bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment
Authors: Kása P. , Dames RW. , Rakonczay Z. , Gulya K. , Joó F. , Wolff RJ. R.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long‐lasting GABA microinfusion into the SCG in vivo apparently resulted in a “functional denervation.”This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p < 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl‐coenzyme A:choline‐O‐acetyl‐transferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The “hyperin‐nervation”of the ganglia led to increases in the AChE activity (to 142.5%, p < 0.01) and the 16S molecular form (to 200%, p < 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.
Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters
Authors: Poli A. , Contestabile A. , Migani P. , Rossi L. , Rondelli C. , Virgili M. , Bissoli R. , Barnabei O.
Published: October 2006

Abstract: Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome‐enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+‐dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40–80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D‐[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+‐stimulated release. Kainic acid at 0.1 mM had no clear‐cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier‐mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high‐affinity uptake of L‐glutamate and D‐aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.
Evidence for [D‐Ala2,D‐Leu5]Enkephalin‐Induced Supersensitivity to 5‐Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB‐20)
Authors: Berry‐Kravis Elizabeth , Dawson Glyn
Published: October 2006

Abstract: A neuroblastoma × Chinese hamster embryonic brain explant hybrid cell line (NCB‐20) expressed 5‐hydroxytryptamine (5‐HT1) receptors, linked to adenylate cyclase, which closely resembled 5‐HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5‐HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB‐20 cells produced by 5‐HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5‐HT. The elevation of cyclic AMP levels by either 5‐HT or PGE1 was reversed by [D‐Ala2,D‐Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4‐dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5‐HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5‐HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5‐HT resulted from an increase in receptor affinity for 5‐HT (from a KD of 150 ± 20 nM to one of 20 ± 7 nM), with a reduction in the number of apparent binding sites. The 5‐HT supersensitivity observed in NCB‐20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.
Distinct Subtypes of the Opioid Receptor with Allosteric Interactions in Brain Membranes
Authors: Demoliou‐Mason Catherine D. , Barnard Eric A.
Published: October 2006

Abstract: The binding isotherms of opioid receptors in rat brain membranes with [3H]D‐Ala2‐D‐Leu5‐enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of δ, K, μ1, and μ2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine‐5′‐(β, γ‐imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of “half‐of‐the‐sites” reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous K–δ or μ1–μ2 receptor complexes is required for stabilization of the high‐affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association‐dissociation were observed when these studies were conducted, not in the Tris‐HCl buffer commonly used in opioid binding assays, but in N‐tris[hydroxymethyl]‐methyl‐2‐aminoethanesulphonate (K+) buffer (TES‐KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of –SH/‐SS– bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association‐dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.
Cyclic Nucleotide Phosphodiesterase Activity in Bovine Brain Coated Vesicles
Authors: Silva Walter I. , Schook William , Mittag Thomas W. , Puszkin Saul
Published: October 2006

Abstract: Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 μM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat‐inactivated, formaldehyde‐fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP‐sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1‐methyl‐3‐isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 μM. These compounds also inhibited the cyclic GMP‐stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 μM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.
Ca2+/Diacylglycerol‐Activated, Phospholipid‐Dependent Protein Kinase in the Hermissenda CNS
Authors: Neary J. T. , Naito S. , De Weer A. , Alkon D. L.
Published: October 2006

Abstract In mammalian systems, Ca2+/diacylglycerol‐activated phospholipid‐dependent protein kinase (C‐kinase) appears to play an important role in regulating physiological responses that outlast the transient rise in cytosolic Ca2+. Electrophysiological experiments in neurons of the nudibranch mollusc, Hermissenda crassicornis, have suggested a role for C‐kinase in the long‐lasting reductions in early and late K+ currents that have been observed following associative learning. Accordingly, we have investigated the catalytic properties of C‐kinase in Hermissenda CNS. Following homogenization in Ca2+‐free buffer, C‐kinase can be separated from Ca2+/calmodulin‐dependent protein kinase by centrifugation; C‐kinase activity is found in the supernatant whereas essentially all of the Ca2+/calmodulin‐dependent protein kinase is found in the membrane fraction. Addition of Ca2+, phosphatidylserine, and diacylglycerol to the cytosol results in phosphorylation of at least eight endogenous proteins. The Hermissenda CNS C‐kinase can also phosphorylate lysine‐rich histone, a substrate for mammalian C‐kinase. The molluscan enzyme exhibits phospholipid specificity in that phosphatidylserine is much more effective than phosphatidylethanolamine, phosphatidylcholine, phospha‐tidylinositol, and phosphatidic acid. Addition of diacylglycerol, in the presence of Ca2+ and phosphatidylserine, increases the activity of the C‐kinase. The percentage of activation by diacylglycerol is larger at lower Ca2+ concentrations. Enzyme activity is inhibited by trifluoperazine and polymixin B sulfate. These studies indicate that the Hermissenda C‐kinase is catalytically similar to mammalian C‐kinase.
Phorbol Esters Enhance Neurotransmitter‐Stimulated Cyclic AMP Production in Rat Brain Slices
Authors: Karbon E. W. , Shenolikar S. , Enna S. J.
Published: October 2006

Abstract The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12‐myristate, 13‐acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15‐min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50= 1 μM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.
Thyroid Hormone Actions on a Cholinergic Neuroblastoma Cell Line (S‐20Y)
Authors: Chacon Marco , Max Stephen R. , Kirshner Judith A. , Tildon J. T.
Published: October 2006

Abstract Thyroid hormone (T3) has a multiplicity of effects on the developing nervous system. We have investigated T3 action using a cholinergic neuroblastoma cell line (S‐20Y) as a model. S‐20Y contains a nuclear receptor for T3 with binding properties similar to those of other T3 target tissues. In addition, these cells can carry out 5′‐deiodination, which is necessary to produce active thyroid hormone in vivo. The enzyme involved in this process appears to be a type I deiodinase, based on its reaction kinetics and its susceptibility to inhibition by propylthiouracil. S‐20Y cells maintained in T3‐depleted medium showed decreased choline acetyltransferase (ChAT) activity. ChAT activity was restored to the control level in a dose‐dependent manner by T3 repletion. Neither cell density nor viability was influenced by the hypothyroid state. The presence of a T3 receptor and the enzyme activity for T3 production, together with an effect of T3 on ChAT activity, demonstrate that S‐20Y cells are a target for T3 action and suggest that these cells represent an excellent model system for studies of T3 effects on nervous tissues.
Direct Demonstration of the Activation of UDP‐N‐Acetylgalactosamine: [GM3]N‐Acetylgalactosaminyltransferase by Cyclic AMP
Authors: Scheideler Mark A. , Dawson Glyn
Published: October 2006

Abstract: Treatment of NG 108–15 cells in culture with the opiate peptide [D‐Ala2,D‐Leu5]enkephalin produces maximal inhibition of cyclic AMP synthesis in < 15 min. The activity of [GM3]:N‐acetylgalactosaminyltransferase is similarly inhibited, but maximal inhibition is not observed for at least 30 min following the addition of [D‐Ala2,D‐Leu5]enkephalin. Conversely, the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine rapidly potentiates the intracellular accumulation of cyclic AMP and, in a more gradual fashion, increases [GM3]:N‐acetyl‐galactosaminyltransferase activity. The reductions in the activity of [GM3]:N‐acetylgalactosaminyltransferase that occur following treatment of NG108‐15 cells with indomethacin argues for a direct role of cyclic AMP in the observed changed in [GM3]:N‐acetylgalactosaminyltransferase activity. By adding low concentrations of cyclic AMP (but not cyclic GMP) to microsomes derived from neonatal rat brain, we were able to demonstrate a dose‐dependent phosphorylation of membrane protein and subsequent doubling of [GM3]:N‐acetylgalactosaminyl‐transferase activity.
Distribution of Dolichol and Dolichyl Phosphate in Human Brain
Authors: Andersson M. , Appelkvist E. L. , Kristensson K. , Dallner G.
Published: October 2006

Abstract: Autopsy material from deceased individuals between ages 2 and 90 was used to prepare cerebellum, pons, and other selected regions of the brain, the spinal cord, and peripheral nerves. The concentration of dolichol in these different tissues varied greatly and the increase in concentration during the life span varied between 2.5‐ and 21‐fold. In contrast, dolichyl‐phosphate (dolichyl‐P) was more evenly distributed in these tissues and its concentration increased to a moderate extent only during childhood. The level of cholesterol displayed smaller regional differences and decreased about 15% between ages 35 and 90. Differences in the total phospholipid content were limited. These results demonstrate enrichment and individual regulation of various lipids in specialized regions of the human brain. The independent regulation of dolichol and dolichyl‐P levels in the brain and the possible role of dolichol in the function of the aging nerve cell are also emphasized.
Synaptic Vesicle Proteins and Acetylcholine Levels in Chick Forebrain Nuclei Are Altered by Passive Avoidance Training
Authors: Bullock Sarah , Csillag Andras , Rose Steven P. R.
Published: October 2006

Abstract: In a search for biochemical markers of modified synaptic function following training of day‐old chicks on a passive avoidance task, we have assayed two monoclonal antibodies to synaptic vesicle proteins (anti‐p65 and anti‐SV2) and one raised to postsynaptic densities (411B). We have also measured total acetylcholine (ACh) content. Measurements were made on three forebrain regions known to show metabolic and morphological change consequent on training—the lobus parolfactorius (LPO), paleostriatuni augmentatum (PA), and medial hyperstriatum ventrale (MHV)—in the right and left hemispheres 2 and 24 h after training chicks on a passive avoidance task, in which they learn to avoid pecking a bead coated with methylanthranilate [methylanthranilate‐trained (M‐trained)]. Control chicks were trained on a water‐coated bead [water‐trained (W‐trained)]. Twenty‐four hours after training, 411B levels showed no differences between W‐trained and M‐trained chicks in any region. M‐training reduced the litre of antip65 by 16% in the left PA and 15% in the left MHV and that of anti‐SV2 by 19% in the left PA. M‐trained chicks showed reduced total ACh content in the LPO by up to 40% and in the PA by up to 48% but had no change in ACh level in the MHV. The decreases in antibody titre were not seen in forebrains analysed 2 h after training, but tendencies toward increases in levels in the right PA and MHV were observed with all three antibodies. Significant differences between right and left hemispheric regions, independent of training, were observed for all the antibodies and for ACh content. In the LPO, antibody litres were some 10% lower in the left compared with the right hemisphere and 34% lower for ACh content, but in the PA, the left hemisphere titre was higher by 14–86%. We discuss these results in the context of morphometric and electrophysiological data oblained from W‐trained and M‐trained chicks.
Traumatic Brain Injury in the Rat: Alterations in Brain Lactate and pH as Characterized by 1H and 31P Nuclear Magnetic Resonance
Authors: Mclntosh T. K. , Faden A. I. , Bendall M. R. , Vink R.
Published: October 2006

Abstract: Application of both phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) to the study of brain metabolism permits the noninvasive measurement of intracellular pH and brain lactate level. We have used water‐suppression 1H MRS with novel lactate‐editing techniques, together with 31P MRS, to characterize sequential changes in brain lactate level and pH in vivo over an 8‐h period following fluid‐percussion brain injury of graded severity in the rat. A transient fall in intracellular pH (from 7.09 ± 0.07 at baséline to 6.88 ± 0.09 at 40 min postinjury) occurred in animals subjected to moderate‐(1.5–2.2 atm) and high‐(2.5–3.3 atm) but not low‐level (0.1–1.2 atm) injury; intracellular pH returned to baseline by 90 min postinjury. Transient elevations in brain lactate level were observed that temporally paralleled and were significantly correlated with the pH changes for all injury levels (r= 0.93, p < 0.001). Postinjury alterations in intracellular brain pH and lactate level were identical in magnitude in animals subjected to either moderate or high‐level injury. However, animals subjected to moderate injury had a moderate chronic neurological deficit that persisted up to 4 weeks postinjury, whereas animals subjected to a high level of injury showed greater histopathological damage and a more severe chronic neurological deficit. These data suggest that the extent of posttraumatic intracellular cerebral acidosis in our model of experimental head injury is not directly related to the severity of functional neurological deficit.
Reserpine Increases Chromaffin Cell Enkephalin Stores Without a Concomitant Decrease in Other Proenkephalin‐Derived Peptides
Authors: Wilson Steven P.
Published: October 2006

Abstract: Reserpine increases the levels of enkephalins in adrenal medullary chromaffin cells; however, the origin of the newly apparent pentapeptides has been the subject of debate, because no increase in the levels of proenkephalin mRNA has been observed. The present study was performed for determining if the reserpine‐induced increase in content of enkephalins was derived from processing of preexisting fragments of proenkephalin. Bovine chromaffin cell enkephalins and larger enkephalin‐containing peptides were separated by reversed‐phase HPLC and identified by approximate molecular weight, elution with peptide standards, and enkephalin sequences contained. Treatment of the cells with reserpine increased the levels of enkephalins and of enkephalin‐containing peptides of up to ∼3 kilo‐daltons without reducing the levels of larger enkephalin‐containing peptides. Similar results were obtained with another catecholamine‐depleting drug, tetrabenazine. In contrast, treatment of chromaffin cells with theophylline or forskolin increased the levels of both enkephalins and enkephalin‐containing peptides of all sizes. The results suggest that new synthesis of proenkephalin is required for the effects of reserpine, although proenkephalin processing is also altered by this drug.
An Immunoglobulin M Monoclonal Antibody, Recognizing a Subset of Acetylcholinesterase Molecules from Electric Organs of Electrophorus and Torpedo, Belongs to the HNK‐1 Anti‐Carbohydrate Family
Authors: Bon Suzanne , Méflah Khaled , Musset Frédérique , Grassi †Jacques , Massoulié Jean
Published: October 2006

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec‐39), obtained against asymmetric acetyl‐cholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec‐39 (Elec‐39+ and Elec‐39‐) differ in their electrophoretic mobility under nondenaturing conditions; the Elec‐39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec‐39‐ component is present at the earliest developmental stages examined, an Elec‐39+ component becomes distinguishable only around the 70‐mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2form. In immunoblots, mAt Elec‐39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec‐39 resembles that of a family of anti‐carbohydrate antibodies that includes HNK‐1, L2, NC‐1, NSP‐4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin‐associated glycoprotein did not react with, Elec‐39+ AChE, mAbs HNK‐1, NC‐1, and NSP‐4 showed the same selectivity as Elec‐39 for Torpedo G2 AChE, but differed in the formation of immune complexes.
Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve
Authors: Larrivee Denis C. , Grafstein Bernice
Published: October 2006

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid‐soluble and acid‐precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid‐precipitable material, a maximal velocity of axonal transport of 13–21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two‐dimensional gel electrophoresis revealed that ∼20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from ∼18,000 to 180,000 and in p1 from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth‐associated protein GAP‐43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including α‐tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3–4 weeks after an optic tract lesion, five proteins, including protein 4, s lowed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least bur others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P‐containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.
Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ‐Aminobutyric Acid Release
Authors: Gallo Vittorio , Suergiu Rossana , Giovannini Claudio , Levi Giulio
Published: October 2006

Using cerebellar, neuron‐enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D‐[3H]aspartate release, whereas N‐methyl‐D‐aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20–100 μM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 μM. Kainate and dihydrokainate (20–100 μM) inhibited the initial rate of D‐[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N‐methyl‐DL‐aspartate were ineffective. D‐[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D‐[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50–200 μM) and, to a lesser extent, 2,3‐cis‐piperidine dicarboxylic acid (100–200 μM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20–100 μM) also stimulated γ‐[3H]aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D‐[3H]aspartate neuronal uptake by interfering with the acidic amino acid high‐affinity transport system.
Effects of an Extract of Ginkgo biloba on the 3′,5′‐Cyclic AMP Phosphodiesterase Activity of the Brain of Normal and Triethyltin‐Intoxicated Rats
Authors: Macovschi Olguta , Prigent Annie‐France , Nemoz Georges , Pacheco Henri
Published: October 2006

Abstract: For clarification of the beneficial effects of the extract of Ginkgo biloba (EGB) on triethyltin (TET) toxicity in rats, the phosphodiesterase (PDE) activities of the cerebral tissue were measured under in vitro and ex vivo conditions. Under in vitro conditions, low concentrations of EGB (0.25–4.0 mg/L) activated the enzyme, whereas after higher concentrations (5–250 mg/L), dose‐dependent inhibition of the enzyme activity was observed. In the lower concentration range, the extract also partially restored the high‐affinity PDE activity (measured with 0.25 μM cyclic AMP) of the particulate fraction of the brain inhibited by TET in vitro. In contrast, the inhibitory influence of TET on the low‐affinity PDE activity (measured with 50 μM cyclic AMP) of the particulate fraction was enhanced by the extract. Although treatment with a single large dose of EGB lowered the particulate PDE activities of the brain of normal rats, no effects of the extract could be detected in animals after repeated daily administrations of EGB during a 4‐day period. Curative treatment of the TET‐intoxicated rats with EGB during a 7‐day period accelerated the recovery of the edematous state of the white matter caused by the intoxication and also normalized the lowered PDE activity of the particulate fraction of the edematous brain tissue. Furthermore, when preventively administered, EGB counteracted both the edema formation and the fall in PDE activity observed with treatment by TET alone. These observations strongly suggest that some beneficial effects of EGB might be due to its modulating influences on cellular cyclic AMP levels via activation of membrane‐bound PDE.
Calmodulin‐Binding Proteins Within the Slow Phase of Axonal Transport in the Rabbit Vagus Nerve Per Ekstrom and Martin Kanje
Authors: Ekstrom Per , Kanje Martin
Published: October 2006

Abstract: : Calmodulin‐binding proteins (CBPs) in the rabbit vagus nerve were studied by means of calmodulin‐Sepha‐rose affinity chromatography and polyacrylamide gel electrophoresis. The soluble fraction (105g supernatant) of a nerve homogenate contained four CBPs with molecular weights of 44, 55, 91, and 93 kD, respectively. Slowly transported proteins were recovered in the vagus 3 days after injection of [35S]methionine into the nodose ganglion. Four labelled CBPs with molecular weights of 44, 55, 69, and 83 kD, respectively were found. The nodose ganglion con tained two labelled CBPs, 44 and 55 kD. The 55‐kD CBP was identified as tubulin after immunoblotting. In separate experiments it was also shown that bovine brain tubulin bound to the calmodulin column.
Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment
Authors: Gonzales Rueben A. , Ganz Neil , Crews Fulton T.
Published: October 2006

Abstract: : The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer‐344 rats either were fed a nutritionally complete ethanol‐containing liquid diet for 5 months or pair‐fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30–500 mM) decreased the polarization values in synaptosomes from sucrose‐control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater than that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol‐fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.
Secretion of [Met]Enkephalyl‐Arg6‐Phe7‐Related Peptides and Catecholamines from Bovine Adrenal Chromaffin Cells: Modification by Changes in Cyclic AMP and by Treatment with Reserpine
Authors: Adams Maria , Boarder Michael R.
Published: October 2006

Abstract: Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl‐Arg6‐Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl‐Arg6‐Phe7 immunoreactivity recognises both peptide B, the 31‐amino acid carboxy‐terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl‐Arg6‐Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B‐like immunoreactivity in both control cells and cyclic AMP‐elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does result in increased accumulation of the heptapeptide [Met]enkephalyl‐Arg6‐Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrena‐line and adrenaline. However, the release of [Met]enkephalin‐Arg6‐Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72‐h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl‐Arg6‐Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl‐Arg6‐Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B‐like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl‐Arg6‐Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of doublebasic residue proteolysis.
Deuterium Isotope Effect in γ‐Aminobutyric Acid Transamination: Determination of Rate‐Limiting Step
Authors: Yu P. H. , Durden D. A. , Davis B. A. , Boulton A. A.
Published: October 2006

Abstract: The rate of transamination of γ‐aminobutyric acid (GABA) catalyzed by hog brain γ‐aminobutyrate ami‐notransferase was substantially reduced when the hydrogen at the γ‐carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the γ‐carbon is the rate‐determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.
Expression of a 48‐Kilodalton Growth‐Associated Protein in the Goldfish Retina
Authors: Perrone‐Bizzozero Nora I. , Benowitz Larry I.
Published: October 2006

Abstract: One of the most striking molecular correlates of optic nerve regeneration in the goldfish is the increased labeling of a 48 kilodalton (kD) acidic protein that is conveyed to the developing nerve endings from the retina by rapid axonal transport. The present study examined the biosynthesis and molecular characteristics of this protein. Retinas derived either from intact controls or from goldfish undergoing optic nerve regeneration (10–14 days postcrush) were pulse‐labeled with [3H]proline or [35S]methionine, followed by subcellular fractionation and analysis of protein synthesis patterns by two‐dimensional gel electrophoresis and fluorography. Synthesis of the 48‐kD acidic protein (termed here GAP‐48) was detected only in retinas that were undergoing axonal regeneration. Pulse‐chase labeling experiments demonstrated that the protein undergoes a post‐translational modification that requires 15–20 min. This processing could be selectively blocked by tunicamycin, an inhibitor of protein N‐glycosylation. The protein was also found to incorporate low levels of phosphate in vitro. Thus, the differential appearance of GAP‐48 in regenerating axons might be regulated either at the level of gene expression or by selective posttranslational processing in retinal ganglion cells. By the criteria of molecular weight, isoelectric point, anomalous migration properties on sodium dodecyl sulfate‐polyacrylamide gels, phosphorylation, subcellular distribution, and the pattern of digestion products generated by Staphylococcus aureus V8 protease, GAP‐48 appears to be equivalent to the B‐50 (F‐1) phosphoprotein of the mammalian brain.
Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments
Authors: Glynn Paul , Chantry Andrew , Groome Nigel , Cuzner M. Louise
Published: October 2006

Abstract: Experiments were performed with isolated human myelin membrane preparations to analyse factors that may modulate association of myelin basic protein (MBP) with the membranes and could contribute to demyelinating processes. Transfer of membranes (5 mg protein ml−1) at 37°C and pH 7.4 from a hypotonic medium, in which they were relatively stable, to one of physiological ionic strength produced three major effects: (1) initial dissociation of MBP from the membranes by a nonenzymatic process that was doubled in the presence of millimolar Ca2+/Mg2+; (2) within 10 min, the appearance in the medium of three major MBP fragments (14.4, 10.3, and 8.4 kilodaltons); and (3) progressive acidification of dissociated MBP and its fragments, probably due to deamidation. Between 1 and 6 h a steady state was apparent in which protein equivalent to 15% of the MBP originally bound to the membranes was found in the medium. The three major MBP fragments formed two thirds of this solubilised material and appeared metabolically stable for 24 h. The kinetics of peptide formation suggested that dissociated, rather than membrane‐bound, MBP was cleaved by myelin‐associated neutral proteases. Two‐dimensional electrophoresis and immunoblotting using two monoclonal antibodies indicated that proteolysis occurred in the vicinity of residues 35 and 75. Evidence was also obtained for removal of C‐terminal arginines and relatively rapid deamidation in the C‐terminal half of MBP. These modifications of MBP might also occur if extracellular fluid gained access to the compacted cytoplasmic space of the myelin sheath.
Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea‐Pig Cortex Synaptosomes
Authors: Adam‐Vizi Vera , Ashley R. H.
Published: October 2006

Abstract: [14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free Ca2+concentration ([Ca2+]i) were measured in guinea‐pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s‐1; t1/2= 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+]i, which then fell to a maintained but still‐elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]ishowed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+‐evoked ACh release also had two phases: a fast initial increase (t1/2= 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2= 130 s) over a 10‐min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+‐dependent fraction of the veratridine‐evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.
Occurrence of Lipid Peroxidation in Brain Microsomes in the Presence of NADH and Vanadate
Authors: Patole M. S. , Ramasarma T.
Published: October 2006

Abstract: Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxy‐phenolic compounds, and by hemin in a concentration‐dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased.
Decreased Metabolism of Cerebrosides and Sulfatides in Rat Sciatic Nerve After Intraneural Injection of Colchicine
Authors: Souyri F. , Barguil S. , Bourre J. M.
Published: October 2006

Abstract: To obtain an understanding of the importance of the neuronal cytoskeleton in Schwann cell metabolism, an antimicrotubular agent (colchicine) was injected into the rat sciatic nerve 24 or 48 h before incubation of the nerve with labeled precursor: [35S]sulfate, [14C]galactose, or [3H]‐galactose. Colchicine inhibited the incorporation of 35S radioactivity into sulfatides and, to a lesser extent, into proteins. With galactose as the radioactive precursor, synthesis of cerebrosides was reduced by colchicine injection, whereas incorporation of radioactivity into phosphatidyl‐serine and phosphatidylcholine increased. Intraneural injection of lumicolchicine had no effect. The effects of colchicine on the metabolism of the Schwann cell are discussed in relation to its action on microtubules.
Ganglioside Composition of Synaptic Vesicles from Torpedo Electric Organ
Authors: Ledeen Robert W. , Parsons Stanley M. , Diebler Marie F. , Sbaschnig‐Agler Michele , Lazereg S.
Published: October 2006

Abstract: We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 μg of N‐acetylneuraminic acid/ mg of protein, respectively), owing to the low protein‐to‐lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 μg, of N‐acetylneuraminic acid/ mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled‐pore glass‐bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N‐acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.
Inositol Uptake and Metabolism in Molluscan Neuronal Tissue
Authors: Tuersley M. D. , Best L. , Tomlinson S.
Published: October 2006

Abstract: The uptake of myo‐[3H]inositol into neurones from Lymnaea stagnalis has been demonstrated to be a sodium‐dependent process, saturable with a Km of approximately 50 μM and shown to be linear with time for at least 120 min. The rate of transport of myo‐inositol into the cell appears to influence directly its incorporation into neuronal lipids. Using anion‐exchange high‐performance liquid chromatography, we have demonstrated a high rate of breakdown of phosphatidylinositol 4,5–bisphosphate in Lymnaea nerve under basal conditions. Stimulation with carbamylcholine enhanced production of inositol 1–phosphate, inositol bisphosphate, inositol 1,4,5–trisphosphate, and inositol 1,3,4–trisphosphate. Formation of inositol tetrakisphosphate was not detected. Electrical stimulation also caused an increased formation of inositol phosphates. These results provide evidence for an active myo‐inositol transport system in molluscan neurones and suggest that the hydrolysis of inositol lipids may play a role as an intracellular signalling system in this tissue.
Release of Purines from Postsynaptic Structures of Amphibian Ganglia
Authors: Rubio R. , Bencherif M. , Berne R. M.
Published: October 2006

Abstract: Isolated sympathetic paravertebral ganglia of the frog were incubated for 1 h with [3H]adenosine. Then, after washout of excess label, the contribution of pre‐ and postsynaptic activation on the release of 3H‐labeled purines was studied. The ganglion was superfused with Ringer's solution at room temperature, and extracellular electrodes were used for stimulation and recording. Preganglionic stimulation enhanced overall release of 3H‐labeled purines. At rest, the release of 3H‐labeled purines per minute represented 0.62 ± 0.02% of the total 3H‐label in the ganglion, and this fraction increased depending on the frequency of orthodromic stimulation. Analyses of the effluent from resting and stimulated ganglia showed that in both cases the nonnucleotide fractions constituted >97% of the total counts in the medium: adenosine (58.4 ± 10.1%); inosine (31.7 ± 12.9%); hypoxanthine (7.1 ± 2.4%); and AMP, ADP, and ATP together (1.6 ± 0.9%) (n = 11). Nucleotides were released, but their levels were not increased significantly during stimulation. Inclusion of ectophosphatase inhibitors slightly enhanced nucleotide release (from 1.1 ± 0.5 to 1.8 ± 0.7%; n = 5) but did not alter the amount of nucleosides. Hence, nucleosides are the main products released by the ganglion and do not arise from hydrolysis of extracellular ATP. Preganglionic stimulation enhanced release of labeled purines, which was frequency dependent from 1 to 20 Hz. Atropine (2 μM) and tubocurarine (150 μM) totally blocked the release of 3H‐labeled purines associated with preganglionic stimulation. Antidromic stimulation was accompanied by a significant release of labeled purines. Carbachol (10−6‐10−2 M) induced a dose‐dependent release of 3H‐labeled purines from the ganglion. These results indicate that the release of nucleosides during synaptic transmission is the result of activation of the postsynaptic structures.