bluuebunny/crossref_metadata_2025 huggingface dataset:
Independent Protein Kinases Associated with the Rat Cerebral Synaptic Junction: Comparison with Cyclic AMP‐Dependent and Ca2+/Calmodulin‐Dependent Protein Kinases in the Synaptic Junction
Authors: Suzuki Tatsuo , Fujii Toshihiro , Tanaka Ryo
Published: October 2006

Abstract: Independent protein kinases in the synaptic junction (SJ) isolated from rat cerebrum were characterized. SJ showed a protein kinase activity, phosphorylating intrinsic proteins, even in the absence of cyclic AMP or Ca2+ plus calmodulin (CaM) exogenously added. The activity was affected neither by Ca2+ concentrations in the physiological fluctuation range nor by the addition of specific ligands such as glutamate, aspartate, acetylcholine, and concanavalin A. The activity was not due to cyclic AMP‐dependent protein kinase in SJ, since the activity was not inhibited by an inhibitor protein for cyclic AMP‐dependent protein kinase, and since synapsin I was not specifically phosphorylated whereas cyclic AMP‐dependent kinase appeared to phos‐phorylate selectively the protein in SJ. Phosphorylation of SJ proteins by the independent kinases was about one‐third of that of the Ca2+/CaM‐dependent protein kinase intrinsic to SJ. The apparent Km for ATP was estimated to be 700 μM. Proteins of 16K Mr and 117K Mr were specifically phosphorylated under the basic condition (in the absence of the substances known to activate specifically protein kinases), as well as six other proteins both under the basic conditions and in the presence of Ca2+ and CaM. The phosphorylation of 150K Mr, 60K Mr, 51K Mr, and 16K Mr SJ proteins was enhanced after prephosphorylation of SJ proteins by intrinsic kinase in the presence of Ca2+ and CaM. This fact suggested that a part of the independent kinase activity was attributable to the autonomous form of Ca2+/ CaM‐dependent kinase which was derived from its auto‐phosphorylation, and that the Ca2+/CaM‐dependent auto‐phosphorylation affected the microtubules in the postsyn‐aptic region inasmuch as the 16K Mr protein occurred in both brain cytosol and microtubule protein fraction prepared from rat brain.
Effect of Bicuculline‐Induced Status Epilepticus on Prostaglandins and Hydroxyeicosatetraenoic Acids in Rat Brain Subcellular Fractions
Authors: Birkle Dale L. , Bazan Nicolas G.
Published: October 2006

Abstract: Rat cerebrum, prelabeled in vivo by intraventric‐ular injection of [1‐14C]arachidonic acid, was used to assess cyclooxygenase and lipoxygenase reaction products in total homogenates, cytosol, synaptosomes, and microsomes. Effects of bicuculline‐induced status epilepticus on arachi‐donic acid metabolism in synaptosomes and microsomes were also measured. Lipoxygenase activity, resulting in the synthesis of hydroxyeicosatetraenoic acids (HETEs), and cyclooxygenase activity, resulting in the synthesis of prostaglandins (PGs), were measured by reverse‐phase and normal‐phase HPLC with flow scintillation detection. Endogenous lipoxygenase products in synaptosomes were identified by capillary gas chromatography‐mass spectrometry. PGs and HETEs were detected in all subcellular fractions. The synaptosomal fraction showed the highest lipoxygenase activity, with 5‐HETE, 12‐HETE, and leukotriene B4 as the major products. Following bicuculline‐induced status epilepticus, endogenous free arachidonic acid and other fatty acids accumulated in synaptosomes, but not in microsomes. Incorporation of [1‐l4C]arachidonic acid into synaptosomal and microsomal phospholipids was decreased after bicuculline treatment. Bicuculline‐induced status epilepticus resulted in increased synthesis of HETEs in synaptosomes. PG synthesis increased in the microsomal fraction. When [1‐14C]arachidonic acid‐labeled synaptosomes and microsomes were incubated for 1 h at 37°C the synthesis of eicosa‐noids, particularly PGD2, was increased significantly in bi‐cuculline‐treated rats, as compared with untreated rats. Depolarization (45 mM K+) of synaptosomes induced a loss of [1‐14C]arachidonic acid from phosphatidylinositol, and increased the synthesis of PGD2 and HETEs, an effect that was enhanced in bicuculline‐treated rats. This study localizes changes in arachidonic acid metabolism and lipoxygenase activity resulting from bicuculline‐induced status epilepticus in the brain subcellular fraction enriched in nerve endings.
Effects of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine and 1‐Methyl‐4‐Phenylpyridinium Ion on Activities of the Enzymes in the Electron Transport System in Mouse Brain
Authors: Mizuno Yoshikuni , Sone Nobuhito , Saitoh Tomohiko
Published: October 2006

Abstract: The effects of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahy‐dropyridine (MPTP) and 1‐methyl‐4‐phenylpyridinium ion (MPP+) on activities of enzyme complexes in the electron transport system were studied using isolated mitochondrial preparations from C57BL/6J mouse brains. Both MPTP and MPP+ dose‐dependently inhibited activity of NADH‐ubiquinone oxidoreductase (EC 1.6.5.3). The inhibition was reversible. Preincubation of freeze‐thawed mitochondria with MPTP or MPP+ had no effect on the inhibition; however, when nonfrozen mitochondria were used, NADH‐ubiquinone oxidoreductase activity was reduced to 46% of that in the nonincubated sample after a 5‐min preincubation with MPTP and to 77% of that in the nonincubated sample after a 5‐min preincubation with MPP+. Kinetic analyses revealed that inhibition of MPTP was non‐competitive and that of MPP+ uncompetitive with respect to NADH. On the other hand, inhibition of MPTP was uncompetitive and that of MPP+ noncompetitive with respect to ubiquinone. Succinate – ubiquinone oxidoreductase (complex II), dihydroubiquinone‐cytochrome c oxidoreductase (complex III), and ferrocytochrome c‐oxygen oxidoreductase (EC 1.9.3.1) activities were either slightly inhibited or not inhibited by MPTP or MPP+. The significance of these findings is discussed in relation to the mechanism of MPTP‐induced neuronal degeneration.
High‐Affinity Transport of γ‐Aminobutyric Acid, Glycine, Taurine, L‐Aspartic Acid, and L‐Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis
Authors: Debler Edmund A. , Lajtha Abel
Published: October 2006

Abstract: In a cortical P2 fraction, [14C]γ‐aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high‐affinity transport systems with L‐glutamic acid and L‐aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high‐affinity system, with no second, low‐affinity, transport system detectable. Only one high‐affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low‐affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons‐me‐dulla‐spinal cord) both high‐ and low‐affinity transport processes could be detected. The high‐affinity GABA and high‐affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High‐affinity GABA transport exhibits no overlap with either the high‐affinity glycine or the high‐affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high‐affinity cortical glycine transport and those of high‐affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high‐affinity glycine transport in the cerebral cortex and in the pons‐medulla‐spinal cord show a significant positive correlation with each other; however, high‐affinity glycine uptake in the pons‐medulla‐spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high‐affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.
Evidence for the Participation of a Cytosolic NADP+‐Dependent Oxidoreductase in the Catabolism of γ‐Hydroxybutyrate In Vivo
Authors: Kaufman Elaine E. , Nelson Thomas
Published: October 2006

Abstract: The concentration of γ‐hydroxybutyrate (GHB) in brain, kidney, and muscle as well as the clearance of [1‐14C]GHB in plasma have been found to be altered by the administration of a number of metabolic intermediates and drugs that inhibit the NADP+‐dependent oxidoreductase, “GHB dehydrogenase,” an enzyme that catalyzes the oxidation of GHB to succinic semialdehyde. Administration of valproate, salicylate, and phenylacetate, all inhibitors of GHB dehydrogenase, significantly increased the concentration of GHB in brain; salicylate increased GHB concentration in kidney, and α‐ketoisocaproate increased GHB levels in kidney and muscle. The half‐life of [1‐14C]GHB in plasma was decreased by D‐glucuronate, a compound that stimulates the oxidation of GHB by this enzyme and was increased by a competitive substrate of the enzyme, L‐gulonate. The results of these experiments suggest a role for GHB dehydrogenase in the regulation of tissue levels of endogenous GHB.
Protein Kinase C Agonists Increase the Expression of Angiotensin II Receptors in Neuronal Cultures
Authors: Sumners Colin , Rueth Susan M. , Crews Fulton T. , Raizada Mohan K.
Published: October 2006

Abstract: Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by α1‐adrenergic receptors. Because α1‐adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1‐day‐old rat brains. The active phorbol ester phorbol‐ 12‐myristate‐13‐acetate (TPA) caused time‐ and concentration‐dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester‐induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.
Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme
Authors: Reinhart Peter H. , Pfeiffer Brigitte , Spengler Siegfried , Hamprecht Bernd
Published: October 2006

Abstract: The physiological function in brain of glycogen and the enzyme catalyzing the rate‐limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700‐fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia‐rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron‐rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.
Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes
Authors: Gillespie C. S. , Trapp B. D. , Colman D. R. , Brophy P. J.
Published: October 2006

Abstract: Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a “receptor” molecule, perhaps a membrane‐anchored protein, for association with the cytoplasmic face of the myelin membrane.
Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N‐Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP
Authors: Iuvone P. Michael
Published: October 2006

Abstract: In chicken retinas, melatonin levels and the activity of serotonin N‐acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light‐dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day‐night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light‐dark cycle in ovo only. NAT activity is regulated by a cyclic AMP‐dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven‐to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP‐dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.
The Epitope(s) Recognized by HNK‐1 Antibody and IgM Paraprotein in Neuropathy Is Present on Several N‐Linked Oligosaccharide Structures on Human P0 and Myelin‐Associated Glycoprotein
Authors: Burger Danielle , Simon Maryline , Perruisseau Geneviève , Steck Andreas J.
Published: October 2006

Abstract: The mouse monoclonal antibody HNK‐1 and the human monoclonal IgM antibody present in patients with polyneuropathy both recognize carbohydrate epitope(s) on human myelin‐associated glycoprotein and P0. In the present study, the oligosaccharide structures that bear the antibody epitope(s) were investigated. The extracellular derivative of myelin‐associated glycoprotein (dMAG) was purified by immunoaffinity chromatography. P0 was electroeluted from gel slices. Western blot analysis of whole glycoproteins demonstrated that the epitopes for HNK‐1 and the human monoclonal IgM antibody were different. The glycopeptides obtained by proteolysis of purified dMAG and P0 were separated and characterized by affinity chromatography on concanavalin A‐Sepharose. Both dMAG and P0 displayed heterogeneity in their oligosaccharide structures, i.e., they both contained mainly tri‐ and tetraantennary oligosaccharides (∼80%), although biantennary (10%) and high‐mannose and/or hybrid (10%) oligosaccharides were present. The human monoclonal IgM antibody epitope was present on all types of isolated oligosaccharide structures from either dMAG and P0. The HNK‐1 epitope was present on all types of oligosaccharide structures of dMAG, whereas it was present only on tri‐ and tetraantennary structures of P0.
Determination of N‐Acetylaspartic Acid in Human Cerebrospinal Fluid by Gas Chromatography–Mass Spectrometry
Authors: Swahn Carl‐Gunnar
Published: October 2006

Abstract: N‐Acetyl‐L‐aspartic acid was identified and determined in human cerebrospinal fluid. The concentration in lumbar fluid was about 2 nmol/ml and about 20 nmol/ml in ventricular fluid. There was no difference between healthy subjects and schizophrenic patients.
Identification of Disialosyl Paragloboside and O‐Acetyldisialosyl Paragloboside in Cerebellum and Embryonic Cerebrum
Authors: Chou Denise K. H. , Flores Sally , Jungalwala Firoze B.
Published: October 2006

Abstract: The lacto series of glycolipids are only minor constituents in mammalian CNS and are found mostly during development. Expression of a significant amount (70 μg of neuraminic acid/g dry weight) of disialosyl‐lacto‐N‐neotetraosylceramide (LD1) in adult mouse cerebellum is reported for the first time in the nervous system. The structure of this ganglioside was determined by hydrolysis with various glycosidases, immunochemical tests, sugar and fatty acid analyses after permethylation and capillary GLC‐mass spectrometry, sugar linkage analysis of permethylated alditol acetates, and fast‐atom bombardment‐mass spectrometry of the native ganglioside. The structure of LD1 was determined to be NeuAc‐NeuAcα2‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glcβ1‐1‐ceramide. The major fatty acid was 18:0, and the long‐chain base was C18‐sphingenine. Mouse cerebellum also contained O‐acetyl‐LD1 and several other O‐acetylated gangliosides as recognized by monoclonal antibodies ME311 and 3G5. The levels of LD1 and O‐acetyl‐LD1 in cerebellum increased during postnatal development. During development of the Purkinje cell degeneration mutant, pcd/pcd, the levels of both of these gangliosides in the cerebellum declined with the loss of Purkinje cells, a finding indicating that these gangliosides are primarily associated with Purkinje cells. In the cortex, LD1, O‐acetyl‐LD1, and O‐acetyl GD3, like GD3, are developmentally regulated antigens and are only expressed in the fetal cortex and not to any significant extent in the adult.
Synaptic Vesicular Glutamate Uptake: Modulation by a Synaptosomal Cytosolic Factor
Authors: Lobur Anne T. , Kish Phillip E. , Ueda Tetsufumi
Published: October 2006

Abstract: We have demonstrated previously that L‐glutamate is taken up into isolated synaptic vesicles in an ATP‐dependent manner, supporting the neurotransmitter role of this acidic amino acid. We now report that a nerve terminal cytosolic factor inhibits the ATP‐dependent vesicular uptake of glutamate in a dose‐dependent manner. This factor appears to be a protein with a molecular weight >100,000, as estimated by size exclusion chromatography, and is precipitated by ammonium sulfate (40% saturation). The inhibitory factor is inactivated by heating to 100°C. Proteolytic digestion of the ammonium sulfate fraction by trypsin or chymotrypsin did not reduce, but rather increased slightly, the inhibition of glutamate uptake. Unlike the native factor, the digest retained inhibitory activity after heating, suggesting that proteolytic digestion may generate active fragments. The inhibition of ATP‐dependent vesicular glutamate uptake is not species‐specific, as the factor obtained from both rat and bovine brains produced an equal degree of inhibition of glutamate uptake into vesicles of each species. These observations raise the possibility that vesicular uptake of glutamate may be regulated by an endogenous factor in vivo.
Sodium/Proton Exchange in Cultured Bovine Adrenal Medullary Cells
Authors: Yanagihara Nobuyuki , Yokota Kenji , Kobayashi Hideyuki , Wada Akihiko , Uezono Yasuhito , Izumi Futoshi
Published: October 2006

Abstract: We investigated the presence of Na+/H+ exchange in cultured bovine adrenal medullary cells. The intracellular pH in control cells measured by 5,5‐dimethyl[2‐14C]oxazolidine‐2,4‐dione was 7.13 ± 0.02 (n = 6). Removal of Na+ from the incubation medium shifted the intracellular pH down to 6.67 ± 0.12 (n = 6). Reintroduction of Na+ to the medium caused a rapid recovery in intracellular pH to 7.20–7.30 that was associated with an increase in uptake of 22Na+ by the cells. Both increases in intracellular pH and uptake of 22Na+ were inhibited by amiloride, an inhibitor of Na+/H+ exchange. The recovery of intracellular pH by addition of Na+ was partially inhibited by quinidine, another inhibitor of Na+/H+ exchange, but not by 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulfonic acid, an anion‐exchange (Cl−/HCO−3) inhibitor. Li+ could substitute for Na+ in the recovery of intracellular pH. Carbachol caused an increase in intracellular pH from 7.12 ± 0.01 to 7.21 ± 0.02 (n = 10). This increase in intracellular pH caused by carbachol was inhibited by amiloride. These results suggest the existence of an amiloride‐sensitive Na+/H+ exchange that regulates the intracellular pH in adrenal medullary cells.
Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor
Authors: Haga Kazuko , Haga Tatsuya , Ichiyama Arata
Published: October 2006

Abstract: Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP‐sensitive high‐affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP‐binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP‐binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP‐binding proteins in vitro is not affected by their phosphorylation.
Cyclic GMP Regulates Nicotine‐Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)
Authors: O'Sullivan Antony J. , Burgoyne Robert D.
Published: October 2006

Abstract: Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II. and bradykinin raised cyclic. GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8‐bromo‐cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8‐bromo‐cGMP inhibited secretion. These effects of 8‐bromo‐cGMP were not due to changes in the nicotine‐induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium‐derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.
Production of Platelet‐Activating Factor from Rat Cerebellar Granule Cells in Culture
Authors: Yue Tian‐Li , Lysko Paul G. , Feuerstein Giora
Published: October 2006

Abstract: Platelet‐activating factor (PAF) is a potent lipid mediator implicated in various pathological conditions, including CNS neuronal injury. However, the production of PAF by mammalian CNS neurons has not as yet been demonstrated. In the present study, we demonstrate that PAF is produced by cultured rat cerebellar granule cells. PAF was identified on the basis of chemical and enzymatic characteristics, biological activities with washed rabbit platelets, and behavior on TLC and HPLC. PAF was detected both in the cells and in the incubation medium, a result indicating the release of PAF from cultured neurons. The amount of PAF produced during a 30‐min incubation was as follows: 1.02 ± 0.10 and 0.93 ± 0.09 pmol/ 4 × 107 cells in incubation buffer and cells, respectively (n = 10). The calcium ionophore A23187 (2.5 μM) had only a mild stimulatory effect on PAF production, a finding indicating that the neuron‐generated PAF might be synthesized mainly by the de novo pathway of PAF production.
Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes
Authors: Nicoletti F. , Magrì G. , Ingrao F. , Bruno V. , Catania M. V. , Dell'Albani P. , Condorelli D. F. , Avola R.
Published: October 2006

Abstract: Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate ([3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12‐h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl‐3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12‐tetradecanoate 13‐acetate. Glutamate and ibotenate reduced [methyl‐3H]‐thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2‐amino‐4‐phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective. Inhibition of [methyl‐3H]thymidine incorporation by quisqualate required a lag time of about 4 h and, in cells synchronized to proliferate, occurred when the drug was added during the transition between G0 and G1, but not during the S phase of the mitotic cycle. This suggests that an inducible factor may be involved in the antiproliferative effect of excitatory amino acids. Accordingly, activation of quisqualate receptors led to a rapid and transient increase in mRNA levels of the early inducible gene, c‐fos. These results suggest that activation of a specific class of “quisqualate‐preferring”excitatory amino acid receptors reduces proliferation of astrocytes in primary cultures.
Arachidonic Acid Incorporation and Redistribution in Human Neuroblastoma (SK‐N‐BE) Cell Phospholipids
Authors: Spinedi A. , Pacini L. , Piacentini M. , Melino G. , Luly P.
Published: October 2006

Abstract: The incorporation and redistribution of [1‐14C]arachidonic acid in SK‐N‐BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum‐enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse‐labelled with [1‐14C]arachidonic acid and then reincubated in label‐free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid‐stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid‐stable PtdEtn and alkenylacyl‐PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid‐stable PtdCho to both alkenylacyl‐PtdEtn and acid‐stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.
Characterization of [3H]Hemicholinium‐3 Binding Sites in Human Brain Membranes: A Marker for Presynaptic Cholinergic Nerve Terminals
Authors: Pascual Julio , González Antonio M. , Pazos Angel
Published: October 2006

Abstract: We report here on the binding properties of [3H]hemicholinium‐3, a selective inhibitor of the high‐affinity choline uptake process, to human brain membranes. Under the assay conditions described, the binding of [3H]hemicholinium‐3 exhibited a dependency of physiological conditions on pH, temperature, and NaCl concentrations. Striatal binding proved to be specific, to a single site, saturable, and reversible, with an apparent KD of 10 nM and a Bmax of 82 fmol/mg of protein. [3H]Hemicholinium‐3 specific binding exhibited a pharmacological profile and an ionic dependency suggestive of physiologically relevant interactions and comparable with those reported for the high‐affinity choline uptake. Moreover, specific [3H]hemicholinium‐3 binding exhibited an uneven regional distribution: striatum ≫ nucleus basalis > spinal cord ≫ midbrain = cerebellum ≧ hippocampus > neocortex = anterior thalamus > posterior thalamus ⋙ white matter. This distribution closely corresponds to the reported activity of both enzymatic cholinergic presynaptic markers and high‐affinity choline uptake in mammalian brain. There are no significant differences between these results and those previously found in the rat brain using this radioligand. Our results demonstrate, for the first time, the presence of [3H]hemicholinium‐3 binding sites in human brain and strongly support the proposal that this radioligand binds to the carrier site mediating the high‐affinity choline uptake process on cholinergic neurons. Thus, [3H]hemicholinium‐3 binding may be used in postmortem human brain as a selective and quantifiable marker of the presynaptic cholinergic terminals.
Structural Differences Between Dopamine D2 Receptors Present in a Rat Pituitary Adenoma and in Transplantable Rat Pituitary Tumors 7315a and MtTW15
Authors: Bouvier C. , Lagacé G. , Potier M. , Collu R.
Published: October 2006

Abstract: We have investigated the structure of dopamine (DA) D2 receptors present in an estrone‐induced, prolactin (PRL)‐secreting, DA‐sensitive adenoma and in two PRL‐secreting and DA‐insensitive transplantable tumors 7315, and MtTW15, in order to identify better the anomalies present in DA‐resistant lactotrophs. D2 receptors were found in both a high‐ and a low‐affinity state in adenomatous lactotrophs as shown by displacement studies with the agonist N‐propylnorapomorphine (NPA), but only in the low‐affinity state in the two DA‐resistant tumors. Treatment with the alkylating agent N‐ethylmaleimide induced a disappearance of the high‐affinity state of the D2 receptor in the adenoma and a reduction in receptor concentration, but did not have any effect on the affinity of receptors present in DA‐resistant tumors. Moreover, target size analysis and radiation inactivation studies of D2 receptors, using membranes preincubated with NPA and [3H]spiperone as ligand or using [3H]NPA as ligand on membranes preparations, have shown the presence of distinct structural differences between adenomatous and tumoral D2 receptors and between the two tumoral receptors themselves; these results suggest that the normal functional unit of the D2 receptor is a dimer associated with a guanine nucleotide‐binding protein (G protein) subunit and that tumoral D2 receptors may exist in various polymeric forms unassociated with G proteins. The anomalies found to be present in tumoral D2 receptor complexes may be responsible for the insensitivity of these tumors to dopaminergic agonists’inhibitory activity on PRL release and tumor growth.
Selective Expression of Factors Preventing Cholinergic Dedifferentiation
Authors: DeMello Fernando G. , DeMello Maria C. F. , Hudson Richard , Klein William L.
Published: October 2006

Abstract: Chicken retina neurons from 8–9‐day‐old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high‐affinity, hemicholiniumsensitive transport system, converted [3H]choline to [3H]acetylcholine ([3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high‐affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long‐lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known “in situ”pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat‐sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers. The data suggest that retinal cholinergic neurons must receive external signals at a critical window during development in order to stabilize initial ChAT expression, that intrinsic cells of the retina produce the diffusible cholinergic‐sustaining activity, and that production of activity is dependent upon appropriate cell‐cell interactions.
Acetylcholinesterase in Cocultures of Rat Myotubes and Spinal Cord Neurons: Effects of Collagenase and cis‐Hydroxyproline on Molecular Forms, Intra‐ and Extracellular Distribution, and Formation of Patches at Neuromuscular Contacts
Authors: Vallette François‐Marie , De la Porte Sabine , Koenig Jeanine , Massoulié Jean , Vigny Marc
Published: October 2006

Abstract: Cultures of rat myotubes from 18‐day‐old embryos produce both globular (G) and asymmetric (A) forms of acetylcholinesterase (AChE; EC 3.1.1.7), mostly G1, G4, and A12 and a small proportion of A8. We show that all forms are partly intracellular and partly exposed to the extracellular medium; the A forms and their intra‐ and extracellular distribution are not modified when myotubes are grown in the presence of spinal cord neurons. In these cocultures, however, AChE patches may be detected immunohistochemically at sites of neuromuscular contacts. These patches represent a very minor proportion of AChE activity. We found that collagenase removes AChE patches but not the acetylcholine receptor clusters with which they coincide. This digestion specifically decreases the level of the A12 form. cis‐Hydroxyproline, an inhibitor of collagen synthesis, reduces the level of G1 and blocks the synthesis of A forms.
Studies on the Effect of Insulin‐Like Growth Factor‐I on Catecholamine Secretion from Chromaffin Cells
Authors: Dahmer Mary K. , Hart P. Michelle , Perlman Robert L.
Published: October 2006

Abstract: Chromaffin cells cultured in serum‐free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum‐containing medium. Addition of insulin‐like growth factor‐I (IGF‐I) to serum‐free medium restored high K+‐stimulated catecholamine secretion to the levels seen in serum‐treated cultures. In contrast, addition of IGF‐I to serum‐containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF‐I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF‐I not only enhanced high K+‐stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl‐phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF‐I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF‐I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2+‐dependent catecholamine secretion was slightly, but consistently, greater from IGF‐I‐treated cells than from untreated cells. Our results suggest that IGF‐I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.
Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase
Authors: Yang Jane , Barchi Robert
Published: October 2006

Abstract: Cyclic AMP‐dependent phosphorylation of the rat brain sodium channel was reported to be restricted to five sites within an ˜210 amino acid region of the primary sequence that is deleted in the homologous sodium channel from rat skeletal muscle. We find that, in spite of this deletion, the rat muscle sodium channel α‐subunit is also an excellent substrate for phosphorylation by this kinase both in primary muscle cells in tissue culture and in vitro after isolation from adult muscle. Sodium channel protein purified from adult rat skeletal muscle was readily phosphorylated in vitro by the catalytic subunit of the bovine cyclic AMP‐dependent protein kinase (PKa). Only the 260,000 MW α‐subunit was labeled, with a maximum level of incorporation in vitro of ˜0.5 mol [32P]phosphate per mole of channel protein. The β‐subunit of the channel is not phosphorylated under these conditions. In primary rat skeletal muscle cells in culture, incorporation of phosphate into the channel α‐subunit is stimulated 1.3‐ to 1.5‐fold by treatment of the cells with forskolin. Phosphorylation of the sodium channel isolated from these cells could also be demonstrated in vitro using PKa. This in vitro phosphorylation could be inhibited 80–90% by pretreatment of the cells in culture with forskolin, suggesting that the sites labeled in vitro by PKa were the same as those phosphorylated in the intact cells by the endogenous cyclic AMP‐dependent kinase. In both the adult muscle channel and the channel from muscle cells in culture, phosphorylation by PKa was limited to serine residues. The location of the phosphorylated residue in the primary sequence of the channel was examined by studying the distribution of label among proteolytic fragments generated in situ from the α‐subunit by endogenous proteases. The label in the intact 260‐kDa α‐subunit partitioned with a 130‐kDa polypeptide representing the amino terminal half of the molecule whereas none was associated with fragments derived from the carboxy‐terminal half of the protein. No label was found in the next smaller 90‐kDa amino‐terminal fragment, however, indicating that the phosphorylated residue is located between 90 and 130 kDa from the amino terminus of the subunit, a region that includes the DI–DII interdomain region and the DII repeat domain. Phosphopeptide analysis of the 260‐kDa α‐subunit and of its 130‐kDa amino‐terminal half confirmed that the single major labeled peptide in each had identical migratory characteristics in this two‐dimensional analysis. Electrophoresis of the tryptic digest in a sodium dodecyl sulfate‐urea system indicated a MW of ˜2,500 for the limit phosphopeptide.
Nerve Growth Factor‐Induced Down‐Regulation of Calmodulin‐Dependent Protein Kinase III in PC12 Cells Involves Cyclic AMP‐Dependent Protein Kinase
Authors: Brady Matthew J. , Nairn Angus C. , Wagner John A. , Palfrey H. Clive
Published: October 2006

Abstract: Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin‐dependent protein kinase III (CaM‐PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126‐1B2) that is deficient in cAMP‐dependent protein kinase activity. Control experiments showed that A 126‐1B2 cells retain other NGF‐mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP‐independent event) and contain a complement of CaM‐PK III and its substrate, elongation factor‐2, comparable to that of wild‐type cells. The ability of NGF or forskolin, but not of EGF, to down‐regulate CaM‐PK III was markedly attenuated in A 126‐1B2 compared to wildtype cells. Treatment of wild‐type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP‐dependent protein kinase activity in wild‐type cells was assessed by measurement of the “activation ratio’(‐cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild‐type cells. These experiments support the hypothesis that NGF, but not EGF, achieves its effects on CaM‐PK III by activation of a cAMP‐dependent protein kinase and suggest that other actions of NGF may be mediated by the same mechanism.
Effect of Phospholipase C from Bacillus cereus on the Release of Membrane‐Bound Choline‐O‐Acetyltransferase from Rat Hippocampal Tissue
Authors: Carroll P. T. , Smith L. K.
Published: October 2006

Abstract: Some of the enzyme choline‐O‐acetyltransferase (ChAT) associated with central cholinergic nerve terminals appears to be nonionically associated with membranes. In the present study, we tested the possibility that some membrane‐bound ChAT might be anchored to membranes by a phosphatidylinositol linkage by incubating rat hippocampal tissue with phospholipase C (PLC) from Bacillus cereus. The PLC selectively augmented the release of ChAT; also, the glycosylphosphatidylinositol‐PLC inhibitor, zinc, blocked this increase in release. When control and PLC‐treated hippocampal tissues were subjected to Triton X‐114 phase separation, a procedure that separates amphiphilic from hydrophilic proteins, the detergent‐soluble, membrane‐bound fraction of tissue ChAT appeared to be the source of the ChAT released by PLC into the incubation medium. Zinc also blocked the temperature‐dependent release of ChAT, but not lactic dehydrogenase, from hippocampal tissue. Extracellular membrane‐bound ChAT appeared to be the source of the ChAT released by a low exogenous concentration of PLC, as well as that released by a temperature‐dependent process during tissue incubation. Phosphatidylinositol‐specific PLC from Bacillus thuringiensis released ChAT, but not lactic dehydrogenase, from a crude synaptosomal fraction prepared from rat hippocampal tissue. These results suggest that some of the membrane‐bound ChAT in rat hippocampal tissue may be extracellular and anchored to the membrane by phosphatidylinositol, and also that an endogenous factor in hippocampal tissue may function to remove this extracellular ChAT from the membrane.
Monoamine Receptors in an Animal Model of Affective Disorder
Authors: Martin Joseph V. , Edwards Emmeline , Johnson Joel O. , Henn Fritz A.
Published: October 2006

Abstract: After a relatively mild course of uncontrollable shocks, two distinct groups of rats can be defined in terms of their performance in learning to escape from a controllable stressor. Response‐deficient (RD) rats do not learn to terminate the controllable stressor, whereas nondeficient (ND) rats learn this response as readily as do untreated control rats. The current studies were designed to determine the neu‐rochemical correlates of the behavioral differences between these groups of rats. The major findings concerned postsyn‐aptic β‐adrenergic effects in the hippocampus of RD rats. These included an up‐regulation of β‐adrenergic receptors and, in parallel experiments, an increase in the sensitivity of adenylyl cyclase to stimulation by norepinephrine. There was no difference in brain levels of catecholamines between the three groups of rats. A statistically significant increase in levels of 5‐hydroxytryptamine was noted in the hippocampus and hypothalamus of RD rats as compared to levels in ND rats, but no significant differences were measured between groups of rats in terms of S1 or S2 serotonergic receptor binding. These results implicate both β‐adrenergic and serotonergic mechanisms in the behavioral deficit caused by uncontrollable shock.
Tissue Distribution of Human Monoamine Oxidase A and B mRNA
Authors: Grimsby Joseph , Lan Nancy C. , Neve Rachael , Chen Kevin , Shih Jean C.
Published: October 2006

Abstract: Monoamine oxidase (MAO) A and B play important roles in the metabolism of biogenic amines. Northern analysis using 32P‐labeled subfragments of human liver MAO A and B cDNA clones detected a 5‐ and a 3‐kb transcript, respectively, in most human tissues examined. However, fetal heart and thymus express minute amounts of MAO A transcript, whereas fetal brain, muscle, thymus, spleen, meninges, and placenta express minute amounts of MAO B transcript. Small intestine and placenta express, in addition to the MAO A 5‐kb transcript, a 2‐kb transcript, which may arise from an alternative polyadenylation site. MAO A and B transcripts are expressed in similar regions of adult human brain. The highest concentrations of these transcripts were located in frontal cortex and locus coeruleus. This study demonstrates the tissue‐specific distribution of the MAO genes and will provide insight into the physiological functions of MAO A and B.