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Ion exchange mechanisms for the control of volume and pH in fish and amphibian erythrocytes Tufts, Bruce Laurie 1987

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ION EXCHANGE MECHANISMS FOR THE CONTROL OF VOLUME AND PH IN FISH AND AMPHIBIAN ERYTHROCYTES By BRUCE L. TUFTS B.Sc. (Honours), A c a d i a U n i v e r s i t y , Nova S c o t i a , 1982 M . S c , A c a d i a U n i v e r s i t y , Nova S c o t i a , 1984 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA October 1987 (5) Bruce L. T u f t s , 1987 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of H - O O U O g Y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date " p e c . Q / g ? i i ABSTRACT The c h a r a c t e r i s t i c s of the i o n exchange mechanisms which r e g u l a t e volume and pH i n f i s h and amphibian erythrocytes were investigated and compared. Experiments were c a r r i e d out under steady s t a t e conditions and also following adrenergic stimulation both i n vivo and i n v i t r o . Under steady s t a t e c o n d i t i o n s , a decrease i n e x t r a c e l l u l a r pH caused an increase i n the volume of rainbow t r o u t e r y t h r o c y t e s , and a decrease i n the i n t r a c e l l u l a r pH. These pH-induced volume changes were mainly associated with movements of chloride across the chloride/bicarbonate exchange pathway. The sodium/proton exchange mechanism i s quiescent at a l l pH's studied under steady state conditions. Beta adrenergic s t i m u l a t i o n of rainbow t r o u t erythrocytes promoted c e l l swelling and proton extrusion from the erythrocytes. Amiloride i n h i b i t e d both the volume and pH changes a s s o c i a t e d with adrenergic stimulation i n d i c a t i n g that t h i s response i s associated with an increase i n the a c t i v i t y of the sodium/proton exchange mechanism on the erythrocyte membrane. The adrenergic swelling and pH responses are enhanced by a decrease i n e x t r a c e l l u l a r pH. An increase i n bicarbonate concentration reduces the adrenergic pH response, but i t i s s t i l l s i g n i f i c a n t even at 10 mM bicarbonate. DIDS markedly enhanced the beta adrenergic e f f e c t on the erythrocyte pH, but abolished the increase i n erythrocyte volume. The adrenergic response was independent of temperature between 10 and 18°C. These r e s u l t s support a loosely coupled sodium/proton and chloride/bicarbonate exchange model f o r the adrenergic response i n rainbow trout erythrocytes. The i n c r e a s e s i n e r y t h r o c y t e pH and volume f o l l o w i n g a d r e n e r g i c s t i m u l a t i o n are a s s o c i a t e d with increases i n the haemoglobin:oxygen a f f i n i t y . The oxygen carrying capacity of the blood i s , therefore, increased f o l l o w i n g a d r e n e r g i c s t i m u l a t i o n i n rainbow t r o u t . Carbon dioxide excretion, however, was not s i g n i f i c a n t l y a f f e c t e d by adrenergic s t i m u l a t i o n . The f u n c t i o n a l s i g n i f i c a n c e of the adrenergic response of f i s h erythrocytes may be to o f f s e t the e f f e c t s of the Root s h i f t on the oxygen carrying capacity of the blood during acute changes i n e x t r a c e l l u l a r pH. In c o n t r a s t to f i s h e r y t h r o c y t e s , the i v sodium/proton exchange mechanism i n amphibian erythrocytes i s active under steady state conditions. In the presence of bicarbonate movements, t h i s exchange s i g n i f i c a n t l y a f f e c t s the erythrocyte volume, but not the erythrocyte pH. Similar to f i s h erythrocytes, protons are passively d i s t r i b u t e d i n amphibian erythrocytes under steady state conditions and i n Donnan equilibrium with chloride ions. The erythrocyte volume also increases with decreases i n e x t r a c e l l u l a r pH as i n f i s h erythrocytes, due to changes i n the chloride d i s t r i b u t i o n across the erythrocyte membrane. Adrenergic stimulation does not a f f e c t the volume or pH of amphibian erythrocytes e i t h e r i n vivo or i n v i t r o . These animals, t h e r e f o r e , do not appear to regulate erythrocyte pH adrenergically. Amphibians are able to e f f i c i e n t l y u t i l i z e oxygen s t o r e s v i a both ce n t r a l and peripheral shunting. In addition, the blood of these animals does not have a Root s h i f t . Adrenergic regulation of erythrocyte pH i n order to enhance oxygen transport during fluctuations i n ambient and i n t e r n a l gas tensions, therefore, i s probably l e s s important than i t would be i n f i s h . V TABLE OF CONTENTS Page ABSTACT i i LIST OF TABLES v i i LIST OF FIGURES X ACKNOWLEDGEMENT x i LIST OF ABBREVIATIONS x i i GENERAL INTRODUCTION 1 GENERAL MATERIALS AND METHODS 12 SECTION I : FISH ERYTHROCYTES 19 OVERVIEW I 20 CHAPTER 1. CONTROL OF ERYTHROCYTE VOLUME AND PH 23 IN THE RAINBOW TROUT, SALMO GAIRDNERI I n t r o d u c t i o n 23 M a t e r i a l s and Methods 25 R e s u l t s 27 D i s c u s s i o n 42 Summary 4 6 CHAPTER 2. EFFECT OF BURST SWIMMING AND 48 ADRENALINE INFUSION ON OXYGEN CONSUMPTION AND CARBON DIOXIDE EXCRETION IN THE RAINBOW TROUT, SALMO GAIRDNERI I n t r o d u c t i o n 48 M a t e r i a l s and Methods 50 R e s u l t s 55 D i s c u s s i o n 65 Summary 70 CHAPTER 3. THE EFFECT OF CATECHOLAMINES ON 71 CHLORIDE/BICARBONATE EXCHANGE IN RAINBOW TROUT ERYTHROCYTES. v i I n t r o d u c t i o n 71 M a t e r i a l s and Methods 73 R e s u l t s 80 D i s c u s s i o n 89 Summary 94 SECTION I I : AMPHIBIAN ERYTHROCYTES 95 OVERVIEW I I 96 CHAPTER 4. THE EFFECTS OF FORCED ACTIVITY ON 99 CIRCULATING CATECHOLAMINES AND PH AND WATER CONTENT OF ERYTHROCYTES IN THE TOAD, BUFO MARINUS. I n t r o d u c t i o n 99 M a t e r i a l s and Methods 101 R e s u l t s 105 D i s c u s s i o n 115 Summary 121 CHAPTER 5. THE DISTRIBUTION OF PROTONS AND 122 CHLORIDE IONS I N AMPHIBIAN ERYTHROCYTES. I n t r o d u c t i o n 122 M a t e r i a l s and Methods 124 R e s u l t s 126 D i s c u s s i o n 136 Summary 140 CHAPTER 6. ION EXCHANGE MECHANISMS ON THE 141 ERYTHROCYTE MEMBRANE OF THE AQUATIC SALAMANDER, AMPHIUMA  TRIDACTYLUM. I n t r o d u c t i o n 141 M a t e r i a l s and Methods 143 R e s u l t s 145 D i s c u s s i o n 151 Summary 156 GENERAL DISCUSSION 157 REFERENCES 174 v i i LIST OF TABLES Table 1. E x t r a c e l l u l a r and i n t r a c e l l u l a r pH and the pH gradient across the erythrocyte membrane i n control c e l l s , i n amiloride-treated c e l l s , and i n c e l l s incubated i n the absence of sodium. Table 2. Changes i n the pH gradient across the 40 erythrocyte membrane induced by isoproterenol. Table 3. E f f e c t s of temperature on the 41 adre n e r g i c response of rainbow t r o u t erythrocytes. Table 4. E f f e c t of adrenaline, isoproterenol 88 and noradrenaline on the plasma pH, erythrocyte pH and the pH gradient of rainbow trout blood. Table 5. E f f e c t of forced a c t i v i t y on the 106 c i r c u l a t i n g adrenaline and noradrenaline l e v e l s i n the toad. Page 39 v i i i Table 6. E f f e c t of isoproterenol on the water 113 content, e x t r a c e l l u l a r pH, i n t r a c e l l u l a r pH and on the pH gradient of toad blood e q u i l i b r a t e d with 5% C0 2/95% a i r . Table 7. Overall means of d i s t r i b u t i o n r a t i o s 134 for chloride and for protons as a function of erythrocyte pH determined v i a the freeze-thaw method and by the DMO method. Table 8. Erythrocyte pH determined by the 135 freeze-thaw method, freeze-thaw method with s a l i n e b r i d g e , DMO d i s t r i b u t i o n method and predicted from the e x t r a c e l l u l a r pH and chloride ion d i s t r i b u t i o n . Table 9. E f f e c t s of adrenaline, DIDS and 146 amiloride on the water content, e x t r a c e l l u l a r pH, erythrocyte pH and on the pH gradient of Amphiuma tridactylum erythrocytes e q u i l i b r a t e d with 4% C0 2/96% a i r . Table 10. E f f e c t s of adrenaline, DIDS and 147 amiloride on the water content, e x t r a c e l l u l a r pH, erythrocyte pH and on the pH gradient of Amphiuma tridactvlum erythrocytes e q u i l i b r a t e d with 8% C0 2/92% a i r . Table 11. E f f e c t s of DIDS, amiloride, ouabain 148 and absence of e x t r a c e l l u l a r sodium on the water content, e x t r a c e l l u l a r pH, erythrocyte pH and on the pH g r a d i e n t of Amphiuma t r i d a c t y l u m erythrocytes e q u i l i b r a t e d with 4% C0 2/96% a i r . Table 12. Comparison of the erythrocyte pH 149 determined by the DM0 d i s t r i b u t i o n and the erythrocyte pH predicted from the e x t r a c e l l u l a r pH and the chloride d i s t r i b u t i o n i n Amphiuma  tridactvlum erythrocytes. Table 13. The presence or absence of the adr e n e r g i c response i n e r y t h r o c y t e s of f i s h versus the presence or absence of a Root s h i f t . 171 X LIST OF FIGURES Page Figure 1. E f f e c t of external pH on the c e l l u l a r 29 water conent of rainbow trout erythrocytes. Figure 2. Isoproterenol-induced increase i n 31 c e l l u l a r water content of rainbow t r o u t erythrocytes. Figure 3. I n t r a c e l l u l a r pH versus e x t r a c e l l u l a r 33 pH of rainbow trout erythrocyte suspensions. Figure 4. E f f e c t s of isoproterenol on the 35 i n t r a c e l l u l a r versus e x t r a c e l l u l a r pH of rainbow trout blood. Figure 5. E x t r a c e l l u l a r pH versus i n t r a c e l l u l a r 37 pH i n DIDS-treated rainbow trout erythrocytes before and a f t e r the a d d i t i o n of 10~ 5 M isoproterenol. Figure 6. Oxygen consumption and carbon dioxide 57 excretion of rainbow trout swimming 40 cm s e c - 1 before and a f t e r burst swimming. x i Figure 7. Respiratory exchange r a t i o of rainbow 59 trout swimming 40 cm s e c - 1 before and a f t e r burst swimming. Figure 8. Oxygen consumption and carbon dioxide 61 e x c r e t i o n of rainbow t r o u t before and a f t e r i nfusion of adrenaline. Figure 9. Respiratory exchange r a t i o of rainbow 63 trout before and a f t e r infusion of adrenaline. Figure 10. The e f f e c t of chloride (0-200 mM) on 77 carbon d i o x i d e e v o l u t i o n from rainbow t r o u t erythrocytes. Figure 11. E f f e c t of changing haematocrit on 82 carbon d i o x i d e e v o l u t i o n from rainbow t r o u t erythrocytes. Figure 12. The e f f e c t of SITS, DIDS or 84 acetazolamide on carbon dioxide evolution from rainbow trout erythrocytes. x i i F i g u r e 13. The e f f e c t of a d r e n a l i n e , 86 i s o p r o t e r e n o l and n o r a d r e n a l i n e on carbon d i o x i d e e v o l u t i o n from rainbow t r o u t erythrocytes. Figure 14. Haematocrit values i n eleven toads 108 before forced a c t i v i t y and at 0, 0.5, 1 and 4 h following forced a c t i v i t y . Figure 15. Haematocrit and c e l l water content 110 i n f i v e toads before and immediately following forced a c t i v i t y . Figure 16. Whole blood and erythrocyte pH i n 11 112 toads before forced a c t i v i t y and at 0, 0.5, 1 and 4 h following forced a c t i v i t y . Figure 17. Erythrocyte water content of toad 128 blood as a function of e x t r a c e l l u l a r pH. Figure 18. E f f e c t s of e x t r a c e l l u l a r pH on 130 proton and chloride d i s t r i b u t i o n r a t i o s of toad blood. Figure 19. E f f e c t s of e x t r a c e l l u l a r pH on 132 erythrocyte pH of toad blood. x i i i Figure 20. A: Adrenergically stimulated f i s h 160 erythrocyte. B: Schematic diagram of beta-adrenergic regulation of erythrocyte pH during an e x t r a c e l l u l a r acidosis i n f i s h erythrocytes. Figure 21. A: Adrenergically stimulated 167 amphibian erythrocyte. B: Schematic diagram of changes i n e r y t h r o c y t e pH d u r i n g an e x t r a c e l l u l a r acidosis i n beta-adrenergically stimulated amphibian erythrocytes. x i v ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k D r. D.J. R a n d a l l , my s u p e r v i s o r , f o r h i s g u i d a n c e d u r i n g t h i s w ork. I n a d d i t i o n , I would l i k e t o t h a n k Dr. M. Nikinmaa and Dr. J.F . S t e f f e n s e n who c o l l a b o r a t e d on work i n c h a p t e r s 1, 2 and 6. I am g r a t e f u l l y i n d e b t e d t o Dr. R.G. B o u t i l i e r f o r h i s a s s i s t a n c e d u r i n g t h e p r e p a r a t i o n o f t h i s t h e s i s . F i n a n c i a l s u p p o r t f o r t h i s w o rk was p r o v i d e d by N.S.E.R.C. LIST OF ABBREVIATIONS xv DIDS 4 , 4 - D i i s o t h i o c y a n o s t i l b e n e - 2 , 2 - D i s u l f o n i c a c i d . 2,4-D.N.P. 2 , 4 - D i n i t r o p h e n o l . DMO D i m e t h y l o x a z o l i d i n e - 2 , 4 - d i o n e . p H e E x t r a c e l l u l a r pH. pH^ E r y t h r o c y t e pH. RE R e s p i r a t o r y exchange r a t i o . SITS 4 - a c e t a m i d o - 4 - i s o - t h i o c y a n a t o - s t i l b e n e - 2 , 2 - d i s u l p h o n i c a c i d . V C 0 2 Carbon d i o x i d e e x c r e t i o n . V 0 2 Oxygen consumption. 1 GENERAL INTRODUCTION I n most v e r t e b r a t e s p e c i e s , more t h a n 90% o f t h e t o t a l oxygen c o n t e n t o f t h e b l o o d i s bound t o haemoglobin and changes i n haemoglobin oxygen a f f i n i t y have a l a r g e e f f e c t on t h e amount o f oxygen d e l i v e r e d t o t h e t i s s u e s a t any g i v e n b l o o d f l o w . I t i s n o t s u r p r i s i n g , t h e r e f o r e , t h a t v e r t e b r a t e haemoglobin systems p o s s e s s a d a p t a t i o n s t o r e g u l a t e haemoglobin oxygen a f f i n i t y and e n s u r e a d e q u a t e d e l i v e r y o f o x y g e n t o a e r o b i c a l l y r e s p i r i n g t i s s u e s . The a d a p t a t i o n s w h i c h r e g u l a t e h a e m o g l o b i n o x y g e n a f f i n i t y i n t h e n o n n u c l e a t e d e r y t h r o c y t e s o f mammals a r e w e l l known (Bauer, 1974). The m a j o r i t y o f v e r t e b r a t e s p e c i e s , h o w e v e r , p o s s e s s n u c l e a t e d e r y t h r o c y t e s . R e l a t i v e t o t h e i r n o n n u c l e a t e d c o u n t e r p a r t s , t h e a d a p t a t i o n s r e g u l a t i n g h a e m o g l o b i n o x y g e n a f f i n i t y i n n u c l e a t e d e r y t h r o c y t e s a r e p o o r l y d e s c r i b e d and i n p a r t i c u l a r , v e r y l i t t l e i s known about t h e f u n c t i o n a l d i v e r s i t y i n t h e s e systems. Haemoglobin: The r e s p i r a t o r y pigment found i n t h e c i r c u l a t o r y system o f v e r t e b r a t e s i s haemoglobin. I t s b a s i c f u n c t i o n i s s i m i l a r t o t h a t o f o t h e r c i r c u l a t i n g r e s p i r a t o r y p i g m e n t s w h i c h i s t o co m b i n e w i t h o x y g e n a t t h e r e s p i r a t o r y s u r f a c e and u n l o a d i t a t t h e t i s s u e s . The 2 c a p a c i t y o f a s p e c i f i c h a e m o g l o b i n t o p e r f o r m t h i s f u n c t i o n w i l l depend on i t s a f f i n i t y f o r oxygen. T h i s a f f i n i t y f o r o x y g e n must be h i g h enough t o a l l o w e f f i c i e n t l o a d i n g a t t h e r e s p i r a t o r y s u r f a c e b u t not so h i g h t h a t i t i m p a i r s e f f i c i e n t r e l e a s e o f oxygen a t t h e t i s s u e s . I t has been demonstrated t h a t t h e i n t r i n s i c a f f i n i t y o f haemoglobin f o r oxygen may v a r y depending on e i t h e r t h e s p e c i e s examined o r t h e p a r t i c u l a r haemoglobin component i n m u l t i p l e haemoglobin systems (see r e v i e w s by Johansen and Weber, 1976; R i g g s , 1979; Powers, 1980; Hochachka and Somero, 1984). I t h a s o f t e n b e e n s u g g e s t e d t h a t h a e m o g l o b i n oxygen a f f i n i t y d e c r e a s e s w i t h i n c r e a s i n g dependence on a e r i a l gas exchange (McCutcheon and H a l l , 1937; L e n f a n t and Johansen, 1967; Johansen e t a l . , 1979a,b). Powers (1980) p o i n t s out t h a t , among f i s h , t h i s c o n c l u s i o n may o n l y be v a l i d f o r c l o s e l y r e l a t e d s p e c i e s . A d d i t i o n a l t r e n d s i n haemoglobin oxygen a f f i n i t y a r e a l s o a p p a r e n t . F i s h w h i c h l i v e i n a low oxygen environment ( e x c l u d i n g a i r b r e a t h e r s ) g e n e r a l l y h a v e h a e m o g l o b i n s w i t h h i g h o x y g e n a f f i n i t i e s w h i l e p e l a g i c f i s h l i v i n g i n an e n v i r o n m e n t w i t h a c o n s t a n t h i g h o x y g e n l e v e l h a v e haemoglobins w i t h l o w e r oxygen a f f i n i t i e s ( R i g g s , 1970; 3 Powers, 1980). There i s a l s o a c o r r e l a t i o n between an organisms m e t a b o l i s m and t h e a f f i n i t y o f i t s haemoglobin f o r oxygen. A c t i v e f i s h such as t r o u t and m a c k e r e l t e n d t o have l o w e r haemoglobin oxygen a f f i n i t i e s t h a n l e s s a c t i v e f i s h ( R i g g s , 1970) . T h i s i s t h o u g h t t o be an a d a p t a t i o n w h i c h i n c r e a s e s t h e u n l o a d i n g o f oxygen a t t h e t i s s u e s . H a e m o g l o b i n s w i t h a v e r y l o w t e m p e r a t u r e s e n s i t i v i t y h a v e a l s o b e e n f o u n d i n a v a r i e t y o f v e r t e b r a t e s . B a r c r o f t and K i n g (1909) f i r s t d e monstrated t h a t an i n c r e a s e i n t e m p e r a t u r e w i l l d e c r e a s e t h e a f f i n i t y o f mammalian h a e m o g l o b i n f o r o x y g e n . T h i s t e m p e r a t u r e e f f e c t on haemoglobin oxygen a f f i n i t y has s i n c e been found i n s e v e r a l s p e c i e s o f p o i k i l o t h e r m i c v e r t e b r a t e s ( G i l l e n and R i g g s , 1971, 1972: Greenwald, 1974; Mied and Powers, 1977). I n t h e s e a n i m a l s , t h e r e s u l t i n g problems o f oxygen u p t a k e a r e compounded by t h e f a c t t h a t t h e r i s i n g t e m p e r a t u r e a l s o i n c r e a s e s b o t h m e t a b o l i s m and t h e r e s u l t i n g t i s s u e oxygen r e q u i r e m e n t s . F u r t h e r m o r e , w a t e r b r e a t h e r s f a c e an a d d i t i o n a l problem because o f t h e r e d u c t i o n i n t h e s o l u b i l i t y o f oxygen i n w a t e r a t h i g h e r t e m p e r a t u r e s . I t h a s b e e n shown, however, t h a t i n d i v i d u a l haemoglobins from some s p e c i e s s uch as t h e t r o u t ( B r u n o r i , 1975), chum salmon (Hashimoto e t a l . , 1960) and t u n a ( R o s s i - F a n e l l i and A n t o n i n i , 1960) 4 have a reduced t e m p e r a t u r e s e n s i t i v i t y . The a c c e p t e d v i e w i s t h a t t h e s e t e m p e r a t u r e i n s e n s i t i v e haemoglobins a r e a d a p t a t i o n s found i n e u r y t h e r m a l s p e c i e s e x p e r i e n c i n g l a r g e f l u c t u a t i o n s i n body t e m p e r a t u r e ( J o h a n s e n and Weber, 1976). Most v e r t e b r a t e s a l s o p o s s e s s more t h a n one t y p e o f h a e m o g l o b i n ( H o c h a c h k a and Somero, 1973) . T h i s t e n d e n c y i s more p r o n o u n c e d i n f i s h , r e p t i l e s and a m p h i b i a n s t h a n i n mammals and b i r d s ( G r a t z e r and A l l i s o n , 1960). These d i f f e r e n t haemoglobins can have r a d i c a l l y d i f f e r e n t oxygen b i n d i n g p r o p e r t i e s a l l o w i n g organisms t o m a i n t a i n oxygen t r a n s p o r t i n t h e f a c e o f c h a n g i n g o x y g e n r e q u i r e m e n t s o r a v a i l a b i l i t y . The r e l a t i v e p r o p o r t i o n s o f d i f f e r e n t haemoglobin components may a l s o change d u r i n g t h e l i f e c y c l e o f an organism (Herner and F r i e d e n , 1961; W i l k e n s , 1968). F o r example, G i l e s and R a n d a l l (1980) found t h a t i n coho salmon, 0. k i s u t c h . h a e m o g l o b i n f r o m t h e f r y h a d a much h i g h e r oxygen a f f i n i t y t h a n t h e a d u l t haemoglobin. D i f f e r e n c e s i n t h e o x y g e n r e q u i r e m e n t s o f organisms and i n t h e a v a i l a b l i t y o f e n v i r o n m e n t a l oxygen have r e s u l t e d i n t h e e v o l u t i o n o f b o t h i n t e r s p e c i f i c and i n t r a s p e c i f i c v a r i a t i o n i n t h e i n t r i n s i c a f f i n i t y o f 5 h a e m o g l o b i n f o r o x y g e n . G e n e r a l l y , t h e i n t r i n s i c a f f i n i t y o f s p e c i f i c haemoglobins f o r oxygen a r e s u i t e d t o e f f i c i e n t l y e x t r a c t oxygen from t h e environment and d e l i v e r i t t o t h e r e s p i r i n g t i s s u e s . The oxygen a f f i n i t y o f most haemoglobins i s a l s o s e n s i t i v e t o changes i n t h e p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h e h a e m o g l o b i n e n v i r o n m e n t , h o w e v e r , r e p r e s e n t i n g a n o t h e r l e v e l o f a d a p t a t i o n and t h e r e f o r e v a r i a t i o n i n h a e m o g l o b i n systems. M o d u l a t i o n o f Haemoglobin Oxygen A f f i n i t y : I n r e s p o n s e t o c h a n g i n g demands on t h e oxygen t r a n s p o r t system, haemoglobin oxygen a f f i n i t y may be a d j u s t e d a t t h e c e l l u l a r l e v e l by a l t e r i n g t h e c o n c e n t r a t i o n o f c e r t a i n o r g a n i c p h o s p h a t e compounds w i t h i n t h e e r y t h r o c y t e . These a r e known as n e g a t i v e m o d u l a t o r s o f haemoglobin oxygen a f f i n i t y (Hochachka and Somero, 1973) s i n c e d e c r e a s e s i n t h e c e l l u l a r c o n c e n t r a t i o n s o f t h e s e compounds d e c r e a s e s t h e a f f i n i t y o f haemoglobin f o r oxygen. I n mammalian e r y t h r o c y t e s , t h e o r g a n i c p h o s p h a t e compound w h i c h m o d u l a t e s h a e m o g l o b i n o x y g e n a f f i n i t y i s 2 , 3 - d i p h o s p h o g l y c e r a t e (2,3-DPG; Benesch and Benesch, 1967; Bunn, 1980). The d i s t r i b u t i o n and c o n c e n t r a t i o n s o f t h e o r g a n i c phosphate c o n s t i t u e n t s o f o t h e r v e r t e b r a t e e r y t h r o c y t e s a r e 6 summarized i n a r e v i e w by B a r t l e t t (1980). I n o s i t o l p e ntaphosphate ( I P 5 ) and adenosine t r i p h o s p h a t e (ATP) a r e t h e main i n o r g a n i c phosphates i n b o t h b i r d and r e p t i l e e r y t h r o c y t e s . I n a m p h i b i a n s , t h e most common e r y t h r o c y t i c o r g a n i c phosphates a r e ATP and 2,3-DPG, and i n f i s h , ATP and g u a n o s i n e t r i p h o s p h a t e (GTP) a r e b e l i e v e d t o be i m p o r t a n t i n m o d u l a t i n g haemoglobin oxygen a f f i n i t y . An i n c r e a s e i n e i t h e r t h e p r o t o n o r t h e c a r b o n d i o x i d e p a r t i a l p r e s s u r e o f t h e haemoglobin environment w i l l a l s o cause a r e d u c t i o n i n t h e haemoglobin oxygen a f f i n i t y . The r e d u c t i o n i n a f f i n i t y caused by p r o t o n s i s known as t h e Bohr e f f e c t o r Bohr s h i f t ( D e j o u r s , 1981) . C a r b o n d i o x i d e c a u s e s a r e d u c t i o n i n a f f i n i t y b o t h i n d i r e c t l y by i t s e f f e c t on pH and d i r e c t l y by r e a c t i n g w i t h haemoglobin t o form carbamino compounds (see r e v i e w by R o u g h t o n , 1 9 7 0 ) . A t t h e r e s p i r i n g t i s s u e s , t h e c o m b i n e d e f f e c t s o f p r o t o n s and c a r b o n d i o x i d e , end p r o d u c t s o f r e s p i r a t i o n , on haemoglobin oxygen a f f i n i t y f a c i l i t a t e s t h e u n l o a d i n g o f oxygen. S i m i l a r l y , a t t h e gas exchange o r g a n , removal o f c a r b o n d i o x i d e from t h e b l o o d f a c i l i t a t e s t h e uptake o f oxygen. R o o t and I r v i n g (1943) d e m o n s t r a t e d t h a t a 7 r e d u c t i o n i n t h e pH o f t h e haemoglobin environment i n v i t r o a l s o l o w e r e d t h e oxygen c a p a c i t y o f t h e b l o o d i n t e l e o s t f i s h . T h i s 'Root' e f f e c t i s i m p o r t a n t i n t h e d e l i v e r y o f oxygen t o t h e swim b l a d d e r (Berg and S t e e n , 1968) and p o s s i b l y a l s o t o t h e r e t i n a l t i s s u e i n some f i s h ( W i t t e n b e r g and W i t t e n b e r g , 1974). Such an e f f e c t , h o w e v e r , may a l s o l i m i t d e l i v e r y o f o x y g e n t o t h e r e s p i r i n g t i s s u e s under c o n d i t i o n s o f low b l o o d pH and, t h e r e f o r e , l i m i t a e r o b i c performance. J o n e s (1971), f o r example, showed t h a t a 30-40% r e d u c t i o n i n b l o o d oxygen c o n t e n t caused by h y p o x i a l e d t o a 30% r e d u c t i o n i n maximum s u s t a i n a b l e swimming speed i n t h e r a i n b o w t r o u t , Salmo q a i r d n e r i . I t has been h y p o t h e s i z e d t h a t t h e Root e f f e c t may even be a f a c t o r c o n t r i b u t i n g t o t h e d e a t h o f h y p e r a c t i v e f i s h ( B l a c k , 1958). Some s p e c i e s o f f i s h h a v i n g m u l t i p l e haemoglobin c o m p o n e n t s have p a r t i c u l a r c o mponents w h i c h a r e i n s e n s i t i v e t o pH (Hashimoto e t a l . , 1960; Powers, 1972; B r u n o r i , 1975). T h i s i n t r i n s i c a d a p t a t i o n may a c t as a s a f e g u a r d t o m a i n t a i n a minimum l e v e l o f oxygen t r a n s p o r t i m m e d i a t e l y f o l l o w i n g h y p e r a c t i v i t y i n t h e s e s p e c i e s ( H o c h a c h k a and Somero, 1 9 8 4 ) . The r e d u c t i o n i n t h e a f f i n i t y o f t h e pH s e n s i t i v e components, however, would s t i l l r e s u l t i n a r e d u c t i o n i n t o t a l a e r o b i c c a p a c i t y . 8 I n summary, t h e e f f e c t s o f o r g a n i c p h o s p h a t e s , hydrogen i o n s and c a r b o n d i o x i d e on haemoglobin oxygen a f f i n i t y r e p r e s e n t an i m p o r t a n t l e v e l f o r a d a p t a t i o n o f v e r t e b r a t e oxygen t r a n s p o r t systems. A t t h i s l e v e l , t h e i n t r i n s i c a f f i n i t y o f haemoglobin f o r oxygen may be r e -a d j u s t e d a c c o r d i n g t o e n v i r o n m e n t a l o r o r g a n i s m i c c h a l l e n g e t o enhance i t s a b i l i t y t o t r a n s p o r t oxygen. B o h r and R o o t s h i f t s a l s o h a v e t h e p o t e n t i a l t o n e g a t i v e l y i n f l u e n c e oxygen t r a n s p o r t i n some s p e c i e s by r e d u c i n g t h e t o t a l oxygen c a r r y i n g c a p a c i t y o f t h e b l o o d . Some a n i m a l s , h o w e v e r, a p p e a r t o h a v e e v o l v e d e r y t h r o c y t i c a d a p t a t i o n s t o a m e l i o r a t e t h i s p o t e n t i a l p roblem. R e g u l a t i o n o f E r y t h r o c y t e pH: B u r s t swimming i n t h e rainbow t r o u t (Primmett e t a l . , 1986) and t h e s t r i p e d b ass (Nikinmaa e t a l . , 1984) c auses a d e c r e a s e i n t h e e r y t h r o c y t i c transmembrane pH g r a d i e n t ( a s t h e e x t r a c e l l u l a r pH f a l l s , t h e i n t r a c e l l u l a r pH i s m a i n t a i n e d o r s l i g h t l y i n c r e a s e d ) . A s i m i l a r r e s p o n s e o c c u r s i n v i v o f o l l o w i n g a c i d i n f u s i o n i n r a i n b o w t r o u t ( B o u t i l i e r e t a l . , 1986b) o r i n v i t r o , upon a d d i t i o n o f c a t e c h o l a m i n e s t o e r y t h r o c y t e s (Nikinmaa and H u e s t i s , 1984; Heming e t a l . , 1987). I t has been 9 c o n c l u d e d t h a t b e t a - a d r e n e r g i c s t i m u l a t i o n o f t h e s e e r y t h r o c y t e s r e s u l t s i n a n e t l o s s o f p r o t o n s from t h e e r y t h r o c y t e . T h i s r e sponse i s t h o u g h t t o h e l p m a i n t a i n b l o o d o x y g e n c a r r y i n g c a p a c i t y i n t h e p r e s e n c e o f a plasma a c i d o s i s (Nikinmaa e t a l . , 1984; Primmett e t a l . . 1986) . The a d d i t i o n o f c a t e c h o l a m i n e s t o rainbow t r o u t e r y t h r o c y t e s i n v i t r o n ot o n l y causes a n e t l o s s o f p r o t o n s from t h e e r y t h r o c y t e s , b u t a l s o a pronounced c e l l s w e l l i n g ( B a r o i n e t a l . , 1984a,b; Nikinmaa and H u e s t i s , 1984 ; Heming e t a l . . , 1 9 8 7 ) . The c e l l s w e l l i n g i s a p p a r e n t l y due t o an i n c r e a s e i n e r y t h r o c y t e sodium and c h l o r i d e l e v e l s w i t h w a t e r f o l l o w i n g p a s s i v e l y ( B a r o i n e t a l . , 1984a; Heming e t a l . , 1987). The e x a c t mechanism by w h i c h a d r e n e r g i c s t i m u l a t i o n e l e v a t e s t h e e r y t h r o c y t e pH and w a t e r c o n t e n t , however, have n o t been d e t e r m i n e d . The pH o f lamprey e r y t h r o c y t e s i s a l s o r e g u l a t e d , b u t t h e c h a r a c t e r i s t i c s o f pH r e g u l a t i o n i n t h e s e e r y t h r o c y t e s a r e q u i t e d i f f e r e n t t h a n i n t e l e o s t f i s h . I n t h e lamprey, p r o t o n s and c h l o r i d e i o n s a r e n o t p a s s i v e l y d i s t r i b u t e d a s i n most o t h e r v e r t e b r a t e s ( N i k i n m a a , 1986). I n s t e a d , t h e r e i s a g r a d i e n t f o r p r o t o n s w h i c h i s a c t i v e l y m a i n t a i n e d s u c h t h a t t h e e r y t h r o c y t e pH i s h i g h e r t h a n t h e plasma pH i n most i n s t a n c e s (Nikinmaa and 10 Weber, 1984; Nikinmaa, 1986). E v i d e n t l y , t h i s g r a d i e n t i s m a i n t a i n e d by h a v i n g a c o n t i n u o u s l y and h i g h l y a c t i v e s o d i u m / p r o t o n exchange mechanism (Nikinmaa e t a l . , 1986) c o u p l e d t o a c h l o r i d e / b i c a r b o n a t e e x c h a n g e r w i t h e x t r e m e l y low l e v e l s o f a c t i v i t y (Nikinmaa, p e r s . comm.). I n d e e d , N i k i n m a a (1986) h a s s u g g e s t e d t h a t t h e f u n c t i o n a l s i g n i f i c a n c e o f pH r e g u l a t i o n i n l a m p r e y e r y t h r o c y t e s i s t o o f f s e t t h e l a r g e B o h r e f f e c t o f lamprey haemoglobin d u r i n g changes i n e x t r a c e l l u l a r pH. A v i a n e r y t h r o c y t e s a l s o e x h i b i t s e v e r a l u n i q u e c h a r a c t e r i s t i c s . These c e l l s do not have a sodium/proton exchange mechanism c a p a b l e o f i n f l u e n c i n g t h e volume o r pH o f t h e c e l l (Nikinmaa and H u e s t i s , 1984) . However, t h e r e i s a s o d i u m / p o t a s s i u m / c h l o r i d e c o t r a n s p o r t system on t h e membrane o f a v i a n e r y t h r o c y t e s w h i c h r e g u l a t e s t h e c e l l volume ( P a l f r e y and Greengard, 1981). W h i l e t h i s mechanism i s s e n s i t i v e t o b e t a - a d r e n e r g i c s t i m u l a t i o n , i t w i l l o n l y cause t h e e r y t h r o c y t e s t o s w e l l i n t h e p r e s e n c e o f e l e v a t e d e x t r a c e l l u l a r p o t a s s i u m and i t d o e s n o t i n f l u e n c e t h e pH o f t h e c e l l ( R i d d i c k e t a l . , 1971; P a l f r e y and Greengard, 1981; Nikinmaa and H u e s t i s , 1984). The i o n i c c h a r a c t e r i s t i c s o f volume r e g u l a t i o n h a v e a l s o been d e s c r i b e d i n a m p h i b i a n e r y t h r o c y t e s . 11 A l t h o u g h t h e s e e r y t h r o c y t e s p o s s e s s a s o d i u m / p r o t o n exchange mechanism w h i c h i s c a p a b l e o f i n f l u e n c i n g t h e c e l l volume ( C a l a , 1980, 1985; Rudolph and Greengard, 1980; P a l f r e y and Greengard, 1981), t h e a b i l i t y o f t h i s exchanger t o i n f l u e n c e t h e e r y t h r o c y t e pH has n o t been i n v e s t i g a t e d . I t i s p o s s i b l e , t h e r e f o r e , t h a t t h i s exchanger a l s o r e g u l a t e s e r y t h r o c y t e pH as i n f i s h and lamprey e r y t h r o c y t e s . Summary: The p r e c e d i n g d i s c u s s i o n has a t t e m p t e d t o o u t l i n e t h e documented i n t e r - and i n t r a s p e c i f i c v a r i a t i o n i n t h e o x y g e n t r a n s p o r t c h a r a c t e r i s t i c s o f n u c l e a t e d e r y t h r o c y t e s . I t has r e c e n t l y been demonstrated t h a t t h e i n t e r s p e c i f i c v a r i a t i o n must a l s o i n c l u d e d i f f e r e n c e s i n i o n e x c h a n g e mechanisms on t h e e r y t h r o c y t e membrane. These mechanisms have t h e p o t e n t i a l t o i n f l u e n c e t h e oxygen, t r a n s p o r t c h a r c t e r i s t i c s o f t h e s e c e l l s by a l t e r i n g t h e c h e m i c a l s t a t e o f t h e h a e m o g l o b i n environment. The h y p o t h e s i s o f t h i s s t u d y i s t h a t t h e i o n exchange mechansims on t h e e r y t h r o c y t e membrane o f f i s h and amphibian e r y t h r o c y t e s w i l l each e x h i b i t u n i q u e a d a p t i v e f e a t u r e s whose f u n c t i o n a l s i g n i f i c a n c e w i l l be a t t r i b u t a b l e t o b o t h e n v i r o n m e n t a l and e v o l u t i o n a r y p r e s s u r e s . 12 GENERAL MATERIALS AND METHODS A n i m a l s and S u r g i c a l P r o c e d u r e s : Rainbow t r o u t (Salmo q a i r d n e r i ) , w e i g h i n g between 285 and 700g were o b t a i n e d from t h e Sun V a l l e y T r o u t Farm ( M i s s i o n , B.C.) and m a i n t a i n e d i n l a r g e o u t d o o r t a n k s . Water t e m p e r a t u r e v a r i e d from 10°C t o 15°C. The f i s h were r e g u l a r l y f e d and i n good h e a l t h . I n e x p e r i m e n t s w h i c h r e q u i r e d c a n n u l a t e d t r o u t , t h e a n i m a l s were a n a e s t h e t i z e d i n an a e r a t e d and pH b a l a n c e d s o l u t i o n o f t r i c a i n e methane s u l p h o n a t e (66.7 mg L " 1 MS-222 and 133.3 mg L - 1 NaHC0 3). The c a n n u l a t i o n was t h e n p e r f o r m e d on a s u r g i c a l t a b l e where a l i g h t e r dose of t h e a e r a t e d a n a e s t h e t i c s o l u t i o n (50 mg L - 1 MS-222 and 100 mg L - 1 NaHC0 3) was r e c i r c u l a t e d o v e r t h e g i l l s . A c a t h e t e r ( S o v e r e i g n i n d w e l l i n g c a n i n e c a t h e t e r , 2 i n c h , 18 gauge) was used t o make a b l i n d p u n c t u r e i n t o t h e d o r s a l a o r t a t h r o u g h t h e mouth o f t h e f i s h . The p u n c t u r e was made on a 45 degree a n g l e ( i n t h e c a u d a l d i r e c t i o n ) a t t h e m i d l i n e o f t h e b r a n c h i a l b a s k e t between t h e f i r s t and second g i l l a r c h e s . A c a n n u l a o f p o l y e t h y l e n e t u b i n g (PE-50) was f e d i n t o t h e a o r t a t h r o u g h t h e i m p l a n t e d c a t h e t e r . The c a n n u l a was t h e n l e d out o f t h e r o o f o f t h e mouth v i a a f l a n g e d s e c t i o n o f t u b i n g ( P E - 2 0 0 ) . 13 F o l l o w i n g t h e c a n n u l a t i o n , t h e f i s h was r e c o v e r e d i n a l i g h t p r o o f Perspex box f o r a t l e a s t 48h p r i o r t o t h e ex p e r i m e n t . The c a n n u l a was f i l l e d w i t h h e p a r i n i z e d (20 U.S.P. u n i t s m L - 1 ) C o r t l a n d s a l i n e ( W o l f , 1963) and f l u s h e d d a i l y i n o r d e r t o m a i n t a i n p a t e n c y . I n c e r t a i n i n s t a n c e s , f i s h b l o o d was a l s o o b t a i n e d v i a c a r d i a c o r c a u d a l p u n c t u r e . I n t h e s e i n s t a n c e s , t h e f i s h were l i g h t l y a n a e s t h e t i z e d i n t h e MS-222 s o l u t i o n o r stunned by a blow t o t h e head. B l o o d was t h e n s l o w l y withdrawn i n t o h e p a r i n i z e d s y r i n g e s i n s e r t e d v e n t r a l l y i n t o t h e c a u d a l v e s s e l o r t h e h e a r t . A d u l t t o a d s , Bufo ma r i n u s . o f b o t h sexes (250-500g) were o b t a i n e d from C h a r l e s D. S u l l i v a n Co. I n c . ( N a s h v i l l e , TN, U.S.A.). The a n i m a l s were k e p t i n l a r g e f i b e r g l a s s a q u a r i a w i t h 2-4 cm o f d e c h l o r i n a t e d t a p wat e r a t t h e e x p e r i m e n t a l t e m p e r a t u r e (22°C) f o r a t l e a s t a week p r i o r t o t h e e x p e r i m e n t s . Toads were a n a e s t h e t i z e d by immersion i n an MS-222 s o l u t i o n (1.5 g L - 1 MS-222 and 3 g L - 1 NaHC0 3) b e f o r e s u r g e r y . The f e m o r a l c a n n u l a t i o n i n v o l v e d making a 2-3 cm i n c i s i o n i n t h e d o r s a l s k i n o f t h e r i g h t l e g and e x p o s i n g t h e f e m o r a l a r t e r y between t h e t r i c e p s f e m o r i s and semimembranous mu s c l e s . A s e c t i o n o f t h e v e s s e l was clamped o f f and a s m a l l h o l e was made downstream from t h e clamp. P o l y e t h y l e n e t u b i n g (PE-60) 14 was i n s e r t e d v i a t h i s h o l e upstream a p p r o x i m a t e l y 1-2 cm. T h i s c a n n u l a was f i l l e d a t a l l t i m e s w i t h h e p a r i n i z e d (125 U.S.P. u n i t s mL - 1) MacKenzie's amphibian s a l i n e (de l a Lande e t a l . , 1962) t o p r e v e n t c l o t t i n g . Once i n p l a c e , t h e c a n n u l a was s e c u r e d t o t h e d o r s a l s k i n and s u r r o u n d i n g m u s c u l a t u r e w i t h s u r g i c a l s u t u r e . F o l l o w i n g s u r g e r y , t h e a n i m a l s were t r a n s f e r r e d t o l i g h t - p r o o f P e r s p e x boxes w i t h 500 mL o f d e c h l o r i n a t e d t a p w a t e r and a l l o w e d t o r e c o v e r f o r 24h. S p e c i m e n s o f Amphiuma t r i d a c t v l u m (450-1600g) were a l s o o b t a i n e d from C h a r l e s D. S u l l i v a n Co. I n c . ( N a s h v i l l e , TN, U.S.A.). The salamanders were k e p t i n l a r g e f i b e r g l a s s a q u a r i a a t 25°C f o r a t l e a s t a week p r i o r t o t h e e x p e r i m e n t s . A n i m a l s were a n a e s t h e t i z e d by immersion i n an MS-222 s o l u t i o n (5 g L - 1 MS-222 and 10 g L - 1 NaHC0 3). A m i d - v e n t r a l i n c i s i o n was made and a c a n n u l a o f p o l y e t h y l e n e t u b i n g (PE-50) was i n s e r t e d i n t o one o f t h e c e l i a c a r t e r i e s . The c a n n u l a was pushed f o r w a r d i n t o t h e d o r s a l a o r t a a p p r o x i m a t e l y 10 - 15 cm, t i e d o c c l u s i v e l y i n t o t h e c e l i a c a r t e r y and s e c u r e d t o t h e s u r r o u n d i n g m u s c u l a t u r e w i t h s u r g i c a l s u t u r e . The i n c i s i o n was t h e n c l o s e d w i t h s u t u r e and t h e a n i m a l was r e c o v e r e d i n a l i g h t - p r o o f g l a s s aquarium i n 10 L o f 15 w a t e r f o r a t l e a s t 24h. C a l c u l a t i o n s : The c e l l w a t e r c o n t e n t was d e t e r m i n e d by w e i g h i n g t h e wet c e l l p e l l e t , d r y i n g i t t o a c o n s t a n t w e i g h t a t 90°C and r e w e i g h i n g i t . The wa t e r c o n t e n t was t h e n c a l c u l a t e d u s i n g t h e f o l l o w i n g f o r m u l a : %H 2O=100-(lOOxdry weight/wet w e i g h t ) . The i n t r a c e l l u l a r pH may be d e t e r m i n e d from t h e d i s t r i b u t i o n o f t h e weak a c i d 5 , 5 - d i m e t h y l - 2 , 4 -o x a z o l i d i n e d i o n e (DMO) (Waddell and B u t l e r , 1959). I n t h e p r e s e n t e x p e r i m e n t s , t h e e r y t h r o c y t e pH (pH^) was c a l c u l a t e d f r o m t h e d i s t r i b u t i o n o f DMO a c r o s s t h e e r y t h r o c y t e membrane. I n i t i a l l y , 10 uL o f 1 u C i mL - 1  1 4C-DMO (New England N u c l e a r , s p e c i f i c a c t i v i t y 50 mCi mmol - 1) was added t o t h e s u s p e n s i o n ( e r y t h r o c y t e s i n plasma o r s a l i n e ) . F o l l o w i n g t h e i n c u b a t i o n p e r i o d , 0.4 mL samples o f b l o o d were t a k e n and c e n t r i f u g e d . A 100 uL sample o f e x t r a c e l l u l a r f l u i d (plasma o r s a l i n e ) was t a k e n f o r t h e d e t e r m i n a t i o n o f t h e e x t r a c e l l u l a r DMO c o n c e n t r a t i o n and t h e r e m a i n i n g e x t r a c e l l u l a r f l u i d d i s c a r d e d . The e r y t h r o c y t e p e l l e t was saved f o r t h e d e t e r m i n a t i o n o f t h e i n t r a c e l l u l a r DMO c o n c e n t r a t i o n . B oth t h e e x t r a c e l l u l a r and i n t r a c e l l u l a r DMO samples were 16 d e p r o t e i n i z e d w i t h 0.2 mL o f 0.6 M p e r c h l o r i c a c i d t o reduce quenching and l e f t f o r 24h. The r e s u l t i n g s u s p e n s i o n s were c e n t r i f u g e d and 0.1 mL o f s u p e r n a t a n t was added t o s c i n t i l l a t i o n v i a l s c o n t a i n i n g 7 mLs o f aqueous c o u n t i n g s c i n t i l l a n t (Amersham c o r p , 111., U.S.A.). The samples were t h e n a n a l y s e d f o r 1 4 C by a Beckman LS 9000 L i q u i d S c i n t i l l a t i o n S y s t e m (Beckman I n s t r u m e n t s , I n c . , CA, U.S.A.). I t s h o u l d be n o t e d t h a t t h e d e p r o t e i n i z e d s a m p l e s were a l s o u s e d f o r t h e d e t e r m i n a t i o n o f t h e c h l o r i d e c o n c e n t r a t i o n o f t h e e x t r a c e l l u l a r f l u i d and t h e e r y t h r o c y t e s . C h l o r i d e c o n c e n t r a t i o n s were d e t e r m i n e d w i t h a Radiometer CMT 10 c h l o r i d e t i t r a t o r . The pH^ was c a l c u l a t e d f r o m t h e d i s t r i b u t i o n o f DMO u s i n g t h e f o l l o w i n g f o r m u l a : P H i = p K D M 0 + l o g ( [ D M O ] A / [ D M O ] e ( l + 1 0 e x p ( P H e - p K D M 0 ) ) - 1 ) ( H e i s l e r , 1975; A l b e r s and Goetz, 1985) where pH^ and p H e a r e t h e i n t r a c e l l u l a r and e x t r a c e l l u l a r pH r e s p e c t i v e l y and DMO^ and DMOe a r e t h e i n t r a c e l l u l a r and e x t r a c e l l u l a r DMO c o n c e n t r a t i o n s r e s p e c t i v e l y . The p K D M 0 w a s t a k e n as 6. 377 f o r Salmo g a i r d n e r i a t 10°C, 6. 307 f o r Salmo  q a i r d n e r i a t 18°C, 6.272 f o r Bufo marinus and 6.245 f o r Amphiuma t r i d a c t y l u m a c c o r d i n g t o A l b e r s e t a l . , (1971). 17 The i n t r a c e l l u l a r and e x t r a c e l l u l a r c o n c e n t r a t i o n o f DMO and c h l o r i d e were c a l c u l a t e d a s : c i = [ ( ( f w c x wet wt) + PCA)/(fwc x wet w t ) ] x C m C e = [ ( ( f w c x S v o l ) + PCA)/(fwc x S v o l ) ] x C m where C^, C e and C m a r e t h e i n t r a c e l l u l a r , e x t r a c e l l u l a r and measured sample c o n c e n t r a t i o n s r e s p e c t i v e l y , fwc i s t h e f r a c t i o n a l w a t e r c o n t e n t (%H2O/100) o f t h e sample, wet wt i s t h e wet w e i g h t (mg) o f t h e e r y t h r o c y t e s , PCA i s t h e volume (uL) o f p e r c h l o r i c a c i d added t o t h e sample and S v o ^ i s t h e s a m p l e v o l u m e (uL) o f e x t r a c e l l u l a r f l u i d . I t s h o u l d be n o t e d t h a t t r a p p e d e x t r a c e l l u l a r f l u i d was n o t t a k e n i n t o a c c o u n t i n t h e i n t r a c e l l u l a r c o n c e n t r a t i o n c a l c u l a t i o n . T h i s v a l u e has been found t o be 2-3% i n f i s h e r y t h r o c y t e s (Houston, 1985) and 2-3% i n amphibian e r y t h r o c y t e s ( E m i l i o and S h e l t o n , 1980). I t i s w e l l e s t a b l i s h e d t h a t c h l o r i d e i o n s a r e p a s s i v e l y d i s t r i b u t e d a c r o s s t h e membrane o f v e r t e b r a t e e r y t h r o c y t e s ( L a s s e n , 1977) and t h e membrane p o t e n t i a l i n t h e s e c e l l s i s e q u a l t o t h e c h l o r i d e e q u i l i b r i u m p o t e n t i a l ( F o r t e s , 1977). The e r y t h r o c y t e pH (pH^) was, t h e r e f o r e , c a l c u l a t e d from t h e e x t r a c e l l u l a r pH ( p H e ) , 18 and t h e i n t r a c e l l u l a r [Cl 'jJ and e x t r a c e l l u l a r [ C l ~ e ] c h l o r i d e c o n c e n t r a t i o n s a s s u m i n g a Donnan e q u i l i b r i u m d i s t r i b u t i o n f o r p r o t o n s and c h l o r i d e i o n s u s i n g t h e f o l l o w i n g e q u a t i o n : p H i = P H e + l o g C C l ' i ] - l o g [ C l ~ e ] ( A l b e r s and Goetz, 1985). S E C T I O N I : F I S H E R Y T H R O C Y T E S 20 OVERVIEW Under normal s t e a d y s t a t e c o n d i t i o n s , t h e pH o f t h e body compartments a r e k e p t c o n s t a n t i n v e r t e b r a t e s . T h i s d o e s n o t i n c l u d e t r a n s i e n t pH c h a n g e s n o r m a l l y o c c u r r i n g i n t h e b l o o d a t t h e gas exchange organ and a t t h e t i s s u e s w h i c h enhance oxygen l o a d i n g and u n l o a d i n g r e s p e c t i v e l y . S e v e r a l t y p e s o f s t r e s s , however, may r e s u l t i n d e v i a t i o n s i n pH away from o p t i m a l s t e a d y s t a t e l e v e l s b o t h i n t h e b l o o d and o t h e r body compartments. I n f i s h , e n v i r o n m e n t a l f a c t o r s commonly c a u s i n g a c i d - b a s e d i s t u r b a n c e s i n c l u d e t e m p e r a t u r e changes, h y p e r c a p n i a and a c i d w a t e r s . I n a d d i t i o n , b u r s t a c t i v i t y ( s u p p o r t e d by a n a e r o b i c g l y c o l y s i s i n c o n t r a c t i n g w h i t e muscle f i b e r s ) i s a f r e q u e n t cause o f a c i d o s i s due t o t h e i n c r e a s e d p r o d u c t i o n o f l a c t a t e and a s s o c i a t e d p r o t o n s (Hochachka and Somero, 1984). F i s h have a l a r g e p r o p o r t i o n o f p o o r l y p e r f u s e d w h i t e m u s c l e ( H e i s l e r , 1 9 8 4 ) , and t h e r e f o r e , b u r s t a c t i v i t y o f t e n r e s u l t s i n a l a r g e number o f p r o t o n s added t o t h e c i r c u l a t i o n ( H o l e t o n e t a l . , 1983; Nikinmaa e t a l . , 1984; Primmett e t a l . , 1986). The a c i d - b a s e s t r e s s imposed on an a n i m a l by e x c e s s H + o r OH -i o n s may be reduced by t h e body b u f f e r systems u n t i l e x c r e t e d . B i o l o g i c a l b u f f e r s y s t e m s c o n s i s t o f 21 n o n b i c a r b o n a t e and C 0 2 / b i c a r b o n a t e b u f f e r s . T h e r e i s c o n s i d e r a b l y l e s s b i c a r b o n a t e i n t h e e x t r a c e l l u l a r space o f w a t e r b r e a t h i n g f i s h t h a n o t h e r v e r t e b r a t e s (Rahn and G a r e y , 1973; H e i s l e r , 1 9 8 0 ) . I n a d d i t i o n , t h e n o n b i c a r b o n a t e b u f f e r v a l u e i s r e l a t i v e l y low i n f i s h ( A l b e r s , 1970). The i n t r a c e l l u l a r compartments i n f i s h a r e b e t t e r b u f f e r e d t h a n t h e e x t r a c e l l u l a r s p a c e ( H e i s l e r , 1984). However, t h e s e a r e s t i l l l o w e r t h a n i n s i m i l a r mammalian t i s s u e s ( H e i s l e r and P i i p e r , 1971). I n d e e d , t h e t o t a l b u f f e r i n g c a p a c i t y i n f i s h i s l o w compared t o o t h e r v e r t e b r a t e s . Thus, r e l a t i v e l y l a r g e a c i d o s e s a r e p r o b a b l y common i n f i s h . I t h a s b e e n d e m o n s t r a t e d t h a t d u r i n g a c u t e e x t r a c e l l u l a r a c i d o s e s i n f i s h , t h e pH o f t h e e r y t h r o c y t e i s r e g u l a t e d a d r e n e r g i c a l l y and b l o o d oxygen c a r r y i n g c a p a c i t y i s , t h e r e f o r e , m a i n t a i n e d ( N i k i n m a a e t a l . . 1984; Primmett e t a l . , 1986; B o u t i l i e r e t a l . , 1986a). Indeed, R a n d a l l e t a l . , (1987) found t h a t Chinook salmon were a b l e t o p e r f o r m a e r o b i c e x e r c i s e as w e l l a f t e r a s b e f o r e a b u r s t swim. However, t h e mechanism by w h i c h a d r e n e r g i c s t i m u l a t i o n e l e v a t e s t h e w a t e r c o n t e n t and pH i n t e l e o s t f i s h e r y t h r o c y t e s i s not c l e a r . I t h a s b e e n d e m o n s t r a t e d t h a t a d r e n e r g i c s t i m u l a t i o n e f f e c t s i o n movements a c r o s s b o t h 22 sodium/proton and c h l o r i d e / b i c a r b o n a t e mechanisms on t h e e r y t h r o c y t e membrane (Nikinmaa and H u e s t i s , 1984; B a r o i n e t a l . , 1984a,b; Wood and P e r r y , 1985; Heming e t a l . , 1987). I n f i s h , t h e r a t e o f c a r b o n d i o x i d e e x c r e t i o n i s dependent i n l a r g e p a r t on t h e c a t a l y s e d d e h y d r a t i o n o f p l a s m a b i c a r b o n a t e by e r y t h r o c y t i c c a r b o n i c a n h y d r a s e ( R a n d a l l and Daxboeck, 1 9 8 4 ) . As i n mammalian e r y t h r o c y t e s , plasma b i c a r b o n a t e e n t e r s f i s h e r y t h r o c y t e s v i a a c h l o r i d e / b i c a r b o n a t e e x c h a n g e mechanism on t h e e r y t h r o c y t e membrane (Cameron, 1978; Obaid e t a l . , 1979; Heming e t a l . , 1986). A d r e n e r g i c e f f e c t s on e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e e x c h a n g e w o u l d , t h e r e f o r e , be e x p e c t e d t o a l t e r t h e r a t e o f c a r b o n d i o x i d e e x c r e t i o n i n t h e i n t a c t a n i m a l . There a r e no documented s t u d i e s , however, w h i c h i n v e s t i g a t e t h e e f f e c t s o f c a t e c h o l a m i n e s on c a r b o n d i o x i d e e x c r e t i o n i n f i s h i n v i v o . The purpose o f t h i s s e c t i o n i s t o d e s c r i b e t h e c h a r a c t e r i s t i c s o f t h e i o n exchange mechanisms wh i c h a r e i n v o l v e d i n t h e a d r e n e r g i c r e s p o n s e i n f i s h e r y t h r o c y t e s . I n a d d i t i o n , t h i s s e c t i o n w i l l examine t h e e f f e c t o f c a t e c h o l a m i n e s on car b o n d i o x i d e t r a n s p o r t and e x c r e t i o n i n f i s h b o t h i n v i v o and i n v i t r o . 23 CHAPTER.1: CONTROL OF ERYTHROCYTE VOLUME AND pH IN THE RAINBOW TROUT, SALMO GAIRDNERI I n t r o d u c t i o n : N i k i n m a a and H u e s t i s (1984) d e s c r i b e d t h e mechanism o f t h e a d r e n e r g i c r e s p o n s e i n rain b o w t r o u t e r y t h r o c y t e s as a l o o s e l y c o u p l e d s o d i u m / p r o t o n and c h l o r i d e / b i c a r b o n a t e exchange. I n c o n t r a s t , Heming e t a l . , (1987) proposed t h a t t h e a d r e n e r g i c s w e l l i n g and pH changes were caused by s t i m u l a t i o n o f t i g h t l y c o u p l e d s o d i u m / p r o t o n and c h l o r i d e / b i c a r b o n a t e e x c h a n g e m e c h a n i s m s . F i n a l l y , a c c o r d i n g t o B a r o i n e t a l . , ( 1 9 8 4 a ) , t h e a d r e n e r g i c r e s p o n s e i n r a i n b o w t r o u t e r y t h r o c y t e s i n v o l v e d s t i m u l a t i o n o f a s o d i u m / p r o t o n e x c h a n g e mechanism and a s o d i u m : c h l o r i d e c o - t r a n s p o r t m e chanism. The e x p e r i m e n t a l c o n d i t i o n s were v e r y d i f f e r e n t i n t h e s e s t u d i e s . The e x p e r i m e n t s o f Heming e t a l . , (1987) were c a r r i e d o u t a t 10°C on e r y t h r o c y t e s s u s p e n d e d i n p l a s m a and t h e pH a d j u s t e d w i t h c a r b o n d i o x i d e . The e x t r a c e l l u l a r pH i n t h e i r s t u d y was never l o w e r t h a n 7.5. Nikinmaa and H u e s t i s (1984) on t h e o t h e r hand, p e r f o r m e d t h e i r e x p e r i m e n t s a t 22°C and a t low e x t r a c e l l u l a r pH (7.2 - 7.3) i n HEPES b u f f e r e d media. F i n a l l y , i n t h e e x p e r i m e n t s o f B a r o i n e t a l . , (1984a), 24 t h e t e m p e r a t u r e was 15°C and t h e pH was a p p r o x i m a t e l y 7.8 ( a d j u s t e d w i t h HEPES b u f f e r ) . C l e a r l y , t h e v a r i a b l e c o n c l u s i o n s o f t h e s e s t u d i e s may have o c c u r r e d as a r e s u l t o f d i f f e r e n t e f f e c t s o f t e m p e r a t u r e , e x t r a c e l l u l a r pH o r e x t r a c e l l u l a r b i c a r b o n a t e i o n c o n c e n t r a t i o n on t h e a d r e n e r g i c r e s p o n s e . The purpose o f t h i s s t u d y was, t h e r e f o r e , t o examine t h e e f f e c t s o f t h e s e v a r i a b l e s on t h e c h a r a c t e r i s t i c s o f t h e a d r e n e r g i c r e s p o n s e i n rainbow t r o u t e r y t h r o c y t e s i n an a t t e m p t t o r e s o l v e t h e s e a p p a r e n t d i s c r e p a n c i e s . 25 M a t e r i a l s and Methods Rainbow t r o u t , Salmo g a i r d n e r i . o f b o t h sexes (200-300g) were o b t a i n e d f r o m Sun V a l l e y H a t c h e r y , M i s s i o n , B.C., and were h e l d o u t d o o r s a t 13-15°C. B l o o d was t a k e n i n t o h e p a r i n i z e d s y r i n g e s from stunned f i s h v i a c a u d a l o r c a r d i a c p u n c t u r e , and t h e r e d c e l l s washed t w i c e i n R i n g e r ' s s o l u t i o n . The R i n g e r ' s s o l u t i o n used had t h e f o l l o w i n g c o m p o s i t i o n : 145 mM N a C l , 4 mM KC1, 1.3 mM C a C l 2 , 1.2 mM M g C l 2 , 5 mM g l u c o s e and 5 o r 10 mM NaHC0 3. The pH o f t h e e x t r a c e l l u l a r medium was a d j u s t e d by v a r y i n g t h e c a r b o n d i o x i d e t e n s i o n , w h i c h ranged from a t m o s p h e r i c (pH 8.2; i n t h i s c a s e 10 mM HEPES was added t o t h e b u f f e r t o s t a b i l i z e t h e pH) t o 4% C 0 2 t o o b t a i n t h e d e s i r e d pH o f 7.1 i n 10 mM HC0 3". F o l l o w i n g t h e washes, t h e c e l l s were resuspended t o a h a e m a t o c r i t o f 15% and t h e sample was d i v i d e d i n t o f o u r p o r t i o n s . Two o f t h e subsamples were i n c u b a t e d f o r 3 0 m i n u t e s i n a s h a k i n g tonometer i n t h e R i n g e r ' s a l o n e , i n a n o t h e r s a m p l e 0.1 mM DIDS ( 4 , 4 - D i i s o t h i o c y a n -o s t i l b e n e - 2 , 2 - D i s u l f o n i c a c i d ) was a d d e d , and i n t h e f o u r t h sample 1 mM a m i l o r i d e was added. The c e l l s were i n c u b a t e d a t d i f f e r e n t e x t r a c e l l u l a r pH v a l u e s ( 7 . 1 - 8 . 2 ) , 26 b i c a r b o n a t e c o n c e n t r a t i o n s (5 and 10 mM) , and t e m p e r a t u r e s (10 and 18°C). A f t e r t h e 30 min i n c u b a t i o n , 0.4 ml samples were t a k e n f o r e x t r a - and i n t r a c e l l u l a r pH d e t e r m i n a t i o n s , and d e t e r m i n a t i o n s o f c e l l u l a r w a t e r c o n t e n t . I m m e d i a t e l y a f t e r s a m p l i n g , 1 0 ~ 5 M ( f i n a l c o n c e n t r a t i o n ) i s o p r o t e r e n o l , f r e s h l y made f o r e a c h e x p e r i m e n t , was added t o t h e DIDS and a m i l o r i d e i n c u b a t i o n s and one o f t h e i n c u b a t i o n s w i t h R i n g e r ' s s o l u t i o n a l o n e . The c e l l s were t h e n i n c u b a t e d f o r an a d d i t i o n a l 30 min. A t t h i s p o i n t , a n o t h e r s e t o f samples was t a k e n f o r t h e pH and c e l l w a t e r d e t e r m i n a t i o n s . S i m i l a r e x p e r i m e n t s were c a r r i e d out i n w h i c h e i t h e r c h o l i n e was s u b s t i t u t e d f o r sodium, o r i n w h i c h 0.1 mM 2 , 4 - d i n i t r o p h e n o l (a p r o t o n o p h o r e , s e e M c L a u g h l i n & D i l g e r , 1980) was added t o t h e R i n g e r ' s s o l u t i o n . The p r o c e d u r e f o r t h e a n a l y s i s o f t h e samples i s d e s c r i b e d i n t h e g e n e r a l m a t e r i a l s and methods s e c t i o n . 27 R e s u l t s B e l o w t h e e x t r a c e l l u l a r pH o f 7.6, t h e c e l l v o l u m e (% w a t e r c o n t e n t ) i n c r e a s e d l i n e a r l y w i t h d e c r e a s i n g e x t r a c e l l u l a r pH by 1%/0.1 pH u n i t (from 67-68 % a t pH 7.6 t o 72-73 % a t pH 7.1; F i g . l ) . The volume o f a m i l o r i d e - t r e a t e d c e l l s changed as t h a t o f c o n t r o l c e l l s , w h e r e a s DIDS e f f e c t i v e l y a b o l i s h e d t h e pH d e p e n d e n t c h a n g e s i n c e l l v o l u m e . A t e a c h pH s t u d i e d , i s o p r o t e r e n o l caused an i n c r e a s e i n t h e c e l l volume. T h i s e f f e c t was a l s o pH dependent and i n c r e a s e d a t low pH ( F i g . 2 ) . The i s o p r o t e r e n o l - i n d u c e d i n c r e a s e i n c e l l volume was s i m i l a r i n b o t h 5 and 10 mM b i c a r b o n a t e . The c e l l w a t e r c o n t e n t a t e x t r a c e l l u l a r pH 7.10-7.15 was 72.4+0.4% i n 5 mM b i c a r b o n a t e and 72.3+0.5% i n 10 mM b i c a r b o n a t e b e f o r e t h e a d d i t i o n o f i s o p r o t e r e n o l , and i n c r e a s e d t o 78.2 + 0.3% i n 5 mM b i c a r b o n a t e and t o 78.4+0.3 % i n 10 mM b i c a r b o n a t e a f t e r t h e a d d i t i o n o f i s o p r o t e r e n o l (N = 8 i n each c a s e ) . T o g e t h e r , t h e b e t a -a d r e n e r g i c and p H - i n d u c e d c h a n g e s r e p r e s e n t a 15 % i n c r e a s e i n c e l l u l a r w a t e r c o n t e n t between pH 8.2 and 7.1. The b e t a - a d r e n e r g i c c e l l s w e l l i n g was a b o l i s h e d by a d d i t i o n o f DIDS o r a m i l o r i d e ( F i g . 2 ) . 28 F i g u r e 1. E f f e c t o f e x t e r n a l pH on t h e c e l l u l a r w a t e r c o n t e n t (%) o f rainbow t r o u t e r y t h r o c y t e s , open c i r c l e s = c o n t r o l c e l l s , t r i a n g l e s = a m i l o r i d e (1 mM)-treated c e l l s , c l o s e d c i r c l e s = DIDS (0.1 mM)-treated c e l l s . L i n e s a r e f i t t e d by eye. Each p o i n t r e p r e s e n t s a mean o f 8 d e t e r m i n a t i o n s . The c e l l w a t e r c o n t e n t o f c o n t r o l and a m i l o r i d e t r e a t e d c e l l s i n c r e a s e d s i g n i f i c a n t l y ( u n p a i r e d t - t e s t ; p<0.05) below pH 7.6. No s i g n i f i c a n t changes were o b s e r v e d i n D I D S - t r e a t e d c e l l s . 29 H 2 0 7 0 65 L 9> \ Or \ O • o \ • _Q_ \ \ \ \ \ \ J I I L J I 7.2 7.6 8 . 0 p H e 30 F i g u r e 2. I s o p r o t e r e n o l - i n d u c e d i n c r e a s e i n c e l l u l a r w a t e r c o n t e n t o f r a i n b o w t r o u t e r y t h r o c y t e s . E r y t h r o c y t e s were i n i t i a l l y i n c u b a t e d f o r 30 min and t h e i r w a t e r c o n t e n t (%H20) d e t e r m i n e d . T h e r e a f t e r , 1 0 - 5 M i s o p r o t e r e n o l was added t o t h e medium and t h e water c o n t e n t d e t e r m i n e d f o l l o w i n g a f u r t h e r 30 min o f i n c u b a t i o n ( % H 2 0 ^ S O p r o t ^ ) . The change i n w a t e r c o n t e n t was c a l c u l a t e d a s : dH 20 (%) = ( % H 2 0 i s o p r o t > - % H 2O)/%H 20 L i n e i s f i t t e d by e y e . E a c h p o i n t r e p r e s e n t s 8 d e t e r m i n a t i o n s , open c i r c l e s = c o n t r o l c e l l s ( 1 0 - 5 M i s o p r o t e r e n o l o n l y ) , t r i a n g l e s = a m i l o r i d e c e l l s (1 mM a m i l o r i d e + 1 0 ~ 5 M i s o p r o t e r e n o l ) , c l o s e d c i r c l e s = DIDS-t r e a t e d c e l l s (0.1 mM DIDS + 1 0 " 5 M i s o p r o t e r e n o l ) . 31 d H 2 0 \ \ \ \ \ \ o \ \ \ 0 L • • I I l I I I I 7.2 7.6 8 . 0 p H e 32 F i g u r e 3. I n t r a c e l l u l a r pH (pH^) v s e x t r a c e l l u l a r pH (pH e) o f rainbow t r o u t e r y t h r o c y t e s u s p e n s i o n s . Each p o i n t r e p r e s e n t s a mean o f 3 t o 8 d e t e r m i n a t i o n s , open c i r c l e s = v a l u e s measured u s i n g t h e DMO method; t r i a n g l e s = v a l u e s c a l c u l a t e d f r o m t h e p H e and t h e c h l o r i d e d i s t r i b u t i o n a c r o s s t h e e r y t h r o c y t e membrane. The d e p e n d e n c e o f i n t r a c e l l u l a r pH on e x t r a c e l l u l a r pH, c a l c u l a t e d f o r i n d i v i d u a l d a t a p o i n t s , i s g i v e n by t h e f o l l o w i n g r e g r e s s i o n l i n e s : DMO method; pH^ = 0.725 x p H e + 1.80, r = 0.93 (n=116). C h l o r i d e d i s t r i b u t i o n method; pH^ = 0.821 x p H e + 1.05, r = 0.95, (n=77). 33 pH 7.7 7.3 I o o B 7.2 7.6 p H e 8 . 0 34 F i g u r e 4. E f f e c t s o f i s o p r o t e r e n o l on t h e i n t r a c e l l u l a r v s . e x t r a c e l l u l a r pH o f r a i n b o w t r o u t e r y t h r o c y t e s . E r y t h r o c y t e s were f i r s t i n c u b a t e d f o r 30 min and t h e i r pH measured (open c i r c l e s = c o n t r o l ) . T h e r e a f t e r , 10~ 5M i s o p r o t e r e n o l was added and d e t e r m i n a t i o n s were r e p e a t e d a f t e r a n o t h e r 30 m i n ( c l o s e d c i r c l e s = c o n t r o l + i s o p r o t e r e n o l ) . L i n e s were f i t t e d by eye. Each p o i n t r e p r e s e n t s a mean o f 8 d e t e r m i n a t i o n s . A p a r t from pH 8.2, i s o p r o t e r e n o l caused a s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d e c r e a s e i n t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane a t e v e r y pH s t u d i e d . 36 F i g u r e 5. E x t r a c e l l u l a r pH (pH e) v s . i n t r a c e l l u l a r pH (pH^) i n D I D S - t r e a t e d rainbow t r o u t e r y t h r o c y t e s b e f o r e (open c i r c l e s ) and a f t e r ( c l o s e d c i r c l e s ) t h e a d d i t i o n o f 1 0 ~ 5 M i s o p r o t e r e n o l . L i n e s f i t t e d by eye. Each p o i n t r e p r e s e n t s t h e mean o f 8 d e t e r m i n a t i o n s . 38 The i n t r a c e l l u l a r pH o f t h e u n t r e a t e d c e l l s , m e a s u r e d by t h e DMO method and t h e i n t r a c e l l u l a r pH c a l c u l a t e d from t h e d i s t r i b u t i o n o f c h l o r i d e (assuming a Donnan e q u i l i b r i u m ) were n o t s i g n i f i c a n t l y d i f f e r e n t a t pH v a l u e s above 7.3 ( F i g . 3 ) . A t l o w e r e x t r a c e l l u l a r pH v a l u e s , however, t h e DMO method gave c o n s i s t e n t l y h i g h e r v a l u e s f o r t h e i n t r a c e l l u l a r pH. The i n t r a c e l l u l a r pH o f n o n - t r e a t e d c e l l s a t l o w e x t r a c e l l u l a r pH was n o t a f f e c t e d by a m i l o r i d e o r by removing sodium from t h e i n c u b a t i o n medium (T a b l e 1) s u g g e s t i n g t h a t s o d i u m / p r o t on exchange does n o t o c c u r under t h e s e c o n d i t i o n s . I s o p r o t e r e n o l caused a s i g n i f i c a n t r e d u c t i o n i n t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane a t e v e r y pH s t u d i e d e x c e p t pH 8.2 ( F i g . 4 ) . T h i s r e d u c t i o n was i n h i b i t e d by a m i l o r i d e ( T a b l e 2) . I n c o n t r a s t , t h i s a d r e n e r g i c r e s p o n s e was enhanced by DIDS ( T a b l e 2, F i g . 5) . The magnitude o f t h e combined e f f e c t o f DIDS and i s o p r o t e r e n o l was d e p e n d e n t on t h e b i c a r b o n a t e c o n c e n t r a t i o n . I n 10 mM b i c a r b o n a t e , t h e c e l l u l a r a l k a l i n i z a t i o n was o n l y h a l f o f t h a t a t 5 mM b i c a r b o n a t e ( T a b l e 2 ) . The a d r e n e r g i c response was independent o f t e m p e r a t u r e o v e r t h e range t e s t e d . The volume change o b s e r v e d a t 10°C was not s i g n i f i c a n t l y d i f f e r e n t t h a n t h a t o b s e r v e d a t 18°C, n o r was t h e pH change ( T a b l e 3 ) . 39 T a b l e 1. E x t r a c e l l u l a r and i n t r a c e l l u l a r pH and t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane i n c o n t r o l c e l l s , i n a m i l o r i d e (1 mM)-treated c e l l s , and i n c e l l s i n c u b a t e d i n t h e absence o f sodium. t r e a t m e n t p H e pH^ dpH C o n t r o l 7.231+0.018 7.051+0.030 0.180 (N=4) A m i l o r i d e 7.262+0.008 7.070+0.012 0.192 (N=4) No Sodium 7.207+0.009 7.037+0.012 0.170 V a l u e s a r e means + one S.E.M. 40 T a b l e 2. Changes i n t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane i n d u c e d by i s o p r o t e r e n o l . 5 mM H C 0 3 10 mM HCO3 dpHO dpHISO Change dpHO dpHISO Change p H e 7.1-7.2 ISO o n l y 0.172 0.102 -0.070 0.131 0.088 -0.043 A m i l 0.155 0.126 -0.029 0.121 0.112 -0.009 DIDS -0.111 -0.393 -0.282 -0.149 -0.307 -0.158 p H e 7.3-7.4 ISO o n l y 0.220 0.150 -0.070 A m i l 0.247 0.243 -0.006 DIDS 0.203 -0.100 -0.303 p H e 7.5-7.6 ISO o n l y 0.377 0.267 -0.110 A m i l 0.299 0.228 -0.071 DIDS 0.505 0.174 -0.159 0.159 0.013 -0.146 V a l u e s a r e means + one S.E.M. The c e l l s were i n i t i a l l y i n c u b a t e d f o r 30 min i n t h e R i n g e r ' s s o l u t i o n o r i n t h e p r e s e n c e o f t h e t r a n s p o r t i n h i b i t o r s (O) . T h e r e a f t e r , 10 M i s o p r o t e r e n o l was a d d e d t o t h e i n c u b a t i o n s and t h e c e l l s i n c u b a t e d f o r an a d d i t i o n a l 3 0 min (ISO) . A t b o t h t i m e s (O and ISO) , t h e p H e and pH± were d e t e r m i n e d , and t h e pH g r a d i e n t (dpH=pH e-pH^) c a l c u l a t e d . The change i n pH g r a d i e n t (Change=dpHISO-dpHO) shows t h e e f f e c t o f i s o p r o t e r e n o l i n t h e d i f f e r e n t t r e a t m e n t s . 41 T a b l e 3. E f f e c t s o f t e m p e r a t u r e on t h e a d r e n e r g i c r e s p o n s e o f rainbow t r o u t e r y t h r o c y t e s . t r e a t m e n t 18°C Cont pH, P H i dpH H 20 10°C Cont 7.539+0.019 7.286+0.027 0.216 70.4+0.3 I s o p r o t 7.491±0.025 7.364±0.019* 0.127 73.3+0.2* 7.428+0.005 7.402+0.011 7.208+0.013 0.220 70.0+0.3 7.253+0.041* 0.150 73.6+0.23* I s o p r o t V a l u e s a r e means + one S.E.M. A s t e r i s k denotes s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e s between i s o p r o t e r e n o l and c o n t r o l v a l u e s . 42 D i s c u s s i o n The e r y t h r o c y t e volume o f rainbow t r o u t i n c r e a s e s as a r e s u l t o f e x t r a c e l l u l a r a c i d i f i c a t i o n below a pH o f 7.6 ( F i g . l ) . D u r i n g e x t r a c e l l u l a r a c i d i f i c a t i o n , t h e i n t r a c e l l u l a r pH a l s o d e c r e a s e s , d e c r e a s i n g t h e n e g a t i v e c h a r g e on t h e h a e m o g l o b i n . To m a i n t a i n e l e c t r o n e u t r a l i t y , t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f c h l o r i d e i n c r e a s e s and w a t e r f o l l o w s o s m o t i c a l l y ( F o r r e v i e w , see e.g. H l a d k y and R i n k , 1977). The volume changes a s s o c i a t e d w i t h changes i n pH c o u l d be p r e v e n t e d by b l o c k i n g t h e a n i o n exchange pathway w i t h DIDS. When a n i o n movements a r e i n h i b i t e d i n t h i s manner, t h e n e t i n f l u x e s ( a t low pH) o r e f f l u x e s ( a t h i g h pH) o f C I - and w a t e r do n o t o c c u r . Thus, a t low pH, t h e volume o f DIDS-t r e a t e d c e l l s i s s m a l l e r and a t h i g h pH g r e a t e r t h a n t h a t o f c o n t r o l c e l l s . C a l a (1983, 1985) has p o i n t e d out t h a t t h e n e t movements o f c h l o r i d e a r e i m p o r t a n t i n t h e c e l l volume changes. The o b s e r v e d d e c r e a s e i n i n t r a c e l l u l a r pH d i d not a c t i v a t e t h e a m i l o r i d e - s e n s i t i v e s o d i u m / p r o t o n exchange. Thus, t h e sodium/proton exchanger o f t r o u t e r y t h r o c y t e s 43 d i f f e r s from t h a t o f lymphocytes ( G r i n s t e i n e t a l . , 1986) and lamprey r e d c e l l s (Nikinmaa e t a l . , 1986) i n wh i c h a c i d i f i c a t i o n o f t h e i n t r a c e l l u l a r compartment a c t i v a t e s t h i s e x c h a n g e r . A d r e n e r g i c a g o n i s t s a c t i v a t e s o d i u m / p r o t o n exchange i n t r o u t e r y t h r o c y t e s i n v i t r o (Nikinmaa and H u e s t i s , 1984; B a r o i n e t a l . , 1984b). I n n o m i n a l l y b i c a r b o n a t e f r e e m e d i a , t h i s a c t i v a t i o n i n c r e a s e s t h e i n t r a c e l l u l a r pH by 0.1-0.2 u n i t s (Nikinmaa and H u e s t i s , 1984). Heming e t a l . , (1987) showed t h a t an a d r e n e r g i c a l l y - i n d u c e d i n c r e a s e i n i n t r a c e l l u l a r pH a l s o o c c u r s i n e r y t h r o c y t e s i n c u b a t e d i n plasma. Our s t u d y c o n f i r m s t h a t t h e i n t r a c e l l u l a r pH may i n c r e a s e when a b i c a r b o n a t e / c a r b o n d i o x i d e system i s used as a b u f f e r . F u r t h e r m o r e , t h i s i n c r e a s e i s s i g n i f i c a n t e v e n a t a b i c a r b o n a t e c o n c e n t r a t i o n o f 10 mM. The magnitude o f t h i s r e s p o n s e , h o w e v e r , a p p e a r s t o d e c r e a s e w i t h i n c r e a s i n g b i c a r b o n a t e c o n c e n t r a t i o n . A m i l o r i d e i n h i b i t s t h i s i n t r a c e l l u l a r a l k a l i n i z a t i o n . T o g e t h e r , t h e s e d a t a s t r o n g l y s u g g e s t t h a t a d r e n a l i n e - a c t i v a t e d s odium/proton e x c h a n g e p l a y s a s i g n i f i c a n t r o l e i n t h e c o n t r o l o f c y t o p l a s m i c pH i n t r o u t e r y t h r o c y t e s . The p r e s e n t r e s u l t s show t h a t DIDS i n h i b i t s t h e c e l l volume i n c r e a s e i n i s o p r o t e r e n o l - t r e a t e d c e l l s , b u t 44 causes a marked i n t r a c e l l u l a r a l k a l i n i z a t i o n . Thus, t h e a c t i v i t y o f t h e sodium/proton exchange does n o t appear t o be a l t e r e d by t h e i n h i b i t i o n o f t h e c h l o r i d e / b i c a r b o n a t e e x c h a n g e r . I n d e e d , t h e a d r e n e r g i c a l l y s t i m u l a t e d s o d i u m / p r o t o n exchange causes a l a r g e r i n t r a c e l l u l a r pH change i n t h e absence o f b i c a r b o n a t e movements. T h i s f i n d i n g s u p p o r t s t h e r e s u l t s o f Nikinmaa and H u e s t i s (1984) who d e s c r i b e t h e s e i o n exchange mechanisms as l o o s e l y c o u p l e d . The a d r e n e r g i c r e s p o n s e s o f t r o u t e r y t h r o c y t e s a r e more pronounced a t low e x t r a c e l l u l a r pH i n d i c a t i n g t h a t t h e a d r e n e r g i c a l l y a c t i v a t e d s o dium/proton t r a n s p o r t e r i s s e n s i t i v e t o p r o t o n s . These f i n d i n g s a r e c o n s i s t e n t w i t h t h e r e s u l t s o f Nikinmaa (1983) and Heming e t a l . , (1986) f o r c e l l s i n c u b a t e d i n TRIS-HC1 b u f f e r e d s a l i n e o r plasma, r e s p e c t i v e l y . S t r e s s i s i n v a r i a b l y a s s o c i a t e d w i t h a d e c r e a s e i n plasma pH i n f i s h ( H o l e t o n & R a n d a l l , 1967; S o i v i o and N i k i n m a a , 1981; H o l e t o n e t a l . , 1983; J e n s e n e t a l . , 1983; Nikinmaa e t a l . , 1984; Primmett e t a l . , 1986). The p r e s e n t r e s u l t s , t h e r e f o r e , i n d i c a t e t h a t t h e a d r e n e r g i c r e s p o n s e p a r t i a l l y o f f s e t s t h e d e t r i m e n t a l e f f e c t s w h i c h s e v e r e e x e r c i s e and low pH would o t h e r w i s e have on e r y t h r o c y t i c oxygen t r a n s p o r t . The a d r e n e r g i c r e s p o n s e was n o t a f f e c t e d by 45 t e m p e r a t u r e i n t h e range 10 t o 18°C. T h i s f i n d i n g shows t h a t t h e i n v i v o d i f f e r e n c e s i n t h e e r y t h r o c y t e r e s p o n s e s t o a d r e n a l i n e o r s t r e n u o u s e x e r c i s e between d i f f e r e n t t e m p e r a t u r e s (Nikinmaa,1982b) o r d i f f e r e n t seasons a r e n o t caused by a s i m p l e t e m p e r a t u r e dependence o f t h e a d r e n e r g i c r e s p o n s e . C o s s i n s and R i c h a r d s o n (1985) h a v e d o c u m e n t e d t h a t t h e c e l l u l a r a l k a l i n i z a t i o n i n t r o u t e r y t h r o c y t e s f o l l o w i n g a d r e n e r g i c a c t i v a t i o n i s caused by s t i m u l a t i o n o f s odium/proton exchange w i t h a s t o i c h i o m e t r y o f 1. I n a d d i t i o n , B o r g e s e et. a l . . , (1986) h a v e r e p o r t e d t h a t n i t r a t e i n h i b i t s s o d i u m / p r o t o n e x c h a n g e . The c o -t r a n s p o r t mechanism f o r a d r e n e r g i c s w e l l i n g i n t r o u t e r y t h r o c y t e s p r o posed by B a r o i n e t a l . , (1984a) can, t h e r e f o r e , be r e j e c t e d . B a r o i n e t a l . , (1984a) based t h e i r argument on t h e a s s u m p t i o n t h a t n i t r a t e does not i n h i b i t s o dium/proton exchange. T o g e t h e r , t h e s e more r e c e n t s t u d i e s have p r o v i d e d f u r t h e r s u p p o r t f o r t h e d o u b l e exchanger mechanism f o r t h e a d r e n e r g i c r e s ponse w h i c h i s p r o p o s e d i n t h i s s t u d y . 46 Summary 1. A d e c r e a s e i n e x t r a c e l l u l a r pH caused an i n c r e a s e i n t h e volume o f rainbow t r o u t e r y t h r o c y t e s , and a d e c r e a s e i n t h e i n t r a c e l l u l a r pH. 2. These pH-induced volume changes a r e m a i n l y a s s o c i a t e d w i t h movements o f c h l o r i d e a c r o s s t h e c h l o r i d e / b i c a r b o n a t e exchange pathway. 3. The sodium/proton exchanger i s q u i e s c e n t a t a l l pH's s t u d i e d . 4. The a d r e n e r g i c d r u g , i s o p r o t e r e n o l , promoted c e l l s w e l l i n g and p r o t o n e x t r u s i o n even i n t h e p r e s e n c e o f 10 mM b i c a r b o n a t e , a l t h o u g h t h e magnitude o f t h e a d r e n e r g i c pH r e s p o n s e d e c r e a s e s w i t h i n c r e a s i n g b i c a r b o n a t e c o n c e n t r a t i o n . 5. The a d r e n e r g i c r e s p o n s e was enhanced by a d e c r e a s e i n e x t r a c e l l u l a r pH. 6. DIDS markedly enhanced t h e e f f e c t o f i s o p r o t e r e n o l on t h e i n t r a c e l l u l a r pH, b u t a b o l i s h e d t h e i n c r e a s e i n e r y t h r o c y t e volume. 7. A m i l o r i d e i n h i b i t e d b o t h t h e v o l u m e and t h e pH changes a s s o c i a t e d w i t h a d r e n e r g i c s t i m u l a t i o n . 8. The a d r e n e r g i c r e s p o n s e was i n d e p e n d e n t o f t e m p e r a t u r e between 10 and 18°C. 9. The r e s u l t s s u p p o r t a l o o s e l y c o u p l e d s o d ium/proton and c h l o r i d e / b i c a r b o n a t e . e x c h a n g e model f o r t h e a d r e n e r g i c r e s p o n s e i n rainbow t r o u t e r y t h r o c y t e s . 48 CHAPTER.2: EFFECT OF BURST SWIMMING AND ADRENALINE INFUSION ON OXYGEN CONSUMPTION AND CARBON DIOXIDE EXCRETION IN THE RAINBOW TROUT, SALMO GAIRDNERI. I n t r o d u c t i o n : Wood and P e r r y (1985) h a v e r e p o r t e d t h a t a d r e n a l i n e i n h i b i t s b i c a r b o n a t e e n t r y i n t o r ainbow t r o u t e r y t h r o c y t e s .in v i t r o . I n t h e i n t a c t a n i m a l , t h i s i n h i b i t i o n w o u l d r e s u l t i n t h e r e t e n t i o n o f p l a s m a b i c a r b o n a t e d u r i n g b r a n c h i a l b l o o d t r a n s i t , and t h e r e f o r e a r e d u c t i o n i n c a r b o n d i o x i d e e x c r e t i o n . A t p r e s e n t , however, t h e r e i s l i t t l e e v i d e n c e t h a t c a t e c h o l a m i n e s modulate c a r b o n d i o x i d e e x c r e t i o n i n t h e i n t a c t a n i m a l . Van den T h i l l a r t e t a_ l . , (1983) have r e p o r t e d l o w r e s p i r a t o r y exchange r a t i o s (carbon d i o x i d e r e t e n t i o n ) i n e x e r c i s i n g coho salmon (Oncorhynchus k i s u t c h ) w h i c h would i n d i c a t e a r e d u c t i o n i n e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange. The e f f e c t o f t h e i r e x p e r i m e n t a l p r o t o c o l on c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s i n t h e s e f i s h , however, i s unknown. The purpose o f t h e s e e x p e r i m e n t s was t o de t e r m i n e i f c a t e c h o l a m i n e s modulate c a r b o n d i o x i d e e x c r e t i o n and t h e r e f o r e t h e r e s p i r a t o r y exchange r a t i o i n t h e rainbow t r o u t i n v i v o . B u r s t swimming i s known t o cause a l a r g e 49 i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s and an a c i d - b a s e d i s t u r b a n c e i n t h e r a i n b o w t r o u t Salmo q a i r d n e r i (Primmett e t a l . , 1986). I have, t h e r e f o r e , examined t h e e f f e c t o f 1) b u r s t swimming and 2) a d r e n a l i n e i n f u s i o n on c a r b o n d i o x i d e e x c r e t i o n and t h e r e s p i r a t o r y exchange r a t i o i n t h e s e a n i m a l s . 50 M a t e r i a l s and Methods The f i r s t s e r i e s o f e x p e r i m e n t s were p e r f o r m e d a t 15°C i n a B r e t t - t y p e swimming r e s p i r o m e t e r ( B r e t t , 1964) w i t h a t o t a l w a t e r v o l u m e o f 37.5 l i t e r s . Oxygen c o n s u m p t i o n was d e t e r m i n e d a t 20 m i n i n t e r v a l s by m e a s u r i n g t h e d e c l i n e i n o x y g e n t e n s i o n o f t h e r e c i r c u l a t i n g w a t e r i n t h e c l o s e d r e s p i r o m e t e r , and t h e amount o f p u r e o x y g e n i n j e c t e d i n t o t h e s y s t e m , as d e s c r i b e d by van den T h i l l a r t e t a l . , (1983). Oxygen t e n s i o n o f t h e w a t e r was m e a s u r e d c o n t i n u o u s l y by r e c i r c u l a t i n g a s m a l l f r a c t i o n o f t h e w a t e r t h r o u g h an oxygen e l e c t r o d e mounted i n a c u v e t t e (Radiometer, E-5046 and D-616), as d e s c r i b e d by S t e f f e n s e n e t a l . (1984). The oxygen e l e c t r o d e was c o n n e c t e d t o a Radiometer PHM 71 a c i d - b a s e a n a l y z e r and a c h a r t r e c o r d e r . C a r b o n d i o x i d e e x c r e t i o n o f t h e f i s h was c a l c u l a t e d from t h e d i f f e r e n c e i n t o t a l c a r b o n d i o x i d e c o n t e n t o f t h e w a t e r , d e t e r m i n e d e v e r y 20 m i n by a n a l y z i n g w a t e r s a m p l e s u s i n g a C a r l e S e r i e s 111 A n a l y t i c a l Gas Chromatograph w i t h a Poropak Q column. T o t a l c a r b o n d i o x i d e c o n t e n t was d e t e r m i n e d as d e s c r i b e d by B o u t i l i e r e t a _ l . , (1985) w i t h t h e f o l l o w i n g m o d i f i c a t i o n s . A w a t e r sample o f 2.0 ml was i n j e c t e d 51 i n t o a g l a s s s y r i n g e (10 ml) c o n t a i n i n g p u r e n i t r o g e n , and a c i d i f i e d w i t h 50 u l o f 1.0 N HC1. A f t e r 2 min o f s h a k i n g , a t l e a s t 6 ml o f t h e gas i n t h e s y r i n g e was i n j e c t e d i n t o t h e gas chromatograph l o o p (volume = 1.0 ml) t o a s s u r e complete washout o f t h e l o o p . The car b o n d i o x i d e c o n c e n t r a t i o n was c a l c u l a t e d by i n t e g r a t i n g t h e s i g n a l from t h e gas chromatograph w i t h a HP 3497 Data A c q u i s i t i o n / C o n t r o l U n i t and a HP 9135A computer. The w a t e r pH i n t h e swimming r e s p i r o m e t e r was k e p t c o n s t a n t a t 6.80. The wat e r pH was c o n t i n u o u s l y m o n i t o r e d w i t h a pH e l e c t r o d e ( C a n l a b , GK2401C) p e r m a n e n t l y mounted i n t h e s y s t e m , c o n n e c t e d t o a Radiometer PHM 71 a c i d - b a s e a n a l y z e r and a comparator. The r e c o r d e r o u t p u t was c o n n e c t e d t o a c i r c u i t c o n t r o l l i n g a H a r v a r d L i n e a r d i s p l a c e m e n t pump i n j e c t i n g 0.25 M NaOH, as d e s c r i b e d by van den T h i l l a r t e t a l . (1983) . When t h e pH o f t h e wat e r d e c r e a s e d below 6.80, t h e pump was a c t i v a t e d and NaOH i n j e c t e d u n t i l a pH o f 6.80 was r e - e s t a b l i s h e d . The f i s h were a c c l i m a t e d i n t h e r e s p i r o m e t e r f o r 24 h b e f o r e t h e ex p e r i m e n t . D u r i n g t h i s p e r i o d , t h e r e s p i r o m e t e r was c o n t i n u o u s l y f l u s h e d w i t h t h e r m o s t a t t e d a e r a t e d w a t e r h a v i n g a pH o f 6.80. The ex p e r i m e n t was s t a r t e d by c l o s i n g t h e r e s p i r o m e t e r . C o n t r o l oxygen consumption and car b o n d i o x i d e e x c r e t i o n were measured o v e r 20 m i n p e r i o d s f o r 80 m i n a t t h e a c c l i m a t i o n swimming speed (40 cm/sec). The swimming speed was t h e n i n c r e a s e d t o 80 - 85 cm/sec f o r 10 min c a u s i n g t h e f i s h t o b u r s t swim t o e x h a u s t i o n . B u r s t swimming causes an i n c r e a s e i n plasma c a t e c h o l a m i n e s i n t r o u t (Primmett e t a l . , 1986) . A f t e r 10 min, t h e speed was r e t u r n e d t o 4 0 cm/sec and t h e same parameters were measured d u r i n g t h e f o l l o w i n g 80 m i n , a t w h i c h p o i n t t h e e x p e r i m e n t was t e r m i n a t e d . I n t h e second s e r i e s o f e x p e r i m e n t s , f i s h were a n a e s t h e t i z e d w i t h MS-222 and t h e d o r s a l a o r t a c a n n u l a t e d w i t h PE-50 p o l y e t h y l e n e t u b i n g as d e s c r i b e d by S o i v i o and O i k a r i (1976). The c a n n u l a was used t o sample b l o o d f o r t h e d e t e r m i n a t i o n o f e r y t h r o c y t e pH (pH^) v i a t h e f r e e z e -thaw method o f Z e i d l e r and Kim (1977) w i t h a Radiometer PHM 71 a c i d - b a s e a n a l y z e r and a micro-pH u n i t . Oxygen consumption, c a r b o n d i o x i d e e x c r e t i o n and e r y t h r o c y t e pH (pH^) were measured i n r e s t i n g f i s h b e f o r e and a f t e r i n f u s i o n o f a d r e n a l i n e (0.25 ml o f 10~ 4M a d r e n a l i n e s o l u t i o n ) . The f i s h were h o u s e d i n a f l o w t h r o u g h r e s p i r o m e t e r w i t h a v o l u m e o f 3.0 l i t e r s . The r e s p i r o m e t e r was c o n s t r u c t e d w i t h a r e c i r c u l a t i n g c i r c u i t 53 t o a s s u r e adequate m i x i n g . Water t e m p e r a t u r e was 10°C. The "mega" dose o f a d r e n a l i n e was used t o make s u r e t h a t t h e b e t a - a d r e n e r g i c r e c e p t o r s were s t i m u l a t e d and s a t u r a t e d . The i n c r e a s e d pH^ i n d i c a t e d t h a t t h e b o l u s a c t e d on t h e b e t a - a d r e n e r g i c r e c e p t o r s o f t h e e r y t h r o c y t e . The a d r e n a l i n e s o l u t i o n was p r e p a r e d l e s s t h a n 10 min b e f o r e t h e i n f u s i o n . A f t e r each f i s h was a c c l i m a t e d f o r a t l e a s t 24 h i n t h e r e s p i r o m e t e r , c o n t r o l measurements were t a k e n a t 10 min i n t e r v a l s f o r 70 m i n . A d r e n a l i n e was t h e n i n j e c t e d and t h e measurements c o n t i n u e d f o r t h e f o l l o w i n g 60 min. Oxygen t e n s i o n , P 0 2 , and t o t a l C 0 2 were measured e v e r y 10 min on samples o f wa t e r e n t e r i n g and l e a v i n g t h e r e s p i r o m e t e r . Oxygen consumption was c a l c u l a t e d from t h e measured i n c u r r e n t and e x c u r r e n t P 0 2 and w a t e r f l o w (V) a c c o r d i n g t o t h e f o l l o w i n g e q u a t i o n : V 0 2 = B X ( ( P i 0 2 - p E 0 2 t = l / 2 ) x V + < P E 0 2 / x V ) b w _ 1 ( U l t s c h e t a l . , 1980) where B = s o l u b i l i t y o f oxygen i n w a t e r ; P J Q2 = p a r t i a l p r e s s u r e o f oxygen i n t h e i n c u r r e n t w a t e r , P£02 = p a r t i a l p r e s s u r e i n t h e e x c u r r e n t w a t e r ; p E 0 2 t = l / 2 = P a r - t ; i - a l p r e s s u r e o f oxygen i n t h e e x c u r r e n t w a t e r a f t e r t h e e l a p s e t i m e o f one h a l f t h e t i m e i n t e r v a l 54 t . V = volume o f r e s p i r o m e t e r . bw = body w e i g h t . Carbon d i o x i d e e x c r e t i o n was c a l c u l a t e d i n a s i m i l a r manner. I n b o t h s e r i e s o f e x p e r i m e n t s , t h e ambient oxygen t e n s i o n was always k e p t above 110 mmHg. 55 R e s u l t s M e a s u r e m e n t s o f o x y g e n c o n s u m p t i o n and C 0 2 e x c r e t i o n f o r 6 f i s h swimming a t 40 cm/sec b e f o r e and a f t e r b u r s t swimming a r e i l l u s t r a t e d i n F i g u r e 6. C o n t r o l oxygen consumption a t a swimming speed o f 40 cm/sec ranged from 118.3 t o 135.6 um k g - 1 m i n - 1 . C 0 2 e x c r e t i o n v a r i e d from 86.7 t o 102.1 um kg _ 1 min - 1 . The c a l c u l a t e d r e s p i r a t o r y e x c h a n g e r a t i o (RE; V C 0 2 / V 0 2 ) v a r i e d from 0.68 t o 0.80 ( F i g . 7 ) . A f t e r b u r s t swimming f o r 10 m i n , o x y g e n consumption i n c r e a s e d s i g n i f i c a n t l y (71%) t o 217.3 + 32.1 um k g - 1 m i n - 1 d u r i n g t h e f i r s t 2 0 min o f r e c o v e r y . C 0 2 e x c r e t i o n i n c r e a s e d 104% t o 192.1 + 49.9 um k g " 1 m i n - 1 , t h u s RE i n c r e a s e d s i g n i f i c a n t l y (17%) t o 0.87 ± 0.11. 20 - 40 min a f t e r b u r s t e x e r c i s e 0 2 consumption and C 0 2 e x c r e t i o n were s t i l l s i g n i f i c a n t l y e l e v a t e d as compared t o t h e c o n t r o l v a l u e s , 32% (167 + 26.9 um k g - 1 m i n - 1 ) and 50% (141.6 + 23.8 um k g - 1 m i n - 1 ) , r e s p e c t i v e l y . The r e s p i r a t o r y exchange r a t i o was not s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l . D u r i n g t h e f o l l o w i n g 20 min, 0 2 consumption was 152.8 + 25.7 um k g - 1 m i n - 1 and C 0 2 e x c r e t i o n was 118.4 + 34.5 um k g - 1 m i n " 1 , o r 21% and 26% h i g h e r t h a n c o n t r o l , 56 F i g u r e 6. Oxygen c o n s u m p t i o n and c a r b o n d i o x i d e e x c r e t i o n o f rainbow t r o u t swimming 40 cm s e c - 1 b e f o r e and a f t e r b u r s t swimmming. V a l u e s a r e means + s t a n d a r d e r r o r (N=6). P a i r e d t - t e s t was used t o compare v a l u e s f o l l o w i n g t h e b u r s t swim t o t h e mean o f t h e c o n t r o l v a l u e s . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . v a burst swimming 1 57 2 0 0 -7 1 0 0 c i a) v c o -2 0 0 . 1 0 0 . I i 1 20 min t ime 58 F i g u r e 7. R e s p i r a t o r y exchange r a t i o o f rai n b o w t r o u t swimming 40 cm s e c " 1 b e f o r e and a f t e r b u r s t swimming. V a l u e s a r e means + s t a n d a r d e r r o r (N=6). P a i r e d t - t e s t was used t o compare v a l u e s f o l l o w i n g a b u r s t swim t o t h e average o f t h e c o n t r o l v a l u e s . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 59 0.8 0.6 . 0.4 _ 0.2 . 0 L i 1 20 min burst swimming i t ime 6 0 F i g u r e 8. Oxygen c o n s u m p t i o n and c a r b o n d i o x i d e e x c r e t i o n o f rainbow t r o u t b e f o r e and a f t e r i n f u s i o n o f a d r e n a l i n e . V a l u e s a r e means + s t a n d a r d e r r o r (N=9). P a i r e d t - t e s t was used t o compare p o s t - i n f u s i o n v a l u e s t o t h e average o f a l l p r e - i n f u s i o n v a l u e s . D i f f e r e n c e s were a c c e p t e d a s s i g n i f i c a n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . c E 50 25 adrenaline infusion 61 v c o 2 50 25 10 min t ime 62 F i g u r e 9. R e s p i r a t o r y exchange r a t i o o f rai n b o w t r o u t b e f o r e and a f t e r i n f u s i o n o f a d r e n a l i n e . V a l u e s a r e means + s t a n d a r d e r r o r (N=9). P a i r e d t - t e s t was used t o compare p o s t - i n f u s i o n v a l u e s t o t h e mean o f a l l p r e -i n f u s i o n v a l u e s . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t l y d i f f e r e n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . R E 0.8 . 0.6 . 0.4 . 0.2 . 63 a d r e n a l i n e i n f u s i o n 0 L 10 min t i m e 6 4 r e s p e c t i v e l y . RE was not s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l . D u r i n g t h e l a s t p e r i o d (60 - 80 m i n ) , none o f t h e measured v a r i a b l e s were s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l s . The e f f e c t o f i n f u s i n g a d r e n a l i n e i n t o 9 r e s t i n g r a i n b o w t r o u t i s shown i n F i g u r e s 8 and 9. Oxygen consumption d u r i n g t h e 70 min c o n t r o l p e r i o d was 47.9 ± 8.0 urn k g - 1 m i n " 1 , C 0 2 e x c r e t i o n was 35.2 + 6.7 urn k g - 1 m i n - 1 , and RE c o n s e q u e n t l y 0.74 + 0.09. E r y t h r o c y t e pH measured p r i o r t o a d r e n a l i n e i n f u s i o n was 7.391 + 0.017. A d r e n a l i n e had no s i g n i f i c a n t e f f e c t on e i t h e r 0 2 consumption, C 0 2 e x c r e t i o n o r RE d u r i n g t h e f o l l o w i n g 60 min. Ten min a f t e r i n j e c t i o n o f a d r e n a l i n e , pH^ had s i g n i f i c a n t l y (P<0.05) i n c r e a s e d t o 7.447+0.034 and r e m a i n e d e l e v a t e d (7.432+0.051) f o r 60 m i n a f t e r a d r e n a l i n e i n f u s i o n . 65 D i s c u s s i o n S e v e r a l t y p e s o f s t r e s s i n c r e a s e t h e l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s i n f i s h (Nakano and T o m l i n s o n , 1968; Mazeaud and Mazeaud, 1981; B o u t i l i e r e t a l . , 1986a; P e r r y , 1986). R e c e n t l y , Primmett e t a l . , (1986) have d o c u m e n t e d 25 t o 35 f o l d i n c r e a s e s i n c i r c u l a t i n g a d r e n a l i n e and n o r a d r e n a l i n e l e v e l s i n t h e rainbow t r o u t f o l l o w i n g a b u r s t swim. I t was assumed, t h e r e f o r e , t h a t t h e b u r s t swim i n t h e p r e s e n t e x p e r i m e n t s would cause a s i m i l a r i n c r e a s e i n t h e l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s . I t h a s b e e n s u g g e s t e d t h a t c a t e c h o l a m i n e s modulate C 0 2 t r a n s p o r t by i n h i b i t i o n o f b i c a r b o n a t e f l u x t h r o u g h t h e e r y t h r o c y t e (Wood and P e r r y , 1985). T h i s i n h i b i t i o n o f e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange c o u l d e x p l a i n t h e low RE v a l u e s r e p o r t e d by van den T h i l l a r t e t a l . , (1983) i n t h e coho salmon (Oncorhvncus  k i s u t c h ) . I n coho salmon e x e r c i s e d i n s e a w a t e r a t pH 7.0, t h e RE was found t o be 0.21. van den T h i l l a r t e t a l . , (1983) a l s o m e a s u r e d V 0 2 and V C 0 2 a f t e r b u r s t swimming i n normal seawater, bu t o n l y as mean r a t e s f o r a 6h p e r i o d " s i n c e most r a t e s d i d n o t change v e r y much 66 d u r i n g each r u n " . They d e t e r m i n e d RE t o be 0.64 (N = 4 ) . I t a p p e a r s , however, t h a t i n t h e i r r e p r e s e n t a t i v e f i s h ( F i g . 3 ) , t h e b i c a r b o n a t e e x c r e t i o n d u r i n g t h e f i r s t hour was o n l y 1/15 o f t h e f o l l o w i n g 3 h o u r s . A c c o r d i n g l y , RE must have been a p p r o x i m a t e l y 0.1 d u r i n g t h e f i r s t hour. The r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s , however, p r o v i d e no e v i d e n c e t h a t i n c r e a s e d c i r c u l a t i n g c a t e c h o l a m i n e s l e a d t o a r e d u c t i o n i n c a r b o n d i o x i d e e x c r e t i o n i n t h e whole a n i m a l . I n f u s i o n o f a d r e n a l i n e i n t o r e s t i n g r a i n b o w t r o u t i n v i v o c a u s e d no s i g n i f i c a n t c h a n g e s i n e i t h e r C 0 2 e x c r e t i o n o r 0 2 consumption and c o n s e q u e n t l y t h e RE v a l u e d i d n o t change s i g n i f i c a n t l y from i t s c o n t r o l v a l u e o f 0.74. I n a d d i t i o n , b u r s t swimming, w h i c h i s a s s o c i a t e d w i t h an i n c r e a s e i n b l o o d c a t e c h o l a m i n e s (Primmett e t a l . , 1986) caused an i n c r e a s e i n b o t h 0 2 consumption (71%) and c a r b o n d i o x i d e e x c r e t i o n (104%) d u r i n g t h e r e c o v e r y p e r i o d . T h u s , i n c o n t r a s t t o t h e " C 0 2 r e t e n t i o n " p r o p o s a l o f Wood and P e r r y (1985), t h e r e was, i n f a c t , a s i g n i f i c a n t i n c r e a s e i n t h e r e s p i r a t o r y exchange r a t i o t o 0.87. The i n c r e a s e d RE, f o l l o w i n g t h e b u r s t swim i s l i k e l y a t t r i b u t a b l e as i n o t h e r a n i m a l s t o t h e t i t r a t i o n o f t h e b l o o d b i c a r b o n a t e p o o l by p r o t o n s e n t e r i n g t h e b l o o d from t h e e x e r c i s i n g t i s s u e s . 67 I t i s p o s s i b l e t h a t t h e absence o f an e f f e c t o f c a t e c h o l a m i n e s on CC»2 e x c r e t i o n i n t h i s s t u d y c o u l d have b e e n due t o s e a s o n a l v a r i a t i o n i n i n h i b i t i o n o f e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e e x c h a n g e due t o s e a s o n a l v a r i a t i o n i n B - r e c e p t o r a c t i v i t y . F o r example, i t has been documented t h a t t h e g i l l s and h e a r t o f e e l s may l o s e B - a d r e n e r g i c s e n s i t i v i t y d u r i n g t h e w i n t e r ( P e y r a u d - W a i t z e n e g g e r e t a_l . , 1980) . S i m i l a r l y , N ikinmaa and J e n s e n (1986) have s u g g e s t e d t h a t t h i s may a l s o be t h e case f o r t h e e r y t h r o c y t e s o f t h e rainbow t r o u t . The f a c t , h o w e v e r , t h a t an e l e v a t i o n i n c a t e c h o l a m i n e l e v e l s caused a s i g n i f i c a n t i n c r e a s e i n t h e e r y t h r o c y t e pH, as shown i n o t h e r s t u d i e s i n . v i t r o ( N ikinmaa, 1983; Nikinmaa and H u e s t i s , 1984; Heming e t a l . , 1987) and i n i n v i v o , i n d i c a t e s t h a t t h e B-a d r e n e r g i c r e c e p t o r s were s t i l l f u n c t i o n a l i n t h e t r o u t used i n t h i s s t u d y . Thus, t h e absence o f an e f f e c t o f c a t e c h o l a m i n e s on C 0 2 e x c r e t i o n i n t h e p r e s e n t s t u d y c a n not be a t t r i b u t e d t o reduced a c t i v i t y o f e r y t h r o c y t i c B - a d r e n e r g i c r e c e p t o r s . There i s a l s o a d i s c r e p a n c y between t h e p r e s e n t e x p e r i m e n t s and t h o s e o f van den T h i l l a r t e t a l . , (1983) c o n c e r n i n g oxygen uptake f o l l o w i n g b u r s t swimming. The 68 l a t t e r r e p o r t e d no change i n oxygen consumption d u r i n g t h e a p p r o x i m a t e l y 4h r e c o v e r y p e r i o d , whereas i n t h i s s t u d y , 0 2 c o n s u m p t i o n i n i t i a l l y i n c r e a s e d and t h e n d e c r e a s e d t o c o n t r o l l e v e l s i n t h e f o l l o w i n g 80 min. B r e t t (1964) r e p o r t e d an 0 2 debt r e p l a c e m e n t up t o 5h a f t e r f a t i g u e i n y e a r l i n g sockeye salmon (Oncorhynchus n e r k a ) . L i k e w i s e S t e v e n s and R a n d a l l (1967) and S t e f f e n s e n e t a l . , (1984) found t h a t t h e 0 2 consumption o f r a i n b o w t r o u t i n i t i a l l y i n c r e a s e d a f t e r s t r e n u o u s e x e r c i s e and t h e n d e c r e a s e d t o c o n t r o l v a l u e s w i t h i n 0.5 t o 4 h o u r s . Why van den T h i l l a r t e t a l . , (1983) found no s uch i n c r e a s e i n 0 2 consumption a f t e r b u r s t swimming ( i . e repayment o f an oxygen debt) i s n o t c l e a r . An oxygen debt a f t e r a n a e r o b i c e x e r c i s e can be e x p e c t e d , s i n c e t h e end p r o d u c t l a c t a t e must be removed m e t a b o l i c a l l y . H o l e t o n e t a l . , (1983) found s t r e n u o u s e x e r c i s e r e s u l t e d i n a s e v e r e l a c t a c i d o s i s , w h i c h was c o r r e c t e d w i t h i n 4h by a t r a n s i e n t n e t t r a n s f e r o f H + i o n s t o t h e e n v i r o n m e n t a l w a t e r . The l a c t a t e was removed m e t a b o l i c a l l y w i t h i n 6-8h. The c u r i o u s l a c k o f an 0 2 d e b t a f t e r b u r s t swimming i n t h e s t u d y o f v a n den T h i l l a r t e t a l . , (1983) i n d i c a t e s t h a t t h e i r s u p r i s i n g l y l o w RE v a l u e s may i n f a c t be due t o t e c h n i c a l l i m i t a t i o n s . 69 I n c o n c l u s i o n , t h e p r e s e n t e x p e r i m e n t s p r o v i d e no e v i d e n c e t o s u p p o r t t h e v i e w t h a t i n c r e a s e d c a t e c h o l a m i n e l e v e l s i n f i s h w i l l cause a r e d u c t i o n i n c a r b o n d i o x i d e e x c r e t i o n (Wood and P e r r y , 1985; P e r r y , 1986). F u r t h e r , t h e p r e s e n t e x p e r i m e n t s a l s o p r o v i d e no e v i d e n c e t h a t e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange i s i n h i b i t e d by e l e v a t e d c a t e c h o l a m i n e s i n t h e rai n b o w t r o u t i n v i v o , i n c o n t r a s t t o what has been r e p o r t e d i n v i t r o (Wood and P e r r y , 1985). 70 Summary 1. I m m e d i a t e l y f o l l o w i n g b u r s t swimming, t h e o x y g e n c o n s u m p t i o n o f r a i n b o w t r o u t i n c r e a s e d 7 1 % , c a r b o n d i o x i d e e x c r e t i o n i n c r e a s e d 104% and t h e r e s p i r a t o r y exchange r a t i o i n c r e a s e d 17%. 2. I n f u s i o n o f a d r e n a l i n e i n t o r e s t i n g f i s h h a d no s i g n i f i c a n t e f f e c t on o x y g e n c o n s u m p t i o n o r c a r b o n d i o x i d e e x c r e t i o n and t h e r e f o r e , t h e r e was no s i g n i f i c a n t change i n t h e r e s p i r a t o r y exchange r a t i o . 3. T h i s i n f u s i o n o f a d r e n a l i n e d i d cause a s i g n i f i c a n t e l e v a t i o n i n t h e e r y t h r o c y t e pH w h i c h was s t i l l p r e s e n t 80 min l a t e r . 4. The p r e s e n t r e s u l t s a r e i n c o n t r a s t t o t h o s e o f van den T h i l l a r t e t a l . , (1983) who found c a r b o n d i o x i d e r e t e n t i o n i n coho salmon f o l l o w i n g a b u r s t swim. 71 CHAPTER. 3: THE EFFECT OF CATECHOLAMINES ON CHLORIDE/BICARBONATE EXCHANGE I N RAINBOW TROUT ERYTHROCYTES I n t r o d u c t i o n : I n t h e p r e v i o u s c h a p t e r , i t was dem o n s t r a t e d t h a t c a t e c h o l a m i n e s do n o t cause a r e d u c t i o n i n c a r b o n d i o x i d e e x c r e t i o n i n t h e rainbow t r o u t i n v i v o . Wood and P e r r y ( 1 9 8 5 ) , on t h e o t h e r h a n d , h a v e r e p o r t e d t h a t e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange i s i n h i b i t e d by c a t e c h o l a m i n e s i n rainbow t r o u t b l o o d i n v i t r o . Why t h i s a d r e n e r g i c i n h i b i t i o n would be f u n c t i o n a l i n v i t r o and y e t a b s e n t i n v i v o i s u n c l e a r . The e x p e r i m e n t s r e p o r t e d by Wood and P e r r y (1985) were c o n d u c t e d u s i n g t h e m o d i f i e d b o a t a s s a y w h i c h measures t h e r a t e o f b i c a r b o n a t e f l u x t h r o u g h i n t a c t e r y t h r o c y t e s as d e s c r i b e d by Heming and R a n d a l l (1982) and H a s w e l l and R a n d a l l (1976). T h i s a s s a y was o r i g i n a l l y d e s c r i b e d and c r i t i c i z e d by Booth (1938) who c o n c l u d e d t h a t t h e c a r b o n i c a n h y d r a s e a c t i v i t y o f i n t a c t e r y t h r o c y t e s cannot be measured m a n o m e t r i c a l l y due t o enzyme s u b s t r a t e a c c e s s i b i l i t y f a c t o r s . A l t h o u g h r e c e n t i n v e s t i g a t o r s s i n c e Booth (1938) have produced m o d i f i e d v e r s i o n s o f t h e as s a y w h i c h g i v e r e p e a t a b l e r e s u l t s , t h e r e has been no c o n c l u s i v e e v i d e n c e i n t h e s e s t u d i e s t h a t t h e a s s a y i s an a c t u a l measure o f t h e b i c a r b o n a t e f l u x t h r o u g h i n t a c t e r y t h r o c y t e s . I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e a p p a r e n t c o n f l i c t between t h e i n v i t r o (Wood and P e r r y , 1985) and t h e i n v i v o ( c h a p t e r . 2) e x p e r i m e n t s may be a r e s u l t o f t h e t e c h n i c a l l i m i t a t i o n s i n t h e i n v i t r o e x p e r i m e n t s . I t i s a l s o p o s s i b l e t h a t t h e e f f e c t o c c u r s i n v i t r o and i n . v i v o , b u t i s c o u n t e r a c t e d by some o t h e r a d j u stment i n v i v o . I n t h e p r e s e n t s t u d y , a f u r t h e r m o d i f i e d b o a t a s s a y w h i c h measures b i c a r b o n a t e f l u x t h r o u g h i n t a c t e r y t h r o c y t e s i s dem o n s t r a t e d and t h e e f f e c t o f c a t e c h o l a m i n e s on c h l o r i d e / b i c a r b o n a t e e x c h a n g e i n r a i n b o w t r o u t e r y t h r o c y t e s i n v i t r o i s re-examined. 73 M a t e r i a l s and Methods E x p e r i m e n t a l P r o t o c o l : The b l o o d used i n t h e s e e x p e r i m e n t s was o b t a i n e d from l i g h t l y a n a e s t h e t i z e d rainbow t r o u t (150 - 250g) v i a c a u d a l v e s s e l p u n c t u r e w i t h t h e e x c e p t i o n o f t h e e x p e r i m e n t s w h i c h e x a m i n e d t h e e f f e c t s o f a d r e n e r g i c s t i m u l a t i o n . I n t h i s c a s e , b l o o d was o b t a i n e d f r o m r e s t i n g f i s h v i a a d o r s a l a o r t i c c a n n u l a w h i c h had been s u r g i c a l l y i m p l a n t e d 48 h p r i o r t o t h e e x p e r i m e n t (see t h e G e n e r a l M a t e r i a l s and Methods s e c t i o n f o r a d e t a i l e d d e s c r i p t i o n o f t h e s e p r o c e d u r e s ) . I n a l l e x p e r i m e n t s , t h e c o l l e c t e d b l o o d was p o o l e d and e q u i l i b r a t e d i n an i n t e r m i t t e n t l y r o t a t i n g g l a s s tonometer a t 10°C w i t h h u m i d i f i e d 0.2% C 0 2 i n a i r o r 1% C 0 2 i n a i r ( d e l i v e r e d by W o s t h o f f gas m i x i n g pumps). The f i r s t s e r i e s o f e x p e r i m e n t s e x a m i n e d t h e e f f e c t o f h a e m a t o c r i t on c a r b o n d i o x i d e e v o l u t i o n . I n t h e s e e x p e r i m e n t s , t h e b l o o d p o o l was e q u i l i b r a t e d w i t h 0.2% C 0 2 i n a i r f o r a t l e a s t 1 h a t w h i c h t i m e a b l o o d sample t o be a s s a y e d was removed. The h a e m a t o c r i t o f t h e sample was a d j u s t e d w i t h an a p p r o p r i a t e volume o f plasma from t h e same b l o o d p o o l and t h e f i n a l volume o f t h e whole b l o o d sample t o be a s s a y e d was a l w a y s 0.4 ml. 74 F o r each whole b l o o d a s s a y , an a d d i t i o n a l a s s a y was a l s o p e r f o r m e d w h i c h m e a s u r e d t h e r a t e o f c a r b o n d i o x i d e e v o l u t i o n o f a plasma sample from t h e b l o o d p o o l . The volume o f plasma a s s a y e d was e q u i v a l e n t t o t h e plasma volume i n t h e p r e v i o u s whole b l o o d sample. These e x p e r i m e n t s were performed a t h a e m a t o c r i t s o f 10, 15, 20, 25 and 30 %. A s e c o n d s e r i e s o f e x p e r i m e n t s e x a m i n e d t h e e f f e c t o f t h e a n i o n exchange i n h i b i t o r s , DIDS and SITS ( 4 - A c e t a m i d o - 4 - i s o t h i o c y a n a t o s t i l b e n e - 2 , 2 - d i s u l f o n i c a c i d ) , and t h e c a r b o n i c a n h y d r a s e i n h i b i t o r , a c e t a z o l a m i d e on t h e r a t e o f c a r b o n d i o x i d e e v o l u t i o n from i n t a c t e r y t h r o c y t e s . I n t h e s e e x p e r i m e n t s , a 2 ml a l i q u o t o f whole b l o o d (20% h a e m a t o c r i t ) was removed from t h e b l o o d p o o l and e q u i l i b r a t e d i n a s e p a r a t e tonometer i n t h e p r e s e n c e o f 0.1 mM DIDS, SITS o r a c e t a z o l a m i d e f o r 3 0 min p r i o r t o t h e boat a s s a y . A c o n t r o l s e t o f e x p e r i m e n t s was a l s o performed i n wh i c h o n l y t h e s a l i n e v e h i c l e (100 u l o f C o r t l a n d ' s s a l i n e ) f o r t h e above s o l u t i o n s was added t o t h e tonometer. I n t h e f i n a l s e r i e s o f e x p e r i m e n t s , t h e e f f e c t s o f t h e a d r e n e r g i c a g o n i s t s , i s o p r o t e r e n o l (O.lmM), a d r e n a l i n e (O.lmM) and n o r a d r e n a l i n e (O.lmM) on car b o n 75 d i o x i d e e v o l u t i o n f r o m i n t a c t e r y t h r o c y t e s were i n v e s t i g a t e d . T h i s s e r i e s o f e x p e r i m e n t s was s i m i l a r t o t h e second s e r i e s w i t h t h e f o l l o w i n g e x c e p t i o n s : 1) The e q u i l i b r a t i o n gas m i x t u r e was 1% C 0 2 i n a i r s i n c e t h e e f f e c t s o f a d r e n e r g i c a gents on t r o u t e r y t h r o c y t e s a r e e n h a n c e d a t l o w e r pH ( s e e C h a p t e r 1 ) . 2) 1 0 - 4 M i s o p r o t e r e n o l , a d r e n a l i n e o r n o r a d r e n a l i n e were added t o t h e t o n o m e t e r s . 3) The v e h i c l e f o r t h e s e a g o n i s t s was 100 mM p e r c h l o r i c a c i d , d i l u t e d t o 4 v o l % w i t h C o r t l a n d s a l i n e ; f i n a l volume 100 u l . 4) The e x t r a c e l l u l a r and e r y t h r o c y t e pH were a l s o d e t e r m i n e d f o l l o w i n g t h e 3 0 min e q u i l i b r a t i o n p e r i o d . M o d i f i e d Boat Assay: The a s s a y s were performed i n a 50 ml ehrlenmeyer f l a s k w h i c h had been p a r t i t i o n e d a l o n g t h e bottom by a r a i s e d g l a s s r i d g e . Two ml o f b i c a r b o n a t e s o l u t i o n (200 mM NaHC0 3 i n 20 mM NaOH) were p l a c e d on one s i d e o f t h e r i d g e and 2 ml o f phosphate b u f f e r s o l u t i o n (61% 200 mM Na 2HP0 4 and 39% 200 mM KH 2P0 4) were p l a c e d on t h e o t h e r s i d e . I t i s note w o r t h y t h a t i n a r e c e n t s t u d y , Nikinmaa e t a l . , (1986) i n c l u d e d 20 mM NaCl i n t h e b i c a r b o n a t e s o l u t i o n . The e f f e c t o f sodium c h l o r i d e a d d i t i o n was, t h e r e f o r e , examined i n t h e p r e s e n t s t u d y ( F i g . 1 0 ) , b u t was found t o have no s i g n i f i c a n t e f f e c t on t h e assay 76 F i g u r e 10. The e f f e c t o f e x t r a c e l l u l a r c h l o r i d e c o n c e n t r a t i o n on t h e c a r b o n d i o x i d e e v o l u t i o n by rainbow t r o u t e r y t h r o c y t e s . V a l u e s a r e means + s t a n d a r d e r r o r (N=6). 77 C 0 2 Evolved ul/min 150 _ 100 . 50 _ 0 L 50 100 200 CI" (mM) 78 between 0 and 2 00 mM N a C l . Thus, no NaCl was added i n t h e p r e s e n t e x p e r i m e n t s . The m a t e r i a l t o be a s s a y e d was added t o t h e s i d e c o n t a i n i n g t h e b u f f e r s o l u t i o n . The t o p o f t h e b o a t was f i t t e d w i t h a ground g l a s s j o i n t c o n n e c t e d t o a d i f f e r e n t i a l p r e s s u r e t r a n s d u c i n g system ( V a l i d y n e DP 103 w i t h CD 16 c a r r i e r d e m o d u l a t o r ) by l a t e x t u b i n g . The o u t p u t f r o m t h e p r e s s u r e t r a n s d u c i n g system was d i s p l a y e d on a c h a r t r e c o r d e r (Gould TA 600) . The b o a t was a l s o c o n n e c t e d t o a s h a k e r . P a r t i a l immersion o f t h e b o a t i n a c o n s t a n t t e m p e r a t u r e b a t h as i n p r e v i o u s s t u d i e s ( H a s w e l l and R a n d a l l , 1976; Heming and R a n d a l l , 1982) was found t o cause a t e m p e r a t u r e r e l a t e d a r t i f a c t i n t h e system. The e n t i r e system was, t h e r e f o r e , k e p t a t 10°C i n a c o n s t a n t t e m p e r a t u r e room. Upon a d d i t i o n o f a sample t o t h e b u f f e r s o l u t i o n , t h e b o a t was s e a l e d and l e f t 5 - 1 0 min u n t i l t h e o u t p u t f r o m t h e r e c o r d e r s t a b i l i z e d (manual s e a l i n g o f t h e v e s s e l caused a temporary t e m p e r a t u r e r e l a t e d p r e s s u r e change). The s h a k e r was t h e n t u r n e d on and t h e p r e s s u r e i n t h e system was f o l l o w e d as t h e d e h y d r a t i o n r e a c t i o n p r oceeded. The system was c a l i b r a t e d w i t h known volumes o f a i r from 50 t o 400 u l a t t h e e x p e r i m e n t a l t e m p e r a t u r e . 79 The i n c r e a s e i n p r e s s u r e was l i n e a r o v e r t h i s range and t h e r e was no d e t e c t a b l e l e a k a g e i n t h e system. R e a c t i o n R a t e : R e a c t i o n r a t e s (ml C 0 2 e v o l v e d m i n - 1 ) were c a l c u l a t e d from t h e t i m e r e q u i r e d f o r t h e sample m i x t u r e t o e v o l v e 200 uL o f gas. The e r y t h r o c y t e r a t e was o b t a i n e d by s u b t r a c t i o n o f each sample's t r u e plasma r a t e from t h e whole b l o o d r a t e . T h i s e l i m i n a t e d any e r r o r t h a t c o u l d be a t t r i b u t e d t o h a e m o l y s i s i n t h e system. 80 R e s u l t s The r a t e o f C 0 2 e v o l u t i o n was p r o p o r t i o n a l t o t h e number o f e r y t h r o c y t e s i n t h e b o a t ( F i g . 1 1 ) . A t a h a e m a t o c r i t o f 10%, t h e r a t e o f c a r b o n d i o x i d e e v o l u t i o n was 30.9 + 13.8 uL m i n - 1 . As t h e h a e m a t o c i t r i t i n c r e a s e d , t h e r a t e o f C 0 2 e v o l u t i o n a l s o i n c r e a s e d and a t a h a e m a t o c r i t o f 30%, t h e C 0 2 e v o l u t i o n had r e a c h e d 259 + 48.2 u l m i n - 1 . The e f f e c t s o f t h e a n i o n exchange i n h i b i t o r s , DIDS and SITS, and t h e c a r b o n i c anhydrase i n h i b i t o r , a c e t a z o l a m i d e , on t h e r a t e o f C 0 2 e v o l u t i o n a r e shown i n F i g u r e 12. The a n i o n exchange i n h i b i t o r s , DIDS and SITS, caused t h e C 0 2 e v o l u t i o n , t o d e c r e a s e from t h e c o n t r o l v a l u e o f 214.7 + 13.4 u l m i n - 1 t o 93.7 + 17.0 u l m i n - 1 and 126 + 18.9 u l m i n - 1 r e s p e c t i v e l y . DIDS, t h e r e f o r e , c a u s e d a 54% r e d u c t i o n i n C 0 2 e v o l u t i o n and SITS i n h i b i t e d t h e system by 41%. T h i s i n h i b i t i o n was due t o a change i n t h e c h a r a c t e r i s t i c s o f t h e e r y t h r o c y t e s s i n c e t h e plasma r a t e o f C 0 2 e v o l u t i o n was n o t s i g n i f i c a n t l y d i f f e r e n t a f t e r a d d i t i o n o f t h e s e b l o c k e r s . The c a r b o n i c anhydrase i n h i b i t o r , a c e t a z o l a m i d e , d e c r e a s e d t h e r a t e o f C0 2 e v o l u t i o n t o 14.7% (95% i n h i b i t i o n ) . N i e t h e r a d r e n a l i n e , i s o p r o t e r e n o l o r 81 F i g u r e 11. E f f e c t o f c h a n g i n g h a e m a t o c r i t on carbon d i o x i d e e v o l u t i o n by rainbow t r o u t e r y t h r o c y t e s . V a l u e s a r e means + s t a n d a r d e r r o r (N=6 i n a l l c a s e s e x c e p t h a e m a t o c r i t 10 where N=5). C 0 2 Evolved ul/min 82 200 100 o L 10 15 20 2 5 30 haematocrit 83 F i g u r e 12. The e f f e c t o f SITS (0.1 mM), DIDS (0.1 mM) o r a c e t a z o l a m i d e (O.lmM) on t h e car b o n d i o x i d e e v o l u t i o n o f rainbow t r o u t e r y t h r o c y t e s ( h a e m a t o c r i t 2 5 % ) . V a l u e s a r e means + s t a n d a r d e r r o r (N=6). U n p a i r e d t - t e s t s were used t o compare b l o c k e r v a l u e s t o c o n t r o l v a l u e s . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 84 C 0 2 Evolved r ul/min 200 150 100 50 _L Cont SITS DIDS Acet 85 F i g u r e 13. The e f f e c t o f a d r e n a l i n e ( 0 . 1 mM) , i s o p r o t e r e n o l (0.1 mM) and n o r a d r e n a l i n e (0.1 mM) on t h e c a r b o n d i o x i d e e v o l u t i o n o f rainbow t r o u t e r y t h r o c y t e s ( h a e m a t o c r i t 2 5 % ) . V a l u e s a r e means + s t a n d a r d e r r o r (N=8). U n p a i r e d t - t e s t s were used t o compare a g o n i s t v a l u e s t o c o n t r o l v a l u e s . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 86 C 0 2 Evolved ul/min 150 100 50 _ o L Cont Adr Isop Norad 87 n o r a d r e n a l i n e had any s i g n i f i c a n t e f f e c t on t h e r a t e o f CC»2 e v o l u t i o n from t h e e r y t h r o c y t e s ( F i g u r e 13) . The c o n t r o l r a t e was 150.9 + 13.2 u l m i n - 1 w h i l e t h e r a t e s f o r t h e a g o n i s t s were 151.5 + 13.8 u l m i n " 1 , 145.5 +15.8 u l m i n " 1 and 160.5 + 18.1 u l m i n " 1 r e s p e c t i v e l y . These a g o n i s t s d i d , however, have a l a r g e e f f e c t on t h e pH g r a d i e n t s a c r o s s t h e e r y t h r o c y t e membrane ( T a b l e 4) . I n e a c h c a s e , t h e e r y t h r o c y t e pH i n c r e a s e d w h i l e t h e e x t r a c e l l u l a r pH d e c r e a s e d . I n t h e c o n t r o l s i t u a t i o n , t h e e x t r a c e l l u l a r pH was 7.601 + 0.049, t h e i n t r a c e l l u l a r pH was 7.273 + 0.082 and t h e pH g r a d i e n t was 0.328. I n t h e p r e s e n c e o f a d r e n a l i n e , t h e e x t r a c e l l u l a r pH was 7.405 + 0.028, t h e i n t r a c e l l u l a r pH was 7.300 + 0.016 and t h u s , t h e pH g r a d i e n t was 0.105. I s o p r o t e r e n o l caused t h e e x t r a c e l l u l a r pH t o f a l l t o 7.371 + 0.029 and an i n c r e a s e i n t h e i n t r a c e l l u l a r pH t o 7.323 + 0.011, and t h e r e f o r e , t h e pH g r a d i e n t was o n l y 0.048. F i n a l l y , i n t h e c a s e o f t h e c e l l s s t i m u l a t e d w i t h n o r a d r e n a l i n e , t h e e x t r a c e l l u l a r pH was 7.446 + 0.029 and t h e i n t r a c e l l u l a r pH and t h e pH g r a d i e n t were 7.323 + 0.020 and 0.123 r e s p e c t i v e l y . 88 Table 4. E f f e c t of ad r e n a l i n e , i s o p r o t e r e n o l and noradrenaline on the plasma pH (pH e), erythrocyte pH (pHjJ and on the pH gradient (dpH) of rainbow trout blood. treatment P H e PHi dpH control 7.601+0. 049 7.273+0. 082 0.328 adrenaline 7.405+0. 080* 7.300+0. 046 0. 105 isoproterenol 7.371+0. 082* 7.323+0. 030 • 0. 048 noradrenaline 7.446+0. 083* 7.323+0. 056 0.123 Values are means + one S.E.M. Asterisk denotes s i g n i f i c a n t (unpaired t - t e s t ; p<0.05) d i f f e r e n c e from contr o l . 89 D i s c u s s i o n A c c o r d i n g t o B o o t h ( 1 9 3 8 ) , c a r b o n i c a n h y d r a s e a c t i v i t y o f i n t a c t e r y t h r o c y t e s c a n n o t be m e a s u r e d m a n o m e t r i c a l l y due t o enzyme s u b s t r a t e a c c e s s i b i l i t y f a c t o r s . H a s w e l l and R a n d a l l (1976), however, were a b l e t o d e monstrate enzyme a c t i v i t y i n r a t whole b l o o d u s i n g a m o d i f i e d b o a t a s s a y s i m i l a r t o t h a t d e s c r i b e d by Booth (1938). H a s w e l l and R a n d a l l (1976) were a l s o a b l e t o measure enzyme a c t i v i t y from t r o u t e r y t h r o c y t e s suspended i n s a l i n e , b u t found o n l y v e r y o c c a s i o n a l a c t i v i t y i n t r o u t whole b l o o d . These a u t h o r s c o n c l u d e d t h a t t h e absence o f enzyme a c t i v i t y i n t r o u t whole b l o o d was due t o a plasma i n h i b i t o r w h i c h caused e r y t h r o c y t e s t o be b i c a r b o n a t e i m p e r m e a b l e i n . v i v o . L a t e r , Heming and R a n d a l l (1982) demonstrated t h a t t h e absence o f a c t i v i t y i n t h e e x p e r i m e n t s o f H a s w e l l and R a n d a l l (1976) was, i n f a c t , c aused by foaming i n t h e a s s a y and t r o u t whole b l o o d c o u l d c a t a l y z e t h e d e h y d r a t i o n o f p l a s m a b i c a r b o n a t e i n t h e t y p i c a l mammalian f a s h i o n i n t h e p r e s e n c e o f d e f o a m i n g a g e n t s . The m o d i f i e d b o a t t e c h n i q u e d e s c r i b e d by Heming and R a n d a l l (1982) was a l s o used i n t h e e x p e r i m e n t s c i t e d by Wood and P e r r y (198 5) 90 w h i c h d e m o n s t r a t e d t h a t c a t e c h o l a m i n e s i n h i b i t t h e d e h y d r a t i o n o f p l a s m a b i c a r b o n a t e by i n t a c t t r o u t e r y t h r o c y t e s . The p r e s e n t s t u d y was done u s i n g a b o a t t e c h n i q u e s i m i l a r t o t h a t d e s c r i b e d by Heming and R a n d a l l (1982) w i t h f u r t h e r m o d i f i c a t i o n s ( s e e M e t h o d s ) . The d i f f i c u l t i e s e n c o u n t e r e d i n t h e p r e v i o u s l y d e s c r i b e d s t u d i e s made i t i m p e r a t i v e , h o w e v e r , t o t h o r o u g h l y examine t h e system p r i o r i n v e s t i g a t i n g t h e e f f e c t s o f c a t e c h o l a m i n e s on t r o u t e r y t h r o c y t e s . The e v o l u t i o n o f c a r b o n d i o x i d e i n t h e s e e x p e r i m e n t s was c l e a r l y dependent on h a e m a t o c r i t . T h i s i s f u r t h e r c o n f i r m a t i o n t h a t f i s h e r y t h r o c y t e s a r e b i c a r b o n a t e permeable (Cameron, 1978; Obaid e t a l . , 1979; Heming and R a n d a l l , 1982). I n a d d i t i o n , t h i s r e s u l t d e m o n s t r a t e s t h a t t h e system was l i n e a r i n t h e chosen r a n g e o f v o l u m e and h a e m a t o c r i t f o r t h e s u b s e q u e n t e x p e r i m e n t s . I n h i b i t i o n o f t h e C 0 2 e v o l u t i o n from whole b l o o d i n t h e p r e s e n t system by a c e t a z o l a m i d e i n d i c a t e s t h a t t h e a s s a y was, i n f a c t , m e a s u r i n g e r y t h r o c y t i c c a r b o n i c a n h y d r a s e a c t i v i t y . F u r t h e r m o r e , t h e l a r g e degree o f i n h i b i t i o n o b t a i n e d when t h e a n i o n exchange i n h i b i t o r s , DIDS and S I T S , were added t o t h e b l o o d p r o v i d e d e v i d e n c e t h a t t h e r a t e o f c a r b o n d i o x i d e 91 e v o l u t i o n i n t h i s a s s a y was dependent on e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange. T o g e t h e r , t h e s e r e s u l t s i n d i c a t e d t h a t t h e p r e s e n t system c o u l d be employed t o e x a m i n e t h e e f f e c t s o f c a t e c h o l a m i n e s on c h l o r i d e / b i c a r b o n a t e exchange i n t r o u t e r y t h r o c y t e s . I n t h e p r e s e n t s t u d y , c a t e c h o l a m i n e s had no e f f e c t on b i c a r b o n a t e f l u x t h r o u g h t r o u t e r y t h r o c y t e s . T h i s r e s u l t i s i n sha r p c o n t r a s t t o t h e r e s u l t s c i t e d by Wood and P e r r y (1985). A c c o r d i n g t o t h e s e a u t h o r s , t h e dosages o f t h e a d r e n e r g i c a g o n i s t s used i n t h i s s t u d y s h o u l d have caused a l a r g e r e d u c t i o n i n t h e C 0 2 e v o l u t i o n from t h e e r y t h r o c y t e s . These a g o n i s t s d i d , however, have an e f f e c t on t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane as i n p r e v i o u s s t u d i e s (Nikinmaa and H u e s t i s , 1984; Heming e t a l . , 1986; Primmett e t a l . , 1986). The e f f e c t o f c a t e c h o l a m i n e s on t h e pH g r a d i e n t d emonstrated, t h e r e f o r e , t h a t t h e e r y t h r o c y t e s were a d r e n e r g i c a l l y s t i m u l a t e d . The r e a s o n f o r t h e d i s p a r i t y between t h e p r e s e n t r e s u l t s and t h o s e o f Wood and P e r r y (1985) i s u n c l e a r . H a e m o l y s i s i n c r e a s e s t h e c a r b o n i c anhydrase a c t i v i t y o f b l o o d s e v e r a l f o l d (Meldrum and Roughton, 1933; H a s w e l l and R a n d a l l , 1976) . Any h a e m o l y s i s i n t h e bo a t a s s a y would, t h e r e f o r e , be an i m p o r t a n t s o u r c e o f e r r o r . T h i s p r o b l e m was a v o i d e d i n t h e p r e s e n t e x p e r i m e n t s by s u b t r a c t i n g t h e r a t e o f t h e t r u e plasma from t h a t o f whole b l o o d t o o b t a i n t h e e r y t h r o c y t e r a t e . T h i s a l s o e n s u r e d t h a t t h e e f f e c t s o f p h a r m a c o l o g i c a l a g e n t s seen i n t h e a s s a y were a r e s u l t o f c h a n g e s i n t h e c h a r a c t e r i s t i c s o f t h e i n t a c t e r y t h r o c y t e s and n o t mer e l y an e f f e c t o f t h e s e agents on t h e f r e e enzyme i n t h e plasma. I n p r e v i o u s s t u d i e s , o n l y t h e whole b l o o d r a t e was d e t e r m i n e d and t h i s was compared t o a s a l i n e c o n t r o l r a t h e r t h a n t o t h e b l o o d ' s t r u e plasma. Heming and R a n d a l l (1982) m o n i t o r e d h a e m o l y s i s by haemoglobin a s s a y . I n p r e l i m i n a r y e x p e r i m e n t s i n t h i s s t u d y , however, i t was f o u n d t h a t h a e m o l y s i s t h a t was u n d e t e c t a b l e by h a e m o g l o b i n a n a l y s i s was a p p a r e n t i f t h e p l a s m a was ass a y e d i n t h e b o a t . F u r t h e r m o r e , i n t h e p r e s e n t s t u d y , i t was ne v e r p o s s i b l e t o wash e r y t h r o c y t e s w i t h o u t some d e t e c t a b l e h a e m o l y s i s . I t i s t h e r e f o r e p o s s i b l e t h a t t h e r e s u l t s c i t e d by Wood and P e r r y (1985) may have been a r e s u l t o f h a e m o l y s i s i n t h e a s s a y . The s t u d y r e p o r t e d by Wood and P e r r y (1985) a l s o f a i l e d t o m o n i t o r t h e e r y t h r o c y t e pH d u r i n g t h e e x p e r i m e n t . As e x p l a i n e d by Booth (1938) , t h i s v a r i a b l e has a l a r g e e f f e c t on t h e b i c a r b o n a t e f l u x t h r o u g h e r y t h r o c y t e s . The documented 93 a d r e n e r g i c i n h i b i t i o n o f b i c a r b o n a t e f l u x c o u l d , t h e r e f o r e , be r e p r e s e n t a t i v e o f e r y t h r o c y t e pH changes d u r i n g t h e i r e x p e r i m e n t . U n f o r t u n a t e l y , w i t h o u t t h e s e measurements, t h e s o u r c e o f e r r o r i n t h e e x p e r i m e n t s d e s c r i b e d by Wood and P e r r y (1985) i s i m p o s s i b l e t o d e t e r m i n e . R e g a r d l e s s , t h e p r e s e n t r e s u l t s a l s o would e x p l a i n why c a t e c h o l a m i n e s had no e f f e c t on t h e r e s p i r a t o r y exchange r a t i o o f t r o u t in v i v o ( C h a p t e r . 2 ) . 94 Summary 1. A f u r t h e r m o d i f i e d b o a t a s s a y i s d e s c r i b e d . T h i s a s s a y i s d e m o n s t r a t e d t o measure b i c a r b o n a t e f l u x t h r o u g h i n t a c t t r o u t e r y t h r o c y t e s . 2. The r e s u l t s c o n f i r m t h a t e r y t h r o c y t e s i n f i s h a r e b i c a r b o n a t e permeable. 3. I n c o n t r a s t t o r e s u l t s c i t e d by Wood and P e r r y (1985), t h i s s t u d y demonstrated t h a t c a t e c h o l a m i n e s do n o t i n h i b i t b i c a r b o n a t e f l u x t h r o u g h i n t a c t t r o u t e r y t h r o c y t e s . 4. C a t e c h o l a m i n e s d i d , however, cause a r e d u c t i o n i n t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane a s i n p r e v i o u s s t u d i e s . SECTION I I : AMPHIBIAN ERYTHROCYTES 96 OVERVIEW A d r e n e r g i c r e g u l a t i o n o f e r y t h r o c y t e pH and volume has been documented i n s e v e r a l s p e c i e s o f f i s h ( N i k i n m a a and H u e s t i s , 1984 ; N i k i n m a a e t a _ l . , 1984 ; J e n s e n and Weber, 1985a,b; Primmett e t a l . , 1986; Heming e t a l . , 1987). T h i s response i s a b s e n t , however, i n d o m e s t i c g o o s e e r y t h r o c y t e s and t h e i o n e x c h a n g e mechanisms on t h e membrane o f t h e s e n u c l e a t e d e r y t h r o c y t e s a r e q u i t e d i f f e r e n t f r o m t h o s e o f f i s h e r y t h r o c y t e s ( N i k i n m a a and H u e s t i s , 1 9 8 4 ) . I n t h e lamprey, t h e pH o f t h e e r y t h r o c y t e i s a c t i v e l y r e g u l a t e d i n t h e absence o f a d r e n e r g i c s t i m u l a t i o n (Nikinmaa, 1986; N i kinmaa e t a l . , 1986). A t t h e p r e s e n t t i m e , however, i t i s n o t known whether any o t h e r s p e c i e s o f v e r t e b r a t e s r e g u l a t e t h e i r e r y t h r o c y t e pH. The amphibians a r e a d i v e r s e group o f a n i m a l s w h i c h have adapted t o a wide range o f h a b i t a t s . The a n u r a n s p e c i e s a r e a b l e t o e n d u r e p e r i o d s o f e n v i r o n m e n t a l a d v e r s i t y by r e m a i n i n g dormant i n burrows (Lee and M e r c e r , 1967; Seymour, 1973; B o u t i l i e r e t a l . , 1979b). The reduced c a p a c i t y f o r t h e c o n v e c t i o n o f gases under t h e s e c o n d i t i o n s may impose b o t h h y p o x i c and h y p e r c a p n i c s t r e s s upon t h e a n i m a l . O t h e r s , l i k e t h e 97 s e m i - a q u a t i c u r o d e l e s may d a i l y e n c o u n t e r w i d e l y f l u c t u a t i n g oxygen and c a r b o n d i o x i d e l e v e l s i n t h e i r e n v i r o n m e n t ( U l t s c h , 1976; H e i s l e r e t a l . , 1 9 8 2 ) . A l t h o u g h r o u t i n e d i v e s a r e m a i n l y a e r o b i c i n t h e semi-a q u a t i c amphibians (Toews e t a i ., 1971; B o u t i l i e r and S h e l t o n , 1986), extended d i v e s t o a v o i d p r e d a t i o n may a l s o r e s u l t i n h y p o x i c s t r e s s . S t u d i e s on a c i d - b a s e r e g u l a t i o n i n amphibians have r e v e a l e d t h a t t h e s e a n i m a l s g e n e r a l l y t o l e r a t e wide o s c i l l a t i o n s i n e x t r a c e l l u l a r pH i n f a v o u r o f r e g u l a t i n g t h e i n t r a c e l l u l a r pH o f c e r t a i n body compartments (Toews and H e i s l e r , 1982; H e i s l e r e t a l . , 1982). There a r e no s t u d i e s , however, w h i c h f o l l o w c h a n g e s i n t h e e r y t h r o c y t e pH d u r i n g a c i d - b a s e d i s t u r b a n c e s i n t h e s e a n i m a l s . The a b i l i t y t o c o n t r o l h a e m e g l o b i n : o x y g e n a f f i n i t y v i a t h e r e g u l a t i o n o f e r y t h r o c y t e pH may be an a d a p t a t i o n w h i c h e n a b l e s a m p h i b i a n s t o more e f f i c i e n t l y t r a n s p o r t o x y g e n t h r o u g h o u t t h i s b r o a d range o f ambient and i n t e r n a l gas t e n s i o n s . The i o n exchange mechanisms i n v o l v e d i n volume r e g u l a t i o n i n amphibian e r y t h r o c y t e s have been d e s c r i b e d i n d e t a i l ( C a l a , 1980, 1985; Rudolph and Greengard, 1980; P a l f r e y and Greengard, 1981). These mechanisms resemble 9 8 t h o s e i n v o l v e d i n volume r e g u l a t i o n f o l l o w i n g a d r e n e r g i c s t i m u l a t i o n i n t e l e o s t e r y t h r o c y t e s ( B a r o i n e t a l . , 1 984a,b; N i k i n m a a and H u e s t i s , 1984 ; Heming e t a l . . 1987). F r o g e r y t h r o c y t e s a l s o e x h i b i t some degree o f a d r e n e r g i c s e n s i t i v i t y . C e l l s w e l l i n g h a s be e n d o c u m e n t e d i n f r o g e r y t h r o c y t e s a f t e r a d d i t i o n o f i s o p r o t e r e n o l , b u t t h i s was o n l y s i g n i f i c a n t i n t h e p r e s e n c e o f a p h o s p h o d i e s t e r a s e i n h i b i t o r (Rudolph and G r e e n g a r d , 1 9 8 0 ) . A d r e n e r g i c s t i m u l a t i o n i n f i s h e r y t h r o c y t e s r e s u l t s i n b o t h a s w e l l i n g and a pH re s p o n s e ( N i k i n m a a and H u e s t i s , 1984 ; Heming e t a _ l . , 1 9 8 7 ) . There a r e no s t u d i e s , however, w h i c h have examined t h e e f f e c t s o f a d r e n e r g i c s t i m u l a t i o n on t h e pH i n amphibian e r y t h r o c y t e s . The p u r p o s e o f t h i s s e c t i o n i s t o d e t e r m i n e whether amphibians a r e a l s o c a p a b l e o f r e g u l a t i n g t h e i r e r y t h r o c y t e pH as i n t h e lamprey and i n f i s h . The a n i m a l s c h o s e n f o r t h e s e s t u d i e s a r e t h e s e m i - t e r r e s t r i a l a n u r a n , B u f o m a r i n u s and t h e s e m i - a q u a t i c u r o d e l e , Amphiuma t r i d a c t y l u m . 99 CHAPTER.4: THE EFFECTS OF FORCED A C T I V I T Y ON CIRCULATING CATECHOLAMINES AND pH AND WATER CONTENT OF ERYTHROCYTES IN THE TOAD, BUFO MARINUS. I n t r o d u c t i o n : E x h a u s t i v e e x e r c i s e i n f i s h r e s u l t s i n an e x t r a c e l l u l a r a c i d o s i s ( H o l e t o n e t a l . , 1983; Nikinmaa e t a l . , 1984; Primmett e t a l . , 1986). I n a d d i t i o n , t h i s t y p e o f a c t i v i t y i s a s s o c i a t e d w i t h an i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s w h i c h p r e s e r v e s t h e o x y g e n c a r r y i n g c a p a c i t y o f t h e b l o o d v i a b e t a - a d r e n e r g i c e f f e c t s on t h e e r y t h r o c y t e ( N i k i n m a a e t a _ l . , 1984 ; Primmett e t a l . , 1986). F o r c e d a c t i v i t y i n t h e s e m i -t e r r e s t r i a l t o a d , Buf o m a r i n u s a l s o r e s u l t s i n an e x t r a c e l l u l a r a c i d o s i s (McDonald e t a l . , 1980). D u r i n g an e x t r a c e l l u l a r a c i d o s i s i n d u c e d by e x p o s u r e t o h y p e r c a p n i a , Toews and H e i s l e r (1982) demonstrated t h a t B u f o m a r i n u s r e g u l a t e d t h e pH o f t h e a n a l y s e d i n t r a c e l l u l a r compartments more t h a n t h e e x t r a c e l l u l a r compartment. There a r e no s t u d i e s , however, w h i c h f o l l o w t h e changes i n t h e e r y t h r o c y t e pH o r t h e l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s d u r i n g an a c i d o s i s i n amp h i b i a n s . Bufo marinus has a s u b s t a n t i a l Bohr e f f e c t (-100 0.230; B o u t i l i e r and Toews, 1 9 8 1 ) . Changes i n e r y t h r o c y t i c pH w i l l , t h e r e f o r e , have a marked e f f e c t on h a e m o g l o b i n : o x y g e n a f f i n i t y . W h i l e f a c i l i t a t i n g t h e u n l o a d i n g o f o x y g e n t o . t h e t i s s u e s u n d e r n o r m a l c i r c u m s t a n c e s , a f a l l i n e r y t h r o c y t e pH c o u l d i n f a c t c o m p r o m i s e o x y g e n u p t a k e i n c o n d i t i o n s where an e x t r a c e l l u l a r a c i d o s i s i s a s s o c i a t e d w i t h h y p o x i a . B o u t i l i e r e t a l . , (1979b) demonstrated t h a t b u r r o w i n g i n t h e s e a n i m a l s r e s u l t s i n b o t h a d e c r e a s e i n t h e e x t r a c e l l u l a r pH and a r e d u c t i o n i n t h e a r t e r i a l P 0 2 . Thus, d u r i n g an e x t r a c e l l u l a r a c i d o s i s , t h e s e a n i m a l s may d e r i v e some b e n e f i t f r o m t h e a b i l i t y t o r e g u l a t e e r y t h r o c y t e pH. The purpose o f t h e s e e x p e r i m e n t s was t o produce an e x t r a c e l l u l a r a c i d o s i s i n t h e t o a d , Bufo marinus and t o d e t e r m i n e i f e r y t h r o c y t e pH and, t h e r e f o r e , t h e c h a r a c t e r i s t i c s o f h a e m o g l o b i n : o x y g e n b i n d i n g , a r e modulated d u r i n g an e x t r a c e l l u l a r a c i d o s i s i n amphibians. 101 Methods I n V i v o E x p e r i m e n t s : A d u l t Bufo marinus were c a n n u l a t e d i n t h e f e m o r a l a r t e r y (see G e n e r a l M a t e r i a l s and Methods). F o l l o w i n g o v e r n i g h t r e c o v e r y , a 1 ml c o n t r o l b l o o d s a m p l e was removed from t h e f e m o r a l c a n n u l a and i m m e d i a t e l y a n a l y s e d f o r pH and h a e m a t o c r i t . The sample was t h e n c e n t r i f u g e d and t h e plasma was removed f o r c a t e c h o l a m i n e a n a l y s i s . The packed e r y t h r o c y t e s were i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n f o r l a t e r a n a l y s i s o f i n t r a c e l l u l a r pH. The a n i m a l was t h e n s u b j e c t e d t o 30 min o f v i g o r o u s e x e r c i s e . T h i s i n v o l v e d t h e manual m a n i p u l a t i o n o f t h e a n i m a l by t h e e x p e r i m e n t e r so t h a t c o n t i n u o u s r i g h t i n g movements were e l i c i t e d . Upon c o m p l e t i o n o f t h i s e x e r c i s e p e r i o d , t h e a n i m a l was r e t u r n e d t o t h e chamber and a n o t h e r b l o o d sample was i m m e d i a t e l y t a k e n . B l o o d samples were a l s o t a k e n 0.5, 1 and 4h a f t e r e x e r c i s e and a n a l y s e d as d e s c r i b e d above f o r t h e c o n t r o l sample. An a d d i t i o n a l s e t o f e x p e r i m e n t s was c o n d u c t e d t o d e t e r m i n e any r e l a t i o n s h i p between changes i n h a e m a t o c r i t and changes i n t h e e r y t h r o c y t e w a t e r c o n t e n t . These two v a r i a b l e s were measured i n f i v e a n i m a l s s u b j e c t e d t o t h e p r e v i o u s l y 102 d e s c r i b e d p r o t o c o l w i t h t h e e x c e p t i o n t h a t o n l y two samples ( c o n t r o l and Oh p o s t - e x e r c i s e ) were t a k e n . I n V i t r o E x p e r i m e n t s : F o l l o w i n g t h e r e c o v e r y p e r i o d from s u r g e r y , b l o o d was c o l l e c t e d from s e v e r a l c a n n u l a t e d t o a d s and p o o l e d . Samples (2.5 ml) o f b l o o d were t h e n t r a n s f e r r e d t o g l a s s t o nometers and e q u i l i b r a t e d w i t h a h u m i d i f i e d 5% C0 2/95% a i r m i x t u r e d e l i v e r e d by Wosthoff gas m i x i n g pumps. T h i s gas m i x t u r e r e s u l t e d i n b l o o d pH v a l u e s c l o s e t o t h o s e o f t h e a n i m a l s i m m e d i a t e l y f o l l o w i n g e x h a u s t i v e e x e r c i s e (see F i g . 1 6 ) . F o l l o w i n g a 90 min e q u i l i b r a t i o n p e r i o d , a 1 ml b l o o d sample was t a k e n from each tonometer and c e n t r i f u g e d ; 0.7 ml i n one t u b e f o r t h e d e t e r m i n a t i o n o f plasma and e r y t h r o c y t e pH and t h e r e m a i n i n g 0.3 ml i n a n o t h e r t u b e f o r t h e d e t e r m i n a t i o n o f c e l l w a t e r c o n t e n t . The pH o f t h e plasma (pH e) was measured i m m e d i a t e l y and t h e r e m a i n i n g p l a s m a f r o m b o t h s a m p l e t u b e s was d i s c a r d e d . The packed e r y t h r o c y t e s from t h e f i r s t sample t u b e were t h e n f r o z e n i n l i q u i d n i t r o g e n f o r l a t e r d e t e r m i n a t i o n o f e r y t h r o c y t e pH w h i l e t h o s e from t h e second sample t u b e were saved f o r t h e d e t e r m i n a t i o n o f c e l l w a t e r c o n t e n t . B a r o i n e t a l . . , (1984a) h a v e d e m o n s t r a t e d t h a t r a i n b o w t r o u t e r y t h r o c y t e s e x h i b i t 103 s i g n i f i c a n t b e t a - a d r e n e r g i c s e n s i t i v i t y f o l l o w i n g 3h o f e q u i l i b r a t i o n i n v i t r o a t 15°C and t h e r e f o r e i t was assumed t h a t t h e e q u i l i b r a t i o n t i m e (90 min) would not a l t e r t h e b e t a - a d r e n e r g i c s e n s i t i v i t y o f t h e c e l l s . A t t h i s p o i n t , each tonometer r e c e i v e d e i t h e r 1 0 ~ 5 mol l - 1 i s o p r o t e r e n o l ( f i n a l c o n c e n t r a t i o n ) o r an e q u i v a l e n t v o l u m e (50 u l ) o f M a c k e n z i e ' s a m p h i b i a n s a l i n e . I s o p r o t e r e n o l was chosen s i n c e t h e r e g u l a t i o n o f pH i n t e l e o s t e r y t h r o c y t e s a p p e a r s t o be a b e t a - a d r e n e r g i c e f f e c t (Nikinmaa e t a l . , 1984; Primmett e t a l . , 1986) and b e t a - a d r e n e r g i c e f f e c t s a r e most p o t e n t l y s t i m u l a t e d by i s o p r o t e r e n o l ( L e f k o w i t z , 1976). The c o n c e n t r a t i o n o f 1 0 ~ 5 mol l - 1 was used i n o r d e r t o f u l l y s a t u r a t e t h e b e t a - a d r e n e r g i c r e c e p t o r s . The s a m p l i n g p r o c e d u r e was t h e n r e p e a t e d f o l l o w i n g a n o t h e r 30 min o f e q u i l i b r a t i o n . I t s h o u l d be n o t e d t h a t i n r e p r e s e n t a t i v e c a s e s (N=3), plasma c a t e c h o l a m i n e measurements were made a t t h i s p o i n t i n t h e e x p e r i m e n t . I n t h e s e c a s e s , plasma a d r e n a l i n e and n o r a d r e n a l i n e l e v e l s from t h e c o n t r o l tonometers were v e r y s i m i l a r t o t h o s e o f r e s t i n g a n i m a l s i n v i v o . A n a l y t i c a l P r o c e d u r e s : Measurements o f pH were made u s i n g a Radiometer PHM 72 a c i d - b a s e a n a l y z e r and a s s o c i a t e d micro-pH u n i t ( R a d i o m e t e r , Copenhagen, Denmark). E r y t h r o c y t i c pH 104 measurements were made u s i n g t h e f r e e z e - t h a w method o f Z e i d l e r and Kim (1977) . P l a s m a a d r e n a l i n e and n o r a d r e n a l i n e l e v e l s were d e t e r m i n e d by h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y ( S p e c t r a P h y s i c s , m o d e l SP8700) w i t h e l e c t r o c h e m i c a l ( B i o a n a l y t i c a l Systems) d e t e c t i o n (Woodward, 1982; P r i m m e t t e t a l . , 1 9 8 6 ) . The d e t e r m i n a t i o n o f t h e e r y t h r o c y t e w a t e r c o n t e n t has been d e s c r i b e d i n t h e G e n e r a l M a t e r i a l s and Methods. 105 R e s u l t s E x h a u s t i v e e x e r c i s e i n t h e t o a d was a s s o c i a t e d w i t h l a r g e i n c r e a s e s i n c i r c u l a t i n g l e v e l s o f a d r e n a l i n e and n o r a d r e n a l i n e ( T a b l e 5) . F o l l o w i n g t h e e x e r c i s e p e r i o d , t h e c i r c u l a t i n g a d r e n a l i n e l e v e l had s i g n i f i c a n t l y i n c r e a s e d from t h e c o n t r o l v a l u e o f 2.2 + 0.4 nmol l " 1 t o 36.5 + 3.2 nmol l - 1 . S i m i l a r l y , n o r a d r e n a l i n e l e v e l s s i g n i f i c a n t l y i n c r e a s e d from 0.3 + 0.1 nmol 1 ^ t o 2.7 + 0.5 nmol 1 x i m m e d i a t e l y a f t e r e x e r c i s e . B o t h o f t h e s e p a r a m e t e r s h a d r e t u r n e d t o l e v e l s w h i c h were not s i g n i f i c a n t l y d i f f e r e n t from t h e i r r e s p e c t i v e c o n t r o l l e v e l s a f t e r 30 min o f r e c o v e r y . The e x e r c i s e p e r i o d a l s o caused an i n c r e a s e i n t h e h a e m a t o c r i t ( F i g . 14) . The c o n t r o l h a e m a t o c r i t was 22.3 + 1.4. I m m e d i a t e l y f o l l o w i n g e x e r c i s e , t h i s i n c r e a s e d s i g n i f i c a n t l y t o 33.5 + 1.4 and a f t e r 30 min o f r e c o v e r y , i t was s t i l l s i g n i f i c a n t l y e l e v a t e d (26.2 + 1.8). A f t e r l h o f r e c o v e r y , t h e h a e m a t o c r i t had r e t u r n e d t o a v a l u e (22.1 + 1.4) w h i c h was n o t s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l . T h i s i n c r e a s e i n h a e m a t o c r i t was n o t a s s o c i a t e d w i t h an i n c r e a s e i n t h e c e l l w a t e r 106 T a b l e 5. E f f e c t o f f o r c e d a c t i v i t y on t h e c i r c u l a t i n g a d r e n a l i n e and n o r a d r e n a l i n e l e v e l s (nanomoles/L) i n t h e t o a d . Time (h) C 0 0.5. 1 4 adr 2.2+0.4 36.5+3.2* 1.7+0.2 1.7+0.2 1.4+0.3 (N= l l ) nor 0.3+0.1 2.7+0.5* 0.2+0.1 0.1+0.1 0.1+0.1 (N=6) C = c o n t r o l ; 0, 0.5, 1 and 4 h = h o u r s f o l l o w i n g 30 m i n u t e s o f f o r c e d a c t i v i t y . V a l u e s a r e means + one S.E.M. A s t e r i s k d e n o t e s s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from c o n t r o l . 107 F i g u r e 14. H a e m a t o c r i t (%) v a l u e s i n e l e v e n t o a d s b e f o r e (C) f o r c e d a c t i v i t y and a t 0, 0.5, 1 and 4 h f o l l o w i n g f o r c e d a c t i v i t y . V a l u e s a r e means + s t a n d a r d e r r o r . P a i r e d t - t e s t was used t o compare p o s t a c t i v i t y v a l u e s t o c o n t r o l v a l u e . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t l y d i f f e r e n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 108 %RBC „ 40 30 20 10 _L_ I C 0 0.5 1 4 time (h) 109 F i g u r e 15. H a e m a t o c r i t (hatched) and c e l l w a t e r c o n t e n t ( s o l i d ) i n f i v e t o a d s b e f o r e (C) and i m m e d i a t e l y f o l l o w i n g (E) f o r c e d a c t i v i t y . V a l u e s a r e means + s t a n d a r d e r r o r . P a i r e d t - t e s t was used t o compare t h e p o s t a c t i v i t y v a l u e t o t h e c o n t r o l v a l u e . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t l y d i f f e r e n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 110 80 60 40 20 . o L control exercise I l l F i g u r e 16. Whole b l o o d ( c i r c l e s ) and e r y t h r o c y t e ( t r i a n g l e s ) pH i n 11 t o a d s b e f o r e (C) f o r c e d a c t i v i t y and a t 0, 0.5, 1 and 4h f o l l o w i n g f o r c e d a c t i v i t y . V a l u e s a r e means + s t a n d a r d e r r o r . P a i r e d t - t e s t was used t o compare t h e p o s t a c t i v i t y v a l u e s t o t h e c o n t r o l v a l u e . D i f f e r e n c e s were a c c e p t e d as s i g n i f i c a n t l y d i f f e r e n t a t t h e p<0.05 l e v e l and a r e i n d i c a t e d by an a s t e r i s k . 112 113 T a b l e 6. E f f e c t o f i s o p r o t e r e n o l on t h e w a t e r c o n t e n t (%), e x t r a c e l l u l a r pH ( p H e ) , i n t r a c e l l u l a r pH (pH^) and on t h e pH g r a d i e n t (dpH) o f t o a d b l o o d e q u i l i b r a t e d w i t h 5% C0 2/95% a i r . 90 min t r e a t m e n t 120 min c o n t r o l (N=9) c e l l w a t e r 69.1+0.4 s a l i n e 67.9+1.4* p H e 7.516+0.017 7.511+0.016 pEi 7.124+0.010 7.134+0.017 dpH +0.392 +0.377 e x p e r i m e n t a l (N=9) c e l l w a t e r 68.5+0.52 i s o p r o t . 67.9+0.4* p H e 7.514+0.016 7.511±0.015 p H i 7.128+0.011 (10 ~ 5 ) 7.140+0.018 dpH +0.386 +0.371 V a l u e s a r e means + one S.E.M. A s t e r i s k d enotes s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from 90 min v a l u e . 114 c o n t e n t ( F i g . 15) . F o l l o w i n g t h e e x e r c i s e p e r i o d , t h e c e l l w a t e r c o n t e n t was i d e n t i c a l t o t h e v a l u e p r i o r t o e x e r c i s e (68.4 + 1.0). The e f f e c t o f f o r c e d a c t i v i t y on t h e whole b l o o d and e r y t h r o c y t e pH i s i l l u s t r a t e d i n F i g u r e 16. Both o f t h e s e p a r a m e t e r s s i g n i f i c a n t l y d e c r e a s e d f o l l o w i n g t h e a c t i v i t y p e r i o d . The m a g n i t u d e o f t h e d r o p i n e r y t h r o c y t e pH (0.270 pH u n i t s ) was l e s s t h a n t h e drop i n e x t r a c e l l u l a r pH (0.429 pH u n i t s ) . T h u s , t h e pH d i f f e r e n c e a c r o s s t h e e r y t h r o c y t e membrane was 0.443 pH u n i t s a t r e s t and was reduced t o 0.284 pH u n i t s a f t e r e x e r c i s e . By t h e 4h r e c o v e r y sample, t h e e x t r a c e l l u l a r pH was s t i l l s i g n i f i c a n t l y r e d u c e d , b u t t h e e r y t h r o c y t e pH had r e t u r n e d t o a v a l u e w h i c h was n o t s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l v a l u e . I s o p r o t e r e n o l had no e f f e c t on t h e pH o r w a t e r c o n t e n t o f e r y t h r o c y t e s e q u i l i b r a t e d i n v i t r o ( T a b l e 6 ) . There was a s m a l l , b u t s i g n i f i c a n t d e c r e a s e i n c e l l w a t e r c o n t e n t i n v i t r o a t 120 min b o t h i n e r y t h r o c y t e s t r e a t e d w i t h s a l i n e and i n t h o s e t r e a t e d w i t h i s o p r o t e r e n o l . The cause o f t h i s e f f e c t i s not c l e a r . 115 D i s c u s s i o n C o n t r o l ( r e s t i n g ) l e v e l s o f a d r e n a l i n e and n o r a d r e n a l i n e f o r Bufo marinus were s l i g h t l y l o w e r t h a n r e s t i n g l e v e l s r e c o r d e d f o r Bufo arenarum (Donoso and Segura, 1965). W h i l e t h e r e a r e no v a l u e s a v a i l a b l e from t h e l i t e r a t u r e f o r c a t e c h o l a m i n e l e v e l s f o l l o w i n g a c t i v i t y i n amphibians, t h e magnitudes o f t h e i n c r e a s e s i n t h e p r e s e n t e x p e r i m e n t s was s i m i l a r t o t h a t found i n t h e rainbow t r o u t f o l l o w i n g e x h a u s t i v e e x e r c i s e (Primmett e t a l . , 1986). I n t h e p r e s e n t e x p e r i m e n t s t h e s e l e v e l s had r e t u r n e d t o t h e c o n t r o l l e v e l s w i t h i n 30 min. I n c o n t r a s t , 4h o f r e c o v e r y were r e q u i r e d i n t h e rainbow t r o u t b e f o r e c a t e c h o l a m i n e l e v e l s had r e t u r n e d t o v a l u e s n e a r t h e c o n t r o l l e v e l s . B o u t i l i e r e t a l . , (1986a) have dem o n s t r a t e d t h a t i n c r e a s e s i n c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s i n t h e rainbow t r o u t a r e p r o p o r t i o n a l t o a c u t e d e c r e a s e s i n b l o o d pH caused by a c i d i n f u s i o n . The r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s i n d i c a t e , however, t h a t i n t h e t o a d , i n c r e a s e s i n c a t e c h o l a m i n e l e v e l s a r e n o t p r o p o r t i o n a l t o t h e degree o f m e t a b o l i c a c i d o s i s s i n c e whole b l o o d pH was s t i l l s i g n i f i c a n t l y r educed a f t e r 4h o f r e c o v e r y and c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s 116 had r e t u r n e d t o normal w i t h i n 30 min. The i n c r e a s e i n h a e m a t o c r i t i n t h e p r e s e n t s t u d y was s i m i l a r t o t h a t documented by McDonald e t a l . , (1980) f o r Bufo marinus f o l l o w i n g f o r c e d a c t i v i t y . I n Rana  c a t e s b e i a n a . i t has a l s o been shown t h a t i n f u s i o n o f a d r e n a l i n e o r n o r a d r e n a l i n e (Mbangkollo and deRoos, 1983; Herman, 1977) o r a p e r i o d o f h a n d l i n g ( M b a n g k o l l o and deRoos, 1983) w i l l cause an i n c r e a s e i n t h e h a e m a t o c r i t . There a r e s e v e r a l f a c t o r s w h i c h may have c o n t r i b u t e d t o t h i s i n c r e a s e i n h a e m a t o c r i t . I n t e l e o s t s , i n c r e a s e d h a e m a t o c r i t l e v e l s f o l l o w i n g e x h a u s t i v e e x e r c i s e a r e due t o e r y t h r o c y t e s w e l l i n g as w e l l as an i n c r e a s e i n t h e c i r c u l a t i n g number o f e r y t h r o c y t e s r e l a t i v e t o t h e plasma volume (Nikinmaa e t a l . , 1984; Primmett e t aJL. , 1986). I n B u f o , t h e a b s e n c e o f e r y t h r o c y t e s w e l l i n g i n t h e p r e s e n c e o f i n c r e a s e d c a t e c h o l a m i n e l e v e l s i n v i v o ( T a b l e 6) i s i n t e r e s t i n g i n v i e w o f t h e s t u d y o f Rudolph and Greengard (1980) i n w h i c h c a t e c h o l a m i n e s were found t o s t i m u l a t e s w e l l i n g i n f r o g e r y t h r o c y t e s i n v i t r o . The i o n e x c h a n g e s i n v o l v e d i n t h e a d r e n e r g i c s w e l l i n g r e s p o n s e i n f r o g e r y t h r o c y t e s ( P a l f r e y and Greengard, 1981) resemble t h o s e i n t e l e o s t s ( B a r o i n e t a l . , 1984a,b; N i k i n m a a and H u e s t i s , 1984 ; Heming e t a _ l . , 1 9 8 7 ) . 117 S w e l l i n g has o n l y been documented i n f r o g e r y t h r o c y t e s i n v i t r o . h o w e v e r , i f t h e c e l l s a r e a d r e n e r g i c a l l y s t i m u l a t e d i n t h e p r e s e n c e o f a p h o s p h o d i e s t e r a s e i n h i b i t o r . The p r e s e n t e x p e r i m e n t s i n d i c a t e t h a t t h e s e i o n exchange p r o c e s s e s a r e n o t c a p a b l e o f r a i s i n g t h e e r y t h r o c y t e w a t e r c o n t e n t i n t h e t o a d d u r i n g b e t a -a d r e n e r g i c s t i m u l a t i o n i n v i v o . T h i s may be due t o o t h e r f a c t o r s t h a t a r e c a p a b l e i n v i v o o f i n f l u e n c i n g e r y t h r o c y t e w a t e r c o n t e n t , s u c h as p l a s m a o s m o t i c p r e s s u r e . However, t h e b e t a - a d r e n e r g i c a g o n i s t i s o p r o t e r e n o l c a u s e d no i n c r e a s e i n t h e c e l l w a t e r c o n t e n t i n . v i t r o . T o g e t h e r , t h e s e r e s u l t s seem t o i n d i c a t e t h a t b e t a - a d r e n e r g i c s t i m u l a t i o n o f t o a d e r y t h r o c y t e s does not r e s u l t i n changes i n w a t e r c o n t e n t u n d e r p h y s i o l o g i c a l c o n d i t i o n s . The i n c r e a s e i n h a e m a t o c r i t i n t h e p r e s e n t e x p e r i m e n t s i s , t h e r e f o r e , p r o b a b l y due t o an i n c r e a s e i n t h e c i r c u l a t i n g number o f e r y t h r o c y t e s r e l a t i v e t o t h e plasma volume. A c c o r d i n g t o B o u t i l i e r e t a l . , (1986b), u p t a k e o f plasma w a t e r by o s m o t i c a l l y e n r i c h e d m u s c l e c e l l s may a c c o u n t f o r a p o r t i o n o f t h e h a e m a t o c r i t i n c r e a s e . E r y t h r o c y t e r e c r u i t m e n t may a l s o have been a c o n t r i b u t i n g f a c t o r , s i n c e N i l s s o n and Grove (1974) have dem o n s t r a t e d t h a t c o n s t r i c t i o n o f t h e s p l e e n may be i n d u c e d i n t e l e o s t s by 118 p e r f u s i o n o f t h e s p l e n i c a r t e r y w i t h a d r e n a l i n e o r n o r a d r e n a l i n e . The a c i d o s i s i n w h o l e b l o o d f o l l o w i n g f o r c e d a c t i v i t y i n Bufo marinus i s s i m i l a r t o t h a t found by McDonald e t a l . , (1980). I n a d d i t i o n , t h e r e s u l t s o f t h e p r e s e n t s t u d y i n d i c a t e t h a t t h e e r y t h r o c y t e pH a l s o f a l l s d u r i n g t h i s p e r i o d . H l a d k y and R i n k (1977) e x p l a i n t h a t a r e d u c t i o n i n pH r e d u c e s t h e n e t c h a r g e on t h e haemoglobin i n s i d e t h e e r y t h r o c y t e . T h i s would r e s u l t i n a r e d u c t i o n i n t h e e r y t h r o c y t e p r o t o n c o n c e n t r a t i o n ( r e l a t i v e t o t h e e x t r a c e l l u l a r c o n c e n t r a t i o n ) and, t h e r e f o r e , i n t h e e r y t h r o c y t e transmembrane pH g r a d i e n t . The e r y t h r o c y t e transmembrane pH g r a d i e n t a l s o d e c r e a s e s w i t h d e c r e a s i n g pH i n r a i n b o w t r o u t e r y t h r o c y t e s e q u i l i b r a t e d i n v i t r o i n t h e absence o f c a t e c h o l a m i n e s (Heming e t a l . , 1987). I t has been demonstrated t h a t w h i l e plasma pH f a l l s a f t e r e x h a u s t i v e e x e r c i s e i n t e l e o s t s , an i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s c a u s e s t h e e r y t h r o c y t e pH t o be m a i n t a i n e d (Nikinmaa e t a l . , 1984) o r i n c r e a s e d (Primmett e t a l . , 1986). B e t a - a d r e n e r g i c a g o n i s t s a l s o cause an i n c r e a s e i n t h e pH o f t e l e o s t e r y t h r o c y t e s i n . v i t r o ( N i k i n m a a and H u e s t i s , 1984; 119 C o s s i n s and R i c h a r d s o n , 1985; Heming e t a l . . , 1987 ; C h a p t e r 1) . N e v e r t h e l e s s , t h e e r y t h r o c y t e pH o f t o a d s f e l l s i g n i f i c a n t l y i n t h e p r e s e n t e x p e r i m e n t s , e v e n though c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s i n c r e a s e d . I n a d d i t i o n , i s o p r o t e r e n o l had no e f f e c t on t h e pH o f t o a d e r y t h r o c y t e s e q u i l i b r a t e d i n v i t r o ( T a b l e 6 ) . The e f f e c t o f c a t e c h o l a m i n e s on t h e pH o f t o a d e r y t h r o c y t e s i s , t h e r e f o r e , q u i t e d i f f e r e n t from t h e e f f e c t s w h i c h have been documented i n t e l e o s t s . I t i s p o s s i b l e t h a t t h e r e g u l a t i o n o f e r y t h r o c y t e pH v i a a d r e n e r g i c mechanisms i s an a d a p t a t i o n w h i c h may be s p e c i f i c t o w a t e r - b r e a t h i n g v e r t e b r a t e s . The d i f f e r e n c e i n t h e a d r e n e r g i c e f f e c t s on t h e e r y t h r o c y t e between t h e t o a d and t e l e o s t f i s h may a l s o be e x p l a i n e d i f t h e subsequent e f f e c t s on oxygen c a r r y i n g c a p a c i t y i n t h e b l o o d o f each o f t h e s e a n i m a l s a r e c o n s i d e r e d . I n t e l e o s t f i s h , a f a l l i n t h e e r y t h r o c y t e pH l o w e r s t h e o x y g e n - c a r r y i n g c a p a c i t y o f t h e b l o o d because o f t h e Root s h i f t (Cameron, 1971; Nikinmaa e t a l . , 1984; B o u t i l i e r e t a l . , 1986a). Amphibians, however, do n o t p o s s e s s a Root s h i f t ( B r i d g e s e t a l . , 1985) and a f a l l i n t h e e r y t h r o c y t e pH w o u l d n o t l o w e r t h e o x y g e n c a r r y i n g c a p a c i t y o f t h e b l o o d o f Bufo marinus as i t does i n t e l e o s t s . The f u n c t i o n a l s i g n i f i c a n c e o f t h e a d r e n e r g i c 120 r e s p o n s e i n t e l e o s t s may, t h e r e f o r e , be t o o f f s e t t h e Root s h i f t . 1 2 1 Summary 1. C i r c u l a t i n g c a t e c h o l a m i n e l e v e l s i n c r e a s e i n t h e t o a d f o l l o w i n g f o r c e d a c t i v i t y . 2. The a c t i v i t y p e r i o d a l s o c a u s e d an i n c r e a s e i n h a e m a t o c r i t and a plasma a c i d o s i s . 3. The i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s was n o t p r o p o r t i o n a l t o t h e d r o p i n e x t r a c e l l u l a r pH as documented i n f i s h . 4. The i n c r e a s e i n h a e m a t o c r i t was not a s s o c i a t e d w i t h any i n c r e a s e i n t h e e r y t h r o c y t e w a t e r c o n t e n t . 5. T h e r e was an a b s e n c e o f any b e t a - a d r e n e r g i c r e g u l a t i o n o f pH o r w a t e r c o n t e n t i n t h e e r y t h r o c y t e s o f t h e t o a d . T h i s i s i n s h a r p c o n t r a s t t o t h e r e s p o n s e o f t e l e o s t f i s h e r y t h r o c y t e s t o b e t a - a d r e n e r g i c s t i m u l a t i o n and may be due t o t h e f a c t t h a t amphibians do n o t have a Root s h i f t . 122 CHAPTER.5: THE DISTRIBUTION OF PROTONS AND CHLORIDE IONS IN AMPHIBIAN ERYTHROCYTES I n t r o d u c t i o n : The d i s t r i b u t i o n o f p r o t o n s i s i n Donnan e q u i l i b r i u m w i t h t h e c h l o r i d e i o n d i s t r i b u t i o n i n n o n n u c l e a t e d mammalian e r y t h r o c y t e s ( H l a d k y and R i n k , 1977). S i m i l a r l y , i n n u c l e a t e d e r y t h r o c y t e s , i t has been demonstrated t h a t p r o t o n s and c h l o r i d e i o n s may be i n Donnan e q u i l i b r i u m ( A l b e r s and Goetz, 1985; Heming e t a l . , 1986) . However, i n t h e n u c l e a t e d e r y t h r o c y t e s o f t h e l a m p r e y ( N i k i n m a a , 1986) and t h e e e l ( S t e e n and T u r i t z i n , 1968), p r o t o n s and c h l o r i d e i o n s a r e not i n Donnan e q u i l i b r i u m . Indeed, Nikinmaa e t a l . , (1986) has f u r t h e r d e m o n s t r a t e d t h a t t h e e r y t h r o c y t e pH i n t h e lamprey i s a c t i v e l y r e g u l a t e d by a s odium/proton exchange mechanism. A l b e r s and Goetz (1985) have shown t h a t t h e s e t y p e s o f s t u d i e s may be c o m p l i c a t e d by d i f f e r e n c e s e n c o u n t e r e d between t h e DMO method and t h e f r e e z e - t h a w method ( Z e i d l e r and Kim, 1977), b o t h f r e q u e n t l y used f o r t h e measurement o f e r y t h r o c y t e pH. M c D o n a l d e t a _ l . , (1980) h a v e d e t e r m i n e d t h a t c h l o r i d e i o n s and b i c a r b o n a t e i o n s a r e i n Donnan e q u i l i b r i u m i n t h e e r y t h r o c y t e s o f t h e a m p h i b i a n , Bufo 123 m a r i n u s . There have been no s t u d i e s , however, w h i c h s i m u l t a n e o u s l y d e t e r m i n e t h e p r o t o n and c h l o r i d e d i s t r i b u t i o n r a t i o s o v e r t h e p h y s i o l o g i c a l pH range i n a mphibian e r y t h r o c y t e s . I t was, t h e r e f o r e , t h e purpose o f t h i s s t u d y t o d e t e r m i n e i f p r o t o n s and c h l o r i d e i o n s a r e d i s t r i b u t e d a c c o r d i n g t o a Donnan e q u i l i b r i u m a c r o s s t h e e r y t h r o c y t e membrane i n t h e a mphibian, Bufo m a r i n u s. The r e l a t i o n s h i p between t h e DMO method and t h e f r e e z e -thaw method f o r measuring t h e e r y t h r o c y t e pH i n t h e s e c e l l s was a l s o i n v e s t i g a t e d . 124 Methods A r t e r i a l b l o o d was c o l l e c t e d f r o m s e v e r a l c a n n u l a t e d t o a d s and p o o l e d . A l i q u o t s (2 mL) o f b l o o d were t h e n t r a n s f e r r e d t o g l a s s t o n o m e t e r s and e q u i l i b r a t e d f o r one hour w i t h a h u m i d i f i e d 1% C0 2/99% a i r m i x t u r e ( d e l i v e r e d by Wosthoff gas m i x i n g pumps) and 0, 50, 100, 150 o r 200 uL o f 0.2N HC1. I n a d d i t i o n , 10 uL o f 1 u C i m l " 1 1 4C-DM0 (New England N u c l e a r , s p e c i f i c a c t i v i t y 50 mCi mmol - 1) was added f o r l a t e r d e t e r m i n a t i o n o f i n t r a c e l l u l a r pH. F o l l o w i n g t h e e q u i l i b r a t i o n p e r i o d (2h) , t h e b l o o d was removed f r o m t h e t o n o m e t e r and d i s t r i b u t e d among f o u r eppendorf sample t u b e s . The f i r s t t u b e r e c e i v e d 0.8 mL o f b l o o d w h i l e each o f t h e r e m a i n i n g t h r e e t u b e s r e c e i v e d 0.4 mL. The t u b e s were t h e n c e n t r i f u g e d and t h e plasma pH was measured from t h e f i r s t sample t u b e i m m e d i a t e l y u s i n g a Radiometer PHM 72 a c i d -base a n a l y s e r and a s s o c i a t e d micro-pH u n i t (Radiometer, Copenhagen, Denmark). The r e m a i n i n g plasma from t h i s sample t u b e was d i s c a r d e d and t h e packed e r y t h r o c y t e s w ere f r o z e n i n l i q u i d n i t r o g e n f o r t h e l a t e r d e t e r m i n a t i o n o f e r y t h r o c y t e pH. The pH o f t h i s sample was d e t e r m i n e d v i a t h e f r e e z e - t h a w method o f Z i e d l e r and Kim (1977) w i t h t h e e x c e p t i o n t h a t , i n one s e r i e s o f 125 e x p e r i m e n t s , t h i s measurement was a l s o made on a s e p a r a t e sample w i t h a b r i d g e o f p h y s i o l o g i c a l s a l i n e between t h e sample and t h e s a t u r a t e d KC1 s o l u t i o n o f t h e pH e l e c t r o d e ( S i g a a r d - A n d e r s e n , 1961; 1974; B o u t i l i e r e t a l . , 1985). T h i s s a l i n e b r i d g e e l i m i n a t e s t h e j u n c t i o n p o t e n t i a l w h i c h may d e v e l o p between t h e sample and t h e K C l s o l u t i o n o f t h e r e f e r e n c e e l e c t r o d e d u r i n g t h e measurement o f pH i n s l u r r i e s (1974) . A 100 uL sample o f plasma and t h e packed e r y t h r o c y t e s were saved from each o f t h e o t h e r t h r e e sample t u b e s f o r t h e d e t e r m i n a t i o n o f w a t e r c o n t e n t and c h l o r i d e and DMO c o n c e n t r a t i o n s r e s p e c t i v e l y . The p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f b o t h t h e e r y t h r o c y t e pH v i a t h e DMO method and t h e e r y t h r o c y t e w a t e r c o n t e n t have been d e s c r i b e d i n t h e G e n e r a l M a t e r i a l s and Methods. C h l o r i d e a n a l y s e s were performed u s i n g a Radiometer CMT 10 c h l o r i d e t i t r a t o r (Radiometer, Copenhagen, Denmark). 126 R e s u l t s The w a t e r c o n t e n t o f t o a d e r y t h r o c y t e s i n c r e a s e d l i n e a r l y w i t h d e c r e a s i n g pH ( F i g u r e 17) . The minimum w a t e r c o n t e n t was 68% a t a p H e o f 7.948 and t h i s i n c r e a s e d t o 75.5% a t a p H e o f 6.913. T h i s r e p r e s e n t e d an 11% change i n t h e wa t e r c o n t e n t o v e r t h e pH range s t u d i e d o r 1.1%/0.1 pH u n i t . The r e g r e s s i o n l i n e s g e n e r a t e d f o r t h e d i s t r i b u t i o n r a t i o s o f c h l o r i d e i o n s ( r C l ~ ) and p r o t o n s w i t h t h e e r y t h r o c y t e pH d e t e r m i n e d v i a b o t h t h e DMO method ( r H + D M 0 ) and t h e f r e e z e - t h a w method ( r H + F T ) a r e d i s p l a y e d i n F i g u r e 18. Both t h e d i s t r i b u t i o n r a t i o s f o r p r o t o n s v a r y i n v e r s e l y t o t h e c h l o r i d e d i s t r i b u t i o n r a t i o w i t h changes i n e x t r a c e l l u l a r pH. The s l o p e o f each l i n e , however, i s s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from t h a t o f t h e o t h e r 2 l i n e s . The y - i n t e r c e p t s o f r H +DMO v s r H + F T a n d r H + F T v s r C 1 _ a r e a l s o s i g n i f i c a n t l y d i f f e r e n t (p<0.05).. The r e l a t i o n s h i p between t h e o v e r a l l d i s t r i b u t i o n r a t i o s ( n e g l e c t i n g t h e e f f e c t s o f e x t r a c e l l u l a r pH) a r e shown i n T a b l e 7. The o v e r a l l d i s t r i b u t i o n r a t i o f o r r C l " i s 0.607 + 0.025 w h i l e t h e v a l u e s f o r rH"*"™ and r H + n v r n a r e 0.470 + 0.023 and 0.635 + 127 F i g u r e 17. E r y t h r o c y t e w a t e r c o n t e n t v e r s u s e x t r a c e l l u l a r pH (pH e) o f t o a d b l o o d . The dependence o f e r y t h r o c y t e w a t e r c o n t e n t on p H e i s g i v e n by t h e f o l l o w i n g r e g r e s s i o n l i n e : %H 20 = -5.706 x p H e + 114.0, r = 0.89 (n=32). 128 % H 2 0 _ v 7 0 6 5 7.0 . X . 7.4 p H e 7.8 129 F i g u r e 18. P r o t o n and C h l o r i d e d i s t r i b u t i o n r a t i o s ( r ) v s e x t r a c e l l u l a r pH (pH e) o f t o a d b l o o d , t r i a n g l e s = p r o t o n d i s t r i b u t i o n r a t i o u s i n g t h e DMO method f o r pH^; a s t e r i c e s = p r o t o n d i s t r i b u t i o n r a t i o u s i n g t h e f r e e z e -thaw method f o r pH^; c i r c l e s = c h l o r i d e d i s t r i b u t i o n r a t i o . The dependence o f t h e d i s t r i b u t i o n r a t i o s on e x t r a c e l l u l a r pH i s g i v e n by t h e f o l l o w i n g r e g r e s s i o n l i n e s : DMO method f o r p f ^ ; r H + = -0.432 x p H e + 3.878, r = 0.97 (n=32) . Freeze-thaw method f o r pH^; r H + = -0. 350 x p H e + 3.101, r = 0.97 (n=32). C h l o r i d e d i s t r i b u t i o n ; r C l " = -0.390 x p H e + 3.535, r = 0.99 (n=32). The s l o p e o f each l i n e i s s i g n i f i c a n t l y (p<0.05) d i f f e r e n t from t h e t h a t o f t h e o t h e r 2 l i n e s . The y i n t e r c e p t o f r H + D M 0 v s r H + F T and r H + F T v s r C l " a r e a l s o s i g n i f i c a n t l y (p<0.05) d i f f e r e n t . 130 0.8 0.6 0.4 . 4 * 4 • *** 7.0 7.4 7.8 p H e 131 F i g u r e 19. E r y t h r o c y t e pH (pH^) v e r s u s e x t r a c e l l u l a r pH (pH e) o f t o a d b l o o d . E r y t h r o c y t e pH was d e t e r m i n e d v i a t h e DMO method. The dependence o f e r y t h r o c y t e pH on t h e e x t r a c e l l u l a r pH i s g i v e n by t h e f o l l o w i n g r e g r e s s i o n l i n e : p H i = 0.706 x p H e + 1.998, r = 0.99 (n=32). pHi 7.4 7.0 • v L —I 1 1 -I I I 7.0 74 7.8 p H e 133 0.029 respectively. The difference between the r C l " and the *"H + D M Q i s , therefore, only 20% of the difference between r C l " and r H + D M 0 . The discrepancy between the d i s t r i b u t i o n r a t i o s for protons r e s u l t s from a consistent difference obtained for the erythrocyte pH between the DMO method and the freeze-thaw method (Table 8) . The difference i n these two values was 0.13 pH units at an e x t r a c e l l u l a r pH of 7.732. This table also shows the e f f e c t of a saline bridge during pH^ measurements v i a the freeze-thaw method on t h i s difference. The addition of the sa l i n e bridge c l e a r l y eliminated t h i s difference and the pH^ values were no longer s i g n i f i c a n t l y d i f f e r e n t . The rela t i o n s h i p between pH e and erythrocyte pH (pHjJ i s p l o t t e d i n Figu r e 19. The e r y t h r o c y t e pH increases l i n e a r l y with increasing e x t r a c e l l u l a r pH. T a b l e 7. O v e r a l l means o f d i s t r i b u t i o n r a t i o s f o r c h l o r i d e ( r C l " ) and f o r p r o t o n s w i t h e r y t h r o c y t e pH d e t e r m i n e d v i a t h e f r e e z e - t h a w method ( r H + F T ) and by t h e DMO method ( r H + D M Q ) . Mean S.E.M. 0.025 0.023 0.029 r C l r H + FT rH + DMO 0.607 0.470 0.635 135 T a b l e 8. E r y t h r o c y t e pH (pH^) d e t e r m i n e d by t h e f r e e z e -thaw method ( F T ) , f r e e z e - t h a w method w i t h s a l i n e b r i d g e ( F T + s a l ) , DMO d i s t r i b u t i o n method (DMO) and p r e d i c t e d from t h e e x t r a c e l l u l a r pH and t h e c h l o r i d e d i s t r i b u t i o n ( C I " ) . P H 6 P H i ( F T ) P Hi(DMO) P H i ( C l - ) 7.732+0.012 7.731+0.018 7.449+0.012* 7.443+0.010* P H e P H i ( F T + s a l ) P Hi(DMO) P H i ( C l - ) 7.738+0.010 7.469+0.023 7.447+0.011 7.455+0.015 V a l u e s (n=9) a r e means + one S.E.M. A s t e r i s k i n d i c a t e s s i g n i f i c a n t ( u n p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from t h e f r e e z e - t h a w o r f r e e z e - t h a w + s a l i n e v a l u e . 136 D i s c u s s i o n The w a t e r c o n t e n t o f Bufo marinus e r y t h r o c y t e s i n c r e a s e s w i t h d e c r e a s i n g pH ( F i g . 17) as i t does i n t h e rain b o w t r o u t , Salmo g a i r d n e r i (Heming e t a l . , 1986). I n t h e t r o u t , t h e i n c r e a s e i n w a t e r c o n t e n t i s a s s o c i a t e d w i t h an i n c r e a s e i n t h e c h l o r i d e r a t i o ( C l ~ i n / C l ~ o u + . ) a c r o s s t h e e r y t h r o c y t e membrane (Heming e t a l . , 1986). The p r e s e n t s t u d y demonstrates t h a t t h e c h l o r i d e r a t i o a c r o s s t h e e r y t h r o c y t e membrane i n Bufo marinus a l s o i n c r e a s e s w i t h d e c r e a s i n g pH. S i m i l a r t o mammalian n o n n u c l e a t e d e r y t h r o c y t e s , t h e n u c l e a t e d e r y t h r o c y t e s o f a m p h i b i a n s p o s s e s s a c h l o r i d e / b i c a r b o n a t e e x c h a n g e mechanism ( C a l a , 1980; B o u t i l i e r and Toews, 1981), and c h l o r i d e and b i c a r b o n a t e a r e p a s s i v e l y d i s t r i b u t e d a c r o s s t h e e r y t h r o c y t e membrane ( M c D o n a l d e t a _ l . , 1980) . A c c o r d i n g t o Hl a d k y and R i n k (1977), d e c r e a s i n g pH i n n u c l e a t e d e r y t h r o c y t e s causes c h l o r i d e t o e n t e r t h e c e l l i n r e s p o n s e t o charge changes on t h e haemoglobin and wa t e r i s drawn i n t o t h e c e l l o s m o t i c a l l y . The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t t h e wa t e r c o n t e n t o f amphibian e r y t h r o c y t e s i s i n f l u e n c e d by s i m i l a r mechanisms. The d i s t r i b u t i o n r a t i o f o r p r o t o n s ( r H + ) v a r i e s 137 i n v e r s e l y t o t h e c h l o r i d e d i s t r i b u t i o n r a t i o ( r C l ~ ) w i t h c h a n g e s i n e x t r a c e l l u l a r pH. The r e g r e s s i o n l i n e s g e n e r a t e d f o r r H + D M 0 v e r s u s p H e and r C l " v e r s u s p H e a r e s i g n i f i c a n t l y d i f f e r e n t o v e r t h e e n t i r e pH range s t u d i e d . I n t h e p h y s i o l o g i c a l pH range (above pH 7.0), however, t h e s e l i n e s a r e i d e n t i c a l . These r e s u l t s i n d i c a t e t h a t p r o t o n s and c h l o r i d e i o n s a r e i n a Donnan e q u i l i b r i u m i n t h e p h y s i o l o g i c a l pH range i n Bufo marinus e r y t h r o c y t e s and p r o t o n s a r e a l s o p a s s i v e l y d i s t r i b u t e d . I n c o n t r a s t , t h e r e g r e s s i o n l i n e g e n e r a t e d f o r r H + F T v e r s u s p H e i s s i g n i f i c a n t l y d i f f e r e n t t h a n t h e r e g r e s s i o n l i n e s f o r r C l " and r H + D M 0 v e r s u s pH e. T h i s r e s u l t i n d i c a t e s t h a t p r o t o n s may not be p a s s i v e l y d i s t r i b u t e d i n amphibian e r y t h r o c y t e s . The s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e l i n e s g e n e r a t e d f o r t h e d i s t r i b u t i o n r a t i o s f o r p r o t o n s i s a r e s u l t o f t h e c o n s i s t e n t d i f f e r e n c e o b t a i n e d f o r t h e e r y t h r o c y t e pH between t h e DMO method and t h e f r e e z e - t h a w method. W h i l e t h e s e two methods g i v e s i m i l a r v a l u e s f o r t h e e r y t h r o c y t e pH i n n o n n u c l e a t e d e r y t h r o c y t e s (Waddel and B a t e s , 1969; Roos and Boron, 1981) and i n n u c l e a t e d t r o u t e r y t h r o c y t e s ( M i l l i g a n and Wood, 1984), a s i m i l a r d i s c r e p a n c y o c c u r s i n t h e e r y t h r o c y t e s o f t h e c a r p ( A l b e r s and Goetz, 1985). I n t h e l a t t e r , e r y t h r o c y t e pH 138 d e t e r m i n e d by t h e f r e e z e - t h a w method was found t o be 0.15 pH u n i t s l o w e r t h a n t h a t o b t a i n e d from t h e DMO method. A t an e x t r a c e l l u l a r pH o f 7.732, t h e d i f f e r e n c e between t h e s e two v a l u e s i n t h i s s t u d y was 0.13 pH u n i t s . Trapped e x t r a c e l l u l a r f l u i d i n t h e packed e r y t h r o c y t e s was n o t r e s p o n s i b l e f o r t h i s d i f f e r e n c e . I n d e e d , c a l c u l a t i o n s which c o r r e c t f o r t r a p p e d e x t r a c e l l u l a r f l u i d p r e d i c t a g r e a t e r pH d i f f e r e n c e . A l b e r s and Goetz (1985) h y p o t h e s i z e d t h a t t h i s d i s c r e p a n c y c o u l d be due t o a ' s u s p e n s i o n e f f e c t ' a t t h e l i q u i d j u n c t i o n p o t e n t i a l between t h e sample and t h e KC1 s o l u t i o n o f t h e r e f e r e n c e e l e c t r o d e . T h i s p o t e n t i a l i s caused by t h e suspended n e g a t i v e l y c h a r g e d p a r t i c l e s i n t h e haemolysate and i s s i m i l a r t o t h e ' h a e m o l y s i s e f f e c t ' d e s c r i b e d i n t h e measurement o f whole b l o o d pH ( S i g g a a r d - A n d e r s e n , 1961; 1974; Maas, 1970; S a i l i n g and S i g g a a r d - A n d e r s e n , 1971; B o u t i l i e r e t a _ l . , 1985) . I n w h o l e b l o o d , S i g g a a r d -Andersen (1961) demonstrated t h a t t h i s e f f e c t d i s a p p e a r e d when p h y s i o l o g i c a l s a l i n e was p l a c e d b e t w e e n t h e s a t u r a t e d KC1 s o l u t i o n and t h e s a m p l e i n t h e pH e l e c t r o d e . Presumably, t h e s a l i n e b r i d g e e l i m i n a t e s t h e c h a r g e d i f f e r e n c e and, t h e r e f o r e , t h e p o t e n t i a l between t h e sample and t h e K C l s o l u t i o n . When t h i s t e c h n i q u e was 139 employed i n t h e p r e s e n t s t u d y t o measure t h e pH o f t h e e r y t h r o c y t e h a e m o l y s a t e , t h e s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two methods o f pH^ measurement was e l i m i n a t e d . Thus, t h e d i f f e r e n c e i s a p p a r e n t l y due t o a ' s u s p e n s i o n e f f e c t ' a t t h e l i q u i d j u n c t i o n p o t e n t i a l o f t h e pH e l e c t r o d e . T h us, p r o t o n s a r e i n Donnan e q u i l i b r i u m w i t h c h l o r i d e i o n s i n amphibian e r y t h r o c y t e s and a r e p a s s i v e l y d i s t r i b u t e d . The s l o p e o f t h e e r y t h r o c y t e pH v e r s u s p H e r e g r e s s i o n l i n e f o r Bufo marinus (0.706) b l o o d i s s i m i l a r t o t h a t r e p o r t e d by Nikinmaa e t a l . , (1987) f o r rainbow t r o u t b l o o d (0.721). Both o f t h e s e v a l u e s , however, a r e q u i t e d i f f e r e n t f r o m t h e s l o p e (0.471) f o r t h i s r e l a t i o n s h i p r e p o r t e d by Heming e t al., ( 1 9 8 6 ) . E r y t h r o c y t i c NTP l e v e l s i n f l u e n c e t h e n e t c h a rge w i t h i n t h e e r y t h r o c y t e and, t h e r e f o r e , t h e d i s t r i b u t i o n o f p r o t o n s a c r o s s t h e e r y t h r o c y t e membrane i f p r o t o n s a r e p a s s i v e l y d i s t r i b u t e d . Indeed, Wood e t a l . , (1975) has d e m o n s t r a t e d t h a t t h e b l o o d ATP c o n c e n t r a t i o n a f f e c t s t h e pH^ v e r s u s p H e r e l a t i o n s h i p . I t i s t h e r e f o r e p o s s i b l e t h a t d i f f e r e n c e s i n e r y t h r o c y t i c NTP l e v e l s r e s u l t i n g f r o m d i f f e r e n t e x p e r i m e n t a l p r o t o c o l s c a u s e d t h e d i f f e r e n c e s i n t h e s l o p e s o f e r y t h r o c y t e pH v e r s u s p H e i n t h e s e s t u d i e s . 140 Summary 1. The w a t e r c o n t e n t o f B u f o m a r i n u s e r y t h r o c y t e s i n c r e a s e s w i t h d e c r e a s i n g e x t r a c e l l u l a r pH. 2. P r o t o n s and c h l o r i d e i o n s a r e i n Donnan e q u i l i b r i u m and a r e b o t h p a s s i v e l y d i s t r i b u t e d a c r o s s t h e e r y t h r o c y t e membrane. 3. There i s a c o n s i s t e n t d i f f e r e n c e i n t h e measurement o f t h e e r y t h r o c y t e pH i n t h i s s p e c i e s between t h e DMO method and t h e f r e e z e - t h a w method. 4. T h i s d i f f e r e n c e i s a p p a r e n t l y due t o a ' s u s p e n s i o n e f f e c t ' a t t h e l i q u i d j u n c t i o n p o t e n t i a l o f t h e pH e l e c t r o d e e n c o u n t e r e d when measuring t h e haemolysate pH. 141 CHAPTER.6: ION EXCHANGE MECHANISMS ON THE ERYTHROCYTE MEMBRANE OF THE AQUATIC SALAMANDER, AMPHIUMA TRIDACTYLUM I n t r o d u c t i o n : The a q u a t i c s a l a m a n d e r , Amphiuma l i v e s i n an environment w h i c h i s p a r t open w a t e r and p a r t v e g e t a t i o n c o v e r e d w a t e r i n w h i c h w a t e r PC0 2's o f 50-60 mm Hg may be a s s o c i a t e d w i t h h y p o x i a ( U l t s c h , 1976; H e i s l e r e t a l . , 1982). A c c o r d i n g t o Toews e t a l . , (1971), most v o l u n t a r y d i v e s i n t h e s e a i r b r e a t h i n g a n i m a l s a r e p r o b a b l y a e r o b i c . A v o i d a n c e o f p r e d a t o r s i n t h i s t y p e o f e n v i r o n m e n t , h o w e v e r , u n d o u b t e d l y i n v o l v e s d i v e s o f l o n g e r d u r a t i o n d u r i n g w h i c h t i m e oxygen s t o r e s may be d e p l e t e d . These a n i m a l s a l s o have a s u b s t a n t i a l Bohr e f f e c t (-0.205; L e n f a n t and Johansen, 1967) w h i c h may e x a c e r b a t e t h e problems o f oxygen t r a n s p o r t under t h e s e c o n d i t i o n s . As i n B u f o m a r i n u s . t h e s e a n i m a l s p r e f e r e n t i a l l y d e f e n d t h e pH o f t h e i n t r a c e l l u l a r c o m p a r t m e n t d u r i n g a c i d - b a s e d i s t u r b a n c e s ( H e i s l e r e t a l . , 1982). I t seems t h a t Amphiuma might a l s o b e n e f i t from a mechanism wh i c h r e g u l a t e d t h e e r y t h r o c y t e pH as i n s a l m o n i d f i s h and t h e lamprey. A g a i n , however, t h e pH o f t h e e r y t h r o c y t e has n o t been m o n i t o r e d d u r i n g an a c i d o s i s i n Amphiuma. 142 C a l a (1980, 1985) has d e s c r i b e d t h e i o n exchange mechanisms i n v o l v e d i n vo l u m e r e g u l a t i o n i n Amphiuma e r y t h r o c y t e s . Volume r e g u l a t o r y d e c r e a s e a f t e r o s m o t i c s w e l l i n g i s a s s o c i a t e d w i t h a n e t l o s s o f e r y t h r o c y t e p o t a s s i u m and c h l o r i d e whereas volume r e g u l a t o r y i n c r e a s e a f t e r o s m o t i c s h r i n k a g e i n v o l v e s l o o s e l y c o u p l e d s o d i u m / p r o t o n and c h l o r i d e / b i c a r b o n a t e e x c h a n g e mechanisms. D u r i n g such volume r e g u l a t o r y i n c r e a s e s , t h e e x t r a c e l l u l a r medium i s a c i d i f i e d . Thus, t h e o b s e r v e d v o l u m e i n c r e a s e may be a s s o c i a t e d w i t h c h a n g e s i n e r y t h r o c y t e pH as i n f i s h e r y t h r o c y t e s . The e f f e c t s o f t h e s e p r o c e s s e s on t h e pH i n t h e s e e r y t h r o c y t e s , however, has n o t been documented. T h i s s t u d y examined t h e c h a r a c t e r i s t i c s o f t h e i o n exchange mechanisms i n Amphiuma e r y t h r o c y t e s by u s i n g d i f f e r e n t t r a n s p o r t and m e t a b o l i c i n h i b i t o r s , and i o n s u b s t i t u t i o n s . The s e n s i t i v i t y o f t h e s e i o n exchange mechanisms t o b e t a - a d r e n e r g i c s t i m u l a t i o n was a l s o i n v e s t i g a t e d . The purpose o f t h e s e e x p e r i m e n t s was t o d e t e r m i n e i f Amphiuma i s c a p a b l e o f r e g u l a t i n g e r y t h r o c y t e pH as documented i n f i s h and lamp r e y s . 143 Methods Samples o f a r t e r i a l b l o o d were c o l l e c t e d from c a n n u l a t e d salamanders and c e n t r i f u g e d i n h e p a r i n i z e d 1.5 mL eppendorf t u b e s . The plasma was d i s c a r d e d f o l l o w i n g c e n t r i f u g a t i o n . The c e l l s w ere t h e n washed i n M a c k e n z i e ' s a m p h i b i a n s a l i n e a d j u s t e d t o 30 mM b i c a r b o n a t e w i t h t h e e x c e p t i o n t h a t i n one s e t o f e x p e r i m e n t s i n w h i c h t h e s a l i n e c o n t a i n e d t h e impermeant c a t i o n c h o l i n e i n s t e a d o f sodium. The p r o t o c o l f o r t h e w a s h i n g o f t h e c e l l s was v a r i a b l e d e p e n d i n g on t h e e x p e r i m e n t . I n t h e e x p e r i m e n t s i n w h i c h c h o l i n e r e p l a c e d sodium, t h e c e l l s were l e f t suspended f o r 10 minutes d u r i n g each wash t o improve t h e removal o f sodium. I f o u a b a i n (10~ 4M) o r 2 , 4 - d i n i t r o p h e n o l (2,4-D.N.P.; 10~ 4M) were t o be used i n t h e e x p e r i m e n t , t h e d r u g would be p r e s e n t i n a l l washings. I f a m i l o r i d e (10~ 3M) o r DIDS (1 0 ~ 4 ) were t o be used, t h e y were o n l y added t o t h e f i n a l s u s p e n s i o n . I n a l l c a s e s , t h e e r y t h r o c y t e s were resuspended t o a p p r o x i m a t e l y 15% h a e m a t o c r i t f o l l o w i n g t h e second wash i n t h e a p p r o p r i a t e i n c u b a t i o n media and 10 uL o f 1 uCi/mL 1 4C-DM0 (New E n g l a n d N u c l e a r , s p e c i f i c a c t i v i t y 50 mCi/mmol) was added t o t h e s u s p e n s i o n f o r 144 l a t e r d e t e r m i n a t i o n o f i n t r a c e l l u l a r pH. The s u s p e n s i o n s were t h e n i n c u b a t e d i n 50 mL tonometers a t 25°C and g a s s e d w i t h h u m i d i f i e d m i x t u r e s o f e i t h e r 4% c a r b o n d i o x i d e / 9 6 % a i r o r 8% c a r b o n d i o x i d e / 9 2 % a i r . The c a r b o n d i o x i d e m i x t u r e s were d e l i v e r e d by W o s t h o f f gas m i x i n g pumps (Wosthoff Digamix, Type M-300a and M-30f, Bochum, FRG) . F o l l o w i n g a 30 minute e q u i l i b r a t i o n p e r i o d , two 0.4 mL s a m p l e s were t a k e n f r o m t h e t o n o m e t e r and c e n t r i f u g e d . The pH o f t h e e x t r a c e l l u l a r f l u i d was t h e n measured i m m e d i a t e l y from t h e f i r s t sample t u b e u s i n g a R a d i o m e t e r PHM 72 and a s s o c i a t e d m i c r o - p H u n i t (Radiometer, Copenhagen, Denmark). A 100 uL sample o f e x t r a c e l l u l a r f l u i d was t a k e n from t h e second sample t u b e f o r t h e d e t e r m i n a t i o n o f DMO l e v e l s f o r s u b s e q u e n t c a l c u l a t i o n o f p H i . The e r y t h r o c y t e s from t h e f i r s t sample t u b e were saved f o r t h e p H i d e t e r m i n a t i o n and t h o s e from t h e second sample t u b e were saved f o r t h e d e t e r m i n a t i o n o f c e l l u l a r w a t e r c o n t e n t . A t t h i s p o i n t , t h e e x p e r i m e n t was e i t h e r t e r m i n a t e d , o r i n t h o s e e x p e r i m e n t s i n w h i c h t h e e f f e c t s o f a d r e n a l i n e were t o be a s s e s s e d , 5 xlO~ 5M a d r e n a l i n e was added t o a l l tonometers e x c e p t t h e c o n t r o l . I n t h e a d r e n a l i n e e x p e r i m e n t s , t h e s a m p l i n g p r o c e d u r e was t h e n r e p e a t e d f o l l o w i n g a n o t h e r 30 minutes o f e q u i l i b r a t i o n . 145 R e s u l t s The pH and v o l u m e o f Amphiuma t r i d a c t y l u m e r y t h r o c y t e s were n ot i n f l u e n c e d by t h e i n c u b a t i o n t i m e s i n t h e range o f 30 t o 60 min. The p r e s e n c e o f t h e a n i o n e x c h a n g e i n h i b i t o r DIDS, h o w e v e r , c a u s e d a marked i n t r a c e l l u l a r a l k a l i n i z a t i o n and d e c r e a s e i n t h e e r y t h r o c y t e w a t e r c o n t e n t a t b o t h c a r b o n d i o x i d e t e n s i o n s s t u d i e d ( T a b l e 9, 10). The i n t r a c e l l u l a r a l k a l i n i z a t i o n c o u l d be p r e v e n t e d o r reduced by (1) removing sodium from t h e i n c u b a t i o n medium (2) t r e a t i n g t h e c e l l s w i t h a m i l o r i d e (3) b l o c k i n g t h e sodium/potassium pump w i t h o u a b a i n ( T a b l e 1 1 ) . These r e s u l t s i n d i c a t e t h a t t h e r e i s a sodium/proton exchange mechanism on t h e e r y t h r o c y t e membrane w h i c h i s f u n c t i o n a l u n d e r s t e a d y - s t a t e c o n d i t i o n s . T h i s e x c h a n g e i s d r i v e n by t h e s o d i u m g r a d i e n t produced by t h e sodium/potassium pump. I n t h e absence o f b i c a r b o n a t e movements as o c c u r s i n t h e DIDS e x p e r i m e n t s ( T a b l e 9, 1 0 ) , t h e i n t r a c e l l u l a r pH i s c o n t r o l l e d by t h e sodium/proton exchange. The p r e s e n c e o f a m i l o r i d e a l o n e i n t h e i n c u b a t i o n medium caused o n l y s l i g h t changes i n t h e pH g r a d i e n t a c r o s s t h e e r y t h r o c y t e membrane, b u t d i d c a u s e a 146 T a b l e 9. E f f e c t s o f a d r e n a l i n e , DIDS and a m i l o r i d e on t h e w a t e r c o n t e n t , e x t r a c e l l u l a r pH ( p H e ) , e r y t h r o c y t e pH (pH^) and on t h e pH g r a d i e n t (dpH) o f Amphiuma  t r i d a c t v l u m e r y t h r o c y t e s e q u i l i b r a t e d w i t h 4% C 0 2 / 9 6 % a i r . i n c u b a t i o n medium 30 min t r e a t m e n t 60 min s a l i n e ( c o n t . ) H 20 74.0+0.6 (8) A 74.0+0.7 (8) p H e 7.62+0.01 (10) 7.63+0.02 (10) 7.39+0.02 (10) 7.37+0.02 (10) dpH +0.23 (10) +0.26 (10) s a l i n e H 20 73.2+0.6 (7) ^  B 75.3+0.09 (7) p H e 7.64+0.01 (9) 7.66+0.01 (9) pHj^ 7.40+0.02 (9) 7.42+0.01 (9) + dpH +0.24 (9) +0.24 (9) s a l i n e + DIDS H 20 70.8+0.4 (8)+ B 72.0+0.8 ( 8 ) * p H e 7.64+0.02 (9) 7.64+0.02 (9) pHj^ 7.64 + 0.03 (9)+ 7.62+0.03 (9) + dpH +0.01 (9) +0.02 (9) s a l i n e + a m i l . H 20 68.5+0.8 (8)+ B 70.5+0.9 (8)+ p H e 7.68+0.02 (10)+ 7.66+0.01 (10) pUi 7.39±0.03 (10) 7.42+0.03 (10) dpH +0.29 (10) +0.24 (10) V a l u e s a r e means + one S.E.M. (n) . A s t e r i s k d enotes s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from 3 0 min v a l u e . + denotes s i g n i f i c a n t ( u n p a i r e d t -t e s t ; p<0.05) d i f f e r e n c e from t r e a t m e n t A. Treatment A = c o n t r o l , t r e a t m e n t B = 5 x 1 0 ~ 5 M a d r e n a l i n e . 147 T a b l e 10. E f f e c t s o f a d r e n a l i n e , DIDS and a m i l o r i d e on t h e w a t e r c o n t e n t , e x t r a c e l l u l a r pH ( p H e ) , e r y t h r o c y t e pH (pH^) and on t h e pH g r a d i e n t (dpH) o f Amphiuma  t r i d a c t y l u m e r y t h r o c y t e s e q u i l i b r a t e d w i t h 8% C 0 2 / 9 2 % a i r . i n c u b a t i o n medium 30 min t r e a t m e n t 60 min s a l i n e ( c o n t . ) H 20 p H e P H i dpH s a l i n e H 20 p H e P H ± dpH s a l i n e + DIDS H 20 p H e P H i dpH s a l i n e + a m i l . H 20 p H e P H i dpH 72.6+0.4 (7) 7.43+0.02 (9) 7.26+0.02 (9) +0.17 (9) 73.0+0.6 (8) B 7.45+0.02 (10) 7.31+0.04 (10) +0.14 (10) 72.0+0.02 (8) B 7.43+0.03 (10) 7.48+0.02 (10)+ -0.05 (10) 71.7+0.6 (8) B 7.48+0.02 (10) 7.29+0.04 (10) +0.19 (10) 73.8+0.2 (7) 7.44+0.02 (9) 7.32+).04 (9) +0.11 (9) 74.1+0.3 (8) 7.43+0.02 (10) 7.27+0.02 (10) +0.16 (10) 72.4+0.4 (8)+ 7.41+0.02 (10) 7.45+0.02 (10)+ -0.04 (10) 71.8+0.8 (8)+ 7.46+0.02 ( 1 0 ) * 7.31+0.03 (10) +0.14 (10) V a l u e s a r e means + one S.E.M. (n) . A s t e r i s k d e notes s i g n i f i c a n t ( p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from t r e a t m e n t A. Treatment A = c o n t r o l , t r e a t m e n t B = 5 x 1 0 ~ 5 M a d r e n a l i n e . 148 T a b l e 11. E f f e c t s o f DIDS, a m i l o r i d e , o u a b a i n and absence o f e x t r a c e l l u l a r sodium on t h e w a t e r c o n t e n t , e x t r a c e l l u l a r pH ( p H e ) , i n t r a c e l l u l a r pH (pH^) and on t h e pH g r a d i e n t (dpH) o f Amphiuma t r i d a c t v l u m e r y t h r o c y t e s e q u i l i b r a t e d w i t h 4% C0 2/96% a i r . H 20 p H e P H i dpH s a l i n e c o n t r o l 73.8+0.6 (4) 7.54+0.01 (4) 7.36+0.02 (4) +0.17 (4) s a l i n e + DIDS 70.9+0, 7.51+0, 7.53+0, 6 ( 4 ) * 04 (4) 05 ( 4)' -0.02 (4) s a l i n e , DIDS + a m i l o r i d e 67.0+1.0 ( 8 ) * 7.54+0.01 (8) 7.35+0.03 (8) +0.20 (8) H 20 p H e P H i dpH H 20 p H e P H i dpH s a l i n e c o n t r o l 75.5+1.1 (6) 7.52+0.01 (6) 7.32+0.04 (6) +0.20 (6) s a l i n e (no Na +) c o n t r o l 67.7+0.4 (20) 7.63+0.03 (4) 7.32+0.06 (4) +0.32 (4) s a l i n e + o u a b a i n 73.9+0.6 (6) 7.54±0.01 (6) 7.24+0.03 (6) +0.29 (6) s a l i n e , o u a b a i n + DIDS 72.2+0.7 ( 6 ) * 7.46+0.01 ( 6 ) * 7.33+0.02 (6) +0.13 (6) s a l i n e (no Na +) + DIDS 67.9+0.4 (24) 7.66+0.01 (8) 7.32±0.03 (8) +0.34 (8) V a l u e s a r e means + one S.E.M. (n) A s t e r i s k d e n o t e s s i g n i f i c a n t ( u n p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e from s a l i n e c o n t r o l . 149 T a b l e 12. Comparison o f t h e e r y t h r o c y t e pH d e t e r m i n e d by t h e DMO d i s t r i b u t i o n ( P H i ( D M 0 ) ) a n d t h e e r y t h r o c y t e P H p r e d i c t e d from t h e e x t r a c e l l u l a r pH and t h e c h l o r i d e d i s t r i b u t i o n ( P H i ( c i - ) ) ^ n Amphiuma t r i d a c t y l u m e r y t h r o c y t e s . i n c u b a t i o n medium P Hi(DMO) P H i ( C l - ) 4% C0 2/96% a i r s a l i n e 7.37+0.02 (10) 7.32+0.03 ( 1 0 ) * s a l i n e + DIDS 7.60+0.04 (6) 7.08+0.05 ( 6 ) * s a l i n e + a m i l o r i d e 7.38+0.04 (6) 7.32+0.05 ( 6 ) * s a l i n e + 2,4-D.N.P. 7.26+0.02 (7) 7.28+0.02 (7) 8% C0 2/92% a i r s a l i n e 7.23+0.02 (10) 7.16+0.03 ( 1 0 ) * s a l i n e + DIDS 7.43+0.01 (6) 6.93±0.08 ( 6 ) * s a l i n e + a m i l o r i d e 7.22+0.03 (6) 7.14±0.06 (6) V a l u e s a r e means + one S.E.M. ( n ) . A s t e r i s k d e n o t e s s i g n i f i c a n t ( u n p a i r e d t - t e s t ; p<0.05) d i f f e r e n c e between p H i ( D M 0 ) and p H i ( c l _ ) . 150 r e d u c t i o n i n t h e e r y t h r o c y t e water c o n t e n t ( T a b l e . 9 , 10). T h e s e r e s u l t s i n d i c a t e t h a t t h e c h l o r i d e / b i c a r b o n a t e exchange i s c a p a b l e o f e q u i l i b r a t i n g a c i d e q u i v a l e n t s under normal c i r c u m s t a n c e s . The s o d i u m / p r o t o n e x c h a n g e mechanism on t h e e r y t h r o c y t e membrane d i d not appear t o be s e n s i t i v e t o b e t a - a d r e n e r g i c s t i m u l a t i o n . A d r e n a l i n e a l o n e had no s i g n i f i c a n t e f f e c t s on any o f t h e measured v a r i a b l e s . I n t h e p r e s e n c e o f DIDS o r a m i l o r i d e , s i g n i f i c a n t d i f f e r e n c e s were s e e n b e t w e e n c o n t r o l and a d r e n a l i n e s a m p l e s . T h e s e d i f f e r e n c e s , h o w e v e r , were n o t c o n s i s t e n t . T a b l e 12 compares t h e e r y t h r o c y t e pH d e t e r m i n e d by t h e DMO method i n s e v e r a l e x p e r i m e n t s w i t h t h e e r y t h r o c y t e pH p r e d i c t e d by t h e d i s t r i b u t i o n o f c h l o r i d e a c r o s s t h e e r y t h r o c y t e membrane u s i n g t h e Donnan e q u a t i o n . T h e s e r e s u l t s showed t h a t t h e r e was a s i g n i f i c a n t d i f f e r e n c e (0.05-0.07 pH u n i t s ) between t h e two d e t e r m i n a t i o n s , even i n t h e c o n t r o l s w h i c h was not a f f e c t e d by a m i l o r i d e . T h i s d i f f e r e n c e was e x a c e r b a t e d (0.50-0.52 pH u n i t s ) i n t h e p r e s e n c e o f DIDS, and c o m p l e t e l y a b o l i s h e d when t h e i o n o p h o r e 2,4-DNP was added t o t h e medium. 151 D i s c u s s i o n C a l a (1980, 1985) has demonstrated t h a t Amphiuma e r y t h r o c y t e s u s e l o o s e l y c o u p l e d , e l e c t r i c a l l y s i l e n t s o d i u m / p r o t o n and c h l o r i d e / b i c a r b o n a t e e x c h a n g e m echanisms t o r e - e s t a b l i s h c e l l v o l u m e a f t e r o s m o t i c s h r i n k a g e . D u r i n g t h i s p r o c e s s , sodium and c h l o r i d e e n t e r t h e c e l l i n exchange f o r p r o t o n s and b i c a r b o n a t e i o n s r e s p e c t i v e l y and c e l l volume i s r e s t o r e d p a s s i v e l y due t o o s m o t i c a l l y o b l i g a t e d w a t e r f l o w . From t h e p r e s e n t e x p e r i m e n t s , i t a l s o appears t h a t sodium/proton and c h l o r i d e / b i c a r b o n a t e e x c h a n g e r s a r e i n v o l v e d i n s t e a d y - s t a t e c e l l v o l u m e r e g u l a t i o n . I n h i b i t i o n o f e i t h e r o f t h e s e exchanger mechanisms causes s i g n i f i c a n t r e d u c t i o n s i n c e l l v o l u m e w h i c h a r e most p r o n o u n c e d (9.2%) when b o t h i o n exchange pathways a r e b l o c k e d . The l a r g e d e c r e a s e i n t h e transmembrane pH g r a d i e n t i n t h e p r e s e n c e o f DIDS ( T a b l e 10, 11) a l s o i n d i c a t e s t h a t t h e sodium/proton a n t i p o r t e r i s c o n s t a n t l y f u n c t i o n i n g and removing p r o t o n s from t h e c e l l i n t e r i o r i n exchange f o r sodium. DIDS b l o c k a d e o f c h l o r i d e / b i c a r b o n a t e exchange e n a b l e s t h i s p r o t o n e x t r u s i o n mechanism t o a l k a l i n i z e t h e i n t r a c e l l u l a r compartment r e l a t i v e t o t h e e x t r a c e l l u l a r 152 medium s i n c e b i c a r b o n a t e can no l o n g e r e q u i l i b r a t e a c r o s s t h e e r y t h r o c y t e membrane. Replacement o f sodium w i t h t h e impermeant c a t i o n c h o l i n e o r t h e p r e s e n c e o f a m i l o r i d e i n t h e i n c u b a t i o n medium i n h i b i t s t h i s i n t r a c e l l u l a r a l k a l i n i z a t i o n . T h i s s u p p o r t s t h e h y p o t h e s i s t h a t t h e i n t r a c e l l u l a r a l k a l i n i z a t i o n o c c u r s due t o s o d i u m dependent p r o t o n e x t r u s i o n as i s t h e case i n t e l e o s t e r y t h r o c y t e s f o l l o w i n g b e t a - a d r e n e r g i c s t i m u l a t i o n (Nikinmaa and H u e s t i s , 1984). The mechanism o f p r o t o n e x t r u s i o n i n Amphiuma e r y t h r o c y t e s i s a l s o o u a b a i n s e n s i t i v e . The sodium/proton exchange mechanism t h e r e f o r e seems t o be dependent on t h e sodium/potassium pump. The a s s o c i a t i o n between t h e s e two mechanisms has a l s o been documented i n t h e f r o g ( P a l f r e y and Greengard, 1981) . I t has been s u g g e s t e d ( P a l f r e y and G r e e n g a r d , 1981) t h a t b e t a -a d r e n e r g i c s t i m u l a t i o n o f sodium/proton exchange i n t h e f r o g e r y t h r o c y t e r e s u l t s i n subsequent s t i m u l a t i o n o f t h e sodium/potassium pump due t o sodium e n t r y i n t o t h e c e l l . Our e x p e r i m e n t s i n d i c a t e t h a t t h e i n t r a c e l l u l a r a l k a l i n i z a t i o n o c c u r r i n g i n Amphiuma e r y t h r o c y t e s i n t h e pr e s e n c e o f DIDS i s a l s o dependent on t h e c o n t i n u o u s c y c l i n g o f t h e s e two mechanisms. 153 There i s a s i g n i f i c a n t d i f f e r e n c e a t b o t h c a r b o n d i o x i d e t e n s i o n s between t h e measured e r y t h r o c y t e pH and t h a t c a l c u l a t e d from t h e d i s t r i b u t i o n o f c h l o r i d e (Table 12) . T h i s d i f f e r e n c e may be removed by t r e a t m e n t w i t h t h e p r o t o n o p h o r e 2,4 DNP, b u t n o t by t r e a t m e n t w i t h a m i l o r i d e i n d i c a t i n g t h a t i t i s p r o b a b l y n o t due t o t h e s o dium/proton exchange mechanism. The s m a l l magnitude o f t h i s d i f f e r e n c e i n d i c a t e s t h a t i t i s p r o b a b l y n o t caused by a d i s e q u i l i b r i u m o f p r o t o n s between t h e c y t o p l a s m and t h e e x t r a c e l l u l a r space. I t i s p o s s i b l y due t o t h e f a c t t h a t p r o t o n s a r e not p a s s i v e l y d i s t r i b u t e d a c c o r d i n g t o a Donnan e q u i l i b r i u m i n a l l t h e i n t r a c e l l u l a r compartments. Upon b e t a - a d r e n e r g i c s t i m u l a t i o n , t e l e o s t e r y t h r o c y t e s show a marked i n c r e a s e i n b o t h c e l l w a t e r c o n t e n t and pH (Nikinmaa, 1982; B a r o i n e t a l . , 1 9 8 4 a , b ; Nikinmaa and H u e s t i s , 1984; Heming e t a l . , 1987). These a d r e n e r g i c e f f e c t s c o n t r i b u t e t o an i n c r e a s e i n haemoglobin o x y g e n - a f f i n i t y d u r i n g s t r e s s (Nikinmaa e t a l . , 1984; Primmett e t a l . , 1986). There have been v e r y few s t u d i e s , however, documenting t h e e f f e c t s o f b e t a -a d r e n e r g i c s t i m u l a t i o n o f amphibian e r y t h r o c y t e s . R u d o l f and G r e e n g a r d (1980) h a v e f o u n d t h a t Rana p i p i e n s e r y t h r o c y t e s w i l l s w e l l i f exposed t o i s o p r o t e r e n o l i n t h e p r e s e n c e o f a p h o s p h o d i e s t e r a s e i n h i b i t o r . The 154 r e s u l t s o f t h e p r e s e n t i n v e s t i g a t i o n , however, i n d i c a t e t h a t b e t a - a d r e n e r g i c s t i m u l a t i o n does n o t a f f e c t e i t h e r t h e c e l l w a t e r c o n t e n t o r t h e pH o f e r y t h r o c y t e s from t h e a q u a t i c a i r - b r e a t h i n g u r o d e l e , Amphiuma t r i d a c t y l u m . T h e s e r e s u l t s a r e s i m i l a r t o t h o s e f o u n d i n t o a d s ( C h a p t e r 5) . The f u n c t i o n a l s i g n i f i c a n c e o f t h e d i f f e r e n c e i n b e t a - a d r e n e r g i c s e n s i t i v i t y b e t w e e n amphibian and t e l e o s t e r y t h r o c y t e s i s n o t c l e a r . Both Amphiuma and Bufo a r e a i r b r e a t h e r s . I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e b e t a - a d r e n e r g i c r e s p o n s e may be an a d a p t a t i o n found o n l y among e x c l u s i v e w a t e r b r e a t h e r s . The d i f f e r e n c e between amphibian and t e l e o s t e r y t h r o c y t e s may a l s o be r e l a t e d t o d i f f e r e n c e s i n t h e o x y g e n t r a n s p o r t c h a r a c t e r i s t i c s o f t h e b l o o d . I n t e l e o s t s , a r e d u c t i o n i n t h e e r y t h r o c y t e pH l o w e r s t h e o x y g e n c a r r y i n g c a p a c i t y o f t h e b l o o d , known as t h e Root s h i f t ( R o o t and I r v i n g , 1943) . T h e r e f o r e , i t w o u l d be b e n e f i c i a l f o r t e l e o s t s t o r e g u l a t e e r y t h r o c y t e pH v i a a d r e n e r g i c mechanisms d u r i n g s t r e s s i n o r d e r t o m a i n t a i n t h e oxygen c a r r y i n g c a p a c i t y o f t h e b l o o d . Amphibian b l o o d does n o t appear t o have a Root s h i f t ( B r i d g e s e t a l . , 1985) and a r e d u c t i o n i n e r y t h r o c y t e pH does not a f f e c t t h e t o t a l o x y g e n c a r r y i n g c a p a c i t y o f t h e e r y t h r o c y t e . 156 Summary 1. T h i s s t u d y s u p o r t s t h e v i e w t h a t t h e membrane o f t h e Amphiuma e r y t h r o c y t e c o n t a i n s s o d i u m / p r o t o n and c h l o r i d e / b i c a r b o n a t e exchange mechanisms. 2. These i o n exchange mechanisms a r e i n v o l v e d i n s t e a d y -s t a t e volume r e g u l a t i o n . 3. The s e c o n d a r i l y a c t i v e s odium/proton exchanger may c a u s e a marked i n t r a c e l l u l a r a l k a l i n i z a t i o n i n t h e absence o f b i c a r b o n a t e movements. 4. I n c o n t r a s t t o t h e sodium/proton exchanger on t h e t e l e o s t e r y t h r o c y t e membrane, t h e sodium/proton exchange mechanism on t h e Amphiuma e r y t h r o c y t e membrane does n ot e x h i b i t b e t a - a d r e n e r g i c s e n s i t i v i t y , b u t i s a c t i v e under s t e a d y - s t a t e c o n d i t i o n s . 157 GENERAL DISCUSSION P r o t o n s a r e p a s s i v e l y d i s t r i b u t e d i n b o t h f i s h ( A l b e r s and Goetz, 1985; Heming e t a l . , 1986; C h a p t e r 1) and amphibian e r y t h r o c y t e s ( C h apter 5, 6) as t h e y a r e i n mammalian e r y t h r o c y t e s ( F i t z s i m o n s and S e n d r o y , 1961; H l a d k y and R i n k , 1 9 7 7 ) . The e r y t h r o c y t e pH w i l l , t h e r e f o r e , be i n f l u e n c e d by t h e e x t r a c e l l u l a r pH and t h e n e t charge on t h e impermeable i o n s w i t h i n t h e e r y t h r o c y t e ( S t e e n and T u r i t z i n , 1 9 6 8 ) . H a e m o g l o b i n and o r g a n i c phosphates a r e t h e predominant impermeable e r y t h r o c y t i c i o n s . I n n u c l e a t e d e r y t h r o c y t e s , i t has r e c e n t l y become ap p a r e n t t h a t t h e e r y t h r o c y t e pH may a l s o be d e t e r m i n e d by t h e a c t i v i t y o f t h e sodium/proton exchange mechanism on t h e e r y t h r o c y t e membrane (Nikinmaa and H u e s t i s , 1984; N i k i n m a a , 1986; Heming e t a l . , 1987 C h a p t e r 1 ) . N a + / H + Exchange i n F i s h E r y t h r o c y t e s : Under s t e a d y - s t a t e c o n d i t i o n s , t h e sodium/proton exchanger i n f i s h e r y t h r o c y t e s i s q u i e s c e n t and i t does n o t a f f e c t t h e volume o r pH o f t h e e r y t h r o c y t e s (Nikinmaa and H u e s t i s , 1984; C h a p t e r 1 ) . B e t a - a d r e n e r g i c s t i m u l a t i o n , however, causes an i n c r e a s e i n t h e a c t i v i t y o f t h i s exchanger and sodium e n t e r s t h e c e l l i n exchange f o r p r o t o n s (Nikinmaa and H u e s t i s , 1984; C o s s i n s and 158 R i c h a r d s o n , 1985; Chapter 1; F i g . 2 0 ) . T h i s exchanger i s s e c o n d a r i l y a c t i v e and i s d e p e n d e n t on t h e s o d i u m g r a d i e n t e s t a b l i s h e d by t h e s o d i u m / p o t a s s i u m A T P a s e . Indeed, Bourne and C o s s i n s (1982) have demonstrated t h a t b e t a - a d r e n e r g i c s t i m u l a t i o n o f rainbow t r o u t e r y t h r o c y t e s r e s u l t s i n a 100-250% i n c r e a s e i n t h e a c t i v i t y o f t h e s o d i u m / p o t a s s i u m A T P a s e . The r e s p o n s e a l s o i n v o l v e s movement o f c h l o r i d e i o n s i n t o t h e c e l l v i a t h e c h l o r i d e / b i c a r b o n a t e exchange mechanism. The i n c r e a s e d c o n c e n t r a t i o n s o f b o t h sodium and c h l o r i d e i o n s causes w a t e r t o be drawn i n t o t h e c e l l o s m o t i c a l l y and t h e e r y t h r o c y t e v o l u m e i n c r e a s e s . T h e r e i s a l s o a s i g n i f i c a n t i n c r e a s e i n t h e e r y t h r o c y t e pH. T h i s i n c r e a s e i n e r y t h r o c y t e pH i s c l e a r l y dependent on t h e a c t i v a t i o n o f t h e sodium/proton exchanger (Nikinmaa and H u e s t i s , 1984; C o s s i n s and R i c h a r d s o n , 1985; Ch a p t e r 1 ) . The o b s e r v e d i n t r a c e l l u l a r a l k a l i n i z a t i o n may be b o t h a d i r e c t and an i n d i r e c t e f f e c t o f t h e a c t i v a t i o n o f t h i s e xchanger. I n a d d i t i o n , i t has been demonstrated t h a t a d r e n e r g i c s t i m u l a t i o n o f f i s h e r y t h r o c y t e s r e s u l t s i n a d e c r e a s e i n c e l l u l a r NTP l e v e l s (Ferguson and B o u t i l i e r , 1987) and ATP (Nikinmaa, 1983) . Ferguson and B o u t i l i e r (1987) propose t h a t t h i s d e c r e a s e i n NTP c o n c e n t r a t i o n i s due t o t h e i n c r e a s e d demand f o r NTP e q u i v a l e n t s by 159 F i g u r e 20. A: Model o f a d r e n e r g i c a l l y s t i m u l a t e d f i s h e r y t h r o c y t e . 1. A d r e n a l i n e b i n d s t o b e t a r e c e p t o r . 2. B e t a a d r e n e r g i c s t i m u l a t i o n causes an i n c r e a s e i n cAMP. 3. N a + / H + exchange i s a c t i v a t e d . 4. The C0 2 h y d r a t i o n r e a c t i o n i s s h i f t e d towards f o r m a t i o n o f p r o t o n s and b i c a r b o n a t e . 5. The a c t i v i t y o f t h e Na +/K + ATPase i s i n c r e a s e d . 6. C h l o r i d e moves i n t o t h e c e l l i n exchange f o r b i c a r b o n a t e . 7. There i s a n e t p r o t o n e x c r e t i o n . 8. There i s a n e t i n c r e a s e i n sodium and c h l o r i d e i n t h e c e l l . 9. There i s a n e t i n f l u x o f w a t e r . B: S c h e m a t i c o f b e t a - a d r e n e r g i c r e g u l a t i o n o f e r y t h r o c y t e pH d u r i n g an e x t r a c e l l u l a r a c i d o s i s i n f i s h e r y t h r o c y t e s . t i m e 161 a d r e n e r g i c a l l y - e n h a n c e d membrane i o n exchanger a c t i v i t y . T h i s d e c r e a s e i n NTP would reduce t h e c o n c e n t r a t i o n o f impermeable a n i o n s w i t h i n t h e c e l l and t h e r e b y e l e v a t e t h e pH. The a b i l i t y o f t h e sodium/proton exchanger t o i n f l u e n c e t h e pH o f t h e e r y t h r o c y t e d i r e c t l y v i a t h e e x t r u s i o n o f c y t o p l a s m i c p r o t o n s i s d i f f i c u l t t o d e t e r m i n e . A c c o r d i n g t o B a r o i n e t a l . , (1984b), t h e s o d i u m / p r o t o n e x c h a n g e mechanism w o u l d be u n a b l e t o i n f l u e n c e t h e e r y t h r o c y t e pH d i r e c t l y because o f t h e p r e s e n c e o f t h e h i g h - c a p a c i t y a n i o n exchanger (Romano and Passow, 1984 ; B a r o i n e t a l . . , 1 9 8 4 a ; C o s s i n s and R i c h a r d s o n , 1985; Heming e t a l . , 1986). P r o t o n s a r e p a s s i v e l y d i s t r i b u t e d i n rainbow t r o u t e r y t h r o c y t e s 30 m i n u t e s subsequent t o a d r e n e r g i c s t i m u l a t i o n (Heming e t a l . , 1987). C o s s i n s and R i c h a r d s o n have demonstrated, h o w e v e r , t h a t t h e a c t i v i t y o f t h e a d r e n e r g i c a l l y s t i m u l a t e d s odium/proton exchanger does approach t h a t o f t h e c h l o r i d e / b i c a r b o n a t e exchanger. F u r t h e r m o r e , Borgese e t a l . , (1986) have found t h a t c h l o r i d e / b i c a r b o n a t e e x c h a n g e l a g s b e h i n d s o d i u m / p r o t o n e x c h a n g e i n t r o u t e r y t h r o c y t e s a t t h e o n s e t o f a d r e n e r g i c s t i m u l a t i o n . I t i s t h e r e f o r e p o s s i b l e t h a t t h i s exchanger may d i r e c t l y i n f l u e n c e t h e pH o f t h e c e l l even i n t h e p r e s e n c e o f t h e 162 c h l o r i d e / b i c a r b o n a t e exchange mechanism v i a t h e e x t r u s i o n o f p r o t o n s and a l s o by a s h i f t i n t h e c a r b o n d i o x i d e h y d r a t i o n r e a c t i o n towards t h e f o r m a t i o n o f b i c a r b o n a t e . The e x a c t c o n t r i b u t i o n o f each o f t h e s e p r o c e s s e s t o t h e i n c r e a s e i n e r y t h r o c y t e pH i s n o t known. The combined e f f e c t o f t h e s e p r o c e s s e s i s , however, t o i n c r e a s e t h e pH o f t h e c e l l even i n t h e p r e s e n c e o f 10 mM b i c a r b o n a t e ( C h a p t e r 1) Thus, t h i s e l e v a t i o n i n e r y t h r o c y t e pH c o n t r i b u t e s t o t h e i n c r e a s e i n h a e m o g l o b i n o x y g e n a f f i n i t y (Nikinmaa e t a l . , 1984; C o s s i n s and R i c h a r d s o n , 1985; Primmett e t a l . , 1986; B o u t i l i e r e t a l . , 1986a) i n t h e i n t a c t a n i m a l a f t e r an i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s . I n a d d i t i o n , t h i s r e s p o n s e i s enhanced a t l o w e r pH v a l u e s (Chapter 1) wh i c h i n d i c a t e s t h a t i t p r o v i d e s t h e h i g h e s t b e n e f i t t o a n i m a l s w h i c h have been s t r e s s e d and i n whi c h b l o o d pH i s reduc e d . The c e l l u l a r mechanisms i n v o l v e d i n t h e b e t a -a d r e n e r g i c s t i m u l a t i o n o f t h e s o d i u m / p r o t o n e x c h a n g e mechanism i n f i s h e r y t h r o c y t e s have n o t been c l e a r l y d e f i n e d . Mahe e t a l ., (1985) have de m o n s t r a t e d t h a t b e t a - a d r e n e r g i c s t i m u l a t i o n o f rainbow t r o u t e r y t h r o c y t e s r e s u l t s i n an i n c r e a s e i n t h e b e t a - a d r e n e r g i c second messenger 3', 5 ' - c y c l i c AMP (cAMP). A c t i v a t i o n o f s p e c i f i c p r o t e i n k i n a s e s i s mediated by l e v e l s cAMP i n 163 o t h e r t i s s u e s (Cohen, 1982) and, t h e r e f o r e , t h i s system may a l s o be o p e r a t i v e i n f i s h e r y t h r o c y t e s . G r i n s t e i n e t a l . , (1985) p r o p o s e s t h a t t h e i n c r e a s e i n volume and i n t r a c e l l u l a r a l k a l i n i z a t i o n o c c u r r i n g i n l y m p h o c y t e s f o l l o w i n g p h o r b o l e s t e r t r e a t m e n t i s a r e s u l t o f s t i m u l a t i o n o f a c e l l u l a r p r o t e i n k i n a s e w h i c h r e g u l a t e s t h e a c t i v i t y o f t h e s o d i u m / p r o t o n e x c h a n g e r . I t i s p o s s i b l e , t h e r e f o r e , t h a t cAMP l e v e l s a l s o r e g u l a t e t h e a c t i v i t y o f t h e s o d i u m / p r o t o n e x c h a n g e r i n f i s h e r y t h r o c y t e s v i a s p e c i f i c p r o t e i n k i n a s e s . T h i s system, however, r e q u i r e s f u r t h e r s t u d y . C1~/HC0 3~ Exchange i n F i s h E r y t h r o c y t e s : G e n e r a l l y , a i r b r e a t h i n g v e r t e b r a t e s r e g u l a t e body pH by an i n i t i a l v e n t i l a t o r y a d j u s t m e n t o f body C 0 2 l e v e l s f o l l o w e d by a l o n g term r e n a l a d j u stment o f body b i c a r b o n a t e l e v e l s ( D a v e n p o r t , 1974; Woodbury, 1 9 7 4 ) . Changes i n v e n t i l a t i o n i n w a t e r b r e a t h i n g f i s h , however, have l e s s o f an e f f e c t on b l o o d P C0 2 o r pH ( J a n s s e n and R a n d a l l , 1975; Iwama e t a l . , 1 9 8 7 ) . I n s t e a d , w a t e r b r e a t h i n g f i s h r e g u l a t e b l o o d pH d u r i n g a c i d - b a s e d i s t u r b a n c e s by a d j u s t m e n t s i n body b i c a r b o n a t e l e v e l s (Cameron and R a n d a l l , 1972; R a n d a l l and Cameron, 1973; Eddy e t a l . , 1977; Cameron, 1978). Wood and P e r r y (1985) 164 have r e p o r t e d t h a t a d r e n a l i n e i n h i b i t s b i c a r b o n a t e e n t r y i n t o r a i n b o w t r o u t e r y t h r o c y t e s i n v i t r o . Based on t h i s d a t a , P e r r y (1986) has proposed t h a t a d r e n e r g i c c o n t r o l o f e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e exchange may p l a y a r o l e i n t h i s i n c r e a s e i n i n t e r n a l b i c a r b o n a t e s t o r e s i n f i s h and, t h e r e f o r e , i n a c i d - b a s e r e g u l a t i o n . An a d r e n e r g i c a l l y m e d i a t e d r e d u c t i o n i n t h e a c t i v i t y o f t h e c h l o r i d e / b i c a r b o n a t e e x c h a n g e p a t h w a y w o u l d a l s o be e x p e c t e d t o r e s u l t i n a d e c r e a s e i n c a r b o n d i o x i d e e x c r e t i o n by t h e i n t a c t a n i m a l . I n t h e p r e s e n t e x p e r i m e n t s , however, t h e r e was no r e d u c t i o n i n c a r b o n d i o x i d e e x c r e t i o n o r i n t h e r e s p i r a t o r y exchange r a t i o a f t e r b u r s t swimming (known t o c a u s e an i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s ; Primmett e t a l . , 1986) o r a d r e n a l i n e i n f u s i o n . F u r t h e r m o r e , i n t h e p r e s e n t s t u d y , n e i t h e r b e t a n o r a l p h a a g o n i s t s c o u l d reduce t h e r a t e o f c h l o r i d e / b i c a r b o n a t e e x c h a n g e i n r a i n b o w t r o u t e r y t h r o c y t e s i n v i t r o whereas t h i s exchange c o u l d be l a r g e l y i n h i b i t e d by a c e t a z o l a m i d e and t h e a n i o n exchange b l o c k e r s DIDS and SITS. Based on t h e s e r e s u l t s , i t can be c o n c l u d e d t h a t c a t e c h o l a m i n e s do n o t i n h i b i t e r y t h r o c y t i c c h l o r i d e / b i c a r b o n a t e e x c h a n g e i n f i s h . Hence, i n c r e a s e s i n plasma b i c a r b o n a t e i n r e s p o n s e t o a c i d o s e s i n t h e s e a n i m a l s a r e n o t a c h i e v e d by a d r e n e r g i c 165 r e g u l a t i o n a t t h e l e v e l o f t h e e r y t h r o c y t e . N a + / H + and C1~/HC0 3~ Exchange i n Amphibian E r y t h r o c y t e s : I n c o n t r a s t t o t h e e x c h a n g e r i n f i s h e r y t h r o c y t e s , t h e s o d i u m / p r o t o n e x c h a n g e mechanism i n a m p h i b i a n e r y t h r o c y t e s i s a c t i v e u n d e r s t e a d y s t a t e c o n d i t i o n s ( C h a p t e r 6; F i g . 2 1 ) . Sod i u m i o n s c o n t i n u o u s l y e n t e r t h e c e l l i n exchange f o r p r o t o n s . I n t h e p r e s e n c e o f b i c a r b o n a t e movements, t h i s e x c h a n g e s i g n i f i c a n t l y a f f e c t s t h e e r y t h r o c y t e volume, b u t n o t t h e c y t o p l a s m i c pH. A c c o r d i n g t o Hl a d k y and R i n k (1977), t h e c h l o r i d e d i s t r i b u t i o n a c r o s s t h e e r y t h r o c y t e membrane i s an i n d i c a t o r o f t h e n e t charge on t h e impermeable i o n s w i t h i n t h e e r y t h r o c y t e s i n c e c h l o r i d e i o n s a r e p a s s i v e l y d i s t r i b u t e d a c r o s s t h e e r y t h r o c y t e membrane. Changes i n t h e a c t i v i t y o f t h e sodium/proton exchange mechanism i n amphibian e r y t h r o c y t e s a r e not a s s o c i a t e d w i t h changes i n t h e c h l o r i d e d i s t r i b u t i o n (Chapter 6) , and, t h e r e f o r e , a r e n o t a s s o c i a t e d w i t h changes i n t h e n e t charge i n amphibian e r y t h r o c y t e s . I n c o n t r a s t , changes i n t h e a c t i v i t y o f t h e s o d i u m / p r o t o n e x c h a n g e r i n f i s h e r y t h r o c y t e s a r e a s s o c i a t e d w i t h changes i n t h e c h l o r i d e d i s t r i b u t i o n a c r o s s t h e e r y t h r o c y t e membrane (Heming e t a l . . 1987) and c h a n g e s i n t h e c o n c e n t r a t i o n s o f 166 F i g u r e 21. A: M o d e l o f a d r e n e r g i c a l l y s t i m u l a t e d a m p h i b i a n e r y t h r o c y t e . 1. A d r e n a l i n e b i n d s t o b e t a r e c e p t o r . 2. The N a + / H + e x c h a n g e mechanism i s c o n t i n u o u s l y a c t i v e , b u t t h i s a c t i v i t y i s not a l t e r e d by a d r e n e r g i c s t i m u l a t i o n . 3. The N a + / K + ATPase e s t a b l i s h e s t h e s o d i u m g r a d i e n t w h i c h d r i v e s t h e c o n t i n u o u s l y a c t i v e N a + / H + exchange. 4. The s t e a d y -s t a t e c e l l volume i s r e g u l a t e d by t h e N a + / H + and C l ~ /HC0 3~ exchange mechanisms, b u t t h e c e l l volume i s not a f f e c t e d by a d r e n e r g i c s t i m u l a t i o n . B: Sc h e m a t i c o f changes i n e r y t h r o c y t e pH d u r i n g an e x t r a c e l l u l a r a c i d o s i s i n b e t a - a d r e n e r g i c a l l y s t i m u l a t e d a mphibian e r y t h r o c y t e s . t i m e 168 i m p e r m e a b l e a n i o n s w i t h i n t h e e r y t h r o c y t e ( N i k i n m a a , 1983; Ferguson and B o u t i l i e r , 1987). These r e s u l t s s u g g e s t t h a t t h e i n f l u e n c e o f t h e sodium/proton exchange m echanism i n d e t e r m i n i n g t h e e r y t h r o c y t e pH may be r e l a t e d t o c h a n g e s i n i m p e r m e a b l e i o n c o n c e n t r a t i o n w i t h i n t h e c e l l . T h u s , t h e i n a b i l i t y o f t h e sodium/proton exchanger t o i n f l u e n c e t h e pH i n amphibian c e l l s may be due t o t h e l a c k o f an a s s o c i a t i o n between t h e a c t i v i t y o f t h i s exchanger and t h e impermeable i o n c o n c e n t r a t i o n s w i t h i n t h e s e c e l l s . P o s s i b l y , t h e number o r t h e a c t i v i t y o f sodium/proton exchangers i n amphibian c e l l s may be reduced r e l a t i v e t o f i s h c e l l s s u ch t h a t t h e a c t i v i t y o f t h e s e e x c h a n g e r s d o e s n o t s i g n i f i c a n t l y e f f e c t c e l l u l a r NTP l e v e l s a s i n f i s h e r y t h r o c y t e s . These r e s u l t s f u r t h e r s u g g e s t t h a t much o f t h e pH change i n f i s h e r y t h r o c y t e s f o l l o w i n g a d r e n e r g i c s t i m u l a t i o n may be due t o c h a n g e s i n c e l l u l a r NTP l e v e l s and a r e d i s t r i b u t i o n o f p r o t o n s r a t h e r t h a n a d i r e c t e f f e c t o f t h e sodium/proton exchange on t h e i n t r a c e l l u l a r p r o t o n c o n c e n t r a t i o n . A d r e n e r g i c s t i m u l a t i o n d i d n o t a f f e c t t h e pH o r v o l u m e o f e r y t h r o c y t e s f r o m e i t h e r Amphiuma o r B u f o ( C h a p t e r 4, 6 ) . These a n i m a l s , t h e r e f o r e , do n o t appear t o r e g u l a t e e r y t h r o c y t e pH a d r e n e r g i c a l l y . I n c o n t r a s t 1 6 9 t o t h e u n d i v i d e d c i r c u l a t o r y system i n f i s h , amphibians p o s s e s s a p a r t i a l l y d i v i d e d d o u b l e c i r c u l a t i o n . T h i s system a l l o w s s e l e c t i v e d i s t r i b u t i o n o f b l o o d between pulmonary and cutaneous c i r c u i t s v i a c a r d i a c s h u n t i n g ( S h e l t o n , 1985). I n a d d i t i o n , B o u t i l i e r e t a l . , (1986c) have demonstrated t h a t t h e r e l a t i v e d i s t r i b u t i o n o f t h e pulmocutaneous h e a r t o u t p u t between l u n g s and s k i n i n Rana c a t e s b e i a n a may be a d j u s t e d depending on t h e p a t t e r n o f e n v i r o n m e n t a l o x y g e n p a r t i a l p r e s s u r e . T h us, amphibians a r e a b l e t o e f f i c i e n t l y u t i l i z e oxygen s t o r e s v i a b o t h c e n t r a l and p e r i p h e r a l s h u n t i n g . A d r e n e r g i c r e g u l a t i o n o f e r y t h r o c y t e pH i n o r d e r t o enhance oxygen t r a n s p o r t d u r i n g f l u c t u a t i o n s i n ambient and i n t e r n a l gas t e n s i o n s , t h e r e f o r e , i s p r o b a b l y l e s s i m p o r t a n t t h a n i t would be i n f i s h . F u n c t i o n a l S i g n i f i c a n c e o f A d r e n e r g i c pH R e g u l a t i o n : What i s t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e a d r e n e r g i c r e g u l a t i o n o f pH and v o l u m e i n n u c l e a t e d e r y t h r o c y t e s ? Root and I r v i n g (1943) demonstrated t h a t a r e d u c t i o n i n t h e pH o f t h e haemoglobin environment i n v i t r o a l s o l o w e r s t h e oxygen c a p a c i t y o f t h e b l o o d i n c e r t a i n s p e c i e s o f f i s h . T h i s e f f e c t i s i m p o r t a n t i n t h e d e l i v e r y o f oxygen t o t h e swimbladder (Berg and S t e e n , 170 1968) w h i c h c o n t r o l s buoyancy i n some s p e c i e s o f f i s h . G l y c o l y s i s o c c u r s i n t h e s e c r e t o r y e p i t h e l i u m o f t h e swimbladder and a c i d i f i e s t h e b l o o d as i t p a s s e s t h r o u g h t h e r e t e s t r u c t u r e i n i t s w a l l . C o n s e q u e n t l y , an i n c r e a s e i n b l o o d P 0 2 r e s u l t s as oxygen i s r e l e a s e d from haemoglobin due t o t h e Root e f f e c t . Thus, a P 0 2 g r a d i e n t i s e s t a b l i s h e d between t h e b l o o d and t h e swim b l a d d e r and oxygen d i f f u s e s i n t o t h e swimbladder. The Root e f f e c t has a l s o been i m p l i c a t e d i n t h e d e l i v e r y o f oxygen t o t h e r e t i n a l t i s s u e i n f i s h ( W i t t e n b e r g and W i t t e n b e r g , 1974). T h i s e f f e c t , however, i f g e n e r a l t h r o u g h o u t t h e body may a l s o l i m i t t h e b l o o d o x y g e n c a r r y i n g c a p a c i t y u n d e r c o n d i t i o n s o f l o w pH and t h e r e b y l i m i t a e r o b i c p e r f o r m a n c e . I t has been s u g g e s t e d (Chapter 4, 6) t h a t t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e a d r e n e r g i c r e s p o n s e i n f i s h e r y t h r o c y t e s may be t o o f f s e t t h e d e t r i m e n t a l e f f e c t s t h a t t h e Root e f f e c t would have on t h e oxygen c a r r y i n g c a p a c i t y o f f i s h b l o o d . I n t h e f a c e o f an e x t r a c e l l u l a r a c i d o s i s , t h i s r e s ponse would m a i n t a i n t h e e r y t h r o c y t e pH and, t h e r e f o r e , t h e o x y g e n c a r r y i n g c a p a c i t y o f t h e b l o o d . I n a d d i t i o n , oxygen d e l i v e r y t o t h e swim b l a d d e r c o u l d c o n t i n u e a t t h e same r a t e as i n u n s t r e s s e d f i s h . I t can be seen from T a b l e 13 t h a t t h e a d r e n e r g i c r e s p o n s e h a s n o t been documented i n any 171 T a b l e 13. The p r e s e n c e o r absence o f t h e a d r e n e r g i c r e s p o n s e i n e r y t h r o c y t e s o f f i s h v e r s u s t h e p r e s e n c e o r absence o f a Root s h i f t . S p e c i e s A d r e n e r g i c Response Root S h i f t Yes/No Yes/No Salmo  q a i r d n e r i Salmo s a l a r C v p r i n u s  c a r p i o S q u a l u s  s u c k l e v i R a j a sp. T i n e a  t i n e a Yes, Nikinmaa, 1982a; Nikinmaa and H u e s t i s , 1984; B a r o i n e t a l . , 1984a,b; Heming e t a l . , 1987; C h a p t e r 1. No, Nikinmaa and J e n s e n , 1986 Yes, Ferguson and B o u t i l i e r , 1987 Yes, Nikinmaa e t a l . , 1987 Yes, Cameron 1971 No, T u f t s and R a n d a l l , unpubl, No, T u f t s and R a n d a l l , unpubl, Yes, T u f t s and R a n d a l l , u n p u b l . Oncorhynchus No, T u f t s and R a n d a l l , t s h a w t s c h a u n p u b l . Yes B o r j e s o n and Haglund, 1976 Yes, Tan e t a l . , 1972 No, L e n f a n t and Johansen 1966 No, L e n f a n t and Johansen 1966 Yes, J e n s e n Weber, 1982 Yes, R a n d a l l e t a l . , 1987 P l a t i c h t h v s Yes, F u g e l l i and f l e s s u s R e i e r s e n , 1978 Yes, Weber and W i l d e , 1975 172 s p e c i e s o f f i s h w h i c h does n o t have a R o o t e f f e c t . E q u a l l y , t h e response i s not found i n a l l s p e c i e s w h i c h do have a Root e f f e c t . T h i s absence o f an a d r e n e r g i c r e s p o n s e i n some s p e c i e s may be due, however, t o t h e l a b i l e n a t u r e o f t h e b e t a a d r e n e r g i c r e c e p t o r s . B e t a -a d r e n e r g i c r e c e p t o r systems may do w n - r e g u l a t e and become d e s e n s i t i z e d (Harden, 1983; S i b l e y and L e f k o w i t z , 1985). I t h a s be e n documented t h a t e e l g i l l s ( P e y r a u d -W a i t z e n e g g e r e t a l . , 1980) and t h e e e l h e a r t (Pennec and Peyraud, 1983) l o s e b e t a a d r e n e r g i c s e n s i t i v i t y d u r i n g t h e w i n t e r . Indeed, Nikinmaa and J e n s e n (1986) found t h a t t h e e r y t h r o c y t e s f r o m t h e r a i n b o w t r o u t , Salmo  g a i r d n e r i may be i n s e n s i t i v e t o a d r e n e r g i c s t i m u l a t i o n under c e r t a i n c o n d i t i o n s . I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e absence o r p r e s e n c e o f t h e a d r e n e r g i c r e s p o n s e may be d e t e r m i n e d by o t h e r v a r i a b l e s such as season o r t e m p e r a t u r e . C e r t a i n l y , i t would be c o n c e i v a b l e t h a t t h i s e n e r g y - r e q u i r i n g p r o c e s s may o n l y be f u n c t i o n a l d u r i n g p e r i o d s when i t would b e n e f i t t h e a n i m a l , such as d u r i n g t h e spawning m i g r a t i o n i n S a l m o n i d s p e c i e s . Thus, t h e e v i d e n c e t o d a t e i s c o n s i s t e n t w i t h t h e v i e w t h a t t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e a d r e n e r g i c r e s p o n s e may be t o o f f s e t t h e Root e f f e c t , b u t i t i s a l s o a p p a r e n t t h a t 173 o t h e r f a c t o r s may i n f l u e n c e t h i s r e s p o n s e . I n summary, i n t e r s p e c i f i c v a r i a t i o n i n t h e i o n i c e x c h a n g e mechanisms on t h e membrane o f n u c l e a t e d e r y t h r o c y t e s i s more w i d e s p r e a d t h a n p r e v i o u s l y t h o u g h t . T h i s v a r i a t i o n i s a p p a r e n t l y y e t a n o t h e r l e v e l o f a d a p t a t i o n f o r m o d u l a t i n g h a e m o g l o b i n o x y g e n a f f i n i t y and, t h e r e f o r e , b l o o d oxygen t r a n s p o r t . I t would seem t h a t t h i s l e v e l o f a d a p t a t i o n may have a r i s e n due t o d i f f e r e n t s e l e c t i v e p r e s s u r e s on t h e e r y t h r o c y t e i o n exchange p r o c e s s e s caused by haemoglobin h e t e r o g e n i e t y as w e l l as d i f f e r e n t s t r a t e g i e s among a n i m a l s f o r t h e r e g u l a t i o n o f a c i d - b a s e b a l a n c e and b l o o d o x y g e n t r a n s p o r t . 174 REFERENCES A l b e r s , C. ( 1 9 7 0 ) . 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