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Recovery from exhaustive exercise in rainbow trout white muscle : a model for studies of the control… Schulte, Patricia Marita 1990

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RECOVERY FROM EXHAUSTIVE EXERCISE IN RAINBOW TROUT WHITE MUSCLE: A MODEL FOR STUDIES OF THE CONTROL OF ENERGY METABOLISM IN VIVO By P A T R I C I A MARITA B.Sc,  SCHULTE  The U n i v e r s i t y o f B r i t i s h C o l u m b i a ,  1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f Zoology)  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF B R I T I S H COLUMBIA September, <£)  Patricia  Marita  1990 Schulte,  1990  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  i i  ABSTRACT Recovery from exhaustive  exercise  i n the white muscle of  rainbow t r o u t (Oncorhynchus mvkiss) was used t o examine the r o l e o f the  adenylates  i n the  c o n t r o l of energy metabolism and to  assess the v a l i d i t y o f e q u i l i b r i u m models of the behaviour of the h i g h energy  phosphates.  The d i f f i c u l t y of o b t a i n i n g muscle samples from f i s h makes detailed analysis phosphates infusion cannula,  of the behaviour of the  complex.  of  a  The use  lethal  of  dose  of  l a b i l e h i g h energy  a new sampling p r o c e d u r e , anaesthetic  via  the  an i n d w e l l i n g  minimized t h i s problem.  At e x h a u s t i o n  [ATP] and [PCr] were depressed by 75 and 80%  r e s p e c t i v e l y r e l a t i v e to the r e s t i n g v a l u e s .  [ATP] d e p l e t i o n was  m i r r o r e d by a s t o i c h i o m e t r i c i n c r e a s e i n [IMP]. During recovery [PCr]  r e t u r n e d t o the r e s t i n g l e v e l w i t h i n 2 hours, but [ATP]  recovery  was  exercise.  In c o n t r a s t , energy charge and R  the  free  and  adenylate  anything, exercise.  slow  higher  not  pool  than  complete  until A T P  phosphorylated  the  resting  values  24  hours  post  ( t h e p r o p o r t i o n of  to by  ATP) 2  were,  hours  if post  T h e r e f o r e , [ATP] and energy s t a t u s can be d i s s o c i a t e d  i n t i s s u e s l i k e f i s h white muscle because of the a c t i o n of the purine nucleotide At  2  hours  cycle. post  exercise  the  calculated  free  c o n c e n t r a t i o n dropped to l e s s than one t e n t h the v a l u e at As a r e s u l t the  [ATP] / [ADP]  f r e e  ADP rest.  r a t i o i n c r e a s e d by n e a r l y 6 f o l d .  i i i  T h i s c o n d i t i o n may  be  r e q u i r e d f o r glycogen  resynthesis  from  l a c t a t e i n muscle. S e v e r a l s i m i l a r e q u i l i b r i u m models of the behaviour of the adenylates and PCr were a p p l i e d t o the f i s h white muscle system. In g e n e r a l ,  the models w e l l d e s c r i b e the r e l a t i o n s h i p between  the h i g h energy phosphates. However, the d e f i n i t i o n of the high energy phosphate p o o l i n t r o d u c e s some c o m p l i c a t i o n s includes  the  deaminase the  total [ATP]  [ATP].  Because  of  the  since t h i s  action  c o n c e n t r a t i o n can change without  of  AMP  measurable  changes i n the energy s t a t u s , which i s not c o n s i d e r e d i n any the models. As long as the extent of IMP models can be a p p l i e d , but  formation  s i n c e the formation  of  i s known the  of IMP  may  vary  from f i s h t o f i s h or with e x e r c i s e regime the models l o s e much of t h e i r p r e d i c t i v e power.  iv TABLE OF CONTENTS ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  x  INTRODUCTION  1  MATERIALS AND METHODS  17  Animals  17  Swimming assessment  17  S u r g i c a l procedure  18  Exercise protocol  19  Sampling and muscle d i s s e c t i o n  20  Muscle homogenization  21  B i o c h e m i c a l analyses  23  I n t r a c e l l u l a r pH  26  Calculations  30  S t a t i s t i c a l analysis  30  Cytosolic  31  redox  I n t r a c e l l u l a r f r e e magnesium  32  Model c a l c u l a t i o n s  35  RESULTS  39  General  39  Adenylates  39  Phosphocreatine  42  A r t e r i a l and i n t r a c e l l u l a r pH  42  Carbohydrate metabolism  44  L a c t a t e / p y r u v a t e r a t i o s and redox  45  Plasma l a c t a t e  45  and g l u c o s e  T i s s u e water  49  I n t r a c e l l u l a r f r e e magnesium  49  Model c a l c u l a t i o n s  49  DISCUSSION The r e s t i n g  59 fish  59  ATP overshoot  64  ATP d e p l e t i o n  66  Phosphocreatine and energy metabolism  70  Carbohydrate metabolism  72  I n t r a c e l l u l a r pH  78  Free ADP  80  Free AMP  85  R  86  AXP  and energy charge  SUMMARY AND CONCLUSIONS REFERENCES  94 97  vi  LIST OF TABLES Table 1 .  Magnesium b i n d i n g s i t e s and b i n d i n g 34  constants i n s k e l e t a l muscle Table 2 .  E q u i l i b r i u m constants  Table 3.  A r t e r i a l and i n t r a c e l l u l a r pH estimated  used  by the DMO and homogenate  38  techniques  p r i o r t o and d u r i n g recovery f o l l o w i n g exhaustive e x e r c i s e Table 4.  40  Adenylate c o n c e n t r a t i o n s i n white muscle p r i o r t o and d u r i n g the recovery from exhaustive e x e r i c s e  Table 5.  i n rainbow t r o u t  C o n c e n t r a t i o n s of phosphocreatine,  41  creatine 43  and i n o r g a n i c phosphate i n white muscle Table 6.  C o n c e n t r a t i o n of l a c t a t e , glycogen and glucose  pyruvate,  i n rainbow t r o u t 46  white muscle Table 7 . L a c t a t e / p y r u v a t e r a t i o and c y t o s o l i c Table 8. Plasma l a c t a t e Table 9 .  and glucose  redox . . . 4 7  concentrations  T o t a l t i s s u e water content  . . . . 48  and f l u i d  distribution  50  Table 10. Magnesium bound ATP, bound ATP as a f r a c t i o n of the t o t a l c r e a t i n e  (Fa) and as a f r a c t i o n  of the t o t a l adenylates charge  (Ratp); c r e a t i n e  (Fc) and n o r m a l i z e d p o t e n t i a l  energy p o o l  55  vii Table 11. F r e e ADP c o n c e n t r a t i o n c a l c u l a t e d without r e f e r e n c e to i o n b i n d i n g , or u s i n g the model equations concentrations  and s e v e r a l  different  of f r e e magnesium  56  Table 12. Free AMP c o n c e n t r a t i o n c a l c u l a t e d at differing  f r e e magnesium c o n c e n t r a t i o n s  Table 13. Energy charge c a l c u l a t e d u s i n g the concentrations  57  total  of ADP and AMP and a c c o r d i n g  to the model d e f i n i t i o n s  58  v i i i  LIST OF FIGURES F i g u r e 1.  Model o f the c o n c e n t r a t i o n s  of PCr,  ATP, ADP, AMP and P i i n the presence of c r e a t i n e kinase and adenylate kinase  as ATP i s h y d r o l y z e d  6  F i g u r e 2. C a l c u l a t e d e q u i l i b r i u m c o n c e n t r a t i o n s of phosphocreatine and adenosine phosphates as high energy phosphate i s discharged  8  F i g u r e 3. Normalized m e t a b o l i t e  concentrations 9  as a f u n c t i o n of energy content F i g u r e 4. ATP c o n c e n t r a t i o n d u r i n g recovery exhaustive  from  e x e r c i s e i n rainbow t r o u t  14  F i g u r e 5. C a l c u l a t e d t o t a l magnesium c o n c e n t r a t i o n at r e s t and at exhaustion  for a variety  of f r e e magnesium c o n c e n t r a t i o n s  from  0 t o 30 mM  51  F i g u r e 6. 3 Carbon u n i t s  ([lactate] +  2[glycogen])  i n rainbow t r o u t white muscle f o l l o w i n g exhaustive  exercise  74  F i g u r e 7. R e s u l t s of model c a l c u l a t i o n s a p p l i e d t o rainbow t r o u t white muscle f o l l o w i n g exhaustive  exercise  88  ix  Figure  8.  The v a r i a t i o n i n c r e a t i n e charge w i t h the n o r m a l i z e d p o t e n t i a l energy p o o l i n rainbow t r o u t white muscle  following  e x h a u s t i v e e x e r c i s e : the e f f e c t of the purine nucleotide  cycle  89  ACKNOWLEDGEMENTS I would l i k e t o thank Dr. Peter Hochachka f o r h i s support and e s p e c i a l l y f o r p r o v i d i n g a s t i m u l a t i n g academic environment which  i s , i n part,  a t t r i b u t a b l e t o everyone  i n t h e l a b but  d e r i v e s i n l a r g e measure from h i s own boundless enthusiasm. A s p e c i a l thanks goes t o my f r i e n d s and c o l l e a g u e s i n the l a b , i n p a r t i c u l a r : C h r i s Moyes, Tim West and Peter A r t h u r without whose help and suggestions  t h i s t h e s i s would not have been p o s s i b l e .  I would a l s o l i k e t o thank Dr. Dave Randall  and h i s group  f o r a l l o w i n g me f o r t h e use of t h e i r c a p i l l a r y pH e l e c t r o d e and the swim t u n n e l . S p e c i a l thanks t o Yong Tang f o r t e a c h i n g me the homogenate  pH  technique.  Thanks  also  t o Dr. Carefoot f o r  a l l o w i n g me t o use one of h i s d r y i n g ovens.  1  INTRODUCTION C e l l u l a r energy metabolism can be c o n c e p t u a l l y d i v i d e d i n t o two d i s t i n c t components, ATP consuming systems and ATP producing systems. One o f the fundamental c h a r a c t e r i s t i c s of metabolic o r g a n i z a t i o n i s the c o u p l i n g o f energy supply t o energy demand. Muscle t i s s u e p r o v i d e s  an e x c e l l e n t system f o r the study of the  c o u p l i n g between these two components because o f i t s wide range of  energy  demand. The metabolic  rate  o f a muscle  cell  can  i n c r e a s e many f o l d over the t r a n s i t i o n from r e s t t o work. For example, energy turnover by  nearly  1,000  contraction The  fold  (Krisanda  ATP  i n the f r o g s a r t o r i u s muscle i n c r e a s e s i n the f i r s t  e_t .al.,  consuming  of t e t a n i c  1988) .  reactions  actomyosin ATPases as well  few seconds  i n muscle  cells  include  as a v a r i e t y of ATP-dependent i o n  pumps. I t i s g e n e r a l l y considered that ATP demand i s the d r i v i n g force, ATP  or independent v a r i a b l e , i n metabolic c o u p l i n g .  demand i n c r e a s e s  due t o , f o r example,  increased  So, as r a t e of  c o n t r a c t i o n i n a muscle, ATP p r o d u c t i o n w i l l a l s o r i s e . However, it  should  be noted t h a t  cause and e f f e c t can be d i f f i c u l t t o  determine and i t may be that the ATP producing systems of the c e l l are simply the  ATP  initiate production  responding t o the same s i g n a l t h a t upregulates  demand. a  For example,  contraction (Connett,  could  the changes also  serve  in to  [Ca ] 2+  speed  which up  ATP  1990). The c o n t r o l of the ATP consuming  r e a c t i o n s i n the muscle c e l l has been e x t e n s i v e l y s t u d i e d , but  2 for  the purposes of t h i s d i s c u s s i o n i t i s s u f f i c i e n t t o regard  these r e a c t i o n s as a "black box" of the general ATP  vADP + P  form:  ±  which can be s e t t o a v a r i e t y o f d i f f e r e n t l e v e l s o f ATP demand. The  systems which supply  t h r e e main sub-systems the  glycolytic  system  (1) the m i t o c h o n d r i a l sub-system  sub-system  (Connett,  energy can be broken down  and (3) the phosphate  into , (2)  energy  sub-  1990) . The m i t o c h o n d r i a l and g l y c o l y t i c  sub-  systems c o n t a i n the energy producing f u n c t i o n s of the c e l l . Both o x i d a t i v e p h o s p h o r y l a t i o n and g l y c o l y s i s share many o f the same control  signals.  Two main  controllers  are redox balance  and  p h o s p h o r l y a t i o n s t a t e , which i s e s s e n t i a l l y some measure of the degree  t o which  the adenylates  are phosphorylated  t o ATP.  E r e c i n s k a e_t a_l. (1977) have shown t h a t aerobic ATP s y n t h e s i s responds t o changes Glycolysis  i n the p h o s p h o r y l a t i o n s t a t e o f the c e l l .  i s a l s o s e n s i t i v e t o phosphorylation  state largely  because of the a l l o s t e r i c c o n t r o l of PFK through ATP i n h i b i t i o n and  deinhibition  Somero, changes  by ADP,  1984) . These in  the  AMP  systems  rate  of  and phosphate  (Hochachka  a c t as feed back ATP  production  loops will  and since  affect  phosphorylation state. M i t o c h o n d r i a l redox s t a t e i s a l s o i n v o l v e d i n the c o n t r o l of  oxidative phosphorylation  al., be  (Koretsky  et a l . , 1984; Katz et  1988) and c y t o s o l i c redox s t a t e i s g e n e r a l l y accepted t o an  brought  important about  controlling  by a network  factor  of c e l l u l a r  metabolism  of near e q u i l i b r i u m r e a c t i o n s i n  3  which p y r i d i n e n u c l e o t i d e s Veech,  1969) . The  p a r t i c i p a t e as c o f a c t o r s  i n t e r a c t i o n of  cytosolic.'. and  (Krebs  and  mitochondrial  redox i s complex. The  role  greatest  of  the  importance  phosphate to  the  energy  present  sub-system  study.  i s of  This  the  sub-system  c o n s i s t s of the energy supply d i r e c t l y a v a i l a b l e i n the c y t o s o l , t h a t i s , the c y t o s o l i c p o o l s of c r e a t i n e phosphate and ATP. is  the  direct  processes  substrate  i n the  cell.  for  most  + PCr  the  Phosphocreatine  thought to p a r t i c i p a t e i n only one ADP  of  + H* ,  -1 ATP  on  energy the  requiring  other hand i s  reaction: + creatine  c a t a l y z e d by the enzyme c r e a t i n e phosphokinase (CPK). The function  of  the  CPK  suggested t h a t PCr  reaction  and  ATP  is controversial.  c r e a t i n e may  It  actual  has  been  f u n c t i o n as a s h u t t l e f o r  the t r a n s p o r t of high energy phosphates between compartments of adenylates shuttle  within  includes  muscle three  mitochondrion, one  cells.  basic  The  parts,  phosphocreatine one  i n the  i n the area of the m y o f i b r i l and  " s h u t t l e " through the c y t o s o l between the two. theory,  at  the  area  myofibril  utilized  the  actual  A c c o r d i n g to the to  produced by the actomyosin ATPase.  The  r e s u l t i n g c r e a t i n e enters the bulk phase of the c y t o s o l . At  the  mitochondria  CPK  t h i s creatine  is  of  CPK  r e p h o s p h o r y l a t e the ADP  PCr  the  energy  is utilized  by  by  an  bound  isozyme  which i s bound c l o s e to the adenylate t r a n s l o c a s e according  t o the theory, the c o n d i t i o n s  formation  of  PCr  from t h i s c r e a t i n e  s i t e . Here,  are favourable  and  the  ATP  of  f o r the  produced  by  4 o x i d a t i v e p h o s p h o r y l a t i o n . The PCr then e n t e r s the bulk phase of the c y t o s o l  and d i f f u s e s t o the m y o f i b r i l  where the  cycle  begins a g a i n . The b a s i c evidence f o r the theory i s o u t l i n e d i n Bessman and Savabi In  apparent  f u n c t i o n of CPK  (1990) . c o n t r a s t , the  classic  theory  regarding  i s t o act as a b u f f e r t o [ATP] d u r i n g  mismatching  between  ATP  between PCr  and  adenylates  the  demand  and are  supply. such  The  that  the  temporary equilibria  when net  high  energy phosphate h y d r o l y s i s occurs the PCr p o o l i s d e p l e t e d t o a g r e a t e r extent than the ATP pool (McGilvery and Murray, A l l e n and Orchard,  1987;  Connett,  1988) . T h i s a n a l y s i s  1974; treats  the contents of the c y t o s o l as e s s e n t i a l l y homogenous and does not r e q u i r e the assumption of compartmentation phosphocreatine equilibrium founded  shuttle.  everywhere  Rather  i n the  i t assumes  cell  necessary f o r the that  CPK  which appears  t o be  (Meyer et a l . , 1984) . As formulated, these two  appear t o be mutually e x c l u s i v e .  Meyer et. al..  is in well  hypotheses  (1984) showed  t h a t they are compatible. They showed that the p r o p e r t i e s of CPK which  make i t a temporal  function  as  a spatial  buffer  buffer  to  of  [ATP]  also  allow  [ATP]. However, the  i t to spatial  b u f f e r i n g f u n c t i o n should not be regarded as a s h u t t l e per se since t h i s  r e q u i r e s some s o r t  of metabolic  compartmentation.  Rather, Meyer et a l . (1984) suggest that t h i s s p a t i a l b u f f e r i n g i s an example of f a c i l i t a t e d d i f f u s i o n , e q u i v a l e n t i n p r i n c i p l e t o the r o l e of myoglobin d i f f e r e n t approach  Jacobus  i n oxygen t r a n s p o r t . Using a t o t a l l y (1985) reaches a s i m i l a r c o n c l u s i o n  5 based on a d i f f u s i o n r e s t r i c t i o n  on ADP.  These hypotheses  based on viewing energy metabolism as a s e r i e s of  are  equilibria.  A l l of the c o n s t i t u e n t s of the phosphate energy sub-system are i n t e r r e l a t e d v i a the f o l l o w i n g  equations:  (1)  Cr + MgATP y-  * MgADP" + PCr " +  (2)  AMP " + M g A T P = F = = ^ ADP " + MgADP"  2  2  2  Reaction  (1), the  (2) , c a t a l y z e d by  CPK  H  2  3  r e a c t i o n was  adenylate kinase  discussed  above.  sets  the  and AdK  are  at or near e q u i l i b r i u m i n s k e l e t a l muscle.  The  c a p a c i t y of CPK always  be  i n s k e l e t a l muscle i s such that i t should almost  near  equilibrium.  support t h i s c o n c l u s i o n  Direct  measurements  and Murrray, 1974)  by  P-NMR  31  (Meyer et a l . , 1985). Adenylate  i s probably a l s o near e q u i l i b r i u m i n s k e l e t a l muscle  of CPK  Reaction  (myokinase), AdK,  r a t i o s of the v a r i o u s adenine n u c l e o t i d e s . Both CPK thought t o be  +  kinase  (McGilvery  s i n c e the enzyme c a p a c i t y i s s i m i l a r to t h a t  i n s k e l e t a l muscle  (Connett, 1988) .  I f these r e a c t i o n s are assumed to be at e q u i l i b r i u m then it  is  possible  concentrations  of  to  model  the  adenylates  phosphate i s hydrolyzed. model  the  Figure  changes  in  the  and  as  "high  PCr  energy"  1 shows the r e s u l t s of such a  ( A l l e n and Orchard, 1987) . I t assumes t h a t the  reactions  relative  following  occur: ATP PCr  + ADP^  > ADP + Pi ^ ATP + Cr  2ADP^ changes i n pH were n e g l e c t e d occur at pH = 7.0.  ATP  +  and  a l l r e a c t i o n s were assumed to  However, the AdK  AMP  and CPK  r e a c t i o n s do  not  6  F i g u r e 1: Model of c o n c e n t r a t i o n s of P C r , ATP, f r e e ADP, and AMP and phosphate i n the presence of c r e a t i n e kinase and adenylate kinase as ATP i s h y d r o l y z e d . See A l l e n and Orchard (1987) . H y d r o l y s i s of ATP was regarded as i r r e v e r s i b l e . R e a c t i o n s were assumed t o occur at pH =7.0 and changes i n pH were i g n o r e d . S t a r t i n g c o n c e n t r a t i o n s were [ATP]= 7mM; [PCr]= 25mM; [ADP]=[AMP]=[Pi]=0 [ATP][Cr]/[ADP][PCr] = 200 [ADP] /[ATP][AMP] = 1 2  7 occur  i n v i v o as they  are shown above. Rather,  only the Mg  2+  c h e l a t e s of the adenylates  are i n v o l v e d . In order to p r o p e r l y  apply  experimentally  these  equilibria  to  measurements some estimate of both H  derived  metabolite  and magnesium b i n d i n g to  +  the adenylates should be taken i n t o account. T h i s approach first  o u t l i n e d by McGilvery  Connett  and Murray  (1974),  see f i g u r e  was 2.  (1988) f u r t h e r r e f i n e d t h i s by s c a l i n g a l l parameters  to  the t o t a l c r e a t i n e p o o l  of  changes i n pH and  (PCr+Cr) and c o n s i d e r i n g the e f f e c t s  [Mg ] . Since PCr p a r t i c i p a t e s i n only the 2+  CPK r e a c t i o n the t o t a l c r e a t i n e p o o l i s e s s e n t i a l l y constant f o r one  t i s s u e over a l l metabolic  widely  from t i s s u e  states. Total  to t i s s u e  (Connett,  [creatine] varies  1988)  so  scaling a l l  parameters i n t h i s way allows d i r e c t comparison between t i s s u e s . As  can  be  behaviour  seen of  the  from  figures  adenylates  1,2  and  and PCr  as  3  which  outline  phosphate  the  energy  is  h y d r o l y z e d a c c o r d i n g to each model, the c a r e f u l c o n s i d e r a t i o n of  i o n b i n d i n g does not make a great d e a l of d i f f e r e n c e to t h e i r  g e n e r a l p r e d i c t i o n s . There are c l e a r l y two d i s t i n c t phases i n the d e p l e t i o n of the h i g h energy phosphate p o o l . In the  first  phase the adenine n u c l e o t i d e p o o l i s p r o t e c t e d and only  [PCr]  decreases  with l i t t l e  (1988) terms t h i s  the  or no  change i n the adenylates.  b u f f e r i n g phase.  In the  Connett  second phase,  energy i s s u p p l i e d at the expense of [ATP] and readjustments  to  the r e l a t i v e p r o p o r t i o n s of the adenine n u c l e o t i d e s are brought about as a r e s u l t of the AdK  e q u i l i b r i u m . One obvious but o f t e n  overlooked p o i n t t h a t i s made c l e a r by these models i s t h a t  8  24  36  32  28  24  20  16  TOTAL HIGH ENERGY PHOSPHATE Figure  12  8  4  0  (mmoles/kg)  2: C a l c u l a t e d e q u i l i b r i u m c o n c e n t r a t i o n s o f p h o s p h o c r e a t i n e and t h e a d e n o s i n e p h o s p h a t e s as h i g h e n e r g y p h o s p h a t e i s d i s c h a r g e d . T o t a l C r = 30 mmoles/kg F r e e [ATP]+[ADP]+[AMP] = 6 mmoles/kg pH assumed t o b e c o n s t a n t a t 7.0 f r e e Mg « 0.5mM 2+  [ATP] [ C r ] / [ H ] [ADP]= 1 . 5 1 x l 0 [AMP] [ATP] / [ADP] = 0.364 +  8  M"  1  2  After  McGilvery  and M u r r a y  (1974)  9  1 .0  0  0.2  0.4  NORMALIZED  (PCr  Figure  3:  0.6  POTENTIAL  +  2ATP  0.8 ENERGY  1.0  POOL  + ADP)  N o r m a l i z e d m e t a b o l i t e c o n c e n t r a t i o n s as a f u n c t i o n of "energy content", t o t a l a d e n y l a t e s = [ATP]+[ADP]+[AMP] t o t a l c r e a t i n e = [PCr]+[Cr] c r e a t i n e charge (Fc) = [ P C r ] / t o t a l creatine e n e r g y c h a r g e (EC)= f A T P 1 + 0 . 5 fADP1 total adenylates Ratp = [ATP]/ t o t a l adenylates Radp = [ADP]/ t o t a l adenylates Ramp = [ A M P ] / t o t a l adenylates A l l v a l u e s were c o m p u t e d u s i n g [Mg ] =lmM pH = 7 . 0 total adenylates/ total creatine =0.2 A f t e r Connett (1988) 2+  10 l a r g e changes i n [ P J can a r i s e without s u b s t a n t i a l [ATP]  changes i n  ( A l l e n and Orchard, 1987) . Note that i f [PCr] f a l l s by 20%  [ADP]  will  free  increase  by 200% and [ P J by 160% without any  change i n [ATP]. T h i s aspect o f the CPK r e a c t i o n probably p l a y s an important p h y s i o l o g i c a l  r o l e i n the muscle  i s a known a c t i v a t o r o f both g l y c o g e n o l y s i s , phosphorylase  (Morgan and Parmeggiani,  through t h e a c t i v a t i o n  cell.  Phosphate  through a c t i o n on  1964) and g l y c o l y s i s  o f PFK (Hochachka,  1980) and at h i g h  l e v e l s i s known t o be an i n h i b i t o r o f actomyosin ATPase and Pate,  1990).  As mentioned takes  into  b e f o r e , Connett's a n a l y s i s  account  the e f f e c t s  o f changing  Changing pH a l t e r s the extent o f the f i r s t , of  high  cytosol  energy results  depletion  (Cooke  phosphate i n less  depletion.  effective  (figure  3) a l s o  [Mg ]  and pH.  2+  or b u f f e r i n g ,  Alkalinization  buffering  o f [ATP]) while a c i d i f i c a t i o n  phase  of  ( i e : more  the  rapid  (as i s seen i n working  s k e l e t a l muscle) leads t o an i n c r e a s e i n the range of b u f f e r i n g . Changes  in  [Mg ] 2+  have  similar  effects  with  high  [Mg ] 2+  d e c r e a s i n g the extent of the b u f f e r i n g phase. When energy charge or R  AXP  ( [ATP ] / ( [ATP ] + [ADP ]  free  + [AMP ]  free  ) values are h i g h (>0.5) a  f a c t o r o f two change i n [Mg ] r e s u l t s i n a l e s s than 10% change 2+  in  t h e v a l u e o f t h e energy  charge  or R . These ATP  e f f e c t s are  g r e a t e r when energy charge i s low. As a r e s u l t changes i n [Mg ] 2+  are probably only important under c o n d i t i o n s o f extreme depletion fall.  when [ATP] and hence energy charge and R  Since ATP i s a major  flTP  intracellular  magnesium  energy  start to chelator,  11 l a r g e changes The  i n [Mg ] 2+  mathematical  approach  allow  free  would be expected at t h a t time.  and  chemical  him t o make  precision  several  o f Connett's  interesting  statements  r e g a r d i n g the phosphate energy sub-system of the c e l l . Among the most  startling  of these i s t h a t  the f r a c t i o n  o f t h e adenine  n u c l e o t i d e p o o l i n the form of ATP and, by analogy, i n the form of AMP amd IMP i s simply a f u n c t i o n of t h e " c r e a t i n e charge" (the f r a c t i o n a l p h o s p h o r y l a t i o n of c r e a t i n e •( [PCr] / [Cr] ) ) , the T  pH,  [Mg ] , and [K ] . 2+  +  I t i s d i f f i c u l t t o adequately t e s t these models i n v i v o i n mammalian s k e l e t a l muscle seldom  falls  events  that  examined been  systems s i n c e even at f a t i g u e [ATP]  by l e s s than 20% (Wilkie, are analogous  1981) . T h e r e f o r e only  t o the b u f f e r i n g  phase  have  i n any d e t a i l . In f i s h white muscle, however,  shown t h a t  1988).  subsequently Hochachka,  i t has  [ATP] may be d e p l e t e d by up t o 90% f o l l o w i n g  exhaustive e x e r c i s e Hochachka,  been  (Dobson  and Hochachka,  In a d d i t i o n  rainbow  1987; Mommsen and  trout  r e c o v e r over a p e r i o d of about  are known t o  24h (Mommsen and  1988; M i l l i g a n and Wood, 1986) . T h i s r e c o v e r y c o u l d  p r o v i d e an i d e a l system i n which t o examine models such  as t h a t  of Connett (1988) s i n c e the system s t a r t s out with low p o t e n t i a l energy  ([PCr]+2[ATP]+[ADP])  g r a d u a l l y r e t u r n i n g t o a s t a t e of  h i g h p o t e n t i a l energy over a time course s u f f i c i e n t l y  slow t o  make sampling f o r c o n v e n t i o n a l b i o c h e m i c a l analyses f e a s i b l e . In  fish  reactions  white  involving  muscle  there  i s an a d d i t i o n a l  s e r i e s of  t h e adenylates which must be c o n s i d e r e d .  12 These r e a c t i o n s are c o l l e c t i v e l y cycle  (Lowenstein,  c a l l e d the p u r i n e n u c l e o t i d e  1972).  (1) AMP : > IMP + NH (2) IMP + a s p a r t a t e " + GTP " 2-  2-  3  2-  1  > a d e n y l o s u c c i n a t e + GDP  4  3-  + Pi  (3) a d e n y l o s u c c i n a t e Reaction  •  > fumarate + AMP  2  (1) c a t a l y z e d by AMP deaminase (AMP aminohydrolase,  EC  3.5.4.6) i s thought t o be the only enzyme o f the c y c l e a c t i v a t e d d u r i n g e x e r c i s e i n many muscle types i n c l u d i n g t r o u t white muscle (2),  (Flanagan  et a l . , 1986),  (Mommsen e t a l . , 1988). Reactions  c a t a l y z e d by adenylosuccinate  synthase  (IMP: L - a s p a r t a t e  l i g a s e (GDP), EC 6.3.4.4) and (3), c a t a l y z e d by a d e n y l o s u c c i n a t e lyase  (adenylosuccinate AMP  l y a s e EC 4.3.2.2), are thought t o  be a c t i v e only d u r i n g recovery. The a c t i o n of AMP deaminase i s probably not o f s i g n i f i c a n c e d u r i n g the b u f f e r i n g phase of phosphate energy d e p l e t i o n s i n c e at  rest  [AMP]  free  i s on the order  of 15nM. The Km  of the AMP  deaminase r e a c t i o n f o r AMP i s about 0.4mM (Smiley and S u e l t e r , 1967), [AMP]  f  very  much  greater  than  i n c r e a s e s t o approximately  the i n v i v o  concentrations.  3 uM as PCr i s d e p l e t e d t o  about 10% o f i t s i n i t i a l l e v e l (assuming an e q u i l i b r i u m constant of  1 f o r AdK at pH 7.0 and [MgATP] of 8mM)  1990)  still  (Krisanda et a l . ,  s u b s t a n t i a l l y lower than the Km. During the second  phase of h i g h energy phosphate d e p l e t i o n the adenylate r e a c t i o n becomes important and [AMP] r i s e s r a p i d l y  kinase  ( f i g u r e s 1,2  and 3) . I t i s d u r i n g t h i s phase t h a t the adenine n u c l e o t i d e pool w i l l be d e p l e t e d by the a c t i o n s of AMP deaminase. The muscle mass of f i s h p r o v i d e s an e x c e l l e n t system f o r  13 i n v e s t i g a t i n g muscle energy metabolism  f o r a number of reasons.  Most important i s the o r g a n i z a t i o n of the myotomal mass of f i s h . F i s h muscle i s s p a t i a l l y (Johnston, into  two  separated i n t o groups by  1981) . In the t r o u t main  fibre  types,  fibre  type  the myotome i s d i f f e r e n t i a t e d  white  and  red,  on  the  b a s i s of  h i s t o c h e m i c a l s t a i n i n g f o r the a e r o b i c enzymes and m y o f i b r i l l a r ATPase. The red, slow t w i t c h , muscle mass i s found p r i m a r i l y i n a narrow l o n g i t u d i n a l  strip  running beneath  the  lateral  line  while the white, f a s t t w i t c h , muscle comprises the bulk of the muscle mass  (Johnston,  1981) . The  white muscle mass i n t r o u t  does i n c l u d e a s m a l l percentage of red type f i b r e s , but t h i s i s n e g l i g i b l e compared t o the h i g h l y heterogenous mammalian  of  have been the  several  recovery  Hochachka,  1988,  s t u d i e s which  period following  ( M i l l i g a n and Wood, 1986; and  of most  muscles.  There nature  nature  Pearson  in  the  phosphate energy  context  sub-system  Mommsen  et a l . , 1990). However, no  of  the  the  exhaustive e x e r c i s e  Dobson and Hochachka, 1987;  study has measured a l l the parameters metabolism  investigated  needed t o assess models  of  the  discussed e a r l i e r .  one  energy  cytosolic  In f a c t ,  the  time course and nature of the changes i n the c o n c e n t r a t i o n s of PCr  and  the adenylates vary  experiment these rest  (see f i g u r e 4). Two  figures at  substantially  r e p o r t an  some p o i n t  from  experiment  of the t h r e e s t u d i e s o u t l i n e d i n  a c t u a l i n c r e a s e i n [ATP]  during  to  recovery.  This  relative  behaviour  is  to not  c o n s i s t e n t with the m e t a b o l i c models d i s c u s s e d e a r l i e r s i n c e  14  F i g u r e 4. ATP c o n c e n t r a t i o n d u r i n g recovery from exhuastive e x e r c i s e i n rainbow t r o u t . The ATP overshoot. A l l c o n c e n t r a t i o n s were converted to umoles/g wet weight u s i n g data a v a i l a b l e i n the o r i g i n a l papers.  restexh 2h 4h  24h  restexh 2h 4h  rwtexh 2h 4h  recovery time  16 they  suggest  t h a t t h e r e may  have been some s y n t h e s i s of  adenylate s k e l e t o n s t o enhance the ATP Adenylate  s y n t h e s i s can  occur  new  pool.  via either  a de  novo  salvage pathway, with p h o s p h o r i b o s y l pyrophosphosphate  or  (PRPP)  as a common i n t e r m e d i a t e . P o s s i b l e sources of s u b s t r a t e f o r the salvage pathway c o u l d i n c l u d e both adenylate breakdown products such as adenosine,  and any breakdown products of RNA  s i n c e the  n u c l e o t i d e s are f r e e l y i n t e r c o n v e r t i b l e . However, h y p o t h e s i z i n g that  the  [ATP]  overshoot  seen  d u r i n g recovery  i s due  s y n t h e s i s by e i t h e r pathway p r e s e n t s a paradox.  to  ATP  Both of these  a n a b o l i c pathways are e n e r g e t i c a l l y expensive and, i n f a c t , are a c t i v a t e d by h i g h energy charge cell  can  (Kunjara et a l . ,  engage i n these processes  reserves  at a time  1987). How  the  when i t s energy  ( i n c l u d i n g ATP and hence energy charge) are c r i t i c a l l y  d e p l e t e d i s an open q u e s t i o n . The  aim  of  the  present  study  is  two-fold:  first,  to  c h a r a c t e r i z e the metabolic events o c c u r r i n g i n f i s h white muscle during  recovery  reference  to  from  the  exhaustive  high  breakdown  products,  existence  and  energy  in  order  of  compounds  further  particular and  their  investigate  p r e v i o u s l y observed  the [ATP]  (1988). The f i s h system i s i d e a l f o r  t h i s purpose s i n c e i t i s one which a profound  to  with  t o t e s t a number of e q u i l i b r i u m models, i n  p a r t i c u l a r t h a t of Connett  seen, which may  phosphate  p o s s i b l e causes  overshoots; second,  exercise,  of the few v e r t e b r a t e systems i n  [ATP] d e p l e t i o n f o l l o w e d by a f u l l recovery i s allow the models t o be t e s t e d t o t h e i r  limits.  17  MATERIALS AND METHODS Animals  Rainbow t r o u t weight  (Oncorhynchus  560 ± 8 9g  mykiss)  of both sexes  (SEM) ) were o b t a i n e d from West Creek  (mean Trout  Ponds, A l d e r g r o v e , B r i t i s h Columbia. F i s h were h e l d outdoors i n one  2m  diameter  circular  tank  supplied  with  flow-through  d e c h l o r i n a t e d Vancouver t a p water between 8 and 12°C. F i s h were f e d by hand d a i l y t o s a t i a t i o n with Oncor P a c i f i c  Salmon Feed  (Moore - C l a r k e ) .  Swimming  Assessment  In the evening i n d i v i d u a l f i s h were t r a n s f e r e d t o a B r e t t type  swim t u n n e l  (1964) and the next day U  crlt  was determined  a c c o r d i n g t o the procedure o u t l i n e d i n (Beamish,  1978).  Three  uncannulated and one cannulated f i s h of t h i s stock were used. U c r i t was c a l c u l a t e d a c c o r d i n g t o the f o l l o w i n g formula: Ucrit= Ui + ( t i / t i i where  x Uii)  U i = the h i g h e s t v e l o c i t y maintained f o r the p r e s c r i b e d time. U i i = v e l o c i t y increment (lOcm/s) t i = time the f i s h swam at the " f a t i g u e " v e l o c i t y (min) t i i = p r e s c r i b e d time p e r i o d at each v e l o c i t y (10 min)  18 This v e l o c i t y was then c o r r e c t e d f o r the presence of the f i s h i n the flume as f o l l o w s : U  c  = U (l + Ai/Aii) s  where  U  c  Uc = Us = Ai = Aii=  corrected velocity v e l o c i t y i n absence of f i s h c r o s s s e c t i o n a l area of f i s h (/0.5d/0.5w) c r o s s s e c t i o n a l area of the t u n n e l  was found t o be 2.8 6 ± 0.03 body lengths/second (mean ±  SEM) f o r the uncannulated f i s h and 2.82 body l e n g t h s per second for  the cannulated f i s h .  I t i s important t o know U  stock o f f i s h i f you wish t o study metabolism  crlt  f o r any  i n white muscle  because white muscle i n f i s h i s p o o r l y r e c r u i t e d at low swimming speeds  (below U  crit  ) (Johnston, 1981) while h i g h e r speeds  result  i n the r e c r u i t m e n t of white f i b r e s and the onset of f a t i g u e .  Surgical  Procedure  Trout  (starved  f o r one  day)  were  b u f f e r e d (NaHC0 ) tricanemethanesulphonate 3  a  c o n c e n t r a t i o n o f 1:6,000  g/L  and then  anaesthetized  in a  (MS-222) s o l u t i o n at transferred  t o the  o p e r a t i n g t a b l e where they were maintained under a n a e s t h e t i c by use  of f o r c e d v e n t i l a t i o n  concentration  of 1:16,000  of a b u f f e r e d MS-222 s o l u t i o n chilled  t o approximately  at a  15 °C and  bubbled with oxygen. D o r s a l a o r t i c cannulae were implanted u s i n g a m o d i f i c a t i o n  of the technique of Sovio e_t a_l. (1972) . In b r i e f , two were put sleeve and  (PE 200)  out  fitted  i n p l a c e i n the r o o f of the mouth and  stitches  a heat  flared  run through the f r o n t of the r o o f of the mouth  i n the  area  of the nares.  Sharpened g u i t a r wire  i n s i d e a p i e c e of p o l y e t h y l e n e t u b i n g  (PE 50)  was  about  25  cm long and then used t o p i e r c e the d o r s a l a o r t a s u p e r f i c i a l l y at the l e v e l of the f i r s t or second g i l l a r c h . The wire was removed and the cannula advanced (up t o about 5cm).  The  then  cannula  was then run out through the implanted s l e e v e and f i x e d i n p l a c e on the r o o f of the mouth u s i n g the s u t u r e s . F i s h were allowed to  recover  from  surgery  f o r at  least  beginning  of experimentation  fed.  P r i o r t o experimentation,  48  hours p r i o r  d u r i n g which time cannulated  to  the  they were not f i s h were h e l d  i n an opaque b l a c k p l e x i g l a s s box d i v i d e d i n t o s i x compartments each  just  sufficiently  s u p p l i e d with which was  large to  flow through  between 8 and  h o l d the  fish.  The  box  d e c h l o r i n a t e d Vancouver tap  12 °C throughout  was  water  the d u r a t i o n of the  experiment.  Exercise Protocol  F i s h were t r a n s f e r r e d by net t o a B r e t t - t y p e swim t u n n e l and allowed t o f a m i l i a r i z e themselves  with the t u n n e l f o r 15 t o  20 minutes with the water f l o w i n g at low speed. Flow was i n c r e a s e d t o the maximum the f i s h c o u l d a t t a i n  then  ( g e n e r a l l y about  130% U U  crit  crlt  ) . Any f i s h which c o u l d not a t t a i n a t l e a s t 120% of the  f o r the stock were d i s c a r d e d . The speed was h e l d constant  u n t i l t h e f i s h c o u l d no longer maintain t h i s v e l o c i t y minutes)  a t which p o i n t  percent.  This  speed  the speed  was h e l d  (about 3  was decreased by about 30  f o r about  5 minutes  and then  g r a d u a l l y i n c r e a s e d and the c y c l e begun again. The flow speed was c o n t i n u a l l y o s c i l l a t e d i n t h i s way as the maximum a t t a i n a b l e speed  gradually  decreased  until  the f i s h  could  no  longer  maintain i t s p o s i t i o n  i n the swim t u n n e l even at t h e slowest  speeds.  the f i s h  At t h i s  grasped  by  the i n v e s t i g a t o r .  unresponsive stiff. and  point  but capable  no longer responded When  exhausted,  of v e n t i l a t i o n  experiments  was  a  fish  the f i s h i s  and i s f l a c c i d , not  In only one case out of over a hundred  other  t o being  driven  f i s h used i n t h i s into  rigor  at  e x h a u s t i o n . The e n t i r e e x e r c i s e procedure took approximately 2530 minutes  t o complete.  F i s h were e i t h e r k i l l e d immediately f o l l o w i n g e x e r c i s e (at exhaustion)  or t r a n s f e r r e d back t o the h o l d i n g box. F i s h were  then randomly a s s i g n e d t o a treatment group and k i l l e d a f t e r 2, 4, 8, or 24 hours of recovery as r e q u i r e d . R e s t i n g f i s h were kept i n the h o l d i n g box f o r at l e a s t 48 hours p r i o r t o sampling.  Sampling  and Muscle  Resting  fish  Dissection  are n o t o r i o u s l y  difficult  t o sample. Any  21 struggling  results  in a  phosphates, p a r t i c u l a r l y  rapid PCr  depletion  (Dobson  of the  high  energy  and Hochachka, 1987) . To  reduce t h i s problem f i s h were a n a e t h e t i z e d p r i o r t o sampling. At  the s p e c i f i e d sampling time 2mL  of b l o o d was  withdrawn  and  p l a c e d on i c e . T h i s volume was r e p l a c e d with a 65mg/mL s o l u t i o n of sodium p e n t o b a r b i t o l  (Somnotol). This dose r a p i d l y k i l l s the  f i s h with minimal s t r u g g l i n g . As soon as v e n t i l a t i o n ceased (30 seconds t o 3 minutes)  the f i s h was  a 1 cm t h i c k steak was the  dorsal  f i n using  1972). The steak was  sliced  removed from the water  from the area immediately behind  a double-bladed hatchet  steak  section under  steak was  nitrogen  kept i n l i q u i d  deproteinized.  Muscle Homoqenization,  A  (Faupel et a l . ,  r a p i d l y freeze-clamped i n l i q u i d  with p r e - c o o l e d aluminium tongs. The nitrogen u n t i l  and  E x t r a c t i o n and N e u t r a l i z a t i o n  of e p a x i a l  liquid  white muscle  nitrogen  and  was  quickly  removed from transferred  the  to  a  p r e c o o l e d mortar. I t i s c r i t i c a l t o m a i n t a i n the t i s s u e sample at low temperature t o a v o i d changes i n the c o n c e n t r a t i o n s of the h i g h energy phosphates.  To t h i s end the mortar was  styrofoam c o o l e r which was  filled  kept i n a  with l i q u i d n i t r o g e n at a l l  times such t h a t the mortar was n e a r l y submerged. The mortar  was  s i m i l a r l y nearly f i l l e d  with l i q u i d n i t r o g e n . In t h i s way  the  sample  in  the  was  submerged  liquid  nitrogen  throughout  22 procedure. The muscle was then c o a r s e l y ground with the mortar and p e s t l e and any p i e c e s of s k i n and bone removed. The n i t r o g e n was muscle was  then r e p l e n i s h e d i f necessary and the  liquid  remaining  ground t o a very f i n e powder.  Approximately pre-weighed perchloric  l g of the powder was  chilled acid  test  (PCA)  tube  then t r a n s f e r r e d t o a  c o n t a i n i n g lmL  which was  quickly  of i c e c o l d  re-weighed,  remaining l i q u i d n i t r o g e n had b o i l e d o f f . Then 3mL  7%  once  any  of c o l d  PCA  was added and the mixture was homogenized u s i n g an U l t r a t u r r a x t i s s u e g r i n d e r . The t i s s u e was ground f o r 15 seconds t h r e e times separated by  one minute t o allow the t i p t o c o o l and  prevent  e x c e s s i v e h e a t i n g of the homogenate. Throughout  homogenization  the  and  tube  was  temperature Two stored  held  remainder  uL  aliquots °C  was  centrifuged  base  reached  for  between  supernatant was  titrated  of  with  later  0  salt  water  i c e at  determination at  and  12,000g 2  a  °C.  of the homogenate were removed  -80  temperature  TRIZMA  slurry  of between -5 and -10  200 at  in a  °C.  A  of  for  9  known  and  glycogen. minutes volume  The at  of  a  the  removed and 500 uL of a s a t u r a t e d s o l u t i o n of  was 10M  added  as  KOH  until  a  buffer. a pH  i n order t o p r e c i p i t a t e  This  mixture  of between 7.0 the  was  and  then  7.2  was  KC10 . T h i s mixture  was  4  again c e n t r i f u g e d i n the c o l d at 12,000g f o r 3 minutes pH of the supernatant checked and a d j u s t e d with 10M KOH  and the or HCl  as necessary. The n e u t r a l i z e d e x t r a c t was s t o r e d at -80 °C u n t i l assayed.  Extraction efficiency energy phosphates was In  the  first  simulated lmg/mL)  validation  by  using  a  above and  percentage  (mean ±  SEM  study  the  tissue  homogenate  solution  of  bovine  aliquot  of  concentrated  c o n t a i n i n g an T h i s mixture  %  high  assessed u s i n g a v a r i e t y of methods.  solution.  6.36  of t h i s method f o r the l a b i l e  was  then  serum  e x t r a c t e d and  albumin PCr  recovery  was  (BSA  standard  neutralized  recovery assessed. Recovery was n=3) . PCr  was  also  as  91.77  assessed  + by  adding a known amount of PCr standard t o a muscle homogenate i n PCA.  Recovery  was  repeated with ATP  100.84 ± 5.73%  (n=3) . T h i s  supplementation.  Recovery was  procedure 103.3  ±  was 4.84%  <n=4) .  Biochemical  Analyses  Chromatography: High performance l i q u i d chromatography measure ATP,  ADP,  AMP  and  IMP.  The  (HPLC) was  procedure  was  used to  carried  out  u s i n g an LKB 2152 HPLC c o n t r o l l e r and 2150 t i t a n i u m pump coupled to  a 2220 r e c o r d i n g i n t e g r a t o r . The s e p a r a t i o n was performed  an Aquapore AX-300 7 um The s e p a r a t i o n was a m o d i f i c a t i o n of  weak anion exchanger  (Brownlee l a b s ) .  s i m i l a r t o t h a t of Harmsen et_ aT. t h a t used  by  Parkhouse  et  (1982) and  a_l. (1987) . To  b r i e f l y o u t l i n e , e l u t i o n was  i s o c r a t i c f o r the f i r s t  of  4  the run u s i n g 60 mM  gradient  from  60mM KH P0 2  KH P0 2  4  (pH  (pH 3.2)  3.2)  f o l l o w e d by  t o 750  mM  on  KH P0 2  4  5 minutes a  linear  (pH  3.5)  24 over for  10  minutes.  12  This  minutes.  c o n c e n t r a t i o n and  The  column  was  flow  r a t e was  ATP  i s favoured  low  [KH P0J  are  2  IMP.  The  the  light  buffer  relevant  concentrations  4  the  required HPLC  (r  were  visualized  exercise  and  in on  grade,  low  expensive. et  which  of  I utilized to  of  of a  the  through  UV  for a l l metabolites  less of  i n the the  salts  are  than 2  UV  the  coefficient 5  KH P0  buffer of  amount  salts thus  solution compounds  available,  a m o d i f i c a t i o n of the  pre-purify analytical  over  the  in  the  60mM range  used  for  provided under a l l variation  percent.  4  range,  of  ultra  monitor.  solution  this  and  difference  throughout  since  AMP  absorbance of  a mixed  and  reequilibration  with  linear  pH  of  compromise  o f n e u t r a l i z e d e x t r a c t was  always  dectection  low  result  by  was  while  separation  flow  c o n d i t i o n s . The  absorbance  a l . (1984)  preparing were  minutes  elution  for a l l metabolites  s c a l e peaks  of  6  Rapid  required for  a l l cases  absorb  pH  increases  preparations  absorbance  sensitivity  the  a BIO-RAD  curves  recovery  Commercial  baseline  which  by  b e t w e e n d u p l i c a t e s was  impurities  the time  = 0.99). 20uL  determination  easily  adequate  constructed  standard 2  high  s o l u t i o n s . D e t e c t i o n was  curves  2  for  (254nm) u s i n g  maintained  r u n . Column t e m p e r a t u r e  and  4  s e l e c t e d were  Standard  KH P0 .  2  f a c t o r s and  b e t w e e n t h e two violet  [KH P0 ]  required  starting  then  2mL/minute t h r o u g h o u t .  high  conditions  between these to  by  was  re-equilibrated for  w i t h s t a r t i n g b u f f e r before the next 55°C a n d  pH  and of but  can  increasing  the  reducing  the  interest. are  technique  reagent  contain  HPLC  extremely of  Reiss  grade KH P0 2  4  to  25 the r e q u i r e d q u a l i t y . A  IM  stock s o l u t i o n  column, BIO RAD an  anion  was  passed  laboratories  exhchange  resin  5cm  (AG1  through  a column  (Econo  diameter X 30cm) packed  X8,  chloride  form),  a  with  cation  exchanger (chelex 100, sodium form) and a c t i v a t e d c h a r c o a l (1460  mesh). Chelex  has  high  selectivity  for divalent  cations,  p a r t i c u l a r l y copper, i r o n and heavy metals. I t i s used t o remove the t r a c e metal contamination present i n the a n a l y t i c a l salt.  The  activated  c h a r c o a l served  contaminants which may of  phosphate  was  to  remove  any  to  use  kept  appropriate technique  the pH  the  stock  and  aromatic  l e a c h from the r e s i n s . The stock s o l u t i o n at  4  °C  and  constantly  recirculated  through the column by means of a p e r s i t a l t i c pump. prior  grade  buffer  vacuum  was  filtered  diluted,  brought  (0.22  b a s e l i n e d i s t u r b a n c e caused  present i n the h i g h c o n c e n t r a t i o n b u f f e r was  Immediately  um). by  the  to  With  the this  impurities  reduced by  74%.  Spectrophotometry: A l l d e t e r m i n a t i o n s were performed on a Perkin-Elmer Lambda 2  UV/visible  spectrophotometer.  Lactate,  pyruvate,  phosphocreatine and glucose were measured u s i n g r o u t i n e NAD/NADH linked  assays,  essentially  as  i n Bergemeyer  (1974) .  Muscle  glycogen was measured by d i g e s t i n g n e u t r a l i z e d homogenate with amyloglucosidase. T o t a l glucose the  free  glucose  pool)  was  then  ( l i b e r a t e d from glycogen p l u s measured  c o n t r i b u t i o n of f r e e glucose s u b t r a c t e d out  as  before  and  the  (Bergmeyer, 1974).  26 Inorganic phosphate  was  determined  technique of Black and Jones  c o l o r i m e t r i c a l l y u s i n g the  (1983).  A l l assays were performed i n d u p l i c a t e . I f the c o e f f i c i e n t of v a r i a t i o n between these d u p l i c a t e s was t h i r d run was observed  performed  g r e a t e r than  6%  a  and the o u t l i e r d i s c a r d e d . Because of  wide v a r i a t i o n  i n the  case  of the phosphate  assay,  t r i p l i c a t e s were r o u t i n e l y performed and i f the c o e f f i c i e n t of v a r i a t i o n between these was g r e a t e r than 6% a f o u r t h was run and the  outlier  discarded.  All  assays  were  validated  with  a p p r o p r i a t e standards.  Intracellular Mean methods:  pH  intracellular the  pH  distribution  d i m e t h y l o x a z o l i d i n e 2,4  dione  technique of P o r t n e r et. a_l.  DMO  (pHJ of (DMO)  was  determined the  weak  using acid  two 5,5  and the t i s s u e homogenate  ( i n press) .  distribution: Use of the weak o r g a n i c a c i d DMO  pHi has been widespread Butler in vivo  as an In v i t r o marker f o r  s i n c e i t s i n t r o d u c t i o n by Waddell  and  (1959) . T h i s technique has, more r e c e n t l y , been employed i n the rainbow  trout  t o estimate pH  ±  in a variety  of  t i s s u e s f o l l o w i n g exhaustive e x e r c i s e ( M i l l i g a n and Wood, 1986) . These authors c o n s i d e r t h i s technique capable of r e f l e c t i n g changes  in  trout  white  muscle  ( M i l l i g a n and Wood, 1985) .  on  the  scale  of  15  pH  ±  minutes  27 Approximately injected  through  12 the  hours  prior  dorsal aortic  to  sampling  cannula  trout  with  10  were  uCi  3  H-  mannitol and 2.5 uCi C-DMO (New England Nuclear) i n C o r t l a n d ' s 14  f i s h p h y s i o l o g i c a l s a l i n e t o a t o t a l volume of 500 of the a r t e r i a l b l o o d sample c o l l e c t e d injection  was  measured  microelectrode  (type  pH  j u s t p r i o r t o somnotol  immediately  E5021)  uL. The  using  maintained  a  at  Radiometer 10°C  via  a  r e c i r c u l a t i n g water bath and l i n k e d t o a Radiometer 2 6 pH meter. White  muscle  levels  of  measured i n lmL of the PCA using  Amersham  ACSII  3  H  and  14  C  radioactivity  e x t r a c t used  aqueous  fluor.  f o r metabolite This  method  were assays  has  the  disadvantage t h a t the PCA e x t r a c t i o n i n c l u d e d an at l e a s t 6 f o l d d i l u t i o n of the sample n e c e s s i t a t i n g counting a l a r g e volume of the  limited  extract.  However, t h i s  method y i e l d s  low  c o l o r l e s s samples which i s i t s main advantage over NCS which  yields  corrected  in  highly two  coloured  ways.  samples.  First,  the  This  samples  digestion  colour can  may  be  introduce  gross  inaccuracies, especially  e s t i m a t i o n of dpms f o r low energy It  acetic  so long as the a p p r o p r i a t e quench curve i s generated  adequately c o r r e c t f o r the c o l o u r . T h i s approach,  i s also  possible to  with  respect to  samples  to  however, can  beta e m i t t e r s l i k e  d e c o l o r i z e the  be  counted  d i r e c t l y a f t e r being n e u t r a l i z e d with 60 uL of g l a c i a l acid,  quench  with  the  tritium. benzoyl  p e r o x i d e as suggested by the manufacturer. However, i n t h i s case h i g h chemical quench i s induced and again estimates of dpm tritiated  samples  can  be  compromised.  To  for  decrease  28 chemiluminescence overnight,  or  acceptable  a l l samples were s t o r e d  until  level,  chemiluminescence  and  then counted on  i n the dark  was an  LKB  reduced 1214  least to  an  rackbeta  l i q u i d s c i n t i l l a t i o n counter using dual l a b e l quench c o r r e c t i o n . T o t a l t i s s u e water was determined by d r y i n g a l - 2 g sample of t i s s u e taken from an area adjacent t o the main sampling s i t e to constant weight i n a d r y i n g oven at 75 °C. Tissue  extracellular  fluid  volume  (ECFV,  mL/g)  was  c a l c u l a t e d a c c o r d i n g t o the equation: ECFV = T i s s u e  [ H] mannitol  (dpm/g)  Plasma  [ H] mannitol  (dpm/g)/ plasma water (mL/g)  3  3  I n t r a c e l l u l a r f l u i d volume was then determined as: ICFV = t o t a l t i s s u e water - ECFV T i s s u e pHi was c a l c u l a t e d according t o the e q u a t i o n : pHi = pK  DM0  + l o g ( [DMO], [DMO]  where  [DMO]  e  and  [DMO],  .  (iO<e -e He  kDMO  >  + 1) -1)  e  represent  extracellular  and  i n t r a c e l l u l a r DMO c o n c e n t r a t i o n r e s p e c t i v e l y and are c a l c u l a t e d as: [DMO] = plasma C DMO/ (dpm/mL) (dpm/mL)  plasma water  [DMO] i - ( t i s s u e  - (ECFV.DMOJ  14  e  pK  DM0  14  C DMO  was taken from Malan et. a l .  / ICFV)  (1976) .  Some c a u t i o n must be e x e r c i s e d with r e s p e c t t o the use of mannitol as an e x t r a c e l l u l a r space marker. Recent s t u d i e s have shown  that  various  mannitol  tissues  i s slowly  yielding  taken up  increasing  and  metabolized  estimates  of  in  ECFV  29 (decreasing pE ) L  over time. However, t h i s e f f e c t has been shown  t o be minor i n t r o u t white muscle over the time course employed i n t h i s study  (Chris Wood, p e r s . comm.)  Measurement of Homogenate pH The e s t i m a t i o n of i n t r a c e l l u l a r pH by the measurement of the pH of a t i s s u e homogenate has been f r e q u e n t l y used f o r human muscle b i o p s i e s  (see f o r example, C o s t i l l et a l . ,  measurement of pH has DMO.  It  a number of advantages  eliminates  respresentative  the  assumption  of the pH  of the  1982) . D i r e c t  over the use  that  blood  pH  is  fluid  and  the  interstitial  dependance on the accurate measurement of t h i s pH. principle,  register  very  rapid  pH  transients  problems of p a r t i a l e q u i l i b r a t i o n which may technique.  However,  i t is difficult  t o be  of  I t can, i n  without  the  complicate the assured  measured pH bears any r e l a t i o n s h i p t o the a c t u a l  that  DMO the  intracellular  pH. Recently P o r t n e r et a_l. (in press) have developed "homogenate  technique"  which  they  have  applied  a  to  new the  measurement of muscle i n t r a c e l l u l a r pH i n , among other animals, rainbow  trout.  approximately  The  method  200mg  sample  is of  very muscle,  simple. ground  In  brief,  under  an  liquid  n i t r o g e n t o a f i n e powder, was added t o an Eppendorf tube which was then q u i c k l y f i l l e d with a s o l u t i o n of 160mM KF and ImM ( n i t r i l o t r i a c e t i c a c i d , disodium s a l t ) . The mixture was briefly  with a needle  and  capped.  The  insoluble  NTA  stirred  fraction  was  30 spun  down  (3-5  temperature). repeated  seconds  in  a  microcentrifuge  Then a l i q u o t s of the supernatant  measurement  of  pH  in  a  capillary  at  were taken f o r pH  electrode  t h e r m o s t a t t e d t o 10°C. V a r i a t i o n i n pH between r e p l i c a t e s the supernatant  room  from  was u s u a l l y below +0.005pH u n i t s and from the  same muscle powder below ±0.02 pH u n i t s .  Calculations  S t a t i s t i c a l Analysis A l l data are r e p o r t e d as means + standard e r r o r o f the mean (SEM). M e t a b o l i t e d i f f e r e n c e s between r e s t and/or the v a r i o u s recovery times were assessed using a one way ANOVA f o l l o w e d by Tukey's HSD on those parameters which showed a s i g n i f i c a n t F r a t i o . In a l l cases B a r t l e t t ' s t e s t f o r homogeneity of v a r i a n c e s was used t o v e r i f y t h a t the assumptions of the above t e s t s were not v i o l a t e d . When the assumptions of homogeneity of v a r i a n c e and n o r m a l i t y were not met simple l o g a r i t h m i c t r a n s f o r m a t i o n of the data proved s u f f i c i e n t t o remove the problem. In a l l cases p=0.05 was taken t o be the l i m i t above t e s t s were performed statistical In way  package  of s i g n i f i c a n c e . A l l of the  on a microcomputer u s i n g the SYSTAT  (SYSTAT i n c . ) .  the case of pH, although the data i s r e p o r t e d i n t h i s  for  statistical  ease  of  comprehension  analyses were performed  does, i n f a c t , make l i t t l e  and  interpretation, a l l  using calculated  [H ]. +  It  d i f f e r e n c e when the pH range being  31  considered i s t h i s  small (see B o u t i l l i e r e t al.., 1980 f o r f u l l  analysis). The  comparison  intracellular distribution  of the two techniques  pH poses technique  particular  problems  f o r estimating because  i s known t o produce pH estimates  g r e a t e r v a r i a n c e than the homogenate technique comm.).  the DMO with  (Y. Tang p e r s .  I f t h i s i s the case with the data from t h i s study then  p a r a m e t r i c techniques  of a n a l y s i s are i n a p p r o p r i a t e . However,  since  a pair  i n most  cases  of data  points  (1 DMO  and 1  homogenate) was taken f o r each f i s h , a number o f q u i t e powerful non p a r a m e t r i c t e s t s are a v a i l a b l e . The s i g n t e s t , signed rank t e s t and Wilcoxon signed rank t e s t were a l l performed t o compare these data. C y t o s o l i c Redox: The lactate  cytosolic  NAD /NADH  dehydrogenase  +  ratio  equilibrium  was c a l c u l a t e d (Williamson  u s i n g the  et .al.,  1967;  Veech et. al.., 1969) a c c o r d i n g t o the f o l l o w i n g e x p r e s s i o n : NADVNADH = ( [pyruvate] x [H ] )/( [ l a c t a t e ] xKeq) +  where  lactate  and pyruvate  concentrations  denote  the t o t a l  measured c o n t e n t s . The e q u i l i b r i u m constant (Keq) was c a l c u l a t e d to be l . l x l O assuming a  - 1 2  M" at 10°C, pH 7.0 a t an i o n i c s t r e n g t h of 0.25, 1  H° of +14 kcal/mol  (Hakala e t .al., 1956) . The use of  t h i s method r e q u i r e s the assumption maintained  t h a t the LDH r e a c t i o n i s  at near e q u i l i b r i u m i n working muscle.  32  I n t r a c e l l u l a r f r e e Magnesium: I n t r a c e l l u l a r f r e e magnesium [Mg ] i s an important 2+  i n the understanding  factor  of r e a c t i o n s i n the c y t o s o l which i n v o l v e  ATP and the other phosphorylated  i n t e r m e d i a t e s . However, the  value of t h i s parameter i s u n c e r t a i n . A v a r i e t y of techniques have been used t o estimate the f r e e l y d i f f u s i b l e p o o l of [Mg ] 2+  including microelectrodes al.,1982) and NMR  (Hess et al.., 1982) , dyes  ( K i r k e l s e t a l . , 1989). Values  f o r mammalian  s k e l e t a l muscle f a l l i n t o two ranges: approximately 5mM  (Connett,  1985). Connett  (Baylor et  ImM amd 3-  (1985) has a p p l i e d a b i n d i n g s i t e  approach t o the e s t i m a t i o n of [Mg ] i n the dog g r a c i l i s muscle. 2+  Skeletal  muscle  contains  a  number  of  proteins  and  other  compounds which are known t o b i n d magnesium and f o r which the magnesium b i n d i n g constants are known. I f the c o n c e n t r a t i o n s of these compounds and the t o t a l magnesium i s known f o r a t i s s u e then the magnesium bound t o the substances  can be c a l c u l a t e d .  Assuming t h a t these are the major c h e l a t o r s of magnesium then the f r e e magnesium can be c a l c u l a t e d by s u b t r a c t i o n . As a r e s u l t of t h i s assumption the [Mg ]  f  a l i k e l y maximum f o r [Mg ]  .  2+  2+  f  c a l c u l a t e d i n t h i s way r e p r e s e n t s  The major p r o t e i n s known t o b i n d magnesium along with t h e i r c o n c e n t r a t i o n s i n s k e l e t a l muscle and t h e i r b i n d i n g are  listed  contributors  i n table  1.  In a d d i t i o n ATP  t o magnesium  binding  and PCr are major  the a p p r o p r i a t e  b i n d i n g constants are l i s t e d i n t a b l e 2.  constants  magnesium  33 B i n d i n g i s estimated as: [Mg ]  = [Mg ] {l+ S [X] .K V (B + K  2+  XM  2+  x  f  XMg  T  [Mg ] 2+  f  where X = the c o n c e n t r a t i o n of the compound o f i n t e r e s t ( p r o t e i n or metabolite) B = 1 for proteins B = 1 + KJJ [ H ] +  subcripts: K  f  f o r phosphorylated  compounds  T = t o t a l concentration f = free concentration  = [MgX]/[Mg ] [X] 2+  Mg  [MgX]  = c o n c e n t r a t i o n of magnesium bound form of X  T o t a l magnesium was c a l c u l a t e d f o r values of [Mg ] between 2+  0 and 20mM. These were then concentration measured  of rainbow  compared t o the t o t a l magnesium  trout  white  as 38.6 + 1.7 mEq/L at r e s t  immediately  muscle  which  has been  and 37.0 ± 0.4 mEq/L  f o l l o w i n g exhaustive e x e r c i s e , or approximately  19mM  (Parkhouse, 1987) . Since t h i s approach y i e l d s only an estimate effects  of  reasonable  c a l c u l a t i o n s were  variations  assessed.  in  [Mg ] 2+  f  of [Mg ] t h e 2+  f  on  the model  34  Table  1 : Magnesium b i n d i n g s i t e s and b i n d i n g constants i n s k e l e t a l muscle a f t e r Connett (1985)  Compound  Concentration i n muscle (mM)  B i n d i n g constant (M") 1  Myosin  0.32  3xl0  5  Troponin  0.32  5xl0  4  Parvalbumin+ Calmodulin  0 .14  lxlO  4  35 Model  Calculations: The  scaled  creatine  kinase  model  (Connett,  1988) was  a p p l i e d t o the data from t h i s study. 1) i o n b i n d i n g : The b i n d i n g intracellular  of ions was handled as with the c a l c u l a t i o n of  [Mg ]  . So the b i n d i n g  2+  of any phosphorylated  [ H ] , [Mg ] , and [ K ] can be  compound with the ions  +  +  2+  described  as: K  = [XY " ] / [X ] [Y ] n  m  m+  n_  where X r e f e r s t o the c a t i o n b i n d i n g f o r example Mg , H , K 2+  +  +  and  Y r e f e r s t o the a n i o n i c phosphate compound (ATP, ADP, PCr e t c . ) . The  total  concentration  of any of these anions can be w r i t t e n  as t h e sum of the f r e e i o n i c and the bound forms: [Y] = [ Y H .B, = 1 + £K£ . x  where  m+  and the s u b s c r i p t i r e f e r s t o the p a r t i c u l a r Y: c =PCr, t =ATP, d =ADP, m =AMP, p =P . ±  The  enzyme e q u i l i b r i a  and the i o n i c b i n d i n g  can then be  combined i n t o two v a r i a b l e s : RCPK ^Adk  2)  =  =  B B  t  T  . •  K ^ J p /( B . B D  .  K Q| . A  K ' |[H ] +  C  A  K  T  A D K  /  . (K  K  A T P  C P K  .  B  2 D  )  C a l c u l a t i o n of f r e e c y t o s o l i c ADP and AMP: The v a r i a b l e s d e f i n e d above can be used i n c o n j u n c t i o n  the  d e f i n i t i o n of the e q u i l i b r i a  to calculate  [AMP] f r e e l y a v a i l a b l e i n t h e c y t o s o l :  with  the [ADP]  and  36 [AMP] = [ADP] [ADP]  2  .R  /  AdK  [ATP]  = [ATP] . [Cr] .R^ /  [PCr]  Since a l a r g e f r a c t i o n o f both ADP and AMP i s known t o be bound to,  f o r example, p r o t e i n i n v i v o i t i s important  and  [AMP]  f  t o know [ADP]  £  i n order t o c a l c u l a t e v a r i a b l e s l i k e energy charge  and t o a c c u r a t e l y assess the energy s t a t u s o f the c e l l .  3) D e f i n i t i o n o f i n t e r m e d i a t e p o o l s : Connett  (1988) d e f i n e s a number o f p o o l s o f i n t e r m e d i a t e s  which serve t o d e s c r i b e t h e phosphate energy subsystem o f t h e cell. to  A l l c o n c e n t r a t i o n terms used i n these d e f i n i t i o n s  the free  cytosolic  pools  involved  refer  i n the equilibrium  r e a c t i o n s as s e t out i n the i n t r o d u c t i o n . ; The  total  creatine pool  [ C r ] = [PCr] + [Cr] i s constant T  for  a t i s s u e over a l l metabolic s t a t e s and i s thus used t o s c a l e  all  other p o o l s t o o b t a i n dimensionless The  [AMP],  total ([Ad] ) T  c y t o s o l . Since to  equations.  f r e e adenine n u c l e o t i d e p o o l ,  [ATP] + [ADP]  i s the r e a c t i v e adenine n u c l e o t i d e p o o l i n t h e [Ad] may or may not be constant i t i s necessary T  d e f i n e the v a r i a b l e s R  AXP  =  [AXP]/[Ad]  T  .  The phosphate bond or p o t e n t i a l energy p o o l : P  e  = [PCr] + 2[ATP] + [ADP]  r e f l e c t s t h e p o o l o f h i g h energy phosphate bonds a v a i l a b l e i n the  tissue.  4) D e f i n i t i o n o f dimensionless Since  +  [ C r ] i s constant T  concentrations: f o r any one t i s s u e  a l l other  37 concentrations  can be s c a l e d t o [ C r ] t o produce a number of T  dimensionless c o n c e n t r a t i o n s : Fc = [ P C r ] / [ C r ] F = [Ad] /[Cr] F = P /[Cr] F = [Pil/[Cr] a  Pe  T  e  T  P1  The  T  T  T  adenylate  energy charge  (Atkinson, 1977) can a l s o be  c a l c u l a t e d u s i n g the f r e e c o n c e n t r a t i o n s of ADP and AMP. EC = 0.5 ((2 [ATP] Connett  + [ADP])/[Ad]  T  )  a l s o d e r i v e s equations t o show t h a t R  can be c a l c u l a t e d i f F , R ^ and R c  AdK  ATP  , F  a  are known. These  , and E.C. equations  were a p p l i e d t o the data from the present study and the r e s u l t s compared t o the R , F , and EC c a l c u l a t e d u s i n g the d e f i n i t i o n s ATP  a  shown above. 5) E q u i l i b r i u m c o n s t a n t s : In order t o p r o p e r l y apply Connett's model t o the data from the  present  study,  values  a p p r o p r i a t e ' t o in v i v o certain  c o n d i t i o n s must  amount o f v a r i a t i o n  constants  of the e q u i l i b r i u m  i n the values  reported i n the l i t e r a t u r e .  s e l e c t e d by Connett  be used.  s i n c e these  values  There  i sa  of the e q u i l i b r i u m  I have used the values were determined  c o n d i t i o n s i n which a l l b i n d i n g was accounted were c o r r e c t e d t o c o n d i t i o n s o f i o n i c  constants  under  f o r . The values  strength equivalent to  t h a t i n v i v o (u=0.17-0.2) and wherever p o s s i b l e t o a temperature of 10°C u s i n g the Van't Hoff In The  (Kz/KJ  equation:  = (- /JH/RT) ( (1/T )-(1/'T ) 2  1  (T i n °K)  e q u i l i b r i u m constants used are l i s t e d i n t a b l e 2.  TableZ  E q u i l i b r i u m Constants used. A l l v a l u e s are c o r r e c t e d t o i o n i c s t r e n g t h u = 0.17-0.2 and 10°C except where noted. constant 2 6.69  K  M A  T P  AH  mM"  1  1.13xl0  3.3  M"  7  (Kcal/mole)  1  0 .087  K&„  550.69 M"  1.5  K *„  5.84 M"  6.0  1534 M"  3.3  A  1  1  1  K * A  KJ^ K*  6.10xl0  P  M"  6  83.99 M"  1. 0  2.92 M"  6..0  1  H  1  P  K &  2 0 M"  K "  3.31x10* M"  P  0.522  1  1  *  1  C r  O  4 6.05 M'  5.59  1  Kp ,  8 . 91x10  4  6  M"  1  1.7 6 M"  6 .14  1  K$ K*,  36.66 M"  3.24  0.99 M"  6.0  J  P  1  P  K  C P K  K  A d K  * at 25°C ** at 38°C  -11.59  4 . 86xl0 8 .1  9  M"  1  -2.4  39  RESULTS  ;  General The homogenate technique of Portner et a l . ( i n press) gave estimates  of  intracellular  distinguishable distribution associated estimates  from  the  technique  with were  the  pH  which  were  estimates  (see t a b l e  not  statistically  yielded  by  3) . However, the  e x e r c i s e group means taken  statistically  the  different  from  variation  from the  DMO  the  DMO  variation  w i t h i n groups d e r i v e d from homogenate measurements. Since the homogenate technique  appeared  to y i e l d  greater p r e c i s i o n a l l  subsequent c a l c u l a t i o n s with and d i s c u s s i o n s of  intracellular  pH w i l l be based on estimates of i n t r a c e l l u l a r pH d e r i v e d from measurements of the pH i n the homogenates.  Adenylates Changes i n the c o n c e n t r a t i o n s of the adenylates and IMP are o u t l i n e d i n t a b l e 4. Immediately f o l l o w i n g exhaustive e x e r c i s e [ATP]  fell  t o 24%  of the  r e s t i n g value while  n e a r l y 1 0 - f o l d . N e i t h e r [AMP] Since TAN  also  accounted  for  d i d not by  the  nor  [ADP]  in  increased  changed s i g n i f i c a n t l y .  change, the decrease increase  [IMP]  [IMP]  in  [ATP]  and  no  can  be  adenine  n u c l e o t i d e s were l o s t from the c e l l as a r e s u l t of the e x e r c i s e protocol. During the f i r s t two hours of recovery t h e r e was  a nearly  40  Table 3 : A r t e r i a l pH and i n t r a c e l l u l a r pH estimated by the DMO and homogenate techniques p r i o r t o and d u r i n g recovery f o l l o w i n g exhaustive e x e r c i s e . pH : a r t e r i a l pH; pH^: i n t r a c e l l u l a r pH (mean ± SEM). A  PH  A  pHi (Homog)  p ^ (DMO)  rest  7.77±.01  7.27+.03  7 .14±.01  exh  7.20±.07**  6.66+.03"  6.78±.ll  2h  7.36±.07*  6.57+.03**  6.52±.13  4h  7.31±.07*  6.57±.02**  6.49±.24  8h  7.63+.17  6.89+.11**  7.01+.19  24h  7.77±.05  7.21+.02  7.22+.04  n = 5 rest;  7 exh.; 5 2h; 5 4h; 4 8h; 6 24h.  significantly different value at p<0.001 significantly different value at p<0.005  from  corresponding  resting  from  corresponding  resting  41  Table 4 :  A d e n y l a t e c o n c e n t r a t i o n s i n w h i t e m u s c l e p r i o r t o and d u r i n g r e c o v e r y from e x h a u s t i v e e x e r c i s e i n rainbow t r o u t , ( u m o l e s / g wet w e i g h t ; Dean 1 SEM). TAN - t o t a l a d e n i n e n u c l e o t i d e s - [ATPJ+[ADP]+[AMP]+[IMP) [ATP]  [ADP)  [AMP ]  [IMP]  rest  7.5210.49  0.76810.069  0.07010.013  0.5410.19  8.9010. 2  exhaustion  1.8210.19"  0.65410.065  0.062+0.016  5.2810.47"  7 . 8 2 1 0 . 60  2h  3.7210.56"  0.61410.068  0.05710.016  2.98*0.51"  7 . 3 7 i 0 . 52  4h  3.5710.52"  0.602*0.045  0.04910.021  2.7710.45"  6 . 7 8 i 0 . 26  8h  3.4510.55"  0.49210.022  0.06510.015  2.77+0.45"  6 . 7 8 1 0 . 26  24h  7.0210.62  0.70910.049  0.05310.011  0.2810.07  8 . O 6 1 O . 63  significantly n -  5 at r e s t ,  different  from c o r r e s p o n d i n g r e s t i n g v a l u e  2h, 4 h ; 4 at 8 h ; 7 at e x h . ; 6 at 24h  [TAN]  at  p<0.001  two-fold  i n c r e a s e i n [ATP] f o l l o w e d by a long p l a t e a u phase  d u r i n g which there was l i t t l e change. However, even at 8h there was no s t a t i s t i c a l l y exhaustion.  By  significant  24h  [ATP]  had  d i f f e r e n c e from the [ATP] at fully  recovered.  recovery the changes i n [ATP] were d i r e c t l y  Throughout  r e f l e c t e d by the  changes i n [IMP]. No s i g n i f i c a n t changes were observed i n [ADP] or  [AMP]  and thus  [TAN] was  conserved  at a l l p o i n t s during  recovery..  Phosphocreatine Changes i n the c o n c e n t r a t i o n s of the c r e a t i n e c o n t a i n i n g compounds and i n o r g a n i c phosphates are o u t l i n e d i n t a b l e 5. At exhaustion value, while  [PCr] decreased  to less  than  2,0% o f the  resting  [Cr] i n c r e a s e d more than 2 f o l d . As was the case  with the adenylates, t o t a l c r e a t i n e remained unchanged, with the decrease  i n [PCr] being accounted  f o r by the i n c r e a s e i n [ C r ] .  The c o n c e n t r a t i o n of i n o r g a n i c phosphate i n c r e a s e d i n p a r a l l e l with  [Cr] as a r e s u l t of the h y d r o l y s i s of PCr. In c o n t r a s t t o [ATP],  exercise  [PCr] i s f u l l y recovered by 2h post  (see t a b l e s 4 and 5) and does not change s i g n i f i c a n t l y  t h e r e a f t e r . As before  [Cr] and [P±] r e f l e c t e d these changes.  A r t e r i a l and I n t r a c e l l u l a r pH Changes i n pH  A  and pR  L  are shown i n t a b l e 3. A r t e r i a l pH  dropped by n e a r l y 0.6 of a pH u n i t and i n t r a c e l l u l a r pH by very s l i g h t l y more at exhaustion. A r t e r i a l pH showed a gradual  43  Table  5  :  C o n c e n t r a t i o n s o f phosphocreatine, c r e a t i n e and inorganic phosphate i n white muscle prior t o and during the recovery from exhaustive exercise in rainbow t r o u t ( u m o l e s / g wet w e i g h t ; mean + S E M ) . total  Cr  [PCr]  [Cr]  rest  22.62±2.69  19.,36±2 .30  2 8 . 5 8 ± 4 . 96  41.98+2.21  (5)  exh  4.26±1.14"  40..7 6+0 .68**  50.12+2. 66*  45.03+1.23  (7)  2h  25.37+3.26  19..82+2 .10  30.28+3. 82  45.19+2.22  4h  20.44+3.49  21.,04+0 .96  28.83+3. 61  41.47+2.69  (5)  8h  21.97+3.76  20.,80±2 .76  2 8 . 4 0 ± 3 . 38  39.87±0.62  (4)  24h  26.23+1.01  15..74±2 .13  1 9 . 8 2 ± 2 . 95  41.97+1.26  (6)  significantly a t p<0.005 significantly a t p<0.001  (n)  different  from corresponding  resting  value  different  from corresponding  resting  value  (5)  44 recovery over the 24h time not  significantly  I n t r a c e l l u l a r pH, 4h  post  course and by  different  from  8h post e x e r c i s e the  resting  was  value.  on the other hand, remained low f o r at l e a s t  exercise  (in f a c t  showing  a  slight non-significant  d e c l i n e over the f i r s t two hours) . However, by 8h post e x e r c i s e pH  A  had i n c r e a s e d s i g n i f i c a n t l y over t h a t of the exhausted s t a t e  and by 24h post e x e r c i s e was  not  significantly  different  from  the r e s t i n g v a l u e .  Carbohydrate  Metabolism  Changes glycogen  and  [Lactate] increase  in  the  glucose  concentrations i n white  i n c r e a s e d more than c o u l d be  accounted  of  muscle  lactate,  are  shown  pyruvate,  in table  1 0 - f o l d at exhaustion f o r by  the  decrease  in  and  this  glycogen  content, although the l a r g e v a r i a t i o n s i n r e s t i n g glycogen the f a c t t h a t the sampling very  rigorous  was  a n a l y s i s of  the  6.  and  t e r m i n a l and not s e r i a l makes a conservation  of  carbon  units  w i t h i n white muscle i m p o s s i b l e . Recovery of [ l a c t a t e ] to r e s t i n g l e v e l s was  r e l a t i v e l y slow (approximately 2 umoles/g/h) and not  s t a t i s t i c a l l y s i g n i f i c a n t u n t i l 8 hours when i t had d e c l i n e d by 50%. By 24h  [ l a c t a t e ] had r e t u r n e d to r e s t i n g l e v e l s . There  was  no a p p r e c i a b l e gylcogen replenishment d u r i n g the f i r s t two hours of  recovery,  significantly  but  4h  different  exhaustion. At 24h different  by  [glycogen]  from e i t h e r  [glycogen]  from the value  was  was  not  [glycogen]  statistically  at exhaustion.  statistically at r e s t  or at  significantly  Because of the l a r g e  variation  in  [glycogen]  from f i s h t o f i s h  p r e c i s e l y d e s c r i b e the time course Total  tissue  glucose  in  to  of i t s recovery.  increased  direct  i t is difficult  contrast  nearly  exhaustion  and,  to  [glycogen],  remained e l e v a t e d throughout the  five  both  fold  at  [lactate]  and  recovery  period.  Pyruvate c o n c e n t r a t i o n i n c r e a s e d at exhaustion to more than twice the r e s t i n g value and remained e l e v a t e d over the f i r s t hours  of  recovery  after  r e c o v e r i n g e n t i r e l y by  Lactate/pyruvate  which  point  i t rapidly  two  decreased,  8h post e x e r c i s e .  r a t i o s and  redox  The l a c t a t e / p y r u v a t e r a t i o i n c r e a s e d at exhaustion and over 8h  of  recovery  significantly by  24h  i t was  such  different fully  that  by  4h  post  exercise  from the r e s t i n g value  recovered.  c a l c u l a t e d NADVNADH r a t i o  The  it  (table 7 ) .  v a r i a t i o n between f i s h  (redox) was  so l a r g e t h a t there  no s i g n i f i c a n t change from the r e s t i n g values at exhaustion during  But in was or  recovery.  Plasma L a c t a t e and  Glucose  Plasma [ l a c t a t e ] i n c r e a s e d by n e a r l y 2 0 - f o l d at (table 8) and 24h  was  post  exhaustion  remained e l e v a t e d through 8h of recovery  e x e r c i s e i t was  not  significantly different  but  by  from the  r e s t i n g v a l u e . Plasma [glucose] i n c r e a s e d s i g n f i c a n t l y l a t e i n recovery and was  s t i l l e l e v a t e d at 24h post e x e r c i s e (table 8).  46  Table 6 :  Concentration of lactate, pyruvate, glycogen and glucose in rainbow trout white muscle prior to and during the recovery from exhaustive exercise, (mean 1 SEM) .  (n)  [Lactate]  [pyruvate]  [glycogen]  [glucose]  rest  (5)  3.92±0.94  0.11310.060  33.9013.63  0.5410.22  exh.  (7)  41.7212.63"  0.28910.044" 10.4815.47*  2h  (5)  36.65±4.62"  0.30510.062' 9.4313.52*  2.2610.34  4h  (5)  34.91±1.94"  0.16610.047  15.9014.79  2.3910.18  8h  (4)  20.6512.81"  0.06810.020  19.0613.84  2.9610.36  24h  (6)  4.85+1.29  0.06910.022  25.8114.08  3.9810.95  UNITS:  * **  2.48+0.48  umoles/g wet weight, except glycogen: umoles glucosyl units/g wet weight.  significantly different from corresponding resting value at p<0.05 significantly different from corresponding resting value at p<0.001  Table 7  L a c t a t e / p y r u v a t e r a t i o and c y t o s o l i c redox p r i o r t o and d u r i n g the recovery from e x h a u s t i v e e x e r c i s e i n rainbow t r o u t white muscle (mean ± SEM).  (n)  [lactate]/ [pyruvate]  redox  (6)  55.06±15.72  1361.901435.55  exh. (7)  159.55±19.53  1404.411144.06  2h  (5)  227.421135.84  1772.591474.99  4h  (5)  360.76±161.83*  1171.581332.60  8h  (4)  359.06±86.55*  336.29134.97  24h  (5)  104.75144.56  845.461173.91  rest  * significantly different at p<0.05 redox = NADVNADH  from corresponding r e s t i n g  ([pyruvate] / [ l a c t a t e ] ) x ([H ]/Keq) +  value  Table 8 : Plasma l a c t a t e and glucose c o n c e n t r a t i o n s (mM) d u r i n g recovery from exhaustive e x e r c i s e i n rainbow t r o u t (0. mvkiss) . Mean ± SEM. time  glucose  lactate  n  rest  1 0 . 4 9 ± 2 .22  0.69±0.27  5  exh.  14 . 0 3 ± 0 .69  13.05±2.64  7  2h  1 3 . 8 5 ± 1 .43  12.04±1.43  5  4h  18.2914 .11  17.12±2.85  5  8h  1 9 . 4 8 ± 5 .81  12.22±8.58  4  24h  1 9 . 4 7 ± 2 .21  1.11±0.38  6  49  T i s s u e Water Changes i n t i s s u e water and f l u i d d i s t r i b u t i o n are shown i n t a b l e 9. No changes i n any of these parameters  relative to  the r e s t i n g s t a t e were observed at any p o i n t d u r i n g r e c o v e r y .  I n t r a c e l l u l a r Free Magnesium T o t a l magnesium at a v a r i e y of [Mg ]  from 0 t o 20 mM i s  2+  f  shown i n f i g u r e s 5 and 6, c a l c u l a t e d with the a p p r o p r i a t e [ATP], PCr, and pH f o r r e s t and exhaustion. These two p o i n t s would be expected t o represent the extremes of [Mg ] 2+  f  s i n c e ATP i s the  major magnesium c h e l a t o r and the g r e a t e s t change i n [ATP] occurs between r e s t and exhaustion. From t h i s f i g u r e i t i s c l e a r t h a t at  rest  [Mg ] 2+  i s approximately  lOmM while  at exhaustion i t  c o u l d be as high as 15mM. Therefore a l l model c a l c u l a t i o n s w i l l be performed with [Mg ] =10mM and a v a r i e t y of [Mg ] 2+  2+  f  20mM w i l l  be c a l c u l a t e d  t o assess  f  the s e n s i t i v i t y  from 1 t o of these  c a l c u l a t i o n s t o changes i n f r e e magnesium.  Model C a l c u l a t i o n s In  general the e f f e c t s of changing  e f f e c t s o f changing  [Mg ] 2+  f  on Energy  [Mg ] were s m a l l . The 2+  charge  f r e e AMP and f r e e  ADP are shown i n t a b l e s 11, 12 and 13. A l l other parameters  were  a f f e c t e d by l e s s than 1% by changes i n [Mg ] over the range 5 2+  f  20 mM  (data not shown). Changes i n t h e c a l c u l a t e d parameters  with e x e r c i s e and  Table 9:  T o t a l t i s s u e water content and f l u i d d i s t r i b u t i o n i n rainbow t r o u t white muscle p r i o r t o and during recovery from exhaustive exercise. mL/kg wet weight (mean ± SEM). Total tissue water  ECFV  ICFV  rest  0.767±.007  0.046±.008  0.720±.007  exhaustion  0.768±.006  0.034±.002  0.7281.004  2h  0.763±.014  0.046±.008  0.722±.014  4h  0.7771.005  0.0511.004  0.730±.010  8h  0.7841.005  0.0751.016  0.7001.012  24h  0.7791.006  0.0771.015  0.7031.012  no s i g n i f i c a n t d i f f e r e n c e s  were d e t e c t e d .  51  f i g u r e 5: C a l c u l a t e d t o t a l magnesium c o n c e n t r a t i o n at r e s t and at exhaustion f o r a v a r i e t y f r e e magnesium c o n c e n t r a t i o n s from 0 t o 30 mM based on an estimate of magnesium b i n d i n g . T o t a l t i s s u e magnesium f o r rainbow t r o u t white muscle from Parkhouse et al., 1987.  52  free m a g n e s i u m  (mM)  53 recovery are shown i n t a b l e s 10 - 13. As was [ATP] , F  declined  a  to  about  20%  of  the  the case  resting  with  value  at  exhaustion and s l o w l y recovered so t h a t by 24h post e x e r c i s e F was  not  significantly  contrast, R  decreased  ATP  post e x e r c i s e was As  expected,  declining  different  only s l i g h t l y  in F  resting  value.  In  at exhaustion  and by  2h  paralleled  c  approximately  16%  exhaustion. By 2h post e x e r c i s e F Potential  the  e l e v a t e d over the r e s t i n g value  changes  to  from  energy  (F  Pe  of  the the  (table  changes  in  resting  value  had f u l l y  c  a  10).  [PCr], at  recovered.  ) d e c l i n e d to approximately  20%  of  the r e s t i n g value at exhaustion but by 2h post e x e r c i s e i t had recovered to n e a r l y 80% recovery was with  F. a  of the value at r e s t  (table 10). This  f o l l o w e d by a long p l a t e a u phase, as was  By  24h  post  exercise F  was  Pe  not  different  the  case  from  the  r e s t i n g value. [ADP]  d i d not change s i g n i f i c a n t l y with e x e r c i s e , but  f  2h of recovery i t had d e c l i n e d by an order of magnitude 11).  [ADP]  recovery  gradually  f  period  significantly calculated  so  increased  that  different  without  by from  reference  magnitude to f r e e ADP  over  24h  post  the to  calculated  the  remainder  exercise  resting  ion binding assuming  was  [Mg ] 2+  f  (table of  i t was  value.  by  the not  Free  ADP  similar  in  = lOmM.  The  d i s c r e p a n c y between the values was g r e a t e s t d u r i n g the recovery period. [AMP] i n c r e a s e d s i g n i f i c a n t l y at exhaustion but by 2h post f  exercise  was  almost  two  orders  of  magnitude  less  than  the  54 r e s t i n g value  (table 12).  As was  the case with  [ADP] ,  [AMP]  f  f  g r a d u a l l y i n c r e a s e d -over the course of the recovery p e r i o d but by  24h  post e x e r c i s e was  value.  Since  intracellular  it  is  [Mg ] 2+  f  still  l e s s than h a l f  difficult the  actual  to  accurately changes  in  of the  resting  estimate free  AMP  the are  difficult  t o assess s i n c e [AMP] i s g r e a t l y a f f e c t e d by changes  in  (table 12) .  [Mg ] 2+  f  f  Energy charge  (EC) estimated u s i n g the model c a l c u l a t i o n s  decreased  slightly  recovered  by  2h  but  post  significantly exercise.  In  at  exhaustion  contrast,  EC  but  calculated  without t a k i n g i n t o account t h a t l a r g e f r a c t i o n s of the ADP AMP  p o o l are bound i n v i v o decreased s i g n i f i c a n t l y  had  and  at exhaustion  and remained depressed u n t i l 24h post e x e r c i s e (see t a b l e  13).  55  Table  10  :  Magnesium bound ATP, bound ATP as a f r a c t i o n o f t h e t o t a l c r e a t i n e (Fa) and as a f r a c t i o n o f t h e t o t a l a d e n y l a t e s (Ratp), c r e a t i n e charge (Fc) and n o r m a l i z e d p o t e n t i a l energy p o o l c a l c u l a t e d a c c o r d i n g t o C o n n e t t (1988) at [Mg *] =0.10mM. (umoles/g wet weight, mean ± SEM) 2  time  MgATP  rest  7.50±.49  exh  Ratp  Fc  .1984±.0145  .9972 + .0006  .4926±.0525  .8893±.0562  1.81±.19  .0418±.0048  .9872±.0039  .0776±.0194  .1610±.0181  2h  3.71 + .55  .0907+.0124  .9995±.0001  .5114±.0547  .6929+.0606  4h  3.55±.52  .0949±.0140  .9992±.0003  .4324±.0630  .6222±.0597  8h  3.42±.56  .0980+.0179  . 9989+. 0003  24h  7.00±.61  .1855±.0128  .99841.0003  n = 5 rest;  Fa  7 exh.; 5 2h; 5 4h; 4 8h; 6 24h.  .4966±.0950 .5881+.0452  Fpe  . 6927±.108O .9591±.0340  56  T a b l e 1 1 : F r e e ADP c o n c e n t r a t i o n c a l c u l a t e d w i t h o u t r e f e r e n c e t o i o n b i n d i n g o r u s i n g the model (Connett 1988) e q u a t i o n s and s e v e r a l d i f f e r e n t concentrations o f free magnesium. Mean t S E M . time  T r e e ADP c a l c u l a t e d without reference to ion binding Keq - 4 . 8 x 1 0 '  Free  ADP c a l c u l a t e d a c c o r d i n g t o C o n n e t t f r e e Magnesium (mM) 5  (1988)  10  20  rest  0.02681.0054  0.01491.0031  0.02091.0043  0.02061.0038  0.01831.0037  exh.  0.02651.0078  0.01171.0034  0.01871.0055  0.01901.0056  0.01731.0051  2h  0.00281.0008  O.OOllt.0003  0.00181.0004  0.00191.0005  0.00171.0004  4h  0.003B1.0015  0.00161.0006  0.00261.0010  0.00271.0011  0.00251.0010  0.00421.0019  0.00381.0017  0.01191.0026  0.01061.0025  8h  0.00601.0024  0.00291.0003  0.00421.0019  24b  0.01551.0034  0.00851.0019  0.01201.0026  n at  rest-5;  exh.-7;  2h-5; 4h-5; Bh-4; 24h-6.  57  T a b l e 12 :  F r e e AMP concentration calculated at d i f f e r i n g c o n c e n t r a t i o n s , (umoles/g wet weight; mean ± SEM)  time  lOmM  f r e e [AMP] magnesium c o n c e n t r a t i o n 15mM  rest  0.0016±.0005  0.0016±.O005  exh.  0.00571.0024  0.0058±.0024  2h  0.00003+.00001  0.00002±. 00001  4h  0.00007±.00005  0.00007+.00005  24h  0.00055±.0002  0.00055±.0005  n = 5 rest;  7 exh.; 5 2h; 5 4h; 4 8h; 6 24h.  free  magnesium  20mM  0.0033±.0014 '.  0.00004±. 00003 0.00008±.00006  58  T a b l e 13 :  Energy charge c a l c u l a t e d u s i n g t h e t o t a l c o n c e n t r a t i o n s o f ADP and AMP and a c c o r d i n g t o t h e model d e f i n i t i o n s (Connett, 1988) at [Mg *]free = lOmM and 15mM. (mean ± SEM) 2  time  Energy Charge ( t o t a l ADP and AMP)  Energy Charge Magnesium C o n c e n t r a t i o n lOmM 15mM  rest  0.9455+.0044  0.9998±.0001  0.9998±.0001  exh.  0.8434±.0116  0. 9969±.001'4  0.9969+.0014  2h  0.9124±.0124  0.9999±.0000  0.9999±.0000  4h  0.9138±.O080  0.9999±.0000  l.OOOOt.OOOO  8h  0.9190±.0083  0.9999±.0000  0.9999±.0000  24h  0.9474±.0016  0.9999±.0000  0.99991.0000  n = rest  5; exh. 7; 2h 5; 4h 5; 8h 4; 24h 6.  59  DISCUSSION  The R e s t i n g F i s h The resting  difficulty fish  literature  i s shown by  values  particular,  i n o b t a i n i n g r e p r e s e n t a t i v e samples  as  phosphocreatine  70%  Similarly,  after ATP  large variation  for a variety  Dobson and Hochachka much  the  as  was  i n the r e p o r t e d  of m e t a b o l i t e s . ATP  few  as  3 tail  shown  to  decrease  flaps by  during  [ATP],  in  labile.  c o u l d decrease  as  sampling.  nearly  sampling p r o t o c o l used i n the present experiment these problems so t h a t  and,  are w e l l known to be very  (1987) showed t h a t PCr  from  30%  .  The  l a r g e l y avoided  [PCr] and glycogen were as high  or h i g h e r than p r e v i o u s r e p o r t s . Literature umoles/g t i s s u e  values  for  resting  [PCr]  range  (Mommsen and Hochachcka, 1988)  from  13.05  t o approximately  25.6 umoles/g t i s s u e ( M i l l i g a n and Wood, 1986) . However, c a u t i o n must  be  e x e r c i s e d i n comparing  c r e a t i n e c o n c e n t r a t i o n may body s i z e .  [PCr] i t s e l f may  of  normalized  the  [PCr]/[PCr]+[Cr], al.  these  results  not be e a s i l y compared, but the use  parameter  the  creatine  charge,  avoids t h i s problem. For example, Dobson et  (1987) r e p o r t a r e s t i n g  [ C r ] , at 31.89  total  vary between stocks of t r o u t or with  [PCr]  of 27.03 umoles/g  which i s h i g h e r than t h a t measured i n t h i s study but  because  umoles/g,  was  tissue,  (see t a b l e I I ) ,  a l s o e l e v a t e d . So c r e a t i n e  60 charge  was  0.459 versus  0.539 i n the  present  study.  (1988) r e p o r t s c r e a t i n e charges ranging from 0.35  Connett  t o 0.84  in a  v a r i e t y of s k e l e t a l muscles of a number of mammals from r a t s t o humans. U n f o r t u n a t e l y very few s t u d i e s of e x e r c i s e i n f i s h have reported  [Cr] so i n many cases t h i s comparison cannot be made.  Even i n the present study i t seems l i k e l y t h a t a c e r t a i n amount of  PCr  h y d r o l y s i s occurred  during  freezing.  However,  i t is  impossible t o estimate the extent of t h i s h y d r o l y s i s or t o "back c a l c u l a t e " t o the t r u e r e s t i n g  animal.  Inorganic phosphate c o n c e n t r a t i o n i s p a r t i c u l a r l y h i g h i n the r e s t i n g t r o u t r e l a t i v e t o measurements made f o r muscles of other animals however, i t i s very s i m i l a r t o p r e v i o u s estimates of  [P ]  in  A  resting  fish  ( f o r example,  25.72+1.93  umoles/g  t i s s u e , Dobson et a l . (1987)) . T h i s method has been v a l i d a t e d for  trout  white  muscle by Dobson  u s i n g P NMR and i t appears ±  p r e s e n t . The  (1986)  t o y i e l d accurate estimates of the  31  P  (1987) and Parkhouse  high P, observed  is likely  an a r t i f a c t  of the  sampling or e x t r a c t i o n process because in. v i v o P NMR of white 31  muscle i n both the r e s t i n g carp and g o l d f i s h has shown P almost  undetectable  (Van  den  Thillart  suggests t h a t the t r u e r e s t i n g P tissue.  The  question  then  ±  et  ±  to be  a l . , 1989).  This  i s somewhere around 1 umole/g  i s : what  is  the  source  of  the  is  the  phosphate measured i n the t i s s u e e x t r a c t ? The  first  and  most  likely  source  of  phosphate  h y d r o l y s i s of PCr. However, the measured r e s t i n g 1.5  times  the  resting  [Cr]  (see  table  5).  [PJ i s n e a r l y So,  while  PCr  61 hydrolysis  during  freezing  may  be  a  major  source  phosphate t h i s cannot be the only phosphate r e l e a s e Hydrolysis  of ATP  i s probably not  was  in  fact  the  the  occurring.  a major c o n t r i b u t o r to  h i g h r e s t i n g phosphate s i n c e even i f a l l of the ADP, measured  of  result  of  ATP  AMP  the  and  hydrolysis  IMP  during  sampling the most phosphate t h a t c o u l d p o s s i b l y be accounted f o r i s roughly  2 umoles/g t i s s u e . One  l a r g e p o s s i b l e source of f r e e  phosphate i s c e l l u l a r p r o t e i n s . In c o n t r a s t to phosphate r e l e a s e by  ATP  or  PCr  likely  be  a  hydrolysis,  result  sampling time. The  of  this  the  release  extraction  from  proteins  protocol  would  rather  than  p e r c h l o r i c a c i d e x t r a c t i o n method used  to  p r e c i p i t a t e the p r o t e i n s i n the sample may be s u f f i c i e n t l y harsh t o s t r i p phosphate groups from a wide v a r i e t y of p r o t e i n s .  Since  many c e l l u l a r p r o t e i n s are known to be phosphorlyated t h i s i s a p o t e n t i a l l y l a r g e source of f r e e phosphate. Even though measured [Pi]  [PJ  i s l i k e l y to be  f a l s e l y elevated  from r e s t with e x e r c i s e and recovery  s i n c e a l l the As was resting umoles/g tissue  reported  tissue  changes i n  should s t i l l be  samples were handled i n the same  the case with  [ATP]  the  valid  way.  [PCr], there are l a r g e v a r i a t i o n s i n  i n the  ( M i l l i g a n and  literature Wood,  ranging  1986)  to  from about 4 7.33  umoles/g  (Dobson e_t a_l., 1987) . Again, these v a r i a t i o n s may  attributable nucleotides  to  either  the  variations  in  the  total  be  adenine  (TAN= ATP+ADP+AMP+IMP) or to d i f f e r e n c e s i n sampling  technique. Since TAN  i s seldom measured d i s t i n g u i s h i n g between  these a l t e r n a t i v e s i s o f t e n i m p o s s i b l e .  TAN  seems to be c l o s e l y  62 r e g u l a t e d w i t h i n a stock of f i s h s i n c e , i n the present study the c o e f f i c i e n t o f v a r i a t i o n f o r r e s t i n g TAN was l e s s than 7%. But i n t e r stock v a r i a t i o n s  can be l a r g e .  F o r example, i n a s i n g l e  study by Dobson et al_. (1987) one stock of f i s h had a r e s t i n g TAN  =4.99 umoles/g t i s s u e while another had TAN =8.44 umole/g  t i s s u e . T h i s d i f f e r e n c e may be due t o s i z e s i n c e the f i r s t of f i s h were small,  60-70g, and the second  250g. However, t h i s i s not a c o n s i s t e n t Hochachka a  stock  (1988) report of small  underline creatine be  a resting  fish,  30-55g.  the importance and TAN s i n c e  stock  much l a r g e r , 200-  t r e n d s i n c e Mommsen and  TAN=8.09 umole/g t i s s u e f o r These  differences  of measuring  factors  serve t o like  total  c o n c e n t r a t i o n s of ATP or PCr alone may  misleading. Reported r e s t i n g [ l a c t a t e ] i s p a r t i c u l a r l y v a r i a b l e ranging  as h i g h as approximately 12 umoles/g t i s s u e  ( M i l l i g a n and Wood,  1986) . Pearson et. al.. (1990) have r e p o r t e d the lowest [lactate] orally  t o date, approximately 1.5 umole/g t i s s u e , by u s i n g  administered  previously  resting  observed  increase  [lactate]  possible  that  diazepam. to cause  i n rat liver  the observed  Sodium a  pentobarbitol  glycolytic  has been  activation  and  (Seitz et. al.., 1973). So i t i s  resting  [lactate],  3.92 umoles/g  t i s s u e i s s l i g h t l y e l e v a t e d over the t r u e r e s t i n g v a l u e . The as  large variations  8.41 umoles/g t i s s u e  a r e s u l t of d i f f e r e n c e s protocol.  i n r e p o r t e d r e s t i n g glycogen, as low (Mommsen and Hochachka, 1988), may be  i n f e e d i n g h i s t o r y r a t h e r than sampling  In p r e l i m i n a r y  experiments  resting  white  muscle  [glycogen] was measured on a d i f f e r e n t batch of the same stock of  fish  used  i n this  study,  but which had not been f e e d i n g  p r o p e r l y f o r s e v e r a l months. The mean r e s t i n g glycogen f o r these fish  was  15.06 +1.45  umole  glucosyl  units"/g  tissue  (n=3) ,  s i g n i f i c a n t l y lower (p=0.05) than the r e s t i n g glycogen from f i s h of the same stock which had been f e e d i n g (see t a b l e 6). The f i s h used i n t h i s experiment r e p o r t e d f o r rainbow It  is  concentrations  have the h i g h e s t r e s t i n g glycogen yet  trout.  difficult  to  are t r u l y  say  what  set  of  metabolite  r e p r e s e n t a t i v e of a " r e s t i n g "  trout  s i n c e d i f f e r e n t stocks may i n f a c t show c o n s i d e r a b l e v a r i a t i o n . However, i t i s c l e a r t h a t the f i s h i n the present study are as c l o s e , or c l o s e r , t o a " r e s t i n g " s t a t e than any t h a t have been studied previously. goldfish  However,  suggest t h a t  31  P NMR  studies  some changes,  of the carp and  particularly  i n [PCr], P  x  and pH may have o c c u r r e d . Using P NMR, Van den T h i l l a r t et a l . 31  (198 9) showed that these parameters are remarkably s e n s i t i v e i n f i s h muscle. Even something so r o u t i n e as MS-222 a n a e s t h e t i a can cause  a  depression  phosphate  i n PCr and a  and decrease  i n pH  concommitant  increase i n  (to below 7.0 i n some cases) .  ±  These changes were not r e v e r s e d as long as the animal was kept anaesthetized. experiment  I t would  on rainbow  be  trout  useful i n order  to  perform  a  similar  t o compare metabolite  c o n c e n t r a t i o n s o b t a i n e d from f r e e z e clamped t i s s u e s with those collected  in  y  v i v o ' with P 31  NMR.  64 ATP  overshoot In  contrast  exhaustive  to previous  studies  e x e r c i s e no overshoot  r e c o v e r y . The reason  of recovery  following  i n [ATP] was observed  f o r t h i s d i s c r e p a n c y most l i k e l y  during lies in  the values f o r the r e s t i n g f i s h . R e s t i n g [ATP] i n t h i s study was n e a r l y double t h a t r e p o r t e d by both Dobson and Hochachka (1987) and  Milligan  and  Wood  (1986) .  In  addition,  i t was  not  s i g n i f i c a n t l y d i f f e r e n t (p=0.05) from the h i g h e s t [ATP] observed d u r i n g the "overshoot" i n e i t h e r of the p r e v i o u s s t u d i e s ( f i g u r e 4) .  This  strongly  depressed.  suggests  that  the r e s t i n g  [ATP] may  be  Since IMP, and hence TAN, was not measured i n e i t h e r  of these s t u d i e s , one cannot e l i m i n a t e the p o s s i b i l i t y t h a t the low r e s t i n g  [ATP] i s simply a r e s u l t of low TAN r a t h e r than the  r e s u l t of s t r e s s r e l a t e d h y d r o l y s i s of ATP with a concommitant increase  i n IMP; however,  i f this  i s the case  then  the high  [ATP] observed d u r i n g the overshoot must be a r e s u l t of de novo synthesis unlikely  or  salvage.  This  i s theoretically  p o s s i b l e , but  because o f the h i g h energy c o s t . Synthesis of ATP from  IMP v i a the p u r i n e n u c l e o t i d e c y c l e r e q u i r e s 1 GTP while the formation of ATP de novo r e q u i r e s 5 ATP and 1 GTP. ATP can be formed  from  reaction  adenosine  and p h o s p h o r i b o s y l  t h a t ' i s termed  the salvage  pyrophosphate  pathway  of  in a  adenylate  s y n t h e s i s which i s a much l e s s c o s t l y a l t e r n a t i v e . However, PRPP i s present i n muscle c e l l s at very low c o n c e n t r a t i o n s 1 and 10 nmoles/g t i s s u e , H i s a t a , 1975; Ipata e t a l . , the  formation  of  PRPP  requires  2  ATP.  The  (between 1987) and  alternative  65 hypothesis,  t h a t measured r e s t i n g  [ATP]  was  depressed due  to  some sampling a r t i f a c t has a l r e a d y been shown t o occur i n f i s h muscle  (see  above)  explanation  and  i s thus  f o r the observed  f a r more  likely  to  "overshoot". In t h i s  be  the  case i t i s  merely necessary t o e x p l a i n why s t r e s s r e l a t e d h y d r o l y s i s of ATP is  less  likely  i n samples  from  a trout  at  12  or  24h  after  exhaustive e x e r c i s e than at " r e s t " . Even 24h a f t e r exhaustive exercise  the  fish  i n the  present  study  were  markedly  responsive t o stimulus than the " r e s t i n g " f i s h . I t was to  less  possible  handle the post e x e r c i s e f i s h without any s t r u g g l i n g while  i n the case of the r e s t i n g f i s h even the s l i g h t e s t d i s t u r b a n c e c o u l d cause a g i t a t i o n .  In t h i s case i t seems l i k e l y t h a t  the  high  both  and  [ATP]  Milligan  r e p o r t e d by  and  respective  Wood  resting  (1986) values  Dobson and  during are  Hochachka  recovery  the  (1987)  relative  result  of  the  to  their  refractory  c o n d i t i o n of the f i s h d u r i n g recovery combined with the great difficulty  i n o b t a i n i n g samples from r e s t i n g  fish.  In Dobson and Hochachka (1987) r e s t i n g c r e a t i n e charge  was  low at only 0.388 as opposed t o 0.539 i n the present study. This suggests t h a t both the low a certain  [ATP]  amount of s t r u g g l i n g  and and  [PCr] may stress  be a r e s u l t  related  hydrolysis  d u r i n g sampling. T h i s i s an i n t e r e s t i n g o b s e r v a t i o n s i n c e might  expect PCr h y d r o l y s i s t o be more e x t e n s i v e b e f o r e  began t o decrease. The a c t i v i t y of AMP is  high so  i t may  be  possible  t o see  of  one [ATP]  deaminase i n t h i s t i s s u e a decline  in  [ATP]  in  p a r a l l e l with the d e c l i n e i n [PCr] . In f a c t , t h i s phenomenon was  66 observed but not d i s c u s s e d by Dobson and Hochachka  (1987) .  The data o f M i l l i g a n and Wood (1986) are more d i f f i c u l t t o explain since resting 25  [PCr] i s r e l a t i v e l y h i g h at approximately  umoles/g wet weight. The f i r s t  possibility  i s that  total  c r e a t i n e was u n u s u a l l y high i n these f i s h , perhaps as a r e s u l t of d i e t . This would cause a s i t u a t i o n i n which although [PCr] was  h i g h at r e s t c r e a t i n e charge would be low. However, s i n c e  t o t a l c r e a t i n e was not measured t h i s hypothesis can n e i t h e r be accepted nor r e j e c t e d . F a i l i n g t h i s i s should be noted t h a t a s i t u a t i o n i n which [ATP] i s low and [PCr] is  high  i s not p h y s i o l o g i c a l l y  (or c r e a t i n e charge)  impossible.  p r e c i s e l y the s i t u a t i o n of the f i s h  In f a c t ,  i n the present  this i s  experiment  at 2, 4 and even 8h post e x e r c i s e . I t may be t h a t the " r e s t i n g " fish  used  by  physiological  Milligan state.  and Wood  This  would  (1986) also  were  help  in a  similar  t o e x p l a i n the  u n u s u a l l y low glycogen and h i g h l a c t a t e observed i n the r e s t i n g fish i n their  ATP  study.  d e p l e t i o n and Purine N u c l e o t i d e C y c l i n g As  has been  Hochachka, 1976)  shown  i n previous  studies  1988; Dobson et al_., 1987; D r e d z i c  (Mommsen  and  and Hochachka,  when ATP i n f i s h white muscle i s d e p l e t e d with e x e r c i s e  t h e r e i s a s t o i c h i o m e t r i c accumulation  of IMP  T h i s s t o i c h i o m e t r y i s maintained throughout  (see t a b l e 4) .  the recovery p e r i o d  which suggests t h a t the AMP 5' n u c l e o t i d a s e route of adenylate depletion  (to adenosine,  and hence i n o s i n e and hypoxanthine) i s  67 not of importance i n t r o u t white muscle. In a d d i t i o n , Mommsen and  Hochachka  (1988) have suggested  t h a t the two arms of the  p u r i n e n u c l e o t i d e c y c l e a r e temporally deamination  occurs  only  during  separated  so t h a t AMP  e x e r c i s e and IMP  reamination  occurs only d u r i n g recovery. The r e s u l t s of t h i s study  support  this conclusion. Large  decreases  i n [ATP] are o f t e n  d e l e t e r i o u s . In mammalian systems f a l l i n g be a s i g n of impending c e l l death may  considered  t o be  [ATP] i s thought t o  (Nayler, 1983) . However, there  be a number of b e n e f i t s t o p u r i n e n u c l e o t i d e c y c l i n g  e x e r c i s e which may outweigh p o s s i b l e disadvantages  with  caused by  l a r g e changes i n [ATP]. F o r example, i t has been suggested t h a t AMP  removal  adenylate  from  the t i s s u e  pool  could  serve  kinase r e a c t i o n i n the ATP producing  t o draw the  d i r e c t i o n , and  second, t h a t the accompanying i n c r e a s e i n ammonium ions c o u l d regulate g l y c o l y t i c 1988  f l u x at the l e v e l of PFK (see Van Waarde,  f o r review). Mommsen and Hochachka have suggested t h a t the  ammmonia  formed by AMP deamination  buffering. anaerobic  This  could  e x e r c i s e i n which  protons. One important may  be  +  important there  under  conditions  like  i s a l a r g e p r o d u c t i o n of  f u n c t i o n of the purine n u c l e o t i d e c y c l e  be t h e c o n s e r v a t i o n  p u r i n e bases,  may c o n t r i b u t e t o H i o n  o f adenylate  backbones.  IMP i s not permeable t o the c e l l  U n l i k e the  membrane thus  p r e v e n t i n g l o s s of adenlyate backbones t o the b l o o d and reducing the cost of adenylate r e p l e t i o n  (see p r e v i o u s  section).  Another 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 may be t o  68 maintain adenylate energy charge (Flanagen e_t a l . , 1986) . Energy charge i s only s l i g h t l y perturbed d u r i n g e x e r c i s e and recovery i n s p i t e o f the very low [ATP]  (see t a b l e 11). I t i s probably  more accurate t o t h i n k o f parameters l i k e R i n m a i n t a i n i n g c e l l v i a b i l i l t y than red  muscle  tissues  which  have  ATP  being  important  [ATP] per se. In mammalian  relatively  p u r i n e n u c l e o t i d e c y c l e enzymes decreases  low a c t i v i t i e s o f i n [ATP]  result i n  i n c r e a s e s o f [ADP] and [AMP] and a r e s u l t a n t decrease  inR . ATP  While i n white type muscles the a c t i o n of AMP deaminase prevents the  accumulation  charge)  o f AMP and ADP and causes  R  AXP  (and energy  t o remain h i g h . Thus [ATP] may not be important  maintainance  of v i a b i l i t y  AMP  deamination  i n the  i n t h i s type o f c e l l . t o IMP i s c l e a r l y  a rapid  process,  able t o keep pace with t h e demands o f e x e r c i s e . However, IMP reamination i s p r o t r a c t e d , r e q u i r i n g up t o 24h to complete. The reason f o r t h i s s l u g g i s h n e s s may be r e l a t e d t o the a c t i v i t y o f adenylosuccinate synthetase and adenylosuccinate l y a s e , although these  enzymes  have  never  been  measured  i n fish  muscle.  A l t e r n a t i v e l y , the slow recovery may be a r e s u l t o f the l i m i t e d a v a i l a b i l i t y of both GTP and a s p a r t a t e . A s p a r t a t e c o n c e n t r a t i o n s i n f i s h muscle f o l l o w i n g exhaustive e x e r c i s e are low (0.055 + 0.008 umoles/g t i s s u e , Mommsen and Hochachka, 1988), f a r below the K  m  of a d e n l y o s u c c i n a t e  synthetase,  0.4mM. [GTP]  has seldom  been measured i n f i s h but has been r e p o r t e d t o be very low i n the  muscle  of fatigued f i s h  (Dredzic and Hochachcka,  1976).  D i f f e r e n c e s i n , f o r example, a s p a r t a t e a v a i l a b i l i t y c o u l d help  69 t o e x p l a i n some p u z z l i n g d i f f e r e n c e s i n the time course of [ATP] recovery  i n t r o u t between s t u d i e s .  The f i s h of M i l l i g a n and  Wood (1986), Mommsen and Hochachka (1988), and the present a l l r e q u i r e around 24h of recovery  before  study  [ATP] r e t u r n s t o the  r e s t i n g l e v e l . However, Dobson and Hochachka (1987), u s i n g of s i m i l a r s i z e t o those used by Mommsen and Hochachka showed f u l l recovery  of [ATP] w i t h i n 2 hours  p o s s i b l e t h a t t h i s discrepancy of purine n u c l e o t i d e  fish  (1988)  ( f i g u r e 4). I t i s  may be due simply  t o the speed  c y c l i n g ( i e : the a v a i l b i l i t y of a s p a r t a t e  or GTP) . T h i s p o s s i b l i t y r e q u i r e s f u r t h e r i n v e s t i g a t i o n . It never  i s often  falls  advanced  stated that  below  stages  about  mammalian s k e l e t a l muscle [ATP]  75% of r e s t i n g values  of muscle  fatigue  even  (see f o r example,  in  the  Wilkie,  1981) . However, t h i s i s probably an over g e n e r a l i z a t i o n even f o r mammalian s k e l e t a l muscle and i s c l e a r l y untrue f o r t r o u t white muscle s i n c e  [ATP] decreases t o l e s s than 25% of the r e s t i n g  value a t exhaustion in  the white  Terjung  (see t a b l e I ) . The s i t u a t i o n may be s i m i l a r  s k e l e t a l muscle  of mammals as w e l l .  Meyer and  (1980) r e p o r t a g r e a t e r than 50% decrease i n [ATP]  with  i n t e n s e s t i m u l a t i o n In s i t u o f the s u p e r f i c i a l white s e c t i o n of the  gastrocnemius i n the r a t . In r e d type muscles ATP content  i s maintained near normal even d u r i n g i n t e n s e s t i m u l a t i o n which produces r a p i d f a t i g u e .  The response of [ATP] t o e x e r c i s e may  represent a b a s i c d i f f e r e n c e between red and white type s k e l e t a l muscles.  It i s possible  that  reductions  i n [ATP] are seldom  observed i n s k e l e t a l muscle i n mammals because of the d i f f i c u l t y  70 i n o b t a i n i n g an unmixed muscle sample as a r e s u l t of the h i g h l y heterogenous nature of most mammalian muscles. T h i s has not been a problem with s t u d i e s on t r o u t because of the c l e a r s e p a r a t i o n of f i b r e t y p e s .  Phosphocreatine  and Energy  Metabolism  The quick recovery i n phosphocreatine  when [ATP]  is s t i l l  s u b s t a n t i a l l y below the r e s t i n g l e v e l i s one-of the fundamental observations  of  this  p r e v i o u s l y observed stated  that  "ATP  study.  and,  This  in fact,  replenishment  phenomenon  has  never  Dobson and Hochachka  must be  completed  been (1987)  before  PCr  c o n c e n t r a t i o n s can be returned t o p r e - e x e r c i s e v a l u e s " . T h i s i s clearly  not  the  study. Although  case,  at l e a s t  with  respect to  the  present  [ATP] recovers very slowly, by 2h post e x e r c i s e  energy charge has l a r g e l y recovered even when c a l c u l a t e d on the b a s i s of t o t a l m e t a b o l i t e pools (see t a b l e 13) . T h i s was not the case  with  energy  the  charge  results  of Dobson  (calculated  on  pools) d i d not recover u n t i l As models  the [ATP]  and  Hochachka  basis  metabolism  charge) i s r e l a t e d t o energy  suggest charge  total  recovery was  d i s c u s s e d i n the i n t r o d u c t i o n , of energy  of  and R  ATP  [PCr]  metabolite  complete.  a number of  that  the adenylates phosphorylated t o ATP).  (1987) where  different  (or c r e a t i n e  (the p r o p o r t i o n of  In t i s s u e s i n which the  t o t a l adenylate p o o l (ATP+ADP+AMP) remains more or l e s s constant ( i e : l i t t l e IMP or adenosine  i s formed) energy charge and  [ATP]  t r a c k each other. However, i n t i s s u e s i n which the s i z e of the  71 adenylate p o o l changes, such as f i s h white muscle, energy charge and  [ATP]  can  be  dissociated  as  i n the present  t h e r e f o r e more accurate to s t a t e t h a t resting cannot index  levels be  until  R  overemphasized  of metabolic  or  ATP  EC  have  t h a t the  [PCr]  usefulness  to of  limited,  t i s s u e s l i k e f a s t t w i t c h s k e l e t a l muscle  It i s  cannot r e t u r n to  returned  s t a t e i s probably  study.  normal. I t [ATP]  as  an  especially  in  i n which the s i z e of  the adenylate p o o l v a r i e s so g r e a t l y . The  reason  why  energy  charge  and  hence  phosphocreatine  r e c o v e r s so q u i c k l y i n these f i s h r e l a t i v e to the f i s h used i n other experiments i s more d i f f i c u l t to a s s e s s . F i r s t  i t should  be noted t h a t i t i s impossible to c a l c u l a t e the t r u e EC the f r e e c o n c e n t r a t i o n s of ADP  and AMP  using  because no p r e v i o u s study  of recovery from exhaustive i n f i s h has measured the components necessary calculated  to c a l c u l a t e the  f r e e ADP  u s i n g the t o t a l  or AMP.  However, even  c o n c e n t r a t i o n s recovers much more  q u i c k l y i n t h i s study than i n p r e v i o u s experiments. for  this  discrepancy  may  have  to  glycogen of t h i s stock of f i s h . In  do  with  the  The  high  reason resting  Mommsen and Hochachka (1988)  and Dobson and Hochachka (1987) energy charge at exhaustion 0.398 and 0.511  EC  respectively  was  (based on the t o t a l c o n c e n t r a t i o n s  of ADP  and AMP),  much lower than i n the present study  (see t a b l e  13).  In  other  fish  both  experiments  the  exhausted  had  s u b s t a n t i a l l y l e s s than 1 umole g l u c o s y l u n i t s / g wet weight i n the muscle and i t i s suggested  t h a t exhaustion was  f u e l d e p l e t i o n . However, i n the present  a r e s u l t of  study glycogen  levels  at exhaustion were, i n f a c t , h i g h e r than the r e s t i n g of both Dobson and Hochachka  glycogens  (1987) and Mommsen and Hochachka  (1988) . T h i s suggests t h a t e i t h e r the f i s h i n the present study were  not  fully  exhausted  or  that  factors  other  than  fuel  d e p l e t i o n were the cause of the exhaustion. There which  i s a l a r g e body of evidence  suggests  (lactate,  that  Pi and pH  the  build  up  from mamalian s t u d i e s  of metabolic  i n p a r t i c u l a r ) may  be  end  causal  products  factors i n  muscle f a t i g u e . For example, M i l l e r e_t a_l. (1988) showed t h a t Pi and  [H ] are a s s o c i a t e d with muscle f a t i g u e i n v i v o i n human +  b e i n g s . In an elegant s e r i e s of experiments fibres  on skinned muscle  i t has been shown t h a t the a p p l i c a t i o n of phosphate can  prevent muscle c o n t r a c t i o n and cause f a t i g u e (see Cooke and Pate 1990  f o r r e v i e w ) . I t i s p o s s i b l e t h a t the f i s h i n the present  study became "exhausted" and c o u l d no longer swim because of end product accumulation r a t h e r than l a c k of f u e l . In t h i s case i t would not be s u r p r i s i n g t o see l e s s change i n energy charge. And this  difference  in  energy  depletion could  d i f f e r e n t r a t e of recovery i n energy  Carbohydrate Two  account  charge and hence  for  the  [PCr].  Metabolism  main  questions  dominate  current  interest  in  carbohydrate metabolism d u r i n g recovery from severe e x e r c i s e i n f i s h white muscle: what i s the f a t e of l a c t a t e ? and, what i s the source of the glycogen r e s y n t h e s i z e d ? In p r i n c i p l e there are a number of p o s s i b l e f a t e s of l a c t a t e : 1) e f f l u x from the t i s s u e  73 (followed  either  and/or glycogen  by  o x i d a t i o n or  i n the  oxidation i n situ,  liver),  2)  by  recoversion to  glucose  c o n v e r s i o n t o pyruvate  and  3) c o n v e r s i o n t o pyruvate and then a l a n i n e  (which i s then metabolised along with the amino a c i d p o o l ) ,  and  4)  the  r e c o n v e r s i o n t o glycogen  in situ.  As can be  seen  from  above, the p o s s i b l e sources of glycogen are i n t i m a t e l y i n v o l v e d with the p o s s i b l e  fates  of l a c t a t e .  formed from l a c t a t e i n s i t u . via  Cori  muscle  cycling.  is  carried  In the in  glycogen  c o u l d be  Second, glycogen c o u l d be  Cori  the  First,  cycle,  blood  to  lactate the  formed  formed  liver  i n the  where  i t is  r e c o n v e r t e d t o g l u c o s e . T h i s glucose i s then c a r r i e d back t o the muscle where i t can be converted t o glycogen. C o r i c y c l i n g been thought t o be necessary t o muscle because of the  has  apparent  absence of some of the enzymes needed t o convert pyruvate  to  glycogen i n t h a t t i s s u e . However, t h e r e i s a l s o a great d e a l of evidence t o suggest t h a t C o r i c y c l i n g i s not necessary, at l e a s t for  some  skeletal  muscles.  Opie  Crabtree e_t a l . (1972) demonstrated  and  Newsholme  (1967)  and  the presence of both m a l i c  enzyme and PEPCK i n rodent and f r o g s k e l e t a l muscles. McLane and Holloszy form  (1979) demonstrated  glycogen  muscle fibres,  i n both  fast  rapid u t i l i z a t i o n t w i t c h red and  i n the p e r f u s e d h i n d limb on  the  other hand,  of the  d i d not  fast  of l a c t a t e twitch  r a t . Slow  utilize  lactate  to  white  oxidative to  form  glycogen. M i l l i g a n and McDonald (1988) have suggested that C o r i c y c l i n g p l a y s only a small r o l e i n glycogen r e s y n t h e s i s i n coho salmon white muscle f o l l o w i n g exhaustive e x e r c i s e s i n c e g r e a t e r  74  Figure 6: 3 carbon units ([lactate] +2[glycogen]) in rainbow trout white muscle following exhaustive exercise (umoles/g wet weight).  20  rest exh 2h  4h  8h recovery time  24h  76 than  80%  of the t o t a l  [ C ] - l a c t a t e was  blood a c t i v i t y  after  the  injection  of  recovered as l a c t a t e . In a p a r a l l e l study  14  have confirmed t h i s suggestion by  we  showing t h a t glucose uptake  i n t o t r o u t white muscle can account f o r no more than 10% of the glycogen r e p l e t i o n observed f o l l o w i n g exhaustive e x e r c i s e (West et a l . i n p r e p a r a t i o n ) . On t h i s b a s i s I would suggest t h a t the m a j o r i t y of the l a c t a t e formed d u r i n g exhaustive e x e r c i s e (see t a b l e 6)  i s r e t a i n e d i n the t i s s u e and r e c o n v e r t e d t o glycogen.  However, examination of f i g u r e 7 suggests t h a t t h e r e i s a s m a l l , non +2  s i g n i f i c a n t decrease i n the t o t a l 3 carbon u n i t s lactate)  deficit  from  r e s t t o the f u l l y  combined with  glycogen  reformed  recovered  the p o s s i b i l i t y  could  have  come  (24h)  t h a t up  from  fish.  to  glucose  (glycogen  10% (see  This  of  the  above)  suggests t h a t t h e r e has been at l e a s t some l o s s of l a c t a t e  from  the t i s s u e . The two most l i k e l y f a t e s of t h i s l a c t a t e are e f f l u x to  the  blood  or  oxidation  in  situ.  It  is  impossible  to  d i s t i n g u i s h between these options on the b a s i s of the data from t h i s study. However, i n a s e r i e s of p a r a l l e l experiments we have found white muscle mitochondria t o d i f f e r s u b s t a n t i a l l y from red muscle m i t o c h o n d r i a with respect t o pyruvate  o x i d a t i o n (Moyes  et a l . i n p r e p a r a t i o n ) . For example, d u r i n g the e a r l y phases of recovery t h e r e are l a r g e i n c r e a s e s i n [pyruvate]  (see t a b l e 7)  and white muscle mitochondria have been shown t o be s e n s i t i v e to  [pyruvate] over t h i s  range.  M i l l i g a n and Wood (1986) suggested phases  of  recovery  in trout  the  extremely  t h a t d u r i n g the  majority  of the  early  lactate  is  77 o x i d i z e d i n s i t u because d u r i n g t h i s time t h e r e i s no i n c r e a s e i n glycogen be  and the decrease  accounted  f o r based  on  i n [ l a c t a t e ] which i s seen cannot the  increase  in  [lactate]  in  the  b l o o d . This a l s o appears to be the case with the t r o u t used i n the present study. During the f i r s t two hours of recovery t h e r e was  no apparent  s y n t h e s i s of glycogen  but  [lactate]  decreased  by approximately 5 umoles/g muscle. Roughly 60% of the body mass of a t r o u t i s white muscle  (Stevens,  1968)  so f o r a 1kg  trout  t h i s would be 600g of white muscle and a whole body l a c t a t e l o a d of  about  3,000 umoles. Trout  whole body  extracellular  fluid  volume has been measured as about 240 mL/kg ( M i l l i g a n and Wood, 1986b) . I f we assume a l l of the l a c t a t e appeared i n the ECF would y i e l d an i n c r e a s e i n ECF t r o u t of roughly 12.5 mM. i n plasma l a c t a t e static  and  [ l a c t a t e ] i n the h y p o t h e t i c a l 1kg  During t h i s time t h e r e was no i n c r e a s e  (table 8). But the b l o o d l a c t a t e p o o l i s not  lactate  i s continually  lost  from the p o o l ,  o x i d i z e d or converted to glucose and/or glycogen so  even  though  there  l a c t a t e there was the  blood.  The  approximately exhaustive  12  was  no  measureable  rate  of  lactate  umol/min/kg  exercise  the  i n the  increase  in  turnover the  coho  is  liver  i n plasma  known  salmon  ( M i l l i g a n and McDonald, 1988)  the r e l e v a n t time p e r i o d (2h). of  either  l i k e l y at l e a s t some e f f l u x of l a c t a t e  correspond to a l o s s of roughly 6mM  half  this  decrease  in tissue  [lactate] . It  be  following  which would  l a c t a t e from the ECF  This c o u l d account  to  into  during  f o r perhaps i s , however,  d i f f i c u l t to make f i r m c o n c l u s i o n s based on c a l c u l a t i o n s of t h i s  78 sort  because  assumptions.  of  the  questionable  nature  of  some  of  For example, i t i s u n l i k e l y t h a t a l l of the  muscle i s e q u a l l y a c t i v e during e x e r c i s e or b u i l d s up  the white  lactate  t o the same e x t e n t . On the other hand i f b l o o d l a c t a t e and  ECF  l a c t a t e are not the same then i t might be more a p p r o p r i a t e to use b l o o d volume r a t h e r than ECFV i n the c a l c u l a t i o n above. I f the b l o o d volume of a 1kg t r o u t i s approximately decrease i n t i s s u e  150mL then the  [ l a c t a t e ] would be e q u i v a l e n t t o an i n c r e a s e  i n blood [ l a c t a t e ] of about 20mM c l e a r l y much g r e a t e r than t h a t a c t u a l l y observed. In t h i s case the amount of l a c t a t e o x i d a t i o n r e l a t i v e t o the amount of e f f l u x i n t o the b l o o d would g r e a t l y increase.  While  fine  scale  conclusions  about  the  fate  of  l a c t a t e d u r i n g the e a r l y phases of recovery are i m p o s s i b l e , i t i s c l e a r t h a t these data  support the c o n t e n t i o n of M i l l i g a n and  Wood (1986a) t h a t the m a j o r i t y of the decrease  i n white muscle  [ l a c t a t e ] i s l i k e l y due t o i n s i t u o x i d a t i o n d u r i n g the  first  two hours of recovery i n which t h e r e i s no glycogen s y n t h e s i s . Because of the f a i r l y l a r g e v a r i a t i o n from f i s h t o f i s h i n both  [glycogen]  and  [lactate] i t i s d i f f i c u l t  firm  c o n c l u s i o n s about the f a t e of l a c t a t e  and  t o make r e a l l y the o r i g i n  of  glycogen d u r i n g recovery. An experimental design i n which s e r i a l samples were taken from the same f i s h would be an i d e a l way i n v e s t i g a t e t h i s problem  Intracellular  to  further.  pH  The l a r g e v a r i a t i o n i n i n t r a c e l l u l a r pH a s s o c i a t e d with the  79 DMO  estimates  derived  pH  (see t a b l e 3) i s e a s i l y understood s i n c e the  is  a  calculated  parameter  measurements each of which i s subject directional influenced  errors. by  In  addition,  involving  7  separate  to a wide v a r i e t y of  the  DMO  technique  marker e q u i l i b r a t i o n time and  DMO  may  non be  e x t r a c t i o n method  as w e l l as a v a r i e t y of other f a c t o r s . The homogenate method on the other hand i s a d i r e c t measurement of pH,  although how  the  measured pH i s r e l a t e d to the t r u e i n t r a c e l l u l a r pH i s a matter open t o press)  question. includes  The a  o r i g i n a l technique  complex  protons c o n t r i b u t e d  calculation  to  capacity  contribution negligable, pH  of  of  this  the  ECF  in  small  trout  compartment  into  al.. i n account  to  volume and  white  the  muscle  measured  was low the  pH  is  e s s e n t i a l l y o u t s i d e the l i m i t s of r e s o l u t i o n of  the  meter. That anaerobic e x e r c i s e  pH  take  et  by the e x t r a c e l l u l a r compartment which  n e g l e c t e d here. However, because of the buffering  (Portner  i s well  known and  present study  the  i s associated  with a decrease i n  magnitude of the  pH  decrease i n  the  (table 3) i s s i m i l a r to that observed by M i l l i g a n  and Wood (1986) f o l l o w i n g an exhaustive e x e r c i s e bout i n rainbow trout.  But  the  complex and that  the  actual  source  of  t h i s pH  c o n t r o v e r s i a l . Mommsen and  relative contribution  of  H  +  decrease  Hochachka  a l s o with  these protons i s e q u a l l y u n c l e a r .  Heisler  majority  of  the  protons  are  both  (1988) show  from g l y c o l y s i s and  h y d r o l y s i s v a r i e s with the pH and  the  is  [Mg ] . The 2+  ATP  f a t e of  (1984) suggests that  transferred  to  the  water  80 f o l l o w i n g severe e x e r c i s e . In c o n t r a s t , i t has been found that over 90% of the protons are r e t a i n e d i n the white muscle a f t e r exhaustive  exercise  i n trout  (Y. Tang, u n p u b l i s h e d ) .  It i s  p o s s i b l e t h a t glycogen r e p l e t i o n i s the primary sink f o r protons during recovery. Immediately  following  exhaustion t h e r e i s a s l i g h t ,  non  s i g n i f i c a n t drop i n the pH. While t h i s may simply be an a r t i f a c t of the small sample s i z e ,  i t should be noted t h a t d u r i n g t h i s  time the complete  recovery of phosphocreatine o c c u r s . In t h i s  d i r e c t i o n the CPK  r e a c t i o n produces  7.0  a proton  (at and above pH  e x a c t l y 1 proton i s r e l e a s e d per Cr phosphorylated (Lawson  and Veech,  1979)). A s i m i l a r d e c l i n e  i n i n t r a c e l l u l a r pH  was  observed by Van den T h i l l a r t e_t al_. (1989) d u r i n g recovery from anoxia i n carp and g o l d f i s h u s i n g P-NMR. In t h i s case the pH 31  d e c l i n e was  Free  a l s o c o i n c i d e n t with the r e s y n t h e s i s of PCr.  ADP Recent  ([ADP] )  evidence  suggests t h a t  free  [ADP]  cytosol  i s at i n much lower c o n c e n t r a t i o n s than those that are  f  measured e n z y m a t i c a l l y . Estimates of r e s t i n g f a l l i n the range of 1-10 umol/g  i n the  (Jacobus e t a l . ,  [ADP]  % of the measured ADP 1982;  Shoubridge et a l . ,  f  generally  (less than 1984;  0.01  Challis  et a l . , 1989) t o approximately 0.07 umol/g (Dawson e t a l . ,  1977;  W i l k i e , 1981; Meyer et a l . , 1985)) . In the present study r e s t i n g f r e e ADP  ranges from l e s s than 3% of the measured t o t a l ADP  t o about 7%  up  (see t a b l e s 4 and 11) depending upon the technique  81 used  f o r the c a l c u l a t i o n , w e l l w i t h i n the range of p r e v i o u s l y  computed v a l u e s . Free ADP when c a l c u l a t e d without r e f e r e n c e to i o n b i n d i n g i s about 1.5 f o l d g r e a t e r than f r e e ADP c a l c u l a t e d a c c o r d i n g t o the model equations  at [Mg ]  = lOmM  2+  f  (see t a b l e  11) . C o n s i d e r i n g the u n c e r t a i n t y of the value o f some of the e q u i l i b r i u m constants used t h i s d i s c r e p a n c y i s remarkably s m a l l . The  Keq used  equations  to calculate  was determined  the [ADP] at excess  1989) . I f a Keq determined  at ImM  1979),  c o r r e c t e d t o 10°C, i s used  tissue  at r e s t ,  f  without [Mg ]  (Eldar  2+  f  [Mg ]  very much h i g h e r than  and Degani,  (Lawson  2+  then  u s i n g the model  [ADP]  f  and Veech,  = 0.054 umol/g  t h a t d e r i v e d from the  model c a l c u l a t i o n s . T h i s v a r i a t i o n i n the measured values of the e q u i l i b r i u m constants i n t r o d u c e s a degree of u n c e r t a i n t y i n t o the estimates of absolute c o n c e n t r a t i o n s of f r e e ADP and AMP. However, the r e l a t i v e changes should be r e l i a b l e . It  i s likely  that  the c a l c u l a t e d  "resting"  [ADP]  f  is  f a l s e l y e l e v a t e d as a r e s u l t of PCr h y d r o l y s i s on f r e e z i n g . NMR s t u d i e s on mammalian white s k e l e t a l muscles suggest t h a t roughly 85% of t o t a l c r e a t i n e content i s present as PCr at r e s t et. al.., 1985, Shoubridge carp  also  show  approximately  that  et a l . , resting  (Meyer  1984). NMR measurements i n the pH  A  in fish  white  muscle i s  7.4 . I f these c o n d i t i o n s are assumed t o be the  case at r e s t i n the t r o u t then f r e e ADP can be c a l c u l a t e d u s i n g the model equations. In t h i s case umol/g t i s s u e ,  [ADP]  f  would be about 0.002  s i m i l a r t o the value c a l c u l a t e d f o r the 2h f i s h  (table 11). So i t i s l i k e l y t h a t the estimated " r e s t i n g "  free  82 ADP  i s somewhat e l e v a t e d  relative  to  the  true  resting  because of PCr h y d r o l y s i s during f r e e z i n g . T h i s may  value  explain  why  [ADP] d i d not i n c r e a s e s i g n i f i c a n t l y from r e s t t o exhaustion as f  expected. T h i s a l s o may during  have been a problem with the  recovery.  For  example,  considering  samples taken  j u s t the  two  hour  p o i n t : i f the measured [PCr] i s " c o r r e c t " ( i e , no PCr h y d r o l y s i s occurred  during  freezing),  estimated  "resting"  complete,  although  Alternatively, i f  [ADP]  f  then and  ATP/ADP  the  [ADP]  recovery ratio  measured  h y d r o l y s i s on f r e e z i n g then the  PCr  occurred  to  assume that  similar  is  low  be  the  disturbed.  because  " c o r r e c t " [ADP]  f  of  at t h i s  PCr point  i n the t a b l e . I t seems  s i m i l a r amounts of ATP  i n samples from r e s t i n g and r e c o v e r i n g  samples were t r e a t e d i n the same way.  to  o f " t h i s parameter i s would  would be even lower than that r e p o r t e d reasonable  is  f  hydrolysis  f i s h since a l l  I f so, the trends  observed  should be s i m i l a r although the absolute c o n c e n t r a t i o n s  would be  proportionally [ADP] may f  lower. have important p h y s i o l o g i c a l s i g n i f i c a n c e s i n c e  at these extremely low  l e v e l s there  i s a strong  dependence of  m i t o c h o n d r i a l oxygen uptake on [ADP]. Bishop and Atkinson showed t h a t  at  [ADP]  equivalent  to  that  calculated  r e s t i n g t r o u t white muscle, oxygen uptake was the  maximal  greater  than  rate 200,  in vitro. as  is  S i m i l a r l y at the  case  in  (1984) for  l e s s than 10%  [ATP]/[ADP] the  trout  at  the of  ratios rest,  r e s p i r a t o r y r a t e i s l e s s than 10% of the s t a t e three r a t e s . This  83 suggests t h a t t r o u t white muscle mitochondria are f a r from t h e i r p o t e n t i a l maximum oxygen uptake at r e s t . I t should be noted that the experiments  of Bishop and A t k i n s o n  (1984) were performed on  mitochondria from r a t h e a r t s and as such may d i f f e r from t r o u t white muscle mitochondria; however, no data are a v a i l a b l e f o r the behaviour of non mammalian or white muscle mitochondria at such  low l e v e l s  declines  of ADP.  relative  More i n t e r e s t i n g l y ,  t o the r e s t i n g  level  f  actually  at 2h post  exercise  (table 11) and as a consequence the [ATP]/[ADP] enormously t o over Under  these  2, 000 i n s p i t e  circumstances  [ADP]  r a t i o increases  f  of the decrease  the data  of Bishop  i n [ATP].  and  Atkinson  suggest t h a t i f anything the oxygen uptake by the white muscle mitochondria recovery  would  from  be  less  exhaustive  than  exercise  i n c r e a s e d over the b a s a l l e v e l , is  possible  that  this  that  seen  energy  at r e s t . demand  During  should  be  so how i s t h i s demand met? I t  additional  demand  may  be  met  g l y c o l y t i c a l l y but t h i s i s u n l i k e l y s i n c e t r o u t white muscle i s probably a c t i v e l y gluconeogenic d u r i n g recovery from exhaustive e x e r c i s e as was d i s c u s s e d e a r l i e r . What i s more l i k e l y i s t h a t factors  other  than  [ADP]  f  are  affecting  mitochondrial  metabolism. There are many p o s s i b l e candidates f o r f a c t o r s which can a f f e c t m i t o c h o n d r i a l metabolism. For example changes i n redox s t a t e are known t o be a c o n t r o l l e r of m i t o c h o n d r i a l metabolism in  mammalian h e a r t s  (Koretsky  et a l . , 1989). Connett  (1987)  suggests t h a t m i t o c h o n d r i a l and c y t o s o l i c redox are l i n k e d . I f  84  t h i s i s the case i t seems u n l i k e l y t h a t redox p l a y s an important r o l e s i n c e c y t o s o l i c redox d i d not change s i g n i f i c a n t l y over the course  of  exercise  and  recovery  (table  11) .  However,  the  r e l a t i o n s h i p between m i t o c h o n d r i a l and c y t o s o l i c redox i s s t i l l u n c l e a r so i t i s i m p o s s i b l e t o r u l e out a r o l e f o r m i t o c h o n d r i a l redox.  Changes i n pH  uptake  in  are w e l l known t o a f f e c t  isolated  mitochondria  from  mitochondria.  the  In  carp a decrease  maximal oxygen  isolated i n pH  red  from  s t i m u l a t e d s t a t e 3 oxygen uptake by n e a r l y two  muscle  7.3  fold  to  6.5  (Moyes et  a l . , 1988) . Increases i n phosphate c o n c e n t r a t i o n are a l s o known to s t i m u l a t e oxygen uptake by i s o l a t e d mitochondria Atkinson,  1984) . Although  there  was  no  (Bishop and  increase  in  total  phosphate from r e s t t o 2h post e x e r c i s e (table 5) f r e e phosphate i s very l i k e l y t o have i n c r e a s e d . [ATP] d e c l i n e d by roughly from  rest  increase  4mM  t o 2h post e x e r c i s e , a s s o c i a t e d with an e q u i v a l e n t i n IMP.  phosphate  per  phosphate  from  previously,  T h i s would i n p r i n c i p l e  mole rest  of to  resting free  ATP,  or  2h  post  an  r e l e a s e 2 moles  increase  of  up  e x e r c i s e . I f , as  [phosphate]  i s l e s s than  to  8  of mM  discussed  ImM  this i s  a p o t e n t i a l l y l a r g e s i g n a l t o i n c r e a s e oxygen uptake over b a s a l levels. The  q u e s t i o n then  i s why  maintain  [ATP]/[ADP] r a t i o s  at  such h i g h l e v e l s d u r i n g recovery. One p o s s i b l i t y r e l a t e s to the fact  that  the  tissue  is  gluconeogenic  at  that  time.  [ATP]/[ADP] r a t i o s c o u l d be r e q u i r e d t o maintain f l u x  High  through  the pyruvate kinase back r e a c t i o n . Although r e v e r s i b l e , pyruvate  85 kinase  normally  catalyzes  the  reation  in  the  glycolytic  d i r e c t i o n ; r e v e r s a l r e q u i r e s h i g h [ATP] and [pyruvate] and very low [ADP] and [PEP] . The f a c t t h a t t h i s may a l s o l i m i t o x i d a t i v e metabolism may not be of importance.  Free  AMP The p o t e n t i a l d e p r e s s i o n of the [PCr] measurements  due t o  PCr h y d r o l y s i s may a l s o have r e s u l t e d i n the o v e r e s t i m a t i o n the c a l c u l a t e d c o n c e n t r a t i o n of AMP;  but whatever the f r e e  at r e s t i t i s c l e a r l y very low. The Km of AMP is  on the order  of 0. 4mM  (Smiley and S u e l t e r ,  1967)  than even the g r e a t e s t the order  of 6 uM.  f o r the enzyme  [AMP]  The  [AMP],  deaminase f o r AMP  from r a b b i t  muscle  s e v e r a l orders of magnitude  higher  f  c a l c u l a t e d f o r the f i s h muscle: on  activity  of AMP  deaminase  i n rainbow  t r o u t white muscle i s approximately 40 umoles/minute/g ( F i j i s a w a and Yoshino, 1987) . I f t h i s  enzyme was  at V  this  max  would  be  s u f f i c i e n t c a p a c i t y t o produce about 1 mmole/g muscle over the 30 minute e x e r c i s e bout used i n t h i s experiment. However,  [AMP]  is  first  far  below  the  K  m  for  the  enzyme.  If,  as  approximation, we assume M i c h a e l i s Menten k i n i e t i c s , observed  [AMP]  f  a  f  then at the  at exhaustion t h e r e would be the c a p a c i t y t o  produce about 9 umoles IMP/g muscle over the 30 minute p r o t o c o l , c l o s e t o the observed i n c r e a s e  i n [IMP] of about 5 umoles/g.  T h i s c a l c u l a t i o n makes the assumption that  [AMP]  f  i s constant at  the h i g h e s t l e v e l recorded throughout the e x e r c i s e bout and that IMP p r o d u c t i o n i s constant, n e i t h e r of which i s l i k e l y t o be the  86 case. However, i t i s c l e a r that the c a p a c i t y of AMP deaminase is  sufficient  t o account  f o r the observed  IMP p r o d u c t i o n at  p h y s i o l o g i c a l l e v e l s of [AMP ] . On the other hand at the [AMP ] f  calculated  f  f o r the 2h post e x e r c i s e group  (table  12) a t most  0.042 umoles IMP/g t i s s u e could be formed over the same t h i r t y minute p e r i o d , This  l e s s than  illustrates  0.5% of the c a p a c i t y  the p o i n t  that  AMP  at exhaustion.  deaminase  function i s  e x q u i s i t e l y s e n s i t i v e t o changes i n [AMP] . f  RaTP  and Energy Although  Charge both  R  ATP  and  EC  decline  significantly  exhaustion the a c t u a l d i f f e r e n c e between groups  at  was extremely  small and recovery was complete by 2h post e x e r c i s e . When EC i s c a l c u l a t e d without t a k i n g account the f a c t t h a t the m a j o r i t y of ADP  and AMP are bound in v i v o these d i f f e r e n c e s are m a g n i f i e d  and recovery appears slower (table 13) . T h i s r a i s e s the q u e s t i o n of  the g e n e r a l r e l e v a n c e of i n d i c e s l i k e energy  cell.  The i n i t i a l  f o r m u l a t i o n of the energy  charge t o the  charge concept by  A t k i n s o n (1977) d e a l t with the t o t a l p o o l s of ADP and AMP. Since then i t has become widely recognized that the m a j o r i t y of ADP and AMP are bound t o p r o t e i n s i n v i v o and thus only the f r e e forms should be c o n s i d e r e d i n c a l c u l a t i o n s  of energy  charge.  C a l c u l a t i n g energy charge from the t o t a l c o n c e n t r a t i o n s of ADP and AMP can have the e f f e c t of magnifying changes i n the energy charge  as can be seen with the r e s u l t s  from t h i s  study. When  r i g o r o u s l y c a l c u l a t e d i t seems that d e v i a t i o n s i n energy  charge  87 are  very small even under c o n d i t i o n s o f recovery from exhaustive  e x e r c i s e . The energy demands o f the c e l l must vary a great deal under  these c o n d i t i o n s  and i t seems reasonable t o expect a  p u t a t i v e c o n t r o l parameter t o vary as w e l l . Energy charge seems to  be a f a r from i d e a l c o n t r o l s i g n a l since., at l e a s t i n t h i s  t i s s u e , i t does not seem t o undergo measurable changes over the physiological closely  operating  range,  rather  conserved. I t may be t h a t  calculated  i t seems t o be q u i t e  energy  r a t i o s may serve as i n d i c e s  charge  of c e l l  or s i m i l a r  v i a b i l i t y but  t h e i r relevance t o c e l l u l a r metabolism remains open t o q u e s t i o n . The d i s c r e p a n c y between t h e energy charge c a l c u l a t e d with the t o t a l and f r e e p o o l s o f ADP and AMP underscores the importance of  dealing  with only  the reactive  a s s e s s i n g t h e energy s t a t u s  p o o l s o f m e t a b o l i t e s when  of the c e l l .  Since t h i s  approach  r e q u i r e s an estimate of i n t r a c e l l u l a r pH very few of the s t u d i e s on e x e r c i s e metabolism i n f i s h white muscle can be r i g o r o u s l y analyzed with r e s p e c t t o energy metabolism. The  action  maintenance times.  o f AMP  both R  I f [ATP]  ATP  deaminase  and EC c l o s e  had d e c l i n e d  i s responsible to resting  for  levels  at  the all  by 75% without t h e concomitant  p r o d u c t i o n o f IMP or adenosine t h e c o n c e n t r a t i o n s ADP and AMP would have i n c r e a s e d many f o l d causing R the  and EC t o f a l l and  c e l l t o enter the d e p l e t i n g phase o f h i g h energy phosphate  metabolism both  AXP  R  ATP  ( f i g u r e 3) . Because  o f t h e a c t i o n o f AMP  and EC are p r e s e r v e d and the c e l l  deaminase  stays  in  the  b u f f e r i n g phase. Connett (1988) s t a t e s t h a t "because AMP i s the  88  Figure  7: R e s u l t s of the model c a l c u l a t i o n s (Connett, 1988) a p p l i e d t o rainbow t r o u t white muscle following exhaustive exercise. Fc = c r e a t i n e charge = [ P C r ] / t o t a l c r e a t i n e Raxp = [AXP]/([ATP] + [ADP] +[AMP] EC = energy charge f  f  89  6  O rest  0.2  0.0  0.4  0.6  0.8  1.0  Normofized poterttio! energy pool (Fpe)  Figure  8.  The v a r i a t i o n i n c r e a t i n e c h a r g e w i t h the normalized p o t e n t i a l energy p o o l i n rainbow t r o u t white muscle following exhaustive exercise: the effect of the purine nucleotide cycle. Fc = c r e a t i n e c h a r g e = [ P C r ] / t o t a l c r e a t i n e  Fpe  = [PCr] + 2[ATP] + [ADP]  1.2  90  s u b s t r a t e f o r the deamination r e a c t i o n s t h a t d e p l e t e the adenine nucleotide pool  i t i s only during t h i s  [depleting] phase that  the adenine n u c l e o t i d e pool w i l l be depleted".  T h i s i s not t h e  case i n t r o u t white muscle s i n c e even at exhaustion  R  and EC  ATP  do not d e c l i n e s u b s t a n t i a l l y while there i s c l e a r l y a great deal of  IMP formed. At most t h e f i s h  have reached t h e t r a n s i t i o n  between t h e d e p l e t i n g and b u f f e r i n g phases. The model ( f i g u r e 3)  a l s o p r e d i c t s how F  c  should  change with changing p o t e n t i a l  energy as high energy phosphates are depleted.  It i s possible  to apply the same a n a l y s i s to the r e s u l t s from the present  study  ( f i g u r e 7 ) . I t seems c l e a r from t h i s f i g u r e t h a t at a l l times d u r i n g t h e e x e r c i s e and recovery  p e r i o d the muscle was i n the  b u f f e r i n g phase o f high energy phosphate d e p l e t i o n . Since t h e cell  remains  i n the b u f f e r i n g  difficult  to test  estimates  R  ATP  the d e r i v e d  and EC simply  phase  i t becomes  equations  with  on the b a s i s of F  much  which  more  Connett  (and pH and i o n  c  concentration) . Agreement seems to be f a i r l y "close but s i n c e the range observed i s so small i t i s impossible t o come t o any f i r m conclusion. If  t h e data  on t h e r e l a t i o n s h i p between F  c  arid F  P e  are  examined i n another way i t becomes apparent that there are some d i s c r e p a n c i e s between the p r e d i c t e d and the observed behaviour. F i g u r e 8 again shows the r e l a t i o n s h i p beween F and F , however, c  each group has been  analyzed  separately.  Pe  From t h i s  i t  is  c l e a r t h a t the data f a l l i n t o two separate groups: one i n c l u d i n g the  24h recovery  group  and t h e r e s t i n g f i s h  and a second  91 c o n t a i n i n g the 2,4  and 8h recovery groups. The reason f o r t h i s  d i f f e r e n c e l i e s i n the d e f i n i t i o n of p o t e n t i a l energy: P [ADP] it  = PCr + 2ATP +  e  ADP  i s very low at a l l times  can be n e g l e c t e d .  r e l a t i v e to  [PCr] and  [ATP]  [PCr] i s roughly constant, between 20  25 umoles/g wet weight, i n a l l groups except the exhausted and  so  [ATP]  groups.  The  groupings the 2,4  i s the  reason  major  for  factor  the  influencing  discrepancy  i s therefore " h i s t o r i c a l " .  and  P  between  It l i e s  fish  in  e  these  these  nucleotide groups. constant  cycling  Since F  Connett's  = 0.2  a  results  with  in F  the  slow  differing  a  original  model  two  exhaustive  e x e r c i s e which decreased the s i z e of the t o t a l adenylate combined  and  i n the f a c t t h a t  8h post e x e r c i s e groups have undergone  (ATP+ADP+AMP) . This  so  rate  of  between  was  purine the  computed  i t i s hardly s u r p r i s i n g that a  pool  two  for  a  discrepancy  appears. What remains t o be seen i s whether t h i s c o n s t i t u t e s a b a s i c flaw i n the model.  F  i s used i n t h i s context almost  Pe  as  an index of work done or umoles of phosphate energy h y d r o l y z e d . As  long  as  the  approximation This  will  certainly  of  be  adenylates  adenylate  i s v a l i d and F  a c t i v i t y of AMP t h e r e may  total  be  the  pool  w i l l be  c  case  deaminase i s lower.  s u f f i c i e n t AMP even when  in  remains  constant  this  l i n e a r l y r e l a t e d to red  muscles  where  Pe  the  In white muscles, however,  deaminase to cause a c e r t a i n  [PCr]  F .  i s h i g h . And  certainly  loss  during  recovery the s i t u a t i o n becomes complex beause of the v a r i a b l e r a t e of p u r i n e n u c l e o t i d e c y c l i n g so t h a t i t may  be  impossible  92 to d e s c r i b e energy metabolism i n t h i s way As  a result  one  u s i n g a s i n g l e curve.  of the p r e d i c t i o n s of the model, t h a t  energy  s t a t e can be assessed simply by knowing the c r e a t i n e charge and the pH,  cannot h o l d f o r white muscles s i n c e the a c t i o n of  AMP  deaminase must a l s o be taken i n t o account. There i s an a d d i t i o n a l c o m p l i c a t i o n to be addressed i f the model i s to adequately  represent events in. v i v o and t h a t i s the  effect  already  of pH.  decreasing phase for  I t has  the  pH  decreasing  shown t h a t  i s to i n c r e a s e the  (see i n t r o d u c t i o n ) .  example,  been  [AMP]  oppose the  there  c o u l d be  f r e e AMP.  a very  changes i n  [AMP]  [PCr]  d e c l i n e d with  large increase  with e x e r c i s e would be  for by  and AMP.  f  AMP  [PCr]  little  the will  and pH.  For  change i n  pH  concentration  of  was  that  of  the  [PCr]  pH  concentrations consequences  t r o u t . For  even t r a n s i e n t l y e l e v a t e d i n t o the  deaminase  dropping  seen mostly i n the f r e e  such as  effects  of decreases i n [PCr] and  T h i s c o u l d have extremely important  white muscle  [AMP]  exercise  c o n c e n t r a t i o n would s t a r t to d e c l i n e . The  of d i f f e r e n c e s i n the time course  in  caused by  with  i n the  on,  I f pH then began t o d e c l i n e with l i t t l e change i n the  [PCr] f r e e AMP  of ADP  of  of the b u f f e r i n g  increases  depend upon the r e l a t i v e r a t e s of change of example i f i n i t i a l l y  effect  The e f f e c t of d e c r e a s i n g the pH  i s to  [PCr] . So the  extent  the  example, i f  range of  the  (which can be accomplished under t h i s model by  about  muscle) a great deal of IMP  70%  without  could be  changing  formed. Since  pH  in  fish  fish white  muscle i s g l y c o l y t i c a l l y very a c t i v e , [PCr] would q u i c k l y s t a r t  93 to r i s e pool  again and pH  to f a l l  but by t h i s p o i n t the  c o u l d have been d e p l e t e d by  the  formation  of  adenylate IMP.  This  would cause c o m p l i c a t i o n s f o r the a p p l i c a t i o n of the model i n white  muscles  since  the  adenylate  pool  size  could  vary  u n p r e d i c t a b l y . What must be examined i s the r e l a t i o n s h i p between changes  i n pH  exercise.  Until  and  PCr  this  and  the  i s done  adenylates" i n muscle i t becomes  very  a c c u r a t e l y model energy metabolism p a r t i c u l a r l y muscles.  during  difficult i n white  to type  94 SUMMARY AND  CONCLUSIONS  The o b s e r v a t i o n  t h a t t r o u t white muscle c e l l s are never out  of the " b u f f e r i n g zone" of the models of h i g h energy phosphate d e p l e t i o n , even immediatly a f t e r exhaustive e x e r c i s e , h i g h l i g h t s an important c h a r a c t e r i s t i c of these models of c e l l u l a r energy state:  that  they  are best  applied  to transient  events.  The  adenylates and phosphocreatine behave i n the way o u t l i n e d i n f i g u r e s 1 through 3 over a very short time s c a l e such that one is,  i n princple,  u n l i k e l y t o be able  t o measure the s i t u a t i o n  described  f o r the " d e p l e t i n g "  phase of h i g h energy phosphate  depletion  under normal p h y s i o l o g i c a l  muscle c e l l . Any p e r t u r b a t i o n s will  be q u i c k l y  conditions  i n a healthy  i n the energy s t a t e o f the c e l l  compensated f o r and the c e l l  r e t u r n e d t o the  b u f f e r i n g zone. For example, when energy demand i n c r e a s e s [PCr] transiently  decreases  "depleting"  phase,  aerobic  conditions  oxygen  consumption  restore  the  anaerobic  and even  t o meet the new  [PCr]/[total like  Cr] r a t i o . fish  g l y c o l y t i c rate also increase  increasing  phase  of  demand  enters the  ( f i g u r e 1) . Under  t h i s would serve t o i n c r e a s e  capacity,  depleting  the c e l l  [ADP] r i s e s d r a m a t i c a l l y  mitochondrial  and as a r e s u l t ,  In t i s s u e s  white muscle,  more complex . With an i n c r e a s e  the  before  with  high  the s i t u a t i o n i s  i n [ADP], oxygen consumption or  as b e f o r e . As the c e l l approaches  high  energy  c o n c e n t r a t i o n s of AMP  phosphate  metabolism,  a c t i v a t e AMP deaminase.  This  p u l l s the adenylate kinase r e a c t i o n i n the ATP forming d i r e c t i o n and  quickly  returns  the  cell  to  the  buffering  zone  95 s i m u l t a n e o u s l y reducing the adenylate p o o l s i z e  (ATP+ADP+AMP)  by the formation of IMP. So even though the energy s t a t e of the c e l l i s r e t u r n e d t o normal,  [ATP] i s depressed. By viewing the  system i n t h i s way  i t i s easy t o see how  muscle  depressed  could  [PCr]/[total  be  Cr] r a t i o  [ATP] i n f i s h  during  sampling  is relatively  h i g h . The  even  white though  action  of  AMP  deaminase i s a l s o r e s p o s i b l e f o r d e v i a t i o n from the behaviour p r e d i c t e d by the models The  observation  (figure 8 ) .  that  [PCr]  recovers  before  [ATP]  is a  r e s u l t of the slow a c t i o n of the reaminating arm of the p u r i n e nucleotide  cycle.  This  results  in  a  dissociation  of  ATP  c o n c e n t r a t i o n from energy s t a t u s which i s not p r e d i c t e d by the models  of  high  energy  phophate  metabolism.  One  of  fundamental d i f f e r e n c e s between white and r e d type muscles  the may  l i e i n t h i s d i s s o c i a t i o n of [ATP] from the energy charge or R . ATP  T h i s c h a r a c t e r i s t i c of white muscle has an important impact on  the  overall  metabolism  of the  fish  during  recovery from  exhaustive e x e r c i s e . A c c o r d i n g t o the c a l c u l a t i o n s , f r e e ADP i s in  a range that  c o u l d be l i m i t i n g  to mitochondrial  oxidative  metabolism. At r e s t the mitochondria may be working at l e s s than 10% of t h e i r p o t e n t i a l maximum r a t e . Although not measured, i t i s reasonable t o assume that with e x e r c i s e t h e i r r a t e of oxygen consumption would r a p i d l y i n c r e a s e as [ADP]  f  i n c r e a s e s , or the  ATP/ADP r a t i o decreases, but the energy demands are such t h a t maximal c a p a c i t y  is insufficient  and the t i s s u e must r e l y  on  anaerobic sources of energy. From exhaustion t o 2h post e x e r c i s e  96 energy  status  (as  reflected  by  [PCr]/total  q u i c k l y r e s t o r e d . T h i s , i n combination down the f r e e ADP [ADP]  f  or h i g h  ratio)  c o n c e n t r a t i o n s to below r e s t i n g l e v e l s .  [ATP]/[ADP]  f  ratios  severely l i m i t  be complicated by e f f e c t s of pH,  although  [ATP]/[ADP]  up c o n d i t i o n s which are f a v o u r a b l e f o r glycogen l a c t a t e . This suggests  the this  f r e e phosphate  ( m i t o c h o n d r i a l or c y t o s o l i c ) . The high  t h a t t h e r e may  is  with the low pH d r i v e s  mitochondrial oxidative phosphorylation, may  [Cr]  f  Low  r a t e of effect  and redox r a t i o s set  s y n t h e s i s from  be a t r a d e o f f i n v o v l e d  i n which r a t e of glycogen s y n t h e s i s i s optimized r e l a t i v e to the r a t e 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 and v i c e v e r s a . The r e t e n t i o n of hydrogen ions which i s c h a r a c t e r i s t i c of t r o u t white muscle may be necessary t o set the r e q u i r e d [ATP]/[ADP] r a t i o r e q u i r e d f o r gluconeogenesis.  [ATP] replenishment per se must be  thought  of as an e n t i r e l y seperate phenomenon with no d i r e c t b e a r i n g on energy metabolism or carbohydrate  metabolism.  Energy metabolism and carbohydrate metabolism i n f i s h white muscle  f o l l o w i n g exhaustive  e x e r c i s e must be  regarded  as  an  i n t e g r a t e d system responding to a number of c o n t r o l l i n g f a c t o r s including  f r e e ADP,  i n o r g a n i c phosphate c o n c e n t r a t i o n and  pH.  Without c o n s i d e r i n g a l l of these parameters i t i s impossible to accurately  assess  the  behaviour  of  these  systems.  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