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The role of fish erythrocytes in transport and excretion of carbon dioxide Heming, Thomas Arthur 1984

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THE ROLE OF FISH ERYTHROCYTES IN TRANSPORT AND EXCRETION OF CARBON DIOXIDE by THOMAS A. HEMING B.Sc. (Honours), U n i v e r s i t y of Guelph, O n t a r i o , 1976 M.Sc. , U n i v e r s i t y of V i c t o r i a , E<ritish Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1984 © T h o m a s A. Heming, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of "Z.QQ\O The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date o < , ( ift / &4 i i ABSTRACT I n v o l v e m e n t o-F t h e r e d b l o o d c e l l s o f - f i s h i n CDs e x c h a n g e was i n v e s t i g a t e d by e x a m i n a t i o n o f t h e C0 r s t r a n s p o r t p r o p e r t i e s of - f i s h b l o o d , o f i o n movements (HCOrc", C I " , H"*") a c r o s s t h e r e d c e l l membrane, and of e r y t h r o c y t 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 . A d r e n e r g i c m o d u l a t i o n o f e r y t h r o c y t e f u n c t i o n i n vitro and 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 b r a n c h i a l CDs> e x c h a n g e in vivo a l s o were s t u d i e d , . A p p r o x i m a t e l y 92°/. o f the? C 0 S c o n t e n t o f v e n o u s b l o o d o f r a i n b o w t r o u t (Salma gairdneri) was HCO3"'", w h i c h a t an h a e m a t o c r i t o f '257* was d i s t r i b u t e d b e t w e e n t h e p l a s m a w a t e r s p a c e and t h e r e d c e l l w a t e r s p a c e i n a r a t i o o f a b o u t 9^1. F'lasraa H C O 3 - a c c o u n t e d f o r some 827. o f t h e COr.-? e x c r e t e d 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 , w h i l e e r y t h r o c y t e HCO3""" a c c o u n t e d f o r o n l y 97.. The r e m a i n d e r o f CO.-*: e x c r e t e d a t t h e g i l l s was d e r i v e d from,, i n d e s c e n d i n g o r d e r o f i m p o r t a n c e , c a r b a m i n o compounds (R—NHCOs>~) , m o l e c u l a r CD ; a, and C O s 3 " . The e r y t h r o c y t e o f r a i n b o w t r o u t was f r e e l y p e r m e a b l e t o HCD3'~, CI"" and H"", a l l o f w h i c h were d i s t r i b u t e d p a s s i v e l y a c r o s s t h e r e d c e l l membrane. HCD 3~ t r a v e r s e d t h e e r y t h r o c y t e membrane i n an o n e - f o r — o n e e x c h a n g e w i t h CI"" v i a a S I T S - s e n s i t i v e m e c hanism a n a l o g o u s t o t h e Band 3 a n i o n e x c h a n g e p a t h w a y o f mammalian r e d c e l l s . The t r a n s m e m b r a n e e q u i l i b r i u m d i s t r i b u t i o n s o f H C O 3 " , C l ' ~ and H'", h o w e v e r , were c o m p l i c a t e d by t h e p r e s e n c e o f a c e l l n u c l e u s . The n u c l e a r c o m p a r t m e n t o f t r o u t e r y t h r o c y t e s i i i a p p e a r e d t o be more a c i d i c and t o c o n t a i n l e s s H C 0 3 ~ and CI™ t h a n t h e c y t o s o l ,. The k i n e t i c s o f t h e u n c a t a l y s e d HCOas™s COs» c o n v e r s i o n were •found t o be at. l e a s t one o r d e r o f m a g n i t u d e t o o s l o w t o a c c o u n t f o r t h e o b s e r v e d b r a n c h i a l CDs. movements- F i s h e r y t h r o c y t e s h o w e v e r , c o n t a i n e d s u f f i c i e n t 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 t o c a t a l y s e t h e i n t e r c o n v e r s i on of HC0 3" _ and CQs, i n c r e a s i n g t h e r a t e o f r e a c t i o n by s e v e r a l o r d e r s o f m a g n i t u d e . F i s h p l a s m a c o n t a i n e d i n h i b i t o r s o f c a r b o n i c a n h y d r a s e w h i c h were a c t i v e a g a i n s t t h e enzyme a c t i v i t y of b o t h e r y t h r o c y t e and g i l l h o m o g e n a t e s . T h e s e i n h i b i t o r s l a c k e d a c c e s s t o i n t r a c e l l u l a r c a r b o n i c a n h y d r a s e and had no d i r e c t e f f e c t on membrane t r a n s p o r t . o f a n i o n s . I t i s s u g g e s t e d t h a t t h e s e i n h i b i t o r s p r o b a b l y f u n c t i o n t o i m m o b i l i z e c a r b o n i c a n h y d r a s e r e l e a s e d i n t o t h e p l a s m a d u r i n g t h e n o r m a l d e s t r u c t i o n o f e r y t h r o c y t e s o r d u r i n g i n j u r y , b u t h a v e no e f f e c t , on i n t a c t r e d e e l Is„ T h e s e d a t a , t o g e t h e r w i t h e v i d e n c e t h a t t h e b a s o l a t e r a l membrane of t h e g i l l i s l a r g e l y i m p e r m e a b l e t o H C 0 3 " ( P e r r y et al- 1 9 8 2 ) , c l e a r l y i n d i c a t e t h a t t h e p r i n c i p a l p a t h w a y f o r C O K e x c r e t i o n i n f i s h i s v i a t h e movement o f p l a s m a H C 0 3 ~ i n t o t h e r e d c e l l by way o f a ' c h l o r i d e s h i f t ' . T h i s b i c a r b o n a t e t h e n i s r a p i d l y d e h y d r a t e d t o f o r m CQ^ i n t h e p r e s e n c e o f e r y t h r o c y t e c a r b o n i c a n h y d r a s e . The r e s u l t a n t d i f f u s e s down i t s c o n c e n t r a t i o n g r a d i e n t o u t o f t h e r e d c e l l and a c r o s s t h e g i l l e p i t h e l i u m . C0Z l o a d i n g o f f i s h b l o o d d u r i n g t i s s u e c a p i l l a r y t r a n s i t i n v o l v e s a s i m p l e r e v e r s a l o f t h e s e t r a n s p o r t and c h e m i c a l m e c h a n i s m s . The p r e s e n t i n f o r m a t i o n c o n c l u s i v e l y i v r e f u t e s t h e g i l l model o f COss e x c r e t i o n ( H a s w e l l et al . 1980) w h i c h a s s e r t s t h a t f i s h e r y t h r o c y t e s h a v e no f u n c t i o n a l r o l e i n b r a n c h i a l COrs e x c h a n g e . E v i d e n c e was f o u n d w h i c h i n d i c a t e d t h a t t h e o r i g i n a l s t u d i e s o f H a s w e l l and c o w o r k e r s s u f f e r e d f r o m t e c h n i q u e - r e l a t e d a r t i f a c t s . C a t e c h o l a m i n e s had p r o f o u n d e f f e c t s on b o t h e r y t h r o c y t e f u n c t i o n and b r a n c h i a l C0^ e x c h a n g e . B e t a - a d r e n e r g i c a g o n i s t s a p p e a r e d t o s t i m u l a t e c o u p l e d Na^/H* and C1"~/HC0 3- e x c h a n g e r s on t h e c e l l membrane o f t r o u t e r y t h r o c y t e s , s i m i l a r t o t h e i o n e x c h a n g e r s i n v o l v e d i n v o l u m e r e g u l a t o r y i o n movements i n a m p h i b i a n r e d c e l l s (Ca'la 1980),. The a d r e n e r g i c r e s p o n s e s 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 i n c l u d e d a n e t c e l l u l a r g a i n o f Na*, C l ~ and HscO,, a n e t c e l l u l a r l o s s o f H"" and H C 0 - 5 ' ~ , a p r o n o u n c e d c e l l s w e l l i n g , and a f u n c t i o n a l r e d u c t i o n i n n e t H C 0 3 ~ " f l u x t h r o u g h t h e r e d c e l l . In vii'o,, t h e s e a d r e n e r g i c r e s p o n s e s were a c c o m p a n i e d by a t r a n s i e n t r e d u c t i o n i n CO,^  e x c r e t i o n , an i n c r e a s e i n body C0s> s t o r e s , and a d i s r u p t i o n o f t h e H C 0 3 - s C O ^ c h e m i c a l e q u i l i b r i u m i n a r t e r i a l b l o o d i m m e d i a t e l y d o w n s t r e a m o f t h e g i l l . Oxygen u p t a k e was u n a f f e c t e d by a d r e n a l i n e . The a d r e n e r g i c r e s p o n s e s o f f i s h r e d c e l l s p r o b a b l y a r e i m p o r t a n t i n r e g u l a t i n g e r y t h r o c y t e pH d u r i n g p e r i o d s o f s t r e s s , and h e n c e s e r v e t o m a i n t a i n 0 2 t r a n s p o r t t o t h e t i s s u e s u n d e r s u c h c o n d i t i o n s . A t t h e same t i m e , t h e s e r e s p o n s e s s l o w n e t H C 0 3 ~ f l u x t h r o u g h t h e r e d c e l l s 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 u s s e r v e t o m a i n t a i n an e x t r a c e l l u l a r p o o l o f H C O 3 " " i n s t r e s s w h i c h t h e n may be u s e d t o e n h a n c e t h e i n t r a c e l l u l a r b u f f e r i n g c a p a c i t i e s o f o t h e r t i s s u e s . V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v L I S T OF TABLES v i i L I S T OF FIGURES ;•: ACKNOWLEDGEMENTS K v i i i GENERAL. INTRODUCTION 1 GENERAL. MATERIALS AND METHODS 7 C h a p t e r 1. C O s TRANSPORT PROPERTIES OF BLOOD OF SftLMQ GAIRDNERI I n t r o d u c t i o n 9 M a t e r i a l s and M e t h o d s A. S o l u b i l i t y o-f C 0 3 and A p p a r e n t D i s s o c i a t i o n 13 ' C o n s t a n t o f H^CO^ B„ In vitro R e s p i r a t o r y and A c i d - b a s e 14 C h a r a c t e r i s t i c s o-f B l o o d C. A r t e r i a l and Venou s T r a n s p o r t o f COm In Vivo 16 R e s u l t s 18 D i s c u s s i o n 46 C h a p t e r 2. A C T I V I T Y AND I N H I B I T I O N OF CARBONIC ANHYDRASE IN FISHES I n t r o d u c t i o n 55 M a t e r i a l s and M e t h o d s A» M a n o m e t r i c M e t h o d : M o d i f i e d B o a t A s s a y 58 B. C o l o u r i m e t r i c M e t h o d : E s t e r a s e A s s a y 59 C„ E l e c t r o m e t r i c Method: pH--stat A s s a y 59 v i D» Ch em :i. c a 1 S t ud i e s 63 E. P h y s i o l oq i c a l S t u d i e s 63 R e s u l t s A„ C h e m i c a l 66 B. P h y s i o l o g i c a l 73 D i s c u s s i o n SO C h a p t e r 3. ION TRANSPORT AND E Q U I L I B R I A ACROSS THE MEMBRANE OF SftLHQ GfilRDNERI ERYTHROCYTES I n t r o d u c t i o n 90 M a t e r i a l s and M e t h o d s A. C h l o r i d e T r a n s p o r t i n Red C e l l s 93 B. B i c a r b o n a t e T r a n s p o r t i n Red C e l l s 95 C„ E f f e c t s o-f A d r e n a l i n e on I o n i c . E q u i l i b r i a 97 A c r o s s t h e Red C e l l Membrane R e s u l t s 99 D i s c u s s i o n 120 C h a p t e r 4. EFFECTS OF ADRENALINE ON C 0 3 EXCRETION AND ACID-BASE STATUS OF SQL HQ GftlRDNERI, IN VIQQ I n t r o d u c t i o n 131 M a t e r i a l s and M e t h o d s 133 R e s u l t s 135 D i s c u s s i o n ' 147 SENERAL DISCUSSION 153 ABBREVIATIONS AND SYMBOLS 159 REFERENCES 160 L I S T OF TABLES T a b l e 1. L e a s t s q u a r e s r e g r e s s i o n l i n e s d e s c r i b i n g t h e e f f e c t o f t e m p e r a t u r e <T, C) on C 0 = s o l u b i l i t y <aC0s;, umol • L - 1 - t o r r " 1 ) o f w a t e r and Sslmo gairdneri p l a s m a , where <3C0s» = a + bT + c T 2 . R e g r e s s i o n c o e f f i c i e n t s a r e p r e s e n t e d + 9 5 % c o n f i d e n c e i n t e r v a l s < n ~ 18). T a b l e 2. In vivo r e s p i r a t o r y and i o n i c c h a r a c t e r i s t i c s o f Sal mo gairdneri p l a s m a a t 10 C. A r t e r i a l and v e n o u s v a l u e s a r e a r i t h m e t i c means + 1 S.E. ( n ) . ' ' A r t e r i a l m i n u s v e n o u s ' v a l u e s a r e p o p u l a t i o n mean d i f f e r e n c e s + 9 5 % c o n f i d e n c e i n t e r v a l s . P o p u l a t i o n mean d i f f e r e n c e s d e n o t e d w i t h * a r e d i f f e r e n t , s i g n i f i c a n t l y f r o m z e r o a t P<0.05 ( p a i r e d t - t e s t ) . n e t , h a e m a t o c r i t . T a b l e 3. P h y s i o l o g i c a l b u f f e r i n g c a p a c i t y i n s l y k e s (mM b i c a r b o n a t e - p H u n i t " 1 ) o f o x y g e n a t e d w h o l e b l o o d , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s o f S a l B O gairdneri d e t e r i m i n e d in vitro b e t w e e n 5 and 15 C. V a l u e s a r e means _•+• 1 S.E. (n=5-7 ) . Temp, t e m p e r a t u r e . Hct., h a e m a t o c r i t . T a b l e 4. Least, s q u a r e s r e g r e s s i o n l i n e s d e s c r i b i n g t h e r e l a t i o n s h i p b e t w e e n b l o o d pl-l ( p H e ) „ and i n t r a c e l l u l a r pH ( p H i ) , R H + ( [ p l a s m a ] / [ ' e r y t h r o c y t e ] ) , R C i - ( [ e r y t h r o c y t e ] / [ p l a s m a ] ) , and R H C O S - ( [ e r y t h r o c y t e ] / [ p l a s m a ] ) o f Salmo gairdneri b l o o d b e t w e e n 5 and 15 C. A l s o shown i s t h e r e l a t i o n s h i p b e t w e e n c a r b a m i n a h a e m o g l o b i n c o n c e n t r a t i o n ( c a r b , mmol-L r e d c e l l - 1 ) and b l o o d P C O B ( t o r r ) , , R e g r e s s i o n c o e f f i c i e n t s a r e p r e s e n t e d + 9 5 % c o n f i d e n c e i n t e r v a l s . Temp, t e m p e r a t u r e ( C ) . T a b l e 5. A model o f CO^ t r a n s p o r t i n a r t e r i a l and v e n o u s b l o o d o f Salmo gairdneri a t 10 C. See t e x t f o r d e t a i l s . V a l u e s a r e umol. T a b l e 6. 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 i n t i s s u e s o f s e v e r a l f i s h e s . M e a s u r e m e n t s were made u s i n g t h e e s t e r a s e a s s a y a t 25 C, pH 7.500 and 3 mM pNA and t h e p H - s t a t a s s a y art a m b i e n t w a t e r t e m p e r a t u r e s ( 9 -11 C) , pH 7.500 and 50 mM NaHC0 3. V a l u e s a r e means + 1 S.E. eu„ enzyme u n i t . ND, none d e t e c t e d . T a b l e 7/. E f f e c t o f p l a s m a on 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 i n t i s s u e s o f s e v e r a l f i s h e s . V a l u e s d e n o t e d by # 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 f r o m z e r o a t P<0„05 ( p a i r e d t - - t e s t ) . R e s t o f c a p t i o n a s i n T a b l e 6,. T a b l e 8. H a l f — t i m e ( s e c ) f o r H C 0 3 ~ d e h y d r a t i o n i n an u n c a t a l y s e d o p p o s e d c a r b o n d i OK i de: b i c a r b o n a t e e q u i l i b r i u m i n a q u e o u s s o l u t i o n , s h o w i n g t h e e f f e c t s o-f pl--l and t e m p e r a t u r e . See t e x t -for d e t a i l s . ""data of- K e r n (1960) a t I==0. E , d a t a o f p r e s e n t s t u d y a t 1=0.15. T a b l e 9. Met H C 0 3 ~ 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 (eu-mL r e d c e l l " " 1 ) o f Sal mo gairdneri, s h o w i n g e f f e c t s o f p l a s m a , a c e t a z o l a m i d e , S I T S , f o a m i n g a g e n t s , and d e f o a m i n g a g e n t s . V a l u e s a r e means + 1 S.E. (n=8). T a b l e 10. E f f e c t s o f a l p h a a d r e n e r g i c and b e t a -a d r e n e r g i c a g o n i s t s and a n t a g o n i s t s on t h e n e t HC0 3~ f l u x (eu-mL r e d c e l l " " 1 ) 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 of Sal mo g a i r d n e r i . V a l u e s a r e means + 1 S.E. (n=6). T a b l e 11„ R e s p i r a t o r y and a c i d - b a s e c h a r a c t e r i s t i c s of a r t e r i a l p l a s m a o f Sal mo gairdneri? s h o w i n g e f f e c t s o f a b o l u s i n j e c t i o n o f 0,5 mL o f C o r t l a n d s a l i n e . V a l u e s a r e means + 1 S.E. (n=5). H c t , h a e m a t o c r i t.. L I S T OF FIGURES F i g u r e 1. P h y s i c o - c h e m i c a l p a r a m e t e r s p e r t a i n i n g t o CO 35 t r a n s p o r t i n Sal mo gai. rdner i b l o o d . P a n e l A. C0;,2 s o l u b i l i t y (umol ~ L~ : L •• t o r r " 1 ) o f w a t e r and t r o u t p l a s m a a t 5., 10 and 15 C. V a l u e s a r e means + 1 S.E. <n=6). L e a s t s q u a r e s r e g r e s s i o n l i n e s a r e p r e s e n t e d i n Table! 1. P a n e l B. E f f e c t , o f pH on thee a p p p a r e n t d i s s o c i a t i o n c o n s t a n t o-f c a r b o n i c : a c i d ( p K , p P ) i n t r o u t p l a s m a a t 10 C. L e a s t s q u a r e s r e g r e s s i o n l i n e i s p K a p p = 6.974 0.098 pi-l <r=~-1.000, n=3) • Valuers a r e means + 1 S.E. (n=8). F i g u r e 2. CO^ d i s s o c i a t i o n c u r v e s and C0%> c a p a c i t a n c e s o-f o x y g e n a t e d w h o l e b l o o d o-f S a l wo gairdneri d e t e r m i n e d in v i t r o . P a n e l A. E f f e c t o f t e m p e r a t u r e on CO^ d i s s o c i a t i o n c u r v e s . V a l u e s a r e means + 1 S.E. (n=5 - 7 ) . P a n e l B. E f f e c t of h a e m a t o c r i t on mean C 0 a c a p a c i t a n c e ( 6 C 0 ^ = (/CCo2/rfPco2) b e t w e e n 2 and 7 t o r r F'cos a t t e m p e r a t u r e s o f 5 ( + ) , 10 <•«•) and 15 (o) C. L e a s t s q u a r e s r e g r e s s i o n l i n e , i g n o r i n g t e m p e r a t u r e , i s fcCOs. = 0.232 •+• 0.022 h a e m a t o c r i t ( r = 0 . 7 5 7 , n=18) . F i g u r e 3. CO^ c a p a c i t a n c e s <hCQ : s d i s s o c i a t i on c u r v e s and mean - rfCcoa / dPcosin between 2 and 7 t o r r ) o f o x y g e n a t e d w h o l e b l o o d (mean h a e m a t o c r i t + 1 S.E. =: 20.1 +. 1 - 7 ) , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s o f Salmo gai rdneri d e t e r m i n e d i n vitro a t 10 C . V a l u e s a r e means + 1 S.E. (n=6--7) . Dashed l i n e r e p r e s e n t s t h e amount of d i s s o l v e d CQ K p r e s e n t . F i g u r e 4„ B u f f e r l i n e s f o r o x y g e n a t e d w h o l e b l o o d (mean h a e m a t o c r i t + 1 S.E. = 2 0 . 1 + 1 . 7 ) , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s o f Salmo gairdneri d e t e r m i n e d i n vitro a t 10 C. V a l u e s a r e means + 1 S.E. (n=6--7) .. F i g u r e 5. Donnan r a t i o s of H"'" ( R M H , = [ p l a s m a ] / [ e r y t h r o c y t e ] ) , C1"" (Re*-- = [ e r y t h r o c y t e ] / [plasma.]) and b H C 0 3 " ( R H C D S - " [ e r y t h r o c y t e ] / [ p l a s m a ] ) o-f- Salmo gairdneri b l o o d , when b l o o d pH was t i t r a t e d w i t h CO as in vitro a t 5 ( + ) , 10 (#) and 15 (o) C. Least, s q u a r e s r e g r e s s i o n l i n e s a r e g i v e n i n T a b l e 4. F i g u r e 6. R e l a t i o n s h i p b e t w e e n t h e amount of b i c a r b o n a t e added t o p l a s m a f r o m r e d c e l l s and t h e amount of c h l o r i d e a dded t o r e d c e l l s f r o m p l a s m a d u r i n g i n vitro COa e q u i l i b r a t i o n s o f Salmo gairdneri b l o o d a t 10 C. A l l c o n c e n t r a t i o n s were? c o r r e c t e d f o r w a t e r c o n t e n t and h a e m a t o c r i t . L e a s t s q u a r e s r e g r e s s i o n l i n e i s c/[bHC03*""] = 0.155 + 0.943 dCC1 ™3 <r=0»802, n=42) ,. Dashed l i n e i s x=y. F i g u r e 7. Water c o n t e n t and r e l a t i v e v o l u m e o f Sal mo gairdneri e r y t h r o c y t e s i n r e l a t i o n t o P o o s a t 5 <•+•), 10 (#) and 15 (o) C V a l u e s a r e means + 1 S.E. <n==5-7)» F i g u r e 8,. R e l a t i o n s h i p b e t w e e n t h e Donnan CI" - r a t i o (Ren- = [ e r y t h r o c y t e ] / C p l a s m a l l ) , and t h e Donnan r a t i o s o f H* (R,-,.,.. - [ p l a s m a ] / [ e r y t h r o c y t e ] ) and bHC0 .3~ ( R H C D S - =- C e r y t h r o c y t e 3 / L *plasma3 ) of Sal mo gairdneri b l o o d a t 5 <-+•) , 10 (•») and 15 ia) C„ V a l u e s a r e means + 1 S.E. (n=5--7) . L e a s t s q u a r e s r e g r e s s i o n o f R H + on R C 1 . _ h a s s l o p e o f 0.741 + 0.350 and y - i n t e r c e p t o f 0.094 + 0.160 (+ 9 5 % c o n f i d e n c e i n t e r v a l s , n=12, r ^ O . 8 3 1 ) . L e a s t s q u a r e s r e g r e s s i o n o f R H C O S - on R C i - h a s s l o p e o f 0.878 + 0.302 and y-i n t e r cep t o f 0.347 + 0.138 < + 9 5 % c o n f i den c e i n t e r v a l s , n=12, r ~ 0 . 8 9 8 ) . Dashed l i n e s a r e x--y. F i g u r e 9. C a r b a m i n o h a e m o g i o b i n f o r m a t i o n i n Salmo gai rdneri b l o o d s h o w i n g e f f e c t s o f P C Q 2 and t e m p e r a t u r e . V a l u e s a r e means + 1 S.E. <n=5-~7) . L e a s t s q u a r e s r e g r e s s i o n l i n e s a r e g i v e n i n T a b l e 4. F i g u r e 10. U n c a t a l y s e d r a t e o f H C 0 3 ~ d e h y d r a t i o n ( s o l i d c i r c l e s ) and c a t a l y s e d r a t e u s i n g 1.0 ug of b o v i n e CA (open c i r c l e s ) d e t e r m i n e d e l e c t r o m e t r i c a l 1 y a t 25 C, pH 7.500 and 50 mM NaHC0 3, s h o w i n g e f f e c t o f t h e f l o w r a t e o-f CQ^—free n i t r o g e n g a s t h r o u g h t h e r e a c t i o n v e s s e l . V a l u e s a r e means + 1 S„E. (n=5). F i g u r e 11. A c e t a z o l a m i d e - s e n s i t i v e e s t e r a s e a c t i v i t y o f p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d c o l o u r i m e t r i c a l 1 y a t pH 7.500 and 3 mM p-n i t r o p h e n y l a c e t a t e (pNA), s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e and enzyme c o n t e n t . V a l u e s a r e means + 1 S.E. <n~5). F i g u r e 12. C a t a l y s i s o f CQffi e v o l u t i o n f r o m a b u f f e r e d N a H C 0 3 s o l u t i o n by p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d m a n o m e t r i c a l 1 y a t pH 6.800 and 200 mM MaHCD 3, s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e and enzyme c o n t e n t . V a l u e s a r e means + 1 S.E. (n=5). F i g u r e 13. C a t a l y s i s o f b i c a r b o n a t e d e h y d r a t i o n by p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d e l e c r o m e t r i c a l 1 y a t pH 7.500 and 50 mM NaHC0 3, s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e and enzyme c o n t e n t . V a l u e s a r e means + 1 S.E. (n=5). F i g u r e 14. D e h y d r a t i o n r a t e c o n s t a n t , kHcD3, and c o r r e s p o n d i n g h a l f - t i m e f o r t h e u n o p p o s e d r e a c t i o n x i v H C O 3 - + H~ -•-> COa, -i- H A0 a t 1=0. 15, s h o w i n g e f f e c t s of r e a c t i o n t e m p e r a t u r e and pH. V a l u e s a r e means + 1 S.E. (n=15). F i g u r e 15. E f f l u x o f 3 < s > c h 1 o r i de f r o m e r y t h r o c y t e s 100 of S a l wo gai rdneri a t 1 C, s h o w i n g e f f e c t s o f a c e t a z o l a m ide < 10~" s M) « S I T S (IQ-"* M) and p l a s m a ( 1 0 % by v o l u m e ) . P e r c e n t r e c o v e r y was c a l c u l a t e d a s a c t i v i t y o f -^"Cl""' i n e x t r a c e l l u l a r medium a t t i m e i d i v i d e d by a c t i v i t y i n medium a f t e r 30 min e q u i l i b r a t i o n , m u l t i p l i e d by 100 ( s e e t e x t f o r d e t a i l s ) . P o i n t s r e p r e s e n t. d ait a f r om 3--• 4 t r i a l s . F i g u r e 16. E f f e c t 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 104 on net. 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 of Salmo g a i r d n e r i . V a l u e s a r e means + 1 S.E. (n=4--6 5 . F i g u r e 17. P l a s m a pH and i n t r a e r y t h r o c y t i c pH o f 109 Salmo gairdneri b l o o d a s a f u n c t i o n o f Peas* d e t e r m i n e d in vitro at. 10 C, s h o w i n g e f f e c t s o f a d r e n a l i n e . A d r e n a l i n e c o n c e n t r a t i o n s were 3.1 "10""* mol"L plasma"" 1 i n c o n t r o l b l o o d and 4.6-10"* mol •  L plasma""* i n a d r e n a l i n e b l o o d . V a l u e s a r e means + 1 S.E. (n=S). A d r e n a l i n e v a l u e s d e n o t e d w i t h # a r e d i f f e r e n t s i g n i f i c a n t l y f r o m c o r r e s p o n d i n g c o n t r o l v a l u e s a t P<0.05 ( p a i r e d t -1est)„ F i g u r e 18. Donnan r a t i o s o f !•-!""', Na"*", l<-, C l ~ , and bl-itX)3""' i n Salmo gairdneri b l o o d a s a f u n c t i o n o f F'co2 d e t e r m i n e d in vitro a t 10 C , s h o w i n g e f f e c t s o f a d r e n a l i n e . A n i o n r a t i o s a r e [ e r y t h r o c y t e 1 / II p 1 asma] w h i l s t c a t i o n r a t i o s a r e [ p l asma"J / L e r y t h r o c y t e II., Rest, o f c a p t i o n a s i n F i g u r e 17. F i g u r e 19. bHC0 3"" c o n c e n t a t i o n s i n o x y g e n a t e d w h o l e blood,, p l a s m a , and e r y t h r o c y t e s o f Salmo gairdneri a s a f u n c t i o n o f Pesos* d e t e r m i n e d in vitro a t 10 C , s h o w i n g e f f e c t s o f a d r e n a l i n e . R e s t o f c a p t i o n a s i n F i g u r e 17,. F i g u r e 20. Mean c e l l s i z e ( h a e m a t o c r i t / C h a e m a g l a b i n D ) and w a t e r c o n t e n t o f Salmo gairdneri e r y t h r o c y t e s a s a f u n c t i o n o f P C o 2 d e t e r m i n e d in vitro a t 10 C , s h o w i n g e f f e c t s o f a d r e n a l i n e . R e s t of c a p t i o n a s i n F i g u r e 17. F i g u r e 2 1 . Donnan H* r a t i o ( [ p l a s m a ] / [ e r y t h r o c y t e ] ) and mean c e l l s i z e o f e r y t h r o c y t e s o f S<almo gai rdneri a s a f u n c t i o n o f P C o s » d e t e r m i n e d in vitro a t 10 C , s h o w i n g e f f e c t s o f a d d i t i o n o f a d r e n a l i n e ? (5 - 10"* M) and e i t h e r p r o p r a n o l o l (IO""* M) o r S I T S ( 1 0 — * M). V a l u e s a r e means + 1 S.E. (n-4) „ F i g u r e 22. A r t e r i a l - v e n o u s d i f f e r e n c e s i n pH. C c o s a x v i and F'coa a c r o s s t h e g i l l s o-f Salmo gairdneri, s h o w i n g t h e e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e a s a b o l u s <5- 10"":l 1 mol-g f i s h " " : t ) and a s a c o n t i n u o u s s t r e a m (5- 10 -" 0 mol • h~ : L) „ A r t e r i a l v a l u e s a r e shown by open c i r c l e s . V e n o u s v a l u e s a r e shown by s o l i d c i r c l e s . V a l u e s a r e means + 1 B.E. ( n ^ S ) . V a l u e s d e n o t e d w i t h + a r e d i f f e r e n t s i g n i f i c a n t l y f r o m t h e t i m e z e r o v a l u e a t F*<0«G5 ( p a i r e d t - t e s t ) . A r t e r i a l v a l u e s d e n o t e d w i t h * a r e d i f f e r e n t s i g n i f i c a n t l y f r am t h e car resp on d i n g ven o u s va1ue a t P< 0.05 ( p a i r e d t - t e s t ) . F i g u r e 23. A r t e r i a l - v e n o u s d i f f e r e n c e s i n C o s , P a s 138 and h a e m a t o c r i t a c r o s s t h e g i l l s o f Sal mo gairdneri, s h o w i n g t h e e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e a s a b o l u s (5- 1 0 ~ 1 * m o l - g f i s h " 1 ) and a s a c o n t i n u o u s s t r e a m (5-10'-° mol - h " 1) „ R e s t o f c a p t i o n a s i n F i g u r e 22'.. F i g u r e 24. Gas e c h a n g e r a t i o (R -•- d p 1 asmaCcoa/d 141 b i o o d C o a ) o f Sal mo gairdneri„ s h o w i n g t h e e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e a s a b o l u s (5- 1 0 " 1 1 m o l - g f i s h " 1 ) and a s a c o n t i n u o u s s t r e a m (5- 10~m mol - h""1 ) . V a l u e s a r e means +• 1 S!>„E. (n=3). V a l u e s d e n o t e d by + a r e d i f f e r e n t s i g n i f i c a n t l y f r o m t h e t i m e z e r o v a l u e a t P<0.05 ( p a i r e d t - t e s t ) . x v i i F i g u r e 25. E f f e c t s o f a d r e n a l i n e i n j e c t i o n a s a 144 b o l u s (5- 1 0 " 1 1 mol-g f i s h " " * ) and a s a c o n t i n u o u s s t r e a m (5-10"° mol - h"" 1)' on a r e a mean b l o o d p r e s s u r e , h e a r t r a t e and v e n t i l a t i o n r a t e o f Salmo g a i r d n e r i . R e s t o f c a p t i o n a s i n F i g u r e 22. x v i i i ACKNOWLEDGEMENTS Many p e o p l e d e s e r v e t o be a c k n o w l e d g e d f o r t h e i r s u p p o r t o-f my work. I n p a r t i c u l a r ; , I t h a n k Dr. D, J . R a n d a l l f o r h i s e n c o u r a g e m e n t and a s s i s t a n c e , and f o r g i v i n g me t h e o p p o r t u n i t y t o u n d e r t a k e t h i s p r o j e c t . My l a b o r a t o r y c r o n i e s a r e t h a n k e d f o r t h e i r p o s i t i v e a d v i c e and a s s i s t a n c e . C e r t a i n p o r t i o n s o f t h i s t h e s i s were done i n c o l l a b o r a t i o n w i t h o t h e r s : p a r t o f c h a p t e r 1 w i t h Dr. R. G. B o u t i l i e r and G. K. Iwarna? p a r t o f c h a p t e r 2 w i t h Dr. T. A. Watson? c h a p t e r 4 w i t h Dr. S„ F» P e r r y . I r e c e i v e d f i n a n c i a l s u p p o r t f o r t h i s work t h r o u g h N.S.E.R.C. p o s t g r a d u a t e s c h o l a r s h i p s and axn U.B.C, u n i v e r s i t y g r a d u a t e f e l l o w s h i p . I r e c e i v e d m o r a l s u p p o r t f r o m my f a m i l y , L u c i n d a , K i r s t e n and D e r e k . 1 GENERAL INTRODUCTION C a r b o n d i o x i d e i s p r o d u c e d by t i s s u e s a s an e n d - p r o d u c t o f o x i d a t i v e m e t a b o l i s m . T r a n s p o r t o f C0.s i n b l o o d and i t s e x c h a n g e a t t h e g a s e x c h a n g e o r g a n i s more c o m p l e x c h e m i c a l l y t h a n t h a t o-f o t h e r r e s p i r a t o r y g a s e s <0s>, NH 3) and i s l e s s w e l l u n d e r s t o o d , e s p e c i a l l y i n t h e l o w e r v e r t e b r a t e s . The a i m o f t h i s t h e s i s was t o e x a m i n e t h e m e c h a n i s m s u n d e r l y i n g t h e t r a n s p o r t and e x c r e t i o n of COj. i n f i s h , w i t h e m p h a s i s on t h e r o l e o f t h e r e d b l o o d c e l l i n t h o s e p r o c e s s e s . COss i s b o t h r e l a t i v e l y s o l u b l e and r e a d i l y h y d r a t e d i n a q u e o u s s o l u t i o n s . C a r b o n i c a c i d , f a r m e d by CO.-.* h y d r a t i o n , d i s s o c i a t e s t o y i e l d h y d r o g e n , b i c a r b o n a t e and c a r b o n a t e ? i o n s ( f o r r e v i e w s e e E d s a i l 1969) a s f o l l o w s (1 ) COs + H:.BO 4 H a C 0 3 H* + H C 0 3 ~ H 2H* + CO:*35". The r e a c t i o n (2) C 0 3 -i- OH- h HC0 3- 4 H* + C 0 3 a -a l s o i s i m p o r t a n t a t pH v a l u e s o f S and a b o v e , a s a r e f o u n d i n f i s h p l a s m a . M o r e o v e r , COas b i n d s d i r e c t l y t o t h e t e r m i n a l a m ino g r o u p s o f p r o t e i n s , i n c l u d i n g h a e m o g l o b i n , f o r m i n g c a r b a m i n o compounds (R NHCO a~™) „ B i c a r b o n a t e i o n i s t h e p r e d o m i n a n t s p e c i e s p r e s e n t w i t h i n t h e p h y s i o l o g i c a l pH r a n g e o f v e r t e b r a t e b l o o d . 2 As a r e s u l t , o-f t h e a q u e o u s CQnt-bi c a r b o n a t e - c a r b o n a t e e q u i l i b r i u m , c a r b o n d i o x i d e b e h a v e s a s a weak a c i d i n b i o l o g i c a l s y s t e m s ? i n c r e a s e s i n C0a t.ensi on ( Pcoas) dec:rease pH wh i 1 s t d e c r e a s e s i n Peas i n c r e a s e pH« A b a l a n c e b e t w e e n CO^ p r o d u c t i o n and e x c r e t i o n i s n e c e s s a r y , t h e r e - f o r e , t o m a i n t a i n a s t a b l e i n t e r n a l pH. R e g u l a t i o n o-f i n t e r n a l a c i d - b a s e s t a t u s i s c r i t i c a l i n p r e s e r v i n g t h e ' c o r r e c t ' n e t c h a r g e s t a t e of p r o t e i n s ( R e e v e s 1 9 7 7 ) , w h i c h i n t u r n i s c r u c i a l t o t h e b i o c h e m i c a l f u n c t i o n i n g of p r o t e i n s . I n g e n e r a l , v e r t e b r a t e s r e g u l a t e t h e i r a c i d - b a s e s t a t u s by a d j u s t m e n t s t o b l o o d C 0 2 l e v e l s . A i r - b r e a t h i n g v e r t e b r a t e s t y p i c a l l y r e g u l a t e b l o o d pH i n t h e s h o r t t e r m by m o d u l a t i n g b l o o d Poos v i a c h a n g e s i n v e n t i l a t i o n ( f o r r e v i e w s e e Comroe 1974 and D a v e n p o r t 1 9 7 4 ) , H y p e r v e n t i l a t i o n i n c r e a s e s t h e d i f f u s i o n g r a d i e n t f o r d i s s o l v e d C0ri-> b e t w e e n a v e o l a r g a s and p u l m o n a r y b l o o d , and t h u s d e c r e a s e s b l o o d Pcoss and r a i s e s b l o o d pH. H y p o v e n t i l a t i o n h a s t h e o p p o s i t e e f f e c t . L o n g - t e r m pH r e g u l a t i o n i n a i r - b r e a t h e r s i s a c h i e v e d by a d j u s t m e n t s t o b l o o d HC0 3~ l e v e l s m a i n l y v i a r e n a l f u n c t i o n ( D a v e n p o r t 1 9 7 4 ) , A s i m i l a r t i g h t c o u p l i n g b e t w e e n v e n t i l a t i o n , COra e x c r e t i o n and a c i d - b a s e r e g u l a t i o n i s a b s e n t i n 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 o x y g e n c a p a c i t a n c e c o e f f i c i e n t and h e n c e t h e o x y g e n c o n c e n t r a t i o n o f w a t e r i s o n l y a b o u t 0.03-0.05 t i m e s t h a t of a i r , w h i l e t h e COa c a p a c i t a n c e c o e f f i c i e n t of w a t e r i s some 20-30 t i m e s g r e a t e r t h a n t h a t of 0ffi ( D e j o u r s 1 9 7 5 ) . C o n s e q u e n t l y , t h e v e n t i l a t o r y s t r a t e g i e s of w a t e r - b r e a t h e r s a r e d i r e c t e d t o w a r d m a i n t e n a n c e o f a d e q u a t e 0 a u p t a k e r a t h e r t h a n m o d u l a t i o n o f C0 ra e x c r e t i o n ( f o r t h e o r e t i c a l d i s c u s s i o n s e e Rahn 1 9 6 6 ) . I n d e e d , b l o o d 0^ l e v e l s s e t t h e main v e n t i l a t o r y d r i v e i n 3 w a t e r - b r e a t h e r s ( D e j o u r s 1973, R a n d a l l and J o n e s 1 9 7 3 ) , r a t h e r t h a n b l o o d COs> l e v e l s a s o c c u r s i n a i r - b r e a t h e r s (Comroe 1974)= As a d i r e c t c o n s e q u e n c e o f t h e d i f f e r e n c e b e t w e e n t h e o x y g e n and c a r b o n d i o x i d e c a p a c i t a n c e c o e f f i c i e n t s o f w a t e r , v e n t i l a t o r y w a t e r f l o w s t h a t a r e a d e q u a t e t o s a t i s f y 0 K demands h y p e r v e n t i l a t e t h e g i l l s w i t h r e s p e c t t o C 0 2 e x c r e t i o n . I n o t h e r w o r d s , d i s s o l v e d CQz>. i s washed o u t o f f i s h b l o o d 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 . As a r e s u l t , b l o o d F'cos l e v e l s i n f i s h a r e e x t r e m e l y l o w (2-5 t o r r a b o v e a m b i e n t w a t e r l e v e l s , Rahn 1966, R a n d a l l 1 9 7 0 a ) , a r e d e p e n d e n t p r i m a r i l y upon d i f f u s i o n and r e a c t i o n k i n e t i c s a t t h e g i l l , and a r e i n d e p e n d e n t of a l l b u t immense c h a n g e s i n v e n t i l a t o r y f l o w (Cameron and F'olhemus 1974, Wood and J a c k s o n 1 9 8 0 ) . A c o r o l l a r y o f t h i s l o o s e c o u p l i n g b e t w e e n v e n t i l a t i o n and COs* e x c r e t i o n i s t h a t f i s h r e g u l a t e b l o o d pH by m o d u l a t i n g b l o o d HC0 3 ~" l e v e l s w h i l e a l l o w i n g b l o o d Pcoa t o p a r a l l e l e n v i r o n m e n t a l l e v e l s . R e t e n t i o n o f p l a s m a H C 0 3 -i i n c r e a s e s e x t r a c e l l u l a r pH w h i l e d e c r e a s e s i n p l a s m a H C 0 3 ~ h a v e t h e o p p o s i t e e f f e c t . The a s s o c i a t e d r e s p i r a t o r y p r o t o n s ( s e e e q u a t i o n 1) a r e f r e e t o c r o s s t h e g i l l e p i t h e l i u m ( M c W i l l i a m s and P o t t s 1978) and t h e y do s o i n d e p e n d e n t of C 0 3 movements (van den T h i l l a r t e t al- 1 9 8 3 ) . R e g u l a t i o n of p l a s m a H C 0 3 " l e v e l s a s a means o f pH r e g u l a t i o n h a s been o b s e r v e d i n r e s p o n s e t o t e m p e r a t u r e c h a n g e s ( R a n d a l l and Cameron 1 9 7 3 ) , e n v i r o n m e n t a l h y p e r c a p n i a (Cameron and R a n d a l l 1972, Eiddy e t a l . 1 9 7 7 ) , and a c i d s t r e s s ( M c D o n a l d e t al„ 1 9 8 0 ) . The m e c h a n i s m s i n v o l v e d i n r e t e n t i o n a n d / o r r e s o r p t i o n o f HCO3'"" a r e n o t y e t f u l l y u n d e r s t o o d . M o r e o v e r , i t . i s u n c e r t a i n w h e t h e r t h e r e g u l a t o r y 4 mechanism a c t s upon H C O 3 " , O H ™ , H~, Nl-U"'" o r a c o m b i n a t i o n o f t h e s e i o n s . E x c r e t i o n o f H* o r r e t e n t i o n o f O H a r e f u n c t i o n a l l y t h e same a s r e t e n t i o n o f HC0 3™ ( s e e e q u a t i o n s 1 and 2 ) . E x c r e t i o n o f NhU* e f f e c t i v e l y r e m o v e s a H* s i n c e ammonia i s p r o d u c e d m e t a b o l i c a l 1 y a s N H - . The g i l l i s t h e p r i n c i p a l s i t e o f C O s s e x c r e t i o n i n t r o u t . Thus, t h e g i l l p l a y s a p a r a m o u n t r o l e i n a c i d - b a s e rand i o n i c r e g u l a t i o n (Cameron 1978a, R a n d a l l et al* 1982, H e i s l e r 1 9 8 4 ) . O t h e r o r g a n s , i n c l u d i n g t h e k i d n e y , g u t and s k i n , a r e g e n e r a l l y of l i t t l e i m p o r t a n c e i n pH r e g u l a t i o n . Two c o n f l i c t i n g m o d e l s h a v e been d e v e l o p e d t o d e s c r i b e t h e p a t h w a y f o r CO53 e x c r e t i o n i n f i s h , and h e n c e t h e r e l a t i o n s h i p b e t w e e n b r a n c h i a l C O s s movements and i o n i c / p H r e g u l a t i o n . The o r i g i n a l t h e o r y , t h e r e d c e l l model o f C 0 S e x c r e t i o n , c o m b i n e s t h e a c c e p t e d mammalian i n f o r m a t i o n r e g a r d i n g C0 f f l e x c r e t i o n ( c f . Rough t o n 1964) w i t h t h e known Wa^/H"*" (Nl-U"1') and C I " / H C O 3 " ( O H ™ ) e x c h a n g e r s p r e s e n t on t h e a p i c a l membrane o f t h e g i l l e p i t h e l i u m (Maetz 1971, E v a n s 1 9 7 5 ) . I n t h i s m o d e l , p l a s m a H C O - ™ e n t e r s t h e r e d c e l l 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 , w h ere i t i s c a t a l y t i c a l 1 y c o n v e r t e d t o C Q s s i n t h e p r e s e n c e o f e r y t h r o c y t e c a r b o n i c anhydrase., The r e s u l t a n t . C O a. d i f f u s e s down i t s c o n c e n t r a t i o n g r a d i e n t o u t o f t h e r e d c e l l and a c r o s s t h e g i l l e p i t h e l i u m . A s m a l l p r o p o r t i o n o f t h i s C0r.E (3-47., Cameron 1976) i s b a c k - c o n v e r t e d t o HC0 3"~ and H* i n t h e p r e s e n c e o f g i l l c a r b o n i c a n h y d r a s e . T h e s e i o n s t h e n s e r v e a s c o u n t e r i o n s f o r t h e a p i c a l i o n e x c h a n g e r s . M o d u l a t i o n o f t h e s e e x c h a n g e r s i s b e l i e v e d t o b e t he p r i m a r y p H - r e g u 1 a t i n g (it e c h a n i s m i n f i s h (Cameron 1978a, H e i s l e r 1 9 8 4 ) . The r a t e - l i m i t i n g s t e p i n C0 = ; 5 e x c r e t i o n i n t h i s model i s t h e e n t r y o f p l a s m a H C 0 3 " ~ i n t o t h e e r y t h r o c y t e . The p o s s i b i l i t y t h a t m o d u l a t i o n o f t h i s s t e p , b i c a r b o n a t e e n t r y i n t o t h e r e d c e l l , a l s o i s i n v o l v e d i n a c i d -b a s e r e g u l a t i o n l a r g e l y h a s been i g n o r e d . , A more r e c e n t t h e o r y , t h e g i l l model o f CO^ e x c r e t i o n ( H a s w e l l and R a n d a l l 1978, H a s w e l l e t a l . 1 9 8 0 ) , h a s been d e v e l o p e d a r o u n d t h e a s s e r 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 f u n c t i o n a l l y i m p e r m e a b l e t o H C 0 3 ' ~ in vivo ( H a s w e l l and R a n d a l l 1976, H a s w e l l et a i , 1 9 7 8 ) . In t h i s m o d e l , p l a s m a H C 0 3 ~ and H"1" e n t e r t h e g i l l e p i t h e l i u m d i r e c t l y and t h e n e i t h e r s e r v e a s c o u n t e r i o n s f o r t h e a p i c a l i o n e x c h a n g e r s o r a r e c a t a l y t i c a l 1 y c o n v e r t e d t o CO..,-; by g i l l c a r b o n i c a n h y d r a s e a n d - e x c r e t e d a s e u c h . The r a t e - l i m i t i n g s t e p i n CO^ e x c r e t i o n i s p r o p o s e d t o be t h e e n t r y o f p l a s m a H C 0 3 - i n t o t h e g i l l e p i t h e l i u m ; C0s» e x c r e t i o n i n t h i s model i s i n d e p e n d e n t o f e r y t h r o c y t e f u n c t i o n . A c i d - b a s e r e g u l a t i o n i n v o l v e s a c o m b i n a t i o n o f m o d u l a t i o n o f t h e a p i c a l i o n e x c h a n g e r s and t h e e n t r y o f p l a s m a H C 0 3 ~ a c r o s s t h e b a s a l membrane o f t h e g i l l . The o b j e c t i v e s o f t h e p r e s e n t s t u d i e s were t o e x a m i n e CO^ 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 i n l i g h t o f t h e r e d c e l l and g i l l t h e o r i e s o f C 0 2 e x c h a n g e . In s o d o i n g , t h e r o l e of t h e e r y t h r o c y t e i n c a t a l y s i n g t h e i n t e r c o n v e r s i o n o f H C 0 3 ~ and C 0 S was s t u d i e d . The f u n c t i o n i n g o f t h e r e d b l o o d c e l l i n t h i s r e g a r d e n t a i l s t h e n e t movement o f HCQ 3™ a c r o s s t h e r e d c e l l membrane, a s w e l l a s , t h e a c t i v i t y o f i n t r a c e l l u l a r c a r b o n i c a n h y d r a s e . The r o l e o f c a t e c h o l a m i n e s i n m o d u l a t i n g t h e n e t f l u x o f HCQ 3" t h r o u g h t h e r e d c e l l , and h e n c e i n r e g u l a t i o n o f a c i d -6 b a s e s t a t u s , a l s o was i n v e s t i g a t e d ir? vitro and in v i v o . O v e r a l l , t h e s e s t u d i e s s h e d l i g h t on t h e r e l a t i o n s h i p b e t w e e n C 0 : Z movements and a c i d - b a s e r e g u l a t i o n i n l o w e r v e r t e b r a t e s . 7 GENERAL MATERIALS AND METHODS Rainbo w t r o u t (S&lwo gairdneri), w e i g h i n g b e t w e e n 200 and 500 g, 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 . ) . The f i s h were h e l d o u t d o o r s a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a i n n a t u r a l l i g h t i n f l o w i n g , d e c h l o r i n a t e d V a n c o u v e r t a p w a t e r . T e m p e r a t u r e o f t h i s w a t e r v a r i e d s e a s o n a l l y b e t w e e n 6 and 13 C. The f i s h were f e d t o s a t i a t i o n d a i l y w i t h a c o m m e r c i a l f i s h f o o d . Food was w i t h h e l d f o r ait l e a s t 24 h p r i o r t o , a s w e l l a s , d u r i n g a l l s t u d i e s . E x p e r i m e n t a l 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 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 m g - L - 1 MS-222, pH b u f f e r e d t o a b o u t 7.5 w i t h 133.3 mg-L™ 1 NaHC0 3) m a i n t a i n e d a t t h e axmbient w a t e r t e m p e r a t u r e . The a n i m a l s t h e n w e re t r a n s f e r r e d t o a s u r g i c a l t a b l e where an 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 t r i c a i n e methane s u l p h o n a t e , 100 mg-L"'"1 NaHCOs) a t 5-10 C 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 . The d o r s a l a o r t a a n d , i n some c a s e s , t h e v e n t r a l a o r t a were c a n n u l a t e d t h r o u g h t h e mouth . 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 gage) was u s e d t o make a b l i n d p u n c t u r e o f t h e d o r s a l a o r t a i n t h e c a u d a d 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 basket. b e t w e e n t h e f i r s t and s e c o n d 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 s u r g i c a l t u b i n g (PE-50) was f e d down t h e c a t h e t e r and i n t o t h e a o r t a . The c a t h e t e r t h e n was removed l e a v i n g t h e c a n n u l a c h r o n i c a l l y i m p l a n t e d i n t h e a o r t a , . The c a n n u l a e x i t e d t h e f i s h t h r o u g h t h e r o o f o f t h e mouth i n f r o n t o f t h e n a r e s by way o f a 8 • f l a n g e d s e c t i o n o f s u r g i c a l t u b i n g ( P E - 2 0 0 ) . The v e n t r a l a o r t a was c a n n u l a t e d i n a s i m i l a r manner. C a n n u l a t i o n o f t h e v e n t r a l a o r t a i n v o l v e d a b l i n d p u n c t u r e o f t h e v e s s e l made l a t e r a l l y b e n e a t h t h e t o n g u e b e t w e e n t h e f i r s t and s e c o n d g i l l a r c h e s . V e n t r a l a o r t i c c a n n u l a e e x i t e d t h e f i s h t h r o u g h t h e f l o o r o f t h e mouth by way of a f l a n g e d s e c t i o n o f s u r g i c a l t u b i n g ( P E - 2 0 0 ) . B o t h d o r s a l and v e n t r a l a o r t i c c a n n u l a e were s e c u r e d w i t h s u t u r e s t o t h e w a l l s of t h e o r a l c a v i t y . C a n n u l a e were f l u s h e d d a i l y w i t h C o r t l a n d s a l i n e (Wolf 1963, 10,000 U.S.P. u n i t s - L - 1 s o d i u m h e p a r i n ) t o m a i n t a i n t h e i r p a t e n c y . F o l l o w i n g s u r g e r y , t h e f i s h were t r a n s f e r r e d t o l i g h t - p r o o f a c r y l i c b o x e s s u p p l i e d w i t h a 3 L - r n i n - 1 f l o w o f a e r a t e d w a t e r . F i s h were a l l o w e d a t l e a s t 48 h of p o s t - o p e r a t i v e rmcovery b e f o r e s t u d i e s were u n d e r t a k e n . OliL'k§. ICS#.t m.ilQt s\Q?i P r e s e n t a t i o n S t a t i s t i c a l t r e a t m e n t o f d a t a i n c l u d e d r e g r e s s i o n a n a l y s i s by t h e method o f l e a s t s q u a r e s , a n a l y s i s o f c o v a r i a n c e t o t e s t h o m o g e n e i t y of r e g r e s s i o n c o e f f i c i e n t s , and s t a n d a r d and p a i r e d S t u d e n t ' s t - t e s t s ( S t e e l and T o r r i e 1960, S n e d e c o r and C o c h r a n 1 9 6 7 ) . I n a l l c a s e s , d i f f e r e n c e s p r e s e n t a t t h e 57. l e v e l o f p r o b a b i l i t y (P < 0.05) were j u d g e d t o be s i g n i f i c a n t . E x p e r i m e n t a l d a t a a r e p r e s e n t e d a s a r i t h m e t i c means + 1 s t a n d a r d e r r o r ( S . E . ) , w h e r e a s s t a t i s t i c a i l y — d e r i v e d d a t a ( i e . r e g r e s s i o n c o e f f i c i e n t s ) a r e p r e s e n t e d w i t h t h e i r 957. c o n f i d e n c e i n t e r v a l s . Mean pH v a l u e s were c a l c u l a t e d d i r e c t l y , a s recommended by B o u t i l i e r and S h e l t o n ( 1 9 8 0 ) , w i t h o u t t r a n s f o r m a t i o n b a c k t o H"1" c on c en t r a t i on s. 9 C h a p t e r 1„ COs> TRANSPORT PROPERTIES OF BLOOD OF SAL MO GAIRDNERI A p r i m a r y p h y s i o l o g i c a l f u n c t i o n of v e r t e b r a t e b l o o d i s t r a n s p o r t . o f t h e r e s p i r a t o r y g a s e s , c a r b o n d i o x i d e and o x y g e n , b e t w e e n t h e g a s e x c h a n g e o r g a n ( s ) and t h e t i s s u e s . The e f f e c t i v e n e s s of t h i s t r a n s p o r t i s d e t e r m i n e d n o t o n l y by t h e f l o w of e x c h a n g e m e d i a ( a i r / w a t e r and b l o o d ) t h r o u g h t h e g a s e x c h a n g e o r g a n ( s ) , b u t a l s o by t h e t r a n s p o r t p r o p e r t i e s of t h e b l o o d u n d e r c o n s i d e r a t i o n . The o b j e c t i v e o f t h i s c h a p t e r i s t o e l u c i d a t e t h e COsa t r a n s p o r t p r o p e r t i e s o f Salmo gairdneri b l o o d i n o r d e r t o p r o v i d e a f r a m e w o r k upon w h i c h s u b s e q u e n t e x a m i n a t i o n s of p i s c i n e g a s e x c h a n g e f u n c t i o n c a n be made. C a r b o n d i o x i d e i s t r a n s p o r t e d i n b l o o d p r i m a r i l y i n c h e m i c a l l y - b o u n d s t a t e s , a s d i s s o c i a t e d s a l t s o f c a r b o n i c a c i d . B i c a r b o n a t e i s t h e p r e d o m i n a n t s p e c i e s p r e s e n t w i t h i n t h e p h y s i o l o g i c a l pH r a n g e o f v e r t e b r a t e b l o o d . By c o n v e n t i o n , t h e r e l a t i o n s h i p b e t w e e n d i s s o l v e d and bound CO32 i s e x p r e s s e d i n t h e f o r m of a H e n d e r s o n H a s s e l b a c h e q u a t i o n <3) pH = p K f f l p p + l o g (Ccoa / ( a C 0 2 " P C O 2 ) - 1) where p K « p p i s t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t o f c a r b o n i c a c i d , Ccoa i s t o t a l CO 3- c o n t e n t , a CDs i s t h e s o l u b i l i t y c o e f f i c i e n t o f CO3., and Pcosa i s C D 2 p a r t i a l p r e s s u r e . The d e r i v a t i o n and l i m i t a t i o n s o f t h i s e q u a t i o n a r e d e a l t w i t h a t 10 l e n g t h by A l b e r s ( 1 9 7 0 ) . Bound COa i s r e p r e s e n t e d by t h e d i f f e r e n c e b e t w e e n C C 0 2 and C s C G s s - P ^ a x l , and g e n e r a l l y i s t e r m e d s i m p l y HCQ3~". F o r t h e p u r p o s e o f t h i s t h e s i s , h o w e v e r , bound CQs> i s r e f e r r e d t o a s a p p a r e n t b i c a r b o n a t e o r bHCGs" s i n c e i t i n c l u d e s a l l s p e c i e s o f c h e m i c a l l y - b o u n d C0s». G i v e n a p p r o p r i a t e v a l u e s f o r p K „ p p and sCOa., e q u a t i o n 3 c a n be u s e d t o c a l c u l a t e CCJOSSJ bHC0;-s"", Pcoa o r pH when any two o f C Coa, P Coa and pH a r e me a s u r e d s i m u l t a n e o u s l y . V a l u e s o f pK„ P P, and sCOa h a v e been m e a s u r e d f o r mammalian p l a s m a w i t h g r e a t a c c u r a c y ( S e v e r i n g h a u s e t al. 1956, B a r t e l s and W r b i t z k y 1960, S i g g a a r d - A n d e r s e n 1962, A u s t i n e t al. 1963, R i s p e n s e t al, 1968, among o t h e r s ) . S i m i l a r v a l u e s a r e a b s e n t f o r most f i s h s p e c i e s . S i m p l e e x t r a p o l a t i o n o f mammalian v a l u e s i s n o t s a t i s f a c t o r y (Mi c o l e t a l * 1983, B o u t i l i e r e t al. 1 9 8 4 a ) , c o n s i d e r i n g t h e l e n g t h o f t e m p e r a t u r e e x t r a p o l a t i o n r e q u i r e d and t h e d i f f e r e n c e s i n i o n i c s t r e n g t h b e t w e e n f i s h p l a s m a ( 0 , 1 5 0.70, s e e A l b e r s 1970 f o r r e f e r e n c e s ) and human p l a s m a ( 0 , 1 6 7 ) . CDs* b e h a v e s a s a weak a c i d i n a q u e o u s s o l u t i o n i n a s much a s i n c r e a s e s i n CO^ t e n s i o n n e c e s s a r i l y r e d u c e pH v i a e q u a t i o n 3, w h i l s t r e d u c t i o n s i n CDs t e n s i o n n e c e s s a r i l y i n c r e a s e s pH. A c o r o l l a r y o f t h i s COssSpH r e l a t i o n s h i p i s t h a t b l o o d i s a c i d i f i e d d u r i n g t i s s u e c a p i l l a r y t r a n s i t and i s a l k a l i n i z e d d u r i n g b r a n c h i a l / p u l m o n a r y c a p i l l a r y t r a n s i t . V a r i a t i o n s i n b l o o d pH r e s u l t i n g f r o m C 0 S t r a n s p o r t a r e b u f f e r e d p r i m a r i l y by b l o o d p r o t e i n s , t h e most i m p o r t a n t , o f w h i c h i s h a e m o g l o b i n ( A l b e r s 1970, Comroe 1 9 7 4 ) . T h i s b u f f e r i n g a c t i o n f a c i l i t a t e s f o r m a t i o n of t h e d i s s o c i a t e d m o i e t y o f c a r b o n i c a c i d v i a t h e 'Law of Mass A c t i o n ' , and t h u s i s an i m p o r t a n t d e t e r m i n a n t o f b l o o d C 0 S 11 t r a n s p o r t p r o p e r t i e s . The b u f f e r i n g c a p a c i t y o f h a e m o g l o b i n e n h a n c e s COssSHCO^."" c o n v e r s i o n i n s i d e t h e r e d b l o o d c e l l . In mammalian e r y t h r o c y t e s , i n t r a c e l l u l a r CO^ r e a c t i o n s a r e f o l l o w e d by e x c h a n g e o f HCQ-~ b e t w e e n t h e r e d c e l l and e x t r a c e l l u l a r p l a s m a ( R o u g h t o n 1 9 6 4 ) . Mammalian r e d c e l l s a r e f r e e l y p e r m e a b l e t o most a n i o n s and H"", a l l o f w h i c h a r e d i s t r i b u t e d p a s s i v e l y a c r o s s t h e c e l l membrane a c c o r d i n g t o a G i b b s - D o n n a n e q u i l i b r i u m ( J a c o b s and S t e w a r t 1947, Gunn e t al„ 1973, Freedman and H o f f m a n 1 9 7 9 ) . L i t t l e i s known of t h e Donnan e q u i l i b r i u m i n f i s h e r y t h r o c y t e s , , Donnan r a t i o s f o r c h l o r i d e a n d / o r bHC0 3~" h a v e been d e t e r m i n e d i n a s m a l l number of f i s h e s ( F e r g u s o n e t al. 1938, F e r g u s o n and B l a c k 1941, A l b e r s e t al. 1969, H a s w e l l e t al. 1978, Wood e t al. 1 9 8 1 ) . As i n mammalian e r y t h r o c y t e s , t h e Donnan r a t i o s o f f i s h e r y t h r o c y t e s v a r y i n v e r s e l y w i t h p l a s m a pH. F o r t h e most p a r t , h o w e v e r , Donnan r a t i o s o f f i s h r e d c e l l s do n o t a g r e e wel1 w i t h v a l u e s p r e d i c t e d on t h e b a s i s o f a p a s s i v e d i s t r i b u t i o n ( J a c o b s and S t e w a r t 1947, Freedman and H o f f m a n 1 9 7 9 ) . A p a s s i v e d i s t r i b u t i o n o f a n i o n s and H"1" i n f i s h e r y t h r o c y t e s t h u s i s open t o q u e s t i o n , e s p e c i a l l y i n l i g h t o f t h e p o l e m i c a l work o f H a s w e l l and R a n d a l l ( 1976, 1978) i n d i c a t i n g t h a t f i s h r e d c e l l s a r e f u n c t i o n a l l y i m p e r m e a b l e t o HC03"" . S t u d i e s i n t h i s c h a p t e r e x a m i n e d t h e CO- t r a n s p o r t p r o p e r t i e s o f r a i n b o w t r o u t b l o o d i n t e r m s o f in vitro d i s s o c i a t i o n c u r v e s , t h a t i s , t h e r e l a t i o n s h i p b e t w e e n C0-„-» c o n t e n t and p a r t i a l p r e s s u r e . The p h y s i c o - c h e m i c a l p r o p e r t i e s of b l o o d w i t h r e s p e c t t o CO- t r a n s p o r t , p K „ p p and a C 0 2 , a l s o w e re 12 d e t e r m i n e d . P a r t i t i o n i n g o f C c o s s b e t w e e n p l a s m a and e r y t h r o c y t e s , and t h e e f f e c t s o f C O ^ on t r a n s m e m b r a n e i o n e q u i l i b r i a were i n v e s t i g a t e d i n d e t a i l . F i n a l l y , i n vivo d e t e r m i n a t i o n s o f a r t e r i a l and v e n o u s C O a and a c i d - b a s e s t a t e s were c o u p l e d w i t h r e s u l t s o f t h e i n vitro s t u d i e s t o p r o d u c e a model o f a r t e r i a l and v e n o u s C O ^ t r a n s p o r t i n r a i n b o w t r o u t . 13 MATERIALS AND METHODS A. § £ 3 . L u b.iii.ty o f C O s s and A g g a r e n t D i s s o c i a t i^on C o n s t a n t o f H 3 C O 3 C O H s o l u b i l i t y c o e f f i c i e n t s were d e t e r m i n e d u s i n g s e p a r a t e d p l a s m a o b t a i n e d by c e n t r i f u g a t i o n o f b l o o d w i t h d r a w n f r o m t h e d o r s a l a o r t a s o f c h r o n i c a l l y c a n n u l a t e d r a i n b o w t r o u t ( s e e G e n e r a l M a t e r i a l s and M e t h o d s ) . One--mL a l i q u o t s o f t h i s p l a s m a were a c i d i f i e d s l o w l y t o a b o u t pH 2.5 by a d d i t i o n o f c o n c e n t r a t e d l a c t i c a c i d and t h e n were t r a n s f e r r e d t o 50 ml... t o n o m e t e r f l a s k s . A l l s a m p l e s w ere k e p t on i c e u n t i l u s e d . A c i d i f i e d s a m p l e s were e q u i l i b r a t e d at. 5, 10 and 15 C w i t h a h u m i d i f i e d g a s m i x t u r e c o n t a i n i n g 407. COs* i n a i r d e l i v e r e d by a W o s t o f f g a s m i x i n g pump. S a m p l e s were g a s s e d f o r a t l e a s t 60 min. T o t a l C 0 S c o n t e n t i C C 0 2 ) o f t h e p l a s m a t h e n was m e a s u r e d i n d u p l i c a t e w i t h a C0;> e l e c t r o d e ( R a d i o m e t e r , t y p e E5036) u s i n g t h e methods o f Cameron ( 1 9 7 1 ) . P a r t i a l p r e s s u r e o f CO^ ( P C D a ) i n t h e p l a s m a was c a l c u l a t e d f r o m t h a t i n t h e g a s m i x t u r e t a k i n g i n t o a c c o u n t t h e p r e v a i l i n g b a r o m e t r i c p r e s s u r e , w a t e r v a p o u r p r e s s u r e , e q u i l i b r a t i o n t e m p e r a t u r e and CQrs c o n t e n t o f a i r (0.0337., G l u e c k a u f 1 9 5 1 ) . C0^ s o l u b i l i t y c o e f f i c i e n t s (aCOra) were c a l c u l a t e d f r o m H e n r y ' s Law, where (4) aCO* = Ccoz / Poos . As a c h e c k o f t h e t e c h n i q u e , C0s» s o l u b i l i t y c o e f f i c i e n t s o f n o r m a l d i s t i l l e d w a t e r and of d i s t i l l e d w a t e r a c i d i f i e d a s a b o v e 1 4 w i t h l a c t i c a c i d a l s o were m e a s u r e d . The a p p a r e n t d i s s o c i a t i o n c o n s t a n t o f c a r b o n i c a c i d <pK» P p) was d e t e r m i n e d u s i n g a m i x e d p o o l o f w h o l e b l o o d o b t a i n e d f r o m t h e d o r s a l a o r t i c c a n n u l a s o f s e v e r a l r a i n b o w t r o u t , Two-mL a l i q u o t s o f p o o l e d b l o o d w e re d i s t r i b u t e d t o 50-mL t o n o m e t e r f l a s k s on i c e and t h e n were e q u i l i b r a t e d a t 10 C w i t h h u m i d i f i e d g a s m i x t u r e s c o n t a i n i n g 0,2, 0.5 and 1.07, C 0 a i n a i r ( W a s t o f f pumps). S a m p l e s were g a s s e d f o r a t l e a s t 60 min and t h e n were c e n t r i f u g e d a n a e r o b i c a l l y a t room t e m p e r a t u r e f o r 2-4 m i n . P l a s m a pH was m e a s u r e d a n a e r o b i c a l 1 y w i t h a g l a s s c a p i l l a r y e l e c t r o d e ( R a d i o m e t e r , t y p e G297/G2) t h e r m o s t a t t e d a t t h e e q u i l i b r a t i o n t e m p e r a t u r e (10 C) and c a l i b r a t e d b e f o r e and a f t e r -e a c h pl-l d e t e r m i n a t i o n w i t h p r e c i s i o n b u f f e r s (Radiometer,, S I 5 0 0 , S 1 5 1 0 ) , P l a s m a Ccus and P C o 2 were d e t e r m i n e d a s b e f o r e . V a l u e s of p K a p p were c a l c u l a t e d f r o m m e a s u r e d pH, Ccos , P<=O;K and <3C03 v a l u e s u s i n g a r e a r r a n g e m e n t o f t h e R e n d e r s o n - H a s s e l b a c h e q u a t i o n f o r c a r b o n i c a c i d (5) pK^p. = pH - 1 og < CCooa/ (<3C0s> •• Poos?) 3 - 1) . B. I.R QLtLLQ.. R g s p _ i _ r a t g r y and A c i d - b a s e C h a r a c t e r i s t i c s o f B l o o d B l o o d s a m p l e s w e re w i t h d r a w n f r o m t h e d o r s a l a o r t i c c a n n u l a e o f s e v e r a l r a i n b o w t r o u t and were p o o l e d on i c e . Two-mL a l i q u o t s o f p o o l e d b l o o d were e q u i l i b r a t e d a t 5, 10 and 15 C w i t h h u m i d i f i e d g a s m i x t u r e s c o n t a i n i n g 0.25, 0,50, 1.00 and 2.00% C 0 a i n a i r ( W o s t o f f pumps). S a m p l e s were g a s s e d f o r a t l e a s t 45 min b e f o r e t h e i r r e s p i r a t o r y and a c i d - b a s e s t a t e s were a s s e s s e d . B l o o d pH was m e a s u r e d w i t h R a d i o m e t e r g l a s s c a p i l l a r y 15 e l e c t r o d e s t h e r m o s t a t t e d a t t h e p r e v a i l i n g e q u i l i b r a t i o n t e m p e r a t u r e and c a l i b r a t e d a s d e s c r i b e d p r e v i o u s l y . I n t r a e r y t h r o e y t i c pH was m e a s u r e d on an a n a e r o b i c a l 1 y - o b t a i n e d p e l l e t o-f r e d c e l l s u s i n g t h e f r e e z e - t h a w t e c h n i q u e s o f Z e i d l e r and Kim (1977) and e m p l o y i n g t h e same pH e l e c t r o d e a s a b o v e . B l o o d P c o s was m e a s u r e d u s i n g a CO- e l e c t r o d e ( R a d i o m e t e r , t y p e E5036) t h e r m o s t a t t e d a t t h e p r e v a i l i n g e q u i l i b r a t i o n t e m p e r a t u r e and p r e c o n d i t i o n e d t o t h e e x p e c t e d P c o s v a l recommended by B o u t i l i e r et al . ( 1 9 7 8 ) . E a c h P C o a d e t e r m i n a t i o n was b r a c k e t e d w i t h c a l i b r a t i o n g a s e s d e l i v e r e d v i a W o s t o f f pumps.. CCos» o-f b l o o d and o f p l a s m a o b t a i n e d -from t h e b l o o d by a n a e r o b i c c e n t r i f u g a t i o n a t room t e m p e r a t u r e was m e a s u r e d a s d e s c r i b e d i n t h e COa s o l u b i l i t y s t u d y . The a p p a r e n t b i c a r b o n a t e c o n c e n t r a t i o n ( b HC0 3") o f s a m p l e s was c a l c u l a t e d f r o m m e a s u r e d C C O B J F'CO- and aCOja v a l u e s a s (6) b H C O s " = C c o a ( a C O a • P e a s ) . I t s h o u l d be n o t e d t h a t bHC0 3"' c a l c u l a t e d i n t h i s way d o e s n o t d i s t i n g u i s h b e t w e e n HCQ3"_, C 0 3 - ~ o r c a r b a m i n o (R-NHCO^-) . H a e m a t o c r i t m e a s u r e m e n t s were made i n t r i p l i c a t e . W ater c o n t e n t s of b l o o d and p l a s m a were d e t e r m i n e d by w e i g h i n g s a m p l e s t o t h e n e a r e s t 0.1 mg, f i r s t wet and t h e n a g a i n a f t e r t h e s a m p l e s had been d r i e d t o c o n s t a n t w e i g h t a t 60 C. A d d i t i o n a l a l i q u o t s o f e q u i l i b r a t e d b l o o d and p l a s m a were s t o r e d f r o z e n f o r l a t e r a n a l y s e s o f i o n s . C l ~ c o n c e n t r a t i o n s o f b l o o d and p l a s m a were m e a s u r e d w i t h a B u t 1 e r - C o t 1 o v e a m p e r o m e t r i c t i t r a t o r u s i n g t h e 16 m e t h o d s o-f C o t l o v e ( 1 9 6 3 ) . P l a s m a Na*, K"*', Mg 5 8* and C a 2 + c o n c e n t r a t i o n s were m e a s u r e d tay flame; p h o t o m e t r y a s d e s c r i b e d by A n n i o (1964) u s i n g a P e r k i n - E l m e r a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r (model 2 3 8 0 ) . L a c t a t e was a s s a y e d e n z y m a t i c a l 1 y (Sigma p r o c e d u r e no. 826-UV) ii b l o o d s a m p l e s u s e d i n l a c t a t e a s s a y s were d e p r o t e i n a t e d w i t h c h i l l e d p e r c h l o r i c a c i d i m m e d i a t e l y upon s a m p l i n g . I n t r a e r y t h r o c y t i c l e v e l s ( I D o f CI-~, bHCOrs™ and w a t e r were c a l c u l a t e d f r o m t h e r e l a t i o n , (7) I L = [ b l o o d v a l u e --• ( p l a s m a v a l u e ) (1 -- h a e m a t o c r i t " lO" - 3 5) 3 / ( h a e m a t o c r i t " 10"~s) . I o n c o n c e n t r a t i o n s were e x p r e s s e d p e r k i l o g r a m o f r e d c e l l o r p l a s m a w a t e r i n c a l c u l a t i o n o f Donnan i o n r a t i o s . The C 0 2 - c o m b i n i n g p r o p e r t i e s o f s e p a r a t e d p l a s m a were e x a m i n e d i n an i n d e p e n d e n t s e r i e s o f e x p e r i m e n t s . S e p a r a t e d p l a s m a was o b t a i n e d by c e n t r i f u g a t i o n o f b l o o d w i t h d r a w n f r o m d o r s a l a o r t i c c a n n u l a e . One-mL a l i q u o t s o f s e p a r a t e d p l a s m a were e q u i l i b r a t e d a t 10 C w i t h h u m i d i f i e d g a s m i x t u r e s c o n t a i n i n g 0.2, 1.0 and 2.07. C 0 K i n a i r ( W o s t o f f pumps). S a m p l e s were g a s s e d f o r a t l e a s t 60 min. P l a s m a pH and C o o ^ t h e n were m e a s u r e d a s d e s c r i b e d a b o v e . P l a s m a Pcrasa was c a l c u l a t e d f r o m t h a t i n t h e g a s m i x t u r e s a s d e s c r i b e d i n t h e C 0 S s o l u b i l i t y s t u d y . C° 6CJr_§!li.§-L §Q£! y.e.D.2y§ ICS0.§E9!l£ 2 f QQs l T l ilii'.Q. B l o o d s a m p l e s were w i t h d r a w n s i m u l t a n e o u s l y f r o m b o t h t h e d o r s a l and v e n t r a l a o r t a s o f c a n n u l a t e d r a i n b o w t r o u t . A m b i e n t w a t e r t e m p e r a t u r e was 10 C. A p o r t i o n o f e a c h b l o o d s a m p l e was 17 u s e d t o m e a s u r e h a e m a t o c r i t i n d u p l i c a t e . The r e m a i n d e r was c e n t r i f u g e d a n a e r o b i c a l l y a t room t e m p e r a t u r e f o r 1-2 m i n . A l l s u b s e q u e n t m e a s u r e m e n t s were made on p l a s m a . A r t e r i a l and v e n o u s v a l u e s o f C Ca2 and pH were m e a s u r e d a s be-fore. Poor . ? v a l u e s were c a l c u l a t e d f r o m m e a s u r e d C C D2, pH, pK«,p,p and aCO^> v a l u e s u s i n g a r e a r r a n g e m e n t o f t h e H e n d e r s o n - H a s s e l b a c h e q u a t i o n f o r c a r b o n i c a c i d , where <8> Poo2 - Ceo- / (C 1 0 p H - p H : * > f ° | a •+• 13 - aCOs) . V a l u e s o f bHC03~" were c a l c u l a t e d u s i n g e q u a t i o n 6. 18 RESULTS COs s o l u b i l i t y c o e f f i c i e n t s <aCO K) of r a i n b o w t r o u t p l a s m a and d i s t i l l e d w a t e r a r e p r e s e n t e d i n F i g u r e 1 and T a b l e 1. The mean m e a s u r e d v a l u e s f o r w a t e r were w i t h i n 2.7 umol • L ™ 1 - t o r r " " 1 of t h o s e r e p o r t e d by M u r r a y and R i l e y (1971), C o m p a r a b l e d a t a f o r t h e <3C0s< o f t r o u t p l a s m a were n o t f o u n d i n t h e l i t e r a t u r e . COs* s o l u b i l i t y was a f u n c t i o n o f t e m p e r a t u r e and i o n i c s t r e n g t h , a C Q 2 d e c r e a s e d when e i t h e r t e m p e r a t u r e o r i o n i c s t r e n g t h i n c r e a s e d ( d i s t i l l e d w a t e r , 1=05 p l a s m a , 1=0.15). C o m p a r i s o n o f a C O a v a l u e s o f n o r m a l and a c i d i f i e d d i s t i l l e d w a i t e r s <Fig„ 1, T a b l e 1) s u g g e s t s t h a t , t h e a c i d i f i c a t i o n p r o c e s s had l i t t l e d i r e c t e f f e c t on m e a s u r e d aCOss v a l u e s . However, P l e s c h k a and W i t t e n b r o c k (1971) n o t e d t h a t a c i d i f i c a t i o n o f t h e p l a s m a o f d o g f i s h ( S c y l iorhinus canicul a S . stelJarzs) r e s u l t e d i n p r e c i p i t a t i o n o f p l a s m a p r o t e i n s , a l t e r i n g p l a s m a o s m o l a r i t y and h e n c e i t s CO® s o l u b i l i t y . In t h e p r e s e n t s t u d y , i n i t i a l a t t e m p t s t o a c i d i f y t r o u t p l a s m a a l s o r e s u l t e d i n p r e c i p i t a t i o n o f p r o t e i n s w h i c h o c c u r r e d s p o r a d i c a l l y , i n d e p e n d e n t o f t h e r a t e o f a c i d i f i c a t i o n o r p l a s m a t e m p e r a t u r e . P r o t e i n p r e c i p i t a t i o n was f o u n d t o be r e l a t e d t o t h e u s e of h e p a r i n a s an a n t i c o a g u l a n t . In an i n d e p e n d e n t e x p e r i m e n t , i t was f o u n d t h a t a c i d i f i c a t i o n o f p l a s m a c o n t a i n i n g more t h a n 10,000 U.S.P. u n i t s - L ~ * s o d i u m h e p a r i n r e s u l t e d i n p r e c i p i t a t i o n o f p r o t e i n s , w h i l e no p r e c i p i t a t i o n was p r e s e n t i n p l a s m a c o n t a i n i n g l e s s t h a n t h a t c o n c e n t r a t i o n o f h e p a r i n . T h i s e f f e c t p r o b a b l y was a t t r i b u t a b l e 19 F i g u r e 1= P h y s i c o - c h e m i c a l p a r a m e t e r s p e r t a i n i n g t o CO^ t r a n s p o r t i n Salmo gairdneri b l o o d . P a n e l A. CO^ s o l u b i l i t y (umol*L™ 1-torr" 1) o f w a t e r and t r o u t p l a s m a a t 5, 10 and 15 C . V a l u e s a r e means + 1 S.E. (n™6)„ L e a s t s q u a r e s r e g r e s s i o n l i n e s a r e p r e s e n t e d i n T a b l e 1. P a n e l B. E f f e c t o f pH on t h e ap p p a r en t d i ssoc: :L a t i on c on st. an t o f c a r b on i c: ac i d (p K^p, p ) i n t r out. p l a s m a a t 10 C. L e a s t s q u a r e s r e g r e s s i o n l i n e i s p K ^ p , = 6.974 0.098 pH <r=--1.000, n=3) . V a l u e s a r e means + 1 S.E. (n=8) . 20 30 8 0 o co CM o o 7 0 G 0 plasma normal • acidified A 5 0 OJ TEMPERATURE, C G . 4 R B. G . 3 G . 2 I-G . 1 G . 0 •3 IT) r v CD PLASMA pH 0J CD 21 T a b l e i . L e a s t s q u a r e s r e g r e s s i o n l i n e s d e s c r i b i n g t h e e f f e c t o f t e m p e r a t u r e (T, C) on C O ^ s o l u b i l i t y (aCOa, umol • L""1 • t o r r - 1 ) of w a t e r and Salmo gai rdneri plasma,, where a CQ 2 := a + bT + c P . R e g r e s s i o n c o e f f i c i e n t s a r e p r e s e n t e d + 95°/, c o n f i d e n c e i n t e r v a l s ( n = l S ) . a b c r d i s t i 1 1 e d w a t e r 110.2 + 2.6 5.87 + 0.59 0.163 + 0.029 -0.998 a c i d i f i ed w a t e r 107.5 + 4 . 7 26 + 1.07 0.131 + 0.053 -0.995 p I asma 100.5 + 3.0 -5.08 -i- 0.68 0.132 + 0.034 -0.997 22 t o t h e a c t i o n o-f h e p a r i n on t h e s t r u c t u r e o f plasmax l i p o p r o t e i n a g g r e g a t e s (Loewe et a i * 1 9 5 3 ) . Use of h e p a r i n was m i n i m i z e d i n s t u d i e s r e p o r t e d h e r e and no p r o t e i n p r e c i p i t a t i o n was e v i d e n t . The a p p a r e n t d i s s o c i a t i o n c o n s t a n t o f c a r b o n i c a c i d ( p K « p p = - l o g CbHC0 3 ~ 3CH*] / C f r e e CDs]) i n t r o u t p l a s m a v a r i e d w i t h p l a s m a pH ( F i g . 1)5 pK«,p,p d e c r e a s e d when p l a s m a pH i n c r e a s e d . T h i s p H - e f f e c t was r e l a t i v e l y s m a l l (-0.098 p K « p p u n i t s p e r pH u n i t ) , b u t i t was s i g n i f i c a n t s t a t i s t i c a l l y ( p a i r e d t — t e s t s d i f f e r e n c e b e t w e e n p K „ p p a t pH 8.1 and pH 7.9, t = 2 . 9 5 5 w i t h 7 d . f . , P<0.02 s i g n c o n s i d e r e d ! ! d i f f e r e n c e b e t w e e n p K „ p p a t pH 8.1 and pH 7.6, t=-'3.371 w i t h 7 d . f . , P<0.01 s i g n c o n s i d e r e d ) . T r u e d i s s o c i a t i o n c o n s t a n t s a r e n o t i n f l u e n c e d by t h e c o n c e n t r a t i o n s of t h e i r r e a c t a n t s o r p r o d u c t s . Hence, t h e f i n d i n g t h a t p K « p p was p H — d e p e n d e n t e m p h a s i z e s t h a t pK,» p p ) h a s no t h e r m o d y n a m i c m e a n i n g , i t i s n o t h i n g more t h a n an e m p i r i c a l l y d e r i v e d f a c t o r w h i c h r e l a t e s p l a s m a pH, f r e e C0s» and bHCO^™. The CQsa-comb i n i ng p r o p e r t i e s o f r a i n b o w t r o u t b l o o d were a f u n c t i o n o f b l o o d t e m p e r a t u r e and h a e m a t o c r i t ( F i g . 2 ) . A t a c o n s t a n t P C O a , c o l d b l o o d had a h i g h e r CCo:^ t h a n warmer b l o o d . Red c e l l s e n h a n c e d t h e C0 r a-combi n i ng a f f i n i t y o f b l o o d ? t h e mean COa c a p a c i t a n c e , 6 C 0 S , c a l c u l a t e d by l i n e a r r e g r e s s i o n o f Ccoa a g a i n s t P Coa o v e r t h e p h y s i o l o g i c a l r a n g e o f P C oa i n t r o u t b l o o d (2-7 t o r r , T a b l e 2 ) , v a r i e d d i r e c t l y w i t h h a e m a t o c r i t . ( F i g . 2 ) . CDs c a p a c i t a n c e r e f l e c t s C O ^ - c o m b i n i n g a f f i n i t y i n t h e same way t h a t t h e s l o p e o f an o x y g e n s a t u r a t i o n curves r e f l e c t s O a - b i n d i n g a f f i n i t y . E f f e c t s o f t e m p e r a t u r e on 6CO53 were n o t e v i d e n t ( F i g . 2) . 23 F i g u r e 2. C 0 2 d i s s o c i a t i o n c u r v e s and CO- c a p a c i t a n c e s o f o x y g e n a t e d w h o l e b l o o d o f Salmo gairdneri d e t e r m i n e d in v i t r o . P a n e l A. E f f e c t o f t e m p e r a t u r e on COra d i s s o c i a t i o n c u r v e s . V a l u e s a r e means + 1 S.E. <n=5-7) . P a n e l B. E f f e c t o f h a e m a t o c r i t on mean CO- c a p a c i t a n c e (6C0- = rfCcoa/rfPcoa) b e t w e e n 2 and 7 t o r r P C o 2 a t t e m p e r a t u r e s o f 5 ( + > , 10 <*) and 15 (o) C. L e a s t s q u a r e s r e g r e s s i o n l i n e , i g n o r i n g t e m p e r a t u r e , i s 6C0— -= 0.232 + 0.022 h a e m a t o c r i t ( r = 0 . 7 5 7 , n=18). 24 H A E M A T O C R I T , % 25 T a b l e 2- In vivo r e s p i r a t o r y and i o n i c c h a r a c t e r i s t i c s o f Salmo gairdneri p l a s m a a t 10 C. A r t e r i a l and v e n o u s v a l u e s a r e a r i t h m e t i c means + 1 S.E. ( n ) . ' A r t e r i a l m i n u s v e n o u s ' v a l u e s a r e p o p u l a t i o n mean d i f f e r e n c e s 957. c o n f i d e n c e i n t e r v a l s . P o p u l a t i o n mean d i f f e r e n c e s d e n o t e d w i t h * a r e d i f f e r e n t s i g n i f i c a n t l y f r o m z e r o a t P<0»05 ( p a i r e d t - t e s t ) . hot,, h a e m a t o c r i t . A r t e r i a l V e n o u s A r t e r i a l m i n u s v e n o u s pH 7.93 + 0. 0 3 ( 8 ) 7.88 + 0.03(8) 0.05 + 0.01* CCc»- < mM) 8.08 + 0.87(8) 9.20 + 0. 9 7 ( 8 ) -1„ 11 + 0.23* Pcosr. ( t o r r ) 2.31 + 0. 1 7 ( 8 ) 2.99 + 0. 1 9 ( 8 ) -0.68 + 0 . 1 1 * bHC0 3-(mM) 7„93 + 0.85(8) 9.01 + 0„96(8) -1.08 + 0.23* hct(7.) 22.6 + 3. 0 ( 8 ) 23.8 + 3. 1 ( 8 ) -1.2 + 0.8* Cl™(mM) 121.61 + 2 . 4 4 ( 1 8 ) Na*(mM) 154.04 + 2 . 2 6 ( 1 8 ) K-(mM) 4. 34 + 0. 2 6 ( 1 8 ) Ca--(mM) 2.96 + 0.07 ( 1 8 ) Mg s*(mM) 0.70 + 0. 0 3 ( 1 8 ) b1ood 0.47 + 0„08(15) l a c t a t e (mM) 26 CD's d i s s o c i a t i o n c u r v e s o-f w h o l e b l o o d , t r u e p l a s m a ( p l a s m a e q u i l i b r a t e d i n t h e p r e s e n c e o f r e d c e l l s ) , s e p a r a t e d p l a s m a ( p l a s m a e q u i l i b r a t e d i n t h e a b s e n c e o f r e d c e l l s ) and e r y t h r o c y t e s at. 10 C a r e co m p a r e d i n F i g u r e 3. Di reset c o m p a r i s o n of t h e a b s o l u t e C c a 2 v a l u e s i n F i g u r e 3 was c o m p l i c a t e d by t h e f a c t . t h a t s e p a r a t e d p l a s m a v a l u e s were d e t e r m i n e d u s i n g a d i f f e r e n t b l o o d p o o l f r o m t h a t u s e d t o m e a s u r e t h e o t h e r v a l u e s . N o n e t h e l e s s , s u c h a c o m p a r i s o n was deemed v a l i d b e c a u s e t h e b l o o d p o o l s u s e d were i n s i m i l a r r e s p i r a t o r y and a c i d - b a s e s t a t e s when t h e b l o o d was w i t h d r a w n i n v i v o . T h e o r e t i c a l l y , t h e d i s s o c i a t i o n c u r v e s o f s e p a r a t e d and t r u e p l a s m a f r o m a s i n g l e b l o o d s o u r c e c r o s s a t t h e P c o a a t w h i c h c e n t r i f u g a t i o n f o r s e p a r a t e d p l a s m a t o o k p l a c e , w h i c h u n d e r a n a e r o b i c c o n d i t i o n s s h o u l d r e f l e c t t h e Pco2 i n vivo. S e p a r a t e d and t r u e p l a s m a l i n e s i n F i g u r e 3 c r o s s e d a t a P c o a ( 2 , 3 t o r r ) c l o s e t o t h e p h y s i o l o g i c a l P C o a of a r t e r i a l b l o o d ( T a b l e 2 ) , M o r e o v e r , t h e m e a s u r e d bC0z> f o r s e p a r a t e d p l a s m a (0.24) was c l o s e t o t h e v a l u e p r e d i c t e d f o r w h o l e b l o o d a t z e r o h a e m a t o c r i t ( 0 , 2 3 , F i g . 2 ) . T r u e p l a s m a had t h e h i g h e s t C C o s » a t P o o a a b o v e 4 t o r r f o l l o w e d by, i n d e s c e n d i n g o r d e r , w h o l e b l o o d , s e p a r a t e d p l a s m a and r e d c e l l s ( F i g . 3 ) , D i f f e r e n c e s i n t h e C0 B-combining p r o p e r t i e s o f t r u e p l a s m a and s e p a r a t e d p l a s m a were a t t r i b u t a b l e t o t h e p r e s e n c e o f r e d c e l l s d u r i n g C0=» e q u i l i b r a t i o n , and r e s u l t e d f r o m movements o f and / o r H C 0 3 ~ b e t w e e n r e d c e l l s and plasma.. The amount o f HCD 3™ w h i c h c r o s s e d t h e r e d c e l l membrane was e s t i m a t e d by c o m p a r i n g t h e c h a n g e s i n bHCGV~ o f t r u e and s e p a r a t e d p l a s m a o v e r an i d e n t i c a l r a n g e of P c o a ( F i g . 3 ) . Th e s e e s t i m a t e s i n d i c a t e d that., o v e r a r a n g e of P c a a f r o m 2 t o 7 t o r r , 27 F i g u r e 3. C O a d i s s o c i a t i o n c u r v e s and mean c a p a c i t a n c e s (frCOrs = t/Ccos> / ( / P C o 2 j b e t w e e n 2 and 7 t o r r ) o f o x y g e n a t e d w h o l e b l o o d (mean h a e m a t o c r i t + 1 S.E. 20.1 + 1 . 7 ) , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s of Salmo gairdneri d e t e r m i n e d in vitro a t 10 C. V a l u e s a r e means + 1 S.E. (n=6-7). Dashed l i n e r e p r e s e n t s t h e amount of d i s s o l v e d COsa p r e s e n t . 1 2 8 CM O O O \ ^ true plasma „ whole blood i _ separated plasma erythrocyte £C0 2, mM/torr whole blood true plasma separated plasma erythrocyte 0.67 + 0.04 0.73 ± 0.05 0.24 ± 0.03 0.64 ± 0.05 0 co pj ID OJ OJ to c » P c o 2 , torr 29 6 2 % o-f t h e i n c r e a s e i n t r u e p l a s m a C - o s a was due t o H C 0 3 ~ added t o t h e p l a s m a f r o m r e d c e l l s . S i n c e r e d c e l l s c o n s t i t u t e d o n l y a b o u t 2 0 % of t h e b l o o d v o l u m e u n d e r c o n s i d e r a t i o n , t h i s i n d i c a t e s t h a t t h e m a j o r i t y o f C D b h y d r a t i o n o c c u r r e d i n s i d e t h e r e d c e l l . F o r t h e most p a r t , p a r t i t i o n i n g o f C C o 3 » among t h e v a r i o u s b l o o d c o m p a r t m e n t s r e f l e c t e d d i f f e r e n c e s i n b u f f e r i n g c a p a c i t y ( F i g . 4, 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 b u f f e r i n g c a p a c i t y were n o t e v i d e n t ( T a b l e 3 ) . I n t e r e s t i n g l y , r e d c e l l s had t h e h i g h e s t b u f f e r i n g c a p a c i t y (1.6 t i m e s t h a t , o f t r u e p l a s m a , T a b l e 3 ) , b u t had t h e l o w e s t C o o s a a t any g i v e n P C o 2 and an i n t e r m e d i a t e ? hCQz>„ S i n c e r e d c e l l s were e q u i l i b r a t e d i n t h e p r e s e n c e o f p l a s m a , t h i s p r o b a b l y was a t t r i b u t a b l e t o t h e same H"*" a n d / o r H C 0 3™ movements b e t w e e n r e d c e l l s and p l a s m a t h a t w e r e r e s p o n s i b l e f o r d i f f e r e n c e s b e t w e e n t h e CO^-combi n i ng p r o p e r t i e s o f t r u e and s e p a r a t e d p l a s m a . I n t r a c e l l u l a r pH v a r i e d l i n e a r l y w i t h p l a s m a pH o v e r t h e pH r a n g e e x a m i n e d ( T a b l e 4 ) . T e m p e r a t u r e had l i t t l e e f f e c t on t h i s r e l a t i o n s h i p . I n c r e a s e s i n t h e P C o 2 o f t r o u t b l o o d w e r e a c c o m p a n i e d by a n e t i n f l u x o f CI"' i n t o t h e r e d c e l l ? t h e Donnan C l ~ r a t i o ( R C i - •--[ e r y t h r o c y t e ] / [ p l a s m a ] ) was r e l a t e d i n v e r s e l y t o b l o o d pH ( F i g . 5 ) . The amount o f CI™ g a i n e d by r e d c e l l s f r o m p l a s m a d u r i n g CO^. e q u i l i b r a t i o n s was s t o i c h i o m e t r i c a l 1 y e q u i v a l e n t t o t h e amount o f H C O 3 ' " ' g a i n e d by t r u e p l a s m a f r o m r e d c e l l s ( F i g . 6 ) . T h i s i n d i c a t e s t h a t b i c a r b o n a t e movements i n t r o u t r e d c e l l s i n v o l v e d a o n e — f o r one c o u n t e r e x c h a n g e o f b i c a r b o n a t e and c h l o r i d e s i m i l a r t o t h e c h l o r i d e s h i f t f o u n d i n mammalian e r y t h r o c y t e s 30 F i g u r e 4. B u f f e r l i n e s f o r o x y g e n a t e d w h o l e b l o o d (mean h a e m a t o c r i t + 1 S.E. = 20.1 + 1 . 7 ) , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s o f Salwo gairdneri d e t e r i m i n e d in vitro a t 10 C . V a l u e s a r e means + 1 S.E. (n~6--7) . 32 T a b l e 3. P h y s i o l o g i c a l b u f f e r i n g c a p a c i t y i n s l y k e s (mlvl b i c a r b o n a t e - p H u n i t ' " 1 ) o f o x y g e n a t e d w h o l e b l o o d , t r u e p l a s m a , s e p a r a t e d p l a s m a and e r y t h r o c y t e s o f Sal mo gairdneri d e t e r m i n e d in vitro b e t w e e n 5 and 15 C, V a l u e s a r e means + 1 S.E. (n=5-~7) . Temp, t e m p e r a t u r e . H c t , h a e m a t o c r i t . B u f f e r i n g c a p a c i t y Temp H c t w h o l e e r y t h r o c y t e t r u e s e p a r a t e d (C) <"/.) b l o o d p l a s m a p l a s m a 5 22.2 ± 1 . 6 9.4 + 0.3 16.1 + 2.4 9.7 + 0.5 10 20.1 + 1.7 9.9 + 0.9 16.5 + 2.0 10.5 + 1.0 2.2 + 0.1 15 19.0 + 1 . 4 9 . 1 + 0 . 5 1 4 . 1 + 1 . 6 9 . 4 + 0 . 6 33 T a b l e 4. L e a s t s q u a r e s r e g r e s s i o n l i n e s d e s c r i b i n g t h e r e l a t i o n s h i p b e t w e e n b l o o d pH ( p H E ) , and i n t r a c e l l u l a r pH <pH x), R H * ( Cpl asmall / [ e r y t h r o c y t e ! ! ) , F * o i - ( [ e r y t h r o c y t e ] / l l p l asmall) , and R H C O S - < [ e r y t h r o c y t e ] / l l p l asmall) o f Salmo gai rdneri b l o o d b e t w e e n 5 and 15 C. A l s o shown i s t h e r e l a t i o n s h i p b e t w e e n c a r b a m i nohaemogl o b i n c o n c e n t r a t i o n ( c a r b , mmol - L r e d c e l l " ~ : l ) and b l o o d Pens ( t o r r ) . R e g r e s s i o n c o e f f i c i e n t s a r e p r e s e n t e d + 957. c o n f i d e n c e i n t e r v a l s . Temp, t e m p e r a t u r e ( C ) . R e l a t i on Temp pHj = a + b pH- 5 3.476 + 0.572 0.501 10 2.708 + 0.580 0.595 15 3.566 + 0.631 0.479 + 0.074 + 0.076 + 0.083 24 27 19 0. 949 0. 955 0. 947 R'M-I- = a + b pHer 5 4.254 + 0.644 10 3.500 + 0.604 15 4.218 +• 0.655 -0.494 + 0.083 •0.403 •0. 500 0. 079 0. 086 27 19 0. 935 0. 903 0. 948 R c i - = a + b pH- 5 4.064 + 1.684 -0.473 + 0.217 10 3.858 + 1.375 -0.450 + 0.181 15 3.764 + 2.619 -0.429 + 0.344 18 0.713 0.732 0 „ 552 Rv a + b pH 10 15 4.499 + 1.282 4.698 + 1.697 -0.493 +• 0.165 22 0.812 -0.486 + 0.174 27 0.754 -0.520 + 0.223 18 0.778 c a r b = a + b 1 og P e a s ; 10 15 1. 305 O. 847 0. 745 0. 816 0. 371 0.544 1.616 1. 574 1. 538 1. 045 0. 457 0.696 27 19 0. 575 0. 8.1.8 0. 749 34 F i g u r e 5, Donnan r a t i o s o-f H-1" ( R M - = [ p l a s m a ] / [ e r y t h r o c y t e ] ) , CI '" ( R C j - = [ e r y t h r o c y t e ] / [ p l a s m a ] ) and bHCQ 3- ( R H O O S - = [ e r y t h r o c y t e ] / [ p l a s m a ] ) o-f Sal mo gairdneri b l o o d , when b l o o d pH was t i t r a t e d w i t h GOs» in vitro a t 5 (•+•), 10 (#) and 15 (o) C. L e a s t s q u a r e s r e g r e s s i o n l i n e s a r e g i v e n i n T a b l e 4. 35 36 F i g u r e 6. R e l a t i o n s h i p b e t w e e n t h e amount o f b i c a r b o n a t e added t o p l a s m a f r o m r e d c e l l s and t h e amount o f c h l o r i d e added t o r e d c e l l s f r o m p l a s m a d u r i n g in vitro CDs: e q u i l i b r a t i o n s o f Sal mo gairdneri b l o o d a t 10 C» A l l c o n c e n t r a t i o n s were c o r r e c t e d f o r w a t e r c o n t e n t and h a e m a t o c r i t . L e a s t s q u a r e s r e g r e s s i o n l i n e i s rfCbi-ICQ-,"-] = 0.155 + 0.943 dLCl~l ( r = 0 . S 0 2 , n=42) . Dashed l i n e i s 37 IOUJUJ ' loOHP 38 (Rough t o n 1 9 6 4 ) . F o r m a t i o n o-f i n t r a c e l l u l a r HC03"~ and t h e s u b s e q u e n t b i c a r b o n a t e ; : c h l o r i de exchange? r e s u l t e d i n n e t movement o-f w a t e r i n t o t h e r e d c e l l and c e l l s w e l l i n g ( F i g . 7 ) . W a t e r c o n t e n t of r e d c e l l s i n c r e a s e d f r o m 55. S + 2. .9 % t o 63.2 ± 1.9 "/. o v e r t h e r a n g e of Pesos* f r o m 2.3 t o 14.1 t o r r . Over t h e same r a n g e o f F'oo=>, p l a s m a w a t e r c o n t e n t d e c r e a s e d f r o m 94.0 + 0.5 % t o 93.7 + 0.6 %. T e m p e r a t u r e a p p e a r e d t o h a v e no e f f e c t on t h e s e w a t e r movements o r on t h e r e s u l t a n t c h a n g e s i n c e i l s i z e ( F i g . 7) „ Changes i n t h e Donnan r a t i o s o f b o t h b H C 0 3 - ( R H C D S - = [ e r y t h r o c y t e ] / [ p l a s m a ] ) and H* (R M+ = [ p l a s m a ] / [ e r y t h r o c y t e ] ) p a r a l l e l e d c h a n g e s i n R C i ~ when b l o o d pl~i was t i t r a t e d w i t h C 0 S (F"ig. 5 and 8 ) . A s s u m i n g t h a t C l ~ was d i s t r i b u t e d p a s s i v e l y a c r o s s t h e r e d c e i l membrane, t h i s i n d i c a t e s t h a t bHCOa™ and H"1" a l s o were d i s t r i b u t e d p a s s i v e l y . However, R H C D S - v a l u e s were c o n s i s t e n t l y g r e a t e r t h a n R C i - v a l u e s by 0.30 t o 0.35 u n i t s ( F i g . 8) . T h i s i n d i c a t e s t h a t a c t u a l i n t r a c e l l u l a r b H C 0 3 — v a l u e s were h i g h e r t h a n what c o u l d be a c c o u n t e d f o r on t h e b a s i s o f a B i b b s -Don n an e q u i I i b r i u m f o r C1 ~. T h i s :' u n a c c o u n t e d - f o r ' i n t r a c e 11 u 1 a r b H C 0 3 " v a r i e d f r o m 1.30 + 0.56 mmol -L r e d c e l l " d - (2.4 t o r r Pesos:, 15 C) t o 3.24 + 0.88 mmol - L r e d c e l l " " 1 ( 1 3 . 8 t o r r P c o a , 5 C) ( F i g . 9 ) . As s u c h , i t r e p r e s e n t e d 3 3 - 5 7 % of t h e m e a s u r e d e r y t h r o c y t i c b H C 0 3-. I t must be remembered, h o w e v e r , t h a t bHC0 3™ d o e s n o t d i s t i n g u i s h b e t w e e n HC0 3~, CQ 3= ;™ o r c a r b a m i n o compounds. The c o n c e n t r a t i o n o f ' u n a c c o u n t e d - f o r 1 ' i n t r a c e l l u l a r b H C 0 3 ~ f a r e x c e e d e d t h e c o n c e n t r a t i o n o f Ctfa 3""' p r e d i c t e d f r o m m e a s u r e d b H C0 3~ c o n c e n t r a t i o n s ( 6 . 7 - 7 . 0 czmol-L r e d c e l l " 1 ) u s i n g t h e t r u e s e c o n d d i s s o c i a t i o n c o n s t a n t o f c a r b o n i c a c i d ( R o b i n s o n and 39 F i g u r e 7. Water c o n t e n t and r e l a t i v e v o l u m e o f Sal mo gairdneri e r y t h r o c y t e s i n r e l a t i o n t o P C 0 2 a t 5 (+), 10 <*) and 15 <o) C. V a l u e s a r e means + 1 S . E . (n=5-7). C E L L WATER C O N T E N T , % 41 F i g u r e 8„ R e l a t i o n s h i p b e t w e e n t h e Donnan CI™ r a t i o < R C i - = [ e r y t h r o c y t e ] / [ p l a s m a ] ) , and t h e Donnan r a t i o s o f H* (RM* -[ p l a s m a ] / [ e r y t h r o c y t e ] ) and bHCO-~ (RHCOS- = [ e r y t h r o c y t e 3 / Cplasma3) o f Salmo gairdneri b l o o d a t 5 <+), 10 (*) and 15 <o) C. V a l u e s a r e means + 1 B.E. (n=5-7). L e a s t s q u a r e s r e g r e s s i o n o f RHH- on R c i - h a s s l o p e o f 0.741 + 0.350 and y - i n t e r c e p t o f 0.094 + 0.160 < + 95"/. c o n f i d e n c e i n t e r v a l s , n™12, r = 0 . 8 3 1 ) . L e a s t s q u a r e s r e g r e s s i o n o f R H c o 3 - on Rc%- h a s s l o p e o f 0.878 + 0.302 and y-~ i n t e r c e p t o f 0.347 + 0.138 < + 957. c o n f i d e n c e i n t e r v a l s , n-~12, r••=0.898). Dashed l i n e s a r e x~y. 4 2 43 F i g u r e 9. C a r b a m i nohaemogl o b i n - f o r m a t i o n i n Salmo gai rdneri b l o o d s h o w i n g e - f f e c t s o-f P c o a and t e m p e r a t u r e . V a l u e s a r e means + 1 S.E. (n~5--7) „ L e a s t s q u a r e s r e g r e s s i o n l i n e s a r e g i v e n i n T a b l e 4. IAIW 'NiaO19OlAI3VHONIIAIVacJV0 45 S t o k e s 1 9 5 9 ) . R a t h e r , t h i s ' 'unaccounted—f or :' i n t r a c e l l u l a r b H C 0 3 ~ p r o b a b l y r e p r e s e n t e d c a r b a m i nohaemogl ob i n, t h a t i s , COr.t bound d i r e c t l y t o h a e m o g l o b i n . S i n c e r a i n b o w t r o u t b l o o d c o n t a i n s a b o u t 4.33 mmol haemogl ob i n - L r e d c e l l " - 1 (Heming, u n p u b l i s h e d d a t a ) , t h i s c a r b a m i n o f o r m a t i o n r e p r e s e n t e d b i n d i n g of o n l y 0,30-0.75 mol C0 r 2 p e r mol h a e m o g l o b i n . T h e o r e t i c a l l y , • f i s h h a e m o g l o b i n s h o u l d be c a p a b l e o f b i n d i n g a maximum o f 2 mol C 0 S p e r mol h a e m o g l o b i n ( F a r m e r 1 9 7 9 ) . F o r m a t i o n of c a r b a m i n o h a e m o g l o b i n d e m o n s t r a t e d s a t u r a t i o n k i n e t i c s and p e r h a p s was t e m p e r a t u r e - d e p e n d e n t ( F i g . 9 ) . A l t h o u g h t h e c o n c e n t r a t i o n o f c a r b a m i n o h a e m o g l o b i n a t a g i v e n P C Q 2 t e n d e d t o i n c r e a s e a t l o w e r t e m p e r a t u r e s , t h e d i f f e r e n c e s were n o t s i g n i f i c a n t s t a t i s t i c a 11 y. 46 D I S C U S S I O N C O a l o a d i n g o-f Salmo gairdneri b l o o d in vitro, and p r e s u m a b l y in vivo, was c o n s i s t e n t w i t h t h e c l a s s i c a l mammalian model o-f R o u g h t o n < 1 9 6 4 ) . D u r i n g C O s l o a d i n g , t h e m a j o r i t y o-f C 0 3 d i f f u s i n g i n t o t h e b l o o d h y d r a t e d and t h e n d i s s o c i a t e d t o •form H C 0 3 ' " ' and H""„ R e l a t i v e l y s m a l l p r o p o r t i o n s o f C 0 2 r e m a i n e d as t h e d i s s o l v e d g a s o r were bound by h a e m o g l o b i n . H y d r a t i o n of C O a o c c u r r e d p r e d o m i n a n t l y i n s i d e t h e r e d c e l l . However, v e r y l i t t l e o f t h e r e s u l t a n t . H C 0 3 ~ r e m a i n e d i n s i d e t h e e r y t h r o c y t e . The m a j o r i t y o f t h i s HCQ 3™ t r a v e r s e d t h e c e l l membrane i n an one f o r - o n e e x c h a n g e w i t h C i " " , r e m i n i s c e n t o f t h e c h l o r i d e s h i f t i n mammalian e r y t h r o c y t e s . F o r m a t i o n of i n t r a c e l l u l a r b i c a r b o n a t e and t h e s u b s e q u e n t H C O . 3 " " : C I ~ e x c h a n g e was a c c o m p a n i e d by i n f l u x o f w a t e r and c e l l s w e l l i n g . A l t h o u g h n o t e x a m i n e d , COs» u n l o a d i n g of r a i n b o w t r o u t b l o o d was p r e s u m e d t o i n v o l v e t h e same c h e m i c a l and t r a n s p o r t p r o c e s s e s , o p e r a t i n g i n r e v e r s e . The Donnan d i s t r i b u t i o n s o f b H C 0 3 ~ and H"1" p a r a l l e l e d t h a t of C l ~ " a s p l a s m a p H was t i t r a t e d w i t h C 0 : a , s u g g e s t i n g t h a t t h e t r a n s m e m b r a n e d i s t r i b u t i o r i s o f b H C 0 3 " , H"1" and C l " ~ w e r e d e p e n d e n t upon s i m i l a r m e c h a n i s m s . Numerous s t u d i e s o f mammalian e r y t h r o c y t e s h a v e i n d i c a t e d t h a t . c h l o r i d e i s d i s t r i b u t e d p a s s i v e l y a c r o s s t h e r e d c e l l membrane. Mammalian e r y t h r o c y t e s a r e e x t r e m e l y p e r m e a b l e t o C l ~ (Gunn et al„ 1 9 7 3 , H o f f m a n and L a r i s 1 9 7 4 , K n a u f e t al. 1 9 7 7 ) and t h e v a r i a t i o n i n R C i - w i t h pH i n mammalian b l o o d i s c o n s i s t e n t w i t h t h a t p r e d i c t e d on the? b a s i s 47 of a p a s s i v e C I " d i s t r i b u t i o n ( H a r r i s and M a i z e l s 1 9 5 2 ) . The most, c o n v i n c i n g e v i d e n c e -for a p a s s i v e C l ~ d i s t r i b u t i o n , h o w e v e r , i s t h e e x c e l l e n t a g r e e m e n t b e t w e e n t h e m e a s u r e d s t e a d y - s t a t e membrane p o t e n t i a l o-f mammalian e r y t h r o c y t e s and t h e c a l c u l a t e d N e r n s t 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 ( L a s s e n 1972., H o f f m a n and L a r i s 1974, Ronne and L a s s e n 1 9 7 7 ) . C h l o r i d e t r a n s p o r t i n f i s h e r y t h r o c y t e s i s a t l e a s t a s r a p i d a s t h a t i n mammalian r e d c e l l s u n d e r s i m i l a r c o n d i t i o n s (Cameron :1978b, O b a i d et al , 1 9 7 9 ) . M o r e o v e r , H a s w e l l et al. (1978) h a v e shown t h a t t h e r e l a t i o n s h i p b e t w e e n R o i - and p l a s m a pl-l i n e r y t h r o c y t e s o f t h e t e l e o s t f i s h , Tilapia mossamhica, i s v i r t u a l l y i d e n t i c a l t o t h a t i n human r e d c e l l s . T h e s e s t u d i e s s t r o n g l y s u g g e s t t h a t C I " i s d i s t r i b u t e d p a s s i v e l y i n f i s h e r y t h r o c y t e s . I t f o l l o w s f r o m t h e p r e s e n t i n vitro d a t a t h e n t h a t H C0 3™ and H* a l s o a r e d i s t r i b u t e d p a s s i v e l y i n r e d c e l l s o f 5„ gairdneri. In i t s e l f , a p a s s i v e HCO—~ d i s t r i b u t i o n i n t r o u t e r y t h r o c y t e s d o e s n o t c o n t r a d i c t t h e g i l l model of CO^ e x c r e t i o n ( H a s w e l l and R a n d a l l 1976, 1978, H a s w e l l et al* 1 9 8 0 ) , w h i c h a s s e r t s t h a t f i s h r e d c e l l s a r e f u n c t i o n a l l y i m p e r m e a b l e t o b i c a r b o n a t e i n vivo. F u n c t i o n a l i m p e r m e a b i l i t y c o u l d be a t t a i n e d in vivo by p l a s m a 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 a r b o n i c a n h y d r a s e . However, t i m e c o u r s e s t u d i e s of t h e c h l o r i d e s h i f t i n t e l e o s t and e l a s m o b r a n c h b l o o d (Cameron 1978b, O b a i d et a i - 1979) c l e a r l y d e m o n s t r a t e t h a t p l a s m a h a s no e f f e c t on t h e a c t i v i t y o f 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 . T h e s e l a t t e r s t u d i e s p r o v i d e s t r o n g e v i d e n c e a g a i n s t t h e g i l l model o f C0a> e x c r e t i o n . The p r e s e n t R C i - v a l u e s 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 do n o t a g r e e s a t i s f a c t o r i l y w i t h o b s e r v e d ( D i l l et al. 1932, F i t z s i m m o n s and S e n d r o y 1961, Sunn et al. 1973, R e e v e s 1976a, H a s w e l l et al, 48 1978) o r t h e o r e t i c a l l y p r e d i c t e d ( J a c o b s and S t e w a r t 1947, Freedman and H o f f m a n 1979) r a t i o s i n mammalian e r y t h r o c y t e s . V a r i a t i o n i n R C i - w i t h p l a s m a pH i n S» gairdneri e r y t h r o c y t e s was s u c h t h a t mammalian and t r o u t R C i - v a l u e s w e re i n good a g r e e m e n t o n l y at. a b o u t pH 7.4. However, t h e s l o p e o f t h e r e l a t i o n s h i p b e t w een R C i - and pH i n t r o u t e r y t h r o c y t e s (-0.429 t o -0.473) was much s t e e p e r t h a n t h a t i n mammalian r e d c e l l s (-0.290 t o -0.344: D i l l e i al. 1932, F i t z s i m m o n s and S e n d r o y 1961, Sunn e t al. 1973, R e e v e s 1976a, H a s w e l l e t al. 1 9 7 8 ) . T h i s a l s o h o l d s t r u e f o r t h e r e l a t i o n s h i p s b e t w e e n rfRH-n / d p h (-0.403 t o -0.500) and O'RHCQS- / dpH (-0.486 t o -0.520) i n t r o u t e r y t h r o c y t e s . T h e s e v a l u e s a r e v i r t u a l l y i d e n t i c a l t o t h o s e o b t a i n e d by Wood e t a l * (1981) f o r ^ R H C O S - / c/pH (-0.43) i n S. gairdneri r e d c e l l s . T h u s , w i t h i n t h e p h y s i o l o g i c a l pH r a n g e of t r o u t b l o o d , t h e m e a s u r e d Donnan r a t i o s o f t r o u t r e d c e l l s a r e much l e s s t h a n mammalian v a l u e s . D i f f e r e n c e s b e t w e e n t h e Donnan C I " r a t i o s of mammalian and f i s h e r y t h r o c y t e s p r o b a b l y a r e a c o n s e q u e n c e o f t h e p r e s e n c e o f a n u c l e u s i n non-mammalian r e d c e l l s . The n u c l e u s 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 o c c u p i e s a b o u t 187. o f t h e c e l l v o l u m e (Eddy 197/7). D i r e c t measurement of t h e membrane p o t e n t i a l o f n u c l e a t e d r e d c e l l s h a s shown c o n s i s t e n t l y t h a t t h e N e r n s t 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 u n d e r e s t i m a t e s t h e m e a s u r e d i n t r a c e l l u l a r v o l t a g e , and t h a t t h i s d i s c r e p a n c y i n c r e a s e s a s R b i - d e c r e a s e s a t h i g h e r pl-l v a l u e s ( L a s s e n 1977, S t o n e r and Kregenow 1 9 8 0 ) . D i f f e r e n c e s b e t w e e n t h e m e a s u r e d and c a l c u l a t e d membrane p o t e n t i a l s o f n u c l e a t e d r e d c e l l s and h e n c e b e t w e e n t h e m e a s u r e d and p r e d i c t e d Donnan r a t i o s i n s u c h c e l l s a p p e a r t o be an i n h e r e n t 49 c h a r a c t e r i s t i c o-f n u c l e a t e d e r y t h r o c y t e s . T h e s e d i s c r e p a n c i e s may be due t o e x c l u s i o n o f C I " f r o m t h e n u c l e u s ( L a s s e n 1972, H o f f m a n u n p u b l i s h e d o b s e r v a t i o n c i t e d i n B t o n e r and Kregenow 1 9 8 0 ) . N u c l e a r e x c l u s i o n o f c h l o r i d e w o u l d e f f e c t i v e l y r e d u c e t h e mean i o n i c a c t i v i t y c o e f f i c i e n t , o f CI"" i n n u c l e a t e d r e d c e l l s . In c o n t r a s t , e n u c l e a t e mammalian r e d c e l l s a r e a d e q u a t e l y d e s c r i b e d a s w e l l - m i x e d s i n g l e c o m p a r t m e n t s w i t h mean i o n i c a c t i v i t y c o e f f i c i e n t s o f u n i t y (Freedman and H o f f m a n 1 9 7 9 ) . T h u s , t h e p r e s e n t r e s u l t s i n f e r t h a t t h e n u c l e a r c o m p a r t m e n t o f f i s h e r y t h r o c y t e s i s more a c i d i c and c o n t a i n s l e s s C l ~ and H C G 3 " " t h a n t h e c y t o s o l i c c o m p a r t m e n t . I t f a l l o w s t h e n t h a t m e a s u r e m e n t s o f e r y t h r o c y t e pH made u s i n g t h e f r e e z e - t h a w t e c h n i q u e , o r any o t h e r method t h a t y i e l d s an a v e r a g e pH f o r t h e e n t i r e , c e l l , u n d e r e s t i m a t e c y t o s o l pH. The a s s u m p t i o n o f c y t o s o l i c and n u c l e a r d i s c o n t i n u i t y i n n u c l e a t e d r e d c e l l s f a i l s t o e x p l a i n t h e r e p o r t e d s i m i l a r i t y i n R C i ~ b e t w e e n n u c l e a t e d Tilapia r e d c e l l s and e n u c l e a t e human c e l l s ( H a s w e l l et al* 1978)5 p e r h a p s Tilapia r e d c e l l s p o s s e s s a r e l a t i v e l y s m a l l n u c l e a r c o m p a r t m e n t when c o m p a r e d t o o t h e r n u c l e a t e d erythrocytes„ The s i g n i f i c a n t d i f f e r e n c e s b e t w e e n R H c o s - and R c i - • i n t h e p r e s e n t s t u d y were assumed t o r e f l e c t f o r m a t i o n o f c a r b a m i n o h a e r a o g 1 o b i n i n S- gairdneri r e d c e l l s ? t h e t e c h n i q u e u s e d t o m e a s u r e b i c a r b o n a t e d i d n o t d i s t i n g u i s h b e t w e e n t h e v a r i o u s s p e c i e s o f c h e m i c a l l y - b o u n d C03»» T h i s a s s u m p t i o n i s s u p p o r t e d by t h e o b s e r v e d s a t u r a t i o n k i n e t i c s o f c a r b a m i n o • f o r m a t i o n w i t h i n c r e a s i n g P C 0 2 . S i m i l a r d i f f e r e n c e s b e t w e e n R H C O S - and R c i _ h a v e been n o t e d i n e r y t h r o c y t e s o f S. gairdneri 50 ( F e r g u s o n and B l a c k 1941) and t h e d o g f i s h , Mastelus canis ( F e r g u s o n et al* 1 9 3 8 ) . In t h e c a r p , Cyprinus carpio? t h e r a t i o s a r e a p p r o x i m a t e l y e q u i v a l e n t ( F e r g u s o n and E^lack 1 9 4 1 ) . D i f f e r e n c e s b e t w e e n t h e p r e s e n t R H c o s - and R c l- c a n be a c c o u n t e d f o r by a s s u m i n g t h a t a t most 37.57. o f t h e a v a i l a b l e b e t a - c h a i n t e r m i n a l s o f t r o u t h a e m o g l o b i n had bound C0 f f i. The t e r m i n a l amino g r o u p s on t h e a l p h a - c h a i n s o f r a i n b o w t r o u t h a e m o g l o b i n a r e a c e t y l a t e d ( R i g g s 1979, P o w e r s 1980) and t h u s o n l y t h e t e r m i n a l s of t h e b e t a - c h a i n s a r e a v a i l a b l e t o b i n d C0 r a (Weber and L y k k e b o e 1978, F a r m e r 1 9 7 9 ) . S i n c e t h e b e t a - c h a i n t e r m i n a l s a l s o b i n d o r g a n i c p h o s p h a t e s ( R i g g s 1970, J o h a n s e n et al. 1976, G r e a n e y and P o w e r s 1977) w h i c h a r e p r e s e n t i n f i s h e r y t h r o c y t e s i n h i g h e r c o n c e n t r a t i o n s t h a n C 0 S ( A l b e r s et al * 1 9 8 3 ) , one w o u l d e x p e c t a low b i n d i n g e f f i c i e n c y f o r CO^. I t i s i n t e r e s t i n g t o s p e c u l a t e a b o u t t h e p o s s i b l e enhancement o f c a r b a m i n o h a e m o g l o b i n f o r m a t i o n a t l o w e r t e m p e r a t u r e s . F o r m a t i o n o f c a r b a m i n o compounds i s i n c r e a s e d a t l o w e r t e m p e r a t u r e s ( S t a d i e and O ' B r i e n 1937, A l b e r s et al. 1 9 8 3 ) . As w e l l , Eddy (1974) h a s shown t h a t t h e H a l d a n e e f f e c t i n S. gairdneri b l o o d , w h i c h i s d e p e n d e n t i n p a r t on c a r b a m i n o f o r m a t i o n , i s i n v e r s e l y r e l a t e d t o t e m p e r a t u r e . R e a s o n s f o r a t e m p e r a t u r e e f f e c t on c a r b a m i n o f o r m a t i o n p r o b a b l y i n c l u d e t h e e f f e c t s o f t e m p e r a t u r e on b l o o d pl-l (Eddy 1 9 7 4 ) , on b i n d i n g c o n s t a n t s o f o r g a n i c p h o s p h a t e s ( P o w e r s 1 9 8 0 ) , and on 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 o r g a n i c p h o s p h a t e s . The p r e s e n t R H C O S - v a l u e s a r e c o n s i s t e n t l y g r e a t e r t h a n t h o s e r e p o r t e d by Wood et al « ( 1 9 8 1 ) . T h i s d i f f e r e n c e i n R H c o 3 -v a l L i e s c a n be l a r g e l y i f n o t w h o l l y a t t r i b u t e d t o Wood and 51 c o w o r k e r s ' u s e of l i t e r a t u r e v a l u e s -from Eddy (1974) f o r r e d c e l l and p l a s m a w a t e r c o n t e n t s o-f 707. and 957, r e s p e c t i v e l y . Eddy (1974) c l e a r l y s t a t e s t h a t t h o s e w a t e r c o n t e n t v a l u e s a r e assumed v a l u e s and n o t d i r e c t d e t e r m i n a t i o n s . T h o s e w a t e r c o n t e n t s d i f f e r s u b s t a n t i a l l y f r o m t h e v a l u e s m e a s u r e d by t h e p r e s e n t a u t h o r and i g n o r e t h e known e f f e c t o f p l a s m a pH on r e d c e l l w a t e r c o n t e n t ( F i g . 7 t h i s s t u d y , Gunn e t al. 1973, Freedman and H o f f m a n 1 9 7 9 ) . R e c a l c u l a t i o n of t h e R H C o 3 - v a l u e s of Wood et a l . ( 1 9 8 1 ) , u s i n g t h e p r e s e n t v a l u e s f o r r e d c e l l ( 55,8 t o 63.27.) and p l a s m a (94.0 t o 93.77.) w a t e r c o n t e n t s , i n c r e a s e s t h e R H c o 3 -v a l u e s of Wood and c o w o r k e r s by some 10 t o 257. and b r i n g s t h e i r v a l u e s i n t o g e n e r a l a g r e e m e n t w i t h t h e p r e s e n t R H C O S - v a l u e s . E f f e c t s o f t e m p e r a t u r e and h a e m a t o c r i t on t h e C 0 K t r a n s p o r t p r o p e r t i e s of f i s h b l o o d a r e w e l l d e s c r i b e d ( A l b e r s 1970, R a n d a l l 1970a, R i g g s 1 9 7 0 ) . In g e n e r a l , t h e CO^? c a r r y i n g c a p a c i t y o f b l o o d i s an i n v e r s e f u n c t i o n of t e m p e r a t u r e ; C G E s o l u b i l i t y i n c r e a s e s a t l o w e r t e m p e r a t u r e s and the? d i s s o c i a t i o n c o n s t a n t s of b o t h p r o t e i n b u f f e r s and c a r b o n i c a c i d d e c r e a s e ( A l b e r s 1970, B o u t i l i e r e t a l . 1984a) s h i f t i n g t h e CO^S I-ICO3" e q u i l i b r i u m i n t h e d i r e c t i o n o f t h e d i s s o c i a t e d m o i e t y . T e m p e r a t u r e had no e f f e c t on t h e b u f f e r i n g c a p a c i t y of t r o u t b l o o d , p l a s m a o r e r y t h r o c y t e s . T h i s i s c o n s i s t e n t w i t h t h e f i n d i n g s o f Eddy ( 1 9 7 4 ) . R e e v e s ( 1 9 7 6 a , b) h a s d e m o n s t r a t e d t h a t t h i s t e m p e r a t u r e - i n d e p e n d e n c y a r i s e s f r o m t h e v i r t u a l a b s e n c e of s i g n i f i c a n t , w a t e r and i o n movements b e t w e e n r e d c e l l s and p l a s m a cis t e m p e r a t u r e v a r i e s a t c o n s t a n t CCo=>. As a r e s u l t , t h e n e t c h a r g e s t a t e o f r e d c e l l and p l a s m a p r o t e i n b u f f e r s i s u n a f f e c t e d by t e m p e r a t u r e . The p r e s e n c e o f e r y t h r o c y t e s i n b l o o d i n f l u e n c e d C 0 S 52 t r a n s p o r t p r o p e r t i e s o f r a i n b o w t r o u t b l o o d by s e r v i n g a s a p r o t o n sink,, Filed c e l l s and t h e i r c o m p l e m e n t o f h a e m o g l o b i n e n h a n c e d t h e b u f f e r i n g c a p a c i t y o f b l o o d and t h u s f a v o u r e d 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 d u r i n g C D s ? l o a d i n g . B i n d i n g o f H" 1" by h a e m o g l o b i n i n f l u e n c e s t h e o x y g e n b i n d i n g a f f i n i t y o f h a e m o g l o b i n (Bohr e f f e c t ) and i t s 0:a c a r r y i n g . c a p a c i t y ( R o o t e f f e c t ) ( f o r r e v i e w s e e R i g g s 1 9 7 0 ) . T h i s c r e a t e s a f u n c t i o n a l l i n k b e t w e e n CO^ and O 3 . t r a n s p o r t . C 0 3 t r a n s p o r t i n a r t e r i a l and v e n o u s b l o o d o f r a i n b o w t r o u t wais m o d e l l e d u s i n g in vivo d a t a ( T a b l e 2) and r e s u l t s o f t h e C Q ; a e q u i l i b r a t i o n s t u d i e s . T h i s model i s p r e s e n t e d i n T a b l e 5. The f o l l o w i n g a s s u m p t i o n s were made d u r i n g c o n s t r u c t i o n o f T a b l e 5. C a r b a m i n o f o r m a t i o n i n p l a s m a was t a k e n a s 0.14% o f p l a s m a C C o 2 ( v a l u e f o r human p l a s m a f r o m A l b r i t t o n 1 9 5 2 ) , and C0 = ; s o l u b i l i t y o f r e d c e l l s was t a k e n a s 8 6 % o f p l a s m a a C 0 a ( v a l u e f o r ox e r y t h r o c y t e s f r o m Van S l y k e and S e n d r o y 1 9 2 8 ) . C o m p a r a b l e d a t a f o r f i s h b l o o d a r e n o t a v a i l a b l e i n t h e l i t e r a t u r e , , C 0 --~ l e v e l s w ere c a l c u l a t e d u s i n g a pK- f o r c a r b o n i c : a c i d a t 10 C of 10.4906 ( R o b i n s o n and S t o k e s 1 9 5 9 ) . T h i s i g n o r e s t h e known e f f e c t s o f i o n i c s t r e n g t h on t h e d i s s o c i a t i o n c o n s t a n t s o f weak a c i d s and c o n s e q u e n t l y s l i g h t l y o v e r e s t i m a t e s CO3"'™ l e v e l s . The ox yg c-3nat i on s t a t e o f b 1 ood was n o t c o n s i cler ed i n c a 1 c u 1 a t i on o f c a r b a m i n o f o r m a t i o n , n o r was t h e l e v e l o f e r y t h r o c y t e o r g a n i c p h o s p h a t e s . As a c o n s e q u e n c e , t h e c a r b a m i n o l e v e l s e s t i m a t e d f o r v e n o u s b l o o d i n T a b l e 5 p r o b a b l y u n d e r e s t i m a t e t h e a c t u a l l e v e l s p r e s e n t . L a m e l l a r b l o o d t r a n s i t t i m e i n r a i n b o w t r o u t , i s a b o u t 3 s a t 53 T a b l e 5 . A model o f C O a t r a n s p o r t i n a r t e r i a l and v e n o u s b l o o d of Sal mo gairdneri a t 1 0 C . Bee t e x t -for d e t a i l s . V a l u e s a r e a mol . Venous m i n u s arterial Venous A r t e r i a l umol % of t o t a l W H O L E B L O O D < 1 0 0 0 mL) T o t a l C O r a c o n t e n t 7 8 0 0 6 8 1 0 9 9 0 1 0 0 . 0 0 PLASMA ( 7 5 0 mL) T o t a l C 0 s > c o n t e n t a s H C 0 3 -a s C 0 3 ' - ~ a s C0 a a s c a r b a m i n o 6 9 0 0 6 7 3 2 . 7 1 6 . 6 1 4 1 . 0 • 9 . 7 6 0 6 0 5 9 2 5 . 6 1 6 . 4 1 0 9 . 5 8 . 5 8 4 0 8 0 7 . 1 0 . 2 3 1 . 5 1 . 2 8 4 . 8 5 8 1 „ 5 3 0 . 0 2 3 . 1 8 0 . 1 2 E R Y T H R O C Y T E S ( 2 5 0 mL) T o t a l C 0 a c o n t e n t a s H C O 3 -a s C 0 3 S ! - " a s C 0 a a s c a r b a m i n o 9 0 0 4 4 5 . 8 0 . 7 4 0 . 4 4 1 3 . 2 7 5 0 3 5 7 . 4 0 . 6 3 1 . 4 3 6 0 . 6 1 5 0 8 8 . 4 0 . 1 9 . 0 5 2 . 6 1 5 . 1 5 8 „ 9 3 0 . 0 1 0 . 9.1. 5 . 3 1 54 r e s t . ( R a n d a l l 1 9 8 2 a ) . D u r i n g t h a t t r a n s i t , , b l o o d C C o 2 d e c r e a s e s by a b o u t 1 mM. The d a t a o f T a b l e 5 i n d i c a t e s t h a t a p p r o x i m a t e l y 827. o f t h e CO- e x c r e t e d i s p l a s m a H C O 3 " " . F r e e CO- a c c o u n t s f o r o n l y a b o u t 47. o f C0r.2 e x c r e t i o n . The r e m a i n i n g 147. i s d i v i d e d a l m o s t e q u a l l y b e t w e e n r e d c e l l b i c a r b o n a t e and r e d c e l l c a r b a m i n o . The c o n t r i b u t i o n s o f C Q 3 ™ " and p l a s m a c a r b a m i n o t o CO- e x c r e t i o n a r e n e g l i b l e . In c o n c l u s i o n , COs> l o a d i n g o f S, gai rdneri b l o o d p r o c e e d s i n a t y p i c a l l y mammalian f a s h i o n . Red c e l l s p l a y a p r e d o m i n a n t r o l e i n C G 3 2 : HCO.3"" c o n v e r s i o n and d i s p l a y a t y p i c a l c h l o r i d e s h i f t . T h i s c h l o r i d e s h i f t i s s i m i l a r i n i t s t i m e c o u r s e and i t s d e p e n d e n c e on i n t r a c e l l u l a r c a r b o n i c a n h y d r a s e t o t h a t o f mammalian c e l l s (Cameron 1978b, O b a i d et al. 1 9 7 9 ) . The a c t i v i t y and p l a s m a 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 a r b o n i c a n h y d r a s e i s d e a l t w i t h i n t h e f o l l o w i n g c h a p t e r . HCO-", C I " and H * a r e d i s t r i b u t e d p a s s i v e l y i n f i s h r e d c e l l s in vitro a n d , t h e r e f o r e , , a r e f r e e l y p e r m e a b l e u n d e r t h o s e c o n d i t i o n s . F i s h r e d c e l l s c a n n o t be c o n s i d e r e d t o be a s i n g l e homogenous c o m p a r t m e n t , h o w e v e r . The n u c l e u s o f f i s h e r y t h r o c y t e s may be mare a c i d i c and c o n t a i n l e s s c h l o r i d e and b i c a r b o n a t e t h a n t h e c y t o s o l . D u r i n g l a m e l l a r b l o o d t r a n s i t , a b o u t 1 mM o f C 0 S i s e x c r e t e d of w h i c h 827. i s d e r i v e d f r o m p l a s m a H C O 3 " . S i n c e t h e b a s o l a t e r a l membrane o f t r o u t g i l l s i s l a r g e l y i m p e r m e a b l e t o H C 0 3 " ( P e r r y et al* 1 9 8 2 ) , t h i s r e d u c t i o n i n p l a s m a b i c a r b o n a t e must, n e c e s s i t a t e i t s c o n v e r s i o n b ack t o C 0 E , T h i s H C Q s " : CO-- c o n v e r s i o n p r o b a b l y i n v o l v e s a s i m p l e r e v e r s a l o f t h e c h e m i c a l and t r a n s p o r t p r o c e s s e s t h a t , o p e r a t e d u r i n g C O s a l o a d i n g . 55 C h a p t e r 2,. A C T I V I T Y AND I N H I B I T I O N OF CARBONIC ANHYDRASE IN FISHES The i n t e r c o n v e r s i o n o f b i c a r b o n a t e and c a r b o n d i o x i d e i s c a t a l y s e d by t h e enzyme c a r b o n i c a n h y d r a s e (CA) (EC 4 . 2 . 1 . 1 . ) . S i n c e t h e p i o n e e r i n g work o-f Mel drum and R o u g h t o n (1933) w i t h ox e r y t h r o c y t e s , t h e d i s t r i b u t i o n and p h y s i o l o g i c a l - f u n c t i o n o f CA i n a w i d e v a r i e t y o f a n i m a l t i s s u e s h a s been w e l l d o c u m e n t e d (Maren 1967, L i n d s k o g e t a l „ 1971, C a r t e r 1972, E<auer e t a l * 1980) . CA h a s been f o u n d i n t h e e r y t h r o c y t e s o f a l l v e r t e b r a t e s e x a m i n e d (Maren 1 9 6 7 ) , w i t h t h e e x c e p t i o n o f t h e a m p h i b i a n mudpuppy, Necturus maculosas? (Toews et al- 1978, Maren and A z a r 1981) and p e r h a p s t h e f l o u n d e r , Platichthys flestts ( M a s h i t e r and Morgan 1 9 7 5 ) . E r y t h r o c y t e CA c l a s s i c a l l y h a s been a s s i g n e d t h e p r e d o m i n a n t r o l e i n c a t a l y s i n g t h e i n t e r c o n v e r s i on of p l a s m a HCOas" and COs , ( R o u g h t o n 1 9 6 4 ) . CA a l s o i s p r e s e n t , i n t h e t i s s u e s o f most g a s e x c h a n g e o r g a n s , i n c l u d i n g i n v e r t e b r a t e g i l l s (Maren 1967, R a n d a l l and Wood 1981, H e n r y and Cameron 1 9 8 2 ) , v e r t e b r a t e g i l l s (Maren 1967, H a s w e l l and R a n d a l l 1978, Toews e t al. 19785, and v e r t e b r a t e l u n g s ( C r a n d a l l and 0 : ' B r a s k y 1978, E i f f r o s e t a l , 1978, K l o c k e 1 9 7 8 ) . L i t t l e i s known o f t h e enzyme's d i s t r i b u t i o n among t h e a i r - b r e a t h i n g o r g a n s of f i s h , a l t h o u g h i t s p r e s e n c e a p p e a r s t o be l i m i t e d t o t h o s e o r g a n s w h i c h a r e d e r i v e d embryon i c a l 1 y f r o m g i l l t i s s u e ( B u r g g r e n and H a s w e l l 1979, Daxboeck and Heming 1 9 8 2 ) , CA i n v e r t e b r a t e b r a n c h i a l e p i t h e l i a i s i n v o l v e d i n i o n t r a n s p o r t (Maetz 1956a, Maren 1 9 6 7 ) . The r o l e 56 o-f CA i n p u l m o n a r y e p i t h e l i a i s u n c l e a r , b u t t h e enzyme h a s been i m p l i c a t e d i n s e c r e t i o n o f l u n g f l u i d i n t h e - f e t u s (Adamson and Waxman 1 9 7 6 ) , i n d a m p e n i n g o-f CO35 t r a n s i e n t s b e t w e e n a l v e o l a r g a s and a r t e r i a l b l o o d ( F a r h i e t al. 1 9 7 6 ) , and i n a t t a i n m e n t o f c h e m i c a l e q u i l i b r i u m i n b l o o d d u r i n g p u l m o n a r y c a p i l l a r y t r a n s i t ( C r a n d a l l and O ' B r a s k y 1978, E f f r o s e t al. 1978, K l o c k e 1978)„ The e p i t h e l i a l CA o f b o t h g i l l s and l u n g s p l a y s a m i n o r r o l e i f a n y , h o w e v e r , i n o v e r a l l e x c r e t i o n of CO^ ( C r a n d a l l and B i d a n i 1981, P e r r y e t al. 1 9 8 2 ) . A number o f a s s a y t e c h n i q u e s h a v e been d e v e l o p e d t o f o l l o w t h e c a t a l y s e d COs»s HC0 3"" c o n v e r s i o n ( f o r r e v i e w s e e D a v i s 1 9 6 3 ) . E a c h method h a s i t s own i n h e r e n t a d v a n t a g e s and d i s a d v a n t a g e s , and no s i n g l e t e c h n i q u e h a s emerged a s a s t a n d a r d f o r m e a s u r i n g CA a c t i v i t y . The o r i g i n a l t e c h n i q u e o f M e l d r u m and R o u g h t o n ( 1 9 3 3 ) , t h e m o d i f i e d b o a t a s s a y , was the? method o f c h o i c e i n e a r l y s t u d i e s and has; s u r v i v e d t o t h e p r e s e n t , w i t h a number of m o d i f i c a t i o n s ( G i a c o b i n i 1962, H o f f e r t 1966, H a s w e l l and R a n d a l l 1976, G i r a u d 1 9 8 1 ) . The b a s i c r e a c t i o n i s e v o l u t i o n o f C 0 2 f r o m a b u f f e r s o l u t i o n c o n t a i n i n g NaHCQa. T h i s t e c h n i q u e s i s s u s c e p t i b l e , h o w e v e r , t o p r o b l e m s r e l a t e d t o the? d i f f u s i o n o f C 0 3 o u t cD-f s o l u t i o n (Heming and R a n d a l l 1 9 8 2 ) . A v a s t a r r a y CDf methods h a v e e v o l v e d a r o u n d measurement o f t h e pH c h a n g e s a s s o c i a t e d w i t h COsi; HC0 3"~ c o n v e r s i o n , i n c l u d i n g s i m p l e i n d i c a t o r c h a n g e s (Maren 1 9 6 0 ) , c o n t i n u o u s e l e c t r o m e t r i c r e c o r d i n g ( D a v i s 1 9 6 3 ) , and t h e use? o f p H - s t a t t i t r a t i o n (Hansen and M a g i d 1 9 6 6 ) . T h e s e t e c h n i q u e s h a v e t h e a d v a n t a g e o f b e i n g a p p l i c a b l e t o c o n t i n u o u s and s t o p - f l o w s y s t e m s ( F o r s t e r and C r a n d a l l 1975, C r a n d a l l -et a l , 1 9 7 1 ) . 5 7 A r e c e n t d e v e l o p m e n t i n CA measurement t e c h n i q u e s h a s been t h e d i s c o v e r y t h a t t h e enzyme c a t a l y s e s t h e h y d r o l y s i s o f many e s t e r s , n o t a b l y p - n i t r o p h e n y l a c e t a t e , and t h e h y d r a t i o n o f a c e t a l d e h y d e and r e l a t e d c a r b o n y l compounds ( S c h n e i d e r and L i e f l a n d e r 1963, P a c k e r and Meany 1965, P o c k e r and S t o n e 1 9 6 5 ) . T h e s e r e a c t i o n s c a n be f o l l o w e d c a 1 o u r i m e t r i c a 1 1 y ( A r m s t r o n g et al . 1966, H o u s t o n and M c C a r t y 1 9 7 8 ) . However, none o f t h e s e new s u b s t r a t e s i s p h y s i o l o g i c a l „ Moreover,, c e r t a i n t i s s u e CA l a c k s e s t e r a s e a c t i v i t y ( T a s h i a n 1965) and s o i s u n d e t e c t a b l e u s i n g e s t e r a s e a s s a y t e c h n i q u e s . The u s e of t h e s e v a r i o u s a s s a y t e c h n i q u e s , t h e r e s u l t s o f w h i c h a r e n o t a l w a y s i n t e r c o n v e r t a b l e o r p h y s i o l o g i c a l l y i n t e r p r e t a b l e , h a s made i t d i f f i c u l t t o compare r e s u l t s o f v a r i o u s CA s t u d i e s ; . W i t h t h a t i n m i n d , t h e p r e s e n t s t u d y e x a m i n e d t h e r e l a t i v e m e r i t s and u s e f u l n e s s i n p h y s i o l o g i c a l s t u d i e s o f t h r e e a s s a y t e c h n i q u e s ! t h e m o d i f i e d boat, a s s a y ? t h e e s t e r a s e a s s a y ? t h e p H - s t a t a s s a y . The l i n e a r i t y , s e n s i t i v i t y and d e t e c t i o n l i m i t s o f e a c h a s s a y were d e t e r m i n e d i n o r d e r t o d e v e l o p a r e l i a b l e CA a s s a y t h a t c o u l d be u s e d i n s t u d i e s w i t h c r u d e t i s s u e h o m o g e n a t e s . B e c a u s e o f t h e i m p o r t a n c e o f CA i n COs> e x c r e t i o n and i o n i c r e g u l a t i o n i n f i s h , a d i s t r i b u t i o n a l s t u d y o f c a r b o n i c a n h y d r a s e i n f i s h t h e n was u n d e r t a k e n u s i n g b o t h t h e e s t e r a s e a s s a y and t h e p H - s t a t a s s a y . 58 MATERIALS AND METHODS A, M.§D92)§t.r.iE Me.thgd.J_ M o d i f i e d B o a t jAssay E l v o l u t i o n of- C 0 S -from a p h o s p h a t e b u f f e r c o n t a i n i n g N a H C 0 3 was m e a s u r e d m a n o m e t r i c a l 1 y a s d e s c r i b e d by H a s w e l l and R a n d a l l ( 1 9 7 6 ) , A r e a c t i o n v e s s e l o r ? b o a t : ' was c o n s t r u c t e d f r o m a 50-mL E h r l e n m e y e r - f l a s k , p a r t i t i o n e d a l o n g i t s b o t t o m by a r a i s e d g l a s s r i d g e . Two mL of b i c a r b o n a t e s o l u t i o n (200 mM NaHCO;* i n 20 mM NaOH, pH > 8.00) were p l a c e d on one s i d e o f t h e rid g e s and 2 mL o f p h o s p h a t e b u f f e r ( m i x t u r e o f 200 mM NaHPQ.* and 200 mM KHs-PO.*, pH 6.80 art t h e p r e v a i l i n g a s s a y t e m p e r a t u r e ) were p l a c e d on t h e o t h e r s i d e ? 0.2 mL o f m a t e r i a l t o be a s s a y e d were a d d e d t o t h e b u f f e r . The b o a t was c o n n e c t e d t o a p r e s s u r e t r a n s d u c e r ( S t a t h a m P23Db) by way of p o l y e t h y l e n e t u b i n g (PE--160) and a 3-way s t o p c o c k , and t h e n was imm e r s e d i n a s h a k i n g t e m p e r a t u r e b a t h . The i m m e r s e d b o a t was l e f t open t o t h e a t m o s p h e r e f o r 3--5 min d u r i n g w h i c h t i m e t h e t e m p e r a t u r e o f i t s c o n t e n t s e q u i l i b r a t e d w i t h t h a t o f t h e b a t h . The b o a t t h e n was s e a l e d , t h e s h a k e r was t u r n e d o n, and t h e i n t e r n a l g a s p r e s s u r e was m o n i t o r e d a s t h e d e h y d r a t i o n r e a c t i o n p r o c e e d e d . The r a t e o f i n c r e a s e i n g a s p r e s s u r e was t a k e n a s b e i n g d i r e c t l y p r o p o r t i o n a l t o t h e r a t e o f HC0 3"" d e h y d r a t i o n . The p r e s s u r e t r a n s d u c e r was c a l i b r a t e d w i t h i n j e c t i o n s o f known v o l u m e s o f a i r made a t t h e p r e v a i l i n g assay-t e m p e r a t u r e . R e a c t i o n r a t e s (mL C0^ e v o l v e d - min"" 1) were c a l c u l a t e d f r o m c h a n g e s i n p r e s s u r e e q u i v a l e n t t o c h a n g e s i n 59 v o l u m e o f f r o m 0.1 t o 0.3 mL; t h e i n c r e a s e i n g a s p r e s s u r e wais l i n e a r o v e r t h i s r a n g e o f v o l u m e s . B« Qp.L9MCLm.§/fe.l'll.<-I d.§.t:ilQ.S!.l. E s t e r a s e Assay. C a t a l y s i s o f p — n i t r o p h e n y l a c e t a t e (pNA) h y d r o l y s i s by CA was m e a s u r e d s p e c t r o p h o t o m e t r i c a l 1 y a s d e s c r i b e d by H o u s t o n and M c C a r t y (1978) and Watson e t al „ ( 1 9 8 2 ) . Two mL o f • f r e s h l y -p r e p a r e d pNA (3 mM pNA i n 3% a c e t o n e ) were added t o a t h e r m o s t a t t e d c u v e t t e . T h i s was f o l l o w e d by e i t h e r 1.8 mL of T r i s b u f f e r (125 mM T r i s ( h y d r o m e t h y l ) m e t h y l a m i n e , pH t i t r a t e d t o 7.50 a t t h e p r e v a i l i n g a s s a y t e m p e r a t u r e w i t h c o n c e n t r a t e d H s e S O , * ) t o m e a s u r e t o t a l e s t e r a s e a c t i v i t y o r 1.8 mL of a c e t a z o l a m i d e s o l u t i o n (500 mM a c e t a z o l a m i d e i n an i d e n t i c a l T r i s b u f f e r ) t o m e a s u r e a c e t a z o l a m i d e ~ i n s e n s i t i v e a c t i v i t y . F i n a l l y , 0.2 mL. of m a t e r i a l t o be a s s a y e d were a d d e d . The c h a n g e i n a b s o r b a n c e a t 348 nm t h e n was f o l l o w e d f o r a t l e a s t 3 min u s i n g a Pye U n i c a m s p e c t r o p h o t o m e t e r (model S P 8 — 2 0 0 ) . The r a t e o f c h a n g e i n a b s o r b a n c e was c o n v e r t e d t o mmol pNA-min™ 1 u s i n g an e x t i n c t i o n c o e f f i c i e n t o f 4.69-mmol~ 1 - cm- 1 (Watson e t a l . 1 9 8 2 ) . A c e t a z o l a m i d e - s e n s i t i v e e s t e r a s e a c t i v i t y was c a l c u l a t e d a s t h e d i f f e r e n c e b e t w e e n t h e t o t a l e s t e r a s e a c t i v i t y and t h e a c e t a z o l a m i d e - i n s e n s i t . i v e a c t i v i t y . A c e t a z o l ami d e - s e n s i t i ve e s t e r a s e a c t i v i t y was assumed t o r e f l e c t CA a c t i v i t y . C« E.lec t r g m e t r i c Met hod J. E . b z s t a t A s s a y B i c a r b o n a t e d e h y d r a t i o n i n a p h o s p h a t e b u f f e r was f o l l o w e d e l e c t r o m e t r i c a l 1 y w i t h an a u t o t i t r a t i o n a s s e m b l y ( R a d i o m e t e r , 60 PHM64 r e s e a r c h pH m e t e r , TTT80 t i t r a t o r , , ABU 12 a u t o b u r e t t e , TTA80 t i t r a t i o n a s s e m b l y ) u s i n g a m o d i f i c a t i o n of t h e methods o f H ansen and i i a g i d ( 1 9 6 6 ) , a s d e s c r i b e d by H e n r y and Cameron ( 1 9 8 3 ) , I n t h e p r e s e n t s t u d y , a r e a c t i o n v e s s e l was c o n s t r u c t e d f r o m a t h e r m o s t a t t e d 30-mL. g l a s s v e s s e l w i t h a 30-mm d i a m e t e r p o r o u s (medium p o r o s i t y ) g l a s s d i s c embedded i n i t s b o t t o m . N i t r o g e n g a s was p a s s e d t h r o u g h a C O ^ - t r a p ( s a t u r a t e d KOH s o l u t i o n ) and b u b b l e d t h r o u g h t h e g l a s s d i s c a t a r a t e e x c e e d i n g 500 ml_ mi n -' 1 t o p u r g e CQ a f r o m t h e r e a c t i o n m i x t u r e a s C0: S was f o r m e d . The r a t e of H C 0 3 ~ d e h y d r a t i o n was i n d e p e n d e n t o f g a s f l o w r a t e , a t f l o w r a t e s a b o v e 400 mL-mi n""1 ( F i g . 1 0 ) . G l a s s pH and c a l o m e l r e f e r e n c e e l e c t r o d e s ( R a d i o m e t e r , t y p e s G2040C and K4040, r e s p e c t i v e l y ) were u s e d t o m o n i t o r pH of thee r e a c t i o n m i x t u r e . A c o n s t a n t pH was m a i n t a i n e d by t h e t i t r a t o r s e t t o i t s p H - s t a t mode? t i t r a t i o n was done u s i n g 0.125 0.400 N HC1 d e l i v e r e d by t h e a u t o b u r e t t e . The r e a c t i o n m i x t u r e c o n s i s t e d of a p h o s p h a t e b u f f e r ( m i x t u r e o f 15 mM Na^HPO^. and 15 mM KH^PO^, pH 0,30 u n i t s l e s s t h a n t h e d e s i r e d a s s a y pH a t t h e p r e v a i l i n g a s s a y t e m p e r a t u r e ) and a b i c a r b o n a t e s o l u t i o n (20 60 mM NaHC0-s) „ I n some a s s a y s , s u f f i c i e n t N a C l was added t o t h e b i c a r b o n a t e s o l u t i o n t o r a i s e t h e o v e r a l l i o n i c s t r e n g t h o f t h e r e a c t i o n m i x t u r e t o 0.15 M. T h r e e mL o f b u f f e r were added t o t h e r e a c t i o n v e s s e l f i r s t and were a l l o w e d t o e q u i l i b r a t e w i t h t h e C O s r - f r e e n i t r o g e n g a s u n t i l a s t a b l e pH was a c h i e v e d . T h r e e mL o f b i c a r b o n a t e s o l u t i o n t h e n were added t o g e t h e r w i t h 0.01--0.10 mL of m a t e r i a l t o be a s s a y e d d e l i v e r e d a s 0.10 mL i n C o r t l a n d s a l i n e (Wolf 1 9 6 3 ) . T i t r a t i o n was i n i t i a t e d when t h e pH o f t h e m i x t u r e r e a c h e d t h e d e s i r e d 61 F i g u r e 10. U n c a t a l y s e d r a t e o f HC0 3"" d e h y d r a t i o n ( s o l i d c i r c l e s ) and c a t a l y s e d r a t e u s i n g 1.0 ag of b o v i n e CA (open c i r c l e s ) d e t e r m i n e d e l e c t r o m e t r i c a l 1 y a t 25 C, pH 7.500 and 50 mM NaHCOs, s h o w i n g e f f e c t o f t h e f l o w r a t e o f C 0 z - f r e e n i t r o g e n g a s t h r o u g h t h e r e a c t i o n v e s s e l . V a l u e s a r e means + 1 S.E. ( n ^ S ) . 62 2 0 0 r 150 c E o E LU 1 0 0 o !5 a: o >-x LLI Q 5 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 NITROGEN F L O W R A T E , m L / min 63 a s s a y pH., The r e a c t i o n t h e n was f o l l o w e d f o r 15-30 s e c , a f t e r w h i c h t i m e t h e v o l u m e o f a c i d added was r e a d f r o m t h e a u t o b u r e t t e t o t h e n e a r e s t 1.0 uL. The r a t e of a c i d a d d i t i o n (mmol H*-min" 1) was t a k e n a s b e i n g e q u i v a l e n t t o t h e r a t e of b i c a r b o n a t e d e h y d r a t i o n (mmol- m i n - 1 ) s i n c e t h e s t o i c h i o m e t r y of H+.HC0 3~ i n t h e d e h y d r a t i o n r e a c t i o n i s 111. QJl§?XQL£a-L Studi.e _ 5 The l i n e a r r a n g e , d e t e c t i o n l i m i t s and s e n s i t i v i t y o f e a c h a s s a y t e c h n i q u e were d e t e r m i n e d u s i n g p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e ( S i g m a C — 7 5 0 0 ) . A l a c k o f d e f i n i t i v e d a t a on t h e k i n e t i c s o f t h e u n c a t a l y s e d C0_a r e a c t i o n s u n d e r t h e p h y s i o l o g i c a l c o n d i t i o n s o f t r o u t b l o o d p r o m p t e d t h e i r measurement. The u n c a t a l y s e d d e h y d r a t i o n r a t e c o n s t a n t , k H c o 3 , of t h e r e a c t i o n H C 0 3 ~ + H* COsa + H35O i n a b u f f e r e d N a C l s o l u t i o n (1=0.15) was d e t e r m i n e d u s i n g t h e p H - s t a t . a s s a y . B i c a r b o n a t e d e h y d r a t i o n i n t h e p H - s t a t a s s a y was an u n o p p o s e d f i r s t o r d e r r e a c t i o n s i n c e t h e r e a c t i o n p r o d u c t , C0 : ; ;, was p u r g e d c o n t i n u a l l y f r o m t h e r e a c t i o n m i x t u r e . The d e h y d r a t i o n r a t e c o n s t a n t was c a l c u l a t e d f r o m m e a s u r e d r a t e s o f H C 0 3 " d e h y d r a t i o n u n d e r s p e c i f i c c o n d i t i o n s of known pH ( 7 . 0 0 0 , 7.250, 7.500, 7.750, 8 . 0 0 0 ) , t e m p e r a t u r e ( 5 , 10, 15 C) and b i c a r b o n a t e c o n c e n t r a t i o n ( 2 0 , 40, 60 mM) a s d e s c r i b e d by Maren et al. ( 1 9 7 6 ) . E» Phy.si,ol.QQ.i.?Li.L St.iidi.es CA a c t i v i t y i n f i s h t i s s u e s was m e a s u r e d u s i n g b o t h t h e 64 e s t e r a s e a s s a y a t 25 C, pH 7.500 and 3 mM pNA, and t h e p H - s t a t a s s a y at. a m b i e n t w a t e r t e m p e r a t u r e s (9-13. C) , pH 7.500 and 50 mM NaHCQs. A n i m a l s u s e d i n t h i s s t u d y were o b t a i n e d a s f o l l o w s : ; r a i n b o w t r o u t (Salmo gairdneri) 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 a 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;; c u t t h r o a t t r o u t (Salmo clarki) f l a r g e s c a l e s u c k e r s (Catostomas macrocheiI as) and brown b u l l h e a d s (Ictalarus nehalosas) were c a p t u r e d i n D e v i l : ' s L a k e (B.C.) and were h e l d a t Simon F r a s e r U n i v e r s i t y ( B u r n a b y , B.C.) i n d e c h l o r i n a t e d B u r n a b y t a p water:; b o w f i n s (f)mia calva) were c a p t u r e d i n L a k e E r i e ( O n t a r i o ) and were a i r l i f t e d t o U.E-i.C. where t h e y were m a i n t a i n e d i n f a c i l i t i e s i d e n t i c a l t o t h o s e d e s c r i b e d f o r r a i n b o w t r o u t , b l a c k p r i c k l e b a c k s (K i phi ster atroparpareus)) were c a p t u r e d n e a r t h e B a m f i e l d M a r i n e S t a t i o n ( B a r n f i e l d , B.C.) and were t r a n s p o r t e d t o U.B.C. where t h e y were m a i n t a i n e d i n r e c i r c u l a t e d s e a - w a t e r ( s a l i n i t y 2 8 - 3 3 % . ) . E r y t h r o c y t e s a m p l e s were o b t a i n e d by c e n t r i f u g a t i o n o f b l o o d w i t h d r a w n f r o m t h e c a u d a l b l o o d v e s s e l s o f a n a e s t h e t i z e d a n i m a l s ( s e e G e n e r a l M a t e r i a l s and M e t h o d s ) . T h e s e r e d c e l l s w e r e washed t w i c e i n C o r t l a n d s a l i n e and t h e n were l y s e d u s i n g s a p o n i n . The r e s u l t i n g l y s a t e was c e n t r i f u g e d a t 5,000 g and 1 C ( S o r v a l - s u p e r s p e e d RC2-B c e n t r i f u g e ) f o r 5 min t o remove n u c l e a r d e b r i s (Watson et al. 1 9 8 2 ) . The s u p e r n a t e n t was r e t a i n e d on i c e f o r CA a s s a y s . F i s h were c l e a r e d o f r e m a i n i n g r e d c e l l s by i n f u s i n g C o r t l a n d s a l i n e a t p r e s s u r e s o f b e t w e e n 60 and 80 cm HraO t h r o u g h a c a n n u l a i n s e r t e d i n t h e v e n t r i c l e . T i s s u e s t o be a s s a y e d were e x c i s e d and h o m o g e n i z e d ( h o m o g e n i z i n g medium: 250 mM s u c r o s e ? 40 mM T r i s ( h y d r o m e t h y l ) m e t h y l amine? S mM e t h y l e n e 65 d i a m i n e t e t r a a c e t a t e (EDTA) ; pH a d j u s t e d t o 7.50 w i t h Hs_S0_*) . T h e s e h o m o g e n a t e s were c e n t r i f u g e d a t 900 g and 1 C -for 10 min t o remove c e l l u l a r d e b r i s and n u c l e i (Watson et al , 1 9 8 2 ) . The s u p e r n a t e n t was r e t a i n e d on i c e f o r CA a s s a y s . A l i q u o t s (0.01 mL) o f C o r t l a n d s a l i n e o r s e p a r a t e d p l a s m a were added t o some a s s a y s t o d e t e r m i n e t h e e f f e c t s o f p l a s m a on CA a c t i v i t y . A l l m e a s u r e m e n t s were made a t l e a s t i n d u p l i c a t e . Where p o s s i b l e , CA a c t i v i t e s were c o n v e r t e d t o a r b i t r a r y enzyme u n i t s ( e u ) , a s (9) eu = ( c a t a l y s e d r e a c t i o n r a t e - u n c a t a l y s e d r a t e ) / u n c a t a l y s e d r a t e . The r e a c t i o n r a t e o f C o r t l a n d s a l i n e was t a k e n t o r e p r e s e n t t h e u n c a t a l y s e d r a t e i n t h e s e c a l c u l a t i o n s . H a e m o g l o b i n l e v e l s were m e a s u r e d ( a l k a l i n e h a e m a t i n method of A n t h o n y 1961) i n s e p a r a t e d p l a s m a and t i s s u e s a m p l e s t o o b t a i n a m e a s u r e o f t h e c o n t a m i n a t i o n o f t h e s e s a m p l e s by e r y t h r o c y t e CA. No d e t e c t a b l e c o n t a m i n a t i o n was p r e s e n t i n any of t h e r e p o r t e d s t u d i e s . 66 R E S U L T S A., C h e m i c a l . A l l t h r e e t e c h n i q u e s showed a l i n e a r r e l a t i o n s h i p b e t w e e n r e a c t i o n r a t e and enzyme c o n t e n t o v e r a t l e a s t some p o r t i o n of t h e r a n g e o f b o v i n e CA c o n c e n t r a t i o n s u s e d ( F i g . 11, 12 and 1 3 ) . The e s t e r a s e a s s a y was l i n e a r f o r a l l amounts o f bovine; CA u s e d f r o m 5 ug and a b o v e . A l t h o u g h n o t shown i n F i g u r e 11, l i n e a r i t y was p r e s e r v e d up t o 1000 ug b o v i n e CA o v e r t h e e n t i r e t e m p e r a t u r e r a n g e u s e d (10-37 C ) . The l o w e r d e t e c t i o n l i m i t o f t h i s a s s a y was a b o u t 2.5 ug CA|i t h e r a t e o f r e a c t i o n s ; u s i n g l e s s t h a n t h a t amount o f enzyme c o u l d n o t be d i s t i n g u i s h e d s t a t i s t i c a l l y f r o m t h e b a c k g r o u n d a c e t a z o l ami d e - i ris e n s i t i ve r a t e , , The e s t e r a s e a s s a y was c h a r a c t e r i z e d by v e r y l o w r e a c t i o n r a t e s ( 0.002 t o 0.117 umol pNA-mi n"~1 ) even a t t h e r e l a t i v e l y h i g h enzyme l e v e l s and h i g h t e m p e r a t u r e s u s e d ( F i g . 1 1 ) . In a d d i t i o n , t h i s a s s a y had a low s e n s i t i v i t y , a s i n d i c a t e d by t h e s l o p e o f t h e r e g r e s s i o n o f r e a c t i o n r a t e a g a i n s t enzyme c o n c e n t r a t i o n (0.00095 umol - m i n " 1 - ug CA' - 1 a t 10 C t o 0.00314 umol - mi n " 1 - ug CA" 1 at. 37 C) . I t was n o t p o s s i b l e t o c o n v e r t . t h e s e s e n s i t i v i t y v a l u e s t o eu•ug CA" 1 s i n c e by d e f i n i t i o n t h e u n c a t a l y s e d r a t e o f a c e t a z o l ami d e - s e n s i t i ve; pMA h y d r o l y s i s i s z e r o . The m o d i f i e d b o a t a s s a y g a v e a l i n e a r r e s p o n s e f r o m 0 t o a b o u t 2 ug b o v i n e CA a t t e m p e r a t u r e s f r o m 10 t o 25 C ( F i g . 1 2 ) . A t 37 C, h o w e v e r , t h e u p p e r l i m i t o f t h i s l i n e a r r a n g e was r e d u c e d t o a p p r o x i m a t e l y 1 ug b o v i n e CA„ The l o w e r d e t e c t i o n 67 F i g u r e 1 1 . A c e t a z o l a m i d e — s e n s i t i v e e s t e r a s e a c t i v i t y o f p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d c o l o u r i m e t r i c a l 1 y a t pH 7.500 and 3 mM p - n i t r o p h e n y l a c e t a t e (pNA)„ s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e and enzyme c o n t e n t . V a l u e s a r e means + 1 S.E. (n=5). R A T E , nrnol pNA /m i n b b b b - — jo J> <~ CD o M 69 F i g u r e 12. C a t a l y s i s o-f C0 r a e v o l u t i o n f r o m a b u f f e r e d N aHC0 3 s o l u t i o n by p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d m a n o m e t r i c a l l y at. pH 6.800 and 200 mM MaHC0 3 f l s h o w i n g e f f e c t s of r e a c t i o n t e m p e r a t u r e and enzyme c o n t e n t . V a l u e s a r e means RATE, mL C 0 2 / min o o _ - !» A co ro o> o 71 F i g u r e 13. C a t a l y s i s o-f b i c a r b o n a t e d e h y d r a t i o n by p u r i f i e d b o v i n e c a r b o n i c a n h y d r a s e m e a s u r e d e l e c r o m e t r i c a l 1 y a t pH 7.500 and 50 mil NaHC0», s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e ? and enzyme c o n t e n t . V a l u e s a r e means + 1 B.E. <n=5). RATE , mmol H C 0 3 / min 73 l i m i t o f t h e a s s a y was a b o u t 0.1 ug CA i n a l l c a s e s . S e n s i t i v i t y o-f t h e m o d i f i e d b o a t a s s a y v a r i e d i n v e r s e l y w i t h t e m p e r a t u r e f r o m 0.26 mL-min" 1-ug CA""1 a t 10 C t o 0.17 mL-mi n""1 - ug CA™ 1 a t 37 C, e q u i v a l e n t t o 0.6 and 0,1 eu-ug CA""1, r e s p e c t i v e l y . The most s a t i s f y i n g r e s u l t s i n t e r m s o f d e t e c t i o n and s e n s i t i v i t y w e r e o b t a i n e d u s i n g t h e p H - s t a t a s s a y . The p H - s t a t a s s a y a t pH 7.500 and 50 mli NaHCD.3 g a v e a l i n e a r r e s p o n s e f r o m 0 t o 2 ug b o v i n e CA a t a l l t e m p e r a t u r e s u s e d (10-37 C) ( F i g . 1 3 ) . The l o w e r d e t e c t i o n l i m i t o f t h i s a s s a y was a b o u t 0.02 ug CA when 0.125 M HC1 was u s e d a s t h e t i t r a n t and c o u l d h a v e been r e d u c e d s t i l l f u r t h e r by u s i n g a more d i l u t e t i t r a n t . S e n s i t i v i t y o f t h e p H - s t a t a s s a y v a r i e d f r o m 47,1 umol - mi n""1 - ug CA" 1 a t 10 C t o 146,6 umol - m i n " 1 - ug CA""1 a t 37 C, e q u i v a l e n t t o an a v e r a g e s e n s i t i v i t y o f 2.2 + 0.2 eu-ug CA""1 (n=5) o v e r t h e t e m p e r a t u r e range? 10 t o 37 C. T h i s r e p r e s e n t e d b e t w e e n a 3.7 t o 2 2 - f o l d i m p r o v e m e n t i n s e n s i t i v i t y o v e r t h e m o d i f i e d b o a t a s s a y . The u n c a t a l y s e d H C 0 3 " d e h y d r a t i o n r e a c t i o n was s e n s i t i v e t o pH and t e m p e r a t u r e ( F i g . 1 4 ) . The u n o p p o s e d d e h y d r a t i o n r a t e c o n s t a n t i n c r e a s e d when pH d e c r e a s e d o r when t e m p e r a t u r e i n c r e a s e d . However, t h e m e a s u r e d k H o o 3 v a l u e s were s u r p r i s i n g l y small 1 and i n d i c a t e d h a l f - t i m e s f o r t h e u n o p p o s e d d e h y d r a t i o n r e a c t i o n r a n g i n g f r o m 3,8 min (15 C, pl-l 7,000) t o 105,0 min (5 C, pH 8 . 0 0 0 ) . B« p h y s i o l o g i c a l . T a b l e 6 s u m m a r i z e s t h CA a c t i v i t y i n t i s s u e s o f s e v e r a l f i s h e s , a s d e t e r m i n e d u s i n g t h e e s t e r a s e a s s a y a t 25 C and t h e p H - s t a t a s s a y a t ambient, w a t e r t e m p e r a t u r e s (9-11 C) „ A t t e m p t s 7 4 F i g u r e 14. D e h y d r a t i o n r a t e c o n s t a n t , k H c o 3 , and c o r r e s p o n d i n g h a l f - t i m e f o r t h e u n o p p o s e d r e a c t i o n H C O 3 " " + H* •-•-> CO33 + H=.0 a t 1=0.15, s h o w i n g e f f e c t s o f r e a c t i o n t e m p e r a t u r e and pH. V a l u e s a r e means + 1 S.E. (n=15). 75 76 T a b l e 6. 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 i n t i s s u e s o f s e v e r a l f i s h e s . M e a s u r e m e n t s were made u s i n g t h e e s t e r a s e a s s a y a t 25 C, pH 7.500 and 3 mM pNA and t h e p H - s t a t a s s a y a t a m b i e n t w a t e r t e m p e r a t u r e s (9-11 C) , pH 7.500 and 50 mM NaHC0 : 3 i. V a l u e s a r e means + 1 S.E. e u , enzyme u n i t . ND, none d e t e c t e d . T i s s u e S ample E s t e r a s e a s s a y p H - s t a t a s s a y s i z e (umol - m i n - 1 • g ™ 1 ) ( e u - g - * ) >almo gairdneri e r y t h r o c y t e g i l l s w i m b l a d d e r s k i n 16 16 4 6 1.44 + 0.12 0.72 + 0.06 0. 04 + 0. 02! ND 882 + 154 773 + 60 5 ± 2 ND Salmo clarki e r y t h r o c y t e 7 2.14 + 0.38 858 + 148 g i l l 7 1.28 ± 0.34 478 + 53 Catostomus macrochei1 as e r y t h r o c y t e 6 2.33 + 0.49 1370 ± 167 g i l l 6 0.64 + 0.10 752 + 80 Ictalaras nebul osas e r y t h r o c y t e g i 11 1.51 + 0.25 0.28 + 0.10 750 ±. 47 + 138 Xiphister atroparpareas e r y t h r o c y t e 6 2.41 + 0.48 1004 + 200 g i l l 6 0.50 + 0.12 396 + 97 s k i n 6 ND ND flmia calva e r y t h r o c y t e g i 11 s w i m b l a d d e r 4 4 1.31 + 0.35 0.52 + 0.08 0. 17 + 0.04 210 + 144 + 14 40 51 77 t o c o n d u c t t h e e s t e r a s e a s s a y a t a m b i e n t w a t e r t e m p e r a t u r e s p r o v e d u n s u c c e s s f u l i n my h a n d s , p r e s u m a b l y due t o t h e e f f e c t o f t e m p e r a t u r e on s e n s i t i v i t y and t h e i n a b i l i t y o f t h i s a s s a y t o d e t e c t s m a l l amounts o f CA <F i g . 1 1 ) . The r a t i o o f CA a c t i v i t y a s m e a s u r e d u s i n g t h e p H - s t a t a s s a y t o t h a t d e t e r m i n e d by t h e e s t e r a s e a s s a y v a r i e d by an o r d e r o f m a g n i t u d e among t h e d i f f e r e n t t i s s u e s ( T a b l e 6 ) . S i n c e t h e two a s s a y s were c o n d u c t e d a t w i d e l y d i f f e r e n t t e m p e r a t u r e s , h o w e v e r , t h i s v a r i a t i o n i n t h e a c t i v i t y r a t i o p r o b a b l y r e f l e c t s d i f f e r e n t t h e r m a l c o e f f i c i e n t s a n d / o r t h e r m a l s e n s i t i v i t i e s f o r c a r b o n i c a n h y d r a s e o f d i f f e r e n t t i s s u e s . N o n e t h e l e s s , t h e two d i f f e r e n t t e c h n i q u e s y i e l d e d s i m i l a r t r e n d s i n CA a c t i v i t y among t h e v a r i o u s t i s s u e s . B o t h t e c h n i q u e s d e m o n s t r a t e d t h a t , on a wet t i s s u e w e i g h t b a s i s , e r y t h r o c y t e s had h i g h e r CA a c t i v i t y t h a n g i l l t i s s u e . T e l e o s t e a n r e d c e l l s p o s s e s s e d f r o m 858 t o 1370 eu-gm t i s s u e " " 1 . H o l o e s t e a n e r y t h r o c y t e s , t h o s e o f Ami a c a l ' / a , d e m o n s t r a t e d l e s s CA a c t i v i t y (210 eu-gm t i s s u e - 1 ) - . I n g e n e r a l , t h e r e s u l t s i n d i c a t e t h a t f i s h b l o o d c o n t a i n s s u f f i c i e n t c a r b o n i c a n h y d r a s e p e r mL. t o a c c e l e r a t e H C 0 3 - d e h y d r a t i o n by some 53 t o 343 t i m e s , a s s u m i n g an h a e m a t o c r i t . o f 2 5 % (Cameron and D a v i s 1 9 7 0 ) . S w i m b l a d d e r h o m o g e n a t e s had s u b s t a n t i a l l y l e s s CA a c t i v i t y t h a n g i l l t i s s u e , b u t n o n e t h e l e s s p o s s e s s e d s i g n i f i c a n t , a c t i v i t y . S k i n h o m o g e n a t e s l a c k e d d e t e c t a b l e CA a c t i v i t y . The p l a s m a o f most f i s h e s e x a m i n e d c o n t a i n e d an e n d o g e n o u s i n h i b i t o r o f CA w h i c h was a c t i v e a g a i n s t b o t h r e d c e l l and g i l l CA ( T a b l e 7 ) . CA a c t i v i t i e s were r e d u c e d f r o m b e t w e e n 14 t o 887. by a d d i t i o n o f p l a s m a t o t h e a s s a y s , i n d e p e n d e n t o f t h e t e c h n i q u e 78 T a b l e 7„ E f f e c t o-f p l a s m a on c a r b o n i c : a n h y d r a s e a x c t i v i t y i n t i s s u e s o f s e v e r a l f i s h e s . V a l u e s d e n o t e d by # 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 f r o m z e r o a t P<0.05 ( p a i r e d t t e s t ) . R e s t o f c a p t i o n a s i n T a b l e 6. P e r c e n t c h a n g e f r o m c o n t r o l a c t i v i t y T i s s u e S a m p l e s i z e E s t e r a s e a s s a y p H - s t a t a s s a y Sa I mo gairdneri -— s e x u a l l y i mmature e r y t h r o c y t e 6 -53 + 18* 30 + 1 3 * g i l l 4 -66 + 7* -60 + 10* Saimo gairdneri — s e x u a l l y m a t u r e e r y t h r o c y t e 6 19 + 16* 24 + 1 3 * Salmo clarki — s e x u a l l y i m m a t u r e e r y t h r o c y t e 4 -26 + 1 3 * 14 + 6* Salmo clarki — s e x u a l l y m a t u r e e r y t h r c o y t e 4 14 + 13 15 + 11 Catostomus mac roc heilus e r y t h r o c y t e 6 -19 +. 7* -17 + 10* Ictalurus nebulosus e r y t h r o c y t e 2 -32 + 8* 22 + 18 Xiphister atropurpureus e r y t h r o c y t e 6 -49 + 17* -88 + 8* Pimia cal va e r y t h r o c y t e 4 7 + 3* 5 + 4 g i l l 4 9 + 8 15 + 6* 79 u s e d „ S i n c e d i f f us:i. ve p r o c e s s e s a r e n o t i mpor t an t - i n t h e e s t e r a s e a s s a y , s u c h p l a s m a i n h i b i t i o n c o u l d n o t h a v e been t h e r e s u l t of d i - f - f u s i o n - r e l a t e d a r t i f a c t s (Heminq and R a n d a l l 1 9 8 2 ) . M o r e o v e r , p l a s m a of a l l s e x u a l l y m a t u r e S a l mo s p . and of t h e h o l o e s t e a n f i s h fimia calva e i t h e r had no e f f e c t . o r s l i g h t l y e l e v a t e d t h e m e a s u r e d CA a c t i v i t i e s o f t i s s u e h o m o g e n a t e s ( T a b l e 7 ) . P l a s m a of t h o s e f i s h must h a v e l a c k e d a f u n c t i o n a l i n h i b i t o r o f CA. 80 DISCUSSION E a c h o-f t h e t h r e e CA a s s a y t e c h n i q u e s t e s t e d p r o v i d e d v a l i d m e a s u r e m e n t s o-f CA a c t i v i t y . . The p H - s t a t a s s a y was j u d g e d t o be t h e more - f a v o u r a b l e f o r u s e w i t h t i s s u e h o m o g e n a t e s , h o w e v e r , b e c a u s e i t e x h i b i t e d t h e lowest, l i m i t o f d e t e c t i o n and t h e h i g h e s t d e g r e e o f s e n s i t i v i t y ( F i g . 1 3 ) . I t a l s o p e r f o r m e d w e l l a t p h y s i o l o g i c a l l e v e l s o f pl~l, t e m p e r a t u r e and b i c a r b o n a t e c o n c e n t r a t i o n . M o r e o v e r , i t . d i r e c t l y m e a s u r e d t h e r a t e o f b i c a r b o n a t e d e h y d r a t i o n . On t h e o t h e r h a n d , t h e m o d i f i e d b o a t a s s a y m e a s u r e d t h e r a t e o f C 0 S e v o l u t i o n i n a c l o s e d v e s s e l a n d , c o n s e q u e n t l y , was dependent, upon t h e d i f f u s i v e e q u i l i b r i u m o f CO^ between an a q u e o u s and a g a s e o u s p h a s e . As w e l l , s i n c e C 0 a l e v e l s were a l l o w e d t o i n c r e a s e i n t h e r e a c t i o n m i x t u r e o f t h e m o d i f i e d b o a t a s s a y , t h e m e a s u r e d r e a c t i o n r a t e was d e p e n d e n t upon t h e o p p o s e d C O s s H C 0 3 - e q u i l i b r i u m , t h a t i s , t h e d i f f e r e n c e b e t w e e n b i c a r b o n a t e d e h y d r a t i o n and s i m u l t a n e o u s COa h y d r a t i o n . In c o n t r a s t , t h e p H - s t a t a s s a y m e a s u r e d t h e u n o p p o s e d f i r s t order-r e a c t i o n . The d e c r e a s e i n s e n s i t i v i t y o f t h e m o d i f i e d b o a t a s s a y a t h i g h e r t e m p e r a t u r e s ( F i g . 12) p r o b a b l y r e s u l t e d f r o m t h e i n a b i l i t y o f t h e s h a k i n g p r o c e s s t o e n s u r e r a p i d d i f f u s i v e e q u i l i b r i u m o f CO® a t t h e h i g h e r r e a c t i o n r a t e s , c o u p l e d w i t h t h e d i f f e r e n t i a l e f f e c t o f t e m p e r a t u r e on d e h y d r a t i o n and h y d r a t i o n r a t e c o n s t a n t s ( E d s a l l 1969) w h i c h f a v o u r s COa h y d r a t i o n a t h i g h e r t e m p e r a t u r e s . B i r a u d (1981) was a b l e t o i n c r e a s e t h e s e n s i t i v i t y o f t h e m o d i f i e d b o a t a s s a y t o l e v e l s c o m p a r a b l e t o 81 t h o s e of t h e p r e s e n t p H - s t a t a s s a y by m a x i m i z i n g t h e s u r f a c e t o v o l u m e r a t i o o f t h e r e a c t i o n m i x t u r e t h r o u g h u s e o f s m a l l r e a g e n t v o l u m e s and a. s m a l l r e a c t i o n v e s s e l , and by c o n d u c t i n g t h e a s s a y a t 0 C. An i n h e r e n t a d v a n t a g e of t h e m o d i f i e d b o a t a s s a y , h o w e v e r , i s i t s p o t e n t i a l f o r u s e w i t h i n t a c t c e l l s ( C h a p t e r 3 t h i s s t u d y , B o o t h 1938a, l i a r e n 1967, H a s w e l l and R a n d a l l 1976, H a s w e l l et al. 1978, Heming and R a n d a l l 1 9 8 2 ) . The e s t e r a s e a s s a y p o s s e s s e d a w i d e r u s a b l e r a n g e of enzyme c o n c e n t r a t i o n s t h a n e i t h e r t h e m o d i f i e d b o a t o r p H - s t a t a s s a y ( F i g . 1 1 ) , and was by f a r t h e s i m p l e s t o f t h e t h r e e t e c h n i q u e s t o u s e . However, i t was c h a r a c t e r i z e d by an e x t r e m e l y s l o w r e a c t i o n r a t e and a s a c o n s e q u e n c e had p o o r d e t e c t i o n l i m i t s and s e n s i t i v i t y , e s p e c i a l l y a t l o w e r t e m p e r a t u r e s . A r m s t r o n g et al. (1966) h a v e shown t h a t t h e CA c a t a l y s e d r a t e o f pNA h y d r o l y s i s p r o c e e d s a t o n l y IO" 5 5 t i m e s t h e r a t e o f C 0 3 h y d r a t i o n . . The i n a b l i l i t y o f t h i s a s s a y t o d e t e c t s m a l l q u a n t i t i e s o f c a r b o n i c a n h y d r a s e h a s been c i t e d a s a p o s s i b l e e x p l a n a t i o n f o r t h e f i n d i n g o f M a s h i t e r and Morgan (1975) t h a t e r y t h r o c y t e s o f f l o u n d e r (Platzchthys fleas) l a c k CA a c t i v i t y ( H a s w e l l 1 9 7 7 ) . N o n e t h e l e s s , t h e e s t e r a s e a s s a y d o e s p r o v i d e an e x c e l l e n t c o n t r o l t e c h n i q u e f o r t h e o t h e r two a s s a y s s i n c e , u n l i k e t h e o t h e r two t e c h n i q u e s , d i f f u s i v e p r o c e s s e s a r e n o t i m p o r t a n t i n t h e e s t e r a s e a s s a y u The u n c a t a l y s e d d e h y d r a t i o n of b i c a r b o n a t e was s u r p r i s i n g l y s l o w u n d e r t h e c o n d i t i o n s t e s t e d w i t h h a l f - t i m e s f o r t h e u n o p p o s e d r e a c t i o n i n t h e r a n g e of 3.8 t o 105.0 min ( F i g . 14),, Swenson and Maren (1978) r e p o r t a h a l f - t i m e o f 1.6 min ( k H c o 3 = 0.0073 s""1) a t 37 C, pH 7.1 and 1-0.15. R e c a l c u l a t i o n o f 82 K n o c h e ' s d a t a (1980)., u s i n g t h e r e l a t i o n k H c o s = k H 2 C D 3 / 1 0 f H " P K (Swenson and Maren 1 9 7 8 ) , y i e l d s a h a l f - t i m e o f 1.7 min ( k H c o s -0.0070 s~*) a t 25 C, pH 7.1 and 1=0. An A r r h e n i u s p l o t ( l o g a r i t h u m of k H o o s v e r s u s r e c i p r o c a l o f a b s o l u t e t e m p e r a t u r e ) of t h e p r e s e n t d a t a ( F i g . 14) a t pl-1 7.10 p r e d i c t s h a l f - t i m e s o f 0.8 min ( k H C o 3 -- 0.0136 s ™ 1 ) a t 37 C and 2.0 min ( k H C o 3 = 0.0057 s " 1 ) a t 25 C. T h e s e v a l u e s a r e i n good a g r e e m e n t w i t h t h o s e of Swenson and Maren (1978) and K noche (1980) c o n s i d e r i n g t h e l e n g t h of t e m p e r a t u r e e x t r a p o l a t i o n i n v o l v e d and t h e d i f f e r e n c e s i n i on i c: s t r eng t h. The u n c a t a l y s e d H C O 3 " " d e h y d r a t i o n r e a c t i o n o c c u r r e d f a r t o o s l o w l y t o account, f o r t h e o b s e r v e d a r t e r i a l - v e n o u s d e c r e a s e i n p l a s m a b i c a r b o n a t e a c r o s s t h e t r o u t g i l l (Tabbies 2 and 5 ) . The h a l f — t i m e f o r u n o p p o s e d H C 0 3 ~ d e h y d r a t i o n ( H C 0 3 ~ — > CO^) a t p h y s i o l o g i c a l pH v a l u e s of t r o u t b l o o d (7.75 8.00) v a r i e d f r o m 22 t o 105 min ( F i g . 1 4 ) . Ey way o f c o m p a r i s o n , t h e r e s i d e n c e t i m e of b l o o d i n t h e g i l l r a n g e s f r o m a maximum o f 3 s a t r e s t t o a b o u t 0.5 s d u r i n g e x e r c i s e (Cameron and P o l h e m u s 1974, R a n d a l l 1 9 8 2 a ) . The c o m p l e t e c i r c u l a t i o n t i m e i n r a i n b o w t r o u t a t r e s t i s o n l y a b o u t 48 t o 96 s ( D a v i s 1 9 7 0 ) . However, when- e x a m i n i n g b i o l o g i c a l s y s t e m s t h e a p p o s e d r e a c t i o n <HCQ 3~ # CDs*) must be t a k e n i n t o a c c o u n t b e c a u s e o f t h e a p p r e c i a b l e amount o f f r e e C0 J 2 p r e s e n t i n b l o o d . The h a l f - t i m e ( t i / 2 ) f o r H C 0 3 ~ d e h y d r a t i o n i n an o p p o s e d C0s»: HCO3"" e q u i l i b r i u m was c a l c u l a t e d a s (10) t t / 2 = I n 2 / ( k C D 2 + kowC0H~3 + a k ' H a c o a ; + a' k Hco;s) 83 where a and a' a r e t h e - f r a c t i o n s o f bound C O ^ p r e s e n t a s HH>CQ 3 and H C 0 . 3 ™ r e s p e c t i v e l y ( K e r n i 9 6 0 ) . The v a r i a b l e s a and a' were assumed t o be 0 and 1 r e s p e c t i v e l y , s i n c e t h e r a t i o EHCuasTl t o ( C f r e e C D = 3 •+• C H A C 0 3 3 ) i n b l o o d i s a b o u t 2 0 : 1 ( K n o c h e 1 9 8 0 ) . V a l u e s o-f kCo=> and k 0 M a t 1=0.15 were o b t a i n e d f r o m P i n s e n t et al . ( 1 9 5 6 ) . The b i o l o g i c a l a p p l i c a b i l i t y o f t j . / 2 v a l u e s c a l c u l a t e d i n t h i s way i s open t o q u e s t i o n . F o r i n s t a n c e , t h e c a l c u l a t i o n s i g n o r e t h e f o r m a t i o n o f C O n s 3 5 - and c a r b a m i no compounds, t h e c a t a l y s i s o f COJB h y d r a t i o n by o x y a n i o n s ( e g . i - l P O ^ . 2 " " , H s - P O - * - ) ( E d s a l l 1 9 6 9 ) , and t h e i n v e r s e r e l a t i o n s h i p b e t w e e n k C D 2 and b u f f e r i n g c a p a c i t y ( G r a y 1 9 7 1 ) . N o n e t h e l e s s , g i v e n t h e c h e m i c a l c o m p l e x i t y o f f i s h b l o o d , t h e s e t i / s v a l u e s ( T a b l e 8) p r o b a b l y a r e t h e b e s t e s t i m a t e s a v a i l a b l e . H a l f - - t i m e s f o r H C O 3 " d e h y d r a t i o n i n 'an o p p o s e d c a r b o n d i o x i d e ! b i c a r b o n a t e e q u i l i b r i u m ( T a b l e 8) were much s m a l l e r t h a n t h o s e o f t h e u n o p p o s e d r e a c t i o n ( F i g . 14) b u t s t i l l e x c e e d e d t h e maximum l a m e l l a r b l o o d t r a n s i t t i m e (3 s a t r e s t , R a n d a l l 1982a) by more t h a n 17 f o l d . Hence, e x c r e 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 g i l l t r a n s i t by way o f t h e u n c a t a l y s e d c o n v e r s i o n o f b i c a r b o n a t e t o c a r b o n d i o x i d e p r o b a b l y i s n e g l i b l e u n d e r n o r m a l c i r c u m s t a n c e s . Red c e l l , g i l l and s w i m b l a d d e r i n v a r i a b l y c o n t a i n e d c a r b o n i c a n h y d r a s e ( T a b l e 6 ) , i n k e e p i n g w i t h t h e w e l l d o c u m e n t e d r o l e o f t h e enzyme i n C 0 3 e x c r e t i o n , i o n t r a n s p o r t and g a s g l a n d f u n c t i o n i n g (Maetz 1956a, F a n g e 1966, Maren 1967, C a r t e r 1 9 7 2 ) . F i s h s k i n , l i k e t h a t o-f many a m p h i b i a n s (Maren 1967, S m i t h 1974, Toews et al. 1 9 7 8 ) , l a c k e d d e t e c t a b l e c a r b o n i c a n h y d r a s e . I n t e r m s o f d i s t r i b u t i o n and a c t i v i t y l e v e l s , t h e p r e s e n t r e s u l t s a r e i n good a g r e e m e n t w i t h t h o s e of Maren (1967) and H o u s t o n and 84 T a b l e 8. H a l f - t i m e ( s e c ) -for H C 0 3 " d e h y d r a t i o n i n an u n c a t a l y s e d o p p o s e d c a r b o n d i o x i d e 2 b i c a r b o n a t e e q u i l i b r i u m i n a q u e o u s s o l u t i o n , s h o w i n g t h e e f f e c t s o f pH and t e m p e r a t u r e . See t e x t f o r d e t a i l s , A d a t a o f K e r n ( i 9 6 0 ) a t 1=0. , s , d a t a o f p r e s e n t s t u d y a t 1=0.15. T e m p e r a t u r e (C) 0* 10* 7.00 240 136 82 48 26 7.50 - 165 91 55 8.00 300 165 90 52 25 85 M c C a r t y ( 1 9 7 8 ) , T e l e o s t b l o o d i n t h e p r e s e n t s t u d y c o n t a i n e d s u f f i c i e n t CA. t o a c c e l e r a t e b i c a r b o n a t e d e h y d r a t i o n by some 2115 t o 343 t i m e s p e r mL b l o o d - T h i s e x c e e d s t h e c a t a l y t i c p o t e n t i a l d e t e r m i n e d by H a s w e l l and R a n d a l l (1976) w i t h t h e m o d i f i e d b o a t a s s a y (-60 90 f o l d p e r mL b l o o d ) , w h i c h i s t o be e x p e c t e d c o n s i d e r i n g t h e d i f f e r e n c e s i n s e n s i t i v i t y b e t w e e n t h e m o d i f i e d b o a t and p H - s t a t a s s a y s . T e l e o s t b l o o d , t h e r e f o r e , h a s o n l y s l i g h t l y l e s s c a t a l y t i c p o t e n t i a l t h a n mammalian b l o o d ; mammalian b l o o d i s c a p a b l e o f a c c e l e r a t i n g C0_a r e a c t i o n s by some 440 t o 800 f o l d , a s s u m i n g an h a e m a t o c r i t o f 407. (Maren 1 9 6 7 ) . The - s w i m b l a d d e r o f t h e b o w f i n , an o r g a n w h i c h i s a d a p t e d f o r a e r i a l r e s p i r a t i o n , c o n t a i n e d a p p r o x i m a t e l y 2.5 t i m e s t h e CA a c t i v i t y o f t h e n o n r e s p i r a t o r y s w i m b l a d d e r o f r a i n b o w t r o u t ( T a b l e 6 ) . B o w f i n s d i f f e r i n t h i s r e s p e c t f r o m Calamoichthys calabancus., t h e o n l y o t h e r b i modal l y b r e a t h i n g f i s h i n w h i c h t h e CA a c t i v i t y o f a r e s p i r a t o r y s w i m b l a d d e r h a s been measured? C. calabancus s w i m b l a d d e r l a c k s d e t e c t a b l 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 (Efurggren and H a s w e l l 1 9 7 9 ) . The p r o p o s a l , t h e r e f o r e , t h a t " t h e e p i t h e l i u m o f t h e a i r — b r e a t h i n g o r g a n (of f i s h ) d o e s n o t c o n t a i n c a r b o n i c a n h y d r a s e e x c e p t i n t h o s e o r g a n s d e r i v e d f r o m g i l l t i s s u e " ( R a n d a l l et al. 1981) must be r e c o n s i d e r e d . CDs e x c r e t i o n a c r o s s t h e a i r - b r e a t h i n g o r g a n s o f f i s h a p p e a r s t o be l i m i t e d d u r i n g a i r e x p o s u r e t o t h e u n c a t a l y s e d r a t e of b i c a r b o n a t e d e h y d r a t i o n ( R a n d a l l et al. 1978, Daxboeck and Heming 1 9 8 2 ) . The p r e s e n t f i n d i n g s i n d i c a t e t h a t f a c t o r s o t h e r t h a n t h e p r e s e n c e o r a b s e n c e o f e p i t h e l i a l CA must be i n v o l v e d i n t h i s r e s p o n s e . E p i t h e l i a l c a r b o n i c a n h y d r a s e i n mammalian t i s s u e s h a s 86 been i m p l i c a t e d i n a t t a i n m e n t o f c h e m i c a l e q u i l i b r i u m i n b l o o d d u r i n g c a p i l l a r y t r a n s i t ( C r a n d a l l and O ' B r a s k y 1978, E f f r a s et al . 1978, K l o c k e 1978) and i n - f a c i l i t a t i o n o f CO a d i f f u s i o n ( K a w a s h i r o and S c h e i d 1976)-" p e r h a p s t h e CA i n r e s p i r a t o r y s w i m b l a d d e r s h a s a s i m i l a r r o l e . The f u n c t i o n a l p r e s e n c e o f p l a s m a i n h i b i t o r s o f c a r b o n i c a n h y d r a s e was b o t h s p e c i e s - s p e c i f i c and t e m p o r a l l y - s p e c i f i c ( T a b l e 7 ) . A l l t e l e o s t e a n f i s h e s e x a m i n e d p o s s e s s e d e n d o g e n o u s CA i n h i b i t o r s d u r i n g a t l e a s t some p o r t i o n o f t h e i r l i f e h i s t o r y . The i n h i b i t o r was a b s e n t i n s e x u a l l y m a t u r e i n d i v i d u a l s , a l t h o u g h o n l y s e x u a l l y m a t u r e s a l m o n i d s were a v a i l a b l e f o r s t u d y . Pimia calva a l s o l a c k e d an i n h i b i t o r when e x a m i n e d . However, c o n s i d e r i n g t h e t e m p o r a l v a r i a t i o n s ; f o u n d i n t h e t e l e o s t i n h i b i t o r , one c a n n o t c o n c l u d e t h a t t h e a b s e n c e o f an i n h i b i t o r i n Omia p l a s m a i s a p e r m a n e n t c o n d i t i o n . P l a s m a i n h i b i t o r s o f CA ha v e been known f o r some t i m e . I n h i b i t o r s h a v e been f o u n d i n t h e p l a s m a o f a v a r i e t y o f f i s h and mammals ( B o o t h 1938b, l i a e t z 1956b, L e i n e r et al, 1962) and more r e c e n t l y i n humans (E.D. C r a n d a l l , p e r s o n a l c o m m u n i c a t i o n ) . E v i d e n c e o f an i n h i b i t o r i n o t h e r v e r t e b r a t e p h y l a h a s n o t been f o u n d . P l a s m a i n h i b i t o r s o f CA a r e b e l i e v e d t o be g l o b u l a r p r o t e i n s ( B o o t h 1938b, L e i n e r et al„ 1962, H a s w e l l and R a n d a l l 1 9 7 6 ) , a l t h o u g h d e f i n a t i v e c h a r a c t e r i z a t i o n o f t h e i n h i b i t o r h a s n o t been p u b l i s h e d . Hence, t e m p o r a l v a r i a t i o n s i n t h e t e l e o s t i n h i b i t o r may h a v e r e s u l t e d f r o m v a r i a t i o n s i n p l a s m a p r o t e i n c o m p o s i t i o n . The e l e c t r o p h o r e t i c p r o t e i n p a t t e r n o f r a i n b o w t r o u t p l a s m a h a s been o b s e r v e d t o v a r y q u a l i t a t i v e l y ( H a i d e r 1970, B o r c h a r d 1978) and q u a n t i t a t i v e l y ( S c h l o t f e l d t 1975) d u r i n g t h e c o u r s e o f 87 d e v e l o p m e n t . However, p l a s m a p r o t e i n p a t t e r n s a l s o a r e i n f l u e n c e d by s t o c k d i f f e r e n c e s ( L a n g h o l t z and D i n k l a g e 1 9 7 0 ) , s t a r v a t i o n ( K a w a t s u 1 9 7 4 ) , s e a s o n a l t e m p e r a t u r e v a r i a t i o n s ( S c h l o t f e l d t 1 9 7 5 ) , and d i s e a s e ( S n i e s z k o et a l . 1966, L o w e - J i n d e 1 9 7 9 ) . The p o s s i b i l i t y t h a t , one o r more of t h e s e l a t t e r f a c t o r s i n f l u e n c e d t h e p r e s e n t r e s u l t s c a n n o t be r u l e d o u t c o m p l e t e l y , a l t h o u g h s i g n s o f d i s e a s e and s t a r v a t i o n were n o t e v i d e n t . What p o s s i b l e a d v a n t a g e s w o u l d be c o n f e r r e d by t e m p o r a l v a r i a t i o n o f p l a s m a i n h i b i t i o n o f CA? E<ooth (1938b) o r i g i n a l l y p r o p o s e d t h a t p l a s m a i n h i b i t o r s o f CA f u n c t i o n e d t o i m m o b i l i z e c a r b o n i c a n h y d r a s e r e l e a s e d i n t o t h e p l a s m a f r o m r e d c e l l s d u r i n g t h e c o u r s e o f t h e c e l l s " n o r m a l d e s t r u c t i o n o r d u r i n g i n j u r y . The p r e s e n t s t u d y s u p p o r t s B o o t h s ' p r o p o s a l i n a s much a s t h e p l a s m a o f some i n d i v i d u a l s n o t o n l y l a c k e d an i n h i b i t o r , b u t a l s o i n c r e a s e d t h e me a s u r e d c a r b o n i c a n h y d r a s e a u r t i v i t y o f t i s s u e h o m o g e n a t e s ( T a b l e 7 ) . S i n c e e n d o g e n o u s a c t i v a t o r s o f CA a r e d o u b t f u l ( C l a r k and P e r r i n 1 9 5 1 ) , t h e p l a s m a o f t h o s e i n d i v i d u a l s p r o b a b l y c o n t a i n e d f r e e CA. T h i s p l a s m a a c t i v i t y c o u l d n o t be a t t r i b u t a b l e t o h a e m o l y s i s d u r i n g s a m p l e p r e p a r a t i o n s i n c e no h a e m o l y s i s was d e t e c t e d , , The aid v a n t a g e of h a w i n g CA c o n f i n e d i n s i d e e r y t h r o c y t e s i s t h a t t h e j u x a p o s i t i on o f C0__: HCQs-c o n v e r s i o n and h a e m o g l o b i n f a c i l i t a t e s E<ohr and R o o t e f f e c t s . The p r e s e n c e o f CA i n p l a s m a c a n c e l vaxbly w o u l d n e g a t e t h i s a d v a n t a g e and s o m i g h t be d i s a d v a n t a g e o u s . However, u n d e r s u c h c i r c u m s t a n c e s , t h e i n t r a c e l l u l a r r e a c t i o n s t i l l w o u l d be e x p e c t e d t o d o m i n a t e b e c a u s e o f t h e g r e a t e r b u f f e r i n g c a p a c i t y o f r e d c e l l s o v e r p l a s m a . The p r e s e n c e o f e x t r a c e l l u l a r CA d u r i n g 88 p e r i o d s of d e c r e a s e d i n h i b i t o r a c t i v i t. y w o u l d - f a c . i l i t. a t e a t t a i n m e n t o f c h e m i c a l e q u i l i b r i u m b e t w e e n r e d c e l l s and p l a s m a d u r i n g t r an s i t t h r oug h p u 1 mon a r y an d t i s s u e c ap i 11 a r i e s (Cr an d a .11 and O ' B r a s k y 1978, E f f r o s et al , 1978, K l o c k e 1 9 7 8 ) , S e v e r a l s t u d i e s h a v e c l a i m e d t h a t t h e i n h i b i t o r i s a c t i v e a g a i n s t c a r b o n i c a n h y d r a s e i n s i d e i n t a c t e r y t h r o c y t e s (Maetz 1956b, H a s w e l l and R a n d a l l 1976) and t h u s v a r i a t i o n s i n i n h i b i t o r a c t i v i t y m i g h t m o d u 1 a t e i n t r a c e l l u 1 a r C A a c t i v i t y. H o w e v e r ,, m o r e r e c e r i t s t u d i e s h a v e been unab 1 63 t o c o n f i rm t h o s e e a r I i e r r e s u 11s (Cameron 1978b, • b a i d et aI„ 1 9 7 9 ) , and t h e v a 1 i d i t y of t h e o r i g i n a l t e c h n i q u e u s e d h a s been q u e s t i o n e d (Heming and R a n d a l l 1 9 8 2 ) , An other" p a s s i b i 1 i t y i s t h a t t he i n h i b i t o r s mod u 1 a t e t. h e a c t i v i t y o-f e n d o t h e l i a l CA, s u c h a s i s p r e s e n t i n t h e c a p i l l a r i e s o f v e r t e b r a t e l u n g s ( C r a n d a l l and O ' B r a s k y 1978, E f f r o s e t al. 1978, K l o c k e 1 9 7 8 ) , The f u n c t i o n s o f t h i s e n d o t h e l i a l CA a r e u n c l e a r a t p r e s e n t , I n c o n c 1 u s i o n , t h e un c a t a 1 y s e d r a t e of CO.^: l-ICOr*"" c on v e r s i on o c c u r s t o o s l o w l y u n d e r t h e p h y s i o l o g i c a l c o n d i t i o n s of f i s h b l o o d t o a c c o u n t f o r t h e o b s e r v e d a r t e r i a l — v e n o u s d i f f e r e n c e i n p1 asma b i c a r b o n a t e . I n s t ead , t h e i n t e r c o n v e r s i o n o f p1 a sma HC0 3"" and C D s o c c u r s p r e d o m i n a n t l y i n s i d e t h e e r y t h r o c y t e s ( C h a p t e r 1 ) , w h i c h c o n t a i n s u f f i c i e n t c a r b o n i c a n h y d r a s e t o a c c e l e r a t e COs-U H C O c s " r e a c t i o n s by s e v e r a l o r d e r s o f m a g n i t u d e . Movement, of H C O 3 - between, r e d c e l l s and p l a s m a i s e x a m i n e d i n t h e f a l l o w i n g c h a p t e r . CA a l s o i s p r e s e n t , i n t h e e p i t h e l i u m o f t h e g i l l s and s w i m b l a d d e r . However, t h i s e p i t h e l i a l CA p l a y s o n l y a m i n o r r o l e i n c a t a l y s i n g t h e d e h y d r a t i o n of p l a s m a H C Q 3 ~ , i n a s much a s t h e b a s a l a t . e r a l membrane o f t h e g i l l i s l a r g e l y i m p e r m e a b l e t o H C D 3 ~ 89 ( P e r r y et a l . 19B2) . F i s h p l a s m a c o n t a i n s an i n h i b i t o r o-f CA w h i c h p r o b a b l y f u n c t i o n s t o s u p p r e s s CA r e l e a s e d f r o m e r y t h r o c y t e s d u r i n g t h e i r n o r m a l d e s t r u c t i o n . The a c t i v i t y o f t h i s i n h i b i t o r shows t e m p o r a l v a r i a t i o n s and p e r h a p s s p e c i e s v a r i a t i oris,, 90 C h a p t e r 3. ION TRANSPORT AND E Q U I L I B R I A ACROSS THE MEMBRANE OF SALMO GAIRDNERI ERYTHROCYTES The s l o w e s t s t e p i n c a p i l l a r y C 0 2 e x c h a n g e i s HC0 3""/C1~ t r a n s p o r t a c r o s s t h e r e d c e l l membrane ( F o r s t e r and C r a n d a l l 1975, Chow e t al. 1977. C r a n d a l l and B i d a n i 1981, F o r s t e r 1 9 8 2 ) . Even t h i s step,, h o w e v e r , i s r a p i d w i t h a h a l f - t i m e i n mammalian e r y t h r o c y t e s a t 37 C o f a b o u t 0.1 s ( D a i m a r k 1972, K l o c k e 1976, Chow e t al„ 19 77, O b a i d and C r a n d a l l 1 9 7 9 ) . A l t h o u g h t h e m e c h a n i c s and k i n e t i c s o f a n i o n t r a n s p o r t i n f i s h e r y t h r o c y t e s h a v e r e c e i v e d s c a n t a t t e n t i o n , t h e y h a v e been s t u d i e d e x t e n s i v e l y i n mammalian r e d c e l l s . A n i o n e x c h a n g e i n mammalian r e d c e l l s i n v o l v e s a c a r r i e r - m e d i a t e d t r a n s p o r t m e chanism (Gunn e t ai, 1973, D a i m a r k 1976, Chow et al. 1977, C r a n d a l l et al. 1978) w h i c h i s a s s o c i a t e d w i t h a t r a n s m e m b r a n e p r o t e i n c h a n n e l , t h e Band 3 a n i o n e x c h a n g e p a t h w a y ( R o t h s t e i n e t al„ 1976, 1978, C a b a n t c h i k e t a l * 197E!) , T h i s c a r r i e r s y s t e m i s i n h i b i t e d s e l e c t i v e l y by d i s u l p h o n i c s t l i b e r i e d e r i v a t i v e s l i k e DIDS and S I T S ( C a b a n t c h i k and R o t h s t e i n 1972, Gunn e t al, 1973, R o t h s t e i n e t al, 1 9 7 6 ) . F o r t h e most p a r t , t h e t r a n s p o r t m e chanism i s a o n e - f a r one e x c h a n g e o f a n i o n s f r o m one s i d e o f t h e c e l l membrane t o t h e o t h e r . The c a r r i e r s y s t e m a l s o i s c a p a b l e o f n e t a n i o n movements (Gunn 1 9 7 7 ) , a l b e i t a t r a t e s a p p r o x i m a t e l y 1 0 3 t i m e s s l o w e r t h a n t h o s e of a n i o n e x c h a n g e (Knauf e t al. 1 9 7 7 ) . T h e r e i s c o n f l i c t i n g e v i d e n c e a s t o t h e a p p l i c a b i l i t y o f t h i s t r a n s p o r t . 91 mechanism t o a n i o n movements i n f i s h r e d c e l l s . S e v e r a l a u t h o r s h ave d e m o n s t r a t e d t h a t t e l e o s t and e l a s m o b r a n c h e r y t h r o c y t e s p o s s e s s a HC03~"/C1"" e x c h a n g e r t h a t i s c o m p a r a b l e t o t h a t i n mammalian r e d c e l l s (Cameron 1978b, O b a i d et al * 1979)., S i m i l a r l y , H a s w e l l et al „ (1978) f o u n d t h a t CI '"'/acetate e x c h a n g e i n Tilapia e r y t h r o c y t e s i s c o m p a r a b l e t o t h a t i n human r e d c e l l s . H owever, H a s w e l l (1978) h a s s t a t e d t h a t CI™ s e l f - 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 s u n a f f e c t e d by S I T S - - i n h i b i t i o n of t h e Band 3 a n i o n exchange? p a t h w a y . M o r e o v e r , s t u d i e s o f t h e CA a c t i v i t y o f i n t a c t f i s h r e d c e l l s h a v e s u g g e s t e d t h a t f i s h p l a s m a c o n t a i n s a f a c t o r w h i c h r e n d e r s f i s h r e d c e l l s f u n c t i o n a l l y i m p e r m e a b l e t o HCG 3~ in vivo ( H a s w e l l and R a n d a l l 1976, H a s w e l l et al, 1 9 7 8 ) . The r o l e o f t h e r e d c e l l i n CGS. e x c h a n g e u l t i m a t e l y w i l l be i n f l u e n c e d by any f a c t o r w h i c h a l t e r s e i t h e r a n i o n e x c h a n g e a c r o s s t h e c e l l membrane o r t h e a c t i v i t y of c e l l u l a r CA. A d r e n a l i n e , f o r e x a m p l e , h a s been shown t o a c t i v a t e s t h e c a r b o n i c a n h y d r a s e i n a v i a n e r y t h r o c y t e s and t h u s may e n h a n c e C 0 E e x c r e t i o n d u r i n g s t r e n u o u s f l i g h t ( Siegmund et al. 1 9 7 4 ) . As w e l l , b e t a - a d r e n e r g i c a g e n t s h a v e been d e m c j n s t r a t e c i t o s t i m u l a t e i o n t r a n s p o r t and t o i n d u c e s w e l l i n g i n e r y t h r o c y t e s of f i s h ( M i k i n m a a 19E32, 1 9 8 3 ) , a m p h i b i a n s ( R u d o l p h and G r e e n g a r d 1980),, b i r d s ( R i d d i c k et al. 1971, Kregenow 1973, A l p e r et ai. 1980, P a l f r e y et al. 1 9 8 0 ) , and mammals (Rasmussen et al„ 1975, Q u i s t and R o u f o g a l i s 1 9 7 7 ) . B o t h a d r e n e r g i c s w e l l i n g and n o n - h o r m o n a l volume r e g u l a t o r y i o n movements a r e a s s o c i a t e d w i t h n e t t r a n s p o r t of a n i o n s v i a m e c h a n i s m s s u c h a s Na*/IC""/CI~ c o t r a n s p o r t (Kregenow 1973, A l p e r et al . 1980) o r c o u p l e d a l k a l i m e t a l / H * , Cl"/HCuV"-92 e x c h a n g e r s ( C a l a 1 9 8 0 ) - A d r e n e r g i c r e s p o n s e s o f e r y t h r o c y t e s a l s o i n c l u d e a d e c r e a s e i n c e l l u l a r o r g a n i c p h o s p h a t e l e v e l s ( N i k i n m a a 1983, N i k i n m a a et a l « 1984) and an i n c r e a s e i n c e l l u l a r Ca2;"'" l e v e l s (Rasmussen et al . 1 9 7 4 ) . B o t h d e p l e t i o n o-f c e l l u l a r o r g a n i c p h o s p h a t e s and i n c r e a s e d c e l l u l a r Ca 3*' haive been f o u n d t o d e c r e a s e a n i o n p e r m e a b i 1 i t i y i n r e d c e l l s v i a s t r u c t u r a l r e a r r a n g e m e n t o f t h e membrane ( M o t a i s et al . 1981)., E f f e c t s of c a t e c h o l a m i n e s on a n i o n t r a n s p o r t and on t h e c e l l u l a r CA a c t i v i t y o f f i s h r e d c e l l s h a v e n o t b een i n v e s t i g a t e d p r e v i o u s l y . The o b j e c t i v e s o f s t u d i e s i n t h i s c h a p t e r were t o e x a m i n e a n i o n t r a n s p o r t and t h e c e l l u l a r CA a c t i v i t y o f e r y t h r o c y t e s of Sal mo g a i r d n e r i . C h l o r i d e t r a n s p o r t was f o l l o w e d u s i n g " ^ c h l o r i d e a s a t r a c e r . B i c a r b o n a t e t r a n s p o r t was m o n i t o r e d by e x a m i n i n g t h e c a p a c i t y o f i n t a c t r e d c e 11 s f o r c a t ax 1 y s i n g 11 "i e deh y d r at. :i. on o f ex t r a c e 11 u 1 a r l-lC0 3-. T h i s r e a c t i an i n vo 1 v e d i n f l u x o f ex t r a c e l l u l a r H C 03"!" i n t o t h e c e 11 ,, c a t a 1 y s e d H C 0 3 -• d e h y d r a t i o n and d i f f u s i o n o f t h e r e s u l t a n t CCfe o u t o f t h e c e l l . B i c a r b o n a t e t r a n s p o r t i n t o t h e r e d c e l l was p r e s u m e d t o be t h e r a t e l i m i t i n g s t e p i n t h i s r e a c t i o n . The s e n s i t i v i t y o f c h l o r i d e and b i c a r b o n a t e t r a n s p o r t t o SITS;, a c e t a z o l ami de and b l o o d p l a s m a were e l u d i c a t e d . As w e l l , t h e r e s p o n s e of f i s h e r y t h r o c y t e s t o a d r e n a l i n e was e x a m i n e d i n d e t a i l . 93 MATERIALS AND METHODS A. C h l o r i d e T r a n s p o r t i n Red C e l l s Red c e l l s were o b t a i n e d f r o m b l o o d s a m p l e s w i t h d r a w n f r o m t h e d o r s a l a o r t a s o f c h r o n i c a l l y c a n n u l a t e d r a i n b o w t r o u t ( s e e G e n e r a l M a t e r i a l s and Methods).. These; b l o o d s a m p l e s were d i l u t e d t o t h r e e t i m e s t h e i r o r i g i n a l v o l u m e w i t h i c e - c o l d medium A (medium A: 140.0 mM NaCLii 5.0 mM KC1 5 1.3 mM CaCU-sii 1.2 mM MgClrsj 5.0 mM D - g l u c o s e 5 27.0 mM g l y c y l g l y c i n e j pH t i t r a t e d on i c e t o 7.80 w i t h 1.0 N NaOH). The e r y t h r o c y t e s were washed t w i c e i n i c e c o l d medium A and f i n a l l y were s u s p e n d e d t o 257. h a e m a t o c r . i t i n 5 mL o f medium A, B, C o r D. M e d i a B, C and D c o n s i s t e d o f medium A c o n t a i n i n g iO-= M a c e t a z o l a m i d e , 10~"* M S I T S ( 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 ) and 10 v o l 7. p l a s m a , r e s p e c t i v e l y . P l a s m a u s e d i n medium D had been e q u i l i b r a t e d a t room t e m p e r a t u r e w i t h h u m i d i f i e d room a i r ( P o o s = 0.0337., G l u e c k a u f 1951) and had a CCDH o f l e s s t h a n 2 mM ( s e e C h a p t e r 1 f o r d e t a i l s o f measurement t e c h n i q u e ) . Red c e l l s u s p e n s i o n s were l o a d e d w i t h ~*»ci~ by a d d i t i o n o f 5 uL o f 118 mM N a 3 < s , C l (0.66 mCi/mol , Amersham) and t h e n were a l l o w e d t o e q u i l i b r a t e on i c e f o r a p p r o x i m a t e l y 30 m i n . F i n a l l y , t h e s u s p e n s i o n s were t r a n s f e r r e d t o c h i l l e d , p l a s t i c t u b e s ( h e a t s e a l e d b a r r e l s o f 1-mL t u b e r c u l i n s y r i n g e s ) and c e n t r i f u g e d a t 12,000 g and 1 C f o r - 1 5 min ( S o r v a l s u p e r s p e a d RC2-B c e n t r i f u g e ) . A p p r o x i m a t e l y 0.2 mL of p a c k e d r e d c e l l s were i s o l a t e d f r o m e a c h t u b e by c u t t i n g t h e t u b e b e l o w t h e c e l l - m e d i u m i n t e r f a c e . I t was 94 assumed t h a t t h i s p e l l e t o-f p a c k e d r e d c e l l s c o n t a i n e d 27. t r a p p e d 3 A C 1 ~--I aden medium (Gunn e t a l . 1 9 7 3 ) . P a c k e d r e d c e l l s , a l o n g w i t h a 5 mL r i n s e o-f t h e a p p r o p r i a t e 3 < S ,C1 ~ ~ f r e e medium, were i n j e c t e d i n t o a s h a k i n g t o n o m e t e r f l a s k c o n t a i n i n g 4.E5 mL of t h e same 3 d i C l " " - f r e e medium, t h e r m o s t a t t e d a t 1 C. The r e s u l t i n g h a e m a t o c r i t was b e t w e e n 1 and 27.,, A t known t i m e i n t e r v a l s , 0.. 4 mL s a m p l e s o f t h i s f i n a l s u s p e n s i o n were w i t h d r a w n , l a y e r e d o n t o O.S mL o f i c e - c o l d d i b u t y l p h t h a l a t e and i m m e d i a t e l y c e n t r i f u g e d f o r 30 s e c i n a F i s h e r M i c r o - c e n t r i f u g e (model 2 3 5 ) . Warming o f s a m p l e s d u r i n g t h i s c e n t r i f u g a t i o n was m i n i m i z e d by s t o r i n g t h e c e n t r i f u g e r o t o r i n a f r e e z e r a t -4 C u n t i l j u s t p r i o r t o i t s u s e . 3 < S*CI~" a c t i v i t y i n t h e r e c o v e r e d medium was d e t e r m i n e d u s i n g a N u c l e a r C h i c a g o I s o c a p l i q u i d s c i n t i l l a t i o n c o u n t e r . T h i s a c t i v i t y was c o r r e c t e d f o r t r a p p e d a c t i v i t y and t h e n was e x p r e s s e d a s a 7. r e c o v e r y , w here (11) 7. r e c o v e r y = ( c o u n t s a t t i m e t / c o u n t s a t 30 min) - 100. T h i r t y m i n u t e s c o r r e s p o n d e d t o a\ p e r i o d of t i m e w h i c h was a b o u t •300 t i m e s l o n g e r t h a n t h e e x p e c t e d h a l f - t i m e o f t h e c h l o r i d e e x c h a n g e . In s t u d i e s w i t h S I T S (medium C ) , t h e 30 min v a l u e o f t h e c o n t r o l (medium A) was u s e d i n d e t e r m i n a t i o n o f t h e 7. r e c o v e r y . E x t r a c o r p u s c u l a r h a e m o g l o b i n l e v e l s were m e a s u r e d ( A n t h o n y 1961) t o m o n i t o r h a e m o l y s i s ; none was d e t e c t e d i n any of t h e r e p o r t e d s t u d i e s . 95 Bi~.sLCbona.te T r a n s p o r t . i n . Red Cel_l_s B i c a r b o n a t e t r a n s p o r t i n t r o u t e r y t h r o c y t e s was a s s e s s e d by m e a s u r i n g t h e CA a c t i v i t y o f i n t a c t , r e d c e l l s s u s p e n d e d i n a p h o s p h a t e b u f f e r c o n t a i n i n g NaHC0 3. C a t a l y s i s o f H C O 3 " " d e h y d r a t i o n by i n t a c t , r e d c e l l s was f o l l o w e d manometr i c a l 1 y u s i n g t h e 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 i n C h a p t e r 2. The o n l y d i f f e r e n c e i n t h e d e s i g n o f a s s a y s w i t h i n t a c t c e l l s was t h e i n c l u s i o n o f 50 mM N a C l i n t h e b i c a r b o n a t e s o l u t i o n t o f a c i l i t a t e H C 0 3~:C1~ e x c h a n g e a c r o s s t h e r e d e e l 1 membrane. Two s e r i e s o f e x p e r i m e n t s w e re u n d e r t a k e n : ( i ) s t u d i e s o f t h e e f f e c t o f b l o o d p l a s m a i l ( i i ) s t u d i e s o f t h e e f f e c t o f p h a r m a c e u t i c a l a g e n t s . Red c e l l s ; u s e d i n p l a s m a s t u d i e s ; were o b t a i n e d f r o m b l o o d w i t h d r a w n f r o m t h e c a u d a l b l o o d v e s s e l s o f a n a e s t h e t i z e d r a i n b o w t r o u t ( s e e G e n e r a l M a t e r i a l s and M e t h o d s ) . H a s w e l l and R a n d a l l (1976) s t a t e t h a t a n a e s t h e s i a w i t h MS--222 h a s no e f f e c t on HCO .3™ movements i n i n t a c t t r o u t e r t h y r o c y t e s . Red c e l l s u s e d i n a l l o t h e r s t u d i e s were o b t a i n e d f r o m b l o o d w i t h d r a w n f r o m t h e d o r s a l a o r t a s o f u n a n a e s t h e ' t i z e d c a n n u l a t e d t r o u t . R e g a r d l e s s o f t h e method of s a m p l i n g , a l l b l o o d s a m p l e s were d i l u t e d t o t h r e e times.'' t h e i r o r i g i n a l v o l u m e w i t h i c e - c o l d C o r t l a n d s a l i n e . E r y t h r o c y t e s w e re washed t w i c e i n C o r t l a n d s a l i n e and f i n a l l y w e r e s u s p e n d e d t o 10% h a e m a t o c r i t i n e i t h e r C o r t l a n d s a l i n e ( ' C o r t l a n d b l o o d " ) o r p l a s m a ('whole b l o o d ' ' ) . A l l s a m p l e s were k e p t on i c e u n t i l u s e d . A s s a y s were c o n d u c t e d a t t h e a m b i e n t w a t e r t e m p e r a t u r e (8-11 C)„ In s t u d i e s w i t h p l a s m a , 0.2 mL o f e i t h e r C o r t l a n d b l o o d o r w h o l e b l o o d w e re a s s a y e d a l o n g w i t h 0.1 mL. o f e i t h e r C o r t l a n d s a l i n e , a f o a m i n g a g e n t o r a d e f o a m i n g a g e n t . F o a m i n g a g e n t s 96 u s e d were b o v i n e s e r u m a l b u m i n (5 mg"/., Sig m a Mo. A-9647) and a common c o l l o i d a l o s m o t i c f i l l e r , p o l y v i n y l - p y r r o l i d i none; K-30 (5 mg"/.) . De-foaming a g e n t s u s e d were o c t a n o l (0.5 mL%) and a c o m m e r c i a l s i 1 i c o n e - b a s e d d e f oamer, No—foam (0.2 mL"/., A r g e n t F--1 4 0 2 - S ) . I n p h a r m a c e u t i c a l s t u d i e s , 0.2 mL o f C o r t l a n d b l o o d were a s s a y e d a l o n g w i t h 0.04 mL of e i t h e r C o r t l a n d s a l i n e o r a p h a r m a c e u t i c a l a g e n t . A g e n t s u s e d were ( - ) - a d r e n a l i n e , L-n o r a d r e n a l i n e , an a l p h a - a d r e n e r g i c a n t a g o n i s t p h e n t o l a m i n e ( R e g i t i n e ) , and a b e t a - a d r e n e r g i c a n t a g o n i s t D L - p r o p r a n o I o l HC1. Th e s e r e a g e n t s were p r e p a r e d i n C o r t l a n d s a l i n e w i t h i n 30 min of t h e i r u s e t o e n s u r e h i g h a c t i v i t y . E f f e c t s o f t h e a n i o n t r a n s p o r t . i n h i b i t o r , S I T S (10~^ M) , and t h e CA i n h i b i t o r , a c e t a z o l ami de ( 1 0 - ! S M) , a l s o were e x a m i n e d . Red c e l l s u s e d i n t h e s e s t u d i e s w e re p r e t r e a t e d w i t h t h e a p p r o p r i a t e p h a r m a c e u t i c a l a g e n t f o r a t l e a s t 10 min p r i o r t o t h e i r u s e i n a s s a y s . CA a c t i v i t y o f i n t a c t r e d c e l l s was c o n v e r t e d t o a r b i t r a r y enzyme u n i t s (eu) u s i n g e q u a t i o n 9 ( C h a p t e r 2 ) . The r e a c t i o n r a t e o f C o r t l a n d s a l i n e was t a k e n t o r e p r e s e n t t h e u n c a t a l y s e d r a t e f o r C o r t l a n d b l o o d , w h e r e a s t h e r e a c t i o n - r a t e o f p l a s m a was t a k e n t o r e p r e s e n t t h e u n c a t a l y s e d r a t e f o r w h o l e b l o o d . P l a s m a u s e d i n e x p e r i m e n t s r e p o r t e d i n t h i s s t u d y d i d n o t c o n t a i n c a r b o n i c : a n h y d r a s e ? r e a c t i o n r a t e s o f t h i s p l a s m a were c o n s i s t e n t l y l e s s t h a n t h a t o f C o r t l a n d s a l i n e . Ex t r ac o r p use u l a r h a e m o g l o b i n 1 eve; I s were m e a s u r e d ( A n t h o n y 1S'61) i n a l l r e a c t i o n m i x t u r e s t o m o n i t o r h a e m o l y s i s . No h a e m o l y s i s was d e t e c t e d i n a n y of t h e r e p o r t e d a s s a y s . 97 C. E f f e c t g f A d r e n a l i n e on I o n i c E f l u i M l i b r i a A c r o s s t h e Red C e l l . Membrane EUood s a m p l e s were w i t h d r a w n f r o m t h e d o r s a l a o r t i c c a n n u l a e of s e v e r a l r a i n b o w t r o u t and w e re p o o l e d on i c e . Two-mL a l i q u o t s of p o o l e d b l o o d were e q u i l i b r a t e d a t 10 C w i t h h u m i d i f i e d g a s m i x t u r e s c o n t a i n i n g 0.2, 0.5 and 1.0"/. C O m i n a i r ( W o s t o f f pumps). B l o o d s a m p l e s were g a s s e d f o r 30 min and t h e n 0.05 mL o f e i t h e r s a l i n e (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 0.4 vol"/, w i t h C o r t l a n d s a l i n e , pH 7.71) o r a d r e n a l i n e (10 mg-mL" 1 ( - ) - a d r e n a l i n e i n 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 0.4 v o l 7. w i t h C o r t l a n d s a l i n e , pH 7.69) were a d d e d . 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 w i t h i n 10 min o f i t s u s e . S a m p l e s t h e n were? g a s s e d f o r a n o t h e r 30 min b e f o r e t h e i r r e s p i r a t o r y and a c i d - b a s e s t a t e s were a s s e s s e d . A p o r t i o n o f e a c h e q u i l i b r a t e d b l o o d s a m p l e was us;ed t o m e a sure h a e m a t o c r i t i n q u a d r u p l i c a t e , b l o o d w a t e r c o n t e n t i n d u p l i c a t e and b l o o d C c r ass. A s e c o n d p o r t i o n of e a c h s a m p l e was c e n t r i f u g e d a n a e r o b i c a l 1 y a t room t e m p e r a t u r e f o r 5 m i n . The r e s u l t i n g p l a s m a and r e d c e l l p e l l e t were u s e d t o m e a s u r e p l a s m a pH, i n t r a e r y t h r o c y t i c pH, p l a s m a w a t e r c o n t e n t , i n d u p l i c a t e and p l a s m a C C o ^ l^Ta^er c o n t e n t s and pH were m e a s u r e d a s d e s c r i b e d p r e v i o u s l y . Coos-; v a l u e s were m e a s u r e d g a s o m e t r i c a l 1 y w i t h a C a r l e a n a l y t i c a l g a s c h r o m a t o g r a p h (model 0111) a s d e s c r i b e d by B o u t i l i e r et al . (1984b) „ A d r e n a l i n e and n o r a d r e n a l i n e c o n c e n t r a t i o n s were m e a s u r e d on 0.5 mL a l i q u o t s of p l a s m a 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 w i t h a S p e c t r a - P h y s i c s H.P.L.C. (model SP8700) u s i n g t h e methods o f Woodward ( 1 9 8 2 ) . F u r t h e r -a l i q u o t s of e q u i l i b r a t e d b l o o d and p l a s m a w ere s t o r e d f r o z e n f o r 98 l a t e r a n a l y s e s o f C I " , K*, Na* and h a e m o g l o b i n . I o n c o n c e n t r a t i o n s were m e a s u r e d a s d e s c r i b e d i n C h a p t e r 1. H a e m o g l o b i n c o n c e n t r a t i o n s were m e a s u r e d a s i r o n by f l a m e p h o t o m e t r y w i t h a P e r k i n - E l m e r a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r (model 2380) u s i n g t h e m e t h o d s of Z e t t n e r and Mensch ( 1 9 6 7 ) . P c o s v a l u e s were c a l c u l a t e d f r o m t h o s e i n t h e g a s m i x t u r e s a s d e s c r i b e d i n t h e C 0 K s o l u b i l i t y s t u d y ( C h a p t e r 1 ) . V a l u e s o f b H C 0 3 - were c a l c u l a t e d u s i n g e q u a t i o n 6 ( C h a p t e r 1 ) . I n t r a c e l l u l a r l e v e l s o f bHC0 3™, CI™, Na*, K* and w a t e r were c a l c u l a t e d u s i n g e q u a t i o n 7 ( C h a p t e r 1 ) . Ion c o n c e n t r a t i o n s were e x p r e s s e d p e r k i l o g r a m o f r e d c e l l o r p l a s m a w a t e r i n c a l c u l a t i o n s o f Donnan i o n r a t i o s . I n 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 , s i m i l a r b l o o d s a m p l e s were e q u i l i b r a t e d a t 0.2 and 1.0 % CQst i n a i r i n t h e p r e s e n c e o f 5-10™* M a d r e n a l i n e and e i t h e r 10™"* M D L - p r o p r a n o l o l HC1 o r 10"""* M SITES. O n l y h a e m a t o c r i t , p l a s m a pH, i n t r a e r y t h r o c y t i c pl-l and b l o o d h a e m o g l o b i n c o n t e n t w e re m e a s u r e d i n t h i s s e r i e s o f ex p e r i m e r i t s . 99 RESULTS T r a n s p o r t o-f c h l o r i d e was e x t r e m e l y r a p i d a c r o s s t h e p l a s m a membrane o f Salmo gairdneri e r y t h r o c y t e s . C h l o r i d e ; s e l f - e x c h a n g e b e t w e e n 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 m p a r t m e n t s i n t h e c o n t r o l s i t u a t i o n was c o m p l e t e d w i t h i n 60 s (Fi g , . 1 5 ) . The h a l f -t i m e f o r c h l o r i d e s e l f - e x c h a n g e , c a l c u l a t e d - f r o m t h e r e g r e s s i o n of t i m e a g a i n s t t h e n a t u r a l l o g a r i t h m o f [ p e r c e n t r e c o v e r y + 12 f o r t h e f i r s t 60 s o f the; r e a c t i o n , was 6 . 1 + 0 . 4 s ( + 9 5 % c o n f i d e n c e i n t e r v a l ) . I n h i b i t i o n o f c a r b o n i c a n h y d r a s e w i t h a c e t a z o l a m i d e had no e f f e c t on c h l o r i d e movements ( F i g . 1 5 ) . The h a l f - t i m e f o r C I - s e l f - e x c h a n g e i n t h e p r e s e n c e o f a c e t a z o l a m i d e was 6.4 + 0.4 s. S i m i l a r l y , a d d i t i o n o f p l a s m a ( C c o a l e s s t h a n 2 mM) had no e f f e c t on c h l o r i d e t r a n s p o r t ( F i g . 1 5 ) . The h a l f - t i m e f o r c h l o r i d e t r a n s p o r t i n t h e p r e s e n c e o f p l a s m a was 6.2 + 0.5 s. I n h i b i t i o n o f t h e membrane a n i o n e x c h a n g e p a t h w a y w i t h S I T S , h o w e v e r , v i r t u a l l y a b o l i s h e d c h l o r i d e movements a c r o s s t h e r e d c e l l membrane (Fig , , 1 5 ) . R a i n b o w t r o u t e r y t h r o c y t e s were p e r m e a b l e t o b i c a r b o n a t e , a s d e m o n s t r a t e d by t h e i r a b i l i t y t o c a t a l y z e t h e c o n v e r s i o n o f e x t r a c e l l u l a r HC0 3"" t o C 0 3 . I n t a c t r e d c e l l s s u s p e n d e d i n C o r t l a n d s a l i n e ? ( ' C o r t l a n d b l o o d ' ) c a t a l y s e d H C 0 3 ~ d e h y d r a t i o n by 43.12 f o l d p e r mL r e d c e l l s ( T a b l e 9 ) . T h i s v a l u e i s not. c o m p a r a b l e d i r e c t l y t o t h o s e m e a s u r e d i n C h a p t e r 2 b e c a u s e oi d i f f e r e n c e s i n s e n s i t i v i t y b e t w e e n t h e pH s t a t and mcDdified b o a t a s s a y s . The c a r b o n i c anhydrase? 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 , 100 F i g u r e 15. E f f l u x o-f - " " c h l o r i d e f r o m e r y t h r o c y t e s o f Salmo gairdneri a t 1 C, s h o w i n g e f f e c t s o f a c e t a z o l ami de (10~ s M) , S I T S (10—* M) and p l a s m a (107. by v o l u m e ) . P e r c e n t r e c o v e r y was c a l c u l a t e d a s a c t i v i t y o f ~ * c i - i n e x t r a c e l l u l a r medium a t t i m e t d i v i d e d by a c t i v i t y i n medium a f t e r 30 min e q u i l i b r a t i o n , m u l t i p l i e d by 100 ( s e e t e x t f o r d e t a i l s ) . P o i n t s r e p r e s e n t d a t a f rom 3 •-4 t r i a l s . 102 T a b l e 9. Net H C O 3 - " 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 leu-mL r e d c e l l " " ' - ) o f Salmo gairdneri, s h o w i n g e f f e c t s o f p l a s m a , a c e t a z o l a m i d e , S I T S , f o a m i n g a g e n t s , and d e f o a m i n g a g e n t s . V a l u e s a r e means + 1 S.E. (n=S>. Whole b l o o d C o r t l a n d b l o o d ( c e l l s i n p l a s m a ) ( c e l l s i n s a l i n e ) C o n t r o l C o r t l a n d s a l i n e 9.93 ± 0.33 43.12 + 1.76 A c e t a z o l a m i de 0» 00 + 0.01 S I T S 0.01 + 0.01 F o a m i n g a g e n t s b o v i n e s e r u m a l b u m i n 1.63 + 0.03 20.09 + 1.21 p o l y v i n y l - p y r r o l i d i none 0.73 + 0.02 10.09 + 0,. 40 D e f o a m i n g a g e n t s octanoi 101.46 + 3.96 108.82 + 5.8: No-foam 91.50 + 2.97 99.32 •+• 3.45 103 and h e n c e t h e n e t flu;-; o f H C 0 3 - t h r o u g h i n t a c t r e d c e l l s , was a b o l i s h e d by a c e t a z o l a m i d e and by S I T S ( T a b l e 9 ) . P l a s m a s i g n i f i c a n t l y i n f l u e n c e d t h e n e 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 r e d c e l l s ( T a b l e 9 ) , S u s p e n s i o n s o f r e d c e l l s i n p l a s m a ('whole b l o o d ' ) d e m o n s t r a t e d o n l y 23,07, o f t h e n e t f l u x o f r e d c e l l s s u s p e n d e d i n s a l i n e ( ' C o r t l a n d b l o o d ' ) , , However, s u b s t a n t i a l f o a m i n g o f w h o l e b l o o d was o b s e r v e d d u r i n g t h e a s s a y p r o c e d u r e . S i m i l a r f o a m i n g was n o t e v i d e n t when C o r t l a n d b l o o d was a s s a y e d . I n v e s t i g a t i o n o f t h e c o n s e q u e n c e s o f r e a g e n t f o a m i n g i n d i c a t e d t h a t f o a m i n g c o u l d i n i t s e l f a c c o u n t f o r t h e o b s e r v e d i n h i b i t o r y e f f e c t s o f p l a s m a . A d d i t i o n o f f o a m i n g a g e n t s ( b o v i n e s e r u m a l b u m i n , p o l y v i n y l - p y r r o l i d i n o n e ) s i g n i f i c a n t l y r e d u c e d t h e m e a s u r e d b i c a r b o n a t e f l u x e s i n b o t h w h o l e and C o r t l a n d b l o o d ( T a b l e 9 ) . F o a m i n g a g e n t s r e d u c e d t h e HCO.3""' f l u x i n C o r t l a n d b l o o d t o n e a r t h e l e v e l o f c o n t r o l w h o l e blood,, On t h e o t h e r h a n d , a d d i t i o n o f d e f o a m i n g a g e n t s ( o c t a n o l , No-foam) s i g n i f i c a n t l y i n c r e a s e d t h e m e a s u r e d n e t H C 0 3 ~ f l u x and n e g a t e d any d i f f e r e n c e s i n n e t f l u x b e t w e e n w h o l e b l o o d and C o r t l a n d b l o o d ( T a b l e 9 ) . T h u s , d e f o a m i n g a g e n t s c o u n t e r e d t h e e f f e c t s o f p l a s m a on f l u x o f H C 0 3 ~ t h r o u g h r e d c e l l s . N e t H C O 3 - 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 was i n f l u e n c e d s i g n i f i c a n t l y by 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 and n o r a d r e n a l i n e b o t h i n h i b i t e d t h e n e 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 r e d c e l l s i n a manner w h i c h was d o s e - d e p e n d e n t (Fi g , , 1 6 ) . A d r e n a l i n e had a g r e a t e r e f f e c t t h a n n o r a d r e n a l i n e . A d r e n e r g i c i n h i b i t i o n o f n e t H C 0 3 " ~ f l u x was n e g l i g i b l e a t t h e p h y s i o l o g i c a l l e v e l s o f a d r e n a l i n e p r e s e n t i n u n s t r e s s e d f i s h (lO -"*' l i , s e e b e l o w ) , b u t was r e s p o n s i b l e f o r a 31.77. r e d u c t i o n i n n e t 104 F i g u r e 16. E f f e c t 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 on n e 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 e r y t h r o c y t e s o f Salvia g a i r d n e r i . V a l u e s a r e means + 1 S.E„ ( n ~ 4 ~ & ) . % CHANGE IN NET H C O 3 FLUX SOT 106 b i c a r b o n a t e - f l u x a t t h e p h y s i o l o g i c a l l e v e l s p r e s e n t i n s t r e s s e d t r o u t (10~ < f e M, Mazeaud and Mazeaud 1 9 8 1 ) . A d r e n e r g i c e f f e c t s were n o t an a r t i f a c t o f r e a g e n t f o a m i n g , u s e of No-foam i n f o u r t r i a l s d i d n o t a l t e r t h e i n h i b i t o r y e f f e c t o f IO" 8 3 M a d r e n a l i n e . Nor w e r e t h e y due t o d i r e c t i n h i b i t i o n o f t h e CA enzyme!! n e i t h e r -a d r e n a l i n e o r i s o p r o t e r o n o l had any s i g n f i c a n t e f f e c t on t h e CA a c t i v i t y o f t r o u t h a e m o l y s a t e s , a s m e a s u r e d i n f o u r t r i a l s w i t h t h e p H - s t a t a s s a y ( s e e C h a p t e r 2 f o r d e t a i l s o f a s s a y method) I n h i b i t i o n o f n e t HCO3'"' f l u x by a d r e n a l i n e ? and n o r a d r e n a l i n e was a b o l i s h e d by s i m u l t a n e o u s b e t a - b l o c k a d e w i t h p r o p r a n o l o l ( T a b l e 1 0 ) , i n d i c a t i n g t h a t t h e i n h i b i t i o n was m e d i a t e d by b e t a -a d r e n e r g i c s i t e s . On t h e o t h e r h a n d , t h e i n h i b i t o r y e f f e c t s o f n o r a d r e n a l i n e were e n h a n c e d by s i m u l t a n e o u s a l p h a - b l o c k a d e w i t h p h e n t o l a m i n e ( T a b l e 1 0 ) . S i n c e n o r a d r e n a l i n e c o n t a i n s b o t h a l p h a - and b e t a - a d r e n e r g i c a c t i v i t y , t h i s r a i s e s t h e p o s s i b i l i t y t h a t a l p h a - a d r e n e r g i c a g o n i s t s e n h a n c e l-IC0 3" f l u x a n d , c o n s e q u e n t l y , a n t a g o n i z e b e t a a d r e n e r g i c e f f e c t s . E f f e c t s o f a d r e n a l i n e on i o n movements a c r o s s t h e r e d c e l l membrane were i n v e s t i g a t e d f u r t h e r i n a s e r i e s o f C O a . e q u i l i b r a t i o n s t u d i e s . In t h e s e s t u d i e s , t h e a d r e n a l i n e c o n c e n t r a t i o n o f w h o l e b l o o d was i n c r e a s e d f r o m l e v e l s p r e s e n t in vivo i n u n s t r e s s e d f i s h (3.1 + 0.5 - I Q - ~ m o l - L p l a s m a " * , n=24) t o 4.6 + 0.3 - I O " * mo.l-L p l a s m a " 1 (n=24) . N o r a d r e n a l i n e c o n c e n t r a t i o n s r e m a i n e d u n c h a n g e d a t 1.2 +• 0.1 - IO""'"* m o l - L p l a s m a " * (n=48). The r e s u l t s , a s p r e s e n t e d b e l o w , d e m o n s t r a t e t h a t a d r e n a l i n e s i g n i f i c a n t l y i n f l u e n c e d t h e e q u i l i b r i u m d i s t r i b u t i o n s o f H~, Na* and C I " , a s w e l l a s , HC0 3". 107 T a b l e 10. E f f e c t s o-f a l p h a - a d r e n e r g i c: and b e t a - a d r e n e r g i c a g o n i s t s and a n t a g o n i s t s on t h e n e t HCO3™ f l u ; - ; (eu-mL. r e d c e l l " - 1 ) 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 o f Sal mo gairdneri. V a l u e s a r e means -1- 1 S.E. (n==6). Net b i c a r b o n a t e f l u x 7, c h a n g e f r o m c o n t r o l C o n t r o l C o r t l a n d s a l i n e 1 0 - 8 5 ti a d r e n a l i n e 10-® l i a d r e n a l i n e + 1 0 — * M p r o p r a n o l o l 10™-» (vj n o r a d r e n a l i n e 10™° l i n o r a d r e n a l i n e + 1 0 — * l i p r o p r a n o l o l 10~™ l i n o r a d r e n a l i n e + 10"-* l i p h e n t o l a m i ne 41.27 ± 2.00 0.0 25.69 ± 2 . 3 7 -37.8 42.92 ± 1.65 4.0 31.89 ± 1.22 -22.7 43.58 ± 0.41 5.6 22.08 + 0.82 -46.5 108 A d r e n e r g i c s t i m u l a t i o n r e s u l t e d i n a n e t l o s s o-f HH" f r o m t r o u t e r y t h r o c y t e s ( F i g . 17)s a d r e n a l i n e - t r e a t e d b l o o d had a h i g h e r i n t r a c e l l u l a r pH and a l o w e r p l a s m a pH a t e a c h F'OOK l e v e l t h a n c o n t r o l b l o o d . The m a g n i t u d e o f t h e a d r e n a l i n e e f f e c t was r e l a t e d d i r e c t l y t o b l o o d P C Q a ! a d r e n a l i n e - i n d u c e d c h a n g e s i n i n t r a c e l l u l a r l - l * c o n c e n t r a t i o n s i n c r e a s e d f r o m 2.05 + 0.35 nM a t 1.8 t o r r P c o a t o 6.59 + 0.62 nM a t 7.8 t o r r P C 0 3.. T h e s e H* movements i n c r e a s e d t h e Donnan H* r a t i o , R H + , i n a d r e n a l i n e -t r e a t e d b l o o d ( F i g . 1 8 ) . A d r e n e r g i c s t i m u l a t i o n a l s o r e s u l t e d i n a n e t c e l l u l a r g a i n of C l ~ and Na"", b u t had no e f f e c t on t h e d i s t r i b u t i o n o f K"*" ( F i g . 1 8 ) . R c i - ( [ e r y t h r o c y t e ] / l l p l asmall) i n c r e a s e d w i t h a d r e n a l i n e , RN«+ ( [ p l a s m a ] / [ e r y t h r o c y t e ] ) d e c r e a s e d , and R K+ < [ p l a s m a ] / [ e r y t h r o c y t e ] ) r e m a i n e d u n c h a n g e d . R C 1 - v a l u e s o f b o t h c o n t r o l and a d r e n a l i n e - t r e a t e d b l o o d s were v i r t u a l l y i d e n t i c a l t o RM.*. v a l u e s , and b o t h R C i - and RM-t- were i n good a g r e e m e n t w i t h t h e d a t a o f F i g u r e 5 ( C h a p t e r 1 ) . T h i s i n d i c a t e s t h a t CI™ and H"1" r e m a i n e d p a s s i v e l y d i s t r i b u t e d f o l l o w i n g t r e a t m e n t w i t h a d r e n a l i n e . I n a l l c a s e s t r o u t e r y t h r o c y t e s m a i n t a i n e d a l o w 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 Na"" and a h i g h 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 K"", r e l a t i v e t o p l a s m a l e v e l s , w i t h Donnan r a t i o s v a s t l y d i f f e r e n t f r o m t h o s e o f H"1", C I " and HCQV", and f r o m e a c h o t h e r . The d i s t r i b u t i o n o f Na* and K* i n t h e s e r e d c e l l s was m a i n t a i n e d by t h e known Na*/K'"*" pump on t h e r e d c e l l membrane. A d r e n a l i n e had a s i g n i f i c a n t e f f e c t on t h e C 0 2 - c a r r y i n g c a p a c i t y o f t r o u t b l o o d s a d r e n a l i n e - t r e a t e d b l o o d c o n t a i n e d l e s s bHC0:s'"" a t e a c h Pcoa t h a n c o n t r o l b l o o d ( F i g . 19) . T h i s d e c r e a s e i n b l o o d bHC0 3™ was due p r i m a r i l y t o a d e c r e a s e i n c e l l u l a r 109 F i g u r e 17= P l a s m a pH and i n t r a e r y t h r o c y t i c pH o f Salmo gairdneri b l o o d a s a - f u n c t i o n o-f P C o 2 d e t e r m i n e d in vitro a t 10 C, s h o w i n g e f f e c t s o f a d r e n a l i n e . A d r e n a l i n e c o n c e n t r a t i o n s w e re 3.1- 10""°' mol-L plasma'"" 1 i n c o n t r o l b l o o d and 4.6- 10" A mol - L plasma"'" 1 i n a d r e n a l i n e b l o o d . V a l u e s a r e means + 1 S.E. <n=8)„ A d r e n a l i n e v a l u e s d e n o t e d w i t h * a r e d i f f e r e n t s i g n i f i c a n t l y f r o m c o r r e s p o n d i n g c o n t r o l v a l u e s a t P<0.05 ( p a i r e d t - t e s t ) . 1 1 0 P c o 2 , t o r r I l l F i g u r e 18. Donnan r a t i o s o f H*, N a * , K*, CI™, and bHCQ 3™ i n Salmo gairdneri b l o o d a s a f u n c t i o n o f Pees a d e t e r m i n e d in vitro a t 10 C, s h o w i n g e f f e c t s o f a d r e n a l i n e . A n i o n r a t i o s a r e C e r y t h r o c y t e 3 / C p l a s m a ' J w h i l s t c a t i o n r a t i o s a r e C p l a s m a H / C e r y t h r o c y t e ] , R e s t o f c a p t i o n a s i n F i g u r e 17. Pco 2 , torr Pco 2 , torr 113 F i g u r e 19. b H C 0 3 ~ c o n c e n t a t i o n s i n o x y g e n a t e d w h o l e b l o o d , p l a s m a , and e r y t h r o c y t e s o-f Salmo gairdneri a s a f u n c t i o n o f P M 2 d e t e r m i n e d in vitro a t 10 C, s h o w i n g e f f e c t s o f a d r e n a l i n e . R e s t of c a p t i o n a s i n F i g u r e 17. 114 P c o 2 , t o r r 115 bl-IC0 3" _; p l a s m a bHCQ 3~ w a i s n o t a f f e c t e d s i g n i f i c a n t l y ( F i g . 1 9 ) . As a r e s u l t , a d r e n a l i n e p r o d u c e d a s i g n i f i c a n t , d e c r e a s e i n t h e Donnan b H C 0 3 ~ r a t i o ( F i g . 1 8 ) . T h i s d e c r e a s e i n R H c o s - was i n s h a r p c o n t r a s t t o t h e a d r e n a l i n e - i n d u c e d i n c r e a s e i n R H+ and Rc* ••-( F i g . 1 8 ) . A d r e n a l i n e i n d u c e d i o n movements were a c c o m p a n i e d by 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 mean c e l l s i z e o f e r y t h r o c y t e s ( m e a s u r e d a s t h e q u o t i e n t o f h a e m a t o c r i t (7.) and h a e m o g l o b i n c o n c e n t r a t i o n , g-L blood"" 3!! F i g . 2 0 ) and by n e t movement of w a t e r i n t o t h e c e l l ( F i g . 2 0 ) . T h e s e e f f e c t s were a b o l i s h e d by b l o c k a d e o f b e t a - a d r e n e r g i c s i t e s w i t h p r o p r a n o l o l and by S I T S ( F i g . 2 1 ) . A d r e n e r g i c r e d i s t r i b u t i o n o f H* a l s o was s e n s i t i v e t o b l o c k a d e o f b e t a - a d r e n e r g i c s i t e s ? a d r e n a l i n e had no e f f e c t on RM+ when b e t a - s i t e s w e re b l o c k e d w i t h p r o p r a n o l o l ( F i g . 2 1 ) . As w e l l , a d r e n e r g i c e f f e c t s on pH were s e n s i t i v e t o i n h i b i t i o n o f t h e membrane a n i o n e x c h a n g e p a t h w a y w i t h S I T S ( F i g . 2 1 ) . 116 F i g u r e 20. Mean c e l l s i z e ( h a e m a t o c r i t / [ h a e m o g l o b i n ] ) and w a t e r c o n t e n t o f Salmo gai rdneri e r y t h r o c y t e s a s a f u n c t i o n o f F'co2 d e t e r m i n e d in vitro a t 10 C, s h o w i n g e f f e c t s o f a d r e n a l i n e . R e s t o f c a p t i o n a s i n F i g u r e 17. MEAN C E L L VOLUME LU 118 F i g u r e 21- Donnan H* r a t i o ( [ p l asmaTJ / [ e r y t h r o c y t e J ) and mean c e l l s i z e o f e r y t h r o c y t e s o f Salmo gairdneri a s a f u n c t i o n o-f Pco2 d e t e r m i n e d in vitro a t 10 C, s h o w i n g e f f e c t s o f a d d i t i o n o f a d r e n a l i n e (5 - 10"~- M) and e i t h e r p r o p r a n o l o l ( 1 0 — * li) o r S I T S (10""* li ) . V a l u e s a r e means + 1 S-E- (n=4) -0.6 r 0.4 h 0.2 r 0' ' ' " > 1 " ' ' 0 I 1 1 1 1 1 1 1 1 0 2 4 6 8 Pco 2 , torr P c o 2 , torr 1 2 0 DISCUSSION T r o u t e r y t h r o c y t e s were r e a d i l y p e r m e a b l e t o c h l o r i d e , w i t h a h a l f — t i m e f o r CI"" s e l f - e x c h a n g e a t 1 C (6.1 s) w h i c h was c o m p a r a b l e t o t h a t o f mammalian r e d b l o o d c e l l s u n d e r s i m i l a r c o n d i t i o n s (7-8 s, W i e t h and B j e r r u m 1 9 8 2 ) . T h i s l a r g e p e r m e a b i l i t y i s f u r t h e r e v i d e n c e f o r a p a s s i v e C l ~ d i s t r i b u t i o n . S i n c e H C 0 3 ~ h a s been shown t o be a s good an e x c h a n g e p a r t n e r f o r C I " a s C I " i t s e l f ( D a i m a r k 19721), t h e p r e s e n t CI"" s e l f - e x c h a n g e d a t a a l s o s u g g e s t t h a t f i s h r e d c e l l s a r e r e a d i l y p e r m e a b l e t o b i c a r b o n a t e . The f i n d i n g s t h a t t r o u t e r y t h r o c y t e s w e re c a p a b l e of c a t a l y z i n g t h e d e h y d r a t i o n o f e x t r a c e l l u l a r H C0 3™ was d i r e c t , e v i d i e n c e o f t h e i r H C 0 3 ~ p e r m e a b i l i t y . F l u x e s of b o t h CI™ and H C 0 3 ~ t h r o u g h t r o u t r e d c e l l s were i n h i b i t e d by S I T S , a s o c c u r s i n mammalian r e d c e l l s ( C a b a n t c h i k and R o t h s t e i n 1 9 7 2 ) . S I T S s p e c i f i c a l l y i n h i b i t s t h e Band 3 a n i o n e x c h a n g e p a t h w a y r e s p o n s i b l e f o r t h e c h l o r i d e s h i f t i n mammalian e r y t h r o c y t e s ( C a b a n t c h i k e t a l . 1978) and p r o b a b l y h a s an i d e n t i c a l e f f e c t on f i s h e r y t h r o c y t e ' s . Membrane t r a n s p o r t of a n i o n s i n t r o u t r e d c e l l s was i n d e p e n d e n t o f e r y t h r o c y t i c CA a c t i v i t y . However, a s one m i g h t e x p e c t , 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 was d e p e n d e n t upon a n i o n t r a n s p o r t . I n h i b i t i o n o f a n i o n e x c h a n g e d u r i n g C 0 S l o a d i n g e f f e c t i v e l y i s o l a t e s i n t r a c e l l u l a r CA f r o m p l a s m a b i c a r b o n a t e , i t s m a j o r s o u r c e o f s u b s t r a t e , w h i l s t i n h i b i t i o n d u r i n g CO^ l o a d i n g s l o w s C 0 E h y d r a t i o n v i a t h e 'Law of Mass A c t i o n ' a s H C 0 3 ~ a c c u m u l a t e s i n s i d e thee c e l l . I n t h i s way, i n h i b i t i o n o f a n i o n e x c h a n g e i s f u n c t i o n a l l y i d e n t i c a l t o 1 2 1 i n h i b i t i o n o f e r y t h r o c y t i c : CA. B l o o d p l a s m a had no s p e c i f i c e f f e c t on a n i o n t r a n s p o r t o r on i n t r a c e l l u l a r C A - a c t i v i t y . T h i s i s c o n s i s t e n t w i t h e v i d e n c e t h a t t e l e o s t and e l a s m o b r a n c h r e d c e l l s p o s s e s s a m a m m a l i a n - l i k e c h l o r i d e s h i f t w h i c h i s u n a f f e c t e d by p l a s m a (Cameron 1978b, O b a i d e t a l . 1 9 7 9 ) . H a s w e l l and R a n d a l l (1976) and H a s w e l l e t al. ( 1 9 7 8 ) , h o w e v e r , f o u n d t h a t f i s h p l a s m a r e n d e r s r e d c e l l s f u n c t i o n a l l y i m p e r m e a b l e t o b i c a r b o n a t e u s i n g a manometic t e c h n i q u e i d e n t i c a l t o t h a t u s e d i n t h e p r e s e n t s t u d y . The p r e s e n t r e s u l t s i n d i c a t e t h a t t h i s p l a s m a i n h i b i t i o n i s a n o n -s p e c i f i c a r t i f a c t r e l a t e d t o r e a g e n t f o a i m i n g d u r i n g t h e m o d i f i e d b o a t a s s a y , and n o t t h e a c t i o n o f a s p e c i f i c t r a n s p o r t / e n z y m e i n h i b i t o r . S i m i l a r d e c r e a s e s i n i n t r a c e l l u l a r CA a c t i v i t y w e r e o b s e r v e d i n t h e p r e s e n c e of b o v i n e s e r u m a l b u m i n and p o l y v i n y l -p y r r o l i d i n o n e . T h e s e e f f e c t s , i n c l u d i n g t h a t o f p l a s m a , w e re c o u n t e r e d by u s e o f d e f o a m e r s . Hence, COse ' p r o d u c e d by HCO»"~ d e h y d r a t i o n d u r i n g t h e m o d i f i e d b o a t a s s a y i s r e t a i n e d i n foam. T h i s f o a m i n g s l o w e d t h e d i f f u s i v e e q u i l i b r i u m o f C O a b e t w e e n t h e l i q u i d and g a s e o u s p h a s e s o f t h e a s s a y . C a r e must be t a k e n , t h e r e f o r e , t o m i n i m i z e o r t o s t a n d a r d i z e r e a g e n t f o a m i n g i n o r d e r t o o b t a i n v a l i d r e s u l t s w i t h t h e m o d i f i e d b o a t a s s a y . Use o f d e f o a r n e r s c a n n o t be recommended, h o w e v e r , u n l e s s t h e e f f e c t s o f t h e i r p r e s e n c e on t h e CA enzyme and on membrane t r a n s p o r t p r o p e r t i e s a r e u n d e r s t o o d . The e q u i l i b r i u m d i s t r i b u t i o n s o f most m a j o r i o n s i n S. gairdneri b l o o d were i n f l u e n c e d s i g n i f i c a n t l y by c a t e c h o l a m i n e s . 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 r o u t r e d c e l l s r e s u l t e d i n a n e t c e l l u l a r g a i n o f Na*, C l ~ and H a 0 , a n e t c e l l u l a r l o s s o f H* and 122 H C O s - , and a p r o n o u n c e d c e l l s w e l l i n g . The e q u i l i b r i u m d i s t r i b u t i o n o-f K* r e m a i n e d v i r t u a l l y u n c h a n g e d . B e t a - a d r e n e r g i c s w e l l i n g and an a s s o c i a t e d H* e x t r u s i o n have? been n o t e d p r e v i o u s l y i n e r y t h r o c y t e s o f r a i n b o w t r o u t and s t r i p e d b a s s , Morone s a x i t i l i s ( N i k i n m a a 1982, 1983, N i k i n m a a et al. 1 9 8 4 ) . M o r e o v e r , N i k i n m a a and H u e s t i s (1984) have; shown t h a t a d r e n e r g i c s w e l l i n g and t h e a s s o c i a t e d pl-l c h a n g e s i n r a i n b o w t r o u t r e d e e l I s a r e i n d e p e n d e n t o f e x t r a c e l l u l a r K*". and i n s e n s i t i v e t o f u r o s e m i d e , b u t a r e d e p e n d e n t upon e x t r a c e l l u l a r Na* and s e n s i t i v e tc3 DIDS and a m i l o r i d e . A d r e n e r g i c s w e l l i n g o f f i s h e r y t h r o c y t e s t h u s a p p e a r s t o i n v o l v e a d i f f e r e n t m o l e c u l a r m echanism t h a n t h a t i n v o l v e d i n a v i a n r e d c e l l s . In a v i a n e r y t h r o c y t e s , a d r e n e r g i c s w e l l i n g i n v c j l v e s Na '*"/KH"/Cl"'" c o t r a n s p o r t i n t o t h e c e l l , r e q u i r e s h i g h , n o n p h y s i o l o g i c a l l e v e l s o f e x t r a c e l l u l a r K*', and i s s e n s i t i v e t o f u r o s e m i d e b u t i n s e n s i t i v e t o d i s u l p h o n i c s t i l b e n e d e r i v a t i v e s ( R i d d i c k e t a l , 1971, Kregenow 1973, P a l f r e y e t a l » 1 9 8 0 ) . In t e r m s cjf a d r e n e r g i c s w e l l i n g , f i s h e r y t h r o c y t e s b e h a v e more l i k e a m p h i b i a n r e d c e l l s , i n a s much a s t h e r e g u l a t o r y v o l u m e i n c r e a s e s o f a m p h i b i a n e r y t h r o c y t e s i n h y p e r t o n i c m e d i a a l s o do n o t r e q u i r e e x t r a c e l l u l a r K* t o m a i n t a i n u p t a k e o f Na'*" and H^O (Kregenow 1 9 8 1 ) . C a l a (1980) f o u n d t h a t a m p h i b i a n e r y t h r o c y t e s g a i n s o d i u m , c h l o r i d e and w a t e r d u r i n g r e g u l a t o r y v o l u m e i n c r e a s e s i n h y p e r t o n i c m e d i a . T h e s e i o n f l u x e s were e l e c t r i c a l l y s i l e n t , were s e n s i t i v e t o medium b i c a r b o n a t e - ; l e v e l s and DIDS, artci w e r e a s s o c i a t e d w i t h H* e x t r u s i o n . T h i s i n f o r m a t i o n l e d C a l a (1980) t o p r o p o s e t h a t v o l u m e r e g u l a t o r y i o n movements i n a m p h i b i a n r e d 123 c e l l s i n v o l v e o b l i g a t o r i l y c o u p l e d a l k a l i metal/H" 1" and C 1 ~ / H C 0 3 ~ ex c h a n g e r s . The p r e s e n t d a t a i n d i c a t e t h a t s i m i l a r e x c h a n g e r s e x i s t i n - f i s h e r y t h r o c y t e s and t h a t t h e y a r e 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 n i s o t o n i c m e d i a . A c c o r d i n g l y , b i n d i n g of b e t a - a g o n i s t s t o membrane r e c e p t o r s on 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 Na* and C l ~ i n f l u x e s i n e x c h a n g e f o r H* and HCO3-5 t h e l a t t e r i o n p a i r do n o t e x e r t o s m o t i c p r e s s u r e when i n e q u i l i b r i u m w i t h C 0 a and Hs»0. The n e t r e s u l t i s an i n c r e a s e i n t h e number o f o s m o t i c a l l y — a c t i v e p a r t i c l e s i n 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 a n d , c o n s e q u e n t l y , u p t a k e o f e x t r a c e l l u l a r w a t e r and c e l l s w e l l i n g . T h e s e i o n e x c h a n g e r s p r o b a b l y a r e i n v o l v e d i n v o l u m e r e g 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 d u r i n g o s m o t i c p e r t u r b a t i o n s a s w e l l . The m o l e c u l a r mechanism i n v o l v e d i n s w e l l i n g of a v i a n r e d c e l l s i s s e n s i t i v e t o b o t h osmotic-p e r t u r b a t i o n s and 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 (Kregenow 1 9 7 3 ) . M o r e o v e r , C a l a (1977) h a s shown t h a t r e g u l a t o r y i n c r e a s e s i n e r y t h r o y c t e v o l u m e a f t e r o s m o t i c p e r t u r b a t i o n of f l o u n d e r (Plearonectes flesas) r e d c e l l s i s a c c o m p a n i e d by a n e t c e l l u l a r g a i n of s o d i u m , c h l o r i d e and w a t e r , w i t h l i t t l e c h a n g e i n p o t a s s i um. A l t e r n a t i v e e x p l a n a t i o n s of t h e m o l e c u l a r m e c h a n i s m u n d e r l y i n g a d r e n e r g i c s w e l l i n g o f f i s h e r y t h r o c y t e s do n o t a c c o u n t f o r t h e o b s e r v e d r e s u l t s a s w e l l a s c o u p l e d Na*/H*, HCO3™ / C I " e x c h a n g e r s . C o u p l e d Na*/H*, c o u p l e d N a * / C l " and Na*/HCQ 3-/ C l ~ c o t r a n s p o r t m e c h a n i s m s h a v e been f o u n d i n a number of i n v e r t e b r a t e and v e r t e b r a t e t i s s u e s ( f o r r e v i e w s e e Roos and B o r o n 1981, M u r e r e t al, 1983, P a l f r e y and Rao 1 9 8 3 ) , and w o u l d a p p e a r t o be t h e most o b v i o u s a l t e r n a t i v e s . However, e a c h of 1 2 4 t h e s e m e c h a n i s m s c a n be d i s c o u n t e d , Na^/H* e x c h a n g e r s a r e i n s e n s i t i v e t o d i s u l p h o n i c s t i l b e n e d e r v i a t i v e s ( S I T S , DIDS) , w h i l s t c o u p l e d t r a n s p o r t o f N a * / H C 0 3 ~ / C l " r e s u l t s i n a n e t c e l l u l a r g a i n o f Na* and HC0 3~, and a l o s s o f C I " . N a * / C l ~ e x c h a n g e r s a r e s e n s i t i v e t o f u r o s e m i d e , w h e r e a s N i k i n m a a and H u e s t i s (1984) h a v e d e m o n s t r a t e d 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 o f f i s h e r y t h r o c y t e s i s n o t . In human e r y t h r o c y t e s , , a d r e n e r g i c s w e l l i n g h a s been p r o p o s e d t o i n v o l v e a c a l c i u m - m e d i a t e d c h a n g e i n t h e submembrane c y t o s k e l e t o n o f t h e r e d c e l l (Rasmussen e t a l , 1 9 7 5 ) . T h i s c y t o s k e l e t o n , t h e m i c r o f i I a m e n t o u s s p e c t r i n c o m p l e x , i s t h o u g h t t o c o n t r o l c e l l s h a p e and s i z e ( S t e c k 1 9 7 4 ) . S w e l l i n g due t o s i m p l e s t r u c t u r a l c h a n g e s c o u l d c o n c e i v a b l y i n c r e a s e s o d i u m l e a k a g e down i t s c o n c e n t r a t i o n g r a d i e n t i n t o t h e c e l l and w o u l d a l t e r t h e Donnan d i s t r i b u t i o n o f p a s s i v e l y p e r m e a n t i o n s by a f f e c t i n g t h e n e t c h a r g e s t a t e o f h a e m o g l o b i n (Gary-E<obo and Solomon 1 9 7 1 ) . Under s u c h c i r c u m s t a n c e s , h o w e v e r , one a l s o w o u l d e x p e c t s i g n i f i c a n t l e a k a g e o f K* o u t o f t h e e e l 1„ M o r e o v e r , c h a n g e s i n t h e n e t c h a r g e s t a t e o f h a e m o g l o b i n w o u l d a f f e c t R H+, R c i - and R H C Q 3 - a l l i n t h e same d i r e c t i o n , w h e r e a s i n t h e p r e s e n t s t u d y a d r e n a l i n e c a u s e d an i n c r e a s e i n RH-*- and R C i - b u t a d e c r e a s e i n RHCOS-. A t t h i s p o i n t , h o w e v e r , t h e d u b i o u s n a t u r e of t h e p r e s e n t R H c o 3 - e s t i m a t e s n e e d s t o be c o n s i d e r e d . I t was a r g u e d i n C h a p t e r 1 t h a t t h e p r e s e n t R H c o 3 - v a l u e s a r e b i a s e d by c a r b a m i n o h a e m o g l o b i n f o r m a t i o n . I t i s c o n c e i v a b l e , t h e r e f o r e , t h a t a d r e n e r g i c - i n d u c e d d e c r e a s e s i n R H c o s - r e f l e c t a d e c l i n e i n c a r b a m i n o f o r m a t i o n r a t h e r t h a n b i c a r b o n a t e e x t r u s i o n . T h i s i s an u n l i k e l y e x p l a n a t i o n f o r t h e f o l l o w i n g r e a s o n . A d r e n e r g i c 125 s w e l l i n g o-f f i s h e r y t h r o c y t e s i s 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 c e l l u l a r n u c l e o s i d e t r i p h o s p h a t e l e v e l s ( N i k i n m a a 1 9 8 3 , N i k i n m a a et st.l . 1 9 8 4 ) a n d , s i n c e n u c l e o s i d e t r i p h o s p h a t e s c o m p e t e w i t h C 0 2 •for t h e b e t a - c h a i n t e r m i n a l s o-f t r o u t h a e m o g l o b i n ( G r e a n e y and P o w e r s 1 9 7 7 ) , one w o u l d e x p e c t a d r e n a l i n e t o e n h a n c e c a r b a m i n o f o r m a t i o n i f a n y t h i n g . The p r e s e n t r e s u l t s d e m o n s t r a t e t h a t t h e mechanism i n v o l v e d i n a d r e n e r g i c s w e l l i n g o f t r o u t r e d c e l l s i s s e n s i t i v e t o S I T S and t h u s i n v o l v e s t h e Band 3 a n i o n e x c h a n g e p a t h w a y o r an a n a l o g o u s s t r u c t u r e . The r e s u l t s a l s o i n d i c a t e t h a t t h e c a t i o n and a n i o n e x c h a n g e r s a r e l i n k e d t i g h t l y i n a s much a s i n h i b i t i o n o f t h e a n i o n p a t h w a y a l s o i n h i b i t s c a t i o n movements. In c o n t r a s t , N i k i n m a a and H u e s t i s ( 1 9 8 4 ) f o u n d t h a t , while? DIDS i n h i b i t e d a d r e n e r g i c s w e l l i n g i n r a i n b o w t r o u t r e d c e l l s , DIDS had no e f f e c t on H"1" e x t r u s i o n . They c o n c l u d e d f r o m t h i s i n f o r m a t i o n t h a t t h e c a t i o n and a n i o n e x c h a n g e r s i n v o l v e d i n a d r e n e r g i c s w e l l i n g o f f i s h e r y t h r o c y t e s a r e l i n k e d o n l y l o o s e l y . T h e i r r e s u l t s , h o w e v e r , were o b t a i n e d u n d e r e x t r e m e l y a d v e r s e c o n d i t i o n s i n w h i c h t r o u t r e d c e l l s were s u s p e n d e d i n an a i r -e q u i l i b r a t e d HEPES b u f f e r s o l u t i o n a t pH 7 . 1 4 3 - 7 . 2 7 7 and 2 3 C , and t h u s may h a v e been i n f l e n c e d by d e g r a d a t i o n o f t h e r e d c e l l s . Eddy ( 1 9 7 7 ) , f o r e x a m p l e , h a s d e m o n s t r a t e d t h a t t h e n o r m a l m e t a b o l i c f u n c t i o n s 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 d e t e r i o r a t e atbove 2 0 C . Can H*, C I " and H C O r j s " t r u l y be c o n s i d e r e d 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 r e d c e l l membrane i n l i g h t o f t h e p r e s e n t i n f o r m a t i o n ? The a d r e n e r g i c r e s p o n s e s o f f i s h r e d c e l l s a r e m i n i m a l a t r e s t i n g c a t e c h o l a m i n e l e v e l s ( N i k i n m a a 1 9 8 2 , F i g . 1 6 126 t h i s s t u d y) a n cl u n d e r t h o s e c: o n d i t i o n s t h e e q u i 1 i b r i u m d i s t r i b u t i o n s o f H~, C l " and HCa 3" a p p e a r t o be a d e q u a t e l y d e s c r i b e d a s p a s s i v e ( C h a p t e r 1 ) . Even a t e l e v a t e d c a t e c h o l a m i n e l e v e l s , a\ p a s s i v e d i s t r i b u t i o n a d e q u a t e l y d e s c r i b e s t h e o b s e r v e d e q u i 1 i b r i um d i s t r i b u t i o n s o f H•+• and C1". The d i s t r i b u t i on of HCO3'""., h o w e v e r , r e m a i n s i n q u e s t i o n . Whereas, one w o u l d h a v e e x p e c t e d an i n c r e a s e i n Rue:03- when p l a s m a pH d e c r e a s e d i n a d r e n a l i n e , t h e o b s e r v e d R H c o 3 - a c t u a l l y d e c l i n e d . To a c e r t a i n e x t e n t , t h i s d e c r e a s e i n R H c o s - p r o b a b l y r e f l e c t s c h a n g e s i n c h e m i c a l l y - b o u n d s p e c i e s o t h e r t h a n H C 0 3 " ( i e . CQ 3"~, c a r b a m i n o ) . However, i t i s d o u b t f u l t h a t t h e m a g n i t u d e o f t h o s e c h a n g e s was s i g n i f i c a n t t o p r o d u c e t h e o b s e r v e d d e c l i n e i n r e d c e l l b H C 0 3~. I n d e e d , a p a s s i v e d i s t r i b u t i o n f o r H C 0 3 " may n o t h o l d when a d r e n a l i n e l e v e l s a r e e l e v a t e d . D e f i n i t i v e s t a t e m e n t s a b o u t t h e t r a n s m e m b r a n e d i s t r i b u t i o n o f HCa 3™, h o w e v e r , a r e d i f f i c u l t t o make f r o m t h e p r e s e n t d a t a b e c a u s e o f t h e i n c l u s i o n o f more; t h a n one c h e m i c a l s p e c i e s i n t h e t e r m 'bHC0 3~' and b e c a u s e o f t h e p o s s i b i l i t y o f n u c l e a r : c y t o s o l i c d i s c o n t i n u i t i e s i n s i d e thee r e d c e l l . A s t r i k i n g f e 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 s p o n s e o f t r o u t r e d c e l l s was t h e c o n c u r r e n t i n h i b i t i o n o f n e t H C 0 3 ~ f l u x t h r o u g h t h e r e d c e l l s , a s i n d i c a t e d by t h e f u n c t i o n a l d e c r e a s e i n c e l l u l a r CA a c t i v i t y . T h i s i n h i b i t i o n o f n e t b i c a r b o n a t e f l u x d i d n o t r e p r e s e n t a d i r e c t e ^ f f e c t on t h e CA enzyme i t s e l f ? b e t a -a d r e n e r g i c a g o n i s t s had no e f f e c t on the; CA a c t i v i t y o f t r o u t h a e m a l y s a t e s . , Nor was i t . due t o a g e n e r a l a n i o n i m p e r m e a b i l i t y . In f a c t , i n h i b i t i o n o f a n i o n t r a n s p o r t w i t h S I T S a l s o i n h i b i t e d 127 t h e a d r e n e r g i c r e s p o n s e , a t l e a s t a s r e g a r d s c e l l s w e l l i n g . S e v e r a l m e c h a n i s m s c o u l d be i n v o l v e d i n t h i s a d r e n e r g i c i n h i b i t i o n o f n e t b i c a r b o n a t e f l u x . F i r s t l y , t h e K M of t h e e r y t h r o c y t e CA of f i s h i s l a r g e ( 1 2-300 mM, Maren and W i l e y 1966, G i r a r d and I s t i n 1975, Ha s w e l 1 1978) r e l a t i v e t o 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 b i c a r b o n a t e a t p h y s i o l o g i c a l C0s» t e n s i o n s ( 2 -5 mM, F i g . 3 and 20, T a b l e 5 ) . Thus, i n t r a c e l l u l a r COa.HCO3™ r e a c t i o n s p r o b a b l y e x h i b i t f i r s t o r d e r k i n e t i c s . As s u c h , d e c r e a s e s i n t h e c e l l u l a r 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 r e s p o n s e t o a d r e n a l i n e w o u l d d e p r e s s t h e c a t a l y s e d r a t e o f HCO3 d e h y d r a t i o n . S e c o n d l y , numerous s t u d i e s h a v e d e m o n s t r a t e d t h a t t h e s l o w e s t s t e p d u r i n g c a p i l l a r y COa e x c h a n g e i s H C 0«~/C1~ t r a n s p o r t a c r o s s t h e r e d c e l l membrane ( F o r s t e r and C r a n d a l l 1975, C r a n d a l l and B i d a n i 1981, F o r s t e r 1 9 8 2 ) . A d r e n e r g i c e x t r u s i o n o f HCO3"" f r o m t h e e r y t h r o c y t e d u r i n g C 0 a u n l o a d i n g o f b l o o d c o u l d c o n c e i v a b l y s l o w t h e n e t i n f l u x o f p l a s m a HCO3™ t o t h e c e l l and h e n c e d e p r e s s c e l l u l a r CA a c t i v i t y . I t i s i n t e r e s t i n g t o n o t e t h a t b o t h o f t h e s e m e c h a n i s m s w o u l d o p e r a t e m a x i m a l l y d u r i n g b r a n c h i a l c a p i l l a r y t r a n s i t when p l a s m a HCO3'"" i s t h e s u b s t r a t e o f p r i m a r y i m p o r t a n c e , w h e r e a s t h e y w o u l d h a v e l i t t l e i f any e f f e c t d u r i n g t i s s u e c a p i l l a r y t r a n s i t when C 0 Z i s t h e p r i m a r y s u b s t r a t e . What. i s t h e p o s s i b l e a d a p t a t i v e 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 o f f i s h e r y t h r o c y t e s ? The r e s t i n g l e v e l s o f p l a s m a c a t e c h o l a m i n e s m e a s u r e d i n t h e p r e s e n t s t u d y (3.1 nM a d r e n a l i n e , 1.2 nM n o r a d r e n a l i n e ) a r e among t h e l o w e s t y e t r e p o r t e d f o r f i s h ( l l a z e a u d and Maseaud 1981) and a r e c o m p a r a b l e t o t h e s e n o t e d by R i s t o r i et al. ( 1 9 7 9 ) ( 2 . 9 5 nM a d r e n a l i n e , 2.30 128 nM n o r a d r e n a l i n e ) -for r a i n b o w t r o u t . A c h a r a c t e r i s t i c o f t h e a d r e n e r g i c r e s p o n s e o f f i s h t o s t r e s s f u l s t i m u l i , i n c l u d i n g s t r e n u o u s e x e r c i s e , p h y s i c a l d i s t u r b a n c e , h y p o x i a and i n j u r y , i s a r a p i d and l a r g e e l e v a t i o n i n p l a s m a c a t e c h o l a m i n e l e v e l s , commonly t o l e v e l s e x c e e d i n g 1000 nM ( f o r r e v i e w s e e Mazeaud and Mazeaud 1 9 8 1 ) . Such s t r e s s f u l s t i m u l i g e n e r a l l y a r e a c c o m p a n i e d by l a c t a t e a c i d e m i a i n f i s h . The a d r e n e r g i c r e s p o n s e s o f f i s h r e d c e l l s p r o b a b l y e n h a n c e r e g u l a t i o n o f i n t r a c e l l u l a r pH and t h u s a m e l i o r a t e t h e e-f-fects o f t h i s m e t a b o l i c a c i d o s i s . N i k i n m a a ( 1 9 8 3 ) , f o r i n s t a n c e , h a s d e m o n s t r a t e d 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 s 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 n h a n c e D K - h a e m o g l o b i n a f f i n i t i y , p r i m a r i l y a s a r e s u l t o f H* e x t r u s i o n . M o r e o v e r , N i k i n m a a et al . (1984) h a v e shown 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 s o f s t r i p e d b a s s r e d c e l l s s e r v e t o m a i n t a i n a r t e r i a l o x y g e n c o n t e n t i n t h e f a c e o f m e t a b o l i c a c i d o s i s f a l l o w i n g p e r i o d s o f s t r e n u o u s e x e r c i s e . O b v i o u s l y , t h e d i s a d v a n t a g e s t o 0 S u p t a k e and r e l e a s e c o n f e r r e d by r e d u c t i o n i n t h e s u r f a c e a r e a t o v o l u m e r a t i o o f s w o l l e n e r y t h r o c y t e s ( J o n e s 1979) a r e o u t w e i g h e d by t h e a d v a n t a g e s t o 0^ a f f i n i t y c o n f e r r e d by a d r e n e r g i c H""' e x t r u s i o n . A d r e n e r g i c H C 0 3 ~ e x t r u s i o n w o u l d do l i t t l e t o a l t e r p l a s m a HC0 3"" l e v e l s d i r e c t l y i n a s much a s r e d c e l l s n o r m a l l y c o n t a i n v e r y l i t t l e H C 0 3 ~ and c o n s t i t u t e l e s s t h a n h a l f o f t h e b l o o d v o l u m e . However, t h e a s s o c i a t e d r e d u c t i o n i n n e t 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 w o u l d r e s u l t i n r e t e n t i o n o f p l a s m a H C O 3 " " 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 , i n d e p e n d e n t o f t h e e x a c t m o l e c u l a r m echanism i n v o l v e d i n t h a t r e d u c t i o n . HC0 3~" i s t h e p r i m a r y n o n - p r o t e i n b u f f e r i n b i o l o g i c a l 129 s y s t e m s ( A l b e r s 1970, Comroe 1 9 7 4 ) . Numerous s t u d i e s h a v e d e m o n s t r a t e d t h a t m y o c a r d i a l c e l l s , n e u r a l c e l l s and CSF u p t a k e H C 0 3 ~ d u r i n g p e r i o d s o f a c i d o s i s a n d / o r a d r e n e r g i c s t i m u l a t i o n ( R i e g l e and C l a n c y 1975, B o r o n and DeWeer 1976, F e n t o n et al , 1978, G o n z a l e z and C l a n c y 1981, Thomas 1982, Ahmad and L o e s c h c k e 1 9 8 3 ) , and t h u s i n c r e a s e t h e i r p h y s i o l o g i c a l b u f f e r i n g c a p a c i t i e s . M o r e o v e r , p l a s m a HCa3"~ f a c i l i t a t e s l a c t a t e r e l e a s e f r o m some s k e l e t a l m u s c l e i n a c i d o s i s ( H i r c h e et al. 1 9 7 5 ) . Thus, a d r e n e r g i c s w e l l i n g and t h e a c c o m p a n y i n g r e d u c t i o n i n n e t b i c a r b o n a t e f l u ; - : t h r o u g h f i s h e r y t h r o c y t e s c a n be c o n s i d e r e d an ' a l t r u i s t i c ' ' b e h a v i o u r by w h i c h t h e b e n e f i t s o f HC0 3"~ b u f f e r i n g a r e s h a r e d w i t h o t h e r c e l l s v i a t h e m i l i e u i n t e r i e u r . T h i s e x e m p l i f i e s t h e g e n e r a l p r i o r i t y i n v e r t e b r a t e s o f i n t r a c e l l u l a r pH r e g u l a t i o n o v e r e x t r a c e l l u l a r . In c o n c l u s i o n , f i s h e r y t h r o c y t e s a r e r e a d i l y p e r m e a b l e t o c h l o r i d e and b i c a r b o n a t e w h i c h t r a v e r s e t h e c e l l membrane v i a t h e Band 3 a n i o n e x c h a n g e p a t h w a y o r an a n a l o g o u s S I T S - s e n s i t i v e structure„ The i n h i b i t o r s o f CA p r e s e n t i n f i s h pI asma ( s e e C h a p t e r 2) h a v e no e f f e c t on e i t h e r membrane t r a n s p o r t o f a n i o n s o r on i n t r a c e l l u l a r CA. I o n t r a n s p o r t i n f i s h r e d c e l l s i s s i g n i f i c a n t l y i n f l u e n c e d , h o w e v e r , by c a t e c h o l a m i n e s . E-feta--a d r e n e r g i c a g o n i s t s a p p e a r t o s t i m u l a t e c o u p l e d Na HVH*", H C a 3 ~ / C l ~ e x c h a n g e r s , s i m i l a r t o t h o s e i n v o l v e d i n v o l u m e r e g u l a t o r y i o n movements i n a m p h i b i a n e r y t h r o c y t e s ( C a l a 1 9 8 0 ) . 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 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 n e t c e l l u l a r g a i n of Na*, C l " and' H 2 0 , a n e t c e l l u l a r l o s s o f H* and HC0 3-, a p r o n o u n c e d c e l l s w e l l i n g , and a . r e d u c t i o n i n n e t 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 r e d c e l l . U nder t h e s e c o n d i t i o n s , t h e t r a n s m e m b r a n e 130 d i s t r i b u t i o n o f H C 0 3 ~ may n o t be p a s s i v e . The a d r e n e r g i c r e s p o n s e s o f f i s h e r y t h r o c y t e s p r o b a b l y a r e i m p o r t a n t i n m a i n t a i n i ng 0 a t r an sp o r t an d i n enhanc i n g i n t r a ce11u1 a r b uf f e r i n g i n s t r e s s . The e f f e c t s o f a d r e n a l i n e ir> viva a r e i n v e s t i g a t e d i n t h e f o l l o w i n g c h a p t e r . 131 C h a p t e r 4„ EFFECTS O F A D R E N A L I N E O N C O a E X C R E T I O N AND ACID-BASE STATUS O F SALMO GAIRDNERI, IN VIVO The p r i n c i p a l p a t h w a y f o r C O a e x c r e t i o n i n f i s h , , a s i n o t h e r v e r t e b r a t e s , i s v i a movement of p l a s m a b i c a r b o n a t e i n t o t h e r e d b l o o d c e l l w h ere t h e b i c a r b o n a t e i s r a p i d l y d e h y d r a t e d t o f o r m C O a i n t h e p r e s e n c e o f c a r b o n i c a n h y d r a s e . T h i s C O a t h e n d i f f u s e s down i t s c o n c e n t r a t i o n g r a d i e n t f r o m t h e r e d c e l l i n t o t h e g a s e x c h a n g e medium ( w a t e r / a i r ) . C a t e c h o l a m i n e s i n f l u e n c e t h e C O a c a r r y i n g c a p a c i t y o f b l o o d in vitro, a l t e r t h e t r a n s m e m b r a n e d i s t r i b u t i o n o f H C 0 3 ~ i n r e d b l o o d c e l l s , and r e d u c e thee a c t i v i t y o f e r y t h r o c y t e ? CA csf r a i n b o w t r o u t b l o o d ( C h a p t e r 3 ) , As s u c h , a d r e n e r g i c s t i m u l a t i o n w o u l d be e x p e c t e d t o d e c r e a s e e x c h a n g e o f C O a i n r a i n b o w t r o u t in vivo.. The p r i m a r y reesponse t o many f o r m s of s t r e s s i n f i s h i s a r a p i d and o f t e n e x t r e m e l y l a r g e 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 l e v e l s ( N a k a n o and T o m l i n s o n 1967, Mazeaud e t a l . 1977, B u t l e r e t al„ 197S, 1 9 7 9 ) , The s e c o n d a r y e f f e c t s o f t h i s n e u r o - h o r m o n a l r e s p o n s e v a r y i n t h e i r d u r a t i o n and p a t t e r n a c c o r d i n g t o the? f i s h s p e c i e s u n d e r c o n s i d e r a t i o n and t h e n a t u r e o f t h e s t r e s s ( f o r r e v i e w s e e Mazeaud and Mazeaud 1 9 8 1 ) , In g e n e r a l , h o w e v e r , t r a n s p o r t o f b l o o d g a s e s i s i n f l u e n c e d by c a t e c h o l a m i n e s v i a c a r d i o v a s c u l a r e f f e c t s i n c l u d i n g i n c r e a s e d c a r d i a c : o u t p u t ( J o n e s and R a n d a l l 1978, R a n d a l l 1982a, b ) , l a m e l l a r r e c r u i t m e n t ( H o l b e r t e t a l . 1 9 7 9 ) , and i n c r e a s e d p e r m e a b i l i t y o f g i l l 132 e p i t h e l i u m ( I s a i a , e t al. 1978a, b). As w e l l , O s - h a e m o g l o b i n a f f i n i t y i n c r e a s e s i n s t r e s s due t o a d r e n e r g i c H"1* e x t r u s i o n f r o m t h e r e d c e l l and a r e d u c t i o n i n e r y t h r o c y t i c n u c l e o s i d e t r i p h o s p h a t e l e v e l s ( N i k i n m a a 1983, N i k i n m a a e t al, 1984)= Thus, c a t e c h o l a m i n e s w o u l d a p p e a r t o m o d u l a t e t r a n s p o r t and e x c h a n g e o f b o t h o x y g e n and c a r b o n d i o x i d e i n s t r e s s v i a n o t o n l y c a r d i o v a s c u l a r e f f e c t s , b u t a l s o v i a d i r e c t m o d u l a t i o n o f b l o o d r e s p i r a t o r y p r o p e r t i e s . The a d r e n e r g i c r e s p o n s e s of f i s h e r y t h r o c y t e s w o u l d t e n d t o f a v o u r 0 3 e x c h a n g e i n s t r e s s , b u t t o d i s f a v o u r C D s * e x c h a n g e . The o b j e c t i v e o f t h e p r e s e n t s t u d y was t o e x a m i n e t h e e f f e c t s o f a d r e n a l i n e on t h e t r a n s p o r t and exchangee o f COs» and Q s i n Sal mo gairdneri„ I n j e c t i o n o f c a t e c h o l a m i n e s i n t o t h e c i r c u l a t o r y s y s t e m o f f i s h e l i c i t s s e c o n d a r y e f f e c t s v i r t u a l l y i d e n t i c a l t o t h o s e a s s o c i a t e d w i t h s t r e s s ( P e y r a u d - W a i t z e n e g g e r 1979, Mazeaud and l i a z e a u d 1981, N i k i n m a a 1 9 8 2 ) . I n t h e p r e s e n t s t u d y , t h e r e s p i r a t o r y and a c i d - b a s e s t a t e s o f v e n o u s and a r t e r i a l b l o o d s w e re a s s e s s e d s i m u l t a n e o u s l y i n o r d e r t o d e t e r m i n e the-; e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e on •__ u p t a k e and C O s a e x c r e t i o n . 133 MATERIALS AND METHODS Ra i n b o w t r o u t (300-400 g) were i m p l a n t e d w i t h b o t h d o r s a l and v e n t r a l a o r t i c c a n n u l a e a 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 M e t h o d s . F i s h a l s o w e re f i t t e d w i t h a b u c c a l c a n n u l a . B u c c a l c a n n u l a e were c o n s t r u c t e d f r o m s e c t i o n s o f p o l y e t h y l e n e s u r g i c a l t u b i n g (PE-90) w h i c h had been h e a t - f l a r e d a t one end. The f l a r e d end was i m p l a n t e d i n t h e b u c c a l c a v i t y . B u c c a l c a n n u l a e e x i t e d t h e f i s h v i a a f l a n g e d s e c t i o n o f s u r g i c a l t u b i n g (PE-200) w h i c h p a s s e d t h r o u g h t h e r o o f o f t h e mouth i n f r o n t o f t h e n a r e s . A l l c a n n u l a e were c o n n e c t e d t o p r e s s u r e s t r a n s d u c e r s (Statha\m P23Db) by way o f 3-way s t o p c o c k s t o a l l o w c o n t i n u o u s r e c o r d i n g o f b l o o d and b u c c a l p r e s s u r e s . The 3-way s t o p c o c k s p r o v i d e d s a m p l i n g and i n j e c t i o n p o r t s . Some f i s h weere g i v e n a 0.5 m L ~ b o l u s i n j e c t i o n o f e i t h e r C o r t l a n d s a l i n e o r <•-••) - a d r e n a l i ne ( 5 - 1 0 " 1 1 mol - g f i s h " " 1 i n C o r t l a n d s a l i n e ) v i a t h e d o r s a l a o r t i c c a n n u l a . T h i s i n j e c t i o n p r o d u c e d an e s t i m a t e d 10 _ < s* M i n c r e a s e i n 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 s , a s s u m i n g t h e b l o o d v o l u m e t o be 57. o f body weight. ( S m i t h 1 9 6 6 ) . O t h e r f i s h were i n j e c t e d c o n t i n u o u s l y f o r a p e r i o d o f 1 h and a t a r a t e o f 0.5 mL-h" 1 w i t h ( - ) - a d r e n a l i ne < 10~"* M i n C o r t l a n d s a l i n e ) v i a t h e d o r s a l a o r t i c c a n n u l a . T h i s i n j e c t i o n p r o d u c e d an e s t i m a t e d :l.0~"<& M i n c r e a s e i n b l o o d a d r e n a l i n e ? l e v e l s w i t h i n 24 min and an e s t i m a t e d 2.5-10"* M i n c r e a s e a f t e r 1 h. A m b i e n t w a t e r t e m p e r a t u r e was 10 C. At known t i m e i n t e r v a l s , b l o o d s a m p l e s were w i t h d r a w n 134 s i m u l t a n e o u s l y f r o m b o t h t h e d o r s a l and v e n t r a l a o r t i c c a n n u l a s . T h ese s a m p l e s were r e p l a c e d w i t h an e q u i v a l e n t v o l u m e o f C o r t l a n d s a l i n e . A p o r t i o n o f e a c h b l o o d s a m p l e was u s e d t o m e a s u r e h a e m a t o c r i t i n d u p l i c a t e and t o t a l Oss c o n t e n t <Coa;) » C02 was m e a s u r e d w i t h an o x y g e n e l e c t r o d e ( R a d i o m e t e r , t y p e E 5 0 4 6 ) u s i n g t h e m e t h o d s o f T u c k e r ( 1 9 6 7 ) . The r e m a i n d e r o f t h e s a m p l e s were c e n t r i f u g e d a t room t e m p e r a t u r e f o r 1 - 2 m i n . A l l s u b s e q u e n t m e a s u r e m e n t s were made on p l a s m a . P a r s and pH v a l u e s were m e a s u r e d u s i n g an I n s t r u m e n t a t i o n L a b o r a t o r y M i c r o 1 3 , p H / b l o o d g a s a n a l y s e r w i t h a s s o c i a t e d e l e c t r o d e s and c a l i b r a t i o n s t a n d a r d s t h e r m o s t a t t e d a t 1 0 C. C C o a v a l u e s were m e a s u r e d u s i n g t h e methods o f Cameron ( 1 9 7 1 ) a s d e s c r i b e d p r e v i o u s l y . P e o a v a l u e s were c a l c u l a t e d u s i n g e q u a t i o n 8 and t h e v a l u e s o f aC0s> and p K « p p o b t a i n e d i n C h a p t e r 1 . A r e a mean b l o o d p r e s s u r e , h e a r t r a t e and v e n t i l a t i o n r a t e were t a k e n f r o m t h e pressure-? r e c o r d s . 135 R E S U L T S I n j e c t i o n o f a d r e n a l i n e i n t o t h e d o r s a l a o r t a o f Salmo gai rdneri p r o d u c e d s i g n i f i c a n t c h a n g e s i n t h e r e s p i r a t o r y and a c i d - b a s e s t a t e s o f t h e f i s h ( F i g . 22 and 2 3 ) . I n j e c t i o n o f an e q u i v a l e n t v o l u m e o f s a l i n e was w i t h o u t e f f e c t ( T a b l e 1 1 ) . E l e v a t i o n o f 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 s was a s s o c i a t e d w i t h a s i g n i f i c a n t t r a n s i e n t d e c r e a s e i n t h e g a s e x c h a n g e r a t i o (R -rfCcoa/dCos)(Fig, 2 4 ) . I t s h o u l d be n o t e d , h o w e v e r , t h a t s i n c e t h e r e p o r t e d R v a l u e s w e r e c a l c u l a t e d u s i n g t r u e p l a s m a C c o a , t h e r e p o r t e d v a l u e s o v e r e s t i m a t e t h e a c t u a l R v a l u e s ? t r u e p l a s m a C Co2 i s n e c e s s a r i l y g r e a t e r t h a n w h o l e b l o o d C C o = ( s e e F i g u r e 3 ) . N o n e t h e l e s s , t h e r e p o r t e d v a l u e s d e m o n s t r a t e t h a t t h e r a t e o f C0:s e x c r e t i o n d e c r e a s e d , r e l a t i v e t o t h e r a t e o f Ors u p t a k e , f o l l o w i n g a d r e n a l i n e i n j e c t i o n . The r e d u c t i o n i n R was f u l l y c o m p e n s a t e d •for w i t h i n 60 min o f the? i n j e c t i o n . R e d u c t i o n s i n R were a c c o m p a n i e d by a s i g n i f i c a n t i n c r e a s e i n p l a s m a Ceora and P M 2 , and a s i g n i f i c a n t d e c r e a s e i n p l a s m a pH ( F i g . 2 2 ) . C h a n g e s i n Cco2, F ' c o z and pH were f u l l y c o m p e n s a t e d f o r w i t h i n t h e 120—min t i m e f r a m e o f c o n t i n u o u s - i n j e c t i o n s t u d i e s , b u t d i d n o t r e t u r n t o p r e t r e a t m e n t l e v e l s w i t h i n t h e s h o r t e r t i m e f r a m e (45 min) o f b o l us>-i n j e c t i on s t u d i e s . I n j e c t i o n o f a d r e n a l i n e e i t h e r r e v e r s e d t h e d i r e c t i o n o f o r a b o l i s h e d a r t e r i a l - v e n o u s d i f f e r e n c e s i n pH and P Co2 ( F i g . 2 2 ) . P r i o r t o i n j e c t i o n o f a d r e n a l i n e , a r t e r i a l p l a s m a was more b a s i c and c o n t a i n e d l e s s f r e e C 0 S ( P C D E ) t h a n i t s v e n o u s c o u n t e r p a r t . 136 F i g u r e 22- A r t e r i a l - v e n o u s d i f f e r e n c e s i n pH, C C o 2 and P o o r s a c r o s s t h e g i l l s o f Sal mo gairdneri, s h o w i n g t h e e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e a s a b o l u s < 5 - 1 G"~ *-l m o l - g f i s h - 1 ) and a s a c o n t i n u o u s s t r e a m ( 5 - 1 0 " - r a m o l - h - 1 ) , , A r t e r i a l v a l u e s a r e shown by open c i r c l e s - V enous v a l u e s a r e shown by s o l i d c i r c l e s - V a l u e s a r e means + 1 S.E. ( n ~ 3 ) . V a l u e s d e n o t e d w i t h + a r e d i f f e r e n t s i g n i f i c a n t l y f r o m t h e t i m e zero v a l u e a t P < 0 - 0 5 ( p a i r e d t - t e s t ) . A r t e r i a l v a l u e s d e n o t e d w i t h * a r e d i f f e r e n t s i g n i f i c a n t l y f r o m t h e c o r r e s p o n d i n g v e n o u s v a l u e a t P < 0 . 0 5 ( p a i r e d t - t e s t ) . 137 T I M E , min 138 F i g u r e 23, A r t e r i a l - v e n o u s d i f f e r e n c e s i n C o s , P 0 2 and h a e m a t o c r i t a c r o s s t h e g i l l s o f Sal mo gairdneri, s h o w i n g t h e e f f e c t s o f i n j e c t i o n o f a d r e n a l i n e a s a b o l u s ( 5 " 1 0 ~ 1 1 mol-g f i s h - 1 ) and a s a c o n t i n u o u s s t r e a m ( 5 - I 0 ~ r a m o l - h - 1 ) . F^est of c a p t i o n a s i n F i g u r e 22. 139 T I M E , min 140 T a b l e 11. R e s p i r a t o r y and a c i d - b a s e c h a r a c t e r i s t i c s o-f a r t e r i a l p l a s m a o f Salmo g a i r d n e r i , s h o w i n g e f f e c t s o f a b o l u s i n j e c t i o n of 0„5 mL o f C o r t l a n d s a l i n e . V a l u e s a r e means ± 1 S.E. (n=5). H c t , h a e m a t o c r i t . Time p o s t - i n j e c t i o n (min) 15 90 pH 8.046 +• 0.019 8.049 + 0.027 8.057 + 0.027 (mM) 9.21 + 0.43 9.33 + 0.45 9.05 + 0.32 F ' c 0 2 ( t o r r ) !.00 + 0.05 2.02 + 0. 11 1.92 + 0,07 H c t 17.9 + 1.6 16.6 + 1.5 14.7 + 1.1 141 F i g u r e 24. Gas e x c h a n g e r a t i o (R = d plasmaC Coz/d bloodCos*) o-f Salmo gairdneri, s h o w i n g t h e e f f e c t s o-f i n j e c t i o n o-f a d r e n a l i n e a s a b o l u s <5- 10~':t 1 mol-g - f i s h " " 1 ) and a s a c o n t i n u o u s s t r e a m (5-10~° m o l - h " * ) . V a l u e s a r e means + 1 S.E. <n=3). V a l u e s d e n o t e d by •+• a r e d i f f e r e n t s i g n i f i c a n t l y f r o m t h e t i m e z e r o v a l u e a t P<0.05 ( p a i r e d t - t e s t ) . GAS EXCHANGE RATIO zvi 143 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 , , h o w e v e r , a r t e r i a l p l a s m a t e n d e d t o be more a c i d i c t h a n and had about, t h e same P C o z a s v e n o u s p l a s m a , d e s p i t e t h e o b s e r v a t i o n t h a t f i s h a t a l l t i m e s m a i n t a i n e d a n e g a t i v e a r t e r i a l - - v e n o u s d i f f e r e n c e i n C C o a , t h a t i s , p o s i t i v e e x c r e t i o n o f COa. A r t e r i. a l - v e n o u s d i f f e r e n c e s i n pH and F'caz r e t u r n e d t o p r e t r e a t m e n t c o n d i t i o n s w i t h i n 60 min f o l l o w i n g t h e i n j e c t i on. The s i g n i f i c a n t a r t e r i a l a c i d o s i s a s s o c i a t e d w i t h e l e v a t e d a d r e n a l i n e l e v e l s was n o t a c c o m p a n i e d by a c o r r e s p o n d i n g d e c r e a s e i n b l o o d Coa ( F i g . 23) w h i c h one m i g h t h a v e e x p e c t e d due t o B o h r and Root. e f f e c t s . E<lood o x y g e n c o n t e n t a c t u a l l y t e n d e d t o i n c r e a s e a s p l a s m a pH f e l l , a l t h o u g h t h e c h a n g e s i n Coa were n o t s i g n i f i c a n t s t a t i s t i c a l l y . T h i s a b s e n c e of a pH e f f e c t on C p 2 was n o t a t t r i b u t a b l e t o an e f f e c t o f a d r e n a l i n e on b l o o d PoaS a r t e r i a l and v e n o u s F'osa l e v e l s r e m a i n e d u n c h a n g e d t h r o u g h o u t t h e s t u d i e s ( F i g . 2 3 ) . I t i s c o n c e i v e a b l e t h a t t h e a b s e n c e of a pH e f f e c t was a t t r i b u t a b l e t o i n c r e a s e d Oa a f f i n i t y o f b l o o d ( c f . N i k i n m a a 1983, N i k i n m a a e t al, 1 9 8 4 ) . However, r e c r u i t m e n t o f a d d i t i o n a l e r y t h r o c y t e s i n t o t h e c i r c u l a t i n g b l o o d c a n n o t be r u l e d o u t , s i n c e h a e m a t o c r i t a l s o t e n d e d t o i n c r e a s e i n c o n c e r t w i t h t h e d e c r e a s e i n p l a s m a pH ( F i g . 2 3 ) . A d r e n a l i n e had s i g n i f i c a n t e f f e c t s on t h e c a r d i o v a s c u l a r s y s t e m of Salmo gairdneri ( F i g . 2 5 ) . B o l u s i n j e c t i o n o f a d r e n a l i n e p r o d u c e d an i m m e d i a t e i n c r e a s e i n a r t e r i a l and v e n o u s b l o o d p r e s s u r e s . B l o o d p r e s s u r e s p e a k e d w i t h i n 3 min of t h e b o l u s i n j e c t i o n and t h e n s l o w l y d e c r e a s e d , r e t u r n i n g t o p r e t r e a t m e n t l e v e l s w i t h i n 45 min o f t h e i n j e c t i o n . C o n t i n u o u s i n j e c t i o n o f a d r e n a l i n e a l s o p r o d u c e d an i n c r e a s e i n b l o o d 144 F i g u r e 25- E f f e c t s o f a d r e n a l i n e i n j e c t i o n a s a b o l u s ( 5 - 1 0 " " 1 1 mol-g f i s h - 1 ) and a s a c o n t i n u o u s s t r e a m (5-10"~~ mol-h™' 1) on a r e a mean b l o o d p r e s s u r e , h e a r t r a t e and v e n t i l a t i o n r a t e o f Salmo gairdneri. R e s t o f c a p t i o n a s i n F i g u r e 22. 145 T I M E , min 146 p r e s s u r e s , a l b e i t t h i s i n c r e a s e o c c u r r e d a t a much s l o w e r r a t e . In t h e s e s t u d i e s , b l o o d p r e s s u r e s c o n t i n u e d t o i n c r e a s e s l o w l y t h r o u g h o u t t h e e n t i r e 6 0-min i n j e c t i o n p e r i o d and r e m a i n e d e l e v a t e d f o r more t h a n 60 min f o l l o w i n g t h a t p e r i o d . , A d r e n a l i n e had no d e t e c t a b l e e f f e c t , on v e n t i l a t i o n r a t e and o n l y a m o d e r a t e e f f e c t on h e a r t r a t e p r o d u c i n g a m i l d b r a d y c a r d i a w h i c h was c o m p e n s a t e d f o r w i t h i n 60 min of t h e i n j e c t i o n ( F i g . 2 5 ) . 147 DISCUSSION E l e v a t i o n o f b l o o d a d r e n a l i n e l e v e l s i n Salmo gai rdneri was a s s o c i a t e d w i t h a t r a n s i e n t r e d u c t i o n i n CO^ c l e a r a n c e and an i n c r e a s e i n body COs> s t o r e s . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h an a d r e n e r g i c r e d u c t i o n i n n e t HCQ3-~ f l u x t h r o u g h t h e r e d c e l l 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 . S i m i l a r i n c r e a s e s i n b l o o d C 0 0 2 and F'co2 h a v e been o b s e r v e d f o l l o w i n g w h o l e - b o d y i n h i b i t i o n o f c a r b o n i c a n h y d r a s e i n d o g f i s h (Squalus acanthias)(Maren 1 9 6 2 ) , l a k e t r o u t (Salvelin us namaycusb) ( H o f f e r t and Fromm 1 9 6 6 ) , and r a i n b o w t r o u t ( H o f f e r t and Fromm 1 9 7 3 ) . S e l e c t i v e i n h i b i t i o n o f e r y t h r o c y t e ? CA w i t h an a m i n o t h i a d i a z o l e d e r i v a t i v e s (CL 1 1 , 3 6 6 ) , w h i c h r e p o r t e d l y l a c k s a c c e s s t o g i l l CA, h a s shown t h a t i n c r e a s e s i n body COs s t o r e s f o l l o w i n g CA i n h i b i t i o n ares due s o l e l y t o r e d c e l l e f f e c t s (Maren and Maren 1 9 6 4 ) . P e r r y et al. (1982) h a v e d e m o n s t r a t e d w i t h a b l o o d - p e r f u s e d r a i n b o w t r o u t p r e p a r a t i o n t h a t S I T S - i n h i b i t i o n o f t h e Band 3 a n i o n e x c h a n g e p a t h w a y i n e r y t h r o c y t e s a l s o c a u s e s a r e d u c t i o n i n C 0 3 e x c r e t i o n and an i n c r e a s e i n body C O a s t a r e s . T h i s r e i t e r a t e s t h a t i n h i b i t i o n o f e r y t h r o c y t e a n i o n t r a n s p o r t h a s t h e same f u n c t i o n a l e f f e c t on b r a n c h i a l CO^ e x c h a n g e a s i n h i b i t i o n o f e r y t h r o c y t e CA ( C h a p t e r 3 ) . A d r e n e r g i c r e d u c t i o n s i n CO^ e x c r e t i o n p r o b a b l y were r e l a t e d t o a d r e n e r g i c H C 0 3 ~ e x t r u s i o n f r o m t h e r e d c e l l , r a t h e r t h a n t h e r e s u l t , o f g e n e r a l a n i o n i m p e r m e a b i l i t y o r d i r e c t i n h i b i t i o n o f t h e CA enzyme ( C h a p t e r 3 ) . A d r e n e r g i c HCQ 3~ e x t r u s i o n f r o m t h e r e d c e l l w o u l d r e d u c e n e t H C 0 3 ~ i n f l u x c i u r i n g 148 b r a n c h i a l b l o o d t r a n s i t when p l a s m a HCO3™ i s t h e s u b s t r a t e o-f c o n c e r n -for e r y t h r o c y t e C A , b u t w o u l d h a v e l i t t l e i-f any e f f e c t on C O a e x c h a n g e d u r i n g t i s s u e b l o o d t r a n s i t when C O a i s t h e s u b s t r a t e o f c o n c e r n . P e r r y et al* (1982) n o t e d t h a t i n h i b i t i o n o f e r y t h r o c y t e H C 0 3 - / C 1 ~ e x c h a n g e s i g n i f i c a n t l y r e d u c e d O a u p t a k e i n a b l o o d -p e r f u s e d r a i n b o w t r o u t p r e p a r a t i o n . P r e s u m a b l y , i n h i b i t i o n o f HCO3™ i n f l u x t o t h e r e d c e l l and t h e r e s u l t a n t r e d u c t i o n i n t h e i n t r a c e l l u l a r l--IC03~ d e h y d r a t i o n r e a c t i o n a l l o w e d o x y - l a b i l e H * " t o a c c u m u l a t e s u f f i c i e n t l y w i t h i n t h e r e d c e l l t o s l o w B o h r - o n and R o o t - o n s h i f t s 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 . U nder t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s , h o w e v e r , a d r e n e r g i c r e d u c t i o n o f n e t HCO3"" f l u x t h r o u g h S. gairdneri e r y t h r o c y t e s had no e f f e c t on 0 S l o a d i n g o f b l o o d . T h i s e m p h a s i z e s t h a t t h e o b s e r v e d r e d u c t i o n i n C O a e x c r e t i o n was n o t due s i m p l y t o an a d r e n e r g i c r e s p o n s e o f t h e c a r d i o v a s c u l a r s y s t e m i n a s much a s c a r d i o v a s c u l a r r e s p o n s e s w o u l d a l t e r C O a e x c r e t i o n and O a u p t a k e i n t h e same d i r e c t i o n . O a u p t a k e i n t h e p r e s e n t in vivo s t u d y p r o b a b l y was m a i n t a i n e d i n t h e f a c e o f a r e d u c t i o n i n C O a e x c r e t i o n by a c o m b i n a t i o n o f f a c t o r s . a d r e n e r g i c H" 1 " e x t r u s i o n f r o m t h e r e d c e l l s w h i c h w o u l d p a r t i a l l y o f f s e t t h e r e d u c t i o n i n e r y t h r o c y t e b i c a r b o n a t e b u f f e r i n g c a p a c i t y ? a d e c r e a s e i n e r y t h r o c y t e n u c l e o s i d e t r i p h o s p h a t e l e v e l s i n r e s p o n s e t o a d r e n a l i n e w h i c h e n h a n c e s 0 a -h a e m o g l o b i n b i n d i n g a f f i n i t y ( N i k i n m a a 1983, N i k i n m a a et a l „ 1984)5 h a e m o c o n c e n t r a t i o n due t o s p l e n i c c o n t r a c t i o n and t h e r e l e a s e o f a d d i t i o n a l e r y t h r o c y t e s , and t o d i u r e s i s ( S t e v e n s 1968, Wood and R a n d a l l 1973, N i l s s o n et a l . 197 /5)5 and a d r e n e r g i c r e s p o n s e s o f thee c a r d i o v a s c u l a r s y s t e m ( f o r r e v i e w s e e 149 J a n e s and R a n d a l l 1978, R a n d a l l 1982a, b ) . M a i n t e n a n c e o-f O a l o a d i n g when c a t e c h o l a m i n e s a r e r e l e a s e d i n s t r e s s w o u l d e n s u r e O a t r a n s p o r t t o t h e t i s s u e s i n t h o s e s i t u a t i o n s . A t t h e same t i m e , r e t e n t i o n o f p l a s m a H C 0 3 ~ w o u l d m a i n t a i n an e x t r a c e l l u l a r HC0 3"" p o o l w h i c h t h e n w o u l d be a v a i l a b l e t o e n h a n c e t h e p h y s i o l o g i c a l b u f f e r i n g c a p a c i t y o f i n t r a c e l l u l a r c o m p a r t m e n t s . R e s p i r a t o r y H * a s s o c i a t e d w i t h t h i s H C 0 3 ~ r e t e n t i o n c o u l d r e m a i n i n t h e e x t r a c e l l u l a r f l u i d s o r be e x c r e t e d a c r o s s t h e g i l l s . F i s h g i l l e p i t h e l i u m i s v e r y p e r m e a b l e t o H* ( M c W i l l i a m s and P o t t s 1978) and r e c e n t e v i d e n c e (van den T h i l l a r t et ai. 1983) i n d i c a t e s t h a t C O a and H"1" e x c r e t i o n a c r o s s t h e g i l l v a r y i n d e p e n d e n t l y o f e a c h o t h e r . The r e s p o n s e s o f r a i n b o w t r o u t t o e x o g e n o u s a d r e n a l i n e i n c l u d e d a t r a n s i e n t d i s r u p t i o n o f t h e arterial-venous d i f f e r e n c e s i n p l a s m a pH and F ' c o a a c r o s s t h e g i l l . T y p i c a l l y , a r t e r i a l p l a s m a h a s a l o w e r C O a c o n t e n t , a l o w e r C O a t e n s i o n and i s more b a s i c t h a n i t s v e n o u s c o u n t e r p a r t . F o l l o w i n g i n j e c t i o n of a d r e n a l i n e , h o w e v e r , arterial p l a s m a had a s i m i l a r P C o a a s and t e n d e d t o be more a c i d i c t h a n v e n o u s p l a s m a , d e s p i t e t h e o b s e r v a t i o n t h a t a net. l o s s o f C O a was s t i l l m a i n t a i n e d 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 . T h e s e r e s u l t s c o u l d h a v e b e e n o b t a i n e d o n l y f r o m b l o o d t h a t was f a r f r o m C O s : H C 0 3 _ c h e m i c a l e q u i l i b r i u m a t t h e t i m e t h e b l o o d was w i t h d r a w n in vivo. When- i n c h e m i c a l e q u i l i b r i u m , an a q u e o u s s o l u t i o n w i t h a l o w e r C C o a a l s o h a s a l o w e r F'co2 and a h i g h e r p H t h a n a s o l u t i o n w i t h a h i g h e r Cco2. In t h e p r e s e n t s t u d y , n e t interconversion o f C C O a + H a 0 3 and CHCO3'"" + H"*"3 must h a v e c o n t i n u e d t o o c c u r d u r i n g the? b l o o d 150 s a m p l i n g and measurement p r o c e d u r e s . T h u s , t h e o b s e r v e d pH and Pcoa v a l u e s d i d n o t r e f l e c t t h e a c t u a l pl-l and Poos o f a r t e r i a l p l a s m a a t t h e s i t e o f w i t h d r a w a l ir> vivo. A c h e m i c a l d i s e q u i l i b r i u m i n a r t e r i a l b l o o d a l s o i s c o n s i s t e n t w i t h an a d r e n e r g i c i n h i b i t i o n o f n e t HC0 3"" f l u x t h r o u g h t h e r e d c e l l 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 . F i s h g i l l e p i t h e l i u m i s v e r y p e r m e a b l e t o C 0 E b u t r e l a t i v e l y i m p e r m e a b l e t o H C 0 3 ~ ( P e r r y et al . 19B.2) , and c o n s e q u e n t l y t h e b u l k o f CO-: e s x c r e t e d 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 e x i t s t h e b l o o d a s C0 f f i ( s e e T a b l e 5 ) . In t h e a b s e n c e o f c a t e c h o l a m i n e e f f e c t s , CO^; l-IC0 3~ c h e m i c a l e q u i l i b r i u m i s m a i n t a i n e d d u r i n g b r a n c h i a l CG^ exchanges v i a c a t a l y s i s o f COs*:; H C 0 3 " r e a c t i o n s by e r y t h r o c y t e CA, The u n c a t a l y s e d r a t e o f C0=»: HCQ3"~ r e a c t i o n s i s a t 1 e a s t one o r d e r o f m a g n i t u d e t o o s l o w t o e n a b l e c h e m i c a l e q u i l i b r i u m t o be a c h i e v e d 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 ( C h a p t e r 2 ) . 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 o f f i s h e r y t h r o c y t e s , h o w e v e r , t h e f u n c t i o n a l a c t i v i t y o f e r y t h r o c y t e CA i s r e d u c e d and c o n s e q u e n t l y t h e r a t e o f H C 0 3 ~ d e h y d r a t i o n 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 c a n no l o n g e r keep p a c e w i t h d i f f u s i o n o f d i s s o l v e d COs. As a r e s u l t , H C 0 3 " i s r e t a i n e d i n t h e p l a s m a and t h e b l o o d i s no l o n g e r a b l e t o a t t a i n COaSHCOrs"" c h e m i c a l e q u i l i b r i u m d u r i n g b r a n c h i a l t r a n s i t . C h e m i c a l e q u i l i b r i u m w o u l d be a t t a i n e d i n v e s s e l s d o w n s t r e a m o f t h e g i l l , and w o u l d be a c c o m p a n i e d t h e o r e t i c a l l y by an i n c r e a s e i n b o t h a r t e r i a l Poos and pH. A r t e r i a l pH u n d e r s u c h c i r c u m s t a n c e s , h o w e v e r , a l s o w o u l d be i n f l u e n c e d by H* movements a c r o s s t h e g i l l ( M c W i l l i a m s and P o t t s 1978, v a n den T h i l l a r t et al . 1983) and by a d r e n e r g i c H"*" e x t r u s i o n f r o m r e d c e l l s ( C h a p t e r 3) . 151 The e f f e c t s o f a d r e n a l i n e were t r a n s i e n t i n n a t u r e . T h i s i s t o be e x p e c t e d f o r s e v e r a l r e a s o n s . F i r s t l y , a r e d u c t i o n i n C 0 S e x c r e t i o n c a u s e s t h e d i f f u s i o n g r a d i e n t f o r d i s s o l v e d C O a a t t h e g a s e x c h a n g e o r g a n t o i n c r e a s e . E v e n t u a l l y , t h e p o i n t w i l l be r e a c h e d a t w h i c h s i m p l e d i f f u s i o n o f C O a m a t c h e s m e t a b o l i c p r o d u c t i o n o f COs» and a s t e a d y - s t a t e i s r e - e s t a b l i s h e d . Swenson and Maren <1978) h a v e e x a m i n e d t h e e f f e c t s of t o t a l i n h i b i t i o n o f e r y t h r o c y t e C A on C0 f f i e x c r e t i o n i n mammals. They f o u n d t h a t , a t r e s t i n t h e p r e s e n c e o f e r y t h r o c y t i c C A a c t i v i t y , 7E37. o f e x c r e t e d COSJ was d e r i v e d f r o m 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 H C0 3~, 107. was f r o m d i s s o l v e d C0 : a, and 1 1 % was f r o m c a r b a m i n o compounds ( s e e T a b l e 5 f o r f i s h v a l u e s ) . T o t a l i n h i b i t i o n o f e r y t h r o c y t e C A had no e f f e c t on t h e s t e a d y - s t a t e rates o f C O s e x c r e t i o n . In t h e a b s e n c e of C A , h o w e v e r , 807. o f e x c r e t e d C O - , was d e r i v e d f r o m d i s s o l v e d C D s , 4% was f r o m u n c a t a l y s e d H C 0 3 ~ d e h y d r a t i o n , and 1 1 % was f r o m c a r b a m i n o compounds. S e c o n d l y , w h i l e c a t e c h o l a m i n e s a r e r e l a t i v e l y s t a b l e i n f i s h b l o o d ( h a l f - t i m e s o f 65.8 min and 163 min f o r a d r e n a l i n e and n o r a d r e n a l i n e , r e s p e c t i v e l y , (Mazeaud 1 9 7 9 ) , c a t e c h o l a m i n e s g e n e r a l l y a r e r a p i d l y d e a c t i v a t e d , a t l e a s t i n mammalian b l o o d (Goodman and G i l m a n 1 9 5 6 ) . L a s t l y , a d r e n a l i n e i n j e c t i o n s i n i n t a c t a n i m a l s c a u s e a m u l t i t u d e o f c a r d i o v a s c u l a r and m e t a b o l i c e f f e c t s w h i c h t h e n l e a d t o t h e i n i t i a t i o n o f r e f 1 ex e s t o c oun t e r ac t t hem. The c a r d i o v a s c u l a r r e s p o n s e s o f f i s h t o c a t e c h o l a m i n e s h a v e been r e v i e w e d by R a n d a l 1 (1970b, 1982a, b) and by J o n e s and R a n d a l l ( 1 9 7 8 ) . The p r e s e n t r e s u l t s a r e c o n s i s t e n t w i t h t h e known e f f e c t s o f a d r e n a l i n e on t h e c a r d i o v a s c u l a r s y s t e m o f f i s h : 152 a p o s i t i v e i n o t r o p i c e f f e c t o f t h e h e a r t ? a d i l a t i o n o f g i l l v a s c u l a t u r e and d e c r e a s e i n g i l l r e s i s t a n c e ? and a r i s e i n b l o o d p r e s s u r e w h i c h e l i c t s a b a r o r e c e p t o r r e - f l e x c a u s i n g s i m u l t a n e o u s v a g a l b r a d y c a r d i a . In c o n c l u s i o n , t h e a d r e n e r g i c r e s p o n s e s o f i n t a c t r a i n b o w t r o u t a t r e s t i n c l u d e d a t r a n s i e n t , r e d u c t i o n i n C O a e x c r e t i o n , an i n c r e a s e i n b ody C O a s t o r e s , and a d i s r u p t i o n o f a r t e r i a l - v e n o u s d i f f e r e n c e s i n p l a s m a pH and F ' c o a . A l l t h e s e r e s u l t s a r e c o n s i s t e n t w i t h an a d r e n e r g i c r e d u c t i o n i n n e t H C O 3 - f l u x t h r o u g h t h e r e d c e l l 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 . O a u p t a k e by b l o o d was u n a f f e c t e d by t h i s r e t e n t i o n of C 0 3 . N i k i n m a a et s i . (1984) h a v e d e m o n s t r a t e d 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 s of f i s h e r y t h r o c y t e s ' s e r v e t o m a i n t a i n a r t e r i a l O a c o n t e n t i n s t r e s s . The p r e s e n t s t u d y i n d i c a t e s t h a t t h e s e a d r e n e r g i c r e s p o n s e s a l s o s e r v e t o m a i n t a i n an e x t r a c e l l u l a r p o o l of H C O 3 - i n s t r e s s w h i c h t h e n c a n be u s e d t o e n h a n c e i n t r a c e l l u l a r b u f f e r i n g c a p a c i t i e s . 1 5 3 GENERAL DISCUSSION T h i s t h e s i s h a s e x a m i n e d t h e t r a n s p o r t and e x c r e t i o n o f c a r b o n d i o x i d e i n f i s h , , and t h e r o l e o f t h e r e d b l o o d c e l l i n t h a t p r o c e s s . The r e s u l t s d e m o n s t r a t e t h a t C O a e x c r e t i o n i n f i s h p r o c e e d s i n a t y p i c a l l y mammalian f a s h i o n ( c f . R o u g h t o n 1 9 6 4 ) . C O a i s t r a n s p o r t e d i n t h e b l o o d p r i m a r i l y a s HC0 3™ (90-957. o f C c o a ) , w h i c h at. an h a e m a t o c r i t o f 257. i s d i s t r i b u t e d b e t w e e n t h e p l a s m a w a t e r s p a c e and t h e r e d c e l l w a t e r s p a c e i n a r a t i o o f a b o u t 92 1. R e l a t i v e l y s m a l l p r o p o r t i o n s o f t h e C O a c o n t e n t o f b l o o d a r e p r e s e n t a s d i s s o l v e d C O a (2-37.) o r a s carbami.no compounds (5-67.). I n r e s t i n g f i s h , t h e r e d c e l l membrane i s r e a d i l y p e r m e a b l e t o H C 0 3 ~ , C I " and H*, a l l o f w h i c h a r e d i s t r i b u t e d p a s s i v e l y a c r o s s t h e c e l l membrane. D u r i n g b r a n c h i a l C O a e x c h a n g e , p l a s m a HC0 3™ t r a v e r s e s t h e e r y t h r o c y t e membrane i n an o n e — f o r — o n e e x c h a n g e w i t h c e l l u l a r C I " . T h i s ' c h l o r i d e s h i f t ' i s s i m i l a r i n i t s t i m e c o u r s e and i t s s e n s i t i v i t y t o d i s u l p h o n i c s t i l b e n e d e r i v a t i v e s and a c e t a z o l ami de a s i s H C 0 3 ™ / C 1 ~ e x c h a n g e i n mammalian e r y t h r o c y t e s (Cameron 1978b,, O b a i d et al * 1 9 7 9 ) , and t h u s p r e s u m a b l y i n v o l v e s a t r a n s p o r t mechanism s i m i l a r t o t h e mammalian Band 3 a n i o n e x c h a n g e p a t h w a y . F i s h e r y t h r o c y t e s c o n t a i n s u f f i c i e n t c a r b o n i c a n h y d r a s e t o c a t a l y s e t h e i n t e r c o n v e r s i on of C O a and HC0 3"" by s e v e r a l o r d e r s o f m a g n i t u d e . Thus, upon e n t r y t o the; r e d c e l l , p l a s m a HCQ 3™ i s r a p i d l y d e h y d r a t e d t o f o r m C O a . T h i s C O a t h e n d i f f u s e s down i t s c o n c e n t r a t i o n g r a d i e n t o u t o f t h e r e d c e l l and a c r o s s t h e g i l l 154 e p i t h e l i u m . T h i s p a t h w a y f a r C D a e x c r e t i o n i s u n a f f e c t e d by e n d o g e n o u s i n h i b i t o r s o f c a r b o n i c a n h y d r a s e p r e s e n t i n f i s h p l a s ma? t h e s e i n h i b i t o r s l a c k a c c e s s t o t h e i n t r a c e l l u l a r enzyme and h a v e no e f f e c t on t h e a n i o n t r a n s p o r t m e chanism i n f i s h e r y t h r o c y t e s . Hence, t h e g i l l model o f C Q - j e x c r e t i o n w h i c h a s s e r t s t h a t f i s h e r y t h r o c y t e s a r e f u n c t i o n a l l y i m p e r m e a b l e t o HCOrs" ir> vivo and t h a t t h e b u l k o f C O r a c r o s s e s t h e g i l l e p i t h e l i u m d i r e c t l y a s H C O 3 " ( H a s w e l l and R a n d a l l 1 9 7 8 , H a s w e l l et al 1 9 8 0 ) no l o n g e r c a n be c o n s i d e r e d c o r r e c t . I n d e e d , i n l i g h t o f r e c e n t e v i d e n c e t h a t t h e b a s o l a t e r a l membrane o f t h e f i s h g i l l i s r e l a t i v e l y i m p e r m e a b l e t o HCDV"" ( P e r r y et al . 1 9 8 2 ) , t h e b u l k o f CDs l e a v i n g t h e b l o o d d u r i n g b r a n c h i a l c a p i l l a r y t r a n s i t must do s o a s d i s s o l v e d C 0 ^ , and t h e r e f o r e must, e x i t v i a t h e r e d b l o o d c e l l . The c o n t r i b u t i o n o f u n 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 H C 0 3 ~ t o o v e r a l l C 0 : s e x c r e t i o n i s n e g l i g i b l e , t h e u n c a t a l y s e d r e a c t i o n p r o c e e d s f a r t o o s l o w l y t o be o f any i m p o r t a n c e d u r i n g l a m e l l a r b l o o d t r a n s i t . The f l u x o f p l a s m a H C 0 3 ™ t h r o u g h t h e r e d c e l l i s i m p o r t a n t i n m a i n t a i n i n g 0 2 u p t a k 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 ( P e r r y et a l « 1 9 8 2 ) , p r e s u m a b l y due t o b i c a r b o n a t e b u f f e r i n g o f o x y - l a b i l e H* and f a c i l i t a t i o n o f B o h r — o n and R o o t — o n s h i f t s . C a r b o n d i o x i d e b e h a v e s l i k e a weak a c i d i n a q u e o u s s o l u t i o n ? i n c r e a s e s i n CO3. t e n s i o n n e c e s s a r i l y d e c r e a s e pH w h i l e d e c r e a s e s i n CDs t e n s i o n n e c e s s a r i l y i n c r e a s e pH. M a i n t e n a n c e o f a s t a b l e i n t e r n a l pH i n b i o l o g i c a l s y s t e m s , t h e r e f o r e , r e q u i r e s a b a l a n c i n g o f C 0 2 p r o d u c t i o n and e x c r e t i o n . I n g e n e r a l , v e r t e b r a t e s r e g u l a t e t h e i r i n t e r n a l a c i d - b a s e s t a t u s by a d j u s t m e n t s t o b l o o d C Q s * l e v e l s . F i s h , and p e r h a p s most o t h e r 155 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 , r e g u l a t e b l o o d pH by m o d u l a t i n g b l o o d H C O 3 l e v e l s w h i l e a l l o w i n g b l o o d P o o r s t o p a r a l l e l e n v i r o n m e n t a l 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, J a n s s e n and R a n d a l l 1975, Eddy et al « 1 9 7 7 ) , E i l e v a t i o n o f p l a s m a H C 0 3 " l e v e l s i n c r e a s e s b l o o d pH w h i l e d e c r e a s e s i n p l a s m a H C 0 3 " l e v e l s h a v e t h e o p p o s i t e e f f e c t . T h i s r e l a t i o n s h i p i s p o s s i b l e b e c a u s e t h e f i s h g i l l i s r e l a t i v e l y i m p e r m e a b l e t o H C 0 3 " ( P e r r y e t al. 1982) b u t i s r e a d i l y p e r m e a b l e t o H"'" (McWi 11 i ams and P o t t s 1978) and C O a , w h i c h a r e e x c r e t e d a c r o s s t h e g i l l e p i t h e l i u m i n d e p e n d e n t o f one a n o t h e r (van den T h i 1 l a r t e t al. 1 9 8 3 ) , W h i l e t h e r e i s l i t t l e d o u b t t h a t t h e Na*'/H"1" (Ml-iv*') and C I ' " " /HCO»~(OH -) e x c h a n g e r s p r e s e n t on t h e a p i c a l membrane o f g i l l e p i t h e l i u m a r e i n v o l v e d i n a c i d - b a s e r e g u l a t i o n (Cameron 1978a, H e i s l e r 1 9 8 4 ) , t h e p r e s e n t s t u d i e s c l e a r l y i n d i c a t e t h a t e x t r a c e l l u l a r H C 0 3 " c o n c e n t r a t i o n s i n f i s h a r e c o n t r o l l e d a t l e a s t i n p a r t a t t h e l e v e l o f t h e r e d b l o o d c e l l . F i s h e r y t h r o c y t e s p o s s e s s Na^/H* and C1 -/HC0»~ e x c h a n g e r s t h a t a r e 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 a g o n i s t s and p e r h a p s t o o s m o t i c p e r t u r b a t i o n s o f t h e r e d c e l l . 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 r e d c e l l s r e s u l t s i n a n e t c e l l u l a r g a i n o f Na"", C I " " and H 3 O , a n e t c e l l u l a r l o s s o f H* and HC03"", a p r o n o u n c e d c e l l s w e l l i n g , and a r e d u c t i o n i n t h e f u n c t i o n a l a c t i v i t y o f e r y t h r o c y t e c a r b o n i c a n h y d r a s e . In vivo, t h e s e r e s p o n s e s s l o w t h e r a t e o f C O a e x c r e t i o n 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 r e s u l t i n r e t e n t i o n o f p l a s m a l-IC03"'". R e t e n t i o n o f p l a s m a HC0 3"" p r o b a b l y i s i m p o r t a n t d u r i n g p e r i o d s o f s t r e s s i n c o m p e n s a t i n g f o r 156 c a r d i o v a s c u l a r e f f e c t s s u c h a s l a m e l l a r r e c r u i t m e n t ( H o l b e r t et al . 1979) w h i c h c o n c e i v a b l y c o u l d r e s u l t i n washout, of b l o o d C0 3» As w e l l , m a i n t e n a n c e o f an e x t r a c e l l u l a r p o o l o f HC03~ i n s t r e s s makes p l a s m a H C 0 3 ~ a v a i l a b l e t o e n h a n c e t h e i n t r a c e l l u l a r b u f f e r i n g c a p a c i t i e s ; o f o t h e r t i s s u e s . T h i s p o o l of e x t r a c e l l u l a r b i c a r b o n a t e , a l t h o u g h s m a l l , w o u l d h a v e a c o n s i d e r a b l e e f f e c t cm i n t r a c e l l u l a r pH c o m p e n s a t i o n b e c a u s e o f t h e i n i t i a l l y l o w i n t r a c e l l u l a r b i c a r b o n a t e l e v e l s o f 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 (1-5 mil, s e e T a b l e 5 ) . The a d r e n e r g i c r e s p o n s e s o f f i s h e r y t h r o c y t e s a l s o s e r v e t o m a i n t a i n 0 3 u p t a k e a t t h e g i l l s i n t h e f a c e o f t h e a c c o m p a n y i n g n e t r e d u c t i o n i n H CC 3~ f l u x t h r o u g h t h e r e d c e l l . The r e d u c t i o n i n i n t r a e r y t h r o c y t i c b i c a r b o n a t e b u f f e r i n g o f o x y - l a b i l e H**" i n s t r e s s i s p a r t i a l l y c o m p e n s a t e d f o r by H*" e x t r u s i o n f r o m t h e r e d c e l l and by r e d u c t i o n s i n e r y t h r o c y t e o r g a n i c p h o s p h a t e l e v e l s w h i c h e n h a n c e G s s-haemogl o b i n b i n d i n g a f f i n i t y ( N i k i n m a a 1983, N i k i n m a a et al. 1 9 8 4 ) , The a d r e n e r g i c r e s p o n s e s of f i s h r e d c e l l s p r o b a b l y a r e t r a n s i e n t i n n a t u r e and e x e r t t h e i r m a x i m a l e f f e c t on a c i d - b a s e r e g u l a t i o n s h o r t l y a f t e r a pH d i s t u r b a n c e o c c u r s . As s u c h , t h e y c a n be c o n s i d e r e d a ' f i r s t l i n e o f d e f e n c e ' a g a i n s t f l u c t u a t i o n s i n i n t e r n a l pH. L o n g - t e r m pH r e g u l a t i o n most l i k e l y i s a c h i e v e d v i a g i l l f u n c t i o n . The a d r e n e r g i c r e s p o n s e s of f i s h e r y t h r o c y t e s p r o b a b l y e x p l a i n t h e a b s e n c e o f f u n c t i o n a l e r y t h r o c y t e CA a c t i v i t y i n b i modal .1. y - b r e a t h i ng f i s h d u r i n g a i r e x p o s u r e . S e v e r a l s t u d i e s ( R a n d a l l et al. 1978, D a xboeck and Heming 1982) h a v e f o u n d t h a t , w h i l e b i m o d a l l y ~ b r e a t . h i n g f i s h a r e c a p a b l e o f m a i n t a i n i n g 0:,?. u p t a k e d u r i n g a i r e x p o s u r e , C 0 Z e x c r e t i o n i n a i r i s l i m i t e d t o 157 t h e u n c a t a l y s e d r a t e o f HC0 3"" d e h y d r a t i o n . The g a s e x c h a n g e r a t i o s of t h e s e f i s h t y p i c a l l y were 0.2-0.4 d u r i n g a i r e x p o s u r e . Whole-body i n h i b i t i o n o f CA was w i t h o u t e f f e c t on C0 : a e x c r e t i o n d u r i n g a i r e x p o s u r e o f t h e s e a n i m a l s , w h e r e a s i n j e c t i o n o f e x o g e n o u s c a r b o n i c a n h y d r a s e s i g n i f i c a n t l y i n c r e a s e d CO^ e x c r e t i o n i n a i r and r e t u r n e d t h e g a s e x c h a n g e r a t i o t o a b o u t u n i t y . The e r y t h r o c y t e s o f b i m o d a l I y - b r e a t h i n g f i s h c o n t a i n s i g n i f i c a n t amounts o f c a r b o n i c a n h y d r a s e ( B u r g g r e n and H a s w e l l 1979, D a xboeck and Heming 1982) and i t i s u n l i k e l y t h a t t h e s e e r y t h r o c y t e s a r e g e n e r a l l y i m p e r m e a b l e t o a n i o n s . More l i k e l y , t h e s t r e s s o f h a n d l i n g and a i r e x p o s u r e i n t h e a b o v e s t u d i e s c a u s e d r e l e a s e o f c a t e c h o l a m i n e s i n t o t h e b l o o d w h i c h i n t u r n r e d u c e d t h e f u n c t i o n a l a c t i v i t y o f e r y t h r o c y t e CA v i 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 r e d c e l l s . A v a l uafo 1 e and i nf o r mat. i v e t e c h n i que f o r quan t i f y i ng t h e movements o f a c i d - b a s e r e l e v a n t i o n s i n f i s h r e c e n t l y h a s been d e v e l o p e d w h i c h i n v o l v e s b a l a n c i n g t h e u p t a k e and e x c r e t i o n o f COrs, H* and c o u n t e r - e x c h a n g e i o n s ( Na*, Cl"~) b e t w e e n w a t e r and t h e ex t r a c : e 11 u 1 a r (p 1 asma) c o m p a r t m e n t o f f i s h ( f o r r e v i e w s e e H e i s l e r 1 9 8 4 ) . Use of t h i s t e c h n i q u e h a s d e m o n s t r a t e d t h a t movements o f HCLT-s" b e t w e e n t h e i n t r a c e l l u l a r and ex t r a c e ? ! 1 u l a r c o m p a r t m e n t s , and b e t w e e n t h e e x t r a c e l l u l a r c o m p a r t m e n t and t h e w a t e r o c c u r d u r i n g pH c o m p e n s a t i o n . However, t h e p o s s i b i l i t y o f a s t r e s s - r e l a t e d r e d i s t r i b u t i o n o f Na*, C l " " , H* and HC0 3"' a c r o s s t h e r e d c e l l membrane s e l d o m h a s been c o n s i d e r e d i n t h e s e i o n b a l a n c i n g s t u d i e s ; . S i n c e t h e r e d c e l l s o f f i s h t y p i c a l l y o c c u p y 2.5-307. o f t h e t o t a l b l o o d v o l u m e , t h e a d r e n e r g i c r e s p o n s e s ; o f 158 f i s h e r y t h r o c y t e s p r o b a b l y a r e a s i g n i f i c a n t s o u r c e o f e r r o r i n i o n b a l a n c i n g s t u d i e s . , T h i s i s e s p e c i a l l y t r u e i n s t u d i e s i n w h i c h i o n movements a c r o s s t h e g i l l a r e i n f e r r e d s o l e l y f r o m m e a s u r e m e n t s o f p l a s m a i o n l e v e l s i n t h e a b s e n c e of m e a s u r e m e n t s of w a t e r i o n l e v e l s <cf» W i l k e s e t a l , 1981) „ 159 ABBREVIATIONS AND SYMBOLS a C O a -- C O a s o l u b i l i t y c o e f f i c i e n t 6 C 0 a ~ mean C O a c a p a c i t a n c e o r c / C c o s / r f P c o z bl- I C O a ; - - app a r e n t , b i c a r b o n a t e o r c h e m i c a l l y - b o u n d C O a C A - c a r b o n i c : a n h y d r a s e C o a - t o t a l o x y g e n c o n t e n t CCOH ~ 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 tf'X - d e l t a X o r c h a n g e i n any v a r i a b l e X,, f o r e x a m p l e , tfCl~ P 0 2 - p a r t i a l p r e s s u r e o f o x y g e n P C Q 2 - p a r t i a l p r e s s u r e o f c a r b o n d i o x i d e R - g a s e x c h a n g e r a t i o o r d C c o s / o ' C a s R x - Donnan r a t i o o f any i o n X, f o r e x a m p l e , R C J . -160 REFERENCES Adamson, T.M. and B.P. 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