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Ammonia stores and excretion in fish : relationship to pH Wright, Patricia Anne 1987

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AMMONIA STORES AND EXCRETION IN FISH: RELATIONSHIP TO pH by PATRICIA ANNE WRIGHT B . S c , McMaster U n i v e r s i t y , 1982 THESIS SUBMITTED IN PARTIAL FUFILMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 (c) P a t r i c i a Anne Wright, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. DE-6(3/81) ABSTRACT The d i s t r i b u t i o n and t r a n s f e r of ammonia between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments of f i s h and the e x t e r n a l water environment was i n v e s t i g a t e d . In, vivo and i n v i t r o experiments were performed on the freshwater rainbow t r o u t (Salmo g a i r d n e r i ) and the i n t a c t , seawater lemon sole (Parophrvs  v e t u l u s ) . The d i s t r i b u t i o n of ammonia and H+ ions were compared between red c e l l s and plasma (in. vivo and i n v i t r o ) taken from rainbow t r o u t at r e s t and during hypercapnia. At r e s t (in, v i v o and i n v i t r o ) . measured i n t r a c e l l u l a r ammonia l e v e l s were equal to those p r e d i c t e d by the plasma to red c e l l pH gradient. The same was not true during hypercapnia, where measured red c e l l ammonia l e v e l s were greater than p r e d i c t e d l e v e l s . The a d d i t i o n of the Na+/K+ ATPase i n h i b i t o r , ouabain, had no e f f e c t on ammonia accumulation during hypercapnia. I t was concluded that ammonia i s p a s s i v e l y d i s t r i b u t e d according to plasma-to-red c e l l H+ ion d i s t r i b u t i o n i n blood at r e s t i n g pH values, but under hypercapnic c o n d i t i o n s , ammonia accumulation must be due to some other a c t i v e uptake mechanism. The d i s t r i b u t i o n of ammonia and l^C-DMO w e r e compared i n white muscle, heart, b r a i n , red c e l l s , and plasma of lemon sole ( i n v ivo) at r e s t , during hypercapnia, and f o l l o w i n g e x e r c i s e . The red c e l l ammonia d i s t r i b u t i o n at r e s t and during an e x t r a c e l l u l a r a c i d o s i s (hypercapnia and e x e r c i s e ) was s i m i l a r to that found i n rainbow t r o u t . Red c e l l s are unusual i n that H+ ions are p a s s i v e l y d i s t r i b u t e d according to membrane p o t e n t i a l (Em), whereas i n other t i s s u e s , t h i s i s not the case. In white muscle, heart, and b r a i n under a l l experimental c o n d i t i o n s , i n t r a c e l l u l a r ammonia l e v e l s f a r exceeded those p r e d i c t e d by transmembrane pH gr a d i e n t s . C a l c u l a t e d E J JH4+ values i n these t i s s u e s were very c l o s e to published r e s t i n g values of Em. I t was concluded that NH4.1. i s permeable across c e l l membranes and that i n t r a c e l l u l a r ammonia stores are not determined by transmembrane pH gradients i n lemon s o l e . The pH of i n t e r l a m e l l a r water was i n v e s t i g a t e d i n rainbow t r o u t by f o l l o w i n g changes i n the downstream pH of expired water using a stopped-flow method. As water flowed over the g i l l s of c o n t r o l f i s h , there was a s i g n i f i c a n t decrease i n water pH. Acetazolamide (carbonic anhydrase (CA) i n h i b i t o r ) added to the water increased the CO2 d i s e q u i l i b r i u m , while CA e l i m i n a t e d the CO2 d i s e q u i l i b r i u m r e l a t i v e to c o n t r o l water. Mucus excreted by the f i s h was found to c o n t a i n CA a c t i v i t y by the pH-stat technique. I t was concluded that water a c i d i f i c a t i o n i s due to the conversion of excreted CO2 to H C O 3 - and H+ at the g i l l s u r face. A p o s s i b l e f u n c t i o n of CA at the e x t e r n a l g i l l surface i s to f a c i l i t a t e carbon d i o x i d e and ammonia e x c r e t i o n . Acetazolamide or CA added to the water d i d not a l t e r carbon d i o x i d e (.HQ02^ o r 0 ammonia (Mft m m) e x c r e t i o n i n i n t a c t rainbow t r o u t . Methazolamide (CA i n h i b i t o r ) or methazolamide + am i l o r i d e (Na-f-i v uptake i n h i b i t o r ) added to the water had no e f f e c t on plasma NH3 tensions ( P N H 3 > ' D u t increased M ^ M M s l i g h t l y compared to c o n t r o l f i s h . In general, methazolamide r e s u l t e d i n an increase i n the d i f f u s i n g c a p a c i t y of ammonia. The i n t e r p r e t a t i o n of these r e s u l t s was complicated by the f a c t that r a p i d s e r i a l blood sampling r e s u l t e d i n a u n i v e r s a l blood a l k a l o s i s . The i n t a c t r e s t i n g f i s h i s u n s u i t a b l e f o r studying the i n t e r a c t i o n between water boundary la y e r chemistry and e x c r e t i o n across the g i l l . With the blood-perfused t r o u t head p r e p a r a t i o n i t was demonstrated that M c o 2 a n (^ MAmm a r e l i n k e d through chemical r e a c t i o n s i n the e x t e r n a l water boundary l a y e r adjacent to the g i l l . P r e - i n c u b a t i o n of blood with acetazolamide reduced MQ02 and M ^ M M i n the blood-perfused head. Increasing the b u f f e r i n g c a p a c i t y of i n s p i r e d water, s i g n i f i c a n t l y reduced M ^ M M , but M Q Q 2 w a s unaffected. Each of these experimental treatments s i g n i f i c a n t l y reduced the a c i d i f i c a t i o n of v e n t i l a t o r y water flowing over the g i l l s . I t i s proposed that the c a t a l y s e d conversion of excreted CO2 to form H C O 3 - and H + ions i n the g i l l boundary l a y e r provides a c o n t i n u a l supply of H + ions needed for the removal of NH3 to N H 4 + , which reduces water NH3 l e v e l s and f a c i l i t a t e s ammonia e x c r e t i o n . Gas t r a n s f e r v a r i a b l e s i n the blood-perfused head p r e p a r a t i o n were compared to i n t a c t cannulated f i s h with and without o r a l masks. Oxygen uptake (MQ2> and M Q O 2 w e r e lower, and M ^ M M , higher i n the blood-perfused head compared to in, v i v o values. V these d i s c r e p a n c i e s were due to d i f f e r e n c e s i n venous O 2 , C O 2 , and ammonia l e v e l s , which determine mean grad i e n t s across the g i l l s . I t was concluded that the blood-perfused head i s a * s u i t a b l e p r e p a r a t i o n f o r studying the i n t e r a c t i o n between MrjQ2 and H^m r n because the o v e r a l l e f f i c i e n c y of t r a n s f e r of N H 3 : C 0 2 was very s i m i l a r between in. v i t r o and i n v i v o p r e p a r a t i o n s , v i TABLE OF CONTENTS Page # ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i GENERAL INTRODUCTION 1 GENERAL MATERIALS AND METHODS 7 Chapter 1. The d i s t r i b u t i o n of ammonia and H+ ions between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments i n Salmo  g a i r d n e r i and Parophrvs v e t u l u s . I n t r o d u c t i o n 17 M a t e r i a l s and Methods 21 Results 33 Di s c u s s i o n 54 Chapter 2. Downstream pH changes i n water flowing over the g i l l s of Salmo g a i r d n e r i I n t r o d u c t i o n 74 M a t e r i a l s and Methods 76 Results 81 Di s c u s s i o n 91 Chapter 3. E f f e c t s of i n h i b i t i o n of e x t e r n a l g i l l c arbonic anhydrase on carbon d i o x i d e and ammonia e x c r e t i o n i n i n t a c t Salmo g a i r d n e r i . I n t r o d u c t i o n 99 M a t e r i a l and Methods 103 Results 109 Di s c u s s i o n 124 Chapter 4. The li n k a g e between carbon d i o x i d e and ammonia e x c r e t i o n i n the i s o l a t e d blood-perfused t r o u t head pr e p a r a t i o n . I n t r o d u c t i o n 133 M a t e r i a l s and Methods 137 Resu l t s 152 Di s c u s s i o n 165 GENERAL DISCUSSION 177 APPENDIX I 197 I I 198 REFERENCES 204 v i i i LIST OF TABLES Table 1. Measured pHe, red c e l l pHi, plasma and red c e l l 34 Tamm,predicted red c e l l pHi and Tamm, and c a l c u l a t e d EJJH4 + and E H + i n rainbow t r o u t (in. v i t r o and in. vivo) during c o n t r o l and hypercapnia. Table 2. Measured pHe, red c e l l pHi, plasma and red c e l l 37 Tamm, p r e d i c t e d red c e l l pHi and Tamm, and c a l c u l a t e d EJJH4 + and E H + i n rainbow t r o u t (in. v i t r o ) during hypercapnia, with and without the a d d i t i o n of ouabain. Table 3. Acid-base s t a t u s i n sole during c o n t r o l , hyper- 39 capnia,and e x e r c i s e regimes. Table 4. Blood haemoglobin, haematocrit, mean c e l l u l a r 41 haemoglobin c o n c e n t r a t i o n and red c e l l and plasma water content i n sole during c o n t r o l , hypercapnia, and e x e r c i s e regimes. Table 5. F l u i d volume d i s t r i b u t i o n i n various t i s s u e s of 43 lemon so l e during c o n t r o l , hypercapnia, and ex e r c i s e treatments. Table 6. Measured pHe, red c e l l pHi, plasma and red c e l l 44 Tamm,predicted red c e l l pHi and Tamm, and c a l c u l a t e d EJJH4 + and EH+ i n lemon so l e during c o n t r o l , hypercapnia, and ex e r c i s e regimes. Table 7. Measured pHe, muscle pHi, plasma and muscle Tamm, 45 pre d i c t e d muscle pHi and Tamm, and c a l c u l a t e d ENH4+ i n lemon sole during c o n t r o l , hypercapnia, and ex e r c i s e regimes. Table 8. Measured pHe, heart pHi, plasma and heart Tamm, 46 pr e d i c t e d heart pHi and Tamm, and c a l c u l a t e d E[jH4 + i n lemon sole during c o n t r o l , hypercapnia, and ex e r c i s e regimes. Table 9. Measured pHe, b r a i n pHi, plasma and b r a i n Tamm, 47 pr e d i c t e d b r a i n pHi and Tamm, and c a l c u l a t e d EJJH4 + i n lemon sole during c o n t r o l , hypercapnia, and e x e r c i s e regimes. Table 10. I n s p i r e d , mixed e x p i r e d , and e q u i l i b r a t e d expired 87 water pH and h a l f - t i m e values for water C02:HC03~ i n t e r -conversions i n rainbow t r o u t i n c o n t r o l water and a f t e r the a d d i t i o n of acetazolamide to the water. Table 11. I n s p i r e d , mixed e x p i r e d , and e q u i l i b r a t e d expired 88 water pH and h a l f - t i m e values for water C02:HC03~ i n t e r -conversions i n rainbow t r o u t i n c o n t r o l water and a f t e r the a d d i t i o n of carbonic anhydrase to the water. i x Table 1 2 . V e n t i l a t i o n and carbon d i o x i d e e x c r e t i o n r a t e s 8 9 i n t r o u t exposed to c o n t r o l waters and e i t h e r acetazolamide or carbonic anhydrase i n the water. Table 1 3 . CO2 dehydration r e a c t i o n r a t e i n c a t a l y s e d ( f i s h mucus) and uncatalysed s o l u t i o n s . Table 1 4 . Plasma CQQ2, P C 0 2 ' A N D H C 0 3 ~ i n t r o u t under c o n t r o l and methazolamide regimes. 9 0 1 2 0 1 2 1 Table 1 5 . Plasma C C Q 2 , P C 0 2 ' a n d H C O 3 - i n t r o u t under methazolamide and methazolamide + a m i l o r i d e regimes. 1 5 8 - 1 5 9 Table 1 6 . Comparison of gas t r a n s f e r v a r i a b l e s i n vivo and i s o l a t e d blood-perfused t r o u t head based on a r t e r i a l -venous d i f f e r e n c e s . Table 1 7 . Comparison of gas t r a n s f e r v a r i a b l e s i j i v i v o and 1 6 1 i s o l a t e d blood-perfused t r o u t head based on i n s p i r e d -e x p i r e d d i f f e r e n c e s . Table 1 8 . Comparison of gas t r a n s f e r between a r t e r i a l - 1 6 2 venous d i f f e r e n c e s and inspired-mixed ex p i r e d d i f f e r e n c e s i n the i s o l a t e d blood-perfused head pr e p a r a t i o n . 1 6 4 Table 1 9 . Comparison of t r a n s f e r f a c t o r s f o r C O 2 , O 2 , ammonia, C O 2 / O 2 , ammonia/02, ammonla/C02 between i n v i v o , i n v i t r o , and t h e o r e t i c a l values. Table 2 0 . Estimated g i l l water boundary l a y e r pH f o r t r o u t 1 8 8 i n freshwater and sol e i n seawater. Table 2 1 . Values used to c a l c u l a t e the thickness of the 1 9 9 g i l l water boundary l a y e r (Appendix I I ) . LIST OF FIGURES Figure 1. The d i f f e r e n c e between i n s p i r e d and mixed expired 9 water pH i n t r o u t with opercular cannulae. 12 35 Figure 2. Two-chambered p l e x i g l a s s experimental chamber. Figure 3. Red c e l l and plasma P(jH3 * n t r o u t (in, v i t r o and i n vivo) under c o n t r o l and hypercapnic regimes. Figure 4. Red c e l l and plasma P N H 3 * n lemon sole ( i n vivo) under c o n t r o l , hypercapnia, and e x e r c i s e regimes. 49 51 Figure 5. White muscle, heart, b r a i n , and plasma P N H 3 l e v e l s i n lemon s o l e under c o n t r o l , hypercapnia, and e x e r c i s e c o n d i t i o n s . 62 Figure 6. T h e o r e t i c a l r e l a t i o n s h i p between tissuerplasma ammonia concentrations versus the r e l a t i v e p e r m e a b i l i t i e s of NH 3 and NH4+. 69 Figure 7. Model of ammonia movements between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments i n f i s h . 82 Figure 8. E f f e c t s of mixing on the t j / 2 of the CO2 h y d r a t i o n r e a c t i o n . Figure 9. Changes i n e x p i r e d water pH over time: e f f e c t s of acetazolamide and carbonic anhydrase. .' 0 Figure 10. Carbon d i o x i d e and ammonia e x c r e t i o n and Vw i n r e s t i n g f i s h and those exposed to acetazolamide. Figure 11. Carbon d i o x i d e and ammonia e x c r e t i o n and-Vw i n r e s t i n g f i s h and those exposed to carbonic anhydrase. Figure 12. Blood pHe, ammonia, and NH3 l e v e l s i n f i s h at r e s t and exposed to methazolamide i n the water. Figure 13. Blood pHe, ammonia, and NH3 l e v e l s i n f i s h exposed to a m i l o r i d e or methazolamide + a m i l o r i d e i n the water. Figure 14. Ammonia e x c r e t i o n r a t e s i n r e s t i n g f i s h , those exposed to methazolamide, a m i l o r i d e , and methazolamide + a m i l o r i d e i n the water. Figure 15. Model of chemical r e a c t i o n s i n g i l l water boundary l a y e r . Figure 16. Experiemntal apparatus f o r the blood-perfused 142 t r o u t head p r e p a r a t i o n . 85 110 112 114 116 118 134 x i F igure 17. Carbon d i o x i d e and ammonia e x c r e t i o n , oxygen 153 uptake, and blood pHa-pHv i n blood-perfused head under c o n t r o l , a m i l o r i d e , a m i l o r i d e + acetazolamide, and a m i l o r i d e + T r i s b u f f e r c o n d i t i o n s . 155 Figure 18. Water pHj-pHE and Vw i n the blood-perfused head under c o n t r o l , a m i l o r i d e , a m i l o r i d e + acetazolamide, and a m i l o r i d e + T r i s c o n d i t i o n s . Figure 19. Schematic r e p r e s e n t a t i o n of g i l l water boundary 181 l a y e r thickness i n t r o u t . 185 Figure 20. R e l a t i o n s h i p between water PQ02 a n d P H i n Vancouver and Ottawa freshwater and Bamfield seawater. Figure 21. Schematic r e p r e s e n t a t i o n of g i l l e p i t h e l i u m and 190 boundary water l a y e r showing p r e d i c t e d ammonia l e v e l s . Figure 22. P l o t of e q u i l i b r a t i o n i n e f f i c i e n c y (£) versus 201 e q u i l i b r a t i o n r e s i s t e n c e index if). ACKNOWLEDGEMENTS To my su p e r v i s o r . Dr. David R a n d a l l , I wish to express my si n c e r e thanks for h i s encouragement and guidance throughout the course of t h i s p r o j e c t . I would l i k e to acknowledge i n p a r t i c u l a r : Dr. CM. Wood, McMaster U n i v e r s i t y , f o r h i s t i r e l e s s enthusiasm and c o l l a b o r a t i o n on the lemon s o l e p r o j e c t at the Bamfield Marine S t a t i o n . Dr. S.F. Per r y , U n i v e r s i t y of Ottawa, f o r h i s r e l i a b l e optimism and c o l l a b o r a t i o n with experiments comprising Chapter 4. My f e l l o w labmates are thanked f o r t h e i r humour and understanding, with s p e c i a l thanks to Dennis Mense f o r great moral support, and La r r y F i d l e r f o r the software programes used i n Chapter 2 and f o r many s t i m u l a t i n g d i s c u s s i o n s . I g r a t e f u l l y acknowledge the t e c h n i c a l a s s i s t a n c e of Steve Hunger, McMaster U n i v e r s i t y . I rec e i v e d f i n a n c i a l support from the Natural Sciences and Engineering Research Council (NSERC) and a UBC Teaching A s s i s t a n t s h i p . GENERAL INTRODUCTION 1 Carbon d i o x i d e , oxygen, and ammonia 1 are the three r e s p i r a t o r y gases t r a n s f e r r e d across f i s h g i l l s . Oxygen i s e x t r a c t e d from water flowing over the g i l l s and i s c a r r i e d i n the blood to the t i s s u e s . Carbon d i o x i d e and ammonia are metabolic endproducts which are c a r r i e d away from the t i s s u e s i n the blood and are e l i m i n a t e d across the g i l l s . Gas t r a n s f e r across the g i l l s i s modified by var y i n g blood and water flow, the c h a r a c t e r i s t i c s of the g i l l e p i t h e l i u m , the d i s t r i b u t i o n of blood w i t h i n the g i l l s , and the p r o p e r t i e s and numbers of red blood c e l l s (Randall & Daxboeck, 1984). The d i f f e r e n t p h y s i c a l and chemical p r o p e r t i e s of oxygen, carbon d i o x i d e , and ammonia have numerous p h y s i o l o g i c a l consequences. The r a t e of molecular d i f f u s i o n through a given medium i s r e l a t e d to the molecular weight of the molecules inv o l v e d . Carbon d i o x i d e i s s l i g h t l y l a r g e r than oxygen, so that i n the gas phase, the d i f f u s i o n c o e f f i c i e n t f o r CO2 i s somewhat lower. In an aqueous medium, however, t r a n s f e r of a molecule i s a l s o r e l a t e d to i t ' s s o l u b i l i t y . The Krogh's permeation c o e f f i c i e n t (K= d i f f u s i o n c o e f f i c i e n t x s o l u b i l i t y ) f or CO2 i s much greater than that for O2 because CO2 i s about 30 times 1. The term ammonia or Tamm w i l l be used to i n d i c a t e the t o t a l ammonia c o n c e n t r a t i o n , while NH4+ and NH3 w i l l r e f e r to ammonium i o n and nonionic ammonia, r e s p e c t i v e l y . 2 more s o l u b l e . Ammonia's molecular weight i s lower than that of e i t h e r CO2 and O2 and i n water, i s about 900 times more s o l u b l e than CO2 and 24,000 times more s o l u b l e than O 2 . The Krogh's permeation c o e f f i c i e n t f o r N H 3 , t h e r e f o r e , w i l l be s e v e r a l orders of magnitude l a r g e r than that f o r e i t h e r CO2 or O 2 . The d i f f u s i o n of gases through an aqueous medium may be i n f l u e n c e d by f a c t o r s other than d i f f u s i o n c o e f f i c i e n t s and s o l u b i l i t y f a c t o r s . The f a c i l i t a t e d d i f f u s i o n of O2 and CO2 modifies the e f f e c t i v e value of the Krogh's constant. The d i f f u s i o n of 0 2 through water i s enhanced by the presence of haemoglobin or myoglobin i n the water s o l u t i o n (Scholander, 1960). The enzyme carbonic anhydrase, which c a t a l y s e s the i n t e r c o n v e r s i o n r e a c t i o n of C02:HC03~, f a c i l i t a t e s CO2 d i f f u s i o n through water (Longmuir §_£. aL., 1966). The chemical p r o p e r t i e s of oxygen, carbon d i o x i d e , and ammonia i n s o l u t i o n are q u i t e d i s s i m i l a r . Oxygen i s a nonpolar gas, while carbon d i o x i d e and ammonia are p o l a r molecules which undergo r e v e r s i b l e h y d r a t i o n r e a c t i o n s i n water. The apparent o pK' of the C02:HC03~ r e a c t i o n at f i s h temperatures (5-15 C) i s about 6 ( B o u t i l i e r ejt. a i - , 1985), whereas the pK f o r the NH3:NH4+ r e a c t i o n i s about 10 (Cameron & H e i s l e r , 1983). The pH of f i s h plasma i s midway between these two values such that the C O 2 / H C O 3 - and NH3/NH4+ r a t i o s are very s m a l l , as described by the Henderson-Hasselbalch equation: pH = pK + l o g [NH 3]/tNH 4+] (1) pH = pK + log [HC0 3-]/tC0 2J (2) C e l l membranes are permeable to the un-ionized CO2 and N H 3 , but r e l a t i v e l y impermeable to the ionized forms of these compounds, NH4+ and H C O 3 - . The fact that body pH i s intermediate between the pK of the CC>2:HC03- and N H 3 : N H 4 + reactions ensures adequate transfer of the un-ionized forms and minimizes the stores of both metabolic endproducts (Wright & Randall, 1987). Accumulation of ammonia or H+ ( 0 H - ) ions w i l l have deleterious e f f e c t s to the animal, while H C O 3 - , C 0 2 , and 0 2 are not p a r t i c u l a r l y toxic. Increases i n body ammonia lev e l s i n t e r f e r e with neural function and may cause convulsions and death i n vertebrates (Visek, 1968). Large fluctuations i n body pH w i l l a l t e r the i o n i z a t i o n state of proteins and other weak e l e c t r o l y t e s , which may i n h i b i t enzyme a c t i v i t y , a l t e r c e l l membrane function, and disrupt protein subunit aggregations. The theory of nonionic d i f f u s i o n (Jacobs & Stewart, 1936; Milne at al.., 1958; P i t t s , 1973) describes the d i s t r i b u t i o n of weak e l e c t r o l y t e s , such as ammonia and carbon dioxide, between body compartments. In terms of ammonia, the theory states that c e l l membranes are r e l a t i v e l y impermeable to NH4+ ions, but highly permeable to the non-ionic form of ammonia, NH3. The transfer of ammonia between body compartments, therefore, w i l l depend on NH3 d i f f u s i o n gradients. NH3 l e v e l s w i l l be larger i n a high pH compartment compared to a low pH compartment, and hence, ammonia le v e l s increase i n the low pH compartment as NH3 e n t e r s , combines with a H+ i o n and i s trapped i n the impermeant NH4+ form. Thus, the d i s t r i b u t i o n of ammonia between t i s s u e compartments w i l l be determined by i n t r a c e l l u l a r - t o - e x t r a c e l l u l a r g r a d i e n t s , described by the equation: ( 3 ) [ i n t r a c e l l u l a r ammonia! = 1 + 10(PK ~ PH A M t r a c e l l i U a r ) [ e x t r a c e l l u l a r ammonia] 1 + 10<PK " P H e x t r a c e l l u l a r ) As the pH of most i n t r a c e l l u l a r compartments i s lower than e x t r a c e l l u l a r f l u i d , t i s s u e s t o r e s of ammonia w i l l be l a r g e . The pH dependence of the CO2 d i s t r i b u t i o n i s the reverse of th a t of ammonia, tha t i s , a c i d c o n d i t i o n s trap N H 4 + , while a l k a l i n e c o n d i t i o n s trap H C O 3 - . The above theory of non-ionic d i f f u s i o n describes t r a n s f e r of carbon d i o x i d e and ammonia across membranes w i t h i n the f i s h . Due to oxygen's nonpolar nature, O2 t r a n s f e r between body compartments and the environment i s dependent on 0 2 p a r t i a l pressure g r a d i e n t s . Ammonia and carbon d i o x i d e t r a n s f e r between the body and the environment are a l s o dependent on p a r t i a l pressure g r a d i e n t s , as w e l l as, NH4+ and H C O 3 -e l e c t r o c h e m i c a l g r a d i e n t s . Ion exchange processes at the g i l l s (eg., Na+/NH4+, C I - / H C O 3 - ) are e s s e n t i a l f o r acid-base balance and i o n r e g u l a t i o n i n f i s h . The r e l a t i v e b r a n c h i a l e x c r e t i o n r a t e s of NH3 versus NH4+ and CO2 versus H C O 3 -v a r i e s under d i f f e r e n t c o n d i t i o n s . In freshwater f i s h , NH3 5 excretion may be between 100% (Cameron & Heisler, 1983) and less than 50% (Wright & Wood, 1985), with the remainder of ammonia eliminated as NH4+. Carbon dioxide i s primarily excreted as CO2 (Perry §£ aJL.., 1982), with less than 10% as H C O 3 -(Cameron, 1976; Perry, 1986). The r a t i o of carbon dioxide production to oxygen consumption in resting f i s h i s near unity (0.7-1.0). In contrast, ammonia production i s considerably lower than oxygen consumption, with a r a t i o of about 0.1 i n starved f i s h (Brett & Zala, 1975). Blood l e v e l s of oxygen, carbon dioxide, and ammonia are not only determined by uptake or production rates, but also by the e f f i c i e n c y of transfer between the body and water environment. In f i s h , a large volume of water must come in contact with the g i l l s to maintain adequate 0 2 uptake because of the low s o l u b i l i t y of O2 i n aqueous media. Consequently, blood carbon dioxide and ammonia concentrations w i l l be comparatively low because of their greater s o l u b i l i t y c o e f f i c i e n t s . The chemical composition and rate of flow of environmental water also strongly influences gas transfer across the g i l l . Water qu a l i t y may vary greatly i n freshwater systems, both s p a t i a l l y and temporally, i n terms of water pH, gas tensions ( O 2 , C 0 2 , ammonia), and temperature. In general, an increase i n the convection of water over the g i l l s w i l l enhance d i f f u s i o n gradients ( 0 2 * C O 2 , NH3) across the g i l l by renewing water at the branchial surface. Next to the branchial surface there i s thin layer of mucus and an associated layer of unstirred water or 6 boundary l a y e r (Scheid & P i i p e r , 1971). Increases In v e n t i l a t o r y flow which d i m i n i s h the thickness of the boundary l a y e r , w i l l enhance gas t r a n s f e r across the g i l l ( P i i p e r §_t §2.., 1986). The o b j e c t i v e s of the present study were to i n v e s t i g a t e the t r a n s f e r of ammonia i n f i s h , i n l i g h t of i t s unique chemical and p h y s i c a l p r o p e r t i e s . The f o l l o w i n g experiments can be d i v i d e d i n t o two p a r t s ; those designed to i n v e s t i g a t e the t r a n s f e r of ammonia between t i s s u e compartments i n the body and those designed to explore ammonia t r a n s f e r between the body and the environment. In the f i r s t s e c t i o n , I propose that the theory of non-ionic d i f f u s i o n d e s c r i b e s the d i s t r i b u t i o n of ammonia between various t i s s u e compartments i n f i s h . More s p e c i f i c a l l y , the hypothesis t e s t e d was that i n t r a c e l l u l a r ammonia sto r e s w i l l be determined by transmembrane pH g r a d i e n t s . In the second s e c t i o n on ammonia t r a n s f e r across the g i l l s of f i s h , the experiments were concerned with two problems. F i r s t , i t was known that ammonia e x c r e t i o n i n f i s h was i n f l u e n c e d by environmental pH (Wright & Wood, 1985), but i t was not known i f water i n contact with the g i l l surface was r e p r e s e n t a t i v e of bulk water. The proposed hypothesis was that water l e a v i n g the g i l l becomes p r o g r e s s i v e l y more a c i d i c as excreted CO2 i s hydrated to form H C O 3 - and H+ ions i n the ex p i r e d water. The second problem concerning ammonia t r a n s f e r across the g i l l s developed from the dis c o v e r y that carbonic anhydrase was present on the e x t e r n a l g i l l s u rface. The f i n a l hypothesis t e s t e d was that the c a t a l y s e d conversion of CO2 to form H+ and H C O 3 - ions i n the g i l l water 7 boundary l a y e r f a c i l i t a t e s ammonia e x c r e t i o n by c h e m i c a l l y removing NH3 from the water next to the g i l l s urface. 8 GENERAL MATERIALS AND METHODS S u r g i c a l Procedures Trout d o r s a l a o r t i c c a n n u l a t i o n Trout were anaesthetized i n a buffered (NaHC03) tricanemethanesulphonate (MS222) s o l u t i o n at a c o n c e n t r a t i o n of 1:10,000 and then t r a n s f e r r e d to the operating t a b l e where they were maintained at a MS222 co n c e n t r a t i o n of 1:20,000. Dorsal a o r t i c (DA) cannulae were implanted using the technique of Sovio et a l . (1972). In t h i s method a sharpened g u i t a r wire was f i t t e d i n s i d e a piece (~ 20cm) of PE50 tubing and used to p i e r c e the DA s u p e r f i c i a l l y , at the l e v e l of the f i r s t or second d o r s a l g i l l arch. The wire was then removed and the DA cannula was advanced before being secured to the roof of the mouth with two s t i t c h e s . The cannula was l e d out of the buccal c a v i t y through a h e a t - f l a r e d sleeve (PE 160). Trout V e n t r a l A o r t i c c a n n u l a t i o n F i s h were anaesthetized, as described above and the v e n t r a l a o r t a (VA) was cannulated by one of the f o l l o w i n g two techniques. A t r o c a r (needle gage 20) was used to puncture the VA at an angle through the s o f t t i s s u e at the side of the tongue at the l e v e l of the second v e n t r a l g i l l arch. Upon removing the t r o c a r needle, a s t r e t c h e d piece of PE 50 tubing was i n s e r t e d i n t o the VA through the t r o c a r c a t h e t e r (gage 18). The t r o c a r 9 catheter was then c a r e f u l l y removed and the cannula was secured to the side of the tongue with two s t i t c h e s . A l t e r n a t i v e l y , the VA was cannulated with a sharpened g u i t a r wire i n s i d e a s t r e t c h e d piece of PE50 tubing. The VA was punctured from the d o r s a l surface of the tongue at the l e v e l of the f i r s t g i l l arch. As before, the VA was secured i n place with two s t i t c h e s and was l e d out of the buccal c a v i t y v i a a h e a t - f l a r e d sleeve (PE160). The o v e r a l l success r a t e of these two techniques was approximately equal: the surgery was more d i f f i c u l t i n the t r o c a r technique, while there was a lower r a t e of s u r v i v a l f o r the g u i t a r wire technique. Trout opercular cannulae In order to measure pH of exp i r e d water j u s t l e a v i n g the opercular c a v i t y , a polyethylene cannula (PE 90) was s t i t c h e d i n p o s i t i o n under and midway along the opercular opening. The presence of the cannula j u s t i n s i d e the opercular c a v i t y d i d not appear to prevent proper c l o s u r e of the opercular valve and to our knowledge there was no i n t e r f e r e n c e with the normal pumping of water over the g i l l s . I t has been suggested that the opercular c a t h e t e r i z a t i o n method i s a poor method f o r sampling mean ex p i r e d water i n t r o u t (Davis & Watters, 1970; Davis & Cameron, 1971). I t was important, t h e r e f o r e , to determine i f water pH v a r i e d with the p o s i t i o n of the opercular cannulae. S i x f i s h were s u r g i c a l l y f i t t e d with three opercular cannula which were p o s i t i o n e d i n d i f f e r e n t l o c a t i o n s along the opercular valve 10 Figure 1. Trout were s u r g i c a l l y f i t t e d with 3 cannulae placed j u s t under the opercular valve (dotted l i n e ) . The d i f f e r e n c e between i n s p i r e d water pH and expired water pH at e q u i l i b r i u m (equilibrium= water pH 8 min a f t e r sample was taken from the opercular chamber, see chapter 2), (ApH), were measured on s i x f i s h . Numbers represent means +. standard e r r o r s . 11 ( F i g . 1). The d i f f e r e n c e between i n s p i r e d water pH and expired water pH at e q u i l i b r i u m (equilibrium= water pH 8 min a f t e r c o l l e c t i o n ) were measured from i n d i v i d u a l cannulae. The unpaired t - t e s t f o r two means was employed to compare the three p o s s i b l e p a i r s of mean values, and pH d i d not vary with the p o s i t i o n of the cannula. In Chapter 2 and 3, f i s h were f i t t e d with one opercular cannula ( r i g h t g i l l ) , while i n Chapter 4, r i g h t and l e f t o percular cannulae were used i n the blood-perfused t r o u t head p r e p a r a t i o n and i n i n t a c t f i s h . The r a t e of water flow over the g i l l s and the r a t e of gas exchange between g i l l t i s s u e s and the environment are approximately matched between the r i g h t and l e f t g i l l s i n t e l e o s t f i s h . Thus, there i s probably l i t t l e d i f f e r e n c e between water samples c o l l e c t e d from s i n g l e or double opercular cannulated f i s h . Trout rubber dam F i s h were f i t t e d with rubber dams prepared from l a t e x surgeon gloves using the technique of Cameron and Davis (1970). This technique i n v o l v e s c u t t i n g the thumb of the glove to form a snug mask, that i s sewn p o s t e r i o r to the mouth and a n t e r i o r to the g i l l s of the f i s h . F o l l o w i n g surgery, f i s h were i n s e r t e d i n t o a narrow black perspex box i n the p o s t e r i o r end of a two chambered box ( F i g . 2). The rubber dam was secured to a d i v i d i n g perspex O-ring so as to separate the f r o n t compartment ( i n s p i r e d water) from the back compartment (expired water), i n t h i s continuous 13 Figure 2. F i s h were placed i n the two-chambered p l e x i g l a s s box. A l a t e x dam was s t i t c h e d around the mouth of the f i s h to separate i n s p i r a t o r y water from mixed e x p i r a t o r y water. The opercular cannula was s t i t c h e d against the body of the f i s h and p o s i t i o n e d j u s t i n s i d e the opercular cavity.The s m a l l , g l a s s stopped-flow chamber contained a pH el e c t r o d e and magnetic s t i r r i n g bar. Expired water flowed through the opercular cannula, past the pH e l c t r o d e and out through the o u t l e t valve by g r a v i t a t i o n a l f o r c e s . Stopped-f low apparatus 15 flow-through apparatus. F i s h were l e f t to recover i n the experimental apparatus f o r 48 hrs f o l l o w i n g surgery. Sole caudal a r t e r y c a n n u l a t i o n Sole were f i t t e d with caudal a r t e r y cannulae (PE50) a f t e r a n a e s t h e t i z a t i o n i n MS222 (1:15,000), as described by Matters and Smith (1973). B r i e f l y , a 2-3 cm i n c i s i o n was made j u s t above the caudal l a t e r a l l i n e . The a r t e r y l y i n g beneath the vertebrae was p r i c k e d with a 26 gage needle and a s t r e t c h e d piece of PE50 tubing (-30 cm) was fed i n t o the a r t e r y i n the c e p h a l i c d i r e c t i o n . A small amount of o x y t e t r a c y c l i n e HC1, a general f i s h a n t i b i o t i c , was placed i n each wound before the wound was c l o s e d with s u r g i c a l s i l k . A f t e r surgery, the sole were placed i n small p l a s t i c chambers (approx. 8 L) covered with sand, and allowed to recover i n f l o w i n g seawater f o r 72 hours p r i o r to experimentation. To r e - e s t a b l i s h r e s t i n g c o n d i t i o n s , f l a t f i s h r e q u i r e long recovery periods and a v a i l a b l e sand i n which to bury themselves (Hood §_t ajL.., 1979). A n a l y t i c a l techniques Gas chromatography Plasma and water samples were analysed f o r t o t a l carbon d i o x i d e content (Crj02^ with a C a r l e gas chromatograph (Model I I I ) c o n t a i n i n g a CO2 d i s c r i m i n a t i n g column (porapak Q) coupled to a data a q u i s i t i o n u n i t ( O l i v e t t i PC). The p r i n c i p l e of the 16 technique i s given by Lenfant and Aucutt (1966) and B o u t i l i e r e_£. a l . (1985). Plasma samples were prepared by i n j e c t i n g 50 u l of plasma (or standard) i n t o a gas t i g h t Hamilton shyringe c o n t a i n i n g 7 ml of N 2 and 1ml of ^ - e q u i l i b r a t e d 0 . 1 N H C 1 . A l t e r n a t i v e l y , 1 ml water samples (or standard) were mixed with 7 ml of U2 and 50 u l of 0 . 1 N H C 1. The samples were a u t o m a t i c a l l y shaken f o r 3 min i n order to l i b e r a t e the CO2 i n t o the gas phase and ensure e q u i l i b r a t i o n of the gas and l i q u i d phase. The gas phase (6 ml) was then introduced i n t o a d r y i n g f i l t e r and flushed through a 1ml sample loop, ensuring complete washout of the previous sample. The area under each peak was i n t e g r a t e d and compared to standard peak areas. Plasma CO2 tensions ( P c 0 2 * a n d H C O 3 - concentrations were c a l c u l a t e d by manipulating the Henderson-Hasselbalch equation as f o l l o w s : plasma P C 0 2 = plasma O n ? (4) ( t C C 0 2 ) ' t l + a n t i l o g (pH - pK)] (5) plasma H C O 3 - = C C 0 2 - ( * C 0 2 * P C 0 2 > where pK i s the apparent pK of plasma and<<co2 * s t n e s o l u b i l i t y of CO2 i n plasma taken from B o u t i l i e r aj.. (1984). S t a t i s t i c s Data are presented as means +. 1 S.E.M. (N). The Student's p a i r e d and unpaired t - t e s t was employed, where a p p r o p r i a t e , to compare the d i f f e r e n c e between mean values (P< 0.05). 18 CHAPTER 1 The d i s t r i b u t i o n of ammonia and H+ ions between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments i n Salmo g a i r d n e r i and Parophrvs v e t u l u s 19 INTRODUCTION Ammonia i s the endproduct of protein, amino acid, and adenylate catabolism i n f i s h . Ammonia formed i n the tissues i s either r e - u t i l i z e d jLn. s i t u or enters the blood where i t remains u n t i l excreted across the g i l l s . There i s a substantial body of knowledge on the mechanisms of ammonia excretion across the g i l l s of various f i s h (eg. Maetz & Garcia Romeu, 1964; Maetz, 1973, Evans, 1977; Payan, 1978; Cameron & Heisler, 1983; Wright & Wood, 1985). However, l i t t l e i s known about ammonia stores in body tissues of f i s h . Ammonia i s a weak base that exists i n solut i o n as ammonia ( N H 3 ) and ammonium ions ( N H 4 + ) . With a pK of 9.7 at 10°C, the majority of ammonia in body f l u i d s (pH= 6.5-8.0) w i l l be i n the ionized form, as NH4+. Movement of ammonia between tissue compartments i s thought to be primarily dependent on NH3 p a r t i a l pressure gradients (P N H3*' because b i o l o g i c a l membranes are highly permeable to NH3 (Castell & Moore, 1971; Klocke f_t a i . . , 1972; Boron, 1980; Lockwood gJt aL., 1980; see also Good & Knepper, 1985). Despite NH3*s greater permeability, there i s evidence that NH4+ electrochemical gradients also play a r o l e i n ammonia transfer across c e l l membranes (Thomas, 1974; Boron & DeWeer, 1976; Aickin & Thomas, 1977). Transfer of ammonium ions w i l l depend on the d i f f u s i v e permeability of the membrane for N H 4 + , the a v a i l a b i l i t y of ion c a r r i e r s i n the membrane, and the i r a f f i n i t y for NH4+ ions. A s i g n i f i c a n t d i f f u s i v e permeability to NH4+ i s now recognized or hypothesized i n many b i o l o g i c a l membranes (Schwartz & Tripolone, 1983; Arruda §_£, aJL., 1984; Evans & Cameron, 1986). Ammonium ions can substitute for K+ i n the Na+/K+/Cl- cotransporter (Kinne §_t a l • . 1986), the Na+/K+ ATPase pump (eg. Post & J o l l y , 1957; Skou, 1960; Robinson, 1970; Aickin & Thomas, 1977; Sorensen, 1981; Kurtz & Balaban, 1986), and for H+ ions i n the Na+/H+ ion exchange mechanism (eg. K i n s e l l a & Aronson, 1981, Aronson, 1983). As well, NH4+ can replace K+ i n nerve K+ channels (Binstock & Lecar, 1969; H i l l e , 1973). Thus, i t appears that transfer of ammonia between tissue compartments may involve both NH3 and NH4+ movements. In a closed system at equilibrium, the d i s t r i b u t i o n of ammonia across b i o l o g i c a l membranes w i l l be determined by the pH gradient across the membrane, as long as the e f f e c t i v e permeability to NH3 i s much greater than that to N H 4 + , and there i s no chemical binding of the species. However, the greater the e f f e c t i v e NH4+ permeability, the greater w i l l be the influence of the e l e c t r i c a l gradient on the equilibrium d i s t r i b u t i o n . Living animals are c e r t a i n l y not closed systems at equilibrium. Nevertheless, the assumption has often been made that the d i s t r i b u t i o n of ammonia between i n t r a c e l l u l a r and e x t r a c e l l u l a r tissue compartments i s largely a function of the i n t r a c e l l u l a r to e x t r a c e l l u l a r pH gradient, in both mammals (eg. Visek, 1968; P i t t s , 1973; Meyer §_£. a l - , 1980; Mutch & Bannister, 1983; Remesy ejt a l . , 1986) and f i s h (Randall & Wright, 1987; Dobson & Hochachka, 1987). The purpose of t h i s study was to determine whether ammonia was d i s t r i b u t e d between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments according to transmembrane pH g r a d i e n t s . The f i r s t set of experiments were performed on blood of rainbow t r o u t (Salmo gairdner i , i n v i v o and in. v i t r o ) . to i n v e s t i g a t e whether the d i s t r i b u t i o n of ammonia followed the pH gradient between red c e l l s and plasma at r e s t , to e s t a b l i s h the s i t u a t i o n under c o n d i t i o n s as c l o s e to steady s t a t e as p o s s i b l e , and during a perturbed s i t u a t i o n , e x t r a c e l l u l a r a c i d o s i s . Red c e l l s are unusual compared with many other t i s s u e s i n that H+ i o n d i s t r i b u t i o n i s passive and f o l l o w s the membrane p o t e n t i a l over a range of pH values (Heming et. a l . . , 1986; Nikinmaa §_£. a l . . , 1987; Lassen, 1977). Thus, i f the ammonia d i s t r i b u t i o n does f o l l o w the red c e l l - t o - p l a s m a pH g r a d i e n t , t h i s w i l l then i n d i c a t e that the d i s t r i b u t i o n i s dependent on transmembrane H+ i o n gra d i e n t s or e l e c t r i c a l g r a d i e n t s , or both. To d i s t i n g u i s h between the two i t i s necessary to look at t i s s u e s other than red c e l l s , where H+ ions are not p a s s i v e l y d i s t r i b u t e d (see Roos & Boron, 1981). I f ammonia movements across c e l l membranes are s o l e l y dependent on I>NH3 g r a d i e n t s , that i s , NH4+ movements are n e g l i b l e , then one would expect the d i s t r i b u t i o n of ammonia between i n t r a - and e x t r a c e l l u l a r compartments to f o l l o w transmembrane pH gr a d i e n t s . On the other hand, i f NH4+ i s permeant, then one would expect the ammonia d i s t r i b u t i o n to f o l l o w the e l e c t r o c h e m i c a l NH4+ gradient between i n t r a - and e x t r a c e l l u l a r compartments. A second set of experiments was performed on lemon so l e (Parophrvs v e t u l u s ) to i n v e s t i g a t e the d i s t r i b u t i o n of ammonia and H+ ions across white muscle, heart, and b r a i n , as w e l l as red blood c e l l s . Measurements were made at r e s t , and under two treatments designed to perturb the normal acid-base s t a t u s by very d i f f e r e n t mechanisms- r e s p i r a t o r y a c i d o s i s (hypercapnia) and metabolic a c i d o s i s (exhaustive e x e r c i s e ) . Lemon sol e were chosen f o r these experiments as f l a t f i s h have a d i s c r e t e white muscle mass uncontaminated by pink or red f i b r e s , i n c o n t r a s t to salmonids (Mosse, 1979), and because t h e i r blood pH i s somewhat lower (approx. 0.3 u n i t s ) than i n t r o u t . I n s t r u c t i v e comparisons could t h e r e f o r e be made with the f i r s t set of experiments on ammonia d i s t r i b u t i o n i n t r o u t blood, as w e l l as with a previous t h e o r e t i c a l a n a l y s i s of ammonia d i s t r i b u t i o n i n t r o u t muscle (Randall & Wright, 1987). 23 MATERIALS & METHODS Experimental animals Rainbow t r o u t (Salmo a a i r d n e r i ) weighing approximately 300g were obtained from Sun V a l l e y Trout Hatchery ( M i s s i o n , B.C.) and transported to the U n i v e r s i t y of B r i t i s h Columbia. F i s h were held i n outdoor, f i b e r g l a s s tanks s u p p l i e d with decholorinated Vancouver tapwater (pH approx. 7, [Na+]= 40 u e q u i v . l - * , [Cl-]= _ i ° 20 u e q u i v . l A, hardness= 12 ppm CaC03, temperature= 7-9 C), and fed a d i e t of commercial t r o u t p e l l e t s . Lemon sol e (Paraphrvs v e t u l u s ) weighing 292 +. 14 grams were c o l l e c t e d by two f i f t e e n minute o t t e r t r a w l s i n Barclay Sound, B r i t i s h Columbia (August). F i s h were held at the Bamfield Marine S t a t i o n i n a sandy-bottomed, outdoor, f i b e r g l a s s tank s u p p l i e d with flow through seawater (pH approx. 7.8, s a l i n i t y approx. 31 o/oo, temperature= 11° C) f o r at l e a s t 7 days before experimentation. Trout were starved a minimum of 72 hours p r i o r to experimentation and s o l e were not fed during c a p t i v i t y to e l i m i n a t e v a r i a t i o n s i n body ammonia l e v e l s due to feeding h i s t o r y (Fromm, 1963; B r e t t & Z a l a , 1975). The experiments i n t h i s chapter can be d i v i d e d i n t o two s e c t i o n s . S e c t i o n A d e s c r i b e s the ammonia d i s t r i b u t i o n across rainbow t r o u t red c e l l s , and s e c t i o n B covers the d i s t r i b u t i o n of ammonia between muscle, heart, b r a i n , red c e l l s , and plasma of lemon s o l e . SECTION A: Trout red c e l l s 24 Experimental P r o t o c o l In the i n v i t r o experiment blood was c o l l e c t e d from s e v e r a l cannulated donor f i s h (see General M a t e r i a l s and Methods), pooled, and then d i v i d e d between tonometers (4 ml/tonometer). Each tonometer r e c e i v e d e i t h e r a 0.2% CO2 ( c o n t r o l ) or a 1.0% CO2 (hypercapnia) h u m i d i f i e d gas mixture i n a i r and was shaken fo r 90 min i n a 9° C water bath before the measurements were taken. In one experiment, blood was e q u i l i b r a t e d with 1.0% CO2 and the Na+/K+ ATPase i n h i b i t o r , ouabain (10~ 4 M) f o r 90 min before the measurements were taken. Blood was analysed f o r whole blood pH (pHe), red c e l l pH ( p H i ) , whole blood and plasma ammonia concentrations (Tamm), plasma and red c e l l water content, and haematocrit ( h c t ) . In the i n v i v o t r o u t experiment, cannulated f i s h were placed i n i n d i v i d u a l , low volume (2 1) flow-through chambers to recover f o r 48 h. In the 30 min p r i o r to sampling, i n f l o w water was turned o f f and f i s h chambers were aerated with e i t h e r 100% a i r (water pH= 7.0, c o n t r o l ) or switched to 1% CO2 i n a i r (water pH= 5.6, hypercapnia). A 2 ml blood sample was withdrawn from each f i s h at the end of 30 min and analysed f o r pHe, pHi, whole blood and plasma Tamm, plasma and red c e l l water content, and hct. A n a l y t i c techniques and c a l c u l a t i o n s A r t e r i a l pHe was measured immediately upon c o l l e c t i o n using a 25 Radiometer microelectrode (type E5021) and acid-base analyzer (PHM 72), maintained at 9° C. Red c e l l pHi was d i r e c t l y measured by the freeze-thaw technique ( Z e i d l e r & Kim, 1977) where a 400 u l whole blood sample was c e n t r i f u g e d i n a b u l l e t tube and then immediately frozen i n e i t h e r l i q u i d N2 or dry i c e chips i n ethanol. The frozen packed red c e l l s were then cut away from the plasma, thawed, and the pH of the red c e l l s l u r r y was measured using the Radiometer pH microelectrode assembly. Haematocrit was determined a f t e r c e n t r i f u g a t i o n (5000 G). Plasma and red c e l l water content were c a l c u l a t e d by dr y i n g to constant weight i n an oven (100° C). Whole blood and plasma samples (250 u l ) were assayed f o r Tamm a f t e r d e p r o t e i n i z a t i o n i n 500 uL of iced 8% H C I O 3 . Samples were then c e n t r i f u g e d and the supernatent was n e u t r a l i z e d with 2 M K H C O 3 . Tamm of t h i s n e u t r a l i z e d supernatent was measured by the glutamate dehydrogenase enzymatic assay (Kun & Kearney, 1971). I n t e r n a l ammonia standards were r o u t i n e l y made i n whole blood because i t was found t h a t d i s t i l l e d water standards d i d not agree with the i n t e r n a l standards. Whole blood i n t e r n a l standards were found to give the same r e s u l t s as plasma i n t e r n a l standards, and t h e r e f o r e , whole blood was used as the reference medium. Plasma Tamm was co r r e c t e d f o r water content, and then red c e l l ammonia l e v e l s were c a l c u l a t e d with the f o l l o w i n g formula: Red c e l l Tamm=whole blood Tamm - [(l - h c t / 1 0 0 ) x plasma Tamml (6) hct/100 Red c e l l Tamm c a l c u l a t e d above was then c o r r e c t e d f o r red c e l l water content, and the f i n a l c o n c e n t r a t i o n (^measured') was expressed as u m o l . l - * c e l l water. Plasma NH3 c o n c e n t r a t i o n ( [ N H 3 ] ) was c a l c u l a t e d by the f o l l o w i n g manipulation of the Henderson-Hasselbalch equation, using pK values given by Cameron and H e i s l e r (1983): Plasma [ N H 3 ] = plasma Tamm • ( a n t i l o a (pHe-pK)) ( 7 ) 1 + ( a n t i l o g (pHe-pK)) I n t r a c e l l u l a r [ N H 3 ] could a l s o be c a l c u l a t e d from red c e l l Tamm and pHi by a s i m i l a r equation: I n t r a c e l l u l a r [ N H 3 ] ^ i n t r a c e l l u l a r Tamm • ( a n t i l o g (pHi-pK)) 1 + ( a n t i l o g (pHi-pK)) (8) Ammonium ion c o n c e n t r a t i o n ( [ N H 4 + ] ) i n e i t h e r compartment could be c a l c u l a t e d as: [NH4+] = Tamm - [ N H 3 ] (9) Henry's law was a p p l i e d to c a l c u l a t e the p a r t i a l pressure of NH3 i n plasma and fed c e l l s (PuH3 i n uTorr: 1 Torr = 133.32 Pa), using the appropriate s o l u b i l i t y c o e f f i c i e n t K N H3' Cameron and H e i s l e r , 1983). 27 P N H 3 = (10) NH3 I f ammonia i s d i s t r i b u t e d between plasma and red c e l l s e n t i r e l y according to the pH g r a d i e n t , then plasma [ N H 3 ] w i l l equal red c e l l [ N H 3 ] and red c e l l pHi can be p r e d i c t e d from measured plasma and red c e l l Tamm using the Henderson-Hasselbalch equation: (11) p r e d i c t e d i n t r a c e l l u l a r pHi = pK + l o g i n t r a c e l l u l a r [ N H 3 I i n t r a c e l l u l a r [NH4+] P r e d i c t e d red c e l l Tamm could a l s o be c a l c u l a t e d by a f u r t h e r manipulation of the Henderson-Hasselbalch equation, employing the measured i n t r a c e l l u l a r pH and again assuming that plasma [ N H 3 ] was equal to red c e l l [ N H 3 ] . (12) p r e d i c t e d red c e l l Tamm = [ N H 3 ] + [ N H 3 ] a n t i l o g (measured pHi-pK) This ^ p r e d i c t e d ' red c e l l Tamm was then compared to ^measured* red c e l l Tamm determined from the d i r e c t measurement of plasma and whole blood ammonia (equation 6). Data are expressed as mean + 1 S.E.M. (N), where N equals the number of animals sampled (in. v i v o ) or the number of tonometers c o n t a i n i n g blood ( i a v i t r o ) . 28 The Student's t w o - t a i l e d p a i r e d and unpaired t - t e s t was employed to evaluate the s i g n i f i c a n c e of d i f f e r e n c e s between mean values, where appropriate (P<0.05). SECTION B: Sole red c e l l s , muscle, v e n t r i c l e , and b r a i n t i s s u e Experimental P r o t o c o l Lemon so l e with caudal a r t e r y cannulae (see General M a t e r i a l s and Methods) were placed i n i n d i v i d u a l , flow-through, sandy-bottomed chambers (~ 8 1). Three experimental c o n d i t i o n s were s t u d i e d : ( i ) A c o n t r o l i n which f i s h were subjected to u n a l t e r e d , aerated seawater (pH= 7.8) p r i o r to sampling. i i ) A second group of f i s h were placed i n a seawater bath e q u i l i b r a t e d to approximately 1% CO2 (pH= 6.7) f o r 30 minutes before sampling i n order to r a p i d l y a l t e r e x t r a c e l l u l a r and i n t r a c e l l u l a r pH v i a high Pco2 l e v e l s ( r e s p i r a t o r y a c i d o s i s ) . ( i i i ) The t h i r d group of f i s h were t r a n s f e r r e d to a lar g e p l a s t i c tank and chased f o r 20 minutes. The aim i n s u b j e c t i n g f i s h to exhaustive e x e r c i s e was to induce a metabolic a c i d o s i s i n both e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments. These f i s h were allowed to recover f o r 30 min p r i o r to sampling. In order to determine i n t r a c e l l u l a r pH (pHi) by the DMO (5,5 dimethyl-2,4-oxazolidinedione) d i s t r i b u t i o n technique (Waddell & B u t l e r , 1959), f i s h were i n j e c t e d with 1 ml.kg -* of 5 u C i . m l - 1 [ 1 4 C ] DMO (New England Nuclear, s p e c i f i c a c t i v i t y 50 mCi.mmol-*) and 20 u C i . m l - ^ of the e x t r a c e l l u l a r marker, [ 3H] mannitol (New England Nuclear, s p e c i f i c a c t i v i t y 27.4 mCi.mmol-!) i n Cort l a n d s a l i n e (adjusted to 160 mmol.l -! NaCl) approximately 12 h p r i o r to sampling ( M i l l i g a n & Hood, 1986a,b). At sampling, 2 ml of blood were withdrawn from each f i s h and replaced with an equal volume of s a l i n e . Blood was immediately analysed f o r pHe, pHi, plasma and whole blood t o t a l carbon d i o x i d e content (CQQ2) and hct. Samples were a p p r o p r i a t e l y f i x e d and stored f o r l a t e r determination of whole blood and plasma Tamm, whole blood l a c t a t e ((La-]) and hemoglobin ([Hb]) concentrations and plasma and red c e l l water content. In l e s s than 5 minutes a f t e r blood withdrawal, f i s h were removed from the water and the s p i n a l cord q u i c k l y severed with a s c a l p e l . E p a x i a l white muscle samples were ex c i s e d and a t h i n s l i c e of t i s s u e immediately frozen with freeze-clamp tongs, cooled by immersion i n t o dry i c e chi p s . The frozen samples were st o r e d on dry i c e f o r l a t e r determination of i n t r a c e l l u l a r Tamm and [ L a - ] , while the unfrozen t i s s u e samples were l a t e r analysed f o r [*4C] DMO and [^H] mannitol. The heart v e n t r i c l e was then q u i c k l y removed. Part of the v e n t r i c l e was freeze-clamped for subsequent a n a l y s i s of i n t r a c e l l u l a r Tamm, while the remaining t i s s u e was 30 analysed f o r t ] DM0, [^H] mannitol, and water content. B r a i n t i s s u e was then c o l l e c t e d a f t e r c u t t i n g through the cranium and, as before, part of the t i s s u e was immediately frozen and l a t e r assayed for i n t r a c e l l u l a r Tamm, while the r e s t of the t i s s u e was reserved f o r [ 1 4 c ] DM0, [ 3H] mannitol, and water content determination. The e n t i r e t i s s u e e x c i s i o n and freeze-clamping procedure took approximately 1-2 minutes. At the end of t h i s procedure, l a r g e samples of white muscle t i s s u e were taken for water content a n a l y s i s . A n a l y t i c a l Techniques and C a l c u l a t i o n s A r t e r i a l pHe was measured immediately upon c o l l e c t i o n using a Radiometer microelectrode (type E5021) maintained at 11° C and l i n k e d to a Radiometer PHM71 acid-base analyzer. Plasma and whole blood C Q O2 were measured on 50 u l samples by the technique of Cameron (1971), using a Radiometer E50 36 CO2 e l e c t r o d e and the same acid-base analyser. Plasma Prj02 a n d H C O 3 - were c a l c u l a t e d by standard manipulations of the Henderson-Hasselbalsch equation (see General M a t e r i a l s & Methods). Red c e l l pHi was d i r e c t l y measured by the freeze-thaw technique of Z e i d l e r & Kim (1977) using the same microle c t r o d e assembly. The freeze-thaw method and not the DM0 method was used to measure red c e l l pHi because the former technique i s a more d i r e c t method f o r pHi determination of i s o l a t e d c e l l s , i t i s a simpler method, and there are no s i g n i f i c a n t d i f f e r e n c e s between red c e l l pHi values from the two techniques i n f i s h blood ( M i l l i g a n & Wood, 1985). Plasma and red c e l l water content were c a l c u l a t e d by dry i n g to constant weight i n an oven at 100° C. Lactate l e v e l s ([La-] ) were assayed e n z y m a t i c a l l y ( L - l a c t i c dehydrogenase/ NADH method, Sigma reagents) a f t e r d e p r o t e i n i z i n g 100 u l whole blood i n 200 u l 8 % H C I O 3 , or homogenizing 100 mg white muscle i n 1 ml 8 % H C I O 3 with a g l a s s homogenizer ( c f . Turner e_t aj^., 1983). Muscle i n t r a c e l l u l a r [La-] were expressed as mmol.l -! of ICF as o u t l i n e d f o r muscle i n t r a c e l l u l a r Tamm below (equation 9). hct was determined by c e n t r i f u g a t i o n (5000 G f o r 5 min) and Hb by the cyanmethaemoglobin method of B l a x h a l l and O a i s l e y (1973) using Sigma reagents. Whole blood and plasma samples (250 u l ) were assayed f o r Tamm as described above for t r o u t blood. Red c e l l Tamm, plasma [ N H 3 ] , red c e l l [ N H 3 ] , PtfH3' p r e d i c t e d red c e l l pHi, and red c e l l Tamm were c a l c u l a t e d using equations 6-12 (see above). I n t r a c e l l u l a r muscle, heart, and b r a i n Tamm were determined i n the f o l l o w i n g manner. Frozen t i s s u e samples were weighed, d e p r o t e i n i z e d i n ic e d 8% H C I O 3 (1:20 d i l u t i o n ) , and then homogenized (Tekmar Tissumizer with microprobe head). Samples were c e n t r i f u g e d , and the supernatant was e x t r a c t e d and n e u t r a l i z e d with saturated T r i s b u f f e r . The enzymatic assay technique was i d e n t i c a l to that described above fo r blood, except that muscle t i s s u e e x t r a c t provided the reference medium for i n t e r n a l standards. (There were no d i f f e r e n c e s between i n t e r n a l standards made up i n muscle, heart, and b r a i n t i s s u e e x t r a c t s . ) Muscle, heart, and b r a i n l e v e l s of [ 3HJ and t ^ C ] r a d i o a c t i v i t y were measured by d i g e s t i n g 50-150 mg of t i s s u e i n 2 ml NCS t i s s u e s o l u b i l i z e r (Amersham) f o r 1-2 days u n t i l the s o l u t i o n was c l e a r . E x t r a c e l l u l a r f l u i d [ 3H] mannitol and [14c] DMO a c t i v i t y were determined i n the same manner, except 100 uL of plasma were added to 2 ml of t i s s u e s o l u b i l i z e r . The t i s s u e d i g e s t s were n e u t r a l i z e d with 60 u l g l a c i a l a c e t i c a c i d and 10 ml of f l u o r (OCS; Amersham) was added. To decrease chemiluminescence, a l l samples were kept i n the dark overnight before being counted on a l i q u i d s c i n t i l l a t i o n counter. D u a l - l a b e l quench c o r r e c t i o n was performed using quench standards prepared from each type of t i s s u e and the e x t e r n a l standard r a t i o method (Kobayashi & Maudsley, 1974). Tissue e x t r a c e l l u l a r f l u i d volume (ECFV, ml.g - 1) was c a l c u l a t e d according to the equation: (13) ECFV=Tissue [ 3 H 1 mannitol (d.p.m.q - 1)  Plasma [ 3 H 3 mannitol (d.p.m.g - 1)/plasma H 2 0 ( m l . g - 1 ) Tot a l t i s s u e water was determined as p r e v i o u s l y described f o r plasma and whole blood samples. I n t r a c e l l u l a r f l u i d volume (ICFV, ml.g - 1) was c a l c u l a t e d as the d i f f e r e n c e between t o t a l t i s s u e water and ECFV. I n t r a c e l l u l a r Tamm was determined by f i r s t accounting f o r ammonia trapped i n the e x t r a c e l l u l a r f l u i d and then c o r r e c t i n g f o r i n t r a c e l l u l a r water, as f o l l o w s : 33 I n t r a c e l l u l a r Tamm = Tissue Tamm - (plasma Tamm • ECFV) (14) ICFV Tissue pHi was c a l c u l a t e d according to the equation: (15) pHi = P K H M O + l o g ([DMO]* • (10(pHe-pKDMO) + p - p [ D M 0 ] e where pKrjMo w a s taken from Malan §_t §i. (1976) and [DMO]e and [DMOli represent e x t r a c e l l u l a r and i n t r a c e l l u l a r [DMO], r e s p e c t i v e l y . These two values were c a l c u l a t e d as: (16) [DMO]e (d.p.m.ml ) = plasma [1*C] DMO (d.p.m.ml-1) plasma H2O (ml.g -*) and (17) [ D M 0 ] i = t i s s u e [14C]DM0 (d.p.m.ml ) - (ECFV . [DMO]*,) (d.p.m.ml - 1) ICFV Tissue [ N H 3 ] , [ N H 4 + ] , and P u H 3 l e v e l s were c a l c u l a t e d as d escribed p r e v i o u s l y using equations ( 8 ) , ( 9 ) , and (10), r e s p e c t i v e l y . To determine whether the d i s t r i b u t i o n of ammonia between e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments i n various t i s s u e s was according to the pH gr a d i e n t , t i s s u e pHi's were p r e d i c t e d 34 from the measured ammonia d i s t r i b u t i o n by equation (11), assuming i n t r a c e l l u l a r [ N H 3 ] = plasma [ N H 3]. These p r e d i c t e d pHi's were compared to the measured pHi's determined from the DMO d i s t r i b u t i o n s . A p r e d i c t e d i n t r a c e l l u l a r Tamm could a l s o be c a l c u l a t e d by equation (12), again assuming i n t r a c e l l u l a r [ N H 3 ] = plasma [ N H 3 ] . To determine whether the d i s t r i b u t i o n of ammonia between e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments i n various t i s s u e s could a l t e r n a t i v e l y be a f u n c t i o n of the membrane p o t e n t i a l , E N H 4 + w a s c a l c u l a t e d from the Nernst equation: E N H 4 + = E l In [ N H 4 + ] - (18) ZF [NH 4+]i where R,T,Z, and F have t h e i r usual va l u e s , and [ N H 4 + ] e and [ N H 4+]i represent plasma and i n t r a c e l l u l a r l e v e l s based on measured e x t r a c e l l u l a r and i n t r a c e l l u l a r pH and Tamm values (equation 8,9,14, and 15). Data are presented as means + 1 S.E.M.(N). The Student's t w o - t a i l e d unpaired t - t e s t was used to determine the s i g n i f i c a n c e of d i f f e r e n c e s between mean values (P<0.05). 35 RESULTS A. Rainbow t r o u t S l i g h t q u a n t i t a t i v e d i f f e r e n c e s between rainbow t r o u t i n  v i t r o and in, vivo data are shown i n Table 1, but the o v e r a l l r e s u l t s and conclusions are the same whether blood was held i n tonometers (in. v i t r o ) or i n l i v e animals (in. vivo) p r i o r to a n a l y s i s . Red c e l l ammonia l e v e l s are c o n s i s t e n t l y higher than plasma l e v e l s , r e s u l t i n g i n ammonia c o n c e n t r a t i o n r a t i o s of between 0.29 and 0.40 (plasma-to-red c e l l , Table 1). Red c e l l pHi p r e d i c t e d from the plasma-to-red c e l l ammonia d i s t r i b u t i o n was not s i g n i f i c a n t l y d i f f e r e n t from measured pHi i n the c o n t r o l experiment, but during hypercapnia, p r e d i c t e d pHi was s i g n i f i c a n t l y l e s s than measured pHi (Table 1). NH3 was i n e q u i l i b r i u m between plasma and red c e l l at r e s t , but not during hypercapnia ( F i g . 3 ) . The Pj jH3 gradient from red c e l l - t o - p l a s m a during hypercapnia was 17 uTorr, in. v i t r o , and 20 uTorr, in. v i v o ( F i g . 3 ) . Our c a l c u l a t i o n s of P N H 3 l e v e l s assume an e q u i l i b r i u m between NH3 and NH4 + i n the red c e l l as w e l l as the plasma, because the NH3 NH4+ r e a c t i o n i s considered instantaneous (<50 msec, Stumm fie Morgan, 1981). I f there i s an NH3 gradient from red c e l l to plasma and i f H+ ions are p a s s i v e l y d i s t r i b u t e d across red c e l l membranes as commonly be l i e v e d (Lassen, 1977; Heming e_t al.., 1986; Nikinmaa et a l . , 1987), then there must a l s o be an e l e c t r o c h e m i c a l g r a d i e n t f o r NH4+. As red c e l l NH3 l e v e l s are e l e v a t e d , NH4+ TABLE 1. Measured pHe, red c e l l pHi, plasma and red c e l l Tamm. and the r a t i o of plasmarred c e l l Tamm, i a v i tro and in vivo., under c o n t r o l and hypercapnic conditions. Predicted red c e l l pHi c a l c u l a t e d from the r a t i o of NHu:NH-.+ in the red c e l l using equation 11 i s compared to measured pHi (freeze-thaw method). Predicted red c e l l Tamm (equation 12) i s compared to measured red c e l l Tamm. ENM-.+ and E M+ were c a l c u l a t e d from equation 18. Means + S.E.H. TREATMENT HEft?VREP pHe pHi PLASMA (fr e e z e - Tamm thaw (umoll -*) method) RED CELL AMMONIA Tamm CONCENTRATION (umoll- 1) RATIO pHi PREPICTEP CftLCVLnTEP RED CELL Tamm (umoll-') (mV) E« + (mV) CONTROL l a v i t r o (N=6) 8.03 + .05 7.48 + .02 304 ± 8 1048 ± 81 . 29 7.46 + .05 1018 + 124 -30.0 + 1.7 -31. 2 + 2.8 in vivo (N=7) 8.02 + .03 7.50 + .01 311 ±19 782 +55 .40 7.51 + .04 943 + 86 -24.9 + 2.2 -29. 3 + 1.6 HYPERCAPNIA l a v i t r o (N=7) 7.63 + .02 7.25 + .01 318 ± 9 969 + 52 , 33 7.11 + .04 704 + 36 -27. 8 + 1.4 -21.3 + 1.1 In vivo (N=7) 7.55 + .02 * » 7. 28 + .01 323 t 3 872 + 30 37 7.09 + .01 560 ±34 -24. 2 + 0.9 -15.3 + 1.6 » s i g n i f i c a n t l y d l f f r e n t from in. v i tro c o n t r o l , paired t - t e s t •* s i g n i f i c a n t l y d i f f e r e n t from in. vivo c o n t r o l , unpaired t - t e s t * s i g n i f i c a n t l y d i f f e r e n t from measured pHi, unpaired t - t e s t t s i g n i f i c a n t l y d i f f e r e n t from "measured" red c e l l Tamm, unpaired t - t e s t , (P < 0.05) 37 F i g u r e 3. P l a s m a PNHS ( s t i p l e d b a r s ) i s compared t o r e d c e l l PNHS (open b a r s ) i n c o n t r o l and h y p e r c a p n i c t r e a t m e n t s o f b o t h i n v i t r o and i n . v i v o e x p e r i m e n t s . • d e n o t e s s i g n i f i c a n t d i f f e r e n c e f rom p l a s m a PNHS. A d e n o t e s s i g n i f i c a n t d i f f e r e n c e f rom c o n t r o l Ptsms. means + 1 S . E . M . 130 in vitro in vivo in vitro in vivo CONTROL HYPERCAPNIA CO TABLE 2. Measured pHe, red c e l l pHi, plasma and red c e l l Tamm, and the r a t i o of plasma:red cell.Tamm, in. v i t r o , under hypercapnic c o n d i t i o n s . Values for c o n t r o l (no ouabain) and ouabain (10~* N) are shown. See Table 1 for d e t a i l s . MEASURED TREATMENT pHe pHi PLASMA ( f r e e z e - Tamm thaw (umoll- 1) method) RED CELL AMMONIA Tamm CONCENTRATION (umoll- 1) RATIO pHi PREDICTED RED CELL Tamm (u m o l l - * CALCULATED E N H 4 + (mV) E M + (mV) c o n t r o l (N=7) 7.64 + .03 7. 25 + .03 346 + 17 1016 + 61 34 7.13 + .03 777 +45 -26. 3 + 1.1 -22.1 •1-4 ouabain (N=7) 7.64 + .01 7. 24 + .01 354 + 16 1000 + 82 35 7.16 + .03 808 • 40 -25.1 11.7 -22.6 •0.4 * s i g n i f i c a n t l y d i f f e r e n t from measured pHi, unpaired t - t e s t f s i g n i f i c a n t l y d i f f e r e n t from "measured" red c e l l Tamm, unpaired t - t e s t , (P < 0.05) ID ions w i l l a l s o be elevated as NH3 molecules combine with H+ to form N H 4 + . In hypercapnia, t h e r e f o r e , there i s a net d i f f u s i o n g r adient f o r both NH3 and NH4+ out of the red c e l l . This i s i l l u s t r a t e d by c a l c u l a t i o n s of E J JH4+ a n d &H+ from the Nernst equation (equation 18) i n Table 1. EflH4 + and EH+ agreed under c o n t r o l c o n d i t i o n s , however, EH+ becomes l e s s negative i n the expected manner (eg. Lassen, 1977) during hypercapnia, while EjjH4 + d i d not change. Incubation of the blood with the Na +/K + ATPase i n h i b i t o r , ouabain, d i d not a l t e r the ammonia d i s t r i b u t i o n d uring hypercapnia (Table 2). Lemon Sole Acid-Base and F l u i d Volume changes The e x t r a c e l l u l a r acid-base s t a t u s of the three experimental groups i s given i n Table 3A. The lower a r t e r i a l pHe a s s o c i a t e d with hypercapnia was due to a r e s p i r a t o r y a c i d o s i s . Elevated plasma Pco2 w a s a s s o c i a t e d with a s i g n i f i c a n t l y greater plasma [ H C O 3 - ] r e l a t i v e to c o n t r o l f i s h . This elevated [ H C O 3 - ] was equal to the product of the d i f f e r e n c e i n pH and the b u f f e r value (B, taken from in. v i t r o f l a t h e a d s o l e data, Turner a l . , 1983b), which i n d i c a t e s t h a t the higher plasma [ H C O 3 - ] l e v e l s were probably due to the elevated Pco2 alone and not to other f a c t o r s . The lower pHe i n e x e r c i s e d f i s h was the r e s u l t of a 41 TABLE 3. Acid-base s t a t u s i n s o l e d u r i n g c o n t r o l , hypercapnia and e x e r c i s e regimes. Means +_ S.E.M. A TREATMENT pHe PLASMA P C Q 2 t o r r PLASMA [HCO»-I mM/L WHOLE BLOOD [La-] mM/L MUSCLE [La-] mM/L CONTROL (N=10) 7.73 + .01 1.94 + .08 4.64 + .19 0 . 11 + .0 3 (N=6) 3. 22 + .09 (N=6) HYPERCAP. (N=9) 7. 32 + .04 7.44 + .74 5.92 + . 14 0 .19 + . 05 (N=7) 6.00 + 2. 16 (N=7) EXERCISE (N=9) 7. 48 + .04 1.99 + .35 2.40 + .23 0. 98 + . 16 (N=10) 19. 57 + 1.55 (N=10) TREATMENT RED CELL pHi WHITE MUSCLE pHi HEART pHi BRAIN pHi CONTROL (N=10) 7. 25 + .02 7. 29 + .03 7. 32 + .06 7. +. 75 03 HYPERCAP. (N=9) 7.01 + .02 6.95 + .03 7. + . 10 03 44 02 EXERCISE (N=9) 7. 22 + .0 2 7.12 + .03 7. 27 + .07 7.71 + .05 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l v a lue, t - t e s t p < 0.05 metabolic a c i d o s i s , because plasma ( H C O 3 - ] was s i g n i f i c a n t l y lower, but PQ02 remained unchanged from c o n t r o l values. There was a s i g n i f i c a n t e l e v a t i o n of blood [La-] i n the e x e r c i s e group over r e s t i n g l e v e l s (+ 0.9 mmol.l -*), but the absolute d i f f e r e n c e was small compared to the d i f f e r e n c e s i n muscle [La-] l e v e l s (+ 16.3 mmol.l - 1. Table 3A). The e l e v a t i o n of blood [La-] (+0.9 mmol.l -!) w a s a i s o small r e l a t i v e to the metabolic a c i d load (H+ m = 3.0 mmol.l -!) which can be c a l c u l a t e d from the d i f f e r e n c e s i n pHe and [ H C O 3 - ] ( c f . Turner et a l . . , 1983a). Of the various t i s s u e s examined, pHi was by f a r the highest i n b r a i n , with a value approximately equal to pHe (Table 3B). Red c e l l s , white muscle, and heart v e n t r i c l e a l l maintained a pHe-pHi gradient of 0.3-0.4 pH u n i t s . Hypercapnia r e s u l t e d i n lower pHi i n a l l t i s s u e s r e l a t i v e to c o n t r o l f i s h , with greater decreases i n b r a i n and white muscle (0.3 u n i t s ) than i n red c e l l s or heart (0.2 u n i t s ) . The a c i d i f y i n g e f f e c t of exhaustive e x e r c i s e was much more s e l e c t i v e , with pHi s i g n i f i c a n t l y lower only i n the working muscle. The acid-base s t a t u s of the other t i s s u e s was unaffected. The changes i n haematological v a r i a b l e s a f t e r hypercapnia were very s i m i l a r to those a f t e r e x e r c i s e : [Hb] remained unchanged, while hct was s i g n i f i c a n t l y g r e a t e r , r e l a t i v e to c o n t r o l values (Table 4). Mean c e l l u l a r haemoglobin c o n c e n t r a t i o n (MCHC= [Hb]/hct) was s i g n i f i c a n t l y l e s s i n the hypercapnia and e x e r c i s e groups, which suggests that the red 43 TABLE 4. Blood haemoglobin, haematocrit, mean c e l l u l a r haemoglobin c o n c e n t r a t i o n (MCHC) and red c e l l and plasma water content i n three groups of lemon s o l e ; c o n t r o l f i s h , hypercapnic f i s h , and f i s h swum to exhaustion. Means +. S.E.M. TREATMENT Hb (g/100 mL blood) Hct (%) MCHC (g/mL c e l l s ) Red c e l l water (%) Plasma water (%) CONTROL (N=10) 3.0 + 0.2 11.0 + 1.0 0. 28 + 0.01 76. 3 ± 1.4 97. 4 + 0.3 HYPERCAPNIA 3.7 15.9 0.24 73.6 96.6 (N=9) +0.2 + 1.1 + 0.01 + 0.5 + 0.0 EXERCISE (N=9) 3.7 + 0.6 17. 2 + 2.2 0. 24 + 0.00 73.9 + 1.4 95. 9 + 0.2 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , unpaired t - t e s t , P<0.05 44 c e l l s swelled. However, t h i s was not confirmed by the red c e l l water content measurements, which d i d not d i f f e r s i g n i f i c a n t l y amongst groups (Table 4). Plasma % water was s i g n i f i c a n t l y l e s s i n both e x e r c i s e d and hypercapnic f i s h . The higher hct values i n the hypercapnic and e x e r c i s e d f i s h r e l a t i v e to c o n t r o l f i s h could be explained by e i t h e r inherent d i f f e r e n c e s between the groups or the e f f e c t s of the experimental treatments. ECFV was much greater i n the heart v e n t r i c l e t i s s u e than i n b r a i n or white muscle, while ICFV demonstrated the opposite trend (Table 5). Hypercapnia had no e f f e c t on ECFV AND ICFV. Foll o w i n g exhaustive e x e r c i s e , however, ECFV was s i g n i f i c a n t l y lower and ICFV, higher i n white muscle, suggesting a net water f l u x i n t o the c e l l s , probably i n response to the osmotic e f f e c t of the increased i n t r a c e l l u l a r [La-] (Table 3A). T o t a l water contents (ECFV + ICFV) d i d not vary s i g n i f i c a n t l y amongst the three t i s s u e s , nor as a r e s u l t of hypercapnia and e x e r c i s e . Ammonia d i s t r i b u t i o n The d i s t r i b u t i o n of ammonia between plasma and t i s s u e s under c o n t r o l , hypercapnia, and e x e r c i s e c o n d i t i o n s i s presented for red c e l l s i n Table 6, white muscle i n Table 7, heart v e n t r i c l e i n Table 8, and b r a i n i n Table 9. In hypercapnic f i s h , plasma ammonia l e v e l s were s i g n i f i c a n t l y lower than i n the c o n t r o l group by about 25%, while t i s s u e s showed g e n e r a l l y s m a l l e r , n o n - s i g n i f i c a n t r e d u c t i o n s . In e x e r c i s e d f i s h , i n t r a c e l l u l a r TABLE 5. F l u i d volume d i s t r i b u t i o n (g rLsO/g t i s s u e ) i n v a r i o u s t i s s u e s of lemon s o l e d uring c o n t r o l , hypercapnia, and e x e r c i s e regimes. T o t a l H 20 r e f e r s to the sum of ECFV and IVFV. Means * S.E.N. TREATMENTS ECFV M U S C L E ICFV TOTAL H 20 ECFV HEflRT. ICFV TOTAL H a0 ECFV ICFV TOTAL H 30 CONTROL 0.0997 0.7174 0.8171 (N=10) +.0113 +.0129 +.0029 0.2594 +.0243 0.5529 +.0241 0.8123 +.0042 0.1206 +.0149 0.6901 +.0171 0.8107 +.0045 HYPERCAPNIA 0.0875 0.7253 0.8129 (N=9) +.0192 +.0197 +.0012 0.2765 +.0103 0.5348 +.0141 0.8110 +.0041 0.1077 +.0051 0.6832 +.0118 0.7909 +.0090 EXERCISE (N=9) 0.0599 +.0090 0.7552 +.0097 0.8152 +.0027 0.2837 +.0278 0.5237 +.0261 0.8074 +.0043 0.1235 +.0234 0.6716 +.0277 0.7951 +.0080 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , unpaired t - t e s t P < 0.05. TABLE 6. Measured pHe, red c e l l pHi, plasma and red c e l l Tamm, and the r a t i o of plasma:red c e l l Tamm at r e s t , during hyper-capnia, and following exercise i n lemon sole. Predicted red c e l l pHi c a l c u l a t e d from the r a t i o of NH3:NH.»+ i n the red c e l l using equation 11 i s compared to measured pHi (freeze-thaw method). Predicted red c e l l Tamm (equation 12) i s compared to measured red c e l l Tamm. E N M « + and E M - were c a l c u l a t e d from equation 18. Means ± S.E.H. HEflSVFEP PREDICTED CALCULATED TREATMENT pHe pHi PLASMA RED CELL AMMONIA pHi RED CELL E N H ^ I E« + ( f r e e z e - Tamm Tamm CONCENTRATION Tamm (mV) (mV) thaw uM/L uM/L RATIO (umol/L) method) CONTROL (N=10) 7.73 1-01 7.25 + .02 192 + 13 657 +93 29 7. 23 + .07 588 +52 -29.1 £3.8 -27.9 ±1-1 HYPERCAPNIA 7.32 7.01 142 (N=9) +.04 +.02 +11 573 +96 25 6.79 + .08 302 + 24 -31.1 15.1 -17.6 11.8 EXERCISE (N=9) 7.48 + .04 7. 22 1.02 228 131 1029 1213 22 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , P<0.05 ~ s i g n i f i c a n t l y d i f f e r e n t from measured pHi, P<0.05 ** s i g n i f i c a n t l y d i f f e r e n t from "measured" red c e l l Tamm, P<0.05. 6.89 + .05 453 +90 -38.0 + 3.6 -15.7 11.3 TABLE 7. Measured pHe, muscle pHi, plasma and muscle Tamm, and the r a t i o of plasma:muscle Tamm at r e s t , during hypercapnia, and f o l l o w i n g e x e r c i s e i n lemon s o l e . P r e d i c t e d muscle pHi c a l c u l a t e d from the r a t i o of NH a:NH«+ i n muscle us i n g equation 11 i s compared to measured pHi (DMO method). P r e d i c t e d muscle Tamm (equation 12) i s compared to measured muscle Tamm. EIMM*+ was c a l c u l a t e d from equation 18. HEASJiBEB P R E D I C T E D C A L C U L A T E D TREATMENT pHe pHi PLASMA MUSCLE AMMONIA pHi MUSCLE E»»+Z (DHO) Tamm Tamm CONCENTRATION Tamm (mV) uM/L uM/L RATIO (uM/L) CONTROL (N=9) 7.73 + .01 7. 29 + .03 192 + 13 6005 +577 .03 6.24 ±..05 550 +35 -83 + 3 HYPERCAPNIA 7.32 6.95 142 (N=9) +.04 +.0 3 +11 4623 +516 03 5.83 ±..06 343 + 26 -83 +- 3 EXERCISE (N=9) 7.48 + .04 7.12 + .03 228 + 31 9344 + 1256 02 5.84 + . 12 560 + 112 -88 + 4 *, ** see TABLE 4 TABLE 8. Measured pHe, heart pHi, plasma and heart Tamm, and the r a t i o of plasma:heart Tamm at r e s t , during hypercapnia, and f o l l o w i n g e x e r c i s e i n lemon s o l e . P r e d i c t e d heart pHi c a l c u l a t e d from the r a t i o of NH3:NrU+ i n heart u s i n g equation 11 i s compared to measured pHi (DMO method). P r e d i c t e d heart Tamm (equation 12) i s compared to measured heart Tamm. ENM<*+ was c a l c u l a t e d from equation 18. HEftSVREP PREDTPTRn * ' ' CALCULATEn' TREATMENT pHe pHi PLASMA HEART AMMONIA pHi HEART E (DMO) Tamm Tamm CONCENTRATION T a m m " (uM/L) (uM/L) RATIO u M / L (mV) CONTROL (N=9) 7.73 + .01 7. 32 + .06 192 + 13 7891 +598 0.02 6.11 + .05 543 +78 -90 + 3 HYPERCAPNIA 7.32 7.10 142 (N=9) +.04 +.03 +11 6941 +998 0.02 5.67 + .08 254 + 33 -94 + 4 EXERCISE (N=9) 7. 48 + .04 7. 27 + .07 228 + 31 12,355 + 1071 0.02 5.70 + . 10 403 £81 -96 £ 2 *, *, ** see TABLE 4 TABLE 9. Measured pHe, b r a i n pHi, plasma and b r a i n Tamm, and the r a t i o of plasma:brain Tamm at r e s t , during hypercapnia, and f o l l o w i n g e x e r c i s e i n lemon s o l e . P r e d i c t e d b r a i n pHi c a l c u l a t e d from the r a t i o of NH 3:NH-»+ i n b r a i n using equation 11 i s compared to measured pHi (DMO method). P r e d i c t e d b r a i n Tamm (equation 12) i s compared to measured b r a i n Tamm. E N M « + was c a l c u l a t e d from equation 18. HEftSVREP PREDICTED CALCULATED TREATMENT pHe pHi PLASMA BRAIN AMMONIA p H i BRAIN E N H < (DMO) Tamm Tamm CONCENTRATION Tamm (mV) uM/L uM/L RATIO (uM/L) CONTROL (N=9) 7.73 + .01 7.75 + .03 192 + 13 3419 +434 0.06 6.48 + .06 195 + 21 -69 + 3 HYPERCAPNIA 7.32 (N=9) + .04 7.44 + .02 142 + 11 2787 +948 0.05 6.19 1-11 115 + 11 -65 +6 EXERCISE (N=9) 7.48 + .04 7.71 + .05 228 + 31 4970 +864 0.05 6. 12 + . 12 144 +2 •72 +4 *, A , ** see TABLE 4 ammonia l e v e l s were elevated by about 55% over c o n t r o l l e v e l s , an e f f e c t which was s i g n i f i c a n t i n a l l t i s s u e s except b r a i n . Plasma ammonia l e v e l s were not s i g n i f i c a n t l y a l t e r e d . As i n t r o u t red c e l l s (Table 1), ammonia was d i s t r i b u t e d between red c e l l s and plasma i n s o l e according to pH g r a d i e n t s , at r e s t , but not during an e x t r a c e l l u l a r a c i d o s i s (hypercapnia and e x e r c i s e . Table 6). Absolute Tamm l e v e l s i n the red c e l l and plasma were lower i n s o l e compared to t r o u t by about 16% and 40%, r e s p e c t i v e l y . Despite these d i f f e r e n c e s , p r e d i c t e d red c e l l pHi and Tamm were not s i g n i f i c a n t l y d i f f e r e n t from measured values i n the c o n t r o l experiment, but were s i g n i f i c a n t l y l e s s during an e x t r a c e l l u l a r a c i d o s i s (hypercapnia and e x e r c i s e . Table 6 ) , as i n t r o u t . As w e l l , NH3 was i n e q u i l i b r i u m between plasma and red c e l l s at r e s t , but not during e i t h e r hypercapnia and e x e r c i s e ( F i g . 4 ) . The P N H 3 g r a d i e n t from red c e l l - t o - p l a s m a during hypercapnia was 10 uTorr and f o l l o w i n g e x e r c i s e about 30 uTorr. &NH4+ a n d EH+ agreed under c o n t r o l c o n d i t i o n s , however, was l e s s negative during both a c i d o t i c treatments, while E J J H 4 + was more negative. Ammonia d i s t r i b u t i o n r a t i o s between muscle and plasma ( c o n t r o l r a t i o = 0.03, Table 7 ) , heart and plasma ( c o n t r o l r a t i o = 0.02, Table 8 ) , and b r a i n and plasma ( c o n t r o l r a t i o = 0.06, Table 9) were c o n s i d e r a b l y lower than those between red c e l l s and plasma ( r a t i o s > 0.20, Table 6). Ammonia was c l e a r l y not d i s t r i b u t e d across t i s s u e compartments according to the H+ d i s t r i b u t i o n . In a l l t i s s u e s and treatments, i n t r a c e l l u l a r pHi 51 Figure 4. Plasma PNHS ( s t i p l e d bars) i s compared to red c e l l PNHS (open bars) i n cont r o l f i s h (C), f i s h exposed to hyper-capnia (H), and exercised f i s h (E). • denotes s i g n i f i c a n t d i f f e r e n c e from pi 3SIB3 PNH3 • A denotes s i g n i f i c a n t d i f f e r e n c e from c o n t r o l P • means + 1 S.E.M. 52 1 :¥>:::¥::::::x::::::::::::::: ,,.,.w.,.,.,.,.,.,.,.,.'.|.,.|.,.,.:.i x-:-:-:*-:*:-^^ m$m mm® UJ O J L J I I L o 00 I o 53 F i g u r e 5.. White muscle, v e n t r i c u l a r h e a r t muscle, and b r a i n P N H S l e v e l s (open b a r s ) are compared to plasma PNHS l e v e l s ( s t i p l e d b a r s ) i n c o n t r o l f i s h ( C ) , f i s h exposed to h y p e r -c a p n i a ( H ) , and e x e r c i s e d f i s h ( E ) . • denotes s i g n i f i c a n t d i f f e r e n c e from plasma PNHS. A denotes s i g n i f i c a n t d i f f e r e n c e from c o n t r o l P MMa. means +1 S.E.M. 1400 pTorr Muscle I • • 1 c h e Heart B r a i n I I I h e c a l c u l a t e d from the plasma-to-tissue ammonia d i s t r i b u t i o n was s i g n i f i c a n t l y l e s s (by 1.0-2.0 pH u n i t s ) than pHi c a l c u l a t e d from the [ 1 4 C ] DMO d i s t r i b u t i o n (Table 7, 8, 9). As w e l l , p r e d i c t e d t i s s u e Tamm was s i g n i f i c a n t l y l e s s than measured t i s s u e Tamm i n each treatment (Table 7, 8, 9). The t i s s u e i n t r a c e l l u l a r - t o - e x t r a c e l l u l a r PtfH3 g r a d i e n t s ( F i g . 5) were an order of magnitude greater than red c e l l - t o - p l a s m a gradients ( F i g . 4). As s t a t e d above for red c e l l s , our c a l c u l a t i o n s of PjjH3 l e v e l s assume an e q u i l i b r i u m between NH3 and NH4+ i n each t i s s u e compartment. The d i f f e r e n c e between red c e l l s and other i n t r a c e l l u l a r compartments, however, i s that H+ io n d i s t r i b u t i o n i s not passive (Roos & Boron, 1981). Hence, NH3 g r a d i e n t s can e x i s t i n the absence of NH4+ e l e c t r o c h e m i c a l g r a d i e n t s , i n a s i t u a t i o n where NH4 + i s p a s s i v e l y d i s t r i b u t e d across the membrane, but H+ ions are not (see D i s c u s s i o n ) . C o n t r o l P N H 3 g r a d i e n t s were smaller between muscle and plasma (410 u T o r r ) , than heart (630 uTorr) or b r a i n (640 uTorr) and plasma. In a l l t i s s u e s , P N H 3 w a s s i g n i f i c a n t l y reduced during hypercapnia, l a r g e l y because of i n t r a c e l l u l a r a c i d o s i s , although i n t r a c e l l u l a r Tamm d i d decrease s l i g h t l y i n a l l t i s s u e s . There were no s i g n i f i c a n t changes i n PflH3 gradients a f t e r e x e r c i s e ( F i g . 5 ) , although Tamm grad i e n t s from t i s s u e to plasma were g r e a t l y increased i n a l l t i s s u e s (Table 7, 8, 9). The e q u i l i b r i u m p o t e n t i a l s f o r NH4+ ( ENH4+) across muscle, heart, and b r a i n c e l l membranes were c a l c u l a t e d from the Nernst equation and are presented i n Tables 7, 8, and 9, 56 r e s p e c t i v e l y . These values were 2-3 f o l d greater than those i n c a l c u l a t e d red blood c e l l s ( c f . Table 6). There were no s i g n i f i c a n t d i f f e r e n c e s i n EJJH4 + between treatments. 57 DISCUSSION E v a l u a t i o n of methods The accuracy of red c e l l Tamm c a l c u l a t i o n s depends l a r g e l y on hct, i n c r e a s i n g with i n c r e a s i n g hct (equation 6). In the t r o u t experiments hct values were greater than 23%, and there f o r e the c a l c u l a t i o n e r r o r was n e g l i g i b l e . However, i n the c o n t r o l s o l e experiment, the e r r o r i n red c e l l Tamm may be as great as 7% i f hct i s o f f by 1%, at the mean hct value of 11%. A 7% e r r o r i n 'measured' red c e l l Tamm would not a l t e r the conclusions that ammonia i s d i s t r i b u t e d according to the H+ i o n d i s t r i b u t i o n across red c e l l membranes at r e s t . The s i g n i f i c a n c e of t h i s problem diminishes i n the s o l e hypercapnia (hct= 16%) and ex e r c i s e (hct= 17%) treatments, where the e r r o r i n red c e l l Tamm i s at most 4% for a 1% d i g r e s s i o n i n hct. The p o t e n t i a l f o r e r r o r i n 'measured' red c e l l Tamm c a l c u l a t i o n s i n the hypercapnia and ex e r c i s e experiments i s small r e l a t i v e to the larg e discrepancy between 'measured' and 'p r e d i c t e d ' red c e l l Tamm. The p o t e n t i a l for e r r o r i n 'measured' i n t r a c e l l u l a r Tamm for muscle, heart, and b r a i n t i s s u e (equation 18) i s even s m a l l e r ; f o r ins t a n c e , f o r a 1% d i g r e s s i o n i n ICFV, the e r r o r i n t i s s u e Tamm i s l e s s than 2%. Thus, ICFV i s not a c r i t i c a l v a r i a b l e i n the c a l c u l a t i o n of i n t r a c e l l u l a r Tamm. The accuracy and l i m i t a t i o n s of the 1 4C-DMO/ 3H-mannitol technique f o r i n t r a c e l l u l a r pH measurements i n f i s h have been assessed i n d e t a i l p r e v i o u s l y ( M i l l i g a n & Wood, 1985, 1986a,b). 58 and w i l l not be discussed i n the present paper. Acid-base changes i n the s o l e : comparisons to other s t u d i e s Resting blood acid-base parameters i n lemon so l e were very s i m i l a r to those measured by McDonald e_£. §JL.. (1982) on the same specie s . Hypercapnia r e s u l t e d i n a plasma a c i d o s i s that was t y p i c a l of other s a l t w a t e r species (Conger conger, Toews e_t a l . , 1983; S c v l i o r h i n u s s t e l l a r i s , H e i s l e r g i aj,.., 1976) when compared over the same p e r i o d of time. The metabolic a c i d o s i s i n c u r r e d a f t e r exhaustive e x e r c i s e i n lemon so l e was t y p i c a l of other b e n t h i c , sluggish-type f i s h swum to exhaustion, with very low blood [La-] d e s p i t e high muscle [La-] and a considerable blood metabolic a c i d load (Hood & Perry, 1985). In other f i s h , however, a r e s p i r a t o r y component (Pc02 e l e v a t i o n ) o f t e n p e r s i s t s f o r 1-2 h f o l l o w i n g e x e r c i s e , which was not seen here. Hence, i f lemon s o l e d i d s u f f e r a r e s p i r a t o r y a c i d o s i s i n a d d i t i o n to the metabolic a c i d o s i s f o l l o w i n g e x e r c i s e , f u l l recovery must have occurred w i t h i n 30 min. Hhite muscle, b r a i n , and red c e l l pHi i n r e s t i n g lemon s o l e were i d e n t i c a l to those reported f o r another f l a t f i s h , the s t a r r y flounder ( P l a t i c h t h v s s t e l l a t u s ) , using the same DMO and freeze-thaw techniques (Hood & M i l l i g a n , 1987), while heart pHi was approximately 0.2 pH u n i t s lower. Exhaustive e x e r c i s e i n lemon so l e r e s u l t e d i n a l e s s marked i n t r a c e l l u l a r a c i d o s i s i n white muscle, and no a c i d o s i s i n heart or b r a i n t i s s u e , i n c o n t r a s t to the s t a r r y flounder. This d i f f e r e n c e undoubtedly r e f l e c t s the lack of a r e s p i r a t o r y component i n sol e r e l a t i v e to the l a r g e Pco2 e l e v a t i o n i n flounder at a comparable time a f t e r e x e r c i s e . Thus, i n the s o l e , the i n t r a c e l l u l a r a c i d o s i s was l i m i t e d to the working muscle i t s e l f and was completely metabolic i n nature, as s i g n a l l e d by the la r g e [La-] e l e v a t i o n i n white muscle. I am aware of no comparable s t u d i e s on the i n t r a c e l l u l a r responses to hypercapnia i n f i s h over t h i s time p e r i o d . Red C e l l Ammonia D i s t r i b u t i o n : t r o u t and s o l e The r e s u l t s show that ammonia i s d i s t r i b u t e d according to the pH gra d i e n t across red c e l l s under r e s t i n g c o n d i t i o n s i n t r o u t and s o l e , which agrees with a study on human red c e l l s (Bone e_£. a l . . 1976). I f NH4+ t r a n s f e r across red c e l l membranes i s n e g l i b l e , and ammonia d i s t r i b u t i o n i s s o l e l y dependent on P N H 3 g r a d i e n t s , then one would expect the red c e l l - t o - p l a s m a ammonia d i s t r i b u t i o n to f o l l o w the H + d i s t r i b u t i o n , as f o r other weak ac i d s and bases with impermeant i o n forms. Red c e l l s , however, are unusual i n that H + ions are p a s s i v e l y d i s t r i b u t e d across the membrane according to the membrane p o t e n t i a l (Lassen, 1977; Heming al.., 1986; Nikinmaa g_£. al.-» 1987), u n l i k e other t i s s u e s (see Roos & Boron, 1981). Therefore, e x a c t l y the same, d i s t r i b u t i o n would occur i f the membrane was permeable only to N H 4 + , and the ammonia d i s t r i b u t i o n was set e n t i r e l y by the membrane p o t e n t i a l . He conclude that ammonia i s p a s s i v e l y d i s t r i b u t e d between red c e l l s and plasma under c o n t r o l c o n d i t i o n s , however, we cannot determine i f ammonia f l u x across 60 red c e l l membranes depends upon P N H 3 g r a d i e n t s s o l e l y , or i f NH4+ e l e c t r o c h e m i c a l g r a d i e n t s are a l s o important. Ammonia i s not d i s t r i b u t e d across red c e l l membranes according to the pH gradient during an e x t r a c e l l u l a r a c i d o s i s (hypercapnia i n t r o u t (in. v i t r o and i n v i v o ) . hypercapnia and ex e r c i s e i n s o l e ) , which agrees with data on avian red c e l l s i n  v i t r o over the pH range 7-8 (Bone aJL.., 1976). Ammonia accumulation i n the red c e l l , could be maintained by the a c t i v e uptake of NH4+ i n the face of NH3 d i f f u s i o n out of the red c e l l down the P N H 3 gradient and NH4+ e l c t r o c h e m i c a l g r a d i e n t s . The a b i l i t y of NH4+ to replace K+ i n Na+/K+ ATPase i s w e l l e s t a b l i s h e d i n many t i s s u e s , i n c l u d i n g red c e l l membranes (Post & J o l l y , 1957; Sorensen, 1981). We test e d the p o s s i b i l i t y t hat NH4+ was r e p l a c i n g K+ i n the Na+/K+ ATPase by adding the s p e c i f i c Na+/K+ ATPase i n h i b i t o r , ouabain, to hypercapnic t r o u t blood, in. v i t r o (Table 2). The a d d i t i o n of ouabain d i d not a l t e r the d i s t r i b u t i o n of ammonia between red c e l l s and plasma and red ce l l - t o - p l a s m a P N H 3 g r a d i e n t s were not abol i s h e d . This i m p l i e s that even i f Na+/K+ ATPase plays a r o l e i n ammonia accumulation w i t h i n the red c e l l during hypercapnia, i t cannot be a major one. Furthermore, t r o u t red c e l l membrane Na+/H+ exchange mechanism i s known to be a c t i v e during an a c i d o s i s (Nikinmaa §_t a l . . 1987). I f NH4+ ions can replace H + i n exchange f o r Na+, as i n other c e l l s (eg. Maetz & Gar c i a Romeu, 1964; K i n s e l l a & Aronson, 1981; Wright & Wood, 1985), then red c e l l ammonia stores would be depleted during hypercapnia. Instead, we observed an 61 accumulation of ammonia during hypercapnia and t h e r e f o r e , NH4+ s u b s t i t u t i o n for H+ i n Na+/H+ exchange cannot be inv o l v e d . Ammonia gra d i e n t s during hypercapnia may develop between i n t r a - and e x t r a c e l l u l a r compartments because of high r a t e s of ammonia production. We te s t e d t h i s p o s s i b i l i t y i n t r o u t blood i n  v i t r o , by f o l l o w i n g whole blood Tamm l e v e l s over time during hypercapnic exposure and found that ammonia l e v e l s d i d not change. Thus, i n t r a c e l l u l a r ammoniogenesis i s not a f a c t o r i n the development of P N H 3 gradients during hypercapnia i n t r o u t , and because of the s i m i l a r i t y of the t r o u t and sol e data, i t i s probably not a f a c t o r i n s o l e , as w e l l . I t i s p o s s i b l e that changes i n pH and water content, which w i l l lead to changes i n ammonia d i s t r i b u t i o n , may have caused the development of ammonia gra d i e n t s between red c e l l and plasma during blood a c i d o s i s . In t r o u t , whole blood pH remained s t a b l e a f t e r 30 min. of hypercapnia jjn v i t r o . Red c e l l water content increased s i g n i f i c a n t l y between c o n t r o l (in. v i t r o , 65.1% +_ . 4 , i n  v i v o , 65.7% +, .3) and hypercapnia (in. v i t r o . 68 .4% +, .3, in. v i v o . 68.9% + .3) experiments. I t seems l i k e l y that water content of red c e l l s was s t a b l e f o l l o w i n g 90 min of exposure to hypercapnia i n v i t r o . Thus i t appears that nonsteady s t a t e s for pH and water content cannot account red c e l l - t o - p l a s m a PtfH3 gr a d i e n t s i n v i t r o during hypercapnia. I t a l s o seems to be an u n l i k e l y e x p l a n a t i o n of the t r o u t and sol e i n . v i v o r e s u l t s because of the s i m i l a r i t y of the jLn. v i t r o and in , v i v o data. Another p o s s i b i l e e x p l a n a t i o n f o r red c e l l - t o - p l a s m a ammonia 62 g r a d i e n t s under a c i d c o n d i t i o n s i s bi n d i n g of ammonia to p r o t e i n s (Barker, 1968) i n the red c e l l . I t seems u n l i k e l y , however, that d i f f e r e n t i a l b i nding would occur, that i s , b i n d i n g at low c e l l pHi values (hypercapnia and e x e r c i s e ) but not at c o n t r o l pH values. Furthermore there i s evidence that ammonia binds i n some mammalian i n t r a c e l l u l a r compartments (see below), but we are unaware of any re p o r t s of ammonia bin d i n g i n red c e l l s . F i n a l l y , Bone §_£. ai.. (1976) proposed that ammonia and DMO would d i s t r i b u t e d i f f e r e n t l y between the c y t o s o l and the a c i d i c nucleus, r e s u l t i n g i n a discrepancy between pHi c a l c u l a t e d from the DMO d i s t r i b u t i o n versus the ammonia d i s t r i b u t i o n . Roos and Boron (1981), however, reanalysed t h e i r data and found t h a t i n t r a c e l l u l a r compartmentalization could not f u l l y account f o r the discrepancy between the two c a l c u l a t e d pHi values. In c o n c l u s i o n , ammonia i s p a s s i v e l y d i s t r i b u t e d according to the plasma-to-red c e l l H+ d i s t r i b u t i o n i n blood at r e s t i n g pH values, but not during an e x t r a c e l l u l a r a c i d o s i s . Various p o s s i b i l i t e s f o r ammonia accumulation during blood a c i d o s i s have been discussed. I propose that some other a c t i v e NHq + uptake process must be operating to maintain red c e l l Tamm l e v e l s above those expected from pH c o n s i d e r a t i o n s . Muscle, Heart, and B r a i n Ammonia D i s t r i b u t i o n Ammonia was not d i s t r i b u t e d between plasma and muscle, heart, and b r a i n t i s s u e according to the H+ d i s t r i b u t i o n at r e s t , during hypercapnia, and f o l l o w i n g e x e r c i s e i n lemon s o l e . These f i n d i n g s are c o n s i s t e n t with s t u d i e s on mammalian hepatocytes where at p h y s i o l o g i c a l pH and ammonia co n c e n t r a t i o n s , i n t r a c e l l u l a r ammonia l e v e l s are much higher than p r e d i c t e d from the H+ d i s t r i b u t i o n (Sainsbury, 1980; Remesy §_£. a l . . , 1986). Ammonia a l s o accumulates i n mammalian b r a i n t i s s u e ( H i n d f e l t , 1975; Benjamin, 1982) and colon (Bown f_t a l - , 1975) i n concentrations above those expected from e x t r a c e l l u l a r ammonia and pH c o n s i d e r a t i o n s . These s t u d i e s and the present r e s u l t s i n d i c a t e t h a t ammonia d i s t r i b u t i o n across t i s s u e membranes cannot be described by the c l a s s i c d i f f u s i o n t rapping model (Visek, 1968; P i t t s , 1973), which p r e d i c t s i n t r a - to e x t r a c e l l u l a r ammonia d i s t r i b u t i o n according to pH g r a d i e n t s , assuming only NH3 i s t r a n s f e r r e d across c e l l membranes and NH4+ t r a n s f e r i s n e g l i b l e . I f we assume that NH4 + p e r m e a b i l i t y i s s i g n i f i c a n t , then the d i s t r i b u t i o n of ammonia at e q u i l i b r i u m w i l l be i n f l u e n c e d by the membrane p o t e n t i a l (Em) as w e l l as the pH g r a d i e n t . The greater the p e r m e a b i l i t y to NH4 + , the greater the i n f l u e n c e of Em. Boron and Roos (1976) and Roos and Boron (1981) derived a general equation d e s c r i b i n g the e q u i l i b r i u m d i s t r i b u t i o n of any weak base fo r which there i s s i g n i f i c a n t p e r m e a b i l i t y to the charged as w e l l as the uncharged form. Adopting t h i s equation for ammonia, and c o r r e c t i n g typographical e r r o r s i n both o r i g i n a l p u b l i c a t i o n s , t h i s equation becomes: 64 (19) [Tamm1„ = (TH-H- + k) x [Tammli {[H+]i + k> (DNH^/pNHn-H) - (F-Em-jT/R-Td-y) ) • (tH+lf/k) (pNH 3/pNH 4 + ) - (F-Em/R-T(l-tf)) • ([H+] e/k) where k i s the NH3/NH4+ d i s s o c i a t i o n constant, PNH3 i s the pe r m e a b i l i t y to N H 3 , PNH4+ i s the p e r m e a b i l i t y to NH4+ and: (20) t= exp • {(Em-F)/(R-T)} I f Em and the pHi-pHe gradient are known, then [Tamm]e/[Tamm]i can be c a l c u l a t e d as a f u n c t i o n of the P N H 3 / P N H 4 + r a t i o using these equations. Em i n f i s h white muscle, as i n most ver t e b r a t e muscle, i s uniformly i n the range of -80 to -85 mV (Hagiwara & Takahashi, 1967; Hidaka & Toida, 1969; Yamamoto, 1972). In F i g 6, Em= -83 mV has been employed along with the measured pHe and pHi values for r e s t i n g white muscle (Table 7) to c a l c u l a t e ITamm]e/[Tamm]1 as a f u n c t i o n of muscle c e l l membrane p e r m e a b i l i t y r a t i o ( P N H 3 / P N H 4 + ). The a n a l y s i s shows that at P N H 3 / P N H 4 + > 10,000, the d i s t r i b u t i o n a s y m p t o t i c a l l y approaches the t h e o r e t i c a l maximum of - 0.36, where i t i s e n t i r e l y a f u n c t i o n of the pH gr a d i e n t , while a t P N H 3 / P N H 4 + < 10, the d i s t r i b u t i o n a s y m p t o y i c a l l y 65 F i g u r e 6. The r e l a t i o n s h i p between the d i s t r i b u t i o n o f ammonia a t e q u i l i b r i u m between e x t r a c e l l u l a r and i n t r a c e l l u l a r com-partments o f r e s t i n g s o l e white muscle ([ Tamm].,/[ Tamm] i ) and the r e l a t i v e p e r m e a b i l i t y (pNH3/pNH.., +) o f the c e l l membrane to NH 3 and N1-U+. A membrane p o t e n t i a l (E,„) o f -83 mV and the measured pHi and pHe v a l u e s o f T a b l e 7 have been assumed. See t e x t f o r a d d i t i o n a l d e t a i l s . .4 I l I I l I 1 10 100 1000 10,000 100,000 P N H 3 / P N H 4 approaches the t h e o r e t i c a l minimum of ~ 0.03, where i t i s e n t i r e l y a f u n c t i o n of the membrane p o t e n t i a l (Em). In between these l i m i t s there i s an approximately a l o g / l i n e a r r e l a t i o n s h i p between P N H 3 / P N H 4 + and e q u i l i b r i u m d i s t r i b u t i o n r a t i o . The measured d i s t r i b u t i o n r a t i o was 0.03 (Table 7), s t r o n g l y suggesting that i t was e n t i r e l y a f u n c t i o n of Em, and therefore P N H 3 / P N H 4 + was low, i . e . , p e r m e a b i l i t y to NH4 + was r e l a t i v e l y high. I t i s probably safe to assume that P N H 3 / P N H 4 + was at l e a s t below 25, where the r a t i o would be < 0.06. Reversing the a n a l y s i s , and using the measured d i s t r i b u t i o n to p r e d i c t Em by the Nernst equation produced the answer E(jH4+= ~83 ± 3 mV (Table 7) i n agreement with l i t e r a t u r e values f o r f i s h white muscle Em. S i m i l a r analyses have been performed with measured and p r e d i c t e d ammonia d i s t r i b u t i o n s i n heart v e n t r i c l e and b r a i n , with the same c o n c l u s i o n s , i . e . , that P N H 3 / P N H 4 + i s low, and the d i s t r i b u t i o n i s almost e n t i r e l y a f u n c t i o n of Em. C a l c u l a t e d ENH4+ £ o r r e s t i n g heart v e n t r i c l e was -90 + 3 mV (Table 8) i n agreement with Jaeger's (1962) measured Em= -84 mV i n Leucisus  r u t i l u s v e n t r i c l e , while c a l c u l a t e d EjjH4+ f° r r e s t i n g b r a i n t i s s u e was -69 + 3 mV, i n c l o s e accord with the g e n e r a l l y accepted value of -70 mV f o r nervous t i s s u e ( H i l l e , 1984). The s i m i l a r i t y between c a l c u l a t e d ENH4+ values for muscle, heart, and b r a i n t i s s u e and published Em values imply that ammonia i s p a s s i v e l y d i s t r i b u t e d according to Em i n these t i s s u e s at r e s t , and that the p e r m e a b i l i t y of t i s s u e membranes to 68 NH4 + ions i s s i g n i f i c a n t . I t i s p o s s i b i l e that NH4+ p e r m e a b i l i t y i s not as great as i s suggested by t h i s a n a l y s i s and that i n t r a c e l l u l a r b i nding of ammonia accounts f o r the high i n t r a c e l l u l a r Tamm concentrations. Wanders §_£. §JL- (1980) presented evidence t h a t a la r g e f r a c t i o n of i n t r a c e l l u l a r ammonia was bound to mitochondrial p r o t e i n i n r a t heart t i s s u e , while smaller f r a c t i o n s of ammonia were as s o c i a t e d with p r o t e i n i n s k e l e t a l muscle and b r a i n . Thus, d e p r o t e i n i z a t i o n of t i s s u e s during the e x t r a c t i o n method would l i b e r a t e t h i s protein-bound ammonia, and would r e s u l t i n an over e s t i m a t i o n of free ammonia i n the c y t o s o l . Mean c e l l u l a r Tamm l e v e l s i n s o l e heart, muscle, and b r a i n have been c a l c u l a t e d assuming that 1) the values given by Wanders e£ al.. (1980) f o r bound ammonia i n mitochondria of r a t are v a l i d for f i s h , 2) that mitochondrial volume i s between 9 and 13% (Hoek §_£. al.. , 1980), 3) that the c y t o s o l c o n s t i t u t e s the major p o r t i o n of the remaining volume, and 4 ) that Tamm l e v e l s i n the c y t o s o l are determined by e x t r a - to i n t r a c e l l u l a r pH g r a d i e n t s . Tamm l e v e l s c a l c u l a t e d with these assumptions c o n s t i t u t e only 8-13% of our measured values, which f a l l s w i t h i n +. 1 S.E.M.. Hence, even i f some f r a c t i o n of c e l l u l a r ammonia binds to mitochondrial p r o t e i n s i n f i s h , i t cannot account for the high Tamm l e v e l s measured i n the present study. Resting l e v e l s of ammonia i n s o l e white muscle measured i n t h i s study, are between published values for cod, Gadus morrhua, (Fraser §_£. a l . . , 1966) and carp, Cvprinus c a r p i o , ( D r e i d z i c & Hochachka, 1976), but are about 4-8 times higher than r e s t i n g l e v e l s i n rainbow t r o u t (Dobson & Hochachka, 1987; Mommsen & Hochachka, unpublished data). In a recent review, Randall & Wright (1987) analysed the d i s t r i b u t i o n of ammonia between t r o u t white muscle and plasma, at r e s t and f o l l o w i n g exhaustive e x e r c i s e (data taken from Mommsen & Hochachka, unpub. and M i l l i g a n & Wood, 1986b). The a n a l y s i s was based on the p r i n c i p l e that ammonia was d i s t r i b u t e d across muscle c e l l s r e l a t i v e to the H+ d i s t r i b u t i o n , assuming that muscle c e l l s were e s s e n t i a l l y impermeable to NH4+. From t h e i r c a l c u l a t i o n s , ammonia was d i s t r i b u t e d according to the H+ d i s t r i b u t i o n i n t r o u t , at r e s t , but not f o l l o w i n g e x e r c i s e , where NH3 g r a d i e n t s e x i s t e d between muscle and blood. I t seems odd that the ammonia d i s t r i b u t i o n i n r e s t i n g t r o u t white muscle apparently f o l l o w s the H+ d i s t r i b u t i o n , while i n sol e i t does not. The problem with t h i s a n a l y s i s i s that the t r o u t muscle Tamm l e v e l s (Mommsen & Hochachka, unpub.) and pHi values ( M i l l i g a n & Wood, 1986b) were obtained from two separate s t u d i e s (see Randall & Wright, 1987). When these measurements are performed on a s i n g l e set of f i s h (C.M.Wood, personnal communication) the t r o u t muscle ammonia d i s t r i b u t i o n p a r a l l e l s the f i n d i n g s of the present study on sol e muscle t i s s u e . At r e s t and f o l l o w i n g e x e r c i s e i n t r o u t , Wood fi n d s that ammonia i s not d i s t r i b u t e d according to H+ ion gradi e n t s and c a l c u l a t e d E ( j H 4 + values ( r e s t -81 £ 9 (N=8), e x e r c i s e -86 + 4 (N=7)) are c l o s e to those c a l c u l a t e d f o r s o l e and s i m i l a r to published muscle Em values. F o l l o w i n g e x e r c i s e , muscle ammonia l e v e l s increased by - 55 % compared to c o n t r o l . An increase i n white muscle ammonia l e v e l s with e x e r c i s e i s c o n s i s t e n t with other s t u d i e s on f i s h (Suyama et. a l . . 1960; Fraser §_£ §JL.., 1966; D r i e d z i c , 1975, D r i e d z i c & Hochachka, 1976; Dobson & Hochachka, 1987) and i s due to deamination of the adenylate pool ( D r i e d z i c & Hochachka, 1976; Dobson & Hochachka, 1987). In mammalian muscle, i t i s recognized that ammonia production and g l y c o l y s i s may be f u n c t i o n a l l y coupled i n s e v e r a l ways. Ammonia may enhance g l y c o l y s i s by a c t i v a t i n g phosphofructokinase, H+ production by g l y c o l y s i s may i n t u r n enhance ammonia production by a c t i v a t i n g AMP deaminase, and ammonia production (as NH3) may be important i n b u f f e r i n g the H+ ions produced by g l y c o l y s i s (Lowenstein, 1972; Mutch & Ban n i s t e r , 1983; Dudley & Terjung, 1985). The q u a n t i t a t i v e importance of the l a t t e r has r e c e n t l y been discounted by the d e t a i l e d study of Katz §_t al.* (1986) on working human l e g muscle, because ammonia production was l e s s than 4% of l a c t a t e production. In the lemon s o l e , however, 30 min a f t e r exhaustive e x e r c i s e , ammonia accumulation i n white muscle (- 3.3 mmol.l - 1. Table 7) was 20% of l a c t a t e accumulation (~ 16.3 mmol.l - 1. Table 3). I d e n t i c a l s t o i c h i o m e t r y was reported by Dobson and Hochachka (1987) i n t r o u t white muscle a f t e r exhaustive e x e r c i s e . Thus i n ammoniotelic t e l e o s t f i s h , d e a m i n a t i o n / g l y c o l y s i s c o u p l i n g may be more important than i n mammals, e s p e c i a l l y i n l i m i t i n g i n t r a c e l l u l a r a c i d o s i s . Ammonia l e v e l s a l s o increased i n heart (~55%) and b r a i n (-45%) t i s s u e f o l l o w i n g strenuous e x e r c i s e , although i t i s u n l i k e l y that ammonia production can account f o r t h i s . Heart i s predominantly an aerobic t i s s u e , and t h e r e f o r e , u n l i k e white muscle, the deamination of adenylates during e x e r c i s e would be n e g l i g i b l e . In nervous t i s s u e , ammonia i s known to be extremely t o x i c (see Randall & Wright, 1987) and t h e r e f o r e , high ammonia production r a t e s are improbable. As w e l l , i t i s doubtful that increased body ammonia l e v e l s a f t e r e x e r c i s e are r e l a t e d to reduced ammonia e x c r e t i o n because i n other t e l e o s t s , ammonia e x c r e t i o n e i t h e r increases or remains unchanged a f t e r strenuous e x e r c i s e (Holeton ejt a l . . , 1983; H e i s l e r , 1984; M i l l i g a n & Wood, 1986a: M i l l i g a n & Wood, 1987). I propose that elevated ammonia l e v e l s i n heart and b r a i n t i s s u e a f t e r e x e r c i s e are simply due to the passive uptake of ammonia from the increased plasma ammonia pool. With hypercapnia there were no s i g n i f i c a n t changes i n muscle, heart, and b r a i n ammonia s t o r e s , although mean l e v e l s decreased i n a l l t i s s u e s , as w e l l as i n the plasma. A r e d u c t i o n i n t o t a l body ammonia s t o r e s may have been due to a decrease i n ammonia production and/or an increase i n ammonia e x c r e t i o n r a t e s . The decrease i n water pH (7.8 to 6.7) caused by hypercapnia would c e r t a i n l y favour increased d i f f u s i v e l o s s of NH3 across the g i l l s . Ammonia e x c r e t i o n r a t e s have been reported to increase with hypercapnia i n some t e l e o s t s ( t r o u t , Lloyd.& S w i f t , 1976; Perry e£. a l . . , 1987; carp, Claiborne & H e i s l e r , 1984), and decrease i n others (Conger conger, Toews g£ al.., 1983; Qpsanus 72 Figure 7. A model of ammonia (NHa and NrU+) movements between the e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments of a h y p o t h e t i c a l t i s s u e where NrU+ ions are p a s s i v e l y d i s t r i b u t e d across the membrane according to the membrane p o t e n t i a l . A. In the i n t r a c e l l u l a r compartment where there i s no ammonia production, the d i r e c t i o n of the NhU+^NHs r e a c t i o n w i l l always be towards the formation of NHs and H* because i n t r a c e l l u l a r H* ions l e v e l s are lower than expected for a passive d i s t r i b u t i o n . The net r e s u l t being an increase i n i n t r a c e l l u l a r H* i o n l e v e l s and PNH3 gradients d i r e c t e d from c e l l to plasma. B. In the a c t i v e s t a t e , there may be both P N H S and NI-U+ gradients from c e l l to plasma. I n t r a c e l l u l a r NH 3 production w i l l trap H+ ions and ammonia l e a v i n g as NrU+ w i l l act to export H"*" ions from the c e l l , while NHs d i f f u s i o n out of the c e l l w i l l not a f f e c t H"* i o n l e v e l s . See t e x t f o r a d d i t i o n a l d e t a i l s . A. Resting state extracellular NH. H + -N H i 7 3 intra cellular > H* 74 beta. Evans, 1982). Based on these s t u d i e s i t i s d i f f i c u l t to p r e d i c t how ammonia e x c r e t i o n would be a f f e c t e d by hypercapnia i n lemon s o l e . Metabolism may be suppressed by high Pc02 l e v e l s i n c e l l c u l t u r e (Folbergrova et a l . , 1972) and i n whole animals ( B i c k l e r , 1986). I t i s l i k e l y , t h e r e f o r e , that a component of decreased Tamm l e v e l s i n t i s s u e s i s due to suppressed metabolism during hypercapnia. The a n a l y s i s of the d i s t r i b u t i o n of ammonia between e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments s t r o n g l y suggests that muscle, heart, and b r a i n c e l l membranes are permeable to NH4+. I t i s assumed that NH3 p e r m e a b i l i t y i s a l s o s i g -n i f i c a n t based on the f a c t that N H 3 : N H 4 + p e r m e a b i l i t y r a t i o s for other t i s s u e s are at l e a s t 5:1 ( C a s t e l l & Moore, 1971; Bown e_t a l . , 1975; K l e i n e r , 1981; Schwatz & T r i p o l o n e , 1983; Arruda a l . , 1984; Cameron & Evans, 1986). Despite the r e l a t i v e l y high p e r m e a b i l i t y of N H 3 , P N H 3 g r a d i e n t s (0.0002-0.001 Torr) do e x i s t between i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments ( F i g . 5). Figure 7 describes a model of N H 3 and N H 4 + + movements across a h y p o t h e t i c a l t i s s u e where NH4 + ions are p a s s i v e l y d i s -t r i b u t e d according to Em, i n t r a c e l l u l a r NH3 and NH4+ are i n e q u i l i b r i u m ( i n t e r c o n v e r s i o n r e a c t i o n r a t e for N H 3 : N H 4 + < 50 msec, Stumm & Morgan, 1981), and ammonia production i s i n s i g -n i f i c a n t ( F i g . 7A). NH3 gradients w i l l form i n the i n t r a -c e l l u l a r compartment as NH4+ ions enter the t i s s u e and d i s s o c i a t e to form NH3 and H + ions. The d i r e c t i o n of t h i s r e a c t i o n w i l l always be towards the formation of NH3 and H + 75 because i n t r a c e l l u l a r H+ ions l e v e l s are lower ( i . e . , pHi higher) than expected f o r a passive d i s t r i b u t i o n (Roos & Boron, 1981). The r a t e of NH3 'back d i f f u s i o n * out of the t i s s u e w i l l depend on the magnitude of the P N H 3 gradient and the NH3 p e r m e a b i l i t y ( P N H 3 ) . As NH3 d i f f u s e s i n t o the e x t r a c e l l u l a r compartment, NH4+ f l u x i n t o the i n t r a c e l l u l a r compartment w i l l be d r i v e n by the transmembrane voltage. The r e s u l t of t h i s c y c l e w i l l be a net t r a n s f e r of H+ ions to the i n t r a c e l l u l a r compartment. The net e f f e c t would be an a c i d i f i c a t i o n of the i n t r a c e l l u l a r compartment and a l k a l i n i z a t i o n of the e x t r a c e l l u l a r compartment, were there not a c t i v e H+ e x t r u s i o n mechanisms which keep [H+]j[ low and w e l l out of e l e c t r o c h e m i c a l e q u i l i b r i u m under steady s t a t e c o n d i t i o n s . With ammonia production by muscle during e x e r c i s e , i t seems l i k e l y that P ( j H 3 a n d NH4+ e l e c t r o c h e m i c a l gradients would form between muscle and plasma ( F i g . 7B). I f ammonia i s produced as N H 3 , then i n t r a c e l l u l a r ammonia w i l l r a i s e pHi by trapping H+ ions (from g l y c o l y s i s ) with an almost 1:1 s t o i c h i o m e t r y . Ammonia may d i f f u s e out of the c e l l as e i t h e r NH3 or N H 4 + . Ammonia l e a v i n g as NH3 w i l l not a f f e c t the H+ ion budget of the i n t r a c e l l u l a r compartment. A l t e r n a t i v e l y , ammonia l e a v i n g as NH4+ would act as a mechanism to export H+ i o n s , which w i l l a l s o help to r e g u l a t e i n t r a c e l l u l a r pH. In summary, the r e s u l t s of t h i s study show that the d i s t r i b u t i o n of ammonia between plasma and muscle, heart, and b r a i n t i s s u e i n lemon so l e i s not dependent on the H+ 76 d i s t r i b u t i o n , which i n d i c a t e s that the c e l l membranes of these t i s s u e s have a s i g n i f i c a n t p e r m e a b i l i t y to NH4+ ions. The ammonia d i s t r i b u t i o n appears to f o l l o w the membrane p o t e n t i a l at r e s t ; c a l c u l a t e d E^H4+ values g e n e r a l l y agree with published values f o r Em i n these t i s s u e s . During hypercapnia and f o l l o w i n g e x e r c i s e NH4 + as w e l l as NH3 g r a d i e n t s may e x i s t between t i s s u e and plasma, p a r t i c u l a r l y i n t i s s u e s where ammonia production i s s t i m u l a t e d , such as, white muscle a f t e r exhaustive e x e r c i s e . CHAPTER 2 Downstream pH changes i n water flowing over the g i l l s of. Salmo g a i r d n e r i 78 INTRODUCTION The apparent mean pH gradient across the g i l l s of f i s h i s u s u a l l y determined as the d i f f e r e n c e between bulk water pH and blood pH. The p r e c i s e g r a d i e n t , however, i s dependent upon p o s s i b l e pH g r a d i e n t s w i t h i n the i n t e r l a m e l l a r space. Water flow i s laminar through the mouth and over the g i l l s (Randall & Daxboeck, 1984). Boundary l a y e r s are undoubtedly present next to the mucous l a y e r covering the g i l l surface since water flow through the g i l l channels i n r e l a t i o n to t h e i r dimensions r e s u l t s i n Reynolds numbers that have been reported by Hughes (1984) to be very small (<10). I t has been suggested (Lloyd & Herbert, 1960; Szumski §_£. a l - , 1982) that the pH of water w i t h i n the g i l l chamber i s s i g n i f i c a n t l y more a c i d i c than that of bulk water due to CO2 e x c r e t i o n and subsequent h y d r a t i o n to H C O 3 - and H+. I f t h i s theory i s c o r r e c t i t would have important i m p l i c a t i o n s to ammonia e x c r e t i o n , which i s pH-dependent (Wright and Wood, 1985). For h y d r a t i o n of excreted CO2 to have such an appreciable e f f e c t on i n t e r l a m e l l a r pH, CO2 h y d r a t i o n must occur r a p i d l y enough to a l t e r the proton a c t i v i t y w i t h i n the i n t e r l a m e l l a r t r a n s i t time of g i l l water (100 to 400 msec, R a n d a l l , 1982a,b). This i s much f a s t e r than the uncatalysed r a t e of C0 2 h y d r a t i o n , which i s on the order of minutes (Kern, 1960) at t y p i c a l f i s h water pH and temperatures. Carbonic anhydrase, the enzyme re s p o n s i b l e f o r c a t a l y z i n g CO2 h y d r a t i o n and dehydration r e a c t i o n s , has been lo c a t e d w i t h i n g i l l e p i t h e l i a l c e l l s of f i s h (Lacy, 1983; Dimberg §_£ aj., 1981). Thus, i t seems p o s s i b l e that the a p i c a l surface of e p i t h e l i a l c e l l s and/or b r a n c h i a l mucus could c o n t a i n carbonic anhydrase. The aim of t h i s study was to i n v e s t i g a t e i n t e r l a m e l l a r water pH of t r o u t (Salmo g a i r d n e r i ) by f o l l o w i n g changes i n the pH of e x p i r e d water (pHg) i n order to determine the c o n t r i b u t i o n of carbonic anhydrase a c t i v i t y to the h y d r a t i o n of CO2 e l i m i n a t e d at the b r a n c h i a l e p i t h e l i u m . Downstream changes i n prig were followed using a stopped-flow apparatus. E f f e c t s of carbonic anhydrase and acetazolamide, a s p e c i f i c i n h i b i t o r of carbonic anhydrase, on downstream pHg changes were a l s o s t u d i e d . In a d d i t i o n , mucus excreted by f i s h was assayed for carbonic anhydrase a c t i v i t y by the pH-stat technique (Henry & Cameron, 1982) . 80 MATERIALS AND METHODS Experimental animals The experiments were d i v i d e d i n t o two s e r i e s . In the f i r s t experiment ( S e r i e s I ) , f i s h were allowed at l e a s t 5 days to acclimate to a t e s t s o l u t i o n of 40 mmol.l - 1 NaCl and 0.5 mmol.l - 1 CaCl2 i n d e c h l o r i n a t e d tapwater (11.7° C + 0.2) with a B value of 81 uequiv.I" 1.pH-1 u n i t , p r i o r to surgery and experimentation. F i s h were starved during t h i s a c c l i m a t i o n p e r i o d and throughout the experiment. This p e r i o d of time i s adequate to allow rainbow t r o u t to r e - e s t a b l i s h i o n i c , osmotic and r e s p i r a t o r y steady-state c o n d i t i o n s a f t e r t r a n s f e r from freshwater to a balanced s a l t s o l u t i o n (Perry & Heming, 1981). The second type of experiment (S e r i e s I I ) was an in. v i t r o experiment where mucus samples were c o l l e c t e d from f i s h held i n d e c h l o r i n a t e d tapwater. Measurements In order to measure pH of exp i r e d water j u s t l e a v i n g the opercular c a v i t y of f i s h i n S e r i e s I , an opercular cannula (PE 90) was s t i t c h e d i n p o s i t i o n under and midway along the opercular opening (see General M a t e r i a l s and Methods). F i s h were then f i t t e d with rubber dams (see General M a t e r i a l s and Methods) and placed i n a two-chambered Perspex box ( F i g . 2), where they were l e f t to recover f o r 48 hrs f o l l o w i n g surgery. In S e r i e s I , water pH was measured at two l o c a t i o n s ; i n s p i r e d 81 water pH was measured continuously with a combination g l a s s pH el e c t r o d e (Radiometer GK2401C) placed i n the f r o n t chamber of the f i s h box, and expired water was drawn from the opercular c a v i t y through the opercular cannula and i n t o a g l a s s 'stopped-flow' chamber (volume, 1 ml), housing a pH e l e c t r o d e (Canlab pH semi-microelectrode, H5503-21) and micro t e f l o n s t i r r i n g bar. The stopped-flow chamber was designed to measure pH changes i n the e x p i r e d water. The opercular cannula fed i n t o the i n l e t port ( i n s i d e diameter 1.27 mm) of the stopped-flow chamber and another cannula (PE 160) i n s e r t e d i n t o the o u t l e t port ( i n s i d e diameter 1.70 mm) c a r r i e d the water out of the chamber. The pH e l e c t r o d e f i t t i g h t l y i n t o a t e f l o n sleeve which then f i t i n t o the g l a s s chamber so as to form a s e a l to ensure the chamber was gas t i g h t . The pH e l e c t r o d e s were c a l i b r a t e d with standard Radiometer b u f f e r s o l u t i o n s at pH 7 and 4. Vancouver tapwater has a low i o n i c s t r e n g t h ( I ~ 0.002 M), r e l a t i v e to other freshwaters, and ther e f o r e the a d d i t i o n of d i s s o l v e d s a l t s to the t e s t water g r e a t l y increased the s t a b i l i t y and decreased the l a g time of the pH e l e c t r o d e response. The e r r o r a s s o c i a t e d with i o n i c strength d i f f e r e n c e s between the commercial b u f f e r s and the t e s t s o l u t i o n , was not c o r r e c t e d f o r and i s estimated to be approximately 0.05 to 0.25 pH u n i t s depending on pH. Although t h i s introduces an e r r o r to the absolute pH values, the r e l a t i v e changes i n pH were of greater importance to t h i s study. The l a g time of the Canlab microelectrode was determined f o r a step change i n water pH at the same i o n i c s t r e n g t h and temperature as the experimental 82 s o l u t i o n . F i s h were exposed to three d i f f e r e n t water regimes: t e s t s o l u t i o n ( c o n t r o l water), acetazolamide (1.6 mmol.l -!) i n t e s t s o l u t i o n , and carbonic anhydrase (6.8 m g . l - 1 or 20,400 Wilbur-Anderson u n i t s . 1 ~ 1 bovine carbonic anhydrase) i n t e s t s o l u t i o n . The b u f f e r values of these s o l u t i o n s were 81, 179, 78 u e q u i v . l ~ l . p H ~ * u n i t , r e s p e c t i v e l y . Two complete sets of measurements were obtained while f i s h were i n c o n t r o l water. The i n f l o w was then changed over to e i t h e r the acetazolamide or carbonic anhydrase t e s t s o l u t i o n ( f i s h were exposed to one or the other, but not both) and the same d u p l i c a t e measurements were repeated a f t e r a 30 minute period. I n s p i r e d and expired water pH were recorded for an hour preceding the experiment to ensure that the water pH was s t a b l e over time. I n s p i r e d water pH (pHj) and expired continuous flow pH (prig) were measured simultaneously and recorded immediately before the flow of water through the stopped-flow chamber was stopped. The ra t e of water flow through the chamber (8-10 ml.min -!) was such that water t r a n s i t time through the opercular cannula to the pH e l e c t r o d e was l e s s than two seconds. Once the flow of water was stopped, e x p i r e d water pH was recorded over an 8 minute p e r i o d with a Hewlett Packard data a c q u i s t i o n u n i t and a s t r i p c h a rt recorder. I f a chemical d i s e q u i l i b r i u m e x i s t e d i n the water, then a change i n pH with time (dpH/dt) was recorded. The f i n a l e q u i l i b r i u m pH a f t e r 8 minutes was r e f e r r e d to as the ex p i r e d stopped-flow pH ( p H s t ) . 83 A f t e r the response time of the e l e c t r o d e was accounted f o r , the h a l f time ( t i / 2 ) of the e q u i l i b r i u m r e a c t i o n was c a l c u l a t e d . To ensure that i n s p i r e d water was i n complete pH e q u i l i b r i u m , water was v i g o r o u s l y bubbled with compressed a i r i n a 100 1 tank before i t flowed i n t o the experimental apparatus. The pH e q u i l i b r i u m of i n s p i r e d water was p e r i o d i c a l l y checked i n the stopped flow chamber as described above. Immediately before the flow of water was stopped i n the chamber, v e n t i l a t i o n (V w) and carbon d i o x i d e content of the i n s p i r e d ( C J C O 2 ) a n d e x p i r e d (Cgco2) water were measured. V w was determined by c o l l e c t i n g the outflow water from the standpipe i n the back chamber of the experimental chamber. C o l l e c t i o n s were made over 1 minute periods and volumes determined by weight. Measurements of water C J C O 2 a n d C E C 0 2 were made by gas chromatography (see General M a t e r i a l s and Methods). The r a t e of carbon d i o x i d e e x c r e t i o n (Kc02^ w a s c a l c u l a t e d by a p p l i c a t i o n of the F i c k equation. In S e r i e s I I , mucus samples taken from the body of t r o u t were assayed for carbonic anhydrase ( C A ) . I n i t i a l l y we t r i e d to o b t a i n mucus samples from the g i l l s u r f a c e ; i t was impossible, however, to c o l l e c t samples that were lar g e enough for the assay technique. The pH s t a t technique was chosen to measure C A a c t i v i t y over other methods (modified boat and esterase assay) because previous work had shown that the pH s t a t method e x h i b i t e d the lowest l i m i t of d e t e c t i o n and the highest degree of s e n s i t i v i t y (Heming, 1984; see a l s o Henry & Cameron, 1982). A 84 mixture of phosphate b u f f e r and a bicarbonate s o l u t i o n i n i t i a t e s the conversion of HCO3 and H+ ions to CO2 gas. As CO2 i s l i b e r a t e d from the r e a c t i o n s o l u t i o n , the pH w i l l increase and the volume of a c i d ( H C 1 ) that i s req u i r e d to maintain the r e a c t i o n mixture at a given pH w i l l be p r o p o r t i o n a l to the production of C O 2 , s i n c e the st o i c h i o m e t r y of H+:CC*2 i s 1 : 1 . The r e a c t i o n r a t e , moles C 0 2/min, w i l l be greater i n the presence of the c a t a l y s t , CA, r e l a t i v e to the uncatalysed r e a c t i o n . Mucus samples ("0.5 ml) were c o l l e c t e d from the body of 6 f i s h by l i g h t l y s t r o k i n g the f i s h with a metal s p a t u l a , and these samples were then assayed f o r CA. The a d d i t i o n of mucus to the r e a c t i o n v e s s e l caused foaming, a problem which was e l i m i n a t e d with a defoaming agent (50 u l oc t a n - 2 - o l ) . The dehydration r e a c t i o n r a t e was not a f f e c t e d by the a d d i t i o n of octan-2-ol. The c a t a l y s e d (mucus) r e a c t i o n r a t e was compared to the uncatalsed r a t e , with or without acetazolamide. Anhydrous acetazolamide (5 mmol.l -!) was added d i r e c t l y to the phosphate b u f f e r s o l u t i o n . To t e s t f o r the presence of c e l l u l a r m a t e r i a l , mucus samples were s t a i n e d with e o s i n and c r y s t a l v i o l e t and examined under a microscope. Determination of h a l f - t i m e values Half-time values ( t i / 2 ) were c a l c u l a t e d by t a k i n g s e v e r a l p o i n t s along i n d i v i d u a l k i n e t i c curves (dpH/dt) and conv e r t i n g 85 these values to percentage change i n pH (assuming that the pH st value at 8 minutes was the e q u i l i b r i u m pH or very c l o s e to i t ) . These p o i n t s were then p l o t t e d on semilog paper as l o g % change i n pH versus time g i v i n g s t r a i g h t l i n e s over the f i r s t 60% of the r e a c t i o n . The ti/2 values were determined g r a p h i c a l l y . Data are presented as means +_ 1 S.E.M. (N). The Student's t w o - t a i l e d p a i r e d t - t e s t was used tp evaluate the s i g n i f i c a n c e of d i f f e r e n c e s between mean values (P<0.05). RESULTS An important f a c t o r i n the l a g time of the pH e l e c t r o d e was the degree of mixing i n the stopped-flow chamber. In order to t e s t the e f f e c t s of mixing under s i m i l a r c o n d i t i o n s to those i n the experiment, a small volume of pure CO2 gas was introduced i n t o the t e s t s o l u t i o n before i t flowed i n t o the stopped-flow chamber. The flow of water past the e l e c t r o d e was then stopped, and the CO2 h y d r a t i o n r e a c t i o n was then followed over time, with and without a s t i r r i n g bar i n the chamber. F i g 8 d e p i c t s the change i n pH with time f o r the mixed ( t i / 2 = 76 sees) and the unmixed ( t i / 2 = 300 sees) s o l u t i o n s . The d i f f e r e n c e of 224 seconds was presumably due to slow d i f f u s i o n of H+ ions through the unmixed s o l u t i o n . To reduce the e f f e c t s of mixing on the l a g time of the pH e l e c t r o d e , a s t i r r i n g bar was placed i n the stopped-flow chamber and a magnetic s t i r r e r maintained a constant r o t a t i o n of the s t i r r i n g bar. This t e s t a l s o demonstrated that 86 F i g u r e 8 . Water f l o w i n t h e s t o p p e d - f l o w chamber was s t o p p e d ( a r r o w ) and t h e k i n e t i c s o f t h e CO^ h y d r a t i o n r e a c t i o n was r e c o r d e d . I n one c a s e , a t e f l o n - c o a t e d , m a g n e t i c s t i r r i n g b a r c o n t i n u o u s l y mixed t h e s o l u t i o n ( m i x e d ) , w h i l e i n t h e o t h e r s o l u t i o n , no s t i r r i n g b a r was p r e s e n t ( u n m i x e d ) . 87 88 the system detects pH d i f f e r e n c e s due to CO2 h y d r a t i o n , and the ti/2 value for the mixed r e a c t i o n i s i n approximate agreement with values given by Kern (1960) at the t e s t pH and temperature. The r e s u l t s from S e r i e s I are presented i n Table 10 and 11. Figure 9 shows r e p r e s e n t a t i v e stopped-flow t r a c e s from two f i s h . As water flowed over the g i l l s of c o n t r o l f i s h , there was a s i g n i f i c a n t decrease from i n s p i r e d water (pHg) to expired water at e q u i l i b r i u m (pH st) (Table 10, 11). Expired c o n t r o l water was almost completely e q u i l i b r a t e d by the time i t reached the pH e l e c t r o d e , because pHg was not s i g n i f i c a n t l y d i f f e r e n t from pH st i n both the f i r s t and second c o n t r o l s e t s . Even though the o v e r a l l e x p i r e d water pH d i s e q u i l i b r i u m i n c o n t r o l water was s m a l l , i n 11 out of 30 stopped-flow pH traces there was a decrease i n pH once water flow was stopped i n the chamber, i n a few cases pH increased a f t e r the flow had been stopped i n the chamber (2/30 stopped-flow pH t r a c e s ) , and i n the remainder of traces there was no measureable change i n water pH (16/30 t r a c e s ) . The a d d i t i o n of acetazolamide s i g n i f i c a n t l y increased the CO2 d i s e q u i l i b r i u m r e l a t i v e to c o n t r o l water ( F i g 9, Table 10). In c o n t r a s t , carbonic anhydrase e l i m i n a t e d the CO2 d i s e q u i l i b r i u m except i n one stopped-flow pH trace where a small pH change (pHj2-pHst = 0.01) was observed ( F i g . 9, Table 11). There was no s i g n i f i c a n t change i n v e n t i l a t i o n and carbon d i o x i d e e x c r e t i o n r a t e s between c o n t r o l I and acetazolamide measurements and the c o n t r o l I I and carbonic anhydrase values 89 F i g u r e 9. R e p r e s e n t a t i v e c h a n g e s i n e x p i r e d water pH o f two f i s h ( c o n t r o l I and a c e t a z o l a m i d e ; c o n t r o l I I and c a r b o n i c a n h y d r a s e ) a f t e r water f l o w p a s t t h e pH e l e c t r o d e was s t o p p e d a t t i m e 0. 6.7 5 H 6.65J CONTROL I 6.45 6.35 i CONTROL II 7.5 H 7.4 i i, ACETAZOLAMIDE 6.39 6.291 —• ' 1 — - J i i 1 2 3 4 5 6 7 8 C A • i i i 1 2 3 4 5 6 7 8 T I M E (min) O 91 TABLE 10. Hater pH values ( i n s p i r e d water, pH x; exp i r e d water immediately l e a v i n g the opercular valves pHe; e q u i l i b r a t e d e x p i r e d water, pH- T, values given as mean + SEM) and h a l f - t i m e values f o r C0 2:HC0--i n t e r c o n v e r s i o n s measured on rainbow t r o u t i n CONTROL water and a f t e r ACETAZOLAMIDE (1.6 mmol.l - 1) was added to the water. CONTROL ACETAZOLAMIDE TEMPERATURE (°C) 10.4+0.1 10.7+0.2 DIRECTION OF 4- (7) t (2) no A (5) (12) pH CHANGE1 (N) pHi 7.41+.01 7.39+.00 7.41+.02 7.42+.02 pH- 6.84+.06 6.70+.05 6.60+.01 7.38+.02* pHe-r 6.82+.06 6.72+.05 6.60 + .01 7.31 + .02* pH e-pHo T 0.03+.00 0.02+.01 0.00+.00 0.07+.01* t i / a (sec) 9 0 + 6 6 6 + 4 - 8 8 + 3 1. 'DIRECTION OF THE pH CHANGE' r e f e r s to whether pH increased or decreased once the flow of water was stopped i n the stopped-flow chamber. * s i g n i f i c a n t l y d i f f e r e n t from pH CONTROL, p i 0.05 92 TABLE 11. Hater pH values ( i n s p i r e d water, pH z; e x p i r e d water immediately l e a v i n g the o p e r c u l a r v a l v e , pH-; e q u i l i -brated e x p i r e d water, p H a T , values given as mean + SEN) and h a l f - t i m e values f o r CO a:HCO»- i n t e r c o n v e r s i o n s , measured on t r o u t i n CONTROL water and a f t e r CARBONIC ANHYDRASE (6.8 mgl" 1) was added to the water. CONTROL CARBONIC ANHYDRASE TEMPERATURE (°C) 12.7 + 0.1 13.1 +0.1 DIRECTION OF * (4) n o £ > ( l l ) no (12) pH CHANGE1 (N) pHi 7.35+.10 7.13+.03 7.19+.06 pH- 6.44+.05 6.35+.03 6.39 + .03 pH - T 6.43+.04 6.35+.03 6.39+.03 pH--pH» T 0.02+.01 0.00+.00 0.00+.00 t i / a (sec) 4 8 + 1 3 1. see TABLE 1 Experimental values were not s t a t i s t i c a l l y s i g n i f i c a n t from c o n t r o l values u s i n g the Student's p a i r e d t - t e s t . 93 TABLE 12. V e n t i l a t i o n ( V W ) and carbon d i o x i d e e x c r e t i o n ( M c « ) r a t e s from experiment one (CONTROL I and ACETAZOLAMIDE) and experiment two (CONTROL II and CARBONIC ANHYDRASE) TEMP. Vw Mcoa (°C) ( l . h - 1 ) (umol.lOOg- 1.h" 1) CONTROL I 10.4+0.1 6.45+0.37 239.39+27.28 ACETAZOLAMIDE 10.7+0.2 6.18+0.18 261.56+28.26 CONTROL II 12.7+0.1 9.95+0.97 380.83+38.96 CARBONIC 13.1+0.1 10.21+0.77 393.11+42.02 ANHYDRASE Experimental values (acetazolamide, c a r b o n i c anhydrase) were not s t a t i s t i c a l l y s i g n i f i c a n t from the r e s p e c t i v e c o n t r o l values using the Student's p a i r e d t - t e s t . 94 TABLE 13. C O 2 Dehydration r e a c t i o n r a t e i n c a t a y l s e d (mucus samples) and uncatalysed s o l u t i o n s , with or without caetazolamide at 22° C. Mucus samples taken from the body of s i x f i s h . Means + S.E.M. (N= 6) CONTROL (COa umol.min-1) ACETAZOLAMIDE (5mM) (COa umol.min-1) CATALYSED (MUCUS) 48.0 + 7.7 20.7 + 0.8 UNCATALYSED 26.0 + 1.6 21.4 + 0.7 * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l # s i g n i f i c a n t l y d i f f e r e n t from uncatalysed c o n t r o l (Table 12). I t i s i n t e r e s t i n g to note, however, that f i s h i n the f i r s t experiment (April-May, 10.5° C) had c o n s i d e r a b l y lower V w and Mco2 r a t e s compared to f i s h i n the second experiment (Sept.-Oct., 13.0° C), and these d i f f e r e n c e s may be r e l a t e d to seasonal v a r i a t i o n s . Mucus excreted by the f i s h contained carbonic anhydrase a c t i v i t y . The a d d i t i o n of mucus doubled the CO2 dehydration r e a c t i o n r a t e r e l a t i v e to the uncatalysed r a t e and acetazolamide reduced the r e a c t i o n r a t e to the uncatalysed r a t e (Table 13). There was a small d i f f e r e n c e i n the r e a c t i o n r a t e between c o n t r o l and acetazolamide uncatalysed r e a c t i o n s . In a d d i t i o n mucus samples were found to c o n t a i n c e l l u l a r m a t e r i a l . DISCUSSION These experiments present the f i r s t documented measurement of the pH change i n water fl o w i n g over the g i l l s of freshwater f i s h . Holeton and Randall (1967) reported a rough estimate of the i n s p i r e d to e x p i r e d water pH d i f f e r e n c e of about 0.2 to 0.5 pH u n i t s , i n s i m i l a r Vancouver tapwater. The a c t u a l d i f f e r e n c e measured i n t h i s study, however, was 0.7-0.9 pH u n i t s . In both cases, these d i f f e r e n c e s were measured at p o i n t sources from the opercular outflow, but p r e l i m i n a r y t e s t s i n t h i s study, to determine i f water pH v a r i e d with the p o s i t i o n of the cannula, showed that there was no s i g n i f i c a n t d i f f e r e n c e s between the pH measured i n three l o c a t i o n s along the opercular valve. Changes i n 96 water sampled from a s i n g l e p o i n t , t h e r e f o r e , are r e p r e s e n t a t i v e of changes i n expired water i n general. D i f f e r e n c e s i n the magnitude of the pH change reported i n our study and that of Holeton and Randall (1967) are probably r e l a t e d to s e v e r a l f a c t o r s , i n c l u d i n g d i f f e r e n c e s i n temperature, the presence or absence of l a t e x masks, f l u c t u a t i o n s i n calcium carbonate and b u f f e r i n g c a p a c i t y of the water, and d i f f e r e n c e s i n the amount of carbon d i o x i d e e l i m i n a t e d by the f i s h . Evidence f o r the presence of carbonic anhydrase i n the mucus i s supported by the f a c t that e x p i r e d water was almost completely e q u i l i b r a t e d as i t l e f t the opercular c a v i t y . Water i s only i n contact with the g i l l f or about 100 to 400 msec (R a n d a l l , 1982a, 1982b) and the time r e q u i r e d f o r complete CO2 h y d r a t i o n i s on the order of minutes (Kern, 1960), t h e r e f o r e , the CO2 r e a c t i o n must be c a t a l y s e d at the g i l l s u r face. Acetazolamide i n h i b i t s CA and increased the e x p i r e d water CO2 d i s e q u i l i b r i u m , whereas, the a d d i t i o n of CA to the water completely e l i m i n a t e d the small d i s e q u i l i b r i u m that was observed i n the expired c o n t r o l water of some f i s h . The cause of t h i s small d i s e q u i l i b r i u m downstream from the g i l l s i n c o n t r o l water w i l l be discussed l a t e r , but i s probably not r e l a t e d to any nonequilibrium s t a t e at the g i l l s u rface. Carbonic anhydrase a c t i v i t y i s present i n mucus covering the epidermis of rainbow t r o u t . Although i t was not f e a s i b l e to measure CA a c t i v i t y i n mucus covering the g i l l s u r f a c e , i t i s 97 probable that CA a c t i v i t y i s present i n the b r a n c h i a l mucus excreted by g i l l e p i t h e l i a l c e l l s . F l e t c h e r et a l (1976) compared epidermal (body) and g i l l f i l a m ent mucus-secreting goblet c e l l s of rainbow t r o u t and found that c e l l d e n s i t y and g l y c o p r o t e i n s composition (predominantly a c i d g l y c o p r o t e i n s ) were very s i m i l a r . B r a n c h i a l e p i t h e l i u m contains high l e v e l s of carbonic anhydrase (Haswell ejb §i, 1980), which appears to be concentrated i n the a p i c a l regions of the e p i t h e l i a l c e l l s (Dimberg e t a l . , 1981). A l s o , CA has been lo c a t e d i n the cytoplasm of c h l o r i d e c e l l s , v e s i c u l a r c e l l s , supportive c e l l s , i n the cytoplasm and mucous granules of goblet c e l l s , and i n the e x t r a c e l l u l a r spaces between v e s i c u l a r and supportive c e l l s of the opercular membrane of the t e l e o s t , Fundulus h e t e r o c l i t u s (Lacy, 1983). I t i s reasonable to assume, t h e r e f o r e , that carbonic anhydrase a c t i v i t y i s a l s o present i n the mucus covering the g i l l f i l a m e n t s of rainbow t r o u t . Mucus samples were found to c o n t a i n c e l l u l a r m a t e r i a l , which i s not s u r p r i s i n g c o n s i d e r i n g that the epidermal c e l l s are r e g u l a r l y sloughed o f f to allow f o r new growth. The presence of c e l l s does not i n v a l i d a t e the experimental f i n d i n g s . Enzyme a c t i v i t y i n the mucus may o r i g i n a t e from CA e x p e l l e d from mucous c e l l s i n t o the mucus covering the g i l l s and body of the f i s h , or from e p i t h e l i a l c e l l fragments which have been sloughed o f f i n t o the mucous l a y e r . The e l i m i n a t i o n r a t e of CO2 f a r exceeds that of other b r a n c h i a l l y excreted molecules, and i t i s w e l l e s t a b l i s h e d that CO2 d i f f u s i o n i s f a c i l i t a t e d i n the presence of CA (Longmuir ej: 98 a l . . 1966; Enns, 1967; Zborowska-Sluis gt §i., 1974; Gutknecht et a l . , 1977; Bur n e t t , 1984). Carbon d i o x i d e i s p r i m a r i l y excreted i n the gaseous form (Perry e t a l . , , 1982), with approximately 10% of t o t a l e x c r e t i o n as H C O 3 - exchanging f o r CI- (Cameron, 1976). Gutknecht et a l (1977) used a r t i f i c i a l l i p i d b i l a y e r membranes to study p r o p e r t i e s of CO2 f l u x and found that at pH 7-8, CA (1-1.6 mg/ml) caused a 10- to 80-f o l d s t i m u l a t i o n of CO2 f l u x . They suggested that the presence of CA i n the u n s t i r r e d l a y e r renders the CO2 hydration-dehydration r e a c t i o n so f a s t that chemical e q u i l i b r i u m between CO2 and H C O 3 - e x i s t s throughout the u n s t i r r e d l a y e r . Furthermore, the r a t e - l i m i t i n g step i n CO2 t r a n s p o r t i n a s i t u a t i o n where H C O 3 - cannot r e a d i l y cross the membrane and the s o l u t i o n s are poorly buffered i s the d i f f u s i o n of H C O 3 - and H+ through the u n s t i r r e d l a y e r . I t i s i n t e r e s t i n g that a small d i s e q u i l i b r i u m was observed i n e xpired c o n t r o l water, even though we p o s t u l a t e complete e q u i l i b r a t i o n of the C O 2 / H C O 3 - r e a c t i o n at the g i l l surface. The f a c t that CA e l i m i n a t e d the pH d i s e q u i l i b r i u m observed downstream of the g i l l s i n d i c a t e s that i t was, i n f a c t , due to a C O 2 / H C O 3 - d i s e q u i l i b r i u m i n the water. CO2 may be i n complete e q u i l i b r i u m with H C O 3 - and H+ i n the mucus and ass o c i a t e d u n s t i r r e d l a y e r next to the g i l l , but the d i f f e r e n t i a l d i f f u s i o n r a t e s of these molecules across the u n s t i r r e d l a y e r may create a small d i s e q u i l i b r i u m i n the mainstream of water flowing over the g i l l s . In a d d i t i o n , mixing of waters of d i f f e r e n t carbon di o x i d e content may r e s u l t i n 99 C O 2 / H C O 3 - d i s e q u i l i b r i u m downstream of the g i l l s , indeed t h i s may account f o r the two tra c e s c o l l e c t e d from one f i s h which showed a reverse r e a c t i o n , that i s , the expired water pH increased s l i g h t l y when the flow of water was stopped i n the chamber. I t may be that r e s p i r a t o r y water at e q u i l i b r i u m was mixed with n o n r e s p i r a t o r y xshunt' water j u s t a f t e r i t entered the opercular c a v i t y . This would r e s u l t i n a small increase i n pH as the shunt water would c o n t a i n a lower H+ a c t i v i t y compared to the r e s p i r a t o r y water. In one t r a c e , slow pH changes were observed i n the presence of C A , presumably due to the uncatalysed CO2 h y d r a t i o n r e a c t i o n . The M C Q2 r a t e was extremely high i n t h i s f i s h and ther e f o r e the l e v e l of C A i n r e l a t i o n to the co n c e n t r a t i o n of CO2 was probably inadequate to completely c a t a l y s e the r e a c t i o n . V a r i a t i o n s i n t ^ / 2 values f o r a given r e a c t i o n are the r e s u l t of d i f f e r e n c e s i n temperatue, i o n i c s t r e n g t h , and b u f f e r i n g c a p a c i t y . I o n i c s t r e n g t h i n f l u e n c e s the i o n i c m o b i l i t y of the s o l u t i o n , as w e l l as e f f e c t i n g the s t a b i l i t y of the pH e l e c t r o d e . The t j / 2 values for the c o n t r o l experiments ranged from 48 to 90 sees. These v a r i a t i o n s i n t ^ / 2 may be due to i o n i c s t r e n g t h d i f f e r e n c e s r e l a t e d to small changes i n the i o n i c compositions of the Vancouver tapwater during d i f f e r e n t times of the year. These seasonal v a r i a t i o n s i n water composition are al s o r e f l e c t e d i n the s l i g h t l y d i f f e r e n t p H i n values between the two c o n t r o l experiments. Increases i n the b u f f e r i n g c a p a c i t y of a s o l u t i o n r e s u l t s i n longer t ^ / 2 values f o r the uncatalysed CC>2 h y d r a t i o n r e a c t i o n (Gray, 1971). Even though acetazolamide increased the B value i n t h i s study, the mean t^/2 value was not a l t e r e d , which may be a r e f l e c t i o n of the complexity of pH changes i n water downstream of the g i l l s . The f i n a l e q u i l i b r i u m pH for acetazolamide-treated f i s h was s i g n i f i c a n t l y higher than the f i n a l c o n t r o l pH. The a d d i t i o n of acetazolamide a l t e r e d the i o n i c s t r e n g t h of the t e s t s o l u t i o n s l i g h t l y . The e r r o r i n the absolute pH value a s s o c i a t e d with i o n i c strength d i f f e r e n c e s was estimated to be no more than 0.01 pH u n i t s , and t h i s can not account f o r the r e l a t i v e l y high expired water pH value. The three parameters that would e f f e c t the e x p i r e d water pH value are M-Q2, V W a n d t n e P value. There were no changes i n e i t h e r M~o2 o r uw a n c^ t n e r e s u l t s can be explained only i n terms of changes i n the B value with the a d d i t i o n of acetazolamide, a weak a c i d . Acetazolamide increased the B value two-fold compared to that of c o n t r o l water. Heming (1985) performed s i m i l a r experiments to t h i s study on rainbow t r o u t acclimated to a s a l i n e t e s t s o l u t i o n ( i n mmol.l - 1: NaCl, 40; KC1, 1.6; C a C l 2 , 0.47; MgS0 4, 0.62; NaHC03, 5.5; NaH^PC^, 0.95 )with d i f f e r e n t r e s u l t s and con c l u s i o n s . He found expired pH was almost i d e n t i c a l to i n s p i r e d pH and water downstream from the g i l l became p r o g r e s s i v e l y more a c i d i c at the uncatalysed r a t e of CO2 h y d r a t i o n , and concluded that CA was not present on the g i l l s urface. An e x p l a n a t i o n for the discrepancy between the two s t u d i e s may be r e l a t e d to the f a c t that the f l u i d i n Heming's stopped-flow chamber was s t a t i c a f t e r water flow stopped, whereas i n the present study the f l u i d was c o n t i n u a l l y mixed v i a a s t i r bar. We have found that the dpH/dt i n s t a t i c f l u i d s has much l a r g e r t ^ / 2 than when mixed and t h i s accounts for the slow responses observed. Carbon d i o x i d e i s not the only molecule e l i m i n a t e d at the g i l l which w i l l i n f l u e n c e water pH. F i s h excrete N H 3 , NH4 + , H+, and H C O 3 - i n a d d i t i o n to CO2 across t h e i r g i l l s . A l l of these molecules w i l l a f f e c t the water pH and the magnitude of the e f f e c t w i l l depend on the r e l a t i v e r a t e s of e x c r e t i o n , the r a t e s of chemical e q u i l i b r i u m , the water-buffering c a p a c i t y , and the ra t e of water flow past the g i l l s . P r o t o n a t i o n of NH3 i s extremely r a p i d , so that NH 3 e x c r e t i o n w i l l r e s u l t i n the immediate e l e v a t i o n of water pH at the g i l l s urface. A l t e r n a t i v e l y , NH4+ e x c r e t i o n w i l l have a n e g l i b l e e f f e c t on water pH. The e f f l u x of H+ (or OH- i n f l u x ) w i l l a l t e r the pH of water instantaneously, but the gradient f o r H+ ions i s g e n e r a l l y very small (H+ gradient from blood (pH 8) to expi r e d water (pH 6) = 9.9 x 10~7 e q u i v . l - 1 ) thus, net e x c r e t i o n of H+, i n exchange f o r Na+ or N H 4 + , w i l l be low under most c o n d i t i o n s , even i f the g i l l s are very permeable to protons (McWilliams & P o t t s , 1978). I t i s c l e a r that i n t e r l a m e l l a r water pH i s s i g n i f i c a n t l y more a c i d i c than that of bulk water i n the g i l l s of rainbow t r o u t . Water a c i d i f i c a t i o n i s due to the c a t a l y s e d conversion of excreted CO2 to H C O 3 - and H+ at the g i l l s u rface. Several authors (Lloyd & Herbert, 1960; Szumski al., 1982) have modelled the observed e f f e c t s of bulk water pH on the a q u a t i c t o x i c i t y of ammonia i n terms of a la r g e C 0 2~induced a c i d i f i c a t i o n of i n t e r l a m e l l a r water. Such models are v a l i d under the water c o n d i t i o n s t e s t e d i n t h i s study, however, water a c i d i f i c a t i o n at the g i l l may not occur i n a l l s i t u a t i o n s . For example, i n very w e l l b u ffered lakes and r i v e r s CO2 e x c r e t i o n may have very l i t t l e e f f e c t on i n t e r l a m e l l a r water pH. Furthermore, the degree of a c i d i f i c a t i o n at the g i l l surface may vary g r e a t l y i n f i s h depending on the environmental water pH, temperature, the r e l a t i v e b r a n c h i a l molecular e x c r e t i o n r a t e s and the r a t e of water flow past the g i l l s . A l l these f a c t o r s must be accounted f o r before one can assess the e f f e c t of CO2 e x c r e t i o n by the f i s h on i n t e r l a m e l l a r water pH i n a given body of water. 103 CHAPTER 3 E f f e c t s of i n h i b i t i o n of e x t e r n a l g i l l carbonic anhydrase on carbon d i o x i d e and ammonia e x c r e t i o n i n i n t a c t Salmo g a i r d n e r i 104 INTRODUCTION The ubquitous enzyme carbonic anhydrase (CA) c a t a l y s e s the i n t e r c o n v e r s i o n of bicarbonate and carbon d i o x i d e i n animal t i s s u e s (Maren, 1967; Lindskog et a l . . , 1971; C a r t e r , 1972; Bauer et a l . . , 1980). I n t r a c e l l u l a r l o c a l i z a t i o n of CA i n f i s h g i l l s i s w e l l known (Haswell g_t a i . . , 1980; Dimberg e t a l . , 1981; Lacy, 1983; Swenson & Maren, 1987), but recent advances suggest that CA i s a l s o present on the e x t e r n a l surface of the g i l l . Wright e_t a l . (1986) concluded that mucus adjacent to the g i l l e p i t h e l i a l surface contained CA a c t i v i t y because 1) water downstream from the g i l l was more a c i d i c than i n s p i r e d water but was i n pH e q u i l i b r i u m and 2) epidermal mucus, which i s chemically s i m i l a r to g i l l mucus, contained CA a c t i v i t y (see Chapter 2, D i s c u s s i o n ) . Rohim, Delaunoy, & Laurent (1987) have demonstrated by immunoelectronmicroscopic techniques that CA molecules are bound to the a p i c a l e p i t h e l i a l membrane of f i s h g i l l s . There are s e v e r a l p o s s i b l e p h y s i o l o g i c a l r o l e s for e x t e r n a l g i l l CA i n the mucous l a y e r or bound to the a p i c a l e p i t h e l i a l membrane. In a l l l i k l i h o o d , e x t e r n a l CA f a c i l i t a t e s CO2 e x c r e t i o n from blood to the environment. I t i s w e l l documented that CA f a c i l i t a t e s CO2 d i f f u s i o n across a v a r i e t y of t i s s u e s , i n c l u d i n g muscle t i s s u e (Zborowska-Sluis et a l . , 1974; Kawashiro & Scheid, 1976), lung t i s s u e (Klocke, 1980; E f f r o s §_£ a l . , 1980; Enns & H i l l , 1983), elasmobranch r e c t a l gland (Swenson & Maren, 105 1984.), and crab g i l l e p i t h e l i u m (McMahon et. al.. *• 1984; Burnett & McMahon, 1985; Henry, 1987). Another p o s s i b l e r o l e of e x t e r n a l CA i s to maintain an a c i d boundary l a y e r next to the g i l l surface to f a c i l i t a t e ammonia e x c r e t i o n . In freshwater f i s h , ammonia i s p r i m a r i l y excreted across the g i l l s by simple d i f f u s i o n of non-ionic NH3 and v i a the Na+/NH4+ ion exchange mechanism (Maetz & Garcia Romeu, 1964; Maetz, 1973; Kirsch n e r et. a l . . , 1973; Evans, 1977; Payan, 1978; Cameron & H e i s l e r , 1983; Wright & Wood, 1985). Sustained NH3 d i f f u s i o n i s dependent upon NH3 removal from the boundary l a y e r because the accumulation of excreted NH3 i n the boundary l a y e r w i l l reduce the blood-to-water NH3 p a r t i a l pressure (P(*H3) d i f f u s i o n gradient. NH3 can be removed from the boundary l a y e r p h y s i c a l l y by d i f f u s i o n i n t o the water fl o w i n g past the g i l l s and/or removed chemically by combining with a H+ ion to form NH4 + i n the boundary l a y e r . The maintenance of PNH3 g r a d i e n t s across the g i l l t h e r e f o r e , w i l l depend, i n p a r t , upon the a v a i l a b i l i t y of H+ ions i n the boundary l a y e r to f a c i l a t a t e i n t e r c o n v e r s i o n of NH3 to NH4+. The r o l e of e x t e r n a l g i l l CA, t h e r e f o r e , i s to c a t a l y s e the conversion of excreted CO2 to H C O 3 - and H+ ions i n the boundary l a y e r to supply H+ ions f o r the N H 3 ' i N H 4 + r e a c t i o n . For e x t e r n a l g i l l CA to a f f e c t b r a n c h i a l carbon d i o x i d e and ammonia e x c r e t i o n the t r a n s f e r of carbon d i o x i d e and ammonia must be d i f f u s i o n l i m i t e d . In other words, no matter how q u i c k l y carbon d i o x i d e or ammonia are d e l i v e r e d to the g i l l s , , t r a n s f e r i s 106 r e s t r i c t e d at one or more p o i n t s along the d i f f u s i o n pathway, that i s , from b l o o d - t o - g i l l epithelium-to-water. On the other hand, i f CO2 and ammonia are p e r f u s i o n l i m i t e d , d i f f u s i o n i s u n r e s t r i c t e d and blood flow r a t e s w i l l be i n v e r s e l y p r o p o r t i o n a l to the r a t e of t r a n s f e r . In the l i t e r a t u r e there are c o n f l i c t i n g r e p o r t s as to whether gas t r a n s f e r i s d i f f u s i o n or p e r f u s i o n l i m i t e d (Daxboeck et. a l . . , 1982; P i i p e r & Scheid, 1982; Part et a l . , 1984; Perry et a l , 1985c). I t i s probable that gas t r a n s f e r across the g i l l i s l i m i t e d by both p e r f u s i o n and d i f f u s i o n components, depending on the p h y s i o l o g i c a l c o n d i t i o n s . I f one i s c o n s i d e r i n g gas d i f f u s i o n i n a simple system, d i f f u s i o n w i l l be more l i m i t i n g i n oxygen t r a n s f e r processes than carbon d i o x i d e , due to (X^'s higher Krogh's permeation c o e f f i c i e n t ( P i i p e r & Scheid, 1982). I t f o l l o w s that ammonia t r a n s f e r w i l l be l e a s t a f f e c t e d by d i f f u s i o n l i m i t a t i o n s because the Krogh's permeation c o e f f i c i e n t f o r ammonia i s cons i d e r a b l y greater than that for O2 or CO2 (see General I n t r o d u c t i o n ) . D i f f u s i o n l i m i t a t i o n s , however, not only include p h y s i c a l d i f f e r e n c e s between gases, but a l s o i n v o l v e r e a c t i o n v e l o c i t i e s (eg., C02:HC03- i n t e r c o n v e r s i o n s ) and i o n t r a n s f e r processes (eg., C I - / H C O 3 - and Na+/NH4+). Hence, i f gas exchange at the g i l l s i s s i g n i f i c a n t l y e f f e c t e d by d i f f u s i o n l i m i t a t i o n s , then enzymatic r e a c t i o n s and e l e c t r o l y t e p e r m e a b i l i t i e s w i l l be important components of gas exchange e f f i c i e n c y ( P i i p e r & Scheid, 1982). The experiments described i n t h i s chapter were designed to 107 t e s t the theory that carbonic anhydrase at the e x t e r n a l g i l l surface maintains an a c i d boundary l a y e r which f a c i l i t a t e s ammonia e x c r e t i o n . The f i r s t set of experiments were designed to i n v e s t i g a t e the e f f e c t s of i n h i b i t i o n of CA at the e x t e r n a l surface of the g i l l on b r a n c h i a l carbon d i o x i d e and ammonia e x c r e t i o n (30 min exposure to acetazolamide or carbonic anhydrase). A second set of experiments were designed to determine the acute e f f e c t s (0-60 min) of i n h i b i t i o n of e x t e r n a l g i l l CA on blood ammonia l e v e l s and b r a n c h i a l ammonia e x c r e t i o n . Methazolamide was used to replace the more common CA i n h i b i t o r , acetazolamide, i n the second set of experiments because methazolamide i s three times more water s o l u b l e and a more potent i n h i b i t o r of CA (Maren, 1967). I f ammonia e x c r e t i o n i s in f l u e n c e d by the pH of boundary l a y e r water, then an i n h i b i t i o n of NH3 d i f f u s i o n may r e s u l t i n a compensatory increase i n NH4+ e f f l u x to maintain net ammonia e x c r e t i o n (Wright & Wood, 1985). To e l i m i n a t e t h i s p o s s i b i l i t y , the Na+ uptake b l o c k e r , a m i l o r i d e , was a l s o added to the water along with methazolamide to e l i m i n a t e NH4+ e x c r e t i o n v i a the Na+/NH4+ i o n exchange mechanism. Passive NH4+ e f f l u x i s of minor q u a n t i t a t i v e importance i n freshwater f i s h (Kormanik & Cameron, 1981a; Wright & Wood, 1985), and t h e r e f o r e , was ignored i n t h i s study. 108 MATERIALS AND METHODS Experiments i n t h i s chapter can be d i v i d e d i n t o two s e c t i o n s , A and B: SECTION A: E f f e c t s of acetazolamide and carbonic anhydrase on MAmm a n d MC02-Experimental animals Rainbow t r o u t (Salmo g a i r d e r n i ) were removed from ho l d i n g tanks (Vancouver tapwater) and held for at l e a s t 5 days i n decholinated tapwater c o n t a i n i n g 40 mmol.l - 1 NaCl and 0.5 mmol.l - 1 CaCl2 (11.7° C +_0.2, see Chapter 2, Experimental animals). A f t e r a n a e s t h e t i z a t i o n , an opercular cannula and o r a l mask were s t i t c h e d i n place (see General M a t e r i a l s and Methods). Fol l o w i n g surgery, f i s h were l e f t to recover i n a two-chambered, flow-through experimental apparatus ( F i g . 2) for 48 h. Experimental P r o t o c o l and Measurements F i s h (mean weight = 257 + 9 g (16)) were exposed to three d i f f e r e n t water regimes: c o n t r o l water, acetazolamide (1.6 mmol.l -!) added to the water, and carbonic anhydrase (6.8 m g . l - 1 or 20,400 Wilbur-Anderson u n i t s . I - 1 bovine carbonic anhydrase) added to the water. Two complete se t s of measurements were obtained while f i s h were i n c o n t r o l water. The i n f l o w water was then changed over to e i t h e r the acetazolamide or the carbonic 109 anhydrase s o l u t i o n ( f i s h were exposed to one or the other, but not to both) and the same d u p l i c a t e measurements were repeated a f t e r 30 min. The r a t e of v e n t i l a t o r y water flow (Vw), carbon d i o x i d e content of the i n s p i r e d (Cj~o2) and expired (C--Q2) water, and the t o t a l ammonia c o n c e n t r a t i o n of i n s p i r e d (Tammj) and t e x p i r e d (Taming) water were measured. Vw was determined by c o l l e c t i n g outflow water from the back chamber standpipe over 1 min i n t e r v a l s and determinig the volume by weight. Measurements of Cjco2 a n ^ ~EC02 were made by gas chromatography (see General M a t e r i a l s and Methods). Tamm l e v e l s i n the water were measured by a m o d i f i c a t i o n of the s a l i c y l a t e - h y p o c h l o r i t e assay (Verdouw §_t a l . . , 1978). The r a t e of carbon d i o x i d e (MCQ2^ a n c ^ ammonia (M^ m m) e x c r e t i o n were c a l c u l a t e d by a p p l i c a t i o n of the Fi c k p r i n c i p l e . Data are presented as means ± 1 S.E.M. Student's t w o - t a i l e d , p a i r e d t - t e s t was used to compare r e l a t i o n s h i p s i n the data (P < 0.05) . SECTION B: E f f e c t s of methazolamide and a m i l o r i d e on M^ m m Experimental animals Rainbow t r o u t (Salmo q a i r d n e r i ) of both sexes were obtained from Spring V a l l e y Trout Farm ( M i s s i o n , B.C.) and held i n outdoor, c i r c u l a r f i b e r g l a s s tanks. The tanks were su p p l i e d with f l o w i n g , aerated and d e c h l o r i n a t e d tapwater (pH approx. 7.0, [Na+]= 40 u e q u i v . I - 1 , [Cl-]= 20 u e q i v . l - 1 , hardness= 12 p.p.m. CaCO^t temperature 9-10° C). F i s h were fed a d i e t of commercial t r o u t p e l l e t s while i n the outdoor ho l d i n g tanks. P r i o r to experimentation (4-5 days) f i s h were t r a n s f e r r e d to the l a b o r a t o r y h o l d i n g f a c i l i t y ( s u p p l i e d with the same water) and were not fed during t h i s period to minimize the i n f l u e n c e of d i e t on ammonia e x c r e t i o n (Fromm, 1963). Trout (mean weight= 300 + 9 g (N=28)) were f i t t e d with d o r s a l a o r t i c cannulae (see General M a t e r i a l s and Methods) and were placed i n i n d i v i d u a l , low volume (2-2.5 1), flow-through chambers to recover f o r at l e a s t 48 h. Experimental P r o t o c o l and Measurements Four experimental c o n d i t i o n s were s t u d i e d : S e r i e s I. A c o n t r o l experiment was performed i n which f i s h were subjected to unaltered freshwater (pH= 7.2). S e r i e s I f i s h served as c o n t r o l animals for S e r i e s I I experiments. S e r i e s I I . In a second group of f i s h , the CA i n h i b i t o r , methazolamide (Neptazane, Lederle L a b o r a t o r i e s , 0.5 mmol.l - 1) was added to the water during the experimental p e r i o d to determine i f CA i n h i b i t i o n at the g i l l e x t e r n a l surface a f f e c t e d ammonia movements across the g i l l . S e r i e s I I I . In the t h i r d group of f i s h , a m i l o r i d e c o n t r o l , the Na+ uptake b l o c k e r , a m i l o r i d e (0.1 mmol.l - 1) was added to the water during the experimental p e r i o d to i n h i b i t NH4+ e x c r e t i o n v i a the b r a n c h i a l Na+/NH4+ ion exchange mechanism. In previous experiments (Wright & Wood, 1985), the same co n c e n t r a t i o n of am i l o r i d e was found to completely a b o l i s h Na+ uptake across g i l l e p i t h e l i u m of rainbow t r o u t . F i s h i n Se r i e s I I I served as c o n t r o l animals for Se r i e s IV experiments. S e r i e s IV. In the f i n a l group of f i s h , methazolamide (0.5 mmol.l -^), w a s added to the water during the experimental period along with a m i l o r i d e (0.1 mmol.l -!). The aim of t h i s experiment was to i n v e s t i g a t e the e f f e c t s of the i n h i b i t i o n of t e x t e r n a l g i l l CA on ammonia e x c r e t i o n , i n the absence of NH4 e x c r e t i o n v i a the Na+/NH4+ i o n exchange. The experimental p r o t o c o l was i d e n t i c a l i n Se r i e s I-IV. F i s h were f i r s t subjected to a 30 min c o n t r o l p e r i o d (unaltered freshwater); water flow was switched o f f so that ammonia e x c r e t i o n could be measured by appearance of ammonia i n the water over time. Water samples (5 ml) f o r ammonia determination were c o l l e c t e d at 0, 15, and 30 min. The water was v i g o r o u s l y aerated throughout the flow-through and c l o s e d - c i r c u i t p e r i o d . P r e l i m i n a r y t e s t s i n the absence of f i s h showed that ammonia was not l o s t from the chamber due to a e r a t i o n alone. A 0.45 ml a r t e r i a l blood sample was c o l l e c t e d at 15 min and replaced with an equal volume of he p a r i n i z e d C o r t l a n d s a l i n e (Wolf, 1963, pH= 112 7.0). F o l l o w i n g the i n i t i a l c o n t r o l p e r i o d , water flow through the box was resumed f o r 1 h to f l u s h through accumulated waste products. F i s h were then subjected to a 1 h experimental p e r i o d . Water flow was switched o f f and i n 3 out of 4 of the experiments a m i l o r i d e and/or methazolamide were added to the water. Ammonia e x c r e t i o n was followed over a 1 h p e r i o d by c o l l e c t i n g water samples every 15 min. A r t e r i a l blood samples (0.45 ml) were taken at 1, 4, 10, 30, and 60 min and were replaced with equal volumes of h e p a r i n i z e d s a l i n e . The volume of blood removed from each f i s h (2.25 ml) represented about 15% of t o t a l blood volume. Whole blood samples were immediately analysed f o r e x t r a c e l l u l a r pH (pHe) and haematocrit ( h c t ) . pHe was determined with a Radiometer microelectrode (type E5021) maintained at 10° C and l i n k e d to a Radiometer PHM72 acid-base analyser. Total carbon d i o x i d e content (Crjo2^ w a s measured on plasma samples by gas chromatography (see General M a t e r i a l s and Methods). Plasma CO2 tensions (Pc02* a n d HCO3- concentrations were c a l c u l a t e d by manipulations of the Henderson-Hasselbalch equation (see General M a t e r i a l s and Methods). Plasma samples (175 u l ) were stored f o r l e s s than 24 h i n 10% t r i c h o l o r o a c e t i c a c i d (350 u l ) at -4° C and then analysed for ammonia content (Tamm), as f o l l o w s . Samples were thawed, c e n t r i f u g e d , and the supernatent was n e u t r a l i z e d with saturated T r i s b u f f e r . Tamm of t h i s n e u t r a l i z e d s o l u t i o n was measured by the glutamate dehydrogenase enzymatic assay (Kun and Kearney, 1971). Plasma NH3 and P^H3 113 l e v e l s were then c a l c u l a t e d (see Chapter 1, M a t e r i a l s and Methods). Water Tamm was determined as described i n S e c t i o n A. Ammonia e x c r e t i o n r a t e s (M^ m r n) were c a l c u l a t e d as: (21) M A m m = (Tammj - Tammf) x V t x W where i and f r e f e r to i n i t i a l and f i n a l c oncentrations i n water i n umol.ml - 1 » V i s the volume of the system i n ml (co r r e c t e d f o r sampling d e f i c i t s ) , t i s the elapsed time i n h, and W i s the f i s h weight i n kg. Water samples for Tamm determination were c o l l e c t e d a t 15 min i n t e r v a l s during the experimental p e r i o d , however, values were averaged over 30 min periods (0-30 min and 30-60 min). Data are presented as means +,1 S.E.M. (N). The Student's t w o - t a i l e d , p a i r e d and unpaired t - t e s t was employed to compare the d i f f e r e n c e between mean values (P < 0.05). A pa i r e d t - t e s t was used to compare i n i t i a l c o n t r o l values with subsequent experimental values w i t h i n a S e r i e s . The unpaired t - t e s t was used to compare values between d i f f e r e n t S e r i e s . RESULTS SECTION A: E f f e c t s of acetazolamide and carbonic anhydrase. Data from the acetazolamide and carbonic anhydrase experiments are presented i n Figures 10 and 11, r e s p e c t i v e l y . There were no s i g n i f i c a n t changes i n v e n t i l a t i o n and carbon d i o x i d e and ammonia e x c r e t i o n r a t e s between c o n t r o l I and acetazolamide and between c o n t r o l I I and carbonic anhydrase values. SECTION B: E f f e c t s of methazolamide and a m i l o r i d e . The r e s u l t s of S e r i e s I-IV are presented i n Figures 12-14 and Tables 14 and 15. The i n i t i a l c o n t r o l values of S e r i e s I-IV were not s t a t i s t i c a l l y d i f f e r e n t from each other. In a l l s e r i e s , pHe of the 4 min blood sample (experimental period) was s i g n i f i c a n t l y higher compared to i n i t i a l c o n t r o l values (+0.13 to +0.18 pH u n i t s . F i g . 12 and 14). This blood a l k a l o s i s was a r e s u l t of a r e s p i r a t o r y a l k a l o s i s , because plasma Prj02 decreased while plasma H C O 3 - l e v e l s remained constant. In S e r i e s I where the chemical composition of the water was not a l t e r e d , Mamm decreased during the 30-60 min experimental p e r i o d by 32% r e l a t i v e to the i n i t i a l c o n t r o l r a t e ( F i g 14). Wright and Wood (1985) a t t r i b u t e the step-wise decrease i n ammonia e x c r e t i o n i n a c l o s e d system to the progressive b u i l d up 115 Figure 10. The rate of carbon dioxide excretion (Mcoa) i n umol. k g - 1 . h - 1 , ammonia excretion (MAmm) i n umol. k g - 1 . h - 1 , and the rate of v e n t i l a t o r y water flow (Vw) i n m l . m i n - 1 . f i s h under control (CI) and acetazolamide (A) treatments. Means +. S.E.M. (N=8) M C 0 2 2400 1600 800 M A mm 300 200 100 w 150 100 50 116 C I 117 Figure 11. The rate of carbon dioxide excretion (Mcoz) i n umol. kg - 1.h"~ 1, ammonia excretion (MAmm) i n umol. k g - 1 . h - 1 , and the rate of v e n t i l a t o r y water flow (Vw) i n m l . m i n - 1 . f i s h measurements under control (CII) and carbonic anhydrase (CA) treatments. Means + S.E.M. (N=8) Mco2 3000 T I 1000 -• M A m m 400 - T T 200 200 -T T 100 -C - II 1 CA 119 Figure 12. Blood pH (pHe), t o t a l ammonia concentration (Tamm) i n umol.l -*, and NH3 tensions ( P N H S ) i n uTorr are shown for f i s h under control conditions (•, N=7) and f i s h exposed to methazolamide (A,N=7). Measurements were made under control conditions ( C ) and throughout the experimental period (0-60 min). * denotes s t a t i s t i c a l d i f ference from the i n i t i a l c o n t r o l value ( C ) . Means +_ S • E • M. TIME (min) 121 Figure 13. Blood pH (pHe), total ammonia concentration (Tamm) in umol.l - 1, and NH3 tensions (P N H S) in uTorr are shown for fish exposed to amiloride ( •, N=8) and to amiloride and methazolamide together (A,N=7). Measure-ments were made under control conditions (C) and throughout the experimental period (0-60 min). * denotes s t a t i s t i c a l difference from the i n i t i a l control value (C). Means ± S« £ • M • 122 1 1 1 1 1 1 I C 0 10 20 30 40 50 60 123 Figure 14. Ammonia excretion rates (M A m m) i n umol.kg-1.h _ 1 are shown f o r , control (Series I (N=7)), methazolamide (Series I I (N=7)), amiloride (Series I I I (N=8)), and amiloride + methazolamide (Series IV (N=7)) experiments. Measurements were made under control conditions (C) and during the experimental period (0-30 min, 30-60 min). * denotes s t a t i s t i c a l d ifference from i n i t i a l c ontrol values, • denotes s t a t i s t i c a l d ifference from Series I cont r o l experiment 0-30 min. • denotes s t a t i s t i c a l s i g n i f i -cance from amiloride Series I I I experiment 0-30 min. Means + S.E.M. 800 Amiloride 400 800 Amilorlde+Methazolamldo 400 i • i EIO-30) E130-60) Control « T Mothazolamldo 1 T T C EIO-30) E (30-60) 125 TABLE 14. Carbon d i o x i d e content (Ccoz), C 0 2 t a n s i o n s (Pcoa), and b i c a r b o n a t e l e v e l s (HCO--) i n plasma o f S e r i e s I ( c o n t r o l , N=7) and S e r i e s II (methazolamide, N=7) f i s h under c o n t r o l and experimental c o n d i t i o n s (0-60 min). Means + 1 S.E.M. TIME Cc02 mmol/l SERIES I Pcoa HCOa-Torr mmol/1 C c mmol/1 SERIES II Pct)2 Torr H C O 3 -mmol/l CONTROL 7.8 2.6 7.7 8.3 2.8 8.1 +. 3 +. 2 +. 3 +. 4 +. 2 +..4 EXPERIMENTAL 1 min 7.7 2.8 7.5 8.2 2.7 8.0 +.4 +.2 +.4 +.5 +.2 +.5 4 min 7.3 + . 1 1.9 ±. 2 7.2 + . 1 8.1 + . 3 1.7 + 2 8.0 + . 3 10 min 7.8 + .6 1.6 + .3 7. +. 9.2 + .5 2.8 + . 3 9.0 + .5 30 min 7.6 + .5 2.0 + . 3 7.4 + .5 9.3 + .5 2.3 + .4 9.2 + .5 60 min 7.6 + . 3 2.4 + .2 7.4 + .3 9.3 + .5 2.6 + . 3 9.0 + .5 * s i g n i f i c a n t l y d i f f e r e n t from i n i t i a l c o n t r o l v a l u e , p a i r e d t - t e s t (P < 0.05). A s i g n i f i c a n t l y d i f f e r e n t from S e r i e s I I I va l u e s a t the same time, unpaired t - t e s t (P < 0.05) 1 2 6 TABLE 15. Carbon d i o x i d e c o n t e n t (Ccoa), C 0 2 t e n s i o n s ( P c o z ) , and b i c a r b o n a t e l e v e l s (HC0 3-) i n plasma o f S e r i e s I I I ( a m i l o r i d e c o n t r o l , N=8) and S e r i e s IV ( a m i l o r i d e + metha-zolamide, N=7) f i s h under c o n t r o l and e x p e r i m e n t a l c o n d i t i o n s (0-60 min). Means + 1 S.E.M. TIME Cc02 mmol/l SERIES I I I Pco2 HCOa-To r r mmol/1 SERIES IV HC03-mmol/1 T o r r mmol/1 CONTROL 7.9 3.0 7.7 7.5 2.5 7.4 +.5 +.2 +.5 +.3 +.3 +.3 EXPERIMENTAL 1 min 8.0 2.9 8.1 7.4 2.2 7.3 +.6 +.2 +.6 +.6 +.4 +.6 4 min 7.9 + .5 2.1 + .2 8.2 + .6 7.7 + . 2 1.8 + . 2 7.6 + . 2 10 min 7.8 + .5 2.6 + .3 7.6 + .5 7.2 + .4 2.6 + .2 7. 30 min 7.5 + .4 2.9 + .3 7.3 + .4 7.1 + .3 2.3 + .4 6. +. 60 min 6.7 + .9 3.0 + .6 6.4 + .9 7.1 ±. 3 2.8 + .4 7. * s i g n i f i c a n t l y d i f f e r e n t from i n i t i a l c o n t r o l v a l u e , p a i r e d t - t e s t (P < 0.05). of ammonia l e v e l s i n the water, which r e s u l t s i n reduced blood-to-water ammonia g r a d i e n t s . Plasma Tamm l e v e l s were depressed by 9-12% during the experimental p e r i o d compared to the i n i t i a l c o n t r o l value, while plasma PN ;H3 l e v e l s d i d not change ( F i g 12). There were no s i g n i f i c a n t changes i n C C Q 2 ' H C O 3 - , and Pc02 between c o n t r o l and experimental values (Table 14). With methazolamide i n the water i n S e r i e s I I , plasma Tamm l e v e l s were reduced by 10-12% and there were no changes i n Pj*H3 l e v e l s ( F i g . 12) compared to i n i t i a l c o n t r o l values. Ammonia e x c r e t i o n increased s l i g h t l y with methazolamide, however, t h i s change was not s i g n i f i c a n t ( F i g . 14). Plasma Pc02 tensions were reduced by 40% at the 4 min sample, but returned to i n i t i a l c o n t r o l l e v e l s a f t e r 10 min. This Prj02 l e v e l at 10 min was s i g n i f i c a n t l y greater than the S e r i e s I values at the same time. Plasma Ccc-2 a n d H C O 3 - values were s i g n i f i c a n t l y greater than both i n i t i a l c o n t r o l ( S e r i e s I I ) and Serie s I values, a f t e r 30 min of the methazolamide exposure. In the a m i l o r i d e c o n t r o l experiment. S e r i e s I I I , a m i l o r i d e depressed ammonia e x c r e t i o n ( F i g . 14), although the decrease was not s t a t i s t i c a l l y s i g n i f i c a n t from the i n i t i a l c o n t r o l period. (The decrease was s t a t i s t i c a l l y d i f f e r e n t r e l a t i v e to Se r i e s I (0-30 min)). Concomittant with the decrease i n M^ m m, plasma Tamm l e v e l s p r o g r e s s i v e l y increased with a m i l o r i d e exposure and at 60 min were 32% greater than the i n i t i a l c o n t r o l l e v e l ( F i g . 13). Plasma P N H3 l e v e l s s i g n i f i c a n t l y increased (+69%) at the 4 min sample, due to a combination of blood a l k a l o s i s and 128 elevated plasma Tamm. Pc02 l e v e l s were s i g n i f i c a n t l y reduced at the 4 min sample r e l a t i v e to i n i t i a l c o n t r o l values. There were no changes i n CrjQ2 o r H C O 3 - l e v e l s (Table 15). In S e r i e s IV, the a d d i t i o n of methazolamide and a m i l o r i d e to the water r e s u l t e d i n very s i m i l a r trends to those observed i n Se r i e s I I I , however, the changes were l e s s marked. The only s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between S e r i e s I I I and IV data was that i n S e r i e s IV, Mamm was 50% greater i n the f i r s t 30 min of the experimental p e r i o d ( F i g . 14). C r j 0 2 ' H C O 3 - , and PfjQ2 were unaltered with the a m i l o r i d e and methazolamide treatment (Table 15). DISCUSSION Pe n e t r a t i o n of CA i n h i b i t o r s Carbonic anhydrase i n h i b i t o r s , such as acetazolamide and methazolamide, are permeable to c e l l membranes. One of the d i f f i c u l t i e s i n adding CA i n h i b i t o r s to the e x t e r n a l water i s that these drugs may enter the f i s h and CA i n h i b i t i o n w i t h i n the f i s h may confound the i n t e r p r e t a t i o n of the r e s u l t s . Henry and Cameron (1983) have measured the time f o r 95% i n h i b i t i o n of b r a n c h i a l e p i t h e l i u m CA i n crabs a f t e r an i n j e c t i o n of acetazolamide and found that there was a l a g of about 4 h. They speculated that the l a g time was equal to the time needed f o r the drug to cross the basal membrane of the g i l l and a f f e c t i n h i b i t i o n . Acetazolamide added to the e x t e r n a l water acheives e q u i l i b r i u m between water and haemolymph of crab i n about 1 h (Burnett g£ aL., 1981). In Se c t i o n A experiments, acetazolamide (1.6 mmol.l -!) was added to the water r e s e r v o i r supplying the flow-through experimental chamber, f o r 30 min p r i o r to sampling. The p e r m e a b i l i t y of g i l l s i s s i g n i f i c a n t l y l e s s i n freshwater than i n seawater (see G i r a r d & Payan, 1980) and t h e r e f o r e , one would expect acetazolamide to d i f f u s e slower across freshwater t r o u t g i l l s compared to seawater crab g i l l s . I f some acetazolamide d i d d i f f u s e i n t o g i l l t i s s u e or the blood, there was no apparent e f f e c t on C62 e x c r e t i o n across the g i l l . Complete i n h i b i t i o n of g i l l e p i t h e l i u m CA does not a f f e c t CO2 130 e x c r e t i o n , while complete i n h i b i t i o n of red c e l l CA causes an increase i n plasma Pc02' s u f f i c i e n t to maintain steady-state CO2 e x c r e t i o n (Swenson & Maren, 1987). There was no e l e v a t i o n of plasma Pc02 during 60 min exposure to methazolamide (Section B). Methazolamide d i f f u s e s more q u i c k l y i n t o t i s s u e s than acetazolamide (Maren, 1967), and t h e r e f o r e , i t can be assumed that acetazolamide d i d not i n h i b i t red c e l l carbonic anhydrase i n the S e c t i o n A experiments. 0 E f f e c t s of Acetazolamide and Carbonic Anhydrase on MQ02 a n d A^mm Acetazolamide or carbonic anhydrase exposure i n t r o u t d i d not a l t e r b r a n c h i a l carbon d i o x i d e or ammonia e x c r e t i o n . The theory proposed was that carbonic anhydrase i n the e x t e r n a l water boundary l a y e r f a c i l i t a t e s the d i f f u s i o n of CO2 and NH3 from the blood compartment to the e x t e r n a l water environment. The r a p i d conversion of excreted CO2 to H C O 3 - and H+ ions i n the g i l l water boundary l a y e r would lower water CO2 l e v e l s and serve to enhance blood-to-water Pc02 d i f f u s i o n g r a d i e n t s . In a d d i t i o n , c a t a l y s e d CO2 h y d r a t i o n i n the boundary l a y e r would f a c i l i t a t e NH3 d i f f u s i o n by p r o v i d i n g H+ ions to combine with NH3 to form N H 4 + , thereby ch e m i c a l l y removing NH3 from the boundary water l a y e r , and enhancing blood-to-water Pj*H3 d i f f u s i o n g r a d i e n t s . There are three p o s s i b l e explanations f o r the observed r e s u l t s . F i r s t , acetazolamide or carbonic anhydrase d i d not contact CA at the e x t e r n a l surface of the g i l l e p i t h e l i u m . Second, carbon d i o x i d e and ammonia e x c r e t i o n are not d i f f u s i o n l i m i t e d under the c o n d i t i o n s t e s t e d i n t h i s study. The t h i r d p o s s i b l i l i t y i s that carbon d i o x i d e and ammonia e x c r e t i o n are d i f f u s i o n l i m i t e d , but compensatory mechanisms were q u i c k l y m o b i l i z e d which masked any e f f e c t s of acetazolamide and carbonic anhydrase at the time of sampling, 30 min l a t e r . F i s h were exposed to acetazolamide or carbonic anhydrase for 30 min, which i s probably s u f f i c i e n t time f o r d i f f u s i o n of these drugs i n t o the g i l l water boundary l a y e r given the r a t e of v e n t i l a t o r y flow (-100-170 ml.min -*) and the r e l a t i v e l y high drug concentrations i n the water (1.6 mmol.l -*, acetazolamide and 6,8 mg.l -*, carbonic anhydrase). Moreover, acetazolamide treatment r e s u l t e d i n an increase i n the pH d i s e q u i l i b r i u m i n downstream water (see Chapter 2), implying that CA l o c a t e d on the e x t e r n a l g i l l surface was i n h i b i t e d . I t i s p o s s i b l e that carbon d i o x i d e and ammonia e x c r e t i o n are not d i f f u s i o n l i m i t e d under the experimental c o n d i t i o n s of the present study. Carbon d i o x i d e t r a n s f e r i s l i m i t e d by the r a t e of C I - / H C O 3 - exchange at the red c e l l membrane, and t h e r e f o r e , there i s an inverse r e l a t i o n s h i p between hct and MrjQ2 (Perry §_t a l . . 1982). Increases i n blood H C O 3 - , however, r e s u l t i n increases i n Mco2 (hct= 11%, Perry e_t a l . . , 1982), i n d i c a t i n g that b r a n c h i a l CO2 t r a n s f e r i s p r i m a r i l y p e r f u s i o n l i m i t e d at higher hct values. I t was suggested by Zborowska-Sluis ejt a l . 1 3 2 ( 1 9 7 4 ) that the f u n c t i o n of e x t r a v a s c u l a r CA was to f a c i l i t a t e CO2 t r a n s p o r t from muscle c e l l s during periods of high muscular a c t i v i t y when CO2 production r a t e s are maximal. During r e s t i n g c o n d i t i o n s , as i n the present study, t h e o r e t i c a l c o n s i d e r a t i o n s suggest that CO2 ( P i i p e r & Scheid, 1 9 8 2 ) and ammonia t r a n s f e r are p e r f u s i o n l i m i t e d . I t may be, however, that under extreme c o n d i t i o n s , such as, high carbon d i o x i d e and ammonia production, elevated blood catecholamine l e v e l s , and water c o n d i t i o n s that reduce blood-to-water d i f f u s i o n g r a d i e n t s , d i f f u s i o n l i m i t a t i o n s may become s i g n i f i c a n t and f a c i l i t a t i o n of CO2 and NH3 t r a n s f e r by e x t e r n a l g i l l CA may play an important r o l e . Whole animal physiology i s o f t e n d i f f i c u l t because of the complexity of p h y s i o l o g i c a l problems due to the multitudinous i n t e r a c t i o n s between body systems. For inst a n c e , i f ammonia e x c r e t i o n i s d i f f u s i o n l i m i t e d , then the proposed theory suggests that i n h i b i t i o n of e x t e r n a l g i l l CA would reduce the a v a i l i b i l i t y of H + ions i n the boundary l a y e r , reduce the r a t e of chemical removal of NH3 from the boundary l a y e r , r a i s e water NH3 l e v e l s , and decrease the blood-to-water P N H 3 gradient. The net r e s u l t would be a decrease i n t o t a l ammonia e x c r e t i o n . In a steady s t a t e system, however, blood-to-water P{*H3 gradients are probably q u i c k l y r e - e s t a b l i s h e d at higher blood Pj*H3 l e v e l s , which would maintain ammonia e x c r e t i o n across the g i l l s . There are other mechanisms which may act to compensate f o r d i f f u s i o n l i m i t a t i o n s . For instance, increases i n g i l l p e r m e a b i l i t y to N H 3 , C O 2 , H + , or NH4+ would a l l play a r o l e i n maintaining 133 ammonia e x c r e t i o n i n the face of an i n i b i t i o n of e x t e r n a l g i l l c arbonic anhydrase. Hormones, such as a d r e n a l i n e , s e l e c t i v e l y increase g i l l c e l l membrane p e r m e a b i l i t y to l i p o p h i l i c substances ( I s a i a , 1984), such as, NH3 and C O 2 . An increase i n CO2 or H+ i o n e x c r e t i o n would s u s t a i n H+ i o n concentrations i n the boundary l a y e r i n a s i t u a t i o n where the CO2 h y d r a t i o n r e a c t i o n was uncatalysed, thereby maintaining P(JH3 g r a d i e n t s across the g i l l s . CO2 e x c r e t i o n was unchanged i n the acetazolamide experiment, however, which r u l e s out t h i s p o s s i b i l i t y . F i n a l l y , an increase i n NH4 + e x c r e t i o n ( v i a the Na+/NH4+ exchange) may occur i n s i t u a t i o n s where NH3 d i f f u s i o n g r a d i e n t s are l e s s favourable (Wright & Wood, 1985). S i m i l a r arguements can be made to e x p l a i n the apparent l a c k of e f f e c t of acetazolamide and CA treatments on carbon d i o x i d e e x c r e t i o n . E f f e c t s of Methazolamide on plasma ammonia l e v e l s and M^ m m Experiments i n t h i s s e c t i o n focused s p e c i f i c a l l y on whether e x t e r n a l g i l l CA acts to f a c i l i t a t e ammonia e x c r e t i o n . These experiments were designed to avoid some of the c o m p l i c a t i n g f a c t o r s inherent i n the design of S e c t i o n A experiments, such as, the 30 min delay f o l l o w i n g exposure to drug, p r i o r to sampling, and NH4+ e x c r e t i o n as an a l t e r n a t e pathway fo r ammonia e x c r e t i o n . Despite these m o d i f i c a t i o n s i n experimental design, i n h i b i t i o n of e x t e r n a l g i l l CA d i d not a l t e r t o t a l ammonia e x c r e t i o n , as expected. Methazolamide added to the water 134 increased the d i f f u s i v i t y of the g i l l s to NH3 (DjjH3 = A^mm/^ NH3 gradient blood-to-water) by about 20% i n the f i r s t 30 min of exposure (4.6 vs 5.4 umoLkg -l.h~l.uTorr -l, S e r i e s I I ) . This increase i n DJJH3 i s unexpected because an i n h i b i t i o n of e x t e r n a l g i l l CA should e l e v a t e water boundary l a y e r pH, elevate NH3 l e v e l s i n the water, and i n the shorterm, reduce NH3 d i f f u s i o n across the g i l l . One p o s s i b l e e x p l a n a t i o n f o r these r e s u l t s i s that a s t i m u l a t i o n of the Na+/NH4+ exchange maintained t o t a l ammonia e x c r e t i o n i n the face of l e s s favourable blood-to-water PfcjH3 g r a d i e n t s . A l k a l i n e waters increase the rat e of Na+/NH4+ exchange r e l a t i v e to NH3 d i f f u s i o n (Wright & Wood, 1985), which i s c o n s i s t e n t with the proposed increase i n boundary water pH with an i n h i b i t i o n of e x t e r n a l g i l l CA. I t should be mentioned, however, that some s t u d i e s r e p o r t an i n h i b i t i o n of Na+ uptake with the CA i n h i b i t o r , acetazolamide (Maetz & Gar c i a Romeu, 1964; Henry & Cameron, 1983), which would argue agai n s t an increase i n Na+/NH4+ exchange i f methazolamide act s i n a s i m i l a r f a s h i o n . To c l a r i f y the r o l e of Na+/NH4+ i n ammonia e x c r e t i o n during methazolamide treatment i t i s of value to look at the experiments where a m i l o r i d e was a l s o added to the water. Am i l o r i d e decreased M A m m by 22% (am i l o r i d e c o n t r o l , mean value over 60 min.), a f i g u r e c o n s i s t e n t with 23% reductions i n i n t a c t rainbow t r o u t (Wright & Wood, 1985) and 30% reductions reported f o r perfused i n v i t r o rainbow t r o u t preparations (Kirschner §_t 3 ! . ., 1973; Payan, 1978). In the present study. 1 3 5 a m i l o r i d e r a p i d l y and p r o g r e s s i v e l y increased plasma Tamm and P{jH3 l e v e l s , r e s u l t i n g i n a 5 0 % r e d u c t i o n i n DJJH3 over the f i r s t 3 0 min. I f i t i s true that the increase i n D [*H3 with methazolamide treatment ( S e r i e s I I ) was due to a s t i m u l a t i o n of Na+/NH4+ exchange, then by adding a m i l o r i d e together with methazolamide D N H3 should equal or be l e s s than the value with a m i l o r i d e alone. This was not the case, D J J H 3 with both drugs i n the water only decreased by 2 0 % ( 5 . 3 vs 4 . 3 u m o l . k g - 1 . h - 1 . u T o r r - 1 ) , compared to the 5 0 % r e d u c t i o n with a m i l o r i d e alone. Hence, the increase i n D J J H 3 with methazolamide cannot be explained by a s t i m u l a t i o n of Na+/NH4+ exchange, and i s due to unknown f a c t o r s . The blood a l k a l o s i s which was observed i n a l l s e r i e s of experiments (4 min, experimental sample) dominated any changes i n blood parameters and complicated i n t e r p r e t a t i o n of the r e s u l t s . The r e s p i r a t o r y a l k a l o s i s (decrease i n Pc02' n o change i n HCO3-) was l i k e l y due to h y p e r v e n t i l a t i o n as a r e s u l t of r a p i d responses ( < 3 min) to blood volume changes. H y p e r v e n t i l a t i o n immediately f o l l o w s the removal of ~ 0 . 5 ml of blood from the d o r s a l a o r t a of cannulated t r o u t under s i m i l a r c o n d i t i o n s (S.F. Perry, personnal communication). The presence and l o c a t i o n of baroreceptors i n f i s h are unknown, but haemorrhage r e s u l t s i n an increase i n r e n i n a c t i v i t y i n f i s h ( B a i l e y & R a n d a l l , 1 9 8 1 ) , as i n other v e r t e b r a t e s . The r e n i n - a n g i o t e n s i n system e f f e c t s many changes i n the body, and i n the present study these changes may be r e l a t e d to the increase i n v e n t i l a t i o n . 136 To summarize, the a d d i t i o n of acetazolamide and carbonic anhydrase to the water d i d not a l t e r CO2 and ammonia e x c r e t i o n i n v i v o . The i n t e r p r e t a t i o n of these r e s u l t s i s complicated by the f a c t that the i n t e r v a l of time p r i o r to sampling may have been too long to observe any short term e f f e c t s of these drugs. Methazolamide r e s u l t e d i n an increase i n the d i f f u s i n g c a p a c i t y of ammonia, unrel a t e d to NH4 + e f f l u x v i a Na+7NH4 + exchange. Rapid s e r i a l blood sampling r e s u l t e d i n a u n i v e r s a l blood a l k a l o s i s , probably as a consequence of h y p e r v e n t i l a t i o n . Because blood pH was not constant during the experiment i t was impossible to determine the v a l i d i t y of the proposed hypothesis. I t i s concluded that the i n t a c t r e s t i n g animal i s un s u i t a b l e for studying the i n t e r a c t i o n between water boundary l a y e r chemistry and e x c r e t i o n across the g i l l . 137 CHAPTER 4 The l i n k a g e between carbon d i o x i d e and ammonia e x c r e t i o n i n the i s o l a t e d , blood-perfused t r o u t head pre p a r a t i o n . 138 INTRODUCTION Transport across e p i t h e l i a l surfaces i s i n f l u e n c e d by the presence of boundary l a y e r s a s s o c i a t e d with the e p i t h e l i u m (Knepper e t a l , , 1984, 1985; Arruda & Dytko, 1985). Boundary l a y e r s are considered " u n s t i r r e d l a y e r s " , and therefore the chemical composition of the boundary l a y e r i s d i s t i n c t from the bulk phase because m a t e r i a l s that are excreted i n t o the boundary l a y e r only s l o w l y d i f f u s e i n t o the bulk phase. E x t e r n a l boundary l a y e r s next to the f i s h g i l l e p i t h e l i u m e x i s t because water flow between g i l l l a mellae i s slow and the p a t t e r n of water flow through the mouth and over the g i l l s i s laminar, rather than t u r b u l e n t (Scheid & P i i p e r , 1971; P i i p e r gjt a l . , 1986). A s i g n i f i c a n t component of ammonia i s e l i m i n a t e d at the g i l l of freshwater f i s h as N H 3 , by d i f f u s i o n down the blood-to-water PNJH3 gradient (deVooys, 1968; Cameron & H e i s l e r , 1983; Wright & Wood, 1985; Cameron, 1986). NH3 l e v e l s may b u i l d up i n the g i l l water boundary l a y e r unless d i f f u s i o n out of the boundary l a y e r i n t o the bulk water i s r a p i d or NH3 i s removed by other means. The e x c r e t i o n of carbon d i o x i d e and subsequent c a t a l y s e d h y d r a t i o n to H C O 3 - and H+ may ensure c o n t i n u a l a v a i l i b i l i t y of H+ ions i n the boundary l a y e r to combine with NH3 to form NH4+. This chemical removal of NH3 would enhance NH3 t r a n s f e r and f u n c t i o n a l l y l i n k carbon d i o x i d e and ammonia e x c r e t i o n across the g i l l ( F i g . 15). To i n v e s t i g a t e the p o s s i b l e l i n k a g e of CO2 and ammonia 139 Figure 15. A s i m p l i f i e d cross-section through the g i l l epithelium showing the bulk water flow, the boundary water layer and associated mucus layer containg carbonic anhydrase molecules represented by The catalysed hydration of C0= i n the boundary layer to HC0 3- + H+ f a c i l i t a t e s ammonia excretion by promoting the interconversion of NH 3 to NH-»+ thereby preventing reductions i n the trans-e p i t h e l i a l NHs d i f f u s i o n gradient due to accumulation of NH 3. The thickness of the arrows denotes the magnitude of the p a r t i c u l a r process i l l u s t r a t e d (not p r e c i s e l y drawn to s c a l e ) . r B U L K BOUNDARY WATER G ILL WATER FLOW L AYE R + MUCUS E P I T H E L I U M L A Y E R B L O O D V Jf N H H C O g <-H + N H c°2 4 H C O , + • H N H 3 ^ N H 1 ] C O N H 1 -U o 141 e x c r e t i o n at the g i l l i t was necessary to employ an XlL v i t r o p r e p a r a t i o n i n which the independent v a r i a b l e s could be p r e c i s e l y c o n t r o l l e d . In chapter 3, i t was impossible to determine whether or not carbonic anhydrase present on the e x t e r n a l surface of the g i l l f a c i l i t a t e d carbon d i o x i d e and/or ammonia e x c r e t i o n because of the d i f f i c u l t i e s i n studying boundary l a y e r phenomena i n i n t a c t animals. With the blood-perfused t r o u t head p r e p a r a t i o n the convective components of gas exchange (blood flow r a t e through the g i l l s and water flow r a t e over the g i l l s ) , as w e l l as the chemical composition of the blood perfusate and v e n t i l a t o r y water, could be c o n t r o l l e d . Perfused preparations are valuable i n e l u c i d a t i n g mechanisms which cannot be e f f e c t i v e l y examined i n the i n t a c t animal. The primary c r i t e r i o n by which to judge a p r e p a r a t i o n i s i t s a b i l i t y to match the p h y s i o l o g i c a l response as i t occurs j j i v i v o . To evaluate the p h y s i o l o g i c a l performance of the blood-perfused t r o u t head p r e p a r a t i o n , blood and water v a r i a b l e s and gas exchange r a t e s were compared between in. v i v o and in. v i t r o p r eparations. MATERIALS & METHODS Experiments were d i v i d e d i n t o two s e c t i o n s . Section A describes the blood-perfused t r o u t head p r e p a r a t i o n used to i n v e s t i g a t e the l i n k a g e between CO2 and NH3 i n the g i l l water boundary l a y e r . S e c t i o n B i n v i v o experiments were designed to emulate the c o n d i t i o n s of the blood-perfused head experiment, so that comparisons between i n v i v o and i n v i t r o parameters could be made. Experimental animals Rainbow t r o u t (Salmo g a i r d n e r i ) of both sexes were obtained from T h i s t l e Springs Trout Farm (Ashton, Ontario) and transported to the U n i v e r s i t y of Ottawa. F i s h were held i n indoor rectangular f i b e r g l a s s tanks s u p p l i e d with f l o w i n g , aerated and d e c h l o r i n a t e d tapwater (pH= 7.0-8.0, [Na+]= 0.10; [Cl-]= 0.10; [Ca++]= 0.35; [K+]= 0.03 m mol.l - 1, temperature= 6-9° C). F i s h were fed a d a i l y d i e t of commercial t r o u t p e l l e t s . S e c t i o n A: Blood-perfused t r o u t head p r e p a r a t i o n Blood c o l l e c t i o n and p r e p a r a t i o n I s o l a t e d t r o u t heads were perfused with whole blood, c o l l e c t e d from f i s h with d o r s a l a o r t i c cannulae implanted 24 h e a r l i e r (see General M a t e r i a l s and Methods). Blood was c o l l e c t e d immediately p r i o r to each experiment. In g e n e r a l , 6-7 cannulated donor f i s h provided enough blood for one perfused head experiment. To increase the volume of blood removed from each doner f i s h , 2-3 ml of h e p a r i n i z e d s a l i n e (ammonium heparin, Sigma 10 u n i t s . m l - 1 ) was i n i t i a l l y i n j e c t e d i n t o each f i s h v i a the d o r s a l a o r t i c cannulae. A f t e r a short time to allow f o r mixing (approx. 1-2 min), blood was withdrawn and pooled. In most cases, 5 to 10 ml of blood could be c o l l e c t e d from each f i s h by t h i s method. In some cases, i t was necessary to augment the t o t a l blood volume for a s i n g l e experiment (50-60 ml) by adding small volumes (3-5 ml) of blood removed by e x t e r n a l puncture from the caudal v e i n - a r t e r y of a few uncannulated f i s h . The haematocrit (hct) of the pooled blood was measured (mean hct= 17.1% +. 0.5) and adjusted to 13-15% (mean= 14.4% £ 0.3) with Cortland s a l i n e (Wolf, 1963) c o n t a i n i n g 2.2% Bovine Serum Albumin (BSA, Sigma). In t r i a l experiments the hct of the pooled blood was adjusted to 10%; however, we found that gas exchange across the g i l l s was c o n s i d e r a b l y l e s s than at a hct of 13-15 %, and the higher hct was adopted. The extent of d i l u t i o n depended on the hct of the pooled donor blood, but plasma osmotic pressure was l i k e l y constant i n a l l experiments since 2.2% Bovine Serum Albumin (BSA, Sigma) was present i n the s a l i n e . In p r e l i m i n a r y experiments, we a l s o found that the a d d i t i o n of the c o l l o i d osmotic f i l l e r , p o l y v i n y l p y r r o l i d o n e (see Perry e_t ai.., 1984a), to the s a l i n e i n place of BSA r e s u l t e d i n extremely high blood pressure i n the 144 i s o l a t e d head pr e p a r a t i o n . Thus, s a l i n e which was used to d i l u t e the blood as w e l l as perfuse the head on the operating t a b l e (see below), only contained BSA. D i l u t e d blood was d i v i d e d i n t o two r e s e r v o i r s and g e n t l y s t i r r e d f o r 90 min i n an i c e bath. The blood was e q u i l i b r t a e d with 0.4% CO2 and 4% 0 3 , balanced with N2 ( PC02 3 Torr, P Q2 30 T o r r ) , for 90 minutes. Blood was gassed f o r 90 min with i d e n t i c a l gas mixtures to simulate venous blood gas tensions. S u r g i c a l procedure f o r preparing i s o l a t e d perfused head Trout (216-353g, mean weight 269g + 6, N = 31) were removed from holding tanks and immediately i n j e c t e d with 1 ml of s a l i n e c o n t a i n i n g 2500 u n i t s . m l - 1 ammonium heparin. F i s h were l e f t f o r approximately 10 minutes to ensure complete mixing of the heparin i n the body, and then removed from the water and q u i c k l y decapitated by c u t t i n g 2cm p o s t e r i o r to the p e c t o r a l f i n s . The head (mean weight 74 g +1) was immediately t r a n s f e r r e d to the operating t a b l e where a tube c o n t a i n i n g flowing tapwater was placed i n the mouth i n order to i r r i g a t e the g i l l s . The pericardium was q u i c k l y torn and an i n c i s i o n was made i n the v e n t r i c l e muscle so as to prevent a i r being pumped i n t o the g i l l v a s c u l a t u r e . The v i s c e r a were removed from the body c a v i t y and a s a l i n e - f i l l e d 4 cm piece of PE50 tubing was introduced i n t o the bulbous a r t e r i o s u s by way of the v e n t r i c u l a r i n c i s i o n and secured i n place with s u r g i c a l s i l k . Next a 3 cm piece of PE 160 tubing was placed i n the d o r s a l aorta (DA), and blood was c l e a r e d from the g i l l s by p e r f u s i n g i n the retrograde d i r e c t i o n f o r approximately 5 sec with f i l t e r e d ( M i l l i p o r e 0.45 urn) s a l i n e c o n t a i n i n g 2.2% BSA and 10~ 7 M epinephrine (Sigma) at a constant pressure of 50 cm H2O (see Appendix I f o r s a l i n e pH, Tamm, C C Q 2 , and p r o t e i n l e v e l s ) . P e r f u s i n g i n the retrograde d i r e c t i o n c l e a r e d any a i r bubbles trapped i n the v e n t r a l a orta (VA). The d i r e c t i o n of p e r f u s i o n was then switched to orthograde by connecting the s a l i n e r e s e v o i r with the VA catheter for the remainder of the s u r g i c a l procedure. The g i l l s were ischemic f o r no longer than 2 minutes. Next the esophagus was f i r m l y l i g a t e d to prevent water contamination i n the body c a v i t y . The body w a l l was then supported by s t i t c h i n g a p l a s t i c s e m i c i r c u l a r c o l l a r (made from a 60 ml disposable shringe) to the i n s i d e of the v e n t r a l body w a l l . The DA catheter was secured by c u t t i n g a h a l f c i r c l e i n the surrounding muscle and s l i p p i n g a piece of s i l k , i n the i n c i s i o n and t y i n g the s i l k , f i r m l y arround the t i s s u e and c a t h e t e r . This completed the c a r d i o v a s c u l a r surgery i n approximately 8 min. Opercular cannulae were a f f i x e d to the i s o l a t e d head i n order to sample mixed ex p i r e d water downstream from the g i l l (see General M a t e r i a l s and Methods). A small tube (Tygon tubing, 0.5 cm diameter) was then s t i t c h e d to the f l o o r of the buccal c a v i t y and the tongue j u s t at the opening of the mouth to ensure proper water flow over the g i l l s during the subsequent experiment. The mouth was then sealed shut around t h i s tube by two l a r g e s t i t c h e s 146 between the upper and lower jaw on e i t h e r s i d e . The opercular openings were l o o s e l y c l o s e d by two s t i t c h e s along the v e n t r a l openings to prevent the opercular valve from f l a r i n g open and r e s u l t i n g i n poor v e n t i l a t o r y flow through the g i l l arches. D i f f e r e n t methods of g i l l i r r i g a t i o n were i n i t i a l l y t r i e d , f or example, an i n f l o w tube was simply placed i n the f i s h ' s mouth without securing the mouth or the opercular valves c l o s e d . This method has been widely used i n other i s o l a t e d head s t u d i e s (eg.. Perry & Wood, 1985; Perry a l . . , 1984c; Bornancin §_t al.., 1985), however, we found that water tended to flow back, out of the mouth and what water d i d pass through the opercular c a v i t y d i d not flow i n a uniform p a t t e r n over the g i l l s . F i n a l l y , a rubber membrane (condom) was f i t t e d around the head by f i r s t making a groove on the e x t e r n a l surface with a heavy piece of s i l k t i e d snuggly around the f i s h , p o s t e r i o r to the p e c t o r a l f i n s . This rubber s e a l prevented mixing of the blood perfusate with the v e n t i l a t o r y water once the f i s h was placed i n the experimental apparatus. Surgery was then complete i n a t o t a l time of approximately 15 minutes. The g i l l s were v i s u a l l y examined to assess the degree of blood clearance and i f l e s s than 75%, the pr e p a r a t i o n was discarded. Experimental apparatus The i s o l a t e d head was immediately t r a n s f e r e d to the small p l a s t i c chamber shown i n the i n s e t of Figure 16 and the condom 147 F i g u r e 16. The e x p e r i m e n t a l a p p a r a t u s f o r the i s o l a t e d head p r e p a r a t i o n i s shown. The i n s e t i l l u s t r a t e s a c l o s e - u p o f the head which was p l a c e d i n the p l a s t i c e x p e r i m e n t a l chamber a f t e r s u r g e r y and s e a l e d w i t h a condom. The placement o f the d o r s a l a o r t i c (DA), venous ( i n p u t ) , and o p e r c u l a r c a n n u l a e i s shown. BR= b l o o d r e s e r v o i r , MS= magnetic s t i r r e r s , CP= c a r d i a c pump, S= s a l i n e , V= one-way v a l v e s , Wk= Wind-k e s s e l , PT= p r e s s u r e t r a n s d u c e r , CR= c h a r t r e c o r d e r , IC= i n p u t c a n n u l a e , DAC= d o r s a l a o r t i c c a n n u l a , 0C= o p e r c u l a r c a n n u l a e , EW or MEW= mixed e x p i r e d water, IW= i n s p i r e d water, WP= water pump. was f i t t e d around the outside of the chamber to make a t i g h t s e a l . Water flow (Vw) over the g i l l s was set to approx. 840 m l . m i n - 1 . k g - 1 (water temp.= 8° C). Tapwater was a i r e q u i l i b r a t e d to ensure that water carbon d i o x i d e l e v e l s were i n e q u i l i b r i u m between water and a i r . The g i l l s were perfused at constant p u l s a t i l e flow (blood flow r a t e , Vb= 11 ml.min -*.kg - 1) with gas e q u i l i b r a t e d f i s h whole blood from one of the two blood r e s e r v o i r s . For d e t a i l s of the modified c a r d i a c pump r e f e r to Davie and Daxboeck (1983). Input ( a f f e r e n t ) pressure ( P i ) was monitored v i a a T-junction i n the input catheter connected to a pressure transducer ( B e l l and Howell) and d i s p l a y e d on a Harvard chart recorder. Pulse pressure was kept constant at 10 cm H2O by a d j u s t i n g the s i z e of a gas space at the top of a wide-bore side-arm (Windkessel) i n the p e r f u s i o n l i n e . The pressure drop across the input catheter was measured with l i g a t u r e s s t i l l i n place a f t e r each experiment. This catheter r e s i s t e n c e (Rs= 13 +, 1 (N=18)) was subtracted from measured t o t a l input pressure (Pt= 81 +_ 3 (N=18)) for each p r e p a r a t i o n to determine c o r r e c t e d input pressure (Pi= 69 + 4 cm H2O (N=18)). Dorsal a o r t i c pressure (Pda) was maintained between 10 and 15 cm H2O (Perry gt §1., 1985a). To t e s t whether water flow was evenly d i s t r i b u t e d between both g i l l s , a non-toxic dye (food c o l o u r ) was added to the i n f l o w water, and the p a t t e r n of flow from each opercular opening was v i s u a l l y asssesed at the onset and t e r m i n a t i o n of each experiment. Preparations were discarded i f the water flow 150 p a t t e r n was not approximately matched on both s i d e s . The head was removed from the chamber at the end of the experiment, and the g i l l s were examined once again to ensure that the p e r f u s i o n reached at l e a s t 75 % of the t i s s u e . The placement of the mouth tube was a l s o checked, along with the p o s i t i o n of the opercular c a t h e t e r s . Experimental P r o t o c o l and measurements Each experiment involved two sets of p a i r e d blood ( a r t e r i a l and venous) and water ( i n s p i r e d and mixed expired) samples. The f i r s t samples ( c o n t r o l ) were c o l l e c t e d 7 minutes a f t e r blood flow was i n i t i a t e d i n the head pr e p a r a t i o n . At 8 min (elapsed time) the blood flow was d e r i v e d from the second blood r e s e r v o i r and depending on the experiment, the chemical composition of the i n f l o w water was a l t e r e d . The second samples (experimental), were c o l l e c t e d 7 min l a t e r or a f t e r 15 min of t o t a l elapsed time. The measurement times were chosen to allow adequate time for s t a b i l i z a t i o n of the head p r e p a r a t i o n (based on p e r f u s i o n pressure measurements) a f t e r the i n i t i a t i o n of blood flow and a f t e r a change i n the composition of blood or water. Four experimental treatments were performed: 1. In the f i r s t experimental treatment the chemical composition of the blood and water was not a l t e r e d ( c o n t r o l ) to provide b a s e l i n e e x c r e t i o n r a t e s , as w e l l as determining the s t a b i l i t y of 151 the head p r e p a r a t i o n over the d u r a t i o n of the experiment. 2 . In subsequent experiments i t was important to i s o l a t e NH3 from NH4+ e x c r e t i o n i n order to determine i f NH3 d i f f u s i o n across the g i l l was l i n k e d to CO2 e x c r e t i o n . In the second experiment, r e f e r r e d to as a m i l o r i d e c o n t r o l , the Na + uptake b l o c k e r , a m i l o r i d e ( 1 0 - 4 M) was added to the water i n both the c o n t r o l and experimental periods to i n h i b i t the e x c r e t i o n of the N H 4 + , v i a the Na+/NH4+ (H+) i o n exchange mechanism (e.g. Kirschner e_t al.. , 1973; Wright & Wood, 1985). In freshwater f i s h , passive NH4+ f l u x across the g i l l i s of minor q u a n t i t a t i v e importance (Kormanik & Cameron, 1981a; Wright & Wood, 1985). The chemical composition of the blood was not a l t e r e d . 3. To determine a p o s s i b l e l i n k between CO2 e x c r e t i o n and NH3 d i f f u s i o n i n the t h i r d experiment one of the two blood r e s e r v o i r s was incubated (90 min) with the carbonic anhydrase i n h i b i t o r , acetzolamide (10~4 M) (Maren, 1977), i n order to i n h i b i t carbon d i o x i d e e x c r e t i o n across the g i l l s . 4 . To t e s t whether the p o s s i b l e l i n k between CO2 and ammonia e x r e t i o n was due, i n f a c t , to chemical r e a c t i o n s i n the boundary l a y e r , a f i n a l experiment was performed i n which the b u f f e r i n g c a p a c i t y of ex p i r e d water was r a i s e d by adding T r i s b u f f e r to the water during the second h a l f of the experiment. A combination of Trizma base (C14H11NO3, Sigma) and Trizma hydrochloride ( C 4 H 1 2 C 1 N 0 3 ' Sigma) was used to make a 4 x 1 0 - 3 M s o l u t i o n at pH= 8 . 0 . Blood pH, oxygen t e n s i o n (Po2^ a n d t o t a l O2 content (CQ2)»- carbon d i o x i d e content (Crjo2)' plasma Tamm, and hct were measured on both a r t e r i a l and venous blood samples, along with Vb and p e r f u s i o n pressure. Plasma epinephrine l e v e l s were measured on venous samples ( 1 . 8 x 1 0 - 7 M +.0.2 x 1 0 ~ 7 ) . Blood pH and P Q 2 were measured with a Radiometer Blood Microsystem (BMS3 Mk2) and as s o c i a t e d acid-base analyser (PHM 7 1 ) , maintained at the experimental temperature. C Q 2 was determined by the Tucker method (Tucker, 1 9 6 7 ) . G Q O 2 w a s measured using a t o t a l CO2 analyser (Corning). Plasma Tamm l e v e l s were determined e n z y m a t i c a l l y as described by Kun & Kearney ( 1 9 7 1 ) . Plasma samples were frozen i n l i q u i d N2 and stored at - 8 0 C for l a t e r determination of epinephrine l e v e l s with a high pressure l i q u i d chromatograph (Woodward, 1 9 8 2 ) . Vb was measured at the beginning and the end of each experiment and these values were averaged, as there were only very small d i f f e r e n c e s between them (< 5%). As w e l l , the s p e c i f i c g r a v i t y (or r e f r a c t i v e index) of plasma was determined with a refractometer ( S c i e n t i f i c Instruments) to assess any d i l u t i o n of the blood by water contamination (2.3% +. 0.0 ( N = 2 3 ) ) . The ra t e s of oxygen uptake (M02) a n d carbon d i o x i d e (HQ02^ a n d ammonia (Mft m m) e x c r e t i o n were determined by the F i c k p r i n c i p l e , where 153 a r t e r i a l - v e n o u s d i f f e r e n c e s were m u l t i p l i e d by Vb. At the same time that blood samples were c o l l e c t e d , i n s p i r e d water pH (pHj) and mixed expired pH (prig) were measured with a F i s c h e r Accumet pH meter (829MP). V e n t i l a t o r y water flow (Vw) was determined by c o l l e c t i n g mixed expired water l e a v i n g the head chamber i n a given period of time and accounting for the small flow (~30 ml.min -*.kg~l) through the opercular cannula. I n s p i r e d and mixed exp i r e d water samples were a l s o c o l l e c t e d f o r t o t a l carbon d i o x i d e and ammonia l e v e l s , as w e l l as oxygen tensions. These values, along with Vw were used to c a l c u l a t e MC02' MAmm' a n d M02 b v t n e F i c k p r i n c i p l e . Although the • • • trends i n the MQ02' MAmm' a n d M02 values c a l c u l a t e d from water parameters were s i m i l a r to those c a l c u l a t e d from blood parameters, there was more s c a t t e r i n the data, which w i l l be discussed as a part of Sec t i o n B. Data are presented as means + 1 S.E.M. The Student's p a i r e d t - t e s t was used to determine s i g n i f i c a n c e between c o n t r o l and experimental values. S e c t i o n B: Trout in. v i v o experiments S u r g i c a l procedures Two groups of f i s h were prepared, those with o r a l masks, opercular cannulae, and v e n t r a l a o r t i c (VA) and d o r s a l a o r t i c (DA) cannulae, and those VA and DA cannulae only. To avoid undue s t r e s s , surgery on the f i r s t group of f i s h was performed i n two ses s i o n s . F i s h were anaesthetized, the VA was cannulated (see General M a t e r i a l s and Methods), and f i s h were l e f t to recover for 24 h i n opaque p l e x i g l a s s chambers (~10 1). I f the VA catheter was patent, f i s h were re-anaethetized, and a DA c a t h e t e r , o r a l mask and opercular cannulae were s t i t c h e d i n place (see General M a t e r i a l s and Methods). F i s h were l e f t to recover i n the two-chambered, flow-through experimental apparatus f o r 48 h f o l l o w i n g surgery. In the second group of f i s h , VA and DA ca t h e t e r s were implanted together and f i s h recovered f o r 24 h i n single-chamber, opaque p l e x i g l a s s chambers. P r o t o c o l and Measurements F i s h with o r a l masks: The aim of t h i s experiment was to i n v e s t i g a t e a r t e r i a l - v e n o u s blood and inspired-mixed expired water d i f f e r e n c e s Xlk v i v o , so as to compare these values with those i n the i s o l a t e d blood-perfused t r o u t head p r e p a r a t i o n . V e n t i l a t o r y water flow (Vw) could be manipulated i n the two-chambered box by a d j u s t i n g the height of the f r o n t compartment standpipe r e l a t i v e to the back compartment standpipe ( F i g 2). A pressure head across the g i l l s was e s t a b l i s h e d i n the a n t e r i o r compartment to r a i s e Vw to l e v e l s comparable to the blood-perfused head (850-900 m l . k g - 1 . m i n - 1 ) . A f t e r 2 h, a r t e r i a l and venous blood samples (0.6 ml) and i n s p i r e d and mixed expired water samples (10 ml) were c o l l e c t e d . A r t e r i a l and venous blood was analysed f o r pH, Po2' ^ 02 ' CQ O 2 ' Tamm, and hct, by methods o u t l i n e d i n Section A. In s p i r e d and expired water samples were analysed for pH (see Se c t i o n A), Tamm (see Chapter 3, S e c t i o n A), CQ Q 2 < s e e General M a t e r i a l and Methods), and Po2- Water PQ 2 was measured with microelectrodes as described for blood i n Section A of t h i s chapter. Vw was measured by c o l l e c t i n g outflow water from the back chamber standpipe over 1 min i n t e r v a l s . F i s h without o r a l masks: The purpose of t h i s experiment was to determine r e s t i n g a r t e r i a l -venous d i f f e r e n c e s i n f i s h unemcumbered by o r a l masks and opercular cannulae. A r t e r i a l and venous blood samples were withdrawn and analysed i n the same manner as o u t l i n e d above. C a l c u l a t i o n s The e f f i c i e n c y of oxygen e x t r a c t i o n i s expressed by the f o l l o w i n g equation: (22) C"2 e x t r a c t i o n e f f i c i e n c y (%) = PaQ2 ~ P v02 Pl02 " P v ° 2 where PaQ2 a n d Pvo2 represent a r t e r i a l and venous O2 t e n s i o n s , r e s p e c t i v e l y , and P102 denotes i n s p i r e d water O2 t e n s i o n . The u t i l i z a t i o n of oxygen i n the water passing over the g i l l was c a l c u l a t e d as f o l l o w s : (23) O2 u t i l i z a t i o n (%) = 100 • [ P s 0 2 - P E Q 2 3 P l 0 2 where P 1 0 2 a n o " ^ E 0 2 represent i n s p i r e d and mixed expired water O2 t e n s i o n s , r e s p e c t i v e l y . The t r a n s f e r f a c t o r (T) i s a measure of the r e l a t i v e a b i l i t y of the g i l l r e s p i r a t o r y surface to exchange gases and i s defined as: (24) To 2 = 1 / 2 ( P I 0 2 + P E 0 2 > " l / 2 ( P a 0 2 + Pv02) Tg = Mg (25) l / 2 ( P a g + Pv g) - l / 2 ( P I 0 2 g + PE029> where g represents CO2 or N H 3 . E x c r e t i o n or uptake r a t e s were c a l c u l a t e d from a r t e r i a l - v e n o u s d i f f e r e n c e s and Vb. A larg e T value for a given gas i n d i c a t e s a more e f f e c t i v e exchange across the g i l l . The r a t i o of t r a n s f e r f a c t o r s f o r two gas species was compared to the r a t i o of Krogh's d i f f u s i o n c o e f f i c i e n t s , where. 157 (26) £gl = d a L ' X c j l Kg2 dg2 ' *92 and gi and g2 are equal to two d i f f e r e n t gases ( C 0 2 r O2, or N H 3 ) , d i s the p h y s i c a l d i f f u s i o n c o e f f i c i e n t i n water, and 0( i s the s o l u b i l i t y c o e f f i c i e n t i n water. D i f f u s i o n c o e f f i c i e n t s were c a l c u l a t e d using Graham's law which s t a t e s that the r a t i o of the d i f f u s i o n of two gases x and y, dx/y, i s i n v e r s e l y r e l a t e d to the square root of t h e i r molar masses. Data are presented as means +_ 1 S.E.M. 158 RESULTS t • S e c t i o n A: Linkage between M C Q2 a n d MAmm The r e s u l t s of the c o n t r o l experiment, where the chemical composition of the blood and water was not a l t e r e d , are d i s p l a y e d i n the f i r s t column of F i g s . 17 and 18. The head pr e p a r a t i o n was st a b l e throughout the experiment because there was no s i g n i f i c a n t d i f f e r e n c e between the values i n the f i r s t ( c o n t r o l ) and second (experimental) measurement periods. The mean c o n t r o l gas exchange values over the two measurement periods were as f o l l o w s : CO2 e x c r e t i o n (Mrjo2^ = 7 9 9 ±. 5 0 u m o l . k g - 1 . h - 1 , oxygen uptake (MQ2)= 324 £ 31 u m o l . k g - 1 . h - 1 , and ammonia e x c r e t i o n ( MAmm ) = 2 6 3 ±- 2 6 u m o l . k g - 1 . h - 1 . The RE value ( M C O2/M Q2^ w a s 2.47 £ .50. The mean a r t e r i a l to venous pH d i f f e r e n c e (pHa-pHv) was 0.07 £ 0.01 and the i n s p i r e d to expired water pH d i f f e r e n c e (pHj-pHE) was 0.28 £0.03. V e n t i l a t i o n was approximately the same i n a l l experiments (-210 ml.min - 1, F i g . 18). In the second treatment where a m i l o r i d e was present i n the water throughout the experiment and blood chemistry was not a l t e r e d ( a m i l o r i d e c o n t r o l ) , there were no changes i n Mcrj2' Mft m m, Mo2»- ( F i g - 17) and pHj-pHE ( F i g . 18) between the c o n t r o l and experimental measurement periods. There was a s m a l l , but s i g n i f i c a n t , decrease i n the pHa-pHv d i f f e r e n c e between the c o n t r o l and the experimental period ( F i g . 17). In general. 159 F i g u r e 17. The f i r s t ( c o n t r o l ) and second ( e x p e r i m e n t a l ) measurements o f c a r b o n d i o x i d e ( M c o = ) and ammonia e x c r e t i o n ( M A m m ) and oxygen uptake ( M 0 2 ) i n u m o l . k g - 1 . h _ 1 , a r e shown a l o n g w i t h pHa-pHv d i f f e r e n c e s , i n the c o n t r o l (N=10), a m i l o r i d e c o n t r o l (N=8), a m i l o r i d e + Diamox (N=6), and a m i l o r i d e + T r i s (N=4) e x p e r i m e n t s . * denotes s t a t i s t i c a l s i g n i f i c a n c e (P<_ 0.05) between the f i r s t and second measurement. Means + S.E.M. I o < P o o — o o o > 3 3 ro o o o o o o 3 T r O o ro o o o o o o o T T CO o o F o o ro ro o o o o control F — amiloride amiloride 4 Diamox amiloride + Tris Figure 18. Water pHi-pH E d i f f e r e n c e and v e n t i l a t i o n (Vw) m l . m i n - 1 . f i s h . See F i g . 16 legend for d e t a i l s TD CD P P P o ro I 1 — i — 1 — r control amiloride amiloride + Diamox amiloride + Tris 1 6 3 * a m i l o r i d e i n the water reduced M A M M by about 3 0 % r e l a t i v e to the i n i t i a l c o n t r o l experiment, and r e s u l t e d i n a s t i m u l a t i o n of M C 0 2 ( - + 7 0 % ) . With a m i l o r i d e i n the water throughout and acetazolamide i n the blood i n the experimental p e r i o d (Diamox experiment), MQ02 * and M A M M were reduced by 6 0 % and 5 5 % , r e s p e c t i v e l y , r e l a t i v e to the c o n t r o l p e r i o d ( F i g . 1 7 ) . As w e l l , there was a s i g n i f i c a n t decrease i n M Q 2 ( 5 0 % , F i g . 1 7 ) . The pHa-pHv d i f f e r e n c e was reversed with the acetazolamide treatment ( F i g . 1 7 ) , while the pHj-pHg d i f f e r e n c e was cut i n h a l f ( F i g . 1 8 ) . In the f o u r t h experiment, T r i s was added to the water i n the experimental p e r i o d ( a m i l o r i d e was present throughout the experiment). T r i s r e s u l t e d i n a s i g n i f i c a n t r e d u c t i o n i n M A mm ( - 3 0 % ) , i n the absence of any change i n Mc02 ( F i g - 1 7 ) . There were no changes i n M Q 2 nor the pHa-pHv d i f f e r e n c e ( F i g . 1 7 ) . The pHj-pHE d i f f e r e n c e was almost e l i m i n a t e d with T r i s b u f f e r i n the water ( F i g . 1 8 ) . S e c t i o n B: Comparisons between the blood-perfused t r o u t head and i n v i v o gas exchange values Table 1 6 compares gas t r a n s f e r v a r i a b l e s i n the blood-perfused head p r e p a r a t i o n (in. v i t r o ) with those i n i n t a c t f i s h (in. v i v o ) with o r a l masks, opercular cannulae, a r t e r i a l and venous cannulae, or i n f i s h with only a r t e r i a l and venous cannulae. MQ2 a n d Cao2~C v02 i n the blood-perfused head 164 T A B L E 1 6 . Comparison o f gas t r a n s f e r v a r i a b l e s JJQ. v i v o and blood perfused t r o u t head based on a r t e r i a l - v e n o u s d i f f e r e n c e s . Data are expressed as means +, S . E . M ( N ) PARAMETER BLOOD-PERFUSED HEAD "HI VIVO e iN m a Vb (ml .min-^g" 1) 1 1 . 1 0 + 0 . 3 9 ( 1 0 ) 1 7 . 6 1 7 . 6 Ca0 2 (mmol.I - 1) 1 . 7 3 + 0 . 0 6 ( 1 0 ) 1 . 8 4 + 0 . 4 2 ( 8 ) 2 . 6 3 + 0 . 2 3 ( 5 ) CvO, (mmol.l" 1) 1 . 2 6 + 0 . 0 5 ( 1 0 ) 0 . 3 1 + 0 . 0 8 ( 8 ) 0 . 5 3 + 0 . 1 2 ( 5 ) CaOa-CvOa (mmol.I- 1) 0 . 4 8 + 0 . 0 6 ( 1 0 ) 1 . 5 3 + 0 . 3 5 ( 8 ) 2 . 1 0 + 0 . 2 5 ( 5 ) Most (mmol.kg- 1h- 1) 0 . 3 3 + 0 . 0 4 ( 1 0 ) X1.B1 ( 8 ) 4 2 . 4 8 ( 5 ) PaO a (Torr) 8 6 . 5 + 7 . 0 ( 1 0 ) 5 2 . 1 + 1 2 . 1 ( 8 ) 8 5 . 3 + 1 2 . 6 ( 6 ) PvOa (Torr) 3 7 . 2 + 1 . 6 ( 1 0 ) 1 2 . 0 + 1 . 1 ( 8 ) 1 3 . 8 + 1 . 5 ( 6 ) PaOa-PvOa (Torr) 4 9 . 0 + 3 . 7 ( 1 0 ) 3 7 . 8 + 1 2 . 3 ( 8 ) 7 3 . 2 + 1 4 . 4 ( 6 ) 0 2 E x t r a c t i o n e f f i c i e n c y (%) 4 1 . 6 ( 1 0 ) 2 8 . 5 ( 8 ) 5 1 . 8 ( 6 ) CaCOa (mmol.l- 1) 9 . 6 8 + 0 . 3 2 ( 1 0 ) 7 . 5 3 + 0 . 7 2 ( 8 ) 6 . 3 5 + 0 . 5 6 ( 6 ) CvCOa (•mol.l-M 1 0 . 8 8 + 0 . 3 0 ( 1 0 ) 8 . 8 1 + 0 . 7 8 ( 8 ) 8 . 6 3 + 0 . 5 0 ( 6 ) CaCOa-CvCOs (mmol.l- 1) 1 . 2 5 + 0 . 0 8 ( 8 ) 1 . 2 9 + 0 . 2 0 ( 8 ) 2 . 0 6 + 0 . 4 5 ( 5 ) 165 TABLE 16. (continued) PARAMETER BLOOD-PERFUSED HEAD *IH VIVO 'IR VIVO Mcos (mmol . k g ^ h - M 0.80 + 0.07 (10) »1.52 (8) *2.43 (5) RE 2.91 + 0.5 (10) 0.84 (8) 0.98 (5) (Tamm]a (nmol.I - 1) 0.74 + 0.07 (10) 0.40 + 0.06 (7) 0.35 + 0.03 (6) [Tamm]v (mmol.I - 1) 1.13 + 0.07 (10) 0.53 + 0.06 (7) 0.51 + 0.05 (6) [Tamm]a-[Tamm]v (mmol.l- 1) 0.40 + 0.05 (10) 0.13 +0.02 (7) 0.16 + 0.03 (6) • MAmm (mmol.kg-^h-*) 0.26 +0.04 (10) *0.15 (7) t0. 18 (6) pHa 8.08 + 0.02 (10) 7.87 • 0.06 (8) 8.01 + 0.03 (6) pHv 8.01 + 0.02 (10) 7.79 + 0.06 (8) 7.85 + 0.06 (6) pHa-pHv 0.07 + 0.02 (10) 0.09 +0.03 (8) 0.15 + 0.05 (6) hct (%) 14.3 +0.3 (10) -20 23.3 + 1.4 (6) water temp C O 9.5 12 12 * Fish were f i t t e d with oral masks, opercular cannulae, and dorsal a o r t i c and ventral a o r t i c cannulae. * Kiceniuk fc Jones (1977) , 1 estimated values based on Vb from Kiceniuk fc Jones (1977). 2 Fish f i t t e d with dorsal and ventral a o r t i c cannulae only. were 70-80% lower and C V Q 2 , 2-4 times higher, than in, vivo values. Oxygen tensions (PaQ2' Pvo2^ a r*d O2 e x t r a c t i o n e f f i c i e n c i e s in. v i t r o and in. vivo were approximately equal. Carbon d i o x i d e contents (CacQ2' C V Q Q2^ i n the blood were s l i g h t l y higher in. v i t r o compared to in. v i v o , and MQ02 w a s 50-70% lower i n the blood-perfused head. The r e s p i r a t o r y exchange r a t i o (RE=Mco2/*-02) i n v i t r o was 2.91, a value that was three times greater compared to the in. v i v o value. A r t e r i a l Tamm, venous Tamm, and the a r t e r i a l - v e n o u s Tamm d i f f e r e n c e were 2-3 times greater i n v i t r o r e l a t i v e to in. v i v o • M^ m m i n v i t r o was 60% greater than i n v i v o values. The pHa-pHv d i f f e r e n c e was s l i g h t l y lower i n v i t r o (-0.02 to -0.08 pH u n i t s ) , as a r e s u l t of the higher pHv value (+0.2 pH u n i t s ) . The hct value was 10% lower i n v i t r o r e l a t i v e to i n v i v o . The water gas t r a n s f e r v a r i a b l e s (in. v i t r o and i n vivo) are given i n Table 17. The in. v i t r o v e n t i l a t i o n r a t e (Vw) was r e l a t i v e l y high (~900 m l . k g - 1 . m i n - 1 ) , but Vw i n vivo was set to a s i m i l a r l e v e l (-1200 m l . k g - 1 . m i n - 1 ) , whereas r e s t i n g i n . v i v o values were con s i d e r a b l y lower (-400 ml. k g - 1 . m i n - 1 , Wright §_t ai.. (1986)). MQ2 AH v i v o was 2-3 f o l d and MrjQ2 about 1.4 f o l d that measured i n the blood-perfused p r e p a r a t i o n . The RE value i n v i t r o was 1.93, while i n v i v o values were approximately 1. M^ m m was approximately 0.6 mmol. k g - 1 . h - 1 i n both i n v i t r o and in. v i v o at s i m i l a r Vw r a t e s , but was 50% l e s s at lower Vw r a t e s i n viv o (Wright et. a l . . , 1986). O2 u t i l i z a t i o n was 6% i n v i t r o , 14% i n 167 TABLE 17. Comparison o f gas t r a n s f e r v a r i a b l e s i n v i v o and pe r f u s e d p r e p a r a t i o n s based on i n s p i r e d - m i x e d e x p i r e d water d i f f e -r ences. Means ± S.E.M. (N) PARAMETER • Vw ( m l k g _ 1 m i n - 1 ) BLOOD-PERFUSED HEAD *IN VIVO 869.0 + 35.3 (7) 1167.0 + 52.7 (10) 1 ' Z I N VIVO 418.3 + 24.0 (8) Mo2 (mmolkg- 1*!- 1) 1.00 + 0.19 (6) 3.14 + 0.32 (10) 1.73 + 0.06 (18) Hco2 1.93 + 0.22 (mmolkg-^- 1) (6) 2.90 + 0.50 (10) 2. 39 + 0.27 (8) RE= Mo2 1.93 0.94 1.38 M « m m 0.57 +0.08 (mmol-kg-*^- 1) (7) 0.64 +0.13 (10) 0.27 + 0.05 (8) P i O a ( T o r r ) 155.9 + 2.4 (7) 144.4 + 2.0 (10) 160.3 + 0.5 U 8 ) P e 0 2 ( T o r r ) 147.1 + 3.0 (7) 124.0 + 3.4 (10) 86.1 + 2.3 (18) 0= U t i l i z a t i o n (%) 5.7 +0.9 (7) 14.0 + 1.4 (10) 46.0 + 1.5 (18) pHx-pH E 0.28 + 0.04 (7) 0.74 + 0.11 d o T 0.69 + 0.04 (8) * F i s h were f i t t e d with o r a l masks, o p e r c u l a r c a t h e t e r s , and d o r s a l a o r t i c and v e n t r a l a o r t i c cannulae. A p r e s s u r e head was e s t a b l i s h e d i n the a n t e r i o r compartment o f the two-chambered box to r a i s e Vw to l e v e l s comparable to the bl o o d - p e r f u s e d head. 1. Data from Wright £_£. aj_. (1986) a l s o see Chapter 2 and 3A. Trout were f i t t e d with o r a l masks and o p e r c u l a r c a t h e t e r s . 2. Data from Cameron and Davis (1970). Trout were f i t t e d with o r a l masks and d o r s a l a o r t i c cannulae. 168 TABLE 18. Comparison between gas t r a n s f e r v a r i a b l e s c a l c u l a t e d from a r t e r i a l - v e n o u s blood d i f f e r e n c e s with those c a l c u l a t e d from i n s p i r e d - e x p i r e d water d i f f e r e n c e s i n the i s o l a t e d b l o o d - p e r f u s e d head p r e p a r a t i o n (in. v i t r o ) and i n i n t a c t f i s h ( i n v i v o ) . Means + S.E.M. (N) I N VITRO WATER BLOOD Vw, Vb ( m l . k g ^ m i n - 1 ) 869.5 + 35.3 (7) 11.61 + 0. 36 (7) Mo 2 (mmol.kg - 1h - 1) 1.00 + 0.19 (6) 0.32 +0.04 (10) HcD2 (mmol.kg - 1h - 1) 1.93 + 0.22 (6) 0.80 + 0.07 (10) RE 1.93 2.42 (mmol.kg - 1h - 1) 0.57 + 0.08 (7) 0.26 +0.04 (10) J J L VI VP Vw, Vb (ml . k g ^ m i n - 1 ) 1167.0 + 52.7 (10) 17.6 1 No2 (mmol.kg - 1h _ x) 3.14 + 0.32 (10) . 1.81 (8) ( mmol.kg - 1h - 1) 2.90 + 0.50 (10) 1.52 (8) RE 0.94 2.42 (mmol.kg - 1h - 1) 0.64 +0.13 (10) 0. 15 (7) 1. e s t i m a t e d v a l u e s based on Vb from K i c e n i u k & Jones (1977) 169 v i v o at high Vw, and 46% at lower Vw r a t e s . The pHj-pHE d i f f e r e n c e was about 0.7 pH u n i t s jjn v i v o , but only 0.3 pH u n i t s i n v i t r o . A comparison between gas t r a n s f e r v a r i a b l e s c a l c u l a t e d from a r t e r i a l - v e n o u s blood d i f f e r e n c e s and inspired-mixed expired water d i f f e r e n c e s i s given i n Table 18 f o r the blood-perfused head p r e p a r a t i o n and i n t a c t t r o u t . M Q 2 ' ^002' a n <^ ^hmm c a l c u l a t e d from water v a r i a b l e s were 2-4 times greater than those values c a l c u l a t e d from blood parameters. Re s p i r a t o r y exchange r a t i o s (RE) c a l c u l a t e d from blood or water c o n s i d e r a t i o n s were s i m i l a r (RE= i n v i t r o 1.93 vs 2.42, i n v i v o 0.94 vs 0.84). In Table 19, t r a n s f e r f a c t o r s were c a l c u l a t e d for C O 2 , 0 2, and N H 3 i n the blood-perfused head p r e p a r a t i o n and i n i n t a c t f i s h . T Q 2 w a s 5 times greater i n i n t a c t f i s h (~0.007) compared to the blood-perfused head (0.0014). ^ C 0 2 AH v i t r o was 0.15 and in, v i v o was 0.22. TJJH3 values were se v e r a l orders of magnitude greater than t r a n s f e r f a c t o r s for O2 and C O 2 . The in, v i t r o value was 288 versus 455, in. v i v o . The in v i t r o r a t i o for T c o 2 / T 0 2 a n o " T N H3^T O2 were 3 times those i n  v i v o , while TjjH3/Trjo2 were approximately equal between the perfused head and i n t a c t animal. 170 TABLE 19. T r a n s f e r f a c t o r , T, f o r 0=., C 0 2 , and ammonia a c r o s s the g i l l , i n m l . m i n - 1 . k g - 1 . h _ 1 . T o r r - 1 . T = ( r a t e o f uptake o r e x c r e t i o n ) / ( m e a n g r a d i e n t a c r o s s the g i l l s ) . T h e o r e t i c a l r a t i o s were c a l c u l a t e d from the r a t i o o f Krogh's d i f f u s i o n c o e f f i c i e n t s f o r r e s p e c t i v e gases i n water (K= d i f f u s i o n c o e f f i c i e n t x s o l u b i l i t y c o e f f i c i e n t s ) . See t e x t f o r d e t a i l s . PARAMETER TcD2 BLOOD PERFUSED IN VIVO TROUT HEAD 0.0014 0.1522 0.0066 0.2247 IN v i V Q i . z . ' 0. 0061 0.02-0.03= 0.0092 3 0.42 3 288 455 T c D 2 109 34 46-58 THEORETICAL 23 Trsna To* 206,000 69,000 32,600 T l M H 3 1890 2020 1400 1. St e v e n s t R a n d a l l (1967), rainbow t r o u t (Salmo q a i r d n e r i ) 2. H o l e t o n & R a n d a l l (1967); R a n d a l l , H o l e t o n , & St e v e n s (1967), rainbow t r o u t (Salmo q a i r d n e r i ) 3. P i i p e r & Baumgarten-Schumann (1968), d o g f i s h ( S c v l i o r h i n u s  s t e l l a r i s ) DISCUSSION 171 E v a l u a t i o n of the blood-perfused t r o u t head p r e p a r a t i o n The present study demonstrates that the blood-perfused t r o u t head p r e p a r a t i o n i s a v i a b l e technique f o r studying the r e l a t i o n s h i p between carbon d i o x i d e and ammonia e x c r e t i o n i n f i s h . Several m o d i f i c a t i o n s have been made to the pr e p a r a t i o n which enhance the preparation's a b i l i t y to simulate i n . v i v o c o n d i t i o n s . In p r e l i m i n a r y experiments, the head was i n i t i a l l y perfused with s a l i n e c o n t a i n i n g the c o l l o i d osmotic f i l l e r , PVP (see M a t e r i a l s & Methods), a l s o used by Wood (1974), Davie (1981), Perry e_t a l . (1984b), and Bornancin e_t a l . (1985). Many problems arose with PVP i n the s a l i n e i n c l u d i n g blood leaks at the g i l l s , incomplete p e r f u s i o n of g i l l arches, and very high blood pressure values. In l a t e r experiments, these problems were e r a d i c a t e d by r e p l a c i n g PVP with the n a t u r a l p r o t e i n , bovine serum albumin (BSA). In terms of v e n t i l a t i o n , i n s p i r e d water was d e l i v e r e d through a mouth tube t i g h t l y secured i n s i d e the buccal chamber, i n a c e n t r a l p o s i t i o n , and at a r a t e comparable to swimming f i s h (Kiceniuk & Jones, 1977). The p a t t e r n of water flow was assessed v i s u a l l y before and a f t e r measurments by adding a nontoxic dye to the i n s p i r e d water to ensure that both opercular chambers were evenly i r r i g a t e d . In other perfused-head preparations (see Perry et a l . , 1984a for review), a mouth tube i s simply placed i n the opening of the mouth. In p r e l i m i n a r y t e s t s (see M a t e r i a l s & Methods), t h i s technique was shown to be f a r l e s s e f f e c t i v e at d e l i v e r i n g an even water flow through both opercular chambers. A l s o , previous i n v e s t i g a t o r s have t y p i c a l l y set Vw at 500-1000 ml.min - 1 (Payan & Matty, 1975; Payan, 1978; Perry et a l . , 1984b; Bornancin et a l . . , 1985; Perry et a l . . , 1985a,b), r a t e s 2.5 times Vw i n the present study and f a r i n excess of the normal range (Kiceniuk & Jones, 1977; Wright e_t a l . . , 1986; Iwama et. a l . , 1987). The perfused head i n the present study was i r r i g a t e d with a l a r g e r e c i r c u l a t i n g water r e s e r v o i r (10 1) or flow-through water ( c o n t r o l ) , i n c o n t r a s t to other s t u d i e s which re p o r t a r e c i r c u l a t i n g volume of 100-200 ml (Payan & Matty, 1975; Payan, 1978; Perry e£ a l . . , 1984b; Bornancin al.. , 1985; Perry e_t a l , 1985a,b). These low volumes are too small to maintain optimal C"2' CO2, and NH3 g r a d i e n t s across the g i l l s over the experimental p e r i o d . The blood-perfused t r o u t head has been shown to be f a r s u p e r i o r to the s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n i n terms of gas t r a n s f e r and acid-base s t a t u s (Perry §_t a l . . , 1985a). I t was the purpose of t h i s study, however, to i n v e s t i g a t e the a b i l i t y of the blood-perfused head p r e p a r a t i o n to exchange gases compared to i n t a c t f i s h . The r a t e of 0 2 uptake i n the blood-perfused p r e p a r a t i o n was c o n s i d e r a b l y lower than i n v i v o , but equal to that reported i n other blood-perfused preparations (Perry §_£. §2.., 1982; Perry al.., 1985a). The a r t e r i a l - v e n o u s P02 d i f f e r e n c e and O2 e x t r a c t i o n e f f i c i e n c y were analagous to 173 i n v i v o values, however, the a r t e r i a l - v e n o u s O2 content d i f f e r e n c e was lower. The lower MQ2 i n the blood-perfused head i s probably not r e l a t e d to impaired d i f f u s i o n because of the normal g i l l e x t r a c t i o n e f f i c i e n c y , but may be due to the low O2 c a r r y i n g c a p a c i t y of the blood. Cameron and Davis (1970) reported that MQ2 i n anaemic f i s h (hct= 8%) was not d i f f e r e n t from normal f i s h (hct= 27%), however, c a r d i a c output was 8 times higher, and 5 times greater compared to Vb i n the present study. Hence, the reduced MQ2 i n the perfused head i s probably due to low blood hct values which r e s u l t e d i n p e r f u s i o n l i m i t a t i o n s under r e s t i n g blood flow (Vb) c o n d i t i o n s . Carbon d i o x i d e e x c r e t i o n was a l s o l e s s i n the blood-perfused head compared to in. v i v o , although s i m i l a r to published values fo r perfused preparations with blood of the same hct (Perry et. a l . , 1982). There i s a strong p o s i t i v e c o r r e l a t i o n between Mco2 a n d nc*- (Perry §_t al.. , 1982), which probably e x p l a i n s the lower in. v i t r o Mrjo2 values i n the present study. The hct value used i n t h i s study (14%) was higher than i n other blood-perfused t r o u t preparations (9-11%, Perry g_t al.. (1984a); Perry g_t a l . (1985a,b)); however, i t i s obvious that to acheive in. v i v o gas exchange r a t e s hct must be c l o s e r to in. v i v o values (20-30%). RE values i n perfused preparations are c o n s i s t e n t l y greater than 1.0 ( t h i s study, 2.9; Perry e£. al.., 1982, 1.7; Perry §_t a l . , 1985a, 2.5), while i n . v i v o values range between 0.8 and 1.0. RE values i n vivo r e f l e c t the r a t e of CO2 production r e l a t i v e to O2 consumption, while those in. v i t r o are g e n e r a l l y r e l a t e d to 1 7 4 t O2 and CO2 venous input l e v e l s which w i l l determine M Q 2 and MC02-In c o n t r a s t to MQ 2 a n d Mc02' ammonia e x c r e t i o n was 60% higher i n the blood-perfused p r e p a r a t i o n compared to in. v i v o , which i s r e l a t e d to the greater venous Tamm l e v e l s . Blood Tamm l e v e l s vary g r e a t l y i n f i s h depending on feeding behaviour, ranging from 0 . 1 3 mmol.l -! i n starved f i s h (Wright & Wood, 1 9 8 5 ) to 0 . 8 5 mmol.l - 1 i n fed f i s h (Perry & Vermette, 1 9 8 7 ) . Venous Tamm l e v e l s were set to a r e l a t i v e l y high l e v e l i n the perfused head to ensure M A t n m was i n the normal range ( 2 0 0 - 3 5 0 umol.kg~l.h~l, McDonald & Wood, 1 9 8 1 ; Cameron & H e i s l e r , 1 9 8 3 ; Wright & Wood, 1 9 8 5 ; Vermette & Perry, 1 9 8 7 ) . In g e n e r a l , the r a t e s of gas t r a n s f e r c a l c u l a t e d from inspired-mixed expired water d i f f e r e n c e s were lower in, v i t r o versus i n . v i v o • As before, the d i f f e r e n c e s were not p r o p o r t i o n a l , that i s , MQ2 w a s most a f f e c t e d , followed by Mcx)2' a n o" t n e n Mft m m. The % u t i l i z a t i o n of O2 from water flowing over the g i l l s i s a r e f l e c t i o n of the amount of water in v o l v e d i n gas exchange. Oxygen u t i l i z a t i o n was low in. v i t r o ( 6 % ) , but was c l o s e r to the high Vw in. vivo value ( 1 4 % ) , than the r e s t i n g Vw value ( 4 6 % ) . Percent u t i l i z a t i o n i s i n v e r s e l y r e l a t e d to Vw i n t r o u t r e s t r a i n e d i n VanDam-type chambers ( s i m i l a r to F i g . 2 ) (Cameron & Davis, 1 9 7 0 ) . Comparisons between the inspired-mixed e x p i r e d water pH d i f f e r e n c e between i n vi v o and in. v i t r o values are d i f f i c u l t . The magnitude of water a c i d i f i c a t i o n w i l l depend on Vw, M C Q 2 ' 175 and M^ m m, among other t h i n g s , and these values were not the same between in. v i t r o and i n v i v o experiments. A comparison between gas t r a n s f e r r a t e s c a l c u l a t e d from water versus blood d i f f e r e n c e s i n the blood-perfused p r e p a r a t i o n and i n t a c t f i s h shows that MQ2' m C 0 2 ' a n d MAmm a r e greater when c a l c u l a t e d from water c o n s i d e r a t i o n s (Table 18). Payan & Matty (1975), i n the s a l i n e - p e r f u s e d t r o u t head, and Cameron & H e i s l e r (1983) i n i n t a c t t r o u t , found that M^ m m was 5-20% greater when c a l c u l a t e d from i n s p i r e d and mixed expired water d i f f e r e n c e s compared to a r t e r i a l - v e n o u s d i f f e r e n c e s . They proposed that the discrepancy between c a l c u l a t e d M^ m m values was due to ammonia production i n g i l l e p i t h e l i u m . The reason for the large d i s c r e p a n c i e s i n the present study cannot be s o l e l y r e l a t e d to endogenous metabolism i n g i l l e p i t h e l i a l t i s s u e and may be r e l a t e d to the sampling technique. Blood samples are assumed to be r e p r e s e n t a t i v e because they are c o l l e c t e d from a s m a l l , homogenous pool. On the other hand, mixed expired water samples are c o l l e c t e d from a l a r g e r p o o l , which may or may not be homogenous. Although the opercular c a n n u l a t i o n technique has been v a l i d a t e d f o r pHj-pHg d i f f e r e n c e s a t r e s t i n g Vw r a t e s i n i n t a c t f i s h (see General M a t e r i a l s & Methods and chapter 2), i n the absence of rhythmical v e n t i l a t o r y movements and at higher v e n t i l a t i o n r a t e s , a greater p r o p o r t i o n of v e n t i l a t o r y water i s shunted past r e s p i r a t o r y surfaces and water t r a n s i t time i n the opercular chamber i s reduced. Thus, samples c o l l e c t e d from the opercular cannula may not always be r e p r e s e n t a t i v e of mixed 176 e x p i r e d water. I t i s assumed, t h e r e f o r e , that c a l c u l a t e d M c o 2 ' MQ2' a n d M ^ M M values are more v a l i d when determined from a r t e r i a l - v e n o u s d i f f e r e n c e s than inspired-mixed expired d i f f e r e n c e s i n the present study. The c a l c u l a t i o n of t r a n s f e r f a c t o r s (T) allows comparisons between the a b i l i t y of a r e s p i r a t o r y surface to exchange gases. Carbon d i o x i d e and ammonia were t r a n s f e r r e d f a r more e f f i c i e n t l y than oxygen. Oxygen i s much l e s s s o l u b l e i n water r e l a t i v e to the two other r e s p i r a t o r y gases, CO2 (0(02/'<*C02= 1/3°) and NH3 (t*02/'**• NH3 = 1/24,000). Transfer i s a l s o dependent on the magnitude of the gradient across the g i l l s and the r a t e of r e a c t i o n s , such as, the 0 2-haemoglobin and HC03~:C02 i n t e r c o n v e r s i o n r e a c t i o n s . In g e n e r a l , for a given gradient between blood and water, l e s s O 2 w i l l be t r a n s f e r r e d compared to CO2 or NH3. This a l s o i m p l i e s that i f gas t r a n s f e r across the g i l l s i s d i f f u s i o n l i m i t e d , then O 2 uptake w i l l be most e f f e c t e d . I t i s a l s o i n t e r e s t i n g to note the tremendous d i f f e r e n c e between T(jH3 and e i t h e r 1Q02 o r T02* Par t of t h i s d i f f e r e n c e may be due to the f a c t that ammonia i s a l s o e l i m i n a t e d at the g i l l s as NH4+, which would overestimate the TJJH3 value. However, even i f 50% of ammonia was excreted as NH4+ and t h i s was accounted f o r , T[jH3 would only be reduced by 1/2 and would s t i l l be s e v e r a l orders of magnitude greater than Trjo2 o r T02- Thus, the l a r g e TJJH3 value i s p r i m a r i l y due to ammonia's greater s o l u b i l i t y i n water compared to O 2 or C0 2. 1 7 7 T h e o r e t i c a l r a t i o s of t r a n s f e r f a c t o r s were c a l c u l a t e d i n Table 1 9 , from the r a t i o s of Krogh's permeation c o e f f i c i e n t s (K= d i f f u s i o n c o e f f i c i e n t x s o l u b i l i t y c o e f f i c i e n t ) . This a n a l y s i s compares t r a n s f e r of gas i n water and does not account f o r d i f f u s i o n through various t i s s u e membranes, t r a n s f e r of i o n spe c i e s , as w e l l as chemical r e a c t i o n s i m p l i c i t i n 03 , CO2, and NH3 t r a n s p o r t . As w e l l , t h i s a n a l y s i s does not account for p e r f u s i o n l i m i t a t i o n s to gas exchange. Gas exchange may be e i t h e r p e r f u s i o n or d i f f u s i o n l i m i t e d , or both, depending on the c o n d i t i o n s f o r t r a n s f e r (see Chapter 3 ) . Any combination of these f a c t o r s may lead to i n e q u a l i t i e s between T f a c t o r s . Despite the s i m p l i c i t y of the a n a l y s i s , i t i s amazing that Trjo2/To2 A N D T J J H 3 / T C O 2 r a t i o s i n t r o u t only vary by a f a c t o r of -1.4 from the t h e o r e t i c a l r a t i o s expected fo r simple d i f f u s i o n i n an " i d e a l " system. P i i p e r and Baumgarten-Schumann ( 1 9 6 7 ) found that i n do g f i s h the T c o 2 / T 0 2 r a t i o was 2 times that expected from a s i m i l a r t h e o r e t i c a l a n a l y s i s . The s i m i l a r i t y between t h e o r e t i c a l and j j i v i v o or £n. v i t r o values i m p l i e s that p h y s i c a l p r o p e r t i e s of gases play a major r o l e i n the e f f i c i e n c y of t r a n s f e r . To summarize, oxygen t r a n s f e r i s l i m i t e d i n the i s o l a t e d blood-perfused head probably because of the low c a r r y i n g - c a p a c i t y of the blood. Carbon d i o x i d e and ammonia t r a n s f e r were q u a n t i t a t i v e l y l e s s i n the perfused head than i n i n t a c t animals, but the r a t i o of T N H 3 / T C Q 2 w a s e Q u a l to that found XlL v i v o . I t can be concluded, t h e r e f o r e , that the blood-perfused t r o u t 178 head i s a s u i t a b l e p r e p a r a t i o n for the study of the r e l a t i o n s h i p between carbon d i o x i d e and ammonia e x c r e t i o n . The lin k a g e between carbon d i o x i d e and ammonia e x c r e t i o n In the perfused head experiments, i t was important to i s o l a t e NH3 from NH4+ e x c r e t i o n i n order to determine i f NH3 d i f f u s i o n across the g i l l was l i n k e d to CO2 e x c r e t i o n . In the am i l o r i d e c o n t r o l experiment, mean ammonia e x c r e t i o n decreased by 30% r e l a t i v e to the c o n t r o l experiment, which equals that reported by Kirschner §_t aj.. (1973) for the s a l i n e perfused t r o u t head p r e p a r a t i o n and compares w e l l with the 23% r e d u c t i o n reported jji vivo (Wright & Wood, 1985; a l s o see Chapter 3 ) . The s t i m u l a t i o n of carbon d i o x i d e e x c r e t i o n with a m i l o r i d e may be r e l a t e d to removal of CO2 from the g i l l e p i t h e l i u m due to a c i d i f i c a t i o n as a r e s u l t of i n h i b i t i o n of H+ e x c r e t i o n . (H+ ions may be excreted d i r e c t l y by Na+/H+ exchange, or may be excreted as N H 4 + , a l s o l i n k e d to Na+ uptake.) The s i g n i f i c a n t r e d u c t i o n i n the pHa-pHv d i f f e r e n c e i n d i c a t e s that the e f f e c t s of the i n h i b i t i o n of H+ ion e x c r e t i o n on the blood compartment were delayed and only apparent i n the second a m i l o r i d e measurement. Acetazolamide d r a m a t i c a l l y reduced Mco2 a n o" ^kmm ( F i g . 17) which demonstrates chemical c o u p l i n g between ammonia and carbon d i o x i d e e x c r e t i o n . The concommittant decrease i n oxygen uptake was probably a d i r e c t r e s u l t of the decrease i n MQO2' because O2 and CO2 gas exchange are r e l a t e d through chemical 179 r e a c t i o n s i n v o l v i n g haemoglobin i n the red c e l l (Maren & Swenson, 1980). The r e v e r s a l of the pHa-pHv d i f f e r e n c e with acetazolamide i s a t t r i b u t a b l e to higher Pc02 l e v e l s i n a r t e r i a l blood due to the i n h i b i t i o n of MQ02' * t should be noted that the decrease i n a r t e r i a l pH d i d not e f f e c t NH3 d i f f u s i o n gradients because the c a l c u l a t e d a r i t h m e t i c a r t e r i a l - v e n o u s mean P(4H3 values d i d not change s i g n i f i c a n t l y between the c o n t r o l (214 +_ 23 uTorr) and acetazolamide (190 + 20 uTorr) measurement. The pHj-pHE d i f f e r e n c e ( F i g . 18) was reduced as a d i r e c t r e s u l t of the depression i n CO2 e x c r e t i o n and subsequent r e d u c t i o n i n CO2 h y d r a t i o n i n the expired water. I t was p o s s i b l e that the l i n k between CO2 and ammonia e x c r e t i o n occurred i n t e r n a l l y , s i m i l a r to the c o u p l i n g between CO2 and O2 i n the red c e l l , r a t h e r than i n the boundary l a y e r on the e x t e r n a l surface of the g i l l . T r i s b u f f e r was added to the water i n the f i n a l experiment to t e s t whether the l i n k between CO2 and ammonia was due to chemical r e a c t i o n s i n the boundary l a y e r . ( I t was assumed that given the flow r a t e of water over the g i l l s and the r e l a t i v e l y small volume of the buccal and opercular chambers, that T r i s i n the bulk phase water reached the boundary l a y e r water i n a r e l a t i v e l y short time.) T r i s caused a s i g n i f i c a n t r e d u c t i o n i n ammonia e x c r e t i o n i n the absence of any change i n CO2 e x c r e t i o n . Since CO2 e x c r e t i o n and O2 uptake d i d not change with the T r i s treatment, i t i s u n l i k e l y that the change i n M A m m was due to d i r e c t e f f e c t s of T r i s on g i l l c e l l membrane p e r m e a b i l i t y . Rather the decrease i n 180 M^ m m was probably because H+ ions normally a v a i l a b l e i n the boundary l a y e r from the CO2 h y d r a t i o n r e a c t i o n were buffered by T r i s . This i s al s o demonstated by the f a c t that the pHj-pHg d i f f e r e n c e was v i r t u a l l y e l i m i n a t e d with T r i s i n the water ( F i g . 18). These experiments e s t a b l i s h that NH3 d i f f u s i o n i s l i n k e d to CO2 e x c r e t i o n i n the g i l l boundary l a y e r , and that H+ ions produced from the c a t a l y z e d CO2 h y d r a t i o n r e a c t i o n are used to protonate N H 3 , which i n t u r n f a c i l i t a t e s NH3 e x c r e t i o n ( F i g . 15). These r e s u l t s do not preclude the p o s s i b i l i t y of an i n t e r n a l l i n k between CO2 and ammonia e x c r e t i o n , however they demonstrate that the e x t e r n a l boundary l a y e r i s a s i t e of li n k a g e between carbon d i o x i d e and ammonia e x c r e t i o n . The CO2 h y d r a t i o n r e a c t i o n i n the e x t e r n a l boundary l a y e r enhances the blood-to-water NH3 d i f f u s i o n gradient and f a c i l i t a t e s ammonia e x c r e t i o n across the g i l l . This f a c i l i t a t i o n of ammonia e x c r e t i o n i n d i c a t e s that under the c o n d i t i o n s of the present study, ammonia t r a n s f e r was d i f f u s i o n l i m i t e d . This does not n e c e s s a r i l y mean that ammonia e x c r e t i o n i s not a l s o p e r f u s i o n l i m i t e d (see Chapter 3 ) . Based on t h e o r e t i c a l c o n s i d e r a t i o n s , one would expect that t r a n s f e r of ammonia would be l a r g e l y p e r f u s i o n l i m i t e d due to ammonia's r e l a t i v e l y high Krogh's permeation c o e f f i c i e n t . This study demonstrates, however, that a s i g n i f i c a n t component of ammonia t r a n s f e r i s d i f f u s i o n l i m i t e d . One would p r e d i c t that the e f f e c t of changes i n CO2 e x c r e t i o n on NH3 d i f f u s i o n w i l l be more pronounced at lower Vw r a t e s because boundary l a y e r t h i c k n e s s w i l l be increased. P i i p e r §_t 181 a l . (1986) have estimated the thickness of the boundary l a y e r i n do g f i s h ( S c v l i o r h i n u s s t e l l a r i s ) at r e s t (Vw= ~200 mLmin~l.kg -l) and during swimming, where Vw r a t e s were s i m i l a r to the present study (Vw= -900 mljmin"~l.kg-l) . They found that at r e s t the thickness of the boundary l a y e r was about 20% greater than that i n swimming f i s h . A 20% increase i n the boundary l a y e r would increase the d i f f u s i o n pathway f o r NH3 and accentuate the importance of chemical removal of NH3 through i n t e r a c t i o n s with the CO2 h y d r a t i o n r e a c t i o n i n the boundary l a y e r . For ammonia e x c r e t i o n to be f a c i l i t a t e d by the c a t a l y s e d CO2 h y d r a t i o n r e a c t i o n i n the g i l l water boundary l a y e r , the a p i c a l e p i t h e l i a l membrane must be r e l a t i v e l y impermeable to i o n species. I f the a p i c a l membrane was h i g h l y permeable to N H 4 + , H C O 3 - , or H+ i o n s , then NH3 l e v e l s i n the water boundary l a y e r would always be low, and there would be no l i n k between carbon d i o x i d e and ammonia e x c r e t i o n . The f a c t that t h i s l i n k a g e has been demonstrated i n d i c a t e s that the g i l l water boundary l a y e r i s a d i s t i n c t "microclimate" and that the a p i c a l membrane i s r e l a t i v e l y impermeable to ions. This f i t s i n with accepted theory of freshwater g i l l e p i t h e l i u m i o n p e r m e a b i l i t i e s (see G i r a r d & Payan, 1980 and P o t t s , 1984 f o r reviews). The s i g n i f i c a n c e of the l i n k between carbon d i o x i d e and ammonia w i l l be g r e a t e s t for f i s h i n a l k a l i n e waters. For in s t a n c e , s e v e r a l species of T i l a p i a i n h a b i t the a l k a l i n e lakes of the Great R i f t V a l l e y i n A f r i c a where water pH may range 182 between 9.6 and 10.5 (Johansen e_t a_l.. , 1975). In these waters, the pK of ammonia w i l l be approximately 9, the NH3:NH4+ r a t i o w i l l be 50% or g r e a t e r , and NH3 l e v e l s i n water near the g i l l s urface may be extremely high (see General D i s c u s s i o n ) . Hence, the r a t e of CO2 e x c r e t i o n may become extremely important i n order to maintain an a c i d boundary l a y e r next to the g i l l which i n t u r n w i l l f a c i l i t a t e ammonia removal from the blood. The i n t e r a c t i o n between carbon d i o x i d e and ammonia e x c r e t i o n would enable these f i s h to s u r v i v e i n such extreme environmental c o n d i t i o n s . In a c i d i c environments, there w i l l be l i t t l e HCO3- formed as CO2 i s excreted i n t o the g i l l water boundary l a y e r . Under these c o n d i t i o n s NH3 e x c r e t i o n may a l k a l i n i z e the boundary l a y e r r e l a t i v e to the bulk water. The t r a n s i t i o n from an a c i d to a more a l k a l i n e water boundary l a y e r w i l l depend on the r e l a t i v e e x c r e t i o n r a t e s of CO2 and NH3 and the pK of these molecules. Is the reverse s i t u a t i o n a l s o t r u e , that i s , that NH3 d i f f u s i o n i n t o the boundary l a y e r f a c i l i t a t e s CO2 e x c r e t i o n by mopping up H+ ions and enhancing the blood-to-water Pc02 g r a d i e n t s ? The f a c t that MrjQ2 d l d n°t change with an increase i n i n s p i r e d water b u f f e r i n g c a p a c i t y by ~ 3 times ( T r i s experiment), i m p l i e s that under these c o n d i t i o n s , increases i n A^mm would probably not a f f e c t the r a t e of CO2 e x c r e t i o n . I t may be, however, that NH3 d i f f u s i o n acts as an important b u f f e r i n the boundary l a y e r under other c o n d i t i o n s , such as, i n poorly buffered waters, at maximal CO2 e x c r e t i o n r a t e s , at low Vw 183 r a t e s , and when boundary l a y e r thickness i s increased. 184 GENERAL DISCUSSION This t h e s i s has examined ammonia sto r e s and e x c r e t i o n i n f i s h , with respect to pH gra d i e n t s w i t h i n the body and between the body and the environment. The r e s u l t s demonstrate that the d i s t r i b u t i o n of ammonia between red c e l l s and plasma i n f i s h i s dependent on transmembrane pH gra d i e n t s under r e s t i n g c o n d i t i o n s . Red c e l l s are the exception, however, and i n other t i s s u e compartments ammonia stores are not determined by pH g r a d i e n t s , i n d i c a t i n g that the general theory of non-ionic d i f f u s i o n f or weak e l e c t r o l y t e s i s not v a l i d i n these t i s s u e s . Although s h i f t s i n i n t r a c e l l u l a r - t o - e x t r a c e l l u l a r pH gradients w i l l not d i r e c t l y a l t e r i n t r a c e l l u l a r ammonia l e v e l s i n these t i s s u e s , changes i n e x t r a c e l l u l a r pH w i l l i n d i r e c t l y a l t e r t i s s u e ammonia sto r e s i f they are accompanied by changes i n plasma ammonia conc e n t r a t i o n s . For example, when f i s h are exposed to a l k a l i n e waters, blood pH and plasma ammonia content i n c r e a s e , while the reverse i s true for f i s h i n a c i d waters (Wright & Wood, 1985). Because ammonia i s p a s s i v e l y d i s t r i b u t e d between plasma and i n t r a c e l l u l a r compartments according to the membrane p o t e n t i a l , a l k a l i n e c o n d i t i o n s w i l l i n c r e a s e , and a c i d c o n d i t i o n s w i l l reduce i n t r a c e l l u l a r ammonia l e v e l s . I t i s commonly s t a t e d i n the l i t e r a t u r e that ammonia, as w e l l as carbon d i o x i d e , are d i s t r i b u t e d i n the body according to pH gradients between t i s s u e compartments (eg. Jacobs & Stewart, 1936; Milne e_t a l . , 1958; P i t t s , 1973; Cameron & H e i s l e r , 1983; 185 M i l l i g a n & Wood, 1986b; Remesey et a l . . , 1986; Randall & Wright, 1987; Wright & R a n d a l l , 1987). Measured muscle, heart, and b r a i n ammonia l e v e l s i n s o l e were 10-20 times higher than those p r e d i c t e d from pH g r a d i e n t s . Bicarbonate t i s s u e s t o r e s were a l s o c a l c u l a t e d from pH gradients assuming that the non-ionic d i f f u s i o n theory describes the d i s t r i b u t i o n of carbon d i o x i d e between body t i s s u e s . Muscle and heart l e v e l s were about 4 times greater than c a l c u l a t e d bicarbonate l e v e l s i n these t i s s u e s (1.5-1.9 m m o l . l - 1 ) , and measured b r a i n ammonia l e v e l s were s l i g h t l y l e s s than c a l c u l a t e d b r a i n bicarbonate concentrations (4.7 m m o l . l - 1 ) . Bicarbonate ions are t r a n s f e r r e d across t i s s u e membranes by ion exchange processes (eg. C I - / H C O 3 - ) , but the r a t e of t r a n s f e r across membranes i s thought to be extremely slow, with the exception of red c e l l membranes (Heming, 1984). I f H C O 3 - ions were more permeable across muscle, heart, and b r a i n t i s s u e membranes than g e n e r a l l y accepted, the membrane p o t e n t i a l would i n f l u e n c e i n t r a c e l l u l a r H C O 3 - l e v e l s . I f t h i s i s the case, the measured t o t a l carbon d i o x i d e t i s s u e l e v e l s w i l l be lower than p r e d i c t e d from pH g r a d i e n t s . I n t u i t i v e l y t h i s makes sense because c e l l s are n e g a t i v e l y charged with respect to e x t r a c e l l u l a r f l u i d (-Em), and t h e r e f o r e c e l l s w i l l sequester NH4+ ions and r e p e l l H C O 3 - ions. Hence, i f H C O 3 -p e r m e a b i l i t y approaches NH4+ p e r m e a b i l i t y i n f i s h t i s s u e s , then the [ N H 4 + ] / [ H C O 3 - ] r a t i o w i l l be as high as 50 f o r heart t i s s u e , and as low as 10 f o r b r a i n t i s s u e , with the r a t i o f o r muscle t i s s u e between these values. The a c t u a l r a t i o s i n f i s h 186 are not known, for H C O 3 - t i s s u e l e v e l s are t y p i c a l l y d erived from the non-ionic d i f f u s i o n theory ( M i l l i g a n & Wood, 1986b) or c a l c u l a t e d from pHi and the i n t r a c e l l u l a r nonbicarbonate b u f f e r values ( H e i s l e r , 1978). Thus, depending on the H C O 3 -p e r m e a b i l i t y value, the [ N H 4+J/ [ H C O 3 - ] w i l l range between 4 and 50 for muscle and v e n t r i c l e , and between 0.7 and 10 i n b r a i n t i s s u e . The r e s u l t s a l s o show that f i s h are able to maintain extremely high r e s t i n g b r a i n ammonia l e v e l s with no t o x i c e f f e c t . Mammalian plasma ammonia concentrations are extremely low (~10 u m o l . l - ! . Mutch & Ban n i s t e r , 1983) and only during disease s t a t e s does ammonia increase i n the body with extremely d e l e t e r i o u s e f f e c t s (Lawerence e_t a l . . , 1957; S t a u f f e r & S c r i b n e r , 1957; Sherlock, 1960). The t o x i c e f f e c t of ammonia i n vert e b r a t e s i s l i n k e d to an i n t e r u p t i o n of neural f u n c t i o n through the d i r e c t a c t i o n of NH4+ ions on e l e c t r i c a l a c t i v i t i e s of neurons (Binstock & Lecar, 1969), di m u n i t i o n of neurotransmitter s y n t h e s i s (Mutch & Ba n n i s t e r , 1983), suppresion of energy-generating processes and d e p l e t i o n of b r a i n ATP l e v e l s ( A r i l l o et a l , 1981; Benjamin, 1982). While profound t o x i c i t y i n mammals (convulsions) occurs at b r a i n ammonia l e v e l s of ~1 mmol.l -! (B a n i s t e r & Singh, 1980), lemon so l e were able to accomodate b r a i n ammonia concentrations of between 2.8 and 5.0 mmol.l -! i n the present study. What accounts f o r t h i s t olerance d i f f e r e n c e between f i s h and mammals? The answer to t h i s i s unknown, however, ammonia i s more t o x i c to some b r a i n 187 c e l l s than others (McGilvery, 1983), and there are vast d i f f e r e n c e s between r e l a t i v e q u a n t i t y and type of neural t i s s u e i n mammal and f i s h . As w e l l , f i s h may be able to withstand l a r g e r f l u c t u a t i o n s i n body ammonia l e v e l s because of e f f i c i e n t d e t o x i f i c a t i o n s mechanisms, such as, glutamine and/or urea synt h e s i s ( A r i l l o §_t ajU, 1981). The d i s t r i b u t i o n of ammonia between body compartments i n f i s h i s dependent on e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s across t i s s u e membranes and t r a n s f e r of ammonia i n the body i s dependent on both NH3 and NH4+ g r a d i e n t s . Studies have shown that t r a n s f e r of ammonia across g i l l e p i t h e l i u m of freshwater f i s h i s al s o i n the NH3 form (deVooys, 1968; Cameron & H e i s l e r , 1983; Wright & Wood, 1985) and NH4+ form (Maetz & Garcia Romeu, 1964; Maetz, 1973; Evans, 1977; Payan, 1978; Wright & Wood, 1985). The r e s u l t s of t h i s t h e s i s demonstrate that ammonia and carbon d i o x i d e e x c r e t i o n are l i n k e d through chemical r e a c t i o n s i n the g i l l water boundary l a y e r . This i n t e r a c t i o n between NH3 d i f f u s i o n and the c a t a l y z e d CO2 h y d r a t i o n r e a c t i o n w i l l be inf l u e n c e d by the depth of the boundary l a y e r at the g i l l s u rface. The thic k n e s s of the boundary l a y e r was c a l c u l a t e d by a method o u t l i n e d i n Appendix I I , f i r s t described by P i i p e r a l . (1986). B r i e f l y , the thickness of the u n s t i r r e d l a y e r next to the g i l l can be determined by the F i c k d i f f u s i o n equation, that i s , the product of the O2 d i f f u s i o n and s o l u b i l i t y c o e f f i c i e n t s and the r a t i o of the secondary l e m a l l a r surface area to the d i f f u s i n g c a p a c i t y of oxygen i n the u n s t i r r e d water l a y e r . F i g . 188 Figure 19. A schematic representation of a cross-section through the channel between two secondary lamellae under two d i f f e r e n t flow conditions; the high flow value i s s i m i l a r to v e n t i l a t o r y flow (Vw) during exhaustive exercise, and the low flow i s representative of r e s t -ing conditions. The thickness of the i n t e r l a m e l l a r distance ( S i d ) , boundary layer ( s b i ) , mucus layer ( S m ) , and epithelium (s«p) are shown, drawn to scale. A 189 B Vw 1750 250 (mLmin-Skg - 1) i n i t i a l St.1 5.7 20.0 (urn) adjusted s b i 4.6 9 .6 (urn) 1/2 s t r f 11.5 11.5 (urn) r a t i o St.i/1/2 s l e, 0.43 0.B7 190 19 i l l u s t r a t e s the r a t i o of the c a l c u l a t e d g i l l water boundary l a y e r to the i n t e r l a m e l l a r distance under high and low water flow c o n d i t i o n s . The g i l l e p i t h e l i u m i s approximately 10 urn thick. ( P i i p e r et a l , , 1986), and the as s o c i a t e d e x t e r n a l mucus l a y e r i s estimated to be about 2 um i n depth. Mucus production v a r i e s under d i f f e r e n t c o n d i t i o n s i n f i s h ( U l t s c h & Gros, 1979) and probably at d i f f e r e n t g i l l l o c a t i o n s , although exact measurements have not been reported. I t i s l i k e l y t hat under normal c o n d i t i o n s , the mucus l a y e r thickness i s i n the range of 1-3 um, because i n f r e e z e - f r a c t u r e methods the mucus l a y e r separates from the e p i t h e l i u m and appears to j u s t cover the morphological heterogeneity of the l a m e l l a r surface ( R a n d a l l , personnal communication). The i n t e r l a m e l l a r distance depends l a r g e l y on the species of f i s h ; i n t r o u t i t i s about 23 um (Hughes & Morgan, 1973; R a n d a l l , 1982a). The boundary l a y e r thickness c a l c u l a t e d at an e x e r c i s e and a r e s t i n g v e n t i l a t i o n r a t e was 5.7 and 20.0 um, r e s p e c t i v e l y . These values are l i k e l y to be overestimates of the true boundary l a y e r thickness because the a n a l y s i s does not account for the f a c t that 20% of the t o t a l surface area of secondary lamellae i s made up of non r e s p i r a t o r y p i l l a r c e l l s ( F a r r e l l et a l . . , 1980). As w e l l , only about 60% of the secondary lamellae are perfused with blood i n r e s t i n g t r o u t (Booth, 1978). Hence, the i n i t i a l c a l c u l a t i o n s were adjusted for these surface area c o n s i d e r a t i o n s , and the adjusted boundary l a y e r thickness ranged from 4.6 to 9.6 um, between r e s t and e x e r c i s e water flow c o n d i t i o n s . At r e s t , the channel f o r free f l o w i n g water between two lamellae i s narrow (-4 urn. F i g . 19). P i i p e r §_t al.. (1986) suggested that d i f f u s i o n across i n t e r l a m e l l a r water i s more important than d i f f u s i o n across the blood-water b a r r i e r i n l i m i t i n g the t r a n s f e r of O2 between water and blood. I t i s not s u r p r i s i n g that at high Vw r a t e s t y p i c a l of e x e r c i s e c o n d i t i o n s there i s a decrease i n the r a t i o of the boundary l a y e r thickness to the i n t e r l a m m e l l a r d i s t a n c e , which undoubtedlty enhances gas t r a n s f e r during increased metabolic demands. P i i p e r g_t al.. (1986) r e p o r t much l a r g e r values f o r estimated boundary l a y e r thickness (32 urn at r e s t , Vw= 195 m l . m i n - 1 . k g - 1 and 26 urn during swimming, Vw= 917 m l . m i n - 1 . k g - 1 ) than those c a l c u l a t e d above. One d i f f e r e n c e between between these s t u d i e s i s that P i i p e r and coworkers used l a r g e S c v l i o r h i n u s s t e l l a r i s (>2 kg), where interlammellar distance was 4 times greater than i n t r o u t . To my knowledge these authors d i d not account f o r the d i f f e r e n c e between f u n c t i o n a l r e s p i r a t o r y surface area and t o t a l surface area, which would overestimate t h e i r c a l c u l a t e d values. Other techniques have been used to estimate boundary l a y e r thickness i n r a t jejunum (Lucus et. al.., 1975) and ileum (Jackson et. al.., 1978), and these values range between 60 and 155 urn. One would expect much l a r g e r boundary l a y e r s a s s o c i a t e d with e p i t h e l i u m l i n i n g i n t e s t i n e , stomach, or kidney tubules because the r a t e of f l u i d flow i s c o n s i d e r a b l y lower r e l a t i v e to f i s h g i l l s . The pH of boundary water at the g i l l surface w i l l be more a c i d i c than expired water. G i l l water boundary l a y e r pH can be 192 Figure 20. The r e l a t i o n s h i p between water carbon dioxide tensions (Pcoz) and pH are given for Vancouver tapwater (10*C, C C o 2 = 0.224 mmol. 1-1), Ottawa tapwater (12*C, C c o 2 = 0.455 mmol.1-1), and Bamfield seawater (11* C, Cco== 2.0 mmol. 1-1). estimated from the f o l l o w i n g assumptions: 1) the CO2 d i f f u s i o n c o e f f i c i e n t i n i n t r a c e l l u l a r f l u i d and i n t e r l a m e l l a r water i s approximately equal, since both media c o n t a i n carbonic anhydrase which f a c i l i t a t e s CO2 d i f f u s i o n (see Chapter 3). 2) that Pc02 l e v e l s decrease from blood-to-water i n p r o p o r t i o n to the di s t a n c e . This l a s t assumption means that because CO2 must d i f f u s e through the g i l l e p i t h e l i u m and the boundary water l a y e r before reaching the bulk, medium, boundary water Pc02 l e v e l s w i l l be equal to about 1/3 blood-to-bulk water mean Pc02 g r a d i e n t . Figure 20 i l l u s t a t e s the r e l a t i o n s h i p between water PC02 l e v e l s and pH f o r three d i f f e r e n t waters; Vancouver tapwater ( CQ O 2 = 0.224 mmol.l -^), Ottawa tapwater (Crjo2 = 0.455 mmol.l -!), and Bamfield seawater (CQQ2= 2.0 mmol.l -!). Mean boundary l a y e r pH (pH^i) was estimated from p r e d i c t e d mean boundary l a y e r Prj02 using F i g . 20; i n r e s t i n g t r o u t pHfci i s 6.75 i n Ottawa tapwater and 6.32 i n Vancouver water. The small d i f f e r e n c e s between these pH values r e l a t e to v a r i a t i o n s i n Prj02 gr a d i e n t s and water b u f f e r i n g c a p a c i t y . In seawater, where the water b u f f e r i n g c a p a c i t y i s r e l a t i v e l y l a r g e mean boundary water pH w i l l be higher; i n lemon sole the value i estimated to be 7.60 (Table 20). Blood Pco2 l e v e l s are known to increase d r a m a t i c a l l y immediately f o l l o w i n g exhaustive e x e r c i s e ( M i l l i g a n & Wood, 1986a; Wood & M i l l i g a n , 1987), and mean water boundary l a y e r pH may be as low as 5.62 (Vancouver) and 6.20 (Ottawa) i n freshwater, and 7.22 i n seawater (Table 20). The consequence of these d i f f e r e n c e s i n boundary water pH 195 TABLE 20. Estimates of g i l l water boundary layer pH (pHoi) from Pco2 gradients across the g i l l at rest and exercise i n freshwater (FW) trout i n two d i f f e r e n t waters (Ottawa, 0.455 mmol.1-1, Vancouver, 0. 224 mmol.l - 1) and i n seawater (SW) sole. The pH of inspi r e d water (pHi) i s also shown. CONDITIONS Pco2 gradients Boundary layer pHi pHt blood-to-water Pco= Trout Ottawa FW Rest 2.53 (Chapter 4) 1.05 8.04 6.75 Exercise 9.00 ( M i l l i g a n & Wood, 1986a; trout) 3.00 8. 04 6.2 Vancouver FW Rest 2.57 (Chapter 3) 1. 34 7.41 6. 32 Exercise 9.00 ( M i l l i g a n & Wood, 1986a; trout) 3.00 7.41 5.62 S2le. Bamfield SW Rest 1.94 (Chapter 1) 0.65 7.80 7. 60 *# Exercise 5.00 (Wood & 1.67 Mi l l i g a n , 1987; SW flounder) 7.80 7. 22 A Pco2 gradient— 1/2 (Pacoz + PVcoz) - 1/2 (P I CD2 T P E C D 2 ) * the Pco2 gradient i s estimated i n these f i s h assuming water Pco2= 0 and PaCOs i s approximately equal to mean blood Pco2. # exercise data from Wood & M i l l i g a n (1987) was used instead of sole exercise data (Chapter 1) because increases i n Pcoz are observed immediately following exhaustive exercise, while the sole e x p e r i -ments samples were taken 30 min following exercise. between freshwater and seawater i s that for f i s h i n seawater the l i n k between NH3 d i f f u s i o n and CO2 e x c r e t i o n across the g i l l s i s l e s s important. In other words, the f a c i l i t a t i o n of NH3 d i f f u s i o n by the CO2 h y d r a t i o n r e a c t i o n i n the boundary l a y e r w i l l be minimal i n seawater where H+ ions w i l l be q u i c k l y absorbed by other b u f f e r s . I t i s not s u r p r i s i n g , t h e r e f o r e , that i n Opsanus beta and Mvoxocephalus octodecimspinosus there i s no evidence for NH3 d i f f u s i o n across the g i l l ( G o l d s t e i n §_t a l . , 1982; Claiborne §£ al.. , 1982). Furthermore, changes i n blood-to-water pH g r a d i e n t s , which would be expected to a l t e r PNH3 g r a d i e n t s , have no a f f e c t on ammonia e x c r e t i o n i n the seawater S_. s t e l l a r i s , S_. c a n i c u l a , and Conger conger (Payan & Maetz, 1973; H e i s l e r , 1984). An a n a l y s i s of ammonia l e v e l s i n g i l l t i s s u e and boundary water l a y e r i s p o s s i b l e using the estimated mean water boundary l a y e r pH values (Figure 21). I f one assumes t h a t , l i k e C O 2 , the NH3 d i f f u s i o n c o e f f i c i e n t s i n water and i n t r a c e l l u l a r f l u i d are s i m i l a r and PjjH3 l e v e l s from blood to bulk phase water decrease with d i s t a n c e from the blood, then for a blood-to-water PNH3 gradient of 124 uTorr, PNH3 i n t n e 9 i H e p i t h e l i u m w i l l be about 83 uTorr and i n the boundary l a y e r , 42 uTorr. G i l l i n t r a c e l l u l a r ammonia l e v e l s are probably determined by the membrane p o t e n t i a l (Em) across the basal membrane, as they are i n other f i s h t i s s u e s (Chapter 1). This i s because the a c t i v i t y of Na+/K+ ATPase i n g i l l c e l l s i s r e l a t i v e l y high (Karnaky, 1980) and the Na+/K+ ATPase i n h i b i t o r , ouabain, decreases ammonia 197 Figure 21. A schematic representation of a cross-section through the g i l l epithelium showing measured mean arterial-venous blood and inspired-expired water parameters (taken from Chapter 4, XL. vivo trout experiments), c a l c u l a t i o n s of boundary water and g i l l c e l l pH, and c a l c u l a t i o n s of ammonia l e v e l s i n the g i l l c e l l and water boundary layer. TEP represents the t r a n s e p i t h e l i a l p o t e n t i a l . mean inspired expired water water boundary layer PH P (Torr) C0 2  TAmm ,r , | l l , , HH3 Em TEP (uTorr) (mV) 7.66 0.2 4 1 6.75 1.1 1446 42 60 -gil l epithelium mean arterial venous blood 7.83 2.7 461 125 to 00 e x c r e t i o n across f i s h g i l l s (Payan, 1978; Claiborne et^ a l . . , 1982; Cameron, 1986), i n d i c a t i n g the replacement of N H 4 + f o r K+ ions i n t h i s a c t i v e exchange mechanism at the basal membrane. To c a l c u l a t e g i l l i n t r a c e l l u l a r ammonia l e v e l s from the Nernst equation (13), an Em value of -40 mV was assumed which i s the d i f f e r e n c e between the measured t r a n s e p i t h e l i a l p o t e n t i a l (blood-to-water= -20 mV; Iwama, 1986) and the measured w a t e r - t o - g i l l c e l l p o t e n t i a l (-60 mV; R a n d a l l , unpublished data). With these values g i l l i n t r a c e l l u l a r ammonia co n c e n t r a t i o n was c a l c u l a t e d to be 2360 umol.l -^-. G i l l c e l l pHi was then c a l c u l a t e d from the r a t i o of N H 3 : N H 4 + with the Henderson-Hasselbalch equation (3). There are no published values of g i l l c e l l pHi to compare with t h i s c a l c u l a t e d value of 6.94, however, because g i l l c e l l s have a r e l a t i v e l y high p e r m e a b i l i t y to H+ ions (McWilliams & P o t t s , 1978) i t i s expected that g i l l e p i t h e l i u m pH w i l l be lower than other i n t r a c e l l u l a r compartments (- 7.2-7.4). Furthermore, i f the estimated Em value of -40 mV i s a good approximation of g i l l c e l l membrane p o t e n t i a l , then t h i s value i s c l o s e r to measured red c e l l Em (-30 mV, Lassen, 1977), where H+ ions are known to be p a s s i v e l y d i s t r i b u t e d (Lassen, 1977; Heming ejt §1., 1986; Nikinmaa et a l . , 1987; Heming gt aj.., 1987), than to other t i s s u e s (Em -70 to -90 mV) where H+ ions are r e l a t i v e l y impermeable (Roos & Boron, 1981). Boundary l a y e r t o t a l ammonia l e v e l s were c a l c u l a t e d assuming that the a p i c a l g i l l membrane i n freshwater f i s h i s r e l a t i v e l y 200 impermeable to NH4+ ions (Kormanik & Cameron, 1981a; Wright & Wood, 1985), as i t i s to other i o n species ( G i r a r d & Payan, 1980). With the estimated mean boundary water pH (pH= 6.75) and mean P N H3 l e v e l ( 4 2 uTorr) the boundary l a y e r ammonia co n c e n t r a t i o n was c a l c u l a t e d with the Henderson-Hasselbalch equation (3) to be 1446 u m o l . l 1 . On the other hand, i f the a p i c a l membrane was h i g h l y permeable to NH4 + i o n s , then boundary water ammonia l e v e l s c a l c u l a t e d from the Nernst equation would be 964 u m o l . l - 1 . As s t a t e d above, however, i t i s u n l i k e l y that a p i c a l membrane NH4+ p e r m e a b i l i t y i s s i g n i f i c a n t i n freshwater f i s h . Moreover, f o r the boundary l a y e r to be maintained as a d i s t i n c t "microclimate", the a p i c a l membrane must be r e l a t i v e l y impermeable to ion species (see Chapter 4). The above a n a l y s i s of ammonia l e v e l s f i t s i n with accepted theory of ammonia t r a n s f e r i n freshwater f i s h . Ammonia may d i f f u s e as NH3 across the g i l l s down the blood-to-water PjjH3 gradient i n t o the i n t e r l a m e l l a r water where an a c i d i c boundary lay e r maintains low NH3:NH4+ r a t i o s . A l t e r n a t i v e l y , NH4+ ions may cross the g i l l s i n exchange for Na+ ions down the NH4 + g i l l c e l l - t o - w a t e r g r a d i e n t . Ammonia concentrations i n the g i l l e p i t h e l i u m would be maintained by a s h u t t l i n g of NH4+ ions across the b a s o l a t e r a l membrane, as ammonia was l o s t to the environment across the a p i c a l membrane. Unl i k e the membrane p o t e n t i a l of most body c e l l s , t r a n s e p i t h e l i a l p o t e n t i a l s across f i s h g i l l s vary widely with changing water c o n d i t i o n s (Iwama, 1986; Ye, 1986; McWilliams & P o t t s , 1978; K e r s t e t t e r et a l . , 201 1970). Changes i n t r a n s e p i t h e l i a l p o t e n t i a l may d i r e c t l y a f f e c t ammonia t r a n s f e r by a l t e r i n g NH4 + e l e c t r o c h e m i c a l g r a d i e n t s , or i n d i r e c t l y a f f e c t ammonia f l u x by a l t e r i n g g i l l i n t r a c e l l u l a r ammonia concentrations. The demonstrated l i n k a g e between ammonia and carbon di o x i d e e x c r e t i o n i n t h i s study makes i t necessary to re-evaluate the experimental i n t e r p r e t a t i o n of e a r l i e r s t u d i e s concerning ammonia e x c r e t i o n where experimental treatments may have induced changes i n CO2 e x c r e t i o n , and the r e f o r e chemical r e a c t i o n s i n the boundary l a y e r . For inst a n c e , Payan and Maetz (1973) i n j e c t e d ammonium s a l t s i n t o d o g f i s h (S. c a n i c u l a ) and observed a decrease i n b r a n c h i a l ammonia e f f l u x a f t e r an i n j e c t i o n of the carbonic anhydrase i n h i b i t o r , acetazolamide. Payan and Matty (1975) removed carbon d i o x i d e from the s a l i n e p e r f u s i n g i s o l a t e d t r o u t heads and found that ammonia e f f l u x was s i g n i f i c a n t l y reduced. In both these s t u d i e s , the r e d u c t i o n i n ammonia e x c r e t i o n may be p a r t l y explained by the i n h i b i t i o n of CO2 e x c r e t i o n across the g i l l s , which would reduce the amount of H+ ions a v a i l a b l e i n the g i l l water boundary l a y e r and eleva t e water P(jH3 tensions. I t i s a l s o f e a s i b l e that the observed decrease i n ammonia e x c r e t i o n i n these s t u d i e s was a l s o p a r t l y due to a r e d u c t i o n i n Na+/NH4+ exchange. Payan and Matty (1975) poin t out that reduced CO2 f l u x across the g i l l s would reduce the a v a i l i b i l i t y of H+ ions i n the g i l l e p i t h e l i u m , which combine with NH3 to supply NH4+ as a counterion for the Na+ uptake mechanism. Although t h i s l a t t e r e x p l a n a t i o n i s a l s o a v i a b l e one, the s i g n i f i c a n c e of the 2 0 2 c a t a l y s e d CO2 h y d r a t i o n r e a c t i o n i n the boundary l a y e r to ammonia e x c r e t i o n was not p r e v i o u s l y recognized. The p h y s i o l o g i c a l s i g n i f i c a n c e of a d i s t i n c t water pH microclimate at the g i l l surface to ammonia e x c r e t i o n w i l l vary depending on the p h y s i o l o g i c a l s t a t e of the animal and water c o n d i t i o n s . For insta n c e , i f water pH at the g i l l surface was suddenly increased to i n s p i r e d water values (pHj= 8 . 0 ) , which might occur i f e x t e r n a l g i l l carbonic anhydrase was removed and the CO2 h y d r a t i o n r e a c t i o n was completely uncatalysed, then PjjH3 l e v e l s i n the g i l l boundary l a y e r would be 7 6 0 uTorr, instead of 4 2 uTorr, as p r e d i c t e d i n F i g . 2 1 . Such a high P(JH3 l e v e l i n the water would make ammonia e x c r e t i o n very d i f f i c u l t . In the short term, a r e v e r s a l of the blood-to-water P N H 3 gradient would r e s u l t i n an uptake of ammonia. To maintain steady s t a t e e x c r e t i o n , however, blood ammonia l e v e l s would have to exceed water ammonia l e v e l s . Blood PjjH3 l e v e l s of 8 0 0 uTorr would be t o x i c to the f i s h (see Randall & Wright, 1 9 8 7 ) , and th e r e f o r e , carbonic anhydrase at the g i l l surface may be of considerable importance to a l l a q u a t i c animals that excrete s i g n i f i c a n t amounts of ammonia. In more a l k a l i n e waters (pH= 9 . 5 , (see chapter 4 ) , P N H 3 l e v e l s i n the boundary l a y e r could be as high as 1 2 , 0 0 0 uTorr (given boundary water Tamm l e v e l s i n F i g . 2 1 ) . One would p r e d i c t that f i s h l i v i n g i n a l k a l i n e waters maintain high r a t e s of carbon d i o x i d e e x c r e t i o n and carbonic anhydrase a c t i v i t y a t the g i l l surface to ensure adequate removal 203 of ammonia from the body. Another i n t e r e s t i n g s i t u a t i o n i s the e f f e c t s of CO2 e x c r e t i o n on ammonia e f f l u x i n s t a r v i n g f i s h . H i l l a b y and Randall (1979) r e p o r t that blood ammonia l e v e l s p r o g r e s s i v e l y increase from day 2 to day 12 of s t a r v a t i o n , a cu r i o u s phenomena i n unfed f i s h . B r e t t and Zala (1975) found that oxygen consumption decreased i n starved f i s h , and t h i s i m p l i e s that there was a l s o a concurrent r e d u c t i o n i n carbon d i o x i d e e x c r e t i o n . I t may be that the increase i n blood ammonia l e v e l s was a compensatory mechanism to increase blood-to-water P N H3 g r a d i e n t s i n the face of reduced CO2 e x c r e t i o n . In c o n c l u s i o n , ammonia t r a n s f e r i n f i s h i s dependent on both NH4+ e l e c t r o c h e m i c a l and NH3 p a r t i a l pressure g r a d i e n t s . Changes i n t i s s u e membrane p o t e n t i a l s or g i l l t r a n s e p i t h e l i a l p o t e n t i a l w i l l a l t e r i n t r a c e l l u l a r ammonia sto r e s and may r e s u l t i n a l t e r a t i o n s of ammonia e f f l u x across the g i l l . Changes i n red c e l l membrane p o t e n t i a l Q X transmembrane pH gradients w i l l a l t e r the i n t r a c e l l u l a r ammonia content, since H + ions are p a s s i v e l y d i s t r i b u t e d between plasma and red c e l l s . Ammonia may be e l i m i n a t e d across the g i l l s of freshwater f i s h as both NH3 and N H 4 + ; the r a t e of NH3 d i f f u s i o n i s i n f l u e n c e d by the a v a i l a b i l i t y of H + ions i n the g i l l water boundary l a y e r . The a v a i l a b i l i t y of H+ ions i s dependent on the r a t e of C O 2 / N H 3 e x c r e t i o n across the g i l l , the rate of the CO2 h y d r a t i o n r e a c t i o n i n the boundary l a y e r , and the b u f f e r i n g c a p a c i t y of the bulk water, among other t h i n g s . The i d e n t i f i c a t i o n of the 204 i n t e r a c t i o n between CO2 h y d r a t i o n and NH3 d i f f u s i o n i n the water boundary l a y e r may have many p h y s i o l o g i c a l consequences and opens new areas of i n v e s t i g a t i o n of gas t r a n s f e r i n f i s h . Furthermore, the g i l l water boundary i s a common component of many t r a n s f e r processes and there are many i n t e r e s t i n g problems to address i n future research concerning the a b i l i t y of aquatic animals to maintain a g i l l microenvironment d i s t i n c t from the general environment. APPENDIX I 205 C h a r a c t e r i s t i c s of the i n i t i a l s a l i n e used to perfuse t r o u t heads while on the operating t a b l e . pH = 7.86 + 0.02 (N=26) CC02 = 5.00 +0.02 (N=28) mmol.l - 1 Tamm = 1028 +44 (N=32) u m o l . l - 1 P r o t e i n = 2.2 + 0.0 (N=28) % 206 APPENDIX I I The thickness of the g i l l water boundary l a y e r , s^]., can be c a l c u l a t e d as o u t l i n e d by P i i p e r et al.. (1986) by the f o l l o w i n g formula: (27) S f c i = d x oc x A/Dw where d i s the O2 d i f f u s i o n c o e f f i c i e n t at 10° C i n water, c< i s the O2 s o l u b i l i t y c o e f f i c i e n t , A i s the secondary lammellar surface area (see Table 21 f o r c o n s t a n t s ) . D w i s the d i f f u s i n g c a p a c i t y of i n t e r l a m e l l a r water, where (28) D w = Vw x <* x In ( 1/ £ ) and Vw i s the v e n t i l a t o r y water flow, and £. i s the e q u i l i b r a t i o n i n e f f i c i e n c y . £ d e f i n e s the f r a c t i o n of r e s p i r a t o r y water that i s shunted past the r e s p i r a t o r y s u r f a c e , while the remainder of water e q u i l i b r a t e s with the secondary lamellae. (29) £ = P E Q 2 ~ PP Pl02 " P° P I 0 2 a n d P I 0 2 a r e t n e oxygen tensions of i n s p i r e d and mixed expired water and Po i s P Q 2 a t t n e secondary l a m e l l a r surface which i s constant. Scheid and P i i p e r (1971) have r e l a t e d £ to 207 TABLE 21. Values used to c a l c u l a t e the boundary layer thickness. See Appendix I I for d e t a i l s . PARAMETER VALUES REFERENCE d 0.0011 cm 2.min - 1 P i i p e r et. al_. , 1986 (extrapolated 10° C) C\ 0.00224 mmol.l- 1.torr- 1 B o u t i l i e r g£ a l . , 1986 A 837.8 cm 2 for 400g f i s h Hughes, 1980 b 0.0115 mm Hughes, 1966 1 0.7 mm " h 0.0 7 mm " N 380,487 calculated from data taken from Hughes & Morgan, 1973 and Hughes, 1980 208 the dimensionless e q u i l i b r a t i o n r e s i s t a n c e i n d e x , ^ • They have shown that the value of (p f o r a c e r t a i n geometry of the secondary lamellae and water v e l o c i t y p r o f i l e through the i n t e r l a m e l l a r channels can be described as a f u n c t i o n of : (30) <p = b 2_ x v 1 x d where b i s one h a l f the i n t e r l a m e l l a r d i s t a n c e , v i s the mean water v e l o c i t y , 1 i s the length of the secondary lamellae at the base. I f water c o n d i t i o n s for 0 2 e q u i l i b r a t i o n across the g i l l are poor, then (p w i l l be l a r g e . F i g . 22 shows t h i s r e l a t i o n s h i p , redrawn from P i i p e r e_t aJL.. (1986). The mean v e l o c i t y was c a l c u l a t e d : (31) v = vw/F F denotes the t o t a l c r o s s - s e c t i o n a l area of the i n t e r l a m e l l a r spaces: (32) F = 2b x h x N where h i s the mean height of the secondary l a m e l l a and N i s the t o t a l number of secondary lamellae. The <p value was c a l c u l a t e d and an approximate £ value was read from F i g . 22 between the curves f o r the two d i f f e r e n t 209 Figure 22. P l o t of ' e q u i l i b r a t i o n i n e f f i c i e n c y ' , ^ (equation 29), against ' e q u i l i b r a t i o n resistance index', <p (equation 30). Abscissa {(p), logarithmic; ordinate (£), l i n e a r . The two curves are for a rectangular lamella (top diagram) and for a trapezoidal lamella, of same base length, but tapering to one-half the base length at the top edge (bottom diagram). Estimates of €. were taken midway between the two curves (see P i i p e r e_£. al.., 1986) for calculated values i n r e s t i n g and active f i s h . Figure redrawn from P i i p e r e_fc. al- - 1986. 210 1 1 • I 1 I • 111 1 1—I l l | i 11 1 i i —] •1 1 10 V geometric gill models proposed by P i i p e r g_t al.. (1986). In dogfish, the measured £ versus Rvalues were situated halfway between the two theoretical geometric models and therefore, it is assumed that similar situation occurs in trout because gas transfer rates between the two species are not unalike. Dw and S b i were then calculated. REFERENCES 212 A i c k i n , C.C. & Thomas, R.C. (1977). An i n v e s t i g a t i o n of the i o n i c mechanisms of i n t r a c e l l u l a r pH r e g u l a t i o n i n mouse soleus muscle f i b r e s . J . P h y s i o l . Lond. 273, 295-316. A r i l l o , A., Margiocco, C , Melodia, F., Mensi, P., & Schenone, G. (1981). 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