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

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METABOLIC ADJUSTMENTS TO ACUTE HYPOXIA IN  THE  AFRICAN LUNGFISH AND RAINBOW TROUT  by J E F F R E Y F. BSc  (Honours), U n i v e r s i t y  DUNN  of B r i t i s h Columbia,  A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology,March,1985) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g required standard  to the  THE UNIVERSITY OF B R I T I S H COLUMBIA  J e f f r e y F.  Dunn,  1978  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia. I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  ^00  t~  K  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  MA&CH ?. Htif  written  i i  ABSTRACT The  inter-tissue  determined (Salmo  in  lungfish  gairdneri).  reduction  in  which t i s s u e , during  or  and  and  Metabolic  using  metabolite  brain  exhibit  but  was  intended  decreased  relatively  in  brain  d e p l e t i o n of  metabolic  throughout b r a i n and  from  that  the  throughout  reduce  heart  blood  isolated  enzymatic monitored  potential.  lactate and  stores,  The  and  (4)  the  from  the  rest  phosphate during  cross-over  and  lactate gradients  released  12 h r of  obtaining  White  fall  glucose  brain  the  not  i n d i c a t e d by  and  that  energy  did  glycogen  (2) t h a t a f t e r  (3)  using  muscle  t h e most o x i d a t i v e  high  heart  a c t i v a t e d as  probably  submergence,  metabolically submergence.  were the  to  oxidative capabilities.  and  endogenous  submergence, heart  rates  metabolic  s u b m e r g e n c e were  Although  Blood-tissue  (1)  a  to determine  reported  H e a r t was  low  inert.  s u b m e r g e n c e , g l y c o l y s i s was  indicated  12 h r  measurements.  accumulation.  with  r e s u l t s w i t h the not  trout  p o t e n t i a l s of t h e h e a r t , b r a i n , w h i t e  e f f e c t s of a  displayed  and  hypoxia  a l s o showed t h e g r e a t e s t a n a e r o b i c  concentrations  glucose  to  were  hypoxia.  muscle remained almost  plots,  hypoxia  i n t h e A f r i c a n l u n g f i s h were e s t i m a t e d  data.  tissue,  It  i n t r o u t , which are  metabolic  liver  rate.  to  aethiopicus),  respond  t h e n compare t h e  rate during  The  (Protopterus  tissues  r e a c t i o n s observed  responses  Lungfish  metabolic  hypoxia,  metabolic  metabolic  a l l liver  submersion their  released  white of  lactate  the  required glucose  muscle body  the  was during  The  l a c k of measurable  concentrations  coupled  changes i n white  t h e s e f i s h e s may  energy the  production.  muscle  to  Instead, i t i s l i k e l y  prevent  H i s t o c h e m i c a l and musculature  the  activation  ultrastructural  of  the  composed  of  Respectively,  mosaic these  average  diameter.  fibres  having  an  low  fibres.  musculature relatively overall  is  geared  (as  was  p r o p o r t i o n of w h i t e  These  rate  data  primarily  exposure  shown  and  23.6  and  lipid  the  to  microns  67.3  suggested  with  a  a b o v e ) may  f i b r e s present  be  in  white  microns.  c o n t e n t were v e r y  suggest  axial  by  fibres.  that  the  axial  function.  of w h i t e muscle i n d i c a t e s  of  the Rainbow t r o u t  and  of  red  succinate  of l i p i d  34.3  for anaerobic  muscle reduced  is  that low.  rate  of  be r e l a t e d t o t h e  The the The ATP large  i n t h e myotome.  T i s s u e m e t a b o l i t e s were m e a s u r e d organism,  was  intermediate  diameter  c a p a c i t y of the muscle t o e x i s t turnover  and  the  s m a l l wedge of  lactate  capillarity  large percentage  metabolic  survival.  The  measured  average  of  during  b u l k of t h e myotome i s composed o f  density,  a l l  of  ability  glycolysis  examination  red  fibres The  of  t r i p h o s p h a t a s e s , and  of  Mitochondrial for  gross  of adenosine a  hypoxia  s t u d i e s were done on  lungfish.  demonstrations  dehydrogenases,  to  t h a t the  to the animal's  c o l o u r e d m u s c l e e v i d e n t upon histochemical  adaptation  leads to  n o t be t h e c a p a c i t y f o r i n c r e a s e d a n a e r o b i c  h y p o x i c d y s o x i a i s t h e key  axial  metabolite  w i t h t h e low enzyme a c t i v i t i e s  t h e s u g g e s t i o n t h a t t h e most s i g n i f i c a n t in  muscle  in  a  hypoxia  sensitive  ( S a l m o q a i r d n e r i ) , b e f o r e and  f o r 3 hr t o i n s p i r e d oxygen t e n s i o n s  of  20  torr  after (at  iv  4°C).  There  were  small  changes i n the b r a i n but the energy  s t a t u s was m a i n t a i n e d .  The r e d m u s c l e was t h e l e a s t  White  phosphate  muscle  creatine  was d e p l e t e d .  Various  i n d i c a t e t h a t t h e w h i t e muscle i s t h e major user substrates  and  the  major  producer  of  of l a c t a t e .  s t r e s s e d a s i n d i c a t e d by a d e c l i n e i n g l y c o g e n , the  total  every  adenylate  indicator  pool.  of  the  fact  white  that  muscle,  liver  metabolic  c o n c e n t r a t i o n s of glycogen The  The  affected. data  glycolytic  The h e a r t i s ATP,  C r P , and  exhibited declines in  homeostasis.  The  liver  d i d not d e c l i n e .  anaerobic while  the  g l y c o l y s i s has been a c t i v a t e d i n muscle  remains  in  metabolic  communication w i t h the other t i s s u e s v i a the blood, supports the suggestion  that  the metabolism  of the white muscle w i l l  p r o n o u n c e d e f f e c t on t h e m e t a b o l i c The  trout  i s maintaining  a c t i v a t i n g anaerobic 'normal' The trout  turnover  in  d i d not  r a t e s of g l u c o s e  i n glucose  that l i v e r  glycogen  uptake w h i l e  attempt  to  s t r e s s as  above.  maintain  to  Glucose  increased  t o 20.6+6.8 M m o l e s / m i n . / k g .  from  The l a c k o f  the  observation  c o n c e n t r a t i o n s do n o t c h a n g e a n d so t h e r e i s flux.  The i n c r e a s e i n l a c t a t e  emphasizes the f a c t that anaerobic t h a t some t i s s u e s a r e o x i d i z i n g problem  animal.  a n d l a c t a t e were m e a s u r e d i n  t u r n o v e r was a t t r i b u t e d  increase i n glucose  The  the  oxygen  change w h i l e l a c t a t e t u r n o v e r  2.8 + 0.4 jumoles/min ./kg increase  of  whole  utilization.  s u b j e c t e d t o t h e same h y p o x i c  turnover  no  i t s rate  glycolysis  r a t e s of energy  s t a t u s of t h e  have a  of  when  a  glycolysis  is  turnover  activated  and  lactate. cell  becomes  hypoxic  and  the  V  r e a c t i o n s of the c e l l (tissue, to  organ,  t o that stress  is  of  the  requirements  metabolism  f o r ATP  anaerobic  The c e l l  energy  the  oxidative  production  may  state  increase  The l u n g f i s h  t o be c a p a b l e o f t h e f o r m e r , t h u s p r e s e r v i n g  other  tissues  formation. major  supplying may  i n r e q u i r e m e n t s , i n w h i c h c a s e t h e r a t e o f ATP  a t t e m p t t o m a i n t a i n ATP p r o d u c t i o n r a t e s .  for  from  synthesis.  p r o d u c t i o n n e e d n o t be a s h i g h a s i n  appears  cell  1  exhibit a decline  conversely,  The  a n i m a l ) h a s two o p t i o n s i f o x y g e r F s u p p l y d r o p s  a l e v e l which prevents o x i d a t i v e  all  addressed.  and  reducing  the  rate  of  or,  i n the muscle  substrates end-product  The t r o u t w h i t e m u s c l e , on t h e o t h e r h a n d , e x e r t s a  influence  upon  the  other  tissues  when  the animal i s  stressed with hypoxia. The not  term  maintain  'energy c o n f o r m e r ' oxygen  uptake  i s a p p l i e d t o animals which  i n the face of a d e c l i n i n g  supply,  and w h i c h a l l o w ATP p r o d u c t i o n t o d e c l i n e c o n c o m i t t a n t l y by activating  g l y c o l y s i s t o a marked d e g r e e .  would a c t i v a t e g l y c o l y s i s rates  of  regulator  ATP  i n the attempt t o  production.  The  trout  not  An e n e r g y  regulator  maintain  oxidative  is  more o f an e n e r g y  t h a n i s t h e l u n g f i s h w i t h t h e main d i f f e r e n c e  c a p a c i t y being i n the white muscle.  do  in  this  vi  TABLE OF CONTENTS  ABSTRACT  ii  L I S T OF TABLES  ...viii  L I S T OF FIGURES  x  L I S T OF ABBREVIATIONS  x i i  ACKNOWLEDGEMENTS .  '.  Introduction Section  xiv 1  1. M e t a b o l i c a d j u s t m e n t s  t o hypoxia  in the A f r i c a n l u n g f i s h . Introduction  16  M a t e r i a l s and Methods Results  21  Discussion  37  Section of  ....18  2. An u l t r a s t r u c t u r a l a n d h i s t o c h e m i c a l  the a x i a l musculature  study  i n the African lungfish.  Introduction  48  Methods  50  Results  53  Discussion  67  Section  3. The m e t a b o l i c a d j u s t m e n t s  environmental  hypoxia  t o acute  i n t h e Rainbow t r o u t .  Introduction  71  M a t e r i a l s and Methods  73  Results  76  Discussion  ,\  90  vi i  Section  4. T u r n o v e r  rates  i n t h e Rainbow t r o u t d u r i n g  of  glucose  acute  and  lactate  hypoxia.  Introduction  103  Methods  105  Results  111  Discussion  128  General Discussion  137  viii  L I S T OF TABLES  Table  1. L u n g f i s h t i s s u e enzyme a c t i v i t i e s .  Table  2. C o n c e n t r a t i o n s  of  22  selected  glycolytic  metabolites i n the l u n g f i s h Table  26  3. C o n c e n t r a t i o n s o f a d e n y l a t e s , c r e a t i n e p h o s p h a t e ,  and c r e a t i n e , a n d t h e e n e r g y c h a r g e i n l u n g f i s h  tissues. 36  Table  4. C o m p a r a t i v e  anaerobic  and  activity  aerobic  ratios  pathways  of  enzymes  from  i n b r a i n s of f i s h and  mammals  38  Table  5. D i a m e t e r o f l u n g f i s h m u s c l e  Table  6. A c o m p a r i s o n  vascularization Table  of  the  fibres  mitochondrial  of v a r i o u s muscle  65 density  and  fibres  66  7. C o n c e n t r a t i o n s o f a d e n y l a t e s , c r e a t i n e p h o s p h a t e ,  and c r e a t i n e , a n d t h e e n e r g y c h a r g e i n t r o u t t i s s u e s . Table  8. S e l e c t e d t r o u t g l y c o l y t i c  during acute hypoxia Table  metabolites at rest  ...77 and  .  79  9. C o r r e l a t i o n s b e t w e e n t i s s u e a n d b l o o d m e t a b o l i t e  concentrations  i n trout  Table  10. G l y c o l y t i c m e t a b o l i t e s t o r e s i n t r o u t  Table  11. The r a t i o s o f b l o o d l a c t a t e t o t i s s u e  82 83 lactate  in  trout Table  12. E q u a t i o n  trout  84 p a r a m e t e r s f o r t h e DPM v s TIME c u r v e s i n 119  ix  Table  13. T r o u t m e t a b o l i t e t u r n o v e r numbers  Table  14. Mass o f t h e r a p i d l y m i x i n g p o o l i n t r o u t  Table  15. A  numbers  compilation .  o f g l u c o s e and l a c t a t e  ....121 ...122  turnover 131  X  L I S T OF FIGURES  Figure  1. C r o s s o v e r p l o t s o f l u n g f i s h t i s s u e m e t a b o l i t e s  the  end o f a submergence  Figure  2. G l y c o g e n c o n t e n t s and  Figure  recovery  3. T i s s u e  27 during  4. T i s s u e  forced  forced  submergence  in lungfish and b l o o d  30  glucose  f o r c e d s u b m e r g e n c e and r e c o v e r y Figure  at  and b l o o d  concentrations in lungfish  lactate  submergence and r e c o v e r y  Figure  5. L u n g f i s h c r o s s - s e c t i o n s  Figure  6. A n t e r i o r  lateral  line  during 32  concentrations  during  in lungfish  34 54  region  s t a i n e d f o r LDH  activity Figure  7. M o s a i c  stained Figure  region  of  the  anterior  lateral  line  f o r LDH a c t i v i t y .  8. M o s a i c  stained Figure  57  region  of  57 the  anterior  lateral  line  f o r ATPase a c t i v i t y  57  9. P o s t e r i o r l a t e r a l - l i n e  region  stained  for  SDH  activity Figure  10. P o s t e r i o r  59 mosaic  region  stained  for  SDH  activity Figure  11. S e c t i o n  59 from  zebra  fish  stained  for  act i v i t y  LDH 59  Figure  12. L a t e r a l s e c t i o n of w h i t e m u s c l e m y o f i b r i l s  62  Figure  13. L a t e r a l s e c t i o n of r e d m u s c l e m y o f i b r i l s  62  Figure  14. T r a n s v e r s e s e c t i o n o f r e d m u s c l e  62  Figure  15. T o t a l  glucose,  glycogen,  and  G6P  stores in  trout  86  Figure  16. T o t a l l a c t a t e  Figure  17. Example o f d e c a y c u r v e  Figure  18. E x a m p l e o f r e c o n s t r u c t e d  glucose  Figure  19. E x a m p l e o f r e c o n s t r u c t e d  lactate curves.  Figure  20. The  relationship  concentrations Figure  stores i n trout  21. The  concentrations  88  reconstruction  between  and g l u c o s e  turnover.  relationship  between  and l a c t a t e  turnover  112  curves  plasma  114 .......116 glucose 123  plasma  lactate 125  L I S T OF ABBREVIATIONS  DTNB  5,5'-dithiobis  PCA  perchloric  Tr i s  t r is(hydroxymethyl)aminomethane  ATP  adenosine  triphosphate  ADP  adenosine  diphosphate  AMP  adenosine  monophosphate  NAD , NADH  nicotinamide  +  (2-nitrobenzoic acid)  acid  adenine d i n u c l e o t i d e  ( o x i d i z e d and reduced) N A D P , NADPH n i c o t i n a m i d e +  adenine d i n u c l e o t i d e  ( o x i d i z e d and reduced) G6P  glucose-6-phosphate  F6P  f ructose-6-phosphate  FBP  fructose-1,6-bisphosphate  G3P  glyceraldehyde-3-phosphate  DHAP  dihydroxyacetone  CrP  creatine  Cr  creat ine  ATPase  adenosine  AAT  aspartate aminotransferase  CPK  creatine  CS  citrate  HK  hexok i n a s e  HOAD  beta-hydroxyacylCoA  LDH  lactate  MDH  malate  phosphate  phosphate  triphosphatase  phosphokinase synthase  dehydrogenase dehydrogenase  dehydrogenase  phosphate  xi i i  PFK  phosphofructokinase  PGI  phosphoglucoisomerase  PK  pyruvate kinase  SDH  s u c c i n a t e dehydrogenase  S.A.  specific  Ra Ms  (-g;-l)  activity  replacement  rate  (glucose;lactate)  mass o f t h e r a p i d l y m i x i n g p o o l  xiv  ACKNOWLEDGEMENTS  D u r i n g t h e y e a r s of work w h i c h received unfaltering family, G.M.O.  and  my  of t h i s  and  M.  wife  P.W.  thesis.  Hochachka.  I t w o u l d be  everyone  who  has  h e l p e d me  Dr.  Thanks a l s o  to  During  s t u d y I was  supported  scholarship,  and  by  a  in  s c h o l a r s h i p f r o m t h e U n i v e r s i t y of B r i t i s h  will  part  McLean  be  able  d u r i n g the  T h o s e whom I have n o t m e n t i o n e d appreciation.  my  f o r m a k i n g t h e Kenyan  satisfying  heartfelt  postgraduate  I have  survivable.  t h a t t h e y have my this  Hickley),  research committee f o r t h e i r c r i t i c i s m s  thesis. thank  (Moire  Guppy t o t h a n k  s t u d y b o t h p o s s i b l e and  t o t h e members of my comments on my  from my  s u p e r v i s o r , Dr.  M a l o i y and D r .  p o r t i o n of t h i s  remember  support  went i n t o . t h i s t h e s i s I have  by  Fraser  Columbia.  to  course  still  an  and  know  NSERC memorial  1  INTRODUCTION  Oxygen i s t h e key t o l i f e kingdom.  This  element  ubiquitous  biological  Such to  provides  engine:  o x y g e n , most a n i m a l s w i l l hypoxia, their  f o r most members  ability  the  the  They  also  which  animal  f o r the Without  When s u b j e c t e d t o  be g r e a t l y  impaired.  may be e x a g g e r a t i o n s , b u t t h e y s e r v e  underscore  i n q u i r e about t h e r o l e which strategies  cornerstone  d i e w i t h i n minutes.  emphasize the severe consequences of  supply.  the  mitochondrion.  to function w i l l  statements  of  may  be  curtailing  the  well  oxygen  t h e r e l e v a n c e of s t u d i e s which  oxygen  plays  utilized  in  when  life,  the  and  supply  the  becomes  limiting. A study of responses forms,  and  within the  so  t o oxygen  may  which  will  meant by a n o x i a , t h e t o t a l  The f i r s t  relative  be  used.  clarify  I t i s apparent  what i s  l a c k of oxygen, but  term which,  i f one l i t e r a l l y  t h e w o r d , means " l o w o x y g e n " . word  can  the  living  The  meaning  of  Hypoxia i s  t r a n s l a t e s t h e r o o t s of  medical  definition  of  the  be s t a t e d a s " l a c k o f an a d e q u a t e amount o f o x y g e n i n  i n s p i r e d a i r such as o c c u r s content  many  step i s to  " h y p o x i a " h a s e x p a n d e d t o encompass many s i t u a t i o n s . a  take  t h e s u b j e c t must be n a r r o w e d a n d d e f i n e d t o f i t  t h e framework o f a t h e s i s .  terminology  limitation  at  high  o r t e n s i o n " (Thomas, 1 9 7 3 ) . i n Denver a r e  concentrations  are  hypoxic lower  does n o t . W i t h i n l i m i t s ,  since  than  altitudes;reduced  oxygen  Does t h i s mean t h a t  people  they  oxygen  exist  where  they are at s e a - l e v e l ?  i n d i v i d u a l s may  adapt  in  a  No, i t fashion  2  that  makes t h e o x y g e n t e n s i o n  them ( D e j o u r s ,  1966).  i n t h e i r environment "normal" f o r  C o n v e r s e l y , an e x e r c i s i n g a n i m a l may have  a s h o r t a g e of oxygen a t t h e t i s s u e oxygen  l e v e l even when t h e  t e n s i o n s a r e " n o r m a l " ( R u s k o and R a h k i l a ,  The  possibility  ( p r o b a b l y many) (Dejours,  researchers  1966,-Jones,  means  that  1979).  f o r m i s u n d e r s t a n d i n g h a s a l r e a d y l e d some to  redefine  1981,-Robin,  ( h y p o x i c ) , when a s s o c i a t e d w i t h a clear,  that  word  "hypoxia"  The  adjective  1980).  noun  subject  the  which  is  the  occurs  depends  cellular  upon  level.  utilization,  there  or  not  effect  the  of  cell  a  e t c ) , has  will  metabolic  react to reaction  the hypoxic state at the  i s the  site  of  must be an i n t r a c e l l u l a r m e t a b o l i c s u p p l y of oxygen  this  before  the  oxygen response  cell  can  be  "hypoxic".  Jones  (1981)  concentration  in  physiological, the  the  Since  c a u s e d by a l i m i t e d termed  Whether  and  However,  n o t n e c e s s a r i l y mean t h a t t h e body, o r c e l l , s t a t e of hypoxia.  logical  (blood, gas, c e l l ,  a v a i l a b l e a l o w e r t h a n n o r m a l amount o f o x y g e n . does  inspired  wrote cells  that,  "Hypoxia i s a subnormal  that  or p a t h o l o g i c a l  causes function."  term " c o n c e n t r a t i o n " which i s  not  This d e f i n i t i o n  the  cell  may  be  may s t i l l  increase  due  too  would  t h e c o n c e p t Of a s u b n o r m a l l e v e l  restrictive  become o x y g e n  "supply"  for  t h e s t a t e m e n t s i n c e o x y g e n c o n c e n t r a t i o n may be l o w when  oxygen  word  force  clarify  In a d d i t i o n ,  The  driving  uses  delivery  i s high.  1977).  biochemical,  oxygen  flux  (Lubbers,  altered  oxygen  of  because, as mentioned above, a  limited  when  oxygen  requirements  t o i n c r e a s e d w o r k , even t h o u g h t h e r a t e o f o x y g e n  3  d e l i v e r y may be n o r m a l . Dejours he  wrote  cells  (1966) p r o b a b l y had a s i m i l a r  "A  hypoxic  t o Jones  when  s t a t e c a n be d e f i n e d a s one i n w h i c h t h e  l a c k o x y g e n , o r more p r e c i s e l y ,  substrates  intent  of the c e l l  in  " l a c k " oxygen."  which  the  oxidizable  This d e f i n i t i o n  encompassing  i n terms of t h e s i t u a t i o n s  because  of  the  oxygen.  However, i n terms o f b i o c h e m i c a l mechanisms, i t i s l e s s  a c c u r a t e because able  to  lack  more  one i s l e f t oxygen.  substrate oxidation oxidizable there  general  statement  to interpret  Under  that  how  by  (Racker, 1976).  i s e v e r enough oxygen  be  the c e l l  a  in a cell  the  availability  I t i s also unlikely to  oxidize  overcome  these  be u s e d :  cellular  hypoxia  delivery  is  than  occurs  when  i n the c e l l  lack  oxygen.  rate  of the c e l l .  h y p o x i a o c c u r s when t h e s u p p l y o f o x y g e n  the  In order t o  of  will oxygen  which o x i d a t i v e metabolism  r e q u i r e t o s u p p l y t h e e n e r g y needs  processes  the  In other  i s such t h a t  would words,  metabolic  have t o be r e o r g a n i z e d t o c o m p e n s a t e  This i s the d e f i n i t i o n which w i l l  of  the  problems t h e f o l l o w i n g m o d i f i e d d e f i n i t i o n  that  is  that  a l l of  f a t , amino a c i d , a n d c a r b o h y d r a t e s t o r e s .  less  to  lacks  substrate  available  of  applied  most c i r c u m s t a n c e s , t h e r a t e o f  i s not c o n t r o l l e d  substrates  i t can  i s more  fora  be u s e d f o r  remainder of the t h e s i s . It  i s i m p o r t a n t t o keep t h i s  discussing because and group  when  t h e r e a r e many s t a t e s w h i c h c a n be t e r m e d t o be  hypoxic  the  work  mind  This i s  may  analysing  in  r e l a t e d t o low oxygen.  which  or  last definition  o r may n o t r e s u l t  causes  of  these  in cellular  states  into  hypoxia.  One may  environmental  or  4  behavioral. ambient  The f o r m e r  oxygen  i n c l u d e s a s c e n d i n g t o a l t i t u d e where t h e  tension  is  lower  than  "normal".  or  exercise  where  i n c l u d e s c a u s e s such as apnea oxygen  tension  apnea  oxygen  hypoxemia  ( D e j o u r s , 1966).  may  cause  even  i f arterial  (Barbee  a_l. ,  1983).  hypoxic  low a r t e r i a l  E x e r c i s e may c a u s e  limitations  oxygen  Thus,  situations.  cell,  hypoxia  will  cellular  oxygen  scenarios  describe  The d u r a t i o n a n d s e v e r i t y o f  d e t e r m i n e whether  (i.e.  0  2  requirements)  a given exposure w i l l  of  elicit a  induced response.  When an a n i m a l oxygen  tension or  t e n s i o n s remain c o n s t a n t  these  e x p o s u r e , a s w e l l a s t h e work r a t e the  ambient  Both ascending t o  and  potentially  the  latter  i s not the d e t e r m i n i n g f a c t o r .  altitude  et  The  tensions,  patterns.  uptake  subjected  r a t e s o f oxygen  Animals  e n v i r o n m e n t a l oxygen metabolic  is  which  to  declining  environmental  u p t a k e may f o l l o w two d i f f e r e n t  maintain  tensions f a l l  their  oxygen  h a v e been  u p t a k e when  called  oxygen  or  r e g u l a t o r s , and t h o s e a n i m a l s i n which r a t e s of oxygen  decline  as  environmental metabolic  oxygen  tensions f a l l  conformers  labelled  oxygen  or  However,  i t is  not p o s s i b l e t o d e t e r m i n e t h e t y p e of c e l l u l a r  r e s p o n s e w h i c h o c c u r s b a s e d upon w h e t h e r r e g u l a t o r o r an o x y g e n expend of  excess  the t i s s u e s  conformer  will  conformer.  may  still  become  regulator  hypoxic  may  oxygen  have  to  u p t a k e a n d s o some (Jones,  have a r e d u c e d s u p p l y o f o x y g e n  1971).  t o some  those t i s s u e s a c t i v a t e  e x h i b i t a decrease i n metabolic rate  1973).  t h e a n i m a l i s an  energy t o m a i n t a i n i t s oxygen  b u t one d o e s n o t know w h e t h e r or  A  (Prosser,  c a n be  (or both).  A  tissues  glycolysis  5  Another a  tissue  caused oxygen,  way o f g r o u p i n g  related  viewpoint.  by e i t h e r an supply  hypoxia  the  oxygen  tissues.  o x y g e n s u p p l y c a n be d i v i d e d  demand  The c a u s e s  into three  or  a  groupings:  Physiological,  eg.  redistribution  2.  Environmental,  eg.  decline in inspired 0  of c a r d i a c output. tension  2  causing a decline i n a r t e r i a l 0 P a t h o l o g i c a l , eg.  decreased  of a r e d u c t i o n i n  1.  3.  i s from'  H e r e , o x y g e n l i m i t a t i o n s may be  increased  to  inducing situations  content.  2  stroke, vascular occlusion  disease, cardiac arrest,e t c . .  E x e r c i s e o r an i n c r e a s e i n t e m p e r a t u r e oxygen l i m i t i n g  situation,  may  induce  the  second  t h a t o f i n c r e a s e d o x y g e n demand.  Now t h a t t h e word h a s been d e f i n e d and t h e s i t u a t i o n s may  c a u s e h y p o x i a h a v e been o u t l i n e d ,  as t o when  the  cell  inadequate  oxygen  itself  supply.  is  the problem  actually  Robin  suffering  impaired.  T h i s t e r m c a n be c l a r i f i e d  oxygen  supply  normal  but  utilization  is  some  limiting  and  is  i n some d e g r e e o f o x y g e n t o x i c i t y  (Robin,  1980).  concerned  with hypoxic  term  "hypoxia"  these  where  "cellular  abnormal  i s elevated  (hyperoxic  definitions,  dysoxia although, or  utilization  causing  where s u p p l y  resulting  the  t h e term  t o d e l i m i t cases  problem  dysoxia),  Using  an  ( h y p o x i c d y s o x i a ) , where s u p p l y i s  intracellular  (normoxic  oxygen  remains from  (1980) h a s p r o p o s e d  d y s o x i a t o d e s c r i b e s i t u a t i o n s where c e l l u l a r is  still  which  this  dysoxia) thesis  f o r pragmatic  hypoxia"  will  is  reasons  be u s e d more  6  regularly. If  one  reactions energy  i g n o r e s a d a p t a t i o n and  in a c e l l  which  production  Bennett,  may  den  follow  production  Thillart,  increases  triphosphate)  in  two  I n one  order  to  supplement  (Anderson,  Wijsman,  and  are  v a r i o u s m e t a b o l i c pathways w i t h i n  which  may  provide  to  yield  lactate  vertebrates and  (Daw  (Shoubridge  1975),  lactate  to  ( H o c h a c h k a and 1982).  1980)  although  is  the causes become  and  glucose  decrease  Somero,  much than  the  when  production,  testing oxygen  kingdom and  glucose  The  l984;Robin,  now  for  yield  Thillart, In the  uptake,  o r ATP  of  rate  or may  n o t be  of  possible the  and  activated.  h y p o x i a and  whether a c e l l  hypoxia. turnover  cell  Jackson,  outline  has  actually  n e e d s an o p e r a t i o n a l d e f i n i t i o n cellular  1982)  case  second  l980;Ultsch  easier to define c e l l u l a r  One  to  a r e t h e m a i n s u b s t r a t e s and  metabolic  i t i s to determine  hypoxic.  1984).  (Hochachka  fermentations  are p o s s i b l e .  I n t h i s c a s e , g l y c o l y s i s may  It  Spehar,  the animal  and H o c h a c h k a , 1981;Van den  glycogen  is  and  i s t h e most common among t h e  i s the c a r b o n - c o n t a i n i n g product.  response  (adenosine  a l . , 1 967 ;McDougal e_t a_l. , 1 968 ;Hochachka  s u c c i n a t e ( T a e g t m e y e r , 1979)  glycolysis,  used  e_t  energy  Somero,  production  see  Somero,  the a n a e r o b i c d e g r a d a t i o n of  (Lehninger,  Somero, l 9 8 4 ; R o b i n ,  ethanol and  Glycolysis,  ATP  and  ATP  l975;Burton  1976;Hochachka  anaerobic  cellular  p a t t e r n , anaerobic  There  Somero, 1 9 8 4 ) .  immediate  patterns (for reviews,  Somero, 1984;Hochachka  1982).  production  l 9 7 l ; D e Zwann and  the  i s s u b j e c t e d t o h y p o x i a , then  1 9 8 2 ; H o c h a c h k a and  l973;Van  looks only at  Measuring r a t e s are  to  be  heat  difficult  7  at  the c e l l u l a r  cell  level  but  are  s u r e ways t o d e t e r m i n e  i s regulating metabolic  rate.  whether  I t i s a l s o p o s s i b l e t o make  i n f e r e n c e s a b o u t c e l l u l a r m e t a b o l i s m b a s e d upon m e t a b o l i t e If  one  can  definition,  determine the c e l l  production  using  glycolysis  is  conclude  in  in  simply  prove  the  that  has  hypoxia  by  of c a r b o h y d r a t e of  criterion not  if  t o an  observed suggested  by  that  (tricarboxylic  The  acid)  information  which  be  cycle.  Even large  the  may  be  occurred prove  that  responded  to  production.  lactate,  the  One  endproduct  this criterion  i t c a n n o t be  way.  J o b s i s and  production may it The  in  is  still  u s e d as t h e  can  excess  be  Stainsby dog  proceed  is  intracellular  have  energy  at  sole  p r o d u c e d e v e n when t h e c e l l  flux than  which  would  cells  of  to  glycolytic  a c t i v a t i o n has  Although  lactate  faster  needed  t o p r o d u c e ATP  There are v a r i o u s  utilized,  glycolytic  produces pyruvate  that  were t o m a i n t a i n i t s  increase in  glycolysis  production  o x y g e n i n any  t h i s aerobic  fold  anaerobic  b e c a u s e l a c t a t e may  limited  18  whether  fermentation.  t h e most w i d e l y  the evidence  the c e l l  changes  indicate  widespread response i s the  proof  induce  glycolytic  activating  ATP  would  been a c t i v a t e d .  c h a n g e s w h i c h may  r a t e s of  availability  l a c k of m e t a b o l i c  glycolysis  by  declined.  oxidation. no  Concurrent  data.  then,  has  i s required for anaerobic  used, to  hypoxic  of m a i n t a i n i n g  provides  flux  r a t e , b e c a u s e up  is  r a t e of t h e c e l l  oxygen  glycolytic  cell  capable  activated  same r a t e as ' g l u c o s e  one  the  pathways.  t h a t the m e t a b o l i c  metabolic flux  i s not  aerobic  not  small reductions changes  that  a  is  (1968)  muscle  and  at a rate which  used  pyruvate  by  the  i s reduced  TCA to  8  lactate.  The  limitation  dehydrogenase chain.  and  not  here the  i s the  activity  activity  of  pyruvate  of the e l e c t r o n  transport  A f l u x o f p y r u v a t e t o l a c t a t e may a l s o be i n d u c e d by  increase  in  concentration  NADH during  (nicotinamide oxidative  adenine  conditions  an  dinucleotide)  (Connett  e_t  al. ,  1984). Atkinson  (1977) d e v e l o p e d t h e c o n c e p t o f t h e e n e r g y  (E.C.) a s an i n d i c a t o r was  determined  by  of c e l l u l a r  energy  status.  This  (adenosine monophosphate) c o n c e n t r a t i o n s , and  these  values  into  his  0.5ADP)/(AMP + ADP + to  test  If  w h i l e energy would as  ATP)).  The  ATP  in"a f a l l  requirements fall  indicator  of  a decline  in may  methods  cellular  equation was  ((ATP +  not  developed  (Vetter  Hodson,  were s e v e r e l y i m p a i r e d ,  constant,  1977).  then  the  E.C.  The p r o b l e m w i t h E.C.  hypoxia  A decline  and  i s that may  t h e r e a r e many  occur  concurrently  i n t h e r a t e s o f ATP p r o d u c t i o n r e g a r d l e s s o f how Furthermore, because t h e c e l l  f o r m a i n t a i n i n g energy charge, and because a  energy charge i n d i c a t e s be  i n t h e E.C.  (Atkinson,  that production i s impaired. many  concept  remained  c a u s e s o f a change i n E . C . with  charge  p r o d u c t i o n by t h e c e l l  necessarily  an  energy  inserting  f o r h y p o x i a , b u t i t h a s been n o t e d t h a t h y p o x i c  e x p o s u r e may r e s u l t 1982).  value  m e a s u r i n g ATP, ADP ( a d e n o s i n e d i p h o s p h a t e ) ,  and AMP  solely  charge  situations  where  severe ATP  intracellular  stress,  has fall there  p r o d u c t i o n may be i m p a i r e d t o a  l i m i t e d e x t e n t w i t h no c o n c o m m i t e n t  fall  in  E.C.  (Atkinson,  1977) . Another  possible  indicator  of  cellular  hypoxia  i s the  9  relative  reduction  reduction  s t a t e of t h e c e l l  state  (nicotinamide respectively),  is adenine  may  because  first  limited  be  useful  change  thereby  K e n n e d y , 1982)., its  The p r o b l e m  1968).  mitochondria which  therefore, What  No  been the  the  cell  indicate  that  to  oxidize  more r e d u c e d  more  the  (Jones and  i s at  oxidized  with when  data  rest  i s that available.  will  hold.  (Jobsis  and  from  isolated  mitochondria  a r e not  i s c o m p l e t e l y reduced.  i s not  hypoxia  i s almost c o m p l e t e l y reduced and  agrees  apparent  oxidized  cytochromes.  i s t h a t when a m u s c l e +  ratio  cellular  the a b i l i t y  becomes  generalization  the c e l l  a  The r e d o x  state,  of hypoxic d y s o x i a . single  indicator  One must c o m b i n e i s , .in f a c t , In  of  various  hypoxic.  order t o determine  i s h y p o x i c one must d e m o n s t r a t e  that there  has  an a d j u s t m e n t o f m e t a b o l i t e c o n c e n t r a t i o n s  indicating  that  aerobic machinery  energy. or  in  i n order t o conclude that the c e l l  whether  and  The  when i t becomes o x y g e n  i s n o t n e c e s s a r i l y an i n d i c a t i o n  hypoxia  single  of  the c e l l  This  t h e NAD*  becomes  cellular data  in  making  when i t t w i t c h e s t h e p o o l  respiring,  NADH/NAD*  reduced  indicators  p o o l o f s j r u t o c h o n d r i a l NAD  Stainsby,  the  dinucleotide,  s h o u l d be a r e d u c t i o n  cytochromes,  by  e_t a_l. , 1 9 8 0 ) .  or the r a t i o of reduced t o o x i d i z e d  These r a t i o s the  indicated  (Chance  an  was n o t a b l e t o s u p p l y a i l o f t h e r e q u i r e d  T h e r e must a l s o be e i t h e r a d e c r e a s e i n increase  criterion  i n energy  i s included to  r e q u i r e m e n t s (or: b o t h ) .  distinguish  from  i n d u c e d by t h e l a c k o f s u b s t r a t e s o t h e r t h a n The  metabolic  oxygen  parameters  mentioned  supply  This last  metabolite  changes  oxygen. above  (lactate  10  production, type  of  hypoxic.  E.C.,  r e d o x ) can  argument  which  The  be  is  grouped together required  In  addition,  one  correlated  with  (Castellini  and  Glycolytic  al., and  increased Somero,  anaerobic  cellular  appended  (Norberg  production.  i n f l u e n c e d by  may may  be  e_t  energy  detrimental  become  for  t h e s i s but  will  a  shortfall the  be  the  in  only  I t i s possible that may  e_t  suggested  is  i s not  a  hypoxic  a s s u m p t i o n i s made t h a t  the  oxidative  s i t e which i s  up  to  20%  extramitochondrial  of  (Robin,  f r a m e w o r k of t h i s t h e s i s , t h e  such a l a r g e p e r c e n t a g e of c e l l u l a r  produce  l967;Dawes  The  However, t h i s  extramitochondrial  and  of  production.  for this  respiration  plots  1975)  has  serve  proviso.  in  a_l. ,  hypoxia w i l l  Although i t i s outside  that  production  Siesjo,  techniques  such a l a c k .  cellular  1980).  (Daw  i s often  Mommsen, 1 9 8 3 ) .  the  m a i n r e s u l t of oxygen l i m i t a t i o n  fact  and  stronger  i n pH  energy and  is  been a c t i v a t e d .  of h y p o x i a and  w i t h one  the  i n d i c a t e d by c r o s s - o v e r s  increasing anaerobic  definition  determining  basal  not,  are a l s o i n d i c a t o r s t h a t the c e l l  i s r e a c t i n g by  energy  o r has  1981;Hochachka  intermediates  1959)  utilized,  because a f a l l  i n glycogen concentrations  The  be  m e a s u r e pH  a c t i v a t i o n as  glycolytic decline  may  the  to prove that a c e l l  more i n d i c a t o r s w h i c h a r e  the argument i s t h a t g l y c o l y s i s has,  to develop  makes i t l i k e l y effects  in  oxygen  utilization  t h a t a l a c k of  areas  other  than  oxygen energy  supply. The  a b o v e d i s c u s s i o n o u t l i n e s some of t h e  may  result  for  s u c h an  in  hypoxic  occurrence.,  dysoxia  and  Now,  one  the may  situations  techniques ask  how  which  used to t e s t the  animal  1  s u r v i v e s t h i s exposure. to  hypoxic  anaerobic  dysoxia  energy  (Hochachka, it  will  hypoxia.  goldfish.  reducing  capacity  i n many ways.  energetically  require  (Daw  et  i.e.  1967,-Dawes  rate  1980),  for either  of  then these  f o r survival during  fermentation tolerate  Finally, may  these  accumulate,  (Castellini  1981).  examined.  proven  may  The t u r t l e  occur  newborns,  t o occur  oxygen  be  augmented  capability  to  varying  The  of  (Hance  e_t  carbohydrate the  cell  to  (such as occurs i n  in  only  reducing  metabolic  rate,  a few o f t h e v e r t e b r a t e s studied vertebrate  when  exposed  to  a  (Jackson and S c h m i d t - N i e l s e n , l 9 6 6 ; L u t z  l980;Robin et a l . ,  other v e r t e b r a t e s which  goldfish  1981).  i s t h e most e x t e n s i v e l y  supply  store  1980).  of  capacity  w i t h t h e c a p a c i t y t o reduce m e t a b o l i c r a t e limiting  may  or  i n mammals  end-products the  a n d Somero,  to  a s do t h e  or i n the t i s s u e s  1959;Shoubridge,  a l t e r n a t e s t r a t e g y , that of  been  They  animals  f l u x may be i n c r e a s e d by i n c r e a s i n g  since  products  few,  fermentations,  turtles,  a s h a s been p r o v e n  1980).  a  i n the l i v e r  et a l . ,  r a t e of g l y c o l y t i c  enzyme t i t r e ,  The  improved  of glycogen  the energy,  al. ,  potential  al.,  activating  metabolic  To name b u t  (Shoubridge and Hochachka,  which  et  of a c e l l  either  increase the c e l l ' s capacity  concentrations  has  by  a n d Somero, 1984,-Robin,  increasing the  high  seals)  or  responses  The s t r a t e g y o f i n c r e a s i n g a n a e r o b i c e n e r g y p r o d u c t i o n  utilize  al.,  encompassed  production  may be a c c o m p l i s h e d may  are  1980;Hochachka  follows that  responses  S i n c e t h e immediate  1  l 9 6 4 ; U l t s c h and J a c k s o n ,  have, or a r e degrees  suggested  are goldfish  to  1982).  Some  have,  this  (Van den T h i l l a r t ,  12  1982), s e a l s  (Kooyman e t a l . ,  Whitten,  1975), toads  Randall,  1 9 7 8 ) , and A f r i c a n  The do  ( F a l e s c h i n i and  1960), s t u r g e o n (Burggren  lungfish  (Lahiri  et a l . ,  to  ATP.  It  i s inversely  (Neely  1970).  among t h e s e a n i m a l s i s t h a t  they  up  f o r the  site  as  in  would  to the rate  Likewise, a concentration by a c t i v a t i n g cells.  Racker  An  when  aerobically  thought  an  respiration  serve  be  to  of  inhibit or  a  oxidative glycolysis.  rise  would a c t i v a t e  The one e x c e p t i o n  ^proceeds  at a rapid  ADP i n t h e c e l l ,  is  rate  cancer  i n these However,  c a u s e d by t h e  A h i g h c o n c e n t r a t i o n of  activation  of  an  ATPase,  cellular  where m e t a b o l i c r a t e  respiration  transfer  glycolytic  i s a l s o not present i n t i s s u e s  i s reduced i n  i s not being a c t i v a t e d  may  conditions.  I t a p p e a r s t h a t t h e i n v e r s e r e l a t i o n s h i p between  the e l e c t r o n  glycolysis  ( R a c k e r , 1976).  control.  activate glycolysis during aerobic  Glycolysis  i n the  s u g g e s t s t h a t t h e r e s p o n s e o f c a n c e r c e l l s may n o t  t o unusual g l y c o l y t i c  and  at  i n ATP c o n c e n t r a t i o n ,  concentration,  PFK ( R a c k e r , 1 9 7 6 ) .  The m a j o r  to  activation  i n o r g a n i c phosphate  glycolysis  cellular  increase  would  ATP  of  is  though oxygen i s a v a i l a b l e  (1976)  due  in  of  Aerobic  c e l l s even  (PFK).  occurs,  fall  of  l 8 6 1 ; R a c k e r , 1976).  glycolysis  occur  phosphorylation  lack  h a s o f t e n been o b s e r v e d t h a t t h e g l y c o l y t i c  related  phosphofructokinase such  make  and Morgan, 1 9 7 4 ; P a s t e u r ,  control  rate  and  not a c t i v a t e anaerobic energy p r o d u c t i o n t o the e x t e n t which  produced  be  (Leivestad,  one common d e n o m i n a t o r  w o u l d be r e q u i r e d  rate  1980), h i b e r n a t o r s  response  to  oxygen  when t h e r a t e o f f l u x  system d e c l i n e s which  makes  i t  lack. through possible  13  that  novel  glycolytic  Conversely,  it  activation increase  is  is  possible  due  i n the  c o n t r o l m e c h a n i s m s may that  the  to i n t r a c e l l u l a r  concentrations  of  lack  the  during hypoxia  has  observation  of  glycolytic  mechanisms w h i c h p r e v e n t  an  activating  or  w h i c h m a i n t a i n c o n c e n t r a t i o n s of g l y c o l y t i c Although  be f u n c t i o n i n g .  modulators,  inhibitors.  that' m e t a b o l i c  r a t e may  been made on t h e w h o l e body l e v e l ,  decline  i t has  yet  t o be d e t e r m i n e d  which t i s s u e or t i s s u e s are r e s p o n s i b l e f o r the  phenomenon.  The  cause of a d e c l i n e i n the m e t a b o l i c  whole a n i m a l  may  be a f a l l  tissues. response This  i n the m e t a b o l i c  Thus, a u s e f u l f i r s t w o u l d be t o i d e n t i f y  may  be  determined  by  r a t e o f one,  s t e p i n our  of  the  a  or many  understanding  the l o c a t i o n of stressing  rate  of  the  phenomenon.  tissues with hypoxia,  and  then  using metabolite data  t o i n f e r w h i c h t i s s u e s have  or  not  exhibited  glycolytic  examining  signs  of  i n t e r - t i s s u e metabolic possible  to  responses  pinpoint  which  i n f l u e n c e upon t h e m e t a b o l i c p o s s i b l e to determine  the  in  activation. this  tissue,  fashion,  i f any,  As  of t h e  i n t e r - t i s s u e metabolic  the  may  be  greatest  I t may  also  patterns  be  which  r a t e t o promote  the  the  lungfish  (Protopterus aethiopicus)  may  have the c a p a c i t y t o reduce i t s m e t a b o l i c resting  value  1970).  Previous  be u n u s u a l  it  animal.  mentioned,  (Protopterus  has  r a t e of t h e a n i m a l .  work i n c o n c e r t w i t h r e d u c t i o n s i n m e t a b o l i c survival  By  have  when  rate  to  f o r c e d t o r e m a i n submerged observations  aethiopicus)  on  the  (Lahiri  African  indicated that this  i n i t s c a p a c i t y to survive hypoxic  20%  of  e_t a l . , lungfish  s p e c i e s may exposure  the  also  (Lahiri  14  et  a_l. ,  1970).  Lungfish  are  bimodal  breathers  u n r e s t r a i n e d , o b t a i n a p p r o x i m a t e l y 90% of  their  from  The  lung  exchange  t e n s i o n s may f a l l forced  (McMahon,  f r o m 50 t o r r  submergence  (Lahiri  1970).  gills  1968).  many h o u r s submersion by  Even t h o u g h  submergence,  the  breathing  observation  oxygen  10  submergence  oxygen  minutes  tensions  of  t o 20  (Johansen  and  uptake  from the  quickly  decline  a r e a b l e t o remain submerged f o r  1970).  l i m i t s oxygen  arterial  t h e r a t e of oxygen  lungfish  (McMahon,  uptake  e t a_l. , 1970) a n d f r o m 40 t o r r  i s low and t h e a r t e r i a l  during  when  oxygen  to 5 torr during  t o r r d u r i n g 5 minutes of u n r e s t r a i n e d Lenfant,  and,  Thus,  the  data  indicate  that  uptake, a s u g g e s t i o n which i s supported  that unrestrained  i n water gassed w i t h  100% 0  lungfish will (Jesse  2  et  cease a i r -  a l . ,  1967).  T h e s e d a t a i n d i c a t e d t h a t t h e l u n g f i s h w o u l d be a g o o d a n i m a l t o use  when  utilizes  examining  t h e m e t a b o l i c r e a c t i o n s o f an a n i m a l w h i c h  t h e s t r a t e g y of m e t a b o l i c r a t e r e d u c t i o n  i n response to  hypoxia. This responses  thesis to  was  designed  cellular  hypoxia  to  elucidate  the  i n an a n i m a l w h i c h d o e s  o v e r a l l m e t a b o l i c r a t e d u r i n g h y p o x i a , a n d an a n i m a l not  been  chosen  reported  to  as t h e s u b j e c t  metabolic metabolic  have  rate reduction. strategy  this  f o r use  metabolic  in  capacity. examining  reduce  which  has  The l u n g f i s h was the  response  of  By s o d o i n g , i t i s i n t e n d e d t h a t t h e  f o r h y p o x i a s u r v i v a l used  by  lungfish  will  become a p p a r e n t . Once  the  intertissue  determined i n the l u n g f i s h ,  m e t a b o l i c r e s p o n s e s t o h y p o x i a were the pattern  was  compared  to  that  15  which  occurs  hypoxia.  in  a  fish  which  is  relatively  The r a i n b o w t r o u t was c h o s e n b e c a u s e t h e s a l m o n i d s a r e  among t h e f i s h e s t h a t a r e most s e n s i t i v e (Doudoroff  and  volume  literature  of  subject  of  Shumway,  trout  1970),  already  physiology  a t t e m p t e d t o e x p o s e t h e two limitation.  Instead,  and  concentration  and  animals  w h i c h was l o n g enough metabolic The  changes  any to  to  During  the  was  not  It  identical  changes  the  in  plasma  animal  oxygen  pattern  was  lactate  m o r t a l i t y , and  of  intracellular  changes i n t h e white  fibres  is likely  muscle.  there  are  Metabolite concentration  In c o n t r a s t , appreciable  when  This  detrimental  remaining  the  the  c a p a c i t y of the l u n g f i s h white  no  a c t i v a t i o n of g l y c o l y s i s prevent  trout  activation  may  tissues  due  detrimental  have to  production.  muscle t o endure hypoxia these  is  muscle i n d i c a t e s  c o m p e t e t i o n f o r s u b s t r a t e s and i n c r e a s e d e n d - p r o d u c t The  metabolic  changes i n the muscle.  changes i n t r o u t a x i a l  upon  r a t e of  tissues are  t h e r e a r e no m e a s u r a b l e  t h a t g l y c o l y s i s h a s been a c t i v a t e d . effects  t o be t h e  i n f l u e n c e on t h e m e t a b o l i c  f o r c e d s u b m e r g e n c e , when o t h e r  s t r e s s e d by h y p o x i a ,  hypoxic,  the  with  e v i d e n c e w h i c h i n d i c a t e s t h a t t h e mass  t i s s u e which has t h e g r e a t e s t  made  concerned  f o r exposure t o hypoxia  of muscle which i s c o m p r i s e d of w h i t e  obviously  i s a large  observable.  thesis provides  lungfish.  deficiency  because t h e r e  concommitant  make  oxygen  metabolism.  a time course  without  to  available  chosen which produced r e p r o d u c i b l e  the  intolerant to  with  effects.  16  SECTION 1  Metabolic adjustments  to hypoxia  i n the A f r i c a n  lunqfish  Introduct ion  The  African  obligate its  air-breathing  resting  Despite  oxygen  a  organs, Thus P a 0  2  of  torr  low  to  blood  after  occurs  in  0  water  skeletal and  tensions  muscle  substrates  fall  d u r i n g the  from  first  occur,  s h o r t p e r i o d s of s u b m e r g e n c e .  lungfish,  for  t i s s u e hypoxia  values  be  It reducing strategy present t.o  has  been  suggested  i t s metabolic for  hypoxic  hypoxia  designed  dysoxia  or  many  the  tissues.  lungfish  of t h e (Lahiri  resting et  lungfish.  r a t e , t h e r e may The  be  become  relatively remain  stress.  i s capable value  as  a _ l . , 1970).  t o e l u c i d a t e the m e t a b o l i c  i n the A f r i c a n  have a d e c l i n e i n m e t a b o l i c one,  that  r a t e t o 20%  surviving  s t u d y was  s h o w i n g s i g n s of m e t a b o l i c  since  hypoxia  h i g h c o m p a r e d w i t h o t h e r v e r t e b r a t e s , s i n c e l u n g f i s h can submerged f o r h o u r s b e f o r e  forced  that,  tissue  t o l e r a n c e may  of  maintained.  t i s s u e s may If  10%  central  10 m i n u t e s of  some  an  et a l . " ,  control  It is possible  is  1970).  (Lahiri  2  l u n g f i s h can  tensions  (McMahan,  ( P a 0 ) a r e not  et. a l . , 1 9 7 0 ) . 2  aethiopicus),  obtains approximately  the  oxygen  torr  (Lahiri  hypoxic  of  oxygen  5  and  from  conserve  i n the A f r i c a n  submergence such  uptake  arterial  (Protopterus  animal  hypoperfusion  1 9 7 0 ) , w h i c h may  50  lungfish  of a The  responses  F o r an a n i m a l  to  a  reduction  in  response  was  p a t t e r n ' of m e t a b o l i c  17  e x a m i n e d on a t i s s u e by whether is  changes  tissue  i n the m e t a b o l i c  instrumental in reducing  animal. tissue  This to  lungfish The  procedure  tissue  s t r a t e g y may  dive.  the  emerge w h i c h may  was  order  r a t e o f one,  metabolic  Thus,  rate  to  determine  o r many, t i s s u e s of  the  to  an  overall  whole  p e r i o d s of  indirectly  measurements  assess and  c o n c e n t r a t i o n s i n t i s s u e s and  to  of  metabolic  h e l p to e x p l a i n the a b i l i t y  to survive during prolonged protocol  in  would a l s o a l l o w f o r a d e s c r i p t i o n  interactions.  p o t e n t i a l s u s i n g enzyme metabolic  basis  of  the  hypoxia. tissue  metabolic  directly  blood through  assess  a 12  hour  18  M a t e r i a l s and  Methods  Experimental  Animals.  aethiopicus) Victoria  were  and  collected  The  lungfish  from  the  t r a n s p o r t e d to Nairobi  i n a q u a r i a a t 22°C. Experiments  African  K a v i r o n d o G u l f i n Lake  where t h e y were k e p t ,  f i s h w e i g h e d 200  were p e r f o r m e d  (Protopterus  b e t w e e n one  to  and  700  grams  unfed, (gm).  t h r e e weeks a f t e r  the  f i s h were c a p t u r e d .  Enzyme  Preparation  from beheaded buffer.  Care  location girdle right  fish was  i n each from  lateral  ventricle  After  Assays.  and  dropped  taken  fish;  the  from  liver  the  to  in  midline from  heart;  for  weighing  most  the had  (roughly  assayed  for  spectrophotometer NADH/NAD  +  The  and t h e p e l l e t  enzyme  pectoral  down t o w a r d s  anterior  0.5  micro-attachment.  was  similar  lobe;  the the  activity  p l u s r e c o r d e r a t 25°C.  c h a n g e s were m o n i t o r e d a t 340  phosphokinase  been s t o r e d a t -90°C.  Omnimixer  10,000 x g f o r 15 min  way  creatine  i n 5 ml o f h o m o g e n i z a t i o n  at  from a  the whole b r a i n , e x c l u d i n g the  were h o m o g e n i z e d with  homogenization  tissues  to half  the  and  cold  j u s t c a u d a l t o the  from l u n g f i s h which  b l o t t i n g d r y and  ice  dissect  T i s s u e s used  were t a k e n  T i s s u e s were q u i c k l y e x c i s e d  white muscle  dorsal  line;  olfactory bulb. assay  and  gm),  the  samples  buffer using a  homogenate was discarded. in  a  nm and  then  spun  Supernatant  Unicam  Reactions  Sorval  SP-1800  coupled  to  those coupled to  19 DTNB were m o n i t o r e d a t 412 All  nm.  R e a c t i o n volumes t o t a l l e d  a s s a y s were c o m p l e t e d w i t h i n Homogenization  KC1,  buffer:  1% t r i t o n - X , pH 7.0 Assay  7.0  buffer:  Buffer  100 mM  values  phosphorylase,  Bergmeyer  i m i d a z o l e , 3.3  mM  MgCl ,  23  2  mM  i m i d a z o l e , 70 mM Citrate  K C 1 , 10 mM  s y n t h a s e was  MgCl ,  pH  2  assayed  i n 50  mM  8.1.  pH  phosphoglucose  33 mM  homogenization.  (adjusted with HC1).  (adjusted with HC1).  T r i s b u f f e r , pH  4 hours of  1 ml.  were  adjusted  at  /3-hydroxyacyl-Coa isomerase  (1974).  The  (PGI)  were  25°C.  Glycogen  dehydrogenase assayed  as  and  described  by  r e m a i n i n g enzymes were a s s a y e d u s i n g t h e  methods o f H o c h a c h k a e_t a l . , ( 1 9 7 8 b ) .  M e t a b o l i t e P r e p a r a t i o n and A s s a y . 5 control for  fish  (with access t o the s u r f a c e ) ,  3 hours, 6 f i s h  for  12  hours  the  below  6  submerged f o r 12 h o u r s , and  fish  submerged  4 fish  submerged  i n d u c e d by p l a c i n g a r e s i n c o a t e d w i r e mesh l i d  aquaria  below  the water  surface.  Water P 0  2  never  fell  120 mmHg. T i s s u e s were removed f r o m b e h e a d e d f i s h and  to  from  and a l l o w e d a c c e s s t o t h e s u r f a c e f o r 12. h o u r s .  Submergence was on  M e t a b o l i t e s were s a m p l e d  -196°C  Clamping were  was  using  aluminum  completed w i t h i n  identical  to  f r o m t h e s e v e r e d vena (PCA).  tongs  90 s e c o n d s  cava  in liquid  (s).  those d e s c r i b e d above.  B l o o d was  on d r y i c e and  frozen  nitrogen.  Regions  i n t o an e q u a l o f 6%  T i s s u e s were g r o u n d  pestle resting  cooled  rapidly  sampled collected  perchloric  t o a f i n e powder u s i n g a m o r t e r further cooled  by  flushing  acid and with  20  liquid  nitrogen.  A b o u t 0.5 gm o f powder was w e i g h e d i n a c o l d  homogenization f l a s k , d i l u t e d homogenized  in a  to  5  ml  with  1.4  with 30  discarded.  The  supernatant  1.4M KOH a n d spun a g a i n . hours  while  PCA, a n d  S o r v a l Omnimixer w i t h m i c r o - a t t a c h m e n t .  homogenate was spun a t 10,000 x g f o r 15 m i n u t e s pellet  M  the  (min.) a n d t h e  was n e u t r a l i z e d t o pH 6.0  M e t a b o l i t e s were m e a s u r e d  samples  were  k e p t on i c e .  Metabolite  the  Measurements were m e a s u r e d by e n z y m a t i c a l l y l i n k i n g  r e a c t i o n s u s i n g NADH/NAD*, o r NADPH/NADP* ( n i c o t i n a m i d e dinucleotide  them t o adenine  phosphate, reduced and o x i d i z e d r e s p e c t i v e l y ) , and  the  reaction  spectrophotometer. amyloglucosidase remaining  crude  i t was s p u n .  Metabolites  following  within  G l y c o g e n was  d e t e r m i n e d on 0.5 m l s a m p l e s w h i c h were removed f r o m homogenate b e f o r e  The  at  Glycogen  technique  metabolites  340  were  nm  on  was  (Keppler measured  and  a  Unicam  SP-1800  measured  using  the  Decker,  1974).  The  with  the  techniques of  Hochachka e t a l . , (1978c). Statistics D a t a were c o m p a r e d u s i n g S t u d e n t s 2 t a i l e d 0.05.  t-test with p  <  21  Results  Enzyme A c t i v i t i e s . the assayed  tissues.  the  and  heart  Table  liver,  occurs low  at  synthase  in  brain  which  present  i n moderate  activities also  has  and both  and  pyruvate  brain. in  any  high  in  and in  Malate  heart.  of  the  other  indicator  (PK) i s s i m i l a r  L a c t a t e dehydrogenase  and  the  in  functions, i s heart  dehydrogenase  and a l d o l a s e a r e s i m i l a r  kinase  aminotransferase  anaerobic  liver  Of  1981),  and h e a r t , b u t o c c u r s a t  (PGI) i s h i g h e s t  but  (MDH)  low  activity  glycolytic  enzymes,  i n the heart;  glycogen  i n t h e h e a r t and muscle; the  heart,  (LDH) i s h i g h e r  muscle  and  i n the heart  than  the muscle.  Metabolites.  Table  2 lists  metabolites  present  in  the  concentrations  control  lungfish  and  of  heart  after  hours  flux  through  of  submergence.  Increased  e q u i l i b r i u m enzyme p h o s p h o f r u c t o k i n a s e substrate  concentration  fell  glycolytic  Figure  r e l a t i v e changes i n b r a i n , m u s c l e , and  the  an o r d e r o f  (HOAD) , an  Aspartate  aerobic  activities  Phosphoglucose isomerase phosphorylase,  than  in liver  muscle.  i n white muscle. very  (CS)  dehydrogenase  highest a c t i v i t i e s  levels  found i n  of t i s s u e s t o o x i d i z e f a t t y a c i d s (Marsh,  (AAT),  is  w i t h heart d i s p l a y i n g almost  /3-Hydroxyacyl-CoA  of t h e a b i l i t y  t h e enzyme a c t i v i t i e s  O x i d a t i v e enzyme a c t i v i t i e s a r e h i g h e s t i n  m a g n i t u d e more c i t r a t e tissues.  1 lists  3  1 shows and  the  (PFK) i s i n d i c a t e d  12  nonsince  below c o n t r o l v a l u e s and t h e  22  Table  1. L u n g f i s h t i s s u e enzyme a c t i v i t i e s . Enzyme a c t i v i t i e s a r e e x p r e s s e d a s Mmoles s u b s t r a t e c o n v e r t e d p e r m i n u t e p e r gm wet w e i g h t o f t i s s u e . Assay c o n d i t i o n s a r e g i v e n i n 'Methods'. Values i n p a r e n t h e s e s a r e p y r u v a t e c o n c e n t r a t i o n i n mM.  23  T a b l e 1.  L u n g f i s h t i s s u e enzyme a c t i v i t i e s . (Mmole/min./gm wet w t . ) Tissue  Glycolytic  n  Mean+S.D.  o r A n a e r o b i c Enzymes  Glycogen Phosphorylase  P h o s p h o q l u c o i somerase  Fructose-1,6bisphosphatase  Aldolase  Pyruvate Kinase  Heart  6  5.15+3.57  Muscle  6  3.94+1,24  Liver  6  1 .61+0.76  Heart  6  100.1+17.0  Muscle  7  57.9+21.9  Liver  6  25.7+ 3.4  Heart  3  <1  Muscle  3  <1  Liver  3  3.14+1.17  Heart  5  15.98+ 4.06  Muscle  5  27.84+13.38  Liver  5  6.74+ 0.52  Heart  5  100.9+17.4  Muscle  6  98.8+20.7  Liver  5  14.0+  Brain  6  1.94  102.9+41.6  Table  1.  Lactate  (continued)  Dehydrogenase  Pyruvate concentration in brackets (mM)  Heart  Muscle  Liver  Brain  Creatine  Phosphokinase  Oxidative  Citrate  or Mixed  Synthase  /G-Hydroxyacyl-CoA Dehydrogenase  g-Glycerol-Pdehydrogenase  (.05)  6  593.4+229.1  (10)  6  402.9+124.8  (.05)  6  256.9+85.0  (10)  6  149.8+65.9  (.05)  6  248.6+168.4  (10)  6  167.8+115.3  (.05)  6  149.4+38.6  (10)  6  116.9+28.7  Muscle  1013 (1083.2-942.8)  F u n c t i o n Enzymes  Heart  5  17.9+1.6  Muscle  5  0.84+0.64  Liver  5  2.44+1.96  Brain  5  2.58+0.59  Heart  6  17.37+3.82  Muscle  6  1.97+0.62  Liver  6  32.89+12.8  Brain  6  3.05+0.59  Heart  3  0.22+0.30  Muscle  3  0.16+0.24  Liver  3  0.41+0.29  Table Malate  1.  (continued)  Dehydrogenase  Aspartate Aminotransferase  Heart  6  939.7+212.0  Muscle  6  195.3+ 74.3  Liver  6  333.8+106.3  Brain  5  262.0+ 73.8  Heart  5  83.4+13.5  Muscle  5  3.66+0.84  Liver  6  29.13+5.40  T a b l e 2.  Tissue Blood  C o n c e n t r a t i o n s of s e l e c t e d g l y c o l y t i c in the lungfish. (/amole/gm wet w t . )  n G l u c o s e G6P 5  F6P  FBP  G3P  metabolites  DHAP  Pyruvate  0.23 + 0.24  White Muscle 5  0..20 0. 20 0..46 0.,13 0.,02 0., 1 4 0. 10 + 0., 12 + 0. 1 3 + 0., 1 1+ 0..04 + 0.,02 + 0.,02 + 0. 05  Heart  5  0..93 0. 1 1 0..25 0..04 0.,02 0..06 + 0..28 + 0. 07 + 0.. 1 2 + 0..02 + 0.,03 + 0,.05  Brain  5  0..72 0. 1 1 0.,25 0.. 1 2 0., 1 1 0.,08 0. 1 3 + 0,.39 + 0. 09 + 0..20 + 0,.05 + 0., 1 2+ 0..07 + 0. 05  Liver  5  2.77 + 1 .99  V a l u e s a r e means + SD. Metabolite abbreviations are explained Abbreviations.  0. 16 + 0. 09  i n the L i s t of  27  Figure  1. C r o s s o v e r p l o t s of l u n g f i s h t i s s u e m e t a b o l i t e s a t t h e end o f a s u b m e r g e n c e . The u p p e r g r a p h r e p r e s e n t s t h e 3 h o u r d i v e and t h e l o w e r g r a p h i l l u s t r a t e s t h e 12 hour d i v e . C o n t r o l v a l u e s a r e l i s t e d i n T a b l e 2. The sample s i z e s a r e i n 'Methods'.  0  1  ) "  I  GLUCOSE  G6P  1 F6P  1 F-I,6BP  1 DHAP  I  G3P  PYR  1"-  LAC  29  product concentration opposite  situation  was e l e v a t e d a b o v e  across  Both  2-4  concentrations recovery.  the  Mean  glycogen,  glycogen  (Fig.  increase  3).  increases during  glucose  concentration  (Fig.  during  the  heart,  b r a i n , and b l o o d recovery  also  After a blood  from  Lactate during  . and  and  3) d i d n o t c h a n g e d u r i n g  hour  in  liver,  the heart  blood,  heart,  indicate that 12  remains  and  During the dive  hour  liver  recovery,  significantly  concentrations  decline  t h e 12 h o u r s a m p l e i n t h e  increases s i g n i f i c a n t l y  4).  pH  lactate  t h e d i v e and t h e n f a l l s  (Fig.  energy s t a t u s of the c e l l  recovery.  Glucose  recovery  h e a r t , and l i v e r .  2), while  data  the dive. in  blood,  (Table  the  d e c l i n e i n the  significantly  These  e l e v a t e d above c o n t r o l v a l u e s .  during  glucose,  concentrations  the dive  glucose  significantly  shift  d u r i n g a 12 h o u r s u b m e r g e n c e a n d a f t e r a 12  concentrations  brain  tissue  The  reduced.  return to control levels after  glucose  the  through t h e remainder of  became more  show  h e a r t , and b r a i n d u r i n g  and  this  t h e e q u i l i b r i u m enzyme LDH i n d i c a t e s t h a t e i t h e r  Figures  The  through  responses i n d i c a t e r e g u l a t o r y s i t e s .  dropped, and/or the c e l l  brain  values.  w i t h HK s u g g e s t s t h a t t h e f l u x  enzyme i s n o t a s r a p i d a s t h e f l u x pathway.  control  Metabolites  the  cellular  the experiment.  i n the  in  each  associated  with  energy  charge  30  Figure  2. G l y c o g e n c o n t e n t s d u r i n g f o r c e d submergence and recovery i n l u n g f i s h . Sample s i z e i n m e t h o d s . Star denotes a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l s t a t e . B a r i n d i c a t e s +1 S.E..  31  o • o £x  220->  White Muscle Heart Brain Liver  180 H  it:  E  1  4  0  3  g 100a O)  i. o  E -  60-  * DIVE  50-  0)  *^ 401 c o  o  R E C O V E R Y —«|  k  c 301 a> o o >>  O  20-  100  0  3  12  Time (hours)  24  32  F i g u r e 3. T i s s u e and b l o o d g l u c o s e c o n c e n t r a t i o n s d u r i n g f o r c e d s u b m e r g e n c e and r e c o v e r y i n l u n g f i s h . Sample s i z e i n methods. Star denotes a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l s t a t e . B a r i n d i c a t e s +1 S.E..  • o • o  DIVE-  Blood White Muscle Heart Brain Liver  RECOVERY-  54 o E c o  •mm  (0  o o  c o  o  o 2  CO  O O  o  0  3  i • 12 Time (hours)  24  34  F i g u r e 4. T i s s u e and b l o o d l a c t a t e c o n c e n t r a t i o n s d u r i n g f o r c e d s u b m e r g e n c e and r e c o v e r y i n l u n g f i s h . Sample s i z e i n methods. Star denotes a s i g n i f i c a n t d i f f e r e n c e from t h e c o n t r o l s t a t e . B a r i n d i c a t e s +1 S.E..  35  • ° • °-  Blood White Muscle Heart Brain  36 T a b l e 3.  C o n c e n t r a t i o n s of a d e n y l a t e s , c r e a t i n e phosphate, and c r e a t i n e , a n d t h e e n e r g y c h a r g e i n lungfish tissues. (Mmole/gm wet w t )  Conditions  n  ATP  Heart Control  5  0 .79 + 0 .36  0. 08 0.83 1 .14 0. 47 0. 15 + 0. 07 + 0. 07 + 0.u8 + 0 .50 + 1 .06  1 .02  3-hr  dive  6  1 .77 0. 36 0.91 1 .82 2. 01 0. 05 + 0 .25 + 0. 07 + 0. 04 + 0-.03 + 0 .29 + 0. 89  2 .18  12-hr  dive  6  1 .58 0. 41 + 0 .31 + 0. 10  1 .52 3. 29 0. 09 0.86 03 + 0.01 + 0 .93 + 1 .45 i°1 .33 0. 28 0. 07 0.88 + 0 .12 + 0. 03 + 0. 01 + 0.02  2 .08  2 .41 0. 33 + 0 .38 + 0. 08  12. 67 ± - 10  2 .76  14. 39 ± - 57  3 .76  6  3 .36 0. 55 0. 04 0.92 7 .94 12. 72 + 0 .37 + 0. 08 + 0. 01 + .004 + 2 .73 + 1 .55  3 .95  Recovery  4  3 .10 0. 54 0. 07 0.91 + 0 .28 + 0. 06 + 0. 02 + .006  Brain Control  5  0.77 0 .59 0. 32 0. 06 0 .52 1 .03 + 0 .22 +0. 08 + 0. 05 + 0.10 + 0 .48 + 0. 45  0 .97  3-hr  dive  6  0 .86 0. 05 0.80 2 . 1 6 3. 16 0. 33 + 0 .24 + 0. 15 + 0. 04 + 0.10 + 2 .13 + 1 .47  1 .24  12-hr  dive  6  0 .83 0.74 0. 36 0. 15 0 .60 3. 31 + 0 .32 + 0. 07 + 0. 04 + 0.09 + 0 .42 + 0. 27.  1 .34  4  0 .87 0. 39 0. 16 0.75 + 0 .29 + 0. 19 + 0. 06 + 0.11  Recovery  4  White muscle Control 5 3-hr 12-hr  dive dive  Recovery  6  ADP  AMP  EC  CrP  0. 02 0.93 1 0.56 01 + 0.01 + 1.68 i°3 . 1 4 0. 57 0. 05 0.91 8 .77 + 0 .42 + 0. 1 1 + 0. 06 + .008 + 1.77  V a l u e s a r e means + SD.  Cr  Total Adenylates  1 .45  2  3  Energy charge a f t e r A t k i n s o n  (1977).  37  Discussion  The metabolic muscle, The  results  from  changes  which  and  blood  this  experiment  occur  during  in  the  allow  one t o e x a m i n e  brain,  heart,  liver,  f o r c e d submergence i n t h e l u n g f i s h .  f o l l o w i n g d i s c u s s i o n o f t h e s e r e s u l t s i s a r r a n g e d by  with  a  concluding  occurring  between  interactions  have  section tissues,  on  the  and  tissue  i n t e r a c t i o n s w h i c h may be  the  upon t h e c a p a c i t y  influence  which  these  of t h e l u n g f i s h t o s u r v i v e  hypoxia.  Brain.  Since  the  energy  supply  for  and  enzymes  metabolism.  assessing  and  breathing ratios  Nordstrom,  are  t h e LDH/CS,  relative  and  and  absolute  to other  activities  in o x i d a t i v e metabolism 1).  observed  The a c t i v i t y  for a  in  both  the  wide  PK/CS  ratios  e_t a l . , and  technique  and  anaerobic ratios  of  Table 4  can  be f o r  two o b l i g a t e a i r -  f i s h e s and mammals.  capacity  for  aerobic  activity  Arapaima q i q a s ,  p o r t i o n o f t h e b r a i n ' s ATP p r o d u c t i o n  (Table  1977 ;McDougal  utilized  i s t o compare  i n d i c a t e an i n c r e a s e d  The  metabolism  o x i d a t i v e enzymes ( K e u l e_t a_l. , 1 9 7 2 ) .  aethiopicus  fish,  glucose  the r e l a t i v e p o t e n t i a l s of a e r o b i c  i n d i c a t e s how h i g h Proto.pterus  upon  B e c a u s e o f t h i s , an i n s t r u c t i v e  metabolism i n the b r a i n glycolytic  relies  (Siesjo  1968), g l y c o l y t i c anaerobic  brain  Such  for providing  a  high large  via glycolysis.  o f two enzymes n o r m a l l y  functional  ( C S , a n d HOAD) a r e l o w i n l u n g f i s h b r a i n o f CS i s o n l y a b o u t variety  of  species  1/10 t h a t including  typically fishes,  38  Table  4,  C o m p a r a t i v e a c t i v i t y r a t i o s o f enzymes f r o m a n a e r o b i c and a e r o b i c pathways i n b r a i n s o f f i s h a n d mammals. L a c t a t e dehydrogenase/Citrate synthase  Spec i e s Air-breathing  Fish  P. a e t h i o p i c u s Arapaima Aquatic  Pyruvate kinase/Citrate synthase Reference  58  qiqas  1 40  Breathing  40 128  This  study  H o c h a c h k a e_t a l . , 1978c.  Fish  Coryphaenoides r u p e s t r i s  14  I1  Coryphaenoides armatus  16  10  S i e b e n a l l e r e_t a_l. , 1 982. ibid  Coryphaenoides l e p t o l e p i s  14  II  ibid  Medialuna c a l i f o r n i e n s i s  12  7  Chromis  12  7  1 1  9  puntipennis  Genyonemus  lineatus  S u l l i v a n a n d .Somero 1980. ibid ibid  Mammals Weddell seal (Leptonychotes Ox  weddelli)  (Bos t a u r i s )  13  12  Murphy e_t a l . , 1980.  10  ibid  39  amphibians,  mammals,  Newsholme, 1975).  and b i r d s  (Murphy e t a l . ,  Thus, t h e p o t e n t i a l  l980;Sugden  for oxidative  i n t h e b r a i n o f P. a e t h i o p i c u s i s d e p r e s s e d , w h i c h t h e b r a i n of Arapaima An also  indication  be  q i q a s (Hochachka,  from  (McDougal e t a l . ,  1968).  are  of t h o s e  turtle,  and a r e about  stores  of  et  al•,  most  1980).  the  size  of  glycogen storage  Brain glycogen l e v e l s i n the Weddell  seal,  i n the  depots  lungfish  f r o g , and d i v i n g  2-4 t i m e s h i g h e r t h a n t h e  brain  glycogen  o t h e r v e r t e b r a t e s (Kerem e t a l . . , 1973 ;McDougal  view of t h e above c o n s i d e r a t i o n s ,  that extended  submergence r e s u l t s  ( F i g . 4).  derived  This  i tis  end-product  f r o m b o t h e n d o g e n o u s g l y c o g e n , s i n c e some  blood  glucose  which,  (Fig. owing  not  surprising  i n lactate accumulation  anaerobic  brain glycogen stores occurs of  i s similar to  1968).  In  brain  metabolism  of t h e a n a e r o b i c c a p a c i t y of b r a i n t i s s u e can  obtained  i n t h e range  and  i nthe  appears  t o be  depletion  of  2 ) , and f r o m t h e u t i l i z a t i o n to  the  blood/brain  glucose  g r a d i e n t , may be t a k e n up d u r i n g t h e e n t i r e  submergence  (Fig.  i n the brain during  3).  submergence indicated  That  glycolysis  i s activated  ( r a t h e r than s i m p l y o p e r a t i n g a t normoxic (a)  by  crossover  p l o t s showing  p h o s p h o f r u c t o k i n a s e r e a c t i o n , and l i m i t a t i o n reaction lactate  (Fig.  indicated  A  good  the  is  at the  hexokinase  1 ) , a n d ( b ) by t h e c o n t i n u o u s a c c u m u l a t i o n o f  i n the brain  Heart.  rates)  facilitation at  period  (Fig. 4).  potential  .for  carbohydrate  metabolism  is  by h i g h g l y c o g e n p h o s p h o r y l a s e , P G I , a l d o l a s e , a n d PK  40  activities, heart  a l l i n about  (Hochachka  et a l . ,  glycolytic  p a t h may  conditions  since  process  and  shuttle.  a-glycerol  significant  these  activities  capacity.  CS  The  former  or salmon  enzyme  phosphate to the  (Table  CS  low  and  ox  are high.  equivilents  This  into  the  malate-aspartate  d o e s not  activity  appear  of  to  be  a-glycerol  also  indicate  2/3  the  level  a good a e r o b i c  similar  (Murphy et: a_l. , 1980)  potential  found  i n the  metabolite data.  i n tuna  of  to  that  while  the  (Guppy et. a l . ,  enzyme  of  i s 1.5  (Hochachka e t a_l. , 1978c), two  times  times  (Mommsen e t aJL. , 1980). of a n a e r o b i c  indicated  main enzyme  higher  LDH,  than  The  i n tuna  heart  than  in  times  Heart  glycogen and  of  lactate.  three  potential  of  which  1) , a p r o p e r t y to  higher  by  indicator  type  (Table  pyruvate  (Hochachka e_t a l . , 1978a), and  indication  is  of m u s c l e  inhibition  favor conversion this  heart  The  pyruvate  another  the  under . a e r o b i c  AAT  in a c t i v i t i e s  lack  salmon h e a r t  that  tuna  1).  shows  heart  assumed  shuttle  i s the a c t i v i t y  of  is  enzymes f u n c t i o n i n t h e  potential  activity  in  (Mommsen e t a l . , 1980).  and  isozymes which  observed  and  reducing  anaerobic a  as  effectively  of MDH  seal  A high anaerobic both  of  i s present  i s about  It  quite  of HOAD and  i n the Weddell  activity  1979)  1978a).  i n t h e h e a r t due  phosphate dehydrogenase The  range  the a c t i v i t i e s  requires a shuttle  The  same  function  mitochondria  found  the  higher  Arapaima than  stores  are high  in are  compared  t o most v e r t e b r a t e s (Dawes e t a_l. , 1959;Kerem e_t a l . , 1973). Compared oxidative:  CS  to other  lungfish  activities  are  tissues, about  20  the times  heart higher  is  very  than  in  41  muscle, 8 times brain  (Table  breathing  1).  fish  organization, heart  by  (Hochachka,  1980).  is  probable  is similar  oxidative  conservation s i n c e a 30%  (Conant,  1975)  supply  indicated  with  a l l of  in  during  endogenous  occur  was  plots  (Bilinski  initiation extended  during  of  by  0  2  demands,  submersion in.  submerged l u n g f i s h  Since  load  during  enough  oxygen  A c t i v a t i o n of g l y c o l y s i s (Fig.  1)  (Fig.  which  show  lactate  4 ) , and  glucose  moves i n  the  most  tissues (McGilvery,  t h a t the h e a r t  may  u t i l i z e plasma g l u c o s e becomes f a v o r a b l e  is  that  accumulation  ( c ) by  depletion  s t o r e s f o l l o w e d by u t i l i z a t i o n  in  gradient  work  receive  a c t i v a t e d , (b) by  submergence  glycogen  stores.  does not  concentration  glucose  aerobic,  i n c a r d i a c energy  cardiac  i t s requirements.  (a) by c r o s s o v e r  the h e a r t  glucose  be  or e x p e r i m e n t a l l y  reductions  phosphofructokinase  of  fully  Upon  may  r a t e can  submergence, however, the h e a r t  in  conditions  et a l . , 1970).  Even  to  in  metabolic  carbohydrate  1972).  m e d i a t e d by a r e d u c t i o n  unrestrained  fishes;  metabolism  d e c l i n e in heart  than  this  under normoxic  f a t and  Jonas,  higher  Given  other of  7 times  i s a l s o found i n other a i r -  that  to  1 9 7 0 ; B i l i n s k i and  submergence,  and  pattern  f a t or.by a mixture  Jonas,  (Lahiri  than i n l i v e r  A similar  it  metabolism  fueled and  higher  of  direction  blood  of  low  1979), i t i s l i k e l y  after  for glucose  the  blood/heart  uptake  (Figs.  2,  3). Lactate those heart  i n the  concentrations b r a i n , and  in  s i n c e the  undoubtedly surpass  those  the  heart  energy of the  reach  roughly  requirements  brain during  of  half the  submergence  42  it  can  be  t e n t a t i v e l y concluded  metabolic (even  rate  still  when submerged t h e  resting  oxygen  metabolic to  is  be  a  hypoxia,  with indications  particularly tuna only  (Hochachka  muscle  in  PK  the  the s k e l e t a l  1978b).  2),  In  an  i n response of  to  anaerobic  enzymes  i s only  display in  (Mommsen e t a l . , 1 9 8 0 ) ,  and  fast  1/13  bicirrhosum)  swimming s p e c i e s  ratio  is  about  a h i g h e r LDH  of  lungfish  exceptionally  This, coupled  occurs at very  low  The  normal range f o r f i s h muscle  white low  (tuna,  20  times  activity  than  muscle  (see  with  capillarity  the  low  t h a t energy p r o d u c t i o n rates.  anaerobic  white muscle.  Moon, 1 9 8 0 ) .  appears  (Guppy e t a l . , 1979,'Mommsen e_t a l . ,  suggests  s u s t a i n e d a e r o b i c and lungfish  there  (Osteoqlossum  LDH  studies  show  activities,  metabolism  The  muscle.  mitochondrial density. enzyme  (McMahon, 1 9 7 0 ) ) .  that  h e a r t a c t u a l l y has  Ultrastructural Section  10%  activation  activity  arawana  i n the l u n g f i s h  Lungfish  the  glycolytic  or salmon  salmon) the white muscle/heart  1980).  metabolism of  metabolism  some  heart  occurred.  1979)  e_t a l . ,  g r e a t e r than  water  anaerobic  low a c t i v i t i e s .  the l e v e l  of  s u p p l i e d by a e r o b i c  the  that  White  (Guppy e t al., 1/5  from  m i x e d a e r o b i c and  muscle.  fraction  here i s , t h e r e f o r e , complex:  e n e r g y p r o d u c t i o n has  White  being  some  f i s h obtains approximately  uptake  situation  that  activity (Johnston  oxidative  from a e r o b i c  Thus t h e c a p a c i t y f o r  energy  production  of CPK,  and  is  low  both in  however, i s i n the  e_t a l . , 1977; J o h n s t o n  T h i s i n d i c a t e s t h a t phosphagen h y d r o l y s i s  is  and the  43  most  likely  source  of energy f o r t h e r a p i d and p o w e r f u l  burst  work o f w h i c h l u n g f i s h m u s c l e i s c a p a b l e . There a r e three energy  utilization  extremely  low.  measurements glycogen  (Fig.  of  Firstly, during  of  evidence  white  muscle  (Fig.  (Fig.  4),  no  suggesting during  d i r e c t evidence  submergence  depletion  accumulation  the  lines  2),  charge  c r e a t i n e phosphate  (Table  no  significant  concentrations  3 ) , a n d no s i g n i f i c a n t Secondly,  suggests  t h a t muscle p e r f u s i o n  has  (Lahiri  et a l . ,  concentrations  The  fallen  during of  i n t h e muscle remain independent of b l o o d  (Figs.  3,  concentration  Since  gradients,  m e t a b o l i t e p o o l s over minimal  both  perfusion  lactate lack  of  12 h o u r s  also  indicates  of t h i s  requirements  and  concentrations move  down  in  these  equilibrium  i n muscle.  that  there  is  Thirdly, whether  The summed  oxygen  o f t h e h e a r t a n d b r a i n i n a submerged l u n g f i s h a r e oxygen uptake,  significant  depression  metabolic  and i t i s  occurs  unlikely  i n these  S i n c e oxygen uptake has d e c l i n e d t o a t l e a s t  resting  glucose  c a n be u s e d t o i n d i c a t e  15% t o 5 0 % o f t h e t o t a l  on).  evidence  t i s s u e d u r i n g submergence.  d e p r e s s i o n has o c c u r r e d  in  submergence  glucose  a  e s t i m a t e s o f oxygen r e q u i r e m e n t s metabolic  and  or  d e p l e t i o n of  indirect  lactate  4).  lactate  change i n l a c t a t e / p y r u v a t e r a t i o s  (CrP) (Table 3 ) .  1970).  metabolite  significant  1 ) , no c h a n g e i n ATP, ADP, o r AMP energy  is  from  show  no  the  submergence  arises  which  that  value  when  the  lungfish  i s submerged  that  organs (see 40%  (unpub.  of  the  obs.),  t h a t w o u l d l e a v e 2 0 % t o 34% o f t h e r e s t i n g o x y g e n u p t a k e f o r t h e musculature.  It i s unlikely  that the  muscle  utilizes  such  a  44  small  percentage  of  the  following calculation. mass,  and  02/gr/hr  if  that  (Gordon,  lungfish  r e s t i n g o x y g e n u p t a k e b a s e d upon  I f muscle muscle  has  a  the  muscle  mmoles  0 /hr.  1968), then  requires  0.173  r e s t i n g o x y g e n u p t a k e o f 0.25 i s more t h a n consistant has  the t o t a l with  occurred  in this  this  Liver.  The  supply  glucose  reduction  whole  delivery  by  e n e r g y p r o d u c t i o n has  main  for  prolonged 3)  are  glycogen  closely  to  of l i v e r  s t o r e s and (Figs.  export  The the  the  which  This  is  depression  conforming the  i n d i c a t e d by  the 2, than  sharp 3).  In  other  indication  liver/blood  glucose  state,  to glucose of  and the  is  the  elevation Liver  (Fig.  in  glucose which  3).  glucose  during  gradients (Fig.  which  implies  liver  perfusion  liver/blood  p e r i o d may  to  marked  i n the b l o o d ,  supply  recovery  to  energy  submergence  to  Reduction  of t h e  be  to other t i s s u e s  resting  glycogen  coordinated.  g r a d i e n t a t t h e end  animal.  reducing  during  must r e m a i n p e r f u s e d  submergence.  conversion  obs.),  of g l y c o l y s i s .  t i s s u e s , as  concentrations  similar  of  occurred.  function  other  favors continued glucose liver  ml  resting  70%  n o t a p p e a r t o d i s p l a y any  c o n c e n t r a t i o n s are s u b s t a n t i a l l y higher  The  kg  is  (unpub.  t h a t t h e m u s c l e may  does  liver's  glucose  mmoles/kg/hr  preventing activation  in liver  1  This  2  body  tissue.  tissue  that anaerobic  a  t h a t some m e t a b o l i c  oxygen  words,  of  the  r a t e of 0.19  suggestion  declining and  metabolic  of  the  indicate  requirements  80%  u p t a k e of  These data the  free  the  comprises  the  be due  to  that are  glucose either  45  increased and  glycogen  subsequent  g l u c o s e l o s s from t h e l i v e r .  brain glycogen recovery  f o r m a t i o n or t o i n c r e a s e d p e r f u s i o n of l i v e r  stores  i t is likely  are  S i n c e t h e h e a r t and  preferentially  that l i v e r  replenished  during  glucose i s s t i l l  going t o those  liver  occurs  tissues. Gluconeogenesis  in  the  probably  during  r e c o v e r y because oxygen as w e l l as s u b s t r a t e s a r e a v a i l a b l e . relatively  high l i v e r  levels  of  LDH/PK  ratio  which  is  systems  observed  (Table 1),  in  (Hochachka,  other  1980).  and  a high  lactate-primed  Lactate i s l i k e l y to  t h e m a j o r s u b s t r a t e due t o i t s a v a i l a b i l i t y .  L u n g f i s h metabolism seals,  during  redistribution  oxygen and s u b s t r a t e s allowing  forced  for  them t o r e m a i n  require  metabolic  (Hochachka, In  the  central  to preserve  organs,  thus  t o h y p o x i c damage, do  adaptations  to  survive  the  brain  hypoxia  and  to  prolonged  not  hypoxia  combined  physiological  occur  body a r e n o t s u f f i c i e n t  show  these adaptations oxygen  l e s s than h a l f  the  the  definitely  T h u s , even t h o u g h t h e c o m b i n e d  oxygen u p t a k e , 1  heart  and u t i l i z e  b r a i n and h e a r t i s p r o b a b l y  in  i s designed  submerged  T h i s means t h a t t h e b r a i n a n d  are sensitive  lungfish,  upon s u b m e r s i o n . the  by  In  1980).  the  adaptations  use  aerobic.  which  major  submergence.  of c a r d i a c output  h e a r t , two o r g a n s  of  c a p a c i t y i s i n d i c a t e d by t h e  fructose-1,6-bisphosphatase  gluconeogenic be  gluconeogenic  A  uptake  of t h e t o t a l  adjustments  which  t o s u p p l y t h i s amount of  4  46  oxygen t o t h e s e  two  organs.  A s t r i k i n g d i f f e r e n c e b e t w e e n s e a l s and blood  glucose  levels  fall  This  pattern  may  develop  rise in  i n d i c a t e d by low enzyme a c t i v i t i e s  (see  Section  continuously in  forced  Thus l i v e r  2).  that  i n the l u n g f i s h ( F i g .  lungfish  p o r t i o n o f t h e body, t h e m y o t o m a l m u s c l e , i s as  is  d u r i n g f o r c e d submergence i n t h e s e a l  (Murphy e_t a_l. , 1 9 8 0 ) , w h e r e a s t h e y 3).  lungfish  Also,  the  because a l a r g e  relatively  inert,  and i t s f i b r e o r g a n i z a t i o n  liver  remains  perfused  s u p p l i e s glucose,, a s i t u a t i o n which does not  and occur  s u b m e r g e n c e i n m a r i n e mammals ( Z a p o l e t a_l. , 1 9 7 9 ) . glucose production  in  the  lungfish  outstrips  the  demand a n d b l o o d g l u c o s e c o n c e n t r a t i o n s i n c r e a s e . These during  data  indicate that a l t e r a t i o n s  submergence  maintain  the  lungfish  may  (a) c o n t i n u e d d e l i v e r y of g l u c o s e  tissues,  and  tissues.  In t h i s  dominating the  in  (b)  i n perfusion patterns be  functioning  from t h e l i v e r  l a c t a t e c l e a r a n c e from t h e l a c t a t e  s c e n a r i o , o x y g e n c o n s e r v a t i o n may  a f u n c t i o n a s i t i s i n a q u a t i c mammals.  tissues  i n the l u n g f i s h  to the  producing  not As a  require a significant  to  be  as  result,  tolerance to  hypox i a . Glycolytic  activation  the h e a r t and b r a i n ,  but  i n response not  in  t o hypoxia  the  muscle.  i s observed i n The  lack  of  ^ f t e r 1 h o u r o f s u b m e r g e n c e , a 500 gm l u n g f i s h h a s an oxygen u p t a k e o f 0.1 mmoles/hr ( u n p u b l i s h e d d a t a ) . The m e t a b o l i c r a t e s of v e r t e b r a t e h e a r t s v a r y f r o m 0.04-0.3 mmoles 0 /gm/hr (Lochner et a l . , 1968;Reeves, 1963). If this range i s a p p l i c a b l e t o l u n g f i s h i t suggests a heart metabolic r a t e o f 0.01 t o 0.05 mmoles G / 0 . 2 3 gm heart/hr f o r a 500 gm f i s h . B r a i n oxygen r e q u i r e m e n t s a r e e s t i m a t e d a t 0.003 mmoles O /0.27 gm brain/hr using data for fish b r a i n f r o m M c D o u g a l e_t a l _ . ( 1 9 6 8 ) . The summed o x y g e n r e q u i r e m e n t s o f t h e h e a r t and b r a i n r a n g e f r o m 15% t o 50% o f t h e o b s e r v e d submerged o x y g e n u p t a k e . 2  2  2  47  response stress  in  the  muscle,  c o n c o m m i t a n t w i t h an o b v i o u s  i n t h e o t h e r t i s s u e s a l l o w s one  s i g n i f i c a n t adaptation to hypoxia capacity  for  tissues.  Instead, i t i s l i k e l y  activation  in  the animal's I t has exhibit  increased  the  anaerobic  f i s h e s may  energy  that  lack  of  dysoxia  most  n o t be  production  the  muscle d u r i n g hypoxic  already  been  80%  decline  submergence  (Lahiri  experiment  supports  Furthermore,  i n these  t h a t the  the  in a l l  glycolytic  i s t h e key  to  survival.  an  metabolic  to suggest  hypoxic  rate it  calculated in  metabolic  e t a_l. , 1 9 7 0 ) .  of is  the the  The  . suggestion lungfish  proposed  that  the  lungfish  rate  during  data  from  that  a  occurs  that metabolic  the  r a t e of t h e w h o l e  animal.  in  the  hypoxia.  depression  w h i t e m u s c l e mass i s t h e m a i n c a u s e of t h e d r o p i n t h e  forced present  decline  during  may  i n the  metabolic  48  SECTION  An  ultrastructural  and  musculature  2  h i s t o c h e m i c a l study i n the  African  of  the  axial  lunqfish  Introduction  As  was  shown  surviving  for  suggested  that  the  capacity  when  oxygen  being  hours  of  upon i t  composition  and  different the  which  wide fibre  pattern  the  of  On  body  a  muscular  slow  so  the  (and  fibre  this  prevent  of  to  the  types  If  i t may in  of  analyse  of  the be  fish  the  be  hypoxic  the  this  fibre  muscle.  potential may  to  of  further muscle  i s capable  possible may  emphasis  lungfish  muscle  to  glycolysis  during  metabolic  was  due  such  of  of  It  was  investigation  metabolism  then  With  properties  an  capable  hypoxia.  muscle  important  such  to  activation  the  on  is  tolerance  limiting.  hypoxia).  of  a  determine  capable  of  such  a  regulation. the  data  i n the  previous  organization for a  movements  entirely or  and  lungfish  subjected  of  literature  of  b a s i s of  Furthermore, almost  of  types  metabolic  the  predict  to  role  deemed  i n metabolism,  of  being  ultrastructural  submergence  reduction  muscle  was  the  component  the  understanding  during  prey  the  1,  concentrations are  situations,  is a  while  a major  placed  There  in Section  involving  restricted swimming.  to  the  either  "Burst"  minimum trunk  burst  muscle  has  section,  one  supply  oxygen.  of  musculature  lunges been  at  may  are  passing  d e s c r i b e d many  49  t i m e s and 1959).  i s comprised  largely  This allows for intense anaerobic  " t h i s c r i t e r i o n as w e l l , one adapted  of w h i t e f i b r e s  (Boddeke  f u n c t i o n and  et a l . thus,  by  would expect a muscular o r g a n i s a t i o n  f o r low o x y g e n , ( B o d d e k e e_t a l . , 1 959; J o h n s t o n ,  1981).  50  Methods  Animals.  African  collected  from  they  Lake  were k e p t i n  within  lungfish  (Protopterus  aquaria.  two weeks o f c a p t u r e  Fish  cooled  on  steel  were k i l l e d  chucks  to i t s freezing  sections  7-1OMm  Slee c r y o s t a t . within  24  The  and  adenosine  removed.  The  with  liquid  various  7-10/im  were n o r m a l l y . u s e d  chloride  tissue  was  isopentane Muscle  for  (1977).  Fresh  a t pH  by  histochemistry  s e c t i o n s used a f t e r  three  were  stained  (ATPase)  for  myofibrillar  using a modified  v e r s i o n of  by D a v i s o n a n d  u n f i x e d s e c t i o n s were p r e i n c u b a t e d  for  10.3 f o l l o w e d by i n c u b a t i o n a t pH 9.4  with  f o r 20  followed  a  activity.  thick  triphosphatase  ATP a s s u b s t r a t e  which  and 16/im t h i c k were t h e n c u t a t -20 °C u s i n g Sections  times  used  period.  nitrogen.  t h e method o f G u t h a n d Samaha ( 1 9 7 0 ) a s d e s c r i b e d Goldspink  this  r a p i d l y frozen using  point  showed enzyme  Sections  were  by d e c a p i t a t i o n , a f t e r  hours of c u t t i n g , although  weeks s t i l l  animals  and were n o t f e d d u r i n g  b l o c k s o f a x i a l m u s c l e were q u i c k l y mounted  were  V i c t o r i a and t r a n s p o r t e d t o N a i r o b i where  seperate  Histochemistry.  aethiopicus)  min..  Further  yellow  ammonium  incubation sulphide  in  cobalt  resulted in a  brown t o b l a c k p r e c i p i t a t e o f c o b a l t s u l p h i d e a t s i t e s o f enzyme activity. white  Using  fibres.  instability  t h i s method, Red  fibres  pink do  not  fibres  stain  stain  at  o f t h e r e d - m u s c l e enzyme a t h i g h  pH.  darker  than  a l l due t o t h e  51  The  activity  demonstrated et a l . shown  dehydrogenase  w h i l e t h a t of l a c t a t e  using  acid  Sudan  Schiff's  s t a i n s were 16jum t h i c k .  Black  B  (1958).  and  technique.  was  (LDH)  Lipid  glycogen  Sections  for  included in a l l staining  rerio  was  deposits  using  the  these  four  S e c t i o n s were c u t -(7-10) jum f r o m  of m u s c l e f r o m t h e t e l e o s t B r a c h y d a n i o were  (SDH)  dehydrogenase  t h e method o f N a c h l a s e t a l .  were l o c a l i z e d periodic  succinic  u s i n g t h e n i t r o - b l u e t e t r a z o l i u m method o f N a c h l a s  (1957), by  of  blocks  (zebrafish).  These  r e a c t i o n s as " c o n t r o l s " t o e n s u r e  t h a t t h e s t a i n i n g p r o c e d u r e s were f u n c t i o n i n g . S t a i n e d s e c t i o n s were u s e d t o i d e n t i f y identified,  diameters  occular eyepiece type  were  of f i v e  of  the  fibres  on a l i g h t m i c r o s c o p e .  counted from both  fibre  types.  were  m e a s u r e d u s i n g an  Fifty  f i b r e s per  fibre  t h e a n t e r i o r and p o s t e r i o r r e g i o n s  fish.  E l e c t r o n microscopy. The f i r s t  Two m e t h o d s o f i n i t i a l  method u s e d a 3% s o l u t i o n  phosphate  buffer  with  fixation  of g l u t a r a l d e h y d e  0.5% s u c r o s e  a t pH 7.5.  was d r i p p e d  onto the t i s s u e  of  were t h e n  tissue  t h e same f i x a t i v e . with  3%  f o r s e v e r a l minutes.  removed a n d f i x e d  The s e c o n d  glutaraldehyde  method  were in  used. 50  mM  A f t e r removing  the s k i n o v e r l y i n g the muscles i n t h e i n t a c t animal  fish  Once  the f i x a t i v e Small  pieces  f o r a f u r t h e r 4 hours i n involved  perfusing  i n phosphate b u f f e r . to  vena c a v a .  i n s e r t e d i n t o the vena cava  and  the  blood  was  flushed  was  out  of  the  the  incision  was made i n t h e a n a e s t h e t i z e d a n i m a l An o c c l u s i v e c a n n u l a  expose  An  the  vascular  heart  and  s y s t e m by  52  perfusing  with  perfusing  isotonic ringer solution.  with  fixative  f o r 15 m i n u t e s .  This  was  followed  Small pieces  by  of muscle  were t h e n removed a n d f i x e d f o r a f u r t h e r 4 h o u r s . All  t i s s u e s were p o s t f i x e d i n 2% osmium t e t r o x i d e a n d  embedded MT-1  in  Epon.  ultramicrotome.  stained with using  uranyl  a Z e i s s EM Capillary  on t h e  grids  microscope  fibres.  acetate  density and  was c a r r i e d o u t on a P o r t e r - B l u m  The s e c t i o n s were  10 e l e c t r o n  slides  d e t e r m i n e d by  and l e a d c i t r a t e .  on  Grids  and  were v i e w e d  microscope.  by  (Mosse,  using 1979).  thin  sections  mounted  on  Mitochondrial  density  was  photoelectronmicrographs  were c u t o u t i n o r d e r  composed o f  grids  was d e t e r m i n e d by s c a n n i n g t h e s e c t i o n s  also  taking  mounted  of  The m i c r o g r a p h s were t h e n w e i g h e d b e f o r e  mitochondria cell  Sectioning  then  mitochondria.  the  muscle  and a f t e r t h e  to f i n d the f r a c t i o n  of  the  53  Results  Gross structure were  of  the  composed  hypaxial  to  lateral colour  mainly  on  the  fibres  could  be  running  towards a  few  skin.  In  up  very  a  the  percentage the  amount  lateral seen  the cells  small  the of  line.  on  each  side  fraction  as  of  of of  a  to  the  cavity  is  small was  lateral  line.  towards  The  to  diameter  the  mainly  in  amount  of  a  (up  to  the of  of  red  below  the  3%).  This  20%)  between the  mass  tail the  of  of  made  total the  band  myoseptum  t i p of  of  wedge  connective  at  muscle  (ca.  the  remainder the  tissue  layer  red  mass  and  large,  its  tail. of  red  at  the  body.  The  tissue  tissue also  at  the  increased  tail. fibres  stained  less  preincubation LDH.  the  located  the  White  because  relative  be  to  transverse  this,  muscle  the  thin  the  of  small  a  body,  region  is still  wedge,  the  of  due  the  posterior  to  red,  immediately  gradually  appears  region  of  and  from  found  increase  number  the  wedge  the  to  this  myotomes  epaxial  be  the  body  into  at  muscle  The  termed  this  Further  thick, could region  muscle  From  a  transversely fish  found  vertebra.  anterior  increase  and  small  running  of  5).  separated  found  muscle  they  muscle  myoseptum  revealed  (Fig.  was  Although  tip  white  lungfish  type  d i s s e c t i o n , was  of  muscle,  "fish"  of  a  the  periphery  The  gross  region  by  the  line.  of  typical  regions  vertebra  the  dissection  The  could  be  identified  intensely  times. fibres  In did  than  addition not  the  using  ATPase  intermediate  they  stain  the  for  failed lipid,  to  stain,  fibers stain  although  as  at  long  for  SDH  they  did  54  F i g u r e 5. Lungfish cross-sections. Note the l i n e of r e d c o l o r e d m u s c l e i n t h e a n t e r i o r c r o s s - s e c t i o n ( r i g h t ) and t h e t h i c k e r wedge i n t h e p o s t e r i o r c r o s s - s e c t i o n (left). Bar = 2cm  55  56  show a s l i g h t myotome  reaction f o r glycogen.  The v a s t m a j o r i t y  was composed o f p u r e w h i t e m u s c l e f i b r e s w i t h no  with the other  types  (Figs.  s e c t i o n a l a r e a , by f a i l u r e  to stain  by t h e i r  (Figs.  6,7),  glycogen  f i b r e s were g e n e r a l l y l a r g e r intensities of this  the  than  small cross-  and  the red  for  lipid. and  SDH  (Figs.  Intermediate  showed  staining  t h a t were i n t e r m e d i a t e b e t w e e n r e d a n d w h i t e  s t a i n s used except  latter  mixing  f o r A T P a s e a t h i g h pH ( F i g .  8 ) , a n d by d i s p l a y i n g t h e s t r o n g e s t r e a c t i o n s LDH  the  6-10).  Red m u s c l e f i b r e s c o u l d be i d e n t i f i e d  9,10),  of  t h a t f o r ATPase ( F i g s .  for a l l  6-10).  r e a c t i o n , t h e i n t e r m e d i a t e f i b r e s were f o u n d  With to  be  t h e most s t a b l e a n d , t h e r e f o r e , t h e most h e a v i l y s t a i n e d a t l o n g preincubation sections  for  times. the  ATPase  sections  for  necessary  t o use  staining  reactions  use  thicker  It  the  were were  sections  in  p a u c i t y of m i t o c h o n d r i a and  stains  obtained  sections  perhaps  reaction  other thick  should  thick  sections difficult  different  results  from the zebra with  the  stained very well teleost division Mitochondria  sections.  including  fish.  obtained  While  11).  of f i b r e s very  into red, evident  w i t h a marked subsarcolemmal  fish  I t was  very  faint  there  and  was  a  lipid.  with the " c o n t r o l " was fish  generally sections  showed t h e t y p i c a l  intermediate  and  i n r e d and i n t e r m e d i a t e  accumulation.  while  the red muscles,  staining  The z e b r a  that  T h i s need t o  that  l u n g f i s h m a t e r i a l , the zebra  (Fig.  were  were  (7-10/im)  the  a l s o t h a t t h e r e were low s t o r e s o f g l y c o g e n Very  here  (16MII0.  of  indicated  i n the c e l l s ,  noted  thin  because  with thin  itself  be  white. fibres  57  Figure  6. A n t e r i o r l a t e r a l l i n e r e g i o n s t a i n e d f o r LDH activity. Most o f t h e f i b r e s a r e " w h i t e " w i t h a t h i n s t r i p of r e d a n d i n t e r m e d i a t e f i b r e s f o r m i n g a p e r i p h e r a l mosaic. Bar = 1.0mm  Figure  7. Mosaic r e g i o n of t h e a n t e r i o r l a t e r a l f o r LDH a c t i v i t y . B a r = 0.5mm  Figure  8. Mosaic r e g i o n of the a n t e r i o r l a t e r a l l i n e s t a i n e d f o r ATPase a c t i v i t y . The d a r k e s t f i b r e s a r e intermediate f i b r e s , moderately s t a i n e d f i b r e s are white f i b r e s , and u n s t a i n e d f i b r e s a r e r e d f i b r e s . Bar = 0. 5mm  line  stained  59  Figure  9. P o s t e r i o r l a t e r a l - l i n e r e g i o n s t a i n e d f o r SDH activity. The a r e a m a r k e d 'u' i s s i m i l a r i n o r g a n i s a t i o n t o the a n t e r i o r mosaic region a l b e i t somewhat more e x t e n s i v e . In a d d i t i o n there i s a second mosaic r e g i o n marked 'x', c o n s i s t i n g m a i n l y of i n t e r m e d i a t e and w h i t e f i b r e s . B a r = 2.0  Figure  10. P o s t e r i o r m o s a i c r e g i o n s t a i n e d f o r SDH (marked 'x' i n F i g u r e 5) B a r = 0.1mm  Figure  11. S e c t i o n f r o m z e b r a f i s h s t a i n e d f o r LDH a c t i v i t y . The s t a i n c o n c e n t r a t e d on t h e c e l l p e r i p h e r y i n d i c a t i o n subsarcolemmal-concentrations of m i t o c h o n d r i a . Note t h a t c o n n e c t i v e t i s s u e s e p a r a t e s t h e f i b r e t y p e s (R = red f i b r e , I = i n t e r m e d i a t e f i b r e , W = white f i b r e ) . Bar = 1.0mm  activity,  60  61  In the found they  in  l u n g f i s h , r e d , i n t e r m e d i a t e , and the  regions  were s e e n as  intermediate  and  regions  were  the  material  with  a  fibres  (Kilarski,  into  of the  for active aerobic Blood  small  6,9).  fixed  long  q u a n t i t i e s great  into In  of  the  a  the work  Most of  the  red  and  three-fibre  composed of w h i t e  cell  containing  and  cylindrical  T h i s was  very  in  post-mortem  fixative  in teleost 1972).  not  had  not  effective,  in  obtained  degenerative reached the even a f t e r  15  The  designed  (Table  demonstrated very w e l l Sections  the  cross-section  Goldspink,  scarce  A  s i t u a t e d at  This applied to a l l three  perfusion.  enough t o be  contractile  g e n e r a l l y found and  were  12,13).  l u n g f i s h m u s c l e was  were  fibres  (Figs.  w i t h the t r i a d s  ribbons  respiration.  the  and  histochemical  muscle  Patterson  showed v e r y m a r k e d that  blends  t h a t the  each  rougly  t h a t the  by  with  A red  fraction  mosaic area  present  capillaries  around red f i b r e s .  types  dissection.  sarcomeres  1967;  micrographs revealed  indicating  a  Here  9,10).  with  s y s t e m was  fishes  material  the  gross  revealed  M y o f i b r i l s were  were  only  (Figs.  arranged  formation  that  (Figs.  body,  further  structure  w i t h no  types.  fibre  extreme p e r i p h e r y  f i n d i n g s of t h e  was  sarcotubular Z-line.  majority  the  E l e c t r o n microscopy in  three  were  dissection.  In the p o s t e r i o r r e g i o n next t o  area  typical  a l l  gross  fibres  f i b r e mosaic at a deeper l e v e l .  of  white  intermediate.  intermediate  of  f i b r e mosaic at the  complimented the  mosaic  up  intermediate  anterior  fibres  mosaic  making  intermediate white  a  termed red d u r i n g  white  fibre  6 ) , even samples from  this  changes, tissues in minutes.  62  Figure  12. L a t e r a l s e c t i o n of w h i t e muscle m y o f i b r i l s . N o t e t h e s h a r p Z - l i n e , l a c k of m i t o c h o n d r i a , l i p i d , and g l y c o g e n , and t h e t r i a d s a t t h e Z - l i n e . B a r = 2.0Mm  Figure  13. L a t e r a l s e c t i o n of r e d muscle m y o f i b r i l s . the l a c k of m i t o c h o n d r i a . B a r = I.OMITI  Figure  14. T r a n s v e r s e s e c t i o n of r e d muscle. Note the i r r e g u l a r shape o f t h e m y o f i b r i l s . B a r = l.tDjum  Note  63  64  The s m a l l p i e c e s o f t i s s u e w h i c h were removed period  were  yellowish  still  their  colour,  "natural"  indicating  after  this  time  c o l o u r and o n l y took  glutaraldehyde  fixation,  on a after  immersion i n f i x a t i v e . In  conjunction  supporting  w i t h t h i s p a u c i t y of c a p i l l a r i e s ,  the evidence  o f t h e h i s t o c h e m i s t r y , were t h e v e r y  numbers o f m i t o c h o n d r i a were  very  low  in  difficult  to find  observed  were  in a l l fibre  red  in  types  (Table  Those  small  mitochondria  muscle.  which  1.5Mirt  low  Numbers  a n d were n o t c o n c e n t r a t e d  Sarcomere l e n g t h s averaged  m u s c l e a n d 2.0MITI f o r w h i t e  6).  m u s c l e , fewer i n i n t e r m e d i a t e and  white.  generally  s u b s a r c o l e m m a l band.  and a l s o  for  very were in a red  65  Table  5. D i a m e t e r of l u n g f i s h m u s c l e  Red N x SD  4.00 23.6  .6.4  Intermediate 400  fibres  White 400  34.3  67.4  9.7  25.0  (urn)  T a b l e 6.  A Comparison of t h e m i t o c h o n d r i a l d e n s i t y and v a s c u l a r i z a t i o n of v a r i o u s muscie f i b r e s . Mitochondrial density (%area o f c r o s s - s e c t i o n ) Red  Reference  InterWhite mediate  S k i p j a c k tuna (Euthynnus pelamis)  35  2  Hulbert  Eel (Anquilla  23  0.1  H u l b e r t a n d Moon (1978)  rostrata)  Shark (Etmopterus Shark (Galeus  spinax)  melastomus)  Coalfish (Gadus v i r e n s )  30  7.2  0.5  Kryvi  (1977)  34  16.3  0.9  Kryvi  (1977)  1.1  P a t t e r s o n and Goldspink (1972)  0.7  P a t t e r s o n and Goldspink (1972)  0.2  This  25  Crucian carp 16.2 (Carassius carassius) Lungf i s h  et a l .  (1979T  5.3  0.2  Capillaries Red Skipjack tuna 4-12 (Ketsuwonus p e l a m i s )  per fibre  study Reference  White 1 .0  Hulbert  3.9 neopilchardus)  1.6  Mosse  (1979)  Mackerel 2.98 (Scomber a u s t r a l a s i c u s )  1.1  Mosse  (1979)  A u s t r a l i a n salmon (Arripis trutta)  0.2  Mosse  (1979)  1.53  Mosse  (1979)  0.02  This  Pilchard (Sardinops  4.2  Y e l l o w t a i l scad 3.1 (Trachurus mccullochi) Lungfish  0.14  et a l .  (19797  -  study  67  Discussion  The A  results  in this  be u s e f u l t o p u t t h e d a t a  shows t h e t y p i c a l Much o f  the  production  types.  of f i s h  muscle  of  1977).  muscle  is  (Gordon, (Table  after  The  and  resting to  the  Goldspink,  is  metabolic  geared  be  fate  roughly  1/5 of  ( B l a c k et_ a l . , 1 9 6 2 ) .  of  for  anaerobic  of  a  that  this  periphery  fish  is normally  reflecting  mitochondria  occurs  separated  This  mitochondria relatively  white  of r e d m u s c l e  muscle  is  R e c o v e r y of w h i t e  the poor b l o o d  poor  (Black et a l . ,  time  muscle  s u p p l y and  the  1962;Johnston  and  as  a  small  wedge  of  cells  o f t h e myotome a t t h e r e g i o n o f t h e l a t e r a l  tissue.  (Johnston  1973).  Red m u s c l e  fibre than  more  shown t h a t t h i s fish  and  11  teleost.  muscle can o n l y f u n c t i o n f o r a s h o r t  f a t i g u e i s slow, numbers  white  The v a s c u l a r i z a t i o n  fatiguing  Figure  d u r i n g swimming a t h i g h s p e e d s  estimated  6),  before  is  energy  1968).  in perspective.  s t r u c t u r e o f t h e swimming m u s c l e of a  muscle  et a l . ,  the  to fibre  s h o r t d e s c r i p t i o n o f t h e f u n c t i o n and l o c a t i o n  fibres will  low  s e c t i o n are a l l r e l a t e d  at  do  from the o t h e r muscle t y p e s type  has a g r e a t e r b l o o d  white  reliant  et a l . , 1977).  f i b r e type  indicating  cruising  involved  speeds  line.  between  red  function  intermediate  and  white  that  it  between  these  two,  is  S t u d i e s have in  (Hudson,  muscle  It  connective  propelling  1973;Johnston  The . t h i r d t y p e , t e r m e d p i n k o r i n t e r m e d i a t e ,  located  the  s u p p l y and more  on a e r o b i c r e s p i r a t i o n .  i s the major normal  fibres,  by  at  is  and a p p e a r s t o have a working  at  speeds  68  slightly 1977). is  above  the  maximum  f o r r e d muscle  D i s t r i b u t i o n o f t h e m u s c l e t y p e v a r i e s between  possibly  related  to  t h e mode o f l i f e  et a l . , 1959;Johnston e t a l . ,  1974).  degree of m i x i n g of i n t e r m e d i a t e These the  the  There  and w h i t e  general  pattern  slightly  (Flood,  1968).  t h r o u g h t h e water u s i n g fibre is  system,  no septum  three  types  T h i s mosaic muscle,  separating  to  and  covering  intermediate  with white  fibres--is  animals.  propel  retained  such  three-  i s somewhat d i f f e r e n t .  There  from  the  rest  and a l l  form a mosaic p a t t e r n of f i b r e s .  i s found a t the p e r i p h e r y  of the muscle  the amphibia,  periphery.  of  the  (Flood,  myotome.  a  mosaic  pattern  towards land l i v i n g  tolerance  since  Arapaima  muscle  (see below).  located  forms  giqas  t o that  around  morphology  and not  e c o l o g i c a l h a b i t s a n d d o e s have t h e f i s h red  White  1 9 6 8 ; T o t l a n d , 1976).  T h i s d e v i a t i o n from t h e " f i s h "  to the trend  hypoxia  with  as  themselves  The A f r i c a n l u n g f i s h m u s c l e h a s a s t r u c t u r e s i m i l a r  due  I ti s  l o c a t e d under t h i s p e r i p h e r a l mosaic l a y e r , s t i l l  the m a j o r i t y  of  some  the  the red f i b r e s  intermingle  usually  red fibres  which  a x i a l m u s c l e have  but t h e morphology  (Boddeke  cephalochordates  Amphibia  and  fibres.  f o r a l a r g e group of a q u a t i c  found i n t e l e o s t s , elasmobranchs and amphioxus  is  w h i t e f i b r e s massed i n t e r i o r l y  sandwiched between—mixed  fish  of the f i s h  three muscle types arranged w i t h  periphery,  fibres  (Johnston e t a l . ,  due  to  has  some  the  is likely  type d e f i n i t i o n  increased similar of  the  The l o s s o f a d i s c r e t e r e d m u s c l e  band  may h a v e a c c o m p a n i e d  the trend  f o r the early Dipnoi  and l e s s on t h e t a i l  f o r much o f t h e r o u t i n e  to rely  movement,  less  relying  69  instead  on  the newly e v o l v e d l i m b s .  However, t h e r e d u c t i o n i n  mass o f a h i g h l y o x i d a t i v e t i s s u e must be of b e n e f i t d u r i n g exposure  to hypoxia.  I n t h e l u n g f i s h , b o t h t h e r e d and relatively  anaerobic  as  indicated  m i t o c h o n d r i a l c o n t e n t and This  is  well  no  teleost  vascularized  in  by  Table  capillary  species.  white muscle types  The  is  for  only  density  a  low  function—much The  well  with  i s less  resting  t o be  little  metabolic  rate  and  more so t h a n o t h e r f i s h w h i t e  rate  short  potential  for  designed  muscle.  r e d m u s c l e band i n t h e l u n g f i s h  this  tissue  for  anaerobic  white f i b r e areas would serve  of  blood  developed.  W h i t e m u s c l e t h e r e f o r e , seems t o be  l a c k of a d i s c r e t e  metabolic  less  the  lungfish  i n white muscle t o supply  t h e r e appears  the predominance of pure the  of  compares  r e d m u s c l e of l u n g f i s h  enough g l y c o g e n  recovery.  both  which  low  lipid.  t h a n t h e w h i t e m u s c l e of o t h e r f i s h w h i l e t h e  a n a e r o b i c b u r s t s and aerobic  appear  vascularity,  intracellular 6  s u p p l y t o l u n g f i s h w h i t e muscle i s even There  low  observable  illustrated  m i t o c h o n d r i a l c o n t e n t and several  any  relatively  to  low.  make  This i s  b e c a u s e t h e m e t a b o l i c r a t e o f w h i t e f i b r e s may  be  that  of  red  measurements,  the  fibres  and,  on  the  basis  of  enzyme  1/5  and  capacity  f o r c o n t i n u o u s a e r o b i c a n d / o r a n a e r o b i c ATP  in  white  the  (Section still  1).  muscle  is  relatively  D u r i n g a b u r s t of  work  low e v e n f o r w h i t e the  metabolic  be h i g h s i n c e i t c a n be p o w e r e d by ATP  c r e a t i n e phosphokinase t h e a n i m a l had  reaction.  a limited  production muscle  rate  may  p r o d u c t i o n v i a the  T h i s d e s i g n w o u l d be of use i f  r e s o u r c e of oxygen which  must s e r v e a l l  70  the  tissues. The  South American a i r - b r e a t h i n g f i s h  similar  habits  t o P.  data are a v a i l a b l e  aethiopicus,  (Hochachka  and some u s e f u l al.,  1978).  African  lungfish, this  believed  t o be r e l a t i v e l y  period.  Both of the muscle types s t u d i e d ,  relatively profiles.  anaerobic  fish  et  i n a c t i v e during  fish  also  has  of  of  this  section  is  t o be o f g r e a t  low.  bearing  cellular  that  fibres.  the  with  the  submerged  r e d and w h i t e ,  appear  benefit  capacity  adjustments  that  would  indicate that and  so  a very  small  the muscle i s would  have  a  of having the bulk f l u x of oxygen i s  t o t h e a n i m a l when  oxygen  supply  t y p e o f f i b r e may a l s o have a  of  1. the  In  Section  muscle  1,  i t was  mass t o r e a c t t o  h y p o x i a w i t h o u t a c t i v a t i n g g l y c o l y s i s was b e n e f i c i a l t o of  that Since  the the  is  lungfish  bulk  this cell  implication  capacity  As  most o f t h e  The s t r a t e g y  The p r e d o m i n a n c e o f t h i s  survival  indicates  the  rate.  upon t h e r e s u l t s o f S e c t i o n  proposed  the  low m e t a b o l i c  t h e myotome a d a p t e d t o u t i l i z e  likely  comparative  limiting.  comprised p r e d o m i n a n t l y of white f i b r e s relatively  has  i s an o b l i g a t e a i r - b r e a t h e r , a n d i s  p r o m o t e s u r v i v a l when o x y g e n was results  giqas  on t h e b a s i s o f f i n e s t r u c t u r e a n d enzyme  Thus, t h i s  The  Arapaima  during  hypoxia.  Section  o f t h e myotome i s c o m p r i s e d o f w h i t e  type predominates i n the a x i a l  that  2  i t is  f o r the observed pattern  this cell  of metabolic  muscle,  type which has t h e control.  71  SECTION 3  The  metabolic adjustments  to acute environmental Rainbow  hypoxia  i n the  trout  Introduction  The  previous s e c t i o n s d e s c r i b e d metabolic responses  African  lungfish to hypoxia.  submergence f o r hours to l e s s than 5 t o r r Under  these  conditions,  et a l . ,  i t was  were a b l e t o a c t i v a t e g l y c o l y s i s rates  of  aerobic  ATP  follow this pattern.  The  toxic  would  the  i n order to  muscle  This  strategy  same  that  a  spares  in  substrates the r a t e  sensitive  inter-tissue metabolic  of  metabolism glycolytic  would ATP  respond  animal used  t o examine t h e s e hypotheses  to  production. was  organism  interactions that  Furthermore,  of  Of  declining  of g l y c o l y s i s  i t reduces  hypoxia  activation  (Salmo  augment  brain  t o be t h e most s i g n i f i c a n t a d a p t a t i o n  were e x e m p l i f i e d by t h e l u n g f i s h . that  1970).  production.  would not e x p e c t  show  fall  shown t h a t t h e h e a r t and  o x y g e n ) f o r o t h e r t i s s u e s and  end-product One  l 9 6 8 ; L a h i r i et a l . ,  l a c k of an a c t i v a t i o n  i n the l u n g f i s h .  (including  oxygen t e n s i o n s  s y n t h e s i s but t h a t w h i t e muscle d i d not  the w h i t e muscle i s l i k e l y to hypoxia  the  These a n i m a l s a r e a b l e t o s u r v i v e  even though a r t e r i a l  (Johansen  of  one  would  hypoxia The the  expect  with  an  experimental rainbow  trout  gairdneri). fishes,  the  s a l m o n i d s a r e among t h e most s e n s i t i v e  to  72  oxygen  deficiency  oxygen  tensions  uptake or  ( D o u d o r o f f and decline  to  below  1970).  40 t o r r ,  may  increase  Goeden, 1 9 8 2 ) .  (Hughes  volume  of  t h e r a t e o f oxygen  and  l967;Hughes  Randall,  and G o e d e n , 1 9 8 2 ) . rise  Goeden, 1 9 8 2 ) . by  with  increases  the trout  though  remains  the  fairly  i s likely  It  responses  which occur section dysoxia  in  the  environmental  total  pumping  (McKim a n d of  oxygen  constant, the actual  supply  rate  to  hypoxia  are not  t h a t muscle and l i v e r  Spehar,  1971).  lactate  However,  the  of each t i s s u e and t h e m e t a b o l i c i n t e r a c t i o n s the  the trout  oxygen  tissues  organ by  by  are organ  subjecting  tensions  not  and  picture  t r o u t d u r i n g exposure  emerges  of  understood.  responses the  then  s p e c i f i c m e t a b o l i t e c o n c e n t r a t i o n changes. overall  the  t o be r e d u c e d .  i s certain  ( B u r t o n and  between  examines  of  (Holeton 1971;McKim  tensions f a l l  The m e t a b o l i c r e s p o n s e s o f t h e t r o u t  concentrations rise  Jones,  requirements  the  concurrently  extraction  and S a u n d e r s 1 - 9 7 0 ;  u p t a k e when o x y g e n  Thus, even  described.  decline,  t h e pumping r a t e a n d may r e a c h 5 0 % o f t h e  t o most o f t h e t i s s u e s  actual  1967)  l970;McKim a n d  tensions  of oxygen  The e n e r g e t i c  r a t e o f oxygen  uptake  Saunders,  w a t e r pumped a c r o s s t h e g i l l s i n the e f f i c i e n c y  mechanism  and  As o x y g e n  with a decline  an  ambient  H o w e v e r , t h i s may n o t mean t h a t t h e t i s s u e s h a v e  a c o n s t a n t s u p p l y of oxygen.  well  As  i n t r o u t may r e m a i n c o n s t a n t ( H o l e t o n a n d R a n d a l l ,  they  total  Shumway,  animal  This  to cellular to  reduced  monitoring  tissue-  From t h e s e  changes,  metabolic organization  to environmental hypoxia.  i n the  73  M a t e r i a l s and Methods  Experimental obtained large  Animals.  Rainbow t r o u t ,  f r o m Sun V a l l e y t r o u t f a r m s  outdoor  tanks  with  natural photoperiod. trout had  pellets until  a  They  during  through water system and a  f e d mid-day  two d a y s p r i o r The  with  commercial  t o the experiment.  experiments  Procedure.  The  fish  p l e x i g l a s s h o l d i n g box one day b e f o r e 6 slatted  tensions  were  The  fish  carried  out  directly  were  placed  in  a  experimentation.  The  were  adjusted  by  bubbling  N  2  Water f l o w e d  i n t o each of t h e f i s h compartments.  from t h i s  an a m p l i f i e r a n d a c h a r t  recorder.  The  torr  after  of  the  mixing chamber  Oxygen t e n s i o n was serially  Water a n d g a s f l o w s  to obtain a reproducible decline i n 0  box.  box  Oxygen  into a central  m o n i t o r e d w i t h a Radiometer oxygen e l e c t r o d e c o n n e c t e d  adjusted  black  c o m p a r t m e n t s , e a c h o f w h i c h h e l d one f i s h .  chamber a t t h e f r o n t o f t h e b o x .  to  were  t h e e a r l y s p r i n g when t h e w a t e r t e m p e r a t u r e was 4°C.  Exper imental  had  qairdneri),  ( M i s s i o n B.C.), and kept i n  flow  were  a mean s i z e o f 320 gm.  (Salmo  2  tension  were  i n the  o x y g e n t e n s i o n d e c l i n e d g r a d u a l l y t o a t e n s i o n o f 20 20 m i n . 3  hour  and was m a i n t a i n e d hypoxic  there  exposure.  f o r the  Preliminary  remainder  tests  were  performed v a r y i n g time of i n i t i a t i o n , the d u r a t i o n of  exposure,  the  of oxygen  availability  tension.  of  surface  access,  T h i s was done t o d e t e r m i n e  a  exposure which would r e p r o d u c i b l y r e s u l t  and t h e l e v e l procedure  for  hypoxic  i n an i n c r e a s e o f b l o o d  74  plasma  lactate  presence  of  blood  experiments cellular In  concentrations  to  lactate  indicate  with  was  no  used  fish in  mortality.  these  The  preliminary  t h a t t h e f i s h were b e i n g  subjected to  hypoxia. t h e major m e t a b o l i t e experiment fish  (without access  s a m p l e s were t a k e n  from  8  control  to  h y p o x i a ) , 4 f i s h exposed f o r 1 h o u r , and 7 f i s h exposed f o r 3  h o u r a t 20 t o r r . of  declining  Timing  oxygen  t o the s u r f a c e but not s u b j e c t e d  began c o n c o m m i t e n t w i t h t h e  tensions.  F i s h were d e n i e d  s u r f a c e by f l o a t i n g wood i n t h e c h a m b e r s . initiated  Preparation  and  Assay.  b e h e a d e d f i s h and f r o z e n a t -196°C  of  w h i c h h a d been c o o l e d  freezing  was  white muscle. the  total  blood,  by  in liquid heart,  and  for  freezing  from  mortar  clamping nitrogen. liver,  PCA.  with  with  aluminum  The u s u a l  r e d m u s c l e , and  a l l t i s s u e s was r o u g h l y locations.  and p l a c e d d i r e c t l y  Tissues  liquid  order  The  and  150 s. anterior  to the g i l l s the  s  and t h e  dorsal f i n  B l o o d was w i t h d r a w n f r o m t h e t r u n k v i a  were  a n d p e s t l e r e s t i n g on  flushing  from  s a m p l e s were f r o m t h e r e g i o n between  caudal v e s s e l puncture 6%  T i s s u e s were removed  the region just caudal  the caudal peduncle.  cold  was  The h e a r t and b r a i n were f r o z e n w i t h i n 60  time  were  posterior  t o the  The g a s b u b b l i n g  brain,  M u s c l e s a m p l e s were removed f r o m two samples  access  a t 10:30 A.M..  Metabolite  tongs  initiation  ground dry  nitrogen.  weighed i n t o a c o l d X o r e x  into  1 volume of i c e -  t o a f i n e powder u s i n g a  i c e and About  c e n t r i f u g e tube  further 0.5  cooled  by  gm o f powder was  containing  1  ml  of  75  1.4M PCA a n d h o m o g e n i z e d w i t h a P o l y t r o n Two  aliquots  subsequent  again.  for  analysis. 15  min.  was n e u t r a l i z e d  A l l metabolites  were m e a s u r e d w i t h i n which  M1 were removed a n d f r o z e n a t -80°C f o r  100  glycogen  10,000 x g supernatant  of  metabolites  The  and  homogenate  the p e l l e t  t o pH 6.7 w i t h except glucose,  was  KOH  glycogen,  measured  and  using  r e a c t i o n a t 340 Glycogen (Keppler  was  NADH/NAD*, nm  on  measured  and Decker,  a  using  1970).  level  were  compared  o f p < 0.05.  spun  order  in  first. them  to  NADPH/NADP , a n d f o l l o w i n g t h e  Unicam  measured w i t h t h e t e c h n i q u e s Data  or  The  w i t h ATP and  M e t a b o l i t e s were m e a s u r e d by e n z y m a t i c a l l y l i n k i n g reactions  at  and l a c t a t e  The  a s s a y e d was k e p t c o n s t a n t  (CrP) being  spun  discarded.  1.4M  9 hours of n e u t r a l i z a t i o n . were  creatine-phosphate  homogenizer-sonicator.  +  SP-1800  spectrophotometer.  the amyloglucosidase The  remaining  technique  metabolites  were  of Hochachka e t a l . , (1978c).  u s i n g one-way ANOVA w i t h  significance  76  Results  P r e l i m i n a r y experiments the  availability  response  surface  to environmental  concentrations hours  of  in control  begining  at  6.85+.65 mM  fish,  fish  had  fish  significantly  those  of g a i n i n g access  These remaining at  d a t a ' were  f o r 3 hours mM,  and  I n a s e r i e s o f 10 f i s h without  access  to  account  to the  hypoxia  while  when d e s i g n i n g t h e  The f o l l o w i n g e x p e r i m e n t s on  trout  that  were  initiated  d i d not  metabolite concentrations f o r the b r a i n , heart,  a n t e r i o r and p o s t e r i o r  Tables  7  and  8.  r e d and w h i t e muscles a r e  Statistical  c o n t r o l and 3 hour samples, samples. exposures  Values  have  very  metabolites  in  concentrations  fell  in  are  comparisons  and a n t e r i o r  listed  liver,  listed  in  w e r e made b e t w e e n  and  for control,  posterior 1 hour,  muscle  and 3 hour  t o hypoxia.  T h e r e was  rise  for 3  to the surface.  The and  hypoxia  4.00+.75  exposed  into  6:30 A.M. a n d were p e r f o r m e d  access  lactate  t o the surface.  taken  experiments.  Plasma  exposed mM,  metabolic  h i g h e r plasma l a c t a t e c o n c e n t r a t i o n s  than b o t h t h e c o n t r o l s and t h e f i s h capable  exposed t o  0.68+.40  f o r 45 m i n . ,  the  i n the t r o u t .  A.M. a n d  A.M. were  affected  (X+S.D., N=5) r e s p e c t i v e l y .  exposed t o hypoxia surface  access  hypoxia  6:30  b e g i n n i n g a t 10:30  i n d i c a t e d t h a t b o t h t i m e o f day a n d  lactate  little the  change  brain  in  the  during  (p = 0.06), c o n c o m i t t e n t  concentration  hypoxia. with a  of  Glycogen significant  c o n c e n t r a t i o n s ( T a b l e 8 . ) . The t o t a l  p o o l of  Table 7. C o n c e n t r a t i o n s of adenylates, c r e a t i n e phosphate, and c r e a t i n e , and the energy charge i n trout t i s s u e s . (x+SD)  Exper imental Condi t ion  ADP  AMP  E.C.  Total  n  ATP  Control  7  0.73+0.18  0.58+0.14  0.39+0.20  1-h  4  0.47+0.14  0.59+0.07  0.35+0.24  3-h  8  O.70+0.23  0.73+0.20  O.26+0.11  0.63+0.09  Control  7  2.43+0.65  0.72+0.27  0.17+0.14  0.84+0.07  1-h  4  2.09+0.32  0.66+0.08  0.15+0.03  3-h  8  1.68+0.58  0.76+0.33  0.13+0.08  0.80+0.08  Control  7  1.35+0.21  0.55+0.38  0.24+0.08  0.76+0.07  1-h  4  0.81+0.45  0.82+0.13  0.41+0.04  3-h  8  0.53+0.23'  0.67+0.09  0.28+0.06  0.58+0.09'  1.48+0.24'  Controi  7  2.85+0.97  0.68+0.22  0.15+0.03  0.86+0.08  3.69+0.84  1-h  4  1.39+0.28  0.68+0.13  0.16+0.06  3-h  8  2.65+0.44  0.82+0.12  0.13+0.05  0.85+0.03  Control  7  3.73+0.90  0.76+0.16  0.15+0.06  0.88+0.05  1-h  4  2.64+0.28  0.80+0.15  0.20+0.13  3-h  8  3.21+0.43  0.83+0.09  O.11+0.04  Creatine  Creatine-P  Total  Brain 0.61+0.11  1.70+0.25  9.68+1.37  0.98+0.53  10.66+0.98  9.26+1.90  0.98+0.53  1.69+0.39  8.55+0.93  1.03+0.46  9.57+0.81  3.33+0.60  3.87+0.78  4.63+0.54  8.51+0.82  4.14+0.56  3.28+0.75  2.57+0.65'  4.55+1.48  2.93+1.23'  7.48+1.61  2.14+0.34  1.20+0.54  0.28+0.22  1.48+0.49  Heart  1  L i ver  0.98+0.19 1.03+0.4 1  < 0.01 0.02+0.02'  1.04+0.41  Ant. R. Muscle 12.43+1.91  5.74+2.93' 18.16+3.51  11.41+3.36  1.89+0.80  3.61+0.40  13.76+4.44  3.57+1.59  17.33+4.19  4.64+0.79  11.65+1.97  9.28+2.56  20.93+4.22  13.35+1.72  5.37+1.19  12.76+2.47  4.72+2.01 17.48+2.72  Post. R\ MUscle  0.88+0.02  4.16+0.51  1  T a b l e 7. cont. Experimental Condit1on  n  ATP  ADP  Control  7  7.42+0.71  1.01+0.20  0.10+0.03  1-h  4  6.57+0.33  1.00+0.08  0.10+0.03  3-h  8  6.78+0.44  1.07+0.16  0.10+0.06  0.92+0.01  7.95+0.49  34.19+4.06  Control  7  7.41+0.86  0.91+0.19  0.09+0.05  0.93+0.02  8.42+0.71  24.47+4.92  1-h  4  5.82+1.49  1.02+0.06  0.13+0.06  3-h  8  6.94+0.35  1.11+0.32  0.11+0.06  AMP  E.C.  Total  Creatine  Creatine-P  Total  Ant. W. Muscle 0.93+0.02  8.54+0.60  25.71+4.94  20.77+2.10' 46.48+5.81  26.51+2.58  18.65+3.17 1  13.65+4. 96'  48.55+2.02  Post. W. Muscle 26.94+5.20  26.65+2.34 0.92+0.02  8.15+0.46  34.92+4.06  51.41+6.27  21.01+1.57 1  13.63+4.96' 48.55+2.02  s i g n i f i c a n t l y d i f f e r s from normoxla (p < 0.05) ' s i g n i f i c a n t l y d i f f e r s from p o s t e r i o r sample (p < 0.05) 1  00  79  Table  8.  S e l e c t e d t r o u t g l y c o l y t i c m e t a b o l i t e s a t r e s t and d u r i n g acute hypoxia. X + S.D. (/xmoles/gm wet wt)  Experimental Condition  n  Glycogen  Glucose  7  3.69+1.78  5.04+3.72  0.08+0.07  2.12+1.23  3.57+0.87  2.16+0.94  0.14+0.05  3.37+0.85  1.83+1.61  4.99 + 2. 14  0. 13+0.05  3.91+1.21  8.82+5.02  0.24+0.07  0.71+0.23  5.19+1.36  0.24+0.04  2.68+0.70  7.53+2.48  0.28+0.17  6. 15 + 3.52  11.9+9.04  0.15+0.14  0.95+0.16  7.1+1.55  0.88+0.33  2.27+0.15  G6P  Lactate  Brain Control 1 -hr  4  3-hr  8  1  Heart Control  7 36.28+19.13  1 -hr  4 33.60+  3-hr  4.53  8 19.27+10.32  1  1  Liver Control  7 133.5+68.66  1-hr  4 189.4+  3-hr  8 142.8+58.3  12.2+4.19  1.21+0.43  Control  7 14.56+6.01  1.71+1.32  0.44+0.11  1-hr  4 18.85+3.61  1.08+0.21  1.33+0.49  3-hr  8 16.52+8.79  1.65+0.33  1.29+0.41  Red  Red  0.44  1  5.40+1.55  1  Ant. Muscle  Post.  2  2.34+1.14 2.97+1.14  1  4.35+2.03  1  Muscle  Control  7 15.26+9.04  1.60+1.05  0.25+0.13  1.73+1.05  1-hr  4 18.33+1.84  0.81+0.23  1.04+0.53  3.85+0.74  3-hr  8 15.80+8.10  1.55+0.39  1.02+0.30  1  4.15+2.21  1  80  T a b l e 8. c o n t . White A n t . Muscle Control  7 15.89+5.04  0.74+0.47  0.80+0.44  6.77+4.85  1-hr  4 15.15+2.31  0.35+0.06  1.03+0.06  5.74+1.78  3-hr  8 12.12+4.94  0.72+0.22  1.40+0.61  10.90+3.88  White Post. Muscle Control  7 18.00+7.58  0.70+0.48  0.57+0.34  5.78+3.76  1 -hr  4 16.49+2.93  0.44+0.15  0.97+0.16  6.76+2.16  3-hr  8 12.28+4.30  0.76+0.24  1.36+0.47  1  11.39+2.92  1  Blood Control  7  12.51+9.45  0.37+0.27  1-hr  4  4.54+2.60  2.34+0.46  3-hr  8  7.30+3.07 •  6.93+1.98  Significantly  differs  from normoxia  significantly  differs  from p o s t e r i o r  2  (p < . 0 5 ) . s a m p l e (p < .05)  1  81  c r e a t i n e and •7.).  CrP  T h i s was  the  l a c t a t e d i d not The  maintained, only  as was  tissue  the energy charge  where  the  showed  while,  concentrations  of  through  significant  [lactate]  ATP,  CrP,  was and  the experiment.  steadily  The  blood.  in  glycogen  rising.  total energy  of  i n the  declines  the  (Table  concentrations  c o r r e l a t e w i t h the c o n c e n t r a t i o n s  heart  concentration  declined  was  The  adenylate charge  pool  did  not  change.  liver  Neither  glycogen  during  hypoxia,  lactate  rose.  The  nor  glucose  while  the  l e v e l s ' of ATP  p o o l s of c r e a t i n e + C r P  and  c o n c e n t r a t i o n s changed i n the concentrations and  adenylates  CrP  fell,  (Tables  charge d e c l i n e d i n d i c a t i n g  that a metabolic  The  lactate  concentrations  of  c o r r e l a t e d w i t h the c o n c e n t r a t i o n s Although no  [G6P]  and  change i n g l u c o s e ,  or g l y c o g e n .  and  the c o n t r o l f i s h w h i l e b l o o d and  a  CrP  [G6P]  higher  in  total energy  occurred. the  (Table  liver  9)  o n l y change i n the in  [CrP].  the  were h i g h e r was  and  i n red muscle, there  The  in  The  s t r e s s has  glucose  fall  differences  c o n c e n t r a t i o n s of ATP  The  was  G6P  as d i d the  7,8).  i n the b l o o d  [ l a c t a t e ] rose  e n e r g y p h o s p a t e compounds anterior-posterior  and  of  red  i n the  There muscle.  tail  region  was high are The of  i n the a n t e r i o r samples.  t i s s u e c o n c e n t r a t i o n s o f l a c t a t e and  glucose  were  significantly correlated. In lactate  white rose  concentrations  m u s c l e , t h e c o n c e n t r a t i o n s of c r e a t i n e , while of  c o n c e n t r a t i o n s may  CrP  that were  be d e c l i n i n g  of  CrP  higher  in  (p = .06)  fell.  Here  the  tail.  (Table  8).  G6P,  and  too,  the  Glycogen  Table  9. C o r r e l a t i o n s b e t w e e n t i s s u e a n d b l o o d concentrations i n trout. (probability  Tissue  Lactate  metabolite  t h a t r = 0, n = 19) Glucose  Blood  Glucose vs Tissue Glycogen  P  r  Brain  .0859  .416  <.  0001  .876  Heart  <.0001  .895  <.  0001  .857  Liver  <. 0001  .973  <.  0001  .906 .  >0 . 1 _  .224  Red A n t .  .0003  .750  <.  0001  .838  >0 . 1  .221  Red  .0014  .693  0001  .838  White Ant.  .0052  .629  0001  .797  >0 . 1  .263  White  .0018  .683  0004  .742  >0 . 1  .238  Post.  Post.  P  <.  r -  p >0 . 1 0 .0498  < .0001  r .225 .469  .463  Table  10. G l y c o l y t i c m e t a b o l i t e s t o r e s i n t r o u t . (Mmoles/500 gm f i s h ) x + S.D.  Tissue  Glycogen  Glucose  Glucose-6-P  Lactate  C  594+305  53 + 40  0.7+0.6  3 hr  635+259  54+1 9  5.4+1.9  Muscle C  5592+2106  238+155  227+117  '3 h r  4025+1491  244+64  455+161  373+176  41+29  8.6+2.5  404+209  40+7.5  29+8.3  C  1 9+1 0  328+299  .15+.05  11+8  3 hr  11+4.4  225+96  .•19+.05  2 1 3 + 61  Liver  White  Red  Muscle C 3 hr  Others  'significantly differs  from c o n t r o l  (p <  4.2+0.7 1  24+6.9  1  2071+1394 1  3677+1087 51+24  1  0.05).  106+52  1  1  1  Table  11.  Tissue  The r a t i o s o f b l o o d trout. (x+SD) Gontrol  lactate  3 hour exposure  to tissue  lactate  Change i n mean r a t i o  Brain  .264+. 270  1 .960+. 871  + 1.696  Heart  .551+. 384  . 1.348+. 589  + 0 .797  Liver  .409+. 313  1 .298+. 184  + 0 .889  Red A n t .  .195+. 141  1 .823+. 692  + 1.628  Red  .283+. 182 .  2 .055+. 908  + 1.772  Post.  White Ant.  .103+. 105  .656+. 1 34  + 0 .553  White  .099+. 081  .627+. 1 92  + 0 .528  Post.  A l l c h a n g e s between c o n t r o l d i f f e r e n t a t p < 0.05.  and 3 hr a r e s i g n i f i c a n t l y  85  The  a v a i l a b l e s u p p l y of s u b s t r a t e s f o r g l y c o l y s i s , and  amount of l a c t a t e p r o d u c e d liver  was  the  concentration contained in  the  significantly  the  amounts  in  t o the energy  store  G6P  the  10..  in  white  white  Although  terms muscle  muscle  s u p p l y as w e l l .  The  of  which  the  absolute  as  a  tissue  G6P  i s present  to  contribute  total  a r e d i a g r a m e d i n F i g u r e 15 t o  of the w h i t e muscle,  s u p p l y of f u e l  Table  g r e a t e s t s u p p l y of c a r b o h y d r a t e .  g l u c o s e , and  impact  glycogen  (133 /xmoles/gm) , t h e  l a r g e enough  glycogen,  main  are l i s t e d  the  pools  of  stress  i s the major s t o r e , as a  for glycolysis.  J u s t a s t h e w h i t e m u s c l e c o n t a i n s most o f t h e s u b s t r a t e , i t a l s o c o n t a i n s most of t h e percentage blood.  changes,  end-product--lactate.  however,  were  in  the  The  greatest  heart, brain,  and  86  Figure  15. T o t a l g l u c o s e , g l y c o g e n , and G6P s t o r e s i n t r o u t . The ' O t h e r s ' a r e t h e b r a i n , h e a r t , and b l o o d . "C" i n d i c a t e s c o n t r o l v a l u e s and "3" i n d i c a t e s v a l u e s f r o m t r o u t exposed t o hypoxia f o r 3 hours.  87  White Muscle d»  6  0  0  0  o o  no  \  m ©  E =x 4000 Ui LU DO —'  v. _J  2000  Red Muscle  o i-  Liver  C 3  C 3  C 3  Others  C 3  88  Figure  16. Total lactate stores in trout. The ' O t h e r s ' a r e t h e b r a i n , h e a r t , and b l o o d . "C" i n d i c a t e s c o n t r o l v a l u e s and "3" i n d i c a t e s v a l u e s f r o m t r o u t e x p o s e d t o hypoxia for 3 hours.  89  C 3  C3  C3  C3  90  Discussion  This discussion responses  of  the  i s devoted trout  to  to  analysing  acute  b e t w e e n a n t e r i o r and p o s t e r i o r m u s c l e first,  the  hypoxia.  The  inter-tissue relationship  s a m p l e s w i l l be  f o l l o w e d by an a n a l y s i s of t h e t i s s u e  discussed  specific  and a d i s c u s s i o n of t h e i n t e r - t i s s u e m e t a b o l i c  responses,  responses.  When n o n - a n a e s t h e t i z e d a n i m a l s a r e s u b j e c t e d t o h y p o x i a possibility  arises  that  exercise  related  effects  caused  s t r u g g l i n g w i l l overshadow the e f f e c t s caused  by  Muscles  to exhibit  would  be  the  most  likely  tissue  the  low  by  oxygen. such a  response. To h e l p c i r c u m v e n t t h i s p r o b l e m , in  the  anterior  r a t i o n a l was  and p o s t e r i o r  t h a t muscle  the muscles  (caudal peduncle)  the  anterior  l i m i t e d number Childress  of  (1980)  i n w h i t e muscle  samples. metabolite  undertook  The  c r e a t i n e phosphate  experiments i n t h e r e d and  d e p e n d e n t upon t h e l o c a t i o n posterior  to  the  dorsal  than those taken a n t e r i o r a difference  enzyme  of P a r a l a b r a x c l a t h r a t u s  present  regions  may  to struggling  than  i s s u p p o r t e d by a  data.  Somero  s e r i a l m e a s u r e m e n t s o f LDH  found a s m a l l i n c r e a s e i n a c t i v i t y fin.  The  T h i s statement and  just showed  sampled  regions.  samples from the p o s t e r i o r  e x h i b i t more marked c o n c e n t r a t i o n c h a n g e s due would  were  and  activity  (a w a t e r - b r e a t h e r ) , and caudal  to  the  dorsal  t h a t c o n c e n t r a t i o n s of  w h i t e muscle  of t r o u t are  i n the f i s h , w i t h the samples  also taken  f i n being higher i n CrP c o n c e n t r a t i o n t o the d o r s a l  f i n . These data  i n the m e t a b o l i c o r g a n i z a t i o n of t h e  suggest  myotome  with  91  the p o s t e r i o r muscle being of  anaerobic  particularly potential  ATP  p o t e n t i a l l y capable  production.  t o l e r e n t to environmental  is  likely  to  swimming o r s t r u g g l i n g .  trout this  Brain.  The  metabolic  such e x e r c i s e as burst  Thus, i f t h e p o s t e r i o r sample e x h i b i t e d change than d i d t h e a n t e r i o r sample,  brain  indicate  and  the  that there  of high energy  that t h i s  oxygen r e q u i r e m e n t s . unaffected  This  by t h e s t r e s s . glycogen  obtain  tissue i s s t i l l  Lactate  concentrations  i n glycogen  significant  changes  remains  that  much  the glycogen  been p r e v i o u s l y n o t e d  stored  glycogen  r e l a t i v e hypoxia 1959;  in  a  i s reported  concentrations. glycogen  as  50%.  concentrations (Section  tissue  brain  have  increased  is  ( p = . 0 6 ) . The because  of  the  I t i s unusual t o t h e mean h a s  the  possibility  may be d e c l i n i n g . I t  1) t h a t t h e c o n c e n t r a t i o n  of  may c o r r e l a t e w i t h t h e t i s s u e ' s  (Daw  et a l . ,  Rerem e t a_l. , 1 9 7 3 ) .  This  tolerance  to  the  The  glycogen  of  l u n g f i s h , which leads t o the suggestion  1967;  Dawes  eta l . ,  i s b e l i e v e d t o be due  c a p a c i t y f o r ATP p r o d u c t i o n  concentration  the  unless  Thus,  tolerance  increased  that  may be f a l l i n g  in  i n the  r e c e i v i n g most o f i t s  to  variation  say  compounds  i s not  concentrations  d e c l i n e d by p e r h a p s a s  i s no s i g n i f i c a n t change i n  phosphate  n o n - s i g n i f i c a n t change i n g l y c o g e n inherent  sample  effects.  observation  the c o n c e n t r a t i o n s  rate  i s not  may i n d i c a t e t h a t most o f t h e c h a n g e i n t h e p o s t e r i o r  was due t o e x e r c i s e  has  the  hypoxia,  be u s e d d u r i n g  a much l a r g e r c o n c e n t r a t i o n it  Since  of a greater  v i a glycolysis.  i n t r o u t b r a i n i s l e s s than  1/2  that  that the trout brain  92  does not have as g r e a t a c a p a c i t y f o r a n a e r o b i c  ATP  generation  as d o e s t h e l u n g f i s h b r a i n . The  concentrations  enough t h a t t h e y  are  following calculation were  probably  supply  gm f i s h  significance.  The  to lactate,  t h e ATP p r o d u c e d  10 m i n u t e s .  would  The b r a i n f r o m a  r e q u i r e s a b o u t 5 ^ m o l e s 0 / h r . , o r 30 y m o l e s A T P / h r . 2  was  3.7  The c o n c e n t r a t i o n o f g l y c o g e n  Mmoles/gm  Mmoles o f g l y c o g e n . via  little  the b r a i n f o r only  (McDougal e t a l . , 1968). brain  of  i n t h e t r o u t b r a i n a r e low  i n d i c a t e s t h a t even i f a l l o f t h e g l y c o g e n  a n a e r o b i c a l l y converted  probably 500  of glycogen  glycolysis.  and  so  i n the  t h e b r a i n would c o n t a i n  T h i s c o u l d p r o d u c e o n l y 4.8 jumoles (brain  and  body  weights  from  of  this  1.6 ATP  study  i n d i c a t e t h a t b r a i n c o m p r i s e s 0.087% o f t h e body. The be  above p o i n t s i n d i c a t e t h a t t h e b r a i n i s not  able  to  requirements  produce  a  significant  from g l y c o l y s i s .  At  proportion  the  same  metabolite concentrations are maintained. the  brain  tissue  metabolism i s remaining  i s p r o t e c t e d from hypoxia  time,  likely  of  to  i t s ATP  the  energy  One may c o n c l u d e  that  l a r g e l y a e r o b i c and t h a t t h e  by  other  (cardiovascular  ?)  m e c h a n i sms.  Heart.  The  intermediate was  no  decline  heart,  as compared t o o t h e r  response t o environmental  t i s s u e s , e x h i b i t e d an  hypoxia.  Although  there  s i g n i f i c a n t c h a n g e i n t h e E.C., t h e r e was a s i g n i f i c a n t i n the t o t a l adenylate  ATP a n d C r P , a n d g l y c o g e n .  p o o l and i n t h e c o n c e n t r a t i o n s of  These  metabolite  changes  t h a t t h e h e a r t m e t a b o l i s m was n o t a b l e t o m a i n t a i n  indicate  c o n t r o l rates  93  of ATP  production v i a aerobic  The w i t h the  decline fall  glycolysis units  i n endogenous g l y c o g e n  i n e n e r g y s t a t u s of  has  been  is  do n o t  decline),  concentrations  cell  endogenous  one  may  glycolysis.  1.22 ATP  f i s h w e i g h s .072  gm  (this  Mmoles of g l u c o s y l u n i t s . and  heart  2.4  Using  17 t o 130  e t a l . , 1 968,'Reeves, 1 9 6 3 ) . ATP  c o m p r i s e d o n l y from  resting heart. If  a l l  This of the  i s not  mean  Since  likely  t o be a n e t  heart  i s not  The  t h a t the  Mmoles ATP/.072 gm Therefore,  high  energy  have  been  an  compounds  increase  in  a  Mmoles of r a t e of  heart/hr  the a n a e r o b i c  o n l y 5.4  a  (Lochner supply  of  r a t e of  the  i n the h e a r t ,  a  33 mM,  the  Mmoles/gm  wet  the heart  was  d u r i n g the experiment.  s t r e s s w e l l as  maintaining  phosphate  in  a l a r g e amount.  lactate exporter  i s not  one  glycogen  the h e a r t  of t h e t o t a l m e t a b o l i c  t h e a c t u a l i n c r e a s e was  tissue  of  metabolic  l a c t a t e produced remained  s u r v i v i n g the  glycogen  values,  wt.  Since  l a c t a t e c o n c e n t r a t i o n w o u l d have i n c r e a s e d by wt..  of g l u c o s y l  T h i s w o u l d p r o d u c e 3.6  1 t o 7%  that  s t u d y ) , i t w o u l d have m o b i l i z e d  Mmoles o f l a c t a t e / h e a r t / 3 h .  i s roughly  evidence  (liver  c a l c u l a t e t h a t t h e r e were 17 mmoles/gm wet  gm  coupled  c a l c u l a t e t h e amount of  ( g l u c o s y l u n i t s ) m o b i l i z e d i n the h e a r t . 500  is  Since the source  probably  p r o d u c e d by a n a e r o b i c  may  the  activated.  for glycolysis  concentrations ATP  metabolism.  i n d i c a t e d by  control  The  the  fact  concentrations  even though t h e r e a p p e a r s rate  of  anaerobic  of to  energy  production.  Liver.  This  tissue  shows t h e most d r a s t i c m e t a b o l i t e  changes  94  during the hypoxic s t r e s s . adenylates,  and  [CrP]  The  energy  a l l decline  charge,  [ATP],  significantly.  total  The  low  c o n c e n t r a t i o n s of t h e s e m e t a b o l i t e s s u p p o r t t h e s u g g e s t i o n t h e r e s y n t h e s i s of g l u c o s e (ie.  lactate  may,  (or glycogen)  o r amino a c i d s ) i s p r o b a b l y e n e r g y  t h e r e f o r e , assume t h a t p r e c u r s o r s f o r  being produced Since  from  the  the  tissue  conversion  does  of  phosphate not  in  the l i v e r  glycogen  odds  (1965).  at  in  the  l a c k o f an  They e x p o s e d  reported  that  mean  with  the  Section  seems  response  to  glycogen  lungfish  1980)  (Section  indicate  tench  i t is  still  There  was  although  depletion  of  to  1  hour  of  concentrations This  the was  liver  Pritchard  hypoxia fell  and  from  70  4 with resultant conclusion that  glycogen  glycogen  paradox  of  further  degree.  phosphorylase  to  hypoxia  M e t a b o l i t e measurements from the  1 ) , and  a decline  flounder  in liver  h y p o x i a , whereas those from c a r p exposed  that  of  was  of l i v e r  be v a r i a b l e .  status  i n d i c a t e that glycogen  not b e i n g m o b i l i z e d t o a measurable The  of  in  energy  does not  r e p o r t o f H e a t h and  Salmo c l a r k i  liver  glucose  value  observed  Mmoles/gm t o l e s s t h a n 5 Mmoles/gm. examined  not  Hbwever, t h e c o n c e n t r a t i o n s  c o n c e n t r a t i o n s would The  is  One  are  do n o t c h a n g e d u r i n g h y p o x i a .  trend  being m o b i l i z e d .  to  the p o s s i b i l i t y  glycogen  n o t even a d e c l i n i n g  glycogen  negate  g l u c o s e t o the b l o o d .  i n G6P  limited.  glycolysis  compounds, t h e  releasing  increase  molecules  gluconeogenesis.  require high-energy  was  from 3-carbon  that  (Demael-Suard  (Jorgensen  and  livers  Mustafa,  glycogen c o n c e n t r a t i o n s with  (Johnston,  e_t a l . , 1974)  1975), do n o t .  and It  3  hr  appears  95  that there glycogen  i s a r e s e a r c h problem i n the c o n t r o l  phosphorylase which awaits  Since  there  concentrations, glucose  as  is  no  then the  i t does i n the  is  decline not  lungfish.  they  are  Red  Muscle.  protected  muscle sampling  as  site  declined  such change.  was  more  exercise  to  the  argument  during  than  which  to was  CrP  i s being  this  rate  in itself  is  from  in  the p o s t e r i o r red  hypoxia,  hypoxic  increase not  the  its  r  but  anterior  dysoxia  higher  CrP  change if  proposed e a r l i e r .  The  one fact  concentration  t h a t the d e c l i n e i n CrP  of  l a c t a t e increased  an  indication  Since i n G6P,  of an  the  only other  one  may  the  s i t e of o r i g i n  r a t e s of ATP  of t h e  production.  i n the  store  red  increase  was of  muscle, in  change i n the  suggest  that  the red  the  red  that  the  f o r the muscle  to  l a c t a t e and  of oxygen to t h i s t i s s u e i s s u f f i c i e n t  maintain  be  f o r work r e l a t e d c a u s e s .  i s not  glycolysis.  m u s c l e i s an  supply  mobilized  concentrations  of  muscle  l a c k of c h a n g e  i n the p o s t e r i o r samples suggests t h a t  The but  a  t h e a n t e r i o r s a m p l e s , and  seen o n l y  tissues  I t i s possible that t h i s  t h a t c o n t r o l p o s t e r i o r s a m p l e s had did  remaining  of  rate.  concentrations  showed no  than  store  to f u e l g l y c o l y s i s i f  i n d i c a t e d by t h e  samples  considers  a  glycogen  concentrations.  Creatine-phosphate  due  liver  r e d m u s c l e , as w i t h t h e b r a i n , a p p e a r s t o  during hypoxia,  metabolite  liver as  Thus, the  h a v e t o r e l y upon e n d o g e n o u s g l y c o g e n  The  in  acting  will  to increase g l y c o l y t i c  fish  further investigation.  apparent liver  of  I t i s worthwhile  noting  96  that  t h i s e x p e r i m e n t was p e r f o r m e d w i t h a  activity. time,  If  capacity  glycogen  to synthesise  capacity  give  stated that some  of  to exercise  may n o t have been  I t was p r e v i o u s l y  endogenous  the  a n i m a l was r e q u i r e d  t h e n t h e oxygen s u p p l y  White muscle. of  the  minimum  muscular  during  this  sufficient.  the  concentrations  i n d i c a t i o n of the r e l a t i v e  ATP v i a g l y c o l y s i s .  By  this  criterion,  of the t r o u t muscle t o a c t i v a t e g l y c o l y s i s exceeds  t h a t o f t h e l u n g f i s h m u s c l e s i n c e t h e t r o u t w h i t e m u s c l e h a s 50% more e n d o g e n o u s g l y c o g e n t h a n does t h e l u n g f i s h w h i t e m u s c l e . The region  c o n t r o l CrP  than they a r e r o s t r a l  i s s i m i l a r to that hypoxia d i f f e r s and  concentrations  are  to the d o r s a l  i n the red muscle).  the c o n c e n t r a t i o n s changes  dysoxia  the  white  which  both  the  anterior  i n ['CrP], a n d a r i s e i n  Thus, u n l i k e  the  red  muscle,  m u s c l e a p p e a r t o be due t o h y p o x i c  concentrations  muscle  but  o f g l y c o g e n show a  the  change  declining  i s not s i g n i f i c a n t .  t h i s change i s  glycogen  Lactate  concentrations.  a  real  necessarily increased.  synthesized  trend  in  It will  be  decline  concentrations  in  increase but  t h e y do i n a l l t i s s u e s a n d , a s p r e v i o u s l y m e n t i o n e d ,  has  tail  However, t h e r e s p o n s e t o  a r g u e d below, however, t h a t  not  the  and n o t t o e x e r c i s e .  The white  in  of c r e a t i n e .  in  f i n (a p a t t e r n  i n the white muscle because  p o s t e r i o r samples e x h i b i t a d e c l i n e  the  higher  this  does  i n d i c a t e that the r a t e of anaerobic g l y c o l y s i s Although i t i s possible  elsewhere  muscle v i a the blood,  in there  the  that  the  lactate  was  body a n d d e l i v e r e d t o t h e w h i t e  are  three  reasons  why  this  is  97  unlikely. First,  i t  intracellular Cr, the  is  t o o b t a i n a rough e s t i m a t e  equilibrium  equation  (Sies jo et a l . ,  calculation  indicates  where a s i g n i f i c a n t occurs.  Second,  r a t i o of blood  decline  the  fact that  respect  phosphokinase  et  1974) .  al. ,  i n the  estimated  the  experiment. inside  Even  with  the white muscle  the i n t r a c e l l u l a r  i f  respect  intracellular  the  quantity  of  that with  the quantity  pH  was  not  to the e x t e r i o r , t h i s l a c t a t e and  I f one t a k e s i n t o a c c o u n t  space i s n e g a t i v e l y  charged  magnitude  Third,  of  with the  i f one t o t a l s t h e  l a c t a t e w h i c h i s p r o d u c e d i n t h e body a n d c o m p a r e s  the  enough s u b s t r a t e  of  available  the  substrate  available  w h i t e m u s c l e , one f i n d s t h a t  concentrations  indicates that  cell  i s synthesizing  i s from t i s s u e t o b l o o d .  in  tissue  remain l e s s than  becomes even more p r o n o u n c e d .  contained  This  the white muscle i s the only  t o the e x t r a c e l l u l a r space, then the  gradient  lactate  1972,-Sahlin  creatine  to tissue lactate concentrations  would i n d i c a t e t h a t gradient  the  into  t i s s u e where t h e  negative  the  that  for  o f ADP  of  the white muscle i s the only  throughout  relatively  of t h e  pH by s u b s t i t u t i n g t h e m e a s u r e d c o n c e n t r a t i o n s  C r P , ATP, a n d 1/10 o f t h e m e a s u r e d c o n c e n t r a t i o n  reaction.  one  possible  to  (Table  white  account  for  10  Figs.  muscle  and  glycogen  which there  the  i s not i s barely  increase 15, 1 6 ) .  stores  are  in This being  mobilized. The a b o v e e v i d e n c e s u p p o r t s t h e p r e m i s e t h a t r a t e has i n c r e a s e d hypoxic.  the g l y c o l y t i c  i n t r o u t w h i t e m u s c l e when t h e m u s c l e becomes  98  Tissue-tissue  interact ions.  there i s a continuous  When  are,  which  t h e l a c t a t e , and  The source  liver of  animal  The  During  for  catabolism  this  to  stress  experiment,  fuel glycolysis  may  be e i t h e r  be  o t h e r endogenous g l y c o g e n  s t a t u s of the proceeding glycogen which  gluconeogenesis  liver,  at  a  stores  one  may  the  the h e a r t .  the  h e a r t can  budgets it  mentioned  however, then  glycogen  glycogen  i s not  then  being  the  or kidney or  source it  may  that gluconeogenesis  i s not  This  leaves  of g l y c o g e n .  The  endogenous only  tissue  concentrations  t h a t the s m a l l s t o r e c o n t a i n e d  lactate  previously,  the t o t a l  i n t h e body o f a 500  gm  umoles  of  decline  the l i v e r  energy  in  load. the  i s t h a t the i n d i v i d u a l v a r i a t i o n  (a  Somero,  d e c l i n e i n glycogen  i s d i f f i c u l t to detect real  used,  the  s u p p l y a l l of t h e p r e c u r s o r s r e q u i r e d f o r t h e  f o r m a t i o n of the r e s u l t i n g As was  a  B a s e d upon t h e low  rate.  source  as  and  i n other t i s s u e s ,  predict  I t i s obvious not  If liver  of  hypoxia.  (Hochachka  however,  stores.  exhibited a significant  was  be  o t h e r t i s s u e s when  i n the l i v e r  significant as  in  the  metabolic  d e p o s i t s a r e u s e d i n many a n i m a l s  c o n c e n t r a t i o n s d i d not d e c l i n e . mobilized  be made a b o u t t h e  i t s component o r g a n s d u r i n g  i s subjected to hypoxic  1984).  w i l l now  t i s s u e o r t i s s u e s a r e t h e main s o u r c e s  glycogen  glucose  a d e p l e t i o n of  q u e s t i o n s which  what i n f e r e n c e s c a n  r a t e of t h e t r o u t and  t r o u t becomes h y p o x i c ,  b u i l d - u p of l a c t a t e and  w h o l e - b o d y s t o r e s of g l y c o g e n . addressed  the  fish  problem  If  mean  p o o l of g l y c o g e n , from  glycogen  i s h i g h and t h e r e f o r e ,  changes.  falls  with  7474  2692 j i m o l e s of 3-C  values  g l u c o s e and Mmoles  units).  to  are G6P 6128  A t t h e same  99  t i m e l a c t a t e r i s e s f r o m 2137 of  1883  decline  Mmoles).  This  in substrates  means  can  be  a  reasonable  be  o x i d i z e d or m e t a b o l i z e d This  to  estimate  Mmoles t o 4020 Mmoles (an that  roughly  a c c o u n t e d f o r as  since  some of  the  c a l c u l a t i o n u n d e r s c o r e s the  be  tissue  is  greatest  The  net  c h a n g e i n l a c t a t e s t o r e s was  If  a l l of the  t a k e n one  step  further  account  for  the  1882  i n a l l of  rise  in  change would s u r e l y r e s u l t i n glycogen only  concentrations  seen i n the  the  muscle  substrate  resultant  lactate  Since  w h i t e m u s c l e has  the  glucose,  i t follows that  observation lactate  phosphorylated  _i_n  situ. that  stores  muscle tissue  This 85%  no  total  which  lactate pool. gm  fish.  w o u l d be  white  lactate concentrations.  Such a  be  differences  in  the  (wherease changes  apparent depleted  G6Pase,  glucosyl  t h e G6P  that  the  are  white  to account f o r  the  of  the  upon  the  and units  therefore  as  r e s u l t i n g f r o m g l y c o g e n must  be  is  supported  observed  white  i n t o the  increase  the  by  the  i n w h o l e body  muscle. out  the  i n f l u e n c e of  whole-body metabolism d u r i n g  providing  cannot blood  statement  o c c u r s i n the  is  determine  The  enough  These c a l c u l a t i o n s s e r v e t o p o i n t white  case,  t i s s u e s except the  significant  must  this  increase.  release  utilized  is  barely  I t becomes  stores  in  Mmoles/500  of t h e s e t i s s u e s  heart).  This  in glycogen.  to  c o n t r i b u t o r t o the  substrates  total  lactate will certainly  necessity,  m u s c l e were c o n v e r t e d t o l a c t a t e , t h e r e  This  lactate.  take i n t o account n o n - s i g n i f i c a n t d e c l i n e s  the  of the  elsewhere.  c a l c u l a t i o n can  to  2/3  increase  major  source  of  the  hypoxia. fuel  for  100  glycolysis, thereby  but  is  also  causing a s i g n i f i c a n t  metabolism load.  i t  of the r e s t  In a d d i t i o n ,  stores  metabolising and d e l e t e r i o u s  a r e somewhat d e p l e t e d  that  the  hypoxic  observed  appeared  as  produced oxygen  lactate  uptake  upon H o l e t o n translates  of  and  then  roughly  in  1967,  totalling  the  rise  assumption  one  g l y c o l y s i s a n d 14.6 produced production metabolic  over  and  and  a  G6P).  In  this  substrate in lactate  Q10  case,  cannot  be  rate  of  the  trout,  resting (based  2.5).  and r o u g h l y glycolysis  3.75 alone  20 m i n . .  accounted  This  Since for  by  stores, i t i s possible.that this  from  mmoles/500 gm  pools  The  of  f o r roughly  obtains a total mmoles  G6P  40 mg/kg/hr  2  the  oxidation  fish/hr) one  oxidized.  o f 2.8 mmoles o f ATP  3 h o u r s i n a 500 gm f i s h .  (5.8  ATP  4308 Mmoles o f ATP w o u l d be  amount o f e i t h e r t h e s u b s t r a t e o r l a c t a t e was this  aerobic  and  mmoles O / 5 0 0 gm f i s h / h r ,  fish/hr..  depleated  energy  in  glucose,  glycogen  be a b l e t o s u s t a i n t h e a n i m a l  1/3 o f t h e  muscle  r a t e of t h e t r o u t changes  a t r o u t a t 4°C i s r o u g h l y  .625  mmoles ATP/500.gm would  glycogen,  Randall,  to  lactate  I f , f o r t h e sake of argument a l l of  in  (3 A T P / g l u c o s y l  the  ATP p r o d u c t i o n .  stress.  decline  upon  a large  white  decline  One may now a s k how t h e m e t a b o l i c the  rn s i t u ,  i n d i c a t e s that the white muscle i s  supplement the  production with anaerobic  with  impact  o f t h e body by p r o d u c i n g  the observation  not a b l e t o c o m p l e t e l y  that fuel  may  of  Since t h i s is  higher  infer  Using from  substrate r a t e o f ATP than  the  that e i t h e r the  metabolic  r a t e o f t h e t r o u t has i n c r e a s e d o r t h a t n o t a l l o f t h e  'missing'  lactate  i soxidized.  101  There i s another r a t e has does  approach to determining  changed d u r i n g h y p o x i a .  not  decline  l982;Holeton  and  Randall,  statement serves produced i n  oxygen  aerobic-anaerobic that  hypoxia.  acute  t h a t the a n i m a l ' s  some  This  metabolic  This hypothesis  been  in  rate  energetically oxygen  to  tensions  increase,  t h e body may  Saunders  then  utilizing  organs d e c l i n e .  a mixed  but  may during  observations i t s metabolism  possible explanation (1970).  When a m b i e n t trout  rates.  This  more  Work by  so  the  must is  an  as  the  ventilatory  of the v e n t i l a t o r y  total  is  From t h i s one  becomes  to d e c l i n e .  s y s t e m i n c r e a s e s , and  i s being  metabolism  v o l u m e of t h e  s t r a t e g y and  This  system  oxygen uptake u t i l i z e d  by  the a c t u a l oxygen d e l i v e r y  T h i s means t h a t  the  bulk  become o x y g e n l i m i t e d e v e n t h o u g h t h e o x y g e n  of  uptake  maintained. Assuming  and  One  extraction  t h e p r o p o r t i o n of t h e  to the remaining  is  maintain  continue  1970).  the combined  the oxygen r e q u i r e m e n t s  the v e n t i l a t o r y  is  oxygen uptake i s m a i n t a i n e d  expensive  system r i s e s ,  the  trout  Goeden,  of a t r o u t i n c r e a s e s  i s b a s e d on  p r o p o s e d by Hughes and  order  of  production.  oxygen t e n s i o n s d r o p , the v e n t i l a t i o n rise  and  Saunders,  animal  i s s h o w i n g s i g n s of o x y g e n l i m i t a t i o n . has  (Mckim  t h a t , even though l a c t a t e  p a t t e r n o f ATP  the  hypoxia  amounts,  based.  metabolic  o x y g e n u p t a k e of t h e  l 9 6 7 ; H u g h e s and  to r e i t e r a t e  increasing  definitely  suggest  during  The  whether  t h a t t h e o x y g e n u p t a k e of t h e  that g l y c o l y s i s the  increased  metabolic by  the  i s used to supplement rate rate  of of  the  the  trout  anaerobic  trout i s  ATP  ATP  w o u l d be  maintained production, effectively  production.  This  1 02  increase in  i n p r o d u c t i o n may  lactate  stores  be c a l c u l a t e d  or the d e c l i n e i n s u b s t r a t e s .  produced would i n c r e a s e the m e t a b o l i c Mmoles ATP/500gm/hour r e s p e c t i v e l y . in  metabolic  number may with  processes  hypoxia  lack  by  summary, in  the  white  a l l  tissues  trout.  t o 36%. t h a t the  of  to  1346  increase  is  and  increasing  The  exhibited liver  a  appears  muscle i s the major source  other  the  thus  the a c t u a l  trout  physiological  coping  metabolic  the  tissues,  other  the  tissues  t h a t the metabolism  response to  body's  fact  the  most  lactate that  of t h i s m e t a b o l i t e .  Even  that in  glycolysis  metabolic supports  been  communication the  suggestion  a  pronounced  effect,  t h e t r o u t i s m a i n t a i n i n g i t s r a t e of o x y g e n u p t a k e w h i l e  'normal'  rates  damage f r o m b o u t s utilizing  of  in  energy u t i l i z a t i o n .  of h y p o x i a  the  products.  whole a n i m a l .  attempt  to  In  maintain  T h i s s t r a t e g y promotes  which are minutes to hours long  substrates quickly without  of t o l e r a t i n g  the  the  has  upon  glycolysis  of  compared  effect  a c t i v a t i n g anaerobic  status  acute  i t appears  v i a the blood  metabolic  to  Although  of t h e w h i t e m u s c l e w i l l h a v e  the  be  r a t e of the w h i t e muscle i s s m a l l  a c t i v a t e d w h i l e the muscle remains with  Although  i n c r e a s e d i n a l l of t h e t i s s u e s ,  though the m e t a b o l i c the  25%  activating  ATP  f r o m 942  b a s e d upon m e t a b o l i t e m e a s u r e m e n t s .  concentrations  to  from  The  requirements.  stressed,  the  of  w h i c h have t h e n e t e f f e c t  metabolic In  rate  r a t e by  increase  T h i s r e p r e s e n t s an  be a r g u e d , i t i s a p p a r e n t  oxygen  b a s e d upon t h e  by  p r o v i d i n g a d e q u a t e methods  103  SECTION 4  Turnover r a t e s of g l u c o s e  a n d l a c t a t e i n t h e Rainbow  during acute  trout  hypoxia  Introduct ion  Metabolism metabolites of  the  i s constantly  being  of  approximations  of r e l a t i v e  The  trout  i s not  1970)  and  conformer  (Holeton  concentration been  increased. will  support  hypoxia  exhibits  no  occurring.  This  only  provide  and u t i l i z a t i o n .  t o l e r a n t (Doudoroff  signs  1967).  of  being  an  The i n c r e a s e  i s evidence  that  i n the turnover  concentrations  i t unlikely  and  oxygen  in lactate  glycolysis  that  hypoxia  has  lactate  active  there  phosphate  gluconeogenesis  that l i v e r  glycogen  is was  and t h a t t i s s u e s had t o r e l y  decline in liver  due t o t h e f a c t t h a t t h e c o n c e n t r a t i o n s that  significantly  energy  stores f o r glycolytic  The l a c k o f a s i g n i f i c a n t  or  r a t e of  do n o t c h a n g e  l e d to the suggestion  mobilized during  stable,  with  hypothesis.  upon e n d o g e n o u s g l y c o g e n  are  flux  The measurement  metabolites  i n t r o u t , and t h e l a c k of h i g h  makes  3).  very  and R a n d a l l ,  glycogen  compounds  being  of  i n many t i s s u e s a n d t h a t l a c t a t e p r o d u c t i o n h a s  this  during hypoxia  state  r a t e s of p r o d u c t i o n  A measured i n c r e a s e  Liver  not  the  during hypoxia  activated  a  c a t a b o l i z e d and a n a b o l i z e d .  concentrations  Shumway,  in  fuel  (see Section  glycogen  of glycogen  i s a large v a r i a b i l i t y  may  be  i n the l i v e r i n glycogen  104  concentrations mobilized,, glucose  between samples.  then  into  one  the  may  blood  predict will  if  i n d i c a t e that  liver  and  l a c k of a s i g n i f i c a n t  hour hypoxic  liver  the  rise  turnover  rates  glycogen i s being  s a m p l e s i s due  glycogen  that  not  Alternately,  t h a t the  glucose  If liver  i s not  being  r a t e of e n t r y during  do  mobilized  of  hypoxia.  increase,  i t will  during  hypoxia  c h a n g e b e t w e e n c o n t r o l and  t o v a r i a t i o n between  samples.  3  105  Methods  The a n i m a l s Experimental that  were f r o m t h e same s o u r c e  techniques  were  the water temperature  30 t o r r .  3,  increases  a  higher  in  Section  was now 9.5°C a n d t h e h y p o x i c  p0  2  was  i n plasma l a c t a t e ) .  3..  a l s o t h e same w i t h t h e e x c e p t i o n  (since the temperature  Section  as  was  higher  required  Trout  than  p0  2  was  i t was  in  t o o b t a i n comparable  were  fed  until  the  day  before cannulation. Cannulae  (PE 50, C l a y Adams) were i n s e r t e d  a o r t a u n d e r MS-222 solution)  (tricaine  anaesthesia  one  methane day  sulfonate,  before  c a n n u l a t i o n s were p e r f o r m e d f o l l o w i n g Randall  (1979).  experimentation flow through recover  the  water  2 3/4  (as i n S e c t i o n  hours  f o r the hypoxic after  the  continued  injection  regime r e s u l t e d  for approximately  Injections containing  The  t h e m e t h o d s o f H i l l a b y and and  during  i n a s l a t t e d b l a c k box w i t h  3).  Fish  were  allowed  to  was  and  s e r i e s were done b e t w e e n 2 1/4  onset  f o r 30 m i n . i n data  of  after  hypoxia. injection  Since  blood  of t r a c e r ,  f r o m t r o u t w h i c h were  this  hypoxic  3 hours.  Sampling.  R o u g h l y 700 nl o f C o u r t l a n d s  10 u n i t s / m l h e p a r i n and no g l u c o s e injected.  Each i n j e c t i o n  and  contained  [ 6- H ] - g l u c o s e , a n d 7 juCi L - [ "C (U) ] - l a c t i c a c i d 3  w/v  experimentation.  cannulation  f i s h were k e p t  sampling  label  1:15,000  f o r 24 h o u r s f o l l o w i n g c a n n u l a t i o n .  Injections and  Following  into the d o r s a l  1  the  roughly (sodium  saline isotope  7 ^ C i Dsalt).  106  The  isotopes  were  reconstituted  in  dried  the  saline.  removed  and  injectate  were  injection  activity.  The  d i s p o s a b l e s y r i n g e , which  Haematocrits Time  0  been i n j e c t e d . immediately label  in  the  After  and  taken  added  of  to  the  taken  before  up  injection  took  preweighed  ul  roughly flush  10 s  the  Blood  (Boyer,  1975),  withdrawals  sampling  were  followed  inhibitor  stock  containing  These i n h i b i t o r s b l o c k e d (Lehninger,  1975),  saturated KC1 into the f i s h v i a the cannula.  f o r 3 min.  on  i c e , reweighed  the weight  40  lactate and the  and  then model  Volume  by 1.09 gm/ml.  c o r r e c t i o n . f a c t o r was o b t a i n e d by s a c r i f i c i n g density.  by  a n d 130  i n a F i s h e r benchtop c e n t r i f u g e ,  b l o o d was c a l c u l a t e d by d i v i d i n g  30  The f i s h were k i l l e d by  P l a s m a was p i p e t t e d o f f a n d f r o z e n a t -80°C.  stock and measuring  began  the next  injecting  235A.  that a l l  The s a m p l e v i a l s c o n t a i n e d 2  enolase  b l o o d v i a l s were p l a c e d  was  1.5 ml m i c r o c e n t r i f u g e v i a l s .  of s a l i n e .  cyanide.  after  and  t o ensure  transport chain respectively.  centrifuged  injection  and  electron  The  i n a 1 ml  o x a l a t e , 50 mg/ml o f s o d i u m f l o u r i d e ,  potassium  dehydrogenase  precise  1/2 o f t h e b o l u s h a d  h a d been i n j e c t e d .  taken,  200  mg/ml o f p o t a s s i u m of  was  the time a t which  u n i t s o f h e p a r i n , a n d 10 M1 o f  mg/ml  obtain  Ten 200 jul s a m p l e s were w i t h d r a w n o v e r  s a m p l e 6 was  injection  to  of the  volumes.  injection  cannula  being  M1 a l i q u o t s  10  counted  before  was w e i g h e d b e f o r e a n d a f t e r  was  Total  Two  f o l l o w e d by a 0.2 ml s a l i n e  10 s h e n c e . min.  were  nitrogen  injection  to o b t a i n p r e c i s e i n j e c t i o n  sampling.  under  of This  4 f i s h o f t h e same  1 07  Assays.  The  g l u c o s e and analysis  lactate  samples using  (Section 3).  scintilation counted  plasma  vials,  Beckman  LS  1900  s t a n d a r d , and calibrated the dark  enzyme  Liquid  with  purchased  f o r at l e a s t  ixl  fluid.  counting  spectrophotometric  (10 /xl) were d r i e d  Counting Counter  previous  in  water  was  and  done on  w i t h an  program  standards.  4 hours  duplicate for  of d i s t i l l e d  Scintilation  label  in  coupled,  i n 500  scintillation  a dual  assayed  A l i q u o t s of p l a s m a  dissolved  i n 8 ml ACS  were  which  a  internal had  been  The  s a m p l e s were k e p t  to  counting  to  in  reduce  chemoluminescence. Ions  were  A m b e r l i t e MB-3 vials. at  The  least  extracted m i x e d bed  from  50  ul  of p l a s m a u s i n g 0.3  e x t r a c t i o n b e a d s i n 1.5  b e a d s were e q u i l i b r a t e d w i t h 1 ml of 18 h o u r s  before adding plasma.  500  in  a  scintilation  M1 d i s t i l l e d  Calculations. replacement et a l . , detailed  The rates  (1974,1981). as  to  mathematical  was  The  method  being s p e c i f i c  and  then  analysis  of  to t h i s  the a d d i t i o n ul  aliquot  of was  reconstituted  in  above.  used  to  obtain  been p r e s e n t e d  analysing  the  by  Katz  data  used t o o b t a i n the  is area  study.  o b t a i n e d by  fitting  i n t e g r a t i n g between 0.1  f o r the s p e c i f i c a c t i v i t y  o r i g i n a l predicted value.  and  done as  f r o m d e c a y c u r v e s has  a r e a u n d e r t h e c u r v e was  equations  required  Counting  dried,  f o l l o w s , w i t h the procedure  under the c u r v e The  water.  vial,  A 50  Mcentrifuge  1 M glucose for  After  p l a s m a t h e v i a l s were s h a k e n f o r 2 h o u r s . placed  ml  gm  (S.A.) t o f a l l  This o r i g i n a l  v a l u e was  the  data  s and  the  time  t o 1%  of  the  calculated  by  108  dividing  d o s e by b l o o d v o l u m e .  o f t h e body w e i g h t The First, at  d a t a were f i t t e d  I n S.A.  least  fluids,  two  and  processes By  (Stevens,  vs.  taking  relatively  t i m e was  was  in  a  plotted.  two  step  process. includes  l o s s of l a b e l due  to d i l u t i o n  into  the  These  two  to m e t a b o l i t e exchange.  which c o n t a i n s at l e a s t  t h e I n of S.A.,  Y =  5%  decay curve  i n a curve  lines  t o be  The  l o s s o f l a b e l due  straight  taken  1968).  to curves  processes,  result  B l o o d volume was  t h e two (Fig.  l i n e s can  17).  The  two  lines."  be v i s u a l i z e d a s  2  equation:  A,X  u s e d t o r e c o n s t r u c t t h e u p p e r p o r t i o n of  the  curve.  The  equat i o n : NX 2  Y = was these  2  u s e d t o f i t t h e d a t a on e q u a t i o n s , Y=  These g e n e r a t e d replacement area  A e  S.A.,  the lower  p o r t i o n of t h e c u r v e .  X = t i m e , and  A and  N are  standardizing  was  c a l c u l a t e d by d i v i d i n g  weight  and  time  t h e d o s e by  (Katz et a l . , 1974).  number g i v e s t h e r a t e a t w h i c h t h e r a d i o a c t i v e l a b e l in the c e n t r a l p o o l . rate  of  turnover  entry  During  a steady  rate, metabolite entry  curves  rate,  is  the r a t e of removal, and  metabolite  The the This  diluted  s t a t e c o n d i t i o n , where  of a m e t a b o l i t e e q u a l s  r a t e , are a l l equal The  constants.  c u r v e s were i n t e g r a t e d t o o b t a i n t h e a r e a .  r a t e (Ra)  In  the the  removal  to each o t h e r .  were t h e n  r e c o n s t r u c t e d by c o m p u t e r and  plotted  109  along  with the o r i g i n a l data  obtained curve.  by  linearly  This  (Fig.  1 7 ) . The  Y  intercept  e x t r a p o l a t i n g the i n i t i a l  portion of the  i n t e r c e p t , when d i v i d e d i n t o t h e d o s e ,  the  mass  of  the r a p i d l y mixing  This  i s t h e mass, o r q u a n t i t y  pool  (in  was  (Ms)(Katz  results  in  e_t a l . , 1 9 7 4 ) .  Mmoles)  of  metabolite  into  in  analysis i s that the  w h i c h t h e d o s e was i n j e c t e d . One  basic  assumption  made  system i s i n a steady s t a t e . state  i s that  The  the metabolite  the  definition  concentration  during sampling.  T h i s a s s u m p t i o n was t e s t e d  first  4  and  series  last  using  ANOVA.  significantly were in  metabolite  different  hypoxic  (Katz, pers.  Statistics. p  <  and f i n a l  ANOVA, u n l e s s  for acceptable  to  examine  Comparisons between  metabolite  was u s e d .  even  just  H o w e v e r , even concentrations  .19 mM, w h i c h i s  a n a l y s i s f o u n d by p r e v i o u s  Model  though  concentration.  significance  two g r o u p s were done  f o r homogeneity  of v a r i a n c e s  level using  indicated  I n t h i s c a s e t h e Mann-  I r e g r e s s i o n a n a l y s i s was u s e d  t h e r e l a t i o n s h i p between  concentration,  was  comm.).  the F test  U-test  run  by o n l y  t h a t t h e v a r i a n c e s were n o t homogeneous. Whitney  the  i n t h e T O sample  lactate  A l l a n a l y s i s were done u s i n g a  0.05.  comparing  difference.  ( p = 0 . 0 5 ) , t h e means d i f f e r e d  well within the l i m i t s  steady  a t p = 0.05 w h i l e t h e r e s t o f t h e s e r i e s  t h e e x p e r i m e n t where i n i t i a l  studies  lactate  this  does n o t change  by  values obtained  f a r from showing a s i g n i f i c a n t  differed  of  One  of  Ra  values  and  metabolite  we c o u l d n o t p r e c i s e l y c o n t r o l t h e This  was  done  because  the  1 10  concentrations Rohlf,  1969).  w i t h ANOVA. the  were  influenced  by t h e e x p e r i m e n t e r ( S o k a l a n d  The s i g n i f i c a n c e o f  the  was  Significance indicates that a s i g n i f i c a n t  variation  in  Y  is  accounted  Comparison of the s l o p e s of r e g r e s s i o n test.  regression  for  by  tested  amount o f  v a r i a t i o n s i n X.  l i n e s was done w i t h an  F  111  Results  The 17.  The  time.  method  upper graph The  b e f o r e and final  of c u r v e  r e c o n s t r u c t i o n i s diagramed i n F i g u r e  shows t h e c u r v e o b t a i n e d u s i n g  e q u a t i o n s o b t a i n e d from after  curve  the i n f l e c t i o n  curves  i n b o t h t h e c o n t r o l and  curves  are  f r o m g l u c o s e and  hypoxic  listed  f r o m one  fish.  The  activity  ( s e e m e t h o d s ) and  u p p e r p o r t i o n of t h e minutes, value. the  needed The  two  for  inflection  equations  is  the  The  the curve  in  normoxic  fish.  which  was  hypoxic variance The the  the  to reach  and  c o n t r o l Ra-1  and  hypoxic  so  fish  normoxic  fish  is  peak  lactate  specific  p o i n t f o r the the  1% o f t h e  time,  initial  i n minutes, The  and  c o n t a i n s data  at  in peak  which  v a l u e s of A and  N  equations.  ( R a - g ) , was over  not  significantly  v a l u e s o b t a i n e d from  T h i s i s i n c o n t r a s t t o the s i g n i f i c a n t  seen i n l a c t a t e  the  experiments  and  initial  time  s h a r e a common p o i n t .  hypoxic  glucose E a c h row  p o i n t i s the time,  t u r n o v e r r a t e of g l u c o s e  elevated  lactate  calculated  1%  are the r e s p e c t i v e c o n s t a n t s f o r the The  12.  i s used as the  curve.  l i n e s t o the p o i n t s  s t a t e a r e shown i n F i g u r e 18  i n Table  peak  vs  graph.  the c o n s t a n t s r e q u i r e d t o r e c o n s t r u c t a l l decay  S.A.  p o i n t were u s e d t o r e c r e a t e  shown i n t h e l o w e r  Representative  fitting  In  turnover rates (Ra-1)(Table  v a l u e s d i d not e x h i b i t  the  increase 13).  The  homogeneity  of  were c o m p a r e d u s i n g t h e M a n n - W h i t n e y U - t e s t . e x h i b i t e d a g r e a t e r v a r i a n c e i n Ra-1  fish.  than  T h i s r e l a t i o n s h i p o c c u r r e d w i t h the  l a c t a t e c o n c e n t r a t i o n d a t a as w e l l .  These d a t a d i d n o t  did blood  exhibit  1 12  Figure  17. Example of decay c u r v e r e c o n s t r u c t i o n . The two graphs a r e from a c o n t r o l g l u c o s e experiment. Top g r a p h : The y - a x i s i s t h e I n o f t h e s p e c i f i c activity. T h i s r e s u l t s i n an i n f l e c t i o n p o i n t where t h e i n f l u e n c e of l a b e l l o s s t h r o u g h d i l u t i o n becomes masked by t h e i n f l u e n c e o f l a b e l l o s s due t o replacement. The i n f l e c t i o n p o i n t i s t a k e n a s t h e p o i n t w h i c h i s s h a r e d by b o t h c u r v e s , and i s i n d i c a t e d by an a r r o w . Bottom g r a p h : T h i s c u r v e c o n t a i n s the d a t a p o i n t s and a l i n e c o n s t r u c t e d by c a l c u l a t i n g t h e b e s t f i t f o r t h e two l i n e s d e t e r m i n e d i n the upper graph. F o r more i n f o r m a t i o n see Methods.  1 14  Figure  18. E x a m p l e of r e c o n s t r u c t e d g l u c o s e c u r v e s . These r e p r e s e n t a t i v e c u r v e s a r e from a c o n t r o l experiment ( t o p ) and a h y p o x i c e x p e r i m e n t ( b o t t o m ) . The c i r c l e s i n d i c a t e d a t a p o i n t s and t h e l i n e s a r e r e c o n s t r u c t e d b a s e d upon t h e t e c h n i q u e o u t l i n e d i n M e t h o d s a n d i n F i g . 17.  130000  TIME (min)  1 16  Figure  19. E x a m p l e of r e c o n s t r u c t e d l a c t a t e c u r v e s . These r e p r e s e n t a t i v e c u r v e s a r e from a c o n t r o l experiment (top) and a h y p o x i c e x p e r i m e n t ( b o t t o m ) . The c i r c l e s i n d i c a t e d a t a p o i n t s and the l i n e s a r e r e c o n s t r u c t e d b a s e d upon t h e t e c h n i q u e o u t l i n e d i n M e t h o d s and i n F i g . 17.  S2  OS  i  I  -OOOOfi  OOOOOI  OOOOCI  0)  > o ^  3  o OOOOOZ  A  luiui) 3WH i  OS i  S2  koooooe  ooooooi  o UOOOOOfil  Z N t 3 o  0000002  118  homogeneity  of  v a r i a n c e , b u t t h e Mann-Whitney  t h a t t h e d a t a were s i g n i f i c a n t l y values,  were  hypoxic  fish,  not  different.  significantly  n o r were t h e i r  The t o t a l mass of m e t a b o l i t e pool  was  not  analysed  The  different  variances  U-test  with  the  therefore, transform usefully  compared  and  the by  glucose  (Ms)  in  the  rapidly  mixing  T h i s was b e c a u s e t h e conditions,  b l o o d volume o f t h e f i s h .  Ms  into  a  number  could  i s 5% o f t h e  body  Assuming t h a t  weight  (Stevens,  1 9 6 8 ) , one c a n d i v i d e t h e mass o f m e t a b o l i t e by t h e b l o o d a r r i v e at a c o n c e n t r a t i o n (Table  14).  v a l u e of g l u c o s e , nor t h e c a l c u l a t e d significantly  calculated in  the  with  concentration  hypoxia.  to  However,  0.24+0.04 i n t h e h y p o x i c  a c t u a l m e t a b o l i t e c o n c e n t r a t i o n i s known, ratio  suggests  different  concentration is  the  i n the hypoxic  concentrations  pool  that  of  the  this  in  The an  fact  that  underestimate  i n d i c a t e s t h a t t h e volume of  lower  than  glucose ratio  of  0.61+0.10  fish.  Since the  discrepency  in  v o l u m e of t h e r a p i d l y m i x i n g p o o l i s  fish.  result  volume  N e i t h e r the measured  t o m e a s u r e d c o n c e n t r a t i o n s do c h a n g e , f r o m  controls  be  c a l c u l a t i n g a p r e d i c t e d c o n c e n t r a t i o n of  the b l o o d volume of a f i s h  varies  but  We d i d ,  which  g l u c o s e and c o m p a r i n g i t w i t h t h e measured v a l u e .  and  and  different.  using s t a t i s t i c s .  weight  blood  between c o n t r o l  mass w o u l d v a r y n o t o n l y w i t h t h e e x p e r i m e n t a l also  indicated  the  predicted  the  volume.  the of  calculated the  actual  rapidly  mixing  This  suggests  peripheral vasoconstriction. F i g u r e 21 obtained  shows  when Ra-1  the  significant  i s regressed  relationship  which  on t h e c o n c e n t r a t i o n o f  is  lactate  1 19  Table  12. E q u a t i o n p a r a m e t e r s f o r t h e DPM v s TIME c u r v e s i n trout. T h e s e p a r a m e t e r s c a n be u s e d t o r e c o n s t r u c t t h e c u r v e s used i n the d a t a a n a l y s i s . "Peak"= c a l c u l a t e d f i r s t p e a k ; " T i m e t o 1%"= t i m e i n m i n u t e s t a k e n t o r e a c h 1% o f t h e i n i t i a l peak a n d i s t h e u p p e r l i m i t o f t u s e d when i n t e g r a t i n g ; " I n f l . Pt."= t i m e a t w h i c h t h e e q u a t i o n s c h a n g e ; ' ^ , " and "A "= c o n s t a n t s f o r t h e r e s p e c t i v e e q u a t i o n s ; " N , " a n d "N "= exponents f o r the r e s p e c t i v e e q u a t i o n s . 2  2  The  equations are l i s t e d  i n Methods.  120  T a b l e 12. trout.  E q u a t i o n p a r a m e t e r s f o r t h e DPM v s TIME c u r v e s i n  Peak (•S.A.) A.  1% Time I n f l . Point  A,  A  N,  2  N  2  Glucose  Fish  No.  Control 1 219781 2 672493 3 80175 4 106363 5 129689 6 1 08672  268 273 261 139 134 • 237  3. 60 5. 1 2 3. 62 4. 50 3. 57 5. 25  276960 170929 . 196362 274081 164000 110511  67933 52092 37069 57844 84078 45889  -1 -0 -1 -1  . 1 603 .7756 .4221 . 1 539 -o .6052 -o .5900  -.0128 -.0075 -.0147 -.0288 -.031 1 -.0158  Hypoxic 1 40850 2 91491 3 1 1 038 4 96882 5 96673 6 100159  11 6 168 182 80 205 210  3. 77 6. 37 7. 33 3. 65 5. 77 3. 00  254824 656336 360283 172051 130927 260521  68370 43228 38951 73081 50598 25530  -0 -1 -1 -0 -0 -2  .9625 .5388 .2017 .7044 .5128 . 1 251  -.0443 -.0230 -.0196 -.0543 -.0193 -.0154  .849 .1413 .481 1 .6844 .9183 .2491  -.0489 -.0344 -.0493 -.0369 -.0371 -.0404  .31 68 .9628 .654 .7542 -o .6287 -1 .4038  -.0601 -.0394 -.0749 -.0267 -.0251 -.0299  B. L a c t a t e Fish  No.  Control 1 1447482 2 1047089 3 460065 4 855803 5 1 052536 6 2282986  74 81 62 85 86 . 63  5. 28 5. 1 2 5. 07 5. 25 5. 40 3. 75  1905315 1036481 850346 2322178 1034190 1625928  544792 171665 9731 5 196618 256875 292877  -0 -1 -1 -1 -0 -1  [ypoxic 1 169397 2 66558 3 423891 4 57251 5 79233 6 63819  51 92 44 132 137 100  3. 77 5. 40 8. 38 10. 1 3 10. 10 4. 98  245955 502923 1696845 92335 89593 91889  35292 25167 1 10986 19463 24965 12636  -1 -1 -1 -0  121  Table  13.  STATE  Trout m e t a b o l i t e Turnover TURNOVER NUMBER (Ra)  Normoxia  10.7+1.9  Hypoxia  10.6+3.3  Menotes s i g n i f i c a n t  (x+S.E.)  Measured [LACTATE] (/amoles/ml)  (Mmoles/min/kg) Glucose  Numbers.  Lactate  2 . 8 +0.4 20.6+6.8  difference  1  0.62+0.13  1  7.02+1.65  from h y p o x i a  (p < 0.05)  122  T a b l e 14.  Mass o f r a p i d l y WT.  mixing pool  in trout.  STATE  FISH gm  Normoxia  356+20.7  63+8  0.61+0.10  Hypoxia  365+26.5  39 + 9  0.24+0.04  MS Mmoles  (x+S.E.)  [GLUCOSE] calculated/ measured 1  Ms i s t h e number o f Mmoles o f s u b s t r a t e w i t h w h i c h t h e i n j e c t a t e was r a p i d l y m i x e d . The c a l c u l a t i o n i s e x p l a i n e d i n Methods. The c a l c u l a t e d g l u c o s e c o n c e n t r a t i o n i s o b t a i n e d by d i v i d i n g t h e p r e d i c t e d b l o o d v o l u m e i n t o t h e Ms. The r a t i o i n d i c a t e s t h a t t h e p r e d i c t e d volume g r e a t l y o v e r e s t i m a t e s t h e a c t u a l volume d u r i n g h y p o x i a . denotes s i g n i f i c a n t d i f f e r e n c e from c o n t r o l (p < 0 . 0 5 ) . 1  123  F i g u r e 20. The r e l a t i o n s h i p b e t w e e n p l a s m a g l u c o s e c o n c e n t r a t i o n s and g l u c o s e t u r n o v e r . The g r a p h c o n t a i n s a l l of the d a t a but the s i g n i f i c a n t l i n e i s f i t only to the c o n t r o l v a l u e s . I f t h e two c i r c l e d p o i n t s a r e o m i t t e d t h e n t h e p o o l e d d a t a r e g r e s s i o n l i n e becomes significant. H y p o x i a p o i n t s a r e r e p r e s e n t e d by a c l o s e d c i r c l e and t h e c o n t r o l p o i n t s a r e r e p r e s e n t e d by an 'x'.  125  Figure  21. The r e l a t i o n s h i p b e t w e e n p l a s m a l a c t a t e c o n c e n t r a t i o n s and l a c t a t e t u r n o v e r . The g r a p h c o n t a i n s a l l of the d a t a . The e q u a t i o n o f t h e l i n e i s 'Y = .12 + 3.034X'.  [Lactate] (mM)  1 27  (Y = 0.12 + 3 . 0 3 4 X ) .  T h i s f i g u r e c o n t a i n s a l l of the d a t a .  r e g r e s s i o n o f c o n t r o l Ra-1 result  in  comparison  a  slope  of h y p o x i c  relationship, 3.416X".  on  which  lactate  concentration  differed  from  unity.  d a t a d i d , however, r e s u l t  The  d i d not The  same  in a significant  w i t h t h e e q u a t i o n of the l i n e being  "Y = -3.361  An a n a l y s i s o f c o v a r i a n c e b e t w e e n t h e p o o l e d d a t a a n d  the hypoxic data  i n d i c a t e s no d i f f e r e n c e  that the c o n t r o l data  i n slope.  This implies  f o l l o w t h e same s l o p e a s t h e h y p o x i c  data.  The r e l a t i o n s h i p b e t w e e n Ra-g a n d g l u c o s e c o n c e n t r a t i o n shown  in  glucose  Figure  data.  20. The  This solid  figure line  also  Analysis  of  the  pooled  r e g r e s s i o n slopes which two  circled  data  and  do n o t d i f f e r  hypoxic  points  the  the hypoxic from  data  unity.  a r e removed, t h e p o o l e d -2.705  larger  have  been  necessary  which  held  during  whether  the  maintained  relationship  during  t h i s was n o t e d , results  hypoxia.  More  f o r both c o n t r o l and hypoxic  temperature  T h i s was due which  +  1.964X).  of g l u c o s e a n d l a c t a t e .  A  to ascertain normoxia  the  same  and  was  the  f i s h were h i g h e r t h a n t h e  possibly  to  the  increase  in  occurred during the intervening period.  A c o r r e l a t i o n a n a l y s i s b e t w e e n Ra-g a n d Ra-1 i n d i c a t e d t h e r e was no s i g n i f i c a n t  data  f i s h were a n a l y s e d a s soon a s  b u t t h e s t o c k was no l o n g e r  o r i g i n a l numbers. water  would  result in  I f , however,  (Y =  size  significant  (Y = -2.236 + 2 . 0 5 5 X ) .  r e g r e s s i o n d o e s become s i g n i f i c a n t sample  is  c o n t a i n s a l l of t h e  represents  regression obtained using control values  the  +  that  r e l a t i o n s h i p between t h e t u r n o v e r r a t e s  128  Di s c u s s i o n  A  metabolite  metabolite enters that  turnover  i n t o a n d i s removed f r o m  metabolite.  the  The  circulatory  intracellular of t r a c e r pool  number d e n o t e s t h e r a t e a t w h i c h a  plus  compartments  other  minor  (Katz e t a l . ,  pool  of  i s injected  extracellular  1974).  i n f l o w i n t o t h e body i s t h r o u g h  the  i n t o the blood)  a s s u m p t i o n s made a b o u t t h e s i t e s be b o t h  central  c e n t r a l p o o l f o r l a c t a t e and g l u c o s e i s  system  (the tracer  could  a  of  tracer  and  The s o l e rapidly  site  mixing  b u t t h e r e a r e no  loss.  from i n s i d e o r o u t s i d e of t h i s p o o l  This  loss  ( K a t z e_t a l . ,  1974). The  r a p i d l y mixing  unknown  volume  calculations from  data  actual  of  pool  the  central pool.  used t o determine which  are  a  shape  large,  but  In t h i s experiment, the  t h e mass o f t h i s  the  pool  of  the  are  made  2-5 m i n . ( t h e decay  curve).  t h e unknown v o l u m e i s t h a t w h i c h t h e l a b e l m i x e s  w i t h i n t h a t p e r i o d of time. is  of  obtained w i t h i n the f i r s t  t i m e d e p e n d i n g upon  Therefore  i s comprised  roughly  90 s  unobstructed  The c i r c u l a t i o n  (Cameron,  throughout  the  1975),  and  vascular  time of  so  bed,  the  into trout  i fc i r c u l a t i o n i s then  the  unknown  volume s h o u l d a p p r o x i m a t e t h e volume o f t h e plasma. In of  the  cases  where v a s o c o n s t r i c t i o n o c c u r s , h o w e v e r , t h e volume  rapidly  volume ( C a s t e l l i n i  mixing  pool  may be l e s s t h a n  e t a_l. , 1 9 8 5 ) .  and t h a t t h e b l o o d w i l l  blood  T h i s i s e x a m i n e d i n T a b l e 13.  B a s e d upon t h e a s s u m p t i o n t h a t t h e t o t a l body w e i g h t  the t o t a l  b l o o d v o l u m e i s 5%  have a u n i f o r m  of  concentration  129  of Ms  the and  m e t a b o l i t e , one the estimated  i s always lower  may  blood volume.  than  that  than  i n t o which the l a b e l  concentrations  the  measured  increase  p e r i p h e r a l v a s o c o n s t r i c t i o n has When t h e  loss,  the  state.  Under t h i s c o n d i t i o n , the  loss  both  state  i s assumed i n these  based  0.5  considered criterion  Glucose  mM  to was  over be  turnover.  believed  to  et al'. ,  1982)  importance  The  is  is  not  increased  glycolysis  is in a  glucose  of  the  pers. and  the r a t e  of  steady  was  comm.).  metabolism  to  (Walton  and  assume  that  that  hypoxia,  turnover  t r o u t d i d not c h a n g e d u r i n g a s i m i l a r  is This  is  ( H o c h a c h k a and argued  during  sampling  lactate.  in fish  during hypoxia  However, t h e  steady  A c h a n g e of  (Katz,  it  pool  i n the blood.  unreasonable  increased  central  ascertained  carbohydrate  section  m e t a b o l i t e measurements.  that  be  be a m a j o r e n e r g y s o u r c e it  metabolite  p r e s e n c e of a can  timecourse  Although  In the p r e v i o u s  through  in the  number.  f o r both  the  r a t e of e n t r y and  acceptable  satisfied  into  c a l c u l a t i o n s and  the  The  indicating  that metabolite  upon m e t a b o l i t e c o n c e n t r a t i o n s  l e s s than  1984)  then  the t u r n o v e r  hypoxia  entry  equals  equal  of  suggests  in a l l cases.  calculated  is  become more p r o n o u n c e d .  r a t e of m e t a b o l i t e rate  injected  This, in turn,  and  during  This indicates  was  s i t e s of v a s c u l a r r e s t r i c t i o n  between  the  calculated concentration  the t o t a l m e t a b o l i t e p o o l .  there are  difference  The  the measured c o n c e n t r a t i o n .  that the m e t a b o l i t e pool smaller  c a l c u l a t e a concentration using  Cowey its  Somero,  the  flux  based  upon  r a t e s of  hypoxic  not  glucose  exposure.  130  There a r e three metabolic with these  observations.  requirements  First,  Secondly, for  they  glycolytic fuel  while  exhibited  a  borne flux  glucose  rely  supply.  i s unlikely  because t h e r e declining  two  that  in  these  to  that  flux  the  third  option  is  that  to lactate  the  cycle.  real  one  s t o r e s i n t h e h e a r t and a  muscle  d e c l i n e then  and  brain.  glycolytic  I f the  activation i s  data.  Thus,  activated turnover  rates f o r glucose  the in  question  of  turnover  (10.6  Mmoles/min./kg)  systems  (Table  This  5 times higher  reported  f o r k e l p bass  i n coho salmon  (Bever  value  i n turnover  is  i n mammalian than  e t a l . , 1977) a n d 4.6 t i m e s  (Huangsheng  i s not  numbers.  t h a t found  15).  whether  a l l the t i s s u e s  0.5 t o 1.2 t i m e s  difference  the  through  i t i s possible  range from a p p r o x i m a t l y  than  increase  augment  produced which i s reduced  white  i s actually  a n s w e r e d by t h e g l u c o s e Control  t i s s u e s which  I t i s not p o s s i b l e t o d i s t i n g u i s h between t h e former  ideas with  glycolysis  requirements  t o t h e p r o p o r t i o n t h a t e n t e r s t h e TCA  c o n c e n t r a t i o n s of g l y c o g e n indicated.  f o r ATP.  those  i s d e p l e t i o n of g l y c o g e n  trend  remained  relative  In t h i s case  their  glycolysis  either  glycogen  their  increase  requirement  i t i s possible  p r o p o r t i o n of pyruvate  It  cohsistant  their  upon e n d o g e n o u s  Thirdly,  increases r e l a t i v e  tissues  decreased  while  g l y c o l y s i s does not i n c r e a s e . the  while others  other  a l l t i s s u e s may m a i n t a i n  blood  are  T h i s w o u l d a l l o w some t i s s u e s t o f u e l  w i t h b l o o d borne g l u c o s e or  which  some t i s s u e s may d e c r e a s e  f o r blood borne glucose  requirements.  aerobic  patterns  e t a_l. ,  1978).  Most  was  higher of  the  r a t e s c a n be a t t r i b u t e d t o t h e v a r i a t i o n  131  T a b l e 15.  A c o m p i l a t i o n o f g l u c o s e and t u r n o v e r numbers.  lactate  Lactate Ra  (^moles/ min/kg)  label  Reference  Animal  21 1  3-trit iated  rat  Katz,  1982  94  3-tritiated  rat  ibid  94  U-carbon-14  rat  " ibid  62  3-tritiated  rat  ibid  79  3-tritiated  rat  ibid  1 78  3-tritiated  rat  ibid  1 78  3-tritiated  rat  Okajama e t a l , 1981  94  3-tritiated  rat  ibid  94  U-carbon-14  rat  ibid  62  U-carbon-14  rat  ibid  44  U-carbon-14  guinea p i g  F r e m i n e t and L e c l e r c , 1980  Glucose Ra  (Mmoles/ min/kg)  label  Reference  Animal  U-carbon-14  Grey  22  6-tritiated  monkey  21  6-tritiated  rabbit  17  U-carbon-14  rabbit  6-tritiated  K e l p bass  .2-15  .6-2.2 2.3  6-tritiated  seal N  Castellini  et a l . , 1985 Armstrong et a l . , 1979 Dunn e t a l . , 1976 ibid  Bever et a l . , 1977 Coho s a l m o n H u a n g s h e n g e t a l . , 1978  Due t o t h e v a r i a t i o n i n t r a c e r , i n j e c t i o n s i t e , and n u t r i t i o n a l s t a t u s , t h e s e g l u c o s e a n d l a c t a t e t u r n o v e r numbers a r e t o be u s e d as a p p r o x i m a t e v a l u e s o n l y .  1 32  in  plasma  glucose  relationship and  concentration.  There  is a  i n t r o u t between t h e c o n t r o l g l u c o s e  t h e plasma  concentration  concentrations  reported  ( F i g . 19).  When  r e l a t i o n s h i p the r e s u l t a n t predicted turnover  3.3  f o r the  turnover  bass  and  r a t e s of glucose  predicted  based  upon  to  why  the  solution  i s likely  control  over  glucose  than  plasma  (Palmer  in  l a b where  glucose  One  in  this  status  that  carbon source  supports  Thus, t h e  to  what  is  glucose  The q u e s t i o n  arises  from t h e t r o u t i n and  concentrations  rainbow  trout  salmon.  in fish.  may  The  Plasma  increase  after  o f 4.5 mM t o a h i g h  Non-fasting  trout  sacrificed  t h e s i s was u n d e r t a k e n h a v e h a d p l a s m a from  1  made i n t h e p r e v i o u s glycogen  It  i n the l i v e r  observation  rates.  are  to  20  mM  (Dunn,  observations).  hypoxia.  of  close  i n the k e l p bass  ranging  c o n c e n t r a t i o n s of l i v e r  occurring  are  concentrations  a n d Ryman, 1 9 7 2 ) .  observation  during  fish  turnover  glucose  concentrations  unpublished  glucose  numbers  f o r t h e salmon.  i n g e s t i o n , from a c o n t r o l l e v e l  of 28 mM the  rate  t o be r e l a t e d t o t h e a p p a r e n t l a c k o f p r e c i s e  concentrations  carbohydrate  the  r e l a t i o n s h i p between plasma  t h e plasma g l u c o s e  t h i s t h e s i s are higher  4.9  i n these  c o n c e n t r a t i o n s and g l u c o s e as  turnover  i n the references are substituted into  this  kelp  significant  do  not  i s unlikely  at this  organ.  time  that due  As a r e s u l t ,  s e c t i o n was t h a t t h e change  significantly  gluconeogenesis to  the  the only  poor  energy  remaining  major  a v a i l a b l e f o r t i s s u e s a r e endogenous s t o r e s . that  glucose  turnover  does n o t r i s e d u r i n g  t h i s p r e d i c t i o n because t h e i n f l u x  of glucose  is  The  hypoxia  into  the  133  central  pool  would  increase  i f the l i v e r  were i n c r e a s i n g i t s  output of glucose.  Lactate  turnover.  I t h a s been  argued  r a t e s a r e i n c r e a s i n g during hypoxia cause  an  increase  lactate utilization lactate fish  lactate  turnover  may i n c r e a s e  (Balinski  readily  and J o n a s ,  Lactate  turnover  increase  because  glycolytic  3 ) , which would  rates. the  In a d d i t i o n ,  availability  r i s e a n d b e c a u s e most  1972;Mommsen,  1984).  rates increase  in  plasma  roughly  lactate  relationship  when  the  3 times  for  concentration,  range  literature  control  data  turnover  in rats.  a  increased  relationship  exists,  i s i n question.  turnover  enough f o r a d e f i n i t e  rise. choice  h y p o t h e s e s b a s e d upon t h e s e  Metabolite  utilization.  The to  of  a  with  the  The mammalian  (1974)  with concentration  with the unit increase  concentrations  a  are analysed  between  Freminet et a l .  linearly  with  lactate  but t h e p r e c i s e form of t h a t  E l d r i d g e e t a l . , (1974) s t a t e s t h a t  is curvilinear lactate  that  and c o n c e n t r a t i o n  relationship that  of observed l a c t a t e c o n c e n t r a t i o n s . indicates  every  2 0 ) . The l a c k  s e p a r a t e l y may be due t o t h e s m a l l s a m p l e s i z e c o u p l e d small  of  o x i d i z e l a c t a t e when o x y g e n i s a v a i l a b l e  r e l a t i o n s h i p o f "Y = 0.12 + 3.034X" ( F i g . significant  tissue  (see Section  i n c r e a s e s as plasma c o n c e n t r a t i o n s  tissues  unit  in  that  be  made  up t o 6 mM  the r e l a t i o n s h i p  i n turnover sample  reported  tailing  size between  o f f as  i s not large these  two  data.  Turnover r a t e s g i v e the r a t e of i n f l u x  1 34  and  efflux  strictest fate  of a m e t a b o l i t e s e n s e t h e y do n o t  of t h a t m e t a b o l i t e .  the p r o p o r t i o n s oxidized. in  of  estimates  (Freminet  proportion w i l l one  turnover then  able  to  metabolic Randall,  gm  the  are  i s oxidized  et a l . ,  1976),  and  metabolic  trout,  50%  the  w o u l d be  state.  of  the  turnover  i s oxidized  contributions  At a g l u c o s e  turnover w o u l d be  Mmoles of ATP 6 m o l e s of  of  these  rate  of  oxidized in  w o u l d be  ATP  lactate  produced.  produced,  then  r e q u i r e d to o x i d i z e the g l u c o s e .  The and  2  1967;adjusted  9°C  1  hour.  i n the c o n t r o l  This  glucose  to  using  t o an o x y g e n u t i l i z a t i o n  oxidization  for  5152  i s consumed p e r  trout during  uptake.  or  r a t e of t h e t r o u t i s 75 Mmoles 0 / m i n . / k g ( H o l e t o n  translates 500  source  r a n g e f r o m 44 t o 48% o x i d i z e d  Mmoles of g l u c o s e  f i s h p e r h o u r and  2  the  which  turnover  of t h e g l u c o s e  rate.  gm  Mmoles of 0  turnover  1974;Issekutz  estimate  a 500  859  lactate  the  45%  M m o l e s / m i n . / k g , 143  2  of t h e  in  have been made, h o w e v e r , of  v a r y w i t h s p e c i e s and  10.6  1 mole of 0  indication  but  S t e e l e e t a l . , 1968;Royle e t a l . , 1982).  assumes t h a t , i n  is  pool,  of t h e l a c t a t e  et a l . ,  metabolites to metabolic  If  and  50%  i s o x i d i z e d and  one  central  Estimates  f r o m mammals f o r g l u c o s e  ( I s s e k u t z e t a l . , 1965;  If  a  g i v e any  glucose  Approximately  mammals  The  from  This  a  Q10  of  r a t e of  1172  would  mean  s t a t e accounts  This  Mmoles 0 that  role  2  in a  glucose  f o r 73% o f t h e  i s u n l i k e l y c o n s i d e r i n g the minor  o x i d a t i o n i n t r o u t and  2.5).  oxygen  suggested  i n d i c a t e s t h a t the percent  t u r n o v e r number w h i c h i s o x i d i z e d i s l o w e r  i n t r o u t than  of in  mammaIs. The  glucose  turnover  r a t e i n the hypoxic  s t a t e i s the  same  135  as  it  is  i n normoxia.  I t i s not p o s s i b l e t o d e t e r m i n e  p r o p o r t i o n of the m e t a b o l i c  rate  which  g l u c o s e o x i d i z a t i o n - c h a n g e s b u t one This  is  based  produced that  upon  the  is  likely  accounted  infer  observation  (and so more g l u c o s e may  it  may  is  i f the for  that i t declines.  that  lactate  i s being  be e n d i n g up a s l a c t a t e ) ,  that lactate oxidation  i n c r e a s e d p r o p o r t i o n of t h e o x y g e n u p t a k e  by  and  i s a c c o u n t i n g f o r an during  hypoxia  (see  below). The result  lactate  i n 714  turnover  Mmoles ATP  17  moles  o f ATP  and  50% o f t h e l a c t a t e  relationship used  produced  result  from  of  ATP  oxidized,  been  oxidation et a l . , The  fish  Using  suggested  be  rate  of  and  l a c t a t e o x i d a t i o n do n o t Glucose  relatively  Cowey,  lactate  minor  (Table  still  possible  f o r the l a c t a t e  rate  of  21  13).  and  119  control the  supports utilize lactate  substrates for  f a t t y a c i d s (Cowey  during  S i n c e the animal t o be o x i d i z e d ?  hypoxia  (Freminet et  rises  i s hypoxic, i s i t With a  Mmole/min./kg, a s s u m i n g .17 A T P / l a c t a t e , and  t h e p r o p o r t i o n o x i d i z e d a t 50%  of  was  1982).  turnover  significantly  same  10% of  lactate  i n f i s h c o m p a r e d t o amino a c i d s and  l977;Walton  i n the  for roughly  oxygen uptake.  to  the  requirement  if  lactate  o x y g e n u t i l i z e d as  T h i s low p e r c e n t o x i d a t i o n of t h a t g l u c o s e and  i n 1 hour  1 mole of  t u r n i n g over  i t would o n l y account  the m a j o r i t y of the t o t a l have  and  a r r i v e s a t an oxygen  s t a t e was  proposal  gm  oxidized.  produced  E v e n i f a l l of t h e l a c t a t e  the  Mmoles/min./kg would  the o x i d a t i o n of  Mmoles.  oxygen u p t a k e .  2.8  i n a 500  turnover i s  between  f o r g l u c o s e , one  rate  al.,  turnover keeping  1974;Issekutz  136  et  a l . , 1976),  one  arrives  at  5355  Mmoles/min./kg  production.  I f 6 m o l e s o f ATP a r e p r o d u c e d p e r mole o f 0  lactate  oxidized,  is  then  the  oxygen  l a c t a t e w o u l d be 895 M m o l e s / m i n . / k g . proportion  of  ATP when  2  needed t o o x i d i z e t h e  This  w o u l d mean  that  the  oxygen uptake d e v o t e d t o l a c t a t e o x i d a t i o n  would  have r i s e n t o a l m o s t 7 5 % o f t h e t o t a l m e t a b o l i c  rate.  As  glucose,  a n d may  indicate  that for  this  the proportion  i s likely  is  of the turnover  now  possible  The f i r s t  decline.  One  production lactate  means  may  production  that  lower  assume,  therefore,  lactate  increased  there  is  does  oxidative  observation  hypoxia.  This  not  energy  is  that  i s strong  increased.  This  some ATP p r o d u c e d a e r o b i c a l l y , a t a r a t e some ATP  a r e t i s s u e s which increase  during  hypoxia,  r a t e of t h e t r o u t i s l i k e l y The  which  liver tissues.  their  r a t e s of  and t h e r e s u l t i s t h a t t h e to  l a c k o f an i n c r e a s e  glucose supports the conclusion  other  that  second  during  uptake  anaerobically.  stress.  the  oxygen  oxygen a t t h e c o n t r o l r a t e s , p l u s  production  metabolic  The  r a t e of  hypoxia over the c o n t r o l  i s that  I n summary, t h e r e  the  is  the metabolic  the r a t e of g l y c o l y s i s has a l s o  which u t i l i z e s i s produced  during  observation  i s maintained.  evidence that  that  which i s o x i d i z e d  t o propose that  t r o u t has a c t u a l l y i n c r e a s e d  values.  this  t o be t o o h i g h  t r o u t t h a n f o r mammals. It  the  value  with  be  increased  during  i n the turnover  r a t e of  made i n S e c t i o n  3, w h i c h  stated  i s n o t a c t i n g a s a s t o r e o f g l u c o s e f o r use i n  1 37  GENERAL DISCUSSION The  goal  understanding  of  this  of  the  thesis  was  to  biochemical  develop  mechanisms  increased hypoxic t o l e r a n c e i n animals. this  goal,  the  environmental which  have  inter-tissue  differing  responses  of  any  cell  plus  the  findings  energy  The  evidence  utilization  survival  survive  introduction. one  point.  is  not  the premise  be more i m p o r t a n t  meanings  hypoxic  the  The  in  developed  requirements,  above  the  and  available  supply  i s the response  data i n t h i s provide  prolonging  with  were  (as  point,  of a t i s s u e its  in  the by  to  of a r e s t i n g a n i m a l  oxygen uptake  would a l l o w o x i d a t i v e metabolism  word  animals in  the  reiterate thesis  increasing i t s  oxygen  exercise).  a r e n o t c a p a b l e of s u p p l y i n g o x y g e n t o t h e allow  of  hypoxia  outlined  requirements, I n s t e a d , the  to c e l l u l a r  In t h i s c a s e , the combined systems of oxygen uptake  would  possible  production.  episodes  therefore  From  that regulation  associated  as a r e s u l t  low  species  t y p e of h y p o x i c d y s o x i a d i s c u s s e d i n t h i s  energy  which  to  fish  about  to  achieve  investigators,  I t i s appropriate, at t h i s  The  that  to  the m u l t i t u d e of s t r a t e g i e s u t i l i z e d during  lead  responses  stress.  other  supporting  may  made  to a hypoxic  Various  " h y p o x i a " , and  subject  were  of  greater  for hypoxia t o l e r a n c e .  than the r e g u l a t i o n of energy  premi se.  to  order  metabolic  capacities  generalizations  increasing  that  o x y g e n t e n s i o n s were e x a m i n e d i n two  these r e s u l t s ,  thesis,  In  a  at  delivery a  rate  t o be m a i n t a i n e d a n d / o r  which  to s a t i s f y  tissues  and  hypoxia.  a l l of the  cellular  1 38  energy  requirements.  There living  are  two p r o c e s s e s w h i c h o c c u r s i m u l t a n e o u s l y i n a l l  organisms—energy  Obviously,  to  changes  in  internal  a d j u s t i n g t h e p r o d u c t i o n r a t e o f ATP. situation  would for not  where,  i f energy  deviate.  cellular  utilization  n o t be a b l e  processes  was c a l l e d  utilization  C o n v e r s e l y , i f oxygen  by  be t r a p p e d i n  a  f o r by an  and p r o d u c t i o n  supply  increased  a n y r e a s o n , l a r g e amounts o f ATP may be p r o d u c e d  when i t i s  required. i s o b v i o u s l y not s u f f i c i e n t  couple the r a t e s of  energy  utilization.  utilization  with  rate.  The t e r m  the  production  requirement  f o r ATP.  i s used  the  rates  of  I t i s important to  i n the context of the a c t u a l I f a muscle  i s contracting,  requirement or  i f the  t u b u l e s a r e pumping i o n s , t h e n t h e r e q u i r e m e n t s o f t h o s e  t i s s u e s a r e e l e v a t e d a b o v e some b a s a l decrease  with  " r e q u i r e m e n t " d o e s n o t mean b a s a l m e t a b o l i c  ATP a t t h a t moment.  kidney  energy  f o r t h e s u r v i v a l of the c e l l  What i s needed i s t h e c a p a c i t y t o m a t c h ATP  s t r e s s t h a t t h e term  for  would  I t would  s t i m u l u s , the r a t e of energy  rapidly  It to  utilization.  the m e t a b o l i c requirements of  I f t h i s were t h e c a s e t h e n t h e c e l l  respond  external  energy  However, i t i s i m p o r t a n t t h a t t h e r a t e o f  p r o d u c t i o n does not d i c t a t e  the c e l l . to  and  f o r c o n t i n u e d w e l l b e i n g , t h e s e two p r o c e s s e s must be  v e r y c l o s e l y matched. energy  production,  during  oxygen  production could decline  limitation i n synchrony,  level. then  If  requirements  both u t i l i z a t i o n  thereby  maintaining  and the  b a l a n c e b e t w e e n t h e s e two p r o c e s s e s .  T h u s , one way t o v i s u a l i z e  the  i s to picture  control  of  energy  metabolism  requirements  139  dictating  the l e v e l  a t w h i c h p r o d u c t i o n and u t i l i z a t i o n  must  be  responses  to  balanced. In  most  of  the  literature  on  metabolic  hypoxia, the s t r a t e g y which i s d e s c r i b e d ATP  production  (Hochachka, Mustafa,  in  the  1975a;Johnston,  et  metabolism  flux  to  maintain  b a l a n c e b e t w e e n ATP p r o d u c t i o n a n d ATP u t i l i z a t i o n .  (Jackson,  1968,-Robin,  l 9 8 0 ; V a n den. T h i H a r t ,  another  metabolism  a way a s t o d e c r e a s e t h e r a t e o f ATP u t i l i z a t i o n  between u t i l i z a t i o n Animals  This  which  energy  term oxygen the  latter  also  utilize  the  i n such  when p r o d u c t i o n  maintain  when t h e c e l l  conformers.  does  not  in  production.  Thus  the  balance  decreasing l i m i t e d may  the be  i s a m o d i f i c a t i o n of t h e preference  preclude  a l l energy  of  i s oxygen  T h i s term  c o n f o r m e r s and i s used term  strategy  r e q u i r e m e n t s a r e b e i n g made up i n w h o l e energy  solution  and p r o d u c t i o n .  requirements of the c e l l called  would  I t has  1982) The o r g a n i s m  may be a b l e t o a d j u s t t h e p a t t e r n o f c e l l u l a r  forced to decline.  and  a l . , 1968) I n o r d e r t o do t h i s , t h e  been p r e v i o u s l y n o t e d , h o w e v e r , t h a t t h e r e i s  is  maintaining  1975b;Jorgensen  o r g a n i s m must be a b l e t o i n c r e a s e g l y c o l y t i c the  of  face of d e c l i n i n g o x i d a t i v e  1982;Johnston,  1980;McDougal  i s one  the  here  because  possibility  that  or i n part v i a anaerobic conformers  are  oxygen  conformers  b u t t h e c o n v e r s e need  n o t be t r u e .  an  conformer  and  anaerobically  t h e energy  s u p p l y a b o v e t h a t w h i c h c o u l d be p r o d u c e d  oxygen  increasing  have  An a n i m a l may be produced  ATP  oxidatively. T h i s s t r a t e g y may be i n v o k e d i n t h e w h o l e  organism, at  the  140  tissue  level,  or a t the c e l l u l a r  level.  I t i s p o s s i b l e t h a t an  a n i m a l may be an e n e r g y c o n f o r m e r a t t h e w h o l e still  contain  drop i t s  relative  metabolic  rate  f a r below  allowing those others  metabolic  rate.  Conversely,  of  other  (or increase)  i t i s p o s s i b l e t h a t many whole  animal  the animal attempts to maintain  tissues while  i s not.  oxygen u p t a k e t o  i n t e r n a l p h y s i o l o g i c a l and/or  adjustments occur which l i m i t other  biochemical  t h e r a t e of oxygen u t i l i z a t i o n  by  tissues.  Responses  by  t h e l u n q f i s h and t r o u t .  been d e f i n e d , evidence  i t i s possible  and  conformity.  examine  The l u n g f i s h  exceeds  that  largely  to  capacity  the  to  of  has the of  Now t h a t  return  i t within  greatly  a  to  the  the  the  experimental  framework  tolerance trout.  the premise has  Is  animal  to  As  was  pointed  out  i n Section  of  energy  hypoxia  this to  2, t h e b u l k  regulate  lungfish  of t h e muscle  and  w h i t e f i b r e s have a lower o x i d a t i v e c a p a c i t y than  that  fish  upon t h e r e l a t i v e l y well  as  the  species  a l l the  (Section 2 ) . This  low m i t o c h o n d r i a l the  relative  muscle  White  types  of most o t h e r  of  ATP  rates?  mass o f t h e l u n g f i s h i s c o m p r i s e d o f w h i t e m u s c l e f i b r e s . fibres are the least o x i d i t i v e  which  t o l e r a n c e due  r e q u i r e m e n t s i n t h e f a c e o f d e c l i n i n g ATP p r o d u c t i o n  as  but  be e x h i b i t e d i n a s i t u a t i o n w h e r e , when e n v i r o n m e n t a l  hypoxia occurs, working  that  to maintain  t i s s u e s a r e c o n f o r m e r s even t h o u g h t h e This could  level  t i s s u e s w h i c h do n o t c o n f o r m ; o r have one t i s s u e  tissues—thereby their  body  statement i s based  d e n s i t y , and  capillarity,  l a c k of o b s e r v a b l e  g l y c o g e n and  141  lipid  deposits  that  the  l u n g f i s h has  a relatively low  This  and  The  are  of o x y g e n a n d ,  of t h e  metabolite low,  peculiarity  are a l s o low.  aerobic  data.  low  usual  r a t e as  i n d i c a t e d by  fermentative  r a t e of s t e a d y  rate  activities  potential  most l i k e l y , has  activities  of  definition thesis,  of  the  the  only  stress  related  of h y p o x i c  dysoxia  would  The  the or  is  Concurrently,  and  are a l l showing s i g n s t h a t the  state.  metabolite  In  to  a  there  be  lower  The  capacity  the for  h e a r t e x h i b i t s a good  the muscle  changes.  muscle  is  which suggests  the  of  is either  becoming  fish  does  Using  an  sees t h a t the b r a i n , h e a r t ,  addition,  concentrations  should  o u t l i n e d i n the b e g i n n i n g that  tissue  i s b a s e d upon  to hypoxia,  conformer. blood  Any  for  matabolism.  metabolite  deficit, one  This  anaerobic  indicate  f r o m an o x y g e n  maintain  utilization.  enzymes.  i s subjected  enzymes  l a c k of a c a p a c i t y  r a t e of energy  elevated.  by  tissue.  of t h e g l y c o l y t i c  energy p r o d u c t i o n .  oxidative is  lungfish  this  suffering  very  oxidative  s t a t e energy p r o d u c t i o n  f o r b o t h a e r o b i c and  show  a  for a glycolytic  of e n e r g y p r o d u c t i o n .  however,  When t h e  hypoxic  survive with  i s p o s s i b l e that the b r a i n a l s o i s adapted  glycolysis,  not  The  i s t h a t the a c t i v i t i e s  a b l e t o s u r v i v e w i t h a low  than  w h i c h can  is  l u n g f i s h white muscle i s supported  as w o u l d be e x p e c t e d  and  w i t h s u c h a low  It  conclusion  I t a p p e a r s t h a t t h e m u s c l e mass can  a low m e t a b o l i c both  One  rate.  picture  enzymatic  fish).  a body m u s c u l a t u r e  small flux  metabolic  enzymes  (compared w i t h o t h e r  the not  energy liver,  i s in a severely  evidence  based  t h a t the white  upon muscle  142  i s hypoperfused hypoxic, supply  during this  the  muscle  time.  does  o f o x y g e n and f u e l  The  bulk  of  n o t e v e n have a c c e s s  the  body  is  to the l i m i t e d  that i s a v a i l a b l e to the rest  of  the  body, and y e t t h e muscle does not e x h i b i t a marked a c t i v a t i o n of glycolysis.  I f oxygen i s t o t a l l y  ATP c y c l i n g which  r a t e t o 1/12 o f t h e  makes  requirement cannot  up  energy  bulk  of  normoxic  be  detected.  s t r o n g l y suggest conforming  rate.  Thus  muscle,  t h e body, has such a low energy  d u r i n g f o r c e d submergence  even  evidence  the  l a c k i n g , t h i s means a d r o p i n  In  that  metabolite  combination,  these  changes p i e c e s of  that the l u n g f i s h white muscle  tissue  when t h e l u n g f i s h  is  an  i s f o r c e d t o water  breath. T i s s u e s other than l a c t a t e producers. are  prime  white  Both  rate  of  ATP p r o d u c e d f r o m a n a e r o b i c converting  tissues. require  from  two  i t s glycogen  tissues  The l i v e r  store into glucose of glycogen  Two m a j o r a d v a n t a g e s o f intertissue  become  fuel,  saturated  the  removing  exchange i s t h a t  the  with  i s responding  f o r use i n o t h e r does  not  the  muscle  mass  ( i ) organs w i t h a small  and ( i i ) n e i t h e r t h e s e with  high  i s b e c o m i n g an e n e r g y  mass b u t w h i c h have h i g h e n e r g y r e q u i r e m e n t s the a v a i l a b l e  remains  to glucose  ATP i t i s p o s s i b l e t h a t t h e l i v e r  the  be  o f a e r o b i c ATP p r o d u c t i o n  metabolism.  Since the conversion  conformer.  to  r e m a i n a c t i v e t o some d e g r e e .  these  enough t o r e q u i r e s u p p l e m e n t a t i o n  by  appear  t i s s u e s d i s p l a y s i g n s of metabolic  t i s s u e s must s t i l l  Thus, the m e t a b o l i c  muscle  Of t h e t i s s u e s e x a m i n e d , t h e b r a i n a n d h e a r t  candidates.  s t r e s s and both  the  c a n have a c c e s s  to  organs nor the plasma  l a r g e q u a n t i t y of u n d e s i r a b l e end-  143  p r o d u c t s w h i c h may The  oxygen t e n s i o n s f a l l .  the white muscle,  white  and  Every  severe  stress  t i s s u e examined,  shows some s i g n o f h y p o x i c  oxidative  of w h i t e  red  o x i d a t i v e and  in  muscle.  stress.  when  including The  trout,  t h e l u n g f i s h , d o e s n o t have a w h i t e m u s c l e mass w h i c h  composed s o l e l y  axial  i n the  t r o u t , on t h e o t h e r h a n d , e x h i b i t s  arterial  unlike  be p r o d u c e d  fibres.  types.  The  t h e i r presence  requirements  of  Instead there i s a mixture  red  fibres  would,  in  t h e m u s c l e mass.  increase  The  fact  within  white  mass  suggests  The  changes i n t h e w h i t e muscle  these  metabolic  accumulation  t h a t t h e y c o u l d n o t be t h e m a j o r  activated  necessary  To to  i n t h e w h i t e m u s c l e and  metabolite  white  is  load.  pools  a r e not  is  the  isolated  only  explain  that  tissue  the  i n the  the l a c t a t e produced  i n the muscle i s a b l e to  efflux  The  l a c t a t e g r a d i e n t s , which  from  the muscle,  muscle glycogen  support  formed,  remain  and  blood  other d u r i n g acute Since  carbohydrate  i t i s probable that enter  the  blood  favorable for lactate  this suggestion.  i s m o b i l i z e d , t h e l a c k of  source  that glycolysis i s  enough  f o r the l a c t a t e  of  end-product  lungfish).  s u b s t r a t e to account  pool.  the  muscle  from each  with  heart  calculations  conclude  ( i n c o n t r a s t to the s i t u a t i o n muscle  but  b r a i n and  suggest  it  hypoxic,  for  response.  were  rate  that  reason  organs  of t h e w h o l e - b o d y l a c t a t e  hypoxia  the  m e t a b o l i t e c o n c e n t r a t i o n s i n the t r o u t  indicate  the  t h a t the red muscle f i b r e s b u r i e d  the white muscle are not, i n themselves,  the observed  be  t h a t the  r e d mass showed l e s s o f a c h a n g e d u r i n g h y p o x i a t h a n  the  of  are considered to itself,  is  Assuming t h a t  glucose-6-phosphatase  144  in  the  except  tissue  the muscle.  results within  precludes  the  use  Thus i t i s u n l i k e l y  from the muscle g l y c o g e n the muscle  rates. in  t h a t the  s i t u a t i o n a t any  Although  surprising,  glucose  since liver  appear  to  blood-borne  The  liver  use  in other  of  the  I t cannot i n d i c a t e  flux  given time.  t u r n o v e r does not  glycogen  in  l u n g f i s h and  rates  of  this  i s activated during rise,  this  f o r an  on  an  differing  are  likely  liver  responses  hypoxia  responses  of l i v e r  of  In  the  this  trout  of  regard,  b e t w e e n t r o u t and most  t r o u t muscle. the of  an  significant  energy  This i n a b i l i t y main acute  hypoxia.  metabolic hypoxia.  store  The  of  glycogen.  contribution In the  to  episode  tissue,  this  by  u n l i k e the  s p e c i e s i s not  ATP-turnover  the  lungfish,  i n the t r o u t i s c o n s i d e r e d , why  not  l u n g f i s h muscle masses  conforming  reason  for  i t i s worth  W h i t e m u s c l e has more  t o l e r a n c e of t h e s e a n i m a l s .  becoming  and  does  does not c o n t a i n the l a r g e s t  organ b a s i s .  t o make t h e  apparently  tolerant  not  increased flux  t h e a x i a l m u s c l e mass i s a b l e t o e n d u r e t h e h y p o x i c  be  is  t h e r e does  t r o u t t o t h e s t r e s s of  function.  t h a t the l i v e r  The  to  lactate  l u n g f i s h gradually releases glucose  t i s s u e s while the have  mentioning  relative  picture  i s n o t m o b i l i z e d and  be a m a j o r r e q u i r e m e n t  of the h y p o x i a  to  glycogen  which  glucose.  muscle  appear  lactate  instantaneous  T h e r e a r e marked d i f f e r e n c e s i n t h e white  tissue  but  trout also indicate that glycolysis  hypoxia.  i n any  s t o r e i s produced anywhere  However, d i r e c t e s t i m a t e s of t u r n o v e r  the  not  fuel  itself.  M e t a b o l i t e d a t a o n l y g i v e an metabolic  of t h i s  rates  here, very (and  145  metabolic  requirements  l u n g f i s h m u s c l e when declines.  This  the  impact  l a c t a t e data  of  Interestingly,  i n samples taken  morning  roughly  of onset  60% of those  Brett  and  Zala  (1975)  (which  was  used  here)  and  artificial  coinciding  with  manipulation  effects  which  capacity  for surviving  a  oxygen  question,  "What  depression  to  depression),  hypoxia  change  that a  roughly  daily  diurnal double  feeding  cycle  of  minimum  Thus,  r a t e showed t h e  near  mid-day  the  time.  The  i n the  from f i s h exposed  causes  reduction i n metabolic  this  beneficial  r a t e may have upon t h e  hypoxia.  cases.  It  would  is  the  J u s t how w i d e - s p r e a d  r a t e i n response  be  survival  invoked  during  importance hypoxia  i tcould s t i l l  adaptations  within  i s the  to a diminished  u s e f u l t o be a b l e t o a n s w e r t h e  relative  I f a l l of the m e t a b o l i c were  i s of  of t h e exposure.  i n d i c a t i o n c a n be p r o v i d e d by t h e f o l l o w i n g  hypoxia  production  depression  to  showed  the  f o r reducing metabolic supply?  taken  of metabolic  Metabolic depression--other capacity  energy  i n the  from f i s h exposed e a r l y  m e t a b o l i c , r a t e w i t h t h e peak b e i n g value  down  t h e c o n c e n t r a t i o n s of  from a s e t exposure  concentrations  feeding  aerobic  f o r metabolic  d e p e n d i n g upon t h e t i m e  mid-day.  switched  o f t h i s c a p a c i t y i s i n d i c a t e d by t h e t r o u t b l o o d  resulting  were  are  for survival.  i n S e c t i o n 3.  blood l a c t a t e markedly  rate  capacity  paramount importance The  f o r ATP)  a  only l a s t  of  or  anoxia?".  One  calculations.  for surviving cell  metabolic  (except  as l o n g as t h e r e  cellular metabolic is  fuel  1 46  for  glycolysis.  the c e l l  For  t h e s a k e o f a r g u m e n t , l e t us assume t h a t  has o p t i m i z e d a l l f a c e t s of a n a e r o b i c energy p r o d u c t i o n  and c a n m e t a b o l i z e u n t i l a l l o f t h e f u e l the s i m p l i f y i n g assumption be  made.  Thus,  i s gone.  that the metabolism  In  addition,  i s anaerobic  will  t h e maximum a n o x i a e x c u r s i o n t i m e i s e q u a l t o  the p e r i o d d u r i n g which  the  glycogen  energy  One  can  requirements.  store  then  can  compare  supply  basal  the t h e o r e t i c a l  maximum w i t h t h e a c t u a l e x c u r s i o n t i m e t o s e e i f t h e r e i s enough available  substrate.  I f t h e maximum e x c u r s i o n  time  is  longer  t h a n t h e c a l c u l a t e d e x c u r s i o n t i m e t h e n m e t a b o l i c d e p r e s s i o n has occurred. For the t u r t l e , are  about  880  liver  glycogen  Mmole/gm  Mmoles  metabolic  of  i n the normoxic  (Daw e t . a l . , 1 9 6 7 ) .  ( e x c l u d i n g t h e s h e l l ) has about 7,600  levels  glycogen  8.6 gm o f l i v e r , (glucosyl  equivalent metabolic  metabolism to  rate,  and t h u s  about  the  assuming  resting  70 mmHg c a n a  Q10  2 ( J a c k s o n , 1971); t h i s  5180  Mmoles  liver  glycogen s t o r e s a r e adequate  100 gm a n o x i c t u r t l e of  of about  turtle  of about  2  be e s t i m a t e d a s 0.036 Mmoles ATP/gm/min., respiratory  A 100 gm  units).  r a t e o f t u r t l e s a t 3°C a n d a P a 0  ATP/100 gm  f o r o n l y about  turtle/day.  2.9 d a y s .  state  for  rate i s At  this  to maintain a  Other  body  stores  g l y c o g e n may e x t e n d t h i s t i m e p e r i o d , b u t i n t i s s u e s s u c h a s  the h e a r t , glycogen s t o r e s a r e hours  of  anoxia  (Daw et.. a _ l . , 1 9 6 7 ) .  a t 24°C, t h e m e t a b o l i c r a t e predive  levels  largely  (Jackson  depleted  after  In s h o r t - d u r a t i o n  i s known t o d r o p  to  about  and S c h m i d t - N i e l s e n , 1966).  results are extrapolated to  the  a  situation  at  3°C,  few  diving 15%  of  I f these then  the  147  anoxia fold,  tolerance i.e.  is  i s the the  the  t u r t l e c o u l d be  t o a b o u t 19 d a y s .  in metabolic•rate than  of  I t i s apparent that  i s f a r more s i g n i f i c a n t  l a r g e s t o r e of l i v e r  observation  that  turtles  at  l o n g as  1982).  metabolic  no  unusual  increased  energetic efficiency,  that  turtle  the  depression.  This  depression t h a n can the  to  be  1/60  survival  time  is left  of  can  extending  g o l d f i s h as a s u b j e c t .  s u r v i v e f r o m 4-5 weeks  Liver  glycogen  1300  mM;  4°C  a  of  total  levels 100  tissues).  is  gm  0.05  The  would  an  conclusion metabolic  a  metabolic  i t i s twenty f o l d alone.  longer  None  of  tolerance could  depression  of  the  be  acclimated  ATP/gm/min. fish/day.  calculations  A cold-acclimated  individual  Hochachka,  1981)  Johanson,  goldfish  a b o u t 6 gm  r e s t i n g metabolic  be a b l e  same  (this w i l l yield  of  1977).  are  liver,  to  about  and  15,600 Mmoles of  r a t e of the g o l d f i s h  thus ATP at  which i s e q u i v i l e n t t o about At  t h i s metabolic  t o s u r v i v e f o r 2.2  5 - f o l d w o u l d be  11 d a y s of a n o x i a ;  do  ( W a l k e r and  g o l d f i s h has  Mmoles  only  anoxia  for winter  7,200 Mmoles ATP/100 gm fish  require  d a y s ( S h o u b r i d g e and  a b o u t 7800 Mmoles g l y c o g e n at the  Jackson,  providing  w i t h the  anoxia  have  effective.  the  several  is  substrate supply  T h e r e i s enough i n f o r m a t i o n t o using  survival  temperature  impressive  would  of r e s t i n g r a t e s and  mechanisms  this  an  7-  Even more r e m a r k a b l e  pathway  one  to  reduction  6 months ( U l t s c h and  undergone  a c c o u n t e d f o r by  other  n e a r l y so  has  a  6-  in prolonging  glycogen.  s u r v i v e d s u b m e r s i o n f o r as Since  e x t e n d e d by  days.  r e q u i r e d f o r the animal  both t h i s metabolic  r a t e and  this  rate, A  the  metabolic to  survive  degree  of  1 48  anoxia  tolerance are w i t h i n  reported estimates for this  species  (Anderson,1975). M y t i l u s s p . , t h e common m u s s e l , i s a l s o a g o o d anaerobe.  If  classical mantle anoxia  we  assume  glycolysis,  and  liver  assume  that  i t i s obtaining  t h e n t h e 550  would  f o r 3 days  that  (De  a l l of  Mmole  Zwann,  rates,  Zwann and W i j s m a n  metabolic  r a t e d r o p s t o 1/20  De  f o r 9 days.  On  in  the  during  1982).  If  we  of r e s t i n g  f r o m 60-150 d a y s ,  depending  the  basis  of  ATP  (1976) e s t i m a t e t h a t  the  rates during anoxia.  At  l o w e r e d m e t a b o l i c r a t e , t h e m u s s e l now  has enough  upon  the  substrate  fermentation  used. Many  hypoxia.  other  animals  Leivestad  undergo  metabolic  that  i t d r o p s t o r o u g h l y 20% o f  become  oxygen  depression during  calorimetry  conformers  (Burggren  the  when of  resting  subjected  level. to  increasing  and R a n d a l l ,  modest  overall  t h o u g h t h e swimming  squirrels  (animals  body t e m p e r a t u r e Whitten,  1975).  measure  capable  when  reduced  of  subjected  are  oxgyen lactate  Kooyman e t . a l . the Weddell  m e t a b o l i c d e p r e s s i o n when muscles  found  Sturgeon  arterial  1978).  (1980) h a v e s u g g e s t e d t h a t a mammalian d i v e r , exhibits  to  r a t e o f a submerged t o a d B u f o m a r i n u s , and  t e n s i o n s b u t show no e v i d e n c e concentrations  metabolic  (1960) u s e d d i r e c t  the  even  from  the glucose i s metabolized to propionate,  turnover  path  glycogen  1983;Hochachka,  last  to survive  i t s energy  supply r e s t i n g metabolic rates  then the animal c o u l d  this  of  facultative  active.  seal,  underwater,  Active  ground  h i b e r n a t i o n ) e x h i b i t a drop i n to  hypoxia  (Faleschini  and  149  These for  cases  metabolic  (Artemia least  r e d u c t i o n e x h i b i t e d by  salina).  This  (Stocco  curtailment metabolic  and  The  currency.  thus  During  energy  This  or,  in  here  a  more  of  mainly  in  itself  the  between  day  embryos per The  day  cell  first  2 weeks.  is  respectively  As was  The  of the  and  total  form  the  1-3  of  of  but i t  depletion  a v e r a g e r a t e of anoxia,  0.037 jumoles  high and  P/100,000  ( S t o c c o e t . a_l. , 1 9 7 2 ) .  oxygen a v a i l a b i l i t y  s t a t e d above, t h i s d e p r e s s i o n  production.  additional  a  cell  has  requirement  two  levels  of  subsequent  below  this  only proceed  minimum  the  t o m a t c h t h e r a t e of energy  needed t o s u r v i v e and  needed t o p e r f o r m  p r o d u c t i o n can  i s wide-  i s a t t a i n e d by  work.  t h a t the s t r a t e g y of r e d u c i n g r e q u i r e m e n t s  r a t e o f ATP fall  The  minimum  requirement  surmise  does not  be  phosphate  i n the  that  days  1.24  i n t h e f a c e of d e c l i n i n g  requirement;  the  sees  between  110,  to  r a t e of metabolism  a d j u s t i n g r a t e s of energy r e q u i r e m e n t s  energy  may  and  words,  above examples i n d i c a t e t h a t the c a p a c i t y f o r m e t a b o l i c  depression spread.  56  low  when one  energy phosphate d e p l e t i o n  other  a n o x i a a b o u t 50%  a  occurs  drastic  recognizable  d5pppp.  becomes e v e n more s t a r t l i n g  a  lipid  (d5pppp) w h i c h i s b r o k e n down t o  months  in  f o r at  or  seems  i s used, mostly  is  embryo  in anoxia  implies  a v a i l a b l e phosphate-bond energy This  shrimp  upon i t s c a r b o h y d r a t e  currency  creating 4  exist  utilization  5'-tetraphosphate  GMP,  brine  can  1972).  substrate  rate.  diguanosine  effect  e_t. a l . ,  of  the  organism  5 months w i t h l i t t l e  reserves  GTP  p a l e , h o w e v e r , when c o m p a r e d w i t h t h e c a p a c i t y  so  f a r as  requirement.  It  any One  t o match production has  been  150  proposed  that  maintaining  ion  g r a d i e n t s a c r o s s membranes  consume a m a j o r p r o p o r t i o n of t h e r e s t i n g cell  ( B r e z i s e_t a l . , 1 9 8 4 a ; B r e z i s  surmise  that i f a c e l l  g r a d i e n t s then be  s h o u l d be  l o w e r and  They  manipulating  i s used which tolerance  a may  these  cell  may  survival  1978;Eveloff  during  normoxic 1981),  exposures.  are  production. rate  still  Thus,  (low  does not d e c l i n e  and  These m a n i p u l a t i o n s  This  perfused  with  the  (Swartz  energy  because  et  utilization are  the  al., i s no during reduced,  requirements  i n t o l i n e w i t h the r a t e of (1984a)  i n c u b a t i n g with polyene  not  reduce  that there  requirements  Conversely, B r e z i s et a l . , of a k i d n e y by  medullary  t r e a t m e n t s do  be p r o p o s e d  when e n e r g y  while  work).  reducing  perfusion  production  this  glomerular  d u r i n g h y p o x i a , b u t do  i t may  j u s t being brought  of  i f a p e r f u s i o n medium  Since these  oxygen uptake  l o n g e r a m a t c h between ATP  ATP  of  damage t o t h e  thus  kidney  rat kidney  rate  filter  exposure,  t h e s o d i u m pump.  et a l . ,  oxygen uptake  the  not  the  f o r the examination  that hypoxic  need  hypoxic  reduce  uptake  that  f u r t h e r e n h a n c e d i f t h e k i d n e y was  of  significantly  shown  (mTAL) i s l e s s s e v e r e  the kidney  was  expenditure  these  of the  isolated, perfused  adjusting  found  limb  before  oxygen  (by  They  thick ascending  ouabain  so one  the c a p a c i t y f o r hypoxia  (1984b) has  s t u d i e d an  work  filtration).  work  requirements  be a good mammalian m o d e l t o use  proposal.  for  e t a l . , 1 9 8 4 b ) , and  of  enhanced.  Work by B r e z i s e t a l . , may  budget  i s a b l e t o t o l e r a t e a r e d u c t i o n of  t h e minimum e n e r g y  correspondingly  energy  may  increased  ATP the  antibiotics.  i n c r e a s e the p e r m e a b i l i t y of c e l l  membranes,  151  which l e a d t o a compensatory i n c r e a s e transport results  (thereby  increasing  in histological  that which  occurs damage  preparations  are  the  rate  oxygen u p t a k e ) .  severe  caused  perfused  hypoxia  and  by  moderate  with  ouabain  of  This  damage w h i c h i s s i m i l a r  with  histological  in  active  procedure  i n appearance t o exacerbates  hypoxia. (thus  the  When  the  reducing  the  activity  o f t h e membrane pumps), o x y g e n u p t a k e d e c l i n e s and t h e  integrity  of t h e kidney  rates  of  i s maintained  (ostensibly  because  energy p r o d u c t i o n a g a i n match t h e energy  of t h e c e l l ) .  These e x p e r i m e n t a l  (requirements)  underscore  manipulations  the  requirements  of  work  rate  the d e t r i m e n t a l e f f e c t s of uncoupling  the r e l a t i o n s h i p between energy r e q u i r e m e n t s  and  the  rates  of  energy p r o d u c t i o n .  Conclusion.  It  i s apparent  that hypoxia  t o l e r a n t animals (or  t i s s u e s ) have much more e f f e c t i v e methods o f survival  during  anaerobic initial  hypoxia  energy  than  production  when  problem w i t h the onset  production  ceases  to  meet  If,  i n order  concurrent  increase  in  maintain  the  utilization.  between  with the hypoxic  energy requirements  required  limiting.  requirements. energy  The energy  However,  utilization  and  t h e energy s t a t u s of t h e c e l l . insult,  energy coupling  t h i s case,  is  i s that aerobic  there i s a l a r g e drop i n  ( i n d i c a t i n g an e n e r g y  anaerobic  In  oxygen  metabolic  to maintain  tissue  increasing the c a p a c i t y f o r  of hypoxia  c e l l s must m a i n t a i n a b a l a n c e production  just  prolonging  conformer),  then  an  p r o d u c t i o n may n o t be n e e d e d t o between  the reduced  ATP  production  and  p r o d u c t i o n o f ATP f r o m  152  aerobic  m e t a b o l i s m may  Conversely, aerobic  be  i f energy requirements  metabolism,  to sustain  sufficient  the  then the  responses  possible  to  the  of  infer  in metabolic  there  the  This  upon t h e to  does  metabolites,  nor  i s n o t e d on  t h e s i s argues that of  have  respiration.  muscle  supplied flux  l e s s than i f  white  no  with  required metabolic  a whole-body vary.  been r a t e of  The  not  an  utilize  which  rate.  uncoupling  It is a  has this  of  the  this  of  the  is most  tissue  rate is  the  d o e s i t p r o d u c e l a r g e q u a n t i t i e s of  it  inverse  response  a large portion  not  decline  the  In  g l y c o l y t i c f l u x and  a d v a n t a g e of  level,  in lungfish,  muscle  whole-body m e t a b o l i c  r e l a t i o n s h i p between the  the  be  i n d i v i d u a l t i s s u e s may  response  appears  cellular  be  t h a t a l l t i s s u e s have r e s p o n d e d w i t h  rate.  metabolic  influence  not  demand.  occurred.  Even when o x y g e n c o n f o r m i t y the  can  amount of a n a e r o b i c  animal would s t i l l  d e p r e s s i o n had  to supply metabolic  of  that  available undesirable  end-products. Trout glycolysis  muscle, during  in  hypoxia.  significant  quantities  accumulated  to  the  contrast,  high  of  a  production which,  body.  l a r g e p e r c e n t a g e of may  lead  when  m e t a b o l i s m and  to  function  T h u s , i n an  i n an  and  the  to  activation muscle  protons,  mass  comprises  r a t e s of  accumulations  of  plasma,  lactate  end-products perturb  the  animal which i s capable o f _ u t i l i z i n g  the  of o t h e r t i s s u e s and  may  if  e f f e c t upon  b o d y , even low  the  of  produces  which,  have a d e l e t e r i o u s  S i n c e the muscle  large  transferred  an  so d o i n g , t h e  lactate  l e v e l s , may  o t h e r organs i n the  such  In  exhibits  organs.  1 53  whole-body  strategy  of r e d u c i n g m e t a b o l i c  to hypoxia,  the metabolic  responses  between t i s s u e s .  Some t i s s u e s may  metabolic  and  rate,  some  may  at the t i s s u e show  tissue  rate  i s the white  signs  exposure  l e v e l may of  vary  maintaining  show s i g n s o f b e i n g c a p a b l e o f  surviving with a reduction i n metabolic the  rate during  rate.  In the  lungfish,  w h i c h shows a c a p a c i t y f o r a r e d u c t i o n i n m e t a b o l i c muscle.  1 54  REFERENCES CITED  A n d e r s o n , J.R. 1975. The a n a e r o b i c r e s i s t a n c e of C a r a s s i u s a u r a t u s . PhD T h e s i s . A u s t r a l i a n N a t i o n a l U n i v e r s i t y , Canberra. A r m s t r o n g , M.K., Romsos, D.R. and L e v e i l l e , G.A. 1979. G l u c o s e t u r n o v e r i n f a s t e d c y n o m o l g u s monkeys (Macaca f a s c i c u l a r i s ) a s m e a s u r e d by ( 2 - H ) , ( 6 - H ) and ( U - C ) g l u c o s e . Comp. B i o c h e m . P h y s i o l . 62A;1014-1015. 3  3  , a  A t k i n s o n , D.E. 1977. 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