Studies on the Physiological Role of Taurine in Mammalian Tissues'
STUDIES ON THE PHYSIOLOGICAL ROLE
OF TAURINE (2-aminoethane sulfonic acid)
IN MAMMALIAN TISSUES
by
MOHAMED AKBERALI REMTULLA
B.Sc, University of British Columbia, 1974
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE.DEGREE OF
DOCTOR OF PHILOSOPHY
in THE FACULTY OF GRADUATE STUDIES in THE DEPARTMENT OF PATHOLOGY (Faculty of Medicine)
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
AUGUST, 1979 (e) Mohamed Akberali Remtulla, 19 79 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5
ii ABSTRACT
'Studies on the Physiological Role of Taurine in Mammalian Tissues'
Mohamed A. Remtulla Ph.D. (Pathology)
Taurine (2-aminoethane sulfonic acid) is one of the most abundant free amino acids found in mammalian brain, heart and muscle. Taurine levels have also been shown to be altered in certain disease states. A physiological role
for taurine in the maintainance of excitatory activity in muscle and nervous tissues has been suggested; however its possible mechanism of action is still uncertain.
Early work on the pharmacological actions of taurine involved its possible conversion to isethionic acid
(2-hydroxyethane sulfonic acid), a strong anion. This con• version was said to lead to the. conductance of cations into the cardiac cell. An analytical technique to measure isethionic acid in mammalian tissues was developed. The method involved extraction, partial purification and methylation with diazomethane, followed by gas-liquid chromatography. With this technique only trace amounts of isethionic acid were detected in rat heart (0.1 mg/lOOg wet weight tissue) and rat brain (0.2 mg per 100 mg wet weight tissue) and none was detected in dog hearts. Recovery of added isethionic acid was between 95 and 100%. The assay was validated using a sample of squid axoplasm. We were also unable to show
14:C-taurine conversion to 14C-isethionic acid in rat heart
slices. Theories on the mode of action of taurine involving
bioconversion to isethionic acid were therefore questioned.
Some recent work suggested that taurine affects
calcium kinetics in perfused guinea-pig hearts and calcium
transport in rat skeletal muscle sarcoplasmic reticulum.
We have investigated the effect of taurine on ATP-dependent
calcium binding and oxalate-dependent calcium uptake in crude preparations of guinea-pig sarcolemma and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine
(5-50 mM) was found to have no significant effect on either 2+
ATP-dependent Ca binding or uptake in both preparations.
This result was observed at all calcium concentrations tested
(0.5-100 u_M) and at all incubation times used (30 seconds to
20 minutes). Taurine (20 mM) neither altered the effect of cyclic AMP-dependent protein kinase on oxalate-dependent calcium uptake nor exerted a stabilization action on calcium transport in these systems.
In a further attempt to determine the possible physiological role of taurine in mammalian tissues, we have investigated the effect of taurine on passive transport of sodium, potassium and calcium in synaptosomal preparations of rat brain. Taurine, in a dose dependent manner, was found to have an inhibitory effect on both calcium- uptake
iv and release in these preparations. Amino acids structurally similar to taurine ( 3- alanine, homotaurine,. hypotaurine and y - aminobutyric acid) were also shown to inhibit calcium uptake in these preparations while a - alanine, proline and valine had no significant effect. Taurine
(20 mM), though, did not alter the permeability of these preparations to either sodium or potassium. It thus appeared that taurine, and chemically related amino acids, can specifically alter calcium movements in these preparations.
It is suggested that this effect is due to the binding of these agents to taurine receptor sites postulated to be present in these membranes. These observations may help to provide an insight into the physiological and pharmacological effects of taurine reported in cardiac and nervous tissues.
Signed
Dr. D.A. Applegarth (Supervisor), Department of Pathology, Faculty of Medicine, U.B.C. and Biochemical Diseases Laboratory, Children's Hospital, Vancouver.
Signed .. . ,
Dr. Sidney Katz (Supervisor) Division of Pharmacology Faculty of Pharmaceutical Sciences, U.B.C.
v TABLE OF CONTENTS
INTRODUCTION
Page
Introduction 1
REVIEW OF THE LITERATURE
I. Historical Review 5
II. Biochemistry of Taurine 6
A. Distribution and Occurrence of Taurine .... 6 B. Taurine Metabolism 11
i. Carbamyltaurine H .ii. Taurocyamine 14 , iii. Isethionic Acid 15
C. Biochemistry of Taurine 17
i. Cysteine Sulfinic Acid Decarboxylase. '19 ii. Cystamine Dioxygenase Pathway ...... ^1 iii. Phosphoadenosine Phosphosulfate Pathway 24
D. Taurine Transport 26
III. Cardiac Disease and Taurine 30
A. Congestive Heart Failure 30 3. Hypertension 32 C. Ischemia 35
IV. Possible Physiological Actions of Taurine in the heart 35
A. Taurine and Arrhythmias 35 3> Taurine and Inotropism 39
vi Page
V. Possible Cardiac Effects of Taurine on Calcium Transport 43
A. Inotropism and Calcium Transport 43 B. Inotropism, Calcium Transport and Taurine 46 C. Calcium Movements and Taurine in Other Tissues 48
Summary 49
VI. Possible Involvement of Taurine in Neurophysiology 50
A. Anticonvulsant Action of Taurine 51 B. Taurine in Retinal Degeneration 5 3 C. Taurine in Brain Development 5 4 D. Effect of Taurine on Endocrine Function.. 57 E. Taurine and Nerve Conduction 58
Summary 60
VII. Rationale 62
MATERIALS AND METHODS
I. Studies with Isethionic Acid 66
A. Development of an Analytical Method for Isethionic Acid by Gas Liquid Chromatography 66
1. Reagents 66 2. Preparation of Isethionic Acid for use as Qualitative Standards 70 3. Methylation of Isethionic Acid 70 4. Silylation of Isethionic Acid 71
vii Page
5. Gas-liquid Chromatography: Flame Ionization Detector . .. 72 6. Gas-liquid Chromatography: Sulfur Detector • 72 7. Gas-liquid Chromatography: Mass Spectrometry 8. Nuclear Magnetic Resonance Spectroscopy.. 73
B. Analysis of Isethionic Acid in Mammalian Tissues 74
1. Reagents '. 74 2. Preparation of Heart, Brain and Other Tissues Used,for the Analysis of Isethionic Acid 75 3. Isolation of ISA from Tissues 77 4. Methylation of the Samples and Preparation of a Standard Curve 82 5. Analysis of Samples by GLC 83
C. Conversion of Taurine to Isethionic Acid ... 83
1. Reagents 83 14 2. Preparation of C-ISA as a marker for the Taurine Bioconversion Studies 86 3. Synthesis of"Isethionic Acid by Rat. Heart Slices ...... ' .—.-. 87
II. Studies on the Effect of Taurine on Ion Transport Processes • •••
A. Effect of Taurine on ATP-dependent Calcium Transport in Guinea-pig Cardiac Muscle 89
1. Reagents 89 2. Preparation of Heart Ventricle Homogenates 95 3. Preparation of Microsomes Enriched in Sarcoplasmic Reticulum 95
viii Page
4. Characterization of Microsomal Preparation Enriched Sarcoplasmic Reticulum 9 8 5. ATP-dependent Calcium Uptake and Binding Assay 6. Assay for Cyclic AMP-dependent Protein Kinase Effect on Calcium Uptake 100 7. Studies on the Effect of Taurine on the Decay of Ca2+ -Transport Activity 101 8. Protein Assay , . 101 9. Calculations 101 10. Statistics 102
B. Studies on the Effect of Taurine on Passive Ion Transport in Rat Brain Synaptosomes 102
1. Reagents 102 2. Preparation of Synaptosomes 104 3. Characterization of Synaptosome Suspension by Electron Microscopy 106 4. Determination of the Osmotic Behaviour of Synaptosomes 106 5. Determination of Sodium and Potassium Permeability 107 6. Determination of Calcium Permeability 107 45 7. Determination of Loss of Ca from Pre• loaded Synaptosomes 108 8. Protein Assay 108 9. Statistics 108
RESULTS
Studies with Isethionic Acid 112
A. Development of an Analytical Method for the Measurement of isethionic acid.._ 112
ix Page
1'. Chromatography of Methylated Isethionic Acid 112
a. Stationary Phases 112 b. Internal Standards 112 c. Mass-Spectrometry and NMR Spectra of Methylated Isethionic Acid 116
2 . Chromatography of Silylated Isethionic Acid. 119
B. Analysis of Isethionic Acid in Tissues 121
1. Isethionic Acid in Rat Heart and Brain Tissues 121 2. Isethionic Acid in Dog Heart Tissues 124 3. Isethionic Acid in "Molluscan Tissues 124 4. Isethionic Acid in Rat Milk Samples 125 C. Bioconversion of Taurine to Isethionic Acid.... 125
Taurine and Ion Transport 129
A. Effect of Taurine on ATP-dependent Calcium Transport in Guinea -pig Cardiac Muscle 129
1. Characterization of Ventricle Heart Homogenate and Sarcoplasmic Reticulum Enriched Preparation 129
2. Effect of Taurine on Calcium Uptake and Binding 133
3. The Effect of Taurine on the Time-course of Calcium Uptake and Binding 136
4. The Effect of Taurine on the Decay of Calcium Uptake Activity 137
5. Effect of Taurine on Cyclic AMP-dependent Protein-kinase Stimulated Calcium Uptake.... 137
x Page
B. Effect of Taurine on Passive Ion Transport in Rat Brain Synaptosomes 141
1. Characterization of Synaptosomal Preparation 141 2. The Osmometric Behaviour of Synaptosomes.. 141 3. The Effect of Taurine on Sodium and Potassium Permeability in Synaptosomal.. , Preparation 144 4. The Effect of Taurine on the Passive Uptake and Release of Calcium in Synaptosomal Preparations 144 5. Dose-dependent Effect of Taurine on Calcium Uptake in Synaptosomal Preparation 146 6. Effect of Other Amino Acids on Calcium Uptake in Synaptosomal Preparations 149
DISCUSSION
I. Bioconversion of Taurine to Isethionic Acid in the
Regulation of Ion Flux 152
II. Taurine and Ion Transport 168
CONCLUSIONS 189 BIBLIOGRAPHY 190
APPENDICES ...... v.-: ...... • • • ...... 225
xi LIST OF TABLES
Page
Table 1: Isethionic acid in tissues analyzed 123
2: Conversion of 14 C-taurin. e to 14 C-isethionic acid by rat heart slices 127 3 3: H-Ouabaih*binding assay of microsomal enriched S.R. preparations 132 4: Effect of taurine on calcium uptake and binding in guinea-pig heart ventricle homogenates and S.R. enriched preparations.. 134
5: The effect of taurine on calcium uptake and binding at various calcium concentrations in guinea-pig heart ventricle homogenates and S.R. enriched preparations 135
6: The effect of taurine on cyclic AMP-dependent protein kinase-stimulated calcium uptake in guinea-pig heart ventricle homogenates and S.R. enriched preparations 140
7: The effect of taurine on sodium (A) and potassium (B) permeability in synaptosomes.. 145 8: The fragmentation and rearrangements of the two methylation products of isethionic acid that were analyzed using an OV-17 column.... 154
xii LIST OF FIGURES
Page
Figure 1: The structure of taurine and its metabolites 12
2: Schematic diagram illustrating the pathway
of taurine biosynthesis in mammals 18
3: Metabolic pathway of cysteine to taurine.... 20
4: Biosynthesis of taurine via cysteamine 22
5: Structure of Coenzyme A 25
6: Flow chart of the isolation of isethionic acid from tissues 78 7: Flow diagram for the preparation of heart microsomes enriched in sarcoplasmic reticulum 96
8: Flow diagram for the preparation of rat brain synaptosomes 106
9: Chromatographic separation of the products of methylation of isethionic acid and salicylic acid using a flame ionization detector 113
10: Chromatographic separation of the products of methylation of isethionic acid and 1-butahe sulfonic,acid using a flame photometric (sulfur) detector
11: Mass spectra of the products of methylation of isethionic acid 117
12: Nuclear magnetic resonance spectra of the products of methylation of isethionic acid (A), 1-butanesulfonic acid (B) and methoxy- ethanol (G) - . v...... 118
13: Time course of chromatographic behaviour of the silylated products of isethionic acid.... 120 14: Calibration curve of methylated isethionic acid 122
14 14 15: Separation of C-taurine, C-isethionic acid and the rat heart slices - l^c-taurine . incubation products by paper chromatography...128
xiii Page
Figure 16: Electron micrograph of microsomal preparations enriched in sarcoplasmic reticulum 130
17: Electron micrograph of the guinea-pig ventricle heart homogenate preparation... 131
18: Time course effect of taurine on calcium uptake and binding in guinea-pig heart ventricle homogenate and sarcoplasmic reticulum enriched preparations 137
19: The effect of taurine on the decay of calcium uptake activity in guinea-pig ventricle homogenates and S.R. enriched preparation 139
20: Electron micrograph of a typical rat brain synaptosomal preparation 14 2
21: The effect of Na2SO^ concentration on the E520 °f a suspension of synaptosomes 143 45 2+ 22: The effect of taurine on (A) Ca uptake
and (B) release of 45ca2+ from preloaded rat synaptosomal preparations 147 23: The effect of various concentrations of taurine on 45caCl2 uPtake in brain synaptosomal preparations 148
24: The effect of various amino acids on calcium uptake in brain synaptosomal preparations 149
25: Mass spectral rearrangements and frag• mentation of dime thy lated- isethionic acid 155
26: Mass spectral rearrangements and frag• mentation of the methylester of isethionic acid 156
xiv ACKNOWLEDGEMENTS
The author wishes to extend his deepest gratitude to Dr. D.A. Applegartha,b and Dr. Sidney Katz° for their encouragement and guidance throughout the course of this study. Without their assistance, this work would have been impossible.
I would like to extend my thanks to Dr. W.L. Dunn ,
Dr. R.H. Pearceb, Dr. P.E. Reidb, Dr. D.E. Brooksb and
L.I. Woolf for their suggestions at the graduate committee meetings and helpful criticism of the Original manuscript.
I wish to thank the staff of the Biochemical
Diseases Laboratory at the Children's Hospital for their cooperation and assistance, during my work (from September
1974 to May 1977) in the laboratory.
Department of Paediatrics, Division of Paediatric Pathology, UBC and Biochemical Diseases Laboratory, Children's Hospital.
Department of Pathology, Faculty of Medicine, UBC.
Division of Pharmacology, Faculty of Pharmaceutical Sciences, UBC.
Division of Neurological Sciences, Department of Psychiatry Faculty of Medicine, UBC.
xv I also wish to thank all members of the Faculty, staff and graduate student body in 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 staff of the Woodward
Biomedical Library of the University of British Columbia for their constant kindness and helpfulness in tracing down references and locating recondite journals.
Financial support in the form of studentship, and for the cost of equipment and chemicals from British
Columbia Heart Foundation to Dr. D.A. Applegarth is gratefully acknowledged.
Finally I would like 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 their helpful comments and assistance.
Faculty of Pharmaceutical Sciences, UBC Department of Paediatrics, Faculty of Medicine, UBC, and Department of Clinical Investigation Facility, Children's Hospital, Vancouver.-
xvi Dr. T.R.C. Boydey for his inspiration.
Dr. Brenda Morrison for her assistance with
Statistical Analysis.
Dr. Frank Abbott1 for his helpful comments and assistance with the mass spectral interpretations.
Mrs. Celine Gunawardene1 for doing an excellent job of typing the final copy of the manuscript.
My parents and brothers for their encouragements and understanding.
y Makerere University, Kampala, Uganda, East Africa. (Present Address: Department of Biochemistry, University of Hong Kong, Hong Kong). k Department of Health Care and Epidemiology, University of British Columbia.
1 Faculty of Pharmaceutical Sciences, U.B.C.
xvii DEDICATED
TO
THE CAUSE
OF
SCIENCE
AND
MEDICINE ABBREVIATIONS USED
ADP adenosine 5--diphosphate ATP adenosine 5'-triphosphate ATPase adenosine triphosphatase BSA N, O-bis-(trimethylsilyl)-acetamide °C degree centigrade Ci Curie u Ci microCurie
2+ 2+ 2+ (Ca +Mg )-ATPase Mg -dependent calcium stimulated ATPase CHF congestive heart failure CNS central nervous system cm centimeters CoA Coenzyme A cpm counts per minute CSA cysteine sulfinic acid Cyclic AMP adenosine 3', 5' - monophosphate DEGS diethylene glycol succinate DMCS dimethylchlorosilane DMF dimethylformamide E520 extinction at 520 nanometers E.C. enzyme classification, established by the commission on enzymes of the International Union of Biochemistry
(Biochim. Biophys. Acta 429:lf 1976) eds. editors EGTA Ethyleneglycol -bis- ( B-Aminoethyl ether) N,N:?-tetra-acetic acid EKG electrocardiogram EM electron microscopy ERG electroretinogram et al. and others ft feet
xix g gram
x g acceleration of gravity
GABA Y -aminobutyric acid
GC-MS gas-liquid chromatography mass spectrometry GLC gas-liquid chromatography HMDS hexamethyldichlorosilane Homotaurine 3-aminopropane sulfonic acid hr. hour
Hypotaurine 2-aminoethylsulfinic acid i.d. internal diameter ISA isethionic acid Isethionic acid 2-hydroxyethane sulfonic acid Kg killogram
K m Michaelis constant yl microliter ml milliliter M molar m milli y micro yM micromolar mEq milliequivalent MEA mercaptoethalamine mg milligram min. minute mM millimolar mm millimeters N normal
NAD:: nicotinamide adenine dinucleotide
Na+,K+-ATPase Sodium-potassium dependent ATPase Na-ISA Sodium salt of Isethionic acid
NMR nuclear magnetic resonance spectroscopy OV-1 methyls!licone OV-17 methyl phenyl silicone
xx P probability
PAPS 3'-phosphoadenosine-5'-phosphosulfate
PEGS polyethylene glycol succinate
Pi orthophosphate
Rf retardation factor
S.E.M. standard error of the mean S.R. sarcoplasmic reticulum
Taurine 2-aminoethane sulfonic acid TMCS trimethylchlorosilane Tris tris(Hydroxymethy1) aminomethane V maximum velocity max J v/v unit of volume per unit of volume w/v unit of weight per unit of volume % percent / per < less than > greater than £ equal to or greater than
xxi INTRODUCTION 2
Introduction
Taurine (2-aminoethanesulfonic acid) is a sulfonic
acid analogue of B- alanine (Figure 1). It is present in
all mammals examined (rat, guinea-pig, mice, dog,- ' ~ .
rabbit, horse, chicken, cow, monkeys,-pig, man, .hamster,
sheep, cat, etc) as a free amino acid (in relatively high
concentrations). It occurs to only a limited extent in plants, primarily in lower forms. It is not utilized for protein synthesis (Banos et al., 1971) nor as a source of
energy (Hayes, 1976). The single clearly defined function
of taurine in animals is the formation of bile salts which
serve as emulsifying agents in the gut to facilitate lipid
digestion.
Taurine is present in high concentrations in the
rat skeletal muscle (14 w moles/g), heart (28 u moles/g)
and, in various areas of the brain (1 to 100 u moles/g) . It
does not seem logical that taurine could be present in the mammalian body with little or no further function. (Gaull,
1971). This line of thought has triggered many workers to
evaluate the possible physiological role of taurine in mammals. The evidence concerning the possible function of
taurine in the heart .(Huxtablerl976a;; „Grosso and Bfessler,
1976) and brain (Sturman et al., 1977a; Barbeau et al., 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 in the literature, do not establish clearly the role of taurine in mammalian tissues. This thesis is concerned with investigations on a possible physiological role of taurine in the regulation of ion transport. REVIEW OF THE LITERATURE 5
I. HISTORICAL REVIEW
As far as it can be established, the name taurine did not appear in the literature until 18 38 where it is found
in a paper by Demarcay(18 38). A .crystalline - material from Ox bile was obtained, similar to a compound called gjaylen-a-sparagin described 11 years earlier by Tiedman and Gmelin (1827) who
termed it taurine, a name which Demarcay credited to Gmelin without indicating when Gmelin had first used that name.
In 1846, Redtenbacher demonstrated that taurine,
contained both sulfur and nitrogen. Its structure at this point was delineated. Evidence for the presence of taurine
in 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 distribution in animals. This view
has been fully supported by numerous reports on the distri-
bution and occurence of taurine published by recent investi•
gators (Huxtable and Bressler, 1974a, 1974b; Grosso and
Bressler, 1976; Kocsis et al., 1976; Perry and Hansen, 1973;
Sturman and Gaull, 19 75). 6
II. BIOCHEMISTRY OF TAURINE
A. Distribution and Occurence of Taurine
In mammals, taurine is present in all tissues of the body, with skeletal muscle accounting for 75 per cent of the total body store (Awapara, 1956). In addition to skeletal 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 rat heart consists of taurine (Awapara et al, 1950; Scharff and
Wool, 1965). The concentration of taurine in muscle or heart ranges from 5 to 40 ymoles/g wet weight and in 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 distribution of taurine in canine ventricle has recently been reported (Kocsis et al., 1976;Crass and -Lombardini, 19 7 8). - Higher taurine concentrations are found in the endocardium than in the epicardium; no evidence of a base-to-apex taurine gradient was found in either the right or left ventricle of the dog. No significant differences were observed.between the mean values for right and left ventricles or right and left auricles. However, differences in taurine levels in auricles and ventricles were found from species to species. For example, the auricles of dog contain twice as much taurine as the ventricles, whereas rats, guinea- 7
pigs and rabbits each showed higher taurine concentration in the ventricles compared to the auricles. The Purkinje tissue of the heart contained the highest concentration of left ventricular taurine.
Taurine levels in 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 in olfactory bulb, cerebral cortex, cerebellum, and striatum; (5 to 11 umoles/g) the lowest in 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 in animal tissues (30-100 pmoles/g) (Guidotti et al., 1972; Vellan, et al., 1970; Pasantes-Morales et al. 1972). Studies on the subcellular distribution of taurine in brain fractions suggest that it is present in synaptosomal fractions
(5 to 26 ymoles/g protein) as well as in the mitochondrial fraction (Lombardini, 1976; Kaczmarek et al.,1971; Sturman et al., 1976b; Rassin et al., 1976, 1977).
Huxtable and Bressler (1972) studied the subcellular 14 . distribution of radioactivity from (1,2 - C) taurine in rat heart after intraperitoneal injection. Of the total activity,
85 per cent of taurine was found in the supernatant fraction of a. homogenized-tissue-preparation. Approximately 12 per cent 8 was bound to the microsomal fraction and less than 2 per cent in the mitochondrial fraction. Measurements of endogenous taurine in beef heart revealed 17.2 umoles/g protein in the microsomal fraction, 1.5 umoles/g protein in the-bulk precipitate and 0.0 ymoles/g protein in the mitochondrial fraction.
35 The distribution of injected S-taurine in different organs has been determined in several species. In vivo 35 experiments in 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 their rates of uptake. The organs that take up taurine rapidly are liver, pancreas and kidney.
Those that take up taurine slowly are skeletal muscle, heart and brain. The organs that accumulate taurine at a slower rate contain maximum amount of the total radioactivity after 3 to 5 days following a single injection. This reflects a redistri• bution of the labeled taurine from the other organs. The loss of radioactivity is also correspondingly slow in skeletal muscle, heart and brain (Hope, 19 55; Awapara, 1957; Huxtable and Bressler, 1972; Sturman, 1973).
Recently studies have shown that there is an effective blood-brain barrier preventing the passage of taurine into the mature brainv This was observed after the in-vivo intraperitoneal injection of massive doses of taurine in a number of species such as rat, mouse, guinea-pig and cat resulted in no significant increases in taurine in most 9 areas of the brain (Battistini et al., 1969; Levi et al., 1967).
However, there does seem to be some exchange of taurine occurring, because after intraperitoneal injection or oral administration of tracer amounts of radioactive taurine to rats, measured amounts of label appear in the brain (Urquart et al., 19 74;
Peck and Awapara, 196 7).
Sturman et al., (1976a) injected humans with 35 .
S-taurine and determined pool sizes by kinetic analysis of plasma, urine and feces. Two pools for taurine were found; one pool was relatively 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 participates in bile acid conjugation and is metabolized to sulfate,carbon dioxide,water and ammonia and probably to isethionic acid,, • by gut bacteria and removed by urinary excretion. Unchanged taurine is also lost from the rapidly exchangeable pool through urinary excretion. Concentrations of: taurine in the slowly exchangeable pool (skeletal muscle, heart and brain)are maintained constant. Much effort has been expended in 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 diet deficient in vitamin Bg (essential substance for the rate limiting enzyme 'cysteine sulfinic acid decarboxylase' activity in the biosynthesis of taurine from methionine or cysteine) were found to have unaltered concentrations of taurine. A similar conservation of taurine in the heart occurs in rats fasted for prolonged periods. Feeding a taurine-rich diet has no effect on cardiac taurine concentrations since the additional taurine is simply excreted in the urine and feces
(Sturman,. 1973; Awapara, 1956; Hope, 1955).
A recent report from Sturman and his group (Knopf et al. , 19 78) and Hayes, .(1976) have indicated that cats maintained on a taurine-deficient diet show marked depletion of taurine in a number of tissues, including the heart.
Carnivores generally obtain a taurine rich diet since taurine occurs in high concentrations and ubiquitously in animal
tissues- Herbivores, on the other hand, receive a poor taurine
diet,.as it is almost absent in the plant world. In this
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, in the absence of
dietary taurine, they are no longer capable of biosynthesizing
sufficient taurine for their needs. 11
B. Taurine Metabolism
In addition to the bile acids the three other
main reported metabolites of taurine are:
(i) Carbamyl taurine (Ureidotaurine)
(ii) Taurocyamine (Guianidinotaurine)
(iii) Isethionic Acid (2-hydroxyethane sulfonic acid)
The structures of these metabolites are shown in Figure 1.
i• Carbamyltaurine
Carbamyltaurine was first reported in human and
canine urine by Salkowski (1873). Since that report,
urinary excretion of carbamyltaurine has, been both
confirmed (Schram and Crokaert, 1957; Thoai, et al.,
1956) and denied (Schmidt and Cerecedo, 1928). Thoai,
et al. postulated in 1958 that the reason for the presence
of carbamyltaurine in mammals was that it formed part
of a modified urea cycle based on taurine in 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
S03H SO3H S03H SO3H
CARBAMYL TAURINE TAUROCYAMINE ISETHIONIC ACID TAURINE
FIGURE 1
THE STRUCTURE OF TAURINE AND ITS METABOLITES O'Keefe and Smith (1973), using, a specific isotope
dilution technique, could not detect carbamyltaurine in human
urine. Since carbamyltaurine was previously reported to occur
in rat urine (Schram and Crokaert, 1957), O'Keefe and Smith
examined homogenates of rat brain and liver for the ability
of these tissues to synthesis carbamyltaurine using carbamyl-
phosphate as carbamylating agent (in systems capable of
synthesizing citrulline or carbamylaspartate). No synthesis
could be detected. The early work on the identification of
carbamyltaurine in the urine of man, rat and dog could possibly have been an artifact of the combination of taurine and urea
to form corresponding ureido acid (as shown by the chemical equation below) occuring in urine after passage through the kidney.
CONH2
NH
NH 7 2 CH / I
CO -I- NH2CH2CH2S03H } NH^ + CH2
UREA TAURINE AMMONIA CARBAMYLTAURINE
This reaction has been reported to occur for the amino acids, tyrosine and phenylalanine in urine giving corresponding ureido amino acids (Schmidt and Allen, 1920).
It is possible that the taurocyamine detected some 100 years 14
ago (Salkowski, 1873) in urine was a result of the process
of isolation of this compound from the urine and not due
to chemical processes occuring in the body as suggested
by Thoai et al., (19 56). At the present time, evidence is
that carbamyltaurine is not a metabolic product of
taurine metabolism in mammalian systems. ii. Taurocyamine
Taurocyamine, is important as its phosphogen
(phosphotaurocyamine) in marine worms where it occurs
consistently in large concentrations in all marine worm
species examined (Thoai and Roche, 19 60, Thoai and Robin
1965). In these species phosphotaurocyamine is believed
to function as part of a phosphagen system analoguous to
the creatine system found in human muscle. Taurocyamine
is believed to function as a phosphate acceptor and
taurocyamine phosphokinase has been isolated from marine
worms (Thoai and Roche 1960). Phosphotaurocyamine has not
been identified in tissues of mammals (Jacobsen and Smith,
196 8) but taurocyamine hasbbeen identified in rat brain
(Blass, 1960) and in the urine of rats and man (Schram
and Crokaert, 1957; Thoai et al., 1954). Mori et al.
(19 74) have quantitated taurocyamine in brain samples of
mice, guinea-pigs, rabbits, rats and man. In these tissues
it occurs at a concentration of 0.002 umole/g, approximately 15
1/1,000th. the taurine concentration. The guanidino
compounds (Mori et al., 19 74) occur in the human brain
in order, according to concentration:
Arginine>guanidinobutyric>acid>glycocyamine>taurocyamine.
The importance of taurocyamine in the brain is not
established but it does not appear to be a quantitatively
important constituent of the tissues examined.
iii.Isethionic Acid
The presence of isethionic acid (ISA) in biological
material was first reported by Koechlin (1955) who found
that it 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 isethionic acid was found in squids
and molluscs generally, but was lacking, or present in
low concentration, in crustacean and vertebrate nerve.
Welty et al., in 1962, reported the isolation and
identification of isethionic acid from dog heart. They
also claimed to show the conversion of cysteine and
taurine to isethionic acid in dog heart slices (Read and
Welty, 1962). More recently, the formation of isethionic
acid from taurine has been described in homogenates of
rat brain (Peck and Awapara, 1966). 16
Several studies have examined the presence of isethionic acid in man: Goodman et al. (1967) have reported the presence of 35S-.isethionic acid in the urine of normal subjects and.children with Downs syndrome given 35
S-taunne. Jacobsen et al. (1967) , using a sensitive and specific double-isotope derivative method, reported the first quantitative data on the urinary excretion of isethionic acid in man. Patients with muscular diseases accompanied by muscle atrophy were found to excrete significantly less isethionic acid compared to levels excreted by patients with honmuscular diseases.
Interest in isethionic acid stemmed from the identification of the compound in dog heart tissue
(Welty et al., 1962) and the claim that dog heart slices were able to convert taurine to isethionic acid
(Read and Welty, 1962). Huxtable (1976a)proposed that the conversion of taurine to isethionic acid proceeds through the intermediary sulfoacetaldehyde. An analogy is the conversion of glycine to glycolate through glyoxylate.
The enzymatic mechanism 'of the conversion has hot been established. An interesting aspect of this transformation of taurine to isethionic acid is that a zwitterionic compound is being converted to a strongly anionic one leading to the proposition that isethionic acid is involved in membrane excitability (Mullins, 19 59) and 17
conductance of cations across cardiac cell membrane
(Read and Welty, 1963, 1965; Chazov et al., 1974). A
detailed mechanism has never been put forward. It has,
however, been suggested that the deamination of taurine
to isethionic acid within the cell would release a
charged group that would attract cations (Welty, 196 3;
Doctoral thesis, University of South Dakota). The
question of the role of isethionic acid in the conductance
of cations in mammalian tissue has been of interest to
numerous workers (Jacobsen et al., 1967; Dietrich and
Diacono, 1971; Guidotti, et al., 1971; I-Iuxtable and
Bressler, 1972; Spaeth and Schneider, 1974; Sturman, 1973),
particularly in its implication in the antiarrhythmic
effects in dog hearts (Read and Welty, 1965; Chazov et al.,
1974). The major obstacle to the elucidation of the
function of isethionic acid has been, the lack of good
analytical procedures.
C. Biosynthesis of T.aurine
Taurine biosynthesis is a complex problem. Several
routes have been established or postulated depending upon the
animal species and tissue of origin. (Fig;2) .The-biosynthesis of
taurine from cysteine has been well characterized in rat
liver (Jacobsen and Smith, 1968). Synthesis of taurine in
cardiac tissue is poorly understood, but does not appear to CO
Figure 2. Schematic Diagram Illustrating the Pathways of Taurine Biosynthesis in Mammals. 19 occur by the same pathway operative in the liver (Jacobsen et al., 1964; Wainer, 1965). The rat, dog and cat heart, and human and cat liver either lack key enzymes necessary for converting cysteine to taurine by the same route used in rat liver (Jacobsen and Smith, 196 8) or can not convert cysteine to taurine in sufficient quantities to maintain tissue pools. i. Cysteine Sulfinic Acid Decarboxylase
In rat liver, cysteine is oxidized to cysteine
sulfinic 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
limiting enzyme in this pathway is cysteine sulfinic
acid decarboxylase. Alternatively, cysteine sulfinic
acid may be oxidized to cysteic acid (Awapara and Wingo,
1953), which is then decarboxylated to taurine.
Available evidence indicates that the preferred pathway
in the liver is via hypotaurine (Awapara, 19 53; Awapara
and Wingo, 19 53), though the oxidation of hypotaurine
to taurine is poorly understood; one brief report has
appeared on the enzymatic conversion of hypotaurine to
taurine in rat liver-(Sumizu, 1962). In this 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. Fiori and Costa (1969)
were unable to reproduce these results; they suggested
that hypotaurine is oxidized by small amounts of hydrogen 20
SH .
^2
CH NH,
COOH
cysteine
(10
S02H SO„H -CO, I2 CH„ CH„ (2) I CH2NH2 CHNH_ i OOH hypotaurine
cysteine sulfinic acid (3) 1(4)
S03H SO_H 1 ~> - C02 H CH, f2 (2)
CHNH, CH2NH2
COOH
cysteic acid Taurine
Figure 3. Metabolic Pathways of Cysteine to Taurine 1. Cysteine dioxygenase (E.C. 1.13.11.20) 2. Cysteine Sulfinic 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 in tissues. However, Oja et al.,(19 7 3)
found that the conversion of hypotaurine to taurine in rat brain slices 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 in vivo administration of potential metabolic precursors
such as cysteine sulfinic acid, cysteic acid or hypotaurine (Huxtable ..arid Bressler, 1976). This is in
contrast to the elevation seen in liver and other tissues
under similar conditions (Jacobsen and Smith, 1968;
Huxtable and Bressler, 1976). Thus taurine may not be
synthesized directly from cysteine in the heart as it
is in liver and brain. A potential alternative source
of taurine in the heart is the Cysteamine Dioxygenase pathway. (Fig.4) During studies on the pharmacology of
cys.teamine, a powerful radioprotectant, it became
evident that this compound could be metabolized to
taurine and that a pathway involving cysteamine as an
intermediate may be functional in heart tissue
(Eldjarn, 1954; Verly • and Koch, 1954). Eighteen 35 hours after the injection of S-cysteamine into mice
or guinea-pigs, radioactivity is present largely as CH. ,-(! —CH -C - NH. CH2 CH2. C02H
OH CH3 OH PANTOTHENATE
H2N-CH2CH2SH CH?— C — CH — C- NH-CH2CH2C02H I-I 1 CYSTEAMINE OPO3H M« OH 4'PHOSPHORANTOTHENATE
H2N-CH-CH2SH
C02H
CYSTEINE
CH-, E—cn—?-NH-CH2CH2C,NH.CH-CH2SH 1 1 1
C02H iP03H Me OH >i' - PHOSPHOPANTOTHENOYL CYSTEINE Q Me 0 0 I I * I -* CH9- C -CH C.NH.CH2CH2 C.NH.r.H2CH2SH CH2— C— CH-C-NH-CH2CH2.CNH.CH2 CH2 SH III OH. Me OH PANTETHEINE OPO^H'Me OH T-PHOSPHOPANTETHEINE
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 in most organs, although in muscle and brain the uptake of cysteamine is extremely low. In vitro, when rat or mouse heart homogenates are incubated in the presence of labeled cysteamine, radioactive taurine is produced (Huxtable and Bressler, 19 76).
The conversion of cysteine to cysteamine, which is required for the cysteamine pathway, has been observed in kidney, heart, brain, liver and skeletal muscles of mice and guinea-pigs. However, cysteine decarboxylase activity has not been detected. Cavallini et al. (1976) have proposed the formation of cysteamine in 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 in this pathway is that cysteamine is a highly toxic compound when it is administered . 24
(2 mmole/kg body weight) exogenously to rats and mice
(Huxtable and Bressler, 1976) .
Alternatively the mercaptoethylamine (MEA)
resulting from the degradation of the pantetheine or
coenzyme A (CoA) may act as a substrate which can be
converted to taurine (Fig. 5) (Dupre et al., 1973).
Cysteine is enzymatically linked to phosphopantothenic
acid by an amide bond to form pantetheine or CoA
(Brown; 1959). However, insufficient information is
available regarding the turnover rate of CoA in the
heart to allow a critical appraisal of this pathway. iii. Phosphoadenosine Phosphosulfate (PAPS) Pathway
The biosynthesis of taurine from inorganic sulfate
in chick (Miraglia et al., 1966) and rat liver (Martin
et al., 19 72) has been reported. Serine has been
proposed as the organic acceptor of inorganic sulfate
for the synthesis of taurine (Sass and Martin, 1972).
Sulfate is believed to be activated by formation of
3'-phosphoadenosine-51-phosphosulfate (PAPS) from
which the sulfate is transfered to a dehydrated serine
intermediate eg. a -aminoacrylic acid. The resultant
cysteic acid is then decarboxylated to taurine while
cysteic acid is in a protein bound form (Martin et al., CN PANTETHEINE
PANTOTHENIC ACID
0 0 ^3 0 0
CH2-0-§-0-^-0-CH2-C-CI/-C-NH-(CH2)2-C-NH-CHo-CH9-SH
H OH
e-MERCAPTOETHYLAMI NE
HO 0-P03H2
ADENOSINE-3'-PHOSPHATE-5'-PYROPHOSPHATE
COENZYME A
FIGURE 5 - Coenzyme A. 26
1972; Gorby and Martin, 1975). The validation of this
pathway was based on paper chromatographic analysis
of products resulting from incubation of tissue 35
fractions with S-PAPS. Other reports have shown that
incorporation of inorganic sulfate to taurine did not
occur in rat heart homogenate (Dziewiatkowski, 19 54;
Green and Robinson, 1960; Huxtable, 1978). Therefore,
at the present time, the significance of this pathway
in the heart is open to doubt.
It had been previously suggested that cats were
able to synthesize taurine directly from sulfate
(Rambaut and Miller, 1966). However, Sturman and his
group (Knopf, et al. , 1978) and Hayes (1976-) found 35
that injection of S-SO^ in cats did not result in
detectable levels of labeled taurine in any tissue
examined even when cats highly deficient in taurine
were used. Hayes (1976) has suggested that taurine is
an essential nutrient in cats and man.
D. Taurine Transport
Circulating taurine derived via biosynthesis or
from a dietary source is taken up by various tissues (Awapara, 1-956 ;
Sturman , -19 73; Hope, .195 5) .. The concentrations of taurine in the cell water of growing Ehrlich tumor
ascites cells and HeLa cells are approximately "1,000i and . 27
7,000 times greater, respectively, than the concentration in the surrounding medium. This suggests the possibility that the gradient is maintained by an active transport process. Kromphardt (1963) has shown that the uptake of taurine by Ehrlich tumor ascites cells is inhibited by
2,4 dinitrophenol and anoxia. A number of reports on in-vivo and in-vitro systems have evaluated the characteristics of taurine uptake in the heart, brain, kidney, platelets, retina (Chesney et al., 1976; Starr and Voaden, 1972;
Edwards, 1977; Ahtee et al., 1974; Gaut and Nauss, 1976;
Lahdesmaki and Oja, 1973; Kaczmarek and Davidson, 1972;
Kontro and Oja, 1978; Hruska et al., 1976). Results of all of these investigations indicate that taurine uptake in the tissues was an active transport process. In these studies, it was also shown that taurine uptake was competitively inhibited by 3 - alanine and hypotaurine and compounds possessing a primary amine and an acidic group, separated by two methylene carbons. Deviation from a close structural resemblance to taurine resulted in decreased inhibition by other amino acids examined.
Two recent reports have characterized active
transport processes of taurine in the isolated perfused
rat myocardium (Chubb and Huxtable, 1978a)and in vitro
cultured foetal mouse hearts (Grosso et al. , 1978-b). . It was demonstrated that in the foetal mouse heart taurine
influx was via a carrier-mediated transport-system and was 28
temperature-dependent, saturable and had structural selecti•
vity for 8-amino acids. In addition, Grosso et al.,(19 78b)
observed that taurine uptake was sodium-dependent, energy-
dependent and that foetal mouse hearts were capable of
accumulating taurine against a concentration gradient. Using
isolated perfused rat hearts, Chubb and Huxtable (1978a)
determined taurine influx over a concentration range of 25 to
400 JJM. Taurine influx was saturable at a concentration of approximately 200 y_M. The transport system had a Michaelis
constant (Km) of 45'yM and a Vmax\ of 32 nmoles/g dry weight /min indicating that taurine influx is mediated by a transport process of relatively high affinity. Specificity and inhibition of taurine influx were examined by perfusing hearts with other radiolabeled amino acids. g-alanine, which is structurally similar to taurine, inhibited taurine influx approximately sevenfold, whereas the a-amino acids: a-aminoisobutyric acid, leucine and serine did not markedly alter taurine influx. These results indicate that at least two types of influx sites for amino acids exist in the heart, one mediating 6-amino acid influx, the other a-amino acid influx (Christensen and Liang, 1956). The slight depression in taurine influx caused by competing a-amino acids may indicate that a small percentage of taurine influx is mediated by a-amino acid sites (Chubb and Huxtable, 19 77) 29
Several workers have reported taurine transport systems in synaptosomes isolated from rat brain (Schmidt et al., 1975; Hruska et al., 1977; Kontro and Oja, 1978;
Lombardini, 1976). Hruska et al. (1977) have described high affinity sodium-dependent transport of taurine into rat brain synaptosomes. Kinetic analysis indicates a high- affinity 1^ value of 3.20 u_M and a.V value of 5.35 nmoles/g protein/minute. The kinetic constants of the low- affinity system were 3340 y_M and 699 nmoles per g protein/ minute. The regional distribution of uptake showed that the midbrain, thalamus and olfactory bulbs had the highest velocity of transport, while the cerebral cortex, spinal cord, and cerebellum had the lowest V : for transport. 30
III. CARDIAC DISEASE AND TAURINE
A. Congestive Heart Failure
Taurine concentrations in the heart are elevated in various states of natural and experimentally-induced cardiac pathology. Peterson, Read and Welty (19 73) demonstrated increased levels of taurine in experimentally- induced right sided congestive heart failure in dogs.
Right ventricular hypertrophy and congestive heart failure
(CHF) were produced in dogs by progressive pulmonary artery stenosis. In this model, the elevation of taurine was only seen in the right ventricle and not the left. Statistically significant increases in free amino acid concentrations of the right ventricle occured only for two compounds, taurine and methionine. There was no elevation of taurine or methionine in the nonhypertrophied left ventricle.
Huxtable and Bressler (1974a, 1974b) reported similar increases in taurine levels in cardiac left ventricle of patients dying of chronic congestive heart failure. 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 in the left ventricle of the heart was doubled in patients who had died of chronic congestive heart failure compared to patients who had died of other causes and had no cardiac pathology.
The increase in taurine was specific to the heart in that no corresponding rise in taurine concentration was observed in aorta or skeletal muscle.
Newman et al. , (19 77) examined the relation between myocardial taurine content and severity of congestive heart failure. Dogs with heart failure were compared to normal controls. Heart failure was induced by creating an infrarenal aortocaval fistula. Taurine concentrations were determined in the left and right ventricles and then related to pulmonary wedge pressure. Pulmonary wedge pressure was significantly increased in dogs with congestive heart failure as was the taurine content in the left and right ventricles:
The wedge pressure in CHF dogs ranged from 6.6 to 28 mm Hg and 2.5 to 7.5 mm Hg in normal dogs. Taurine concentrations in the ventricle ranged from 17 to 153 umoles/g protein in
CHF animals; and 18 to 49 ymoles/g protein in normals. No
significant difference between taurine content of the left and right ventricles of either normal or CHF dogs was observed.
Linear regression analysis of myocardial taurine content of either the left or right ventricle yielded a highly signi•
ficant correlation with the pulmonary wedge pressure. 32
B. Hypertension
Huxtable and Bressler (1974a) also reported a positive correlation between cardiac taurine concentrations and increases in blood pressures averaged over a period of weeks before death. Patients with an average systolic blood pressure of 145 mm had ventricular taurine levels of
8.5 pmole/g., whereas patients averaging 108 mm systolic pressure had taurine levels of 5.0 .pmole/g. A similar relationship was reported for diastolic pressure.
In experimental stress-induced hypertensive rats,, there was a doubling in taurine concentration in the whole heart (expressed relative to protein) with no accompanying taurine concentration changes in muscle or brain (Huxtable and Bressler, 1974b). The hypertensive male rats of the
Okamoto strain (Okamoto and Aoki, 1963) also showed an increase in heart taurine when compared to age-matched
Wistar control rats. However, rats which were stressed and who had developed cardiac hypertrophy but not hypertension,
showed no alteration in the taurine to protein ratio.
Therefore, the increase in cardiac taurine concentration was due entirely to the development of hypertension and not to hypertrophy of the heart.
The significance of these alterations in taurine
levels seen in cardiac disease is unclear. There is,
usually, a general increase in free amino acid content of
the heart in cardiac hypertrophy which is probably related 33
to increased protein turnover and synthesis. Peterson et al.
(1973), for example, found increased levels of all free
amino acids in the right ventricle in right-sided heart
failure in dogs, although statistically significant increases
were observed only in the cases of taurine and methionine.
If the increase in taurine was a response to the development
of congestive heart failure, then it can be postulated that
by increasing taurine levels the heart is making available
an increased amount of an endogenously inotropic agent
(inotropic effects of taurine have been shown in guinea-pig
and rat hearts). The high taurine levels in CHF could
equally indicate that taurine is toxic at such levels.
According to Huxtable (19 76a) the changes in taurine concen•
tration that do occur in 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 influx into heart cells is regulated through
^-activation of the adrenergic system. One of the major
mechanisms whereby the heart increases its output under
work stress is via the 0-adrenergic system. Prolonged
stimulation of this system causes an increase in heart
mass-cardiac hypertrophy-and., if the stress is severe and
long-lasting, eventually congestive heart failure will
occur. Studies were designed to determine whether . .... 34
metabolic or transport processes affecting taurine concen• tration were modified by cardiac stress. Isoproterenol, a ^-adrenergic agonist, was used to produce a high output stress on the heart. Isoproterenol given to rats for periods of up to 10 days produced a cardiac hypertrophy accompanied by a marked increase in total taurine content of the heart (Huxtable, 1976b). No alteration was observed in the rate of taurine synthesis, as measured either by the overall conversion of cysteine to taurine, or by the activity of cysteamine dioxygenase (E.C. 1.13.11,19). However, increases in the rate of taurine influx were observed:
Taurine, in concentrations of 25 to 200 u_M, was perfused through the heart. The Lineweaver-Burke plot of taurine influx showed a Michaelis constant (K_) of 45 uM and a m —
v maximum velocity ( max) of 32 nmole/g of tissue dry weight per minute. Addition of isoproterenol to the perfusion medium resulted in an immediate stimulation of taurine influx
(K^ = 62 u_M; V x = 42 nmoles/g dry weight / minute) . The stimulation of taurine influx was dependent on the concentration of isoproterenol perfused over the range -9 -7 7 x 10 M to 4 x 10 M. Higher concentrations of isoproterenol caused a decreased stimulation of the rate of taurine influx, and led to arrhythmia. C. Ischemia
Crass and Lombardini (1977) have demonstrated a
generalized decrease of taurine content in ischemic muscle.
This loss in cardiac taurine was observed after acute left
ventricular ischemia in the dog (in vivo) and whole heart
anoxia in the perfused rat heart (in vitro). The left
ventricular ischemia in the dog was produced by occluding
the circumflex branch of the left coronary artery. Anoxia,
in isolated rat heart, was produced by perfusing the hearts with a taurine-free and oxygen-deficient buffer. In vivo
experiments after four hours of hypoxia markedly decreased
tissue taurine content; the greatest disappearance of
taurine was observed in the inner zone of the ventricle.
Anoxic perfusion resulted in a similar decrease, in rat ventricular taurine levels.
IV POSSIBLE PHYSIOLOGICAL ACTIONS OF TAURINE IN THE HEART
A. Taurine and Arrhythmias
Read and Welty (1963) were the first to suggest that taurine might influence cardiac activity by affecting ion movements. Their conclusions were based on their 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 cell potassium by dog heart slices exposed to epinephrine and digoxin. Taurine also prevented the epinephrine-induced loss of potassium from the intact heart in 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 in potassium concentration in the coronary sinus and arterial plasma. When taurine was given by itself, it did not affect heart rate, blood pressure, or EKG.
These results, however, were suggested to imply that taurine had potential therapeautic value in controlling cardiac arrhythmias caused by the loss of cellular potassium.
Chazov et al. (19 74) also established a relationship between taurine and potassium in the isolated guinea-pig
.heart. Taurine was shown to be effective in reversing abnormal EKG1s brought on by perfusion with strophanthin-K in K+ free medium. In fibrillating hearts perfused with
K+ -free medium, the addition of K+ (2.8 mM) and taurine, but not K+- alone, eliminated the fibrillation. The effects of taurine on EKG parameters in 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 cell permeability to potassium. 37
The above reports appeared to show that taurine affects potassium ion fluxes in myocardial cells. It has been known for a long time that digitalis and other cardiac glycosides (strophanthin-K and ouabain ) alter transmembrane + + Na , K -fluxes'(Hajdu and Leonard, 1959). Digitalis inhibits the Na+, K+- ATPase enzyme system which is responsible for + + the active Na efflux and active K influx across the cyto• plasmic membranes (Skou, 1965). Cardiac Na+, K+ - ATPase has been reported to be inhibited by as much as 40 per cent in patients receiving therapeutic doses of digitalis (Repke, 1965).20-40 per cent inhibition of Na+, K+- ATPase occurs during intravenous infusion of ouabain in the dog
(Akera et al. , 1969; 1970; Besch et al. ,. 1970). The Na+,K+- . . . . + 2+
ATPase inhibition 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 al., 1974). A logical site of action for taurine
therefore would be on the Na+, K+-ATPase pump system. Recently, Akera, et al., (19 76) reported that taurine,
in concentrations up to 100 mM failed to affect brain
Na+, K+-ATPase activity. In this experiment, a partially purified Na+, K— ATPase preparation obtained from rat brain 3 was used. Taurine was shown not to affect H-ouabam binding to Na+, K+-ATPase or the release of ouabain bound 38
to the enzyme. However, it is possible that taurine may
act on events which follow Na+, K+-inhibition rather than
on the Na-pump inhibition itself.or that the cardiac enzyme
is different from the brain enzyme.
Criticism has recently been made of the interpretations placed on the purported mitigating actions of taurine on drug induced arrhythmias. Hinton, Souza and Gillis (1975)
studied the capacity of taurine to counteract ventricular arrhythmias induced by deslanoside in the cat. This model had previously been successfully employed to determine the efficacy of antiarrhythmic agents. In this model, taurine was found to aggravate the cardiac rhythm disturbances produced by deslanoside. These workers claim that the antiarrhythmic effect, reported previously (Read and Welty,
1965; Chazov et al., 1974), was probably a reflection of the digitalis effect wearing off since no experimental controls were reported to indicate the duration of the arrhythmias when taurine was administered. Presumably, therefore, this critism could also apply to the effect of taurine on K+ efflux.
However, the recent findings of Fujimoto and Iwata
(1975) and Fujimoto, Iwata and Yoneda (1976) confirmed the earlier work of Read and Welty (1965) and Chazov et al.,
(1974) described above. It was shown that infusing taurine together with ouabain intramuscularly in the rat, 39
prevented the development of arrhythmias. They also noted that the myocardial taurine concentration was reduced in ouabain"induced arrhythmias. It was argued that the arrhythmic effect of taurine was likely due to its action on the autonomic nervous system, since propanolol, a fi-adrenergic antagonist, inhibited both ouabain-induced arrhythmia and loss of taurine. This point was further strengthened in their later paper (Fujimoto, Iwata and Yoneda, 1976) where taurine was observed to inhibit the change in response to acetylcholine and ouabain but not to noradrenaline. It was noted that taurine itself did not change either the EKG pattern.: or cardiac responses to acetylcholine and ouabain but did prevent the development of arrhythmias observed in the presence of digitoxin. However, the significance of these data is puzzling, particularly when the authors conclude with the statement that "the effects of digitoxin 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 al.
(1974) must be repeated to allow proper evaluation of the effect 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 to be a species-dependent
phenomenon (Dietrich and Diacono, 1971). Ouabain has a
positive inotropic effect on guinea-pig heart and a negative
inotropic effect on rat heart. Dietrich and Diacono (1971)
have shown that taurine in both normal and low calcium medium
exerts a positive inotropic effect on guinea-pig heart and a
negative inotropic effect on rat heart. Taurine was also
shown to potentiate the inotropic effect of ouabain on
both guinea-pig and rat hearts. Perfusion with ouabain
(10 ^g/ml) caused an increase in contraction in guinea-pig
heart (+9 0 per cent after 5 minutes) and a decrease in the
rat heart (-29 per cent after 3 minutes). Taurine (8mM) was shown to duplicate these actions.
In low calcium medium (0.54 mM CaCl2 instead of
2.16 mM), the negative inotropism'Observed in guinea-pig heart was less pronounced in the presence of taurine (70 per cent after 5 minutes instead of 80 per cent), while in
the presence of taurine the effect of low calcium was more pronounced in rat heart (69 per cent after 5 minutes
instead of 57 per cent).
In the guinea-pig, ouabain progressively reduced
the negative inotropic effect of low calcium medium. Taurine potentiated this effect of ouabain. The reduction of
contraction in the low calcium Tyrode solution was 91 per
(.cent; with ouabain added to the medium it was 58 per cent; with ouabain and taurine together there was only a 36 per cent reduction in contractions. On.the•other hand, in the rat, ouabain and taurine together showed increases in the negative inotropic effect 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 in the presence of ouabain and 92 per cent in the presence of both ouabain and taurine. Taurine thus potentiated the inotropic effects of ouabain in all the experiments quoted above and opposed the inotropic effects of lowered environ• mental calcium.
To evaluate the inotropic effect of taurine,
Schaffer et al.,(1978a)looked at cardiac work (aortic pressure x cardiac output) (Neely et al., 1967) in perfused rat hearts. It was found that taurine (10 mM) mediated a small, but significant, positive inotropic effect, when hearts were perfused with Krebs-Henseleit buffer containing 2+
1.25 mM Ca . These results, however conflict with the results of Dietrich and Diacono (1971). Schaffer et al.
(1978a)argue that their study utilizes a more physiological working heart preparation.
It was also found by Guidotti, Badiani and Giotti
(1971) that perfusion of taurine (8 mM) through isolated guinea-pig auricles increased the contractile 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 effect on the response to strophanthin-K. These workers further showed.that perfusion of auricles with a taurine-free medium1 resulted in a substantial loss of tissue taurine and that perfusion with 8 mM taurine maintained constant tissue levels.
Dolara et al (1978a), studied the effect of taurine on the recovery of contractile force by calcium-depleted guinea-pig ventricle strips. The contractile force of the guinea-pig ventricle strip can;.'.be reduced to very low levels, when perfused with a medium devoid of calcium.
Calcium concentrations in the medium were then increased by steps and the recovery in the contractile force was measured.
It was shown that taurine (4 mM) significantly increased the contractile force at external calcium concentrations of
1.8 and 3.6 mM, but it had no effect at lower calcium concentrations. It was further demonstrated, that taurine could affect the contractility of the heart ventricular strips under conditions of calcium loading. It is known that an increase in the frequency of stimulation of heart rates is accompanied by increased calcium influx in cardiac cells (Grossman and Furchgott, 1964; Winegrad and Shanes,
1962).Taurine in a dose-related-manner was-found to increase the contractility of the heart ventricular strips at a frequency of 120 beats/minute, but was ineffective at 6 0 beats/minute. 43
Iwata and Fujimoto (19 76) have also confirmed that taurine has a positive inotropic effect on electrically driven guinea-pig atria. Taurine (3.0 mM, but not 0.5 mM) was shown to pontentiate the positive inotropic effect of ouabain on myocardium independent of extracellular K+ concentration. The potentiation by taurine of ouabain action was not related to the changes in the myocardial content of taurine. A positive correlation between the potentiation by taurine of the positive 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 positive inotropic effect of ouabain was due, at least in part, to the increased content of calcium in 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 is probable that the inotropic action of digitalis glycosides is due ultimately to an increase in intracellular calcium available for binding to the myofila• ments and particularly to the protein troponin (Katz, 1970). 44
As the free calcium concentration in the cardiac cell rises -7 above approximately 10 M, calcium binds to an increasing number of troponin sites and, by a mechanism not yet defined, removes the troponin inhibition of bridge formation between actin and myosin. Full activation at all available sites is achieved as calcium concentration increases to a value of approximately 5 x 10 ^ M, or some 50 times that of the relaxed state. To attain this concentration and obtain full activation
50 to 60 umoles of calcium ion per kilogram of heart muscle is required to be released upon excitation (Langer,1971).
Frequently, however, the heart is called upon to develop greater tension and to develop it more rapidly without a change in the end-diastolic length of its cells (Bowditch,
1871).
The source of calcium available for excitation- contraction coupling in the presence of the cardiac glycosides is still in dispute. A number of workers are in favour of cardiac glycoside-induced liberation of calcium from intracellular pools, such as sarcoplasmic reticulum (S.R.) mitochondria or microsomes (Chipperfield,
1969; Glitsch et al., 1970; Dutta,et al., 1968). Other workers (Langer,1971; Van Winkel and Schwartz, 1976) have shown that cardiac glycosides could also increase the penetration of calcium into the cell through an active transport mechanism at the sarcolemmal membrane whereby 45 internal sodium is exchanged against external calcium. This transport system is said to be insensitive to cardiac glycosides directly (unlike the Na+, K+ ATPase) (Blaustein and Hodgkin, 1969), but very sensitive to intracellular sodium changes since its activity is proportional to the intracellular sodium ion concentration. The inhibition of the first pump
(Na+, K+ ATPase) by ouabain could then result in an increased entry of calcium into the cells (Langer,1971; Langer, 1968).
It has also been proposed that a third mechanism exists by which internal calcium is exchanged for external sodium
(Baker et al., 1969).
The results of Dietrich and Diacono (1971) concerning the negative inotropic effects on rat heart have been related to the absence in rat of a time lag in the functioning of the Na+, K+-ATPase (Guilbalt et al., 1962;
Blesa,• et al., 1970). The positive inotropic effect of digitalis in the guinea-pig can be attributed to the inability of the sodium pump to extrude all the penetrating sodium, thus leading to an increment in the amount of calcium influx into the myocardial cell. The calcium is thus available to the myofilaments which then leads to a staircase phenomenon. An alternative hypothesis is that the third 2+ pump (Ca extrusion) might be more active in rat than in guinea-pig and thus able to follow more quickly the inward 2+ calcium movements either by extruding Ca from the cells 2+ or by filling the intracellular Ca pools, since the tissue 46 calcium content"is higher in rat heart (4.9 a moles per kg.) than in guinea-pig heart (1.7 a moles/kg). ..
B. Inotropism, Calcium Transport and Taurine
The explanations above remain only theories but it is usual to assume that the digitalis-like effects of taurine are mediated through calcium movements in both rat and guinea-pig hearts. This is also suggested by the work described below.
The probable major intracellular structure for removing and releasing calcium is the sarcoplasmic reticulum.
In sarcoplasmic reticulum (S.R.) isolated from rat skeletal muscle in the presence of 15 mM taurine (Huxtable and
Bressler, 1973), calcium transport was increased by 30 per cent. Isolation procedure in the presence of taurine also led to an increase in the yield of microsomes and S.R. per gram of muscle compared to isolation in the absence of added taurine. Huxtable and Bressler (1973) interpreted these results to indicate that the presence of taurine prevented denaturation and lysis of the isolated organelles.
Taurine was also shown, in their experiments, to slow the rate of loss of calcium transport activity and ATPase activity of S.R. produced by phospholipase C. The data mentioned above suggest a primary role of taurine in 47
affecting (Mg + Ca )-ATPase activity.
There is also a body of work which suggests that taurine can also have an effect on calcium binding to membranes-.,
Dolara, Agresti, Giotti and Pasquini (1973) found that taurine affects calcium kinetics in perfused guinea-pig hearts. Hearts were perfused with Tyrode solution containing
2.7 mM calcium chloride in the control group and calcium chloride plus 8 mM taurine in 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 in the loss of contractile 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 in the controls. Dolara et al.
(1973, 1978a, 1978b) suggested that the calcium salt of taurine had a higher affinity than calcium ion for intra• cellular structures, thereby leading to a larger pool of bound calcium. It was suggested that the protective action of taurine on the rate of loss of contractile force was due to an increase in the amount of calcium available for contraction.
Later Dolara, Agresti, Giotti and Sorace (1976) studied calcium influx in a system where Sarcoplasmic
Reticulum (S.R.) preparations of guinea-pig hearts were 48
contained in a dialysis bag. Equilibrium dialysis has previously been used as a tool for the study of drug-protein interaction and amine uptake processes. In this system it was found that taurine increased total calcium binding to the sarcoplasmic vesicles. Calcium accumulation was due to a marked increase in the calcium influx rate whereas the efflux rate was not appreciably altered.
C. Calcium Movements and Taurine in Other Tissues
Recently, Izumi, Butterworth and Barbeau (19 77) studied the. effect of taurine on calcium binding to microsomes isolated from the rat cerebral cortex. Calcium binding to the microsomes was Shown to be inhibited by taurine in a dose-dependent fashion in the presence of an incubation medium containing 5 mM KC1 and 115 mM NaCl. Taurine was 2+ also found to decrease Ca binding in the medium containing 2+
70 mM KC1, without NaCl. There was no inhibition of Ca binding seen in the medium containing 115 mM KC1 and 5.mM NaCl.
Isethionic acid, Glycine, B-alanine, GABA and L-leucine showed 2+ no effect on Ca binding to the microsomes in the medium containing 70 mM KC1 without NaCl. It was concluded that
taurine has an inhibitory effect "on calcium binding to the microsomes in states of depolarization but is inactive in
the normal resting state. Apparently, this effect is specific
to taurine. 49
Igisu, et al., (1976) have studied the effects of
taurine in vitro on ouabain-inhibited ATPase activity in human
erythrocyte membrane in the presence and absence of calcium.
Ouabain was shown to inhibit the total ATPase activity in the
human erythrocyte membrane in a dose-dependent manner but in 2+
a fashion that was dependent on Ca concentrations. Taurine,
at concentrations of 15 to 60 mM stimulated ATPase activity
to a figure close to that seen without added ouabain. These workers suggested that the effect of taurine was due to a
competition with the membrane for calcium ions thus lowering
the effective calcium concentration. They state, however, without evidence, that taurine does not itself complex
calcium. The effect of taurine in the absence of ouabain, was not reported by these workers. Since total ATPase
activity was measured, it is not clear which type of enzyme,
Na ,K—ATPase, and/or (Ca. -+Mg )-ATPase, is most influenced by taurine. Taurine alone seems not to activate ouabain inhibited microsomal Na+, K+-ATPase activity in rat brain
(Akera et al., 1976; Donaldson et al., 1974).
SUMMARY
The work quoted in the preceding sections suggest that taurine induces changes in ion fluxes particularly those of calcium in cardiac muscle. On the basis of the effects 50
of taurine on inotropism, taurine can be considered to have
its main action on myocardium related to calcium ion transport.
The antiarrhythmic effect of taurine on epinephrine and
digitalis-induced cardiac arrhythmias was / said to involve
the regulation of the efflux of intracellular potassium
ions (Read and Welty, 1965). It is possible that taurine
may be more directly concerned with calcium and only J
indirectly affect potassium movements (based on the results
of Ueda et al. (1961) on the arrhythmogenic effect of
caffeine and epinephrine and those of Nayler (1963) relating 2+ the inotropic effect of caffeine and Ca -movements). More
tangible evidence for the function of taurine in the heart
consists of the observation of an increased taurine concen•
tration in heart muscle of hypertensive rats and in the left
ventricle of the patients who died from congestive heart
failure.
VI. POSSIBLE INVOLVEMENT OF TAURINE IN NEUROPHYSIOLOGY
Interest in the study of taurine and its possible physiological actions has been stimulated in recent years by the observation that taurine tissue levels are altered in certain clinical conditions. 51
A. Anticonvulsant Action of Taurine
The first suggestion of a possible involvement of taurine in seizure activity emanated from the report of van Gelder et al., (1972)>. Lower levels of taurine were found in the epileptogenic focus in human brain in comparison with the surrounding tissue. Decreased taurine levels were also observed in experimental cobalt-induced epileptogenic foci in cats, mice and rats (Craig and Hartman, 1973; Koyama,
1972; vanGelder, 1972). The observation of van-Gelder et al./
(1972) in human brain was not confirmed by Perry et al. (1975).
This could be due to differences in the methodology used: van Gelder et al.,(19 72) compared taurine levels in the same patient and Perry et al., (1975) used other subjects as controls.
Van Gelder (19 72) first reported the anticonvulsant action of taurine in mice and cats with cobalt-induced lesions. Subsequently, taurine has proved efficacious as an anti-convulsant in a variety of experimental models of seizure activity. Taurine has been shown to have a protective effect against seizures induced by ouabain, pentylenetetrazol, and strychnine (Izumi et al.,1973; 1974; Tsukada et al., 1974).
It also has antiepileptic effects on chronic and acute epileptic foci (produced by cobalt, aluminum, penicillin, estrogen, strychnine) and in the photosensitive Papio papio
(Derouaux et al., 1973; Mutani et al., 1974a, 1974b). The failure 52
of taurine to protect against convulsions in some experimental models (Wada et al.,19 75) may be related to a failure of penetration of taurine into the CNS. In genetically determined audiogenic seizures in rats and mice, taurine injected intra- peritoneally failed to protect against seizures, whereas intraventricular injections cause a dose-dependent attenuation of seizure activity (Laird and Huxtable, 1976).
Thus, the differences between models of experimental epilepsy
that respond to intraperitoneal administration of taurine may relate to differences in the penetration of taurine
through the blood-brain barrier. The protective effects of
taurine in human epileptic patients are highly variable
(Barbeau, 1974; Bergamini et al., 1974; Sbarbaro, 1974;
Striano et al., 1974).
The mechanism underlying the anticonvulsant and
antiepileptic effects of taurine is unknown. Its efficacy
against several experimental convulsive models and epilepsies
with differing etiologies suggests that it 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 in ratio of
glutamine/glutamic acid in convulsive states. Glutamate
and (f-aminobutyric acid) GABA levels are decreased in
experimental cobalt-induced and human epilepsies and seem
to return to normal after taurine treatment and cessation of seizures (van Gelder, 19 72; van Gelder et al., 19 75
Joseph and Emson, 19 76). Another proposed explanation for the anticonvulsive effects of taurine is that a reduction in hyperexcitability produced by taurine might be related to its effects on membrane permeability to chloride, which produces hyperpolarization (Gruener and Bryant, 19 75). A more direct action of taurine on calcium and potassium fluxes in myocardial and neural tissues has been described.
(Grosso and Bressler, 1976; Huxtable, 1976a, Pasantes-Morales et al., 1978, Barbeau et al., 1975, Izumi et al., 1977).
B. Taurine in Retinal Degeneration
Recent studies have shown that cats and kittens fed a taurine-free diet with casein as the only source of protein develop-. retinal degeneration which subsequently results in photoreceptor death (Berson et al., 1976; Hayes et al., 1975;
Schmidt et al., 1976, 1977; Rabin et al., 1973). Supplementation of the taurine-free casein diet with methionine, cysteine, inorganic sulfate, vitamin B^ or vitamin Bg with cysteine did not prevent development of retinal taurine deficiency and retinal malfunction (Berson et al., 1976; Schmidt et al.,
1976). A synthetic amino acid diet also results in retinal taurine deficiency and retinal malfunction. Only taurine- containing diets (i.e. chow or casein plus taurine) preserved normal electroretinogram (ERG) amplitude and normal retinal 54
taurine concentrations (Berson et al., 1976; Schmidt et al.,
19 77). These findings have firmly established a role for exogenous taurine in maintaining retinal function in the kitten (Knopf et al., 1978). In models of retinitis pigmentosa (retinal dystrophy) in rats and mice taurine was found to be the only amino acid to be reduced in concentration
(Cohen et al., 1973; Brotherton, 1962).
C. Taurine in Brain Development
A great deal of information indicates that taurine is present in higher concentration at birth and in prenatal brain than in mature animals (Agrawal et al., 1968a, 1968b;
Agrawal and Himwick, 1970; Oja et al., 1968; Sturman and
Gaull, 19 75), the exception being the frog, where small amounts of taurine are found in the mature brain, whereas none is found in the tadpole brain (Roberts et al., 195 8).
However, the concentration of taurine found in frog brain is much smaller than that found in mammalian brain
(Okumura et al., 1959). In all species studied throughout development, the decrease in taurine concentration in brain, from the high values in the new born animal takes place gradually during postnatal development and is complete approximately at weaning. This decrease is in contrast to the concentrations of most amino acids in brain, which either increase or change very little, during development (Agrawal and Himwick, 19 70). The high concentration of taurine in 55
foetal brain and its slow decrease postnatally suggesto that taurine may be associated in some way with brain development
(Sturman and Gaull, 1976; Kaczmarek et al., 1971).
The origin of the very high concentration of taurine in the developing brain is uncertain. The activity of cysteine sulfinic acid decarboxylase is low early in the development of rat brain and increases later (Agrawal et al., 19 71;
Kaczmarek et al., 1970). Therefore, biosynthesis of taurine seems unlikely to account for the large concentrations present in newborn brain. An efficient and highly selective transport system for attaining and maintaining high intracellular concentrations of taurine in brain during development has been suggested, and such a mechanism might account for the large concentrations in the newborn brain (Agrawal et al.,
1971). Sturman et al., (1977b) have recently demonstrated 35 ... that S-taurme injected into a pregnant rat enters foetal brain as rapidly as it enters foetal liver, with maximum values being reached after 12 hours. In contrast, labeled taurine enters adult brain more slowly than it enters adult liver, with maximum values in brain being reached after
5 to 7 days.
The source for the high concentration^of taurine in brain may be dietary. Human milk, unlike bovine milk and artificial formulas derived from it, contains a considerable amount of taurine (Gaull et al., 1977).
Recent work reported by Gaull et al., (19 77) also shows that the concentration of taurine decreases progressively in 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 full-term human infants fed human milk as opposed to bovine milk has also been documented (Jagenburg, 1959; Jonxis, 1951).
As discussed in the previous subsection kittens fed a synthetic diet containing partially purified casein as the source of protein become taurine-deficient and develop retinal degeneration, eventually resulting in blindness.
In this regard, man has a lower capacity for synthesis of
taurine than the cat (Gaull et al., 1977; Knopf et al., 1978).
In light of these studies, it seems possible that there is
a dietary requirement for taurine in the rapidly growing human infant.
Sturman. et al. , (1978) and Hayes (1976) in their
reviews conclude that taurine is probably an essential 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 in human infants, particularly in the etiology
of some forms of sudden infant death syndrome,(SIDS) . "
D. Effect of Taurine on Endocrine Functions:
Although taurine levels are not particularly high
in the hypothalamus (Collins, 1974), this region possesses
a high capacity for taurine biosynthesis and a very effective
mechanism for taurine uptake; it is indeed, the sole region
in which taurine concentration is significantly increased
after intraperitoneal injections (Hruska et al., 1973). On
this basis, a role for taurine in certain neuroendocrine
functions regulated by the hypothalamus has been suggested.
Intracerebral or intraperitoneal administration of
taurine causes hypothermia in rats and mice (Hruska et al.,
1973; Sgaragli and Pavan, 1972). The mechanism mediating this effect is unknown, but taurine does not appear to
interfere with peripheral heat-generating mechanisms. Some evidence suggests that the effects of taurine are mediated by central cholinergic or serotoninergic systems(Sgaragli et al., 1975).
Taurine has been reported to alter conditional eating and drinking functions regulated by hypothalamic structures (Thut et al. , 1976. Hruska, et al. , 1975),. Intraperiotoneally administered taurine, at doses that increase hypothalamic 58
taurine concentrations significantly reduce conditional water and food responses in rats and mice. Further, chronic taurine administration reduces weight gain of genetically obese mice (Taisho et al., 1970).
There is.-.evidence that taurine and some related amino acids, may also be involved in other endocrine functions such as in the regulation of circardian rhythm (Grosso et al.,
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 in the maintainance of excitatory activity
in mammalian nervous tissues. Mandel et al.,; (1975),
Kaczmarek (1976), Phillis (1978), Oja and Kontro (1978) and Guidotti (1978) have raised the possibility that taurine is an inhibitory neurotransmitter.
The inhibitory action of taurine has been demonstrated in a variety of nerve preparations: Taurine administered iontophoretically in the cerebral cortex blocks both spontaneous and chemically-induced firing of cortical neurons (Curtis et al., 1971; Krnjevic and Phillis,
1963). Taurine, microiontophoretically injected, depresses 59
spontaneous neuronal discharge of the evoked field potential in the spinal cord and brainstem neurons (Curtis et al., 1960,
1971; Haas and Hosli, 1973). The microiontophoretic application of taurine in the cerebellar cortex of the rat, produces a dose-dependent depression of the spike frequency of cerebellar neurons (Frederickson et al., 1978). More recently, McBride and Frederickson (19 78) have obtained neurochemical and neurophysiological evidence for the presence of taurinergic neurons in the cerebellar cortex of the rat.
Taurine has also been shown to have a powerful inhibitory effect on the retinal bioelectric response of isolated retinas; taurine added to the perfusion medium induces a rapid and specific depressant effect on the electroretinogram that is totally reversible after washing with taurine-free medium (Urban et al., 1976). Identical results have been obtained after intravitreal injection of taurine in the chicken (Pasantes-morales et al., 1973).
Inhibitory effects of taurine are also observed in muscle. Taurine showed a hyperpolarizing effect on the membrane resting potential of frog and rat muscle fibers and produced changes in characteristics of the action potential,
influencing mainly repolarization. In these studies, the
duration of the action potential is prolonged, and the
interspike intervals are increased. These effects of taurine 60
can be observed either in-vitro on isolated muscle prepara• tions or in-vivo after loading by injection of taurine
(Gruener, and Bryant, 1975; Gruener et al., 1975, 1976).
Summary
What is the possible neurophysiological role for taurine? It is believed that taurine may have a neurotrans• mitter role in the central nervous system. A set of criteria that a substance must fulfill before it can be considered a neurotransmitter has been reported, and taurine appears to fulfill the majority of these requirements (Mandel et al.,
1975; Lahdesmaki and Oja, 1973; Kaczmarek, .1976)...
In addition, its strong inhibitory actions on neuronal activity in cortical areas and spinal cord (Curtis and
Crawford, 1969; Curtis and Watkins, 1960; Crawford and Curtis,
1964; Haas and Hosli, 1973), as well as its antagonistic effects on seizures (Izumi et al., 1974; Kaczmarek and Adey,
1974; Mutani, et al., 1974b), provide further indications that taurine may function as a neurotransmitter or may have a modulatory role in nerve excitability. However, a definitive answer to this question cannot be given on the basis of the presently available knowledge (Oja and Kontro, 1978).
Other investigators (van Gelder, 1972; Gruener et al.,
19 75; Honegger et al., 1973; Barbeau et al., 19 75; Huxtable,
1976a)have argued in favour of -taurine as a neuromodulator. 61
According to Phillis (1978), a substance causing longer lasting alteration to cell excitability may /be called a "modulator".Most appropriately taurine can be described as a neuroeffector, possibly acting on ion fluxes (Hagins and Yoshikama, 1974;
Pasantes-Morales et al., 1978; Izumi et al., 1978) and membrane stabilization of the neurons (Gruener and Bryant, .
1975; Gruener et al. , 1975; Gruener - et al. , 1976).. . ../
The effector role" of taurine may•also'be related to its effects on certain metabolic actions, such as pyruvate dehydrogenase (Izumi et al., 1978) or glutamate regulation
(van Gelder, 1978), and the inhibitory effect on the release . of other neurotransmitters (epinephrine, norepinephrine)
(Kuriyama et al., 1978). The regulatory effect of taurine on the control of epinephrine release from adrenal storage granules, was said to be due to changes in the calcium affinity of these granules or by stabilizing the granular membrane (Kuriyama and Nakagawa, 1976).
The finding of McBride- and Frederickson (1978) and others (Frederickson et al., 1978; McBride et al., 1976;
Nadi et al., 1977) on the presence of the inhibitory stellate cells of the cerebellar cortex, signify that inhibitory taurinergic neurons do indeed exist in certain areas of the brain. In the report of McBride and Frederickson (1978) taurine was shown to inhibit firing rate of Purkinje cells, through the inhibitory stellate cells in the cerebellar cortex. 62
RATIONALE
The multiplicity of taurine effects discussed in the
review of the literature section of thesis suggests the
possibility that taurine has a variety of actions. However,
it is possible that a single basic mechanism (which remains to be clarified) is responsible for all these effects. One such
mechanism might involve taurine alterations in ion flux. A
number of workers have indicated an involvement of taurine in
calcium ion" transport in various tissues (Dolara et al., 19 73;
1976; Dietrich and Diacono, 1972; Huxtable and Bressler, 1973;
Igisu et al. 1976; Izumi et al., 1977; Barbeau et al., 1975;
Kuriyama and Nakagawa, 1976; Kuriyama et al., 1978). The
mechanism by which taurine may act on calcium ion movements
though remains speculative.
Many workers (read and Welty, 1965; Chazov et al.,
1974; Dietrich and Diacono, 1971; Guidotti, et al., 1971;
Jacobsen and Smith, 1964, 1968; Yamaguchi et al., 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 its possible conversion
to isethionic acid (2-hydroxyethane sulfonic acid), a strong
anion. This conversion was said to lead to the conductance
of cations into the cardiac cell. One major obstacle to the
search for possible functions of isethionic acid (ISA), at 63
the time when this work was begun was the lack of a good analytical procedure. The work described in this thesis, therefore, began with a search for a sensitive gas liquid chromatographic method to measure isethionic acid in mammalian tissues. It was, then, necessary to re-examine the enzymatic conversion of "^C-taurine to "^C-ISA in rat heart slices. This work suggested that isethionic acid was not likely to be involved in the mechanism of action of taurine.
Following the results obtained in the above studies, the possibility that taurine itself might affect the transport of calcium ions across membranes was evaluated. Firstly, a study of the effect of taurine on ATP-dependent calcium transport in guinea-pig whole heart homogenates and sarcoplasmic reticulum (S.R.) preparations was undertaken. Secondly, studies on the effect of taurine on the passive diffusion of calcium and other ions were investigated. In the first study guinea-pig hearts were used because most of the previous experiments on cardiac effects of taurine were observed in this animal species. In the later studies, rat brain synaptosomes were used as a model system to study the effect of taurine on the passive diffusion of calcium, sodium and potassium ions. Synaptosomal preparations were used in these studies, since several workers (Kuriyama et al.
1978; Barbeau et al., 1975; Pasantes-Morales et al. 1978; Lahdermaki and Pajunen, 1977) suggested'that the main action of taurine in the CNS may be related to ionic fluxes occurring at the synaptic terminal level. The alteration in calcium ion fluxes in the synaptosomes is well known to be important in the regulation of the excitability 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 salt from Sigma Chemical Co.
St. Louis, Missouri.
- Sodium isethionate was recrystallized with hot
95% ethanol to constant melting point (192°C.)
- Stock solution (5 mM) was prepared in water.
(b) Salicylic Acid:
- Obtained from Sigma Chemical Co. St. Louis,
Missouri.
- Recrystallized to constant melting point (138°C)
from 10% ethanol and dried in a vaccum desicator
over P2°5' - Solution (1 mM) was prepared in methanol.
(c) 1-Butane-Sulfonic Acid:
- Sodium salt, analytical grade, was obtained from
Eastman Kodak Cp., Rochester, N.Y. 67
- Used without further purification, 1. mM stock
solution was prepared in water.
(d) Other Internal Standards:
- Methyl caproate (C5 COOH)
- Methyl caprylate (C? H15 COOH)
- Methyl caprate (Cg H19 COOH)
- Methyl laurate {C^ H23 COOH)
- All obtained from Applied Science Laboratories,
Inc. Penna., 100 mg of each was dissolved in 0.5
ml methanol.
- Benzoic acid, obtained from BDH Canada 7(AnalaR),
1 mM solution was prepared in methanol.
- Acetylsalicylic acid, BDH Pharmaceuticals, Toronto,
Canada; 5 mg/ml solution was prepared in 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
Specialities Ltd. Brockville, Ontario.
- Empty columns, 6 ft by 4 mm (i.d.) glass U-tubes
for Bendix, model 2500 GLC were also obtained
from Gas Chromatographic Specialties. The columns,
packed with either OV-1 or OV-17, were conditioned on
the GLC for one day at 200 with no nitrogen(N2) flow
and then for one day each at '250.and 300°C respectively 68
with carrier (N2) 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 Specialities,
Brockville, Ontario.
-SP-400 on chromosorb W, 80-100 mesh, was obtained
from Supelco, Inc. Bellefonte, Pennsyslvania.
Silylating 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: acetonitrile; pyridine and dimethyl•
formamide (DMF)
- All silylation grade, obtained from Pierce Chemical
Company, Rockford, Illinois. 69
(h) Diazomethane;
Diazald and diazald kit were obtained from Aldrich
Chemical Company, Milwaukee, Wisconsin.
Diazald (2.14 g) was dissolved in ether (30 ml).
Diazomethane was distilled after the addition of
potassium hydroxide in ethanol (10ml, 0.04 g/100 ml).
The conventional distillation was carried out using
the diazald kit. The distillate, bright golden-
yellow, was kept cold at all times and stored at
-21°C. This preparative method is essentially
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 distilled water to
neutrality and then regenerated to the hydrogen
form with 5 bed volumes of 2N hydrochloric acido
followed by water washing. The resin was later
washed in methanol three times and suspended in an
equal volume of methanol. 70
(j) Other Reagents:
- Methanol
- Ether
- Ethanol
- All the chemical were reagent grade, obtained from
Fisher Scientific Co., Fair Lawn, New Jersey.
Preparation of Isethionic Acid for Use as Qualitative
Standard. :
A suspension of AG 50, (Hydrogen form) in methanol was pipetted into 5 ml glass stoppered, calibrated, conical centrifuge tubes. After allowing the resin to settle, 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 in water. The volume was adjusted to 4 ml in each tube with methanol, and the resin suspended using a vortex mixer. After centrifugation, the methanol layer from each tube was removed into reaction vials and evaporated to dryness in a vacuum desiccator over sulfuric acid.
Methylation of Isethionic Acid
Isethionic acid preparations were dissolved in 0.1 ml solution of internal standards (salicylic acid, butanesulfonic acid, benzoic acid, acetylsalicylic acid methyl caproate, methyl caprylate, methyl caprate or 71
methyl laurate) in methanol. When butanesulfonic
acid was used as an internal standard, the sodium
salt was added to the resin along with sodium
isethionate. The vials were stoppered using teflon
laminated discs and placed in an ice-bath for
five minutes. Ethereal diazomethane solution was
introduced into the vials slowly, with mixing, until
the yellow color persisted. An additional three drops
of diazomethane solution was added and the mixture
was allowed to stand for about thirty minutes in ice.
The total volume in the vial was 0.2 ml or less.
The methylation reaction was stopped by adding one
drop (0.02 ml) of 50% acetic acid in water (v/v).
4.. Silylation of Isethionic Acid
Isethionic acid (10 mg) was prepared as described
above, and dissolved in 0.75 ml dimethylformamide,
pyridine or acetonitrile, BSA, 0.25 ml, was then
added. The vials were heated on a Reacti-Therm
block (Pierce: Chemical Co., Rockford, Illinois) for
6 minutes at 135°C, and allowed to cool. 1 ul 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 isethionic acid and the same
treatment as for BSA was carried out. Alternatively, 72 isethionic acid (10 mg) was dissolved in 0.5 ml of pyridine, treated with 0.1 ml HMDS and 0.2 ml
TMCS and boiled under reflux for 30 minutes.
Gas-Liquid Chromatography: Flame Ionization Detector
The gas-liquid chromatograph used was a Bendix, model 2500, equipped with a flame ionization detector.
Columns were 6 ft 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 carrier gas at a flow rate of 40 ml/min.
Gas-Liquid Chromatography:Sulphur Detector5
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.,
Falls Church, Virginia). The column used was a
6 ft 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 carried 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 carrier gas with an inlet flow of 30 ml/min.
Column temperature was 100°C. Oxygen flow to the
detector was 10 ml/min and air flow was 30 ml/min.
The internal standard used was 1-butanesulfonic
acid.
7. Gas-Liquid Chromatography; Mass Spectrometry
The mass-spectrometer used was a Hitachi Perkin-Elmer,
operating at an ionization 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 after methylation with
diazomethane. Chromatographic details were as
outlined above for flame ionization detection.
Q 8. Nuclear Magnetic Resonance Spectroscopy
For proton NMR spectroscopy, the methyl esters of
ISA and butane sulfonic acid were prepared in the
following manner: Recrystallized sodium isethionate
(100 mg) and 1-butane sulfonic acid ( sodium salt)
The experiments were performed under the direction 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 British Columbia, Vancouver, B.C. Canada. 74
were each treated with resin (AG50, H form), and
methylated as described above. After thirty minutes,
excess diazomethane was carefully blown off 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 purification, were taken at 100 mHz
in a Varian HA-100 spectrometer. Samples were
dissolved in deuterated dimethylsulfoxide (Merck
Sharpe and Dohme Ltd., Canada) with tetramethyl
silane being used as an internal standard. For the
purposes of comparison, the NMR spectrum of
crystallized, non methylated ISA, also dissolved
in dimethylsulfoxide was obtained.
B. Analysis of Isethionic Acid in Mammalian Tissues i
1. Reagents
(a) Folch Solvent:
- Chloroform and methanol were mixed in 2:1
(v/v) ratio, 5% water was added to the total
volume.
(b) Other Reagents
- Methanol 75
-•Chloroform
- Ethanol
- All chemical used were reagent grade, obtained
from Fisher Scientific Co., Fair 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
sacrificed by a sharp blow to the head. Hearts and
brainswere promptly excised and the tissues rinsed
in normal saline, blotted on Whatman #1 filter paper
and immediately frozen in small plastic vials in
liquid 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 British Columbia,
following the use of these animals for minor
experiments which did not involve the heart. The
dogs were sacrificed 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, Illionis
Institute of Technology, Chicago, Illinois. The
method used for the isolation of axoplasm from the
squid axon is that of Maxfield(19 53). Pre-isolated
material from the squid and Nautilus were obtained
from the above source in freeze-dried form.
(d) Rat Milk Samples
Milk samples were obtained from Dr. John A. Sturman,
Institute of Basic Research in Mental Retardation,
Staten Island, New York, N.Y. Samples of milk
(0.2 ml) were collected from lactating rats of
;.Nelson-Wistar. .s.traih, and freeze-dried. The
samples on arrival from New York were stored at
-20°C. Further details for procuring milk samples
from the rats are given in an earlier paper of
Sturman et al., (1977b). 77
3. Isolation 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 in 50% methanol/water (v/v), (10 ml),
in a Sorvall Omni-mixer (Ivan Sorvall Inc., Norwalk,
Connecticut), using a teflon chamber at 3/4 of the
full speed for five minutes, followed by one minute
of full speed (Figure 6). The homogenate was
transferred to a centrifuge tube and centrifuged at
3 ,000'xgfor five minutes. The homogenizing chamber
was rinsed three times with 5 ml, 50% methanol/water
(v/v), and the rinse washings added to the centri•
fuge pellet which was suspended in the rinsing
solution using a Vortex mixer. The resulting
suspension was then centrifuged again for five
minutes and the supernatant fluid 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 Isolation of
Isethionic Acid From Tissues 78a
TISSUE Minced, Divided into two equal portions To one portion sodium isethionate in water was added To the other portion, the control, no ISA was added, Both portions, were left at room temperature for 5 minutes to equilibrate Both :were homogenized with - 50%-methanol water in a sorvall omni-mixer
HOMOGENATE
Centrifuged —\ Pellet washed three times with 50% methanol Supernatant and washings combined
T SUPERNATANT Added equal volume of Folch solvent Mixed and centrifuged
AQUEOUS LAYER Evaporated to dryness in a rotary evaporator under reduced pressure
RESIDUE - AG 50 (H form, prewashed in methanol) was added to the flask containing residue. - Flask swirled to mix thoroughly - Taken up in methanol - Add internal standard (1-butanesulfonic acid) - Centrifuged - Methanol layer transferred to a vial and desiccated to dryness
DIAZOMETHANE TREATMENT 79
separate the layers. The upper aqueous layer was
removed and evaporated to dryness in a rotary
evaporator under reduced pressure. After evaporation
of the aqueous layer to dryness, 2 ml of a purified
cation exchange resin (AG 50 - X8 , '-50-100 .mesh, form,
suspended in methanol), was added to the flask.
After trituration, 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 in a
vacuum desiccator over sulphuric acid.
(b) Dog Heart:
Pooled dog hearts (400 g) were minced and homogenized
in an equal volume of warm distilled 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 pellet 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
in 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 collected. The column had a void volume of 65 ml. The eluted fractions were pooled in 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 in a rotary evaporator under reduced pressure.
They were then treated with AG 50 (H+form) resin/ methanol (1:1, w/v) as described above for the isolation 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 fraction contained additional sodium isethionate (10 mg/100 g heart wet weight). All the portions were reduced in volume to
5 ml and attempts were made to crystallize 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 in 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 this
mixture treated with an equal volume of Folch
solvent. The rest of the procedure was the same as
that described in Fig. 6 for isolation of ISA from
other tissues.
(d) Rat Milk:
Freeze-dried, milk samples (0.2 ml) were resuspended
in 10 ml deionized water using a glass-glass homogenizer.
Authentic isethionic acid (25 to 150 nmoles) was added
to some of the samples at this 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 isolation 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
ul of 5 mM sodium isethionate solution and 10 u1
of 1 mM aqueous solution of 1-butanesulfonic acid
with AG 50, hydrogen form resin (prewashed in methanol)
as described on page 70., for the "Preparation of
Isethionic Acid". Butanesulfonic acid was used as an
internal standard.
ISA standards, or samples obtained from a tissue
extract were dissolved in 0.1 ml of methanol. In
separate experiments, salicylic acid was used as an
internal standard to confirm the identity of isethionic
acid peaks using OV-1 and OV-17 columns. In these studies
isethionic acid standard consisted of 0, 0.5, 1.0, 1.5,
2.0,"2.5 and 3.0 umoles and salicylic acid (0.1 ml
of 1 mM solution in methanol) was added to the standards
or samples after treatment with AG 50 resin and butane•
sulf onic acid was omitted from the procedure. The
"Methylation of ISA" was thqn followed as described on
page 70. in each case the total volume in the vial was 0.2 83
ml or less.Where the total volume in the vial was more, than
0.2 ml, the excess solvent was carefully evaporated under a stream of dry nitrogen and the sample remethylated for a further thirty minute period. This remethylation procedure was found not to affect the standard calibration curve for ISA.
Analyses of Samples by GLC.
Following methylation, aliquots (5 yl) of the standards and samples were injected into a gas-liquid chromatograph
(GLC) with either a flame ionization detector or sulfur detector. The standards used for the GLC with sulphur detector were 1/10 th of the concentration used for the flame ionization detector, and 1-butane sulfonic acid was used as an internal standard.
Conversion of Taurine to ISA
Reagents
(a) Isethionic Acid
Same as on page 66 .
(b) Taurine:
Obtained from Sigma Chemical Co., St. Louis Missouri. 84
(c) Radioactive Taurine (1,2xt *C) :
- Obtained from New England Nuclear.
Specific activity was 50 mCi/mmole.
(d) Scintillation Fluid:
- Toluene, scintillation grade, Fisher Scientific
Co., New Jersey.
- Methyl cellosolve (PIERSOLVE, ethylene
glycol monomethyl ether, sequanal grade,
Pierce, Rockford, Illionois)
- PPO (2, 5 diphenyioxanole), scintillation
grade, Kent Laboratories Ltd, Canada).
- POPOP (1,4-bis(2-(5-phenyloxazolyl)benzene,
Scintillation 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,
stirred for 2 hrs to dissolve and used within
24 hrs after preparation.
(e) Acridine:
- Obtained from Aldrich Chem. Co. Milwaukee, Wis.
- 0.1% solution prepared in absolute ethanol.
(f) Ninhydrin:
- Obtained from Fisher Scientific Co., New Jersey.
- 0.2% Solution prepared in absolute ethanol in 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 al (1974). The ingredients
were:
0.9 % NaCl 100 parts
1.15% KC1 . 4 parts
1.22% CaCl2 3 parts
2.11% KH2P04 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.
All reagents used were analytical grade; obtained
from Fisher Scientific Co., New Jersey.
(h) ATP (0.01 M).: .
- Obtained from Sigma Chemical Company, St. Louis,
Missouri as a disodium salt.
- 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 nitrite (NaNO,,) 20% aqueous solution.
- Hydrochloric acid
- Ethanol
- t-Butanol
- Pyridine
- Methanol
- Sulfuric Acid
- All reagents were analytical grade, obtained
from Fisher Scientific Co., New Jersey.
14
2. Preparation of C-ISA as Marker for the Taurine
Bioconversion Studies:
Radioactive (1,214 C) taurine (100 yl, lOyCi, 50 yCi/mole)
was dissolved in 20% sodium nitrite (0.4 ml). The
reaction was carried out on a water bath at 60°C by
adding concentrated HC1 dropwise (0.2 ml) until
effervesence stopped. The reaction mixture was
centrifuged to remove sodium chloride precipitate.
Sodium isethionate (40 mg) was added to the supernatant,
and sodium isethionate crystallized with hot 95%
ethanol. The resulting crystals (shiny, colorless,
rhombic plates) melted at 192°C (uncorrected) and
the mixed melting point with authentic sodium isethionate
crystals 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 strips (1" x 20") were cut out and
developed by spraying with riinhydrin or acridine in ethanol.
The paper chromatogram of authentic isethionic acid, and
taurine showed an Rf value of 0.71 and 0.59, respectively.
With radioactive isethionate, both acid and its sodium
salt, an Rf value of 0.71 was obtained. A strip counter 14 14 was not.available. C-ISA and C-taurine after
chromatography, were detected by cutting 0.5" x 1"
pieces from the spotting origin and counting the pieces
in scintillation fluid (10 ml) using standard liquid
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 isethionic acid. The yield from this preparation 14 .
was more than 50% of the radioactivity of the C-taurine
substrate.
3. Synthesis of Isethionic Acid by Rat Heart Slices:
The method used was that described by Read and Welty
(1962). Rats, Wistar strain, weighing 200-250 g were
sacrificed by cervical dislocation. The hearts were
promptly removed, cut into four pieces, washed in ice- 88
cold saline, blotted on filter paper and transfered to
ice-cold incubation medium containing Krebs-Henseleit
phosphate buffer, pH 7.3 and 0.01 M glucose. The ventri•
cles were sliced (0.5 mm thick) using a Stadie-Riggs
microtome (Arthur H. Thomas Co. Phila, P.A., USA). The
slices were weighed out (0.1 to 0.15 g) after light drying
on filter paper and then resuspended in fresh incubation
medium and incubated at 37°C in a shaker water bath for
30 minutes. At the end of the incubation, the slices 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 yl). The slices 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 chilled homogenizing tubes and homogenized using a Polytron homogenizer, at full
speed for 1 minute. The homogenate was centrifuged at
3,00 0 x g for five minutes. The pellet was resuspended three times in 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 isolation of ISA from rat heart and brain tissue ( page 77). The methanol residue, dried in a vacuum desiccator over sulphuric acid, was 89
made up to 0.1 ml with distilled water. The solution
(5 yl)was then used for paper chromatography.
II: STUDIES ON-'THE EFFECT OF TAURINE ON ION TRANSPORT PROCESSES
A. Effect of Taurine on ATP-dependent Calcium Transport
in Guinea-pig Cardiac Muscle:
1. Reagents:
(a) Sodium Bicarbonate Buffer (lOmM):
- Sodium bicarbonate (NaHCO^) (lOmM), 5 mM sodium
azide (NaN^) and 0.2 mM ascorbic acid were
dissolved in water, pH 6.8 (adjusted with IN HC1.)
(b) KC1 Solution (0.6M):
- Potassium chloride andimM magnesium chloride
(MgCl2.6H20) were dissolved in 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) Sucrose Solution (40%), 40 mM Tris-HCl buffer:
-Sucrose solution (60%), was treated with AG 50
(Na+ form) to remove cation contaminants (Carsten,
1964) and then 100 ml of the solution was diluted
with 15 ml of 0.4 M Tris-HCl buffer. The pH was
adjusted to 7.2 before making up to 150 ml volume
with distilled water.
(e) Taurine:
-Obtained from Sigma Chemical Co., St. Louis,
Missouri. •
-0.5 M aqueous solution.
(f) Calcium-45:
-Obtained in the chloride form in aqueous solution
from the Radiochemical Centre, Amersham, England:
(CES.3), specific activity was 10-40 mCi/mg calcium.
Diluted 1 in 5 with water and stored at -20°C.
(g) Calcium Chloride Solution (100 mM):
-Aqueous solution. Serial dilutions were made to
obtain desired concentration.
(h) EGTA Solution (100 -mM) :
-EGTA (Ethyleneglycol-bis-( 3 - aminoethyl ether)
N,N'-tetra acetate) was obtained from Sigma
Chemical Co. (# E3251)
-Aqueous solution, pH 6.8 (adjusted with 2M Tris-
base). 91
(i) Determination of Free Calcium Concentrations Present
in Ca-EGTA Buffers:
- The equation of Katz et al. (19 70) was used to
calculate the desired free calcium concentrations:
([CaCl,] - 5.41 [Ca2+] ) — '' = • 4.4 x lO5
2+ 2+ [Ca 1 . (\Z_. - ([CaCl2] - 5.41 [Ca ]))
Solving above equation for Z;
2+ ([CaCl2]-5.41 [Ca ]) Z = ([CaCl,] - 5.41 [Ca2+]) + 5 2+ 4.4 x 10° [Ca ]
Substituting the desired values for [CaCl2]and 2+ [Ca ] will give Z, the amount of EGTA to be added 2+ to achieve the desired free Ca concentration. For example, the amount of EGTA needed to prepare 2+ a Ca-EGTA buffer containing 1.0 yM free Ca
in a solution containing 125 y_M_CaCl2 will be:
125 x 10~6 - 5.41 x 10"6 Z= + (125 x 10~6 - 5.41 x 10~6) 4.4 x 105 x 10~6
= 0.391 mM EGTA.
2+
Therefore, to obtain 1.0 pM free Ca , 0.391 mM
EGTA and 0.125 mM CaCl2 are used. 92
- The amount of CaCl2 and EGTA used in each case to prepare
solutions of various concentrations of free Ca2+ in a
total volume of 0.3 ml were as follows:
Desired Free
2+ ( M) 45 Ca U 10 mM EGTA 10 mM CaCl~ Water z Ca 0.5 .203 .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 diluted 1 in 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 Tris base.
(1) Tris-ATP (0.1 M):
-Adenosine 5' - triphosphate (tris (hydroxymethyl)-
amino-methane salt from Equine muscle) was obtained
from Sigma Chemical Co., St. Louis, Missouri. 93
- Aqueous solution, pH adjusted to 6.8 with 2 M Tris
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 in 1 ml
water, and stored at -20°C. For use the suspension
was diluted 1 in 2 (final concentration: 2.5 mg/ml).
(p) Cyclic AMP:
- Obtained from Sigma Chemical Co., St. Louis,
Missouri.
- 10 mM aqueous solution.
(q) Sorensen's Glutaraldehyde Fixative:
- 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 in veronal-acetate buffer, pH 7.3-7.5,
according to Glauert (1965).
(h) Uranyl Acetate:
-2% aqueous solution.
(i) .] Reynold's Lead Citrate. :
-Prepared according to Reynolds (196 3).
3' (j) H-Ouabain .
- obtained in a solution of ethanol/benzene (9:1 v/v)
from New England Nuclear, Boston, Mass. •
Specific Activity was 10-20 Ci/mmole,catalogue # NET-211
(k) Other Reagents:
- Sodium bicarbonate (NaHCO^), Fisher Scientific Co.,
New Jersey.
- Sodium azide (NaN^), Sigma Chemical Co.
- Ascorbic acid, BDH, Canada.
- Potassium chloride, Sigma Chemical Co.
- Sucrose, Fisher Scientific Co.
- Calcium chloride (CaCl2.2H20), Mallinckrodt, St. Louis,
Missouri.
- Magnesium chloride, (MgCl2•6H20), Sigma Chemical Co.
- Aquasol, New England Nuclear, Boston, Mass. (Cat.
# NEN-934).
- Other "reagents used in electron microscopic work were
all obtained from Fisher Scientific 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 sacrificed by a blow to the head. The hearts were promptly excised, washed in saline and the aorta and connective tissue removed. The ventricular muscle was then promptly frozen (within 30 seconds of sacrifice) in 2-methyl-butane and dry ice. The frozen hearts were wrapped in tin foil and stored at -80°C. The tissue could be stored in this way for up to 3 months without significant loss in calcium transport activity.
For preparation of heart ventricle homogenates a piece
(0.1-0.2 g) was cut from a frozen heart preparation and homogenized in 5 ml of a medium consisting of 40% sucrose and 40 mM Tris-Cl, pH 7.2 using a Polytron P20 homogenizer
(3 strokes of 5 sec. duration at setting 5).
Preparation of Microsomes Enriched in Sarcoplasmic
Reticulum:
The method of Harigaya and Schwartz (1969) was followed
with slight modifications (Figure 7-) . Frozen heart
preparations, from two guinea-pigs were cut into small R pieces with a razor blade and placed in Corex Figure 7
Flow diagram for the preparation of heart microsomes enriched in sarcoplasmic reticulum 96a
HEART TISSUE
Frozen preparation was cut into small pieces. Homogenized in sodium bicarbonate buffer, pH 7.2. Using Polytron P20 at setting 5, three fast strokes. Care taken not to denature the preparation at this step. HOMOGENATE
Centrifuged at 12,000 x g for 15 minutes at 4°C. SUPERNATANT Centrifuged at 45,000 x g for 1 hour at 4°C. PELLET Suspended in 10 ml of 0.6 M/KC1 solution, using a glass-teflon hand homogenizer (1-2 strokes).
Centrifuged at 45,000 x g for 1 hour at 4°C.
PELLET Resuspended in 10 ml of 10% Tris-HCl buffer, pH 7.2
Centrifuged at 45,000 x g for 1 hour at 4°C.
PELLET Resuspended in 40% sucrose and 40 mM Tris-HCl buffer,pH 7.2, using glass-teflon hand homogenizer (2-3 strokes).
•MICROSOMAL PREP. ENRICHED IN,S.R. 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 rest interval of about 15 to 20 seconds.
The entire procedure was carried out in crushed ice.
The resulting 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 precipitate was
resuspended in a glass homogenizer with a teflon pestle
in 10 ml of 0.6 M KC1 solution. The resulting suspension::, was then centrifuged at 45,000 x g for 1 hour to remove
solubilized actomyosin. The precipitate was again
suspended in 10 ml of 10 mM Tris-HCl buffer, pH 7.2
and centrifuged at 45,000 x g. The harvested precipitate was then suspended in a small volume of a medium
consisting of 40% sucrose and 40 mM Tris-HCl;,pH 7.2. The resulting microsomal preparation is enriched in sarcoplasmic
reticulum. Storage in 40% sucrose reduced the loss of
calcium transport activrty noted when these preparations were maintained at 4°C (Carsteh, 1964). Unless otherwise
indicated, all experiments were conducted within 2 hours
of preparation of this fraction. 98
4. Characterization of Microsomes Enriched
Sarcoplasmic Reticulum
(a) Electron Microscopy:
A suspension of enriched sarcoplasmic reticulum in 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 pellet was treated like blocks of
whole tissues, fixed in Sorensen's glutaraldehyde
(2.5%) fixative for 24 hours at 4°C. It was then
post-fixed in Palade's fixative, buffered 1% osmium
tetraoxide, for l-l^s hrs. at room temperature. The
tissue was then washed in water, dehydrated in :
ethanol and cut into small pieces. The pieces
were dehydrated in acetone and embedded in maraglas.
Sections were stained on the grid with uranylacetate
and 'lead citrate and observations were carried out on
a Siemens, Elmiskop !•> model, electron microscope.
3 (b) .H-Ouabain Binding Assay:
The method of Gelbart and Goldman (19 77) was
utilized. 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 British Columbia, „ Vancouver, Canada. . 99
with 10 M [ H] ouabain (specific activity 12.7
C\/mol) in the presence of 3. mM MgCl2, —
NaCl, 1 mM EGTA, 25 mM Tris-HCl, pH 7.4, in the
presence and absence of 3 mM Tris-ATP. Following
10 min of incubation at 37°, an aliquot of the
reaction mixture was filtered through a Millipore 3
filter (0.45 y_M) to separate unbound [ H]-ouabain
from tissue-bound [^H]-ouabain. The filters were
washed and dried, dissolved in Aquasol counting
medium and assayed using liquid scintillation 3
counting techniques. Non-specific [ H]-ouabain
binding, determined in the absence of ATP, was
subtracted from the total binding to determine the
degree of ATP-dependent binding.
5. ATP-dependent Calcium Uptake and Binding Assay
The method of Tada et al. (1974) was followed with a
few modifications. Oxalate-facilitated calcium uptake
was determined in 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
MgCl2, 110 mM KC1, 5 mM Tris-ATP, 2.5 mM Tris-oxalate 4 5 5 and CaCl2 containing CaCl2 (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 in a total
volume of 0.5 ml. The reaction was terminated by
filtering an aliquot (0.4 ml) of the reaction mixture
through a millipore filter (HA 45, Millipore Co.).
The filter was then washed twice with 15 ml of 40 mM
Tris-HCl, pH 7.2, then dried and counted for radio*
activity in 10 ml Aquasol using standard liquid
scintillation counting techniques
ATP-dependent calcium binding was studied under
identical conditions except that Tris-oxalate was
omitted from the reaction medium.
6. Assay for Cyclic-AMP-dependent Protein Kinase Effect
on Calcium Uptake:
The effect of cyclic AMP-dependent protein kinase on
calcium uptake was assayed under the identical conditions
used to measure ATP-dependent calcium uptake. Cyclic
AMP-dependent protein kinase was added to the incubation
medium in a concentration of 50 yg/ml along with 1.0 uM
cyclic AMP at least 7 minutest'prior to the start of the
reaction. 101
7. Studies on the Effect of Taurine on the Decay 2+ of Ca Transport Activity:
The heart ventricle homogenate and the microsomal
preparation enriched in S.R. were prepared as outlined
on pages 95 and 96 respectively, except that the
preparations were stored in 2 mM Tris-HCl buffer, pH 7.2
instead of a medium containing 40% sucrose and 40 mM
Tris-Cl 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 intervals
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 al.
(19 51) using bovine serum albumin as a standard.
2+ 2+
9. Calculations: Ca uptake and Ca binding were expressed
in nmoles/mg/min.
Example: 2+ Ca-Uptake for 1.0 uM free Ca : 102
13 9 cpm on Millipore 9C.a 62.5 nmoles CaZ Uptake / : " filter X x Ca2+
(nmoles/mg/min/ y/ ) , .. £ .. c , cpm of the . . c . , d Standard ; x m^ Proteln x 5 minutes
a. Dilution factor: 0.4 ml of the reaction mixture of
0.5 ml total volume was passed through a millipore filter;
s*. a dilution factor of 1.25.
b. Calcium content in each tube was 62.5 nmoles.
c. Radioactivity of Ca-EGTA buffer (50 ui) was determined
in 10 ml Aquasol.
d. Calcium uptake was determined for 5 minutes of
incubation time.
10.Statistics:
Statistical analysis was done by Students "t" test for
unpaired and common variance (Wonnacott and Wonnacott,
1977). A probability of p<0.05 was taken as the criterion
for significance. Standard Error of the Mean (S.E.M.)
was used as a measure of variation.
B- Studies on the Effect of Taurine on Passive Ion
Transport in Rat,,Brain Synaptosomes:
1. Reagents:
(a) Sucrose Solution: 103
-Obtained from Fisher Scientific Co., analytical 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 final 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 resin was treated twice with
1 volume of >1N NaOH to generate the sodium form
and washed with distilled water until the pH of the
wash was neutral?. (7.0-7.5). , .
- 3-alanine , A grade, Calbiochem, Los Angeles, California. a B-alanine, hypotaurine and homotaurine were kindly provided by Dr.. Thomas Perry, Pharmacology Department, University of British 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, California.
- Methionine, Nutritional Biochemicals Corporation,
Cleveland, Ohio.
- a-alanine, Nutritional Biochemical Corporation,
Cleveland, Ohio.
- Proline, Nutritional Biochemical Corporation,
Cleveland, Ohio.
- Valine, Nutritional Biochemical Corporation,
Cleveland, Ohio.
- Sodium sulphate13, Matheson Coleman & Bell, Norwood Ohio.
- Sodium acetate*3, BDH Chemicals, Canada.
- Potassium acetate*3, Matheson Coleman & Bell.
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 killed by
cervical dislocation and the whole brain removed to ice-
cold 0.32 M sucrose. The brains were homogenized in 10 vol.
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 British Columbia, Vancouver, B.C.
400 mM Aqueous stock solutions. Figure 8
Flow diagram for the preparation
rat brain; synaptosomes 105a
RAT BRAIN (from 2 rats)
Washed in ice-cold 0.32 M sucrose. — t HOMOGENIZED In 0.32 M sucrose (10 vol.) using teflon-glass motor t driven homogenizer CENTRIFUGED At 1,0 00 x g for 10 minutes. t SUPERNATANT Recentrifuged at 12,000 x g for 20 minutes.
PELLET Resuspended in 10 ml of 0.32 M sucrose. — T DISCONTINUOUS DENSITY GRADIENT Layered, 5 ml suspension on a gradient containing 15 ml of 0.8 M sucrose and 15 ml of 1.2 M sucrose.
CENTRIFUGED 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. t PELLET Resuspended in 4 ml of a medium containing 0.32 M sucrose and 10 mM Tris-HCl buffer,pH 7.2
SYNAPTOSOME SUSPENSION 106
800 rev./min.)The homogenate was centrifuged at 1000 x g
for 10 minutes and the resulting supernatant recentrifuged at 12,000 x g for 20 minutes. The pellets were resuspen• ded in 10 ml of 0.32 M sucrose and 5ml carefully layered onto each of two discontinuous density gradients consis• ting 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 in an SW 27 swing-out head using a Beckman L2-65 ultra- centrifuge. The material at the 0.8 M-1.2 M interface was removed, diluted with 0.32 M sucrose and centrifuged at
20,000 x g for 30 minutes. The resultant pellets were resuspended in 4 ml in 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 in solutions of 25-300 mM Na2SC>4 (0.95 ml) in a microcuvette. The extinction of the suspension was
recorded at Eq9n 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-cold 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 in medium containing 0.3 M sucrose and 10 mM
Tris-HCl.buffer,pH 7.2, in the presence or absence of taurine (20 mM), in a total volume of 3 ml. Following a preincubation of 1 hour, the reaction was started by 45 5 the addition of 10 yM CaCl2 (5 x 10 cpm/sample).
Aliquots (0.2 ml) of the incubation mixture were then removed at intervals and passed through a millipore filter (HA 45, Millipore, Co.) The filter was washed twice with 5 ml of 10 mM Tris-HCl buffer, pH 7.2 in 0.3 M sucrose then dried and counted for radioactivity in Aquasol using standard liquid scintillation counting techniques. 108
45 Determination of Loss of Ca from Preloaded Synaptosomes
Synaptosomal suspensions (0.2 mg protein/ml) were all 45 loaded to a similar extent with 10 y_M CaC^ 5 o (5 x 10 cpm/sample) at 2 C in conditions similar to that described above for the determination of calcium permeability. After 1 hour, an aliquot (0.2 ml) was passed through a millipore filter. The remaining incubation medium was centrifuged at 12,00 0 x g for
10 minutes. The resulting pellet was resuspended in
3 ml of ice-cold media containing 0.3 M sucrose and
10 mM Tris-Cl buffer, pH 7.2 in 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 millipore filter. The rate of calcium release was then calculated by the following equation: (cpm in filter after 1 hour preloading)'-
4C. (cpm in filter at sampling time) Ca Release (%) = (cpm in filter after 1 hour preloading)
Protein Assay: same as on page 101
Statistics:
The slope or regression coefficient(b) of the line, its intercept (a) and correlation coefficient (r) were 109
obtained using a standard calculator, Texas Instruments, model T-55.
The sum of squares of deviations, Edy x2 is the basis for an estimate of error in fitting the line. The corresponding degrees of freedom are n-2.
d y>x = where Y = Y + b (X-X)
dy.x2 = (Y^>2
2 Z<3 v2 = E(Y-Y)
then, S 2 =Edy.x2/n-2. y. x
where S 2 is the 'mean square deviation from regression' X= independent variable and Y = dependent variable. The resulting sample standard deviation from regression is
S = l/S~ ~2 y.x y.x
The standard deviation of the slope is then obtained:
2 2 2 Sh = S. / Ex , where Ex = E(X.-X)
A test of significance between two slopes (obtained for the data with the presence and absence of taurine) is given by student t-test
bl " b2
, d. f. = n-^ + n2 - 4
fbl)2 +(Sb2)2 110
The estimated standard error of Y is:
/ 2 2 S£ = Sy>x (l/n) + (x / Ex );
where n = number of observations
2 - 2 x = (X-X)**
x2=E(X-X)2
A test of significance between two intercepts is given by:
— a„ 1 2 t = ' ; d.f = n^^ + n2 - 4 \ )2 +(S )2 yal a2
All statistical formulae used were obtained from Snedecor and Cochran, 1967. Ill
RESULTS 112
I. STUDIES WITH ISETHIONIC ACID
Development of an Analytical Method-for the Measurement of Isethionic Acid:
1. Chromatography of Methylated Isethionic Acid:
a. Stationary Phases:
The chromatography of the methylated isethionic
acid on OV-1 and OV-17 column is shown in
figure 9. Using a column of 5% OV-1, a
single peak with a retention time of 1.6 minutes
was obtained from methylated isethionic 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 isethionic
acid were obtained.
b. Internal Standards:
Salicylic acid was found to be a convenient
internal standardMn that it could be used
for both OV-1 and OV-17 columns at the same
time during gas-chromatography with a flame
ionization detector. Separation of methylated 113
Figure 9.
Chromatographic separation of the products of methylation of isethionic acid and salicylic acid
using flame ionization 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 salicylic acid and isethionic acid on 5% OV-1 and
5% OV-17 columns are shown in figure 9A and 9C respectively.
1-Butanesulfonic acid was used as an internal standard when gas chromatography was conducted on a flame photometric (sulfur) detector, with OV-17 columns. Two peaks for methylated isethionic acid were obtained using sulfur detector and an OV-17 column
(Figure 1.0) , similar to that seen in figure 9B and 9C.
1-Butanesulfonic acid was found to co-chromatograph with isethionic acid on OV-1 columns; but, it served as a good internal standard on an OV-17 column (Figure 1
Other compounds tested as internal standards for the isethionic acid assay were found to be unsatisfactory: These compounds as separated on OV-1 columns relative to methylated isethionic acid, were either too close to the solvent peak (example, methyl caprylate) or had a retention time undesirable for the assay purpose (acetylsalicylic acid and methyl laurate) . Methyl benzoa-te was found to co-chromatograph with methylated isethionic acid on the OV-1 column. 115
Figure 10
Chromatographic separation of the products of methylation of isethionic 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 details refer to'Materials and Methods' section of the text (page 72).
Isethionic acid (I) was identified as the methylester, methylether derivative.Isethionic acid (II) was
identified as the methylester (see discussion) 115a
Recorder Pen Response
Butanesulfonic Acid
Isethionic Acid
Flame Response Isethionic Acid
T T" n r t 2 4 6 8 0 Min. 116
c. Mass-Spectrometry and NMR spectra of Methylated
Isethionic acid:
The identities of; the- two. peaks "of methylated
isethionic acid obtained on OV-17 column were
established by the use of gas chromatography mass-
spectrometry (GC-MS) (figure 11) and nuclear
magnetic resonance spectroscopy (NMR) (figure 12).
Interpretation of the mass-spectrometry fragmentation
pattern of the two peaks obtained after OV-17 gas
chromatography is shown in figure 11. The large
peak (Peak II) was the methylester of the isethionic
acid, while the small peak (Peak I) was the
methylether, methylester of isethionic acid.
The NMR spectra of methylated isethionic acid,
1-butanesulfonic acid and methoxyethanol are shown
in figure 12A, 12B,, and 12C respectively. The
1-butanesulfonic acid and methoxyethanol spectra
were used to identify the CH^ singlet peaks of
methylester (3.88 ppm) and methylether (3.38 ppm),
respectively. The integration of the spectrum
indicated an approximate methylester CH^ to
methylether CH-. ratio of 20:1. Figure 11
Mass spectra of the products of methylation
of isethionic acid.
A. Mass spectrum of the peak I from figure 9 B.
B. Mass spectrum of peak II from figure 9 B.
Experimental conditions and interpretation of data are described in 'Materials and Methods'
(page 73.) and 'Discussions' (page 152) sections the text. 117a
100 Spectrum A
i2OCH3
+ £cH3OCH=CH2J H2S03CH3
Methylester methylether of Isethionic Acid
124
+ ^£cH2SO(OCH3|^J 118
Figure 12
Nuclear magnetic resonance spectra of the products of methylation of isethionic acid(A),
1-butanesulf onic acid (B) and methoxyethanol (C) .
The NMR spectra of the methylation products of isethionic acid and 1-butanesulfonic acid were obtained on a Varian HA-100 spectrometer at 100 mHz, dissolved in deuterated dimethyl- sulfoxide. Methoxyethanol spectruma 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 British Columbia. 118a
Hz i—i—i—i—|—i—i—i—r" 1 1 1 1 1 i—i—[—i—l—i—i—I—i—l—r- ' I I I I I I I 1 1 I 1 ' | I M I I I I i I ; i | i | I i i 1 i i i | i | i 500 4001 300 200 1 I001 1 0
J
CHj-O-CHj CH-OH
2-Methoxyethanol
-a- ' I ' i I i i I nj 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 Silylated Isethionic Acid
Chromatography of isethionic acid on OV-17 after
silylation with BSA in DMF solvent is shown in figure
13. It can be seen that the mixtures obtained
after silylation of isethionic acid are extraordinarily
complex and the products appear to be labile.
Silylation of isethionic acid in the presence of
BSA and DMF solvent gave peaks of varying number,
size and retention times during the course of
subsequent injection of the same sample (Figure 13).
1-Butanesulfonic acid was also found to behave in a
similar manner to isethionic acid. Salicylic acid
and caprylic acid always gave only one peak and this
behaviour was reproducible.
Pyridine and acetonitrile were substituted as solvents
for DMF during silylation of isethionic acid with
BSA. Similar chromatographic behaviour of silylation
products to that found using DMF as solvent was again
obtained.
Other silylating agents were also used. Silylation
of Isethionic acid with HMDS/TMCS on chromatography
yielded different multiple peaks. TMSIM yielded
no peaks for Isethionic acid on the OV-17 column. 120 •
Figure 13
Time course injection of silylated
products of isethionic acid
Isethionic acid (10 mg) was dissolved in
0.75 ml DMF and then treated with 0.5 ml
BSA. The reaction was carried out at
135°C for 6 minutes. The sample was injected (l;.ul) on a GLC column, after storage in ice-bath, at intervals of 15 minutes (A), 45 minutes (B), 75 minutes(C) and after 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 isethionic acid silylation studies.
The stationary phases used were PEGS and SP-400. On
neither of these two columns could isethionic acid
peaks be seen.
B. Analysis of Isethionic Acid in Tissues;
A typical standard curve for methylated isethionic
acid using flame ionization detection-gas chromatography
on OV-17 columns is shown in 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 this work was undertaken.
Identical standard curves were obtained on OV-1 columns.
The standard curve obtained using a sulfur detector was
10 times more sensitive than the flame ionization detector.
Similar standard curve obtained on sulfur detector and
1-butanesulfonic acid was used to quantitate isethionic
acid in mammalian and arthropod tissues. The results
of the analyses are shown in Table 1.
1. Isethionic Acid in Rat Heart and Brain Tissues:
In rat brain, isethionic 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
tissue. The value of isethionic acid for rat heart is
only an estimate since at this level the method is Figure 14
A typical calibration curve of methylated isethionic acid obtained on a column of 5%
OV-17 flame ionization detector.
Similar standard curve was obtained with sulfur detector and 1-butanesulfonic acid was an internal standard.
The operating parameters are quoted, in the
'Materials and Methods' section on page 72 . 9.0
0.5 1.0 1.5 2.0 2.5 3.0 jimole Isethionic Acid/Vial :.i2 3
TABLE 1
Isethionic Acid in Tissues Analyzed
Rat Heart 0.00 8 ymole/g wet weight' (0.1mg/100 g wet weight)
Rat Brain 0.016 ymole/g wet weight (0.2 mg/100 g wet weight)
Rat Milk - None was Detected*3
Dog Heart - None was Detected
Squid Axoplam '* cl - 150 y mble/g wet weight (18.6 mg/g wet weight)
Squid Ganglion - 34.09 pmole/g wet weight (4.28 mg/g wet weight)
Nautilus Ganglion - 2.9 8 Mmole/g wet weight (0.37 mg/g wet weight)
The tissue were analyzed by Gas-liquid chromatography
(GLC) after extraction and methylation as described in
"Materials and Methods" section on pages 75 to 83. An
appropriate standard curve was used. The recovery of
authentic isethionic acid added to the tissue was always between 95-100% (see text).
a. The value given is only an estimate because at* this level the method is approximately at the limit of sensitivity of the assay technique. The limit of detectibility of the method with sulfur detector was approximately .00 8 ymoles ISA/g(0.1 mg ISA/lOOg) wet weight tissue.
b. The sensitivity of the method (Flame photometric, sulfur detector) was <20 nmoles ISA/ml milk. c. The GLC sensitivity, using Flame Ionization detector was .008ymoles ISA/g (0.1 mg ISA/lOOg) of heart tissue.
d. The identity of the ISA peaks on GLC were confirmed on Gas chromatography mass spectrometers. 124
approximately at the limit of its sensitivity. The recovery
of isethionic acid (2.0 or 0.2 ymole/g of tissue) added
to rat brain or heart tissue was always between 95 and 100%
The sensitivity of isethionic acid detection in rat heart
and brain tissue, using a flame ionization detector was
approximately 0.2 pmole isethionic acid per g tissue
extracted (approximately 2.5 mg/100 g). Using the sulfur
detector, the sensitivity of the imethod was approximately
0.008 umole/g (0.1 mg/100 g tissue).
2.Isethionic Acid in Dog Heart Tissue
A large scale extract of dog heart tissue (400g)
was examined for isethionic acid following the method of
Welty and Read (1962). It was found that the extract
did not yield any crystals of isethionate. Furthermore,
analysis of an aliquot of the extract on gas chromatography
revealed ho evidence of isethionic acid. The sensitivity
of the gas liquid chromatographic portion of this experi•
ment was approximately 0.008 u mole/lOOg (0.1 mg/100g) of
heart tissue.
3. Isethionic Acid in Molluscan Tissues
In squid axoplasm, isethionic acid was found at a
concentration of 150 u mole per g axoplasm. Squid ganglion and Nautilus ganglion were found to contain
isethionic acid at 34.09 umoles and 2.9 8 umoles isethionic
acid per g (wet weight) ganglion respectively. The
identify of the peak was confirmed by gas chromato•
graphy arid mass spectrometry.
Isethionic acid in Rat Milk Samples:
The samples of rat milk analyzed showed no trace of
isethionic acid. The recovery of authentic isethionic
acid added to the milk samples was always 100% and the
sensitivity of the^method using a flame photometric
(sulfur) detector was such that 20 nmoles isethionic
acid/ml milk could have been easily detected.
Bioconversion of Taurine to Isethionic acid
Samples from dog heart and rat heart slices 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 slices showed that 90 per cent of the radioactivity 14 was recovered as C-taurine and up to 10 per cent was converted to a radioactive compound which behaved chromatographically like isethionic acid. The taurine to apparent "Isethionic acid" conversion was found riot to 126
increase with incubation time. The same result was obtained, 14 when C-taurine was added to heart slxces in Kreb-Henseleit phosphate buffer and the tissues were extracted immediately with methanol. A typical experimental result on rat heart slices is shown in Table 2. The chromatographic separation of radioactive taurine and isethionic acid on Whatman #1 14 paper relative to the separation of C-taurine after incu• bation with rat heart slices is shown in figure 15.
In order to evaluate the possible metabolism of isethionic acid or conversion to taurine, radioactively labeled isethionic acid was added to the tissue slices in 14 an incubating medium similar to that used in the C- taurine-heart slices experiments. All the radioactivity added to the tissue was recovered intact as (100%) isethionic 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 Slices
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 isethionic acid by rat
heart slices (150 mg wet weight) was carried out in
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 yl, 1.4 yM, 50 mCi/mmole)
in a total volume of 1.5 ml. The reaction was carried
out at 37°C in 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 isolated on ascending
paper chromatography. 128
Figure 15
14 14 Separation of C-taurine, C-isethionic acid 14 and the rat heart slices- 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 pi samples were spotted 1" apart and radioactivity, after chromatography, was detected by cutting 0.5" x 1" pieces and counting them in scintillation fluid.
Paper chromatographic separation of radioactive taurine (o o) and isethionic acid (o o) is shown. The chromatogram is interposed with the separation of the incubation product of 14
C-taurine with rat heart slices for the incubation times of:
1. 0 minutes (controls, left bars with solid
blocks).
2. 30 minutes (middle bars with oblique
striations) and
3. 60 minutes (right bars with straight
striations). 128 a 129
II. TAURINE AND ION TRANSPORT
A. Effect of Taurine on ATP-dependent Calcium Transport in Guinea-pig Cardiac Muscle;- _
1. Characterization of Ventricle Heart Homogenate and Sarcoplasmic Reticulum Enriched Preparation:
The electron micrographs of both microsomes enriched in
sarcoplasmic reticulum (S.R.) and ventricle homogenate
preparations are shown in figures 16 and 17 respectively.
The S.R. preparation revealed a high degree of smooth
membrane vesiculation, heavily contaminated with small
dark granules similar in texture to glycogen granules.
The smooth membrane vesicles were mainly sarcoplasmic
reticulum and possibly transverse tubules. Some
sarcolemmal membrane vesicles were also present as
revealed by ouabain binding assay measurements (see
Table 3). Occasionally, lysosomal bodies were
identified. No mitochondrial contamination could be
seen. Analyses using chemical markers (cytochrome
C oxidase) were not done to rule out the possibility of
mitochondrial fragment contamination.
Electron micrographs of the ventricle heart homogenates
showed intact mitochondria and membrane vesicles. Figure 16
Electron Micrograph .of Microsomal preparations enriched in sarcoplasmic Reticulum.
Micrographs were made by the standard procedure as described in the 'Materials and Methods' section, page 98. The micro-organelles identified in the micrographs were smooth membrane vesicles
(siav) , small dark granules similar in texture to glycogen granules (gly), and some Lysosomes (lys).
Magnification approximately 45,000 x. 130a 131
Figure 17
Electron micrograph of guinea-pig ventricle
heart homogenate preparation.
Electron micrographs were made by the standard procedure as described in the 'Material and
Methods' section, page 9 8 . The micro-organelles
identified were intact mitochondria (mito) and
the vesicles (ves) of different sizes. 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. Purified on sucrose density gradient 0.045 + 0.002 a'b
Microsomes enriched in 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 purified S.R. preparation were assayed for ouabain binding as described on page 98. ; _ a. The results shown --are a Mean +V.S.E.M. of 4 experiments from separate microsomal preparations. b. This value of ouabain binding to purified S.R. is in agreement with the observation of Gelbart and "Goldman (1977). 133
Occasionally swollen mitochondria were observed. It is doubt•
ful if the preparation has undergone significant irreversible
pathological changes,since the- homogenate preparation
consistently exhibited ah ATP-dependent calcium binding
activity as well as calcium uptake in the presence
of oxalate. The enzymatic activity of these calcium
transport processes are known to be labile at room
temperature or with prolonged or extensive homogenization
(Katz and Repke, 1967) .
2. Effect of Taurine on Calcium Uptake and Binding:
The effect of varying taurine concentrations on calcium
uptake and binding in both ventricular homogenate
and sarcoplasmic reticulum enriched preparations is
shown in Table 4. The free calcium concentration used
in these studies was 1.0 y_M. Taurine in concentrations
of 5 to 50 mM had no significant effect on calcium
uptake or binding in either of these preparations.
The effect of 20 mM taurine on calcium uptake and binding
in 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
Effect of Taurine on Calcium Uptake and Binding in Guinea-pig Heart Ventricle 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° 0.16 ± 0.03 12.40 ± 0.79 0.71 ± 0.10 (2.36 ± 0.40)b (0.16 ± 0.03) (12.72 t 0.89) (0.75 ± 0.09)
10 2.39 ± 0.15 0.17 ± 0.02 12.93 t 1.27 0.76 t 0.05 (2.32 ± 0.29) (0.16 ± 0.02) (13.19 t 1.19) (0.75 ± 0.04)
20 2.68 i 0.10 0.16 ± 0.02 12.72 ± 1.37 0.77 ± 0.05 (2.56 ± 0.14) (0.18 1 0.02) (12.18 ± 1.33) (0.81 t 0.02)
30 2.89 i 0.14 0.17 ± 0.01 14.79 ± 1.83 0.81 1 0.04 (2.89 i 0.13) (0.19 ± 0.02) (14.09 t 0.02) (0.74 ± 0.03)
40 2.62 t 0.22 0.18 ± 0.01 11.84 t 1.79 0.76 ± 0.02 2.70 ± 0.14 0.17 ± 0.02 11.27 t 1.72 0.76 ± 0.05
50 2.59 0.17 0.16 0.02 12.5S 2.05 0.70 0.08 (2.56 1 0.34) (0.17 ± 0.01) (12.52 ± 0.01) (0.75 t 0.09)
Guinea-pig heart ventricle homogenates (200-300 yg protein) or sarcoplasmic reticulum enriched preparations (40-50 wg protein) were incubated for 5 min with and without taurine in medium containing 40 mM histidine-HCl, pH6.8, 5 mM MgCl,, 5.mM ATP, 110 mM KC1, 2.5 mM Tris-oxalate, and 1.0 uM free Ca ~7l25 y_M CaCl2 containing~T5caCl2 (10 Ci/mole) and~391 uM EGTA). The reaction was carried out at 30°C in a total volume of 0.5 ml. Calcium binding was determined in an identical reaction mixture, except that 2.5 mM Tris-oxalate was omitted.
a. The results are a Mean t S.E.M. of at least 3 observations each performed in duplicate. b. The values in parentheses are controls (taurine omitted from the reaction medium). TABLE 5
The Effect of Taurine on Calcium Uptake and Binding at Various Calcium Concentrations in Guinea-pig Heart Ventricle Homogenates and Sarcoplasmic Reticulum Enriched Preparations
Calcium Homogenate Preparation Sarcoplasmic Reticulum Preparation Cone. (vK) Calcium Uptake Calcium Binding Calcium Uptake Calcium Binding (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min)
0.5 1.07 ± 0.03a 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)
5 100.0 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 5 0.12)
Guinea-pig heart ventricle homogenates (200-300 ug protein) or sarcoplasmic reticulum enriched preparations (40-50 ug protein) were incubated for 5 min with and without 20 mM taurine as described in Table 4 in the presence of various concentrations of free calcium. Calcium binding was measured under identical conditions in the absence of 2.5 mM Tris-oxalate.
a. The results are a Mean ± S.E.M. of at least 3 observations each performed in duplicate.
b. The values in parentheses are controls (taurine omitted from the incubation medium). 136 concentrations higher than 10 u_M were inhibitory. This 2+ profile of the Ca concentration effect on calcium transport was similar in the homogenate preparation. 2+
Taurine (20 mM) at all free Ca concentrations studied had no significant effect on calcium uptake or binding in either of these preparations.
The Effect of Taurine on the Time-course of Calcium
Uptake and Bindings:
The time course of calcium uptake and binding in homogenate and sarcoplasmic reticulum enriched, preparations is shown in figure 18A and 18B/ respectively. Calcium uptake in S.R. enriched preparations was linear for the first 10 minutes of incubation following which the rates of calcium uptake declined slightly. Maximum calcium binding was observed at 5 minutes of incubation. Taurine was observed to have no significant effect on calcium uptake or binding at all the incubation times studied.
In the heart ventricle homogenate preparation, maximum calcium binding was observed at 10 minutes of incubation. Again, no significant effect of taurine was observed on calcium binding or uptake either at the initial time (30 seconds) or at longer Figure 18
Time course effect of 20 mM Taurine (0,A), on calcium uptake (solid lines) and binding
(dotted lines) in Guinea-pig heart ventricle homogenates (A) and sarcoplasmic reticulum enriched preparations (B) . The.;%e-r-tic£e.Hlines.-,i represent ± S.E.M. of 3 determinations each performed in 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 in the presence of 1.0 yM free calcium as described in
Table 4 (Page 134). Calcium binding was measured under identical conditions in the absence of j 2.5 mM Tris-oxalate.
138 periods of incubation.
The Effect of Taurine on the Decay of Calcium Uptake
Activity:
Both the homogenate and the sarcoplasmic reticulum enriched preparations decreased rapidly in calcium uptake activity when kept at 4°C in the absence of 2+
40% sucrose. Ca -uptake activity of the homogenate decreased in a curvilinear fashion with time. This activity in sarcoplasmic reticulum enriched preparation exhibited a linear decay. Addition of 15 mM taurine to these preparations under these conditions did not alter this steady decline in calcium uptake activity
(figure 19). In the presence and absence of taurine, the respective coefficients in the regression equations were not significantly different (using-a computer program; UBC- SLTEST.)
Effect of Taurine on Cyclic AMP-dependent Protein-
Kinase Stimulated Calcium Uptake:
When the sarcoplasmic reticulum enriched preparation was incubated with protein kinase and cyclic-AMP in
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 Effect of Taurine on the decay of calcium uptake activity in guinea-pig ventricle homogenates and sarcoplasmic reticulum enriched preparations. Homo• genate (solid lines, 200-300 pg protein) or sarcoplasmic reticulum preparations (dotted lines, 40-50 ug protein) were maintained in 2 mM Tris-Cl, pH 7.2 in the presence
(0,A) and absence (•, •) of 15 mM taurine. Calcium uptake was measured at specified times as described in table 4: . The regression line for the sarcoplasmic reticulum was fitted 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 in duplicates. 139 a
Ca++ Uptake: nmoles/mg S.R.protein/min (D,A)
*»• po so o- o
P P — .— rsj oo ro o- o
Ca++ Uptake: nmoles/mg Homogenate Protein/min [;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 With Without With cAMP-dependent cAMP-dependent cAMP-dependent cAMP-dependent Protein kinase Protein kinase Protein kinase Protein kinase
a b - 2.39 t 0.29 ' 3.38 t 0.14° 9.72 t 1.02 16.86 ± 1.51d
e + 3.30 t 0.17 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 cyclic AMP (1.0 wM) and cyclic 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 Ca2+-uptake In the absence of cyclic AMP-dependent protein kinase. e. Not significant compared to the activity seen without taurine in the presence of cyclic AMP-dependent protein kinase. 141
Similarly, the rate of calcium uptake by the homogenate
also increased in the presence of cyclic AMP-dependent
protein kinase (p<0.05). Taurine (20 mM) had no
significant effect on the cyclic AMP-dependent protein
kinase stimulation of calcium uptake in both of these
preparations.
B. Effect of Taurine on Passive Ion Transport in Rat
Brain Synaptosomes
1. : Characterization of Synaptosomal Preparations
Electron micrographs of a typical synaptosomal
preparation is shown in Figure 20. The synaptosomal
suspension were found to consist of synaptic vesicles
and mitochondria enclosed within membranes to form
nerve ending particles. The electron micrographs
were consistent with those originally obtained by
Gray and Whittaker (1962) and De.Robertis et al.(1961).
2. The Osmometric Behaviour of Synaptosomes
of tne The optical extinction (E52Q) synaptosomal
preparations suspended in solutions of Na2SO^ was
found to increase with the molarity of the Na2S04
solution (Figure 21A). The reciprocal plot of
extinction (1/E52Q) against l/Na2SC>4 (figure 21B) Figure 20
Electron micrograph of a typical rat brain
synaptosomal preparation:
Electron micrographs were made by the standard
procedure as described in the 'Materials and
Methods' section, page 9 8 .-' The fraction
obtained between the sucrose gradient (0.8 M and
1.2 M sucrose) consisted of synaptic vesicles
(SV) and mitochondria (M) enclosed within membrane
(tm) to form a nerve ending particle. The electron micrograph is similar to those of Gray and Whittaker
(1962). Magnification approximately 45,000x. 142 a Figure 21
The effect of Na2SC>4 concentration on the' E52Q of a suspension of synaptosomes:
In (A) the data are plotted as E52Q against
Na2S04 while in (B) l/Na2SC>4 is plotted against
1/E^2Q. Results are shown as Mean ±S.E.M. of .
3 different synaptosomal preparations. 143 a
•520 144
showed a linear relationship. These results confirm
the observations of Keen and White (1970) and show that
the synaptosomal preparations behave as osmometers
conforming to Boyle and Van't Hoff's law. (Keen and
White, 1970).
3. The Effect of Taurine on Sodium and Potassium
Permeability in Synaptosomal Preparation
The permeability of the synaptosomal preparations to
sodium and potassium ions in the presence or absence
of taurine is shown in Table 8A and 8B, respectively.
Synaptosomal preparations preincubated with 2 0 mM
taurine and suspended in 100-200 mM sodium or
potassium acetate solutions containing 20 mM taurine
showed no significant change in E520 w^en compared
to results obtained in the absence of taurine.
4. The Effect of Taurine on the Passive Uptake and
Release of Calcium in Synaptosomal Preparations:
The time course of uptake and release of calcium in
an isotonic sucrose medium is shown in figure 22A
and 22B, respectively. The calcium concentration
used in this study was 10 y_M. Under these conditions,
synaptosomal preparations were initially observed , TABLE 7 " '
Effect of Taurine on Sodium (A) and Potassium (B) Permeability in Synaptosomes.
+ ! + A. Na Acetate B. ; K. Acetate
E520 . . E520 mM Na+ — mM K+ 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 E^g °f a synapto• somal membrane suspension (50 pi) in acetate salts in the presence (TAURINE) or absence (CONTROL) of 20 mM taurine. Each value is the mean ± S.E.M. of three separate synaptosomal preparations. 146
to take up calcium rapidly. After 2 minutes in 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 all time points studied.
This difference was significant (p<0.001; t-test of
the difference between the intercepts on the ordinate).
The slope of the lines were not significantly different.
Calcium release from preloaded synaptosomes is shown
in figure 22B. In the absence of taurine, after 2 . .
minutes of incubation, about 50% of the calcium load
was released from the synaptosomes. In the presence
of 20 mM taurine, calcium efflux from the preloaded
synaptosomes was reduced at all incubation .times
tested. Curves fitted by linear regression had
intercepts on the ordinates which were significantly
different (p <0.001).
Dose-dependent Effect of Taurine on Calcium Uptake
in 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 carried out where taurine was substituted for an equimolar Figure 22
The effect of taurine on (A) 4JCa';T uptake and 45 2+
(B) release of Ca from preloaded rat synaptosomal
preparations. Calcium uptake and release were
determined in the presence (o o) or absence
(• •) of 20 mM taurine in a medium containing
0.3 M sucrose and 10 mM Tris-HCl, pH 7.2 as described
in the 'Materials and Methods" section (pages 107, to 108).
Effect of taurine on calcium uptake is expressed as 45 2+ 2+
Ca uptake relative to the value of Ca uptake
observed at 30 minutes incubation time in the absence
of taurine. Calcium release from preloaded synaptosomes was calculated as % release as described in the /
'Materials and Methods' section, page 108. Lines were derived by a linear regression analysis of the data.
Each time.point represents determination from three
separate synaptosomal membrane preparations. 147a
Relative 45 Ca2+Uptake (%) Figure 23
The effect of various concentrations of taurine 45
on CaCl2 uptake in brain synaptosomal preparations
(solid bars). Control experiments were done in the
presence of equimolar concentrations of choline
chloride (middle bars with oblique striations) and
in the presence of neither taurine nor choline
chloride (left bars with straight striations).
Calcium uptake was determined at 2°C for 30 minutes
in medium containing synaptosomes (0.2 mg/ml), 0.3 M
45 sucrose, 10 mM Tris-HCl, pH 7.2 and 10 y_M CaCl2 5 (5 x 10 cpm/sample) in a total volume of 0.3 ml. Each bar represents the Mean ± S.E.M. of three
separate experiments relative to the value of the 45
CaC^-uptake of controls measured in 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 in the incubation medium. No change in
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 in calcium
uptake was observed. Choline chloride (on a molar
basis) was more potent than taurine in lowering
synaptosomal calcium uptake in concentrations greater
than 10.0 mM.
6. Effect of Other Amino Acids on Calcium Uptake in
Synaptosomal Preparations
A number of compounds, in a concentration of 20 mM,
were tested for their effect on passive calcium
uptake in synaptosomal preparations (figure 24).
Homotaurine, hypotaurine, 3 - alanine and GABA
exhibited similar effects to'.'taurine, significantly
decreasing the degree of calcium uptake observed in ;
the absence of these agents (p<0.05). a - alanine, :did
not significantly affect calcium transport in this
preparation. Methionine, proline and"valine (not shown)
did not significantly affect calcium transport in this
preparation. :-~ • Figure 24
Effect of various amino acids on calcium uptake in brain synaptosomal preparatons. Calcium uptake was measured for 30 minutes under the same conditions as that described in Figure -'23' in 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 in each case. ^Ca2+-Uptake: nmoles/mg protein
o to Ln o O 61 T T 1 Water
Taurine
j3-Al a nine
GABA
Homotaurine
Hypotaurine
Of-Alanine
o 151
DISCUSSION 152
I. BYCONVERSION. OF TAURINE TO ISETHIONIC ACID
IN THE REGULATION OF ION FLUX
Methylation of isethionic 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 ratio of these two compounds produced by methylation
is approximately 20:1 with the methylester derivative being predominant.
This ratio was confirmed by proton NMR spectro•
scopy (figure 12A). The NMR spectra of both methylated
isethionic acid (figure 12A) and 1-butanesulfonic acid
(figure 12B) showed the CH^ peak of the methylester as a
singlet at 3.88 ppm. A small singlet at 3.30 ppm occurring
'in the spectra of methylated isethionic acid was assigned
as the CH2 group of the methylether of isethionic acid on the basis of the known chemical shift value of 3.30 ppm of the methylether singlet in methoxyethanol (figure 12C).
The assignment of structures to the two methylated derivatives of isethionic acid was also carried out using
GC-MS. The assignment of structures to the mass spectra of
the peaks by methylation of isethionic acid (figure 11) is in conformity with known fragmentation pattern of alkyl 153
alkanesulfonates (Truce et al., 1967). The fragmentation and
rearrangements of the two methylated isethionic acid compounds are shown in Table 8;. Some possible mechanisms for fragmen•
tation structures are shown in figures 25 and 26. The parent ions .'. [M] t were not seen in the methylated isethionic
acid mass spectra. Truce et al. (1967) claim that these ions are scarce for most of the alkyl 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 isethionic acid at m/e=139.
The gas-liquid chromatographic-methy.lation technique was used for the analyses of isethionic acid in mammalian tissues. Welty, Read and Shaw (196 2) quoted a
figure of 42.6 mg isethionic acid per 100 g rat heart tissue and 12.9 mg per 100 g dog heart. These amounts would have been quite easily detectable with my method. In heart, using the sulfur detector,which in this particular case .was ten times more sensitive than the flame ionization detector,' only a very small peak, roughly 0.10 mg/lOOg wet weight tissue (0.008 y moles/g) in the position of isethionic acid, could be seen.Insufficient material was available to confirm that this small amount of material was truly isethionic acid. As much as 400 g of dog heart tissue was extracted to search for isethionic acid, following exactly the procedure of Welty, Read and Shaw (1962). In this large TABLE <
The FragBentation and Kaarrangeaents of the Two Methylation Product* of laethlonic Acid that were Analysed Using an OV-17 Column
FRAGMENTS STRUCTURE FRAGMENTATION AND REARRANGEMENT "»/e PROCESS
A. Methylester atethvlether of Isethionic acid
31 OCUj* a cleavage
45 CHS* A rearrangement process shown by high resolution Measurements.
CHjO - CHj* Cleavage typical of aliphatic ether
se CHjOCH - CHj B- hydrogen rearrangement (Pig. 25) (A e- hydrogen transfer vith a-cleavage).
+ a - cleavage 59 CBjCBjOCH3
Rearrangement ion derived from the 79 B02 CH3* aUtane group of the sulfonate ester.
so CH * o - cleavage 95 3 3
+ t - hydrogen rearrangement (Fig.25). 96 BOS02CH3 (A e- hydrogen transfer with a - cleavage)
CBJSOIOCHJ) * McLafferty rearrangement (Fig.25) 124 2 (methyl group transfer with e- cleavage)
CH3OCB2CM2S02a* a' cleavage with hydrogen transfer
B. Methylester of Isethionic Acid
31 OCHj* o cleavage
44 CHjCHOH* 8 - hydrogen rearrangement (Fig. 26) CA 6 - hydrogen transfer with a - cleavage)
45 HOCHjCBj* o - cleavage
CHS* A rearrangement process shown by high resolution measurements
+ 79 CH3S02 Rearrangement ion derived from the alkane group of the sulfonate ester
80 CHJSOJH* a' cleavage with transfer of s $' hydrogen
+ 95 S03CH3 Q- cleavage
(OH) SOCH + 97 2 3 Two 6 - hydrogen transfer with a - cleavage
110 + McLafferty Rearrangement (Fig. 26) CH2SO(OH)OCH3 (o - hydrogen transfer with fl -cleavage).
o' cleavage with BOCHJCHJSOJH* hydrogen transfer
OB(CH CH + 139 2)2S03 2 |M - 1) *
The fragnentations and rearrangements undergoing mass spectral conditions are
explained by sjachanilIB S established from the work of Truce et al. (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 CQ- Cft bond, u" cleavage refers to the 8-OR bond. A substituent referred to as an a vubetitusnt will 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 al., 1967). The mass ions obtained for
Methylether, Methylester due to these rearrangements were relatively abundant on the spectrum (Figure 11)
and were important in the assignment of its
structure. The convention proposed by Budzikiewicz et al.^
1964 for denoting election shifts 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, Calif., p. xii. 155a
MASS SPECTRA OF METHYLATED ISA — FRAGMENTATION & REARRANGEMENT (PEAK I of Fig. 9Band SPECTRUM A of Fig. 11)
OCH o+ 3
O 1 I / OCH^ _^ Q=CH2 + CH2=S — OCH3| 0\- II O ^ CH2 m/e = 124
Methylester, Methylether [CH0] + of Isethionic Acid m/e = 29
McLAFFERTY REARRANGEMENT
OH
S —OCH, CH3OCH =CH2 + [CH3OCH=CHJ
Methylester, Methylether m/e - 58 m/e = 96 of Isethionic Acid
(3- HYDROGEN REARRANGEMENT
(TRUCE, et. al. J. Org. Chem. 32: 308, 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 al., 1967). These massiions obtained for the methylester of isethionic acid were relatively abundant on the spectrum (figure 11) and were important in the assignment of its structure. *
The convention proposed by Budzikiewicz et al.
1964 for denoting electron shifts 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, Calif., p. xii. 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
McLAFFERTY REARRANGEMENT
[TlOCH=CH7|t m/e 96 Methylester of Isethionic Acid m/e = 44
HYDROGEN REARRANGEMENT
(TRUCE, et. al. J. Org. Chem. 32: 308, 1967) 157
experiment, neither the crystals of sodium isethionate
(that they reported) nor gas chromatographic evidence of
the presence of isethionic acid were obtained. The
sensitivity of the gas-liquid chromatographic portion of
this 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 isethionic acid on ion-exchange chromatography
reported by Welty, Read and Shaw (196 2) was not correct.
Isethionic acid, a strong sulfonic acid was reported to be
retained on an acidic cation exchange resin. The validity
of results of these workers is questionable.
When the methylation technique was used to analyze
isethionic acid in rat brain, a small peak in the position of
isethionic acid was detected at a concentration of
approximately 0.2 mg/100 g of wet weight tissue (0.016 pmole/g).
Insufficient material was available to confirm the identity of this material by mass spectrometry. The analytical procedure was always monitored by adding isethionic 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 isethionic acid in mammalian tissue was confirmed using squid giant axon where isethionic acid is found in high concentrations.
The value obtained for the concentration of isethionic acid in the squid giant axon compares favourably with other data 158 obtained using different less sensitive methods (Deffner and
Hafter, 196 0; Hoskin and Brande, 1973). It remains possible, however, that isethionic acid is irreversibly bound to tissue membranes. The addition of methanol during homogenization lowers the dielectric constant and this could theoretically suppress its ionization to some extent. Any effects of methanol on isethionic acid in tissues could theoretically, cause binding of isethionic acid to tissues and not necessarily affect binding of added isethionic acid. There is a precedent for such a possibility in the binding of phosphoinositides to brain tissues during extraction procedures (Rouser et al.,
1967). However, Schaffer et al. (1978b) recently reported measurement of isethionic acid levels in rat hearts. Isethionic acid in their studies was extracted with 3% perchloric acid after the rat hearts were lyophilized in liquid nitrogen.
Only trace amounts of isethionic acid (1.03 pmole/g dry weight) were reported to be present in rat heart. This concentration is about 50-fold less than the values previously reported by
Rosei et al. (1974) and Welty et al. (1962). In my studies, I found even lower concentrations of isethionic acid in rat heart and brain tissues than those reported by Schaffer.
However Schaffer did not confirm the fact that their gas chroma• tographic peaks contained >only isethionic acid and did not use a sulfur detector in their work. The use of a sulfur detector was especially important in my work because it has a much greater sensitivity than a flame ionization detector. However, the findings of Schaffer et al. (1978b) confirm our results in that 159
isethionic acid in rat and dog hearts and rat brain is a minor anion in these tissues.
During the course of analysis of isethionic acid in
tissues, it was usually necessary to use two columns, one
of OV-1 and another of OV-17, and occasionally two methods of
detection, flame ionization and flame photometry (for sulfur).
This was necessary because all of the mammalian tissues
studied gave a peak in the position of isethionic acid in 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 isethionic acid per 100 g heart or brain.
Confirmation that this peak was not isethionic acid depended on
rechromatography of the same extract on OV-17 columns and
verification that this peak did not contain sulfur. The large
peak from heart and brain extracts seen on the OV-1 column
when reassessed during the OV-17 column and the sulfur detector
proved to contain only very small amounts of isethionic acid.
This use of a sulfur detector- was necessary because routine
access to GC-MS was not possible at the time.
During the course of developing an analytical
method for the analysis of isethionic acid, Rosei et al.
(1974) reported a gas-liquid chromatographic detection
of isethionic acid in guinea-pig heart. Silylation of
isethionic acid using the method of Rosei et al.(1974) 16 0 proved to give a very complex mixture. Thus, silylation was found not to be feasible in our hands. Accordingly, this technique was abandoned. Recently, Schaffer et al. (1978b) and Fellman et al. (1978) have reported a gas-chromatographic method to measure isethionic acid in heart muscles ;using a silylation procedure different from that of Rosei et al.
(1974). Both Fellman and Schaffer reported that isethionic acid is present in very small concentrations or almost absent in heart tissue, thus confirming our own results
(Applegarth et al., 1976; Remtulla et al., 1977).
To complete our studies of isethionic acid in heart tissue, it was logical to repeat the taurine to isethionic acid conversion experiments reported by Read and Welty (19 62) using rat heart slices. Utilizing their 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 slices and extracted immediately with methanol, a radioactive compound was obtained which behaved chromatographically (on Whatman
#1 paper) like isethionic acid. It is possible that the compound Read and Welty (1962) reported is an artifact 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 identity of this compound. However, Fellman et al. (1978) recently showed that taurine is not converted to isethionic acid by heart, brain or liver (This work of Fellman will be enlarged upon later). 161
Recently, Sturman, Rassin and Gaull (1977c) reported the presence of a radioactive compound in extracts of rat milk, 35 following the injection of S-taurine to lactatmg rats. This compound co-chromatographed with authentic isethionic acid.
A possibly similar compound was obtained by Huxtable and
Bressler (1972) during their studies on the distribution and interconversion of radioactive taurine and isethionic acid in the rat. The compound obtained in these studies was not characterized. It was merely identified as a radioactive compound in the area of a thin layer chromatography (TLC) or ion-exchange column separation normally occupied by isethionic acid. However, the report of the presence of isethionic acid in these studies was puzzling. It was of interest to determine whether or not isethionic acid was present as a natural constituent of milk. Milk samples from lactating rats were kindly provided by Dr. John Sturman and analyzed for isethionic acid by our technique. The samples analyzed showed no trace of isethionic acid. Recovery of authentic isethionic acid added to the milk sample was 100% and the sensitivity of the method was such that 20 nmoles isethionic acid/ml milk could have been easily detected. In the report of Sturman,
Rassin and Gaull (1977c), the radioactive compound in milk which co-chromatographed with authentic isethionic comprised 30-40% of the total radioactivity (the rest of 162
which was present as taurine). The present assay could
easily have detected isethionic acid present at 10% of the
taurine concentration.
A clue to the resolution of the discrepancy in
these observations comes from the recent report of Fellman
et al. (1978). In this 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 in the same
fractions as did authentic isethionic acid. Fellman et al. (1978) also reported the synthesis 3
of (2- H)-taurine of high specific activity and tested it as
a precursor for isethionic acid synthesis in dog heart
slices, rat heart and brain slices and rat heart and brain homogenates. The conditions used in this study were also
those described by Read and Welty (1962). The assay consisted
of the measurement of the release of tritiated water during
the formation of isethionic acid via a sulfonic-acetaldehyde
intermediate. The mammalian tissues tested were found not
to convert taurine to isethionic acid in any significant quantity. The limit of detectability of the isethionic
acid conversion was less than 4.5 x 10 u. moles, of isethionic
acid. On the other hand, the bacterium, Pseudomonas
(Toyama et al., 1973; Yonaha et al.:,, 1976) and samples from 163
rat feces and gut washings were found to release tritiated 3 water when incubated with (2- H)-taurine.
Hoskin and Kordik (1977) and Cavallini et al. (1978)
provided further evidence that isethionic acid is not a
metabolite of taurine in squid and mammalian tissues. In
squid giant axon, where isethionic acid is found in high
concentration, hydrogen sulfide and not taurine was shown
to be the precursor for the synthesis of isethionic acid
(Hoskin and Brande, 1973; Hoskin et al., 1975). The enzyme
involved in this pathway was said to be 'rhodanese'
(Hoskin and Kordik, 1975; Hoskin, 1976) .
In mammalian tissues, taurine and isethionic acid have been postulated to be derived from a common precursor
along the cysteine to taurine pathway (Cavallini et al.,
19-78) . Studies carried out by Cavallini 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 isethionic acid, starting
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 sulfinic acid, which then undergoes further oxidation to isethionic acid. The identification of isethionic acid in 164
these experiments was based on paper electrophoresiscand 35 paper chromatographic techniques. When S-labeled mercaptoethanol was injected into rats, 91% of the radio• activity was excreted in the urine within 24 hours. A small amount of this radioactivity was identified as isethionic acid (Federici et al.,19 76). The acceptance of this hypothesis as the true synthetic pathway requires further studies using either isotope dilution analysis or more sensitive methods for the detection of isethionic acid.
The trace amounts of isethionic acid present in heart and brain tissues reported in this thesis and elsewhere
(Remtulla et al., 1977; Schaffer et al., 19 78b)could either have originated from the intestinal tract bacterial-deamination of taurine to isethionic acid (Fellman et al., 1978) or have been produced by the alternate pathway described by
Cavallini et al. (1978). Further research may clarify this point.
Isethionic acid, even if not a product of tissue bioconversion of taurine, may possibly have a function of its own. Bourke et al. (19 70,. 1970) have provided evidence for the potential usefulness of isethionic acid in the reduction of cerebral edema. Jacobsen et al.
(1967) using isotope techniques,claimed to find isethionic 165
acid in human urine and plasma > The average urinary excretion of isethionic acid was 13.7 mg/24 hours (Jacobsen et al., 1967) and the average concentration in blood was
32.6 yg/100 ml plasma (Jacobsen., - 1968). 'From these values, the renal clearance for isethionic acid was calculated to be 30 ml/minute assuming that isethionic acid is . not bound to plasma proteins and passes freely across the glomerular membrane (Jacobsen, 196 8). The mammalian nephron must therefore contain mechanisms, whereby 70-80% of the isethionic acid, filtered through the glomerular membrane, is 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 significant decrease in the concentration of K+ and Cl~ in the serum and Cl~ in liver and kidney were observed. No overt toxicity was evident on hematological and histopathological study of these tissues.
The elucidation of the function of isethionic acid awaits further evidence.
In summary, the results described in this thesis clearly demonstrate that isethionic acid is present in only trace amounts in mammalian brain and heart and .taurine was found not to be metabolized to isethionic acid as was originally claimed by Read and Welty (1962). 166
In view of these results, it is pertinent to re-examine the original proposition of Read and Welty
(1965) that ion translocation in myocardial cells involves
the bioconversion of taurine to isethionic acid. : Read and
Welty (1963) were the first to claim that taurine prevented premature ventricular contraction of the dog heart, caused by digoxin or epinephrine. Welty and Read (196 4) suggested
that this effect of taurine was mediated by a precursor of
its metabolite isethionic acid. Welty (Ph.D. thesis,
South Dakota, 196 3) hypothesized that taurine, under physiological conditions, existed in part as a ring structure due to ionic bonding between the sulfonic acid group and amino group. He proposed that such a cyclic molecule would carry, no charge and would be relatively permeable through the cell membrane. Within the cell, deamination of taurine to isethionic
acid'"would produce a negatively charged molecule- capable of
cation attraction. It was suggested that this would result
in asymmetric distribution of sodium and potassium inside
and outside the cell which in turn would result in the
development of an electric potential across the cell membrane.
During cardiac arrhythmias a large efflux of potassium has been shown to occur in experimental animals given toxic
doses of epinephrine (Melville et al., 19 55; Daniel et al.,
1957) or cardiac glycosides (Holland and Dunn, 1954;
Sarnoff et al., 1963; Tuttle et al., 1962). The deamination
of taurine to isethionic acid was said to release a charged 167
anion group which served to retard or prevent the efflux of cellular potassium which accompanied cardiac arrhythmias.
There are a number of problems with this proposition:
(1) Isethionic acid was said to act solely by attraction of
cations. Therefore, a stoichiometric relationship would
exist between the excess cations retained and the iseth•
ionic acid produced. However, even in Read and Welty's
work only a small fraction of the taurine pool (1.4%)
was claimed to be converted to isethionic acid over a
30 minute period (Read and Welty, 1962).
(2) The mechanism for potassium retention in the cell was
said to be by charge neutralization. The changes in
membrane potential due to the presence of a negatively
charged species (isethionic acid) in the myocardial
cell should not-affect the selective permeability to ions.
(3) The hypothesis also overlooks the necessity of
potassium flux across the cell membrane for excitation-
contraction coupling to occur.
Evidence in the literature on the time course of the action of taurine in the heart muscle (Read and Welty,
1965; Chazov et al., 1974) indicated an effect of taurine per se and not the effects of a slowly produced metabolite.
I therefore, next looked at actions of taurine itself in biological preparations. 168
II TAURINE AND ION TRANSPORT
Further evidence in the literature (Dolara et al.,
1973, 1976; Huxtable and Bressler, 1973) implied that- : in the heart .taurine had effect on calcium transport. The work of Dolara et al. , (1973) in perfused guinea-pig hearts and in S.R. preparations of guinea-pig hearts (Dolara et al. , 1976) suggested that taurine had an effect on calcium binding and transport in these preparations/
The field of taurine physiology is prone to contradictory results and it seemed prudent to re-assess the effects of taurine on calcium binding and transport in heart preparations.
The studies on the possible effect of taurine on ATP-dependent calcium transport in 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 in the presence of
ATP and oxalate. In the latter process, calcium oxalate has been shown to precipitate within the membrane vesicle
(Hasselbach and Makinose, 1962)-. The calcium accumulation in the S.R. occurs by transport of calcium through the membrane into the vesicle lumen when precipitating agents such as oxalate or phosphate are present in the solution
(Kanazawa et al., 1970; Tonomura, 1973;MacLennan, 1975). 169
In the mammalian heart, full contractile activity occurs when 50-100 nmole calcium per. g wet Weight of ventricular tissue are made available for binding to troponin, the calcium receptor protein of the contractile system (Katz, 1970).
During each cardiac cycle at maximal contractility, this amount of calcium must bind to and then be removed from the regulatory sites of troponin. Removal of this calcium from the contractile unit is controlled by the sarcoplasmic reticulum (Langer, 1973), a membranous intracellular structure which surrounds the myofibrils. Preparations of cardiac microsomes that are enriched in fragmented sarcoplasmic reticulum have been shown to accumulate calcium against a 2+ concentration gradient in the presence of ATP and Mg
(Carsten, 1964; Katz and Repke, 1967). Calcium accumulation by cardiac microsomes is coupled to ATP hydrolysis via 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 al.,
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 al., 1978; Ebashi and Endo, 1968).
Calcium uptake by sarcoplasmic reticulum is critical to the control of the rate of relaxation of cardiac and skeletal muscle. The degree of accumulation of calcium is also 170
believed to relate to the force of contraction of subsequent
beats (Tada et_al., 1975).
Results obtained on the ATP-dependent calcium
uptake and binding parameters of the S.R.-enriched preparation
used in this study were similar to those noted by other
workers (Tada et al., 1974; 1976; Repke and Katz, 1972;
Nayler et al., 1975). In separate experiments (not shown
in this 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 is 2+
ln close agreement with other studies (0.75-1.10 pM Ca ;
Hicks-arid Katz, 1979; Tada et al. , 1978). The sarcoplasmic
reticulum enriched preparation used in this study is a
routine preparation used in the laboratory where this 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 activity as well as calcium uptake in the presence of oxalate; these activities, though, were lower than those noted in the S.R. enriched preparations. The calcium concentration dependency
(Table 5) and the time course of calcium uptake and binding were similar for both enriched S.R. and whole heart 171
ventricle homogenate preparations (Figure 18). Similar
profiles for the decay of calcium uptake activity were
also seen in these preparations (Figure 19). Furthermore,
cAMP-dependent protein kinase was found to stimulate calcium
uptake activity in whole heart ventricle homogenate
preparations to the same extent (approximately 170% of
the activity seen in the absence of cAMP-dependent
protein kinase) to that observed in the enriched S.R.
preparation (Table 6, Tada et al. 19 74). Neither of
these preparations exhibited enhancement of calcium binding activity in the presence of cAMP-dependent protein kinase (results not shown; Tada et al., 1974; 1976).
It is possible that calcium uptake activity noted in the whole heart homogenate is due mainly to sarcolemmal membrane vesicles. It has been reported that cAMP- dependent protein kinase stimulates sarcolemmal membrane
calcium uptake in the presence of oxalate (Sulakhe et al.,
1976). 172
In the present study, it was demonstrated that
taurine (5-50 mM) did not significantly affect ATP-
dependent calcium transport in guinea-pig cardiac ventricle
homogenates or in cardiac preparations enriched in
sarcoplasmic reticulum. Various parameters of calcium
uptake and binding were examined; Taurine (20 mM) was
found to have no significant effect 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 in homogenate prepara•
tions or in the cardiac preparations enriched in S.R. did
not affect the enhancement of calcium transport produced by cyclic AMP-dependent protein kinase. Lack of an
effect on this system indicates that taurine does not
act as a modulator of calcium transport through this
cyclic AMP-mediated pathway.
Recently, Schaffer et al. (19 78a)reported that
the positive inotropic effect of taurine was not mediated by changes in cyclic nucleotide levels. These workers used standard working perfused rat heart preparations as described by Neely et al. (1967); Perfusion with both
taurine and epinephrine caused a rapid increase in cAMP
levels to the same extent as that observed in the presence of epinephrine alone. 17:3
Entman et al. (19 77) recently reported that taurine had no effect on calcium transport in canine cardiac S.R. preparations. These workers used the spectrophotometric murexide dye technique for the measurement of calcium transport 45 whereas in our studies CaC^ and the millipore filteration technique were utilized. Present studies thus confirm those of Entman et al. (1977) using both an S.R. enriched and crude homogenate preparation of heart tissue in a species in which the pharmacological effects of taurine in cardiac tissue have been noted (Guidotti et al. , 19 7-1; Dietrich and Diacono, 1971). The possibility that taurine may alter calcium uptake or binding in cellular organelles (Sarcolemmal membrane, mitochondria) other than S.R. tends ...to be ruled out by the lack of an effect on calcium uptake or binding in the homogenate preparations used in this study.
Huxtable and Bressler (1973) reported that calcium uptake by S.R. isolated from rat skeletal muscle could be increased by the isolation of the S.R. in 15 mM taurine.
Exposure of the S.R. to taurine throughout"the isolation procedure also resulted in an increased yield of sarcoplasmic reticulum. In our present studies, it was observedtthat when cardiac microsomal preparations enriched in S.R. were stored in -the absence of 40% sucrose at 4°C, the calcium accumulating capacity decreased rapidly. In this study, using these conditions, taurine had no effect on the decay process on 174
either preparation. The lack of. an effect of taurine on 2+
this decay in Ca -transport was also noted by Entman et al., (1977) under different experimental conditions.
The results obtained in these present studies differ from those of Huxtable and Bressler (1973). A number of possible reasons for this discrepancy are apparent.
Firstly, these present studies employed cardiac muscle preparations and those of Huxtable and Bressler, skeletal muscle. There are a number of anatomical and electro• physiological differences between skeletal and cardiac tissues: Compared to skeletal muscle, the heart cell is smaller, has a slower rate of contraction and has a less 3 extensive sarcoplasmic reticulum. Secondly, it should also be noted that the isolation techniques for S.R. and the incubation conditions for measuring calcium accumulation were different in these present studies compared to those used by Huxtable and Bressler. Thirdly, there was a difference in the animal species used in these two studies. The present experiments, using these same conditions should therefore be repeated in skeletal muscle in order to verify the results of Huxtable and Bressler (1973).
Guinea-pig heart muscle preparations were used in the present studies, because most of the previous experiments on the cardiac effects of taurine were observed in this animal species (Dietrich and-Diacono, 1971; Giotti and 175
Guidotti, 1969; Guidotti et al. 1971; Polara et al.,
1973; 1976). Microsomal preparations enriched in S.R. used in this present study have previously been used by other investigators to determine the in-vitro effects on calcium transport activity of a number of agents XCaffeine, verapamil, lanthanum, ionophores, epinephrine, glucagon, cardiac glycosides; Nayler et al. 19 75; Katz et al., 19 77; Katz and
Repke, 1973. Entman et al., 1969a; 1969b; 1973; Lee and Choi,
1966; Kirchberger et al., 1972; Tada et al., 1978, Weller and Laing, 1979). Similar preparations have been used successfully in identifying S.R. abnormalities in congestive heart failure (Harigaya and Schwartz, 196 9; Murr, et al.,
1970) and ischemia (Lee et al., 1967; Schwartz et al. 1973).
We have recently reported (Katz and Remtulla, 19 78) that ".a phosphodiesterase activator protein isolated from bovine brain stimulated calcium transport in similar microsomal preparations enriched in S.R. isolated from canine hearts.
More recently, Chubb and Huxtable (1978b)reported 2+ that taurine (20 mM) had no effect on either Ca -binding 2+ 2+ or (Ca + Mg )-ATPase activity in S.R. preparations isolated from hearts of normotensive Wistar and Okamoto spontaneously hypertensive rats. These workers commented that their findings do not agree with those of Dolara et al. (19 76) who observed that taurine increased the calcium content of guinea- pig cardiac sarcoplasmic reticulum. It was argued that the 176
discrepancy .in these results was due to differences in the method of isolation of S.R. in the animal species used and in the techniques used for the measurement of calcium transport. However, Chubb and Huxtable (1978b) did not point out that their own results were at variance with those reported earlier in studies using rat skeletal muscle sarcoplasmic reticulum preparations (Huxtable and
Bressler, 1973).
In summary, the results obtained in this thesis clearly show that taurine does not affect ATP-dependent calcium transport in either a microsomal preparation enriched in S.R. or in a crude homogenate preparation containing a number of cellular organelles that might be implicated in a taurine effect. In this study as well, no effect of taurine was noted on the decay of calcium accumulating capacity in either preparation;. These results corroborate those of Entman et al. (19 77) and have recently been substantiated by Chubb and Huxtable
(1978).
Chovan et al. (1979X'.-ha-ve recently reported that taurine (10 mM) increased calcium binding to rat heart sarcolemmal membranes. This report differs from the present studies on guinea-pig heart preparations in that the calcium binding studies by Chovan were carried out in the absence 177
of ATP. The possibility that taurine may exert its
effects in mammalian cells by altering the passive (ATP-
independent) transport of ions was evaluated independently
in this thesis work and has been reported elsewhere
(Remtulla et al., 1979).
The effect of taurine on ATP-independent calcium
transport was studied in rat brain synaptosomal preparations.
Cardiac sarcoplasmic reticulum vesicles 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 in calcium permeability due to the phosphory- lation of the synaptosomal membrane has also been reported
(Weller and Morgan, 19 77). Taurine has been implicated in. a role in ion flux at the synaptic terminal level(Kuriyama et al., 1978; Lahdesmaki and Pajunen, 1977). It was there• fore appropriate to use synaptosomes as a model system to study the possible effect of taurine on passive ion transport in mammalian tissues.
Before attempting to determine the permeability of the synaptosomes to ions it was first necessary to ensure that the preparation was in the form of sealed vesicles
(Keen and White, 1970; Koch, 1961, Tedeschi and Harris, 1955). 178
Such vesicles should behave as osmometers and obey Boyles and van't Hoff's law; that is, their volumes should be inversely proportional to the osmotic strength of the medium in which they are suspended. The volume of particles
in suspension is inversely proportional to the optical extinction of the suspension since smaller particles scatter more light. Initial experiments, indicated that
the E^2u °^ synaptosomes suspended in a solution of Na2SC>4
increased with the strength of the solution and that when
was 1/E52Q plotted against l/Na2S04 -a linear 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 in the form of sealed vesicles.
Synaptosomal preparations similar 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 al.,
1968), 5-hydroxytryptamine (5-Ht) (Bogdanski et al.,
1968), choline (Marchbanks, 1968) and GABA (Weinstein et al., 1965). Keen and White (1970) used this technique to 179 measure the permeability of synaptosomes to various ions.
They observed that acetate ions were more permeable than chloride ions; acetate salts of Na+ and K+ were therefore used in these present experiments. It was found that there was no effect of taurine (20 mM) on the permeability of the synaptosomal membranes suspended in solutions of sodium and potassium acetate (100 to 200 mM). Therefore, taurine did not affect the permeability of synaptosomes to Na+ or K+ ions. Lahdesmaki and Pajunen (1977), using a different technique, reported a reduced outflow of Na and K ions from synaptosomes in the presence of 5 mM taurine. Their experiments were performed in sodium- and potassium-free . . medium containing choline, calcium and magnesium. It is possible that this effect of taurine was secondary to an effect on the permeability of calcium. The possible effect of taurine on the passive permeability of calcium in this preparation was therefore evaluated.
Initial experiments showed no detectable decrease in E^Q on suspension of synaptosomes in solutions of calcium acetate indicating that the permeability of 2+ . . Ca was too low to be measured by this technique. The rate of calcium permeability in the synaptosomes. was there- 45 2+ . . . fore determined using Ca as a monitor since this 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 after 20 minutes of
incubation. The synaptosomal preparations used in this
study were found to be more active in that maximal calcium
uptake (3.5 nmoles'/mg protein) was reached ^after 4 5 minutes
incubation in the absence of taurine. In these experiments
it was demonstrated that taurine had an inhibitory effect on both calcium uptake and release in synaptosomal prepar• ations suspended in isotonic 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 significant effect of taurine on calcium uptake. The discrepancy in the calcium uptake results of this study and that of
Kuriyama et al., (1978) could be due to the differences in the experimental procedures utilized; 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 in S.R. described earlier in this thesis and . .elsewhere (Remtulla et al., 1978) . In these studies as well, we found no;effect of taurine on either
ATP-dependent calcium uptake or calcium binding.
In the present studies in brain synaptosomes, the inhibitory effect 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 effect. Hue et al.,(1978) in studies on the insect central nervous system also demonstrated that taurine at concentrations lower than 10 mM had no effect on the sensitivity of post - synaptic neurotransmission.
It should be noted that in these experiments, choline chloride in concentrations of 10 mM or greater produced a marked inhibition of calcium uptake. This inhibition was greater than that observed for equimolar concentrations of taurine.Choline chloride was used to control possible tonicity changes caused by added concentrations of taurine. Measurement of the osmolality of the controls in the absence of choline and those containing taurine were not observed to be significantly different.Although the mechanism of this effect of choline was not further investi• gated it should be stated that choline cannot be substituted for other ions in this or similar studies as it markedly inhibits ion movements in its own right. This finding is in agreement with
Jones et al., (1977) who reported that choline ions (100 mM) 2+ had an inhibitory effect on the rate of Ca -uptake in cardiac
S.R. membrane vesicles.
In these present studies, it was also shown that homotaurine, $-alanine and GABA inhibited calcium uptake in synaptosomal preparations in the same concentration as that observed to produce the inhibitory effect of taurine. This indicates that the inhibitory effect on calcium 182 uptake in these preparations was not specific to taurine.
Byington (thesis, South Dakota) in 1964 reported similar results; homotaurine, (3-amino propanesulfonic acid), the three carbon analogue of taurine and a close structural 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 inhibitors of impulse transmission in stretch receptor neurons of cray fish (McLennan and Hagen, 1963) and in mammalian neurons
(Curtis and Watkins; 1961). They also act as depressants, causing both loss of muscle tone and gross incoordination, when injected intraventricularly into the brain of mice
(Crawford, 1963).
A number of studies, reviewed in detail by Curtis and Watkins (196 0; 196 5) and Usherwood (19 78) indicate that
GABA is a physiological inhibitor of impulse transmission in 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 in the brain. In vertebrate and invertebrate preparations it has been shown that GABA and taurine mimic the action of one another (Krnjevic^and Puil/ .1976.; Enna-and
Snyder, 1975; Edward and Kuffler, 1959). From the results obtained in this present study and from previous studies, one might suggest that the mechanism underlying the effect of taurine, GABA and homotaurine on'excitability of neuronal 183
and cardiac tissues is common to all.
Taurine receptor sites have recently been reported
to be present in synaptosomal preparations (Lahdesmaki
et al., 19 77) and in heart ventricular sarcolemmal preparations
(Kulakowski et al., 1978). Lahdesmaki et al. (1977) have
reported that taurine binding to synaptosomes was inhibited
by hypotaurine, 3 -alanine and GABA. The binding sites appear
to have a strict requirement for a particular chemical
structure as in the present study only those amino acids
which were chemically related to taurine were found to have
inhibitory effects on the passive permeability to calcium
ions. It was also seen that these same compounds inhibited
calcium uptake to the same extent as taurine suggesting
that this effect may be due to the binding of these compounds
to the taurine receptor sites.
Chubb and Huxtable (1978b) perfused rat heart for 45
15 minutes with Krebs-Henseleit . buffer containing 2.5 mM Ca
in the absence or presence of 8 mM taurine followed by washout
with a calcium-free medium. It was found that the amount of
calcium washed from the heart, and that remaining following
washout, was significantly greater in the taurine-treated
hearts. They interpreted these results 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 effect on either sarcoplasmic 184
2+ 2+ 2+ reticulum ATP-dependent Ca -binding or (Ca +Mg )-ATPase
activity. These data thus indicate that the taurine effect on calcium accumulation in the heart was not due to an effect on the S.R. calcium pump.
More recently, Chovan et al. (19 79) reported that taurine (10 mM) increased calcium binding to low affinity sites in isolated rat heart sarcolemmal membranes. Taurine was also shown to antagonize the inhibition of calcium binding to the sarcolemma caused by both verapamil and lanthanum. These results differ from the present studies on guinea pig heart muscle preparations (Remtulla et al.,
1978) where no effect of taurine on calcium binding was noted. These latter studies were done in the presence of
ATP, whereas those of Chovan et al. (19 79) were done in the absence of ATP.
Both the report of Chubb and Huxtable 1978b) and that of Chovan et al. (19 79) agree with these present studies on brain synaptosomes. In all these studies a taurine effect on calcium binding or permeability involved an ATP-independent process.
The precise mode of action of taurine in the regulation of passive calcium transport in mammalian tissues remains to be elucidated. Several theories have evolved. Huxtable (1976a)suggested that taurine, a 185
stable zwitterion with a high dipole moment and present in heart cells in large amounts, interacts with the zwitterion phospholipid structure of membranes and cause a conformational change by virtue of1 stabilizing charge separation1. It was further suggested that due to membrane conformational changes, the ion flux and cation affinity would be altered.
Kulakowski, et al. (1978) identified taurine receptor binding sites in the cardiac ventricular sarcolemma. They found that taurine binding to these receptors exhibit positive 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 al., 19 79) showed that membrane fluidity changes due to taurine binding could not be detected using the spin label ESR probe 2N14. The probability that extremely; localized membrane structural changes or protein-protein interaction not affecting membrane fluidity were occuring was not ruled out by these
ESR probe observations. These results of Chovan et al.
(1979), however, suggest that close association exists 2+ between taurine and Ca binding sites. Further work is necessary to understand the physio-chemical properties of the taurine-receptor sites in mammalian tissues. 186
It is well known that calcium plays an important
role in the regulation of excitability of neuronal tissue.
A decrease in the calcium permeability of the synaptic membrane could alter the calcium concentration in the synaptic
terminal and effect neurotransmitter release (Hubbard, 1970;
Katz and Miledi, 1970). The dependence of transmitter 2+
release upon Ca influx has also been noted in synaptosomal preparations in-vitro (Levy et al. , 1974) . Kuriyama et al. ,.
(1976; 1978) has provided evidence that in both central and peripheral nervous system, taurine acts as a modulator of membrane excitability by inhibiting the release of other neurotransmitters such as epinephrine and acetylcholine.
Reuter (1972) showed that long-term changes in membrane permeability to calcium could change the potential
gradient across the membrane leading to hyperpolarization.
T.he resultant hyperpolarization will reduce the potassium
current, and the efficacy of synaptic activation. Taurine has consistently been shown to produce a hyperpolarization
(Curtis and Johnston, 1974; Krnjevic, 1974) in excitable membranes, probably by modifying membrane permeability to potassium and chloride ions (Gruener et al., 1976).
It is conceiveable that the anticonvulsant action
of taurine reported in epilepsy (Barbeau and Donaldson,
1974; Mutani et al. ,• -19 74a; 1974b) might be a result of 187 changes in the intracellular calcium concentrations. This suggestion is supported by the observation;that pretreatment of epileptic animals with sodium edetate (a calcium chelator) significantly diminished the protective effect of taurine against pentylenetetrazol induced seizure (Izumi et al.,
19 75). Taurine may possibly be involved in the restoration of the normal distribution of calcium in membrane compartments.
Hagins and Yoshikami (19 74) have suggested, that calcium bound to the outer segment of retinal photoreceptors is released on illumination and diffuses to the outer membrane, thus modifying permeability to sodium. Redistribution of calcium in its original state occurs during dark adaptation.
Pasantes-Morales et al. (19 78) have shown that taurine
(30 mM) affects calcium uptake and distribution in the..retina.
It thus might be considered that taurine affects ion conductance in photoreceptors and thereby exerts an influence on synaptic function at the photoreceptor terminal. These present results of a taurine effect on passive calcium transport in synaptosomes corroborate this conclusion.
These present studies and those reviewed in this thesis imply that the possible cardiac action of taurine are related to alterations in cardiac calcium permeability.
Evidence for an effect of taurine on changes in cardiac calcium levels has been reported. It has been shown that 188
myocardial calcium is markedly elevated in cardiomyopathic
hamsters (Bajusz et al., 1969). McBroom and Welty (1977)
showed that cardiomyopathic hamsters given 0.1 M taurine in
tap water ad libitum did not manifest the dramatic calcium
increases observed in the hearts of the untreated control
counterparts; the heart taurine content, plasma calcium and
plasma taurine concentration were found to be no different
in the control and taurine treated groups. It is possible
that the exogenous administration of taurine alters both
taurine and calcium kinetics in the heart muscle of
cardiomyopathic hamsters such that the rate of turnover is
increased, thereby augmenting the tissue flux without
increasing the cardiac pool size of either taurine or calcium.
From the evidence present in the literature and
the results obtained in this thesis work, it is not possible to ascertain whether taurine is involved in only one or in several aspects of mammalian physiology. However, on the basis of the known effects of taurine in excitable membranes, one may consider that the' -main:action "of: taurine in mammalian tissues is related to calcium ion fluxes. It has been found that taurine affects calcium permeability in a number of tissues (synaptosomes,Remtulla et al., 1979; heart muscle, Chubb and Huxtable, 1978b ; and retina, -a 1
Pasantes-Morales et al., 1978). Regulation of the intra• cellular calcium concentration is recognized as an 189
important determinant of such cellular processes as :
cell division, cellular excitability, neurotransmitter and
hormone release and muscle contraction. A role for taurine
in the modulation of calcium permeability might account for
its possible effects on a number of these systems.
CONCLUSIONS
The purpose of this thesis work has been to
evaluate the possible mode of action of taurine in mammalian physiology. The evidence presented indicates that this mode of action does not involve the bioconversion of taurine
to isethionic acid as was originally suggested. It was
further shown that the reported cardiac effects of taurine do not involve an effect on ATP-dependent calcium transport.
However, evidence was presented that taurine is involved in the passive (ATP-independent) movement of calcium in a
synaptosomal preparation. This effect together with the demonstrated effect on calcium permeability in perfused heart and retinal preparations (Chubb and Huxtable, 1978b;
Pasantes-Morales et al., 1978) suggests that an important physiological role for taurine in mammalian tissues is related to calcium ion translocation. 190
BIBLIOGRAPHY 191
t The numbers in parantheses refer to the pages in the thesis, where the author(s) is cited.
Agrawal, H.C, Havis, J.M. and Himwick, W.A. (1968a) : Maturational changes in amino acids in CNS of different mammalian species. In: Recent advances in biolog!ca1 Psychiatry, vol. 10 edited by J. Wortis, pp. 258-265. Plenum. Press, N.Y. (54)t._-
Agrawal, H.C, Davis, J.M. and Himwick, W.A. (1968b): Development changes in mouse brain: Weight, water content and free amino acids. J. Neurochem., 15:917-923(54).
Agrawal, H.C. Davison, A.N. and Kaczmarek, L.K. (1971): Subcellular distribution of taurine and cysteinesulphinate decarbozylase in developing rat brain. Biochem. J., 122: 759-763.(55).
Agrawal, H.C, and Himwick, W.A. (1970): Amino acids, proteins and monoamines of: developing brain. In: Deve1opmenta1 neurobiology, edited by W.A. Himwick, pp. 287-310, Charles C. Thomas, Springfield, Illinois. (54).
Ahtee, L., Boullin, D.J.,and Paasonen, M.K. (1974): Transport of Taurine by normal human blood platelets. Br. J. Pharmacol., 52: 245:251.(27).
Akera, T., Ku, D., and Brody, T.M. (1976): Alteration of ion movements as a mechanism of drug-induced arrhythmias and inotropic response. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 121-134, Raven Press, N.Y. (37,49).
Akera, T., Larsen, F.S. and Brody, T.M. (1969): The effect of ouabain on sodium and potassium activated adenosine triphosphatase from the heart of several mammalian species. J. Pharmacol. Exp. Ther., 170: 17-26. (37).
Akera, T. , Larsen, F.S., and Brody,' T.M. (1970): Correlation of cardiac sodium and potassium-activated adenosine triphosphate activity with ouabain-induced inotropic stimulation. J. Pharmacol. Exp. Ther., 173: 145-151.(37)
Applegarth, D.A., Remtulla, M.. and Williams, I.H. (1976): Does isethionic acid occur in heart and brain tissue- Abstract. Clin. Res., 24: 646A.(160).
Austin, L. and Morgan, I.G. (1967): Incorporation of 14c-labelled leucine into synaptosomes from rat cerebral cortex in-vitro. J. Neurochem., 14: 377-387.(177). 192
Awapara, J. (1953): 2-Aminoethanesulfonic acid: An intermediate in the oxidation of cysteine in-vivo. J. Biol. Chem., 203: 183-188. (19).
Awapara, J. (19 56): The taurine concentration of organs from fed and fasted rats. J. Biol. Chem., 218: 571-576. (6,10,26). 35 Awapara, J. (1957): Absorption of injected taurine-S by rat organs. J. Biol. Chem., 225 : 877-882. (8). Awapara, J. and Berg, M. (1976): Uptake of taurine by slices of rat heart and kidney. In: Taurine, pp. 135-144 edited by R. Huxtable and A. Barbeau, Raven Press, N.Y. (27) .
Awapara, J., Landua, A.J., and Fuerst, R. (1950): Distribution of free amino acids and related substances in organs of the rat. Biochem. Biophys. Acta., 5: 45 7-462.(6).
Awapara, J. and Wingo, W.J. (1953): On the mechanism of taurine formation from cysteine in the rat. J. Biol. Chem., 2,03:. 189-194. (19) .
Bajusz, E. (1969): Hereditary cardiomyopathy: A new disease model. Am. Heart J., 77 : 686-696. (188).
Baker, P.F., Blaustein, M.P., Hodgkin, A.L., and Steinhard, R.A. (1969): The influence of calcium on sodium efflux in squid axons. J. Physiol., 200 431-458.(45).
Banos, G., Daniel, P.M. Love, E.R., and Moorhouse, S.W. (1971): The entry of amino acids into the myocardium of the growing rat in-vivo. J. Physiol. (London), 216: 64-65P.(2)
Barbeau, A. (1974): Zinc, taurine and epilepsy, Arch. Neurol., 30: 52-58. (52, 186).=
Barbeau, A., and Huxtable, R. (Eds.) (1978): Taurine and Neurological Disorders. Raven Press, N.Y. (3)
Barbeau, A., Inoue, N., Tsukada, Y. and Butterworth, R.F. (1975): The neuropharmacology of taurine. Life Sci., 17: 669-678.(2,53,60,62,63). Baskin, S.I. and Dagirmanjian, R. (1973): Possible involvement of taurine in the genesis of muscular dystrophy. Nature (Lond.), 245: 464-465. (58).
Baskin, S.I., Hinkamp, D.L., Marquis, W.L., and Tilson, H.A. (1974): Effects of taurine on psychomotor activity in the rat. Neuropharmacol., 13: 591-594. (60). 193
Battistini, L. , Grynbaun, A., and Lajtha, A. (1969): Distribution and uptake of amino acids in various regions of the cat brain in vitro. J. Neurochem., 16: 1459-1468.(9) .
Bennett, J. and Dave, C. (1974): Effect of intravenous infusion of isethionate on rats. Proc. Soc. Expt. Biol. Med., 146: 170-175. (165).
Bergamini, L., Mutani, R., Delsedime, M., and Durrelli, L. (1974): First clinical experience on the antiepileptic action of taurine. Eur. Neurol., 11: 261-269. (52).
Berson E.L., Hayes, K.C., Rabin, A.R., Schmidt, S.Y., and Watson, G. (1976) : Retinal degeneration in cats fed Casein: II. Supplementation with methionine, cysteine and taurine. Invest. Ophthalmol., 15: 52-58. (53,54).
Besch, H.R. Jr., Allen, J.C, Glick, G. , and Schwartz, A. (19 70): Correlation between the inotropic action of ouabain and its effects on subcellular enzyme systems from canine myocardium. J. Pharmacol. Expt. Ther., 171: 1-12 (37).
Blass, J.P. (1960) : The simple monosubstituted guanidines of mammalian brain. Biochem. J., 77: 484-489.(14).
Blaustein, M.P., and Hodgkin, A.L. (1969): The effect of cyanide on the efflux of calcium-from squid axons. J. Physiol., 200: 49 7-527.(45).
Blesa, E.S., Langer, G.A., Brady, A.J. and Serena (1970) Potassium exchange in rat ventricular myocardium: its relation to rate of stimulation Amer. J. Physiol. 219: 747-754.(45).
Bogdanski, D.F., Tissari A. and Brodie, B.B. (1968): Role of sodium, potassium, ouabain and Reserpine in uptake, storage and metabolism of biogenic amines in synaptosomes. Life Sci., 7 : 419-428. (178). 194
Bourke, R.S., Gebelnick, H.L., and Young 0. (1970): Mediated transport of chloride from blood into cerebrospinal fluid. Exp. Brain. Res., 10: 17-38.(164).
Bourke, R.S., Nelson, K.M., Naumann, R.A. and Young, O.M. (1970): Studies of the production and subsequent reduction of swelling in primate cerebral cortex under isosmetic conditions in-vivo. Expt. Brain Res., 10: 427-246.(164).
Bowditch, H.P. (1871): Ueber die Eigenthumlichkeiten der Reizbarkeit, Welche die Muskelfasern des Herzens Zeigen. Arbeit. Physiol. Anst. (Leipzig), 6 :. 139-176 . (44) .
Brenton, D.P.; Cusworth, D.C., and Gaull, G.E. (1965): Homocystinuria: Biochemical studies of tissues including a comparison with cystathioninuria. Pediatrics, 35: 50-56. (2)
Brotherton, J. (1962): Studies on the metabolism of the rat retina with special reference to retinitis pigmentosa. II Amino acid content as shown by chromatography. Exp. EyeRes., ] : 246-252.(54).
Brown, G.M. (1959): The metabolism of Pantothenic acid. J. Biol. Chem., 234: 370-378. (24).
Byington, K.H. (1964): Aminosulfonic acids and cardiac arrhythmais. Thesis, State University of South Dakato. pp. 40-46.(182).
Carsten, M.E. (1964): The cardiac pump. Proc. Natl. Acad. Sci. U.S.A., 52.: 1456-1462.(90,97,103,169).
Cavallini, D., Dupre, S., Federici, G., Solinas, S., Ricci, G. and Matarese, M. (1978): Isethionic acid as a taurine co-metabolite. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 29-34, Raven Press, N.Y. (163, 164).
Cavallini, D., Scandurra, R., Dupre, S., Federicci, G., Santoro, L. Ricci, G., and Barra, D. (1976): Alternative pathways of taurine biosynthesis. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 59-66, Raven Press, N.Y. (23).
Chanda, R., and Himwich, W.A. (1970): Taurine levels in developing rabbit brain and other organs. Dev. Psychobiol., 3: 191-196.(6). 195
Chapeville, F., and Fromageot, P. (1955): La Formation de l'acide cysteinesulfinique a partir de la cystine chez le rat. Biochim. Biophys. Acts., 17: 275-276.(19).
Chazov, E.I., Malchidova, L.S., Lipina, N.V., Assfov, G.B., and Smirnov, V.N. (1974): Taurine and the Electrical activity of the heart. Circ. Res., 34 & 35: Suppl. Ill, 11-21.(17,36,37,38, 39,62,16 7) .
Chesney, R.W., Scriver, CR. , and Mohyliddin, F. (1976): Localization of the membrane defect in trasepithelial transport of taurine by parallel studies in vivo and in-vitro in hypertaurinuric mice, J. Clin. Invest. 57: 183-193.(27).
Chipperfield, D. (1969): The effect of Ouabain on Mitochondrial calcium in vitro. Life Sci., 8: 39-44.(44).
Chovan, J.P., Kulakowski, E.C, Benson, B.W. , and - Schaffer, S.W. (1979): Taurine enhancement of calcium binding to rat heart sarcolemma. Biochim. Biophys. Acts., 551: 129-136. (176,184,1851*1 Christensen, H.N. (1964): Relations in the transport of 3 - alanine and the L-amino acids in the Ehrich cell. J. Biol. Chem., 239: 3584-3589.(28).
Christensen, H.N. and Liang, M. (1956): An amino acid transport system of unassigned function in'the Ehrlich Ascites tumor cell. J. Biol. Chem., 240: 3601-3607.(28).
Chubb, J. and Huxtable, R. (1977): Taurine influx in the perfused rat heart. Proc. West. Pharmacol., Soc, 20: 245-251. (28) .
Chubb, J. and Huxtable, R. (1978a) Isoproterenol-stimulated taurine influx in the perfused rat heart. Eur. J. Pharmacol. , 48: 369-376. (27,28) .
Chubb, J., and Huxtable, R.J. (1978b): Transport and Biosynthesis of taurine in the stressed heart. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 161-178, Raven Press, N.Y. (175,176,183,184,188,189). Cohen, A.I., McDaniel, M. and Orr, H.T. (1973): Absolute levels of some free amino acids in normal and biologir, cally fractionated retinas. Invest. Ophthalmos. 12: 686-693. (54) . 196
Colburn, R.W., Goodwin, F.K., Murphy, D.L., Bunney, W.E. and Davis, J.M. (1968): Quantitative studies of norepinephrine uptake by synaptosomes, Biochem. Pharmacol., 17: 957-964.(178).
Collins,' G.G.S. (1974): The rate of synthesis, uptake and disappearance of 14c-taurine in eight areas of the rat central nervous system. Brain, Res., 76 : 447-459.(57).
Craig, CR. and Hartmann, E.R. (1973): Concentration of amino acids in the brain of cobalt epileptic rat. Epilepsia (Amst.), 14: 409-414.(51)
Crass, M.F. Ill and Lombardini, J.B. (1977): Loss of cardiac muscle taurine after acute left ventricular ischemia. Life Sci., 21: 951-958.(35).
Crass, M.F. Ill and Lombardini, J.B. (1978): Release of tissue taurine from the oxygen-deficient perfused rat heart. Proc. Soc. Exp. Biol. Med., 157 : 486-488.(6).
Crawford, J.M. (1963): The effect upon mice of intraventri• cular injection of excitant and depressant amino acids. Biochem. Pharmacol., 12: 1443-1444.(182).
Crawford,. J.M. and Curtis, D.R. (1964): The excitation and depression-of mammalian cortical.neurons by jamino acids. Br. J. Pharmacol.' 23: 313-329.(60).'
Curtis, D.R. and Crawford, J..M. (1969) : Central synaptic transmission-microelectrophoretic studies. Ann. Rev. 'Pharmacol., 9: 209-240.(60). - -
Curtis, D.R., Duggan, A.W., Felix, D., Johnston, G.A.R., and Mclennan, H. (1971): Antagonism between bicuculline and GABA in the cat brain. Brain Res. , 33: 57- 73.(58,59).
Curtis, D.R. and Johnston, G.A.R. (1974): Amino acid transmitters in the mammalian central nervous system. Ergeb. Physiol., 6.9: 97-188.(186).
Curtis, D.R., and Watkins, J.C. (1960): The excitation and depression of spinal neurons by structurally related amino acids. J. Neurochem., 6: 117-141.(59,60,182).
Curtis, D.R. and Watkins, J.C. (1961): Analogues of glutamic acid, x - amino-n-butyri-c acids having potent actions on mammalian neurons. Nature (London), 191: 1010-^1011. (182) . 197
Curtis, D.R., and Watkins, J.C. (1965): The pharmacology of amino acids related to Gamma-aminobutyric• acid. Pharmacol. Rev., 17: 347-391. (182).
Daniel, E.E., Dawkins, 0. and Hunt, J. (1957): Selective depletion of rat aorta potassium by small pressor doses of norepinephrine. Am. J. Physiol. 190: 67-70. (166).
Deffner, G.G.J. (1961): The dialyzable free organic constituents of squid blood; a comparison with nerve axoplasm. Biochim. Biophys. Acta. 47: 378-388. (15) .
Deffner, G.G.J, and Hafter, R.E. (1959): Chemical investi• gation of the giant nerve fibers of the squid. II. Detection and identification of cysteic acid amide (B -suloalaninamide) in squid nerve axoplasm. Biochim. Biophys. Acta., 35: 334-340. (15).
Deffner, G.G.J, and Hafter, R.E. (1960): Chemical investigations of the giant nerve fibers of the squid IV. Acid-base balance in axoplasm. Biochim. Biophys. Acta, 42: 202*205.(15, 158).
Demarcay, H. (1838): Uber die Natur der Galle. Anne, der Pharmacie, 27: 270-291. (5).
Derouaux, M. , Puil, E., and Naquet, R. (1973):. Electro. . Encephalogr.: Antiepileptic effect of taurine.in -. photosensitive epilepsyClin. Neurophysiol., 34: 770.(51). ~~ 198
Dietrich, J., and Diacono J. (1971): Comparison between ouabain and taurine effects on isolated rat and guinea-pig hearts in low calcium medium. Life Sci. 10: 499-501.(17, 40,41,45,62,173,174). : :
Dietrich, J., and Diacono, J. (1972): Comparative action of ouabain and taurine in the presence of lanthanum and lithium ions on isolated hearts of rat and guinea-pig. Therapy, 27: 861-871.(62).
De Robertis, E., Pellegrino De Iraldi, A., Rodriguez, G., and Gomez, C.J. (1961): On the Isolation of nerve endings and synaptic vesicles. J^Biophys. Biochem. Cytol. 9: 229-235.(141) : '
Dolara, P., Agresti, A., Giotti, A., Pasquini, G. (1973): Effect of taurine on calcium kinetics of guinea-pig heart. Eur. J. Pharmac 24: 352-358.(47,6 2,16 8,175).
Dolara, P., Agresti, A., Giotti, A., and Sorace, E. (1976): The effect of taurine on calcium exchange of sarcoplasmic reticulum of guinea-pig heart studied by means of dialysis kinetics. Can. J. Physiol. Pharmacol. 54: 529-533. (47,62,168,175).
Dolara, P., Ladda, F., Mugelli A.,Mantelli, L., Zilletti, L., Franconi, F. and Giotti, A. (19 78a): Effect of taurine on calcium, inotropism and electrical activity of the heart. In: Taurine.and Neurological Disorders, edited by Barbeau, A. and R.J. Huxtable, pp. 151-159, Raven Press, N.Y.(42,47).
Dolara, P., Franconi, F., Giotti A., Basosi, R., Valensin, G. (1978b): Taurine-calcium interaction measured by means of 13C-nuclear magnetic resonance. Biochem. Pharmacol. 27: 803-804. (47).
Donaldson, J., Tsukada, Y., Inoe, N. and Barbeau, A.(1974): Amino acid effects on ATPase and %-ouabain binding in membrane fractions from rat brain. Abstract. Proc. Can. Fed. Biol. Soc, 17: 313.(49)
Dowson, R.M.C., Elliott, D.C, Eliott, W.H. , and Jones, K.M. (1969): Physiological Media. In: Data for Biochemical Research, 2nd edition, p. 507. Oxford University Press, N.Y. and Oxford.(85)
Dupre, S., Rosei, M.A., Bellussi, L., Grosso, E.D., and Cavallini, D. (1973): The substrate specificity of pantethinase'. Eur. J. Biochem. , 40: 103-107.(2 4). 199
Dutta, S., Goswami, S., Lindower, J.O., and Marks, B.H. (196 8): Subcellular distribution of digoxin-H^ in isolated guinea-pig and rat heart. J. Pharmacol. Exptl. Therap., 159: 324-334. (44)
Dziewietkowski, D.D. (1954): Utilization of sulfate sulphur in the rat for the synthesis of cysteine, J. Biol. Chem., 207: 181-186. (26)
Ebashi, S., and Endo, M. (1968): Calcium ion and muscle contraction. Progress in Biophysics and Molecular Biology, 18: 123-183.(169)
Edward, C., and Kuffler, S.W. (1959): The blocking effect of y-aminobutyric acid (GABA) and the action of related compounds on single nerve cells. J. Neurochem.,4: 19-30.(182)
Edwards, R.B. (1977): Accumulation of taurine by cultured retinal pigment epithelium of the rat. Invest. Opthalmol. Vis. Sci., 16: 201-208.(27).
Eldjarn, L. (1954): The conversion of cystinamine to taurine in rat, rabbit and man. J. Biol. Chem. 206: 483-490.(21).
Encrantz, J.C, and Sjorall, J. (1959): On the bile acids in duodenal contents of infants and children. Clin. Chim. Acta. 4: 793-799.(2)
Enna, S.J. and Snyder, S.H. (1975): Properties of y-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res. 100: 81-97.(182).
Entman, M.L., Bornet, E.P. and Bressler, R. (1977): The effect of taurine on cardiac sarcoplasmic reticulum. Life Sci., 21; 543-550.(173, 174,176).
Entman, M.L., Hansen, J.L., and Cook, J.W. Jr. (1969a): Calcium metabolism in cardiac microsomes incubated with lanthanum ion, Biochem. Biophys. Res. Commun., 35: 258-264. (175).
Entman, M.L., Levey, G.S. and Epstein, S.E. (1969b): Mechanism of action of epinephrine and glucagon on the canine heart: Evidence for increase in sarcotubular calcium stores mediated" by cyclic 3', 5' - AMP. Circ. Res. 25: 429-438.(175). 200
Entman, M.L., Snow, T.R., Freed,D. and Swartz, A. (1973): analysis of calcium binding and release by canine cardiac relaxing system (sarcoplasmic reticulum): The use of specific inhibitors to construct a two- component model for calcium binding and transport. J. Biol. Chem., 248: 7762-7772.(175)
Escueta, A.V. and Appel, S.H. (1969): Biochemical studies of synapses in vitro. II. Potassium transport. Biochemistry, 8: 725-733. (177,178) .
Federici, G., Ricci, G., Dupre, S., Antonucci, A., Cavallini, D. (19 76): The metabolism of mercaptoethanol by the living rat. Biochem. Exp. Biol., 12: 341-345.(164).
Fellman, J.H., Roth, E.S. and Fujita, T.S. (1978): Taurine is not metabolized to isethionic acid in mammalian tissue. In: Taurine and Neurological Disorder, edited by A. Barbeau and R.J. Huxtable, pp. 19-22, Raven Press, N.Y. (160,162,164).
Fiori, A. and Costa, M. (1969): Oxidation of hypotaurine by pyroxide. Acta Vitamin, 23: 204-207 (19).
Frederickson, R.C.A., Neuss, M., Morzorati, S.L., and McBride W.J. (1978): A comparison of the inhibitory effects of taurine and GABA on identified purkinje cells and other neurons in the cerebellar cortex of the rat. Brain Res. 145: 117-126. (59,61).
Fujimoto, S. and Iwata, H. (1975): In Japanese, Folia. Pharmacol, japon. (38).
Fujimoto, S., Iwata, H. and Yoneda, Y. (1976): Effect of taurine on responses to noradrenaline, acetylcholine and ouabain in isolated auricles from digitalized guinea-pigs. Japan J. Pharmacol., 26: 105-110. (38,39).
Gaull, G.E. (1971): The biochemistry of taurine in developing rat brain (comment). In: Inherited Disorders of Sulphur Metabolism, edited by N.A.J. Carson and D.N. Raine, p.69 Churchill Livingston, London. (2).
Gaull, G.E., Rassin, D.K., Raiha, N.C.R., and Heinonen, K. (1977): Milk protein quantity and quality in low-birth weight infants. 3. Effect on sulfur amino acids in plasma and urine. J. Pediatr. 90: 348-355.(56).
Gaut, Z.N., Nauss, C.B. (19 76): Uptake of taurine by human blood platelets: a possible model for brain. In: Taurine, edited by R. Huxtable and A. Barbeau, pp 91-98 Raven Press, N.Y.(27) . 201
Gelbert, A. and Goldman^ R.H. (1977): Correlation between microsomal (Na +K )-AtPase Activity and(3H) Ouabain binding to heart-.tissue homogenates. Biochem. Biophys. Acta 481: 689-694. (98,132).
Giotti, A., and Guidotti, A.(1969) ?: Digitalis inotropic effect on the auricular miocardium of taurine treated guinea-pig hearts in low calcium medium. In: Medicaments et Metabolisme du Myocarde et du muscle strie, Symp. Int., Edited by Nancy, M. Lamarche et R. Royal, pp. 487-490. (174).
Glauert, A.M. (1965): The fixation and embedding of biological specimens. In; Techniques for Electron Microscopy, edited by D.H. Kay, 2nd edition, pp. 166-212. Blackwell Scientific Publishers, Oxford. (94).
Glitsch, H.G., Reuter, H., and Scholz, H. (1970): The efflux of the internal sodium concentration on calcium fluxes in isolated guinea-pig auricles. J. Physiol., 209: 25-43. (44).
Goodman, H.O., Wainer, A., King, J.S., and Thomas, J.J. (1967): Hydroxyethanesulfonic acid (isethionic acid) in urine of human subjects given 35s-taurine. Proc. Soc. Exp. Biol. Med. 125: 109-113 (16).
Gorby, W.G., and Martin W.G. (1975): The synthesis of taurine from sulfate. VIII. The effect of potassium. Proc. Soc. Exp. Biol. Med. 14 8: 544-549. (26).
Gray, E.G. and Whittaker, V.P. (1962): The isolation of nerve endings from brain: An electron-microscopic study of cell fragments derived by homogenization and centrifu• gation. J. Anat. (London) 96: 79-88.(104,141, 142):
Green, J.P., and Robinson, J.D. (1960): Cerebroside sulfate (sulfatide A) in some organs of the rat and in mast cell tumor. J. Biol. Chem. 235: 1621-1624. (26).
Gregory, J.D. and Robbins, P.W. (1960): Metabolism of Sulfur compounds (Sulfate metabolism). Ann. Rev. Biochem. 29: 347-364.(24).
Grossman, A. and Furchgott, R.F. (196 4): The effect of external calcium concentration on the distribution ~ and exchange of calcium in resting and beating guinea-pig auricles. J. Pharmacol. Exp. Ther., 143: 107-119. (42).
Grosso, D.S. and Bressler, R. (1976): Commetry-Taurine and cardiac physiology. Biochem. Pharmacol., 25: 2227-2232. (2,5,53). 202
Grosso, D.S., Bressler, R. , and Benson, B. (1978a) : Circardian rhythm and uptake of taurine by the rat pineal gland. Life Sci., 22: 1789-1798.(58).
Grosso, D.S. Roeske, W.R., and Bressler, R. (1978b=) : Characterization of a carrier-mediated transport system for taurine in the fetal mouse heart in vitro., J. Clin. Invest., 61: 944-952.(27,28).
Gruener, R. and Bryant, H.J. (1975): Excitability modulation by taurine, action on axon membrane permeabilities. J. Pharmacol. Exp. Ther., 194: 514-521.(53,6 0,61).
Gruener, R., Bryant, H., Markovitz, D., Huxtable, R., and Bressler, R. (1976): Ionic actions of taurine on nerve and muscle membranes: Electrophysiological studies. In: Taurine, edited by Huxtable, R. and Barbeau, A. pp.225-242. Raven Press, N.Y.(60,61,186).
Gruener, R., Markovitz, D., Huxtable, R. and Bressler, R. (1975): Excitability modulation by taurine: Trasmembrane measurements of nuromuscular trans• mission. , J. Neurol. Sci., 24: 351-360. (60.61)
Guidotti, A. (1978): Role of taurine in brain function. In: Taurine and Neurological Disorders, edited by A.Barbeau and R. Huxtable, pp. 237-248. Raven Press, N.Y(58) .
Guidotti, A., Badiani, G., and Giotti, A. (1971): Potentiation by taurine of inotropic effect of strophanthin-K on guinea-pig isolated auricles. Pharmac. Res. Commun., 3: 29-38. (17,41,62,173,175).
Guidotti, A., Badiani, G., and Pepeu, G. (1972): Taurine distribution in cat brain. J. Neurochem., 19: 431-435.(7) .
Hajdu, S., and Leonard, E. (1959): The cellular basis of cardiac glycoside action. Pharmacol. Rev. 11:173-209.(37
Haas, H.L., and Hosli, L. (1973): The depression of brain stem neurons by taurine and its interaction with strychnine and bicuculline. Brain. Res. 52: 399-402.(59,6
Hagins, W.A., and Yoshikami, S. (1974): Role for calcium in excitation of retinal rods and cones. Exp. Eye Res., 18: 299-305. (61,187). 203
Harigaya, S. and Schwartz, A. (1969): Rate of calcium binding and uptake in normal animal and failing cardiac muscle. Circ. Res., 25: 781-794 (95,175).
Hasselbach, W. and Makinose, M. (1962): ATP and active trasport. Biochem. Biophys. Res. Commun., 7: 132-136.(168).
Hayes, K.C. (1976): A review on the biological function of taurine. Nutr. Rev., 34: 161-165.(2,10,26,56).
Hayes, K.C, Carey, R.E., and Schmidt, S.Y. (1975): Retinal degeneration associated with taurine deficiency in the cat., Science, 188: 949-951.(53).
Hicks, and Katz A.M. (19 79): Mechanism by which cyclic Adenosine 3': 5' - monophosphate-dependent protein kinase stimulates calcium transport in cardiac sarcoplasmic reticulum. Circ. Res.,44: 384-391.(170).
Hinton, J.P., Souza, J.D., and Gillis, R.A. (1975): Deleterious effects of taurine in cats with digitalis-induced arrhythmias. Eur. J. Pharmacol., 33: 383-387. (38).
Holland, W.C, and Dunn, C.E. (1954): Role of the cell membrane and mitochondria in the phenomenon of ion transport in cardiac muscle. Am. J. Physiol., 179: 486-490.(166).
Honegger, C.G., Krepelka, L.M., Steiner, M., and Van Hann, H.P. (1973): Kinetics and subcellular destribution of 35S-taurine uptake in rat cerebral cortex slices. Experientia (Bassel), 29: 1235-1237. (60).
Hope, D.B. (1955) : p.yridoxal phosphate as the co-enzyme of the mammalian decarboxylase for L-cysteine sulphinate and L-cysteic acid. Biochem. J., 59: 497-50018,10,26).
Hoskin, F.C.G. (1976): Distribution of diisopropylphosphoro- fluoridate-hydrolyzing enzyme between sheath and axoplasm of squid giant axon. J. Neurochem., 26: 1043-1045. (163) .
Hoskin, F.C.G., and Brande, M. (1973): An improved sulphur assay applied to a problem of isethionate metabolism in squid axon and other nerves. J. Neurochem., 20: 1317-1327. (158, 163).
Hoskin, F.C.G., and Kordik, E.R. (1975): Rhodanese and DFPase in relation to isethionate-^-in squid nerve. Biological Bulletin., 149: 429-430. (163). 204
Hoskin, F.C.G., and Kordik, E.F. (1977): Hydrogen sulfide as a precursor for the synthesis of isethionate in squid giant axon. Arch. Biochem. Biophys., 180- 583-586.(163).
Hoskin, F.C.G., Pollock, M.L., and Prusch, R.D. (1975): An improved method for the measurement of 14C02 applied to a problem of cysteine metabolism in squid nerve. J. Neurochem., 25: 445-449.(163)
Hruska, R.E., Huxtable, R.J..,. Bressler, R. and Yamamura, H. (1976) : Sodium dependent high affinity transport of taurine into rat brain synaptosomes. Proc. West. Pharmacol. Soc., 19: 152-156.(27).
Hruska, R.E., Thut, P.D., Huxtable, R., and Bressler, R. (1973): Taurine, hypothermic effect on mice. Pharmacologist., 14: 301. (57).
Hruska, R.E., Thut, P.D., Huxtable, R.J., and Bressler, R. (1975): Suppression of conditioned drinking by taurine and related compounds. Pharmac. Biochem. Behav., 3: 593-599.(57).
Hruska, R.E., Padjen, A., Bressler, R., and Yamamura, H.I. (1977) : Taurine: Sodium-dependent high-affinity transport into rat brain synaptosomes. Mol. Pharmacol., 14: 77-85. (29).
Hubbard, J.I. (1970): Mechanism of transmitter release. Prog. Biophys. Mol. Biol., 21: 33-124.(186).
Hue, B., Pahhate, M. and Chanelet, J. (1978): Sensitivity of postsynaptic neurons of the insect central nervous system to externally applied taurine. In: Taurine and Neurological Disorders, edited by A. Barbeau, and R. Huxtable, pp. 225-236. Raven Press, N.Y. (181).
Huxtable, R. (1976a): Metabolism and function of taurine in the heart. In: Taurine, edited by R.J. Huxtable and A. Barbeau, pp. 99-119. Raven Press, N. Y. (2,16,33,53, 60,184,185). Huxtable, R. (1976b): Taurine. Circ. Res.,38 (Supp. 1): 112-112.(34).
Huxtable, R.J. (19 78): Regulation of taurine in the heart. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 5-17, Raven Press, N.Y. (10,26). 205
Huxtable, R. and Barbeau, A. (Editors) (1976) : Taurine, Raven Press, N.Y.(3)
Huxtable, R., and Bressler, R. (1972): Taurine and isethionic acid: Distribution and interconversion in the rat. J. Nutr. 102: 805-814.(7,8,17,62,161).
Huxtable, R. and Bressler, R. (1973): Effect of taurine on a muscle intracellular membrane. Biochem. Biophys. Acta. 323: 573-583.(46,62,168,173,174,176).
Huxtable, R. and Bressler, R. (1974a): Elevation of taurine in human congestive heart failure. Life Sci. 14: 1353-1359. (5,30,32).
Huxtable, R. and Bressler, R. (1974b): Taurine concentrations in congestive heart failure. Sci. (N.Y.), 184: 1187-1188.(5,30,32).
Huxtable, R. and Bressler, R. (1976): The metabolism of cysteamine to taurine. In: Taurine, edited by R.J. Huxtable and A. Barbeau, pp. 45-57. Raven Press, N.Y . (21,23,24). Huxtable, R. and Chubb, J. (1977): Adrenergic stimulation of taurine transport by heart. Sci. ,198: 409-411.(33).
Igisu, H., Izumi, K., Goto, I., and Kina, K. (1976): Effect of taurine on the ATPase activity in the human erythrocyte membranes. Pharmacology, 14: 362-366.(49,62).
Iwatta, H. and Fujimoto, S. (1976): Potentiation by taurine of the inotropic effect of ouabain and the content of intracellular Ca^+ and taurine in the heart. Experientia, 32: 1559-1560. (43).
Izumi, K., Butterworth, R.F. and Barbeau, A. (1977): Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex. Life Sci., 20: 943-940.(48,53,62).
Izumi, K. , Donaldson-, ,. J. , Munnich, J.L., and Barbeau, A. (1973): Ouabain induced seizures in rats: Suppressive effects of taurine and y-aminobutyric acid. Can. J. Physiol. Pharmacol., 51: 885-889. (51)
Izumi, K., Igisu, H. and Fukuda, J. (1974): Suppression of seizures by taurine-specific or nonspecific? Brain. Res., 76: 171-173.(51,60).
Izumi, K., Igisu, H., and Fukuda, T. (1975): Effect of edetate on seizure suppressing actions of taurine and GABA. Brain Res., 88: 576-579.(187). 206
Izumi, K. , Ngo, -T.T. and Barbeau, A. (1978): Metabolic modulation in the central nervous system by taurine. In: Taurine and Neurological Disorders, edited by A. Barbeau and R.J. Huxtable, pp. 137-150. Raven Press, N.Y.(61) .
Jacobsen, J.C. (1968): Studies on taurine: Occurance, biosynthesis, metabolic fate and physiological role in mammal, Nyt Nordisk. Forlag, Copenhagen. (16 5) .
Jacobsen, J.C, Collins, L.L., and Smith, L.H. (1967): Urinary excretion of isethionic acid in man. Nature, 214: 1247-1248. (16,17,164,165).
Jacobsen, J.G., and Smith, L.H. Jr. (1968): Biochemistry and physiology and taurine and taurine derivatives. Physiol. Rev., 48: 424-511.(3,6,14,17,19,21,62).
Jacobsen, J.G., Thomas, L.L.and Smith, L.H. Jr. (1964): Properties and distribution of mammalian cysteine sulfinate carboxylases. Biochim. Biophys. Acta., 85: 103-116.(19,62). Jagenberg, O.R. (1959): The urinary excretion of free amino acids and other amino compounds by the human. Scand. J. Clin. Lab. Invest. 11: (Suppl. 43), 3-183. (56).
Jones, L.R. , Besch, H.R. , Watanabe, A.M. (197.7): Monovalent cation stimulation of Ca2+ uptake by cardiac membrane vesicles; correlation with stimulation of Ca2+ - ATPase activity. J. Biol. Chem., 252:: 3315-3323.(181).
Jonxis, J.H.P. (1951): The influence of differences in food on the amino acid excretion. Arch. Dis. Child, 26: 272. (56).
Joseph, M.H., and Emson, P.C. (1976): Taurine and Cobalt induced epilepsy in the rat: A biochemical and electrocorticographic study. J. Neurochem., 27: 1495-1501.(53).
Kaczmarek, L.K. (1976): A comparison of evidence for taurine and GABA as neurotransmitters. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 2 83-292, Raven Press, N.Y. (7,58,60,182). Kaczmarek, L.K., and Adey, W.R. (1974): Factors affecting the release of 14c-taurine from cat brain; The electrical effects of taurine on normal and seizure prone cortex. Brain Res.,.76: 83-94.(60).
Kaczmarek, L.K., Agrawal, H.C. and Davison, A.N. (1970): Biochemical studies of taurine in the developing rat brain. Biochem. J. 119: 45P-46P. (55). 207
Kaczmarek, L.K., Agrawal, H.C. and Davidson, A.N. (1971): The Biochemistry of taurine in developing rat brain. In: Inherited disorders of sulfur metabolism, edited, by N.A.J. Carson and D.N. Raine, Churchill Livingstone, London, pp. 63-69.(7,55).
Kaczmarek, L.K., and Davidson, A.N. (1972): Uptake and release of taurine from rat brain slices. J. Neurochem. 19: 2355-2362. (27).
Kanazawa, T., Yamada, S., and Tonomura, Y. (1970): ATP formation from ADP and a phosphorylated inter• mediate of Ca2+-dependent ATPase in fragmented sarcoplasmic reticulum. J. Biochem. 68: 593-595 .(168).
Katz, A.M. (1970): Contractile proteins of the heart. Physiol.Rev., 50: 63-158.(43,169).
Katz, A.M., Repke D.I. (1967): Quantitative aspects of dog cardiac microsomal calcium binding and calcium uptake. Circ. Res.,21: 153-162 and 31: 308-316.(133,169).
Katz, A.M., and Repke, D.I. (1973): Calcium-membrane interactions in the myocardium: Effect of ouabain, epinephrine and 3 ', 5' - cyclic adenosine monophosphate. Am. J. Cardiol., 31: 193-201. (175).
Katz, A.M., Repke, D.I., and Hasselbach, W. (1977): Dependence of ionophores- and caffeine-induced release from sarcoplasmic vesicles on external and internal calcium ion concentration. J. Biol. Chem., 252: 1938-1949. (175).
Katz, A.M., Repke, D.E., Upshaw, J.E., and Polazak, M.A. (1970): Characterization of dog cardiac microsomes: Use of zonal centrifugation to fractionate fragmented sarcoplasmic reticulum, (Na++K+)-activated ATPase and mitochondrial transports. Biochem. Biophys. Acta, 205: 473-490. (91).
Katz, B., and Miledi, R. (1970): Membrane noise produced by acetylcholine. Nature, 226: 962-96 3. (186).
Katz, s . , Dobovicnik B. (1979): The possible localization of adenylate cylase in microsomal fraction of cardiac muscle enriched in sarcoplasmic reticulum. In Press. Proc. Western Pharm. Society. (170).
Katz, S., and Remtulla, M.A. (1978): Phosphodiesterase protein activator stimulates calcium transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum. Biochem. Biophys. Res. Commun., 83: 1373-1379. (170,175) . 208
Katz, S. and Reynolds, S. (1978): The role of cyclic AMP- dependent protein kinase in sarcoplasmic reticulum calcium transport. Can. Fed. Biol. Soc. Proc. 21:75.(170)
Keen, P. and White, T.D. (197$): A light-scattering technique for study of the permeability of rat brain synaptosomes in vitro. J. Neurochem., 17: 565-571(104,106,144,177,178)
Kirchberger, M.A., Tada, M., Repke, D.I., and Katz, A.M. (1972): Cyclic Adenosine 3', 5'-monophosphate- dependent protein kinase stimulation of calcium microsomes. J. Mol. Cell. Cardiol. 4: 673-680.(175).
Knopf, K., Sturman, J.A., Armstrong, M., and Hayes, K.C. (1978): Taurine: An essential nutrient for the cat. J. Nutr., 108: 773-778.(10,26,54,56).
Koch, A.L. (1961): Some calculations on the turbidity of mitochondria and bacteria. Biochem. Biophys. Acta. 51: 429-44. (177).
Kochakian, CD. (1973): Regulation of production of seminal vesicle and prostate of guinea-pig by testosterone. Nature, 241: 202-203.(58).
Kochakian, CD. (1976a): Taurine related sulfur compounds in the reproductive tract of marine animals. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 375-378. Raven Press, N.Y.(58).
Kochakian, CD. (1976b): Influence of testosterone on the concentration of hypotaurine and taurine in the reproductive tract of the male guinea-pig, rat and mouse. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 327-334, Raven Press, N.Y.(58)
Kocsis, J.J., Kostos, V.J., and Baskin, S.I. (1976): Taurine levels in the heart tissues of various species. In: Taurine,edited by R. Huxtable and A. Barbeau, pp. 145-154. Raven Press, N.Y.(5,6).
Koechlin, B.A. (1955): On the chemical composition of the axoplasm of squid giant nerve fibre with particular reference to its ion pattern. J. Biophysic. Biochem. Cytol. , 1: 511-529. (15,62).
Kontro, P..and Oja, S.S. (1978): Taurine uptake by rat brain synaptosomes. J. Neurochem., 30: 1297-1304.(27,29).
Koyama. I. (1972): Amino acids in the cobalt induced epiletogenic and non-epileptogenic cats cortex. Can. J. Physiol. Pharmacol. 50: 740-752.(51). 209
Kromphardt, H. (1963): Die Aufnahme von Taurine in Ehrlich- Ascites-Tumorzellen. Biochem. Z., 339: 233-254.(27) .
Krnjevic, K. (1974): Chemical nature of synaptic transmission in vertebrates. Physiol. Rev., 54: 418-540. (186).
Krnjevic, K., and Phillis, J.W. (1963): Iontophoretic studies of neurones in the mammalian cerebral cortex. J. Physiol. (London), 165: 274-304.(58) .
Krnjevic, K., and Puil, ,E. (1976): Electrophysiological studies on actions of taurine. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 179-189. Raven Press, N.Y. (182) .
Krukenberg, C.F.W. (1881a): Physiologisch-Chemischem Unter- suchungen. Vergl. Physiol. Studien Reihel. Abt., 4: 29-64. (5).
Krukenberg, C.F.W. (1881b): Beitrape zur Kenntnis Derverbreitung des Harnstoffs und der Amidosauren bei Winbellosen. Verg. Physiol. Studien Reihel. Abt., 2: 31-36.(5).
Krukenberg, C.F.W. (1882): Weotese Untersuchungen zur Vergleichenden Muskelchemie. Vergl. Physiol. Studien. Reihe Z. Abt., 1: .143-147.(5) .
Krukenberg, C.F.W., and Wagner, H. (1885): Uber Bresonderheiten des Chemischen baues Contractile Gewebe. Z. Biol., 21: 25-40.(5) .
Kulakowski, E.C, Maturo, J. and Shaffer, S.W. (1978): The identification of taurine receptors from rat heart sarcolemma. Biochem. Biophys. Res. Commun. 80: 936-941.(183, 185).
Kuriyama, K., Muramatsu, M., Nakagawa, K., and Kakito, K. (1978): Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissues. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 201-216, Raven Press, N.Y. (61,62,63,177,180,186).
Kuriyama, K. and Nakagawa, K. (1976): Role of taurine in adrenal gland: A preventive effect of stress induced release of catecholamines from chromaffin granules. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 335-345. Raven Press, N.Y.(58,61,62,186). 210
Lahdesmaki, P., Kumplainen, E., Raasakka, O., and Kyski, P. (1977): Interaction of taurine, GABA and Glutamic acid with synaptic membranes. J. Neurochem., 29: 819-826.(183)
Lahdesmaki, P. and Oja, S.S. (1972): Neurotransmitter or modulator: Does taurine qualify?.Abstract. Scand. J. Clin. Lab. Invest., 29 (Suppl. 122:): 71. (60) .
Lahdesmaki, P. and Oja, S.S. (1973): On the mechanism of taurine transport at brain cell membrane. J. Neurochem., 20: 1411-1417.(27).
Lahdesmaki, P. and Pajunen, A. (1977): Effect of taurine, GABA and glutamate on the distribution of Na+ and K+ ions between the isolated synaptosomes and incubation media. Neurosci. Letters, 4: 167-170. (64,177,179).
Laird, H.E., and Huxtable, R. (1976): Effect of taurine on audiogenic seizure response in rats. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 267-273. Raven Press, N.Y. (52) .
Langer, G.A. (1971): The intrinsic control of myocardial contraction-ionic factors. New Eng. J. Med., 285: 1065-1071.(44,45).
Langer, G.A. (1968): Ion fluxes in cardiac excitation and contraction and their relation to myocardial contractility. Physiol. Rev., 48: 708-757. (45).
Langer, G.A. (1973): Heart: Excitation Contraction coupling. Annu. Rev. Physiol. 35: 55-86.(169).
Lee, K.S., and Choi, S.J. (1966): Effect of the cardiac glycosides on the Ca2+-uptake of cardiac sarcoplasmic reticulum. J. Pharmacol. Exp. Therap. , 153: 114-120.(1757".
Lee, K.S., and Klaus, W. (1971): The subcellular basis for mechanism of inotropic action of cardiac glycosides. Pharmacol. Rev., 23: 193-261. (175).
Lee, K.S., Ladinsky, H. and Stuckey, J.H. (1967): Decreased Ca2+-uptake by sarcoplasmic reticulum after coronary artery occlusion for 60 and 90 minutes. Circ. Res., 21: 439-444.(175). 211
Levi, G. , Kandera, J., and Lajtha, A. (1967): Control of cerebral metabolite levels. I. Amino acid uptake and levels in various species. Arch. Biochem. Biophys., 119: 303-311.(9).
Levy, W.B., Haycock, J.W., and Cotman, C.W. (1974): Effect of polyvalent cations on stimulus-coupled secretion of (l^C)-Y-aminobutyric acid from isolated brain synaptosomes. Molec. Pharmacol., . 10: 438-449. (186).
Limbricht, H. (1863): Vorlaufige Notif Uber Einige Bestandtheile der Fleischflussigkeit von Fischen. Ann Chem. Pharmakol., 127: 185-189.(5).
Limbricht, H. (18 65): Uber Einige Bestandtheile der Fleischflussigkeit. Ann. Chem. Pharmacol., 133:293-305. (5) .
Ling. CM., and Abdel-Latif, A.A. (1968): Studies on sodium transport in rat brain nerve-ending particles. J. Neurochem., 15: 721-729. (177,178).
Lombardini, J.B. (1976): Regional subcellular studies on . taurine in the rat central nervous system. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 311-326. Raven Press, N.Y. (7,29).
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with the folin phenol reagent. J. Biol.Chem. , 193: 265-275. (1Q1);.
Maclennan, D.H. (19 75): Resolution of the calcium transport system of sarcoplasmic reticulum. Can. J. Biochem., 53: 251-261. (168).
MacLennen, D.H. and Holland, P.C. (19 75): Calcium transport in Sarcoplasmic reticulum. Annu. Rev. Biophys. Bioeng., 4: 377-404.(168).
Mandel, P., and Pasantes-Morales, H. (1978): Taurine in the nervous system. In: Reviews of Neurosciences, edited by S. Ehrenpreis and I. Kopin, Vol. 3~7 pp. 157-193. Raven Press, N.Y. (2).
Mandel, P., Pasantes-Morales, H., and Urban, P.F. (1975): Taurine, a putative transmitter in retina. In: Transmitters in the visual process, edited by S.L. Bontmg, pp. 89-108, Pergamon Press, Oxford. (5.8,60) . 212
14 Marchbanks, R.M. (196 8): The uptake of C-choline into synaptosomes in-vitro. Biochem. J., 110: 533-541.(178).
Martin, W.G., Sass, N.L., Hill, L., Tarka, S., and Truex, R. (1972): The synthesis of taurine from sulfate. IV. An alternate pathway for taurine synthesis by the rat. Proc. Soc. Exp. Biol. Med., 141: 6 32-6 33.(24).
Maxfield, M. (1953): Axoplasmic proteins of the squid giant nerve fibre with particular reference .to the fibrous protein., General Physiol., 37: 201-217.(76).
Melville, K., Mazurkiewicy, I. and Korol, B. (1955): Potassium disequilibrium as a factor in production of cardiac irregularities following epinephrine and nor-epinephrine. Fed. Proc., 14: 369.(166).
McBride,. W.J. , and Frederickson, R.C.A. (1978): Neurochemical and neurophysiological evidence for a role of taurine as an inhibitory neurotransmitter in the cerebellum of the rat. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 415-428. Raven Press N.Y. (59,61).
McBride, W.J., and Nadi, N.S., Altman, J., and Apprison, M.H. (1976): Effects of selective doses of x-irra- diation on the levels of several amino acids in the cerebellum of the rat. Neurochem. Res., 1: 141-152.(61).
McBroom, M.J., and Welty, J.D.(1977): Effects of taurine on heart calcium in the cardiomyopathic hamster. J. Mol. Cell. Cardiol., 9: 85 3-858.(188).
McLennan, H. and Hagen, B.A. (1963): On the response of the stretch receptor neurones of crayfish to 3-OH-tryptamine and other compounds. Comp. Biochem. Physiol., 8: 219- 222. (182).
Miraglia, R.J., Martin, W.G., Spaeth, D.G., and Patrick, H. (1966): On the synthesis of taurine from sulfate by the chick: I. Influential dietary factors. Proc. Soc. Exp. Biol. Med., 123: 725-730. (24).
Mori, A., Hosotani, M., and Choong, T.L. (1974): Studies on brain guanidino compounds by automatic liquid chromatography. Biochem. Med., 10: 8-14. (14,15). 213
Muir, J.R., Dhalla, N.S., Orteza, J.M., and Olson, R.E. (1970): Energy-linked calcium transport in subcellular fractions of the failing rat heart. Circ. Res., 26: 429-438. (175).
Mullins, L.J. (1959): An analysis of conductance changes in squid axon. J. Gen. Physiol., 42: 1013-1035.(16).
Mutani, R., Bergamini, L., Delsedime, M., and Durrelli, L. (19 74a): Effect of taurine in chronic experimental epilepsy. Brain. Res., 79: 330-332.(5.1,186).
Mutani, R., Bergamini, L., Feriello, R., and Delsedime, M. (1974b): Effect of taurine on cortical acute epileptic foci. Brain Res., 70: 170-173.(51,60,186).
Nadi, N.S., McBride, W.J., and Aprison, M.H. (1977): Distribution of several amino acids in regions of the cerebellum of the rat. J. Neurochem., 28: 453-455.(61).
Nayler, W.G. (1963): Effect of caffeine on cardiac contractile activity and radio-calcium movement. Am. J. Physiol. 204: 969-974.(50).
Nayler, W.G., Dunnett, J., and Berry, D. (1975): The calcium accumulating activity of subcellular fractions isolated from rat and guinea-pig heart muscle. J. Mol. Cell. Cardiol., 7: 275-288. (170,175).
Neely, J.R., Liebermeister, H., Battersby, E.J., and Morgan, H.E. (1967): Effect of pressure development on oxygen consumption by isolated rat heart. Am. J. Physiol., 210: 804-814. (41,172).
Nenhoff, V., and Tonge, S.R. (1973): Some pharmacological inductions of a role for taurine in the regulation of pituitary activity. J. Pharm. Pharmacol., 25 (Suppl.) : 138-139.(58) .
Newman, W.H., Frangakis, C.J., Gross, D.S., and Bressler,R. (19 77): A relationship between myocardial taurine contentand pulmonary wedge pressure in dogs with heart failure. Physiol. Chem. Physics, 9: 259-263. (31). 214
Oja, S.S., Karvonen, M.L. , and Lahdesmaki, P. (1973): Biosynthesis of taurine and enhancement of decar• boxylation of cysteine sulfinate and glutamate by electrical stimulation of rat brain slices. Brain Res., 55: 173-178.(21).
Oja, S.S.,' and Kontro, P. (1978): Neurotransmitter actions of:" taurine in the central nervous system. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 181-200. Raven Press, N.Y. (58, 60).
Oja, S.S., Unsitalo, A.J., Vehvelainen, M., and Piha, R.S. (196 8): Changes in cerebral and hepatic amino acids in the rat and guinea-pig during development. Brain Res., 11: 655-661. (54).
Okamoto, K., and Aoki, K. (1963): Development of a strain of spontaneously hypertensive rats. Jap. Circ. J., 27: 282-293. (32) .
O'keefe, CM., Smith, Jr. L.H. (1973): Carbamyltaurine: Absence of a role in taurine metabolism. Res. Commun. Chem. Path. Pharm. 6: 755-758. (13).
Okumura, N., Orsuki, S., and Aoyama, J. (1959): Studies on the free amino acids and related compounds in the brain of fish, amphibia, reptile, aves and mammal by ion exchange chromatography. J. Biochem. 46: 207-212. (54).
Pasantes-Morales, H., Bonaventure, N., Wioland, N., and Mandel, P. (1973): Effect of intravitreal injections of taurine and GABA on Chicken ERG. Int. J. Neurosci. 5: 235-241.(59).
Pasantes-Morales, H., Kleth, J., Ledig, M., and Mandel, P. (19 72) : Free amino acids in chicken and rat retina. Brain Res. 41: 494-497.(7). 215
Pasantes-Morales, H., Salceda, R., and Lopez Colome, A. (19 78): Taurine in normal retina. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 265-280. Raven Press, N.Y. (53,61,63,: 187,188,189).
Peck, E.J., and Awapara, J. (1966): Metabolism of sulfur amino acids in rat brain. Federation Proc., 25: 642.(15).
Peck, E.J., and Awapara, J. (196 7) : Formation of taurine and isethionic acid in rat brain. Biochem. Biophys. Acta, 141: 499-506. (9,62).
Perry, T.L., and Hansen, S. (1973): Quantitation of free amino compounds of.rat brain: Identification of hypotaurine. J. Neurochem., 21: 1009-1011. (5,6,16).
Perry, T.L., Hansen, S. and Kennedy, M.O. (1975): Amino acids in human epileptogenic foci. Arch. Neurol. 32: 752-754.(51)
Peterson, M.B., Mead, R.J., and Welty, J.D. (1973): Free amino acids in congestive heart failure. J. Molec. Cell. Cardiol. 5: 139-147. (30,33).
Phillis, J.W. (1978): Overview of neurochemical and neurophysiological action of taurine. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 289-304. Raven Press, N.Y. (58, 61).
Rabin, A.R., Hayes, K.C., and Berson, E.L. (1973): Cone and rod responses in nutritionally induced retinal degeneration in the cat. Invest. Opthalmol., 12: 694-704. (53).
Rambaut, P., and Miller, S. (1966): Studies in feline sulfur amino acids metabolism. Proc. Seventh Internatl. Congr. Nutrition, Hamberg, p. 16 4. Pergamon Press, Oxford. (26).
Rassin, D.K., Sturman, J.A., and Gaull, G.E. (1976): Taurine: Subcellular distribution in brain of the developing rat-Abstract. Trans. Amer. Soc. Neurochem., 7: 163.(7). 216
Rassin, D.K., Sturman, J.A., and Gaull, G.E. (1977): Taurine in developing rat brain: Subcellular distribution and association with synaptic vesicles of (35s) taurine in maternal, fetal and neonatal rat brain. J. Neurochem. 28: 41-50.(7).
Read, W.O., and Welty, J.D. (1962): Synthesis of taurine and isethionic acid by dog heart slices. J. Biol. Chem., 237: 1521-1522.(15,16,87,160,162, 165,16 7). Read, W.O., and Welty, J.D. (1963): Effect of taurine on epinephrine- and digoxin-induced irregularities of dog heart. J. Pharmacol. Exp. Therap. , 139: 283-289. (17, 35 ,16"6~T~
Read, W.O., and Welty, J.D. (1965): Taurine as a regulator of cell potassium in the heart. Electrolytes Cardiovasc. Diseases, 1: 70-85. (17, 36 , 37, 38 , 39 , 50 ,6 2,. 166,167) . : Redtenbacher, J. (1846): Uber die Zusammensetzung des Taurins. Ann. Chem. Pharmakol., 57: 170-174.(5).
Remtulla, M.A., Applegarth, D.A., Clark, D.G., and Williams, I.H. (1977): Analysis of isethionic acid in mammalian tissues. Life Sci. 20: 2029-2036.(160,164).
Remtulla, M.A., Katz, S., and Applegarth, D.A. (1978): Effect of taurine on ATP-dependent calcium transport in guinea-pig cardiac muscle. Life Sci. 23: 383-390. (180,184). /
Remtulla, M.A., Katz, S., and Applegarth, D.A. (1979): Effect of taurine on passive ion transport in rat brain synaptosomes. Life Sci. (in press), volume 24. (177,188).
Repke, D.I., and Katz, A.M. (1972): Calcium-binding and calcium-uptake by cardiac microsomes: A kinetic analysis. J. Mol. Cell. Cardiol. 4: 401-416.(170).
Repke, K. (1965): Effect of Digitalis on membrane adenosine triphosphatase of cardiac muscle. In: Drugs and Enzymes, Proceedings of the Second Inter• national Pharmacology meeting, edited by Brodie, B.B. and J.R. Gillette, pp. 65-87. Pergamon Press, Oxford.(37). 217
Reuter, H. (1972): Divalent cations as charge carriers in excitable membranes. Prog. Biophys. Mol. Biol., 26: 3-43. (186).
Reynolds, E.S. (1963): The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol. 17: 208-213.(94).
Roberts, E., Lowe, I.P., Guth, L., and Jelinger, B. (1958) Distribution of y-aminobutyric acid and other amino acids in nervous tissues of various species. J. Exp. Zool., 138: 313-328.(54).
Rosei, M.A., Orlando, M. and Bellussi, L. (1974): Gas- chroma to graphic detection of isethionic acid in pig heart. Ital. J. Biochem. 23: 67.71. (158,159,160).
Rouser, G., Kritchevsky, G., Galli, C., Yamamoto, A. and Knudson, A.G. (196 7): In: Inborn Errors of Sphingolipid Metabolism, Edited by S.M. Aronson and B.W. Volk, p. 303. Pergamon Press, Oxford. (158).
Salkowski, E. (1873): Ueber die Entstehung der Schwefelsaure and das Verhalten des Taurins im Thierischen Organismus. Verchows Arch. Pathol. Anat. Klin. Med., 58: 460-508; 580. (11,14).
Sarnoff, S.J., Gilmore, J.O., Mitchell, J.H., and Remensnyder, J.P. (196 3): Potassium changes in the heart during homeometric autoregulation and acetyl strophanthidin. Am. J. Med. 34: 440-451. (166)
Sass, N.L., and Martin, W.G. (1972): The synthesis of taurine from sulfate. III. Further evidence for the enzymatic pathway in chick liver. Proc. Soc. Exp. Biol. Med. 139: 755-761. (24).
Sbarbaro, V. (1974): Electroclinical effects of taurine in some epileptic patients; preliminary results. Acta Neurol. (Napoli), 29: 33-37. (52).
Schaffer, S.W., Chovan, J.P., and Wekman, R.F. (1978): Dissociation of cAMP changes and myocardial contra• ctility in taurine perfused rat heart. Biochem. Biophys. Res. Commun. 81: 248-253.(41,172).
Schaffer, S.W. , Severn, A., and Chovan, J. (1978): New chromatographic procedure for measuring isethionic acid levels in tissue. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 25-28 Raven Press, N. Y. (158,160,164).
Scharff, R., and Wool, I.G. (1965): Accumulation of amino acids in muscle of perfused rat heart. Biochem. J. 97: 257-271. (§) . 218
Schmidt, R., Sieghart, W., and Karobath, M. (1975): Taurine uptake in synaptosomal fractions of rat cerebral cortex. J. Neurochem. 25: 5-9. (29).
Schmidt, C.L.A., and Allen, E.G. (1920): Further studies on the elimination of taurine administered to man. J. Biol. Chem. 42: 55-58. (13) .
Schmidt, C.L.A., and Cerecedo, L.K. (1928): Further experiments on the fate of taurine in dogs. Proc. Soc. Exp. Biol. Med. 25: 270-271.(11).
Schimidt, S.Y., Berson, E.L., and Hayes, K.C. (1976): Retinal degeneration in cats-fed casein. I. Taurine deficiency. Invest. Ophthalmol. 15: 47-52. (53).
Schimidt. S.Y., Berson, E.L., Watson, G., and Huang, C. (1977): Retinal degeneration in cats fed casein III. Taurine defeciency and ERG amplitudes. Invest. Ophthalmol. Vis. Sci., 16: 673-678.(53,54).
Schram, E. and Crokaert, R. (1957): L'identification de la Guianido-et al Carbamyltaurine dans 1"urine par chromatographie sur echangeurs d'ions. Bull Soc. Chim. Biol. 39: 561-568. (11, 13; 14).
Schwartz, A., Wood, J.M. , Allen, J.C, Bornet, E.P., Entman. M.L., Goldstein, M.A., Sordahl, L.A. and Suzuki, M. (1973): Biochemical and morphological correlates of cardiac ischemia. 1. Membrane system. Am. J. Cardiol., 32: 46-61. (175).
Sgaragli, CP. and Pavan, F. (1972): Effect of amino acid compounds injected into cerebrospinal fluid spaces on colonic temperature, arterial blood pressure and behaviour of the rat. Neuropharmacology, 11: 45-56. (57).
Sgaragli, CP., Pavan, F. , and Gelli, A. (1975): Is taurine induced hypothermia in the rat mediated by 5-Ht? NaunynSchmiedebergs Arch. Pharmacol., 288: 179-184. (57).
Skou, T.C _£1965) :+Enzymatic basis for active transport of Na and K across cell membrane. Physiol. Rev., 45: 596-617. (37).
Snedecor, G.W., and Cochram, W.G. (1967): Statistical Methods, 6th edition, pp. 138-171. The Iowa State University Press, Ames, Iowa, U.S.A. (110). 219
Spaeth, D.G., and Schneider, D.L. (1974): Taurine synthesis, concentration, and bile salt conjugation in rat, guinea-pig and rabbit. Proc. Soc. Exp. Biol. Med., 147: 855-858.(6,17,62).
Staedeller, G., and Frerichs, F.T. (1858): Ueber das Vorkommen von Hams toff, Taurin and Scyllit in den Organen der Plagiostomen. J. Prakt. Chem., 73: 48-55.(5).
Starr, M.S., and Voaden, M.J. (1972): The uptake, metabolism and release of 14c-taurine by rat retina in vitro. Vision Res., 12: 1261-2169.(27).
Striano, S., Grasso, A., Buscaino, G.A., and Perretti,P. (1974): Primi Eusultati sugali effetti della Taurins Nella Epilessia Umana. Acta. Neurol. (Nepoli), 29: 537-542.(52).
Sturman, J.A. (1973): Taurine pool sizes in the rat: Effect
of vitamin Bfi deficiency and high taurine diet. J.Nutr. 103: 1566-15 80. (8,10,17,26,62) .
Sturman, J.A., and Gaull, G.E. (1975): Taurine in the brain and liver of the developing human and monkey. J. Neurochem.: 25: 831-835.(5,6,54).
Sturman, J.A., and Gaull, G.E. (1976): Taurine in the brain and liver of the developing human and Rhesus monkey. In: Taurine,,. edited by R. Huxtable and A. Barbeau, pp. 73-84. Raven Press. N.Y.(55).
Sturman, J.A., Hepner, G.W., Hoffman, A.F., and Thomas, P.J. (1976a): Taurine pool sizes in man: Studies with ?5s-taurine. In: Taurine, edited, by -R; Huxtable and A. Barbeau, pp. 21-34. Raven Press, N.Y.(9).
Sturman, J.A. , Rassin, D.K. , and Gaull, G.E. (1976b)^: origin and Subcellular localization of taurine in developing rat brain. Proc. Int. Congr. Biochem. P. 553.(7).
Sturman, J.A., Rassin, D.K., and Gaull, G.E. (1977a): Taurine in development. Life,Sci., 21: 1-22.(2).
Sturman, J.A., Rassin, D.K., and Gaull, G.E. (1977b): Taurine in developing rat brain: Maternal-fetal transfer of 35S-taurine and its fate in the neonate. J. Neurochem., 28: 31-39.(55,76). 220
Sturman, J.A., Rassin, D.K., and Gaull, G.E. (1977c): Taurine in developing rat brain: Transfer of (-^S) taurine to pups via the milk. Pediat. Res., 11: 28-33. (161). Sturman, J.A., Rassin, D.K., and Gaull, G.E. (1978): Taurine in the development of the CNS. In: Taurine and Neurological Disorders, edited by A. Barbeau and R: Huxtable, pp. 49-71. Raven Press, N.Y. (56).
Sumizu, K. (196 2): Oxidation of hypotaurine in rat liver. Biochim.Biophys. Acta, 63: 210-212119,62).
Sulakhe, P.V., Leung, N.L-K., and St. Louis, P.J. (1976): Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase. Can. J. Biochem. 54: 438-445. (171) .
Tada, M., Kirchberger, M.A., and Katz, A.M. (1976): Regulation of calcium transport in cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase. In: Recent Advances in Studies on Cardiac Structure and Metabolism, volume 9, The Sarcolemma, edited by Roy, P.E. and Dhalla, N.S., pp. 225-239. University Park Press, Baltimore. • (170.171). Tada, M., Kirchberger, M.A., and Katz, A.M. (1975): Phosphorylation of a 22,000 dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5' - monophosphate - dependent protein kinase. J. Biol. Chem. 250: 2640-2647.(170).
Tada, M., Kirchberger, M.A. Repke, D.I., and Katz, A.M. (1974): The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3': 5' - monophosphate-dependent protein kinase. J. Biol. Chem., 249: 6174-6180.(99,169,170,171).
Tada, M., Yamakoto, T., and Tonomura, Y. (1978): Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Phys. Rev., 58: 1-79.(169,170,175).
Taisho, Y., Fujihira, E., Takahashi, H., and Nakazawa, M. (1970): Effect of long term feeding of taurine in hereditary hyperglycemic obese mice. Chem. Pharm. Bull. (Tokyo), 18: 1636-1642.(58).
Tedeschi, H., and Harris, D.L. (1955): The osmotic behaviour and permeability to non-electrolyte of mitochondria. Arch. Biochem. Biophys., 58: 52-56. (177).
Thoai, N.V., Roche, J., and Olomucki, A. (1958): Sur la Presence de la Taurocyamine (Guanidotaurine) dans L'urine de Rat et sa Signification Biochimique dans L'excretion Azotee. Biochem. Biophys. Acta., 14: 448 (11) 221
Thoai, N.V., Olomucki, A., Robin, Y., Prodel, L.A., and Roche, J. (1956): Sur le Presence de Nombreux Derives Carbamyles et Guanidiques dans les Urines et sur leur Signification Biologique. Compt. Rend. Soc. Biol., 150: 2160-2164.(11,14).
Thoai, N.V., and Roche, J. (1960): Phosphagens of marine animals. Ann. N.Y. Acad, Sci., 90: 923-928.(14).
Thoai, N.V., Roche, J. and Olomuchi, A. (1954): Sur la Presence de la Taurocyamine (guanidotaurine) dans Taurine de Rat et sa Signification Biologique dans L'excretion Azotee. Biochim. Biophys. Acta, 14: 448.(14).
Thoai, N.V., and Robin, Y. (1965): Distribution of phosphagens in errant and sedentary polychaeta. In: Studies in Comparative Biochemistry, edited by Munday, K.A., pp. 152-161. Pergamon Press, London'; .(14) .
Thut, P.D., Hruska, E., Huxtable, R., and Bressler, R. (1976): Effect of taurine on eating and drinking behaviour. In: Taurine, edited by R. Huxtable and A. Barbeau, pp. 357-364. Raven Press, N.Y. (57.).
Tiedman, F., and Gmelin, L. (1827): Einige neur Bestandtheilen der Galle des Ochsen. Ann. Pysik. Chem., 9: 326-337. (5) 2+ 2+ Tonomura, Y. (19 73): The Ca -Mg -dependent ATPase and the uptake of Ca2+ by the fragmented sarcoplasmic reticulum. In: Muscle protein, muscle contraction and cation, transport, edited by Y. Tonomura, pp. 305-356. University Park Press, Bait., London, Tokyo. (168). Toyama, S. , Miyasato, K. , Yasuda, M. , and Soda, K.' (1973) : Occurance of taurine-pyruvate eminotransferase in bacterial extracts. Agr. Biol. Chem.,37: 2939-2941. (162).
Truce, W.E., Campbell, E.W., and Madding, G.D. (1967) Mass spectral study of alkyl alkanesulfonates. J. Org. Chem. , 32: 308-317. (153,154 ,155 ,156) y;--;
Tsukada, Y., Inoue, N., Donaldson, J., and Barbeau, A. (1974): Suppressive effects of various amino acids against ouabain-induced seizures in rats. Can. J. Neurol. Sci., 1: 214-221. (51) . 222
Tuttle, R., Witt, P., and Farah, A. (1962): Therapeutic and toxic effects of ouabain on K+ fluxes in rabbit atria. J. Pharmacol. Exp. Therap., 137: 24-30. (166).
Ueda, I., Loehining, R.W., and Ueyama, H. (1961): Relationship between sympathomimetic amines and methylxanthines including cardiac arrhythmias. Anesthesiology, 22: 926-932.(50).
Urban, P.F., Dreyfus, H., and Mandel, P. (1976): Influence of various amino acids on the bioelectrical response to light stimulation of a superfused frog retina. Life Sci., 18: 473-480.(59).
Urquhart, N.M., Perry, T.L., Hansen, S., and Kennedy, J. (1974): Passage of taurine into adult mammalian brain. J. Neurochem. 22: 871-872.(9).
Usherwood, P.N.R. (1978): Amino acids as neurotransmitters. In: Advances in Comparative Physiology and Biochemistry, edited by O. Lowenstein, pp. 227-310. Academic Press., N.Y. (182).
Valenciennes, A., and Fremy. (1855): Recherches sur la Composition des muscle dans la serie des animaux. Compt. Rend. 41: 735-741(5).
Valenciennes, A. and Fremy. (1857): Recherches sur la Composition des Oeufs et des Muscles dans la serie des animaux. Ann. Chim. Phys., 3rd. Ser., 50: 129-178. (5).
Van Gelder, N.M. (1972): Antagonism by taurine of cobalt induced epilepsy in cat and mouse. Brain Res., 47: 157-168. (51,53,60).
Van Gelder, N.M. (1976): Rectification of abnormal glutamic acid levels by taurine. In: Taurine, edited by R. Huxtable and A. Barbeau, PP. 293-302. Raven Press. N.Y. (52).
Van Gelder, N.M. (1978): Glutamic acid and epilepsy: The action of Taurine. In: Taurine and Neurological Disorders, edited by A. Barbeau and R. Huxtable, pp. 387-402. Raven Press. N.Y. (61).
Van Gelder, N.M., Sherwin, A.L. and Rasmussen, T. (1972): Amino acid content of epileptogenic human brain: Focal versus surrounding regions. Brain Rds,. , 40: 385-393. (51,60) . 223
Van Gelder, N.M., Sherwin, A.L., Sacks, C. , and Anderman, F. (1975): Biochemical observations following administer- ation of taurine to patients with epilepsy. Brain Res., 94: 297-306. (53) .
Van Winkle, W.B., and Schwartz, A. (1976): Ions and inotropy. Ann. Rev. Physiol., 38: 247-272. (44).
Vellan, E.J., Gjessihn, L.R., and Stalsberg, H. (1970): Free amino acids in the pineal and pituitary glands of human brain. J. Neurochem. , 17: 699-701.(7)
Verly, W.G., and Koch, G. (1954): Metabolism of 0-mercatoethy- lamine. 2. In the dog. Biochem. J., 58: 663-665. (21)
Vogel, A.I. (1964) Diazomethane. In: A textbook of Practical Organic Chemistry, including qualitative organic analysis, 3rd, edition, p. 971. Longmans, London.(69)
Wada, J.A., Osawa, T., Wake, A. and Carcoran, M.E. (1975): Effects of taurine on kindled amygdaloid seizures in rats, cats, and photosensitive baboons. Epilepsia, 16: 229-234. (52).
Wainer, A. (1965): The production of Cysteinesulfinic acid from cysteine in-vitro. Biochem. Biophys. Acta, 104: 405-412. (19).
Weller, M., and Laing, W. (1979): The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum. Biochem. Biophys. Acta, 551: 406-419.(175).
Weller, M., and Morgan, I.G. (1977): A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability. Biochim. Biophys. Acta, 465: 527-534.(177,178,179).
Welty, J.D. (1963): The role of taurine in cardiac excitability. Ph.D.Thesis: University of South Dakota.(17, 166).
Welty, J.D. Jr., Read, W.O., and Shaw,. Jr. , E.H. (1962) : Iolation of 2-hydroxyethanesulfonic and (isethionic acid) from dog heart. J. Biol. Chem., 237: 1160-1161. (15,16,124,15 3,157,15 8^ Welty, J.D., and Read, W.O. (1964): Studies on some cardiac effects 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 14c-Y-aminobutyric acid by subcellular brain particles. Bid chem. Pharmaco1., 14: 273-288. (178).
Winegrad, S., and Shanes, A.M. (1962): Calcium flux and contractility in guinea-pig atria. J. Gen.Physiol., 45: 371-385. (42).
Wonnacott, J.H., and Wonnacott, R.J. (1977): Introductory Statistics, 3rd edition, 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 liver. II. Measurement of cysteine metabolism in vivo and the activation of in vivo activity of cysteine oxidase. Biochim. Biophys. Acta 29 7: 48-59. (62).
Yonada, K., Toyama, S., Yosuda, M., and Soda, K. (19 76); Purification and crystalization of bacterial W-aminoacids-pyruvate aminotransferase. FEBS Lett., 71: 21-24. (162). 225
APPENDICES 226
Life Sciences Vol. 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 Paediatrics, Pathology and Chemistry, The University of British Columbia, Biochemical Diseases Laboratory, Children's Hospital and Government of Canada Agriculture Research Station, Vancouver, British Columbia
(Received in final form May 17, 1977) SUMMARY
A gas-liquid chromatographic assay has been developed to measure isethionic acid after methylation with diazomethane. The identity of the products of methylation has been confirmed by mass-spectro- metry and nuclear magnetic resonance spectroscopy. The method was used to measure isethionic acid in rat heart, dog heart and rat brain. The assay was validated by measuring isethionic acid on squid axoplasm. We have been able to detect only trace amounts of isethionic acid in rat brain (0.2 mg/lOOg) and rat heart (0.1 mg/ lOOg). None was found in dog heart.
Isethionic acid, 2-hydroxy-ethane sulfonic acid, is the deaminated analogue of taurine (1). Its presence in biological material was first reported by Koechlin (2) who found that it was the major anion of the axoplasm from the squid giant axon. He suggested that ISA might indirectly be responsible for the production of electrical phenomena in the nerve. Welty et al. (3) proposed that taurine was the precursor of ISA. These workers apparently isolated sub• stantial quantities of ISA from dog and rat heart tissues, by a gravimetric method involving the crystallization of ISA as its sodium salt from a hot aqueous extract of dog heart or rat heart. They later demonstrated the con• version of 35g_taurine to ^S-ISA by dog heart slices (A). Later Peck and Awapara (5) reported that very small amounts of Isethionic acid could be formed from lsotopically labelled taurine in rat heart and brain tissues.
Other workers (6,7) have suggested that the conversion of taurine to ISA in myocardial cells facilitates the retention of intracellular calcium or potassium ions. The major difficulty of studying the function of ISA has been the lack of a good analytical procedure for its detection and quantitation. Methods used in the past to detect ISA do not offer much accuracy or sensiti• vity and some apparently promising methods have never been published in full (8,9). We therefore report here an analytical method to measure ISA.
MATERIALS AND METHODS
For analysis of rat tissues, we used Wistar rats weighing approximately 200g. Animals were sacrificed by a sharp blow to the head. Hearts and brains were promptly excised and the tissues rinsed ln normal saline, blotted on Whatman
Correspondence to: Derek A. Applegarth, Biochemical Diseases Laboratory, Children's Hospital, 250 West 59th Avenue, Vancouver, B.C. Canada V5X 1X2.
2029 227
2030 Isethionic'Acid In Mammalian Tissues Vol. 20. No. 12, 1977
HI filter paper, and Immediately frozen in small plastic vials ln liquid nitro• gen. This process required less than ten minutes per rat.
For analysis of dog heart, animals were obtained from the Department of Physio• logy at The University of British Columbia, after having been used for open heart surgery. The dogs were sacrificed with 152 potassium chloride (10 ml) and the heart stored at -20 degrees before use. Samples of the giant axon of squid (Loligo pealli) were obtained from Dr. F.C.G. Hoskin of the Department of Biology, Illinois Institute of Technology, Chicago, Illinois.
Isolation of ISA from Heart and Brain Tissues: 5g samples of pooled brain or heart tissues were used for experimentation. The heart tissue was minced before being used and then divided into two equal portions of 2.5g each. To one por• tion was added 1.0 umole of sodium isethionate (Sigma Chemical Company, St. Louis, Missouri). The other portion was used without any addition. Both portions were homogenized in 501 methanol/water (v/v), (10 ml), in a Sorvall omnimixer (Ivan Sorvall Inc., Norwalk, Connecticut), using a teflon chamber at 3/4 of the full speed for five minutes, followed by one minute of full speed. The homogenate was transferred to a centrifuge tube and centrifuged at 3,000g for five minutes. The homogenizing chamber was rinsed three times with 5 ml, 50% methanol/water (v/v), and the rinse washings added to the centrifuge pellet which was suspended in the rinsing solution using a Vortex mixer. The resulting suspension was then centrifuged again for five minutes and the super• natant fluid removed.
To the combined volume of supernatants and rinsings, an equal volume of Folch solvent (chloroform:methanol, 2:1, v/v) was added. The solutions were mixed thoroughly and centrifuged to separate the layers. The upper aqueous layer was removed and evaporated to dryness ln a rotary evaporator under reduced pressure. After evaporation of the aqueous layer to dryness, 2 ml of a puri• fied cation exchange resin (AG-50W-X8, 50-100 mesh, H+ form Bio Rad Laborator• ies, Richmond, California), prewashed ln methanol and suspended in an equal volume of methanol, was added to the flask. After trituration, the mixture was transferred to a 5 ml glass-stoppered conical centrifuge tube. After centri• fugation, the methanol layer was dried in a vacuum desiccator over sulphuric acid.
Isolation of ISA from Axoplasm of the Squid Giant Axon: Axoplasm from the squid giant axon was obtained in freeze-dried form. The sample, 83mg fresh weight axoplasm, was dispersed ln deionized water using a glass homogenizer. The turbid solution so formed was made up to a volume of 10 ml with deionized water. An aliquot of the solution was mixed with an equal volume of absolute methanol and this mixture treated with an equal volume of Folch solvent. The rest of the procedure was the same as that described above, for Isolation of •ISA from heart and brain tissues.
Preparation of Standards: A suspension of Ion exchange resin AG-50 (H+ form in methanol, 1 ml of resin + 1 ml of methanol) was pipetted into each of six, 5 ml glass stoppered, calibrated, conical centrifuge tubes. After allowing the resin to settle, 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 aliquots of 5 mM sodium isethionate solution in water.
Volume was made up to 4 ml in each tube with methanol, and the resin suspended using a Vortex mixer. After centrifugation, the methanol layer from each tube was removed into reaction vials and evaporated to dryness in a vacuum desicca- tor over sulphuric acid. Abbreviations: Throughout the text, isethionic acid is abbreviated as ISA. Nuclear magnetic resonance spectroscopy is abbreviated as NMR. 228
Vol. 20, No. 12, 1977 Isethionic Acid In Mammalian Tissues 2031
Methylation of the Samples and Preparation of a Standard Curve: ISA standards, or samples obtained from a tissue extract, were dissolved in 0.1 ml of a 1 mM solution of salicylic acid (Sigma Chemical Company, St. Louis, Missouri) in methanol. Salicylic acid was used as an internal standard. The vials were stoppered using teflon laminated discs and placed in an ice-bath for five minutes. Ethereal diazomethane solution prepared from diazald (Aldrich Chemical Company, Milwaukee, Wisconsin) was introduced into the vials slowly, with mixing until the yellow color persisted. An additional three drops of diazomethane solution was added and the mixture was allowed to stand for about thirty minutes in ice. The total volume in the vial was 0.2 ml or less. Where the total volume in the vial was more than 0.2 ml, the excess solvent was carefully evap• orated under a stream of dry nitrogen and the sample remethylated for a further thirty minute period. Methylation reactions were all stopped by adding one drop 502 acetic acid in water (v/v). This remethylation procedure was found not to affect the standard calibration curve for ISA.
Gas-Liquid Chromatography: Flame Ionization Detection: The gas-liquid chromato• graph used was a Bendix,Model 2500, equipped with a flame ionization detector. Columns were 6 ft by 4 mm (i.d.) glass U-tubes. The stationary phases used were 5% 0V-1 and 5% OV-17 (Gas Chromatographic Specialities Ltd., Brockville, 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 for 0V-1 was 115 degrees C and for OV-17 was 135 degrees C. Nitro• gen was used as a carrier gas at a flow rate of 40 ml/min.
Gas-Liquid Chromatography: Sulphur Detection: The gas-liquid chromatograph used for such experiments was a Micro Tek 220 equipped with a flame photometric detector, Model FPD 100 (Melpar Inc., Falls Church, Virginia). The column used was a "6 ft 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 carrier gas with an inlet 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 air flow was 30 ml/min. j
Gas-Liquid Chromatography: Mass Spectrometry: The mass-spectrometer used was a Hitachi Perkin-Elmer, operating at an ionization energy of 70 ev. interfaced with a Varian, Model 1400 gas chromatograph. Authentic ISA and butane sulfonic acid samples were analyzed on 0V-17 column after methylation with diazomethane. Chromatographic details were as outlined above for flame ionization detection.
Nuclear Magnetic Resonance Spectroscopy: For proton NMR spectroscopy, the methyl esters of ISA and butane sulfonic acid were prepared in the following manner. Five times recrystallized sodium isethionate (100 mg) in boiling absolute ethanol and 1-butanesulfonic acid sodium salt (100 mg) (Eastman Kodak Company, Rochester, N.Y.) were each dissolved in methanol (1.0 ml) and the samples treated with resin, and methylated as described above. After thirty minutes, excess diazomethane was carefully blown off with a stream of dry nitro• gen and the sample completely dried down under reduced pressure over sulphuric acid. The NMR spectra of the reaction products, without further purification, were taken at 100 mHz in a Varian HA-100 spectrometer. Samples were dissolved in deuterated dimethylsulfoxide (Merck, Sharpe and Dohme Ltd., Canada) with tetramethyl silane being used as an internal standard. For the purposes of comparison, the NMR spectrum of crystallized, non-methylated ISA, also dissolved in dimethylsulfoxide was obtained.
RESULTS
Chromatography of the methylated ISA on 0V-1 and on an OV-17 column is shown in Figure 1. Using a column of 5% 0V-1, a single peak with a retention time of 1.6 minutes was obtained for methylated ISA. Using a column of 5% OV-17, two 229
2032 Isethionic Acid In Mammalian Tissues Vol. 20, No. 12, 1977
peaks at retention times of 3.5 and 4.0 minutes were obtained. The identity 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 after OV-17 gas chromatography is shown in 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 Acid
Salicylic Acid
\ P.ol I
\^^Salicylic l
FIG. 1 Chromatographic separation of the products of methylation of isethionic acid and salicylic acid using flame ionization 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 retention time.
The NMR spectra of both methylated ISA and butanesulfonic acid showed the CH3 peak of the methylester as a singlet at 3.88 ppm. A small singlet at 3.30 ppm occurring in the spectrum of methylated ISA was assigned as the CH3 of the methylether of ISA on the basis of the known chemical shift value of 3.38 ppm of the methylether singlet in methoxyethanol (10). Integration of the spectrum indicated an approximate^ratio of methylester CH3 to methylether CH3 of 20:1. 230
Vol. 20, No. 12, 1977 Isethionic Acid In Mammalian Tissues 2033
Spectrum A CHjOCM,
M*thyt«it«r \0 tMthyUthar of •••thionic Acid
0) rCMjCMjOC^I' >
[co]
J L 1, 1,1
m/e
FIG. 2 Mass spectra.of the products of methylation of isethionic acid. A. Mass spectrum of the Peak I from Figure 1. B. Mass spectrum of Peak II from Figure 1. Experimental conditions and inter• pretation of data are described in the text.
Figure 3 shows a typical standard curve. When the method was used to analyze ISA ln biological samples, a small peak in the position of ISA was detected in rat brain at a concentration of approximately 0.2 mg/lOOg of tissue and in rat heart at a concentration of approximately 0.10 mg/lOOg tissue. This value for rat heart is only an estimate because at this level we are at the approximate limit of sensitivity for the assay technique. We were unable to detect any ISA in dog heart. The analytical procedure was always monitored by adding ISA at concentrations of 2.0 and 0.2 umole/g of tissue to duplicate aliquots of tissue examined. Recovery was always between 95 and 100%. The method as described using flame ionization detection is capable of detecting ISA in tissue as a concentration of approximately 0.2 umole ISA/gram of tissue extracted (approxi• mately 2.5 mg/lOOg). With the sulfur detector the sensitivity of the method was approximately 0.008 umoles/gm (0.1 mg/lOOg tissue).
In squid axoplasm, isethionic acid was found at a concentration of 150 umole per ml axoplasm. The identity of the peaks was confirmed by gas chromato• graphy mass-spectrometry. 2034 Isethionic Acid In Mammalian Tissues Vol. 20, No. 12, 1977
9.0 L
Hmole Isethionic Acid/Vial
FIG. 3 Calibration curves of methylated isethionic acid obtained on a column of 5% OV-17 on H.P. Chromosorb W. Operating parameters are quoted in the text.
DISCUSSION
Methylation of ISA produces two compounds, a methylester and the doubly methyl• ated methylester, methylether derivative. These two compounds co-elute when analyzed by gas-liquid chromatography on a column of OV-1, but can be separated on a column of OV-17 (see Fig. IB). The ratio of these two compounds separated on OV-17 is approximately 20:1 with the methylester derivative being predomi• nant. The ratio of the two peaks was invariant over a wide range of gas chromatographic conditions. The ratio was confirmed by proton NMR spectroscopy.
The assignment of structures to the mass spectra of the peaks by methylation of ISA (Fig. 2) follows the discussion of fragmentation patterns of alkyl alkane• sulfonates by Truce et al. (11). The parent ions {M}. were not seen in the methylated ISA mass spectra. Truce et al. claim that these ions are scarce for most of the alkyl alkanesulfonates. However, the assignment of structures of the two peaks were strengthened by the appearance of the {M-l}t fragment for the methylester of ISA at m/e - 139.
We found that it was usually necessary to use two columns, one of OV-1 and the other of OV-17, and occasionally two methods of detection, flame ionization and flame photometric (for sulfur) to look at analytical extracts of tissues. This was necessary because all of the tissues studied gave a peak in the position of 232
Vol. 20, No. 12, 1977 Isethionic Acid In Mammalian Tissues > 2035
ISA in the crude extracts of rat and dog heart and rat brain 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 brain If they had been ISA. Confirmation that this peak was not ISA depended on rechromatography of the same extract on OV-17, and verification that this peak did not contain sulfur. The large peak from heart and brain extracts seen on OV-1 proved to contain only very small amounts of ISA when reassessed using OV-17 and the sulfur detector.
The value that we obtained for the analysis of ISA in the squid giant axon compares favorably with other data obtained using different, less sensitive methods where 150 umole ISA per g. axoplasm has been reported (12,13).
Welty et al. (3) quoted a figure of 42.6 mg ISA per lOOg rat heart tissue and 12.9 mg per lOOg dog heart. These amounts would have been quite easily detect• able with our method. Using the sulfur detector which, ln this particular case, was ten times more sensitive than the flame ionization detector, only a very small peak in the position of ISA could be seen for rat heart at a concentration of roughly 0.10 mg/lOOg. Insufficient material was available to confirm Its identity by mass spectrometry. In the case of rat brain, a peak could be seen with the sulfur detector at the position of ISA, at a concentration of 0.2 mg/ lOOg of tissue. Again, Insufficient material was available to confirm that this small amount of material was truly ISA. We extracted as much as 400g of dog heart tissue to search for ISA. In this large scale experiment we also found no ISA peak. In this large scale experiment we followed exactly the procedure of Welty, Read and Shaw (3). We obtained neither the crystals of sodium isethionate that they reported nor gas chromatographic evidence of ISA. We can not explain the difference between our results and those of Welty, Read and Shaw (3). The sensitivity for the gas-liquid chromatographic portion of this experi• ment would have been approximately 0.1 mg/lOOg of heart tissue. Our findings cast doubt on theories of the mode of action of taurine which involve bio- conversion of taurine to ISA.
This work was presented at the 2nd International Congress on Taurine in Tucson, Arizona, March 1977, and a portion of it has appeared in abstract form (14).
ACKNOWLEDGEMENTS
We wish to thank the B.C. Heart Foundation for a grant-in-aid. We also wish to thank Mr. Greg Owen, Department of Chemistry, Simon Fraser University for help in obtaining mass spectra.
REFERENCES
1. J. G. JACOBSEN and L. H. SMITH, JR, Physiol. Rev. 48 424-511 (1968). 2. B. A. K0ECHLIN, J. Biophys and Biochem. Cytol. _1 511-529 (1955). 3. J. D. WELTY, W. 0. READ and E. H. SHAW, J. Biol. Chem. 237 1150-1161 (1962). 4. W. 0. READ and J.D. WELTY, J. Biol. 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, Circ. Res. 34-35 III-ll (1974). 7. W. 0. READ and J.D. WELTY, Electrolyte 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 Isethionic Acid In Mammalian Tissues Vol. 20, No. 12, 1977
Street, Philadelphia 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, Clin. Res. XXIV, 646A (1976). 234 Pediat. Res. 12: 732 (1978)
Letter to the Editor: Isethionic Acid and Milk
MOHAMED A. REMTULLA AND DEREK A. APPLEGARTH131
Biochemical Diseases Laboratory, Childrens Hospital, Vancouver, Canada
JOHN A. STURMAN AND GERALD E. GAULL 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 was 100% and the sensitivity of the method was such that 20 nmol rats was transferred via the milk to the pups (2). In this report we isethionic acid/ml milk could have been easily detected. In our noted the presence of another radioactive compound besides earlier report, we noted that the radioactive compound in milk taurine which was present only in extracts of milk and which which cochromatographed with authentic isethionic acid com• cochromatographed with authentic isethionic acid. The presence prised 30-40% of the total radioactivity (the rest of which was of this compound in milk was puzzling and we felt that it was present as taurine). The present assay could easily have detected important to determine whether or not isethionic acid was present isethionic acid present at 10% of the taurine concentration. as a natural constituent of milk. The development of a sensitive We conclude that isethionic acid is not a significant constituent gas-liquid chromatographic assay for isethionic acid (1) allowed of rat milk (if indeed it is present at all). The radioactive compound us to perform such measurements on rat milk samples. which cochromatographed with authentic isethionic acid is either Samples of milk were obtained from lactating rats of the Nelson- another compound or was produced by gut bacteria, reabsorbed, Wistar strain, from 2 days after birth to 6 days after birth, as and secreted in the milk after the ip injection. Of note in this previously described (2). Samples of milk (0.2 ml) were freeze- regard are the results of experiments designed to test the possible dried and resuspended in 10 ml deionized water using a glass-glass bioconversion of taurine to isethionic acid by slices of dog heart 35 homogenizer. Authentic isethionic acid (25-150 nmol) was added and rat heart. We found that [ S]taurine was converted in up to to some of the samples at this stage. The suspension was mixed 10% yield to a radioactive compound which behaved chomato- with an equal volume of methanol (10 ml) and then treated with graphically like isethionic acid. The same result was obtained, an equal volume of Folch solvent (20 ml), mixed thoroughly, and however, when [^SJtaurine was added to heart slices in buffer and centrifuged to separate the layers. The aqueous layer was removed extracted immediately with methanol. We can detect no biocon• and extracted as previously described (1). Methylation with diaz• version of taurine to isethionic acid in heart tissue by the proce• omethane in the presence of butane-sulfonic acid as internal dures described above, feras&k It is likely that the radioactive standard (10 nmol/sample) was carried out as described (1). A compound detected in milk may be the same or a similar chemical thick white precipitate formed in the methylation mixture and was to the radioactive compound detected in heart slices, but it is removed by centrifugation. The supernatant fluid was concen• unlikely to be isethionic acid. 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 REFERENCES AND NOTES column, 130°, equipped with a flame ionization detector). Ise• 1. Remtulla, M. A„ Applegarth, D. A., Clark. D. G.. and Williams, I. H.: Analysis thionic acid standards of 0, 25, 50, 100, and 200 nmol were used 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: for quantification. Transfer of ("Sjtaurine to pups via the milk. Pediat. Res., //: 28 (1977). The samples analyzed showed no trace of isethionic acid. The 3. To whom correspondence should be addressed. recovery of authentic isethionic acid added to the milk samples 4. Received for publication October 5, 1977. '
Copyright © 1978 International Pediatric Research Foundation. Inc. Printed in U.S.A. 0031 -3998/78/ 1206-0732S02.O0/0 v 235
Life Sciences, Vol. 23, pp. 383-390 Pergamon Press Printed in 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
Division of Pharmacology, Faculty of Pharmaceutical Sciences and Departments of Paediatrics & Pathology, Faculty of Medicine, University of British Columbia, and Biochemical Diseases Laboratory, Children's Hospital, Vancouver, B.C., Canada
(Received in final form June 12, 1978)
SUMMARY
The effect of taurine on ATP-dependent calcium transport was examined in guinea-pig cardiac ventricle homogenates and in micro• somal preparations enriched in sarcoplasmic reticulum. Taurine (5-50 mM) did not affect ATP-dependent calcium binding or uptake in either of these preparations or alter the rate of decay of calcium uptake activity. Taurine (20 mM) also did not affect the oxalate-dependent calcium uptake stimulation noted in the presence of cyclic AMP-dependent protein kinase and cyclic AMP. The mech• anism by which taurine alters cardiac function remains to be
elucidated. r
Taurine is present in high concentrations in mammalian hearts represent• ing nearly 50% of the total free amino acid pool (1,2). In congestive heart failure, taurine levels are markedly elevated in several species including humans (3-5). Changes in myocardial taurine concentration have also been reported in experimentally-induced hypertension (6) and ischemia (7).
Taurine has been shown to alter the pharmacological response to cardiac glycosides in guinea-pig and rat heart preparations (8,9 ). Several reports suggest that taurine may play a role in the control of K+ and Ca2+ movements in the heart (8-13) and thereby affect cardiac function. The possible role of /taurine in the regulation of ion movements in the heart remains to be elucidat• ed.
The present investigation was undertaken to determine whether taurine could influence ATP-dependent calcium uptake and binding in guinea-pig cardiac ventricle homogenates and in microsomal preparations enriched in sarcoplasmic reticulum.
MATERIALS AND METHODS
Heart Preparations: Guinea-pigs (200-300 g, albino,Hartley strain) were sacrificed by a blow to the head. The hearts were promptly excised, washed in saline and the aorta, atria and connective tissue removed. The ventricular muscle was then promptly frozen (within 30 sec. of sacrifice) in methyl butane , J * Correspondence to: Dr. Sidney Katz, Division of Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C., c Canada V6T 1W5. 0300-9653/78/0724-0383$02.00/0 Copyright © 1978 Pergamon Press 236
384 Effect of Taurine on Calcium Transport Vol. 23, No. 4, 1978
and dry ice. The frozen hearts were wrapped in tin foil and stored at -80°C. The tissue could be stored in this way for up to 3 months without significant loss in calcium transport activity.
Preparations of Heart Ventricle Homogenates: A piece (0.1-0.2 g) was cut from a frozen heart preparation and homogenized in 5 ml of a medium consisting of 40% sucrose and 40 mM Tris-Cl,pH7.2 using a Polytron P20 homogenizer (3 strokes of 5 sec. duration at setting 5).
Preparations of Microsomes Enriched in Sarcoplasmic Reticulum: The method of Harigaya and Schwartz (14) was'followed with slight modifications. The frozen preparation was homogenized using a Polytron P20 homogenizer (3 strokes of 5 sec duration at setting 5) and the homogenate fractionated by differential centrifugation followed by 0.6 M KC1 treatment to reduce acto- myosin contamination. The final preparation was suspended in a medium consist• ing of 40% sucrose and 40 mM Tris-Cl, pH 7.2. Storage in 40% sucrose reduced the loss of calcium transport activity noted when these preparations were maintained at 4°C (15). Unless otherwise indicated, all experiments were con• ducted within 2 h of the preparation of these fractions.
ATP-dependent Calcium Uptake and Binding Assays: The method of Tada et al. (16) was followed with a few modifications. Oxalate-facilitated calcium uptake was determined in 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 histidine-HCl, pH 6.8, 5 mM MgCl2, 110 mM
KC1, 5 mM Tris-ATP, 2.5 mM Tris-Oxalate and CaCl2 containing 45caCl2 (10 Ci/ 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 in a total volume of 0.5 ml. The reaction was terminated by filtering an aliquot of the reaction mixture through a millipore filter (HA 45, Millipore Co.). The filter was then washed twice with 15 ml of 40 mM Tris-Cl, pH 7.2, then dried and counted for radioactivity in Aquasol (New England Nuclear) using standard liquid scintillation counting techniques. When cyclic AMP- dependent protein kinase was added to the medium, it was present in a concen• tration of 50 ug/ml along with 1.0 ii M cyclic AMP.
ATP-dependent calcium binding was studied under identical conditions except that Tris-oxalate was omitted from the reaction medium.
In studies on the effect of taurine on the decay of calcium transport activity, preparations were stored at 4°C in 2 mM Tris-Cl, pH 7.2 in 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.
Statistics: Student's "t" test for unpaired, common variance data (19) was used as a measure of significance. Standard Error of the Mean (S.E.M.) was used as a measure of variation.
Materials: All chemicals were reagent grade. Tris-ATP, Cyclic AMP, EGTA, and bovine cardiac protein kinase (Type 1) were obtained from Sigma Chem• ical Co., St. Louis, Mo. ^CaCl2 was obtained from the Radiochemical Centre, Amersham, England. 237
Vol. 23, No. 4, 1978 Effect of Taurine on Calcium Transport 385
RESULTS
Effect of Taurine on Calcium Uptake and Binding: The effect of varying taurine concentrations on calcium uptake and binding in both ventricular homo• genates and sarcoplasmic reticulum enriched preparations is shown in Table 1. The free calcium concentration used in these studies was 1.0 uM. Taurine in concentrations of 5-50 mM had no significant effect on calcium uptake or binding in either of these preparations.
TABLE 1
Effect of Taurine on Calcium Uptake and Binding in Guinea-pig Heart Ventricle 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.,1 7 + 0.38a 0.16 + 0.03 12. .40 + 0.79 0. 71 + 0.10 (2.,3 6 0.40)b (0.16 + 0.03) (12.,7 2 + 0.89) (0. 75 + 0.09)
10 2..3 9 + 0.15 0.17 + 0.02 12. ,93 + 1.27 0. 76 + 0.05 (2..3 2 + 0.29) (0.16 + 0.02) (13..1 9 + 1.19) (0. 75 + 0.04)
20 2.,6 8 + 0.10 0.16 + 0.02 12. ,72 + 1.37 0. 77 0.05 (2..5 6 + 0.14) (0.18 + 0.02) (12,.1 8 + 1.33) (0. 81 + 0.02)
30 2.,8 9 + 0.14 0.17 + 0.01 14..7 9 + 1.83 0. 81 + 0.04 (2..8 9 + 0.13) (0.19 + 0.02) (14..0 9 + 0.02) (0. 74 + 0.03)
AO 2..6 2 + 0.22 0.18 + 0.01 11, .84 + 1.79 0. 76 + 0.02 (2..7 0 + 0.14) (0.17 + 0.02) (11.,2 7 + 1.72) (0. 76 + 0.05) '
50 2..5 9 + 0.17 0.16 + 0.02 12, .59 + 2.05 0. 70 + 0.08 (2.,5 6 + 0.34) (0.17 + 0.01) (12..5 2 + 0.01) (0. 75 + 0.09)
Guinea-pig heart ventricle homogenates (200-300 p g protein) or sarcoplasmic reticulum enriched preparations (40-50 yg protein) were incubated for 5 min with and without taurine in medium
containing 40 mM histidine HC1, pH 6.8, 5 mM MgCl2, 5 mM ATP, 110 mM KC1, 2.5 mM Tris-oxalate, and 1.0 u M free Ca"^ (125 M M CaCl2 containing 45CaCl2 (10 Ci/Mole) and 391 MM EGTA). The reaction was carried out at 30°C in a total volume of 0.5 ml. Calcium binding was determined in an identical reaction mixture, except that 2.5 mM Tris-oxalate was omitted. a. The results are a Mean ± S.E.M. of at least 3 observations each performed in duplicate. b; The values in parentheses are controls (taurine omitted). 238
386 Effect of Taurine on Calcium Transport Vol. 23, No. 4, 1978
The effect of 20 mM taurine on calcium uptake and binding in both these preparations was examined at various calcium concentrations (Table 2). The sarcoplasmic reticulum enriched preparation exhibited an increase in calcium uptake and binding with increasing Ca2+ concentration to a maximum at 10 iiM free Ca2+; Ca2+ concentrations higher than 10pM were inhibitory. This profile of the Ca2+ concentration effect on calcium transport was similar in the homo• genate preparation. Taurine (20 mM) at all free Ca2+ concentrations studied had no significant effect on calcium uptake or binding in either of these preparations.
TABLE 2
The Effect of Taurine on Calcium Uptake and Binding at Various Calcium Concentrations in Guinea-pig Heart Ventricle Homogenates and Sarcoplasmic Reticulum Enriched Preparations
Calcium Homogenate Preparation Sarcoplasmic Reticulum Preparation Cone. (M M) Calcium Uptake Calcium Binding Calcium Uptake Calcium Binding (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min) (nmoles/mg/min)
+ a + 0.5 1..0 7 0 .03 0.09 0,.0 1 8 .91 + 1.32 0,.5 4 + 0.11 + (1 .18 0 .03)b (0.11 + 0,.01 ) (8 .77 + 1.24) (0, .58 + 0.08)
+ + 1.0 2,.4 3 0,.0 9 0.14 0..0 1 20, .14 + 3.02 0..6 3 + 0.14 + + (2,.3 6 0,• 17) (0.15 0..01 ) (19..6 1 + 2.94) (0. .65 + 0.14)
5.0 + 7..8 1 1..0 9 0.40 0..0 3 71..4 2 + 14.89 1.,3 3 + 0.25 ± + + (7. ,94 0..52 ) (0.39 0. 03) (71.,5 9 14.00) (1. ,17 + 0.15)
± 10.0 9..6 7 0..9 9 0.79~± 0. 05 84. ,56 + 20.09 1. 42 + 0.11 + + (10. 00 1.•17 ) (0.85 0. 06) (86.,6 4 + 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 ventricle 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 in Table 1 in the presence of various concentrations of free calcium. Calcium binding was measured under identical conditions in the absence of 2.5 mM Tris-oxalate. a. The results are a Mean ± S.E.M. of at least 3 observations each performed in duplicate. b. The values in parentheses are controls (taurine omitted).
The Effect of Taurine on the Time-course of Calcium Uptake and Binding: The time course of calcium uptake and binding in homogenate and sarcoplasmic reticulum enriched preparations is shown in Figure 1A and IB, respectively. Calcium uptake in both these preparations was linear for the first 10 minutes of incubation following which the rates declined slightly. Maximal calcium binding was observed at 10 minutes in the homogenate preparation and at 5 min in the sarcoplasmic reticulum preparation. No significant effect of taurine was observed in these preparations either at the initial time (30 sec) or at longer periods of incubation. 239
Vol. 23, No. 4, 1978 Effect of Taurine on Calcium Transport 387
A 50-i
40 H
30 H
20 H • < c c '55 "5 IOH o o k_ a Q. cn O) E E tn o o E E c B c 400- i 4.0 di (-4— d) c o 360- 3.6 c a 320- T/f r- 3.2 co
280- 2.8 o D u 240- 1 2.4 u
200- • 2.0
160- 1.6
120- 1.2 * 80- > 0.8
0.4
5 10 15 20
Incubation Time (minutes)
FIG. 1 Time course effect of 20 mM taurine (O.A) on calcium uptake (solid lines) and binding (dotted lines) in Guinea-pig heart ventricle homogenates (A) and sarco• plasmic reticulum enriched preparations (B). The vertical lines represent ± S.E.M. of 3 determinations each performed in duplicate. 240
388 Effect of Taurine on Calcium Transport Vol. 23, No. 4, 1978
The Effect of Taurine on the Decay of Calcium Uptake Activity: Both the homogenate and the sarcoplasmic reticulum enriched preparations decreased rapidly in calcium uptake activity when kept at 4°C in the absence of 40% sucrose. Addition of 15 mM taurine to these preparations under these con• ditions did not alter this steady decline in calcium uptake activity (Figure 2).
•J 1 1 1 1 i • 0 12 3 4 5 6
Time After Preparation (hours)
FIG. 2 The effect of taurine on the decay of calcium uptake activity in guinea-pig ventricle homogenates and sarcoplasmic reticulum enriched preparations. Homogenates (solid lines, 200-300 ug protein) or sarcoplasmic reticulum preparations (dotted lines, 40-50ug protein) were maintained in 2 mM Tris-Cl, pH 7.2 in the presence (O, A ) and absence (•, • ) of 15 mM taurine. Calcium uptake was measured at specified times as described in Table 1. The vertical lines represent ± S.E.M. of 3 determin• ations each performed in duplicate.
Effect of Taurine on Cyclic AMP-dependent Protein-kinase Stimulated Calcium Uptake: When the sarcoplasmic reticulum enriched preparation was incubated with protein kinase and cyclic AMP in the presence of 1.0 uM Ca++, calcium uptake was increased more than 2 .fold (p<0.02) (Table 3). Similarly, the rate of calcium uptake by the homogenates also increased in the presence of cyclic AMP-dependent protein kinase (p<0.|05). Taurine (20 mM) had no signifi• cant effect on the cyclic AMP-dependent protein kinase stimulation of calcium uptake in these preparations. 241
Vol. 23, No. 4, 1978 Effect of Taurine on Calcium Transport 389
TABLE 3
Effect of Taurine on Cyclic AMP-dependent Protein Kinase-Stimulated Calcium Uptake in Guinea-pig Heart Ventricle Homogenates and Sarcoplasmic Reticulum Enriched Preparations:
Taurine Homogenate Preparation Sarcoplasmic Reticulum Preparation
(20 mM)\ Without With Without With cAMP-dependent cAMP-dependent cAMP-dependent cAMP-dependent Protein kinase Protein kinase Protein kinase Protein kinase
a,b c - d 2.39 ± 0.29 3.38 ± 0.14 9.72 ± 1.02 16.86 + 1.51
+ e 3.30 ± 0.17 17.89 ± 1.84e
Guinea-pig ventricle homogenates (200-300 ug protein) and sarcoplasmic reticulum enriched preparations (40-50 yg protein) were incubated with and without cyclic AMP (1.0 uM) and cyclic 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 1. In these experiments the free calcium concentration was 1.0 uM and the incubation time was 5 minutes. a. The results are a Mean ± 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 Ca^+-uptake in the absence of cyclic AMP-dependent protein kinase. d. P<0.02 compared to Ca2+-uptake in the absence of cyclic AMP-dependent protein kinase. e. Not significant compared to the activity seen without taurine in the presence of cyclic AMP-dependent protein kinase.
DISCUSSION
Results obtained on the ATP-dependent calcium uptake and binding para• meters of the sarcoplasmic reticulum enriched preparation used in this study were similar to those noted by other workers (16,20,21). The homogenate pre• paration exhibited ATP-dependent Ca^+ binding activity as well as Ca2+-uptake activity in the presence of oxalate; these activities, though, were lower than those noted in the sarcoplasmic reticulum enriched preparation.
In this study we have demonstrated that taurine does not significantly affect ATP-dependent calcium transport in guinea-pig cardiac ventricle homogen• ates or in cardiac preparations enriched with sarcoplasmic reticulum. Entman et al. (22) recently reported that taurine had no effect on calcium transport in canine sarcoplasmic reticulum preparations. These workers used the spectro• photometry murexide dye technique for the measurement of calcium transport whereas in our studies ^-*CaCl2 and the millipore filtration technique were utilized. Our present studies thus confirm those of Entman et al. (22) using both a sarcoplasmic reticulum enriched and crude homogenate preparation of heart tissue in a species in which pharmacological effects of taurine in cardiac tissue have been noted (8,9). The possibility that taurine may alter calcium uptake or binding activity in cellular organelles other than sarcoplasmic reticulum tends to be ruled out by the lack of an effect on calcium uptake or binding in the homogenate preparation used in this study. 242
390 Effect of Taurine on Calcium Transport Vol. 23, No. 4, 1978
Cyclic AMP-dependent protein kinase has been shown to stimulate calcium uptake in preparations of cardiac sarcoplasmic reticulum (21). Taurine did not affect the enhancement of calcium transport by cyclic AMP-dependent protein kinase either in the homogenate preparation or in the sarcoplasmic reticulum preparation. Lack of an effect on this system indicates that taurine does not act as a modulator of calcium transport through this cyclic AMP-mediated path• way.
Previous reports indicated that the decay in calcium transport ability observed in skeletal muscle sarcoplasmic reticulum preparations could be reduc• ed by the presence of taurine (13). In this study the presence of taurine had no effect on this decay process in either the homogenate or sarcoplasmic reticulum enriched preparations. This lack of effect of taurine was also noted by Entman et_ al_. (22) under different experimental conditions.
Taurine has been shown to be of potential importance in cardiac pathology. The possibility still exists that taurine exerts its effects in the cardiac cell by altering the passive diffusion of ions or by affecting calcium release from sarcotubular, mitochrondrial or sarcoplasmic reticulum stores. These possibili• ties are currently under investigation in our laboratory.
ACKNOWLEDGEMENT This work was supported by a grant from the British 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, Life Sciences 14 1353-1359 (1974). 4. M.B. PETERSON, R.J. MEAD, J.D. WELTY, J. Mol. Cell. Cardiol. 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, Life 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, Life Sciences 10 499-507 (1971). 10. W.O. READ and J.D. WELTY, Electrolyte 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, Circ. Res. 25 781-794 (1969). 15. M.E. CARSTEN, Proc. Natl. Acad. Sci. U.S.A. 52_ 1456-1462 (1964). 16. M. TADA, M.A. KIRCHBERGER, D.I. REPKE and A.M. KATZ, J. Biol. 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. Biol. Chem. 193 265-275 (1951). 19. T.H. WONNACOTT and R.J. WONNACOTT, Introductory Statistics, p. 215, John Wiley & Sons, New York, Third Edition (1977). 20. D.I. REPKE and A.M. KATZ, J. Mol. Cell. Cardiol. 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, Life Sciences 21 543-550 (1977). 243
Life Sciences, Vol. 24, pp. 1885-1892 Pergamon Press Printed in the U.S.A.
EFFECT OF TAURINE ON PASSIVE ION TRANSPORT IN RAT BRAIN SYNAPTOSOMES
Mohamed A. Remtulla, Sidney Katz and Derek,A. Applegarth
Division of Pharmacology, Faculty of Pharmaceutical Sciences and Departments of Paediatrics & Pathology, Faculty of Medicine, University of British Columbia, and Biochemical Diseases Laboratory, Children's Hospital Vancouver, B.C., Canada
(Received in final form April 2, 1979)
Summary
Taurine, in concentrations greater than 10 mM, was found to have an inhibitory effect on passive calcium uptake and release in rat brain synaptosomal preparations. Amino acids similar to that of taurine in chemical structure, 6-alanine, hypotaurine, homotaurine and y-amino- butyric acid were also shown to inhibit calcium uptake in this prepar• ation. Taurine, though, did not alter the permeability of these preparations to sodium or potassium. It thus appears that taurine and chemically related amino acids can alter calcium movements in these preparations. It is postulated that this effect is due to binding to specific taurine sites in the synaptosomal membranes.
Taurine is known to be present in relatively^ high concentrations in the brain, heart and muscle of mammals (1). Several groups of investigators have reported that taurine concentrations are reduced in experimentally-induced epileptogenic foci of cats (2), mice (3), rats (4) and man (5). Taurine has also been shown to have anti-epileptic effects in both experimentally-induced epilepsy (3,6-8) and in human patients (9,10).
Recently, investigators have suggested a physiological role for taurine in the maintenance of excitatory activity in muscle and nervous tissues (11-15). Others have raised the possibility that taurine is an inhibitory neurotrans• mitter (16,17). However, its possible mechanism of action is still uncertain. Izumi, et al• (18) have suggested that taurine may regulate the free calcium concentration of nervous tissue. We have recently reported (19) that taurine does not affect ATP-dependent calcium transport in guinea-pig whole heart homo• genates and preparations enriched in sarcoplasmic reticulum. In this present work, as part of a study on the possible physiological mechanism of action of taurine, we have studied the effect 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, Division of Pharmacology, Faculty of Pharmaceutical Sciences, University of British 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 in solutions of 25-300 mM Na2S0^ (0.95 ml) in a micro• cuvette. The extinction at E520 was 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 aliquot (0.05 ml) was then added to a microcuvette containing from 100-200 mM ice-cold sodium or potassium acetate (0.95 ml) solution. The content of the microcuvette was rapidly mixed with a pasteur pipette and the extinction 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 in medium containing 0.3 M sucrose, and 10 mM Tris-HCl pK 7.2, in the presence or absence of taurine (20 mM), in a total volume of 3 ml. Following a preincubation of 1 hour, the reaction was started by the addition of 10 uM '45CaCl2 (5 x 105 cpm/sample) . Aliquots (0.2 ml) of the incubation mixture were then removed at intervals and passed through a millipore filter (HA 45, Millipore, Co.). The filter was washed twice with 5 ml of 10 mM Tris- HCl (pH 7.2) in 0.3 M sucrose then dried and counted for radioactivity in Aquasol (New England Nuclear Co.) using standard liquid scintillation counting techniques.
Determination of Loss of ^-*Ca from Preloaded Synaptosomes: Synaptosomal prepar• ations (0.2 mg protein/ml) were loaded with 10 yM ^CaC^ (5 x 10^ cpm/sample) at 2°C in conditions similar to that described above. After 1 hour, an aliquot (0.2 ml) was passed through a millipore filter. The remaining incubation medium was centrifuged at 12,000 x g for 10 minutes. The resulting pellet was resuspended in 3 ml of ice-cold media containing 0.3 M sucrose and 10 mM Tris- HCl, pK 7.2 in the presence and absence of 20 mM taurine. The release of
45caCl2 with time, was determined by passing aliquots (0.2 ml) of the .reaction medium through a millipore filter. The rate of calcium release was then calculated by the following equation: (cpm in filter after 1 hour preloading) - (cpm in filter at sampling time) 45Ca release (%) = (cpm in filter after 1 hour preloading)
Protein Assay: Protein concentrations were measured by the method of Lowry, et al. (31) using bovine serum albumin as a standard. •
Statistics: Statistical analysis was done by Students "t" test for unpaired data. A probability of p<0.05 was taken as the criterion for significance. Standard Error of the Mean (S.E.M.) was used as a measure of variation.
Materials: All chemicals were reagent grade. Taurine was obtained from Sigma
Chemical Co., St. Louis, Mo.. ^5rjaci2 was obtained from the Radiochemical Centre, Amersham, England.
Results
The Osmometric Behaviour of Synaptosomes: The optical extinction (E520) of the synaptosomal preparations suspended in solutions of Na2S04 was found to increase with the strength of the solution (figure 1A). The reciprocal plot of extinction (I/E520) against l/Na2S0z, (figure IB) showed a linear relationship. These results 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
1.2 -i
l.H 1.15H
i.oH l.H
0.9-J 1.05 H
0.8 H ioH
0.95- 0-7 H
0.6 1 1 1 0.9 -1 1 r- 0 100 200 300 .02 005 .01 .015
mM l/Na2SQ,
FIG. 1
The effect of Na2S04 concentration on the E520 of a suspension of synaptosomes:
In (A) the data are plotted as E520 against Na2S0z, while in (B) l/Na2S04 is plotted against l/E52o. Results are shown as Mean S.E.M. 'of 3 different synaptosomal preparations. i
The Effect of Taurine on Sodium and Potassium Permeability in Synaptosomal Preparations: The permeability of the synaptosomal preparations to sodium and potassium ions in the presence or absence of'taurine is shown in Table 1A and IB, respectively. Synaptosomal preparations preincubated with 20 mM taurine and suspended in 100-200 mM sodium or potassium acetate solutions containing 20 mM taurine showed no significant change in E520 when compared to results obtained in the absence of taurine. '
The Effect of Taurine on the Passive Uptake and Release of Calcium in Synapto• somal Preparations: The time course of uptake and release of calcium in an isotonic sucrose medium is shown in figure 2A and 2B, respectively. Under these conditions, passive calcium uptake in synaptosomal preparations was linear with time. The amount of calcium taken up and released from preloaded synaptosomes was lower in the presence of 20 mM taurine. A significant difference in calcium uptake in the presence of taurine was observed after 18 min of incubation (p<0.05). In the calcium release experiments, calcium efflux was significantly reduced in the presence of taurine (20 mM) at all incubation times tested (p<0.01).
Dose-dependent Effect of Taurine on Calcium Uptake in Synaptosomal Preparations: Various concentrations of taurine (0.5 to 50 mM) were studied with respect to synaptosomal calcium uptake (Fig. 3). Control experiments were carried out where taurine was substituted for an equimolar concentration of choline chloride. 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
Effect of Taurine on Sodium (A) and Potassium (B) Permeability in Synaptosomes.
A. Na Acetate B. K Acetate
"520 E520 + mM Na+ mM K CONTROL TAURINE CONTROL TAURINE
100 0..89 8 ± 0.050 0. 913 + 0.044 100 0. 915 ± 0.035 0.,91 8 ± 0.044
125 0.,93 4 ± 0.053 0.,91 6 ± 0.044 ' 125 0..93 4 ± 0.050 0.,94 8 ± 0.041
150 0..96 5 + 0.072 0..99 4 ± 0.041 150 0..96 1 ± 0.045 0..96 6 ± 0.048
175 1..00 0 ± 0.052 1..01 8 ± 0.042 175 1,.018> + 0.033 1..00 1 ± 0.045
200 1..00 0 ± 0.056 1..02 5 ± 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) in acetate salts in the presence (TAURINE) or absence (CONTROL) of 20 mM taurine. Each value is the Mean ± S.E.M. of three separate synaptosomal preparations.
present in the incubation medium. No change in 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 in lowering synaptosomal calcium uptake in concentrations greater than 10.0 mM.
Effect of Other Amino Acids on Calcium Uptake in Synaptosomal Preparations: A number of compounds, in a concentration of 20 mM, were tested for their effect on passive calcium uptake in synaptosomal preparations (Fig. 4). Homotaurine, hypotaurine, 8-alanine and GABA exhibited similar effects to taurine, signifi• cantly decreasing the degree of calcium uptake observed in the absence of these agents (p<0.05). On the other hand, a-alanine stimulated calcium uptake to a small extent (not significant). Methionine, proline and valine also did not significantly affect calcium transport in this preparation (not shown).
Discussion
In this study we have demonstrated that taurine has an inhibitory effect on both calcium uptake and release in synaptosomal preparation suspended in iso• tonic medium. Similar results have recently been reported on the release of ^calcium from preloaded synaptosomes by .Kuriyama et al. (22). These workers, though, did not show any significant effect of taurine on calcium uptake. The discrepancy in calcium uptake results between this present study and that of Kuriyama et al (22) could be due to the differences in the experimental proce• dures .utilized.
In this present study, the inhibitory effect 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 effect. Hue et al. (23) in studies on the insect central nervous system also demonstrated that taurine at concentrations lower than 10 mM had no effect on the sensitivity of post synaptic neurons.
r 247
Vol. 24, No. 20, 1979 Taurine and Passive Ion Transport 1889
120
100
o o 80
• o 60 u
20
J 0 10 20 30 0 10 20 30
incubation Time-, minutes Incubation Time- minutes
FIG. 2 The effect of taurine on (A)^^Ca^+ uptake and (B) release of ^Ca^+ from preloaded rat synaptosomal preparations. Calcium uptake and release were determined in the presence ( O O ) or absence (9 •) of 20 mM taurine in a. medium containing 0.3 M sucrose and 10 mM Tris-HCl, pH 7.2 as described in the text. Effect of taurine on calcium uptake is expressed as ^-"Ca2+ uptake relative to the value of Ca2+ uptake observed at 30 min incubation time in the absence of taurine. Calcium release from preloaded synaptosomes was calculated as % release as described in the text. The verticle lines represent ± S.E.M. of at least three separate synaptosomal membrane preparations.
In these present studies, it was also noted that homotaurine, hypotaurine, 8-alanine and GABA also inhibited calcium uptake in synaptosomal preparations in the same concentration as that observed to produce the inhibitory effect of taurine. This indicates that the inhibitory effect on calcium uptake in these preparations was not specific to taurine. Other amino acids were shown not to inhibit calcium uptake. Taurine receptor sites have recently been reported to be present in synaptosomal preparations (24) and in heart ventricular sarco• lemmal preparations (25). Lahdesmaki et al. (24) have reported that taurine binding to synaptosomes was inhibited by hypotaurine, 6-alanine and GABA. It thus appears that the binding sites have a strict requirement for a specific chemical structure since only those amino acids which were chemically close to taurine were inhibitory. It also can be seen that these same compounds inhibited calcium uptake to the same extent as taurine suggesting that this effect may be due to the specific binding of these compounds to the taurine receptor sites.
Lahdesmaki and Pajunen (26) reported a reduced outflow of sodium and potassium Ions from synaptosomes in the presence of taurine when experiments were performed in sodium- and potassium-free medium containing choline chloride, calcium and 248
1890 Taurine and Passive Ion Transport Vol. 24, No. 20, 1979
100 r-
2 a O u to >
ot
20.0 30.0 50.0
mM Taurine or Choline chloride
FIG. 3 The effect of various concentrations of taurine on 45"^CaCl. 2 uptake in brain synaptosomal preparations (solid bars). Control experiments were done in the presence of equimolar concentrations of choline chloride (middle bars with oblique striations) and in the presence of neither taurine nor choline chloride (left bars with straight striations). Calcium uptake was determined at 2°C for 30 min in medium containing synaptosomes (0.2 mg/ml), 0.3 M sucrose, 10 mM Tris-
HCl, pH 7.2 and 10 uM 45caCl2 (5 x 105 cpm/sample) in a total volume of 0.3 ml. Each bar represents the Mean ± S.E.M. of three separate experiments relative to
the value of the ^CaCl2-uptake of controls measured in the absence of taurine or choline chloride.
magnesium. In our hands, using a different technique, we were unable to observe a significant effect of taurine on the passive permeability of sodium and potassium ions. It is possible that the effect of taurine on the release of sodium and potassium ions from the synaptosomes as observed by Lahdesmaki and Pajunen (26) was secondary to an effect of taurine on the permeability to calcium.
It should be noted that in these present experiments, choline chloride in concentrations of 10 ml! or greater produced a marked inhibition of calcium uptake. This inhibition was greater than that observed for equimolar concentra• tions of taurine. Although the mechanism of this effect was not further investigated it should be stated that choline cannot be used as a substitute for other ions in this or similar studies as it markedly inhibits ion movements in its own right.
It is well known that calcium plays an important role in the regulation of the excitability of neuronal tissue. A decrease in 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£ 1.5 o E c 1.0 O a Z> A' o 0.5 u e 0) £ c < _c c o "C 3 E O E < 3 5 O O s < E s o .X 8. ^ FIG. 4 Effect of. various amino acids on calcium uptake in brain synaptosomal prepara• tions. Calcium uptake was measured for 30 min under the same conditions as that described in Fig. 3 in the presence of 20 mM concentrations of various amino acids. Controls consisted of the same medium with no amino acid addition. The results shown are the mean ± S.E.M. of at least 3 separate- preparations in each case. the synaptic membrane could alter the calcium concentration in the synaptic terminal and affect neurotransmitter release (27). A similar dependence on calcium has been noted in synaptosomal preparations in-vitro (28). Long-term changes in membrane permeability to calcium could change the potential gradient across the membrane leading to hyperpolarization (29) . Taurine could render the nerve terminal refractory to depolarization or prevent calcium influx, necessary for the co-ordinated release of transmitter. Taurine, present in high concentra• tions in the synaptosomes could serve to modulate neuronal activity (29) . The observations reported here may help to provide an insight into the physiological and pharmacological effects of taurine reported both in cardiac (30) and nervous tissue (15,17). Acknowledgement This work was supported by a Grant from the British Columbia (Canada) Heart Foundation. The authors thank Dr. B.D. Roufogalis for his helpful 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. VAN GELDER, Brain Res. 47 157-165 (1972). 250 1892 Taurine and Passive Ion Transport Vol. 24, No. 20, 1979 4. CR. CRAIG and E.R. HARTMANN, Epilepsia (amst.) 14 409-414 (1973). 5. N.M. VAN GELDER, A.L. SHERWIN and T. RASMUSSEIN, Brain Res. 40 385-393 (1972). 6. R. MUTANI, L. BERGAMINI, M. DELSEDIME and L. DURRELI, Brain Res. 79^ 330- 332 (1974). 7. R. MUTANI, L. BERGAMINI, R. FERIELLO and M. DELSEDIME, Brain Res. 70 170- 173 (1974). 8. M. DEROUAUX, E. PUIL and R. NAQUET, Clin, tteurophysiol • 34 770 (1973). 9. A. BARBEAU and J. DONALDSON, Arch. Neurol. 30 53-58 (1974). 10. L. BERGAMINI, R. MUTANI, M. DELSEDIME and L. DURELLI, Eur, neurol. 11 261- 269 (1974). 11. P. LAHDESMAKI and S.S. OJA, Clin. Lab. Invest. 29 (suppl.) _122 71 (1972). 12. C.G. HONEGGER, L.M. KREPELKA, M. STEINER and H.P. VAN HAHN, Experientia 29 1235-1237 (1973). 13. R. GRUENER and H.J. BRYANT, J. Pharmac. Exp. Ther. 194 514-521 (1975). 14. R. GRUENER, K.J. BRYANT, D. MARKOVITZ, R. HUXTABLE and R. BRESSLER, J^ Neurol. Sci. 24 351-360 (1975). 15. A. BARBEAU, N. INOUE, Y. TSUKADA and R.F. BUTTERWORTH, Life Sci. 17 669- 678 (1975). 16. L.K. KACZMAREK, In Taurine (R. Huxtable and A. Barbeau, Eds.) p. 283-292, Raven Press, New York (1976). 17 J.W. PHILLIS (1978) In Taurine and Neurological Disorders, (A. Barbeau and R. Huxtable, Eds.) p. 289-304, Raven Press, New York (1978). 18 K. IZUMI, R.F. BUTTERWORTH and A. BARBEAU, Life Sci. 20 943-950 (1977). 19. M.A. REMTULLA, S. KATZ and D.A. APPLEGARTH, Life Sci. 23 383-390 (1978). 20. E.G. GRAY and V.P. WHITTAKER, J. Anat. (Lond.), 96 79-87 (1962). 21. P. KEEN and J.D. WHITE, J. Neurochem. 17 565-571 (1970). 22. K. KURIYAMA, M. MURAMATSU, K. NAKACAWA and K. KAKITA, In Taurine and Neurological Disorders, (A. Barbeau and R. Huxtable, Eds.) p. 201-216, Raven Press, New York (1978). 23. B. HUE, M. PELHATE and J. CHANELET, In Taurine and Neurological Disorders, (A. Barbeau and R. Huxtable, Eds.), p. 225-236, Raven Press, New York (1978). 24. P. LAHDESMAKI, E. KUMPLAINEN, O. RAASAKKA and P. KYRKI, J. Neurochem. 29 819-826 (1977). 25. E.C. KALAKOWSKI, J. MATURO and S.W. SCHAFFER, Biochem. Biophys. Res. Commun. 80 936-941 (1978). 26. P. LAHDESMAKI and A. PAJUNEN, Neurosci. Letters 4 167-170 (1977). 27. ' J.I. HUBBARD, Prog. Biophys. Mol. Biol. 21 33-124 (1970). 28. W.B. LEVY, J.W. HAYCOCK and C.W. COTMAN, Molec. Pharmacol. 10 438-449 (1974). 29. J.B. LOMBARDINI, In Taurine, (R. Huxtable. and A. Barbeau, Eds.) p. 311-326, Raven Press, New York (1976), 30. D.S. GROSSO and R. BRESSLER, Biochem. Pharmacol. 25 2227-2232 (1976) 31. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem 193 265-275 (1951). —-