UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 385 - ISSN 0346-6612 From the Department of Pharmacology University of Umeå, Umeå, Sweden Cyanide and Central Nervous System A Study with Focus on Brain Dopamine by Gudrun Cassel University of Umeå Umeå 1993 The drawing on the front cover: Gunilla Guldbrand Copyright © 1993 by Gudrun Cassel ISBN 91-7174-824-5 Printed in Sweden by Tidtryck Umeå 1993 From the Department of Pharmacology University of Umeå, Umeå, Sweden Cyanide and Central Nervous System A Study with Focus on Brain Dopamine AKADEMISK AVHANDLING som för avläggande av doktorsexamen i medicinsk vetenskap vid Umeå Universitet offentligen försvaras i Farmakologiska institutionens föreläsningssal A 5, byggnad6 A fredagen den 29 oktober 1993 kl 09.15 av Gudrun Cassel £ o £ AlV3 Handledare: Professor Sven-Åke Persson Fakultetsopponent: Professor Frode Fonnum ABSTRACT Cyanide and Central Nervous System - a study with focus on brain dopamine. The brain is a major target site in acute cyanide intoxication, as indicated by several symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and inhibited production of energy. In this thesis, some neurotoxic effects of cyanide, in particular, the effects on dopaminergic pathways were studied. In a previous study, decreased levels of striatal dopamine and HVA were found after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine were found in rats showing convulsions after infusion of low doses of cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose). Increased striatal dopamine synthesis was observed in rats after cyanide treatment and in vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose- dependently increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but not that of PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of dopamine. In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine D j- and D2 -receptor binding 1 hour after injection. Increased extracellular levels of striatal dopamine and homovanillic acid were also shown after cyanide 2.0( mg/kg; i.p.). DOPAC and 5-HIAA were slightly decreased. This indicates an increased release or an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Thus the effects of cyanide on dopamine D j- and D2 ~receptors could in part be due to cyanide-induced release of dopamine. Because of reported changes in intracellular calcium in cyanide-treated animals, the effects of cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not to affect the inositol phospholipid breakdown in vitro. The effects of cyanide on the synthesis and metabolism of brain GAB A were also examined. A decreased activity of both GAD and GAB A-T were found in the rat brain tissue. The reduced activity of GAB A-T, but not that of GAD returned to the control value after adding PLP in the incubation media. The cyanide-produced reduction of GABA levels will increase the susceptibility to convulsions, and could partly be due to GAD inhibition. In conclusion, cyanide affects the central nervous system in a complex manner. Some effects are probably direct. The main part, however, appears to be secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide. Keyword: CNS; cyanide; dopaminergic system; convulsions; receptor binding; tyrosine hydroxylase; monoamine oxidase; extracellular release; inositol phosphate; GABA; GAD. ISBN 91-7174-824-5 ABSTRACT Cyanide and Central Nervous System - a study with focus on brain dopamine. The brain is a major target site in acute cyanide intoxication, as indicated by several symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and inhibited production of energy. In this thesis, some neurotoxic effects of cyanide, in particular, the effects on dopaminergic pathways were studied. In a previous study, decreased levels of striatal dopamine and HVA were found after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine were found in rats showing convulsions after infusion of low doses of cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose). Increased striatal dopamine synthesis was observed in rats after cyanide treatment andin vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose-dependently increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but not that of PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of dopamine. In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine D j- and D2- receptor binding 1 hour after injection. Increased extracellular levels of striatal dopamine and homo vanillic acid were also shown after cyanide (2.0 mg/kg; i.p.). DOPAC and 5- HIAA were slightly decreased. This indicates an increased release or an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Thus the effects of cyanide on dopamine D j- and D2-receptors could in part be due to cyanide-induced release of dopamine. Because of reported changes in intracellular calcium in cyanide-treated animals, the effects of cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not to affect the inositol phospholipid breakdown in vitro. The effects of cyanide on the synthesis and metabolism of brain GABA were also examined. A decreased activity of both GAD and GAB A-T were found in the rat brain tissue. The reduced activity of GABA-T, but not that of GAD returned to the control value after adding PLP in the incubation media. The cyanide-produced reduction of GABA levels will increase the susceptibility to convulsions, and could partly be due to GAD inhibition. In conclusion, cyanide affects the central nervous system in a complex manner. Some effects are probably direct. The main part, however, appears to be secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide. Keyword: CNS; cyanide; dopaminergic system; convulsions; receptor binding; tyrosine hydroxylase; monoamine oxidase; extracellular release; inositol phosphate; GABA; GAD. CONTENTS List of papers.....................................................................................................5 Abbrevations......................................................................................................6 Introduction........................................................................................................7 Background......................................................................................................................7 Neurotoxic effects of cyanide........................................................................................ 7 Dopaminergic system......................................................................................................9 Synthesis of dopamine........................................................................................ 9 Storage, release and reuptake of dopamine...................................................... 10 Metabolism of dopamine.................................................................................... 11 Regulation of the dopamine neurons.................................................................11 GABAergic system......................................................................................................... 13 The purpose of the thesis................................................................................. 14 Materials and methods...................................................................................... 15 Animals..............................................................................................................................15 Chemicals......................................................................................................................... 15 Radiochemicals....................................................................................................15 Other chemicals...................................................................................................15 Methods............................................................................................................................15 Methods used to kill the animals.......................................................................15 Selection and handling of brain tissue............................................................... 16 HPLC analysis......................................................................................................16 Convulsive threshold estimation (Paper I)....................................................... 16 In vivo estimation of dopamine synthesis. (Paper II).......................................17 Microdialysis (Paper VI).................................................................................... 17 Dopamine D l- and D2-receptor binding (Paper V )........................................17 Inositol phospholipid breakdown (Paper VI)...................................................18 Assay of TH activity (Paper III)........................................................................ 18 Assay of MAO activity (Paper IV)....................................................................18