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Energy metabolism in carp white muscle Driedzic, William Richard 1975

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ENERGY METABOLISM IN CARP WHITE MUSCLE  by  WILLIAM RICHARD DRIEDZIC B.Sc.  (Hons.), York U n i v e r s i t y , 1970  M.Sc,  U n i v e r s i t y o f T o r o n t o , 1972  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of ZOOLOGY  We a c c e p t t h i s t h e s i s as conforming  to the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  that  it  purposes  for  may  for  of  JULY  gain  ZOOLOGY  1975.  of  Columbia,  British  by  Columbia  for  the  is understood  financial  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  extensive  be g r a n t e d  It  fulfilment  available  permission.  Department  Date  freely  permission  representatives. thesis  partial  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  i i  ABSTRACT  The myotomal muscle of f i s h i s l a r g e l y composed of two u s u a l l y r e f e r r e d to as the r e d and w h i t e f i b e r s .  On  a metabolism which i s predominantly  glycogen burning  as i t s f u e l source and  t h a t w h i t e muscle  a n a e r o b i c a l l y based,  utilizing  t h a t r e d muscle f u n c t i o n s l a r g e l y  f a t s and/or c a r b o h y d r a t e s .  In carp  (Cyprinus c a r p i o L.)  f i b e r s are found as a t h i n s u p e r f i c i a l l a y e r below the s k i n and f i b e r s make up it  the mass of the u n d e r l y i n g myotome.  types,  the b a s i s of h i s t o c h e m i -  c a l and b i o c h e m i c a l p r o p e r t i e s i t i s g e n e r a l l y a c c e p t e d has  tissue  aerobically the r e d  the white  Thus, i n t h i s s p e c i e s  i s p o s s i b l e to r a p i d l y o b t a i n an homogenous sample of w h i t e muscle a l l o w -  i n g the a n a l y s i s of l a b i l e m e t a b o l i t e s . of g l y c o l y s i s i n w h i t e muscle and  T h i s study i n v e s t i g a t e s the  the source and  control  f u n c t i o n of a n a e r o b i c  NH*  p r o d u c t i o n by w h i t e muscle. The  c o n c e n t r a t i o n s o f key m e t a b o l i t e s were determined  e x e r c i s e and  a f t e r maximal a c t i v i t y .  i n l e v e l s o f glucose-6-phosphate,  During  e x e r c i s e t h e r e was  fructose-6-phosphate  phosphate which, along w i t h a d e c r e a s e  i n ATP  i n muscle b e f o r e an i n c r e a s e  and f r u c t o s e - 1 - 6 - d i -  l e v e l s , c o u l d account  i n c r e a s e i n g l u c o l y t i c f l u x by a c t i v a t i o n of p h o s p h o f r u c t o k i n a s e kinase.  I t was  decreased unchanged.  found  t h a t a f t e r maximal a c t i v i t y  by about 65%, ADP Consequently  Simultaneously  t h e r e was  monophosphate) and NH^.  decreased  remained low  decreased  by  l e v e l and  and  (inosine  the decrease  in  s t o i c h i o m e t r y , a r e s u l t showing t h a t  the r e a c t i o n c a t a l y z e d by 5' AMP  However, the i n c r e a s e i n f r e e M l t was  ATP  decreased.  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 of IMP  a d e n y l a t e p o o l were e s s e n t i a l l y i n 1:1 the a d e n y l a t e p o o l was  s l i g h t l y , w h i l e AMP  i n c r e a s e i n IMP  pyruvate  the c o n c e n t r a t i o n o f  the l e v e l of the f r e e a d e n y l a t e p o o l  The  and  f o r the  l e s s than the decrease  i n the  deaminase. adenylate  ii i  pool.  The c o n c e n t r a t i o n  muscle, b e f o r e  o f f r e e amino a c i d s was a l s o determined i n w h i t e  and a f t e r severe environmental h y p o x i a .  D u r i n g the h y p o x i c  p e r i o d the t o t a l amount o f n i t r o g e n i n the amino a c i d p o o l i n c r e a s e d and t h e r e was a tendency f o r an i n c r e a s e i n the t o t a l number  o f f r e e amino a c i d s .  On the b a s i s o f t h i s study i t i s p o s s i b l e to c o n s t r u c t hensive metabolic anaerobic  work.  The energy r e q u i r e d f o r work i s u l t i m a t e l y d e r i v e d When ATP l e v e l s cannot be m a i n t a i n e d t h e c o n t e n t  i n c r e a s e s and as t h i s occurs  the l e v e l o f AMP  e q u i l i b r i u m r e l a t i o n s h i p of the a d e n y l a t e s . exceeds i t s a e r o b i c  c a p a b i l i t i e s GTP  during  from the o f ADP  a l s o i n c r e a s e s due to the As the work l o a d on the t i s s u e  (guanosine t r i p h o s p h a t e )  deaminase i s a c t i v a t e d and the a d e n y l a t e  r e l e a s e d from AMP  compre-  scheme f o r n i t r o g e n metabolism i n c a r p w h i t e muscle  h y d r o l y s i s o f ATP.  5' AMP  a fairly  i s subsequently incorporated  levels  pool i s decreased.  drop, NH^  i n t o the f r e e amino a c i d p o o l .  TABLE OF CONTENTS  Abstract T a b l e o f Contents L i s t of Tables L i s t of F i g u r e s Acknowledgements Chapter I .  Introduction  Chapter I I .  M a t e r i a l s and Methods  Chapter I I I . R e s u l t s Chapter IV.  Discussion  Chapter V.  Concluding  Remarks:  Red-White Muscle D i f f e r e n c e s  and the F u n c t i o n o f the P u r i n e N u c l e o t i d e Chapter VI.  Literature  Appendix I.  Maintenance of Blood L a c t a t e L e v e l s i n  Cited  Free Swimming T r o u t Appendix I I  Enzyme Nomenclature  Cycle  V  LIST OF TABLES Page Table I.  C o n c e n t r a t i o n s o f g l y c o l y t i c i n t e r m e d i a t e s i n white  30  muscle o f carp under two w e l l d e f i n e d c o n d i t i o n s : r e s t i n g and maximally a c t i v e . Table I I .  Concentrations  of the a d e n y l a t e s  and r e l a t e d meta-  31  b o l i t e s i n w h i t e muscle o f carp under two w e l l defined conditions: Table I I I .  r e s t i n g and maximally a c t i v e .  Energy charge and changes  i n c o n c e n t r a t i o n s o f the  32  a d e n y l a t e p o o l and r e l a t e d m e t a b o l i t e s i n white muscle of carp under two w e l l d e f i n e d c o n d i t i o n s : r e s t i n g and maximally T a b l e IV.  Concentrations  active.  of Krebs c y c l e i n t e r m e d i a t e s and  r e l a t e d m e t a b o l i t e s i n w h i t e muscle o f carp two w e l l d e f i n e d c o n d i t i o n s :  33  under  r e s t i n g and m a x i m a l l y  active. T a b l e V.  C o n c e n t r a t i o n s o f m e t a b o l i t e s and the energy charge v a l u e i n w h i t e muscle of carp a f t e r v a r i o u s of  Table VI.  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 carp white muscle  38  stress.  Free amino a c i d c o n c e n t r a t i o n s i n carp white muscle b e f o r e and a f t e r h y p o x i c  Table V I I I .  levels  activity.  b e f o r e and a f t e r h y p o x i c Table V I I .  35  39  stress.  A r t e r i a l and venous 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 of rainbow t r o u t f o l l o w i n g e x e r c i s e to f a t i g u e .  87  vi LIST OF FIGURES Page F i g u r e 1.  Schematic r e p r e s e n t a t i o n o f carp  (Cyprinus  cafpio)  2  i n c r o s s - s e c t i o n a t the l e v e l o f the p o s t e r i o r margin of t h e d o r s a l f i n . F i g u r e 2.  Anaerobic  metabolism i n i n v e r t e b r a t e f a c u l t a t i v e  10  anaerobes. F i g u r e 3.  The percentage a e r o b i c swimming e f f i c i e n c y  versus  45  swimming speed o f g o l d f i s h . F i g u r e 4.  D e p l e t i o n o f the a d e n y l a t e during anaerobic  F i g u r e 5.  p o o l i n white muscle  from a n a e r o b i c  as an energy  Augmentation of Krebs c y c l e i n t e r m e d i a t e s u t i l i z a t i o n o f f a t as an energy  F i g u r e 8.  Blood at  54  work.  Augmentation o f Krebs c y c l e i n t e r m e d i a t e s u t i l i z a t i o n o f carbohydrate  F i g u r e 7.  53  work.  R e g e n e r a t i o n o f the a d e n y l a t e during recovery  F i g u r e 6.  p o o l i n w h i t e muscle  during  59  source. during  63  source.  l a c t a t e l e v e l s o f i n d i v i d u a l swimming t r o u t  s p e c i f i e d swimming speed and f o l l o w i n g f a t i g u e .  84  vii  ACKNOWLEDGEMENTS  I would l i k e throughout  to thank Dr. P. W. Hochachka f o r h i s c o n s t a n t i n s p i r a t i o n  the course o f t h i s study.  I would a l s o l i k e t o express my  thanks  to Drs. B i l i n s k i , B r e t t , Hoar, Jones and R a n d a l l f o r t h e i r comments and c r i t i c i s m s o f the t h e s i s ,  to Dr. G. I . Drummond f o r h i s h e l p i n the e a r l y  development o f the problem,  and to Mr. R. H u r s t f o r i n s t r u c t i o n i n the  e x t r a c t i o n and a n a l y s i s o f v o l a t i l e  acids.  I am g r a t e f u l to Dr. I . P. T a y l o r , Department o f Botany, B r i t i s h Columbia, analyzer.  f o r use of b o t h the gas l i q u i d  U n i v e r s i t y of  chromatograph and amino  The s u c c i n i c t h i o k i n a s e and the TH f o r m y l a s e were g i f t s  from  Dr. W. B r i d g e r , Department o f B i o c h e m i s t r y , U n i v e r s i t y of A l b e r t a , and Dr. J . C. Rabinowitz, Department o f B i o c h e m i s t r y , U n i v e r s i t y o f C a l i f o r n i a , respectively. The study p r e s e n t e d i n Appendix I was done i n c o l l a b o r a t i o n w i t h Mr.  J . W. K i c e n i u k , t o whom I am e s p e c i a l l y g r a t e f u l .  thank Mr. K i c e n i u k f o r c o l l e c t i n g  I would a l s o l i k e to  the animals used i n the swimming  experiments  and f o r e x e r c i s i n g some o f them. Throughout  the study I was the h o l d e r o f a N a t i o n a l Research C o u n c i l o f  Canada Graduate S c h o l a r s h i p . Finally,  I wish  to thank Cath, Adam and Sean f o r h e l p i n g me keep my work  i n i t s proper p e r s p e c t i v e .  1  CHAPTER I .  INTRODUCTION  1  The  s k e l e t a l muscle of v e r t e b r a t e s i s composed of a number of c h a r a c t e r -  i s t i c a l l y d i f f e r e n t f i b e r types. fiber  At the extreme ends o f the spectrum of  types a r e those which make up  red, white, masses.  a  and  the r e d and w h i t e m u s c l e s .  In mammals,  i n t e r m e d i a t e type f i b e r s u s u a l l y o c c u r i n complex mixed muscle  In some i n s t a n c e s , such as the c a t s o l e u s , a r e l a t i v e l y pure r e d  muscle may  be o b t a i n e d , but i n mammalian systems, white muscle per se, does  not e x i s t as a d i s c r e t e t i s s u e .  However, i t i s p o s s i b l e to study white muscle  by u t i l i z i n g lower v e r t e b r a t e s .  In many f i s h s p e c i e s the white f i b e r s , which  c o n s t i t u t e 80-95% o f the swimming musculature, s e p a r a b l e t i s s u e mass.  e x i s t as a d i s c r e t e ,  Where t h i s o c c u r s , the r e d f i b e r s are found  easily as a t h i n  s u p e r f i c i a l l a y e r below the s k i n forming a t h i c k e r t r i a n g l e of muscle a t l e v e l of the l a t e r a l l i n e , w i t h the w h i t e f i b e r s making up u n d e r l y i n g nyotome.  The myotomal musculature  i s s t r u c t u r e d i n t h i s manner ( F i g u r e 1 ) . i n the s i z e of the f i b e r s chemical evidence  of carp  Although  the  the mass o f  the  (Cyprinus c a r p i o  L.)  t h e r e may  be  gradations  (Boddeke j i t a l , 1962), a l l of the a v a i l a b l e  histo-  i n d i c a t e s t h a t w h i t e muscle of carp i s an homogenous t i s s u e  i n terms of energy g e n e r a t i n g p r o p e r t i e s (Ogata  and M o r i , 1964;  B r o t c h i , 1968).  In t h i s s p e c i e s a s m a l l number of i n t e r m e d i a t e type f i b e r s a r e found, these o c c u r between the r e d and w h i t e muscle masses. with very l i t t l e d i f f i c u l t y  but  Thus i t i s p o s s i b l e  to o b t a i n a r e l a t i v e l y pure p r e p a r a t i o n o f white  muscle from c a r p . I t i s now  w e l l e s t a b l i s h e d t h a t the r e d and w h i t e muscle of f i s h have  markedly d i f f e r e n t m e t a b o l i c  capabilities.  On  the b a s i s o f b i o c h e m i c a l  h i s t o l o g i c a l p r o p e r t i e s i t i s g e n e r a l l y accepted metabolism which i s predominantly largely aerobically. of m i t o c h o n d r i a  t h a t white muscle has  and a  a n a e r o b i c , whereas the r e d muscle f u n c t i o n s  Thus, r e d muscle i s c h a r a c t e r i z e d by a h i g h e r  content  (Buttkus, 1963), myoglobin (Hamoir e t a l , 1972), haemoglobin  2  F i g u r e 1.  Schematic r e p r e s e n t a t i o n of carp  (Cyprinus  carpio) i n  c r o s s - s e c t i o n a t the l e v e l of the p o s t e r i o r margin o f the d o r s a l f i n .  Red Muscle White Muscle  3  (Hamoir e t a l , 1972), l i p i d  (Bone, 1966;  L i n e_t a l , 1974), l i p o l y t i c  (George, 1962), Krebs c y c l e enzymes (Bostrom and  Johansson, 1972), and  chrome o x i d a s e (Bostrom and  Johansson, 1972) .  by a h i g h e r  to the t i s s u e (Stevens, 1968)  vascular  supply  to consume oxygen ( L i n e t a l , 1974;  oxygen consumption r a t e  W i t t e n b e r g e r and  and  i s biochemically  t i s s u e has  ( L i n et^ a l , 1974;  cyto-  These p r o p e r t i e s are r e f l e c t e d  muscle, on the o t h e r hand, i s p o o r l y v a s c u l a r i z e d low  enzymes  and  a greater  D i a c i u c , 1965).  capacity White  (Stevens, 1968), shows a  W i t t e n b e r g e r and  geared f o r a n a e r o b i c metabolism.  D i a c i u c , 1965) ,  Consequently,  a h i g h c o n t e n t of g l y c o l y t i c enzymes (Hamoir e_t a l , 1972)  this and  an  extremely a c t i v e l a c t a t e dehydrogenase designed to channel p y r u v a t e i n t o lactate  (Bostrom and  Johansson, 1972).  Studies  on mammalian systems are i n  t o t a l agreement w i t h the above m e t a b o l i c f i n d i n g s and P e t t e and The  Staudte  c o n t r o l of b l o o d  understood. respect  (1973) and  have been reviewed  K e u l e t a l (1972).  f l o w to s k e l e t a l muscle i n f i s h i s s t i l l  S a t c h e l l (1971) has  to b o t h e x e r c i s e and  i n p e r i p h e r a l r e s i s t a n c e and  hypoxia.  E x e r c i s e evokes an o v e r a l l r e d u c t i o n  an i n c r e a s e d b l o o d  flow  flow  through the^vtrunk, r e g i o n .  to t h i s  such a l a r g e p o r t i o n of  tissue increases  D u r i n g h y p o x i a however, 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 o c c u r s I t i s w e l l e s t a b l i s h e d that during hypoxia blood mammals i n reduced  ( I r v i n g , 1964); a l t h o u g h not  appears t h a t the same mechanism f u n c t i o n s  t i s s u e approximates a c l o s e d system d u r i n g The  flow  during  activity.  ( S a t c h e l l , 1971) .  to s k e l e t a l muscle i n  unequivocally  in fish.  poor c i r c u l a t i o n o f white muscle under a e r o b i c this  poorly  reviewed t h i s a r e a of l i t e r a t u r e w i t h  S a t c h e l l argues t h a t s i n c e the w h i t e muscle makes up the body m u s c u l a t u r e , b l o o d  by  established, i t  Moreover, i n l i g h t of  the  conditions, i t i s probable that hypoxia.  m e t a b o l i c c h a r a c t e r i s t i c s of r e d and white muscle a r e r e f l e c t e d by  4  a f u n c t i o n a l d i f f e r e n c e between these two  tissues.  Electrophysiological  s t u d i e s show t h a t d u r i n g slow swimming the p r o p u l s i v e f o r c e i s d e r i v e d e n t i r e l y from the r e d musculature. muscle becomes maximally power f o r l o c o m o t i o n thought  A t the h i g h e s t swimming v e l o c i t i e s  a c t i v e and  (Bone, 1966;  t o g e t h e r w i t h the r e d muscle p r o v i d e s the  Hudson, 1973) .  White muscle was p r e v i o u s l y  to be u t i l i z e d o n l y d u r i n g p e r i o d s o f b u r s t a c t i v i t y  however, i t i s now  the white  (Bone, 1966);  g e n e r a l l y a c c e p t e d , t h a t a t l e a s t i n the t e l e o s t ,  t i s s u e p l a y s a r o l e over a much wider range o f swimming speeds.  this  A l l of the  c u r r e n t d a t a i n d i c a t e t h a t a t some l e v e l i n the t r a n s i t i o n from low t o h i g h swimming speed based on:  t h e r e i s i n c r e a s i n g r e c r u i t m e n t o f the white f i b e r s .  (a) the a c c u m u l a t i o n o f l a c t a t e i n the white muscle o f  trout  ( B l a c k ^ t a l , 1962), carp (Johnston and G o l d s p i n k , 1973a), c o a l f i s h and G o l d s p i n k , 1973b), and mackerel mediate v e l o c i t i e s ;  ( P r i t c h a r d e t a l , 1971)  This i s  (Johnston  worked a t i n t e r -  (b) hypertrophy of white muscle f i b e r s i n c o a l f i s h  to swim a t moderate speeds f o r extended  p e r i o d s of time  (Walker  forced  and P u l l ,  1973);  (c) the repayment o f an oxygen debt by salmon d u r i n g r e c o v e r y from swimming a t all  e l e v a t e d speeds ( B r e t t , 1964)  and  (d) the o b s e r v a t i o n t h a t i n g o l d f i s h  the mass o f the r e d muscle f i b e r s a l o n e i s not g r e a t enough to meet the o v e r a l l power output o f the f i s h  (Smit et^ ail, 1971) .  D u r i n g e x e r c i s e carp w h i t e muscle generates a l a r g e p o r t i o n o f i t s energy by a n a e r o b i c g l y c o l y s i s  (Wittenberger and D i a c i u c , 1965); thus, d u r i n g work  t h e r e i s a decrease i n g l y c o g e n w i t h a concomitant  increase i n lactate.  The  q u a n t i t a t i v e a s p e c t s of t h i s phenomenon, i n numerous s p e c i e s , have r e c e n t l y been reviewed by B i l i n s k i  (1974) and need not be r e p e a t e d here.  A l l o f the  a v a i l a b l e e v i d e n c e i n d i c a t e s t h a t g l y c o g e n i s m e t a b o l i z e d i n f i s h by  the  c l a s s i c a l Embden, Myerhof, Parnas pathway; f o r each mole o f g l y c o g e n - d e r i v e d  5  g l u c o s e m o b i l i z e d , 2 moles of l a c t a t e are formed w i t h a net g a i n of 3 moles of ATP.  Again t h i s area of l i t e r a t u r e has been reviewed  Hochachka (1969) and  T a r r (1972).  i n f i s h white muscle appears  i n depth  by  In many r e s p e c t s the c o n t r o l of  s i m i l a r to other systems s t u d i e d .  m o b i l i z a t i o n i s i n i t i a t e d by glycogen p h o s p h o r y l a s e ,  glycolysis  Thus,  glycogen  a r e g u l a t o r y enzyme,  which i n most t i s s u e s i n under a v a r i e t y of c o n t r o l s i n c l u d i n g hormonal  agents  I|  such as e p i n e p h e r i n e and n o r e p i n e p h e r i n e , (Drummond, 1971).  the a d e n y l a t e s , and  In f i s h white muscle, Ca  appears  not the s o l e r e g u l a t o r o f g l y c o g e n phosphorylase hormonal s i g n a l s a r e minimal  free  Ca  to be the primary i f  s i n c e b l o o d f l o w and  (Pocinwong ^ t a l , 1974).  hence  The next s i t e of  c o n t r o l i n the g l y c o l y t i c pathway o c c u r s a t the r e a c t i o n c a t a l y z e d by phosp h o f r u c t o k i n a s e (PFK) which c a t a l y z e d the c o n v e r s i o n of p l u s ATP  to f r u c t o s e - l , 6 - d i p h o s p h a t e p l u s ADP.  r e p r e s e n t s the f i r s t unique  fructose-6-phosphate  The r e a c t i o n c a t a l y z e d by  step i n g l y c o l y s i s ; hence, i t i s not  PFK  surprising  that the enzyme i s p r e c i s e l y r e g u l a t e d by v a r i o u s m e t a b o l i t e s i n a manner t h a t c o n t r o l s the r a t e o f g l y c o l y s i s i n a c c o r d w i t h the c e l l s ' need f o r energy or g l y c o l y t i c  intermediates.  p r o d u c t s o f the PFK  In a v a r i e t y of t i s s u e s , b o t h s u b s t r a t e s and  r e a c t i o n , as w e l l as o t h e r f a c t o r s , a r e a l l o s t e r i c  m o d i f i e r s o f the enzyme.  Thus, a c t i v a t o r s of PFK  phosphate, f r u c t o s e - 1 , 6 - d i p h o s p h a t e , P^, c r e a t i n e phosphate are i n h i b i t o r s of muscle g l y c o l y s i s ,  the a c t i v i t y  c o n t r o l s i t e i n the pathway: v e r s i o n of phosphoenolpyruvate  and NH^;  i n c l u d e AMP,  fructoses-  whereas ATP,  citrate  (Mansour, 1972) . of PFK  (PyK).  PyK  c a t a l y z e s the  to p y r u v a t e p l u s ATP.  of the r a t e c o n t r o l l i n g enzymes i s a c h i e v e d i n two ways. i n the PFK  activation  i s i n t e g r a t e d w i t h the next major  pyruvate kinase p l u s ADP  To complete the  and  r e a c t i o n s e r v e s as a s u b s t r a t e f o r PyK,  An  con-  integration  F i r s t l y , ADP  formed  and s e c o n d l y , a t l e a s t i n  6  the lower v e r t e b r a t e s ,  fructose-1,6-diphosphate  f u n c t i o n s as a f e e d  a c t i v a t o r of PyK  (Hochachka and  both PFK  from f i s h muscle have been s t u d i e d  and  PyK  Hochachka, 1968; glycolytic  Somero, 1973).  The  k i n e t i c p r o p e r t i e s of (Freed, 1971;  flux i n vivo i n this  t i s s u e had  not been v e r i f i e d . of m e t a b o l i t e s  the o n l y a n a e r o b i c  metabolic  known to  f i s h w h i t e muscle d u r i n g e x e r c i s e . muscle content 1966)  and  Kutty  of NH^  that  pathway i n w h i t e muscle.  l e a s t t h r e e s t u d i e s have shown the m o b i l i z a t i o n of n i t r o g e n o u s  NH^~  Thus, i t  a s u b s t a n t i a l amount of e v i d e n c e which suggests  g l y c o l y s i s i s not  1960).  and  t h i s pathway under v a r y i n g c o n d i t i o n s of energy demand.  There i s now  al,  Somero  Mustafa e t a l , 1971); however, the mechanism c o n t r o l l i n g  seemed worthwhile to determine the c o n c e n t r a t i o n regulate  forward  Thus, w i t h  compounds i n  e x e r c i s e to f a t i g u e , the w h i t e  i n c r e a s e s from about 4 to 7 ymoles/gm i n cod  from about 2.5  (Fraser et  to 7 umoles/gm i n T r i a c h i s s c y l l i u m (Suyama et a l ,  (1972) has h y p o t h e s i z e d ,  on  the b a s i s of oxygen consumption  e x c r e t i o n by T i l a p i a mossambica, t h a t a t l e a s t a p o r t i o n o f the  produced i n the muscle d u r i n g a c t i v i t y i s of a n a e r o b i c this contention  a r i s e s from the o b s e r v a t i o n s  origin.  d u r i n g h y p o x i a (Dejours  et a l , 1968)  of NH^  i n c r e a s e s d u r i n g h y p o x i a (Pequin  and  and  and  NH^~  Support f o r  that g o l d f i s h expire  amounts of NH^ i n carp  At  increased  t h a t the b l o o d  S e r f a t y , 1962).  level  I t would  be of i n t e r e s t to a s c e r t a i n i f there a r e r e a c t i o n s i n w h i t e muscle, f u n c t i o n a l under a n a e r o b i c  c o n d i t i o n s , which c o u l d p r o v i d e  on the b a s i s o f our knowledge of i n t e r m e d i a r y l i k e l y o r i g i n s of a n a e r o b i c  NH^:  a s o u r c e o f NH^~.  There appear,  metabolism, to be o n l y  the f r e e a d e n y l a t e  p o o l and  two  the amino a c i d  pool. I t i s recognized  t h a t the enzyme 5' AMP  c o n t r o l l e d r e l e a s e of NH  A  deaminase i s i n s t r u m e n t a l  i n s k e l e t a l muscle (Lowenstein, 1972) .  5*  i n the AMP  7  deaminase c a t a l y z e s the d e g r a d a t i o n of AMP  to IMP  ( i n o s i n e monophosphate)  p l u s NH^ and o c c u r s i n p a r t i c u l a r l y h i g h t i t r e s i n s k e l e t a l muscle.  Further-  more, the a c t i v i t y of t h i s enzyme i s a t l e a s t 15 times h i g h e r i n carp  white  muscle than i n r e d muscle or h e a r t ( F i e l d s , p e r s o n a l communication).  This  r e a c t i o n was  thus c o n s i d e r e d a l i k e l y  a n a e r o b i c MH^. ate k i n a s e  AMP,  c a n d i d a t e f o r the p r o d u c t i o n of  however, i s i n e q u i l i b r i u m w i t h ATP  and ADP  by the a d e n y l -  reaction: 2 ADP  T h e r e f o r e i f 5' AMP  •AMP  +  ATP  deaminase i s r e s p o n s i b l e f o r the p r o d u c t i o n of a n a e r o b i c  NH£ t h i s s h o u l d be r e f l e c t e d by a decrease  i n the e n t i r e a d e n y l a t e p o o l .  In  i n s e c t f l i g h t muscle, a t i s s u e w i t h no a n a e r o b i c c a p a c i t y , the a d e n y l a t e p o o l in  f a c t remains c o n s t a n t d u r i n g a c t i v i t y  hypoxia  (Ford and Candy, 1972).  more complex p r o b a b l y due  an hypoxic  i n e i t h e r s k e l e t a l muscle  o r h e a r t ( S h a f e r and W i l l i a m s o n , 1973) , whereas a f t e r  s t r e s s i t i s found to d e c r e a s e w i t h a concomitant  increase i n  plus i t s degradation products  (Imai e t a l , 1964;  and  Jones and Murray (1960) found a 1:1  G e r l a c h , 1966).  In f i s h ,  Chaudry e t a l , 1974;  m e t r i c r e l a t i o n s h i p between d e p l e t i o n of adenine n u c l e o t i d e s and of IMP  i n cod muscle a f t e r the animal had been f a t i g u e d .  apparent  IMP  Deuticke  stoichioaccumulation  In l i g h t of the  d i s c r e p a n c i e s i n t h i s a r e a , a study of a d e n y l a t e p o o l a l t e r a t i o n s  i n an organism, was  fibers i n  In working c o n d i t i o n s when oxygen i s not  the a d e n y l a t e p o o l does not decrease  ( E d i n g t o n e_t a l , 1973)  o r imposed  But i n v e r t e b r a t e muscle the s i t u a t i o n i s  to the v a r y i n g content of r e d and w h i t e  d i f f e r e n t mammalian m u s c u l a t u r e s . limiting  (Gerez and K i r s t e n , 1965)  the c a r p , p o s s e s s i n g a l a r g e homogenous white muscle mass  initiated. In a study of a n a e r o b i c NliT p r o d u c t i o n , the p o s s i b i l i t y of the  fermenta-  8  t i o n o f amino a c i d s must a l s o be c o n s i d e r e d . f a c u l t a t i v e anaerobes m o b i l i z e both ly ATP  and t h i s a p p a r e n t l y p r o v i d e s  Many i n v e r t e b r a t e s which a r e  carbohydrates  and amino a c i d s a n a e r o b i c a l -  them w i t h a d d i t i o n a l means o f g e n e r a t i n g  over and above c l a s s i c a l g l y c o l y s i s .  Figure 2 i s a s i m p l i f i e d  representa-  t i o n o f what i s thought t o occur d u r i n g p e r i o d s of oxygen d e p r i v a t i o n by i n v e r t e b r a t e f a c u l t a t i v e anaerobes (Hochachka e t a l , 1973). scheme f o r c a r b o h y d r a t e s  i s the same as t h a t which occurs  as f a r as the l e v e l o f phosphoenolpyruvate. pyruvate  being d i r e c t l y  converted  metabolite  fates.  i n vertebrate  tissue  However, i n s t e a d o f phosphoenol-  to pyruvate  o x a l o a c e t a t e which i s q u i c k l y reduced  The r e a c t i o n  i ti s first  to m a l a t e .  carboxylated  to form  Malate a p p a r e n t l y has two  One r o u t e i n v o l v i n g the r e v e r s a l of Krebs c y c l e r e a c t i o n s  l e a d s to the f o r m a t i o n o f s u c c i n a t e and the concomitant p r o d u c t i o n S u c c i n a t e may accumulate as an end product proprionate, a v o l a t i l e acid, with  of ATP.  or be f u r t h e r c a t a b o l i z e d t o  the f u r t h e r p r o d u c t i o n of ATP and C ^ .  o t h e r d e s t i n a t i o n o f malate i s c o n v e r s i o n t i o n w i t h glutamate t o form a l a n i n e .  to pyruvate  f o l l o w e d by  The  transamina-  I n v e r t e b r a t e s have p a r t i c u l a r l y  high  l e v e l s of f r e e amino a c i d s and i t i s thought t h a t these a r e i n i t i a l l y transaminated a-ketoacids  w i t h a - k e t o g l u t a r a t e to form t h e i r r e s p e c t i v e a - k e t o a c i d s .  formed from amino a c i d s such as l e u c i n e and v a l i n e a r e f u r t h e r  c a t a b o l i z e d to v o l a t i l e a c i d s a g a i n w i t h  the p r o d u c t i o n o f ATP and CX^.  I n t e r e s t i n g l y enough, t h e r e a r e data which l e n d c r e d u l e n c e hypothesis bic  The  to the  t h a t such a r e a c t i o n scheme may occur i n f i s h t i s s u e s .  p r o d u c t i o n o f CO 2 by f i s h has been r e p o r t e d s e v e r a l times.  The anaero-  Thus, g o l d f i s h  i n j e c t e d w i t h l a b e l l e d g l u c o s e produce l a b e l l e d CT^ under a n o x i c c o n d i t i o n s (Hochachka, 1961) and g i l l produces C 0  9  of metabolic  t i s s u e i n c u b a t e d under a n a e r o b i c origin  (Ekberg,  1962).  conditions  T h i s concept  of anaerobic  9  p r o d u c t i o n i s supported  by numerous r e p o r t s of r e s p i r a t o r y q u o t i e n t s  g r e a t e r than 1 d u r i n g both h y p o x i a 1967;  Mathur, 1967).  NH^,  swimming ( K u t t y , 1968,  Furthermore, the a n a e r o b i c  v o l a t i l e a c i d s has been n o t e d and Kopecky, 1961).  and  on at l e a s t two  1972;  Morris,  p r o d u c t i o n of u n i d e n t i f i e d  occasions  ( B l a z k a , 1958;  Perhaps the o b s e r v a t i o n s of the a n a e r o b i c  Blazka  p r o d u c t i o n of  CC>2 and v o l a t i l e a c i d s can be e x p l a i n e d s i n g u l a r l y , but when a l l o f them  are c o n s i d e r e d  ±a t o t o they suggest  t h a t something i s unaccounted f o r i n our  c u r r e n t theory of a n a e r o b i c metabolism i n f i s h and no means be suggest  considered closed.  (1) t h a t l a c t a t e may  t h a t the problem should  In p a r t i c u l a r , the above unusual  not be the s o l e end product  g e n o l y s i s , and/or (2) g l y c o g e n  i s not  the o n l y a n a e r o b i c  observations  of a n a e r o b i c energy  by  glyco-  source  utilized. Although i t a l s o has  w h i t e muscle generates  much o f i t s energy by a n a e r o b i c means,  an a e r o b i c component to i t s metabolism.  t h i s t i s s u e has  Moreover, d u r i n g work  the c a p a c i t y to i n c r e a s e i t s oxygen consumption to a s m a l l  degree (Wittenberger  and D i a c i u c , 1965).  I n the mammalian h e a r t and  skeletal  muscle the t o t a l amount of Krebs c y c l e i n t e r m e d i a t e s i n c r e a s e s as the work load increases  (Shafer and W i l l i a m s o n ,  1973;  E d i n g t o n ' e t ' a l , ' 1973); however,  i t had never been a s c e r t a i n e d whether or not w h i t e muscle per se has c a p a c i t y to do so.  the  Consequently, t h i s work a l s o i n v e s t i g a t e s the a b i l i t y  of  w h i t e muscle to augment the s i z e o f i t s Krebs c y c l e p o o l d u r i n g i n c r e a s e d energy demands. In the p r e s e n t  t h e s i s , two  types of experiments were c a r r i e d out  examine energy metabolism i n carp w h i t e muscle. animals  were e x e r c i s e d i n o r d e r  and Krebs c y c l e a l t e r a t i o n s . s t r e s s and  I n one  to e l u c i d a t e a n a e r o b i c  I n another  to  s e t o f experiments, c o n t r o l mechanisms  study carp were s u b j e c t e d to  changes i n the f r e e amino a c i d p o o l were examined.  hypoxic  10  F i g u r e 2.  Anaerobic  metabolism i n i n v e r t e b r a t e f a c u l t a t i v e anaerobes.  Abbreviations not i n d i c a t e d i n text: phoglycerate;  1,3 DPG, 1,3 d i p h o s -  2-KCA, 2 - k e t o i s o c a p r o a t e ;  a-KGA, a - k e t o g l u t a r -  a t e ; a-KVA, a - k e t o i s o v a l e r a t e ; OXA, o x a l o a c e t a t e ; PEP, phosphoenolpyruvate; 3 PGA, 3 p h o s p h o g l y c e r a t e . from Hochachka ^ t a l (1973).  Modified  GLYCOGEN  \  NAD + -  3 PGA  ADP+P;  NADH-  *-a-KvA  /~-\ NAD'1"  2-KCA  Oj-KGA Pyruvate  maiate •  ADP + Pj  fumarate  FP  red NADP  ATP-  GTPGDP+Pi  succinate  ox  succinyl CoA  J - C o ASH methylmalonylCoA ADP+P; ATP  . -C02  proprionyl CoA ADP+ P; ATP-  -CoASH propnonate  HsobutyrylCoA  NAD+  isobutyrate  11  NADH CQ  7~\  ATP  ADP+Pj isovalerylCoA  ATP -isovalerate  NADH  | a ianine| O  NADPH  11  CHAPTER I I  MATERIALS AND METHODS  lift  Animals Carp  (Cyprinus  Columbia.  c a r p i o L.) were s e i n e d from l o c a l ponds i n s o u t h e r n  F i s h employed i n the swimming experiments were between 12 and 15 cm  i n l e n g t h and weighed between 40 and 60 gm. 12 ±1°C i n a e r a t e d  These animals were m a i n t a i n e d a t  running water, under a s i m u l a t e d  f e d corn ad l i b . The carp u t i l i z e d  n a t u r a l photoperiod  and  i n the h y p o x i a experiment were about 30 cm  i n l e n g t h and weighed about 1000 gm. running  British  These animals were h e l d out o f doors i n  a e r a t e d water a t 12 ±3°C and d u r i n g  the h o l d i n g p e r i o d o f not l o n g e r  than three weeks were n o t f e d . E x e r c i s e Experiments F i s h were e x e r c i s e d i n a swim t u n n e l s i m i l a r to t h a t d e s c r i b e d by B r e t t (1964).  F i s h were i n t r o d u c e d  for 1 hr. periods  Following  i n t o the t u n n e l and f o r c e d t o swim a t 8.6 cm/sec  the i n t r o d u c t o r y phase the f i s h were s u b j e c t e d  o f swimming a t f i x e d v e l o c i t i e s a f t e r which the v e l o c i t y was r a p i d l y  increased.  The v e l o c i t y increment was a p p r o x i m a t e l y 7 cm/sec.  was terminated  The experiment  e i t h e r a f t e r s u c c e s s f u l completion o f the 2nd v e l o c i t y l e v e l  or when the f i s h was unable to remove i t s e l f at the downstream end o f the t u n n e l . f a t i g u e and u s u a l l y o c c u r r e d  during  from an e l e c t r i f i e d  grid  (15 v AC)  T h i s l a t e r b e h a v i o u r was d e f i n e d as the 4th speed increment.  m a i n t a i n e d a t 11°C and 100% a i r s a t u r a t i o n . using  to 10 min  The water was  C r i t i c a l v e l o c i t y was c a l c u l a t e d  the e m p i r i c a l formula o f B r e t t (1964) such t h a t the l a s t v e l o c i t y t h a t  the f i s h s u c c e s s f u l l y m a i n t a i n e d was added t o the v e l o c i t y a t which the f i s h f a t i g u e d , m u l t i p l i e d by the p r o p o r t i o n o f the 10 minute p e r i o d t h a t i t was able t o s u s t a i n t h i s f i n a l speed.  The mean c r i t i c a l v e l o c i t y f o r the animals  o f t h i s experiment was 45 cm/sec. F a i l u r e o f a f i s h t o meet the imposed v e l o c i t y i s due to the l i m i t a t i o n of the oxygen d e l i v e r y system and not e x h a u s t i o n  o f the w h i t e muscle  (Jones,  12  1971;  B r e t t , 1964) .  A f t e r the e x p e r i m e n t a l p e r i o d an animal i s s t i l l  perform b u r s t a c t i v i t y is  i f f o r c e d to do so.  The  day b e f o r e an experiment  A e r a t e d water,  a f i s h was  the evening and uncovered  The  t e s t chamber was  the f o l l o w i n g morning.  flowed  through  covered w i t h b l a c k p l a s t i c The e n t i r e  apparatus  immersed i n a water b a t h which s e r v e d to m a i n t a i n the temperature.  reduced by n i t r o g e n gas to about  the O2  In the  content of the i n f l o w i n g water  10% a i r s a t u r a t i o n as measured by a micro  W i n k l e r t e c h n i q u e (Kent and H a l l , p e r s o n a l communication). the O2 content i n the chamber f e l l  and  j u s t s l i g h t l y l a r g e r than the a n i m a l .  morning, on the day o f an experiment, was  removed from the h o l d i n g tank  about 1 C° h i g h e r than the h o l d i n g temperature,  the chamber a t 180 ±10 ml/min.  was  fatigued.  Experiments  p l a c e d i n a s e a l e d chamber which was  in  Thus, i n the p r e s e n t study t h e r e  no reason to b e l i e v e that the white muscle was  Hypoxia  a b l e to  W i t h i n 45  minutes  to t h i s l e v e l and c o n t i n u e d to drop f o r the  d u r a t i o n o f the experiment which was  terminated a f t e r 4 hours.  For c o n t r o l  f i s h which were kept i n the chamber f o r the same l e n g t h of time as the e x p e r i mentals In  the O2 content never f e l l below 60% o f the a i r s a t u r a t e d l e v e l . an experiment  of t h i s  type i t i s d i f f i c u l t  h y p o x i c s t r e s s to which the animals were exposed.  to q u a n t i t a t e the degree o f Observations of a q u a l i t a -  t i v e n a t u r e , however, i n d i c a t e d beyond q u e s t i o n that the e x p e r i m e n t a l f i s h were indeed s u b j e c t e d t o s e v e r e h y p o x i a . t h a t t h e r e was  Thus, p r e l i m i n a r y s t u d i e s showed  m o r t a l i t y i n animals s u b j e c t e d to e x p e r i m e n t a l c o n d i t i o n s f o r  p e r i o d s any l o n g e r than f o u r hours; whereas, the c o n t r o l f i s h c o u l d s u r v i v e for  a second day and p r o b a b l y l o n g e r .  reduced oxygen l e v e l demonstrated lost equilibrium.  Furthermore,  animals s u b j e c t e d to the  an extreme h y p e r v e n t i l a t i o n and  N e i t h e r of these b e h a v i o u r p a t t e r n s was  frequently  observed i n the  13  c o n t r o l group.  D u r i n g the t e s t p e r i o d both the c o n t r o l and the e x p e r i m e n t a l  f i s h demonstrated v e r y l i t t l e  activity.  Preparation of Tissue f o r Biochemical A n a l y s i s In the e x e r c i s e experiments, the f i s h were removed from the h o l d i n g or the swim t u n n e l and immediately d e c a p i t a t e d .  tank  In the h y p o x i a experiment,  the animals were removed from the e x p e r i m e n t a l chamber and stunned by a blow to the head. 1 gm was  In both s t u d i e s , a p o r t i o n o f w h i t e muscle weighing a p p r o x i m a t e l y  d i s s e c t e d from immediately below the d o r s a l f i n ,  p o s t e r i o r margin and going a n t e r i o r l y .  s t a r t i n g a t the  The t i s s u e sample was  then f r o z e n i n  l i q u i d n i t r o g e n , w i t h i n 20 sec a f t e r removal of the f i s h from the water.  In  the h y p o x i a experiment, a second sample o f t i s s u e , weighing about 30 gm,  was  a l s o d i s s e c t e d out and f r o z e n i n l i q u i d n i t r o g e n . used f o r v o l a t i l e a c i d  T h i s t i s s u e sample  was  analysis.  E x t r a c t i o n of A l l M e t a b o l i t e s Except V o l a t i l e - A c i d s The f r o z e n t i s s u e was  powdered w i t h mortar and p e s t l e which had been  p r e v i o u s l y c o o l e d and then the sample was  p l a c e d i n a 40 ml p l a s t i c  tube i n t o which a T e f l o n p e s t l e c o u l d f i t s n u g l y . a l i q u o t o f c o l d HCIO^ (8% w/v)  i n 40% e t h a n o l .  w i t h a g l a s s rod and the amount o f HCIO^ was The t i s s u e was d u r i n g which  The homogenate was  The sample was mixed  (#23)  same volume of HCIO^ as p r e v i o u s l y used.  K C0^ c o n t a i n i n g 0.5  mixer  maintained i n a d r y - i c e ethanol bath.  saved and the p r e c i p i t a t e was  supernatant s o l u t i o n s were combined  quickly  taken up to 3.5 ml/gm t i s s u e .  spun a t 25,000 g (-4°C) f o r 10 min t o p r e c i p i t a t e  The s u p e r n a t a n t s o l u t i o n was  9  The t e s t tube c o n t a i n e d an  homogenized f o r 2 min at h i g h speed w i t h a V i r t i s  time the t e s t tube was  centrifuge  protein.  resuspended i n the  A f t e r a f u r t h e r c e n t r i f u g a t i o n the  and n e u t r a l i z e d to pH 5.5-6.0 w i t h 3 M  triethanolamine.  The p r e c i p i t a t e d KCIO^ was  removed by  14  c e n t r i f u g a t i o n and and  the supernatant  s o l u t i o n was  s t o r e d a t -20°C  Corkey, 1969).  E x t r a c t i o n of V o l a t i l e  Acids  V o l a t i l e a c i d s were i s o l a t e d by steam d i s t i l l a t i o n l a r g e r p i e c e of f r o z e n t i s s u e was 500  was  A concentrate  Ten volumes of H^O  (Sigma) (Ackman and Noble, 1973)  a d j u s t e d to 2.0-2.5 w i t h 10 N ILjSO^.  of  fluid  KOH  o r i g i n a l l y present  pH=8.  KOH.  The  The  acetate f r e e formic  and  1957).  The  Between 10 and  maintained  f l a s h evaporated  2 drops of  were added and 11 times  i n the d i s t i l l i n g v e s s e l was  pH of the d i s t i l l a t e was  d i s t i l l a t e was  (Baker,  broken i n t o s m a l l p i e c e s and p l a c e d i n a  ml double neck d i s t i l l i n g f l a s k .  Antifoam  of  (Williamson  the  pH  the volume  c o l l e c t e d i n 25  ml  between 8.0-8.5 w i t h 0.1  to dryness  and  taken up  N  i n 200 y l  acid.  Enzymatic A n a l y s i s of M e t a b o l i t e s All  of the m e t a b o l i t e s , w i t h  the e x c e p t i o n of the amino a c i d s and  v o l a t i l e a c i d s , were measured e n z y m a t i c a l l y , i c a n a l y s e s , except  the procedures  Furthermore, each of the enzymat-  f o r formate and  IMP,  absorbance changes of the p y r i d i n e n u c l e o t i d e s a t 340 of cal  the  was  my.  based on The  change i n amount  the p y r i d i n e n u c l e o t i d e s i n ymoles i s c a l c u l a t e d from the e q u a t i o n d e n s i t y . ^ Q ) ( v o l u m e of assay m i x t u r e ) / ? , where E = 6.22  the  (A  bpti-  cm  /ymole, the  e x t i n c t i o n c o e f f i c i e n t of the p y r i d i n e n u c l e o t i d e s a t 340  my.  This value  represents  the amount of  the number of ymoles of m e t a b o l i t e p r e s e n t  per  p r o t e i n f r e e n e u t r a l i z e d e x t r a c t added, i f i n the a n a l y s i s t h e r e i s a r a t i o between the content  of measured m e t a b o l i t e s  t i o n o f the p y r i d i n e n u c l e o t i d e s . SP 1800  and  1:1  the o x i d a t i o n or  reduc-  Assays were c a r r i e d out a t 37°C on a Unicam  d u a l beam spectophotometer connected to a s t r i p  enzymes were purchased from Sigma, S t . L o u i s ,  Mo.  chart recorder.  All  15 A l a n i n e assay: 4.  _L M A T . T J  _L u  p y r u v a t e + NADH + H  +  l a c t a t e dehydrogenase z  e  ^_ . , „._.+ l a c t a t e + NAD  glutamate-pyruvate alanine + a-ketoglutarate  transaminase  p y r u v a t e + glutamate  Reagents Buffer: NADH:  0.5 M t r i s pH 8.1 8 mM i n 1% KHC0 (w/v) 3  a-ketoglutarate:  0.1 M i n 0.1 M t r i s pH 7.4  L a c t a t e dehydrogenase: Glutamate-pyruvate  source - beef h e a r t  transaminase:  source - p i g h e a r t  Procedure LDH  i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NADH (0.17 mM), and  n e u t r a l i z e d p r o t e i n f r e e e x t r a c t t o remove endogenous p y r u v a t e . When the r e a c t i o n i s complete a - k e t o g l u t a r a t e (0.2 mM) i s added. F i n a l l y glutamate-pyruvate i n O D ^ ^ Q i s recorded. preferable to  transaminase i s added and the decrease  The r e a c t i o n i s extremely slow,  t o p l o t an a l a n i n e standard c u r v e and i n t e r p o l a t e  a s c e r t a i n the c o n t e n t of a l a n i n e i n t h e e x t r a c t .  times - p y r u v a t e , 1 min; 1972).  thus i t i s  Reaction  a l a n i n e , 10 min (Lowry and Passonneau,  16  Aspartate  assay: malate dehydrogenase + +• malate + NAD  + o x a l o a c e t a t e + NADH + H  glutamateoxaloacetate transaminase >oxaloacetate aspartate + a-ketoglutarate  + glutamate  Reagents Buffer: NADH:  50 mM i m i d a z o l e pH 7 8 mM i n 1% KHC0  a-ketoglutarate:  3  (w/v)  0.1 M i n 0.1 M t r i s pH 7.4  M a l a t e dehydrogenase:  source - p i g h e a r t  Glutamate-oxaloacetate  transaminase:  source - p i g h e a r t  . Procedure M a l a t e dehydrogenase i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NADH (0.17 mM), a - k e t o g l u t a r a t e free extract.  (0.2 mM), and n e u t r a l i z e d p r o t e i n  When t h e r e a c t i o n i s complete  t a t e i s added and t h e decrease  glutamate-oxaloace-  i n 0D-... i s r e c o r d e d . 340  Reaction  time - 10 min (Lowry and Passonneau, 1972). Citrate  assay:  + o x a l o a c e t a t e + NADH + H citrate  malate dehydrogenase  c i t r a t e lyase  oxaloacetate + acetate  Reagents Buffer:  0.1 M t r i s pH 7.6  NADH:  8 mM i n 1% KHC0  ZnCl :  1.2 mM i n H 0  2  (w/v)  2  M a l a t e dehydrogenase: Citrate lyase:  3  ••malate + NAD  source - p i g h e a r t  source - A e r o b a c t e r  aerogenes  17  Procedure M a l a t e dehydrogenase i s added t o a c u v e t t e c o n t a i n i n g  buffer,  NADH (0.17 mM), Z n C l  free  extract.  2  (40 pM) and n e u t r a l i z e d p r o t e i n  When t h e r e a c t i o n i s complete c i t r a t e l y a s e i s added  and t h e d e c r e a s e i n 0°3^Q r e c o r d e d t o determine c i t r a t e  content.  R e a c t i o n time - 3 min (Lowry and Passonneau, 1972). Formate a s s a y :  ^ . , , , . tetrahydrofolic acid formylase c  J  formate + ATP + t e t r a h y d r o f o l i c a c i d N  ( l O ) - f o r m y l - t e t r a h y d r o f o l i c a c i d + ADP + P H  N (lO)-formyl-tetrahydrofolic acid  + •5,10-methenyltetrahydrofolic acid  Reagents Buffer:  1.0 M t r i e t h a n o l a m i n e pH 8.0  Tetrahydrofolic acid: ATP:  0.01 M pH 7.0 i n 1 M 2-mercaptoethanol  0.05 M i n 1.0 M t r i e t h a n o l a m i n e  MgCl : 2  0.1 M i n H 0 2  Percholic acid:  2% (w/v) i n B^O  T e t r a h y d r o f o l i c a c i d formylase:  source - C l o s t r i d i u m  cylindrosporum  Pr.66edure T e t r a h y d r o f o l i c a c i d f o r m y l a s e i s added to a c e n t r i f u g e tube containing buffer, tetrahydrofolic acid MgCl  2  (0.4 mM), ATP '(1 mM),  (5 mM) and n e u t r a l i z e d p r o t e i n f r e e e x t r a c t .  A f t e r 2 min  a t 37°C 1 volume o f p e r c h l o r i c a c i d i s added and t h e m i x t u r e centrifuged  t o remove p r o t e i n .  The d i f f e r e n c e i n 3 ^ Q between  a b l a n k and a sample i s determined.  0 D  The e x t i n c t i o n c o e f f i c i e n t  18 2 of 5 , 1 0 - m e t h e n y l - t e t r a h y d r o f o l i c a c i d a t 350 mu i s 24.9 cm / ymole  (Rabinowitz and Pr'icer,  1965).  Fructose-6-phosphate and f r u c t o s e - 1 , 6 - d i p h o s p h a t e a s s a y : glucose-6-phosphate glucose-6-phosphate + NADP  dehydrogenase  +  ^ 6-phosphogluconate + NADPH + H  +  phosphoglucose isoniGir3.s6  f ructose-6-phosphate  • glucose-6-phosphate  r^ , ,. , , _ fructose-1,6-diphosphatase , fructose-1,6-diphosphate »- f r u c t o s e - 6 phosphate + P^ n  Reagents Buffer:  1.0 M t r i s pH 8.8  NADP :  10 mM i n IL/)  MgCl :  0.1 M i n H 0  +  2  EDTA:  2  1.2% (w/v) i n H 0 2  Glucose-6-phosphate Phosphoglucose  dehydrogenase:  isomerase:  Fructose-1,6-diphosphatase:  . source - y e a s t  source - y e a s t source-^arabbit l i v e r  Procedure Glucose-6-phosphate b u f f e r , NADP  +  dehydrogenase  (0.33 mM), M g G l  protein free extract.  2  i s added to a c u v e t t e c o n t a i n i n g  (7 mM), EDTA (10 mM), and n e u t r a l i z e d  Glucose-6-phosphate  dehydrogenase i s  added t o remove.endogenous glucose-6-phosphate. i s complete phosphoglucose ^340  "*"  Sr e c o r <  ^  e <  ^  t  o  When t h e r e a c t i o n  isomerase i s added and t h e i n c r e a s e i n  determine t h e c o n t e n t o f f r u c t o s e - 6 - p h o s p h a t e .  F i n a l l y f r u c t o s e - 1 , 6 - d i p h o s p h a t a s e i s added t o determine t h e  19  l e v e l of fructose-l,6-diphosphate.  R e a c t i o n times -  glucose-6-phosphate, 1 min; f r u c t o s e - 6 - p h o s p h a t e , 5 min; f r u c t o s e - l , 6 - d i p h o s p h a t e , 30 min (Racker, 1965). Glucose-6-phosphate and ATP a s s a y : glucose-6-phosphate glucose-6-phosphate + NADP  +  dehydrogenase  ^ 6-phosphogluconate + NADPH + H  ATP + g l u c o s e  hexokinase  ^  +  +  glucose-6-phosphate  Reagents Buffer:  0.05 M t r i e t h a n o l a m i n e pH 7.5  NADP :  10 mM i n H 0  MgCl :  60 mM i n H 0  +  2  2  2  Glucose:  30 mM i n H 0 2  Glucose-6-phosphate dehydrogenase: Hexokinase:  source - yeast  source - yeast  Procedure .Glucose-6-phosphate dehydrogenase i n g b u f f e r , NADP  +  (0.17 mM), M g C l  i s added t o a c u v e t t e c o n t a i n 2  (1 mM), and n e u t r a l i z e d  p r o t e i n f r e e e x t r a c t t o determine t h e c o n t e n t o f g l u c o s e s phosphate. added. OD^Q  When t h e r e a c t i o n i s complete g l u c o s e (1.0 mM) i s  F i n a l l y hexokinase i s i n c l u d e d and t h e i n c r e a s e i n r e c o r d e d t o determine t h e l e v e l of A l P i  R e a c t i o n times -  glucose-6-phosphate, 1 min; ATP, 8 min (Lamprecht and Trautschold,  1965).  20  a-glycerophosphate  assay: glycerophosphate dehydrogenase >  + a - g l y c e r o p h o s p h a t e + NAD  dihydroxyacetonephosphate + NADH + H  Reagents Buffer:  g l y c i n e - h y d r a z i n e pH 9.2 (prepared by Sigma s t o c k #826-6)  NAD : +  10 mM i n H 0 2  a - g l y c e r o p h o s p h a t e dehydrogenase:  source - r a b b i t muscle  Procedure a - g l y c e r o p h o s p h a t e dehydrogenase i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NAD  +  (0.17 mM), and n e u t r a l i z e d p r o t e i n f r e e  and t h e i n c r e a s e i n 0 D 340  o / r i  i s recorded.  extract  The r e a c t i o n i s  extremely slow, thus i t ,is p r e f e r a b l e t o p l o t a s t a n d a r d curve and i n t e r p o l a t e t o a s c e r t a i n t h e content o f a - g l y c e r o p h o s p h a t e i n the extract.  R e a c t i o n time - 10 min (Lowry and Passonneau,  1972).  I n o s i n e monophosphate assay: hypoxanthine + 20„ + 2 H_0  i n o s i n e + P. l  xanthic oxidase >• u r i c a c i d + 2 H„0  nucleoside phosphorylase >hypoxanthine + r i b o s e - 5 phosphate 5' n u c l e o t i d a s e  Reagents Buffer: EDTA:  0.05 M K H P 0 2  0.1 M i n H 0 o  4  pH 7.4  > i n o s i n e + P.  21  xanthic oxidase:  source - b u t t e r m i l k  nucleoside phosphorylase: 5  1  nucleotidase:  source - c a l f s p l e e n  source - C r o t a l u s adamanteus venom  Procedure The  course o f t h e r e a c t i o n i s f o l l o w e d a t 293 mu.  Xanthic  o x i d a s e i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , EDTA (33 mM), and n e u t r a l i z e d p r o t e i n f r e e e x t r a c t . complete  When t h e r e a c t i o n i s  n u c l e o s i d e p h o s p h o r y l a s e i s added t o remove any endo-  genous i n o s i n e . determine  F i n a l l y 5' n u c l e o t i d a s e i s i n c l u d e d t o  t h e c o n t e n t o f IMP.  The e x t i n c t i o n c o e f f i c i e n t f o r  2 uric  a c i d a t 293 my i s 12 cm /umole.  hypoxanthine, Coddington,  1 min;  R e a c t i o n times -  i n o s i n e , 1 min;  IMP, 20 min (adapted  from  1965).  a-ketoglutarate assay: glutamate a - k e t o g l u t a r a t e + NH* + NADH + H  +  d  e  h  y  d  r  o  g  e  n  a  s  e  , i g  u t  amate  + NAD  Reagents Buffer: NADH:  0.5 M t r i s pH 8.0 8 mM i n 1% KHC0  (NH£) S0 : 2  4  Glutamate  3  (w/v)  35 mM i n H 0 2  dehydrogenase:  source - bovine  liver  Procedure GDH i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NADH (0.17 mM), (NH^^SO^ (5 mM), and n e u t r a l i z e d p r o t e i n f r e e e x t r a c t and t h e decrease i n O D ^ Q recorded. and B e r n t , 1965)..  R e a c t i o n time - 8 min (Bergmeyer  +  22  P y r u v a t e , ADP and AMP a s s a y : + p y r u v a t e + NADH + H ADP  +  l a c t a t e dehydrogenase  phosphoenolpyruvate  AMP + ATP  +  *• l a c t a t e + NAD  pyruvate kinase > ATP + p y r u v a t e  adenylate kinase  >. 2 ADP  Reagents Buffer: NADH:  0.05 M t r i e t h a n o l a m i n e pH 7.5 8 mM i n 1% KHC0 (w/v) 3  MgCl :  120 mM i n H 0  2  KCl:  2  750 mM i n H 0 2  Phosphoenolpyruvate: ATP:  15 mM i n 0.05 M t r i e t h a n o l a m i n e pH 7.5  6 mM i n 0.05 t r i e t h a n o l a m i n e pH 7.5  L a c t a t e dehydrogenase: Pyruvate k i n a s e : Adenylate kinase:  source - beef h e a r t  s o u r c e - r a b b i t muscle source - r a b b i t muscle  Procedure LDH  i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NADH (0.17 mM),  MgCl  2  (2 mM), K C l (75 mM), and n e u t r a l i z e d p r o t e i n f r e e  to determine t h e c o n t e n t o f p y r u v a t e . complete  phosphoenolpyruvate  p y r u v a t e k i n a s e to determine  extract  When the r e a c t i o n i s  (0.25 mM) i s added f o l l o w e d by the c o n t e n t o f ADP.  When the  second r e a c t i o n i s complete ATP (0.1 mM) i s i n c l u d e d and the d e c r e a s e i n O D ^ Q r e c o r d e d a f t e r the a d d i t i o n o f a d e n y l a t e kinase.  F o r each mole o f AMP two moles o f NADH a r e o x i d i z e d .  R e a c t i o n times - p y r u v a t e , 1 min; ADP, 2-4 min; AMP, 6 min (Lowry and Passonneau,  1972).  23  S u c c i n a t e assay: ,  *TA™T  U  _L  p y r u v a t e + NADH + H , , phosphoenolpyruvate  +  + l a c t a t e dehydrogenase . H ——2 »- l a c t a t e + NAD  , pyruvate kinase ^ , + ADP — — • p y r u v a t e + ADP A T V r i  , A . ™i ^ A s u c c i n a t e + ATP + CoA  A r > T 1  succinate thiokinase . . _ . , . _ — • s u c c i n y l CoA + ADP + P A T v n  :  i  Reagents Buffer: NADH:  0.05 M t r i e t h a n o l a m i n e , 10 mM MgSO^, 5 mM EDTA pH 7.4 5 mM i n 0.1 M t r i e t h a n o l a m i n e pH 8.2  Phosphoenolpyruvate: ATP: CoA,  0.1 M i n 0.05 M t r i e t h a n o l a m i n e pH 7.4  10 mM i n 0.05 M t r i e t h a n o l a m i n e pH 7.4 lithium salt:  5 mM i n ^ 0  L a c t a t e dehydrogenase: Pyruvate k i n a s e :  source - beef h e a r t  source - r a b b i t muscle  Succinate thiokinase:  source - E^. c o l i  Procedure LDH  i s added to a c u v e t t e c o n t a i n i n g b u f f e r , NADH (0.17 mM), and  n e u t r a l i z e d p r o t e i n f r e e e x t r a c t to remove, endogenous p y r u v a t e . When t h e i n i t i a l reaction i s complete  phosphoenolpyruvate  (1.5  mM)  and p y r u v a t e k i n a s e a r e added t o the c u v e t t e t o remove endogenous ADP.  When the second r e a c t i o n i s complete,  and ATP (0.15 mM) a r e added.  Finally  pyruvate,,1 min; ADP, 3 min; Corkey,  i  R e a c t i o n times -  s u c c i n a t e , 30 min ( W i l l i a m s o n and  1969).  L a c t a t e assay: _L i a A ^  l a c t a t e + NAD  +  mM)  succinate thiokinase i s  added and the decrease i n 0 D i s recorded. 340 o / o  l i t h i u m CoA (0.8  l a c t a t e dehydrogenase  ^ ., _ . + p- p y r u v a t e + NADH + H TA  TT  77  24  Reagents Buffer: NAD :  g l y c i n e - h y d r a z i n e pH 9.2 (prepared, by Sigma-stock #826-6) 10 mM i n H 0  +  2  L a c t a t e dehydrogenase:  source  - beef  heart  Procedure LDH i s added t o a c u v e t t e c o n t a i n i n g b u f f e r , NAD  +  (0.33 mM),  and n e u t r a l i z e d p r o t e i n f r e e e x t r a c t and the i n c r e a s e i n O D ^ ^ Q recorded. Malate  Reaction  time - 45 min (Sigma b u l l e t i n #826).  assay: glutamateoxaloacetate o x a l o a c e t a t e + glutamate T  ^  .  t  r  a  n  s  a  m  l  n  a  s  e  ^ aspartate + a-ketoglutarate  malate dehydrogenase . _ ^ » » ^ T T _L T T ^ • o x a l o a c e t a t e + NADH + H  „,T>+  malate + NAD  3  +  T  Reagents Buffer: NAD :  g l y c i n e - h y d r a z i n e pH 9.2 (prepared by Sigma-stock #826-6) 10 mM i n H 0  +  2  Glutamate:  88 mM i n 0.5 M t r i s  Glutamate-oxaloacetate  transaminase:  Malate dehydrogenase:  source  source  - p i g heart  - p i g heart  Procedure Glutamate-oxaloacetate b u f f e r , NAD  +  transaminase i s added t o a c u v e t t e . c o n t a i n i n g  (0.17 mM), glutamate (10 mM), and n e u t r a l i z e d p r o t e i n  free extract.  When the r e a c t i o n i s complete malate dehydrogenase  i s added and the i n c r e a s e i n O D ^ Q i s r e c o r d e d .  Reaction  time -  60 min (Lowry and Passonneau, 1972) NH^ assay: HTTT  +  NH,  glutamate dehydrogenase . ^ + a - k e t o g l u t a r a t e + NADH + H — - — — • glutamate + NAD+ i  i  ^  T  ^  ~KTATVTT  .  T T  +  25  Reagents Buffer: NADH:  0.5 M t r i s pH 8.0 8 mM i n 1% KHC0 (w/v) 3  a-ketoglutarate:  0.1 M i n 0.1 M t r i s pH 7.4  Glutamate dehydrogenase:  NH^ f r e e , source - b o v i n e  liver  Procedure GDH i s added t o a c u v e t t e a-ketoglutarate and  (Kun Amino A c i d  containing  b u f f e r , NADH (0.17  (10 mM), and n e u t r a l i z e d p r o t e i n f r e e  the decrease i n 0D~ 340  /rt  recorded.  mM) , extract  R e a c t i o n time - 10 min  and Kearney, 1970).  Analysis  P r i o r t o amino a c i d a n a l y s i s the p r o t e i n f r e e e x t r a c t , was s e p a r a t e d from i n t e r f e r i n g substances by a b s o r p t i o n  on a column (1 x 15 cm) o f A m b e r l i t e  IR-120 which had been p r e v i o u s l y . w a s h e d w i t h 5% H C l . extract adjusted  2.0 ml o f p r o t e i n  t o pH 2-3 w i t h 10 N ^ S O ^ were a p p l i e d  to the column.  free The  column was washed w i t h 40 ml o f d i s t i l l e d water and the amino a c i d s were e l u t e d w i t h 40 ml 2 N NH^OH (Williamson ated  et_ a l , 1967).  The e l u a n t was evapor-  t o dryness i n a 1 l i t r e f l a s k taken up i n 5 ml ^ 0 and t r a n s f e r r e d to a  50 ml f l a s k .  The o r i g i n a l 1 l i t r e c o l l e c t i n g f l a s k was washed w i t h 2 ml H^O  which was p l a c e d  i n the 50 ml f l a s k .  The amino a c i d e x t r a c t was a g a i n  evaporated t o dryness and s t o r e d a t -20°C.  Immediately p r i o r t o a n a l y s i s the  sample was taken up i n c i t r a t e b u f f e r pH 2.2. Amino a c i d s i n 200-500 y l a l i q u o t s were separated on a Beckman 120C amino acid analyzer.  The i o n exchange r e s i n was a s u l f o n a t e d  benzene copolymer. and pH  The o p e r a t i n g  temperature was 55°C.  polystyrene-divinyl Lysine, h i s t i d i n e  a r g i n i n e were separated on a 16 cm column by e l u t i o n w i t h 0.35 N Na c i t r a t e 5.25.  The a c i d i c amino a c i d s were separated on a 46 cm column by e l u t i o n  26  w i t h 0.2 N Na c i t r a t e pH 3.25. reagent.  A l l amino a c i d s were d e t e c t e d  by n i n h y d r i n  I n t e r n a l s t a n d a r d s , a^-amino-3-guanidinopropionic a c i d f o r the b a s i c s  column and n o r l e u c i n e  f o r the a c i d i c s column, were employed so t h a t a c o r r e c -  t i o n c o u l d be made f o r the aging o f the n i n h y d r i n moles of each amino a c i d r e s i d u e  reagent.  The number o f  a p p l i e d was determined by comparison w i t h a  standard chromatogram. V o l a t i l e Acid  Analysis  V o l a t i l e a c i d s were separated by gas l i q u i d chromatography. Aereograph (#1700) equipped w i t h a flame i o n i z a t i o n d e t e c t o r  A Varian  was used.  The  columns were 6 f t by 1/8 i n s t a i n l e s s s t e e l packed w i t h Chromsorb 101 80/100 mesh, and were m a i n t a i n e d a t 130°C. of 56 ml/min. volatile  The c a r r i e r gas was N  No attempt was made t o a c c u r a t e l y  2  quantitate  with a flow rate the c o n t e n t o f  acids.  Calculations Both the amino a c i d a n a l y s i s and. the enzymatic a n a l y s i s p r o v i d e d a t a i n ymoles o f s p e c i f i e d substance p e r a l i q u o t o f p r o t e i n f r e e n e u t r a l i z e d The  extract.  a l i q u o t o f n e u t r a l i z e d p r o t e i n f r e e e x t r a c t i s c o n v e r t e d t o the c o r r e s -  ponding v a l u e i n gm f r e s h t i s s u e by m u l t i p l y i n g by  (v )(W) c  (V  where,  V  + V-,) (V d a  + V. )  h'  t o t a l volume o f HC10, added d u r i n g  the e x t r a c t i o n  V. b  amount of water i n the sample o f t i s s u e powder  V  volume o f a l i q u o t used f o r n e u t r a l i z a t i o n  V W The  a  c  c d  volume o f K^CO^ added to n e u t r a l i z e the above a l i q u o t V  c  f r e s h weight o f t i s s u e sample i n grams  water c o n t e n t o f carp w h i t e muscle was determined t o be about 80%.  27  In the r e s u l t s , where a p p r o p r i a t e , v a l u e s a r e expressed as ± standard error  o f the mean.  R e s u l t s were a n a l y z e d s t a t i s t i c a l l y , by a two sampled t -  t e s t f o r data c o l l e c t e d  i n the swimming experiment  U t e s t f o r d a t a o f the hypoxia  experiment.  than 0.05 was c o n s i d e r e d t o be s i g n i f i c a n t .  and by the Mann-Whitney  In a l l c a s e s a p r o b a b i l i t y  of l e s s  28  CHAPTER I I I  RESULTS  28  a  Swimming Experiment C a r r i e d . Out, i n S p r i n g  1974  The c o n c e n t r a t i o n s o f a l l of the g l y c o l y t i c i n t e r m e d i a t e s measured w i t h the e x c e p t i o n of pyruvate  increased during a c t i v i t y  a tendency f o r a-glycerophosphate, pathway, to i n c r e a s e .  The  phosphate), was  1.82  a m e t a b o l i t e a s s o c i a t e d w i t h the  The mass a c t i o n r a t i o of the  i n the r e s t e d f i s h and  o r d e r s of magnitude  (Mahler, and  2.78  the animals  III.  The  were e x e r c i s e d , ATP  L e v e l s of ADP  i n this  a l s o decreased  content  of ATP  support  t h i s decrease l e v e l and  the decrease  decreased  with a c t i v i t y . by about  concentration.  i n the a d e n y l a t e  The  NH^  to IMP  and NH^.  AMP the  increase i n  IMP  p o o l were e s s e n t i a l l y i n 1:1  stoichio-  decreased  I t i s i n t e r e s t i n g to note t h a t  by  The  energy charge,  as d e f i n e d by A t k i n s o n the r e s t e d f i s h , and  (1968a), was  0.83  exercised  i n the e x e r c i s e d group  animals.  [ADP]/[ATP] + [ADP]  h i g h i n both groups of animals,  e q u i l i b r i u m c o n s t a n t o f the a d e n y l a t e r e s t e d and  [ATP] + 0 . 5  (Table I I I ) .  k i n a s e r e a c t i o n was  0.3  The  the  although  c o n c e n t r a t i o n i n c r e a s e d i n working white muscle, the change i s not  l a r g e as f o r IMP.  total  Concomitant w i t h  metry, a r e s u l t c l e a r l y showing t h a t the a d e n y l a t e p o o l was c o n v e r s i o n of AMP  When  65%.  Thus i n t h i s complex way,  d u r i n g the e x e r c i s e p e r i o d .  an i n c r e a s e i n IMP  of  p o o l are summar-  a s m a l l but s i g n i f i c a n t amount; however,  p o o l decreased was  the concept  tissue.  c o n c e n t r a t i o n s were reduced  c o n c e n t r a t i o n s remained low and unchanged. free adenylate  phosphofructo-  of r e a c t i o n by about  r e s u l t s of t h i s study w i t h r e s p e c t to the a d e n y l a t e  i z e d i n T a b l e s I I and  glycolytic  i n the e x e r c i s e d group.  Cordes, 1966), and  a r e g u l a t o r y r o l e of p h o s p h o f r u c t o k i n a s e The  also  (ADP)(fructose-l,6-diphosphate)/(ATP)(fructose-6-  These v a l u e s were d i s p l a c e d from.the e q u i l i b r i u m c o n s t a n t two  There was  g r e a t e s t change o c c u r r e d i n l a c t a t e l e v e l s which  i n c r e a s e d by about 10 ymoles/gm. kinase r e a c t i o n , that i s  (Table I ) .  as +  [AMP],  0.89  in  apparent  i n both  the  29  Of the two amino a c i d s measured, a s p a r t a t e l e v e l s but  s i g n i f i c a n t amount w h i l e  decreased, by a s m a l l  t h e r e was a tendency f o r an i n c r e a s e i n the l e v e l  of a l a n i n e (Table I V ) . C i t r a t e , malate, a - k e t o g l u t a r a t e and o x a l o a c e t a t e , compounds a s s o c i a t e d w i t h the Krebs c y c l e , and i n f a c t  t h e r e was a tendency f o r c i t r a t e t o d e c r e a s e .  t h a t carp white muscle has a l i m i t e d cycle pool.  d i d n o t i n c r e a s e during- a c t i v i t y , I t t h e r e f o r e appears  c a p a c i t y t o augment the s i z e o f i t s Krebs  30  Table  C o n c e n t r a t i o n s o f g l y c o l y t i c i n t e r m e d i a t e s i n w h i t e muscle of  carp under two w e l l d e f i n e d c o n d i t i o n s :  maximally  Metabolite  resting  active.  Resting  Maximally active  Glucose-6-phosphate  0.67  ± 0.05  1.33  ±  Fructose-6-phosphate  0.11  ± 0.01  0.18  ± 0.03*  Fructose-1,6diphosphate  0.85  ± 0.08  1.28  ±  Pyruvate  0.11  ± 0.03  0.12  ±0.03  Lactate  3.71  + 0.17  12.58  +  1.18*  a-glycerophosphate  2.52  + 0.60  4.59  ±  1.30  0.10*  0.10*  A l l v a l u e s a r e e x p r e s s e d i n micromoles/gm of f r e s h t i s s u e N =  7.  *Statistically  and  s i g n i f i c a n t d i f f e r e n c e between  groups.  (±S.E.).  31  Table I I -  C o n c e n t r a t i o n s o f the a d e n y l a t e s and r e l a t e d m e t a b o l i t e s i n white muscle o f carp under two w e l l d e f i n e d c o n d i t i o n s : r e s t i n g and maximally  active.  Maximally Metabolite  Resting  ATP  4.12 ± 0.18  1.87 ± 0.08*  ADP  0.97 ± 0.05  0.73 ± 0.18*  AMP  0.07 ± 0.02  0.08 ± 0.02  IMP  1.38 ± 0.26 3.00 + 0.30  4.01 + 0.16 4.10 ± 0.25*  NHI" 4  A l l v a l u e s are expressed N  =  active  i n micromoles/gm o f f r e s h t i s s u e  7.  *Statistically  s i g n i f i c a n t d i f f e r e n c e between  groups.  (±S.E.M).  32  Table I I I .  Energy charge and p o o l and two  changes i n c o n c e n t r a t i o n s of the  related metabolites  adenylate  i n w h i t e muscle of carp  under  r e s t i n g and maximally  active.  well defined conditions:  Resting  Maximally active  Difference  5.1  2.68  -2.48  IMP  1.38  4.01  +2.63  NH;  3.00  4.10  Energy charge  0.89  0.83  Adenylate  Adenylate  pool  p o o l , IMP  fresh tissue.  N =  and NH^ 7.  a r e expressed  •  i n micromoles/gm o f  +1.10 -0.06  33  Table  TV.  C o n c e n t r a t i o n s of Krebs c y c l e i n t e r m e d i a t e s and  related  m e t a b o l i t e s i n w h i t e muscle of carp under two w e l l d e f i n e d conditions:  r e s t i n g and maximally  Maximally active  Resting  Metabolite  active.  Citrate  0.50  ±  0.06  0.34  +  0.04  Malate  1.12 ±  0.28  1.16  ±  0.09  Oxaloacetate  Undetectable  a-ketoglutarate  <0.10  Undetectable <0.10  Aspartate  0.24  +  0.03  0.14  ± 0.03*  Alanine  2.62  +  0.46  3.12  ±  A l l v a l u e s a r e expressed N =  i n micromoles/gm of f r e s h t i s s u e  7.  *Statistically  s i g n i f i c a n t d i f f e r e n c e between groups.  0.47  (±S.E.).  34  Swimming Experiment C a r r i e d Out  i n Summer  1974  R e s u l t s of a second study i n which f i s h were a l s o e x e r c i s e d a t an mediate speed a r e shown i n T a b l e V.  Although  w h i t e muscle i s a c t i v e a t i n t e r m e d i a t e cult For  t h i s reason  the experiment was  that  speeds, these d a t a , however, a r e  terminated  a f t e r o n l y a few  In l i g h t of the p a u c i t y of r e s u l t s a l l of the raw  Even though the data a r e l i m i t e d  diffi-  i n d i v i d u a l s were  data are  presented.  they a r e i n t e r e s t i n g i n a number of r e s p e c t s .  c o n c e n t r a t i o n of l a c t a t e i n the c o n t r o l and maximally e x e r c i s e d groups  s i m i l a r to t h a t found 12 umoles/gm i n both  i n the s p r i n g study. swimming s t u d i e s .  L a c t a t e reached  l e v e l r e l a t i v e to t h a t found  /gm.  The  l e v e l of NH^  group.  with a c t i v i t y ,  There was  worked muscle.  i n most cases  i n one  As expected  the lowest  h i g h content  be due  l e v e l of ATP;  to e x p e r i m e n t a l  of the a d e n y l a t e p o o l and  95 umoles  times g r e a t e r  than  and ADP. g e n e r a l l y  i n the maximally e x e r c i s e d  The  t h e r e was  of IMP  of the f i s h e x e r c i s e d a t i n t e r m e d i a t e  f i n d i n g may  con-  to note t h a t the energy charge remained h i g h  i n the maximally e x e r c i s e d group a l t h o u g h  unexpected i n l i g h t of the low  NH^  to i n c r e a s e i n the most s t r e n u o u s l y  and r e l a t i v e l y c o n s t a n t a t a l l three work l o a d s .  The  5-6  both ATP  l e v e l s occurred  a tendency f o r AMP  It i s interesting  i n t h i s component.  intermediate i n  i n d i v i d u a l case reached  i n the summer sampled f i s h was  t h a t i n the s p r i n g sampled animals. decreased  a maximum of about  i n r e s t i n g and maximally a c t i v e muscle.  c e n t r a t i o n i n c r e a s e d w i t h a c t i v i t y and  was  L a c t a t e c o n c e n t r a t i o n i n white muscle  sampled from f i s h worked a t moderate speeds was  two  w e l l recognized  to i n t e r p r e t s i n c e the degree of a c t i v i t y of i n d i v i d u a l f i b e r s i s unknown.  sampled.  The  i t i s now  inter-  l e v e l of IMP  was  much i n d i v i d u a l  i n the one  highest  variability  r e s t e d f i s h i s not  however, the low l e v e l of IMP  speeds remains an enigma.  e r r o r s i n c e i n a l l other cases  the c o n c e n t r a t i o n of IMP  the  in  This level  are i n v e r s e l y p r o p o r t i o n a l .  T a b l e V.  C o n c e n t r a t i o n s o f m e t a b o l i t e s and t h e energy  charge v a l u e i n w h i t e muscle,of  carp a f t e r v a r i o u s  l e v e l s of a c t i v i t y .  L e v e l of  Animal number  activity  Metabolite Lactate  Rested  Intermediate -  Maximally active  q-  Energy chargi  ATP  ADP  AMP  IMP 0.92  1  1.70*  16.96  5.17  1.01  0.02  0.78  2  3.26  16.52  2.31  0.72  0.02  2.59  3  2.36  25.26  3.99  0.93  0.03  0.38  0.90  4  4.54  23.13  1.98  0.49  0.02  0.52  0.89  5  7.17  19.12  2.77  0.70  0.02  1.24  0.89  6  9.90  25.11  1.62  0.67  0.06  3.29  0.83  7  12.66  95.10  1.07  0.64  0.14  4.06  0.75  *A11 v a l u e s e x p r e s s e d  i n ymoles/gm f r e s h  tissue.  ...  0.87  36  Hypoxia The  Experiment c o n c e n t r a t i o n of l a c t a t e i n white muscle of. carp.exposed  environmental lar  hypoxia was  about 12 ymoles/gm (Table V I ) .  to t h a t found a f t e r maximal a c t i v i t y by white muscle.  l a c t a t e i n the c o n t r o l f i s h of the hypoxia the e x p e r i m e n t a l s .  study was  to severe  T h i s v a l u e was The  simi-  c o n t e n t of  almost as h i g h as t h a t of  In l i g h t o f p r e v i o u s o b s e r v a t i o n s ( T a b l e s I and V) t h a t the  l a c t a t e l e v e l i n white muscle of r e s t e d f i s h was p r o b a b l e t h a t the e x p e r i m e n t a l f i s h , some degree of a n a e r o b i c s t r e s s .  about 3 ymoles/gm, i t i s  i n the p r e s e n t study, were s u b j e c t e d to  N e v e r t h e l e s s , many p o s i t i v e c o n c l u s i o n s  may  be made from t h i s work. I t has been c l e a r l y shown t h a t the v o l a t i l e a c i d s , a c e t a t e , p r o p r i o n a t e , b u t y r a t e , or v a l e r a t e (Table VI) were not produced i n the white muscle of c a r p .  Accumulation  as a n a e r o b i c end  products  of formate would not be d e t e c t e d  w i t h the a n a l y t i c a l t e c h n i q u e s employed here; however, u s i n g an enzymatic i t had been shown t h a t t h i s a c i d was d u r i n g strenuous e x e r c i s e .  not produced  Furthermore,  i n white muscle of carp  the data show t h a t s u c c i n a t e was  a q u a n t i t a t i v e l y important a n a e r o b i c end p r o d u c t i n carp white The  c o n t e n t of f r e e amino a c i d s may  assay  not  muscle.  be found i n T a b l e V I I .  S i n c e the  c o n t r o l animals were, on the b a s i s of l a c t a t e c o n c e n t r a t i o n , s u b j e c t e d to some degree of a n a e r o b i c s t r e s s , any a l t e r a t i o n i n the f r e e amino a c i d p o o l w i l l minimal  and consequently  difficult  to p i c k up.  w i t h some c o n f i d e n c e t h a t no one amino a c i d was a n a e r o b i c end product  R e g a r d l e s s , i t may a quantitatively  such as a l a n i n e i s i n i n v e r t e b r a t e a n i m a l s .  be  said  important No  single  amino a c i d was  markedly a l t e r e d by the hypoxic c o n d i t i o n s ; a l t h o u g h ,  was  f o r an i n c r e a s e i n the t o t a l f r e e amino a c i d p o o l and a l l of  a tendency  the amino a c i d s w i t h the e x c e p t i o n . o f g l y c i n e . i n r e l a t i o n to amino a c i d metabolism was  The most s i g n i f i c a n t  the amount of n i t r o g e n t h a t  there  finding was  be  37 l o c k e d up i n the f r e e amino a c i d . p o o l .  T h i s i n c r e a s e d by about.6. ymoles/gm  (Table V I I ) and was i n agreement w i t h the tendency f o r f r e e NH^ t o decrease (Table VI) and the t o t a l f r e e amino a c i d p o o l to i n c r e a s e .  A l a c k of  1:1  s t o i c h i o m e t r y between the i n c r e a s e i n amino a c i d s and the i n c r e a s e i n n i t r o g e n i n c o r p o r a t e d i n t o the amino a c i d p o o l occurs  s i n c e h i s t i d i n e , l y s i n e , and  a r g i n i n e c o n t a i n more than 1 n i t r o g e n atom each. porated NH^.  The amount of. n i t r o g e n i n c o r -  i n t o the f r e e amino a c i d p o o l was f a r i n excess of the decrease i n  When these f i n d i n g s a r e c o n s i d e r e d  v o l a t i l e a c i d s i t may be s a f e l y concluded g e n e r a l amino a c i d f e r m e n t a t i o n .  a l o n g w i t h the l a c k of p r o d u c t i o n of t h a t there was n o t an a c t i v e and  G l y c i n e may, however, be an e x c e p t i o n  g e n e r a l r u l e s i n c e i t was the o n l y amino a c i d to decrease d u r i n g  to t h i s  hypoxia.  38  Table  VI.  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 carp w h i t e muscle b e f o r e and a f t e r hypoxic  stress.  Control  Hypoxic  Mean  Range  Mean  . . Range  Lactate  9.60*  6.23-13.50  12.02  6.90-15.12  NH+  4.32  2.18-8.24  3.09  1.90-5.63  Succinate  <0.30  <0.30  Acetate Proprionate Butyrate Valerate-  -3 <0.2 x 10  -3 <0.2 x 10  *A11 v a l u e s a r e expressed  i n ymoles/gm o f f r e s h  tissue.  N = 4.  39  Table  VII.  F r e e amino a c i d a f t e r hypoxic  c o n c e n t r a t i o n i n carp w h i t e muscle b e f o r e and  stress.  Control Mean  Hypoxic Range  Mean  .  Range  Asp  0.15*  (0.08-0.21)  0.09  (0.07-0.11)  Thr  0.47  (0.21-0.70)  0.48  (0.28-0.63)  Ser-Gln  0.73  (0.46-1.09)  1.00  (0.76-1.42)  Glut  0.22  (0.11-0.31)  0.26  (0.19-0.32)  0.17  (0.08-0.23)  0.25  (0.25-0.37)  Gly  6.17  (3.69-9.82)  5.42  (4.31-6.94)  Ala  0.84  (0.42-1.08)  1.41  (0.80-1.99)  Val  0.29  (0.14-0.48)  0.44  (0.29-0.69)  Met  0.08  (0.05-0.12)  0.11  (0.09-0.16)  Isoleu  0.26  (0.12-0.43)  0.38  (0.21-0.38)  Lec  0.37  (0.23-0.57)  0.55  (0.34-0.83)  Tyr  0.06  (0.02-0.11)  0.09  (0.07-0.12)  Phe  0.08  (0.04-0.12)  0.33  (0.08-0.73)  Lys  1.22  (0.64-2.12)  1.97  (0.63-3.40)  Hist  3.71  (2.83-4.66)  4.74  (3.56-6.02)  Arg  0.10  (trace-0.16)  0.23  (0.13-0.32)  Prol  ,  T o t a l amino a c i d s  14.93  (13.12-18.18)  17.75  (17.31-18.67)  N i t r o g e n i n amino acid pool  23.74**  (21.16-26.24)  29.90**  (27.63-31.92)  *A11 v a l u e s a r e expressed i n ymoles/gm f r e s h  tissue.  ^ ^ S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between means•  N = 3.  40  CHAPTER IV  DISCUSSION  40  a  C o n t r o l of G l y c o l y s i s The  s i t u a t i o n w i t h r e s p e c t . t o the c o n t r o l of g l y c o l y s i s i n white muscle  seems r e l a t i v e l y s t r a i g h t forward.  C o n c e n t r a t i o n s of  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 - d i p h o s p h a t e and e x e r c i s e p e r i o d i n d i c a t i n g t h a t , as expected,  glucose-6-phosphate,  l a c t a t e r i s e d u r i n g the  the g l y c o l y t i c c o n t r i b u t i o n to  energy p r o d u c t i o n i s i n c r e a s e d d u r i n g h i g h work r a t e s .  A c t i v a t i o n of the  two  key r e g u l a t o r y enzymes of g l y c o l y s i s , p h o s p h o f r u c t o k i n a s e and p y r u v a t e k i n a s e , may  be e x p l a i n e d on the b a s i s o f the known k i n e t i c p r o p e r t i e s of these enzymes.  Thus s u b s t r a t e and p r o d u c t a c t i v a t i o n  (Freed, 1971)  of p h o s p h o f r u c t o k i n a s e  (by f r u c t o s e - 6 - p h o s p h a t e and f r u c t o s e - 1 , 6 - d i p h o s p h a t e , r e s p e c t i v e l y ) w i t h concomitant  f r u c t o s e - 1 , 6 - d i p h o s p h a t e feed, forward a c t i v a t i o n of p y r u v a t e k i n a s e ,  commonly observed Mustafa  i n f i s h muscle p y r u v a t e k i n a s e s (Somero and Hochachka," 1968;  e t a l , 1971), c o u l d r e a d i l y account  glycolytic rate.  f o r the observed i n c r e a s e i n  Moreover, d e i n h i b i t i o n of these two  as a consequence of f a l l i n g  l e v e l s of ATP  enzymes would be  (Freed, 1971;  Mustafa  expected  e t a l , 1971),  and of c r e a t i n e phosphate ( S t o r e y and Hochachka, 1974), both p r o c e s s e s b e i n g f a c i l i t a t e d by f r u c t o s e - 1 , 6 - d i p h o s p h a t e . carp white muscle g l y c o l y s i s appears s t u d i e d systems. mention.  Two  In these c o n t r o l  to be s i m i l a r to o t h e r more commonly  i n c o n s i s t e n c i e s w i t h the l i t e r a t u r e , however, deserve  F i r s t l y , i t i s e v i d e n t from the d a t a t h a t the energy  e s s e n t i a l l y i d e n t i c a l a t both l e v e l s o f muscle metabolism A l t h o u g h i n v i t r o both p h o s p h o f r u c t o k i n a s e kinase  characteristics,  ( P u r i c h and Fromm, 1973)  i n v i v o i t i s c l e a r t h a t energy  and muscle work.  (Shen e_t a l , 1968)  and  pyruvate  are s t i m u l a t e d by a decrease i n t h i s  parameter,  charge p l a y s o n l y a modest r o l e i n s u s t a i n i n g  the h i g h g l y c o l y t i c r a t e s t h a t support extreme muscle work. i s no evidence whatever t h a t AMP  charge i s  Secondly,  there  c o n s t i t u t e s an u n i q u e l y important m e t a b o l i t e  41  s i g n a l to g l y c o l y s i s i n white muscle, as suggested  by Newsholme (1972) f o r  h e a r t , because i t s c o n c e n t r a t i o n i s s i m i l a r a t the w i d e l y d i f f e r i n g rates.  glycolytic  In c o n t r a s t , i f t h e r e i s a s i n g l e a d e n y l a t e s i g n a l t h a t i s important  to a s u s t a i n e d h i g h l e v e l of g l y c o l y s i s i t presumably i s ATP, o v e r a l l c o n c e n t r a t i o n change i s the g r e a t e s t .  since i t s  However, as s h a l l be  argued  l a t e r , i n order to take advantage of t h i s m e t a b o l i c " s i g n a l " the organism  must  t o l e r a t e an o v e r a l l r e d u c t i o n i n the a d e n y l a t e p o o l . L a c t a t e and Other P o t e n t i a l End I t i s i n t e r e s t i n g to note ed here  Products t h a t i n the t h r e e i n d i v i d u a l experiments  the maximal l e v e l of white muscle l a c t a t e i s c o n s i s t e n t l y about  ymoles/gm.  However, when carp white muscle was  the muscle i t s e l f D i a c i u c , 1965).  f a t i g u e d , l a c t a t e reached The p r e s e n t d a t a may  approaches i n v i v o f o r t h i s s p e c i e s . limit  electrically  Data which support  12  stimulated u n t i l  33 ymoles/gm (Wittenberger  i n d i c a t e an upper l i m i t  report-  and  that l a c t a t e  the concept  of a  to which l a c t a t e i s a l l o w e d to n o r m a l l y accumulate have been o b t a i n e d  w i t h rainbow t r o u t .  Thus, i n t r o u t s t r e n u o u s l y e x e r c i s e d f o r 5 minutes muscle  l a c t a t e i n c r e a s e d from 3 to 47 ymoles/gm.  Yet i n animals  15 minutes of strenuous e x e r c i s e , t h e r e was  sampled a f t e r 9 and  no f u r t h e r i n c r e a s e i n muscle  l a c t a t e above t h a t found i n the animals worked f o r 5 minutes 1962) .  Stevens  and B l a c k  (Black e t a l ,  (1966) and Hammond and Hickman (1966) a l s o o b t a i n e d  r e s u l t s of a s i m i l a r n a t u r e w i t h  trout.  A l l of the a v a i l a b l e b i o c h e m i c a l and h i s t o l o g i c a l evidence carp white muscle i s p a r t i c u l a r l y w e l l designed f a c t , on the b a s i s of hypoxia be a "good anaerobe". ments was  The  suggests t h a t  f o r a n a e r o b i c metabolism.  In  s t u d i e s , one would n o r m a l l y c o n s i d e r carp to  extreme r e s i s t a n c e of t h i s animal to low 0  demonstrated by Mazeaud  (1973) who  2  induced a n o x i a i n carp by  environexpos-  42  i n g them.to a i r f o r p e r i o d s up to water.  to 2 hours w i t h o n l y i n f r e q u e n t s h o r t r e t u r n s  Even a t temperatures as h i g h as 10°C  dissolved  f o r carp was  (Downing and Merkins,  observed  1957).  the lower l e t h a l l e v e l of  to be about 0.5  mg/1  Species c l o s e l y r e l a t e d  i n o r d i n a t e t o l e r a n c e to h y p o x i a .  a i r saturation)  to carp a l s o show an  For i n s t a n c e , Basu (1949) found  g o l d f i s h h e l d f o r 9 hours i n water w i t h an 0^ c o n t e n t This p a r t i c u l a r  (-5%  o f o n l y 0.6  no deaths i n mg/1,  a t 28°C.  s p e c i e s can i n f a c t s u r v i v e t o t a l i n t e r r u p 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 by cyanide p o i s o n i n g  ( F r y , p e r s o n a l communication).  A further  extreme case i s demonstrated by C r u c i a n carp which l i v e i n s m a l l ponds t h a t become i c e l o c k e d , g r a d u a l l y grow a n o x i c and remain 0^ f r e e f o r up  to 2 months  ( B l a z k a , 1958).  t h a t have  These f i n d i n g s a r e v e r y much d i f f e r e n t from those  been o b t a i n e d w i t h many o t h e r f i s h s p e c i e s .  For i n s t a n c e , the salmonids,  which  a r e the most a c t i v e l y s t u d i e d , have been r e p e a t e d l y shown to succumb between 1 and  2 mg/1  dissolved 0  2  (see Doudoroff and  Shumway, 1970,  references).  S t u d i e s of t h i s n a t u r e ,  the a n a e r o b i c  c a p a c i t y of white muscle per se.  estimate exercised The  the a n a e r o b i c  f o r numerous  of course, do not p r o v i d e evidence  of  However, i t i s p o s s i b l e to  c a p a b i l i t i e s of the swimming musculature w i t h f o r c e d  experiments. a e r o b i c e f f i c i e n c y of a working muscle, t h a t i s the energy  to u s e f u l work/energy a v a i l a b l e from consumed oxygen, i s c o n s i d e r e d 20-30% ( H i l l ,  converted to be about  1950); any v a l u e s h i g h e r than t h i s a r e thus i n d i c a t i v e of anaero-  b i c metabolism.  The  percentage a e r o b i c working e f f i c i e n c y of swimming  ( F i g u r e 3) has been determined by Smit e_t al_ (1971) .  At low  goldfish  swimming speeds  the g o l d f i s h demonstrates a low a e r o b i c e f f i c i e n c y , as the animal  swims  faster  the percentage e f f i c i e n c y i n c r e a s e s , and a t v e l o c i t i e s above. 6 l e n g t h s / s e c  the  working e f f i c i e n c y i s g r e a t e r than can be accounted f o r by a e r o b i c means a l o n e .  43  At the h i g h e s t speeds the g o l d f i s h can a t t a i n the working e f f i c i e n c y almost. 100%..  Thus, i t may be s a i d w i t h some c e r t i t u d e t h a t much of the energy  r e q u i r e d f o r i n t e n s e swimming by the g o l d f i s h i s generated and  reaches  i n the extreme case approaches 80% o f the energy  a comparable study employing  by a n a e r o b i c means  output o f the a n i m a l .  In  rainbow t r o u t , Webb (1971) has shown t h a t the  o v e r a l l c o n t r i b u t i o n o f a n a e r o b i c metabolism t o i n t e n s e swimming* i s n e g l i g i b l e . Yet i n t r o u t , e x e r c i s e d s t r e n u o u s l y f o r only, a few minutes,  the c o n c e n t r a t i o n  of muscle l a c t a t e i n c r e a s e s t o 40-50 ymoles/gm from a r e s t i n g v a l u e of about 3 ymoles/gm (Black et_ a l , 1962; Stevens 1966).  and B l a c k , 1966; Hammond and Hickman,  Even i n mammalian muscle, the c o n t e n t o f l a c t a t e n o r m a l l y  35 ymoles/gm d u r i n g a c t i v i t y  (Edington jit a l , 1972).  reaches  S i n c e carp white  muscle  i s a p p a r e n t l y capable o f p e r f o r m i n g h i g h l e v e l s o f a n a e r o b i c work one would p r e d i c t t h a t i t would a l s o produce, high.amounts o f l a c t a t e .  Yet, d e s p i t e the  a n a e r o b i c c a p a b i l i t i e s o f carp white muscle, l a c t a t e a c c u m u l a t i o n  i n this  t i s s u e i s low by v e r t e b r a t e s t a n d a r d s . The phenomenon o f a l a c k o f p r o p o r t i o n a l i t y between the amount o f energy which must be generated observed  a n a e r o b i c a l l y and the accumulation  f o r other s p e c i e s .  F o r i n s t a n c e , B l a z k a and Kopecky (1961) c l a i m e d  t h a t a f t e r 4 hours o f a n o x i a t h e C r u c i a n carp accumulated lactate/gm.  o f l a c t a t e has been  o n l y 0.5 ymoles o f  D u r i n g hypoxic e x c u r s i o n s (ending i n anoxia) b u l l h e a d s  o n l y about 0.7 ymoles l a c t a t e / g m o f muscle; by comparison l a c t a t e  accumulated  levels  i n c r e a s e d by about 15 and 30 ymoles/gm i n rainbow and brown t r o u t , r e s p e c t i v e l y , a l t h o u g h the hypoxic  s t r e s s i n the l a t t e r  (Burton and Spehar, 1971).  two cases was much l e s s  severe  C l e a r l y , white muscle o f some s p e c i e s has a h i g h  a n a e r o b i c c a p a b i l i t y b u t i t does n o t accumulate an e x t r a o r d i n a r y amount o f lactate.  I t i s known t h a t l a c t a t e i s n o t e x c r e t e d d u r i n g a n a e r o b i c work  44  ( P r o s s e r et. a l , hypoxia  1957)  and  ( S a t c h e l l , 1971)  i n l i g h t " o f 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 during i t i s u n l i k e l y t h a t t h e r e c o u l d be an e f f e c t i v e  p o s i t i o n i n o t h e r t i s s u e s such as the l i v e r .  These f i n d i n g s t h e r e f o r e  de-  suggest  t h a t something i s y e t unanswered about the manner i n which carp white muscle d e a l s w i t h low oxygen a v a i l a b i l i t y . The  data of the p r e s e n t study show t h a t carp white muscle i s " not the  of metabolic, pathways which have e v o l v e d i n f a c u l t a t i v e anaerobes. t i o n of v o l a t i l e a c i d s a n a e r o b i c a l l y by t r o u t (Blazka and Kopecky, 1961) f u t e d f o r muscle and  ( B l a z k a , 1958)  had been suggested;  l i v e r by Burton and  f i n d i n g to be an a r t i f a c t .  Spehar (1971) who  T h i s study c o n f i r m s  i n t i s s u e s o t h e r than muscle and  produc-  C r u c i a n carp  however, t h i s f i n d i n g was considered  re-  Blazka's  the work of Burton and  (1971) a t l e a s t f o r muscle; however a t the p r e s e n t time products  and  The  site  Spehar  the f o r m a t i o n o f  l i v e r cannot be r u l e d out.  these  The  f a i l u r e to f i n d v o l a t i l e a c i d s argues s t r o n g l y a g a i n s t the p o s s i b i l i t y of an a c t i v e amino a c i d f e r m e n t a t i o n , s i n c e d u r i n g a n a e r o b i c work i n i n v e r t e b r a t e s it  i s b e l i e v e d t h a t v o l a t i l e end p r o d u c t s a r e d e r i v e d from the c a t a b o l i s m of  amino a c i d s (Hochachka e_t a l , 1973).  The p o s s i b i l i t y of an a c t i v e amino a c i d  f e r m e n t a t i o n i s f u r t h e r negated by the o b s e r v a t i o n t h a t d u r i n g hypoxia i s a tendency f o r the f r e e amino a c i d p o o l to i n c r e a s e not d e c r e a s e .  there Further-  more, t h e r e i s not a r e o r g a n i z a t i o n of the amino a c i d p o o l which c o u l d a p r e f e r e n t i a l u t i l i z a t i o n of some amino a c i d s .  G l y c i n e may  to t h i s r u l e s i n c e t h e r e i s a tendency f o r t h i s amino a c i d hypoxia.  be an  exception  to decrease  I t i s known though t h a t f i s h have an a c t i v e g l y c i n e anabolism  both a e r o b i c and hypoxic some c i r c u m s t a n c e s  c o n d i t i o n s (Demael-Suard e t a l , 1974)  and  indicate  during under  t h a t under  the c o n c e n t r a t i o n o f f r e e g l y c i n e i n c a r p white muscle  be as h i g h as 30 ymoles/gm (Creach, 1966).  C e r t a i n l y the r o l e of g l y c i n e  may  45  F i g u r e 3.  The percentage a e r o b i c swimming e f f i c i e n c y v e r s u s swimming speed o f g o l d f i s h .  A e r o b i c e f f i c i e n c y i s d e f i n e d as energy  r e q u i r e d t o d e v e l o p power/energy a v a i l a b l e from consumed oxygen. (1971).  The graph i s p l o t t e d from the d a t a o f Smit e_t a l  46  metabolism should be the s u b j e c t of future.work.  T h i s study a l s o shows t h a t  s u c c i n a t e i s not a s i g n i f i c a n t a n a e r o b i c end product  i n f i s h white muscle  whereas i n the o y s t e r h e a r t even a f t e r a s h o r t anoxic p e r i o d i t r e a c h e s 5 ymoles /gm  ( C o l l i c u t , p e r s o n a l communication).  In the i n v e r t e b r a t e s , s u c c i n a t e i s  formed a n a e r o b i c a l l y by a r e v e r s a l of the Krebs c y c l e .  One  to occur o n l y under c o n d i t i o n s of complete a n o x i a ; t h i s was the p r e s e n t study, and  would expect  this  not the case i n  i t i s d o u b t f u l whether t h i s ever o c c u r s as a normal  c o u r s e o f events i n the c a r p . In c o n c l u s i o n , i t would be of extreme i n t e r e s t to a s c e r t a i n i f under a n a e r o b i c c o n d i t i o n s glycogen i s q u a n t i t a t i v e l y c o n v e r t e d to l a c t a t e i n carp white muscle.  Stevens  and B l a c k  (1966) have p r o v i d e d s t a t i s t i c a l  t h a t l a c t a t e i s the s o l e end p r o d u c t of a n a e r o b i c  evidence  glycogen c a t a b o l i s m i t t r o u t ;  however, s i m i l a r d a t a do not e x i s t f o r any o t h e r s p e c i e s . NHI" L e v e l s The r e s u l t s of the swimming experiment  c a r r i e d out i n s p r i n g 1974  show  q u i t e c o n c l u s i v e l y t h a t the a d e n y l a t e p o o l i s a source of a n a e r o b i c NH^ d u c t i o n i n carp white muscle. NH^  IMP  In t h a t p a r t i c u l a r . e x p e r i m e n t , the i n c r e a s e i n  content i n muscle d u r i n g a c t i v i t y was  or IMP  increase.  One  pro-  l e s s than the a d e n y l a t e p o o l  e x p l a n a t i o n f o r the l a c k of 1:1  decrease  s t o i c h i o m e t r y between  + + and NH^ i n c r e a s e i s t h a t some NH^ i s b e i n g r e l e a s e d i n t o the b l o o d .  would p r o v i d e an e x p l a n a t i o n f o r the observed p r o d u c t i o n by swimming f i s h hypoxia  study, t h e r e appears  free adenylate pool.  (Kutty, 1972). to be another  phenomenon of a n a e r o b i c  This  NH^  However, on the b a s i s of the f a t e of NH^  r e l e a s e d from  T h i s a s p e c t s h a l l be d i s c u s s e d under amino a c i d  the metabol-  ism. The NH^  c o n t e n t of carp white muscle sampled i n the e x e r c i s e  experiment  47  c a r r i e d out i n summer. 1974  i s most i n t r i g u i n g . .  The v a l u e s are. extremely  high  and i n f a c t appear t o be the h i g h e s t ever r e p o r t e d i n the l i t e r a t u r e f o r s k e l e t a l muscle.  I t i s w e l l known t h a t carp can m o b i l i z e t h e i r muscle p r o t e i n s to  serve as an energy  source  (Creach and  S e r f a t y , 1974)  the animals i n the p r e s e n t study a r e d o i n g .  and  t h i s i s p r o b a b l y what  T h i s concept  i s supported by  o b s e r v a t i o n t h a t NH^  c o n t e n t i n c r e a s e s q u i t e markedly d u r i n g a c t i v i t y .  d a t a f u r t h e r suggest  t h a t c a r p muscle has  amino a c i d s d i r e c t l y as an energy carbohydrates  source i n s i t u and  i n o t h e r t i s s u e s i s not n e c e s s a r y .  t h a t p r i o r c o n v e r s i o n to  I t i s not c l e a r why  the  One  possi-  i s t h a t even though the f i s h were f e d on a d a i l y b a s i s , the s u p p l i e d  d i e t may  have been inadequate.  Otherwise  s w i t c h i n f u e l source as i s thought tion  The  the c a p a c i t y t o t o t a l l y u t i l i z e some  animals were m o b i l i z i n g t h e i r p r o t e i n s t o r e s i n the p r e s e n t study. bility  the  (Saunders,  these f i s h may.undergo a s e a s o n a l  to occur i n salmonids  p e r s o n a l communication).  during s m o l t i f i c a -  C l e a r l y t h i s problem warrants  further  consideration. Adenylate Pool Size The  s t o i c h i o m e t r i c r e l a t i o n s h i p between a d e n y l a t e p o o l d e p l e t i o n and  accumulation  clearly  c a t a l y z e d by 5' AMP  IMP  shows, t h a t the a d e n y l a t e p o o l i s reduced by the r e a c t i o n deaminase.  The r e g u l a t o r y n a t u r e of t h i s enzyme from  carp  white muscle has been w e l l c h a r a c t e r i z e d ( F i e l d s , p e r s o n a l communication; P u r z y c h a - P r e i s and Zydowo, 1969). 0.5  mM)  The  and p o t e n t l y i n h i b i t e d by GTP  enzyme i s a c t i v a t e d by ADP (guanosine  From the p r e s e n t study the enzyme appears removal of GTP  inhibition.  and Murray, 1960;  GTP  i n c r e a s e d u r i n g a c t i v i t y GTP  to be c o n t r o l l e d l a r g e l y by  f o r GTP  uM).  the  low i n f i s h muscle  and as demands f o r h i g h energy  l e v e l s must f a l l ,  about  t r i p h o s p h a t e ) (K^ about 50  l e v e l s are i n i t i a l l y  Gras et_ a l , 1967)  (K  (Jones  phosphates  i s o n l y formed i n  48  e s s e n t i a l l y two ways, f i r s t l y by t r a n s p h o s p h o r y l a t i o n w i t h ATP the Krebs c y c l e r e a c t i o n c a t a l y z e d by s u c c i n a t e . t h i o k i n a s e . a r e reduced reduced.  the r a t e of GTP  secondly  When ATP. l e v e l s  Furthermore, as energy demands a r e p l a c e d on white muscle,  1965), consequently  (Wittenberger  glycolysis  and.Diaciuc,  the p r o p o r t i o n of t r i p h o s p h o r y l a t e d n u c l e o t i d e s t h a t  r e p r e s e n t s must decrease.  by  p r o d u c t i o n by the former r e a c t i o n must a l s o be  i s a c t i v a t e d f a r more than Krebs c y c l e a c t i v i t y  d e t e c t GTP  and  GTP  In f a c t , Jones and Murray (1960) were unable to  i n muscle of f a t i g u e d cod.  Be t h a t as i t may, cance of the reduced e x p l a n a t i o n may  the q u e s t i o n s t i l l remains of the p h y s i o l o g i c a l s i g n i f i -  a d e n y l a t e p o o l d u r i n g h i g h muscle work r a t e s .  be t h a t 5' AMP  servation.  p r o d u c t i o n by a mass a c t i o n e f f e c t .  be a more important  Thus, as ATP  simple  deaminase f u n c t i o n s i n c o n c e r t w i t h the adeny-  l a t e k i n a s e r e a c t i o n to maximize ATP However, there may  One  thermodynamic e x p l a n a t i o n . f o r the  ob-  l e v e l s drop d u r i n g muscle work the r a t i o change of  [ADP][P_^]/[ATP] c o u l d d r a s t i c a l l y reduce the f r e e energy of ATP  hydrolysis  a c c o r d i n g to the f o l l o w i n g r e l a t i o n s h i p :  AG = AG° + RT  In  [ADP][P.] — [ATP]  D u r i n g muscle work, c o n t r o l of t h i s r a t i o may  become i n c r e a s i n g l y d i f f i c u l t  s i n c e not o n l y i s t h e r e a change i n the ADP/ATP r a t i o , i n c r e a s e i n P^ c o n c e n t r a t i o n s  there a l s o occurs  (Hammond and Hickman, 1966) .  an  These c o n s i d e r a -  t i o n s emphasize t h a t i n the absence of e x t e r n a l c o n t r o l l i n g mechanisms l a r g e drops i n ATP concomitantly  c o n c e n t r a t i o n s c o u l d not be w i t h i n c r e a s i n g ADP  that i s c l e a r l y prevented. may  be l e s s c r i t i c a l  t o l e r a t e d because they would  occur  l e v e l s of comparable magnitude, a. s i t u a t i o n  In t h i s c o n n e c t i o n  the r e g u l a t i o n of. energy charge  to g l y c o l y t i c c o n t r o l , than to. the maintenance of a  suit-  49  a b l e r e l a t i o n s h i p between. ADP and ATP l e v e l s . a d j u s t e d by the c o n c e r t e d  a c t i o n of a d e n y l a t e  AMP formed from the a d e n y l a t e i n order  t o minimize ADP  That r e l a t i o n s h i p c o u l d be k i n a s e and AMP deaminase, the  k i n a s e r e a c t i o n b e i n g removed by AMP deaminase  accumulation.  Amino A c i d Metabolism The  r e s u l t s o f the hypoxia  study  show t h a t d u r i n g a n a e r o b i c  n i t r o g e n i s i n c o r p o r a t e d i n t o the f r e e amino a c i d p o o l . n i t r o g e n i s most l i k e l y  f r e e NH^ l i b e r a t e d  known mechanisms f o r f i x i n g  The source  of t h i s  from the a d e n y l a t e p o o l .  The o n l y  f r e e NH^ i n muscle a r e by the r e a c t i o n s c a t a l y z e d  by glutamate dehydrogenase and glutamine c a t a l y z e s the f o r m a t i o n o f glutamine the c o n c e n t r a t i o n o f glutamine out as a major n i t r o g e n o u s  metabolism  synthetase.  The l a t t e r  enzyme  from glutamate and NH^~; however, s i n c e  i s q u i t e low i n f i s h muscle t h i s can be r u l e d  sink.  I t thus  seems l i k e l y ,  genase, which has been shown t o occur i n f i s h muscle  t h a t glutamate dehydro-  (McBean e t a l , 1966),  f u n c t i o n s t o f i x f r e e NH^ i n t o the amino a c i d p o o l by the f o l l o w i n g r e a c t i o n : NH^~  +  a-ketoglutarate  +  NADH  •glutamate  +  NAD  +  Carp white muscle has a v e r y h i g h c a p a c i t y t o u t i l i z e amino a c i d s f o r e n e r g e t i c purposes under a e r o b i c c o n d i t i o n s (Creach and S e r f a t y , 1974).  Thus, g i v e n an  a c t i v e a e r o b i c c a t a b o l i s m of amino a c i d s t h e r e must be a t any g i v e n time a p o o l of  partially  oxidized products.  A wide spectrum o f glutamate transaminase  activity  has been demonstrated i n f i s h muscle ( S i e b e r t e_t a l , 1964) and i t i s  probable  t h a t the s m a l l i n c r e a s e i n a number of.amino a c i d s d u r i n g  metabolism i s due t o t r a n s a m i n a t i o n The  anaerobic  of p r e - e x i s t i n g a-ketoacids with  glutamate dehydrogenase r e a c t i o n c o u l d n o t o n l y serve t o m a i n t a i n  glutamate. low NH^  l e v e l s d u r i n g a n a e r o b i c metabolism b u t may a l s o c o n f e r an e n e r g e t i c advantage to  t h e t i s s u e s i n c e i t would p r o v i d e an a d d i t i o n a l method of o x i d i z i n g NADH.  50  Proposed Scheme o f N i t r o g e n On  the b a s i s o f the h y p o x i a and the swimming experiments i t i s p o s s i b l e to  construct during for  Metabolism  a fairly  comprehensive m e t a b o l i c scheme f o r n i t r o g e n  a n a e r o b i c work i n carp white muscle ( F i g u r e 4 ) .  metabolism  The energy  required  work i s u l t i m a t e l y d e r i v e d from the h y d r o l y s i s o f ATP t o ADP and P^  (reaction a).  When ATP l e v e l s cannot be m a i n t a i n e d by the energy  production  pathways the c o n t e n t o f ADP i n c r e a s e s , and as the ADP l e v e l i n c r e a s e s ATP and AMP a r e formed by the a d e n y l a t e k i n a s e effect  (reaction b).  r e a c t i o n simply  As the work l o a d on. the  b i l i t i e s GTP l e v e l s drop  t i s s u e exceeds i t s a e r o b i c  ( r e a c t i o n c and d ) , AMP deaminase i s a c t i v a t e d  t i o n e) and the a d e n y l a t e p o o l i s d e c r e a s e d . substrate  because o f a mass a c t i o n  to form amino a c i d s  (reaction g).  adenylate pool i s replenished Lowenstein  The q u e s t i o n  i n the r e c o v e r y  period  following fatigue.  (1972) has proposed t h a t the 5' AMP deaminase r e a c t i o n i s one nucleotide  cycle  (Figure 5 ) .  t o Lowenstein, IMP f u r t h e r r e a c t s w i t h GTP and a s p a r t a t e  adenylosuccinate.  acids  then remains as t o how the  step i n a r e a c t i o n span t h a t i s termed the p u r i n e  fumarate.  kinase.  from AMP i s f i x e d i n t o glutamate by glutamate dehydrogenase  ( r e a c t i o n f ) and i s s u b s e q u e n t l y t r a n s f e r r e d t o a v a r i e t y of a-keto  According  (reac-  T h i s i s p o s s i b l e s i n c e AMP,, the  o f the AMP deaminase r e a c t i o n , i s made a v a i l a b l e by a d e n y l a t e  NH^ r e l e a s e d  capa-  The a d e n y l o s u c c i n a t e  to form  i n t u r n i s c o n v e r t e d to AMP  plus  I t has been shown i n homogenates o f mammalian s k e l e t a l muscle t h a t  the c y c l e f u n c t i o n s i n c o n c e r t w i t h g l y c o l y s i s (Tornheim and Lowenstein, 1974). The  theory,'however, p r e d i c t s o n l y a t r a n s i e n t i n c r e a s e  maintenance o f the a d e n y l a t e p o o l .  i n IMP w i t h  general  C l e a r l y the c y c l e p e r se does n o t operate  d u r i n g a c t i v i t y i n carp white muscle s i n c e there i s an a c c u m u l a t i o n o f IMP. Moreover, the c y c l e i s n o t simply  operating  a t a new steady s t a t e  ( i . e . at  51  a l t e r e d l e v e l s of the a d e n y l a t e s and  IMP)  s i n c e d u r i n g a n a e r o b i c metabolism i n  t h i s t i s s u e t h e r e i s no a l t e r a t i o n i n the f r e e amino.acid  pool.  I f the c y c l e  were to become a c t i v e o n l y d u r i n g r e c o v e r y of the a d e n y l a t e p o o l a f t e r e x e r c i s e , the d i s c r e p a n c y between these r e s u l t s and would be apparent  r a t h e r than  those of Tornheim and Lowenstein  (1974)  real.  S i n c e the enzymes of the p u r i n e n u c l e o t i d e c y c l e a p p a r e n t l y a r e p r e s e n t i n white muscle ( F i e l d s , p e r s o n a l communication), i t i s p r o b a b l e  t h a t they  supply a pathway f o r the r e g e n e r a t i o n of the a d e n y l a t e p o o l from IMP r e c o v e r y f o l l o w i n g a n a e r o b i c work.  during  Thus, i n white muscle the r e a c t i o n pathway  shown i n F i g u r e 5 i s a " c y c l e " o n l y i n a f o r m a l sense, because the two arms o f the c y c l e a r e f u n c t i o n a l l y s e p a r a t e d i n time.  One  arm,  i n a s e , i s f o r m a l l y a c a t a b o l i c pathway l e a d i n g to AMP i s f o r m a l l y an a n a b o l i c pathway l e a d i n g . t o AMP c o n t r o l p r o p e r t i e s of AMP  c a t a l y z e d by AMP  deam-  h y d r o l y s i s ; the o t h e r  formation during recovery.  deaminase as w e l l as a d e n y l o s u c c i n a t e  arm The  synthetase,  which c a t a l y z e s the f o r m a t i o n of a d e n y l o s u c c i n a t e , a r e e n t i r e l y c o n s i s t e n t w i t h t h i s model. due  Thus, d u r i n g white muscle work, AMP  to d r o p p i n g c o n c e n t r a t i o n s o f GTP,  are presumably i n c r e a s e d . . GDP thetase  (Muirhead  and Bishop,  a v a i l a b i l i t y of one  and  deaminase would be d e i n h i b i t e d  a t the same time, GDP  i s a p o t e n t i n h i b i t o r of a d e n y l o s u c c i n a t e 1974)  and  t h i s e f f e c t , coupled w i t h  deaminase i s b e i n g s t r o n g l y d e i n h i b i t e d .  The  t h i s arm  f i n a l aspect The  t h a t i s s t o r e d i n the amino a c i d p o o l d u r i n g anaerobic: work can be  p o r a t e d i n t o the a d e n y l a t e p o o l by a s e r i e s of t r a n s a m i n a t i o n s .  of  r a t e a t the  the p i c t u r e i s the r o l e of amino a c i d s . d u r i n g the r e c o v e r y p e r i o d . gen  syn-  reduced  of i t s s u b s t r a t e s (GTP), r e a d i l y e x p l a i n s how  the p u r i n e n u c l e o t i d e c y c l e i n white muscle i s h e l d a t a reduced same time as AMP  concentrations  to  nitroreincor-  In'this re-  spect the fumarate produced by the p u r i n e n u c l e o t i d e c y c l e would p l a y an  52  i n t e g r a l r o l e i n p r o v i d i n g o x a l o a c e t a t e f o r the f o r m a t i o n . o f v a r i e t y of' amino a c i d s .  This metabolic  aspartate with a  scheme, makes good b i o l o g i c a l  sense,  however i t remains to be t e s t e d . Krebs C y c l e P o o l S i z e There i s no i n c r e a s e i n f o u r of the Krebs c y c l e i n t e r m e d i a t e s even when w h i t e muscle i s maximally a c t i v e . very l i t t l e  c a p a c i t y i f any  I t i s thus apparent t h a t t h i s t i s s u e  to augment.the s i z e of i t s Krebs c y c l e p o o l i n  c o n c e r t w i t h i n c r e a s e d energy demands.  T h i s i s v e r y much d i f f e r e n t  mammalian heart. (Shaferrand W i l l i a m s o n ,  1973)  al,  1973)  or s k e l e t a l muscle  from  (Edington et^  i n which the c o n c e n t r a t i o n of Krebs c y c l e i n t e r m e d i a t e s may  e l e v a t e d d u r i n g strenuous  has  work.  In h e a r t b u r n i n g  be h i g h l y  g l u c o s e augmentation of  Krebs c y c l e i n t e r m e d i a t e s i s f u l l y accounted f o r by a s p a r t a t e d e p l e t i o n . A s p a r t a t e i s transaminated mate.  The  w i t h a - k e t o g l u t a r a t e to form o x a l o a c e t a t e and g l u t a -  glutamate then undergoes a second t r a n s a m i n a t i o n w i t h pyruvate  form a l a n i n e and  regenerate  a-ketoglutarate.  Krebs c y c l e i n t e r m e d i a t e s and and W i l l i a m s o n ,  1973).  The  working white muscle of f i s h for  the s m a l l but  Thus a s p a r t a t e carbon appears as  a s p a r t a t e n i t r o g e n appears as a l a n i n e  same phenomenon may s i n c e glycogen  s i g n i f i c a n t decrease  to  occur  (Shafer  to a l i m i t e d e x t e n t i n  i s the f u e l s o u r c e .  i n a s p a r t a t e and  This  accounts  the tendency f o r  a l a n i n e to i n c r e a s e (see Chapter V f o r an extended d i s c u s s i o n of t h i s  point).  53  F i g u r e 4.  D e p l e t i o n of the a d e n y l a t e p o o l i n white muscle d u r i n g a n a e r o b i c work.  53a  (a)  ATP  ->ADP  +  P,  (b)  2 ADP-  -> ATP  +  AMP  ->GDP  +  ATP  GTP  (d)  +  ADP-  s u c c i n y l CoA-  GDP  (e)  (f)  AMP-  NH^ + NADH + a - k e t o g l u t a r a t e  glutamate  •> s u c c i n a t e  GTP  ->IMP  + NH;  -> glutamate  NAD  -> a - k e t o g l u t a r a t e  (g) a-ketoacid  +  amino a c i d  54  F i g u r e 5.  Regeneration  of the a d e n y l a t e p o o l i n white muscle d u r i n g  r e c o v e r y from a n a e r o b i c work. i n text:  ASP,  oxaloacetate.  A b b r e v i a t i o n s not  a s p a r t a t e ; a-KGA, a - k e t o g l u t a r a t e ;  indicated OXA,  f u m a r a t e  m a l a t e  nucleotide a d e n y l o s u c c i n a t e  cycle  GDP+P: GTP  OXA  ASP a-KGA  a m i n o  acid  g l u t a m a t e  a -  ketoacid  55  CHAPTER V  CONCLUDING REMARKS: RED-WHITE MUSCLE DIFFERENCES AND THE FUNCTION OF THE PURINE NUCLEOTIDE CYCLE  55 a  The  s k e l e t a l muscle of v e r t e b r a t e s i s composed of  d i f f e r e n t t i s s u e types.  characteristically  At the extreme ends of the spectrum  which make up the r e d and white muscles.  These two  a r e the  f i b e r types may  fibers be  t i n g u i s h e d by numerous c r i t e r i a such as the content of m i t o c h o n d r i a , haemoglobin and o x i d a t i v e enzymes, b l o o d f l o w and Chapter  1).  dismyoglobin,  consumption r a t e (see  I t i s g e n e r a l l y a c c e p t e d t h a t r e d muscle f u n c t i o n s l a r g e l y  aerobically u t i l i z i n g  f a t s or . c a r b o h y d r a t e s  as i t s f u e l source whereas white  muscle has an e x t r a o r d i n a r y a n a e r o b i c component to i t s metabolism based glycogen u t i l i z a t i o n . r e d muscle  upon  In t h i s r e s p e c t the mammalian h e a r t i s v e r y s i m i l a r t o  (Keul e t a l , 1972).  Meaningful  s t u d i e s a t the m e t a b o l i t e l e v e l of  mammalian r e d or white muscle are e s s e n t i a l l y i m p o s s i b l e s i n c e the two types e x i s t i n mixed bundles; however, i n many f i s h s p e c i e s r e d and muscle occur as d i s c r e t e e a s i l y s e p a r a b l e t i s s u e masses. study I have taken advantage of the unique  fiber  white  In the p r e s e n t  d i s t r i b u t i o n of muscle f i b e r s i n  f i s h to e l u c i d a t e the c o n t r o l of energy metabolism i n white muscle a l o n e . Furthermore,  i n c o n s i d e r i n g the r e s u l t s w i t h i n t h e framework of  red-white  muscle d i f f e r e n c e s i t has been p o s s i b l e to i n t e r p r e t some h e r e t o f o r e i n e x p l i c a b l e . f i n d i n g s i n the  literature.  R e g u l a t i o n of the A d e n y l a t e P o o l S i z e During  strenuous a c t i v i t y by white muscle, when a n a e r o b i c metabolism i s  h i g h l y a c t i v a t e d , t h e r e i s an o v e r a l l r e d u c t i o n i n the a d e n y l a t e p o o l c o n t e n t . T h i s o c c u r s because ATP minimum. decrease  c o n c e n t r a t i o n cannot be m a i n t a i n e d  over a  certain  However, when h e a r t o r r e d muscle i s f o r c e d to work t h e r e i s no i n the t o t a l l e v e l of a d e n y l a t e s s i n c e ATP  once a new  content i s m a i n t a i n e d  steady s t a t e i s a t t a i n e d (Neely et a l , 1972;  Gerez and  Kirsten,  56  1965).  But when e i t h e r of these two  i s a decrease 1966; In  t i s s u e s i s s u b j e c t e d to h y p o x i a  i n the a d e n y l a t e p o o l (Imai e t a l , 1964;  Neely et a l , 1973;  Gerlach,  Chaudry et a l , 1 9 7 4 ) . j u s t as o c c u r s i n white  s k e l e t a l muscle t h i s i s accomplished  and t h e decrease  D e u t i c k e and  there  by a c t i v a t i o n of 5' AMP  muscle.  deaminase  i n the a d e n y l a t e s i s i n .1:1 s t o i c h i o m e t r y w i t h e i t h e r  i n c r e a s e or the sum  of IMP  and  D e u t i c k e and G e r l a c h , 1966).  i t s degradation products I t i s proposed  IMP  (Imai e t a l , 1964;  that f o l l o w i n g recovery  from  a n a e r o b i c work the a d e n y l a t e p o o l i s r e s t o r e d by a r e a c t i o n span known as the p u r i n e n u c l e o t i d e c y c l e  (see Chapter W„  however, the a d e n y l a t e p o o l i s reduced the c o n v e r s i o n of AMP 1973).  to adenosine  F i g u r e 5l)l.I, 2 ) .  In h e a r t ,  by 5' n u c l e o t i d a s e which c a t a l y z e s  p l u s ribose-5-phosphate  (Rubio et_ a l ,  R e g a r d l e s s , i n a l l t h r e e t i s s u e s the phenomenon i s the same:  thus,  when the a n a e r o b i c component pf metabolism i s a c t i v a t e d r e l a t i v e to the a e r o b i c component t h e r e o c c u r s a r e d u c t i o h ' i r i the s i z e of the a d e n y l a t e p o o l . But h e a r t c o n t a i n s the enzymes of the p u r i n e n u c l e o t i d e c y c l e 1972; to  Muirhead and Bishop, 1974)  hypoxia  (Lowenstein,  and moreover r e d muscle i s r a r e l y s u b j e c t e d  i n the normal course of e v e n t s .  The q u e s t i o n then a r i s e s , as t o  what o t h e r f u n c t i o n s the p u r i n e n u c l e o t i d e c y c l e has i n the c e l l . to  the q u e s t i o n comes from a r e c e n t study by Winder e t al,(1974)  i s shown t h a t a d e n y l o s u c c i n a t e , the f i n a l  A clue i n which i t  enzyme i n the p u r i n e n u c l e o t i d e  c y c l e , i s t i g h t l y c o r r e l a t e d w i t h Krebs c y c l e a c t i v i t y and not  glycolysis.  However, b e f o r e p u r s u i n g t h i s r e l a t i o n s h i p f u r t h e r i t i s n e c e s s a r y a g e n e r a l l y u n a p p r e c i a t e d a s p e c t of Krebs c y c l e  to d e s c r i b e  function.  S t r a t e g i e s of Krebs C y c l e A c t i v a t i o n It  i s i n t u i t i v e l y obvious t h a t i n the t r a n s i t i o n fromaa r e s t i n g to a  working s t a t e i n v o l v i n g an i n c r e a s e d r a t e of oxygen consumption there must be  5'7  a concomitant a c t i v a t i o n o f the Krebs c y c l e .  I t i s important  to r e a l i z e  t h a t t h e Krebs c y c l e can be a c t i v a t e d i n but two fundamental ways:  (a) by  i n c r e a s i n g the turnover r a t e o r the r a t e of " s p i n n i n g " , w i t h no change i n p o o l s i z e of i n t e r m e d i a t e s , o r (b) by i n c r e a s i n g the steady  s t a t e l e v e l of  Krebs c y c l e i n t e r m e d i a t e s as w e l l as i n c r e a s i n g " s p i n n i n g " r a t e . first  In the  i n s t a n c e , t h e r e i s no change i n t h e maximum c a t a l y t i c p o t e n t i a l of the  c y c l e ; i n t h e second, t h e r e i s an i n c r e a s e i n c a t a l y t i c p o t e n t i a l of the c y c l e t h a t i s p r o p o r t i o n a l t o the augmentation o f c y c l e i n t e r m e d i a t e s . i n Krebs c y c l e " s p i n n i n g " r a t e i s a p p a r e n t l y a c h i e v e d b o l i t e r e g u l a t i o n o f key c y c l e enzymes. i s now f a i r l y w e l l d e s c r i b e d  through t i g h t meta-  T h i s a s p e c t o f Krebs c y c l e c o n t r o l  ( A t k i n s o n , 1968b; LaNoue and W i l l i a m s o n ,  LaNoue e t a l , 1972) and w i l l n o t be emphasized h e r e . however, t h a t i n any g i v e n m e t a b o l i c  Change  1971;  S u f f i c e to i n d i c a t e ,  s t a t e , merely i n c r e a s i n g a c e t y l C o A  a v a i l a b i l i t y can i n c r e a s e t h e " s p i n n i n g " r ate of the Krebs c y c l e a t the t  initially  low, b a s a l l e v e l s of c y c l e i n t e r m e d i a t e s .  mechanism M  itself  i s usually.inadequate  The main reason why  i s a shortage  of o x a l o a c e t a t e .  B a s a l l e v e l s of o x a l o a c e t a t e a r e v e r y low, on t h i s t h e r e i s widespread ment.  A c t u a l values are d i f f i c u l t  The b e s t a v a i l a b l e e s t i m a t e s ,  i n d i c a t e t h a t o x a l o a c e t a t e c o n c e n t r a t i o n s may be ,as low as 1S5 uM 1973; G a r l a n d  agree-  t o e s t i m a t e , because o f compartmentation  and o f i n s t a b i l i t y d u r i n g , e x t r a c t i o n .  and W i l l i a m s o n ,  this  and Randle, 1964; W i l l i a m s o n ,  however,  (Shafer  1965), and t h i s  low o x a l o a c e t a t e a v a i l a b i l i t y s e v e r e l y l i m i t s the r a t e a t which Krebs c y c l e s p i n n i n g can be i n c r e a s e d .  In f a c t , a t any g i v e n m e t a b o l i c . s t a t e , i t i s a  r e l a t i v e u n a v a i l a b i l i t y of oxaloacetate that probably Krebs c y c l e s p i n n i n g  (LaNoue and W i l l i a m s o n ,  s e t s the b a s a l r a t e of  1971; LaNoue e t a l , 1972) and  t h e r e f o r e , i t i s g e n e r a l l y agreed, l a r g e Krebs c y c l e a c t i v a t i o n r e q u i r e s  58 augmenting the c y c l e i n t e r m e d i a t e s .  How  i s t h i s augmentation  achieved?  Krebs C y c l e Augmentation D u r i n g U t i l i z a t i o n of Carbohydrate Mechanisms f o r augmenting the Krebs c y c l e i n t e r m e d i a t e s a r e dependent upon whether carbohydrate first  or f a t i s b e i n g u t i l i z e d as the f u e l s o u r c e .  examine what o c c u r s d u r i n g the c a t a b o l i s m of carbohydrate  p r o c e s s has been more f u l l y  studied.  s h u t t l e d i n t o the m i t o c h o n d r i a arise:  firstly,  glycolytic  to form a c e t y l C o A ,  and  two  us  this  When g l u c o s e - d e r i v e d p y r u v a t e  t h e r e i s a requirement  newly formed a c e t y l C o A ,  as  Let  i s being  problems t e m p o r a r i l y  f o r more o x a l o a c e t a t e to handle  the  s e c o n d l y , a redox imbalance i s c r e a t e d i n the  path.  Both problems (the requirement  f o r o x a l o a c e t a t e and  the redox imbalance)  are s o l v e d by a s p a r t a t e transaminase c a t a l y z e d m o b i l i z a t i o n of a s p a r t a t e . T h i s s i t u a t i o n i s . p a r t i c u l a r l y w e l l documented i n r a t h e a r t b u r n i n g (Shafer and W i l l i a m s o n ,  glucose  1973), where the augmentation of Krebs c y c l e i n t e r -  mediates i s f u l l y accounted f o r by a s p a r t a t e d e p l e t i o n .  During  the  transi-  t i o n or a c t i v a t i o n p e r i o d , . a s p a r t a t e - d e r i v e d o x a l o a c e t a t e i s reduced malate.in the c y t o s o l , a process r e q u i r e d NAD  t h a t accounts  for sustaining glycolysis  a-glycerophosphate  and  f o r a l a r g e f r a c t i o n of  The malate then moves i n t o  the m i t o c h o n d r i a  where i t i s r e c o n v e r t e d  c i t r a t e synthase  ( F i g u r e 6\)' l )  aspartate-carbon  appears as Krebs c y c l e i n t e r m e d i a t e s , and  Two  c  the  (the o t h e r f r a c t i o n coming from  l a c t a t e dehydrogenases).  ;  to  to oxaloacetate, f o r sparking  p o i n t s to emphasize here a r e  (a) t h a t  (b) t h a t a s p a r t a t e  n i t r o g e n appears as a l a n i n e because the a s p a r t a t e transaminase r e a c t i o n i s coupled  to a l a n i n e transaminase through the c o s u b s t r a t e s glutamate  a-ketoglutarate.  The  l a t t e r two  a r e tumbled between a s p a r t a t e and  transaminase i n t h i s s i t u a t i o n and  t o t a l . a l a n i n e accumulation  and alanine  equals  augmenta-  59  F i g u r e 6.  Augmentation of the Krebs c y c l e i n t e r m e d i a t e s d u r i n g u t i l i z a t i o n of c a r b o h y d r a t e as an energy  source.  59a  glucose  pyruvate  alanine  cc-ketoglutarate  t acetyl C o A glutamate  aspartate  oxaloacetate  60  t i o n of Krebs c y c l e i n t e r m e d i a t e s .  P r e c i s e l y the same mechanism o c c u r s i n  the a c t i v a t i o n of metabolism, i n r e d muscle (Ruderman and Berger, 1974) to a l i m i t e d e x t e n t i n white muscle. from g l u c o s e i s the u n q u e s t i o n a b l e 1974).  and  Under these g§ndit'ion s p y r u v a t e d e r i v e d ;  amino a c c e p t o r  T  (Ruderman and  Berger,  Thus, by t h i s simple p r o c e s s , the Krebs c y c l e i s s e t at a new  and  h i g h e r c a t a l y t i c p o t e n t i a l f o r s u s t a i n i n g a p r o l o n g e d work l o a d . The  l i m i t e d c a p a c i t y of white muscle to augment t h e s i z e of the Krebs  c y c l e p o o l i s r e f l e c t e d by the content of a s p a r t a t e which i n t h i s t i s s u e i s o n l y about 0.25  ymoles/gm.  In h e a r t , however, a s p a r t a t e l e v e l s a r e much  h i g h e r as i s the c a p a c i t y t o i n c r e a s e o x a l o a c e t a t e content 1972) .  The  (Neely et_ a l ,  a s p a r t a t e content of r e d muscle, per se, i s not known but i t has  been shown t h a t d u r i n g a c t i v i t y Krebs c y c l e i n t e r m e d i a t e s i n t h i s may  i n c r e a s e to l e v e l s even h i g h e r than those found  i n heart  tissue  (Edington e t a l ,  1973) . In working w h i t e muscle t h e r e i s a decrease a concomitant  i n c r e a s e i n f r e e NH^".  i n the a d e n y l a t e p o o l w i t h  But i n working h e a r t or r e d muscle, t h e r e  a r e no measurable changes i n the t o t a l a d e n y l a t e p o o l , a l t h o u g h changes i n ATP,  ADP,  and. AMP  c o n c e n t r a t i o n s can occur  transitory  (Neely e t a l , 1972;  + Shafer and W i l l i a m s o n , 1973). i s produced  by working h e a r t  As f a r as the d a t a c u r r e n t l y i n d i c a t e , no (Shafer and W i l l i a m s o n , 1973)  or r e d muscle  (Gerez and K i r s t o n , 1965); the o n l y form of n i t r o g e n o u s "waste" product accumulates  i s a l a n i n e and  NH^  that  i t i s t h e o n l y s i g n i f i c a n t form of n i t r o g e n c a r r i e r  b e i n g r e l e a s e d from muscle d u r i n g a e r o b i c g l u c o s e metabolism.  Glutamine,  an  e q u a l l y important n i t r o g e n c a r r i e r under some c o n d i t i o n s , i s not r e l e a s e d from h e a r t o r r e d muscle b u r n i n g g l u c o s e u n l e s s the system i s s u p p l i e d w i t h exogenous amino a c i d s (Odessey e t a l , 1974).  (In the l a t t e r case,  glutamine  61  i s formed  by mechanisms d i s c u s s e d below.)  G l y c o l y t i c I n h i b i t i o n D u r i n g F a t and Amino A c i d C a t a b o l i s m I f a , g l u c o s e - p e r f u s e d h e a r t i s t r a n s f e r r e d to a c e t a t e as the c h i e f genous carbon s o u r c e , g l u c o s e u t i l i z a t i o n drops to n e a r l y zero 1970), and the same appears t r u e d u r i n g metabolism et a l , 1964)  and o f keto a c i d s formed  (Johnson and C o n n e l l y , 1972).  exo-  (Randle et a l ,  of p a l m i t i c a c i d  from v a l i n e , i s o l e u c i n e , and  (Randle leucine  These a r e c r u c i a l o b s e r v a t i o n s f o r they  i n d i c a t e t h a t when f a t s or amino a c i d s a r e o x i d i z e d , c a r b o h y d r a t e  (the o n l y  major a n a e r o b i c f u e l ) i s b e i n g " s p a r e d " by feedback i n h i b i t o r y l o o p s from m i t o c h o n d r i a l metabolism  to key s t e p s i n g l y c o l y s i s .  Although f u r t h e r  details  c o n c e r n i n g these i n h i b i t o r y i n t e r a c t i o n s undoubtedly w i l l be e l u c i d a t e d , i t i s a l r e a d y known t h a t c r e a t i n e phosphate, ATP,  and c i t r a t e a r e a l l p o t e n t i a l  i n h i b i t o r s of b o t h p h o s p h o f r u c t o k i n a s e and p y r u v a t e k i n a s e ( S t o r e y and Hochachka, 1974), and these s i n g l y or i n combination a r e thought g l y c o l y s i s during active f a t oxidation.  to dampen  In a d d i t i o n , the a-keto a c i d s  formed  from v a l i n e , i s o l e u c i n e , and l e u c i n e a r e b e l i e v e d t o i n h i b i t g l u c o s e u t i l i z a t i o n by competing  w i t h p y r u v a t e f o r p y r u v a t e dehydrogenase (Johnson  C o n n e l l y , 1972).  Whatever the mechanism f o r the i n h i b i t o r y  between m i t o c h o n d r i a l metabolism  interaction  and g l y c o l y s i s , the important p o i n t to bear  i n mind i s t h a t when f a t i s . u t i l i z e d , a source of p y r u v a t e .  and  g l u c o s e becomes l a r g e l y u n a v a i l a b l e as  Thus, Krebs c y c l e p r i m i n g by a s p a r t a t e t r a n s a m i n a t i o n  to o x a l o a c e t a t e , coupled to p y r u v a t e t r a n s a m i n a t i o n to a l a n i n e , as d e s c r i b e d above ( F i g u r e 6:)Y 1 ) , i s a r e a c t i o n span t h a t may substrate  q u i c k l y run out of a key  (pyruvate) under c o n d i t i o n s , f a v o u r i n g f a t metabolism.  i n g l y , the amount o f a l a n i n e formed not account f o r the augmentation  Not  surpris-  i n the h e a r t under these c o n d i t i o n s does  of Krebs c y c l e i n t e r m e d i a t e s (Randle e t a l ,  1970).  Whereas 0^  uptake i n c r e a s e s  s k e l e t a l muscle i n c r e a s e s Sometimes a s p a r t a t e  by  by n e a r l y  10-fold,  l e s s than 2 - f o l d  i s d e p l e t e d , but  alanine  ( F e l i g and  i t s depletion  aspartate  i n f a c t may.be accumulated d u r i n g  (Neely et_ a l , 1972). during  What, then, i s the  activation.of fatty acid  source of Krebs c y c l e  appear to be  amino a c i d c a t a b o l i s m .  of a s y n e r g i s t i c n a t u r e .  p a r t i c u l a r l y v a l i n e , i s o l e u c i n e , and such as o c t a n o a t e  leucine,  (Buse et a l , 1972).  In c o n t r a s t  amino a c i d  oxidation  i s potently  of  to the i n -  catabolism  of amino a c i d s , enhanced by  the  ilt  these amino a c i d s , not  c y c l e during, a c t i v a t i o n of. f a t metabolism. cine) increase  the  l e v e l of  intermediates, oxidizable  aspartate, The  first  generally  succinylCoA,  two  ( v a l i n e and  (acetylCoA),  i n Krebs c y c l e t u r n o v e r r a t e leucine perfusion  It  isoleu-  increasing  augmenting the p o o l s i z e of  while l e u c i n e , along with f a t t y a c i d s , increases  2-carbon s u b s t r a t e  and  (Figure  the cycle  the amount of  thus l e a d s d i r e c t l y to 7>)'i :2)~v  and  t h a t prime the Krebs  c a t a l y t i c p o t e n t i a l of the Krebs c y c l e by  a v a i l a b i l i t y of o x a l o a c e t a t e and  fatty  c a t a b o l i s m of v a l i n e  carbon of l e u c i n e f e e d s i n t o the Krebs c y c l e as a c e t y l C o A .  appears t h a t  the  D u r i n g p e r i o d s of t r a n s i t i o n  i s o l e u c i n e f e e d s carbon i n t o the Krebs c y c l e at the  (a) why  intermediates  f a t t y a c i d or amino a c i d  Thus, the  to h i g h r a t e s of f a t t y a c i d o x i d a t i o n ,  w h i l e the  oxidation  Fat  c a t a b o l i s m , t t h e i n t e r a c t i o n s between f a t t y a c i d and  increase  times,  oxidation?  h i b i t o r y e f f e c t s on g l y c o l y s i s brought about by  from low  At o t h e r  i n s i g h t i n t o the above q u e s t i o n comes from a c o n s i d e r a t i o n  i n t e r a c t i o n between f a t and  acids  account f o r newly  t r a n s i t i o n to a c t i v e f a t  Krebs C y c l e Augmentation D u r i n g M o b i l i z a t i o n of A key  from  Wahren, 1971).  does not  formed Krebs c y c l e intermeddiates (Randle et_ a l , 1970).  release  T h i s model  an  explains  of mammalian muscle l e a d s to a 5 - f o l d drop i n  63  F i g u r e 7.  Augmentation of the Krebs c y c l e i n t e r m e d i a t e s u t i l i z a t i o n of f a t as an energy  source.  during  NHA  •  glutamate GTP  r  GDP+.Pi i  ATP  K-ADP+P;  valine isoleucine ,* -ketoglutarate—  l  f  glutamate  r  j  cycle  fumarate 1  oxal aloacetate "malate  <* -keto acids I CoA derivatives  glutamine  /aspartate /oxaloacetate  cytosol mitochondrion  ^malate ^ tumarate  „  oxaloacetate acetyl CoA  \V V .I  succinate '  - 5 ^ succinyl CoA  •  KREBS  * citrate  C Y C  isocitrate  c - ketoglutarate fatty acids leucine  64  valine released  i n t o the e f f e r e n t c i r c u l a t i o n by  B e r g e r , 1974), and isoleucine  (b) why  ( F e l i g and  exercise leads  t h a t muscle  i n aspartate,  i n alanine.  r e l e a s e s a l a n i n e as the n i t r o g e n  but  leucine enters  the  Krebs  u l t i m a t e l y i n glutamine  U n l i k e muscle b u r n i n g g l u c o s e  (which  c a r r i e r ) , muscle b u r n i n g f a t and/or amino  a c i d s , r e l e a s e s both glutamine and  a l a n i n e , and  of the two,  glutamine appears  the predominant form of "waste" n i t r o g e n , removing from muscle 2-4 much n i t r o g e n / m o l e of amino a c i d as does a l a n i n e .  I f muscle i s  w i t h l e u c i n e . o r v a l i n e , glutamine, as w e l l as s m a l l e r i n t o the e f f e r e n t f l o w  (Ruderman and  times as  perfused  amounts of a l a n i n e ,  again  released  data,  t h e r e f o r e , are c o n s i s t e n t w i t h the f o l l o w i n g r e a c t i o n scheme (see  Figure  and  Wahren, 1971).  c y c l e , the n i t r o g e n appears f i r s t to a l e s s e r extent  and  to a measurable uptake of v a l i n e  Whereas the carbon of v a l i n e , i s o l e u c i n e , and  and  (Ruderman  Berger, 1974).  are  These also  §y : 1):, y  In t h i s view, glutamate and aminase and  a - k e t o g l u t a r a t e . tumble between a s p a r t a t e  transaminases f o r v a l i n e , i s o l e u c i n e , and  leucine  trans-  ( F i g u r e 7)':  65  As a means f o r r e g e n e r a t i n g a - k e t o g l u t a r a t e r e q u i r e d f o r m o b i l i z i n g these amino a c i d s , a s p a r t a t e transaminase i s favoured because of l i m i t i n g a v a i l a b i l i t y of p y r u v a t e ;  over a l a n i n e transaminase  however, any  a l a n i n e which i s  r e l e a s e d from f a t b u r n i n g muscle i s formed by a l a n i n e transaminase and  Berger,  1974), the p y r u v a t e  f o r the r e a c t i o n presumably a r i s i n g from a  r e s i d u a l g l y c o l y t i c a c t i v i t y t h a t can i n f a c t be glucose  i n c r e a s e d w i t h exogenous  (Odessey e t a l , 1974).  The A s p a r t a t e - O x a l o a c e t a t e  Cycle  A c c o r d i n g l y , c y t o s o l i c a s p a r t a t e transaminase f u n c t i o n d u r i n g of  f a t c a t a b o l i s m i s favoured  catabolism. state l e v e l  The  a s p a r t a t e formed t h i s way  (Neely e t a l , 1972)  or i t may  may  Ruderman and  Berger,  1974)  On  by glutamine  +  ATP ( +  C l e a r l y , a source.of genase i s not p r e s e n t  is  f u n c t i o n we  must f i r s t  s t r o n g l y i n d i c a t e s t h a t glutamine ( i n c l u d i n g v a l i n e and  i n q u i r e as  (Hills,  1972;  formation i n  leucine) i s catalyzed  synthetase:  glutamate  primary  steady  (Randle- e t a l , 1970),  t h i s q u e s t i o n , r e c e n t evidence  muscle b u r n i n g v a r i o u s amino a c i d s  just  aerobic.glucose  accumulate to a new  be d e p l e t e d  but f o r a f u l l a p p r e c i a t i o n of i t s f a t e and the o r i g i n of glutamine.  activation  i n the d i r e c t i o n of a s p a r t a t e p r o d u c t i o n ;  the o p p o s i t e , of c o u r s e , o c c u r s at t h i s enzyme l o c u s d u r i n g  to  (Ruderman  NH^  NH^  • glutamine  i s r e q u i r e d f o r t h i s r e a c t i o n . .As  +  ADP  +  P  glutamate dehydro-  i n h e a r t or r e d muscle, i t i s w i d e l y accepted  that  pathway f o r the c o n t r o l l e d r e l e a s e of amino n i t r o g e n i n muscle  the p u r i n e n u c l e o t i d e c y c l e (Lowenstein,  the  tissue  1972).  T h e r e f o r e , i t i s proposed t h a t i n r e d muscle or h e a r t , the r e a c t i o n s t e p s of  the p u r i n e n u c l e o t i d e c y c l e f u n c t i o n as_ jl c y c l e d u r i n g m o b i l i z a t i o n of  amino a c i d s , p a r t i c u l a r l y v a l i n e and  isoleucine.  The  aspartate i n i t i a t i n g  the  66  p u r i n e n u c l e o t i d e c y c l e i n f a c t can be r e g e n e r a t e d by i t ,  from the fumarate formed  s i n c e fumarate can be converted t o o x a l o a c e t a t e .  T h i s scheme, termed  the a s p a r t a t e - o x a l o a c e t a t e c y c l e , p r o v i d e s a source of o x a l o a c e t a t e f o r the a s p a r t a t e transaminase  and  i n effect i s a cyclic,  c a t a l y t i c mechanism  ( i n i t i a t e d by a s p a r t a t e and r e f o r m i n g a s p a r t a t e ) , t h a t primes n i t r o g e n through of carbon ates.  the p u r i n e n u c l e o t i d e c y c l e to glutamine,  (b) the  flow  from i s o l e u c i n e and v a l i n e i n t o the p o o l of Krebs c y c l e i n t e r m e d i -  F o r t h i s reason,  v a l i n e and  and  (a) t h e f l o w of  I p r e d i c t t h a t d u r i n g muscle a c t i v a t i o n , d e p l e t i o n of  i s o l e u c i n e should be i n v e r s e l y p r o p o r t i o n a l to glutamine  and r e l e a s e . f r o m muscle.  formation  For t h i s r e a s o n , t o o , the augmentation o f Krebs  c y c l e i n t e r m e d i a t e s s h o u l d be i n v e r s e l y p r o p o r t i o n a l t o v a l i n e and  isoleucine  depletion. In t h i s view, whether or not a s p a r t a t e i s d e p l e t e d , accumulated, or unchanged d u r i n g t r a n s i t i o n to a c t i v e f a t o x i d a t i o n appears to depend upon the a v a i l a b i l i t y of i s o l e u c i n e and v a l i n e .  C l e a r l y some of the fumarate  formed by t h e a s p a r t a t e - o x a l o a c e t a t e c y c l e c o u l d f e e d i n t o , <and be r e t a i n e d w i t h i n , the p o o l of Krebs c y c l e i n t e r m e d i a t e s ; t h a t amount s h o u l d appear as an a s p a r t a t e d e p l e t i o n . however, a s p a r t a t e may  I f i s o l e u c i n e and v a l i n e r e s e r v e s a r e adequate,  a c t u a l l y accumulate.  In a b a l a n c e d  a s p a r t a t e would n e i t h e r be d e p l e t e d nor accumulated. i n f a c t have been observed  (Neely e t a l , 1972;  situation,  clearly  A l l three a l t e r n a t i v e s  Randle et a l , 1970), though  never p r e v i o u s l y e x p l a i n e d . Thus, i t appears t h a t when r e d muscle and/or h e a r t burn f a t , a c t i v a t i o n of the Krebs c y c l e r e q u i r e s the simultaneous p a r t i c u l a r l y v a l i n e and the "cogs".of  isoleucine.  m o b i l i z a t i o n of amino a c i d s ,  At l e a s t d u r i n g the a c t i v a t i o n p e r i o d ,  the Krebs c y c l e appear to mesh w i t h those of the a s p a r t a t e -  67  o x a l o a c e t a t e c y c l e which primes the f l o w of amino a c i d carbon i n t o the Krebs c y c l e and  the f l o w of amino n i t r o g e n through the p u r i n e n u c l e o t i d e c y c l e to  glutamine  (Figure 7).  organization.  Firstly,  S e v e r a l advantages accrue the o n l y major a n a e r o b i c  from t h i s k i n d of source  r a t e ) i s maximally " s p a r e d " d u r i n g a e r o b i c metabolism.  of energy Secondly,  metabolic (carbohydi f one  compares the energy y i e l d e d d u r i n g v a l i n e (or i s o l e u c i n e ) p r i m i n g v e r s u s gained by  that  the a s p a r t a t e p r i m i n g mechanism, about 10 times as much u t i l i z a b l e  energy ( i n the form of ATP  equivalents) i s obtained  (Krebs,  1964).  Thirdly,  t h i s mechanism a l l o w s amino a c i d s to be used i n muscle both f o r p r i m i n g  the  Krebs c y c l e as w e l l as d i r e c t l y f o r energy p r o d u c t i o n w i t h o u t muscle r e q u i r i n g a u r e a - c y c l e f o r h a n d l i n g "waste" n i t r o g e n . s t i l l u n c l e a r , but p r o b a b l y intermediates,  F i n a l l y , f o r reasons  that are  i n v o l v i n g a g r e a t e r a b s o l u t e augmentation of c y c l e  the maximum degree of Krebs c y c l e a c t i v a t i o n o b t a i n a b l e appears  h i g h e r d u r i n g f a t o x i d a t i o n than i t i s d u r i n g g l u c o s e o x i d a t i o n (Neely e_t a l , 1972).  In c o n c l u s i o n , i t i s i n t e r e s t i n g to note t h a t h e a r t muscle i s capable  of b u r n i n g a v a r i e t y of s u b s t r a t e s , f a t t y a c i d s b e i n g p r e f e r r e d i n v i v o  (Neely  and Morgan, 1974); r e d muscle f i b e r s appear to be r a t h e r s i m i l a r to the h e a r t i n t h i s r e s p e c t , whereas, as we unique dependence upon  have seen, w h i t e muscle d i s p l a y s a s t r o n g  and  carbohydrate.  Summary Much of the data i n r e l a t i o n to energy metabolism i n mammalian muscle i s d i f f i c u l t the p r e s e n t  study  to i n t e r p r e t due  skeletal  to the h e t e r o g e n e i t y of t h i s t i s s u e .  t h i s problem has been circumvented  In  by the u t i l i z a t i o n of a  s p e c i e s of f i s h i n which r e d and white muscle e x i s t as d i s c r e t e e a s i l y separable  t i s s u e masses.  During, t r a n s i t i o n to work i n white muscle, as w e l l  as d u r i n g hypoxic  s t r e s s i n r e d muscle and  the t o t a l content  of the a d e n y l a t e  pool.  heart, there occurs a r e d u c t i o n i n In s k e l e t a l muscle t h i s i s accom-  68  p l i s h e d . by a c t i v a t i o n of 5' AMP  deaminase,, the f i r s t  known as the p u r i n e n u c l e o t i d e c y c l e .  enzyme i n a. r e a c t i o n span  Following recovery  the second arm  of the p u r i n e n u c l e o t i d e c y c l e i s turned  regenerate  and hence the a d e n y l a t e  AMP  pool.  from a n a e r o b i c  on,  work  s e r v i n g to  However, the main f u n c t i o n of  the p u r i n e n u c l e o t i d e c y c l e i n r e d muscle and  h e a r t i s r e l a t e d to a e r o b i c  metabolism. When muscle undergoes a t r a n s i t i o n from a r e s t i n g to a working s t a t e , t h e r e i s an i n c r e a s e i n the s i z e of the Krebs c y c l e p o o l . for  t h i s augmentation p r e s e n t s  f u e l source; under these c a t a l y z e d by a s p a r t a t e aspartate  i t s e l f when c a r b o h y d r a t e s  transaminase I s coupled  s i m p l e s t method  a r e u t i l i z e d as a  conditions, aspartate conversion  transaminase, s e r v e s  The  to  oxaloacetate,  to spark the Krebs c y c l e .  Because  to a l a n i n e transaminase, the i n c r e a s e i n  Krebs c y c l e p o o l s i z e i s q u a n t i t a t i v e l y accounted f o r by a l a n i n e  accumulation.  When muscle burns f a t , however, t h i s augmentation mechanism i s reduced i n importance due pyruvate. favoured ing  to i n h i b i t i o n of g l y c o l y s i s and  to reduced a v a i l a b i l i t y  of  Under these c o n d i t i o n s , the c a t a b o l i s m of v a l i n e and . I s o l e u c i n e i s and  these appear to be  the predominant sources  of carbon f o r augment-  Krebs c y c l e p o o l s i z e . . A l t h o u g h the carbon of these branched c h a i n amino  a c i d s appears as Krebs c y c l e i n t e r m e d i a t e s , a s p a r t a t e , but  then i s r e l e a s e d as NH^  by  the n i t r o g e n appears f i r s t  in  the p u r i n e n u c l e o t i d e c y c l e ; the  then s e r v e s as s u b s t r a t e f o r glutamine s y n t h e t a s e ,  i n which  NH^  circumstance  glutamine becomes the p r i m a r y means f o r removal of amino n i t r o g e n from muscle. 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I n h i b i t i o n o f g l y c o l y s i s by a c e t a t e and p y r u v a t e i n t h e i s o l a t e d p e r f u s e d r a t h e a r t . J . B i o l . Chem. 240: 2308-2321. W i l l i a m s o n , J . R. and Corkey, B. E. 1969. Assays o f i n t e r m e d i a t e s o f t h e c i t r i c , a c i d c y c l e and r e l a t e d compounds by f l u o r o m e t r i c enzyme methods. In Methods i n Enzymology. E d i t e d by J . M. Lowenstein. Academic P r e s s , New York. V o l . 13: 434-512. Winder, W. W., T e r j u n g , R. L., Baldwin, K. M. and H o l l o s z y , J . 0. 1974. E f f e c t o f e x e r c i s e on AMP deaminase and a d e n y l o s u c c i n a t e i n r a t s k e l e t a l muscle. Am. J . P h y s i o l . 227: 1411-1414. W i t t e n b e r g e r , C. and D i a c i u c , I . V. 1965. E f f o r t metabolism o f l a t e r a l muscles i n c a r p . J . F i s h . Res. Bd. Canada 22: 1397-1406.  79  APPENDIX I  BLOOD LACTATE LEVELS IN FREE SWIMMING TROUT BEFORE AND AFTER STRENUOUS EXERCISE RESULTING IN FATIGUE  79  a  INTRODUCTION  The  myotomal musculature o f  f i s h i s l a r g e l y composed o f two systems  commonly r e f e r r e d to as the r e d and white f i b e r s .  The two f i b e r  types may be  d i s t i n g u i s h e d i n numerous ways i n c l u d i n g haemoglobin, myoglobin and mitochond r i a l content, vascular  supply  and enzymatic p r o p e r t i e s .  On the b a s i s o f  these c h a r a c t e r i s t i c s i t i s g e n e r a l l y accepted t h a t r e d muscle has a metabolism t h a t i s a e r o b i c a l l y based b u r n i n g f a t s and/or c a r b o h y d r a t e s whereas white muscle f u n c t i o n s production  l a r g e l y a n a e r o b i c a l l y u t i l i z i n g g l y c o g e n w i t h the concomitant  of l a c t a t e .  D u r i n g slow swimming the p r o p u l s i v e  e n t i r e l y from the r e d m u s c u l a t u r e .  force i s derived  But a t the h i g h e s t  swimming v e l o c i t i e s  the white muscle becomes maximally a c t i v e and together  w i t h the r e d muscle  s u p p l i e s the power f o r l o c o m o t i o n .  A t some p o i n t i n the t r a n s i t i o n from low  to h i g h  swimming speeds, there i s r e c r u i t m e n t  duciton  to t h e s i s f o r r e f e r e n c e s ) .  I t i s w e l l recognized  of the white f i b e r s (see I n t r o -  t h a t some f i s h can m a i n t a i n swimming, speeds j u s t  below t h e i r c r i t i c a l v e l o c i t y f o r extended p e r i o d s During excursions in conditions  o f time ( B r e t t , 1964).  of t h i s n a t u r e new steady s t a t e l e v e l s must be a t t a i n e d and  i n v o l v i n g white muscle a c t i v i t y l a c t a t e cannot be allowed to  accumulate i n t h a t t i s s u e and hence must be e l i m i n a t e d . found t h a t blood  from rainbow t r o u t sampled by c a r d i a c puncture on r e s t r a i n e d  f i s h a f t e r periods (Black,  Numerous workers have  of moderate a c t i v i t y c o n t a i n e d  elevated  l e v e l s of l a c t a t e  1957; B l a c k e t a l , 1960; B l a c k e t a l , 1966; M i l l e r e t a l , 1959).  t h i s study the l a c t a t e c o n t e n t of blood  taken s e r i a l l y  from i n d i v i d u a l rainbow t r o u t (Salmo g a i r d n e r i ) d u r i n g v e l o c i t i e s i s recorded.  I t i s reported  there i s no marked i n c r e a s e  by i n d w e l l i n g  i n the c o n c e n t r a t i o n  catheters  swimming a t w e l l  t h a t under the p r e s e n t of blood  In  defined  conditions,  l a c t a t e d u r i n g the  80 swimming p e r i o d a t any time p r i o r to f a t i g u e ; however, l a c t a t e  concentration  rises rapidly following fatigue. The  f a t e o f l a c t a t e produced i n the white muscle o f f i s h remains an open  question.  I n mammalian systems which have been b e t t e r s t u d i e d i t i s known t h a t  80-90% of the blood the  l a c t a t e i s oxidized to C 0  s i t e of o x i d a t i o n being  1970), l i v e r  heart  and water  2  (Drury and Wick, 1956);  (Keul e t a l , 1972), s k e l e t a l muscle  (Rowell e t a l , 1972) and k i d n e y  (Levy, 1962).  In r e s t i n g  t i o n s a p p r o x i m a t e l y 15% o f the l a c t a t e i s c o n v e r t e d to g l u c o s e 1963)  i n the l i v e r  (Rowell e t a l , 1966); however, d u r i n g  Black,  condi-  (Reichard  activity  ( i . e . the C o r i c y c l e ) may be q u a n t i t a t i v e l y more important  (Jorfeldt,  et_ a l ,  t h i s process  (Keul e t a l , 1972).  on the b a s i s o f h i s s t u d i e s w i t h f i s h , was f o r c e d t o conclude t h a t i n  these animals the C o r i c y c l e i s o f l i t t l e  importance  (Black et^ a l , 1966).  Moreover, f o l l o w i n g a n a e r o b i c s t r e s s g l y c o g e n does n o t r e t u r n t o p r e s t r e s s l e v e l s even a f t e r 24 hours (Black et^ a l , 1962; Heath and P r i t c h a r d , 1965), t h e r e f o r e i t i s p r o b a b l e t h a t i n f i s h , as w i t h mammals, most o f the blood l a c t a t e i s oxidized to C 0 . 2  Bilinski  and Jonas (1972) have shown t h a t the  c a p a c i t y to c h a n n e l l a c t a t e through the c i t r i c a c i d c y c l e p e r u n i t weight o f t i s s u e d e c r e a s e s i n the f o l l o w i n g o r d e r : h e a r t , white muscle. s i g n i f i c a n c e of g i l l and is  as a s i t e o f l a c t a t e u t i l i z a t i o n  removed from the b l o o d activity.  k i d n e y , r e d muscle,  An attempt has been made t o q u a n t i t a t e  a f t e r i t s passage through t h i s t i s s u e .  strenuous  gill,  liver,  the i n v i v o  by sampling blood  before  The r e s u l t s suggest t h a t l a c t a t e  by the g i l l s d u r i n g  the r e c o v e r y  period  following  81  MATERIALS AND  METHODS  Animals Female t r o u t supplier  (Salmo g a i r d n e r i )  (40-53 cm)  were purchased from a  (Trout Lodge, E p h r a t o , Wash., U. S. A.)  and  commercial  t r a n s p o r t e d : t o U. B.  C.  by tank t r u c k , where they were h e l d i n l a r g e c y l i n d r i c a l , tanks (8000 l i t r e s ) . Constant i n f l o w of f r e s h d e c h l o r i n a t e d water times.  (6-7°C) was  maintained at a l l  F i s h used f o r experiments were p h y s i c a l l y t r a i n e d f o r p e r i o d s of a t  l e a s t two weeks p r i o r to use. f i s h to 2000 l i t r e  circular  water j e t s d r i v e n by pumps.  T r a i n i n g was  accomplished by t r a n s f e r r i n g  tanks i n which the water was  kept i n motion  the  by  The water v e l o c i t y v a r i e d from n e a r l y 0 a t the  c e n t r e of the tank to 35-40 cm/sec a t the c i r c u m f e r e n c e . swim c o n s t a n t l y i n the h i g h water v e l o c i t y zone.  The  f i s h tended  to  A l l f i s h were f e d C l a r k ' s  T r o u t P e l l e t s s i x times weekly. Cannulation  Techniques  D o r s a l a o r t i c c a n n u l a t i o n was Bell at  and  (1964) (MS222 a n a e s t h e s i a ) u s i n g a 50 cm l e n g t h of PE60 t u b i n g terminated  the p r o x i m a l end w i t h a 12 mm  c a n n u l a t i o n was  s e c t i o n of 21a Huber p o i n t n e e d l e .  Ventral  accomplished u s i n g a cannula s i m i l a r to the d o r s a l one  t h a t the n e e d l e was was  accomplished by the method of Smith  2 cm l o n g and bent a t 60° 6 mm  except  from the t i p . . T h i s cannula  i n s e r t e d i n t o the v e n t r a l a o r t a through the tongue a t the l e v e l of the  third g i l l arch. the mouth.  The cannula was  s u t u r e d to the tongue and extended  A f t e r p a r t i a l r e c o v e r y on the o p e r a t i n g t a b l e  t o r y frequency) the f i s h was  out of  (constant r e s p i r a -  t r a n s f e r r e d to a water t u n n e l ( B r e t t j  1964),  of  2 126.5 of  cm  c r o s s - s e c t i o n a l a r e a and  35 l i t r e volume, to r e c o v e r f o r a minimum  18 h r s a t a water v e l o c i t y of 9 cm/sec.  82 Experimental The  Design  f i s h were e x e r c i s e d i n a s e r i e s of 60 min  increments, u n t i l f a t i g u e o c c u r r e d . lengths/sec. taken a t min  Blood samples,  saturation.  was  prior  F a t i g u e was  The water was  C r i t i c a l v e l o c i t y was  velocity  about  0.25  to f a t i g u e , from f o u r i n d i v i d u a l ' f i s h , were  from the e l e c t r i f i e d  stream end of the t u n n e l .  Brett  Each v e l o c i t y increment was  60 o f the t e s t p e r i o d .  the f i s h to remove i t s e l f  stepwise i n c r e a s i n g  d e f i n e d as the i n a b i l i t y of  grid  (10-20 v AC)  m a i n t a i n e d a t 6-7°C and  a t the down100% a i r  c a l c u l a t e d u s i n g the e m p i r i c a l formula of  (1964) such t h a t the l a s t v e l o c i t y t h a t the f i s h s u c c e s s f u l l y m a i n t a i n e d  added to the v e l o c i t y a t which the f i s h f a t i g u e d m u l t i p l i e d by the p r o p o r -  t i o n of the 60 minute p e r i o d t h a t i t was  a b l e to s u s t a i n t h i s f i n a l  The mean c r i t i c a l v e l o c i t y f o r the animals of t h i s experiment  was  speed.  1.6  lengths/  sec. Analytical  Techniques  Blood samples (0.3-1.0 ml) were taken from the ends of the cannulae a 1 ml Hamilton  syringe.  In some cases both a r t e r i a l and venous samples were  taken from the same a n i m a l .  When t h i s o c c u r r e d the venous sample was  1 minute b e f o r e the a r t e r i a l sample. ately diluted  0.5  the supernatant was  M triethanolamine.  the supernatant was  Wilcoxon  KCIO^. was  The  of the b l o o d i t was sample was  obtained immedi-  c e n t r i f u g e d to  n e u t r a l i z e d w i t h 3 M K^CO^  containing  removed by c e n t r i f u g a t i o n and an a l i q u o t of  analyzed f o r l a c t a t e enzymatically.  out on a Unicam SP 1800 recorder.  Upon removal  1.0:3.5 v/v w i t h c o l d 8% HCIO^.  remove p r o t e i n and  with  d u a l beam spectophotometer  Assays were c a r r i e d  connected  to a s t r i p c h a r t  A r t e r i a l and venous b l o o d l a c t a t e c o n t e n t were compared w i t h the t e s t f o r p a i r e d o b s e r v a t i o n s and a p r o b a b i l i t y o f l e s s than 0.05  c o n s i d e r e d to be  significant.  was  83 RESULTS AND  DISCUSSION  There i s no change i n l a c t a t e c o n c e n t r a t i o n between a r t e r i a l . a n d venous b l o o d when samples a r e t a k e n . p r i o r to f a t i g u e .  In o r d e r  samples from the same animal,, o n l y a r t e r i a l b l o o d swimming t r o u t b e f o r e and  even though, i n many c a s e s , i s maintained  l a c t a t e l e v e l s of  individual  immediately a f t e r f a t i g u e a r e shown i n F i g u r e  C l e a r l y , t h e r e i s no i n c r e a s e i n b l o o d  and  to a v o i d d u p l i c a t e  l a c t a t e a t any  and v e n t i l a t i o n frequency,  s u s t a i n e d swimming speed,  the e x e r c i s e l e v e l approaches the c r i t i c a l  a t t h a t l e v e l f o r 60 minutes. following a c t i v i t y ,  ment ( K i c e n i u k , p e r s o n a l communication) and  On  Webb (1971) was  velocity  the b a s i s of h e a r t r a t e  on the animals of t h i s e x p e r i -  other  evidence  c i t e d i n Chapter I ,  i t appears t h a t t h e r e must be white muscle involvement a t l e a s t a t the sustainable v e l o c i t i e s .  8.  highest  of the o p i n i o n t h a t i n t r o u t , the  white muscle comes i n t o p l a y a t about.80% of the c r i t i c a l v e l o c i t y , y e t i s no  increase i n blood  S i n c e , i n the p r e s e n t had  l a c t a t e a t speeds up  study b l o o d was  t h a t blood  of the c r i t i c a l  sampled.only a f t e r a steady  been a t t a i n e d i t must be concluded  from white muscle i s e q u a l  to 93%  there  velocity.  state level  t h a t the r a t e of e l i m i n a t i o n of l a c t a t e  to the r a t e of u t i l i z a t i o n elsewhere.  f l o w to the l i v e r i s reduced d u r i n g e x e r c i s e ( S a t c h e l l ,  I t i s thought 1971),  thus i t i s u n l i k e l y t h a t t h i s t i s s u e i s a major s i t e of l a c t a t e d e p o s i t i o n . As  i n d i c a t e d above, the g i l l s are not a major s i t e of l a c t a t e u t i l i z a t i o n  d u r i n g the e x e r c i s e p e r i o d .  I t i s p o s s i b l e t h a t l a c t a t e produced i n the white  muscle i s f u r t h e r o x i d i z e d i n the r e d muscle as W i t t e n b e r g e r and D i a c i u c have suggested occurs  i n the c a r p .  I t would be of i n t e r e s t  t h e r e are d i f f e r e n c e s between t r o u t and  (1965)  to a s c e r t a i n i f  carp i n t h e i r c a p a c i t y to e l i m i n a t e  l a c t a t e produced i n white muscle. Following f a t i g u e there i s a r a p i d increase i n blood  l a c t a t e concen-  84  Figure  8.  Blood  l a c t a t e l e v e l s o f i n d i v i d u a l swimming t r o u t a t s p e c i f i e d  swimming speed and f o l l o w i n g f a t i g u e .  Multiple points at a  given percentage c r i t i c a l v e l o c i t y a r e r e p r e s e n t a t i v e of repeat  runs on d i f f e r e n t days.  A l l blood  d o r s a l a o r t a except one r e p r e s e n t e d ventral aorta.  by  samples taken from which i s from  The c u r v e i s a r e g r e s s i o n l i n e drawn through  a l l p o i n t s p r i o r to f a t i g u e .  84a  85  t r a t i o n to about 2.5  umoles/ml ( F i g u r e 8; T a b l e V I I I ) .  This represents a  to 5 - f o l d i n c r e a s e over the l e v e l a t the h i g h e s t s u s t a i n e d speed. l e v e l s of b l o o d l a c t a t e immediately  Furthermore,  Stevens and B l a c k , 1966;  a l t h o u g h the data over  i n c r e a s e i n 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 , immediately  al,  1962;  hours and  B l a c k e t a l , 1966;  Hammond and  the r e c o v e r y p e r i o d are  l i m i t e d they f i t the g e n e r a l p a t t e r n o f t e n d e s c r i b e d and  r e a c h i n g a maximum i n 2-4  Elevated  f o l l o w i n g strenuous e x e r c i s e have been  r e p e a t e d l y shown ( B l a c k e t a l , 1966; Hickman, 1966).  4-  d i s c u s s e d , of a r a p i d  f o l l o w i n g a n a e r o b i c work,  then s l o w l y r e t u r n i n g to normal (Black e_t  Hammond and Hickman, 1966).  The  f a c t that blood  l a c t a t e i n c r e a s e s to such a degree f o l l o w i n g a c t i v i t y i n d i c a t e s t h a t a l t h o u g h l a c t a t e i s e l i m i n a t e d from white muscle d u r i n g a c t i v i t y a l a r g e amount i s a l s o retained. to how  The q u e s t i o n remains as to how  much i s allowed  much l a c t a t e i s e l i m i n a t e d r e l a t i v e  to accumulate d u r i n g s u s t a i n e d swimming.  The p o s t f a t i g u e l e v e l s of b l o o d l a c t a t e r e p o r t e d here are i n agreement w i t h the f i n d i n g s of o t h e r s i n a b s o l u t e v a l u e and content i n c r e a s e s and  then d e c r e a s e s .  i n the manner i n which  However, i t had been p r e v i o u s l y c l a i m e d  t h a t b l o o d l a c t a t e l e v e l s i n t r o u t e x e r c i s e d a t moderate speeds a r e 2-3 h i g h e r than i n u n e x e r c i s e d f i s h 1959).  ( B l a c k , 1957;  B l a c k e t a l , 1966;  times  M i l l e r et a l ,  I t i s q u i t e c l e a r from F i g u r e 8 t h a t b l o o d l a c t a t e does not i n c r e a s e  markedly even a t h i g h s u s t a i n e d v e l o c i t i e s . p r e s e n t d a t a and  the data of o t h e r s may  In a l l p r e v i o u s s t u d i e s b l o o d was p r e s e n t work blood was appear to y i e l d  The  be due  d i s s i m i l a r i t y between the  to m e t h o d o l o g i c a l  procedures.  o b t a i n e d by c a r d i a c puncture whereas i n the  sampled by i n d w e l l i n g c a t h e t e r s .  The  s i m i l a r r e s u l t s when b l o o d i s sampled a f t e r  two  techniques  the e x e r c i s e  p e r i o d ; however, b l o o d taken from f i s h p r i o r to f a t i g u e by c a r d i a c puncture c o n t a i n s e l e v a t e d l e v e l s of l a c t a t e .  I t i s p o s s i b l e that i n manipulating  the  86  f i s h for cardiac puncture.there.is an i n c r e a s e d for  an.increased  c a r d i a c , output which f l u s h e s  amount of l a c t a t e out of the white muscle.  Another unaccounted  parameter i n sampling a t moderate swimming speeds i s the a c t i v i t y of  a n i m a l per  se d u r i n g  the sampling p e r i o d .  Thus, blood  lactate  increases  f o l l o w i n g c e s s a t i o n of e x e r c i s e i n v o l v i n g white muscle a c t i v i t y but i n c r e a s e when swimming i s a l l o w e d to c o n t i n u e . t i o n ) has  Randall  the  (personal  does not  communica-  suggested t h a t f o l l o w i n g c e s s a t i o n of a c t i v i t y there may  be  a  l o c a l i z e d hyperaemia i n the white musculature which causes a massive f l u s h i n g out of l a c t a t e .  T h i s c o u l d e x p l a i n not  o n l y the f i n d i n g s a t  intermediate  v e l o c i t i e s but a l s o the g e n e r a l phenomenon of a r a p i d i n c r e a s e tate concentration it  f o l l o w i n g strenuous e x e r c i s e .  Whatever the answer may  the animals i n . which both a r t e r i a l and  samples were taken f o l l o w i n g f a t i g u e . showed a n e g a t i v e ymoles/ml.  Of  venous  I t t h e r e f o r e appears t h a t i n the r e c o v e r y  A 1000  gm .trout has  c i r c u l a t i o n time of a p p r o x i m a t e l y 2 min trout t h i s s i z e could After entering  take up  the g i l l  period  about 50 ml  (Randall,.1970).  following  p y r u v a t e , which may  be  of blood  and  a  Thus the g i l l s  of a  or f u r t h e r m e t a b o l i z e d .  a minimal amount of l a c t a t e (Karuppannan, 1972),  i s no r e a s o n to b e l i e v e t h a t f o l l o w i n g e x e r c i s e  i s p r o b a b l e t h a t any  during i t s  on the average about 10 umoles ' l a c t a t e / m i n .  l a c t a t e must e i t h e r be e x c r e t e d  During exercise f i s h excrete there  blood  a r t e r i a l - v e n o u s d i f f e r e n c e which on the average i s about  passage through the g i l l s .  It  be,  the eleven, measurements made, e i g h t  strenuous e x e r c i s e there i s a net uptake of l a c t a t e from the b l o o d  but  lac-  i s c l e a r t h a t t h i s problem warrants r e s o l u t i o n . Table VIII l i s t s  0.4  i n blood  l a c t a t e taken up by further u t i l i z e d  the g i l l s  this level  i s reconverted  i n a v a r i e t y of ways.  c a p a c i t y to o x i d i z e l a c t a t e t o t a l l y to C0„  and  increases. to  G i l l has  water ( B i l i n s k i and  the  Jonas,  1972)  87  Table  VIII...  Arterial  and venous 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 o f r a i n b o w  trout following  Fish  Time f o l l o w i n g f a t i g u e (min)  1  1080  2  120  3  1  30 60 660 4  1 60 90 120 135  exercise  Venous (ventral aorta)  1.1*  .  to f a t i g u e .  Arterial (dorsal aorta)  A-V difference  1.0  -0.1  10.3  9.8  -0.5  3.3 5.4 8.4 1.5  2.8 5.6 7.1 1.8  -0.5 +0.2 -1.3 +0.3  1.8 3.1 4.1 6.4 6.8  0.6 3.0 4.9 5.6 5.3  -1.2 -0.1 +0.8 -0.8 -1.5  Mean  -0.4  * L a c t a t e c o n c e n t r a t i o n i n umoles/ml b l o o d .  88  and  the i n v i v o data o f Rao (1968) i n d i c a t e t h a t the oxygen consumption o f  gill  o f a 1000 gm t r o u t i s g r e a t enough t o c o m p l e t e l y  lactate/min.  Furthermore, a l t h o u g h  o x i d i z e 10 umoles  s t u d i e s on the i n t e r m e d i a r y metabolism o f  the f i s h g i l l a r e n o t a b l y l a c k i n g , i t i s known t h a t the c r u s t a c e a n g i l l has an extremely gill  h i g h gluconeogenic  c a p a c i t y (Thabrew e_t al_,  1971) .  I f the f i s h  i s a t a l l s i m i l a r t o the analagous c r u s t a c e a n t i s s u e , a 1000 gm t r o u t  c o u l d e a s i l y d i r e c t a l a r g e f r a c t i o n o f the l a c t a t e i n t o g l u c o s e . r e s p e c t i t i s i n t e r e s t i n g to note  t h a t f i s h g i l l has p a r t i c u l a r l y  In t h i s high  glycogen d e p o s i t s which l i e i n c l o s e p r o x i m i t y t o an abundant m i t o c h o n d r i a l system (Conte, for  1969).  this tissue.  I t may be t h a t glycogen  to  simply c o n v e r t e d  t h e muscle.  fuel  I t i s a l s o p o s s i b l e that there i s a f u n c t i o n a l e l e c t r o n  s h u t t l e system between white muscle and g i l l ; muscle.is  s e r v e s as the m e t a b o l i c  to p y r u v a t e  such t h a t l a c t a t e formed i n  i n the g i l l and subsequently  returns  I n t h i s case the g i l l would be f u n c t i o n i n g to o x i d i z e NADH  produced from the l a c t a t e dehydrogenase r e a c t i o n .  D u r i n g a bout o f strenuous  a c t i v i t y a 1000 gm t r o u t may accumulate 30,000 umoles o f l a c t a t e i n i t s white muscle  (Black e_t al, 1962; Stevens and B l a c k , 1966; Hammond and Hickman, 1966).  Since i t then takes from 12-24 hours f o r b l o o d l a c t a t e t o r e t u r n to normal i t is  p o s s i b l e t h a t the g i l l p l a y s a h e r e t o f o r e unrecognized  ism o f t h i s m e t a b o l i t e d u r i n g the r e c o v e r y p e r i o d .  r o l e i n the metabol-  89  SUMMARY Rainbow t r o u t , ( S a l m o gairdneri.) were e x e r c i s e d stepwise i n c r e a s i n g v e l o c i t y increments. lactate concentration  a t any time d u r i n g  i n a s e r i e s of 60 minute  There i s no i n c r e a s e  i n blood  the e x e r c i s e p e r i o d ; even though,  some of the animals were e x e r c i s e d a t 93% o f t h e i r c r i t i c a l v e l o c i t y on a sustained production elsewhere.  basis.  The d a t a i n d i c a t e t h a t under these c o n d i t i o n s  of l a c t a t e by white muscle i s e q u a l to i t s r a t e of u t i l i z a t i o n Immediately f o l l o w i n g f a t i g u e blood  D u r i n g the r e c o v e r y gills.  the r a t e of  period  lactate l e v e l rapidly increases.  there appears to be a n e t uptake of l a c t a t e by the  90  APPENDIX I I  ENZYME NOMENCLATURE  90 a  a d e n y l a t e k i n a s e (E.C. 2.7.4.3) adenylosuccinase  (E.C. 4.3.2.2)  a d e n y l o s u c c i n a t e s y n t h e t a s e (E.C. 6.3.4.4) a l a n i n e transaminase  (E.C. 2.6.1.2)  AMP deaminase (E.C. 3.5.4.6) a s p a r t a t e transaminase c i t r a t e lyase  (E.C. 2.6.1.1)  (E.C. 4.1.3.8)  c i t r a t e synthase  (E.C. 4.1.3.7)  f r u c t o s e - 1 , 6 - d i p h o s p h a t a s e (E.C. 3.1.3.11) glutamate dehydrogenase  (E.C. 1.4.1.2)  g l u t a m a t e - o x a l o a c e t a t e transaminase glutamate-pyruvate transaminase  (E.C. 2.6.1.1)  (E.C. 2.6.1.2)  glutamine s y n t h e t a s e (E.C. 6.3.1.2) glucose-6-phosphate  dehydrogenase  a-glycerophosphate dehydrogenase glycogen phosphorylase hexokinase  (E.C. 1.1.1.49) (E.C. 1.1.1.8)  (E.C. 2.4.1.1)  (E.C. 2.7.1.2)  l a c t a t e dehydrogenase  (E.C. 1.1.1.27)  malate dehydrogenase  (E.C. 1.1.1.38)  n u c l e o s i d e p h o s p h o r y l a s e (E.C. 3.2.2.1) 5' n u c l e o t i d a s e (E.C. 3.1.3.5) phosphofructokinase  (E.C. 2.7.1.11)  phosphoglucoisomerase p y r u v a t e dehydrogenase  (E.C. 5.3.1.9) (E.C. 1.2.4.1)  p y r u v a t e k i n a s e (E.C. 2.7.1.40)  91  succinic thiokinase  (E.C. 6.2.1.5)  t e t r a h y d r o f o l i c a c i d formylase xanthic oxidase  (E.C. 1.2.3.2)  (E.C. 3.5.1.10)  

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