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CO₂ excretion and acid-base regulation in the rainbow trout, Salmo gairdneri Haswell, Monty Stephen 1978

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C 0 2 EXCRETION AND ACID-BASE REGULATION IN THE RAINBOW TROUT, SALMO GAIRDNERI by MONTY STEPHEN HASWELL B.S., C a l i f o r n i a S t a t e U n i v e r s i t y , Long Beach 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR.THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH.COLUMBIA June, 1978 (T) Monty Stephen H a s w e l l , 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requ i rement s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f Z o o l o g y The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e 9 August 1978 ABSTRACT The r o l e of carbonic anhydrase i n carbon dioxide excre-t i o n and acid-base regulation i n the rainbow trout, Salmo  gairdneri has been investigated. While a s i g n i f i c a n t amount of carbonic anhydrase was found i n the blood of the trout, calculations based on red c e l l hemolysates suggest that the probable c i r c u l a t i n g l e v e l s of carbonic anhydrase a c t i v i t y i n blood may not be s u f f i c i e n t to account for the observed carbon dioxide excretion. An analysis of carbonic anhydrase a c t i v i t y i n whole blood from the trout revealed that i n t a c t f i s h erythrocytes, unlike mammalian erythrocytes t o t a l l y f a i l to f a c i l i t a t e the dehydration of e x t r a c e l l u l a r bicarbonate. The possible mechanism of t h i s phenomenon has been examined; however the s a l i e n t point was that f i s h red blood c e l l s do not appear capable and therefore by implication apparently not necessary for the excretion of carbon dioxide at the g i l l s of trout. The observed excretion of carbon dioxide i n the trout was found to be accounted f o r by the g i l l s and t h e i r compli-ment of carbonic anhydrase. This finding was based on the following observations. (1) Depletion of c i r c u l a t i n g blood carbonic anhydrase leve l s during severe anemia was without effect on carbon dioxide excretion rates or blood acid-base status. (2) I n t r o d u c t i o n o f the c a r b o n i c anhydrase i n h i b i t o r , diamox i n t o anemic f i s h produced a severe acid-base d i s t u r b a n c e a s -s o c i a t e d w i t h a f a l l i n observed carbon d i o x i d e e x c r e t i o n . (3) I s o l a t e d perfused g i l l p r e p a r a t i o n s excrete carbon d i o x i d e a t r a t e s comparable to those observed i n v i v o from f r e e swim-ming f i s h . (4) Carbon d i o x i d e e x c r e t i o n i n i s o l a t e d g i l l p r e p a r a t i o n s i s a b o l i s h e d by diamox. The e x c r e t i o n of carbon d i o x i d e i n f i s h occurs v i a the movement of plasma b i c a r b o n a t e i n t o the b r a n c h i a l e p i t h e l i u m , where i t i s subsequently dehy-drated i n t o molecular carbon d i o x i d e and excreted. A model i s proposed and s u p p o r t i v e evidence presented to account f o r the c o u p l i n g of i o n i c exchange o c c u r r i n g a c r o s s the g i l l with carbon d i o x i d e e x c r e t i o n . The proposed model d i s t i n g u i s h e s between c o n t r o l o f plasma hydrogen i o n a c t i v i t y and r e g u l a t i o n of plasma t o t a l carbon d i o x i d e c o n c e n t r a t i o n per se. The f u n c t i o n a l s i g n i f i c a n c e of t h i s p a t t e r n of carbon d i o x i d e e x c r e t i o n f o r a q u a t i c animals i s d i s c u s s e d along with the i m p l i c a t i o n s f o r a i r b r e a t h i n g f i s h . : i v TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 CHAPTER 1 "Carbonic Anhydrase i n the Trout" 13 Introduction 14 Methods 19 Results 25 Discussion 28 CHAPTER 2 "Carbonic Anhydrase i n Red Blood C e l l s " 33 Introduction 34 Methods 38 Results 40 Discussion 45 CHAPTER -...3 " C 0 2 Excretion i n the Trout" 54 Introduction 55 Methods 56 Results 61 Discussion 63 CHAPTER 4 "Acid-Base Regulation i n the Trout" 68 Introduction 69 Methods 71 Results 74 Discussion 77 GENERAL DISCUSSION 85 REFERENCES CITED 99 V LIST OF FIGURES FIGURE Page #1 - Open C0 2 System ?a #2 - Carbonic anhydrase isozymes 25a #3 - E f f e c t of Diamox on carbonic anhydrase 26a #4 - T i f o r uncatalyzed dehydration reaction 36a #5 - Tracing from a t y p i c a l carbonic anhydrase assay 40a #6 - Rat blood carbonic anhydrase a c t i v i t y 40c #7 - Washed trout blood enzyme a c t i v i t y 42a #8 - E f f e c t of removing plasma on the dehydration 42c a c t i v i t y of trout blood #9 - Red c e l l a l k a l i n i z a t i o n rates of trout blood 43a #10 - pHa versus Hematocrit i n trout 62c #11 - PaC0 2 versus Hematocrit i n trout 62e #12 - Carbon dioxide excretion i n trout 64a #13 - C0 2 excretion i n the perfused g i l l preparation 74a #14 - E f f e c t of SCN on chloride i n f l u x i n trout 75a #15 - E f f e c t of SCN on branchial carbonic anhydrase 75g #16 - E f f e c t of SITS on chloride i n f l u x i n trout 76a #17 - Pattern of C0 2 and s a l t movement through the 78a teleo s t g i l l presented diagramatically #18 - E f f e c t of infusion of carbonic anhydrase on 96a blood acid-base status i n the a i r breathing f i s h Hoplerythrinus 1 INTRODUCTION 2 The b i o l o g i c a l s i g n i f i c a n c e cf carbon dioxide has long been appreciated and p o s s i b l y has not been more elo q u e n t l y s t a t e d than by L.J. Henderson i n 1913 i n "The F i t n e s s of the Environment" (Chapter 4,p. 133): "Two chemical i n d i v i d u a l s stand alone i n importance f o r the great b i o l o g i c a l c y c l e upon the earth. The one i s water, the ether carbon d i o x i d e . These two simple substances are the common source of every one of the complicated substances which are produced by l i v i n g beings and they are the common endproducts of the wearing away of a l l the c o n s t i t u e n t s of protoplasm, and the d e s t r u c t i o n of these m a t e r i a l s which y i e l d energy to the body." Not only i s C02 a major endproduct of a l l aerobic metabolic pathways, i t al s o forms the b u i l d i n g blocks,of photosynthesis by p l a n t s , which i s v i t a l f o r the replenishment of environmental oxygen. C02 i n the gaseous s t a t e behaves much l i k e any other gas and i s found to obey the p h y s i c a l gas laws. When C02 enters the aguecus medium the e g u i l i b r i u m r e a c t i o n can be described by eguation #1. Equation #1 C02 + H20 = H2C03 = HC03 + H = C03 + H Eguation #1a C02 + H20 = HC03 + H = C03 + H Because the i o n i z a t i o n of H2C03 i s so r a p i d the net eguation can be reduced to eguation # 1a. at n e u t r a l pH's i n buffered s o l u t i o n s , HC03 i s the dominant molecular species comprisinq approximately 95% of the t o t a l CO2 (TC0 2) while the remainder i s p r i m a r i l y molecular C02 with a s m a l l amount of C03. At a c i d i c pH's the dominant species becomes molecular C02 and, conversely at a l k a l i n e pH's the C03 becomes s i g n i f i c a n t , Molecular C02 i s not an a c i d s t r i c t l y speaking; however i n a f u n c t i o n a l sense the 3 a d d i t i o n o r r e m o v a l o f C 0 2 i n s o l u t i o n s a t p h y s i c l o g i c a l p H * s i s e q u i v a l e n t t o a d d i n g o r r e m o v i n g h y d r o g e n i o n s , T h u s , f o r a n y o r g a n i s m , C 0 2 I s n o t m e r e l y t h e m e t a b o l i c e n d p r o d u c t w h i c h m u s t be r e m o v e d , i t i s a l s o a h i g h l y r e a c t i v e c h e m i c a l m o i e t y c a p a b l e o f a l t e r i n g pH a n d i o n i c a n d o s m o t i c s t r e n g t h a s w e l l a s b u f f e r i n g c a p a c i t y . T h e r e f o r e i t i s n ' t s u r p r i s i n g t h a t m o s t o r g a n i s m s e x e r c i s e c o n t r o l o v e r t h e t r a n s p o r t a n d u l t i m a t e e x c r e t i o n o f t h i s m o l e c u l e . M o v e m e n t o f C 0 2 i n t o o r o u t o f a n a q u e o u s s o l u t i o n i n v o l v e s a c h a n g e i n pH. I f C 0 2 i s b u b b l e d i n t o a s o l u t i o n t h e pH w i l l n e c e s s a r i l y f a l l a c c o r d i n g t o e g u a t i o n #1a. E g u a l l y t r u e , t h e w h o l e s a l e r e m o v a l o f C 0 2 f r o m a n y s o l u t i o n w i l l d r i v e t h e pH u p , a l l e t h e r f a c t o r s b e i n g e q u a l . I n b i o l o g i c a l s y s t e m s C 0 2 i s c o n t i n u a l l y b e i n g a d d e d f r o m t h e r e s p i r i n g t i s s u e s ; h o w e v e r i f t h a t q u a n t i t y o f C02 i s r e m o v e d a t a n e q u a l r a t e no n e t c h a n g e i n pH w i l l o c c u r . H o w e v e r i t s h o u l d b e a p p a r e n t t h a t i f C 0 2 o u t p u t f r o m t h e t i s s u e s i s i n c r e a s e d w i t h o u t a c o n c o m i t a n t i n c r e a s e i n t h e r a t e o f C 0 2 e x c r e t i o n a n e t a c i d o s i s w i l l o c c u r . C o n v e r s e l y , a d e c r e a s e i n C 0 2 p r o d u c t i o n o r e x c e s s i v e e x c r e t i o n r a t e w i l l r e s u l t i n a n e t a l k a l o s i s . I f C 0 2 e x c r e t i o n i s m a t c h e d w i t h C 0 2 i n p u t f r o m t h e t i s s u e s pH d i s t u r b a n c e s w i l l b e m i n i m i z e d . T h i s s y s t e m , a s i t o c c u r s i n mammals, i s f a i r l y w e l l c h a r a c t e r i z e d a n d p r o v i d e s a u s e f u l b a s i s f o r f u t u r e c o m p a r i s o n s w i t h o t h e r a n i m a l g r o u p s . I n b l o o d t h e b u l k o f C 0 2 i s c a r r i e d a s b i c a r b o n a t e i n t h e p l a s m a . 9 h e n b l o o d r e a c h e s t h e l u n g s o f mammals o r b i r d s t h e p l a s m a b i c a r b o n a t e i s s h u t t l e d i n t o t h e e r y t h r o c y t e i n e x c h a n g e f o r a c h l o r i d e i o n ( r e f e r r e d t o a s t h e H a m b u r g e r o r c h l o r i d e 4 s h i f t ) , where i t i s r a p i d l y dehydrated i n t o C02 v i a c a r b o n i c anhydrase which r e s i d e s i n the e r y t h r o c y t e . CO 2 then d i f f u s e s out f o l l o w i n g i t s c o n c e n t r a t i o n g r a d i e n t . In a d d i t i o n a p o r t i o n of the C02 i s c a r r i e d i n d i r e c t combination with the hemoglobin, r e f e r r e d t o as carbami.no C02. Estimates of the importance o f carbamino C02 vary but i t probably does not c o n s t i t u t e more than 30% of the t o t a l C02 removed (Bauer ,1972). In examining C02 e x c r e t i o n i n f i s h , fundamental d i f f e r e n c e s from t h e i r mammalian and avian c o u n t e r p a r t s are r e a d i l y apparent. F i r s t , f i s h are c h a r a c t e r i z e d by very low C02 t e n s i o n s i n the blood, t y p i c a l l y only 1-2 mm Hg above i n s p i r e d l e v e l s compared to 30-50 mm Hg f o r mammals and b i r d s . In mammals and b i r d s the C02 g r a d i e n t s from a i r t o blood can be a l t e r e d by changes i n v e n t i l a t i o n . Thus an i n c r e a s e d v e n t i l a t i o n r a t e w i l l tend to i n c r e a s e the C02 g r a d i e n t across the lung, r e s u l t i n g i n an i n c r e a s e d C02 l o s s . The converse i s e g u a l l y t r u e , whereby a decrease i n v e n t i l a t i o n w i l l cause a r e t e n t i o n of C02. The net r e s u l t i s t h a t adjustments i n pH can be accomplished by r e g u l a t i n g a r t e r i a l C02 te n s i o n s (PaC02) v i a changes i n v e n t i l a t i o n . T h i s i s not the case f o r t e l e o s t s , changes i n v e n t i l a t i o n dc not a l t e r the C02 g r a d i e n t s or pH (Randall 6 Jones ,1973; R a n d a l l ,unpublished o b s e r v a t i o n s ) , and pH i s r e g u l a t e d v i a changes i n bicarbonate l e v e l s and not PaC02 (Randall S Cameron ,1973; Janssen S Ra n d a l l ,1975; Cameron & Randall ,1972). A second p o i n t f o r comparison i s t h a t , i n f i s h , at l e a s t a p o r t i o n o f e x p i r e d C02 can be l i n k e d to i o n i c uptake across the g i l l , f o r example HC03/C1 exchange (Maetz et a l ,1976). In a d d i t i o n R a n d a l l e t a l (1976) demonstrated that the 5 d o g f i s h i s capable of taking-up HC03 from seawater across the g i l l i n order to f a c i l i t a t e pH r e g u l a t i o n d u r i n g hypercapnia. Thus i n f i s h i t appears that C02 movement across the r e s p i r a t o r y e p i t h e l i u m may not be as " p a s s i v e " as the mammalian/avian system. F i s h e x e r c i s e some degree of c o n t r o l over the form of excreted C02 (HC03 or C02) and even i n the d i r e c t i o n of movement. However a d e f i n i t i v e answer as to how C02 i s excreted and the s i g n i f i c a n c e of bicarbonate i n o v e r a l l C02 e x c r e t i o n as w e l l as i o n i c and pH r e g u l a t i o n remains obscure. So f a r i t has been pointed out t h a t pfl r e g u l a t i o n occurs and that C02 has an e f f e c t on pH. Only s l i g h t changes i n pH can cause l a r g e f l u c t u a t i o n s i n the r a t e s of chemical r e a c t i o n s , some being depressed while others may be a c c e l e r a t e d . Most chemical r e a c t i o n s c a r r i e d out i n b i o l o g i c a l systems are mediated by enzymes. A l l p r o t e i n s , i n c l u d i n g enzymes, are a f f e c t e d by changing hydrogen i o n c o n c e n t r a t i o n s . The hydrogen io n c o n c e n t r a t i o n , among other v a r i a b l e s , can a l t e r the f u n c t i o n a l s t a t e of enzymes v i a d i s s o c i a t i o n and/or weak bond i n t e r a c t i o n s which u s u a l l y r e s u l t s i n a c o n f o r m a t i o n a l o r s t r u c t u r a l change i n the enzyme. While enzymes may be more or l e s s s u s c e p t a b l e to an a l t e r e d pH, most p r o t e i n s do have a pH where the charge d i s t r i b u t i o n y i e l d s a s p e c i f i c three dimensional conformation where enzyme a c t i v i t y i s optimized. Shen the pH i s changed the degree of d i s s o c i a t i o n may be a l t e r e d so t hat t h i s t hree dimensional c o n f i g u r a t i o n i s v a r i e d , and d e t r i m e n t a l or even l e t h a l e f f e c t s on enzyme a c t i v i t y may occur. However, i t should be noted t h a t unless a constant temperature i s maintained an i n c r e a s e i n hydrogen i o n a c t i v i t y may not 6 n e c e s s a r i l y r e f l e c t an a c i d o s i s jger se. For example, while at 25 C pH 7.0 may r e f l e c t the pH of n e u t r a l i t y , at 10 C pH 7.0 i s no l o n g e r a n e u t r a l s o l u t i o n but r a t h e r a c i d i c . In f a c t i t has been demonstrated that p o i k i l o t h e r m i c animals r e g u l a t e the r e l a t i v e a l k a l i n i t y {OH/H r a t i o ) , not hydrogen ion l e v e l s £er se { Rahn and Baumgardner ,1972; Howell et a l , 1970), Given the s i t u a t i o n where an organism needs to maintain the d i f f e r e n c e between pH and n e u t r a l i t y at a constant l e v e l , what general mechanisms are a v a i l a b l e to i t ? F i r s t , a l l the body f l u i d s are s u p p l i e d with acid-base " b u f f e r systems" which act as the f i r s t l i n e of defence and are a l s o the most r a p i d to respond to a pH s t r e s s . As an example and to examine the importance of b i c a r b o n a t e i n the b u f f e r i n g of b i o l o g i c a l l y a c t i v e systems a b r i e f l o c k at the b u f f e r i n g c a p a c i t y of human blood may be i n f o r m a t i v e . S e v e r a l i n d i v i d u a l b u f f e r i n g systems can be i s o l a t e d w i t h i n blood, such as albumin, b i c a r b o n a t e , g l o b i n s (HHb and Hb02), i n o r g a n i c and o r g a n i c phosphates. A l l these systems combine to give the blood b u f f e r i n g c a p a c i t y . T h i s i s not to imply these b u f f e r i n g c a p a c i t i e s are e q u a l l y d i s t r i b u t e d i n whole blood, that i s plasma and/or e r y t h r o c y t e s . The approximate c o n t r i b u t i o n of each has been computed by Winters 6 D e l l (1965) , see t a b l e #1. .Generally these b u f f e r s are c l a s s i f i e d i n t o two major groups b i c a r b o n a t e b u f f e r s and non-bicarbonate b u f f e r s . Bicarbonate b u f f e r s account f o r approximately 53% of the t o t a l b u f f e r i n g c a p a c i t y i n mammalian blood. C l e a r l y , b e s i d e s the c o n t r i b u t i o n of hemoglobin, almost a l l the b u f f e r i n g c a p a c i t y r e s i d e s i n the b i c a r b o n a t e system. Furthermore almost t o t a l r e g u l a t i o n cf pH ,6a TABLE #1. A p p r o x i m a t e c o n t r i b u t i o n o f i n d i v i d u a l b u f f e r s t o t o t a l b u f f e r i n g i n whole b l o o d . From W i n t e r s & D e l l ( 1 9 6 5 ) . "INDIVIDUAL BUFFERS PERCENT BUFFERING . IN WHOLE BLOOD hemoglobin and Oxyhemoglobin 35 Organic Phosphates 3 Inorganic Phosphates 2 Plasma Proteins 7 "Plasma Bicarbonate 35 Erythrocytic Bicarbonate 18 7 l i e s w i t h i n the bicarb o n a t e system when c o n s i d e r i n g the almost l i m i t l e s s supply of C02 to the blood from the body t i s s u e s . The f u n c t i o n a l r e l a t i o n s h i p can perhaps be b e t t e r a p p r e c i a t e d by l o o k i n g at the Henderson-Hasselbalch eguation (eguation #2), and by c o n s i d e r i n g the C02 system as dep i c t e d i n f i g u r e #1. Equation #2 pH = pK + l o g f HC03 ]/[ C021 By viewing the diagram and the Henderson-Hasselbalch eguation i t can be a p p r e c i a t e d t h a t three mechanisms of a l t e r i n g pH e x i s t s a t s t e a d y - s t a t e C02 production. Namely, e i t h e r an organism can change d i s s o l v e d C02 (C02(d)} content of the system, or i t can a l t e r the bicarbonate c o n c e n t r a t i o n , or both c o u l d be r e g u l a t e d . As s t a t e d p r e v i o u s l y i t appears that b i r d s and mammals a d j u s t CO 2(d) v i a changes i n v e n t i l a t i o n , while f i s h appear to r e g u l a t e b i c a r b o n a t e l e v e l s . The mechanism of t h i s mode of bicarbo n a t e r e g u l a t i o n i s not c l e a r . A n a l y s i s of gas exchange i n an agueous vs a e r i a l medium would suggest t h i s i s probably not a chance occurrence but the only p o s s i b l e mechanism a v a i l a b l e . A i r b r e a t h i n g b i r d s and mammals can r e g u l a t e t h e i r blood pH {within l i m i t s ) by a l t e r i n g t h e i r v e n t i l a t i o n ; however t h i s i s only p o s s i b l e due t o the l a r g e content of oxygen i n a i r . This enables the organism to g r e a t l y a l t e r v e n t i l a t i o n f o r the sake of c o n t r o l l i n g C02 content, without i m p a i r i n g oxygen uptake and hence d e l i v e r y t o the t i s s u e s . In the case of agu a t i c r e s p i r a t i o n t h i s simply i s n ' t p o s s i b l e . F i s h can i l l a f f o r d to a l t e r v e n t i l a t i o n f o r the sake of CO2 c o n t r o l , i n the face of l i m i t e d and v a r i a b l e amounts of a v a i l a b l e oxygen i n water (Bandall S Cameron, 1973). FIGURE #1. Diagramatic r e p r e s e n t a t i o n of an open C 0 2 system. C0 2(d) and C0 2(g) r e f e r to d i s s o l v e d and gaseous C0 2 r e s p e c t -i v e l y . 7b C 0 2 EXCRETION IN EXPIRED GAS Cq 2(d)S======= 5: H 2 C O s H + +HCO3" C 0 2 PRODUCTION FROM TISSUE METABOLISM 8 In f a c t i t might be argued that C02 e x c r e t i o n i n a g u a t i c animals never poses a problem, tut indeed the r e t e n t i o n of s u f f i c i e n t C02 to prevent l o s s of b u f f e r r e s e r v e and t i s s u e a l k a l o s i s may present a more formidable l i m i t a t i o n . The capacitance of carbon d i o x i d e and oxygen i n a i r i s e g u a l ; however the c a p a c i t a n c e r a t i o of carbon d i o x i d e and oxygen i n water i s around 30 (Dejours, 1575). a d d i t i o n a l l y , while the conductance of oxygen and carbon d i o x i d e i n a i r i s n e a r l y egual the permeation c o e f f i c i e n t of carbon d i o x i d e i n water i s much gre a t e r than oxygen. Consequently i t has been argued by Rahn (1966) and others that water con v e c t i o n requirements ac r o s s the g i l l s of f i s h s u f f i c i e n t f o r oxygen uptake r e s u l t s i n a v i r t u a l vacuum f o r C02. Thus with water a c t i n g as a s i n k f o r C02 one can r e a d i l y a p p r e c i a t e why a r t e r i a l C02 t e n s i o n s i n f i s h are low. Thus i n r e a n a l y z i n g C02 e x c r e t i o n across the g i l l s of f i s h one would p r e d i c t t h a t v i r t u a l l y a l l C02 would be l o s t a t p r a c t i c a l l y any v e n t i l a t i o n r a t e i n normocapnic waters. The f a c t t h a t a r t e r i a l C02 t e n s i o n s do not change i n the f a c e of a l t e r e d v e n t i l a t i o n r a t e s , as during hypoxia (Randall & Jones, 1973) , i s t h e r e f o r e p u z z l i n g . E i t h e r g i l l s must provide an e f f e c t i v e d i f f u s i o n b a r r i e r f o r CO2 but not oxygen or the i n t e r c c n v e r s i o n of HC03 to C02 may be a r a t e l i m i t i n g process i n f i s h blood. The r a t e of the r e v e r s i b l e h y d r a t i o n of C02 as i t occurs i n these animals appears to be of i n t e r e s t , The enzyme c a r b o n i c anhydrase c a t a l y z e s the r e a c t i o n depicted by eguation #1a. T h i s enzyme has one of the f a s t e s t turnover r a t e s of any enzyme yet c h a r a c t e r i z e d (Maren,1967). Consequently, where s u f f i c i e n t c a r b o n i c anhydrase e x i s t s the 9 intereconversion of CO2 and HC03 i s never r a t e l i m i t i n g . I t i s t h e r e f o r e e s s e n t i a l to i n v e s t i g a t e the k i n e t i c s of t h i s r e a c t i o n i n f i s h and p a r t i c u l a r i l y important to evaluate how c a r b o n i c anhydrase a f f e c t s these r a t e s i n f i s h , Carbonic anhydrase has been def i n e d by Maren (1967) as any substance t h a t c a t a l y z e s the r e v e r s i b l e r e a c t i o n of H20 + C02 = H2C03 {equation #1a) i n the presence of s u i t a b l e b u f f e r s . I t seems a p p r o p r i a t e that such an enzyme be d e f i n e d l o o s e l y , f o r i t s forms and f u n c t i o n s are i n c r e d i b l y v a r i e d , seemingly even more so than the g l o b i n molecules. Carbonic anhydrase i s found i n p l a n t s as w e l l as a l l groups of the animal kingdom and has even been found i n c e r t a i n groups of b a c t e r i a . Given t h i s p h y l e t i c d i s t r i b u t i o n , e q u a l l y impressive i s the v a r i e d b i o l o g i c a l r c l e s to which c a r b o n i c anhydrase has been l i n k e d . For example c a r b o n i c anhydrase has been found i n the f o l l o w i n q organs or organ systems: blood c e l l s ; kidney; eye; c e r e b r o s p i n a l f l u i d formation; stomach; pancreas; l i v e r and the b i l a r y system; s a l i v a r y glands; sweat glands; t a s t e ; r e p r o d u c t i v e system; avian s a l t gland; r e c t a l gland; a l k a l i n e gland; g i l l s ; swimbladder; i n t e s t i n e ; i n n e r ear; s i c k l i n g i n r e d c e l l s ; a d r e n a l gland and a l s o the t h y r o i d gland. Since t h i s enzyme's i n i t i a l d i s c o v e r y by Meldrum and Eoughton (1933) a l a r g e amount o f l i t e r a t u r e has accumulated ; however only r e c e n t l y have s i g n i f i c a n t s t r i d e s been made concerning the a c t u a l b iochemistry of t h i s enzyme which are d i r e c t l y a p p l i c a b l e t o the p h y s i o l o g i c a l aspects. Most of the e a r l y i n v e s t i g a t i o n s centered around the presence or absence of c a r b o n i c anhydrase i n v a r i o u s systems or organisms, and r e a c t i o n r a t e s and some i n h i b i t o r s t u d i e s , as 10 c l i n i c a l a p p l i c a t i o n s w e r e q u i c k l y r e a l i z e d . T o i l l u s t r a t e how e l u s i v e p r o g r e s s h a s b e e n w i t h c a r b o n i c a n h y d r a s e i t i s i n t e r e s t i n g t o n o t e t h a t t h e a c t u a l p r o d u c t o f t h e h y d r a t i o n r e a c t i o n ( H C 0 3 o r H2C03) r e m a i n s i n d o u b t . M o s t c a r b o n i c a n h y d r a s e s a r e c h a r a c t e r i z e d b y v e r y h i g h t u r n o v e r r a t e s , i n f a c t t h e h i g h t u r n o v e r n u m b e r s o f some m a m m a l i a n c a r b o n i c a n h y d r a s e s c a n n o t b e a c c o u n t e d f o r w i t h e x i s t i n g c a t a l y t i c m o d e l s . K h a l i f a h ( 1 9 7 3 ) t r e a t s t h i s f a s c i n a t i n g s u b j e c t i n g r e a t e r d e t a i l . A s g e n e r a l l y f o u n d i n human e r y t h r o c y t e s , c a r b o n i c a n h y d r a s e e x i s t s a s o n e o f t w o i s o z y m e s ( i s o e n z y m e s ) . T h e r e i s a h i g h l y a c t i v e " C " f o r m , w h i c h r e p r e s e n t s a p p r o x i m a t e l y 1 5 - 2 0 % o f t h e t o t a l c a r b o n i c a n h y d r a s e p r e s e n t , w h i l e a l e s s a c t i v e " B " f o r m c o m p r i s e s t h e r e m a i n d e r . I t s h o u l d b e n o t e d t h a t " l e s s a c t i v e " i n t h i s i n s t a n c e i s a r e l a t i v e t e r m s i n c e t h e "C" f o r m h a s o n e o f t h e h i g h e s t t u r n o v e r r a t e s o f a n y k n o w n e n z y m e ( a p p r o x i m a t e l y o n e m i l l i o n p e r s e c o n d ) , t h e "B" f o r m i s l e s s a c t i v e b u t s t i l l s i g n i f i c a n t a t 1 0 0 , 0 0 0 p e r s e c o n d ( E d s a l l S K h a l i f a h , 1 9 7 2 ) , Due t o t h e r e l a t i v e a b u n d a n c e o f t h e " B " f o r m i t was p u r i f i e d i n w o r k a b l e a m o u n t s f i r s t , a n d c o n s e g u e n t l y was t h e f i r s t c a r b o n i c a n h y d r a s e i s o z y m e w i t h i t s c o m p l e t e a m i n o a c i d s e g u e n c e d e t e r m i n e d . R e c e n t l y , s e v e r a l g r o u p s h a v e r e p o r t e d t h e c o m p l e t i o n o f t h e s e q u e n c i n g o f t h e "C" f o r m a n d a g e n e r a l p i c t u r e o f t h e e n z y m e c a n b e p r e s e n t e d . C a r b o n i c a n h y d r a s e { i n a n i m a l s ) e x i s t s a s a s i n g l e p o l y p e p t i d e c h a i n w i t h a z i n c l i g a n d b o u n d b y h i s t a d y l r e s i d u e s i n t h e a p p r o x i m a t e g e o m e t r i c c e n t e r o f t h e a c t i v e e n z y m e ( W a a r a e t a l , 1 9 7 2 ) . T h e »C» f o r m h a s 2 6 0 a m i n o a c i d r e s i d u e s w h i l e t h e " B " f o r m h a s o n e l e s s a t 2 5 9 ; 11 furthermore L i n & Deutsch (1974) claim that over 60% of the residues i n human "B" and "C" are i d e n t i c a l i n homologous p o s i t i o n s . These i n d i v i d u a l s a l s o claim that most of the d i f f e r e n c e s can be accounted f o r by a s i n g l e base s u b s t i t u t i o n i n the r e s p o n s i b l e codon. As mentioned, carbonic anhydrase i s f u n c t i o n a l l y and p h y l e t i c a l l y v a r i e d ; however i n the f u t u r e d i s c u s s i o n only i t s r o l e i n C02 transport and acid-base r e g u l a t i o n w i l l be discussed. In the l a t e 1920's and e a r l y igaO's there were two main t h e o r i e s as to the mode of C02 tran s p o r t i n the blood. One, C02 was c a r r i e d from organs to the lungs i n the form of bicarbonate, and at the lungs the pr o t e i n s of the blood, a c t i n g as weak a c i d s , converted t h i s bicarbonate to carbonic a c i d which i n turn was dehydrated to C02 plus water , and owing to the v o l a t i l i t y of C02, d i f f u s e d i n t o the gas space of the lung. The second, s t a t e d that i n a d d i t i o n , p a r t , and p o s s i b l y a l l the p h y s i o l o g i c a l l y important C02 i s c a r r i e d i n d i r e c t r e v e r s i b l e combination with the blood p r o t e i n s . From 1917 to 1921 the problems were worked on i n t e n s l y by numerous B r i t i s h p h y s i o l o g i s t s with the r e s u l t that the bicarbonate theory , i n the view of most w r i t e r s , was favored. Op u n t i l about 1925, a t t e n t i o n had been given s o l e l y to e g u i l i b r i u m s t a t e s of the process; however i n t h i s year Hartridge and Houghton, working on the combination and d i s s o c i a t i o n of oxygen with hemoglobin, pointed out the d e s i r a b i l i t y of studying the k i n e t i c s of the carbon dioxide process i n blood. In 1926 Henrigues t a c k l e d t h i s problem with a s t a r t l i n g outcome. According to the bicarbonate theory the d e t a i l e d chemical r e a c t i o n s which l e a d to C02 12 e v o l u t i o n i n the lungs was as f o l l o w s ; (1) HP ( p r o t e i n acid) + NaHC03= NaP ( p r o t e i n s a l t ) + H2C0 3 (2) H2C03 = C02 + H20 Reaction (1) i s p u r e l y i o n i c and could proceed q u i t e r a p i d l y , but (2) was known to be a r a t h e r slow step. So i n the b i c a r b o n a t e theory, r e a c t i o n (2) would be the r a t e l i m i t i n g s t e p . Using the a p p r o p r i a t e v e l o c i t y c o n s t a n t s , Henrigues (1926) c a l c u l a t e d the r a t a at which C02 could be l i b e r a t e d under p h y s i o l o g i c a l c o n d i t i o n s , and found i t to be f a r l e s s than those values a c t u a l l y observed i n v i v o . Benrigues concluded that e i t h e r a c a t a l y s t must e x i s t or t h a t the p h y s i o l o g i c a l t r a n s p o r t of carbon d i o x i d e i n the blood must take place by some mechanism other than the bicarbonate one. However , Henrigues had the misfortune of having the only known case of a hemolyzed blood s o l u t i o n l a c k i n g c a r b o n i c anhydrase and was prevented from d i s c o v e r i n g t h i s enzyme (Maren, 1972). F i n a l l y i n 1932 Meldrum & Roughton i s o l a t e d from ox blood a white substance of which 1 p a r t i n 10 m i l l i o n was a c t i v e i n a c c e l e r a t i n g the r e a c t i o n . So i t was that work on C02 movement l e d to the hypothesis of t h i s enzyme's e x i s t e n c e and e v e n t u a l l y to i t s d i s c o v e r y . Given the huge c a t a l y t i c p o t e n t i a l of c a r b o n i c anhydrase and the importance of c o n t r o l l i n g bicarbonate i o n s , i t seems e s s e n t i a l to take a c l o s e r look a t the t i s s u e s where c a r b o n i c anhydrase could play a r o l e i n f i s h r e s p i r a t i o n and acid-base homecstasis. 13 CHAPTER J - SOME P R O P E E T I E S OF C A R B C N I C ANHYDRASE I N T H E TROUT. 14 INTEODUCTIGN The presence of c a r b o n i c anhydrase i n f i s h , indeed i n a l l a g u a t i c animals, has been known f o r some time, f o r d e t a i l s see the review by Maren (1967). E a r l y i n v e s t i g a t i o n s were concerned with documenting i t s mere presence; however only s i n c e the d i s c o v e r y of potent and s p e c i f i c i n h i b i t o r s of c a r b o n i c anhydrase, f o r example acetazolamide, has i t been p o s s i b l e to i n v e s t i g a t e s p e c i f i c e f f e c t s . I t has been r e p e a t e d l y shown t h a t i n h i b i t i o n of b r a n c h i a l c a r b o n i c anhydrase i n t e r f e r e s with i o n i c movements o c c u r i n g at the g i l l . For example sodium uptake a c r o s s the g i l l i s blocked d u r i n g i n h i b i t i o n of c a r b o n i c anhydrase i n t r o u t ( K e r s t e t t e r & K i r s c h n e r , 1972); g o l d f i s h {Maetz S G a r c i a -ficmeau, 1964) and d o g f i s h (Payan & Maetz, 1 973). Thus b r a n c h i a l c a r b o n i c anhydrase i s g e n e r a l l y thought to be e s s e n t i a l f o r i n i o n i c r e g u l a t i o n i n f i s h {Maetz , 1973; Maetz, 1971). The r o l e of red c e l l c a r b o n i c anhydrase i n the e x c r e t i o n of r e s p i r a t o r y C02 i n mammals i s w e l l documented . A l l v e r t e b r a t e red blood c e l l s , i n c l u d i n g those of f i s h , c o n t a i n a p p r e c i a b l e amounts of c a r b o n i c anhydrase, consequently when a C02 r e t e n t i o n (and r e s u l t a n t a c i d o s i s ) develops i n f i s h during acetazolamide treatment i t has been a t t r i b u t e d to i n h i b i t i o n of red c e l l c a r b o n i c anhydrase ( H o f f e r t , 1966 ; Hodler et a l , 1955; Maren £ Maren, 1964). Indeed Maren & Maren (1964) s t a t e d c a t e g o r i c a l l y that b r a n c h i a l c a r b o n i c anhydrase was not i n v o l v e d i n the e x c r e t i o n of r e s p i r a t o r y C02 i n the d o g f i s h . However two o b s e r v a t i o n s cause problems with a n a l y s i s of b r a n c h i a l versus red c e l l c a r b o n i c anhydrase f u n c t i o n . Table #2 d e p i c t s pooled data from the l i t e r a t u r e g i v i n g r a t i o s of b r a n c h i a l to 15 e r y t h r o c y t i c c a r b o n i c anhydrase present i n s e v e r a l s p e c i e s of f i s h . Due t o v a r i a t i o n s i n the methods i t i s not p o s s i b l e t o express a b s o l u t e l e v e l s f o r comparison between f i s h ; however i t i s c l e a r t h at on a per gram wet weight b a s i s f i s h possess as much c a r b o n i c anhydrase i n the b r a n c h i a l t i s s u e as i n blood. Cameron (1976) demonstrated t h a t i n the a r c t i c g r a y l i n g only 2-H% of the t o t a l e x p i r e d C02 i s u t i l i z e d t o provide the g i l l b i carbonate i o n s (presumibly v i a the hyd r a t i o n of C02 w i t h i n the g i l l ) f o r c h l o r i d e uptake. K e r s t e t t e r & K i r s c h n e r (1972) argue that s u f f i c i e n t bicarbonate i s a v a i l a b l e from plasma to ensure c h l o r i d e uptake at the g i l l e p i t h e l i u m , as i n h i b i t i o n of ca r b o n i c anhydrase was without e f f e c t on c h l o r i d e uptake i n the t r o u t . Why then should the g i l l r e g u i r e an egual or l a r g e r amount of enzyme f o r the h y d r a t i o n of C02 produced by an egual or s m a l l e r amount of c a r b o n i c anhydrase via the dehydration r e a c t i o n i n red c e l l s ? B i o l o g i c a l systems presumably don't operate by such an o v e r k i l l mechanism. Secondly, attempts to d u p l i c a t e the experimental approach of Haren S Haren (1964) on t r o u t have been i n c o n c l u s i v e (D.J. E a n d a l l , personal communication). T h e r e f o r e attempts to c l a s s i f y t r a n c h i a l c a r b o n i c anhydrase as d i s t i n c t l y i o n i c or r e s p i r a t o r y i n a f u n c t i o n a l sense are probably premature. However based on C02. conductance r a t e s and the c a p a c i t a n c e of C02 i n water i t can be argued that c a r b o n i c anhydrase i n aguatic animals was probably i n i t i a l l y r e l a t e d t o i o n i c t r a n s p o r t and not the movement of C02 ^e r se. .'Furthermore under these c o n d i t i o n s C02 e x c r e t i o n would be coupled t o i o n i c r e g u l a t i o n i n such a f a s h i o n that while the l o s s of molecular C02 may be u n c o n t r o l l a b l e , the t r a n s l o c a t i o n 16 of i o n s can be t i g h t l y r e g u l a t e d , Krogh (1941) estimated t h a t animals s m a l l e r than 1mm i n diameter c o u l d s a t i s f y t h e i r oxygen requirements purely on the b a s i s of d i f f u s i o n . Given the huge C02 sink t h a t water pro v i d e s i t i s c l e a r t h a t c y t o p l a s m i c anoxia would r e s u l t long before r e t e n t i o n of C02 (and the a s s o c i a t e d a c i d o s i s ) could ever develop. Given the r e l a t i v e l y slow r a t e s of the uncatalyzed h y d r a t i o n of C02 ( r e s u l t s s e c t i o n #2) t h e l o s s of molecular C02 may not be c o n t r o l l a b l e . Therefore i n order f o r these c e l l s and/or organisms t o e s t a b l i s h a b u f f e r reserve i t would be an advantage t o hydrate metabolic C02 to HC03 plus the proton. T h i s r e a c t i o n would of n e c e s s i t y be c a t a l y z e d by c a r b o n i c anhydrase. The proton could be e l i m i n a t e d by i o n i c exchange , e.g. Na/H exchange on the membrane (Sachs, 1977), with a s i m i l a r exchanger (C1/HC03) c o n t r o l l i n g anion l e v e l s . Thus the u n c o n t r o l l e d l o s s of d i s s o l v e d C02 would be e f f e c t i v e l y prevented by c o u p l i n g C02 l o s s t o i o n i c exchange. Of course an obvious advantage t o t h i s system i s t h a t c o u n t e r i o n s are now a v a i l a b l e f o r the e l e c t r i c a l l y s i l e n t exchange of environmental sodium and c h l o r i d e . That t h i s scheme may be f u n c t i o n a l a t the c e l l u l a r l e v e l i s i n d i c a t e d by the demonstration t h a t i s o l a t e d f r o g o x y n e t i c c e l l s placed i n C02 f r e e media maintain constant c y t o p l a s m i c t o t a l C02 l e v e l s with time while maintaining a higher i n t r a c e l l u l a r pH ( M i c h e l a n g e l i , 1978), I f t h i s i s an accurate d e s c r i p t i o n thus f a r and the l o s s of C02 i s indeed coupled t o i o n i c exchange, then these a g u a t i c organisms would e f f e c t i v e l y r e g u l a t e i n t r a c e l l u l a r pH independent of ab s o l u t e C02 l e v e l s . Thus so l o n g as i n t r a c e l l u l a r pH i s at the " s e t 16a TABLE #2. R a t i o o f b l o o d v e r s u s g i l l c a r b o n i c a n h y d r a s e a c t i v i t i e s . Enzyme a c t i v i t i e s e x p r e s s e d p e r gram t i s s u e (wet w e i g h t ) . 16b BLOOD/GILL ORGANISM CARBONIC ANHYDRASE Lake T r o u t 1 0.86 P e r c h 2 1.32 3 Sea Bass 0.96 3 P a r r o t - f i s h 1.06 D o g f i s h 4 0.36 1= H o f f e r t (1965) 2= Maetz (1956) 3= S m i t h & P a u l s o n (1975) 4= H o d l e r e t a l (1955) 17 p o i n t " t o t a l C02 may f a l l where i t may, with pHi adjustments r e g u l a t e d v i a e i t h e r a n i o n i c or c a t i o n i c membrane exchange processes. Thus i f pHi f a l l s hydrogen i o n s or t h e i r e q u i v a l e n t would be excreted. Conversely an e l e v a t i o n i n pHi may produce a r e d u c t i o n i n proton pumping. T h i s appears t o be the s i t u a t i o n i n i s o l a t e d s i n g l e c e l l s (Boron, 1977; Thomas, 1976; Aiken & Thomas, 1975; ; Boos,1975; R u s s e l l S Boron,1976; Boron S De Beer, 1976a,b, ). I t can be demonstrated that c e l l s s u b j e c t e d to pH p e r t u r b a t i o n s respond with an a p p r o p r i a t e i n c r e a s e or decrease in t r a n s l o c a t i o n of hydrogen ion e q u i v a l e n t s . I t i s t h e r e f o r e i n t e r e s t i n g to note the response of the m u l t i c e l l e d a q u a t i c f i s h t o an a c i d l o a d . I f one c o n s i d e r s the g i l l s u r f a c e area as d e l i n e a t i n g the water membrane or s i t e of c a t i o n i c and a n i o n i c exchange, and i f one views plasma as a very l a r g e cytoplasmic p o o l , the systems are at l e a s t s u p e r f i c i a l l y very s i m i l a r . However i n s t e a d of u t i l i z i n g e i t h e r the a n i o n i c c r the c a t i o n i c exchangers, i t would appear both are i n v o l v e d . For example during hypercapnic a c i d o s i s the a r c t i c g r a y l i n q i n c r e a s e s hydrogen i o n pumping, as evidenced by i n c r e a s e d sodium uptake r a t e s (Cameron, 1976). Sodium uptake has been shown to c o r r e l a t e w e l l with hydrogen i o n e x c r e t i o n (Kirschner et a l , 1973; Maetz, 1973; Payan & Maetz, 1973). I t has a l r e a d y been s t a t e d t h a t hypercapnic a c i d o s i s r e s u l t e d i n the uptake of bicarbonate from seawater i n the d o g f i s h (Randall e t a l , 1S76), Also i t has been demonstrated that the movement of HC03/C1 across the f i s h g i l l i s an e l e c t r i c a l l y s i l e n t 1:1 coupled exchange (Maetz et a l , 1S76). Obviously comparing an i n t a c t f i s h with a w e l l d e f i n e d s i n g l e c e l l system i s pushing the 18 analogy, and very r e a l complicating factors have been ignored; however i t i s int e r e s t i n g that the response of aquatic g i l l breathers may be more closely aligned to aquatic single c e l l systems rather than to the systen employed by a i r breathing mammals and birds. Consequently although the current evidence would suggest plasma bicarbonate i s probably dehydrated within the f i s h red c e l l (catalyzed by erythrocytic carbonic anhydrase) there i s no convincing evidence to exclude the p o s s i b i l i t y that plasma HC03 may move d i r e c t l y into the epithelium where i t may be acted upon by branchial carbonic anhydrase. To further investigate t h i s p o s s i b i l i t y i t i s f i r s t e ssential to l o c a l i z e and characterize the carbonic anhydrase available for t h i s purpose. 19 METHODS C A R B C H I C ANHYDRASE A S S A Y : C a r b o n i c a n h y d r a s e a c t i v i t y w a s m e a s u r e d m a n o m e t r i c a l l y u s i n g a m o d i f i e d b o a t t e c h n i q u e a s d e s c r i b e d b y M e l d r u m & R o u g h t o n ( 1 9 3 3 ) ; R o u g h t o n & B o o t h ( 1 9 4 6 ) a n d a s l a t e r m o d i f i e d b y H c f f e r t ( 1 9 6 6 ) . B a s i c a l l y t h e m e t h o d i s a s f o l l o w s : a s l i g h t l y a l k a l i n e b i c a r b o n a t e s o l u t i o n i s a l l o w e d t o m i x w i t h a b u f f e r e d s o l u t i o n o f a p p r o x i m a t e l y pH 6.8, w h e r e u p o n C 0 2 i s e v o l v e d . T h e r a t e o f t h e C 0 2 e v o l u t i o n c a n b e m e a s u r e d w i t h a n d w i t h o u t c a r b o n i c a n h y d r a s e p r e s e n t a n d t h u s p r o v i d e s t h e b a s i s o f t h e a s s a y . T h e r e a c t i o n v e s s e l o r " b o a t " c o n s i s t s o f a m o d i f i e d 50 m l E r l e n m e y e r f l a s k . T h e b o t t o m o f t h e " b o a t " h a s b e e n p a r t i t i o n e d s u c h t h a t t h e t w o s o l u t i o n s ( b i c a r b o n a t e a n d b u f f e r ) r e m a i n s e p a r a t e d u n t i l s h a k i n g i s c o m m e n c e d , w h e r e u p o n t h e s o l u t i o n s m i x a n d t h e r e a c t i o n p r o c e e d s . T h e u p p e r p o r t i o n o f t h e f l a s k i s p r o v i d e d w i t h a g r o u n d g l a s s j o i n t f o r a t t a c h m e n t t o a G i l s o n D i f f e r e n t i a l R e s p i r o m e t e r . T h e G i l s o n B e s p i r o m e t e r p r o v i d e s t h e s h a k i n g m o t o r a n d t e m p e r a t u r e b a t h , w h i l e C 0 2 e v o l u t i o n i s m e a s u r e d b y H e w l e t t - P a c k a r d 2 6 7 B C d i f f e r e n t i a l p r e s s u r e t r a n s d u c e r s , v i a HP 3 5 0 - 1 1 0 0 c C a r r i e r P r e -A m p l i f i e r s a n d d i s p l a y e d b y a 2 - c h a n n e l B e c k m a n T y p e RS D y n c g r a p h . Two s u c h " b o a t s " a n d a s s o c i a t e d HP t r a n s d u c e r s a n d HP P r e - A m p s a r e u t i l i z e d s u c h t h a t t w o a s s a y s c a n b e r u n s i m u l t a n e o u s l y . P h o s p h a t e B u f f e r s T h e p r i n c i p l e b u f f e r u t i l i z e d h a s b e e n a 0.2 M p h o s p h a t e s o l u t i o n w i t h a pH o f 6.8, T h e b u f f e r i s c o m p o s e d o f 0.2 M N a 2 H E 0 4 a n d 0.2 H K H 2 P 0 4 , t h e r e l a t i v e p r o p o r t i o n s b e i n g 2 0 determined by the r e q u i r e d pH. Bi£15^2fi^te Solutions^. For bicarbonate s o l u t i o n s the sodium s a l t was made by d i s s o l v i n g NaHC03 i n 0.02 M NaOH. A l l r e s u l t s presented were obtained u t i l i z i n g a bicarbonate s o l u t i o n of 200 mM u n l e s s otherwise i n d i c a t e d . £hi§iological S a l i n e Whenever a p h y s i o l o g i c a l s a l i n e was r e q u i r e d C o r t l a n d s a l i n e (wolf, 1963) was u t i l i z e d . T i s s u e Preparations.: Blood: Blcod hemolysates were prepared in the f o l l o w i n g manner: whole blood was c e n t r i f u g e d , the plasma removed and the packed c e l l s washed 3X i n C o r t l a n d s a l i n e . An a l i q u o t of c e l l s was then hemolyzed i n known volumes of d i s t i l l e d water. A s m a l l q u a n t i t y of e i t h e r saponin or T r i t o n X-100 was found to g r e a t l y f a c i l i t a t e hemolysis and was r o u t i n e l y u t i l i z e d f o r t h i s purpose. G i l l homogenatesi F i s h were immobilized by a blow t o the head and a v e n t r a l i n c i s i o n made t o expose the pericardium . The g i l l s were subsequently perfused v i a the v e n t r a l aorta with h e p a r i n i z e d C o r t l a n d s a l i n e to remove trapped e r y t h r o c y t e s . When the g i l l s appeared devoid of e r y t h r o c y t e s the arches were removed and placed i n i c e c o l d C o r t l a n d s a l i n e . The arches were separated from the f i l a m e n t s or u t i l i z e d whole. G i l l t i s s u e s i n known volumes of 300 mM sucrose were homogenized by hand over i c e u t i l i z i n g a g l a s s homogenizing tube. T h i s suspension was then c e n t r i f u g e d t o remove the c e l l u l a r d e b r i s . 21 S u b c e l l u l a r D i s t r i b u t i o n The s u b c e l l u l a r d i s t r i b u t i o n of b r a n c h i a l c a r b o n i c anhydrase was estimated u t i l i z i n g d i f f e r e n t i a l c e n t r i f u g a t i o n . A f t e r homogenization the crude homogenate was c e n t r i f u g e d a t 20,000 X G f o r 35 minutes at 4 C using a S o r v a l l r e f r i g e r a t e d c e n t r i f u g e . The p e l l e t represented the n u c l e a r and m i t o c h o n d r i a l d e b r i s plus the heavy microsomes. The supernatant was c e n t r i f u g e d f o r 20 minutes at 4 C at 100,000 x G using a Beckman U l t r a c e n t r i f u g e to o b t a i n the microsomal p e l l e t . General procedure: The t r a n s d u c e r s were c a l i b r a t e d by a l t e r i n g the volume of the c l o s e d system ( i n c l u d i n g the G i l s o n manometer, the r e a c t i o n v e s s e l and pressure transducer) and r e c o r d i n g the change i n pressure. To perform an assay 2 mis of bicarbonate s o l u t i o n were placed i n t o one chamber of the "boat" while the other was f i l l e d with 2 mis of phosphate b u f f e r p l u s the m a t e r i a l t o be t e s t e d . I f the t e s t m a t e r i a l contained carbonic anhydrase, the uncatalyzed c o n t r o l value was obtained by s u b s t i t u t i n g an egual volume of C o r t l a n d s a l i n e . The "boats" were then attached to the G i l s o n , submerged i n the water bath and allowed t o temperature e q u i l i b r a t e ( u s u a l l y 2-3 minutes) to the bath temperature cf 5 C. When i t was apparent no pressure changes were o c c u r r i n g the shaking motor was turned on and the r e a c t i o n allowed to proceed. A f t e r f u l l d e f l e c t i o n of the pens the v a l v e s were opened and the shaking motor and c h a r t r e c o r d e r turned o f f . The r e a c t i o n v e s s e l s were then removed, washed and a i r d r i e d and ready f o r the next assay. C a l c u l a t i o n s : 22 The r a t e s of the r e a c t i o n are expressed as m i c r o l i t e r s of C02 evolved per second. These values are e a s i l y obtained from the c h a r t r e c o r d e r t r a c i n g . Carbonic anhydrase a c t i v i t y can then be expressed according t o the f o l l o w i n g formula; E = Kc ~ Ko / Ko where, E i s egual to enzyme u n i t s of c a r b o n i c anhydrase a c t i v i t y , Kc i s equal to the r a t e of the c a t a l y z e d r e a c t i o n , and Ko i s egual to the r a t e of the uncatalyzed r e a c t i o n . 11 E n values are a r b i t r a r y u n i t s such that when the c a t a l y z e d r a t e i s e x a c t l y twice the uncatalyzed r a t e "E 1 1 equals 1. The e f f e c t s of acetazolamide (Diamox, Lederle) and c h l o r i d e on c a r b o n i c anhydrase dehydration a c t i v i t y were a l s o i n v e s t i g a t e d . In these i n s t a n c e s i n h i b i t i o n of enzyme a c t i v i t y was c a l c u l a t e d as f o i l c u s : % I n h i b i t i o n = Kc - Kt/Kc X 100 where, Kc i s the same as above and Kt i s the c a t a l y t i c r a t e i n the presence of the a p p r o p r i a t e t e s t s o l u t i o n . E s t i m a t i o n of Km b i c a r b o n a t e : Carbonic anhydrase a f f i n i t y f o r the dehydration s u b s t r a t e HC03 was determined as f o l l o w s : a constant amount of crude heraclysate or g i l l hcmoginate was added to scdium bicarbonate c o n c e n t r a t i o n s of 25, 37 and 71 mM. The r e a c t i o n r a t e s were p l o t t e d g r a p h i c a l l y and the Km f i t t e d by eye. P£2£frl2 d e t e r m i n a t i o n : T o t a l p r o t e i n was determined using a modified b i u r e t method (Accu-Stat, Clay Adams, Parsippany, N.J.) with albumin as a standard. l o c a l i z a t i o n of b r a n c h i a l c a r b o n i c anhydrase 23 H i s t o c h e m i c a l l o c a l i z a t i o n : 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 c a r b o n i c anhydrase was demonstrated using a m o d i f i c a t i o n of the Hansson (1967, 1968) method as d e s c r i b e d by R i d d e r s t r a l e (1976) Autoradiographic l o c a l i z a t i o n : Carbonic anhydrase has been l o c a l i z e d i n the g i l l using the l a b e l e d i n h i b i t o r acetazolamide as d e s c r i b e d by Gay S Mueller (1973). 5.0 MCi of acetazolamide-3H, r e p r e s e n t i n g 6.3 mg of dry powder, was obtained by s p e c i a l order from New England Nuclear (Boston, Mass.). As Gay S Mueller (1973) found i n - v i t r o l a b e l i n g u n s a t i s f a c t o r y , the f o l l o w i n g i n v i v o exposure was u t i l i z e d ; the acetazolamide-3H was suspended i n C o r t l a n d s a l i n e and 3mCi int r o d u c e d v i a a c h r o n i c i n d w e l l i n g c a t h e t e r r e s i d i n g i n the d o r s a l a o r t a , see Methods Chap. I I I . A f t e r s i x hours exposure the animal was s a c r i f i c e d and the g i l l s removed f o r a u t o r a d i o g r a p h i c a n a l y s i s . The s e c t i o n s were f i x e d i n g l u t a r a l d e h y d e and c r i t i c a l point d r i e d . The c r i t i c a l p o i n t d r i e d s e c t i o n s were mounted i n P a r a p l a s t (Sherwood Ind. , St. L o u i s , Mo.) f o r s e c t i o n i n g . 3 micron s e c t i o n s were mounted on c l e a n dry s l i d e s and dipped i n NTB-2 t r a c k i n g g e l (Eastman Kodak, Rochester, n.y.). The dry s l i d e s were placed i n l i g h t and a i r - t i g h t boxes c o n t a i n i n g d r i e r i t e and stored a t 4 c. The autoradiographs were developed f o r 2 minutes i n Kodak Dektol developer (1:2 d i l u t i o n with water), f i x e d f o r 10 minutes and r i n s e d with water f o r 20 minutes. A f t e r d r y i n g the s l i d e s were c o u n t e r s t a i n e d using Nuclear Fast Red. T h i s s t a i n i n g procedure has been found not to induce a d i f f e r e n t i a l s h r i n k i n g or s h i f t i n g of the photographic emulsion which c o u l d otherwise 24 d i s t o r t i t s r e l a t i o n s h i p with u n d e r l y i n g t i s s u e s e c t i o n s (C. S l c n e k e r , Anatomy Dept. Univ. of B.C., personal communication). Carbonic Anhydrase Isozymes,: The presence of m u l t i p l e enzyme forms (isozymes) was determined by c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s u t i l i z i n g a Beckman Hicrozone e l e c t r o p h o r e s i s chamber and a s s o c i a t e d power pack. E l e c t r o p h o r e s i s was c a r r i e d out i n the c o l d u sing b a r b i t a l b u f f e r (0.06 M) at pH 8.6. The v o l t a g e was held constant at 250 V which u s u a l l y r e s u l t e d i n a c u r r e n t of between 8 and 10 milllamps. The l o c a t i o n of c a r b o n i c anhydrase a c t i v i t y a f t e r e l e c t r o p h o r e s i s was determined u t i l i z i n g bromolthymcl blue as an i n d i c a t o r as d e s c r i b e d by Tashian (1969). 25 RESULTS S i g n i f i c a n t q u a n t i t i e s of c a r b o n i c anhydrase are present i n both blood and g i l l t i s s u e . Table #3 presents the enzyme a c t i v i t i e s based on a per gram of t i s s u e and per gram of p r o t e i n b a s i s , as a comparison, using the manometric assay at 5 C, the enzyme a c t i v i t y cf r a t blood was approximately 450 u n i t s per gram t i s s u e , while p u r i f i e d bovine c a r b o n i c anhydrase (Sigma, St L o u i s , Mc.) possessed aprroximately 2,000 Eu per gram of p u r i f i e d enzyme. Carbonic Anhydrase Isozymes; F i g u r e #2 shows that c a r b o n i c anhydrase found i n g i l l and blood i s a combination of two e l e c t r o p h o r e t i c a l l y d i s t i n c t forms. U t i l i z i n g crude g i l l homogenates two d i s t i n c t s t a i n i n g r e g i o n s are e v i d e n t , a densely s t a i n i n g slow migrating f r o n t and a l i g h t e r s t a i n i n g f a s t migrating band. T h i s s t a i n i n g p a t t e r n c o n t r a s t e d with p u r i f i e d bovine c a r b o n i c anhydrase , which c o n t a i n s only one enzyme form, a l s o run as a marker. No f u r t h e r attempt to q u a n t i f y or c h a r a c t e r i z e the r e s p e c t i v e isozymes was attempted. Substrate A f f i n i t y : The apparent Km b i c a r b o n a t e f o r b r a n c h i a l c a r b o n i c anhydrase obtained from three t r o u t ranged from 22-25 mM with a mean of 23.3 mM. The e r y t h r o c y t i c c a r b o n i c anhydrase from 5 rainbow t r o u t ranged from 30-34 mM with a mean of 31.4 mM, At HC03 c o n c e n t r a t i o n s i n excess of 100 mM the enzyme a c t i v i t y {both b r a n c h i a l and e r y t h r o c y t i c ) was depressed. T h i s was not evident i n e i t h e r bovine or r a t carbonic anhydrases at the bicarbonate c o n c e n t r a t i o n u t i l i z e d , 25a FIGURE #2. Diagramatic r e p r e s e n t a t i o n of c a r b o n i c anhydrase isozymes as r e v e a l e d by c e l l u -l o s e a c e t a t e e l e c t r o p h o r e s i s . Bovine c a r b o n i c anhydrase was run as a marker. B O V I N E B L O O D G I L L 2 5 c L TABLE #3. Branchial and erythrocytic carbonic anhydrase activity i n the rainbow trout, (mean 1 S.D.) 25d g. p r b t e i n / 1 0 0 ml Eu/g t i s s u e Eu/g p r o t e i n BLOOD (n=5) 21 ± 6 68 + 23 319 + 47 GILL (n=5) 6 + 1 6 2 + 8 972 + 186 26 Carbonic Anhydrase I n h i b i t i o n by Diamox: Diamox (Acetazolamide, Lederle) was found to s t r o n g l y i n h i b i t b r a n c h i a l and e r y t h r o c y t i c c a r b o n i c anhydrase. F i g u r e #3 i s a p l o t of b r a n c h i a l enzyme a c t i v i t y versus i n c r e a s i n g c o n c e n t r a t i o n s of Diamox. The apparent 150 ( c o n c e n t r a t i o n of i n h i b i t o r to reduce the enzyme a c t i v i t y by ha l f ) i s 4 x 10- 8 M, L o c a l i z a t i o n of B r a n c h i a l Carbonic Anhydrase: The h i s t o c h e m i c a l s t a i n i n g technigue and acetazolaraide-SH autoradiography r e v e a l e d s e v e r a l f e a t u r e s concerning the l o c a l i z a t i o n of ca r b o n i c anhydrase i n t h i s t i s s u e . F i r s t , most of the enzyme appeared to be l o c a l i z e d r a t h e r g e n e r a l l y i n the cytoplasm. Carbonic anhydrase d i d not appear to be sequestered i n any s p e c i f i c c e l l s , e.g. C h l o r i d e c e l l s , but was a s s o c i a t e d p r i n c i p a l l y with r e s p i r a t o r y c e l l s . Secondly, i n a d d i t i o n there appeared to- be some enzyme a c t i v i t y a s s o c i a t e d with the plasma membrane. In most i n s t a n c e s i t appeared the enzyme was a s s o c i a t e d with the a p i c a l border; however i n some s e c t i o n s i t appeared the enzyme was a s s o c i a t e d with the b a s a l l a m i n a l membrane as we l l . I t was not p o s s i b l e to r e s o l v e the s p e c i f i c l o c a t i o n using l i g h t microscopy i n a l l cases. In an attempt t o f u r t h e r t e s t the p o s s i b i l i t y t h a t a f r a c t i o n of b r a n c h i a l c a r b o n i c anhydrase c o u l d be a s s o c i a t e d with the membrane, d i f f e r e n t i a l c e n t r i f u g a t i o n was u t i l i z e d to i s o l a t e the microsomal f r a c t i o n of the b r a n c h i a l t i s s u e . This s u b c e l l u l a r f r a c t i o n i n a d d i t i o n to c o n t a i n i n g the riboscmes c o n t a i n s the plasma membranes. While the bulk of b r a n c h i a l c a r b o n i c anhydrase was found i n the cytoplasm, c a r b o n i c anhydrase was a l s o found i n the microsomal f r a c t i o n of the two 26a FIGURE #3. I n h i b i t i o n of b r a n c h i a l carbonic anhydrase by Diamox. 27 f i s h which were examined. In the second a n a l y s i s s u f f i c i e n t microsomes were c o l l e c t e d to measure p r o t e i n and carbonic anhydrase. The crude homogenate possessed 1,22 0 Eu/g p r o t e i n compared with 1,473 and 441 Eu/g p r o t e i n f o r the supernatant and microsomal f r a c t i o n r e s p e c t i v e l y . Carbonic anhydrase a c t i v i t y of g i l l arches 1 thru 4 from a s i n g l e t r o u t was 52. 9, 52.4, 57.7 and 56.3 Eu/g t i s s u e r e s p e c t i v e l y , i n d i c a t i n g that the enzyme i s probably d i s t r i b u t e d e g u a l l y among the r e s p e c t i v e arches. D i s t r i b u t i o n of Carbonic Anhydrase i n Blood No evidence t o suggest the presence of carbonic anhydrase i n plasma was ever obtained . Consequently i t must be concluded that a l l blood carbonic anhydrase a c t i v i t y r e s i d e s i n the ery t h r o c y t e s . I n i t i a l l y hemolysates were obtained using only d i s t i l l e d water. This crude hemolysate was then c e n t r i f u g e d and carbonic anhydrase measurement based on the supernatant f r a c t i o n ; however a n a l y s i s of the p e l l e t revealed s i g n i f i c a n t carbonic anhydrse a c t i v i t y . When hemolysis was c a r r i e d out with the a d d i t i o n of saponin or T r i t o n x-100 (both agents capable of d i s r u p t i n g membrane i n t e g r i t y ) a homogenous mixture with increased enzyme a c t i v i t y was i n v a r i a b l y obtained. 28 DISCUSSION S i g n i f i c a n t q u a n t i t i e s of c a r b o n i c anhydrase are present i n both blood and g i l l s of the rainbow t r o u t . While the a c t i v i t y of blood and g i l l a r e nea r l y equal when expressed on a per gram t i s s u e b a s i s i t can be seen t h a t the g i l l i s p a r t i c u l a r l y a c t i v e when expressed on a per gram p r o t e i n b a s i s . At l e a s t some e r y t h r o c y t i c c a r b o n i c anhydrase appears to be a s s o c i a t e d with the membrane, i n c o n t r a s t to mammalian red blood c e l l s where i t i s thought t o be e x c l u s i v e l y l o c a l i z e d i n the cytoplasm (Maren, 1967). T h i s c o n c l u s i o n was i n i t i a l l y based on the f i n d i n g t h a t a s i z a b l e f r a c t i o n o f c a r b o n i c anhydrase a c t i v i t y was l o s t upon c e n t r i f u g a t i o n and recovered i n the p e l l e t . Subsequently, u t i l i z a t i o n of membrane detergents such as Saponin or T r i t o n X-100 to achieve hemolysis f u r t h e r i n c r e a s e d c a r b o n i c anhydrase a c t i v i t y and i m p l i e d a f r a c t i o n of e r y t h r o c y t i c c a r b o n i c anhydrase was a s s o c i a t e d with the membrane. T h i s c o n c l u s i o n has subsequently been s u b s t a n t i a t e d (Houston £ McCarty, 1978). The s i g n i f i c a n c e of a membrane bound ca r b o n i c anhydrase remains,obscure. The bulk of c a r b o n i c anhydrase found i n the g i l l i s s o l u b l e and l o c a t e d i n the cytoplasm. A small f r a c t i o n has however been demonstrated t o be present i n the plasma membrane as demonstrated by a u t o r a d i o g r a p h i c and h i s t o c h e m i c a l a n a l y s i s . A d d i t i o n a l l y the presence of s i q n i f i c a n t c a r b o n i c anhydrase a c t i v i t y i n the microsomal f r a c t i o n lends f u r t h e r support to t h i s c o n c l u s i o n . I t i s noteworthy that R i d d e r s t r a l l e (1976) and Wistrand S Kinne , (1977) a l s o found evidence f o r a membrane bound c a r b o n i c anhydrase i n the kidney tubules of the f r o g and r a b b i t . I t would appear that with improved methods and c u r r e n t techniques, Maren's (1967) conte n t i o n t h a t a l l c a r b o n i c anhydrase i s c y t o p l a s m i c may be s u b j e c t to some q u a l i f i c a t i o n s . U n f o r t u n a t e l y i t wasn't always p o s s i b l e u t i l i z i n g l i g h t microscopy to determine whether the membrane bound c a r b o n i c anhydrase was a s s o c i a t e d with the a p i c a l or b a s o l a t e r a l membrane or both. A f u r t h e r i n v e s t i g a t i o n a t the e l e c t r o n microscope l e v e l eight be expected to r e s o l v e t h i s g u e s t i o n . Enzyme K i n e t i c s Due to the presence of m u l t i p l e c a r b o n i c anhydrase isozymes i t i s not r i g o r o u s l y c o r r e c t t o a s s i g n a Km value (Segel, 1975); however i t does r e f l e c t the a f f i n i t y of the t i s s u e i n question f o r the dehydration s u b s t r a t e . The f i n d i n g that b r a n c h i a l c a r b o n i c anhydrase has a g r e a t e r a f f i n i t y f o r b i c a r b o n a t e than the e r y t h r o c y t i c c a r b o n i c anhydrase might imply t h a t , a l l f a c t o r s being egual, p r i n c i p a l l y enzyme/substrate a c c e s s a b i l i t y f a c t o r s , plasma bicar b o n a t e pools might be more e a s i l y dehydrated w i t h i n the g i l l e p i t h e l i u m . M u l t i p l e isozymes have a l s o been demonstrated i n e e l g i l l and blood ( G i r a r d & I s t i n , 1975). € i r a r d & I s t i n a l s o found e e l g i l l c a r b o n i c anhydrase possessed a g r e a t e r a f f i n i t y f o r bicarbonate than the e r y t h r o c y t i c form. The a b s o l u t e Km f o r bicarbonate by f i s h c a r b o n i c anhydrase would appear to be somewhat high. For example the Km f o r bovine c a r b o n i c anhydrase i s around 10 mM or approxiamtely h a l f the b i c a r b o n a t e c o n c e n t r a t i o n s found i n plasma (20-30 mM). On the other hand plasma b i c a r b o n a t e l e v e l s i n the t r o u t are between 6-10 mM while the red c e l l c a r b o n i c anhydrase .Km f o r bicarbonate i s between 30-35 m.M. Th i s value i s 30 approximately 3X the plasma bicarbonate l e v e l s . Trout red c e l l bicarbonate l e v e l s can exceed plasma concentrations ( G i l e s S Haswell, unpublished); however bicarbonate l e v e l s are s t i l l f a r below the observed Km f o r bicarbonate. The s i t u a t i o n f o r the b r a n c h i a l carbonic anhydrase i s seemingly mere f a v o r a b l e with a Km between 20-25 mM. No good estimate of e p i t h e l i a l c e l l bicarbonate l e v e l s i s c u r r e n t l y a v a i l a b l e ; however they would not be expected to g r e a t l y exceed plasma l e v e l s . Two seperate determinations on bovine carbonic anhydrase (Sigma, St. Louis) gave a mean Km bicarbonate of 13 mM. De Voe & K i s t i a k c w s k i {1961) working under s i m i l a r c o n d i t i o n s found a Km of 9.6 mM f o r bovine carbonic anhydrase . Thus although experimentally derived Km's f o r g i l l and blood carbonic anhydrase appear high they are probably r e l i a b l e . G i r a r d & I s t i n (197 5) found apparent Km's i n excess of 100 mM. I t i s not c l e a r why e e l carbonic anhydrase Km's should be so high. However i n t h i s study i t was found that bicarbonate concentrations i n excess of 100 mM depressed apparent carbonic anhydrase a c t i v i t y i n the t r o u t and i t may be po s s i b l e e e l carbonic anhydrase i s l i k e w i s e a f f e c t e d . I f t h i s were the case carbonic anhydrase a c t i v i t y at the higher bicarbonate concentrations would skew a Lineweaver-Burke p l o t (Girard & I s t i n u t i l i z e d t h i s method f o r estimating t h e i r Km values) such as t o generate higher than a c t u a l values. In t h i s study Km's were f i t t e d by eye and bicarbonate concentrations kept w e l l below 100 mM. Both g i l l and e r y t h r o c y t i c carbonic anhydrase were s t r o n g l y i n h i b i t e d by Diamox. The 150 of 10~ a M f o r Diamox i s i n good agreement with the mammalian f a s t forms, e.g. Human "C" where 31 the 150 f o r Diamox i s a l s o 10 ~8 M (Maren e t a l , 1 976). C h l o r i d e c o n c e n t r a t i o n s up t o 200 mM are without e f f e c t on apparent c a r b o n i c anhydrase a c t i v i t y . Human "B" (slow form) i s f u l l y 50% i n h i b i t e d at c h l o r i d e c o n c e n t r a t i o n s of 100 mM. Human "C" r e q u i r e s a c h l o r i d e c o n c e n t r a t i o n of 600 mM to achieve t h i s same i n h i b i t i o n . Tashian e t a l (1977) c l a i m s the appearance of the c a r b o n i c anhydrase slow form i s a f a i r l y r ecent e v o l u t i o n a r y occurence, probably the r e s u l t of a gene d u p l i c a t i o n i n e a r l y mammalian e v o l u t i o n . Thus f a r the slow form has only been demonstrated i n mammals. I t i s p o s s i b l e t h a t the slow form c a r b o n i c anhydrase would be masked i n a s o l u t i o n or t i s s u e p r e p a r a t i o n c o n t a i n i n g both forms. Indeed i n human blood the f a s t form or "C" isozyme r e p r e s e n t s approximately 15% of the molar c o n c e n t r a t i o n of carb o n i c anhydrase present and y e t accounts f o r over 90% of the enzyme a c t i v i t y (Maren, 1 9 6 7 ) C o n s e q u e n t l y the i s o l a t i o n and f u r t h e r p u r i f i c a t i o n of f i s h c a r b o n i c anhydrase w i l l be r e g u i r e d before the e x i s t e n c e or absence of k i n e t i c a l l y d i s t i n c t isozymes and t h e i r p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e can be assessed. However i t i s worth mentioning t h a t while d i s t i n c t isozymes have been found i n t r o u t and e e l , both e u r y h a l i n e s p e c i e s , no evidence of m u l t i p l e enzymes was found i n two s p e c i e s of shark (Maynard, 1971). In summary s i g n i f i c a n t amounts of carbonic anhydrase e x i s t i n the g i l l s and blood of the rainbow t r o u t . T h i s c a r b o n i c anhydrase appears as two e l e c t r o p h o r e t i c a l l y d i s t i n c t bands, suggesting the presence o f f u n c t i o n a l isozymes. The bulk of b r a n c h i a l c a r b o n i c anhydrase i s s o l u b l e and present i n the 32 cytoplasm, although a smaller f r a c t i o n appears to be l o c a l i z e d in the plasma membranes. In blood a larger portion, possibly over 50%, may be incorporated into, or at least associated with, the membrane while the remainder i s cytoplasmic. On a per gram protein basis the g i l l s possess much higher enzyme a c t i v i t i e s than erythrocytes; t h i s f a c t coupled with the greater bicarbonate a f f i n i t y demonstrated by branchial carbonic anhydrase compared with erythrocytic carbonic anhydrase suggests the g i l l tissue may be predisposed to the dehydration of plasma bicarbonate. 33 C H J P T E B I I - C A R B O N I C AN B Y E R A S E A C T I V I T Y I N THE TRQOT BED BLOOD CELL. 34 INTRODUCTION In mammalian blood the i n t e r c o n v e r s i o n of plasma bicarb o n a t e and molecular CO2 i s g r e a t l y f a c i l i t a t e d by red blood c e l l s and t h e i r compliment of c a r b o n i c anhydrase. I t has long been known that s u f f i c i e n t c a r b o n i c anhydrase i s present i n mammalian red blood c e l l s t o c a t a l y z e the observed C02 e x c r e t i o n . Kernohan S Houghton (1966) c a l c u l a t e d 13,0001 more ca r b o n i c anhydrase was present i n red blood c e l l s than c a t a l y t i c a l l y necessary. F o r s t e r and C r a n d a l l (1975) have s i n c e r e v i s e d t h i s estimate to a 6,000X excess. C l e a r l y , the i n t e r c o n v e r s i o n of HC03-C02 w i t h i n the red blood c e l l should not normally be l i m i t i n g . As blood passes through the lung c a p i l l a r i e s molecular CO 2 w i l l d i f f u s e from blood i n t o the lunq space due to the p a r t i a l pressure g r a d i e n t e x i s t i n g between blood and a l v e o l a r gas phases. As approximately 90% of the t o t a l C02 i n blood i s r e s i d e n t i n plasma as bicarbonate, t h i s l o s s of C02 c o u l d c r e a t e an apparent d i s e g u i l i b r i u m s i t u a t i o n should the production of C02 from plasma b i c a r b o n a t e l a g behind. However, due to the r a p i d exchange of plasma bicarbonate f o r red c e l l c h l o r i d e , plasma b i c a r b o n a t e i s r a p i d l y dehydrated to molecular C02 w i t h i n the red c e l l s by c a r b o n i c anhydrase, thus keeping the r e a c t i o n i n apparent e g u i l i b r i u m . Klocke (1976) has demonstrated t h a t the t r a n s l o c a t i o n of plasma bicarbonate i n t o the red c e l l i s never normally the r a t e l i m i t a t i o n i n the production of C02. Thus these two processes (red c e l l c a r b o n i c anhydrase a c t i v i t y and the c h l o r i d e s h i f t ) ensure a r t e r i a l C02 t e n s i o n s equal a l v e c l a r C02 t e n s i o n s even during e x e r c i s e when blood c a p i l l a r y r e s i d e n c e time i s s h o r t e s t (Chinard et a l , 1S60). Thus, i n 35 mammals, at any f i x e d lung blood p e r f u s i o n , i n c r e a s i n g the a r t e r i a l - a l v e o l a r C02 p a r t i a l pressure g r a d i e n t w i l l r e s u l t i n a l a r g e r c o n v e r s i o n of plasma bicarbonate to C02 w i t h i n blood. although s u f f i c i e n t c a r b o n i c anhydrase e x i s t s i n mammalian red blood c e l l s , t h i s may not be the case i n the t r o u t red c e l l . F i g u r e #4 i s a p l o t of the c a l c u l a t e d h a l f - t i m e f o r the dehydration of b i c a r b o n a t e at v a r i o u s temperatures. although t h i s i s a s i m p l i f i c a t i o n of the u n c a t a l y z e d r e a c t i o n , and f a c t o r s such as pH, i o n i c s t r e n g t h , non-bicarbonate b u f f e r s t r e n g t h , and the back r e a c t i o n have been ignored , i t does g i v e an estimate, as w e l l as demonstrating the e f f e c t of temperature on t h i s r e a c t i o n . With these l i m i t a t i o n s i n mind and assuming a h a l f - t i m e of 5 minutes at 15 C, and f u r t h e r assuming a g i l l r e s i d e n c e time of 1-3 seconds (Haswell & R a n d a l l , 1978), the amount of enzyme r e q u i r e d can be c a l c u l a t e d as f o l l o w s : C a t a l y t i c f a c t o r •= Uncatalyzed r a t e / G i l l r e s i d e n c e time U t i l i z i n g the above values a c a t a l y t i c f a c t o r of 20 0 i s r e q u i r e d . Thus f o r each ml of blood passinq through the g i l l s the r e a c t i o n would have to be a c c e l e r a t e d seme 200 times. From t a b l e #3 i t can be seen that t r o u t blood only possesses around 70 enzyme u n i t s or l e s s than h a l f the p r e d i c t e d requirement. Since Houghton's (1935) i n i t i a l estimate of the uncatalyzed r e a c t i o n v e l o c i t y (approximately 1 minute at 37 C) recent i n v e s t i q a t i o n s have found the apparent i n s i t u h a l f - t i m e to be approximately 10 seconds ( H i l l e t al,1977; F o r s t e r S C r a n d a l l , 1975). Thus Rouqhton's o r i q i n a l estimate was too high by a 36 f a c t o r of n e a r l y 10. I f the c a l c u l a t i o n s presented i n f i q u r e #5 are l i k e w i s e too high by a f a c t o r of 10 then the uncatalyzed r a t e at 10 C would be c l o s e r t o 6 0 seconds i n s t e a d of 11.8 minutes. I t i s i n t e r e s t i n g t h e r e f o r e t h a t i n t r o d u c t i o n of a l i g u o t s of sodium bicarbonate i n t o t r o u t plasma generated a r i s e i n PC02 with an apparent h a l f - t i m e of between 60-70 seconds at 10 C . I f 60 seconds i s a b e t t e r estimate of the uncatalyzed r e a c t i o n time the c a t a l y t i c reguirement can now be reduced to 30-60 enzyme u n i t s f o r blood at 10 C. Thus by minimizing uncatalyzed r a t e s i t would appear the reguirement might p o s s i b l y be met by the t r o u t . However i t i s a l s o worth noting t h a t the enzyme a c t i v i t y expressed i n t a b l e #3 was obtained when assay bicarbonate l e v e l s were well i n excess of the b i c a r b o n a t e Km (35 mH). Thus while maximally 70 u n i t s / m l of c a r b o n i c anhydrase a c t i v i t y may e x i s t i n t r o u t blood, at i n v i v o b i c a r b o n a t e c o n c e n t r a t i o n s of 6-10 mM the i n s i t u c a r b o n i c anhydrase a c t i v i t y i s probably f a r l e s s than the c a t a l y t i c reguirement. A d d i t i o n a l l y lower temperatures and e x e r c i s e , among other v a r i a b l e s , might be expected to impose severe l i m i t a t i o n s on C02 e x c r e t i o n , and c e r t a i n l y the huge c a t a l y t i c r e s erve as e v i d e n t i n mammals i s not present i n the t r o u t . Thus i t may be p o s s i b l e t h a t the i n t e r c o n v e r s i o n of HC03-C02 i s a r a t e l i m i t a t i o n i n the production of CO2 i n f i s h blood, and t h i s would provide a p o s s i b l e e x p l a n a t i o n as to why a g r e a t e r washout of C02 does not occur. I t thus became of i n t e r e s t to i n v e s t i g a t e c a r b o n i c anhydrase a c t i v i t y as might be found i n f i s h e r y t h r o c y t e s i n s i t u . I t was t h e r e f o r e e s s e n t i a l to evaluate the dehydration of 36a FIGURE #4. E f f e c t of temperature on the c a l c u l a t e d h a l f - t i m e s f o r the u n c a t a l y z e d p r o d u c t i o n of C02 from HCO^. Adapted from Roughton (1964); r a t e c onstants from E d s a l l (I969). TEMPERATURE fe CO V4 CO o ro o H c ZJ o E 8. t a — - CD ZT *< b i c a r b o n a t e b y i n t a c t f i s h r e d b l o o d c e l l s . 38 M a t e r i a l s and Methods Carj3£I3ic Anhvdrase A c t i v i t y : i n order to c l a r i f y what c o n t r i b u t i o n , i f any, the e r y t h r o c y t i c carbonic anhydrase was making to o v e r a l l C02 e x c r e t i o n , the r a p i d mixing mancmetrie assay as p r e v i o u s l y d e s c r i b e d i n Chapter I has been u t i l i z e d . F i s h blood was obtained v i a d o r s a l a o r t i c puncture on MS-222 an e s t h e t i z e d f i s h or from d o r s a l a o r t i c c a t h e t e r s from f r e e swimming f i s h , u t i l i z i n g h e p a r i n i z e d syringes. The presence or absence of MS-222 d i d not a f f e c t the r e s u l t s obtained. Some whele blood experiments were c a r r i e d out u t i l i z i n g r a t b l o o d , t h i s blood was obtained v i a c a r d i a c puncture on animals a n e s t h e t i z e d with e t h e r . A l l blood was kept on i c e u n t i l r e q u i r e d and u s u a l l y was analyzed w i t h i n 10-15 minutes of removal from the animal . The water bath was maintained at 5 C unless otherwise i n d i c a t e d . C1/HC03 Exchange! As t h i s exchange process forms such an i n t e g r a l part of the C02 e x c r e t i o n mechanism i n mammalian red blood c e l l s i t was of i n t e r e s t to demonstrate i t s presence or absence i n f i s h red blood c e l l s . Red c e l l a 1 k a 1 i n i z a t i p n T h i s i s an i n d i r e c t method r e c e n t l y employed by Z e i d l e r & Kim (1S77) on c a l f r e d blood c e l l s . When red blood c e l l s are placed i n i s o t o n i c sucrose i n t e r n a l c h l o r i d e i s l o s t i n exchange f o r e x t e r n a l bicarbonate. The progress of t h i s r e a c t i o n can be foll o w e d by measuring i n t r a c e l l u l a r pH. Thus as bic a r b o n a t e r e p l a c e s c h l o r i d e w i t h i n the red c e l l pHi w i l l tend to r i s e . In t h i s study i n t r a c e l l u l a r pH's were determined on ethanol/dry i c e 39 f r e e z e thawed c e l l s as d e s c r i b e d by Z e i d l e r S Kim (1977). Sucrose s o l u t i o n s were 300 mM + 5 mM HC03, with pH ad-justed using P04 b u f f e r s . Carbon Dioxide P a r t i t i o n i n g i n Bleed The p a r t i t i o n i n g of C02 between plasma and e r y t h r o c y t e s was determined as f o l l o w s ; Blood suspensions were tonometered a g a i n s t known gas c o n c e n t r a t i o n s f o r a t l e a s t one hour. Gas mixtures were ob t a i n e d from Wostoff Gas Mixing pumps (Bochum, Germany). & blood sample was withdrawn and analyzed f o r t o t a l C02 (see methods Chap III) and h e m a t o c r i t . . a second sample was c e n t r i f u g e d and plasma t o t a l CO 2 measured. E r y t h r o c y t e t o t a l C02 was determined by d i f f e r e n c e s between plasma and whole blood t o t a l CO2, c o r r e c t e d f o r hematocrit. 40 R E S U L T S Rat Elood: Figure #5 demonstrates the change i n dehydration r e a c t i o n v e l o c i t y of a noncatalyzed c o n t r o l (100 u l C o r t l a n d s a l i n e ) and the same r e a c t i o n using 100 u l of whole r a t blood. A decrease i n v e l o c i t y o c c c u r r e d i f the r e a c t i o n was followed t o completion {not d e p i c t e d i n f i g u r e #5); however, unless very s m a l l volumes of whole blood were u t i l i z e d the r a t e of C02 evolved always remained l i n e a r through the f i r s t 200 m i c r o l i t e r s of C02 evolved. Figure #6 shows the change i n enzyme a c t i v i t y by i n c r e a s i n g the volume of r a t blood u t i l i z e d per assay. I t i s apparent t h a t an i n c r e a s e i n blood volume i s accompanied by an i n c r e a s e d enzyme a c t i v i t y . A l l experiments with r a t blood were performed a t 5 C. Whole F i s h Blood: Repeated assays using up to one ml of whole blood {unwashed) from the rainbow t r o u t f a i l e d to demonstrate any s i g n i f i c a n t dehydration a c t i v i t y i n excess of the u n c a t a l y z e d c o n t r o l s . Assays were r o u t i n e l y performed a t 5-6 C, but the f i s h were maintained at water temperatures near 8-10 C, t h e r e f o r e s e v e r a l assays were performed a t 10 C and 15 C. The change i n temperature f a i l e d to demonstrate any i n c r e a s e d dehydration a c t i v i t y over the uncatalyzed c o n t r o l r a t e s . Most assays were performed with the phosphate b u f f e r at or near pH 6.8; however to assess the e f f e c t s of pH on the dehydration a c t i v i t y of the i n t a c t e r y t h r o c y t e , phosphate b u f f e r s with v a r y i n g pH's were a l s o u t i l i z e d . There was no 40a FIGURE #5. T y p i c a l t r a c i n g of u n c a t a l y z e d (100 u l C o r t l a n d s a l i n e ) and the c a t a l y z e d (100 u l of whole r a t b l o o d , upper t r a c e ) d e h y d r a t i o n r e a c t i o n . O 200 c o 2 EVOLVED too o S E C PER DIVISION 4-Oc FIGURE #6. E f f e c t o f i n c r e a s i n g volumes of whole r a t bloo d on enzyme a c t i v i t y , demonstrating l i n e a r k i n e t i c s ( 1 S.D., where n=6). 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 5 "To 15 20 yL OF RAT BLOOD 41 e f f e c t o f i n c r e a s i n g t h e pH o n e n z y m e a c t i v i t y . O c c a s i o n a l l y s m a l l a m o u n t s o f a c t i v i t y w e r e n o t i c e d . U n l i k e r a t b l o o d , i n c r e a s i n g v o l u m e s o f w h o l e f i s h b l o o d w e r e n o t a c c o m p a n i e d b y i n c r e a s i n g e n z y m e a c t i v i t y , a n d e n z y m e a c t i v i t y was a l w a y s a s s o c i a t e d w i t h s o m e r e d c e l l h e m o l y s i s , p r e s u m a b l y r e l e a s i n g c a r b o n i c a n h y d r a s e i n t o t h e p l a s m a . A s s a y s u s i n g w h o l e b l o o d f r o m t h e f o l l o w i n g f i s h a l s o f a i l e d t o d e m o n s t r a t e a n y d e h y d r a t i o n a c t i v i t y : c a r p { C y j a r i n u s ) , l i n g c o d { O p h i o d o n ) , s k i l f i s h { E r i l e n a s ) , f l o u n d e r ( P l a t i c h t h y s ) , a n d t h e m o u t h b r e e d e r { T i l a p i a ) . C a r b o n i c a n h y d r a s e a c t i v i t y o f h e m o l y z e d t r o u t b l o o d : fiainbow t r o u t c o n t a i n a p p r e c i a b l e g u a n t i t i t e s o f c a r b o n i c a n h y d r a s e a s d e m o n s t r a t e d b y a s s a y s on e r y t h r o c y t e s h e m o l y z e d w i t h s a p o n i n { C h a p t e r # 1 ) . One m l o f b l o o d f r o m t h e a v e r a g e r a i n b o w t r o u t c o n t a i n s a p p r o x i m a t e l y 6 0 - 9 0 e n z y m e u n i t s ( t a b l e # 3 ) . T h u s e a c h m l o f r a i n b o w t r o u t b l o o d w i t h i n t a c t r e d c e l l s c o n t a i n s s u f f i c i e n t c a r b o n i c a n h y d r a s e t o p o t e n t i a l l y g e n e r a t e a 6 0 - 9 0 f o l d i n c r e a s e i n d e h y d r a t i o n a c t i v i t y , b u t i t d o e s n o t . T h e r e f o r e , e i t h e r c a r b o n i c a n h y d r a s e i s i n h i b i t e d i n i n t a c t c e l l s o r b i c a r b o n a t e d o e s n o t e n t e r t h e r e d c e l l s a t a p p r e c i a b l e r a t e s . W a s h e d T r o u t E r y t h r o c y t e s : P l a s m a w a s r e p l a c e d by C o r t l a n d s a l i n e ( t h r e e w a s h e s ) a n d t h e n t h i s r e d b l o o d c e l l s u s p e n s i o n a n a l y z e d f o r c a r b o n i c a n h y d r a s e a c t i v i t y . T h e r e d c e l l s u s p e n s i o n was f o u n d t o h a v e p r o n o u n c e d e n z y m e a c t i v i t y w h i c h w a s l o s t i f C o r t l a n d s a l i n e was r e p l a c e d by p l a s m a ( s e e b e l o w ) . T h e r a t e was e g u i v a l e n t t o 3-8 e n z y m e u n i t s / m l , l e s s t h a n t h a t f o r h e m o l y z e d b l o o d , b u t much f a s t e r 42 t h a n e v e r f o u n d u s i n g t h e u n w a s h e d b l o o d , C o r t l a n d s a l i n e w a s h e d b l o o d , t e r m e d " C o r t l a n d B l o o d " , s h o w e d l i n e a r k i n e t i c s { f i g u r e #7) , u n l i k e t h e o c c a s i o n a l a c t i v i t y f o u n d i n u n w a s h e d b l o o d . T h e s u p e r n a t a n t o f C o r t l a n d b l o o d a s s a y s ( o b t a i n e d b y c e n t r i f u g a t i o n ) was a n a l y z e d f o r a c t i v i t y ; i t n e v e r s h o w e d a n y c a r b o n i c a n h y d r a s e a c t i v i t y , i n d i c a t i n g t h a t h e m o l y s i s d i d n o t o c c u r i n t h e s e e x p e r i m e n t s . A t t h i s p o i n t t h e e x i s t e n c e c f some m o d i f i e r i n t h e p l a s m a s e e m e d p o s s i b l e . E v i d e n c e f o r a p l a s m a i n h i b i t o r was c o n f i r m e d i n t h e f o l l o w i n g m a n n e r . T h e r e m a i n i n g b l o o d w a s c e n t r i f u q e d , t h e p l a s m a r e m o v e d a n d k e p t o n i c e f o r f u r t h e r u s e . T h e e r y t h r o c y t e s w e r e t h e n w a s h e d t h r e e t i m e s a n d r e s u s p e n d e d w i t h C o r t l a n d s a l i n e a s o u t l i n e d a b o v e . T h e C o r t l a n d b l o o d was t h e n a s s a y e d . A t t h i s p o i n t t h e r e m a i n i n g C o r t l a n d b l o o d was t h e n c e n t r i f u g e d t o r e m o v e t h e C o r t l a n d s a l i n e . The s u p e r n a t a n t was d i s c a r d e d a n d t h e c e l l s r e s u s p e n d e d t o t h e i n i t i a l h e m a t o c r i t u s i n g t h e o r i g i n a l p l a s m a . T h i s r e c o n s t i t u t e d b l o o d s h o w e d z e r o a c t i v i t y . H e m o l y s i s o f t h e r e m a i n i n g b l o o d a n d s u b s e g u e n t a s s a y o f t h e h e m o l y s a t e c l e a r l y d e m o n s t r a t e d t h a t t h e r e m a i n i n g b l o o d s t i l l p o s s e s s e d c a r b o n i c a n h y d r a s e a c t i v i t y . T h i s a p p r o a c h was r e p e a t e d t h r e e m o r e t i m e s w i t h t r o u t b l o o d w i t h t h e same r e s u l t s . F i g u r e #8 r e p r e s e n t s t h e r e s u l t s o f o n e o f t h e i n v e s t i g a t i o n s . I t i s i n t e r e s t i n g t o n o t e t h a t e x p e r i m e n t s on t h e b l o c d o f f r e s h w a t e r c a r p , m o u t h b r e e d e r , t h e m a r i n e l i n g c o d , a n d t h e e u r y h a l i n e f l o u n d e r , P l a t i c h t h y j s f i e s u s g a v e s i m i l a r r e s u l t s , i n d i c a t i n g a w i d e d i s t r i b u t i o n o f , t h e p l a s m a i n h i b i t o r i n t e l e o s t s . T h i s a p p r o a c h w i t h r a t b l o o d w a s a t t e m p t e d o n l y o n c e ; 42a FIGURE #7. E f f e c t of i n c r e a s i n g volumes of " C o r t l a n d Blood" on enzyme a c t i v i t y , demonstrating l i n e a r k i n e t i c s . ( 1 S.D., where n=6). 42c FIGURE #8. E f f e c t of removing o r i g i n a l plasma on dehy-d r a t i o n a c t i v i t y of t r o u t b l o o d . 43 however, i t appeared that washing the c e l l s was without e f f e c t , and the presence of an e f f e c t i v e i n h i b i t o r a t the whole c e l l l e v e l was not evident. C h l o r i d e Exchange i n Fish Red C e l l s The f i n d i n g that t r o u t red c e l l s f a i l e d to dehydrate e x t r a c e l l u l a r bicarbonate i m p l i e s that e i t h e r c h l o r i d e / b i c a r b o n a t e exchange mechanism does not e x i s t i n t r o u t red c e l l s or a l t e r n a t e l y carbonic anhydrase i s i n h i b i t e d i n s i t u preventing the c a t a l y z e d dehydration r e a c t i o n . Rates of Bed C e l l A l k a l i n i z a t i o n 0.nder these experimental conditions the net e f f l u x of c h l o r i d e i s d i r e c t l y r e l a t e d to the presence and/or a b i l i t y of red c e l l s to take-up bicarbonate as the c e l l s attempt to maintain Donnan e g u i l i b r i u m . Under these c o n d i t i o n s the uptake of bicarbonate £er se i s not measured but r a t h e r the r e s u l t a n t change i n red c e l l pH. Washed t r o u t red c e l l s suspended i n 300 mM sucrose r a p i d l y take-up bicarbonate as r e f l e c t e d by the r a p i d and large r i s e i n red c e l l pH, see f i g u r e #9. This r i s e i n pH continues at a slower r a t e u n t i l the red c e l l s u l t i m a t e l y hemolyse. At 5 C a l l washed c e l l s were completely hemolyzed a f t e r s e v e r a l hours. On the contrary unwashed c e l l s suspended i n sucrose show a small i n i t i a l r i s e i n pHi of approximately .2-.4 pH u n i t s . However the red c e l l pH r i s e s only very slowly or not at a l l a f t e r the f i r s t minute. Blood from two separate f i s h was subjected to sucrose + 50 mM bicarbonate. Even under these elevated plasma bicarbonate l e v e l s red c e l l pH remained s t a b l e , t a b l e #4. In f a c t even a f t e r approximately 24 hours pHi was elevated only .2 u n i t s from the 43a FIGURE #9. E f f e c t o f suspending t r o u t e r y t h r o c y t e s i n 300 mM sucrose + 5 mM NaHCO-^ on i n t r a c e l l u l a r pH. Closed c i r c l e s r e p r e s e n t e r y t h r o c y t e s from whole blood while a l l other c e l l s were obtained from washed c e l l s . See t e x t f o r d e t a i l s . Water temperature was maintained a t 5 C, -3-9-0 8-8 8-6 8-4 pHi 8-2 8-0 A 0 7-8 7-6 7-4 no 7-21 10 20 30 60 90 120 T I M E I N S U C R O S E ( M I N ) 43c TABLE #4. E f f e c t o f suspending red c e l l s from whole t r o u t blood i n 300 mM sucrose + 50 mM NaHCO on i n t r a c e l l u l a r pH. T3 -3" I n i t i a l p H i 1 min. B l o o d Sample "A" 7.6 7.9 B l o o d Sample "B" 7.6 8.1 TIME IN SUCROSE + 50 mM NaHCO^ 8 min. 16 min. 30 m i n . 60 m i n . 24 h o u r s 7.9 7.95 - 8.05 8.0 8.05 8.0 8.0 - 8.3 44 r e a d i n g obtained a f t e r 1 man and hemolysis was never e v i d e n t . When t r o u t blood was tonometered f o r one hour with 1% C02 i n a i r , approximately 90% of the t o t a l C02 was found to be present i n the plasma. P a r t i a l C h a r a c t e r i z a t i o n of Plasma Factor P r e l i m i n a r y attempts t o i s o l a t e and c h a r a c t e r i z e the plasma i n h i b i t o r i n t r o u t plasma have r e v e a l e d the f o l l o w i n g . The plasma f a c t o r i s heat and a c i d l a b i l e . The f a c t o r i s n o n d i a l y z a b l e a g a i n s t e i t h e r d e i o n i z e d water or balanced s a l t s o l u t i o n s . The g r e a t e s t f r a c t i o n of a c t i v i t y i s removed by s a l t i n g out the p r o t e i n s with a 50-75% ammonium s u l f a t e s o l u t i o n . Thus i t would appear the plasma i n h i b i t o r i s probably p r o t e i n i n nature and s u f f i c i e n t l y l a r g e as to be unable t o pass through d i a l y s i s t u b i n g . 4 5 D I S C U S S I O N Booth (1938a) concluded that i t was impossible to measure carbonic anhydrase a c t i v i t y of i n t a c t erythrocytes u t i l i z i n g manometric techniques. Booth, using r a t blood, found t h a t only the i n i t i a l few seconds of the r e a c t i o n r e f l e c t e d true carbonic anhydrase a c t i v i t y , a f t e r which the r a t e f e l l to the uncatalyzed r a t e . He s t a t e d that t h i s was due to enzyme substrate a c c e s s i b i l i t y f a c t o r s . He argued that maximal carbonic anhydrase a c t i v i t y was only p o s s i b l e during the i n i t i a l phase of the r e a c t i o n when i n t r a c e l l u l a r c h l o r i d e would be a v a i l a b l e to exchange with e x t r a c e l l u l a r bicarbonate. In my hands, whole r a t blood and washed f i s h blood c o n s i s t e n t l y gave l i n e a r a c t i v i t y during the e v o l u t i o n of the f i r s t 200 m i c r o l i t e r s of carbon dioxide evolved. Booth also s t a t e d that some methods such as the "blob technigue" (a term coined by Booth whereby the red c e i l s remain aggregated r a t h e r than d i s p e r s i n g randomly) and minimizing temperature e g u i l i b r a t i o n times helped maintain i n t r a c e l l u l a r c h l o r i d e l e v e l s , so that the r a t e of CO2 e v o l u t i o n would be maintained longer. While the times f o r temperature e q u i l i b r a t i o n were kept constant, i t was not p o s s i b l e to demonstrate any d i f f e r e n c e between the "blob technique" and "normal" p i p e t t i n g . The reason f o r the d i s p a r i t y i n Booth's r e s u l t s and mine i s not c l e a r . Booth's c o n c l u s i o n that enzyme substrate a c t i v i t y i s c r u c i a l to carbonic anhydrase a c t i v i t y i n i n t a c t e r y t h r o c y t e s seems unquestionably true. I f a s m a l l q u a n t i t y of s a p o n i f i e d r a t blood i s analyzed f o r carbonic anhydrase a c t i v i t y i t has approximately a 7-10 f o l d greater a c t i v i t y than the unlysed 46 equivalent. This f i n d i n g i s i n agreement with Meldrum and Houghton (1933), who found t h a t l y s e d blood from the ox and the goaf had a 4 and 40 f o l d greater a c t i v i t y than the unlysed eq u i v a l e n t s . The s a l i e n t feature concerning the present assay of r a t blood, i n contrast to Booth's data, i s that the r e a c t i o n r a t e s were measurable, reproducible and obeyed l i n e a r k i n e t i c s . I t i s also i n t e r e s t i n g to note that Meldrum & Roughton make no mention of d i f f i c u l t y i n assaying carbonic anhydrase a c t i v i t y from shole blood . Thus i t seems that the assay methods used could a c c u r a t e l y portray any c a t a l y s i s of bicarbonate to C02 o c c c r i n g w i t h i n the i n t a c t e r y t h r o c y t e s , the r a t e being determined by carbonic anhydrase a c t i v i t y and a c c e s s i b i l i t y of substrate. O r i g i n a l attempts to estimate t r o u t carbonic anhydrase a c t i v i t y were d i f f i c u l t to evaluate. While i t c e r t a i n l y appeared that t r o u t blood lacked any dehydration a c t i v i t y , Booth's arguments seemed compelling. I n i t i a l l y i t was f e l t that due t o the methods used any dehydration a c t i v i t y was simply going undetected. However when even 10-20 m i c r o l i t e r volumes of r a t blood gave obvious dehydration a c t i v i t y i t became harder to understand why m i l l i l i t e r volumes of t r o u t blood lacked a c t i v i t y . The chance f i n d i n g of a plasma i n h i b i t o r generated new confidence i n the method and c e r t a i n l y helped e x p l a i n the d i f f e r e n c e between f i s h blood and r a t blood. That an i n h i b i t o r of e r y t h r o c y t i c dehydration a c t i v i t y e x i s t s i s demonstrated by the r e s u l t s of trout assays included i n f i g u r e #8. The o c c a s i o n a l a c t i v i t y found i n some of the e a r l i e r preparations i s probably a r e f l e c t i o n of a combination 47 of l i b e r a t e d c a r b o n i c anhydrase from hemolyzed c e l l s and/cr a d i l u t i o n of the i n h i b i t o r . fl plasma i n h i b i t o r of ca r b o n i c anhydrase i s not a new f i n d i n g . B o o t h (1938b) found i n h i b i t o r s i n the plasma of sheep,pigs, and r a t s among other s . Haetz (1956b), working with f i s h , a l s o found a plasma i n h i b i t o r . L e i n e r e t a l (1962) p a r t i a l l y p u r i f i e d the plasma i n h i b i t o r o f sheep and evaluated i t s e f f e c t s on c a r b o n i c anhydrase from a v a r i e t y of organisms, i n c l u d i n g f i s h . However a l l plasma i n h i b i t o r s thus f a r e s t a b l i s h e d have been assayed f o r t h e i r i n h i b i t i o n of e i t h e r hemolysates or some form of the p u r i f i e d enzyme. Any e f f e c t s which these i n h i b i t o r s may have had on i n t a c t e r y t h r o c y t e s has not been i n v e s t i g a t e d . I t i s i n t e r e s t i n g t o note, however, that washing r a t blood had no e f f e c t on the dehydration a c t i v i t y (Booth found a plasma i n h i b i t o r i n the blood of the r a t ) . Booth, i n d i s c u s s i n g h i s plasma i n h i b i t o r (he claimed a g l o b u l a r p r o t e i n of some s o r t , l a t e r confirmed by L e i n e r 1 s group), suggested i t s p o s s i b l e f u n c t i o n was i n i m m o b i l i z i n g c a r b c n i c anhydrase l i b e r a t e d by e r y t h r o c y t e s during the course of t h e i r normal d e s t r u c t i o n or during t h e i r i n j u r y . T h i s o r i g i n a l l y a t t r a c t i v e p r o p o s a l was undermined when Booth found that human blood a p p a r e n t l y l a c k e d a plasma i n h i b i t o r . P r e l i m i n a r y attempts to c h a r a c t e r i z e the t r o u t i n h i b i t o r do not exclude the p o s s i b i l i t y t h a t i t i s a l s o a g l o b u l a r p r o t e i n . The plasma i n h i b i t o r i s heat and a c i d l a b i l e and presumably i a a l a r g e molecule as judged by i t s i n a b i l i t y to pass thrugh d i a l y s i s t u b i n g . A l s o the f i n d i n g that the i n h i b i t o r can be s a l t e d out with ammonium s u l f a t e i s c o n s i s t e n t with the 48 suggestion that the plasma i n h i b i t o r i s p r o t i n a c i o u s . The plasma i n h i b i t o r could seemingly operate i n one of two fashions. The i n h i b i t o r could act at the l e v e l of the enzyme jagr se ,or the same e f f e c t could be achieved by i n h i b i t i n g the i n f l u x of bicarbonate ions. A complete explanation of how the tr o u t i n h i b i t o r f u n c t i o n s i s not p o s s i b l e ; however the f o l l o w i n g t e n t a t i v e e x p l a n a t i o n can be o f f e r e d at t h i s time. I f t r o u t blood was hemclyzed, dehydration a c t i v i t y was always observed, even i f the o r i g i n a l plasma was present. While t h i s type of experiment does not c o n c l u s i v e l y demonstrate how the i n h i b i t o r f u n c t i o n s i t does e s t a b l i s h that the i n t e g r i t y of the e r y t h r o c y t i c membrane i s e s s e n t i a l f o r the carbonic anhydrase i n a c t i v a t i o n to occur. Cabantchik and fiothstein (1S74a,b), working with human ery t h r o c y t e s , have demonstrated the presence of anion channels i n the membrane. Furthermore, with the use of d i e t h y l s t i l b e n e d e r i v a t i v e s they found i t p o s s i b l e t o i n h i b i t (in excess of 98%) anion f l u x e s , while c a t i o n f l u x e s remain unaffected. I f t h i s type of channel e x i s t s i n t r o u t erythrocytes a plasma i n h i b i t o r could f u n c t i o n a l l y i n h i b i t the enzyme i n t h i s f a s h i o n , Haswell et a l (1978) using the red blood c e l l s of the mouthbreeder , were unable to demonstrate any e f f e c t of plasma on the r a t e s of c h l o r i d e / c h l o r i d e self-exchange. P r e l i m i n a r y i n v e s t i g a t i o n s suggest plasma i s a l s o without e f f e c t on c h l o r i d e / c h l o r i d e exchange In the rainbow t r o u t red blood c e l l s . Thus i t would appear that the presence or absence of plasma does not confer a generalized anion impermeability to f i s h red c e l l s . In these i n v e s t i g a t i o n s (present study and Haswell et a l , 1978) i t was 49 not p o s s i b l e to study i n d e t a i l the e f f e c t s of plasma on net c h l o r i d e f l u x . I n mammalian red c e l l s the r a t e o f c h l o r i d e s e l f -exchange proceeds at a r a t e some 1,000 times f a s t e r than net movements of c h l o r i d e (Knauf e t a l , 1977). Thus the r a t e s of 3 6 c h l o r i d e e g u i l i b r i u m between plasma and c e l l s proceeds with a h a l f - t i m e of only seconds during c o n d i t i o n s o f sel f - e x c h a n g e . The analogous r e a c t i o n , f o r example C l / A c e t a t e or C l / l a c t a t e , takes of the order of minutes or longer . There i s some evidence that these two processes (self-exchange versus net exchange) may u t i l i z e d i f f e r e n t t r a n s p o r t mechanisms (Gunn et a l ,1973). SITS and DIDS (both s t i l b o n i c a c i d d e r i v a t i v e s ) appear to i n h i b i t both net and self-exchange i n human e r y t h r o c y t e s . However the e f f e c t s of SITS i s v a r i a b l e i n i t s response, f o r example, i n the cow SITS produces only 80-90% i n h i b i t i o n of c h l o r i d e e f f l u x versus 98-99% i n human red c e l l s ( Z e i d l e r , personal communication). A d d i t i o n a l l y SITS was without e f f e c t on C l / C l exchange i n t r o u t red c e l l s , although e f f e c t i v e on red c e l l s of lilUJEia. (Haswell et a l , 1S78) . Thus i t i s p o s s i b l e that a negative e f f e c t of plasma on C l / C l exchange i s not s u f f i c i e n t evidence to r u l e out the p o s s i b l e d i r e c t e f f e c t of plasma on C1/HC03 exchange p_er se. Indeed the r a t e s of red c e l l a l k a J i n i z a t i o n would appear to confirm t h i s s u g g e s t i o n . The adoption of the c e l l a l k a l i n i z a t i o n p r o t o c o l p r o v i d e s a method of f o l l o w i n g the a c t u a l C1/HC03 exchange, a l b e i t under a r t i f i c i a l c o n d i t i o n s . when red blood c e l l s are suddenly suspended i n a c h l o r i d e f r e e medium a l a r g e chemical c o n c e n t r a t i o n g r a d i e n t i s e s t a b l i s h e d . However the movement of anions i n t o e r y t h r o c y t e s i s e l e c t r i c a l l y s i l e n t v i a an exchange 50 d i f f u s i o n process (Gunn et a l , 1973), and the eventual e f f l u x of c h l o r i d e i s only p o s s i b l e i f counter ions of l i k e charge are a v a i l a b l e on the o u t s i d e . Thus , as c h l o r i d e l e a v e s , another anion must enter to maintain e l e c t r o n e u t r a l i t y . In t h i s study a l l s o l u t i o n s were made of a n a l y t i c a l grade or b e t t e r chemicals and the added bicarbonate was e s s e n t i a l l y the only a v a i l a b l e counterion. Under these c o n d i t i o n s , as c h l o r i d e leaves bicarbonate e n t e r s , maintaining Donnan e g u i l i b r i u m . The net consequence i s an i n c r e a s e i n red c e l l pHi. Washed red c e l l s , when suspended i n sucrose, r a p i d l y gained bicarbonate as evidenced by the increased pHi. Hydrogen i o n concentrations continued to f a l l although at a slower r a t e throughout the measurement period. This exchange process would be expected to continue u n t i l c h l o r i d e e g u i l i b r i u m i s eventually reached. A f t e r approximately two hours hemolysis was severe and f u r t h e r pHi determinations were impossible due to i n s u f f i c i e n t packed red c e l l s . These r e s u l t s using t r o u t erythrocytes are g u a l i t a t i v e l y i n agreement with those of Z e i d l e r & Kim (1977) working with c a l f red c e l l s . In c a l f red c e l l s hemolysis occurs before f i n a l e g u i l i b r i u m due to the l o s s of membrane s t r u c t u r a l i n t e g r i t y . These authors found that a l k a l i n e pH caused the l o s s of i n t e g r i t y of a membrane p r o t e i n , the band 3 p r o t e i n , which was r e s p o n s i b l e f o r the hemolysis. Hemolysis was not associated with red c e l l volume changes . In contrast to r e s u l t s obtained with washed er y t h r o c y t e s , u t i l i z i n g c e l l s from whole blood r e s u l t e d i n q u a n t i t a t i v e and g u a l i t a t i v e d i f f e r e n c e s , Although an i n i t i a l r i s e i n pHi of between .2-.4 u n i t s was observed upon exposure to sucrose, the pHi t h e r e a f t e r remained constant or 51 rose only s l i g h t l y . In the extreme s i t u a t i o n where e x t r a c e l l u l a r bicarbonate was elevated to 50 mM the pH change a f t e r 24 hours was only .2 u n i t s higher, a d d i t i o n a l l y no signs of hemolysis were apparent even a f t e r 24 hours. Thus i t would appear that t r o u t c e l l s taken from whole blood are s u s c e p t i b l e to e l e v a t i o n s i n pHi as compared to those c e l l s p r e v i o u s l y washed and devoid of plasma. I t may be the small i n i t i a l r i s e i n pHi i s the r e s u l t of outward d i f f u s i o n of molecular C02 from the c e l l i n t o the e x t r a c e l l u l a r medium and/or associated with a changing red c e l l volume. There can be l i t t l e doubt that t r o u t red c e l l f u n c t i o n i n g i s a f f e c t e d by the presence or absence of plasma. This i s not the only instance where plasma born f a c t o r s are known to a f f e c t red c e l l a c t i v i t y . For example, Seider 6 Kim (1978) demonstrated a f a c t o r r e s i d e n t i n cow plasma which i s capable of modulating red blood c e l l g l y c o l y t i c r a t e s . The apparent nonpermeation of bicarbonate i s s i g n i f i c a n t from the standpoint of C02 e x c r e t i o n , but i t can r e a d i l y be appreciated the a b i l i t y to r a p i d l y a l k a l i n i z e red c e l l pH w i l l a f f e c t oxygen loading and thus may be important i n oxygen t r a n s p o r t as w e l l . Indeed t h i s seems to be the s i t u a t i o n in f i s h blood. Berg S Steen (1968) found that the ra t e of oxygen binding by blood l e a v i n g the re t e s t r u c t u r e of the e e l swimbladder was much slower than the rate of oxygen unloading w i t h i n the rete. This phenomenon has important consequences i n swimbladder f u n c t i o n i n g (Fange, 1973; Steen, 1970). F o r s t e r & Steen (196 9) took a c l o s e r look at oxygen binding k i n e t i c s of f i s h blood, using e e l blood and c l e a r l y demonstrated the asymmetrical nature of the "-Boot" 52 s h i f t . F o r s t e r & Steen found that at 10 C the h a l f - t i m e of oxygen r e l e a s e i n the face of e l e v a t e d PC02 and hydrogen i o n c o n c e n t r a t i o n was around 0.02 seconds. Under the reverse c o n d i t i o n s of decreased hydrogen ion c o n c e n t r a t i o n and e l e v a t e d bicarbonate the r a t e of oxygen r e b i n d i n g was f u l l y 9 seconds or over 100X slower. Under c o n d i t i o n s which should enhance oxygen bindin g (decreased hydrogen i o n c o n c e n t r a t i o n and e l e v a t e d bicarbonate l e v e l s ) i t would be p r e d i c t e d that plasma bicarbonate would s h u t t l e i n t o the red c e l l , combine with a c y t o p l a s m i c proton and form C02 and H20. T h i s C02 molecule would then d i f f u s e out of the c e l l f o l l o w i n g the C02 g r a d i e n t . As p r e v i o u s l y s t a t e d c a r b o n i c anhydrase c a t a l y z e s t h i s r e a c t i o n i n mammalian red c e l l s and a l s o the movement of bicarbonate i s very r a p i d , t h e r e f o r e i t i s not s u r p r i s i n g t h a t the r a t e s of oxygen l o a d i n g and unloading i n mammalian r e d c e l l s are e s s e n t i a l l y egual ( F o r s t e r & Steen, 1968). The asymmetrical nature of oxygen l o a d i n g and unloading i n e e l r e d blood c e l l s i s c o n s i s t e n t with the present a n a l y s i s cf t r o u t red c e l l a l k a l i n i z a t i o n . C l e a r l y the r a t e s of oxygen l o a d i n g may be a f f e c t e d by the plasma f a c t o r Another e x p l a n a t i o n of the data however does e x i s t , As demonstrated i n Chapter #1 a l a r g e p o r t i o n of red c e l l c a r b o n i c anhydrase i s bound to or at l e a s t a s s o c i a t e d with the t r o u t red c e l l membrane. T h i s a l s o appears to be the s i t u a t i o n i n f l o u n d e r e r y t h r o c y t e s (Haswell, 1977) and probably i n a l l f i s h . Thus i f t h i s c a r b o n i c anhydrase i s a c c e s s i b l e to the e x t r a c e l l u l a r environment a plasma born f a c t o r could f u n c t i o n a l l y i n h i b i t the enzyme jaer se . However the h y d r a t i o n r e a c t i o n does occur i n i n t a c t e r y t h r o c y t e s ( F o r s t e r & Steen, 1968). Thus a plasma 5 3 f a c t o r w o u l d o n l y i n h i b i t t h e d e h y d r a t i o n r e a c t i o n a n d n o t t h e h y d r a t i o n r e a c t i o n . C u r r e n t l y , no k n o w n c a r b o n i c a n h y d r a s e i n h i b i t o r i s c a p a b l e o f i n h i b i t i n g o n e r e a c t i o n , e i t h e r d e h y d r a t i o n o r h y d r a t i o n , w i t h o u t a l s o i n h i b i t i n g t h e b a c k r e a c t i o n . I t s e e m s r e a s o n a b l e a t p r e s e n t t o a s s u m e t h e e n z y m e c a n c a t a l y z e e i t h e r r e a c t i o n b u t s u b s t r a t e a v a i l a b i l i t y i s t i g h t l y c o n t r o l l e d . I n s u m m a r y , i t c a n be c o n c l u d e d t h a t w h i l e e n o u g h c a r b o n i c a n h y d r a s e may p o s s i b l y r e s i d e i n f i s h r e d c e l l s t o c a t a l y z e t h e d e h y d r a t i o n o f p l a s m a b i c a r b o n a t e t o C 0 2 , i t i s n o t p o s s i b l e t o d e m o n s t r a t e c a r b o n i c a n h y d r a s e d e h y d r a t i o n a c t i v i t y i n i n t a c t e r y t h r o c y t e s . T h i s i n a b i l i t y o f f i s h r e d c e l l s t o d e h y d r a t e p l a s m a b i c a r b o n a t e i s a p p a r e n t l y t h e r e s u l t o f t h e i n a b i l i t y o f t h i s b i c a r b o n a t e t o g a i n a c c e s s t o r e d c e l l c a r b o n i c a n h y d r a s e . A f a c t o r i n t h e p l a s m a , p r o b a b l y p r o t e i n i n n a t u r e , i s r e s p o n s i b l e f o r t h i s i n a c t i v a t i o n . O v e r 9 0 % o f t h e t o t a l C 0 2 f o u n d i n t r o u t b l o o d r e s i d e s i n t h e p l a s m a { t a b l e # 1 0 ) a n d t h e a b i l i t y t o g e n e r a t e m o l e c u l a r CO2 w i t h i n t h e b l o o d m u s t s u r e l y l i m i t t h e r o l e o f t h e e r y t h r o c y t e s a n d e r y t h r o c y t i c c a r b o n i c a n h y d r a s e i n t h e e x c r e t i o n o f C 0 2 a t t h e g i l l s . 54 C H A P T E R I I I ! C 02 E X C R E T I O N I N THE T R O U T l A ROLE FOR B R A N C H I A L C A R B O N I C ANHYDRASE 55 INTRODUCTION Over 901 of the t o t a l C02 in f i s h blood resides in the plasma and the bulk of excreted CO2 originates as plasma bicarbonate. Furthermore, i t can be demonstrated that the formation of molecular C02 i s a slow process, and carbonic anhydrase i s probably necessary. The significance of this slow uncatalyzed rate would be compounded i f g i l l residence times are r e l a t i v e l y short as i s true of blood i n mammalian lung c a p i l l a r i e s (West, 1974) . Hoffert and Fromm (1973); Hodler et a l ,(1955); Haren & Maren, (1964) have repeatedly demonstrated the requirement of carbonic anhydrase for C02 excretion in f i s h . However th i s study has indicated that (1) f i s h red c e l l s may not possess s u f f i c i e n t carbonic anhydrase to catalyze the dehydration reaction, based s o l e l y on t h e o r e t i c a l qrounds, and (2) trout red c e l l s in v i t r o do not appear to dehydrate ex t r a c e l l u l a r bicarbonate at a l l . Given the high lev e l s of carbonic anhydrase a c t i v i t y i n the q i l l i t may be possible t h i s tissue i s the source of carbonic anhydrase responsible f o r the excretion of C02 at the q i l l s . Certainly the findinq that branchial carbonic anhydrase has a qreater a f f i n i t y for bicarbonate than the erythrocytic carbonic anhydrase provides further support for t h i s p o s s i b i l i t y . The following experiments were desiqned to d i r e c t l y test t h i s hypothesis. 56 MATERIAL & METHODS A l l experiments were performed on rainbow t r o u t ( Salmo g a i r d n e r i } weighing between 200-400 grams. These f i s h were maintained i n l a r g e c i r c u l a r tanks provided with oxygenated d e c h l o r i n a t e d Vancouver tap water. The water temperature during the course of these experiments was between 8-10 C. A l l f i s h were implanted with c h r o n i c i n d w e l l i n g c a t h e t e r s f o r blood sampling. Cannulas were implanted i n the d o r s a l aorta according to Smith S B e l l (19 64) with the f o l l o w i n g m o d i f i c a t i o n s . Instead of u t i l i z i n g a needle lodged i n the d o r s a l a o r t a a l e n g t h of P.E. 50 tubing (Clay-Adams) i s advanced d i r e c t l y i n t o the d o r s a l a o r t a u t i l i z i n g a Sovereign I n d w e l l i n g Catheter and needle assembly (Sherwood Medical, St. L o u i s , Mo.). The c a t h e t e r assembly c o n s i s t s of a 20 gauge needle which f i t s snugly i n t o the t i p of a f l a r e d 2 i n c h long c a t h e t e r . Upon s u c c e s s f u l l o c a t i o n and p e n e t r a t i o n of the d o r s a l a o r t a the needle i s withdrawn l e a v i n g the c a t h e t e r s t i l l i n the a o r t a . Once the needle i s removed a l e n g t h of P.E. 50 t u b i n g can be advanced i n t o the d o r s a l a o r t a . A f t e r i n s e r t i o n of the P.E. 50 t u b i n g i n the d o r s a l a o r t a , the Sovereign c a t h e t e r i s withdrawn from the a o r t a and removed, the P.E. 50 tubing i s then secured with a s i l k l i g a t u r e and p u l l e d through a polyethylene nosecone { Snsith S B e l l , 1964). A m o d i f i c a t i o n of t h i s technique i s a l s o u t i l i z e d to cannulate the v e n t r a l a o r t a . In t h i s case the P.E. 50 tubing was secured with two s i l k l i g a t u r e s i n the tongue, with the P.E. 50 tubing e x i t i n g through a s m a l l p e r f o r a t i o n i n the s o f t t i s s u e i n the jaw. T h i s technique i s s u p e r i o r to the o r i g i n a l method u t i l i z i n g needles i n t h a t the cannulas remain 57 patent f o r much lon g e r p e r i o d s of time. A l l o p e r a t i v e procedures were performed under M.S. 222 a n e s t h e s i a . F i s h u t i l i z e d f o r g i l l p e r f u s i o n s t u d i e s were immobilized by a blow to the head subsequent t o c a n n u l a t i o n , the p e r i c a r d i n a l c a v i t y was exposed by a v e n t r a l i n c i s i o n , and a l e n g t h of P.E. 190 tubing was secured with a l i g a t u r e i n the bulbus a r t e r i o s u s . A l l other f i s h were allowed to recover i n i n d i v i d u a l darkened l u c i t e chambers f o r at l e a s t 24 hours b e f o r e the experiments were i n i t i a t e d . Blood Measurements pH determinations were made u t i l i z i n g a Radiometer PHM-71 Acid/Base Analyzer and a s s o c i a t e d micro pH e l e c t r o d e . In an attempt to improve i n v i t r o measurements the use of a medical mass spectrometer, the "Medspect" (Searle I n s t r . , B a i t . MD.), has been employed. The Medspect i s a c t u a l l y designed f o r i n s i t u blood gas measurements i n c l i n i c a l p r a c t i c e . Gas a n a l y s i s i s obtained by u t i l i z i n g e i t h e r s i l a s t i c or t e f l o n covered hcllow s t a i n l e s s c a t h e t e r s . The t e r m i n a l segment of the c a t h e t e r s {approximately 1 i n c h i n the s i l a s t i c c a t h eter) are p e r f o r a t e d with a s e r i e s of h o l e s over which the s i l a s t i c c o a t i n g provides a d i f f u s i o n b a r r i e r . The c a t h e t e r s {2) are f a s t e n e d to the "Medspect" and the e n t i r e system operates under vacuum {approximately 1 0 - 6 t o r r . ) . Gases flow i n t o the c a t h e t e r s from the membrane covered t i p s ( a i r or l i q u i d phase) to the mass spectrometer f o r subsequent a n a l y s i s . Oxyqen and carbon d i o x i d e p a r t i a l pressures ( mm Hg) are d i s p l a y e d on d i g i t a l meters. C02 t e n s i o n s can be expressed i n 0.01 c f a mm Hg while oxygen i s read t o the nearest mm Hg. The response times are e s s e n t i a l l y independent of temperature and the C02 a n a l y s i s i s s u f f i c i e n t l y 58 sensitive to detect differences between inspired and expired water PC02's from a trout. While the system i s designed for ins e r t i o n i n human a r t e r i e s , due to the size and flow dependence of the s i l a s t i c catheter i t i s not possible to obtain in s i t u gas analysis on trout. Conseguently an i n v i t r o system was constructed. One of the two catheters rests in a small thermostatted cuvette (0.3 ml volume) for determination of oxygen and carbon dioxide p a r t i a l pressure. Although the response times of s i l a s t i c catheters are faster than the teflon catheters (1-2 minutes versus 3-5 minutes) they are flow dependent, Conseguently the cuvette i s provided with a small teflon s t i r r i n g bar and the whole apparatus placed on a magnetic s t i r r e r . The second catheter i n a second cuvette i s u t i l i z e d to determine oxygen and carbon dioxide contents. A degassed solution of a c i d i f i e d potassium ferricyanide (Van Slyke, 1927) i s placed into the content cuvette (approximate volume 2 mis) and the i n i t i a l P02 and PC02 noted. After introduction of a blood sample the f i n a l P02/PC02 are recorded (approximately 3 minutes). The t o t a l 0 2 i s determined i n accordance with the method of Tucker (1967) , while t o t a l C02 i s calculated by u t i l i z i n g NaHC03 standards (Cameron, 1971). Gas mixtures provided by Wostoff gas mixing pumps (Bochum, W. Germ.) are u t i l i z e d f o r c a l i b r a t i o n . Although th i s system s t i l l has a response time of 60-90 seconds i t i s a considerable improvement over C02 electrodes and the increased s t a b i l i t y and s e n s i t i v i t y over the temperature and' C02 range u t i l i z e d i s without comparison. Experimental Protocol 59 A) Anemic F i s h . A f t e r recovery i n i t i a l blood samples were obtained to e s t a b l i s h c o n t r o l l e v e l s f o r pH, PC02, t o t a l C02 content (TC02) and hematocrit (bet. ). Severe anemia was then induced e i t h e r by i n t r a p e r i t o n e a l i n j e c t i o n s of phenylhydrazine (Cameron 6 Davis, 1970) or by repeated b l e e d i n g , the blood l o s t being r e p l a c e d by r e t u r n i n g the plasma plus C o r t l a n d s a l i n e to the f i s h . I t was d i f f i c u l t t o remove a l l e r y t h r o c y t e s by e i t h e r method and the anemic f i s h group had hematocrits of l e s s than 4 percent compared with the c o n t r o l group with hematocrits of 18-25%, Dorsal a o r t i c blood was sampled and pHa, PaC02 and TC02 were measured 24 hours a f t e r anemia had been e s t a b l i s h e d . Diamox d i s s o l v e d i n s a l i n e was i n j e c t e d (10 mg/kg body weight) i n t o the d o r s a l aorta of anemic f i s h . Six hours l a t e r pHa, PaC02 and TC02 were measured i n blood sampled from the d o r s a l a o r t a . Thus pHa, PaC02 and TC02 of a r t e r i a l blood were measured i n normal, anemic, and anemic plus Diamox i n j e c t e d f i s h , The same f i s h made up the anemic and anemic Diamox i n j e c t e d groups of f i s h . C02 E x c r e t i o n Bates The e f f e c t of anemia on C02 e x c r e t i o n r a t e s was measured by s e a l i n g a rainbow t r o u t i n a l u c i t e chamber c l o s e d except f o r a water i n l e t and o u t l e t . The water flow r a t e through the box and the C02 content of i n f l o w i n g and o u t f l o w i n g water sere determined. Anemia was then induced by i n t r a p e r i t o n e a l i n j e c t i o n s of phenylhydrazine and 24 hours l a t e r C02 e x c r e t i o n r a t e s were again determined. T h i s same procedure was a l s o u t i l i z e d ' t o assess the e f f e c t s of Diamox on C02 e x c r e t i o n . Perfused G i l I s : These experiments were c a r r i e d out on 14 rainbow t r o u t . A f i s h 60 was secured v e n t r a l s i d e up i n a l u c i t e chamber and the g i l l s were perfused v i a the v e n t r a l aorta with h e p a r i n i z e d (10 I.0./ml) C o r t l a n d s a l i n e using a Harvard Apparatus motor d r i v e n s y r i n g e pump and a 100 ml g l a s s s y r i n g e . The s a l i n e was e g u i l i b r a t e d with 1% C02 mixed with a i r and held a t water temperature. The s a l i n e passed through the g i l l s , around the body and out through the cut v e n t r i c l e at a r a t e of 4.5 ml/min. The f i r s t 100 mis of p e r f u s a t e was used to wash out e r y t h r o c y t e s . Measurements were made on the second 100 mis of pe r f u s a t e before and a f t e r f l o w i n g through the g i l l s . The p o s t b r a n c h i a l sample was obtained through the i n d w e l l i n g d o r s a l a o r t i c c a t h e t e r . G i l l v e n t i l a t i o n was maintained at 1,000 ml/min from a constant head r e s e r v o i r through a rubber tube i n s e r t e d i n the mouth. This r a t e of water flow should have been adequate t o ensure C02 removal (Davis S Cameron, 1970). Diamox (10 raq/kq body weight) d i s s o l v e d i n s a l i n e was i n j e c t e d i n t r a p e r i t o n e a l l y i n t o e i g h t of the fo u r t e e n f i s h , the remaining s i x a c t i n g as a c e n t r c l group. Diamox was i n j e c t e d s i x hours before any surgery was i n i t i a t e d . The Henderson-Hasselbalch equation was used to c a l c u l a t e bicarbonate c o n c e n t r a t i o n s . In the p e r f u s i o n experiments TC02 content was a l s o c a l c u l a t e d u t i l i z i n g t he f o l l o w i n g equation. TC02 = (alpha x PC02) + (alpha x PC02 x a n t i l o g pH - pK) where, alpha i s the s o l u b i l i t y c o e f f i c i e n t of C02 i n s a l i n e at 10 C and pK values are from A l b e r s (1970) f o r human plasma. 61 RESULTS A) Anemia Anemia did not r e s u l t i n any change in pHa, PaC02, or the C02 content of a r t e r i a l blood (table #5). The addition of Diamox, however, caused a marked drop i n pHa and a near t r i p l i n g of PaC02. The i n j e c t i o n of Diamox into anemic f i s h was often l e t h a l whereas i n j e c t i o n of the same dose into controls was rarely so. Presumably the difference in e f f e c t i s due to the buffering power of hemoglobin. A l l anemic f i s h survived the f i r s t six hours and the values were recorded at t h i s time. Although no further change in pH or PaC02 was apparent after six hours TC02 .frequently continued to r i s e , as evident i n those f i s h surviving for longer periods of time. A r t e r i a l blood pH and PaC02 was unaffected by hematocrit (Figures #10 and #11). C02 excretion rates were also unaffected by anemia (table #6). Anemia was correlated with a decrease i n blood oxygen capacity as expected. B) G i l l Perfusion: Perfusion of the g i l l s with saline eguilibrated with 1$ C02 in a i r resulted in the removal of 12% of the t o t a l C02 content of the perfusate {table #7). Only 5% of the t o t a l C02 present i n the inflowing perfusate was molecular C02, the remainder was bicarbonate. Transit time for saline flow through the g i l l s was 1-3 seconds as judged by the appearance of methylene-blue i n the dorsal a o r t i c catheter. The half-time for the uncatalyzed reaction velocity f o r bicarbonate at 10 C i s around one minute 62 ( s e e C h a p t e r # 2 ) , T r e a t m e n t o f r a i n b o w t r o u t w i t h D i a m o x b e f o r e s a l i n e p e r f u s i o n r e d u c e d C02 e x c r e t i o n t o z e r o i n s a l i n e -p e r f u s e d g i l l s ( t a b l e #7). T h e e x c r e t i o n r a t e s i n u n t r e a t e d g i l l p r e p a r a t i o n s a r e g u i t e s i m i l a r t o t h o s e c a l c u l a t e d f r o m a r t e r i a l - v e n o u s TC02 c o n t e n t d i f f e r e n c e s f o u n d i n f r e e s w i m m i n g f i s h (mean = 11.4% p l u s o r m i n u s 4.3%, when n=5 p l u s o r m i n u s 62a TABLE #5. E f f e c t o f anemia and subsequent c a r b o n i c anhydrase i n h i b i t i o n (Diamox) on d o r s a l a o r t i c pH and P C 0 2 i n the rainbow t r o u t . Water temp-e r a t u r e was 9 C. CM Control period Anemic period Diamox + 6 hrs. pH 7.82 ± .08 7.86 i .08 7.44 * .16 n=11, mean i s. dev.] P C O 2 2.64 i .5 2.55 i .35 6.85 i 1.7 62c » FIGURE # 1 0 . Effect of hematocrit on arterial pH in the rainbow trout. S-Oi • 7-8 9 . . . • • • • 7-6 7-4 72 10 15 20 25 Hematocrit FIGURE #11. E f f e c t of h ematocrit on a r t e r i a l PC0 2 the rainbow t r o u t . 60 T 50 E E 40 8 30 0 ? 20 e 1-0 10 15 20 Hematocrit 62g TABLE #6. C 0 2 e x c r e t i o n f o l l o w i n g p h e n y l h y d r a z i n e t r e a t -ment i n rainbow t r o u t (see t e x t f o r d e t a i l s ) . Values not s i g n i f i c a n t l y d i f f e r e n t . 62h CONTROL ANEMIC E x c r e t i o n Rate (mM/hr) 4.83 t 1.36 3.99 + 1.65 (t S.D., where n=6) 62i TABLE #?. E f f e c t of diamox on C 0 2 e x c r e t i o n i n perfused "trout g i l l s . A l l values depicted f o r c o n t r o l f i s h are s i g n i f i c a n t l y d i f f e r e n t at the 9 5 ^ confidence l e v e l using the students " t " t e s t . Experimental values not s i g n i f i c a n t l y d i f f e r e n t . CN VO CONTROL FI S H (n=6 mean * S.D.) P e r f u s a t e D o r s a l a o r t a sample pH 7.495 t 0.008 7.586 + 0.051 PC0 2 7.5 5.36 + 0.69 C 0 2 (g) (mM) 0.51 0.37 + 0.05 HC0 3 (mM) 9.39 ± 0.18 8.4 + 0.67 T o t a l C 0 2 (mM) 9.9 + 0.18 8.77 + 0.67 EXCRETION (%) 11.5 DIAMOX-INJECTED FISH (n=8 mean ± S.D.) P e r f u s a t e D o r s a l a o r t a sample 7.469 + 0.017 7.419 + 0.085 7.5 8.68 + 2.29 0.51 0.59 + 0.16 8.86 + 0.35 8.81 + 0.65 9.37 + 0.35 9.4 I 0.75 Zero 63 DISCUSSION In i n t a c t rainbow t r o u t the dehydration of plasma bicarbonate provides the majority of excreted CO 2 , and plasma bicarbonate c o n c e n t r a t i o n s are reduced by between 10-30% as the blood passes through the g i l l s . Diamox i n j e c t e d i n t o t r o u t r e s u l t s i n a marked i n c r e a s e i n PaC02 and a r e d u c t i o n i n pHa, i n d i c a t i n g t h a t c a r b o n i c anhydrase i s important i n C02 e x c r e t i o n ( H c f f e r t & Frcmm, 1973). Plasma bicar b o n a t e i s excreted as C02 and the dehydration -reaction i s c a t a l y z e d by c a r b o n i c anhydrase because, f i r s t l y , i n the present study, C02 e x c r e t i o n was reduced to zero i n the s a l i n e - p e r f u s e d g i l l f o l l o w i n g a p p l i c a t i o n of Diamox and, secondly, the t r a n s i t time f o r blood flow through the g i l l s i s of the order of a second , much l e s s than the h a l f - t i m e f o r the dehydration r e a c t i o n . The r e s u l t s of the anemia experiments i n d i c a t e that e r y t h r o c y t e s are unnecessary f o r plasma bicarbonate dehydration, a c o n c l u s i o n c o n s i s t e n t with the o b s e r v a t i o n t h a t f i s h e r y t h r o c y t e s i n v i t r o do not c a t a l y z e plasma bicarbonate dehydration. Normal r a t e s of C02 e x c r e t i o n are maintained d u r i n g anemia and the s a l i n e - p e r f u s e d g i l l i s a b l e to e x c r e t e bicarbonate i o n s i n the absence of e r y t h r o c y t e s . The r a t i o of g i l l e p i t h e l i a l t o e r y t h r o c y t i c c a r b o n i c anhydrase i s around 1 (t a b l e #3) . Carbonic anhydrase from the g i l l has a higher s u b s t r a t e a f f i n i t y than t h a t from e r y t h r o c y t e s ( t h i s study and G i r a r d S I s t i n , 1975) and i t i s concluded that plasma b i c a r b o n a t e i s dehydrated w i t h i n the g i l l e p i t h e l i u m . What then i s the f u n c t i o n of e r y t h r o c y t i c c a r b o n i c anhydrase i n f i s h ? Although f i s h red c e l l s may be impermeable to 64 plasma bicarbonate they are permeable t o C02 and so bicarb o n a t e w i l l be formed i n the e r y t h r o c y t e s as CO 2 enters the blood i n the t i s s u e s , causing a Bohr s h i f t augmenting oxygen t r a n s f e r to the t i s s u e s . The slow Boot-on s h i f t observed by Berg S Steen (196 8) i s c o n s i s t e n t with the hypothesis t h a t the f i s h e r y t h r o c y t e s are impermeable t o the e f f l u x as w e l l as i n f l u x of bicarbonate a c r o s s the red c e l l membrane. I f t h i s i s the case then as blood l e a v e s the t i s s u e s and enters the ve i n s , plasma bicarbonate w i l l be formed a t the uncatalyzed r a t e f o l l o w i n g the r i s e i n plasma C02 l e v e l s i n the t i s s u e s . The blood i n the veins i s a c l o s e d system, hence, as plasma bicar b o n a t e l e v e l s i n c r e a s e , PC02 f a l l s . C02 w i l l d i f f u s e from the e r y t h r o c y t e s i n t o the plasma and red c e l l b i carbonate w i l l be dehydrated. The r a t e l i m i t i n g step w i l l be the un c a t a l y z e d hydration r e a c t i o n v e l o c i t y i n the plasma. Thus some of the bicarbonate formed i n the red c e l l s while blood i s i n the t i s s u e c a p i l l a r i e s w i l l be dehydrated to C02 before blood reaches the g i l l s . Plasma bicarbonate e n t e r s the g i l l e p i t h e l i u m and i s dehydrated t o CO2 before d i f f u s i n g i n t o water f l o w i n g over the g i l l s ( f i g u r e #12). Blood t r a n s i t times are around a minute or two i n f i s h (Davis, 1971) and t h i s plus the e x p e r i m e n t a l l y d e r i v e d h a l f - t i m e of around one minute g i v e support f o r t h i s hypothesis. C a l c u l a t i o n s based on C02 e x c r e t i o n r a t e s and changes i n plasma bicarbonate before and a f t e r the g i l l s i n d i c a t e t h a t the majority of C02 excreted o r i g i n a t e s as plasma bicarbonate. Thus the i n c l u s i o n of ca r b o n i c anhydrase i n f i s h red blood c e l l s i s probably a p r i o r i to produce a r a p i d Bohr and/or "Boot- o f f " s h i f t . Thus e r y t h r o c y t i c c a r b o n i c anhydrase i s u t i l i z e d t o f a c i l i t a t e the 64a FIGURE # 1 2 . Summary of carbon d i o x i d e e x c r e t i o n i n rainbow t r o u t . I I l . l 111 mi rissues CO' P l a s m a c a r b o n a n h y d CO- HCO-c a r b o n i c G i l l a n h y d r a s e e p i t h e l i u m . W a t e r CO. 65 l o a d i n g and unloading of oxygen w i t h i n red blood c e l l s with l i t t l e d i r e c t e f f e c t on C02 t r a n s p o r t . Red blood c e l l s are not r e g u i r e d to e f f e c t CO2 e x c r e t i o n and acid-base r e g u l a t i o n i n f i s h . T h i s becomes even more apparent when one c o n s i d e r s that the i c e f i s h (an a n t a r t i c f i s h c h a r a c t e r i z e d by being t o t a l l y devoid of e i t h e r hemoglobin or e r y t h r o c y t e s and hence probably carbonic anhydrase as t h i s enzyme has never been found e x t r a c e l l u l a r l y ) , i s c h a r a c t e r i z e d by normal r a t e s of oxygen uptake and t h e r e f o r e presumably C02 e x c r e t i o n r a t e s (Holeton , 1970) . As p r e v i o u s l y s t a t e d t r o u t , l i k e a l l other a g u a t i c f i s h l i v e i n a medium r e l a t i v e l y poor i n oxygen, and must c o n t i n u a l l y face the problem of e x t r a c t i n g s u f f i c i e n t environmental oxygen to supply t i s s u e needs. F i s h cannot u t i l i z e changes i n v e n t i l a t i o n to achieve pfl r e g u l a t i o n , as do mammals and b i r d s , without compromising oxygen d e l i v e r y (Randall & Cameron, 1S73). In mammals and b i r d s the dehydration of plasma bicarbonate i s never the r a t e l i m i t i n g step i n the production of d i s s o l v e d C02. In f i s h , water/blood d i f f u s i o n d i s t a n c e s and v e n t i l a t i o n : p e r f u s i o n r a t i o s are optimized to ensure oxygen t r a n s f e r . I f the dehydration r e a c t i o n o c c u r r i n g i n the blood was not the r a t e - l i m i t i n g step, then the c o n t r o l of C02 e x c r e t i o n would not be p o s s i b l e , as the l o s s of molecular C02 would be u n c o n t r o l l a b l e . Therefore i n f i s h the production of molecular C02, as i t occurs i n whole blood, i s p o s s i b l e only at the uncatalyzed r a t e , s i n c e red c e l l c a r b o n i c anhydrase i s u n a v a i l a b l e to plasma b i c a r b o n a t e . Due to the long uncatalyzed r e a c t i o n times ( e s p e c i a l l y a t lower ambient temperatures) and 66 s h o r t r e s i d e n c e times f o r blood i n the q i l l very l i t t l e C02 w i l l be formed from plasma bicarbonate as blood moves through the g i l l . T h i s i s supported by the o b s e r v a t i o n that no e x c r e t i o n of C02 occurred i n the i s o l a t e d perfused g i l l s p r e v i o u s l y t r e a t e d with Diamox. The observed CO2 e x c r e t i o n i n f i s h i s the r e s u l t of the movement of plasma b i c a r b o n a t e i n t o the g i l l e p i t h e l i u m . O n l i k e molecular C02 the movement of bicarbonate can be, and i s , r e g u l a t e d t o achieve c o n t r o l of o v e r a l l C02 e x c r e t i o n . The movement of bicarbonate across the g i l l e p i t h e l i u m i s l i k e l y to be complex, f o r i n s t a n c e , Randall e t a l (1976) have shown that b i c a r b o n a t e f l u x can be rev e r s e d i n d o g f i s h . T h i s observed r e v e r s a l of bicarbonate f l u x a c r o s s the g i l l s modulated the a c i d o s i s caused by e l e v a t e d C02 l e v e l s i n the blood. When bicarbonate e n t e r s the g i l l s from plasma and forms C02 there must be a c o - t r a n s p o r t of hydrogen i o n s i n t o the e p i t h e l i u m . T h i s problem has yet to be r e s o l v e d . The f u n c t i o n a l s i g n i f i c a n c e of t h i s p a t t e r n of C02 e x c r e t i o n compared with t h a t seen i n mammals and b i r d s i s the f o l l o w i n g . F i r s t l y , the formation of plasma bicarbonate by hydration i n the plasma, r a t h e r than bicarbonate d i f f u s i o n from the e r y t h r o c y t e s , r e s u l t s i n an e l e v a t i o n i n e r y t h r o c y t i c pH and a b i n d i n g of oxygen to hemoglobin i n the v e i n s , lowering Pv02 as blood flows from the t i s s u e s to the g i l l s and augmenting oxygen g r a d i e n t s a c r o s s the g i l l s . Secondly, the e x c r e t i o n c f a s i g n i f i c a n t p r o p o r t i o n of t o t a l C02 as plasma bicarbonate v i a the g i l l e p i t h e l i u m a l l o w s f o r the modulation of C02 e x c r e t i o n , and t h e r e f o r e blood pH, independent of oxygen-mediated v e n t i l a t o r y adjustments. 67 I t i s t h e r e f o r e p o s s i b l e t c f u r t h e r s t r e s s t h e a n a l o g y o f a c i d - b a s e r e g u l a t i o n i n f i s h a s c o m p a r e d t o t h a t i n i s o l a t e d s i n g l e c e l l s y s t e m s . H o w e v e r t h e e x a c t r e l a t i o n s h i p b e t w e e n i o n i c c o u p l i n g a n d C 0 2 e x c r e t i o n a c r o s s t h e g i l l r e m a i n s o b s c u r e . 6 8 S S i l U l I I - A C I D ^ B A S E B E G U L A T I O N I N THE B A I N B O H TROJQT l J M O D E L . 69 INT.BODUCT.ION T h e r e c a n now be l i t t l e d o u b t t h a t t h e g i l l o f t h e a q u a t i c t e l e o s t i s t h e s i t e o f p l a s n - a b i c a r b o n a t e d e h y d r a t i o n . F u r t h e r m o r e t h e i n v o l v e m e n t o f b r a n c h i a l c a r b o n i c a n h y d r a s e i n t h i s r e a c t i o n i s now f i r m l y e s t a b l i s h e d , What r e m a i n s o b s c u r e i s t h e u n d e r l y i n g m e c h a n i s m w h i c h u l t i m a t e l y p r o v i d e s t h e d e s i r e d a l k a l i n i t y i n t h e p l a s m a o f t h e f r e e s w i m m i n g f i s h . T h e i s o l a t e d p e r f u s e d g i l l p r e p a r a t i o n s h o u l d p r o v i d e a means o f f u r t h e r c h a r a c t e r i z i n g t h e m o v e m e n t o f b i c a r b o n a t e i n t o t h e g i l l ; h o w e v e r i t i s n o t c l e a r w h a t d e t e r m i n e s t h e a c t u a l l o s s o f C 0 2 f r c m t h i s t i s s u e . A l t h o u g h t h e r a t e o f f o r m a t i o n o f m o l e c u l a r C 02 ( o r t h e r e v e r s e r e a c t i o n ) w i l l n o t be r a t e l i m i t i n g d u e t o t h e p r e s e n c e o f b r a n c h i a l c a r b o n i c a n h y d r a s e , t h e r e l a t i v e l o s s o f C 0 2 may w e l l b e l i m i t e d b y t h e e p i t h e l i a l c e l l c y t o p l a s m i c pH. F o r e x a m p l e a n a l k a l i n i z a t i o n o f t h e e p i t h e l i a l c e l l w o u l d s h i f t e g u a t i o n #1a t o t h e r i g h t r e d u c i n g t h e f o r m a t i o n o f C 0 2 . A l t e r n a t e l y t h e r e l a t i v e a c i d i f i c a t i o n o f t h e e p i t h e l i a l c e l l w o u l d f a v o r t h e f o r m a t i o n o f C 0 2 . I t i s a s s u m e d t h a t a s u f f i c i e n t g r a d i e n t f r o m c e l l t o w a t e r w o u l d e x i s t s o t h a t m o l e c u l a r C 0 2 o n c e f o r m e d w o u l d b e r a p i d l y l o s t t o t h e w a t e r . A s p r e v i o u s l y m e n t i o n e d s i n g l e c e l l s r e g u l a t e i n t r a c e l l u l a r pH by u t i l i z i n g e i t h e r c a t i o n i c o r a n i o n i c e x c h a n q e m e c h a n i s m s o p e r a t i n g a t t h e m e m b r a n e l e v e l . I o n i c e x c h a n q e p r o c e s s e s a r e o f c o u r s e k n o w n t o b e p r e s e n t i n t h e g i l l s o f f i s h ( M a e t z , 1 9 7 1 ; M a e t z e t a l , 1 9 7 6 ) a n d i n d e e d t h e s e e x c h a n g e p r o c e s s e s may b e m o d u l a t e d t o a c h i e v e p f l a d j u s t m e n t s i n f i s h ( C a m e r o n , 1 9 7 6 ; De B e n z i s , 1 9 7 5 ; B o r n a n c i n e t ' a l , 1 S 7 7 ) , T h u s i t may b e p o s s i b l e t h a t g i l l c a t i o n i c a n d a n i o n i c e x c h a n g e p r o c e s s e s a r e m o d u l a t e d 70 i n such a fashion as to achieve either a loss or gain of bicarbonate from plasma by co n t r o l l i n g e p i t h e l i a l c e l l pH. Therefore, i n addition to following bicarbonate movements from plasma into g i l l , i t i s of interest to follow plasma t o t a l C02 and pH as a function of s a l t movements into the g i l l . 71 MATERIALS AND METHODS I s o l a t e d Perfused G i l l P r e p a r a t i o n s C02 e x c r e t i o n as a f u n c t i o n o f per f u s a t e bicarbonate l e v e l s : The e f f e c t of i n c r e a s i n g p e r f u s a t e bicarbonate c o n c e n t r a t i o n s was analyzed using the perfused g i l l p r e p a r a t i o n as d e s c r i b e d i n Chapter I I I . A t o t a l of 15 f i s h were perfused with varying HC03 c o n c e n t r a t i o n s , sodium bicarbonate being added to C o r t l a n d s a l i n e to bring the f i n a l c o n c e n t r a t i o n cf C02 to the d e s i r e d l e v e l . A f t e r the a d d i t i o n of b i c a r b o n a t e pH was adjusted to approximately 7.48-7.5 with e i t h e r HCL or NaOfl. E f f e c t s of SITS and A m i l o r i d e cn C02 e x c r e t i o n : During the p r e v i o u s l y d e s c r i b e d perfused g i l l experiments i t was found t h a t the g i l l p r e p a r a t i o n was s u b j e c t to a r a p i d degradation. Using t h i s p r e p a r a t i o n as described the i n i t i a l CO2 e x c r e t i o n r a t e s i n v a r i a b l y f e l l t o l e v e l s approaching z e r c , or even negative values, a f t e r the f i r s t 3 0-6 0 minutes of p e r f u s i o n . Due to t h i s problem i t was only p o s s i b l e t o u t i l i z e one bicarbonate l e v e l per g i l l p r e p a r a t i o n and to analyze C02 e x c r e t i o n only through the f i r s t 100 mi's of p e r f u s i o n . As a r e s u l t the v a r i a b i l i t y among d i f f e r e n t p r e p a r a t i o n s obscured any tr e n d s . An attempt was t h e r e f o r e made to i n c r e a s e t h e i r s t a b i l i t y , and the f o l l o w i n g a l t e r a t i o n s were found to g r e a t l y i n c r e a s e the u s e f u l l i f e s p a n o f the g i l l p r e p a r a t i o n . To C o r t l a n d s a l i n e 4% PVP { p c l y v i n y l p y r r o l i d i n e , M« = 40,000) was added as an osmotic f i l l e r a f t e r which the s o l u t i o n was f i l t e r e d { 0.45 micron, M i l l i p o r e Corp). The f i l t e r e d p e r f u s a t e was then bubbled with n i t r o g e n to lower the P02 to approximate i n vi v o 72 P02»s (20-50 mm Hg). To t h i s s o l u t i o n NaHC03 was added to b r i n g the f i n a l TC02 c o n c e n t r a t i o n to the d e s i r e d l e v e l . These a l t e r a t i o n s were found t o g r e a t l y improve the p r e p a r a t i o n and s t a b l e C02 e x c r e t i o n r a t e s c o u l d be maintained f o r 4-5 hours. A l l subsequent perfused g i l l experiments i n c o r p o r a t e d these changes. The e f f e c t s of 0.1 mM c o n c e n t r a t i o n s of SITS and Amiloride i n the p e r f u s a t e on C02 e x c r e t i o n r a t e s were determined as f o l l o w s . A f t e r i n i t i a l C02 e x c r e t i o n r a t e s were determined e i t h e r SITS or A m i l o r i d e was added t o the p e r f u s a t e . C02 e x c r e t i o n r a t e s were again determined a f t e r p e r f u s i o n of the f i r s t 100 mis of p e r f u s a t e or approximately 20-25 minutes a f t e r i n t r o d u c t i o n of the drug. Whole Animal Experiments C h l o r i d e Uptake Bates Apparent c h l o r i d e i n f l u x e s were determined by f o l l o w i n g the disappearance of r a d i o c h l o r i d e from the bathing medium i n a s m a l l volume r e c i r c u l a t i n g system of approximately 1.5 L. 36-C h l o r i d e was purchased as Na- 3 6C1 from Hew England Nuclear. Counting was done i n a Nuclear Chicago Isocap l i q u i d s c i n t i l l a t i o n counter. E f f e c t of Na and CI uptake i n h i b i t i o n on blood acid-base balance: At l e a s t 24 hours a f t e r o p e r a t i v e procedures d u p l i c a t e blood samples were withdrawn f o r i n i t i a l d eterminations of TC02, PC02 and plasma C l . A f t e r samplinq, the system, as d e s c r i b e d f o r the c h l o r i d e f l u x r a t e s above, was c l o s e d and the a p p r o p r i a t e druqs were added to the bathinq s o l u t i o n . NaSCN was added to a 73 f i n a l c oncentration of 10 mM. SITS ( B r i t i s h Drug House) was added to a f i n a l c o ncentration of .1 mM. Amiloride, a generous g i f t cf Dr Dorian of Merck F r o s s t .Laboratories, a l s o was added to b r i n g the f i n a l c o n c e n t r a t i o n to .1 mM. A l l values c i t e d represent one hour of exposure to the drugs, unless otherwise s t a t e d , whereupon the animals were returned to freshwater. Experimental values were measured i n d u p l i c a t e . E f f e c t of SCN on B r a n c h i a l Carbonic Anhydrase A c t i v i t y . Due to the s i g n i f i c a n c e of b r a n c h i a l carbonic anhydrase i n the e x c r e t i o n of C02, and the a b i l i t y of SCN to i n h i b i t t h i s enzyme (Maren, 1967), the e f f e c t of SCN on b r a n c h i a l carbonic anhydrase was evaluated (see Chapter I , methods). Values at any given SCN concentration were measured i n t r i p l i c a t e . A n a l y t i c a l Procedures: Due to t e c h n i c a l d i f f i c u l t i e s with the Mass Spectrometer i t wasn't o p e r a t i o n a l during much of the i n v e s t i g a t i v e period reported i n t h i s s e c t i o n . Therefore during the perfused g i l l experiments TC02 was determined u t i l i z i n g the method of Cameron (1971). During a m i l o r i d e and SITS experiments on whole animals plasma TC02 was determined using a Harleco-micro C02 analyzer. Chloride concentrations were measured i n plasma samples u t i l i z i n g a Radiometer CMT-10 c h l o r i d e t i t r a t o r . 74 RESULTS P e r f u s e d G i l l E x p e r i m e n t s : A t o t a l o f f i f t e e n f i s h , r e p r e s e n t i n g t h r e e e x p e r i m e n t a l g r o u p s w e r e p e r f u s e d w i t h e i t h e r 8,14 o r 24 mM b i c a r b o n a t e . C 0 2 e x c r e t i o n r a t e s w i t h l o w b i c a r b o n a t e l e v e l s (8 mM) w e r e a p p r o x i m a t e l y 5%. T h i s was l o w e r t h a n t h e C 0 2 e x c r e t i o n r a t e s o f a p p r o x i m a t e l y 1 0 % o b t a i n e d w i t h m edium b i c a r b o n a t e l e v e l s (14 mM) o r t h e h i g h e s t b i c a r b o n a t e c o n c e n t r a t i o n s e m p l o y e d (24 mM) . T h e s e r e s u l t s a r e s u m m a r i z e d i n f i g u r e # 1 3 . S h e n t h e s e e x p e r i m e n t s w e r e p e r f o r m e d i t was o n l y p o s s i b l e t o o b t a i n o n e e x p e r i m e n t a l p o i n t p e r f i s h , a n d c o n s e g u e n t l y t h e r e i s a l a r g e a m o u n t o f v a r i a b i l i t y i n t h e d a t a . H o w e v e r i t a p p e a r s t h e m o v e m e n t o f b i c a r b o n a t e i n t o t h e e p i t h e l i u m i s d e p e n d e n t c n t h e p e r f u s a t e b i c a r b o n a t e c o n c e n t r a t i o n a t a n y f i x e d pH. T h e a p p a r e n t s a t u r a t i o n o b s e r v e d a t t h e h i g h e s t b i c a r b o n a t e c o n c e n t r a t i o n u t i l i z e d may r e f l e c t a r a t e l i m i t i n g s t e p i n t h e d e h y d r a t i o n r e a c t i o n , f o r e x a m p l e h y d r o g e n i o n a v a i l a b i l i t y , o r a l t e r n a t e l y i t may r e f l e c t s a t u r a t i o n o f t h e a n i o n t r a n s p o r t p r o c e s s . I n t r o d u c t i o n o f .1 mM S I T S i n t o t h e p e r f u s a t e t o t a l l y a b o l i s h e d C02 e x c r e t i o n i n 4 o f 6 f i s h t e s t e d . S I T S c u t C 0 2 e x c r e t i o n i n a n o t h e r f i s h b y 5 0 % b u t was w i t h o u t e f f e c t i n o n e f i s h . T h u s i n h i b i t i o n o f a n i o n t r a n s p o r t t o t a l l y a b o l i s h e d o r g r e a t l y r e d u c e d C02 e x c r e t i o n i n 5 o f 6 f i s h e x a m i n e d . T h e r e a s o n f o r t h e l a c k o f e f f e c t i n t h e o n e f i s h i s n o t c l e a r ; h o w e v e r i t may be p o s s i b l e t h a t t h e S I T S w a s n o t o f s u f f i c i e n t c o n c e n t r a t i o n o r a l t e r n a t e l y may n o t h a v e b l o c k e d a n i o n t r a n s p o r t i n t h i s p a r t i c u l a r a n i m a l . 74a FIGURE #13. E f f e c t o f i n c r e a s i n g p e r f u s a t e b i c a r b o n a t e l e v e l s a t constant pH on COg e x c r e t i o n from the perfused g i l l p r e p a r a t i o n . (mean 1 S.D.) 2<H 15 /0 Total C 0 2 Removed 10 5 0 5 10 15 20 25 30 PERFUSATE H C O 3 " (mM) 75 That the p r i n c i p l e source cf protons f o r the dehydration r e a c t i o n i s from the perfusate, i s demonstrated with the c a t i o n t r a n s p o r t i n h i b i t o r a m i l o r i d e . Amiloride i n the perfusate completely abolished C02 e x c r e t i o n i n a l l three g i l l preparations exposed to a m i l o r i d e . Whole Animal Experiments: When t r o u t were exposed t o 10 mM NaSCN i n the bathing medium, c h l o r i d e uptake was completely i n h i b i t e d i n the three f i s h where f l u x e s were determined. Figure #14 i s t y p i c a l of the r e s u l t s obtained and shows the change i n bath 3 6 - c h l c r i d e counts/min versus time i n c o n t r o l and thiocyanate exposure. Af t e r one hours exposure a l l f i s h (n=6) had a pronounced a l k a l o s i s , c o r r e l a t e d with an increase in TC02. A r t e r i a l C02 tensions were unchanged while plasma CI f e l l (Table #8). In one animal i t was p o s s i b l e to determine VC02 by f o l l o w i n g i n s p i r e d and expired water PC02*s as described i n Chapter I I I . Upon exposure t o SCN, VC02 r a p i d l y f e l l by 22% (Table 9). A f t e r removal of SCN from the bathing s o l u t i o n i n t r o d u c t i o n of Diamox (10 mg/kg IV) produced a reduction i n VC02 of approximately 60%. As b r a n c h i a l carbonic anhydrase plays a most v i t a l r o l e i n C02 ex c r e t i o n the e f f e c t of i n c r e a s i n g SCN concentrations on b r a n c h i a l carbonic anhydrase was i n v e s t i g a t e d , f i g u r e #15. At a concentration of 10 mJ3 SCN the enzyme i s f u l l y 80% i n h i b i t e d , with an apparent 150 cf 0.8 mM. SITS Treatment In the t r o u t , c h l o r i d e uptake i s i n h i b i t e d by SITS i n a dose dependent fashion . Figure #16 shows the t y p i c a l response 75a FIGURE #14. E f f e c t of 10 mM SCN on chloride i n f l u x i n a single f i s h . 75c TABLE #8. E f f e c t o f 10 mM SCN on blood acid-base and c h l o r i d e s t a t u s , (± S.D., where n=6) CONTROL SCN % Change H + 13.3 + 1 10.7 + 1 -19 P C O 2 2.41 + 0.47 2.41 + 0.69 N.C. TC0 2 8.64 + 1.37 10.35 + 0.82 +24.6 75e TABLE #9. E f f e c t o f 10 mM SCN and Diamox on CO e x c r e t i o n r a t e s i n t r o u t #5* CONTROL 10 mM SCN DIAMOX I n s p i r e d Water PCO 1.72 1.71 1.80 E x p i r e d Water P C 0 2 d e l t a P C 0 2 % Change 2.86 1.14 100% 2.60 0.89 22% 2.30 0.50 56% FIGURE #15 . B r a n c h i a l c a r b o n i c anhydrase a c t i v i t y with i n c r e a s i n g SCN c o n c e n t r a t i o n s . o-76 of i n c r e a s i n g c o n c e n t r a t i o n s of SITS on c h l o r i d e uptake i n a s i n g l e f i s h . I n h i b i t i o n was complete at .1 mM i n a l l four f i s h where c h l o r i d e i n f l u x was measured. Dnlike i t s a c t i o n i n mammalian e r y t h r o c y t e s the i n h i b i t o r y a c t i o n of SITS appeared to be r e v e r s i b l e i n the t r o u t g i l l . The e f f e c t of e x t e r n a l SITS a t .1 m i l l i m o l a r on blood acid-base s t a t u s and c h l o r i d e are d e p i c t e d i n t a b l e #10. No s i g n i f i c a n t d i f f e r e n c e was obvious a f t e r one hour of SITS treatment. However i f the SITS treatment was continued f o r 3 hours there was a s i g n i f i c a n t r i s e i n a r t e r i a l pH and TC02, t a b l e #11. T h i s t r e n d was e v i d e n t i n two f i s h where the measurements were continued past the f i r s t hour of exposure. SITS up to 1 m i l l i m o l a r was without e f f e c t on b r a n c h i a l c a r b o n i c anhydrase. Ami l o r i d e Treatment Ki r s c h n e r et a l (1973) demonstrated i n the t r o u t , that a m i l o r i d e at 0.1 mM i n the b a t h i n g water almost completely a b o l i s h e d sodium uptake. This r e d u c t i o n i n sodium uptake was c o r r e l a t e d with a 56% f a l l i n t i t r a t a b l e a c i d e x c r e t e d . In t h i s study a m i l o r i d e a t 0.1 mM r e s u l t e d i n a s i g n i f i c a n t f a l l i n pHa. In f o u r of f i v e f i s h t e s t e d TC02 f e l l a f t e r one hour of exposure, although the means are not s t a t i s t i c a l l y d i f f e r e n t due to the l a r g e i n d i v i d u a l v a r i a t i o n among animals. C h l o r i d e f e l l i n a n o n - s i g n i f i c a n t manner, t a b l e #12. 76a FIGURE #16. E f f e c t of i n c r e a s i n g c o n c e n t r a t i o n s of S I T S i n i n s p i r e d water on c h l o r i d e i n f l u x i n a r e p r e s e n t a t i v e rainbow t r o u t . 76 c TABLE #10. Effect of one hour, exposure to 10 M. SITS on blood acid-base and chloride levels. Values a r e not significantly different. pH T C 0 2 m M Control 7.77 ±0.07 10.3 ± 2.3 10" 4 SITS 7.72 ± 0.11 10.8± 2.5 N = 6 , ± S.D. CI meq 115.2± 2.9 113.9± 3.5 76e TABLE #11. E f f e c t o f 3 hours exposure to SITS on blood acid-base and c h l o r i d e s t a t u s i n an i n d i v i d u a l rainbow t r o u t . p H Tco 2 C f Control 7.79 7.5 113 SITS + 1 SITS + 2 SITS + 7.81 7.82 7.85 7.74 7.82 7.85 112 110 108 76g TABLE # 1 2 . E f f e c t of a m i l o r i d e ( 1 0 ~ M.) on blood acid-base and c h l o r i d e l e v e l s i n t r o u t . P H T C 0 2 CI" Control 7 . 7 8 ± 0 . 0 5 11.55 ±3.3 114.8±2.6 Amiloride 7.67+ 0.09 10 .45±2 .4 113.6±4.2 10'4 M N = 5 ± S.D. 77 DISCUSSION C02 movements through the t e l e o s t g i l l can now be c h a r a c t e r i z e d as f o l l o w s . The u l t i m a t e dehydration cf plasma bicarbonate occurs w i t h i n the g i l l e p i t h e l i u m , t h i s dehydration r e a c t i o n being g r e a t l y dependent upon b r a n c h i a l c a r b o n i c anhydrase. The movement of the bicarbonate i n t o the e p i t h e l i u m i s a p a s s i v e process being governed by the g r a d i e n t between plasma and e p i t h e l i a l c e l l . The a c t u a l t r a n s l o c a t i o n of bicarbonate i n t o the e p i t h e l i a l c e l l s possess many of the c h a r a c t e r i s t i c s of an exchange d i f f u s i o n process s i m i l a r to the anion t r a n s p o r t system found i n the red blood c e l l (Gunn et a l ,1973). For example the t r a n s p o r t of b i c a r b o n a t e i n t o the e p i t h e l i u m i s probably a s a t u r a t a b l e process ( f i g #13) . The f i n d i n g t h a t SITS, a potent anion t r a n s p o r t i n h i b i t o r , i n h i b i t e d C02 e x c r e t i o n i s a l s o c o n s i s t e n t with t h i s c o n c l u s i o n . The maximum CO2 e x c r e t i o n r a t e s may be a f u n c t i o n of the number of a v a i l a b l e a n i o n i c t r a n s p o r t i n g s i t e s a v a i l a b l e f o r b i c a r b o n a t e t r a n s p o r t . Thus f o r any given blood d i s t r i b u t i o n p a t t e r n the C02 e x c r e t i o n r a t e s a t t a i n a b l e w i l l be p r o p o r t i o n a l to the e n t r y of bicarbonate. The u l t i m a t e source of protons f o r the b i c a r b o n a t e dehydration r e a c t i o n comes p r i n c i p a l l y from plasma. This c o n c l u s i o n i s based on the f o l l o w i n g o b s e r v a t i o n s . (1) I.ntreduction of a c i d i n t o e i t h e r the blood or the gut pass through the p l a s m a / g i l l membrane as judged by the s t i m u l a t i o n of sodium uptake on the a p i c a l membrane ( Payan S Maetz ,1973; Maetz ,1973). (2) In t h i s study a m i l o r i d e i n the p e r f u s a t e r e s u l t e d i n the t o t a l l o s s of C02 e x c r e t i o n from the i s o l a t e d V 78 perfused g i l l preparation. Kirschner et a l (1973) have already demonstrated that a m i l o r i d e p o t e n t i a t e d i n h i b i t i o n of sodium uptake from water and a l s o blocked the e x c r e t i o n of hydrogen ion e f f l u x . Thus , i f a m i l o r i d e a l s o blocks hydrogen ion entrance i n t o the g i l l from the perfusate, the f a l l i n CO2 e x c r e t i o n can be i n t e r p r e t e t e d as probably a proton l i m i t a t i o n f o r the dehydration r e a c t i o n occurring w i t h i n the epithelium. NaCl movements across the G i l l : A Model f o r Acid-Base Beaulation jln Aguatic F i s h . In the t r o u t , i n h i b i t i o n of c h l o r i d e uptake r e s u l t e d i n an a l k a l o s i s and r e t e n t i o n of C02. On the c o n t r a r y , i n h i b i t i o n of sodium uptake r e s u l t e d i n a blood a c i d o s i s and lowering of plasma TC02. These r e s u l t s now make i t p o s s i b l e t o formulate a simple model capable of e x p l a i n i n g acid-base r e g u l a t i o n as w e l l as p r e d i c t i n g s a l t movements.,In t h i s model the e x c r e t i o n of C02 i s not c o n t r o l l e d per se ,but rather i s a necessary consequence of the a c t i v e r e g u l a t i o n of hydrogen ion l e v e l s , or more p r e c i s e l y a constant r e l a t i v e a l k a l i n i t y (Howell et a l , 1S70). Thus TC02 i n blood may r i s e or f a l l , but t h i s r i s e and f a l l w i l l be t i g h t l y c o r r e l a t e d to the appropriate bicarbonate l e v e l s necessary to achieve any f i x e d hydrogen i o n c o n c e n t r a t i o n . Foremost to the e f f e c t i v e r e g u l a t i o n of plasma acid-base st a t u s i s the a b i l i t y of f i s h to c o n t r o l hydrogen ion l e v e l s w i t h i n the cytoplasm of e p i t h e l i a l c e l l s . The f o l l o w i n g scheme can now be constructed to e x p l a i n CO 2 e x c r e t i o n through the g i l l e p i t h e l i u m , see f i g u r e #17. The placement of a n i o n i c and c a t i o n i c exchange s i t e s on the a p i c a l membrane i s w e l l FIGURE #17. P a t t e r n o f C 0 2 and s a l t movement t h r o u g h t h e t e l e o s t g i l l p r e s e n t e d d i a g r a m a t i c a l l y . CC H 20 G I L L E P I T H E L I U M B L O O D 79 e s t a b l i s h e d { M a e t z e t a l , 1 9 7 6 ; M a e t z , 1 9 7 1 ) . A t l e a s t p a r t o f t h i s c a t i o n i c e x c h a n g e r i s o u a b a i n s e n s i t i v e a n d r e q u i r e s ATP { P a y a n & M a e t z , 1 9 7 3 ) . T h e C 1 / H C 0 3 e x c h a n g e i s p r o b a b l y a n e x c h a n g e d i f f u s i o n s y s t e m . T h e e x i s t e n c e o f a H C 0 3 - d e p e n d e n t A T P a s e i n f i s h g i l l s h a s b e e n w e l l d o c u m e n t e d (De B e n z i s & B o r n a n c i n , 1 9 7 7 ; K i r s c h n e r S K e r s t e t t e r , 1 9 7 4 ; Van A m e l s v o o r t e t a l , 1 9 7 7 ) ; h o w e v e r i t s f u n c t i o n a l i n v o l v e m e n t i n i o n i c e x c h a n g e h a s y e t t o b e e s t a b l i s h e d u n e q u i v o c a l l y . T h e l o c a t i o n o f t h e i o n i c u p t a k e s i t e s o n t h e b a s o l a t e r a 1 b o r d e r h a v e a l r e a d y b e e n d i s c u s s e d . D u r i n q s t e a d y - s t a t e c o n d i t i o n s t h e f a t e c f b i c a r b o n a t e w i l l d e p e n d o n s e v e r a l f a c t o r s , t h e f o r e m o s t b e i n q c y t o p l a s m i c h y d r o q e n i o n l e v e l s . I f c y t o p l a s m i c h y d r o q e n i o n l e v e l s r i s e e q u a t i o n #1 w o u l d b e d r i v e n t o t h e l e f t , t h u s f a v o r i n g t h e f o r m a t i o n o f C 0 2 , t h e p r e s e n c e o f c a r b o n i c a n h y d r a s e e n s u r i n q t h e r e a c t i o n v e l o c i t y i s n o t r a t e l i m i t i n q . O n c e f o r m e d t h e h a l f - l i f e o f C02 i n t h e c y t o p l a s m w o u l d b e e x t r e m e l y s h o r t a s i t i s l o s t v i a d i f f u s i o n t o e x p i r e d w a t e r . C o n v e r s e l y , s h o u l d h y d r o q e n i o n l e v e l s f a l l i n t h e c y t o p l a s m t h e r e a c t i o n w o u l d b e s h i f t e d t o t h e r i q h t , r e s u l t i n q i n a r e t e n t i o n o f H C 0 3 a s p r o t o n s b e c o m e l i m i t i n q . A m i l o r i d e i n t h e p e r f u s a t e t o t a l l y a b o l i s h e d C 0 2 e x c r e t i o n i n t h e i s o l a t e d p e r f u s e d q i l l p r e p a r a t i o n a n d i s a n e x t r e m e e x a m p l e . T h u s u n d e r s t e a d y - s t a t e c o n d i t i o n s ( c o n s t a n t m e t a b o l i c r a t e a n d m o v e m e n t o f b i c a r b o n a t e a n d p r o t o n s i n t o t h e e p i t h e l i a l c e l l s ) i t i s c l e a r t h a t t h e a v a i l a b i l i t y o f p r o t o n s f o r t h e d e h y d r a t i o n r e a c t i o n w i l l d e t e r m i n e t h e v o l u m e o f C02 p r o d u c e d f r o m t h e d e h y d r a t i o n o f b i c a r b o n a t e , a l b e i t c a t a l y z e d . U n d e r t h e s e s t e a d y - s t a t e 80 c o n d i t i o n s , a l t e r i n g the exchange rates on the w a t e r / e p i t h e l i a l membrane w i l l produce p r e d i c t a b l e e f f e c t s on the cytoplasmic pH. When c h l o r i d e uptake i s i n h i b i t e d at the outer membrane ( a p i c a l ) , as during SITS exposure, bicarbonate (normally excreted i n exchange f o r c h l o r i d e ) accumulates and r e s u l t s i n an accumulation of bicarbonate within the blood (table #17). The f a i l u r e of i n h i b i t i o n of C1/HC03 exchange t c produce a more cbvicus e f f e c t may be r e l a t e d to i t s r e l a t i v e l y small c a p a c i t y , Cameron (1976) c a l c u l a t e d t h a t maximally only 2-4 35 of the TC02 excreted was l i n k e d to the uptake of c h l o r i d e i n the a r c t i c g r a y l i n g . A change of 2-4% i n the plasma TC02 i s probably w i t h i n the experimental e r r o r of the TC02 measurements, so that i t may go undetected during the f i r s t hour. When sodium uptake i s i n h i b i t e d the re t a i n e d hydrogen i o n s produce a f a l l i n blood pH and TC02. De Eenzis (1S75) allowed g o l d f i s h to acclimate (6-9 weeks) i n water devoid of e i t h e r sodium or c h l o r i d e . He found that f i s h placed i n ch o l i n e c h l o r i d e had lower plasma pH values and TC02 l e v e l s when compared to untreated c o n t r o l s . When f i s h were placed i n sodium s u l f a t e they became a l k a l i n e and had elevated TC02 l e v e l s . However i t i s worth noting that these f i s h were allowed to acclimate for long periods of time. The small changes i n acid-base s t a t u s observed during t h i s study with SITS treatment are probably i n d i c a t i v e of the small time allowed f o r t r o u t to manifest these, changes. I t i s predicted t r o u t exposed f o r longer lengths of time would a l s o show a more pronounced acid-base s h i f t as was found i n acclimated g o l d f i s h . The o v e r a l l r e g u l a t i o n of pH i n aguatic f i s h appears q u i t e 81 s i m i l a r to the r e c e n t l y d e s c r i b e d s i n g l e c e l l systems such as i n sguid g i a n t axon ( R u s s e l l and Boron, 1976} and the i s o l a t e d barnacle muscle {Boron, 1977). In the sguid g i a n t axon, r e g u l a t i o n occurs v i a an exchange of c h l o r i d e and b i c a r b o n a t e , while the sodium/hydrogen exchanger does not appear to be i n v o l v e d . On the c o n t r a r y A i k i n & Thomas (1977) has shown that i n the mouse s o l e u s muscle c e l l , r e g u l a t i o n i s predominently v i a a c a t i o n i c exchange process with l i t t l e involvement of anion exchange. In the t r o u t i n h i b i t i o n o f c a t i o n i c exchange a t the g i l l i s capable of producing s i g n i f i c a n t changes i n hydrogen i o n l e v e l s and i t i s tempting to draw the c o n c l u s i o n t h a t t h i s exchanger i s the p r i n c i p l e mechanism u t i l i z e d i n f i s h . While the e f f e c t s of c h l o r i d e i n h i b i t i o n on plasma pH were not as dramatic as during sodium t r a n s p o r t i n h i b i t i o n , a s l i g h t a l k a l o s i s d i d r e s u l t , along with an i n c r e a s e i n TC02. Thus i t would appear the a c t i v i t y of t h i s exchange system may a l s o be e f f e c t i v e i n c o n t r o l l i n g hydrogen i o n l e v e l s . Therefore r e g u l a t i o n of both a n i o n i c and c a t i o n i c f l u x r a t e s may be u t i l i z e d to r e g u l a t e plasma hydrogen i o n l e v e l s i n the f i s h . The d i f f i c u l t y i n the c u r r e n t model a r i s e s when t r y i n g t o assess changes i n the f l u x r a t e s i n the face of imposed acid-base d i s t u r b a n c e s , while the r e s u l t o f s w i t c h i n g pumps o f f completely on acid-base s t a t u s i s now c l e a r , the model does not yet allow one to p r e d i c t the magnitude or even the s p e c i f i c exchanger to be u t i l i z e d . For example, f i s h s u b j e c t e d to an a c i d o s i s compensate by i n c r e a s i n g plasma bicarbonate l e v e l s (Janssen S R a n d a l l , 1975; Ran d a l l et a l , 1976; Bornancin et a l , 1977) , and i n h i b i t i o n of c h l o r i d e uptake produces the a p p r o p r i a t e response, e.g. an a l k a l o s i s 82 achieved v i a i n c r e a s e d bicarbonate l e v e l s . Thus i t would seem that f i s h s u b j e c t e d to an a c i d o s i s would n e c e s s a r i l y reduce c h l o r i d e / b i c a r b o n a t e exchange a c t i v i t y to achieve t h i s end. However t h i s could a l s o be achieved by i n c r e a s i n g sodium/hydrogen exchange. Cameron (1976) induced acid-base d i s t u r b a n c e s i n the a r c t i c g r a y l i n g v i a hypercapnia and r a p i d thermal changes and followed sodium and c h l o r i d e f l u x e s . Cameron found t h a t d u r i n g hypercapnia pH r e g u l a t i o n was a s s o c i a t e d with i n c r e a s e d sodium uptake r a t e s . During an a l k a l o s i s induced v i a an acute i n c r e a s e i n temperature, there was an i n c r e a s e i n c h l o r i d e / b i c a r b o n a t e exchange and i n sodium exchange. Thus i n s i t u the animal may decrease or i n c r e a s e i o n i c exchange r a t e s to a d j u s t e p i t h e l i a l pH and u l t i m a t e l y plasma pH. Despite these d i f f i c u l t i e s i t seems that t h i s model a c c u r a t e l y d e p i c t s the a p p r o p r i a t e d i r e c t i o n of cytoplasmic hydrogen i o n movement w i t h i n the g i l l e p i t h e l i u m i n order to ensure constancy of pH i n the plasma of f i s h . SCN Treatment £ B r a n c h i a l Carbonic Anhydrase: The i n i t i a l experiments u t i l i z i n g SCN r e s u l t e d i n an obvious blood a l k a l o s i s a f t e r only one hour, as p r e d i c t e d ; however the r a p i d and l a r g e f a l l i n VC02 as evident i n f i s h #5 when t r e a t e d with SCN was d i s c o n c e r t i n g . This f a l l i n VC0 2 was much l a r g e r than expected based on Cameron's (1976) c a l c u l a t i o n s f o r g r a y l i n g , and a decrease of only a few percent was a n t i c i p a t e d . The i n h i b i t i o n of c h l o r i d e uptake c o u l d c o n c e i v a b l y generate a l a r g e r r e d u c t i o n i n VC0 2 but t h i s would only be p o s s i b l e a f t e r the g i l l e p i t h e l i u m was s u f f i c i e n t l y a l k a l i n e to s h i f t equation #1 to the r i g h t . C l e a r l y from the SITS 8 3 experiments t h i s i s a slow process and the rapi d f a l l i n VC02 cannot be accounted f o r i n t h i s f a s h i o n . U n f o r t u n a t e l y i t was not p o s s i b l e t o f o l l o w changes i n VC02 with SITS treatment due to t e c h n i c a l d i f f i c u l t i e s . A simple and more s a t i s f a c t o r y e x p l a n a t i o n i s t o assume c a r b o n i c anhydrase a c t i v i t y was a l s o i n h i b i t e d during SCN exposure. At 10 mM, 80% of the enzyme*s a c t i v i t y i s i n h i b i t e d . SCN i s only poorly ta ken-up from freshwater (Epstein et a l , 1975 ), even so a SCN c o n c e n t r a t i o n of 0.8 mM i n h i b i t s the enzyme 50%. I t thus seems probable the bulk of the l a r g e f a l l i n VC02, as evident i n f i s h #5, i s due to the i n h i b i t i o n of b r a n c h i a l c a r b o n i c anhydrase by SCN. I t i s of i n t e r e s t . t o compare the e f f e c t s of c a r b o n i c anhydrase i n h i b i t i o n on acid-base s t a t u s when i n h i b i t o n i s brought about by SCN and Diamox. As can be seen i n t a b l e #14, SCN r e s u l t s i n a r i s e i n a r t e r i a l T C 0 2 , HC03 and pH, PC02 does not change, while VC02 f a l l s . C h l o r i d e uptake i s completely i n h i b i t e d , while sodium f l u x e s are un a f f e c t e d ( K e r s t e t t e r S K i r s c h n e r , 1972). The r e s u l t s of Diamox are s t r i k i n g l y s i m i l a r , TC02 and HC03 r i s e and VCO2 f a l l s ; however, u n l i k e SCN treatment, PaC02 and pH f a l l . Diamox i n h i b i t s sodium uptake, while i t s e f f e c t s on c h l o r i d e i s somewhat v a r i a b l e . In the g o l d f i s h , Diamox i n h i b i t s c h l o r i d e uptake (Maetz S G a r c i a -Romeau, 1964); however i n the t r o u t Diamox i s ap p a r e n t l y without e f f e c t on c h l o r i d e f l u x e s ( K e r s t e t t e r & K i r s c h n e r , 1972). Thus the e f f e c t of both these c a r b o n i c anhydrase i n h i b i t o r s i s s i m i l a r except f o r t h e i r d i f f e r e n t i a l a c t i o n on scdium t r a n s p o r t . The c h l o r i d e t r a n s p o r t i n h i b i t i o n brought about by SCN treatment i s probably independent of i t s c a r b o n i c anhydrase 84 e f f e c t i n l i g h t of SCN's demonstrated e f f e c t on HC03~dependent ATPase (Bornancin S de E e n z i s , 1977). Given the present model and data the d i f f e r e n c e s between SCN and Diamox mediated c a r b o n i c anhydrase i n h i b i t i o n can be f u l l y e x p l a i n e d i n terras of the sodium response, such t h a t i n h i b i t o n of sodium uptake during SCN exposure would produce the blood a c i d o s i s and consequent r i s e i n a r t e r i a l C02 t e n s i o n s e v i d e n t d u r i n g Diamox exposure, D I S C U S S I O N 86 Teleosts represent one of the l a r g e r and more s u c c e s s f u l groups of organisms to i n h a b i t the aguatic environment. Probably the success as evidenced by t e l e o s t s can be l a r g e l y a t t r i b u t e d to the e f f i c i e n c y of the g i l l s . In t e l e o s t s , s a l t balance, water movements and ammonia excr e t i o n occur at the g i l l ; and they a l s o provide the necessary surface area f o r the d i f f u s i o n of r e s p i r a t o r y gases. In aguatic t e l e o s t s the o v e r a l l design as w e l l as the v e n t i l a t i o n and perfusion of the g i l l r e s u l t s i n an e f f i c i e n t means of e x t r a c t i n g environmental oxygen to meet t i s s u e demands. Many f i s h are capable of e x t r a c t i n g oxygen at e f f i c i e n c i e s matching or even exceeding those of mammalian lungs (Randall, 1970). 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 f u r t h e r a t t e s t e d to by the a b i l i t y of some tunas to maintain metabolic rates egual to or even exceeding those of comparably si z e d mammals (Stevens , 1972). Conseguently i n normoxic waters oxygen d e l i v e r y to the t i s s u e s probably never poses a problem; however as e f f i c i e n t as g i l l s may be, most f i s h are extremely s e n s i t i v e and vulnerable to decreased environmental oxygen l e v e l s (Sheltcn,- 1970). For example a nominal decrease i n i n s p i r e d water oxygen tensions (Pi02) from 150 - 110 mm Hg i s s u f f i c i e n t to e l i c i t c a r d i o v a s c u l a r responses from free swimming a c t i v e rainbow t r o u t , and as , Pi02 decreased much below h a l f -s a t u r a t i o n , standard metabolic r a t e s can no longer be maintained i n the t r o u t (Hcleton 8 R a n d a l l , 1967). Thus although f i s h g i l l s are e f f i c i e n t at e x t r a c t i n g oxygen, t h e i r v e n t i l a t i o n and perfusion must be responsive to changes i n environmental oxygen l e v e l s to ensure adeguate r a t e s of oxygen d e l i v e r y to the t i s s u e s . I f f i s h were presented with the added task of 87 c o n t r o l l i n g C02 l e v e l s by v e n t i l a t o r y adjustments, oxygen d e l i v e r y would s u r e l y be compromised. T h i s i d e a as o r i g i n a l l y proposed by Randall & Cameron ( 1973) makes good b i o l o g i c a l sense f o r an a g u a t i c t e l e o s t . While g i l l s f a c i l i t a t e gas t r a n s f e r by g r e a t l y i n c r e a s i n g the a v a i l a b l e s u r f a c e area and decreasing d i f f u s i o n d i s t a n c e s between blood and water, the g i l l cannot be considered simply as a t h i n sheet of blood covered by e p i t h e l i a l c e l l s . The g i l l i s a very complex and m e t a b o l i c a l l y a c t i v e t i s s u e and combines the f u n c t i o n s of the mammalian lung with some of the f u n c t i o n s of the mammalian kidney. Therefore i t r e a l l y i s no more remarkable t h a t acid-base s t a t u s i s u n a f f e c t e d by changes i n Vg (mediated to achieve constant metabolic rates) than the f a c t that s a l t and ammonia homeostasis i s l i k e w i s e u n a f f e c t e d . C l e a r l y changes i n v e n t i l a t i o n and p e r f u s i o n of the t e l e o s t g i l l are p r i m a r i l y f o r the purpose o f ma i n t a i n i n g adequate oxygen uptake at e n e r g e t i c a l l y f a v o r a b l e r a t e s . That the p a t t e r n of C02 e x c r e t i o n and acid-base r e g u l a t i o n i n f i s h i s d i s t i n c t l y u n l i k e the system as e x e m p l i f i e d by b i r d s and mammals there can now te l i t t l e doubt and i n some r e s p e c t s the notion t h a t f i s h red c e l l s c o n t a i n c a r b o n i c anhydrase and hence must f u n c t i o n j u s t l i k e mammalian red c e l l s has h i t h e r t o only served to confuse the s i t u a t i o n of acid-base r e g u l a t i o n i n f i s h . As the d i f f u s i o n of C02 i n an a g u a t i c medium exceeds that fo r oxygen, i t can be a p p r e c i a t e d t h a t any organism capable of procuring s u f f i c i e n t oxygen v i a d i f f u s i o n would not face a problem e x c r e t i n g molecular C02. Thus the e x c r e t i o n of C02 i n small a g u a t i c organisms never presented a problem. Of much g r e a t e r importance would be the maintenance of s a l t and water 88 balance along with c c n t r o l of hydrogen i o n l e v e l s . In these s m a l l a g u a t i c organisms a premium would be on i o n i c exchange mechanisms capable of e f f e c t i v e l y modulating the i n t e r n a l environment with r e s p e c t to s o l u t e and water balance. At ambient a g u a t i c temperatures, l i t t l e b i c a r b o n a t e or hydrogen ions would be formed v i a the uncatalyzed h y d r a t i o n of C02. Consequently the production o f endogenous coun t e r i o n s f o r exchange of sodium and c h l o r i d e would be smal l as the bulk of C02 would be r a p i d l y l o s t v i a d i f f u s i o n i n t o the surrounding environment . The i n c l u s i o n of c a r bonic anhydrase i n these c e l l s would thus g r e a t l y f a c i l i t a t e the h y d r a t i o n r e a c t i o n and hence an e n e r g e t i c a l l y f a v o r a b l e mechanism t o provide the endogencusly r e q u i r e d c o u n t e r i o n s . In a d d i t i o n to p r o v i d i n g c o u n t e r i o n s f o r the a p i c a l exchange process, the c o u p l i n g of C02 e x c r e t i o n to i o n i c exchange would provide a means of c o n t r o l l i n g c e l l u l a r b u f f e r r e s e r v e . Note t h a t the i n c l u s i o n of c a r b o n i c anhydrase i n these c e l l s i s to f a c i l i t a t e i o n i c exchange processes a t the expense of metabolic molecular C02 and not to f a c i l i t a t e the production of molecular C02 (via the dehydration r e a c t i o n ) . As organisms grow i n s i z e and t r a n s i t times and/or d i s t a n c e s i n c r e a s e t i s s u e C02 s t o r e s w i l l b u i l d and the bulk of e x t r a c e l l u l a r CO2 w i l l now be as bi c a r b o n a t e as the r e a c t i o n moves toward e q u i l i b r i u m . Thus at the exchange s i t e e i t h e r bicarbonate (and hydrogen ions) must be excreted d i r e c t l y or a l t e r n a t e l y C02 must be dehydrated from bi c a r b o n a t e and hydrogen i o n s at the exchange s i t e t o avoid a bu i l d - u p of C02. I f normal g i l l f u n c t i o n i n g i s s e v e r e l y impaired in f i s h such as by i n a c t i v a t i o n o f the g i l l s , as when f i s h are exposed t o a i r or i n a c t i v a t i o n of b r a n c h i a l c a r b o n i c anhydrase 89 as during diamox treatment, a r e t e n t i o n of C02 develops along with the a s s o c i a t e d a c i d o s i s . However i t now appears t h a t these cases are not r e a l l y p h y s i o l o g i c a l and the g i l l s are a c t u a l l y a hydrogen i o n e x c r e t i n g and r e g u l a t i n g t i s s u e , r a t h e r than a C02 e x c r e t i n g and r e g u l a t i o n pathway. Obviously C02 e x c r e t i o n does occur at the g i l l ; however t h i s i s a conseguence of the a c t i v e r e g u l a t i o n of hydrogen i o n a c t i v i t y w i t h i n plasma. Only when the input of metabolic CO2 surpasses the c a p a b i l i t i e s of the "proton pumping" mechanism at the g i l l would e x c r e t i o n of C02 per se become s i g n i f i c a n t . Thus so long as plasma hydrogen i o n a c t i v i t y f a l l s near some d e f i n e d " s e t p o i n t " t o t a l C02 would net be expected to be c o n t r o l l e d . T h i s appears to be the s i t u a t i o n i n f i s h , as Randall S Cameron (1973) found that d u r i n g temperature induced acid-base d i s t u r b a n c e s a r t e r i a l C02 t e n s i o n s remained constant while t o t a l C02 rose and f e l l a p p r o p r i a t e l y . Again duri n g hypercapnia hydrogen i o n a c t i v i t y i s r e g u l a t e d independently from a r t e r i a l C02 t e n s i o n s (which remained approximately 2.0 mm Hg above i n s p i r e d l e v e l s ) by i n c r e a s i n g t o t a l C02. As s t a t e d p r e v i o u s l y these changes i n hydrogen ion a c t i v i t y and TC02 are achieved independent of v e n t i l a t o r y changes. Thus while the a b i l i t y to e x c r e t a C02 and/or c o n t r o l l i n g t h e i r a b s o l u t e l e v e l s i n a g u a t i c t e l e o s t s probably r a r e l y poses a problem, hydrogen i o n a c t i v i t y i s t i g h t l y r e g u l a t e d . T h i s r e g u l a t i o n of hydrogen i o n a c t i v i t y i s f a c i l i t a t e d by c o u p l i n g e x p i r e d C02 to s a l t movements across the w a t e r / g i l l membrane (Chapter IV). The mechanisms u t i l i z e d to c o n t r o l plasma hydrogen ion l e v e l s are remarkably s i m i l a r to the responses of s i n g l e c e l l systems exposed to acid-base 9 0 c h a l l e n g e s . T h u s e i t h e r c a t i o n i c , a n i o n i c o r b o t h e x c h a n g e p r o c e s s e s a r e u t i l i z e d t o m o v e h y d r o g e n i o n s o r t h e i r e g u i v a l e n t s i n t h e a p p r o p r i a t e d i r e c t i o n f r o m t h e i n t e r n a l m i l e a u . C a t i o n i c a n d a n i o n i c e x c h a n g e p r o c e s s e s a r e a l s o u t i l i z e d i n t h e t r o u t when f a c e d w i t h a n a c i d - b a s e c h a l l e n g e a n d t h e t o t a l i n h i b i t i o n o f t h e s e e x c h a n g e s y s t e m s r e s u l t s i n a c i d -b a s e d i s t u r b a n c e s i n t h e t r o u t ( C h a p t e r I V ) , T h e e x a c t n a t u r e o f p r o t o n p u m p i n g i n t h e t e l e o s t g i l l a n d t h e m e c h a n i s m o f r e g u l a t i o n r e m a i n s o b s c u r e . H y d r o g e n i o n p u m p i n g i s w e l l d o c u m e n t e d i n n u m e r o u s t i s s u e s a n d o r g a n i s m s a n d p o s s i b l y t w o o f t h e b e t t e r u n d e r s t o o d s y s t e m s a r e t h e a m p h i b i a n a n d r e p t i l i a n u r i n a r y b l a d d e r a n d a c i d s e c r e t i o n i n v e r t e b r a t e g a s t r i c m u c o s a . T h e u r i n a r y b l a d d e r i s c h a r a c t e r i z e d b y t h e a b i l i t y t o a c i d i f y t h e l u m i n a l s i d e s o l u t i o n b o t h i n v i v o a n d i n y i t r o . B e c a u s e t h i s t i s s u e i s m o r p h o l o g i c a l l y a s h e e t l i k e s t r u c t u r e i t i s p o s s i b l e t o m o u n t t i s s u e p r e p a r a t i o n s i n U s s i n g t y p e c h a m b e r s a n d h a s p r o v e d m o s t u s e f u l i n a s s e s s i n g p r o t o n p u m p i n g i n b i o l o g i c a l s y s t e m s . M u c h o f t h e p r e s e n t u n d e r s t a n d i n g o f t h i s t i s s u e i s b a s e d o n t h e w o r k o f S t e i n m e t z , S c h w a r t z a n d t h e i r c o - w o r k e r s a n d t h e f o l l o w i n g a c c o u n t i s b a s e d p r i m a r i l y o n t h e i r w o r k ( S t e i n m e t z , 1 9 6 7 , 1 9 6 9 , 1 9 7 4 ; S t e i n m e t z S L a w s o n , 1 9 7 1 ; S c h w a r t z , 1 9 7 6 ; S c h w a r t z S S t e i n m e t z , 1 9 7 1 ; S c h w a r t z e t a l , 1 9 7 2 ; L e s l i e e t a l , 1 9 7 3 ; A l - A w g a t i e t a l , 1 9 7 6 ; A l - A w g a t i e t a l , 1 9 7 7 ) . A t t h e l u m i n a l b o r d e r o f t h e b l a d d e r a m o l e c u l e o f w a t e r i s c l e a v e d i n s o m e m a n n e r p r o d u c i n g a p r o t o n p l u s a h y d r o x y l i o n . T h e p r o t o n i s e x c r e t e d w i t h t h e a s s o c i a t e d i n w a r d m o v e m e n t o f a s o d i u m i o n m a i n t a i n i n g e l e c t r o n e u t r a l i t y . I t ' s n o t c e r t a i n i f t h i s s o d i u m / h y d r o g e n t r a n s l o c a t i o n i s a n o b l i g a t o r y 9 1 exchange; however removal of luminal side sodium cr exposure to am i l e r i d e decreases a c i d s e c r e t i o n , a l t e r n a t e l y decreasing rates of ac i d s e c r e t i o n a f f e c t the rates of sodium i n f l u x at the lumi n a l border. The hydroxyl remaining from the p h o t o l y s i s of water would now be expected to d r a s t i c a l l y e l e v a t e c e l l u l a r pH unless buffered or excreted. In f a c t i t can be demonstrated that t h i s hydroxyl i o n i s buffered by the hydration of C02 + OH to HC03, with the bicarbonate ion having l i t t l e d i r e c t e f f e c t on c e l l u l a r pHi. This b u f f e r i n g i s c r u c i a l to the a b i l i t y of the bladder to excrete protons,. This conclusion i s based on the f o l l o w i n g . The production of metabolic C02 i s n ' t s u f f i c i e n t to maintain maximal proton pumping r a t e s . I f C02 however i s increased i n a stepwise fashion on the s e r o s a l side of the bladder the increase i n t i t r a t a b l e a c i d on the lum i n a l side i n c r e a s e s u n t i l a maximal r a t e of pumping i s achieved whereupon f u r t h e r increases i n C02 are without e f f e c t {Schwartz, 1976}, The b u f f e r i n g a c t i o n v i a the hydration of C02 i s carbonic anhydrase dependent, as diamox treatment produces a f a l l i n proton pumping. Carbonic anhydrase i n the toad bladder i s found i n the cytoplasm but also seems to be incorporated i n t o the luminal membrane. Diamox i n the luminal bath produces a rapid f a l l i n proton pumping while diamox i n the s e r o s a l bath r e q u i r e s higher concentrations and i s c h a r a c t e r i z e d by a d e f i n i t e time l a g before proton pumping f a l l s (Schwartz, 1976). This type of evidence i s i n t e r p r e t e d to mean the bound carbonic anhydrase i n the luminal membrane i s probably r e s p o n s i b l e f o r the f a l l i n proton pumping during diamox i n h i b i t i o n . I n t e r e s t i n g l y when proton pumping i s i n h i b i t e d i n the t u r t l e bladder with diamox, 92 sodium i n f l u x a l s o f a l l s . In the t r o u t , carbonic anhydrase i s a l s o i n the a p i c a l (='s luminal membrane of the u r i n a r y bladder) membrane and diamox a l s o i n h i b i t s sodium i n f l u x . The c e l l u l a r bicarbonate formed from the h y d r a t i o n of C02 i n the bladder i s excreted i o n i c l y . The bulk cf the bicarbonate moves through the s e r o s a l membrane i n t o the bathing s o l u t i o n . T h i s step can be blocked by SITS. Some bicarbonate a l s o leaves v i a the l u m i n a l membrane as w e l l . The movement of t h i s bicarbonate i s dependent on e x t e r n a l c h l o r i d e and appears to be a t i g h t 1:1 c o u p l i n g . In the u r i n a r y bladder the magnitude of t h i s exchange i s smal l and diamcx doesn't appear to a l t e r c h l o r i d e i n f l u x g r e a t l y . I t may be p o s s i b l e t h a t e p i t h e l i a c h a r a c t e r i z e d by a gr e a t e r c h l o r i d e / b i c a r b o n a t e exchange c a p a c i t y may be more or l e s s s u s c e p t a b l e to i n h i b i t i o n of anion movements during c a r b o n i c anhydrase i n h i b i t i o n . Thus i f one f i s h had a high c a p a c i t y to take-up environmental c h l o r i d e (in exchange f c r bicarbonate) diamcx might a p p r e c i a b l y i n h i b i t t h a t f l u x by i n h i b i t i n g the hy d r a t i o n r e a c t i o n and hence the supply of bicarbonate f r e e t o exchange. I f the magnitude of the pump was small i t might net be a f f e c t e d to any g r e a t extent. I f t h i s i s true then the d i f f e r i n g e f f e c t s o f diamox on c h l o r i d e uptake i n f r e s h water f i s h might be e x p l a i n a b l e . Maetz and Garcia-Eomeau found c h l o r i d e uptake was i n h i b i t e d with diamox i n the g o l d f i s h while diamox was apparently without e f f e c t i n the t r o u t ( K e r s t e t t e r & K i r s c h n e r , 1972). Acid s e c r e t i o n i n the u r i n a r y bladder and the f i s h g i l l are s i m i l a r i n s e v e r a l other r e s p e c t s , a m i l o r i d e blocks sodium uptake ac r o s s the g i l l and b l o c k s hydrogen i o n e x c r e t i o n i n t r o u t (Kirschner et a l , 1973). Ansiloride has the same e f f e c t i n 93 t h e u r i n a r y b l a d d e r . As j u s t m e n t i o n e d a c i d s e c r e t i o n i s s t r o n g l y c o r r e l a t e d t o n u t r i e n t ( s e r o s a l ) s i d e C 0 2 l e v e l s . When t h e o n l y s o u r c e o f C 0 2 f o r t h e h y d r a t i o n r e a c t i o n i s f r o m e n d o g e n o u s m e t a b o l i c C 0 2 p r o d u c t i o n , p r o t o n p u m p i n g i s g r e a t l y r e d u c e d , P a y a n a n d M a t t y ( 1 9 7 5 ) w o r k i n g w i t h p e r f u s e d t r o u t g i l l s m e a s u r e d t h e a c i d i f i c a t i o n o f t h e b a t h i n g s o l u t i o n w i t h t i m e w h e n t h e g i l l s w e r e p e r f u s e d w i t h 5% C02 o r z e r o CO 2 g a s e g u i l i b r a t e d s o l u t i o n s . When t h e p e r f u s a t e l a c k e d C 0 2 t h e a p p a r e n t r a t e o f a c i d i f i c a t i o n o f t h e e n v i r o n m e n t a l w a t e r was a p p r o x i m a t e l y h a l f t h a t f o u n d d u r i n g p e r f u s i o n w i t h 5 % C 0 2 . T h e i n c r e a s e i n a c i d i f i c a t i o n was n o t t h e r e s u l t o f d i f f u s i o n o f m o l e c u l a r C 0 2 a n d r e p r e s e n t e d h y d r o g e n i o n s . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e d a t a f o r t h e t u r t l e b l a d d e r w h e r e h y d r o g e n i o n s e c r e t i o n i s d e p e n d e n t o n s e r o s a l C 0 2 l e v e l s . U n f o r t u n a t e l y w h i l e t h e u r i n a r y b l a d d e r i s e a s i l y t e s t e d e x p e r i m e n t a l l y t h e t e l e o s t g i l l p r o v i d e s much m o r e f o r m i d a b l e t e c h n i c a l d i f f i c u l t i e s . P r e s e n t l y i t c a n o n l y b e s u g g e s t e d t h a t t h e g i l l a p p e a r s r e m a r k a b l y s i m i l a r t o a c i d s e c r e t i o n i n t h e w e l l d e f i n e d a m p h i b i a n a n d r e p t i l i a n u r i n a r y b l a d d e r . C l e a r l y t h i s a r e a r e g u i r e s f u r t h e r w o r k b u t a l s o a p p e a r s t o be a f r u i t f u l a r e a f o r f u t u r e r e s e a r c h . T h a t f i s h a n d many s i n g l e c e l l s y s t e m s r e g u l a t e i n t e r n a l h y d r o g e n i o n l e v e l s when f a c e d w i t h a n a c i d - b a s e c h a l l e n g e i s c l e a r ; h o w e v e r t h e u l t i m a t e c o n t r o l l i n g m e c h a n i s m s r e m a i n s o b s c u r e . I n e x a m i n i n g a c i d s e c r e t i o n i n t h e v e r t e b r a t e g a s t r i c m u c o s a S a c h s ( 1 9 7 8 ) h a s s u g g e s t e d t h a t a t l e a s t t w o l e v e l s o f c o n t r o l e x i s t i n t h i s t i s s u e . F i r s t , b l o o d f l o w o r p e r f u s i o n c a n be u t i l i z e d . I n t h e s t o m a c h a c i d s e c r e t i o n o n l y o c c u r s when t h e 94 acid s e c r e t i n g membranes are perfused. Thus many neural and humoral agents known to stimulate a c i d s e c r e t i o n i n the stomach do so by t h e i r a b i l i t y to i n c r e a s e perfusion of the appropriate a c i d s e c r e t i n g membranes. The second l e v e l cf c o n t r o l r e s i d e s at the c e l l u l a r or biochemical l e v e l . For example i n the t u r t l e bladder aldosterone i n c r e a s e s a c i d s e c r e t i o n by s t i m u l a t i n g sodium uptake at the luminal membrane (Al-Awguati e t a l , 1576), Cyclic-AMP i s l i k e w i s e e f f e c t i v e (Aceves, 1977) , Thus many po s s i b l e c o n t r o l l i n g mechanisms e x i s t at the c e l l u l a r l e v e l i n f i s h . Regulation of a c i d s e c r e t i o n i n the f i s h i s probably normally never perfusion l i m i t e d with r e g u l a t i o n most l i k e l y occuring at the c e l l u l a r l e v e l p o s s i b l y humorally mediated. I f aguatic t e l e o s t s never faced the problem of r e t a i n i n g C02 and the g i l l i s u t i l i z e d to adjust plasma hydrogen ion l e v e l s v i a i o n i c exchange mechanisms across the w a t e r / g i l l membrane what r e s t r i c t i o n s are placed on a e r i a l r e s p i r a t i o n ? In most f i s h t o t a l l y removed from water the a b i l i t y of the g i l l s to f u n c t i o n i s g r e a t l y reduced. The p r i n c i p a l s i t e of environmental exchange i n the t e l e o s t g i l l i s the l e a f - l i k e secondary lamellae . These s t r u c t u r e s are e a s i l y supported i n water; however i n the l e s s dense medium of a i r the secondary lamellae c o l l a p s e under t h e i r own weight. The c o l l a p s e of the secondary lamellae c r e a t e s a huge d i f f u s i o n dead space and undoubtedly an increase i n g i l l vascular r e s i s t a n c e . Thus t y p i c a l l y , g i l l s i n a i r are d i f f u s i o n and p o s s i b l y perfusion l i m i t e d with respect to oxygen and carbon d i o x i d e t r a n s f e r . However i n a d d i t i o n to l i m i t i n g gas t r a n s f e r i o n i c processes oc c u r r i n g i n the presence of water w i l l a l s o be a f f e c t e d . Thus 95 ammonia e x c r e t i o n and i o n i c exchange would a l s o he l i m i t e d i n f i s h denied the a b i l i t y to v e n t i l a t e t h e i r g i l l s with water. Therefore a rainbow t r o u t placed i n a i r would be l i m i t e d with r e s p e c t t o oxygen uptake and carbon d i o x i d e e x c r e t i o n but a l s o the a b i l i t y to r e g u l a t e a r t e r i a l hydrogen ion l e v e l s as well as ammonia and s a l t l e v e l s . Thus f o r the t r o u t i n a i r a b u i l d - u p of C 0 2 would occur and t h i s r e t e n t i o n of C02 would c l e a r l y not be compensated f o r as proton pumping i s not now p o s s i b l e . However i t i s p o s s i b l e that i n the case of a rainbow t r o u t the l i m i t i n g f a c t o r f o r s u r v i v a l may be oxygen procurement and t i s s u e anoxia would prove f a t a l b e f o r e the animal encountered a hypercapnic a c i d o s i s of s u f f i c i e n t magnitude to be l e t h a l . Many f i s h have developed a l t e r n a t e or accessory gas exchange areas to u t i l i z e atmospheric oxygen and do not f a c e anoxia d u r i n g - a i r exposure. Accessory gas exchange organs have evolved i n numerous groups of f i s h i n order to o b t a i n oxygen from a i r . These a i r b r e a t h i n g organs may be m o d i f i e d swimbladders, pharyngeal c a v i t i e s and even stomach and i n t e s t i n e (see Johansen, 1970; Munshi, 1976; Singh, 1976 f o r reviews). These organs are u t i l i z e d f o r oxygen uptake, as i n d i c a t e d by t h e i r t y p i c a l r e s p i r a t o r y q u o t i e n t s of 0.1 - 0.4, The g i l l s are thus r e t a i n e d as the major route f o r carbon d i o x i d e e x c r e t i o n with the method presumably i d e n t i c a l ' t o that i n t r u l y a g u a t i c f i s h such as the t r o u t . Hhen a i r b r e a t h i n g f i s h such as the bladder breather H o p l e r y t h r i n u s u n i t a e n i a t u s are a i r exposed a r t e r i a l oxygen content remains high (Haswell, unpublished o b s e r v a t i o n s ) . Johansen (1966) found t h a t i n Synbranchus blood oxygen c a r r y i n g c a p a c i t y a c t u a l l y i n c r e a s e d from a maximal of 50-60% s a t u r a t i o n , 96 while i n water, to 100% s a t u r a t i o n during a i r exposure. During t h i s same a i r exposure C02 content a c t u a l l y rose and d i d not f a l l u n t i l a g u a t i c v e n t i l a t i o n was i n i t i a t e d . Thus althouqh i n Svmbranchnus a i r exposure and u t i l i z a t i o n o f an accessory gas exchanqe pathway a c t u a l l y enhanced oxygen uptake a r e t e n t i o n of C02 develops. As f i s h red c e l l s f a i l to dehydrate plasma bicarb o n a t e ( t h i s study) and the accessory exchange organs do not possess c a r b o n i c anhydrase (Burggren 6 Haswell, 1978) these animals can ex c r e t e C02 at these exchange s i t e s only at the uncatalyzed r a t e s . That t h i s i s t r u e i s f u r t h e r demonstrated by Randa l l et a l (1978). These authors found that i n f u s i o n of bovine c a r b o n i c anhydrase i n t o air-exposed H o p l e r y t h r i n u s r e s u l t e d i n a doubling o f the bladder r e s p i r a t o r y q u o t i e n t which g r e a t l y a l l e v i a t e d the r i s e i n blood PaC02 and f a l l i n pHa normally evident during a i r exposure i n t h i s f i s h . F igure #18 demonstrates the e f f e c t of a i r exposure and subsequent i n f u s i o n of bovine c a r b o n i c anhydrase on blood acid-base s t a t u s i n a s i n g l e f i s h . Thus i n a i r bre a t h i n g f i s h exposed to a i r the i n a b i l i t y of red c e l l s to p a r t i c i p a t e i n the dehydration of plasma HC03 i s f o r the f i r s t time no longer an advantage but ra t h e r a l i a b i l i t y . Many f i s h such as l u n g f i s h and some e e l s have u t i l i z e d i n c r e a s e d v a s c u l a r i z a t i o n of the s k i n to help excrete C02. T h i s i n c r e a s e d c a p i l l a r y d e n s i t y i n c l o s e proximity with a moist s k i n would thus enhance the l o s s of molecular C02. I t i s a n t i c i p a t e d t h a t i n c r e a s e d temperatures would a l s o enhance the f a c i l i t a t i o n of C02. p r o d u c t i o n from plasma bicarbonate v i a the uncatalyzed r e a c t i o n . Increased c a p i l l a r y d i s t a n c e s may r e s u l t i n long s k i n c a p i l l a r y r e s i d e n c e times and would thus 96a FIGURE #18. Changes i n dorsal a o r t i c pH (pHa) and PCOg (PaCOg) during a i r exposure and the e f f e c t of i n f u s i o n of carbonic anhydrase (C.A.) into the dorsal aorta of an i n d i -vidual Hoplerythrinus. 96b 97 a l s o f a c i l i t a t e the l o s s of C02; however no r e l e v a n t data e x i s t s to draw any r e l i a b l e c o n c l u s i o n s concerning t h i s p o s s i b i l i t y . Although the v a s c u l a r i z a t i o n of the s k i n and i n c r e a s i n g lung volumes may help f a c i l i t a t e the l o s s of C02 the g i l l s i n v a r i a b l y are r e t a i n e d and provide a major pathway f o r the r e l e a s e of C02 and presumibly are s t i l l u t i l i z e d v i a i o n i c exchange processes o c c u r i n g i n the g i l l to r e g u l a t e plasma hydrogen i c n a c t i v i t y . U l t i m a t e l y metabolic r a t e s i n a i r b r e a t h i n g f i s h may be l i m i t e d by t h e i r a b i l i t y t o e x c r e t e C02 without v e n t i l a t i n g t h e i r g i l l s t r u c t u r e s with water. Those a i r b r e a t h i n g f i s h which have become the most " t e r r e s t i a l " may be f o r c e d to pay f o r t h i s freedom by e i t h e r enduring p e r i o d s of a c i d o s i s and/or reducing metabolic C02 p r o d u c t i o n . Consequently e s t i v a t i o n by l u n q f i s h may provide a means of r e d u c i n g CO2 production and a v o i d i n g what would otherwise r e s u l t i n a l e t h a l r i s e i n blood C02 d u r i n q periods when drouqht c o n d i t i o n s may l i m i t e x c r e t i o n of C02 a c r o s s the s k i n (Delaney et a l , 1977). In amphibians w e l l developed lungs are u t i l i z e d to procure oxygen from a i r ; however t h e i r s k i n provides the major s i t e of C02 e x c r e t i o n (Vinegar & Hutchison, 1965; Hutchison et a l , 1968; Emilo & S h e l t o n , 1974). In more t e r r e s t i a l v e r t e b r a t e s such as r e p t i l e s as w e l l as b i r d s and mammals the lungs provide the s i t e f o r oxygen uptake and C02 e x c r e t i o n . I t i s t h e r e f o r e i n t e r e s t i n g t h a t i n t a c t red blood c e l l s from Xencpus , Amphiurna , Bufo and Sana p i p i e n s a l s o appear to l a c k carbonic anhydrase dehydration a c t i v i t y while the red blood c e l l s of the t u r t l e Crv.§ejnes possess obvious c a r b o n i c anhydrase a c t i v i t y . C l e a r l y any mutation e n a b l i n g the red c e l l to p a r t i c i p a t e i n the .98 dehydration of plasma bicarbonate would be expected to g r e a t l y f a c i l i t a t e the movement of animals from aguatic to f u l l y t e r r e s t i a l a i r breathers. These p o s s i b i l i t i e s should provide a rewarding area f o r f u t u r e research. REFERENCES CITED too Aceves, J . 1977. Sodium pump stimulation by oxytocin and c y c l i c AMP i n the isolated epithelium of the frog skin. Pflugers Arch. 221* 211-216. Aickin, C.C., and R.C. Thomas. 1975. Micro-electrode measure-ment of the i n t e r n a l pH.of crab muscle f i b r e s . J. 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