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UBC Theses and Dissertations

Strategies for acid-base regulation in fishes Iwama, George Katsushi 1986

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STRATEGIES FOR ACID-BASE REGULATION IN FISHES by GEORGE KATSUSHI IWAMA B . S c , 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 , 1975 H . S c , 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 , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( Z o o l o g y ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u - i r e d s t a n d a r d THE, UNI^ETTSITY OF B R I T I S H C O L U M B I A J u l y 1986 (s) G e o r g e K a t s u s h i Iwama, 1986 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 available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis 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 r ZOOLOGY The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date August 21, 1986 E-6 (3/81) i i ABSTRACT Three s e t s of i n v i v o experiments were conducted to i n v e s t i g a t e s e v e r a l aspects of acid-base r e g u l a t i o n i n f i s h e s . There are two p o s s i b l e ways that i n v o l v e the g i l l s o f f i s h e s i n which the acid-base r e g u l a t i o n of the e x t r a c e l l u l a r f l u i d can be a d j u s t e d . F i r s t , CO2 e x c r e t i o n can be a d j u s t e d by a l t e r i n g g i l l water flow to i n c r e a s e or decrease the PC02 t e n s i o n s i n the blood. The second mechanism would i n v o l v e the exchange of ions a c r o s s the g i l l e p i t h e l i u m to change the c o n c e n t r a t i o n s of H + , HCO3- or NH4+ i n the blood. The f i r s t two s e t s of experiments were, r e s p e c t i v e l y , designed to i n v e s t i g a t e these two p o s s i b i l i t e s . The t h i r d s e t of experiments i n v e s t i g a t e d the r o l e t h a t plasma catecholamines might p l a y i n r e g u l a t i n g the pH of the e x t r a c e l l u l a r f l u i d as w e l l as the i n t r a c e l l u l a r compartment of the red blood c e l l . Experimental m a n i p u l a t i o n of v e n t i l a t i o n i n rainbow t r o u t i n steady s t a t e showed t h a t g i l l water flow a f f e c t e d CO2 e x c r e t i o n only a t l e v e l s lower than about lOOml/min. Carbon d i o x i d e e x c r e t i o n was r e t a r d e d and blood PC02 p r e s s u r e s i n c r e a s e d a t these l e v e l s of g i l l v e n t i l a t i o n . I n c r e a s i n g g i l l water flow above c o n t r o l l e v e l s e f f e c t e d n e i t h e r O2 or CO2 exchange acr o s s the g i l l . D o g f i s h , s u b j e c t e d to environmental hyperoxia and v a r i o u s l e v e l s of hypercapnia, showed the best c o r r e l a t i o n between g i l l i i i v e n t i l a t i o n and plasma pH. There was a very weak c o r r e l a t i o n with plasma Pco2 t e n s i o n and plasma HCC>3~ c o n c e n t r a t i o n s d i d not a f f e c t v e n t i l a t i o n a t a l l . G i l l v e n t i l a t i o n i n c r e a s e d e x p o n e n t i a l l y as plasma pH d e c l i n e d . Experiments that i n v o l v e d the f r e s h water t r o u t and the sea water conger e e l showed that water s a l i n i t y had a d i r e c t e f f e c t on the acid-base r e g u l a t i o n of the plasma. Recovery o f plasma pH i n both s p e c i e s , a f t e r an i n i t i a l d e c l i n e i n response to exposure to environmental hypercapnia, was dependent on water s a l i n i t y . The recover y was e f f e c t e d by an i n c r e a s e i n plasma HCO3 - c o n c e n t r a t i o n . There was a l s o an a s s o c i a t e d decrease i n plasma C l ~ c o n c e n t r a t i o n i n both s p e c i e s , i n d i c a t i n g the p o s s i b l e involvement of a C1~/HC03~ exchange process. When carp were exposed to environmental hypercapnia, a r e d u c t i o n i n the a c t i v e uptake of water C l ~ , while m a i n t a i n i n g normal e f f l u x r a t e s , caused the r e d u c t i o n of the plasma c o n c e n t r a t i o n o f t h i s i o n . T h e r e f o r e , i t seems t h a t the modulation o f t h i s a c t i v e C1 _/HC03~ exchange process e f f e c t e d the HCC>3~ accumulation i n the carp, and probably a l s o i n the t r o u t and conger i n f r e s h water. C o n s i s t e n t with the data from the above carp experiment, f u r t h e r a n a l yses o f the e l e c t r o c h e m i c a l g r a d i e n t s f o r C l ~ i n t r o u t exposed to environmental hypercapnia a t the three s a l i n i t i e s showed that a c t i v e exchange processes must have accumulated the plasma HCO3 - by the proposed i v C1~/HC03 - mechanism. These analyses a l s o showed t h a t the t r o u t g i l l was about 2.5 times more permeable to Na + than to C l ~ i n steady s t a t e c o n t r o l c o n d i t i o n s . Furthermore, Na + i s maintained out of e l e c t r o c h e m i c a l e q u i l i b r i u m more than C T - by a f a c t o r of about 1.5 - 2.0. T h i s l a t t e r c a l c u l a t i o n was based on the comparison between the measured plasma c o n c e n t r a t i o n s of these ions and the expected c o n c e n t r a t i o n s based on a d i s t r i b u t i o n a c c o r d i n g to the e x i s t i n g e l e c t r o c h e m i c a l gradents Catecholamines are r e l e a s e d i n t r o u t immediately a f t e r a c i d i n f u s i o n . This r e l e a s e i s p r o p o r t i o n a l to the change i n plasma pH r e l a t i v e to c o n t r o l values and f u n c t i o n s to m a i n t a i n the oxygen c a r r y i n g c a p a c i t y of the blood which would otherwise be compromised due to the Root s h i f t . T h i s data supports e x i s t i n g data showing t h a t some o f the e f f e c t s which catecholamines have on the p h y s i o l o g y of f i s h e s i n c l u d e those which enhance the r e g u l a t i o n of the acid-base s t a t u s of the e x t r a c e l l u l a r and red c e l l compartments. This data a l s o suggests t h a t the r e l e a s e of catecholamines d u r i n g b u r s t e x e r c i s e i s due, a t l e a s t p a r t i a l l y , to the excess proton l o a d from the l a c t a c i d o s i s . V TABLE OF CONTENTS Page # A b s t r a c t 1 1 L i s t o f Tables v i L i s t o f F i g u r e s v i i Acknowledgements x i i General I n t r o d u c t i o n 1 General M a t e r i a l s and Methods 6 S e c t i o n 1 18 V e n t i l a t i o n and Acid-Base R e g u l a t i o n i n F i s h e s S e c t i o n 2 60 T r a n s e p i t h e l i a l Ion Fl u x e s f o r Acid-Base R e g u l a t i o n i n F i s h e s S e c t i o n 3 132 Furth e r A n a l y s i s o f the T r o u t - S a l i n i t y -Hypercapnia Experiment S e c t i o n 4 163 Catecholamine Release i n A c i d - I n f u s e d Trout General D i s c u s s i o n 199 References 210 Appendix 222 v i L I S T OF T A B L E S Table 1. G i l l v e n t i l a t i o n volumes of f i s h e x p e r i e n c i n g v a r i o u s head p r e s s u r e s i n a Van Dam apparatus 30 Table 2. Time course f o r pH compensation i n f i s h s t r e s s e d with environmental hypercapnia 63 Table 3. Mean water Na + and C l ~ c o n c e n t r a t i o n s f o r the two expriments i n t h i s s e c t i o n 65 Table 4. A r t e r i a l plasma pH, red c e l l pH, mls02 bound per gram of hemoglobin, a d r e n a l i n e c o n c e n t r a t i o n 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 before and f o l l o w i n g i n t r a - a r t e r i a l i n f u s i o n of 120mM s a l i n e s o l u t i o n i n t r o u t 175 Table 5. Plasma i o n c o n c e n t r a t i o n s i n rainbow t r o u t i n f u s e d with HC1 and s a l i n e 186 Table 6. Water i o n c o n c e n t r a t i o n s f o r rainbow t r o u t i n f u s e d with HC1 and s a l i n e 187 Table 7. R a t i o s of measured to expected plasma c o n c e n t r a t i o n s of Na +, C l ~ , K +, HCO3 -, NH4+, C a + + and Mg + + f o r t r o u t i n f u s e d with HC1 188 Table A . l . I o n i c c o n c e n t r a t i o n s f o r the sea s a l t s used to make up the v a r i o u s s a l i n i t i e s i n experiment 2A i n S e c t i o n 2 223 v i i LIST OF FIGURES F i g u r e GI. Apparatus f o r measurement of t r a n s e p i t h e l i a l p o t e n t i a l s ( TEP) 11 F i g u r e 1. Diagram of experimental apparatus used to conduct r e s p i r a t o r y and acid-base r e g u l a t o r y experiments i n the shark, 27 F i g u r e 2. The r e l a t i o n s h i p between b r e a t h i n g frequency, s t r o k e volume and the volume o f water f l o w i n g over the g i l l s o f t r o u t with a Van Dam apparatus 32 F i g u r e 3. The r e l a t i o n s h i p between a r t e r i a l Pco2 and v e n t i l a t i o n volume over the range o f v e n t i l a t i o n volumes imposed on t r o u t 34 F i g u r e 4. The r e l a t i o n s h i p between a r t e r i a l Pco2 and a r t e r i a l P02 over the range of v e n t i l a t i o n volumes imposed on t r o u t 37 F i g u r e 5. The r e l a t i o n s h i p between the c o n v e c t i o n requirement f o r CO2 and a r t e r i a l PC02 over the range o f v e n t i l a t i o n volumes imposed 39 F i g u r e 6. The r e l a t i o n s h i p between CO2 e x c r e t i o n and O2 uptake a t d i f f e r e n t v e n t i l a t i o n volumes 41 F i g u r e 7. The r e l a t i o n s h i p between v e n t i l a t o r y volume and plasma pH i n the shark undergoing simultaneous exposure to environmental hyperoxia and hypercapnia 43 F i g u r e 8. As i n 7. showing o n l y the data f o r the experiment which i n v o l v e d the exposure to environmental hyperoxia and hypercapnia 45 F i g u r e 9. As i n 7. showing o n l y the data f o r the experiment which i n v o l v e d the exposure to environmental hyperoxia and hypercapnia and where changes i n pH were minimized 47 F i g u r e 10. The best f i t l i n e f o r the aggregate data s e t f o r both data s e t s d e s c r i b e d i n 7. above 49 F i g u r e 11. The r e l a t i o n s h i p between v e n t i l a t o r y volume and a r t e r i a l PC02 i n shark s u b j e c t e d to three e x p e r i m e n t a l ^ p r o t o c o l s d e s c r i b e d i n S e c t i o n 1 51 v i i i F i g u r e 12. As i n 11. except that the r e l a t i o n s h i p between v e n t i l a t o r y volume and a r t e r i a l [HCC>3_J i n mM f o r the three experimental p r o l o c o l s 53 Fi g u r e 13. Experimental apparatus f o r experiment 2A.: Trout - S a l i n i t y - Hypercapnia 68 Fi g u r e 14. Experimental apparatus f o r experiment 2 C : Carp - Isotope - Hypercapnia 76 Fi g u r e 15. Plasma Pco2 i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia 80 Fi g u r e 16. Plasma Pco2 i n conger d u r i n g c o n t r o l , s a l i n i t y change and exposure to 1 % environmental hypercapnia 82 F i g u r e 17. Plasma pH i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia 84 Fi g u r e 18. As F i g . 17. except t h a t a l l values were r e f e r e n c e d to the average value d u r i n g the c o n t r o l p e r i o d 86 Fi g u r e 19. Plasma pH changes from average c o n t r o l values i n conger d u r i n g s a l i n i t y change and exposure to 1 % hypercapnia 88 Fi g u r e 20. E f f e c t o f water s a l i n i t y , r e p r e s e n t e d by [ C l ~ ] , on pH recovery i n conger 90 Fi g u r e 21. Plasma HCO3- c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia 92 Fi g u r e 22. Plasma HCC>3~ changes from average c o n t r o l values i n conger d u r i n g s a l i n i t y change and exposure to 1 % hypercapnia ...94 Fi g u r e 23. E f f e c t o f water s a l i n i t y , r e p r e s e n t e d by [ C l ~ ] , on HCO3- accumulation i n conger 96 Fig u r e 24. Net H + f l u x i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1 % environmental hypercapnia 100 i x F i g u r e 25. Plasma C l ~ c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1 % environmental hypercapnia 102 Fi g u r e 26. Changes i n plasma C l -c o n c e n t r a t i o n s from average c o n t r o l v a l u e s i n conger d u r i n g s a l i n i t y change and exposure to 1 % environmental hypercapnia 104 F i g u r e 27. Plasma Na + c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1 % environmental hypercapnia. 106 F i g u r e 28. Plasma Os m o l a r i t y i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1% environmental hypercapnia f o r 24h and recovered f o r 24h 108 Fi g u r e 29.a.b.c. R e l a t i o n s h i p of plasma ( t N a + ] - ( C l - ] ) to plasma [HC0 3 -] d u r i n g exposure and recovery from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y 110 F i g u r e 30.a.b.c. R e l a t i o n s h i p o f changes i n plasma C l ~ c o n c e n t r a t i o n s to corresp o n d i n g changes i n plasma HCO3 - c o n c e n t r a t i o n s d u r i n g exposure and rec o v e r y from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl, r e s p e c t i v e l y 112 Fi g u r e 31.a.b.c. R e l a t i o n s h i p o f changes i n plasma Na + c o n c e n t r a t i o n s to corresp o n d i n g changes i n plasma HCO3 - c o n c e n t r a t i o n s d u r i n g exposure and recovery from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y 114 Fig u r e 32. R e l a t i o n s h i p o f changes i n plasma C l ~ c o n c e n t r a t i o n s to cor r e s p o n d i n g changes i n plasma HCO3 - c o n c e n t r a t i o n s i n conger exposed to s a l i n i t y changes and exposure to 1 % environmental hypercapnia 116 F i g u r e 33.a.b.c. T r a n s e p i t h e l i a l p o t e n t i a l (TEP) values d u r i n g c o n t r o l , exposure and reco v e r y from 1% environmental hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y 118 X F i g u r e 34. Plasma hematocrit values (Hct i n %) o f t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to 1% environmental hypercapnia f o r 24h and then recovered f o r 24h 121 F i g u r e 35. Net f l u x e s of Na + and C l ~ i n carp d u r i n g exposure to 5% environmental hypercapnia f o r 48h followed by 24h r e c o v e r y 123 F i g u r e 36. As i n F i g . 35, except the e f f l u x o f Na + and C I " are shown 125 F i g u r e 37.a. R e l a t i o n s h i p o f plasma HCO3 -to plasma (Na + - C l ~ ) f o r three s a l i n i t i e s f o r conger. F i g u r e 37.b. R e l a t i o n s h i p o f d e l t a HCO3 -to d e l t a C l ~ f o r plasma data from hypercapnia and r e c o v e r y p e r i o d s . F i g u r e 37.c. R e l a t i o n s h i p o f d e l t a HCO3 - to d e l t a C l ~ f o r plasma data from hypercapnia and r e c o v e r y p e r i o d s 137 F i g u r e 38. H y p o t h e t i c a l a c t i v e and p a s s i v e C l ~ movements a c r o s s the t r o u t g i l l a t three s a l i n i t i e s and the a s s o c i a t e d TEP and plasma HCC>3~ accumulation c h a r a c t e r i s t i c s 139 F i g u r e 39.a. and b. P e r m e a b i l i t e s o f Na + and C l ~ r e l a t i v e to HCC>3~ f o r the g i l l o f t r o u t d u r i n g and a f t e r exposure to environmental hypercapnia 143 F i g u r e 40.a. P e r m e a b i l i t y o f Na + r e l a t i v e to C I - f o r the t r o u t g i l l d u r i n g and a f t e r exposure to environmental hypercapnia. F i g u r e 40.b. Data of 40.a. r e f e r e n c e d to the average c o n t r o l v a l u e s . E x p t l . ( P N a + / P C l ~ ) - C o n t r o l (PNa + /PCl~) 146 F i g u r e 41.a.b.c. R a t i o s of measured to expected plasma Na + c o n c e n t r a t i o n s f o r t r o u t d u r i n g and a f t e r exposure to environmental hypercania a t three water s a l i n i t i e s 150 F i g u r e 42.a.b.c. As 41.a.b.c. r e s p e c t i v e l y except t h a t data i s f o r plasma C l ~ 152 F i g u r e 43.a.b.c. As 41.a.b.c. r e s p e c t i v e l y except that data i s f o r plasma HCO3 - 154 F i g u r e 4 4 . a. A r t e r i a l p l a s m a p H , b. r e d c e l l p H , c . m i s 0 2 b o u n d p e r g r a m o f h a e m o g l o b i n a n d d . p l a s m a c a t e c h o l a m i n e c o n c e n t r a t i o n s f o l l o w i n g i n t r a - a r t e r i a l i n f u s i o n o f HC1 s o l u t i o n o f 120mM s a l i n e i n r a i n b o w t r o u t F i g u r e 4 5 . R e l a t i o n s h i p b e t w e e n t h e c h a n g e s i n p l a s m a p H a n d t h e c o r r e s p o n d i n g c h a n g e s i n p l a s m a a d r e n a l i n e c o n c e n t r a t i o n s b e t w e e n p r e - i n f u s i o n c o n t r o l s a m p l e s a n d t h e + 5 m i n p o s t - i n f u s i o n s a m p l e s f o r e a c h o f 14 a n i m a l s c o n t r i b u t i n g t o t h e m e a n d a t a i n F i g . 44 1 7 7 F i g u r e 4 6 . R e l a t i o n s h i p b e t w e e n 0 2 c a p a c i t y p e r g r a m o f h a e m o g l o b i n a n d r e d b l o o d c e l l p H i n r a i n b o w t r o u t b l o o d e q u i l i b r a t e d i n v i t r o a g a i n s t g a s m i x t u r e s h a v i n g a n o x y g e n p a r t i a l p r e s s u r e o f 1 5 2 mmHg a n d a CO2 p a r t i a l p r e s s u r e o f 2 . 5 mmHg 1 7 9 F i g u r e 4 7 . a . P l a s m a p H a n d b . p l a s m a HCC>3~ c o n c e n t r a t i o n s i n r a i n b o w t r o u t i n f u s e d w i t h HC1 a n d s a l i n e 1 8 2 F i g u r e 4 8 . a . H e m a t o c r i t (%) o f r a i n b o w t r o u t i n f u s e d w i t h HC1 a n d s a l i n e 1 8 4 F i g u r e 4 9 . T r a n s e p i t h e l i a l P o t e n t i a l s ( T E P ) i n r a i n b o w t r o u t i n f u s e d w i t h HC1 s o l u t i o n m a d e u p i n 120mM p h y s i o l o g i c a l s a l i n e a n d t h e s a l i n e a l o n e 1 9 0 F i g u r e 5 0 . a . N e r n s t r a t i o f o r p l a s m a N a + a n d b . p l a s m a N H 4 + i n t r o u t i n f u s e d w i t h HC1 1 9 2 F i g u r e 5 1 . A s i n 5 0 . e x c e p t t h a t i t p e r t a i n s t o C a + + 1 9 4 x i 1 7 3 ACKNOWLEDGEMENTS I g r a t e f u l l y acknowledge the s t i m u l a t i n g guidance o f my s u p e r v i s o r . Dr. David J . Randall i n my s t u d i e s and the p r e p a r a t i o n o f t h i s t h e s i s . I would a l s o l i k e to acknowledge the p a t i e n t t e a c h i n g o f Drs. Robert G. B o u t i l i e r and Norbert H e i s l e r i n both experimental techniques and the fundamentals o f acid-base r e g u l a t i o n . I am pl e a s e d to acknowledge the f o l l o w i n g people f o r t h e i r c o l l a b o r a t i o n i n v a r i o u s experiments i n c l u d e d i n t h i s t h e s i s . Drs. R.G. B o u t i l i e r and T.A. Heming : Experiment IA, The r o l e of v e n t i l a t i o n i n CO2 e x c r e t i o n i n rainbow t r o u t . Drs. N. H e i s l e r , R.G. B o u t i l i e r , J.B. C l a i b o r n e and N. Andersen : Experiment IB, The c o n t r o l o f v e n t i l a t i o n i n the l a r g e r s p o t t e d d o g f i s h . ; Experiment 2B, The e f f e c t o f water s a l i n i t y on acid-base r e g u l a t i o n i n the conger e e l . Drs. N. H e i s l e r , N. Andersen and J.B. C l a i b o r n e : Experiment 2C, U n i d i r e c t i o n a l i o n f l u x e s i n carp d u r i n g hypercapnia. Dr. R.G. B o u t i l i e r : Catecholamine r e l e a s e i n rainbow t r o u t with a c i d i n f u s i o n . x i i i I was supported by funds from the f o l l o w i n g sources: 1) N a t u r a l Sciences and E n g i n e e r i n g Research C o u n c i l (NSERC) Postgraduate S c h o l a r s h i p ; 2) U.B.C. Mclean F r a s e r S c h o l a r s h i p s ; 3) Max-Planck Foundation Research F e l l o w s h i p , F.R.G.; 4) U.B.C, Dept. Zoology Teaching A s s i s t a n t s h i p s ; 5) NSERC o p e r a t i n g grant to D.J. R a n d a l l . I am a l s o g r a t e f u l f o r the t e c h n i c a l a s s i s t a n c e o f Mr. Dennis Mense a t the U n i v e r s i t y o f B r i t i s h Columbia and S. Glage, G. F o r c h t , H. Slama and W. Neusse a t the Max-PLanck I n s t i t u t f u r e x p e r i m e n t e l l e Medizin, Goettingen, F.R.G. I am e s p e c i a l l y g r a t e f u l f o r the love and support o f my f a m i l y , my wife M a r i l y n Joy, and my sons Adam and D a n i e l . For the understanding d u r i n g evenings and weekends t h a t I had to work and f o r keeping my eyes open to the c o r r e c t p r i o r i t i e s i n l i f e , I am deeply t h a n k f u l . 1 GENERAL INTRODUCTION 2 INTRODUCTION F i s h have a t l e a s t three major s t r a t e g i e s f o r r e g u l a t i n g the acid-base s t a t u s o f t h e i r body f l u i d s t h a t i n v o l v e the environment. One i s the adjustment o f the blood P C 0 2 t e n s i o n s and the other two i n v o l v e the t r a n s f e r o f ions between the f i s h and water. These are the i o n exchange processes a c r o s s the g i l l e p i t h e l i u m and those i n v o l v i n g the r e n a l system. S t u d i e s on the r o l e o f the r e n a l system i n acid-base r e g u l a t i o n i n f i s h e s are few by comparison with those on the r o l e s o f v e n t i l a t i o n and i o n t r a n s f e r s a c r o s s the g i l l . The kidney has i n some i n s t a n c e s been seen as the major compensatory process i n a c i d o s e s , i n ot h e r s i t has been concluded that the kidney p l a y s a n e g l i g i b l e r o l e . In summary, the a v a i l a b l e evidence suggests t h a t the r o l e o f the kidney i s g e n e r a l l y minor r e l a t i v e to the p o t e n t i a l o f i o n exchange processes a c r o s s the g i l l e p i t h e l i u m f o r acid-base r e g u l a t i o n (see H e i s l e r 1985). Data r e g a r d i n g the r o l e o f v e n t i l a t i o n i n c o r r e c t i n g acid-base d i s t u r b a n c e s i n f i s h e s are a l s o v a r i a b l e . Some trends are emerging, however, t h a t p o i n t to a minor r o l e i n r e g u l a t i n g acid-base balance i n f i s h e s . As suggested by Randall and Cameron (1973), there are s e v e r a l a p r i o r i reasons why the r e g u l a t i o n o f blood acid-base balance through 3 adjustments i n blood PC02 would be i n a p p r o p r i a t e i n f i s h e s : 1) a very f i n e c o n t r o l o f blood Pco2 would be needed to r e g u l a t e pH due to the l o g / l i n e a r r e l a t i o n s h i p ; 2) low Pco2 t e n s i o n s i n f i s h blood l i m i t the scope of adjustment; and 3) o r i e n t a t i o n o f v e n t i l a t i o n to adjustments of Pco2 may compromise O2 uptake, a disadvantageous c o n d i t i o n i n water where the O2 content i s lower than i n a i r . There i s evidence t h a t the s t i m u l a t i o n o f v e n t i l a t i o n d u r i n g hypercapnia i n f i s h e s f u n c t i o n s to o f f s e t the e f f e c t s o f the Bohr and Root s h i f t s found i n t e l e o s t blood, which lower the oxygen c a r r y i n g c a p a c i t y o f the blood i n a c i d c o n d i t i o n s (Smith and Jones 1982). E x c e p t i o n s to t h i s t r e n d are (1) elasmobranchs, which show a s i m i l a r s t i m u l a t i o n o f v e n t i l a t i o n i n response to a c i d c o n d i t i o n s but show no Bohr and Root s h i f t s c h a r a c t e r i s t i c s i n the blood (Lenfant and Johansen 1966) and i n the t e l e o s t f a m i l y C y p r i n i d a e i n which there i s very l i t t l e s t i m u l a t i o n o f v e n t i l a t i o n i n hypercapnic c o n d i t i o n s (Dejours 1973) but the blood e x h i b i t s both Bohr and Root s h i f t s . The experiments i n S e c t i o n 1. address two s u b j e c t s r e g a r d i n g t h i s broad area of v e n t i l a t i o n and acid-base r e g u l a t i o n i n f i s h e s . The f i r s t experiment examined the r o l e of g i l l water flow i n CO2 e x c r e t i o n . Any l i m i t a t i o n i n t h i s r egard would d e f i n e the scope of a d j u s t i n g the acid-base s t a t u s o f the blood through changes i n PC02 t e n s i o n s by r e g u l a t i n g g i l l water flow. The second experiment addressed 4 the q u e s t i o n o f which acid-base parameters might be i n f l u e n c i n g the s t i m u l a t i o n of v e n t i l a t i o n i n the d o g f i s h , i n a c i d c o n d i t i o n s . A s i g n i f i c a n t r o l e of i o n exchange processes i n the r e g u l a t i o n of acid-base balance i n f i s h e s has been demonstrated i n most s t u d i e s of t h i s s u b j e c t (see review by H e i s l e r 1985). The movement of H +, NH4 + and of HCO3 - ions a c r o s s the g i l l e p i t h e l i u m have been c o r r e l a t e d to the movements of Na + (Maetz and Garcia-Romeu 1964, K e r s t e t t e r e t al_. 1970, Evans 1977, Payan 1978, Maetz 1973) and to C l ~ (Maetz and Garcia-Romeu 1960, De Renzis and Maetz 1973), r e s p e c t i v e l y . The f a c t t h at pH compensation i n f i s h takes longer and i s l e s s complete i n f i s h i n f r e s h waters than i n waters o f g r e a t e r i o n i c s t r e n g t h such as the sea water, f u r t h e r i m p l i e s the e x i s t e n c e of a l i n k between i o n i c groups. The f i r s t two experiments i n S e c t i o n 2 were conducted to study the r e l a t i o n s h i p between water s a l i n i t y and acid-base r e g u l a t o r y performance. A freshwater t e l e o s t was a c c l i m a t e d to higher s a l i n i t i e s and a sea water t e l e o s t was t r a n s f e r r e d to l e s s s a l i n e waters. A c i d o t i c c o n d i t i o n s were induced i n both s p e c i e s a t the v a r i o u s s a l i n i t i e s and changes i n acid-base s t a t u s were foll o w e d . The l a s t experiment i n S e c t i o n 2 was c a r r i e d out to determine u n i d i r e c t i o n a l f l u x e s of Na + and C l ~ i n carp, a l s o exposed to environmental hypercapnia. T h i s was c a r r i e d out i n an attempt to understand the mechanisms by which net changes i n plasma i o n 5 c o n c e n t r a t i o n s observed i n f i s h under s i m i l a r a c i d c o n d i t i o n s were t a k i n g p l a c e . An a c i d o t i c c o n d i t i o n , l i k e other s t r e s s f u l s t a t e s i n f i s h , e l i c i t s a r e l e a s e o f catecholamines (Primmett e t a l . 1986, B o u t i l i e r et. al.. 1986, Perry 1986). While t h i s i n c r e a s e i n catecholamines has potent c a r d i o v a s c u l a r e f f e c t s , some of the e f f e c t s i n c l u d e the modulation of i o n t r a n s p o r t a c r o s s e p i t h e l i a and membranes. Some o f those e f f e c t s , such as the i n h i b i t i o n o f C1"7HCC>3~ exchange and the s t i m u l a t i o n o f Na +/H +(NH4 +) exchange known i n mammalian t i s s u e s have been demonstrated i n the f i s h g i l l (Perry et. al.. 1984, Payan g_t, ai.. 1975, G i r a r d and Payan 1977, Payan 1978). While a l a r g e body o f data suggests t h a t the r o l e o f i o n exchange processes ac r o s s the f i s h g i l l i n the r e g u l a t i o n o f acid-base balance i s a major one, most of the evidence i s d e s c r i p t i v e . Analyses o f the t r o u t - s a l i n i t y - h y p e r c a p n i a experiment of S e c t i o n 2. and the experiments i n S e c t i o n 4. address the s u b j e c t o f the p o s s i b l e c o n t r o l l i n g f a c t o r s f o r acid-base r e g u l a t i o n i n i o n t r a n s f e r processes between blood and water. The f u n c t i o n a l s i g n i f i c a n c e of catecholamines a t the g i l l as w e l l as the red c e l l membrane are a l s o examined. 6 G E N E R A L M A T E R I A L S A N D M E T H O D S 7 GENERAL MATERIALS AND METHODS I. CANNULATIONS A l l experimental animals were f i t t e d with c h r o n i c i n d w e l l i n g cannulae under a n a e s t h e s i a i n s h o r t o p e r a t i o n s l a s t i n g about 10-15 minutes f o r d o r s a l a o r t i c c a n n u l a t i o n s and about 25 minutes f o r c a n n u l a t i o n s of the abdominal d o r s a l a o r t a through an i n t e s t i n a l a r t e r y . A l l f i s h were a n e s t h e t i z e d with T r i c a n e methane sulphonate (MS222) at c o n c e n t r a t i o n s of 1:10,000 to i n i t i a l l y render the animals unconscious and then maintained with a c o n c e n t r a t i o n of 1:20,000 on the o p e r a t i n g t a b l e . Both s o l u t i o n s were b u f f e r e d to about pH 7.5 with NaHC03. The o p e r a t i n g t a b l e c o n s i s t e d of a water t a b l e with an a d j u s t a b l e n e t t i n g which h e l d the f i s h v e n t r a l s i d e up. The g i l l s o f the f i s h were c o n t i n u o u s l y i r r i g a t e d with an a n e s t h e t i c s o l u t i o n . The temperature o f the o p e r a t i n g t a b l e a n e s t h e t i c was maintained a t the same temperature as t h a t used i n the experiments and to which the f i s h had been a c c l i m a t e d . A l l s y r i n g e s , c a t h e t e r s and cannulas were r i n s e d with h e p a r i n i z e d s a l i n e p r i o r to use. Except f o r the experiments on the shark, and conger e e l , a l l f i s h were f i t t e d with cannulae i n the d o r s a l a o r t a by b l i n d puncture i n a n e s t h e t i z e d f i s h . The technique of Smith and B e l l (1964) or the technique of S o i v i o e£..al.. (1972) was 8 used. The s i z e o f the cannula ( e i t h e r PE50 or PE60) depended on the s i z e o f the f i s h and i s s p e c i f i e d i n the M a t e r i a l s and Methods o f each s e c t i o n . In the f i r s t technique, a c a t h e t e r (Sovereign I n d w e l l i n g Canine Catheter 2 i n c h , 18 gauge) was used to make the b l i n d puncture through the m i d l i n e of the r o o f of the mouth, between the f i r s t and second g i l l arches and i n t o the d o r s a l a o r t a . A f t e r the puncture was made, the metal needle i n the c a t h e t e r was removed and a p o l y e t h y l e n e cannula was fed down the c a t h e t e r i n t o the a o r t a . The p l a s t i c c a t h e t e r was then removed l e a v i n g the cannula c h r o n i c a l l y implanted i n the v e s s e l . The cannula was fed through a p i e c e of PE200 cannula f l a n g e d on one end to anchor i t i n the mouth and which passed through the r o o f of the mouth v i a a hole i n f r o n t o f the nares. T i e s with c o t t o n or s i l k thread were made around the PE200 with the i n d w e l l i n g cannula j u s t o u t s i d e the f i s h to add another l e v e l o f s e c u r i t y to the cannula. The second method i n v o l v e d the b l i n d puncture o f the d o r s a l a o r t a u s i n g a sharpened s t e e l wire i n s e r t e d i n t o a l e n g t h o f cannula so t h a t j u s t the t i p of the wire protruded from the cannula. With the same o r i e n t a t i o n f o r e n t r y as the f i r s t method, the wire f u n c t i o n e d to guide the cannula and to enter the w a l l o f the d o r s a l a o r t a . The wire was removed and the cannula was advanced down the a o r t a between 5 and 7 cent i m e t e r s depending on the s i z e o f the f i s h . T h i s i n d w e l l i n g cannula was guided out of the f i s h i n an i d e n t i c a l 9 manner as above. However, s i n c e t h i s cannula was n e c e s s a r i l y s h o r t due to the i n s e r t e d wire, a connector was used to a t t a c h i t to a longer cannula to f a c i l i t a t e sampling. In the experiments which i n v o l v e d the shark and the conger e e l the d o r s a l a o r t a was cannulated through a g a s t r i c or i n t e s t i n a l a r t e r y . The body c a v i t y was opened by a 3-4 cm long m i d - v e n t r a l i n c i s i o n and a PE50 c a t h e t e r was in t r o d u c e d i n t o the g a s t r i c or i n t e s t i n a l a r t e r y and advanced through to the d o r s a l a o r t a . T h i s cannula was s e c u r e l y t i e d to the v e s s e l , was c u t and f i t t e d to a t h i c k - w a l l e d PVC tu b i n g (1mm i . d . , 1.8mm o.d.) through which blood was sampled. The body w a l l was c l o s e d by two l a y e r s o f sut u r e s and the cannula was l e d out of the body c a v i t y by another small i n c i s i o n caudal to the l a r g e r i n c i s i o n . F u r t h e r d e t a i l s o f t h i s surgery are gi v e n by Toews e_t.al.. (1983). F o l l o w i n g surgery, a l l animals recovered i n perspex boxes with ample flow o f aera t e d water. Cannulae were f l u s h e d d a i l y with C o r t l a n d s a l i n e (Wolf 1963) c o n t a i n i n g 10,000 USP u n i t s / L sodium he p a r i n . A l l animals were allowed to recover f o r a t l e a s t 24h p r i o r to s t a r t i n g experimental procedures. I I . MEASUREMENT OF TRANSEPITHELIAL POTENTIALS (TEP) TEP was measured a c r o s s the g i l l e p i t h e l i u m u s i n g p a i r s o f calomel or s i l v e r - s i l v e r c h l o r i d e e l e c t r o d e s . The r e f e r e n c e 10 F i g u r e G l . Apparatus f o r measurement of t r a n s e p i t h e l i a l p o t e n t i a l s ( T E P ) . S t i p l e d 'bridge' cannulae c o n t a i n e d 3M KC1 s e t i n agar. One was i n c o n t a c t with the cannulae i n the d o r s a l a o r t a ( c l e a r ) through a 'T' p i e c e . The other r e f e r e n c e was i n c o n t a c t with the water near the g i l l s . The i n s e r t shows a c l o s e - u p o f the o r i e n t a t i o n o f the r e f e r e n c e b r i d g e with p a r t o f the o p e r c u l a r cover c u t away. The r e f e r e n c e b r i d g e was threaded through another cannula o f l a r g e r diameter which was f l a r e d on one end to anchor i t i n s i d e the o p e r c u l a r c a v i t y . APPARATUS FOR TEP MEASUREMENTS 12 and measuring e l e c t r o d e s were connected to the water and blood by PE50 cannulae f i l l e d with 3M KCL s e t i n agar. The diagram below d e s c r i b e s the apparatus. The r e f e r e n c e e l e c t r o d e was p l a c e d near the g i l l s by t h r e a d i n g i t through a l a r g e r diameter cannula which was sewn i n p l a c e j u s t i n s i d e the operculum. The measuring e l e c t r o d e was i n c o n t a c t with the blood through the s a l i n e which f i l l e d the i n d w e l l i n g cannula. There was no d e t e c t a b l e leak o f KC1 i n t o the blood. The "T" pi e c e allowed the c o n n e c t i o n of the blood to e i t h e r the measuring e l e c t r o d e or to the s y r i n g e which was used f o r blood c o l l e c t i o n or i n f u s i o n procedures. The p o t e n t i a l a c r o s s the e l e c t r o d e s was measured u s i n g v o l t m e t e r s which are s p e c i f i e d i n the M a t e r i a l s and Methods of i n d i v i u a l experiments. The vol t m e t e r s were zeroed by s h o r t i n g the c i r c u i t by co n n e c t i n g the e l e c t r o d e s with a s h o r t cannula c o n t a i n i n g the 3M KCL d e s c r i b e d above. The zero v o l t a g e used f o r the d e t e r m i n a t i o n of the TEP value was t h a t read when both r e f e r e n c e and measuring e l e c t r o d e cannulae were p l a c e d i n the water together. T h i s o f f s e t v o l t a g e was s m a l l e r than 1 mV i n a l l cases. I I I . MEASUREMENT OF ACID-BASE PARAMETERS A. Constants : The apparent f i r s t d i s s o c i a t i o n constant (pKapp) of c a r b o n i c a c i d and the s o l u b i l i t y o f carbon d i o x i d e 13 (CO2) determined by B o u t i l i e r et_. al_. (1985) were used. B. pH : A l l measurements were made a t the temperature o f the animal. pH was measured on whole blood and red c e l l l y s a t e s u s i n g a Radiometer G279/G2 g l a s s c a p i l l a r y e l e c t r o d e and K497 calomel e l e c t r o d e coupled to a PHM 84 pH meter. The red c e l l l y s a t e s were obtained by c e n t r i f u g i n g whole blood to o b t a i n the red c e l l f r a c t i o n which was then twice f r o z e n and thawed as d e s c r i b e d by Z e i d l e r and Kim (1977). Minor d e v i a t i o n s from t h i s procedure are noted i n the M a t e r i a l s and Methods s e c t i o n s of i n d i v i d u a l experiments. Radiometer p r e c i s i o n phosphate b u f f e r s S1500 and S1510 were used i n c a l i b r a t i o n s . Readings were r e f e r e n c e d to the S1510 b u f f e r and adjustments were made by adding or s u b t r a c t i n g one h a l f o f the d r i f t i n the measured b u f f e r value. C. T o t a l CO2 : T o t a l C 0 2 was measured i n water and blood samples u s i n g one of two methods which are s p e c i f i e d i n the M a t e r i a l s and Methods o f each experiment. Standards f o r both techniques were made with d r i e d NaHC03. The f o l l o w i n g procedure was employed when the Capni-Con III(Cameron Instruments Inc., P o r t Aransas, Texas) was used. T h i s i s a technique where HC03~ i s converted to C 0 2 by a c i d i f i c a t i o n and measurement i s made of the change i n PC02 (Cameron 1971). Plasma samples were taken from hematocrit tubes immediately a f t e r d e t e r m i n a t i o n . When a C 0 2 ~ s p e c i f i c 14 gas chromatograph ( C a r l e Model I I I , C a r l e Instruments Inc. U.S.A.) was used, the gaseous CO2 evolved from the a c i d i f i c a t i o n o f the sample was a n a l y s e d by d i f f e r e n t i a l t h e r m a l c o n d u c t i v i t y . Peak h e i g h t s on the output r e c o r d e r were r e f e r e n c e d to standards (Lenfant and Aucutt 1966). D. P C 0 2 1 P c o 2 values e i t h e r were c a l c u l a t e d u s i n g the Henderson/Hasselbach e q u a t i o n or measured with Radiometer e l e c t r o d e s and meters a c c o r d i n g to the recommendations of B o u t i l i e r et.al.(1978,1985). E. HCO3 - : HCO3 - values f o r plasma samples were c a l c u l a t e d by the equation, HCO3- = T C 0 2 - P c o 2 * ( s o l u b i l i t y of C 0 2 ) F. NH4 +/NH3 : Plasma t o t a l ammonia c o n c e n t r a t i o n s were determined by L-glutamic dehydrogenase/NAD enzymatic assays from e i t h e r Sigma (Sigma D i a g n o s t i c s U.S.A.) or Boehringer-Mannheim (Boehringer Mannheim Gmbh D i a g n o s t i c a F.R.G.). Water t o t a l ammonia c o n c e n t r a t i o n s were determined by these methods or by an ammonia e l e c t r o d e as d e s c r i b e d below i n V. f o r experiments t h a t i n v o l v e d a D e l t a Bicarbonate System. 15 IV. ION CONCENTRATIONS Plasma and water Na +, K +, Mg + + and C a + + c o n c e n t r a t i o n s were determined by atomic a b s o r p t i o n spectrophotometry (Perkin-Elmer Model 2380) as d e s c r i b e d byAnnio (1964). C h l o r i d e c o n c e n t r a t i o n s f o r water and plasma were determined by one o f three methods which are s p e c i f i e d i n the d e s c r i p t i o n o f i n d i v i d u a l experiments. In some experiments, plasma and water c h l o r i d e c o n c e n t r a t i o n s were determined with a Buchler C o t l o v e c h l o r i d e t i t r a t o r a c c o r d i n g to the method of Cotlo v e (1963) u s i n g a p p r o p r i a t e standards. In other experiments plasma and water (when c o n c e n t r a t i o n s were g r e a t e r than lOOmM) c h l o r i d e c o n c e n t r a t i o n s were determined by t i t r a t i o n u s i n g a Radiometer CMT10 T i t r a t o r . Water c h l o r i d e c o n c e n t r a t i o n s were determined i n some experiments with a system c o n s i s t i n g o f a s o l i d s t a t e C l ~ s e n s i t i v e e l e c t r o d e and r e f e r e n c e connected to a mi c r o p r o c e s s o r / i o n a n a l y s e r and was c a l i b r a t e d with NaCl standards and r e f e r e n c e d to a p a r t i c u l a r standard between each measurement. V. DELTA BICARBONATE SYSTEM A c l o s e d water r e c i r c u l a t i o n system such as t h a t d e s c r i b e d 16 by H e i s l e r ejt.al.. (1976) was used f o r S a l i n i t y - T r o u t , S a l i n i t y - C o n g e r and the Na +-Cl~-Carp experiments. V a r i a t i o n s of t h i s system which were unique to those experiments are d e s c r i b e d i n the M a t e r i a l s and Methods s e c t i o n s of the r e s p e c t i v e experiments. Such a system t y p i c a l l y c o n s i s t e d of a f i s h box, an oxygenator and bubble t r a p system, and a water c i r c u l a t i o n pump. Th i s system wasclosed f o r a l l n o n - v o l a t i l e substances but open f o r gas exchange. The main advantage of such a system was t h a t changes i n i o n c o n c e n t r a t i o n s due to exchanges between f i s h and water accumulate to l e v e l s t h a t can be measured a c c u r a t e l y The p o t e n t i a l problem of the b u i l d - u p o f t o x i c waste products such as ammonia was avoided by f l u s h i n g the system with f r e s h water thermostatted to the temperature of the system every 24h. T h i s system allowed the continuous measurement o f net HCO3 - changes and an automated way to measure NH4 + c o n c e n t r a t i o n s i n the water. Water from the f i s h chamber was c o n t i n u o u s l y pumped to a pH g l a s s e l e c t r o d e and a double e l e c t r o l y t e b r i d g e (Ag/AgCl) r e f e r e n c e e l e c t r o d e a f t e r e q u i l i b r a t i o n with 1% CO2 and pumped back to the f i s h chamber. A l l e l e c t r o d e s were a c c l i m a t e d to the p a r t i c u l a r s a l i n i t y o f the experiment f o r a t l e a s t 3 weeks. The e l e c t r o d e s were connected to a high impedance i s o l a t i o n a m p l i f i e r (Knick, B e r l i n F.R.G.) and the r e s u l t i n g s i g n a l was output to a r e c o r d e r . At programmed i n t e r v a l s water was 17 pumped from the f i s h chamber along with a s t r o n g base from another r e s e r v o i r to an ammonia e l e c t r o d e (Ingold E l e c t r o d e s Inc., L e xington Mass. U.S.A.). The flow and base c o n c e n t r a t i o n were a d j u s t e d so t h a t the r e s u l t i n g mixture had a pH of about 10. The base converted a l l NH4 + to p h y s i c a l l y d i s s o l v e d ammonia which was sensed by the e l e c t r o d e . The s i g n a l from the e l e c t r o d e was a m p l i f i e d and f i l t e r e d and the r e s u l t i n g peak h e i g h t s on the output r e c o r d e r were r e f e r e n c e d to standards made from NH4CI. VI. GASES A l l gas mixtures were made up from pure gases or a i r mixed with Wostoff (Bokum, F.R.G.) gas mixing pumps. SECTION 1. VENTILATION AND ACID-BASE REGULATION IN FISHES 19 INTRODUCTION V e n t i l a t i o n i n f i s h e s i s s t i m u l a t e d by acid-base d i s t u r b a n c e s . T h i s has been shown f o r f i s h exposed to a c i d waters (Hargis 1976; Hoglund and Persson 1971; D i v e l y e t al.. 1977), f o r f i s h i n f u s e d with a c i d or base (Cunningham 1974; Janssen and Randall 1975) and f o r hypercapnic f i s h (Van Dam 1938; Randall and Jones 1973; Janssen and Rand a l l 1975; Eddy 1976; Smith and Jones 1982). This e f f e c t i s a l s o found i n a wide range of s p e c i e s i n c l u d i n g elasmobranchs, cyclostomes and t e l e o s t s . Evidence i n the l i t e r a t u r e suggests that the predominant cause i n the responses by these s p e c i e s may be d i f f e r e n t . The s t i m u l a t i o n o f v e n t i l a t i o n i n f i s h under a c i d c o n d i t i o n s suggests that the sti m u l u s i s r e l a t e d to a need to a l t e r e i t h e r oxygen uptake or carbon d i o x i d e e x c r e t i o n . There i s a l a r g e body of data s u g g e s t i n g t h a t the i n c r e a s e i n v e n t i l a t i o n i n t e l e o s t s d u r i n g a c i d c o n d i t i o n s i s due to a stimu l u s to i n c r e a s e d oxygen e x t r a c t i o n s i n c e Bohr and Root s h i f t s would r e s u l t i n a lower oxygen c a r r y i n g c a p a c i t y o f the blood by red u c i n g the oxygen-hemoglobin a f f i n i t y . Smith and Jones (1982) have shown t h a t the i n c r e a s e d v e n t i l a t i o n i n t r o u t exposed to environmental hypercapnia was a t t e n t u a t e d by i n c r e a s i n g the oxygen content o f the water. This s t r o n g l y 20 suggests that i n those s p e c i e s showing Bohr and Root s h i f t s , t h e s t i m u l a t i o n o f v e n t i l a t i o n i s o r i e n t e d to i n c r e a s i n g oxygen uptake from the water. There are two ex c e p t i o n s to t h i s , one the elasmobranch, the blood of which shows no Bohr or Root s h i f t c h a r a c t e r i s t i c s (Lenfant and Johansen 1966), but v e n t i l a t i o n i s s t i m u l a t e d i n response to hypercapnic c o n d i t i o n s (Randall e t al.. 1976; H e i s l e r et. a l . 1976). The other i s members of the f a m i l y C y p r i n i d a e which show l i t t l e change i n v e n t i l a t i o n i n s p i t e o f the blood showing evidence of Bohr and Root s h i f t s (Dejours 1983). Randall and Cameron (1973) have suggested three a p r i o r i reasons a g a i n s t the other p o s s i b l e r o l e of i n c r e a s e d v e n t i l a t i o n i n response to hypercapnia, t h a t i s the e x c r e t i o n of CO2 and the consequent r e g u l a t i o n o f blood Pco2 t e n s i o n . F i r s t , the l o g / l i n e a r r e l a t i o n s h i p o f Pco2 to pH would r e q u i r e very f i n e c o n t r o l o f Pco2 t e n s i o n s to c o n t r o l pH. Second, the scope o f adjustment i s l i m i t e d by the low CO2 content of f i s h blood. T h i r d , the o r i e n t a t i o n of v e n t i l a t i o n to adjustments of Pco2 may compromise O2 uptake; a c o n d i t i o n which i s disadvantageous to l i f e i n water where the O2 content i s lower than a i r . The two experiments d e s c r i b e d i n t h i s S e c t i o n were designed to i n v e s t i g a t e two aspects of t h i s broad s u b j e c t of v e n t i l a t i o n and i t s r o l e i n acid-base r e g u l a t i o n i n f i s h e s . The f i r s t experiment i n v e s t i g a t e d the a c t u a l scope of a d j u s t i n g blood CO2 t e n s i o n s by a l t e r i n g g i l l water flow i n 21 t r o u t . Wood and Jackson (1980) estimated the c a p a c i t y of Pco2 adjustments v i a changes i n g i l l water flow to be about 2 mmHg i n t h a t s p e c i e s . G i l l water flow was e x p e r i m e n t a l l y manipulated and C 0 2 e x c r e t i o n was measured d i r e c t l y i n order to d e s c r i b e the r e l a t i o n s h i p between these parameters over a range o f v e n t i l a t i o n volumes. The second experiment i n t h i s S e c t i o n i n v e s t i g a t e d the c o n t r o l o f v e n t i l a t i o n i n the l a r g e r s p o t t e d d o g f i s h . The l a c k of Bohr and Root s h i f t s i n the blood of t h i s elasmobranch emphasizes the p o s s i b i l i t y t h a t the c o n t r o l of v e n t i l a t i o n might be o r i e n t e d towards some parameter or s e t of parameters other than the maintenance of 0 2 s a t u r a t i o n o f the blood. A p o s s i b i l i t y i s the r e g u l a t i o n of pH, P c o 2 t e n s i o n s or HCC>3~ l e v e l s i n the blood by adjustments of blood P c o 2 v i a changes i n g i l l water flow. D i r e c t measurement o f water flow over the g i l l s o f the d o g f i s h d u r i n g simultaneous exposure to environmental hyperoxia and v a r y i n g l e v e l s of hypercapnia enabled the c o r r e l a t i o n of g i l l v e n t i l a t i o n with the three acid-base parameters i n the blood. MATERIALS AND METHODS EXPERIMENT IA. VENTILATION AND C0 2 EXCRETION IN TROUUT FISH 22 Rainbow t r o u t , Sal mo c r a i r d n e r i , between 192 and 353 g were obtained from a commercial hatchery and maintained i n outdoor f i b e r g l a s s tanks s u p p l i e d with a e r a t e d and d e c h l o r i n a t e d Vancouver tap water (5-10°C; pH 6.9-7.1; CaCC>3 4 ppm). They were maintained on a dry t r o u t p e l l e t fed ad 1ibitum with a s e l f feeder. F i s h were s t a r v e d f o r a t l e a s t 48 h p r i o r to s u r g i c a l procedures and subsequent experimentation. SURGERY, APPARATUS AND EXPERIMENTAL PROCEDURES : F i s h were f i t t e d with c h r o n i c i n d w e l l i n g d o r s a l a o r t i c cannulas by the method of Smith and B e l l (1964)(see General M a t e r i a l s and Methods) and with rubber Van Dam masks a c c o r d i n g to the method d e s c r i b e d by Cameron and Davis (1970). A f t e r the o p e r a t i o n each f i s h was i n s e r t e d i n t o a narrow b l a c k box i n one p a r t of a two chambered perspex box. The rubber mask, which was att a c h e d to the mouth and snout o f the f i s h , was secured to the d i v i d e r between the two chambers and acted as a dam t h a t i n s u r e d a l l water p a s s i n g from the f r o n t chamber to the back chamber was v i a the mouth and g i l l s o f the f i s h . I n s p i r e d and mixed e x p i r e d waters were thus separated and v e n t i l a t i o n volumes were i n f l u e n c e d by a d j u s t i n g the water he i g h t i n the i n s p i r e d water chamber a n t e r i o r to the f i s h . T h i s was done by a d j u s t i n g the h e i g h t of the f r o n t chamber overflow standpipe r e l a t i v e to that i n the p o s t e r i o r chamber, which c a r r i e d the overflow to waste. P o s i t i v e , zero and 23 negative pressure heads from the mouth to o p e r c u l a r chambers were e f f e c t e d i n t h i s way. A p e r i o d of 24 h fo l l o w e d the s e t t i n g o f any pre s s u r e head before measurements were taken. Changes i n head pressure were always from p o s i t i v e to negative although the magnitude of change and the number o f changes imposed per f i s h were not c o n s i s t e n t . MEASUREMENTS : V e n t i l a t i o n volume (Vg) was determined by c o l l e c t i n g the outflow water from the standpipe i n the chamber c o n t a i n i n g the trunk o f the f i s h . C o l l e c t i o n s were made over 1 min p e r i o d s and volumes were determined by weight. Concurrent measurements of v e n t i l a t o r y frequency ( f ) were made by coun t i n g o p e r c u l a r movements through a small c l e a r s e c t i o n i n the b l a c k box c o n t a i n i n g the f i s h . S e v e r a l counts per minute were averaged. Stroke volume of the bucca l pump (Vsv) was c a l c u l a t e d as Vg d i v i d e d by f. I n s p i r e d (Pio2 ) a n d mixed e x p i r e d (PeQ 2) water oxygen t e n s i o n s as we l l as a r t e r i a l oxygen t e n s i o n s were measured with Radiometer oxygen e l e c t r o d e s thermostatted to the experimental water temperature. T o t a l oxygen content (CaQ2^ °^ t n e bloocl was measured with a Lex-02-Cont apparatus (Lexington I n s t r . ) . Plasma CO2 t e n s i o n (PC02) was measured with a 24 Radiometer e l e c t r o d e thermostatted to the experimental temperature a c c o r d i n g to the recommendations of B o u t i l i e r e_t a l . (1978). Measurements of t o t a l CO2 i n the i n s p i r e d ( C i c o 2 ) and mixed e x p i r e d (Ceco2) water samples as w e l l i n plasma samples were made by gas chromatography a c c o r d i n g to the method d e s c r i b e d i n General M a t e r i a l s and Methods. Hematocrit was measured a c c o r d i n g to the method of Snieszko (1960). Gas t r a n s f e r r a t e s (M02 and Mco 2) were c a l c u l a t e d by the F i c k p r i n c i p l e . pH of the e x t r a c e l l u l a r (pHe) and red c e l l i n t r a c e l l u l a r (pHi) f l u i d s were measured as d e s c r i b e d i n General M a t e r i a l s and Methods. C o n c e n t r a t i o n s o f the catecholamines, a d r e n a l i n e and n o r a d r e n a l i n e o f 11 plasma samples taken from f i s h a t s e v e r a l head p r e s s u r e s were determined by high pressure l i q u i d chromatography (HPLC) a c c o r d i n g to Woodward (1982). Whole blood l a c t a t e c o n c e n t r a t i o n s were assayed e n z y m a t i c a l l y with Sigma reagents (Sigma b u l l e t i n no. 826-UV). STATISTICS : C o r r e l a t i o n and r e g r e s s i o n analyses were used to d e s c r i b e the r e l a t i o n s h i p s between parameters i n t h i s experiment. A n a l y s i s o f v a r i a n c e (ANOVA) was used to compare the s t a t i s t i c a l s i g n i f i c a n c e among means with a 5 % l e v e l o f r e j e c t i o n . EXPERIMENT IB. SHARK - HYPEROXIA - HYPERCAPNIA 25 ANIMALS : Larger s p o t t e d d o g f i s h , S c v l i o r h i n u s s t e l l a r i s , weighing between 1550 to 2820 g were caught i n the Bay of Naples, I t a l y . They were h e l d i n 200 1 f i b e r g l a s s tanks i n the l a b o r a t o r y without feed u n t i l they were used f o r experimentation. The tanks r e c e i v e d a continuous flow o f ambient sea water (pH 7.9; [HCC>3~] 2.3 mM) . Water temperature was maintained a t 19°C. SURGERY AND APPARATUS : A c h r o n i c i n d w e l l i n g cannula was i n s e r t e d i n t o the d o r s a l a o r t a through an i n t e s t i n a l a r t e r y as d e s c r i b e d i n General M a t e r i a l s and Methods. Latex rubber bags were fashioned from the thumbs of surgeons gloves and attached over the g i l l s l i t s to c o l l e c t the e x p i r e d water. E l e c t r o m a g n e t i c flow probes were connected to the ends of these bags to measure the v e n t i l a t i o n volume. The animals were recovered i n a 'Delta Bicarbonate System' ( F i g . 1) d e s c r i b e d i n General M a t e r i a l s and Methods and i n d e t a i l i n H e i s l e r (1978). There was a recovery p e r i o d o f 24 h experimental procedures were i n i t i a t e d . PROTOCOL AND MEASUREMENTS : 26 F i g u r e 1. Diagram of experimental apparatus used to conduct r e s p i r a t o r y and acid-base r e g u l a t o r y experiments i n the shark, S c v l i o r h i n u s s t e l l a r i s . See t e x t f o r d e t a i l s . 28 A f t e r 3 c o n t r o l samples were taken, the ambient P02 t e n s i o n was e l e v a t e d to 500 mmHg of atmospheric p r e s s u r e . A f t e r a p e r i o d where only the oxygen t e n s i o n was i n c r e a s e d , the PC02 t e n s i o n o f the ambient water was a l s o r a i s e d to va r i o u s l e v e l s f o r each f i s h to induce a range o f aci d o s e s and consequent range i n blood pH, PC02 and HCO3 - l e v e l s . The hypercapnia l e v e l s ranged from 7 to 44 mmHg. Approximately 700 u l of blood was removed throught the cannula a t each sample time f o r the d i r e c t measurement o f blood pH, PC02 and Tco2- Blood HCC>3~ c o n c e n t r a t i o n s were c a l c u l a t e d a c c o r d i n g to the methods d e s c r i b e d i n General M a t e r i a l s and Methods. Blood samples were taken a t 30 min, 1 h and 2 h a f t e r the onset o f hyperoxia and every 30 min du r i n g the combined hyperoxia/ hypercapnia exposure Two a d d i t i o n a l experiments were c a r r i e d out. The p r o t o c o l and sampling procedures were i d e n t i c a l to th a t f o r the above experiment. In the f i r s t , 0.3 M NaHCC>3 or 0.6 M HC1 was in f u s e d through the cannula with each e l e v a t i o n i n P c o 2 i n order to minimize the changes i n blood pH. In the second a d d i t i o n a l p r o t o c o l , the water PC02 was e l e v a t e d to only one l e v e l . The n a t u r a l accumulation of blood HCO3 - was supplemented by i n f u s i o n of NaHCC>3 through the cannula. STATISTICS : C o r r e l a t i o n analyses were used to d e s c r i b e the 29 r e l a t i o n s h i p s between data s e t s . RESULTS EXPERIMENT IA. : ROLE OF VENTILATION IN ACID-BASE REGULATION A l l animals were q u i e s c e n t and i n steady s t a t e with r e s p e c t to gas exchange. The mean plasma 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 11 samples from 5 f i s h e x p e r i e n c i n g v a r i o u s pressure heads was 2.6 + 0.42(mean + 1S.E.) nanomoles/1, a value c o n s i s t e n t with r e s t i n g l e v e l s f o r f i s h . Blood l a c t a t e c o n c e n t r a t i o n s of 8 animals i n Van Dam boxes were a l s o c o n s i s t e n t l y low, 0.58 +. 0.02(means +, 1S.E.) mM, and d i d not vary s i g n i f i c a n t l y with the changes i n head pre s s u r e . The imposed pressure heads e f f e c t e d s i g n i f i c a n t l y d i f f e r e n t v e n t i l a t i o n volumes, although there was c o n s i d e r a b l e o v e r l a p i n the ranges of Vg a t n e g a t i v e , n e u t r a l and p o s i t i v e heads (Table 1). As shown by o t h e r s , f i s h v a r i e d s t r o k e volume of the bucc a l pump r a t h e r than frequency of b r e a t h i n g to a d j u s t v e n t i l a t i o n volume ( F i g . 2) i n the face of imposed pressure heads (Van Dam 1938; Davis and Cameron 1971; Randall and Jones 1973). The r e l a t i o n s h i p between Vg and a r t e r i a l Pco2 was an i n v e r s e power f u n c t i o n ( F i g . 3). Vg and a r t e r i a l P02 showed the r e v e r s e r e l a t i o n s h i p . 30 Table 1. G i l l v e n t i l a t i o n volumes (Vg) of f i s h experiencing various head pressures i n a Van Dam apparatus. RANGE OF HEAD RANGE OF Vg MEANS + STD.ERR. PRESSURES(mm) (ml/min) -8 to 0 26.8 to 131.3 56.28 +6.95 0 42.2 to 139.9 75.13 + 12.27 =j s i g . 0 to +21 48.4 to 342.4 145.92 + 19.39 ~1 "1 n. s.  si'c 7... J n.s. not s i g n i f i c a n t l y d i f f e r e n t by ANOVA ; P<.05 s i g . s i g n i f i c a n t l y d i f f e r e n t by same t e s t i 31 F i g u r e 2. The r e l a t i o n s h i p between b r e a t h i n g frequency ( f ) , the volume o f water pumped each breath or s t r o k e volume ( V s v ) and the volume of water f l o w i n g over the g i l l s (Vg) of t r o u t with a Van Dam apparatus. Best f i t l i n e a r r e g r e s s i o n l i n e s : Vsv = 0.0892 + 0.1456 * Vg; r 2 = 0.9211; N = 42 Vsv = 288.145 - 0.7093 * f; r 2 = 0.0025; N = 39 (ml/min) 33 F i g u r e 3. The r e l a t i o n s h i p between a r t e r i a l PC02 and v e n t i l a t i o n volume (Vg) over the range of v e n t i l a t i o n volumes imposed on t r o u t i n t h i s study. Black dots r e p r e s e n t f i s h which ram v e n t i l a t e d . Best f i t power curve : P c o 2 = 8.95*Vg~°•209. r _ 0.484; N = 43. 34 35 P c o 2 = 8.95 * v g - 0 - 2 0 9 r = 0.484, N = 43 P o 2 = 27.66 * V g 0 - 2 9 2 r = 0.469, N = 31 There was a s i g n i f i c a n t negative c o r r e l a t i o n between P o 2 and P c o 2 : r = -0.784, N = 31 ( F i g . 4). C0 2 e x c r e t i o n , Mco 2, showed a weak but s i g n i f i c a n t n e g a tive c o r r e l a t i o n with a r t e r i a l P c o 2 : r = -0.333, N = 40 Higher c o n v e c t i o n requirements , Vg/Mco 2, were a s s o c i a t d with lower P c o 2 values ( F i g . 5). At low l e v e l s of Vg, both Mo 2 and Mco 2 decreased with g i l l v e n t i l a t i o n volume. The mean r e s p i r a t o r y gas exchange r a t i o , RQ, was 0.87 + 0.04(means £ 1S.E.). L i n e a r r e g r e s s i o n a n a l y s i s of these parameters y i e l d e d a slope o f 0.012 and r 2 = 0.637 ( F i g . 6). EXPERIMENT IB. ROLE OF pH IN VENTILATION Given s u f f i c i e n t oxygen, an e l e v a t i o n of environmental P c o 2 r e s u l t s i n a plasma a c i d o s i s . Blood P c o 2 and HCO3 - are e l e v a t e d and as a consequence pH drops; g i l l v e n t i l a t i o n i n c r e a s e s as w e l l . The i n c r e a s e i n v e n t i l a t i o n i s best c o r r e l a t e d with changes i n plasma pH ( F i g . 7, 8 & 9). Vg i n c r e a s e s e x p o n e n t i a l l y as plasma pH decreases ( F i g . 10). The e l i m i n a t i o n of the pH change d u r i n g t h i s exposure to hypercapnia, causes a c o n s i d e r a b l e decrease i n the c o r r e l a t i o n between P c o 2 and Vg ( F i g . I D a s well as between [HC03~3 and Vg ( F i g . 12) . 36 Fi g u r e 4. The r e l a t i o n s h i p between a r t e r i a l Pco2 and a r t e r i a l P02 over the range o f v e n t i l a t i o n volumes imposed on t r o u t i n t h i s study. PC02 = 5.660 0.019*Po 2; r 2 = 0.6150; N = 31. r a c o 2 (mmHg) 3 P a 0 2 (mmHg) 38 F i g u r e 5. The r e l a t i o n s h i p between the c o n v e c t i o n requirement f o r CO2 (Vg/Mco2) and a r t e r i a l PC02 over the range of v e n t i l a t i o n volumes imposed i n t h i s study. Vg/Mco 2 = 3 . 1 6 5 * P c o 2 ~ ° ' 4 7 7 ; r = 0.333; N = 40). 40 Fi g u r e 6. The r e l a t i o n s h i p between CO2 e x c r e t i o n (HC02) and O2 uptake (M02) a t d i f f e r e n t v e n t i l a t i o n volumes. The gas exchange r a t i o was 0.87 + 0.04. M02 = 0.298 + 1.012*Mco 2; r 2 = 0.637; N = 42. 5 0 1 2 3 4 5 M 0 2 (mM/h/kg) 42 F i g u r e 7. The r e l a t i o n s h i p between v e n t i l a t o r y volume and plasma pH (pHpl) i n S c v l i o r h i n u s s t e l l a r i s undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. V e n t i l a t o r y volume (Vg act./Vg c o n t r . ) i s the r a t i o o f the measured Vg d i v i d e d by the Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the best f i t l i n e f o r a l l the experimental data f o r an i n d i v i d u a l f i s h . The data s e t shown i n c l u d e s two experiments. One i s the combination o f environmental hyperoxia and hypercapnia exposure alone. The other has the added treatment of NaHC03 or HC1 i n f u s i o n to keep the pH ' c o n s t a n t 1 , or to minimize the change i n pH. C o e f f i c i e n t s f o r each l i n e : Vg a c t . / Vg c o n t r . = a * e ( b * P H> a b r 2 5. 3996 * 1 015 -4.9322 0. 950 0. 2600 * IO 9 -2.4640 0. 841 5. 7770 * 10 6 -2.1344 0. 792 2. 2580 * i o i o -3.2478 0.636 4. 7401 * -3.3712 0.696 2. 3955 * I O " -3.8865 0.963 1. 3504 * 1021 -6.5133 0. 934 2. 2187 * I O " -3.4635 0. 846 1. 1901 * I O " -3.4600 0. 830 2. 2668 * 1013 -4.2010 0.978 2. 8936 * I O " -3.5686 0.833 2. 2743 * 1014 -4.4154 0.956 3. 1797 * i o n -3.6072 0.860 43 44 F i g u r e 8. The r e l a t i o n s h i p between v e n t i l a t o r y volume and plasma pH (pHpl) i n S c v l i o r h i n u s s t e l l a r i s undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. V e n t i l a t o r y volume (Vg act./Vg c o n t r . ) i s the r a t i o o f the measured Vg d i v i d e d by the Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the best f i t l i n e f o r a l l the experimental data f o r an i n d i v i d u a l f i s h . T h i s graph shows on l y the data f o r the experiment which i n v o l v e d the exposure to environmental hyperoxia and hypercapnia. 45 46 F i g u r e 9. The r e l a t i o n s h i p between v e n t i l a t o r y volume and plasma pH (pHpl) i n S c v l i o r h i n u s s t e l l a r i s undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. V e n t i l a t o r y volume (Vg act./Vg c o n t r . ) i s the r a t i o o f the measured Vg d i v i d e d by the Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the best f i t l i n e f o r a l l the experimental data f o r an i n d i v i d u a l f i s h . T h i s graph shows on l y the data f o r the experiment which i n v o l v e d the exposure to environmental hyperoxia and hypercapnia and where NaHCC>3 or HC1 was i n f u s e d to minimize changes i n pH due to the f i r s t treatment ( F i g u r e 7). 48 Fi g u r e 10. The best f i t l i n e f o r the aggregate data s e t f o r both data s e t s d e s c r i b e d i n F i g u r e 7. above. Each symbol r e p r e s e n t s an i n d i v i d u a l f i s h . C o e f f i c i e n t s f o r the l i n e : Vg a c t . / Vg c o n t r . = a * e < b * P H> a b r2 5.0476 * 1 0 1 0 -3.353 0.875 49 50 F i g u r e 11. The r e l a t i o n s h i p between v e n t i l a t o r y volume (Vg act./Vg c o n t r . ) and a r t e r i a l Pco2 i n shark, s u b j e c t e d to three experimental p r o t o c o l s : 1.exposure to environmental hyperoxia and hypercapnia (+); 2.exposure to environmental hyperoxia and hypercapnia and i n f u s e d with NaHC03 or HC1 to minimize changes i n pH ( o ) ; 3.exposure to environmental hyperoxia and hypercapnia and i n f u s e d with NaHCC>3 to l e v e l s beyond those accumulated by the animals ( x ) . 51 52 F i g u r e 12. As i n 11. except t h a t the r e l a t i o n s h i p between v e n t i l a t o r y volume (Vg act./Vg c o n t r . ) and a r t e r i a l [HCO3 -] i n mM f o r the three experimental p r o l o c o l s . 53 x X X X X X + • •+ + «• • • • X + + x. s»x~ I I 1 I I I I I —• o O r- U o o u in o 54 Shark accumulate plasma HCO3 - to compensate the f a l l i n plasma pH due to environmental hypercapnia and there seems to be a maximum l e v e l to t h i s accumulation. I n f u s i o n of NaHCC>3 beyond t h i s maximum l e v e l produces no change i n v e n t i l a t i o n ( F i g . 11 & 12). DISCUSSION The animals i n experiment IA were un s t r e s s e d and i n a steady s t a t e as f a r as a l l measured parameters were concerned. T h i s i s supported by the constant RQ and the agreement i n the catecholamine and l a c t a t e c o n c e n t r a t i o n s to those r e p o r t e d by Mazeaud and Mazeaud (1981) and H e i s l e r (1984) f o r r e s t i n g f i s h . Vg l e v e l s below about lOOml/min r e s u l t i n an e l e v a t i o n o f blood Pcx>2 and a decrease i n blood P02. At n e u t r a l heads, or i n the absence of any imposed head p r e s s u r e s , the observed Vg l e v e l s were 42.2 to 139.9ml/min. The lower end of t h i s range i s i n the r e g i o n where Vg e v i d e n t l y l i m i t s gas exchange a c r o s s the g i l l s and a f f e c t s blood gas t e n s i o n s . Higher Vg l e v e l s have no apparent e f f e c t on blood gas t e n s i o n s . The d i f f u s i n g c a p a c i t y o f the g i l l to these gases and / or the r e a c t i o n v e l o c i t i e s i n blood and water with r e s p e c t to these gases presumably have the major determing e f f e c t on PC02 and P02 a t the high Vg l e v e l s . 5 5 These o b s e r v a t i o n s are c o n s i s t e n t with p r e v i o u s s t u d i e s which have shown t h a t hyperoxia, which reduces Vg, i s a s s o c i a t e d with an i n c r e a s e i n a r t e r i a l Pco2 (Wood and Jackson 1980) whereas hypercapnia, which r e s u l t s i n an i n c r e a s e i n Vg, has no e f f e c t on the PC02 d i f f e r e n c e s between blood and water. Thus f i s h can i n c r e a s e g i l l v e n t i l a t i o n d u r i n g hypoxia, m a i n t a i n i n g oxygen d e l i v e r y , without c a u s i n g i n c r e a s e d CO2 e x c r e t i o n and t h e r e f o r e a r e s p i r a t o r y a l k a l o s i s . Wood and Jackson (1980) estimated that there c o u l d be a c o n v e c t i v e component to l i m i t i n g CO2 e x c r e t i o n which would r e s u l t i n an i n c r e a s e i n blood PC02 by 2 mmHg. The data from t h i s study confirms t h a t estimate as the decrease i n CO2 e x c r e t i o n from the lowered Vg r e s u l t e d i n comparable i n c r e a s e s i n blood Pco2-The r e d u c t i o n i n gas t r a n s f e r r a t e s a t the low v e n t i l a t i o n l e v e l s was not expected s i n c e these animals were i n steady s t a t e i n t h i s regard. Rather than r e f l e c t i n g unsteady s t a t e s , the d e c l i n i n g t r a n s f e r r a t e s with v e n t i l a t i o n volumes may r e f l e c t t i s s u e uptake and p r o d u c t i o n of O2 and CO2, r e s p e c t i v e l y . Burggren and Randall (1978) observed decreased 0 2 uptake r a t e s with v e n t i l a t i o n and a r t e r i a l P02 i n the sturgeon and suggested t h a t t i s s u e oxygen u t i l i z a t i o n was determined by the blood : t i s s u e oxygen g r a d i e n t . There was no obvious c o r r e l a t i o n between a r t e r i a l P02 and oxygen uptake i n t h i s experiment, however, both Mo 2 and a r t e r i a l P02 decreased with Vg. The r e d u c t i o n i n a e r o b i c metabolism 56 c o u l d not be r e l a t e d to a r e d u c t i o n i n r e s p i r a t o r y e f f o r t because high Vg was o f t e n a s s o c i a t e d with a p o s i t i v e pressure head and low Vg with a negative head, such t h a t there i s no c l e a r c o r r e l a t i o n between Vg and the e f f o r t expended by the f i s h . The r e d u c t i o n i n M02 was not compensated by an i n c r e a s e i n anaerobic metabolism as the l a c t a t e c o n c e n t r a t i o n s remained low i n a l l f i s h . These data agree with p r e v i o u s s t u d i e s ( S o i v i o 1981, Eddy 1974) that changes i n g i l l water flow can o n l y i n c r e a s e s i g n i f i c a n t l y the blood PC02 l e v e l i n t r o u t by d e c r e a s i n g v e n t i l a t i o n volume. Carbon d i o x i d e e x c r e t i o n cannot be i n c r e a s e d by i n c r e a s i n g g i l l water flow above normal l e v e l s . T h i s i m p l i e s t h a t adjustments of g i l l v e n t i l a t i o n c o u l d o n l y be e f f e c t i v e i n c o r r e c t i n g a l k a l o t i c c o n d i t i o n s i n the blood through i n c r e a s e s i n blood Pco2-Changes i n g i l l water flow i n f i s h e s under a wide range of environmental c o n d i t i o n s are r e p o r t e d i n a l a r g e body of l i t e r a t u r e . The s e n s i t i v i t y of v e n t i l a t i o n i n f i s h to environmental oxygen content i s w e l l documented (Saunders 1962, Holeton and R a n d a l l 1967, Davis and Cameron 1971, Randall and Jones 1973, Dejours §_t aj,.. 1977, Itazawa and Takeda 1978, Wood and Jackson 1980, Smith and Jones 1982). I t i s a l s o known t h a t v e n t i l a t i o n i s s t i m u l a t e d i n f i s h s t r e s s e d with exposure to a c i d waters (Hargis 1976; Hoglund and Persson 1971; D i v e l y et. al.. 1977), i n f u s i o n of a c i d or base (Cunningham 1974; Janssen and Randall 1975), and hypercapnia 57 (Van Dam 1938; Randall and Jones 1973; Janssen and Randall 1975; Eddy 1976; Smith and Jones 1982). I t seems t h a t an i n c r e a s e i n blood Pco2 t e n s i o n s i s important i n the s t i m u l a t i o n of v e n t i l a t i o n . Exposure o f f i s h to a c i d c o n d i t i o n s e x t e r n a l l y or i n t e r n a l l y would cause some degree of hypercapnia through the d e h y d r a t i o n of the HCO3 - i o n . Furthermore, N e v i l l e (1979a,b) has shown that v e n t i l a t i o n i n t r o u t i s not s t i m u l a t e d i n a c i d c o n d i t i o n s u n l e s s accompanied by hypercapnia. A l a r g e p o r t i o n of the r e p o r t e d data on the r e l a t i o n s h i p between v e n t i l a t i o n volumes and acid-base d i s t u r b a n c e s p o i n t to the o r i e n t a t i o n of the c o n t r o l of g i l l water flow to the maintenance of the oxygen uptake and c a r r y i n g c a p a c i t y o f the blood (see review by S h e l t o n e_t al.. 1986). Oxygen seems to have a r e l a t i v e l y g r e a t e r i n f l u e n c e on v e n t i l a t i o n i n f i s h e s compared to CO2 (Randall and Jones 1973; Dejours 1973). A c i d c o n d i t i o n s i n the blood would reduce the c a r r y i n g c a p a c i t y f o r O2 by the Bohr and Root e f f e c t s which have been shown ,to e x i s t i n the blood of f i s h e s (carp. Black and I r v i n g 1937; t r o u t , Cameron 1971, Eddy 1971, B o u t i l i e r e_t al.. 1986; tench, Eddy 1973). V e n t i l a t i o n i s reduced when f i s h are exposed to environmental hyperoxia (Smith and Jones 1982; Randall and Jones 1973) and there i s o n l y a minimal s t i m u l a t i o n of v e n t i l a t i o n with a superimposed c o n d i t i o n of hypercapnia (Babak and Dedek 1907; Peyraud and S e r f a t y 1964; Dejours 1972). Smith and Jones (1982) showed t h a t the 58 s t i m u l a t i o n of v e n t i l a t i o n i n t r o u t exposed to mild environmental hypercapnia c o u l d be a b o l i s h e d with h y p e r o x i c c o n d i t i o n s . There are, however, exc e p t i o n s to t h i s emerging trend i n the p u b l i s h e d data. The blood o f C y p r i n i d f i s h e s e x h i b i t Bohr and Root s h i f t s but show l i t t l e r e s p i r a t o r y s t i m u l u s to hypercapnia (Dejours 1973). Furthermore, while the d o g f i s h , Saualus s u c k l e v i , shows a s t i m u l a t i o n of v e n t i l a t i o n with hypercapnia, Lenfant and Johansen (1966) showed that i t s blood showed no Bohr or Root s h i f t s . The p o s s i b i l i t y t h a t f a c t o r s other than O2 c a r r y i n g c a p a c i t y may be d r i v i n g v e n t i l a t i o n i n the elasmobranch i s supported by the r e s u l t s of experiment IB. The s e n s i t i v i t y of v e n t i l a t i o n to blood PC02 and e s p e c i a l l y pH are more s i m i l a r to the s i t u a t i o n i n other v e r t e b r a t e s than i n the t e l e o s t s . The p o s s i b i l i t y of i n p u t s from sensors of the acid-base s t a t u s of the blood or some other c l o s e l y r e l a t e d compartment i n a d d i t i o n to i n p u t s from the oxygen sensors cannot be ignored. A wide range of f i s h e s , i n c l u d i n g those which e x h i b i t Bohr and Root s h i f t s i n t h e i r blood, show t h i s s e n s i t i v i t y to hypercapnia and while the response can be a t t e n u a t e d with oxygen, a r e s i d u a l response to CO2 always remains. The second reason g i v e n by R a n d a l l and Cameron (1973) a g a i n s t the r e g u l a t i o n of PC02 i n the blood to c o r r e c t a c i d o t i c c o n d i t i o n s i s s u b s t a n t i a t e d by the r e s u l t of the f i r s t experiment. The scope of a d j u s t i n g blood PC02 through changes i n v e n t i l a t i o n i s low and only i n the range of 2-3 59 mmHg. Furthe r experiments that d e l i n e a t e the e f f e c t s o f 0 2 and C 0 2 as well as the acid-base parameters of pH and P c o 2 on the v e n t i l a t o r y response i n f i s h are needed. Since i t i s not o n l y those animals l a c k i n g Bohr and Root s h i f t s t h a t e x h i b i t a s e n s i t i v i t y i n v e n t i l a t i o n to changes i n the acid-base s t a t u s o f the blood, experiments of t h i s nature on s p e c i e s such as those i n the C y p r i n i d a e would make v a l u a b l e c o n t r i b u t i o n s to t h i s f i e l d o f f i s h p h y s i o l o g y . Great gaps e x i s t i n the knowledge of the r e g u l a t i o n o f v e n t i l a t i o n i n f i s h e s . While c l e a r trends seem to be emerging among data s e t s , the e x c e p t i o n are e q u a l l y obvious. SECTION 2 . TRANSEPITHELIAL ION FLUXES FOR ACID-BASE REGULATION IN FISHES 61 INTRODUCTION Under steady s t a t e c o n d i t i o n s , freshwater f i s h o s m o t i c a l l y g a i n water through t h e i r g i l l s and e x c r e t e excess water i n the u r i n e . Body e l e c t r o l y t e s are a l s o l o s t i n t h i s e x c r e t i o n . T h i s l o s s , however, i s coun t e r a c t e d by uptake of sodium (Na +) and c h l o r i d e ( C l ~ ) ions a c r o s s the secondary l a m e l l a e ( G i r a r d and Payan 1977a). The c h l o r i d e c e l l s o f the freshwater f i s h are s m a l l , p o o r l y developed and few i n number. The mitochondria and t u b u l a r systems are a l s o p o o r l y developed. A l l these c h a r a c t e r i s t i c s are o p p o s i t e to those of c h l o r i d e c e l l s i n s a l t w a t e r . In s a l t w a t e r , f i s h d i f f u s i v e l y g a i n Na + and C l ~ through the g i l l and a l s o through the gut as they d r i n k water to r e p l a c e the o s m o t i c a l l y d r i v e n water l o s s a t the g i l l . The excess Na + and C l ~ i s a c t i v e l y pumped out of the blood by the c h l o r i d e c e l l s (Karnaky §_t a l . 1977, F o s k e t t e t al.. 1979, M a r s h a l l and N i s h i o k a 1980, Fo s k e t t and S c h e f f e y 1982) l o c a t e d on the e p i t h e l i u m a t the base of and between the secondary l a m e l l a e . A major way i n which f i s h r e g u l a t e acid-base s t a t u s i s by t r a n s e p i t h e l i a l i o n exchange acr o s s the g i l l (see H e i s l e r 1980, 1982, 1984). Since Krogh (1939) f i r s t suggested that 62 the a c t i v e uptake o f Na + and C l ~ i n the g o l d f i s h was coupled to the movement of the acid-base r e l e v a n t ions H +, NH4 + f o r Na + and HCO3 - f o r Cl~", many s t u d i e s have confirmed the e x i s t e n c e o f these l i n k s . There i s experimental evidence f o r both the Na +/H +(NH4 +) (Maetz and Romeu 1964; K e r s t e t t e r §_t. §JL.. 1970; Cameron 1976; Evans 1977; Payan 1978; Maetz 1973; Perry and Randall 1981; Wood et_ al.. 1984) and the C1~/HC03~ (Maetz and Romeu 1964; DeRenzis and Maetz 1973; Perry and Rand a l l 1981; Perry et_ a l . 1981; Holeton e t a l . 1983) exchange processes i n the f i s h g i l l . There i s c i r c u m s t a n t i a l evidence t h a t suggests t h a t the i o n i c composition o f the water a f f e c t s the r e g u l a t i o n o f e x t r a c e l l u l a r pH i n response to acid-base d i s t u r b a n c e s (Table 2). Perry et. al.. (1981) showed t h a t the recovery o f plasma pH i n t r o u t exposed to environmental hypercapnia was g r e a t e r i n water with a higher c o n c e n t r a t i o n o f Na + than i n the ambient Vancouver tap water. One of the aims of t h i s S e c t i o n was to provide f u r t h e r experimental evidence f o r t h i s r e l a t i o n s h i p . T h i s S e c t i o n r e p o r t s on experiments where f i s h were su b j e c t e d to a c i d o t i c c o n d i t i o n s by exposure to environmental hypercapnia, a c o n d i t i o n which can occur n a t u r a l l y from CO2 p r o d u c t i o n by s u r f a c e p l a n t s such as h y a c i n t h mats ( U l t s c h and Antony 1973) i n f r e s h water as we l l as from the absence of pho t o s y n t h e s i s combined with r e s p i r a t i o n and anaerobic g l y c o l y s i s o f animals and b a c t e r i a attached to f a l l i n g p a r t i c l e s o f o r g a n i c d e b r i s a t depths o f 50-100m i n sea water 63 Table 2. Time course for pH compensation i n f i s h s t r e s s e d with environmental hypercapnia. SPECIES HATER Pco 2 mmHg TIME h REFERENCES Conoer conaer 8 8-10 c Toews eJt al.- 1983 S c v l i o r h i n u s s t e l l a r i s 8 8-10° H e i s l e r ai.. 1976 I c t a l u r u s Dunctatus 10 24P Cameron 1980 Salmo a a i r d n e r i 15 22P Eddy e_t al- 1977 5.2 72 c Janssen and Randall 1975 7.5 7.8 24P(52%)* 24P(88%)** Perry g£ al.. 1981 c complete compensation; p p a r t i a l compensation; * Percent recovery of H + ion concentration before hypercapnia was imposed, i n dechlorinated Vancouver tap water; ** As * except Na + ion concentration i n the water was r a i s e d to 3 itiM. 64 (Harvey 1974). I t was hoped t h a t imposing t h i s acid-base d i s t u r b a n c e would s t i m u l a t e i o n f l u x e s a c r o s s the g i l l e p i t h e l i u m and that the super i m p o s i t i o n of a d d i t i o n a l treatments would r e s u l t i n a b e t t e r understanding of the nature and mechanism of these i o n exchange processes. The acid-base r e g u l a t o r y performance of t r o u t a c c l i m a t e d to waters of d i f f e r e n t i o n i c s t r e n g t h (Table 3) were compared on the hypothesis that a l t e r i n g the c o n c e n t r a t i o n s o f Na + and C I - i n the water should a f f e c t the r e g u l a t i o n of blood pH i f there i s a dependence of the t r a n s e p i t h e l i a l movement of these ions to H + ( N H 4 + ) and HCO3 -, r e s p e c t i v e l y . R a d i o a c t i v e i s o t o p e s of Na + and C l ~ were a l s o i n j e c t e d i n t o carp to determine the d i r e c t i o n a l f l u x e s of these ions d u r i n g r e c o v e r y from a c i d o s i s . MATERIALS AND METHODS EXPERIMENT 2A. SALINITY - TROUT - HYPERCAPNIA ANIMALS : Rainbow t r o u t , Salmo g a i r d n e r i , weighing between 800 and 1535 g were obtained from a commercial hatchery and h e l d indoors under ambient l i g h t c o n d i t i o n s . They were h e l d i n l a r g e g l a s s a q u a r i a at a d e n s i t y of about 150 1 / f i s h which 65 Table 3. Mean water Na + and Cl concentrations for the two experiments in this section. A l l concentrations in mM. Trout - Hypercapnia - 3 S a l i n i t i e s SAMPLING TIME [Na +] [Cl -] [Na+] EC1- [Na+] [ci-CONTROL 2. 32 3. 29 97. 72 113. 82 320. 72 333. 82 HYPERCAPNIA + . 25h 2. 61 3. 32 94. 2 113. 2 330. 0 332. 75 + .5h 2. 45 3. 25 93. 8 113. , 1 323. ,5 331. 17 +lh 2. 19 3. 33 98. 2 114. 2 314. .0 332. 58 +2h 2. 42 3. 42 100 .6 113. 9 318. , 17 332. 25 +4h 2. 54 3. 38 94. 4 111. ,4 318. ,67 327. ,42 +8h 2. 28 3. 58 97. 8 113. ,0 323, ,5 324. .92 +20h 2. 41 3. 58 96. 6 110, .5 319, .0 332. , 33 +24h 2. 38 3. 46 96. 0 110. ,5 333, .0 335. ,4 RECOVERY + . 25h 2. 37 3. ,55 92. 75 114, . 88 325, .5 330, . 25 + .5h 2. 18 3. 58 95. 6 110 , 9 325, .8 332, , 2 +lh 2. 17 3. ,45 94. 2 108. . 8 315. .0 331. .7 +5h 2. 45 3. ,58 97. 4 113, .9 338 .4 334 . 1 +10h 2. 47 3. 25 99. 4 114. .6 330. .6 333, .8 +20h 2. 21 3. ,41 95. 4 114 .8 313, .8 336, .0 + 24h 2. 67 3. , 2 96. 75 117, .0 335, .25 337. . 1 Conger - Hypercapnia - 6 Salnities SAMPLING TIME [Cl~] [Cl~] [Cl~] [Cl -] [ C l - ] [Cl -] i n i t i a l < 3 40 80 140 360 540 tCl~) determ. 66 r e c e i v e d d e c h o l r i n a t e d tap water a t 10 +_ 3°C (mean +_ S.E.). The f i s h were fed to s a t i a t i o n s e v e r a l times a day. A l l f i s h were a c c l i m a t e d to 3 water s a l i n i t i e s f o r a t l e a s t 1 month p r i o r to experimentation. Sea s a l t s were added to ambient f r e s h d e c h l o r i n a t e d water to achieve NaCl c o n c e n t r a t i o n s o f about 3, 100 and 300 mM. The chemical composition of the s a l t s used to make up the v a r i o u s s a l i n i t i e s i s g i v e n i n Table A . l . o f Appendix I. SURGERY AND APPARATUS : A l l animals were f i t t e d with c h r o n i c i n d w e l l i n g cannulas i n the d o r s a l a o r t a by the method of S o i v i o e t al_. (1972) as d e s c r i b e d i n General M a t e r i a l s and Methods. The s a l i n i t y to which the animal was a c c l i m a t e d was maintained i n surgery and i n subsequent experimental procedures. Each animal was recovered i n the experimental chamber shown i n F i g u r e 13. The system i s d e s c r i b e d i n General M a t e r i a l s and Methods as a 'Delta Bicarbonate System'. Experimental procedures were i n i t i a t e d a t l e a s t 24 h a f t e r s u r g i c a l procedures. PROTOCOL : The experimental p r o t o c o l c o n s i s t e d o f exposing f i s h a c c l i m a t e d a t the above s a l i n i t i e s to 24 h of 1 % 67 F i g u r e 13. Experimental apparatus f o r experiment 2A.: Trout - S a l i n i t y - Hypercapnia . A l l components of t h i s diagram are e x p l a i n e d i n General M a t e r i a l s and Methods under the d e s c r i p t i o n s of the 'Delta Bicarbonate System' and the apparatus f o r measuring t r a n s e p i t h e l i a l p o t e n t i a l s (TEP). co 69 environmental hypercapnia and then o b s e r v i n g the recover y from t h i s exposure f o r 24 h a f t e r the high Pco2 t e n s i o n s were e l i m i n a t e d . Blood and water samples were c o l l e c t e d 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 20 h, and 24 h a f t e r the beginning o f the hypercapnia exposure and 15min, 30min, 1 h, 5 h, 10 h, 20 h, and 24 h a f t e r the end of the hypercapnia exposure. The f o l l o w i n g procedure was c a r r i e d out f o r each sampling time. 5 ml of water was removed from the system. The d o r s a l a o r t i c cannula was connected to a •T* p i e c e which enabled c o n t a c t with e i t h e r a KCl/agar b r i d g e f o r t r a n s e p i t h e l i a l p o t e n t i a l (TEP) measurements (see General M a t e r i a l s and Methods) or a s y r i n g e f o r blood c o l l e c t i o n . One ml of blood was c o l l e c t e d ; the volume was r e p l a c e d with h e p a r i n i z e d p h y s i o l o g i c a l s a l i n e and then the v a l v e on the •T' p i e c e was turned so that c o n t a c t with the KCl/agar b r i d g e was made. Contact with the KCl/agar b r i d g e was maintained between most of the sampling times, a l l o w i n g near continuous r e c o r d i n g of TEP d u r i n g the experimental p e r i o d s . Water HC03~ c o n c e n t r a t i o n was a l s o recorded on a continuous b a s i s with the apparatus d e s c r i b e d i n General M a t e r i a l s and Methods. For t h i s and TEP r e c o r d i n g s , values were read o f f the c h a r t a t the sampling times l i s t e d above. MEASUREMENTS : Whole blood pH, t o t a l CO2 (TC02 with the Capni-Con 7 0 I I I ) and hematocrit were measured a c c o r d i n g the methods i n General M a t e r i a l s and Methods. The remaining blood was c e n t r i f u g e d i n Eppendorf v i a l s to o b t a i n separated plasma. An a l i q u o t o f plasma was a c i d i f i e d and f r o z e n f o r t o t a l ammonia a n a l y s i s and the remaining plasma was analyzed f o r Na + and C l ~ c o n c e n t r a t i o n s as w e l l as f o r o s m o l a r i t y . Sodium was measured by spectrophotometry and C l ~ was measured with the Radiometer CMT10 t i t r a t o r (see General M a t e r i a l s and Methods). T o t a l plasma o s m o l a r i t y was measured with a micro-osmometer. Plasma PC02 t e n s i o n s and HCO3 -c o n c e n t r a t i o n s were c a l c u l a t e d u s i n g the measured pH and TC02 values as d e s c r i b e d i n General M a t e r i a l s and Methods. Water samples were analysed f o r Na + and C l ~ c o n c e n t r a t i o n s . The method f o r Na + c o n c e n t r a t i o n d e t e r m i n a t i o n was the same as f o r plasma but C l ~ c o n c e n t r a t i o n s were determined with the e l e c t r o d e method d e s c r i b e d i n General M a t e r i a l s and Methods. T o t a l ammonia c o n c e n t r a t i o n s were measured with an ammonia e l e c t r o d e i n an automated way d e s c r i b e d i n General M a t e r i a l s and Methods. That S e c t i o n a l s o d e s c r i b e s the way i n which continuous r e c o r d i n g s of water HCO3 - c o n c e n t r a t i o n s were determined. Net f l u x r a t e s of ions were c a l c u l a t e d i n u n i t s of umol/Kg body weight/min by c a l c u l a t i n g r a t e s o f change i n the contents ( c o n c e n t r a t i o n * volume) of the p a r t i c u l a r i o n . 71 STATISTICS : A n a l y s i s of v a r i a n c e was used to d i s c e r n s t a t i s t i c a l s i g n i f i c a n c e among the means of any parameter a t three s a l i n i t i e s . P a i r e d Student's t t e s t was used to t e s t s i g n i f i c a n c e of d i f f e r e n c e s between the mean c o n t r o l value and any subsequent experimental value a t any one s a l i n i t y . L i n e a r r e g r e s s i o n a n a l y s i s was used to d e s c r i b e the r e l a t i o n s h i p between c e r t a i n data s e t s . The l e v e l o f r e j e c t i o n i n a l l cases was 5 %. EXPERIMENT 2B. CONGER - SALINITY - HYPERCAPNIA ANIMALS : Conger e e l . Conger conger, weighing between 800 to 1500 g were caught i n the Bay of Naples, I t a l y . They were brought to the l a b o r a t o r y where they were h e l d a t 19°C i n 200 1 f i b e r g l a s s tanks s u p p l i e d with ambient sea water before surgery and subsequent experimentation. The animals were not fed once they were i n the l a b o r a t o r y , a time p e r i o d which v a r i e d from 48 h to 2 weeks. SURGERY AND APPARATUS : A l l animals were f i t t e d with c h r o n i c i n d w e l l i n g d o r s a l 72 a o r t i c cannulas v i a a g a s t r i c or i n t e s t i n a l a r t e r y . The method of o p e r a t i o n i s d e s c r i b e d i n General M a t e r i a l s and Methods. The animals were recovered i n a r e c i r c u l a t i n g water system s i m i l a r to the 'Delta Bicarbonate System' d e s c r i b e d i n General M a t e r i a l s and Methods i n which the experiment was conducted. The chemistry of the water was i d e n t i c a l to the a c c l i m a t i o n water u n t i l the s a l i n i t y changes d e s c r i b e d below were i n i t i a t e d . Water temperature was maintained constant at 20°C. An 8 to 24 h r e c o v e r y p e r i o d was allowed before i n i t i a t i n g experimental procedures. PROTOCOL : There were three p e r i o d s i n the experiment; c o n t r o l , s a l i n i t y change and hypercapnia. Each experiment l a s t e d 8 hours e x c l u d i n g the c o n t r o l p e r i o d . In the f i r s t three hours the s a l i n i t y o f the water was changed to one o f s i x s a l i n i t i e s which were a l l lower than sea water. In the f o l l o w i n g 5 h, the f i s h were exposed to 1 % environmental hypercapnia. Sampling p e r i o d s were as f o l l o w s : 2 c o n t r o l samples; 30 min, 1 h, 2 h and 3 h a f t e r the step change i n s a l i n i t y ; and 30 min, 1 h, 2 h, 4 h and 5 h a f t e r the environmental hypercapnia was imposed. At each sample p e r i o d , 500 u l of whole blood was withdrawn through the cannula and analysed f o r the parameters d e s c r i b e d below. 73 MEASUREMENTS : Whole blood pH, hematocrit and t o t a l CO2 (TC02 with the Capni-Con I I I ) , plasma HCO3 - c o n c e n t r a t i o n and PC02 t e n s i o n and plasma C l " c o n c e n t r a t i o n s were c a l c u l a t e d or determined by t i t r a t i o n u s i n g the Radiometer CMT10 t i t r a t o r as d e s c r i b e d i n General M a t e r i a l s and Methods. STATISTICS : C o r r e l a t i o n and l i n e a r r e g r e s s i o n a n a l y s i s were used to d e s c r i b e r e l a t i o n s h i p s between data s e t s . EXPERIMENT 2C. CARP-HYPERCAPNIA-ISOTOPE ANIMALS : Carp, Cvprinus c a r p i o , weighing 1500 to 2000 g were obtained from a commercial hatchery and maintained i n l a r g e g l a s s indoors a q u a r i a , a t a d e n s i t y o f about 100 1 per f i s h , under n a t u r a l l i g h t and r e c e i v i n g 15°C d e c h l o r i n a t e d tap water. F i s h were fed s e v e r a l times a day to s a t i a t i o n with a p e l l e t e d carp feed. F i s h were a c c l i m a t e d f o r a t l e a s t one month p r i o r to use i n experiments. SUGERY AND APPARATUS 74 A l l f i s h were f i t t e d with c h r o n i c i n d w e l l i n g d o r s a l a o r t i c cannulas u s i n g the method of S i o v i o e t al.. ( 1972) and recovered i n a 'Delta Bicarbonate System' ( F i g . 14) d e s c r i b e d i n d e t a i l by C l a i b o r n e and H e i s l e r (1984). The f o l l o w i n g p r o t o c o l and measurements were c a r r i e d out a f t e r a 24 h rec o v e r y p e r i o d a f t e r surgery. PROTOCOL : One hundered uCi of the i s o t o p e Na +24 and 20 uCi of Cl~35 were i n j e c t e d through the cannula and the f o l l o w i n g procedures were c a r r i e d out. A f t e r 3 pre-exposure c o n t r o l samples were taken, water and blood samples were taken d u r i n g 48 h exposure to 5 % environmental hypercapnia and d u r i n g a 21 h r e c o v e r y p e r i o d a f t e r the high CO2 t e n s i o n was turned o f f . Blood samples were taken a t 0, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 25 h, 29 h, 33 h, 45 h, 46 h, 49 h, 53 h and 69 h a f t e r the onset o f hypercapnia which l a s t e d only 48 of the 69 h sampling regime. Water samples were c o l l e c t e d a t the same sampling times as f o r the blood except that a d d i t i o n a l samples were taken every 4 h whenever p o s s i b l e between the 53 and 69h sampling times. MEASUREMENTS : Water pH a t a s e t Pcx>2 as w e l l as Na + and C l ~ 75 F i g u r e 14. Experimental apparatus f o r experiment 2C.: Carp - Isotope - Hypercapnia. A l l components of t h i s diagram are e x p l a i n e d i n General M a t e r i a l s and Methods under the d e s c r i p t i o n of the 'Delta Bicarbonate System'. The experimental apparatus f o r experiment 2B. was s i m i l a r to t h i s apparatus. Computer Power on-off lonmeter Digital Printer - 5 Thermostat "^_30°C — mm NH^-Electrode Power on - off Water Na" .Cl 3 6 Amplifier • Recorder PH Na.Cl' i nmp Ft! ' Valve W-f—f Temp. Control Heater Cooler — 30 °C <r—j> Plasma L Na n.Cl*. Na*,Cl" Temp. Probe Filter, 1% C0 2 in N2 Air or C02/Air 77 c o n c e n t r a t i o n s were measured by g l a s s e l e c t r o d e s with a p p r o p r i a t e r e f e r e n c e e l e c t r o d e s w i t h i n the 'Delta Bicarbonate System', The Na + and C l ~ e l e c t r o d e s were c a l i b r a t e d with v a r i o u s NaCl s o l u t i o n s . Water HCO3 - c o n c e n t r a t i o n s were c a l c u l a t e d u s i n g the above measurements and methods. Plasma c o n c e n t r a t i o n s o f Na + and C l ~ were determined by spectrophotometry and by t i t r a t i o n u s i n g the Radiometer CMT10 t i t r a t o r r e s p e c t i v e l y . Net f l u x e s of each i o n between blood and water were determined by the system water volume and c o n c e n t r a t i o n changes of each i o n . The u n i d i r e c t i o n a l f l u x e s o f each i n j e c t e d i s o t o p e were determined with the water and plasma a c t i v i t i e s f o r Na +24 and C l ~ 3 5 together with the ' c o l d ' c o n c e n t r a t i o n s and s p e c i f i c a c t i v i t i e s o f the ions. The a c t i v i t i e s o f the i s o t o p e s were counted i n Gamma and Beta counters. The Na +24 a c t i v i t y o f each sample was counted f i r s t . A f t e r a p e r i o d o f 3 weeks, a p e r i o d exceeding the h a l f - l i f e o f t h a t i s o t o p e , the C l _ 3 g a c t i v i t y was counted. C o r r e c t i o n s f o r decay, background, co u n t i n g time and e f f i c i e n c y were automated i n the machines. D i s i n t i g r a t i o n s per minute (dpm) were then c a l c u l a t e d . STATISTICS : Student's t t e s t s were used to compare the mean c o n t r o l f l u x e s o f these ions to the mean f l u x e s a f t e r the treatments 78 were imposed. The 5 % l e v e l o f r e j e c t i o n was a p p l i e d i n a l l cases. RESULTS EXPERIMENT 2A & 2B. EFFECTS OF WATER SALINITY ON ACID-BASE REGULATION IN FISHES I. A c i d - Base R e g u l a t i o n . An e l e v a t i o n o f water Pco2 p r e s s u r e s caused plasma P C 0 2 to r i s e 4-6 times the c o n t r o l values ( F i g . 15 & 16). In t r o u t and i n the conger, the i n i t i a l drop i n pHe induced by exposure to environmental hypercapnia was compensated i n v a r y i n g degrees over the time course o f the exposure p e r i o d depending on the e x t e r n a l water s a l i n i t y ( F i g . 17,18 & 19). There was a p o s i t i v e c o r r e l a t i o n between the degree o f compensation and water s a l i n i t y ( F i g . 18 & 20). In both s p e c i e s compensation o f pHe was e f f e c t e d by accumulation o f plasma HC03~ ( F i g . 21 & 22). HCO3 -accumulation i n t r o u t a c c l i m a t e d to 100 and 300mM NaCl was gr e a t e r than those a c c l i m a t e d to 3mM ( F i g . 21). There was a p o s i t i v e c o r r e l a t i o n between water s a l i n i t y and HC03~ accumulation i n the conger ( F i g . 23). Net H + f l u x changed from an i n f l u x d u r i n g c o n t r o l 79 F i g u r e 15. Means + one standard e r r o r (S.E.) of plasma PC02 i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia. Time course shown i n c l u d e s c o n t r o l , 1 % hypercapnia and recovery p e r i o d s . A l l u n i t s i n mmHg. ( D H U J U I ) '"'-'d 81 gure 16. Plasma PC02 i n conger d u r i n g c o n t r o l , s a l i n i t y -change and exposure to 1 % environmental hypercapnia. Each l i n e r e p r e s e n t s an i n d i v i d u a l f i s h . C o n t r o l p e r i o d was i n sea water. S a l i n i t i e s were changed i n one step to those noted and f i s h were exposed to the hypercapnia a t those s a l i n i t i e s . P l a s m a P c o 2 f o r I n d i v i d u a l F i s h Cummulatlve Time (h) 83 gure 17. Means +_ S.E. of a c c l i m a t e d to 3, 100 and environmental hypercapnia. c o n t r o l , 1 % hypercapnia and plasma pH i n rainbow t r o u t 300 mM NaCl and exposed to Time course shown i n c l u d e s r e c o v e r y p e r i o d s . 85 ure 18. As F i g . 17 except that a l l values were r e f e r e n c e d to the average value d u r i n g the c o n t r o l p e r i o d . Means. • 3mM Cummulative Time (h) + 100 mM o 300 mM oo 0^ 1 87 gure 19. Plasma pH changes from average c o n t r o l values i n conger d u r i n g s a l i n i t y change and exposure to 1 % hypercapnia. Each l i n e r e p r e s e n t s an i n d i v i d u a l f i s h . A c t u a l average c o n t r o l values f o r pH were : 360mM=7.829, 140mM=7.736, 155mM=7.847, 0.005mM=8.843, 2.6mM=7.816, 80mM=7.783(mean of 6), 40mM=7.835(mean of 2). Cumm. Time (h) co CO 89 gure 20. E f f e c t of water s a l i n i t y , r e p r e s e n t e d by [ C l ~ ] , on pH r e c o v e r y i n conger. Percent r e c o v e r y to the pH value measured p r i o r to exposure to 1 % environmental hypercapnia was c a l c u l a t e d by d i v i d i n g the pH value a t the end of the exposure p e r i o d by the pre-exposure value and m u l t i p l y i n g by 100. R e s u l t s of l i n e a r r e g r e s s i o n a n a l y s i s shown. E f f e c t o f W a t e r S a l i n i t y o n p H R e c o v e r y (post—/pre—hypercapnia) * 100 100 - i — , P 0 200 400 600 [Cl"]water (mM) 91 F i g u r e 21. Means +_ S.E. of plasma HCO3- c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia. Time course shown i n c l u d e s c o n t r o l , 1 % hypercapnia and recovery p e r i o d s . A l l u n i t s i n mM. 92 93 F i g u r e 22. Plasma H C O 3 - changes from mean c o n t r o l values i n conger d u r i n g s a l i n i t y change and exposure to 1 % hypercapnia. Each l i n e r e p r e s e n t s an i n d i v i d u a l f i s h . A c t u a l average c o n t r o l values f o r H C O 3 - were : 0.005mM=3.68, 2.6mM=3.52, 40mM=2.92(mean of 2), 80mM=3.15(mean of 5), 140mM=3.07, 155=2.63, 360=3.40. A l l values i n mM. D e l t a [ H C O " ] f o r I n d i v i d u a l F i s h 0 2 4 6 8 Cummulatlve Time (h) vo 95 F i g u r e 23. E f f e c t o f water s a l i n i t y , r e p r e s e n t e d by [ C l ~ ] , on H C O 3 - accumulation i n conger. C a l c u l a t i o n s as i n F i g . 20 e x c l u d i n g m u l t i p l i c a t i o n by 100. R e s u l t s o f l i n e a r r e g r e s s i o n a n a l y s i s shown. E f f e c t o f W a t e r S a l i n i t y o n [ H C O ~ ] p l (post—/pre—hypercapnia) 1 r 1 1 1 —r 1 1 1 0 100 200 300 400 [Cr]water (mM) vo 97 c o n d i t i o n s to an e f f l u x d u r i n g the p e r i o d o f exposure to water hypercapnia i n a l l groups o f t r o u t ( F i g . 24.). D i f f e r e n c e s among groups o f f i s h were not d i f f e r e n t . Net H + i n f l u x or HCO3 - e f f l u x was resumed a t the end of the rec o v e r y p e r i o d . I I . Plasma Ions. Plasma C l - c o n c e n t r a t i o n s i n both the t r o u t ( F i g . 25) and conger ( F i g . 26) decreased d u r i n g the exposure p e r i o d compared to c o n t r o l c o n c e n t r a t i o n s . These changes were marked a f t e r lOh of exposure i n the t r o u t . Plasma Na + c o n c e n t r a t i o n s were i n c r e a s e d i n the t r o u t a c c l i m a t e d to 100 and 300mM NaCl d u r i n g the f i r s t lOh o f exposure i n the t r o u t ( F i g . 27). Plasma Na + c o n c e n t r a t i o n s were not measured i n the conger experiment. Plasma o s m o l a r i t y i n t r o u t a c c l i m a t e d to 3mM was reduced compared to c o n t r o l values d u r i n g exposure to hypercapnia. There were no s i g n i f i c a n t trends i n the 100 and 300mM groups ( F i g . 28) I I I . Acid-Base and Ion C o r r e l a t i o n s : There were weak but s i g n i f i c a n t c o r r e l a t i o n s between plasma (Na + - C l ~ ) and plasma HCC>3~ c o n c e n t r a t i o n s i n t r o u t a t a l l three s a l i n i t i e s ( F i g . 29 a . b . c ) . The reason 98 f o r u s i n g the parameter (Na + - C l ~ ) was t h a t the t r a n s e p i t h e l i a l f l u x o f both ions c o u l d have r e s u l t e d i n the accumulation of plasma HC03~ v i a Na +/H + and C1~/HC03~ exchanges. The ions were s u b t r a c t e d s i n c e the f l u x e s of Na + and C I - would have to be i n o p p o s i t e d i r e c t i o n s to accumulate the HCO3 -. Therefore (Na + C l ~ ) r e p r e s e n t s the net e f f e c t o f these combined f l u x e s . F u r t h e r a n a l y s i s showed that the c o r r e l a t i o n above was l a r g e l y due to the C1~/HCC>3~ e f f e c t r a t h e r than the Na +/H +. Increases i n plasma HC03 - were c o r r e l a t e d with changes i n plasma C l ~ ( F i g . 30). There was no change i n plasma Na + a s s o c i a t e d with the changes i n plasma HCO3 - ( F i g . 31). There was a l s o a negative c o r r e l a t i o n between changes i n plasma Cl~" and plasma HCO3 - i n conger ( F i g . 32). IV. T r a n s e p i t h e l i a l P o t e n t i a l s (TEP) TEP values were ne g a t i v e , near zero and p o s i t i v e i n t r o u t a c c l i m a t e d to 3, 100 and 300 mM ( F i g . 33 a . b . c ) , r e s p e c t i v e l y . There was a d e p o l a r i z i n g trend i n the i n i t i a l sampling p e r i o d s i n f i s h i n a l l s a l i n i t i e s . TEP values g e n e r a l l y i n c r e a s e d d u r i n g the exposure to hypercapnia i n a l l groups as w e l l . There was a t r e n d towards c o n t r o l values d u r i n g the recovery p e r i o d s although there was c o n s i d e r a b l e v a r i a b i l i t y . 99 gure 24. Means +. S.E. of a c c l i m a t e d to 3, 100 and environmental hypercapnia. c o n t r o l , 1 % hypercapnia and i n umol/Kg/min. net H + f l u x i n rainbow t r o u t 300mM NaCl and exposed to 1 % Time course shown i n c l u d e s reco v e r y p e r i o d s . A l l values 1 0 0 101 F i g u r e 25. Plasma C l ~ c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1 % environmental hypercapnia. Time course shown i n c l u d e s c o n t r o l , 1 % hypercapnia and recover y p e r i o d s . A l l values i n mM. Means + S.E. 102 O (WUJ) gaicJOIHO VlrMSVHd 103 gure 26. Changes i n plasma C l ~ c o n c e n t r a t i o n s from average c o n t r o l values i n conger d u r i n g s a l i n i t y change and exposure to 1 % environmental hypercapnia. Each l i n e r e p r e s e n t s an i n d i v i d u a l f i s h . S a l i n i t y l e v e l s shown next to each l i n e as C l ~ c o n c e n t r a t i o n . A c t u a l average c o n t r o l [ C I - ] were : 0.005=150.25, 2.6=148.75, 80=151.88(mean of 2), 140=165.25, 155=155.25, 360=153.75. A l l values i n mM. D e l t a [CI ] p l v s T i m e f o r Ind. F i s h 0 2 4 6 8 Cummulatlve Time (h) 105 F i g u r e 27. Means +. S.E. of plasma Na + c o n c e n t r a t i o n s i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1 % environmental hypercapnia. Time course shown i n c l u d e s c o n t r o l , 1 % hypercapnia and re c o v e r y p e r i o d s . A l l values i n mM. P L A S M A S O D I U M ( m M ) 901 107 F i g u r e 28. Means +_ S.E. of plasma Osmolarity i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl and exposed to 1% environmental hypercapnia f o r 24h and recovered f o r 24h. A l l values i n mOsmol. TIME (h) 109 F i g u r e 29.a.b.c. R e l a t i o n s h i p o f plasma ( [ N a + ] - [ C l ~ ] ) to plasma [HCO3 -] d u r i n g exposure and recovery from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y . A l l values i n mM. Best f i t r e g r e s s i o n l i n e s shown. C o r r e l a t i o n c o e f f i c i e n t r n 3mM 0.5281 64 lOOmM 0.3676 57 300mM 0.3503 85 C o e f f i c i e n t s f o r b e s t - f i t l i n e ( [ N a + ] - [ C l _ ] ) = a + b ( [ H C 0 3 - ] ) a b 0.9430 0.7222 1.4437 26. 62 34.66 11. 27 110 I l l F i g u r e 30.a.b.c. R e l a t i o n s h i p o f changes i n plasma C l ~ c o n c e n t r a t i o n s to corresponding changes i n plasma HCC>3~ c o n c e n t r a t i o n s d u r i n g exposure and rec o v e r y from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl, r e s p e c t i v e l y . A l l values i n mM. Best f i t r e g r e s s i o n l i n e shown. C o r r e l a t i o n c o e f f i c i e n t r n 3mM 0.5084 58 lOOmM 0.3986 48 300mM 0.4139 74 C o e f f i c i e n t s f o r b e s t - f i t l i n e ( [ N a + ] - [ C l ~ ] ) = a + b ([HCO3-]) a b -0.8470 -0.5439 -0.7463 -1.0584 -0.0905 -0.4684 112 (WUi) [-10] V 113 F i g u r e 31.a.b.c. R e l a t i o n s h i p o f changes i n plasma Na + c o n c e n t r a t i o n s to corres p o n d i n g changes i n plasma HCO3 - c o n c e n t r a t i o n s d u r i n g exposure and recovery from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y . A l l values i n mM. Best f i t r e g r e s s i o n l i n e shown. C o r r e l a t i o n C o e f f i c i e n t s f o r b e s t - f i t l i n e c o e f f i c i e n t ( [ N a + ] - [ C l - 3 ) = a + b ( [ H C 0 3 - ] ) r n a b 3mM 0.0541 61 -0.2275 -1.0536 lOOmM 0.0255 59 -0.0424 0.1704 300mM 0.0361 74 0.0960 -0.3126 114 -40 A H C O ; - T T 1 1 , — , 3 -8 -4 O 4 8 12 115 gure 32. R e l a t i o n s h i p of changes i n plasma C l -c o n c e n t r a t i o n s to corr e s p o n d i n g changes i n plasma HCO3 - c o n c e n t r a t i o n s i n conger exposed to s a l i n i t y changes ( f i l l e d c i r c l e s ) a n d exposure to 1 % environmental hypercapnia. A l l values i n mM. 116 2 0 , O < 20-, 10. -10 -- 2 0 --30 . 20 -10 -I 3mM a. • i r-o ^ o <co ° ° c P ° ° . 1 1 1 r o o - o o - i 1 r -10 -I -20A - 3 0 . —r--4 i r 4 lOOmM b. 3 0 0 m M c. 12 A H C O 3 (mM) 117 F i g u r e 33.a.b.c. Means £ S . E . of t r a n s - e p i t h e l i a l p o t e n t i a l (TEP) v a l u e s d u r i n g c o n t r o l , exposure and r e c o v e r y from 1% environmental hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM NaCl, r e s p e c t i v e l y . A l l v a l u e s i n mV. 118 119 Hematocrit i n c r e a s e d i n i t i a l l y with the onset of hypercapnia and then d e c l i n e d t h e r e a f t e r with sampling i n a l l groups of f i s h . Hematocrit values were higher and more v a r i a b l e i n the group of f i s h a c c l i m a t e d i n 3mM ( F i g . 34). EXPERIMENT 2C. CARP ISOTOPE EXPERIMENT There was a net f l u x of C l ~ from the blood to the water d u r i n g the exposure of the f i s h to 5% environmental hypercapnia. T h i s trend was r e v e r s e d a f t e r the C 0 2 was turned o f f and a e r a t i o n with a i r was resumed ( F i g . 35). There was no change i n the net f l u x of Na + over the course of the experiment r e l a t i v e to the t r e n d f o r c o n t r o l f i s h ( F i g . 35). There was no change i n the u n i d i r e c t i o n a l e f f l u x of C l ~ r e l a t i v e to the c o n t r o l t rend over the e n t i r e course of the experiments ( F i g . 36). U n i d i r e c t i o n a l Na + e f f l u x was lower than the c o n t r o l t r e n d from about the 24h mark, d u r i n g exposure to environmental hypercapnia. T h i s trend continued througout the hypercapnia p e r i o d as w e l l as through the r e c o v e r y p e r i o d ( F i g . 36). The net f l u x r e s u l t s can o n l y be e x p l a i n e d by r e d u c t i o n of C l ~ uptake from the water i n response to the exposure to environmental hypercapnia and the acid-base d i s t u r b a n c e . Once the reco v e r y p e r i o d s t a r t e d , the uptake r a t e o f C I - from the water r e t u r n e d to c o n t r o l l e v e l s . With the y e t unchanged 120 gure 34. Means £ S.E. of plasma hematocrit values (Hct i n %) of t r o u t a c c l i m a t e d to 3, 100 and 300 mM NaCl and exposed to 1% environmental hypercapnia f o r 24h and then recovered f o r 24h. 121 122 F i g u r e 35. Means + S « E - o f n e t f l u x e s o f N a + ( f i l l e d c i r c l e s ) and Cl~(open c i r c l e s ) i n carp d u r i n g exposure to 5% environmental hypercapnia f o r 48h fo l l o w e d by 24h recover y . Fluxes are r e f e r e n c e d to the c o n t r o l p o i n t ( h a l f f i l l e d c i r c l e ) . C o n t r o l net f l u x e s f o r Na + and C l - are shown by the and the l i n e s r e s p e c t i v e l y and are e x t r a p o l a t i o n s o f net f l u x trends p r i o r to the exposure p e r i o d extended over the e n t i r e time course o f the experiment. A l l data shown are f o r the water. Therefore p o s i t i v e v a l u e s i n d i c a t e e f f l u x from the f i s h and negative values i n d i c a t e uptake by the f i s h . NET FLUX A N a + A C l " (mmol/ kg fish) o — o Na + Na+conirol •—• cr Cl~ control i A / A \ No Na + J L 0 20 AO T . f u l 60 Time (h) t\5 CO 124 F i g u r e 36. Means +. S.E. of e f f l u x r a t e s f o r N a + ( f i l l e d c i r c l e s ) and Cl~(open c i r c l e s ) i n carp d u r i n g exposure to 5% environmental hypercapnia f o r 48h fo l l o w e d by 24h recover y . Fluxes are r e f e r e n c e d to the c o n t r o l p o i n t ( h a l f f i l l e d c i r c l e ) . C o n t r o l e f f l u x e s f o r Na + and C l - are shown by the and the l i n e s r e s p e c t i v e l y and are e x t r a p o l a t i o n s o f e f f l u x trends p r i o r to the exposure p e r i o d extended over the e n t i r e time course o f the experiment. 15 EFFLUX 10 A N a + ACI" (mmol/ kg fish) —o Na + •••• Na+control - • Cl" — Cl" control 0 i Na + 4 c r o 20 AO Time (h) 60 126 e f f l u x o f C l ~ from the blood, t h i s would r e s u l t i n the observed d e c l i n e n net f l u x from blood to water. Na + uptake from the water must have a l s o been reduced to match the reduced e f f l u x i n order t h a t an unchanged net f l u x from c o n t r o l trends c o u l d r e s u l t . DISCUSSION Environmental hypercapnia caused a plasma a c i d o s i s i n t r o u t and conger and the i n i t i a l f a l l i n plasma pH was fo l l o w e d by a trend towards recove r y with an a s s o c i a t e d accumulation of plasma HCO3 -. These trends are c h a r a c t e r i s t i c o f f i s h exposed to environmental hypercapnia i n f r e s h water (carp, C l a i b o r n e and H e i s l e r 1984; rainbow t r o u t , Janssen and Randall 1975, Eddy e £ al.. 1977, L l o y d and White 1967, Cameron and Randall 1972, Eddy 1976) and sea water ( d o g f i s h . Cross et. al.. 1969; s p o t t e d d o g f i s h , H e i s l e r et. a l . 1976,1980, Randall e t a l . 1976; coho salmon, Bubien and Meade 1979). Water s a l i n i t y had a p o s i t i v e e f f e c t i n c o r r e c t i n g the a c i d o s e s i n t r o u t a c c l i m a t e d to higher s a l i n i t i e s as w e l l as i n marine conger e e l c o n d i t i o n e d to d i l u t e waters. This supports the c i r c u m s t a n t i a l evidence f o r the p o s i t i v e c o r r e l a t i o n between the i o n i c content of the water and the c o r r e c t i o n of acid-base d i s t u r b a n c e s from exposure to 127 environmental hypercapnia. Because the H C O 3 -c o n c e n t r a t i o n , and thus b u f f e r i n g , i n the water was c o n t r o l l e d so t h a t they were n e a r l y e q u i v a l e n t a t the three s a l i n i t i e s , the data was analysed with regard to the Na + and C l -c o n c e n t r a t i o n s i n the water. The accumulation of plasma H C O 3 - was c o r r e l a t e d with r e d u c t i o n s i n plasma C l - i n both t r o u t and i n conger. T h i s r e l a t i o n s h i p suggests t h a t a C1-/HCC>3- exchange mechanism p l a y s a major r o l e i n the compensation o f the a c i d o s i s i n t r o u t and conger caused by exposure to environmental hypercapnia. The link, between C l - and HC03~ i n t r a n s e p i t h e l i a l i o n movements has been documented i n numerous s t u d i e s (Maetz and Garcia-Romeu 1964, De Renzis and Maetz 1973, De Renzis 1975, K e r s t e t t e r and K i r s h n e r 1972, Kormanik and Evans 1979). Cameron (1976) and C l a i b o r n e and H e i s l e r (1984) have shown t h a t a C1~/HCC>3~ exchange process i n the a r c t i c g r a y l i n g and carp, r e s p e c t i v e l y , p l a y s an important r o l e i n the compensation of plasma pH d u r i n g environmental hypercapnia. While these s t u d i e s a l s o demonstrated Na +/H +(NH4 +) exchanges to p l a y a s i g n i f i c a n t r o l e , net changes i n i o n c o n c e n t r a t i o n s i n the t r o u t experiment r e p o r t e d here d i d not show a s i g n i f i c a n t r o l e of t h i s exhange process i n the accumulation of plasma H C O 3 - and the consequent pH recovery. I t i s p o s s i b l e that t h i s exchange was present but not d e t e c t e d because of a Na + leak from plasma to water which would have had to be roughly 128 e q u i v a l e n t to the a c t i v e uptake o f t h i s i o n i n exchange f o r the e f f l u x o f H + to the water. While there were changes i n plasma [Na +] i n t r o u t d u r i n g the e a r l y hours a f t e r exposure to hypercapnia, these changes were not c o r r e l a t e d to the changes i n plasma HCO3 -. The r e d u c t i o n i n plasma C l ~ c o n c e n t r a t i o n s a s s o c i a t e d with the HCO3- accumulation i n the c o r r e c t i o n of plasma pH i n carp exposed to environmental hypercapnia was e f f e c t e d by a r e d u c t i o n i n the r a t e s of Cl~~ uptake from the water while m a i n t a i n i n g normal e f f l u x r a t e s . T h i s , of course, p e r t a i n s to the c o n d i t i o n i n f r e s h water and the e q u i v a l e n t f l u x e s i n f i s h a t higher s a l i n i t i e s are unknown. The l i m i t a t i o n s to i n v e s t i g a t i o n s of t h i s nature a t higher s a l i n i t i e s are p a r t i a l l y t e c h n i c a l s i n c e the r e s o l u t i o n of net f l u x e s through c o n c e n t r a t i o n changes i s obscured by the high background c o n c e n t r a t i o n s o f i o n s . While u n r e l a t e d to any acid-base d i s t u r b a n c e , Kormanik and Evans (1979) demonstrated i n c r e a s e d C l ~ e f f l u x from the marine toad f i s h , Qpsanus  beta, by i n c r e a s i n g water HCO3 -. The process of C1~/HC03~ exchange i s t h e r e f o r e a v a i l a b l e to f i s h i n s a l t water as a mechanism f o r HCO3 - accumulation. I t i s not known, however, how t h i s exchange process i s modulated to e f f e c t the HCO3 - accumulation. C h l o r i d e e f f l u x c o u l d be s t i m u l a t e d or there c o u l d be a decrease i n the p a s s i v e i n f l u x o f C l ~ which would r e s u l t i n the accumulation of plasma HCC>3~ through t h i s exchange process. 129 There may be a g e n e r a l phenomenon of r e d u c i n g a c t i v e uptake of ions from the water because i t seems t h a t Na + uptake from the water i n carp was a l s o reduced upon exposure to environmental hypercapnia. I n h i b i t i o n of a c t i v e Na + uptake from the water has a l s o been observed i n t r o u t exposed to a c i d waters (Wright and Wood 1985; Shaw 1960; McWilliams and P o t t s 1978; Packer and Dunson 1972; Ye, unpublished d a t a ) . The i r r e g u l a r i t y here was t h a t while normal C l ~ uptake r a t e s are probably resumed d u r i n g r e c o v e r y from environmental hypercapnia, reduced Na + uptake from the water continued d u r i n g r e c o v e r y . Modulation of the a c t i v e C l ~ uptake step to accumulate the plasma HCO3"" i n freshwater carp p r o v i d e s i n v i v o evidence s u p p o r t i n g p r e v i o u s in. v i t r o s t u d i e s showing the e x i s t e n c e of an anion s t i m u l a t e d ATPase i n the f i s h g i l l ( K e r s t e t t e r and K i r s c h n e r 1974, DeRenzis and Bornancin 1977, Bornancin et, al.. 1980). S i m i l a r enzymes have been found i n mammalian pancreas (Simon e t aj.. 1972), i n t e s t i n a l and r e n a l brush border membranes (Humphreys et. al.. 1979, Van Os et. a l . 1977, K i n n e - S a f f r a n and Kinne 1974, L i a n g and Sacktor 1976), and i n the g i l l s o f Necturus (Wiebelhaus e t a_l.. 1971), e e l (Morisawa and U t i d a 1976) and crab (Lee 1982). The microsomal l o c a l i z a t i o n o f t h i s enzyme i n t r o u t ( K e r s t e t t e r and K i r s c h n e r 1974) and crab (Lee 1982) g i l l s i m p l i c a t e s i t s f u n c t i o n a l r o l e to a t l e a s t i n c l u d e anion exchanges r e l a t e d to i o n o - and acid-base r e g u l a t i o n . 130 The l a c k of c o n s i s t e n t change i n the o s m o l a r i t y of t r o u t d u r i n g hypercapnia a t 100 and 300 mM suggests t h a t the i o n i c exhanges were t a k i n g p l a c e i n balance as f a r as i o n i c charges were concerned. This i s c o n s i s t e n t with the f i n d i n g s o f H e i s l e r et_ aJU (1976) and Toews e t §JL. (1983) f o r the s p o t t e d d o g f i s h and conger, r e s p e c t i v e l y , t h a t o s m o l a r i t y remained con s t a n t i n these f i s h d u r i n g exposure to environmental hypercapnia. The r e d u c t i o n i n o s m o l a r i t y i n t r o u t exposed to hypercapnia i n 3 mM suggests an imbalance i n the i o n i c changes d u r i n g the experiment. The i n c r e a s e i n hematocrit v a l u e s and the d e p o l a r i z i n g trend i n the TEP d u r i n g the i n i t i a l sampling p e r i o d s a f t e r the onset of hypercapnia i n t r o u t may have been due to the e f f e c t s of catecholamines which may have been present d u r i n g t h a t time. T h i s i s l i k e l y s i n c e Perry (1986) has shown t h a t catechlamines are r e l e a s e d i n t r o u t i n response to exposure to environmental hypercapnia. Nikinmma and H e u s t i s (1984) and B a r o i n e t al.. (1984) r e p o r t e d red c e l l s w e l l i n g which c o u l d e x p l a i n the i n c r e a s e i n hematocrit. I s a i a e t al.. (1978), furthermore, observed i n c r e a s e d water p e r m e a b i l i t y i n response to catecholamines i n f i s h . An i n c r e a s e i n water p e r m e a b i l i t y of the g i l l s suggests an i n c r e a s e i n the general p e r m e a b i l i t y to ions i f the movement of water molecules are o c c u r r i n g through i o n channels i n membranes. Given these o b s e r v a t i o n s , the d e p o l a r i z i n g trend observed here might be expected from the r e d u c t i o n of c o n c e n t r a t i o n g r a d i e n t s between blood and water. 131 Reports of TEP values i n the l i t e r a t u r e are v a r i a b l e but the general trend of i n c r e a s i n g values with the s a l i n i t y of the a c c l i m a t i o n medium i s c o n s i s t e n t with r e p o r t e d s t u d i e s ( K e r s t e t t e r et, al_. 1970; P o t t s and Eddy 1973; House and Maetz 1974; Eddy 1975; McWilliams and P o t t s 1978). These values a l s o i n c r e a s e d u r i n g hypercapnia and r e t u r n towards c o n t r o l values d u r i n g recovery from hypercapnia. The e n t i r e data s e t i n t h i s S e c t i o n demonstrates t h a t the i o n i c s t r e n g t h o f the water has a p o s i t i v e i n f l u e n c e on the acid-base r e g u l a t o r y performance of f i s h d u r i n g exposure to environmental hypercapnia over a wide range of s a l i n i t i e s , i n c l u d i n g hypotonic, near i s o t o n i c and h y p e r t o n i c media. A d d i t i o n a l l y , C1 -/HC03~ p l a y s a dominant r o l e i n t h i s r e g u l a t i o n through the accumulation o f plasma H C O 3 - . T h i s accumulation has been shown to be a modulation of the a c t i v e C l ~ uptake process i n freshwater (Cameron 1976; Wood e_t a l . 1984.). T h i s i s i n c o n t r a s t to the r e s u l t s o f Perry e_t a l . (1981) and K e r s t e t t e r and Mize (1976) who found no s i g n i f i c a n t change i n the i n f l u x r a t e s o f Na + and C l ~ i n t r o u t undergoing an a c i d o s i s . The reasons f o r t h i s d i s c r e p e n c y are not known. SECTION 3. FURTHER ANALYSIS OF THE TROUT-SALINITY-HYPERCAPNIA EXPERIMENT INTRODUCTION Th i s S e c t i o n p r e s e n t s f u r t h e r a n a l y s i s o f the i o n i c c o n c e n t r a t i o n and t r a n s e p i t h e l i a l p o t e n t i a l (TEP) data from the Trout - S a l i n i t y - Hypercapnia experiment i n S e c t i o n 2. I t was the general aim of these analyses to g a i n more understanding about the observed d i s t r i b u t i o n o f ions between the water and blood i n t r o u t d u r i n g exposure to and recover y from environmental hypercapnia. A c c o r d i n g l y , three analyses were c a r r i e d out. F i r s t , the e l e c t r o c h e m i c a l g r a d i e n t between blood and water f o r C l - was analysed i n a q u a l i t a t i v e manner f o r the three s a l i n i t y c o n d i t i o n s of the experiment. The observed c o r r e l a t i o n between changes i n plasma C l - and plasma HCO3 - accumulation d u r i n g exposure to hypercapnia i m p l i e d that modulations of a C1~/HC03~ exchange process played an important r o l e i n e x t r a c e l l u l a r pH r e g u l a t i o n i n t r o u t i n t h i s type of a c i d o s i s . Data from the carp -hypercapnia experiment i n d i c a t e d t hat the a c t i v e C1 _/HC03 _ exchange process was being i n h i b i t e d d u r i n g exposure to environmental hypercapnia to r e s u l t i n the observed HCO3"" accumulation. The aim of t h i s a n a l y s i s was to determine whether these i o n g r a d i e n t s were being maintained by p a s s i v e or a c t i v e processes. Second, the apparent p e r m e a b i l i t y of the g i l l e p i t h e l i u m 134 to Na + and C l ~ was determined by s o l v i n g f o r these v a r i a b l e s i n the Goldman equation (Goldman 1943). The three data s e t s , one f o r each s a l i n i t y , which i n c l u d e d TEP and the c o n c e n t r a t i o n s of Na + and C l ~ f o r plasma and water enabled the s o l u t i o n . F i n a l l y , the Nernst e q u a t i o n was a p p l i e d to c a l c u l a t e the expected plasma c o n c e n t r a t i o n s based on a p a s s i v e d i s t r i b u t i o n o f ions a c c o r d i n g to t h e i r e l e c t r o c h e m i c a l g r a d i e n t s . Comparisons of these expected c o n c e n t r a t i o n s to the measured plasma c o n c e n t r a t i o n s gave i n d i c a t i o n s of the r e l a t i v e magnitude of a c t i v e processes i n m a i n t a i n i n g the observed c o n c e n t r a t i o n s of each i o n b e f o r e , d u r i n g and a f t e r exposure to environmental hypercapnia. MATERIALS AND METHODS The methods by which the v a l u e s used i n t h i s S e c t i o n were measured or c a l c u l a t e d have been d e s c r i b e d i n the M a t e r i a l s and Methods of S e c t i o n 2. S e v e r a l new parameters were d e r i v e d based on some of those v a l u e s : the p e r m e a b i l i t y of g i l l to Na + (PNa+) and to C I - (PCI -) and the Nernst R a t i o f o r Na + , C l ~ and HCC>3~. The Nernst R a t i o i s the r a t i o of the measured c o n c e n t r a t i o n of an i o n d i v i d e d by the expected c o n c e n t r a t i o n based on c a l c u l a t i o n s u s i n g the Nernst equation. A value of 1.0, t h e r e f o r e , means t h a t the i o n i s 135 d i s t r i b u t e d a c c o r d i n g to the e x i s t i n g e l e c t r o c h e m i c a l g r a d i e n t s . The equations presented i n Appendix II and Appendix I I I were used to c a l c u l a t e the Nersnt R a t i o f o r Na +, C l ~ and HCO3 - ions as we l l as the r e l a t i v e p e r m e a b i l i t i e s , PNa+/PHC03-, PCI-/PHCO3- and PNa +/PCl". RESULTS I. ELECTROCHEMICAL GRADIENT ANALYSIS OF C I - . Trout a c c l i m a t e d to 3, 100 and 300mM NaCl and then exposed to 1% hypercapnia showed an i n i t i a l plasma a c i d o s i s and trends towards recovery i n plasma pH over the 24h exposure p e r i o d . pH recovery was g r e a t e r i n the 100 and 300mM a c c l i m a t e d f i s h than those a c c l i m a t e d i n 3mM. The degree of rec o v e r y was p r o p o r t i o n a l to the degree o f plasma HCO3 - accumulation. There were changes i n plasma i o n c o n c e n t r a t i o n s a s s o c i a t e d with the changes i n plasma HCO3 - c o n c e n t r a t i o n s . Increases i n plasma HCO3"" c o n c e n t r a t i o n s were most s t r o n g l y c o r r e l a t e d to decreases i n plasma C l ~ c o n c e n t r a t i o n s ( F i g . 37 a . b . c ) . At 3mM NaCl, the C l ~ g r a d i e n t was from blood to water ( F i g . 38 a . ) . On the b a s i s of a p o s s i b l e C1~/HC03~ exchange process, the pa s s i v e 136 F i g u r e 37.a. R e l a t i o n s h i p o f plasma HCO3 - to plasma (Na + - C l - ) f o r three s a l i n i t i e s . Best f i t r e g r e s s i o n l i n e s r e p r e s e n t data f o r t r o u t d u r i n g hypercapnia and recovery p e r i o d s . F i g u r e 37.b. R e l a t i o n s h i p o f d e l t a H C 0 3 - to d e l t a C l -f o r plasma data from hypercapnia and recovery p e r i o d s . F i g u r e 37.c. R e l a t i o n s h i p o f d e l t a H C 0 3 - to d e l t a Na + f o r plasma data from hypercapnia and recovery p e r i o d s . For F i g u r e s 37a.b.c. : 3 mM; 100 mM; 300 mM. A l l u n i t s i n mM. C o r r e l a t i o n C o e f f i c i e n t s f o r b e s t - f i t l i n e c o e f f i c i e n t Y = a + b * X a. r n a b 3mM 0.5281 64 0.9430 26.62 lOOmM 0.3676 57 0.7222 34.66 300mM 0.3503 85 1.4437 b. 11. 27 r n a b 3mM 0.5084 58 -0 .8470 -1.0584 lOOmM 0.3986 48 -0.5439 -0.0905 300mM 0.4139 74 -0.7463 c. -0.4684 r n a b 3mM 0.0541 61 -0.2275 -1.0536 lOOmM 0.0255 59 -0.0424 0.1704 300mM 0.0361 74 0.0960 -0 .3126 138 F i g u r e 38. H y p o t h e t i c a l a c t i v e and p a s s i v e C l ~ movements acro s s the t r o u t g i l l a t three s a l i n i t i e s and the a s s o c i a t e d t r a n s e p i t h e l i a l p o t e n t i a l (TEP) and plasma HCO3 - accumulation c h a r a c t e r i s t i c s . A l l data from t r o u t exposed to environmental hypercapnia a t three water s a l i n i t i e s . The symbols w and b r e p r e s e n t the water and blood compartments, r e s p e c t i v e l y . S o l i d and broken l i n e s with arrows r e p r e s e n t the net d i r e c t i o n a l movement of ions a c r o s s the g i l l e p i t h e l i u m which i s re p r e s e n t e d by the double v e r t i c a l l i n e s . 139 1Q0mM C l " Cl" cr .... _^HCO: NEAR ZERO HIGH 3 0 0 m M POSITIVE HIGH t > Passive +—..W.~.~Z... Active 140 e f f l u x o f blood C l ~ c o u l d have caused the observed accumulation o f plasma HCO3 -. The negative TEP values a l s o would have enhanced the p a s s i v e e f f l u x of C l ~ . At lOOmM, the g r a d i e n t f o r plasma C l ~ , i n the same d i r e c t i o n as i n 3 mM, was very small ( F i g . 38 b . ) . I t i s u n l i k e l y t h a t the pa s s i v e e f f l u x o f C l ~ along i t s small c o n c e n t r a t i o n g r a d i e n t c o u l d have e f f e c t e d the observed l a r g e accumulation o f plasma HCO3 -. S i m i l a r l y , the near zero p o t e n t i a l a c r o s s the g i l l e p i t h e l i u m a t t h i s s a l i n i t y would have had a minor e f f e c t i n the C l ~ and r e l a t e d HCO3 - movements. At 300mM, the g r a d i e n t f o r C l ~ was from water to blood ( F i g . 38 c ) . On the b a s i s o f p a s s i v e movements and a C1~/HC03~ exchange mechanism, HCO3 - would have been l o s t i n s t e a d o f gained. The i n s i d e - p o s i t i v e p o t e n t i a l would have enhanced C l ~ uptake from the water on a p a s s i v e b a s i s as w e l l . Th i s a n a l y s i s of the p o s s i b l e movements of C l ~ shows that the degree of HCO3 - accumulation i n the f i s h a c c l i m a t e d to 100 and 300mM cannot be a t t r i b u t e d to p a s s i v e C I - movements. On the b a s i s o f p a s s i v e C I - movements, the data s e t would p r e d i c t the g r e a t e s t r e d u c t i o n i n plasma C I - and consequent HCO3 - accumulation i n 3mM, the l e a s t i n lOOmM and a l o s s o f HCO3 - i n 300mM. Since the observed t r e n d o f HC03~ accumulation was i n the opp o s i t e d i r e c t i o n a c t i v e processes must have accumulated the plasma HCO3 - i n f i s h exposed to hypercapnia a t 100 and 300mM. Although the f i s h i n 3mM c o u l d have accumulated the plasma 141 HCO3 - through a C1~/HCC>3- exchange process l i n k e d to the p a s s i v e e f f l u x o f C l ~ , experiment 2 C , which i n v o l v e d s t u d y i n g the acid-base r e g u l a t i o n i n carp exposed to environmental hypercapnia, showed t h a t a c t i v e i o n exchange processes i n t h a t f i s h i n freshwater e f f e c t e d the accumulation of plasma HC03~. I I . PERMEABILITY OF THE GILL TO Na+ (PNa +) AND C l " (PC1~). PNa + was about 0.4 of PHCO3 - whereas PC1~ was about 0.2 of PHCO3 - i n t r o u t before experimental procedures were i n i t i a t e d ( F i g . 39 a.b.). Exposure to environmental hypercapnia caused s l i g h t t r a n s i e n t changes i n r e l a t i v e p e r m e a b i l i t i e s which were g r e a t e s t j u s t a f t e r exposure and decreased to c o n t r o l values at the end of the 24h exposure. To a sm a l l e r degree, s i m i l a r t r a n s i e n t s were seen a f t e r recovery from exposure to hypercapnia was s t a r t e d . Both PNa +/PHC0 3 - and PC1 -/PHC0 3- f o r the 3-300mM data s e t showed l a r g e changes w i t h i n the f i r s t 2h a f t e r the beginning o f hypercapnia ( F i g . 39 a.b.). Both values changed from c o n t r o l values to l a r g e negative values and then changed to about 4 times c o n t r o l values a t the l h sampling p e r i o d . PNa +/PHC03~ c a l c u l a t e d f o r the data s e t s : 3-100mM and 100-300mM showed s i m i l a r values as w e l l as trends 142 Fi g u r e 39.a. and b. Permeabil i t e s o f Na + (PNa + /PHCC>3-) and C l - (PC1~/PHC03~) r e l a t i v e to H C 0 3 _ f o r the g i l l o f t r o u t d u r i n g and a f t e r exposure to environmental hypercapnia. These two parameters were d e r i v e d by simultaneous s o l u t i o n o f the Goldman eq u a t i o n f o r three unique p a i r s of data from the three water s a l i n i t i e s i n v e s t i g a t e d . The three s e t s o f s o l u t i o n s from t h i s a n a l y s i s are represented by the three l i n e s on each graph. 143 CUMMULATIVE TIME (h) 144 throughout the experiment ( F i g . 39 a . ) . These d i f f e r e d from values d e r i v e d from the 3-300mM data s e t . They were lower d u r i n g exposure to hypercapnia and higher d u r i n g recovery compared to 3-300mM va l u e s . During exposure to hypercapnia, PC1~/PHCC>3- values were lowest when the 3-100mM data s e t was used ( F i g . 39 b . ) . They were h i g h e s t when the 3-300mM data s e t was used and intermediate f o r the 100-300mM data s e t . The d i f f e r e n c e s were approximately equal d u r i n g hypercapnia which g r a d u a l l y decreased over the 24 h exposure p e r i o d . D e v i a t i o n s from the range o f c o n t r o l values d u r i n g hypercapnia were i n the PC1~/PHC0 3~ values f o r the 3-100mM data s e t . During recovery, PC1~/PHC03~ c a l c u l a t e d f o r the 100-300 and 3-300mM data s e t s were s i m i l a r i n value and trend and were lower than those values c a l c u l a t e d f o r the 3-300mM data s e t . There was an in c r e a s e i n the PC1-/PHCC>3- values i n the f i r s t hour of recovery f o r the 3-100mM data s e t . The PNa +/PCl~ values averaged about 2.5 f o r the c o n t r o l p e r i o d ( F i g . 40 a.b.). T h i s r a t i o was changed d u r i n g and a f t e r exposure to environmental hypercapnia. Changes which o c c u r r e d d u r i n g hypercapnia were a b o l i s h e d by the end of the 24h exposure. The r a t i o s of PNa +/PCl~ f o r the data s e t s : 100-300mM and 3-300mM were s i m i l a r i n t h e i r response d u r i n g the hypercapnia p e r i o d . Those values decreased r e l a t i v e to average c o n t r o l values a f t e r onset o f hypercapnia and 145 F i g u r e 40.a. P e r m e a b i l i t y of Na + r e l a t i v e to C T - f o r the t r o u t g i l l d u r i n g and a f t e r exposure to environmental hypercapnia. C a l c u l a t e d by d i v i s i o n o f PNa +/PHC03~ by PC1~/PHCC>3"; see f i g u r e legends 39a. and 39b. above. F i g u r e 40.b. Data of 40.a. r e f e r e n c e d to the average c o n t r o l v a l u e s . E x p t l . ( P N a + / P C l _ ) - A v e . C o n t r o l ( P N a + / P C l - ) . 146 CUMMULATIVE TIME (h) 147 i n c r e a s e d g r a d u a l l y to c o n t r o l l e v e l s by the 24h sampling time. In c o n t r a s t , PNa +/PCl~ f o r the 3-100mM data s e t in c r e a s e d r e l a t i v e to c o n t r o l l e v e l s immediately a f t e r onset of hypercapnia and g e n e r a l l y d e c l i n e d back to c o n t r o l values by the 24h sampling time except f o r a p e r i o d between 2-4h when there was a t r a n s i e n t d e c l i n e to near c o n t r o l l e v e l s . The end of hypercapnia caused the PNa +/PCl~ r a t i o s f o r the 100-300mM and 3-300mM data s e t s to i n c r e a s e and remain higher than c o n t r o l l e v e l s f o r the remainder o f the experiment. T h i s was caused by lower PC1 _/PHC03~ values r e l a t i v e to PNa + /PHCC>3- values f o r t h i s p e r i o d . The lower PNa +/PCl~ val u e s i n the i n i t i a l sampling p e r i o d s i n recovery from hypercapnia f o r the 3-100 mM data s e t was due to in c r e a s e d PC1~/PHCC>3~ values r e l a t i v e to PNa+/PHCC>3- values which stayed near c o n t r o l l e v e l s . Other than t h i s change, PNa +/PCl~ r a t i o s remained near c o n t r o l values d u r i n g the recover y p e r i o d f o r the 3-100 mM data s e t . The PNa +/PCl~ r a t i o f o r the 3-300 mM data s e t decreased s h a r p l y 1 h a f t e r the end o f hypercapnia a f t e r which i t i n c r e a s e d to near c o n t r o l l e v e l s f o r the remainder o f the experiment. The d i f f e r e n c e i n PNa +/PCl~ values between the 3-100 mM data s e t and the other data s e t s , 3-300 and 100-300 mM, suggests that the apparent p e r m e a b i l i t y to these ions changed over the s a l i n i t y range i n v e s t i g a t e d and o c c u r r e d i n the range of 3-100 mM. The s i m i l a r i t y i n trends i n data whenever the 148 300 mM data s e t was i n c l u d e d i n the c a l c u l a t i o n s i n d i c a t e s that some aspect o f t h i s high s a l i n i t y had a st r o n g i n f l u e n c e on the parameters i n these c a l c u l a t i o n s . I I I . NERNST RATIO ANALYSIS The Nernst r a t i o f o r plasma Na + d u r i n g the c o n t r o l p e r i o d averaged about 34, 1.3 and 0.7 f o r f i s h i n 3mM, lOOmM and 300mM r e s p e c t i v e l y ( F i g . 41 a . b . c ) . There was a general i n c r e a s e i n t h i s r a t i o d u r i n g exposure to hypercapnia i n a l l three s a l i n i t i e s . Values d u r i n g the recovery p e r i o d were g e n e r a l l y v a r i a b l e and showed no c o n s i s t e n t d i f f e r e n c e from the hypercapnia p e r i o d except f o r the 300mM group o f f i s h where there was a c l e a r t rend towards c o n t r o l v a l u e s . The average Nernst r a t i o s f o r plasma C l ~ were about 20, 0.85 and 0.58 f o r 3mM, lOOmM and 300mM r e s p e c t i v e l y ( F i g . 42 a . b . c ) . There was l i t t l e change i n t h i s r a t i o at 3mM d u r i n g hypercapnia and recovery p e r i o d s . The r a t i o s a t lOOmM d u r i n g hypercapnia were g e n e r a l l y higher than c o n t r o l values but v a r i a b i l i t y masked the d i f f e r e n c e s . There was a general t r e n d towards the c o n t r o l value d u r i n g r e c o v e r y . There was a s i g n i f i c a n t i n i t i a l d e c l i n e i n Nernst r a t i o a t 300mM a f t e r which there was a recovery back to the c o n t r o l value by the end of the 24h. The end of hypercapnia caused an i n c r e a s e which subsequently d e c l i n e d to the c o n t r o l value. The average Nernst r a t i o s f o r plasma HCO3 - were about 149 F i g u r e 41.a.b.c. Means + S.E. o f r a t i o s of measured to expected plasma Na + c o n c e n t r a t i o n s f o r t r o u t d u r i n g and a f t e r exposure to environmental hypercania a t three water s a l i n i t i e s . 'Expected' values c a l c u l a t e d from the Nernst equation. F i l l e d c i r c l e s are the average c o n t r o l p o i n t s . 150 1 5 1 F i g u r e 42.a.b.c. Means + S.E. o f r a t i o s of measured to expected plasma C l ~ c o n c e n t r a t i o n s f o r t r o u t d u r i n g and a f t e r exposure to environmental hypercania a t three water s a l i n i t i e s . 'Expected' values c a l c u l a t e d from the Nernst equation. F i l l e d c i r c l e s are the average c o n t r o l p o i n t s . 152 153 F i g u r e 43.a.b.c. Means +_ S.E. o f r a t i o s o f measured to expected plasma HC0~3 c o n c e n t r a t i o n s f o r t r o u t d u r i n g and a f t e r exposure to environmental hypercania a t three water s a l i n i t i e s . 'Expected' values c a l c u l a t e d from the Nernst equation. F i l l e d c i r c l e s are the average c o n t r o l p o i n t s . 154 155 8, 13, and 13 f o r 3mM, lOOmM and 300mM r e s p e c t i v e l y ( F i g . 43 a . b . c ) . U n l i k e the r a t i o s f o r Na + and C l ~ , trends i n the Nernst r a t i o s f o r HCO3 - showed c o n s i s t e n t trends d u r i n g both hypercapnia and recovery p e r i o d s . The r a t i o s i n c r e a s e d 4-5 times d u r i n g the hypercapnia p e r i o d and r e t u r n e d to n e a r - c o n t r o l l e v e l s by the end of the recovery p e r i o d . DISCUSSION The analyses i n t h i s S e c t i o n provide a b a s i s f o r a s e r i e s of q u a l i t a t i v e statements about the nature o f the d i s t r i b u t i o n of Na + and C l ~ between blood and water i n t r o u t a c c l i m a t e d to three water s a l i n i t i e s and exposed to environmental hypercapnia. A c t i v e r a t h e r than p a s s i v e processes i n v o l v i n g a C l ~ f o r HCO3 - exchange e f f e c t e d the accumulation o f plasma HCO3 - i n t r o u t exposed to 1% environmental hypercapnia a t a l l three s a l i n i t i e s i n v e s t i g a t e d . T h i s agrees with the r e s u l t s o f the carp hypercapnia study o f S e c t i o n 2 which showed t h a t not onl y was t h i s exchange process important i n r e g u l a t i o n o f plasma pH d u r i n g a hypercapnic a c i d o s i s i n carp but a l s o t h a t i t was the i n h i b i t i o n o f t h i s process t h a t was r e s p o n s i b l e f o r plasma HCO3 - accumulation i n f r e s h water. The analyses i n t h i s S e c t i o n suggest that the C1~/HC03~ exchange process was s t i m u l a t e d i n t r o u t a c c l i m a t e d to the two higher s a l i n i t i e s i n order to accumulate the plasma HCO3-"; 156 that i s , an a c t i v e step was i n v o l v e d i n the e f f l u x of C l ~ from blood to water and a l s o i n the l i n k e d uptake of HCO3 -from water to blood. Cameron (1976) showed t h a t an a c t i v e C1 -/HC03~ exchange process i n the g i l l o f Thvmallus  a r c t i c u s played an important r o l e i n the r e g u l a t i o n o f e x t r a c e l l u l a r acid-base balance when s u b j e c t e d to environmental hypercapnia or to a step change i n temperature, both of which d i s t u r b e d plasma pH. The d i s c u s s i o n i n S e c t i o n 2 presented the p u b l i s h e d i n f o r m a t i o n f o r the e x i s t e n c e of an anion s t i m u l a t e d ATPase i n the f i s h g i l l which would be the r e g u l a t e d enzyme i n t h i s case. The d e v i a t i o n s of Nernst r a t i o s f o r HCO3 - a l s o support the c o n c l u s i o n that a c t i v e processes were i n v o l v e d i n accumulation of plasma HCO3 - d u r i n g hypercapnia i n t r o u t a c c l i m a t e d a t a l l s a l i n i t i e s . Nernst r a t i o s f o r HCO3"" d u r i n g c o n t r o l p e r i o d s as well as f o r experimental p e r i o d s were s i m i l a r f o r f i s h a c c l i m a t e d to 100 and 300 mM NaCl, both of which were g r e a t e r than r a t i o s f o r f i s h a c c l i m a t e d to 3 mM at a l l sampling times. These r e s u l t s are a l s o c o n s i s t e n t with the degree of HC03~ accumulation i n f i s h a t the three s a l i n i t i e s r e p o r t e d i n S e c t i o n 2. Plasma HCO3"" accumulation was s i m i l a r i n f i s h a c c l i m a t e d a t 100 and 300 mM NaCl, both of which were g r e a t e r than the accumulation at 3 mM. The proposed a c t i v e nature of the C1~/HCG"3~ exchange mechanism i n f i s h a c c l i m a t e d to the three s a l i n i t i e s i s 157 f u r t h e r supported by another aspect of the Nernst r a t i o data The Nernst r a t i o s f o r C l ~ were h i g h e s t f o r the f i s h a c c l i m a t e d i n 3 mM and near u n i t y f o r those a c c l i m a t e d a t 100 and 300 mM. The Nernst r a t i o s f o r HCO3 -, on the other hand, were lowest f o r the f i s h a c c l i m a t e d to 3 mM and h i g h e s t f o r those a c c l i m a t e d a t the two high s a l i n i t i e s . Because the Nernst r a t i o i s d e f i n e d as the r a t i o of the measured plasma c o n c e n t r a t i o n of an i o n over t h a t expected a c c o r d i n g to a p a s s i v e d i s t r i b u t i o n o f the i o n between blood and water, the proposed l i n k between C l ~ and HCO3 - by an a c t i v e exchange process would p r e d i c t the observed r e s u l t s f o r each i o n which complemented each other. The Nernst r a t i o s f o r Na +, C l ~ and HCO3 - a l l suggest that there were two groups of f i s h with regard to i o n d i s t r i b u t i o n between blood and water. One group was f i s h a c c l i m a t e d to 3 mM NaCl which showed l a r g e p o s i t i v e r a t i o s and the other c o n s i s t e d of the f i s h a c c l i m a t e d to 100 and 300 mM, both of which showed r a t i o s near one. Both Na + and C l ~ Nernst r a t i o s showed t h a t plasma c o n c e n t r a t i o n s of these ions were a c t i v e l y maintained higher than that expected on the b a s i s o f p a s s i v e d i s t r i b u t i o n a c c o r d i n g to e x i s t i n g e l e c t r o c h e m i c a l g r a d i e n t s i n f i s h a c c l i m a t e d to 3 mM. For f i s h a c c l i m a t e d a t 100 and 300 mM, Nernst r a t i o s of Na + and C l ~ were c l o s e to one r e l a t i v e to the f i s h a t 3 mM. F i s h i n 300 mM NaCl showed Nernst r a t i o s f o r Na + and C l ~ which were l e s s than one. The r e c i p r o c a l of the Nernst r a t i o s 158 d u r i n g the c o n t r o l p e r i o d s show t h a t plasma Na + and CT~ c o n c e n t r a t i o n s were maintained about 1.43 and 1.72 times lower than c o n c e n t r a t i o n s expected on the b a s i s of a p a s s i v e d i s t r i b u t i o n of i o n s . While i t i s tempting to a s c r i b e the s t i m u l a t i o n of C l ~ and the a s s o c i a t e d HCO3 - uptake to c h l o r i d e c e l l s , a t l e a s t f o r the f i s h a c c l i m a t e d i n 300 mM, there are s e v e r a l reasons t h a t argue a g a i n s t involvement of c e l l u l a r r e c r u i t m e n t or even s y n t h e s i s of new a n i o n - s t i m u l a t e d ATPase f o r i o n pumping as shown by Lee (1982) i n the blue crab. C a l l i n e c t e s  sapidus, a c c l i m a t e d to d i l u t e waters, i n the C1~/HCC>3~ exchange observed i n t h i s experiment. F i r s t , f i s h a c c l i m a t e d to 100 mM NaCl, a medium which i s not h y p e r t o n i c to body f l u i d s , showed a s i m i l a r r a t e o f HCO3 - accumulation as f i s h i n 300 mM. Since c h l o r i d e c e l l s have been shown to develop i n f i s h i n h y p e r t o n i c waters and f u n c t i o n to r e g u l a t e body i o n c o n c e n t r a t i o n s they would not be expected to develop i n the f i s h a c c l i m a t e d i n 100 mM. An e x c e p t i o n would be the p o s s i b i l i t y o f t h e i r development i n response to a s t i m u l u s from an acid-base d i s t u r b a n c e such as i n t h i s experiment. There i s , however, no evidence f o r such a response. Another argument a g a i n s t involvement of c e l l u l a r and molecular r e c r u i t m e n t i n a c t i v e C1~/HC03 - exchange i n f i s h a c c l i m a t e d i n 100 and 300 mM i s the d i f f e r e n c e i n the time course of these processes. The time course of net changes i n plasma io n s , i n the order of hours, s t r o n g l y 159 suggests t h a t there was s t i m u l a t i o n o f e x i s t i n g t r a n s p o r t mechanisms r a t h e r than p o s s i b l e development of c e l l u l a r and molecular s t r u c t u r e s , which r e q u i r e time spans i n the order of days. I t i s p o s s i b l e t h a t s t i m u l a t i o n o f the t r a n s p o r t mechanisms present i n the g i l l o f f i s h i n 100 mM NaCl was s u f f i c i e n t to accumulate plasma HCO3 - observed i n f i s h a t both high s a l i n i t i e s . The graded accumulation o f plasma HCO3 - i n the conger, r e p o r t e d i n S e c t i o n 2, a c c o r d i n g to the water s a l i n i t y supports t h i s p o s s i b i l i t y which p r e c l u d e s the requirement f o r c h l o r i d e c e l l development to e f f e c t the degree of plasma C l ~ and HCO3 - c o n c e n t r a t i o n changes. The conger were not a c c l i m a t e d to the new s a l i n i t i e s l e v e l s f o r more than three hours, a p e r i o d too s h o r t f o r such c e l l u l a r r e c r u i t m e n t to take p l a c e . Both Na + and C l ~ are l e s s permeable than HCO3 -a c r o s s the g i l l . The r e l a t i v e p e r m e a b i l i t e s o f PNa +/PHC0 3 _ and PC1-/PHC0 3 _ d i f f e r e d i n f i s h i n the d i f f e r e n t s a l i n i t i e s d u r i n g exposure to hypercapnia and du r i n g r e c o v e r y as shown by the d i f f e r e n c e s when d i f f e r e n t data s e t s were used to d e r i v e these v a l u e s . Both p e r m e a b i l i t i e s were s l i g h t l y g r e a t e r than the average c o n t r o l values when the 3-300mM data s e t was used. The reasons f o r l a r g e f l u c t u a t i o n s i n these p e r m e a b i l i t i e s d u r i n g the i n i t i a l sampling times cannot be e x p l a i n e d . The PNa +/PCl~ data f o r the 3-300 mM data s e t show t h a t l a r g e f l u c t u a t i o n s i n both PNa + /PHCC>3~ and PC1~/PHCC>3~ oc c u r r e d i n c o n c e r t ; 160 i . e . PNa +/PCl~ valu e s changed very l i t t l e . PC1~/PHC03~ was reduced when the 3-100mM data s e t was used. While i t i s not p o s s i b l e to say how i n d i v i d u a l p e r m e a b i l i t i e s were changed to g i v e the observed r e s u l t s , these data were u s e f u l i n e x p l a i n i n g some aspects o f the subsequently c a l c u l a t e d r a t i o PNa +/PCl~. The t r o u t g i l l i s about 2.5 times more permeable to Na + than to C l - under steady s t a t e c o n d i t i o n s . R e l a t i v e p e r m e a b i l i t y estimates of near u n i t y have been r e p o r t e d by I s a i a and Masoni (1976) f o r the e e l and by McWilliams and P o t t s (1978) f o r brown t r o u t . These r e s u l t s are supported by p r e v i o u s s t u d i e s showing a g r e a t e r p e r m e a b i l i t y of the f i s h g i l l to Na + than to C l ~ . While I s a i a and Masoni (1976) showed the p e r m e a b i l i t i e s of these ions to be approximately equal i n the e e l , A n g u i l l a a n q u i l l a , i n f r e s h water and d i s t i l l e d water changes i n C a + + and magnesium (Mg + +) had a l a r g e r e f f e c t on PNa + than P C I - . T h i s suggests a g r e a t e r Na + p e r m e a b i l i t y s i n c e d i v a l e n t ions are knwon to mainly a f f e c t p a s s i v e i o n p e r m e a b i l i t y c h a r a c t e r i s t i c s of the g i l l . Eddy (1975) a l s o found brown t r o u t g i l l s to be more permeable to Na + than to C l - . The Nernst r a t i o s f o r Na + were c o n s i s t e n t l y g r e a t e r than those f o r C l " by about 1.5-2.0 f o r the s a l i n i t y range i n t h i s study. The agreement o f t h i s estimate with the estimate of PNa +/PCl~ of about 2.5 i m p l i e s that the p a s s i v e leak of Na + was being compensated by a c t i v e processes under steady s t a t e c o n d i t i o n s . 161 The r e l a t i v e p e r m e a b i l i t y o f the g i l l , PNa +/PCl~, changed with s a l i n i t y when exposed to hypercapnia and d u r i n g recovery. An assumption i n these c a l c u l a t i o n s i s the con s t a n t p e r m e a b i l i t y o f HCO3 - to both Na + and C l ~ . I t i s c l e a r from c a l c u l a t i o n s of r e l a t i v e p e r m e a b i l i t i e s i n t h i s S e c t i o n t h a t these terms r e p r e s e n t apparent p e r m e a b i l i t i e s , and t h e r e f o r e do not i n d i c a t e the mechanisms by which the observed d i s t r i b u t i o n o f ions took p l a c e . An i n c r e a s e i n the PNa+/PHCC>3- v a l u e , f o r example, f o r f i s h i n freshwater may l e a d to r e d u c t i o n s i n plasma Na + c o n c e n t r a t i o n s toward the water v a l u e s . However, i t does not s p e c i f y how t h i s i s t a k i n g p l a c e . The p a s s i v e leak o f Na + may be i n c r e a s i n g with normal Na + uptake r a t e s , the p a s s i v e leak o f Na + from blood to water may be constant while the a c t i v e uptake step i s reduced or both events might be o c c u r r i n g s i m u l t a n e o u s l y . The PNa +/PCl~ values a l s o tend to be very s e n s i t i v e to changes i n e i t h e r PNa +/PHC0 3- or PC1 -/PHC0 3- v a l u e s . T h i s can be seen i n the two p o i n t s i n the 3-100 mM data s e t which f a l l below the 0 c o n t r o l l e v e l e a r l y i n the hypercapnia p e r i o d . Those values are low because the i n c r e a s e i n PC1~/PHCC>3- i s s l i g h t l y g r e a t e r than the i n c r e a s e i n PNa +/PHC03~. Con c l u s i o n s were t h e r e f o r e only based on c o n s i s t e n t trends i n t h i s parameter. The 3-100 mM data s e t showed g e n e r a l l y higher PNa +/PCl~ values compared to c o n t r o l v a l u e s . T h i s was due to a reduced PC1~/PHC03~ values r e l a t i v e to 162 corresponding PNa + /PHCC>3- v a l u e s . T h i s reduced apparent C l - p e r m e a b i l i t y can be e x p l a i n e d f o r the 3 mM s a l i n i t y data s e t by the C l ~ f l u x observed i n the carp hypercapnia experiment, assuming t h a t s i m i l a r f l u x e s would have o c c u r r e d i n t r o u t a c c l i m a t e d a t 3 mM and exposed to environmental hypercapnia. On that b a s i s , the apparent p e r m e a b i l i t y of C l - may have been due to the reduced uptake o f C l ~ without any change i n the p a s s i v e leak from blood to water. The two data s e t s , 100-300 mM and 3-300 mM both showed lower PNa +/PCl~ values d u r i n g hypercapnia. I t was the g r e a t e r r e d u c t i o n i n PNa +/PHC03~ valu e s compared to the PC1~/PHC03~ t h a t produced t h i s r e s u l t i n the 100-300 mM data s e t . In c o n t r a s t , i t was i n c r e a s e d PC1 -/PHC03 -over the PNa +/PHC0 3~ which r e s u l t e d i n low PNa +/PCl~ values d u r i n g hypercapnia i n the 3-300 mM s a l i n i t y . In s p i t e of these d i f f e r e n c e s , i t i s tempting to s p e c u l a t e t h a t some aspect o f the 300 mM s a l i n i t y , such as the a c t i v e e f f l u x of C l ~ from blood to water, i n f l u e n c e d t h i s parameter. T h i s would r e s u l t i n the decreased apparent PC1~/PHC03~ value over PNa +/PHC03~ such as was the case f o r the 300 mM data s e t . S E C T I O N 4. C A T E C H O L A M I N E R E L E A S E I N A C I D I N F U S E D TROUT 164 INTRODUCTION An i n c r e a s e i n c i r c u l a t i n g catecholamines c h a r a c t e r i z e s the acute s t r e s s response i n f i s h (Nakano and Tomlinson 1967) and can have v a r i e d e f f e c t s on blood flow p a t t e r n s (Richards and Fromm 1969; Bergman, Olson and Fromm 1974; Wood 1974, 1975; Payan and G i r a r d 1977; Booth 1978), i o n i c f l u x e s ( G i r a r d and Payan 1977; Perry 1981), and general p e r m e a b i l i t y o f the f i s h g i l l ( I s a i a ejt al_. 1978). Acid-base d i s t u r b a n c e s are no e x c e p t i o n to t h i s phenomenon and plasma catecholamines i n f i s h have been observed to i n c r e a s e i n response to severe metabolic (Primmett ejt al_. 1986; Ye, i n prep.) and r e s p i r a t o r y (Perry 1986) a c i d o s e s . S e v e r a l e f f e c t s o f catecholamines on i o n t r a n s f e r processes a c r o s s the e r y t h r o c y t e membrane and g i l l e p i t h e l i u m of f i s h e s are known. In freshwater, beta r e c e p t o r s t i m u l a t i o n i n h i b i t s C1~/HC0 3~ exchange (Perry e_t al.. 1984) and s t i m u l a t e s Na +/H +(NH4 + ) exchange (Payan et_ al.. 1975; G i r a r d and Payan 1977; Payan 1978). The r e s u l t o f the modulation o f these i o n exchange processes i s to r e s t o r e the acid-base balance o f the i n t r a c e l l u l a r and e x t r a c e l l u l a r f l u i d s . The i n t r a c e l l u l a r pH o f the t r o u t red c e l l i s i n c r e a s e d i n v i t r o with the a d d i t i o n o f catecholamines (C o s s i n s and Richardson 1985). T h i s serves to o f f s e t the Bohr 165 and Root s h i f t s t h a t are e x h i b i t e d and thus p r o t e c t s the oxygen c a r r y i n g c a p a c i t y of the blood. T h i s e f f e c t has been shown in, v i v o f o r t r o u t a f t e r a bout of a naerobic e x e r c i s e which causes a plasma a c i d o s i s (Primmett e t al_. 1986). Holeton e_t al.. (1983) a l s o showed that a m etabolic a c i d o s i s of s i m i l a r magnitude to t h a t of Primmett et. al.. (1986) induced net i o n exchanges acr o s s the g i l l e p i t h e l i u m of t r o u t t h a t was a s s o c i a t e d with c o r r e c t i o n of the acid-base d i s t u r b a n c e i n the blood. I t i s not c l e a r , however, whether the catecholamine r e l e a s e i n response to m etabolic a c i d o s e s i n the above experiments was due to the acid-base d i s t u r b a n c e or to some other aspect of the e x e r c i s e . The two in. v i v o experiments r e p o r t e d i n t h i s S e c t i o n t e s t e d the hypothesis t h a t catecholamines are r e l e a s e d i n t r o u t d u r i n g a bout of anaerobic e x e r c i s e i n response to i n c r e a s e i n the a c i d l o a d of the blood and t h a t the f u n c t i o n a l s i g n i f i c a n c e of t h i s r e l e a s e i s to m a i ntain e r y t h r o c y t i c pH and thus the oxygen c a r r y i n g c a p a c i t y of the blood i n face of the plasma a c i d o s i s . MATERIALS AND METHODS This S e c t i o n d e s c r i b e s two s e r i e s of experiments with i d e n t i c a l treatment but with d i f f e r e n t measurements and s l i g h t l y d i f f e r e n t sampling times. The f i r s t experiment was 166 designed to i n v e s t i g a t e the p o s s i b i l i t y o f catecholamine r e l e a s e with a c i d i n f u s i o n and i t s r o l e i n red c e l l pH r e g u l a t i o n and the second i n v e s t i g a t e d e f f e c t s t h i s treatment had on i o n o r e g u l a t i o n . EXPERIMENT 4A. CATECHOLAMINE RELEASE AND RED CELL pH REGULATION : ANIMALS : Rainbow t r o u t , Salmo g a i r d n e r i . weighing from 221.5 to 460 g were o b t a i n e d from a commercial hatchery and h e l d outdoors i n f i b e r g l a s s tanks s u p p l i e d with f l o w i n g d e c h l o r i n a t e d Vancouver tap water (8-10°C; pH 6.9-7.1; CaC03 4 ppm). F i s h were fed ad. 1ibitum from s e l f feeders c o n t a i n i n g dry t r o u t p e l l e t s . In the l a b o r a t o r y , f i s h were kept i n blackened perspex a q u a r i a a t 10°C and s t a r v e d f o r 48 h p r i o r to s u r g i c a l o p e r a t i o n s and experimentation. SURGERY : The d o r s a l a o r t a o f a l l experimental animals were c h r o n i c a l l y cannulated with PE50 cannulas by the procedures of Smith and B e l l (1964) 167 PROTOCOL : Fouteen animals were i n f u s e d through the d o r s a l a o r t i c cannula with 5 ml*Kg~l body weight of a 0.05 N HC1 s o l u t i o n made up i n 120 mM p h y s i o l o g i c a l s a l i n e . F i v e f i s h t r e a t e d i n an i d e n t i c a l f a s h i o n were i n f u s e d with the s a l i n e alone and served as c o n t r o l s . I n f u s i o n times averaged about 5 minutes. A r t e r i a l blood samples of 500 u l were taken before the i n f u s i o n and a t time p e r i o d s of 5, 30, 60 and 120 min p o s t - i n f u s i o n . P o r t i o n s of each blood sample were analyzed f o r pHe, t o t a l oxygen content and hemoglobin c o n c e n t r a t i o n ; the remainder of the blood sample 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 . The r e s u l t i n g plasma was taken up i n t o a c h i l l e d s y r i n g e and t r a n s f e r r e d to an Eppendorf v i a l f o r storage a t -40°C. Stored plasma was subsequently analyzed for catecholamines. The red c e l l f r a c t i o n a f t e r c e n t r i f u g a t i o n was used f o r i n t r a c e l l u l a r pH measurement. ROOT EFFECT DETERMINATION : Blood samples were drawn from q u i e s c e n t f i s h f i t t e d with d o r s a l a o r t i c cannulas as d e s c r i b e d above. Blood samples were immediately pooled, t r a n s f e r r e d to an i n t e r m i t t e n t l y r o t a t i n g tonometer, and e q u i l i b r a t e d a g a i n s t h u m i d i f i e d gas mixtures c o n t a i n i n g e i t h e r 0.2 or 1.0 % C0 2 i n a i r (mixed with Wosthoff pumps). A f t e r 30-40 min of tonometry a t 10°C, 168 blood was taken up i n t o a p o s i t i v e displacement g a s - t i g h t s y r i n g e (Hamilton) and measurements were made of blood and red c e l l pH, hemoglobin c o n c e n t r a t i o n and oxygen content. 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 measured i n the plasma of a 1 ml sample of the blood pool taken d u r i n g the i n i t i a l stages o f the e q u i l i b r a t i o n procedure. ANALYTICAL PROCEDURES : The procedures f o r whole blood and red c e l l pH det e r m i n a t i o n s as well as f o r Pc02 measurements are d e s c r i b e d i n General M a t e r i a l s and Methods. Hemoglobin c o n c e n t r a t i o n s were determined s p e c t r o p h o t o m e t r i c a l l y (Sigma b u l l e t i n no. 525). Blood oxygen contents were measured with the Lex-02~Cont apparatus (Lexington Instruments, Mass., USA). Plasma a d r e n a l i n e and n o r a d r e n a l i n e c o n c e n t r a t i o n s were measured u s i n g high pressure l i q u i d chromatography by a method d e s c r i b e d by Woodward (1982). STATISTICAL PROCEDURES : The Student's t t e s t ( p a i r e d and unpaired as a p p r o p r i a t e ) was used to d i s c e r n s t a t i s t i c a l s i g n i f i c a n c e between means with a 5 % l e v e l o f r e j e c t i o n . Various data s e t s were a l s o d e s c r i b e d by l i n e a r r e g r e s s i o n a n a l y s i s . 169 EXPERIMENT 4B. ION REGULATION AFTER ACID INFUSON : The M a t e r i a l s and Methods f o r t h i s experiment were s i m i l a r to t h a t f o r the experiment 4A. Only the d i f f e r e n c e s are noted below. ANIMALS AND PREPARATION : The Rainbow t r o u t , Salmo g a i r d n e r i , weighed between 280 to 378 g. A l l f i s h i n t h i s experiment were f i t t e d with u r i n a r y c a t h e t e r s through which u r i n e was c a r r i e d out o f the r e c i r c u l a t i n g water system to waste. The u r i n a r y c a t h e t e r s were made of PE 60 which were l e d up the u r e t e r j u s t past the s p h i n c t e r muscle. Ti s s u e glue was a p p l i e d to the o u t s i d e o f the c a t h e t e r w a l l to secure i t i n p l a c e . As an a d d i t i o n a l s e c u r i t y , c o t t o n sutures were anchored to the two f l a p s which cover the u r i n a r y p a p i l l a e and wrapped around the cather and f i r m l y t i e d i n p l a c e . The end of the c a t h e t e r was p o s i t i o n e d at a s l i g h t n e g a t i v e head r e l a t i v e to i t s o r i g i n . The experimental chamber c o n s i s t e d of a bl a c k perspex box for a s i n g l e f i s h connected to a pump and a r e s e r v o i r . The system c o n s t i t u t e d a r e c i r c u l a t i n g c l o s e d system f o r n o n - v o l a t i l e ions and chemicals. An a e r a t i o n stone mixed water i n the r e s e r v o i r and e q u i l i b r a t e d the system with a i r . The e n t i r e water volume was approximately 10 times the weight o f the f i s h . I t was hoped t h a t t h i s system would be s e n s i t i v e 170 enough to r e f l e c t the net t r a n s f e r s o f ions between f i s h and water. The system water was renewed s e v e r a l times a day but not d u r i n g the sh o r t experimental p r o t o c o l d e s c r i b e d below. PROTOCOL : E i g h t animals were i n f u s e d with HC1 and 5 were sham i n j e c t e d with the s a l i n e . The d o r s a l a o r t i c cannula was connected to the KCl/agar b r i d g e f o r a t r a n s e p i t h e l i a l p o t e n t i a l (TEP) re a d i n g . Once s t a b l e r e a d i n g was obtained (< 1 min), the co n n e c t i o n was broken and a 300 u l whole blood sample was c o l l e c t e d f o r the measurements below. Samples were c o l l e c t e d before i n f u s i o n , which served as another c o n t r o l v a l u e , and a t time p e r i o d s o f 5, 15, 40, 60 and 120 min a f t e r the i n f u s i o n o f e i t h e r a c i d or s a l i n e was complete. MEASUREMENTS : Plasma i o n c o n c e n t r a t i o n s o f Na +, C l - , K +, C a + + and Mg + + were determined by spectrophotometry. Plasma NH4 + c o n c e n t r a t i o n was measured by a m o d i f i c a t i o n o f the Solarzano method. Ion c o n c e n t r a t i o n s above were measured f o r each water sample by spectrophotometry. Water HCO3 - c o n c e n t r a t i o n s were determined by e q u i l i b r a t i n g each sample with 1 % CO2 (Wosthoff pump) and de t e r m i n i g the TC02 content o f that 171 sample. Water H C O 3 c o n c e n t r a t i o n was c a l c u l a t e d u s i n g these values and the s o l u b i l i t y c onstant from B o u t i l i e r §_t a l . (1985). TEP values were determined a c c o r d i n g to the methods d e s c r i b e d i n General M a t e r i a l s and Methods and Nernst r a t i o s as d e s c r i b e d i n S e c t i o n 3. were c a l c u l a t e d f o r plasma i o n s . STATISTICS : P a i r e d and unpaired Student's t t e s t s were used to d i s c e r n s t a t i s t i c a l s i g n i f i c a n c e between means us i n g 5 % as the r e j e c t i o n l e v e l . RESULTS EXPERIMENT 4A. CATECHOLAMINE RELEASE & RED CELL pH REGULATION The maximum change i n plasma pH o c c u r r e d i n the f i r s t sampling p e r i o d which was 5min a f t e r i n f u s i o n of a c i d ended. Plasma pH f e l l 0.167 u n i t s below t h a t of p r e - i n f u s i o n c o n t r o l l e v e l s at t h a t time ( F i g . 44 a ) . At the same time as plasma a d r e n a l i n e c o n c e n t r a t i o n s i n c r e a s e d by 6 - f o l d whereas plasma n o r a d r e n a l i n e i n c r e a s e d only s l i g h t l y ( F i g . 44 d ) . Red c e l l pH d e c l i n e d s l i g h t l y a t the 5min mark. ( F i g . 44 b) . In 4 out of the 14 f i s h , red c e l l pH i n c r e a s e d r e l a t i v e to p r e - i n f u s i o n 172 F i g u r e 44. Means +_ S.E. of a. A r t e r i a l plasma pH (pHe), b. red c e l l pH ( p H i ) , c. mis 0 2 bound per gram of haemoglobin (mis 0 2*gHg - 1) and d. plasma catecholamine c o n c e n t r a t i o n s f o l l o w i n g i n t r a - a r t e r i a l i n f u s i o n o f a 5 ml * K g _ 1 body weight 0.05N HC1 s o l u t i o n of 120mM s a l i n e i n 14 rainbow t r o u t . Shaded v e r t i c a l bar r e p r e s e n t s the i n f u s i o n time p e r i o d . Shaded h o r i z o n t a l bars ( l a b e l l e d shams) r e p r e s e n t £ 1 standar e r r o r o f the combined mean data o f f i v e c o n t r o l experiments designed to examine the i n f l u e n c e o f the s a l i n e v e h i c l e alone ( i . e . 5 ml * K g - 1 of 120mM s a l i n e i n j e c t i o n s ) . The shading i s r e p r e s e n t a t i v e o f +_ 1 S.E.M. f o r both a d r e n a l i n e and no r a d r e n a l i n e l e v e l s i n the bottom pane l . C = p r e - i n f u s i o n c o n t r o l . (N = 14). 173 Time (min) 174 l e v e l s i n the face of a f a l l i n plasma pH. R e l a t i v e to the c o n t r o l animals there was no change i n O2 content per gram of hemoglobin throughout the course of the experiments ( F i g . 44 c ) . In the 2h f o l l o w i n g the 5min mark, of maximum change i n these parameters, plasma pH and catecholamine l e v e l s g r a d u a l l y r e t u r n e d toward c o n t r o l values ( F i g . 44 a.d). S a l i n e i n f u s e d c o n t r o l f i s h showed no s i g n i f i c a n t p o s t - i n f u s i o n changes i n any of the measured v a r i a b l e s r e l a t i v e to t h e i r p r e - i n f u s i o n values ( F i g . 44 a.b.c.d. and Table 4). Changes i n c i r c u l a t i n g catecholamine c o n c e n t r a t i o n s are p r o p o r t i o n a l to changes i n pHe i n a c i d i n f u s e d f i s h o f the present experiment. The g r e a t e r the decrease i n pHe, the g r e a t e r the i n c r e a s e i n catecholamine c o n c e n t r a t i o n ( F i g . 45). Rainbow t r o u t blood e x h i b i t a Root s h i f t . Reduction of plasma pH i n v i t r o by e q u i l i b r a t i o n with high CO2 t e n s i o n s caused a r e d u c t i o n of red c e l l pH i n the absence o f i n c r e a s e d catecholamine l e v e l s . The amount of oxygen bound to hemoglobin decreased as w e l l . I t i s assumed that a c i d i f i c a t i o n by the a d d i t i o n o f a c i d and i n the absence of e l e v a t e d catecholamine l e v e l s would have had the same e f f e c t ( F i g . 46). EXPERIMENT 4B. PLASMA AND WATER IONS AND TEP CHANGES AFTER HC1 INFUSION As i n the p r e v i o u s experiment, plasma pH a l s o d e c l i n e d 175 Table 4. Means + S.E. of a r t e r i a l plasma pH(pHe), red c e l l pH(pHi), mis O2 bound per gram of hemoglobin(mls02*gHb _ 1), adr e n a l i n e ( [ A ] ) and noradrenaline([NA]) concentrations before and fo l l o w i n g i n t r a - a r t e r i a l i n f u s i o n of a 5 ml*Kg~ 1 120mM s a l i n e s o l u t i o n i n f i v e animals. None of the means values are s i g n i f i c a n t l y d i f f e r e n t from those of the p r e - i n f u s i o n c o n t r o l s . PARAMETER PRE-INFUSION CONTROL + 5min POST-INFUSION + 30min + 60min + 120min pHe 7.948 + 0.034 7.963 + 0.033 7.954 + 0.028 7.947 + 0.026 7. 947 + 0.030 pHi 7.413 + 0.027 7. 418 + 0.032 7.400 + 0.018 7. 382 + 0.019 7. 415 + 0.029 mls0 2*gHb- 1 1.04 + 0.03 1.04 + 0.03 1.03 + 0.04 1.03 + 0.05 1. 04 + 0.04 [A] nmol*L -l 0. 31 + 0.11 0.45 + 0.12 0.34 + 0.11 0.76 + 0.36 0.74 + 0.17 [NA] nmol*L~l 0. 27 + 0.04 0.23 + 0.05 0. 22 + 0.03 0.17 + 0.03 0. 23 + 0.03 176 F i g u r e 45. R e l a t i o n s h i p between the changes i n plasma pH and the corresponding changes i n plasma a d r e n a l i n e c o n c e n t r a t i o n s between p r e - i n f u s i o n c o n t r o l samples and the + 5min p o s t - i n f u s i o n samples f o r each of 14 animals c o n t r i b u t i n g to the mean data i n F i g . 2.1. L i n e a r r e g r e s s i o n a n a l y s i s of the data p o i n t s were used to generate the best f i t l i n e . D e l t a [ A d r e n a l i n e ] = 42.46 -2.28*Delta pHe, r 2 = 0.92. 177 178 gure 46. R e l a t i o n s h i p between 02 c a p a c i t y per gram o f haemoglobin and red blood c e l l pH i n rainbow t r o u t blood e q u i l i b r a t e d in. v i t r o (10°C) a g a i n s t gas mixtures having an oxygen p a r t i a l pressure o f 152 mmHg and a C02 p a r t i a l p ressure o f 2.5 mmHg. Data are i n d i v i d u a l measurements on a s i n g l e blood pool from three animals. Haemoglobin c o n c e n t r a t i o n o f the blood pool = 8.4 gHb*100ml~l. The l i n e o f best f i t was generated by l i n e a r r e g r e s s i o n a n a l y s i s where mis 0 2 * g _ 1 [ H b ] = -5.005 + 0.800*pHi (r = 0.94). 179 Red cell pH 180 about 0.17 u n i t s r e l a t i v e to the p r e - i n f u s i o n c o n t r o l value ( F i g . 47 a ) . Plasma pH values f o r s a l i n e i n f u s e d sham c o n t r o l f i s h d i d not change i n the e a r l y sampling p e r i o d s d u r i n g which maximum changes i n the a c i d i n f u s e d f i s h o c c u r r e d . There was a s l i g h t and t r a n s i e n t i n c r e a s e i n plasma pH of the sham c o n t r o l s a t 40 and 60min p o s t - i n f u s i o n . By 120min, at the end of the experiment, both a c i d and s a l i n e i n f u s e d f i s h showed plasma pH values which were not d i f f e r e n t from t h e i r r e s p e c t i v e p r e - i n f u s i o n c o n t r o l l e v e l s . Plasma pH of the a c i d i n f u s e d f i s h was not s i g n i f i c a n t l y d i f f e r e n t from va l u e s of the s a l i n e i n f u s e d f i s h . Plasma HCO3- c o n c e n t r a t i o n s i n s a l i n e i n f u s e d f i s h were v a r i a b l e and showed i n c r e a s i n g trends d u r i n g the 5, 15 and 40min sampling p e r i o d s ( F i g . 47 b ) . Mean v a l u e s , however, d e c l i n e d c l o s e to c o n t r o l l e v e l s by the 120min mark. Plasma HCO3- c o n c e n t r a t i o n s i n HC1 i n f u s e d f i s h d e c l i n e d i n the f i r s t 5min f o l l o w i n g i n f u s i o n and g r a d u a l l y i n c r e a s e d toward p r e - i n f u s i o n l e v e l s over the course of the experiment. While the 120min HCO3- c o n c e n t r a t i o n f o r a c i d - i n f u s e d f i s h was not s i g n i f i c a n t l y d i f f e r e n t from e i t h e r the p r e - i n f u s i o n l e v e l or the l e v e l f o r f i s h i n f u s e d with s a l i n e , the mean value was 0.56mM lower than the p r e - i n f u s i o n c o n t r o l value. There was no d i f f e r e n c e i n the Hct values between s a l i n e and a c i d i n f u s e d f i s h throughout the course of the experiment. Values i n both groups d e c l i n e d with sampling throughout the experiment ( F i g . 48). There were no 181 F i g u r e 47. Means + S.E. of a. Plasma pH and b. plasma HCO3 - c o n c e n t r a t i o n s i n rainbow t r o u t i n f u s e d with HC1 and s a l i n e i n the d o r s a l a o r t a v i a a c h r o n i c i n d w e l l i n g c a t h e t e r (at the 0 time mark which r e p r e s e n t s the end of a 5 m i n i n f u s i o n r o u t i n e ) over the time course o f the experiments. [HCC>3~] i n mM. 182 TIME (min) 183 gure 48. Means + S.E. of a. Hematocrit (%) of rainbow t r o u t i n f u s e d with HC1 and s a l i n e i n the d o r s a l a o r t a v i a a c h r o n i c i n d w e l l i n g c a t h e t e r (at the 0 time mark which r e p r e s e n t s the end of a 5min i n f u s i o n r o u t i n e ) over the time course of the experiments. 184 2 7 , 0 30 70 110 TIME (min) 185 s i g n i f i c a n t trends i n measured plasma i o n c o n c e n t r a t i o n s f o r the d u r a t i o n o f the course o f the experiment f o r e i t h e r s a l i n e or a c i d i n f u s e d f i s h (Table 5). No c o n s i s t e n t trends were observed i n any of the measured water i o n c o n c e n t r a t i o n s i n e i t h e r s a l i n e or a c i d i n f u s e d f i s h (Table 6). T r a n s e p t h e l i a l p o t e n t i a l values were h i g h l y v a r i a b l e f o r both s a l i n e and a c i d i n f u s e d f i s h throughout the experiment. R e l a t i v e to i t s own p r e - i n f u s i o n c o n t r o l v a l u e , there was a t r a n s i e n t i n c r e a s e i n TEP i n the f i r s t 15min f o l l o w i n g a c i d i n f u s i o n . TEP f o r a c i d i n f u s e d f i s h f l u c t u a t e d about val u e s fo r the s a l i n e i n f u s e d f i s h f o r the reminder o f the experiment. These l a t t e r values were not s i g n i f i c a n t l y d i f f e r e n t from the p r e - i n f u s i o n mean ( F i g . 49). Table 7 shows Nernst r a t i o s c a l c u l a t e d f o r Na +, C l - , K +, HC0 3 -, NH4+, C a + + , and Mg++ f o r each sampling p e r i o d throughout the experiment. The r e s u l t s were g e n e r a l l y v a r i a b l e and no s i g n i f i c a n t trends were observed f o r C l - , HCO3 -, K + and Mg + + r a t i o s . Na+ r a t i o s i n c r e a s e d above p r e - i n f u s i o n c o n t r o l values throughout the experiment ( F i g . 50 a ) . There was a r a p i d d e c l i n e i n NH4 + r a t i o i n the f i r s t 15min f o l l o w i n g a c i d i n f u s i o n a f t e r which the values remained s t a b l e to the end of the experiment ( F i g . 50 b ) . C a + + Nernst r a t i o s d e c l i n e d to l e v e l s below c o n t r o l values a t the 60 and 120min sampling p e r i o d s ( F i g . 51). Nernst r a t i o s f o r a l l ions are not equal to u n i t y . 186 Table 5. Means +. S.E. of plasma ion concentrations f or rainbow trout infused with a-HCKN = 8) and s - s a l ine (N = 5). A l l concentrations i n mM. ION CONTROL + 5min + 15min + 40min + 60min + 120min a-Na + s-Na+ a - C l ~ s - C l -a-K + s-K + a - C a + + s - C a + + a-Mg + + s-Mg + + a-NH 4 + 143.52 + 3.94 119.76 + 9.62 114.15 + 6.30 107.94 + 12.25 3.69 + 0. 35 4.93 + 0.38 1.24 + 0. 17 1.57 + 0.41 0.57 + 0.08 0.60 + 0.09 0.04 + 0.01 144.68 + 5.56 120.10 + 8.02 114.86 + 6. 27 110.41 + 12.78 3.48 + 0. 22 4.14 + 0. 37 1.08 + 0.10 1.35 + 0.26 0.56 + 0.06 0.55 + 0.05 0.04 + 0.01 149.97 + 5.81 121.07 + 8.83 111.16 + 5.23 110.20 + 12.30 3. 92 + 0.53 4.44 + 0.54 0.91 + 0.10 1.21 + 0.24 0.52 + 0.09 0.54 + 0.05 0.03 + 0.01 145.90 + 5.27 116.41 + 9.42 116.17 + 6.59 106.28 + 12.69 3. 38 + 0. 15 4.97 + 1.27 1.03 + 0.11 1.13 + 0.29 0.59 + 0.09 0.54 + 0.06 0.03 + 0.01 147.12 + 5.64 120.32 + 7.88 115.78 + 6.21 114.03 + 12.01 3.46 + 0. 15 4. 16 + 0.40 1.05 + 0.14 1. 18 + 0.26 0.58 + 0.07 0.55 + 0.04 0.04 + 0.01 149.03 + 4.16 118.57 + 7.64 111.16 + 5. 27 113.12 + 12.34 3.92 + 0.53 4. 15 + 0.39 0. 91 + 0.10 1. 16 + 0.25 0.52 + 0.09 0.59 0.04 0.03 + 0.01 187 Table 6. Means +_ S.E. of water ion concentrations for rainbow trout infused with a-HCKN = 8) and s-sa l i n e ( N = 5). A l l concentrations i n mM. ION CONTROL + 5min + 15min + 40min + 60min + 120min a-HC03" s-HC03-0.61 + 0.11 0. 27 + 0. 12 0.52 + 0.11 0. 22 + 0.05 0.57 + 0.11 0.43 + 0.23 0.57 + 0.10 0. 34 + 0. 14 0.51 + 0.12 0. 34 + 0.12 0.57 + 0.11 0.63 + 0. 32 »-Na + s-Na+ 0.89 + 0.06 0.90 + 0.07 0. 84 + 0.04 0.85 + 0.08 0.77 + 0.04 0.84 + 0.07 0.79 + 0.03 0.80 + 0.07 0.83 + 0.04 0. 85 + 0.08 0.77 + 0.04 0.84 + 0.07 a - C l -s-Cl" 0.92 + 0.04 0. 88 + 0.03 0.91 + 0.04 0. 85 + 0.02 0.90 + 0.05 0.85 + 0.04 0. 89 + 0.04 0. 85 + 0.05 0.09 + 0.04 0.85 + 0.05 0.90 + 0.05 0.85 + 0.04 a-K + 5-K+ 0.03 + 0.01 0.04 + 0.01 0.03 + 0.01 0.05 + 0.01 0.04 + 0.01 0.04 + 0.01 0.04 + 0.01 0.05 + 0.01 0.04 + 0.01 0.05 + 0.01 0.04 + 0.01 0.05 + 0.01 »-Ca + + s - C a + + 0.03 + 0.01 0.02 + 0.01 0.03 + 0.01 0.02 + 0.01 0.03 + 0.01 0.02 + 0.01 0.03 + 0.01 0.01 + 0.01 0.03 + 0.01 0.02 + 0.01 0.03 + 0.01 0.02 + 0.01 a-Mg s-Mg ++ 0.01 0.01 0.01 +. v a r i a b i l i t y undetectable 0.01 0.01 £ v a r i a b i l i t y undetectable 0.01 0.01 0.01 0.01 0.01 0.01 a-NH 4 +0.03 0.32 0.36 0.31 0.35 0.36 +0.13 +0.14 +0.15 + 0.13 + 0.15 + 0.15 188 Table 7. Means + S.E. of plasma r a t i o s of : Measured [ion] / Expected [ion] by the Nernst equation for t r o u t infused with HC1. ION CTRL. +5min +15min +40min +60min +120min Na + 82.682 84.475 95.660 90.071 100.221 108.297 + 10.861 + 8.113 + 9.187 + 6.662 + 11.696 + 11.712 CI" 226.910 261.204 254.701 271.775 266.697 250.564 + 28.901 + 21.257 + 25.862 + 34.589 + 39.165 + 31.741 K + 60.181 66.312 71.870 73.194 79.919 94.506 + 24.384 + 21.032 + 18.538 + 21.961 + 22.469 + 54.794 HC0 3- 24.934 29.144 33.043 30.043 22.956 28.542 +4.548 +4.970 + 5.312 + 6.694 + 4.379 + 7.212 NH4+ 0.108 0.080 0.060 0.060 0.064 0.061 + 0.020 + 0.020 + 0.010 + 0.010 + 0.010 + 0.009 C a + + 10.075 12.262 12.518 9.095 6.905 7.640 + 2.398 + 2.304 + 2.111 + 0.963 + 1.354 + 1.043 Mg + + 15.948 14.318 17.086 17.890 11.219 14.829 +1.266 +2.094 +4.272 +5.664 +2.915 +4.266 189 F i g u r e 49. Means + S.E. of t r a n s - e p i t h e l i a l p o t e n t i a l s (TEP) i n rainbow t r o u t i n f u s e d with 5ml*Kg x body weight of a 0.05 N HC1 s o l u t i o n made up i n 120mM p h y s i o l o g i c a l s a l i n e and the s a l i n e alone. TRANSEPITHELIAL POTENTIALS (TEP) 191 F i g u r e 50. Means +. S.E. of a. nernst r a t i o f o r plasma Na + i n t r o u t i n f u s e d with 5ml*Kg - 1 body weight 0.05 N HC1. F i l l e d c i r c l e i s the p r e - i n f u s i o n c o n t r o l p o i n t , b. As a. except t h a t i t p e r t a i n s to NH/j"1". 1 9 2 193 Fi g u r e 51. Means +. S.E. of the nernst r a t i o f o r plasma C a + + i n t r o u t i n f u s e d with 5ml*Kg -^ body weight 0.05 N HC1. F i l l e d c i r c l e i s the p r e - i n f u s i o n c o n t r o l p o i n t . 194 195 DISCUSSION These a c i d i n f u s i o n experiments demonstrate t h a t the i n c r e a s e d a c i d l o a d i n t r o u t d u r i n g a bout of anaerobic e x e r c i s e e l i c i t s a s i g n i f i c a n t r e l e a s e o f the catecholamines, a d r e n a l i n e and n o r a d r e n a l i n e . T h i s evidence suggests t h a t the r e l e a s e o f catecholamines i n f i s h i n response to a c i d o s e s (eg. Primmett ejb al.. 1986) i s , a t l e a s t p a r t i a l l y , due to the excess H + l o a d i n the c i r c u l a t o r y system. T h i s i n c r e a s e i n catecholamine c o n c e n t r a t i o n i n e x t r a c e l l u l a r f l u i d o f f i s h e s reduces the change i n e r y t h r o c y t i c pH and t h i s i n t u r n maintains the 02~Hb a f f i n i t y and s a t u r a t i o n o f the blood i n face o f the plasma a c i d o s i s . T h i s in. v i v o evidence supports the r e c e n t in. v i t r o study by Co s s i n s and Richardson (1985) which showed t h a t t h i s maintenance of 02~Hb a f f i n i t y was due to a t t e n u a t i o n o f the Bohr and Root s h i f t s shown by the blood of t r o u t . The p r o p o r t i o n a l r e l a t i o n s h i p between c i r c u l a t i n g catecholamines and the change i n plasma pH from c o n t r o l values f u r t h e r h i g h l i g h t s the apparent f u n c t i o n a l s i g n i f i c a n c e o f t h i s phenomenon i n the maintenance of blood 0 2-Hb a f f i n i t y and c a r r y i n g c a p a c i t y . The need f o r t h i s p r o t e c t i o n i s g r e a t e s t when the degree of a c i d o s i s , or d e l t a pHe, i s a l s o maximum. As expected, the nature of t h i s i n c r e a s e i n catecholamine c o n c e n t r a t i o n i s a t r a n s i e n t phenomenon. T h i s i s c o n s i s t e n t 196 with the acute s t r e s s response i n f i s h (Nakano and Tomlinson 1967) and a l s o with a v a i l a b l e data where t r a n s i e n t i n c r e a s e s i n catecholamine c o n c e n t r a t i o n s have been observed i n t r o u t d u r i n g a b u r s t swim (Primmett et. al.. 1986). The r e s u l t s of experiment IA i n S e c t i o n l . , i n which animals i n steady s t a t e c o n d i t i o n s of a c i d o s i s 24h a f t e r an a l t e r a t i o n i n v e n t i l a t o r y volume and i n which catecholamine l e v e l s were c o n s i s t e n t with r e s t i n g v a l u e s , a l s o suggests t h a t i n c r e a s e i n catecholamines i n response to an acid-base d i s t u r b a n c e i s a t r a n s i e n t phenomenon. While Holeton et. al.. (1983) showed a number of net changes i n plasma and water i o n c o n c e n t r a t i o n s i n response to metabolic a c i d o s i s induced i n e x c e r c i s e d t r o u t , no s i g n i f i c a n t changes i n i o n c o n c e n t r a t i o n s were observed i n the present experiments. The a c i d o s i s induced i n the two experiments i n t h i s S e c t i o n r e p r e s e n t about one h a l f the change i n pH demonstrated by other s t u d i e s (Holeton et_ al.. 1983 and Primmett et. al.. 1986) where metabolic a c i d o s e s have been e x p e r i m e n t a l l y induced; although the p a t t e r n of acid-base d i s t u r b a n c e was s i m i l a r to those s t u d i e s . Changes i n pH i n the order of 0.5 and 0.3 u n i t s from the c o n t r o l v a l u e s were recorded f o r the Holeton study above where t r o u t were prodded to exhaustion and where t r o u t were swum to exhaustion (Primmett e t al_. 1986), r e s p e c t i v e l y . The i n s u l t i n our study may have been too m i l d to induce net i o n changes i n a magnitude that c o u l d be d e t e c t e d i n the water, g i v e n the 197 f i s h / w a t e r volume r a t i o . I t i s a l s o p o s s i b l e that the acid-base d i s t u r b a n c e t h a t was induced e f f e c t e d changes i n t r a n s e p i t h e l i a l i o n f l u x e s which were o f f s e t by other compensatory i o n exchanges caused by some aspect o f the treatment, such as the i n c r e a s e i n c i r c u l a t i n g catecholamines. The i n c r e a s e s i n the Nernst r a t i o s f o r Na + d u r i n g the experiment i n d i c a t e t h a t there was a s t i m u l a t i o n o f the a c t i v e uptake of Na + with a c i d i n f u s i o n . On t h i s b a s i s , the f a c t t h a t there were no s i g n i f i c a n t net changes i n [Na +] i n the blood suggests t h a t the p e r m e a b i l i t y o f the g i l l e p i t h e l i u m i n c r e a s e d to match i n c r e a s e d a c t i v i t y o f the i o n pump. This proposed process c o u l d t h e o r e t i c a l l y reduce the excess H + l o a d o f the plasma through a Na +/H +(NH4 +) exchange acro s s the g i l l e p i t h e l i u m . There i s evidence f o r a Na +/NH4 + exchange process i n f i s h (Maetz and Garcia-Romeu 1964, Evans 1977, Payan 1978) and Maetz (1973) suggested t h a t NH4 + and H + may compete f o r s i m i l a r s i t e s on a t r a n s p o r t v e h i c l e i n exchange f o r Na +. Such a s t i m u l a t i o n has been r e p o r t e d f o r t r o u t i n response to a d r e n a l i n e through the s t i m u l a t i o n of beta a d r e n e r g i c r e c e p t o r s (Payan e £ al.. 1975; G i r a r d and Payan 1977; Payan 1978). The reduced Nernst r a t i o f o r NH4 + means t h a t i t was being a c t i v e l y pumped out of the animal a g a i n s t the e x i s t i n g e l e c t r o c h e m i c a l g r a d i e n t s and i t supports the p o s s i b i l i t y t h a t t h i s a c t i v e exchange process was being s t i m u l a t e d through the a c t i o n o f catecholamines i n response to 198 the a c i d i n f u s i o n . Two c o n d i t i o n s must accompany the above hy p o t h e s i s of an a c t i v e Na +/H +(NH4 +) exchange p l a y i n g a major r o l e i n the r e d u c t i o n of H + from the blood of t r o u t a f t e r a c i d i n f u s i o n ; that i s the i n c r e a s e i n g i l l p e r m e a b i l i t y to Na + and the concomitant i n c r e a s e i n the e f f l u x of an anion or an i n f l u x of another c a t i o n to maintain e l e c t r i c a l n e u t r a l i t y . I s a i a e i . al.. (1978) demonstrated t h a t there was an i n c r e a s e i n the g i l l p e r m e a b i l i t y to water i n response to catecholamines. T h i s suggests t h a t there was a g e n e r a l i n c r e a s e i n p e r m e a b i l i t y to ions as w e l l . The analyses i n S e c t i o n 3. suggested t h a t Na + was more permeable than C l - a c r o s s the t r o u t g i l l e p i t h e l i u m . The proposed s t i m u l a t i o n of the c a t i o n exchange c o u l d have taken p l a c e , assuming a general i n c r e a s e i n p e r m e a b i l i t y and t h i s d i f f e r e n t i a l between PNa + and P C I - . The p e r m e a b i l i t y o f the g i l l to C l - would a l s o have i n c r e a s e d , but the a c t i v e uptake of t h i s i o n to compensate the i n c r e a s e d leak may have had to be minimal g i v e n the m i l d degree of a c i d o s i s i n g e n e r a l . Given the l a c k o f evidence t h a t C l - was i n v o l v e d f o r charge balance i n t h i s proposed hy p o t h e s i s , i t i s p o s s i b l e that C a + + might have been taken from the water a g a i n s t i t s e l e c t r o c h e m i c a l g r a d i e n t to balance the negative c a t i o n balance i n the blood ( F i g . 51). GENERAL DISCUSSION 200 GENERAL DISCUSSION F i s h r e g u l a t e blood pH i n response to an acid-base d i s t u r b a n c e (see H e i s l e r 1985 f o r review). That i s , pH i s r e t u r n e d toward r e s t i n g l e v e l s over time e i t h e r f o l l o w i n g a s h o r t - t e r m p e r t u r b a t i o n such as a b u r s t swim or d u r i n g a prolonged treatment such as the exposure to environmental hypercapnia. A s t i m u l a t i o n of v e n t i l a t i o n as w e l l as i o n exchange processes a c r o s s the g i l l e p i t h e l i u m c h a r a c t e r i s e the response of f i s h e s to a c i d o t i c c o n d i t i o n s . Acid-base d i s t u r b a n c e s a l s o e l i c i t a g e n e r a l i z e d s t r e s s response and s e v e r a l a c i d o t i c c o n d i t i o n s have been shown to r a i s e l e v e l s of catecholamines above r e s t i n g l e v e l s i n f i s h (Primmett e t a l . 1986; Perry 1986). The r e s u l t s o f the s t u d i e s i n t h i s t h e s i s c o n f i r m these general statements r e g a r d i n g the response o f f i s h to acid-base d i s t u r b a n c e s and d e s c r i b e some s p e c i f i c p rocesses which r e s u l t i n the r e s t o r a t i o n o f the acid-base s t a t u s of the blood. The data from the experiments r e p o r t e d i n t h i s t h e s i s suggest t h a t there i s a g r e a t e r p o t e n t i a l f o r r e g u l a t i n g the acid-base s t a t u s of the blood i n f i s h e s through i o n exchange processes between blood and water r a t h e r than through r e g u l a t i n g the Pco2 t e n s i o n of the blood by adjustments of v e n t i l a t i o n volume. While v e n t i l a t i o n i n c r e a s e s i n response to a c i d 201 c o n d i t i o n s , the l i m i t e d range f o r a d j u s t i n g the Pco2 t e n s i o n i n the blood reduces i t s p o t e n t i a l i n c o r r e c t i n g acid-base d i s t u r b a n c e s . The f i r s t experiment i n S e c t i o n 1. showed t h a t i n steady s t a t e c o n d i t i o n s , Pco2 can o n l y be i n c r e a s e d by about 2 mmHg by r e d u c i n g v e n t i l a t i o n below normal l e v e l s . Pco2 t e n s i o n s probably r i s e i n any a c i d o s i s . T h i s w i l l occur d u r i n g exposure to environmental hypercapnia due to the d i f f u s i o n o f CO2 from water to blood. An excess l o a d of H + ions of a m e tabolic o r i g i n w i l l a l s o t i t r a t e blood HCO3 - to molecular C 0 2 and thereby i n c r e a s e PC02 t e n s i o n s . The f u n c t i o n a l r o l e of the s t i m u l a t i o n of v e n t i l a t i o n i n a c i d o t i c c o n d i t i o n s i s l i m i t e d to those circumstances where the Pcx>2 i s endogenous, such as i n the case o f HCO3 - t i t r a t i o n by H + from metabolism. The PC02 t e n s i o n might be reduced i n t h a t case toward normal valu e s by i n c r e a s e d g i l l water flow. A l k a l o t i c c o n d i t i o n s might a l s o be c o r r e c t e d by r e d u c i n g v e n t i l a t i o n and thereby l i m i t i n g CO2 e x c r e t i o n . Given t h i s l i m i t e d range f o r a d j u s t i n g C 0 2 e x c r e t i o n by v e n t i l a t i o n , the p o s s i b i l i t y becomes a t t r a c t i v e t h a t the f u n c t i o n a l s i g n i f i c a n c e of the s t i m u l a t i o n of v e n t i l a t i o n i n response to a c i d c o n d i t i o n s i s the enhancement of oxygen uptake to o f f s e t Bohr and Root s h i f t s . While the s e n s i t i v i t y o f v e n t i l a t i o n i n f i s h to oxygen i s w e l l documented, t h i s simple r u l e has many ex c e p t i o n s . Elasmobranchs which i n c r e a s e v e n t i l a t i o n under a c i d c o n d i t i o n s have blood which does not 202 e x h i b i t e i t h e r Bohr or Root s h i f t s . C y p r i n i d s have been r e p o r t e d to show l i t t l e v e n t i l a t o r y response to exposure to hypercapnia while t h e i r blood shows Bohr and Root s h i f t s . I t has a l s o been shown t h a t the r e d u c t i o n i n blood oxygen c a r r y i n g c a p a c i t y i n a c i d o t i c c o n d i t i o n s can be o f f s e t by catecholamines ( B o u t i l i e r §_t al_. 1986, C o s s i n s and Richardson 1986) which are r e l e a s e d i n f i s h under such c o n d i t i o n s ( B o u t i l i e r e t al_. 1986, Primmett §_t al.. 1986, P e r r y 1986, i n p r e p . ) . The mechanism by which t h i s e f f e c t i s o f f s e t i s the a l k a l i n i z a t i o n o f the e r y t h r o c y t e c y t o s o l . The a c i d i n f u s i o n study i n S e c t i o n 4. demonstrated t h i s e f f e c t in. v i v o by showing no s i g n i f i c a n t r e d u c t i o n i n blood oxygen c a r r y i n g c a p a c i t y i n s p i t e of a plasma a c i d o s i s . The s t i m u l a t i o n of v e n t i l a t i o n i n a c i d o t i c c o n d i t i o n s i s , t h e r e f o r e , not c l e a r l y a t t r i b u t e d to r e d u c t i o n s i n the blood oxygen c a r r y i n g c a p a c i t y as might be demonstrated i n v i t r o . While i n v i v o s t u d i e s have shown an a t t e n u a t i o n of t h i s e f f e c t with h y p e r o x i c c o n d i t i o n s (Smith and Jones 1982), some r e s i d u a l s e n s i t i v i t y to the a c i d o t i c c o n d i t i o n seems to e x i s t . The v e n t i l a t i o n study of the d o g f i s h i n S e c t i o n 1 showed th a t the s t i m u l a t i o n o f v e n t i l a t i o n was most s e n s i t i v e to blood pH. Given the l a c k o f Bohr and Root s h i f t s and the e q u i v o c a l nature of the d i r e c t r e l a t i o n s h i p between v e n t i l a t o r y s t i m u l a t i o n under a c i d c o n d i t i o n s and the Bohr and Root s h i f t s shown by t e l e o s t blood, i t i s p o s s i b l e t h a t a s e n s i t i v i t y o f v e n t i l a t i o n to the a c t u a l acid-base s t a t u s of 203 the blood i n t e l e o s t s e x i s t s . Some i n c o n s i s t e n c i e s i n the p u b l i s h e d data must, however, be r e c o n c i l e d . One obvious q u e s t i o n i s why r a i s i n g the oxygen content o f water would attenuate the i n c r e a s e d v e n t i l a t i o n i n t r o u t exposed to hypercapnic c o n d i t i o n s (Smith and Jones 1982) when i t has been shown t h a t catecholamines o f f s e t the Bohr and Root s h i f t s i n  v i v o ( B o u t i l i e r et: a l . 1985; Primmett §_t al.. 1986) and i n  v i t r o (Cossins and Richardson 1986) and t h a t catecholamines are r e l e a s e d i n t h a t s p e c i e s exposed to environmental hypercapnia (Perry 1986). The p o t e n t i a l f o r c o r r e c t i n g acid-base d i s t u r b a n c e s i n g e n e r a l and the a c i d o t i c c o n d i t i o n s r e p o r t e d i n the experiments i n t h i s t h e s i s i s c l e a r l y g r e a t e r through adjustments of i o n exchange processes between blood and water than through changes i n g i l l water flow. With a few e x c e p t i o n s (Perry et. al.. 1981b., K e r s t e t t e r and Mize 1976) net changes i n i o n t r a n s f e r r a t e s (Cameron 1976, carp experiment i n t h i s t h e s i s ) and blood i o n c o n c e n t r a t i o n s ( C l a i b o r n e and H e i s l e r 1984, Holeton et. al.. 1983, conger and t r o u t experiments i n t h i s t h e s i s ) change i n f i s h i n v a r i o u s s i t u a t i o n s of a c i d o s e s . T r a n s e p i t h e l i a l i o n t r a n s f e r s seem to predominate over a r e n a l f u n c t i o n i n t h i s regard (see review by H e i s l e r 1985). The accumulation o f blood HC03~ c h a r a c t e r i z e s the r e c o v e r y o f blood pH a f t e r the i n i t i a l f a l l i n a c i d o t i c c o n d i t i o n s . In the t r o u t , conger and c a r p , a c t i v e C1 -/HC03~ 204 exchange processes between the blood and water were observed to reduce blood C l - and i n c r e a s e blood HCO3 -c o n c e n t r a t i o n s i n response to environmental hypercapnia. T h i s i s c o n s i s t e n t with the r e s u l t s o f other s t u d i e s where f i s h have been exposed to environmental hypercapnia ( L l o y d and White 1967; Cross e t aJL. 1969; Cameron and Rand a l l 1975; Janssen and Randall 1975; H e i s l e r e t al.. 1976; Borjeson 1976; Ran d a l l e £ al.. 1976; Bubien and Meade 1979; H e i s l e r §_t a l . 1980; C l a i b o r n e and H e i s l e r 1984). These i o n i c exchanges r e s u l t e d i n plasma pH valu e s r e t u r n i n g towards c o n t r o l v a l u e s . I t i s c l e a r from the experiments i n t h i s t h e s i s t h a t a c t i v e r a t h e r than p a s s i v e processes were i n v o l v e d i n the accumulation o f plasma HCO3 - through exchange processes between blood and water with C l - . The u n i d i r e c t i o n a l i o n f l u x study with the carp showed t h a t i t was an i n h i b i t i o n o f the a c t i v e C l - uptake from the water which e f f e c t e d the net changes i n t h a t i o n and the a s s o c i a t e d accumulation o f plasma HCO3 - i n response to hypercapnia. I t i s l i k e l y t h a t s i m i l a r processes were prese n t i n conger and t r o u t under f r e s h water or near f r e s h water c o n d i t i o n s . T h i s i n h i b i t i o n can occur r a p i d l y over a few hours and s i n c e t h i s mechanism o f C l - uptake i s r e p o r t e d l y p r e s e n t on the r e s p i r a t o r y c e l l s o f the g i l l e p i t h e l i u m ( G i r a r d and Payan 1977), i t i s l i k e l y t h a t t h i s c o u l d have o c c u r r e d even i n the conger which were tempered f o r onl y three hours i n f r e s h water. I t was concluded from the experiments i n S e c t i o n 2 on t r o u t t h a t 205 a c t i v e processes must have e f f e c t e d accumulation o f plasma HCC>3~ i n f i s h a c c l i m a t e d to 100 mM and 300 mM NaCl. I t seemed most l i k e l y t h a t t h i s i n v o l v e d the s t i m u l a t i o n o f C l ~ e f f l u x f o r HCO3 - uptake through t r a n s p o r t mechanisms a l r e a d y present on the g i l l r a t h e r than involvement o f c h l o r i d e c e l l r e c r u i t m e n t or s y n t h e s i s o f new t r a n s p o r t p r o t e i n s p e c i f i c a l l y f o r t h i s process. T h i s h y p o t h e s i s might a l s o be extended to i n c l u d e the conger e e l which a l s o showed a graded response i n recover y from hypercapnic a c i d o s i s a c c o r d i n g to environmental s a l i n i t y . F u r t h e r support f o r the a c t i v e nature o f the C1~/HC03~ exchange a t a l l three s a l i n i t i e s i n v e s t i g a t e d i n the t r o u t study was seen i n the Nernst r a t i o a n a l y s i s f o r these i o n s . The Nernst r a t i o s f o r C l ~ were higher a t 3 mM than those f o r f i s h i n 100 mM and 300 mM whereas Nernst r a t i o s f o r HCO3 - was lower i n f i s h a t 3 mM than those f o r 100 mM and 300 mM. While net changes i n plasma i o n c o n c e n t r a t i o n s were not observed i n t r o u t i n f u s e d with a c i d , as r e p o r t e d i n S e c t i o n 4, Nernst r a t i o a n a l y s i s suggested t h a t there might have been an a c t i v e Na +/H +(NH4 +) exchange mechanism working to reduce the H + l o a d o f the plasma, and thereby c o r r e c t i n g blood pH. I t i s hypothesized t h a t t h i s a c t i v e uptake o f Na + from water to blood was matched with an e q u i v a l e n t i n c r e a s e i n a p a s s i v e leak f o r t h i s i o n , s i n c e a net accumulation o f Na + was not observed i n the plasma. There was general agreement between p e r m e a b i l i t y estimates 206 of the g i l l and degree o f a c t i v e net f l u x f o r Na + and C l ~ . Under steady s t a t e c o n d i t i o n s i n t r o u t p r i o r to exposure to environmental hypercapnia, there was good agreement between the estimate f o r PNa +/PCl~ of 2.5 to the Nernst r a t i o f o r Na + which was 1.5 to 2.0 times g r e a t e r than those r a t i o s f o r C l ~ a t a l l three s a l i n i t y l e v e l s . The apparent p e r m e a b i l i t y of C l ~ was lower than t h a t f o r Na + over the 3-100 mM s a l i n i t y range i n t r o u t d u r i n g exposure to hypercapnia. This a l s o agreed with the o b s e r v a t i o n i n carp t h a t i n f r e s h water, C l ~ i n f l u x was reduced i n response to exposure to environmental hypercapnia. Both the decrease i n the i n f l u x r a t e s g i v e n normal e f f l u x r a t e s , would r e s u l t i n the observed net r e d u c t i o n i n plasma C l ~ c o n c e n t r a t i o n . The i n v i v o r e l e a s e of catecholamines i n t r o u t i n response to a c i d c o n d i t i o n s has been r e p o r t e d by Perry (1986) f o r exposure to hypercapnia, by B o u t i l i e r et. aJL- (1986) and by Primmett et_ al.. (1986) f o r b u r s t e x e r c i s e . The i n c r e a s e d h e m a t o c r i t and d e p o l a r i z i n g t r e n d i n the t r o u t exposed to hypercapnia a t a l l three s a l i n i t i e s a l s o p o i n t e d to the r e l e a s e of catecholamines. The i n f u s i o n of a c i d i n the t r o u t , r e p o r t e d i n S e c t i o n 4, a l s o showed an i n c r e a s e i n catecholamines i n p r o p o r t i o n to the change i n plasma pH. These r e s u l t s should be expected s i n c e a s i g n i f i c a n t change i n the acid-base s t a t u s o f the blood p e r t u r b s many p h y s i o l o g i c a l f u n c t i o n s which i n c l u d e c a r d i o v a s c u l a r and i o n t r a n s p o r t systems. An i n h i b i t i o n of C1 -/HC03~ exchange (Perry et_ 207 a l . 1984) and an i n c r e a s e i n Na +/H +(NH4 + ) exchange through beta r e c e p t o r s t i m u l a t i o n (Payan e t al_. 1975; G i r a r d and Payan 1977; Payan 1978) has been r e p o r t e d f o r the f i s h g i l l . T h i s may e x p l a i n i n h i b i t i o n o f C1~/HCC>3~ exchange seen i n the carp i n f r e s h water exposed to hypercapnia, although an a c t u a l i n c r e a s e i n catecholamines i n t h a t s p e c i e s under s i m i l a r c o n d i t i o n s has not been r e p o r t e d . While s t i m u l a t i o n o f Na +/H + exchange was suggested f o r a c i d - i n f u s e d t r o u t i n S e c t i o n 4 where catecholamine l e v e l s were shown to be high, such a s t i m u l a t i o n i n the t r o u t study of S e c t i o n 2 was not seen. There i s a p o s s i b i l i t y t h a t i t was present and masked as i n the a c i d - i n f u s e d t r o u t . Nernst r a t i o s f o r Na + i n t r o u t exposed to environmental hypercapnia s i g n i f i c a n t l y d e v i a t e d from u n i t y a t a l l sampling times and i n c r e a s e d d u r i n g hypercapnia, e s p e c i a l l y a t the 100 mM and 300 mM l e v e l s . There was no c l e a r agreement i n the PNa +/PCl~ data with t h i s p o s s i b l e s t i m u l a t i o n o f net Na + f l u x between blood and water. The Na + - C l ~ would have been expected to i n c r e a s e a t these s a l i n i t i e s , s p e c i f i c a l l y due to an in c r e a s e i n the PNa + component over P C I - i n order to mask any net accumulation o f Na + i n the blood as a r e s u l t o f i n c r e a s e d Na + uptake. Another known e f f e c t o f catecholamines i s the i n h i b i t i o n of Na + and C l ~ f l u x e s from c h l o r i d e c e l l s by an alpha and beta r e c e p t o r s t i m u l a t i o n ( P i c ejt al.. 1975; G i r a r d 1976; Payan and G i r a r d 1978; Shuttleworth 1978; Degnan §_t_ ai.. 1977). T h i s 208 supports one c o n c l u s i o n of S e c t i o n 2 t h a t the a c t i v e e f f l u x of C l ~ from blood to water t h a t e f f e c t e d the HCO3-accumulation i n t r o u t a t 100 mM and 300 mM s a l i n i t i e s was not due to the r e c r u i t m e n t o f c h l o r i d e c e l l s . The l i k e l y presence o f e l e v a t e d catecholamines would have i n h i b i t e d C l ~ e f f l u x r a t h e r than s t i m u l a t e d i t a t the h i g h s a l i n i t i e s . T h i s t h e r e f o r e , suggests t h a t the s e t of mechanisms t h a t was a c t u a l l y r e s p o n s i b l e f o r the i n c r e a s e d C l ~ e f f l u x and consequent HCO3- uptake would have had to overcome t h i s i n h i b i t i o n of C l ~ e f f l u x from the c h l o r i d e c e l l s and e f f e c t the net r e d u c t i o n i n plasma c h o r i d e c o n c e n t r a t i o n . T h i s a l s o suggests e i t h e r t h a t there are l o c a t i o n s f o r the a n i o n - s t i m u l a t e d ATPase on s i t e s other than the r e p o r t e d one on the c h l o r i d e c e l l or t h a t other mechanisms f o r a c t i v e C l -e f f l u x b e s i d e s c h l o r i d e c e l l s e x i s t . I t should a l s o be noted t h a t the r e s u l t s o f experiments on the e f f e c t s o f catecholamines on i o n t r a n s p o r t a c r o s s b i o l o g i c a l b a r r i e r s are not c o n s i s t e n t . For example, the i n h i b i t i o n o f C l ~ t r a n s p o r t by catecholamines has been r e p o r t e d f o r the r a b b i t c o l o n by Halm e t aJL.. (1983) but as t h a t author p o i n t s out, t h a t r e s u l t seems anomalous s i n c e beta a d r e n e r g i c a g o n i s t s are b e l i e v e d to a c t through c y c l i c AMP (cAMP) and exogenous cAMP i s a s t i m u l a n t of c o l o n i c C I ~ t r a n s p o r t i n the r a t ( F o s t e r §_t al.. 1983). While i t i s p o s s i b l e that a c t i o n s o f a secondary messenger such as C a + + i s s t i m u l a t i n g the C l ~ t r a n s p o r t i n the presence o f 209 exogenous cAMP and thus masking the beta a d r e n e r g i c e f f e c t , the r e s u l t s are not d e f i n i t i v e . While the data s e t on the movements o f H +, HC03 - and NH-4+ a c r o s s the f i s h g i l l e p i t h e l i u m shows t h a t these movements are modulated d u r i n g r e c o v e r y from acid-base d i s t u r b a n c e s and t h e i r l i n k to the movements of Na + and C l - are w e l l documented, the f a c t o r s d etermining these exchange processes are not well understood. S t u d i e s on acid-base r e g u l a t i o n i n f i s h e s have been l a r g e l y d e s c r i p t i v e and inroads are s t a r t i n g to be made, such as i n the e f f e c t of catecholamines on these exchange pr o c e s s e s , i n t o the mechanisms by which these movements occur. The l i n k a g e o f these two groups of ions p l a c e s obvious demands on the r e g u l a t o r y processes i n t h a t both osmoregulation and acid-base r e g u l a t i o n are a f f e c t e d by these i o n t r a n s f e r s . 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R e s p i r a t o r y c o n t r o l o f a r t e r i a l pH as temperature changes i n rainbow t r o u t Salmo  q a i r d n e r i . Am. J . P h y s i o l . 225, 997-1002. R a n d a l l , D.J., N. H e i s l e r and F. Drees. 1976. V e n t i l a t o r y response to hypercapnia i n the l a r g e r s p o t t e d d o g f i s h S c v l i o r h i n u s s t e l l a r i s . Am. J . P h y s i o l . 230, 590-594. R a n d a l l , D.J. and D.R. Jones. 1973. The e f f e c t o f d e a f f e r e n t a t i o n o f the pseudobranch on the r e s p i r a t o r y response to hypoxia and hyperoxia i n the t r o u t (Salmo  q a i r d n e r i ) . R e s p i r . P h y s i o l . 17, 291-301. Ri c h a r d s , B.D. and P.O. Fromm. 1969. P a t t e r n s o f blood flow through f i l a m e n t s and l a m e l l a e o f i s o l a t e d p e r f u s e d t r o u t g i l l s . Am. Z o o l . 8, 766-771. 220 Saunders, R.L. 1962. The i r r i g a t i o n o f the g i l l s i n f i s h e s . I I . E f f i c i e n c y o f oxygen uptake i n r e l a t i o n to r e s p i r a t o r y flow a c t i v i t y and c o n c e n t r a t i o n s o f oxygen and carbon d i o x i d e . Can. J . Zo o l . 40, 817 -862. Shaw, J . 1960. A b s o r b t i o n o f sodium ions by the c r a y f i s h Astacus p a l l i p e s L. I I I . E f f e c t o f other c a t i o n s i n the e x t e r n a l s o l u t i o n . J . Exp. B i o l . 37, 548-556. She l t o n , G., D.R. Jones and W.K. Milsom. 1986. C o n t r o l o f b r e a t h i n g i n e c t o t h e r m i c v e r t e b r a t e s . In: Handbook of  Ph y s i o l o g y . p857-909. Shutt l e w o r t h , T.J. 1978. The e f f e c t o f a d r e n a l i n e on p o t e n t i a l s i n the g i l l s o f the fl o u n d e r ( P l a t i c h t h v s f l e s u s L . ) . J . Comp. P h y s i o l . 124, 129-136. Simon, B., R. Kinne and G. Sachs. 1972. The presence o f a HC03~-ATPase i n p a n c r e a t i c t i s s u e . Biochim. Biophys. Acta. 282, 293-300. Smith, F.M. and D.R. Jones. 1982. The e f f e c t o f changes i n blood oxygen-carrying c a p a c i t y on v e n t i l a t i o n volume i n rainbow t r o u t (Salmo g a i r d n e r i ) . J . exp. B i o l . 97, 325-334. Smith, L.S. and G.R. B e l l . 1964. A technique f o r prolonged blood sampling i n freeswimming salmon. J . F i s h . Res. Bd. Canada. 23, 1439-1446. Snieszko, S.F. 1960. Microhematocrit as a t o o l i n f i s h e r y r e s e a r c h and management. U.S. F i s h W i l d l . Serv. Spec. S c i . Rep.-Fish. 341, 15p. S o i v i o , A., Westman, K. and Nyholm, K. 1972. Improved method of d o r s a l a o r t a c a t h e t e r i z a t i o n : h aematological e f f e c t s f o l l o w e d f o r three weeks i n rainbow t r o u t (Salmo  g a i r d n e r i ) . F i n n i s h F i s h Res. 1, 11-21. S o i v i o , A., M. Nikinmaa, K. Nyholm and K. Westman. 1981. the r o l e o f g i l l s i n the responses o f Salmo g a i r d n e r i d u r i n g moderate hypoxia. Comp. Biochem. P h y s i o l . 70A, 133-139. Toews, D.P., Holeton, G.F. and H e i s l e r , N. 1983. R e g u l a t i o n o f the acid-base s t a t u s d u r i n g environmental hypercapnia i n the marine t e l e o s t f i s h Conger conger. J . Exp. B i o l . 107, 9-20. U l t s c h , G.R. and D.S. Antony. 1973. The r o l e o f a q u a t i c exchange of carbon d i o x i d e i n the ecology o f the water h y a c i n t h , E i c h o r n i a c r a s s i p e s . F l o r i d a S c i e n t . 36, 16-22. 221 Van Dam, L. 1938. On the u t i l i z a t i o n o f oxygen and r e g u l a t i o n of b r e a t h i n g i n some a q u a t i c animals. Ph.D. T h e s i s . U n i v e r s i t y o f Groningen, Groningen, The Netherlands. Van Os, C.H., A.K. M i r c h e f f and E.M. Wright. 1977. D i s t r i b u t i o n o f b i c a r b o n a t e - s t i m u l a t e d ATPase i n r a t i n t e s t i n e e p i t h e l i u m . J . C e l l B i o l . 73, 257-260. Wiebelhaus, V.D., C P . Sung, H.F. Helander, G. Shah, A.L. Blum and G. Sachs. 1971. S o l u b i l i z a t i o n o f anion ATPase from Necturus o x y n t i c c e l l s . Biochim. Biophys. Acta. 241, 49-56. Wolf, K. 1963. P h y s i o l o g c a l s a l i n e s f o r freshwater t e l e o s t s . Prog. F i s h C u l t . 25, 135-140. Wood, CM. 1974. A c r i t i c a l examination o f the p h y s i c a l and a d r e n e r g i c f a c t o r s a f f e c t i n g blood flow through the g i l l s o f rainbow t r o u t . J . Exp. B i o l . 60, 241-265. Wood, CM. 1975. A pharmacological a n a l y s i s o f the a d r e n e r g i c and c h o l i n e r g i c mechanisms r e g u l a t i n g b r a n c h i a l v a s c u l a r r e s i s t a n c e i n the rainbow t r o u t (Salmo q a i r d n e r i ) . Can. J . Zo o l . 53, 1569-1577. Wood, CM. and E.B. Jackson. 1980. Blood acid-base r e g u l a t i o n d u r i n g environmental hyperoxia i n the rainbow t r o u t Salmo  q a i r d n e r i . R e s p i r . P h y s i o l . 42, 351-372. Wood, CM., M.G. Wheatly and H. Hobe. 1984. The mechanisms of acid-base and i o n o r e g u l a t i o n i n the freshwater rainbow t r o u t d u r i n g environmental hyperoxia and subsequent normoxia. I I I . B r a n c h i a l exchanges. R e s p i r . P h y s i o l . 55, 175-192. Woodward, J . J . 1982. Plasma catecholamines i n r e s t i n g t r o u t , Salmo q a i r d n e r i Richardson, by high pressure l i q u i d chromatography. J . F i s h . B i o l . 21, 429-432. Wright, P. A. and CM. Wood. 1985. an a n a l y s i s o f b r a n c h i a l ammonia e x c r e t i o n i n the freshwater rainbow t r o u t : e f f e c t s of environmental pH change and sodium uptake blockade. J . Exp. B i o l . 114, 329-353. Z e i d l e r , R. and Kim, H.D. 1977. P r e f e r e n t i a l hemolysis of p o s t n a t a l c a l f red c e l l s induced by i n t e r n a l a l k a l i n i z a t i o n . J . Gen. P h y s i o l . 70, 385-401. 222 APPENDICES 223 APPENDIX I. Table A . l . Ionic concentratioi various s a l i n i t i e s i n experimen purchased from Wiegandt GmbH & F.R.G. A l l concentrations i n mM. ION CONCENTRATION Na + 468.0 K + 10.0 Mg + + 53.3 C a + + 10.4 Sr 0.2 F 0.07 is for the sea s a l t used to make up the 2A. i n Section 2. These s a l t s were Co., Sterkenhofweg 13, D-4150 K r e f e l d 1, ION CONCENTRATION C l " 545.7 B r - 0.8 I~ 0.003 S0 4- 28.1 H C O 3 - 2.4 H 3 B O 3 25 224 APPENDIX I I . NERNST RATIO CALCULATIONS RT [cationlw or [ a n i o n l p l TEP = — * In ZF [ c a t i o n l p l or [anionlw TEP i n v o l t s ; Concentrations i n moles; In = na t u r a l l og To c a l c u l a t e what the plasma c a t i o n concentration, f o r example, would be given the TEP and [cationlw and assuming the co n d i t i o n s f or the Nernst equation, e-TEP/(RT/ZF) = [cationlw / [ c a t i o n ] p l [ c a t i o n l p l * = (cationlw / e ~ T E P / ( R T / 2 F ) NERNST RATIO = [ c a t i o n l p l measured / I c a t i o n ] p l expected* 225 APPENDIX I I I . PERMEABILITY CALCULATIONS RT PNa +[Na +]w + P C l - [ C l _ ] p l + P H C 0 3 - [ H C 0 3 ~Jpl TEP = — * In ZF PNa +[Na +]pl + PCI - [Cl -]w + PHC0 3 _ [HC0 3 -]w C = e*< T E p/ R T/ Z F ,> C (PNa+[Na + ]pl + P C l _ [ C l - ) i » + PHC0 3 _ [HC0 3 _ ) w ) * (PNa +tNa +]w + P C l - t C l " ] p l + P H C 0 3~lH C 0 3 -]pl) d i v i d e by P H C 0 3 -C(PNa + /PHC0 3~[Na +]pl + PCl _ /PHC0 3-{Cl-]w + [HC03-Jw) = (PNa +/PHC0 3-[Na +]w + P C l - / P H C 0 3 - [ C l _ ] p l + [HC0 3~]pl) set to 0 PNa + /PHC0 3 _(C[Na +]pl - [Na+]w) + PCl - /PHC0 3-(C[Cl-]w - tCl - ] p l ) + (C[HC03"]w - [HC0 3-]pD - 0 PNa + /PHC0 3 _(C[Na +]pl - [Na +]tO + PCl _ / PHC0 3-(CfCl - J i » - [Cl-Jpl) -- (C[HC0 3 -]w - (HC0 3-Jpl) SOLVED FOR PNa + /PHC0 3 - & P C 1 - / P H C 0 3 _ FOR 2 SALINITIES 3 EXPERIMENTAL SALINITIES ALLOWED 3 UNIQUE SALINITY PAIRS WHICH ALLOWED 3 ESTIMATES OF EACH OF THESE PARAMETERS PNa +/PCl - •> PNa + /PHC0 3 _ / P C 1 - / P H C 0 3 _ THESE ESTIMATES WERE MADE FOR EACH SAMPLING TIME THROUGH THE EXPERIMENT PUBLICATIONS Heming, T . A . , D.J. R a n d a l l , R.G. B o u t i l i e r , G.K. Iwama and D.N. Primmett. 1985. I o n i c e q u i l i b r i a i n red blood c e l l s o f rainbow t r o u t . In Press i n R e s p i r a t i o n Physiology. Iwama, G.K., G.L. Greer and D.J. R a n d a l l . 1986. Changes i n s e l e c t e d hematological parameters i n j u v e n i l e Chinook, salmon su b j e c t e d to a b a c t e r i a l c h a l l e n g e and a t o x i c a n t . J . F i s h B i o l . 28, 563-572. B o u t i l i e r , R.G., G.K. Iwama and D.J. R a n d a l l . 1986. Acute e x t r a c e l l u l a r a c i d o s e s promote catecholamine r e l e a s e i n rainbow t r o u t (Salmo g a i r d n e r i ) : i n t e r a c t i o n s between red c e l l pH and 02~Hb c a r r y i n g c a p a c i t y . J . Exp. B i o l . 123, 145-157. B o u t i l i e r , R.G., G.K. Iwama, T.A. Heming and D.J. R a n d a l l . 1985. The apparent pK of c a r b o n i c a c i d i n rainbow t r o u t blood plasma between 5 and 15 C. Resp. P h y s i o l . 61, 237-254. B o u t i l i e r , R.G., T .A. Heming and G.K. Iwama. 1985. Physicochemical parameters f o r use i n f i s h r e s p i r a t o r y p h y s i o l o g y . In F i s h P h y s i o l o g y V o l . XA Appendix, ed. W.S. Hoar and D.J. R a n d a l l , pg.403-430. Academic Press. N.Y. Iwama, G.K. and G.L. Greer. 1982. M o r t a l i t y i n j u v e n i l e chinook salmon exposed to sodium pentachlorophenate and undergoing p r o g r e s s i v e symptomatic b a c t e r i a l kidney d i s e a s e . Can. Tech. Rep. F i s h . Aquat. S c i . No. 1100, 9p. Iwama, G.K. and A.F. Tautz. 1981. A simple growth model f o r salmonids i n h a t c h e r i e s . Can. J . F i s h . Aquat. S c i . 38, 649-656. Iwama, G.K. 1980. In c u b a t i o n times r e s u l t i n g from experimental i n j e c t i o n s o f kidney d i s e a s e b a c t e r i a i n t o j u v e n i l e coho salmon. Prog. F i s h . C u l t . 42(2), 182-183. Iwama, G.K. and G.L. Greer. 1980. E f f e c t o f a b a c t e r i a l i n f e c t i o n on the t o x i c i t y o f sodium pentachlorophenate to j u v e n i l e coho salmon. Trans. Am. F i s h . Soc. 109(3), 290-292. Iwama, G.K. 1981. Comment on "Simple growth model f o r salmonids i n h a t c h e r i e s . Can. J . F i s h e r i e s Aquat. S c i . 39(8), 1220-1221. Iwama, G.K. and G.L. Greer. 1979. T o x i c i t y o f sodium pentachlorophenate to j u v e n i l e chinook salmon under c o n d i t i o n s of high l o a d i n g d e n s i t y and continuous flow exposure. B u l l . E n v i r o n . Contam. T o x i c o l . 23, 711-716. Iwama, G.K., G.L. Greer and P.A. L a r k i n . 1976. Changes i n some hemat o l o g i c a l c h a r a c t e r i s t i c s o f coho salmon (Oncorhvnchus  k i s u t c h ) i n response to acute exposure to d e h y d r o a b i e t i c a c i d DHAA a t d i f f e r e n t e x e r c i s e l e v e l s . J . F i s h . Res. Board Can. 33, 285-289. Iwama, G.K. 1979. "One-Eye", a d i s e a s e o f rainbow t r o u t (Salmo  g a i r d n e r i ) a t the Kootenay Trout Hatchery, B r i t i s h Columbia. B r i t . C o l . F i s h and W i l d l i g f e Br. Tech. C i r . No. 42, l i p . Iwama, G.K., C.Y. Cho and J . D . Hynes ( E d i t o r s ) . 1981. Handbook of F i s h C u l t u r e . Government of On t a r i o Publ. O n t a r i o M i n i s t r y o f Na t u r a l Resources. ISBN 0-7743-6343-6. 

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