STUDIES ON THE PHYSIOLOGICAL ROLE OF TAURINE (2-aminoethane sulfonic acid) IN MAMMALIAN TISSUES by MOHAMED AKBERALI REMTULLA B.Sc, University of British Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE.DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES in THE DEPARTMENT OF PATHOLOGY (Faculty of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1979 (e) Mohamed Akberali Remtulla, 19 79 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ii ABSTRACT 'Studies on the Physiological Role of Taurine in Mammalian Tissues' Mohamed A. Remtulla Ph.D. (Pathology) Taurine (2-aminoethane sulfonic acid) is one of the most abundant free amino acids found in mammalian brain, heart and muscle. Taurine levels have also been shown to be altered in certain disease states. A physiological role for taurine in the maintainance of excitatory activity in muscle and nervous tissues has been suggested; however its possible mechanism of action is still uncertain. Early work on the pharmacological actions of taurine involved its possible conversion to isethionic acid (2-hydroxyethane sulfonic acid), a strong anion. This con• version was said to lead to the. conductance of cations into the cardiac cell. An analytical technique to measure isethionic acid in mammalian tissues was developed. The method involved extraction, partial purification and methylation with diazomethane, followed by gas-liquid chromatography. With this technique only trace amounts of isethionic acid were detected in rat heart (0.1 mg/lOOg wet weight tissue) and rat brain (0.2 mg per 100 mg wet weight tissue) and none was detected in dog hearts. Recovery of added isethionic acid was between 95 and 100%. The assay was validated using a sample of squid axoplasm. We were also unable to show 14:C-taurine conversion to 14C-isethionic acid in rat heart slices. Theories on the mode of action of taurine involving bioconversion to isethionic acid were therefore questioned. Some recent work suggested that taurine affects calcium kinetics in perfused guinea-pig hearts and calcium transport in rat skeletal muscle sarcoplasmic reticulum. We have investigated the effect of taurine on ATP-dependent calcium binding and oxalate-dependent calcium uptake in crude preparations of guinea-pig sarcolemma and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine (5-50 mM) was found to have no significant effect on either 2+ ATP-dependent Ca binding or uptake in both preparations. This result was observed at all calcium concentrations tested (0.5-100 u_M) and at all incubation times used (30 seconds to 20 minutes). Taurine (20 mM) neither altered the effect of cyclic AMP-dependent protein kinase on oxalate-dependent calcium uptake nor exerted a stabilization action on calcium transport in these systems. In a further attempt to determine the possible physiological role of taurine in mammalian tissues, we have investigated the effect of taurine on passive transport of sodium, potassium and calcium in synaptosomal preparations of rat brain. Taurine, in a dose dependent manner, was found to have an inhibitory effect on both calcium- uptake iv and release in these preparations. Amino acids structurally similar to taurine ( 3- alanine, homotaurine,. hypotaurine and y - aminobutyric acid) were also shown to inhibit calcium uptake in these preparations while a - alanine, proline and valine had no significant effect. Taurine (20 mM), though, did not alter the permeability of these preparations to either sodium or potassium. It thus appeared that taurine, and chemically related amino acids, can specifically alter calcium movements in these preparations. It is suggested that this effect is due to the binding of these agents to taurine receptor sites postulated to be present in these membranes. These observations may help to provide an insight into the physiological and pharmacological effects of taurine reported in cardiac and nervous tissues. Signed Dr. D.A. Applegarth (Supervisor), Department of Pathology, Faculty of Medicine, U.B.C. and Biochemical Diseases Laboratory, Children's Hospital, Vancouver. Signed .. , Dr. Sidney Katz (Supervisor) Division of Pharmacology Faculty of Pharmaceutical Sciences, U.B.C. v TABLE OF CONTENTS INTRODUCTION Page Introduction 1 REVIEW OF THE LITERATURE I. Historical Review 5 II. Biochemistry of Taurine 6 A. Distribution and Occurrence of Taurine .... 6 B. Taurine Metabolism 11 i. Carbamyltaurine H .ii. Taurocyamine 14 , iii. Isethionic Acid 15 C. Biochemistry of Taurine 17 i. Cysteine Sulfinic Acid Decarboxylase. '19 ii. Cystamine Dioxygenase Pathway ....... ^1 iii. Phosphoadenosine Phosphosulfate Pathway 24 D. Taurine Transport 26 III. Cardiac Disease and Taurine 30 A. Congestive Heart Failure 30 3. Hypertension 32 C. Ischemia 35 IV. Possible Physiological Actions of Taurine in the heart 35 A. Taurine and Arrhythmias 35 3> Taurine and Inotropism 39 vi Page V. Possible Cardiac Effects of Taurine on Calcium Transport 43 A. Inotropism and Calcium Transport 43 B. Inotropism, Calcium Transport and Taurine 46 C. Calcium Movements and Taurine in Other Tissues 48 Summary 49 VI. Possible Involvement of Taurine in Neurophysiology 50 A. Anticonvulsant Action of Taurine 51 B. Taurine in Retinal Degeneration 5 3 C. Taurine in Brain Development 5 4 D. Effect of Taurine on Endocrine Function.. 57 E. Taurine and Nerve Conduction 58 Summary 60 VII. Rationale 62 MATERIALS AND METHODS I. Studies with Isethionic Acid 66 A. Development of an Analytical Method for Isethionic Acid by Gas Liquid Chromatography 66 1. Reagents 66 2. Preparation of Isethionic Acid for use as Qualitative Standards 70 3. Methylation of Isethionic Acid 70 4. Silylation of Isethionic Acid 71 vii Page 5. Gas-liquid Chromatography: Flame Ionization Detector . .. 72 6. Gas-liquid Chromatography: Sulfur Detector • 72 7. Gas-liquid Chromatography: Mass Spectrometry 8. Nuclear Magnetic Resonance Spectroscopy.. 73 B. Analysis of Isethionic Acid in Mammalian Tissues 74 1. Reagents '. 74 2. Preparation of Heart, Brain and Other Tissues Used,for the Analysis of Isethionic Acid 75 3. Isolation of ISA from Tissues 77 4. Methylation of the Samples and Preparation of a Standard Curve 82 5. Analysis of Samples by GLC 83 C. Conversion of Taurine to Isethionic Acid ... 83 1. Reagents 83 14 2. Preparation of C-ISA as a marker for the Taurine Bioconversion Studies 86 3. Synthesis of"Isethionic Acid by Rat. Heart Slices . .. ..' .—.-. 87 II. Studies on the Effect of Taurine on Ion Transport Processes • ••• A. Effect of Taurine on ATP-dependent Calcium Transport in Guinea-pig Cardiac Muscle 89 1. Reagents 89 2. Preparation of Heart Ventricle Homogenates 95 3. Preparation of Microsomes Enriched in Sarcoplasmic Reticulum 95 viii Page 4. Characterization of Microsomal Preparation Enriched Sarcoplasmic Reticulum 9 8 5. ATP-dependent Calcium Uptake and Binding Assay 6. Assay for Cyclic AMP-dependent Protein Kinase Effect on Calcium Uptake 100 7. Studies on the Effect of Taurine on the Decay of Ca2+ -Transport Activity 101 8. Protein Assay , . 101 9. Calculations 101 10. Statistics 102 B. Studies on the Effect of Taurine on Passive Ion Transport in Rat Brain Synaptosomes 102 1. Reagents 102 2. Preparation of Synaptosomes 104 3. Characterization of Synaptosome Suspension by Electron Microscopy 106 4. Determination of the Osmotic Behaviour of Synaptosomes 106 5. Determination of Sodium and Potassium Permeability 107 6. Determination of Calcium Permeability 107 45 7. Determination of Loss of Ca from Pre• loaded Synaptosomes 108 8. Protein Assay 108 9. Statistics 108 RESULTS Studies with Isethionic Acid 112 A. Development of an Analytical Method for the Measurement of isethionic acid.._ 112 ix Page 1'. Chromatography of Methylated Isethionic Acid 112 a. Stationary Phases 112 b. Internal Standards 112 c. Mass-Spectrometry and NMR Spectra of Methylated Isethionic Acid 116 2 . Chromatography of Silylated Isethionic Acid. 119 B. Analysis of Isethionic Acid in Tissues 121 1. Isethionic Acid in Rat Heart and Brain Tissues 121 2. Isethionic Acid in Dog Heart Tissues 124 3. Isethionic Acid in "Molluscan Tissues 124 4. Isethionic Acid in Rat Milk Samples 125 C. Bioconversion of Taurine to Isethionic Acid.... 125 Taurine and Ion Transport 129 A. Effect of Taurine on ATP-dependent Calcium Transport in Guinea -pig Cardiac Muscle 129 1. Characterization of Ventricle Heart Homogenate and Sarcoplasmic Reticulum Enriched Preparation 129 2. Effect of Taurine on Calcium Uptake and Binding 133 3. The Effect of Taurine on the Time-course of Calcium Uptake and Binding 136 4. The Effect of Taurine on the Decay of Calcium Uptake Activity 137 5. Effect of Taurine on Cyclic AMP-dependent Protein-kinase Stimulated Calcium Uptake.... 137 x Page B. Effect of Taurine on Passive Ion Transport in Rat Brain Synaptosomes 141 1. Characterization of Synaptosomal Preparation 141 2. The Osmometric Behaviour of Synaptosomes.. 141 3. The Effect of Taurine on Sodium and Potassium Permeability in Synaptosomal.. , Preparation 144 4. The Effect of Taurine on the Passive Uptake and Release of Calcium in Synaptosomal Preparations 144 5. Dose-dependent Effect of Taurine on Calcium Uptake in Synaptosomal Preparation 146 6. Effect of Other Amino Acids on Calcium Uptake in Synaptosomal Preparations 149 DISCUSSION I. Bioconversion of Taurine to Isethionic Acid in the Regulation of Ion Flux 152 II. Taurine and Ion Transport 168 CONCLUSIONS 189 BIBLIOGRAPHY 190 APPENDICES . ...v.-: ............ .. .. • • • ........ 225 xi LIST OF TABLES Page Table 1: Isethionic acid in tissues analyzed 123 2: Conversion of 14 C-taurin.