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Metabolic adjustments to acute hypoxia in the African lungfish and rainbow trout Dunn, Jeffrey Frank 1985

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METABOLIC ADJUSTMENTS TO ACUTE HYPOXIA IN THE AFRICAN LUNGFISH AND RAINBOW TROUT by JEFFREY F. DUNN BSc (Honours), U n i v e r s i t y of B r i t i s h Columbia, 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,March,1985) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J e f f r e y F. Dunn, In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia. I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^00 t~ K The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date MA&CH ?. Htif DE-6 (3/81) i i ABSTRACT The i n t e r - t i s s u e m e t a b o l i c responses t o h y p o x i a were d e t e r m i n e d i n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) , and t r o u t (Salmo g a i r d n e r i ) . L u n g f i s h respond t o h y p o x i a w i t h a r e d u c t i o n i n m e t a b o l i c r a t e . I t was i n t e n d e d t o determine which t i s s u e , or t i s s u e s e x h i b i t d e c r e a s e d m e t a b o l i c r a t e s d u r i n g h y p o x i a , and then compare the r e s u l t s w i t h the m e t a b o l i c r e a c t i o n s observed i n t r o u t , which a r e not r e p o r t e d t o reduce m e t a b o l i c r a t e d u r i n g h y p o x i a . The m e t a b o l i c p o t e n t i a l s of the h e a r t , b r a i n , w h i t e muscle and l i v e r i n the A f r i c a n l u n g f i s h were e s t i m a t e d u s i n g enzymatic d a t a . M e t a b o l i c e f f e c t s of a 12 hr submergence were m o n i t o r e d u s i n g m e t a b o l i t e measurements. Hear t was the most o x i d a t i v e t i s s u e , but a l s o showed the g r e a t e s t a n a e r o b i c p o t e n t i a l . The b r a i n d i s p l a y e d r e l a t i v e l y low o x i d a t i v e c a p a b i l i t i e s . White muscle remained almost i n e r t . A l t h o u g h h i g h energy phosphate c o n c e n t r a t i o n s i n b r a i n and h e a r t d i d not f a l l d u r i n g submergence, g l y c o l y s i s was a c t i v a t e d as i n d i c a t e d by c r o s s - o v e r p l o t s , d e p l e t i o n of endogenous g l y c o g e n s t o r e s , and l a c t a t e a c c u m u l a t i o n . B l o o d - t i s s u e l a c t a t e and g l u c o s e g r a d i e n t s i n d i c a t e d (1) t h a t the h e a r t and b r a i n r e l e a s e d l a c t a t e t h r o u g h o u t submergence, (2) t h a t a f t e r 12 hr of submersion the b r a i n and h e a r t were p r o b a b l y o b t a i n i n g a l l t h e i r r e q u i r e d g l u c o s e from the b l o o d (3) t h a t the l i v e r r e l e a s e d g l u c o s e t h r o u g h o u t submergence, and (4) the w h i t e muscle was m e t a b o l i c a l l y i s o l a t e d from the r e s t of the body d u r i n g submergence. The l a c k of measurable changes i n w h i t e muscle m e t a b o l i t e c o n c e n t r a t i o n s c o u p l e d w i t h the low enzyme a c t i v i t i e s l e a d s t o the s u g g e s t i o n t h a t the most s i g n i f i c a n t a d a p t a t i o n t o h y p o x i a i n t hese f i s h e s may not be the c a p a c i t y f o r i n c r e a s e d a n a e r o b i c energy p r o d u c t i o n . I n s t e a d , i t i s l i k e l y t h a t the a b i l i t y of the muscle t o p r e v e n t the a c t i v a t i o n of g l y c o l y s i s d u r i n g h y p o x i c d y s o x i a i s the key t o the a n i m a l ' s s u r v i v a l . H i s t o c h e m i c a l and u l t r a s t r u c t u r a l s t u d i e s were done on the a x i a l m u s c u l a t u r e of the l u n g f i s h . The s m a l l wedge of red c o l o u r e d muscle e v i d e n t upon g r o s s e x a m i n a t i o n was shown by h i s t o c h e m i c a l d e m o n s t r a t i o n s of l a c t a t e and s u c c i n a t e dehydrogenases, of adenosine t r i p h o s p h a t a s e s , and of l i p i d t o be composed of a mosaic of r e d and i n t e r m e d i a t e f i b r e s . R e s p e c t i v e l y , t h e s e f i b r e s measured 23.6 and 34.3 m i c r o n s i n average d i a m e t e r . The b u l k of the myotome i s composed of w h i t e f i b r e s h a v i n g an average d i a m e t e r of 67.3 m i c r o n s . M i t o c h o n d r i a l d e n s i t y , c a p i l l a r i t y and l i p i d c o n t e n t were v e r y low f o r a l l f i b r e s . These d a t a suggest t h a t the a x i a l m u s c u l a t u r e i s geared p r i m a r i l y f o r a n a e r o b i c f u n c t i o n . The r e l a t i v e l y l a r g e p e r c e n t a g e of w h i t e muscle i n d i c a t e s t h a t the o v e r a l l m e t a b o l i c r a t e of the a x i a l muscle i s low. The c a p a c i t y of the muscle t o e x i s t w i t h a reduced r a t e of ATP t u r n o v e r (as was suggested above) may be r e l a t e d t o the l a r g e p r o p o r t i o n of w h i t e f i b r e s p r e s e n t i n the myotome. T i s s u e m e t a b o l i t e s were measured i n a h y p o x i a s e n s i t i v e o rganism, the Rainbow t r o u t (Salmo q a i r d n e r i ) , b e f o r e and a f t e r exposure f o r 3 hr t o i n s p i r e d oxygen t e n s i o n s of 20 t o r r (at i v 4°C). There were s m a l l changes i n the b r a i n but the energy s t a t u s was m a i n t a i n e d . The r e d muscle was the l e a s t a f f e c t e d . White muscle c r e a t i n e phosphate was d e p l e t e d . V a r i o u s d a t a i n d i c a t e t h a t the w h i t e muscle i s the major user of g l y c o l y t i c s u b s t r a t e s and the major p r o d u c e r of l a c t a t e . The h e a r t i s s t r e s s e d as i n d i c a t e d by a d e c l i n e i n g l y c o g e n , ATP, CrP, and the t o t a l a d e n y l a t e p o o l . The l i v e r e x h i b i t e d d e c l i n e s i n ever y i n d i c a t o r of m e t a b o l i c h o m e o s t a s i s . The l i v e r c o n c e n t r a t i o n s of gl y c o g e n d i d not d e c l i n e . The f a c t t h a t a n a e r o b i c g l y c o l y s i s has been a c t i v a t e d i n the w h i t e muscle, w h i l e the muscle remains i n m e t a b o l i c communication w i t h the o t h e r t i s s u e s v i a the b l o o d , s u p p o r t s the s u g g e s t i o n t h a t the met a b o l i s m of the whi t e muscle w i l l have a pronounced e f f e c t on the m e t a b o l i c s t a t u s of the whole a n i m a l . The t r o u t i s m a i n t a i n i n g i t s r a t e of oxygen uptake w h i l e a c t i v a t i n g a n a e r o b i c g l y c o l y s i s i n the attempt t o m a i n t a i n 'normal' r a t e s of energy u t i l i z a t i o n . The t u r n o v e r r a t e s of g l u c o s e and l a c t a t e were measured i n t r o u t s u b j e c t e d t o the same h y p o x i c s t r e s s as above. G l u c o s e t u r n o v e r d i d not change w h i l e l a c t a t e t u r n o v e r i n c r e a s e d from 2.8 + 0.4 jumoles/min ./kg t o 20.6+6.8 Mmoles/min./kg. The l a c k of i n c r e a s e i n g l u c o s e t u r n o v e r was a t t r i b u t e d t o the o b s e r v a t i o n t h a t l i v e r g l y c o g e n c o n c e n t r a t i o n s do not change and so t h e r e i s no i n c r e a s e i n g l u c o s e f l u x . The i n c r e a s e i n l a c t a t e t u r n o v e r emphasizes the f a c t t h a t a n a e r o b i c g l y c o l y s i s i s a c t i v a t e d and t h a t some t i s s u e s a re o x i d i z i n g l a c t a t e . The problem of when a c e l l becomes h y p o x i c and the V r e a c t i o n s of the c e l l t o t h a t s t r e s s i s a d d r e s s e d . The c e l l ( t i s s u e , organ, a n i m a l ) has two o p t i o n s i f oxygerF 1 s u p p l y drops t o a l e v e l which p r e v e n t s o x i d a t i v e m e t abolism from s u p p l y i n g a l l of the r e q u i r e m e n t s f o r ATP s y n t h e s i s . The c e l l may e x h i b i t a d e c l i n e i n r e q u i r e m e n t s , i n which case the r a t e of ATP p r o d u c t i o n need not be as h i g h as i n the o x i d a t i v e s t a t e o r , c o n v e r s e l y , a n a e r o b i c energy p r o d u c t i o n may i n c r e a s e i n the attempt t o m a i n t a i n ATP p r o d u c t i o n r a t e s . The l u n g f i s h muscle appears t o be ca p a b l e of the former, thus p r e s e r v i n g s u b s t r a t e s f o r o t h e r t i s s u e s and r e d u c i n g the r a t e of end-product f o r m a t i o n . The t r o u t w h i t e muscle, on the o t h e r hand, e x e r t s a major i n f l u e n c e upon the o t h e r t i s s u e s when the a n i m a l i s s t r e s s e d w i t h h y p o x i a . The term 'energy conformer' i s a p p l i e d t o a n i m a l s which do not m a i n t a i n oxygen uptake i n the f a c e of a d e c l i n i n g s u p p l y , and which a l l o w ATP p r o d u c t i o n t o d e c l i n e c o n c o m i t t a n t l y by not a c t i v a t i n g g l y c o l y s i s t o a marked degree. An energy r e g u l a t o r would a c t i v a t e g l y c o l y s i s i n the attempt t o m a i n t a i n o x i d a t i v e r a t e s of ATP p r o d u c t i o n . The t r o u t i s more of an energy r e g u l a t o r than i s the l u n g f i s h w i t h the main d i f f e r e n c e i n t h i s c a p a c i t y b e i n g i n the w h i t e muscle. v i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES . . . v i i i L IST OF FIGURES x LIST OF ABBREVIATIONS x i i ACKNOWLEDGEMENTS . '. x i v I n t r o d u c t i o n 1 S e c t i o n 1. M e t a b o l i c a d j u s t m e n t s t o h y p o x i a i n the A f r i c a n l u n g f i s h . I n t r o d u c t i o n 16 M a t e r i a l s and Methods ....18 R e s u l t s 21 D i s c u s s i o n 37 S e c t i o n 2. An u l t r a s t r u c t u r a l and h i s t o c h e m i c a l study of the a x i a l m u s c u l a t u r e i n the A f r i c a n l u n g f i s h . I n t r o d u c t i o n 48 Methods 50 R e s u l t s 53 D i s c u s s i o n 67 S e c t i o n 3. The m e t a b o l i c a d j u s t m e n t s t o a c u t e e n v i r o n m e n t a l h y p o x i a i n the Rainbow t r o u t . I n t r o d u c t i o n 71 M a t e r i a l s and Methods 73 R e s u l t s 76 D i s c u s s i o n ,\ 90 v i i S e c t i o n 4. Turnover r a t e s of g l u c o s e and l a c t a t e i n the Rainbow t r o u t d u r i n g acute h y p o x i a . I n t r o d u c t i o n 103 Methods 105 R e s u l t s 111 D i s c u s s i o n 128 G e n e r a l D i s c u s s i o n 137 v i i i L IST OF TABLES Table 1. L u n g f i s h t i s s u e enzyme a c t i v i t i e s . 22 T a b l e 2. C o n c e n t r a t i o n s of s e l e c t e d g l y c o l y t i c m e t a b o l i t e s i n the l u n g f i s h 26 T a b l e 3. C o n c e n t r a t i o n s of a d e n y l a t e s , c r e a t i n e phosphate, and c r e a t i n e , and the energy charge i n l u n g f i s h t i s s u e s . 36 Table 4. Comparative a c t i v i t y r a t i o s of enzymes from a n a e r o b i c and a e r o b i c pathways i n b r a i n s of f i s h and mammals 38 Tab l e 5. Diameter of l u n g f i s h muscle f i b r e s 65 T a b l e 6. A comparison of the m i t o c h o n d r i a l d e n s i t y and v a s c u l a r i z a t i o n of v a r i o u s muscle f i b r e s 66 Tab l e 7. C o n c e n t r a t i o n s of a d e n y l a t e s , c r e a t i n e phosphate, and c r e a t i n e , and the energy charge i n t r o u t t i s s u e s . ...77 Tab l e 8. S e l e c t e d t r o u t g l y c o l y t i c m e t a b o l i t e s a t r e s t and d u r i n g a c u t e h y p o x i a . 79 Tab l e 9. C o r r e l a t i o n s between t i s s u e and b l o o d m e t a b o l i t e c o n c e n t r a t i o n s i n t r o u t 82 Tab l e 10. G l y c o l y t i c m e t a b o l i t e s t o r e s i n t r o u t 83 Tab l e 11. The r a t i o s of b l o o d l a c t a t e t o t i s s u e l a c t a t e i n t r o u t 84 Tab l e 12. E q u a t i o n parameters f o r the DPM vs TIME c u r v e s i n t r o u t 119 i x T a b l e 13. Tr o u t m e t a b o l i t e t u r n o v e r numbers ....121 Ta b l e 14. Mass of the r a p i d l y m i x i n g p o o l i n t r o u t ...122 Ta b l e 15. A c o m p i l a t i o n of g l u c o s e and l a c t a t e t u r n o v e r numbers . 131 X LIST OF FIGURES F i g u r e 1. C r o s s o v e r p l o t s of l u n g f i s h t i s s u e m e t a b o l i t e s at the end of a submergence 27 F i g u r e 2. Glycogen c o n t e n t s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h 30 F i g u r e 3. T i s s u e and b l o o d g l u c o s e c o n c e n t r a t i o n s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h 32 F i g u r e 4. T i s s u e and b l o o d l a c t a t e c o n c e n t r a t i o n s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h 34 F i g u r e 5. L u n g f i s h c r o s s - s e c t i o n s 54 F i g u r e 6. A n t e r i o r l a t e r a l l i n e r e g i o n s t a i n e d f o r LDH a c t i v i t y 57 F i g u r e 7. Mosaic r e g i o n of the a n t e r i o r l a t e r a l l i n e s t a i n e d f o r LDH a c t i v i t y . 57 F i g u r e 8. Mosaic r e g i o n of the a n t e r i o r l a t e r a l l i n e s t a i n e d f o r ATPase a c t i v i t y 57 F i g u r e 9. P o s t e r i o r l a t e r a l - l i n e r e g i o n s t a i n e d f o r SDH a c t i v i t y 59 F i g u r e 10. P o s t e r i o r mosaic r e g i o n s t a i n e d f o r SDH a c t i v i t y 59 F i g u r e 11. S e c t i o n from z e b r a f i s h s t a i n e d f o r LDH a c t i v i t y 59 F i g u r e 12. L a t e r a l s e c t i o n of w h i t e muscle m y o f i b r i l s 62 F i g u r e 13. L a t e r a l s e c t i o n of r e d muscle m y o f i b r i l s 62 F i g u r e 14. T r a n s v e r s e s e c t i o n of r e d muscle 62 F i g u r e 15. T o t a l g l u c o s e , g l y c o g e n , and G6P s t o r e s i n t r o u t 86 F i g u r e 16. T o t a l l a c t a t e s t o r e s i n t r o u t 88 F i g u r e 17. Example of decay c u r v e r e c o n s t r u c t i o n 112 F i g u r e 18. Example of r e c o n s t r u c t e d g l u c o s e c u r v e s 114 F i g u r e 19. Example of r e c o n s t r u c t e d l a c t a t e c u r v e s . .......116 F i g u r e 20. The r e l a t i o n s h i p between plasma g l u c o s e c o n c e n t r a t i o n s and g l u c o s e t u r n o v e r . 123 F i g u r e 21. The r e l a t i o n s h i p between plasma l a c t a t e c o n c e n t r a t i o n s and l a c t a t e t u r n o v e r 125 LIST OF ABBREVIATIONS DTNB PCA Tr i s ATP ADP AMP NAD +, NADH NADP +, NADPH G6P F6P FBP G3P DHAP CrP Cr ATPase AAT CPK CS HK HOAD LDH MDH 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) p e r c h l o r i c a c i d t r is(hydroxymethyl)aminomethane adenosine t r i p h o s p h a t e adenosine d i p h o s p h a t e adenosine monophosphate n i c o t i n a m i d e adenine d i n u c l e o t i d e ( o x i d i z e d and reduced) n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate ( o x i d i z e d and reduced) glucose-6-phosphate f r u c t o s e - 6 - p h o s p h a t e f r u c t o s e - 1 , 6 - b i s p h o s p h a t e g l y c e r a l d e h y d e - 3 - p h o s p h a t e d i h y d r o x y a c e t o n e phosphate c r e a t i n e phosphate c r e a t ine adenosine t r i p h o s p h a t a s e a s p a r t a t e a m i n o t r a n s f e r a s e c r e a t i n e phosphokinase c i t r a t e s y n t h a s e hexok i n a s e b e t a - h y d r o x y a c y l C o A dehydrogenase l a c t a t e dehydrogenase malate dehydrogenase x i i i PFK p h o s p h o f r u c t o k i n a s e PGI phosphoglucoisomerase PK p y r u v a t e k i n a s e SDH s u c c i n a t e dehydrogenase S.A. s p e c i f i c a c t i v i t y Ra (-g;-l) replacement r a t e ( g l u c o s e ; l a c t a t e ) Ms mass of the r a p i d l y m i x i n g p o o l x i v ACKNOWLEDGEMENTS D u r i n g the y e a r s of work which went i n t o . t h i s t h e s i s I have r e c e i v e d u n f a l t e r i n g support from my w i f e (Moire H i c k l e y ) , my f a m i l y , and my s u p e r v i s o r , Dr. P.W. Hochachka. I have Dr. G.M.O. M a l o i y and Dr. M. Guppy t o thank f o r making the Kenyan p o r t i o n of t h i s s tudy both p o s s i b l e and s u r v i v a b l e . Thanks a l s o t o the members of my r e s e a r c h committee f o r t h e i r c r i t i c i s m s and comments on my t h e s i s . I t would be s a t i s f y i n g t o be a b l e t o remember and thank everyone who has h e l p e d me d u r i n g the c o u r s e of t h i s t h e s i s . Those whom I have not mentioned w i l l s t i l l know t h a t they have my h e a r t f e l t a p p r e c i a t i o n . D u r i n g t h i s study I was s u p p o r t e d i n p a r t by an NSERC p o s t g r a d u a t e s c h o l a r s h i p , and by a McLean F r a s e r memorial s c h o l a r s h i p from the U n i v e r s i t y of B r i t i s h Columbia. 1 INTRODUCTION Oxygen i s the key t o l i f e f o r most members of the an i m a l kingdom. T h i s element p r o v i d e s the c o r n e r s t o n e f o r the u b i q u i t o u s b i o l o g i c a l e n g i n e : the m i t o c h o n d r i o n . Without oxygen, most a n i m a l s w i l l d i e w i t h i n m i n u t e s . When s u b j e c t e d to h y p o x i a , t h e i r a b i l i t y t o f u n c t i o n w i l l be g r e a t l y i m p a i r e d . Such statements may be e x a g g e r a t i o n s , but they s e r v e w e l l t o emphasize the severe consequences of c u r t a i l i n g t he oxygen s u p p l y . They a l s o u n d e r s c o r e the r e l e v a n c e of s t u d i e s which i n q u i r e about the r o l e which oxygen p l a y s i n l i f e , and the s t r a t e g i e s which may be u t i l i z e d when the s u p p l y becomes l i m i t i n g . A study of responses t o oxygen l i m i t a t i o n may t a k e many forms, and so the s u b j e c t must be narrowed and d e f i n e d t o f i t w i t h i n the framework of a t h e s i s . The f i r s t s t e p i s t o c l a r i f y t he t e r m i n o l o g y which w i l l be used. I t i s apparent what i s meant by a n o x i a , the t o t a l l a c k of oxygen, but the meaning of " h y p o x i a " has expanded t o encompass many s i t u a t i o n s . Hypoxia i s a r e l a t i v e term which, i f one l i t e r a l l y t r a n s l a t e s the r o o t s of the word, means "low oxygen". The m e d i c a l d e f i n i t i o n of the word can be s t a t e d as " l a c k of an adequate amount of oxygen i n i n s p i r e d a i r such as o c c u r s a t h i g h a l t i t u d e s ; r e d u c e d oxygen c o n t e n t or t e n s i o n " (Thomas, 1973). Does t h i s mean t h a t p e o p l e l i v i n g i n Denver are h y p o x i c s i n c e they e x i s t where oxygen c o n c e n t r a t i o n s a re lower than t h e y a r e a t s e a - l e v e l ? No, i t does n o t . W i t h i n l i m i t s , i n d i v i d u a l s may adapt i n a f a s h i o n 2 t h a t makes the oxygen t e n s i o n i n t h e i r environment "normal" f o r them ( D e j o u r s , 1966). C o n v e r s e l y , an e x e r c i s i n g a n i m a l may have a s h o r t a g e of oxygen a t the t i s s u e l e v e l even when the i n s p i r e d oxygen t e n s i o n s a re "normal" (Rusko and R a h k i l a , 1979). The p o s s i b i l i t y f o r m i s u n d e r s t a n d i n g has a l r e a d y l e d some ( p r o b a b l y many) r e s e a r c h e r s t o r e d e f i n e the word " h y p o x i a " ( D e j o u r s , 1966,-Jones, 1981,-Robin, 1980). The a d j e c t i v e ( h y p o x i c ) , when a s s o c i a t e d w i t h a noun which i s l o g i c a l and c l e a r , means t h a t t h a t s u b j e c t ( b l o o d , gas, c e l l , e t c ) , has a v a i l a b l e a lower than normal amount of oxygen. However, t h i s does not n e c e s s a r i l y mean t h a t the body, or c e l l , w i l l r e a c t t o the s t a t e of h y p o x i a . Whether or not a m e t a b o l i c r e a c t i o n o c c u r s depends upon the e f f e c t of the h y p o x i c s t a t e a t the c e l l u l a r l e v e l . S i n c e the c e l l i s the s i t e of oxygen u t i l i z a t i o n , t h e r e must be an i n t r a c e l l u l a r m e t a b o l i c response caused by a l i m i t e d s u p p l y of oxygen b e f o r e the c e l l can be termed " h y p o x i c " . Jones (1981) wrote t h a t , "Hypoxia i s a subnormal oxygen c o n c e n t r a t i o n i n c e l l s t h a t causes a l t e r e d b i o c h e m i c a l , p h y s i o l o g i c a l , or p a t h o l o g i c a l f u n c t i o n . " T h i s d e f i n i t i o n uses the term " c o n c e n t r a t i o n " which i s not the d r i v i n g f o r c e f o r oxygen d e l i v e r y ( L ubbers, 1977). The word " s u p p l y " would c l a r i f y t he statement s i n c e oxygen c o n c e n t r a t i o n may be low when f l u x i s h i g h . In a d d i t i o n , the concept Of a subnormal l e v e l of oxygen may be too r e s t r i c t i v e because, as mentioned above, a c e l l may s t i l l become oxygen l i m i t e d when oxygen r e q u i r e m e n t s i n c r e a s e due t o i n c r e a s e d work, even though the r a t e of oxygen 3 d e l i v e r y may be normal. D e j o u r s (1966) p r o b a b l y had a s i m i l a r i n t e n t t o Jones when he wrote "A hy p o x i c s t a t e can be d e f i n e d as one i n which the c e l l s l a c k oxygen, or more p r e c i s e l y , i n which the o x i d i z a b l e s u b s t r a t e s of the c e l l " l a c k " oxygen." T h i s d e f i n i t i o n i s more encompassing i n terms of the s i t u a t i o n s i t can be a p p l i e d t o because of the more g e n e r a l statement t h a t the c e l l l a c k s oxygen. However, i n terms of b i o c h e m i c a l mechanisms, i t i s l e s s a c c u r a t e because one i s l e f t t o i n t e r p r e t how a s u b s t r a t e i s a b l e t o l a c k oxygen. Under most c i r c u m s t a n c e s , the r a t e of s u b s t r a t e o x i d a t i o n i s not c o n t r o l l e d by the a v a i l a b i l i t y of o x i d i z a b l e s u b s t r a t e s (Racker, 1976). I t i s a l s o u n l i k e l y t h a t t h e r e i s ever enough oxygen i n a c e l l t o o x i d i z e a l l of the a v a i l a b l e f a t , amino a c i d , and c a r b o h y d r a t e s t o r e s . In o r d e r t o overcome t h e s e problems the f o l l o w i n g m o d i f i e d d e f i n i t i o n w i l l be used: c e l l u l a r h y p o x i a o c c u r s when the r a t e of oxygen d e l i v e r y i s l e s s than t h a t which o x i d a t i v e m e t abolism would r e q u i r e t o s u p p l y the energy needs of the c e l l . In o t h e r words, h y p o x i a o c c u r s when the s u p p l y of oxygen i s such t h a t m e t a b o l i c p r o c e s s e s i n the c e l l have t o be r e o r g a n i z e d t o compensate f o r a l a c k of oxygen. T h i s i s the d e f i n i t i o n which w i l l be used f o r the remainder of the t h e s i s . I t i s i m p o r t a n t t o keep t h i s l a s t d e f i n i t i o n i n mind when d i s c u s s i n g or a n a l y s i n g work r e l a t e d t o low oxygen. T h i s i s because t h e r e a r e many s t a t e s which can be termed t o be h y p o x i c and which may or may not r e s u l t i n c e l l u l a r h y p o x i a . One may group the causes of these s t a t e s i n t o e n v i r o n m e n t a l or 4 b e h a v i o r a l . The former i n c l u d e s a s c e n d i n g t o a l t i t u d e where the ambient oxygen t e n s i o n i s lower than "normal". The l a t t e r i n c l u d e s causes such as apnea or e x e r c i s e where the ambient oxygen t e n s i o n i s not the d e t e r m i n i n g f a c t o r . Both a s c e n d i n g t o a l t i t u d e and apnea may cause low a r t e r i a l oxygen t e n s i o n or hypoxemia ( D e j o u r s , 1966). E x e r c i s e may cause c e l l u l a r oxygen l i m i t a t i o n s even i f a r t e r i a l oxygen t e n s i o n s remain c o n s t a n t (Barbee et a_l. , 1983). Thus, these s c e n a r i o s d e s c r i b e p o t e n t i a l l y h y p o x i c s i t u a t i o n s . The d u r a t i o n and s e v e r i t y of exposure, as w e l l as the work r a t e ( i . e . 0 2 r e q u i r e m e n t s ) of the c e l l , w i l l d e termine whether a g i v e n exposure w i l l e l i c i t a h y p o x i a i n d u c e d response. When an a n i m a l i s s u b j e c t e d t o d e c l i n i n g e n v i r o n m e n t a l oxygen t e n s i o n s , r a t e s of oxygen uptake may f o l l o w two d i f f e r e n t p a t t e r n s . A n i m a l s which m a i n t a i n t h e i r oxygen uptake when e n v i r o n m e n t a l oxygen t e n s i o n s f a l l have been c a l l e d oxygen or m e t a b o l i c r e g u l a t o r s , and those a n i m a l s i n which r a t e s of oxygen uptake d e c l i n e as e n v i r o n m e n t a l oxygen t e n s i o n s f a l l can be l a b e l l e d oxygen or m e t a b o l i c conformers ( P r o s s e r , 1973). However, i t i s not p o s s i b l e t o determine the type of c e l l u l a r response which o c c u r s based upon whether the a n i m a l i s an oxygen r e g u l a t o r or an oxygen conformer. A r e g u l a t o r may have t o expend e x c e s s energy t o m a i n t a i n i t s oxygen uptake and so some of the t i s s u e s may s t i l l become h y p o x i c (Jones, 1971). A conformer w i l l have a reduced s u p p l y of oxygen t o some t i s s u e s but one does not know whether those t i s s u e s a c t i v a t e g l y c o l y s i s or e x h i b i t a decrease i n m e t a b o l i c r a t e (or b o t h ) . 5 Another way of g r o u p i n g h y p o x i a i n d u c i n g s i t u a t i o n s i s from' a t i s s u e r e l a t e d v i e w p o i n t . Here, oxygen l i m i t a t i o n s may be caused by e i t h e r an i n c r e a s e d oxygen demand or a de c r e a s e d oxygen, s u p p l y t o the t i s s u e s . The causes of a r e d u c t i o n i n oxygen s u p p l y can be d i v i d e d i n t o t h r e e g r o u p i n g s : 1. P h y s i o l o g i c a l , eg. r e d i s t r i b u t i o n of c a r d i a c o u t p u t . 2. E n v i r o n m e n t a l , eg. d e c l i n e i n i n s p i r e d 0 2 t e n s i o n c a u s i n g a d e c l i n e i n a r t e r i a l 0 2 c o n t e n t . 3. P a t h o l o g i c a l , eg. s t r o k e , v a s c u l a r o c c l u s i o n d i s e a s e , c a r d i a c a r r e s t , e t c . . E x e r c i s e or an i n c r e a s e i n temperature may induce the second oxygen l i m i t i n g s i t u a t i o n , t h a t of i n c r e a s e d oxygen demand. Now t h a t the word has been d e f i n e d and the s i t u a t i o n s which may cause h y p o x i a have been o u t l i n e d , the problem s t i l l remains as t o when the c e l l i t s e l f i s a c t u a l l y s u f f e r i n g from an inad e q u a t e oxygen s u p p l y . Robin (1980) has proposed the term d y s o x i a t o d e s c r i b e s i t u a t i o n s where c e l l u l a r oxygen u t i l i z a t i o n i s i m p a i r e d . T h i s term can be c l a r i f i e d t o d e l i m i t c a s e s where oxygen s u p p l y i s l i m i t i n g ( h y p o x i c d y s o x i a ) , where s u p p l y i s normal but some i n t r a c e l l u l a r problem i s c a u s i n g abnormal u t i l i z a t i o n (normoxic d y s o x i a ) , and where s u p p l y i s e l e v a t e d r e s u l t i n g i n some degree of oxygen t o x i c i t y ( h y p e r o x i c d y s o x i a ) ( R o b i n , 1980). U s i n g t h e s e d e f i n i t i o n s , t h i s t h e s i s i s concerned w i t h h y p o x i c d y s o x i a a l t h o u g h , f o r p r a g m a t i c reasons the term " h y p o x i a " or " c e l l u l a r h y p o x i a " w i l l be used more 6 r e g u l a r l y . I f one i g n o r e s a d a p t a t i o n and l o o k s o n l y a t the immediate r e a c t i o n s i n a c e l l which i s s u b j e c t e d t o h y p o x i a , then c e l l u l a r energy p r o d u c t i o n may f o l l o w two p a t t e r n s ( f o r r e v i e w s , see B e n n e t t , 1982;Hochachka and Somero, 1984;Hochachka and Somero, l973;Van den T h i l l a r t , 1982). In one p a t t e r n , a n a e r o b i c energy p r o d u c t i o n i n c r e a s e s i n o r d e r t o supplement ATP (adenosine t r i p h o s p h a t e ) p r o d u c t i o n (Anderson, l 9 7 5 ; B u r t o n and Spehar, l 9 7 l ; D e Zwann and Wijsman, 1976;Hochachka and Somero, 1984). There a r e v a r i o u s m e t a b o l i c pathways w i t h i n the a n i m a l kingdom which may p r o v i d e a n a e r o b i c ATP p r o d u c t i o n (Hochachka and Somero, 1984). G l y c o l y s i s , the a n a e r o b i c d e g r a d a t i o n of g l u c o s e to y i e l d l a c t a t e ( L e h n i n g e r , 1975), i s the most common among the v e r t e b r a t e s (Daw e_t a l . , 1 967 ;McDougal e_t a_l. , 1 968 ;Hochachka and Somero, l 9 8 4 ; R o b i n , 1980) a l t h o u g h f e r m e n t a t i o n s t o y i e l d e t h a n o l (Shoubridge and Hochachka, 1981;Van den T h i l l a r t , 1982) and s u c c i n a t e (Taegtmeyer, 1979) are p o s s i b l e . In the case of g l y c o l y s i s , g l y c o g e n and g l u c o s e are the main s u b s t r a t e s and l a c t a t e i s the c a r b o n - c o n t a i n i n g p r o d u c t . The second p o s s i b l e response i s t o decrease the m e t a b o l i c r a t e of the c e l l (Hochachka and Somero, l 9 8 4 ; R o b i n , l 9 8 0 ; U l t s c h and J a c k s o n , 1982). In t h i s c a s e , g l y c o l y s i s may or may not be a c t i v a t e d . I t i s much e a s i e r t o d e f i n e c e l l u l a r h y p o x i a and o u t l i n e the causes than i t i s t o d e t e r m i n e whether a c e l l has a c t u a l l y become h y p o x i c . One now needs an o p e r a t i o n a l d e f i n i t i o n t o be used when t e s t i n g f o r c e l l u l a r h y p o x i a . Measuring heat p r o d u c t i o n , oxygen uptake, or ATP t u r n o v e r r a t e s are d i f f i c u l t 7 a t the c e l l u l a r l e v e l but a r e sure ways t o determine whether a c e l l i s r e g u l a t i n g m e t a b o l i c r a t e . I t i s a l s o p o s s i b l e t o make i n f e r e n c e s about c e l l u l a r m e t a b o l i s m based upon m e t a b o l i t e d a t a . I f one can determine t h a t the c e l l i s h y p o x i c t h e n , by d e f i n i t i o n , the c e l l i s not c a p a b l e of m a i n t a i n i n g r a t e s of ATP p r o d u c t i o n u s i n g a e r o b i c pathways. C o n c u r r e n t p r o o f t h a t g l y c o l y s i s i s not a c t i v a t e d p r o v i d e s the e v i d e n c e needed t o c o n c l u d e t h a t the m e t a b o l i c r a t e of the c e l l has d e c l i n e d . Even s m a l l r e d u c t i o n s i n oxygen a v a i l a b i l i t y would induce l a r g e changes i n g l y c o l y t i c f l u x i f the c e l l were t o m a i n t a i n i t s m e t a b o l i c r a t e , because up t o an 18 f o l d i n c r e a s e i n g l y c o l y t i c f l u x i s r e q u i r e d f o r a n a e r o b i c g l y c o l y s i s t o produce ATP a t the same r a t e as 'glucose o x i d a t i o n . The i n f o r m a t i o n which may be used, t o prove t h a t no g l y c o l y t i c a c t i v a t i o n has o c c u r r e d i s s i m p l y the l a c k of m e t a b o l i c changes which would prove t h a t g l y c o l y s i s has been a c t i v a t e d . There are v a r i o u s i n t r a c e l l u l a r changes which may i n d i c a t e whether c e l l s have responded t o h y p o x i a by a c t i v a t i n g a n a e r o b i c energy p r o d u c t i o n . One w i d e s p r e a d response i s the p r o d u c t i o n of l a c t a t e , the endproduct of c a r b o h y d r a t e f e r m e n t a t i o n . A l t h o u g h t h i s c r i t e r i o n i s s t i l l one of the most w i d e l y u t i l i z e d , i t cannot be used as the s o l e c r i t e r i o n because l a c t a t e may be produced even when the c e l l i s not l i m i t e d by oxygen i n any way. J o b s i s and S t a i n s b y (1968) observed t h i s a e r o b i c l a c t a t e p r o d u c t i o n i n dog muscle and suggested t h a t g l y c o l y t i c f l u x may p r o ceed a t a r a t e which produces p y r u v a t e f a s t e r than i t can be used by the TCA ( t r i c a r b o x y l i c a c i d ) c y c l e . The excess p y r u v a t e i s reduced t o 8 l a c t a t e . The l i m i t a t i o n here i s the a c t i v i t y of p y r u v a t e dehydrogenase and not the a c t i v i t y of the e l e c t r o n t r a n s p o r t c h a i n . A f l u x of p y r u v a t e t o l a c t a t e may a l s o be induced by an i n c r e a s e i n NADH ( n i c o t i n a m i d e adenine d i n u c l e o t i d e ) c o n c e n t r a t i o n d u r i n g o x i d a t i v e c o n d i t i o n s (Connett e_t a l . , 1984). A t k i n s o n (1977) d e v e l o p e d the concept of the energy charge (E.C.) as an i n d i c a t o r of c e l l u l a r energy s t a t u s . T h i s v a l u e was d e t e r m i n e d by measuring ATP, ADP (adenosine d i p h o s p h a t e ) , and AMP (adenosine monophosphate) c o n c e n t r a t i o n s , and i n s e r t i n g t hese v a l u e s i n t o h i s energy charge e q u a t i o n ((ATP + 0.5ADP)/(AMP + ADP + A T P ) ) . The concept was not de v e l o p e d s o l e l y t o t e s t f o r h y p o x i a , but i t has been noted t h a t h y p o x i c exposure may r e s u l t i n " a f a l l i n the E.C. ( V e t t e r and Hodson, 1982). I f ATP p r o d u c t i o n by the c e l l were s e v e r e l y i m p a i r e d , w h i l e energy r e q u i r e m e n t s remained c o n s t a n t , then the E.C. would n e c e s s a r i l y f a l l ( A t k i n s o n , 1977). The problem w i t h E.C. as an i n d i c a t o r of c e l l u l a r h y p o x i a i s t h a t t h e r e a r e many causes of a change i n E . C . A d e c l i n e may oc c u r c o n c u r r e n t l y w i t h a d e c l i n e i n the r a t e s of ATP p r o d u c t i o n r e g a r d l e s s of how t h a t p r o d u c t i o n i s i m p a i r e d . F u r t h e r m o r e , because the c e l l has many methods f o r m a i n t a i n i n g energy charge, and because a f a l l i n energy charge i n d i c a t e s severe i n t r a c e l l u l a r s t r e s s , t h e r e may be s i t u a t i o n s where ATP p r o d u c t i o n may be i m p a i r e d t o a l i m i t e d e x t e n t w i t h no concommitent f a l l i n E.C. ( A t k i n s o n , 1977) . Another p o s s i b l e i n d i c a t o r of c e l l u l a r h y p o x i a i s the 9 r e l a t i v e r e d u c t i o n s t a t e of the c e l l (Chance e_t a_l. , 1980). The r e d u c t i o n s t a t e i s i n d i c a t e d by the NADH/NAD* r a t i o ( n i c o t i n a m i d e adenine d i n u c l e o t i d e , reduced and o x i d i z e d r e s p e c t i v e l y ) , or the r a t i o of reduced t o o x i d i z e d cytochromes. These r a t i o s may be u s e f u l i n d i c a t o r s of c e l l u l a r h y p o x i a because the f i r s t change i n the c e l l when i t becomes oxygen l i m i t e d s h o u l d be a r e d u c t i o n i n the a b i l i t y t o o x i d i z e the cytochromes, t h e r e b y making the c e l l more reduced (Jones and Kennedy, 1982)., The problem i s t h a t when a muscle i s a t r e s t i t s p o o l o f s j r u t o c h o n d r i a l NAD + i s almost c o m p l e t e l y reduced and when i t t w i t c h e s the p o o l becomes more o x i d i z e d ( J o b s i s and S t a i n s b y , 1968). T h i s agrees w i t h d a t a from i s o l a t e d m i t o c h o n d r i a which i n d i c a t e t h a t when m i t o c h o n d r i a a r e not r e s p i r i n g , the NAD* i s c o m p l e t e l y reduced. The redox s t a t e , t h e r e f o r e , i s not n e c e s s a r i l y an i n d i c a t i o n of h y p o x i c d y s o x i a . What becomes apparent i s t h a t a s i n g l e i n d i c a t o r of c e l l u l a r h y p o x i a i s not a v a i l a b l e . One must combine v a r i o u s d a t a i n o r d e r t o c o n c l u d e t h a t the c e l l i s , .in f a c t , h y p o x i c . No s i n g l e g e n e r a l i z a t i o n w i l l h o l d . In o r d e r t o determine whether the c e l l i s h y p o x i c one must demonstrate t h a t t h e r e has been an adjustment of m e t a b o l i t e c o n c e n t r a t i o n s i n d i c a t i n g t h a t the a e r o b i c machinery was not a b l e t o su p p l y a i l of the r e q u i r e d energy. There must a l s o be e i t h e r a decrease i n oxygen s u p p l y or an i n c r e a s e i n energy r e q u i r e m e n t s (or: b o t h ) . T h i s l a s t c r i t e r i o n i s i n c l u d e d t o d i s t i n g u i s h from m e t a b o l i t e changes induced by the l a c k of s u b s t r a t e s o t h e r than oxygen. The m e t a b o l i c parameters mentioned above ( l a c t a t e 10 p r o d u c t i o n , E.C., redox) can be grouped t o g e t h e r t o d e v e l o p the t y pe of argument which i s r e q u i r e d t o prove t h a t a c e l l i s h y p o x i c . The more i n d i c a t o r s which are u t i l i z e d , the s t r o n g e r the argument i s t h a t g l y c o l y s i s has, or has n o t , been a c t i v a t e d . In a d d i t i o n , one may measure pH because a f a l l i n pH i s o f t e n c o r r e l a t e d w i t h i n c r e a s e d a n a e r o b i c energy p r o d u c t i o n ( C a s t e l l i n i and Somero, 1981;Hochachka and Mommsen, 1983). G l y c o l y t i c a c t i v a t i o n as i n d i c a t e d by c r o s s - o v e r s i n p l o t s of g l y c o l y t i c i n t e r m e d i a t e s (Norberg and S i e s j o , 1975) and a d e c l i n e i n g l y c o g e n c o n c e n t r a t i o n s (Daw e_t a_l. , l967;Dawes e_t a l . , 1959) are a l s o i n d i c a t o r s t h a t the c e l l has become h y p o x i c and i s r e a c t i n g by i n c r e a s i n g a n a e r o b i c energy p r o d u c t i o n . The d e f i n i t i o n of h y p o x i a and the t e c h n i q u e s suggested f o r d e t e r m i n i n g c e l l u l a r h y p o x i a w i l l s e r v e f o r t h i s t h e s i s but w i l l be appended w i t h one p r o v i s o . The assumption i s made t h a t the main r e s u l t of oxygen l i m i t a t i o n i s a s h o r t f a l l i n o x i d a t i v e energy p r o d u c t i o n . However, t h i s i s not the o n l y s i t e which i s i n f l u e n c e d by such a l a c k . I t i s p o s s i b l e t h a t up t o 20% of b a s a l c e l l u l a r r e s p i r a t i o n may be e x t r a m i t o c h o n d r i a l ( R o b i n , 1980). A l t h o u g h i t i s o u t s i d e the framework of t h i s t h e s i s , the f a c t t h a t such a l a r g e p e r c e n t a g e of c e l l u l a r oxygen u t i l i z a t i o n may be e x t r a m i t o c h o n d r i a l makes i t l i k e l y t h a t a l a c k of oxygen may produce d e t r i m e n t a l e f f e c t s i n a r e a s o t h e r than energy s u p p l y . The above d i s c u s s i o n o u t l i n e s some of the s i t u a t i o n s which may r e s u l t i n h y p o x i c d y s o x i a and the t e c h n i q u e s used t o t e s t f o r such an occurrence., Now, one may ask how the a n i m a l 1 1 s u r v i v e s t h i s exposure. S i n c e the immediate responses of a c e l l t o h y p o x i c d y s o x i a a r e encompassed by e i t h e r a c t i v a t i n g a n a e r o b i c energy p r o d u c t i o n or r e d u c i n g m e t a b o l i c r a t e (Hochachka, 1980;Hochachka and Somero, 1984,-Robin, 1980), then i t f o l l o w s t h a t i n c r e a s i n g the c a p a c i t y f o r e i t h e r of these responses w i l l i n c r e a s e the c e l l ' s c a p a c i t y f o r s u r v i v a l d u r i n g h y p o x i a . The s t r a t e g y of i n c r e a s i n g a n a e r o b i c energy p r o d u c t i o n may be a c c o m p l i s h e d i n many ways. To name but a few, a n i m a l s may u t i l i z e e n e r g e t i c a l l y improved f e r m e n t a t i o n s , as do the g o l d f i s h . (Shoubridge and Hochachka, 1981). They may s t o r e h i g h c o n c e n t r a t i o n s of gly c o g e n i n the l i v e r or i n the t i s s u e s which r e q u i r e the energy, i . e . t u r t l e s , newborns, or g o l d f i s h (Daw e t a l . , 1967,-Dawes e t a l . , 1959;Shoubridge, 1980). The p o t e n t i a l r a t e of g l y c o l y t i c f l u x may be i n c r e a s e d by i n c r e a s i n g enzyme t i t r e , as has been proven t o occur i n mammals (Hance e_t a l . , 1980). F i n a l l y , s i n c e e n d-products of c a r b o h y d r a t e f e r m e n t a t i o n may accu m u l a t e , the c a p a c i t y of the c e l l t o t o l e r a t e t h e s e p r o d u c t s may be augmented (such as o c c u r s i n s e a l s ) ( C a s t e l l i n i and Somero, 1981). The a l t e r n a t e s t r a t e g y , t h a t of r e d u c i n g m e t a b o l i c r a t e , has been proven t o oc c u r i n o n l y a few of the v e r t e b r a t e s examined. The t u r t l e i s the most e x t e n s i v e l y s t u d i e d v e r t e b r a t e w i t h the c a p a c i t y t o reduce m e t a b o l i c r a t e when exposed t o a l i m i t i n g oxygen s u p p l y (Jackson and S c h m i d t - N i e l s e n , l 9 6 6 ; L u t z et a l . , l 9 8 0 ; R o b i n et a l . , l 9 6 4 ; U l t s c h and J a c k s o n , 1982). Some ot h e r v e r t e b r a t e s which have, or a r e suggested t o have, t h i s c a p a b i l i t y t o v a r y i n g degrees a r e g o l d f i s h (Van den T h i l l a r t , 1 2 1982), s e a l s (Kooyman et a l . , 1980), h i b e r n a t o r s ( F a l e s c h i n i and W h i t t e n , 1975), toads ( L e i v e s t a d , 1960), s t u r g e o n (Burggren and R a n d a l l , 1978), and A f r i c a n l u n g f i s h ( L a h i r i e t a l . , 1970). The one common denominator among the s e a n i m a l s i s t h a t they do not a c t i v a t e a n a e r o b i c energy p r o d u c t i o n t o the e x t e n t which would be r e q u i r e d t o make up f o r the l a c k of a e r o b i c a l l y produced ATP. I t has o f t e n been observed t h a t the g l y c o l y t i c r a t e i s i n v e r s e l y r e l a t e d t o the r a t e of c e l l u l a r r e s p i r a t i o n (Neely and Morgan, 1974;Pasteur, l 8 6 1 ; R a c k e r , 1976). The major c o n t r o l s i t e i n g l y c o l y s i s i s thought t o be at p h o s p h o f r u c t o k i n a s e (PFK). An i n c r e a s e i n ATP c o n c e n t r a t i o n , such as would occur when an a c t i v a t i o n of o x i d a t i v e p h o s p h o r y l a t i o n o c c u r s , would s e r v e t o i n h i b i t g l y c o l y s i s . L i k e w i s e , a f a l l i n ATP c o n c e n t r a t i o n , or a r i s e i n the c o n c e n t r a t i o n of i n o r g a n i c phosphate would a c t i v a t e g l y c o l y s i s by a c t i v a t i n g PFK (Racker, 1976). The one e x c e p t i o n i s can c e r c e l l s . A e r o b i c g l y c o l y s i s ^proceeds at a r a p i d r a t e i n the s e c e l l s even though oxygen i s a v a i l a b l e (Racker, 1976). However, Racker (1976) suggests t h a t the response of c a n c e r c e l l s may not be due t o unusual g l y c o l y t i c c o n t r o l . A h i g h c o n c e n t r a t i o n of ADP i n the c e l l , caused by the a c t i v a t i o n of an ATPase, may a c t i v a t e g l y c o l y s i s d u r i n g a e r o b i c c o n d i t i o n s . I t appears t h a t the i n v e r s e r e l a t i o n s h i p between g l y c o l y t i c r a t e and c e l l u l a r r e s p i r a t i o n i s a l s o not p r e s e n t i n t i s s u e s where m e t a b o l i c r a t e i s reduced i n response t o oxygen l a c k . G l y c o l y s i s i s not b e i n g a c t i v a t e d when the r a t e of f l u x t h r ough the e l e c t r o n t r a n s f e r system d e c l i n e s which makes i t p o s s i b l e 1 3 t h a t n o v e l g l y c o l y t i c c o n t r o l mechanisms may be f u n c t i o n i n g . C o n v e r s e l y , i t i s p o s s i b l e t h a t the l a c k of g l y c o l y t i c a c t i v a t i o n i s due t o i n t r a c e l l u l a r mechanisms which pr e v e n t an i n c r e a s e i n the c o n c e n t r a t i o n s of a c t i v a t i n g m o d u l a t o r s , or which m a i n t a i n c o n c e n t r a t i o n s of g l y c o l y t i c i n h i b i t o r s . A l t h o u g h the o b s e r v a t i o n that' m e t a b o l i c r a t e may d e c l i n e d u r i n g h y p o x i a has been made on the whole body l e v e l , i t has y e t t o be d e t e r m i n e d which t i s s u e or t i s s u e s are r e s p o n s i b l e f o r the phenomenon. The cause of a d e c l i n e i n the m e t a b o l i c r a t e of a whole a n i m a l may be a f a l l i n the m e t a b o l i c r a t e of one, or many t i s s u e s . Thus, a u s e f u l f i r s t s t e p i n our u n d e r s t a n d i n g of the response would be t o i d e n t i f y the l o c a t i o n of the phenomenon. T h i s may be determined by s t r e s s i n g t i s s u e s w i t h h y p o x i a , and then u s i n g m e t a b o l i t e d a t a t o i n f e r which t i s s u e s have or have not e x h i b i t e d s i g n s of g l y c o l y t i c a c t i v a t i o n . By examining i n t e r - t i s s u e m e t a b o l i c responses i n t h i s f a s h i o n , i t may be p o s s i b l e t o p i n p o i n t which t i s s u e , i f any, has the g r e a t e s t i n f l u e n c e upon the m e t a b o l i c r a t e of the a n i m a l . I t may a l s o be p o s s i b l e t o determine the i n t e r - t i s s u e m e t a b o l i c p a t t e r n s which work i n c o n c e r t w i t h r e d u c t i o n s i n m e t a b o l i c r a t e t o promote the s u r v i v a l of the a n i m a l . As mentioned, the l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) may have the c a p a c i t y t o reduce i t s m e t a b o l i c r a t e t o 20% of the r e s t i n g v a l u e when f o r c e d to remain submerged ( L a h i r i e_t a l . , 1970). P r e v i o u s o b s e r v a t i o n s on the A f r i c a n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) i n d i c a t e d t h a t t h i s s p e c i e s may a l s o be unusual i n i t s c a p a c i t y t o s u r v i v e h y p o x i c exposure ( L a h i r i 14 et a_l. , 1970). L u n g f i s h a re bimodal b r e a t h e r s and, when u n r e s t r a i n e d , o b t a i n a p p r o x i m a t e l y 90% of t h e i r oxygen uptake from l u n g exchange (McMahon, 1970). The a r t e r i a l oxygen t e n s i o n s may f a l l from 50 t o r r t o 5 t o r r d u r i n g 10 minutes of f o r c e d submergence ( L a h i r i e t a_l. , 1970) and from 40 t o r r t o 20 t o r r d u r i n g 5 minutes of u n r e s t r a i n e d submergence (Johansen and L e n f a n t , 1968). Even though the r a t e of oxygen uptake from the g i l l s i s low and the a r t e r i a l oxygen t e n s i o n s q u i c k l y d e c l i n e d u r i n g submergence, l u n g f i s h a r e a b l e t o remain submerged f o r many hours (McMahon, 1970). Thus, the d a t a i n d i c a t e t h a t submersion l i m i t s oxygen uptake, a s u g g e s t i o n which i s su p p o r t e d by the o b s e r v a t i o n t h a t u n r e s t r a i n e d l u n g f i s h w i l l cease a i r -b r e a t h i n g i n water gassed w i t h 100% 0 2 ( J e s s e e t a l . , 1967). These d a t a i n d i c a t e d t h a t the l u n g f i s h would be a good a n i m a l t o use when examining the m e t a b o l i c r e a c t i o n s of an a n i m a l which u t i l i z e s t he s t r a t e g y of m e t a b o l i c r a t e r e d u c t i o n i n response to h y p o x i a . T h i s t h e s i s was d e s i g n e d t o e l u c i d a t e the m e t a b o l i c responses t o c e l l u l a r h y p o x i a i n an a n i m a l which does reduce o v e r a l l m e t a b o l i c r a t e d u r i n g h y p o x i a , and an a n i m a l which has not been r e p o r t e d t o have t h i s c a p a c i t y . The l u n g f i s h was chosen as the s u b j e c t f o r use i n examining the response of m e t a b o l i c r a t e r e d u c t i o n . By so d o i n g , i t i s i n t e n d e d t h a t the m e t a b o l i c s t r a t e g y f o r h y p o x i a s u r v i v a l used by l u n g f i s h w i l l become a p p a r e n t . Once the i n t e r t i s s u e m e t a b o l i c responses t o h y p o x i a were de t e r m i n e d i n the l u n g f i s h , the p a t t e r n was compared t o t h a t 15 which o c c u r s i n a f i s h which i s r e l a t i v e l y i n t o l e r a n t t o h y p o x i a . The rainbow t r o u t was chosen because the salmonids a re among the f i s h e s t h a t a re most s e n s i t i v e t o oxygen d e f i c i e n c y (Doudoroff and Shumway, 1970), and because t h e r e i s a l a r g e volume of l i t e r a t u r e a l r e a d y a v a i l a b l e c o n c e r n e d w i t h the s u b j e c t of t r o u t p h y s i o l o g y and metabolism. I t was not attempted t o expose the two a n i m a l s t o the i d e n t i c a l oxygen l i m i t a t i o n . I n s t e a d , a time c o u r s e f o r exposure t o h y p o x i a was chosen which produced r e p r o d u c i b l e changes i n plasma l a c t a t e c o n c e n t r a t i o n w i t h o u t any concommitant a n i m a l m o r t a l i t y , and which was l o n g enough t o make the p a t t e r n of i n t r a c e l l u l a r m e t a b o l i c changes o b s e r v a b l e . The t h e s i s p r o v i d e s e v i d e n c e which i n d i c a t e s t h a t the mass of muscle which i s comprised of w h i t e f i b r e s i s l i k e l y t o be the t i s s u e which has the g r e a t e s t i n f l u e n c e on the m e t a b o l i c r a t e of the l u n g f i s h . D u r i n g f o r c e d submergence, when o t h e r t i s s u e s a re o b v i o u s l y s t r e s s e d by h y p o x i a , t h e r e a re no measurable m e t a b o l i c changes i n the w h i t e muscle. In c o n t r a s t , when the t r o u t i s made h y p o x i c , t h e r e a re a p p r e c i a b l e changes i n the muscle. M e t a b o l i t e c o n c e n t r a t i o n changes i n t r o u t a x i a l muscle i n d i c a t e s t h a t g l y c o l y s i s has been a c t i v a t e d . T h i s a c t i v a t i o n may have d e t r i m e n t a l e f f e c t s upon the r e m a i n i n g t i s s u e s due t o co m p e t e t i o n f o r s u b s t r a t e s and i n c r e a s e d end-product p r o d u c t i o n . The c a p a c i t y of the l u n g f i s h w h i t e muscle to endure h y p o x i a w i t h no a c t i v a t i o n of g l y c o l y s i s p r e v e n t these d e t r i m e n t a l e f f e c t s . 16 SECTION 1 M e t a b o l i c a d j u s t m e n t s t o h y p o x i a i n the A f r i c a n l u n q f i s h  I n t r o d u c t i o n The A f r i c a n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) , i s an o b l i g a t e a i r - b r e a t h i n g a n i m a l and o b t a i n s a p p r o x i m a t e l y 10% of i t s r e s t i n g oxygen uptake from the water (McMahan, 1970). D e s p i t e a h y p o p e r f u s i o n of s k e l e t a l muscle ( L a h i r i e t a l . " , 1970), which may c o n s e r v e oxygen and s u b s t r a t e s f o r c e n t r a l o r g a n s , a r t e r i a l oxygen t e n s i o n s ( P a 0 2 ) a r e not m a i n t a i n e d . Thus Pa0 2 i n the A f r i c a n l u n g f i s h can f a l l from c o n t r o l v a l u e s of 50 t o r r t o 5 t o r r d u r i n g the f i r s t 10 minutes of f o r c e d submergence ( L a h i r i et. a l . , 1970). I t i s p o s s i b l e t h a t , s i n c e such low b l o o d 0 2 t e n s i o n s o c c u r , some t i s s u e s may become hy p o x i c a f t e r s h o r t p e r i o d s of submergence. I f t i s s u e h y p o x i a o c c u r s i n l u n g f i s h , t i s s u e h y p o x i a t o l e r a n c e may be r e l a t i v e l y h i g h compared w i t h o t h e r v e r t e b r a t e s , s i n c e l u n g f i s h can remain submerged f o r hours b e f o r e showing s i g n s of m e t a b o l i c s t r e s s . I t has been suggested t h a t the l u n g f i s h i s c a p a b l e of r e d u c i n g i t s m e t a b o l i c r a t e t o 20% of the r e s t i n g v a l u e as a s t r a t e g y f o r s u r v i v i n g h y p o x i a ( L a h i r i e t a _ l . , 1970). The p r e s e n t s t u d y was d e s i g n e d t o e l u c i d a t e the m e t a b o l i c responses t.o h y p o x i c d y s o x i a i n the A f r i c a n l u n g f i s h . For an a n i m a l t o have a d e c l i n e i n m e t a b o l i c r a t e , t h e r e may be a r e d u c t i o n i n one, or many t i s s u e s . The p a t t e r n ' of m e t a b o l i c response was 17 examined on a t i s s u e by t i s s u e b a s i s i n o r d e r t o determine whether changes i n the m e t a b o l i c r a t e of one, or many, t i s s u e s i s i n s t r u m e n t a l i n r e d u c i n g the m e t a b o l i c r a t e of the whole a n i m a l . T h i s procedure would a l s o a l l o w f o r a d e s c r i p t i o n of t i s s u e t o t i s s u e i n t e r a c t i o n s . Thus, an o v e r a l l m e t a b o l i c s t r a t e g y may emerge which may h e l p t o e x p l a i n the a b i l i t y of the l u n g f i s h t o s u r v i v e d u r i n g p r o l o n g e d p e r i o d s of h y p o x i a . The p r o t o c o l was t o i n d i r e c t l y a s s e s s t i s s u e m e t a b o l i c p o t e n t i a l s u s i n g enzyme measurements and t o d i r e c t l y a s s e s s m e t a b o l i c c o n c e n t r a t i o n s i n t i s s u e s and b l o o d through a 12 hour d i v e . 18 M a t e r i a l s and Methods E x p e r i m e n t a l A n i m a l s . A f r i c a n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) were c o l l e c t e d from the K a v i r o n d o G u l f i n Lake V i c t o r i a and t r a n s p o r t e d t o N a i r o b i where they were k e p t , u n f e d , i n a q u a r i a a t 22°C. The f i s h weighed 200 t o 700 grams (gm). E x p e r i m e n t s were performed between one and t h r e e weeks a f t e r the f i s h were c a p t u r e d . Enzyme P r e p a r a t i o n and Assays. T i s s u e s were q u i c k l y e x c i s e d from beheaded f i s h and dropped i n i c e c o l d h o m o g e n i z a tion b u f f e r . Care was taken to d i s s e c t t i s s u e s from a s i m i l a r l o c a t i o n i n each f i s h ; w h i t e muscle j u s t c a u d a l t o the p e c t o r a l g i r d l e from the d o r s a l m i d l i n e t o h a l f way down towards the r i g h t l a t e r a l l i n e ; l i v e r from the most a n t e r i o r l o b e ; the v e n t r i c l e from the h e a r t ; and the whole b r a i n , e x c l u d i n g the o l f a c t o r y b u l b . T i s s u e s used f o r the c r e a t i n e phosphokinase assay were t a k e n from l u n g f i s h which had been s t o r e d a t -90°C. A f t e r b l o t t i n g d r y and w e i g h i n g ( r o u g h l y 0.5 gm), the samples were homogenized i n 5 ml of homogenization b u f f e r u s i n g a S o r v a l Omnimixer w i t h m i c r o - a t t a c h m e n t . The homogenate was then spun at 10,000 x g f o r 15 min and the p e l l e t d i s c a r d e d . Supernatant was assayed f o r enzyme a c t i v i t y i n a Unicam SP-1800 s p e c t r o p h o t o m e t e r p l u s r e c o r d e r a t 25°C. R e a c t i o n s c o u p l e d t o NADH/NAD+ changes were m o n i t o r e d a t 340 nm and t h o s e c o u p l e d t o 19 DTNB were m o n i t o r e d a t 412 nm. R e a c t i o n volumes t o t a l l e d 1 ml. A l l a s s a y s were completed w i t h i n 4 hours of h o m o g e n i z a t i o n . Homogenization b u f f e r : 33 mM i m i d a z o l e , 3.3 mM M g C l 2 , 23 mM K C 1 , 1% t r i t o n - X , pH 7.0 ( a d j u s t e d w i t h H C 1 ) . Assay b u f f e r : 100 mM i m i d a z o l e , 70 mM K C 1 , 10 mM M g C l 2 , pH 7.0 ( a d j u s t e d w i t h H C 1 ) . C i t r a t e synthase was a s s a y e d i n 50 mM T r i s b u f f e r , pH 8.1. B u f f e r pH v a l u e s were a d j u s t e d a t 25°C. Glycogen p h o s p h o r y l a s e , /3-hydroxyacyl-Coa dehydrogenase and phosphoglucose isomerase (PGI) were assayed as d e s c r i b e d by Bergmeyer (1974). The r e m a i n i n g enzymes were a s s a y e d u s i n g the methods of Hochachka e_t a l . , (1978b). M e t a b o l i t e P r e p a r a t i o n and Assay. M e t a b o l i t e s were sampled from 5 c o n t r o l f i s h ( w i t h a c c e s s t o the s u r f a c e ) , 6 f i s h submerged f o r 3 h o u r s , 6 f i s h submerged f o r 12 h o u r s , and 4 f i s h submerged f o r 12 hours and a l l o w e d a c c e s s t o the s u r f a c e f o r 12. h o u r s . Submergence was induced by p l a c i n g a r e s i n c o a t e d w i r e mesh l i d on the a q u a r i a below the water s u r f a c e . Water P 0 2 never f e l l below 120 mmHg. T i s s u e s were removed from beheaded f i s h and r a p i d l y f r o z e n t o -196°C u s i n g aluminum tongs c o o l e d i n l i q u i d n i t r o g e n . Clamping was completed w i t h i n 90 seconds ( s ) . Regions sampled were i d e n t i c a l t o those d e s c r i b e d above. B l o o d was c o l l e c t e d from the s e v e r e d vena cava i n t o an e q u a l of 6% p e r c h l o r i c a c i d (PCA). T i s s u e s were ground t o a f i n e powder u s i n g a morter and p e s t l e r e s t i n g on dry i c e and f u r t h e r c o o l e d by f l u s h i n g w i t h 20 l i q u i d n i t r o g e n . About 0.5 gm of powder was weighed i n a c o l d h o m o g e n i z a t i o n f l a s k , d i l u t e d t o 5 ml w i t h 1.4 M PCA, and homogenized i n a S o r v a l Omnimixer w i t h m i c r o - a t t a c h m e n t . The homogenate was spun at 10,000 x g f o r 15 minutes (min.) and the p e l l e t d i s c a r d e d . The s u p e r n a t a n t was n e u t r a l i z e d t o pH 6.0 w i t h 1.4M KOH and spun a g a i n . M e t a b o l i t e s were measured w i t h i n 30 hours w h i l e the samples were kept on i c e . Glycogen was det e r m i n e d on 0.5 ml samples which were removed from the crude homogenate b e f o r e i t was spun. M e t a b o l i t e Measurements M e t a b o l i t e s were measured by e n z y m a t i c a l l y l i n k i n g them t o r e a c t i o n s u s i n g NADH/NAD*, or NADPH/NADP* ( n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate, reduced and o x i d i z e d r e s p e c t i v e l y ) , and f o l l o w i n g the r e a c t i o n a t 340 nm on a Unicam SP-1800 s p e c t r o p h o t o m e t e r . Glycogen was measured u s i n g the a m y l o g l u c o s i d a s e t e c h n i q u e ( K e p p l e r and Decker, 1974). The re m a i n i n g m e t a b o l i t e s were measured w i t h the t e c h n i q u e s of Hochachka et a l . , (1978c). S t a t i s t i c s Data were compared u s i n g S t u d e n t s 2 t a i l e d t - t e s t w i t h p < 0.05. 21 R e s u l t s Enzyme A c t i v i t i e s . T a b l e 1 l i s t s the enzyme a c t i v i t i e s found i n the ass a y e d t i s s u e s . O x i d a t i v e enzyme a c t i v i t i e s a r e h i g h e s t i n the h e a r t and l i v e r , w i t h h e a r t d i s p l a y i n g almost an o r d e r of magnitude more c i t r a t e synthase (CS) than any of the o t h e r t i s s u e s . /3-Hydroxyacyl-CoA dehydrogenase (HOAD) , an i n d i c a t o r of the a b i l i t y of t i s s u e s t o o x i d i z e f a t t y a c i d s (Marsh, 1981), o c c u r s a t h i g h e s t a c t i v i t i e s i n l i v e r and h e a r t , but o c c u r s a t low l e v e l s i n b r a i n and muscle. A s p a r t a t e a m i n o t r a n s f e r a s e (AAT), which has b o t h a e r o b i c and a n a e r o b i c f u n c t i o n s , i s p r e s e n t i n moderate a c t i v i t i e s i n l i v e r and h e a r t but low a c t i v i t i e s i n wh i t e muscle. M a l a t e dehydrogenase (MDH) a c t i v i t y i s a l s o v e r y h i g h i n h e a r t . Of the g l y c o l y t i c enzymes, Phosphoglucose isomerase (PGI) i s h i g h e s t i n the h e a r t ; g l y c o g e n p h o s p h o r y l a s e , and a l d o l a s e are s i m i l a r i n the h e a r t and muscle; and p y r u v a t e k i n a s e (PK) i s s i m i l a r i n the h e a r t , muscle and b r a i n . L a c t a t e dehydrogenase (LDH) i s h i g h e r i n the h e a r t than i n t he muscle. M e t a b o l i t e s . Table 2 l i s t s the c o n c e n t r a t i o n s of g l y c o l y t i c m e t a b o l i t e s p r e s e n t i n c o n t r o l l u n g f i s h and F i g u r e 1 shows r e l a t i v e changes i n b r a i n , muscle, and h e a r t a f t e r 3 and 12 hours of submergence. I n c r e a s e d f l u x t h r ough the non-e q u i l i b r i u m enzyme p h o s p h o f r u c t o k i n a s e (PFK) i s i n d i c a t e d s i n c e the s u b s t r a t e c o n c e n t r a t i o n f e l l below c o n t r o l v a l u e s and the 22 Tab l e 1. L u n g f i s h t i s s u e enzyme a c t i v i t i e s . Enzyme a c t i v i t i e s a r e e x p r e s s e d as Mmoles s u b s t r a t e c o n v e r t e d per minute per gm wet weight of t i s s u e . Assay c o n d i t i o n s a r e g i v e n i n 'Methods'. V a l u e s i n p a r e n t h e s e s a r e p y r u v a t e c o n c e n t r a t i o n i n mM. 23 T a b l e 1. L u n g f i s h t i s s u e enzyme a c t i v i t i e s . (Mmole/min./gm wet wt.) T i s s u e n Mean+S.D. G l y c o l y t i c or An a e r o b i c Enzymes Glycogen P h o s p h o r y l a s e Heart Muscle L i v e r 6 6 6 5.15+3.57 3.94+1,24 1 .61+0.76 P h o s p h o q l u c o i somerase Heart Muscle L i v e r 6 7 6 100.1+17.0 57.9+21.9 25.7+ 3.4 F r u c t o s e - 1 , 6 -b i s p h o s p h a t a s e Heart Muscle L i v e r 3 3 3 <1 <1 3.14+1.17 A l d o l a s e Heart Muscle L i v e r 5 5 5 15.98+ 4.06 27.84+13.38 6.74+ 0.52 P y r u v a t e K i n a s e Heart Muscle L i v e r B r a i n 5 6 5 6 100.9+17.4 98.8+20.7 14.0+ 1.94 102.9+41.6 Table 1. (continued) L a c t a t e Dehydrogenase Pyruvate c o n c e n t r a t i o n i n b rackets (mM) Heart (.05) 6 (10) 6 Muscle (.05) 6 (10) 6 L i v e r (.05) 6 (10) 6 B r a i n (.05) 6 (10) 6 593.4+229.1 402.9+124.8 256.9+85.0 149.8+65.9 248.6+168.4 167.8+115.3 149.4+38.6 116.9+28.7 C r e a t i n e Phosphokinase Muscle 1013 (1083.2-942.8) O x i d a t i v e or Mixed F u n c t i o n Enzymes C i t r a t e Synthase Heart Muscle L i v e r B r a i n 5 5 5 5 17.9+1.6 0.84+0.64 2.44+1.96 2.58+0.59 /G-Hydroxyacyl-CoA  Dehydrogenase Heart Muscle L i v e r B r a i n 6 6 6 6 17.37+3.82 1.97+0.62 32.89+12.8 3.05+0.59 g - G l y c e r o l - P -dehydrogenase Heart Muscle L i v e r 3 3 3 0.22+0.30 0.16+0.24 0.41+0.29 Table 1. ( c o n t i n u e d ) M a l a t e Dehydrogenase Heart Muscle L i v e r B r a i n 6 6 6 5 939.7+212.0 195.3+ 74.3 333.8+106.3 262.0+ 73.8 A s p a r t a t e A m i n o t r a n s f e r a s e Heart Muscle L i v e r 5 5 6 83.4+13.5 3.66+0.84 29.13+5.40 Table 2. C o n c e n t r a t i o n s of s e l e c t e d g l y c o l y t i c m e t a b o l i t e s i n the l u n g f i s h . (/amole/gm wet wt.) T i s s u e n Gl u c o s e G6P F6P FBP G3P DHAP P y r u v a t e B l o o d 5 0.23 + 0.24 White Muscle 5 0. .20 0. 20 0. .46 0. ,13 0. ,02 0. , 1 4 0. 10 + 0. , 12 + 0. 1 3 + 0. , 1 1 + 0. .04 + 0. ,02 + 0. ,02 + 0. 05 Heart 5 0. .93 0. 1 1 0. .25 0. .04 0. ,02 0. .06 0. 16 + 0. .28 + 0. 07 + 0. . 1 2 + 0. .02 + 0. ,03 + 0, .05 + 0. 09 B r a i n 5 0. .72 0. 1 1 0. ,25 0. . 1 2 0. , 1 1 0. ,08 0. 1 3 + 0, .39 + 0. 09 + 0. .20 + 0, .05 + 0. , 1 2 + 0. .07 + 0. 05 L i v e r 5 2.77 + 1 .99 V a l u e s are means + SD. M e t a b o l i t e a b b r e v i a t i o n s a r e e x p l a i n e d i n the L i s t of A b b r e v i a t i o n s . 27 F i g u r e 1. C r o s s o v e r p l o t s of l u n g f i s h t i s s u e m e t a b o l i t e s a t the end of a submergence. The upper graph r e p r e s e n t s the 3 hour d i v e and the lower graph i l l u s t r a t e s the 12 hour d i v e . C o n t r o l v a l u e s a r e l i s t e d i n T a b l e 2. The sample s i z e s a r e i n 'Methods'. 0 1 ) " I 1 1 1 I 1"-GLUCOSE G6P F6P F-I,6BP DHAP G3P PYR LAC 29 p r o d u c t c o n c e n t r a t i o n was e l e v a t e d above c o n t r o l v a l u e s . The o p p o s i t e s i t u a t i o n w i t h HK su g g e s t s t h a t the f l u x through t h i s enzyme i s not as r a p i d as the f l u x t h r ough the remainder of the pathway. Both responses i n d i c a t e r e g u l a t o r y s i t e s . The s h i f t a c r o s s the e q u i l i b r i u m enzyme LDH i n d i c a t e s t h a t e i t h e r the pH dropped, and/or the c e l l became more reduced. F i g u r e s 2-4 show g l y c o g e n , g l u c o s e , . and l a c t a t e c o n c e n t r a t i o n s d u r i n g a 12 hour submergence and a f t e r a 12 hour r e c o v e r y . Mean g l y c o g e n c o n c e n t r a t i o n s d e c l i n e i n the l i v e r , h e a r t , and b r a i n d u r i n g the d i v e ( F i g . 2 ) , w h i l e the h e a r t and b r a i n r e t u r n t o c o n t r o l l e v e l s a f t e r r e c o v e r y . D u r i n g the d i v e g l u c o s e c o n c e n t r a t i o n s i n c r e a s e s i g n i f i c a n t l y i n b l o o d , h e a r t , and b r a i n ( F i g . 3 ) . These d a t a a l s o i n d i c a t e t h a t l i v e r g l u c o s e i n c r e a s e s d u r i n g the d i v e . A f t e r a 12 hour r e c o v e r y , g l u c o s e c o n c e n t r a t i o n i n the b l o o d remains s i g n i f i c a n t l y e l e v a t e d above c o n t r o l v a l u e s . G l u c o s e c o n c e n t r a t i o n s d e c l i n e s i g n i f i c a n t l y d u r i n g r e c o v e r y from the 12 hour sample i n the b l o o d , h e a r t , and l i v e r . L a c t a t e i n c r e a s e s s i g n i f i c a n t l y i n the h e a r t , b r a i n , and b l o o d d u r i n g the d i v e and then f a l l s i n each t i s s u e d u r i n g r e c o v e r y ( F i g . 4 ) . M e t a b o l i t e s a s s o c i a t e d w i t h the energy s t a t u s of the c e l l and the c e l l u l a r energy charge (T a b l e 3) d i d not change d u r i n g the e x p e r i m e n t . 30 F i g u r e 2. Glycogen c o n t e n t s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h . Sample s i z e i n methods. S t a r denotes a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l s t a t e . Bar i n d i c a t e s +1 S.E.. 31 220-> 180 H i t : 1 4 0 E 3 g 100-a O) i. 60-o * E - 50-0) *^ c o o c a> o o >> O 401 301 20-10-0 k DIVE o White Muscle • Heart o Brain £x Liver R E C O V E R Y —«| 0 3 12 Time (hours) 24 32 F i g u r e 3. T i s s u e and b l o o d g l u c o s e c o n c e n t r a t i o n s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h . Sample s i z e i n methods. S t a r denotes a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l s t a t e . Bar i n d i c a t e s +1 S.E.. • Blood o White Muscle • Heart o Brain Liver DIVE- RECOVERY-54 o E c o •mm (0 o o c o o o 2 CO O O o 0 3 i • 12 Time (hours) 24 34 F i g u r e 4. T i s s u e and b l o o d l a c t a t e c o n c e n t r a t i o n s d u r i n g f o r c e d submergence and r e c o v e r y i n l u n g f i s h . Sample s i z e i n methods. S t a r denotes a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l s t a t e . Bar i n d i c a t e s +1 S.E.. 35 • Blood ° White Muscle • Heart °- Brain 36 T a b l e 3. C o n c e n t r a t i o n s of a d e n y l a t e s , c r e a t i n e phosphate, and c r e a t i n e , and the energy charge i n l u n g f i s h t i s s u e s . (Mmole/gm wet wt) C o n d i t i o n s n ATP ADP AMP EC CrP Cr T o t a l A d e n y l a t e s Heart C o n t r o l 5 0 + 0 .79 .36 0. + 0. 15 07 0. + 0. 08 07 0.83 + 0.u8 1 + 0 .14 .50 0. + 1 . 47 06 1 .02 3-hr d i v e 6 1 + 0 .77 .25 0. + 0. 36 07 0. + 0. 05 04 0.91 + 0-.03 1 + 0 .82 .29 2. + 0. 01 89 2 .18 12-hr d i v e 6 1 + 0 .58 .31 0. + 0. 41 10 0. i°-09 03 0.86 + 0.01 1 + 0 .52 .93 3. + 1 . 29 45 2 .08 Recovery 4 1 + 0 .33 .12 0. + 0. 28 03 0. + 0. 07 01 0.88 + 0.02 1 .45 White muscle C o n t r o l 5 2 + 0 .41 .38 0. + 0. 33 08 0. i°-02 01 0.93 + 0.01 1 0 + 1 .56 .68 12. ± 2-67 10 2 .76 3-hr d i v e 6 3 + 0 . 1 4 .42 0. + 0. 57 1 1 0. + 0. 05 06 0.91 + .008 8 + 1 .77 .77 14. ± 3-39 57 3 .76 12-hr d i v e 6 3 + 0 .36 .37 0. + 0. 55 08 0. + 0. 04 01 0.92 + .004 7 + 2 .94 .73 12. + 1 . 72 55 3 .95 Recovery 4 3 + 0 .10 .28 0. + 0. 54 06 0. + 0. 07 02 0.91 + .006 B r a i n C o n t r o l 5 0 + 0 .59 .22 0. +0. 32 08 0. + 0. 06 05 0.77 + 0.10 0 + 0 .52 .48 1 . + 0. 03 45 0 .97 3-hr d i v e 6 0 + 0 .86 .24 0. + 0. 33 15 0. + 0. 05 04 0.80 + 0.10 2 + 2 . 1 6 .13 3. + 1 . 16 47 1 .24 12-hr d i v e 6 0 + 0 .83 .32 0. + 0. 36 07 0. + 0. 15 04 0.74 + 0.09 0 + 0 .60 .42 3. + 0. 31 27. 1 .34 Recovery 4 0 + 0 .87 .29 0. + 0. 39 19 0. + 0. 16 06 0.75 + 0.11 V a l u e s a r e means + SD. Energy charge a f t e r A t k i n s o n (1977). 37 D i s c u s s i o n The r e s u l t s from t h i s experiment a l l o w one t o examine m e t a b o l i c changes which occur i n the b r a i n , h e a r t , l i v e r , m uscle, and b l o o d d u r i n g f o r c e d submergence i n the l u n g f i s h . The f o l l o w i n g d i s c u s s i o n of the s e r e s u l t s i s a r r a n g e d by t i s s u e w i t h a c o n c l u d i n g s e c t i o n on the i n t e r a c t i o n s which may be o c c u r r i n g between t i s s u e s , and the i n f l u e n c e which these i n t e r a c t i o n s have upon the c a p a c i t y of the l u n g f i s h t o s u r v i v e h y p o x i a . B r a i n . S i n c e the b r a i n r e l i e s upon g l u c o s e metabolism f o r energy s u p p l y ( S i e s j o and Nordstrom, 1977 ;McDougal e_t a l . , 1968), g l y c o l y t i c enzymes a r e u t i l i z e d i n both a e r o b i c and a n a e r o b i c m e t a b o l i s m . Because of t h i s , an i n s t r u c t i v e t e c h n i q u e f o r a s s e s s i n g the r e l a t i v e p o t e n t i a l s of a e r o b i c and a n a e r o b i c m etabolism i n the b r a i n i s t o compare the a c t i v i t y r a t i o s of g l y c o l y t i c and o x i d a t i v e enzymes ( K e u l e_t a_l. , 1972). T a b l e 4 i n d i c a t e s how h i g h the LDH/CS, and PK/CS r a t i o s can be f o r Proto.pterus a e t h i o p i c u s and Arapaima q i q a s , two o b l i g a t e a i r -b r e a t h i n g f i s h , r e l a t i v e t o o t h e r f i s h e s and mammals. Such h i g h r a t i o s i n d i c a t e an i n c r e a s e d c a p a c i t y f o r p r o v i d i n g a l a r g e p o r t i o n of the b r a i n ' s ATP p r o d u c t i o n v i a g l y c o l y s i s . The a b s o l u t e a c t i v i t i e s of two enzymes n o r m a l l y f u n c t i o n a l i n o x i d a t i v e metabolism (CS, and HOAD) are low i n l u n g f i s h b r a i n (Table 1 ) . The a c t i v i t y of CS i s o n l y about 1/10 t h a t t y p i c a l l y o bserved f o r a wide v a r i e t y of s p e c i e s i n c l u d i n g f i s h e s , 38 T a b l e 4, Comparative a c t i v i t y r a t i o s of enzymes from a n a e r o b i c and a e r o b i c pathways i n b r a i n s of f i s h and mammals. Spec i e s L a c t a t e dehydro-g e n a s e / C i t r a t e synthase P y r u v a t e k i n a s e / C i t r a t e synthase R e f e r e n c e A i r - b r e a t h i n g F i s h P. a e t h i o p i c u s  Arapaima q i q a s A q u a t i c B r e a t h i n g F i s h 58 1 40 40 T h i s study 128 Hochachka e_t a l . , 1978c. Coryphaenoides r u p e s t r i s 14 Coryphaenoides armatus 16 Coryphaenoides l e p t o l e p i s 14 Medi a l u n a c a l i f o r n i e n s i s 12 1 2 Chromis p u n t i p e n n i s  Genyonemus l i n e a t u s I 1 S i e b e n a l l e r e_t a_l. , 1 982. 10 i b i d II i b i d 7 S u l l i v a n and .Somero 1980. i b i d 1 1 7 9 i b i d Mammals Weddell s e a l 13 (L e p t o n y c h o t e s w e d d e l l i ) 12 Murphy e_t a l . , 1980. Ox (Bos t a u r i s ) 10 i b i d 39 amphibians, mammals, and b i r d s (Murphy et a l . , l980;Sugden and Newsholme, 1975). Thus, the p o t e n t i a l f o r o x i d a t i v e m e t abolism i n the b r a i n of P. a e t h i o p i c u s i s de p r e s s e d , which i s s i m i l a r t o the b r a i n of Arapaima q i q a s (Hochachka, 1980). An i n d i c a t i o n of the a n a e r o b i c c a p a c i t y of b r a i n t i s s u e can a l s o be o b t a i n e d from the s i z e of g l y c o g e n s t o r a g e depots (McDougal e t a l . , 1968). B r a i n g l y c o g e n l e v e l s i n the l u n g f i s h are i n the range of tho s e i n the Weddell s e a l , f r o g , and d i v i n g t u r t l e , and are about 2-4 times h i g h e r than the b r a i n g l y c o g e n s t o r e s of most o t h e r v e r t e b r a t e s (Kerem e t al . . , 1973 ;McDougal et a l • , 1968). In view of the above c o n s i d e r a t i o n s , i t i s not s u r p r i s i n g t h a t extended submergence r e s u l t s i n l a c t a t e a c c u m u l a t i o n i n the b r a i n ( F i g . 4 ) . T h i s a n a e r o b i c end-product appears t o be d e r i v e d from both endogenous g l y c o g e n , s i n c e some d e p l e t i o n of b r a i n g l y c o g e n s t o r e s o c c u r s ( F i g . 2 ) , and from the u t i l i z a t i o n of b l o o d g l u c o s e which, owing t o the b l o o d / b r a i n g l u c o s e g r a d i e n t , may be taken up d u r i n g the e n t i r e submergence p e r i o d ( F i g . 3 ) . That g l y c o l y s i s i s a c t i v a t e d i n the b r a i n d u r i n g submergence ( r a t h e r than s i m p l y o p e r a t i n g a t normoxic r a t e s ) i s i n d i c a t e d (a) by c r o s s o v e r p l o t s showing f a c i l i t a t i o n a t the p h o s p h o f r u c t o k i n a s e r e a c t i o n , and l i m i t a t i o n a t the h e x o k i n a s e r e a c t i o n ( F i g . 1 ) , and (b) by the c o n t i n u o u s a c c u m u l a t i o n of l a c t a t e i n the b r a i n ( F i g . 4 ) . H e a r t . A good p o t e n t i a l . f o r c a r b o h y d r a t e metabolism i s i n d i c a t e d by h i g h g l y c o g e n p h o s p h o r y l a s e , PGI, a l d o l a s e , and PK 40 a c t i v i t i e s , a l l i n about the same range as observed i n tuna heart (Hochachka et a l . , 1978a). I t i s assumed that the g l y c o l y t i c path may f u n c t i o n q u i t e e f f e c t i v e l y under . a e r o b i c c o n d i t i o n s s i n c e the a c t i v i t i e s of MDH and AAT are high. T h i s process r e q u i r e s a s h u t t l e of reducing e q u i v i l e n t s i n t o the mitochondria and these enzymes f u n c t i o n i n the malate-aspartate s h u t t l e . The a - g l y c e r o l phosphate s h u t t l e does not appear to be s i g n i f i c a n t i n the heart due to the low a c t i v i t y of a - g l y c e r o l phosphate dehydrogenase (Table 1). The a c t i v i t i e s of HOAD and CS a l s o i n d i c a t e a good a e r o b i c c a p a c i t y . The former i s present i n a c t i v i t i e s s i m i l a r to that found in the Weddell s e a l and ox (Murphy et: a_l. , 1980) while the CS a c t i v i t y i s about 2/3 the l e v e l found i n tuna (Guppy et. a l . , 1979) or salmon (Mommsen et a l . , 1980). A high anaerobic p o t e n t i a l i n the heart i s i n d i c a t e d by both enzyme and m e t a b o l i t e data. The main enzyme i n d i c a t o r of anaerobic p o t e n t i a l i s the a c t i v i t y of muscle type LDH, which shows a lack of pyruvate i n h i b i t i o n (Table 1) , a property of isozymes which favor c o n v e r s i o n of pyruvate to l a c t a t e . The a c t i v i t y of t h i s enzyme i s 1.5 times higher than i n tuna heart (Hochachka et a_l. , 1978c), two times higher than i n Arapaima heart (Hochachka e_t a l . , 1978a), and three times higher than i n salmon heart (Mommsen et aJL. , 1980). Heart glycogen s t o r e s are another i n d i c a t i o n of anaerobic p o t e n t i a l and are high compared to most v e r t e b r a t e s (Dawes et a_l. , 1959;Kerem e_t a l . , 1973). Compared to other l u n g f i s h t i s s u e s , the heart i s very o x i d a t i v e : CS a c t i v i t i e s are about 20 times higher than i n 41 muscle, 8 t i m e s h i g h e r than i n l i v e r and 7 times h i g h e r than i n b r a i n ( T a b l e 1 ) . A s i m i l a r p a t t e r n i s a l s o found i n o t h e r a i r -b r e a t h i n g f i s h (Hochachka, 1980). Given t h i s m e t a b o l i c o r g a n i z a t i o n , i t i s p r o b a b l e t h a t under normoxic c o n d i t i o n s h e a r t m e t abolism i s s i m i l a r t o o t h e r f i s h e s ; f u l l y a e r o b i c , f u e l e d by f a t or.by a m i x t u r e of f a t and c a r b o h y d r a t e ( B i l i n s k i and Jonas, 1 9 7 0 ; B i l i n s k i and J o n a s , 1972). Upon i n i t i a t i o n of submergence, o x i d a t i v e m e t a b o l i s m may be extended by 0 2 c o n s e r v a t i o n mediated by a r e d u c t i o n i n c a r d i a c energy demands, s i n c e a 30% d e c l i n e i n h e a r t r a t e can occur d u r i n g submersion i n . u n r e s t r a i n e d (Conant, 1975) or e x p e r i m e n t a l l y submerged l u n g f i s h ( L a h i r i et a l . , 1970). Even w i t h r e d u c t i o n s i n c a r d i a c work l o a d d u r i n g submergence, however, the h e a r t does not r e c e i v e enough oxygen to s u p p l y a l l of i t s r e q u i r e m e n t s . A c t i v a t i o n of g l y c o l y s i s i s i n d i c a t e d (a) by c r o s s o v e r p l o t s ( F i g . 1) which show t h a t p h o s p h o f r u c t o k i n a s e was a c t i v a t e d , (b) by l a c t a t e a c c u m u l a t i o n i n the h e a r t d u r i n g submergence ( F i g . 4 ) , and (c) by d e p l e t i o n of endogenous g l y c o g e n s t o r e s f o l l o w e d by u t i l i z a t i o n of b l o o d g l u c o s e s t o r e s . S i n c e g l u c o s e moves i n the d i r e c t i o n of low c o n c e n t r a t i o n i n most t i s s u e s ( M c G i l v e r y , 1979), i t i s l i k e l y t h a t the h e a r t may u t i l i z e plasma g l u c o s e a f t e r the b l o o d / h e a r t g l u c o s e g r a d i e n t becomes f a v o r a b l e f o r g l u c o s e uptake ( F i g s . 2, 3 ) . L a c t a t e c o n c e n t r a t i o n s i n the h e a r t reach r o u g h l y h a l f those i n the b r a i n , and s i n c e the energy r e q u i r e m e n t s of the h e a r t u ndoubtedly s u r p a s s those of the b r a i n d u r i n g submergence 42 i t can be t e n t a t i v e l y c o n c l u d e d t h a t some f r a c t i o n of h e a r t m e t a b o l i c r a t e i s s t i l l b e i n g s u p p l i e d by a e r o b i c metabolism (even when submerged the f i s h o b t a i n s a p p r o x i m a t e l y 10% of the r e s t i n g oxygen uptake from the water (McMahon, 1970)). The m e t a b o l i c s i t u a t i o n here i s , t h e r e f o r e , complex: t h e r e appears t o be a mixed a e r o b i c and a n a e r o b i c metabolism i n response to h y p o x i a , w i t h i n d i c a t i o n s t h a t some a c t i v a t i o n of a n a e r o b i c energy p r o d u c t i o n has o c c u r r e d . White muscle. White muscle g l y c o l y t i c enzymes d i s p l a y p a r t i c u l a r l y low a c t i v i t i e s . PK a c t i v i t y i s o n l y 1/13 t h a t i n tuna (Guppy et al., 1979) or salmon (Mommsen et a l . , 1980), and o n l y 1/5 t h e l e v e l i n the arawana (Osteoqlossum b i c i r r h o s u m ) (Hochachka e_t a l . , 1978b). In f a s t swimming s p e c i e s ( t u n a , salmon) the white m u s c l e / h e a r t LDH r a t i o i s about 20 t i m e s g r e a t e r than i n the l u n g f i s h (Guppy et a l . , 1979,'Mommsen e_t a l . , 1980). L u n g f i s h h e a r t a c t u a l l y has a h i g h e r LDH a c t i v i t y than the s k e l e t a l muscle. U l t r a s t r u c t u r a l s t u d i e s of l u n g f i s h w h i t e muscle (see S e c t i o n 2 ) , show an e x c e p t i o n a l l y low c a p i l l a r i t y and m i t o c h o n d r i a l d e n s i t y . T h i s , c o u p l e d w i t h the low o x i d a t i v e enzyme a c t i v i t i e s , s u g g e s t s t h a t energy p r o d u c t i o n from a e r o b i c m e t a b o l i s m o c c u r s a t v e r y low r a t e s . Thus the c a p a c i t y f o r both s u s t a i n e d a e r o b i c and a n a e r o b i c energy p r o d u c t i o n i s low i n l u n g f i s h w h i t e muscle. The a c t i v i t y of CPK, however, i s i n the normal range f o r f i s h muscle (J o h n s t o n e_t a l . , 1977; J o h n s t o n and Moon, 1980). T h i s i n d i c a t e s t h a t phosphagen h y d r o l y s i s i s the 43 most l i k e l y source of energy f o r the r a p i d and p o w e r f u l b u r s t work of which l u n g f i s h muscle i s c a p a b l e . There a r e t h r e e l i n e s of e v i d e n c e s u g g e s t i n g t h a t the energy u t i l i z a t i o n of w h i t e muscle d u r i n g submergence i s e x t r e m e l y low. F i r s t l y , d i r e c t e v i d e n c e a r i s e s from m e t a b o l i t e measurements d u r i n g submergence which show no s i g n i f i c a n t g l y c o g e n d e p l e t i o n ( F i g . 2 ) , no s i g n i f i c a n t l a c t a t e a c c u m u l a t i o n ( F i g . 4 ) , no change i n l a c t a t e / p y r u v a t e r a t i o s ( F i g . 1 ) , no change i n ATP, ADP, or AMP c o n c e n t r a t i o n s or i n the energy charge ( T a b l e 3 ) , and no s i g n i f i c a n t d e p l e t i o n of c r e a t i n e phosphate (CrP) (Table 3 ) . Se c o n d l y , i n d i r e c t e v i d e n c e s u g g e s t s t h a t muscle p e r f u s i o n has f a l l e n d u r i n g submergence ( L a h i r i e t a l . , 1970). The c o n c e n t r a t i o n s of g l u c o s e and l a c t a t e i n the muscle remain independent of b l o o d c o n c e n t r a t i o n s ( F i g s . 3, 4 ) . S i n c e both l a c t a t e and g l u c o s e move down c o n c e n t r a t i o n g r a d i e n t s , a l a c k of e q u i l i b r i u m i n these m e t a b o l i t e p o o l s over 12 hours a l s o i n d i c a t e s t h a t t h e r e i s min i m a l p e r f u s i o n of t h i s t i s s u e d u r i n g submergence. T h i r d l y , e s t i m a t e s of oxygen r e q u i r e m e n t s can be used t o i n d i c a t e whether m e t a b o l i c d e p r e s s i o n has o c c u r r e d i n muscle. The summed oxygen r e q u i r e m e n t s of the h e a r t and b r a i n i n a submerged l u n g f i s h are 15% t o 50% of the t o t a l oxygen upta k e , and i t i s u n l i k e l y t h a t s i g n i f i c a n t m e t a b o l i c d e p r e s s i o n o c c u r s i n t h e s e organs (see on ) . S i n c e oxygen uptake has d e c l i n e d t o a t l e a s t 40% of the r e s t i n g v a l u e when the l u n g f i s h i s submerged (unpub. o b s . ) , t h a t would l e a v e 20% t o 34% of the r e s t i n g oxygen uptake f o r the m u s c u l a t u r e . I t i s u n l i k e l y t h a t the muscle u t i l i z e s such a 44 s m a l l p e r c e n t a g e of the r e s t i n g oxygen uptake based upon the f o l l o w i n g c a l c u l a t i o n . I f muscle comprises 80% of the body mass, and i f t h a t muscle has a m e t a b o l i c r a t e of 0.19 ml 0 2 / g r / h r (Gordon, 1968), then the muscle of a 1 kg r e s t i n g l u n g f i s h r e q u i r e s 0.173 mmoles 0 2 / h r . T h i s i s 70% of the r e s t i n g oxygen uptake of 0.25 mmoles/kg/hr (unpub. o b s . ) , which i s more than the t o t a l uptake of the whole a n i m a l . T h i s i s c o n s i s t a n t w i t h the s u g g e s t i o n t h a t some m e t a b o l i c d e p r e s s i o n has o c c u r r e d i n t h i s t i s s u e . These d a t a i n d i c a t e t h a t the muscle may be c o n f o r m i n g t o the d e c l i n i n g oxygen d e l i v e r y by r e d u c i n g the energy r e q u i r e m e n t s and p r e v e n t i n g a c t i v a t i o n of g l y c o l y s i s . In o t h e r words, t h i s t i s s u e does not appear t o d i s p l a y any i n d i c a t i o n t h a t a n a e r o b i c energy p r o d u c t i o n has o c c u r r e d . L i v e r . The l i v e r ' s main f u n c t i o n d u r i n g submergence i s t o s u p p l y g l u c o s e f o r o t h e r t i s s u e s , as i n d i c a t e d by the marked r e d u c t i o n i n l i v e r g l y c o g e n s t o r e s and the s h a r p e l e v a t i o n i n f r e e g l u c o s e c o n c e n t r a t i o n s ( F i g s . 2, 3 ) . L i v e r g l u c o s e c o n c e n t r a t i o n s are s u b s t a n t i a l l y h i g h e r than i n the b l o o d , which f a v o r s c o n t i n u e d g l u c o s e e x p o r t t o o t h e r t i s s u e s ( F i g . 3 ) . The l i v e r must remain p e r f u s e d t o s u p p l y g l u c o s e d u r i n g p r o l o n g e d submergence. The l i v e r / b l o o d g l u c o s e g r a d i e n t s ( F i g . 3) a r e s i m i l a r t o the r e s t i n g s t a t e , which i m p l i e s t h a t c o n v e r s i o n of l i v e r g l y c o g e n t o g l u c o s e and l i v e r p e r f u s i o n a r e c l o s e l y c o o r d i n a t e d . R e d u c t i o n of the l i v e r / b l o o d g l u c o s e g r a d i e n t a t the end of the r e c o v e r y p e r i o d may be due t o e i t h e r 45 i n c r e a s e d g l y c o g e n f o r m a t i o n or t o i n c r e a s e d p e r f u s i o n of l i v e r and subsequent g l u c o s e l o s s from the l i v e r . S i n c e the h e a r t and b r a i n g l y c o g e n s t o r e s a re p r e f e r e n t i a l l y r e p l e n i s h e d d u r i n g r e c o v e r y i t i s l i k e l y t h a t l i v e r g l u c o s e i s s t i l l g o i n g t o those t i s s u e s . G l u c o n e o g e n e s i s i n the l i v e r p r o b a b l y o c c u r s d u r i n g r e c o v e r y because oxygen as w e l l as s u b s t r a t e s a r e a v a i l a b l e . A r e l a t i v e l y h i g h l i v e r g l u c o n e o g e n i c c a p a c i t y i s i n d i c a t e d by the l e v e l s of f r u c t o s e - 1 , 6 - b i s p h o s p h a t a s e ( T a b l e 1 ) , and a h i g h LDH/PK r a t i o which i s observed i n o t h e r l a c t a t e - p r i m e d g l u c o n e o g e n i c systems (Hochachka, 1980). L a c t a t e i s l i k e l y t o be the major s u b s t r a t e due t o i t s a v a i l a b i l i t y . L u n g f i s h metabolism d u r i n g f o r c e d submergence. In submerged s e a l s , r e d i s t r i b u t i o n of c a r d i a c output i s d e s i g n e d t o p r e s e r v e oxygen and s u b s t r a t e s f o r use by the c e n t r a l o r g a n s , thus a l l o w i n g them t o remain a e r o b i c . T h i s means t h a t the b r a i n and h e a r t , two organs which a r e s e n s i t i v e t o h y p o x i c damage, do not r e q u i r e major m e t a b o l i c a d a p t a t i o n s t o p r o l o n g e d h y p o x i a (Hochachka, 1980). In the l u n g f i s h , the b r a i n and h e a r t d e f i n i t e l y show a d a p t a t i o n s t o s u r v i v e h y p o x i a and u t i l i z e t hese a d a p t a t i o n s upon submersion. Thus, even though the combined oxygen uptake of the b r a i n and h e a r t i s p r o b a b l y l e s s than h a l f of t h e t o t a l oxygen u p t a k e 1 , the combined p h y s i o l o g i c a l a d j u s t m e n t s which occur i n the body are not s u f f i c i e n t t o s u p p l y t h i s amount of 4 46 oxygen t o the s e two organs. A s t r i k i n g d i f f e r e n c e between s e a l s and l u n g f i s h i s t h a t b l o o d g l u c o s e l e v e l s f a l l d u r i n g f o r c e d submergence i n the s e a l (Murphy e_t a_l. , 1980), whereas they r i s e i n the l u n g f i s h ( F i g . 3 ) . T h i s p a t t e r n may d e v e l o p i n l u n g f i s h because a l a r g e p o r t i o n of the body, the myotomal muscle, i s r e l a t i v e l y i n e r t , as i n d i c a t e d by low enzyme a c t i v i t i e s and i t s f i b r e o r g a n i z a t i o n (see S e c t i o n 2 ) . A l s o , the l i v e r remains p e r f u s e d and c o n t i n u o u s l y s u p p l i e s g l u c o s e , , a s i t u a t i o n which does not occur i n f o r c e d submergence i n marine mammals (Za p o l e t a_l. , 1979). Thus l i v e r g l u c o s e p r o d u c t i o n i n the l u n g f i s h o u t s t r i p s the demand and b l o o d g l u c o s e c o n c e n t r a t i o n s i n c r e a s e . These d a t a i n d i c a t e t h a t a l t e r a t i o n s i n p e r f u s i o n p a t t e r n s d u r i n g submergence i n the l u n g f i s h may be f u n c t i o n i n g t o m a i n t a i n (a) c o n t i n u e d d e l i v e r y of g l u c o s e from the l i v e r t o the t i s s u e s , and (b) l a c t a t e c l e a r a n c e from the l a c t a t e p r o d u c i n g t i s s u e s . In t h i s s c e n a r i o , oxygen c o n s e r v a t i o n may not be as d o m i n a t i n g a f u n c t i o n as i t i s i n a q u a t i c mammals. As a r e s u l t , the t i s s u e s i n the l u n g f i s h r e q u i r e a s i g n i f i c a n t t o l e r a n c e t o hypox i a . G l y c o l y t i c a c t i v a t i o n i n response t o h y p o x i a i s observed i n the h e a r t and b r a i n , but not i n the muscle. The l a c k of ^ f t e r 1 hour of submergence, a 500 gm l u n g f i s h has an oxygen uptake of 0.1 mmoles/hr ( u n p u b l i s h e d d a t a ) . The m e t a b o l i c r a t e s of v e r t e b r a t e h e a r t s v a r y from 0.04-0.3 mmoles 0 2/gm/hr (Lochner et a l . , 1968;Reeves, 1963). I f t h i s range i s a p p l i c a b l e t o l u n g f i s h i t su g g e s t s a h e a r t m e t a b o l i c r a t e of 0.01 t o 0.05 mmoles G 2/0.23 gm h e a r t / h r f o r a 500 gm f i s h . B r a i n oxygen r e q u i r e m e n t s a re e s t i m a t e d a t 0.003 mmoles O 2/0.27 gm b r a i n / h r u s i n g d a t a f o r f i s h b r a i n from McDougal e_t a l _ . (1968). The summed oxygen r e q u i r e m e n t s of the h e a r t and b r a i n range from 15% to 50% of the observed submerged oxygen uptake. 47 response i n the muscle, concommitant w i t h an o b v i o u s h y p o x i c s t r e s s i n the o t h e r t i s s u e s a l l o w s one t o suggest t h a t the most s i g n i f i c a n t a d a p t a t i o n t o h y p o x i a i n these f i s h e s may not be the c a p a c i t y f o r i n c r e a s e d a n a e r o b i c energy p r o d u c t i o n i n a l l t i s s u e s . I n s t e a d , i t i s l i k e l y t h a t the l a c k of g l y c o l y t i c a c t i v a t i o n i n the muscle d u r i n g h y p o x i c d y s o x i a i s the key t o the a n i m a l ' s s u r v i v a l . I t has a l r e a d y been c a l c u l a t e d t h a t the l u n g f i s h may e x h i b i t an 80% d e c l i n e i n m e t a b o l i c r a t e d u r i n g f o r c e d submergence ( L a h i r i et a_l. , 1970). The data from the p r e s e n t experiment s u p p o r t s the . s u g g e s t i o n t h a t a d e c l i n e i n the m e t a b o l i c r a t e of t h e l u n g f i s h o c c u r s d u r i n g h y p o x i a . F u r t h e r m o r e , i t i s proposed t h a t m e t a b o l i c d e p r e s s i o n i n the w h i t e muscle mass i s the main cause of the drop i n the m e t a b o l i c r a t e of the whole a n i m a l . 48 SECTION 2 An u l t r a s t r u c t u r a l and h i s t o c h e m i c a l s t u d y of t h e a x i a l  m u s c u l a t u r e i n t h e A f r i c a n l u n q f i s h I n t r o d u c t i o n As was shown i n S e c t i o n 1, t h e l u n g f i s h i s c a p a b l e o f s u r v i v i n g f o r h o u r s w h i l e b e i n g s u b j e c t e d t o h y p o x i a . I t was s u g g e s t e d t h a t a major component o f t h i s t o l e r a n c e was due t o t h e c a p a c i t y of t h e m u s c l e t o p r e v e n t a c t i v a t i o n of g l y c o l y s i s when oxygen c o n c e n t r a t i o n s a r e l i m i t i n g . W i t h s u c h e m p h a s i s b e i n g p l a c e d upon t h e r o l e of t h e m u s c l e d u r i n g h y p o x i c s i t u a t i o n s , i t was deemed i m p o r t a n t t o a n a l y s e t h e f i b r e c o m p o s i t i o n and u l t r a s t r u c t u r a l p r o p e r t i e s of t h i s m u s c l e . T h e r e i s a wide body of l i t e r a t u r e on t h e m e t a b o l i c p o t e n t i a l o f d i f f e r e n t f i b r e t y p e s and so s u c h an i n v e s t i g a t i o n may f u r t h e r t h e u n d e r s t a n d i n g of t h e m e t a b o l i s m of t h e l u n g f i s h m u s c l e d u r i n g submergence (and h y p o x i a ) . I f t h e m u s c l e i s c a p a b l e o f a r e d u c t i o n i n m e t a b o l i s m , t h e n i t may be p o s s i b l e t o d e t e r m i n e w h i c h o f t h e f i b r e t y p e s i n f i s h may be c a p a b l e of s u c h a p a t t e r n o f m e t a b o l i c r e g u l a t i o n . On t h e b a s i s o f t h e d a t a i n t h e p r e v i o u s s e c t i o n , one may p r e d i c t a m u s c u l a r o r g a n i z a t i o n f o r a minimum s u p p l y of o xygen. F u r t h e r m o r e , movements i n v o l v i n g t h e t r u n k m u s c u l a t u r e a r e a l m o s t e n t i r e l y r e s t r i c t e d t o e i t h e r b u r s t l u n g e s a t p a s s i n g p r e y or slow swimming. " B u r s t " m u s c l e has been d e s c r i b e d many 49 t i m e s and i s comprised l a r g e l y of w h i t e f i b r e s (Boddeke et a l . 1959). T h i s a l l o w s f o r i n t e n s e a n a e r o b i c f u n c t i o n and t h u s , by "this c r i t e r i o n as w e l l , one would expect a muscular o r g a n i s a t i o n adapted f o r low oxygen, (Boddeke e_t a l . , 1 959; J o h n s t o n , 1981). 50 Methods A n i m a l s . A f r i c a n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) were c o l l e c t e d from Lake V i c t o r i a and t r a n s p o r t e d t o N a i r o b i where they were kept i n s e p e r a t e a q u a r i a . The a n i m a l s were used w i t h i n two weeks of c a p t u r e and were not f e d d u r i n g t h i s p e r i o d . H i s t o c h e m i s t r y . F i s h were k i l l e d by d e c a p i t a t i o n , a f t e r which b l o c k s of a x i a l muscle were q u i c k l y removed. The t i s s u e was mounted on s t e e l chucks and r a p i d l y f r o z e n u s i n g i s o p e n t a n e c o o l e d t o i t s f r e e z i n g p o i n t w i t h l i q u i d n i t r o g e n . Muscle s e c t i o n s 7-1OMm and 16/im t h i c k were then c u t a t -20 °C u s i n g a S l e e c r y o s t a t . S e c t i o n s were n o r m a l l y . u s e d f o r h i s t o c h e m i s t r y w i t h i n 24 hours of c u t t i n g , a l t h o u g h s e c t i o n s used a f t e r t h r e e weeks s t i l l showed enzyme a c t i v i t y . S e c t i o n s 7-10/im t h i c k were s t a i n e d f o r m y o f i b r i l l a r a denosine t r i p h o s p h a t a s e (ATPase) u s i n g a m o d i f i e d v e r s i o n of the method of Guth and Samaha (1970) as d e s c r i b e d by Davison and G o l d s p i n k (1977). F r e s h u n f i x e d s e c t i o n s were p r e i n c u b a t e d f o r v a r i o u s times a t pH 10.3 f o l l o w e d by i n c u b a t i o n a t pH 9.4 w i t h ATP as s u b s t r a t e f o r 20 min.. F u r t h e r i n c u b a t i o n i n c o b a l t c h l o r i d e f o l l o w e d by y e l l o w ammonium s u l p h i d e r e s u l t e d i n a brown t o b l a c k p r e c i p i t a t e of c o b a l t s u l p h i d e a t s i t e s of enzyme a c t i v i t y . U s i n g t h i s method, p i n k f i b r e s s t a i n d a r k e r than w h i t e f i b r e s . Red f i b r e s do not s t a i n a t a l l due t o the i n s t a b i l i t y of the red-muscle enzyme a t h i g h pH. 51 The a c t i v i t y of s u c c i n i c dehydrogenase (SDH) was demonstrated u s i n g the n i t r o - b l u e t e t r a z o l i u m method of N a c h l a s et a l . (1957), w h i l e t h a t of l a c t a t e dehydrogenase (LDH) was shown by the method of N a c h l a s e t a l . (1958). L i p i d d e p o s i t s were l o c a l i z e d u s i n g Sudan B l a c k B and g l y c o g e n u s i n g the p e r i o d i c a c i d S c h i f f ' s t e c h n i q u e . S e c t i o n s f o r the s e f o u r s t a i n s were 16jum t h i c k . S e c t i o n s were c u t -(7-10) jum from b l o c k s of muscle from the t e l e o s t B r a c h y d a n i o r e r i o ( z e b r a f i s h ) . These were i n c l u d e d i n a l l s t a i n i n g r e a c t i o n s as " c o n t r o l s " t o ensure t h a t the s t a i n i n g p r o c e d u r e s were f u n c t i o n i n g . S t a i n e d s e c t i o n s were used t o i d e n t i f y f i b r e t y p e s . Once i d e n t i f i e d , d i a m e t e r s of the f i b r e s were measured u s i n g an o c c u l a r e y e p i e c e on a l i g h t m i c r o s c o p e . F i f t y f i b r e s per f i b r e type were counted from both t h e a n t e r i o r and p o s t e r i o r r e g i o n s of f i v e f i s h . E l e c t r o n m i c r o s c o p y . Two methods of i n i t i a l f i x a t i o n were used. The f i r s t method used a 3% s o l u t i o n of g l u t a r a l d e h y d e i n 50 mM phosphate b u f f e r w i t h 0.5% s u c r o s e a t pH 7.5. A f t e r removing the s k i n o v e r l y i n g the muscles i n the i n t a c t a n i m a l the f i x a t i v e was d r i p p e d onto the t i s s u e f o r s e v e r a l m i n u t e s . S m a l l p i e c e s of t i s s u e were then removed and f i x e d f o r a f u r t h e r 4 hours i n the same f i x a t i v e . The second method i n v o l v e d p e r f u s i n g the f i s h w i t h 3% g l u t a r a l d e h y d e i n phosphate b u f f e r . An i n c i s i o n was made i n the a n a e s t h e t i z e d a n i m a l t o expose the h e a r t and vena c a v a . An o c c l u s i v e c a n n u l a was i n s e r t e d i n t o the vena cava and the b l o o d was f l u s h e d out of the v a s c u l a r system by 52 p e r f u s i n g w i t h i s o t o n i c r i n g e r s o l u t i o n . T h i s was f o l l o w e d by p e r f u s i n g w i t h f i x a t i v e f o r 15 m i n u t e s . S m a l l p i e c e s of muscle were then removed and f i x e d f o r a f u r t h e r 4 h o u r s . A l l t i s s u e s were p o s t f i x e d i n 2% osmium t e t r o x i d e and then embedded i n Epon. S e c t i o n i n g was c a r r i e d out on a P o r t e r - B l u m MT-1 u l t r a m i c r o t o m e . The s e c t i o n s were mounted on g r i d s and s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e . G r i d s were viewed u s i n g a Z e i s s EM 10 e l e c t r o n m i c r o s c o p e . C a p i l l a r y d e n s i t y was d e t e r m i n e d by s c a n n i n g the s e c t i o n s on the g r i d s and a l s o by u s i n g t h i n s e c t i o n s mounted on microscope s l i d e s (Mosse, 1979). M i t o c h o n d r i a l d e n s i t y was det e r m i n e d by t a k i n g p h o t o e l e c t r o n m i c r o g r a p h s of the muscle f i b r e s . The m i c r o g r a p h s were then weighed b e f o r e and a f t e r the m i t o c h o n d r i a were c u t out i n o r d e r t o f i n d the f r a c t i o n of the c e l l composed of m i t o c h o n d r i a . 53 R e s u l t s G r o s s d i s s e c t i o n of t h e l u n g f i s h r e v e a l e d a m u s c l e s t r u c t u r e of t h e t y p i c a l " f i s h " t y p e ( F i g . 5 ) . The myotomes were composed m a i n l y o f w h i t e m u s c l e s e p a r a t e d i n t o e p a x i a l and h y p a x i a l r e g i o n s by a myoseptum r u n n i n g t r a n s v e r s e l y f r o m th e v e r t e b r a t o the p e r i p h e r y of t h e f i s h a t t h e r e g i o n of t h e l a t e r a l l i n e . The s m a l l amount of m u s c l e termed r e d , due t o i t s c o l o u r on g r o s s d i s s e c t i o n , was f o u n d as a wedge of t i s s u e a t t h e r e g i o n o f t h e l a t e r a l l i n e . From t h i s wedge, a t h i n band of f i b r e s c o u l d be seen on e a c h s i d e o f t h e t r a n s v e r s e myoseptum r u n n i n g t o w a r d s the v e r t e b r a . F u r t h e r t o t h i s , a l a y e r o f r e d m u s c l e , a few c e l l s t h i c k , c o u l d be f o u n d i m m e d i a t e l y below t h e s k i n . In t h e a n t e r i o r r e g i o n of t h e body, t h e r e d m u s c l e made up a v e r y s m a l l f r a c t i o n of t h e m u s c l e mass ( c a . 3 % ) . T h i s p e r c e n t a g e was f o u n d t o i n c r e a s e g r a d u a l l y (up t o 20%) between t h e p o s t e r i o r r e g i o n of t h e body c a v i t y and t h e t i p of t h e t a i l . A l t h o u g h t h i s number a p p e a r s t o be l a r g e , t h e t o t a l mass of r e d m u s c l e i s s t i l l s m a l l b e c a u s e t h e d i a m e t e r of t h e t a i l a t t h e t i p i s s m a l l r e l a t i v e t o t h e r e m a i n d e r o f t h e body. The i n c r e a s e was l o c a t e d m a i n l y i n t h e wedge of t i s s u e a t t h e l a t e r a l l i n e . The amount of c o n n e c t i v e t i s s u e a l s o i n c r e a s e d t o w a r d s t h e t a i l . W h i t e f i b r e s c o u l d be i d e n t i f i e d u s i n g t h e ATPase s t a i n , as t h e y s t a i n e d l e s s i n t e n s e l y t h a n t h e i n t e r m e d i a t e f i b e r s a t l o n g p r e i n c u b a t i o n t i m e s . In a d d i t i o n t h e y f a i l e d t o s t a i n f o r SDH and LDH. The f i b r e s d i d not s t a i n f o r l i p i d , a l t h o u g h t h e y d i d 54 F i g u r e 5. L u n g f i s h c r o s s - s e c t i o n s . Note the l i n e of r e d -c o l o r e d muscle i n the a n t e r i o r c r o s s - s e c t i o n ( r i g h t ) and the t h i c k e r wedge i n t h e p o s t e r i o r c r o s s - s e c t i o n ( l e f t ) . Bar = 2cm 55 56 show a s l i g h t r e a c t i o n f o r g l y c o g e n . The v a s t m a j o r i t y of the myotome was composed of pure w h i t e muscle f i b r e s w i t h no m i x i n g w i t h the o t h e r types ( F i g s . 6-10). Red muscle f i b r e s c o u l d be i d e n t i f i e d by t h e i r s m a l l c r o s s -s e c t i o n a l a r e a , by f a i l u r e t o s t a i n f o r ATPase a t h i g h pH ( F i g . 8 ) , and by d i s p l a y i n g the s t r o n g e s t r e a c t i o n s f o r SDH ( F i g s . 9,10), LDH ( F i g s . 6,7), g l y c o g e n and l i p i d . I n t e r m e d i a t e f i b r e s were g e n e r a l l y l a r g e r than the red and showed s t a i n i n g i n t e n s i t i e s t h a t were i n t e r m e d i a t e between r e d and w h i t e f o r a l l of the s t a i n s used except t h a t f o r ATPase ( F i g s . 6-10). With t h i s l a t t e r r e a c t i o n , the i n t e r m e d i a t e f i b r e s were found t o be the most s t a b l e and, t h e r e f o r e , the most h e a v i l y s t a i n e d a t l o n g p r e i n c u b a t i o n t i m e s . I t s h o u l d perhaps be noted here t h a t s e c t i o n s f o r the ATPase r e a c t i o n were t h i n (7-10/im) w h i l e s e c t i o n s f o r the o t h e r s t a i n s were t h i c k (16MII0. I t was n e c e s s a r y t o use t h i c k s e c t i o n s because of the v e r y f a i n t s t a i n i n g r e a c t i o n s o b t a i n e d w i t h t h i n s e c t i o n s . T h i s need t o use t h i c k e r s e c t i o n s i n i t s e l f i n d i c a t e d t h a t t h e r e was a p a u c i t y of m i t o c h o n d r i a i n the c e l l s , i n c l u d i n g the r e d m u s c l e s , and a l s o t h a t t h e r e were low s t o r e s of g l y c o g e n and l i p i d . Very d i f f e r e n t r e s u l t s were o b t a i n e d w i t h the " c o n t r o l " s e c t i o n s from the zebra f i s h . W h i l e s t a i n i n g was g e n e r a l l y d i f f i c u l t w i t h the l u n g f i s h m a t e r i a l , the z e b r a f i s h s e c t i o n s s t a i n e d v e r y w e l l ( F i g . 11). The z e b r a f i s h showed the t y p i c a l t e l e o s t d i v i s i o n of f i b r e s i n t o r e d , i n t e r m e d i a t e and w h i t e . M i t o c h o n d r i a were v e r y e v i d e n t i n r e d and i n t e r m e d i a t e f i b r e s w i t h a marked subsarcolemmal a c c u m u l a t i o n . 57 F i g u r e 6. A n t e r i o r l a t e r a l l i n e r e g i o n s t a i n e d f o r LDH a c t i v i t y . Most of the f i b r e s a re " w h i t e " w i t h a t h i n s t r i p of r e d and i n t e r m e d i a t e f i b r e s f o r m i n g a p e r i p h e r a l mosaic. Bar = 1.0mm F i g u r e 7. Mosaic r e g i o n of the a n t e r i o r l a t e r a l l i n e s t a i n e d f o r LDH a c t i v i t y . Bar = 0.5mm F i g u r e 8. Mosaic r e g i o n of the a n t e r i o r l a t e r a l l i n e s t a i n e d f o r ATPase a c t i v i t y . The d a r k e s t f i b r e s a re i n t e r m e d i a t e f i b r e s , m o d e r a t e l y s t a i n e d f i b r e s a r e w h i t e f i b r e s , and u n s t a i n e d f i b r e s are r e d f i b r e s . Bar = 0. 5mm 59 F i g u r e 9. P o s t e r i o r l a t e r a l - l i n e r e g i o n s t a i n e d f o r SDH a c t i v i t y . The a r e a marked 'u' i s s i m i l a r i n o r g a n i s a t i o n t o the a n t e r i o r mosaic r e g i o n a l b e i t somewhat more e x t e n s i v e . In a d d i t i o n t h e r e i s a second mosaic r e g i o n marked 'x', c o n s i s t i n g m a i n l y of i n t e r m e d i a t e and w h i t e f i b r e s . Bar = 2.0 F i g u r e 10. P o s t e r i o r mosaic r e g i o n s t a i n e d f o r SDH a c t i v i t y , (marked 'x' i n F i g u r e 5) Bar = 0.1mm F i g u r e 11. S e c t i o n from z e b r a f i s h s t a i n e d f o r LDH a c t i v i t y . The s t a i n c o n c e n t r a t e d on the c e l l p e r i p h e r y i n d i c a t i o n s u b s a r c o l e m m a l - c o n c e n t r a t i o n s of m i t o c h o n d r i a . Note t h a t c o n n e c t i v e t i s s u e s e p a r a t e s the f i b r e t y p e s (R = red f i b r e , I = i n t e r m e d i a t e f i b r e , W = w h i t e f i b r e ) . Bar = 1.0mm 60 61 In the l u n g f i s h , r e d , i n t e r m e d i a t e , and w h i t e f i b r e s were found i n the r e g i o n s termed red d u r i n g g r o s s d i s s e c t i o n . Here they were seen as a mosaic of a l l t h r e e f i b r e t y p e s w i t h i n t e r m e d i a t e making up the m a j o r i t y ( F i g s . 6,9). A r e d and i n t e r m e d i a t e f i b r e mosaic at the extreme p e r i p h e r y b l e n d s i n t o a w h i t e and i n t e r m e d i a t e f i b r e mosaic at a deeper l e v e l . In the a n t e r i o r r e g i o n s of the body, the h i s t o c h e m i c a l work complimented the f i n d i n g s of the g r o s s d i s s e c t i o n . Most of the f i b r e s were w h i t e w i t h o n l y a s m a l l f r a c t i o n of r e d and i n t e r m e d i a t e . In the p o s t e r i o r r e g i o n next t o the t h r e e - f i b r e mosaic a r e a was a f u r t h e r mosaic area composed of w h i t e and i n t e r m e d i a t e f i b r e s ( F i g s . 9,10). E l e c t r o n m i c r o s c o p y r e v e a l e d t h a t the muscle f i b r e s were t y p i c a l i n s t r u c t u r e w i t h each c e l l c o n t a i n i n g c o n t r a c t i l e m a t e r i a l a r r a n g e d i n t o sarcomeres ( F i g s . 12,13). A s a r c o t u b u l a r system was p r e s e n t w i t h the t r i a d s s i t u a t e d a t the Z - l i n e . M y o f i b r i l s were r o u g l y c y l i n d r i c a l i n c r o s s - s e c t i o n w i t h no f o r m a t i o n of the l o n g r i b b o n s g e n e r a l l y found i n t e l e o s t f i s h e s ( K i l a r s k i , 1967; P a t t e r s o n and G o l d s p i n k , 1972). The m i c r o g r a p h s r e v e a l e d t h a t the l u n g f i s h muscle was not d e s i g n e d f o r a c t i v e a e r o b i c r e s p i r a t i o n . T h i s a p p l i e d t o a l l t h r e e f i b r e t y p e s . B l o o d c a p i l l a r i e s were v e r y s c a r c e (Table 6 ) , even around red f i b r e s . T h i s was demonstrated v e r y w e l l i n samples t h a t were f i x e d by p e r f u s i o n . S e c t i o n s o b t a i n e d from t h i s m a t e r i a l showed v e r y marked post-mortem d e g e n e r a t i v e changes, i n d i c a t i n g t h a t the f i x a t i v e had not reached the t i s s u e s i n q u a n t i t i e s g r e a t enough t o be e f f e c t i v e , even a f t e r 15 m i n u t e s . 62 F i g u r e 12. L a t e r a l s e c t i o n of w h i t e muscle m y o f i b r i l s . Note the s h a r p Z - l i n e , l a c k of m i t o c h o n d r i a , l i p i d , and g l y c o g e n , and the t r i a d s a t the Z - l i n e . Bar = 2.0Mm F i g u r e 1 3 . L a t e r a l s e c t i o n of r e d muscle m y o f i b r i l s . Note the l a c k of m i t o c h o n d r i a . Bar = I.OMITI F i g u r e 1 4 . T r a n s v e r s e s e c t i o n of r e d muscle. Note the i r r e g u l a r shape of the m y o f i b r i l s . Bar = l.tDjum 63 64 The s m a l l p i e c e s of t i s s u e which were removed a f t e r t h i s time p e r i o d were s t i l l t h e i r " n a t u r a l " c o l o u r and o n l y took on a y e l l o w i s h c o l o u r , i n d i c a t i n g g l u t a r a l d e h y d e f i x a t i o n , a f t e r immersion i n f i x a t i v e . In c o n j u n c t i o n w i t h t h i s p a u c i t y of c a p i l l a r i e s , and a l s o s u p p o r t i n g the e v i d e n c e of the h i s t o c h e m i s t r y , were the v e r y low numbers of m i t o c h o n d r i a i n a l l f i b r e t y p e s ( T a b l e 6 ) . Numbers were v e r y low i n r e d muscle, fewer i n i n t e r m e d i a t e and v e r y d i f f i c u l t t o f i n d i n w h i t e . Those m i t o c h o n d r i a which were observed were g e n e r a l l y s m a l l and were not c o n c e n t r a t e d i n a subsarcolemmal band. Sarcomere l e n g t h s averaged 1.5Mirt f o r r e d muscle and 2.0MITI f o r w h i t e muscle. 65 Table 5. Diameter of l u n g f i s h muscle f i b r e s (urn) Red Intermediate White N 4.00 400 400 x 23.6 3 4 . 3 67.4 SD . 6 . 4 9.7 25.0 T a b l e 6. A Comparison of the m i t o c h o n d r i a l d e n s i t y and v a s c u l a r i z a t i o n of v a r i o u s muscie f i b r e s . M i t o c h o n d r i a l d e n s i t y (%area of c r o s s - s e c t i o n ) Red I n t e r - White mediate R e f e r e n c e S k i p j a c k tuna (Euthynnus p e l a m i s ) E e l ( A n q u i l l a r o s t r a t a ) Shark (Etmopterus s p i n a x ) Shark (Galeus melastomus) C o a l f i s h (Gadus v i r e n s ) C r u c i a n c a r p ( C a r a s s i u s c a r a s s i u s ) Lungf i s h 35 2 23 0.1 30 7.2 0.5 34 16.3 25 1.1 16.2 0.7 5.3 0.2 0.2 H u l b e r t e t a l . (1979T H u l b e r t and Moon (1978) K r y v i (1977) 0.9 K r y v i (1977) P a t t e r s o n and G o l d s p i n k (1972) P a t t e r s o n and G o l d s p i n k (1972) T h i s study C a p i l l a r i e s per f i b r e R e f e r e n c e S k i p j a c k tuna (Ketsuwonus p e l a m i s ) Red 4-12 P i l c h a r d 3.9 ( S a r d i n o p s n e o p i l c h a r d u s ) M a c k e r e l 2.98 (Scomber a u s t r a l a s i c u s ) A u s t r a l i a n salmon ( A r r i p i s t r u t t a ) 4.2 Y e l l o w t a i l scad 3.1 (Tr a c h u r u s m c c u l l o c h i ) L u n g f i s h 0.14 White 1 .0 H u l b e r t e t a l . (19797-1.6 Mosse (1979) 1.1 Mosse (1979) 0.2 Mosse (1979) 1.53 Mosse (1979) 0.02 T h i s study 67 D i s c u s s i o n The r e s u l t s i n t h i s s e c t i o n are a l l r e l a t e d t o f i b r e t y p e s . A s h o r t d e s c r i p t i o n of the f u n c t i o n and l o c a t i o n of f i s h muscle f i b r e s w i l l be u s e f u l t o put the d a t a i n p e r s p e c t i v e . F i g u r e 11 shows the t y p i c a l s t r u c t u r e of the swimming muscle of a t e l e o s t . Much of the muscle i s w h i t e and i s geared f o r a n a e r o b i c p r o d u c t i o n of energy d u r i n g swimming at h i g h speeds (J o h n s t o n et a l . , 1977). The r e s t i n g m e t a b o l i c f a t e of a f i s h w h i t e muscle i s e s t i m a t e d t o be r o u g h l y 1/5 t h a t of r e d muscle (Gordon, 1968). The v a s c u l a r i z a t i o n of t h i s muscle i s poor ( T a b l e 6 ) , and the muscle can o n l y f u n c t i o n f o r a s h o r t time b e f o r e f a t i g u i n g ( B l a c k et_ a l . , 1962). Recovery of white muscle a f t e r f a t i g u e i s slow, r e f l e c t i n g the poor b l o o d supply and the low numbers of m i t o c h o n d r i a ( B l a c k et a l . , 1962;Johnston and G o l d s p i n k , 1973). Red muscle o c c u r s as a s m a l l wedge of c e l l s a t the p e r i p h e r y of the myotome a t the r e g i o n of the l a t e r a l l i n e . I t i s n o r m a l l y s e p a r a t e d from the o t h e r muscle t y p e s by c o n n e c t i v e t i s s u e . T h i s f i b r e type has a g r e a t e r b l o o d s u p p l y and more m i t o c h o n d r i a than do w h i t e f i b r e s , i n d i c a t i n g t h a t i t i s r e l a t i v e l y more r e l i a n t on a e r o b i c r e s p i r a t i o n . S t u d i e s have shown t h a t t h i s i s the major f i b r e type i n v o l v e d i n p r o p e l l i n g the f i s h a t normal c r u i s i n g speeds (Hudson, 1973;Johnston e t a l . , 1977). The . t h i r d t y p e , termed p i n k or i n t e r m e d i a t e , i s l o c a t e d between red and w h i t e muscle and appears t o have a f u n c t i o n i n t e r m e d i a t e between these two, w o r k i n g a t speeds 68 s l i g h t l y above the maximum f o r r e d muscle (Johnston e t a l . , 1977). D i s t r i b u t i o n of the muscle type v a r i e s between f i s h and i s p o s s i b l y r e l a t e d t o the mode of l i f e of the f i s h (Boddeke et a l . , 1959;Johnston et a l . , 1974). There i s u s u a l l y some degree of m i x i n g of i n t e r m e d i a t e and w h i t e f i b r e s . These t h r e e muscle t y p e s a r r a n g e d w i t h r e d f i b r e s c o v e r i n g the p e r i p h e r y , w h i t e f i b r e s massed i n t e r i o r l y and i n t e r m e d i a t e f i b r e s sandwiched b e t w e e n — m i x e d s l i g h t l y w i t h w h i t e f i b r e s - - i s the g e n e r a l p a t t e r n f o r a l a r g e group of a q u a t i c a n i m a l s . I t i s found i n t e l e o s t s , elasmobranchs and c e p h a l o c h o r d a t e s such as amphioxus ( F l o o d , 1968). Amphibia which p r o p e l t h e m s e l v e s t h r o u g h the water u s i n g a x i a l muscle have r e t a i n e d the t h r e e -f i b r e system, but the morphology i s somewhat d i f f e r e n t . There i s no septum s e p a r a t i n g the red f i b r e s from the r e s t and a l l t h r e e t y p e s i n t e r m i n g l e t o form a mosaic p a t t e r n of f i b r e s . T h i s mosaic i s found a t the p e r i p h e r y of the myotome. White muscle, l o c a t e d under t h i s p e r i p h e r a l mosaic l a y e r , s t i l l forms the m a j o r i t y of the muscle ( F l o o d , 1968;Totland, 1976). The A f r i c a n l u n g f i s h muscle has a s t r u c t u r e s i m i l a r t o t h a t of the a m p h i b i a , w i t h a mosaic p a t t e r n l o c a t e d around the p e r i p h e r y . T h i s d e v i a t i o n from the " f i s h " morphology i s l i k e l y due t o the t r e n d towards l a n d l i v i n g and not due t o i n c r e a s e d h y p o x i a t o l e r a n c e s i n c e Arapaima g i q a s has some s i m i l a r e c o l o g i c a l h a b i t s and does have the f i s h type d e f i n i t i o n of the r e d muscle (see b e l o w ) . The l o s s of a d i s c r e t e r e d muscle band may have accompanied the t r e n d f o r the e a r l y D i p n o i t o r e l y l e s s and l e s s on the t a i l f o r much of the r o u t i n e movement, r e l y i n g 69 i n s t e a d on the newly e v o l v e d l i m b s . However, the r e d u c t i o n i n mass of a h i g h l y o x i d a t i v e t i s s u e must be of b e n e f i t d u r i n g any exposure t o h y p o x i a . In the l u n g f i s h , b o t h the red and w h i t e muscle t y p e s appear r e l a t i v e l y a n a e r o b i c as i n d i c a t e d by low v a s c u l a r i t y , low m i t o c h o n d r i a l c o n t e n t and no o b s e r v a b l e i n t r a c e l l u l a r l i p i d . T h i s i s w e l l i l l u s t r a t e d i n T a b l e 6 which compares the m i t o c h o n d r i a l c o n t e n t and c a p i l l a r y d e n s i t y of l u n g f i s h w i t h s e v e r a l t e l e o s t s p e c i e s . The r e d muscle of l u n g f i s h i s l e s s v a s c u l a r i z e d than the w h i t e muscle of o t h e r f i s h w h i l e the b l o o d s u p p l y t o l u n g f i s h w h i t e muscle i s even l e s s w e l l d e v e l o p e d . There i s o n l y enough g l y c o g e n i n w h i t e muscle t o s u p p l y s h o r t a n a e r o b i c b u r s t s and t h e r e appears t o be l i t t l e p o t e n t i a l f o r a e r o b i c r e c o v e r y . White muscle t h e r e f o r e , seems t o be d e s i g n e d f o r b oth a low r e s t i n g m e t a b o l i c r a t e and f o r a n a e r o b i c f u n c t i o n — m u c h more so than o t h e r f i s h w h i t e muscle. The l a c k of a d i s c r e t e r e d muscle band i n the l u n g f i s h and the predominance of pure w h i t e f i b r e a r e a s would s e r v e t o make the m e t a b o l i c r a t e of t h i s t i s s u e r e l a t i v e l y low. T h i s i s because the m e t a b o l i c r a t e of w h i t e f i b r e s may be 1/5 t h a t of red f i b r e s and, on the b a s i s of enzyme measurements, the c a p a c i t y f o r c o n t i n u o u s a e r o b i c and/or a n a e r o b i c ATP p r o d u c t i o n i n the w h i t e muscle i s r e l a t i v e l y low even f o r w h i t e muscle ( S e c t i o n 1 ) . D u r i n g a b u r s t of work the m e t a b o l i c r a t e may s t i l l be h i g h s i n c e i t can be powered by ATP p r o d u c t i o n v i a the c r e a t i n e phosphokinase r e a c t i o n . T h i s d e s i g n would be of use i f the a n i m a l had a l i m i t e d r e s o u r c e of oxygen which must s e r v e a l l 70 the t i s s u e s . The South American a i r - b r e a t h i n g f i s h Arapaima g i q a s has s i m i l a r h a b i t s t o P. a e t h i o p i c u s , and some u s e f u l c o m p a r a t i v e d a t a a re a v a i l a b l e (Hochachka et a l . , 1978). As w i t h the A f r i c a n l u n g f i s h , t h i s f i s h i s an o b l i g a t e a i r - b r e a t h e r , and i s b e l i e v e d t o be r e l a t i v e l y i n a c t i v e d u r i n g most of the submerged p e r i o d . Both of the muscle t y p e s s t u d i e d , r e d and w h i t e , appear r e l a t i v e l y a n a e r o b i c on the b a s i s of f i n e s t r u c t u r e and enzyme p r o f i l e s . Thus, t h i s f i s h a l s o has a d j u s t m e n t s t h a t would promote s u r v i v a l when oxygen was l i m i t i n g . The r e s u l t s of t h i s s e c t i o n i n d i c a t e t h a t the muscle i s com p r i s e d p r e d o m i n a n t l y of w h i t e f i b r e s and so would have a r e l a t i v e l y low m e t a b o l i c r a t e . The s t r a t e g y of h a v i n g the b u l k of the myotome adapted t o u t i l i z e a v e r y s m a l l f l u x of oxygen i s l i k e l y t o be of g r e a t b e n e f i t t o the animal when oxygen s u p p l y i s low. The predominance of t h i s type of f i b r e may a l s o have a b e a r i n g upon the r e s u l t s of S e c t i o n 1. In S e c t i o n 1, i t was proposed t h a t the c a p a c i t y of the muscle mass t o r e a c t t o c e l l u l a r h y p o x i a w i t h o u t a c t i v a t i n g g l y c o l y s i s was b e n e f i c i a l t o the s u r v i v a l of the l u n g f i s h d u r i n g h y p o x i a . S e c t i o n 2 i n d i c a t e s t h a t the b u l k of the myotome i s co m p r i s e d of w h i t e f i b r e s . S i n c e t h i s c e l l type predominates i n the a x i a l muscle, the i m p l i c a t i o n i s t h a t i t i s t h i s c e l l type which has the c a p a c i t y f o r the obser v e d p a t t e r n of m e t a b o l i c c o n t r o l . 71 SECTION 3 The m e t a b o l i c a d j u s t m e n t s t o acute e n v i r o n m e n t a l h y p o x i a i n the Rainbow t r o u t I n t r o d u c t i o n The p r e v i o u s s e c t i o n s d e s c r i b e d m e t a b o l i c responses of the A f r i c a n l u n g f i s h t o h y p o x i a . These a n i m a l s a r e a b l e t o s u r v i v e submergence f o r hours even though a r t e r i a l oxygen t e n s i o n s f a l l t o l e s s than 5 t o r r (Johansen e t a l . , l 9 6 8 ; L a h i r i e t a l . , 1970). Under these c o n d i t i o n s , i t was shown t h a t the h e a r t and b r a i n were a b l e t o a c t i v a t e g l y c o l y s i s i n o r d e r t o augment d e c l i n i n g r a t e s of a e r o b i c ATP s y n t h e s i s but t h a t w h i t e muscle d i d not f o l l o w t h i s p a t t e r n . The l a c k of an a c t i v a t i o n of g l y c o l y s i s i n the w h i t e muscle i s l i k e l y t o be the most s i g n i f i c a n t a d a p t a t i o n t o h y p o x i a i n the l u n g f i s h . T h i s s t r a t e g y s p a r e s s u b s t r a t e s ( i n c l u d i n g oxygen) f o r o t h e r t i s s u e s and i t reduces the r a t e of t o x i c end-product p r o d u c t i o n . One would not expect t h a t a h y p o x i a s e n s i t i v e organism would show the same i n t e r - t i s s u e m e t a b o l i c i n t e r a c t i o n s t h a t were e x e m p l i f i e d by the l u n g f i s h . F u r t h e r m o r e , one would expect t h a t muscle m e t a b o l i s m would respond t o h y p o x i a w i t h an a c t i v a t i o n of g l y c o l y t i c ATP p r o d u c t i o n . The e x p e r i m e n t a l a n i m a l used t o examine these hypotheses was the rainbow t r o u t (Salmo g a i r d n e r i ) . Of f i s h e s , the salmonids a r e among the most s e n s i t i v e t o 72 oxygen d e f i c i e n c y ( Doudoroff and Shumway, 1970). As ambient oxygen t e n s i o n s d e c l i n e t o below 40 t o r r , the r a t e of oxygen uptake i n t r o u t may remain c o n s t a n t ( H o l e t o n and R a n d a l l , 1967) or they may i n c r e a s e (Hughes and Saunders, l970;McKim and Goeden, 1982). However, t h i s may not mean t h a t the t i s s u e s have a c o n s t a n t s u p p l y of oxygen. As oxygen t e n s i o n s d e c l i n e , the volume of water pumped a c r o s s the g i l l s i n c r e a s e s c o n c u r r e n t l y w i t h a d e c l i n e i n the e f f i c i e n c y of oxygen e x t r a c t i o n ( H o l e t o n and R a n d a l l , l967;Hughes and S a u n d e r s 1 - 9 7 0 ; J o n e s , 1971;McKim and Goeden, 1982). The e n e r g e t i c r e q u i r e m e n t s of the pumping mechanism r i s e w i t h t h e pumping r a t e and may re a c h 50% of the t o t a l r a t e of oxygen uptake when oxygen t e n s i o n s f a l l (McKim and Goeden, 1982). Thus, even though the t o t a l r a t e of oxygen uptake by the t r o u t remains f a i r l y c o n s t a n t , the a c t u a l s u p p l y t o most of the t i s s u e s i s l i k e l y t o be reduced. The m e t a b o l i c r e s p o n s e s of the t r o u t t o h y p o x i a are not w e l l d e s c r i b e d . I t i s c e r t a i n t h a t muscle and l i v e r l a c t a t e c o n c e n t r a t i o n s r i s e ( B u r t o n and Spehar, 1971). However, the a c t u a l r e sponses of each t i s s u e and the m e t a b o l i c i n t e r a c t i o n s which o c c u r between the t i s s u e s a r e not u n d e r s t o o d . T h i s s e c t i o n examines the organ by organ r e s p o n s e s t o c e l l u l a r d y s o x i a i n the t r o u t by s u b j e c t i n g the a n i m a l t o reduced e n v i r o n m e n t a l oxygen t e n s i o n s and then m o n i t o r i n g t i s s u e -s p e c i f i c m e t a b o l i t e c o n c e n t r a t i o n changes. From th e s e changes, an o v e r a l l p i c t u r e emerges of m e t a b o l i c o r g a n i z a t i o n i n the t r o u t d u r i n g exposure t o e n v i r o n m e n t a l h y p o x i a . 73 M a t e r i a l s and Methods E x p e r i m e n t a l A n i m a l s . Rainbow t r o u t , (Salmo q a i r d n e r i ) , were o b t a i n e d from Sun V a l l e y t r o u t farms ( M i s s i o n B.C.), and kept i n l a r g e outdoor ta n k s w i t h a f l o w through water system and a n a t u r a l p h o t o p e r i o d . They were f e d mid-day w i t h commercial t r o u t p e l l e t s u n t i l two days p r i o r t o the ex p e r i m e n t . The f i s h had a mean s i z e of 320 gm. The e x p e r i m e n t s were c a r r i e d out d u r i n g the e a r l y s p r i n g when the water temperature was 4°C. Exper i m e n t a l P r o c e d u r e . The f i s h were p l a c e d i n a b l a c k p l e x i g l a s s h o l d i n g box one day b e f o r e e x p e r i m e n t a t i o n . The box had 6 s l a t t e d compartments, each of which h e l d one f i s h . Oxygen t e n s i o n s were a d j u s t e d by b u b b l i n g N 2 i n t o a c e n t r a l m i x i n g chamber a t the f r o n t of the box. Water flo w e d from t h i s chamber d i r e c t l y i n t o each of the f i s h compartments. Oxygen t e n s i o n was m o n i t o r e d w i t h a Radiometer oxygen e l e c t r o d e c o n n e c t e d s e r i a l l y t o an a m p l i f i e r and a c h a r t r e c o r d e r . Water and gas f l o w s were a d j u s t e d t o o b t a i n a r e p r o d u c i b l e d e c l i n e i n 0 2 t e n s i o n i n the box. The oxygen t e n s i o n d e c l i n e d g r a d u a l l y t o a t e n s i o n of 20 t o r r a f t e r 20 min. and was m a i n t a i n e d t h e r e f o r the remainder of the 3 hour h y p o x i c e x p o s u r e . P r e l i m i n a r y t e s t s were performed v a r y i n g time of i n i t i a t i o n , the d u r a t i o n of exposure, the a v a i l a b i l i t y of s u r f a c e a c c e s s , and the l e v e l of oxygen t e n s i o n . T h i s was done t o de t e r m i n e a proce d u r e f o r h y p o x i c exposure which would r e p r o d u c i b l y r e s u l t i n an i n c r e a s e of b l o o d 74 plasma l a c t a t e c o n c e n t r a t i o n s w i t h no f i s h m o r t a l i t y . The presence of b l o o d l a c t a t e was used i n the s e p r e l i m i n a r y e x p e r i m e n t s t o i n d i c a t e t h a t the f i s h were b e i n g s u b j e c t e d t o c e l l u l a r h y p o x i a . In the major m e t a b o l i t e experiment samples were taken from 8 c o n t r o l f i s h ( w i t h o u t a c c e s s t o the s u r f a c e but not s u b j e c t e d t o h y p o x i a ) , 4 f i s h exposed f o r 1 hour, and 7 f i s h exposed f o r 3 hour a t 20 t o r r . T iming began concommitent w i t h the i n i t i a t i o n of d e c l i n i n g oxygen t e n s i o n s . F i s h were d e n i e d a c c e s s t o the s u r f a c e by f l o a t i n g wood i n the chambers. The gas b u b b l i n g was i n i t i a t e d a t 10:30 A.M.. M e t a b o l i t e P r e p a r a t i o n and Assay. T i s s u e s were removed from beheaded f i s h and f r o z e n a t -196°C by c l a m p i n g w i t h aluminum tongs which had been c o o l e d i n l i q u i d n i t r o g e n . The u s u a l o r d e r of f r e e z i n g was b l o o d , h e a r t , b r a i n , l i v e r , r e d muscle, and whit e muscle. The h e a r t and b r a i n were f r o z e n w i t h i n 60 s and the t o t a l time f o r f r e e z i n g a l l t i s s u e s was r o u g h l y 150 s. Muscle samples were removed from two l o c a t i o n s . The a n t e r i o r samples were from the r e g i o n j u s t c a u d a l t o the g i l l s and the p o s t e r i o r samples were from the r e g i o n between the d o r s a l f i n and the c a u d a l p e d u n c l e . B l o o d was withdrawn from the t r u n k v i a c a u d a l v e s s e l puncture and p l a c e d d i r e c t l y i n t o 1 volume of i c e -c o l d 6% PCA. T i s s u e s were ground t o a f i n e powder u s i n g a mortar and p e s t l e r e s t i n g on d r y i c e and f u r t h e r c o o l e d by f l u s h i n g w i t h l i q u i d n i t r o g e n . About 0.5 gm of powder was weighed i n t o a c o l d X o r e x c e n t r i f u g e tube c o n t a i n i n g 1 ml of 75 1.4M PCA and homogenized w i t h a P o l y t r o n h o m o g e n i z e r - s o n i c a t o r . Two a l i q u o t s of 100 M1 were removed and f r o z e n a t -80°C f o r subsequent glycogen a n a l y s i s . The homogenate was spun at 10,000 x g f o r 15 min. and the p e l l e t d i s c a r d e d . The s u p e r n a t a n t was n e u t r a l i z e d t o pH 6.7 w i t h 1.4M KOH and spun a g a i n . A l l m e t a b o l i t e s e xcept g l u c o s e , g l y c o g e n , and l a c t a t e were measured w i t h i n 9 hours of n e u t r a l i z a t i o n . The or d e r i n which m e t a b o l i t e s were a s s a y e d was kept c o n s t a n t w i t h ATP and c r e a t i n e - p h o s p h a t e (CrP) b e i n g measured f i r s t . M e t a b o l i t e s were measured by e n z y m a t i c a l l y l i n k i n g them t o r e a c t i o n s u s i n g NADH/NAD*, or NADPH/NADP+, and f o l l o w i n g the r e a c t i o n a t 340 nm on a Unicam SP-1800 s p e c t r o p h o t o m e t e r . Glycogen was measured u s i n g the a m y l o g l u c o s i d a s e t e c h n i q u e ( K e p p l e r and Decker, 1970). The r e m a i n i n g m e t a b o l i t e s were measured w i t h the t e c h n i q u e s of Hochachka et a l . , (1978c). Data were compared u s i n g one-way ANOVA w i t h s i g n i f i c a n c e l e v e l of p < 0.05. 76 R e s u l t s P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t both time of day and the a v a i l a b i l i t y of s u r f a c e a c c e s s a f f e c t e d the m e t a b o l i c response t o e n v i r o n m e n t a l h y p o x i a i n the t r o u t . Plasma l a c t a t e c o n c e n t r a t i o n s i n c o n t r o l f i s h , f i s h exposed to h y p o x i a f o r 3 hours b e g i n i n g a t 6:30 A.M. and f i s h exposed f o r 3 hours b e g i n n i n g a t 10:30 A.M. were 0.68+.40 mM, 4.00+.75 mM, and 6.85+.65 mM (X+S.D., N=5) r e s p e c t i v e l y . In a s e r i e s of 10 f i s h exposed t o h y p o x i a f o r 45 min., thos e w i t h o u t a c c e s s t o the s u r f a c e had s i g n i f i c a n t l y h i g h e r plasma l a c t a t e c o n c e n t r a t i o n s than both the c o n t r o l s and the f i s h exposed t o h y p o x i a w h i l e c a p a b l e of g a i n i n g a c c e s s t o the s u r f a c e . These d a t a ' were taken i n t o account when d e s i g n i n g the r e m a i n i n g e x p e r i m e n t s . The f o l l o w i n g e x p e r i m e n t s were i n i t i a t e d a t 6:30 A.M. and were performed on t r o u t t h a t d i d not have a c c e s s t o the s u r f a c e . The m e t a b o l i t e c o n c e n t r a t i o n s f o r the b r a i n , h e a r t , l i v e r , and a n t e r i o r and p o s t e r i o r r e d and w h i t e muscles a r e l i s t e d i n T a b l e s 7 and 8. S t a t i s t i c a l comparisons were made between c o n t r o l and 3 hour samples, and a n t e r i o r and p o s t e r i o r muscle samples. V a l u e s a r e l i s t e d f o r c o n t r o l , 1 hour, and 3 hour exp o s u r e s t o h y p o x i a . There was v e r y l i t t l e change i n the c o n c e n t r a t i o n of m e t a b o l i t e s i n the b r a i n d u r i n g h y p o x i a . Glycogen c o n c e n t r a t i o n s f e l l (p = 0.06), c o n c o m i t t e n t w i t h a s i g n i f i c a n t r i s e i n l a c t a t e c o n c e n t r a t i o n s ( T a b l e 8.). The t o t a l p o o l of Table 7. Concentrations of adenylates, creatine phosphate, and creatine, and the energy charge in trout tissues. (x+SD) Exper imental Condi t ion n Brain Control 7 1-h 4 3-h 8 Heart Control 7 1-h 4 3-h 8 L i ver Control 7 1-h 4 3-h 8 Ant. R. Muscle Controi 7 1-h 4 3-h 8 Post. R\ MUscle Control 7 1-h 4 3-h 8 ATP 0.73+0.18 0.47+0.14 O.70+0.23 2.43+0.65 2.09+0.32 1.68+0.581 1.35+0.21 0.81+0.45 0.53+0.23' 2.85+0.97 1.39+0.28 2.65+0.44 3.73+0.90 2.64+0.28 3.21+0.43 ADP 0.58+0.14 0.59+0.07 0.73+0.20 0.72+0.27 0.66+0.08 0.76+0.33 0.55+0.38 0.82+0.13 0.67+0.09 0.68+0.22 0.68+0.13 0.82+0.12 0.76+0.16 0.80+0.15 0.83+0.09 AMP E.C. Total Creatine Creatine-P Total 0.39+0.20 0.61+0.11 1.70+0.25 9.68+1.37 0.98+0.53 10.66+0.98 0.35+0.24 0.15+0.03 0.41+0.04 0.16+0.06 0.20+0.13 9.26+1.90 0.98+0.53 O.26+0.11 0.63+0.09 1.69+0.39 8.55+0.93 1.03+0.46 9.57+0.81 0.17+0.14 0.84+0.07 3.33+0.60 3.87+0.78 4.63+0.54 8.51+0.82 4.14+0.56 3.28+0.75 0.13+0.08 0.80+0.08 2.57+0.65' 4.55+1.48 2.93+1.23' 7.48+1.61 0.24+0.08 0.76+0.07 2.14+0.34 1.20+0.54 0.28+0.22 1.48+0.49 0.98+0.19 < 0.01 0.28+0.06 0.58+0.09' 1.48+0.24' 1.03+0.4 1 0.02+0.02' 1.04+0.41 0.15+0.03 0.86+0.08 3.69+0.84 12.43+1.91 5.74+2.93' 18.16+3.51 11.41+3.36 1.89+0.80 0.13+0.05 0.85+0.03 3.61+0.40 13.76+4.44 3.57+1.59 17.33+4.19 0.15+0.06 0.88+0.05 4.64+0.79 11.65+1.97 9.28+2.56 20.93+4.22 13.35+1.72 5.37+1.19 O.11+0.04 0.88+0.02 4.16+0.51 12.76+2.47 4.72+2.01117.48+2.72 Table 7. cont. Experimental n Condit1on Ant. W. Muscle Control 7 1-h 4 ATP 7.42+0.71 6.57+0.33 ADP AMP E.C. 3-h 8 6.78+0.44 1.01+0.20 0.10+0.03 0.93+0.02 1.00+0.08 0.10+0.03 1.07+0.16 0.10+0.06 0.92+0.01 Post. W. Muscle Control 7 1-h 4 3-h 8 7.41+0.86 5.82+1.49 6.94+0.35 0.91+0.19 1.02+0.06 1.11+0.32 0.09+0.05 0.13+0.06 0.11+0.06 0.93+0.02 0.92+0.02 1 s i g n i f i c a n t l y d i f f e r s from normoxla (p < 0.05) ' s i g n i f i c a n t l y d i f f e r s from posterior sample (p < 0.05) Total Creatine Creatine-P Total 8.54+0.60 25.71+4.94 26.51+2.58 7.95+0.49 34.19+4.061 8.42+0.71 24.47+4.92 26.65+2.34 8.15+0.46 34.92+4.061 20.77+2.10' 46.48+5.81 18.65+3.17 13.65+4. 96' 48.55+2.02 26.94+5.20 51.41+6.27 21.01+1.57 13.63+4.96' 48.55+2.02 00 79 Table 8. S e l e c t e d t r o u t g l y c o l y t i c m e t a b o l i t e s at r e s t and d u r i n g acute hypoxia. X + S.D. (/xmoles/gm wet wt) Experimental C o n d i t i o n n Glycogen Glucose B r a i n C o n t r o l 7 1 -hr 4 3-hr 8 Heart C o n t r o l 7 1 -hr 4 3-hr 8 L i v e r C o n t r o l 7 1-hr 4 3-hr 8 Red Ant. Muscle C o n t r o l 7 1-hr 4 3-hr 8 Red Post. Muscle C o n t r o l 7 1-hr 4 3-hr 8 3.69+1.78 3.57+0.87 1.83+1.61 36.28+19.13 33.60+ 4.53 19.27+10.321 133.5+68.66 189.4+ 0.44 142.8+58.3 14.56+6.01 18.85+3.61 16.52+8.79 15.26+9.04 18.33+1.84 15.80+8.10 5.04+3.72 2.16+0.94 4.99 + 2. 14 8.82+5.02 5.19+1.36 7.53+2.48 11.9+9.04 7.1+1.55 12.2+4.19 1.71+1.32 1.08+0.21 1.65+0.33 1.60+1.05 0.81+0.23 1.55+0.39 G6P L a c t a t e 0.08+0.07 0.14+0.05 0. 13+0.05 0.24+0.07 0.24+0.04 0.28+0.17 0.15+0.14 0.88+0.33 2.12+1.23 3.37+0.85 3.91+1.211 0.71+0.23 2.68+0.70 6. 15 + 3.52 1 0.95+0.16 2.27+0.15 1.21+0.431 5.40+1.551 0.44+0.11 2 2.34+1.14 1.33+0.49 2.97+1.14 1.29+0.411 4.35+2.031 0.25+0.13 1.04+0.53 1.73+1.05 3.85+0.74 1.02+0.301 4.15+2.211 80 T a b l e 8. c o n t . White Ant. Muscle C o n t r o l 7 15.89+5.04 1-hr 4 15.15+2.31 3-hr 8 12.12+4.94 White P o s t . M u s c l e C o n t r o l 7 18.00+7.58 1 -hr 3-hr B l o o d C o n t r o l 1-hr 3-hr 4 16.49+2.93 8 12.28+4.30 7 4 8 0.74+0.47 0.35+0.06 0.72+0.22 0.70+0.48 0.44+0.15 0.76+0.24 12.51+9.45 4.54+2.60 7.30+3.07 • 0.80+0.44 1.03+0.06 1.40+0.61 0.57+0.34 0.97+0.16 6.77+4.85 5.74+1.78 10.90+3.88 5.78+3.76 6.76+2.16 1.36+0.47 1 11.39+2.92 1 0.37+0.27 2.34+0.46 6.93+1.98 1 S i g n i f i c a n t l y d i f f e r s from normoxia (p < .05). 2 s i g n i f i c a n t l y d i f f e r s from p o s t e r i o r sample (p < .05) 81 c r e a t i n e and CrP was m a i n t a i n e d , as was the energy charge (Table •7.). T h i s was the o n l y t i s s u e where the c o n c e n t r a t i o n s of l a c t a t e d i d not c o r r e l a t e w i t h the c o n c e n t r a t i o n s i n the b l o o d . The h e a r t showed s i g n i f i c a n t d e c l i n e s i n g l y c o g e n c o n c e n t r a t i o n w h i l e , [ l a c t a t e ] was s t e a d i l y r i s i n g . The c o n c e n t r a t i o n s of ATP, CrP, and the t o t a l a d e n y l a t e p o o l d e c l i n e d through the e x p e r i m e n t . The energy charge d i d not change. N e i t h e r g l y c o g e n nor g l u c o s e c o n c e n t r a t i o n s changed i n the l i v e r d u r i n g h y p o x i a , w h i l e the c o n c e n t r a t i o n s of G6P and l a c t a t e r o s e . The l e v e l s ' of ATP and CrP f e l l , as d i d the t o t a l p o o l s of c r e a t i n e + C r P and a d e n y l a t e s ( T a b l e s 7,8). The energy charge d e c l i n e d i n d i c a t i n g t h a t a m e t a b o l i c s t r e s s has o c c u r r e d . The c o n c e n t r a t i o n s of l a c t a t e and g l u c o s e i n the l i v e r c o r r e l a t e d w i t h the c o n c e n t r a t i o n s i n the b l o o d ( T a b l e 9) A l t h o u g h [G6P] and [ l a c t a t e ] r o s e i n r e d muscle, t h e r e was no change i n g l u c o s e , or g l y c o g e n . The o n l y change i n the h i g h energy phospate compounds was a f a l l i n [ C r P ] . There are a n t e r i o r - p o s t e r i o r d i f f e r e n c e s i n the r e d muscle. The c o n c e n t r a t i o n s of ATP and CrP were h i g h e r i n the t a i l r e g i o n of the c o n t r o l f i s h w h i l e [G6P] was h i g h e r i n the a n t e r i o r samples. The b l o o d and t i s s u e c o n c e n t r a t i o n s of l a c t a t e and g l u c o s e were s i g n i f i c a n t l y c o r r e l a t e d . In w h i t e muscle, the c o n c e n t r a t i o n s of c r e a t i n e , G6P, and l a c t a t e r o s e w h i l e t h a t of CrP f e l l . Here t o o , the c o n c e n t r a t i o n s of CrP were h i g h e r i n the t a i l . Glycogen c o n c e n t r a t i o n s may be d e c l i n i n g (p = .06) ( T a b l e 8 ) . T a b l e 9. C o r r e l a t i o n s between t i s s u e and b l o o d m e t a b o l i t e c o n c e n t r a t i o n s i n t r o u t . ( p r o b a b i l i t y t h a t r = 0, n = 19) T i s s u e L a c t a t e G l u c o s e B l o o d G l u c o s e vs T i s s u e Glycogen P r P r - p r B r a i n .0859 .416 <. 0001 .876 >0 . 1 .225 Heart <.0001 .895 <. 0001 .857 0 .0498 .469 L i v e r <. 0001 .973 <. 0001 .906 . >0 . 1 _ .224 Red Ant. .0003 .750 <. 0001 .838 >0 . 1 .221 Red P o s t . .0014 .693 0001 .838 < .0001 .463 White Ant. .0052 .629 <. 0001 .797 >0 . 1 .263 White P o s t . .0018 .683 0004 .742 >0 . 1 .238 T a b l e 10. G l y c o l y t i c m e t a b o l i t e s t o r e s i n t r o u t . (Mmoles/500 gm f i s h ) x + S.D. T i s s u e Glycogen G l u c o s e Glucose-6-P L a c t a t e L i v e r C 3 hr White Muscle C '3 hr Red Muscle C Others 3 hr C 3 hr 594+305 635+259 373+176 404+209 1 9+1 0 11+4.4 53 + 40 54+1 9 4025+1491 244+64 41+29 40+7.5 328+299 225+96 0.7+0.6 5.4+1.9 1 5592+2106 238+155 227+117 4.2+0.7 24+6.9 1 2071+1394 455+161 1 3677+1087 1 8.6+2.5 51+24 29+8.3 1 106+52 1 .15+.05 11+8 .•19+.05 2 1 3 + 61 1 ' s i g n i f i c a n t l y d i f f e r s from c o n t r o l (p < 0.05). T a b l e 11. The r a t i o s of b l o o d l a c t a t e t o t i s s u e l a c t a t e t r o u t . (x+SD) T i s s u e G o n t r o l 3 hour Change i n exposure mean r a t i o B r a i n .264+. 270 1 .960+. 871 + 1 .696 He a r t .551+. 384 . 1 .348+. 589 + 0 .797 L i v e r .409+. 313 1 .298+. 184 + 0 .889 Red Ant. .195+. 141 1 .823+. 692 + 1 .628 Red P o s t . .283+. 182 . 2 .055+. 908 + 1 .772 White Ant. .103+. 105 .656+. 1 34 + 0 .553 White P o s t . .099+. 081 .627+. 1 92 + 0 .528 A l l changes between c o n t r o l and 3 hr a r e s i g n i f i c a n t l y d i f f e r e n t a t p < 0.05. 85 The a v a i l a b l e s u p p l y of s u b s t r a t e s f o r g l y c o l y s i s , and the amount of l a c t a t e produced are l i s t e d Table 10.. A l t h o u g h the l i v e r was the main g l y c o g e n s t o r e i n terms of a b s o l u t e c o n c e n t r a t i o n (133 /xmoles/gm) , the w h i t e muscle as a t i s s u e c o n t a i n e d the g r e a t e s t s u p p l y of c a r b o h y d r a t e . G6P i s p r e s e n t i n l a r g e enough amounts i n the w h i t e muscle t o c o n t r i b u t e s i g n i f i c a n t l y t o the energy s u p p l y as w e l l . The t o t a l p o o l s of g l y c o g e n , g l u c o s e , and G6P are diagramed i n F i g u r e 15 t o s t r e s s the impact of the w h i t e muscle, which i s the major s t o r e , as a s u p p l y of f u e l f o r g l y c o l y s i s . J u s t as the w h i t e muscle c o n t a i n s most of the s u b s t r a t e , i t a l s o c o n t a i n s most of the e n d - p r o d u c t - - l a c t a t e . The g r e a t e s t p e r c e n t a g e changes, however, were i n the h e a r t , b r a i n , and b l o o d . 86 F i g u r e 15. T o t a l g l u c o s e , g l y c o g e n , and G6P s t o r e s i n t r o u t . The 'Others' a r e the b r a i n , h e a r t , and b l o o d . "C" i n d i c a t e s c o n t r o l v a l u e s and "3" i n d i c a t e s v a l u e s from t r o u t exposed t o h y p o x i a f o r 3 h o u r s . 87 d» 6 0 0 0 o o no \ m © E =x 4000 Ui LU DO v . — ' _ J o i -2000 White Muscle C 3 Red Muscle Liver Others C 3 C 3 C 3 88 F i g u r e 16. T o t a l l a c t a t e s t o r e s i n t r o u t . The 'Others' a r e t h e b r a i n , h e a r t , and b l o o d . "C" i n d i c a t e s c o n t r o l v a l u e s and "3" i n d i c a t e s v a l u e s from t r o u t exposed t o h y p o x i a f o r 3 h o u r s . 89 C 3 C 3 C 3 C 3 90 D i s c u s s i o n T h i s d i s c u s s i o n i s devoted t o a n a l y s i n g t h e i n t e r - t i s s u e r e s ponses of the t r o u t t o a c u t e h y p o x i a . The r e l a t i o n s h i p between a n t e r i o r and p o s t e r i o r muscle samples w i l l be d i s c u s s e d f i r s t , f o l l o w e d by an a n a l y s i s of the t i s s u e s p e c i f i c r e s p o n ses, and a d i s c u s s i o n of the i n t e r - t i s s u e m e t a b o l i c r e s p o n s e s . When n o n - a n a e s t h e t i z e d a n i m a l s a r e s u b j e c t e d t o h y p o x i a the p o s s i b i l i t y a r i s e s t h a t e x e r c i s e r e l a t e d e f f e c t s caused by s t r u g g l i n g w i l l overshadow the e f f e c t s caused by low oxygen. M u s c l e s would be the most l i k e l y t i s s u e t o e x h i b i t such a response. To h e l p c i r c u m v e n t t h i s problem, the muscles were sampled i n the a n t e r i o r and p o s t e r i o r ( c a u d a l p e d u n c l e ) r e g i o n s . The r a t i o n a l was t h a t muscle samples from the p o s t e r i o r r e g i o n s may e x h i b i t more marked c o n c e n t r a t i o n changes due t o s t r u g g l i n g than would the a n t e r i o r samples. T h i s statement i s s u p p o r t e d by a l i m i t e d number of m e t a b o l i t e and enzyme d a t a . Somero and C h i l d r e s s (1980) undertook s e r i a l measurements of LDH a c t i v i t y i n w h i t e muscle of P a r a l a b r a x c l a t h r a t u s (a w a t e r - b r e a t h e r ) , and found a s m a l l i n c r e a s e i n a c t i v i t y j u s t c a u d a l t o the d o r s a l f i n . The p r e s e n t e x p e r i m e n t s showed t h a t c o n c e n t r a t i o n s of c r e a t i n e phosphate i n the red and w h i t e muscle of t r o u t are a l s o dependent upon the l o c a t i o n i n the f i s h , w i t h t h e samples taken p o s t e r i o r t o the d o r s a l f i n b e i n g h i g h e r i n CrP c o n c e n t r a t i o n than t h o s e taken a n t e r i o r t o the d o r s a l f i n . These d a t a suggest a d i f f e r e n c e i n the m e t a b o l i c o r g a n i z a t i o n of the myotome w i t h 91 the p o s t e r i o r muscle b e i n g p o t e n t i a l l y c a p a b l e of a g r e a t e r r a t e of a n a e r o b i c ATP p r o d u c t i o n . S i n c e the t r o u t i s not p a r t i c u l a r l y t o l e r e n t t o e n v i r o n m e n t a l h y p o x i a , t h i s m e t a b o l i c p o t e n t i a l i s l i k e l y t o be used d u r i n g such e x e r c i s e as b u r s t swimming or s t r u g g l i n g . Thus, i f the p o s t e r i o r sample e x h i b i t e d a much l a r g e r c o n c e n t r a t i o n change than d i d the a n t e r i o r sample, i t may i n d i c a t e t h a t most of the change i n the p o s t e r i o r sample was due t o e x e r c i s e e f f e c t s . B r a i n . The o b s e r v a t i o n t h a t t h e r e i s no s i g n i f i c a n t change i n the c o n c e n t r a t i o n s of h i g h energy phosphate compounds i n the b r a i n i n d i c a t e t h a t t h i s t i s s u e i s s t i l l r e c e i v i n g most of i t s oxygen r e q u i r e m e n t s . T h i s i s not t o say t h a t the b r a i n i s u n a f f e c t e d by the s t r e s s . L a c t a t e c o n c e n t r a t i o n s have i n c r e a s e d and the gl y c o g e n c o n c e n t r a t i o n s may be f a l l i n g (p = .06). The n o n - s i g n i f i c a n t change i n gl y c o g e n i s r e p o r t e d because of the i n h e r e n t v a r i a t i o n i n gl y c o g e n c o n c e n t r a t i o n s . I t i s unusual t o o b t a i n s i g n i f i c a n t changes i n g l y c o g e n u n l e s s the mean has d e c l i n e d by perhaps as much as 50%. Thus, the p o s s i b i l i t y remains t h a t the gly c o g e n c o n c e n t r a t i o n s may be d e c l i n i n g . I t has been p r e v i o u s l y noted ( S e c t i o n 1) t h a t the c o n c e n t r a t i o n of s t o r e d g l y c o g e n i n a t i s s u e may c o r r e l a t e w i t h the t i s s u e ' s r e l a t i v e h y p o x i a t o l e r a n c e (Daw e t a l . , 1967; Dawes et a l . , 1959; Rerem e t a_l. , 1973). T h i s t o l e r a n c e i s b e l i e v e d t o be due to the i n c r e a s e d c a p a c i t y f o r ATP p r o d u c t i o n v i a g l y c o l y s i s . The g l y c o g e n c o n c e n t r a t i o n i n t r o u t b r a i n i s l e s s than 1/2 t h a t of l u n g f i s h , which l e a d s t o the s u g g e s t i o n t h a t the t r o u t b r a i n 92 does not have as g r e a t a c a p a c i t y f o r a n a e r o b i c ATP g e n e r a t i o n as does the l u n g f i s h b r a i n . The c o n c e n t r a t i o n s of g l y c o g e n i n the t r o u t b r a i n a re low enough t h a t they a re p r o b a b l y of l i t t l e s i g n i f i c a n c e . The f o l l o w i n g c a l c u l a t i o n i n d i c a t e s t h a t even i f a l l of the gl y c o g e n were a n a e r o b i c a l l y c o n v e r t e d t o l a c t a t e , the ATP produced would p r o b a b l y s u p p l y the b r a i n f o r o n l y 10 minutes. The b r a i n from a 500 gm f i s h r e q u i r e s about 5 ^moles 0 2 / h r . , or 30 ymoles ATP/hr. (McDougal et a l . , 1968). The c o n c e n t r a t i o n of gly c o g e n i n the b r a i n was 3.7 Mmoles/gm and so the b r a i n would c o n t a i n 1.6 Mmoles of g l y c o g e n . T h i s c o u l d produce o n l y 4.8 jumoles of ATP v i a g l y c o l y s i s . ( b r a i n and body w e i g h t s from t h i s study i n d i c a t e t h a t b r a i n c o m p r i s e s 0.087% of the body. The above p o i n t s i n d i c a t e t h a t the b r a i n i s not l i k e l y t o be a b l e t o produce a s i g n i f i c a n t p r o p o r t i o n of i t s ATP re q u i r e m e n t s from g l y c o l y s i s . At the same t i m e , the energy m e t a b o l i t e c o n c e n t r a t i o n s a re m a i n t a i n e d . One may c o n c l u d e t h a t the b r a i n metabolism i s r e m a i n i n g l a r g e l y a e r o b i c and t h a t the t i s s u e i s p r o t e c t e d from h y p o x i a by o t h e r ( c a r d i o v a s c u l a r ?) mechani sms. H e a r t . The h e a r t , as compared t o o t h e r t i s s u e s , e x h i b i t e d an i n t e r m e d i a t e response t o e n v i r o n m e n t a l h y p o x i a . A l t h o u g h t h e r e was no s i g n i f i c a n t change i n the E.C., t h e r e was a s i g n i f i c a n t d e c l i n e i n the t o t a l a d e n y l a t e p o o l and i n the c o n c e n t r a t i o n s of ATP and CrP, and g l y c o g e n . These m e t a b o l i t e changes i n d i c a t e t h a t the h e a r t metabolism was not a b l e t o m a i n t a i n c o n t r o l r a t e s 93 of ATP p r o d u c t i o n v i a a e r o b i c m e tabolism. The d e c l i n e i n endogenous g l y c o g e n c o n c e n t r a t i o n s c o u p l e d w i t h the f a l l i n energy s t a t u s of the c e l l i s e v i d e n c e t h a t g l y c o l y s i s has been a c t i v a t e d . S i n c e the source of g l u c o s y l u n i t s f o r g l y c o l y s i s i s p r o b a b l y endogenous ( l i v e r g l y c o g e n c o n c e n t r a t i o n s do not d e c l i n e ) , one may c a l c u l a t e the amount of ATP produced by a n a e r o b i c g l y c o l y s i s . U s i n g mean v a l u e s , one may c a l c u l a t e t h a t t h e r e were 17 mmoles/gm wet wt. of g l y c o g e n ( g l u c o s y l u n i t s ) m o b i l i z e d i n the h e a r t . S i n c e the h e a r t i n a 500 gm f i s h weighs .072 gm ( t h i s s t u d y ) , i t would have m o b i l i z e d 1.22 Mmoles of g l u c o s y l u n i t s . T h i s would produce 3.6 Mmoles of ATP and 2.4 Mmoles of l a c t a t e / h e a r t / 3 h . The m e t a b o l i c r a t e of a h e a r t i s r o u g h l y 17 t o 130 Mmoles ATP/.072 gm h e a r t / h r (Lochner et a l . , 1 968,'Reeves, 1963). T h e r e f o r e , the a n a e r o b i c s u p p l y of ATP c o m p r ised o n l y from 1 t o 7% of the t o t a l m e t a b o l i c r a t e of a r e s t i n g h e a r t . T h i s i s not a l a r g e amount. I f a l l of the l a c t a t e produced remained i n the h e a r t , the l a c t a t e c o n c e n t r a t i o n would have i n c r e a s e d by 33 Mmoles/gm wet wt.. S i n c e the a c t u a l i n c r e a s e was o n l y 5.4 mM, the h e a r t was l i k e l y t o be a net l a c t a t e e x p o r t e r d u r i n g the e x p e r i m e n t . The h e a r t i s not s u r v i v i n g the s t r e s s w e l l as i n d i c a t e d by the f a c t t h a t the t i s s u e i s not m a i n t a i n i n g c o n t r o l c o n c e n t r a t i o n s of h i g h energy phosphate compounds even though t h e r e appears t o have been an i n c r e a s e i n the r a t e of a n a e r o b i c energy p r o d u c t i o n . L i v e r . T h i s t i s s u e shows the most d r a s t i c m e t a b o l i t e changes 94 d u r i n g the h y p o x i c s t r e s s . The energy c h a r g e , [ATP], t o t a l a d e n y l a t e s , and [CrP] a l l d e c l i n e s i g n i f i c a n t l y . The low c o n c e n t r a t i o n s of these m e t a b o l i t e s support the s u g g e s t i o n t h a t the r e s y n t h e s i s of g l u c o s e (or g l y c o g e n ) from 3-carbon m o l e c u l e s ( i e . l a c t a t e or amino a c i d s ) i s p r o b a b l y energy l i m i t e d . One may, t h e r e f o r e , assume t h a t p r e c u r s o r s f o r g l y c o l y s i s are not b e i n g produced from g l u c o n e o g e n e s i s . S i n c e the c o n v e r s i o n of g l y c o g e n t o g l u c o s e does not r e q u i r e h i g h - e n e r g y phosphate compounds, the energy s t a t u s of the t i s s u e does not negate the p o s s i b i l i t y t h a t i t i s s t i l l r e l e a s i n g g l u c o s e t o the b l o o d . Hbwever, the c o n c e n t r a t i o n s of g l y c o g e n i n the l i v e r do not change d u r i n g h y p o x i a . There was not even a d e c l i n i n g t r e n d i n the mean v a l u e a l t h o u g h the i n c r e a s e i n G6P c o n c e n t r a t i o n s would i n d i c a t e t h a t g l y c o g e n was b e i n g m o b i l i z e d . The l a c k of an o b s e r v e d d e p l e t i o n of l i v e r g l y c o g e n i s a t odds w i t h the r e p o r t of Heath and P r i t c h a r d (1965). They exposed Salmo c l a r k i t o 1 hour of h y p o x i a and r e p o r t e d t h a t l i v e r g l y c o g e n c o n c e n t r a t i o n s f e l l from 70 Mmoles/gm t o l e s s than 5 Mmoles/gm. T h i s paradox was f u r t h e r examined i n S e c t i o n 4 w i t h r e s u l t a n t c o n c l u s i o n t h a t g l y c o g e n was not b e i n g m o b i l i z e d t o a measurable degree. The response of l i v e r g l y c o g e n p h o s p h o r y l a s e t o h y p o x i a seems t o be v a r i a b l e . M e t a b o l i t e measurements from the l i v e r s of l u n g f i s h ( S e c t i o n 1), and f l o u n d e r (Jorgensen and M u s t a f a , 1980) i n d i c a t e a d e c l i n e i n l i v e r g l y c o g e n c o n c e n t r a t i o n s w i t h h y p o x i a , whereas those from c a r p ( J o h n s t o n , 1975), and 3 hr exposed t e n c h (Demael-Suard e_t a l . , 1974) do n o t . I t appears 95 t h a t t h e r e i s a r e s e a r c h problem i n the c o n t r o l of f i s h l i v e r g l y c o g e n p h o s p h o r y l a s e which a w a i t s f u r t h e r i n v e s t i g a t i o n . S i n c e t h e r e i s no apparent d e c l i n e i n l i v e r g l y c o g e n c o n c e n t r a t i o n s , then the l i v e r i s not a c t i n g as a s t o r e of g l u c o s e as i t does i n the l u n g f i s h . Thus, the r e m a i n i n g t i s s u e s w i l l have t o r e l y upon endogenous gly c o g e n t o f u e l g l y c o l y s i s i f they a r e t o i n c r e a s e g l y c o l y t i c r a t e . Red M u s c l e . The red muscle, as w i t h the b r a i n , appears t o be p r o t e c t e d d u r i n g h y p o x i a , as i n d i c a t e d by the l a c k of change i n m e t a b o l i t e c o n c e n t r a t i o n s . C r e a t i n e - p h o s p h a t e c o n c e n t r a t i o n s from the p o s t e r i o r r e d muscle s a m p l i n g s i t e d e c l i n e d d u r i n g h y p o x i a , r but a n t e r i o r samples showed no such change. I t i s p o s s i b l e t h a t t h i s change was due more t o e x e r c i s e than t o h y p o x i c d y s o x i a i f one c o n s i d e r s the argument which was proposed e a r l i e r . The f a c t t h a t c o n t r o l p o s t e r i o r samples had a h i g h e r CrP c o n c e n t r a t i o n than d i d the a n t e r i o r samples, and t h a t the d e c l i n e i n CrP was seen o n l y i n the p o s t e r i o r samples suggests t h a t the s t o r e of CrP i s b e i n g m o b i l i z e d f o r work r e l a t e d c a u s e s . The c o n c e n t r a t i o n s of l a c t a t e i n c r e a s e d i n the r e d muscle, but t h i s i n i t s e l f i s not an i n d i c a t i o n of an i n c r e a s e i n the r a t e of g l y c o l y s i s . S i n c e the o n l y o t h e r change i n the red muscle i s an i n c r e a s e i n G6P, one may suggest t h a t the red muscle i s not the s i t e of o r i g i n of the l a c t a t e and t h a t the s u p p l y of oxygen to t h i s t i s s u e i s s u f f i c i e n t f o r the muscle t o m a i n t a i n i t s r a t e s of ATP p r o d u c t i o n . I t i s w o r t h w h i l e n o t i n g 96 t h a t t h i s experiment was performed w i t h a minimum of muscular a c t i v i t y . I f the a n i m a l was r e q u i r e d t o e x e r c i s e d u r i n g t h i s t i m e , then the oxygen s u p p l y may not have been s u f f i c i e n t . White muscle. I t was p r e v i o u s l y s t a t e d t h a t the c o n c e n t r a t i o n s of endogenous g l y c o g e n g i v e some i n d i c a t i o n of the r e l a t i v e c a p a c i t y t o s y n t h e s i s e ATP v i a g l y c o l y s i s . By t h i s c r i t e r i o n , the c a p a c i t y of the t r o u t muscle t o a c t i v a t e g l y c o l y s i s exceeds t h a t of the l u n g f i s h muscle s i n c e the t r o u t white muscle has 50% more endogenous g l y c o g e n than does the l u n g f i s h w h i t e muscle. The c o n t r o l CrP c o n c e n t r a t i o n s a re h i g h e r i n the t a i l r e g i o n than they a re r o s t r a l t o the d o r s a l f i n (a p a t t e r n which i s s i m i l a r t o t h a t i n the red m u s c l e ) . However, the response t o h y p o x i a d i f f e r s i n the w h i t e muscle because both the a n t e r i o r and p o s t e r i o r samples e x h i b i t a d e c l i n e i n ['CrP], and a r i s e i n the c o n c e n t r a t i o n s of c r e a t i n e . Thus, u n l i k e the red muscle, the changes i n the w h i t e muscle appear t o be due t o h y p o x i c d y s o x i a and not t o e x e r c i s e . The c o n c e n t r a t i o n s of gly c o g e n show a d e c l i n i n g t r e n d i n w h i t e muscle but the change i s not s i g n i f i c a n t . I t w i l l be argued below, however, t h a t t h i s change i s a r e a l d e c l i n e i n g l y c o g e n c o n c e n t r a t i o n s . L a c t a t e c o n c e n t r a t i o n s i n c r e a s e but they do i n a l l t i s s u e s and, as p r e v i o u s l y mentioned, t h i s does not n e c e s s a r i l y i n d i c a t e t h a t the r a t e of a n a e r o b i c g l y c o l y s i s has i n c r e a s e d . A l t h o u g h i t i s p o s s i b l e t h a t the l a c t a t e was s y n t h e s i z e d elsewhere i n the body and d e l i v e r e d t o the w h i t e muscle v i a the b l o o d , t h e r e a re t h r e e reasons why t h i s i s 97 u n l i k e l y . F i r s t , i t i s p o s s i b l e t o o b t a i n a rough e s t i m a t e of the i n t r a c e l l u l a r pH by s u b s t i t u t i n g the measured c o n c e n t r a t i o n s of C r , CrP, ATP, and 1/10 of the measured c o n c e n t r a t i o n of ADP i n t o the e q u i l i b r i u m e q u a t i o n f o r the c r e a t i n e phosphokinase r e a c t i o n . ( S i e s j o et a l . , 1972,-Sahlin et a l . , 1974) . T h i s c a l c u l a t i o n i n d i c a t e s t h a t the w h i t e muscle i s the o n l y t i s s u e where a s i g n i f i c a n t d e c l i n e i n the e s t i m a t e d i n t r a c e l l u l a r pH o c c u r s . Second, the w h i t e muscle i s the o n l y t i s s u e where the r a t i o of b l o o d t o t i s s u e l a c t a t e c o n c e n t r a t i o n s remain l e s s than one throughout the e x p e r i m e n t . Even i f the c e l l was not r e l a t i v e l y n e g a t i v e i n s i d e w i t h r e s p e c t t o the e x t e r i o r , t h i s would i n d i c a t e t h a t the w h i t e muscle i s s y n t h e s i z i n g l a c t a t e and the g r a d i e n t i s from t i s s u e t o b l o o d . I f one t a k e s i n t o account the f a c t t h a t the i n t r a c e l l u l a r space i s n e g a t i v e l y charged w i t h r e s p e c t t o the e x t r a c e l l u l a r space, then the magnitude of the g r a d i e n t becomes even more pronounced. T h i r d , i f one t o t a l s the q u a n t i t y of l a c t a t e which i s produced i n the body and compares t h a t w i t h the q u a n t i t y of s u b s t r a t e a v a i l a b l e which i s not c o n t a i n e d i n the w h i t e muscle, one f i n d s t h a t t h e r e i s b a r e l y enough s u b s t r a t e a v a i l a b l e t o account f o r the i n c r e a s e i n l a c t a t e c o n c e n t r a t i o n s (Table 10 and F i g s . 15, 16). T h i s i n d i c a t e s t h a t the w h i t e muscle g l y c o g e n s t o r e s a re b e i n g m o b i l i z e d . The above e v i d e n c e s u p p o r t s the premise t h a t the g l y c o l y t i c r a t e has i n c r e a s e d i n t r o u t w h i t e muscle when the muscle becomes h y p o x i c . 98 T i s s u e - t i s s u e i n t e r a c t i o n s . When the t r o u t becomes h y p o x i c , t h e r e i s a c o n t i n u o u s b u i l d - u p of l a c t a t e and a d e p l e t i o n of the whole-body s t o r e s of g l y c o g e n . The q u e s t i o n s which w i l l now be a d d r e s s e d a r e , which t i s s u e or t i s s u e s are the main s o u r c e s of the l a c t a t e , and what i n f e r e n c e s can be made about the m e t a b o l i c r a t e of the t r o u t and i t s component organs d u r i n g h y p o x i a . The l i v e r g l y c o g e n d e p o s i t s are used i n many a n i m a l s as a source of g l u c o s e f o r c a t a b o l i s m i n o t h e r t i s s u e s when the a n i m a l i s s u b j e c t e d t o h y p o x i c s t r e s s (Hochachka and Somero, 1984). D u r i n g t h i s e x p e r i m e n t , however, the l i v e r g l y c o g e n c o n c e n t r a t i o n s d i d not d e c l i n e . I f l i v e r g l y c o g e n i s not b e i n g m o b i l i z e d t o f u e l g l y c o l y s i s i n o t h e r t i s s u e s , then the s o u r c e may be e i t h e r g l u c o n e o g e n e s i s i n the l i v e r or kidney or i t may be o t h e r endogenous g l y c o g e n s t o r e s . Based upon the low energy s t a t u s of the l i v e r , one may p r e d i c t t h a t g l u c o n e o g e n e s i s i s not p r o c e e d i n g a t a s i g n i f i c a n t r a t e . T h i s l e a v e s endogenous gl y c o g e n s t o r e s as the s o u r c e of g l y c o g e n . The o n l y t i s s u e which e x h i b i t e d a s i g n i f i c a n t d e c l i n e i n g l y c o g e n c o n c e n t r a t i o n s was the h e a r t . I t i s o b v i o u s t h a t the s m a l l s t o r e c o n t a i n e d i n the h e a r t can not s u p p l y a l l of the p r e c u r s o r s r e q u i r e d f o r the f o r m a t i o n of the r e s u l t i n g l a c t a t e l o a d . As was mentioned p r e v i o u s l y , the problem w i t h g l y c o g e n budgets i s t h a t the i n d i v i d u a l v a r i a t i o n i s h i g h and t h e r e f o r e , i t i s d i f f i c u l t t o d e t e c t r e a l changes. I f mean v a l u e s are used, however, then the t o t a l p o o l of g l y c o g e n , g l u c o s e and G6P i n the body of a 500 gm f i s h f a l l s from 7474 Mmoles t o 6128 umoles (a d e c l i n e of 2692 jimoles of 3-C u n i t s ) . At the same 99 time l a c t a t e r i s e s from 2137 Mmoles t o 4020 Mmoles (an i n c r e a s e of 1883 Mmoles). T h i s means t h a t r o u g h l y 2/3 of the t o t a l d e c l i n e i n s u b s t r a t e s can be a ccounted f o r as l a c t a t e . T h i s i s a r e a s o n a b l e e s t i m a t e s i n c e some of the l a c t a t e w i l l c e r t a i n l y be o x i d i z e d or m e t a b o l i z e d e l s e w h e r e . T h i s c a l c u l a t i o n u n d e r s c o r e s the n e c e s s i t y , i n t h i s c a s e , t o take i n t o account n o n - s i g n i f i c a n t d e c l i n e s i n g l y c o g e n . The c a l c u l a t i o n can be t aken one s t e p f u r t h e r t o determine which t i s s u e i s the g r e a t e s t c o n t r i b u t o r t o the t o t a l l a c t a t e p o o l . The net change i n l a c t a t e s t o r e s was 1882 Mmoles/500 gm f i s h . I f a l l of the s u b s t r a t e s i n a l l of the t i s s u e s except the w h i t e muscle were c o n v e r t e d t o l a c t a t e , t h e r e would be b a r e l y enough t o account f o r the r i s e i n l a c t a t e c o n c e n t r a t i o n s . Such a change would s u r e l y r e s u l t i n s i g n i f i c a n t d i f f e r e n c e s i n the g l y c o g e n c o n c e n t r a t i o n s of these t i s s u e s (wherease changes are o n l y seen i n the h e a r t ) . I t becomes apparent t h a t the w h i t e muscle s u b s t r a t e s t o r e s must be d e p l e t e d t o account f o r the r e s u l t a n t l a c t a t e i n c r e a s e . S i n c e w h i t e muscle has no G6Pase, and t h e r e f o r e cannot r e l e a s e the p h o s p h o r y l a t e d g l u c o s y l u n i t s i n t o the b l o o d as g l u c o s e , i t f o l l o w s t h a t the G6P r e s u l t i n g from g l y c o g e n must be u t i l i z e d _i_n s i t u . T h i s statement i s s u p p o r t e d by the o b s e r v a t i o n t h a t 85% of the observed i n c r e a s e i n whole body l a c t a t e s t o r e s o c c u r s i n the w h i t e muscle. These c a l c u l a t i o n s s e r v e t o p o i n t out the i n f l u e n c e of the w h i t e muscle upon the whole-body metabolism d u r i n g h y p o x i a . T h i s t i s s u e i s p r o v i d i n g the major source of f u e l f o r 100 g l y c o l y s i s , but i t i s a l s o m e t a b o l i s i n g t h a t f u e l rn s i t u , t h e r e b y c a u s i n g a s i g n i f i c a n t and d e l e t e r i o u s impact upon the metabolism of the r e s t of the body by p r o d u c i n g a l a r g e l a c t a t e l o a d . In a d d i t i o n , the o b s e r v a t i o n t h a t w h i t e muscle energy s t o r e s are somewhat d e p l e t e d i n d i c a t e s t h a t the w h i t e muscle i s not a b l e t o c o m p l e t e l y supplement the d e c l i n e i n a e r o b i c ATP p r o d u c t i o n w i t h a n a e r o b i c ATP p r o d u c t i o n . One may now ask how the m e t a b o l i c r a t e of the t r o u t changes w i t h the hypoxic s t r e s s . I f , f o r the sake of argument a l l of the observed d e c l i n e i n g l y c o g e n , g l u c o s e , and G6P p o o l s appeared as l a c t a t e then r o u g h l y 4308 Mmoles of ATP would be produced (3 A T P / g l u c o s y l i n g l y c o g e n and G6P). The r e s t i n g oxygen uptake of a t r o u t a t 4°C i s r o u g h l y 40 mg/kg/hr (based upon H o l e t o n and R a n d a l l , 1967, and a Q10 of 2.5). T h i s t r a n s l a t e s t o .625 mmoles O 2/500 gm f i s h / h r , and r o u g h l y 3.75 mmoles ATP/500.gm f i s h / h r . . In t h i s c a s e , g l y c o l y s i s a l o n e would be a b l e t o s u s t a i n the a n i m a l f o r r o u g h l y 20 min.. S i n c e 1/3 of the d e p l e a t e d s u b s t r a t e cannot be accounted f o r by t o t a l l i n g the r i s e i n l a c t a t e s t o r e s , i t i s p o s s i b l e . t h a t t h i s amount of e i t h e r the s u b s t r a t e or l a c t a t e was o x i d i z e d . U s i n g t h i s assumption one o b t a i n s a t o t a l of 2.8 mmoles of ATP from g l y c o l y s i s and 14.6 mmoles from the o x i d a t i o n of s u b s t r a t e produced over 3 hours i n a 500 gm f i s h . S i n c e t h i s r a t e of ATP p r o d u c t i o n (5.8 mmoles/500 gm f i s h / h r ) i s h i g h e r than the m e t a b o l i c r a t e of the t r o u t , one may i n f e r t h a t e i t h e r the m e t a b o l i c r a t e of the t r o u t has i n c r e a s e d or t h a t not a l l of the ' m i s s i n g ' l a c t a t e i s o x i d i z e d . 101 There i s another approach t o d e t e r m i n i n g whether m e t a b o l i c r a t e has changed d u r i n g h y p o x i a . The oxygen uptake of the t r o u t does not d e c l i n e d u r i n g a c u t e h y p o x i a (Mckim and Goeden, l 9 8 2 ; H o l e t o n and R a n d a l l , l967;Hughes and Saunders, 1970). T h i s statement s e r v e s t o r e i t e r a t e t h a t , even though l a c t a t e i s b e i n g produced i n i n c r e a s i n g amounts, some of the m e tabolism i s d e f i n i t e l y oxygen based. T h i s a n i m a l i s u t i l i z i n g a mixed a e r o b i c - a n a e r o b i c p a t t e r n of ATP p r o d u c t i o n . From t h i s one may suggest t h a t the m e t a b o l i c r a t e of a t r o u t i n c r e a s e s d u r i n g h y p o x i a . T h i s h y p o t h e s i s i s based on the combined o b s e r v a t i o n s t h a t the a n i m a l ' s oxygen uptake i s m a i n t a i n e d but i t s m e tabolism i s showing s i g n s of oxygen l i m i t a t i o n . One p o s s i b l e e x p l a n a t i o n has been proposed by Hughes and Saunders (1970). When ambient oxygen t e n s i o n s d r op, the v e n t i l a t i o n volume of the t r o u t must r i s e i n o r d e r t o m a i n t a i n e x t r a c t i o n r a t e s . T h i s i s an e n e r g e t i c a l l y e x p e n s i v e s t r a t e g y and becomes more so as the oxygen t e n s i o n s c o n t i n u e t o d e c l i n e . Work by the v e n t i l a t o r y system r i s e s , the oxygen r e q u i r e m e n t s of the v e n t i l a t o r y system i n c r e a s e , the p r o p o r t i o n of the t o t a l oxygen uptake u t i l i z e d by the v e n t i l a t o r y system i n c r e a s e s , and the a c t u a l oxygen d e l i v e r y t o the r e m a i n i n g organs d e c l i n e . T h i s means t h a t the b u l k of the body may become oxygen l i m i t e d even though the oxygen uptake i s m a i n t a i n e d . Assuming t h a t the oxygen uptake of the t r o u t i s m a i n t a i n e d and t h a t g l y c o l y s i s i s used t o supplement the ATP p r o d u c t i o n , then the m e t a b o l i c r a t e of the t r o u t would be e f f e c t i v e l y i n c r e a s e d by the r a t e of a n a e r o b i c ATP p r o d u c t i o n . T h i s 1 02 i n c r e a s e i n p r o d u c t i o n may be c a l c u l a t e d based upon the i n c r e a s e i n l a c t a t e s t o r e s or the d e c l i n e i n s u b s t r a t e s . The ATP thus produced would i n c r e a s e the m e t a b o l i c r a t e by from 942 t o 1346 Mmoles ATP/500gm/hour r e s p e c t i v e l y . T h i s r e p r e s e n t s an i n c r e a s e i n m e t a b o l i c r a t e of from 25% t o 36%. A l t h o u g h the a c t u a l number may be argued, i t i s apparent t h a t the t r o u t i s c o p i n g w i t h oxygen l a c k by a c t i v a t i n g p h y s i o l o g i c a l and m e t a b o l i c p r o c e s s e s which have the net e f f e c t of i n c r e a s i n g the body's m e t a b o l i c r e q u i r e m e n t s . In summary, a l l t i s s u e s e x h i b i t e d a response t o a c u t e h y p o x i a i n the t r o u t . The l i v e r appears t o be the most s t r e s s e d , based upon m e t a b o l i t e measurements. A l t h o u g h l a c t a t e c o n c e n t r a t i o n s i n c r e a s e d i n a l l of the t i s s u e s , i t appears t h a t the w h i t e muscle i s the major sou r c e of t h i s m e t a b o l i t e . Even though the m e t a b o l i c r a t e of the w h i t e muscle i s s m a l l compared t o the o t h e r t i s s u e s , the f a c t t h a t g l y c o l y s i s has been a c t i v a t e d w h i l e the muscle remains i n m e t a b o l i c communication w i t h the o t h e r t i s s u e s v i a the b l o o d s u p p o r t s the s u g g e s t i o n t h a t the metabolism of the w h i t e muscle w i l l have a pronounced e f f e c t upon the m e t a b o l i c s t a t u s of the whole a n i m a l . In e f f e c t , the t r o u t i s m a i n t a i n i n g i t s r a t e of oxygen uptake w h i l e a c t i v a t i n g a n a e r o b i c g l y c o l y s i s i n the attempt t o m a i n t a i n 'normal' r a t e s of energy u t i l i z a t i o n . T h i s s t r a t e g y promotes damage from bouts of h y p o x i a which a r e minutes t o hours l o n g by u t i l i z i n g s u b s t r a t e s q u i c k l y w i t h o u t p r o v i d i n g adequate methods of t o l e r a t i n g the p r o d u c t s . 103 SECTION 4 Turnover r a t e s of g l u c o s e and l a c t a t e i n the Rainbow t r o u t d u r i n g a c u t e h y p o x i a I n t r o d u c t i o n M e t a b o l i s m i s c o n s t a n t l y i n a s t a t e of f l u x w i t h m e t a b o l i t e s b e i n g c a t a b o l i z e d and a n a b o l i z e d . The measurement of the c o n c e n t r a t i o n s of the m e t a b o l i t e s o n l y p r o v i d e a p p r o x i m a t i o n s of r e l a t i v e r a t e s of p r o d u c t i o n and u t i l i z a t i o n . The t r o u t i s not v e r y h y p o x i a t o l e r a n t (Doudoroff and Shumway, 1970) and e x h i b i t s no s i g n s of b e i n g an oxygen conformer (Ho l e t o n and R a n d a l l , 1967). The i n c r e a s e i n l a c t a t e c o n c e n t r a t i o n d u r i n g h y p o x i a i s e v i d e n c e t h a t g l y c o l y s i s has been a c t i v a t e d i n many t i s s u e s and t h a t l a c t a t e p r o d u c t i o n has i n c r e a s e d . A measured i n c r e a s e i n the t u r n o v e r r a t e of l a c t a t e w i l l s u p p o r t t h i s h y p o t h e s i s . L i v e r glycogen c o n c e n t r a t i o n s do not change s i g n i f i c a n t l y d u r i n g h y p o x i a i n t r o u t , and the l a c k of h i g h energy phosphate compounds makes i t u n l i k e l y t h a t a c t i v e g l u c o n e o g e n e s i s i s o c c u r r i n g . T h i s l e d t o the s u g g e s t i o n t h a t l i v e r g l y c o g e n was not b e i n g m o b i l i z e d d u r i n g h y p o x i a and t h a t t i s s u e s had t o r e l y upon endogenous gly c o g e n s t o r e s f o r g l y c o l y t i c f u e l (see S e c t i o n 3 ) . The l a c k of a s i g n i f i c a n t d e c l i n e i n l i v e r g l y c o g e n may be due t o the f a c t t h a t the c o n c e n t r a t i o n s of g l y c o g e n i n the l i v e r a r e s t a b l e , or t h a t t h e r e i s a l a r g e v a r i a b i l i t y i n gly c o g e n 104 c o n c e n t r a t i o n s between samples. I f l i v e r g l y c o g e n i s not b e i n g m o b i l i z e d , , then one may p r e d i c t t h a t the r a t e of e n t r y of g l u c o s e i n t o the b l o o d w i l l not r i s e d u r i n g h y p o x i a . A l t e r n a t e l y , i f g l u c o s e t u r n o v e r r a t e s do i n c r e a s e , i t w i l l i n d i c a t e t h a t l i v e r g l y c o g e n i s b e i n g m o b i l i z e d d u r i n g h y p o x i a and t h a t the l a c k of a s i g n i f i c a n t change between c o n t r o l and 3 hour h y p o x i c l i v e r samples i s due t o v a r i a t i o n between samples. 105 Methods The a n i m a l s were from the same source as i n S e c t i o n 3.. E x p e r i m e n t a l t e c h n i q u e s were a l s o the same w i t h the e x c e p t i o n t h a t the water temperature was now 9.5°C and the h y p o x i c p 0 2 was 30 t o r r . ( s i n c e the temp e r a t u r e was h i g h e r than i t was i n S e c t i o n 3, a h i g h e r p 0 2 was r e q u i r e d t o o b t a i n comparable i n c r e a s e s i n plasma l a c t a t e ) . T rout were f e d u n t i l the day b e f o r e c a n n u l a t i o n . Cannulae (PE 50, C l a y Adams) were i n s e r t e d i n t o the d o r s a l a o r t a under MS-222 ( t r i c a i n e methane s u l f o n a t e , 1:15,000 w/v s o l u t i o n ) a n a e s t h e s i a one day b e f o r e e x p e r i m e n t a t i o n . The c a n n u l a t i o n s were performed f o l l o w i n g the methods of H i l l a b y and R a n d a l l (1979). F o l l o w i n g c a n n u l a t i o n and d u r i n g e x p e r i m e n t a t i o n the f i s h were kept i n a s l a t t e d b l a c k box w i t h f l o w t h r o u g h water (as i n S e c t i o n 3 ) . F i s h were a l l o w e d t o r e c o v e r f o r 24 hours f o l l o w i n g c a n n u l a t i o n . I n j e c t i o n s f o r the h y p o x i c s e r i e s were done between 2 1/4 and 2 3/4 hours a f t e r the onset of h y p o x i a . S i n c e b l o o d s a m p l i n g c o n t i n u e d f o r 30 min. a f t e r i n j e c t i o n of t r a c e r , t h i s i n j e c t i o n regime r e s u l t e d i n d a t a from t r o u t which were h y p o x i c f o r a p p r o x i m a t e l y 3 h o u r s . I n j e c t i o n s and S a m p l i n g . Roughly 700 nl of C o u r t l a n d s s a l i n e c o n t a i n i n g 10 u n i t s / m l h e p a r i n and no g l u c o s e and the i s o t o p e l a b e l was i n j e c t e d . Each i n j e c t i o n c o n t a i n e d r o u g h l y 7 ^ C i D-[ 6-3H ]-glu c o s e , and 7 juCi L-[ 1 "C (U) ] - l a c t i c a c i d (sodium s a l t ) . 106 The i s o t o p e s were d r i e d under n i t r o g e n b e f o r e b e i n g r e c o n s t i t u t e d i n the s a l i n e . Two 10 M1 a l i q u o t s of the i n j e c t a t e were removed and counted t o o b t a i n the p r e c i s e i n j e c t i o n a c t i v i t y . The i n j e c t i o n was taken up i n a 1 ml d i s p o s a b l e s y r i n g e , which was weighed b e f o r e and a f t e r i n j e c t i o n t o o b t a i n p r e c i s e i n j e c t i o n volumes. H a e m a t o c r i t s were taken b e f o r e i n j e c t i o n and a f t e r s a m p l i n g . Time 0 was the time a t which 1/2 of the b o l u s had been i n j e c t e d . T o t a l i n j e c t i o n took r o u g h l y 10 s and was imm e d i a t e l y f o l l o w e d by a 0.2 ml s a l i n e f l u s h t o ensure t h a t a l l l a b e l i n the ca n n u l a had been i n j e c t e d . B l o o d s a m p l i n g began 10 s hence. Ten 200 jul samples were withdrawn over the next 30 min. and added t o preweighed 1.5 ml m i c r o c e n t r i f u g e v i a l s . A f t e r sample 6 was t a k e n , the w i t h d r a w a l s were f o l l o w e d by i n j e c t i o n of 200 u l of s a l i n e . The sample v i a l s c o n t a i n e d 2 u n i t s of h e p a r i n , and 10 M1 of i n h i b i t o r s t o c k c o n t a i n i n g 40 mg/ml of p o t a s s i u m o x a l a t e , 50 mg/ml of sodium f l o u r i d e , and 130 mg/ml of p o t a s s i u m c y a n i d e . These i n h i b i t o r s b l o c k e d l a c t a t e dehydrogenase (Boyer, 1975), e n o l a s e ( L e h n i n g e r , 1975), and the e l e c t r o n t r a n s p o r t c h a i n r e s p e c t i v e l y . The f i s h were k i l l e d by i n j e c t i n g s a t u r a t e d KC1 i n t o the f i s h v i a the c a n n u l a . The b l o o d v i a l s were p l a c e d on i c e , reweighed and then c e n t r i f u g e d f o r 3 min. i n a F i s h e r benchtop c e n t r i f u g e , model 235A. Plasma was p i p e t t e d o f f and f r o z e n a t -80°C. Volume of b l o o d was c a l c u l a t e d by d i v i d i n g the weight by 1.09 gm/ml. T h i s c o r r e c t i o n . f a c t o r was o b t a i n e d by s a c r i f i c i n g 4 f i s h of the same s t o c k and measuring d e n s i t y . 1 07 A s s a y s . The plasma samples were assayed i n d u p l i c a t e f o r g l u c o s e and l a c t a t e u s i n g enzyme c o u p l e d , s p e c t r o p h o t o m e t r i c a n a l y s i s ( S e c t i o n 3 ) . A l i q u o t s of plasma (10 /xl) were d r i e d i n s c i n t i l a t i o n v i a l s , d i s s o l v e d i n 500 ixl of d i s t i l l e d water and counted i n 8 ml ACS s c i n t i l l a t i o n f l u i d . C o u n t i n g was done on a Beckman LS 1900 L i q u i d S c i n t i l a t i o n Counter w i t h an i n t e r n a l s t a n d a r d , and a d u a l l a b e l c o u n t i n g program which had been c a l i b r a t e d w i t h purchased s t a n d a r d s . The samples were kept i n the dark f o r at l e a s t 4 hours p r e v i o u s to c o u n t i n g t o reduce chemoluminescence. Ions were e x t r a c t e d from 50 u l of plasma u s i n g 0.3 gm A m b e r l i t e MB-3 mixed bed e x t r a c t i o n beads i n 1.5 ml M c e n t r i f u g e v i a l s . The beads were e q u i l i b r a t e d w i t h 1 ml of 1 M g l u c o s e f o r a t l e a s t 18 hours b e f o r e a d d i n g plasma. A f t e r the a d d i t i o n of plasma the v i a l s were shaken f o r 2 h o u r s . A 50 u l a l i q u o t was p l a c e d i n a s c i n t i l a t i o n v i a l , d r i e d , and r e c o n s t i t u t e d i n 500 M1 d i s t i l l e d water. C o u n t i n g was done as above. C a l c u l a t i o n s . The m a t h e m a t i c a l a n a l y s i s used t o o b t a i n replacement r a t e s from decay c u r v e s has been p r e s e n t e d by Katz et a l . , (1974,1981). The method of a n a l y s i n g the d a t a i s d e t a i l e d as f o l l o w s , w i t h the p r o c e d u r e used t o o b t a i n the area under the c u r v e b e i n g s p e c i f i c t o t h i s s tudy. The a r e a under the c u r v e was o b t a i n e d by f i t t i n g the data t o e q u a t i o n s and then i n t e g r a t i n g between 0.1 s and the time r e q u i r e d f o r the s p e c i f i c a c t i v i t y (S.A.) t o f a l l t o 1% of the o r i g i n a l p r e d i c t e d v a l u e . T h i s o r i g i n a l v a l u e was c a l c u l a t e d by 108 d i v i d i n g dose by b l o o d volume. Bl o o d volume was taken t o be 5% of the body weight ( S t e v e n s , 1968). The d a t a were f i t t e d t o c u r v e s i n a two s t e p p r o c e s s . F i r s t , In S.A. v s . time was p l o t t e d . The decay curve i n c l u d e s a t l e a s t two p r o c e s s e s , l o s s of l a b e l due t o d i l u t i o n i n t o the f l u i d s , and l o s s of l a b e l due t o m e t a b o l i t e exchange. These two p r o c e s s e s r e s u l t i n a c u r v e which c o n t a i n s a t l e a s t two l i n e s . " By t a k i n g the In of S.A., the two l i n e s can be v i s u a l i z e d as 2 r e l a t i v e l y s t r a i g h t l i n e s ( F i g . 17). The e q u a t i o n : Y = A,X was used t o r e c o n s t r u c t the upper p o r t i o n of the c u r v e . The equat i o n : N 2X Y = A 2e was used t o f i t the d a t a on the lower p o r t i o n of the c u r v e . In these e q u a t i o n s , Y= S.A., X = t i m e , and A and N are c o n s t a n t s . These g e n e r a t e d c u r v e s were i n t e g r a t e d t o o b t a i n the a r e a . The replacement r a t e (Ra) was c a l c u l a t e d by d i v i d i n g the dose by the a r e a s t a n d a r d i z i n g weight and time (Katz et a l . , 1974). T h i s number g i v e s the r a t e a t which the r a d i o a c t i v e l a b e l i s d i l u t e d i n the c e n t r a l p o o l . D u r i n g a steady s t a t e c o n d i t i o n , where the r a t e of e n t r y of a m e t a b o l i t e e q u a l s the r a t e of removal, the t u r n o v e r r a t e , m e t a b o l i t e e n t r y r a t e , and m e t a b o l i t e removal r a t e , a r e a l l e q u a l t o each o t h e r . The c u r v e s were then r e c o n s t r u c t e d by computer and p l o t t e d 109 a l o n g w i t h the o r i g i n a l d a t a ( F i g . 17). The Y i n t e r c e p t was o b t a i n e d by l i n e a r l y e x t r a p o l a t i n g the i n i t i a l p o r t i o n of the c u r v e . T h i s i n t e r c e p t , when d i v i d e d i n t o the dose, r e s u l t s i n the mass of the r a p i d l y m i x i n g p o o l ( M s ) ( K a t z e_t a l . , 1974). T h i s i s the mass, or q u a n t i t y ( i n Mmoles) of m e t a b o l i t e i n t o which the dose was i n j e c t e d . One b a s i c assumption made i n the a n a l y s i s i s t h a t the system i s i n a steady s t a t e . The d e f i n i t i o n of t h i s s t e a d y s t a t e i s t h a t the m e t a b o l i t e c o n c e n t r a t i o n does not change d u r i n g s a m p l i n g . T h i s assumption was t e s t e d by comparing the f i r s t and l a s t 4 m e t a b o l i t e v a l u e s o b t a i n e d i n the T O sample s e r i e s u s i n g ANOVA. One h y p o x i c l a c t a t e run was j u s t s i g n i f i c a n t l y d i f f e r e n t a t p = 0.05 w h i l e the r e s t of the s e r i e s were f a r from showing a s i g n i f i c a n t d i f f e r e n c e . However, even i n the experiment where i n i t i a l and f i n a l l a c t a t e c o n c e n t r a t i o n s d i f f e r e d (p = 0.05), the means d i f f e r e d by o n l y .19 mM, which i s w e l l w i t h i n the l i m i t s f o r a c c e p t a b l e a n a l y s i s found by p r e v i o u s s t u d i e s ( K a t z , p e r s . comm.). S t a t i s t i c s . A l l a n a l y s i s were done u s i n g a s i g n i f i c a n c e l e v e l of p < 0.05. Comparisons between two groups were done u s i n g ANOVA, u n l e s s the F t e s t f o r homogeneity of v a r i a n c e s i n d i c a t e d t h a t the v a r i a n c e s were not homogeneous. In t h i s case the Mann-Whitney U - t e s t was used. Model I r e g r e s s i o n a n a l y s i s was used to examine the r e l a t i o n s h i p between Ra v a l u e s and m e t a b o l i t e c o n c e n t r a t i o n , even though we c o u l d not p r e c i s e l y c o n t r o l the m e t a b o l i t e c o n c e n t r a t i o n . T h i s was done because the 1 10 c o n c e n t r a t i o n s were i n f l u e n c e d by the e x p e r i m e n t e r ( S o k a l and R o h l f , 1969). The s i g n i f i c a n c e of the r e g r e s s i o n was t e s t e d w i t h ANOVA. S i g n i f i c a n c e i n d i c a t e s t h a t a s i g n i f i c a n t amount of the v a r i a t i o n i n Y i s a c c o u n t e d f o r by v a r i a t i o n s i n X. Comparison of the s l o p e s of r e g r e s s i o n l i n e s was done w i t h an F t e s t . 111 R e s u l t s The method of c u r v e r e c o n s t r u c t i o n i s diagramed i n F i g u r e 17. The upper graph shows the curve o b t a i n e d u s i n g In S.A. vs t i m e . The e q u a t i o n s o b t a i n e d from f i t t i n g l i n e s t o the p o i n t s b e f o r e and a f t e r the i n f l e c t i o n p o i n t were used t o r e c r e a t e the f i n a l c u r v e shown i n the lower graph. R e p r e s e n t a t i v e c u r v e s from g l u c o s e and l a c t a t e experiments i n both the c o n t r o l and h y p o x i c s t a t e are shown i n F i g u r e 18 and the c o n s t a n t s r e q u i r e d t o r e c o n s t r u c t a l l g l u c o s e and l a c t a t e decay c u r v e s are l i s t e d i n Table 12. Each row c o n t a i n s data from one f i s h . The peak i s the c a l c u l a t e d peak s p e c i f i c a c t i v i t y (see methods) and i s used as the i n i t i a l p o i n t f o r the upper p o r t i o n of the c u r v e . The 1% time i s the t i m e , i n minutes, needed f o r the c u r v e t o r e a c h 1% of the i n i t i a l peak v a l u e . The i n f l e c t i o n p o i n t i s the t i m e , i n m i n u t e s , at which the two e q u a t i o n s share a common p o i n t . The v a l u e s of A and N are the r e s p e c t i v e c o n s t a n t s f o r the e q u a t i o n s . The t u r n o v e r r a t e of g l u c o s e (Ra-g), was not s i g n i f i c a n t l y e l e v a t e d i n the h y p o x i c f i s h over v a l u e s o b t a i n e d from the normoxic f i s h . T h i s i s i n c o n t r a s t t o the s i g n i f i c a n t i n c r e a s e which was seen i n l a c t a t e t u r n o v e r r a t e s ( R a - 1 ) ( T a b l e 13). The h y p o x i c and c o n t r o l Ra-1 v a l u e s d i d not e x h i b i t homogeneity of v a r i a n c e and so were compared u s i n g the Mann-Whitney U - t e s t . The h y p o x i c f i s h e x h i b i t e d a g r e a t e r v a r i a n c e i n Ra-1 than d i d the normoxic f i s h . T h i s r e l a t i o n s h i p o c c u r r e d w i t h the b l o o d l a c t a t e c o n c e n t r a t i o n d a t a as w e l l . These d a t a d i d not e x h i b i t 1 12 F i g u r e 17. Example of decay c u r v e r e c o n s t r u c t i o n . The two graphs a r e from a c o n t r o l g l u c o s e e x p e r i m e n t . Top graph: The y - a x i s i s the In of the s p e c i f i c a c t i v i t y . T h i s r e s u l t s i n an i n f l e c t i o n p o i n t where the i n f l u e n c e of l a b e l l o s s t h r o u g h d i l u t i o n becomes masked by the i n f l u e n c e of l a b e l l o s s due t o r e p l a c e m e n t . The i n f l e c t i o n p o i n t i s taken as the p o i n t which i s shared by both c u r v e s , and i s i n d i c a t e d by an arrow. Bottom graph: T h i s c u r v e c o n t a i n s the d a t a p o i n t s and a l i n e c o n s t r u c t e d by c a l c u l a t i n g the best f i t f o r the two l i n e s d e t e r m i n e d i n the upper graph. For more i n f o r m a t i o n see Methods. 1 14 F i g u r e 18. Example of r e c o n s t r u c t e d g l u c o s e c u r v e s . These r e p r e s e n t a t i v e c u r v e s a r e from a c o n t r o l experiment (top) and a h y p o x i c experiment (bottom). The c i r c l e s i n d i c a t e d a t a p o i n t s and the l i n e s a r e r e c o n s t r u c t e d based upon the t e c h n i q u e o u t l i n e d i n Methods and i n F i g . 17. 130000 TIME (min) 1 16 F i g u r e 19. Example of r e c o n s t r u c t e d l a c t a t e c u r v e s . These r e p r e s e n t a t i v e c u r v e s a r e from a c o n t r o l experiment (top) and a h y p o x i c experiment ( b o t t o m ) . The c i r c l e s i n d i c a t e d ata p o i n t s and the l i n e s a r e r e c o n s t r u c t e d based upon the t e c h n i q u e o u t l i n e d i n Methods and i n F i g . 17. i O S I S2 - O O O O f i OOOOOI 0) > o OOOOCI ^ 3 o OOOOOZ A luiui) 3WH i O S i S2 k o o o o o e ooooooi U O O O O O f i l o Z N t 3 o 0 0 0 0 0 0 2 118 homogeneity of v a r i a n c e , but the Mann-Whitney U - t e s t i n d i c a t e d t h a t the d a t a were s i g n i f i c a n t l y d i f f e r e n t . The b l o o d g l u c o s e v a l u e s , were not s i g n i f i c a n t l y d i f f e r e n t between c o n t r o l and h y p o x i c f i s h , nor were t h e i r v a r i a n c e s d i f f e r e n t . The t o t a l mass of m e t a b o l i t e (Ms) i n the r a p i d l y m i x i n g p o o l was not a n a l y s e d u s i n g s t a t i s t i c s . T h i s was because the mass would v a r y not o n l y w i t h the e x p e r i m e n t a l c o n d i t i o n s , but a l s o w i t h the weight and b l o o d volume of the f i s h . We d i d , t h e r e f o r e , t r a n s f o r m the Ms i n t o a number which c o u l d be u s e f u l l y compared by c a l c u l a t i n g a p r e d i c t e d c o n c e n t r a t i o n of g l u c o s e and comparing i t w i t h the measured v a l u e . Assuming t h a t the b l o o d volume of a f i s h i s 5% of the body weight ( S t e v e n s , 1968), one can d i v i d e the mass of m e t a b o l i t e by the b l o o d volume and a r r i v e a t a c o n c e n t r a t i o n ( T a b l e 14). N e i t h e r the measured v a l u e of g l u c o s e , nor the c a l c u l a t e d c o n c e n t r a t i o n of g l u c o s e v a r i e s s i g n i f i c a n t l y w i t h h y p o x i a . However, the r a t i o of c a l c u l a t e d t o measured c o n c e n t r a t i o n s do change, from 0.61+0.10 i n the c o n t r o l s t o 0.24+0.04 i n the h y p o x i c f i s h . S i n c e the a c t u a l m e t a b o l i t e c o n c e n t r a t i o n i s known, t h i s d i s c r e p e n c y i n r a t i o s u g g e s t s t h a t t h e volume of the r a p i d l y m i x i n g p o o l i s d i f f e r e n t i n the h y p o x i c f i s h . The f a c t t h a t the c a l c u l a t e d c o n c e n t r a t i o n s r e s u l t i n an u n d e r e s t i m a t e of the a c t u a l c o n c e n t r a t i o n i n d i c a t e s t h a t the volume of the r a p i d l y m i x i n g p o o l i s lower than the p r e d i c t e d volume. T h i s s u g g e s t s p e r i p h e r a l v a s o c o n s t r i c t i o n . F i g u r e 21 shows the s i g n i f i c a n t r e l a t i o n s h i p which i s o b t a i n e d when Ra-1 i s r e g r e s s e d on the c o n c e n t r a t i o n of l a c t a t e 1 19 Table 12. E q u a t i o n parameters f o r the DPM vs TIME c u r v e s i n t r o u t . These parameters can be used t o r e c o n s t r u c t the c u r v e s used i n the d a t a a n a l y s i s . "Peak"= c a l c u l a t e d f i r s t peak;"Time t o 1%"= time i n minutes taken t o re a c h 1% of the i n i t i a l peak and i s the upper l i m i t of t used when i n t e g r a t i n g ; " I n f l . Pt."= time a t which the e q u a t i o n s change;'^," and "A2"= c o n s t a n t s f o r the r e s p e c t i v e e q u a t i o n s ; " N , " and "N2"= exponents f o r the r e s p e c t i v e e q u a t i o n s . The e q u a t i o n s a r e l i s t e d i n Methods. 1 2 0 T a b l e 12. E q u a t i o n parameters f o r the DPM vs TIME c u r v e s i n t r o u t . Peak 1% Time I n f l . A, A 2 N, N 2 (•S.A.) P o i n t A. G l u c o s e F i s h No. C o n t r o l 1 219781 268 3. 60 276960 67933 -1 . 1 603 -.0128 2 672493 273 5. 1 2 170929 52092 -0 .7756 -.0075 3 80175 261 3. 62 . 196362 37069 -1 .4221 -.0147 4 106363 139 4. 50 274081 57844 -1 . 1 539 -.0288 5 129689 134 • 3. 57 164000 84078 -o .6052 -.031 1 6 1 08672 237 5. 25 110511 45889 -o .5900 -.0158 Hypoxic 1 40850 1 1 6 3. 77 254824 68370 -0 .9625 -.0443 2 91491 168 6. 37 656336 43228 -1 .5388 -.0230 3 1 1 038 182 7. 33 360283 38951 -1 .2017 -.0196 4 96882 80 3. 65 172051 73081 -0 .7044 -.0543 5 96673 205 5. 77 130927 50598 -0 .5128 -.0193 6 100159 210 3. 00 260521 25530 -2 . 1 251 -.0154 B. L a c t a t e F i s h No. C o n t r o l 1 1447482 74 5. 28 1905315 544792 -0 .849 -.0489 2 1047089 81 5. 1 2 1036481 171665 -1 .1413 -.0344 3 460065 62 5. 07 850346 9731 5 -1 .481 1 -.0493 4 855803 85 5. 25 2322178 196618 -1 .6844 -.0369 5 1 052536 86 5. 40 1034190 256875 -0 .9183 -.0371 6 2282986 . 63 3. 75 1625928 292877 -1 .2491 -.0404 [ypoxic 1 169397 51 3. 77 245955 35292 -1 .31 68 -.0601 2 66558 92 5. 40 502923 25167 -1 .9628 -.0394 3 423891 44 8. 38 1696845 1 10986 -1 .654 -.0749 4 57251 132 10. 1 3 92335 19463 -0 .7542 -.0267 5 79233 137 10. 10 89593 24965 -o .6287 -.0251 6 63819 100 4. 98 91889 12636 -1 .4038 -.0299 121 Table 13. T r o u t m e t a b o l i t e Turnover Numbers. (x+S.E.) STATE TURNOVER NUMBER (Ra) (Mmoles/min/kg) Gluc o s e L a c t a t e Measured [LACTATE] (/amoles/ml) Normoxia Hypoxia 10.7+1.9 10.6+3.3 2 . 8 +0.4 1 20.6+6.8 0.62+0.13 1 7.02+1.65 M e n o t e s s i g n i f i c a n t d i f f e r e n c e from h y p o x i a (p < 0.05) 1 2 2 T a b l e 14. Mass of r a p i d l y m i x i n g p o o l i n t r o u t . (x+S.E.) STATE FISH WT. gm MS Mmoles [GLUCOSE] c a l c u l a t e d / measured Normoxia 356+20.7 63+8 0.61+0.10 1 Hypoxia 365+26.5 39 + 9 0.24+0.04 Ms i s the number of Mmoles of s u b s t r a t e w i t h which the i n j e c t a t e was r a p i d l y mixed. The c a l c u l a t i o n i s e x p l a i n e d i n Methods. The c a l c u l a t e d g l u c o s e c o n c e n t r a t i o n i s o b t a i n e d by d i v i d i n g the p r e d i c t e d b l o o d volume i n t o the Ms. The r a t i o i n d i c a t e s t h a t the p r e d i c t e d volume g r e a t l y o v e r e s t i m a t e s the a c t u a l volume d u r i n g h y p o x i a . 1 d e n o t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l (p < 0.05). 123 F i g u r e 20. The r e l a t i o n s h i p between plasma g l u c o s e c o n c e n t r a t i o n s and g l u c o s e t u r n o v e r . The graph c o n t a i n s a l l of the d a t a but the s i g n i f i c a n t l i n e i s f i t o n l y t o the c o n t r o l v a l u e s . I f the two c i r c l e d p o i n t s a r e o m i t t e d then the p o o l e d d a t a r e g r e s s i o n l i n e becomes s i g n i f i c a n t . Hypoxia p o i n t s a r e r e p r e s e n t e d by a c l o s e d c i r c l e and the c o n t r o l p o i n t s are r e p r e s e n t e d by an 'x'. 125 F i g u r e 21. The r e l a t i o n s h i p between plasma l a c t a t e c o n c e n t r a t i o n s and l a c t a t e t u r n o v e r . The graph c o n t a i n s a l l of the d a t a . The e q u a t i o n of the l i n e i s 'Y = .12 + 3.034X'. [Lactate] (mM) 1 27 (Y = 0.12 + 3.034X). T h i s f i g u r e c o n t a i n s a l l of the d a t a . The r e g r e s s i o n of c o n t r o l Ra-1 on l a c t a t e c o n c e n t r a t i o n d i d not r e s u l t i n a s l o p e which d i f f e r e d from u n i t y . The same comparison of h y p o x i c d a t a d i d , however, r e s u l t i n a s i g n i f i c a n t r e l a t i o n s h i p , w i t h the e q u a t i o n of the l i n e b e i n g "Y = -3.361 + 3.416X". An a n a l y s i s of c o v a r i a n c e between the p o o l e d d a t a and the h y p o x i c d a t a i n d i c a t e s no d i f f e r e n c e i n s l o p e . T h i s i m p l i e s t h a t the c o n t r o l data f o l l o w the same s l o p e as the h y p o x i c d a t a . The r e l a t i o n s h i p between Ra-g and g l u c o s e c o n c e n t r a t i o n i s shown i n F i g u r e 20. T h i s f i g u r e a l s o c o n t a i n s a l l of the g l u c o s e d a t a . The s o l i d l i n e r e p r e s e n t s the s i g n i f i c a n t r e g r e s s i o n o b t a i n e d u s i n g c o n t r o l v a l u e s (Y = -2.236 + 2.055X). A n a l y s i s of the p o o l e d d a t a and the h y p o x i c d a t a r e s u l t i n r e g r e s s i o n s l o p e s which do not d i f f e r from u n i t y . I f , however, the two c i r c l e d h y p o x i c p o i n t s a r e removed, the p o o l e d d a t a r e g r e s s i o n does become s i g n i f i c a n t (Y = -2.705 + 1.964X). A l a r g e r sample s i z e would have been n e c e s s a r y t o a s c e r t a i n whether the r e l a t i o n s h i p which h e l d d u r i n g normoxia was m a i n t a i n e d d u r i n g h y p o x i a . More f i s h were a n a l y s e d as soon as t h i s was n o t e d , but the s t o c k was no l o n g e r the same and the r e s u l t s f o r both c o n t r o l and h y p o x i c f i s h were h i g h e r than the o r i g i n a l numbers. T h i s was due p o s s i b l y t o the i n c r e a s e i n water temperature which o c c u r r e d d u r i n g the i n t e r v e n i n g p e r i o d . A c o r r e l a t i o n a n a l y s i s between Ra-g and Ra-1 i n d i c a t e d t h a t t h e r e was no s i g n i f i c a n t r e l a t i o n s h i p between the t u r n o v e r r a t e s of g l u c o s e and l a c t a t e . 128 Di s c u s s i o n A m e t a b o l i t e t u r n o v e r number denotes the r a t e a t which a m e t a b o l i t e e n t e r s i n t o and i s removed from a c e n t r a l p o o l of t h a t m e t a b o l i t e . The c e n t r a l p o o l f o r l a c t a t e and g l u c o s e i s the c i r c u l a t o r y system p l u s o t h e r minor e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments (Katz e t a l . , 1974). The s o l e s i t e of t r a c e r i n f l o w i n t o the body i s thr o u g h the r a p i d l y m i x i n g p o o l (the t r a c e r i s i n j e c t e d i n t o the blood) but t h e r e a re no assumptions made about the s i t e s of t r a c e r l o s s . T h i s l o s s c o u l d be both from i n s i d e or o u t s i d e of t h i s p o o l (Katz e_t a l . , 1974). The r a p i d l y m i x i n g p o o l i s com p r i s e d of a l a r g e , but unknown volume of the c e n t r a l p o o l . In t h i s e x p e r i m e n t , the c a l c u l a t i o n s used t o dete r m i n e the mass of t h i s p o o l a r e made from d a t a which a re o b t a i n e d w i t h i n the f i r s t 2-5 min. (the a c t u a l time depending upon the shape of the decay c u r v e ) . T h e r e f o r e the unknown volume i s t h a t which the l a b e l mixes i n t o w i t h i n t h a t p e r i o d of t i m e . The c i r c u l a t i o n time of the t r o u t i s r o u g h l y 90 s (Cameron, 1975), and so i f c i r c u l a t i o n i s u n o b s t r u c t e d throughout the v a s c u l a r bed, then the unknown volume s h o u l d approximate the volume of the plasma. In c a s e s where v a s o c o n s t r i c t i o n o c c u r s , however, the volume of the r a p i d l y m i x i n g p o o l may be l e s s than the t o t a l b l o o d volume ( C a s t e l l i n i e t a_l. , 1985). T h i s i s examined i n Ta b l e 13. Based upon the assumption t h a t the t o t a l b l o o d volume i s 5% of body weight and t h a t the b l o o d w i l l have a u n i f o r m c o n c e n t r a t i o n 129 of the m e t a b o l i t e , one may c a l c u l a t e a c o n c e n t r a t i o n u s i n g the Ms and the e s t i m a t e d b l o o d volume. The c a l c u l a t e d c o n c e n t r a t i o n i s always lower than the measured c o n c e n t r a t i o n . T h i s i n d i c a t e s t h a t the m e t a b o l i t e p o o l i n t o which the l a b e l was i n j e c t e d i s s m a l l e r than the t o t a l m e t a b o l i t e p o o l . T h i s , i n t u r n , s u g g e s t s t h a t t h e r e a r e s i t e s of v a s c u l a r r e s t r i c t i o n i n a l l c a s e s . The d i f f e r e n c e between the measured and c a l c u l a t e d m e t a b o l i t e c o n c e n t r a t i o n s i n c r e a s e d u r i n g h y p o x i a i n d i c a t i n g t h a t p e r i p h e r a l v a s o c o n s t r i c t i o n has become more pronounced. When the r a t e of m e t a b o l i t e e n t r y i n t o the c e n t r a l p o o l e q u a l s the r a t e of l o s s , then t h a t m e t a b o l i t e i s i n a steady s t a t e . Under t h i s c o n d i t i o n , the r a t e of e n t r y and the r a t e of l o s s both e q u a l the t u r n o v e r number. The presence of a steady s t a t e i s assumed i n these c a l c u l a t i o n s and can be a s c e r t a i n e d based upon m e t a b o l i t e c o n c e n t r a t i o n s i n the b l o o d . A change of l e s s than 0.5 mM over the t i m e c o u r s e of the sampling i s c o n s i d e r e d t o be a c c e p t a b l e ( K a t z , p e r s . comm.). T h i s c r i t e r i o n was s a t i s f i e d f o r both g l u c o s e and l a c t a t e . G l u c o s e t u r n o v e r . A l t h o u g h c a r b o h y d r a t e metabolism i s not b e l i e v e d t o be a major energy so u r c e i n f i s h (Walton and Cowey et a l ' . , 1982) i t i s not u n r e a s o n a b l e t o assume t h a t i t s importance i s i n c r e a s e d d u r i n g h y p o x i a (Hochachka and Somero, 1984) In the p r e v i o u s s e c t i o n i t was argued t h a t the f l u x t hrough g l y c o l y s i s i n c r e a s e d d u r i n g h y p o x i a , based upon m e t a b o l i t e measurements. However, the t u r n o v e r r a t e s of g l u c o s e i n the t r o u t d i d not change d u r i n g a s i m i l a r h y p o x i c exposure. 130 There are t h r e e m e t a b o l i c p a t t e r n s which a r e c o h s i s t a n t w i t h t h e s e o b s e r v a t i o n s . F i r s t , some t i s s u e s may decrease t h e i r r e q u i r e m e n t s f o r b l o o d borne g l u c o s e w h i l e o t h e r s i n c r e a s e t h e i r r e q u i r e m e n t s . T h i s would a l l o w some t i s s u e s t o f u e l g l y c o l y s i s w i t h b l o o d borne g l u c o s e w h i l e o t h e r t i s s u e s e i t h e r remained a e r o b i c or they e x h i b i t e d a d e c r e a s e d requirement f o r ATP. Sec o n d l y , a l l t i s s u e s may m a i n t a i n t h e i r r e l a t i v e r e q u i r e m e n t s f o r b l o o d borne g l u c o s e w h i l e those t i s s u e s which i n c r e a s e g l y c o l y t i c f l u x r e l y upon endogenous g l y c o g e n t o augment the f u e l s u p p l y . T h i r d l y , i t i s p o s s i b l e t h a t f l u x through g l y c o l y s i s does not i n c r e a s e . In t h i s case i t i s p o s s i b l e t h a t the p r o p o r t i o n of p y r u v a t e produced which i s reduced t o l a c t a t e i n c r e a s e s r e l a t i v e t o the p r o p o r t i o n t h a t e n t e r s the TCA c y c l e . I t i s u n l i k e l y t h a t the t h i r d o p t i o n i s the r e a l one because t h e r e i s d e p l e t i o n of g l y c o g e n s t o r e s i n the h e a r t and a d e c l i n i n g t r e n d i n w h i t e muscle and b r a i n . I f the c o n c e n t r a t i o n s of gl y c o g e n d e c l i n e then g l y c o l y t i c a c t i v a t i o n i s i n d i c a t e d . I t i s not p o s s i b l e t o d i s t i n g u i s h between the former two i d e a s w i t h these d a t a . Thus, the q u e s t i o n of whether g l y c o l y s i s i s a c t u a l l y a c t i v a t e d i n a l l the t i s s u e s i s not answered by the g l u c o s e t u r n o v e r numbers. C o n t r o l r a t e s f o r g l u c o s e t u r n o v e r (10.6 Mmoles/min./kg) range from a p p r o x i m a t l y 0.5 t o 1.2 t i m e s t h a t found i n mammalian systems ( T a b l e 15). T h i s v a l u e i s 5 t i m e s h i g h e r than was r e p o r t e d f o r k e l p bass (Bever et a l . , 1977) and 4.6 t i m e s h i g h e r than i n coho salmon (Huangsheng et a_l. , 1978). Most of the d i f f e r e n c e i n t u r n o v e r r a t e s can be a t t r i b u t e d t o the v a r i a t i o n 1 3 1 T a b l e 15. A c o m p i l a t i o n of g l u c o s e and l a c t a t e t u r n o v e r numbers. L a c t a t e Ra (^moles/ min/kg) l a b e l A nimal R e f e r e n c e 21 1 3 - t r i t i a t e d r a t K a t z , 1982 94 3 - t r i t i a t e d r a t i b i d 94 U-carbon-14 r a t " i b i d 62 3 - t r i t i a t e d r a t i b i d 79 3 - t r i t i a t e d r a t i b i d 1 78 3 - t r i t i a t e d r a t i b i d 1 78 3 - t r i t i a t e d r a t Okajama e t a l , 1981 94 3 - t r i t i a t e d r a t i b i d 94 U-carbon-14 r a t i b i d 62 U-carbon-14 r a t i b i d 44 U-carbon-14 G l u c o s e g u i n e a p i g Frem i n e t and L e c l e r c , 1980 Ra (Mmoles/ min/kg) l a b e l A n imal R e f e r e n c e .2-15 22 21 17 U-carbon-14 6 - t r i t i a t e d 6 - t r i t i a t e d U-carbon-14 Grey s e a l monkey N r a b b i t r a b b i t C a s t e l l i n i e t a l . , 1985 Armstrong et a l . , 1979 Dunn et a l . , 1976 i b i d .6-2.2 2.3 6 - t r i t i a t e d 6 - t r i t i a t e d K e l p bass Bever e t a l . , 1977 Coho salmon Huangsheng e t a l . , 1978 Due t o the v a r i a t i o n i n t r a c e r , i n j e c t i o n s i t e , and n u t r i t i o n a l s t a t u s , t h e s e g l u c o s e and l a c t a t e t u r n o v e r numbers a r e t o be used as approximate v a l u e s o n l y . 1 32 i n plasma g l u c o s e c o n c e n t r a t i o n . There i s a s i g n i f i c a n t r e l a t i o n s h i p i n t r o u t between the c o n t r o l g l u c o s e t u r n o v e r r a t e and the plasma c o n c e n t r a t i o n ( F i g . 19). When the g l u c o s e c o n c e n t r a t i o n s r e p o r t e d i n the r e f e r e n c e s a r e s u b s t i t u t e d i n t o t h i s r e l a t i o n s h i p the r e s u l t a n t p r e d i c t e d t u r n o v e r numbers a r e 3.3 f o r the k e l p bass and 4.9 f o r the salmon. Thus, the t u r n o v e r r a t e s of g l u c o s e i n these f i s h a r e c l o s e t o what i s p r e d i c t e d based upon the r e l a t i o n s h i p between plasma g l u c o s e c o n c e n t r a t i o n s and g l u c o s e t u r n o v e r r a t e s . The q u e s t i o n a r i s e s as t o why the plasma g l u c o s e c o n c e n t r a t i o n s from the t r o u t i n t h i s t h e s i s a re h i g h e r than i n the k e l p bass and salmon. The s o l u t i o n i s l i k e l y t o be r e l a t e d t o the apparent l a c k of p r e c i s e c o n t r o l over plasma g l u c o s e c o n c e n t r a t i o n s i n f i s h . Plasma g l u c o s e c o n c e n t r a t i o n s i n rainbow t r o u t may i n c r e a s e a f t e r c a r b o h y d r a t e i n g e s t i o n , from a c o n t r o l l e v e l of 4.5 mM t o a h i g h of 28 mM (Palmer and Ryman, 1972). N o n - f a s t i n g t r o u t s a c r i f i c e d i n the l a b where t h i s t h e s i s was undertaken have had plasma g l u c o s e c o n c e n t r a t i o n s r a n g i n g from 1 t o 20 mM (Dunn, u n p u b l i s h e d o b s e r v a t i o n s ) . One o b s e r v a t i o n made i n the p r e v i o u s s e c t i o n was t h a t the c o n c e n t r a t i o n s of l i v e r g l y c o g e n do not change s i g n i f i c a n t l y d u r i n g h y p o x i a . I t i s u n l i k e l y t h a t g l u c o n e o g e n e s i s i s o c c u r r i n g i n the l i v e r a t t h i s t ime due t o the poor energy s t a t u s of t h a t organ. As a r e s u l t , the o n l y r e m a i n i n g major carbon s o u r c e a v a i l a b l e f o r t i s s u e s a r e endogenous s t o r e s . The o b s e r v a t i o n t h a t g l u c o s e t u r n o v e r does not r i s e d u r i n g h y p o x i a s u p p o r t s t h i s p r e d i c t i o n because the i n f l u x of g l u c o s e i n t o the 133 c e n t r a l p o o l would i n c r e a s e i f the l i v e r were i n c r e a s i n g i t s o u t p u t of g l u c o s e . L a c t a t e t u r n o v e r . I t has been argued t h a t t i s s u e g l y c o l y t i c r a t e s a r e i n c r e a s i n g d u r i n g h y p o x i a (see S e c t i o n 3 ) , which would cause an i n c r e a s e i n l a c t a t e t u r n o v e r r a t e s . In a d d i t i o n , l a c t a t e u t i l i z a t i o n may i n c r e a s e because the a v a i l a b i l i t y of l a c t a t e i n c r e a s e s as plasma c o n c e n t r a t i o n s r i s e and because most f i s h t i s s u e s r e a d i l y o x i d i z e l a c t a t e when oxygen i s a v a i l a b l e ( B a l i n s k i and Jonas, 1972;Mommsen, 1984). L a c t a t e t u r n o v e r r a t e s i n c r e a s e r o u g h l y 3 tim e s f o r every u n i t i n c r e a s e i n plasma l a c t a t e c o n c e n t r a t i o n , w i t h a r e l a t i o n s h i p of "Y = 0.12 + 3.034X" ( F i g . 20). The l a c k of a s i g n i f i c a n t r e l a t i o n s h i p when the c o n t r o l d a t a are a n a l y s e d s e p a r a t e l y may be due t o the s m a l l sample s i z e c o u p l e d w i t h the s m a l l range of observed l a c t a t e c o n c e n t r a t i o n s . The mammalian l i t e r a t u r e i n d i c a t e s t h a t a r e l a t i o n s h i p between l a c t a t e t u r n o v e r and c o n c e n t r a t i o n e x i s t s , but the p r e c i s e form of t h a t r e l a t i o n s h i p i s i n q u e s t i o n . F r e m i n e t et a l . (1974) r e p o r t e d t h a t t u r n o v e r i n c r e a s e d l i n e a r l y w i t h c o n c e n t r a t i o n up t o 6 mM i n r a t s . E l d r i d g e et a l . , (1974) s t a t e s t h a t the r e l a t i o n s h i p i s c u r v i l i n e a r w i t h the u n i t i n c r e a s e i n t u r n o v e r t a i l i n g o f f as l a c t a t e c o n c e n t r a t i o n s r i s e . The sample s i z e i s not l a r g e enough f o r a d e f i n i t e c h o i c e t o be made between the s e two hypotheses based upon these d a t a . M e t a b o l i t e u t i l i z a t i o n . Turnover r a t e s g i v e the r a t e of i n f l u x 1 34 and e f f l u x of a m e t a b o l i t e from a c e n t r a l p o o l , but i n the s t r i c t e s t sense they do not g i v e any i n d i c a t i o n of the s o u r c e or f a t e of t h a t m e t a b o l i t e . E s t i m a t e s have been made, however, of the p r o p o r t i o n s of g l u c o s e and l a c t a t e t u r n o v e r which are o x i d i z e d . A p p r o x i m a t e l y 50% of the l a c t a t e t u r n o v e r i s o x i d i z e d i n mammals (Freminet e t a l . , 1 9 7 4;Issekutz e t a l . , 1976), and e s t i m a t e s from mammals f o r g l u c o s e range from 44 t o 48% o x i d i z e d ( I s s e k u t z et a l . , 1965; S t e e l e e t a l . , 1968;Royle et a l . , 1982). The p r o p o r t i o n w i l l v a r y w i t h s p e c i e s and m e t a b o l i c s t a t e . I f one assumes t h a t , i n the t r o u t , 50% of the l a c t a t e t u r n o v e r i s o x i d i z e d and 45% of the g l u c o s e t u r n o v e r i s o x i d i z e d then one i s a b l e t o e s t i m a t e the c o n t r i b u t i o n s of these m e t a b o l i t e s t o m e t a b o l i c r a t e . At a g l u c o s e t u r n o v e r r a t e of 10.6 Mmoles/min./kg, 143 Mmoles of g l u c o s e would be o x i d i z e d i n a 500 gm f i s h per hour and 5152 Mmoles of ATP would be produced. I f 1 mole of 0 2 i s consumed per 6 moles of ATP produced, then 859 Mmoles of 0 2 would be r e q u i r e d t o o x i d i z e the g l u c o s e . The m e t a b o l i c r a t e of the t r o u t i s 75 Mmoles 0 2/min./kg ( H o l e t o n and R a n d a l l , 1967;adjusted t o 9°C u s i n g a Q10 of 2.5). T h i s t r a n s l a t e s t o an oxygen u t i l i z a t i o n r a t e of 1172 Mmoles 0 2 i n a 500 gm t r o u t d u r i n g 1 hour. T h i s would mean t h a t g l u c o s e o x i d i z a t i o n i n the c o n t r o l s t a t e a c c o u n t s f o r 73% of the oxygen uptake. T h i s i s u n l i k e l y c o n s i d e r i n g the minor r o l e suggested f o r g l u c o s e o x i d a t i o n i n t r o u t and i n d i c a t e s t h a t the p e r c e n t of the t u r n o v e r number which i s o x i d i z e d i s lower i n t r o u t than i n mammaIs. The g l u c o s e t u r n o v e r r a t e i n the h y p o x i c s t a t e i s the same 135 as i t i s i n normoxia. I t i s not p o s s i b l e t o d e t e r m i n e i f the p r o p o r t i o n of the m e t a b o l i c r a t e which i s a c c o u n t e d f o r by g l u c o s e o x i d i z a t i o n - c h a n g e s but one may i n f e r t h a t i t d e c l i n e s . T h i s i s based upon the o b s e r v a t i o n t h a t l a c t a t e i s b e i n g produced (and so more g l u c o s e may be ending up as l a c t a t e ) , and t h a t i t i s l i k e l y t h a t l a c t a t e o x i d a t i o n i s a c c o u n t i n g f o r an i n c r e a s e d p r o p o r t i o n of the oxygen uptake d u r i n g h y p o x i a (see b e l o w ) . The l a c t a t e t u r n o v e r r a t e of 2.8 Mmoles/min./kg would r e s u l t i n 714 Mmoles ATP produced i n a 500 gm f i s h i n 1 hour i f 17 moles of ATP r e s u l t from the o x i d a t i o n of 1 mole of l a c t a t e and 50% of the l a c t a t e t u r n o v e r i s o x i d i z e d . U s i n g the same r e l a t i o n s h i p between ATP produced and oxygen u t i l i z e d as was used f o r g l u c o s e , one a r r i v e s a t an oxygen r e q u i r e m e n t of 119 Mmoles. Even i f a l l of the l a c t a t e t u r n i n g over i n the c o n t r o l s t a t e was o x i d i z e d , i t would o n l y account f o r r o u g h l y 10% of the oxygen upt a k e . T h i s low p e r c e n t o x i d a t i o n of l a c t a t e s u p p o r t s the p r o p o s a l t h a t g l u c o s e and l a c t a t e o x i d a t i o n do not u t i l i z e the m a j o r i t y of the t o t a l oxygen uptake. G l u c o s e and l a c t a t e have been suggested t o be r e l a t i v e l y minor s u b s t r a t e s f o r o x i d a t i o n i n f i s h compared t o amino a c i d s and f a t t y a c i d s (Cowey et a l . , l977;Walton and Cowey, 1982). The r a t e of l a c t a t e t u r n o v e r d u r i n g h y p o x i a r i s e s s i g n i f i c a n t l y (Table 13). S i n c e the a n i m a l i s h y p o x i c , i s i t s t i l l p o s s i b l e f o r the l a c t a t e t o be o x i d i z e d ? W i t h a t u r n o v e r r a t e of 21 Mmole/min./kg, assuming .17 A T P / l a c t a t e , and keeping the p r o p o r t i o n o x i d i z e d a t 50% (Freminet et a l . , 1974;Issekutz 136 e t a l . , 1976), one a r r i v e s a t 5355 Mmoles/min./kg ATP p r o d u c t i o n . I f 6 moles of ATP are produced per mole of 0 2 when l a c t a t e i s o x i d i z e d , then the oxygen needed t o o x i d i z e the l a c t a t e would be 895 Mmoles/min./kg. T h i s would mean t h a t the p r o p o r t i o n of oxygen uptake devoted t o l a c t a t e o x i d a t i o n would have r i s e n t o almost 75% of the t o t a l m e t a b o l i c r a t e . As w i t h g l u c o s e , t h i s v a l u e i s l i k e l y t o be too h i g h and may i n d i c a t e t h a t the p r o p o r t i o n of the t u r n o v e r which i s o x i d i z e d i s lower f o r t r o u t than f o r mammals. I t i s now p o s s i b l e t o propose t h a t the m e t a b o l i c r a t e of the t r o u t has a c t u a l l y i n c r e a s e d d u r i n g h y p o x i a over the c o n t r o l v a l u e s . The f i r s t o b s e r v a t i o n i s t h a t oxygen uptake does not d e c l i n e . One may assume, t h e r e f o r e , t h a t o x i d a t i v e energy p r o d u c t i o n i s m a i n t a i n e d . The second o b s e r v a t i o n i s t h a t l a c t a t e p r o d u c t i o n i n c r e a s e d d u r i n g h y p o x i a . T h i s i s s t r o n g e v i d e n c e t h a t the r a t e of g l y c o l y s i s has a l s o i n c r e a s e d . T h i s means t h a t t h e r e i s some ATP produced a e r o b i c a l l y , a t a r a t e which u t i l i z e s oxygen a t the c o n t r o l r a t e s , p l u s some ATP which i s produced a n a e r o b i c a l l y . In summary, t h e r e a r e t i s s u e s which i n c r e a s e t h e i r r a t e s of l a c t a t e p r o d u c t i o n d u r i n g h y p o x i a , and the r e s u l t i s t h a t the m e t a b o l i c r a t e of the t r o u t i s l i k e l y t o be i n c r e a s e d d u r i n g t h i s s t r e s s . The l a c k of an i n c r e a s e i n the t u r n o v e r r a t e of g l u c o s e s u p p o r t s the c o n c l u s i o n made i n S e c t i o n 3, which s t a t e d t h a t the l i v e r i s not a c t i n g as a s t o r e of g l u c o s e f o r use i n the o t h e r t i s s u e s . 1 37 GENERAL DISCUSSION The g o a l of t h i s t h e s i s was t o d e v e l o p a g r e a t e r u n d e r s t a n d i n g of the b i o c h e m i c a l mechanisms t h a t l e a d t o i n c r e a s e d h y p o x i c t o l e r a n c e i n a n i m a l s . In o r d e r t o a c h i e v e t h i s g o a l , the i n t e r - t i s s u e m e t a b o l i c responses t o low e n v i r o n m e n t a l oxygen t e n s i o n s were examined i n two f i s h s p e c i e s which have d i f f e r i n g c a p a c i t i e s f o r h y p o x i a t o l e r a n c e . From t h e s e r e s u l t s , g e n e r a l i z a t i o n s were made about the p o s s i b l e responses of any c e l l t o a h y p o x i c s t r e s s . The data i n t h i s t h e s i s , p l u s the f i n d i n g s of o t h e r i n v e s t i g a t o r s , p r o v i d e i n c r e a s i n g e v i d e n c e s u p p o r t i n g the premise t h a t r e g u l a t i o n of energy u t i l i z a t i o n may be more i m p o r t a n t i n p r o l o n g i n g h y p o x i a s u r v i v a l than the r e g u l a t i o n of energy p r o d u c t i o n . The premi se. V a r i o u s meanings a s s o c i a t e d w i t h the word " h y p o x i a " , and the m u l t i t u d e of s t r a t e g i e s u t i l i z e d by a n i m a l s t o s u r v i v e d u r i n g h y p o x i c e p i s o d e s were o u t l i n e d i n the i n t r o d u c t i o n . I t i s a p p r o p r i a t e , a t t h i s p o i n t , t o r e i t e r a t e one p o i n t . The type of h y p o x i c d y s o x i a d i s c u s s e d i n t h i s t h e s i s i s not t h a t d e v e l o p e d as a r e s u l t of a t i s s u e i n c r e a s i n g i t s energy r e q u i r e m e n t s , and t h e r e f o r e i t s oxygen r e q u i r e m e n t s , above the a v a i l a b l e s u p p l y (as i n e x e r c i s e ) . I n s t e a d , the s u b j e c t i s the response of a r e s t i n g a n i m a l t o c e l l u l a r h y p o x i a . In t h i s c a s e , the combined systems of oxygen uptake and d e l i v e r y a r e not c a p a b l e of s u p p l y i n g oxygen t o the t i s s u e s at a r a t e which would a l l o w oxygen uptake t o be m a i n t a i n e d and/or which would a l l o w o x i d a t i v e m e t abolism t o s a t i s f y a l l of the c e l l u l a r 1 38 energy r e q u i r e m e n t s . There a r e two p r o c e s s e s which occur s i m u l t a n e o u s l y i n a l l l i v i n g o r g a n i s m s — e n e r g y p r o d u c t i o n , and energy u t i l i z a t i o n . O b v i o u s l y , f o r c o n t i n u e d w e l l b e i n g , t h e s e two p r o c e s s e s must be v e r y c l o s e l y matched. However, i t i s im p o r t a n t t h a t the r a t e of energy p r o d u c t i o n does not d i c t a t e the m e t a b o l i c r e q u i r e m e n t s of the c e l l . I f t h i s were the case then the c e l l would not be a b l e t o respond t o changes i n i n t e r n a l c e l l u l a r p r o c e s s e s by a d j u s t i n g the p r o d u c t i o n r a t e of ATP. I t would be t r a p p e d i n a s i t u a t i o n where, i f energy u t i l i z a t i o n was c a l l e d f o r by an e x t e r n a l s t i m u l u s , the r a t e of energy u t i l i z a t i o n and p r o d u c t i o n would r a p i d l y d e v i a t e . C o n v e r s e l y , i f oxygen s u p p l y i n c r e a s e d f o r any r e a s o n , l a r g e amounts of ATP may be produced when i t i s not r e q u i r e d . I t i s o b v i o u s l y not s u f f i c i e n t f o r the s u r v i v a l of the c e l l t o c o u p l e the r a t e s of energy p r o d u c t i o n w i t h the r a t e s of energy u t i l i z a t i o n . What i s needed i s the c a p a c i t y t o match ATP u t i l i z a t i o n w i t h the requirement f o r ATP. I t i s i m p o r t a n t t o s t r e s s t h a t the term " r e q u i r e m e n t " does not mean b a s a l m e t a b o l i c r a t e . The term i s used i n the c o n t e x t of the a c t u a l r equirement f o r ATP a t t h a t moment. I f a muscle i s c o n t r a c t i n g , or i f the ki d n e y t u b u l e s a r e pumping i o n s , then the r e q u i r e m e n t s of those t i s s u e s a r e e l e v a t e d above some b a s a l l e v e l . I f r e q u i r e m e n t s d e c r e a s e d u r i n g oxygen l i m i t a t i o n then both u t i l i z a t i o n and p r o d u c t i o n c o u l d d e c l i n e i n synchrony, t h e r e b y m a i n t a i n i n g the ba l a n c e between t h e s e two p r o c e s s e s . Thus, one way t o v i s u a l i z e the c o n t r o l of energy metabolism i s t o p i c t u r e r e q u i r e m e n t s 139 d i c t a t i n g the l e v e l a t which p r o d u c t i o n and u t i l i z a t i o n must be b a l a n c e d . In most of the l i t e r a t u r e on m e t a b o l i c responses t o h y p o x i a , the s t r a t e g y which i s d e s c r i b e d i s one of m a i n t a i n i n g ATP p r o d u c t i o n i n the f a c e of d e c l i n i n g o x i d a t i v e m e t a b o l i s m (Hochachka, 1982;Johnston, 1975a;Johnston, 1975b;Jorgensen and M u s t a f a , 1980;McDougal et a l . , 1968) In o r d e r t o do t h i s , the organism must be a b l e t o i n c r e a s e g l y c o l y t i c f l u x t o m a i n t a i n the b a l a n c e between ATP p r o d u c t i o n and ATP u t i l i z a t i o n . I t has been p r e v i o u s l y n o t e d , however, t h a t t h e r e i s an o t h e r s o l u t i o n ( J a c k s o n , 1968,-Robin, l980;Van den. T h i H a r t , 1982) The organism may be a b l e t o a d j u s t the p a t t e r n of c e l l u l a r m etabolism i n such a way as t o de c r e a s e the r a t e of ATP u t i l i z a t i o n when p r o d u c t i o n i s f o r c e d t o d e c l i n e . T h i s would a l s o m a i n t a i n the b a l a n c e between u t i l i z a t i o n and p r o d u c t i o n . A n i m a l s which u t i l i z e the s t r a t e g y of d e c r e a s i n g the r e q u i r e m e n t s of the c e l l when the c e l l i s oxygen l i m i t e d may be c a l l e d energy c o n f o r m e r s . T h i s term i s a m o d i f i c a t i o n of the term oxygen conformers and i s used i n p r e f e r e n c e here because the l a t t e r term does not p r e c l u d e the p o s s i b i l i t y t h a t r e q u i r e m e n t s a r e b e i n g made up i n whole or i n p a r t v i a a n a e r o b i c energy p r o d u c t i o n . Thus a l l energy conformers a r e oxygen conformers but the conv e r s e need not be t r u e . An a n i m a l may be an oxygen conformer and have a n a e r o b i c a l l y produced ATP i n c r e a s i n g the energy s u p p l y above t h a t which c o u l d be produced o x i d a t i v e l y . T h i s s t r a t e g y may be in v o k e d i n the whole organism, a t the 140 t i s s u e l e v e l , or a t the c e l l u l a r l e v e l . I t i s p o s s i b l e t h a t an a n i m a l may be an energy conformer at the whole body l e v e l but s t i l l c o n t a i n t i s s u e s which do not conform; or have one t i s s u e d rop i t s r e l a t i v e m e t a b o l i c r a t e f a r below t h a t of o t h e r t i s s u e s — t h e r e b y a l l o w i n g t h o s e o t h e r s t o m a i n t a i n (or i n c r e a s e ) t h e i r m e t a b o l i c r a t e . C o n v e r s e l y , i t i s p o s s i b l e t h a t many t i s s u e s a re conformers even though the whole a n i m a l i s n o t . T h i s c o u l d be e x h i b i t e d i n a s i t u a t i o n where, when e n v i r o n m e n t a l h y p o x i a o c c u r s , the a n i m a l a t t e m p t s t o m a i n t a i n oxygen uptake t o working t i s s u e s w h i l e i n t e r n a l p h y s i o l o g i c a l and/or b i o c h e m i c a l a d j u s t m e n t s o c c u r which l i m i t the r a t e of oxygen u t i l i z a t i o n by o t h e r t i s s u e s . Responses by the l u n q f i s h and t r o u t . Now t h a t the premise has been d e f i n e d , i t i s p o s s i b l e t o r e t u r n t o the e x p e r i m e n t a l e v i d e n c e and examine i t w i t h i n the framework of energy c o n f o r m i t y . The l u n g f i s h has a t o l e r a n c e t o hyp o x i a which g r e a t l y exceeds t h a t of the t r o u t . I s t h i s t o l e r a n c e due l a r g e l y t o the c a p a c i t y of the a n i m a l t o r e g u l a t e ATP r e q u i r e m e n t s i n the face of d e c l i n i n g ATP p r o d u c t i o n r a t e s ? As was p o i n t e d out i n S e c t i o n 2, the bulk of the muscle mass of the l u n g f i s h i s c o m p r i s e d of whi t e muscle f i b r e s . White f i b r e s a r e the l e a s t o x i d i t i v e of a l l the muscle types and l u n g f i s h w h i t e f i b r e s have a lower o x i d a t i v e c a p a c i t y than t h a t of most o t h e r f i s h s p e c i e s ( S e c t i o n 2 ) . T h i s statement i s based upon the r e l a t i v e l y low m i t o c h o n d r i a l d e n s i t y , and c a p i l l a r i t y , as w e l l as the the r e l a t i v e l a c k of o b s e r v a b l e glycogen and 141 l i p i d d e p o s i t s (compared w i t h o t h e r f i s h ) . One c o n c l u s i o n i s t h a t the l u n g f i s h has a body m u s c u l a t u r e which can s u r v i v e w i t h a r e l a t i v e l y s m a l l f l u x of oxygen and, most l i k e l y , has a v e r y low m e t a b o l i c r a t e . T h i s p i c t u r e of the l u n g f i s h w h i t e muscle i s s u p p o r t e d by e n z y m a t i c and m e t a b o l i t e d a t a . The a c t i v i t i e s of o x i d a t i v e enzymes are low, as would be e x p e c t e d f o r a g l y c o l y t i c t i s s u e . The p e c u l i a r i t y i s t h a t the a c t i v i t i e s of the g l y c o l y t i c enzymes are a l s o low. I t appears t h a t the muscle mass can o n l y m a i n t a i n a low m e t a b o l i c r a t e as i n d i c a t e d by the l a c k of a c a p a c i t y f o r both a e r o b i c and f e r m e n t a t i v e energy p r o d u c t i o n . Any t i s s u e w i t h such a low r a t e of s t e a d y s t a t e energy p r o d u c t i o n s h o u l d be a b l e t o s u r v i v e w i t h a low r a t e of energy u t i l i z a t i o n . I t i s p o s s i b l e t h a t the b r a i n a l s o i s adapted t o a lower than u s u a l r a t e of energy p r o d u c t i o n . T h i s i s based upon the low a c t i v i t i e s of the o x i d a t i v e enzymes. The c a p a c i t y f o r g l y c o l y s i s , however, i s e l e v a t e d . The h e a r t e x h i b i t s a good p o t e n t i a l f o r both a e r o b i c and a n a e r o b i c m a tabolism. When the l u n g f i s h i s s u b j e c t e d t o h y p o x i a , the muscle does not show s t r e s s r e l a t e d m e t a b o l i t e changes. U s i n g the d e f i n i t i o n of h y p o x i c d y s o x i a o u t l i n e d i n the b e g i n n i n g of the t h e s i s , t h i s would i n d i c a t e t h a t the muscle i s e i t h e r not s u f f e r i n g from an oxygen d e f i c i t , or i s becoming an energy conformer. C o n c u r r e n t l y , one sees t h a t the b r a i n , h e a r t , l i v e r , and b l o o d a r e a l l showing s i g n s t h a t the f i s h i s i n a s e v e r e l y h y p o x i c s t a t e . In a d d i t i o n , t h e r e i s e v i d e n c e based upon m e t a b o l i t e c o n c e n t r a t i o n s which s u g g e s t s t h a t the w h i t e muscle 142 i s h y p o p e r f u s e d d u r i n g t h i s t i m e . The b u l k of the body i s h y p o x i c , the muscle does not even have a c c e s s t o the l i m i t e d s u p p l y of oxygen and f u e l t h a t i s a v a i l a b l e t o the r e s t of the body, and y e t the muscle does not e x h i b i t a marked a c t i v a t i o n of g l y c o l y s i s . I f oxygen i s t o t a l l y l a c k i n g , t h i s means a drop i n ATP c y c l i n g r a t e to 1/12 of the normoxic r a t e . Thus muscle, which makes up the b u l k of the body, has such a low energy requ i r e m e n t d u r i n g f o r c e d submergence t h a t m e t a b o l i t e changes cannot even be d e t e c t e d . In c o m b i n a t i o n , these p i e c e s of ev i d e n c e s t r o n g l y suggest t h a t the l u n g f i s h w h i t e muscle i s an energy c o n f o r m i n g t i s s u e when the l u n g f i s h i s f o r c e d t o water b r e a t h . T i s s u e s o t h e r than the w h i t e muscle appear t o be the l a c t a t e p r o d u c e r s . Of the t i s s u e s examined, the b r a i n and h e a r t a r e prime c a n d i d a t e s . Both t i s s u e s d i s p l a y s i g n s of m e t a b o l i c s t r e s s and both t i s s u e s must s t i l l remain a c t i v e t o some degree. Thus, the m e t a b o l i c r a t e of the s e two t i s s u e s remains h i g h enough t o r e q u i r e s u p p l e m e n t a t i o n of a e r o b i c ATP p r o d u c t i o n w i t h ATP produced from a n a e r o b i c m e t a b o l i s m . The l i v e r i s r e s p o n d i n g by c o n v e r t i n g i t s g l y c o g e n s t o r e i n t o g l u c o s e f o r use i n o t h e r t i s s u e s . S i n c e the c o n v e r s i o n of g l y c o g e n t o g l u c o s e does not r e q u i r e ATP i t i s p o s s i b l e t h a t the l i v e r i s becoming an energy conformer. Two major advantages of removing the muscle mass from the i n t e r t i s s u e exchange i s t h a t ( i ) organs w i t h a s m a l l mass but which have h i g h energy r e q u i r e m e n t s can have a c c e s s t o the a v a i l a b l e f u e l , and ( i i ) n e i t h e r these organs nor the plasma become s a t u r a t e d w i t h the l a r g e q u a n t i t y of u n d e s i r a b l e end-143 p r o d u c t s which may be produced i n the muscle. The t r o u t , on the o t h e r hand, e x h i b i t s s e v e r e s t r e s s when a r t e r i a l oxygen t e n s i o n s f a l l . E v ery t i s s u e examined, i n c l u d i n g the w h i t e muscle, shows some s i g n of h y p o x i c s t r e s s . The t r o u t , u n l i k e the l u n g f i s h , does not have a w h i t e muscle mass which i s composed s o l e l y of w h i t e f i b r e s . I n s t e a d t h e r e i s a m i x t u r e of w h i t e and r e d t y p e s . The r e d f i b r e s are c o n s i d e r e d t o be o x i d a t i v e and t h e i r p r e s e nce would, i n i t s e l f , i n c r e a s e the o x i d a t i v e r e q u i r e m e n t s of the muscle mass. The f a c t t h a t the a x i a l r e d mass showed l e s s of a change d u r i n g h y p o x i a than t h a t i n the w h i t e mass s u g g e s t s t h a t the r e d muscle f i b r e s b u r i e d w i t h i n the w h i t e muscle a r e n o t , i n t h e m s e l v e s , the reason f o r the o b s e r v e d changes i n t h e w h i t e muscle r e s p o n s e . The m e t a b o l i t e c o n c e n t r a t i o n s i n the t r o u t b r a i n and h e a r t i n d i c a t e t h e s e organs were h y p o x i c , but c a l c u l a t i o n s of m e t a b o l i c r a t e suggest t h a t they c o u l d not be the major source of the whole-body l a c t a t e l o a d . To e x p l a i n the end-product a c c u m u l a t i o n i t i s n e c e s s a r y t o c o n c l u d e t h a t g l y c o l y s i s i s a c t i v a t e d i n the w h i t e muscle and t h a t the muscle and b l o o d m e t a b o l i t e p o o l s a r e not i s o l a t e d from each o t h e r d u r i n g acute h y p o x i a ( i n c o n t r a s t t o the s i t u a t i o n i n the l u n g f i s h ) . S i n c e w h i t e muscle i s the o n l y t i s s u e w i t h enough c a r b o h y d r a t e s u b s t r a t e t o account f o r the l a c t a t e formed, i t i s p r o b a b l e t h a t the l a c t a t e produced i n the muscle i s a b l e t o e n t e r the b l o o d p o o l . The l a c t a t e g r a d i e n t s , which remain f a v o r a b l e f o r l a c t a t e e f f l u x from the muscle, support t h i s s u g g e s t i o n . Assuming t h a t muscle g l y c o g e n i s m o b i l i z e d , the l a c k of glucose-6-phosphatase 144 i n the t i s s u e p r e c l u d e s the use of t h i s f u e l i n any t i s s u e except the muscle. Thus i t i s u n l i k e l y t h a t the l a c t a t e which r e s u l t s from the muscle g l y c o g e n s t o r e i s produced anywhere but w i t h i n the muscle i t s e l f . M e t a b o l i t e data o n l y g i v e an i n s t a n t a n e o u s p i c t u r e of the m e t a b o l i c s i t u a t i o n a t any g i v e n t i m e . I t cannot i n d i c a t e f l u x r a t e s . However, d i r e c t e s t i m a t e s of t u r n o v e r r a t e s of l a c t a t e i n the t r o u t a l s o i n d i c a t e t h a t g l y c o l y s i s i s a c t i v a t e d d u r i n g h y p o x i a . A l t h o u g h g l u c o s e t u r n o v e r does not r i s e , t h i s i s not s u r p r i s i n g , s i n c e l i v e r g l y c o g e n i s not m o b i l i z e d and t h e r e does not appear t o be a major requirement f o r an i n c r e a s e d f l u x of blood-borne g l u c o s e . There a r e marked d i f f e r e n c e s i n the responses of l i v e r and w h i t e muscle i n l u n g f i s h and t r o u t t o the s t r e s s of h y p o x i a . The l i v e r of the h y p o x i a l u n g f i s h g r a d u a l l y r e l e a s e s g l u c o s e f o r use i n o t h e r t i s s u e s w h i l e the l i v e r of the t r o u t does not appear t o have t h i s f u n c t i o n . In t h i s r e g a r d , i t i s worth m e n t i o n i n g t h a t the l i v e r does not c o n t a i n the l a r g e s t s t o r e of g l y c o g e n on an organ b a s i s . White muscle has more g l y c o g e n . The d i f f e r i n g responses between t r o u t and l u n g f i s h muscle masses are l i k e l y t o make the most s i g n i f i c a n t c o n t r i b u t i o n t o the r e l a t i v e h y p o x i a t o l e r a n c e of these a n i m a l s . In the l u n g f i s h , the a x i a l muscle mass i s a b l e t o endure the h y p o x i c e p i s o d e by a p p a r e n t l y becoming an energy c o n f o r m i n g t i s s u e , u n l i k e the t r o u t muscle. T h i s i n a b i l i t y i n the t r o u t i s c o n s i d e r e d , h e r e , t o be the main m e t a b o l i c reason why t h i s s p e c i e s i s not v e r y t o l e r a n t of acute h y p o x i a . The ATP-turnover r a t e s (and 145 m e t a b o l i c r e q u i r e m e n t s f o r ATP) are s w i t c h e d down i n the l u n g f i s h muscle when the r a t e of a e r o b i c energy p r o d u c t i o n d e c l i n e s . T h i s c a p a c i t y f o r m e t a b o l i c d e p r e s s i o n i s of paramount importance f o r s u r v i v a l . The impact of t h i s c a p a c i t y i s i n d i c a t e d by the t r o u t b l o o d l a c t a t e d a t a i n S e c t i o n 3. I n t e r e s t i n g l y , the c o n c e n t r a t i o n s of b l o o d l a c t a t e r e s u l t i n g from a s e t exposure t o h y p o x i a change markedly depending upon the time of onset of t h e exposure. The c o n c e n t r a t i o n s i n samples taken from f i s h exposed e a r l y i n the morning were r o u g h l y 60% of those taken from f i s h exposed near mid-day. B r e t t and Z a l a (1975) showed t h a t d a i l y mid-day f e e d i n g (which was used here) causes a d i u r n a l c y c l e of met a b o l i c , r a t e w i t h the peak b e i n g r o u g h l y d o u b l e the minimum v a l u e and c o i n c i d i n g w i t h the f e e d i n g t i m e . Thus, t h i s a r t i f i c i a l m a n i p u l a t i o n of m e t a b o l i c r a t e showed the b e n e f i c i a l e f f e c t s which a r e d u c t i o n i n m e t a b o l i c r a t e may have upon the c a p a c i t y f o r s u r v i v i n g h y p o x i a . M e t a b o l i c d e p r e s s i o n - - o t h e r c a s e s . J u s t how wide - s p r e a d i s the c a p a c i t y f o r r e d u c i n g m e t a b o l i c r a t e i n response t o a d i m i n i s h e d oxygen s u p p l y ? I t would be u s e f u l t o be a b l e t o answer the q u e s t i o n , "What i s the r e l a t i v e i mportance of m e t a b o l i c d e p r e s s i o n t o s u r v i v a l d u r i n g h y p o x i a or a n o x i a ? " . One i n d i c a t i o n can be p r o v i d e d by the f o l l o w i n g c a l c u l a t i o n s . I f a l l of the m e t a b o l i c a d a p t a t i o n s f o r s u r v i v i n g c e l l u l a r h y p o x i a were i n v o k e d w i t h i n a c e l l ( e x c e pt m e t a b o l i c d e p r e s s i o n ) , i t c o u l d s t i l l o n l y l a s t as l o n g as t h e r e i s f u e l 1 46 f o r g l y c o l y s i s . For the sake of argument, l e t us assume t h a t the c e l l has o p t i m i z e d a l l f a c e t s of a n a e r o b i c energy p r o d u c t i o n and can m e t a b o l i z e u n t i l a l l of the f u e l i s gone. In a d d i t i o n , the s i m p l i f y i n g assumption t h a t the metabolism i s a n a e r o b i c w i l l be made. Thus, the maximum a n o x i a e x c u r s i o n time i s e q u a l t o the p e r i o d d u r i n g which the g l y c o g e n s t o r e can s u p p l y b a s a l energy r e q u i r e m e n t s . One can then compare the t h e o r e t i c a l maximum w i t h the a c t u a l e x c u r s i o n time t o see i f t h e r e i s enough a v a i l a b l e s u b s t r a t e . I f the maximum e x c u r s i o n time i s l o n g e r than the c a l c u l a t e d e x c u r s i o n time then m e t a b o l i c d e p r e s s i o n has o c c u r r e d . For the t u r t l e , l i v e r g l y c o g e n l e v e l s i n the normoxic s t a t e a r e about 880 Mmole/gm (Daw e t . a l . , 1967). A 100 gm t u r t l e ( e x c l u d i n g the s h e l l ) has about 8.6 gm of l i v e r , and thus about 7,600 Mmoles of g l y c o g e n ( g l u c o s y l u n i t s ) . the r e s t i n g m e t a b o l i c r a t e of t u r t l e s a t 3°C and a P a 0 2 of about 70 mmHg can be e s t i m a t e d as 0.036 Mmoles ATP/gm/min., assuming a Q10 f o r r e s p i r a t o r y metabolism of about 2 ( J a c k s o n , 1971); t h i s r a t e i s e q u i v a l e n t t o 5180 Mmoles ATP/100 gm t u r t l e / d a y . At t h i s m e t a b o l i c r a t e , l i v e r g l y c o g e n s t o r e s a re adequate t o m a i n t a i n a 100 gm a n o x i c t u r t l e f o r o n l y about 2.9 days. Other body s t o r e s of g l y c o g e n may extend t h i s t i me p e r i o d , but i n t i s s u e s such as the h e a r t , g l y c o g e n s t o r e s a r e l a r g e l y d e p l e t e d a f t e r a few hours of a n o x i a (Daw et.. a_l. , 1967). In s h o r t - d u r a t i o n d i v i n g a t 24°C, the m e t a b o l i c r a t e i s known t o drop t o about 15% of p r e d i v e l e v e l s (Jackson and S c h m i d t - N i e l s e n , 1966). I f these r e s u l t s a r e e x t r a p o l a t e d t o the s i t u a t i o n a t 3°C, then the 147 a n o x i a t o l e r a n c e of the t u r t l e c o u l d be extended by 6- t o 7-f o l d , i . e . t o about 19 days. I t i s apparent t h a t a r e d u c t i o n i n m e t a b o l i c • r a t e i s f a r more s i g n i f i c a n t i n p r o l o n g i n g s u r v i v a l than i s the l a r g e s t o r e of l i v e r g l y c o g e n . Even more remarkable i s the o b s e r v a t i o n t h a t t u r t l e s a t t h i s t emperature have s u r v i v e d submersion f o r as l o n g as 6 months ( U l t s c h and J a c k s o n , 1982). S i n c e no unusual m e t a b o l i c pathway i s p r o v i d i n g an i n c r e a s e d e n e r g e t i c e f f i c i e n c y , one i s l e f t w i t h the c o n c l u s i o n t h a t the t u r t l e has undergone an i m p r e s s i v e m e t a b o l i c d e p r e s s i o n . T h i s s u r v i v a l time would r e q u i r e a m e t a b o l i c d e p r e s s i o n t o 1/60 of r e s t i n g r a t e s and i t i s twenty f o l d l o n g e r than can be accounted f o r by s u b s t r a t e s u p p l y a l o n e . None of the o t h e r mechanisms of e x t e n d i n g a n o x i a t o l e r a n c e c o u l d be n e a r l y so e f f e c t i v e . There i s enough i n f o r m a t i o n t o do the same c a l c u l a t i o n s u s i n g the g o l d f i s h as a s u b j e c t . A c o l d - a c c l i m a t e d i n d i v i d u a l can s u r v i v e from 4-5 days (Shoubridge and Hochachka, 1981) t o s e v e r a l weeks of t o t a l a n o x i a (Walker and Johanson, 1977). L i v e r g l y c o g e n l e v e l s f o r w i n t e r a c c l i m a t e d g o l d f i s h a r e about 1300 mM; a 100 gm g o l d f i s h has about 6 gm of l i v e r , and thus about 7800 Mmoles glycogen ( t h i s w i l l y i e l d 15,600 Mmoles of ATP a t the t i s s u e s ) . The r e s t i n g m e t a b o l i c r a t e of the g o l d f i s h at 4°C i s 0.05 Mmoles ATP/gm/min. which i s e q u i v i l e n t t o about 7,200 Mmoles ATP/100 gm f i s h / d a y . At t h i s m e t a b o l i c r a t e , the f i s h would o n l y be a b l e t o s u r v i v e f o r 2.2 days. A m e t a b o l i c d e p r e s s i o n of 5 - f o l d would be r e q u i r e d f o r the a n i m a l t o s u r v i v e 11 days of a n o x i a ; both t h i s m e t a b o l i c r a t e and t h i s degree of 1 48 a n o x i a t o l e r a n c e a r e w i t h i n r e p o r t e d e s t i m a t e s f o r t h i s s p e c i e s ( A n d e r s o n , 1 9 7 5 ) . M y t i l u s sp., the common mussel, i s a l s o a good f a c u l t a t i v e anaerobe. I f we assume t h a t i t i s o b t a i n i n g i t s energy from c l a s s i c a l g l y c o l y s i s , then the 550 Mmole of g l y c o g e n i n the mantle and l i v e r would s u p p l y r e s t i n g m e t a b o l i c r a t e s d u r i n g a n o x i a f o r 3 days (De Zwann, 1983;Hochachka, 1982). I f we assume t h a t a l l of the g l u c o s e i s m e t a b o l i z e d t o p r o p i o n a t e , then the a n i m a l c o u l d l a s t f o r 9 days. On the b a s i s of ATP t u r n o v e r r a t e s , De Zwann and Wijsman (1976) e s t i m a t e t h a t the m e t a b o l i c r a t e drops t o 1/20 of r e s t i n g r a t e s d u r i n g a n o x i a . At t h i s lowered m e t a b o l i c r a t e , the mussel now has enough s u b s t r a t e t o s u r v i v e from 60-150 days, depending upon the f e r m e n t a t i o n p a t h used. Many o t h e r a n i m a l s undergo m e t a b o l i c d e p r e s s i o n d u r i n g h y p o x i a . L e i v e s t a d (1960) used d i r e c t c a l o r i m e t r y t o measure the m e t a b o l i c r a t e of a submerged to a d Bufo m a r i n u s , and found t h a t i t drops to r o u g h l y 20% of the r e s t i n g l e v e l . Sturgeon become oxygen conformers when s u b j e c t e d t o reduced oxgyen t e n s i o n s but show no e v i d e n c e of i n c r e a s i n g a r t e r i a l l a c t a t e c o n c e n t r a t i o n s (Burggren and R a n d a l l , 1978). Kooyman e t . a l . (1980) have suggested t h a t a mammalian d i v e r , the Weddell s e a l , e x h i b i t s modest o v e r a l l m e t a b o l i c d e p r e s s i o n when underwater, even though the swimming muscles a r e a c t i v e . A c t i v e ground s q u i r r e l s ( a n i m a l s c a p a b l e of h i b e r n a t i o n ) e x h i b i t a drop i n body temperature when s u b j e c t e d t o h y p o x i a ( F a l e s c h i n i and W h i t t e n , 1975). 149 These cases p a l e , however, when compared w i t h the c a p a c i t y f o r m e t a b o l i c r e d u c t i o n e x h i b i t e d by the b r i n e shrimp embryo ( A r t e m i a s a l i n a ) . T h i s organism can e x i s t i n a n o x i a f o r a t l e a s t 5 months w i t h l i t t l e e f f e c t upon i t s c a r b o h y d r a t e or l i p i d r e s e r v e s ( S t o c c o e_t. a l . , 1972). T h i s i m p l i e s a d r a s t i c c u r t a i l m e n t of s u b s t r a t e u t i l i z a t i o n o r , i n o t h e r words, m e t a b o l i c r a t e . The energy c u r r e n c y here seems t o be d i g u a n o s i n e 5 ' - t e t r a p h o s p h a t e (d5pppp) which i s broken down to GTP and GMP, thus c r e a t i n g a more r e c o g n i z a b l e phosphate c u r r e n c y . D u r i n g 4 months of a n o x i a about 50% of the t o t a l a v a i l a b l e phosphate-bond energy i s used, m o s t l y i n the form of d5pppp. T h i s i s i n i t s e l f a low r a t e of metabolism but i t becomes even more s t a r t l i n g when one sees t h a t the d e p l e t i o n o c c u r s m a i n l y i n the f i r s t 2 weeks. The average r a t e of h i g h energy phosphate d e p l e t i o n between days 1-3 of a n o x i a , and between day 56 and 110, i s 1.24 and 0.037 jumoles P/100,000 embryos per day r e s p e c t i v e l y ( S t o c c o e t . a_l. , 1972). The above examples i n d i c a t e t h a t the c a p a c i t y f o r m e t a b o l i c d e p r e s s i o n i n the face of d e c l i n i n g oxygen a v a i l a b i l i t y i s wide-s p r e a d . As was s t a t e d above, t h i s d e p r e s s i o n i s a t t a i n e d by the c e l l a d j u s t i n g r a t e s of energy r e q u i r e m e n t s t o match the r a t e of energy p r o d u c t i o n . The c e l l has two l e v e l s of energy r e q u i r e m e n t ; a minimum re q u i r e m e n t needed t o s u r v i v e and any a d d i t i o n a l requirement needed t o pe r f o r m subsequent work. One may s u r m i s e t h a t the s t r a t e g y of r e d u c i n g r e q u i r e m e n t s t o match the r a t e of ATP p r o d u c t i o n can o n l y proceed so f a r as p r o d u c t i o n does not f a l l below t h i s minimum r e q u i r e m e n t . I t has been 150 proposed t h a t m a i n t a i n i n g i o n g r a d i e n t s a c r o s s membranes may consume a major p r o p o r t i o n of the r e s t i n g energy budget of a c e l l ( B r e z i s e_t a l . , 1 9 8 4 a ; Brezis et a l . , 1984b), and so one may s u r m i s e t h a t i f a c e l l i s a b l e t o t o l e r a t e a r e d u c t i o n of t h e s e g r a d i e n t s then the minimum energy r e q u i r e m e n t s of the c e l l may be c o r r e s p o n d i n g l y lower and the c a p a c i t y f o r h y p o x i a s u r v i v a l s h o u l d be enhanced. Work by B r e z i s et a l . , (1984b) has shown t h a t the k i d n e y may be a good mammalian model t o use f o r the e x a m i n a t i o n of t h i s p r o p o s a l . They s t u d i e d an i s o l a t e d , p e r f u s e d r a t k i d n e y w h i l e m a n i p u l a t i n g work (by a d j u s t i n g the r a t e of g l o m e r u l a r f i l t r a t i o n ) . They found t h a t h y p o x i c damage t o the m e d u l l a r y t h i c k a s c e n d i n g l i m b (mTAL) i s l e s s s e v e r e i f a p e r f u s i o n medium i s used which the kidney need not f i l t e r (low w o r k ) . T h i s t o l e r a n c e was f u r t h e r enhanced i f the k i d n e y was p e r f u s e d w i t h ouabain b e f o r e h y p o x i c exposure, thus r e d u c i n g the energy e x p e n d i t u r e of the sodium pump. S i n c e t h e s e t r e a t m e n t s do not s i g n i f i c a n t l y reduce oxygen uptake d u r i n g h y p o x i a , but do reduce oxygen uptake d u r i n g normoxic p e r f u s i o n (Swartz e t a l . , 1 9 7 8 ; E v e l o f f e t a l . , 1981), i t may be proposed t h a t t h e r e i s no l o n g e r a match between ATP p r o d u c t i o n and u t i l i z a t i o n d u r i n g t h e s e e x p o s u r e s . Thus, when energy r e q u i r e m e n t s a r e reduced, oxygen uptake s t i l l does not d e c l i n e because the r e q u i r e m e n t s f o r ATP a r e j u s t b e i n g brought i n t o l i n e w i t h the r a t e of ATP p r o d u c t i o n . C o n v e r s e l y , B r e z i s et a l . , (1984a) i n c r e a s e d the work r a t e of a kidney by i n c u b a t i n g w i t h p o l y e n e a n t i b i o t i c s . These m a n i p u l a t i o n s i n c r e a s e the p e r m e a b i l i t y of c e l l membranes, 151 which l e a d t o a compensatory i n c r e a s e i n the r a t e of a c t i v e t r a n s p o r t ( t h e r e b y i n c r e a s i n g oxygen u p t a k e ) . T h i s procedure r e s u l t s i n h i s t o l o g i c a l damage which i s s i m i l a r i n appearance t o t h a t which o c c u r s w i t h severe h y p o x i a and e x a c e r b a t e s the h i s t o l o g i c a l damage caused by moderate h y p o x i a . When the p r e p a r a t i o n s a re p e r f u s e d w i t h ouabain (thus r e d u c i n g the a c t i v i t y of the membrane pumps), oxygen uptake d e c l i n e s and the i n t e g r i t y of the kid n e y i s m a i n t a i n e d ( o s t e n s i b l y because the r a t e s of energy p r o d u c t i o n a g a i n match the energy r e q u i r e m e n t s of the c e l l ) . These e x p e r i m e n t a l m a n i p u l a t i o n s of work r a t e ( r e q u i r e m e n t s ) u n d e r s c o r e the d e t r i m e n t a l e f f e c t s of u n c o u p l i n g the r e l a t i o n s h i p between energy r e q u i r e m e n t s and the r a t e s of energy p r o d u c t i o n . C o n c l u s i o n . I t i s apparent t h a t h y p o x i a t o l e r a n t a n i m a l s (or t i s s u e s ) have much more e f f e c t i v e methods of p r o l o n g i n g t i s s u e s u r v i v a l d u r i n g h y p o x i a than j u s t i n c r e a s i n g the c a p a c i t y f o r a n a e r o b i c energy p r o d u c t i o n when oxygen i s l i m i t i n g . The i n i t i a l problem w i t h the onset of h y p o x i a i s t h a t a e r o b i c energy p r o d u c t i o n ceases t o meet m e t a b o l i c r e q u i r e m e n t s . However, c e l l s must m a i n t a i n a b a l a n c e between energy u t i l i z a t i o n and p r o d u c t i o n i n o r d e r t o m a i n t a i n the energy s t a t u s of the c e l l . I f , c o n c u r r e n t w i t h the h y p o x i c i n s u l t , t h e r e i s a l a r g e drop i n energy r e q u i r e m e n t s ( i n d i c a t i n g an energy c o n f o r m e r ) , then an i n c r e a s e i n a n a e r o b i c energy p r o d u c t i o n may not be needed t o m a i n t a i n the r e q u i r e d c o u p l i n g between ATP p r o d u c t i o n and u t i l i z a t i o n . In t h i s c a s e , the reduced p r o d u c t i o n of ATP from 152 a e r o b i c m e t a b o l i s m may be s u f f i c i e n t t o s u pply m e t a b o l i c demand. C o n v e r s e l y , i f energy r e q u i r e m e n t s can not be s u p p l i e d w i t h a e r o b i c m e tabolism, then the amount of a n a e r o b i c f l u x r e q u i r e d t o s u s t a i n the a n i m a l would s t i l l be l e s s than i f no m e t a b o l i c d e p r e s s i o n had o c c u r r e d . Even when oxygen c o n f o r m i t y i s noted on a whole-body l e v e l , the responses of the i n d i v i d u a l t i s s u e s may v a r y . I t i s not p o s s i b l e t o i n f e r t h a t a l l t i s s u e s have responded w i t h a d e c l i n e i n m e t a b o l i c r a t e . T h i s t h e s i s argues t h a t i n l u n g f i s h , i t i s the m e t a b o l i c response of w h i t e muscle which has the most i n f l u e n c e upon the whole-body m e t a b o l i c r a t e . In t h i s t i s s u e t h e r e appears t o have been an u n c o u p l i n g of the i n v e r s e r e l a t i o n s h i p between the r a t e of g l y c o l y t i c f l u x and the r a t e of c e l l u l a r r e s p i r a t i o n . The advantage of t h i s response i s t h a t the muscle does not u t i l i z e a l a r g e p o r t i o n of the a v a i l a b l e m e t a b o l i t e s , nor does i t produce l a r g e q u a n t i t i e s of u n d e s i r a b l e e n d - p r o d u c t s . T r o u t muscle, i n c o n t r a s t , e x h i b i t s an a c t i v a t i o n of g l y c o l y s i s d u r i n g h y p o x i a . In so d o i n g , the muscle produces s i g n i f i c a n t q u a n t i t i e s of l a c t a t e and p r o t o n s , which, i f a ccumulated t o h i g h l e v e l s , may have a d e l e t e r i o u s e f f e c t upon the o t h e r organs i n the body. S i n c e the muscle mass com p r i s e s such a l a r g e p e r c e n t a g e of the body, even low r a t e s of l a c t a t e p r o d u c t i o n may l e a d t o l a r g e a c c u m u l a t i o n s of end-products which, when t r a n s f e r r e d t o the plasma, may p e r t u r b the m etabolism and f u n c t i o n of o t h e r t i s s u e s and o rgans. Thus, i n an i n an a n i m a l which i s c a p a b l e o f _ u t i l i z i n g the 1 53 whole-body s t r a t e g y of r e d u c i n g m e t a b o l i c r a t e d u r i n g exposure t o h y p o x i a , the m e t a b o l i c responses a t the t i s s u e l e v e l may v a r y between t i s s u e s . Some t i s s u e s may show s i g n s of m a i n t a i n i n g m e t a b o l i c r a t e , and some may show s i g n s of b e i n g c a p a b l e of s u r v i v i n g w i t h a r e d u c t i o n i n m e t a b o l i c r a t e . In the l u n g f i s h , the t i s s u e which shows a c a p a c i t y f o r a r e d u c t i o n i n m e t a b o l i c r a t e i s the w h i t e muscle. 1 54 REFERENCES CITED Anderson, J.R. 1975. The a n a e r o b i c r e s i s t a n c e of C a r a s s i u s  a u r a t u s . PhD T h e s i s . A u s t r a l i a n N a t i o n a l U n i v e r s i t y , C a n b e r r a . A r m s t r o n g , M.K., Romsos, D.R. and L e v e i l l e , G.A. 1979. Gl u c o s e t u r n o v e r i n f a s t e d cynomolgus monkeys (Macaca  f a s c i c u l a r i s ) as measured by ( 2 - 3 H ) , (6- 3H) and ( U - , a C ) g l u c o s e . Comp. Biochem. P h y s i o l . 62A;1014-1015. A t k i n s o n , D.E. 1977. C e l l u l a r energy metabolism and i t s r e g u l a t i o n . Academic P r e s s . New York. Barbee, R.W., S t a i n s b y , W.N. and C h i r t e l , S.J. 1983. Dynamics of 0 2 , C 0 2 , l a c t a t e , and a c i d exchange d u r i n g c o n t r a c t i o n s and r e c o v e r y . J . A p p l . P h y s i o l . : R e s p i r a t . E n v i r o n . E x e r c i s e P h y s i o l . 54:1687-1692. B e n n e t t , A.F. 1982. The e n e r g e t i c s of r e p t i l i a n a c t i v i t y . I n : B i o l o g y of the R e p t i l i a . (Cans, C. e d . ) , Academic P r e s s , New York, N.Y.. Bergmeyer, H.V. 1974. Methods of enzymatic a n a l y s i s . V o l . 1. Academic P r e s s . New York. Bever, K., Chenoweth, M. and Dunn, A. 1977. Gl u c o s e t u r n o v e r i n k e l p bass ( P a r a l a b r a x s p . ) : i n v i v o s t u d i e s w i t h [ 6 - 3 H , 6-1 " C ] g l u c o s e . Am. J . P h y s i o l . 232:R66-R72. B i l i n s k i , E. and Jonas, R.E.E. 1972. O x i d a t i o n of l a c t a t e t o car b o n d i o x i d e by rainbow t r o u t (Salmo g a i r d n e r i ) t i s s u e s . J . F i s h . Res. Bd. Canada. 29:1467-1471. B i l i n s k i , E. and Jonas, R.E.E. 1970. E f f e c t s of coenzyme A and c a r n i t i n e on f a t t y a c i d o x i d a t i o n by rainbow t r o u t m i t o c h o n d r i a . J . F i s h . Res. Bd. Canada. 27:857-864. B l a c k , E.C., Connor, A.R., Lam, K.C. and C h i u , W.G. 1962. Changes i n g l y c o g e n , p y r u v a t e , and l a c t a t e i n rainbow t r o u t (Salmo g a i r d n e r i ) d u r i n g and f o l l o w i n g muscular a c t i v i t y . J . F i s h Res. Bd. Canada 19:409-436. 1 55 Boddeke, R., S l i j p e r , E . J . and van der S t e l t , A. 1959. H i s t o l o g i c a l c h a r a c t e r i s t i c s of the body m u s c u l a t u r e of f i s h e s i n c o n n e c t i o n w i t h t h e i r mode of l i f e . Ned. Ak. Wetwesch Ser. C 62:576-588. Boyer P.D. 1975. The enzymes. V o l . X I . O x i d a t i o n - R e d u c t i o n P a r t A. 3ed. Academic P r e s s . New York. B r e t t , J.R. and Z a l a , C.A. 1975. D a i l y p a t t e r n of n i t r o g e n e x c r e t i o n and oxygen consumption of sockeye salmon (Oncorhynchus nerka) under c o n t r o l l e d c o n d i t i o n s . J . F i s h . Res. Board Can. 32:2497-2486. B r e z i s , M., Rosen, S., S i l v a , P., Spokes, K. and E p s t e i n , F.H. 1984a. Polyene t o x i c i t y i n r e n a l m e d u l l a : i n j u r y mediated by t r a n s p o r t a c t i v i t y . S c i e n c e 224:66-68. B r e z i s , M., Rosen, S., Spokes, K., S i l v a , P. and E p s t e i n , F.H. 1984b. Transport-dependent a n o x i c c e l l i n j u r y i n the i s o l a t e d p e r f u s e d r a t k i d n e y . Am. J . P a t h o l . 116:327-341 . Burggren, W.W., and R a n d a l l , D.J. 1978. Oxygen uptake and t r a n s p o r t d u r i n g h y p o x i c exposure i n the s t u r g e o n A c i p e n s e r transmontanus. Resp. P h y s i o l . 34:171-183. B u r t o n , D.T. and Spehar, A.M. 1971. A r e - e v a l u a t i o n of the a n a e r o b i c end-products of f r e s h - w a t e r f i s h exposed t o e n v i r o n m e n t a l h y p o x i a . Comp. Biochem. P h y s i o l . 40A:945-954. Cameron, J.N. 1975. B l o o d f l o w d i s t r i b u t i o n as i n d i c a t e d by t r a c e r m i c r o s p h e r e s i n r e s t i n g and h y p o x i c A r c t i c G r a y l i n g (Thymallus a r c t i c u s ) . Comp. Biochem. P h y s i o l . 52A:441-444. C a s t e l l i n i , M.A., Murphy, B . J . , Fedak, K.R., G o f t o n , N. and Hochachka, P.W. 1985. P o t e n t i a l l y c o n f l i c t i n g m e t a b o l i c demands of d i v i n g and e x e r c i s e i n s e a l s . J . A p p l . P h y s i o l . 58 {]_) : i n p r e s s . C a s t e l l i n i , M.A., and Somero, G.N. 1981. B u f f e r i n g c a p a c i t y of v e r t e b r a t e muscle: c o r r e l a t i o n s w i t h p o t e n t i a l s f o r a n a e r o b i c f u n c t i o n . J . Comp. P h y s i o l . 143:191-198. 156 Chance, B., S i l b e r s t e i n , B.R. and May.evski, A. 1980. H e t e r o g e n e i t y of m e t a b o l i c s t a t e s of the c e r e b r a l c o r t e x i n v i v o . I_n: C e r e b r a l m e t a b o l i s m and n e u r a l f u n c t i o n . (Passonneau, J.V., Hawkins, R.A., L u s t , W.D. and Welsh, F.A. e d s . ) . W i l l i a m s and W i l k i n s , B a l t i m o r e , Md.. pp. 77-84. Conant, E.B. 1975. O b s e r v a t i o n s on c a r d i a c and r e s p i r a t o r y f u n c t i o n i n the A f r i c a n l u n g f i s h , P r o t o p t e r u s . B i o s . 46: 21-37. Conn e t t , R.J., G a y e s k i , E . J . and H o n i g , C R . 1984. L a c t a t e a c c u m u l a t i o n i n f u l l y a e r o b i c , w o r k i ng dog g r a c i l i s muscle. Am. J . P h y s i o l . 246:H12Q-H128. Cowey, C.B., de l a H i g u e r a , M. and Adron, J.W. 1977. The e f f e c t of d i e t a r y c o m p o s i t i o n and of i n s u l i n on g l u c o n e o g e n e s i s i n rainbow t r o u t (Salmo g a i r d n e r i ) . B r . J . N u t r . 38:385-395. Daw, J.C., Wenger, D.P. and Berne; R.M. 1967. R e l a t i o n s h i p between c a r d i a c g l y c o g e n and t o l e r e n c e t o a n o x i a i n the Western P a i n t e d T u r t l e (Chrysemys p i c t a b e l l i i ) . Comp. Biochem. P h y s i o l . 22:69-73. Dawes, G.S., Mo t t , J.C. and S h e l l e y , H.J. 1959. The importance of c a r d i a c g l y c o g e n f o r the maintenance of l i f e i n f o e t a l lambs and new-born a n i m a l s d u r i n g a n o x i a . J . P h y s i o l . 146: 516-538. De Zwann, A. 1983. Ca r b o h y d r a t e c a t a b o l i s m i n b i v a l v e s . I n : B i o c h e m i s t r y of the M o l l u s c a . (Hochachka, P.W. ed .TT Academic P r e s s , New York. De Zwaan, A. and Wijsman, T.C.M. 1976. A n a e r o b i c m e t a b o l i s m i n B i v a l v i a ( M o l l u s c a ) . Comp. Biochem. P h y s i o l . 54b:313-324. D e j o u r s , P. 1966. R e s p i r a t i o n . O x f o r d U n i v . P r e s s , O x f o r d . Demael-Suard, A., G a r i n , D., B r i c h o n , G., Mure, M., and P e r e s , G. 1974. Neoglycogenese a p a r t i r de l a g l y c i n e 1 f lC chez l a tanche ( T i n e a v u l g a r i s L.) au c o u r s de l ' a s p h y x i e . Comp. Biochem. P h y s i o l . 47A:1023-1033. 157 D o u d o r o f f , P. and Shumway, D.L. 1970. D i s s o l v e d oxygen re q u i r e m e n t s of f r e s h w a t e r f i s h e s . F.A.O. F i s h e r i e s T e c h n i c a l Paper No. 86. Dunn, A., K a t z , J . , Golden, S. and Chenoweth, M. 1976. E s t i m a t i o n of g l u c o s e t u r n o v e r and r e c y c l i n g i n r a b b i t s u s i n g v a r i o u s [ 3H, 1 4 C ] g l u c o s e l a b e l s . Am. J . P h y s i o l . 230:1159-1162. E l d r i d g e , F.L. 1974. R e l a t i o n s h i p between l a c t a t e t u r n o v e r r a t e and b l o o d c o n c e n t r a t i o n i n hemorragic shock. J . A p p l . P h y s i o l . 37:321-323. E v e l o f f , J . , B a y e r d o r f f e r , E., S i l v a , P. and K i n n e , R. 1981. S o d i u m - c h l o r i d e t r a n s p o r t i n the t h i c k a s c e n d i n g l i m b of Henl e ' s l o o p . P f l u g e r s A r c h . 389:263-270. F a l e s c h i n i , R.J. and W h i t t e n , B.K. 1975. Comparative h y p o x i c t o l e r a n c e i n the S c i u r i d a e . Comp. Biochem. P h y s i o l . 52A: 217-221. F l o o d , P.R. 1968. S t r u c t u r e of the segmental t r u n k muscles i n amphioxus. Z. Z e l l f o r s c h . 84:389-416. F r e m i n e t , A., Bursaux, E. and P o y a r t , C.F. 1974. E f f e c t of e l e v a t e d l a c t a t a e m i a on the r a t e s of l a c t a t e t u r n o v e r and o x i d a t i o n i n r a t s . P f l u g e r s A r c h . 346:75-86. F r e m i n e t , A. and L e c l e r c L. 1980. E f f e c t of f a s t i n g on g l u c o s e , l a c t a t e , and a l a n i n e t u r n o v e r i n r a t s and g u i n e a - p i g s . Comp. Biochem. P h y s i o l . 65B:363-367. Gordon, M.S. 1968. Oxygen consumption of red and w h i t e muscles from tuna f i s h e s . S c i e n c e . 159:87-90. Guppy, M., H u l b e r t , W.C. and Hochachka, P.W. 1979. M e t a b o l i c s o u r c e s of heat and power i n tuna muscles I I . Enzyme and m e t a b o l i t e p r o f i l e s . J . Exp. B i o l . 82:303-320. Guth, L. and Samaha, F . J . 1970. Procedure f o r the h i s t o c h e m i c a l d e m o n s t r a t i o n of ac t o m y o s i n ATPase. E x p t l . N e u r o l . 28:365-367. 158 Hance, A . J . , R o b i n , E.D., Simon, L.M., A l e x a n d e r , S., Herzenberg, L.A. and Theodore, J . 1980. R e g u l a t i o n of g l y c o l y t i c enzyme a c t i v i t y d u r i n g c h r o n i c h y p o x i a by changes i n r a t e - l i m i t i n g enzyme c o n t e n t . J . C l i n . I n v e s t . 66:1258-1264. Heath, A.G. and P r i t c h a r d , A.W. 1965. E f f e c t s of severe h y p o x i a on c a r b o h y d r a t e energy s t o r e s and m e t a b o l i s m i n two s p e c i e s of f r e s h - w a t e r f i s h . P h y s i o l . Z o o l . 38:325-334. H i l l a b y , B.A., and R a n d a l l , D.J. 1967. The e f f e c t of h y p o x i a upon the p a r t i a l p r e s s u r e of gases i n the b l o o d and water a f f e r e n t and e f f e r e n t t o the g i l l s of rainbow t r o u t . J . Exp. B i o l . 46:621-629. Hochachka, P.W. 1982. A n a e r o b i c m e tabolism: l i v i n g w i t h o u t oxygen. I_n: A Companion t o Animal P h y s i o l o g y . ( T a y l o r , C.R., Johansen, K. and B o l i s L. e d s . ) , Cambridge U n i v . P r e s s , Cambridge, U.K.. Hochachka, P.W. 1980. L i v i n g w i t h o u t oxygen. H a r v a r d U n i v . P r e s s , Cambridge Mass.. Hochachka, P.W., Guppy, M., G u d e r l e y , H., S t o r e y , K.B. and H u l b e r t , W.C. 1978a. M e t a b o l i c b i o c h e m i s t r y of water v s . a i r - b r e a t h i n g o s t e o g l o s s i d s : h e a r t enzymes and u l t r a s t r u c t u r e . Can. J . Z o o l . 56:759-768. Hochachka, P.W., Guppy, M., G u d e r l y , H., S t o r e y , K.B. and HulberfJ, W.C. 1978b. M e t a b o l i c b i o c h e m i s t r y of water- v s . a i r - b r e a t h i n g f i s h e s : muscle enzymes and u l t r a s t r u c t u r e . Can. J . Z o o l . 56:736-750. Hochachka, P.W., H u l b e r t , W.C. and Guppy, M. 1978c. The tuna power p l a n t and f u r n a c e . I_n: The P h y s i o l o g i a l E c o l o g y of Tuna. (Sharp, G.D. and D i z o n A.E. e d s . ) , Academic P r e s s , New York. Hochachka, P.W. and Mommsen, T. 1983. P r o t o n s and a n a e r o b i o s i s . S c i e n c e 219:1391-1397. 159 Hochachka, P.W. and Somero, G. 1984. B i o c h e m i c a l A d a p t a t i o n . P r i n c e t o n U. P r e s s , P r i n c e t o n , N.J.. Hochachka, P.W. and Somero, G.N. 1973. B i o c h e m i c a l a d a p t a t i o n s to the environment. W.B. Saunders Co. P h i l a d e l p h i a , ' Pa. . H o l e t o n , G.F. and R a n d a l l , D.J. 1967. The e f f e c t of h y p o x i a upon the p a r t i a l p r e s s u r e of gases i n the b l o o d and water a f f e r e n t and e f f e r e n t t o the g i l l s of Rainbow t r o u t . J . Exp. B i o l . 46:317-327. Huangsheng, L., Romsos, D.R., Tack, P . I . and L e v e i l l e , G.A. 1978. D e t e r m i n a t i o n of g l u c o s e u t i l i z a t i o n i n coho salmon [Oncorhynchus k i s u t c h (Walbaum)] w i t h ( 6 - 3 H ) - and ( U - 1 " C ) -g l u c o s e . Comp. Biochem. P h y s i o l . 5_9A: 189-191 . Hudson, R.C.L. 1973. On the f u n c t i o n of the w h i t e muscle i n t e l e o s t s a t i n t e r m e d i a t e swimming speeds. J . Exp. B i o l . 58:509-522. Hughes, G.M. and Saunders, R.L. 1970. Responses of the r e s p i r a t o r y pumps t o h y p o x i a i n the rainbow t r o u t (Salmo  g a i r d n e r i ) . J . Exp. B i o l . 53:529-545. H u l b e r t , W.C. and Moon, T.W. 1978. A h i s t o c h e m i c a l , l i g h t , and e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n of e e l , Anqui11a r o s t r a t a , w h i t e muscle. J . F i s h . B i o l . 13:527-533. H u l b e r t , W.C, Guppy, M. , Murphy, B. and Hochachka,'P.W. 1979. M e t a b o l i c s o u r c e s of heat and power i n tuna muscles. 1. Muscle f i n e s t r u c t u r e . J . Exp. B i o l . 82:289-301. I s s e k u t z , B. J r . , M i l l e r , H.I-., P a u l , P. and R o d a h l , K. 1965. E f f e c t of l a c t i c a c i d on f r e e f a t t y a c i d s and g l u c o s e o x i d a t i o n i n dogs. Am. J . P h y s i o l . 209:1137-1144. I s s e k u t z , B. J r . , Shaw, W.A.S. and I s s e k u t z , A.C. 1976. L a c t a t e m e t a b o l i s m i n r e s t i n g and e x e r c i s i n g dogs. J . A p p l . P h y s i o l . 40:312-319. 160 J a c k s o n , D.C. 1971. The e f f e c t of temperature on v e n t i l a t i o n i n the t u r t l e , Pseudemys s c ^ i p t a e l e q a n s . Resp. P h y s i o l . 12; 131-140. J a c k s o n , D.C. 1968. M e t a b o l i c d e p r e s s i o n and oxygen d e p l e t i o n i n the d i v i n g t u r t l e . J . A p p l . P h y s i o l . 24:503-509. J a c k s o n , D.C. and S c h m i d t - N i e l s e n , K. 1966. Heat p r o d u c t i o n d u r i n g d i v i n g i n the f r e s h water t u r t l e , Pseudemys  s c r i p t a . J . C e l l . P h y s i o l . 67:225-232. J e s s e , M.J., Shub, C. and Fishman, A.P. 1967. Lung and g i l l v e n t i l a t i o n of the A f r i c a n l u n g f i s h . Resp. P h y s i o l . 3^267-287. J o b s i s , F.F. and S t a i n s b y , W.N. 1968. O x i d a t i o n of NADH d u r i n g c o n t r a c t i o n s of c i r c u l a t e d mammalian s k e l e t a l . Resp. P h y s i o l . 4:292-300. Johansen, K. and L e n f a n t , C. 1968. R e s p i r a t i o n i n the A f r i c a n l u n g f i s h , P r o t o p t e r u s a e t h i o p i c u s 2. C o n t r o l of b r e a t h i n g . J . Exp. B i o l . 49:453-468. J o h n s t o n , I.A. 1981. S p e c i a l i z a t i o n of f i s h muscle. I n : Development and S p e c i a l i z a t i o n of M u s c l e . ( G o l d s p i n k , D.F. e d . ) . Cambridge U n i v . P r e s s , Cambridge. J o h n s t o n , I.A. 1975a. A n a e r o b i c metabolism i n the c a r p ( C a r a s s i u s c a r a s s i u s L . ) . Comp. Biochem. P h y s i o l . 51B:235-241 . J o h n s t o n , I.A. 1975b. S t u d i e s on the swimming metabolism of the rainbow t r o u t I I . Muscle metabolism d u r i n g severe h y p o x i a . J . F i s h . B i o l . 7:459-467. J o h n s t o n , I.A., D a v i s o n , W. and G o l d s p i n k , G. 1977. Energy m e t a b o l i s m of c a r p swimming musc l e s . J . Comp. P h y s i o l . 114:203-216. J o h n s t o n , I.A. and G o l d s p i n k , G. 1973. A study of the swimming performance of the c r u c i a n c a r p C a r a s s i u s c a r a s s i u s i n r e l a t i o n t o the e f f e c t s of e x e r c i s e and r e c o v e r y on b i o c h e m i c a l changes i n the myotomal muscles and l i v e r . J . F i s h . B i o l . 5:249-260. 161 J o h n s t o n , I.A. and Moon, T.W. 1980. E x e r c i s e t r a i n i n g i n s k e l e t a l muscle of brook t r o u t ( S a l v e l i n u s f o n t i n a l i s ) . J . Exp. B i o l . 87:177-194. J o h n s t o n , I.A., P a t t e r s o n , S., Ward, P. and G o l d s p i n k , G. 1974. The h i s t o c h e m i c a l d e m o n s t r a t i o n of m y o f i b r i l l a r a denosine t r i p h o s p h a t a s e a c t i v i t y i n f i s h muscle. Can. J . Z o o l . 52: 871-877. Jones, D. T971. T h e o r e t i c a l a n a l y s i s of f a c t o r s which may l i m i t the maximum oxygen uptake of f i s h . The oxygen c o s t of the c a r d i a c and b r a n c h i a l pumps. J . Theor. B i o l . 32:341-349. Jones, D.P. 1981. Hypoxia and drug m e t a b o l i s m . Biochem. Pharmacol. 30: 10-19-1023. Jones, D.P. and Kennedy, F.G. 1982. I n t r a c e l l u l a r oxygen s u p p l y d u r i n g h y p o x i a . Am. J . P h y s i o l . 243:C247-C253• J o r g e n s e n , J.B. and M u s t a f a , T. 1980. The e f f e c t of h y p o x i a on c a r b o h y d r a t e metabolism i n f l o u n d e r ( P l a t i c h t h y s f l e s u s L.) 2. High energy phosphate compounds and the r o l e of g l y c o l y t i c and g l u c o n e o g e n e t i c enzymes. Comp. Biochem. P h y s i o l . 67B:249-256. K a t z , J . , Dunn, A., Chenoweth, M. and Golden, S. 1974. D e t e r m i n a t i o n of s y n t h e s i s , r e c y c l i n g , and body mass of g l u c o s e i n r a t s and r a b b i t s i n v i v o w i t h 3H- and Re-l a b e l l e d g l u c o s e . Biochem. J . 142:171-183. K a t z , J . , Rostami, H. and Dunn, A. 1974. E v a l u a t i o n of g l u c o s e t u r n o v e r , body mass, and r e c y c l i n g w i t h r e v e r s i b l e and i r r e v e r s i b l e t r a c e r s . Biochem. J . 142:161-170. K a t z , J . , Okajima, F., Chenoweth, M. and Dunn, A. 1981. The d e t e r m i n a t i o n of l a c t a t e t u r n o v e r in v i v o w i t h 3H- and 1 * C - l a b e l l e d l a c t a t e . Biochem. J . 194:513-524. K a t z , Joseph 1982. Importance of s i t e s of t r a c e r a d m i n i s t r a t i o n and s a m p l i n g i n t u r n o v e r s t u d i e s . F e d e r a t i o n P r o c . 41:123-128. 162 K e p p l e r , D. and Decker, K. 1974. Glycogen d e t e r m i n a t i o n w i t h a m y l o g l u c o s i d a s e . I_n: Methods of Enzymatic A n a l y s i s . (Bergmeyer, H.V. e d . ) , Academic P r e s s . , New York, N.Y.. Kerem, D., Hammond, D. and E i s n e r , R. 1973. T i s s u e glycogen l e v e l s i n the Weddell s e a l L e p t o n y c h o t e s w e d d e l l i : A p o s s i b l e a d a p t a t i o n t o a s p h y x i a l h y p o x i a . Comp. Biochem. P h y s i o l . 45A.731-736. K e u l , J . , D o l l , E. and K e p p l e r , D. 1972. M e d i c i n e and S p o r t . V o l . 7. U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md.. K i l a r s k i , W. 1967. T h e , f i n e s t r u c t u r e of s t r i a t e d muscles i n t e l e o s t s . Z. Z e l l f o r s c h . 79:562-580. Kooyman, G.L., Wahrenbrock, E.A., C a s t e l l i n i , M.A., D a v i s , R.W. and S i n n e t t , E.E. 1980. A e r o b i c and a n a e r o b i c metabolism d u r i n g v o l u n t a r y d i v i n g i n Weddell s e a l s : E vidence of p r e f e r r e d pathways from b l o o d c h e m i s t r y and b e h a v i o r . J . Comp. P h y s i o l . 138:335-346. K r y v i , H. 1977. U l t r a s t r u c t u r e of the d i f f e r e n t f i b r e t y p e s i n a x i a l muscles of t h e sh a r k s Etmopterus s p i n a x and Galeus melastomus. C e l l T i s s u e . Res. 184:287-300. L a h i r i , S., S z i d o n , J.P. and Fishman, A.P. 1970. P o t e n t i a l r e s p i r a t o r y and c i r c u l a t o r y a d j u s t m e n t s t o h y p o x i a i n the A f r i c a n l u n g f i s h . Fed. P r o c . 29:1141-1148. L e h n i n g e r , A.L. 1975. B i o c h e m i s t r y . 2ed.. Worth Pub. I n c . , New York, N.Y.. L e i v e s t a d , H. 1960. The e f f e c t of p r o l o n g e d submersion on the metabolism and the h e a r t r a t e i n the t o a d (Bufo b u f o ) . U n i v . Bergen A r b . mat.-Naturv. R. Nr. 5. Bergen Loch n e r , W., A r n o l d , G. and M u l l e r - R u c k h o l t z , E.R. 1968. Me t a b o l i s m of the a r t i f i c i a l l y a r r e s t e d h e a r t and of the g a s - p e r f u s e d h e a r t . Am. J . C a r d i o l . 22:299-311. 1 63 Lubb e r s , D.W. 1977. Q u a n t i t a t i v e measurement and d e s c r i p t i o n of oxygen s u p p l y t o the t i s s u e . I_n: Oxygen and P h y s i o l o g i c a l F u n c t i o n . ( J o b s i s , F.F. e d . ) . P r o f e s s i o n a l I n f o r m a t i o n L i b r a r y , D a l l a s , Texas, pp. 254-276. L u t z , P.L., LaManna, J.C., Adams, M.R. and R o s e n t h a l , M. 1 9 8 0 . C e r e b r a l r e s i s t a n c e t o a n o x i a i n the marine t u r t l e . Resp. P h y s i o l . 4 J _ : 2 4 1 - 2 5 1 . Marsh, R.L. 1 9 8 1 . C a t a b o l i c enzyme a c t i v i t i e s i n r e l a t i o n t o p r e m i g r a t o r y f a t t e n i n g and muscle h y p e r t r o p h y i n the gray c a t b i r d (Dumetella c a r o l i n e n s i s ) . J . Comp. P h y s i o l . 1 4 1 B : 4 1 7 - 4 2 4 . McDougal J r . , D.B., Holowack, J . , Howe, M.C., J o n e s , E.M. and Thomas, C.A. 1968. The e f f e c t s of a n o x i a upon energy s o u r c e s and s e l e c t e d m e t a b o l i c i n t e r m e d i a t e s i n the b r a i n s of f i s h , f r o g , and t u r t l e . J . Neurochem. 15:577-588. M c G i l v e r y , R.W. and G o l d s t e i n , G. 1979. B i o c h e m i s t r y , a f u n c t i o n a l approach. 2ed. W.B. Saunders Co., P h i l a d e l p h i a P.A. . McKim, J.M. and Goeden, H.M. 1982. A d i r e c t measure of the uptake e f f i c i e n c y of a x e n o b i o t i c c h e m i c a l a c r o s s the g i l l s of brook t r o u t ( S a l v e l i n u s f o n t i n a l i s ) under normoxic and hy p o x i c c o n d i t i o n s . Comp. Biochem. P h y s i o l . 72C.65-74. McMahon, B.R. 1970. The r e l a t i v e e f f i c i e n c y of gaseous exchange a c r o s s the lun g s and g i l l s of an A f r i c a n l u n g f i s h , P r o t o p t e r u s a e t h i o p i c u s . J . exp. B i o l . 52:1-15. Mommsen T.P. 1984. M e t a b o l i s m of the f i s h g i l l . lr\: F i s h P h y s i o l o g y . V o l . XB, (Hoar, W. and R a n d a l l D. e d s . ) . Academic P r e s s , New York, N.Y.. pp. 203-238 Mommsen, T.P., Fr e n c h , C.J. and Hochachka, P.W. 1980. S i t e s and p a t t e r n s of p r o t e i n and amino a c i d u t i l i z a t i o n d u r i n g the spawning m i g r a t i o n of salmon. Can. J . Z o o l . 58:1785-1799. 164 Mosse, P.R.L. 1979. C a p i l l a r y d i s t r i b u t i o n and m e t a b o l i c h i s t o c h e m i s t r y of the l a t e r a l p r o p u l s i v e m u s c u l a t u r e of p e l a g i c t e l e o s t f i s h . C e l l T i s s u e Res. 203:141-160. Murphy, B., Z a p o l , W.M. and Hochachka, P.W. 1980. M e t a b o l i c a c t i v i t i e s of h e a r t , l u n g , and b r a i n d u r i n g d i v i n g and r e c o v e r y i n the Weddell s e a l . J . A p p l . P h y s i o l . 48:596-605. N a c h l a s , M.M., Tsou, K.C., De Sonza, E., Cheng, C S . and S e l i g m a n , A.M. 1957. C y t o c h e m i c a l d e m o n s t r a t i o n of s u c c i n i c dehydrogenase by the use of a new p - n i t r o p h e n y l s u b s t i t u t e d d i t e t r a z o l e . J . Histochem. Cytochem. 5_:420-436. N a c h l a s , M.M., Walker, D.G. and S e l i g m a n , A.M. 1958. A h i s t o c h e m i c a l method f o r the d e m o n s t r a t i o n of d i a p h o s p h o p y r i d i n e n u c l e o t i d e d i a p h o r a s e . J . B i o p h y s . Biochem. C y t o l . 4:29-38. N e e l y , J.R. and Morgan H.E. 1974. R e l a t i o n s h i p between c a r b o h y d r a t e and l i p i d m e t a b o l i s m and the energy b a l a n c e of h e a r t muscle Ann. Rev. P h y s i o l . 36:413-459. Nor b e r g , K., and B.K. S i e s j o . 1975. C e r e b r a l m e t a b o l i s n i n h y p o x i c h y p o x i a . I . P a t t e r n of a c t i v a t i o n of g l y c o l y s i s : A r e - e v a l u a t i o n . B r a i n Res. 86:31-44. Okajima, F., Chenoweth, M., Rognstad, R., Dunn, A. and K a t z , J . 1981. M e t a b o l i s m of 3-H and 1 " C - l a b e l e d l a c t a t e i n s t a r v e d r a t s . Biochem. J . 194:525-540. Palmer, T.N. and Ryman, B.E. S t u d i e s on o r a l g l u c o s e i n t o l e r a n c e i n f i s h . . J . F i s h B i o l . 4:311-319. P a s t e u r , M.L. 1861. E x p e r i e n c e s e t vues n o u v e l l e s s u r l a n a t u r e des f e r m e n t a t i o n s . C. R. Hebd. Seances Acad. S c i . 52:1260-1 264. P a t t e r s o n , S. and G o l d s p i n k , G. 1972. The f i n e s t r u c t u r e of red and w h i t e myotomal muscle f i b r e s of the c o a l f i s h (Gadus v i r e n s ) . Z. Z e l l f o r s c h . 133:463-474. 165 P r o s s e r , C.L. 1973. Comparative a n i m a l p h y s i o l o g y . V o l . 1. W.B. Saunders Co., P h i l a d i l p h i a , Pa.. Ra c k e r , E 1976. A new loo k a t mechanisms i n b i o e n e r g e t i c s . Academic P r e s s , New York. Reeves, R.B. 1963. Energy c o s t of work i n a e r o b i c and a n a e r o b i c t u r t l e h e a r t muscle. Am. J . P h y s i o l . 205;17-22. R o b i n , E.D., V e s t e r , J.W., Murdaugh, H.V. J r . and M i l l e n , J.E. 1964. P r o l o n g e d a n a e r o b i o s i s i n a v e r t e b r a t e : a n a e r o b i c m e t abolism i n the f r e s h w a t e r t u r t l e . J . C e l l . Comp. P h y s i o l . 63:287-297. R o b i n , E.D. 1980. Of men and m i t o c h o n d r i a : c o p i n g w i t h h y p o x i c d y s o x i a . Am. Rev. Resp. D i s e a s e 122:517-531. R o y l e , G.T., Wolfe, R.R. and Burke, J.F. 1982. Gluc o s e and f a t t y a c i d k i n e t i c s i n f a s t e d r a t s : e f f e c t s of p r e v i o u s p r o t e i n i n t a k e . M e t a b o l i s m 31:279-283. Rusko, H. and R a h k i l a , P. 1979. Maximum oxygen uptake, a n a e r o b i c t h r e s h o l d , and s k e l e t a l muscle enzymes i n male a t h l e t e s . I n : E x e r c i s e and Spo r t B i o l o g y , (Komi, P.V. e d . ) , Human k i n e t i c s P u b l . , Champaign, I l l i n o i s . S a h l i n , K., H a r r i s , R.C. and E. Hultman 1974. C r e a t i n e k i n a s e e q u i l i b r i u m and l a c t a t e c o n t e n t compared w i t h muscle pH i n t i s s u e samples o b t a i n e d a f t e r i s o m e t r i c e x e r c i s e . I n : Muscle p h y s i o l o g y . ( C a r l s o n , F.D. and W i l k i e , D.R. e d s . ) . P r r e n t i c e - H a l l I n c . , Englewood C l i f f s , New J e r s e y , pp. 92-99. S i e b e n a l l e r , J.F., Somero, G.N. and H a e d r i c h R.L. 1982. B i o c h e m i c a l c h a r a c t e r i s t i c s of m a c r o u r i d f i s h e s d i f f e r i n g i n t h e i r depths of d i s t r i b u t i o n . B i o l . B u l l . 163:240-249. S h o u b r i d g e , E.A. 1980. The m e t a b o l i c s t r a t e g y of the a n o x i c g o l d f i s h . PhD. T h e s i s . U n i v e r s i t y of B r i t i s h Columbia, Vancouver. B.C.. 166 S h o u b r i d g e , E.A. and Hochachka, P.W. 1981. The o r i g i n and s i g n i f i c a n c e of m e t a b o l i c carbon d i o x i d e p r o d u c t i o n i n the a n o x i c g o l d f i s h . M o l e c . P h y s i o l . J_: 31 5-338 . S i e s j o , B.R., F o l b e r g r o v a , J . and M a c M i l l a n , V. 1972. The e f f e c t of h y p e r c a p n i a upon i n t r a c e l l u l a r pH i n the b r a i n , e v a l u a t e d by the b i c a r b o n a t e - c a r b o n i c a c i d method and from the c r e a t i n e phosphokinase e q u i l i b r i u m . J . Neurochem. 19: 2483-2495. S i e s j o , B.K. and Nordstrom, C.H. 1977. B r a i n metabolism i n r e l a t i o n to oxygen s u p p l y . I n : Oxygen and P h y s i o l o g i c a l F u n c t i o n . ( J o b s i s , F.F. ed .TT P r o f e s s i o n a l I n f o r m a t i o n L i b r a r y , D a l l a s , Texas. S o k a l , R.R. and R o h l f , F . J . 1969. B i o m e t r y . The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l r e s e a r c h . W.H. Freeman and Co. San F r a n c i s c o , C a l i f o r n i a . Somero, G.N. and C h i l d r e s s , J . J . 1980. A v i o l a t i o n of the m e t a b o l i s m - s i z e s c a l i n g paradigm: a c t i v i t i e s of g l y c o l y t i c enzymes i n muscle i n c r e a s e i n l a r g e r - s i z e f i s h . P h y s i o l . Z o o l . 53:322-337. S t e e l e , R., W i n k l e r , B., Rathgeb, I . , B j e r k n e s , C. and A l t s z u l u r , N. 1968. Plasma g l u c o s e and f r e e f a t t y a c i d m e t a bolism i n normal and l o n g - f a s t e d dogs. Am. J . P h y s i o l . 214:313-319. Ste v e n s , E.D. 1968. The e f f e c t of e x e r c i s e on the d i s t r i b u t i o n of b l o o d to v a r i o u s organs i n rainbow t r o u t . Comp. Biochem. P h y s i o l . 25:615-625. S t o c c o , D.M., B e e r s , P.C. and Warner, A.H. 1972. E f f e c t of a n o x i a on n u c l e o t i d e metabolism i n e n c y s t e d embryos of the b r i n e shrimp. Dev. B i o l . 27:479-493. Sugden, P.H. and Newsholme, E.A. 1975. A c t i v i t i e s of c i t r a t e s y n t h a s e , NAD+-linked and NADP+-linked i s o c i t r a t e dehydrogenases, g l u t a m a t e dehydrogenases, a s p a r t a t e a m i n o t r a n s f e r a s e s , and a l a n i n e a m i n o t r a n s f e r a s e i n nervous t i s s u e s from v e r t e b r a t e s and i n v e r t e b r a t e s . Biochem. J . 150:105-111 . 1 67 S u l l i v a n K.M. and Somero G.N. 1980. Enzyme a c t i v i t i e s of f i s h s k e l e t a l muscle and b r a i n as i n f l u e n c e d by depth of occurence and h a b i t s of f e e d i n g and l o c o m o t i o n . Marine B i o l . 60:91-99. S w a r t z , R.D., S i l v a , P., H a l l a c , R. and E p s t e i n , F.H. 1978. The r e l a t i o n between sodium t r a n s p o r t and oxygen consumption i n i s o l a t e d p e r f u s e d r a t k i d n e y . I_n: C u r r e n t Problems i n C l i n i c a l B i o c h e m i s t r y . (Guder, W.G. and Schmidt U. e d s . ) , Hans Huber Pub., Bern. pp. 123-132. Taegtmeyer, H. 1979. M e t a b o l i c responses t o c a r d i a c h y p o x i a . I n c r e a s e d p r o d u c t i o n of s u c c i n a t e by r a b b i t p a p i l l a r y muscles C i r c . Res. 43:80.8-815. Thomas, C.L. 1973. Taber's c y c l o p e d i c m e d i c a l d i c t i o n a r y . V o l . 12., F.A. D a v i s Co., P h i l a d e l p h i a . T o t l a n d , G.K. 1976. Three muscle f i b r e t y p e s i n the a x i a l muscle of A x o l o t l (Ambystoma mexicanum Shaw). A q u a n t i t a t i v e l i g h t and e l e c t r o n m i c r o s c o p e s t u d y . C e l l T i s s u e Res. 168: 65-78. U l t s c h , G.R. and J a c k s o n , D.C. 1982. Long-term submergence at 3°C of the t u r t l e , Chrysemys p i c t a , i n normoxic and h y p o x i c water. I . s u r v i v a l , gas exchange and a c i d - b a s e s t a t u s . J . Exp. B i o l . 96:11-28. Van den T h i l l a r t , G. 1982. A d a p t a t i o n s of f i s h energy metabolism t o h y p o x i a and a n o x i a . M o l . P h y s i o l . 2:49-61. V e t t e r , R.D. and Hodson, R.E. 1982. Use of a d e n y l a t e c o n c e n t r a t i o n s and a d e n y l a t e energy charge as i n d i c a t o r s of h y p o x i c s t r e s s i n e s t u a r i n e f i s h . Can. J . F i s h . Aquat. S c i . 39:535-541. Walker, R.M. and Johanson, P.H. 1977. A n a e r o b i c metabolism i n g o l d f i s h C a r a s s i u s a u r a t u s . Can. J . Z o o l . 55: 1304-1311. Walton, M.J. and Cowey, C.B. 1982. A s p e c t s of i n t e r m e d i a r y metabolism i n s a l m o n i d f i s h . Comp. Biochem. P h y s i o l . 73B: 59-79. 168 Z a p o l , W.M., L i g g i n s , G.C., S c h n e i d e r , R.C., Q v i s t , J . , S n i d e r , M.T., Creas y , R.K. and Hochachka, P.W. 1979. R e g i o n a l b l o o d f l o w d u r i n g s i m u l a t e d d i v i n g i n the c o n s c i o u s Weddell s e a l . J . A p p l . P h y s i o l . £7:968-973. 

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