"Science, Faculty of"@en . "Zoology, Department of"@en . "DSpace"@en . "UBCV"@en . "Iwama, George Katsushi"@en . "2010-08-06T13:54:10Z"@en . "1986"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Three sets of in vivo experiments were conducted to investigate several aspects of acid-base regulation in fishes. There are two possible ways that involve the gills of fishes in which the acid-base regulation of the extracellular fluid can be adjusted. First, CO\u00E2\u0082\u0082 excretion can be adjusted by\r\naltering gill water flow to increase or decrease the PCO\u00E2\u0082\u0082 tensions in the blood. The second mechanism would involve the exchange of ions across the gill epithelium to change the concentrations of H\u00E2\u0081\u00BA, HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB or NH\u00E2\u0082\u0084\u00E2\u0081\u00BA in the blood. The first two sets of experiments were, respectively, designed to investigate these two possibilites. The third set of experiments investigated the role that plasma catecholamines might play in regulating the pH of the extracellular fluid as well as the intracellular compartment of the red blood cell.\r\nExperimental manipulation of ventilation in rainbow trout in steady state showed that gill water flow affected CO\u00E2\u0082\u0082 excretion only at levels lower than about 100ml/min. Carbon dioxide excretion was retarded and blood PCO\u00E2\u0082\u0082 pressures increased at these levels of gill ventilation. Increasing gill water flow above control levels effected neither O\u00E2\u0082\u0082 or CO\u00E2\u0082\u0082 exchange across the gill.\r\nDogfish, subjected to environmental hyperoxia and various levels of hypercapnia, showed the best correlation between gill ventilation and plasma pH. There was a very weak correlation with plasma PCO\u00E2\u0082\u0082 tension and plasma HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB concentrations did not affect ventilation at all. Gill ventilation increased exponentially as plasma pH declined.\r\nExperiments that involved the fresh water trout and the sea water conger eel showed that water salinity had a direct effect on the acid-base regulation of the plasma. Recovery of plasma pH in both species, after an initial decline in response to exposure to environmental hypercapnia, was dependent on water salinity. The recovery was effected by an increase in plasma HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB concentration. There was also an associated decrease in plasma Cl\u00E2\u0081\u00BB concentration in both species, indicating the possible involvement of a Cl\u00E2\u0081\u00BB/ HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB exchange process. When carp were exposed to environmental hypercapnia, a reduction in the active uptake of water Cl\u00E2\u0081\u00BB, while maintaining normal efflux rates, caused the reduction of the plasma concentration of this ion. Therefore, it seems that the modulation of this active Cl\u00E2\u0081\u00BB/ HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB exchange process effected the HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB accumulation in the carp, and probably also in the trout and conger in fresh water.\r\nConsistent with the data from the above carp experiment, further analyses of the electrochemical gradients for Cl\u00E2\u0081\u00BB in trout exposed to environmental hypercapnia at the three salinities showed that active exchange processes must have accumulated the plasma HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB by the proposed Cl\u00E2\u0081\u00BB/ HCO\u00E2\u0082\u0083\u00E2\u0081\u00BB mechanism. These analyses also showed that the trout gill was about 2.5 times more permeable to Na\u00E2\u0081\u00BA than to Cl\u00E2\u0081\u00BB in steady state control conditions. Furthermore, Na\u00E2\u0081\u00BA is maintained out of electrochemical equilibrium more than Cl\u00E2\u0081\u00BB by a factor of about 1.5 - 2.0. This latter calculation was based on the comparison between the measured plasma concentrations of these ions and the expected concentrations based on a distribution according to the existing electrochemical gradents\r\nCatecholamines are released in trout immediately after acid infusion. This release is proportional to the change in plasma pH relative to control values and functions to maintain the oxygen carrying capacity of the blood which would otherwise be compromised due to the Root shift. This data supports existing data showing that some of the effects which catecholamines have on the physiology of fishes include those which enhance the regulation of the acid-base status of the extracellular and red cell compartments. This data also suggests that the release of catecholamines during burst exercise is due, at least partially, to the excess proton load from the lactacidosis."@en . "https://circle.library.ubc.ca/rest/handle/2429/27114?expand=metadata"@en . "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\u00C2\u00A3..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\u00C2\u00B0C; 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^ \u00C2\u00B0^ 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\u00C2\u00B0C. 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~\u00C2\u00B0\u00E2\u0080\u00A2209. 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 \u00C2\u00A3 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 ~ \u00C2\u00B0 ' 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 + \u00E2\u0080\u00A2 \u00E2\u0080\u00A2+ + \u00C2\u00AB\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 X + + x. s\u00C2\u00BBx~ I I 1 I I I I I \u00E2\u0080\u0094\u00E2\u0080\u00A2 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 \u00C2\u00A7_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 \u00C2\u00A7_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 \u00C2\u00A7_t. \u00C2\u00A7JL.. 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\u00C2\u00B0 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\u00C2\u00A3 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\u00C2\u00B0C (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 \u00E2\u0080\u00A2T* 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 \u00E2\u0080\u00A2T' 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\u00C2\u00B0C 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\u00C2\u00B0C. 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\u00C2\u00B0C 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\u00C2\u00B0C \u00E2\u0080\u0094 mm NH^-Electrode Power on - off Water Na\" .Cl 3 6 Amplifier \u00E2\u0080\u00A2 Recorder PH Na.Cl' i nmp Ft! ' Valve W-f\u00E2\u0080\u0094f Temp. Control Heater Cooler \u00E2\u0080\u0094 30 \u00C2\u00B0C 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. \u00E2\u0080\u00A2 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\u00E2\u0080\u0094/pre\u00E2\u0080\u0094hypercapnia) * 100 100 - i \u00E2\u0080\u0094 , 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\u00E2\u0080\u0094/pre\u00E2\u0080\u0094hypercapnia) 1 r 1 1 1 \u00E2\u0080\u0094r 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 , \u00E2\u0080\u0094 , 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. \u00E2\u0080\u00A2 i r-o ^ o 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 \u00C2\u00A7JL. (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 +\u00E2\u0080\u0094..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\u00C2\u00B0C; 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\u00C2\u00B0C 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\u00C2\u00B0C. 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\u00C2\u00B0C, 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 \u00C2\u00A7_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 \u00C2\u00A3 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\u00C2\u00B0C) 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 \u00C2\u00BB-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 \u00C2\u00BB-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 \u00C2\u00A3 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 \u00C2\u00A3 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 \u00C2\u00A7_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 \u00C2\u00A7_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 \u00C2\u00A7_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 \u00C2\u00A3 al.. 1976; Bubien and Meade 1979; H e i s l e r \u00C2\u00A7_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 \u00C2\u00A7_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 \u00C2\u00A7_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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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. 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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 = \u00E2\u0080\u0094 * 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 = \u00E2\u0080\u0094 * 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 \u00C2\u00BB + 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 \u00C2\u00BB - [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 - \u00E2\u0080\u00A2> 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 . 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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. "@en . "Thesis/Dissertation"@en . "10.14288/1.0097288"@en . "eng"@en . "Zoology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Strategies for acid-base regulation in fishes"@en . "Text"@en . "http://hdl.handle.net/2429/27114"@en .