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Studies on the physiological role of taurine (2-aminoethane sulfonic acid) in mammalian tissues Remtulla, Mohamed Akberali 1979

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STUDIES ON THE PHYSIOLOGICAL ROLE OF TAURINE (2-aminoethane su l f o n i c acid) IN MAMMALIAN TISSUES by MOHAMED AKBERALI REMTULLA B.Sc, University of B r i t i s h 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 t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1979 (e) Mohamed Akberali Remtulla, 19 79 In present ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Co lumbia, I agree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy. I f u r t h e r agree that permiss ion f o r ex tens ive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s en t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n pe rm i s s i on . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 i i ABSTRACT 'Studies on the Physiological Role of Taurine i n Mammalian Tissues' Mohamed A. Remtulla Ph.D. (Pathology) Taurine (2-aminoethane su l f o n i c acid) i s one of the most abundant free amino acids found i n mammalian brain, heart and muscle. Taurine levels have also been shown to be altered i n c e r t a i n disease states. A physiological role for taurine i n the maintainance of excitatory a c t i v i t y i n muscle and nervous tissues has been suggested; however i t s possible mechanism of action i s s t i l l uncertain. Early work on the pharmacological actions of taurine involved i t s possible conversion to i s e t h i o n i c acid (2-hydroxyethane sulfonic acid), a strong anion. This con-version was said to lead to the. conductance of cations into the cardiac c e l l . An a n a l y t i c a l technique to measure i s e t h i o n i c acid i n mammalian tissues was developed. The method involved extraction, p a r t i a l p u r i f i c a t i o n and methylation with diazomethane, followed by gas-liquid chromatography. With thi s technique only trace amounts of i s e t h i o n i c acid were detected i n 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 i n dog hearts. Recovery of added i s e t h i o n i c acid was between 95 and 100%. The assay was validated using a sample of squid axoplasm. We were also unable to show 1 4:C-taurine conversion to 1 4 C - i s e t h i o n i c acid i n r a t heart s l i c e s . Theories on the mode of action of taurine involving bioconversion to i s e t h i o n i c acid were therefore questioned. Some recent work suggested that taurine a f f e c t s calcium k i n e t i c s i n perfused guinea-pig hearts and calcium transport i n r a t s k e l e t a l muscle sarcoplasmic reticulum. We have investigated the e f f e c t of taurine on ATP-dependent calcium binding and oxalate-dependent calcium uptake i n crude preparations of guinea-pig sarcolemma and in microsomal preparations enriched i n sarcoplasmic reticulum. Taurine (5-50 mM) was found to have no s i g n i f i c a n t e f f e c t on either 2+ ATP-dependent Ca binding or uptake i n both preparations. This r e s u l t was observed at a l l calcium concentrations tested (0.5-100 u_M) and at a l l incubation times used (30 seconds to 20 minutes). Taurine (20 mM) neither altered the e f f e c t of c y c l i c AMP-dependent protein kinase on oxalate-dependent calcium uptake nor exerted a s t a b i l i z a t i o n action on calcium transport i n these systems. In a further attempt to determine the possible physiological role of taurine i n mammalian tissues, we have investigated the e f f e c t of taurine on passive transport of sodium, potassium and calcium i n synaptosomal preparations of rat brain. Taurine, i n a dose dependent manner, was found to have an i n h i b i t o r y e f f e c t on both calcium- uptake i v and release i n these preparations. Amino acids s t r u c t u r a l l y similar to taurine ( 3 - alanine, homotaurine,. hypotaurine and y - aminobutyric acid) were also shown to i n h i b i t calcium uptake i n these preparations while a - alanine, proline and valine had no s i g n i f i c a n t e f f e c t . Taurine (20 mM), though, did not a l t e r the permeability of these preparations to either sodium or potassium. I t thus appeared that taurine, and chemically related amino acids, can s p e c i f i c a l l y a l t e r calcium movements i n these preparations. It i s suggested that t h i s e f f e c t i s due to the binding of these agents to taurine receptor s i t e s postulated to be present i n these membranes. These observations may help to provide an insight into the physiological and pharmacological e f f e c t s of taurine reported i n 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. H i s t o r i c a l Review 5 II. Biochemistry of Taurine 6 A. D i s t r i b u t i o n and Occurrence of Taurine .... 6 B. Taurine Metabolism 11 i . Carbamyltaurine H . i i . Taurocyamine 14 , i i i . Isethionic Acid 15 C. Biochemistry of Taurine 17 i . Cysteine S u l f i n i c Acid Decarboxylase. '19 i i . Cystamine Dioxygenase Pathway ....... ^1 i i i . Phosphoadenosine Phosphosulfate Pathway 2 4 D. Taurine Transport 26 III. Cardiac Disease and Taurine 30 A. Congestive Heart Failu r e 30 3 . Hypertension 32 C. Ischemia 35 IV. Possible Physiological Actions of Taurine i n the heart 35 A. Taurine and Arrhythmias 35 3> Taurine and Inotropism 39 v i 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 i n Other Tissues 48 Summary 49 VI. Possible Involvement of Taurine i n Neurophysiology 50 A. Anticonvulsant Action of Taurine 51 B. Taurine i n Retinal Degeneration 5 3 C. Taurine i n Brain Development 5 4 D. Ef f e c t 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 A n a l y t i c a l 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. S i l y l a t i o n of Isethionic Acid 71 v i i 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 i n Mammalian Tissues 74 1. Reagents '. 74 2. Preparation of Heart, Brain and Other Tissues Used,for the Analysis of Isethionic Acid 75 3. I s o l a t i o n 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 S l i c e s . . .. ..' .—.-. 87 II. Studies on the E f f e c t of Taurine on Ion Transport Processes • ••• A. E f f e c t of Taurine on ATP-dependent Calcium Transport i n Guinea-pig Cardiac Muscle 89 1. Reagents 89 2. Preparation of Heart V e n t r i c l e Homogenates 95 3. Preparation of Microsomes Enriched i n Sarcoplasmic Reticulum 9 5 v i i i Page 4. Characterization of Microsomal Preparation Enriched Sarcoplasmic Reticulum 9 8 5. ATP-dependent Calcium Uptake and Binding Assay 6 . Assay for C y c l i c AMP-dependent Protein Kinase E f f e c t on Calcium Uptake 100 7. Studies on the E f f e c t of Taurine on the Decay of Ca2+ -Transport A c t i v i t y 101 8. Protein Assay , . 101 9 . Calculations 101 10. S t a t i s t i c s 102 B. Studies on the E f f e c t of Taurine on Passive Ion Transport i n 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 . S t a t i s t i c s 108 RESULTS Studies with Isethionic Acid 112 A. Development of an A n a l y t i c a l Method for the Measurement of i s e t h i o n i c acid.._ 112 i x 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 S i l y l a t e d Isethionic Acid. 119 B. Analysis of Isethionic Acid i n Tissues 121 1. Isethionic Acid i n Rat Heart and Brain Tissues 121 2. Isethionic Acid i n Dog Heart Tissues 124 3. Isethionic Acid i n "Molluscan Tissues 124 4. Isethionic Acid i n Rat Milk Samples 125 C. Bioconversion of Taurine to Isethionic Acid.... 125 Taurine and Ion Transport 129 A. E f f e c t of Taurine on ATP-dependent Calcium Transport i n Guinea -pig Cardiac Muscle 129 1. Characterization of V e n t r i c l e Heart Homogenate and Sarcoplasmic Reticulum Enriched Preparation 129 2. E f f e c t of Taurine on Calcium Uptake and Binding 133 3. The E f f e c t of Taurine on the Time-course of Calcium Uptake and Binding 136 4. The E f f e c t of Taurine on the Decay of Calcium Uptake A c t i v i t y 137 5. E f f e c t of Taurine on C y c l i c AMP-dependent Protein-kinase Stimulated Calcium Uptake.... 137 x Page B. E f f e c t o f T a u r i n e on P a s s i v e I o n T r a n s p o r t i n R a t B r a i n Synaptosomes 141 1. C h a r a c t e r i z a t i o n o f S y n a p t o s o m a l P r e p a r a t i o n 141 2. The Os m o m e t r i c B e h a v i o u r o f S y n a p t o s o m e s . . 141 3. The E f f e c t o f T a u r i n e on Sodium and P o t a s s i u m P e r m e a b i l i t y i n S y n a p t o s o m a l . . , P r e p a r a t i o n 144 4. The E f f e c t o f T a u r i n e on t h e P a s s i v e U p t a k e and R e l e a s e o f C a l c i u m i n S y n a p t o s o m a l P r e p a r a t i o n s 144 5. D o s e - d e p e n d e n t E f f e c t o f T a u r i n e on C a l c i u m U p t a k e i n S y n a p t o s o m a l P r e p a r a t i o n 146 6. E f f e c t o f O t h e r Amino A c i d s on C a l c i u m U p t a k e i n S y n a p t o s o m a l P r e p a r a t i o n s 149 DISCUSSION I. B i o c o n v e r s i o n o f T a u r i n e t o I s e t h i o n i c A c i d i n t h e R e g u l a t i o n o f I o n F l u x 152 I I . T a u r i n e and I o n T r a n s p o r t 1 6 8 CONCLUSIONS 1 8 9 BIBLIOGRAPHY 1 9 0 APPENDICES . . . . . . v . - : . . . . . . . . . . . . .. .. • • • . . . . . . . . 225 x i LIST OF TABLES Page Table 1: Isethionic acid i n tissues analyzed 1 2 3 14 . 14 2: Conversion of C-taurine to C-isethionic acid by rat heart s l i c e s 127 3 3: H-Ouabaih*binding assay of microsomal enriched S.R. preparations 132 4: E f f e c t of taurine on calcium uptake and binding i n guinea-pig heart v e n t r i c l e homogenates and S.R. enriched preparations.. 134 5: The e f f e c t of taurine on calcium uptake and binding at various calcium concentrations i n guinea-pig heart v e n t r i c l e homogenates and S.R. enriched preparations 135 6: The e f f e c t of taurine on c y c l i c AMP-dependent protein kinase-stimulated calcium uptake i n guinea-pig heart v e n t r i c l e homogenates and S.R. enriched preparations 140 7: The e f f e c t of taurine on sodium (A) and potassium (B) permeability i n synaptosomes.. 145 8: The fragmentation and rearrangements of the two methylation products of i s e t h i o n i c acid that were analyzed using an OV-17 column.... 154 x i i LIST OF FIGURES Page Figure 1: The structure of taurine and i t s metabolites 1 2 2 : Schematic diagram i l l u s t r a t i n g the pathway of taurine biosynthesis i n mammals 1 8 3 : Metabolic pathway of cysteine to taurine.... 2 0 4: Biosynthesis of taurine v i a cysteamine 2 2 5 : Structure of Coenzyme A 2 5 6: Flow chart of the i s o l a t i o n of i s e t h i o n i c acid from tissues 7 8 7 : Flow diagram for the preparation of heart microsomes enriched i n sarcoplasmic reticulum 9 6 8 : Flow diagram for the preparation of rat brain synaptosomes 1 0 6 9: Chromatographic separation of the products of methylation of i s e t h i o n i c acid and s a l i c y l i c acid using a flame i o n i z a t i o n detector 1 1 3 1 0 : Chromatographic separation of the products of methylation of i s e t h i o n i c acid and 1-butahe s u l f o n i c , a c i d using a flame photometric (sulfur) detector 1 1 : Mass spectra of the products of methylation of i s e t h i o n i c acid 1 1 7 1 2 : Nuclear magnetic resonance spectra of the products of methylation of i s e t h i o n i c acid (A), 1-butanesulfonic acid (B) and methoxy-ethanol (G) - . v. . . . ... . . . . . . . 1 1 8 1 3 : Time course of chromatographic behaviour of the s i l y l a t e d products of i s e t h i o n i c acid.... 1 2 0 1 4 : C a l i b r a t i o n curve of methylated i s e t h i o n i c acid 1 2 2 14 1 4 1 5 : Separation of C-taurine, C-isethionic acid and the rat heart s l i c e s - l ^ c - t a u r i n e . incubation products by paper chro m a t o g r a p h y . . . 1 2 8 x i i i Figure 16: Electron micrograph of microsomal preparations enriched i n sarcoplasmic reticulum 17: Electron micrograph of the guinea-pig v e n t r i c l e heart homogenate preparation... 131 18: Time course e f f e c t of taurine on calcium uptake and binding i n guinea-pig heart v e n t r i c l e homogenate and sarcoplasmic reticulum enriched preparations 137 19: The e f f e c t of taurine on the decay of calcium uptake a c t i v i t y i n guinea-pig v e n t r i c l e homogenates and S.R. enriched preparation 139 20: Electron micrograph of a t y p i c a l rat brain synaptosomal preparation 14 2 21: The e f f e c t of Na2SO^ concentration on the E520 °f a suspension of synaptosomes 143 45 2+ 22: The e f f e c t of taurine on (A) Ca uptake and (B) release of 4 5 c a 2 + f r o m preloaded rat synaptosomal preparations 147 23: The e f f e c t of various concentrations of taurine on 45caCl2 uPtake i n brain synaptosomal preparations 148 24: The e f f e c t of various amino acids on calcium uptake i n brain synaptosomal preparations 149 25: Mass spectral rearrangements and frag-mentation of dime thy lated- i s e t h i o n i c acid 155 26: Mass spectral rearrangements and frag-mentation of the methylester of i s e t h i o n i c acid 156 Page 130 xiv ACKNOWLEDGEMENTS The author wishes to extend his deepest gratitude to Dr. D.A. A p p l e g a r t h a , b and Dr. Sidney Katz° for t h e i r encouragement and guidance throughout the course of t h i s study. Without t h e i r assistance, t h i s work would have been impossible. I would l i k e to extend my thanks to Dr. W.L. Dunn , Dr. R.H. Pearce b, Dr. P.E. Reid b, Dr. D.E. Brooks b and L.I. Woolf for t h e i r suggestions at the graduate committee meetings and he l p f u l c r i t i c i s m of the Ori g i n a l manuscript. I wish to thank the s t a f f of the Biochemical Diseases Laboratory at the Children's Hospital for t h e i r cooperation and assistance, during my work (from September 1974 to May 1977) i n the laboratory. Department of Paediatrics, D i v i s i o n of Paediatric Pathology, UBC and Biochemical Diseases Laboratory, Children's Hospital. Department of Pathology, Faculty of Medicine, UBC. Divisi o n of Pharmacology, Faculty of Pharmaceutical Sciences, UBC. Divisi o n of Neurological Sciences, Department of Psychiatry Faculty of Medicine, UBC. xv I also wish to thank a l l members of the Faculty, s t a f f and graduate student body i n the Faculty of Pharmaceutical Sciences for making my stay (from May 1977 to December 1978) at the Faculty very enjoyable. My thanks also go to the s t a f f of the Woodward Biomedical Library of the University of B r i t i s h Columbia for t h e i r constant kindness and helpfulness i n tracing down references and locating recondite journals. Fi n a n c i a l support i n the form of studentship, and for the cost of equipment and chemicals from B r i t i s h Columbia Heart Foundation to Dr. D.A. Applegarth i s gr a t e f u l l y acknowledged. F i n a l l y I would l i k e to acknowledge a number of other people for the help they have given me during the course of my graduate studies: e f Dr. B.D. Roufogalis' and Dr. A.G.F. Davidson for t h e i r h e l p f u l comments and assistance. Faculty of Pharmaceutical Sciences, UBC Department of Paediatrics, Faculty of Medicine, UBC, and Department of C l i n i c a l Investigation F a c i l i t y , Children's Hospital, Vancouver.-xvi Dr. T.R.C. Boyde y f o r his i n s p i r a t i o n . Dr. Brenda Morrison for her assistance with S t a t i s t i c a l Analysis. Dr. Frank Abbott 1 for his h e l p f u l comments and assistance with the mass spectral interpretations. Mrs. Celine Gunawardene1 for doing an excellent job of typing the f i n a l copy of the manuscript. My parents and brothers for t h e i r encouragements and understanding. y Makerere University, Kampala, Uganda, East A f r i c a . (Present Address: Department of Biochemistry, University of Hong Kong, Hong Kong). k Department of Health Care and Epidemiology, University of B r i t i s h Columbia. 1 Faculty of Pharmaceutical Sciences, U.B.C. x v i i DEDICATED TO THE CAUSE OF SCIENCE AND MEDICINE ABBREVIATIONS USED ADP ATP ATPase BSA °C Ci u Ci (Ca 2 ++Mg 2 +)-ATPase CHF CNS cm CoA cpm CSA C y c l i c AMP DEGS DMCS DMF E520 E.C. eds. EGTA EKG EM ERG et a l . f t adenosine 5--diphosphate adenosine 5'-triphosphate adenosine triphosphatase N, O-bis-(trimethylsilyl)-acetamide degree centigrade Curie microCurie 2+ Mg -dependent calcium stimulated ATPase congestive heart f a i l u r e central nervous system centimeters Coenzyme A counts per minute cysteine s u l f i n i c acid adenosine 3', 5' - monophosphate diethylene g l y c o l succinate dimethylchlorosilane dimethylformamide extinction at 520 nanometers enzyme c l a s s i f i c a t i o n , established by the commission on enzymes of the International Union of Biochemistry (Biochim. Biophys. Acta 429:l f 1976) editors Ethyleneglycol - b i s - ( B-Aminoethyl ether) N,N:?-tetra-acetic acid electrocardiogram electron microscopy electroretinogram and others feet xix g gram x g a c c e l e r a t i o n o f g r a v i t y GABA Y - a m i n o b u t y r i c a c i d GC-MS g a s - l i q u i d c h r o m a t o g r a p h y mass s p e c t r o m e t r y GLC g a s - l i q u i d c h r o m a t o g r a p h y HMDS h e x a m e t h y l d i c h l o r o s i l a n e H o m o t a u r i n e 3-aminopropane s u l f o n i c a c i d h r . h o u r H y p o t a u r i n e 2 - a m i n o e t h y l s u l f i n i c a c i d i . d . i n t e r n a l d i a m e t e r ISA i s e t h i o n i c a c i d I s e t h i o n i c a c i d 2 - h y d r o x y e t h a n e s u l f o n i c a c i d Kg k i l l o g r a m K M i c h a e l i s c o n s t a n t m y l m i c r o l i t e r ml m i l l i l i t e r M m o l a r m m i l l i y m i c r o yM m i c r o m o l a r mEq m i l l i e q u i v a l e n t MEA m e r c a p t o e t h a l a m i n e mg m i l l i g r a m m in. m i n u t e mM m i l l i m o l a r mm m i l l i m e t e r s N n o r m a l NAD:: n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e N a + , K + - A T P a s e S o d i u m - p o t a s s i u m d e p e n d e n t ATPase Na-ISA Sodium s a l t o f I s e t h i o n i c a c i d NMR n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y OV-1 m e t h y l s ! l i c o n e OV-17 m e t h y l p h e n y l s i l i c o n e xx P p r o b a b i l i t y PAPS 3 ' - p h o s p h o a d e n o s i n e - 5 ' - p h o s p h o s u l f a t e PEGS p o l y e t h y l e n e g l y c o l s u c c i n a t e P i o r t h o p h o s p h a t e Rf r e t a r d a t i o n f a c t o r S.E.M. s t a n d a r d e r r o r o f t h e mean S.R. s a r c o p l a s m i c r e t i c u l u m T a u r i n e 2-aminoethane s u l f o n i c a c i d TMCS t r i m e t h y l c h l o r o s i l a n e T r i s t r i s ( H y d r o x y m e t h y 1 ) aminomethane V maximum v e l o c i t y max J v / v u n i t o f volume p e r u n i t o f volume w/v u n i t o f w e i g h t p e r u n i t o f volume % p e r c e n t / p e r < l e s s t h a n > g r e a t e r t h a n £ e q u a l t o o r g r e a t e r t h a n x x i I N T R O D U C T I O N 2 Introduction Taurine (2-aminoethanesulfonic acid) i s a su l f o n i c acid analogue of B - alanine (Figure 1). I t i s present i n a l l mammals examined (rat, guinea-pig, mice, dog,- ' ~ . rabbit, horse, chicken, cow, monkeys,-pig, man, .hamster, sheep, cat, etc) as a free amino acid (in r e l a t i v e l y high concentrations). I t occurs to only a limited extent i n plants, primarily i n lower forms. I t i s not u t i l i z e d for protein synthesis (Banos et a l . , 1971) nor as a source of energy (Hayes, 1976). The single c l e a r l y defined function of taurine i n animals i s the formation of b i l e s a l t s which serve as emulsifying agents i n the gut to f a c i l i t a t e l i p i d digestion. Taurine i s present i n high concentrations i n the rat s k e l e t a l muscle (14 w moles/g), heart (28 u moles/g) and, i n various areas of the brain (1 to 100 u moles/g) . I t does not seem l o g i c a l that taurine could be present i n the mammalian body with l i t t l e or no further function. (Gaull, 1971). This l i n e of thought has triggered many workers to evaluate the possible physiological role of taurine i n mammals. The evidence concerning the possible function of taurine i n the heart .(Huxtablerl976a;; „Grosso and Bfessler, 1976) and brain (Sturman et a l . , 1977a; Barbeau et a l . , 1975; Mandel and Pasantes-Morales, 19 7 8) has recently been reviewed. Evidence for a functional role of taurine as a neurotransmitter 3 or as a regulator of calcium and potassium fluxes have also been dealt with (Huxtable and Barbeau, eds, 1976; Barbeau and Huxtable, eds. 1978; Jacobsen and Smith, 1968). At present, the available data i n the l i t e r a t u r e , do not e s t a b l i s h c l e a r l y the role of taurine i n mammalian tissues. This thesis i s concerned with investigations on a possible physiological role of taurine i n the regulation of ion transport. REVIEW OF THE LITERATURE 5 I. HISTORICAL REVIEW As far as i t can be established, the name taurine did not appear i n the l i t e r a t u r e u n t i l 18 38 where i t i s found i n a paper by Demarcay(18 38). A .crystalline - material from Ox b i l e was obtained, similar to a compound c a l l e d gjaylen-a-sparagin described 11 years e a r l i e r by Tiedman and Gmelin (1827) who termed i t taurine, a name which Demarcay credited to Gmelin without i n d i c a t i n g when Gmelin had f i r s t used that name. In 1846, Redtenbacher demonstrated that taurine, contained both s u l f u r and nitrogen. Its structure at t h i s point was delineated. Evidence for the presence of taurine i n tissues from a number of vertebrate species gradually accumulated over the next decades (Krukenberg, 1881a, 1882, 1885; Limbricht, 1863, 1865; Staedler and Frerichs, 1858; Valenciennes and Fremy, 1855, 1857) and towards the end of the 19th century, surveys by Krukenberg (1881a, 1881b) indicated that taurine had a wide d i s t r i b u t i o n i n animals. This view has been f u l l y supported by numerous reports on the d i s t r i -bution and occurence of taurine published by recent i n v e s t i -gators (Huxtable and Bressler, 1974a, 1974b; Grosso and Bressler, 1976; Kocsis et a l . , 1976; Perry and Hansen, 1973; Sturman and Gaull, 19 75). 6 II. BIOCHEMISTRY OF TAURINE A. D i s t r i b u t i o n and Occurence of Taurine In mammals, taurine i s present i n a l l tissues of the body, with s k e l e t a l muscle accounting for 75 per cent of the t o t a l body store (Awapara, 1956). In addition to s k e l e t a l muscle, the heart, brain and spleen also contain high concen-trations of taurine (Jacobsen and Smith, 196 8). As much as 50 per cent of the free amino acid content of the dog and r a t heart consists of taurine (Awapara et a l , 1950; Scharff and Wool, 1965). The concentration of taurine i n muscle or heart ranges from 5 to 40 ymoles/g wet weight and i n brain from 1 to 11 pmoles/g (Chanda and Himwich, 1970; Spaeth and Schneider, 1974; Perry and Hansen, 1973; Sturman and Gaull, 1975). The regional d i s t r i b u t i o n of taurine i n canine v e n t r i c l e has recently been reported (Kocsis et a l . , 1976;Crass and -Lombardini, 19 7 8 ) . - Higher taurine concentrations are found i n the endocardium than i n the epicardium; no evidence of a base-to-apex taurine gradient was found i n either the r i g h t or l e f t v e n t r i c l e of the dog. No s i g n i f i c a n t differences were observed.between the mean values for ri g h t and l e f t v e n t r i c l e s or ri g h t and l e f t a u r i c l e s . However, differences i n taurine levels i n auricles and ve n t r i c l e s were found from species to species. For example, the auricles of dog contain twice as much taurine as the v e n t r i c l e s , whereas rats, guinea-7 pigs and rabbits each showed higher taurine concentration i n the v e n t r i c l e s compared to the a u r i c l e s . The Purkinje tissue of the heart contained the highest concentration of l e f t v e n t r i cular taurine. Taurine levels i n various areas of the central nervous system (CNS) have also been measured (Kaczmarek, 1976; Lombardini, 1976). In the rat the highest amounts of taurine reported are found i n olfactory bulb, cerebral cortex, cerebellum, and striatum; (5 to 11 umoles/g) the lowest i n hypothalamus, medulla and spinal cord (1 to 3 umoles/g). However, according to Sturman (personal communication), the retina; may contain even higher levels of taurine (30 - 50 ymoles/g). The neurohypophysis and pineal glands contain the highest concentrations of taurine found i n animal tissues (30-100 pmoles/g) (Guidotti et a l . , 1972; Vellan, et a l . , 1970; Pasantes-Morales et a l . 1972). Studies on the subcellular d i s t r i b u t i o n of taurine i n brain fractions suggest that i t i s present i n synaptosomal fractions (5 to 26 ymoles/g protein) as well as i n the mitochondrial f r a c t i o n (Lombardini, 1976; Kaczmarek e t al.,1971; Sturman et a l . , 1976b; Rassin et a l . , 1976, 1977). Huxtable and Bressler (1972) studied the subcellular 14 . d i s t r i b u t i o n of r a d i o a c t i v i t y from (1,2 - C) taurine i n rat heart a f t e r i ntraperitoneal i n j e c t i o n . Of the t o t a l a c t i v i t y , 85 per cent of taurine was found i n the supernatant f r a c t i o n of a. homogenized-tissue-preparation. Approximately 12 per cent 8 was bound to the microsomal f r a c t i o n and less than 2 per cent i n the mitochondrial f r a c t i o n . Measurements of endogenous taurine i n beef heart revealed 17.2 umoles/g protein i n the microsomal f r a c t i o n , 1.5 umoles/g protein i n the-bulk p r e c i p i t a t e and 0.0 ymoles/g protein i n the mitochondrial f r a c t i o n . 35 The d i s t r i b u t i o n of injected S-taurine i n d i f f e r e n t organs has been determined i n several species. In vivo 35 experiments i n which S-taurine was injected into rats indicated that the rates of taurine uptake vary and organs can be divided into two groups depending on t h e i r rates of uptake. The organs that take up taurine rapidly are l i v e r , pancreas and kidney. Those that take up taurine slowly are s k e l e t a l muscle, heart and brain. The organs that accumulate taurine at a slower rate contain maximum amount of the t o t a l r a d i o a c t i v i t y a f t e r 3 to 5 days following a single i n j e c t i o n . This r e f l e c t s a r e d i s t r i -bution of the labeled taurine from the other organs. The loss of r a d i o a c t i v i t y i s also correspondingly slow i n s k e l e t a l muscle, heart and brain (Hope, 19 55; Awapara, 1957; Huxtable and Bressler, 1972; Sturman, 1973). Recently studies have shown that there i s an e f f e c t i v e blood-brain b a r r i e r preventing the passage of taurine into the mature brainv This was observed af t e r the in-vivo intraperitoneal i n j e c t i o n of massive doses of taurine i n a number of species such as r a t , mouse, guinea-pig and cat resulted i n no s i g n i f i c a n t increases i n taurine i n most 9 areas of the brain ( B a t t i s t i n i et a l . , 1969; Levi et a l . , 1967). However, there does seem to be some exchange of taurine occurring, because a f t e r i n t r a p e r i t o n e a l i n j e c t i o n or o r a l administration of tracer amounts of radioactive taurine to rats, measured amounts of la b e l appear i n the brain (Urquart et a l . , 19 74; Peck and Awapara, 196 7). Sturman et a l . , (1976a) injected humans with 35 . S-taurine and determined pool sizes by k i n e t i c analysis of plasma, urine and feces. Two pools for taurine were found; one pool was r e l a t i v e l y small and turned over rapidly; the other was much larger, but turned over at a much slower rate. According to Sturman, dietary taurine enters the rapidly exchangeable taurine pool, biosynthesis (in some species) being of the same order of magnitude as the dietary input. The rapidly, exchangeable pool then exchanges with the larger slowly exchangeable pool. The rapidly exchangeable taurine pool p a r t i c i p a t e s i n b i l e acid conjugation and i s metabolized to sulfate,carbon dioxide,water and ammonia and probably to is e t h i o n i c acid,, • by gut bacteria and removed by urinary excretion. Unchanged taurine i s also l o s t from the rapidly exchangeable pool through urinary excretion. Concentrations of: taurine i n the slowly exchangeable pool (skeletal muscle, heart and brain)are maintained constant. Much e f f o r t has been expended i n attempting to manipulate cardiac taurine concentrations. The taurine 10 concentrations of man, rats and other animals remain invariant under a range of experimental conditions. Rats fed a d i e t d e f i c i e n t i n vitamin Bg (essential substance for the rate l i m i t i n g enzyme 'cysteine s u l f i n i c acid decarboxylase' a c t i v i t y i n the biosynthesis of taurine from methionine or cysteine) were found to have unaltered concentrations of taurine. A s i m i l a r conservation of taurine i n the heart occurs i n rats fasted for prolonged periods. Feeding a t a u r i n e - r i c h diet has no e f f e c t on cardiac taurine concentrations since the additional taurine i s simply excreted i n the urine and feces (Sturman,. 1973; Awapara, 1956; Hope, 1955). A recent report from Sturman and his group (Knopf et a l . , 19 78) and Hayes, .(1976) have indicated that cats maintained on a taurine-deficient d i e t show marked depletion of taurine i n a number of tissues, including the heart. Carnivores generally obtain a taurine r i c h d i e t since taurine occurs i n high concentrations and ubiquitously i n animal tissues- Herbivores, on the other hand, receive a poor taurine diet,.as i t i s almost absent i n the plant world. In t h i s regard, Huxtable (1978) postulated that herbivores need to maintain active biosynthetic mechanisms for taurine, whereas carnivores, and to a lesser extent omnivores, not having such a need have evolved to the point that, i n the absence of dietary taurine, they are no longer capable of biosynthesizing s u f f i c i e n t taurine for t h e i r needs. 11 B. Taurine Metabolism In addition to the b i l e acids the three other main reported metabolites of taurine are: (i) Carbamyl taurine (Ureidotaurine) ( i i ) Taurocyamine (Guianidinotaurine) ( i i i ) Isethionic Acid (2-hydroxyethane su l f o n i c acid) The structures of these metabolites are shown i n Figure 1. i • Carbamyltaurine Carbamyltaurine was f i r s t reported i n human and canine urine by Salkowski (1873). Since that report, urinary excretion of carbamyltaurine has, been both confirmed (Schram and Crokaert, 1957; Thoai, et a l . , 1956) and denied (Schmidt and Cerecedo, 1928). Thoai, et a l . postulated i n 1958 that the reason for the presence of carbamyltaurine i n mammals was that i t formed part of a modified urea cycle based on taurine i n which the following series of reactions took place: CO 2 * Carbamyltaurine Taurocyamine N H o N H o I I C = 0 G = N H N H N H I I C H 2 C H 2 C H 2 0 H C H 2 N H C H o C H C H o C H 9 S 0 3 H S O 3 H S 0 3 H S O 3 H CARBAMYL TAURINE TAUROCYAMINE ISETHIONIC ACID TAURINE FIGURE 1 THE STRUCTURE OF TAURINE AND ITS METABOLITES O'Keefe and Smith (1973), using, a s p e c i f i c isotope d i l u t i o n technique, could not detect carbamyltaurine i n human urine. Since carbamyltaurine was previously reported to occur i n r at urine (Schram and Crokaert, 1957), O'Keefe and Smith examined homogenates of rat brain and l i v e r for the a b i l i t y of these tissues to synthesis carbamyltaurine using carbamyl-phosphate as carbamylating agent (in systems capable of synthesizing c i t r u l l i n e or carbamylaspartate). No synthesis could be detected. The early work on the i d e n t i f i c a t i o n of carbamyltaurine i n the urine of man, rat and dog could possibly have been an a r t i f a c t of the combination of taurine and urea to form corresponding ureido acid (as shown by the chemical equation below) occuring i n urine a f t e r passage through the kidney. CONH2 NH 7N H2 CH / I CO -I- NH 2CH 2CH 2S0 3H } NH^ + CH 2 UREA TAURINE AMMONIA CARBAMYLTAURINE This reaction has been reported to occur for the amino acids, tyrosine and phenylalanine i n urine giving corresponding ureido amino acids (Schmidt and Al l e n , 1920). It i s possible that the taurocyamine detected some 100 years 14 ago (Salkowski, 1873) i n urine was a r e s u l t of the process of i s o l a t i o n of this compound from the urine and not due to chemical processes occuring i n the body as suggested by Thoai et a l . , (19 56). At the present time, evidence i s that carbamyltaurine i s not a metabolic product of taurine metabolism i n mammalian systems. i i . Taurocyamine Taurocyamine, i s important as i t s phosphogen (phosphotaurocyamine) i n marine worms where i t occurs consistently i n large concentrations i n a l l marine worm species examined (Thoai and Roche, 19 60, Thoai and Robin 1965). In these species phosphotaurocyamine i s believed to function as part of a phosphagen system analoguous to the creatine system found i n human muscle. Taurocyamine i s believed to function as a phosphate acceptor and taurocyamine phosphokinase has been i s o l a t e d from marine worms (Thoai and Roche 1960). Phosphotaurocyamine has not been i d e n t i f i e d i n tissues of mammals (Jacobsen and Smith, 196 8) but taurocyamine hasbbeen i d e n t i f i e d i n r a t brain (Blass, 1960) and i n the urine of rats and man (Schram and Crokaert, 1957; Thoai et a l . , 1954). Mori et a l . (19 74) have quantitated taurocyamine i n brain samples of mice, guinea-pigs, rabbits, rats and man. In these tissues i t occurs at a concentration of 0.002 umole/g, approximately 15 1/1,000th. the taurine concentration. The guanidino compounds (Mori et a l . , 19 74) occur i n the human brain i n order, according to concentration: Arginine>guanidinobutyric>acid>glycocyamine>taurocyamine. The importance of taurocyamine i n the brain i s not established but i t does not appear to be a q u a n t i t a t i v e l y important constituent of the tissues examined. i i i . I s e t h i o n i c Acid The presence of i s e t h i o n i c acid (ISA) i n b i o l o g i c a l material was f i r s t reported by Koechlin (1955) who found that i t was the major anion of the axoplasm from the squid giant axon. These observations were subsequently confirmed and extended by Deffner and Hafter (1959, 1960, 1961) who noted that i s e t h i o n i c acid was found i n squids and molluscs generally, but was lacking, or present i n low concentration, i n crustacean and vertebrate nerve. Welty et a l . , i n 1962, reported the i s o l a t i o n and i d e n t i f i c a t i o n of i s e t h i o n i c acid from dog heart. They also claimed to show the conversion of cysteine and taurine to i s e t h i o n i c acid i n dog heart s l i c e s (Read and Welty, 1962). More recently, the formation of i s e t h i o n i c acid from taurine has been described i n homogenates of rat brain (Peck and Awapara, 1966). 16 Several studies have examined the presence of i s e t h i o n i c acid i n man: Goodman et a l . (1967) have reported the presence of 3 5S-.isethionic acid i n the urine of normal subjects and.children with Downs syndrome given 35 S-taunne. Jacobsen et a l . (1967) , using a sensitive and s p e c i f i c double-isotope derivative method, reported the f i r s t quantitative data on the urinary excretion of i s e t h i o n i c acid i n man. Patients with muscular diseases accompanied by muscle atrophy were found to excrete s i g n i f i c a n t l y less i s e t h i o n i c acid compared to l e v e l s excreted by patients with honmuscular diseases. Interest i n i s e t h i o n i c acid stemmed from the i d e n t i f i c a t i o n of the compound in dog heart tissue (Welty et a l . , 1962) and the claim that dog heart s l i c e s were able to convert taurine to i s e t h i o n i c acid (Read and Welty, 1962). Huxtable (1976a)proposed that the conversion of taurine to i s e t h i o n i c acid proceeds through the intermediary sulfoacetaldehyde. An analogy i s the conversion of glycine to glycolate through glyoxylate. The enzymatic mechanism 'of the conversion has hot been established. An i n t e r e s t i n g aspect of t h i s transformation of taurine to i s e t h i o n i c acid i s that a zwitterionic compound i s being converted to a strongly anionic one leading to the proposition that i s e t h i o n i c acid i s involved i n membrane e x c i t a b i l i t y (Mullins, 19 59) and 17 conductance of cations across cardiac c e l l membrane (Read and Welty, 1963, 1965; Chazov et a l . , 1974). A detailed mechanism has never been put forward. I t has, however, been suggested that the deamination of taurine to i s e t h i o n i c acid within the c e l l would release a charged group that would a t t r a c t cations (Welty, 196 3; Doctoral thesis, University of South Dakota). The question of the role of i s e t h i o n i c acid i n the conductance of cations i n mammalian tissue has been of i n t e r e s t to numerous workers (Jacobsen et a l . , 1967; D i e t r i c h and Diacono, 1971; Guidotti, et a l . , 1971; I-Iuxtable and Bressler, 1972; Spaeth and Schneider, 1974; Sturman, 1973), p a r t i c u l a r l y i n i t s implication i n the antiarrhythmic effects i n dog hearts (Read and Welty, 1965; Chazov et a l . , 1974). The major obstacle to the elucidation of the function of i s e t h i o n i c acid has been, the lack of good a n a l y t i c a l procedures. C. Biosynthesis of T.aurine Taurine biosynthesis i s a complex problem. Several routes have been established or postulated depending upon the animal species and tissue of o r i g i n . (Fig;2) .The-biosynthesis of taurine from cysteine has been well characterized i n rat l i v e r (Jacobsen and Smith, 1968). Synthesis of taurine i n cardiac tissue i s poorly understood, but does not appear to CO Figure 2. Schematic Diagram I l l u s t r a t i n g the Pathways of Taurine Biosynthesis i n Mammals. 19 occur by the same pathway operative i n the l i v e r (Jacobsen et a l . , 1964; Wainer, 1965). The rat, dog and cat heart, and human and cat l i v e r either lack key enzymes necessary for converting cysteine to taurine by the same route used in rat l i v e r (Jacobsen and Smith, 196 8) or can not convert cysteine to taurine i n s u f f i c i e n t quantities to maintain tissue pools. i . Cysteine S u l f i n i c Acid Decarboxylase In rat l i v e r , cysteine i s oxidized to cysteine s u l f i n i c acid (Chapeville and Fromageot, 1955) which may then be decarboxylated (Awapara, 19 5 3) to hypotaurine followed by oxidation to taurine. (Fig.3) The rate l i m i t i n g enzyme i n t h i s pathway i s cysteine s u l f i n i c acid decarboxylase. A l t e r n a t i v e l y , cysteine s u l f i n i c acid may be oxidized to cysteic acid (Awapara and Wingo, 1953), which i s then decarboxylated to taurine. Available evidence indicates that the preferred pathway i n the l i v e r i s v i a hypotaurine (Awapara, 19 53; Awapara and Wingo, 19 53), though the oxidation of hypotaurine to taurine i s poorly understood; one b r i e f report has appeared on the enzymatic conversion of hypotaurine to taurine i n rat liver-(Sumizu, 1962). In t h i s study, the formation of taurine appeared to be catalyzed by a reductase, since no uptake of oxygen occured, and the reaction required NAD+ as a cofactor. F i o r i and Costa (1969) were unable to reproduce these r e s u l t s ; they suggested that hypotaurine i s oxidized by small amounts of hydrogen 20 SH . ^ 2 CH NH, COOH cysteine (10 S0 2H CH„ I CHNH_ -CO, (2) i OOH cysteine s u l f i n i c acid 1(4) S0 3H fH2 CHNH, COOH cysteic acid - C02 (2) SO„H I 2 CH„ CH2NH2 hypotaurine (3) SO_H 1 ~> CH, CH 2NH 2 Taurine Figure 3. Metabolic Pathways of Cysteine to Taurine 1. Cysteine dioxygenase (E.C. 1.13.11.20) 2. Cysteine S u l f i n i c acid decarboxylase (E.C. 4.1.1.29) 3. Hypotaurine dehydrogenase (E.C. 1.8.1.3) 4. Cysteine Sulfinate dehydrogenase (E.C. 1.8.1-) peroxide present i n tissues. However, Oja et al.,(19 7 3) found that the conversion of hypotaurine to taurine i n rat brain s l i c e s requires NAD+ as a cofactor, thus providing further evidence for the enzymatic nature of the reaction. Cysteamine Dioxygenase Pathway Cardiac taurine levels are not altered by the i n vivo administration of p o t e n t i a l metabolic precursors such as cysteine s u l f i n i c acid, cysteic acid or hypotaurine (Huxtable ..arid Bressler, 1976). This i s i n contrast to the elevation seen i n l i v e r and other tissues under s i m i l a r conditions (Jacobsen and Smith, 1968; Huxtable and Bressler, 1976). Thus taurine may not be synthesized d i r e c t l y from cysteine i n the heart as i t i s i n l i v e r and brain. A p o t e n t i a l alternative source of taurine i n the heart i s the Cysteamine Dioxygenase pathway. (Fig.4) During studies on the pharmacology of cys.teamine, a powerful radioprotectant, i t became evident that t h i s compound could be metabolized to taurine and that a pathway involving cysteamine as an intermediate may be functional i n heart tissue (Eldjarn, 1954; Verly • and Koch, 1954). Eighteen 35 hours aft e r the i n j e c t i o n of S-cysteamine into mice or guinea-pigs, r a d i o a c t i v i t y i s present largely as CH. ,-(! —CH -C - NH. OH CH3 OH CH ?— C — CH — C - NH-CH2CH2C02H I - I 1 OPO3H M« OH 4'PHOSPHORANTOTHENATE CH2 CH2. C02H PANTOTHENATE H2N-CH-CH2SH C02H CYSTEINE CH-, E—cn—?-1 1 1 iP03H Me OH NH-CH2CH2C,NH.CH-CH2SH C02H >i' - PHOSPHOPANTOTHENOYL CYSTEINE Q Me 0 0 I I * I C H 2 — C — C H - C - N H - C H 2 C H 2 . C N H . C H 2 CH 2 SH H2N-CH2CH2SH CYSTEAMINE OPO^ H'Me OH T-PHOSPHOPANTETHEINE -* CH9- C -CH I I I OH. Me OH C.NH.CH2CH2 C.NH.r.H2CH2SH PANTETHEINE HS. CH2CH. NH2 >HS. CH2CH2. NH2 > H02. S. CH2CH2. NH2 *H03S, CH2CH2. NH2 "I CO^ CYSTEINE © © CYSTEAMINE HYPOTAURINE TAURINE FIGURE 4. BIOSYNTHESIS OF TAURINE VIA CYSTEAMINE. ,„ „ 1) pantothenate kinase (E.C. 2.7.1.33); (2) phosphopantothenoyl cysteine synthetase (E.C. 6.3.Z.b); 3) phosphopantothenoyl cysteine decarboxylase (E.C. 4.1.1.36); (4) phosphatase(unknown); 5) pantetheinase (E.C.3.5.1. ); (6) Cysteamine dioxygenase (E.C. 1.13.11.19) 7) Hypotaurine dehydrogenase-!^. C. 1.8.1.3). 23 taurine i n most organs, although i n muscle and brain the uptake of cysteamine i s extremely low. In v i t r o , when rat or mouse heart homogenates are incubated i n the presence of labeled cysteamine, radioactive taurine i s produced (Huxtable and Bressler, 19 76). The conversion of cysteine to cysteamine, which i s required for the cysteamine pathway, has been observed i n kidney, heart, brain, l i v e r and s k e l e t a l muscles of mice and guinea-pigs. However, cysteine decarboxylase a c t i v i t y has not been detected. C a v a l l i n i et a l . (1976) have proposed the formation of cysteamine i n the course of the biosynthesis of phospho-pantetheine and CoA. The route of cysteine catabolism by this pathway to taurine requires the following steps: 1. Synthesis of pantothenyl cysteine or phosphopantothenyl-cysteine. 2. Decarboxylation of phospho-pantothenylcysteine to form phospho-pantetheine. 3. Formation of cysteamine from pantetheine. 4. Oxygenation of the sulfhydryl group of cysteamine to produce hypotaurine. 5. Oxidation of hypotaurine to taurine. The drawback i n this pathway i s that cysteamine i s a highly toxic compound when i t i s administered . 24 (2 mmole/kg b o d y w e i g h t ) e x o g e n o u s l y t o r a t s a nd m i c e ( H u x t a b l e a n d B r e s s l e r , 1976) . A l t e r n a t i v e l y t h e m e r c a p t o e t h y l a m i n e (MEA) r e s u l t i n g f r o m t h e d e g r a d a t i o n o f t h e p a n t e t h e i n e o r coenzyme A (CoA) may a c t a s a s u b s t r a t e w h i c h c a n be c o n v e r t e d t o t a u r i n e ( F i g . 5) (Dupre e t a l . , 1 9 7 3 ) . C y s t e i n e i s e n z y m a t i c a l l y l i n k e d t o p h o s p h o p a n t o t h e n i c a c i d b y an amide b o n d t o f o r m p a n t e t h e i n e o r CoA (Brown; 1 9 5 9 ) . However, i n s u f f i c i e n t i n f o r m a t i o n i s a v a i l a b l e r e g a r d i n g t h e t u r n o v e r r a t e o f CoA i n t h e h e a r t t o a l l o w a c r i t i c a l a p p r a i s a l o f t h i s p a t h w a y . i i i . P h o s p h o a d e n o s i n e P h o s p h o s u l f a t e (PAPS) P a t h w a y The b i o s y n t h e s i s o f t a u r i n e f r o m i n o r g a n i c s u l f a t e i n c h i c k ( M i r a g l i a e t a l . , 1966) and r a t l i v e r ( M a r t i n e t a l . , 19 72) h a s b e e n r e p o r t e d . S e r i n e h a s b e e n p r o p o s e d a s t h e o r g a n i c a c c e p t o r o f i n o r g a n i c s u l f a t e f o r t h e s y n t h e s i s o f t a u r i n e ( S a s s a n d M a r t i n , 1 9 7 2 ) . S u l f a t e i s b e l i e v e d t o be a c t i v a t e d by f o r m a t i o n o f 3 ' - p h o s p h o a d e n o s i n e - 5 1 - p h o s p h o s u l f a t e (PAPS) f r o m w h i c h t h e s u l f a t e i s t r a n s f e r e d t o a d e h y d r a t e d s e r i n e i n t e r m e d i a t e e g . a - a m i n o a c r y l i c a c i d . The r e s u l t a n t c y s t e i c a c i d i s t h e n d e c a r b o x y l a t e d t o t a u r i n e w h i l e c y s t e i c a c i d i s i n a p r o t e i n b o u n d f o r m ( M a r t i n e t a l . , CN PANTETHEINE PANTOTHENIC ACID 0 0 ^ 3 0 0 C H 2 - 0 - § - 0 - ^ - 0 - C H 2 - C - C I / - C - N H - ( C H 2 ) 2 - C - N H - C H o -CH9 - S H H OH e-MERCAPTOETHYLAMI NE HO 0-P0 3 H 2 ADENOSINE-3'-PHOSPHATE-5'-PYROPHOSPHATE COENZYME A FIGURE 5 - Coenzyme A. 26 1972; Gorby and Martin, 1975). The v a l i d a t i o n of t h i s pathway was based on paper chromatographic analysis of products r e s u l t i n g from incubation of tissue 35 fractions with S-PAPS. Other reports have shown that incorporation of inorganic sulfate to taurine did not occur i n r a t heart homogenate (Dziewiatkowski, 19 54; Green and Robinson, 1960; Huxtable, 1978). Therefore, at the present time, the significance of t h i s pathway i n the heart i s open to doubt. I t had been previously suggested that cats were able to synthesize taurine d i r e c t l y from sulfate (Rambaut and M i l l e r , 1966). However, Sturman and his group (Knopf, et a l . , 1978) and Hayes (1976-) found 35 that i n j e c t i o n of S-SO^ i n cats did not r e s u l t i n detectable levels of labeled taurine i n any tissue examined even when cats highly d e f i c i e n t i n taurine were used. Hayes (1976) has suggested that taurine i s an e s s e n t i a l nutrient i n cats and man. D. Taurine Transport C i r c u l a t i n g taurine derived v i a biosynthesis or from a dietary source i s taken up by various tissues (Awapara, 1-956 ; Sturman , -19 73; Hope, .195 5) .. The concentrations of taurine i n the c e l l water of growing E h r l i c h tumor ascites c e l l s and HeLa c e l l s are approximately "1,000i and . 27 7,000 t i m e s g r e a t e r , r e s p e c t i v e l y , t h a n t h e c o n c e n t r a t i o n i n t h e s u r r o u n d i n g medium. T h i s s u g g e s t s t h e p o s s i b i l i t y t h a t t h e g r a d i e n t i s m a i n t a i n e d b y an a c t i v e t r a n s p o r t p r o c e s s . K r o m p h a r d t (1963) h a s shown t h a t t h e u p t a k e o f t a u r i n e by E h r l i c h t u m o r a s c i t e s c e l l s i s i n h i b i t e d b y 2,4 d i n i t r o p h e n o l a n d a n o x i a . A number o f r e p o r t s o n i n - v i v o a n d i n - v i t r o s y s t e m s h a v e e v a l u a t e d t h e c h a r a c t e r i s t i c s o f t a u r i n e u p t a k e i n t h e h e a r t , b r a i n , k i d n e y , p l a t e l e t s , r e t i n a ( C h e s n e y e t a l . , 1976; S t a r r a n d V o a d e n , 1972; E d w a r d s , 1977; A h t e e e t a l . , 1974; G a u t a n d N a u s s , 1976; L a h d e s m a k i and O j a , 1973; K a c z m a r e k a n d D a v i d s o n , 1972; K o n t r o a n d O j a , 1978; H r u s k a e t a l . , 1 9 7 6 ) . R e s u l t s o f a l l o f t h e s e i n v e s t i g a t i o n s i n d i c a t e t h a t t a u r i n e u p t a k e i n t h e t i s s u e s was an a c t i v e t r a n s p o r t p r o c e s s . I n t h e s e s t u d i e s , i t was a l s o shown t h a t t a u r i n e u p t a k e was c o m p e t i t i v e l y i n h i b i t e d b y 3 - a l a n i n e and h y p o t a u r i n e a nd compounds p o s s e s s i n g a p r i m a r y amine a n d an a c i d i c g r o u p , s e p a r a t e d b y two m e t h y l e n e c a r b o n s . D e v i a t i o n f r o m a c l o s e s t r u c t u r a l r e s e m b l a n c e t o t a u r i n e r e s u l t e d i n d e c r e a s e d i n h i b i t i o n b y o t h e r a m ino a c i d s e x a m i n e d . Two r e c e n t r e p o r t s h a v e c h a r a c t e r i z e d a c t i v e t r a n s p o r t p r o c e s s e s o f t a u r i n e i n t h e i s o l a t e d p e r f u s e d r a t m y o c a r d i u m (Chubb a n d H u x t a b l e , 1 9 7 8 a ) a n d i n v i t r o c u l t u r e d f o e t a l mouse h e a r t s ( G r o s s o e t a l . , 1978-b). . I t was d e m o n s t r a t e d t h a t i n t h e f o e t a l mouse h e a r t t a u r i n e i n f l u x was v i a a c a r r i e r - m e d i a t e d t r a n s p o r t - s y s t e m a n d was 28 temperature-dependent, saturable and had s t r u c t u r a l s e l e c t i -v i t y for 8-amino acids. In addition, Grosso et al.,(19 78b) observed that taurine uptake was sodium-dependent, energy-dependent and that f o e t a l mouse hearts were capable of accumulating taurine against a concentration gradient. Using i s o l a t e d perfused rat hearts, Chubb and Huxtable (1978a) determined taurine i n f l u x over a concentration range of 25 to 400 J J M . Taurine i n f l u x was saturable at a concentration of approximately 200 y_M. The transport system had a Michaelis constant (K m) of 45'yM and a V m a x \ of 32 nmoles/g dry weight /min i n d i c a t i n g that taurine i n f l u x i s mediated by a transport process of r e l a t i v e l y high a f f i n i t y . S p e c i f i c i t y and i n h i b i t i o n of taurine i n f l u x were examined by perfusing hearts with other radiolabeled amino acids. g-alanine, which i s s t r u c t u r a l l y similar to taurine, i n h i b i t e d taurine i n f l u x approximately sevenfold, whereas the a-amino acids: a-aminoisobutyric acid, leucine and serine did not markedly a l t e r taurine i n f l u x . These results indicate that at least two types of i n f l u x s i t e s for amino acids e x i s t i n the heart, one mediating 6-amino acid i n f l u x , the other a-amino acid i n f l u x (Christensen and Liang, 1956). The s l i g h t depression in taurine i n f l u x caused by competing a-amino acids may indicate that a small percentage of taurine i n f l u x i s mediated by a-amino acid s i t e s (Chubb and Huxtable, 19 77) 29 Several workers have reported taurine transport systems i n synaptosomes i s o l a t e d from rat brain (Schmidt et a l . , 1975; Hruska et a l . , 1977; Kontro and Oja, 1978; Lombardini, 1976). Hruska et a l . (1977) have described high a f f i n i t y sodium-dependent transport of taurine into r at brain synaptosomes. Ki n e t i c analysis indicates a high-a f f i n i t y 1^ value of 3.20 u_M and a.V value of 5.35 nmoles/g protein/minute. The k i n e t i c constants of the low-a f f i n i t y system were 3340 y_M and 699 nmoles per g protein/ minute. The regional d i s t r i b u t i o n of uptake showed that the midbrain, thalamus and ol f a c t o r y bulbs had the highest v e l o c i t y of transport, while the cerebral cortex, spinal cord, and cerebellum had the lowest V : for transport. 30 II I . CARDIAC DISEASE AND TAURINE A. Congestive Heart Fail u r e Taurine concentrations i n the heart are elevated i n various states of n a t u r a l and experimentally-induced cardiac pathology. Peterson, Read and Welty (19 73) demonstrated increased levels of taurine i n experimentally-induced r i g h t sided congestive heart f a i l u r e i n dogs. Right v e n t r i c u l a r hypertrophy and congestive heart f a i l u r e (CHF) were produced i n dogs by progressive pulmonary artery stenosis. In t h i s model, the elevation of taurine was only seen i n the right v e n t r i c l e and not the l e f t . S t a t i s t i c a l l y s i g n i f i c a n t increases i n free amino acid concentrations of the right v e n t r i c l e occured only for two compounds, taurine and methionine. There was no elevation of taurine or methionine i n the nonhypertrophied l e f t v e n t r i c l e . Huxtable and Bressler (1974a, 1974b) reported si m i l a r increases i n taurine levels i n cardiac l e f t v e n t r i c l e of patients dying of chronic congestive heart f a i l u r e . This increase was seen regardless of whether taurine content was calculated on a wet-weight tissue basis, on the basis of acid-precipitable weight or per weight of protein. The concentration of taurine i n the l e f t v e n t r i c l e of the heart was doubled i n patients who had died of chronic congestive heart f a i l u r e compared to patients who h a d d i e d o f o t h e r c a u s e s and h a d no c a r d i a c p a t h o l o g y . The i n c r e a s e i n t a u r i n e was s p e c i f i c t o t h e h e a r t i n t h a t no c o r r e s p o n d i n g r i s e i n t a u r i n e c o n c e n t r a t i o n was o b s e r v e d i n a o r t a o r s k e l e t a l m u s c l e . Newman e t a l . , (19 77) e x a m i n e d t h e r e l a t i o n b etween m y o c a r d i a l t a u r i n e c o n t e n t and s e v e r i t y o f c o n g e s t i v e h e a r t f a i l u r e . Dogs w i t h h e a r t f a i l u r e were compared t o n o r m a l c o n t r o l s . H e a r t f a i l u r e was i n d u c e d by c r e a t i n g an i n f r a r e n a l a o r t o c a v a l f i s t u l a . T a u r i n e c o n c e n t r a t i o n s were d e t e r m i n e d i n t h e l e f t and r i g h t v e n t r i c l e s and t h e n r e l a t e d t o p u l m o n a r y wedge p r e s s u r e . P u l m o n a r y wedge p r e s s u r e was s i g n i f i c a n t l y i n c r e a s e d i n dogs w i t h c o n g e s t i v e h e a r t f a i l u r e as was t h e t a u r i n e c o n t e n t i n t h e l e f t and r i g h t v e n t r i c l e s : The wedge p r e s s u r e i n CHF dogs r a n g e d f r o m 6.6 t o 28 mm Hg and 2.5 t o 7.5 mm Hg i n n o r m a l dogs. T a u r i n e c o n c e n t r a t i o n s i n t h e v e n t r i c l e r a n g e d f r o m 17 t o 153 u m o l e s / g p r o t e i n i n CHF a n i m a l s ; and 18 t o 49 y m o l e s / g p r o t e i n i n n o r m a l s . No s i g n i f i c a n t d i f f e r e n c e between t a u r i n e c o n t e n t o f t h e l e f t and r i g h t v e n t r i c l e s o f e i t h e r n o r m a l o r CHF dogs was o b s e r v e d . L i n e a r r e g r e s s i o n a n a l y s i s o f m y o c a r d i a l t a u r i n e c o n t e n t o f e i t h e r t h e l e f t o r r i g h t v e n t r i c l e y i e l d e d a h i g h l y s i g n i -f i c a n t c o r r e l a t i o n w i t h t h e p u l m o n a r y wedge p r e s s u r e . 32 B. Hypertension Huxtable and Bressler (1974a) also reported a p o s i t i v e c o r r e l a t i o n between cardiac taurine concentrations and increases i n blood pressures averaged over a period of weeks before death. Patients with an average s y s t o l i c blood pressure of 145 mm had v e n t r i c u l a r taurine levels of 8.5 pmole/g., whereas patients averaging 108 mm s y s t o l i c pressure had taurine levels of 5.0 .pmole/g. A si m i l a r r e l a t i o n s h i p was reported for d i a s t o l i c pressure. In experimental stress-induced hypertensive rats,, there was a doubling i n taurine concentration i n the whole heart (expressed r e l a t i v e to protein) with no accompanying taurine concentration changes i n muscle or brain (Huxtable and Bressler, 1974b). The hypertensive male rats of the Okamoto s t r a i n (Okamoto and Aoki, 1963) also showed an increase i n heart taurine when compared to age-matched Wistar control ra t s . However, rats which were stressed and who had developed cardiac hypertrophy but not hypertension, showed no a l t e r a t i o n i n the taurine to protein r a t i o . Therefore, the increase i n cardiac taurine concentration was due e n t i r e l y to the development of hypertension and not to hypertrophy of the heart. The significance of these alterations i n taurine levels seen i n cardiac disease i s unclear. There i s , usually, a general increase i n free amino acid content of the heart i n cardiac hypertrophy which i s probably related 33 to increased protein turnover and synthesis. Peterson et a l . (1973), for example, found increased levels of a l l free amino acids i n the r i g h t v e n t r i c l e i n right-sided heart f a i l u r e i n dogs, although s t a t i s t i c a l l y s i g n i f i c a n t increases were observed only i n the cases of taurine and methionine. If the increase i n taurine was a response to the development of congestive heart f a i l u r e , then i t can be postulated that by increasing taurine levels the heart i s making available an increased amount of an endogenously inotropic agent (inotropic e f f e c t s of taurine have been shown i n guinea-pig and rat hearts). The high taurine levels i n CHF could equally indicate that taurine i s toxic at such l e v e l s . According to Huxtable (19 76a) the changes i n taurine concen-t r a t i o n that do occur i n CHF are"induced at-ah advanced stage of the disease-indicating that "the changes are reactive to -the disease process"rather than causal. Recent evidence (Huxtable and Chubb, 1977) suggests that taurine i n f l u x into heart c e l l s i s regulated through ^-activation of the adrenergic system. One of the major mechanisms whereby the heart increases i t s output under work stress i s v i a the 0-adrenergic system. Prolonged stimulation of t h i s system causes an increase i n heart mass-cardiac hypertrophy-and., i f the stress i s severe and long-lasting, eventually congestive heart f a i l u r e w i l l occur. Studies were designed to determine whether . .... 34 m e t a b o l i c or t r a n s p o r t processes a f f e c t i n g t a u r i n e concen-t r a t i o n were m o d i f i e d by c a r d i a c s t r e s s . I s o p r o t e r e n o l , a ^-adrenergic a g o n i s t , was used t o produce a h i g h output s t r e s s on the h e a r t . I s o p r o t e r e n o l g i v e n t o r a t s f o r p e r i o d s of up to 10 days produced a c a r d i a c hypertrophy accompanied by a marked i n c r e a s e i n t o t a l t a u r i n e content of the h e a r t (Huxtable, 1976b). No a l t e r a t i o n was observed i n the r a t e of t a u r i n e s y n t h e s i s , as measured e i t h e r by the o v e r a l l c o n v e r s i o n of c y s t e i n e t o t a u r i n e , or by the a c t i v i t y o f cysteamine dioxygenase (E.C. 1.13.11,19). However, i n c r e a s e s i n the r a t e of t a u r i n e i n f l u x were observed: T a u r i n e , i n c o n c e n t r a t i o n s of 25 t o 200 u_M, was p e r f u s e d through the h e a r t . The Lineweaver-Burke p l o t of t a u r i n e i n f l u x showed a M i c h a e l i s c o n s t a n t (K_) o f 45 uM and a m — maximum v e l o c i t y ( v m a x ) of 32 nmole/g o f t i s s u e dry weight per minute. A d d i t i o n o f i s o p r o t e r e n o l t o the p e r f u s i o n medium r e s u l t e d i n an immediate s t i m u l a t i o n of t a u r i n e i n f l u x (K^ = 62 u_M; V x = 42 nmoles/g dry weight / minute) . The s t i m u l a t i o n o f t a u r i n e i n f l u x was dependent on the c o n c e n t r a t i o n o f i s o p r o t e r e n o l p e r f u s e d over the range -9 -7 7 x 10 M t o 4 x 10 M. Higher c o n c e n t r a t i o n s o f i s o p r o t e r e n o l caused a decreased s t i m u l a t i o n o f the r a t e of t a u r i n e i n f l u x , and l e d to arrhythmia. C. Ischemia Crass and Lombardini (1977) have demonstrated a generalized decrease of taurine content i n ischemic muscle. This loss i n cardiac taurine was observed af t e r acute l e f t v e n t r i cular ischemia i n the dog (in vivo) and whole heart anoxia i n the perfused rat heart (in v i t r o ) . The l e f t v e n t r i c u l a r ischemia i n the dog was produced by occluding the circumflex branch of the l e f t coronary artery. Anoxia, i n i s o l a t e d rat heart, was produced by perfusing the hearts with a taurine-free and oxygen-deficient buffer. In vivo experiments a f t e r four hours of hypoxia markedly decreased tissue taurine content; the greatest disappearance of taurine was observed i n the inner zone of the v e n t r i c l e . Anoxic perfusion resulted i n a s i m i l a r decrease, i n rat v e n t r i c u l a r taurine l e v e l s . IV POSSIBLE PHYSIOLOGICAL ACTIONS OF TAURINE IN THE HEART A. Taurine and Arrhythmias Read and Welty (1963) were the f i r s t to suggest that taurine might influence cardiac a c t i v i t y by a f f e c t i n g ion movements. Their conclusions were based on t h e i r observations of a mitigating action of taurine towards 36 epinephrine or digoxin-induced arrhythmias. These same investigators (Welty and Read, 1964; Read and Welty, 1965) also reported that taurine prevented the loss of c e l l potassium by dog heart s l i c e s exposed to epinephrine and digoxin. Taurine also prevented the epinephrine-induced loss of potassium from the i n t a c t heart i n both fed and fasted dogs. Furthermore, feeding taurine to dogs, led to an increased uptake of potassium by the heart. This was shown by changes i n potassium concentration i n the coronary sinus and a r t e r i a l plasma. When taurine was given by i t s e l f , i t did not a f f e c t heart rate, blood pressure, or EKG. These r e s u l t s , however, were suggested to imply that taurine had pot e n t i a l therapeautic value i n c o n t r o l l i n g cardiac arrhythmias caused by the loss of c e l l u l a r potassium. Chazov et a l . (19 74) also established a r e l a t i o n s h i p between taurine and potassium i n the i s o l a t e d guinea-pig .heart. Taurine was shown to be e f f e c t i v e i n reversing abnormal EKG 1s brought on by perfusion with strophanthin-K i n K + free medium. In f i b r i l l a t i n g hearts perfused with K + -free medium, the addition of K + (2.8 mM) and taurine, but not K+- alone, eliminated the f i b r i l l a t i o n . The e f f e c t s of taurine on EKG parameters i n the presence of strophanthin-K and low potassium were said to be evidence for a protective action by taurine on the heart through regulation of c e l l permeability to potassium. 37 The above reports appeared to show that taurine af f e c t s potassium ion fluxes i n myocardial c e l l s . I t has been known for a long time that d i g i t a l i s and other cardiac glycosides (strophanthin-K and ouabain ) a l t e r transmembrane + + Na , K -fluxes'(Hajdu and Leonard, 1959). D i g i t a l i s i n h i b i t s the Na +, K+- ATPase enzyme system which i s responsible for + + the active Na e f f l u x and active K i n f l u x across the cyto-plasmic membranes (Skou, 1965). Cardiac Na +, K + - ATPase has been reported to be i n h i b i t e d by as much as 40 per cent i n patients receiving therapeutic doses of d i g i t a l i s (Repke, 1965).20-40 per cent i n h i b i t i o n of Na +, K +- ATPase occurs during intravenous infusion of ouabain i n the dog (Akera et a l . , 1969; 1970; Besch et a l . ,. 1970). The Na +,K +-. . . . + 2+ ATPase i n h i b i t i o n however, causes accumulation of Na or Ca and a cumulative loss of K + by the myocardium. This loss of K + by the myocardium has been -related to the arrhythmogenic actions of the cardiac glycosides (Read and Welty, 1965; Chazov et a l . , 1974). A l o g i c a l s i t e of action for taurine therefore would be on the Na +, K +-ATPase pump system. Recently, Akera, et a l . , (19 76) reported that taurine, i n concentrations up to 100 mM f a i l e d to a f f e c t brain Na +, K+-ATPase a c t i v i t y . In t h i s experiment, a p a r t i a l l y p u r i f i e d Na +, K— ATPase preparation obtained from rat brain 3 was used. Taurine was shown not to a f f e c t H-ouabam binding to Na +, K+-ATPase or the release of ouabain bound 38 to the enzyme. However, i t i s possible that taurine may act on events which follow Na +, K + - i n h i b i t i o n rather than on the Na-pump i n h i b i t i o n i t s e l f . o r that the cardiac enzyme i s d i f f e r e n t from the brain enzyme. C r i t i c i s m has recently been made of the interpretations placed on the purported mitigating actions of taurine on drug induced arrhythmias. Hinton, Souza and G i l l i s (1975) studied the capacity of taurine to counteract v e n t r i c u l a r arrhythmias induced by deslanoside i n the cat. This model had previously been successfully employed to determine the ef f i c a c y of antiarrhythmic agents. In t h i s model, taurine was found to aggravate the cardiac rhythm disturbances produced by deslanoside. These workers claim that the antiarrhythmic e f f e c t , reported previously (Read and Welty, 1965; Chazov et a l . , 1974), was probably a r e f l e c t i o n of the d i g i t a l i s e f f e c t wearing o f f since no experimental controls were reported to indicate the duration of the arrhythmias when taurine was administered. Presumably, therefore, t h i s c r i t i s m could also apply to the e f f e c t of taurine on K + e f f l u x . However, the recent findings of Fujimoto and Iwata (1975) and Fujimoto, Iwata and Yoneda (1976) confirmed the e a r l i e r work of Read and Welty (1965) and Chazov et a l . , (1974) described above. I t was shown that infusing taurine together with ouabain intramuscularly i n the rat, 39 prevented the development of arrhythmias. They also noted that the myocardial taurine concentration was reduced i n ouabain"induced arrhythmias. I t was argued that the arrhythmic e f f e c t of taurine was l i k e l y due to i t s action on the autonomic nervous system, since propanolol, a fi-adrenergic antagonist, i n h i b i t e d both ouabain-induced arrhythmia and loss of taurine. This point was further strengthened i n t h e i r l a t e r paper (Fujimoto, Iwata and Yoneda, 1976) where taurine was observed to i n h i b i t the change i n response to acetylcholine and ouabain but not to noradrenaline. It was noted that taurine i t s e l f did not change either the EKG pattern.: or cardiac responses to acetylcholine and ouabain but did prevent the development of arrhythmias observed i n the presence of d i g i t o x i n . However, the significance of these data i s puzzling, p a r t i c u l a r l y when the authors conclude with the statement that "the e f f e c t s of d i g i t o x i n on the heart are complicated, mechanisms for the antiarrhy-thmic action of taurine remain unelucidated" (Fujimoto, Iwata and Yoneda, 1976). The work of Read and Welty (196 5) and Chazov et a l . (1974) must be repeated to allow proper evaluation of the e f f e c t of taurine on arrhythmia and potassium loss. B. Taurine and Inotropism The:nature of the inotropic response of the heart 40 to ouabain has been demonstrated t o be a species-dependent phenomenon ( D i e t r i c h and Diacono, 1971). Ouabain has a p o s i t i v e i n o t r o p i c e f f e c t on gu i n e a - p i g h e a r t and a n e g a t i v e i n o t r o p i c e f f e c t on r a t h e a r t . D i e t r i c h and Diacono (1971) have shown t h a t t a u r i n e i n both normal and low c a l c i u m medium e x e r t s a p o s i t i v e i n o t r o p i c e f f e c t on guinea-pig h e a r t and a negative i n o t r o p i c e f f e c t on r a t hea r t . Taurine was a l s o shown to p o t e n t i a t e the i n o t r o p i c e f f e c t of ouabain on both gu i n e a - p i g and r a t h e a r t s . P e r f u s i o n with ouabain (10 ^g/ml) caused an i n c r e a s e i n c o n t r a c t i o n i n g u i n e a - p i g h e a r t (+9 0 per cent a f t e r 5 minutes) and a decrease i n the r a t h e a r t (-29 per cent a f t e r 3 minutes). Taurine (8mM) was shown to d u p l i c a t e these a c t i o n s . In low ca l c i u m medium (0.54 mM C a C l 2 i n s t e a d of 2.16 mM), the ne g a t i v e inotropism'Observed i n g u i n e a - p i g h e a r t was l e s s pronounced i n the presence of t a u r i n e (70 per cent a f t e r 5 minutes i n s t e a d of 80 per c e n t ) , while i n the presence o f t a u r i n e the e f f e c t o f low c a l c i u m was more pronounced i n r a t h e a r t (69 per cent a f t e r 5 minutes i n s t e a d of 57 per c e n t ) . In the gu i n e a - p i g , ouabain p r o g r e s s i v e l y reduced the negative i n o t r o p i c e f f e c t o f low c a l c i u m medium. Taurine p o t e n t i a t e d t h i s e f f e c t o f ouabain. The r e d u c t i o n o f c o n t r a c t i o n i n the low c a l c i u m Tyrode s o l u t i o n was 91 per (.cent; w i t h ouabain added to the medium i t was 58 per cent; with ouabain and taurine together there was only a 36 per cent reduction i n contractions. On.the•other hand, i n the rat, ouabain and taurine together showed increases i n the negative inotropic e f f e c t caused by low calcium medium. In the low calcium medium, the contraction of rat heart was reduced by 30 per cent; the reduction was 75 per cent i n the presence of ouabain and 92 per cent i n the presence of both ouabain and taurine. Taurine thus potentiated the inotropic effects of ouabain i n a l l the experiments quoted above and opposed the inotropic e f f e c t s of lowered environ-mental calcium. To evaluate the inotropic e f f e c t of taurine, Schaffer et al.,(1978a)looked at cardiac work (aortic pressure x cardiac output) (Neely et a l . , 1967) i n perfused rat hearts. I t was found that taurine (10 mM) mediated a small, but s i g n i f i c a n t , p o s i t i v e inotropic e f f e c t , when hearts were perfused with Krebs-Henseleit buffer containing 2+ 1.25 mM Ca . These r e s u l t s , however c o n f l i c t with the results of Di e t r i c h and Diacono (1971). Schaffer et a l . (1978a)argue that t h e i r study u t i l i z e s a more phys i o l o g i c a l working heart preparation. I t was also found by Guidotti, Badiani and G i o t t i (1971) that perfusion of taurine (8 mM) through i s o l a t e d guinea-pig auricles increased the c o n t r a c t i l e responses to stophanthin-k. Perfusion of strophanthin-k (0.2 mg/ml) for 42 15 minutes caused an increase of 48 per cent (p<0.01). Homotaurine (3-aminopropane sulfonic acid) had no e f f e c t on the response to strophanthin-K. These workers further showed.that perfusion of auricles with a taurine-free medium1 resulted i n a substantial loss of tissue taurine and that perfusion with 8 mM taurine maintained constant tissue l e v e l s . Dolara et a l (1978a), studied the e f f e c t of taurine on the recovery of c o n t r a c t i l e force by calcium-depleted guinea-pig v e n t r i c l e s t r i p s . The c o n t r a c t i l e force of the guinea-pig v e n t r i c l e s t r i p can;.'.be reduced to very low le v e l s , when perfused with a medium devoid of calcium. Calcium concentrations i n the medium were then increased by steps and the recovery i n the c o n t r a c t i l e force was measured. It was shown that taurine (4 mM) s i g n i f i c a n t l y increased the co n t r a c t i l e force at external calcium concentrations of 1.8 and 3.6 mM, but i t had no e f f e c t at lower calcium concentrations. It was further demonstrated, that taurine could a f f e c t the c o n t r a c t i l i t y of the heart v e n t r i c u l a r s t r i p s under conditions of calcium loading. I t i s known that an increase i n the frequency of stimulation of heart rates i s accompanied by increased calcium i n f l u x i n cardiac c e l l s (Grossman and Furchgott, 1964; Winegrad and Shanes, 1962).Taurine i n a dose-related-manner was-found to increase the c o n t r a c t i l i t y of the heart v e n t r i c u l a r s t r i p s at a frequency of 120 beats/minute, but was i n e f f e c t i v e at 6 0 beats/minute. 43 Iwata and Fujimoto (19 76) have also confirmed that taurine has a po s i t i v e inotropic e f f e c t on e l e c t r i c a l l y driven guinea-pig a t r i a . Taurine (3.0 mM, but not 0.5 mM) was shown to pontentiate the posit i v e inotropic e f f e c t of ouabain on myocardium independent of e x t r a c e l l u l a r K + concentration. The potentiation by taurine of ouabain action was not related to the changes i n the myocardial content of taurine. A p o s i t i v e c o r r e l a t i o n between the potentiation by taurine of the posit i v e inotropic action of ouabain and the uptake of calcium ion by the heart was observed. The authors concluded that the potentiation by taurine of the posit i v e inotropic e f f e c t of ouabain was due, at le a s t i n part, to the increased content of calcium i n the heart, though the mode of action of taurine remained unexplained. V. POSSIBLE CARDIAC EFFECT OF TAURINE ON CALCIUM TRANSPORT A. Inotropism and Calcium Transport It i s probable that the inotropic action of d i g i t a l i s glycosides i s due ultimately to an increase i n i n t r a c e l l u l a r calcium available for binding to the myofila-ments and p a r t i c u l a r l y to the protein troponin (Katz, 1970) . 44 As the free calcium concentration i n the cardiac c e l l r i s e s -7 above approximately 10 M , calcium binds to an increasing number of troponin s i t e s and, by a mechanism not yet defined, removes the troponin i n h i b i t i o n of bridge formation between acti n and myosin. F u l l a c t i v a t i o n at a l l available s i t e s i s achieved as calcium concentration increases to a value of approximately 5 x 10 ^ M, or some 50 times that of the relaxed state. To at t a i n t h i s concentration and obtain f u l l a c t i v a t i o n 50 to 60 umoles of calcium ion per kilogram of heart muscle i s required to be released upon e x c i t a t i o n (Langer,1971). Frequently, however, the heart i s c a l l e d upon to develop greater tension and to develop i t more rapidly without a change i n the end-diastolic length of i t s c e l l s (Bowditch, 1871). The source of calcium available for e x c i t a t i o n -contraction coupling i n the presence of the cardiac glycosides i s s t i l l i n dispute. A number of workers are i n favour of cardiac glycoside-induced l i b e r a t i o n of calcium from i n t r a c e l l u l a r pools, such as sarcoplasmic reticulum (S.R.) mitochondria or microsomes (Chipperfield, 1969; G l i t s c h et a l . , 1970; Dutta,et a l . , 1968). Other workers (Langer,1971; Van Winkel and Schwartz, 1976) have shown that cardiac glycosides could also increase the penetration of calcium into the c e l l through an active transport mechanism at the sarcolemmal membrane whereby 45 i n t e r n a l sodium i s exchanged against external calcium. This transport system i s said to be in s e n s i t i v e to cardiac glycosides d i r e c t l y (unlike the Na +, K + ATPase) (Blaustein and Hodgkin, 1969), but very sensitive to i n t r a c e l l u l a r sodium changes since i t s a c t i v i t y i s proportional to the i n t r a c e l l u l a r sodium ion concentration. The i n h i b i t i o n of the f i r s t pump (Na +, K + ATPase) by ouabain could then r e s u l t i n an increased entry of calcium into the c e l l s (Langer,1971; Langer, 1968). It has also been proposed that a t h i r d mechanism exists by which i n t e r n a l calcium i s exchanged for external sodium (Baker et a l . , 1969). The results of D i e t r i c h and Diacono (1971) concerning the negative inotropic e f f e c t s on rat heart have been related to the absence i n r a t of a time lag i n the functioning of the Na +, K+-ATPase (Guilbalt et a l . , 1962; Blesa,• et a l . , 1970). The pos i t i v e inotropic e f f e c t of d i g i t a l i s i n the guinea-pig can be attributed to the i n a b i l i t y of the sodium pump to extrude a l l the penetrating sodium, thus leading to an increment i n the amount of calcium i n f l u x into the myocardial c e l l . The calcium i s thus available to the myofilaments which then leads to a staircase phenomenon. An alternative hypothesis i s that the t h i r d 2+ pump (Ca extrusion) might be more active i n rat than i n guinea-pig and thus able to follow more quickly the inward 2+ calcium movements either by extruding Ca from the c e l l s 2+ or by f i l l i n g the i n t r a c e l l u l a r Ca pools, since the tissue 46 calcium content"is higher i n rat heart (4.9 a moles per kg.) than i n guinea-pig heart (1.7 a moles/kg). .. B. Inotropism, Calcium Transport and Taurine The explanations above remain only theories but i t i s usual to assume that the d i g i t a l i s - l i k e e f f e c t s of taurine are mediated through calcium movements i n both rat and guinea-pig hearts. This i s also suggested by the work described below. The probable major i n t r a c e l l u l a r structure for removing and releasing calcium i s the sarcoplasmic reticulum. In sarcoplasmic reticulum (S.R.) i s o l a t e d from rat s k e l e t a l muscle i n the presence of 15 mM taurine (Huxtable and Bressler, 1973), calcium transport was increased by 30 per cent. I s o l a t i o n procedure i n the presence of taurine also led to an increase i n the y i e l d of microsomes and S.R. per gram of muscle compared to i s o l a t i o n i n the absence of added taurine. Huxtable and Bressler (1973) interpreted these results to indicate that the presence of taurine prevented denaturation and l y s i s of the i s o l a t e d organelles. Taurine was also shown, i n t h e i r experiments, to slow the rate of loss of calcium transport a c t i v i t y and ATPase a c t i v i t y of S.R. produced by phospholipase C. The data mentioned above suggest a primary role of taurine i n 47 a f f e c t i n g (Mg + Ca )-ATPase a c t i v i t y . There i s also a body of work which suggests that taurine can also have an e f f e c t on calcium binding to membranes-., Dolara, Agresti, G i o t t i and Pasquini (1973) found that taurine affects calcium k i n e t i c s i n perfused guinea-pig hearts. Hearts were perfused with Tyrode solution containing 2.7 mM calcium chloride i n the control group and calcium chloride plus 8 mM taurine i n the experimental group. After 15 minutes of perfusion, the hearts were washed out,then perfused with calcium-free Tyrode solution. The group which had been perfused with taurine showed a decrease i n the loss of c o n t r a c t i l e force. After 1 minute of perfusion with calcium-free medium, the taurine-pretreated hearts had a calcium content of 5.9 ± 0.1 mEq. per kg. of wet weight, compared to only 3.19 ± 0.1 i n the controls. Dolara et a l . (1973, 1978a, 1978b) suggested that the calcium s a l t of taurine had a higher a f f i n i t y than calcium ion for i n t r a -c e l l u l a r structures, thereby leading to a larger pool of bound calcium. I t was suggested that the protective action of taurine on the rate of loss of c o n t r a c t i l e force was due to an increase i n the amount of calcium available for contraction. Later Dolara, Agresti, G i o t t i and Sorace (1976) studied calcium i n f l u x i n a system where Sarcoplasmic Reticulum (S.R.) preparations of guinea-pig hearts were 48 contained i n a d i a l y s i s bag. Equilibrium d i a l y s i s has previously been used as a to o l for the study of drug-protein int e r a c t i o n and amine uptake processes. In t h i s system i t was found that taurine increased t o t a l calcium binding to the sarcoplasmic v e s i c l e s . Calcium accumulation was due to a marked increase i n the calcium i n f l u x rate whereas the e f f l u x rate was not appreciably altered. C. Calcium Movements and Taurine i n Other Tissues Recently, Izumi, Butterworth and Barbeau (19 77) studied the. e f f e c t of taurine on calcium binding to microsomes is o l a t e d from the rat cerebral cortex. Calcium binding to the microsomes was Shown to be i n h i b i t e d by taurine i n a dose-dependent fashion i n the presence of an incubation medium containing 5 mM KC1 and 115 mM NaCl. Taurine was 2+ also found to decrease Ca binding i n the medium containing 2+ 70 mM KC1, without NaCl. There was no i n h i b i t i o n of Ca binding seen i n the medium containing 115 mM KC1 and 5.mM NaCl. Isethionic acid, Glycine, B-alanine, GABA and L-leucine showed 2+ no e f f e c t on Ca binding to the microsomes i n the medium containing 70 mM KC1 without NaCl. It was concluded that taurine has an i n h i b i t o r y e f f e c t "on calcium binding to the microsomes i n states of depolarization but i s inactive i n the normal resting state. Apparently, this e f f e c t i s s p e c i f i c to taurine. 49 I g i s u , e t a l . , (1976) have s t u d i e d the e f f e c t s of t a u r i n e i n v i t r o on o u a b a i n - i n h i b i t e d ATPase a c t i v i t y i n human e r y t h r o c y t e membrane i n the presence and absence of calcium. Ouabain was shown to i n h i b i t the t o t a l ATPase a c t i v i t y i n the human e r y t h r o c y t e membrane i n a dose-dependent manner but i n 2+ a f a s h i o n t h a t was dependent on Ca c o n c e n t r a t i o n s . T a u r i n e , a t c o n c e n t r a t i o n s of 15 to 60 mM s t i m u l a t e d ATPase a c t i v i t y to a f i g u r e c l o s e to t h a t seen without added ouabain. These workers suggested t h a t the e f f e c t of t a u r i n e was due to a c o m p e t i t i o n with the membrane f o r c a l c i u m ions thus lowering the e f f e c t i v e c a l c i u m c o n c e n t r a t i o n . They s t a t e , however, without evidence, t h a t t a u r i n e does not i t s e l f complex calcium. The e f f e c t of t a u r i n e i n the absence of ouabain, was not r e p o r t e d by these workers. Since t o t a l ATPase a c t i v i t y was measured, i t i s not c l e a r which type of enzyme, Na ,K—ATPase, and/or (Ca. -+Mg )-ATPase, i s most i n f l u e n c e d by t a u r i n e . Taurine alone seems not to a c t i v a t e ouabain i n h i b i t e d microsomal Na +, K +-ATPase a c t i v i t y i n r a t b r a i n (Akera e t a l . , 1976; Donaldson e t a l . , 1974). SUMMARY The work quoted i n the p r e c e d i n g s e c t i o n s suggest t h a t t a u r i n e induces changes i n i o n f l u x e s p a r t i c u l a r l y those of c a l c i u m i n c a r d i a c muscle. On the b a s i s of the e f f e c t s 50 of taurine on inotropism, taurine can be considered to have i t s main action on myocardium related to calcium ion transport. The antiarrhythmic e f f e c t of taurine on epinephrine and d i g i t a l i s - i n d u c e d cardiac arrhythmias was / said to involve the regulation of the e f f l u x of i n t r a c e l l u l a r potassium ions (Read and Welty, 1965). I t i s possible that taurine may be more d i r e c t l y concerned with calcium and only J i n d i r e c t l y a f f e c t potassium movements (based on the r e s u l t s of Ueda et a l . (1961) on the arrhythmogenic e f f e c t of caffeine and epinephrine and those of Nayler (1963) r e l a t i n g 2+ the inotropic e f f e c t of caffeine and Ca -movements). More tangible evidence for the function of taurine i n the heart consists of the observation of an increased taurine concen-t r a t i o n i n heart muscle of hypertensive rats and i n the l e f t v e n t r i c l e of the patients who died from congestive heart f a i l u r e . VI. POSSIBLE INVOLVEMENT OF TAURINE IN NEUROPHYSIOLOGY Interest i n the study of taurine and i t s possible physiological actions has been stimulated i n recent years by the observation that taurine tissue levels are altered i n certain c l i n i c a l conditions. 51 A. Anticonvulsant Action of Taurine The f i r s t suggestion of a possible involvement of taurine i n seizure a c t i v i t y emanated from the report of van Gelder et a l . , (1972)>. Lower leve l s of taurine were found i n the epileptogenic focus i n human brain i n comparison with the surrounding tissue. Decreased taurine le v e l s were also observed i n experimental cobalt-induced epileptogenic f o c i i n cats, mice and rats (Craig and Hartman, 1973; Koyama, 1972; vanGelder, 1972). The observation of van-Gelder et a l . / (1972) i n human brain was not confirmed by Perry et a l . (1975). This could be due to differences i n the methodology used: van Gelder et al.,(19 72) compared taurine le v e l s i n the same patient and Perry et a l . , (1975) used other subjects as controls. Van Gelder (19 72) f i r s t reported the anticonvulsant action of taurine i n mice and cats with cobalt-induced lesions. Subsequently, taurine has proved e f f i c a c i o u s as an anti-convulsant i n a variety of experimental models of seizure a c t i v i t y . Taurine has been shown to have a protective e f f e c t against seizures induced by ouabain, pentylenetetrazol, and strychnine (Izumi et al.,1973; 1974; Tsukada et a l . , 1974). It also has a n t i e p i l e p t i c e f f e c t s on chronic and acute e p i l e p t i c f o c i (produced by cobalt, aluminum, p e n i c i l l i n , estrogen, strychnine) and i n the photosensitive Papio papio (Derouaux et a l . , 1973; Mutani et a l . , 1974a, 1974b). The f a i l u r e 52 of taurine to protect against convulsions i n some experimental models (Wada et al.,19 75) may be related to a f a i l u r e of penetration of taurine i n t o the CNS. In genetically determined audiogenic seizures i n rats and mice, taurine injected i n t r a -peritoneally f a i l e d to protect against seizures, whereas i n t r a v e n t r i c u l a r i n j e c t i o n s cause a dose-dependent attenuation of seizure a c t i v i t y (Laird and Huxtable, 1976). Thus, the differences between models of experimental epilepsy that respond to i n t r a p e r i t o n e a l administration of taurine may relate to differences i n the penetration of taurine through the blood-brain b a r r i e r . The protective e f f e c t s of taurine i n human e p i l e p t i c patients are highly variable (Barbeau, 1974; Bergamini et a l . , 1974; Sbarbaro, 1974; Striano et a l . , 1974). The mechanism underlying the anticonvulsant and a n t i e p i l e p t i c e f f e c t s of taurine i s unknown. Its e f f i c a c y against several experimental convulsive models and epilepsies with d i f f e r i n g e t i o l o g i e s suggests that i t has a nonspecific effect.Van Gelder (1976) proposed a biochemical mechanism for the anticonvulsant properties of taurine, involving the restoration to normal of the disturbances i n r a t i o of glutamine/glutamic acid i n convulsive states. Glutamate and (f-aminobutyric acid) GABA le v e l s are decreased i n experimental cobalt-induced and human epilepsies and seem to return to normal a f t e r taurine treatment and cessation of seizures (van Gelder, 19 72; van Gelder et a l . , 19 75 Joseph and Emson, 19 76 ) . Another proposed explanation for the anticonvulsive e f f e c t s of taurine i s that a reduction in hyperexcitability produced by taurine might be related to i t s e f f e c t s on membrane permeability to chloride, which produces hyperpolarization (Gruener and Bryant, 19 75). A more d i r e c t action of taurine on calcium and potassium fluxes i n myocardial and neural tissues has been described. (Grosso and Bressler, 1976; Huxtable, 1976a, Pasantes-Morales et a l . , 1978, Barbeau et a l . , 1975, Izumi et a l . , 1977). B. Taurine i n Retinal Degeneration Recent studies have shown that cats and kittens fed a taurine-free d i e t with casein as the only source of protein develop-. r e t i n a l degeneration which subsequently results i n photoreceptor death (Berson et a l . , 1976; Hayes et a l . , 1975; Schmidt et a l . , 1976, 1977; Rabin et a l . , 1973). Supplementation of the taurine-free casein d i e t with methionine, cysteine, inorganic sulfate, vitamin B^ or vitamin Bg with cysteine did not prevent development of r e t i n a l taurine deficiency and r e t i n a l malfunction (Berson et a l . , 1976; Schmidt et a l . , 1976). A synthetic amino acid d i e t also results i n r e t i n a l taurine deficiency and r e t i n a l malfunction. Only taurine-containing diets ( i . e . chow or casein plus taurine) preserved normal electroretinogram (ERG) amplitude and normal r e t i n a l 54 taurine concentrations (Berson et a l . , 1976; Schmidt et a l . , 19 77). These findings have firmly established a role for exogenous taurine i n maintaining r e t i n a l function i n the k i t t e n (Knopf et a l . , 1978). In models of r e t i n i t i s pigmentosa ( r e t i n a l dystrophy) i n rats and mice taurine was found to be the only amino acid to be reduced i n concentration (Cohen et a l . , 1973; Brotherton, 1962). C. Taurine i n Brain Development A great deal of information indicates that taurine i s present i n higher concentration at b i r t h and i n prenatal brain than i n mature animals (Agrawal et a l . , 1968a, 1968b; Agrawal and Himwick, 1970; Oja et a l . , 1968; Sturman and Gaull, 19 75), the exception being the frog, where small amounts of taurine are found i n the mature brain, whereas none i s found i n the tadpole brain (Roberts et a l . , 195 8). However, the concentration of taurine found i n frog brain i s much smaller than that found i n mammalian brain (Okumura et a l . , 1959). In a l l species studied throughout development, the decrease i n taurine concentration i n brain, from the high values i n the new born animal takes place gradually during postnatal development and i s complete approximately at weaning. This decrease i s i n contrast to the concentrations of most amino acids i n brain, which either increase or change very l i t t l e , during development (Agrawal and Himwick, 19 70). The high concentration of taurine i n 55 f o e t a l brain and i t s slow decrease postnatally suggesto that taurine may be associated i n some way with brain development (Sturman and Gaull, 1976; Kaczmarek et a l . , 1971). The o r i g i n of the very high concentration of taurine i n the developing brain i s uncertain. The a c t i v i t y of cysteine s u l f i n i c acid decarboxylase i s low early i n the development of rat brain and increases l a t e r (Agrawal et a l . , 19 71; Kaczmarek et a l . , 1970). Therefore, biosynthesis of taurine seems unlikely to account for the large concentrations present i n newborn brain. An e f f i c i e n t and highly s e l e c t i v e transport system for attaining and maintaining high i n t r a c e l l u l a r concentrations of taurine i n brain during development has been suggested, and such a mechanism might account for the large concentrations i n the newborn brain (Agrawal et a l . , 1971). Sturman et a l . , (1977b) have recently demonstrated 35 . . . that S-taurme injected into a pregnant rat enters f o e t a l brain as rapidly as i t enters f o e t a l l i v e r , with maximum values being reached af t e r 12 hours. In contrast, labeled taurine enters adult brain more slowly than i t enters adult l i v e r , with maximum values i n brain being reached af t e r 5 to 7 days. The source for the high concentration^of taurine i n brain may be dietary. Human milk, unlike bovine milk and a r t i f i c i a l formulas derived from i t , contains a considerable amount of taurine (Gaull et a l . , 1977). Recent work reported by Gaull et a l . , (19 77) also shows that the concentration of taurine decreases progressively i n plasma and urine of human pre-term infants fed casein-synthetic formulas derived from bovine milk. In contrast, pre-term human infants fed pooled human milk did not have such decreases. Greater excretion of taurine by ful l - t e r m human infants fed human milk as opposed to bovine milk has also been documented (Jagenburg, 1959; Jonxis, 1951). As discussed i n the previous subsection kittens fed a synthetic d i e t containing p a r t i a l l y p u r i f i e d casein as the source of protein become taurine-deficient and develop r e t i n a l degeneration, eventually r e s u l t i n g i n blindness. In this regard, man has a lower capacity for synthesis of taurine than the cat (Gaull et a l . , 1977; Knopf et a l . , 1978). In l i g h t of these studies, i t seems possible that there i s a dietary requirement for taurine i n the rapidly growing human infant. Sturman. et a l . , (1978) and Hayes (1976) i n th e i r reviews conclude that taurine i s probably an e s s e n t i a l nutrient.for humans-and that babies maintained on, , commercial milk formulas may be suffering from taurine deprivation. These reviewers further comment: "Only systematic investigation can possibly reveal the importance 57 of taurine i n human infants, p a r t i c u l a r l y i n the etiology of some forms of sudden infant death syndrome,(SIDS) . " D. E f f e c t of Taurine on Endocrine Functions: Although taurine levels are not p a r t i c u l a r l y high i n the hypothalamus (Coll i n s , 1974), th i s region possesses a high capacity for taurine biosynthesis and a very e f f e c t i v e mechanism for taurine uptake; i t i s indeed, the sole region i n which taurine concentration i s s i g n i f i c a n t l y increased after intraperitoneal injections (Hruska et a l . , 1973). On this basis, a role for taurine i n certain neuroendocrine functions regulated by the hypothalamus has been suggested. Intracerebral or intraperitoneal administration of taurine causes hypothermia i n rats and mice (Hruska et a l . , 1973; Sgaragli and Pavan, 1972). The mechanism mediating t h i s e f f e c t i s unknown, but taurine does not appear to interf e r e with peripheral heat-generating mechanisms. Some evidence suggests that the effects of taurine are mediated by central cholinergic or serotoninergic systems(Sgaragli et a l . , 1975). Taurine has been reported to a l t e r conditional eating and drinking functions regulated by hypothalamic structures (Thut et a l . , 1976. Hruska, et a l . , 1975),. Intraperiotoneally administered taurine, at doses that increase hypothalamic 58 taurine concentrations s i g n i f i c a n t l y reduce conditional water and food responses i n rats and mice. Further, chronic taurine administration reduces weight gain of genetically obese mice (Taisho et a l . , 1970). There is.-.evidence that taurine and some related amino acids, may also be involved i n other endocrine functions such as i n the regulation of c i r c a r d i a n rhythm (Grosso et a l . , 19 78a;Nenhoff and Tonge, 1973; Baskin and Dagirmanjian, 19 73); reproductive functions (Kochakian, 1973, 1976a, 1976b,. and adrenal function (Kuriyama and Nakagawa, 1976). E. Taurine and Nerve Conduction Some investigators have suggested a physiological role for taurine i n the maintainance of excitatory a c t i v i t y i n mammalian nervous tissues. Mandel et a l . , ; (1975), Kaczmarek (1976), P h i l l i s (1978), Oja and Kontro (1978) and Guidotti (1978) have raised the p o s s i b i l i t y that taurine i s an i n h i b i t o r y neurotransmitter. The i n h i b i t o r y action of taurine has been demonstrated i n a variety of nerve preparations: Taurine administered iontophoretically i n the cerebral cortex blocks both spontaneous and chemically-induced f i r i n g of c o r t i c a l neurons (Curtis et a l . , 1971; Krnjevic and P h i l l i s , 1963). Taurine, microiontophoretically injected, depresses 59 spontaneous neuronal discharge of the evoked f i e l d p o t e n t i a l i n the spinal cord and brainstem neurons (Curtis et a l . , 1960, 1971; Haas and H o s l i , 1973). The microiontophoretic application of taurine in the cerebellar cortex of the r a t , produces a dose-dependent depression of the spike frequency of cerebellar neurons (Frederickson et a l . , 1978) . More recently, McBride and Frederickson (19 78) have obtained neurochemical and neurophysiological evidence for the presence of taurinergic neurons i n the cerebellar cortex of the rat. Taurine has also been shown to have a powerful i n h i b i t o r y e f f e c t on the r e t i n a l b i o e l e c t r i c response of i s o l a t e d retinas; taurine added to the perfusion medium induces a rapid and s p e c i f i c depressant e f f e c t on the electroretinogram that i s t o t a l l y reversible a f t e r washing with taurine-free medium (Urban et a l . , 1976) . Identical results have been obtained a f t e r i n t r a v i t r e a l i n j e c t i o n of taurine i n the chicken (Pasantes-morales et a l . , 1973). Inhibitory e f f e c t s of taurine are also observed i n muscle. Taurine showed a hyperpolarizing e f f e c t on the membrane resting p o t e n t i a l of frog and rat muscle fi b e r s and produced changes i n c h a r a c t e r i s t i c s of the action p o t e n t i a l , influencing mainly r e p o l a r i z a t i o n . In these studies, the duration of the action p o t e n t i a l i s prolonged, and the interspike i n t e r v a l s are increased. These ef f e c t s of taurine 60 can be observed either i n - v i t r o on i s o l a t e d muscle prepara-tions or in-vivo a f t e r loading by i n j e c t i o n of taurine (Gruener, and Bryant, 1975; Gruener et a l . , 1975, 1976). Summary What i s the possible neurophysiological role for taurine? I t i s believed that taurine may have a neurotrans-mitter role i n the central nervous system. A set of c r i t e r i a that a substance must f u l f i l l before i t can be considered a neurotransmitter has been reported, and taurine appears to f u l f i l l the majority of these requirements (Mandel et a l . , 1975; Lahdesmaki and Oja, 1973; Kaczmarek, .1976)... In addition, i t s strong i n h i b i t o r y actions on neuronal a c t i v i t y i n c o r t i c a l areas and spinal cord (Curtis and Crawford, 1969; Curtis and Watkins, 1960; Crawford and Cur t i s , 1964; Haas and Hos l i , 1973), as well as i t s antagonistic effects on seizures (Izumi et a l . , 1974; Kaczmarek and Adey, 1974; Mutani, et a l . , 1974b), provide further indications that taurine may function as a neurotransmitter or may have a modulatory role i n nerve e x c i t a b i l i t y . However, a d e f i n i t i v e answer to thi s question cannot be given on the basis of the presently available knowledge (Oja and Kontro, 1978). Other investigators (van Gelder, 1972; Gruener et a l . , 19 75; Honegger et a l . , 1973; Barbeau et a l . , 19 75; Huxtable, 1976a)have argued i n favour of -taurine as a neuromodulator. 61 According to P h i l l i s (1978), a substance causing longer l a s t i n g a l t e r a t i o n to c e l l e x c i t a b i l i t y may /be c a l l e d a "modulator".Most appropriately taurine can be described as a neuroeffector, possibly acting on ion fluxes (Hagins and Yoshikama, 1974; Pasantes-Morales et a l . , 1978; Izumi et a l . , 1978) and membrane s t a b i l i z a t i o n of the neurons (Gruener and Bryant, . 1975; Gruener et a l . , 1975; Gruener - et a l . , 1976).. . ./ The ef f e c t o r role" of taurine may•also'be related to i t s e f f e c t s on certain metabolic actions, such as pyruvate dehydrogenase (Izumi et a l . , 1978) or glutamate regulation (van Gelder, 1978), and the i n h i b i t o r y e f f e c t on the release . of other neurotransmitters (epinephrine, norepinephrine) (Kuriyama et a l . , 1978). The regulatory e f f e c t of taurine on the control of epinephrine release from adrenal storage granules, was said to be due to changes i n the calcium a f f i n i t y of these granules or by s t a b i l i z i n g the granular membrane (Kuriyama and Nakagawa, 1976). The finding of McBride- and Frederickson (1978) and others (Frederickson et a l . , 1978; McBride et a l . , 1976; Nadi et a l . , 1977) on the presence of the i n h i b i t o r y s t e l l a t e c e l l s of the cerebellar cortex, s i g n i f y that i n h i b i t o r y taurinergic neurons do indeed e x i s t i n certain areas of the brain. In the report of McBride and Frederickson (1978) taurine was shown to i n h i b i t f i r i n g rate of Purkinje c e l l s , through the i n h i b i t o r y s t e l l a t e c e l l s i n the cerebellar cortex. 62 RATIONALE The m u l t i p l i c i t y of taurine effects discussed i n the review of the l i t e r a t u r e section of thesis suggests the p o s s i b i l i t y that taurine has a variety of actions. However, i t i s possible that a single basic mechanism (which remains to be c l a r i f i e d ) i s responsible for a l l these e f f e c t s . One such mechanism might involve taurine alterations i n ion fl u x . A number of workers have indicated an involvement of taurine i n calcium ion" transport i n various tissues (Dolara et a l . , 19 73; 1976; D i e t r i c h and Diacono, 1972; Huxtable and Bressler, 1973; Igisu et a l . 1976; Izumi et a l . , 1977; Barbeau et a l . , 1975; Kuriyama and Nakagawa, 1976; Kuriyama et a l . , 1978). The mechanism by which taurine may act on calcium ion movements though remains speculative. Many workers (read and Welty, 1965; Chazov et a l . , 1974; D i e t r i c h and Diacono, 1971; Guidotti, et a l . , 1971; Jacobsen and Smith, 1964, 1968; Yamaguchi et a l . , 1973; Sumizu, 1962; Peck and Awapara, 1967; Huxtable and Bressler, 1972; Koechlin, 1955; Spaeth and Schneider, 1974; Sturman, 1973) have stated or repeated the argument that the pharmacological actions of taurine (a zwitterion) involve i t s possible conversion to i s e t h i o n i c acid (2-hydroxyethane sulfonic acid), a strong anion. This conversion was said to lead to the conductance of cations into the cardiac c e l l . One major obstacle to the search for possible functions of i s e t h i o n i c acid (ISA), at 63 the time when t h i s work was begun was the lack of a good a n a l y t i c a l procedure. The work described i n t h i s t h e s is, therefore, began with a search for a sensitive gas l i q u i d chromatographic method to measure i s e t h i o n i c acid i n mammalian tissues. I t was, then, necessary to re-examine the enzymatic conversion of "^C-taurine to "^C-ISA i n rat heart s l i c e s . This work suggested that i s e t h i o n i c acid was not l i k e l y to be involved i n the mechanism of action of taurine. Following the results obtained i n the above studies, the p o s s i b i l i t y that taurine i t s e l f might a f f e c t the transport of calcium ions across membranes was evaluated. F i r s t l y , a study of the e f f e c t of taurine on ATP-dependent calcium transport i n guinea-pig whole heart homogenates and sarcoplasmic reticulum (S.R.) preparations was undertaken. Secondly, studies on the e f f e c t of taurine on the passive d i f f u s i o n of calcium and other ions were investigated. In the f i r s t study guinea-pig hearts were used because most of the previous experiments on cardiac effects of taurine were observed i n t h i s animal species. In the l a t e r studies, rat brain synaptosomes were used as a model system to study the e f f e c t of taurine on the passive d i f f u s i o n of calcium, sodium and potassium ions. Synaptosomal preparations were used i n these studies, since several workers (Kuriyama et a l . 1978; Barbeau et a l . , 1975; Pasantes-Morales et a l . 1978; Lahdermaki and Pajunen, 1977) suggested'that the main action of taurine i n the CNS may be related to i o n i c fluxes occurring at the synaptic terminal l e v e l . The a l t e r a t i o n i n calcium ion fluxes i n the synaptosomes i s well known to be important i n the regulation of the e x c i t a b i l i t y of neuronal tissue. MATERIALS AND METHODS I: STUDIES WITH ISETHIONIC ACID Development of - an/Analytical Method- for Isethionic Acid  by-Gas Liquid Chromatography. Reagents (a) Isethionic Acid: - Obtained as sodium s a l t from Sigma Chemical Co. St. Louis, Missouri. - Sodium isethionate was r e c r y s t a l l i z e d with hot 95% ethanol to constant melting point (192°C.) - Stock solution (5 mM) was prepared i n water. (b) S a l i c y l i c Acid: - Obtained from Sigma Chemical Co. St. Louis, Missouri. - Re c r y s t a l l i z e d to constant melting point (138°C) from 10% ethanol and dried i n a vaccum desicator over P2°5' - Solution (1 mM) was prepared i n methanol. (c) 1-Butane-Sulfonic Acid: - Sodium s a l t , a n a l y t i c a l grade, was obtained from Eastman Kodak Cp., Rochester, N.Y. 67 - Used without further p u r i f i c a t i o n , 1. mM stock solution was prepared i n water. (d) Other Internal Standards: - Methyl caproate (C 5 COOH) - Methyl caprylate (C ? H 1 5 COOH) - Methyl caprate (C g H 1 9 COOH) - Methyl laurate {C^ H 2 3 COOH) - A l l obtained from Applied Science Laboratories, Inc. Penna., 100 mg of each was dissolved i n 0.5 ml methanol. - Benzoic acid, obtained from BDH Canada 7(AnalaR), 1 mM solution was prepared i n methanol. - A c e t y l s a l i c y l i c acid, BDH Pharmaceuticals, Toronto, Canada; 5 mg/ml solution was prepared i n ethanol. (e) OV-1 and OV-.17 Columns: - OV-1, 5% and 5% OV-17 on II. P. chromosorb W, 80-100 mesh; were obtained from Gas Chromatographic S p e c i a l i t i e s Ltd. B r o c k v i l l e , Ontario. - Empty columns, 6 f t by 4 mm (i.d.) glass U-tubes for Bendix, model 2500 GLC were also obtained from Gas Chromatographic S p e c i a l t i e s . The columns, packed with either OV-1 or OV-17, were conditioned on the GLC for one day at 200 with no nitrogen(N 2) flow and then for one day each at '250.and 300°C respectively 68 with c a r r i e r (N 2) gas flows of 50 ml ../minute. Other Columns Used: -DEGS, 5% (diethylene glycol-succinate) chrosorb W (AW-DMCS) , 60-80 mesh was obtained / from Western Chromatographic Supplies, Vancouver. -PEGS, 2% (polyethylene glycol succinate) on chromosorb W, acid washed and DMCS treated, 80-100 mesh was obtained from Chromatographic S p e c i a l i t i e s , B r o c k v i l l e , Ontario. -SP-400 on chromosorb W, 80-100 mesh, was obtained from Supelco, Inc. Bellefonte, Pennsyslvania. S i l y l a t i n g Agents: -"BSA" N,0-Bis-(trimethylsilyl)-acetamide. - TRI-SIL/BSA, Formula P (in pyridine solvent) Formula D (in dimethylformamide solvent). - Hexamethyldisilazane (HMDS) and trimethylchloro-silane (TMCS). - Solvents: a c e t o n i t r i l e ; pyridine and dimethyl-formamide (DMF) - A l l s i l y l a t i o n grade, obtained from Pierce Chemical Company, Rockford, I l l i n o i s . 69 (h) Diazomethane; Diazald and diazald k i t were obtained from A l d r i c h Chemical Company, Milwaukee, Wisconsin. Diazald (2.14 g) was dissolved i n ether (30 ml). Diazomethane was d i s t i l l e d a f t e r the addition of potassium hydroxide i n ethanol (10ml, 0.04 g/100 ml). The conventional d i s t i l l a t i o n was carried out using the diazald k i t . The d i s t i l l a t e , bright golden-yellow, was kept cold at a l l times and stored at -21°C. This preparative method i s e s s e n t i a l l y that of Vogel (1964). (i) Resin: - AG 50-X8, 50-100 mesh, hydrogen form, Bio-Rad Laboratories. Before use, the resin was treated with 5 bed volumes of 1 N NaOH, washed with d i s t i l l e d water to n e u t r a l i t y and then regenerated to the hydrogen form with 5 bed volumes of 2N hydrochloric acido followed by water washing. The r e s i n was l a t e r washed i n methanol three times and suspended i n an equal volume of methanol. 70 (j) Other Reagents: - Methanol - Ether - Ethanol - A l l the chemical were reagent grade, obtained from Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey. Preparation of Isethionic Acid for Use as Qualitative  Standard. : A suspension of AG 50, (Hydrogen form) i n methanol was pipetted into 5 ml glass stoppered, calibrated, conical centrifuge tubes. After allowing the resin to s e t t l e , the methanol layer was aspirated and discarded. To each tube was added 0.5 ml or 1.0 ml of 5 mM sodium isethionate solution i n water. The volume was adjusted to 4 ml i n each tube with methanol, and the r e s i n suspended using a vortex mixer. After centrifugation, the methanol layer from each tube was removed into reaction v i a l s and evaporated to dryness i n a vacuum desiccator over s u l f u r i c acid. Methylation of Isethionic Acid Isethionic acid preparations were dissolved i n 0.1 ml solution of i n t e r n a l standards ( s a l i c y l i c acid, butanesulfonic acid, benzoic acid, a c e t y l s a l i c y l i c acid methyl caproate, methyl caprylate, methyl caprate or 71 methyl laurate) i n methanol. When butanesulfonic acid was used as an i n t e r n a l standard, the sodium s a l t was added to the resi n along with sodium isethionate. The v i a l s were stoppered using t e f l o n laminated discs and placed i n an ice-bath for fiv e minutes. Ethereal diazomethane solution was introduced into the v i a l s slowly, with mixing, u n t i l the yellow color persisted. An additional three drops of diazomethane solution was added and the mixture was allowed to stand for about t h i r t y minutes i n i c e . The t o t a l volume i n the v i a l was 0.2 ml or less. The methylation reaction was stopped by adding one drop (0.02 ml) of 50% acetic acid i n water (v/v). 4.. S i l y l a t i o n of Isethionic Acid Isethionic acid (10 mg) was prepared as described above, and dissolved i n 0.75 ml dimethylformamide, pyridine or a c e t o n i t r i l e , BSA, 0.25 ml, was then added. The v i a l s were heated on a Reacti-Therm block (Pierce: Chemical Co., Rockford, I l l i n o i s ) for 6 minutes at 135°C, and allowed to cool. 1 u l was then injected on to an OV-17, PEGS or SP-400 column. When Tri-Sil/BSA (either formula P or D) was used, 1 ml was added to 10 mg i s e t h i o n i c acid and the same treatment as for BSA was carr i e d out. A l t e r n a t i v e l y , 72 is e t h i o n i c acid (10 mg) was dissolved i n 0.5 ml of pyridine, treated with 0.1 ml HMDS and 0.2 ml TMCS and boiled under re f l u x for 30 minutes. Gas-Liquid Chromatography: Flame Ionization Detector The gas - l i q u i d chromatograph used was a Bendix, model 2500, equipped with a flame i o n i z a t i o n detector. Columns were 6 f t by 4 mm (i.d.) glass U-tubes. The stationary phases used were 5% OV-1 and 5% OV-17. Analyses were performed isothermally at temperatures ranging from 100°C to 150°C unless otherwise indicated: The optimal temperature for OV-1 was 115°C and for OV-17 was 135°C. Nitrogen was used as a c a r r i e r gas at a flow rate of 40 ml/min. Gas-Liquid Chromatography:Sulphur Detector 5 The gas-liquid chromatograph used for such experi-ments was a Micro Tek 220 equipped with a flame photometric detector, Model FPD 100 (Melpar Inc., F a l l s Church, V i r g i n i a ) . The column used was a 6 f t by 2 mm (i.d.) glass U-tube with 5% OV-17 on 80-100 mesh H.P. chromosorb W. Nitrogen was used a The analysis were ca r r i e d out at the laboratories of the Government of Canada Agriculture Research Station, Vancouver, B.C., under the supervision of Mr. Ian H. Williams. 73 as the c a r r i e r gas with an i n l e t flow of 30 ml/min. Column temperature was 100°C. Oxygen flow to the detector was 10 ml/min and a i r flow was 30 ml/min. The i n t e r n a l standard used was 1-butanesulfonic acid. 7. Gas-Liquid Chromatography; Mass Spectrometry The mass-spectrometer used was a Hitachi Perkin-Elmer, operating at an i o n i z a t i o n energy of 70 eV interfaced with a Varian ;, Model 1400 gas chroma-tograph. Authentic ISA and butane sulfonic acid were analyzed on OV-17 column aft e r methylation with diazomethane. Chromatographic d e t a i l s were as outlined above for flame i o n i z a t i o n detection. Q 8. Nuclear Magnetic Resonance Spectroscopy For proton NMR spectroscopy, the methyl esters of ISA and butane su l f o n i c acid were prepared i n the following manner: Rec r y s t a l l i z e d sodium isethionate (100 mg) and 1-butane sulfonic acid ( sodium salt) The experiments were performed under the d i r e c t i o n of Mr. Greg Owen at the Department of Chemistry, Simon Fraser University, Burnaby, B.C., Canada cThe NMR was kindly determined and interpreted by Dr. Donald G. Clark, Department of Chemistry, University of B r i t i s h Columbia, Vancouver, B.C. Canada. 74 were each treated with re s i n (AG50, H form), and methylated as described above. After t h i r t y minutes, excess diazomethane was c a r e f u l l y blown o f f with a stream of dry nitrogen and the sample completely dried in- a vacuum dessicator over sulphuric acid. The NMR spectra of the reaction products, without further p u r i f i c a t i o n , were taken at 100 mHz i n a Varian HA-100 spectrometer. Samples were dissolved i n deuterated dimethylsulfoxide (Merck Sharpe and Dohme Ltd., Canada) with tetramethyl silane being used as an i n t e r n a l standard. For the purposes of comparison, the NMR spectrum of c r y s t a l l i z e d , non methylated ISA, also dissolved i n dimethylsulfoxide was obtained. B. Analysis of Isethionic Acid i n Mammalian Tissues i 1. Reagents (a) Folch Solvent: - Chloroform and methanol were mixed i n 2:1 (v/v) r a t i o , 5% water was added to the t o t a l volume. (b) Other Reagents - Methanol 75 -•Chloroform - Ethanol - A l l chemical used were reagent grade, obtained from Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey.' 2. Preparation of Heart, Brain and Other Tissues Used for  the Analyses of Isethionic Acid. (a) Rat Heart and Brain Preparation: Wistar rats weighing approximately 200 g were s a c r i f i c e d by a sharp blow to the head. Hearts and brainswere promptly excised and the tissues rinsed i n normal saline, blotted on Whatman #1 f i l t e r paper and immediately frozen i n small p l a s t i c v i a l s i n l i q u i d nitrogen. This preparative process required less than ten minutes per rat. (b) Dog Heart Preparation: Mongrel dog hearts were obtained from the Department of Physiology University of B r i t i s h Columbia, following the use of these animals for minor experiments which did not involve the heart. The dogs were s a c r i f i c e d with 15% potassium chloride (10 ml) and the heart stored at -20°C before use. 76 (c) Axoplasm from Squid Giant Axon, Squid Ganglion  and Nautilus Ganglion. Samples of the giant squid axon, squid ganglion and Nautilus ganglion were obtained from Dr. Frank C. Hoskin of the Department of Biology, I l l i o n i s I n s t i t u t e of Technology, Chicago, I l l i n o i s . The method used f o r the i s o l a t i o n of axoplasm from the squid axon i s that of Maxfield(19 53). Pre-isolated material from the squid and Nautilus were obtained from the above source i n freeze-dried form. (d) Rat Milk Samples Milk samples were obtained from Dr. John A. Sturman, Institute of Basic Research i n Mental Retardation, Staten Island, New York, N.Y. Samples of milk (0.2 ml) were co l l e c t e d from l a c t a t i n g rats of ;.Nelson-Wistar. .s.traih, and freeze-dried. The samples on a r r i v a l from New York were stored at -20°C. Further d e t a i l s for procuring milk samples from the rats are given i n an e a r l i e r paper of Sturman et a l . , (1977b). 77 3. Is o l a t i o n of ISA from Tissues (a) Rat Heart and Brain Tissues: Samples (5 g) of pooled rat brain or heart were used for experimentation.The heart or brain tissues were minced before being used and then divided into two equal portions of 2.5 g each. To one portion was added O.5.-umole of sodium isethionate. The other portion was used without any addition. Both portions were homogenized i n 50% methanol/water (v/v), (10 ml), in a S o r v a l l Omni-mixer (Ivan S o r v a l l Inc., Norwalk, Connecticut), using a t e f l o n chamber at 3/4 of the f u l l speed for f i v e minutes, followed by one minute of f u l l speed (Figure 6). The homogenate was transferred to a centrifuge tube and centrifuged at 3 ,000'xgfor f i v e minutes. The homogenizing chamber was rinsed three times with 5 ml, 50% methanol/water (v/v), and the rinse washings added to the c e n t r i -fuge p e l l e t which was suspended i n the r i n s i n g solution using a Vortex mixer. The r e s u l t i n g suspension was then centrifuged again for f i v e minutes and the supernatant f l u i d removed. To the combined volume of supernatants and rinsings, an equal volume of Folch solvent was added. The solutions were mixed thoroughly and centrifuged to Figure 6 Flow Chart of the I s o l a t i o n of Isethionic Acid From Tissues 78a TISSUE HOMOGENATE T SUPERNATANT AQUEOUS LAYER RESIDUE Minced, Divided into two equal portions To one portion sodium isethionate i n water was added To the other portion, the control, no ISA was added, Both portions, were l e f t at room temperature for 5 minutes to eq u i l i b r a t e Both :were homogenized with - 50%-methanol water i n a s o r v a l l omni-mixer Centrifuged —\ P e l l e t washed three times with 50% methanol Supernatant and washings combined Added equal volume of Folch solvent Mixed and centrifuged Evaporated to dryness i n a rotary evaporator under reduced pressure - AG 50 (H form, prewashed i n methanol) was added to the flask containing residue. - Flask swirled to mix thoroughly - Taken up i n methanol - Add i n t e r n a l standard (1-butanesulfonic acid) - Centrifuged - Methanol layer transferred to a v i a l and desiccated to dryness DIAZOMETHANE TREATMENT 79 separate the layers. The upper aqueous layer was removed and evaporated to dryness i n a rotary evaporator under reduced pressure. After evaporation of the aqueous layer to dryness, 2 ml of a p u r i f i e d cation exchange r e s i n (AG 50 - X8 , '-50-100 .mesh, form, suspended i n methanol), was added to the f l a s k . After t r i t u r a t i o n , the mixture was transferred to a 5 ml glass-stoppered conical centrifuge tubes containing 0.1 pinole butanesulfonic acid. After centrifugation, the methanol layer was dried i n a vacuum desiccator over sulphuric acid. (b) Dog Heart: Pooled dog hearts (400 g) were minced and homogenized i n an equal volume of warm d i s t i l l e d water (6 0°C). An equal volume of absolute ethanol was then added and the mixture centrifuged for 10 minutes at 3,000 x g. The p e l l e t was washed three times with 50% ethanol and the washings added to the supernatant. The supernatant and washings were mixed with three volumes of chloroform. The aqueous portion was removed and evaporated to approximately 25 ml volume i n a rotary evaporator, and then passed through an AG 50 (H + form, 135 ml resin) column (82 x 1.75 cm). The column was eluted with deionized water. 45 80 fractions of 5 ml volume were co l l e c t e d . The column had a void volume of 65 ml. The eluted fractions were pooled i n three groups as pretaurine, taurine, and posttaurine. The pooled fractions were neutralized to pH 7.0.Taurine fractions (18 to 28) were detected by paper chromatography and ninhydrin as described on pages 86 to 87. Aliquots of the pooled fractions were evaporated to dryness i n a rotary evaporator under reduced pressure. They were then treated with AG 50 (H+form) r e s i n / methanol ( 1 : 1 , w/v) as described above for the i s o l a t i o n of ISA from rat heart and brain tissues and were analyzed by GLC (pp. 82-83.) The remainder of the pooled fractions were divided into two portions. The duplicate f r a c t i o n contained additional sodium isethionate (10 mg/100 g heart wet weight). A l l the portions were reduced i n volume to 5 ml and attempts were made to c r y s t a l l i z e sodium isethionate with warm 95% ethanol. Axoplasm from the Squid Giant Axon, Squid Ganglion  and Nautilus Ganglion. Materials from squid giant axon (83 mg fresh weight) 81 squid ganglion and Nautilus ganglion (500 mg fresh) weight), were each dispersed i n deionized water using a glass homogenizer. The turbid solution so formed was made up to 10 ml with deionized water. An aliquot of the solution was mixed with an equal volume of absolute methanol and t h i s mixture treated with an equal volume of Folch solvent. The rest of the procedure was the same as that described i n F i g . 6 for i s o l a t i o n of ISA from other tissues. (d) Rat Milk: Freeze-dried, milk samples (0.2 ml) were resuspended i n 10 ml deionized water using a glass-glass homogenizer. Authentic i s e t h i o n i c acid (25 to 150 nmoles) was added to some of the samples at t h i s stage. The suspension was mixed with an equal volume of Folch solvent (20 ml), mixed thoroughly, and centrifuged to separate the layers. The aqueous layer was removed and extracted as described, for i s o l a t i o n of ISA from rat heart and brain tissues. \ 82 4. Methylation of the Sample and Preparation of  a Standard Curve. Isethionic acid standards were obtained following treatment of 0, 10, 20, 30, 40, 50 and 60 u l of 5 mM sodium isethionate solution and 10 u1 of 1 mM aqueous solution of 1-butanesulfonic acid with AG 50, hydrogen form re s i n (prewashed i n methanol) as described on page 70., for the "Preparation of Isethionic Acid". Butanesulfonic acid was used as an i n t e r n a l standard. ISA standards, or samples obtained from a tissue extract were dissolved i n 0.1 ml of methanol. In separate experiments, s a l i c y l i c acid was used as an i n t e r n a l standard to confirm the i d e n t i t y of i s e t h i o n i c acid peaks using OV-1 and OV-17 columns. In these studies i s e t h i o n i c acid standard consisted of 0, 0.5, 1.0, 1.5, 2.0,"2.5 and 3.0 umoles and s a l i c y l i c acid (0.1 ml of 1 mM solution i n methanol) was added to the standards or samples a f t e r treatment with AG 50 r e s i n and butane-su l f onic acid was omitted from the procedure. The "Methylation of ISA" was thqn followed as described on page 70. i n each case the t o t a l volume i n the v i a l was 0.2 83 ml o r l e s s . W h e r e t h e t o t a l volume i n t h e v i a l was more, t h a n 0.2 ml, t h e e x c e s s s o l v e n t was c a r e f u l l y e v a p o r a t e d u n d e r a s t r e a m o f d r y n i t r o g e n and t h e sample r e m e t h y l a t e d f o r a f u r t h e r t h i r t y m i n u t e p e r i o d . T h i s r e m e t h y l a t i o n p r o c e d u r e was f o u n d n o t t o a f f e c t t h e s t a n d a r d c a l i b r a t i o n c u r v e f o r ISA. A n a l y s e s o f Samples by GLC. F o l l o w i n g m e t h y l a t i o n , a l i q u o t s (5 y l ) o f t h e s t a n d a r d s and s a m p l e s were i n j e c t e d i n t o a g a s - l i q u i d c h r o m a t o g r a p h (GLC) w i t h e i t h e r a f l a m e i o n i z a t i o n d e t e c t o r o r s u l f u r d e t e c t o r . The s t a n d a r d s u s e d f o r t h e GLC w i t h s u l p h u r d e t e c t o r were 1/10 t h o f t h e c o n c e n t r a t i o n u s e d f o r t h e f l a m e i o n i z a t i o n d e t e c t o r , and 1-butane s u l f o n i c a c i d was u s e d as an i n t e r n a l s t a n d a r d . C o n v e r s i o n o f T a u r i n e t o ISA  R e a g e n t s (a) I s e t h i o n i c A c i d Same as on page 66 . (b) T a u r i n e : O b t a i n e d f r o m Sigma C h e m i c a l Co., S t . L o u i s M i s s o u r i . 84 (c) Radioactive Taurine (1,2 xt*C) : - Obtained from New England Nuclear. Spe c i f i c a c t i v i t y was 50 mCi/mmole. (d) S c i n t i l l a t i o n F l u i d : - Toluene, s c i n t i l l a t i o n grade, Fisher S c i e n t i f i c Co., New Jersey. - Methyl cellosolve (PIERSOLVE, ethylene g l y c o l monomethyl ether, sequanal grade, Pierce, Rockford, I l l i o n o i s ) - PPO (2, 5 diphenyioxanole), s c i n t i l l a t i o n grade, Kent Laboratories Ltd, Canada). - POPOP (1,4-bis(2-(5-phenyloxazolyl)benzene, S c i n t i l l a t i o n Grade, Kent Laboratories Ltd. Canada. - To a 5 00 ml mixture of toluene and cellosolve (1:1, v/v), 25 mg POPOP and 2 g PPO were added, s t i r r e d for 2 hrs to dissolve and used within 24 hrs after preparation. (e) Acridine: - Obtained from A l d r i c h Chem. Co. Milwaukee, Wis. - 0.1% solution prepared i n absolute ethanol. (f) Ninhydrin: - Obtained from Fisher S c i e n t i f i c Co., New Jersey. - 0.2% Solution prepared i n absolute ethanol i n 85 the presence of 1% pyridine. (g)Kreb-Henseleit /Phosphate Buffer, pH 7.3: Krebs-Henseleit buffer, pH 7.3 was prepared as described by Dowson et a l (1974). The ingredients were: 0.9 % NaCl 100 parts 1.15% KC1 . 4 parts 1.22% C a C l 2 3 parts 2.11% KH 2P0 4 1 part 3.8% MgS04.7H20 1 part 1.3% NaHCC>3, gassed with CC>2 for 1 hour 21 parts To 100 ml of the mixture, 0.18 g glucose was added. The osmolality of the buffer was 300 mOsmoles/kg. A l l reagents used were a n a l y t i c a l grade; obtained from Fisher S c i e n t i f i c Co., New Jersey. (h) ATP (0.01 M).: . - Obtained from Sigma Chemical Company, St. Louis, Missouri as a disodium s a l t . - Aqueous solution, pH 7.3 (adjusted with NaOH) (i) Pyridine-HCl: - obtained from Sigma Chemical Company, St. Louis, Missouri - 10 mg/ml aqueous solution. 86 (j) Other Reagents: - Sodium n i t r i t e (NaNO,,) 20% aqueous solution. - Hydrochloric acid - Ethanol - t-Butanol - Pyridine - Methanol - S u l f u r i c Acid - A l l reagents were a n a l y t i c a l grade, obtained from Fisher S c i e n t i f i c Co., New Jersey. 14 2. Preparation of C-ISA as Marker for the Taurine  Bioconversion Studies: Radioactive (1,2 1 4C) taurine (100 y l , l O y C i , 50 yCi/mole) was dissolved i n 20% sodium n i t r i t e (0.4 ml). The reaction was carr i e d out on a water bath at 60°C by adding concentrated HC1 dropwise (0.2 ml) u n t i l effervesence stopped. The reaction mixture was centrifuged to remove sodium chloride p r e c i p i t a t e . Sodium isethionate (40 mg) was added to the supernatant, and sodium isethionate c r y s t a l l i z e d with hot 95% ethanol. The r e s u l t i n g c r y s t a l s (shiny, c o l o r l e s s , rhombic plates) melted at 192°C (uncorrected) and the mixed melting point with authentic sodium isethionate c r y s t a l s was 192°C. Ascending paper chromatography was performed for 17 hours on Whatman No.3 paper 87 (7" x 20") using t-butanol, pyridine and water (1:1:1) as solvent. The s t r i p s (1" x 20") were cut out and developed by spraying with riinhydrin or acridine i n ethanol. The paper chromatogram of authentic i s e t h i o n i c acid, and taurine showed an R f value of 0.71 and 0.59, respectively. With radioactive isethionate, both acid and i t s sodium s a l t , an R f value of 0.71 was obtained. A s t r i p counter 14 14 was not.available. C-ISA and C-taurine a f t e r chromatography, were detected by cutting 0.5" x 1" pieces from the spotting o r i g i n and counting the pieces i n s c i n t i l l a t i o n f l u i d (10 ml) using standard l i q u i d counting techniques. Analysis by paper chromatography showed less than 2 per cent radioactive \impurities. The R^ value of the 'major' impurity was greater than that of i s e t h i o n i c acid. The y i e l d from t h i s preparation 14 . was more than 50% of the r a d i o a c t i v i t y of the C-taurine substrate. 3. Synthesis of Isethionic Acid by Rat Heart S l i c e s : The method used was that described by Read and Welty (1962). Rats, Wistar s t r a i n , weighing 200-250 g were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n . The hearts were promptly removed, cut into four pieces, washed i n i c e -88 cold s a l i n e , blotted on f i l t e r paper and transfered to i c e - c o l d incubation medium containing Krebs-Henseleit phosphate buffer, pH 7.3 and 0.01 M glucose. The v e n t r i -cles were s l i c e d (0.5 mm thick) using a Stadie-Riggs microtome (Arthur H. Thomas Co. P h i l a , P.A., USA). The s l i c e s were weighed out (0.1 to 0.15 g) a f t e r l i g h t drying on f i l t e r paper and then resuspended i n fresh incubation medium and incubated at 37°C i n a shaker water bath for 30 minutes. At the end of the incubation, the s l i c e s were transfered to a preincubated medium (37°C) containing 2.0 ml ofKrebs-Henseleit phosphate buffer pH 7.3, 0.15 ml of 0.01 M ATP, 0.05 ml of pyridoxine-HCl (10 mg/ml) and 14 0.1 ml of C-taunne (100 y l ) . The s l i c e s were incubated at 37°C for 0, 30 or 60 minutes. The reaction was stopped by the addition of 2.5 ml methanol. The contents were transfered to c h i l l e d homogenizing tubes and homogenized using a Polytron homogenizer, at f u l l speed for 1 minute. The homogenate was centrifuged at 3,00 0 x g for f i v e minutes. The p e l l e t was resuspended three times i n 2 ml, 50% methanol/water (v/v) using a vortex mixer. To the combined volume of supernatant and rinsings, an equal volume of Folch solvent (5 ml) was added. The rest of the procedure was the same as that described for the i s o l a t i o n of ISA from rat heart and brain tissue ( page 77). The methanol residue, dried i n a vacuum desiccator over sulphuric acid, was 89 made up to 0.1 ml with d i s t i l l e d water. The solution (5 yl)was then used for paper chromatography. II: STUDIES ON-'THE EFFECT OF TAURINE ON ION TRANSPORT PROCESSES A. E f f e c t of Taurine on ATP-dependent Calcium Transport  i n Guinea-pig Cardiac Muscle: 1. Reagents: (a) Sodium Bicarbonate Buffer (lOmM): - Sodium bicarbonate (NaHCO^) (lOmM), 5 mM sodium azide (NaN^) and 0.2 mM ascorbic acid were dissolved i n water, pH 6.8 (adjusted with IN HC1.) (b) KC1 Solution (0.6M): - Potassium chloride andimM magnesium chloride (MgCl 2.6H 20) were dissolved i n 2 mM Tris-HCl buffer, pH 7.2. (c) Tris-HClbuffer, pH 7.2 (40 mM): - Trizma (base), reagent grade, obtained from Sigma Chemical,. Company, St. Louis, Missouri, Catalogue # T-1503 - Aqueous solution, pH 7.2(adjusted with hydrochloric acid) 90 (d) S u c r o s e S o l u t i o n ( 4 0 % ) , 40 mM T r i s - H C l b u f f e r : - S u c r o s e s o l u t i o n ( 6 0 % ) , was t r e a t e d w i t h AG 50 ( N a + form) t o remove c a t i o n c o n t a m i n a n t s ( C a r s t e n , 1964) a n d t h e n 100 m l o f t h e s o l u t i o n was d i l u t e d w i t h 15 m l o f 0.4 M T r i s - H C l b u f f e r . The pH was a d j u s t e d t o 7.2 b e f o r e m a k i n g up t o 150 m l v o l u m e w i t h d i s t i l l e d w a t e r . (e) T a u r i n e : - O b t a i n e d f r o m S i g m a C h e m i c a l Co., S t . L o u i s , M i s s o u r i . • -0.5 M a q u e o u s s o l u t i o n . ( f ) C a l c i u m - 4 5 : - O b t a i n e d i n t h e c h l o r i d e f o r m i n a q u e o u s s o l u t i o n f r o m t h e R a d i o c h e m i c a l C e n t r e , Amersham, E n g l a n d : ( C E S . 3 ) , s p e c i f i c a c t i v i t y was 10-40 mCi/mg c a l c i u m . D i l u t e d 1 i n 5 w i t h w a t e r a n d s t o r e d a t -20°C. (g) C a l c i u m C h l o r i d e S o l u t i o n (100 mM): -Aqueous s o l u t i o n . S e r i a l d i l u t i o n s w e r e made t o o b t a i n d e s i r e d c o n c e n t r a t i o n . (h) EGTA S o l u t i o n (100 -mM) : -EGTA ( E t h y l e n e g l y c o l - b i s - ( 3 - a m i n o e t h y l e t h e r ) N , N ' - t e t r a a c e t a t e ) was o b t a i n e d f r o m S i g m a C h e m i c a l Co. (# E3251) -Aqueous s o l u t i o n , pH 6.8 ( a d j u s t e d w i t h 2M T r i s -b a s e ) . 91 (i) Determination of Free Calcium C o n c e n t r a t i o n s Present i n Ca-EGTA B u f f e r s : - The equation of Katz e t a l . (19 70) was used to c a l c u l a t e the d e s i r e d f r e e c a l c i u m c o n c e n t r a t i o n s : ([CaCl,] - 5.41 [ C a 2 + ] ) — '' = • 4.4 x l O 5 [Ca 2 + 1 . (\Z_. - ( [ C a C l 2 ] - 5.41 [ C a 2 + ] ) ) S o l v i n g above equation f o r Z; ([ C a C l 2 ] - 5 . 4 1 [ C a 2 + ] ) Z = ([CaCl,] - 5.41 [ C a 2 + ] ) + 5 2+ 4.4 x 10° [Ca ] S u b s t i t u t i n g the d e s i r e d v a l u e s f o r [ C a C l 2 ] a n d 2+ [Ca ] w i l l g i v e Z, the amount of EGTA to be added 2+ to achieve the d e s i r e d f r e e Ca c o n c e n t r a t i o n . For example, the amount of EGTA needed t o prepare 2+ a Ca-EGTA b u f f e r c o n t a i n i n g 1.0 yM f r e e Ca i n a s o l u t i o n c o n t a i n i n g 125 y_M_CaCl2 w i l l be: 125 x 10~ 6 - 5.41 x 10" 6 Z= + (125 x 10~ 6 - 5.41 x 10~ 6) 4.4 x 10 5 x 10~ 6 = 0.391 mM EGTA. 2+ Th e r e f o r e , to o b t a i n 1.0 pM f r e e Ca , 0.391 mM EGTA and 0.125 mM C a C l 2 are used. 92 - The amount of CaCl 2 and EGTA used i n each case to prepare solutions of various concentrations of free C a 2 + i n a t o t a l volume of 0.3 ml were as follows: D e s i r e d Free C a 2 + (UM) 10 mM EGTA 10 mM CaCl~ 45 Ca Water 0.5 .203 z .038 .030 .029 1.0 . 117 .038 .030 .115 5.0 . 043 .038 . 030 . 189 10. 0 . 026 .038 .030 . 206 50. 0 .000 .015 .030 . 255 100. 0 . 000 .030 .030 . 240 These preparations were d i l u t e d 1 i n 10 during the assay procedure described on page 99.  (j)Histidine-HCl (0.11M): - L-Histidine (free base) was obtained from Sigma Chemical Company, St. Louis, Missouri. - Aqueous solution, pH 6.8 (Adjusted with IN HC1.) (k)Tris-Oxalate (0.5 M): -TRIZMA OXALATE, was obtained from Sigma Chemical Co., St. Louis, Missouri, reagent grade (# T-7258). -Aqueous solution, pH 6.8 adjusted with 2M T r i s base. (1) Tris-ATP (0.1 M): -Adenosine 5' - triphosphate ( t r i s (hydroxymethyl)-amino-methane s a l t from Equine muscle) was obtained from Sigma Chemical Co., St. Louis, Missouri. 93 - Aqueous solution, pH adjusted to 6.8 with 2 M T r i s base. (m) Potassium Chloride (2 M): - Aqueous solution. (n) Magnesium Chloride (1M): - Aqueous solution. (o) Protein Kinase - Bovine cardiac protein kinase (type 1) was obtained from Sigma Chemical Co., St. Louis, Missouri. (Cat # P-4890). - The content of a 5 mg bottle was suspended i n 1 ml water, and stored at -20°C. For use the suspension was di l u t e d 1 i n 2 ( f i n a l concentration: 2.5 mg/ml). (p) C y c l i c AMP: - Obtained from Sigma Chemical Co., St. Louis, Missouri. - 10 mM aqueous solution. (q) Sorensen's Glutaraldehyde F i x a t i v e : - Gultaraldehyde was purchased from Ladd Research Industries, Burlington, Vermont. - Gultaraldehyde solution, 2.5% was prepared inO.lM Sodium phosphate buffer, pH 7.2. (r) Palade's Buffered Osmium Tetroxide Fixative - Osmium tetrbx'ide " was obtained from Stevens 94 Metallurgical Corporation, New York, N.Y. -0.1% solution prepared i n veronal-acetate buffer, pH 7.3-7.5, according to Glauert (1965). (h) Uranyl Acetate: -2% aqueous solution. (i) .] Reynold's Lead Cit r a t e . : -Prepared according to Reynolds (196 3). 3' (j) H-Ouabain . - obtained i n a solution of ethanol/benzene (9:1 v/v) from New England Nuclear, Boston, Mass. • S p e c i f i c A c t i v i t y was 10-20 Ci/mmole,catalogue # NET-211 (k) Other Reagents: - Sodium bicarbonate (NaHCO^), Fisher S c i e n t i f i c Co., New Jersey. - Sodium azide (NaN^), Sigma Chemical Co. - Ascorbic acid, BDH, Canada. - Potassium chloride, Sigma Chemical Co. - Sucrose, Fisher S c i e n t i f i c Co. - Calcium chloride (CaCl 2.2H 20), Mallinckrodt, St. Louis, Missouri. - Magnesium chloride, (MgCl 2•6H 20), Sigma Chemical Co. - Aquasol, New England Nuclear, Boston, Mass. (Cat. # NEN-934). - Other "reagents used i n electron microscopic work were a l l obtained from Fisher S c i e n t i f i c Co., New Jersey. - Maraglas was purchased from Ernest, F. Fullam, Inc., Schenectady, N.Y. Preparation of Heart Ventricle Homogenate,: Guinea-pigs (200-300 g, albino, Hartley strain) were s a c r i f i c e d by a blow to the head. The hearts were promptly excised, washed i n saline and the aorta and connective tissue removed. The v e n t r i c u l a r muscle was then promptly frozen (within 30 seconds of s a c r i f i c e ) i n 2-methyl-butane and dry i c e . The frozen hearts were wrapped i n t i n f o i l and stored at -80°C. The tissue could be stored i n t h i s way for up to 3 months without s i g n i f i c a n t loss i n calcium transport a c t i v i t y . For preparation of heart v e n t r i c l e homogenates a piece (0.1-0.2 g) was cut from a frozen heart preparation and homogenized i n 5 ml of a medium consisting of 40% sucrose and 40 mM T r i s - C l , pH 7.2 using a Polytron P20 homogenizer (3 strokes of 5 sec. duration at setting 5). Preparation of Microsomes Enriched i n Sarcoplasmic  Reticulum: The method of Harigaya and Schwartz (1969) was followed with s l i g h t modifications (Figure 7-) . Frozen heart preparations, from two guinea-pigs were cut into small R pieces with a razor blade and placed i n Corex Figure 7 Flow diagram for the preparation of heart microsomes enriched i n sarcoplasmic reticulum 96a HEART TISSUE HOMOGENATE SUPERNATANT PELLET P E L L E T P E L L E T •MICROSOMAL PREP. ENRICHED IN,S.R. Frozen preparation was cut into small pieces. Homogenized i n sodium bicarbonate buffer, pH 7.2. Using Polytron P20 at setting 5, three fast strokes. Care taken not to denature the preparation at th i s step. Centrifuged at 12,000 x g for 15 minutes at 4°C. Centrifuged at 45,000 x g for 1 hour at 4°C. Suspended i n 10 ml of 0.6 M/KC1 solution, using a gl a s s - t e f l o n hand homogenizer (1-2 strokes). Centrifuged at 45,000 x g for 1 hour at 4°C. Resuspended i n 10 ml of 10% Tris-HCl buffer, pH 7.2 Centrifuged at 45,000 x g for 1 hour at 4°C. Resuspended i n 40% sucrose and 40 mM Tris-HCl buffer,pH 7.2, using g l a s s - t e f l o n hand homogenizer (2-3 strokes). 97 centrifuge tubes containing 5 ml of sodium bicarbonate buffer (pH 7.2). The suspension was homogenized three times for 5 seconds using a Polytron (P20, Brinkman Instruments Co.), with a rheostat setting of 5, with a re s t i n t e r v a l of about 15 to 20 seconds. The entire procedure was carried out i n crushed i c e . The r e s u l t i n g homogenate was centrifuged at 12,00 0 x g for 5 minutes at 4°C. The supernatant was recentrifuged at 45,000 x g for 1 hour at 4°C. The p r e c i p i t a t e was resuspended i n a glass homogenizer with a t e f l o n pestle i n 10 ml of 0.6 M KC1 solution. The r e s u l t i n g suspension::, was then centrifuged at 45,000 x g for 1 hour to remove s o l u b i l i z e d actomyosin. The p r e c i p i t a t e was again suspended i n 10 ml of 10 mM Tris-HCl buffer, pH 7.2 and centrifuged at 45,000 x g. The harvested p r e c i p i t a t e was then suspended i n a small volume of a medium consisting of 40% sucrose and 40 mM Tris-HCl;,pH 7.2. The r e s u l t i n g microsomal preparation i s enriched i n sarcoplasmic reticulum. Storage i n 40% sucrose reduced the loss of calcium transport activrty noted when these preparations were maintained at 4°C (Carsteh, 1964). Unless otherwise indicated, a l l experiments were conducted within 2 hours of preparation of t h i s f r a c t i o n . 98 4. Characterization of Microsomes Enriched  Sarcoplasmic Reticulum (a) Electron Microscopy: A suspension of enriched sarcoplasmic reticulum i n a medium containing 40%sucrose and 40 mM Tris-HCl buffer was centrifuged at 45,000 x g for 1 hour at 4°C. The p e l l e t was treated l i k e blocks of whole tissues, fixed i n Sorensen's glutaraldehyde (2.5%) f i x a t i v e for 24 hours at 4°C. I t was then post-fixed i n Palade's f i x a t i v e , buffered 1% osmium tetraoxide, for l-l^s hrs. at room temperature. The tissue was then washed i n water, dehydrated i n : ethanol and cut into small pieces. The pieces were dehydrated i n acetone and embedded in maraglas. Sections were stained on the g r i d with uranylacetate and 'lead c i t r a t e and observations were carr i e d out on a Siemens, Elmiskop !•> model, electron microscope. 3 (b) .H-Ouabain Binding Assay: The method of Gelbart and Goldman (19 77) was u t i l i z e d . Membranes (1.0-2.0 mg/ml) were incubated The Electonmicroscopic work:was_carried out at the Shaughnessy Hospital, .Vancouver, -B.C.., .under, the guidance and help of Dr. A. B.Y.'Magil.. .-_The electronmicrographs were kindly interpreted by- Dr. ''B.J. Crawford, Department of. Anatomy.", University of B r i t i s h Columbia, „ Vancouver, Canada. . 99 with 10 M [ H] ouabain ( s p e c i f i c a c t i v i t y 12.7 C\/mol) i n the presence of 3. mM MgCl 2, — NaCl, 1 mM EGTA, 25 mM Tris-HCl, pH 7.4, i n the presence and absence of 3 mM Tris-ATP. Following 10 min of incubation at 37°, an aliquot of the reaction mixture was f i l t e r e d through a M i l l i p o r e 3 f i l t e r (0.45 y_M) to separate unbound [ H]-ouabain from tissue-bound [^H]-ouabain. The f i l t e r s were washed and dried, dissolved i n Aquasol counting medium and assayed using l i q u i d s c i n t i l l a t i o n 3 counting techniques. Non-specific [ H]-ouabain binding, determined i n the absence of ATP, was subtracted from the t o t a l binding to determine the degree of ATP-dependent binding. 5. ATP-dependent Calcium Uptake and Binding Assay The method of Tada et a l . (1974) was followed with a few modifications. O x a l a t e - f a c i l i t a t e d calcium uptake was determined i n the presence or absence of taurine using either 40-60 pg of the S.R. preparation or 200-300 yg of the homogenate preparation. The incubation medium contained 40 mM histidine-HCl, pH 6.8, 5 mM MgCl 2, 110 mM KC1, 5 mM Tris-ATP, 2.5 mM Tris-oxalate 4 5 5 and CaCl 2 containing C a C l 2 (5 x 10 cpm/sample) with the desired free calcium concentration maintained by 100 Ca-EGTA buffer (50 a 1) (see page 91) .-Following a preincubation of 7 minutes at 30°C the reaction was started by the 45 addxtion of CaC^. Unless otherwise indicated, the time of incubation was 5 minutes at 30°C i n a t o t a l volume of 0.5 ml. The reaction was terminated by f i l t e r i n g an aliquot (0.4 ml) of the reaction mixture through a m i l l i p o r e f i l t e r (HA 45, M i l l i p o r e Co.). The f i l t e r was then washed twice with 15 ml of 40 mM Tris-HCl, pH 7.2, then dried and counted for radio* a c t i v i t y i n 10 ml Aquasol using standard l i q u i d s c i n t i l l a t i o n counting techniques ATP-dependent calcium binding was studied under i d e n t i c a l conditions except that Tris-oxalate was omitted from the reaction medium. 6. Assay for Cyclic-AMP-dependent Protein Kinase E f f e c t  on Calcium Uptake: The e f f e c t of cyclic AMP-dependent protein kinase on calcium uptake was assayed under the i d e n t i c a l conditions used to measure ATP-dependent calcium uptake. C y c l i c AMP-dependent protein kinase was added to the incubation medium i n a concentration of 50 yg/ml along with 1.0 uM c y c l i c AMP at least 7 minutest'prior to the s t a r t of the reaction. 101 7. Studies on the E f f e c t of Taurine on the Decay 2+ of Ca Transport A c t i v i t y : The heart v e n t r i c l e homogenate and the microsomal preparation enriched i n S.R. were prepared as outlined on pages 95 and 96 respectively, except that the preparations were stored i n 2 mM Tris-HCl buffer, pH 7.2 instead of a medium containing 40% sucrose and 40 mM T r i s - C l buffer. The preparations were divided into two equal portions. To one portion was added taurine (15 mM); the other portion was used without any addition. ATP-dependent calcium uptake was determined at i n t e r v a l s between 0-6 hours as outlined above on page 99 . 8. Protein Assay: Protein concentrations of the homogenate and S.R. preparations were measured by the method of Lowry et a l . (19 51) using bovine serum albumin as a standard. 2+ 2+ 9. Calculations: Ca uptake and Ca binding were expressed i n nmoles/mg/min. Example: 2+ Ca-Uptake for 1.0 uM free Ca : 102 9 cpm on M i l l i p o r e 9 C.a 62.5 nmoles1 3 Ca Z Uptake / : " f i l t e r X x C a 2 + (nmoles/mg/min) , .. £ .. c , / y / cpm of the . . c . , d Standard ; x m ^ P r o t e l n x 5 minutes a. D i l u t i o n factor: 0.4 ml of the reaction mixture of 0.5 ml t o t a l volume was passed through a m i l l i p o r e f i l t e r ; s*. a d i l u t i o n factor of 1.25. b. Calcium content i n each tube was 62.5 nmoles. c. Radioactivity of Ca-EGTA buffer (50 u i ) was determined i n 10 ml Aquasol. d. Calcium uptake was determined for 5 minutes of incubation time. 1 0 . S t a t i s t i c s : S t a t i s t i c a l analysis was done by Students " t " te s t for unpaired and common variance (Wonnacott and Wonnacott, 1977). A p r o b a b i l i t y of p<0.05 was taken as the c r i t e r i o n for s i g n i f i c a n c e . Standard Error of the Mean (S.E.M.) was used as a measure of v a r i a t i o n . B- Studies on the E f f e c t of Taurine on Passive Ion  Transport i n Rat,,Brain Synaptosomes: 1. Reagents: (a) Sucrose Solution: 103 -Obtained from Fisher S c i e n t i f i c Co., a n a l y t i c a l grade. -2.4 M aqueous solution treated with AG 50 (Na + form) to remove impurities (Carsten 1964). -Diluted with equal volume of 20 mM Tris-HCl buffer, pH 7.2 to give a f i n a l solution of 1.2 M sucrose/10 mM Tris-HCl. (b) Calcium-45: -Same as on page 90. (d) Taurine: -Same as on page 90. (e) Choline Chloride: -Obtained from Sigma Chemical Co., St. Louis, Mo. (Cat # C-1879). -500 mM aqueous solution. (f) Other Reagents: -AG 50-X8, 200-400 mesh, hydrogen form, Bio Rad Laboratories. The resi n was treated twice with 1 volume of >1N NaOH to generate the sodium form and washed with d i s t i l l e d water u n t i l the pH of the wash was neutral?. (7.0-7.5). , . - 3-alanine , A grade, Calbiochem, Los Angeles, C a l i f o r n i a . a B-alanine, hypotaurine and homotaurine were kindly provided by Dr.. Thomas Perry, Pharmacology Department, University of B r i t i s h Columbia, Vancouver, B.C. 104 - Hypotaurine , 0-aminoethylsulfinic acid, Calbiochem. - Homotaurine , 3-aminopropane sulfonic acid, K & K Laboratories,Plainview, N.Y. - GABA,. Calbiochem, San Diego, C a l i f o r n i a . - Methionine, N u t r i t i o n a l Biochemicals Corporation, Cleveland, Ohio. - a-alanine, N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio. - Proline, N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio. - Valine, N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio. - Sodium sulphate 1 3, Matheson Coleman & B e l l , Norwood Ohio. - Sodium acetate* 3, BDH Chemicals, Canada. - Potassium acetate* 3, Matheson Coleman & B e l l . 2. Preparation of Synaptosomes The procedure used was based on that of Gray and Whittaker (1962) as modified by Keen and White (1970) (Figure 8). Two male Wistar rats (weighing 200-250 g) were k i l l e d by c e r v i c a l d i s l o c a t i o n and the whole brain removed to i c e -cold 0.32 M sucrose. The brains were homogenized i n 10 v o l . of 0.32 M sucrose by twenty strokes of a teflon-glass homogenizer with a motor-driven pestle (0.25 mm clearance, B-alanine, hypotaurine and homotaurine were kindly provided by vDr. Thomas Perry, Pharmacology Department, University of B r i t i s h Columbia, Vancouver, B.C. 400 mM Aqueous stock solutions. F i g u r e 8 Flow d iagram f o r the p r e p a r a t i o n r a t b r a i n ; synaptosomes 105a RAT BRAIN (from 2 rats) t HOMOGENIZED t CENTRIFUGED t SUPERNATANT PELLET T DISCONTINUOUS DENSITY GRADIENT CENTRIFUGED t PELLET SYNAPTOSOME SUSPENSION Washed i n ic e - c o l d 0.32 M sucrose. — In 0.32 M sucrose (10 vol.) using teflon-glass motor driven homogenizer At 1,0 00 x g for 10 minutes. Recentrifuged at 12,000 x g for 20 minutes. Resuspended i n 10 ml of 0.32 M sucrose. — Layered, 5 ml suspension on a gradient containing 15 ml of 0.8 M sucrose and 15 ml of 1.2 M sucrose. At 95,000 x g for 90 minutes. Interface material was removed. Diluted with 0.32 M sucrose. Centrifuged at 20,0 00 x g for 30 minutes. Resuspended i n 4 ml of a medium containing 0.32 M sucrose and 10 mM Tris-HCl buffer,pH 7.2 1 0 6 800 rev./min.)The homogenate was centrifuged at 1000 x g for 10 minutes and the r e s u l t i n g supernatant recentrifuged at 12,000 x g for 20 minutes. The p e l l e t s were resuspen-ded i n 10 ml of 0.32 M sucrose and 5ml c a r e f u l l y layered onto each of two discontinuous density gradients consis-ti n g of 15 ml 0.8 M sucrose and 15 ml 1.2 M sucrose. The gradients were centrifuged at 95,000 x g for 90 minutes i n an SW 27 swing-out head using a Beckman L2-65 u l t r a -centrifuge. The material at the 0.8 M-1.2 M interface was removed, dil u t e d with 0.32 M sucrose and centrifuged at 20,000 x g for 30 minutes. The resultant p e l l e t s were resuspended i n 4 ml i n medium containing 0.32 M sucrose and 10 mM Tris-HCl buffer,pH 7.2 to form the "synaptosomal suspension". Characterization of Synaptosome Suspension by Electron  Microscopy: Same as on page 98 . Determination of the Osmometric Behaviour of Synaptosomes: The method of Keen and White (1970) was followed: 0.05 ml of the synaptosomal suspension (5 mg protein/ml) was suspended i n solutions of 25-300 mM Na2SC>4 (0.95 ml) i n a microcuvette. The extinction of the suspension was recorded at E q 9 n for a period of 15 minutes at room 107 temperature using a Beckman recording spectrophotometer (model 25). Determination of Sodium and Potassium Permeability: Synaptosomal suspensions were preincubated with and without taurine (20 mM) for 1 hour at 2°C. An aliquot (0.05 ml) was then added to a microcuvette containing from 100-200 mM ice - c o l d sodium or potassium acetate (0.95 ml) solution. The content of the microcuvette was rapidly mixed with a pasteur pipette and the extinction at recorded over a 5 minute period. Determination of Calcium Permeability: The synaptosomal suspension (0.2 mg/ml) was preincubated at 2°C i n medium containing 0.3 M sucrose and 10 mM Tris-HCl.buffer,pH 7.2, i n the presence or absence of taurine (20 mM), i n a t o t a l volume of 3 ml. Following a preincubation of 1 hour, the reaction was started by 45 5 the addition of 10 yM CaCl 2 (5 x 10 cpm/sample). Aliquots (0.2 ml) of the incubation mixture were then removed at i n t e r v a l s and passed through a m i l l i p o r e f i l t e r (HA 45, M i l l i p o r e , Co.) The f i l t e r was washed twice with 5 ml of 10 mM Tris-HCl buffer, pH 7.2 i n 0.3 M sucrose then dried and counted for r a d i o a c t i v i t y i n Aquasol using standard l i q u i d s c i n t i l l a t i o n counting techniques. 108 45 Determination of Loss of Ca from Preloaded Synaptosomes Synaptosomal suspensions (0.2 mg protein/ml) were a l l 45 loaded to a s i m i l a r extent with 10 y_M CaC^ 5 o (5 x 10 cpm/sample) at 2 C i n conditions similar to that described above for the determination of calcium permeability. After 1 hour, an aliquot (0.2 ml) was passed through a m i l l i p o r e f i l t e r . The remaining incubation medium was centrifuged at 12,00 0 x g for 10 minutes. The r e s u l t i n g p e l l e t was resuspended i n 3 ml of i c e - c o l d media containing 0.3 M sucrose and 10 mM T r i s - C l buffer, pH 7.2 i n the presence or absence of 20 mM taurine and incubated at 2°C. The 45 release of CaC^ with time, was determined by passing aliquots (0.2 ml) of the reaction medium through a mil l i p o r e f i l t e r . The rate of calcium release was then calculated by the following equation: (cpm i n f i l t e r a f t e r 1 hour preloading)'-4 C. (cpm i n f i l t e r at sampling time) Ca Release (%) = (cpm i n f i l t e r after 1 hour preloading) Protein Assay: same as on page 101 S t a t i s t i c s : The slope or regression coefficient(b) of the l i n e , i t s intercept (a) and correlation c o e f f i c i e n t (r) were 1 0 9 obtained using a standard c a l c u l a t o r , Texas Instruments, model T-55. The sum of squares of d e v i a t i o n s , Edy x2 i s the b a s i s f o r an estimate of e r r o r i n f i t t i n g the l i n e . The corresponding degrees of freedom are n-2. dy > x = where Y = Y + b (X-X) d y . x 2 = ( Y ^ > 2 Z<3 v2 = E ( Y - Y ) 2 then, S 2 =Edy.x 2/n-2. y. x where S 2 i s the 'mean square d e v i a t i o n from r e g r e s s i o n ' X= independent v a r i a b l e and Y = dependent v a r i a b l e . The r e s u l t i n g sample standard d e v i a t i o n from r e g r e s s i o n i s S = l/S~ ~2 y.x y.x The standard d e v i a t i o n of the slope i s then obtained: S h = S. / Ex 2 , where Ex 2 = E ( X . - X ) 2 A t e s t of s i g n i f i c a n c e between two s l o p e s (obtained f o r the data with the presence and absence of t a u r i n e ) i s g i v e n by student t - t e s t b l " b2 , d. f . = n-^  + n 2 - 4 f b l ) 2 + ( S b 2 ) 2 110 The estimated standard error of Y i s : S£ = S y > x / ( l / n ) + ( x 2 / E x 2 ) ; where n = number of observations 2 - 2 x = (X-X)** x 2 = E ( X - X ) 2 A test of significance between two intercepts i s given by: — a„ 1 2 t = ' ; d.f = n^ ^ + n 2 - 4 \ ) 2 +(S ) 2  y a l a2 A l l s t a t i s t i c a l formulae used were obtained from Snedecor and Cochran, 1967. I l l R E S U L T S 112 I. STUDIES WITH ISETHIONIC ACID Development of an A n a l y t i c a l Method-for the Measurement  of Isethionic Acid: 1. Chromatography of Methylated Isethionic Acid: a. Stationary Phases: The chromatography of the methylated i s e t h i o n i c acid on OV-1 and OV-17 column i s shown i n figure 9. Using a column of 5% OV-1, a single peak with a retention time of 1.6 minutes was obtained from methylated i s e t h i o n i c acid. Using a column of 5% OV-17, two peaks at retention times of 3.5 and 4.0 minutes were obtained. The peak area of the large peak, on OV-17 column was approximately 20 times that of the smaller peak. Using a column of 5% DEGS, no peaks for methylated i s e t h i o n i c acid were obtained. b. Internal Standards: S a l i c y l i c acid was found to be a convenient i n t e r n a l standardMn that i t could be used for both OV-1 and OV-17 columns at the same time during gas-chromatography with a flame i o n i z a t i o n detector. Separation of methylated 113 Figure 9. Chromatographic separation of the products of methylation of i s e t h i o n i c acid and s a l i c y l i c a cid using flame i o n i z a t i o n detection A. The column used was a 5% OV-1 column; oven temperature 115 degrees C. B. The column used was a 5% OV-17 column; oven temperature 135 degrees C. C. The column used was a 5% OV-17 column at an oven temperature of 135 degrees C. The ordinates shows detector response. The abscissa shows retention time. 114 s a l i c y l i c acid and i s e t h i o n i c acid on 5% OV-1 and 5% OV-17 columns are shown i n figure 9A and 9C respectively. 1-Butanesulfonic acid was used as an i n t e r n a l standard when gas chromatography was conducted on a flame photometric (sulfur) detector, with OV-17 columns. Two peaks for methylated i s e t h i o n i c acid were obtained using sulf u r detector and an OV-17 column (Figure 1.0) , s i m i l a r to that seen i n figure 9B and 9C. 1-Butanesulfonic acid was found to co-chromatograph with i s e t h i o n i c acid on OV-1 columns; but, i t served as a good i n t e r n a l standard on an OV-17 column (Figure 1 Other compounds tested as i n t e r n a l standards for the i s e t h i o n i c acid assay were found to be unsatisfactory: These compounds as separated on OV-1 columns r e l a t i v e to methylated i s e t h i o n i c acid, were either too close to the solvent peak (example, methyl caprylate) or had a retention time undesirable for the assay purpose ( a c e t y l s a l i c y l i c acid and methyl laurate) . Methyl benzoa-te was found to co-chromatograph with methylated i s e t h i o n i c acid on the OV-1 column. 115 Figure 10 Chromatographic separation of the products of methylation of i s e t h i o n i c acid and 1-butanesulfonic acid using, flame'. Photometric - (-sulfur-)--detection The column used was a 5% OV-17 column; oven temperature was 100°C. For further d e t a i l s refer to'Materials and Methods' section of the text (page 72). Isethionic acid (I) was i d e n t i f i e d as the methylester, methylether derivative.Isethionic acid (II) was i d e n t i f i e d as the methylester (see discussion) 115a Flame Response t 0 Recorder Pen Response Butanesulfonic Acid Isethionic Acid Isethionic Acid T 2 T" 4 Min. n r 6 8 116 c. Mass-Spectrometry and NMR s p e c t r a o f Methylated  I s e t h i o n i c a c i d : The i d e n t i t i e s of; the- two. peaks "of methylated i s e t h i o n i c a c i d o b t a i n e d on OV-17 column were e s t a b l i s h e d by the use of gas chromatography mass-spectrometry (GC-MS) ( f i g u r e 11) and n u c l e a r magnetic resonance spectroscopy (NMR) ( f i g u r e 12). I n t e r p r e t a t i o n o f the mass-spectrometry fragmentation p a t t e r n of the two peaks o b t a i n e d a f t e r OV-17 gas chromatography i s shown i n f i g u r e 11. The l a r g e peak (Peak II) was the me t h y l e s t e r of the i s e t h i o n i c a c i d , while the s m a l l peak (Peak I) was the methylether, m e t h y l e s t e r of i s e t h i o n i c a c i d . The NMR s p e c t r a of methylated i s e t h i o n i c a c i d , 1 - b u t a n e s u l f o n i c a c i d and methoxyethanol are shown i n f i g u r e 12A, 12B,, and 12C r e s p e c t i v e l y . The 1-but a n e s u l f o n i c a c i d and methoxyethanol s p e c t r a were used t o i d e n t i f y the CH^ s i n g l e t peaks of me t h y l e s t e r (3.88 ppm) and methylether (3.38 ppm), r e s p e c t i v e l y . The i n t e g r a t i o n o f the spectrum i n d i c a t e d an approximate m e t h y l e s t e r CH^ to methylether CH-. r a t i o o f 20:1. F i g u r e 11 Mass s p e c t r a o f t h e p r o d u c t s o f m e t h y l a t i o n o f i s e t h i o n i c a c i d . A. Mass s p e c t r u m o f t h e peak I f r o m f i g u r e 9 B. B. Mass s p e c t r u m o f peak I I f r o m f i g u r e 9 B. E x p e r i m e n t a l c o n d i t i o n s and i n t e r p r e t a t i o n o f d a t a a r e d e s c r i b e d i n ' M a t e r i a l s and Methods' (page 73.) and ' D i s c u s s i o n s ' (page 152) s e c t i o n s t h e t e x t . 1 1 7 a 100 Spectrum A £ c H 3 O C H = C H 2 J + i 2 O C H 3 H 2 S 0 3 C H 3 Methylester methylether of Isethionic Ac id 1 2 4 ^£cH 2SO(OCH3 |^ J + 118 Figure 12 Nuclear magnetic resonance spectra of the products of methylation of i s e t h i o n i c acid(A), 1-butanesulf onic acid (B) and methoxyethanol (C) . The NMR spectra of the methylation products of i s e t h i o n i c acid and 1-butanesulfonic acid were obtained on a Varian HA-100 spectrometer at 100 mHz, dissolved i n deuterated dimethyl-sulfoxide. Methoxyethanol spectrum a was obtained from 'The Sadtler Standard Nuclear Magnetic Resonance Spectra:' (Spectrum # 32M Sadtler Research Inc., Pub. by Sadtler Res. Laboratories, 3316 Spring Gardens Street, Philadelphia, PA 19104, USA, 1967). Methoxyethanol spectrum was reproduced at the Biomedical Communication Department, Faculty of Medicine, University of B r i t i s h Columbia. 118a Hz ' I 1 500 I 1 1 I 1 I I 1 4001 i — i — i — i — | — i — i — i — r " I I 1 1 I 1 ' | I i — i—[— i— l— i — i — I — i— l— r -1 M I I I i i I i i | i | I ; i | i | I i i i 200 1 I001 1 0 300 C H j - O - C H j C H - O H 2-Methoxyethanol -a-J ' I ' i I i i I n j i i I i I I M | . i I i i | i i I i i | i i I i i | i i I ' ^ I \ i | x ' ' 8-0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 PPM(6) 119 2. Chromatography of S i l y l a t e d Isethionic Acid Chromatography of i s e t h i o n i c acid on OV-17 afte r s i l y l a t i o n with BSA i n DMF solvent i s shown i n figure 13. I t can be seen that the mixtures obtained afte r s i l y l a t i o n of i s e t h i o n i c acid are ex t r a o r d i n a r i l y complex and the products appear to be l a b i l e . S i l y l a t i o n of i s e t h i o n i c acid i n the presence of BSA and DMF solvent gave peaks of varying number, size and retention times during the course of subsequent i n j e c t i o n of the same sample (Figure 13). 1-Butanesulfonic acid was also found to behave i n a sim i l a r manner to i s e t h i o n i c acid. S a l i c y l i c acid and c a p r y l i c acid always gave only one peak and t h i s behaviour was reproducible. Pyridine and a c e t o n i t r i l e were substituted as solvents for DMF during s i l y l a t i o n of i s e t h i o n i c acid with BSA. Similar chromatographic behaviour of s i l y l a t i o n products to that found using DMF as solvent was again obtained. Other s i l y l a t i n g agents were also used. S i l y l a t i o n of Isethionic acid with HMDS/TMCS on chromatography yielded d i f f e r e n t multiple peaks. TMSIM yielded no peaks for Isethionic acid on the OV-17 column. 120 • Figure 13 Time course i n j e c t i o n of s i l y l a t e d products of i s e t h i o n i c acid Isethionic acid (10 mg) was dissolved i n 0.75 ml DMF and then treated with 0.5 ml BSA. The reaction was c a r r i e d out at 135°C for 6 minutes. The sample was injected (l;.ul) on a GLC column, aft e r storage i n ice-bath, at i n t e r v a l s of 15 minutes (A), 45 minutes (B), 75 minutes(C) and a f t e r 3 hours (D). The column used was 5% OV-17 at an oven temperature of 120°C. 121 Different stationary phases were also used during the course of i s e t h i o n i c acid s i l y l a t i o n studies. The stationary phases used were PEGS and SP-400. On neither of these two columns could i s e t h i o n i c acid peaks be seen. B. Analysis of Isethionic Acid i n Tissues; A t y p i c a l standard curve for methylated i s e t h i o n i c acid using flame i o n i z a t i o n detection-gas chromatography on OV-17 columns i s shown i n figure 14. The standard curve shows a non-linear response. This could be due to measurement of peak height as detector response since the smaller peaks were wider. An integrator was not available at the time t h i s work was undertaken. Identical standard curves were obtained on OV-1 columns. The standard curve obtained using a s u l f u r detector was 10 times more sensitive than the flame i o n i z a t i o n detector. Similar standard curve obtained on sulfu r detector and 1-butanesulfonic acid was used to quantitate i s e t h i o n i c acid i n mammalian and arthropod tissues. The results of the analyses are shown i n Table 1. 1. Isethionic Acid i n Rat Heart and Brain Tissues: In r at brain, i s e t h i o n i c acid was detected at a concentration of approximately 0.2 mg/lOOg tissue and rat heart at a concentration of approximately 0.1 mg/lOOg tiss u e . The value of i s e t h i o n i c acid for rat heart i s only an estimate since at t h i s l e v e l the method i s Figure 14 A t y p i c a l c a l i b r a t i o n curve of methylated i s e t h i o n i c acid obtained on a column of 5% OV-17 flame i o n i z a t i o n detector. Similar standard curve was obtained with su l f u r detector and 1-butanesulfonic acid was an i n t e r n a l standard. The operating parameters are quoted, i n the 'Materials and Methods' section on page 72 . 9.0 0.5 1.0 1.5 2.0 2.5 3.0 j i m o l e Isethionic A c i d / V i a l :.i2 3 TABLE 1 Isethionic Acid i n Tissues Analyzed Rat Heart Rat Brain Rat Milk Dog Heart Squid Axoplam Squid Ganglion Nautilus Ganglion 0.00 8 ymole/g wet weight' (0.1mg/100 g wet weight) 0.016 ymole/g wet weight (0.2 mg/100 g wet weight) - None was Detected* 3 - None was Detected '* cl - 150 y mble/g wet weight (18.6 mg/g wet weight) - 34.09 pmole/g wet weight (4.28 mg/g wet weight) - 2.9 8 Mmole/g wet weight (0.37 mg/g wet weight) The tissue were analyzed by Gas-liquid chromatography (GLC) aft e r extraction and methylation as described i n "Materials and Methods" section on pages 75 to 83. An appropriate standard curve was used. The recovery of authentic i s e t h i o n i c acid added to the tissue was always between 95-100% (see te x t ) . a. The value given i s only an estimate because at* t h i s l e v e l the method i s approximately at the l i m i t of s e n s i t i v i t y of the assay technique. The l i m i t of d e t e c t i b i l i t y of the method with sulf u r detector was approximately .00 8 ymoles ISA/g(0.1 mg ISA/lOOg) wet weight t i s s u e . b. The s e n s i t i v i t y of the method (Flame photometric, sulfu r detector) was <20 nmoles ISA/ml milk. c. The GLC s e n s i t i v i t y , using Flame Ionization detector was .008ymoles ISA/g (0.1 mg ISA/lOOg) of heart tissue. d. The i d e n t i t y of the ISA peaks on GLC were confirmed on Gas chromatography mass spectrometers. 124 approximately at the l i m i t of i t s s e n s i t i v i t y . The recovery of i s e t h i o n i c acid (2.0 or 0.2 ymole/g of tissue) added to rat brain or heart tissue was always between 95 and 100% The s e n s i t i v i t y of i s e t h i o n i c acid detection i n rat heart and brain tissue, using a flame i o n i z a t i o n detector was approximately 0.2 pmole i s e t h i o n i c acid per g tissue extracted (approximately 2.5 mg/100 g). Using the sulfu r detector, the s e n s i t i v i t y of the imethod was approximately 0.008 umole/g (0.1 mg/100 g ti s s u e ) . 2.Isethionic Acid i n Dog Heart Tissue A large scale extract of dog heart tissue (400g) was examined for i s e t h i o n i c acid following the method of Welty and Read (1962). I t was found that the extract did not y i e l d any cr y s t a l s of isethionate. Furthermore, analysis of an aliquot of the extract on gas chromatography revealed ho evidence of i s e t h i o n i c acid. The s e n s i t i v i t y of the gas l i q u i d chromatographic portion of t h i s experi-ment was approximately 0.008 u mole/lOOg (0.1 mg/100g) of heart t i s s u e . 3. Isethionic Acid i n Molluscan Tissues In squid axoplasm, i s e t h i o n i c acid was found at a concentration of 150 u mole per g axoplasm. Squid ganglion and Nautilus ganglion were found to contain i s e t h i o n i c acid at 34.09 umoles and 2.9 8 umoles i s e t h i o n i c acid per g (wet weight) ganglion respectively. The i d e n t i f y of the peak was confirmed by gas chromato-graphy arid mass spectrometry. Isethionic acid i n Rat Milk Samples: The samples of rat milk analyzed showed no trace of i s e t h i o n i c acid. The recovery of authentic i s e t h i o n i c acid added to the milk samples was always 100% and the s e n s i t i v i t y of the^method using a flame photometric (sulfur) detector was such that 20 nmoles i s e t h i o n i c acid/ml milk could have been e a s i l y detected. Bioconversion of Taurine to Isethionic acid Samples from dog heart and r a t heart s l i c e s incubated 14 for 30 or 60 minutes with radioactive C-taurine were extracted with methanol and then chromatographed on Whatman #1. paper. The experiments with both dog and rat heart s l i c e s showed that 90 per cent of the r a d i o a c t i v i t y 14 was recovered as C-taurine and up to 10 per cent was converted to a radioactive compound which behaved chromatographically l i k e i s e t h i o n i c acid. The taurine to apparent "Isethionic acid" conversion was found riot to 126 increase with incubation time. The same r e s u l t was obtained, 14 when C-taurine was added to heart slxces i n Kreb-Henseleit phosphate buffer and the tissues were extracted immediately with methanol. A t y p i c a l experimental r e s u l t on rat heart s l i c e s i s shown i n Table 2. The chromatographic separation of radioactive taurine and i s e t h i o n i c acid on Whatman #1 14 paper r e l a t i v e to the separation of C-taurine aft e r incu-bation with r a t heart s l i c e s i s shown i n figure 15. In order to evaluate the possible metabolism of is e t h i o n i c acid or conversion to taurine, radioactively labeled i s e t h i o n i c acid was added to the tissue s l i c e s i n 14 an incubating medium similar to that used i n the C-taurine-heart s l i c e s experiments. A l l the r a d i o a c t i v i t y added to the tissue was recovered i n t a c t as (100%) i s e t h i o n i c acid (results not shown) and no metabolism or conversion to taurine was observed. 127 TABLE 2 14 14 Conversion of C-taurine to C-Isethionic Acid by Rat Heart S l i c e s Incubation Taurine Isethionic Acid % Time CPM/lOOmg wet weight tissue Conversion 0 92,,,5 38 10,112 9.85 % 30 94,145 11,506 10.9 % 60 107,070 12,447 10.4 % 14 . . Conversion of C-taurme to i s e t h i o n i c acid by r a t heart s l i c e s (150 mg wet weight) was car r i e d out i n a medium containing krebs-Heinseleit phosphate ..buffer (2.0 ml), pH 7.3, 0.05 ml pyridine-HCl (10 mg/ml). 14 and 0.1 ml of C-taurine (100 y l , 1.4 yM, 50 mCi/mmole) i n a t o t a l volume of 1.5 ml. The reaction was c a r r i e d out at 37°C i n a shaker water bath for 0, 30 and 60 minutes. The reaction was stopped by the addition of 2.5 ml methanol. Isethionic acid and taurine were then extracted with Folch solvent and i s o l a t e d on ascending paper chromatography. 128 Figure 15 14 14 Separation of C-taurine, C-isethionic acid 14 and the rat heart s l i c e s - C-taurme incubation products by paper chromatography. Ascending paper chromatography was developed with t-butanol, pyridine and water (1:1:1) as solvent, for 17 hours on Whatman No.3 paper (7" x 20"). 5 p i samples were spotted 1" apart and r a d i o a c t i v i t y , a f t e r chromatography, was detected by cutting 0.5" x 1" pieces and counting them i n s c i n t i l l a t i o n f l u i d . Paper chromatographic separation of radioactive taurine (o o) and i s e t h i o n i c acid (o o) i s shown. The chromatogram i s interposed with the separation of the incubation product of 14 C-taurine with rat heart s l i c e s for the incubation times of: 1. 0 minutes (controls, l e f t bars with s o l i d blocks). 2. 30 minutes (middle bars with oblique s t r i a t i o n s ) and 3. 60 minutes (right bars with s t r a i g h t s t r i a t i o n s ) . 128 a 129 II. TAURINE AND ION TRANSPORT A. E f f e c t of Taurine on ATP-dependent Calcium Transport i n Guinea-pig Cardiac Muscle;- _ 1. Characterization of Ven t r i c l e Heart Homogenate and  Sarcoplasmic Reticulum Enriched Preparation: The electron micrographs of both microsomes enriched i n sarcoplasmic reticulum (S.R.) and v e n t r i c l e homogenate preparations are shown i n figures 16 and 17 respectively. The S.R. preparation revealed a high degree of smooth membrane vesi c u l a t i o n , heavily contaminated with small dark granules s i m i l a r i n texture to glycogen granules. The smooth membrane vesicl e s were mainly sarcoplasmic reticulum and possibly transverse tubules. Some sarcolemmal membrane ves i c l e s were also present as revealed by ouabain binding assay measurements (see Table 3). Occasionally, lysosomal bodies were i d e n t i f i e d . No mitochondrial contamination could be seen. Analyses using chemical markers (cytochrome C oxidase) were not done to rule out the p o s s i b i l i t y of mitochondrial fragment contamination. Electron micrographs of the v e n t r i c l e heart homogenates showed i n t a c t mitochondria and membrane ve s i c l e s . Figure 16 Electron Micrograph .of Microsomal preparations enriched i n sarcoplasmic Reticulum. Micrographs were made by the standard procedure as described i n the 'Materials and Methods' section, page 98. The micro-organelles i d e n t i f i e d i n the micrographs were smooth membrane vesicl e s (siav) , small dark granules similar i n texture to glycogen granules (gly), and some Lysosomes ( l y s ) . Magnification approximately 45,000 x. 130a 131 Figure 17 Electron micrograph of guinea-pig v e n t r i c l e heart homogenate preparation. Electron micrographs were made by the standard procedure as described i n the 'Material and Methods' section, page 9 8 . The micro-organelles i d e n t i f i e d were i n t a c t mitochondria (mito) and the v e s i c l e s (ves) of d i f f e r e n t s i z e s . Magnification approximately 4 5,00Ox. 131a 132 TABLE 3 Ouabain Binding Assay of Microsomal Enriched S.R. Preparation. Ouabain binding (pmoles/mg Protein/min) S.R. Enriched Preparation 0.067 + 0.002 a S.R. P u r i f i e d on 0.002 a ' b sucrose density gradient 0.045 + Microsomes enriched i n S.R. were prepared as described on page 96. The microsomes were further fractionated on a discontinuous sucrose density gradient according to Katz and Dobovicnik (1979). Both the crude and p u r i f i e d S.R. preparation were assayed for ouabain binding as described on page 98. ; _ a. The res u l t s shown --are a Mean +V.S.E.M. of 4 experiments from separate microsomal preparations. b. This value of ouabain binding to p u r i f i e d S.R. i s i n agreement with the observation of Gelbart and "Goldman (1977). 133 Occasionally swollen mitochondria were observed. It i s doubt-f u l i f the preparation has undergone s i g n i f i c a n t i r r e v e r s i b l e pathological changes,since the- homogenate preparation consistently exhibited ah ATP-dependent calcium binding a c t i v i t y as well as calcium uptake i n the presence of oxalate. The enzymatic a c t i v i t y of these calcium transport processes are known to be l a b i l e at room temperature or with prolonged or extensive homogenization (Katz and Repke, 1967) . 2. E f f e c t of Taurine on Calcium Uptake and Binding: The e f f e c t of varying taurine concentrations on calcium uptake and binding i n both v e n t r i c u l a r homogenate and sarcoplasmic reticulum enriched preparations i s shown i n Table 4. The free calcium concentration used i n these studies was 1.0 y_M. Taurine i n concentrations of 5 to 50 mM had no s i g n i f i c a n t e f f e c t on calcium uptake or binding i n either of these preparations. The e f f e c t of 20 mM taurine on calcium uptake and binding i n both these preparations was examined at various calcium concentrations (Table 5). The sarcoplasmic reticulum enriched preparation exhibited an increase 2+ in calcium uptake and binding with increasing Ca 2+ concentration to a maximum of 10 uM free Ca ; Calcium TABLE 4 E f f e c t of Taurine on Calcium Uptake and Binding i n Guinea-pig Heart V e n t r i c l e Homogenates and Sarcoplasmic Reticulum Enriched Preparations. Taurine Homogenate Preparation Sarcoplasmic Reticulum Preparation Cone. — — <2!i> Calcium Uptake Calcium Binding Calcium Uptake Calcium Binding (nmoles/mg/min) (nraoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) 2.17 ± 0.38° (2.36 ± 0.40) b 0.16 ± 0.03 (0.16 ± 0.03) 12.40 ± 0.79 (12.72 t 0.89) 0.71 ± 0.10 (0.75 ± 0.09) 10 2.39 ± 0.15 (2.32 ± 0.29) 0.17 ± 0.02 (0.16 ± 0.02) 12.93 t 1.27 (13.19 t 1.19) 0.76 t 0.05 (0.75 ± 0.04) 20 2.68 i 0.10 (2.56 ± 0.14) 0.16 ± 0.02 (0.18 1 0.02) 12.72 ± 1.37 (12.18 ± 1.33) 0.77 ± 0.05 (0.81 t 0.02) 30 2.89 i 0.14 (2.89 i 0.13) 0.17 ± 0.01 (0.19 ± 0.02) 14.79 ± 1.83 (14.09 t 0.02) 0.81 1 0.04 (0.74 ± 0.03) 40 2.62 t 0.22 2.70 ± 0.14 0.18 ± 0.01 0.17 ± 0.02 11.84 t 1.79 11.27 t 1.72 0.76 ± 0.02 0.76 ± 0.05 50 2.59 0.17 (2.56 1 0.34) 0.16 0.02 (0.17 ± 0.01) 12.5S 2.05 (12.52 ± 0.01) 0.70 0.08 (0.75 t 0.09) Guinea-pig heart v e n t r i c l e homogenates (200-300 yg protein) or sarcoplasmic reticulum enriched preparations (40-50 wg protein) were incubated f o r 5 min with and without taurine i n medium containing 40 mM h i s t i d i n e - H C l , pH6.8, 5 mM MgCl,, 5.mM ATP, 110 mM KC1, 2.5 mM Tri s - o x a l a t e , and 1.0 uM free Ca ~7l25 y_M CaCl2 containing~T5caCl2 (10 Ci/mole) and~391 uM EGTA). The reaction was c a r r i e d out at 30°C i n a t o t a l volume of 0.5 ml. Calcium binding was determined i n an i d e n t i c a l reaction mixture, except that 2.5 mM Tris-oxa l a t e was omitted. a. The r e s u l t s are a Mean t S.E.M. of at l e a s t 3 observations each performed i n duplicate. b. The values i n parentheses are controls (taurine omitted from the reaction medium). TABLE 5 The E f f e c t o f T a u r i n e on C a l c i u m Uptake and B i n d i n g a t V a r i o u s C a l c i u m C o n c e n t r a t i o n s i n G u i n e a - p i g H e a r t V e n t r i c l e Homogenates and S a r c o p l a s m i c R e t i c u l u m E n r i c h e d P r e p a r a t i o n s C a l c i u m Homogenate P r e p a r a t i o n S a r c o p l a s m i c R e t i c u l u m P r e p a r a t i o n Cone. ( v K ) C a l c i u m Uptake C a l c i u m B i n d i n g C a l c i u m U p t a k e C a l c i u m B i n d i n g (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) 0.5 1.07 ± 0 . 0 3 a (1.18 ± 0 . 0 3 ) b 1.0 2.43 ± 0.09 (2.36 ± 0.17 5.0 7.81 ± 1.09 (7.94)± 0.52) 10.0 9.67 + 0.99 (10.00 ± 1.17) 50.0 8.56 ± 1.58 (8.62 ± 1.74 100.0 7.34 ± 1.34 (7.02 ± 1.22) 0.09 ± 0.01 8.91 (0.11 ± 0.01) (8.77 0.14 ± 0.01 20.14 (0.15 ± 0.01) (19.61 0.40 ± 0.03 71.42 (0.39 ± 0.03) (71.59 0.79 ± 0.05 84.56 (0.85 ± 0.06) (86.64 0.74 ± 0.02 69.84 (0.75 ± 0.04) (70.15 1.01 ± 0.10 81.29 (1.04 ± 0.08) (81.21 ± 1.32 0.54 ± 0.11 ± 1.24) (0.58 ± 0.08) ± 3.02 0.63 ± 0.14 ± 2.94) (0.65 ± 0.14) ± 1 4 . 8 9 1.33 ± 0.25 ± 14.00) (1.17 ± 0.15) ± 20.09 1.42 ± 0.11 ± 2 0 . 3 4 ) (1.44 ± 0.10) ± 10.36 1.14 ± 0.11 ± 12.67) (1.19 ± 0.15) ± 26.61 1.26 5 0.14 ± 29.37) (1.34 5 0.12) G u i n e a - p i g h e a r t v e n t r i c l e homogenates (200-300 ug p r o t e i n ) o r s a r c o p l a s m i c r e t i c u l u m e n r i c h e d p r e p a r a t i o n s (40-50 u g p r o t e i n ) were i n c u b a t e d f o r 5 min w i t h and w i t h o u t 20 mM t a u r i n e as d e s c r i b e d i n T a b l e 4 i n t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f f r e e c a l c i u m . C a l c i u m b i n d i n g was measured u n d e r i d e n t i c a l c o n d i t i o n s i n t h e absence o f 2.5 mM T r i s - o x a l a t e . a. The r e s u l t s a r e a Mean ± S.E.M. o f a t l e a s t 3 o b s e r v a t i o n s e a c h p e r f o r m e d i n d u p l i c a t e . b. The v a l u e s i n p a r e n t h e s e s a r e c o n t r o l s ( t a u r i n e o m i t t e d f r o m t h e i n c u b a t i o n medium). 136 concentrations higher than 10 u_M were i n h i b i t o r y . This 2+ p r o f i l e of the Ca concentration e f f e c t on calcium transport was similar i n the homogenate preparation. 2+ Taurine (20 mM) at a l l free Ca concentrations studied had no s i g n i f i c a n t e f f e c t on calcium uptake or binding i n either of these preparations. The E f f e c t of Taurine on the Time-course of Calcium  Uptake and Bindings: The time course of calcium uptake and binding i n homogenate and sarcoplasmic reticulum enriched, preparations i s shown i n figure 18A and 18B/ respectively. Calcium uptake i n S.R. enriched preparations was l i n e a r for the f i r s t 10 minutes of incubation following which the rates of calcium uptake declined s l i g h t l y . Maximum calcium binding was observed at 5 minutes of incubation. Taurine was observed to have no s i g n i f i c a n t e f f e c t on calcium uptake or binding at a l l the incubation times studied. In the heart v e n t r i c l e homogenate preparation, maximum calcium binding was observed at 10 minutes of incubation. Again, no s i g n i f i c a n t e f f e c t of taurine was observed on calcium binding or uptake either at the i n i t i a l time (30 seconds) or at longer Figure 18 Time course e f f e c t of 20 mM Taurine (0,A), on calcium uptake (s o l i d lines) and binding (dotted lines) i n Guinea-pig heart v e n t r i c l e homogenates (A) and sarcoplasmic reticulum enriched preparations (B) . The.;%e-r-tic£e.Hlines.-,i represent ± S.E.M. of 3 determinations each performed i n duplicate. Guinea-pig whole heart homogenate (200-300 ug protein) and enriched sarcoplasmic reticulum preparation (40-60 yg protein) were incubated .: with and without taurine for various incubation periods. Calcium uptake was measured i n the presence of 1.0 yM free calcium as described i n Table 4 (Page 134). Calcium binding was measured under i d e n t i c a l conditions i n the absence of j 2.5 mM Tris-oxalate. 138 periods of incubation. The E f f e c t of Taurine on the Decay of Calcium Uptake  A c t i v i t y : Both the homogenate and the sarcoplasmic reticulum enriched preparations decreased rapidly i n calcium uptake a c t i v i t y when kept at 4°C i n the absence of 2+ 40% sucrose. Ca -uptake a c t i v i t y of the homogenate decreased i n a c u r v i l i n e a r fashion with time. This a c t i v i t y i n sarcoplasmic reticulum enriched preparation exhibited a l i n e a r decay. Addition of 15 mM taurine to these preparations under these conditions did not a l t e r t h i s steady decline i n calcium uptake a c t i v i t y (figure 19). In the presence and absence of taurine, the respective c o e f f i c i e n t s i n the regression equations were not s i g n i f i c a n t l y d i f f e r e n t (using-a computer program; UBC- SLTEST.) E f f e c t of Taurine on C y c l i c AMP-dependent Protein- Kinase Stimulated Calcium Uptake: When the sarcoplasmic reticulum enriched preparation was incubated with protein kinase and cyclic-AMP i n the presence of 1.0 y M free calcium, calcium uptake was increased approximately "two folds (p<0.02) • (Table 6) 139 Figure 19 The E f f e c t of Taurine on the decay of calcium uptake a c t i v i t y i n guinea-pig v e n t r i c l e homogenates and sarcoplasmic reticulum enriched preparations. Homo-genate ( s o l i d l i n e s , 200-300 pg protein) or sarcoplasmic reticulum preparations (dotted l i n e s , 40-50 ug protein) were maintained i n 2 mM T r i s - C l , pH 7.2 i n the presence (0,A) and absence (•, •) of 15 mM taurine. Calcium uptake was measured at sp e c i f i e d times as described i n table 4: . The regression l i n e for the sarcoplasmic reticulum was f i t t e d by the method of least squares, .. and that for homogenate by the method of 3rd. degree polynomial least squares using a computer program (UBC-OLQF)-. Each point represents mean + S.E.M. of 3 determinations each performed i n duplicates. 139 a C a + + U p t a k e : n m o l e s / m g S . R . p r o t e i n / m i n ( D , A ) *»• po so o- o P P — .— rsj oo ro o- o C a + + U p t a k e : n m o l e s / m g H o m o g e n a t e P r o t e i n / m i n [;o) TABLE 6 Effect of Taurine on Cyclic AMP-dependent Protein Kinase-Stimulated Calcium Uptake in Guinea-pig Heart Ventricle Homogenates and Sarcoplasmic Reticulum Enriched Preparations. w«*u™ Taurine Homogenate Preparation Sarcoplasmic Reticulum Preparation (20mM) Without cAMP-dependent Protein kinase With cAMP-dependent Protein kinase Without cAMP-dependent Protein kinase With cAMP-dependent Protein kinase - 2.39 t 0.29 a' b 3.38 t 0.14° 9.72 t 1.02 16.86 ± 1.51d + 3.30 t 0.17e 17.89 ± 1.84e Guinea-pig ventricle homogenates (200-300 jig protein) and sarcoplasmic reticulum enriched preparations (40-50 ug protein) were incubated with and without c y c l i c AMP (1.0 wM) and cy c l i c AMP-dependent protein kinase (50 ug/ml, Sigma grade type 1) in the presence and absence of 20 mM taurine. Calcium uptake was measured as described in Table 4. In these experiments the free calcium concentration was 1.0 pM and the incubation time was 5 minutes. a. The results are a Mean t S.E.M. of at least 3 observations each performed in duplicate. b. Calcium uptake activity expressed as nmoles/mg/min. c. P<0.05 compared to Ca2*"-uptake in the absence of cyclic AMP-dependent protein kinase. d. P<0.02 compared to Ca 2 +-uptake In the absence of cyclic AMP-dependent protein kinase. e. Not significant compared to the activity seen without taurine i n the presence of cyclic AMP-dependent protein kinase. 141 S i m i l a r l y , t h e r a t e o f c a l c i u m u p t a k e by t h e h omogenate a l s o i n c r e a s e d i n t h e p r e s e n c e o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e ( p < 0 . 0 5 ) . T a u r i n e (20 mM) h a d no s i g n i f i c a n t e f f e c t on t h e c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e s t i m u l a t i o n o f c a l c i u m u p t a k e i n b o t h o f t h e s e p r e p a r a t i o n s . B. E f f e c t o f T a u r i n e on P a s s i v e I o n T r a n s p o r t i n R a t  B r a i n S y n a p t o s o m e s 1. : C h a r a c t e r i z a t i o n o f S y n a p t o s o m a l P r e p a r a t i o n s E l e c t r o n m i c r o g r a p h s o f a t y p i c a l s y n a p t o s o m a l p r e p a r a t i o n i s shown i n F i g u r e 20. The s y n a p t o s o m a l s u s p e n s i o n w e r e f o u n d t o c o n s i s t o f s y n a p t i c v e s i c l e s a n d m i t o c h o n d r i a e n c l o s e d w i t h i n membranes t o f o r m n e r v e e n d i n g p a r t i c l e s . The e l e c t r o n m i c r o g r a p h s w e r e c o n s i s t e n t w i t h t h o s e o r i g i n a l l y o b t a i n e d by G r a y a n d W h i t t a k e r (1962) a n d D e . R o b e r t i s e t a l . ( 1 9 6 1 ) . 2. The O s m o m e t r i c B e h a v i o u r o f S y n a p t o s o m e s The o p t i c a l e x t i n c t i o n ( E 5 2 Q ) o f t n e s y n a p t o s o m a l p r e p a r a t i o n s s u s p e n d e d i n s o l u t i o n s o f N a 2 S O ^ was f o u n d t o i n c r e a s e w i t h t h e m o l a r i t y o f t h e N a 2 S 0 4 s o l u t i o n ( F i g u r e 2 1 A ) . The r e c i p r o c a l p l o t o f e x t i n c t i o n ( 1 / E 5 2 Q ) a g a i n s t l/Na 2SC> 4 ( f i g u r e 21B) Figure 20 Electron micrograph of a t y p i c a l r a t brain synaptosomal preparation: Electron micrographs were made by the standard procedure as described i n the 'Materials and Methods' section, page 9 8 .-' The f r a c t i o n obtained between the sucrose gradient (0.8 M and 1.2 M sucrose) consisted of synaptic v e s i c l e s (SV) and mitochondria (M) enclosed within membrane (tm) to form a nerve ending p a r t i c l e . The electron micrograph i s sim i l a r to those of Gray and Whittaker (1962). Magnification approximately 45,000x. 1 4 2 a Figure 21 The e f f e c t of Na2SC>4 concentration on the' E 5 2 Q of a suspension of synaptosomes: In (A) the data are plotted as E 5 2 Q against N a 2S0 4 while i n (B) l/Na2SC>4 i s plotted against 1 / E ^ 2 Q . Results are shown as Mean ±S .E.M. of . 3 d i f f e r e n t synaptosomal preparations. 143 a •520 144 showed a l i n e a r r e l a t i o n s h i p . These r e s u l t s c o n f i r m the o b s e r v a t i o n s of Keen and White (1970) and show t h a t the synaptosomal p r e p a r a t i o n s behave as osmometers conforming to Boyle and Van't H o f f ' s law. (Keen and White, 1970). 3. The E f f e c t of Taurine on Sodium and Potassium  P e r m e a b i l i t y i n Synaptosomal P r e p a r a t i o n The p e r m e a b i l i t y of the synaptosomal p r e p a r a t i o n s to sodium and potassium i o n s i n the presence or absence of t a u r i n e i s shown i n Table 8A and 8B, r e s p e c t i v e l y . Synaptosomal p r e p a r a t i o n s p r e i n c u b a t e d w i t h 2 0 mM t a u r i n e and suspended i n 100-200 mM sodium or potassium a c e t a t e s o l u t i o n s c o n t a i n i n g 20 mM t a u r i n e showed no s i g n i f i c a n t change i n E520 w ^ e n compared to r e s u l t s o b t a i n e d i n the absence of t a u r i n e . 4. The E f f e c t of Taurine on the P a s s i v e Uptake and  Release of Calcium i n Synaptosomal P r e p a r a t i o n s : The time course of uptake and r e l e a s e of c a l c i u m i n an i s o t o n i c sucrose medium i s shown i n f i g u r e 22A and 22B, r e s p e c t i v e l y . The c a l c i u m c o n c e n t r a t i o n used i n t h i s study was 10 y_M. Under these c o n d i t i o n s , synaptosomal p r e p a r a t i o n s were i n i t i a l l y observed , TABLE 7 " ' E f f e c t o f Ta u r i n e on Sodium (A) and Potassium (B) P e r m e a b i l i t y i n Synaptosomes. + + A. Na Aceta t e B. ;! K. Acetate E520 . . E520 mM N a + — mM K + CONTROL TAURINE CONTROL TAURINE 100 0.898 + 0. 050 0.913 + 0. 044 -, 100 0.915 0.934 + 0.035 0. 918 + 0. 044 125 0.934 + 0. 053 0.916 + 0. 044 125 + 0.050 0. 948 + 0. 041 150 0.965 + 0. 072 0.994 + 0. 041 150 0.961 + 0.045 0. 966 0. 048 175 1.000 + 0. 052 1.018 + 0. 042 175 1.018 + 0.033 1. 001 + 0. 045 200 1.000 + 0. 056 1.025 + 0. 045 200 1.024 + 0.034 1. 020 + 0. 045 The p e r m e a b i l i t y was measured as a f u n c t i o n of the change i n E ^ g °f a synapto-somal membrane suspension (50 pi) i n acetate s a l t s i n the presence (TAURINE) or absence (CONTROL) o f 20 mM t a u r i n e . Each value i s the mean ± S.E.M. of t h r e e separate synaptosomal p r e p a r a t i o n s . 146 to take up calcium rapidly. After 2 minutes i n the absence of taurine, the amount of calcium taken up was almost 45% of that taken up at 30 minutes. Addition of 20 mM taurine to the incubation medium, lowered the amount of calcium taken up at a l l time points studied. This difference was s i g n i f i c a n t (p<0.001; t - t e s t of the difference between the intercepts on the ordinate). The slope of the lines were not s i g n i f i c a n t l y d i f f e r e n t . Calcium release from preloaded synaptosomes i s shown i n figure 22B. In the absence of taurine, a f t e r 2 . . minutes of incubation, about 50% of the calcium load was released from the synaptosomes. In the presence of 20 mM taurine, calcium e f f l u x from the preloaded synaptosomes was reduced at a l l incubation .times tested. Curves f i t t e d by l i n e a r regression had intercepts on the ordinates which were s i g n i f i c a n t l y d i f f e r e n t (p <0.001). Dose-dependent E f f e c t of Taurine on Calcium Uptake  i n Synaptosomal Preparations Various concentrations of taurine (0.5 to 50 mM) were studied with respect to synaptosomal calcium uptake (figure 2 3). Control experiments were c a r r i e d out where taurine was substituted for an equimolar Figure 22 The e f f e c t of taurine on (A) 4 JCa' ; T uptake and 45 2+ (B) release of Ca from preloaded rat synaptosomal preparations. Calcium uptake and release were determined i n the presence (o o) or absence (• •) of 20 mM taurine i n a medium containing 0.3 M sucrose and 10 mM Tris-HCl, pH 7.2 as described i n the 'Materials and Methods" section (pages 107, to 108). E f f e c t of taurine on calcium uptake i s expressed as 45 2+ 2+ Ca uptake r e l a t i v e to the value of Ca uptake observed at 30 minutes incubation time i n the absence of taurine. Calcium release from preloaded synaptosomes was calculated as % release as described i n the / 'Materials and Methods' section, page 108. Lines were derived by a l i n e a r regression analysis of the data. Each time.point represents determination from three separate synaptosomal membrane preparations. 1 4 7 a Relative 4 5 C a 2 + U p t a k e (%) Figure 23 The e f f e c t of various concentrations of taurine 45 on CaCl2 uptake i n brain synaptosomal preparations (s o l i d bars). Control experiments were done i n the presence of equimolar concentrations of choline chloride (middle bars with oblique s t r i a t i o n s ) and i n the presence of neither taurine nor choline chloride ( l e f t bars with straight s t r i a t i o n s ) . Calcium uptake was determined at 2°C for 30 minutes i n medium containing synaptosomes (0.2 mg/ml), 0.3 M sucrose, 10 mM Tris-HCl, pH 7.2 and 10 y_M 4 5 C a C l 2 5 (5 x 10 cpm/sample) i n a t o t a l volume of 0.3 ml. Each bar represents the Mean ± S.E.M. of three separate experiments r e l a t i v e to the value of the 45 CaC^-uptake of controls measured i n the absence of taurine or choline chloride. 0.5 1.0 5.0 10.0 20.0 30.0 50.0 mM Taurine or Choline chloride 149 concentration of choline chloride. Conditions were also studied where neither taurine nor choline chloride were present i n the incubation medium. No change i n calcium uptake could be detected at.lower concentra-tions of taurine (0.5 to 5.0 mM)-; thereafter, as taurine concentrations were increased a decline i n calcium uptake was observed. Choline chloride (on a molar basis) was more potent than taurine i n lowering synaptosomal calcium uptake i n concentrations greater than 10.0 mM. 6. E f f e c t of Other Amino Acids on Calcium Uptake i n  Synaptosomal Preparations A number of compounds, i n a concentration of 20 mM, were tested for t h e i r e f f e c t on passive calcium uptake i n synaptosomal preparations (figure 24). Homotaurine, hypotaurine, 3 - alanine and GABA exhibited similar e f f e c t s to'.'taurine, s i g n i f i c a n t l y decreasing the degree of calcium uptake observed i n ; the absence of these agents (p<0.05). a - alanine, :did not s i g n i f i c a n t l y a f f e c t calcium transport i n t h i s preparation. Methionine, proline and"valine (not shown) did not s i g n i f i c a n t l y a f f e c t calcium transport i n t h i s preparation. :-~ • Figure 24 E f f e c t of various amino acids on calcium uptake i n brain synaptosomal preparatons. Calcium uptake was measured for 30 minutes under the same conditions as that described i n Figure -'23' i n the presence of 20 mM concentrations of various amino acids. Controls consisted of similar smedium with no amino acid addition. The results shown are the mean ± S.E.M. of 3 to 4 separate preparations i n each case. ^ C a 2 + - U p t a k e : nmoles/mg protein W a t e r Taur ine j3 -Al a nine G A B A H o m o t a u r i n e H y p o t a u r i n e o Ln T o O T t o 61 1 O f - A l a n i n e o 1 5 1 DISCUSSION 152 I. BYCONVERSION. OF TAURINE TO ISETHIONIC ACID IN THE REGULATION OF ION FLUX Methylation of i s e t h i o n i c acid produces two compounds, a methylated methylester and a dimethylated, methylester, methylether derivative. These two compounds when analyzed by gas-liquid chromatography co-elute on a column of OV-1, but can be separated on a column of OV-17 (see figures 9 and 10). The r a t i o of these two compounds produced by methylation i s approximately 20:1 with the methylester derivative being predominant. This r a t i o was confirmed by proton NMR spectro-scopy (figure 12A). The NMR spectra of both methylated i s e t h i o n i c acid (figure 12A) and 1-butanesulfonic acid (figure 12B) showed the CH^ peak of the methylester as a sin g l e t at 3.88 ppm. A small si n g l e t at 3.30 ppm occurring 'in the spectra of methylated i s e t h i o n i c acid was assigned as the CH2 group of the methylether of i s e t h i o n i c acid on the basis of the known chemical s h i f t value of 3.30 ppm of the methylether s i n g l e t i n methoxyethanol (figure 12C) . The assignment of structures to the two methylated derivatives of i s e t h i o n i c acid was also c a r r i e d out using GC-MS. The assignment of structures to the mass spectra of the peaks by methylation of i s e t h i o n i c acid (figure 11) i s i n conformity with known fragmentation pattern of a l k y l 153 alkanesulfonates (Truce et a l . , 1967). The fragmentation and rearrangements of the two methylated i s e t h i o n i c acid compounds are shown i n Table 8;. Some possible mechanisms for fragmen-tatio n structures are shown i n figures 25 and 26. The parent ions .'. [M] t were not seen i n the methylated i s e t h i o n i c acid mass spectra. Truce et a l . (1967) claim that these ions are scarce for most of the a l k y l alkanesulfonates. However, the assignment of structures to the two peaks are strengthened by .the appearance of the [M-l]t fragment for the methylester of i s e t h i o n i c acid at m/e=139. The gas-liquid chromatographic-methy.lation technique was used for the analyses of i s e t h i o n i c acid i n mammalian tissues. Welty, Read and Shaw (196 2) quoted a figure of 42.6 mg i s e t h i o n i c acid per 100 g rat heart tissue and 12.9 mg per 100 g dog heart. These amounts would have been quite e a s i l y detectable with my method. In heart, using the s u l f u r detector,which i n t h i s p a r t i c u l a r case .was ten times more sensitive than the flame i o n i z a t i o n detector,' only a very small peak, roughly 0.10 mg/lOOg wet weight tissue (0.008 y moles/g) i n the p o s i t i o n of i s e t h i o n i c acid, could be seen.Insufficient material was available to confirm that t h i s small amount of material was t r u l y i s e t h i o n i c acid. As much as 400 g of dog heart tissue was extracted to search for i s e t h i o n i c acid, following exactly the procedure of Welty, Read and Shaw (1962). In t h i s large TABLE < The FragBentation and Kaarrangeaents of the Two Methylation Product* of laethlonic Acid that were Analysed Using an OV-17 Column FRAGMENTS "»/e STRUCTURE FRAGMENTATION AND REARRANGEMENT PROCESS A. Methylester atethvlether of Isethionic acid 31 OCUj* a cleavage 45 CHS* CHjO - CHj* A rearrangement process shown by high resolution Measurements. Cleavage typical of aliphatic ether se CHjOCH - CHj B - hydrogen rearrangement (Pig. 25) (A e - hydrogen transfer vith a-cleavage). 59 CBjCBjOCH3+ a - cleavage 79 B02 C H 3 * Rearrangement ion derived from the aUtane group of the sulfonate ester. 95 s o 3 C H 3 * o - cleavage 96 BOS0 2 C H 3 + t - hydrogen rearrangement ( F i g . 2 5 ) . (A e - hydrogen transfer with a - cleavage) 124 C B J S O I O C H J ) 2 * CH3OCB2CM2S02a* McLafferty rearrangement (Fig.2 5 ) (methyl group transfer with e - cleavage) a' cleavage with hydrogen transfer B. Methylester of Isethionic Acid 31 OCHj* o cleavage 44 CHjCHOH* 8 - hydrogen rearrangement (Fig. 2 6 ) CA 6 - hydrogen transfer with a - cleavage) 45 HOCHjCBj* CHS* o - cleavage A rearrangement process shown by high resolution measurements 79 CH 3S0 2 + Rearrangement ion derived from the alkane group of the sulfonate ester 80 C H J S O J H * a' cleavage with transfer of s $' hydrogen 95 S0 3CH 3 + Q- cleavage 97 ( O H ) 2 S O C H 3 + Two 6 - hydrogen transfer with a - cleavage 110 CH2SO(OH)OCH3+ B O C H J C H J S O J H * McLafferty Rearrangement (Fig. 2 6 ) (o - hydrogen transfer with fl -cleavage). o' cleavage with hydrogen transfer 139 O B ( C H 2 ) 2 S 0 3 C H 2 + |M - 1) * The fragnentations and rearrangements undergoing mass spectral conditions are explained by sjachanil IBS established from the work of Truce et a l . (1967). In referring to various bonds undergoing clesvsge in the fragmentation, the following schene was used. 0 C - O - C - C - S - - 0 - C - H 0 t y fl a a e a cleavage means cleavage of C fl- cleavage refers to the C Q- C f t bond, u" cleavage refers to the 8-OR bond. A substituent referred to as an a vubetitusnt w i l l be borne on the a carbon. 155. Figure 25 Mass Spectral Rearrangements and Fragmentation of Methylether, Methylester of Isethionic acid. The most common fragmentation and rearrangement of alkanesulfonates on mass spectrometer are due to McLafferty and a-hydrogen rearrangement (Truce et a l . , 1967). The mass ions obtained for Methylether, Methylester due to these rearrangements were r e l a t i v e l y abundant on the spectrum (Figure 11) and were important i n the assignment of i t s structure. The convention proposed by Budzikiewicz et al.^ 1964 for denoting e l e c t i o n s h i f t s was used (A fishhook (/"*) indicates the movement of a single electron) . Budsikiewics, H., Djerassi, C., and Williams, D.E..(1964) "Interpretation of Mass Spectra of Organic Compounds," Holden-Day, Inc., San Francisco, C a l i f . , p. x i i . 155a MASS SPECTRA OF METHYLATED ISA — FRAGMENTATION & REARRANGEMENT (PEAK I of Fig. 9Band SPECTRUM A of Fig. 11) o+ O ^ CH 2 / 1 0\- OCH^ _ ^ Q = C H 2 + Methylester, Methylether of Isethionic Acid [ C H 0 ] + m/e = 29 O C H 3 I C H 2 = S — OCH 3 | II O m/e = 124 M c L A F F E R T Y R E A R R A N G E M E N T Methylester, Methylether of Isethionic Acid CH3OCH = C H 2 [ C H 3 O C H = C H J m/e - 58 + OH S — O C H , m/e = 96 (3- H Y D R O G E N R E A R R A N G E M E N T (TRUCE, et. a l . J . O r g . C h e m . 32 : 3 0 8 , 1967) 156:. Figure 26 Mass Spectral Rearrangements and Fragmentation of Methylester of Isethionic acid. The most common fragmentation and rearrangements of alkanesulfonates on Mass Spectrometer are due to McLafferty and ^-hydrogen rearrangement processes (Truce et a l . , 1967). These massiions obtained for the methylester of i s e t h i o n i c acid were r e l a t i v e l y abundant on the spectrum (figure 11) and were important i n the assignment of i t s structure. * The convention proposed by Budzikiewicz et a l . 1964 for denoting electron s h i f t s was used (A fishhook ( ) indicates the. movement of a single electron). Budsikiewics, H., Djerassi, C., and Williams, D.H. (1964) "Interpretation of Mass Spectra of Organic Compounds," Holden-Day, Inc., San Francisco, C a l i f . , p. x i i . 156 a MASS SPECTRA OF METHYLATED ISA — FRAGMENTATION & REARRANGEMENT (PEAK II of Fig. 9B and SPECTRUM B of Fig. 11) H Methylester of Isethionic Acid m/e = 29 M c L A F F E R T Y R E A R R A N G E M E N T [TlOCH=CH7|t m/e 96 Methylester of Isethionic Acid m/e = 44 H Y D R O G E N R E A R R A N G E M E N T (TRUCE, et. a l . J . O r g . C h e m . 32: 3 0 8 , 1967) 157 experiment, neither the c r y s t a l s of sodium isethionate (that they reported) nor gas chromatographic evidence of the presence of i s e t h i o n i c acid were obtained. The s e n s i t i v i t y of the gas-liquid chromatographic portion of t h i s experiment would have been approximately 0.1 mg/lOOg. The differences between my results and those of Welty, Read and Shaw (1962) can not be understood. However, the behaviour of i s e t h i o n i c acid on ion-exchange chromatography reported by Welty, Read and Shaw (196 2) was not correct. Isethionic acid, a strong s u l f o n i c acid was reported to be retained on an a c i d i c cation exchange r e s i n . The v a l i d i t y of results of these workers i s questionable. When the methylation technique was used to analyze i s e t h i o n i c acid i n r a t brain, a small peak i n the p o s i t i o n of i s e t h i o n i c acid was detected at a concentration of approximately 0.2 mg/100 g of wet weight tissue (0.016 pmole/g). I n s u f f i c i e n t material was available to confirm the i d e n t i t y of t h i s material by mass spectrometry. The a n a l y t i c a l procedure was always monitored by adding i s e t h i o n i c acid at concentrations of 0.2 and 2.0 pmole/g of tissue to duplicate portions of tissue. Recovery was always between 9 5 and 100%. Furthermore, the procedure for the analysis of i s e t h i o n i c acid i n mammalian tissue was confirmed using squid giant axon where i s e t h i o n i c acid i s found i n high concentrations. The value obtained for the concentration of i s e t h i o n i c acid i n the squid giant axon compares favourably with other data 158 obtained using d i f f e r e n t less sensitive methods (Deffner and Hafter, 196 0; Hoskin and Brande, 1973). I t remains possible, however, that i s e t h i o n i c acid i s i r r e v e r s i b l y bound to tissue membranes. The addition of methanol during homogenization lowers the d i e l e c t r i c constant and thi s could t h e o r e t i c a l l y suppress i t s i o n i z a t i o n to some extent. Any ef f e c t s of methanol on i s e t h i o n i c acid i n tissues could t h e o r e t i c a l l y , cause binding of i s e t h i o n i c acid to tissues and not necessarily a f f e c t binding of added i s e t h i o n i c acid. There i s a precedent for such a p o s s i b i l i t y i n the binding of phosphoinositides to brain tissues during extraction procedures (Rouser et a l . , 1967). However, Schaffer et a l . (1978b) recently reported measurement of i s e t h i o n i c acid le v e l s i n rat hearts. Isethionic acid i n t h e i r studies was extracted with 3% perchloric acid aft e r the rat hearts were l y o p h i l i z e d i n l i q u i d nitrogen. Only trace amounts of i s e t h i o n i c acid (1.03 pmole/g dry weight) were reported to be present i n rat heart. This concentration i s about 50-fold less than the values previously reported by Rosei et a l . (1974) and Welty et a l . (1962). In my studies, I found even lower concentrations of i s e t h i o n i c acid i n r a t heart and brain tissues than those reported by Schaffer. However Schaffer did not confirm the fact that t h e i r gas chroma-tographic peaks contained >only i s e t h i o n i c acid and did not use a sulfu r detector i n t h e i r work. The use of a su l f u r detector was espe c i a l l y important i n my work because i t has a much greater s e n s i t i v i t y than a flame i o n i z a t i o n detector. However, the findings of Schaffer et a l . (1978b) confirm our results i n that 159 i s e t h i o n i c acid i n rat and dog hearts and rat brain i s a minor anion i n these tissues. During the course of analysis of i s e t h i o n i c acid i n tissues, i t was usually necessary to use two columns, one of OV-1 and another of OV-17, and occasionally two methods of detection, flame io n i z a t i o n and flame photometry (for s u l f u r ) . This was necessary because a l l of the mammalian tissues studied gave a peak i n the p o s i t i o n of i s e t h i o n i c acid i n the crude extracts of rat and dog heart and rat brain when,an OV-1 column was used. The peaks on OV-1 chromatography corresponded to an amount of material that could have been approximately 10-12 mg i s e t h i o n i c acid per 100 g heart or brain. Confirmation that t h i s peak was not i s e t h i o n i c acid depended on rechromatography of the same extract on OV-17 columns and v e r i f i c a t i o n that t h i s peak did not contain s u l f u r . The large peak from heart and brain extracts seen on the OV-1 column when reassessed during the OV-17 column and the s u l f u r detector proved to contain only very small amounts of i s e t h i o n i c acid. This use of a s u l f u r detector- was necessary because routine access to GC-MS was not possible at the time. During the course of developing an a n a l y t i c a l method for the analysis of i s e t h i o n i c acid, Rosei et a l . (1974) reported a gas-liquid chromatographic detection of i s e t h i o n i c acid i n guinea-pig heart. S i l y l a t i o n of i s e t h i o n i c acid using the method of Rosei et al.(1974) 16 0 proved to give a very complex mixture. Thus, s i l y l a t i o n was found not to be feasible i n our hands. Accordingly, t h i s technique was abandoned. Recently, Schaffer et a l . (1978b) and Fellman et a l . (1978) have reported a gas-chromatographic method to measure i s e t h i o n i c acid i n heart muscles ;using a s i l y l a t i o n procedure d i f f e r e n t from that of Rosei et a l . (1974). Both Fellman and Schaffer reported that i s e t h i o n i c acid i s present i n very small concentrations or almost absent i n heart tissue, thus confirming our own results (Applegarth et a l . , 1976; Remtulla et a l . , 1977). To complete our studies of i s e t h i o n i c acid i n heart tissue, i t was l o g i c a l to repeat the taurine to i s e t h i o n i c acid conversion experiments reported by Read and Welty (19 62) using rat heart s l i c e s . U t i l i z i n g t h e i r procedures, I was 14 not able to detect any bioconversion of C-taurine to 14 xsethionic acid. When C-taurine was added to the heart s l i c e s and extracted immediately with methanol, a radioactive compound was obtained which behaved chromatographically (on Whatman #1 paper) l i k e i s e t h i o n i c acid. It i s possible that the compound Read and Welty (1962) reported i s an a r t i f a c t of the same or a similar chemical that was found during our experi-ments, since these workers did not report such control experi-ments. No further work was undertaken to evaluate the id e n t i t y of thi s compound. However, Fellman et a l . (1978) recently showed that taurine i s not converted to i s e t h i o n i c acid by heart, brain or l i v e r (This work of Fellman w i l l be enlarged upon l a t e r ) . 161 Recently, Sturman, Rassin and Gaull (1977c) reported the presence of a radioactive compound i n extracts of rat milk, 35 following the i n j e c t i o n of S-taurine to l a c t a t m g rats. This compound co-chromatographed with authentic i s e t h i o n i c acid. A possibly s i m i l a r compound was obtained by Huxtable and Bressler (1972) during t h e i r studies on the d i s t r i b u t i o n and interconversion of radioactive taurine and i s e t h i o n i c acid i n the rat. The compound obtained i n these studies was not characterized. I t was merely i d e n t i f i e d as a radioactive compound i n the area of a thin layer chromatography (TLC) or ion-exchange column separation normally occupied by i s e t h i o n i c acid. However, the report of the presence of i s e t h i o n i c acid i n these studies was puzzling. I t was of i n t e r e s t to determine whether or not i s e t h i o n i c acid was present as a natural constituent of milk. Milk samples from l a c t a t i n g rats were kindly provided by Dr. John Sturman and analyzed for i s e t h i o n i c acid by our technique. The samples analyzed showed no trace of i s e t h i o n i c acid. Recovery of authentic i s e t h i o n i c acid added to the milk sample was 100% and the s e n s i t i v i t y of the method was such that 20 nmoles i s e t h i o n i c acid/ml milk could have been e a s i l y detected. In the report of Sturman, Rassin and Gaull (1977c), the radioactive compound i n milk which co-chromatographed with authentic i s e t h i o n i c comprised 30-40% of the t o t a l r a d i o a c t i v i t y (the rest of 162 which was present as taurine). The present assay could e a s i l y have detected i s e t h i o n i c acid present at 10% of the taurine concentration. A clue to the resolution of the discrepancy i n these observations comes from the recent report of Fellman e t a l . (1978). In t h i s study a time-dependent accumulation 35 3 of a product of the radioautolysis of both S- and H-labeled taurine was observed. This product was said to behave as an anion and appeared on Bio-Rad AG-50 columns i n the same fractions as did authentic i s e t h i o n i c acid. Fellman et a l . (1978) also reported the synthesis 3 of (2- H)-taurine of high s p e c i f i c a c t i v i t y and tested i t as a precursor for i s e t h i o n i c acid synthesis i n dog heart s l i c e s , r at heart and brain s l i c e s and rat heart and brain homogenates. The conditions used i n t h i s study were also those described by Read and Welty (1962). The assay consisted of the measurement of the release of t r i t i a t e d water during the formation of i s e t h i o n i c acid v i a a sulfonic-acetaldehyde intermediate. The mammalian tissues tested were found not to convert taurine to i s e t h i o n i c acid i n any s i g n i f i c a n t quantity. The l i m i t of d e t e c t a b i l i t y of the i s e t h i o n i c acid conversion was less than 4.5 x 10 u. moles, of i s e t h i o n i c acid. On the other hand, the bacterium, Pseudomonas (Toyama et a l . , 1973; Yonaha et al.:,, 1976) and samples from 163 rat feces and gut washings were found to release t r i t i a t e d 3 water when incubated with (2- H)-taurine. Hoskin and Kordik (1977) and C a v a l l i n i et a l . (1978) provided further evidence that i s e t h i o n i c acid i s not a metabolite of taurine i n squid and mammalian tissues. In squid giant axon, where i s e t h i o n i c acid i s found i n high concentration, hydrogen s u l f i d e and not taurine was shown to be the precursor for the synthesis of i s e t h i o n i c acid (Hoskin and Brande, 1973; Hoskin et a l . , 1975). The enzyme involved i n t h i s pathway was said to be 'rhodanese' (Hoskin and Kordik, 1975; Hoskin, 1976) . In mammalian tissues, taurine and i s e t h i o n i c acid have been postulated to be derived from a common precursor along the cysteine to taurine pathway ( C a v a l l i n i et a l . , 19-78) . Studies car r i e d out by C a v a l l i n i consisted of a system of enzymes (diamine oxidase, E.C. 1.4.3.6; alcohol dehydrogenase, E.C. 1.1.1.1; and cysteamine dioxygenase, E.C. 1.13.11.19) that produced i s e t h i o n i c acid, s t a r t i n g from cysteamine, a common sulfur containing compound, and using mercaptoethanol as an intermediate: The enzymes, diamine oxidase and alcohol dehydrogenase "'convert cystamine to a mixture of cysteamine and mercaptoethanol. Cysteamine dioxygenase then converts mercaptoethanol to a s u l f i n i c acid, which then undergoes further oxidation to i s e t h i o n i c acid. The i d e n t i f i c a t i o n of i s e t h i o n i c acid i n 164 these experiments was based on paper electrophoresiscand 35 paper chromatographic techniques. When S-labeled mercaptoethanol was injected into rats, 91% of the radio-a c t i v i t y was excreted i n the urine within 24 hours. A small amount of t h i s r a d i o a c t i v i t y was i d e n t i f i e d as i s e t h i o n i c acid (Federici et al.,19 76). The acceptance of th i s hypothesis as the true synthetic pathway requires further studies using either isotope d i l u t i o n analysis or more sensitive methods for the detection of i s e t h i o n i c acid. The trace amounts of i s e t h i o n i c acid present i n heart and brain tissues reported i n t h i s thesis and elsewhere (Remtulla et a l . , 1977; Schaffer et a l . , 19 78b)could either have originated from the i n t e s t i n a l t r a c t bacterial-deamination of taurine to i s e t h i o n i c acid (Fellman et a l . , 1978) or have been produced by the alternate pathway described by C a v a l l i n i et a l . (1978). Further research may c l a r i f y t h i s point. Isethionic acid, even i f not a product of tissue bioconversion of taurine, may possibly have a function of i t s own. Bourke et a l . (19 70,. 1970) have provided evidence for the p o t e n t i a l usefulness of i s e t h i o n i c acid i n the reduction of cerebral edema. Jacobsen et a l . (1967) using isotope techniques,claimed to f i n d i s e t h i o n i c 165 acid i n human urine and plasma > The average urinary excretion of i s e t h i o n i c acid was 13.7 mg/24 hours (Jacobsen et a l . , 1967) and the average concentration i n blood was 32.6 yg/100 ml plasma (Jacobsen., - 1968). 'From these values, the renal clearance for i s e t h i o n i c acid was calculated to be 30 ml/minute assuming that i s e t h i o n i c acid i s . not bound to plasma proteins and passes fre e l y across the glomerular membrane (Jacobsen, 196 8). The mammalian nephron must therefore contain mechanisms, whereby 70-80% of the i s e t h i o n i c acid, f i l t e r e d through the glomerular membrane, i s reabsorbed. However, when Bennett and Dave (19 74) infused 115 mM sodium isethionate along with 4.0 mM potassium isethionate intravenously to rats (at the rate of 0.5 ml/hour or 1.5 ml/hour) a s i g n i f i c a n t decrease i n the concentration of K + and C l ~ i n the serum and C l ~ i n l i v e r and kidney were observed. No overt t o x i c i t y was evident on hematological and histopathological study of these tissues. The elucidation of the function of i s e t h i o n i c acid awaits further evidence. In summary, the results described i n this thesis c l e a r l y demonstrate that i s e t h i o n i c acid i s present i n only trace amounts i n mammalian brain and heart and .taurine was found not to be metabolized to i s e t h i o n i c acid as was o r i g i n a l l y claimed by Read and Welty (1962). 166 In view of these r e s u l t s , i t i s pertinent to re-examine the o r i g i n a l proposition of Read and Welty (1965) that ion translocation i n myocardial c e l l s involves the bioconversion of taurine to i s e t h i o n i c acid. : Read and Welty (1963) were the f i r s t to claim that taurine prevented premature ventricular contraction of the dog heart, caused by digoxin or epinephrine. Welty and Read (196 4) suggested that t h i s e f f e c t of taurine was mediated by a precursor of i t s metabolite i s e t h i o n i c acid. Welty (Ph.D. thesis, South Dakota, 196 3) hypothesized that taurine, under physiological conditions, existed i n part as a rin g structure due to i o n i c bonding between the sul f o n i c acid group and amino group. He proposed that such a c y c l i c molecule would carry, no charge and would be r e l a t i v e l y permeable through the c e l l membrane. Within the c e l l , deamination of taurine to i s e t h i o n i c acid'"would produce a negatively charged molecule- capable of cation a t t r a c t i o n . I t was suggested that t h i s would r e s u l t i n asymmetric d i s t r i b u t i o n of sodium and potassium inside and outside the c e l l which i n turn would r e s u l t i n the development of an e l e c t r i c p o t e n t i a l across the c e l l membrane. During cardiac arrhythmias a large e f f l u x of potassium has been shown to occur i n experimental animals given toxic doses of epinephrine (Melville et a l . , 19 55; Daniel et a l . , 1957) or cardiac glycosides (Holland and Dunn, 1954; Sarnoff et a l . , 1963; Tuttle et a l . , 1962). The deamination of taurine to i s e t h i o n i c acid was said to release a charged 167 anion group which served to retard or prevent the e f f l u x of c e l l u l a r potassium which accompanied cardiac arrhythmias. There are a number of problems with t h i s proposition: (1) Isethionic acid was said to act s o l e l y by a t t r a c t i o n of cations. Therefore, a stoichiometric r e l a t i o n s h i p would exis t between the excess cations retained and the i s e t h -i o n i c acid produced. However, even i n Read and Welty's work only a small f r a c t i o n of the taurine pool (1.4%) was claimed to be converted to i s e t h i o n i c acid over a 30 minute period (Read and Welty, 1962). (2) The mechanism for potassium retention i n the c e l l was said to be by charge n e u t r a l i z a t i o n . The changes i n membrane poten t i a l due to the presence of a negatively charged species (isethionic acid) i n the myocardial c e l l should not-affect the selective permeability to ions. (3) The hypothesis also overlooks the necessity of potassium flux across the c e l l membrane for e x c i t a t i o n -contraction coupling to occur. Evidence i n the l i t e r a t u r e on the time course of the action of taurine i n the heart muscle (Read and Welty, 1965; Chazov et a l . , 1974) indicated an e f f e c t of taurine per se and not the e f f e c t s of a slowly produced metabolite. I therefore, next looked at actions of taurine i t s e l f i n b i o l o g i c a l preparations. 168 II TAURINE AND ION TRANSPORT Further evidence i n the l i t e r a t u r e (Dolara et a l . , 1973, 1976; Huxtable and Bressler, 1973) implied that- : in the heart .taurine had e f f e c t on calcium transport. The work of Dolara et a l . , (1973) i n perfused guinea-pig hearts and i n S.R. preparations of guinea-pig hearts (Dolara et a l . , 1976) suggested that taurine had an e f f e c t on calcium binding and transport i n these preparations/ The f i e l d of taurine physiology i s prone to contradictory r e s u l t s and i t seemed prudent to re-assess the e f f e c t s of taurine on calcium binding and transport i n heart preparations. The studies on the possible e f f e c t of taurine on ATP-dependent calcium transport i n guinea-pig heart muscle was therefore undertaken. In these studies, two events of the calcium transport process were evaluated: ATP-dependent calcium binding to the outer surface of the membrane (Maclennan and Holland, 1975) and calcium uptake i n the presence of ATP and oxalate. In the latter process, calcium oxalate has been shown to p r e c i p i t a t e within the membrane v e s i c l e (Hasselbach and Makinose, 1962)-. The calcium accumulation i n the S.R. occurs by transport of calcium through the membrane into the v e s i c l e lumen when p r e c i p i t a t i n g agents such as oxalate or phosphate are present i n the solution (Kanazawa et a l . , 1970; Tonomura, 1973;MacLennan, 1975). 169 In the mammalian heart, f u l l c o n t r a c t i l e a c t i v i t y occurs when 50-100 nmole calcium per. g wet Weight of v e n t r i c u l a r tissue are made available for binding to troponin, the calcium receptor protein of the c o n t r a c t i l e system (Katz, 1970). During each cardiac cycle at maximal c o n t r a c t i l i t y , t h i s amount of calcium must bind to and then be removed from the regulatory s i t e s of troponin. Removal of t h i s calcium from the c o n t r a c t i l e unit i s controlled by the sarcoplasmic reticulum (Langer, 1973), a membranous i n t r a c e l l u l a r structure which surrounds the myofibrils. Preparations of cardiac microsomes that are enriched i n fragmented sarcoplasmic reticulum have been shown to accumulate calcium against a 2+ concentration gradient i n the presence of ATP and Mg (Carsten, 1964; Katz and Repke, 1967). Calcium accumulation by cardiac microsomes i s coupled to ATP hydrolysis v i a a 2+ 2+ membrane-bound Ca stimulated and Mg -dependent ATPase. A stoichiometric relationship of 2:1 between the amount of calcium taken up and ATP hydrolyzed exists (Tada et a l . , 1974). The sarcoplasmic reticulum preparations have widely been used to characterize events occuring during the excitation-contraction coupling of the mammalian cardiac muscle (Tada . et a l . , 1978; Ebashi and Endo, 1968). Calcium uptake by sarcoplasmic reticulum i s c r i t i c a l to the control of the rate of relaxation of cardiac and s k e l e t a l muscle. The degree of accumulation of calcium i s also 170 believed to relate to the force of contraction of subsequent beats (Tada e t _ a l . , 1975). Results obtained on the ATP-dependent calcium uptake and binding parameters of the S.R.-enriched preparation used i n t h i s study were similar to those noted by other workers (Tada et a l . , 1974; 1976; Repke and Katz, 1972; Nayler et a l . , 1975). In separate experiments (not shown in t h i s thesis work) the k,. for calcium uptake diss c and binding of the sarcoplasmic reticulum enriched 2+ preparation was found to be 0.82 y_M Ca . This value i s 2+ l n close agreement with other studies (0.75-1.10 pM Ca ; Hicks-arid Katz, 1979; Tada et a l . , 1978). The sarcoplasmic reticulum enriched preparation used i n t h i s study i s a routine preparation used i n the laboratory where th i s work was conducted (. Katz and Reynolds, 1978 j^Katz' and Dobovicnik, 19 79;-Katz.and.Remtulla; 1978.) The homogenate preparation exhibited ATP-dependent calcium binding a c t i v i t y as well as calcium uptake i n the presence of oxalate; these a c t i v i t i e s , though, were lower than those noted i n the S.R. enriched preparations. The calcium concentration dependency (Table 5) and the time course of calcium uptake and binding were sim i l a r for both enriched S.R. and whole heart 171 v e n t r i c l e homogenate preparations (Figure 18). Similar p r o f i l e s for the decay of calcium uptake a c t i v i t y were also seen i n these preparations (Figure 19). Furthermore, cAMP-dependent protein kinase was found to stimulate calcium uptake a c t i v i t y i n whole heart v e n t r i c l e homogenate preparations to the same extent (approximately 170% of the a c t i v i t y seen i n the absence of cAMP-dependent protein kinase) to that observed i n the enriched S.R. preparation (Table 6, Tada et a l . 19 74). Neither of these preparations exhibited enhancement of calcium binding a c t i v i t y i n the presence of cAMP-dependent protein kinase (results not shown; Tada et a l . , 1974; 1976). It i s possible that calcium uptake a c t i v i t y noted i n the whole heart homogenate i s due mainly to sarcolemmal membrane v e s i c l e s . I t has been reported that cAMP-dependent protein kinase stimulates sarcolemmal membrane calcium uptake i n the presence of oxalate (Sulakhe et a l . , 1976). 172 In the present study, i t was demonstrated that taurine (5-50 mM) did not s i g n i f i c a n t l y a f f e c t ATP-dependent calcium transport i n guinea-pig cardiac v e n t r i c l e homogenates or i n cardiac preparations enriched i n sarcoplasmic reticulum. Various parameters of calcium uptake and binding were examined; Taurine (20 mM) was found to have no s i g n i f i c a n t e f f e c t on either calcium uptake or binding at the various free calcium concentra-tions (0.5 to 100 y_M) or incubation times (0.5 to 20 minutes) studied. Taurine, either i n homogenate prepara-tions or i n the cardiac preparations enriched i n S.R. did not a f f e c t the enhancement of calcium transport produced by c y c l i c AMP-dependent protein kinase. Lack of an e f f e c t on thi s system indicates that taurine does not act as a modulator of calcium transport through t h i s c y c l i c AMP-mediated pathway. Recently, Schaffer et a l . (19 78a)reported that the p o s i t i v e inotropic e f f e c t of taurine was not mediated by changes i n c y c l i c nucleotide l e v e l s . These workers used standard working perfused rat heart preparations as described by Neely et a l . (1967); Perfusion with both taurine and epinephrine caused a rapid increase i n cAMP levels to the same extent as that observed i n the presence of epinephrine alone. 17:3 Entman et a l . (19 77) recently reported that taurine had no e f f e c t on calcium transport i n canine cardiac S.R. preparations. These workers used the spectrophotometric murexide dye technique for the measurement of calcium transport 45 whereas i n our studies CaC^ and the m i l l i p o r e f i l t e r a t i o n technique were u t i l i z e d . Present studies thus confirm those of Entman et a l . (1977) using both an S.R. enriched and crude homogenate preparation of heart tissue i n a species i n which the pharmacological e f f e c t s of taurine i n cardiac tissue have been noted (Guidotti et a l . , 19 7-1; D i e t r i c h and Diacono, 1971). The p o s s i b i l i t y that taurine may a l t e r calcium uptake or binding i n c e l l u l a r organelles (Sarcolemmal membrane, mitochondria) other than S.R. tends ...to be ruled out by the lack of an e f f e c t on calcium uptake or binding i n the homogenate preparations used i n t h i s study. Huxtable and Bressler (1973) reported that calcium uptake by S.R. is o l a t e d from rat s k e l e t a l muscle could be increased by the i s o l a t i o n of the S.R. i n 15 mM taurine. Exposure of the S.R. to taurine throughout"the i s o l a t i o n procedure also resulted i n an increased y i e l d of sarcoplasmic reticulum. In our present studies, i t was observedtthat when cardiac microsomal preparations enriched i n S.R. were stored i n -the absence of 40% sucrose at 4°C, the calcium accumulating capacity decreased rapidly. In thi s study, using these conditions, taurine had no e f f e c t on the decay process on 174 eithe r preparation. The lack of. an e f f e c t of taurine on 2+ t h i s decay i n Ca -transport was also noted by Entman et a l . , (1977) under d i f f e r e n t experimental conditions. The results obtained i n these present studies d i f f e r from those of Huxtable and Bressler (1973). A number of possible reasons for t h i s discrepancy are apparent. F i r s t l y , these present studies employed cardiac muscle preparations and those of Huxtable and Bressler, s k e l e t a l muscle. There are a number of anatomical and e l e c t r o -physiological differences between sk e l e t a l and cardiac ti s s u e s : Compared to s k e l e t a l muscle, the heart c e l l i s smaller, has a slower rate of contraction and has a less 3 extensive sarcoplasmic reticulum. Secondly, i t should also be noted that the i s o l a t i o n techniques for S.R. and the incubation conditions for measuring calcium accumulation were d i f f e r e n t i n these present studies compared to those used by Huxtable and Bressler. Thirdly, there was a difference i n the animal species used i n these two studies. The present experiments, using these same conditions should therefore be repeated i n s k e l e t a l muscle i n order to v e r i f y the results of Huxtable and Bressler (1973). Guinea-pig heart muscle preparations were used i n the present studies, because most of the previous experiments on the cardiac e f f e c t s of taurine were observed i n t h i s animal species ( D i e t r i c h and-Diacono, 1971; G i o t t i and 175 Guidotti, 1969; Guidotti et a l . 1971; Polara et a l . , 1973; 1976). Microsomal preparations enriched i n S.R. used i n t h i s present study have previously been used by other investigators to determine the i n - v i t r o e f f e c t s on calcium transport a c t i v i t y of a number of agents XCaffeine, verapamil, lanthanum, ionophores, epinephrine, glucagon, cardiac glycosides; Nayler et a l . 19 75; Katz et a l . , 19 77; Katz and Repke, 1973. Entman et a l . , 1969a; 1969b; 1973; Lee and Choi, 1966; Kirchberger et a l . , 1972; Tada et a l . , 1978, Weller and Laing, 1979). Similar preparations have been used successfully i n i d e n t i f y i n g S.R. abnormalities i n congestive heart f a i l u r e (Harigaya and Schwartz, 196 9; Murr, et a l . , 1970) and ischemia (Lee et a l . , 1967; Schwartz et a l . 1973). We have recently reported (Katz and Remtulla, 19 78) that ".a phosphodiesterase activator protein i s o l a t e d from bovine brain stimulated calcium transport i n s i m i l a r microsomal preparations enriched i n S.R. i s o l a t e d from canine hearts. More recently, Chubb and Huxtable (1978b)reported 2+ that taurine (20 mM) had no e f f e c t on either Ca -binding 2+ 2+ or (Ca + Mg )-ATPase a c t i v i t y i n S.R. preparations i s o l a t e d from hearts of normotensive Wistar and Okamoto spontaneously hypertensive rats. These workers commented that t h e i r findings do not agree with those of Dolara et a l . (19 76) who observed that taurine increased the calcium content of guinea-pig cardiac sarcoplasmic reticulum. I t was argued that the 176 discrepancy .in these results was due to differences i n the method of i s o l a t i o n of S.R. i n the animal species used and i n the techniques used for the measurement of calcium transport. However, Chubb and Huxtable (1978b) did not point out that t h e i r own results were at variance with those reported e a r l i e r i n studies using rat s k e l e t a l muscle sarcoplasmic reticulum preparations (Huxtable and Bressler, 1973). In summary, the results obtained i n t h i s thesis c l e a r l y show that taurine does not a f f e c t ATP-dependent calcium transport i n either a microsomal preparation enriched i n S.R. or i n a crude homogenate preparation containing a number of c e l l u l a r organelles that might be implicated i n a taurine e f f e c t . In t h i s study as well, no e f f e c t of taurine was noted on the decay of calcium accumulating capacity i n either preparation;. These results corroborate those of Entman et a l . (19 77) and have recently been substantiated by Chubb and Huxtable (1978). Chovan et a l . (1979X'.-ha-ve recently reported that taurine (10 mM) increased calcium binding to rat heart sarcolemmal membranes. This report d i f f e r s from the present studies on guinea-pig heart preparations i n that the calcium binding studies by Chovan were carr i e d out i n the absence 177 of ATP. The p o s s i b i l i t y that taurine may exert i t s ef f e c t s i n mammalian c e l l s by a l t e r i n g the passive (ATP-independent) transport of ions was evaluated independently i n this thesis work and has been reported elsewhere (Remtulla et a l . , 1979). The e f f e c t of taurine on ATP-independent calcium transport was studied i n rat brain synaptosomal preparations. Cardiac sarcoplasmic reticulum v e s i c l e s are generally recognized as 'leaky membranes'.it was therefore not possible to use these preparations for the study of the passive permeability to ions. Rat brain synaptosomes have previously been used for measurement of permeability to ions and other substances (Ling and Abdel-Latif-, 1968; Escueta and Appel, 1969; Keen and White, 1970). Changes i n calcium permeability due to the phosphory-l a t i o n of the synaptosomal membrane has also been reported (Weller and Morgan, 19 77). Taurine has been implicated in. a r o l e i n ion flux at the synaptic terminal level(Kuriyama et a l . , 1978; Lahdesmaki and Pajunen, 1977). I t was there-fore appropriate to use synaptosomes as a model system to study the possible e f f e c t of taurine on passive ion transport i n mammalian tissues. Before attempting to determine the permeability of the synaptosomes to ions i t was f i r s t necessary to ensure that the preparation was i n the form of sealed v e s i c l e s (Keen and White, 1970; Koch, 1961, Tedeschi and Harris, 1955). 178 Such vesic l e s should behave as osmometers and obey Boyles and van't Hoff's law; that i s , t h e i r volumes should be inversely proportional to the osmotic strength of the medium i n which they are suspended. The volume of p a r t i c l e s i n suspension i s inversely proportional to the o p t i c a l extinction of the suspension since smaller p a r t i c l e s scatter more l i g h t . I n i t i a l experiments, indicated that the E^2u °^ synaptosomes suspended i n a solution of Na2SC>4 increased with the strength of the solution and that when 1/E 52Q w a s plotted against l/Na 2S0 4 -a l i n e a r relationship was found (figure 21). These results confirm the observations of Keen and White (19 70) and show that the preparations of synaptosomes used behave as osmometers and thus are i n the form of sealed v e s i c l e s . Synaptosomal preparations s i m i l a r to those used in the present work have previously been used to measure the permeability to Na + (Ling and Abdel-Latif, 1968), K + 2+ (Escueta and Appel, 1969)and Ca (Weller and Morgan, 1977) and the movement across the synaptic membrane of neuro-trasmitter substances such as noradrenaline (Colburn et a l . , 1968), 5-hydroxytryptamine (5-Ht) (Bogdanski et a l . , 1968), choline (Marchbanks, 1968) and GABA (Weinstein et a l . , 1965). Keen and White (1970) used th i s technique to 179 measure the permeability of synaptosomes to various ions. They observed that acetate ions were more permeable than chloride ions; acetate s a l t s of Na + and K + were therefore used i n these present experiments. It was found that there was no e f f e c t of taurine (20 mM) on the permeability of the synaptosomal membranes suspended i n solutions of sodium and potassium acetate (100 to 200 mM). Therefore, taurine did not a f f e c t the permeability of synaptosomes to Na + or K + ions. Lahdesmaki and Pajunen (1977), using a d i f f e r e n t technique, reported a reduced outflow of Na and K ions from synaptosomes i n the presence of 5 mM taurine. Their experiments were performed i n sodium- and potassium-free . . medium containing choline, calcium and magnesium. I t i s possible that t h i s e f f e c t of taurine was secondary to an e f f e c t on the permeability of calcium. The possible e f f e c t of taurine on the passive permeability of calcium i n t h i s preparation was therefore evaluated. I n i t i a l experiments showed no detectable decrease i n E ^ Q o n suspension of synaptosomes i n solutions of calcium acetate i n d i c a t i n g that the permeability of 2+ . . Ca was too low to be measured by t h i s technique. The rate of calcium permeability i n the synaptosomes. was there-45 2+ . . . fore determined using Ca as a monitor since t h i s was a more sensitive technique. Weller and Morgan (1977) have 180 also employed synaptosomes for the measurement of calcium ion permeability. They reported a maximum calcium uptake 2+ of 2.0 nmoles Ca /mg protein af t e r 20 minutes of incubation. The synaptosomal preparations used i n t h i s study were found to be more active i n that maximal calcium uptake (3.5 nmoles'/mg protein) was reached ^after 4 5 minutes incubation i n the absence of taurine. In these experiments i t was demonstrated that taurine had an i n h i b i t o r y e f f e c t on both calcium uptake and release i n synaptosomal prepar-ations suspended i n isot o n i c sucrose media. Similar 45 2+ results have been reported on the release of Ca from preloaded synaptosomes by Kuriyama et al.,(197 8). These workers, however, did not show any s i g n i f i c a n t e f f e c t of taurine on calcium uptake. The discrepancy i n the calcium uptake results of t h i s study and that of Kuriyama et a l . , (1978) could be due to the differences i n the experimental procedures u t i l i z e d ; The calcium-uptake described by Kuriyama was an ATP-dependent process. These studies can be more appropriately compared to studies on ATP-dependent calcium uptake and binding to microsomal preparations enriched i n S.R. described e a r l i e r i n t h i s thesis and . .elsewhere (Remtulla et a l . , 1978) . In these studies as well, we found n o ; e f f e c t of taurine on either ATP-dependent calcium uptake or calcium binding. In the present studies i n brain synaptosomes, the i n h i b i t o r y e f f e c t of taurine on calcium uptake was 181 observed to be dose dependent at taurine concentrations greater than 10 mM. Lower taurine concentrations (0.5 to 5.0 mM) had no e f f e c t . Hue et al.,(1978) i n studies on the insect central nervous system also demonstrated that taurine at concentrations lower than 10 mM had no e f f e c t on the s e n s i t i v i t y of post -synaptic neurotransmission. I t should be noted that i n these experiments, choline chloride i n concentrations of 10 mM or greater produced a marked i n h i b i t i o n of calcium uptake. This i n h i b i t i o n was greater than that observed for equimolar concentrations of taurine.Choline chloride was used to control possible t o n i c i t y changes caused by added concentrations of taurine. Measurement of the osmolality of the controls i n the absence of choline and those containing taurine were not observed to be s i g n i f i c a n t l y different.Although the mechanism of t h i s e f f e c t of choline was not further i n v e s t i -gated i t should be stated that choline cannot be substituted for other ions i n t h i s or s i m i l a r studies as i t markedly i n h i b i t s ion movements i n i t s own right. This finding i s i n agreement with Jones et a l . , (1977) who reported that choline ions (100 mM) 2+ had an i n h i b i t o r y e f f e c t on the rate of Ca -uptake i n cardiac S.R. membrane v e s i c l e s . In these present studies, i t was also shown that homotaurine, $-alanine and GABA i n h i b i t e d calcium uptake i n synaptosomal preparations i n the same concentration as that observed to produce the i n h i b i t o r y e f f e c t of taurine. This indicates that the i n h i b i t o r y e f f e c t on calcium 182 uptake i n these preparations was not s p e c i f i c to taurine. Byington (thesis, South Dakota) i n 1964 reported s i m i l a r r e s u l t s ; homotaurine, (3-amino propanesulfonic acid), the three carbon analogue of taurine and a close s t r u c t u r a l analogue of y -aminobutyric acid (GABA) , and taurine were found to abolish digoxin-induced cardiac arrhythmias. Taurine and homotaurine are also known to be potent i n h i b i t o r s of impulse transmission i n stretch receptor neurons of cray f i s h (McLennan and Hagen, 1963) and i n mammalian neurons (Curtis and Watkins; 1961). They also act as depressants, causing both loss of muscle tone and gross incoordination, when injected i n t r a v e n t r i c u l a r l y into the brain of mice (Crawford, 1963). A number of studies, reviewed i n d e t a i l by Curtis and Watkins (196 0; 196 5) and Usherwood (19 78) indicate that GABA i s a physiological i n h i b i t o r of impulse transmission i n the central nervous system of both vertebrates and invertebrates. Kaczmarek (19 76) has reviewed and compared the evidence for the role of taurine and GABA as neuro-transmitters i n the brain. In vertebrate and invertebrate preparations i t has been shown that GABA and taurine mimic the action of one another (Krnjevic^and P u i l / .1976. ; Enna-and Snyder, 1975; Edward and Ku f f l e r , 1959). From the res u l t s obtained i n th i s present study and from previous studies, one might suggest that the mechanism underlying the e f f e c t of taurine, GABA and homotaurine on'excitability of neuronal 183 and cardiac tissues i s common to a l l . Taurine receptor s i t e s have recently been reported to be present i n synaptosomal preparations (Lahdesmaki et a l . , 19 77) and i n heart v e n t r i c u l a r sarcolemmal preparations (Kulakowski et a l . , 1978). Lahdesmaki et a l . (1977) have reported that taurine binding to synaptosomes was i n h i b i t e d by hypotaurine, 3 -alanine and GABA. The binding s i t e s appear to have a s t r i c t requirement for a p a r t i c u l a r chemical structure as i n the present study only those amino acids which were chemically related to taurine were found to have i n h i b i t o r y e f f e c t s on the passive permeability to calcium ions. I t was also seen that these same compounds i n h i b i t e d calcium uptake to the same extent as taurine suggesting that t h i s e f f e c t may be due to the binding of these compounds to the taurine receptor s i t e s . Chubb and Huxtable (1978b) perfused rat heart for 45 15 minutes with Krebs-Henseleit . buffer containing 2.5 mM Ca i n the absence or presence of 8 mM taurine followed by washout with a calcium-free medium. I t was found that the amount of calcium washed from the heart, and that remaining following washout, was s i g n i f i c a n t l y greater i n the taurine-treated hearts. They interpreted these r e s u l t s to indicate that taurine increases the amount of calcium taken up into the heart. In the same report (Chubb and Huxtable, 1978b)taurine was found to have no e f f e c t on either sarcoplasmic 184 2+ 2+ 2+ r e t i c u l u m A T P - d e p e n d e n t Ca - b i n d i n g o r (Ca +Mg )-AT P a s e a c t i v i t y . T h e s e d a t a t h u s i n d i c a t e t h a t t h e t a u r i n e e f f e c t on c a l c i u m a c c u m u l a t i o n i n t h e h e a r t was n o t due t o a n e f f e c t on t h e S.R. c a l c i u m pump. More r e c e n t l y , C h o v a n e t a l . (19 79) r e p o r t e d t h a t t a u r i n e (10 mM) i n c r e a s e d c a l c i u m b i n d i n g t o l o w a f f i n i t y s i t e s i n i s o l a t e d r a t h e a r t s a r c o l e m m a l membranes. T a u r i n e was a l s o shown t o a n t a g o n i z e t h e i n h i b i t i o n o f c a l c i u m b i n d i n g t o t h e s a r c o l e m m a c a u s e d b y b o t h v e r a p a m i l a n d l a n t h a n u m . T h e s e r e s u l t s d i f f e r f r o m t h e p r e s e n t s t u d i e s on g u i n e a p i g h e a r t m u s c l e p r e p a r a t i o n s ( R e m t u l l a e t a l . , 1978) w h e r e n o e f f e c t o f t a u r i n e o n c a l c i u m b i n d i n g was n o t e d . T h e s e l a t t e r s t u d i e s w e r e done i n t h e p r e s e n c e o f ATP, w h e r e a s t h o s e o f C h o v a n e t a l . (19 79) w e r e done i n t h e a b s e n c e o f ATP. B o t h t h e r e p o r t o f Chubb a n d H u x t a b l e 1978b) and t h a t o f C h o v a n e t a l . (19 79) a g r e e w i t h t h e s e p r e s e n t s t u d i e s o n b r a i n s y n a p t o s o m e s . I n a l l t h e s e s t u d i e s a t a u r i n e e f f e c t o n c a l c i u m b i n d i n g o r p e r m e a b i l i t y i n v o l v e d a n A T P - i n d e p e n d e n t p r o c e s s . The p r e c i s e mode o f a c t i o n o f t a u r i n e i n t h e r e g u l a t i o n o f p a s s i v e c a l c i u m t r a n s p o r t i n mammalian t i s s u e s r e m a i n s t o be e l u c i d a t e d . S e v e r a l t h e o r i e s h a v e e v o l v e d . H u x t a b l e ( 1 9 7 6 a ) s u g g e s t e d t h a t t a u r i n e , a 185 stable zwitterion with a high dipole moment and present i n heart c e l l s i n large amounts, interacts with the zwitterion phospholipid structure of membranes and cause a conformational change by virtue of 1 s t a b i l i z i n g charge separation 1. I t was further suggested that due to membrane conformational changes, the ion flux and cation a f f i n i t y would be altered. Kulakowski, et a l . (1978) i d e n t i f i e d taurine receptor binding s i t e s i n the cardiac v e n t r i c u l a r sarcolemma. They found that taurine binding to these receptors e x h i b i t p o s i t i v e cO-operativity. However, they suggested that taurine binds to protein receptors rather than interacts with membrane phospholipids as suggested by Huxtable (19 76a). More recently, the same group of workers (Chovan et a l . , 19 79) showed that membrane f l u i d i t y changes due to taurine binding could not be detected using the spin l a b e l ESR probe 2N14. The p r o b a b i l i t y that extremely; l o c a l i z e d membrane s t r u c t u r a l changes or protein-protein i n t e r a c t i o n not a f f e c t i n g membrane f l u i d i t y were occuring was not ruled out by these ESR probe observations. These results of Chovan et a l . (1979), however, suggest that close association exists 2+ between taurine and Ca binding s i t e s . Further work i s necessary to understand the physio-chemical properties of the taurine-receptor s i t e s i n mammalian tissues. 186 I t i s w e l l known t h a t c a l c i u m p l a y s an i m p o r t a n t r o l e i n t h e r e g u l a t i o n o f e x c i t a b i l i t y o f n e u r o n a l t i s s u e . A d e c r e a s e i n t h e c a l c i u m p e r m e a b i l i t y o f t h e s y n a p t i c membrane c o u l d a l t e r t h e c a l c i u m c o n c e n t r a t i o n i n t h e s y n a p t i c t e r m i n a l and e f f e c t n e u r o t r a n s m i t t e r r e l e a s e ( H u b b a r d , 1970; K a t z a n d M i l e d i , 1 9 7 0 ) . The d e p e n d e n c e o f t r a n s m i t t e r 2+ r e l e a s e upon Ca i n f l u x h a s a l s o b e e n n o t e d i n s y n a p t o s o m a l p r e p a r a t i o n s i n - v i t r o ( L e v y e t a l . , 1974) . K u r i y a m a e t a l . ,. (1976; 1978) h a s p r o v i d e d e v i d e n c e t h a t i n b o t h c e n t r a l and p e r i p h e r a l n e r v o u s s y s t e m , t a u r i n e a c t s as a m o d u l a t o r o f membrane e x c i t a b i l i t y b y i n h i b i t i n g t h e r e l e a s e o f o t h e r n e u r o t r a n s m i t t e r s s u c h a s e p i n e p h r i n e and a c e t y l c h o l i n e . R e u t e r (1972) showed t h a t l o n g - t e r m c h a n g e s i n membrane p e r m e a b i l i t y t o c a l c i u m c o u l d c h a n g e t h e p o t e n t i a l g r a d i e n t a c r o s s t h e membrane l e a d i n g t o h y p e r p o l a r i z a t i o n . T.he r e s u l t a n t h y p e r p o l a r i z a t i o n w i l l r e d u c e t h e p o t a s s i u m c u r r e n t , a n d t h e e f f i c a c y o f s y n a p t i c a c t i v a t i o n . T a u r i n e h a s c o n s i s t e n t l y b e e n shown t o p r o d u c e a h y p e r p o l a r i z a t i o n ( C u r t i s a n d J o h n s t o n , 1974; K r n j e v i c , 1974) i n e x c i t a b l e membranes, p r o b a b l y by m o d i f y i n g membrane p e r m e a b i l i t y t o p o t a s s i u m a n d c h l o r i d e i o n s ( G r u e n e r e t a l . , 1 9 7 6 ) . I t i s c o n c e i v e a b l e t h a t t h e a n t i c o n v u l s a n t a c t i o n o f t a u r i n e r e p o r t e d i n e p i l e p s y ( B a r b e a u and D o n a l d s o n , 1974; M u t a n i e t a l . ,• -19 74a; 1974b) m i g h t be a r e s u l t o f 187 changes i n the i n t r a c e l l u l a r calcium concentrations. This suggestion i s supported by the observation;that pretreatment of e p i l e p t i c animals with sodium edetate (a calcium chelator) s i g n i f i c a n t l y diminished the protective e f f e c t of taurine against pentylenetetrazol induced seizure (Izumi et a l . , 19 7 5 ) . Taurine may possibly be involved i n the restoration of the normal d i s t r i b u t i o n of calcium i n membrane compartments. Hagins and Yoshikami (19 74) have suggested, that calcium bound to the outer segment of r e t i n a l photoreceptors i s released on ill u m i n a t i o n and diffuses to the outer membrane, thus modifying permeability to sodium. Redistribution of calcium i n i t s o r i g i n a l state occurs during dark adaptation. Pasantes-Morales et a l . (19 78) have shown that taurine (30 mM) affects calcium uptake and d i s t r i b u t i o n i n the..retina. I t thus might be considered that taurine affects ion conductance i n photoreceptors and thereby exerts an influence on synaptic function at the photoreceptor terminal. These present results of a taurine e f f e c t on passive calcium transport i n synaptosomes corroborate t h i s conclusion. These present studies and those reviewed i n this thesis imply that the possible cardiac action of taurine are related to alterations i n cardiac calcium permeability. Evidence for an e f f e c t of taurine on changes i n cardiac calcium levels has been reported. I t has been shown that 188 myocardial calcium i s markedly elevated i n cardiomyopathic hamsters (Bajusz et a l . , 1969). McBroom and Welty (1977) showed that cardiomyopathic hamsters given 0.1 M taurine i n tap water ad libitum did not manifest the dramatic calcium increases observed i n the hearts of the untreated control counterparts; the heart taurine content, plasma calcium and plasma taurine concentration were found to be no d i f f e r e n t i n the control and taurine treated groups. I t i s possible that the exogenous administration of taurine a l t e r s both taurine and calcium k i n e t i c s i n the heart muscle of cardiomyopathic hamsters such that the rate of turnover i s increased, thereby augmenting the tissue flux without increasing the cardiac pool size of either taurine or calcium. From the evidence present i n the l i t e r a t u r e and the results obtained i n t h i s thesis work, i t i s not possible to ascertain whether taurine i s involved i n only one or i n several aspects of mammalian physiology. However, on the basis of the known ef f e c t s of taurine i n excitable membranes, one may consider that the' -main:action "of: taurine i n mammalian tissues i s related to calcium ion fluxes. I t has been found that taurine a f f e c t s calcium permeability i n a number of tissues (synaptosomes,Remtulla et a l . , 1979; heart muscle, Chubb and Huxtable, 1978b ; and r e t i n a , -a 1 Pasantes-Morales et a l . , 1978). Regulation of the i n t r a -c e l l u l a r calcium concentration i s recognized as an 189 important determinant of such c e l l u l a r processes as : c e l l d i v i s i o n , c e l l u l a r e x c i t a b i l i t y , neurotransmitter and hormone release and muscle contraction. A role for taurine i n the modulation of calcium permeability might account for i t s possible e f f e c t s on a number of these systems. CONCLUSIONS The purpose of t h i s thesis work has been to evaluate the possible mode of action of taurine i n mammalian physiology. The evidence presented indicates that t h i s mode of action does not involve the bioconversion of taurine to i s e t h i o n i c acid as was o r i g i n a l l y suggested. I t was further shown that the reported cardiac e f f e c t s of taurine do not involve an e f f e c t on ATP-dependent calcium transport. However, evidence was presented that taurine i s involved i n the passive (ATP-independent) movement of calcium i n a synaptosomal preparation. This e f f e c t together with the demonstrated e f f e c t on calcium permeability i n perfused heart and r e t i n a l preparations (Chubb and Huxtable, 1978b; Pasantes-Morales et a l . , 1978) suggests that an important physiological role for taurine i n mammalian tissues i s related to calcium ion translocation. 190 B I B L I O G R A P H Y 1 9 1 t The numbers i n p a r a n t h e s e s r e f e r to the pages i n the t h e s i s , where the a u t h o r ( s ) i s c i t e d . Agrawal, H.C, Havis, J.M. and Himwick, W.A. ( 1 9 6 8 a ) : Maturational changes i n amino acids i n CNS of d i f f e r e n t mammalian species. In: Recent advances i n  biolog!ca1 Psychiatry, v o l . 10 edited by J. Wortis, pp. 258-265. Plenum. Press, N.Y. (54)t ._-Agrawal, H.C, Davis, J.M. and Himwick, W.A. 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(1977): A possible role of the phosphorylation of synaptic membrane proteins i n the control of calcium ion permeability. Biochim. Biophys. Acta, 465: 527-534.(177,178,179). Welty, J.D. (1963): The role of taurine i n cardiac e x c i t a b i l i t y . Ph.D.Thesis: University of South Dakota.(17, 166). Welty, J.D. J r . , Read, W.O., and Shaw,. J r . , E.H. (1962) : Iolat i o n of 2-hydroxyethanesulfonic and (isethionic acid) from dog heart. J. B i o l . Chem., 237: 1160-1161. (15,16,124,15 3,157,15 8^ Welty, J.D., and Read, W.O. (1964): Studies on some cardiac e f f e c t s of taurine, J. Pharmac. Exp. Ther., 144: 110-115.(36,166). 224 Weinstein, H., Varon, S., Muhleman, D.R., and Roberts, E. (1965), A carrier-mediated transfer model for the accumulation of 1 4 c - Y - a m i n o b u t y r i c acid by subcellular brain p a r t i c l e s . Bid chem. Pharmaco1., 14: 273-288. (178). Winegrad, S., and Shanes, A.M. (1962): Calcium f l u x and c o n t r a c t i l i t y i n guinea-pig a t r i a . J. Gen.Physiol., 45: 371-385. (42). Wonnacott, J.H., and Wonnacott, R.J. (1977): Introductory S t a t i s t i c s , 3rd ed i t i o n , p. 215. John Wiley & Sons, N.Y. (102). Yamaguchi, K., Sakakibara, S., Abamizu, J., and Ueda, I. (19 73): Induction and activation of cysteine oxidase of rat l i v e r . I I . Measurement of cysteine metabolism i n vivo and the activation of i n vivo a c t i v i t y of cysteine oxidase. Biochim. Biophys. Acta 29 7: 48-59. (62). Yonada, K., Toyama, S., Yosuda, M., and Soda, K. (19 76); P u r i f i c a t i o n and c r y s t a l i z a t i o n of b a c t e r i a l W-aminoacids-pyruvate aminotransferase. FEBS Lett., 71: 21-24. (162). 225 APPENDICES 226 L i f e Sciences V o l . 20, pp. 2029-2036, 1977 Printed In The U.S.A. Pergamon Press ANALYSIS OF ISETHIONIC ACID IN MAMMALIAN TISSUES Mohamed A. Remtulla, Derek A. Applegarth, Donald G. Clark and Ian H. Williams Departments of P a e d i a t r i c s , Pathology and Chemistry, The U n i v e r s i t y of B r i t i s h Columbia, Biochemical Diseases Laboratory, Children's H o s p i t a l and Government of Canada A g r i c u l t u r e Research Station, Vancouver, B r i t i s h Columbia (Received i n f i n a l form May 17, 1977) SUMMARY A g a s - l i q u i d chromatographic assay has been developed to measure i s e t h i o n i c acid a f t e r methylation with diazomethane. The i d e n t i t y of the products of methylation has been confirmed by mass-spectro-metry and nuclear magnetic resonance spectroscopy. The method was used to measure i s e t h i o n i c acid i n r a t heart, dog heart and r a t b r a i n . The assay was va l i d a t e d by measuring i s e t h i o n i c acid on squid axoplasm. We have been able to detect only trace amounts of i s e t h i o n i c acid i n r a t b r a i n (0.2 mg/lOOg) and r a t heart (0.1 mg/ lOOg). None was found i n dog heart. I s e t h i o n i c a c i d , 2-hydroxy-ethane s u l f o n i c a c i d , i s the deaminated analogue of taurine (1). I t s presence i n b i o l o g i c a l m a terial was f i r s t reported by Koechlin (2) who found that i t was the major anion of the axoplasm from the squid giant axon. He suggested that ISA might i n d i r e c t l y be responsible f o r the production of e l e c t r i c a l phenomena i n the nerve. Welty et a l . (3) proposed that taurine was the precursor of ISA. These workers apparently i s o l a t e d sub-s t a n t i a l q u a n t i t i e s of ISA from dog and r a t heart t i s s u e s , by a gravimetric method inv o l v i n g the c r y s t a l l i z a t i o n of ISA as i t s sodium s a l t from a hot aqueous extract of dog heart or r a t heart. They l a t e r demonstrated the con-v e r s i o n of 35g_taurine to ^S-ISA by dog heart s l i c e s (A). Later Peck and Awapara (5) reported that very small amounts of I s e t h i o n i c acid could be formed from l s o t o p i c a l l y l a b e l l e d taurine i n r a t heart and b r a i n t i s s u e s . Other workers (6,7) have suggested that the conversion of taurine to ISA i n myocardial c e l l s f a c i l i t a t e s the r e t e n t i o n of i n t r a c e l l u l a r calcium or potassium ions. The major d i f f i c u l t y of studying the function of ISA has been the lack of a good a n a l y t i c a l procedure f o r i t s detection and q u a n t i t a t i o n . Methods used i n the past to detect ISA do not o f f e r much accuracy or s e n s i t i -v i t y and some apparently promising methods have never been published i n f u l l (8,9). We therefore report here an a n a l y t i c a l method to measure ISA. MATERIALS AND METHODS For analysis of r a t t i s s u e s , we used Wistar r a t s weighing approximately 200g. Animals were s a c r i f i c e d by a sharp blow to the head. Hearts and brains were promptly excised and the t i s s u e s rinsed l n normal s a l i n e , b l o t t e d on Whatman Correspondence to: Derek A. Applegarth, Biochemical Diseases Laboratory, Children's H o s p i t a l , 250 West 59th Avenue, Vancouver, B.C. Canada V5X 1X2. 2029 227 2030 Is e t h i o n i c ' A c i d In Mammalian Tissues V o l . 20. No. 12, 1977 HI f i l t e r paper, and Immediately frozen i n small p l a s t i c v i a l s l n l i q u i d n i t r o -gen. This process required l e s s than ten minutes per r a t . For analysis of dog heart, animals were obtained from the Department of Physio-logy at The U n i v e r s i t y of B r i t i s h Columbia, a f t e r having been used f o r open heart surgery. The dogs were s a c r i f i c e d with 152 potassium chlo r i d e (10 ml) and the heart stored at -20 degrees before use. Samples of the giant axon of squid (Loligo p e a l l i ) were obtained from Dr. F.C.G. Hoskin of the Department of Biology, I l l i n o i s I n s t i t u t e of Technology, Chicago, I l l i n o i s . I s o l a t i o n of ISA from Heart and Brain Tissues: 5g samples of pooled brain or heart tissues were used f o r experimentation. The heart t i s s u e was minced before being used and then divided into two equal portions of 2.5g each. To one por-t i o n was added 1.0 umole of sodium isethionate (Sigma Chemical Company, St. Louis, M i s s o u r i ) . The other portion was used without any a d d i t i o n . Both portions were homogenized i n 501 methanol/water (v/v), (10 ml), i n a S o r v a l l omnimixer (Ivan S o r v a l l Inc., Norwalk, Connecticut), using a t e f l o n chamber at 3/4 of the f u l l speed for f i v e minutes, followed by one minute of f u l l speed. The homogenate was transferred to a centrifuge tube and centrifuged at 3,000g for f i v e minutes. The homogenizing chamber was rinsed three times with 5 ml, 50% methanol/water (v/v), and the r i n s e washings added to the centrifuge p e l l e t which was suspended i n the r i n s i n g s o l u t i o n using a Vortex mixer. The r e s u l t i n g suspension was then centrifuged again f o r f i v e minutes and the super-natant f l u i d removed. To the combined volume of supernatants and r i n s i n g s , an equal volume of Folch solvent (chloroform:methanol, 2:1, v/v) was added. The solutions were mixed thoroughly and centrifuged to separate the l a y e r s . The upper aqueous layer was removed and evaporated to dryness l n a rotary evaporator under reduced pressure. A f t e r evaporation of the aqueous layer to dryness, 2 ml of a p u r i -f i e d cation exchange r e s i n (AG-50W-X8, 50-100 mesh, H+ form Bio Rad Laborator-i e s , Richmond, C a l i f o r n i a ) , prewashed l n methanol and suspended i n an equal volume of methanol, was added to the f l a s k . A f t e r t r i t u r a t i o n , the mixture was transferred to a 5 ml glass-stoppered c o n i c a l centrifuge tube. A f t e r c e n t r i -fugation, the methanol layer was dried i n a vacuum desiccator over sulphuric a c i d . I s o l a t i o n of ISA from Axoplasm of the Squid Giant Axon: Axoplasm from the squid giant axon was obtained i n freeze-dried form. The sample, 83mg fresh weight axoplasm, was dispersed l n deionized water using a glass homogenizer. The t u r b i d s o l u t i o n so formed was made up to a volume of 10 ml with deionized water. An a l i q u o t of the s o l u t i o n was mixed with an equal volume of absolute methanol and t h i s mixture treated with an equal volume of Folch solvent. The r e s t of the procedure was the same as that described above, f o r I s o l a t i o n of •ISA from heart and b r a i n t i s s u e s . Preparation of Standards: A suspension of Ion exchange r e s i n AG-50 (H + form i n methanol, 1 ml of r e s i n + 1 ml of methanol) was pipetted i n t o each of s i x , 5 ml glass stoppered, c a l i b r a t e d , c o n i c a l centrifuge tubes. A f t e r allowing the r e s i n to s e t t l e , the methanol layer was aspirated and discarded. To each tube was added 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml a l i q u o t s of 5 mM sodium isethionate s o l u t i o n i n water. Volume was made up to 4 ml i n each tube with methanol, and the r e s i n suspended using a Vortex mixer. A f t e r c e n t r i f u g a t i o n , the methanol layer from each tube was removed i n t o reaction v i a l s and evaporated to dryness i n a vacuum desicca-tor over sulphuric a c i d .  Abbreviations: Throughout the text, i s e t h i o n i c a c i d i s abbreviated as ISA. Nuclear magnetic resonance spectroscopy i s abbreviated as NMR. 228 V o l . 20, No. 12, 1977 I s e t h i o n i c Acid In Mammalian Tissues 2031 Methylation of the Samples and Preparation of a Standard Curve: ISA standards, or samples obtained from a ti s s u e extract, were dissolved i n 0.1 ml of a 1 mM s o l u t i o n of s a l i c y l i c acid (Sigma Chemical Company, St. Louis, Missouri) i n methanol. S a l i c y l i c a c i d was used as an i n t e r n a l standard. The v i a l s were stoppered using t e f l o n laminated discs and placed i n an ice-bath f o r f i v e minutes. Ethereal diazomethane s o l u t i o n prepared from d i a z a l d ( A l d r i c h Chemical Company, Milwaukee, Wisconsin) was introduced i n t o the v i a l s slowly, with mixing u n t i l the yellow color p e r s i s t e d . An a d d i t i o n a l three drops of diazomethane s o l u t i o n was added and the mixture was allowed to stand f o r about t h i r t y minutes i n i c e . The t o t a l volume i n the v i a l was 0.2 ml or l e s s . Where the t o t a l volume i n the v i a l was more than 0.2 ml, the excess solvent was c a r e f u l l y evap-orated under a stream of dry nitrogen and the sample remethylated f o r a further t h i r t y minute period. Methylation reactions were a l l stopped by adding one drop 502 a c e t i c acid i n water (v/v). This remethylation procedure was found not to a f f e c t the standard c a l i b r a t i o n curve f o r ISA. Gas-Liquid Chromatography: Flame Ioni z a t i o n Detection: The g a s - l i q u i d chromato-graph used was a Bendix,Model 2500, equipped with a flame i o n i z a t i o n detector. Columns were 6 f t by 4 mm (i.d.) glass U-tubes. The stationary phases used were 5% 0V-1 and 5% OV-17 (Gas Chromatographic S p e c i a l i t i e s L td., B r o c k v i l l e , Ontario) on 80-100 mesh H.P. Chromosorb W. Analyses were performed isothermally at temperatures ranging from 100 degrees C to 150 degrees C. The optimal temperature f o r 0V-1 was 115 degrees C and f o r OV-17 was 135 degrees C. N i t r o -gen was used as a c a r r i e r gas at a flow rate of 40 ml/min. Gas-Liquid Chromatography: Sulphur Detection: The g a s - l i q u i d chromatograph used f o r such experiments was a Micro Tek 220 equipped with a flame photometric detector, Model FPD 100 (Melpar Inc., F a l l s Church, V i r g i n i a ) . The column used was a "6 f t by 2 mm (i.d.) glass U-tube with 5% OV-17 on 80-100 mesh H.P. Chromo-sorb W. Nitrogen was used as the c a r r i e r gas with an i n l e t flow of 30 ml/min. Column temperature was 100 degrees C. Oxygen flow to the detector was 10 ml/min hydrogen flow was 70 ml/min and a i r flow was 30 ml/min. j Gas-Liquid Chromatography: Mass Spectrometry: The mass-spectrometer used was a H i t a c h i Perkin-Elmer, operating at an i o n i z a t i o n energy of 70 ev. in t e r f a c e d with a Varian, Model 1400 gas chromatograph. Authentic ISA and butane s u l f o n i c acid samples were analyzed on 0V-17 column a f t e r methylation with diazomethane. Chromatographic d e t a i l s were as outlined above f o r flame i o n i z a t i o n detection. Nuclear Magnetic Resonance Spectroscopy: For proton NMR spectroscopy, the methyl esters of ISA and butane s u l f o n i c a c i d were prepared i n the following manner. Five times r e c r y s t a l l i z e d sodium isethionate (100 mg) i n b o i l i n g absolute ethanol and 1-butanesulfonic a c i d sodium s a l t (100 mg) (Eastman Kodak Company, Rochester, N.Y.) were each dissolved i n methanol (1.0 ml) and the samples treated with r e s i n , and methylated as described above. A f t e r t h i r t y minutes, excess diazomethane was c a r e f u l l y blown o f f with a stream of dry n i t r o -gen and the sample completely dried down under reduced pressure over sulphuric a c i d . The NMR spectra of the reaction products, without further p u r i f i c a t i o n , were taken at 100 mHz i n a Varian HA-100 spectrometer. Samples were dissolved i n deuterated dimethylsulfoxide (Merck, Sharpe and Dohme Ltd., Canada) with tetramethyl s i l a n e being used as an i n t e r n a l standard. For the purposes of comparison, the NMR spectrum of c r y s t a l l i z e d , non-methylated ISA, also dissolved i n dimethylsulfoxide was obtained. RESULTS Chromatography of the methylated ISA on 0V-1 and on an OV-17 column i s shown i n Figure 1. Using a column of 5% 0V-1, a si n g l e peak with a retention time of 1.6 minutes was obtained f o r methylated ISA. Using a column of 5% OV-17, two 229 2032 Iset h i o n i c Acid In Mammalian Tissues V o l . 20, No. 12, 1977 peaks at re t e n t i o n times of 3.5 and 4.0 minutes were obtained. The i d e n t i t y of these two peaks was established by the use of gas chromatography mass-spectro-metry (Figure 2) and nuclear magnetic resonance spectroscopy. On OV-17 the peak area of the large peak was approximately 20 times that of the smaller peak. Interpretation of the mass-spectrometry fragmentation pattern of the two peaks obtained a f t e r OV-17 gas chromatography i s shown i n Figure 2. The large peak (Peak II) was the methylester of the ISA, while the small peak (Peak I) was the methylether, methylester of ISA. - lt«thionic A c i d \ ^ ^ S a l i c y l i c l \ P . o l I Sal icyl ic A c i d FIG. 1 Chromatographic separation of the products of methylation of i s e t h i o n i c a c i d and s a l i c y l i c acid using flame i o n i z a t i o n detection. A. The column used was a 5? OV-1 column; oven temperature 115 degrees C. B. • The column used was a 52OV-17 column; oven temperature 135 degrees C. C. The column used was a 5% OV-17 column at an oven temperature of 135 degrees C. The ordinate shows detector response. The abscissa shows re t e n t i o n time. The NMR spectra of both methylated ISA and butanesulfonic acid showed the CH3 peak of the methylester as a s i n g l e t at 3.88 ppm. A small s i n g l e t at 3.30 ppm occurring i n the spectrum of methylated ISA was assigned as the CH3 of the methylether of ISA on the basis of the known chemical s h i f t value of 3.38 ppm of the methylether s i n g l e t i n methoxyethanol (10). Integration of the spectrum indicated an approximate^ratio of methylester CH3 to methylether CH3 of 20:1. 230 V o l . 20, No. 12, 1977 I s e t h i o n i c Acid In Mammalian Tissues 2033 \0 0) > [ c o ] J L Spectrum A CHjOCM, M*thyt«it«r tMthyUthar of •••thionic A c i d rCMjCMjOC^I' 1, 1,1 m/e FIG. 2 Mass spectra.of the products of methylation of i s e t h i o n i c a c i d . A. Mass spectrum of the Peak I from Figure 1. B. Mass spectrum of Peak II from Figure 1. Experimental conditions and i n t e r -p r e t a t i o n of data are described i n the text. Figure 3 shows a t y p i c a l standard curve. When the method was used to analyze ISA l n b i o l o g i c a l samples, a small peak i n the p o s i t i o n of ISA was detected i n r a t b r a i n at a concentration of approximately 0.2 mg/lOOg of t i s s u e and i n r a t heart at a concentration of approximately 0.10 mg/lOOg t i s s u e . This value f o r r a t heart i s only an estimate because at t h i s l e v e l we are at the approximate l i m i t of s e n s i t i v i t y f o r the assay technique. We were unable to detect any ISA i n dog heart. The a n a l y t i c a l procedure was always monitored by adding ISA at concentrations of 2.0 and 0.2 umole/g of t i s s u e to duplicate a l i q u o t s of t i s s u e examined. Recovery was always between 95 and 100%. The method as described using flame i o n i z a t i o n detection i s capable of detecting ISA i n t i s s u e as a concentration of approximately 0.2 umole ISA/gram of t i s s u e extracted (approxi-mately 2.5 mg/lOOg). With the s u l f u r detector the s e n s i t i v i t y of the method was approximately 0.008 umoles/gm (0.1 mg/lOOg t i s s u e ) . In squid axoplasm, i s e t h i o n i c a c i d was found at a concentration of 150 umole per ml axoplasm. The i d e n t i t y of the peaks was confirmed by gas chromato-graphy mass-spectrometry. 2034 Ise t h i o n i c Acid In Mammalian Tissues V o l . 20, No. 12, 1977 9.0 L Hmole Isethionic Acid/Vial FIG. 3 C a l i b r a t i o n curves of methylated i s e t h i o n i c acid obtained on a column of 5% OV-17 on H.P. Chromosorb W. Operating parameters are quoted i n the text. DISCUSSION Methylation of ISA produces two compounds, a methylester and the doubly methyl-ated methylester, methylether d e r i v a t i v e . These two compounds co-elute when analyzed by g a s - l i q u i d chromatography on a column of OV-1, but can be separated on a column of OV-17 (see F i g . IB). The r a t i o of these two compounds separated on OV-17 i s approximately 20:1 with the methylester d e r i v a t i v e being predomi-nant. The r a t i o of the two peaks was i n v a r i a n t over a wide range of gas chromatographic conditions. The r a t i o was confirmed by proton NMR spectroscopy. The assignment of structures to the mass spectra of the peaks by methylation of ISA ( F i g . 2) follows the discussion of fragmentation patterns of a l k y l alkane-sulfonates by Truce et a l . (11). The parent ions {M}. were not seen i n the methylated ISA mass spectra. Truce et a l . claim that these ions are scarce f o r most of the a l k y l alkanesulfonates. However, the assignment of structures of the two peaks were strengthened by the appearance of the {M-l}t fragment f o r the methylester of ISA at m/e - 139. We found that i t was usually necessary to use two columns, one of OV-1 and the other of OV-17, and occasionally two methods of detection, flame i o n i z a t i o n and flame photometric (for s u l f u r ) to look at a n a l y t i c a l extracts of t i s s u e s . This was necessary because a l l of the t i s s u e s studied gave a peak i n the p o s i t i o n of 232 V o l . 20, No. 12, 1977 Is e t h i o n i c Acid In Mammalian Tissues > 2035 ISA i n the crude extracts of r a t and dog heart and r a t b r a i n studied when an OV-1 column was used. The peaks on OV-1 chromatography corresponded to an amount of material that would have been approximately 10-12 mg ISA/lOOg heart or b r a i n I f they had been ISA. Confirmation that t h i s peak was not ISA depended on rechromatography of the same extract on OV-17, and v e r i f i c a t i o n that t h i s peak did not contain s u l f u r . The large peak from heart and b r a i n extracts seen on OV-1 proved to contain only very small amounts of ISA when reassessed using OV-17 and the s u l f u r detector. The value that we obtained for the analysis of ISA i n the squid giant axon compares favorably with other data obtained using d i f f e r e n t , l e s s s e n s i t i v e methods where 150 umole ISA per g. axoplasm has been reported (12,13). Welty et a l . (3) quoted a f i g u r e of 42.6 mg ISA per lOOg ra t heart t i s s u e and 12.9 mg per lOOg dog heart. These amounts would have been quite e a s i l y detect-able with our method. Using the s u l f u r detector which, l n t h i s p a r t i c u l a r case, was ten times more s e n s i t i v e than the flame i o n i z a t i o n detector, only a very small peak i n the p o s i t i o n of ISA could be seen f o r r a t heart at a concentration of roughly 0.10 mg/lOOg. I n s u f f i c i e n t material was a v a i l a b l e to confirm I t s i d e n t i t y by mass spectrometry. In the case of r a t b r a i n , a peak could be seen with the s u l f u r detector at the p o s i t i o n of ISA, at a concentration of 0.2 mg/ lOOg of t i s s u e . Again, I n s u f f i c i e n t material was a v a i l a b l e to confirm that t h i s small amount of material was t r u l y ISA. We extracted as much as 400g of dog heart tissue to search for ISA. In t h i s large scale experiment we also found no ISA peak. In t h i s large scale experiment we followed exactly the procedure of Welty, Read and Shaw (3). We obtained neither the c r y s t a l s of sodium isethionate that they reported nor gas chromatographic evidence of ISA. We can not explain the difference between our r e s u l t s and those of Welty, Read and Shaw (3). The s e n s i t i v i t y for the g a s - l i q u i d chromatographic portion of t h i s experi-ment would have been approximately 0.1 mg/lOOg of heart t i s s u e . Our fin d i n g s cast doubt on theories of the mode of action of taurine which involve b i o -conversion of taurine to ISA. This work was presented at the 2nd Inte r n a t i o n a l Congress on Taurine i n Tucson, Arizona, March 1977, and a portion of i t has appeared i n abstract form (14). ACKNOWLEDGEMENTS We wish to thank the B.C. Heart Foundation f o r a grant-in-aid. We also wish to thank Mr. Greg Owen, Department of Chemistry, Simon Fraser U n i v e r s i t y f o r help i n obtaining mass spectra. REFERENCES 1. J . G. JACOBSEN and L. H. SMITH, JR, Ph y s i o l . Rev. 48 424-511 (1968). 2. B. A. K0ECHLIN, J . Biophys and Biochem. C y t o l . _1 511-529 (1955). 3. J . D. WELTY, W. 0. READ and E. H. SHAW, J . B i o l . Chem. 237 1150-1161 (1962). 4. W. 0. READ and J.D. WELTY, J . B i o l . Chem. 237 1521-1522 (1962). 5. E. J . PECK, JR, and J . AWAPARA, Biochem. Biophys. Acta 141 499-506 (1967). 6. E. I. CHAZOV, L. S. MALCHIK0VA, N. V. LIPINA, G. B. ASAF0V and V. N. SMIRN0V, C i r c . Res. 34-35 I I I - l l (1974). 7. W. 0. READ and J.D. WELTY, E l e c t r o l y t e and Cardiovascular Diseases, E. BAJUSZ, Ed. pp. 70-85, S. KARGER, Basel/New York (1965). 8. J . G. JACOBSEN, L. L. COLLINS and L. H. SMITH, JR, Nature 214 1247-1248 (1967). 9. I. LEHTINEN and R. S. PIHA, Comm. 9th Inter. Congr. of Biochem. p. 446 (1973). 10. THE SADTLER STANDARD NUCLEAR MAGNETIC RESONANCE SPECTRUM, 032M. Sadtler Research Inc., Pub. by Sadtler Research Laboratories, 3316 Spring Gardens r 233 2036 I s e t h i o n i c Acid In Mammalian Tissues V o l . 20, No. 12, 1977 Street, P h i l a d e l p h i a PA. 19104, USA (1967). 11. W. E. TRUCE, E. W. CAMPBELL and G. D. MADDING, J . Org. Chem. 32 308-317 (1967). 12. G. G. J . DEFFNER and R. E. HAFTER, Biochem. Biophys. Acta 42 200-205 (1960). 13. F. C. G. HOSKIN and M. BRANDE, J . Neurochem. 20 1317-1327 (1973) 14. D. A. APPLEGARTH, M. REMTULLA and I. H. WILLIAMS, C l i n . Res. XXIV, 646A (1976). Pediat. Res. 12: 732 (1978) 234 Letter to the Editor: Isethionic Acid and Milk M O H A M E D A. R E M T U L L A A N D D E R E K A. A P P L E G A R T H 1 3 1 Biochemical Diseases Laboratory, Childrens Hospital, Vancouver, Canada JOHN A. S T U R M A N A N D G E R A L D E. G A U L L Department of Human Development and Genetics, Institute of Basic Research in Mental Retardation, Slaten Island, and Department of Pediatrics, Mount Sinai School of Medicine of The City University of New York, New York, New York, USA We recently reported that [MS]taurine injected into lactating rats was transferred via the milk to the pups (2). In this report we noted the presence of another radioactive compound besides taurine which was present only in extracts of milk and which cochromatographed with authentic isethionic acid. The presence of this compound in milk was puzzling and we felt that it was important to determine whether or not isethionic acid was present as a natural constituent of milk. The development of a sensitive gas-liquid chromatographic assay for isethionic acid (1) allowed us to perform such measurements on rat milk samples. Samples of milk were obtained from lactating rats of the Nelson-Wistar strain, from 2 days after birth to 6 days after birth, as previously described (2). Samples of milk (0.2 ml) were freeze-dried and resuspended in 10 ml deionized water using a glass-glass homogenizer. Authentic isethionic acid (25-150 nmol) was added to some of the samples at this stage. The suspension was mixed with an equal volume of methanol (10 ml) and then treated with an equal volume of Folch solvent (20 ml), mixed thoroughly, and centrifuged to separate the layers. The aqueous layer was removed and extracted as previously described (1). Methylation with diaz-omethane in the presence of butane-sulfonic acid as internal standard (10 nmol/sample) was carried out as described (1). A thick white precipitate formed in the methylation mixture and was removed by centrifugation. The supernatant fluid was concen-trated to 20 /il under a stream of dry nitrogen and samples (3.5 /il) were injected into the gas chromatograph (Bendix 2500, ov-17 column, 130°, equipped with a flame ionization detector). Ise-thionic acid standards of 0, 25, 50, 100, and 200 nmol were used for quantification. The samples analyzed showed no trace of isethionic acid. The recovery of authentic isethionic acid added to the milk samples Copyright © 1978 International Pediatric Research Foundation. Inc. 0031 -3998/78/ 1206-0732S02.O0/0 was 100% and the sensitivity of the method was such that 20 nmol isethionic acid/ml milk could have been easily detected. In our earlier report, we noted that the radioactive compound in milk which cochromatographed with authentic isethionic acid com-prised 30-40% of the total radioactivity (the rest of which was present as taurine). The present assay could easily have detected isethionic acid present at 10% of the taurine concentration. We conclude that isethionic acid is not a significant constituent of rat milk (if indeed it is present at all). The radioactive compound which cochromatographed with authentic isethionic acid is either another compound or was produced by gut bacteria, reabsorbed, and secreted in the milk after the ip injection. Of note in this regard are the results of experiments designed to test the possible bioconversion of taurine to isethionic acid by slices of dog heart and rat heart. We found that [35S]taurine was converted in up to 10% yield to a radioactive compound which behaved chomato-graphically like isethionic acid. The same result was obtained, however, when [^ SJtaurine was added to heart slices in buffer and extracted immediately with methanol. We can detect no biocon-version of taurine to isethionic acid in heart tissue by the proce-dures described above, feras&k It is likely that the radioactive compound detected in milk may be the same or a similar chemical to the radioactive compound detected in heart slices, but it is unlikely to be isethionic acid. REFERENCES AND NOTES 1. Remtulla, M. A„ Applegarth, D. A., Clark. D. G.. and Williams, I. H.: Analysis of isethionic acid in mammalian tissues. Life Sci., 20: 2029 (1977). 2. Sturman, J. A., Rassin, D. K , and Gaull, G. E.: Taurine in developing rat brain: Transfer of ("Sjtaurine to pups via the milk. Pediat. Res., //: 28 (1977). 3. To whom correspondence should be addressed. 4. Received for publication October 5, 1977. ' Printed in U.S.A. v 2 3 5 L i f e Sciences, V o l . 23, pp. 383-390 Pergamon Press Printed i n the U.S.A. EFFECT OF TAURINE ON ATP-DEPENDENT CALCIUM TRANSPORT IN GUINEA-PIG CARDIAC MUSCLE Mohamed A. Remtulla, Sidney Katz* and Derek A. Applegarth D i v i s i o n of Pharmacology, Faculty of Pharmaceutical Sciences and Departments of Pae d i a t r i c s & Pathology, Faculty of Medicine, U n i v e r s i t y of B r i t i s h Columbia, and Biochemical Diseases Laboratory, Children's H o s p i t a l , Vancouver, B.C., Canada (Received i n f i n a l form June 12, 1978) SUMMARY The e f f e c t of taurine on ATP-dependent calcium transport was examined i n guinea-pig cardiac v e n t r i c l e homogenates and i n micro-somal preparations enriched i n sarcoplasmic reticulum. Taurine (5-50 mM) did not a f f e c t ATP-dependent calcium binding or uptake i n either of these preparations or a l t e r the rate of decay of calcium uptake a c t i v i t y . Taurine (20 mM) also did not a f f e c t the oxalate-dependent calcium uptake stimulation noted i n the presence of c y c l i c AMP-dependent protein kinase and c y c l i c AMP. The mech-anism by which taurine a l t e r s cardiac function remains to be elucidated. r Taurine i s present i n high concentrations i n mammalian hearts represent-ing nearly 50% of the t o t a l free amino acid pool (1,2). In congestive heart f a i l u r e , taurine l e v e l s are markedly elevated i n several species including humans (3-5). Changes i n myocardial taurine concentration have also been reported i n experimentally-induced hypertension (6) and ischemia (7). Taurine has been shown to a l t e r the pharmacological response to cardiac glycosides i n guinea-pig and rat heart preparations (8,9 ). Several reports suggest that taurine may play a r o l e i n the cont r o l of K+ and Ca2+ movements i n the heart (8-13) and thereby a f f e c t cardiac function. The possible r o l e of /taur i n e i n the regulation of ion movements i n the heart remains to be e l u c i d a t -ed. The present i n v e s t i g a t i o n was undertaken to determine whether taurine could influence ATP-dependent calcium uptake and binding i n guinea-pig cardiac v e n t r i c l e homogenates and i n microsomal preparations enriched i n sarcoplasmic reticulum. MATERIALS AND METHODS Heart Preparations: Guinea-pigs (200-300 g, albino,Hartley s t r a i n ) were s a c r i f i c e d by a blow to the head. The hearts were promptly excised, washed i n sa l i n e and the aorta, a t r i a and connective t i s s u e removed. The v e n t r i c u l a r muscle was then promptly frozen (within 30 sec. of s a c r i f i c e ) i n methyl butane , J * Correspondence to: D r . Sidney Katz, D i v i s i o n of Pharmacology, Faculty of Pharmaceutical Sciences, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C., c Canada V6T 1W5. 0300-9653/78/0724-0383$02.00/0 Copyright © 1978 Pergamon Press 236 384 E f f e c t of Taurine on Calcium Transport V o l . 23, No. 4, 1978 and dry i c e . The frozen hearts were wrapped i n t i n f o i l and stored at -80°C. The tissue could be stored i n t h i s way for up to 3 months without s i g n i f i c a n t loss i n calcium transport a c t i v i t y . Preparations of Heart V e n t r i c l e Homogenates: A piece (0.1-0.2 g) was cut from a frozen heart preparation and homogenized i n 5 ml of a medium c o n s i s t i n g of 40% sucrose and 40 mM Tris-Cl,pH7.2 using a Polytron P20 homogenizer (3 strokes of 5 sec. duration at s e t t i n g 5). Preparations of Microsomes Enriched i n Sarcoplasmic Reticulum: The method of Harigaya and Schwartz (14) was'followed with s l i g h t modifications. The frozen preparation was homogenized using a Polytron P20 homogenizer (3 strokes of 5 sec duration at s e t t i n g 5) and the homogenate fractionated by d i f f e r e n t i a l c e n t r i f u g a t i o n followed by 0.6 M KC1 treatment to reduce acto-myosin contamination. The f i n a l preparation was suspended i n a medium c o n s i s t -ing of 40% sucrose and 40 mM T r i s - C l , pH 7.2. Storage i n 40% sucrose reduced the loss of calcium transport a c t i v i t y noted when these preparations were maintained at 4°C (15). Unless otherwise indicated, a l l experiments were con-ducted within 2 h of the preparation of these f r a c t i o n s . ATP-dependent Calcium Uptake and Binding Assays: The method of Tada et a l . (16) was followed with a few modifications. O x a l a t e - f a c i l i t a t e d calcium uptake was determined i n the presence or absence of taurine using either 40-60 ug of the S.R. preparation or 200-300 v g of the homogenate preparation. The incubation medium contained 40 mM h i s t i d i n e - H C l , pH 6.8, 5 mM MgCl2, 110 mM KC1, 5 mM Tris-ATP, 2.5 mM Tris-Oxalate and CaCl2 containing 45c aCl 2 (10 C i / Mole) with the desired free calcium concentration maintained by the addition of ethylene-bis ( &-aminoethyl ether) N,N'-tetra acetate (EGTA). The free Ca^"1" concentrations were determined by the equations of Katz et^ al_. (17). Following a preincubation of 7 min.at 30°C the reaction was started by the addition of ^^CaCl2. Unless otherwise indicated, the time of incubation was 5 min at 30°C i n a t o t a l volume of 0.5 ml. The reaction was terminated by f i l t e r i n g an aliquot of the reaction mixture through a m i l l i p o r e f i l t e r (HA 45, M i l l i p o r e Co.). The f i l t e r was then washed twice with 15 ml of 40 mM T r i s - C l , pH 7.2, then dried and counted for r a d i o a c t i v i t y i n Aquasol (New England Nuclear) using standard l i q u i d s c i n t i l l a t i o n counting techniques. When c y c l i c AMP-dependent protein kinase was added to the medium, i t was present i n a concen-t r a t i o n of 50 ug/ml along with 1.0 ii M c y c l i c AMP. ATP-dependent calcium binding was studied under i d e n t i c a l conditions except that Tris-oxalate was omitted from the reaction medium. In studies on the e f f e c t of taurine on the decay of calcium transport a c t i v i t y , preparations were stored at 4°C i n 2 mM T r i s - C l , pH 7.2 i n the presence and absence of 15 raM taurine. , Protein Assay: Protein concentrations were measured by the method of Lowry et_ al^. (18) using bovine serum albumin as a standard. S t a t i s t i c s : Student's " t " test for unpaired, common variance data (19) was used as a measure of s i g n i f i c a n c e . Standard Error of the Mean (S.E.M.) was used as a measure of v a r i a t i o n . M a t e r i a l s : A l l chemicals were reagent grade. Tris-ATP, C y c l i c AMP, EGTA, and bovine cardiac protein kinase (Type 1) were obtained from Sigma Chem-i c a l Co., St. Louis, Mo. ^ C a C l 2 was obtained from the Radiochemical Centre, Amersham, England. 237 Vol. 23, No. 4, 1978 E f f e c t of Taurine on Calcium Transport 385 RESULTS Effe c t of Taurine on Calcium Uptake and Binding: The e f f e c t of varying taurine concentrations on calcium uptake and binding i n both v e n t r i c u l a r homo-genates and sarcoplasmic reticulum enriched preparations i s shown i n Table 1. The free calcium concentration used i n these studies was 1.0 uM. Taurine i n concentrations of 5-50 mM had no s i g n i f i c a n t e f f e c t on calcium uptake or binding i n either of these preparations. TABLE 1 E f f e c t of Taurine on Calcium Uptake and Binding in Guinea-pig Heart V e n t r i c l e Homogenates and Sarcoplasmic Reticulum Enriched Preparations. ' Taurine Homogenate Preparation Sarcoplasmic Reticulum Preparation Cone. Calcium Uptake Calcium Binding Calcium Uptake Calcium Binding (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) 5 2. ,17 + 0.38a 0.16 + 0.03 12. .40 + 0.79 0. 71 + 0.10 (2. ,36 0.40) b (0.16 + 0.03) (12. ,72 + 0.89) (0. 75 + 0.09) 10 2. .39 + 0.15 0.17 + 0.02 12. ,93 + 1.27 0. 76 + 0.05 (2. .32 + 0.29) (0.16 + 0.02) (13. .19 + 1.19) (0. 75 + 0.04) 20 2. ,68 + 0.10 0.16 + 0.02 12. ,72 + 1.37 0. 77 0.05 (2. .56 + 0.14) (0.18 + 0.02) (12, .18 + 1.33) (0. 81 + 0.02) 30 2. ,89 + 0.14 0.17 + 0.01 14. .79 + 1.83 0. 81 + 0.04 (2. .89 + 0.13) (0.19 + 0.02) (14. .09 + 0.02) (0. 74 + 0.03) AO 2. .62 + 0.22 0.18 + 0.01 11, .84 + 1.79 0. 76 + 0.02 (2. .70 + 0.14) (0.17 + 0.02) (11. ,27 + 1.72) (0. 76 + 0.05) ' 50 2. .59 + 0.17 0.16 + 0.02 12, .59 + 2.05 0. 70 + 0.08 (2. ,56 + 0.34) (0.17 + 0.01) (12. .52 + 0.01) (0. 75 + 0.09) Guinea-pig heart v e n t r i c l e homogenates (200-300 p g protein) or sarcoplasmic reticulum enriched preparations (40-50 yg protein) were incubated for 5 min with and without taurine i n medium containing 40 mM h i s t i d i n e HC1, pH 6.8, 5 mM MgCl 2, 5 mM ATP, 110 mM KC1, 2.5 mM T r i s - o x a l a t e , and 1.0 u M free Ca"^ (125 M M CaCl2 containing 4 5 C a C l 2 (10 Ci/Mole) and 391 MM EGTA). The reaction was c a r r i e d out at 30°C i n a t o t a l volume of 0.5 ml. Calcium binding was determined i n an i d e n t i c a l r e a c t i o n mixture, except that 2.5 mM Tris-oxalate was omitted. a. The r e s u l t s are a Mean ± S.E.M. of at l e a s t 3 observations each performed i n duplicate. b; The values i n parentheses are controls (taurine omitted). 238 386 E f f e c t of Taurine on Calcium Transport Vol. 23, No. 4, 1978 The e f f e c t of 20 mM taurine on calcium uptake and binding i n both these preparations was examined at various calcium concentrations (Table 2). The sarcoplasmic reticulum enriched preparation exhibited an increase i n calcium uptake and binding with increasing Ca2+ concentration to a maximum at 10 iiM free Ca2+; Ca2+ concentrations higher than 10pM were i n h i b i t o r y . This p r o f i l e of the Ca2+ concentration e f f e c t on calcium transport was s i m i l a r i n the homo-genate preparation. Taurine (20 mM) at a l l free Ca2+ concentrations studied had no s i g n i f i c a n t e f f e c t on calcium uptake or binding i n e i t h e r of these preparations. TABLE 2 The E f f e c t of Taurine on Calcium Uptake and Binding at Various Calcium Concentrations i n Guinea-pig Heart V e n t r i c l e Homogenates and Sarcoplasmic Reticulum Enriched Preparations Calcium Cone. (M M) Calcium Uptake Homogenate Preparation Sarcoplasmic Reticulum Preparation Calcium Binding Calcium Uptake Calcium Binding (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) 0.5 1. .07 + 0 .03 a 0.09 + 0, .01 8 .91 + 1.32 0, .54 + 0.11 (1 .18 + 0 .03)b (0.11 + 0, .01) (8 .77 + 1.24) (0, .58 + 0.08) 1.0 2, .43 + 0, .09 0.14 + 0. .01 20, .14 + 3.02 0. .63 + 0.14 (2, .36 + 0, • 17) (0.15 + 0. .01) (19. .61 + 2.94) (0. .65 + 0.14) 5.0 7. .81 1. .09 0.40 + 0. .03 71. .42 + 14.89 1. ,33 + 0.25 (7. ,94 ± 0. .52) (0.39 + 0. 03) (71. ,59 + 14.00) (1. ,17 + 0.15) 10.0 9. .67 ± 0. .99 0.79~± 0. 05 84. ,56 + 20.09 1. 42 + 0.11 (10. 00 + 1. •17) (0.85 + 0. 06) (86. ,64 + 20.34) (1. 44 + 0.10) 50.0 8. 56 + 1. 58 0.74 + 0. 02 69. 84 + 10.36 1. 14 + 0.11 (8. 62 + 1. 74) (0.75 + 0. 04) (70. 15 + 12.67) (1. 19 + 0.15) 100.0 7. 34 ± 1. 34 1.01 + 0. 10 81. 29 + 26.61 1. 26 + 0.14 < 7 - 02 + 1. 22) (1.04 + 0. 08) (81. 21 + 29.37) (1. 34 + 0.12) Guinea-pig heart v e n t r i c l e homogenates (200-300 ug protein) or sarcoplasmic reticulum enriched preparations (40-50 pg protein) were incubated for 5 min with and without 20 mM taurine as described i n Table 1 i n the presence of various concentrations of free calcium. Calcium binding was measured under i d e n t i c a l conditions i n the absence of 2.5 mM T r i s - o x a l a t e . a. The r e s u l t s are a Mean ± S.E.M. of at lea s t 3 observations each performed i n dup l i c a t e . b. The values i n parentheses are controls (taurine omitted). The E f f e c t of Taurine on the Time-course of Calcium Uptake and Binding: The time course of calcium uptake and binding i n homogenate and sarcoplasmic reticulum enriched preparations i s shown i n Figure 1A and IB, r e s p e c t i v e l y . Calcium uptake i n both these preparations was l i n e a r for the f i r s t 10 minutes of incubation following which the rates declined s l i g h t l y . Maximal calcium binding was observed at 10 minutes i n the homogenate preparation and at 5 min in the sarcoplasmic reticulum preparation. No s i g n i f i c a n t e f f e c t of taurine was observed i n these preparations e i t h e r at the i n i t i a l time (30 sec) or at longer periods of incubation. 239 V o l . 23, No. 4, 1978 A 5 0 - i 4 0 H E f f e c t of Taurine on Calcium Transport 387 c "5 o Q. O) E tn o E c di o a o u 3 0 H 2 0 H IOH 4 0 0 -3 6 0 -3 2 0 -2 8 0 -2 4 0 -2 0 0 -1 6 0 -120-8 0 -B i (-4— T/f r-1 • > * 4 . 0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0 .8 0 .4 • < c '55 o k_ a cn E o E c d) c c co D u 5 10 15 2 0 Incubation Time (minutes) FIG. 1 Time course e f f e c t of 20 mM taurine (O.A) on calcium uptake ( s o l i d l i n e s ) and binding (dotted l i n e s ) in Guinea-pig heart v e n t r i c l e homogenates (A) and sarco-plasmic reticulum enriched preparations (B). The v e r t i c a l l i n e s represent ± S.E.M. of 3 determinations each performed in duplicate. 240 388 E f f e c t of Taurine on Calcium Transport V o l . 23, No. 4, 1978 The Effect of Taurine on the Decay of Calcium Uptake A c t i v i t y : Both the homogenate and the sarcoplasmic reticulum enriched preparations decreased r a p i d l y i n calcium uptake a c t i v i t y when kept at 4°C i n the absence of 40% sucrose. Addition of 15 mM taurine to these preparations under these con-d i t i o n s did not a l t e r t h i s steady decline i n calcium uptake a c t i v i t y (Figure 2). •J 1 1 1 1 i • 0 1 2 3 4 5 6 Time After P repara t i on (hours) FIG. 2 The e f f e c t of taurine on the decay of calcium uptake a c t i v i t y in guinea-pig v e n t r i c l e homogenates and sarcoplasmic reticulum enriched preparations. Homogenates ( s o l i d l i n e s , 200-300 ug protein) or sarcoplasmic reticulum preparations (dotted l i n e s , 40-50ug protein) were maintained i n 2 mM T r i s - C l , pH 7.2 i n the presence (O, A ) and absence (•, • ) of 15 mM taurine. Calcium uptake was measured at s p e c i f i e d times as described in Table 1. The v e r t i c a l l i n e s represent ± S.E.M. of 3 determin-ations each performed i n duplicate. Effect of Taurine on C y c l i c AMP-dependent Protein-kinase Stimulated  Calcium Uptake: When the sarcoplasmic reticulum enriched preparation was incubated with protein kinase and c y c l i c AMP i n the presence of 1.0 uM C a + + , calcium uptake was increased more than 2 .fold (p<0.02) (Table 3). S i m i l a r l y , the rate of calcium uptake by the homogenates also increased in the presence of c y c l i c AMP-dependent protein kinase (p<0.|05). Taurine (20 mM) had no s i g n i f i -cant e f f e c t on the c y c l i c AMP-dependent protein kinase stimulation of calcium uptake i n these preparations. 241 Vol. 23, No. 4, 1978 E f f e c t of Taurine on Calcium Transport 389 TABLE 3 E f f e c t of Taurine on C y c l i c AMP-dependent Protein Kinase-Stimulated Calcium Uptake i n Guinea-pig Heart V e n t r i c l e Homogenates and Sarcoplasmic Reticulum Enriched Preparations: Taurine Homogenate Preparation Sarcoplasmic Reticulum Preparation (20 mM)\ Without cAMP-dependent Protein kinase With cAMP-dependent Protein kinase Without cAMP-dependent Protein kinase With cAMP-dependent Protein kinase - 2.39 ± 0.29 a , b 3.38 ± 0.14c 9.72 ± 1.02 16.86 + 1.51 d + 3.30 ± 0.17 e 17.89 ± 1.84 e Guinea-pig v e n t r i c l e homogenates (200-300 ug protein) and sarcoplasmic reticulum enriched preparations (40-50 yg protein) were incubated with and without c y c l i c AMP (1.0 uM) and c y c l i c AMP-dependent protein kinase (50 ug/ml, Sigma grade type 1) i n the presence and absence of 20 mM taurine. Calcium uptake was measured as described i n Table 1. In these experiments the free calcium concentration was 1.0 uM and the incubation time was 5 minutes. a. The r e s u l t s are a Mean ± S.E.M. of at lea s t 3 observations each performed i n duplicate. b. Calcium uptake a c t i v i t y expressed as nmoles/mg/min. c. P<0.05 compared to Ca^ +-uptake i n the absence of c y c l i c AMP-dependent protein kinase. d. P<0.02 compared to Ca2+-uptake i n the absence of c y c l i c AMP-dependent protein kinase. e. Not s i g n i f i c a n t compared to the a c t i v i t y seen without taurine i n the presence of c y c l i c AMP-dependent pr o t e i n kinase. DISCUSSION Results obtained on the ATP-dependent calcium uptake and binding para-meters of the sarcoplasmic reticulum enriched preparation used i n t h i s study were s i m i l a r to those noted by other workers (16,20,21). The homogenate pre-paration exhibited ATP-dependent Ca^ + binding a c t i v i t y as well as Ca2+-uptake a c t i v i t y in the presence of oxalate; these a c t i v i t i e s , though, were lower than those noted i n the sarcoplasmic reticulum enriched preparation. In thi s study we have demonstrated that taurine does not s i g n i f i c a n t l y a f f e c t ATP-dependent calcium transport i n guinea-pig cardiac v e n t r i c l e homogen-ates or in cardiac preparations enriched with sarcoplasmic reticulum. Entman et a l . (22) recently reported that taurine had no e f f e c t on calcium transport i n canine sarcoplasmic reticulum preparations. These workers used the spectro-photometry murexide dye technique for the measurement of calcium transport whereas i n our studies ^-*CaCl2 and the m i l l i p o r e f i l t r a t i o n technique were u t i l i z e d . Our present studies thus confirm those of Entman et a l . (22) using both a sarcoplasmic reticulum enriched and crude homogenate preparation of heart tissue i n a species i n which pharmacological e f f e c t s of taurine i n cardiac tissue have been noted (8,9). The p o s s i b i l i t y that taurine may a l t e r calcium uptake or binding a c t i v i t y i n c e l l u l a r organelles other than sarcoplasmic reticulum tends to be ruled out by the lack of an e f f e c t on calcium uptake or binding i n the homogenate preparation used i n t h i s study. 242 390 E f f e c t of Taurine on Calcium Transport V o l . 23, No. 4, 1978 Cy c l i c AMP-dependent protein kinase has been shown to stimulate calcium uptake i n preparations of cardiac sarcoplasmic reticulum (21). Taurine did not af f e c t the enhancement of calcium transport by c y c l i c AMP-dependent protein kinase either in the homogenate preparation or i n the sarcoplasmic reticulum preparation. Lack of an e f f e c t on t h i s system indicates that taurine does not act as a modulator of calcium transport through t h i s c y c l i c AMP-mediated path-way. Previous reports indicated that the decay i n calcium transport a b i l i t y observed i n s k e l e t a l muscle sarcoplasmic reticulum preparations could be reduc-ed by the presence of taurine (13). In this study the presence of taurine had no eff e c t on t h i s decay process i n either the homogenate or sarcoplasmic reticulum enriched preparations. This lack of e f f e c t of taurine was also noted by Entman et_ al_. (22) under d i f f e r e n t experimental conditions. Taurine has been shown to be of p o t e n t i a l importance i n cardiac pathology. The p o s s i b i l i t y s t i l l e x i s t s that taurine exerts i t s e f f e c t s in the cardiac c e l l by a l t e r i n g the passive d i f f u s i o n of ions or by a f f e c t i n g calcium release from sarcotubular, mitochrondrial or sarcoplasmic reticulum stores. These p o s s i b i l i -t i e s are currently under i n v e s t i g a t i o n i n our laboratory. ACKNOWLEDGEMENT This work was supported by a grant from the B r i t i s h Columbia (Canada) Heart Foundation. REFERENCES 1. J.G. JACOBSEN and L.H. SMITH, JR., Physiol. Rev. 48 424-511 (1968). 2. J. AWAPARA, A. LANDUA, R. FUERST, Biochim. Biophys. Acta 5 457-462 (1950). 3. R. HUXTABLE and R. BRESSLER, L i f e Sciences 14 1353-1359 (1974). 4. M.B. PETERSON, R.J. MEAD, J.D. WELTY, J. Mol. C e l l . C a r d i o l . 5 139-147 (1973). 5. W.H. NEWMAN, C.J. FRANGAKIS, D.S. GROSSO and R. BRESSLER, Physiol. Chem. Phys. 9 259-263 (1977). 6. R. HUXTABLE and R. BRESSLER, Science 184 1187-1188 (1974). 7. M.F. CRASS III and J.B. LOMBARDINI, L i f e Sciences 21 951-958 (1977). 8. A.' GUIDOTTI, G. BANDIAN1 and A. GIOTTI, Pharmacol. Res. Communications 3 29-38 (1971). 9. J . DIETRICH and J . DIACONO, L i f e Sciences 10 499-507 (1971). 10. W.O. READ and J.D. WELTY, E l e c t r o l y t e and Cardiovascular Diseases (E. Bajuzz, ed.) p. 70, S.P. Karger, Basel New York (1965). 11. D.S. GROSSO and R. BRESSLER, Biochem. Pharm. 25 2227-2232 (1976). 12. P. DOLARA, A. AGRESTI, A. GIOTTI and G. PASQUINI, Eur. J . Pharmacol. 24 352-358 (1973). 13. R. HUXTABLE and R. BRESSLER, Biochim. Biophys. Acta 323 573-583 (1973). 14. S. HARIGAYA and A. SCHWARTZ, C i r c . Res. 25 781-794 (1969). 15. M.E. CARSTEN, Proc. Natl. Acad. S c i . U.S.A. 52_ 1456-1462 (1964). 16. M. TADA, M.A. KIRCHBERGER, D.I. REPKE and A.M. KATZ, J. B i o l . Chem. 249 6174-6180 (1974). 17. A.M. KATZ, D.I. REPKE, J.E. UPSHAW and M.A. POLASCIK, Biochim. Biophys. Acta 205 473-490 (1970). 18. O.H. LOWRY, N.J. R0SEBR0UGH, A.L. FARR and R.J. RANDALL, J . B i o l . Chem. 193 265-275 (1951). 19. T.H. WONNACOTT and R.J. WONNACOTT, Introductory S t a t i s t i c s , p. 215, John Wiley & Sons, New York, Third E d i t i o n (1977). 20. D.I. REPKE and A.M. KATZ, J . Mol. C e l l . C a r d i o l . 4_ 401-416 (1972). 21. M. TADA, M.A. KIRCHBERGER, and A.M. KATZ, Recent Advances in Studies on  Cardiac Structure and Metabolism, volume 9_ (P.E. Roy and N.S. Dhalla, Eds.) p. 225, University Park Press, Baltimore, (1976). 22. M.L. ENTMAN, E.P. BORNET and R. BRESSLER, L i f e Sciences 21 543-550 (1977). 243 L i f e Sciences, Vol. 24, pp. 1885-1892 Pergamon Press Printed i n the U.S.A. EFFECT OF TAURINE ON PASSIVE ION TRANSPORT IN RAT BRAIN SYNAPTOSOMES Mohamed A. Remtulla, Sidney Katz and Derek,A. Applegarth D i v i s i o n of Pharmacology, Faculty of Pharmaceutical Sciences and Departments of Paediatrics & Pathology, Faculty of Medicine, U n i v e r s i t y of B r i t i s h Columbia, and Biochemical Diseases Laboratory, Children's Hospital Vancouver, B.C., Canada (Received i n f i n a l form A p r i l 2, 1979) Summary Taurine, i n concentrations greater than 10 mM, was found to have an i n h i b i t o r y e f f e c t on passive calcium uptake and release i n rat brain synaptosomal preparations. Amino acids s i m i l a r to that of taurine i n chemical structure, 6-alanine, hypotaurine, homotaurine and y-amino-butyric acid were also shown to i n h i b i t calcium uptake in t h i s prepar-ation. Taurine, though, did not a l t e r the permeability of these preparations to sodium or potassium. It thus appears that taurine and chemically related amino acids can a l t e r calcium movements i n these preparations. I t i s postulated that t h i s e f f e c t i s due to binding to s p e c i f i c taurine s i t e s i n the synaptosomal membranes. Taurine i s known to be present i n r e l a t i v e l y ^ high concentrations in the brain, heart and muscle of mammals (1). Several groups of i n v e s t i g a t o r s have reported that taurine concentrations are reduced i n experimentally-induced epileptogenic f o c i of cats (2), mice (3), rats (4) and man (5). Taurine has also been shown to have a n t i - e p i l e p t i c e f f e c t s i n both experimentally-induced epilepsy (3,6-8) and i n human patients (9,10). Recently, investigators have suggested a p h y s i o l o g i c a l r o l e for taurine i n the maintenance of excitatory a c t i v i t y i n muscle and nervous tissues (11-15). Others have raised the p o s s i b i l i t y that taurine i s an i n h i b i t o r y neurotrans-mitter (16,17). However, i t s possible mechanism of action i s s t i l l uncertain. Izumi, et a l • (18) have suggested that taurine may regulate the free calcium concentration of nervous ti s s u e . We have recently reported (19) that taurine does not a f f e c t ATP-dependent calcium transport i n guinea-pig whole heart homo-genates and preparations enriched in sarcoplasmic reticulum. In thi s present work, as part of a study on the possible p h y s i o l o g i c a l mechanism of action of taurine, we have studied the e f f e c t of taurine on the passive transport of sodium, potassium and calcium in preparations of rat brain. Materials and Methods Preparation of Synaptosomes: r Synaptosomes were prepared from brain of male Wistar rats (250-300 g) following the method of Gray and Whittaker (20) as modified by k^en and White (21). Correspondence to: Dr. S. Katz, D i v i s i o n of Pharmacology, Faculty of Pharmaceutical Sciences, University of B r i t i s h Columbia, Vancouver, B.C Canada V6T 1W5 0024-3205/79/201885-08$02.00/0 'Copyright (c) 1979 Pergamon Press Ltd 244 1886 Taurine and Passive Ion Transport Vol. 24, No. 20, 1979 Determination of the Osmometric Behaviour of Synaptosomes: The method of Keen and White (21) was followed: 0.05 ml of the synaptosomal preparation (5 mg protein/ml) was suspended i n solut i o n s of 25-300 mM Na2S0^ (0.95 ml) i n a micro-cuvette. The e x t i n c t i o n at E520 w a s followed for a period of 15 minutes at room temperature using a Beckman recording spectrophotometer (model 25). Determination of Sodium and Potassium Permeability: Synaptosomal preparations were preincubated with and without taurine (20 mM) for 1 hour at 2°C. An aliq u o t (0.05 ml) was then added to a microcuvette containing from 100-200 mM ice - c o l d sodium or potassium acetate (0.95 ml) s o l u t i o n . The content of the microcuvette was r a p i d l y mixed with a pasteur pipette and the e x t i n c t i o n at F.520 recorded over a 5 minute period. Determination of Calcium Permeability: The synaptosomal preparation (0.2 mg/ml) was preincubated at 2°C i n medium containing 0.3 M sucrose, and 10 mM Tris-HCl pK 7.2, i n the presence or absence of taurine (20 mM), i n a t o t a l volume of 3 ml. Following a preincubation of 1 hour, the reaction was started by the addition of 10 uM ' 4 5CaCl2 (5 x 10 5 cpm/sample) . Aliquots (0.2 ml) of the incubation mixture were then removed at i n t e r v a l s and passed through a m i l l i p o r e f i l t e r (HA 45, M i l l i p o r e , Co.). The f i l t e r was washed twice with 5 ml of 10 mM T r i s -HCl (pH 7.2) i n 0.3 M sucrose then dried and counted for r a d i o a c t i v i t y i n Aquasol (New England Nuclear Co.) using standard l i q u i d s c i n t i l l a t i o n counting techniques. Determination of Loss of -^*Ca from Preloaded Synaptosomes: Synaptosomal prepar-ations (0.2 mg protein/ml) were loaded with 10 yM ^ C a C ^ (5 x 10^ cpm/sample) at 2°C i n conditions s i m i l a r to that described above. After 1 hour, an a l i q u o t (0.2 ml) was passed through a m i l l i p o r e f i l t e r . The remaining incubation medium was centrifuged at 12,000 x g for 10 minutes. The r e s u l t i n g p e l l e t was resuspended i n 3 ml of i c e - c o l d media containing 0.3 M sucrose and 10 mM T r i s -HCl, pK 7.2 i n the presence and absence of 20 mM taurine. The release of 45caCl 2 with time, was determined by passing a l i q u o t s (0.2 ml) of the .reaction medium through a m i l l i p o r e f i l t e r . The rate of calcium release was then calculated by the following equation: (cpm i n f i l t e r a f t e r 1 hour preloading) -(cpm i n f i l t e r at sampling time) 4 5 C a release (%) = (cpm i n f i l t e r a f t e r 1 hour preloading) Protein Assay: Protein concentrations were measured by the method of Lowry, et a l . (31) using bovine serum albumin as a standard. • S t a t i s t i c s : S t a t i s t i c a l a nalysis was done by Students " t " test f o r unpaired data. A p r o b a b i l i t y of p<0.05 was taken as the c r i t e r i o n for s i g n i f i c a n c e . Standard Error of the Mean (S.E.M.) was used as a measure of v a r i a t i o n . Materials: A l l chemicals were reagent grade. Taurine was obtained from Sigma Chemical Co., St. Louis, Mo.. ^ 5 r j a c i 2 was obtained from the Radiochemical Centre, Amersham, England. Results The Osmometric Behaviour of Synaptosomes: The o p t i c a l e x t i n c t i o n (E520) of the synaptosomal preparations suspended i n solutions of Na2S04 was found to increase with the strength of the s o l u t i o n ( f i g u r e 1A). The r e c i p r o c a l p l o t of e x t i n c t i o n (I/E520) against l/Na2S0z, (fi g u r e IB) showed a l i n e a r r e l a t i o n s h i p . These r e s u l t s confirm the observations of Keen and White (21) and show that the synaptosomal preparations behave as osmometers confirming to Boyle and Van't Hoff's law. 245 Vol. 24, No. 20, 1979 Taurine and Passive Transport 1887 l . H i.oH 0.9-J 0.8 H 0-7 H 0.6 1.2 - i 1 .15H l.H 1.05 H ioH 0.95-1 1 1 0 100 200 300 0.9 -1 1 r-005 .01 .015 .02 m M l / N a 2 S Q , FIG. 1 The e f f e c t of Na 2S0 4 concentration on the E 5 2 0 of a suspension of synaptosomes: In (A) the data are plotted as E 5 2 0 against Na2S0z, while i n (B) l/Na 2S04 i s plotted against l/E52o. Results are shown as Mean i S.E.M. 'of 3 d i f f e r e n t synaptosomal preparations. The E f f e c t of Taurine on Sodium and Potassium Permeability i n Synaptosomal  Preparations: The permeability of the synaptosomal preparations to sodium and potassium ions i n the presence or absence of'taurine i s shown in Table 1A and IB, r e s p e c t i v e l y . Synaptosomal preparations preincubated with 20 mM taurine and suspended i n 100-200 mM sodium or potassium acetate solutions containing 20 mM taurine showed no s i g n i f i c a n t change in E520 when compared to r e s u l t s obtained i n the absence of taurine. ' The E f f e c t of Taurine on the Passive Uptake and Release of Calcium i n Synapto- somal Preparations: The time course of uptake and release of calcium i n an i s o t o n i c sucrose medium i s shown i n f i g u r e 2A and 2B, r e s p e c t i v e l y . Under these conditions, passive calcium uptake i n synaptosomal preparations was l i n e a r with time. The amount of calcium taken up and released from preloaded synaptosomes was lower i n the presence of 20 mM taurine. A s i g n i f i c a n t d i f f erence i n calcium uptake i n the presence of taurine was observed a f t e r 18 min of incubation (p<0.05). In the calcium release experiments, calcium e f f l u x was s i g n i f i c a n t l y reduced i n the presence of taurine (20 mM) at a l l incubation times tested (p<0.01). Dose-dependent E f f e c t of Taurine on Calcium Uptake i n Synaptosomal Preparations: Various concentrations of taurine (0.5 to 50 mM) were studied with respect to synaptosomal calcium uptake (Fi g . 3). Control experiments were c a r r i e d out where taurine was substituted for an equimolar concentration of choline c h l o r i d e . Conditions were also studied where neither taurine nor choline chloride were 246 1888 Taurine and Passive Ion Transport. Vol. 24, No. 20, 1979 TABLE 1 E f f e c t of Taurine on Sodium (A) and Potassium (B) Permeability i n Synaptosomes. A. Na Acetate B. K Acetate mM Na + "520 mM K + E520 CONTROL TAURINE CONTROL TAURINE 100 0. .898 ± 0.050 0. 913 + 0.044 100 0. 915 ± 0.035 0. ,918 ± 0.044 125 0. ,934 ± 0.053 0. ,916 ± 0.044 ' 125 0. .934 ± 0.050 0. ,948 ± 0.041 150 0. .965 + 0.072 0. .994 ± 0.041 150 0. .961 ± 0.045 0. .966 ± 0.048 175 1. .000 ± 0.052 1. .018 ± 0.042 175 1, .018>+ 0.033 1. .001 ± 0.045 200 1. .000 ± 0.056 1. .025 ± 0.045 200 1 .024 ± 0.034 1 .020 ± 0.045 The permeability was measured as a function of the change in E520 of a synapto-somal membrane suspension (50 ul) i n acetate s a l t s i n the presence (TAURINE) or absence (CONTROL) of 20 mM taurine. Each value i s the Mean ± S.E.M. of three separate synaptosomal preparations. present i n the incubation medium. No change i n calcium uptake could be detected at lower concentrations of taurine (0.5 to 5.0 mM); thereafter, a decline in calcium uptake was observed as taurine concentrations were increased. Choline chloride was more potent than taurine i n lowering synaptosomal calcium uptake i n concentrations greater than 10.0 mM. Ef f e c t of Other Amino Acids on Calcium Uptake i n Synaptosomal Preparations: A number of compounds, i n a concentration of 20 mM, were tested for t h e i r e f f e c t on passive calcium uptake i n synaptosomal preparations (Fig. 4). Homotaurine, hypotaurine, 8-alanine and GABA exhibited s i m i l a r e f f e c t s to taurine, s i g n i f i -cantly decreasing the degree of calcium uptake observed i n the absence of these agents (p<0.05). On the other hand, a-alanine stimulated calcium uptake to a small extent (not s i g n i f i c a n t ) . Methionine, proline and vali n e also did not s i g n i f i c a n t l y a f f e c t calcium transport i n this preparation (not shown). Discussion In thi s study we have demonstrated that taurine has an i n h i b i t o r y e f f e c t on both calcium uptake and release i n synaptosomal preparation suspended in i s o -tonic medium. Similar r e s u l t s have recently been reported on the release of ^ c a l c i u m from preloaded synaptosomes by .Kuriyama et a l . (22). These workers, though, did not show any s i g n i f i c a n t e f f e c t of taurine on calcium uptake. The discrepancy i n calcium uptake r e s u l t s between t h i s present study and that of Kuriyama et a l (22) could be due to the differences i n the experimental proce-dures . u t i l i z e d . In th i s present study, the i n h i b i t o r y e f f e c t of taurine on calcium uptake was observed at taurine concentrations greater than 10 mM. Lower taurine concentra-tions (0.5-5.0 mil) had no e f f e c t . Hue et a l . (23) i n studies on the insect c e n t r a l nervous system also demonstrated that taurine at concentrations lower than 10 mM had no e f f e c t on the s e n s i t i v i t y of post synaptic neurons. r 247 Vol. 24, No. 20, 1979 Taurine and Passive Ion Transport 1889 120 100 o o • o 60 u 80 20 J 0 10 20 30 incubation Time-, minutes 0 10 20 30 Incubation Time- minutes FIG. 2 The e f f e c t of taurine on (A)^^Ca^ + uptake and (B) release of ^Ca^+ from preloaded r a t synaptosomal preparations. Calcium uptake and release were determined i n the presence ( O O ) or absence ( 9 • ) of 20 mM taurine i n a. medium containing 0.3 M sucrose and 10 mM T r i s - H C l , pH 7.2 as described i n the text. E f f e c t of taurine on calcium uptake i s expressed as ^-"Ca2+ uptake r e l a t i v e to the value of Ca2 + uptake observed at 30 min incubation time i n the absence of taurine. Calcium release from preloaded synaptosomes was calculated as % release as described i n the text. The v e r t i c l e l i n e s represent ± S.E.M. of at l e a s t three separate synaptosomal membrane preparations. In these present studies, i t was also noted that homotaurine, hypotaurine, 8-alanine and GABA also i n h i b i t e d calcium uptake i n synaptosomal preparations i n the same concentration as that observed to produce the i n h i b i t o r y e f f e c t of taurine. This indicates that the i n h i b i t o r y e f f e c t on calcium uptake i n these preparations was not s p e c i f i c to taurine. Other amino acids were shown not to i n h i b i t calcium uptake. Taurine receptor s i t e s have recently been reported to be present i n synaptosomal preparations (24) and i n heart v e n t r i c u l a r sarco-lemmal preparations (25). Lahdesmaki et a l . (24) have reported that taurine binding to synaptosomes was i n h i b i t e d by hypotaurine, 6-alanine and GABA. It thus appears that the binding s i t e s have a s t r i c t requirement f o r a s p e c i f i c chemical structure since only those amino acids which were chemically close to taurine were i n h i b i t o r y . It also can be seen that these same compounds i n h i b i t e d calcium uptake to the same extent as taurine suggesting that t h i s e f f e c t may be due to the s p e c i f i c binding of these compounds to the taurine receptor s i t e s . Lahdesmaki and Pajunen (26) reported a reduced outflow of sodium and potassium Ions from synaptosomes i n the presence of taurine when experiments were performed i n sodium- and potassium-free medium containing choline c h l o r i d e , calcium and 2 4 8 1890 Taurine and Passive Ion Transport V o l . 24, No. 20, 1979 2 a O u to > ot 100 r-20.0 30.0 50.0 m M Taurine or Choline chloride 45. FIG. 3 The e f f e c t of various concentrations of taurine on " ^ C a C l 2 uptake i n brain synaptosomal preparations ( s o l i d bars). Control experiments were done i n the presence of equimolar concentrations of choline chloride (middle bars with oblique s t r i a t i o n s ) and i n the presence of neither taurine nor choline chloride ( l e f t bars with s t r a i g h t s t r i a t i o n s ) . Calcium uptake was determined at 2°C for 30 min i n medium containing synaptosomes (0.2 mg/ml), 0.3 M sucrose, 10 mM T r i s -HCl, pH 7.2 and 10 uM 45c aCl 2 (5 x 105 cpm/sample) i n a t o t a l volume of 0.3 ml. Each bar represents the Mean ± S.E.M. of three separate experiments r e l a t i v e to the value of the ^CaCl 2-uptake of controls measured i n the absence of taurine or choline chloride. magnesium. In our hands, using a d i f f e r e n t technique, we were unable to observe a s i g n i f i c a n t e f f e c t of taurine on the passive permeability of sodium and potassium ions. It i s possible that the e f f e c t of taurine on the release of sodium and potassium ions from the synaptosomes as observed by Lahdesmaki and Pajunen (26) was secondary to an e f f e c t of taurine on the permeability to calcium. It should be noted that in these present experiments, choline chloride i n concentrations of 10 ml! or greater produced a marked i n h i b i t i o n of calcium uptake. This i n h i b i t i o n was greater than that observed for equimolar concentra-tions of taurine. Although the mechanism of this e f f e c t was not further investigated i t should be stated that choline cannot be used as a substitute for other ions i n thi s or s i m i l a r studies as i t markedly i n h i b i t s ion movements in i t s own r i g h t . It i s well known that calcium plays an important role in the regulation of the e x c i t a b i l i t y of neuronal ti s s u e . A decrease i n the calcium permeability of 249 Vol. 24, No. 20, 1979 Taurine and Passive Ion Transport 1891 2.5 r 1 9 2.0 E J£ o E c O a Z> A' o u <o 1.5 1.0 0.5 £ o 3 O e c < E < < FIG. 4 0) _c "C 3 O O E o . X c 5 E s s 8. ^ E f f e c t of. various amino acids on calcium uptake i n brain synaptosomal prepara-tions. Calcium uptake was measured for 30 min under the same conditions as that described i n F i g . 3 i n the presence of 20 mM concentrations of various amino acids. Controls consisted of the same medium with no amino acid a d dition. The r e s u l t s shown are the mean ± S.E.M. of at least 3 separate- preparations i n each case. the synaptic membrane could a l t e r the calcium concentration i n the synaptic terminal and a f f e c t neurotransmitter release (27). A s i m i l a r dependence on calcium has been noted in synaptosomal preparations i n - v i t r o (28). Long-term changes i n membrane permeability to calcium could change the p o t e n t i a l gradient across the membrane leading to hyperpolarization (29) . Taurine could render the nerve terminal r e f r a c t o r y to de p o l a r i z a t i o n or prevent calcium i n f l u x , necessary for the co-ordinated release of transmitter. Taurine, present i n high concentra-tions in the synaptosomes could serve to modulate neuronal a c t i v i t y (29) . The observations reported here may help to provide an ins i g h t into the p h y s i o l o g i c a l and pharmacological e f f e c t s of taurine reported both i n cardiac (30) and nervous tissue (15,17). Acknowledgement This work was supported by a Grant from the B r i t i s h Columbia (Canada) Heart Foundation. The authors thank Dr. B.D. Roufogalis for his h e l p f u l comments. References 1. J.G. JACOBSEN and L.H. SMITH, JR., Physiol. Rev. 4j3 424-451 (1968). 2. I. KOYAMA, Can. J. Physiol. Pharmacol. 50 740-752 (1972). 3. N.M. 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