bluuebunny/crossref_metadata_2025 huggingface dataset:
Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain
Authors: Simon J. , Szücs M. , Benyhe S. , Borsodi A. , Zeman P. , Wollemann M.
Published: December 2006

Abstract: Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, and Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane‐bound and the solubilized receptors showed weak binding of enkephalin and μ‐specific drugs, suggesting that they are predominantly of the k‐type. The membrane‐bound and the soluble receptors showed the same distribution of subtypes, i.e., 70%k, 13%μ, and 17%δ for the membrane‐bound and 71%k, 17%μ, and 12%δ for the soluble receptors
Subcellular Localization of 5′‐Nucleotidase in Rat Brain
Authors: Heymann Dagmar , Reddington Martin , Kreutzberg Georg W.
Published: December 2006

Abstract: The subcellular distribution of the ectoenzyme, 5′‐nucleotidase, in cerebral cortex and cerebellum of the rat was studied both biochemically and cytochemically. The fractions were characterized biochemically by marker enzymes. The localization of 5′‐nucleotidase activity was also investigated cytochemically in the myelin, synaptosomal, mitochondrial, and microsomal fractions. Biochemically 5′‐nucleotidase was found to be enriched in the membrane‐containing fractions, i.e., myelin, synaptosomal, and microsomal fractions. Cytochemistry showed the reaction product in the myelin fraction to be associated with myelin profiles. In the synaptosomal fraction reaction product could occasionally be seen at synaptosomal membranes, although it could not be attributed unequivocally to the synaptosome itself, since in positions with reaction product unidentifiable membrane structures could always be seen attached. Mitochondria were virtually without any reaction product. In the microsomal fraction 5′‐nucleotidase activity was associated with unidentifiable membrane structures. It is concluded that 5′‐nucleotidase is associated with myelin profiles and that the high activity found in the synaptosomal fraction is probably not associated with nerve ending plasma membranes.
Presence of Phospholipid‐N‐Methyltransferases and Base‐Exchange Enzymes in Rat Central Nervous System Axolemma‐Enriched Fractions
Authors: Hattori Hiroshi , Bansal Vinay S. , Orihel Danka , Kanfer Julian N.
Published: December 2006

Abstract: Rat CNS myelinated axons were fractionated by sucrose density gradient centrifugation with a zonal rotor. Fraction VI, obtained at 28–30% sucrose, appeared, on the basis of the presence of related marker enzymes, to be enriched in axolemma. Phospholipid‐N‐methyltransferases (PMTs) and base‐exchange enzymes were associated with fraction VI. PMT activity was significantly stimulated by the addition of either phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine but the PMT activity of the homogenate or the myelinated axons was unresponsive. Recoveries of the ethanolamine, serine, and choline base‐exchange activities were 14.4%, 13.8%, and 3.4%, respectively, of that present in the myelinated axons. The myelin‐rich fraction obtained simultaneously seems contaminated with other membrane fractions.
Regional and Cortical Laminar Distributions of Serotonin S2, Benzodiazepine, Muscarinic, and Dopamine D2 Receptors in Human Brain
Authors: Luabeya Mesu'a K. , Maloteaux Jean‐Marie , Laduron Pierre M.
Published: December 2006

Abstract: Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in the human brain but were present in all cortical areas. The laminar distributions of [3H]ketanserin, [3H]diazepam, and [3H]quinuclidinylbenzilate were investigated in the temporal cortex and revealed a high density in the IIIrd and IVth layers. Dopamine D2 receptors were not detected in the cortex.
Enzymes of Energy Metabolism in the Mudpuppy Retina
Authors: Dick Evan
Published: December 2006

Abstract: The distributions of glycogen phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase, glucose‐6‐phosphate dehydrogenase, citrate synthase, malate dehydrogenase, β‐hydroxyacyl CoA dehydrogenase, and adenylokinase were determined in the mudpuppy retina. Distinct differences were found in regard to the glycolytic and oxidative capacities of the various layers. In the outer retina, citric acid cycle enzymes were high while glycolytic enzymes were low. Synaptic zones were distinctly enriched in all energy‐producing enzymes. Mudpuppy photoreceptors were found to be rich in phosphorylase but poor in glucose‐6‐phosphate dehydrogenase, suggestive of some evolutionary divergence from mammals in the metabolic machinery which is used to support the visual process.
Cellular Distribution of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Staggerer Mutant
Authors: Seyfried Thomas N. , Bernard David J. , Yu Robert K.
Published: December 2006

Abstract: The distribution of cerebellar gangliosides was studied in staggerer (sg/sg) mutant mice, where the majority of granule cells die after completing their migration across the molecular layer. In addition, the external granule cell layer in (sg/sg) mice persists longer than in normal mice. Moreover, in the sg/sg cerebellum, Purkinje cells are significantly reduced in number, and almost none have tertiary branchlet spines. The loss of Purkinje cells and granule cells in sg/sg mice is accompanied by an early‐onset reactive gliosis that continues through adulthood. By correlating changes in ganglioside composition with the well‐documented histological events of cerebellar development in normal and sg/sg mice, we obtained strong evidence for a nonrandom cellular distribution of gangliosides. The sharpest reduction in the GD1a content of sg/sg cerebellum occurred after 15 days of age, coincident with granule cell loss. GT1a, on the other hand, was significantly reduced from 15 through 150 days in the sg/sg mice. GD3 is a major ganglioside of the undifferentiated granule cell, but it becomes rapidly displaced by the more complex gangliosides with the onset of granule cell maturation. In the sg/sg mice, GD3 persisted at abnormally high levels from 15 to 28 days and then accumulated through adulthood. These findings, and those from other cerebellar mouse mutants, suggest that GD1a is enriched in granule cells and that GT1a is enriched in Purkinje cells. Our findings also suggest that GT1a is more concentrated in branchlet spines than in other regions of the Purkinje cell membrane. GT1b appears to be enriched in both granule cells and Purkinje cells, whereas GM1 appears to be enriched in myelin. Furthermore, the apparent persistence of the embryonic ganglioside GD3 in sg/sg mice results from an early‐onset reactive gliosis, together with a partial retardation in granule cell maturation. The accumulation of GD3 beyond 28 days reflects the continued accretion of GD3 in reactive glia.
pH Selectivity of N‐Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes
Authors: Childers Steven R. , Jackson Jerry L.
Published: December 2006

Abstract: N‐Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]d‐Ala2‐Met5‐enkephalinamide (d‐Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100–250 μm NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM‐reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 μm of NEM.
Change in the Distribution of Acetylcholinesterase Molecular Forms in Hirschsprung's Disease
Authors: Rakonczay Z. , Németh P.
Published: December 2006

Abstract: Density gradient ultracentrifugation shows that two molecular forms of acetylcholinesterase (4S and 10S) can be distinguished in the bowels of both normal subjects and Hirschsprung's disease patients. In this disease, besides the very large elevation of acetylcholinesterase activity, the relative distribution of the heavy and light forms was also changed. In the affected bowel the 10S/4S ratio was 2.5 times higher than the normal value. It is assumed that the accumulation of the 10S form might be a response of the intestine to this pathological state. It is also suggested that the increase in the heavy form is closely connected with the nerve fibre proliferation in the aganglionic megacolon.
Molecular Genetic Analysis of Myelin‐Deficient Mice: Shiverer Mutant Mice Show Deletion in Gene(s) Coding for Myelin Basic Protein
Authors: Kimura Minoru , Inoko Hidetoshi , Katsuki Motoya , Ando Asako , Sato Tadayuki , Hirose Tadaaki , Takashima Hideaki , Inayama Seiichi , Okano Hideyuki , Takamatsu Ken , Mikoshiba Katsuhiko , Tsukada Yasuzo , Watanabe Itaru
Published: December 2006

Abstract: The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3‐week‐old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.
Cell‐Free Synthesis of the D2‐Cell Adhesion Molecule: Evidence for Three Primary Translation Products
Authors: Hansen Ole C. , Nybroe Ole , Bock Elisabeth
Published: December 2006

Abstract: The D2‐cell adhesion molecule (D2‐CAM) is a membrane glycoprotein that is involved in cell‐cell adhesion in the nervous system. To study the biosynthesis of D2‐CAM we have translated free and membrane‐bound polysomes from rat brain in vitro in the rabbit reticulocyte lysate system. D2‐CAM was exclusively synthesized on membrane‐bound polysomes. The primary translation products of D2‐CAM were three polypeptides of apparent molecular weights 187,000, 134,000, and 112,000. No interconversion between these polypeptides was detected. In contrast to previous suggestions, we conclude that all three D2‐CAM polypeptides are primary translation products. When translating polysomes from embryonic and postnatal rat brain, we found that the relative amounts of the three polypeptides synthesized varied with age. Their molecular weights, however, were not age‐dependent.
Measurement of Substance P Metabolites in Rat CNS
Authors: Sakurada T. , Le Grevés P. , Stewart J. , Terenius L.
Published: December 2006

Abstract: A procedure based on ion‐exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P(SP), the SP(1–7), and C‐terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50‐ to 100‐fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1–7) comigrated with the authentic standard whereas practically all activity isolated as C‐terminal fragments comigrated with SP(5–11). The levels of C‐terminal fragments in rat brain areas rich in SP and in spinal cord were 1–2% of those of parent compound. The levels of SP(1–7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1–7) levels fell more rapidly than those of SP or C‐terminal fragments.
Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System
Authors: Briggs Clark A. , Horwitz Joel , McAfee Donald A. , Tsymbalov Sophia , Perlman Robert L.
Published: December 2006

Abstract: The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomie tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.
Phospholipid‐Induced Changes of γ‐Aminobutyric Acid Transport in Cortex Grey Matter in Culture
Authors: Giesing M. , Gerken U. , Kastrup H.
Published: December 2006

Abstract: The function of membrane phospholipids (PL) in the regulation of γ‐aminobutyric acid (GABA) transport and GABA carrier binding has been investigated in organized cultures of rat cerebral cortex. The cellular lipid composition has been changed by growing the cells in a delipidated nutrient solution or by short‐term exposure of the cells to PL emulsions. Introduction of PL into the cellular matrix was monitored by analysis of biologically active fluorescently labeled phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Parinaroyl and dansyl derivatives were used. Conditions of maintenance as well as exogenously given PL affected the transport of GABA. Two transport systems were observed, one first‐order system and one cooperative system. Saturated species of PC or PE reduced first‐order GABA uptake with increase in chain length of the fatty acid residues. The effects of unsaturated PL were dependent upon the polar head. Unsaturated PC enhanced the capacity of the first‐order transport of the amino acid. In comparison to cultures grown in lipid‐free medium, introduction of diarachinoyl‐PC into the cells increased the density of the first‐order active transport sites by a factor of 8 and the affinity constant by a factor of 17. Diarachinoyl‐PE reduced both kinetic parameters. GABA uptake via the cooperative system was enhanced by the unsaturated PE, not by PC. The role of endogenous PL and their asymmetric distribution was studied by application of phospholipase A2, C, and D. Stimulation of carrier activity was induced by hydrolysis of PL on the external leaflet. Inhibition occurred upon enzymatic degradation of external and cytoplasmic PL. Lipolysis also affected GABA receptor binding, suggesting that the effects observed represent the activity of both classes of binding sites, the carrier and the receptor. However the latter accounted for a small fraction of the binding. Transport of the amino acid was temperature sensitive. The temperature curve was shifted within two discontinuities, appearing in the Arrhenius plot as a function of membrane lipids. The results suggest a partitioning of the proteins between fluid and ordered lipid domains. Displacement of the protein may govern the rate constants and/or the effective protein concentration.
Effect of Light on the Metabolism of Lipids in the Rat Retina
Authors: Anderson Robert E. , Maude Maureen B. , Pu Glen A‐Wen , Hollyfield Joe G.
Published: December 2006

Abstract: The effect of light on the in vitro incorporation of a variety of radioactive precursors into glycerolipids was tested in isolated retinas of albino rats. There was an increase in the incorporation of [2‐3H]myo‐inositol, 32Pi, [2‐3H]glycerol, and [methyl‐3H]choline into retinal phospholipids in light compared to that in darkness. [2‐3H]myo‐inositol was incorporated primarily into phos‐phatidylinositol. 32Pi was incorporated primarily into the phosphoinositides, although there were significant increases in the specific activities of all retinal phospholipids in light compared to those in darkness. Likewise, [2‐3H]glycerol incorporation into all retinal phospholipids and diglycerides was greater in light than in the dark. There was no effect of light on the incorporation of [2‐3H]ethanolamine into phosphatidylethanolamine or of [3‐3H]serine into phosphatidylserine, although these phospholipids were labeled to a greater extent in light with [2‐3H]glycerol. There was no effect of light on the incorporation of [3H]palmitic acid into diglycerides and phospholipids, with the exception of phosphatidylino‐sitol. Light also had no effect on the uptake of [2‐3H]glycerol, [2‐3H]inositol, or [methyl‐3H]choline into the retina. We conclude from these studies that light stimulates the phosphoinositide effect in the rat retina. Although some of the results are consistent with a stimulation of de novo synthesis of all lipid classes, our studies with [3H]palmitate, [2‐3H]ethanolamine, and [3‐3H]serine do not support this conclusion. A possible explanation for the increased incorporation of [2‐3H]glycerol, or 32Pi, and [methyl‐3H]choline into retinal phospholipids is that the metabolic events that follow photon capture alter the size and/or turnover rate of the choline, choline phosphate, ATP, and glycerol‐3‐phosphate pools, thus altering the specific radioactivities of these precursors. This would result in different specific activities of the lipid products in the absence of any direct effect of light on the rate of synthesis.
Myelin‐Associated Glycoprotein and Other Proteins in Trembler Mice
Authors: Inuzuka Takashi , Quarles Richard H. , Heath John , Trapp Bruce D.
Published: December 2006

Abstract: The myelin‐associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20‐day‐old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/μg of total protein in the controls to 1.4 ng/μg protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20‐day‐old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age‐matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt‐Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous sytem (PNS) of the Trembler mice may contribute to the pathology in this mutant.
Incorporation of Intracisternally Administered l‐[methyl‐3H]Methionine and S‐Adenosyl‐l‐[methyl‐3H]‐Methionine into Rat Brain Phospholipids
Authors: Zawad John S. , Brown Fountaine C.
Published: December 2006

Abstract: The incorporation of intracisternally injected l‐[methyl‐3H]methionine ([3H]Met) or S‐adenosyl‐l‐[methyl‐3H]methionine (Ado[3H]Met) into rat brain AdoMet and phospholipid pools was examined. When [3H]Met was administered, both AdoMet and phospholipid pools were labeled. However, exogenously injected Ado[3H]Met did not serve as a substrate for phospholipid‐N‐methyltransferases. It was concluded that only Ado[3H]Met formed in situ was utilized to methylate phospholipids and that this process was initiated on the cytoplasmic side of the membrane. The apparent biological half‐life in brainstem of phosphatidyl‐N‐monomethylethanolamine and phosphatidyl‐N,N‐dimethylethanolamine formed from [3H]Met was 1.4 and 1.7 days, respectively. The half‐life of phosphatidylcholine could not be determined due to interference from peripheral sources.
Altered Composition of Polyunsaturated Fatty Acyl Groups in Phosphoglycerides of Down's Syndrome Fetal Brain
Authors: Brooksbank B. W. L. , Martinez Manuela , Balazs R.
Published: December 2006

Abstract: We have observed recently that in vitro lipoperoxidation is enhanced in Down's syndrome brain homogenates of prenatal age. As this may relate to the composition of polyunsaturated acyl groups (PUFA) in phospholipids, we have examined the PUFA of ethanolamine and serine phosphoglycerides (EPG and SPG), which are particularly rich in PUFA, in the same series of cerebral cortex specimens of Down's syndrome and age‐matched control fetuses. Although the total percentages of PUFA in the two phosphoglycerides were not altered, compared with controls the ratio of PUFA of the (n‐3) series to those of the (n‐6) series was very significantly elevated in Down's syndrome, from 0.32 to 0.55 in EPG and from 0.60 to 0.97 in SPG. In particular, docosahexaenoyl, 22:6(n‐3), groups were uniformly increased in Down's syndrome compared with controls by 54% and 33% in EPG and SPG, respectively, while arachidonoyl, 20:4(n‐6), groups were decreased by 16% and 30%, respectively. Similar changes occur during normal development, but the (n‐3) to (n‐6) ratio of PUFA in these phosphoglycerides of Down's syndrome at the fifth month of gestation resembled that of normal human cerebral grey matter at term. However, other developmental indices related to PUFA composition were not significantly affected. It seems therefore that in the developing Down's syndrome brain there may be a distortion of the normal transformations of essential fatty acids and of their incorporation into phosphoglycerides. The disproportion between docosahexaenoyl and arachidonoyl groups in membrane phosphoglycerides during prenatal development in Down's syndrome may also result in disturbances of the proper functioning, and the ontogenetic integration, of membrane enzymes and transport processes.
β‐N‐Oxalylamino‐l‐Alanine: Action on High‐Affinity Transport of Neurotransmitters in Rat Brain and Spinal Cord Synaptosomes
Authors: Ross Stephen M. , Roy D. N. , Spencer Peter S.
Published: December 2006

Abstract: β‐N‐Oxalylamino‐l‐alanine (BOAA) is a dicarboxylic diamino acid present in Lathyrus sativus (chickling pea). Excessive oral intake of this legume in remote areas of the world causes humans and animals to develop a type of spastic paraparesis known as lathyrism. BOAA is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal vacuolation and necrosis. The present study investigates the action of BOAA in vitro on CNS high‐affinity transport systems for glutamate, γ‐aminobutyric acid (GABA), aspartate, glycine, and choline and in the activity of glutamate decarboxylase (GAD), the rate‐limiting enzyme in the decarboxylation of glutamate to GABA. Crude synaptosomal fractions (P2) from rat brain and spinal cord were used for all studies. [3H]Aspartate transport in brain and spinal cord synaptosomes was reduced as a function of BOAA concentration, with reductions to 40 and 30% of control values, respectively, after 15‐min preincubation with 1 mM BOAA. Under similar conditions, transport of [3H]glutamate was reduced to 74% (brain) and 60% (spinal cord) of control values. High‐affinity transport of [3H]GABA, [3H]glycine, and [3H]choline, and the enzyme activity of GAD, were unaffected by 1 mM BOAA. While these data are consistent with the excitotoxic (convulsant) activity of BOAA, their relationship to the pathogenesis of lathyrism is unknown.
Dopamine Inhibits Calcium‐Independent γ‐[3H]Aminobutyric Acid Release Induced by Kainate and High K+ in the Fish Retina
Authors: Kato Satoru , Negishi Koroku , Teranishi Tsunenobu
Published: December 2006

Abstract: Kainic acid (KA) at micromolar concentrations stimulated the release of γ‐[3H]aminobutyric acid ([3H]GABA) from a participate fraction of the carp (Cyprinus carpio) retina. The KA action was dose‐dependent but Ca2+‐independent. A similar response was elicited by another glutamate receptor agonist, quisqualic acid, and high K+, but not by an aspartate agonist, N‐methyl‐d‐aspartic acid. The stimulatory action of KA on the [3H]GABA release was selectively blocked by the KA blockers γ‐d‐glutamylglycine and cis‐2,3‐piperidine dicarboxylic acid. Dopamine (DA), which is contained in DA interplexiform cells in the carp retina, inhibited the [3H]GABA release induced by KA and high K+ in a dose‐dependent manner. 5‐Hydroxytryptamine and two well‐known GABA antagonists, bicuculline (Bic) and picrotoxin (Pic), also mimicked the DA effect on the GABA release at a comparable concentration. This inhibitory effect of DA as well as Bic and Pic on the [3H]GABA release evoked by KA was clearly antagonized by a DA blocker, haloperidol. The action of these agents (KA, DA, GABA antagonist) belonging to three different receptor categories on the GABAergic neurons (possibly external horizontal cells; H1 cells) is discussed in relation to other electrophysiological studies on the lateral spread of S‐potentials between H1 cells.
Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study
Authors: Werz W. , Fischer G. , Schachner M.
Published: December 2006

Abstract: Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to <5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.
Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol
Authors: Cavalla David , Neff Norton H.
Published: December 2006

Abstract: Muscimol is one of the most potent agonist ligands at the γ‐aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]‐muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 ± 3%. Dose‐dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l‐glutamate, and diazepam exerted no effect at high concentrations. SDS‐PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular‐weight species. One failed to enter the separating gel, implying a molecular weight >250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).
Choline Acetyltransferase Depletion in the Rat Retina After Intraocular Injection of Neurotoxins
Authors: Gómez‐Ramos Pilar , Estrada Carmen , Pérez‐Rico Consuelo
Published: December 2006

Abstract: The effects of kainic acid (KA), quisqualic acid (QA), and ibotenic acid (IBO) on histology of the retina and on the retinal choline acetyltransferase (ChAT) activity were studied in the rat. KA produced the highest number of altered cells in the ganglion cell layer (GCL) and in the inner nuclear layer (INL), with an almost complete depletion of ChAT activity. QA was less effective than KA in terms of both the number of altered cells and in ChAT depletion. In contrast, retinas injected with IBO showed the mildest morphological lesions together with the highest reduction in the enzyme activity. These results indicate that IBO affects nearly all the cholinergic neurons in the rat retina, whereas other populations, sensitive to KA or QA, are spared. Because of this higher specificity toward the cholinergic subpopulation, IBO may be a useful tool when cholinergic cells need to be destroyed in the retina.
[3H]1‐[2‐(4‐Aminophenyl)Ethyl]‐4‐(3‐Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5‐HT1A Receptors in Rat Brain
Authors: Ransom Richard W. , Asarch Kenneth B. , Shih Jean C.
Published: December 2006

Abstract: 1‐[2‐(4‐Aminophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5‐hydroxytryptamine (5‐HT, serotonin) binding to 5‐HT1A and 5‐HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5‐HT1A receptor radioligand was evaluated. With either 10 μM 5‐HT or 1 μM 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d‐Lysergic acid diethylamide and 5‐HT, two nonselective inhibitors of [3H]5‐HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5‐HT1 receptors. Spiperone and 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, two compounds that discriminate [3H]5‐HT binding to 5‐HT1A and 5‐HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5‐HT1A receptor subtype. Furthermore, the ability of N‐(m‐trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5‐HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin‐sensitive [3H]PAPP sites correlated with the densities of 5‐HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5‐HT1A receptor sites in rat brain.
Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ
Authors: Momoi Mariko Yoshida , Lennon Vanda A.
Published: December 2006

Abstract: Acetylcholine receptor (AChR) purified from human skeletal muscle was affinity‐alkylated with bromoacetyl[methyl‐3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the α‐subunit. The binding of [125I]α‐bungarotoxin to AChR was completely inhibited by affinity‐alkylation, indicating that the human AChR's binding site for α‐bungarotoxin is closely associated with the α‐subunit's acetylcholine binding site. Structures in the vicinity of the α‐bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of α‐bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the α‐bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's α‐bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.
Polyamine Regulation of the Microtubule‐Associated Protein Kinase
Authors: Nickerson Jeffrey A. , Wells William W.
Published: December 2006

Abstract: Microtubule protein prepared by cycles of assembly‐disassembly contains a cyclic AMP‐dependent protein kinase that phosphorylates the high‐molecular‐weight microtubule‐associated protein MAP‐2. The polyamine spermine at 2 mM affected the phosphorylation of MAP‐2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10−6M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10−6M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP‐dependent phosphorylation but did not affect the protein substrate specificity of the microtubule‐associated protein kinase. MAP‐2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.
Human Skeletal Muscle Cells Synthesise a Neuronotrophic Factor Reactive with Spinal Neurons
Authors: Doherty Patrick , Dickson John G. , Flanigan Thomas P. , Walsh Frank S.
Published: December 2006

Abstract: Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell‐type‐specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7‐day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 μg/ml, with a dose‐response relationship yielding maximal and half‐maximal effects at 4 and 1 μg/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate twofold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.
Postnatal Development of Proteins Associated with Different Benzodiazepine Receptors
Authors: Eichinger A. , Sieghart W.
Published: December 2006

Abstract: The postnatal development of several proteins irreversibly labeled by [3H]flunitrazepam in membranes from rat cerebral cortex was investigated. It was demonstrated that in the early postnatal days proteins with apparent molecular weights 55,000 and 59,000 were predominantly labeled whereas irreversible labeling of a protein with apparent molecular weight 51,000 started to predominate only in the second postnatal week. Irreversible labeling of another protein with apparent molecular weight 62,000 was weak throughout development. All these proteins seem to be associated with central benzodiazepine receptors. Irreversible labeling at various time points after birth seems to parallel the postnatal development of these proteins, and the different time course of development and different binding properties of theindividual proteins support the hypothesis that these proteins are associated with separate and distinct benzodiazepine receptor subtypes. The pharmacological properties of the individual receptor subtypes seem to be fully developed in the early postnatal days, and therefore newborn animals seem to be a good model system for the investigation of properties and function of these various benzodiazepine receptor subtypes.
Dopamine Metabolites in Rat Cisternal Cerebrospinal Fluid: Major Contribution from Extrastriatal Dopamine Neurones
Authors: Hutson P. H. , Curzon G.
Published: December 2006

Abstract: The interpretation of central 3,4‐dihydroxy‐phenylethylamine (dopamine, DA) metabolism, as indicated by determinations in rat cisternal CSF, was investigated using intrastriatal injection of the DA neurotoxin 6‐hydroxydopamine (6‐OHDA) and intraperitoneal injection of the noradrenergic neurotoxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP4). DA turnover was subsequently determined by measurement of the rate of accumulation of total 3,4‐dihydroxyphenyIacetic acid and homovanillic acid (DOPAC + HVA) in the CSF after probenecid was given. Two days later the rats were killed, and metabolism of DA and 5‐hydroxytryptamine (5‐HT) was investigated by determining levels of the amines and their metabolites in brain regions. Although 6‐OHDA greatly decreased striatal DA metabolism, this was not paralleled by DA turnover as indicated by CSF, as this fell only moderately and approximately in parallel with results for the brain as a whole. 5‐HT metabolism was essentially unaltered. DSP4 considerably depleted nor‐adrenaline and caused smaller decreases of 5‐HT metabolism in some regions. However, DA metabolism was not significantly affected, either in brain or CSF, which suggests that noradrenaline neurones make only a small contribution to central DA metabolism. Results as a whole suggest that DOPAC and HVA concentrations in rat cisternal CSF reflect whole brain DA metabolism and derive predominantly from DA neurones in extrastriatal regions of the brain.
Differential Effect of Subchronic Treatment with Various Neuroleptic Agents on Serotonin2 Receptors in Rat Cerebral Cortex
Authors: Andree Terrance H. , Mikuni Masahiko , Tong Ching Y. , Koenig James I. , Meltzer Herbert Y.
Published: December 2006

Abstract: In addition to antidepressant drugs, some neuroleptic (NL) drugs reduce serotonin, (5‐HT2) receptor binding sites after chronic administration. The present study was undertaken to characterize further this property of NL drugs. Scatchard analysis of [3H]spiperone binding in rat cerebral cortex revealed that 21‐day treatment with chlorpromazine (CPZ), cis‐flupenthixol, and thioridazine reduced 5‐HT2 radioligand binding density by 60, 27, and 18%, respectively. The more selective dopamine‐D2 antagonists haloperidol and sulpiride were totally ineffective in this regard. No reduction in 5‐HT2 ligand binding sites occurred after 1 day of treatment with CPZ but 3‐days of treatment was effective and this reduction persisted, although diminished, for at least 72 h after the last injection. cis‐Flupenthixol and d‐butaclamol were also effective after 3 days of treatment but trans‐flupenthixol and l‐butaclamol were not, indicating stereo‐specificity of the response mechanism. Female rats showed the same response to CPZ as did male rats. Central 5,7‐dihydroxytryptamine‐induced lesions of 5‐HT neurons demonstrated that intact 5‐HT neurons were not required for the reduction of 5‐HT2 receptor ligand binding by CPZ. Since CPZ has high affinity for many receptors, including α1, histamine1, and muscarinic receptors, the role of these effects in producing 5‐HT2 receptor down‐regulation was considered by studying the effects of prazosin, atropine, and pyrilamine administration on 5‐HT2 radioligand binding. Results indicate that no one of these actions appears to account for the down‐regulation of 5‐HT, receptors by CPZ. Several of these effects, in combination, or some unique mechanism, may be involved.
Studies on the Role of α2‐Adrenoceptors in the Control of Synaptosomal [3H]5‐Hydroxytryptamine Release: Effects of Antidepressant Drugs
Authors: Ellison David W. , Campbell Iain C.
Published: December 2006

Abstract: The sensitivity of α2‐adrenoceptors on 5‐hydroxytryptamine (5‐HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21–28 days) or clorgyline (1 mg/kg/day for 21–28 days). [3H]5‐HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5‐HT nerve terminals. The sensitivity of K+‐stimulated release of [3H]5‐HT to α2‐adrenoceptor occupancy was assessed in a superfusion system by means of the dose‐dependent inhibition of [3H]5‐HT release by clonidine. This is blocked by yohimbine (1 μM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these α2‐heteroreceptors. The effect of addition of citalopram (1 μM) to superfusates suggests that some reuptake of [3H]5‐HT occurs during superfusion. Of the tritium released into superfusates during “background” and K+‐stimulated release, 17 and 90%, respectively is [3H]5‐HT. The attenuation of K+‐stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter α2‐adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on α2‐adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA. A significant decrease in sensitivity to clonidine occurred with DSP4 alone. This study with drugs from the two major classes of antidepressants suggests that antidepressant drugs do not alter α2‐heteroreceptors on serotonergic neurones.
Contribution of Noradrenergic Neurons to the Regulation of Dopaminergic (D1) Receptor Denervation Supersensitivity in Rat Prefrontal Cortex
Authors: Tassin J. P. , Studler J. M. , Hervé D. , Blanc G. , Glowinski J.
Published: December 2006

Abstract: 3,4‐Dihydroxyphenylethylamine (DA, dopamine) levels in the rat prefrontal cortex were selectively decreased by 52%, leaving noradrenaline (NA) levels unaffected, 4 weeks following restricted bilateral electrolytic lesions of the ventral mesencephalic tegmentum (VMT). These lesions also induced a significant increase in DA‐sensitive, but not isoproterenol‐sensitive, adenylate cyclase activity in tissue homogenates (+ 38%). We had shown previously that chemical (6‐hydroxydo‐pamine, 6‐OHDA) lesions of the VMT destroy both ascending DA and NA fibers but do not alter the D1‐receptor density in the prefrontal cortex. In this study, electrolytic lesions of the VMT were combined with bilateral injections of 6‐OHDA made laterally in the pedunculus cerebellaris superior to assess the role of NA fibers in the development of D1‐receptor supersensitivity. This combined treatment produces a large decrease of cortical NA levels (‐95%), an increase in β‐adrenergic‐sensitive adenylate cyclase activity (+110%), and a decrease in DA levels (‐ 60%), but does not alter D1‐receptor density in the prefrontal cortex. These results indicate that the development of D1‐receptor supersensitivity in the prefrontal cortex following electrolytic lesion of the VMT depends on the presence of an intact NA innervation.
Interactions Between Vasoactive Intestinal Peptide and Dopamine in the Rabbit Retina: Stimulation of a Common Adenylate Cyclase
Authors: Pachter Jonathan A. , Lam Dominic Man‐Kit
Published: December 2006

Abstract: Although 3,4‐dihydroxyphenylethylamine (dopamine, DA) and vasoactive intestinal peptide (VIP) have been reported to stimulate adenylate cyclase activity in the rabbit retina, possible interactions between VIP‐sensitive and DA‐sensitive adenylate cyclase systems have not been previously investigated. To elucidate the interactions between these two putative transmitter‐stimulated cyclase systems, the effects of VIP, DA, and VIP + DA on the conversion of [α‐32P]ATP to [32P]cyclic AMP in rabbit retinal homogenates were measured. VIP stimulated adenylate cyclase activity in a biphasic manner, suggesting that two classes of VIP receptors may be involved in the induction of cyclic AMP formation. DA was less potent than VIP, and stimulated cyclase activity with a monophasic dose‐response curve. When assayed together, these stimulations were partially nonadditive, implying the existence of a common adenylate cyclase pool that may be stimulated by both putative neurotransmitters. The dopaminergic antagonist (+)‐butaclamol completely blocked dopaminergic stimulation, but had no significant effect on VIP‐induced stimulation. indicating that VIP interacts with specific VIP receptor sites, which are distinct from the dopaminergic receptor sites. Furthermore, the specific D‐2 dopaminergic receptor agonist LY141865 demonstrated no inhibitory effect on adenylate cyclase activity, suggesting that the interaction between the VIP‐ and DA‐sensitive adenylate cyclase systems does not result from a D‐2 receptor‐mediated cyclase inhibition in the rabbit retina. Finally, at maximally effective concentrations, DA and VIP were less potent than fluoride or forskolin in the stimulation of cyclic AMP formation, suggesting that adenylate cyclase pools that are not sensitive to DA and VIP may also be present in this retina. Our studies demonstrate the existence of several adenylate cyclase pools in the rabbit retina, at least one of which is sensitive to stimulation by both VIP and DA.