bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Anti‐Conjugated Acetylcholine Antibody and Immunohistochemical Applications in Rat Nervous System
Authors: Chagnaud Jean‐Luc , Souan Marie‐Laure , Charrier Marie‐Christine , Geffard Michel
Published: October 2006

Abstract: Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme‐linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline‐glutaryl‐bovine sej‐um albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/0/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti‐conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti‐conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline‐glutaryl‐protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh‐immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Pread‐sorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.
Kinetic Analysis of A23187‐Mediated Polyphosphoinositide Breakdown in Rat Cortical Synaptosomes Suggests that Inositol Bisphosphate Does Not Arise Primarily by Degradation of Inositol Trisphosphate
Authors: Brammer Michael , Weaver Keith
Published: October 2006

Abstract: The kinetics of polyphosphoinositide breakdown and inositol phosphate formation have been studied in rat cortical synaptosomes labelled in vitro with myo‐[2‐3H]inositol. Intrasynaptosomal Ca2+ concentrations have been varied by the use of Ca‐EGTA buffers or by adding the onophore A23187 in the presence and absence of 1 mM Ca2+. The former studies have revealed that, at very low (20 nM) intrasynaptosomal free Ca2+ levels, inositol pisphos‐phate, but not inositol monophosphate levels are reduced. Addition of A23187 in the absence of added Ca2+ gives rise to greatly enhanced inositol bisphosphate accumulation, which is further enhanced if 1 mM Ca2+ is present in the extrasynaptosomal medium. At all time points examined (down to 2 s after adding ionophore), the ratio of inositol trisphosphate/inositol bisphosphate accumulation does not exceed 0.2, and calculations based on inositol bis‐ and trisphosphate breakdown rates in synaptosomal lysates suggest that only a minority of the inositol bisphosphate arises from degradation of inositol trisphosphate. Addition of ionophore in the presence (but not in the absence) of 1 mM Ca2+ leads to rapid breakdown of phosphatidylinositol 4,5‐bisphosphate (PtdInsP2) and ATP and slower breakdown of phosphatidylinositol 4‐phosphate (PtdInsP). The rates of loss of PtdinsP2 and ATP are very highly correlated, suggesting that polyphosphoinositide resynthesis may be limited by ATP availability at high Ca2+ levels. Analysis of 32P‐labelled synaptosomes also reveals that A23187 produces Ca2+‐dependent losses of PtdInsP2, PtdlnsP, ATP, and GTP radioactivity and a marked increase in the radioactivity of a compound distinct from nucleotides or any of the lipid breakdown products tested. This compound may be a hexose bisphosphate arising from stimulation of glycolysis by A23187.
Effects of l‐Methyl‐4‐Phenyl‐l,2,3,6‐Tetrahydropyridine in the Dog: Effect of Pargyline Pretreatment
Authors: Johannessen J. N. , Chiueh C. C. , Bacon J. P. , Garrick N. A. , Burns R. S. , Weise V. K. , Kopin I. J. , Parisi J. E. , Markey S. P.
Published: October 2006

Abstract: Adult beagle dogs of either sex were injected with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐HCl (2.5 mg/kg, i.v.) alone or after pretreatment with pargyline (5.0 mg/kg, s.c, twice), with pargyline alone, or were unin‐jected. Groups were killed 2 h, 3 weeks, or 3 months after injection, and several brain areas were assayed for biogenic amines and their synthetic and degradative enzymes. MPTP caused a massive and permanent loss of striatal dopamine, tyrosine hydroxylase, and 3,4‐dihydroxyphenylalanine decarboxylase activities and the loss of cells within the substantia nigra pars compacta. Dopamine and norepinephrine also were depleted to various degrees in cortex, olfactory bulb, and hypothalamus; however, dopamine β‐hydroxylase activity in cortex was normal. There was no cell loss in the ventral tegmental area or locus ceruleus. The activities of monoamine oxidase (MAO)‐A and MAO‐B in cortex and caudate were not affected by MPTP. Despite a permanent loss of the ni‐grostriatal system, the dogs exhibited only a transient hypokinesia lasting 1‐2 weeks. Pargyline pretreatment prevented the loss of striatal dopamine and cells from the substantia nigra, but did not prevent a prolonged but reversible decrease in the concentration of dopamine metabolites. It is argued that this apparent inhibition of MAO is due not to suicide inactivation of the enzyme by MPTP, but to reversible inhibition by accumulation of the pyridinium metabolite, 1‐methyl‐4‐phenylpyridinium, selectively in aminergic terminals.
α2‐Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK‐N‐SH Human Neuroblastoma Cells
Authors: Baron Bruce M. , Siegel Barry W.
Published: October 2006

Abstract: Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK‐N‐SH cells. The α2‐adrenergic agonist UK 14304 decreased forskolin‐stimulated cyclic AMP levels by 40 ± 2%, with an EC50 of 83 ± 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50= 1 ng/ml) or by the α2‐antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 ± 0.1, 2.2 ± 0.3, and 1.4 ± 0.1 μM, respectively. This finding suggests the presence of normal inhibitory coupling of SK‐N‐SH cell α2‐adrenergic receptors to adenylate cyclase via the inhibitory GTP‐binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK‐N‐SH cells, muscarinic agonists synergistically increased (67‐95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremo‐rine facilitation of the forskolin response were 1.2 ± 0.2 and 0.3 ± 0.1 μM, respectively. Pharmacological studies using the muscarinic receptor subtype‐preferring antagonists 4‐di‐phenylacetoxy‐N‐methylpiperidine, pirenzepine, and AF‐DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 ± 1, 16,857 ± 757, and 148,043 ± 16,209 nM, respectively. CCh‐elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK‐N‐SH cell membranes. Our results indicated the presence of a single CCh binding site with an apparent affinity (Ki= 5 ± 1 μM) consistent with that concentration of agonist required to evoke biochemical responses in the intact cell.
Stimulation‐Induced Release of Coexistent Transmitters in the Prefrontal Cortex: An In Vivo Microdialysis Study of Dopamine and Neurotensin Release
Authors: Bean Andrew J. , During Matthew J. , Roth Robert H.
Published: October 2006

Abstract: Extracellular fluid levels of dopamine and neurotensin in the rat prefrontal cortex were measured using in vivo microdialysis. Electrical stimulation of the median forebrain bundle resulted in increased release of both dopamine and neurotensin from the prefrontal cortex. Thus, stimulation of neurons in which dopamine and neurotensin are colocalized can evoke the in vivo release of both substances.
Immunological Identification of Multiple α‐Like Subunits of the γ‐Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex
Authors: Sato Thomas N. , Neale Joseph H.
Published: October 2006

Abstract: Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174‐203 of the bovine γ‐aminobutyric acidA (GABAA) receptor α1‐subunit. The antibodies recognized this synthetic α1 ‐peptide, but failed to react with the homologous peptide sequence, 170‐199, of the bovine βl‐subunit. On Western blots, anti‐α1‐subunit antibody recognized a 50‐kilodalton (kDa) protein in affinity‐purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the antiα1‐antibody reacted with 50‐kDa, 53‐54‐kDa, and 59‐kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50‐kDa and 59‐kDa subunits of affinity‐purified neonatal receptor, which were photoaffinity‐labeled with [3H]fluni‐trazepam, were immunoprecipitated to different extents by α‐subunit antibody. These data suggest the existence in GA‐BAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to αl‐subunit of bovine GABAA receptor. The presence of an α‐ and a β‐like subunit with similar mobility on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53‐54‐kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple α‐like sub‐units may be related to the presence of a relatively high concentration of type IIGABA receptor in this tissue.
In Human Brain Two Subtypes of D1 Dopamine Receptors Can Be Distinguished on the Basis of Differences in Guanine Nucleotide Effect on Agonist Binding
Authors: de Keyser Jacques , Walraevens Hiljle , Ebinger Guy , Vauquelin Georges
Published: October 2006

Abstract: D1 dopamine receptors were identified in membranes of human nucleus caudatus, nucleus accumbens, amygdala, and globus pallidus, by the specific binding of [3H](+)‐R‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐lH‐benzazepine‐7‐ol ([3H]SCH 23390). In these four brain regions, dopamine/[3H]SCH 23390 competition binding curves were computer‐analyzed to a two‐site model, distinguishing a high‐ (RH) and low‐ (RL) affinity site for dopamine. The ability of guanine nucleotides (0.4 mM GTP or 0.1 mM 5′‐guanylylimidodiphosphate) to provoke a conversion of RH into RL was different between these brain regions. In amygdala, a complete conversion was seen, whereas there was no guanine nucleotide‐effect on RH in globus pallidus. In nucleus caudatus and nucleus accumbens, guanine nucleotides provoked only a partial conversion of RH into RL, suggesting that these brain regions may contain guanine nucleotide‐sen‐sitive and ‐insensitive receptors. Heating of the membranes at 60°C for 5 min had the same effect as guanine nucleotides. The pharmacological profiles of the guanine nucleotide‐sensitive and ‐insensitive D1 receptors were similar, suggesting that D1 receptors in human brain are heterogeneous only with respect to their effector‐coupling mechanism: guanine nucleotide‐sensitive receptors, which are capable of undergoing functional coupling with Gs, and guanine nucleotide‐insensitive receptors, which are not. A lesion of the cortico‐striatal pathways had no effect on the D1 receptors in nucleus caudatus, whereas a lesion of the mesostriatal pathways resulted in an up‐regulation of the guanine nucleotide‐sensitive, as well as the guanine nucleotide‐insensitive receptors, suggesting that in nucleus caudatus both receptor types are located postsynaptically to the mesostriatal axons, probably on intrinsic striatal neurons.
The GTP‐Binding Proteins, Go and Gi2, of Neural Cloned Cells and Their Changes During Differentiation
Authors: Asano Tomiko , Morishita Rika , Sano Mamoru , Kato Kanefusa
Published: October 2006

Abstract: The levels of the α‐subunits of two GTP‐binding proteins, Go and Gi2, were determined in neural and non‐neural cloned cells by immunoassays. Goα was detected in all neural cells and some of nonneural cells, but not in HL‐60 leukemic cells and PYS‐2 teratocarcinoma‐derived cells. The level of Goα was highest in the PC12 pheochroihocytoma cells. Gi2α was present in all types of cells tested, arid its level was highest in the HL‐60 cells and relatively high in glioma ells. Treatment of PC 12 cells and neuroblastoma X glioma hybrid NG108‐15 cells with nerve growth factor and forskolin, respectively, caused the extension of neuronal‐like processes and increase in the level of Goα by 60‐80%, but small changes in the levels of Gi2α.
Purification and Subunit Composition of a Cholinergic Synaptic Vesicle Glycoprotein, Phosphointermediate‐Forming ATPase
Authors: Yamagata Susan K. , Noremberg Krystyna , Parsons Stanley M.
Published: October 2006

Abstract: A glycoprotein ATPase in cholinergic synaptic vesicles of Torpedo electric organ was solubilized with octa‐ethylene glycol dodecyl ether detergent. Study of potential stabilizing factors identified crude brain phosphatidylserine, glycerol, dithiothreitol, and protease inhibitors as of value in maintaining activity. The ATPase was purified from the solubilized, stabilized material by glycerol density gradient band sedimentation velocity ultracentrifugation, and hydroxyl‐apatite, wheat germ lectin affinity, and size exclusion chromatographies. The pure ATPase had a specific activity of about 37 μmol ATP hydrolyzed/min/mg protein. After sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the purified material typically exhibited three polypeptides of molecular masses 110, 104, and 98 kilodaltons (kDa) and a fourth diffuse polypeptide of 60 kDa. This composition suggests that the ATPase is a member of the P‐type, or phos‐phointermediate‐forming, family, but it was shown to be distinct from the ouabain‐sensitive Na+,K+‐ and Ca2+‐stimulated Mg2+‐ATPases. The purified vesicle enzyme was rapidly phosphorylated by [γ‐32P]ATP on about 14% of the subunits with molecular weights of 98,000‐110,000. About 16% of the ATPase was phosphorylated in whole‐vesicle ghosts in a manner consistent with formation of a phosphointermediate, thus confirming the P‐type nature of this enzyme
Metabolism of Exogenous Gangliosides in Cerebellar Granule Cells, Differentiated in Culture
Authors: Ghidoni Riccardo , Riboni Laura , Tettamanti Guido
Published: October 2006

Abstract: The metabolism of exogenous gangliosides in the CNS has been investigated using cerebellar granule cells in culture as a model. For this purpose, GM2 and GM1, both isotopically radiolabeled at the level of the terminal sugar residue or of the long chain base moiety, were administered to differentiated cells for a 15‐h pulse, and their metabolic fate was followed in a time course protocol. At each time investigated (1,2, and 4 days after the pulse), several compounds, besides the ones administered, were detected: (a) GM2 (only after GM1 was given), GM3, lactosylceramide, glucosylceramide, and ceramide, all products of ganglioside stepwise catabolism; (b) GM 1 (only after GM2 was given), GDI a, GDlb, O‐Ac‐GTlb, and GTlb, that is, gangliosides more complex than the one administered; and (c) sphingomyelin. The compounds derived from ganglioside catabolism and sphingomyelin were detected only after administration of long chain base‐labeled precursors, whereas the others were found regardless of the labeling position of the precursor. In addition, radioactivity was incorporated in the delipidized residue when sugar‐labeled gangliosides were given to cells. Besides qualitative differences, quantitative ones were found after administration of the different precursors. Comparison of differences in the radioactivity incorporation values and calculation of the specific radioactivity of the individual sugar components of the GDI a and GTlb formed led to the conclusions that exogenous gangliosides (a) are easily taken up and metabolized by cerebellar granule cells, (b) undergo catabolism following the conventional pathway, (c) can be utilized directly as substrates for glycosylating reactions, thus giving rise to more complex gangliosides, and (d) give rise to fragments (of either saccharide or lipid nature) that can escape complete degradation and be reutilized for the resynthesis of gangliosides and sphingomyelin. This recycling process seems to be more efficient for lipid‐carrying fragments than for sugar‐containing fragments.
5′ Flanking DNA Sequences Direct Cell‐Specific Expression of Rat Tyrosine Hydroxylase
Authors: Cambi Franca , Fung Brenda , Chikaraishi Dona
Published: October 2006

Abstract: Tyrosine hydroxylase (TH) is selectively expressed in cat‐echolaminergic neurons and in chromaffin cells of the adrenal medulla. Constructs in which 5’flanking sequences of the rat TH gene directed expression of bacterial chloramphenicol acetyltransferase (CAT) were transfected into cell lines and assayed for transient expression of CAT. In most nonexpressing cell lines, CAT levels were < 5% of that found in a TH‐positive pheochromocytoma line (PC8b). In two lines described here, a rat anterior pituitary (pell line (GH4) and a rat fibroblast line (Fr3T3), CAT expression reached 12 and 20%, respectively, of the PC8b level. Greater than 90% of the PC8b activity was lost when sequences between ‐212 and ‐ 187 (in relation to the transcriptional initiation site) were deleted. Further deletions that removed the cyclic AMP response element (CRE)|(‐45) and the TATA box at ‐29 reduced transcriptional activity to background in all three lines. These data suggest that 212 nucleotides of the 5’sequence are sufficient for pheochromocytoma expression and that information between —212 and —187, which includes an API site (‐206 to —200), is essential for full transcriptional activity. In addition, sites for other protein transcription factors (AP2, POU/Oct, SP1, and CRE) reside between ‐221 and ‐38 and are largely conserved between the human and rat gene.
Solubilization of Sodium Channel from Human Brain
Authors: de Rycker Cécile , Grandfils Christian , Bettendorff Lucien , Schoffeniels Ernest
Published: October 2006

Abstract: [3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0°C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P‐40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.
Inhibition of Monoamine Oxidase Within Monoaminergic Neurons in the Rat Brain by (E)‐β‐Fluoromethylene‐m‐Tyrosine (MDL 72394)
Authors: Fagervall Ingrid , Ross Svante B.
Published: October 2006

Abstract: The irreversible inhibition of the monoamine oxidase (MAO) activity within monoaminergic neurons in the rat brain 24 h after single or repeated administration of (E)‐β‐fluoromethylene‐m‐tyrosine (FMMT, MDL 72394) was examined. The enzyme activity was determined by incubating synaptosome‐rich homogenates of hypothalamus or striatum with low concentrations of 5‐[14C]hydroxytryptamine (5‐HT), [14C]noradrenaline (NA), or [14C]dopamine (DA) in the absence and presence of the selective amine uptake inhibitors citalopram (5‐HT), maprotiline (NA), and GBR 12909 (DA). After a single subcutaneous injection of FMMT, the inhibition of MAO within the noradrenergic and dopaminergic neurons was significant but only slightly greater than that outside these neurons. The opposite relationship was observed for the serotonergic neurons. After 7 days’ treatment of rats with carbidopa, 20 mg/kg p.o., + FMMT once daily, the preference for the inhibition of MAO within the noradrenergic and dopaminergic neurons was accentuated further. The inhibition outside the serotonergic neurons was still greater than within these neurons. The NA uptake inhibitor CPP 199 antagonized the selective inhibition of MAO within the noradrenergic neurons, which indicates that this preference is due to the accumulation of the active metabolite (E)‐β‐fluoromethylene‐m‐tyramine by the NA transporter.
Divergent Effects of Acute Depolarization on Somatostatin Release and Protein Synthesis in Cultured Fetal and Neonatal Rat Brain Cells
Authors: De Los Frailes Maria Teresa , Cacicedo Lucinda , Lorenzo Maria Jesús , Sánchez‐Franco Franco
Published: October 2006

Abstract: The influence of membrane depolarization on somatostatin secretion and protein synthesis by fetal and neonatal cerebrocortical neurons was studied. Cortical cells obtained by mechanical dispersion were maintained as monolayer cultures for 8 days. The ability of fetal cerebrocortical and hypothalamic cells to release immunoreactive somatostatin (IR‐SRIF) was confirmed. Total protein synthesis was determined by the incorporation of [3H]phenylalanine into trichloroacetic acid‐precipitable proteins. To study the effect of acute depolarization on protein synthesis, cells were incubated for 30 min with [3H]phenylalanine or [3H]leucine and the depolarizing agent. In fetal cerebrocortical cells, potassium (30 and 56 mM) decreased protein synthesis and RNA levels and increased IR‐SRIF release. Depolarization by veratridine, a sodium channel activator, induced a similar effect. The effect of veratridine on IR‐SRIF and protein synthesis was reversed by tetrodotoxin, a sodium channel blocker, or verapamil, a calcium channel blocker. These findings suggest that protein synthesis by cerebrocortical cells is decreased in fetal brain cells by membrane depolarization and is dependent on Na+ and Ca2+ entry into cells. In postnatal (day 7) cerebrocortical cells, depolarization induced by high potassium concentrations led to a concomitant increase in protein synthesis, RNA content, and somatostatin release. These findings indicate that depolarization of the cellular membrane is coupled to an increase in protein synthesis in neonatal, but not in fetal, dispersed brain cells.
Characterization, by Size, Density, Osmotic Fragility, and Immunoaffinity, of Acetylcholine‐ and Vasoactive Intestinal Polypeptide‐Containing Storage Particles from Myenteric Neurones of the Guinea‐Pig
Authors: Agoston D. V. , Whittaker V. P.
Published: October 2006

Abstract: When cytoplasmic extracts of guinea‐pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg2+‐activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle‐exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense‐cored, enkephalin‐ and noradrenaline‐containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor–product relationship with the classic synaptic vesicles containing the small‐molecular‐mass transmitters and found in the same nerve terminals.
Regional Variations in α1‐Adrenergic Receptor Subtypes in Rat Brain
Authors: Wilson Karen M. , Minneman Kenneth P.
Published: October 2006

Abstract: α1‐Adrenergic receptor subtypes were differentiated by their affinities for the competitive antagonist WB 4101 and their sensitivities to inactivation by chlorethylclonidine (CEC) in eight rat brain regions. WB 4101 showed low Hill coefficients for inhibition of specific 125I‐[2‐β‐(4‐hydroxy‐phenyl)ethylaminomethyl]tetralone (125IBE) binding in all regions. Nonlinear regression analysis showed that there were two binding sites with different affinities for WB 4101 in each region. The proportions of these sites varied among regions, although the affinity of WB 4101 for each site remained constant. Thalamus and cerebral cortex had the highest proportion of low‐affinity sites, whereas hippocampus and pons‐medulla had the highest proportion of high‐affinity sites. Pre‐treatment with CEC in hypotonic buffer significantly reduced the density of 125IBE binding sites in all brain regions. Cerebral cortex and cerebellum had the highest proportion of CEC‐sensitive sites, whereas hippocampus and spinal cord had the highest proportion of CEC‐insensitive sites. There was a significant correlation between the proportion of binding sites with a low affinity for WB 4101 and those sensitive to inactivation by CEC
Hydrodynamic Molecular Weight of Solubilized Cholinergic Synaptic Vesicle Glycoprotein ATPase
Authors: Yamagata Susan K. , Parsons Stanley M.
Published: October 2006

Abstract: The Torpedo californica electric organ synaptic vesicle glycoprotein ATPase was solubilized with octaethyleneglycoldodecyl ether and stabilized with phosphatidylserine. The complex was analyzed by size exclusion chromatography and band sedimentation velocity ultracentrifugation in water/glycerol and deuterium oxide/glycerol density gradients. The complex was found to have a Stokes' radius of 79 ± 0.7 Å, a sedimentation velocity coefficient at 20±C in water of 6.8 ± 0.2S, a partial specific volume of 0.81 ± 0.01 cm3/g, and a frictional coefficient of 1.6. The molecular weight of the solubilized complex was calculated to be 320,000 ± 7,000 and that of the protein 210,000 ± 9,000. The relationship of this latter value to the major transport ATPase types is discussed.
Carbachol‐Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells
Authors: Okazaki Masahiro , Yanagihara Nobuyuki , Izumi Futoshi , Nakashima Yasuhide , Kuroiwa Akio
Published: October 2006

Abstract: The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 × 106/dish) contained 100.4 ± 6.0 fmol of immunoreactive ANP (IR‐ANP) and 207.3 ± 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR‐ANP and catecholamines corresponding to the ratio of IR‐ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol‐stimulated secretion of IR‐ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR‐ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse‐phase HPLC analysis showed that both stored and secreted IR‐ANP consisted of two components, which eluted at the position of ANP(99–126) or ANP(1–126). These results indicate that ANPs are stored as ANP(99–126) and ANP(1–126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+‐dependent manner by the stimulation of nicotinic acetylcholine receptors.
Acute Effect of Protein or Carbohydrate Breakfasts on Human Cerebrospinal Fluid Monoamine Precursor and Metabolite Levels
Authors: Teff Karen L. , Young Simon N. , Marchand Luc , Botez Mihai I.
Published: October 2006

Abstract: Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3‐methyoxy‐4‐hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5‐hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5‐hydroxytryptamine synthesis.
Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial‐Derived Factors and Voltage‐Dependent Ion Channels
Authors: Simantov Rabi , Levy Rivka
Published: October 2006

Abstract: The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum‐containing medium, KCl had only a small and transient effect in serum‐free cultures of both types. The basal receptor density in serum‐free cultures was similar to the receptor density in KCl‐treated serum‐containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum‐free cultures. The effect of KCl in serum‐containing forebrain cultures was enhanced by the voltage‐dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose‐dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only δ type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage‐dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.
tele‐Methylhistamine Levels and Histamine Turnover in Nuclei of the Rat Hypothalamus and Amygdala
Authors: Itoh Yoshinori , Oishi Ryozo , Nishibori Masahiro , Saeki Kiyomi
Published: October 2006

An HPLC method using fluorescence detection for the determination of tele‐methylhistamine! (t‐MH) was improved to a sensitivity level which enabled the detection of 0.05 pmol of tissue t‐MH. The t‐MH contents and the histamine turnover rates in various nuclei of the rat hypothalamus and amygdala were subsequently measured. The histamine turnover rates were estimated from pargyline‐induced t‐MH accumulation. Both the t‐MH levels and the histamine turnover rates were shown to be relatively high in the nuclei dorsomedialis and premammillaris ventralis of the hypothalamus, and also in the nucleus medialis of the amygdala. The steady‐state t‐MH levels in various nuclei of the hypothalamus and amygdala correlated well with the histamine turnover rates in these nuclei.
Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium‐Dependent Excitatory Amino Acid Release
Authors: McMahon Harvey T. , Nicholls David G.
Published: October 2006

Guinea‐pig cerebral cortical synaptosomes were preincubated for 60 min with 100 μMd‐aspartate, l‐aspartate, or l‐glutamate. The total d‐ plus l‐aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+‐dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (d‐aspartate), 149% (l‐aspartate), or 168% (l‐glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+‐dependent glutamate release. Thus the Ca2+‐dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1–0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extra‐synaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase‐derived glutamate. No increase was seen in the extent of Ca2+‐dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with “free'’brain mitochondria, confirming the origin of the glutamine‐derived glutamate.
Human Neuroectoderm‐Derived Cell Line Secretes Fibronectin that Shares the HNK‐1/10C5 Carbohydrate Epitope with Neural Cell Adhesion Molecules
Authors: Harper John R. , Perry Susan K. , Davis Roxanne M. , Laufer David M.
Published: October 2006

A human malignant melanoma cell line, Melur, secretes several glycoproteins that contain a unique carbohydrate epitope shared by neural cell adhesion molecules and recognized by the monoclonal antibodies HNK‐1, L2, and 10C5. In this report, we present evidence that one of the major melanoma glycoproteins containing the HNK‐1/10C5 epitope is the cell adhesion molecule, fibronectin, or a fibronectin‐like molecule. Melanoma‐derived fibronectin was isolated from serum‐free conditioned medium by gelatin‐Sepharose affinity adsorption and shown to react with monoclonal antibodies HNK‐1 and 10C5 in Western blot analysis. HNK‐1‐containing fibronectin was purified on a gelatin‐Sepharose column followed by an affinity column using a monoclonal antibody against the HNK‐1 carbohydrate. The purified HNK‐1‐fibronectin then could be incorporated into the extracellular matrix of hamster fibroblasts in vitro, and such a matrix was detectable using the HNK‐1 monoclonal antibody in an immunofluorescence assay. Of the seven neuroectoderm‐derived tumor cell lines tested, only the Melur melanoma cell secreted fibronectin containing the HNK‐1 carbohydrate. Identification of human neuroectoderm‐derived fibronectin as a potential carrier of the HNK‐1 carbohydrate suggests a new role for fibronectin in neural development and regeneration, and represents a new model for studying the function of this carbohydrate domain in neural cell adhesion.
Multiple Pathways of N‐Kinase Activation in PC12 Cells
Authors: Rowland‐Gagné Elizabeth , Greene Lloyd A.
Published: October 2006

Past work established a cell‐free assay for a nerve growth factor (NGF)‐activated protein kinase activity (designated N‐kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well‐characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N‐kinase activity is regulated in PC12 rat pheochromocytoma cells. N‐kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N‐kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down‐regulation of protein kinase C by phorbol ester pretreatment prevents N‐kinase activation by phorbol ester, but not by the other agents. A PC12 cell‐derived line deficient in cyclic AMP‐dependent protein kinase II activity exhibits N‐kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N‐kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N‐kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N‐kinase can be activated via multiple second‐messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses to various extracellular signals.
Regional Difference in Responsiveness of Norepinephrine‐Sensitive Cyclic AMP‐Generating Systems of Rat Cerebral Cortex with Iron‐Induced Epileptic Activity
Authors: Hattori Yukio , Moriwaki Akiyoshi , Hayashi Yasushi , Hori Yasuo
Published: October 2006

Responsiveness of norepinephrine (NE)–sensitive cyclic AMP (cAMP)‐generating systems was determined in slices from different areas of the rat cerebral cortex in which FeCl2 solution was injected unilaterally into the sensorimotor cortex to induce epileptic activity. In anterior cortical areas of rats in which the appearance of electrographic isolated spikes was dominant either ipsilaterally or contralaterally to the injection site 8–10 days after the injection, the cAMP accumulations elicited by NE and an NE‐phentolamine combination were greater on the side of dominant spike activity than on the other. In anterior cortical areas of rats showing dominant spike activity on either side of the cortex 31–60 days after the injection, the cAMP accumulation elicited by NE was smaller on the dominant side than on the other. In anterior cortical areas of rats showing nearly equal spike activity on the two sides 31–60 days after the injection, the cAMP accumulations elicited by NE and an NE‐phentolamine combination were greater on the side ipsilateral to the injection site than on the other. In anterior and posterior cortical areas of rats in which the appearance of spike and wave complexes, as well as isolated spikes, was detected 31–60 days after the injection, the cAMP accumulations elicited by NE and combinations of NE and phentolamine or propranolol were greater on the side ipsilateral to the injection site than on the other. The elicitation by an NE‐propranolol combination, but not by an NE‐phentolamine combination, of cAMP accumulation was almost completely inhibited by 8‐phenyltheophylline. These results suggest that alterations in NE‐sensitive cAMP‐generating systems of the cortex involving α‐ and β‐adrenoceptors and adenosine receptors are related to the neurochemical process of iron‐induced epilepsy.
μ‐Opioid Receptors and Not K‐Opioid Receptors Are Coupled to the Adenylate Cyclase in the Cerebellum
Authors: Polastron J. , Boyer M.‐J. , Quertermont Y. , Thouvenot J.‐P. , Meunier J.‐C. , Jauzac Ph.
Published: October 2006

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (>75%μ‐opioid receptors) and the guinea pig cerebellum (>80%K‐opioid receptors). In the μ‐preparation, but not in the K‐preparation, opioids inhibited the basal and the forskolin‐stimulated adenylate cyclase activity in a dose‐dependent manner and stereospecifically. The inhibition was in the 20–30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a μ‐opioid binding site, with the effect being elicited by nonselective (etorphine and morphine) and μ‐selective (Tyr‐D‐Ala‐Gly‐Me‐Phe‐Gly‐ol) agonists, whereas δ‐ and K‐selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP‐binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.
6,7‐Dichloroquinoxaline‐2,3‐Dione is a Competitive Antagonist Specific to Strychnine‐Insensitive [3H]Glycine Binding Sites on the N‐Methyl‐D‐Aspartate Receptor Complex
Authors: Ogita Kiyokazu , Yoneda Yukio
Published: October 2006

Multiple binding sites on the N‐methyl‐D‐aspartate (NMDA) receptor complex were examined using rat brain synaptic membranes treated with Triton X‐100. Binding of [3H](+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,dcyclohepte5,10‐imine ([3H]MK‐801), a noncompetitive NMDA antagonist, in the presence of 10 μM L‐glutamate not only was inhibited by different types of antagonists, such as 6,7‐dichloro‐3‐hydroxy‐2‐quinoxaline‐carboxylate, 7‐chlorokynurenate, and 6,7‐dichloroquinoxaline‐2,3‐dione (DCQX), but also was abolished by non‐NMDA antagonists, including 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and 6,7‐dinitroquinoxaline‐2,3‐dione. The inhibition of [3H]MK‐801 binding by these compounds was invariably reversed or attenuated by addition of 10 μM glycine. Among these novel antagonists with an inhibitory potency on [3H]MK‐801 binding, only DCQX abolished [3H]glycine binding without inhibiting [3H]glutamate and [3H](±)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonate bindings. Other antagonists examined were all effective as displacers of the latter two bindings. These results suggest that DCQX is an antagonist highly selective to the strychnine‐insensitive glycine binding sites with a relatively high affinity.
Production and Characterization of Monoclonal Antibodies to the Major Peripheral Myelin Glycoprotein P0
Authors: Bollensen Edgar , Scheidt Peter , Schachner Melitta
Published: October 2006

Several monoclonal antibodies were generated against the major glycoprotein P0 of human peripheral nervous system myelin. Antibodies were selected for their reactivity with P0 in Western blots. The antibodies were of the immunoglobulin G subclass and reacted with the glycopeptidase F‐treated P0, indicating that the reactive epitope resides in the protein backbone. In fresh frozen and paraffin‐embedded sections of central and peripheral nervous system of rat and human, P0 antibody 592 reacted with myelin sheaths of peripheral, but not central, nervous system.
Comparison of Transmitter Amino Acid Levels in Rat Globus Pallidus and Neostriatum During Hypoglycemia or After Treatment with Methionine Sulfoximine or γ‐Vinyl γ‐Aminobutyric Acid
Authors: Fonnum F. , Paulsen R. E.
Published: October 2006

The levels of amino acids in globus pallidus, a structure heavily innervated with γ‐aminobutyric acid (GABA)‐ergic terminals but few glutamergic terminals, were compared with the levels in neostriatum, a structure richly innervated with glutamergic terminals but intermediate in GABAergic terminals. The level of glutamate in neostriatum was twice as high as in globus pallidus whereas the level of GABA in globus pallidus was three times higher than in neostriatum. The level of aspartate was similar in both regions whereas the level of glutamine was correlated with the level of glutamate. Methionine sulfoximine, a glutamine synthetase inhibitor, reduced the level of glutamine to 10‐20% of control in both structures. This reduction was accompanied by the largest decrease in the level of glutamate in neostriatum, indicating that transmitter glutamate turns over more rapidly than other glutamate pools. Likewise, insulin decreased the levels of glutamate and glutamine more in neostriatum than in globus pallidus. γ‐Vinyl GABA increased the level of GABA in globus pallidus more than in neostriatum although the percent increase was largest in neostriatum. Treatment with γ‐vinyl GABA was accompanied by a large reduction in the level of glutamine that was correlated with the increase in the level of GABA, indicating that a substantial proportion of the glutamine pool is linked to GABA metabolism.
Effects of 1‐Methyl‐4‐Phenyl‐ 1,2,3,6‐Tetrahydropyridine and 1 ‐Methyl‐4‐Phenylpyridinium Ion on ATP Levels of Mouse Brain Synaptosomes
Authors: Scotcher Kevin P. , Irwin Ian , DeLanney Louis E. , Langston J. William , Di Monte Donato
Published: October 2006

Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its toxic metabolite 1‐methyl‐4‐phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10–100 γM) similar to those it reaches in the brain in vivo. MPTP‐induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.