bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in Lysolecithin Content of Brain Microsomes in Phenobarbitone‐Administered Rats and Its Relation to Lipid Peroxidation
Authors: Awasthi Sita , Kakkar Poonam , Viswanathan P. N. , Bhardwaj R.
Published: October 2006

Abstract: Administration of phenobarbitone caused a marked increase in the capacity of rat brain microsomes to produce thiobarbituric acid‐reactive substances in vitro. Enzymatic peroxidation of lipids was more affected than the nonenzy‐matic processes occurring in heat‐inactivated preparations. Analysis of the phospholipid profile showed a drastic decrease in phosphatidylcholine and total phospholipid contents in the exposed animals, but about a fivefold increase in the lysophosphatidylcholine fraction. Data for in vivo incorporation of [14C]choline showed a similar pattern of high radioactivity in lysolecithin. The increase in lipid peroxidation could be related to the higher level of lysolecithin and the accompanying structural and functional changes in microsomes resulting from the neurotoxic effects of phenobarbitone.
Expression of Transferrin mRNA in the CNS of Normal and Jimpy Mice
Authors: Bartlett William P. , Li Xiao‐Su , Connor James R.
Published: October 2006

Abstract: Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22–25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population.
Synaptic Concentration of Dopamine in the Mouse Striatum in Relationship to the Kinetic Properties of the Dopamine Receptors and Uptake Mechanism
Authors: Ross Svante B.
Published: October 2006

Abstract: The concentration of dopamine (DA) in the synaptic cleft in the mouse striatum in vivo was estimated from the competition between the synaptic DA and the 3H‐labelled DA D2 receptor agonists N‐n‐propylnorapomorphine (NPA) or N,N‐diethyl‐N′‐[(3α,4aα,10β)‐1,2,3,4,4a,5,10,10a‐octahydro‐7‐hydroxyl‐1‐propyl‐3‐benzo (g) quinolinyl]sulfamide (Sandoz 205–501) injected intravenously in tracer doses. Knowing the inhibitor constant for DA in inhibiting the binding of these receptor agonists in vitro, attempts were made to calculate the changes in the synaptic DA concentration from the changes in the in vivo binding of the receptor agonists evoked by various pharmacological agents. Inhibiting the firing of the dopaminergic neurons by γ‐butyrolactone (GBL) increased the binding of the receptor agonists corresponding to a decrease in the synaptic DA concentration of 55 ± 2 nM in the experiments with [3H]Sandoz 205–501 and 48 ± 3 nM in the experiments with tracer doses of [3H]NPA. These values may therefore approximate the normal DA concentration in the synaptic cleft in the mouse striatum. With this technique it was also possible to determine the synaptic concentration of NPA by its competition with [3H]Sandoz 205–501 for the DA D2 receptors in the striatum of GBL‐treated mice in vivo. To compare the estimated synaptic concentration of DA with the affinity of DA to D1 and D2 receptors and to the DA transporter in the mouse striatum the kinetic parameters were determined at 37°C in vitro. The Ki value for DA in inhibiting the binding of ligands to the agonist sites of the D1 receptors (30–40 nM) was about one‐third of that of the D2 receptors (80–120 nM). The Km value of the initial uptake of [3H]DA into synaptosomes from the mouse striatum was 209 ± 20 nM (n = 3) and the Vmax was 7.00 ± 1.29 nmol/g tissue/min. Comparison of the affinities of DA for the D1 and D2 receptors with the synaptic DA concentrations shows that a considerable proportion of the receptors is occupied by DA under these conditions, whereas the uptake sites are occupied to a lesser degree.
Brain Histamine in Rats with Hepatic Encephalopathy
Authors: Fogel W. Agnieszka , Andrzejewski Wojciech , Maslinski Czeslaw
Published: October 2006

Abstract: Chronic liver failure induced by portocaval anastomosis (PCA) in Wistar rats resulted in a dramatic increase in histamine concentration in hypothalamus and a smaller, but clearly pronounced, elevation in the rest of brain. Between 10 and 120 days following surgery, shunted rats exhibited a histamine level 2.4‐ to 13‐fold higher in hypothalamus and 1.5‐ to 2.5‐fold higher in the rest of brain as compared to their control, sham‐operated pairs. There were no significant changes in histamine concentration in the other examined tissues. The increase in brain histamine could not be attributed to the inhibition of its degradation, because activity of histamine N‐methyltransferase remained unchanged for at least 40 days. Although the activity of histidine decarboxylase also remained unchanged when measured at a saturating concentration of L‐histidine, the increase in histamine content in brain seems to be due to its enhanced synthesis brought about by increased availability of L‐histidine in the tissue, as indicated by two to four times higher concentrations of this amino acid in PCA rats.
Glutamatergic Control of Dopamine Release in the Rat Striatum: Evidence for Presynaptic N‐Methyl‐D‐Aspartate Receptors on Dopaminergic Nerve Terminals
Authors: Krebs M. O. , Desce J. M. , Kemel M. L. , Gauchy C. , Godeheu G. , Cheramy A. , Glowinski J.
Published: October 2006

Abstract: The N‐methyl‐D‐aspartate (NMDA) receptor‐mediated regulation of the release of newly synthesized [3H]dopamine ([3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2+‐free medium conditions, the NMDA (5 × 10−5M)‐evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX‐resistant stimulatory effect of NMDA was blocked by either Mg2+ (10−3M) or the noncompetitive antagonist MK‐801 (10−6M). In addition, the TTX‐resistant NMDA‐evoked response could be potentiated by glycine (10−6M) in the presence of strychnine (10−6M). The coapplication of NMDA (5 × 10−5M) and glycine (10−6M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10−3M) or MK‐801 (10−5M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electro‐physiological studies for postsynaptic NMDA receptors.
Nerve Growth Factor Mediates Monosialoganglioside‐Induced Release of Fibronectin and J1/Tenascin from C6 Glioma Cells
Authors: Yavin Ephraim , Gabai Avi , Gil Shosh
Published: October 2006

Abstract: C6 rat glioma cells incubated in serum‐free medium with D‐[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270‐, 220‐, and 69‐kDa Gp. Several growth factors, hormones, phorbol ester, and disialo‐ and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of ∼220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF‐stimulated release and also blocked MSG‐evoked release. The 220‐kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15‐min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220‐kDa Gp labeled by D‐[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF‐like molecule also secreted by the C6 glioma cells.
Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain
Authors: Kaul Rajinder , Casanova Jose , Johnson Anne B. , Tang Peter , Matalon Reuben
Published: October 2006

Abstract: Canavan disease, an autosomal recessive disorder, is characterized biochemically by N‐acetylaspartic aciduria and aspartoacylase (N‐acyl‐L‐aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N‐acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of ∼ 19,000–20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58‐kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or β‐mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 × 10−4M and a Vmax of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl‐L‐aspartate and amino derivatives of D‐aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N‐acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N‐acetylaspartic acid in normal brain biology.
Mutations in the Myelin Proteolipid Protein Gene Alter Oligodendrocyte Gene Expression in Jimpy and Jimpymsd Mice
Authors: Macklin Wendy B. , Gardinier Minnetta V. , Obeso Zaida O. , King Kit D. , Wight Patricia A.
Published: October 2006

Abstract: The mouse myelin proteolipid protein (PLP) gene has been studied in normal and jimpymsd mice. Potential upstream regulatory regions of the normal gene have been cloned and mapped, but when these regions were studied in jimpymsd mice by Southern blots, no alterations were observed, relative to the normal gene. To assess whether the low ratio of PLP to DM20 proteins in this mutant reflected an altered PLP/DM20 ratio mRNAs, S1 nuclease analyses were undertaken, which demonstrated that at all ages studied in both jimpy and jimpymsd mice, PLP mRNA was elevated above DM20 mRNA. When exon 3 (the site of the alternative splice signal for DM20 mRNA) of the jimpymsd PLP gene was sequenced, no mutation was identified. The transcription of the PLP gene in normal and mutant animals was studied. The transcription rate increases in normal animals with development, and in very young jimpymsd or jimpy mice, the transcription rate of the PLP gene was close to that of agematched normal animals. However, by 10 days of age, the transcription rate of this gene in both mutants was significantly below that of age‐matched controls. The transcription rate of the myelin basic protein (MBP) gene was also reduced, indicating that expression of both genes is affected by this mutation. In contrast, the transcription rate of the glycerol phosphate dehydrogenase (GPDH) gene, an early marker of oligodendrocytes, is equal to or greater than normal in both mutants. We have confirmed an earlier report of a point mutation in exon 6 of the jimpymsd PLP gene, which converts an alanine to a valine. This mutation apparently alters oligodendrocyte metabolism such that the cell can differentiate to express early oligodendrocyte genes such as GPDH, but it cannot differentiate to a stage where it expresses the PLP and MBP genes at normal high levels.
Electrical Stimulation of the Substantia Nigra and Changes of 2‐Phenylethylamine Synthesis in the Rat Striatum
Authors: Juorio A. V. , Paterson I. A. , Zhu M. Y. , Matte G.
Published: October 2006

Abstract: In rats pretreated with deprenyl (2 mg/kg), electrical stimulation of the left substantia nigra produced an increase in the concentrations of 3,4‐dihydroxyphenylacetic acid and homovanillic acid in the left striatum by 57 and 45%, but the levels of 2‐phenylethylamine and p‐tyramine decreased by 22 and 41%, respectively, as compared with those in the right striatum. The administration of α‐methyl‐p‐tyrosine (1.25 mg/kg, i.p.), a tyrosine hydroxylase inhibitor, 1 h before nigral stimulation, did not affect the concentration of 2‐phenylethylamine in unstimulated striata but prevented the stimulation‐induced decrease in the concentration of 2‐phenylethylamine. Neither stimulation nor α‐methyl‐p‐tyrosine affected the activity of monoamine oxidase A or B, and stimulation did not produce any change in striatal blood flow, a finding demonstrating that the changes in the rate of accumulation of 2‐phenylethylamine were not due to changes in catabolism or removal of 2‐phenylethylamine from the brain. These experiments demonstrate that the rate of synthesis of striatal 2‐phenylethylamine is decreased following nigral stimulation and that this effect is blocked after partial inhibition of tyrosine hydroxylase. This suggests that 2‐phenylethylamine is present in tyrosine hydroxylase‐containing neurons and therefore supports the coexistence of 2‐phenylethylamine and dopamine in the nigrostriatal pathway.
The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5‐Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes
Authors: Harel‐Dupas Catherine , Cloëz Isabelle , Fillion Gilles
Published: October 2006

Abstract: The effect of the serotonergic receptor agonist 1‐(m‐trifluoromethylphenyl)piperazine (TFMPP) was studied on the K+‐evoked [3H]acetylcholine ([3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5–1,000 μM) inhibited the evoked ACh release in a dose‐dependent manner (IC50= 81.8 μM). The inhibitory effect of TFMPP was mimicked by CGS‐12066B (10, 30, and 100 μM), a 5‐hydroxytryptamine1B (5‐HT1B)/5‐HT1D receptor agonist; 1‐(m‐chlorophenyl)piperazine (100 μM), a 5‐HT1C/5‐HT1B receptor agonist; and 5‐carboxamidotryptamine (10 μM), a nonselective 5‐HT1 receptor agonist. 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin (10 and 100 μM), a 5‐HT1A receptor agonist, and quipazine (10 and 100 μM), a 5‐HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 μM), metergoline (0.1 μM), methysergide (0.5 and 1 μM), or yohimbine (1 and 10 μM), blocked the TFMPP effect dose‐dependently. In contrast, methiotepine (0.3 and 1 μM), propranolol (1 μM), ketanserin (0.1 μM), mesulergine (0.1 μM), ICS 205930 (0.1 and 1 μM), and spiroperidol (1 and 7 μM) did not affect the TFMPP‐induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K+‐evoked ACh release is modulated by a 5‐HT1 receptor distinct from the 5‐HT1A, 5‐HT1B, and 5‐HT1C subtypes.
Effect of Precursor Loading on the Synthesis Rate and Release of Dopamine and Serotonin in the Striatum: A Microdialysis Study in Conscious Rats
Authors: Westerink Ben H. C. , De Vries Jan B.
Published: October 2006

Abstract: The effects of systemic administration of tyrosine and phenylalanine on the extracellular levels of tyrosine and dopamine were determined by microdialysis in the striatum of awake rats. In addition, the effects of these precursors on in vivo 3,4‐dihydroxyphenylalanine (DOPA) formation were determined during continuous infusion of a decarboxylase inhibitor. Both precursors increased the dialysate levels of tyrosine sixfold, but only phenylalanine administration stimulated DOPA formation. However, neither precursor affected the release of dopamine. When the precursor administration was repeated in rats in which the release of dopamine was stimulated by haloperidol pretreatment, again no effect was seen on the release of dopamine. Systemic administration of tryptophan (100 mg/kg, i.p.) during continuous infusion of a decarboxylase inhibitor induced a threefold increase in the formation of 5‐hydroxytryptophan and caused an increase in the release of serotonin during infusion of an uptake inhibitor to about 150% of controls. Finally, we investigated whether dietary precursors were able to influence neurotransmitter formation and release. Rats trained to consume their daily food in a period of 2 h were implanted with microdialysis probes. Scheduled eating induced a small increase in the extracellular levels of tyrosine (135% of controls), but the release of dopamine and the formation of 5‐hydroxytryptophan during continuous infusion of a decarboxylase inhibitor were not affected.
Enzyme Immunoassay for Tachykinin‐Like Immunoreactivity in the Guinea Pig Spinal Cord
Authors: Renzi Daniela , Maillet Sylvie , Grassi Jacques , Mantellini Paola , Santicioli Paolo , Theodorsson Elvar , Surrenti Calogero , Geppetti Pierangelo
Published: October 2006

Abstract: A solid‐phase enzyme immunoassay for quantitation of tachykinin‐like immunoreactivity (TK‐LI) is presented. Because the antiserum K‐12 recognizes various tachykinins, such as neurokinin A (100%), kassinin (103%), eledoisin (51%), neurokinin B (18%), physalaemin (0.7%), and substance P (0.7%), the immunoreactivity detected in this enzyme immunoassay has been termed TK‐LI. The assay was performed on 96‐well microtiter plates coated with a mouse monoclonal second antibody. After preincubation of soluble neurokinin A or samples and K‐12 antiserum for 3 h at room temperature, acetylcholinesterase‐labelled neurokinin A was allowed to react overnight at 4°C. Samples were finally incubated with Ellman's reagent for 2 h and the absorbance was measured at 414 nm. The threshold for detection of TK‐LI was 2 fmol/well. TK‐LI release from guinea pig dorsal spinal cord slices was evoked by capsaicin or high K+ medium. The capsaicin‐evoked TK‐LI release was increased in the presence of thiorphan, but not in that of captopril.
Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin‐Dependent Protein Kinase II
Authors: Yanagihara Nobuyuki , Tachikawa Eiichi , Izumi Futoshi , Yasugawa Setsuko , Yamamoto Hideyuki , Miyamoto Eishichi
Published: October 2006

Abstract: We investigated the effect of staurosporine on Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10–100 nM) inhibited the activity of CaM kinase II. The half‐maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (β‐casein). (c) Staurosporine suppressed the autophosphorylation of α‐ and β‐subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+ calmodulin‐independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP‐binding site or substrate‐binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.
Chronic Electroconvulsive Treatment Augments Coupling of the GTP‐Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs
Authors: Ozawa Hiroki , Rasenick Mark M.
Published: October 2006

Abstract: A significant increase of guanylylimidodiphosphate (GppNHp)‐, fluoride‐, and forskolin‐stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3‐(4‐azidoanilido)‐P1‐5′‐GTP ([32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs‐mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.
Presynaptic Mechanism of Action of 4‐Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B‐50 (GAP‐43) Phosphorylation
Authors: Heemskerk F. M. J. , Schrama L. H. , Ghijsen W. E. J. M. , De Graan P. N. E. , Lopes da Silva F. H. , Gispen W. H.
Published: October 2006

Abstract: Recently we have shown that 4‐aminopyridine (4‐AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B‐50 (GAP‐43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca2+. Hence, we were interested in the relationship between changes induced by 4‐AP in the intracellular free Ca2+ concentration ([Ca2+]i) and B‐50 phosphorylation in synaptosomes. 4‐AP (100 μM) elevates the [Ca2+]i (as determined with fura‐2) to approximately the same extent as depolarization with 30 mM K+ (from an initial resting level of 240 nM to ∼480 nM after treatment). However, the underlying mechanisms appear to be different: In the presence of 4‐AP, depolarization with K+ still evoked an increase in [Ca2+]i, which was additive to the elevation caused by 4‐AP. Several Ca2+ channel antagonists (CdCl2, LaCl3, and diphenylhydantoin) inhibited the increase in B‐50 phosphorylation by 4‐AP. It is interesting that the increase in [Ca2+]i and the increase in B‐50 phosphorylation by 4‐AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na+ channels in this action. These results suggest that 4‐AP (indirectly) stimulates both Ca2+ influx and B‐50 phosphorylation through voltage‐dependent channels by a mechanism dependent on Na+ channel activity.
Effect of Monomethylethanolamine, Dimethylethanolamine, Gangliosides, Isoproterenol, and 2‐Hydroxyethylhydrazine on the Conversion of Ethanolamine to Methylated Products by Cultured Chick Brain Neurons
Authors: Andriamampandry C. , Freysz L. , Kanfer J. N. , Dreyfus H. , Massarelli R.
Published: October 2006

Abstract: The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline‐containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water‐soluble ethanolamine‐containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10−8M and 10−5M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2‐hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10−6M and 10−5M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 × 10−5M propranolol.
Neurite‐Promoting Activities of Phosphatidylinositol and Other Lipids on Fetal Rat Septal Neurons in Culture
Authors: Arakawa Yoshihiro , Isahara Kyoko , Tachibana Shinro
Published: October 2006

Abstract: Neurite‐promoting activities of lipids were assessed using serum‐free cultures of fetal rat septal neurons. The most potent one was phosphatidylinositol (PI), followed by PI 4‐phosphate, phosphatidylserine, sphingomyelin, and phosphatidylcholine. The EC50 value for PI was 1.5 μg/ml (1.8 μM), and activity was maximal at 4 μg/ml (56% of total cells had neurites after 24 h). Cerebroside, sulfatide, and di‐ and triacylglycerols showed relatively low activities. Synthetic dipalmitoyl phosphatidylcholine was also active, with a maximal activity (47%) at 100 μg/ml, a finding implying that the unsaturated fatty acid moiety is not released and further used as substrate for the arachidonic acid cascade. Lysophospholipids, phosphatidylglycerol, and cardiolipin were rather cytotoxic and lacked activity, an observation suggesting that membrane perturbation is not responsible for the neuritepromoting activity. Treatment with a protein kinase C inhibitor, H‐7, or an Na+,K+‐ATPase inhibitor, ouabain, inhibited the PI‐induced neurite outgrowth, but the cyclic AMP‐ and cyclic GMP‐dependent protein kinase inhibitor HA1004 did not inhibit this activity, a result indicating that multiple elements (protein kinase C and Na+,K+‐ATPase) are involved in the induction of neurites. Because phospholipids can be provided either as lipid vesicles or as lipoproteins produced by macrophages at regeneration sites, they may play an important role in the regeneration of certain populations of neurons.
Localization of 2‐[125I]Iodomelatonin Binding Sites in Mammalian Retina
Authors: Blazynski Christine , Dubocovich Margarita L.
Published: October 2006

Abstract: Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2‐[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2‐[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2‐[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40–60% of the binding was specific, as determined with both the agonist 6‐chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2‐[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2‐[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2‐[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.
Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum
Authors: Madden John , Evans Christopher J. , Tyler Andrew N. , Esch Frederick S. , Böhlen Peter , Makk George , Weber Eckard
Published: October 2006

Abstract: The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both γ‐ and k‐subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino‐terminal Tyr‐Gly‐Gly‐Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl‐Arg6‐Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl‐Arg6‐Arg7‐Val8‐NH2 (metorphamide) or [Met5]enkephalyl‐Arg6‐Gly7‐Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.
Purification of the γ‐Aminobutyric AcidA/Benzodiazepine Receptor Complex by Immunoaffinity Chromatography
Authors: Park Dongeun , Vitorica Javier , Tous Guillermo , de Blas Angel L.
Published: October 2006

Abstract: The bovine γ‐aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62‐3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7‐1986/1. High‐resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of the immunoaffinity‐purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7‐1986/1 affinity‐purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS‐PAGE.
Postmortem Changes in Rat Brain Extracellular Opioid Peptides Revealed by Microdialysis
Authors: Maidment Nigel T. , Siddall Brenda , Rudolph Veronica D. , Evans Christopher J.
Published: October 2006

Abstract: Microdialysis combined with a solid‐phase radioimmunoassay was used to monitor changes in extracellular opioid peptide levels in the rat globus pallidus/ventral pallidum as a result of terminal brain ischemia. Ischemia was induced by anesthetic overdose or by severance of blood vessels supplying the brain. In control animals the recovered immunoreactivity increased an average of 13‐fold in the 30‐min sample following anesthetic overdose. Perfusion of a calcium‐free, 10 mM EGTA‐containing medium through the dialysis probe significantly attenuated the amplitude of this response, with the average increase being only threefold. Shorter sampling intervals (5 min) indicated that release of opioid peptide material into the extracellular environment occurs within the first 5 min of ischemia resulting from severance of the blood supply to the brain. HPLC analysis identified the majority of the postmortem‐induced immunoreactive material as Met‐ and Leu‐enkephalin.
L‐Phosphoserine, a Metabolite Elevated in Alzheimer's Disease, Interacts with Specific L‐Glutamate Receptor Subtypes
Authors: Klunk William E. , McClure Richard J. , Pettegrew Jay W.
Published: October 2006

Abstract: L‐Phosphoserine is one of the phosphomonoesters elevated in Alzheimer's disease brain and has close structural similarity to L‐glutamate. This study attempts to define precisely the actions of L‐phosphoserine at L‐glutamate receptor subtypes. L‐Phosphoserine is shown to bind to N‐methyl‐D‐aspartate and kainic acid receptor subtypes, but not to the quisqualic acid subtype. Studies of [3H]MK‐801 binding in the presence and absence of L‐glutamate and glycine show L‐phosphoserine to be a competitive N‐methyl‐D‐aspartate antagonist. The IC50 of L‐phosphoserine in these studies varies from 373 to 721 μM. This may indicate a physiologically relevant action of L‐phosphoserine in Alzheimer's disease brain because whole brain concentrations may reach over 1 mM.
G Proteins Coupled to Phospholipase C: Molecular Targets of Long‐Term Ethanol Exposure
Authors: Simonsson Per , Rodriguez F. David , Loman Niklas , Alling Christer
Published: October 2006

Abstract: Long‐term ethanol exposure is known to inhibit bradykinin‐stimulated phosphoinositide hydrolysis in cultures of neuroblastoma × glioma 108–15 cells. In the present study, [3H]bradykinin binding, GTP‐binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5′‐O‐(3‐thiotriphosphate) [GTP(S)]‐ and, to a lesser extent, NaF/AlCl3‐stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5‐bisphosphate‐specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol‐containing medium. The results indicate that long‐term ethanol exposure exerts its effects on receptor‐stimulated phosphoinositide hydrolysis primarily at the level of the GTP‐binding protein.
β‐Adrenergic Effects on Plasma and Brain Large Neutral Amino Acids Are Unaltered by Chronic Administration of Antidepressants
Authors: Paetsch Paul R. , Greenshaw Andrew J.
Published: October 2006

Abstract: Effects of isoproterenol (3 mg kg−1, i.p. for 60 min) and salbutamol (3, 10 mg kg−1, i.p. for 60 min) on large neutral amino acid concentrations in rat plasma and brain were assessed. Phenylalanine, leucine, isoleucine, and valine were measured by gas chromatography with electron‐capture detection; tyrosine and tryptophan were measured by HPLC with electrochemical detection. These drugs induced increases in brain tryptophan, tyrosine, phenylalanine, and valine and decreases in plasma tryptophan, tyrosine, leucine, isoleucine, and valine. Effects of salbutamol (3 mg kg−1, i.p. for 60 min) were assessed following chronic administration of phenelzine sulfate and desipramine · HCl (each drug 10 mg kg−1 per day, s.c. via Alzet 2ML4 osmotic minipumps for 28 days). There were no effects of these antidepressants on basal levels of large neutral amino acids in brain and plasma. In both brain and plasma, salbutamol‐induced changes in large neutral amino acids were unaffected by these antidepressants. The results indicate that β‐adrenoceptor‐regulated availability of plasma and brain large neutral amino acids is unaffected by chronic administration of tricyclic or monoamine oxidase inhibitor antidepressants.
Conditions Restricting Depolarization‐Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65
Authors: Gómez‐Puertas Paulino , Martínez‐Serrano Alberto , Blanco Pablo , Satrústegui Jorgina , Bogónez Elena
Published: October 2006

Abstract: Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High‐K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K+‐stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na+‐based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 μM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.
Membrane Lipids in the Aging Human Brain
Authors: Svennerholm L. , Boström K. , Helander C. G. , Jungbjer B.
Published: October 2006

Abstract: The membrane lipid composition of human brain has been studied in 21 men and 18 women 60–97 years of age. This brain tissue series is unique, because it has been obtained only from individuals who lived a normal social life in their own homes and died suddenly and unexpectedly from arteriosclerotic heart disease or ruptured aortic aneurysms. They had no history of neurological or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The percentage of solids diminished continuously during the whole period, but the marked individual differences suggested large variations in the hydration of the brain. The content of membrane lipids also diminished continuously up to 90 years of age, when a marked diminution in level of gangliosides and cerebrosides occurred, a result indicating a rapid reduction in amount of neuronal membranes and myelin. The clinical implications of the variation in brain hydration and the rapid loss of membrane lipids after 90 years of age are discussed.
Cholesterol from Degenerating Nerve Myelin Becomes Associated with Lipoproteins Containing Apolipoprotein E
Authors: Goodrum Jeffry F.
Published: October 2006

Abstract: Apolipoprotein E is synthesized and secreted by rat sciatic nerve consequent to several types of injury. It has been proposed that endoneurial apolipoprotein E, in analogy to its role in systemic cholesterol transport, is involved in the salvage and reutilization of myelin cholesterol during degeneration and regeneration. To test this hypothesis, nerve lipids were prelabeled via intraneural injection of [3H]acetate. Four weeks later the nerves were crushed. From 1 to 12 weeks later, crushed nerves were examined for extracellular lipoprotein‐bound cholesterol label. By 2 weeks after injury, 10% of the endoneurial lipid label was in a soluble form that was releasable into incubation medium. This released fraction was enriched in labeled cholesterol, and its labeled lipid composition was constant, in contrast to the changing distribution of label in the nerve with time after injury. On a KBr gradient, the released lipid label cofractionated with the released apolipoprotein E at densities similar to that of lipoproteins. These data indicate that at least some myelin cholesterol in injured nerve becomes associated with apolipoprotein E‐containing lipoproteins and thus is available for reutilization via the hypothesized model.
Receptor‐Coupled Phosphoinositide Hydrolysis in Human Retinal Pigment Epithelium
Authors: Feldman Eva L. , Randolph Ann E. , Johnston Gregory C. , DelMonte Monte A. , Greene Douglas A.
Published: October 2006

Abstract: Carbachol and histamine stimulated phosphoinositide (PPI) hydrolysis in cultured human retinal pigment epithelium (RPE), as reflected by an accumulation of 3H‐inositol phosphates in the presence of 10 mM Li+. Carbachol increased PPI hydrolysis to greater than 600% of basal with an EC50 of 60 μM; stimulation was linear up to 60 min. This activation likely occurred via the M3 muscarinic cholinergic receptor based on the IC50 values for 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (0.47 nM), pirenzepine (280 nM), and 11‐[[2‐[(diethylamino)methyl]‐1‐piperidinyl]‐acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one (1.4 μM). Carbachol‐mediated PPI hydrolysis was decreased by 80% in the absence of extracellular Ca2+. Histamine stimulated PPI turnover in a linear manner by 180% with an EC50 of 20 μM by the H1 histaminergic receptor. Serotonin, glutamate, norepinephrine, and dopamine were inactive. In human RPE, the resting cytoplasmic Ca2+ concentration, as determined by fura‐2 fluorescence, was 138 ± 24 nM. On the addition of carbachol, there was a 180% increase in peak intracellular Ca2+; addition of histamine increased intracellular Ca2+ by 187%. These results suggest receptor‐mediated, inositol lipid hydrolysis is coupled to intracellular Ca2+ flux in human RPE.
Pretreatment of Cerebellar Granule Cells with Concanavalin A Potentiates Quisqualate‐Stimulated Phosphoinositide Hydrolysis
Authors: Raulli R. , Danysz W. , Wroblewski J. T.
Published: October 2006

Abstract: The hydrolysis of phosphoinositides (PI) elicited in cerebellar granule cell cultures by agonists of metabolotropic glutamate receptors, glutamate and quisqualate, was enhanced when the cells were pretreated with concanavalin A (Con‐A). A similar effect was produced by wheat germ agglutinin, but not by several other lectins tested. Con‐A produced a dose‐dependent effect (EC50= 3 μM) and increased the efficacy but not the potency of the agonists. In contrast, Con‐A failed to enhance PI hydrolysis evoked by N‐methyl‐D‐aspartate, kainate, carbachol, the calcium ionophore A23187, or 50 mM K+. The Con‐A stimulatory effect was prevented by simultaneous pretreatment with the agonists of ionotropic quisqualate receptors quisqualate, kainate, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, but not by the antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). CNQX, which did not inhibit quisqualate‐stimulated PI hydrolysis in untreated cells, abolished the component of quisqualate response enhanced by Con‐A pretreatment. The pretreatment with Con‐A also increased the influx of 45Ca2+ in granule cells stimulated by quisqualate. This increase was inhibited by CNQX. Moreover, the potentiation of PI hydrolysis by Con‐A, but not the response to quisqualate alone, was abolished in the absence of Ca2+ and Na+. Pretreatment of granule cells with pertussis toxin inhibited PI hydrolysis stimulated by the metabolotropic quisqualate receptor and the Con‐A‐potentiated response by the same percentage, but Ca2+ influx induced by quisqualate was not affected. Hence, Con‐A appears to enhance PI hydrolysis by acting at the ionotropic quisqualate receptor which results in an enhanced Ca2+ influx and a subsequent activation of phospholipase C through a pertussis toxin‐sensitive G protein. This may indicate the existence of a functional cooperativity between the metabolotropic and ionotropic quisqualate receptors.
Endothelial Cells from Human Fetal Brain Microvessels May Be Cholinoceptive, but Do Not Synthesize Acetylcholine
Authors: Kasa P. , Pakaski M. , Joó F. , Lajtha A.
Published: October 2006

Abstract: Brain homogenate, cerebral microvessels, and endothelial cells (ECs) were prepared from 15–18‐week‐old human fetuses and analyzed biochemically for the presence of elements of the cholinergic system [acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and butyrylcholinesterase]. The ECs were cultured, and their purity was checked by light microscopic immunohistochemistry with the application of anti‐human factor VIII and glial fibrillary acidic protein. The highest activity of ChAT was found in the brain homogenate and the lowest in the microvessel fraction. No ChAT activity could be detected in the cultured ECs, despite the presence of high AChE activity. It is suggested that human brain ECs may be under the control of acetylcholine released from cholinergic nerve terminals but that the cells do not produce the transmitter itself. In coculture experiments, when ECs were plated on the upper surface of a polycarbonate filter and glial cells were seeded on the lower surface, the electric resistance was measured. During the culture period, the resistance first increased up to 5 days in vitro (297 ± 17 ohm·cm2) but later gradually declined. These results demonstrate that human ECs cocultured with glial cells provide a useful model for study of the function of the blood‐brain barrier in vitro.
Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation
Authors: Klein Jochen , Köppen Andrea , Löffelholz Konrad
Published: October 2006

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high‐choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (>96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet‐induced changes of the net uptake and release of choline.