by Mano Arumanayagam. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 249-265. / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Transport Pathways of Sodium in Human Erythrocytes --- p.2 / Chapter 1.2 --- Active Transport - the Sodium Pump --- p.2 / Chapter 1.2.1 --- Effects of Sodium and Potassium Ions --- p.3 / Chapter 1.2.2 --- Molecular Weight and Subunit Structure --- p.4 / Chapter 1.2.3 --- Inhibitors of the Sodium Pump --- p.6 / Chapter 1.2.4 --- "Measurements of the Sodium Pump/Na+,K+-ATPase" --- p.8 / Chapter 1.3 --- The Passive Fluxes of Sodium & Potassium --- p.13 / Chapter 1.3.1 --- The Na-K-Cl Co-transport System (SPC) --- p.14 / Chapter 1.3.2 --- Sodium-Lithium Countertransport (SLC) --- p.16 / Chapter 1.3.3 --- Ouabain & Frusemide Insensitive Na+ 'leak' --- p.17 / Chapter 1.3.4 --- Methods for the Determination of Passive Na Fluxes --- p.18 / Chapter 1.4 --- Erythrocyte Sodium Transport and Thyroid Hormones --- p.19 / Chapter 1.5 --- Thyroid Hormones and the Na Pump in Other Cells --- p.22 / Chapter 1.6 --- "Mechanism of T3 Induced Increments of the Sodium Pump/Na+,K+-ATPase Activity" --- p.25 / Chapter 1.7 --- Aims of the Project --- p.28 / Chapter CHAPTER 2 --- "ERYTHROCYTE SODIUM FLUXES, OBS AND NA+,K+-ATPASE ACTIVITY IN HYPERTHYROIDISM 29" / Chapter 2.1 --- PREAMBLE --- p.30 / Chapter 2.2 --- MATERIALS & METHODS --- p.31 / Chapter 2.2.1 --- Materials --- p.31 / Chapter 2.2.2 --- Subjects --- p.31 / Chapter 2.2.3 --- Blood Specimen --- p.32 / Chapter 2.2.4 --- Separation of Erythrocytes from Whole Blood --- p.32 / Chapter 2.2.5 --- Erythrocyte Naic and Kic --- p.32 / Chapter 2.2.6 --- Ouabain-sensitive sodium transport --- p.33 / Chapter 2.2.7 --- "Determination of Na+,K+-ATPase activity" --- p.34 / Chapter 2.2.8 --- "Determination SPC, SLC and Na+ 'leak'" --- p.35 / Chapter 2.2.9 --- Determination of OBS and its Kd --- p.37 / Chapter 2.2.10 --- Plasma Thyroid Hormone Analyses --- p.41 / Chapter 2.2.11 --- "Determination of Plasma Concentrations of Sodium, Potassium, Urea and Creatinine" --- p.41 / Chapter 2.2.12 --- "Determination of Hb concentration, Leucocyte and Erythrocyte Counts, and MCV" --- p.41 / Chapter 2.2.13 --- Assessment of the Precision of the Methods --- p.41 / Chapter 2.3 --- STATISTICS --- p.42 / Chapter 2.4 --- RESULTS --- p.44 / Chapter 2.4.1 --- Determination of the Concentration of Ouabain in a Stock of 3H-ouabain --- p.44 / Chapter 2.4.1.1 --- Effect of Incubation Time on Binding of Ouabain to Erythrocytes --- p.46 / Chapter 2.4.1.2 --- Scatchard Plots of Bound Against Ratio of Bound to Free --- p.48 / Chapter 2.4.2 --- "Establishment of the Method for the Simultaneous Determination of SLC, SPC and Na+'leak'" --- p.48 / Chapter 2.4.3 --- Descriptive Statistics of the Subjects in the Study --- p.54 / Chapter 2.4.4 --- "Plasma concentrations of T4, T3, free T4, and free T3 in control subjects and hyperthyroid patients" --- p.54 / Chapter 2.4.5 --- "Plasma Concentrations of Sodium, Potassium, Urea, Creatinine and Ratios of Creatinine to Urea in Control Subjects and Hyperthyroid Patients" --- p.54 / Chapter 2.4.6 --- "Leucocyte Count, Erythrocyte Count, Hb Concentration, MCV and Derived Values for PCV, MCHC and MCH in Control Subjects and Hyperthyroid Patients" --- p.58 / Chapter 2.4.7 --- "Intracellular Sodium Naic, potassium Kic, ouabain-sensitive efflux rate (fo), ouabain-sensitive efflux rate constant (ko), OBS and its dissociation constant (Kd), and Na+,K+-ATPase activity in control subjects and hyperthyroid patients" --- p.58 / Chapter 2.4.8 --- SPC and its Rate Constant --- p.58 / Chapter 2.4.9 --- SLC and its rate constant --- p.63 / Chapter 2.4.10 --- Na+ 'leak' and its rate constant --- p.63 / Chapter 2.4.11.1 --- The Spearman Rank Coefficient of Correlation Matrix for the Characteristics of Sodium Transport Before Treatment --- p.63 / Chapter 2.4.11.2 --- Partial Coefficient of Correlation Between the Rate Constant for SLC and OBS and Naic --- p.69 / Chapter 2.4.11.3 --- Partial Coefficients of Correlation Between the Rate Constants for SLC and SPC with Naic Held Constant --- p.71 / Chapter 2.4.11.4 --- "Partial Coefficients of Correlation (r123) Between Na+ 'leak' and OBS, Na+,K+-ATPase activity and efflux rate constant with Naic Held Constant" --- p.71 / Chapter 2.4.12 --- Spearman Rank Coefficient of Correlation for Plasma Thyroid Function Tests with Sodium Transport Variables --- p.71 / Chapter 2.4.13 --- Effect of Treatment on 11 of the 18 Subjects --- p.74 / Chapter 2.4.13.1 --- "Effect of Treatment on Body Weight, Systolic and Diastolic BP's" --- p.74 / Chapter 2.4.13.2 --- "Plasma Concentrations of Sodium, Potassium, Urea, Creatinine and Ratio of Urea to Creatinine After Treatment" --- p.74 / Chapter 2.4.13.3 --- Hb Concentrations and Other Blood Indices Before and After Treatment --- p.74 / Chapter 2.4.13.4 --- Plasma Concentrations of T4 and Free T3 in patients before and after treatment --- p.78 / Chapter 2.4.13.5 --- The Characteristics of the Sodium Pump --- p.78 / Chapter 2.4.13.6 --- Passive Fluxes of Sodium After Treatment --- p.78 / Chapter 2.4.14 --- "Longitudinal Assessment of Plasma Thyroid Function Tests, Naic, Kic, OBS, Na+,K + -ATPase Activity and Sodium Fluxes in Patients Undergoing Treatment" --- p.87 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter CHAPTER 3 --- "THE EFFECT OF HYPERTHYROIDISM ON IN VIVO AGING OF ERYTHROCYTE OUABAIN BINDING SITES, INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS 105" / Chapter 3.1 --- Review of Literature --- p.106 / Chapter 3.1.1 --- Physical Changes and Methods of Separation --- p.106 / Chapter 3.1.2 --- Biochemical Changes --- p.108 / Chapter 3.1.2.1 --- "Naic,Kic and the Transport of Sodium" --- p.108 / Chapter 3.1.2.2 --- Changes in Other Enzymes/Proteins --- p.111 / Chapter 3.2 --- MATERIALS & METHODS --- p.116 / Chapter 3.2.1 --- Materials --- p.116 / Chapter 3.2.2 --- Subjects --- p.116 / Chapter 3.2.3 --- Plasma Thyroid Hormone Analyses --- p.116 / Chapter 3.2.4 --- Separation of Erythrocytes According to Age --- p.117 / Chapter 3.2.5 --- Determination of MCV & MCHC --- p.118 / Chapter 3.2.6 --- Erythrocyte Creatine Concentration --- p.118 / Chapter 3.2.7 --- Determination of Naic and Kic --- p.119 / Chapter 3.2.8 --- Maximum Number of OBS --- p.119 / Chapter 3.3 --- STATISTICS --- p.119 / Chapter 3.4 --- RESULTS --- p.120 / Chapter 3.4.1 --- "Establishment of the Method for the Separation of Young, Middle and Old Cells" --- p.120 / Chapter 3.4.2 --- Descriptive Statistics of the Subjects in the Study --- p.122 / Chapter 3.4.4 --- Effect of Erythrocyte Age on Markers of Cell Age --- p.124 / Chapter 3.4.4 --- "Effect of Erythrocyte Age on Naic, Kic and OBS" --- p.127 / Chapter 3.5 --- DISCUSSION --- p.133 / Chapter CHAPTER 4 --- THYROID HORMONES AND OUABAIN BINDING SITES OF RETICULOCYTES --- p.140 / Chapter 4.1.1 --- Review of the Literature --- p.141 / Chapter 4.1.2 --- Intracellular Organelles --- p.143 / Chapter 4.1.2.1 --- Ribosomes & RNA --- p.143 / Chapter 4.1.2.2 --- Mitochondria --- p.143 / Chapter 4.1.2.3 --- Other Organelles --- p.144 / Chapter 4.1.3 --- Changes in the Sodium Pump --- p.145 / Chapter 4.1.4 --- Changes in Other Membrane Proteins --- p.146 / Chapter 4.1.5 --- Aim of the Study --- p.147 / Chapter 4.2 --- METHODS --- p.148 / Chapter 4.2.1 --- Animals --- p.148 / Chapter 4.2.2 --- Induction of Reticulocytosis --- p.148 / Chapter 4.2.3 --- Identification of Reticulocytes --- p.148 / Chapter 4.2.4 --- Separation of Reticulocytes from erythrocytes --- p.149 / Chapter 4.2.5 --- Treatment of guinea pigs --- p.150 / Chapter 4.2.6 --- Oxygen Consumption --- p.150 / Chapter 4.2.7 --- Determination of Number of OBS --- p.154 / Chapter 4.2.8 --- STATISTICS --- p.154 / Chapter 4.3 --- RESULTS --- p.155 / Chapter 4.3.1 --- Establishment of the Method for Determinating the Number of OBS --- p.155 / Chapter 4.3.2 --- Scatchard Analysis of Binding of Ouabain to Erythrocytes --- p.155 / Chapter 4.3.3 --- Induction of Reticulocytosis and Treatment of animals --- p.160 / Chapter 4.3.4 --- Weight Loss and O2 Consumption in Control and T3 Treated Guinea Pigs --- p.160 / Chapter 4.3.5 --- Determination of OBS and Kd of Reticulocytes from Guinea Pigs Treated with T3 and Control --- p.164 / Chapter 4.4 --- DISCUSSION --- p.168 / Chapter CHAPTER 5 --- "THYROID HORMONES AND NA+,K+- ATP ASE ACTIVITY OF ERYTHROID CELLS" --- p.171 / Chapter 5.1 --- Review of the Literature --- p.172 / Chapter 5.1.1 --- Erythroid Differentiation and Maturation --- p.172 / Chapter 5.1.2 --- Methods of Separation of Erythroid Cells --- p.178 / Chapter 5.1.3 --- Biochemical Changes During Erythropoiesis --- p.179 / Chapter 5.1.4 --- In vitro Models of Differentiation and the Biochemical Changes During Haemoglobin Synthesis --- p.182 / Chapter 5.1.5 --- Aim of the Study --- p.185 / Chapter 5.2 --- MATERIALS & METHODS --- p.186 / Chapter 5.2.1 --- Materials --- p.186 / Chapter 5.2.2 --- Sterilisation of glassware --- p.186 / Chapter 5.2.3 --- Solutions --- p.186 / Chapter 5.2.4 --- Heat Inactivation of Foetal Bovine Serum --- p.187 / Chapter 5.2.4.1 --- Preparation of Cell Culture Medium (CCM) --- p.187 / Chapter 5.2.4.2 --- Sterility Test of CCM --- p.188 / Chapter 5.2.5 --- Growth of K562 cells --- p.188 / Chapter 5.2.5.1 --- Assessment of Cell Viability --- p.189 / Chapter 5.2.5.2 --- Subculture of Cells --- p.190 / Chapter 5.2.6 --- "Effect of T3 on Na+,K+-ATPase activity of K562 cell line" --- p.190 / Chapter 5.2.7 --- Induction of Haemoglobin Synthesis --- p.191 / Chapter 5.2.8 --- Benzidine Staining for Haemoglobin --- p.192 / Chapter 5.2.9 --- "The Determination of Na+,K+-ATPase activity" --- p.192 / Chapter 5.2.10 --- Determination of Protein --- p.194 / Chapter 5.3 --- RESULTS --- p.195 / Chapter 5.3.1 --- "Effect of Deoxycholate concentration on unmasking Na+,K+-ATPase activity" --- p.195 / Chapter 5.3.2 --- "Effect of varying saponin concentration on Na+, K+-ATPase activity" --- p.195 / Chapter 5.3.3 --- "Effect of Varying Incubation Time on Na+,K+-ATPase activity" --- p.198 / Chapter 5.3.4 --- "Effect of Varying the Amount of Protein on Na+,K+-ATPase activity" --- p.198 / Chapter 5.3.5 --- "Effect of varying ouabain concentration on Na+,K+-ATPase activity" --- p.201 / Chapter 5.3.6 --- Intra- and Inter-Assay Precision --- p.201 / Chapter 5.3.7 --- "Effect of T3 on the Na+,K+-ATPase Activity of K562 Cells" --- p.201 / Chapter 5.3.8 --- "T3 Concentration-Response Relationship for Na+,K+-ATPase Activity of K562 Cells" --- p.204 / Chapter 5.3.9 --- "Time course of Effect of T3 on Na+,K+-ATPase Activity of K562 cell line" --- p.207 / Chapter 5.3.10 --- Induction of Haemoglobin Synthesis in K562 cells --- p.210 / Chapter 5.3.11 --- Effect of Differentiation on T3 Stimulated K562 Cells --- p.210 / Chapter 5.4 --- DISCUSSION --- p.212 / Chapter CHAPTER 6 --- THYROID HORMONES AND THE ATP-DEPENDANT PROTEOLYTIC SYSTEM --- p.217 / Chapter 6.1 --- The ATP-Dependant Proteolytic System --- p.218 / Chapter 6.1.1 --- The Ubiquitin Pathway --- p.219 / Chapter 6.1.2 --- The Non-Ubiquitin Pathway --- p.222 / Chapter 6.1.3 --- ATP-dependant Proteolysis and Erythropoiesis --- p.223 / Chapter 6.1.4 --- Methods Used for Determining Proteolysis --- p.226 / Chapter 6.1.5 --- Aim of the Study --- p.227 / Chapter 6.2 --- MATERIALS & METHODS --- p.228 / Chapter 6.2.1 --- Materials --- p.228 / Chapter 6.2.2 --- Induction of Reticulocytosis and T3 Treatment --- p.228 / Chapter 6.2.3 --- Culture of K562 Cells and Effect of T3 --- p.229 / Chapter 6.2.4 --- Preparation of K562 Cell Lysate --- p.229 / Chapter 6.2.5 --- Preparation of Reticulocyte Extracts --- p.229 / Chapter 6.2.6 --- Iodination of Lysozyme --- p.230 / Chapter 6.2.7 --- Determination of Proteolytic Activity --- p.231 / Chapter 6.3 --- RESULTS --- p.234 / Chapter 6.3.1 --- Proteolytic Activity of Reticulocyte and K562 Cell Lysates --- p.234 / Chapter 6.3.2 --- Induction and Separation of Reticulocytes --- p.234 / Chapter 6.3.3 --- Effect of T3 Treatment on Guinea Pigs --- p.234 / Chapter 6.3.4 --- ATP-dependant proteolytic system of Reticulocytes and K562 Cells Treated with T3 and Controls --- p.239 / Chapter 6.4 --- DISCUSSION --- p.241 / Chapter CHAPTER 7 --- OVERVIEW & FUTURE WORK --- p.244 / Chapter 7.1 --- Overview & Future Work --- p.245 / REFERENCES --- p.249

by Mui Kin Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 97-113). / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- ION TRANSPORT SYSTEMS IN HUMAN ERYTHROCYTES --- p.6 / Chapter 2.1.1 --- "Sodium Pump (Na+,K+-ATPase)" --- p.6 / Chapter 2.1.2 --- Passive Sodium Transport Systems --- p.9 / Chapter 2.1.2.1 --- Sodium-potassium-chloride cotransport system --- p.9 / Chapter 2.1.2.2 --- Sodium-lithium Countertransport --- p.13 / Chapter 2.1.3 --- Ouabain- and Frusemide-Resistant Passive Effluxes --- p.17 / Chapter 2.2 --- ERYTHROCYTE SODIUM TRANSPORT SYSTEMS IN ESSENTIAL HYPERTENSION --- p.17 / Chapter 2.2.1 --- "Sodium Pump (Na+, K+-ATPase) in Essential Hypertension" --- p.18 / Chapter 2.2.2 --- Sodium-Potassium-Chloride Cotransport in Essential Hypertension --- p.20 / Chapter 2.2.3 --- Sodium-Lithium Countertransport in Essential Hypertension --- p.23 / Chapter 2.2.4 --- Passive Ion Fluxes in Essential Hypertension --- p.26 / Chapter 2.2.5 --- Intracellular Sodium Concentration in Essential Hypertension --- p.26 / Chapter 2.3 --- ERYTHROCYTE SODIUM TRANSPORT SYSTEMS IN DIABETES MELLITUS --- p.27 / Chapter CHAPTER 3: --- MATERIALS & METHODS --- p.29 / Chapter 3.1 --- MATERIALS --- p.30 / Chapter 3.1.1 --- Choline Wash Solution (CWS) --- p.30 / Chapter 3.1.2 --- Lithium Loading Solution --- p.31 / Chapter 3.1.3 --- Choline Wash Solution with Ouabain (CWS-O) --- p.31 / Chapter 3.1.4 --- Sodium Containing Medium (SCM) --- p.31 / Chapter 3.1.5 --- Sodium Free Medium (SFM) --- p.31 / Chapter 3.1.6 --- Sodium Free Medium with Bumetanide (SFM-B) --- p.32 / Chapter 3.1.7 --- Preservation Solution --- p.32 / Chapter 3.2 --- STUDY POPULATION --- p.32 / Chapter 3.2.1 --- Control Subjects --- p.35 / Chapter 3.2.2 --- Patients with Essential Hypertension --- p.35 / Chapter 3.2.3 --- Diabetic Patients --- p.35 / Chapter 3.3 --- DETERMINATION OF ERYTHROCYTE INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS (Naic/Kic --- p.36 / Chapter 3.3.1 --- Preparation of Erythrocytes --- p.36 / Chapter 3.3.2 --- Preparation of Haemolysates --- p.38 / Chapter 3.3.3 --- Determination of Sodium and Potassium Concentrations in Haemolysates --- p.38 / Chapter 3.3.4 --- Determination of Haemoglobin Concentration in Haemolysates --- p.38 / Chapter 3.3.5 --- Evaluation of Erythrocyte Intracellular Sodium and Potassium Concentrations --- p.39 / Chapter 3.4 --- DETERMINATION OF ERYTHROCYTE PASSIVE POTASSIUM EFFLUX --- p.39 / Chapter 3.4.1 --- Determination of Potassium Concentrations in Supernatant --- p.40 / Chapter 3.4.2 --- Evaluation of Passive Potassium Efflux --- p.40 / Chapter 3.5 --- DETERMINATION OF ERYTHROCYTE SODIUM-LITHIUM COUNTERTRANSPORT (SLC) AND LITHIUM-POTASSIUM COTRANSPORT (LPC) --- p.41 / Chapter 3.5.1 --- Lithium Loading --- p.42 / Chapter 3.5.2 --- Determination of Haematocrit --- p.42 / Chapter 3.5.3 --- Preparation of Haemolysates --- p.42 / Chapter 3.5.4 --- Determination of the Lithium Concentration in Haemolysates --- p.43 / Chapter 3.5.5 --- Determination of Lithium Efflux --- p.43 / Chapter 3.5.6 --- Evaluation of Lithium Efflux Rate --- p.43 / Chapter 3.5.7 --- Evaluation of Intracellular Lithium Concentration --- p.44 / Chapter 3.6 --- VALIDATION OF METHODOLOGY FOR DETERMINATION OF ERYTHROCYTE SODIUM TRANSPORT SYSTEMS --- p.45 / Chapter 3.6.1 --- Effect of Time Course of Lithium Efflux --- p.45 / Chapter 3.6.2 --- Intracellular Potassium Concentration and Its Effect on Ouabain- and Frusemide-Resistant Passive Potassium Efflux --- p.45 / Chapter 3.7 --- PRESERVATION OF ERYTHROCYTES FOR DETERMINATION OF SODIUM TRANSPORT SYSTEMS --- p.51 / Chapter 3.8 --- PRECISION OF THE METHOD --- p.51 / Chapter 3.9 --- STATISTICS --- p.52 / Chapter CHAPTER 4: --- RESULTS --- p.56 / Chapter 4.1 --- POPULATION CHARACTERISTICS --- p.57 / Chapter 4.2 --- ERYTHROCYTE INTRACELLULAR LITHIUM CONCENTRATIONS AFTER LITHIUM LOADING --- p.57 / Chapter 4.3 --- RELATIONSHIP BETWEEN ERYTHROCYTE ION TRANSPORT PARAMETERS AND OTHER VARIABLES --- p.58 / Chapter 4.4 --- ERYTHROCYTE SODIUM TRANSPORT SYSTEMS IN ESSENTIAL HYPERTENSION --- p.64 / Chapter 4.5 --- ERYTHROCYTE SODIUM TRANSPORT SYSTEMS IN PATIENTS WITH DIABETES MELLITUS --- p.64 / Chapter 4.5.1 --- NIDDM Patients without Hypertension --- p.64 / Chapter 4.5.2 --- NIDDM Patients with Hypertension --- p.65 / Chapter 4.5.3 --- NIDDM Patients with and without Hypertension --- p.65 / Chapter 4.6 --- ERYTHROCYTE SODIUM TRANSPORT SYSTEMS IN DIABETES MELLITUS PATIENTS WITH PROTEINURIA --- p.65 / Chapter 4.6.1 --- Clinical Features and Biochemistry Indices --- p.69 / Chapter 4.6.2 --- Ion Transport Systems and NIDDM Patients with Proteinuria --- p.69 / Chapter 4.7 --- EFFECTS OF TREATMENTS ON ERYTHROCYTE ION TRANSPORT SYSTEMS IN DIABETIC HYPERTENSIVE PATIENTS --- p.70 / Chapter 4.7.1 --- Effects of Diuretic Therapy --- p.70 / Chapter 4.7.2 --- Effects of Enalapril and Nifedipine Therapy --- p.74 / Chapter 4.7.3 --- Effects of Enalapril Therapy --- p.74 / Chapter 4.7.4 --- Effects of Nifedipine Therapy --- p.75 / Chapter 4.7.5 --- Comparison of the Effects of Enalapril and Nifedipine Therapy --- p.75 / Chapter CHAPTER 5: --- DISCUSSION --- p.81 / Chapter 5.1 --- SODIUM TRANSPORT IN ESSENTIAL HYPERTENSION --- p.82 / Chapter 5.1.1 --- Erythrocyte Sodium-Lithium Countertransport in Essential Hypertension --- p.82 / Chapter 5.1.2 --- Erythrocyte Sodium-Potassium Cotransport in Essential Hypertension --- p.86 / Chapter 5.1.3 --- Erythrocyte Intracellular Concentration of Sodiumin Essential Hypertension --- p.87 / Chapter 5.1.4 --- Erythrocyte Passive Potassium Efflux in Essential Hypertension --- p.90 / Chapter 5.2 --- SODIUM TRANSPORT SYSTEMS IN NON-INSULIN- DEPENDENT DIABETES MELLITUS (NIDDM) --- p.91 / Chapter 5.2.1 --- Sodium-Lithium Countertransport in Non-Insulin-Dependent Diabetes Mellitus --- p.91 / Chapter 5.2.2 --- Erythrocyte Lithium-Potassium Cotransport and Intracellular Sodium Concentration in Non-Insulin-Dependent Diabetes Mellitus --- p.93 / Chapter 5.3 --- EFFECT OF ANTIHYPERTENSIVE AGENTS ON ERYTHROCYTE SODIUM TRANSPORT SYSTEMS --- p.95 / REFERENCES --- p.98

The characteristics of the ion exchange mechanisms which regulate volume and pH in fish and amphibian erythrocytes were investigated and compared. Experiments were carried out under steady state conditions and also following adrenergic stimulation both in vivo and in vitro. Under steady state conditions, a decrease in extracellular pH caused an increase in the volume of rainbow trout erythrocytes, and a decrease in the intracellular pH. These pH-induced volume changes were mainly associated with movements of chloride across the chloride/bicarbonate exchange pathway. The sodium/proton exchange mechanism is quiescent at all pH's studied under steady state conditions. Beta adrenergic stimulation of rainbow trout erythrocytes promoted cell swelling and proton extrusion from the erythrocytes. Amiloride inhibited both the volume and pH changes associated with adrenergic stimulation indicating that this response is associated with an increase in the activity of the sodium/proton exchange mechanism on the erythrocyte membrane. The adrenergic swelling and pH responses are enhanced by a decrease in extracellular pH. An increase in bicarbonate concentration reduces the adrenergic pH response, but it is still significant even at 10 mM bicarbonate. DIDS markedly enhanced the beta adrenergic effect on the erythrocyte pH, but abolished the increase in erythrocyte volume. The adrenergic response was independent of temperature between 10 and 18°C. These results support a loosely coupled sodium/proton and chloride/bicarbonate exchange model for the adrenergic response in rainbow trout erythrocytes. The increases in erythrocyte pH and volume following adrenergic stimulation are associated with increases in the haemoglobin:oxygen affinity. The oxygen carrying capacity of the blood is, therefore, increased following adrenergic stimulation in rainbow trout. Carbon dioxide excretion, however, was not significantly affected by adrenergic stimulation. The functional significance of the adrenergic response of fish erythrocytes may be to offset the effects of the Root shift on the oxygen carrying capacity of the blood during acute changes in extracellular pH. In contrast to fish erythrocytes, the sodium/proton exchange mechanism in amphibian erythrocytes is active under steady state conditions. In the presence of bicarbonate movements, this exchange significantly affects the erythrocyte volume, but not the erythrocyte pH. Similar to fish erythrocytes, protons are passively distributed in amphibian erythrocytes under steady state conditions and in Donnan equilibrium with chloride ions. The erythrocyte volume also increases with decreases in extracellular pH as in fish erythrocytes, due to changes in the chloride distribution across the erythrocyte membrane. Adrenergic stimulation does not affect the volume or pH of amphibian erythrocytes either in vivo or in vitro. These animals, therefore, do not appear to regulate erythrocyte pH adrenergically. Amphibians are able to efficiently utilize oxygen stores via both central and peripheral shunting. In addition, the blood of these animals does not have a Root shift. Adrenergic regulation of erythrocyte pH in order to enhance oxygen transport during fluctuations in ambient and internal gas tensions, therefore, is probably less important than it would be in fish. / Science, Faculty of / Zoology, Department of / Graduate