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Carbon dioxide excretion and acid-base regulation in the freshwater rainbow trout (Salmo gairdneri) :… Perry, Steve F. 1981

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CARBON DIOXIDE EXCRETION AND ACID-BASE REGULATION IN THE FRESHWATER RAINBOW TROUT ( Salmo gairdneri) : INVOLVEMENT OF THE BRANCHIAL EPITHELIUM AND RED BLOOD CELL by STEVE F. PERRY B.Sc. (hons. ) , Concordia U n i v e r s i t y , Montreal , 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1981 © S t e v e F. Per ry , 1981 In present ing t h i s t h e s i s in 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 Un ivers i ty of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I fur ther agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representa t ives . It i s understood that copying or p u b l i c a t i o n of t h i s thes is for f i n a n c i a l gain s h a l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date \\)Vy 7 lj W / " 7 Q V ABSTRACT Carbon dioxide excret ion and acid-base regulat ion were studied in the freshwater rainbow t r o u t , Salmo gairdneri., Two d i f f e r e n t models for C 0 9 excret ion (M p n ) were inves t iga ted . One model proposes that erythrocytes are not invo lved , and that the r a t e - l i m i t i n g step in CO2 excret ion i s the entry of plasma b i c a r b -onate (HCO-j ) in to the g i l l epi thel ium where i t i s dehydrated to molecular CO2 by branchia l carbonic anhydrase. According to t h i s model, the counter i o n s , HCO^ and H + , for branchia l a p i c a l — — + + + CI /HCO^ and Na /H (NH4 ) exchanges, a r i se from plasma. The other model adopts a t y p i c a l mammalian ro le for f i s h ery throcytes , whereby HCO^ i s converted to CO2 by e ry throcy t ic carbonic anhyd-rase . In t h i s scheme carbon dioxide i s thought to enter the g i l l epi thel ium as molecular CO2 where i t i s c a t a l y t i c a l l y hydrated to HCO^ and H + thereby supplying the counter ions for CI /HCO^ + + + and Na /H (NH^ ) exchanges. Experiments using an i s o l a t e d , sa l ine -per fused head prep-arat ion demonstrated that branchia l CI /HCO^ exchange i s re la ted only to the p a r t i a l pressure of CO2 ( p c 0 ) -*-n the perfusate and not to HCO^ concentrat ion. These r e s u l t s ind ica te that CO2 entry and not HCO^ entry in to the g i l l epithel ium i s the dominant pathway. Furthermore, invest iga t ions using var ious other s a l i n e -perfused g i l l preparat ions have shown that M C Q i s not re la ted to perfusate HCO concentrat ion, but only to P_,^  . These resu l ts 3 CO2 show that entry of plasma bicarbonate in to the g i l l epithel ium i s not a major pathway for CO2 excre t ion . Measurements of a constant i i i trans-membrane po ten t ia l (basal membrane of g i l l e p i t h e l i a l c e l l s ) at a l l concentrat ions of perfusate HC0 3 support the conclusion that the basal membrane i s impermeable to t h i s i o n . A spontaneously v e n t i l a t i n g , blood-perfused t rout prepar-at ion was developed to study the ro le of the red blood c e l l i n CC>2 excre t ion . Unl ike sa l ine -per fused preparat ions, b lood-perfused f i s h excreted CG>2 at rates comparable to in vivo va lues . The importance of the red blood c e l l for CC>2 excret ion was demon-strated by per fusing with blood of var ious haematocrits (Hcts) as wel l as plasma. A l i n e a r r e l a t i o n s h i p was observed between Hct and M C Q while plasma perfusion completely abol ished M C Q . 2 2 CC>2 excret ion in blood-perfused f i s h was st imulated by increased blood HC0 3~ concentrat ion. This was due to increased entry of HCO^ in to the erythrocyte and not in to the g i l l ep i the l ium. S i m i l a r l y , SITS, an anion transport i n h i b i t o r , reduced M C Q as a r e s u l t of reduced entry of HCO^ into erythrocytes . C l e a r l y , HCO^" entry into the red blood c e l l i s the r a t e - l i m i t i n g step in CC>2 excre t ion . + The branchia l ion exchange processes (CI /HCO^ and Na /H (NH^+)) have been impl icated in both maintainance of in te rna l pH and regula t ion of acid-base d isturbances. Studies using pharm-a c o l o g i c a l i n h i b i t o r s of these exchange processes indeed have demonstrated that branchia l ion exchange i s extremely important in maintaining steady-state i n t e r n a l acid-base s ta tus . I have postulated that proton movement from plasma in to the g i l l e p i t h -elium i s con t ro l l ed by i n t r a c e l l u l a r pH, which in turn i s governed by the rates of a p i c a l ion exchange. Thus, perturbat ions of these i v 'pumps' a f fec t blood acid-base status by a l t e r i n g proton movement in to the g i l l ep i the l ium. Branchia l CI /HCO^ exchange in sa l ine -per fused heads i s c o n t r o l l e d by l eve ls of c i r c u l a t i n g catecholamines. St imulat ion of adrenergic 0 receptors i n h i b i t CI uptake while st imulat ion of ck receptors st imulate CI uptake. These are d i r e c t e f fec ts and are not due to accompanying haemodynamic changes. Despite the l i k e l i h o o d that catecholamine l eve ls increase during hypercapnia and the r e l a t i o n s h i p that ex is ts between i o n i c exchange and a c i d -base balance, I was unable to demonstrate that modulations of these exchanges are involved in the regulat ion of hypercapnic a c i d o s i s . Another p o s s i b i l i t y for acid-base regula t ion during hypercapnic ac idos is i s a reduct ion of CO^ excre t ion . Because the g i l l epi thel ium i s permeable to protons, a reduction of C0 2 exc-re t ion w i l l not a f fec t H + ion excret ion and w i l l r e s u l t i n accum-u l a t i o n of plasma HCO^ . Inh ib i t ion of HCO^ entry in to ery thro-cytes was observed using l e v e l s of adrenaline associated with s t ress in f i s h . Control of t h i s pathway by catecholamines, l e a d -ing to a reduction of CC^ excre t ion , may be an important process in the regulat ion of hypercapnic a c i d o s i s . V TABLE OF CONTENTS Abstract i i L i s t of Tables v i i i L i s t of Figures x Acknowledgements xiy. General Introduction 1 Chapter I. An Invest igat ion of CO2 excre t ion , Ion Exchange and Acid-Base Regulation Using S a l i n e -Perfused G i l l Preparat ions: A Test of Two Models . . . 16 Introduction 17 Mater ia ls and Methods . . 19 Experimental Animals 19 Experimental Protocol 20 Results 35 Iso la ted , Sa l ine-Per fused Holobranchs, T o t a l l y Sa l ine-Per fused Rainbow Trout and Coho Salmom . . . 35 Iso la ted , Sa l ine-Per fused Rainbow Trout Head Preparation 39 Discussion 49 Chapter II. CO2 Excret ion in a Spontaneously V e n t i l a t i n g Blood-Perfused Rainbow Trout Preparation 59 Introduction 6 0 Mater ia ls and Methods 62 Surg ica l Procedures 63 Blood-Perfusion 69 v i Blood Sampling and Analys is . . . . . . . . . . . . . . . . . . . . 70 Pressure and Flow Recording . . . . . . . . . . . . . . . . . . . . 71 Experimental Protocol : . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Results . 73 Character iza t ion of the 'Normal 1 Spontaneously V e n t i l a t i n g , Blood-Perfused Rainbow Trout . . . . . . 73 Experimental Manipulations 79 Discussion 88 Experimental Manipulations 91 Chapter III. Branchia l Ionic Uptake and Acid-Base Regulation 96 Introduction 97 Mater ia l and Methods 99 Experimental Animals 99 Blood Sampling and Analys is 99 Sodium and Chlor ide Inf luxes 100 Experimental Protocol 101 (1) Chemically Treated F i s h 101 (2) Hypercapnia 101 (3) Hypercapnia and Elevated External Sodium or Bicarbonate 102 Results 103 (1) Chemically Treated F i s h 103 (2) Hypercapnia 118 (3) Hypercapnia and Elevated External Sodium or Bicarbonate 127 v i i Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 134 Chapter IV. Control of CC^ Excret ion and Acid-Base Regulation 143 Introduct ion 144 Mater ia ls and Methods . . . 146 Adrenergic Control , of Branchia l Chlor ide Transport 14 6 Control of CO,, Movements Through Red Blood C e l l s . . . 146 Results 151 Discussion 163 General Discussion 171 Abbreviat ions and Symbols . . . . . . . . . . . . 182 L i te ra tu re Ci ted 184 Biography 201 v i i i LIST OF TABLES Table 1. Chemical composition of two d i f f e r e n t p h y s i o l o g i c a l sa l ines used for s a l i n e - p e r f u s i o n studies 21 Table 2. The e f f e c t of P C Q on CO2 excret ion and acid-base status of sa l ine -per fused holobranchs 36 Table 3. The e f fec t of P C Q on CO2 excret ion and acid-base status of sa l ine -per fused rainbow trout 37 Table 4. The e f f e c t of [ H C 0 3 ] on C 0 2 excret ion and a c i d -base status of sa l ine -per fused Coho salmon 38 Table 5. Summary of blood var iab les for the normal b lood-perfused rainbow trout 77 Table 6. Summary of the d i f fe rences in blood var iab les across the g i l l and systemic c i r c u l a t i o n s in the normal blood-perfused rainbow trout 78 Table 7. Summary of blood resp i ra tory and acid-base status of blood-perfused rainbow trout at three d i f f e r e n t haematocrits 8 0 Table 8 T The e f fec t of ^HCO^ ] on resp i ra tory and acid-base status of blood-perfused rainbow t rout 8 5 Table 9. The e f fec t of SITS on resp i ra tory and acid-base status of blood-perfused rainbow trout 86 Table 10. The .e f fec t of P c o on resp i ra tory and acid-base status of blood-perfused rainbow t rou t . - 87 Table 11. The e f fec t of external drug exposure on acid-base status of rainbow trout 108 ix Table 12. The e f fec ts of SITS and amilor ide on branchia l i o n i c uptake in rainbow trout 116 Table 13. The e f fec t of external bicarbonate on blood acid-base status of rainbow trout 117 Table 14. The e f f e c t of external hypercapnia on blood acid-base status of freshwater rainbow trout and seawater Coho salmon 128 Table 15. The e f fec t of hypercapnia and elevated external sodium on blood acid-base status of rainbow trout 12 9 Table 16. The e f fec t of external bicarbonate and hypercap-n ia on blood acid-base status of rainbow trout 133 X LIST OF FIGURES Figure 1. Graphical representat ion of C0 2 and s a l t move-ments through a branchia l e p i t h e l i a l c e l l in freshwater. I 12 Figure 2. Graphical representat ion of C0 2 and s a l t move-ments through a branchia l e p i t h e l i a l c e l l in freshwater. II 14 Figure 3. Schematic representat ion of apparatus used for i s o l a t e d , sa l ine -per fused holobranch preparat ion 23 Figure 4. Schematic representat ion of apparatus used for t o t a l l y sa l ine -per fused rainbow trout and Coho salmon 2 6 Figure 5.,,, Diagram of catheter and p l a s t i c c o l l a r pos i t ions for the i s o l a t e d , sa l ine -per fused head preparat ion 29 Figure 6. Schematic representat ion of apparatus used for i s o l a t e d , sa l ine -per fused head preparat ion 31 Figure 7. The e f fec t of perfusate bicarbonate concentrat ion on ch lor ide in f lux in the i s o l a t e d , sa l ine -per fused head of rainbow t rout 40 Figure 8. The e f fec t of perfusate bicarbonate concentrat ion on ch lor ide i n f l u x in the i s o l a t e d , sa l ine -per fused head of rainbow t rout fo l lowing addi t ion of e i ther SITS or Diamox to the perfusate or external thiocyanate 42 Figure 9. The e f fec t of perfusate bicarbonate concentrat ion on input pressure in the i s o l a t e d , sa l ine -per fused head of rainbow t rout 44 x i Figure 10. The e f fec t of perfusate P p n on ch lor ide in f lux in the i s o l a t e d , perfused head of rainbow t rout 47 Figure 11. The e f fec t of decreasing input pressure on st imulat ion of ch lor ide in f lux in the i s o l a t e d , perfused head of rainbow t rout . 55 Figure 12. Diagram of catheter pos i t ions in the heart of t rout used for b lood-per fusion experiments. 65 Figure 13. Schematic representat ion of b lood-per fus ion of rainbow trout 67 Figure 14. Record of pressure and flows from spontaneously v e n t i l a t i n g , blood-perfused rainbow trout 74 Figure 15. The e f fec t of haematocrit on A) CC^ excret ion and B) 0^ uptake in the spontaneously v e n t i l a t i n g , b lood-perfused rainbow trout preparat ion •••81 Figure 16. The e f fec t of external amilor ide on branchia l sodium uptake in rainbow t rout . -104 Figure 17. The e f fec t of external SITS on branchia l ch lo r ide uptake in rainbow trout .106 Figure 18. The e f f e c t s of external amilor ide and SITS on branchia l sodium and ch lor ide uptakes in rainbow t rout . 110 Figure 19. The e f fec t of external furosemide on branchia l ch lor ide in f lux in rainbow t rou t . 112 Figure 20. The e f fec t of external sodium bicarbonate on branchia l ch lor ide uptake in rainbow t rou t . 114 Figure 21. The e f fec t of 24 h hypercapnia on branchia l x i i sodium uptake in rainbow trout 119 Figure 22. The e f fec t of 24 h hypercapnia on branchia l ch lor ide uptake in rainbow trout 121 Figure 23. The e f fec t of 30 min hypercapnia on branchia l sodium uptake in rainbow trout 1^3 Figure 24. The e f fec t of 30 min hypercapnia on branchia l ch lor ide uptake in rainbow trout 1 2 5 Figure 25. The e f fec t of exposure to hypercapnia for 24 h and a l te red external environment on plasma sodium and ch lor ide l e v e l s in rainbow t rout 130 Figure 26. Redrawn traces from a t y p i c a l carbonic anhydrase assay 1 49 Figure 27. The e f f e c t s of var ious catecholamines on branchia l ch lor ide uptake in the i s o l a t e d , perfused head of rainbow t rout . I 5 3 Figure 28. The e f fec t of A) switching from ^ to A st imulat^ ion and B) from ck to £ st imulat ion on ch lor ide uptake and input pressure in the i s o l a t e d , perfused head of rainbow trout 155 Figure 29. The e f f e c t of A)£ antagonists and B)<* antagonists on ch lor ide uptake and input pressure in the i s o l a t e d , perfused head of rainbow trout I57 Figure 30. The r e l a t i o n s h i p between input pressure and ch lor ide uptake using four d i f f e r e n t catecholamines l,5g Figure 31. The e f fec t of increas ing concentrat ions of adrenaline on carbonic anhydrase a c t i v i t y of washed red x i i i blood c e l l s of rainbow t rout . . . 161 Figure 32. The e f fec t of or 0 receptor st imulat ion on ch lor ide i n f l u x , external sodium and ch lor ide concentrat -ions and input pressure in an i n d i v i d u a l i s o l a t e d head of rainbow trout 165 Figure 33. A diagramatic representat ion of carbon dioxide excret ion in the rainbow trout 17 3 x iv ACKNOWLEDGEMENTS I would l i k e to acknowledge a number of people who have helped make the l a s t four years an enjoyable learning experience. I am e s p e c i a l l y g r a t e f u l to my superv isor , Dr. David Randal l , who provided encouragement and support and who was always there to keep me pointed in the r i g h t d i r e c t i o n . To Dr. Chuck Daxboeck go my s incere thanks for being a great f r i end a l l these years . The spontaneously v e n t i l a t i n g , blood-perfused preparat ion was developed, and the experiments performed, in co l l abora t ion with Dr. Daxboeck and Dr. Peter Davie. I am most appreciat ive of Dr. Michel Bornancin who arranged for me to do research at the Univer-s i t y of Nice and who made sure our stay in Nice was a pleasant one. Experiments using the i s o l a t e d head preparat ion were performed in the "Laboratoire de Physio logie C e l l u l a i r e et Comparee" with Dr. Pa t r ick Payan and Dr. Jean-Pier re G i r a r d . I am a lso g ra te fu l to Drs. David Jones, Chr is Wood and Brian Eddy for providing extensive c r i t i c a l comment on t h i s t h e s i s . A l s o , I must acknowledge Dr. David Pr ieur and Dr . -Per ry Kupatch who always were w i l l i n g to continue st imulat ing d iscussions u n t i l the ear ly hours of the morning. To Tanja go my h e a r t - f e l t thanks for providing moral support and understanding. Simply, I could not have done i t a lone. During my stay at U .B .C . I was supported by an N . S . E . R . C . 1967 Science Scholarsh ip . Research costs were made ava i lab le through an N . S . E . R . C . operating grant to D . J . Randal l . 1 GENERAL INTRODUCTION 2 F i s h must maintain a constant production and e l iminat ion of carbon dioxide or a disturbance of the i n t e r n a l acid-base e q u i l i b -rium w i l l develop. Acid-base disturbances can be defined as a l l fac tors which cause an a l t e r a t i o n of the hydrogen ion concentrat -ion (CH+3) in the body f l u i d s . An increase in hydrogen ion concentrat ion i s termed ac idos is while a decrease i s termed a l k a l o s i s . The importance of regulatory mechanisms responding to acid-base disturbances cannot be overstated and these mechanisms are c e r t a i n l y e s s e n t i a l for s u r v i v a l . The fo l lowing equations w i l l help to i l l u s t r a t e the e f fec t of C0 2 on blood acid-base balance. C0 2 in aqueous so lut ions d isp lays the fo l lowing e q u i l -ibrium r e a c t i o n : C0 2 + H 2 0 ^ H 2 C0 3 ^ H + + HC0 3~ (1) Carbonic ac id formed from the hydrat ion of C 0 2 , d i s s o c i a t e s to y i e l d hydrogen and bicarbonate (HC03 ) i o n s . It can be shown t h e o r e t i c a l l y and confirmed experimental ly that the r e l a t i o n s h i p between substances represented in equation 1 can be explained by the mass act ion law (Davenport, 1974). Thus, an increase i n d i s s -olved CC>2 w i l l s h i f t the equi l ibr ium towards the r igh t r e s u l t i n g i n an increase in the hydrogen ion concentrat ion or a decrease in pH. I f the buf fer ing capacity of the so lu t ion i s not suffic^-ient to neut ra l i ze the excess hydrogen ions then an ac idos is w i l l develop. S i m i l a r l y , an increase in bicarbonate ion concentrat ion (tHC03"^l) w i l l d isp lace the react ion to the l e f t causing a l k a l o s i s . A reorganizat ion of equation 1 gives the wel l known Henderson-3 Hasselbalch equat ion: . . HCO ~ pH = pK'+ log (2) * C 0 2 ^ c o / pK' i s the negative logarithm of the equi l ibr ium constant/ K, for the C0 2 equ i l ibr ium react ion and <*> i s the s o l u b i l i t y c o e f f i c i e n t of carbon d iox ide . It i s c lea r from t h i s r e l a t i o n s h i p that pH i s a f fected by both d isso lved molecular C0 2 (<*C0 2(P c o )) and HC0 3~ . The necessi ty of maintaining constant i n t e r n a l pH i s due to the e f f e c t of pH on the d i s s o c i a t i o n of enzymatic and s t r u c t -u r a l p ro te ins . Consider the Henderspn-Hasselbalch equat ion: pH = pK + log —— (3) HA Where A i s a prote in residue and pK* i s the negative logarithm of the d i s s o c i a t i o n constant , K. C l e a r l y , any change in pH without an equal change in pK' w i l l a f fec t the d i s s o c i a t i o n r a t i o of the prote in (A /HA) . Thus, the net charge on p r o t e i n s , an important determinant of f u n c t i o n , w i l l be a f fec ted by changes i n pH. The a b i l i t y to e f f e c t i v e l y regulate acid-base disturbances i s therefore v i t a l to enzyme funct ion and hence to an organisms s u r v i v a l . The two most exstensive ly studied factors leading to a c i d -base disurbances in f i s h are temperature (Howell, Baumgardner, Bondi & Rahn, 1970; Randall & Cameron, 1973; Cameron, 1976; Cameron, 1978) and hypercapnia (Lloyd & White, 1967; Cameron & 4 Randal l , 1972; Janssen & Randa l l , 1975; Cameron, 1976; Eddy, 1976; Randa l l , He is le r & Drees, 1976; Eddy, Lomholt, Weber & Johansen, 1977; Cameron, 1978; Eddy, Smart & Bath, 1979). A l t h -ough only hypercapnia i s dealt with in t h i s t h e s i s , a b r i e f d i s -cussion of thermal acid-base disturbances i s inc luded , for completeness. F i s h a r e . p o i k i l i o t h e r m i c ver tebrates , therefore any change in ambient temperature w i l l be r e f l e c t e d by changes in body temperature. An inverse r e l a t i o n s h i p e x i s t s between . temperature and blood pH in f i s h (Rahn & Baumgardner, 1972; Randall & Cameron, 19 73)„and r e s u l t s from the e f fec t of temperature on the d i s s o c i a t i o n of p ro te ins . This e f f e c t of temperature can be o f f s e t by changes in pH such that the d i s s o c i a t i o n state of prote ins remains unal tered. Consider equation 3; i f dpH/dT = dpK' /dT then pH - pK 1 w i l l be constant over a range of temper-atures and log A /HA also w i l l be constant. Therefore as temp-erature v a r i e s , changes in p K i : a r e n u l l i f i e d by changes in pH and the d i s s o c i a t i o n of prote ins remain constant (Rahh.y. 1966)s. The e f fec t of temperature on the pK' of the C0 2 equi l ibr ium react' 1 . , ion (equation 1) i s much smaller than the change in blood pH. Reexamining equation 2, one can see that i f the change in pH with temperature does not equal the change in p K 1 , then the r a t i o of HCO., / P r n must change with temperature. As temperature inc reases , pH and CC^ decrease (Albers, 1971). This means that e i ther ("HCO- 1 must a lso decrease or P_,n must r i s e in order that L U 2 pH changes in proport ion to the pK' of blood pro te ins . Randall & Cameron (1973) and Cameron (1978) have shown that with i n c r e a s -ing temperature, a r t e r i a l P r n ( P a m ^ does not change and that 5 [HC03"i] decreases - in rainbow t rou t . 3 Hypercapnia describes the p h y s i o l o g i c a l state in which the CC^ tension of blood i s elevated above normal. This condi t ion i s achieved experimental ly by increas ing ambient P C Q thereby reducing the gradient for C0 2 excre t ion . From equation 1 i t i s c l e a r that the net r e s u l t of increased Pa- C 0 i s a decrease in pH. Various invest iga tors (Cameron & Randal l , 1972; Janssen & Randal l , 1975; Randall e t a l . , 1976; Eddy et a l . , 1977; Cameron, 1978) have studied the e f fec ts of environmental hypercapnia on blood pH in f i s h . The t y p i c a l response i s a rapid decrease in pH followed by a long per iod of gradual recovery usual ly complete a f te r 3-4 days. In a l l cases the compensatory increase in blood pH i s accompanied by a p a r a l l e l e levat ion of plasma HCO^ . It appears that f i s h are unable to adjust P a _ n by changes in v e n t i l a t i o n c u 2 under condi t ions of steady-state CC>2 production as in mammals (Cameron, 1978). F i s h there fore , unl ike mammals, regulate blood pH during hypercapnia by adjust ing plasma bicarbonate l e v e l s . In mammals, CC>2 excret ion i s governed by the d i f f u s i o n of molecular CC^ from the plasma into the a lveo la r gas. The rate at which CG^ d i f fuses across the lung epithel ium therefore i s dependent on the P gradient between plasma and a lveo lar gas. c u 2 Factors which a l t e r t h i s gradient w i l l a f fec t o v e r a l l CC^ excre-t i o n . Probably the most important factor regulat ing the gradient i s the rate of v e n t i l a t i o n . An increased ven t i l a to ry rate tends to increase the P ^ Q gradient across the lung epi the l ium, r e s u l t i n g in greater C0 2 excre t ion . S i m i l a r l y , hypovent i la t ion causes C0 2 re ten t ion . The net r e s u l t i s that adjustments of blood pH can 6 be accomplished in short periods of time by regulat ing P a C Q v i a changes in v e n t i l a t i o n . Due to the high s o l u b i l i t y of oxygen in a i r , oxygen uptake i s not impaired during periods of v e n t i l -atory adjustment. Lacking information to the contrary , i t was assumed prev^-ious ly that f i s h would regulate in te rna l pH by ven t i l a to ry cont ro l of Pa__ as has been wel l es tab l ished for mammals and a i r -b rea th ing poik i l io therms (Jackson, Palmer & Meadows, 1974; Davies, 1975). Experimental evidence (Randall & Cameron, 1973; Janssen & Randal l , 1975) l a t e r showed that v e n t i l a t i o n i s not involved in pH and P a _ n r egu la t ion . A s im i l a r conclusion i s a r r ived at using theor-e t i c a l cons idera t ions . The r a t i o of the oxygen s o l u b i l i t i e s of blood and water i s very h igh , therefore the v e n t i l a t i o n - p e r f u s i o n r a t i o (V /Q) must be very high in water breathers in order to have an e f f i c i e n t oxygen uptake system. Values for V /Q are t y p i c a l l y about 10 for te leos ts and elasmobranchs (Cameron & Davis , 1970; Cameron, Randall & Davis , 1971; P i i p e r & Schumann, 1967; Hanson & Johansen, 1970). Since the s o l u b i l i t y of CC^ in f r e s h -water i s about the same as b lood , t h i s means that the g i l l i s hypervent i la ted with respect to CC^ and that Pa(-.Q w i l l be rather i n s e n s i t i v e to va r ia t ions of v e n t i l a t i o n . In other words, f i s h cannot a f ford to a l t e r v e n t i l a t i o n for the sake of CC^ c o n t r o l , i n the face of l im i ted and var iab le amounts of ava i lab le oxygen in water. It i s c lea r from the preceeding d iscuss ion that bicarbonate leve ls are adjusted to compensate for pH changes caused by thermal perturbat ions or hypercapnia. What remains unclear are the mech-7 anisms involved in the adjustment of plasma HCO^ l e v e l s . F i s h posess three poss ib le s i t e s of regu la t ion . These are the gut , the kidney and the g i l l . I am pr imar i l y in terested in the ro le of the g i l l and w i l l only discuss the other two b r i e f l y . At t h i s time the involvement of the gut in regulat ing acid-base d i s t u r b -ances i s unknown but should probably not be discounted u n t i l fur ther research i s done in t h i s area. The poss ib le ro le of the kidney in acid-base balance commonly i s ignored or discounted (Hickman & Trump, 1969). In recent years , var ious invest iga tors have reported c o n f l i c t i n g r e s u l t s concerning th is ro le fo r the p isc ine kidney. Cross , Packer, L i n t a , Murdaugh & Robin (1969) found that the elasmobranch kidney plays an i n s i g n i f i c a n t ro le in regulat ing acute experimental a c i d o s i s . In cont ras t , Wood & Caldwell (1978) have concluded that the t o t a l e levat ion in ur inary ac id excret ion over 72 h was such that no other mech-anism besides renal funct ion i s needed to expla in the ult imate compensation of an imposed ac id load in rainbow t rou t . Cameron & Wood (1978) studying renal ac id excret ion in two Amazonian f i s h , Hopl'ias .malabaricus '(a water breather) and Hoplerythrinus unitaeniatus (an a i r breather ) , found that metabolic ac idos is produced no compensation whereas resp i ra tory ac idos is caused increased renal ac id excret ion in Hoplerythrinus but not in Hoplias. S i m i l a r l y , Cameron (1978) observed only a 10% increase in ur inary ac id excret ion fo l lowing ac id load in the c a t f i s h , ictalurus punctatus. As w e l l , Cameron (1980) has concluded that in the c a t f i s h , about 14% of the whole body bu i ld -up i s due to renal ac id excret ion during compensation of hypercapnic a c i d o s i s . With the paucity of 8 data that ex is t at t h i s time i t i s d i f f i c u l t to def ine a ro le for the f i s h kidney in acid-base balance. It appears that-i t may be less important in t h i s funct ion than the kidney of higher vertebrates where i t plays a major ro le in long-term r e g -u l a t i o n of chronic acid-base disturbances although Kobayashi & Wood (19 80) have concluded that the response of the t rout kidney to true metabolic ac idos is i s s i m i l a r to that of the mammalaian kidney. The g i l l i s the p r i n c i p l e organ responsible for C0 2 removal in f i s h . While the branchia l epithel ium cons is ts of three c e l l types; mucus c e l l s , ch lor ide c e l l s and resp i ra tory c e l l s , i t i s the l a t t e r that are involved in C0 2 excret ion (Girard & Payan, 1980) . Although the ro le of the g i l l in i o n i c regula t ion has been f i rmly e s t a b l i s h e d , few data ex is t concerning i t s funct ion as an acid-base regula t ing structure and the data that are a v a i l -able are often confusing and c o n f l i c t i n g . S i m i l a r l y , while i t i s known that the g i l l serves as the primary pathway for CC>2 removal, the o v e r a l l pattern and the mechanisms involved in C0 2 excret ion remain obscure at t h i s t ime. In freshwater, the g i l l i s responsible for i o n i c reg -u l a t i o n . Sodium (Na+) and ch lor ide (CI ) ions which are d i f f u s i n g constant ly into the d i l u t e environment are replaced v i a branchia l ac t ive transport mechanisms (Maetz, 1971). Under steady-state cond i t ions , the act ive in f lux of these ions equals the passive e f f lux such that the net f lux i s approximately zero. The Na + and C l ~ absorbt ion mechanisms are independent of each other (Romeu & Maetz, 1964; Kerstet ter & K i rschner , 1972) and for t h i s 9 reason i t i s necessary that Na and CI ions be exchanged for counter ions in order that e l e c t r o n e u t r a l i t y be maintained. It i s genera l ly agreed that HCO^ i s exchanged for CI (Maetz & Romeu, 1964; Kers te t ter & K i rschner , 1972; de Renzis & Maetz, 1973; de Renzis , 1975) and H + and/or NH^ + ions are exchanged for N a + (Maetz7& Romeu, 1964; K e r s t e t t e r , Kirschner & Rafuse, 1970; Maetz, 1973; Payan & Matty, 1975; Payan, 1978), these exchanges occurr ing at the a p i c a l membrane of g i l l e p i t h e l i a l c e l l s . It i s apparent then, that the f i s h g i l l i s an i d e a l s i t e for a c i d -base regula t ion and that i o n i c movements across the branchia l epi thel ium are int imate ly coupled to CC^ movements. While these fac ts are u n i v e r s a l l y agreed upon, the mechanisms by which the counter ions (HCO^ and H +(NH^ +)) are generated, and the form in which carbon dioxide i s excreted (CC^ or HC0 3 ) are under debate. Present ly , two widely contrast ing theor ies e x i s t surround-ing- the pattern of CC^ excret ion in f i s h and the r e l a t i o n s h i p between branchia l CC^ movements and i o n i c movements. These theor-ies need be discussed in some d e t a i l as ideas inherent in each form a large port ion of t h i s t h e s i s . The more widely accepted model (F ig . 1) has stemmed from the pioneering research of Jean Maetz, F . Garcia Romeu and c o -workers. In t h i s model, plasma carbon dioxide (as molecular CC^) i s thought to enter the g i l l epithel ium where the enzyme carbonic anhydrase cata lyses the hydrat ion r e a c t i o n , forming HCG^ and H + . HCO^ then i s exchanged for C l at the a p i c a l membrane'and H + i s exchanged for Na + or combines with ammonia (NH )^ forming ammonium ion XNH.+) which exchanges for N a + . 10 Na and CI ions move in to the plasma across the basal membrane where e l e c t r o n e u t r a l i t y i s maintained by potassium movements, inherent i n t h i s model i s that the ro le of the f i s h erythrocyte in CC>2 excret ion i s s imi la r to that in mammals and b i r d s . That i s , when blood reaches the resp i ra tory epi the l ium, plasma HCO^ i s shut t led in to the erythrocyte in exchange for a CI ion (the ch lor ide s h i f t ) . CC^ then d i f fuses out of the erythrocyte accord-ing to the concentrat ion grad ients . A more recent theory (Haswell & Randal l , 19 76; Haswell , 1978; Haswell & Randal l , 1978; Haswel l , Randall & Perry , 1980) suggests that f i s h erythrocytes are f u n c t i o n a l l y impermeable to plasma HCO^ making e ry throcy t ic carbonic anhydrase unavai lable to cata lyse the dehydration r e a c t i o n . Instead, these authours propose that plasma HCO^ enters the g i l l epi thel ium where i t i s c a t a l y t i c a l l y dehydrated by branchia l carbonic anhydrase (F ig . 2) . CC>2 formed from t h i s react ion d i f fuses across the ap ica l membrane into the water and accounts for the majori ty of carbon dioxide excreted. The counter ions (HCO^ and H + (NH 4 + ) ) exchanged for CI and Na + r e s p e c t i v e l y , a r ise from plasma and cross the basal membrane a lso in exchange for CI and Na + i ons . A fundamental d i f fe rence between these two models i s that in one (F ig . 1) the r a t e - l i m i t i n g step in CC>2 excret ion i s the entry of plasma HCO^ in to red blood c e l l s whereas in the other (F ig . 2) the r a t e - l i m i t i n g step i s the entry of plasma HCO^ in to the g i l l ep i the l ium. Both models l i n k carbon dioxide movements t o . i o n i c movements and imply that acid-base disturbances may be regulated by modulations of the a p i c a l ion exchange processes 11 thereby leading to accumulation of plasma bicarbonate. In t h i s thes is I have attempted to v e r i f y experimental ly e i ther of these models and by doing so 1) c l a r i f y the pattern of G0 2 excret ion in f i s h , 2) def ine the r e l a t i o n s h i p between branchia l ion exchange and acid-base regu la t ion , 3) determine the r e l a t i v e involvements of the erythrocyte and branchia l epithel ium in C0 2 excret ion and acid-base regula t ion and 4) i d e n t i f y contr -o l l i n g mechanisms for blood acid-base balance and C0„ excre t ion . 12 Figure 1. Graphical representat ion of carbon dioxide and s a l t movements through a branchia l e p i t h e l i a l c e l l i n freshwater. This model i s based on the research of J . Maetz and co-workers and has been redrawn from Maetz (1971). (CA) carbonic anhydrase. 13 14 Figure 2. Graphical representat ion of carbon dioxide and s a l t movement through a branchia l e p i t h e l i a l c e l l i n f r e s h -water. This drawing has been modif ied s l i g h t l y from Haswell et a l . (1980). See text for fur ther d e t a i l s . 15 16 CHAPTER I AN INVESTIGATION OF C0 2 EXCRETION, ION EXCHANGE AND ACID-BASE REGULATION USING SALINE-PERFUSED G I L L PREPARATIONS: A TEST OF TWO MODELS 17 INTRODUCTION According to the model of Haswell et al. (1980; F i g . 2) , the entry of plasma HCO^ in to the g i l l epi thel ium i s the ra te -l i m i t i n g step in CO2 excre t ion . Haswell & Randall (1976, 1978) a lso have proposed that the f i s h erythrocyte i s f u n c t i o n a l l y impermeable to plasma HCO^ and postulated that branchia l carbonic anhydrase replaces the funct ion of e ry throcyt ic carbonic anhyd-rase; the dehydration of plasma HCO^ . These conclusions have ar isen from in vitro studies of red blood c e l l s (Haswell & Randal l , 1976) and in vivo experiments comparing normal and anaemic f i s h (Haswell & Randal l , 1978). This theory contrasts with themore wide accepted view that f i s h erythrocytes are permeable to HCO^ and funct ion as t y p i c a l mammalian rbe ' s ; generating molecular C 0 2 from the cata lysed dehydration of plasma HCO^ . In t h i s model (Maetz, 1972; F i g . 1) , branchia l carbonic anhydrase i s assigned the ro le of rehydrat ing plasma C 0 2 thereby furn ish ing the counter i o n s , HCO^" and H + (NH^ + ) , to be exchanged for CI and Na + r e s p e c t i v e l y . Two methods of inves t iga t ion were i n i t i a t e d to tes t these c o n f l i c t i n g hypotheses. The f i r s t l i n e of inves t iga t ion involved monitoring C 0 2 excret ion in var ious sa l ine -per fused preparat ions. If the model of Haswell (1978) i s c o r r e c t , and red blood c e l l s are not involved in C 0 2 excre t ion , then one would expect s a l i n e -perfused preparat ions to excrete C 0 2 at a normal ra te . Fur ther -more, C 0 2 excret ion should be propor t ional to the concentrat ion of HCO-3 in the per fusate . Conversely, i f the entry of HCO^ 18 into the g i l l epithel ium i s not the r a t e - l i m i t i n g step in CO^ excret ion then one would expect only perfusate P ^ to a f fec t CC>2 excre t ion . The second l i n e of inves t iga t ion involved examining bran- :' c h i a l CI i n f l u x . By monitoring CI i n f l u x under a va r ie ty of condi t ions while varying perfusate ^HCO^"] and P C Q , one can det-ermine whether HCO^ exchanged for CI a r ises d i r e c t l y from the plasma(Fig. 2) or from the hydration of plasma CG^ (F ig . 1) . If the model of Maetz (1971; F i g . 1) i s c o r r e c t , I would expect only perfusate P C Q to a f fec t CI i n f l u x . A l t e r n a t i v e l y , i f the model of Haswell - et al. (1980; F i g . 2) i s c o r r e c t , I would expect only perfusate £HC03~^J t o a f fec t CI i n f l u x . The carbonic anhy-drase i n h i b i t o r , acetazolamide (Diamox) and the anion transport i n h i b i t o r , SITS (4 -ace tamido-4 ' - i so - th iocyanatos t i lbene-2 ,2 ' d i s u l f o n i c acid) a lso w i l l be used in an attempt to v e r i f y these models. It i s hoped that r e s u l t s from these experiments w i l l help c l a r i f y the manner in which carbon dioxide t raverses the branchia l ep i the l ium, and i n d i r e c t l y assign a ro le to the erythrocyte in CO n excre t ion . 19 MATERIALS AND METHODS Experimental animals .. Rainbow trout ( Salmo gairdneri ) . weighing between 200-400 g (for t o t a l l y sa l ine -per fused preparations) and 900-1200 g (for i s o l a t e d , sa l ine -per fused holobranchs) were obtained from Sun Va l l ey Trout Farm (Mission, B r i t i s h Columbia) and P a c i f i c E n v i r -onment Ins t i tu te (West Vancouver, B r i t i s h Columbia) r e s p e c t i v e l y . They were kept in large c i r c u l a r f i b r e g l a s s tanks suppl ied with aerated, dechlor inated Vancouver tap water: (Na + = 40 u e q u i v . / l , C l ~ = 20 u e q u i v . / l , hardness = 12 ppm CaCO^) and kept at ambient temperature (7-12°C) and photoperiod. F i s h were fed d a i l y with a commercial pe l l e ted t rout d ie t (Moore-Clarke C o . ) . They were not fed 4 8 h p r i o r t o , or during experiments. Experiments invo lv ing seawater were performed at Bamfield Marine Stat ion (Bamfield, B r i t i s h Columbia). Coho salmon ( Onchorynchus kisutch ) weighing between 300-500 g were obtained from P a c i f i c B i o l o g i c a l . S t a t i o n (Nanaimo, B r i t i s h Columbia). They were maintained in running seawater in a manner s im i l a r to rainbow t rou t . I so la ted , sa l ine -per fused head studies were performed at the Un ivers i ty of Nice (Nice, France) in co l labora t ion with Drs. Payan, Gi rard and Bornancin. Rainbow trout weighing between 150-2 50 g were obtained from a ' loca l hatchery and maintained in f lowing tap water (temperature = 15-16°C) for periods never exceeding three weeks. F i s h were not fed during these per iods . 20 Experimenta1 protocol (1) Isolated, saline-perfused rainbow trout holbbranch preparation Approximately 30 min p r i o r to s u r g i c a l procedures, f i s h were in jec ted i n t r a p e r i t o n e a l l y with sodium heparin (5000 USP u n i t s / k g ) . Fol lowing t h i s pe r iod , f i s h were anaesthet ised in a s o l -ut ion of 1:15 , 000 MS 222 (trimethane sulphonate; ,-pH adjusted to 7.0-7.5 with NaHCO^) and t ransfer red to an operat ing table (Smith & B e l l , 1967) where 1:20,000 MS 222 (aerated and c h i l l e d to 4°C) was r e c i r c u l a t e d over the g i l l s . The heart and vent ra l aorta were exposed by a v e n t r a l , midl ine i n c i s i o n . The bulbus a r t e r i o s u s / vent ra l aorta was cannulated with a short length of polyethylene tubing (PE 160; ID 1.14 mm, OD 1.57 mm) and secured in p lace . The g i l l s were c leared of blood by per fus ing manually with f i l t -ered (Mi l l ipore I n c . , 0 . 4 5 uM) modif ied Cort land sa l ine (Wolf, 1963; see Table 1). The branchia l basket was removed and i n d i -v idua l holobranchs c a r e f u l l y d issected free and stored in aerated sa l ine on ice u n t i l requ i red . Af ferent ( p r e - g i l l ) and e f ferent ( p o s t - g i l l ) arch vessels were exposed and cannulated with blunt 2 0 or 21 gauge hypodermic needles depending on the diameter of the vesse ls ( F a r r e l l , 19 79; F a r r e l l , Daxboeck & Randal l , 1979). Holobranchs remained sub-merged i n i c e - c o l d sa l ine during these procedures. The g i l l arch was l iga ted as near to the catheter as p o s s i b l e , leaving a min-imal amount of cut t i ssue being perfused. Cannulated holobranchs were suspended in an aerated, well-mixed water bath at constant temperature and perfused at constant flow (2.2 ml /min/arch/kg 21 Table 1. Chemical composition of two d i f f e r e n t p h y s i o l o g i c a l sa l ines used for s a l i n e - p e r f u s i o n s tud ies . Cort land 1 's (Wolf, 1963) Payan & Matty (1975) Molar i ty (mM) g / i Molar i ty (mM) NaCl 7.25 124.1 6.59 112.8 KC1 0.38 5.1 0.31 4.2 a Na 2 HP0 4 0.41 2.9 0.14 1.0 MgS04 0.23 1.9 0.14 1.2 C a C l 2 0.16 1.4 0.14 1.3 NaHC03 1.00 11.9 1.10 c 13.1 (NH 4 ) 2 S0 4 - - 0.02 0.1 KH 2 P0 4 - - 0.05 0.4 Glucose 1.00 - 1.00 -bPVP 40.0 4% 40.0 4% Heparin 10,000 USP u n i t s / 1 5,000 USP un i ts /1 The o r i g i n a l Cort land sa l ine recipe used NaH2PC>4. I found using Na 2 HP0 4 resul ted i n a more r e a l i s t i c pH (approximately 8.0) . PVP (po lyv iny l -pyrro l id inone) i s not included in the o r i g i n a l Cort land sa l ine r e c i p e . I t was f i r s t added by Wood (1974) as an osmotic f i l l e r . The sa l ine of Payan & Matty (1975) was modif ied to 13.1 mM NaHCO^ instead of the o r i g i n a l 2 6 mM NaHCO^. 22 body weight) with gas -equ i l ib ra ted Cort land sa l ine approximating venous blood (0.5% C0 2 i n 40% a i r , remainder N 2 ; see Haswell , Perry & Randal l , 1978) with a syringe pump (Harvard) (see F i g . 3) . Input pressure was monitored with a Harvard pressure t r a n -sducer and chart recorder while e f ferent flow (Q )^ was monit-ored with a drop counter. Experiments d id not commence u n t i l input pressure had s t a b i l i s e d (usual ly 20-30 min). Protocol consis ted of sampling af ferent and e f ferent sa l ine while per fus ing with sa l ine e q u i l i b r a t e d with normal (0.5%) and high (2.0%) C 0 2 . Gas mixtures were suppl ied by gas-mixing pumps (Wosthoff). To ta l C0 2 content ( C C Q ) was determined using the method of Cameron (1971) with a Radiometer PHM-71 acid-base analyser and associated C0 2 e lectrode (E5036/0) maint-ained at 45°C to speed electrode response t ime. pH measurements were made u t i l i s i n g the same acid-base analyser and micro pH e lectrode (G297/G2) thermostatted to ambient temperature. P a r t i a l pressure of C0 2 (PQQ ) and bicarbonate concentrat ion ( [HCO^ 3) were ca lcu la ted using the measured pH and C C Q values and a reorganizat ion of the Henderson-Hasselbalch equation as fo l lows: CCO P = 2 ( 4 ) 2 a n t i l o g (pH-pK') (<*C02) + ot C0 2 [HC0 3 ] = C C 0 2 - ( « P C 0 2 ) (5) The operat ional pK* values of carbonic ac id were obtained from Severinghaus, Stupfe l & Bradley (1956) and the s o l u b i l i t y 23 Figure 3. Schematic representat ion of apparatus used for i s o l a t e d , sa l ine -per fused holobranch preparat ion. Sal ine i s pumped from a reservo i r through a cool ing chamber and into a holobranch suspended in a constant . _ temperature water bath. 2 4 25 c o e f f i c i e n t s of C0 2 (<*C02) fo r human plasma were obtained from Albers (1971). Human values of * C 0 2 are a good approximation of t rout plasma values because of the s i m i l a r i t y in i o n i c s t rentgh. (2) Totally saline-perfused rainbow trout and Coho salmon T o t a l l y perfused preparat ions were prepared as described by Wood, McMahon & McDonald (1978). B r i e f l y , the vent ra l aorta of pre-hepar in ised f i s h (2000 USP u n i t s / f i s h ) was exposed, s e c t -ioned and cannulated i n the orthograde and retrograde d i r e c t i o n s using polyethylene tubing (PE 190; ID 1.19 mm, OD,1.70 mm). F i l t e r e d (Mi l l ipore I n c . , 0.45 uM) Cort land sa l ine was infused orthograde with a 100 ml syringe u n t i l the venous e f f luen t appeared free of blood (approximately 10 min). A tube for vent-i l a t i o n was placed in to the mouth and sewn in to p o s i t i o n . F i s h then were t ransfer red to a darkened Perspex box and perfused with Cort land s a l i n e (equi l ibra ted with 0.4% C0 2 i n 40 % a i r , remain-der N 2) at constant flow (17 ml /min/kg; Kiceniuk & Jones, 1977) using a p u l s a t i l e pump (Watson-Marlow) and ven t i l a ted a r t i f i c i a l l y at 500 ml H 2 0/min. Input samples were taken from a T junct ion in the in fus ion l i n e near the point of i n s e r t i o n in to the vent ra l aorta and a r t e r i a l samples were taken from a dorsa l a o r t i c cannula (Smith & B e l l , 1964; Smith, 1978) implanted 24 h p rev ious ly . (see F i g . 4 for a diagramatic representat ion of the perfusion apparatus). C n and pH were determined as described above. P n c u 2 2 was determined only i n sa l ine -per fused Coho salmon, using a Radiometer PHM-71 acid-base analyser and associated C>2 e lectrode (E5046/0) thermostatted to seawater temperature. 26 Figure 4. Schematic representat ion of apparatus used for t o t a l l y sa l ine -per fused rainbow trout and Coho salmon. Sal ine i s pumped from a reservo i r through a coo l ing chamber and in to the f i s h v i a a vent ra l a o r t i c catheter . P o s t - g i l l sa l ine i s c o l l e c t e d from a catheter implanted in the dorsa l aor ta . 27 28 (3) Isolated, saline-perfused rainbow trout head preparation Isolated head preparations were prepared according to: Payan & Matty (1975) as fo l lows. F i s h were in jec ted i n t r a p e r i t o n e a l l y with sodium heparin (2500 USP u n i t s / f i s h ) . Fol lowing a 20 min p e r i o d , the unaesthetised f i s h was decapitated just caudal of the pectora l f i n s . The head was placed on an operating table and the g i l l s i r r i g a t e d with freshwater. Immediately, the p e r i -cardium was cut and the v e n t r i c l e severed to prevent a i r from being pumped into the g i l l s . V i s c e r a were removed from the body cav i ty and a catheter was inser ted in to the bulbus ar te r iosus through the severed v e n t r i c l e and t i e d in place (see F i g . 5) . The g i l l s were c leared of blood by per fusing with f i l t e r e d ( M i l l -ipore I n c . , 0.22 uM) sa l ine (modified from Payan & Matty, 1975; see Table 1) at a constant pressure of 50 cm H^O. A t i g h t l y f i t t i n g s e m i - c i r c u l a r p l a s t i c c o l l a r was placed ins ide the abd-ominal cav i ty and sutured in to place thus making the body wal l r i g i d (F ig . 5) . A catheter was inser ted in to the dorsa l aorta for c o l l e c t i o n of p o s t - g i l l s a l i n e . The operation las ted approx-imately 8 min and during t h i s per iod the head exhib i ted frequent coughing movements. Fol lowing removal of the head from the operating t a b l e , i t was placed in to a c y l i n d r i c a l p l a s t i c box and held by a th in rubber bag in order to prevent leakage of the external medium contained i n s i d e . ( F i g . 6) . The g i l l s were i r r i g a t e d with a 1 mM NaCl so lu t ion by a tube placed in to the mouth and were perfused at constant p u l s a t i l e flow (4.5 ml/min) from a double reservo i r 29 Figure 5. Diagram of catheter and p l a s t i c c o l l a r pos i t ions for the i s o l a t e d , sa l ine -per fused rainbow t rout head preparat ion. 0 3-0 PLASTIC COLLAR INPUT CATHETER DA. CATHETER VENTRAL AORTA BULBUS ARTERIOSUS VENTRICLE 31 Figure 6. Schematic representat ion of apparatus used for i s o l a t e d , sa l ine -per fused head preparat ion. Sal ine i s pumped from reservo i rs through a coo l ing chamber and into the head v i a a vent ra l a o r t i c catheter . Post -g i l l sa l ine i s c o l l e c t e d v i a a catheter implanted in the dorsa l aor ta . 32 -COOLING CHAMBER -PRESSURE TRANSDUCER -CARDIAC PUMP 33 with gas equ i l ib ra ted sa l ine (0.3% CC^ in a i r ) containing 10 M noradrenaline (Arterenol) using a cardiac pump. Gas mixtures were suppl ied by gas mixing pumps (Godart). Input pressure was monitored v i a a T junct ion in the input catheter connected to a Statham P23dB pressure transducer and chart recorder . Isolated heads which exhibi ted i n i t i a l input pressures greater than 70 cm R^O usual ly were i n d i c a t i v e of trapped a i r bubbles in the branchia l vasculature and were d iscarded. Heads were perfused for a per iod of 5-10 min (or u n t i l input pressure had s tab i l i sed ) p r i o r to experimentation. Experiments consisted of monitoring ch lor ide i n f l u x ( J ^ n C l ) and net f lux ( J n e t ) while adjust ing P C Q and £HC0 3~3 or adding the drugs, SITS ( B r i t i s h Drug House) and acetazolamide (Diamox; hederle) to the per fusate . Changing from one experimental con-d i t i o n to another was accomplished by switching to the other sa l ine reservo i r by means of a 3-way tap (see F i g . 5) . P ^ was changed by adjust ing the gas mixing pumps. SITS and Diamox - 3 were added to f i n a l concentrat ions of 10 M. J ^ C l was determined by fo l lowing the disappearance of 3 6 — CI from the external medium and i t s appearance in the dorsa l 3 6 — aor ta . Approximately 8 micro Curies of CI were added to the external medium and fo l lowing a 5 min mixing p e r i o d , simultaneous samples were taken from the external medium and dorsal aorta 3 6 every 5 min fo r the durat ion of the experimental per iod . CI a c t i v i t y was determined using l i q u i d s c i n t i l l a t i o n count ing. Correct ion using a quench curve was not necessary as a l l samples 34 showed i d e n t i c a l counting, e f f i c i e n c y . External ch lor ide conc-entrat ion was determined using a Technico autoanalyser. J \ n C l , expressed as u equ iv . / lOOg/h was ca lcu la ted using the fo l lowing equation. (Payan, 197 8) : CPM D.A. x Q x C l ~ ext . J . C l ~ = (6) i n CPM ext. A l l experimental values are presented in tables and f igures as means ± standard error from the mean. Results were s t a t i s t -i c a l l y analysed using Student 's t tes t between sample means where appropriate and 5% was taken as the f i d u c i a l l i m i t of s i g -n i f i c a n c e . S t a t i s t i c a l treatment of pH values i s not v a l i d and for t h i s reason t tes ts were performed on only . For c l a r i t y , both and pH values are reported in t a b l e s . 35 RESULTS (1) Isolated, saline-perfused holobranchs, totally saline-perfused rainbow trout and Coho salmon No sa l ine -per fused .prepara t ion displayed measureable C 0 2 excret ion at p h y s i o l o g i c a l pH and P _ except t o t a l l y perfused 2 Coho salmon and t h i s was not s i g n i f i c a n t (Tables 2-4) . In fact C n usua l ly was higher in p o s t - g i l l sa l ine i n d i c a t i n g a net L ° 2 ' uptake of CO.,. Only when perfusate P C Q was increased to 2% C 0 2 (15 mm Hg; perfused holobranchs) or 1.2% (9 mm Hg; t o t a l l y perfused rainbow t r o u t ) , was M C Q measureable. The d i f fe rences in C^Q across the g i l l between normal and high P ^ groups are h ighly s i g n i f i c a n t . Increasing the concentrat ion of H C 0 3 in • the perfusate had no e f fec t on M in t o t a l l y perfused Coho C U 2 salmon (Table 4 ) . The trans-membrane po ten t ia l (basal membrane of g i l l e p i t h e l i a l c e l l s ) , as measured with microelectrodes (Maetz & Campanini, 1 9 6 6 ) , a lso d id not vary from -21 mV ( inside of c e l l negative) as HCO^ was r a i s e d . In the Coho salmon preparat ion a P N increase of approximately 2 0 mm Hg was observed U 2 across the g i l l s (Table 4) and t h i s a lso d id not vary with i n c -reased [HCO^ " J . Under normal cond i t ions , both perfused arches and perfused rainbow trout showed net H + ion movements from water to blood (Tables 2 & 3 ) . When input P C Q was elevated to 2% C 0 2 i n perfused holobranchs, a s i g n i f i c a n t change in d i r e c t i o n occurred; net H + ion movement now was from blood to water (Table 2 ) . 36 Table 2. The e f fec t of perfusate P c o on CO,, excret ion (M C Q ) 2 2 and acid-base status of i s o l a t e d , sa l ine -per fused rainbow trout holobranchs. Mean values ± S .E .M . (n = 6 holobranchs). Af ferent Ef ferent M C Q sa l ine sa l ine A ' c o 2 P C 0 2 P c o (mmHg) 1 5 . 4 0 ± 0 . 8 1 3 . 1 4 ± 0 . 6 - 2 . 2 6 ± 0 . 4 * \e +] (nM) • 55.6411.7 48.3311.8 -7.3111.5* pH 7.25 7.32 +0.07 2 I Normal C c o (mM) 8.7110.4 8 .88±0 .5 +0.1710.1 - 0 . 2 7 a P C 0 2 P C Q (mmHg) 4.0110.3 4.7610.5 +0.7510.2 [H"] (nM) 19.0410.9 21. 8211.1 +2.7810.8 pH 7.72 7.66 -0.06 I I High C n _ (mM) 10.8210.6 10.5010.6 -0.32+0.1 0.51 a Negat ive value for M C Q ind ica tes net uptake of CO by the e f ferent 2 2 s a l i n e . * S i g n i f i c a n t l y d i f f e r e n t from normal value at 5% confidence l e v e l . 37 Table 3. The e f fec t of perfusate P c o on CC>2 excret ion (M C Q ) and acid-base status of t o t a l l y sa l ine -per fused rainbow t rou t . Mean values ± S .E .M . (n = 5 f i s h ) . Input Dorsal M A 2 sa l ine aorta / \ I Normal C c 0 (mM) 10.5710.3 11.6110.3 +1.0410.3 - 1 . 8 7 a P C 0 2 P C 0 2 (mmHg) 3 ' 1 4 ± 0 - 2 4.8010.1 +1.6610.1 H + (nM) 12.0810.6 16.3710.8 +4.2910.5 pH 7.92 7.79 -0.13 II High Cnn (nM) 11.0010.4 10.4610.3 -0.5410.4 0.97 P ' c o 2 C 0 2 P C0 (m m H5) 9.16+0.2 9.8810.2 +0.7210.2 H + (nM) 32.2811.1 36.4811.3 +4.20+0.9 pH 7.49 7.44 -0.05 * a * Negative value for M^ 0 ind ica tes a net uptake of C0 2 in to the dorsa l aor ta . * S i g n i f i c a n t l y d i f f e r e n t from normal value at 5% cofidence l e v e l . 38 Table 4. The e f f e c t of perfusate HCO^ concentrat ion on CG^ « excret ion ( M c 0 ), resp i ra tory and acid-base status and trans-membrane p o t e n t i a l (TMP) in t o t a l l y s a l i n e -perfused Coho salmon. Mean values ± S . E . M . (n = 5 f ish) Input Dorsal M sa l ine aorta / \ Normal c c a (mM) 1 1 . 8 6 ± 0 . 4 1 1 . 7 2 ± 0 . 4 - 0 . 1 4 ± 0 . 2 0.16 HCO. 2 HC03~(mM) 1 1 . 2 7 ± 0 . 4 1 1 . 1 3 ± 0 . 4 -0.1410.2 P n (mmHg) 59.27+2.1 79.3011.9 +20.112.3 2 TMP (mV) -21 II High CQQ (mM) 33.1510.7 33.3010.8 +O.1510.2 - 0 . 1 7 a HCO. HC0 3 (mM) 31.4910.7 31.6410.8 +0.1510.2 P 0 (mmHg) 58.4011.9 80.0011.7 +21.612.2 u 2 TMP (mV) -21 a . • Negative value for M ind icates net uptake of CO in to the ^ u2 2 dorsa l aor ta . 39 (2) Isolated, saline-perfused rainbow trout head preparation At f i r s t considerable d i f f i c u l t y was encountered in the measurement of J . CI in the i s o l a t e d , sa l ine -per fused head i n ' ^ preparat ion. I n i t i a l i n f l u x values often were s i m i l a r to those reported from in vivo studies but in every instance J \ n C l dec-reased rap id ly with time and fo l lowing 10-15 min of pe r fus ion , was near zero. Eventual ly i t was dicovered that J . CI remained J i n constant i f noradrenaline (10 M) f i r s t was added to the per fus -ate . Thus, a l l subsequent experiments using t h i s preparat ion were performed with 10 ^ M noradrenaline in the s a l i n e . This phenomenon of catecholamine st imulat ion of ch lor ide i n f l u x w i l l be discussed in greater d e t a i l i n Chapter IV. F i g . 7 summarises the e f fec t of perfusate [HCO^^] on ch lor ide i n f l u x . Increasing the concentrat ion of HCO^ from zero to 5 mM and then 13 mM resul ted in s i g n i f i c a n t 50% and 85% increases in ch lor ide i n f l u x r e s p e c t i v e l y . I n te res t ing ly , r a i s i n g QjCO^^j from 13 mM to 26 mM caused a reduct ion in J . C l . S imi la r s t im-m u l a t i o n of J \ C l by HCO^ a lso was observed fo l lowing addi t ion -3 of 10 M SITS (an anion transport inh ib i to r ) or Diamox (a c a r -_3 bonic anhydrase i n h i b i t o r ; , M a r e n , 1 9 7 7 ) . Adding 10 M t h i o c y -anate (a ch lor ide transport inh ib i to r ) to the external medium also d id not abo l ish the st imulatory e f fec t of HCO_ on J . C l 2 3 i n (F ig . 8) although i t d id i n h i b i t J ^ n C l in HCO^ - f r e e s a l i n e (F ig . 8 ) . The addi t ion of HCO^ to HCO^ - f r e e sa l ine caused a large increase in pH (HC0 3 ~ - f r e e s a l i n e , pH = 6.92±0.08; 13 mM HC0 3~ 40 Figure 7. The e f fec t of perfusate bicarbonate concentrat ion on ch lor ide in f lux in the i s o l a t e d , perfused head of rainbow t rou t . n = 4 for each curve shown. 41 42 Figure 8. The e f fec t of perfusate bicarbonate concentrat ion on ch lor ide i n f l u x in the i s o l a t e d , perfused head -3 of rainbow trout fo l lowing addi t ion of e i ther 10 M _3 SITS (n = 4) or 10 M Diamox (n = 3) i n the perfusate _3 or 10 M external thiocyanate (n = 3). 43 TIME, min 44 Figure 9. The e f fec t of perfusate bicarbonate concentrat ion on input pressure in the i s o l a t e d , sa l ine -per fused head of rainbow trout (n = 5). 45 46 s a l i n e , pH = 8.04+0.4) and was associated with a profound decr -ease i n input pressure . F i g . 9 i l l u s t r a t e s the e f f e c t of chang-ing from HCO^ - f r e e sa l ine to 13 mM HCO^ sa l ine on input pressure . Changing to 5 mM sa l ine caused a l esser increase in pH and was associated with a smaller decrease of input pressure . Switching from 13 mM to 26 mM HCO^ sa l ine however, was without e f fec t on input pressure although once again pH was increased (8 .1-8 .3) . F i g . 10 d isp lays the e f fec t of sa l ine P c 0 on J ^ n C l . Changing from P C Q 7.5 mm Hg (1% CG^) to P C Q zero caused a large reduct ion in J \ n C l which was p a r t i a l l y restored to i n i t i a l l e v e l s by switching back to P ^ 7.5 mm Hg. In contrast to the previous experiments, adjustments of P p n d id not s i g n i f i c a n t l y c u 2 a f fec t input pressure and changes in sa l ine pH were minimal. 47 Figure 10. The e f f e c t of perfusate P _ on ch lor ide i n f l u x in the i s o l a t e d , perfused head of rainbow trout (n = 3) . CHLORIDE INFLUX, uEq-lOOg.h 49 DISCUSSION In recent years var ious invest iga tors have employed i s o l a t e d perfused g i l l preparat ions (Kirschner, 1969; Rankin & Maetz, 1971; Shutt leworth, 1972, 1978; Shuttleworth & Freeman, 1973; Kers te t ter & Kee ler , 1976; Haswell & Randal l , 1978; Wood et al., 1978) or perfused head preparat ions (Kirschner, 1969; Payan & Matty, 1975; Gi rard & Payan, 1977; I s a i a , Maetz & Haywood, 1978; Cla i rborne & Evans, 1980) for studying i o n i c and acid-base regu-l a t i o n or gas t ransfer in f i s h e s . Three major problems must be solved before a perfused g i l l preparat ion can be used for p h y s i o l o g i c a l s tud ies . F i r s t , i t i s e s s e n t i a l to provide adequate s t i r r i n g of the external - medium to reduce boundary layers in the f l u i d in d i r e c t contact with the g i l l epithel ium and allow i t to reach the act ive s i t e of ion transport between the lamellae (Maetz, 1971). Secondly, i t i s necessary to master the complex problems of the haemodynamics of the per fusion f l u i d . T h i r d l y , ca the te r i za t ion of a f ferent and ef ferent blood vesse ls (espec ia l ly in preparat ion of perfused holobranchs) i s often d i f f i c u l t and leakage of e f f luent f l u i d in to the bathing medium leads to inaccuracies in f lux measurements. It qu ick ly became apparent that both sa l ine -per fused h o l o -branchs and t o t a l l y sa l ine -per fused f i s h were not su i tab le for branchia l ion f lux determinations. Leakage of perfusate in to the r e c i r c u l a t i n g water was a major problem with these prepar-a t ions . Q q in sa l ine -per fused holobranchs was at best 75% of 50 meaning that contamination of the external medium w i l l qu ick ly increase the concentrat ions of ions thereby lowering the s p e c i f i c a c t i v i t y . F l u i d leakage in t o t a l l y sa l ine -per fused preparat ions was not as severe but s t i l l prevented accurate ion f lux deter -minat ions. For these reasons, these preparat ions were used only to study excret ion and acid-base regu la t ion . The i s o l a t e d , perfused head preparat ion was used for a l l branchia l ion exchange experiments. This preparat ion i s wel l sui ted for f lux determin-at ions owing to i t s 'non- leak iness ' and the small external volume needed. A small external volume means that small changes in ion concentrat ion can be detected e a s i l y . Other advantages of t h i s preparat ion are that no anaesthetic i s used and the surgery i s r a p i d . Prolonged periods of branchia l ischemia are e l iminated by perfusing with sa l ine during the operative procedures. D i s -advantages of the i so la ted head include the lack of dorsa l a o r t i c back pressure which may a f fec t the pattern of lamel lar per fus ion , and the rapid de te r io ra t ion of t h i s preparat ion (approximately 1 hour) . It i s evident that sa l ine -per fused preparat ions do not excrete C 0 9 at p h y s i o l o g i c a l P_,n and pH. Although t h i s may be due, i n par t , to increased d i f f u s i o n bar r i e rs to gas t r a n s f e r , I f e e l t h i s i s u n l i k e l y to explain the absence of M C Q for the fo l lowing reason. T o t a l l y sa l ine -per fused Coho salmon displayed no s i g n i f i c a n t CO2 excret ion from the perfusion f l u i d but d id show an increase in dorsa l a o r t i c P Q of approximately 2 0 mm Hg. Considering that C 0 2 d i f fuses more e a s i l y than (Dejours, 1975) one would expect CO- to be excreted at an even greater rate than 51 C>2. Nor i s the absence of in sa l ine -per fused preparat ions due to a r t i f i c i a l v e n t i l a t i o n as and oxygen uptake (MQ ) i n blood-perfused f i s h are unaffected by a r t i f i c i a l v e n t i l a t i o n (see Chapter I I ) . I t i s l i k e l y that the lack of C0 2 excret ion i s due soley to the absence of red blood c e l l s in the per fusion f l u i d . That perfusate HCO^ concentrat ion i s without e f f ec t on M in sa l ine -per fused f i s h ind icates that the branchia l e p i t h -elium i s impermeable to HCO^ and that i t s movement from plasma to epithel ium does not const i tu te a pathway for C0 2 excre t ion . Measurement of a constant trans-membrane p o t e n t i a l of e p i t h e l i a l c e l l s at a l l HCO^.: concentrat ions support t h i s conc lus ion . Of course i t i s poss ib le that HCO^ may not contr ibute to the t rans -membrane po ten t ia l i f i t s movement in to the epithel ium i s coupled to CI entry in to the plasma. However, that increased perfusate HCO^ i s without e f f ec t on M C Q and that SITS does not e f f ec t CI i n f l u x , ind ica te that t h i s i s u n l i k e l y . These r e s u l t s oppose the hypothesis that o v e r a l l C0 2 excret ion i s governed by the rate of HCO^ entry into branchia l e p i t h e l i a l c e l l s (Haswell, 1978; Haswell & Randal l , 1978; Haswell et a l . , 1980) and supply i n d i r e c t evidence for a major ro le of the f i s h erythrocyte in t h i s process. Haswell & Randall (1978) a lso perfused f i s h with s a l i n e , and in contrast to the r e s u l t s of t h i s study, observed s i g n i f i c a n t C0 2 excre t ion . This observation contr ibuted supporting evidence to the i r hypothesis of non-involvement of the red blood c e l l in C0 2 excre t ion . Inspection of the i r data reveals that f i s h were perfused with sa l ine equ i l ib ra ted with 1% C0 2 in a i r ( p c 0 "7.5 mm Hg, pH 7.5) . This may account for the discrepancy between 52 the i r r e s u l t s and the r e s u l t s of the present study. Whereas sa l ine [j^ CO^  ] had no e f fec t on M C Q , increas ing P C Q in s a l i n e -perfused holobranchs and t o t a l l y sa l ine -per fused t rout d id s t im-ulate M r n which was s i g n i f i c a n t l y d i f f e r e n t from normal va lues . Thus, increas ing the amount of d isso lved CG^ in sa l ine can ." increase M C Q , simply by enhanced d i f f u s i o n of molecular CC^ across the branchia l epi thel ium. Therefore the r e s u l t s of Haswell & Randall ,(197 8) may not be representat ive of CC^ excr - .:' e t ion in normal f i s h . An i n t e g r a l component of the model proposed by Haswell et al. (198 0; see F i g . 2) i s that branchia l carbonic anhydrase (CA) f u n c t i o n a l l y replaces ery throcyt ic CA.„ These authours propose that branchia l CA cata lyses the dehydration of plasma HCO^ thereby producing molecular CC^ which d i f fuses in to the water. In t h i s manner the majori ty of CC^ i s excreted. The model a lso shows that the counter i o n s , HCO^ and H + , exchanged for C l and N a + at the branchia l a p i c a l membrane, a r i se from plasma. This theory contrasts an e a r l i e r model (Maetz & Romeu, 1964; Maetz, 1971; Maetz, Payan & de Renzis , 1976) in which branchia l CA cata lyses the hydration react ion of plasma C0 2 thereby furn ish ing the counter ions , HCO^ and H+(NH^"1") , to be exchanged for C l and Na + r espec t ive ly (F ig . 1) . Experiments were performed using the i s o l a t e d head prep-arat ion in an attempt to v e r i f y one of the above models. The r e s u l t s show that branchia l C l i n f lux i s d i r e c t l y re la ted to the concentrat ion of HCO^ in the per fusate . At f i r s t glance t h i s r e s u l t supports the hypothesis of Haswell & Randall (1978) that 53 HCO-j entry into the g i l l epi thel ium i s an important step in a p i c a l CI /HCO^ exchange. That Diamox was without e f f e c t on CI i n f l u x ind ica tes that the dehydration of CC^ by branchia l CA i s not important in generating exchangeable HCO^ . Diamox pre -v i o u s l y has been found to i n h i b i t both Na + and CI uptake in g o l d f i s h (Maetz & Romeu, 1964) and Na + uptake on ly , in rainbow trout (Kerstetter & K i rschner , 1972). The d i s u l f o n i c s t i lbene d e r i v a t i v e , SITS, has been shown to i n h i b i t anion movements (HCO^ and CI ) i n red blood c e l l s (Knauf & Rothste in , 1971; Cabantchik & Rothste in , 1972; Haswell & Kim, 1978; Shami, Rothstein & Knauf, 1978; Cabantchik, Knauf & Rothste in , 1978), t u r t l e bladder (Ehrenspeck & Brodsky, 1976; Cohen, Mueller & Steinmetz, 1977), squid axon (Russel l & Boron, 1976) and Apiysia neurons" (Russe l l , 1978). The fact that SITS has no e f fec t on HCO^ stimulated CI i n f l u x i s s u r p r i s i n g and d i f f i c u l t to expla in given the previous data. One must e i ther assume that SITS i s i n e f f e c t i v e i n b locking HCO^ entry in to the g i l l epi thel ium or that t h i s process does not take place to any large degree. Furthermore, th iocyanate, a potent i n h i b i t o r of branchia l CI /HCO^ exchange in f i s h (Epstein , Maetz & de Renzis , 1973; Kers te t ter & K i rschner , 1974; de Renzis, 1975) a lso was without e f f ec t on HCO^ stimulated CI i n f lux although i t d id i n h i b i t J . CI under normal condit ions and i n HCO_. - f r e e in 3 s a l i n e . Given these r e s u l t s , how does one expla in the s t imula t -ory e f f ec t of HCO^ on CI uptake? A poss ib le explanation i s the increased pH of the perfusate associated with HCO^ a d d i t i o n . 54 Accompanying t h i s pH change i s a sharp decrease in per fus ion pressure. Thus, i t i s poss ib le that C l i n f l u x i s enhanced simply by an increase in g i l l funct iona l surface area, assuming that a decrease i n input pressure r e f l e c t s a greater surface area perfused. Depending on the concentrat ion of HCO^ used, perfused preparat ions d isplayed varying degrees of input pressure reduct ion . F i g . 11 descr ibes the r e l a t i o n s h i p between the reduc-t ion in input pressure fo l lowing HCO^ a d d i t i o n , and st imulat ion of C l uptake. It i s apparent that J j L n c l i- s st imulated to the greatest extent when P^ n i s reduced to the greatest"extent . I n te res t ing ly , changing the concentrat ion of perfusate HC0 3~ from 13 mM to 26 mM caused no change in input pressure and C l uptake a lso was unaf fected. In one instance pH was kept constant while switching from HC0 3 - f r e e sa l ine to 13 mM HCC»3 s a l i n e . As expected, input pressure d id not change and unl ike previous experiments C l uptake a lso remained unchanged. The above r e s u l t s ind ica te that s t imulat ion of branchia l C l uptake by perfusate HCC»3 i s due to accompanying haemodynamic changes and not to increased entry of HC0 3 in to the g i l l e p i -thel ium. Once again , the r e s u l t s of the present study oppose the model of Haswell & Randall (1978). On the other hand, the r e s u l t s do support the more-widely accepted model (F ig . 1) wherein branchia l CA cata lyses the hydration of plasma C 0 2 . The s t im-ulatory e f f e c t of perfusate P C Q on C l ~ in f lux can be explained by increased production of HC0 3 by CA cata lysed C0 2 hydrat ion. S imi la r conf irmat ion for t h i s r o l e of branchia l CA came from the 55 Figure 11. The e f fec t of decreasing input pressure on st imulat ion of ch lor ide i n f l u x i n the i s o l a t e d , perfused head of rainbow t rou t . The decreases in input pressure were caused by adding var ious concentrat ions of HCO., to the per fusate . 56 57 + + study of Payan & Matty (1975) who showed that Na /NH^ exchange in the perfused head i s propor t ional to perfusate P p n . The only r e s u l t which remains d i f f i c u l t to in terpre t i s the lack of an i n h i b i t o r y e f f ec t of Diamox on branchia l C l uptake while i n h i b i t i n g Na + uptake. One poss ib le explanation i s the large d i f fe rence in concentrat ions of HCO^ and H + ions in e p i t h e l i a l c e l l s . Although t h e i r concentrat ions have never been determined i t i s l i k e l y they resemble plasma values of approximately 10 mM • -5 + + HCO^ and 10 mM H . The very low pool s ize of H compared to HCO^ may mean that Na + /H + (NH 4 + ) exchange i s sens i t i ve to a red -uct ion in [H^J caused by carbonic anhydrase i n h i b i t i o n while C l /HCO^ exchange remains unaffected by a s imi la r reduct ion in HCO^ • In t h i s study, C l i n f lux was monitored for 15-20 min fo l lowing addi t ion of Diamox. It i s poss ib le that C l i n f lux i s a f fected only a f te r i n t r a c e l l u l a r HC0 3 l eve ls have been depleted s i g n i f i c a n t l y , which may take longer than 15-2 0 min of CA i n h i b i t i o n . Another poss ib le explanation i s that Diamox i n h i b i t s Na + /H + (NH 4 + ) exchange by in te rac t ing d i r e c t l y with the transport s i t e rather than by i n h i b i t i o n of carbonic anhydrase. This explanation i s favoured for the fo l lowing reasons. The basal,membrane of g i l l e p i t h e l i a l c e l l s i s permeable to H + ions (McWilliams & P o t t s , 1978) implying a cont inual supply of exchan-geable protons. Furthermore, subsequent experiments (see Chapter III) have shown that C l uptake i s sens i t i ve to immediate red -uct ions of i n t r a c e l l u l a r HCO^ . Therefore the argument of d i f f e r e n t pool s izes of HCO^ and H + i s l i k e l y i n c o r r e c t . This study, while showing that the branchia l epithel ium 58 i s impermeable to HCO^ , and that the entry of HCO^ into the epithel ium i s not important for CC>2 excret ion or branchia l ion uptake, has only i n d i r e c t l y provided evidence for an important involvement of the erythrocyte in 'CC^ excre t ion . The fo l lowing chapter w i l l deal s p e c i f i c a l l y with the ro le of the red blood .. c e l l in C0 9 excret ion and acid-base regu la t ion . 59 CHAPTER II C0 2 EXCRETION IN A SPONTANEOUSLY VENTILATING, BLOOD-PERFUSED RAINBOW TROUT PREPARATION 60 INTRODUCTION The previous chapter'.showed that the entry of plasma HCO^ in to the g i l l epi thel ium i s not important for C0 2 excret ion or branchia l ion exchange, unl ike the theory of Haswell et al. (1980). The r e s u l t s ind icated i n d i r e c t l y that the red blood c e l l may have a major involvement in C0 2 excre t ion , also contrary to the theory of Haswell & Randall (197 8). In order to evaluate d i r -e c t l y , the cont r ibut ion of the erythrocyte to C0 2 excret ion i n f i s h , i t i s necessary to work with l i v e in tac t animals or with blood-perfused preparat ions. Studies with l i v e in tac t f i s h are l im i ted by the d i f f i c u l t y i n obta in ing appropriate simultaneous blood samples and low vascular volumes of f i s h (Holmes & Donald-son, 1970) . Low vascular volumes mean that only a few small blood samples can be withdrawn from a s ing le animal at one t ime. Moreover, with in vivo studies i t i s often d i f f i c u l t to d i s t i n g -u ish between the s p e c i f i c e f f ec ts of an experimental manipulation or a secondary e f f e c t , such as card iac output adjustments. Frequently i t i s necessary to a l t e r blood chemistry or add drugs when examining C0 2 excre t ion . This presents numerous problems using l i v e i n t a c t f i s h which can be overcome e a s i l y by b lood-perf usion . This chapter descr ibes a spontaneously v e n t i l a t i n g , b lood-perfused rainbow trout preparat ion which overcomes many of the problems associated with in vivo and s a l i n e - p e r f u s i o n s tud ies . The preparat ion i s charac ter ised , and i t s s u i t a b i l i t y for the 61 study of C0 2 excret ion i n f i s h assessed. Once having determined the s u i t a b i l i t y of t h i s preparat ion, experiments were designed s p e c i f i c a l l y to test the two c o n f l i c t i n g theor ies of C0 2 excret -ion out l ined p rev ious ly . The primary ob ject ive of t h i s i n v e s t -iga t ion was to assess the importance of the red blood c e l l i n C0 2 excre t ion . It was hoped that a comparison of r e s u l t s from b lood-per fus ion and s a l i n e - p e r f u s i o n (Chapter I) studies would enable me to determine the r e l a t i v e contr ibut ions of the branchia l epi thel ium and red blood c e l l to C0 9 excre t ion . 62 MATERIAL AND METHODS Experimental animals Rainbow trout ( Salmo gairdneri ) weighing between 278-378 g were obtained from Sun Va l ley Trout Farm (Mission, B r i t i s h C o l -umbia) and maintained in a s im i l a r manner as described in Chapter I. Blood c o l l e c t i o n and preparat ion Donor f i s h were anaesthetised with 1:15,000 MS 222 (pH adjusted to 7.0-7.5) and then t ransfer red to an operating table (Smith & B e l l , 1967) where 1:20,000 MS 222 (aerated and cooled to 4°C) was r e c i r c u l a t e d over the g i l l s . To f a c i l i t a t e blood withdrawl, f i s h were implanted with chronic indwel l ing dorsa l a o r t i c cannulae (Smith, 1978) and allowed to recover for a t . l eas t 24 h in darkened Perspex boxes. Genera l ly , 12 f i s h were cannulated and would supply enough blood for two per fusion exp-eriments. Blood was c o l l e c t e d from donor f i s h immediately p r i o r to each per fusion in the fo l lowing manner: Approximately 3 ml of Cort land sal ine(Wol f , 1963) containing 2000 USP uni ts of heparin were in jected in to the dorsa l a o r t i c cannula and fo l lowing a f i v e min mixing p e r i o d , blood was withdrawn anaerob ica l ly . T y p i c a l l y , 10 ml of blood could be obtained from each f i s h using t h i s technique. A f i n a l blood volume of 100 ml usua l ly was r e -quired for a s ing le perfusion preparat ion and was prepared by d i l u t i n g donor blood with sa l ine to a desi red Hct of 10-12%. Blood then was d iv ided into three or four tonometer f l a s k s . 63 These were shaken cont inuosly (Burre l l wr ist act ion shaker) and e q u i l i b r a t e d with gas mixtures (0.4% C0 2 i n 40% a i r , remainder N 2) c l o s e l y resembling t rout venous blood gas tens ions . These mixtures were suppl ied by gas mixing pumps (Wosthoff). Surg ica l procedures A f i s h cannulated the previous day and which had a patent dorsa l a o r t i c cannula was anaesthetised as before . A second cannula (PE 50, ID 0.58 mm, OD 0.97 mm) was implanted in the buccal cav i ty to monitor v e n t i l a t o r y movements as described by Saunders ' (1961). The f i s h was t ransfer red to an operating table where the g i l l s were i r r i g a t e d throughout the operat ion with aer-ated 1:20,000 MS 222 so lu t ion e i ther retrograde from tubes placed in the opercular openings or orthograde from a tube in the mouth. The f i s h was l a i d supine and the pericardium exposed by cut t ing the skin above the heart and c a r e f u l l y par t ing the hyp-a x i a l musculature down the mid l ine . Any small vesse ls which bled into the opening were cauter ised with a B i r tcher e l e c t r o -s e c t i l i s uni t (Bir tcher C o r p . , Los Angeles) . The pericardium was opened and heparin (2000 USP uni ts in two ml sal ine) was in jec ted into the blood v i a the dorsa l a o r t i c cannula and allowed to c i r c u l a t e for f i v e min. As much blood as poss ib le was then withdrawn from the dorsa l aorta and discarded because i t contained anaesthet ic . The vent ra l a o r t i c (input) catheter consis ted of 2.5 cm of s i l a s t i c rubber tubing (ID 1.45 mm, OD 2.45 mm) attached to a 13 gauge hypodermic needle shaf t . The catheter was connected 64 to a reservo i r of Cort land sa l ine to which 10 J M noradrenaline (Arterenol) had been added. Sal ine was f i l t e r e d through m i l l i p o r e d i s c s (Mi l l ipore Inc . , 0.45 uM) before use. The bulbus was cut just caudal of the midpoint and the vent ra l a o r t i c catheter inser ted (F ig . 12) and t i e d in p lace . The per fusion flow was star ted and the animal was perfused at a pressure of 50 cm fi^O to c lea r a l l vesse ls of b lood. Af ter exsanguinat ion, the perfusion flow was reduced and the r e t r o -grade (venous return) catheter inser ted and secured. The venous return catheter was a h e a t - f l a r e d piece of PE 200 tubing (ID 1.4 mm, OD 1.9 mm) which was inser ted into the bulbus through the same cut as the input catheter (F ig . 12). Perfusion was resumed while catheters were anchored to the body wal l and the i n c i s i o n closed with sutures. The f i s h was t ransfer red to a van Dam type box (see Davis & Cameron, 1971) where b lood-per fus ion was star ted (F ig . 13). Water flow over the g i l l s was maintained during recovery from the operat ion by a tube in the mouth. The operat ion took, on average, 45 min. Interuptions to perfusion with e i ther blood or sa l ine were less than a minute. During s a l i n e - p e r f u s i o n on the operat ing t a b l e , f i s h often exhib i ted v e n t i l a t o r y movements, p a r t i c u l a r i l y coughing. Once placed into the box and perfused with b lood, equi l ibr ium was regained and regular v e n t i l a t i o n resumed wi th in 30 min. F i s h were l e f t for 2-3 h to recover from the acute e f fec ts of anaesthesia before any experiments were s ta r ted . 65 Figure 12. Diagram of catheter pos i t ions in the heart of t rout used for b lood-per fusion experiments. A, atrium; B, bulbus a r t e r i o s u s ; IC, input catheter ; VRC, venous return catheter . 67 Figure 13. Schematic representat ion of b lood-per fus ion of rainbow t rou t . Q, flow record d isplayed on chart recorder; S, sampling s i t e s ; VR, venous return to tonometer f l a s k s ; W, windkessel to adjust pulse pressure . 6 8 69 Blood per fusion Blood was held in three or four tonometer f l a s k s . Each tonometer, containing approximately 30 ml of b lood, had a po ly -ethylene tube (PE 160, ID 1.14 mm, OD 1.57 mm) leading to a set of three-way taps enabling blood to be drawn in to a cardiac pump. The three-way taps allowed switching from one f l a s k to any other without in terupt ion of per fus ion . The cardiac pump (Harvard p u l s a t i l e blood pump, model 1405) was modif ied as described by Davie & Daxboeck (1981). The flow ra te , Q, ca lcu la ted by mult-i p l i c a t i o n of stroke volume (SV) and frequency (F) , was accurate to within 1% of independent gravimetr ic measurements and was not pressure s e n s i t i v e . This pump allowed independent adjustments of SV and F without flow in te rup t ion . The f ixed phase of the pump act ion (1/3 s y s t o l e , 2/3 d iasto le ) made i t idea l for b lood-per fusion studies where the act ion of the heart i s to be simu-lated . Pulse pressure was adjusted by changing the s ize of a gas space at the top of a wide-bore sidearm in the perfusion l i n e (F ig . 13). The blood was pumped into the ventra l aorta v i a the input catheter and c i r c u l a t e d through the ent i re body. Despite two catheters in the bulbus, v e n t r i c u l a r contract ions were maintained and pumped venous return blood back to the ton-ometers.via a wide-bore s i l a s t i c rubber tube (ID 1.97 mm, OD 3.05 mm). Perfusion flow was adjusted by a l t e r i n g SV at a cardiac pump frequency of 4 0 st rokes/min, to that necessary to maintain 70 dorsa l aor t i c pressure (DAP) at 40 cm H^O. This flow rate was always about 16-17 ml/min/kg and was taken as the 'normal' value for that preparat ion. Blood sampling and ana1ysis Approximately 0.7 ml of blood was withdrawn simultaneously from each of the three sampling s i t e s ( input, dorsa l aorta and venous re tu rn ) . Samples were sealed and stored on ice during the ana lys is per iod . Genera l ly , input blood, was analysed f i r s t , fol lowed by dorsa l a o r t i c blood and venous re turn . Ana lys is was completed wi th in 15 min of sampling and no measured blood var iab le was found to change during t h i s pe r iod . pH and P Q measurements were made u t i l i s i n g an Instrument Laborator ies Micro 13 pH/blood gas analyser . C c o was determined as described in Chapter I and t o t a l oxygen content (C ) was determined using U 2 the method of Tucker (1967) with a Radiometer PHM-71 d i g i t a l ac id-base analyser and associated 0 9 e lec t rode . A l l pH and P measurements were performed at ambient water temperature, while corresponding C C Q and C Q determinations were performed at 45°C to speed up the response time of the e lec t rodes . P C Q and [H C 03^] were ca lcu la ted using the measured pH and C values and a r e -organizat ion of the Henderson-Hasselbalch equation (see Mater ia ls and Methods, Chapter I ) . For s i m p l i c i t y only C p n values are 2 reported in the r e s u l t s while [H c 03^] values have been omitted. At p h y s i o l o g i c a l pH, {^HC03^] comprises approximately 95% of blood C C Q and as such C C Q i s a reasonable approximation of [HCO^^J Following a n a l y s i s , a small por t ion of blood was used to determine 71 haematocrit (Hct) and any remaining blood was returned to the tonometer or centr i fuged and frozen so that plasma samples could be analysed at a l a te r date. Plasma [ c i^j was determined with a Buchler-Cot love amperometric t i t r a t o r and osmolari ty was meas-ured using an Osmette f reez ing point depression osmometer. Pressure and: flow recording Pressure ( input, dorsa l a o r t i c , v e n t r i c u l a r and buccal) a l l were measured using Statham P23Db pressure t ransducers, manometrically c a l i b r a t e d against a s t a t i c column of water. Mean pressure was ca lcu la ted as d i a s t o l i c + 1/3 pulse pressure (Burton, 1972). The pressure drop across the input catheter was measured, with l iga tures s t i l l i n p lace , a f ter each exper i -ment. Catheter res is tance was used to cor rec t measured input pressures fo r each preparat ion. Inflow was measured with an IVM blood flow transducer . Each of the pressure and flow s igna ls was ampl i f ied and displayed on a Brush s ix channel recorder . Experimental protocol Experiments involved manipulation of input blood Hct, P^Q and C _ n as wel l as the addi t ion of the anion transport i n h i b i t o r , c u 2 SITS. T y p i c a l l y , 'normal' blood samples were withdrawn from the tonometer ( input) , dorsa l aorta and venous return and analysed immediately for C C Q , C Q , pH, P Q and Hct. Input blood then 2 2 2 was changed by switching to another tonometer which had been prepared appropr ia te ly . Fol lowing a f i v e min adjustment p e r i o d , 72 blood samples again were withdrawn and analysed. A 'normal' per iod always preceded and followed any experimental pe r iod . Input blood Hct was adjusted e i ther by adding known volumes of red b lood. .ce l ls or plasma, obtained from donor f i s h . Three categor ies of blood were u t i l i s e d ; normal Hct (approximately 10%), high Hct (approximately 20%) and low Hct (approximately 4%). P _ was doubled by changing the gas mixture e q u i l i b r a t i n g the blood from 0.4% to 0.8% CO,, in 40% a i r , the remainder N 2 « These mixtures were provided by gas mixing pumps (Wosthoff). Blood C C Q was increased by the addi t ion of known quant i t ies 2 -4 of NaHCO^. SITS was added to a f i n a l concentrat ion of 10 M. A l l experimental values are presented in tables as means ± standard error from the mean. Results were s t a t i s t i c a l l y analysed using Student 's t tes t where appropriate between sample means and 5 or 10% was taken as the f i d u c i a l l i m i t of s i g n i f -icance . These experiments were performed in co l l abora t ion with Dr. P. Davie and C. Daxboeck. RESULTS Character iza t ion of the 'normal' spontaneously v e n t i l a t i n g , blood-perfused rainbow trout preparat ion F i s h star ted to v e n t i l a t e spontaneously fo l lowing 20-30 min of blood per fus ion . Once v e n t i l a t i o n became regu lar , the mouth tube which ass is ted water flow over the g i l l s was removed and f i s h i r r i g a t e d the i r g i l l s at 69 ± 1 v e n t i l a t i o n s / m i n . By t h i s time f i s h had regained t h e i r r i g h t i n g and v i s u a l t rack ing re f lexes l o s t during anaesthesia. Some f i s h became agi tated and attempted to swim during the i n i t i a l recovery per iod . V i s u a l disturbances from the surroundings were e l iminated , in par t , by masking the box with black p l a s t i c sheets. T y p i c a l simultaneous recordings of c a r d i o - r e s p i r a t o r y var iab les from a preparat ion are shown in F i g . 14. Vent i l a to ry in te rac t ions on the input pressure t race were frequent ly observed, e s p e c i a l l y during a resp i ra tory 'cough' (Hughes & Ardeny, 1977) (see F i g . 14a). This pulse of increased pressure , whether in phase with the cardiac pump cyc le or not , usua l ly was transmitted through the g i l l vasculature to some extent and was evident in the dorsa l a o r t i c pressure t race (also see Wood & Shel ton, 1980a). F i g . 14b shows a por t ion of input pressure trace in which pulse pressure had been r a i s e d . Bradycardia i s associated with exposure of t rout to hypoxic water and i s one of the better described card iovascular re f lexes in f i s h (Daxboeck & Holeton, 1978; Smith & Jones, 1978). Blood-perfused f i s h showed t y p i c a l 74 Figure 14. Record of pressure and flows from spontaneously v e n t i l a t i n g blood-perfused rainbow t rou t . (a) . Simultaneous records of flows and pressures from a. preparat ion which d isplayed c a r d i o - r e s p i r a t o r y i n t e r a c t i o n s . Arrow heads show in te rac t ions of resp i ra tory movements on the pressure and flow t races . Q, per fusion flow ra te ; VAP, vent ra l a o r t i c pressure; DAP, dorsa l a o r t i c pressure; BP, buccal pressure record of v e n t i l a t o r y movements; VP, v e n t r i c u l a r pressure record of i n t r i n s i c heart a c t i v i t y . (b). Record of VAP during increased pulse pressure , (c) . Record of VP during exposure to hypoxic water showing the 'on ' response (bradycardia) a f ter 150 sec to hypoxic water and the ' o f f response (post-hypoxic tachycardia) a f ter resumption of normoxic water f low. 75 ml-min A 10|-,1 5 Oi ioor VAP cmH20 50| 0 ioor DAP cmH20 S 0! BUCCAL R c m ^ O B -TIME.SEC N2 ON 180 SEC. T T < OQ 111 X y (0 z E z 10 SEC 10 SEC UU www1 INTRINSIC^ HEART BEAT V/ V V V VV V 10 SEC. A AIR ON + 280 SEC. TIME.SEC. 76 hypoxic bradycardia responses when N 2 was bubbled through the insp i red water (F ig . 14c). Both the 'on ' response (bradycardia) and the ' o f f response (post-hypoxic tachycardia) were observed. These r e s u l t s ind icate that blood-perfused f i s h have operat ional a f ferent and ef ferent re f l ex pathways ava i lab le by which c a r d i o -vascular adjustments may be made. The var iab les measured from undisturbed res t ing b lood-perfused t rout are summarised in Tables 5 and 6. These data are considered to be normal as def ined by the c r i t e r i a set out e a r l i e r (see Mater ia ls and Methods). Under these condi t ions a l l f i s h showed oxygen uptake and carbon dioxide excret ion across the g i l l s . Across the systemic c i r c u l a t i o n , oxygen was extracted and carbon dioxide produced by the t i s s u e s . Branchia l vascular res is tance comprised 43% of the t o t a l vascular res is tance of the f i s h (Tables 5 and 6). In fact a l l preparations behaved as would be expected from data ava i lab le from l i v e in tac t res t ing rainbow t rout . Only [H"^J appeared to change in an unexpected manner across the g i l l and the systemic c i r c u i t s . One might expect opposite r e s u l t s to those shown in Tables 5 and 6; that i s an increase in [H^] across the body and a decrease across the g i l l s consistent with C0 2 production and excret ion r e s p e c t i v e l y . Kiceniuk & Jones (1977) however, reported changes in £H +^J which are s im i l a r to those presented here. Table 5 ind icates that blood was d i lu ted across the g i l l s . Hct f e l l by 14% and plasma osmolari ty decreased by 3.5%. These changes could have been caused by a number of fac tors inc lud ing c e l l volume changes due to M_ n , water uptake across the g i l l s Table 5. Summary of blood variables for the normal, resting state of the spontaneously v e n t i l a t i n g , blood-perfused rainbow trout (n = 15 f i s h ) . X 1.62 + S.E.M. 0.03 n observations 45 X ± S.E.M. n observations 58.8 2:0 45 34.8 1.0 45 Pulse Hct H 4 P°2 C° 2 PC0 2 c c o 2 (cmH20) (%) (nM/1) (mmHg) (mM) (mmHg) (mM) INPUT BLOOD 10.7 10.3 17.7 , 24.9 0.9 3.63 10. 3 (7.76) 0:2 0.2 0.4 1.1 0.1 0.2 0.2 45 45' 44 44 44 44 45 DORSAL AORTIC BLOOD 2.1 R.8 19.7 103.4 1.6 3.66 9.0 (7.72) 0.1 0.2 0.7 2.4 0.1 0.2 0.2 45 42 44 44 44 44 45 Plasma osmolarity (mOsm) 275.4 0.6 5 265.7 0.3 5 9 1 (mM) 111.9 0.7 5 115.8 0.6 5 VENOUS RETURN BLOOD F IHB g (/min) (/min) X 69.4 48.5 9.4 17.6 13.0 0.4 3.65 10.0 273.8 108.2 (7.76) S.E.M. — — 1.0 1.0 0.6 0.8 0.5 0.1 0.2 : 0.2 0.7 1.6 observations 42 37 39 42 41 39 41 42 5 5 * Correspoding pH value I n t r i n s i c heart beat. TABLE 6. Summary of the difference i n blood variables across the g i l l ( input-dorsal aorta) and the systemic ( dorsal aorta-venous return) c i r c u l a t i o n s i n the re s t i n g state of the spontaneously v e n t i l a t i n g , blood-perfused rainbow trout (n = 15 f i s h ) . A INPUT-DORSAL AORTA H + P ° 2 c o 2 PCO 2 c c o 2 p g R g *9C0 2 (nM) (mmHg) (mM) (mmHg) (mM) (cmH20) (uM/lOOg/min XuM/lOOg/min) X +2.1 + 78.8 +0.7 + 0.05 -1.25 23.1 14.2 1.17 2.05 + S.E.M. 0.6 2.4 0.1 0.1 0.1 1.8 1.1 0.1 0.2 n observations 44 44 44 43 45 45 44 44 44 A DORSAL AORTA-VENOUS RETURN P R s s °2 CO2 X -1.4 -89.3 -1.2 + 0.07 +1.01 31.1 19.2 1.97 1.63 + S.E.M. 0.6 2.2 0.1 0.1 0.1 1.0 0.9 0.1 0.09 n observations 41 41 39 41 42 45 44 39 44 79 or simply an a r t i f a c t of sampling. Experimenta1 manipulations The e f fec t of Hct on resp i ra tory and acid-base status in the spontaneously v e n t i l a t i n g , blood-perfused rainbow t rout are shown in F i g . 15 and Table 7. Both CC^ excret ion and uptake increased in a s im i l a r manner as Hct was r a i s e d , while the RQ « • value across the g i l l s (M_,n /M_ ) decreased. Although input blood c u 2 2 P^ . decreased with increasing Hct, dorsa l a o r t i c P„ and the °2 °2 change in P n across the g i l l s (&Pn ) d id not decrease s i g n i f -°2 U 2 i c a n t l y . As expected, input blood C_ was re la ted d i r e c t l y to U 2 Hct (Table 7) . Input blood C p n however, remained v i r t u a l l y c u 2 constant in a l l three groups ind ica t ing l i t t l e or no carbamino-CC>2 formation in the blood. Net hydrogen ion f lux (A.H+) in a l l cases was in the d i r e c t i o n of water to blood but decreased s i g -n i f i c a n t l y in the high Hct group. Regardless of whether high or low Hct blood was used, ven t ra l a o r t i c pressure(VAP) was increased to the same l e v e l above 'normal' (Table.7) . Conversely dorsa l a o r t i c pressure (DAP) in both instances f e l l to s imi la r l eve ls below 'normal' (Table 7) . These changes in VAP and DAP were associated with increases of g i l l res is tance (R ) both during low and high Hct per fus ion . These s imi la r changes in branchia l haemodynamics cannot expla in the d i f f e r e n t i a l e f f ec ts of low and high Hct blood on blood resp i ra tory and acid-base s ta tus . In two instances f i s h were perfused with plasma; during t h i s condi t ion M r n was abol ished completely while dorsa l a o r t i c c u 2 Table 7. Summary of blood resp i ra tory and acid-base status in the spontaneously v e n t i l a t i n g , blood-perfused trout perfused with three d i f f e r e n t Hcts; low, normal and high. n = 6 f i s h ± S .E .M. I. Low II. Normal III. High I. Low II. Normal III. High Hct (%) 4 .3±0 .4 11 .3±0 .5 20.211.6 3.910.5 9.310.5 16.511.2 C ° 2 (mM) 10.8710.6 10.7310.5 11.3010.6 °2 (mM) INPUT BLOOD 0.5910.03 0.9510.21 1.3710.26 G2 (mm Hg) 39.414.2 20.312.5 16.312.6 DORSAL AORTIC BLOOD 10.0610.5 9.1210.4 8.9010.3 0. 8710.12 1. 7710.20 2.8810.40 108.715.8 95.316.6 91.619.9 H + (nM) 15.7811.1 (7.81) 16.8711.3(7.78) 18.3911.4(7.75) 19.5812.2 (7.72) 18.2712.0 (7.75) 18.4211.9 (7.75) VAP (cm H20) 76.2 60.6 76.1 •: DAP (cm H20) 28.3 40.2 33.8 A INPUT - DORSAL AORTA I. Low II. Normal III. High Hct (%) -0.4 -2.0 -5.2 M c o 2 (uM/lOOg/min) 1.3410.3 2.6210.3 3.8710.7 \ (uM/lOOg/min) 0.4610.2 1.3410.2 2.4510.4 O, (mm Hg) 69.417.9 75.015.9 75.3112.1 H + (pM/lOOg/min) 6.0013.5 2.0711.5 0.1311.7 Rg 29.6 12.6 26.1 Corresponding pH value 81 Figure 15. The e f fec t of haematocrit on A) CO,, excret ion and B) oxygen uptake in the spontaneously v e n t i l a t i n g , blood-perfused rainbow t rout . See text for fur ther d e t a i l s . 8 2 HAEMATOCRIT, °ja 83 P n remained unchanged. 2 Increasing HCO^ concentrat ion of input blood to approxi -mately 25 mM s i g n i f i c a n t l y increased M _ four f o l d (Table 8) . c u 2 C Q was not a f fected although M Q appeared to decrease (not s i g -n i f i c a n t l y ) . Increased J H C O ^ a lso was associated with s i g n i f -icant decreases in dorsa l a o r t i c P_ f ^ p o and A H + across the °2 °2 g i l l s (Table 8). Branchia l haemodynamics and v e n t i l a t i o n were unaffected by HCO^ treatment. The s t i l b o n i c ac id der iva t ive SITS has been used to i n h i b i t ch lor ide transport in red blood c e l l s as wel l as other t ranspor t -ing t i ssues (Cabantchik & Rothste in , 1974; Shami et al., 1978). -4 The e f f e c t s of SITS (10 M) on blood resp i ra tory and acid-base status are shown in Table 9. M an M decreased s i g n i f i c a n t l y C 2 2 fo l lowing addi t ion of SITS to the input b lood. Dorsal a o r t i c P 0 and [H"^| both increased s i g n i f i c a n t l y although o n l y A P p and 2 2 n o t A H + was s i g n i f i c a n t l y d i f f e r e n t from normal va lues . Occasion-a l l y , per fusion was switched back to SITS-free blood and in these instances (three f ish) M ^ and M^ were restored to normal l e v e l s . SITS treatment caused no s i g n i f i c a n t e f f e c t s on branchia l haemodynamics or v e n t i l a t i o n . Increasing P_,n of input blood by 1.7 times (3.3 mm Hg to 5.6 mm Hg) (Table 10) s i g n i f i c a n t l y increased M but was wi th-L U 2 out e f f ec t on 0„ t ransfer although input blood P n increased 1 U 2 s i g n i f i c a n t l y . As in other experiments, normal f i s h showed net H + movements from water to blood. When input P r n was e levated, a s i g n i f i c a n t change in d i r e c t i o n occurred; net H movement now 84 was from blood to water. S i m i l a r l y , P C Q across the g i l l s . changed from a s l i g h t increase to a s i g n i f i c a n t decrease. P red ic tab ly , high input P C Q increased dorsa l a o r t i c blood [ H + J and usua l ly was associated with a smal l , slow increase in dorsa l a o r t i c pressure . None of the above experiments produced any v i s u a l signs of s t ress in blood-perfused f i s h except plasma p e r f u s i o n . „ This protocol evoked a v i o l e n t s t ruggl ing response and b r i e f pauses (2-3 sec) in v e n t i l a t o r y movements and i n t r i n s i c heart beat. Due to the severe nature of these responses, t h i s l ine of i nves t -iga t ion was d iscont inued. Table 8. E f f e c t of blood [HCO^ ^  on blood resp i ra tory and ac id-base status in the spontaneously v e n t i l a t i n g , blood-perfused rainbow t rou t . n = 6 f i s h ± S .E .M, C 0 2 (mM) °2 (mM) °2 (mm Hg) INPUT BLOOD c o 2 (mm Hg) H + (nM) I. Normal II. High HCO, 9.86±0.5 24.74±l.o' ** 1.18±0 .2 1.20+0.3 30.1±4.4 23.8±4.7 3.54±0.2 5.50±0.45 18.32±1.7(7.74) 11.36±1.1(7.96) ** DORSAL AORTIC BLOOD I. Normal II. High HC03" I. Normal II. High HC0 3 ' 8.85±0.4 20.58±l.o' ** M CO, 1.86+0.2 1.71±0.3 109.0+3.9 89.9±4.7 5 * * A INPUT - DORSAL AORTA M 0, (uM/lOOg/min)(uM/lOOg/min) 1.63+0.3 6.50±0.4: ** 1.13±0.3 0.84±0.3 °2 (mm Hg) 78.9+6.5 66.2±6.9: 4.36±0.7 5.03±0.7 c o 2 (mm Hg) 0.82±0.5 •0.53±0.8 23.91±3.4(7.64) 12.22±1,7(7.93) H (pM/lOOg/min) 8.71±3.8 1.11±2.7' * * RQg 1.4 7.7 * * Corresponding pH value S i g n i f i c a n t l y d i f f e ren t from normal value at 5% confidence l e v e l S i g n i f i c a n t l y d i f f e ren t from normal value at 10% confidence l e v e l -4 Table 9. E f f e c t of SITS (10 M) on blood resp i ra tory and acid-base status in the spontaneously v e n t i l a t i n g , blood-perfused rainbow t rou t , n = 5 f i s h 1S.E.M, C 0 2 (mM) °2 (mM) (mm Hg) INPUT BLOOD c o 2 (mm Hg) H (nM) I. II, I. II I. II, Normal 10~ 4 M SITS Normal 10~ 4 M SITS 11.5110.3 1 0 . 5 0 ± 0 . 4 ' 9.84+0.3 9 .97±0 .4 M CO, 1 .06±0 .2 1 .22±0 .2 2 0 . 9 ± 1 . 9 2 7 . 0 ± 3 . 2 DORSAL AORTIC BLOOD 1.8310.4 1.6910.3 87.715.8 89.316.0 A INPUT - DORSAL AORTA M '0, (uM/lOOg/min) (uM/lOOg/min) (mm Hg) Normal 10~ 4 M SITS 2.5710.3 0.8010.2' 1.2110.3 0.7010.3' 65.615.6 62.316.2 2.9810.3 3.1610.3 3.2710.3 4.1110.3' ** c o 2 (mm Hg) 0.2910.3 0.9510.3' 12.7111.2(7.90) 14.7110.9 (7.83) 16.1611.0(7.79) 19.6510.9(7.71) H + (pM/lOOg/min) 2.0510.5 4.9411.5 Corresponding pH value ** S i g n i f i c a n t l y d i f fe ren t from normal value at 5% confidence l e v e l * S i g n i f i c a n t l y d i f f e ren t from normal value at 10% confidence l e v e l Table 10. The e f fect of blood on blood resp i ra tory and acid-base status in the spontaneously v e n t i l a t i n g , blood-perfused rainbow t r o u t . n = 6 f i s h 1S.E.M C 0 2 (mM) °2 (mM) °2 (mm Hg) c o 2 (mm Hg) H + (nM) I. Normal II. High P CO, I. Normal II. High P CO, I. Normal II. High P 12 .35±0 .8 13 .28±0 .5 10 .80±0 .7 10.8710.5 M M C ° 2 (uM/lOOg/min) 2.5610.6 CO, 3.9710.4 ** INPUT. BLOOD 0.8710.2 19.813.1 0.9510.2 30.112.5 DORSAL AORTIC BLOOD 1.7810.3 94.118.1 1.6810.3 101.015.3 A INPUT M 3.3110.1 5.6010.3' ** 3.4910.3 4. 4610. A °2 (uM/lOOg/min) 1.5010.2 1.2010.3 DORSAL AORTA P, °2 (mm Hg) 74.317.6 70.915.9 c o 2 (mm Hg) 0.1910.3 -1.1310.4' 13.4110.7(7.88) 20.3811.2(7.69) 15.6910.9(7.81) 19.6811.2(7.71) H + (pM/lOOg/min) 3.6811.3 ** -1.26 2.2 * * ** Corresponding pH value S i g n i f i c a n t l y d i f f e ren t from normal value at 5% confidence l e v e l v 88 DISCUSSION Information in Tables 5 and 6 enable comparisons to be made with data from in vivo and sa l ine -per fused preparat ions (Chapter I ) . They a lso allow one to character ise the preparat ion in terms of card iovascular dynamics and gas exchange. Preparations were maintained for up to 18 h. F a i l u r e always was due to equipment f a i l u r e rather than de te r io ra t ion of the f i s h . Blood used for perfusion was taken from donor f i s h which had recovered for at l eas t 14 h from the e f fec ts of cannulat ion. I n i t i a l l y , cardiac puncture was used to c o l l e c t blood for pe r f -us ion . Blood c o l l e c t e d in th is manner proved unsat is fac tory as pH was always between 7.5-7.7 and probably contained s i g n i f i c a n t amounts of MS 222. Blood from the experimental animal was discarded for the same reasons. T y p i c a l l y , experiments las ted 4-6 h, which, when combined with 2-3 h recovery, meant that measurements were taken from f i s h perfused for no more than 9 h. Over t h i s time period there appeared to be no appreciable de te r io ra t ion of the b lood, as ind icated by l a c k . o f c e l l l y s i s , or of the f i s h , as indicated by stable oxygen uptake and carbon dioxide excret ion r a t e s . Three 0.7 ml blood samples were withdrawn simultaneously from the tonometer, dorsa l aorta and venous re turn . No d i f f e r -ences were observed in pH or gas tensions of blood from tonom-eters or input catheter . An advantage of extracorporeal r e s e r -89 v o i r s i s that repeated sampling does not deplete blood volume of the animal . Consequently more experiments can be performed on blood-perfused preparat ions than on in tac t f i s h . Hct of blood used for perfusion was lower than that meas-ured in in tac t f i s h . Blood was d i l u t e d with sa l ine so that fewer f i s h had to be s a c r i f i c e d for each preparat ion . Trout are capable of surv iv ing with Hcts as low as 3% and values of 15% are not uncommon (see Wood & Shel ton, 1980a). Thus a Hct of 10-12% was considered a reasonable compromise between economy and simulat ion of in vivo cond i t ions . Of course i t would have been better to d i l u t e blood with plasma instead of s a l i n e , unfortunately t h i s was not poss ib le due to the large number of f i s h that would have to be s a c r i f i c e d . Haswell & Randall (1976) reported a plasma i n h i b i t o r , in t rout blood which was impl icated in rendering erythrocytes impermeable to HCO^ . A d iscuss ion of t h i s putat ive plasma i n h i b i t o r w i l l fo l low subsequently. It i s h ighly u n l i k e l y that d i l u t i o n of blood with sa l ine instead of plasma in any way a f fected the o v e r a l l r e s u l t s of these experim-ents . Blood-perfused f i s h were capable of maintaining equ i l ib r ium, v i s u a l t r a c k i n g , swimming, and d isplayed a pronounced bradycardia in response to hypoxia. Occas iona l ly , bleeding was observed from sutures which c losed the i n c i s i o n or anchored catheters to the body w a l l . Small leaks usual ly c l o t t e d , while la rger leaks usua l ly ind icated a dis lodged catheter . Cannulation of the dorsa l aorta 24 h p r i o r to experimentation el iminated leakage 90 from around the point of i n s e r t i o n . In no preparat ion was leak-age so large as to require addi t ion of more blood to the tonom-e te rs . The e f f e c t s of anaesthesia on the f i s h would have been apparent a f te r only 2-3 h recovery (Houston, Madden, Wood & M i l e s , 1971). This l i k e l y was not a problem in blood-perfused f i s h because f i s h were perfused•with blood contain ing no MS 222. The spontaneously v e n t i l a t i n g , blood-perfused preparat ion d isplayed card iovascular dynamics which are s imi la r to those of in tac t animals. Gas exchange at the g i l l s and t i ssues a lso i s s imi la r to data ava i lab le from in vivo measurements (see Tables 5 & 6) . Only branchia l vascular res is tance (Rg) and branchia l H + ion movements d i f f e r e d from t y p i c a l in vivo va lues . Mean Rg of blood-perfused f i s h was 14.2 + 1.1 cm HjO ml 1 min 100 g - 1 (Table 6). In vivo Rg values are t y p i c a l l y around 6 cm E^O ml ^ min 100 g " 1 (6.02, Kiceniuk & Jones, 1977; 5.3, Stevens & Randal l , 1967; 3.4, Wood & Shel ton, 1980). In te res t ing ly , 'normal' blood-perfused f i s h displayed net uptake of H + ions across thr g i l l . The movement of protons across the g i l l appeared to be re la ted to blood pH. When blood pH was lowered, net H + ion uptake a lso was reduced. Occas iona l ly blood-perfused f i s h showed net H + excret ion at very low blood pH's as was observed in sa l ine -per fused preparations (Chapter I ) . These resu l ts ind icate that branchia l proton movements are inde-pendent of C0 2 movemnts but are re la ted to the H + ion gradient 91 between blood and water. McWilliams and Potts (1978) ar r ived at a s im i l a r conclusion by measuring the t r a n s - e p i t h e l i a l po ten t ia l of rainbow trout g i l l s in vivo at var ious external pH's . In conc lus ion , t h i s preparat ion i s su i tab le f o r , and o f f e r s unique oppurtuni t ies to examine a va r i e ty of problems in f i s h card ioresp i ra tory physiology where Q and venous blood gas tensions and chemistry are experimental parameters rather than measured or estimated v a r i a b l e s . Experimental manipulations The r e s u l t s of studies using the spontaneously v e n t i l a t i n g , blood-perfused preparat ion c l e a r l y demonstrate an important involvement of t rout erythrocytes in C C ^ exc re t ion . As such, the r e s u l t s oppose the theory of Haswell & Randall (1978) that the te leos t red blood c e l l i s f u n c t i o n a l l y impermeable to plasma bicarbonate. Much of the evidence supporting non-involvement of the te leos t erythrocyte has come from in vivo studies using anaemic f i s h (Haswell, 1978; Haswell & Randal l , 1978). These experiments showed that CC>2 excre t ion , a r t e r i a l pH and P ^ Q d id not vary fo l lowing 24 h of severe anaemia in rainbow t rou t . The present r e s u l t s however, show a h ighly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between Hct and M and M_ i n d i c a t i n g a common pathway through L U 2 2 the erythrocyte . The d i f fe rences between the two studies are probably due to the profound cardiovascular adjustments associated with severely anaemic f i s h , p a r t i c u l a r i l y increased cardiac output (Q) (Wood, McMahon & McDonald, 1979; Wood & Shel ton, 1980) 92 due to increased stroke volume (Cameron & Davis , 1980). These responses, by increasing the de l i ve ry of p h y s i c a l l y d isso lved CO2 to the g i l l s , would maintain net thereby masking any e f fec ts of anaemia on t h i s process. In experiments using the spontaneously v e n t i l a t i n g , blood-perfused t r o u t , I was able to maintain Q constant thereby e l iminat ing the e f fec ts of c a r d i o -vascular changes on M _ . Branchia l -vascu lar res is tance i n c r -2 eased both during low and high Hct experiments so i t i s u n l i k e l y that changes in branchia l haemodynamics contr ibuted to the o v e r a l l r e s u l t s . Thus I am conf ident that these r e s u l t s r e f l e c t only the concentrat ion of c i r c u l a t i n g erythrocytes . Recently, Wood, McDonald & McMahon (1981) observed that severe experimental anaemia (1-5% Hct) of s tar ry f lounder , Platichthys stellatus and rainbow trout caused resp i ra tory ac idos is (decreased pH, increased P C Q ) supporting the conclusion that plasma bicarbonate i s de-hydrated within erythrocytes in a t y p i c a l mammalian fashion (Cameron & Polhemus, 19 74). Unl ike the present study however, Wood et al. (1981) found no e f f e c t u n t i l a Hct of 5-10% was reach-ed. Again , t h i s i s probably a t t r ibu tab le to card iovascular adjustments which can maintain M n during mild anaemia in in tac t f i s h . At Hcts below 10% i t i s l i k e l y that these compensatory adjustments are no longer s u f f i c i e n t to maintain M _ and blood L U 2 acid-base s ta tus . Perfusion of spontaneously v e n t i l a t i n g , blood-perfused f i s h with plasma abol ished M but was without e f f ec t on dorsa l L U 2 a o r t i c P_ . This fur ther substant iates the argument that the ° 2 d i f fe rences between sa l ine -per fus ion (Chapter I) and b lood-per f -93 usion are due to the presence or absence of erythrocytes and not to d i f fe rences in d i f f u s i o n bar r i e rs to gas t rans fe r . Increasing the concentrat ion of HCO^ i n the input blood of perfused f i s h caused a dramatic increase in M^Q . This i s due to increased f lux of HCO^ in to red blood c e l l s and not in to the g i l l ep i the l ium. I conclude t h i s for two reasons; f i r s t because of the accompanying e f fec t on oxygen transport and sec-ondly, because of the lack of an e f fec t of increased HCO^ in t o t a l l y sa l ine -per fused f i s h (Chapter I ) . That HCO^ i s without e f f e c t in sa l ine -per fused f i s h ind ica tes that the branchia l epi thel ium i s impermeable to HCO^ and that i t s movement from plasma to epithel ium cannot const i tu te a major pathway for CC^ excre t ion . The decrease in dorsa l a o r t i c P n in blood-perfused 2 f i s h during high HCO^ perfusion cannot be re la ted to lower input P Q because f luc tua t ions of input P Q in t h i s range H10 mm Hg) d id not a f fec t dorsa l a o r t i c P_ of normal f i s h . An a l ternate 2 explanation i s increased entry of HCO^ in to the red blood c e l l r a i s i n g pH and thereby f a c i l i t a t i n g 0^ binding to haemoglobin, and reducing the amount d isso lved in s o l u t i o n . Results from t h i s study and others (McWilliams & Pot ts , 19 78; van den T h i l l a r t & Randal l , in preparation) have shown that net H + ion movement across the g i l l i s re la ted to the H + ion gradient between blood and water. Increasing HCO^ of input blood c e r t a i n l y increases t h i s grad ient , yet net H + ion i n f l u x i s reduced s i g n i f i c a n t l y (Table 8). This can be explained by enhanced H + ion excret ion v i a combination with HCO^ forming 94 CC>2 which d i f f u s e s in to the water. Normally, binding to haemoglobin w i l l provide protons to maintain an RQ of 0.7 i f a l l CC>2 i s der ived from HCO^ (German & Wyman, 1937). RQg chan- . ged from 1.4 to 7.7 during high HCO^ per fus ion . C l e a r l y , to maintain an RQg of 7.7 requires a source of protons other than that der ived from haemoglobin oxygenation. Two poss ib le sources are those re leased from pro te ins , e s p e c i a l l y haemoglobin, i f pH r i s e s , and secondly protons that d i f f u s e in to the blood from other compartments. In t h i s instance a l i k e l y candidate i s the water. Blood pH d id not r i s e during passage.through the g i l l s , thus the proton source must have come from another comp-artment, e i ther the g i l l t i ssue or the water. Given that the g i l l epithel ium i s h ighly permeable to H + ions plus the large number of protons requ i red , i n f lu x from the water i s the most probable explanat ion. SITS i s a potent i n h i b i t o r of anion movements across mammalian red blood c e l l s (Cabantchik & Rothste in , 1974; Caban-tch ik et al., 1978; Shami e t a l . , 1978). Again , the i n h i b i t o r y act ion of SITS on i s due to i n h i b i t i o n of e ry throcy t ic HCO^ /CI exchange (chloride sh i f t ) and not to i n h i b i t i o n of HCO_ movement into g i l l e p i t h e l i a l c e l l s . The decrease in M n 3 u 2 associated with SITS treatment must be due to a b o l i t i o n of the ch lor ide s h i f t which decreases red blood c e l l pH, thereby caus-ing haemoglobin to remain in the non-d issoc ia ted state (HbH). The increase in input blood P n also-".can be a t t r ibu ted to a Bohr 2 s h i f t (due to decreased rbc pH) and therefore decreased 0^ binding to haemoglobin. 95 Cameron (1978) demonstrated that te leos t blood (red snapper and rainbow trout) d isplayed a t y p i c a l ch lor ide s h i f t (HCC>3~/C1~ exchange) which was abol ished by the addi t ion of the carbonic anhydrase i n h i b i t o r , acetazolamide. Obaid, C r i t z & Crandal l (1979) a lso have demonstrated HCO^ / C l " exchange i n dogf ish e r -ythrocytes which was blocked by SITS. These r e s u l t s together with the f ind ings of t h i s study, present overwhelming evidence opposing the theory of Haswell & Randall (19 78). Results from in vitro experiments, which indicated the presence of a plasma i n h i b i t o r rendering e ry throcyt ic carbonic anhydrase unavai lable to cata lyse plasma HC0 3~ dehydration (Haswell & Randal l , 1976), can be a t t r ibu ted to methodological problems. The manometric assay u t i l i s e d by Haswell & Randall (1976T measures the change in pressure in a sealed react ion vesse l as HC0 3~ i s coverted to gaseous C 0 2 . Recently i t has been shown that the evolut ion of C0 2 in to the gas phase i s l im i ted by foaming on the surface of the react ion mixture (Heming & Randal l , in preparat ion) . The i n h i b i t i o n of carbonic anhydrase a c t i v i t y of blood suspended in plasma i s due to foaming by plasma prote ins and not to imperm-e a b i l i t y of the erythrocyte to HCC>3 as thought p rev ious ly . Treatment of plasma with de-foaming agents abol ishes the i n h i b -i t o r y e f f e c t s observed using the manometric assay. Having now assessed the r e l a t i v e contr ibut ions of the branchia l epi thel ium and erythrocyte to CC>2 excre t ion , the f o l l -owing chapter w i l l address the problem of acid-base regulat ion in f i s h . 96 CHAPTER III BRANCHIAL IONIC UPTAKE AND ACID-BASE REGULATION 97 INTRODUCTION As discussed p rev ious ly , f i s h regulate acid-base d i s t u r b -ances by slow adjustment of plasma HCO^ l e v e l s and not by vent-i l a t o r y manipulation of Pa-,- as in mammals. For plasma HCO., to increase s u f f i c i e n t l y to restore pH, i t i s c lea r that CO,, excret ion must be reduced or H + ion excret ion enhanced during hypercapnic a c i d o s i s . The presence of Na + /H + (NH 4 + ) and C l /HCO^ exchange pro -cesses in g i l l e p i t h e l i a l c e l l s i s wel l documented (Krogh, 1939; Maetz & Romeu, 1964; Kerste t ter et al., 197 0; Kers te t ter & K i rschner , 1972; Maetz, 1973; de Renzis , 1975; Payan, 1978). The exact l o c a t i o n , whether on the a p i c a l or serosa l side of the g i l l epi thel ium i s l ess c l e a r ; Maetz et al. (1976) suggest a loca t ion of these exchange processes on the a p i c a l membrane. It has been proposed that the ac t ive regulat ion of these pumps may be u t i l i s e d to adjust acid-base status in f i s h (Payan & Maetz, 1973; de Renzis , 1975; Cameron, 1976; Bornancin, de Renzis & Maetz, 1977). The only d i r e c t evidence for the involvement of branchia l exchange processes in the regulat ion of in te rna l pH disturbances comes from the work of Cameron (1976). He showed that hypercapnic A r c t i c gray l ing d isplayed an increased r a t i o of Na + to C l uptake, the response one would pred ic t i n terms of plasma HCO^ adjustment. In cont ras t , Kers te t ter & Mize (1976) found that depression of blood pH by 0.4 un i ts had no e f f e c t on the i n f l u x rates of e i ther Na + or C l and concluded 98 that the g i l l ion uptake sytems of t rout do not respond rap id ly to blood pH changes. In an attempt to resolve t h i s matter I a lso attempted to determine i f and how Na + /H + (NH 4 + ) and CI /HCO^ exchange processes are modulated during acid-base regulat ion in f i s h . In t h i s study, movements of CC^ and H + through the branchi epithel ium are considered. Data are presented from experiments invo lv ing manipulation of the external (water) environment, i n which the rates of ion uptake and acid-base status of f i s h were monitored e i ther as water P C Q , HC0 3 and/or Na + l eve ls were ra ised or fo l lowing addi t ion of c a t i o n i c and anionic uptake i n -h i b i t o r s to the water. De Renzis & Maetz (1973) studied the e f fec ts of environmental N a + and CI removal on blood acid-base status of f i s h a f te r a per iod of several days. The present experiments have been designed to fo l low the time course of ac id base changes over a per iod of hours fo l lowing manipulation of the environment. 99 MATERIALS AND METHODS Experimental animals Rainbow trout ( Salmo gairdneri ) weighing between 200-400 g were obtained from Sun Va l ley Trout Farm (Mission, B r i t i s h C o l -umbia) . They were held in large c i r c u l a r f i b r e g l a s s tanks suppl ied with aerated, dechlor inated Vancouver tap water and kept at ambient temperature (10-12°C) and photoperiod. F i s h were fed d a i l y with a commercial pe l l e ted t rout d ie t (Moore-Clarke Co.) . They were not fed 48 h p r i o r to or during exper-iments. Seawater experiments were performed at Bamfield Marine Stat ion (Bamfield, B r i t i s h Columbia). Coho salmon ( onchorynchus kisutch ) weighing between 300-500 g were obtained from P a c i f i c B i o l o g i c a l Stat ion (Nanaimo, B r i t i s h Columbia). They were main-tained in a s imi la r manner to rainbow t rou t . Blood sampling and ana lys is F i s h were anaesthetised with 1:15,000 MS 222 (pH adjusted to 7-7.5 with NaHCO^) and then t ransfer red to an operating table where 1:20,000 MS 222 was r e c i r c u l a t e d over the g i l l s . To f a c i l -i t a t e the sampling of b lood, f i s h were implanted with chronic indwelling'eannuiae (Smith, 1978) and then allowed to recover in the experimental chamber for at least 24 h before any experiments were begun (Houston et al., 1971). Approximately 0.4 ml of blood was withdrawn at each sampling time and replaced by an equivalent volume of Cort land sa l ine (Wolf, 1963). A port ion of t h i s blood 100 was used to determine Hct while a l l subsequent measurements were made on plasma. Approximately 0.1 ml of plasma was frozen for l a t e r ana lys is of ions . pH and P C Q measurements were made using a Radiometer PHM-71 d i g i t a l acid-base analyser and associated micro pH (G297/G2) and P C Q (E5036/0) e lec t rodes . Tota l carbon dioxide ( C C Q ) was determined using, the method of Cameron (1971) with a PHM-71 acid-base analyser and associated CO2 e lec t rode . A l l pH and ^CQ measurements were performed at ambient temper-ature while C_, n measurements were performed at 45°C to speed up c u 2 the response time of the C0 2 e lec t rode . P C Q (rainbow trout only) and plasma [ H C 0 3 J | were ca lcu la ted using the measured pH and C ^ Q values and a reorganizat ion of the Henderson-Hasselbalch equation as described i n Chapter I. Plasma ch lor ide was deter -mined with a Buchler-Cot love amperometric t i t r a t o r and sodium l e v e l s were measured on d i l u t e d plasma (10 u l plasma:2 ml H20) using a Techtron 120 flame photometer. Sodium and ch lor ide in f luxes Apparent sodium and ch lor ide in f luxes were determined by 22 3 6 fo l lowing the diappearance of Na or C l (New England Nuclear) from the bathing water in an aerated, r e c i r c u l a t i n g system of approximately 2 l i t r e s . The isotope was added to the water and a mixing time of f i v e min was allowed before dupl icate 500 u l 22 3 6 water samples were removed at known i n t e r v a l s . Na and C l a c t i v i t i e s were determined using a Nuclear Chicago Isocap l i q u i d s c i n t i l l a t i o n counter. Apparent rather than absolute in f lu x rates were determined for two reasons. F i r s t l y , Vancouver water 1 0 1 contains very l i t t l e sodium and I d id not want to increase water JjNa'^J to a detectable l e v e l i n a l l experiments because th is was found to a f f e c t the acid-base status of the f i s h , and secondly, I was in terested in changes in f lux rates rather than absolute l eve ls and these could be detected by measuring apparent in f lu x r a t e s . T h e o r e t i c a l l y , the apparent in f lu x rate w i l l show a log r e l a t i o n s h i p with time. I detected no d i f fe rence in f lux rates whether data were p lo t ted in a l i near or log fash ion . Experimental protocol ( 1 ) Chemically treated fish Af ter i n i t i a l blood sampling, the r e c i r c u l a t i n g system was closed and the appropriate chemicals added to the water. A m i l - . o r i d e , a generous g i f t of Dr.^W.D. Dorian of Merck Frost L a b s . , or SITS ( B r i t i s h Drug House) were added to f i n a l concentrat ions -4 of 1 0 M. Sodium bicarbonate (NaHCO^) was added to a f i n a l concentrat ion of 1 2 mM. Upon the addi t ion of these chemicals, blood acid-base status was monitored throughout an experimental period a f te r which f i s h were returned to normal water. The e f fec t of each of these chemicals on branchia l s a l t i n f lu x was determined on uncannulated f i s h . Af ter a cont ro l f lux p e r i o d , drugs were added to the water with the f lux cont inuing uninterupted. F i s h that struggled during the i n i t i a l cont ro l per iod were re jec ted . ( 2 ) Hypercapnia F i s h (rainbow trout and Coho salmon) were made hypercapnic by rep lac ing the a i r supplying the r e c i r c u l a t i n g chambers with 1% C O 2 i n a i r ( P c 0 = 7 . 5 mm Hg). This mixture was suppl ied by 1 02 gas mixing pumps (Wosthoff). Blood acid-base status was monit-ored during a 24 h hypercapnic per iod (at 1, 4 and 24 h) and, in two experimental s e r i e s , a lso at 6 h fo l lowing the return to normocapnic water. The branchia l i n f l u x of sodium or ch lor ide ions was determined on uncannulated f i s h (rainbow trout only) p r i o r to hypercapnia, 30 min or 24 h a f te r . the i n i t i a t i o n of hypercapnia, and 6 h fol lowing return to normocapnic water. (3) Hypercapnia and elevated external sodium or bicarbonate in freshwater Sodium (as Na2SC>4) was added to the bathing water to a f i n a l concentrat ion of 3 mM and bicarbonate (as NaHCO^) to 12 mM. F i s h were made hypercapnic and blood acid-base status mon-i to red as before No f i s h was exposed to more than one experimental treatment. A l l experimental values were measured in dupl icate and are pres -ented in f igures and tables as means ± standard error from the mean. Results were s t a t i s t i c a l l y analysed using Student 's t tes t where appropriate between sample means and 5% was taken as the f i d u c i a l l i m i t of s i g n i f i c a n c e . 103 RESULTS (1) Chemically treated fish The d i u r e t i c compound, ami lor ide , added to the external medium caused a rapid 7 8% reduct ion in the rate of sodium uptake in unrestrained f i s h (F ig . 16). S imi lar r e s u l t s were obtained by K i rschner , Greenwald & Kerstet ter (1973) using res t ra ined and l i g h t l y anaesthetised rainbow t rou t . Table 11 shows the e f fec t of amilor ide exposure on blood acid-base status fo l lowing three and s ix h exposure. Plasma [H~^J increased s i g n i f c a n t l y through-out the exposure per iod while C c o and [HCJC^^] were s i g n i f i c a n t l y reduced a f ter s ix h. Water l e v e l s remained saturated and f i s h showed no signs of s t ruggl ing during the experimental pe r iod , but to confirm that the ac idos is was a r e s u l t of amilor ide and not s t r e s s , a cont ro l group was examined. Control f i s h d isplayed no s i g n i f i c a n t changes in any blood acid.-base. var iab le tested (Table 11). The s t i l b o n i c ac id der iva t ive SITS has been used to i n h i b i t ch lor ide t ransport i n red blood c e l l s as wel l as other t ranspor-t ing t i ssues (Cabantchik & Rothste in , 19.74; Shami et al., 1978). Chlor ide uptake was i n h i b i t e d by 66% upon the" addi t ion of SITS to the external medium (F ig . 17). SITS treatment, l i k e amilor ide d id not produce any v i s u a l signs of s t ress and t rout survived continued exposure (up to 24 h) without apparent i l l e f f e c t s . SITS exposure for three h had no e f fec t on plasma acid-base status or ch lor ide l eve ls (Table 11). However, a f ter s ix h there 1.04 Figure 16. The e f fec t of 10 M external amilor ide on branchia l sodium uptake in rainbow t rou t . •- • , t reated f i s h (n = 6); o O , cont ro l f i s h (n = 9). A common l i n e has been drawn for the f i r s t 2 5 min as the slopes descr ib ing the two groups are not s i g n i f -i c a n t l y d i f f e r e n t . Amilor ide caused a 78% reduct ion in the rate of sodium uptake. This value i s obtained by comparing the slopes of the f i n a l 30 min of the cont ro l group (-0.525) with the f i n a l 35 min of the amilor ide t reated group (-0.117). V e r t i c a l bars represent ± 1 S . E . M . 10 5 TIME (min.) 10 6 Figure 17. The e f fec t of 10 M external SITS on branchia l ch lor ide uptake in rainbow t rou t . • • , t reated f i s h (n = 4); o O , cont ro l f i s h (n = 8) . A common' l i n e has been drawn for the f i r s t 25 min as the slopes descr ib ing the two groups are not s i g n -i f i c a n t l y d i f f e r e n t . SITS causes a 66% reduct ion in the rate of ch lor ide uptake. This value i s ob-tained by comparing the slopes of the f i n a l 30 min of the cont ro l group (-0.472) with the f i n a l 35 min of the SITS treated group (-0.159). V e r t i c a l bars represent ± 1 S .E .M . 107 I I L_ 20 40 60 TIME (min.) 10.8 Table 11. The e f fec t of external drug exposure on acid-base status of rainbow t rou t . Time H + C C 0 2 HCO3- P C 0 2 C l " (h) (nM) (mM) (mM) (mm Hg) (mM) CONTROL (n = 6) 0 1 2 . 8 3 ± 0 . 8 8.4511.0 8.2911.0 2.5810.1 126.812.7 (7.90)* 3 12.1610.9 8.52+1.0 8.3811.0 2.4610.2 129.313.1 (7.91) 6 13.6211.6 8.5311.2 8.3611.2 2.8010.5 127.512.0 (7.87) 0 14.4711.5 (7.85) * 3 18.1911.3 (7.75) * 6 22.5412.5 (7.66) AMILORIDE (10 M; n = 6) 10.6210.5 10.3810.5 8.9210.9 8.05+0.7 8.6810.9 7.7710.7 3.4310.4 3.6610.5 4.10+0.5 0 13.5510.9 (7.87) 3 12.1811.3 (7.93) * 6 11.5011.2 (7.95) SITS (10~4M; n = 6) 7.8710.4 7.7210.4 8.3710.7 9.0710.6 8.2310.7 8.9310.6 2.5810.1 2.4310.2 2.4910.2 129.0+2.2 128.812.3 129.211.8 Corresponding pH value S i g n i f i c a n t l y d i f f e r e n t from value at zero.t ime 109 was a s i g n i f i c a n t a l k a l o s i s accompanied by increases in C _ n 2 and [HC03"^ (Table 11). A l l f i s h exposed to SITS and returned to f resh water survived and although only two f i s h were examined they were found to absorb ch lor ide at the cont ro l i n f lu x rate a f te r being r insed for 30 min. Thus, unl ike i t s ac t ion on mamm-a l i a n erythrocytes (Shami et al., 1978), the i n h i b i t o r y act ion of SITS on the t rout g i l l may be r e v e r s i b l e . Amilor ide and SITS, as wel l as i n h i b i t i n g Na + and C l in f luxes r e s p e c t i v e l y , a lso i n h i b i t e d the c o n t r a l a t e r a l exchange processes. That i s , the addi t ion of amilor ide to the bathing water a lso resu l ted in a rapid 54% i n h i b i t i o n in the rate of C l i n f lux (F ig . 18). This surpr is ing i n h i b i t o r y act ion of amilor ide was maintained fo l lowing three (55% inhib i t ion) ' and s ix h (41% inh ib i t ion ) continuous exposure (F ig . 18, Table 12). S i m i l a r l y , SITS caused an 82% reduction in the rate of Na + uptake which again was maintained throughout the s ix h treatment per iod (F ig . 18, Table 12)'. These e f fec ts of amilor ide and SITS appear to be r e v e r s i b l e . Rinsing the experimental chamber with f resh water for only 20 min was s u f f i c i e n t to return the f lux rates to pre -treatment l e v e l s . Furosemide, a s p e c i f i c i n h i b i t o r of sodium coupled ch lor ide transport was without e f fec t on branchia l C l i n f l u x (F ig . 19). The addi t ion of NaHCO^ to the external medium resu l ted in a 50% i n h i b i t i o n of C l 'uptake (F ig . 20). Table 13 summarises the e f f e c t of external NaHCO^ on blood acid-base status during a 25 h exposure per iod . As in the case of SITS treatment, plasma 110 -4 The e f fec t of 10 M external SITS on branchia l Na + i n f lux in rainbow t rou t . Na + f luxes were measured immediately fo l lowing SITS add i t ion (0 time) as wel l as 3 and 6 h l a t e r . # # = p re -SITS; O O = post-SITS. -4 The e f f e c t of 10 M external ami lor ide on branchia l C l ~ i n f l u x in rainbow t rou t . • • = pre -ami lor ide ; O O = post -ami lor ide . V a r i a b i l i t y i s ind icated at representat ive times as ± 1 S .E .M . I l l 112 Figure 19. The e f fec t of 10 M external furosemide on branchia l ch lor ide in f lux in rainbow t rout . • -M = cont ro l group (n = 4); • • = t reated group (n = 4). V e r t i c a l bars represent ± 1 S .E .M . 113 TIME(min.) 114 Figure 20. The e f fec t of 12 mM external sodium bicarbonate on branchia l ch lor ide uptake in rainbow t rout . • • , t reated f i s h (n = 5); O O , cont ro l f i s h (n = 5). Sodium bicarbonate causes a 50% reduct ion in the rate of ch lor ide uptake. This value i s obtained by comparing the slopes of the f i n a l 30 min of the cont ro l group (-0.304) with the f i n a l 35 min of the sodium bicarbonate treated group (-0.158). V e r t i c a l bars represent ± 1 S .E .M. 115 TIMECmin.) 116 Table 12. The i n h i b i t o r y act ion of external amilor ide (10 M) -4 and SITS (10 M) on branchia l sodium and ch lor ide in f luxes immediately a f ter addi t ion (0 time) as wel l as 3 and 6 hours fo l lowing . n numbers are indicated in parentheses. % Inh ib i t ion Sodium in f lu x Chlor ide in f lu x 0 3h 6h 0 3h 6h Amiloride 84 (5) - 92 (5) 54 (5) 55 (5) 41 (6) SITS 82 (4) 65 (4) 75 (5) 71 (4) - 60 (4) 117 Table 13. The e f fec t of external bicarbonate (12 mM) on blood acid-base status of rainbow t rou t . (mean ± S . E . M . , n = 8) . Time (h) 0 1.5 3 4.5 6 9 25 H (nM) 12.93+0.6 7.89)^ 8 : 2 1 1 0 : 8 ' 8.09) 6 . 6 1 ± 0 . 61 8.18) 6.8610.4' 8.16) 5.7910.6' 8.24) 5.9910.4' 8.22) 4.7510.4' 8. 32) C 0 2 (mM) 9 . 1 2 ± 0 . 3 1 3 . 7 1 ± 0 . 5 1 2 . 8 5 ± 0 . 5 1 4 . 8 7 ± 0 . 3 13.8610.5 1 6 . 9 0 ± 0 . 9 20.7510.9 HC03 (mM) 8.9510.3 13.5210.5' 12.7310.5 : 14.7310.3' 13.7510.5' 16.6410.9' 20.4110.9' c o 2 (mm Hg) 2.5910.1 2.2710.2 1.98+0.1' 1.9810.l ' 1 . 7 0 ± 0 . l ' 2.0010.2' 1.8910.l ' Corresponding pH value * S i g n i f i c a n t l y d i f f e r e n t from value at zero time 118 was s i g n i f i c a n t l y lowered at three h and remained lowered decreased s i g n i f i c a n t l y while Cnn and HCO,~ increased. 2 throughout the treatment per iod . It i s worth noting that while NaHC03 i n h i b i t s C l ~ uptake less than SITS (F igs. 17 & 20), i t has a greater and much more.rapid e f f ec t on blood acid-base status (Tables 11 &13). (2) Hypercapnia Na + and CI in f luxes were measured pre-hypercapnia, 24 h fo l lowing the i n i t i a t i o n of hypercapnia (1% CC^), and post -hypercapnia. The e f fec ts of hypercapnic ac idos is on Na + uptake are summarised in F i g . 21. The s l i g h t reduct ion (12%) of uptake between the pre-hypercapnic and hypercapnic periods was not s i g n i f i c a n t nor was the s l i g h t increase in the post-hypercapnic per iod . CI uptake a lso was not s i g n i f i c a n t l y a l te red during hypercapnia (F ig . 22) although once again there was a s l i g h t reduct ion (16%) of uptake during hypercapnia and a s l i g h t i n -crease in post-hypercapnia. The r a t i o s of Na + to CI uptake a lso d i d not change s i g n i f i c a n t l y , these being 1.6, 1.7 and 1.5 r e s p e c t i v e l y . Measurements of N a + and CI in f luxes were repeated at 30 min fo l lowing i n i t i a t i o n of hypercapnia because i t was f e l t that any poss ib le changes in f lux rates may have gone undetected by wait ing 2 4 h. F i g s . 2 3 and 2 4 summarise the short- term e f f e c t s of hypercapnic ac idos is on Na + and CI in f luxes respect -i v e l y . Na + uptake was reduced s i g n i f i c a n t l y by 22% while CI uptake was increased s i g n i f i c a n t l y by 4 4%. The r a t i o s of Na + 119 Figure 21. The e f fec t of 24 h hypercapnia (1% C0 2) on branchia l sodium uptake in rainbow trout (n = 5). See text for fur ther d e t a i l s . 120 PRE-HYPERCAPNIA HYPERCAPNIA POST-HYPERCAPNIA T I M E (MIN.) 121 Figure 22. The e f fec t of 24 h hypercapnia (1% C0 2) on branchia l ch lor ide uptake in rainbow trout (n = 5) . See text for fur ther d e t a i l s . 122 123 Figure 23. The e f fec t of 30 min hypercapnia (1% CC^) on branchia l sodium uptake in rainbow t rou t . • • , cont ro l f i s h (n = 5); • • , hypercapnic f i s h (n = 5). V e r t i c a l bars represent ± 1 S . E . M . . See text for fur ther d e t a i l s . 124 125 Figure 24. The e f fec t of 3 0 min hypercapnia (.1% CO,,) on branchia l ch lor ide uptake in rainbow t rout . • • , cont ro l f i s h (n = 5); • • , hypercapnic f i s h (n = 5). V e r t i c a l bars represent ± 1 S .E .M. See text for fur ther d e t a i l s . 1 27 to CI uptake during pre-hypercapnia and hypercapnia were 1.2 and 0.6 r e s p e c t i v e l y . The e f f e c t s of external hypercapnia on blood acid-base status of freshwater rainbow trout and seawater Coho salmon are shown in Table 14. The r e s u l t s fo r rainbow t rout are q u a l i t a t -i v e l y s im i l a r to those of Janssen & Randall (1975). Hypercapnia induced a rap id ac idos is which slowly returned towards normal as plasma HCO^ l e v e l s increased. P C Q reached a new steady-state a f te r one h and remained constant for the remainder of the exposure per iod . Plasma [ e l ]j d i d not change and the 4% increase in plasma [Na+] was not s i g n i f i c a n t (F ig . 25). Blood a c i d o s i s in Coho salmon was not near ly as severe as in rainbow trout and i blood pH was almost returned to normal fo l lowing 2 4 h exposure. The more rapid regula t ion of blood pH in Coho salmon i s due to a greater and more rap id accumulation of plasma HCC>3 (Table 14) . As in the case of rainbow t rou t , Pa^g reached a new steady-state a f ter one h. (3) Hypercapnia and elevated external sodium or bicarbonate The concentrat ion of Na + in Vancouver water i s extremely low (40 u e q u i v . / l ) and for t h i s reason the hypercapnia exper-iments were repeated with the l e v e l of external Na + ra ised to 3 mM with Na 2 S0 4 (SO^ i s an impermeant i o n ) . Under t h i s cond-i t i o n the hypercapnic ac idos is was much less pronounced and a f te r 24 h plasma [H 4 ] was nearly restored to the pre-hypercapnic value (Table 15). S i m i l a r l y , the increase in C c o and J H C O ^ J were almost twice as great with elevated external N a 2 S 0 4 . When 12.8 Table 14. E f f e c t of external hypercapnia (1% C0 2) on blood acid-base status of freshwater rainbow trout and Time (h) 0 1 4 24 Post hyper seawater Coho salmon. Means ± S.E.M. HCC 3 (mM) H (nM) C ^CO-(mMT 1 2 . 9 7 ± 0 . 7 (7.89)a 30.4512.4' (7.52) 28.1011.9' (7.55) 2 5 . 0 7 ± l . o ' (7.60) 13.1510.8' (7.88) RAINBOW TROUT (n = 8) 9 . 4 1 ± 0 . 5 9.2210.5 11.0310.4 12.2410.7 13.8310.5 10.6510.5 10.4910.4 11.6910.7 13.2710.5 10.4210.5 CO-(mm Hg) 2.5610.2 7.3610.6 7.3710.3 7.5810.4 2.9110.2 0 1 4 24 Post hyper 14.8310.5 (7.83) 21.9311.5' (7.66) 16.7911.3' (7.78) 15.8510.9 (7.80) 13.6110.6 (7.87) COHO SALMON (n = 6) 10.2911.0 9.8111.0 16.8011.2 21.49+1.2 24.1912.2 12.7910.7 16.3011.2 20.8511.2 23.4612.2 12.4110.7 3.4910.4 7.9311.0 8.1410.4 8.7911.1 3.4810.2 Corresponding pH value S i g n i f i c a n t l y d i f f e r e n t from value at zero time 12 9 Table 15. The e f fec t of hypercapnia (1% C0 2) and elevated external sodium (3 mM) on blood acid-base status of rainbow t rou t . Means ± S .E .M. Time (h) 0 1 4 24 H' (nM) C (mMJ HC03 (mM) 11.7110.8 (7.94)a 10.3911.0 (7.99) 9.7810.9' (8.02) 8.1111.0' (8.10) 3 mM EXTERNAL Na (n = 5) 8.5610.6 8.4110.6 10.0410.7 12.4110.9 14.5911.1 9.8910.7 12.2210.9 14.4211.1 c o 2 (mm Hg) 2.3510.3 2.4710.2 2.9410.3 2.7410.2 0 1 4 24 HYPERCAPNIA AND 3 mM EXTERNAL Na + (n 9.0610.9 8.8910.9 5) 12.9710.8 (7.89) 23.9911. l ' (7. 6.2) 20.8911.2 (7.68) 14.7910.8* (7.83) 11.8111.2 14.3511.2 22.5211.2 11.3311.2 13.8911.2 22.0011.2 2.9110.3 7.2310.8 7.7110.9 8.4510.7 Corresponding pH value S i g n i f i c a n t l y d i f f e r e n t from value at zero time 13 0 Figure 25. The e f fec t of exposure to hypercapnia (1% CO^) for 24 h, and a l te red external environment on plasma N a + ( ^ ) and C l ~ ( • ) l e v e l s . * , s t a t i s t i c a l l y d i f f e r e n t change. See text for fur ther d e t a i l s . % CHANGE I + 01 O Ol HYPERCAPNIA EXTERNAL HCO3 HYPERCAPNIA and HC03" 13.2 3 mM Na2S0^ was added to normocapnia water the r e s u l t was a s i g -n i f i c a n t a l k a l o s i s fo l lowing four h, which increased throughout the exposure per iod (Table 15). As w e l l , there were s i g n i f i c a n t increases in C _ n and plasma HCO_ . Plasma Na + was s i g n i f -c u 2 i c a n t l y increased fo l lowing 2 4 h and the combination of hyper-capnia and elevated external Na + evoked no further increases (F ig . 25). Hypercapnia experiments a lso were repeated with elevated external HCC»3 . As in the case of external Na + treatment, the hypercapnic ac idos is was much l ess pronounced. Blood pH was restored to normal fo l lowing 24 h (Table 16). The rapid reg -u la t ion of blood pH was accomplished by increased retent ion of plasma HCO^ . Externa l HCO^ s i g n i f i c a n t l y reduced plasma CI l e v e l s which were not further reduced by the combination of external HCO-. and hypercapnia. (F ig . 25) . 13 3 Table 16. The e f fec t of 12 mM external bicarbonate and hyper-capnia on blood acid-base status of rainbow t rou t . Means ± S .E .M . (n = 5) . Time H + C HC0 3 - P (h) (nM) (mMT (mM) (mm Hg) 0 8.8110.5 7.16+0.6 7.0710.6 2.1410.1 (8 .06) a 1 14.7610.9* 14.9310.9* 14.5910.9* 5.3510.3* (7.83) 4 12.7910.8* 21.3311.1* 20.9111.1* 5.9610.4* (7.89) 24 9.5110.6 27.8411.4* 27.4411.4* 5.9610.4* (8.02) Corresponding pH value * S i g n i f i c a n t l y d i f f e r e n t from value at zero time 13 4 DISCUSSION The d i u r e t i c compound, ami lor ide , has been shown to spec-i f i c a l l y i n h i b i t sodium transport across var ious t i ssue e p i t h e l i a , inc lud ing f rog sk in (E ig le r , Ke l te r & Renner, 1967), toad and ur inary bladders (Bentley, 1968; Wilczewski & Brodsky, 1975) and rabbi t colon ( F r i z z e l l , Koch & Schu l tz , 1976). Moreover, ami lor -ide was shown to i n h i b i t branchia l sodium uptake in in tac t f r e s h -water animals inc lud ing f i s h (Kirschner, 1973), crabs (Cameron, 1978) and c r a y f i s h (Kirschner, Greenwald & Kers te t te r , 1973) without a f f e c t i n g ch lor ide uptake. In f a c t , amilor ide has been one of the too ls used to show that Na + and C l t ransport systems are independent of each other. SITS, while i n h i b i t i n g anion movements in red blood c e l l s (Cabantchik & Rothstein 19 72; Has-w e l l , Ze id ler & Kim, 1978) and t u r t l e bladder (Cohen et a l . , 1978) does not a f fec t sodium t ransport . I have assumed that drugs added to the water act on the a p i c a l surface of the g i l l and that there i s l im i ted penetrat ion in to the animal. This appears to be a reasonable assumption consider ing the large molecular s ize of the drugs (amiloride = C 6 HgClN 7 0 , SITS = C 1 7 H 1 2 N 2 0 7 S 3 N a 2 3 H 2 0 ) . I f e e l therefore that the e f fec ts of these drugs can be explained in terms of an act ion on the external surface of the g i l l s rather than any e f fec t on the kidney. SITS and amilor ide a f fec t Na + and C l f luxes across the g i l l s , presumably v i a i n h i b i t i o n of c a t i o n i c and anionic exchange processes, and as these drugs are large molecules which ; 13 5 probably do not enter e p i t h e l i a , these exchange processes are located probably at the a p i c a l membrane of the g i l l ep i the l ium. It i s c lear from t h i s study that these a p i c a l ion exchange processes (Na/H + (NH 4 + ) and C1~/HC0 3~) are important in mainta in-ing in te rna l acid-base s ta tus . Perturbat ions using i n h i b i t o r y drugs a f fec t the in te rna l acid-base equi l ibr ium over r e l a t i v e l y short periods of time (3-6 h ) . In the model of Haswell et al. (1980) a passive d i s t r i b u t i o n of HCO^ between plasma and bran-^ c h i a l e p i t h e l i a l c e l l s was assumed. As carbonic anhydrase l e v e l s are high in the epithel ium (Maetz, 1956) and C0 2 d i f fuses r a p i d l y , HCO^ entry in to the branchia l epi thel ium could be the r a t e - l i m i t i n g step in CO^ excret ion and an important determinant of blood acid-base s ta tus . Previous studies (Chapters I and II) have shown that HCC>3 entry into the epithel ium i s not, the ra te -l i m i t i n g step in CC>2 excret ion and i s c e r t a i n l y not of a large magnitude. In f a c t , there i s no evidence supporting the hypoth-es is that HC0 3 enters the g i l l from plasma, at a l l . Therefore, the e f f e c t s of amilor ide and SITS on blood acid-base status cannot be explained in terms of a l te red rates of bicarbonate entry in to the g i l l epi thel ium as was postulated prev iously (Perry et . al., 1981). It i s c lea r however, that the branchia l epithel ium i s permeable to H + ions and that the movement of H + ions between plasma and epithel ium w i l l be a major determinant of blood a c i d -base s ta tus . The pH of g i l l e p i t h e l i a l c e l l s i s probably an important fac tor determining H + entry . If i n t r a c e l l u l a r pH i s lowered, then H + f lux from the plasma to g i l l epi thel ium w i l l be reduced. The reverse w i l l be true i f i n t r a c e l l u l a r pH of the 13 6 epithel ium i s r a i s e d . Factors that a f fec t e p i t h e l i a l pH there-f o r e , w i l l a f f ec t H + movement between plasma and g i l l epithel ium which in turn w i l l be r e f l e c t e d by HGO^ leve ls in the b lood. The i n h i b i t i o n of Na + /H + (NH 4 + ) exchange with ami lor ide , according to t h i s model, causes a reduct ion of i n t r a c e l l u l a r pH and therefore a decrease in H + ion entry from the plasma. The net r e s u l t i s a decrease in plasma pH and C_,n . On the other hand, i n h i b i t i o n of C l /HCO^ exchange with SITS causes e p i t h e l -i a l c e l l pH to r i s e thereby increas ing the entry of H + in to the g i l l ep i the l ium. The r e s u l t i s a slow increase in plasma pH and C C Q . Blood acid-base changes s im i l a r to those reported here, 2 + -have been observed in g o l d f i s h exposed to Na or C l free water for periods of severa l days (de Renzis & Maetz, 1973). These r e s u l t s ind ica te that H + ion movement between plasma and the g i l l epi thel ium does occur and i s regulated, to some extent, by the operating rates of the a p i c a l ion exchange proce-sses . Although C0 2 excret ion was not monitored during these experiments, the r e l a t i v e l y small changes in plasma C r n fo l low-er ing a p i c a l ion exchange inh ib i ton ind icate that o v e r a l l C0 2 excret ion was general ly unaf fected. Those r e s u l t s are in d i s -agreement with those of Dejours (196 9) who observed that a b o l i t i o n of C l /HCO^ exchange completely i n h i b i t e d C0 2 excret ion and in some cases led to C0 2 uptake. These r e s u l t s have not been r e -peatable and the explanation remains unclear . The model of Maetz (1971) i s c e r t a i n l y incor rec t in assuming that 100% of carbon dioxide excreted i s in the form of HCO_ and coupled to C l 13 7 i n f l u x . Cameron (19 76) a lso concluded that only a minor por t ion of t o t a l CC>2 excreted i s l inked to a p i c a l C l uptake. If the above conclusion i s v a l i d , that branchia l e p i t h e l i a l c e l l pH i s re la ted to branchia l ion exchange, one would expect amilor ide a lso to i n h i b i t C l uptake (a consequence of lowered e p i t h e l i a l c e l l pH and exchangeable HCO^ ) and SITS to i n h i b i t Na + i n f l u x (a consequence of lowered e p i t h e l i a l c e l l H + ) . Subsequent experiments have indeed shown t h i s to be t rue . The e f fec ts of amilor ide and SITS on the c o n t r a l a t e r a l exchange processes are not i d e n t i c a l however, thus the d i f f e r e n t i a l e f f - . ects of these drugs on acid-base status are r e a d i l y expla ined. Amilor ide i s thought to i n h i b i t e p i t h e l i a l Na + t ransport by competing for Na + t ransport s i t e s (see review by Cuthbert, Edwardson, Aceves & Wi lson, 1979), while SITS i s known to i n h i b i t anion transport by binding to a s p e c i f i c membrane prote in (Shami et al., 1978) . Thus i t seems u n l i k e l y that the e f fec ts of amilor ide and SITS on the c o n t r a l a t e r a l exchange processes are due to d i r e c t i n t e r a c t i o n with transport s i t e s . A l s o , there i s no evidence that Na + and C l t ransport are d i r e c t l y coupled in the branchia l epi thel ium. Furthermore, furosemide, an i n h i b i t o r of Na + coupled C l t ransport (Burg, 1976) i s without e f f ec t on branchia l C l i n f l u x . I f e e l therefore that the e f fec ts of amilor ide and SITS presented here are secondary in nature r e s u l t -ing from a l te red e p i t h e l i a l c e l l pH due to the primary i n h i b i t o r y act ion of these drugs. The e f f e c t s of amilor ide on e p i t h e l i a l c e l l pH and there-13 8 fore C l i n f lux may be t r a n s i t o r y and have a d i f f e r e n t time course to the blocking e f fec t of the drug on Na + /H + (NH 4 + ) exch-ange. This would be due to the act ion of other compensatory f a c t o r s , which are independent of Na + /H + (NH 4 + ) exchange, act ing on i n t r a c e l l u l a r pH. The extent and time course of pH change wi th in the epithel ium might vary with the environmental cond-i t i o n s and the species in quest ion, and an e f f e c t of amilor ide on C l i n f l u x may not be apparent i f the measurement of C l f lux i s made some time a f ter the addi t ion of the drug. These c o n s i d -erat ions may represent a poss ib le explanation of the d i f fe rences observed in t h i s study and those of Kirschner (1973) and Cameron (1978). Cameron (1978) worked on the blue crab and Kirschner (1973) used anaesthetised t rou t . The anaesthet ic , MS 222, i s known to have profound e f fec ts on blood chemistry, g i l l e l e c t r o -ly te composition (Houston et a l . , 1971) and c i r c u l a t i o n (Daxboeck & Holeton, 1980). It i s i n t e r e s t i n g that elevated water NaHCO^ increases plasma pH and C ^ Q more rap id ly and to higher l e v e l s than SITS even though external NaHCO^ i n h i b i t s C l uptake to a lesser . . extent. This i s probably due to a combination of net uptake of HCO^ from the bathing water ind icated by the f a l l in plasma [ c i " [ ] (F ig . 25) , and an increase in H + ion excret ion due to both enhanced N a + / H + ( N H 4 + ) exchange (a r e s u l t of higher external Na +) and passive H + ion e f f lux (a r e s u l t of higher external pH). That 40% of C l uptake s t i l l occurs at t h i s high concentrat ion of HCO^ can perhaps be a t t r ibu ted to C l / C l exchange d i f f u s i o n (Maetz, 1971). 139 The g i l l a p i c a l ion exchange processes are important in maintaining steady-state in te rna l acid-base balance and modul-at ions of these processes can a f fec t blood acid-base status by changing the rate at which plasma H + ions enter the g i l l e p i t h -el ium. During hypercapnic ac idos is blood acid-base status i s regulated by the accumulation of plasma bicarbonate. To test the hypothesis that the a p i c a l ion exchange processes are i n v o l -ved in t h i s regulatory process, branchia l in f lux rates were determined. From the r e s u l t s of t h i s study i t does not appear that g i l l ion exchanges are involved in regulat ing hypercapnic ac idos is as indicated by the constant rates of Na + and C l uptake before , during and a f te r hypercapnia . , These r e s u l t s are in agreement with those of Kerste t ter & Mize (1976), who concluded that acute metabolic ac idos is does not a f fec t the rate of e i ther Na + or C l in f lux in rainbow trout over a short time span (15 min). Their study d id not exclude the p o s s i b i l i t y of a delayed response to a blood pH s h i f t . For t h i s reason I measured Na + and C l uptakes at 24 h fo l lowing the i n i t i a t i o n of hypercapnia. If there i s an adjustment of e i ther Na + or C l uptake i t should be not iceable at 24 h since blood pH i s already being returned to pre-hypercapnic l e v e l s . However, I observed no changes in f lux ra tes . In a l a t e r ser ies of experiments, branchia l ion f luxes were measured 30 min fo l lowing i n i t i a t i o n of hypercapnia. S u r p r i s i n g l y , fo l lowing 30 min of hypercapnia, Na + i n f l u x was s i g n i f i c a n t l y reduced while C l uptake was s i g n i f i c a n t l y increased. These are the exact opposite r e s u l t s one would expect in terms 14 0 of plasma HCO^ adjustment, and would only serve to lower blood pH even more. It i s cer ta in that external hypercapnia reduces water pH (espec ia l ly in poorly buffered water) . McWilliams & Potts (1978) have shown that the g i l l i s extremely permeable to H + ions and that lowered water pH causes H + ions to move from water to blood thereby causing the t r a n s - e p i t h e l i a l po ten t ia l to become more p o s i t i v e . Results from sa l ine and blood-per fus ion studies have a lso shown.that H + movement i s re la ted to the grad-ient between water and blood. It i s poss ib le that the reduct ion in Na + uptake and increase in C l uptake fo l lowing 3 0 min hyper-capnia are due to a s h i f t of the TEP in a p o s i t i v e d i r e c t i o n . Apparently these ion f lux changes are temporary since they are no longer observed at 24 h. McWilliams (1980) a lso reported t rans ient Na + i n f l u x reductions fo l lowing t ransfer of Norwegian brown trout ( Salmo trutta ) to water of low pH. In contrast to t h i s study, Cameron (1976) reported that the r a t i o of Na + to C l uptake increased during hypercapnia in the A r c t i c g r a y l i n g . I f e l t the discrepancy between the two studies might have been due to the low l e v e l s of Na + in Vancouver tap water. Because of the very low s p e c i f i c a c t i v i t y of the water when elevated external Na + i s used, I was unable to measure Na + i n f l u x , but d id compare the e f fec ts of hypercapnia on blood a c i d -base status with and without elevated external N a + . It i s c lea r that f i s h exposed to hypercapnia with elevated external Na + be-come less a c i d o t i c and exh ib i t a more rapid pH recovery. However i t i s not c lea r whether t h i s i s due to actual modulation of N a + / 141 H (NH^ ) exchange during hypercapnia or a r e s u l t of an already increased rate of Na + uptake. I . f e e l that the l a t t e r i s probably the case as f i s h exposed to. only high external Na + exh ib i t a pronounced a l k a l o s i s and e levat ion of plasma [jSIa*| . Thus, hyper-capnia appears to have l i t t l e e f fec t on Na + /H + (NH 4 + ) exchange, ind icated by the fact that Na + l eve ls in plasma and N a + i n f l u x are unaffected. A l s o , i f external Na + i s e levated, I observed the same increase in plasma Na + in both normocapnic and hyper-capnic f i s h (F ig . 25). The same argument can be made for the absence of an e f f e c t of hypercapnia on C l /HCC>3~ exchange. When external HCO^ i s elevated I observed the same decrease in plasma C l in both normocapnic and.hypercapnic f i s h . Seawater Coho salmon regulate hypercapnic ac idos is much more e f f i c i e n t l y than freshwater rainbow t rou t . S i m i l a r l y , rainbow trout regulate hypercapnic ac idos is more e f f e c t i v e l y when HCO^ i s added to the water. The rapid regulat ion in sea-water may be due to the d i f fe rence in external buf fer ing capac i ty . It i s ce r ta in that bubbling with 1% C0 2 w i l l cause a greater decrease in pH i n freshwater than in seawater. Therefore in freshwater, pH w i l l decrease more than blood pH and the gradient for H + movement w i l l be in the d i r e c t i o n of water to b lood. In seawater the gradient w i l l be very much reduced and consequently blood pH w i l l not decrease to the same extent. That pH regu la t -ion in freshwater i s fas ter with external HCO^ i s probably due to a combination of increased buf fer ing capaci ty and reduced HCO^ excret ion (perhaps even HC0 3~ uptake). Randall et al. (1976) observed HCO., uptake in hypercapnic dogf ish . 14 2 If modulations of the a p i c a l ion exchange processes do not play a ro le in the regulat ion of hypercapnic ac idos is as i n d i c -ated by t h i s study,, then how i s the compensatory increase in plasma HCO^ achieved? Another poss ib le explanation for e lev -at ion of plasma HCO^ - i s i n h i b i t i o n of carbon dioxide excret ion thereby causing HCO^ re ten t ion . This only can be accomplished by i n h i b i t i o n of HCO^ / C l exchange in red blood c e l l s . The poss ib le cont ro l of e ry throcy t ic HCO^ / C l exchange as wel l as branchia l HCO-, / C l exchange w i l l be evaluated in Chapter IV. 14 3 CHAPTER IV CONTROL OF C0 2 EXCRETION AND ACID-BASE REGULATION 14 4 INTRODUCTION Acid-base disturbances in f i s h , inc lud ing hypercapnic a c i d -o s i s , are regulated by adjustments of plasma bicarbonate l e v e l s . The inorease in plasma j^ HCO^ J during hypercapnic ac idos is must r e s u l t e i ther from increased H + ion excret ion and/or reduced C0 2 excre t ion . Independence of C0 2 and H + ion excret ion in f i s h ensures that reductions in C0 2 excret ion do not fur ther compound the a c i d o t i c condi t ion as would occur in mammals. Results from Chapter III have shown that H + ion movement from plasma into branchia l e p i t h e l i a l c e l l s i s cont ro l l ed by i n t r a c e l l u l a r pH which in turn i s c o n t r o l l e d , in par t , by the a p i c a l ion exchange mechanisms. Thus, H + ion excret ion i s p a r t i a l l y governed by the + + + — — rates of Na /H (NH4 ) and C l /HCO^ exchanges which w i l l there-fore a f fec t blood HCO^ l e v e l s . The r a t e - l i m i t i n g step in C0 2 excret ion i s e ry throcy t ic HCO^ / C l exchange. Thus, adjustments of HCO^ entry into erythrocytes a lso w i l l a f fec t blood HCO^ l e v e l s . G i rard & Payan (1977) have shown that branchia l N a + / H + (NH 4 + ) exchange in freshwater t rout i s a f fected by catecholam-. i n e s . It i s c lea r that l e v e l s of c i r c u l a t i n g catecholamines increase during periods of s t ress in f i s h (Nakano & Tomlinson, 1967). Although blood catecholamine l eve ls have never been measured during hypercapnic a c i d o s i s , i t i s probable that they increase . For these reasons, the e f fec ts of catecholamines on both branchia l and ery throcyt ic C l /HCO^ exchange were examined, in order to invest igate the p o s s i b i l t y that C l /HCO^ exchange 14 5 i s adjusted during acid-base disturbances to regulate blood pH. 14 6 MATERIALS AND METHODS Adrenergic cont ro l of branchia l ch lor ide transport Branchia l ch lor ide transport i n freshwater rainbow trout was studied using an i s o l a t e d , perfused head preparat ion (Payan & Matty, 1975). Experiments were performed in co l l abora t ion with Drs. Payan, Gi rard & Bornancin at the Un ivers i ty of Nice (Nice, France) . The mater ia ls and methods involved in preparing t h i s preparat ion have been discussed in d e t a i l in Chapter I. Chlor ide i n f l u x , external C l and Na + concentrat ions, and input pressure (p^n) were monitored under normal condi t ions and with a va r i e ty of catecholamines in the per fusate . Isoprenal ine ( Isoproterenol) , adrenal ine , noradrenaline (Arterenol) and phenyl --5 ephrine were added to f i n a l concentrat ions of 10 M. Adrenergic blocking agents, propranolol (0 blocker) and phentolamine (Rog--4 i t i n e ; ok blocker) were added to f i n a l concentrat ions of 10 M. Changing from one experimental condi t ion to another was accomp-l i shed by switching sa l ine reservo i rs by means of a three-way tap (F ig . 5) . Control of CO,, movements through red blood c e l l s Carbonic anhydrase a c t i v i t y of red blood c e l l s was measured manometrically using a modif ied boat technique as described by Meldrum & Roughton (1933), Roughton & Booth (1946) and as l a t e r modif ied by Hoffert (1966) and Haswell & Randall (1976). B r i e f l y , a s l i g h t l y a lka l ine bicarbonate so lu t ion i s allowed to mix with a buffered so lu t ion of pH 6.8 in a sealed react ion vesse l where-14 7 upon gaseous CC^ i s evolved. The rate of CO^ evolut ion can be measured with and without CA present and t h i s provides the basis of the assay. C l e a r l y , a fas ter rate, of C0 2 evolut ion impl ies a greater c a t a l y t i c a c t i v i t y of e ry throcyt ic CA, thereby r e f l e c t -ing an enhanced movement of HCO^ into the erythrocyte . The change in pressure in the react ion vesse l as CC^ i s evolved into the gas phase was measured with a Statham P23Db pressure t rans -ducer and d isplayed on a s ing le channel chart recorder (Cole-Parmer) . The transducer was c a l i b r a t e d by a l t e r i n g the volume of the c losed system ( including the G i lson Manometer, react ion vesse l and pressure transducer) and recording the change in pressure . As much blood as poss ib le was withdrawn from the dorsa l aorta of prev ious ly cannulated rainbow t rou t . Blood was cent r -i fuged and washed four times and resuspended using Cort land sa l ine (Wolf, 1963; PVP was not added to t h i s s a l i n e ) . A f i n a l Hct of 10-15% was considered acceptable. Blood was d iv ided into equal a l iquots and stored on ice u n t i l requ i red . To perform an assay, 2 ml of bicarbonate so lu t ion were placed into one chamber of the react ion vesse l while the other chamber was f i l l e d with 2 ml of phosphate buffer plus 200 u l of b lood. The uncatalysed cont ro l value was obtained by s u b s t i t u t -ing blood with an equal-volume of Cort land s a l i n e . The react ion vesse l then was attached to the G i lson Manometer, submerged in a constant temperature bath (ambient temperature) and allowed to temperature e q u i l i b r a t e (3 min). The shaking motor was turned 14 8 on and the react ion allowed to proceed. A f te r completion of the r e a c t i o n , the valves were opened and the shaking motor turned o f f . The rates of the react ion are expressed as u l of C0 2 e v o l -ved /sec . These values are obtained e a s i l y from the l i n e a r por t ion of the t races (F ig . 26) . CA a c t i v i t y i s expressed using the fo l lowing equation: K - K E = - £ * (7) K O E i s equal to enzyme un i ts of CA a c t i v i t y , K C i s equal to the rate of the cata lysed r e a c t i o n , and K q i s equal to the rate of the uncatalysed r e a c t i o n . Carbonic anhydrase a c t i v i t y was determined for normal washed blood and for blood to which adrenal ine had been added prev ious ly (5-10 min). The addi t ion of adrenal ine to washed blood d id not a f f e c t pH and any poss ib le d i l u t i o n e f f e c t s were cont ro l l ed for by adding equal volumes of sa l ine to normal b lood. Percent i n h i b i t i o n of CA a c t i v i t y of red blood c e l l s during adrenal ine treatment was ca lcu la ted using the fo l lowing formula: . K - K % i n h i b i t i o n = — - ~ X 100 (8) K c where K i s the ra te of the normal react ion and K i s the rate of 1 1 a the react ion a f ter adrenaline a d d i t i o n . 14 9 Figure 26. Redrawn traces from a t y p i c a l carbonic anhydrase assay showing the uncatalysed rate (200 u l sal ine) and the cata lysed rate (200 u l washed blood) of C0 2 evolut ion from the HCO^ dehydration r e a c t i o n . CA a c t i v i t y i s determined by comparing the slopes of the l i n e a r port ions ( ) of the cata lysed and uncatalysed reac t ions . See text for fur ther d e t a i l s . 400 0 20 40 60 TIME, sec. 151 RESULTS Perfusing i s o l a t e d rainbow trout heads with p h y s i o l o g i c a l sa l ine resu l ted in extremely low values of J \ n C l (2-4 u equiv / lOOg/h compared to 20 u equiv/ lOOg/h from in vivo determinat ions) . I n i t i a l i n f l u x values often were s im i l a r to those reported from in vivo s tudies but in every instance J ^ C l decreased rap id ly with time and fo l lowing 10-15 min of per fusion J \ n C l was near zero. F a i l u r e of t h i s preparat ion to absorb ch lor ide was not due to t i ssue hypoxia (bubbling the external medium with 0^ had no ef fect ) or the concentrat ion of external C l ( increasing or decreasing external C l a lso was without e f f e c t ) . These r e s u l t s prompted an inves t iga t ion into the ro le of hormones in regulat ing branchia l ch lor ide t ranspor t . Using a va r ie ty of catecholamines in the perfusate i t was found that the rate of C l i n f l u x i s d i r e c t l y propor t iona l to d receptor s t imulat ion and/or inverse ly propor t iona l to /$ receptor s t imulat ion (F ig . 27). Phenylephrine (an tagon is t ) gave the greatest rates of J \ n C l followed by noradrenaline (p r imar i l y * * , some £ ) , adrenal ine (equal «*» and B ) and isoprenal ine ( £ agonist) . Perfusing only with isoprenal ine in the perfusate resul ted in a steady decl ine of J \ C l which was abol ished when perfusion was switched to phenylephrine (F ig . 28a). Conversely, J ^ n C l i n c r -eased s t e a d i l y while perfusing with phenylephrine and was i n h i b -i t ed when per fusion was switched to r inger containing isopren-a l ine (F ig . 28b). T y p i c a l l y , phenylephrine and isoprenal ine 15 2 caused increases and decreases respec t ive ly in P^ n (Pig. 28). The addi t ion of the £ b locker , propranolol to perfusate conta in -ing adrenal ine caused a rapid increase in both J . C l and P. 3 r m m (F ig . 2 9a). Conversely, adding the eA b locker , phentolamine, to perfusate conta in ing noradrenaline caused a sharp decrease in J . C l " but had l i t t l e e f f ec t on P. (F ig . 29b). i n in 3 F i g . 3 0 shows the r e l a t i o n s h i p between J . C l and P. r i n i n under a l l experimental cond i t ions . It i s obvious that there i s no c o r r e l a t i o n between P. and' J . C l e i ther at the onset of i n i n perfusion (F ig . 30a) or 20 min l a t e r (F ig . 30b). The e f f e c t of adrenal ine on red blood c e l l carbonic anhy-drase a c t i v i t y i s i l l u s t r a t e d in F i g . 31. When added to blood -9 at concentrat ions greater than 10 M, adrenaline was found to i n h i b i t e ry th rocy t ic CA a c t i v i t y in a t y p i c a l dose-response manner in in tac t but washed erythrocytes . 153 Figure 27. The e f fec t of var ious catecholamines on branchia l ch lor ide uptake in the i s o l a t e d , perfused head of rainbow t rou t . A) % change during 20 min per fus ion . O O , phenylephrine (n = 5) ; r • rrQ , adrenaline (n = 5); • • , noradrenaline (n = 7 ) ; • • , isoprenal ine (n = 7). B) Absolute ch lor ide in f l ux fo l lowing 20 min perfusion with same four ca techo l -amines. 15 5 Figure 28. The e f fec t of A) Switching from ft to ^ s t i m u l a t i o n (n = 5) and B) From <K to B s t imulat ion (n = 5) on ch lor ide uptake and input pressure in the i s o l a t e d perfused head preparat ion. 156 157 Figure 29. The e f fec t of A) P antagonists ( n = 5) and B) ck antagonists (n = 5) on ch lor ide uptake and input pressure in the i s o l a t e d , perfused head of rainbow t rou t . 158 159 Figure 30. The r e l a t i o n s h i p between input pressure and ch lor ide in f lux using four d i f f e r e n t catecholamines at A) the onset of per fusion and B) 20 min l a t e r . • • , noradrenal ine; A A , adrenal ine; • • , i s o -prenal ine; # • , phenylephrine. 160 L A ><20h 10 K o o O A L - L °o A 20 30 40 50 P j n , mm Hg 40 30 20 10 J I L . B A • 60 20 30 40 50 Pj n ,mm Hg 161 Figure 31. The e f fec t of increas ing concentrat ions of adrenaline on carbonic anhydrase a c t i v i t y of washed red blood c e l l s of rainbow trout (n = 4). 163 DISCUSSION During perfusion of the i s o l a t e d head with p h y s i o l o g i c a l s a l i n e , the rate of C l i n f l u x i s lower than that observed in vivo and invar iab ly dec l ines with time. This pattern suggested the p o s s i b i l i t y of regulat ion of C l i n f l u x by an endogenous humoral or neurohumoral f a c t o r ( s ) . It was apparent from i n i t i a l experiments using adrenergic agonists that ch lor ide i n f l u x i s re la ted d i r e c t l y to cx, receptor st imulat ion and/or inverse ly re la ted to B r e c e p t o r " s t i m u l a t i o n „ according to c r i t e r i a of A l q u i s t (1948). Subsequent experiments using the ««> antagonist , phentolamine and the B antagonist , prop-r a n o l o l showed that in the i s o l a t e d perfused head of rainbow t r o u t , C l i n f l u x i s st imulated by both receptors and i n h i b i t e d by y8 receptors . It i s c lear from t h i s study and others (Richards & Fromm, 1969; Bergman, Olson & Fromm, 1974; Wood, 1974> 1975; Payan & G i r a r d , 1977; Booth, 1978) that catecholamines a f fec t the pattern of blood flow through the g i l l s and may a l t e r funct iona l surface area perfused. Thus, i t i s not a simple matter of separat ing poss ib le s p e c i f i c e f f e c t s of adrenergic st imulat ion on C l i n f lux (permeabil i ty e f f e c t s or d i r e c t i n t e r a c t i o n with transport s i tes) from accompanying haemodynamic a l t e r a t i o n s . I f e e l that the e f fec ts of catecholamines on C l i n f l u x are due to s p e c i f i c a c t -ions rather than to haemodynamic changes for the fo l lowing reas -ons. It has been shown (Bergman et al., 1974) that both <* and B 164 st imulat ion increases funct iona l surface area in i s o l a t e d , per -fused g i l l s of rainbow t rou t , yet I have observed opposite e f fec ts of e*. and B receptor st imulat ion on C l i n f l u x . It i s d i f f i c u l t to imagine these d i f f e r e n t i a l responses being mediated by a com-mon haemodynamic change. Furthermore, phentolamine (<* antagon-is t ) causes a sharp reduct ion in J ^ n C l while only minimally a f f e c t i n g input pressure , i n d i c a t i n g l i t t l e haemodynamic a l t e r -a t ion (F ig . 29). This hypothesis i s fur ther supported by the fac t that no c o r r e l a t i o n ex is ts between input pressure and C l i n f l u x under any experimental condi t ion (F ig . 30). Figure 32 shows the e f fec t of ok or B s t imulat ion on C l i n f l u x , external Na + and C l concentrat ions and input pressure in an i n d i v i d u a l f i s h . As expected, isoprenal ine ( Bagonist) causes i n h i b i t i o n of C l i n f lux and a reduct ion of input pressure while causing increases and decreases in external C l and Na + r e s p e c t i v e l y . It i s c lea r that isoprenal ine i s causing opposite e f f ec ts on C l and Na + net f l u x e s . In a d d i t i o n , Gi rard & Payan (1977) showed that 8 receptor st imulat ion st imulates Na"*- uptake while t h i s study has shown that 8 receptor s t imulat ion causes i n h i b i t i o n of C l uptake. Once again , i t i s d i f f i c u l t to imagine the d i f f -e r e n t i a l e f f e c t s of B receptor st imulat ion on branchia l ion f luxes being caused by a s ingular decrease in per fusion pressure . Catecholamines have been shown to a f fec t ion movements in freshwater and seawater f i s h . In freshwater, adrenal ine st imulates Na + /H + (NH 4 + ) exchange in the secondary lamellae by a £ e f f ec t (Payan, Matty & Maetz, 1975; Girard & Payan, 1977; 165 Figure 32. The e f f e c t s of <^  or $ receptor s t imulat ion on ch lor ide i n f l u x , external sodium and ch lor ide concentrat ions, and input pressure in an i n d i v i d u a l i s o l a t e d head preparat ion. 16T> mm Hg 50 I-40 I -30 20 1300 Ext. Na+ u E q L " 1 1200 | - f 1 0 0 0 H t f E p i Ext. cr I i 9 0 0 ^ 2 0 ^ Jin cr u E q 1 0 0 g " 1 h " 1 1 0 u E q L " -1 1 1 1 1 1 10 20 30 40 TIME (min.) 1.6 7 Payan & G i r a r d , 1978; Payan, 1978) and in seawater i t i n h i b i t s Na + and C l e f f luxes from the ch lor ide c e l l s of the primary lamellae and opercular epithel ium by an e f fec t (Pic , Mayer-Gostan & Maetz, 1975; G i r a r d , 1976; Shutt leworth, 1978; Degnan, Karnaky & Zadunaisky, 1977). Studies using the i s o l a t e d opercular epithel ium (Degnan et al., 1977) are in good agreement with the r e s u l t s of experiments using the perfused head and provide f u r - .. ther evidence that the observed e f fec ts are not caused by acc-ompanying haemodynamic phenomena. Although i t i s ce r ta in that catecholamine e f fec ts on e p i t h e l i a l ion t ransport are not due to haemodynamic changes, the exact mechanism by which the t ransport processes are a f fected remains obscure at t h i s t ime. According to Girard & Payan (1977) the increase of t r a n s e p i t h e l i a l Na + f luxes may be associated with increased permeabi l i ty of the a p i c a l membrane towards t h i s i o n . The hypothesis according to which the rate of ac t ive t ransport i s s t rongly dependent on the e f f e c t i v e permeabi l i ty of the outer membrane has been suggested prev ious ly for frog skin (Curran, Herrera & F lan igan , 1963; Morel & Leblanc, 1975). Act ive N a + t ransport by frog skin epithel ium i s increased when $ adrenergic receptors are st imulated by adrenal ine (Rajer ison, Montegut, Jard & More l , 1972). It i s be l ieved that the c a t e c h o l -amine e f f e c t s are mediated by modi f icat ion in the leve ls of i n t r a c e l l u l a r c y c l i c AMP. C y c l i c AMP and the c y c l i c nucleot ide phosphodiesterase i n h i b i t o r , theophy l l ine , have been shown to st imulate ch lor ide secret ion in the shark r e c t a l gland ( S i l v a , 168 S t o f f , F i e l d , F i n e , Forrest & E p s t e i n , 1977). Recent research of Shuttleworth & Thompson (1980) has shown that c-AMP s t imu l -a t ion of C l secret ion i s due to a passive increase of C l e f f lux as a r e s u l t of an increased gradient for t h i s ion subsequent to an enhanced Na e f f lux caused by an increase in funct iona l Na s i t e s . It i s unknown whether s i m i l a r mechanisms operate in f i s h g i l l s but i t has been shown that adrenaline increases the l e v e l of c-AMP in the g i l l epi thel ium of the freshwater Mul let (personal communication to Girard & Payan, 1977). G i rard & Payan (1977) have postulated that increased act ive t ransport of N a + observed a f te r s t imual t ion of & receptors in the t rout i s depend-ent upon an increased i n t r a c e l l u l a r concentrat ion of c-AMP. In t h i s study, B receptor s t imulat ion i n h i b i t e d b r a n c h i a l - C l i n f l u x . It i s poss ib le that elevated c-AMP leve ls while st imulat ing Na + i n f l u x , i n h i b i t C l i n f l u x . This idea i s supported by the obs-ervat ion of extremely low values of C l i n f lux fo l lowing addi t ion of theopy l l ine or c-AMP to perfusate (only two f i s h were exam-ined) . Stress in f i s h i s associated with elevated l e v e l s of c i r c u l a t i n g catecholamines (Nakano & Tomlinson, 1967). A c i d -base disturbances such as hypercapnic ac idos is most c e r t a i n l y cause f i s h to be stressed and although catecholamine leve ls have never been measured during an acid-base d isturbance, i t i s l i k e l y that they increase . From the r e s u l t s of t h i s study and others (Payan et a l . , 1975; Girard & Payan, 1977) i t i s poss ib le to theor-ize a scheme for acid-base regulat ion invo lv ing adrenergic 169 cont ro l of branchia l C1~/HC0 ~ and Na + /H + (NH. + ) exchange. If catecholamine leve ls do indeed increase during hypercapnic a c i d -o s i s then i t i s poss ib le that C l /HCO^ exchange i s i n h i b i t e d and N a + / H + ( N H 4 + ) exchange enhanced v i a & receptor s t imu la t ion , as discussed prev ious ly . These, of course would be the approp-r i a t e responses in terms of in te rna l pH adjustment, serving to in to the g i l l epi thel ium (see Chapter I I I ) . Thus, catecholamines released in to the blood during per iods of s t r e s s , inc lud ing hyper-capnia , and severe e x e r c i s e , may ameliorate accompanying resp-i r a t o r y and metabolic acid-base disturbances as wel l as augmenting oxygen t ranspor t . Unfortunately , I was unable to demonstrate the appropriate adjustments of Na + or C l i n f l u x during hyper-capnia in rainbow trout (Chapter III) so the s i g n i f i c a n c e of these mechanisms to acid-base regula t ion remains unclear at t h i s t ime. Another poss ib le mechanism for pH regulat ion during hyper-capnic ac idos is which has been overlooked p rev ious ly , i s modulat-ion of e ry throcy t ic HCO^ / C l exchange. The r e s u l t s of t h i s study have shown that e ry throcy t ic carbonic anhydrase a c t i v i t y — 8 — 7 i s i n h i b i t e d by concentrat ions of adrenal ine (10 - 10 M) that have been measured in stressed f i s h (Nakano & Tomlinson, 1967). In other words, adrenal ine decreases the rate of C0 2 evolut ion from red blood c e l l s which of course r e f l e c t s a reduced rate of HCO^ entry into the red blood c e l l . If blood adrenaline l e v e l s do r i s e during hypercapnia then e ry throcy t ic HCO^ / C l exchange w i l l be i n h i b i t e d causing plasma HCO., l e v e l s to i h c r -increase plasma by increas ing the entry of plasma H ions 170 ease thereby res to r ing blood pH to normal. Since the g i l l e p i -elevated as long as e ry throcyt ic HCO^ / C l exchange i s i n h i b i t e d . No attempt was made to determine the receptor responsible for the adrenal ine e f f ec t on e ry throcyt ic HCO^ / C l exchange. However, i f e ry th rocy t ic HCO^ / C l exchange i s s im i l a r to branchia l HCO^ / C l exchange, i t i s poss ib le that B receptor s t imulat ion i s responsib le for the observed i n h i b i t i o n . thelium i s impermeable to HCC> , plasma w i l l remain GENERAL DISCUSSION 172 The pattern of carbon dioxide excret ion and acid-base regulat ion in the rainbow t rou t , Salmo gairdneri, has been inves t -igated in t h i s study. Figure 33 i l l u s t r a t e s the pattern of C 0 2 excret ion i n f i s h as ind icated by the r e s u l t s of t h i s t h e s i s . Carbon dioxide produced by r e s p i r i n g t i ssue enters the plasma and d i f f u s e s in to the erythrocyte according to the concentrat ion gradient . This concentrat ion gradient i s generated as haemoglo-b in unloads oxygen and hydrogen ions are bound on, causing the carbonic anhydrase cata lysed CC^ equi l ibr ium react ion to proceed towards the d i r e c t i o n of HCO^ . HCO^ formed by t h i s react ion enters the plasma in exchange for ch lo r ide ions (the ch lor ide s h i f t ) . As blood enters the g i l l and oxygen binds to haemoglobin, H + ions are unloaded and causes the CC^ equi l ibr ium react ion to s h i f t in the d i r e c t i o n of molecular CC^. This causes plasma HCO3 to enter the erythrocyte , once again in exchange for C l i o n s . Molecular CC^ produced v i a t h i s pathway enters the plasma, d i f f u s e s across the g i l l epithel ium into the water and accounts for the major i ty of t o t a l carbon dioxide excreted. Branchia l carbonic anhydrase cata lyses the conversion of a small por t ion of molecular CC^ to HCO^ and H + ions which ex i t across the a p i c a l membrane in exchange for C l and Na + ions r e s p e c t i v e l y . A l t e r -na te ly , H ions may combine with NH^ and be exchanged for Na in the form of the ammonium i o n , NH^ + . No evidence was gathered to support the hypothesis of Haswell & Randall (1978) that the basal membrane of g i l l e p i -t h e l i a l c e l l s i s permeable to HCO., . In f a c t , the r e s u l t s have Figure 33. A diagramatic representat ion of carbon dioxide excret ion in the rainbow t r o u t , Salmo gairdneri. See text for fur ther d e t a i l s . 1*4 deoxygenati oxygenation C 0 2 i C 0 2 4 *HCCC + H + TISSUES PLASMA COo * » HCOo+H + PLASMA Na + N H 3 basal 175 ind icated that the basal membrane i s impermeable to HCO^ and that i t s movement from the plasma to the g i l l epi thel ium in no way contr ibutes to o v e r a l l ! C 0 2 excret ion or acid-base regu la t ion . On the other hand, i t appears that the g i l l epithel ium ( inc lud-ing the basal membrane) i s very permeable to H + i ons . The move-ment of H + ions across the branchia l epithel ium i s independent of CO2 excret ion and appears to be re la ted to the H + ion grad-ient between the blood and water. It i s suggested that the passive entry of protons in to the g i l l epi thel ium i s in pa r t , governed by e p i t h e l i a l i n t r a c e l l u l a r pH, which in turn i s r e g -ulated by the operating rates of Na + /H + (NH 4 + ) and C1~/HC0 3~ exchange processes. Internal pH, in f i s h as in other animals, i s maintained in part by balanced rates of C 0 2 production and e l im ina t ion . Changes of i n t e r n a l pH have a marked e f fec t on the d i s s o c i a t i o n and conf igurat ion of p ro te ins , and these changes are usua l ly detr imental to the organism. Hence, in the face of a continuous and var iab le product ion, C 0 2 excret ion must be regulated to pre -vent o s c i l l a t i o n s in body pH and the subsequent d is rup t ion of metabolism. It i s important to note that carbon d iox ide , by i t s e l f , i s not very t o x i c , so i t i s l i k e l y that C 0 2 excret ion must be regulated to cont ro l body pH and not the C 0 2 content of the body. Normally, a reduct ion in molecular C0„ l eve ls or an or the excret ion of HCO^ w i l l lower pH. Hypercapnia descr ibes the p h y s i o l o g i c a l condi t ion in which w i l l tend to r a i s e pH, whereas C0„ re tent ion 176 a r t e r i a l P^Q i s elevated above normal l eve ls and i s a conseq-uence of elevated external P P ' . It i s important to r e a l i z e that external hypercapnia i s not only a condi t ion observed in : the laboratory . Carbon dioxide l e v e l s in lakes and oceans vary considerab ly , unl ike atmospheric l e v e l s , which remain very low and s tab le . Metabolic production of CO,,, normally not a problem in wel l mixed water, becomes extremely s i g n i f i c a n t when f i s h are densely stocked and suppl ied with r e c i r c u l a t i n g ground water. Under these cond i t ions , CO., tensions of around 20 mm Hg are not uncommon. Other causes of CO., e levat ion include decay of organic matter (Hynes, 1960), plant r e s p i r a t i o n and discharge of ac id into water of high carbonate content (Doudorhoff & Katz , 1950). The d i f f u s i o n of CO., across the branchia l epi thel ium i s governed by the CO,, gradient between blood and water. During hypercapnia t h i s gradient i s reduced causing blood CO,, tensions to r i s e u n t i l a new steady-state i s reached. F i s h , i f not a l l aquatic animals, regulate bicarbonate concentrat ion rather than molecular CO,, l e v e l s in the blood during hypercapnic a c i d o s i s . High CO., l e v -e l s do cause changes i n g i l l v e n t i l a t i o n in f i s h , but the i n c r -ease in water flow has l i t t l e e f f ec t on e i ther blood pH or P C Q (Janssen & Randal l , 1975). It has been speculated by.these authours that increased g i l l v e n t i l a t i o n during hypercapnia serves to augment oxygen t ranspor t , necessary because of hypox-r emia due to low blood pH. Thus, unl ike the s i t u a t i o n in mammals, manipulation of molecular C0 2 v i a changes in v e n t i l a t i o n i s not u t i l i s e d to regulate pH. 177 The compensatory increase in plasma bicarbonate concent-ra t ion during hypercapnia can be accomplished v i a var ious mech-anisms, inc lud ing non-bicarbonate b u f f e r i n g , an increase in proton excret ion and/or a reduct ion of CC^ excre t ion . The increase in plasma HCO^ i s much larger than can be a t t r ibuted to non-bicarbonate b u f f e r i n g . Poss ib le s i t e s for the cont ro l of proton excret ion are the g i l l and kidney. The poss ib le ro le of the kidney in acid-base regula t ion in f i s h i s usua l ly not considered, or when so considered, i s given a very minor ro le (Cameron & Randal l , 1972; Janssen & Randal l , 1975; Cameron & Wood, 1978). However:/ i t has been recent ly demonstrated that rainbow trout in freshwater w i l l increase the ur inary output of ac id when faced with an ac id load (Wood & Ca ldwel l , 1978; Kobayashi & Wood, 1980). The s i t e of proton pumping has not been l o c a l i s e d in f i s h , but kidney structure i s s i m i l a r to that of Amphibia, so f i s h may be capable of excret ing ac id v i a both the kidney tubule and the bladder. The r o l e of the kidney in the regula t ion of hypercapnic ac idos is has not been examined in t h i s thes is and r e s u l t s of Wood (personal communication) ind ica te that i t i s of l i t t l e s i g n i f i c a n c e . The other major s i t e of H + ion excret ion in f i s h i s the g i l l . Results from these studies have indicated that branchia l H + ion excret ion i s cont ro l l ed by the operating rates + + + — — of a p i c a l Na /H (NH^ ) and C l /HCC>3 exchanges and i s important in the maintainance of a homeostatic i n t e r n a l pH. It has been suggested that these exchange processes are modulated during hypercapnic ac idos is in order to restore blood pH v i a enhanced 178 proton excre t ion . Indeed i t has been demonstrated that these 'pumps' are s e n s i t i v e to catecholamines, C l /HCO^ exchange i n h i b i t e d and Na /H (NH4 ) exchange st imulated by & receptor s t imula t ion . Although i t i s l i k e l y that c i r c u l a t i n g c a t e c h o l -amine l e v e l s increase during hypercapnia, the appror iate ad just -— — + + + ments of C l /HCO^ and Na /H (NH4 ) exchanges have not been ob-served in rainbow t rou t . The only d i r e c t evidence for an i n v o l -vement of branchia l ion exchanges in the regula t ion of acid-base disturbances comes from the work of Cameron (1976). He demon-strated that hypercapnic A r c t i c gray l ing displayed an increased r a t i o of Na + to C l uptake (pr imari ly as a r e s u l t of increased Na + uptake). Assuming that these r e s u l t s r e f l e c t increased H + ion excre t ion , these adjustments would serve to increase plasma bicarbonate concentrat ion and r a i s e blood pH. Another poss ib le mechanism for r a i s i n g plasma bicarbonate l eve ls i s a reduct ion of CC^ excre t ion . In mammals, reductions of CC>2 excret ion normally would lead to ac idos is due to proton re ten t ion . In f i s h , on the other hand, a reduct ion of CC^ exc-re t ion w i l l cause a l k a l o s i s due to HCO^ accumulation. This i s poss ib le because i n f i s h , unl ike in mammals, H + ion excret ion can be independent of CC^ excret ion owing to the permeabi l i ty of the g i l l epi thel ium to protons. Although the g i l l i s the p r i n c i p a l s i t e of CO2 excret ion in f i s h , i t i s u n l i k e l y that t h i s organ i s involved in the cont ro l of CC^ excre t ion . Carbon dioxide t raverses the g i l l p r imar i l y i n the form of molecular C 0 2 (F ig . 3 3)-. Only a small por t ion (approximately 3%) i s exchanged for 179 C l at the a p i c a l membrane. Thus i t i s improbable that the movement of CO^ through the branchia l epithel ium can be contr-'J. o i l e d . The argument of Perry et al. (1981), that the movement of plasma bicarbonate in to the g i l l epithel ium i s an important determinant of blood acid-base status and may be modulated during acid-base d is turbances, can no longer be considered v a l i d . The kidney contr ibutes very l i t t l e to CO2 excret ion and even in . freshwater f i s h , with high urine flow r a t e s , i t i s doubtful that the kidney i s involved i n HCO^ re ten t ion , v i a a reduction of CO., excre t ion . Knowing that the f i s h erythrocyte i s permeable to bicarbonate and that i t s entry in to the rbc from plasma i s the r a t e - l i m i t i n g step in C 0 2 excre t ion , the p o s s i b i l i t y ex is ts that t h i s step i s con t ro l l ed during acid-base d isturbances. It i s in te res t ing that t h i s hypothesis has never been suggested even by invest iga tors who have always assumed that f i s h red blood c e l l s are f u n c t i o n a l l y s im i l a r to mammalian red blood c e l l s . The proposal that f i s h erythrocytes are involved in a c i d - b a s e . r e g u l -at ion has not been tested r igorous ly here. However, i t has been shown that e ry throcy t ic HCO^. /Cl exchange i s sens i t i ve to c a t -echolamines in a manner s imi la r to that observed for the branch-i a l ep i the l ium. C l e a r l y , t h i s area of research warrants fur ther i n v e s t i g a t i o n . Having confirmed that the erythrocyte of rainbow trout i s permeable to bicarbonate and cata lyses the dehydration r e a c t i o n , can one assume that a l l f i shes posess s imi la r mechanisms? Exp-erimental evidence ind icates that a i r -b rea th ing f i s h posess 180 profoundly d i f f e r e n t mechanisms for CO^ excret ion (Randall et al., 1980). Bimodal breathers, when in water, excrete only a s l i g h t amount of carbon dioxide v i a the a i r -b rea th ing organ. This a lone, of course does not ind ica te red blood c e l l imperm-e a b i l i t y because the blood returning from the t i ssues passes f i r s t through the g i l l s where most of the CG^ i s excreted. During a i r exposure however, when the g i l l becomes non- func t iona l , blood P C Q r i s e s and the a i r -b rea th ing organ i s s t i l l unable to excrete s i g n i f i c a n t amounts of. carbon dioxide although oxygen uptake i s unimpaired. T y p i c a l l y , RQ values for the a i r -b rea th ing organs of a i r -b rea th ing f i s h range between 0.1 and 0.3. It has been shown (Randall , F a r r e l l & Haswell , 1978) that i n j e c t i o n of carbonic anhydrase in to blood of a i r exposed Hoplerythrinus unitaen-iatus reduces P a ^ and increases pHa i n d i c a t i n g enhanced CC^ e l im ina t ion . These r e s u l t s ind ica te that red blood c e l l s of a i r -breathing f i s h may be f u n c t i o n a l l y impermeable to HCO^ and hence, play no r o l e in CC^ excre t ion . I t i s d i f f i c u l t to imagine any advantages that impermeable red blood c e l l s would give to a i r -breathing f i s h while the disadvantages are obvious. The l u n g f i s h , Protopterus and Lepidoslren, aest ivate during prolonged periods of a i r exposure. These animals re t reat in to mud burrows and surround themselves with mucus, which dr ies out in to a coccoon. During these per iods , the g i l l and the skin can no longer funct ion in CC>2 excret ion and consequently P a C Q may r i s e as high as 50 mm Hg while pHa decreases sharply (DeLaney, L a h i r i & Fishman, 1974). Even though the f i s h i s in a state of torpor , t h i s a c i d o t i c state 181 must place add i t iona l s t resses on the animal. Any poss ib le advantages of impermeable red blood c e l l s in a i r -b rea th ing f i shes remain unknown at t h i s t ime. C l e a r l y , fur ther research i s warranted in t h i s area. Although i t i s uncertain whether a i r -breathing f i s h have permeable red blood c e l l s , i t i s c lea r that red blood c e l l s of rainbow trout are permeable to bicarbonate and that i t s entry into erythrocytes i s the r a t e - l i m i t i n g step in CC>2 excre t ion . Moreover, i t appears that t h i s pathway i s con t ro l l ed by catecholamines. Inh ib i t ion of e ry throcyt ic HCO^ / C l exchange during hypercapnic ac idos is i s perhaps an important mechanism cont r ibut ing to accumulation of plasma bicarbonate and subsequent pH regu la t ion . On the other hand, although the g i l l ion t ransport systems are important in maintaining in te rna l a c i d -base s ta tus , no evidence was found to support the hypothesis that branchia l ion exchange mechanisms are involved in the regulat ion of hypercapnic a c i d o s i s . 182 ABBREVIATIONS AND SYMBOLS <*C02 S o l u b i l i t y c o e f f i c i e n t of C0 2 Ad Adrenaline CA Carbonic anhydrase [ c i ~ ] Chlor ide ion concentrat ion C n Tota l oxygen content U 2 C c 0 To ta l carbon dioxide content DAP Dorsal a o r t i c pressure 0Epi Phenylephrine F" Cardiac pump frequency [ H + Q Hydrogen ion concentrat ion QjCO-j J Bicarbonate ion concentrat ion Hct Haematocrit Ipr Isoprenal ine J . 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A z r i e l l i Postgraduate Fe l lowship , Concordia Un ivers i ty 1977-81 N.R.C. 1967 Science Scholarship 1981-82 N . S . E . R . C . Postdoctora l Scholarship Publ ica t ions Haswell , M .S . , Perry , S . F . & Randal l , D . J . (1978). The e f fec t of perfusate oxygen l e v e l s on C0 2 excret ion in the perfused g i l l . J . exp. Z o o l . 205, 309-315 Haswell , M .S . , Randal l , D . J . & Perry , S . F . (1980). F i s h g i l l carbonic anhydrase: Acid-base regula t ion or s a l t t ransport? Am. J . P h y s i o l . 238, 240-245. Perry , S . F . , Haswell , M .S . , Randal l , D . J . & F a r r e l l , A . P . (1981). Branchia l i o n i c uptake and acid-base regulat ion in the 2 02 rainbow t r o u t , Salmo g a i r d n e r i . J . exp. B i o l . In p ress . Perry , S . F . & Randal l , D . J . (1981). The e f fec ts of amilor ide and SITS on branchia l ion uptake in rainbow t rou t , Salmo  g a i r d n e r i . J . exp. Zool . 215,. 225-228. Perry , S . F . & Heming, T . A . (1981). Blood ion ic and acid-base status in rainbow t rout (Salmo gairdner i ) fo l lowing rap id t ransfer from freshwater to seawater: E f f e c t of pseudobranchial denervat ion. Can. J . Z o o l . In press . Perry , S . F . , Davie, P . S . , Daxboeck, C. & Randal l , D . J . (1981). A comparison of CC>2 excret ion in a spontaneously v e n t i l -a t i n g , blood-perfused t rout preparat ion and sa l ine -per fused g i l l preparat ions: Contr ibut ion of the branchia l epi thel ium and red blood c e l l . Submitted to J . exp. B i o l . Perry , S . F . , Payan, P. & G i r a r d , J . P . Adrenergic cont ro l of ch lor ide transport in rainbow t r o u t , Salmo g a i r d n e r i . In preparat ion. Perry , S . F . , Davie, P .S . & Daxboeck, C. (1982). Perfusion methods used to study f i s h g i l l physiology. In: F i s h  Physiology (Eds. W.S. Hoar & D . J . Randal l ) , Vo l X, Academic Press , In preparat ion. Randal l , D . J . , Perry , S . F . & Heming, T . A . (1981). Gas t ransfer and acid-base regula t ion in Salmonids. Comp. Biochem. Physiol- . In p ress . Davie, P . S . , Daxboeck, C , Perry , S . F . & Randal l , D . J . A spontaneously v e n t i l a t i n g , blood-perfused t rout preparat ion: A new approach to the study of gas t ransfer in f i s h e s . 2.03 Submitted to J . exp. B i o l . Daxboeck, C , Davie, P . S . , Perry , S . F . & Randal l , D . J . Oxygen uptake in a spontaneously v e n t i l a t i n g , blood-perfused trout preparat ion. Submitted to J . exp. B i o l . 

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