bluuebunny/crossref_metadata_2025 huggingface dataset:
Biexponential Kinetics of (R)‐α‐[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor
Authors: West Robert E. , Zweig Adam , Granzow Russell T. , Siegel Marvin I. , Egan Robert W.
Published: October 2006

Abstract: The H3 histamine receptor is a high‐affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization‐induced histamine release. We have used (R)‐α‐[3H]methylhistamine, a specific, high‐affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD= 0.68 nM; Bmax= 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: Nα‐methylhistamine, (R)‐α‐methylhistamine > histamine, thioperamide > impromidine > burimamide > dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on‐and‐off rates. Density of (R)‐α‐[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex > hypothalamus > brainstem > cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undif‐ferentiated.
Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse
Authors: Shigematsu Hidenari , Morimoto Satoshi , Kishimoto Yasuo , Weiler Solly , Tomich John , Barranger John , Shinohara Mitsuko , Yeager Andrew M. , O'Brien John S.
Published: October 2006

Abstract: The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide β‐galactosidase activity. Saposin A and C, the heat‐stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide β‐galactosidase as well as glucosylceramide β‐glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide β‐glucosidase (measured by hydrolysis of 4‐methylumbelliferyl‐β‐glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide 4bT‐galactosidase as well as 4‐methylum‐belliferyl‐β‐glucoside β‐glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide β‐galactosidase.
Tyrosine Hydroxylase mRNA Concentration in Midbrain Dopaminergic Neurons Is Differentially Regulated by Reserpine
Authors: Pasinetti Giulio M. , Morgan David G. , Johnson Steven A. , Millar Sharon L. , Finch Caleb E.
Published: October 2006

Abstract: Tyrosine hydroxylase (TH)‐mRNA, assayed by in situ hybridization combined with TH immunocytochemistry, showed a selective increase in the ventral tegmental area (A‐10) but not in the substantia nigra (A‐9) midbrain dopaminergic (DAergic) neurons 3 days after reserpine treatment. TH‐mRNA in locus ceruleus noradrenergic (A‐4) neurons was increased by reserpine, as confirmed by RNA blot hybridization. These findings show that TH‐mRNA is differentially regulated in midbrain DAergic neurons in response to reserpine.
Normal Mitochondrial Genome in Brain from Patients with Parkinson's Disease and Complex I Defect
Authors: Lestienne P. , Nelson J. , Riederer P. , Jellinger K. , Reichmann H.
Published: October 2006

Abstract: The mitochondrial genome codes for 13 proteins which are located in the respiratory chain. In postmortem brain of patients with Parkinson's disease, decreased activity of complex I of the respiratory chain could be demonstrated. Because seven subunits of complex I are coded by the mitochondrial genome, we analyzed the mitochondrial DNA of human postmortem substantia nigra, putamen, and frontal cortex by the Southern blot technique. No deletions of the mitochondrial genome could be demonstrated, thus indicating that either subunits which are encoded by the nuclear genome are decreased or enzyme activity is diminished by metabolites, toxins, or increase of Fe3+.
Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain
Authors: Oishi Ryozo , Adachi Naoto , Okada Keishi , Muroi Nobuyuki , Saeki Kiyomi
Published: October 2006

Abstract: To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of rele‐methylhistamine (t‐MH) during the 90‐min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady‐state t‐MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/ kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/ kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.
Antinociceptive Actions of α2‐Adrenoceptor Agonists in the Rat Spinal Cord: Evidence for Antinociceptive α2‐Adrenoceptor Subtypes and Dissociation of Antinociceptive α2‐Adrenoceptors from Cyclic AMP
Authors: Uhlén S. , Persson M.‐L. , Alari L. , Post C. , Axelsson K. L. , Wikberg J. E. S.
Published: October 2006

Abstract: The antinociceptive actions of intrathecal injections of two α2‐adrenergic agonists, UK‐14,304 and guanfacine, were investigated in rats after pretreatment of the animals with the noradrenaline neurotoxin N‐2‐chloroethyl‐N‐ethyl‐2‐bromobenzylamine (DSP4) 14 days in advance. The chronic noradrenaline depletion induced by DSP4 caused a marked increase in sensitivity of the antinociceptive action of UK‐14,304 in the tail‐flick test. By contrast, the antinociceptive effect of guanfacine was not appreciably affected by the DSP4 treatment. The antinociceptive effects of both UK‐14,304 and guanfacine were blocked by intraperitoneal injections of yohimbine, a result indicating that both drugs induced their actions by activating α2‐adrenoceptors. Both UK‐14,304 and guanfacine were found to reduce the production of cyclic AMP (cAMP) in the spinal cord, as determined using an in vitro radioisotopic method. The cAMP inhibitory effects of both agonists were effectively blocked by yohimbine, but not by prazosin, a finding indicating the α2‐adrenergic nature of the response. However, the cAMP inhibitory effect of UK‐14,304 was not potentiated by pretreatment with DSP4, a finding in marked contrast with the strong potentiation of the antinociceptive action of UK‐14,304 induced by the chronic depletion of endogenous noradrenaline. Moreover, intrathecal injections of forskolin, which increased the endogenous levels of spinal cord cAMP fivefold, did not modify the antinociceptive effects of UK‐14,304 or guanfacine in neither normal nor DSP4‐treated animals. It is suggested that there exist pharmacologically differing α2‐adrenergic receptor pathways capable of mediating antinociceptive effects at the level of the spinal cord. The cAMP inhibitory actions of spinal cord α2‐adrenoceptors appear not to be directly linked with the antinociceptive actions of these receptors.
Lipid Droplets in Schwann Cells During Tellurium Neuropathy Are Derived from Newly Synthesized Lipid
Authors: Goodrum Jeffry F. , Earnhardt Todd S. , Goines Nelson D. , Bouldin Thomas W.
Published: October 2006

Abstract: Exposure of weanling rats to a diet containing elemental tellurium results in a peripheral neuropathy characterized by segmental demyelination and minimal axonal degeneration. One of the earliest ultrastructural abnormalities in tellurium neuropathy is an increased number of cytoplasmic lipid droplets in myelinating Schwann cells. The pathogenesis of these lipid droplets was investigated using light and electron microscopic autoradiography. Nerve lipids were either “prelabeled” with [3H]acetate via in vivo intraneural injection 3 days before a 2‐day exposure to tellurium, or “postlabeled” via in vivo intraneural injection or in vitro incubation with [3H]acetate following a 2‐day exposure to tellurium. In the prelabeled nerves, myelin became heavily labeled, but the tellurium‐induced cytoplasmic lipid droplets were rarely labeled. In the postlabeled nerves, the tellurium induced cytoplasmic lipid droplets were the most heavily labeled structures within the nerve. These data indicate that the tellurium‐induced lipid droplets in Schwann cells are derived from newly synthesized lipid rather than from the early breakdown and internalization of myelin lipids. The earliest biochemical abnormality observed in tellurium neuropathy is an inhibition of cholesterol synthesis at the squalene epoxidase step. This leads to an accumulation of squalene within the nerve. We conclude that the cytoplasmic lipid droplets in Schwann cells contain this accumulated lipid.
Subcellular Fractionation of Prohormone Processing Products in the Bag Cell Neurons
Authors: Sweet‐Cordero Alejandro , Fisher Joseph M. , Sossin Wayne , Newcomb Robert , Scheller Richard H.
Published: October 2006

Abstract: Multiple biologically active peptides arising from a common prohormone are sorted into distinct classes of dense core vesicles within the bag cell neurons of Aplysia californica. In this study, pulse‐chase analysis, combined with subcellular fractionation on Percoll gradients, are used to define the location of the prohormone processing events within the secretory pathway. Initial cleavage of the prohormone occurs in a light cellular compartment associated with the Golgi apparatus. The amino‐terminal processing intermediate then accumulates in a denser compartment containing small dense cores enclosed in membranous sacs, as well as larger immature vesicles. After 4 h, amino‐terminai products are found primarily in a much denser compartment which consists of large and small dense core vesicles. These large and small vesicles can be separated from each other using Percoll gradient centrifugation and are found to be enriched in amino‐ and carboxy‐terminal products, respectively. Lastly, membrane association experiments suggest differential binding to membranes, or integral membrane proteins, as a possible mechanism for sorting of amino‐ and carboxy‐terminal products.
Aluminum Inhibits Calpain‐Mediated Proteolysis and Induces Human Neurofilament Proteins to Form ProteaseResistant High Molecular Weight Complexes
Authors: Nixon Ralph A. , Clarke Jane F. , Logvinenko Kimberly B. , Tan Michelle K. H. , Hoult Mary , Grynspan Frida
Published: October 2006

Abstract: We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF‐H, NF‐M, and NF‐L, the high, middle, and low molecular weight subunits. respectively) and other cytoskeletal proteins using calcium‐activated neutral proteinase (calpain) purified from human brain. Calpain‐mediated proteolysis of NF‐L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50= 200 μM) and by aluminum lactate (IC50= 400 μM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 μM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two‐ to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 μM or higher, proportions of NF‐M and NF‐H formed urea‐insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50= 1.2 and 2.1 μM for aluminum lactate and A1C13, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.
Phosphorylation of P0 Glycoprotein in Peripheral Nerve Myelin
Authors: Suzuki Masaru , Sakamoto Yasushi , Kitamura Kunio , Fukunaga Kohji , Yamamoto Hideyuki , Miyamoto Eishichi , Uyemura Keiichi
Published: October 2006

Abstract: The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid‐dependent protein kinase (protein kinase C), calcium/calmodulin‐dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP‐dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.
Interleukin‐1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice
Authors: Kopmels B. , Wollman E. E. , Guastavino J. M. , Delhaye‐Bouchaud N. , Fradelizi D. , Mariani J.
Published: October 2006

Abstract: Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin‐1 (IL‐1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/ or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL‐1 cDNA probes, revealed a clear‐cut hyperexpression of IL‐1 mRNA in staggerer macrophages. An IL‐1 bioassay using the IL‐1‐responsive D10.G4 cell line also revealed a sixfold increase of IL‐1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3‐week to 1‐year‐old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL‐1 production by peripheral macrophages.
Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids
Authors: Remmers Ann E. , Nordby Gordon L. , Medzihradsky Fedor
Published: October 2006

Abstract: In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 μmol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the μ‐selective opioid [3H]Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans‐vaccenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the δ‐opioid [3H](D‐Pen2,D‐Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis‐vaccenic acid abolished the low‐affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the μ‐ and δ‐binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.
Comparison of the In Vitro Receptor Binding Properties of N‐[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes
Authors: Hall Håkan , Wedel Ilse , Halldin Christer , Kopp Jutta , Farde Lars
Published: October 2006

Abstract: The aim of the present investigation was to study and compare the in vitro binding properties of the two ra‐dioligands N‐[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug‐naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5‐hydroxytryptamine2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10‐fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half‐life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The K1 values were, however, two‐ to fourfold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.
Evidence for Functional Activity of Up‐Regulated Nicotine Binding Sites in Rat Striatal Synaptosomes
Authors: Rowell Peter P. , Wonnacott Susan
Published: October 2006

Abstract: A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1‐week administration of the potent nicotinic agonist, (+)‐anatoxin‐a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)‐anatoxin‐a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin‐treated animals compared to control. There was a 43% increase in the activity of 3 μM nicotine to release [3H]dopamine from synaptosomes of anatoxin‐treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)‐anatoxin‐a treatment on the uptake of [3H]dopamine. A strong positive correlation (r= 0.64) was found between the density of [3H]nicotine binding sites and the nicotine‐induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)‐anatoxin‐a, like nicotine, produces an up‐regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.
The Specificity of 130‐kDa Leech Sensory Afferent Proteins Is Encoded by Their Carbohydrate Epitopes
Authors: Bajt Mary Lynn , Cole Robert N. , Zipser Birgit
Published: October 2006

Abstract: From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130‐kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3–2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2–369, Laz7–79, and Laz6–212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6–297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9–84) is located on connective tissue surrounding the CNS. as well as in the peripheral tissues. The hydrophilic‐hydrophobic nature of the 130‐kDa sensory afferent and glial proteins was determined by phase separation with Triton X‐114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130‐kDa glycoproteins with N‐Glycanase or preincubation of their respective mAbs with α‐methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose‐containing Lan3–2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.
Evidence for the Binding of Calmodulin to Endogenous B‐50 (GAP‐43) in Native Synaptosomal Plasma Membranes
Authors: De Graan Pierre N. E. , Oestreicher A. Beate , De Wit Marina , Kroef Marian , Schrama Louise H. , Gispen Willem Hendrik
Published: October 2006

Abstract: The neuron‐specific protein B‐50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B‐50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B‐50/CaM binding, we used the cross‐linker disuccimidyl suberate (DSS) to form a covalent B‐50/CaM complex, which is stable on SDS‐PAGE. Upon addition of DSS, purified B‐50 and calmodulin form a 70‐kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti‐B‐50 and anti‐CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70‐kDa B‐50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B‐50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B‐50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.
Species and Regional Differences in the Expression of Cell‐Type Specific Elements at the Human and Rat Tyrosine Hydroxylase Gene Loci
Authors: Gandelman Kuan‐Yun , Coker George T. , Moffat Mark , O'Malley Karen L.
Published: October 2006

Abstract: The expression of the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), is confined to several different types of neuroendocrine cells. Using a transient assay system, we examined more than 10 kb of the human TH gene and 6.5 kb of 5’ flanking sequences of the rat TH gene for DNA elements that confer cell‐type specific expression. Surprisingly, these elements do not appear to be conserved in position or sequence across species. When plasmids containing DNA sequences —749 bp from the transcription start site of the rat gene were introduced into PC12 cells, up to sixfold higher levels of expression were observed as compared to the same fragments introduced into HepG2 cells or LAN‐1 cells. In contrast to the rat gene, analogous fragments of the human 5’ promoter failed to confer cell‐type specific expression. However, when plasmids containing a truncated thymidine kinase promoter and either orientation of a 760 bp 3’ human TH gene fragment were introduced into PC12 and LAN‐1 cells, we observed a six‐ and 3,5‐fold increase, respectively, over that observed for HepG2 cells. Subsequent deletion of this fragment led to significant activation of transcription in PC12 and HepG2 cell lines. These data indicate the presence of multiple elements contributing to the cell‐type specific expression of tyrosine hydroxylase genes.
Decreased Ferritin Levels in Brain in Parkinson's Disease
Authors: Dexter D. T. , Carayon A. , Vidailhet M. , Ruberg M. , Agid F. , Agid Y. , Lees A. J. , Wells F. R. , Jenner P. , Marsden C. D.
Published: October 2006

Abstract: Ferritin levels were measured in postmortem brain tissue from patients dying with Parkinson's disease [treated with L‐3,4‐dihydroxyphenylalanine (L‐DOPA)] and from control patients. Ferritin levels were decreased in the substantia nigra, caudate‐putamen, globus pallidus, cerebral cortex, and cerebellum when compared with age‐matched control tissues. However, in CSF from L‐DOPA‐treated patients and in serum from L‐DOPA‐treated and untreated parkinsonian patients, ferritin levels were normal. Previous studies have suggested an increased total iron content in substantia nigra of parkinsonian brain. The failure of substantia nigra ferritin formation to be stimulated by increased iron levels suggests some defect in iron handling in this critical brain region in Parkinson's disease. The reason for decreased ferritin levels throughout the parkinsonian brain is not clear but does not seem to reflect a general system deficit in ferritin.
Direct Effects of Chronic Ethanol Exposure on β‐Adrenergic and Adenosine‐Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures
Authors: Rabin Richard A.
Published: October 2006

Abstract: The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both β‐adrenergic and adenosine‐sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2‐chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2‐chloroadenosine. Activation of norepinephrine‐stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2‐chloroadenosine‐stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor‐stimulated cyclic AMP production.
Molecular Cloning of Microtubule‐Associated Protein 1 (MAP1A) and Microtubule‐Associated Protein 5 (MAP1B): Identification of Distinct Genes and Their Differential Expression in Developing Brain
Authors: Garner Craig C. , Garner Abigail , Huber Gerda , Kozak Christine , Matus Andrew
Published: October 2006

Abstract: cDNA clones encoding microtubule‐associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10‐kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11‐kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse‐hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap‐1, and the MAP5 gene to chromosome 13, designated Mtap‐5 MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.
In Vivo Binding to Peripheral Benzodiazepine Binding Sites in Lesioned Rat Brain: Comparison Between [3H]PK11195 and [18F]PK14105 as Markers for Neuronal Damage
Authors: Price G. W. , Ahier R. G. , Hume S. P. , Myers R. , Manjil L. , Cremer J. E. , Luthra S. K. , Pascali C. , Pike V. , Frackowiak R. S. J.
Published: October 2006

Abstract: Peripheral‐type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a “specific signal.”[3H]PK11195 binding peaked at 20–30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18F]PK14105‐specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral‐type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary‐ and remote‐lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.
Regulation by Thyroid Hormones of Glucose Transport in Cultured Rat Myotubes
Authors: Brodie Chaya
Published: October 2006

Abstract: Primary cultures of skeletal muscle obtained from neonatal rats possess a saturable process for active glucose uptake, the myotubes having a relatively high affinity for the substrate with a Km of 1 mM. The expression of the glucose transport system was most apparent after fusion of single myoblasts to multinucleated myotubes [3–4 days in vitro (DIV)], at which time glucose uptake increased sharply to reach plateau values at about 6–8 DIV. Treatment of the cells at age 6 DIV with triiodothyronine or thyroxine caused a marked increase in glucose uptake beginning 4 h after treatment and reaching a maximum at 24 h. Thyroid hormoneinduced increase in glucose uptake was not reduced by either tetrodotoxin or verapamil, thus indicating that the effect was not secondary to the ability of the hormone to increase contractile activity. The effect of thyroid hormones was eliminated completely by inhibition of protein synthesis. The results indicate that thyroid hormones play an important role in regulation of glucose transport in skeletal muscle.
Further Characterization of the Guinea Pig Cerebral Cortex Idazoxan Receptor: Solubilization, Distinction from the Imidazole Site, and Demonstration of Cirazoline as an Idazoxan Receptor‐Selective Drug
Authors: Wikberg Jarl E. S. , Uhlén Staffan
Published: October 2006

Abstract: We have demonstrated previously that [3H]idazoxan, besides being able to bind to α2‐adrenergic receptors, may also bind to a nonadrenergic idazoxan‐receptor site with high affinity. The idazoxan receptor is tightly bound to cellular membranes, and we have now developed a method to solubilize it from the guinea pig cerebral cortex by using the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS). The CHAPS‐solubilized receptor retains its binding properties for drugs: the membrane‐bound, as well as the solubilized, idazcxan receptor shows high affinities for a number of imidazolines (cirazoline, idazoxan, tolazoline, naphazoline, tramazoline, clonidine, and oxymetazoline), some imidazoles (medetomidine, detomidine), and guanfacine. By contrast, catecholamines (adrenaline, noradrenaline, isoprenaline, and dopamine) and a number of other neurotransmitters and neuromodulators (serotonin, histamine, glutamic acid, γ‐aminobutyric acid, glycine, and adenosine) show negligible affinities for the idazoxan receptor. Moreover, the idazoxan receptor shows grossly different binding properties for histamine, cimetidine, and imidazole‐4‐acetic acid compared to what has been described for the nonadrenergic imidazole site labeled by p‐[3H]aminoclonidine, indicating that the two receptor sites are distinct. Radioligand binding data further indicate that cirazoline is an idazoxan receptor‐selective drug (KD= 1 nM) showing a 50–210‐fold selectivity for binding to the idazoxan receptor when compared to α2‐adrenergic receptors and an about 500‐fold selectivity when compared to α1‐adrenergic receptors. We have also reviewed the literature for possible nonadrenergic actions of idazoxan and cirazoline, and we suggest that idazoxan receptors might be involved in the control of prolactin release from the pituitary.
Chronic Treatment of Rats with SCH‐23390 or Raclopride Does Not Affect the Concentrations of DARPP‐32 or Its mRNA in Dopamine‐Innervated Brain Regions
Authors: Grebb Jack A. , Girault Jean‐Antoine , Ehrlich Michelle , Greengard Paul
Published: October 2006

Abstract: DARPP‐32 (dopamine‐ and cyclic AMP‐regulated phosphoprotein, Mr= 32,000, as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) is a neuronal phosphoprotein that is enriched in neurons which possess dopamine D1 receptors, particularly striatonigral neurons. In rat brain slices, the phosphorylation state of DARPP‐32 is regulated by dopamine, acting through the dopamine D1 receptor and the adenylyl cyclase system. This study reports that chronic blockade (21 days) of either dopamine D1 receptors by SCH‐23390 or dopamine D2 receptors by raclopride does not affect the concentrations of DARPP‐32 in specific rat brain regions (striatum, thalamus, hippocampus, frontal cerebral cortical pole). Northern blot analysis indicates that the steady‐state level of DARPP‐32 mRNA in striatum is also unchanged by these treatments.
Arachidonic Acid Increases Inositol Phospholipid Metabolism and Glutamate Release in Synaptosomes Prepared from Hippocampal Tissue
Authors: Lynch M. A. , Voss K. L.
Published: October 2006

Abstract: We have been interested in the possibility that arachidonic acid or one of its 12‐lipoxygenase metabolites may function as a retrograde messenger in long‐term potentiation (LTP) in the dentate gyrus of the hippocampus. One criterion required of a retrograde messenger is that it stimulates presynaptic changes. Here, two possible presynaptic actions of arachidonic acid and its 12‐lipoxygenase metabolites, 12‐hydroxyeicosatetraenoic acid (HETE) and 12‐hydroperoxyeicosatetraenoic acid (HPETE), are examined. We report that arachidonic acid, HETE, and HPETE significantly increase both K+‐stimulated release of [3H]glutamate and [3H]inositol labelling of inositol phosphates in synaptosomes, whereas other biologically important fatty acids (oleic, palmitic, and stearic) failed to induce a similar response. The findings of these experiments are consistent with the hypothesis that arachidonic acid, HETE, or HPETE may play the role of a retrograde messenger in LTP.
Calmodulin Involvement in Stress‐ and Corticosterone‐Induced Down‐Regulation of Cyclic AMP‐Generating Systems in Brain
Authors: Gannon Maureen N. , McEwen Bruce S.
Published: October 2006

Abstract: Manipulation of the hypothalamic‐pituitary‐adrenal axis selectively alters α‐adrenergic potentiation of the cyclic AMP response to β‐adrenergic receptor stimulation in rat cerebral cortex. Calcium has been implicated in this α‐receptor‐mediated response, which may involve activation of phospholipases A2 and C and/or calmodulin‐dependent adenylate cyclase. We therefore investigated the effects of stress and corticosterone (CORT) on membrane calmodulin‐dependent adenylate cyclase and noradrenaline‐stimulated cyclic AMP accumulation in brain slices. Repeated stress for 21 days selectively attenuated the adenylate cyclase response to calcium/calmodulin in cerebral cortex membranes, without affecting basal or forskolin‐stimulated enzyme activity. There was no such effect in hippocampal membranes. The same pattern of response was elicited by daily CORT injection (50 mg/kg s.c.) for 21 days, while vehicle injection had no effect. CORT in the drinking water (400 μg/ml) elicited the same reduction of body weight as CORT injections, but had no effect on calmodulin adenylate cyclase. In parallel with calmodulin adenylate cyclase, cyclic AMP accumulation elicited by noradrenaline in slices of cerebral cortex was suppressed by both stress and daily CORT injections, with smaller effects observed with CORT in the drinking water. Unlike calmodulin adenylate cyclase, noradrenaline‐stimulated cyclic AMP accumulation in hippocampus showed the same suppression as that in cerebral cortex. These results are discussed in relation to the differential mode of coupling of α‐adrenergic receptors to cyclic AMP‐generating systems between brain regions. Glucocorticoid‐mediated suppression of noradrenaline‐stimulated cyclic AMP accumulation and calmodulin‐dependent adenylate cyclase may represent parallel effects of the hormone, but their co‐occurrence in cerebral cortex may also indicate some functional coupling and compartmentalization. The ability of daily CORT injection to mimic the effects of repeated stress, but inability of CORT ingestion at high doses in the drinking water to do so, suggests that the time course of CORT elevation and the coincidence of some type of stress may be important for the suppression of cyclic AMP‐generating systems in brain.
Staggerer Mutant Mouse Purkinje Cells Do Not Contain Detectable Calmodulin mRNA
Authors: Messer Anne , Plummer‐Siegard Julie , Eisenberg Bonnie
Published: October 2006

Abstract: Within the cerebellum calmodulin mRNA is found predominantly in Purkinje cells, with lower levels in granule cells and interneurons. The message shows developmental increases during the first 14 days postnatally. Surviving Purkinje cells of the staggerer (sg/sg) mutant, which are grossly stunted and lack tertiary dendritic spines, contain no detectable calmodulin mRNA, as assayed by Northern blot or an enhanced biotinylated in situ hybridization. This is in contrast to both the Lurcher Purkinje cells and sg/sg granule cells, which express normal levels of this mRNA up until the time they disappear. The sg/sg phenotype can be explained by a defective Purkinje‐cell‐specific regulatory mechanism for calmodulin.
Mechanisms of Toxicity and Cellular Resistance to 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine and 1‐Methyl‐4‐Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures
Authors: Reinhard John F. , Carmichael Stephen W. , Daniels Alejandro J.
Published: October 2006

Abstract: Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and 1‐methyl‐4‐phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19–6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4–1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration‐dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose‐free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days’exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic “buffer.”Furthermore, under conditions where MPP+ impairs mitochondrial respiration, cells survive by increasing their reliance on glycolytic metabolism.
Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla
Authors: Shukla Rakesh , Wakade Arun R.
Published: October 2006

Abstract: Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 μM), partially blocked (50%) by cadmium (100 μM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation‐evoked secretion by 80–90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 μg), nicotine (5 μM), muscarine (50 μg), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine‐and K+‐evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine‐evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetyl‐choline‐evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.