bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding Sites for [3H]Cocaine in Mouse Striatum and Cerebral Cortex Have Different Dissociation Kinetics
Authors: Reith Maarten E. A. , Sershen Henry , Lajtha Abel
Published: December 2006

Abstract: [3H]Cocaine dissociates from its binding sites in the mouse cerebral cortex with a half‐time of 25 s. The dissociation kinetics in the striatum is consonant with the presence of two populations of sites with dissociation half times of 2 s and 27 s, comprising 88% and 12%, respectively, of the total binding sites. On the basis of previous pharmacological characterization of [3H]cocaine binding, we propose that the slowly dissociating component represents the sites associated with 5‐hydroxytryptamine (serotonin) uptake, and the rapidly dissociating component the 3,4 dihydroxyphenylethylamine (dopamine)‐related sites. Evidence is presented that the extremely high dissociation rates do not preclude the measurement of [3H]cocaine binding by rapid filtration. The dissociation of [3H]cocaine from cerebrocortical membranes is slowed to a small but statistically significant extent by serotonin.
Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme
Authors: Ferrand Catherine , Clarous Dominique , Delteil Christine , Weber Michel J.
Published: December 2006

Abstract: The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle‐conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60–80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane‐bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membranebound or entrapped in vesicles originating from, e.g., the Golgi apparatus.
Somatostatin Binding Sites in Human and Monkey Brain: Localization and Characterization
Authors: Beal M. Flint , Tran Vinh T. , Mazurek Michael F. , Chattha Geetinder , Martin Joseph B.
Published: December 2006

Abstract: A radioiodinated analogue of somatostatin 28, 125I[Leu8, D‐Trp22, Tyr25]SS‐28, was used to localize and characterize somatostatin binding sites in both human and monkey brain. High‐affinity binding sites (approximately 1 nM) were found in cerebral cortex. The highest binding was in cerebral cortex with intermediate binding found in hippocampus, striatum, and amygdala and low binding in hypothalamus and brainstem. There was a rough correlation between somatostatin receptor binding and concentrations of somatostatin‐like immunoreactivity (SLI) in human brain. Somatostatin receptors were stable for up to 24 h in an animal model simulating human autopsy conditions and there was no correlation between postmortem interval and receptor binding in human brain. Pharmacologic characterization in human cortex showed that there was a correlation between the inhibition of receptor binding by somatostatin analogues and their known abilities to inhibit growth hormone secretion. These findings demonstrate that a highly specific membrane‐associated receptor for somatostatin is present in both monkey and human brain. Examination of somatostatin receptor binding in Alzheimer's disease and Huntington's disease may improve understanding of the role of somatostatin in both these illnesses.
Binding of Bodian's Silver and Monoclonal Antibodies to Defined Regions of Human Neurofilament Subunits: Bodian's Silver Reacts with a Highly Charged Unique Domain of Neurofilaments
Authors: Autilio‐Gambetti Lucila , Crane Regina , Gambetti Pierluigi
Published: December 2006

Abstract: Cleavage at cysteine and chymotrypsin digestion were applied to two human neurofilament (NF) sub‐units, low‐ and high‐molecular‐weight NF (NF‐L and NF‐H), to locate the regions reacting with Bodian's silver stain and with several monoclonal antibodies, including NF‐specific antibodies and one that recognizes all intermediate filaments (anti‐IFA). Our findings indicate that whereas anti‐IFA recognizes the highly conserved rod domain, all the NF‐specific antibodies, as well as Bodian's silver, react with the carboxy‐terminal tailpiece of NF subunits. The silver binding sites in NF‐L are located in a carboxy‐terminal 12‐Kd chymotrypsin fragment, a highly charged, unique domain of NF.
Ca2+ Sensitivity of Ca2+‐Dependent Protein Kinase Activities Toward Intrinsic Proteins in Synaptosomal Membrane Fragments from Rat Cerebral Tissue
Authors: Gower Hilary , Rodnight Richard , Brammer Michael J.
Published: December 2006

Abstract: The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+‐dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half‐maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation ∼110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+‐dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B‐50 protein, an established substrate of the lipid‐dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50–130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.
In Vitro Metabolism of Mevalonic Acid in the Bovine Retina
Authors: Fliesler Steven J. , Schroepfer George J.
Published: December 2006

Abstract: Bovine retinas were incubated with 3RS‐[5‐3H]‐ mevalonic acid under conditions similar to those previously shown to support opsin biosynthesis in vitro. TLC of the total lipids indicated the formation of numerous radiolabeled components, including sterols, hydrocarbons, and “fatty acid‐like material.” The nonsaponifiable lipids were analyzed by TLC, GLC, and chromatography on columns of silicic acid‐Super Cel, silica gel G‐Super Cel‐silver nitrate, and alumina‐Super Cel‐silver nitrate. The major nonsaponifiable components had the Chromatographic properties of squalene and “methylated sterols” (i.e., C30, C29, and C28 monohydroxy sterols). Cholesterol represented no more than 1% of the total radioactivity in the nonsaponifiable lipid fraction. The “fatty acid‐like material” was derivatized with diazomethane, and the resulting methyl esters were analyzed by GLC before and after catalytic hydrogenation. The radioactivity did not correspond to the normal fatty acids endogenous to the retina, but rather had the Chromatographic properties of C15 and C20 isoprenoid acids. These results obtained with intact retinas are consistent with our previous observations concerning mevalonic acid metabolism in cell‐free homogenates of bovine retinas.
Relation of Cellular Phospholipid Composition to Oligodendroglial Differentiation in C‐6 Glial Cells
Authors: Volpe Joseph J. , Iimori Yuichi , Haven Grant G. , Goldberg Richard I.
Published: December 2006

Abstract: The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C‐6 glial cells. Induction of the oligodendroglial enzyme, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N, N′‐dimethyl‐ethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum‐free medium. The 48‐h exposure to serum‐free medium resulted in untreated C‐6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N, N′‐dimethyl‐ethanolamine, total inhibition of this induction was observed. A graded, concentration‐dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C‐6 cells, (Na++ K+)‐activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum‐free medium recurred after removal of the analogue. N, N′‐Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.
Effects of Kainic Acid on Brain Calcium Fluxes Studied In Vivo and In Vitro
Authors: Lazarewicz Jerzy W. , Lehmann Anders , Hagberg Henrik , Hamberger Anders
Published: December 2006

Abstract: The effect of in vivo administration of kainic acid into the rabbit hippocampus was studied with brain dialysis and subsequent determination of the Ca2+ concentration in the dialysate. When included in the perfusing medium, kainic acid as well as veratridine induced a decrease in extracellular Ca2+. The effect of kainic acid (but not of veratridine) was insensitive to tetrodotoxin. In vitro studies revealed no effect of kainic acid on 45Ca2+ uptake by isolated astrocytes, but showed an enhancement of synaptosomal 45Ca2+ accumulation. This was, however, only 25% of the stimulatory effect of high K+ depolarization. Glutamate activated synaptosomal Ca2+ uptake, whereas dihydrokainate had no effect. The up take evoked by kainate and glutamate was independent of the K+ level in the medium which indicates the involvement of other than voltage‐sensitive Ca2+ channels. The results confirm previous findings that kainic acid promotes the uptake of Ca2+ in brain cells. Kainate affects Ca2+ fluxes pre‐ and postsynaptically. Presynaptic Ca2+ influx may be mediated by chemically gated mechanisms.
Rat and Mouse Monoclonal Antibodies to Human Myelin Basic Protein
Authors: Elfman L. , Kynoch P. A. M. , Siddle K. , Thompson R. J.
Published: December 2006

Abstract: BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N‐ and C‐terminal fragments. Mouse × mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N‐terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1–44 and 45–91. Three monoclonal antibodies, all of the IgM class, were obtained by rat × rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C‐terminal fragment, respectively, each bound to peptide 118–178. The remaining antibody, raised against the N‐terminal fragment, bound to peptide 45–91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immmunohistochemical investigation of the structural relationships of the myelin sheath.
Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System
Authors: Barker David L. , Murray Thomas F. , Siebenaller Joseph F. , Mpitsos George J.
Published: December 2006

Abstract: The selective muscarinic antagonist L‐[3H]‐quinuclidinyl benzilate (L‐[3H]QNB) binds reversibly and with high affinity (KD= 0.3 nM) to a single population (Bmax= 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (‐)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L‐[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L‐[3H]QNB binding than the nicotinic antagonists deca‐methonium and d‐tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L‐[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L‐[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.
Differential Effect of Denervation on Free‐Radical Scavenging Enzymes in Slow and Fast Muscle of Rat
Authors: Asayama Kohtaro , Dettbarn Wolf D. , Burr Ian M.
Published: December 2006

Abstract: To determine the effect of denervation on the free‐radical scavenging systems in relation to the mitochondrial oxidative metabolism in the slow‐twitch soleus and fast‐twitch extensor digitomm longus (EDL) muscles, the sciatic nerve of the rat was crushed in the mid‐thigh region and the muscle tissue levels of five enzymes were studied 2 and 5 weeks following crush. Recently developed radioimmunoassays were utilized for the selective measurement of cuprozinc (cytosolic) and man‐gano (mitochondrial) superoxide dismutases. Total tissue content of cuprozinc superoxide dismutase showed a mild decrease after denervation in slow but not in fast muscle. Manganosuperoxide dismutase and fumarase decreased markedly at 2 weeks and returned toward control levels by 5 weeks, the changes appearing to be greater in slow than in fast muscle. At 2 weeks, cytochrome c oxidase decreased significantly in slow, but not in fast muscle. GSH‐peroxidase at baseline was 10‐fold higher in slow than in fast muscle, markedly decreased at 2 weeks in slow muscle, and returned toward control levels at 5 weeks, whereas the total enzyme activity in fast muscle did not change through 5 weeks. These data represent the first systematic report of free radical scavenging systems in slow and fast muscles in response to denervation. Selective modification of cuprozinc and manganosuper‐oxide dismutases and differential regulation of GSH‐peroxidase was demonstrated in slow and fast muscle.
Inositol Trisphosphate Mobilizes Intracellular Calcium in Permeabilized Adrenal Chromaffin Cells
Authors: Stoehr Sally J. , Smolen James E. , Holz Ronald W. , Agranoff Bernard W.
Published: December 2006

Abstract: Using permeabilized chromaffin cells and the fluorescent probe Quin 2 (an indicator of free Ca2+), we found that inositol trisphosphate (IP3) specifically triggered an immediate and dose‐dependent release of Ca2+ from intracellular stores. Desensitization of the response was observed at nonsaturating concentrations of inositol trisphosphate and resequestration of Ca2+ was not observed. While representing only a small fraction of the total cellular Ca2+, the amount released by IP3 could significantly raise cytosolic Ca2+ and may account for muscarinic effects on Ca2+ metabolism in chromaffin cells.
Triacylglycerol Lipase in Developing Rat Sciatic Nerve
Authors: Yao Jeffrey K.
Published: December 2006

Abstract: Triacylglycerol lipase activity, with a pH optimum of 7.5, was demonstrated in cell‐free homogenates of rat sciatic endoneurium. 1,2‐Diacylglycerol was the major product of triacylglycerol hydrolysis. A rapid decline in lipase activity was found in rats up to 2 months of age. After this time, the decrease continued, but at a much slower rate. Such developmental changes in triacylglycerol lipase activity may, at least in part, account for the slower metabolic turnover of endoneurial triacylglycerol in adult rat sciatic nerve.
Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters
Authors: Reith Maarten E. A.
Published: December 2006

Abstract: It was suggested in a recent report by Phillips et al. [J. Neurochem.43, 479–486 (1984)] that the low‐affinity binding of [3H]imipramme in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 μM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low‐affinity component, in addition to the high‐affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data), (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.
Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors
Authors: Kloog Yoel , Galron Ronit , Sokolovsky Mordechai
Published: December 2006

Abstract: A study of the effects of bisquaternary pyridinium oximes on calcium‐dependent potassium‐evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]‐acetylcholine release in an atropine‐sensitive and dose‐dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre‐ and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1, 1′‐(4‐hydroxy‐iminopyridinium)trimethylene (TMB‐4) (I50= 8 μM) and the weakest were 1‐(2‐hydroxyiminoethylpyridinium) 1‐(3‐cyclohexylcarboxypyridinium) dimethylether (HGG‐42) and 1‐(2‐hydroxyiminoethylpyridinium) 1‐(3‐phenyl‐carboxypyridinium) dimethylether (HGG‐12) (I50= 150 μM). As postsynaptic antagonists, the latter drugs are more potent (K1= 1.3‐3.3 μM) than TMB‐4 (K1= 50 μM). Combined therapy with two drugs such as TMB‐4 and HGG‐12 might be effective in blocking severe hyper‐activity of the cholinergic system.
Differential Sensitivity of “Central” and “Peripheral” Type Benzodiazepine Receptors to Phospholipase A2
Authors: Havoundjian H. , Cohen R. M. , Paul S. M. , Skolnick P.
Published: December 2006

Abstract: The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the “central” and “peripheral” types. PLA2 (0.005–0.1 U/ml) increased [3H]flunitrazepam and [3H]3‐carbo‐ethoxy‐β‐carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither γ‐aminobutyric acid (GABA)‐enhanced [3H]fluni‐trazepam binding nor [3H]Ro 15–1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)‐ and barbiturate (pentobarbital)‐enhanced [3H]flunitrazepam binding and [35S]t‐butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025–0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine‐GABA receptor chloride ionophore complex, which results in a selective loss of allosteric “regulation” between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5–4864 to peripheral‐type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of “central” and “peripheral” types of benzodiazepine receptors.
Effects of Cell Division, Cell Density, and Cyclic Nucleotides on Choline Acetyltransferase Activity in a Cholinergic Neuroblastoma Cell Line (S‐20Y)
Authors: Kirshner Judith A. , Markelonis George J. , Max Stephen R.
Published: December 2006

Abstract: We investigated the effects of a number of experimental perturbations on choline acetyltransferase (ChAT) in a cholinergic mouse neuroblastoma cell line (S‐20Y). ChAT specific activity increased by 4.5‐fold during growth, suggesting that enzyme activity is dependent on increased cell density. This was confirmed by assessing enzyme activity at differential initial seeding densities. ChAT activity was also markedly enhanced by 1 mM di‐butyryl cyclic‐3′,5′‐AMP (dBcAMP), an effect that was blocked by cycloheximide. Confirmation of the dBcAMP effect was achieved with forskolin, a compound known to enhance intracellular cyclic AMP; forskolin (100 μM) caused a significant increase in ChAT activity. After a 20‐h latent interval ChAT activity was also enhanced significantly by cytosine arabinoside. The common element in these diverse effects on ChAT activity may be cessation of cell division, although cell‐cell interactions at the level of the cell membrane may also be important in the control of ChAT in S‐20Y.
Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites
Authors: Stephenson F. Anne , Casalotti Stefano O. , Mamalaki Cleanthi , Barnard Eric A.
Published: December 2006

Abstract: Polyclonal antibodies have been raised against the GABAA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X‐100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I‐la‐belled ‐γ‐aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immuno‐precipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3‐[(3‐cholamidopropyl)dimethylammonio] 1‐propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl‐ß‐carboline‐3‐carboxylate binding, the [35S]t‐butylbicyclophosphorothionate binding (TBPS), the barbiturate‐enhanced [3H]fluni‐trazepam binding, and the GABA‐enhanced [3H]fluni‐trazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the α‐subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.
Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin‐Permeabilized Adrenal Medullary Chromaffin Cells
Authors: Morita Kyoji , Levine Mark , Pollard Harvey B.
Published: December 2006

Abstract: The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4‐dihydroxyphen‐ylethylamine ([3H]dopamine) in calcium‐free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg‐ATP in the medium. These results indicate that the permea‐bilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5–2 mM in the presence of Mg‐ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.
Enzymatic Reconstitution of Brain Membrane and Membrane Opiate Receptors
Authors: Farahbakhsh Zohreh T. , Deamer David W. , Lee Nancy M. , Loh Horace H.
Published: December 2006

Abstract: A new method using lysophosphatide and acyl‐CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl‐CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze‐fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane‐bound enzymes Na+, K − ‐ATPase and Ca2 ‐, Mg2+‐ATPaxe in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.
Monoclonal Antibodies and Polyvalent Antiserum to Chicken Choline Acetyltransferase
Authors: Johnson Carl D. , Epstein Miles L.
Published: December 2006

Abstract: Monoclonal antibodies (mAbs) to chick cho‐line acetyltransferase (ChAT) were obtained from mouse‐hybridoma cultures after immunization with partially purified enzyme isolated from optic lobes. Antibodies that bound active enzyme were detected in 11 hybridoma cultures. The mAbs showed cross‐reactivity to ChAT from quail and beef but not to ChAT from several other species. An affinity column prepared with one of the mAbs was used to purify ChAT to apparent homogeneity. Polyclonal antiserum to mAb affinity‐purified ChAT was produced in a rabbit. This antiserum inhibited chick ChAT activity and quantitatively precipitated ChAT activity from solution. On immunoblots, the antiserum stained ChAT and two other proteins. After preadsorption of the antiserum with effluent from the mAb affinity column, the antiserum became monospecific for ChAT. This an‐tiserum was useful for immunocytochemical localization of ChAT. it selectively stained neuronal cell bodies in chick spinal cord and rat brain at locations known to contain cholinergic neurons.
Binding of the Novel Serotonin Agonist 8‐Hydroxy‐2‐(Di‐n‐Propylamino) Tetralin in Normal and Alzheimer Brain
Authors: Middlemiss D. N. , Palmer A. M. , Edel N. , Bowen D. M.
Published: December 2006

Abstract: Binding of [3H]8‐hydroxy‐2‐(di‐n‐propylamino) tetralin. a putative ligand for the 5‐hydroxytryptamine (5‐ HT, serotonin) 1A recognition site, was measured in neocortex from postmortem human brain. The substance was found to bind to a saturable site with a KD value and pharmacological profile similar to that of rat. Binding to membranes from normal human temporal cortex was found to significantly correlate (inversely) with age. A significant reduction in binding, reflecting decreased density of recognition sites, was observed in the frontal cortex of patients with Alzheimer's disease (48% loss). This region in the dement brains showed unaltered presynaptic 5‐HT function (5‐HT and 5‐hydroxyindoleacetic acid content) whereas 5‐HT concentration was reduced in the temporal cortex.
Structural Microheterogeneity of the Muscarinic Cholinergic Receptor Is Not Related to Functional Diversity Identified by Differences in Affinity for Pirenzepine
Authors: Dadi H. K. , Batteiger D. , Keen M. , Morris R. J.
Published: December 2006

Abstract: The muscarinic receptor for acetylcholine shows a diversity in ligand binding properties and effector mechanisms which have suggested the existence of two subtypes (M1 and M2), to which the selective antagonist pirenzepine binds with markedly different affinities. The receptor from rat brain, covalently labelled with the alkylating antagonist tritiated propylbenzilylcholine mustard, displays a structural microheterogeneity on electrophoresis, covering the region of apparent molecular weight 66,000‐76,000, with dominant components at 68,000 and 73,000. Selective inhibition by pirenzepine of labelling of the M1 receptor with tritiated mustard has been analysed on fluorographs of sodium dodecyl sulphate‐polyacrylamide gels and shown to cause a uniform reduction in radioactive labelling of the broad receptor peak, rather than selectively inhibiting either the high‐ or low‐molecular‐weight regions of the band. It is further shown that although this receptor microheterogeneity is found for each of four brain regions studied, it is not found for the heart receptor, which gives a discrete labelled band of apparent molecular weight 72,000. It is therefore suggested that the structural microheterogeneity is the result of tissue‐specific, posttranslational modification of the molecule, such as glycosylation, and is not directly related to the functional diversity of the receptor.
Turnover of Glial Filaments in Mouse Spinal Cord
Authors: DeArmond Stephen J. , Lee Yuen‐Ling , Kretzschmar Hans A. , Eng Lawrence F.
Published: December 2006

Abstract: Twenty‐day‐old mice received a single tail vein injection of [guanido‐14 C]arginine. The cytoskeleton was extracted from the spinal cords at varying lengths of time thereafter. Glial fibrillary acidic protein (GFAP) formed a distinct, broad band that was widely separated from other protein bands in one‐dimensional polyacrylamide gels. The purity of the GFAP band was verified by Western blot analysis of one‐ and two‐dimensional electrophoretic patterns. In addition, enzyme‐linked immunosorbent assay and quantitative Western blot analysis indicated that 95% of the total spinal cord GFAP was extracted in the cytoskeletal preparation. The specific activity of GFAP was obtained by eluting the protein from the cytoskeletal GFAP band in preparative one‐dimensional gels. Specific activity reached a peak 2 h after injection with [14C]arginine. Forty percent of the incorporated radioactivity was still present in cytoskeletal GFAP at 9 weeks, indicating that a significant proportion of glial filaments turns over relatively slowly in vivo.
Glucose Modulates the Release of Histamine from the Mouse Hypothalamus In Vitro
Authors: Nishibori Masahiro , Oishi Ryozo , Itoh Yoshinori , Saeki Kiyomi
Published: December 2006

Abstract: The effect of glucose concentration on the in vitro release of histamine (HA) was examined, using two different preparations of the mouse hypothalamus. The HA and tele‐methylhistamine released from whole blocks of the hypothalamus into the medium linearly increased during 2‐h incubation in normal Krebs‐Ringer bicarbonate solution in the absence of external depolarizing stimuli. The release of HA from this preparation depended on the temperature and Ca2+ in the medium and was progressively increased with decrease in the glucose concentration from 11.5 to 1 mM. The rate of the HA release was dependent on the absolute concentration of glucose and not on an abrupt change in the concentration. When slices of the hypothalamus were incubated in high K+ medium, a temperature‐ and Ca2+‐dependent HA release was observed. At low concentrations of glucose, the K+ (20 mM)‐induced HA release from the hypothalamic slices was also enhanced. Tetrodotoxin (10 μM) inhibited the enhancing effect of a low glucose concentration (2 mM) on the HA release by 60%, in both preparations of the hypothalamus. The possibility that the release of HA from the mouse hypothalamus is regulated by glucose concentration and that activation of neuronal Na+ channels is involved in the enhancement of the HA release by low glucose concentrations warrants further attention.
Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12
Authors: Lukas Ronald J.
Published: December 2006

Abstract: Studies were conducted on the properties of 125I‐labeled α‐bungarotoxin binding sites on cellular membrane fragments derived from the PC 12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d‐tubocurarine are among the most potent inhibitors of high‐affinity toxin binding. The affinity of high‐affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N‐ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high‐affinity toxin binding to PC 12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high‐affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC 12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti‐nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.
Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes
Authors: Carvalho C. A. M. , Coutinho O. P. , Carvalho A. P.
Published: December 2006

Abstract: The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)‐verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 ± 0.1 nM, a Bmax of 161 ± 27 fmol.mg‐1 protein, and a Hill slope of 1.07, at 25°C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: (1) nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; (2) verapamil and (‐)‐D 600 are partial competitors, with biphasic competition behavior. Thus, (+)verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (‐)‐verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (‐)‐D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50= 430 nM). However, (+)‐verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization‐induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)‐verapamil inhibits Ca2+ influx by 50% at about 500 μM, whereas it inhibits 50% of the binding at concentrations 200‐fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP‐dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d‐cis‐diltiazem, with similar IC50 values and in the same concentration range (10‐5‐10‐3M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high‐affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+‐ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.
Nucleoside Transporter of Cerebral Micro vessels and Choroid Plexus
Authors: Kalaria Rajesh N. , Harik Sami I.
Published: December 2006

Abstract: The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1–0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguano‐sine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleo‐sides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood‐brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.
Failure to Maintain Glycolysis in Anoxic Nerve Terminals
Authors: Kauppinen Risto A. , Nicholls David G.
Published: December 2006

Abstract: Synaptosomal glycolysis is stimulated eight‐to 10‐fold when the respiratory chain is inhibited by cyanide or by anoxia. However, the stimulation is transient and after 15 min declines toward the preanoxic rate. The decline is not seen when Ca2+ is absent or when the respiratory chain is inhibited by rotenone. The decline in glycolysis is reversible, is not due to substrate exhaustion, and is the cause, rather than the effect, of lowered synaptosomal ATP/ADP ratios. The failure to maintain glycolysis when the terminal oxidase of the respiratory chain is inhibited may have relevance to the sensitivity of the brain to anoxic damage.
Incorporation of Glycosylated β‐Galactosidase into Bovine Brain Synaptosomes
Authors: Naoi Makoto , Nagatsu Toshiharu
Published: December 2006

Abstract: Using a synthesized glycoprotein, β‐galactosidase modified with p‐aminophenyl β‐D‐galactopyranoside (β‐D‐Gal β‐gal), the incorporation of the glycoprotein into bovine brain synaptosomes was studied. The uptake was mediated by a specific receptor to β‐D‐galactoside, and was inhibited by GM1 ganglioside. The uptake was found to require energy and to be sensitive to metabolic inhibitors. Kinetic studies on β‐D‐Gal β‐gal uptake indicated the presence of a saturable, carrier‐mediated transport system in synaptosomes. By subcellular fractionation the β‐D‐Gal β‐gal taken up was found in the fractions corresponding to the nucleus and membrane fragments, the soluble cytosomal fractions, and the mitochondria and lysosomes. The uptake was markedly increased by addition of Ca2+ to the incubation medium. The maximal uptake was obtained at pH 8.0 in the presence of 10 mMCa2+ at 37°C. By addition of a Ca2+ ionophore A23187, β‐D‐Gal β‐gal uptake was increased in a dose‐dependent way parallel to the increase in the intrasynaptosomal concentration of Ca2+. Preincubation of synaptosomes with calmodulin antagonists such as trifluoperazine and N‐(6‐aminohexyl)‐5‐chloro‐1 ‐napthalenesulfonamide (W‐7) was found to inhibit the uptake markedly, and diazepam, an inhibitor of Ca2+/calmodulin‐dependent protein kinase, also inhibited the uptake. At a concentration between 1 and 10 μM, 50% inhibition of the uptake was observed with either inhibitor. On the other hand, the addition of dibutyryl cyclic AMP did not affect the uptake of the glycoprotein into synaptosomes. These results suggest that the incorporation of this macromolecule is dependent on a Ca2+/calmodulin‐dependent protein kinase.
Determination of 3‐Methoxytyramine in Rat Brain by HPLC with Electrochemical Detection and Its Correlation with Dopamine Function After Administration of a Monoamine Oxidase Inhibitor and L‐3,4‐Dihydroxyphenylalanine
Authors: Davies C. L. , Heal D. J.
Published: December 2006

Abstract: 3‐Methoxytyramine (3‐MT), normally a minor metabolite of 3,4‐dihydroxyphenylethylamine (dopamine) in brain, becomes the sole product of metabolism following the administration of a monoamine oxidase (MAO) inhibitor. A simplified reverse‐phase HPLC method for 3‐MT employing electrochemical detection is fully described. This method has a detection limit of 0.1 μg/g brain wet weight and is sensitive enough to detect 3‐MT in individual brain regions after rats have been pretreated with an MAO inhibitor. Administration of tranylcypromine (TCP, 10 mg/kg) and L‐3,4‐dihydroxyphenylalanine (L‐DOPA) (10‐50 mg/kg) produced a dose‐dependent linear increase in 3‐MT concentrations in the dopaminergjc brain regions n. cauda‐tus (r = 0.95; p < 0.01) and n. accumbens (r = 0.96; p <0.01). This treatment also produced a dose‐dependent increase in behavioural activity in rats (r = 0.88; p <0.01). Furthermore, a good correlation was found between the activity responses of individual rats and the accumulation of 3‐MT after TCP/L‐DOPA in both n. caudatus (r = 0.76; p <0.01) and n. accumbens (r = 0.84; p <0.01). These data describe a simple and sensitive HPLC analysis technique for 3‐MT and demonstrate that following administration of an MAO inhibitor this metabolite may provide a useful monitor of central dopamine function.
Effect of Decreased Oxygen on In Vitro Release of Endogenous 3,4‐Dihydroxyphenylethylamine from Mouse Striatum
Authors: Freeman Gary B. , Gibson Gary E.
Published: December 2006

Abstract: The effects of hypoxia on release of endogenous 3,4‐dihydroxyphenylethylamine (DA, dopamine) were investigated in mouse striatal slices. Following a 60‐min pre‐incubation, potassium increased DA release 12 times between zero and 1 min. By 10 min, uptake processes exceeded release and DA levels in the media decreased. Hypoxia (low oxygen) and anoxia (no oxygen) increased DA in the media by 120 and 205%, respectively, but did not alter dihydroxyphenylacetic acid concentrations. Under similar conditions, anoxia increased [3H]DA uptake eightfold. For the uptake studies, the amount of DA added to the media was critical; in the presence of high concentrations of DA, anoxia reduced reuptake. Regardless of exogenous DA, hypoxia and anoxia increased extracellular DA, which may play a role in ischemic cell damage.
Phosphatidylcholine Biosynthesis in the Neuroblastoma‐Glioma Hybrid Cell Line NG 108‐15: Stimulation by Phorbol Esters
Authors: Liscovitch Mordechai , Freese Andrew , Blusztajn Jan K. , Wurtman Richard J.
Published: December 2006

Abstract: We have examined the effects of phorbol esters on phosphatidylcholine (PtdCho) metabolism in the neuroblastoma‐glioma hybrid cell line NG 108‐15. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA), 100 nM, stimulated twofold the incorporation of [3H]choline into PtdCho during 2 h of incubation at 37°C. This effect of TPA was concentration dependent, exhibiting an EC50 of 24.5 ± 4.4 nM The effect of TPA was also time dependent and became apparent only after a lag period of 15–30 min. TPA also decreased the incorporation of [3H]choline into water‐soluble cellular constituents in a manner whose concentration and time‐dependence paralleled the changes observed in Ptd‐Cho content. HPLC analysis of this pool revealed that the levels of its major (85–95%) constituent, [3H]phosphocholine, were decreased by 29 ± 5%, whereas those of [3H]glycerophosphocholine (0.5–2% of the pool) were increased by 84 ± 4%. PtdCho labeling was also stimulated when cells were pulse labeled with [3H]choline and chased in the presence of TPA. The incorporation of [3H]inositol, [14C]ethanolamine, or [14C]serine into phospholipids was not affected by TPA. The non‐tumor‐promoting compounds phorbol and 4α‐phorbol‐12, 13‐didecanoate (at 100 nM) were completely ineffective in modulating choline incorporation, whereas the biologically active analogs 4β‐phorbol‐12, 13‐didecanoate and 4β‐phorbol‐12, 13‐dibutyrate were as effective as TPA. We conclude that tumor‐promoting phorbol esters can modulate PtdCho metabolism in neural‐derived cells. The mechanisms mediating this effect and the possible involvement of PtdCho metabolism in normal signal transduction events and in the biological actions of tumor promoters are discussed.