Gnegy Margaret E. , Muirhead Nancy , Harrison Jeffrey K.
Published: December 2006

Abstract: Neural retina from most species contains 3,4‐dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca2+‐binding protein, cal‐modulin, and that calmodulin can increase dopamine‐sen‐sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine‐ and calmodulin‐stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low‐intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca2+ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua‐nosine‐5′‐(β,γ‐imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn2+‐, and GTP‐stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down‐regulation of dopamine receptor activity. The down‐regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.
An N‐Protected γ‐Aminobutyric Acid Dipeptide with Anticonvulsant Action
Authors: Galzigna L. , Bianchi M. , Bertazzon A. , Barthez A. , Quadro G. , Coletti‐Previero M. A.
Published: December 2006

Abstract: N‐Pivaloyl‐leucyl–γ‐aminobutyric acid (PLG) is a synthetic dipeptide with a partition coefficient of 1.67 in an ethyl acetate/water system that partially inhibits the synaptosomal uptake and activates the release of [U‐ 14C]‐γ‐aminobutyric acid ([U‐14C]GABA). The displacement of GAB A from crude synaptic membranes by PLG occurs with an IC50 of 10−5M. The compound has the capacity to cross the blood‐brain barrier and increase central GABA levels. Its ED50 on cardiazol‐induced convulsions is 60‐65 mg/kg. PLG is resistant to hydrolysis by chymotrypsin and partially inhibits the proteolytic activity of trypsin.
Relationship Between Phenolamines and Catecholamines During Rat Brain Embryonic Development In Vivo and In Vitro
Authors: David J. C.
Published: December 2006

Abstract: p‐Octopamine and phenylethanolamine are present in the embryonic rat brain earlier than catecholamines. These phenolamines are localized mainly in the hypothalamus, where the level of p‐octopamine is very high. The parallel developmental study of the activities of dopamine β‐hydroxylase, 3,4‐dihydroxyphenylalanine decarboxylase, tyrosine hydroxylase, and monoamine oxidase shows that phenolamines are present in significant amounts in the hypothalamus until tyrosine hydroxylase and monoamine oxidase become catalytically active. The culture of embryonic hypothalamus at different ages shows that no tyrosine hydroxylase and monoamine oxidase activities can be detected if the tissue is cultured before 15 days. This clearly indicates that all the enzymes related to catecholamine biosynthesis are not triggered at the same time during the development of the rat brain. These results are discussed on the basis of the physiological importance of phenolamines in mammals and of the use of the developing rat brain as a model for the study of the onset of the catecholaminergic system and the decline of the octopamine.
Unilateral Activation of Caudate Tyrosine Hydroxylase During Voluntary Circling Behavior
Authors: Morgan Michael E. , Yamamoto Bryan K. , Freed Curt R.
Published: December 2006

Abstract: We trained rats to circle for a sucrose water reward and found that this behavior is associated with a unilateral increase in the activity of caudate tyrosine hydroxylase, the rate‐limiting enzyme for dopamine synthesis. The increase in tyrosine hydroxylase activity occurs in caudate contralateral to the circling direction and the change is transient, increasing during the first 20 min of circling but then plateauing and falling as turning slows. Enhanced synthetic capacity is followed by increases in the contents of dopamine and dihydroxyphenylacetic acid in the contralateral caudate nucleus. These observations are the first evidence for specific activation of a neurotransmitter synthetic enzyme during voluntary motor behavior.
Role of Creatine Phosphate in the Discharge of the Electric Organ of Torpedo marmorata
Authors: Borroni E.
Published: December 2006

Abstract: The role of creatine phosphate and adenosine triphosphate, as high energy phosphate sources, has been investigated during the discharge and recovery of the electric organ of Torpedo. ATP serves as the immediate source of energy for the biochemical process supporting the electrical activity of the electric organ. Under repetitive stimulation, when the energy demands exceed production, ATP levels are maintained constant at the expense of creatine phosphate. Only when the reservoir of creatine phosphate is depleted do the levels of ATP decrease, and this point corresponds to the state of maximal fatigue of the electric organ. Recovery studies show that the electric organ rapidly recovers the capacity to respond to single pulse stimuli. This recovery is statistically related to the recovery of the levels of ATP and acetylcholine. However, in this phase, the fatiguability of the electric organ is very high since its energy reservoir is still depleted. The complete recovery of the electric organ requires several hours and is closely related to the restoration of the levels of creatine phosphate.
Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons
Authors: Richter‐Landsberg Christiane , Lee Virginia M. , Salton Stephen R. J. , Shelanski Michael L. , Greene Lloyd A.
Published: December 2006

Abstract: Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4‐8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr≲ 230,000. Another major component of apparent Mr= 55,000 as well as minor components of apparent Mr≲ 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr≲ 210,000 was released only by the sympathetic neurons. The soluble released Mr≲ 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF‐inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane‐derived NILE GP. At least several of the other released GPs appeared to be derived from membrane‐bound components with which they share immuno‐crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS‐polyacrylamide gels than their apparent membrane‐bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.
Comparison of the Properties of Semipurified Mitochondrial and Cytosolic Monoamine Oxidases from Rat Brain
Authors: Mayanil C. S. K. , Baquer N. Z.
Published: December 2006

Abstract: Mitochondrial and cytosolic monoamine oxidases were purified 220‐ and 129‐fold, respectively, from rat brain. The purification procedure involved extraction (without the use of detergents for mitochondrial monoamine oxidase), ammonium sulfate precipitation, and chromatography on Sephadex G‐25 and a DEAE‐cellulose column. The properties of both enzymes with kynuramine as substrate, including Km values and pH optima at different kynuramine concentrations; the Rf values on polyacrylamide gel electrophoresis; and the thermal in‐activation patterns were different. 2‐Mercaptoethanol, together with heat treatment, released the flavin and decreased the enzyme activity differentially for the two enzymes. The absorption spectrum showed a “Red shift” in the absorption maxima when the spectra of the non‐Triton‐treated purified preparations were compared with those of the Triton‐treated ones, thus possibly revealing that the mitochondrial and the cytosolic monoamine oxidases may be two different enzyme entities.
Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells
Authors: Trapp Bruce D. , Dubois‐Dalcq Monique , Quarles Richard H.
Published: December 2006

Abstract: The myelin specific protein, P2, was localized immunocytochemically in electron micrographs of 4‐day‐old rat peripheral nerve by a preembedding technique. P2 staining was restricted to Schwann cells that had established a one‐to‐one relationship with an axon. P2 antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P2 antiserum. This cytoplasmic localization of P2 protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P2 antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P2 antiserum stained the major dense line of compact myelin. These results demonstrate that P2 protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P2 to be a peripheral membrane protein.
Identification of Several Forms of the Glial Fibrillary Acidic Protein, or α‐Albumin, by a Specific Monoclonal Antibody
Authors: Gheuens J. , de Schutter E. , Noppe M. , Lowenthal A.
Published: December 2006

Abstract: A monoclonal antibody (mAb), termed UIA/ NEU/I/G1 (G1), that reacted with the astroglial marker glial fibrillary acidic protein (GFAP), or α‐albumin, is described. It was directed against a structural determinant of GFAP. The G1 mAb could be used for quantitative determination of GFAP in two‐site radiometric assays and for histoimmunological demonstration of GFAP. The G1 mAb reacted with the GFAP from rat as well as from man. The presence of several different molecular weight forms of GFAP in aqueous and detergent extracts from human brain was shown with the G1 mAb. The possible meaning of these forms is discussed.
Effects of Ethanol, Temperature, and Endogenous Regulatory Factors on the Characteristics of Striatal Opiate Receptors
Authors: Hoffman Paula L. , Chung Cathie T. , Tabakoff Boris
Published: December 2006

Abstract: Ethanol can alter the affinity of mouse striatal opiate receptors for their ligands, and the present studies were aimed at determining the importance of the receptor microenvironment for this effect of ethanol. Changing the temperature of the binding assay, and thus altering the properties of neuronal membrane lipids, resulted in changes in the observed affinity of striatal binding sites for dihydromorphine (DHM), but not for d‐Ala2, d‐Leu5‐enkephalin (ENK). The changes in temperature also differentially altered the response of the two binding sites to ethanol. Two other factors that regulate opiate receptor affinity, Na+ and GTP, also affected the response to ethanol. High concentrations of ethanol were more effective at decreasing receptor affinity for both DHM and ENK when the binding assays were performed in the presence of GTP or Na+. In addition, at 37°C and in the presence of GTP or Na+, DHM binding, but not ENK binding, was significantly inhibited by a low, physiologically attainable concentration of ethanol. Our results suggest that the response of opiate receptors to ethanol is influenced by the microenvironment of the receptors, including the physical state of the membrane lipids and/or by the nature of the interactions of receptors with “coupling proteins” within the membrane. The differential responses of μ and δ receptors to temperature and to ethanol suggest that these receptors reside in specific membrane environments. Under physiological conditions, several different factors may contribute to a selective action of ethanol on particular subtypes of opiate receptors.
Localisation of the MRC OX‐2 Glycoprotein on the Surfaces of Neurones
Authors: Webb Michael , Barclay A. Neil
Published: December 2006

Abstract: The MRC OX‐2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B‐lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX‐2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double‐immunofluorescence staining of cerebellar interneurone cultures with MRC OX‐2 antibody and tetanus toxin showed that all tetanus‐positive cells (neurones) were MRC OX‐2‐positive. Glial fibrillary acidic protein‐positive astrocytes were not labelled by MRC OX‐2 antibody. Thus OX‐2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy‐1.
Benzodiazepine Binding to Cultured Human Pituitary Cells
Authors: Voigt Mark M. , Davis Leonard G. , Wyche James H.
Published: December 2006

Abstract: Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18‐54,SF) grown in serum‐free medium. Preparations of 18–54,SF whole cells and cell membranes were shown to possess satu‐rable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole‐cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37°C) and when longer incubation times (60 min) were employed at 4°C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5–4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18–54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral‐type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.
Differential Stimulation of Inositol Phospholipid Turnover in Brain by Analogs of Oxotremorine
Authors: Fisher Stephen K. , Figueiredo Joanne C. , Bartus Raymond T.
Published: December 2006

Abstract: Structural analogs of oxotremorine have been employed to examine the relationship between the binding of agonists to muscarinic receptors in guinea pig cerebral cortex and the enhancement of inositol lipid turnover. Large differences were observed in the ability of the analogs to stimulate inositol phospholipid turnover, as measured both by the increase in labeling of phosphatidate and phosphatidylinositol from 32Pi in a nerve‐ending fraction, and by the stimulated release of labeled inositol phosphates from slices of cerebral cortex, a direct measure of inositol lipid breakdown. The quaternary N+ analogs, oxotremorine‐M and its N‐methylacetamide derivative, were five to thirteen times as effective as oxotremorine. In contrast, methyl substitution of the pyrrolidone ring of oxotremorine resulted in a complete loss of agonist activity. Receptor occupancy data obtained from the displacement of labeled quinuclidinyl benzilate bound to receptors in a nerve‐ending fraction indicated that the more efficacious agonists interacted with at least two affinity forms of the muscarinic receptor, whereas the less effective agonists bound to a single affinity form. Dose‐response curves obtained in the presence of oxotremorine‐M for inositol lipid turnover in both the nerve‐ending fraction and slice preparation correlated with the occupancy of a single low‐affinity form of the muscarinic receptor. The results suggest that the differential abilities of analogs of oxotremorine to enhance inositol lipid turnover in brain are closely related to the extent of agonist‐induced conformational change in the muscarinic receptor.
Molecular Forms of Acetylcholinesterase from Human Caudate Nucleus: Comparison of Salt‐Soluble and Detergent‐Soluble Tetrameric Enzyme Species
Authors: Gennari K. , Brodbeck U.
Published: December 2006

Abstract: Extraction of human caudate nucleus under high‐ionic‐strength conditions solubilized 20–30% of total acetylcholinesterase (AChE) activity. Density gradient centrifugation revealed monomeric (5.0 S) and tetrameric (11.0 S) enzyme species. The purified, tetrameric salt‐soluble (SS) AChE sedimented at 10.6 S and did not bind detergents. It showed an immunochemical reaction of identity with the detergent‐soluble (DS) AChE species from human caudate nucleus and human erythrocytes, but did not cross‐react with antibodies raised against human serum cholinesterase. The remaining activity was solubilized under low‐ionic‐strength conditions in the presence of 1.0% Triton X‐100. The purified tetrameric, DS‐AChE sedimented at 10.0 S as detergent‐protein mixed micelle and on extensive removal of the detergent this enzyme formed defined aggregates by self‐micellarization. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing conditions revealed that the salt‐soluble and detergent‐soluble tetrameric enzyme species both contained a heavy and a light dimer; under reducing conditions mainly one band corresponding to the light subunit was seen. Molecular weights of 300,000 dalton and 280,000 dalton were calculated for SS‐AChE and DS‐AChE, respectively. Limited digestion of DS‐AChE with proteinase K led to isolation of an enzyme that no longer bound detergents and lacked the inter‐subunit disulfide bridges.
Methamphetamine‐Induced Depression of Monoamine Synthesis in the Rat: Development of Tolerance
Authors: Schmidt Christopher J. , Somalia Patricia K. , Hanson Glen R. , Peat Michael A. , Gibb James W.
Published: December 2006

Abstract: Animals treated with high doses of amphetamines have been used as a model of schizophrenia due to the similarities between the psychosis associated with this mental disorder and that induced by chronic amphetamine abuse. When administered to naive rats in high doses, the amphetamine‐like CNS stimulant methamphet‐amine produces drastic alterations in the neurochemical parameters of the neostriatal monoaminergic systems. These alterations are characterized by a decrease in the activities of the rate‐limiting enzymes for dopamine and serotonin synthesis, as well as a decrease in the concentrations of both neurotransmitters and their metabolites. However, tolerance develops to these neurochemical effects when drug administration occurs in a pattern similar to that encountered during chronic amphetamine abuse. The results indicate that the neurochemical alterations produced by amphetamines in naive and tolerant animals differ widely. This suggests that the administration of high doses of amphetamine‐like central stimulants to naive rats may not be an appropriate model for studying the neurochemical changes associated with psychosis and amphetamine abuse.
Methionine Sulfoximine Prevents the Accumulation of Large Neutral Amino Acids in Brain of Portacaval‐Shunted Rats
Authors: Rigotti Paolo , Jonung Torbjorn , Peters John C. , James J. Howard , Fischer Josef E.
Published: December 2006

Abstract: Portal‐systemic shunting and hyperammonemia lead to an accumulation of the large neutral amino acids in brain and apparently alter transport of neutral amino acids across the blood‐brain barrier. It has been proposed that portal‐systemic shunting leads to a high brain concentration of glutamine, a product of cerebral ammonia detoxification, and thereby affects the transport of other neutral amino acids across the blood‐brain barrier. To test this hypothesis, rats with a portacaval shunt were treated with l‐methionine‐dl‐sulfoximine (MSO), an inhibitor of glutamine synthesis. Treatment with MSO resulted in lower concentrations of the neutral amino acids in brain of portacaval‐shunted rats and a higher brain ammonia concentration, compared with untreated shunted rats. These results suggest that the accumulation of neutral amino acids in brain after portacaval shunt depends on the increased synthesis of glutamine in brain.
Two Forms of Rat Brain Glutamic Acid Decarboxylase Differ in Their Dependence on Free Pyridoxal Phosphate
Authors: Denner L. A. , Wu J.‐Y.
Published: December 2006

Abstract: There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal‐5′‐phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45°C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonie, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl‐trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of γ‐aminobutyric acid (GABA) level is also discussed.
Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals
Authors: El Mestikawy S. , Glowinski J. , Hamon M.
Published: December 2006

Abstract: The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor‐dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4‐dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under “normal” conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7‐dihydroxy‐N, N‐dimethyl‐2‐aminotetralin (TL‐99), 7‐hydroxy‐N, N‐dipropyl‐2‐aminotetralin (7‐OH‐DPAT), trans‐ (–)‐4,4a,5,6,7,8,8a,9‐octahydro‐ 5 ‐propyl‐2H‐pyrazolo‐3,4‐quinoline, and 3‐(3‐hydroxyphenyl)‐N‐n‐propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol‐containing drugs apomorphine and TL‐99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under “normal” conditions. In contrast, when tissues were incubated in a K+‐enriched (60 mM) medium, (–)‐sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (–)‐sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d‐amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+‐induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7‐OH‐DPAT or the antagonist (–)‐sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP‐dependent phosphorylation of the enzyme.
Coupling of Inositol Phospholipid Metabolism with Excitatory Amino Acid Recognition Sites in Rat Hippocampus
Authors: Nicoletti F. , Meek J. L. , Iadarola M. J. , Chuang D. M. , Roth B. L. , Costa E.
Published: December 2006

Abstract: Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, l‐glutamate, and l‐aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N‐methyl‐d‐aspartate, kainate, quinolinate, and N‐acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above‐mentioned excitatory amino acids is potently and selectively antagonized by dl‐2‐amino‐4‐phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.
Effect of Adrenalectomy on γ‐Aminobutyric Acid Concentrations in the Central Nervous System
Authors: David S. , Kalyankar G. D.
Published: December 2006

Abstract: Pyridoxal‐5′‐phosphate (PLP) plays a crucial role in regulating the steady‐state levels of γ‐aminobutyric acid (GABA) in CNS. Adrenalectomy resulted in decreased conversion of dietary vitamin B6 to PLP. As a consequence of this, GABA levels in cerebral cortex decrease, since synthesis of GABA is determined by glutamate decarboxylase, a PLP‐dependent enzyme. Feeding diet supplemented with vitamin B6 elevated the GABA levels in adrenalectomized animals, because of increased availability of the coenzyme for apodecarbox‐ylase. The data suggest a role for corticosteroids in maintaining GABA levels, through their effects on PLP formation.