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UBC Theses and Dissertations

Strategies for acid-base regulation in fishes Iwama, George Katsushi 1986

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S T R A T E G I E S FOR  ACID-BASE REGULATION  IN FISHES  by GEORGE K A T S U S H I IWAMA B.Sc,  University  of British  Columbia,  1975  H.Sc,  University  of British  Columbia,  1977  A T H E S I S SUBMITTED  I N P A R T I A L F U L F I L M E N T OF  THE R E Q U I R E M E N T S  FOR  DOCTOR OF  THE DEGREE  OF  PHILOSOPHY  in  THE F A C U L T Y OF GRADUATE S T U D I E S (Zoology)  We  accept to  THE,  this  thesis  the requ-ired  UNI^ETTSITY  OF  July  (s) G e o r g e  as  conforming  standard  BRITISH  COLUMBIA  1986  K a t s u s h i Iwama, 1986  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 o f the  requirements f o r an advanced degree a t the U n i v e r s i t y o f 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 o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by t h e head o f my department o r by h i s o r her r e p r e s e n t a t i v e s .  It i s  understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l n o t be allowed without my w r i t t e n  permission.  Department o f  r  ZOOLOGY  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  Date  E-6  (3/81)  August 21, 1986  Columbia  ii  ABSTRACT  Three  sets  investigate There  are  which  the  be  of  several two  gill  water  tensions  in  the  exchange  of  ions  concentrations  to  flow  blood.  these  two  investigated  might  in  as  the  intracellular  steady  dioxide  only  water  at  flow  Dogfish, levels  at  these above  exchange a c r o s s  can  -  in fishes.  of  be  or  fishes  NH4  or  +  in  in can  adjusted  decrease  epithelium  the  the  role  that  pH  the  of  compartment o f  showed  excretion  increased  gill  to  extracellular fluid  possibilites.  manipulation  state  excretion  gills  s e c o n d mechanism would  the  regulating  Experimental in  the  the  increase  HCO3  +  experiments  well  to  the  H ,  conducted  by  PC02  the  involve  the  t o change  the  the  blood.  s e t s o f e x p e r i m e n t s were, r e s p e c t i v e l y , d e s i g n e d  investigate  play  involve  excretion  The  across  of  two  CO2  were  acid-base regulation  r e g u l a t i o n of  First,  altering  experiments  ways t h a t  acid-base  first  vivo  aspects of  possible  adjusted.  The  in  that  levels was  of  retarded  l e v e l s of g i l l control  levels  third  plasma  set  extracellular fluid the  red  water  than about and  blood  flow  effected  trout  affected  PC02  ventilation.  CO2  Carbon pressures  Increasing  neither  as  cell.  lOOml/min.  blood  of  catecholamines  v e n t i l a t i o n i n rainbow  gill  lower  The  O2  or  gill CO2  gill.  subjected  of hypercapnia,  to  environmental  showed the  best  h y p e r o x i a and  various  c o r r e l a t i o n between  gill  iii  ventilation with  and  plasma  did  not  Pco2  affect  exponentially  the  pH  in  that  species,  to  recovery  reduction  were in  maintaining  effected  salinity  was e f f e c t e d was  of  exposed  the  normal  of the  efflux  HCC>3~  Consistent  analyses exposed  accumulated  of to  showed the  i n response to  by an i n c r e a s e an  ion.  plasma  i n d i c a t i n g the  exchange  of  process.  hypercapnia,  water  Therefore,  C1 /HC03~  a  C l ~ , while  in  i t seems t h a t t h e exchange  _  the  carp,  process  and p r o b a b l y  water.  from t h e above c a r p e x p e r i m e n t ,  electrochemical  active  i n plasma  caused the r e d u c t i o n o f the  i n fresh  data  o n water  associated decrease  environmental  environmental  that  decline  to  uptake  effect  Recovery o f plasma  C1~/HC03~  accumulation  the  and t h e s e a  had a d i r e c t  a  active  the  trout  both species,  rates,  and c o n g e r  with  also  in  active  this  i n the t r o u t  salinities  increased  h y p e r c a p n i a , was d e p e n d e n t  concentration of this  modulation  concentrations  ventilation  water  an i n i t i a l  There  involvement  carp  further  water  C l ~ concentration  possible  plasma  HCC>3~  Gill  the f r e s h  after  environmental The  plasma  When  plasma  at a l l .  involved  concentration.  -  trout  and  a s p l a s m a pH d e c l i n e d .  both  salinity.  also  tension  a c i d - b a s e r e g u l a t i o n o f the plasma.  exposure  in  T h e r e was a v e r y weak c o r r e l a t i o n  c o n g e r e e l showed t h a t  on  HCO3  pH.  ventilation  Experiments water  plasma  gradients  hypercapnia  exchange HCO3  -  at  processes by  the  forC l ~ in the must  three have  proposed  iv  C1~/HC03 trout Cl~ is CT  mechanism.  -  gill in  was  by  was  about  steady  state  maintained -  a  out  on  based  on  of a  oxygen  data  carrying  on  the  are  of  more  measured  expected  to Na  +  than  to  plasma  concentrations the  existing  i n t r o u t immediately  after acid  of  to  the  of  the  the  fishes  acid-base  burst  excess proton  to  the  change  functions  blood  i n plasma  to m a i n t a i n  the  w h i c h would o t h e r w i s e  This  data  include  also  e x t r a c e l l u l a r and  suggests that  exercise load  have  those which enhance  the  f r o m the  be  supports e x i s t i n g  e f f e c t s which c a t e c h o l a m i n e s  status of  This data  during  and  Root s h i f t .  some o f  the  to the  is proportional  capacity  that  catecholamines  released  values  compartments.  partially,  the  than  +  latter calculation  the  according  control  physiology  regulation cell  due  showing  and  the  Furthermore,  This  between  ions  to Na  equilibrium  - 2.0.  comparison  release  to  compromised  1.5  that  gradents  This  relative  showed  conditions.  electrochemical  these  Catecholamines  pH  also  t i m e s more p e r m e a b l e  distribution  electrochemical  infusion.  analyses  control  o f about  the  concentrations  2.5  of  factor  based  These  is  the due,  release at  lactacidosis.  the red of  least  V  TABLE OF CONTENTS  Page #  Abstract  1  List  o f Tables  List  of Figures  1  vi v i i  Acknowledgements  x i i  General  Introduction  1  General  M a t e r i a l s and Methods  6  Section  1  18  Ventilation Section  and A c i d - B a s e R e g u l a t i o n  2  60  Transepithelial Regulation Section  Ion Fluxes  i n Fishes 132  A n a l y s i s o f the  Hypercapnia  Trout-Salinity-  Experiment  4  Catecholamine Release General  f o r Acid-Base  3  Further  Section  i n Fishes  Discussion  163 i n Acid-Infused  Trout 199  References  210  Appendix  222  vi LIST  OF  TABLES  T a b l e 1. G i l l v e n t i l a t i o n volumes o f f i s h e x p e r i e n c i n g v a r i o u s head p r e s s u r e s i n a Van Dam a p p a r a t u s  30  T a b l e 2. Time c o u r s e f o r pH c o m p e n s a t i o n i n f i s h s t r e s s e d with environmental hypercapnia  63  T a b l e 3. Mean water N a and C l ~ c o n c e n t r a t i o n s f o r t h e two e x p r i m e n t s in this section  65  +  T a b l e 4. A r t e r i a l p l a s m a pH, r e d c e l l pH, mls02 bound p e r gram o f h e m o g l o b i n , a d r e n a l i n e c o n c e n t r a t i o n and n o r a d r e n a l i n e c o n c e n t r a t i o n b e f o r e and f o l l o w i n g i n t r a - a r t e r i a l i n f u s i o n o f 120mM s a l i n e s o l u t i o n i n t r o u t  175  T a b l e 5. trout  Plasma i o n c o n c e n t r a t i o n s i n rainbow i n f u s e d w i t h HC1 and s a l i n e  186  T a b l e 6. trout  Water i o n c o n c e n t r a t i o n s f o r r a i n b o w i n f u s e d w i t h HC1 and s a l i n e  187  T a b l e 7. R a t i o s o f measured t o e x p e c t e d p l a s m a c o n c e n t r a t i o n s o f Na , C l ~ , K , HCO3 , NH4+, C a and M g for trout infused w i t h HC1  188  Table A . l . I o n i c c o n c e n t r a t i o n s f o r the sea s a l t s u s e d t o make up t h e v a r i o u s s a l i n i t i e s i n e x p e r i m e n t 2A i n S e c t i o n 2  223  +  + +  +  -  + +  vii  L I S T OF  FIGURES  Figure GI. A p p a r a t u s f o r measurement o f t r a n s e p i t h e l i a l p o t e n t i a l s ( TEP)  11  F i g u r e 1. Diagram o f e x p e r i m e n t a l a p p a r a t u s u s e d t o c o n d u c t r e s p i r a t o r y and a c i d - b a s e r e g u l a t o r y e x p e r i m e n t s i n t h e shark,  27  F i g u r e 2. The r e l a t i o n s h i p between b r e a t h i n g frequency, s t r o k e volume and t h e volume o f water f l o w i n g over the g i l l s o f t r o u t w i t h a Van Dam a p p a r a t u s  32  Figure  3.  The  relationship  between  arterial  Pco2 and v e n t i l a t i o n volume o v e r t h e r a n g e o f v e n t i l a t i o n v o l u m e s imposed on  Figure  4.  The  trout  between a r t e r i a l range v e n t i l a t i o n v o l u m e s imposed on t r o u t  relationship  Pco2 and a r t e r i a l P02 o v e r t h e of  34  37  F i g u r e 5. The r e l a t i o n s h i p between t h e c o n v e c t i o n r e q u i r e m e n t f o r CO2 and a r t e r i a l PC02 o v e r t h e r a n g e o f v e n t i l a t i o n v o l u m e s imposed  39  F i g u r e 6. The e x c r e t i o n and volumes  41  r e l a t i o n s h i p between CO2 O2 u p t a k e a t d i f f e r e n t v e n t i l a t i o n  F i g u r e 7. The r e l a t i o n s h i p between v e n t i l a t o r y volume and p l a s m a pH i n t h e s h a r k u n d e r g o i n g simultaneous exposure to environmental h y p e r o x i a and h y p e r c a p n i a  43  F i g u r e 8. As i n 7. s h o w i n g o n l y t h e d a t a f o r t h e experiment which i n v o l v e d the exposure to e n v i r o n m e n t a l h y p e r o x i a and h y p e r c a p n i a  45  F i g u r e 9. As i n 7. s h o w i n g o n l y t h e d a t a f o r t h e e x p e r i m e n t w h i c h i n v o l v e d the e x p o s u r e t o e n v i r o n m e n t a l h y p e r o x i a and h y p e r c a p n i a and where c h a n g e s i n pH were m i n i m i z e d  47  F i g u r e 10. The b e s t f i t l i n e f o r t h e a g g r e g a t e data s e t f o r both data sets d e s c r i b e d i n 7. above  49  F i g u r e 11. The r e l a t i o n s h i p between v e n t i l a t o r y volume and a r t e r i a l PC02 i n s h a r k s u b j e c t e d to three e x p e r i m e n t a l ^ p r o t o c o l s d e s c r i b e d i n Section 1  51  viii F i g u r e 12. As i n 11. e x c e p t t h a t t h e r e l a t i o n s h i p b e t w e e n v e n t i l a t o r y volume and a r t e r i a l [HCC>3 J i n mM f o r t h e t h r e e e x p e r i m e n t a l p r o l o c o l s  53  F i g u r e 13. Experimental apparatus f o r e x p e r i m e n t 2A.: T r o u t - S a l i n i t y - H y p e r c a p n i a  68  F i g u r e 14. 2 C : Carp  76  _  Experimental apparatus f o r - Isotope - Hypercapnia  experiment  F i g u r e 15. P l a s m a Pco2 i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300 mM N a C l a n d exposed to environmental h y p e r c a p n i a  80  F i g u r e 16. P l a s m a Pco2 i n c o n g e r d u r i n g c o n t r o l , s a l i n i t y change a n d e x p o s u r e t o 1 % environmental hypercapnia  82  F i g u r e 17. P l a s m a pH i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300 mM NaCl a n d exposed t o environmental h y p e r c a p n i a  84  F i g u r e 18. As F i g . 17. e x c e p t t h a t a l l v a l u e s were r e f e r e n c e d t o the average value d u r i n g the control period  86  F i g u r e 19. P l a s m a pH c h a n g e s from a v e r a g e c o n t r o l v a l u e s i n c o n g e r d u r i n g s a l i n i t y change and exposure t o 1 % hypercapnia  88  F i g u r e 20. E f f e c t o f water s a l i n i t y , [ C l ~ ] , o n pH r e c o v e r y i n c o n g e r  90  F i g u r e 21. rainbow mM NaCl  r e p r e s e n t e d by  P l a s m a HCO3 c o n c e n t r a t i o n s i n t r o u t a c c l i m a t e d t o 3, 100 and 300 and e x p o s e d t o e n v i r o n m e n t a l h y p e r c a p n i a -  F i g u r e 22. P l a s m a HCC>3~ c h a n g e s from a v e r a g e c o n t r o l v a l u e s i n c o n g e r d u r i n g s a l i n i t y change and e x p o s u r e t o 1 % h y p e r c a p n i a F i g u r e 23. E f f e c t o f water s a l i n i t y , r e p r e s e n t e d by [ C l ~ ] , o n HCO3 a c c u m u l a t i o n i n c o n g e r -  F i g u r e 24. Net H f l u x i n rainbow t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM NaCl and e x p o s e d to 1 % e n v i r o n m e n t a l h y p e r c a p n i a  92  ...94 96  +  100  ix F i g u r e 25. Plasma C l ~ c o n c e n t r a t i o n s i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM NaCl and exposed t o 1 % e n v i r o n m e n t a l hypercapnia  102  F i g u r e 26. Changes i n p l a s m a C l c o n c e n t r a t i o n s from average c o n t r o l v a l u e s i n c o n g e r d u r i n g s a l i n i t y change and e x p o s u r e t o 1 % environmental hypercapnia  104  F i g u r e 27. Plasma N a c o n c e n t r a t i o n s i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM N a C l and e x p o s e d t o 1 % e n v i r o n m e n t a l h y p e r c a p n i a .  106  F i g u r e 28. Plasma O s m o l a r i t y i n rainbow t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM N a C l a n d e x p o s e d t o 1% e n v i r o n m e n t a l h y p e r c a p n i a f o r 24h and r e c o v e r e d f o r 24h  108  F i g u r e 29.a.b.c. R e l a t i o n s h i p o f plasma ( t N a ] - ( C l - ] ) t o plasma [ H C 0 ] d u r i n g e x p o s u r e and r e c o v e r y from 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d t o 3, 100 and 300mM N a C l , r e s p e c t i v e l y  110  -  +  +  -  3  F i g u r e 30.a.b.c. R e l a t i o n s h i p o f changes i n plasma C l ~ c o n c e n t r a t i o n s t o c o r r e s p o n d i n g changes i n plasma H C O 3 concentrations d u r i n g e x p o s u r e and r e c o v e r y f r o m 1 % h y p e r c a p n i a i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 and 300 mM N a C l , respectively 112 -  F i g u r e 31.a.b.c. R e l a t i o n s h i p o f changes i n plasma N a c o n c e n t r a t i o n s t o c o r r e s p o n d i n g changes i n plasma H C O 3 c o n c e n t r a t i o n s d u r i n g exposure and r e c o v e r y from 1 % h y p e r c a p n i a i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM N a C l , respectively  114  F i g u r e 32. R e l a t i o n s h i p o f changes i n plasma C l ~ c o n c e n t r a t i o n s t o c o r r e s p o n d i n g changes i n plasma H C O 3 concentrations i n conger exposed t o s a l i n i t y changes and exposure to 1 % environmental hypercapnia  116  F i g u r e 33.a.b.c. T r a n s e p i t h e l i a l p o t e n t i a l (TEP) v a l u e s d u r i n g c o n t r o l , exposure and r e c o v e r y f r o m 1% e n v i r o n m e n t a l hypercapnia i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 a n d 300mM N a C l , r e s p e c t i v e l y  118  +  -  -  X  F i g u r e 34. P l a s m a h e m a t o c r i t v a l u e s ( H c t i n %) o f t r o u t a c c l i m a t e d t o 3, 100 and 300 mM N a C l and e x p o s e d t o 1% e n v i r o n m e n t a l h y p e r c a p n i a for 24h and t h e n r e c o v e r e d f o r 24h  121  F i g u r e 35. Net f l u x e s o f N a and C l ~ i n c a r p d u r i n g e x p o s u r e t o 5% e n v i r o n m e n t a l hypercapnia f o r 48h f o l l o w e d by 24h r e c o v e r y  123  F i g u r e 36. of Na  125  +  +  and  As i n F i g . 35, e x c e p t C I " a r e shown  the  efflux  F i g u r e 37.a. R e l a t i o n s h i p o f p l a s m a H C O 3 to plasma ( N a - Cl~) for three s a l i n i t i e s for conger. F i g u r e 37.b. R e l a t i o n s h i p o f d e l t a H C O 3 to d e l t a C l ~ f o r plasma d a t a from h y p e r c a p n i a and r e c o v e r y p e r i o d s . F i g u r e 37.c. R e l a t i o n s h i p o f d e l t a H C O 3 to d e l t a C l ~ f o r plasma d a t a from hypercapnia and r e c o v e r y p e r i o d s  137  F i g u r e 38. H y p o t h e t i c a l a c t i v e and p a s s i v e C l ~ movements a c r o s s t h e t r o u t g i l l a t t h r e e s a l i n i t i e s and t h e a s s o c i a t e d TEP and p l a s m a HCC>3~ a c c u m u l a t i o n c h a r a c t e r i s t i c s  139  F i g u r e 39.a. and b. P e r m e a b i l i t e s o f N a and C l ~ r e l a t i v e t o HCC>3~ f o r t h e g i l l o f t r o u t d u r i n g and a f t e r e x p o s u r e t o environmental hypercapnia  143  F i g u r e 40.a. Permeability of Na relative to C I f o r t h e t r o u t g i l l d u r i n g and a f t e r exposure to environmental hypercapnia. F i g u r e 40.b. D a t a o f 40.a. r e f e r e n c e d t o t h e average c o n t r o l values. E x p t l . ( P N a / P C l ~ ) - C o n t r o l (PNa / P C l ~ )  146  F i g u r e 41.a.b.c. R a t i o s o f measured to expected plasma Na concentrations for t r o u t d u r i n g and a f t e r e x p o s u r e t o e n v i r o n m e n t a l h y p e r c a n i a a t t h r e e water s a l i n i t i e s  150  F i g u r e 42.a.b.c. As except that data  41.a.b.c. r e s p e c t i v e l y i s f o r plasma C l ~  152  F i g u r e 43.a.b.c. As except that data  41.a.b.c. r e s p e c t i v e l y i s f o r plasma H C O 3  154  -  +  -  -  +  +  -  +  +  +  -  xi F i g u r e 44. a. A r t e r i a l plasma pH, b. red c e l l pH, c. mis 0 bound per gram o f haemoglobin and d. plasma catecholamine concentrations following intra-arterial i n f u s i o n o f HC1 s o l u t i o n o f 120mM s a l i n e in rainbow trout 2  F i g u r e 45. Rel i n p l a s m a pH in plasma adr pre-infusion post-infusion contributing  a t i o n s h i p between the changes and the c o r r e s p o n d i n g changes enaline concentrations between c o n t r o l samples and the + 5min s a m p l e s f o r e a c h o f 14 animals t o t h e m e a n d a t a i n F i g . 44  F i g u r e 46. R e l a t i o n s h i p between per gram of haemoglobin and red pH i n r a i n b o w t r o u t b l o o d e q u i l a g a i n s t gas m i x t u r e s h a v i n g an p r e s s u r e o f 1 5 2 mmHg a n d a CO2 o f 2 . 5 mmHg  0  capa blood ibrated oxygen partial 2  173  177  city cell in vitro partial pressure 179  F i g u r e 47. a . P l a s m a pH a n d b. plasma HCC>3~ c o n c e n t r a t i o n s i n r a i n b o w trout i n f u s e d w i t h HC1 a n d s a l i n e  182  F i g u r e 48. a. trout infused  184  H e m a t o c r i t (%) of rainbow w i t h HC1 a n d s a l i n e  F i g u r e 49. Transepithelial Potentials (TEP) i n r a i n b o w t r o u t i n f u s e d w i t h HC1 solution made u p i n 120mM p h y s i o l o g i c a l s a l i n e a n d t h e saline alone  190  F i g u r e 50. a n d b. HC1  192  Figure to  51. C a  a. Nernst r a t i o for plasma Na plasma NH4 in trout infused with +  +  As +  +  in  50.  except  that  i t  pertains 194  ACKNOWLEDGEMENTS  I  gratefully  supervisor.  Dr.  preparation  of  the  patient  Heisler of  acknowledge David  both  acid-base  regulation.  I  included  i n this  larger  spotted  salinity  Drs.  Heisler, :  N.  pleased  with a c i d  :  i n rainbow  Experiment  i o n fluxes  :  various  IA, The r o l e  trout.  J.B.  Claiborne  and  N.  o f v e n t i l a t i o n i n the  2B, The e f f e c t o f w a t e r  i n the conger  eel.  N. A n d e r s e n and J . B . C l a i b o r n e  Boutilier  in  Experiment  I B , The c o n t r o l  on a c i d - b a s e r e g u l a t i o n  infusion.  t o acknowledge the  collaboration  Boutilier,  dogfish.;  Unidirectional  R.G.  R.G.  Experiment  Heisler,  t o acknowledge  thesis.  v e n t i l a t i o n i n CO2 e x c r e t i o n  Andersen  Dr.  their  am  R.G. B o u t i l i e r and T.A. Heming  N.  like  and the  t e c h n i q u e s and t h e f u n d a m e n t a l s  experiments  2C,  I would a l s o  studies  experimental  for  Drs.  my  R o b e r t G. B o u t i l i e r and N o r b e r t  people  of  in  g u i d a n c e o f my  o f Drs.  following  Drs.  stimulating  Randall  this thesis.  teaching  in  J.  the  i n carp during  :  Experiment  hypercapnia.  Catecholamine release  i n rainbow  trout  xiii  I  was  Natural  supported  Sciences  Postgraduate  4)  3)  am  Dennis  Mense  Glage,  G.  Institut  I  am  family, the  the  Forcht,  H.  my w i f e  understanding  for  (NSERC)  Mclean  Fraser  Research  Teaching  Fellowship,  A s s i s t a n t s h i p s ; 5)  t h e t e c h n i c a l a s s i s t a n c e o f Mr.  University Slama  fur experimentelle  especially  Council  U.B.C.  Foundation  1)  t o D.J. R a n d a l l .  grateful at  Research  2)  Dept. Z o o l o g y  grant  also  from t h e f o l l o w i n g s o u r c e s :  Engineering  Max-Planck  U.B.C,  NSERC o p e r a t i n g  I  and  funds  Scholarship;  Scholarships; F.R.G.;  by  British  Columbia  and S.  and W. Neusse a t t h e Max-PLanck  Medizin,  grateful  of  Goettingen,  F.R.G.  f o r t h e l o v e and s u p p o r t  M a r i l y n J o y , and my s o n s Adam and D a n i e l . during  e v e n i n g s and weekends t h a t  o f my For  I had t o  work  and f o r k e e p i n g my e y e s o p e n t o t h e c o r r e c t p r i o r i t i e s i n  life,  I am d e e p l y  thankful.  1  GENERAL  INTRODUCTION  2  INTRODUCTION  Fish the  have  at  acid-base  status  environment. tensions  the  the  processes renal  One  and  between  t h r e e major s t r a t e g i e s  of is  their  the  other  fish  across  and  body  adjustment  two  gill  t h a t i n v o l v e the  of  involve  water.  the  fluids  the  These  for regulating  the  blood  transfer  are  PC02  of ions  t h e i o n exchange  e p i t h e l i u m and t h o s e  i n v o l v i n g the  system.  Studies  on  regulation roles  in  of  kidney  has  concluded  kidney  is  regulation Data  trends  generally  Randall  minor  across  emerging, acid-base  and Cameron  the  the  across  been  seen  in  others  a  i n acid-base  gill  those  on t h e  the g i l l . as  the  The major  i t has  negligible  suggests  relative  role in  of  fishes  however, balance  (1973),  regulation  system  been  role.  In  t h a t the r o l e  o f the  to the p o t e n t i a l  of ion  epithelium f o r acid-base  1985).  the  disturbances  regulating  plays  evidence  (see H e i s l e r  are  renal  acidoses,  kidney  available  regarding  acid-base  instances  in  the  the  and i o n t r a n s f e r s  some  processes  of  a r e few by c o m p a r i s o n w i t h  process  the  exchange  role  fishes  in  that  summary,  the  ventilation  compensatory  why  least  of  ventilation are  also  in  correcting  variable.  Some  t h a t p o i n t t o a minor r o l e i n in  fishes.  As  suggested  by  there are several a p r i o r i  reasons  blood  through  acid-base  balance  3  adjustments  in  1)  fine  a  very  blood  control  regulate  pH  due  tensions  in  fish  orientation  PC02  to  of  would of  be i n a p p r o p r i a t e  blood  Pco2  the l o g / l i n e a r  blood l i m i t  would  ventilation  to  adjustments  uptake,  a  where  O2  content  is  that  the  stimulation  of  fishes  functions  to o f f s e t  hypercapnia Bohr  and  in Root  shifts  oxygen  carrying  (Smith  and  Jones  elasmobranchs, in  response  shifts  in  very  little  conditions Root  of  the  show a s i m i l a r  acid  conditions in  the t e l e o s t  the  air.  during  the e f f e c t s  in  acid  to this  is  o f the  lower the conditions  trend  a r e (1)  stimulation of ventilation but  blood  of  There  b l o o d , which  may  i n water  ventilation  show  no Bohr  (Lenfant  family Cyprinidae  stimulation  Pco2  condition  in  blood  Exceptions  of  and Root  and J o h a n s e n  i n which  ventilation  in  ( D e j o u r s 1973) b u t t h e b l o o d e x h i b i t s  there i s  hypercapnic  b o t h Bohr and  shifts. The  experiments  regarding  this  regulation  in  of  than  found i n t e l e o s t  characteristics and  lower  1982).  which to  1966)  disadvantageous  capacity  2) low P c o 2  t h e s c o p e o f a d j u s t m e n t ; and 3)  O2  evidence  be needed t o  relationship;  compromise the  i n fishes:  gill  regard  would  status  of  regulating  broad fishes.  water  in  Section  area  of  The f i r s t  1.  address  ventilation  the gill  blood water  the  scope  through flow.  and  subjects acid-base  e x p e r i m e n t examined  f l o w i n CO2 e x c r e t i o n . define  two  of  Any l i m i t a t i o n adjusting  changes  in  PC02  the r o l e i n this  the acid-base t e n s i o n s by  The s e c o n d e x p e r i m e n t a d d r e s s e d  4  the  question  influencing acid  the  which  of  demonstrated Heisler HCO3  most  1985).  the  et  Cl~  1973),  might  be  i n the d o g f i s h ,  Na  longer  waters  further  1970,  study  and  of  The  i s less  greater  first  two  regulatory  performance.  to  higher  salinities  to  less  saline  species  acid-base Section Na  +  at  was and  hypercapnia.  ionic  in  the were  carried Cl~  in  T h i s was  mechanisms  of a link  by  have b e e n  1978,  De  R e n z i s and  Maetz  followed.  the sea  also  carried  out  which  net  water,  groups.  teleost  salinities  was was  acclimated transferred  were i n d u c e d i n and  last  exposed  changes  in  experiment  in  to  fluxes  environmental  i n an a t t e m p t changes  to  and a c i d - b a s e  out to determine u n i d i r e c t i o n a l carp,  than  2 were c o n d u c t e d  salinity  The  in fish  waters  between i o n i c  Acidotic conditions various  1964, 1973)  A freshwater t e l e o s t and a s e a water  of  Maetz  i n fresh  i n Section  by  correlated  Garcia-Romeu  s u c h as  water  been  and  +  pH c o m p e n s a t i o n  in fish  the  has  NH4  +  1960,  that  in  (see review  H ,  Payan  strength  between  waters.  status 2  complete  experiments  relationship  subject  and  1977,  fact  the e x i s t e n c e  the  both  Evans  fishes  of  (Maetz  (Maetz and G a r c i a - R o m e u  implies  The  of t h i s  epithelium  +  processes  in  movement  of  al_.  exchange  balance  gill  respectively.  takes  ion  studies  the  movements  to  of  The  across  Kerstetter and  role  acid-base  in  ions  -  to  the  parameters  stimulation of v e n t i l a t i o n  significant  regulation  of  acid-base  conditions. A  in  of  in  to understand plasma  ion  5  concentrations were t a k i n g An  observed  acidotic  condition,  elicits  1986,  B o u t i l i e r et. al..  a  release  catecholamines  the  effects  epithelia  and  been  of  in  g_t, ai.. 1975, G i r a r d a  exchange  large  balance  descriptive. experiment address  and the  water. gill  of  1986).  modulation  known the f i s h  across a  Section  (Primmett e t a l .  While  this  increase  e f f e c t s , some o f  of ion transport  and  in  (Perry  across  of  2.  and  gill  one, the  stimulation tissues  et. al..  of have  1984, P a y a n  1978).  suggests that  the f i s h major  the  mammalian  gill  body o f d a t a  is  states i n  Some o f t h o s e e f f e c t s , s u c h a s t h e exchange  Analyses  the  acid-base  conditions  stressful  catecholamines  and P a y a n 1977, P a y a n  processes  acid-base  the  C1"7HCC>3~  demonstrated  other  potent cardiovascular  exchange  +  While  of  membranes.  Na /H (NH4 ) +  like  1986, P e r r y  has  include  inhibition +  under s i m i l a r a c i d  place.  fish,  in  i n fish  the r o l e o f i o n  i n the r e g u l a t i o n o f  most o f t h e e v i d e n c e i s  trout-salinity-hypercapnia  the experiments  i n Section  4.  subject  of  the p o s s i b l e  controlling factors for  regulation  in  ion transfer  p r o c e s s e s between  The f u n c t i o n a l  as w e l l  blood  s i g n i f i c a n c e of catecholamines at  as the r e d c e l l  membrane a r e a l s o  examined.  6  GENERAL  MATERIALS  AND  METHODS  7  GENERAL MATERIALS AND  I.  CANNULATIONS  All  experimental  indwelling  cannulae  lasting about  about 25  aorta  an  unconscious  to  about  of  a  fish  1:10,000  and  on  the  pH  water  then  7.5  with  table  with  operating  had  cannulas  blind  fish  been  for were  puncture Bell  (1964)  with  that  used  The  of  i n the  the  the  were  (MS222) the  at  animals  concentration  of  table consisted  fish The  temperature o f  syringes,  the  cannulae fish.  at  prior  i n the  the  the same  to which  catheters  shark, and  The  the  continuously  e x p e r i m e n t s and  on  technique  were  maintained  All  i n anesthetized or  and  dorsal  fish  render  heparinized saline  with  abdominal  operating  was  experiments  fitted  cannulations  B o t h s o l u t i o n s were b u f f e r e d  gills  acclimated.  the  operations  All  a  chronic  a d j u s t a b l e n e t t i n g which h e l d  anesthetic  were r i n s e d w i t h  Except all  as  short  the  initially  NaHC03.  The  with  sulphonate  anesthetic solution.  table  temperature fish  an  to  an  of  methane  table.  with  in  artery.  maintained  operating  v e n t r a l s i d e up.  irrigated  cannulations  Tricane  of  fitted  for dorsal aortic  intestinal  with  concentrations  1:20,000  for  were  anaesthesia  minutes  minutes  through  animals under  10-15  anesthetized  and  METHODS  to  the and  use.  conger e e l ,  dorsal aorta  technique  of  by  Smith  o f S o i v i o e£..al.. (1972)  was  8  used. on  The  the  size  size  o f the f i s h  Methods o f e a c h In  the  Canine  After was  first  and  and  into  through  the  nares.  polyethylene aorta.  the  The  roof  of  of security  dorsal  aorta  length  of  cannula.  method,  enter  the cannula  indwelling  using  cannula  first  centimeters  method a  catheter  was  implanted  in  a p i e c e o f PE200  via  a hole  then the  cannula passed  i n f r o n t o f the  t h r e a d were made a r o u n d  involved sharpened just  the  blind  steel  puncture  wire  f u n c t i o n e d to guide  advanced on  aorta.  down the  inserted  the t i p o f the wire  W i t h t h e same o r i e n t a t i o n  depending cannula  plastic  down t h e  the  t o add  to the cannula.  o f the d o r s a l  was  fed  cannula j u s t o u t s i d e the f i s h  so t h a t  the wire  wall  was  i t i n t h e mouth and w h i c h  mouth  aorta.  i n the c a t h e t e r  cannula  f e d through  the  the d o r s a l  needle  chronically  with cotton or s i l k  second  the  The  cannula  with the indwelling level  t o make t h e b l i n d  a r c h e s and i n t o  one end t o a n c h o r  Ties  another  the  the  gill  The c a n n u l a was on  PE200  18 gauge) was u s e d  was made, t h e m e t a l  a  leaving  flanged  from  second  removed  vessel.  i n t h e M a t e r i a l s and  t h e m i d l i n e o f t h e r o o f o f t h e mouth, b e t w e e n  puncture  removed  and i s s p e c i f i e d  inch,  the  catheter  PE50 o r PE60) d e p e n d e d  technique, a catheter (Sovereign Indwelling 2  through  first  (either  section.  Catheter  puncture the  o f the c a n n u l a  the size  o f the into a  protruded  f o r e n t r y as the  t h e c a n n u l a and t o  The w i r e was removed and aorta of  between the  was g u i d e d o u t o f t h e f i s h  5 and 7  fish.  i n an  This  identical  9  manner  as  short it  above.  due t o t h e i n s e r t e d  to a longer In  eel  However, s i n c e t h i s  cannula  wire,  the  dorsal  intestinal  aorta  artery.  was  The body c a v i t y incision  into  the  or i n t e s t i n a l  dorsal  vessel, i.d.,  aorta.  was  cut  1.8mm  wall  was  This  closed  through  the  larger  incision.  ample  with  Cortland saline  II.  securely  tied  to the  t o a t h i c k - w a l l e d PVC t u b i n g which b l o o d  by a n o t h e r  was s a m p l e d .  small  details  a l l animals  flow o f a e r a t e d  24h p r i o r  was  through to  (1mm  The body was  i n c i s i o n caudal to of this  surgery are  (1983).  surgery,  with  least  a g a s t r i c or  a r t e r y and a d v a n c e d  Further  g i v e n by Toews e_t.al..  heparin.  through  by two l a y e r s o f s u t u r e s a n d t h e c a n n u l a  o u t o f t h e body c a v i t y  sodium  and t h e c o n g e r  was o p e n e d by a 3-4 cm  cannula  led  Following  to attach  a n d a PE50 c a t h e t e r was i n t r o d u c e d  and f i t t e d  o.d.)  was u s e d  sampling.  cannulated  mid-ventral  the  was n e c e s s a r i l y  i n v o l v e d the shark  long  gastric  a connector  to f a c i l i t a t e  t h e e x p e r i m e n t s which  cannula  (Wolf  water.  i n perspex  boxes  C a n n u l a e were f l u s h e d  daily  1963) c o n t a i n i n g 10,000 USP u n i t s / L  A l l animals to s t a r t i n g  recovered  were a l l o w e d  experimental  to recover  for at  procedures.  MEASUREMENT OF TRANSEPITHELIAL POTENTIALS (TEP)  TEP calomel  was measured a c r o s s or  silver-silver  the g i l l  epithelium using pairs of  chloride electrodes.  The r e f e r e n c e  10  Figure G l . Apparatus f o r measurement of transepithelial potentials(TEP). Stipled ' b r i d g e ' c a n n u l a e c o n t a i n e d 3M KC1 s e t i n agar. One was i n c o n t a c t w i t h t h e c a n n u l a e i n the dorsal aorta ( c l e a r ) t h r o u g h a 'T' p i e c e . The o t h e r reference was i n contact w i t h t h e water n e a r t h e g i l l s . The insert shows a close-up of the o r i e n t a t i o n o f the reference bridge with part of the o p e r c u l a r cover c u t away. The r e f e r e n c e b r i d g e was t h r e a d e d t h r o u g h a n o t h e r cannula of l a r g e r d i a m e t e r w h i c h was f l a r e d o n one end t o anchor i t i n s i d e the o p e r c u l a r c a v i t y .  APPARATUS FOR T E P  MEASUREMENTS  12  and by  m e a s u r i n g e l e c t r o d e s were c o n n e c t e d PE50  below  cannulae  describes  placed  near  diameter  filled the  the  cannula  operculum.  The  with  3M KCL s e t i n a g a r .  apparatus.  gills  by  which  was  The r e f e r e n c e  threading sewn  measuring  the s a l i n e  which  There  was  detectable  l e a k o f KC1  piece  allowed  measuring  or  electrodes  was  the  the  the  were  and  with  measuring  was  electrode  together.  This  short  The z e r o  value  blood  to  The p o t e n t i a l  voltmeters  cannula  voltage  that  read  cannulae  offset  voltage  i n s i d e the with  were  cannula. The "T"  e i t h e r the f o r blood across  experiments.  f o r the  when  the  the  which a r e s p e c i f i e d  by  containing  used  was  larger  the blood.  by s h o r t i n g t h e c i r c u i t  a  a  i n contact  Methods o f i n d i v i u a l  zeroed  above. TEP  using  just  w h i c h was u s e d  procedures.  measured  electrodes  described of  infusion  Materials  voltmeters the  of  electrode  the i n d w e l l i n g into  e l e c t r o d e or to the s y r i n g e  collection  in  connection  filled  blood  The d i a g r a m  through  place  e l e c t r o d e was  through  the  i t  in  blood  no  t o t h e water and  both  placed  was s m a l l e r  The  connecting t h e 3M  KCL  determination reference in  the  t h a n 1 mV  and  water ina l l  cases.  III.  A.  MEASUREMENT OF ACID-BASE  Constants  (pKapp)  of  :  The  carbonic  PARAMETERS  apparent acid  first  dissociation  and t h e s o l u b i l i t y  of carbon  constant dioxide  13  (CO2) d e t e r m i n e d  B.  pH  :  animal. using  was  measured  Radiometer  calomel  electrode  lysates  were  the as  red  of  cell  fraction by Z e i d l e r  individual  o n whole b l o o d a n d r e d c e l l  by  centrifuging  which  S1500  and  lysates  electrode  a n d K497  The r e d c e l l  was t h e n t w i c e f r o z e n a n d thawed Minor  deviations  i n t h e M a t e r i a l s a n d Methods Radiometer  S1510 were u s e d  t o t h e S1510 b u f f e r  adding or s u b t r a c t i n g  o f the  whole b l o o d t o o b t a i n  a n d Kim ( 1 9 7 7 ) .  experiments.  were r e f e r e n c e d by  used.  c o u p l e d t o a PHM 84 pH meter.  procedure a r e noted  buffers  (1985) were  G279/G2 g l a s s c a p i l l a r y  obtained  described  this  et_. al_.  A l l measurements were made a t t h e t e m p e r a t u r e  pH a  by B o u t i l i e r  from  sections  precision  phosphate  i n calibrations.  Readings  and a d j u s t m e n t s  one h a l f o f t h e d r i f t  were made  i n t h e measured  buffer value.  C.  Total  samples  CO2  :  using  Materials  one  and  techniques  Total  was  made  Inc., Port  technique  where  (Cameron tubes  measured  two methods w h i c h  with  employed  Instruments  acidification  was  2  Methods o f e a c h e x p e r i m e n t .  were  procedure  of  C0  and  1971).  immediately  dried when  HC03~  is  measurement Plasma after  is  samples  Standards The  Capni-Con  Texas)  and b l o o d  are specified  NaHC03. the  Aransas,  i n water  f o r both following  III(Cameron  was u s e d .  converted  i n the  This  to  made o f t h e change  C0  2  isa by  i n PC02  were t a k e n from h e m a t o c r i t  determination.  When a C 0 2 ~ s p e c i f i c  14  gas  chromatograph  U.S.A.)  was  (Carle  used,  acidification  recorder  the  of  differentialthermal were  Model  III,  gaseous  the  CO2  sample  conductivity.  referenced  C a r l e Instruments  to  evolved  was  from  the  analysed  Peak h e i g h t s on  standards  Inc.  (Lenfant  by  the  output  and  Aucutt  1966).  D.  PC02  Pco  1  values  2  Henderson/Hasselbach electrodes Boutilier  E.  HCO3  and  meters  :  -  by  HCO3 the  HCO3- = T C 0  2  NH4 /NH3  :  +  determined from  by  F.R.G.).  V.  System.  values  2  measured to  the  total  L-glutamic Sigma  using  with  the  Radiometer  recommendations  plasma  total  experiments  of  of  samples  were  C0 ) 2  ammonia  concentrations  dehydrogenase/NAD (Sigma  (Boehringer  methods o r by  for  for  * (solubility  Plasma  Water  in  calculated  equation,  Boehringer-Mannheim  these  or  according  -  - Pco  either  by  equation  were  et.al.(1978,1985).  calculated  F.  either  enzymatic  Diagnostics Mannheim  were assays  U.S.A.)  Gmbh  ammonia c o n c e n t r a t i o n s were  or  Diagnostica determined  an ammonia e l e c t r o d e as d e s c r i b e d below that  involved  a  Delta  Bicarbonate  15  IV.  ION CONCENTRATIONS  Plasma  and  concentrations  water were  spectrophotometry byAnnio  Na ,  d e t e r m i n e d by one o f t h r e e  the  description  determined to  the  were  with  and  by  than  titration  chloride with  sensitive  electrode  microprocessor/ion standards  a  concentrations  were  (when  a  were  reference and  according standards.  concentrations concentrations  R a d i o m e t e r CMT10  consisting  and  analyser  and r e f e r e n c e d  some  chloride titrator  chloride  using  system  described  In  (1963) u s i n g a p p r o p r i a t e  concentrations  experiments  as  f o r water and plasma  chloride  Cotlove  lOOmM)  + +  absorption  experiments.  e x p e r i m e n t s plasma and water  determined  2380)  Ca  methods w h i c h a r e s p e c i f i e d i n  water  o f Cotlove  and  + +  atomic  Model  individual  a Buchler  greater  Water  of  plasma  method  In other  by  Chloride concentrations  were  experiments,  Mg  +  determined  (Perkin-Elmer  (1964).  K ,  +  was  to a p a r t i c u l a r  determined of  were  Titrator. in  some  a solid  state C l ~  connected  to  calibrated standard  with  a  NaCl  between each  measurement.  V.  DELTA BICARBONATE SYSTEM  A  c l o s e d water r e c i r c u l a t i o n  system such as t h a t  described  16  by  Heisler  ejt.al..  Salinity-Conger Variations  and  of  experiments  are the  typically  consisted  wasclosed  for  exchange. changes and  in  water The  This  to l e v e l s  of  Materials  to  and  Such  pump.  was  a  a v o i d e d by  bubble system  but open f o r gas s y s t e m was  build-up  to  system  This  to exchanges  the  Methods a  that  between  t h a t c a n be measured  of  those  an o x y g e n a t o r and  such  due  thermostatted  allowed and in  pumped  electrolyte  bridge  equilibration  with  All of  were (Knick, a  an  fish  accurately  of toxic  flushing  the  waste system  the  temperature  o f the  continuous  measurement  of net  automated  to  a  Water  pH  1%  CO2  a  at  At  back  least  high  B e r l i n F.R.G.) and  recorder.  measure  and  to  to the 3  impedance  intervals  +  was  a double after  the  fish  particular  weeks.  the r e s u l t i n g  programmed  NH4  chamber  electrode  acclimated  for to  electrode  pumped  were  connected  to  reference  and  experiment  way  from the f i s h  glass  (Ag/AgCl)  electrodes  the  electrodes  the  the water.  continuously  to  unique  substances  advantage  ammonia  experiments.  24h.  concentrations  output  box,  non-volatile  problem  as  the  were  circulation  concentrations  changes  amplifier  fish  water  Salinity-Trout,  experiments.  ion  system  salinity  in  main  water  chamber.  which  The  such  system every  -  a l l  accumulate  fresh  HCO3  a  potential  products with  and  a  for  +  respective of  used  Na -Cl~-Carp  system  described  of  system,  was  the  this  sections  trap  (1976)  The  isolation signal  was  water  was  17  pumped  from  another Inc.,  the  fish  reservoir  to  Lexington  concentration a  pH  of  physically  about  The  VI.  Mass.  U.S.A.).  adjusted 10.  so t h a t  The ammonia  signal  and t h e r e s u l t i n g  were r e f e r e n c e d  along with a strong  a n ammonia e l e c t r o d e  dissolved  electrode. filtered  were  chamber  base  (Ingold  The  converted was  from t h e e l e c t r o d e  and  base  m i x t u r e had  a l l NH4 sensed  by  +  to the  was a m p l i f i e d a n d  peak h e i g h t s on t h e o u t p u t  t o s t a n d a r d s made f r o m  from  Electrodes  flow  the r e s u l t i n g  which  base  recorder  NH4CI.  GASES  All  g a s m i x t u r e s were made up f r o m p u r e g a s e s o r a i r mixed  with Wostoff  (Bokum, F.R.G.) g a s m i x i n g pumps.  SECTION  VENTILATION AND  1.  ACID-BASE  REGULATION IN FISHES  19  INTRODUCTION  Ventilation  in  fishes  disturbances.  This  waters  (Hargis  1976;  1977),  for  Janssen  fish  Randall  1938;  Randall  and  1976;  Smith  and  H o g l u n d and P e r s s o n  Jones  range o f s p e c i e s Evidence  predominant  cause  with  1975) Jones  teleosts.  stimulated  b e e n shown f o r f i s h  infused  and  wide  has  is  by  exposed  to a c i d  1971; D i v e l y e t al..  a c i d o r base  (Cunningham 1974;  and f o r h y p e r c a p n i c  fish  1973; J a n s s e n and R a n d a l l  1982).  acid-base  This e f f e c t  i s also  ( V a n Dam  1975; Eddy found  in a  i n c l u d i n g e l a s m o b r a n c h s , c y c l o s t o m e s and in  in  the  literature  the  responses  of  ventilation  suggests  by t h e s e  that  species  the  may be  different. The  stimulation  conditions  suggests  that  the stimulus  in  e i t h e r oxygen uptake o r c a r b o n d i o x i d e  is  large  body  ventilation stimulus shifts  in  to  by  Jones  (1982)  trout  exposed  increasing  increased  reducing have  the  data  teleosts  would r e s u l t  blood  of  suggesting  during oxygen  acid  under  acid  i s r e l a t e d t o a need t o  alter a  fish  excretion.  that  There  the increase i n  conditions  i s due t o a  e x t r a c t i o n s i n c e Bohr and Root  i n a lower oxygen c a r r y i n g c a p a c i t y o f the the oxygen-hemoglobin a f f i n i t y . shown  that  to environmental oxygen  content  the  increased  hypercapnia of  S m i t h and  ventilation i n  was a t t e n t u a t e d  the water.  This  by  strongly  20  suggests  that  shifts,the  in  those  stimulation  increasing  oxygen  exceptions  to  this,  Bohr  or  shows  no  Johansen  1966),  hypercapnic 1976). show  little  showing  reasons  ventilation of  CO2  in  the  First, very  pH.  Second,  the  ventilation uptake;  of to  a  fine  Heisler  a l . . 1976;  in  spite  et. a l .  scope fish  the b l o o d  role  blood.  which  of  increased  i s the of  priori  excretion  blood  Pco2  r e l a t i o n s h i p o f Pco2 to of Pco2 tensions  o f adjustment  adjustments  condition  of  which  ( D e j o u r s 1983).  regulation  control  and  et  possible  log/linear  which  i n response to  shifts  consequent  two  (Lenfant  (1973) have s u g g e s t e d t h r e e a other  the  require  content  and Root  to  i s stimulated  ventilation  the  would  CO2  in  Root  are  the b l o o d o f  response to h y p e r c a p n i a , t h a t  and  tension.  There  i s members o f t h e f a m i l y C y p r i n i d a e  Cameron  against  water.  and  oriented  characteristics  (Randall  e v i d e n c e o f Bohr and  shift  ventilation  change  Randall  the  Bohr is  the elasmobranch,  Root  other  showing  ventilation  from  one  conditions  The  of  uptake  but  species  of  to  control  by  the  low  orientation  of  compromise  O2  i s limited  Third,  the  Pco2  pH  may  i s disadvantageous to l i f e  i n water  where t h e O2 c o n t e n t i s l o w e r t h a n a i r . The designed  two to  ventilation The  first  adjusting  experiments investigate and  its  role  experiment blood  CO2  described two  in  this  aspects of this  Section  broad s u b j e c t  i n acid-base regulation investigated  tensions  the  by a l t e r i n g  were  actual gill  of  i n fishes. scope  water  of  flow i n  21  trout. Pco2 2  Wood  adjustments  mmHg  in  C0  the  second  control lack  of  emphasizes  relationship  other  than  the  possibility  is  via flow  oriented  levels  changes over  exposure  to  hypercapnia the  in  in  i n order  of 0  regulation  the  blood  gills  of  environmental the  investigated  the  by  flow. the  2  control  elasmobranch  of  ventilation  or s e t of  of  pH,  Pco  adjustments Direct  parameters  dogfish  2  tensions  of  blood  measurement o f during  and  ventilation  METHODS  2  or Pco  2  water  levels  i n the b l o o d .  C0  A  simultaneous  varying  c o r r e l a t i o n of g i l l  EXPERIMENT IA. VENTILATION AND  The  s a t u r a t i o n o f the b l o o d .  hyperoxia  three acid-base parameters  the  spotted dogfish.  some p a r a m e t e r  the  water  Section  larger  that  MATERIALS AND  FISH  about  experimentally  i n the b l o o d o f t h i s  maintenance  enabled  f l o w was  measured d i r e c t l y  this  the  shifts  towards  in gill the  f l o w t o be  between t h e s e p a r a m e t e r s o v e r a  in  possibility  be  water  water  of  volumes.  and Root  might  in gill  Gill  experiment  the  e s t i m a t e d the c a p a c i t y  e x c r e t i o n was  ventilation  o f Bohr  HCC>3~  changes  2  range o f v e n t i l a t i o n The  (1980)  species.  and  describe  Jackson via  that  manipulated to  and  EXCRETION IN TROUUT  of  with  22  Rainbow obtained  trout,  from  fiberglass  a  a commercial  tanks  Vancouver They  S a l mo c r a i r d n e r i ,  tap  were m a i n t a i n e d  self  feeder.  surgical  cannulas  by  Materials to  the  the in  and s u b s e q u e n t  operation  which  pellet  was  described  attached  fish  to  to the d i v i d e r  that  insured  ventilation height This  in was  overflow which  and  chronic  done  rubber  carried  (1964)(see  Van Dam  masks  by Cameron and D a v i s was  the  the  with to  inserted  into  aortic General  according  (1970).  After  a n a r r o w b l a c k box  box.  The r u b b e r  mouth and s n o u t  mask,  o f the f i s h ,  was  b e t w e e n t h e two chambers and a c t e d as a  were  inspired  standpipe  ppm).  48 h p r i o r  from  t h e f r o n t chamber t o o f the  mixed e x p i r e d w a t e r s were t h u s  by  4  indwelling dorsal  o f S m i t h and B e l l  a l l water p a s s i n g  volumes the  CaCC>3  f e d ad 1 i b i t u m  t h e b a c k chamber was v i a t h e mouth and g i l l s Inspired  dechlorinated  experimentation.  p a r t o f a two chambered p e r s p e x  secured dam  with  method  each  6.9-7.1;  i n outdoor  EXPERIMENTAL PROCEDURES :  and M e t h o d s ) and w i t h method  one  pH  and  were s t a r v e d f o r a t l e a s t  fitted the  and m a i n t a i n e d  aerated  on a d r y t r o u t  SURGERY, APPARATUS AND  were  with  (5-10°C;  Fish  procedures  Fish  hatchery  supplied  water  b e t w e e n 192 and 353 g were  influenced water  adjusting relative overflow  by  fish.  s e p a r a t e d and  a d j u s t i n g t h e water  chamber a n t e r i o r  to the f i s h .  the h e i g h t o f the f r o n t to t h a t i n the p o s t e r i o r to  waste.  Positive,  chamber chamber, z e r o and  23  negative  pressure  were e f f e c t e d A  heads  from  t h e mouth t o o p e r c u l a r  chambers  i n t h i s way.  p e r i o d o f 24 h f o l l o w e d t h e s e t t i n g  before  measurements were t a k e n .  always  from  change  and  positive the  to  number  Changes  negative  of  o f any p r e s s u r e  head  i n head p r e s s u r e  were  although  changes  t h e magnitude o f  imposed p e r f i s h  were n o t  consistent.  MEASUREMENTS :  Ventilation outflow trunk  water of  and  from  the  volumes  measurements counting the  volume  were  Collections  were  determined  of  Stroke  a s Vg d i v i d e d  the  (Pio2  tensions  measured  with  as  °^  apparatus Plasma  t  )  volume  n  e  a  n  well  water bloocl  (Lexington CO2  Concurrent  ( f ) were  made  by  a small clear  section i n  Several counts  per minute  o f t h e b u c c a l pump  ( V s v ) was  by f .  Radiometer  experimental  (CaQ2^  movements t h r o u g h  1 min p e r i o d s  weight.  frequency  box c o n t a i n i n g t h e f i s h .  Inspired oxygen  were made o v e r by  ventilatory  by c o l l e c t i n g t h e  i n t h e chamber c o n t a i n i n g t h e  fish.  averaged.  calculated  was d e t e r m i n e d  the standpipe  opercular  black  (Vg)  mixed  d  as  expired  arterial  oxygen  2  oxygen  water  t e n s i o n s were  electrodes thermostatted to  temperature. was  (PeQ )  Total  measured  with  oxygen a  content  Lex-02-Cont  Instr.).  tension  (PC02)  was  measured  with  a  24  Radiometer  electrode  temperature  according  al.  (1978).  (Cico2)  the  mixed  samples  were  Hematocrit  (pHe)  by  the  the  and r e d c e l l  described  taken  determined  by  according  to  experimental  CO2  in  water  the  inspired  samples as w e l l i n  M a t e r i a l s and Methods. according  to  rates  principle.  intracellular  i n General  the  by g a s c h r o m a t o g r a p h y a c c o r d i n g t o  transfer  Fick  catecholamines,  samples  made  Gas  total  (Ceco2)  measured  (1960).  calculated  of  i n General  was  to  t h e r e c o m m e n d a t i o n s o f B o u t i l i e r e_t  expired  method d e s c r i b e d  Snieszko  to  Measurements  and  plasma  thermostatted  (M02  from  fish  at  high  pressure  Woodward  (pHi) f l u i d s  were  assayed  (Sigma b u l l e t i n  no. 826-UV).  of  and M c o ) were 2  were measured a s Concentrations  and n o r a d r e n a l i n e several liquid  (1982).  concentrations  method  pH o f t h e e x t r a c e l l u l a r  M a t e r i a l s and Methods. adrenaline  the  head  o f 11 p l a s m a  pressures  chromatography Whole  enzymatically  with  of  were (HPLC)  blood  lactate  Sigma  reagents  STATISTICS : Correlation the  and  relationships  Analysis  of  statistical  regression analyses between  variance  significance  parameters  (ANOVA) among  were u s e d  to describe  in  experiment.  this  was  used  to  means  with  a  compare 5  the  % level of  rejection.  EXPERIMENT IB. SHARK - HYPEROXIA - HYPERCAPNIA  25  ANIMALS :  Larger between  spotted  1550  Italy.  to  They  dogfish, 2820  were  laboratory  sea  temperature  were  held  without  experimentation. ambient  g  Scvliorhinus  in  feed  The  (pH  was m a i n t a i n e d  200  1  until  tanks  water  caught  weighing  i n t h e Bay o f N a p l e s , fiberglass  they  received 7.9;  stellaris,  tanks  were  a  used  continuous  [HCC>3~]  2.3  i nthe for  flow o f  mM) .  Water  a t 19°C.  SURGERY AND APPARATUS :  A  chronic  indwelling  aorta  through  an  Materials the to were  and  intestinal  Methods.  thumbs o f s u r g e o n s collect  the  connected  to  volume.  Bicarbonate  System'  Methods  recovery  and  period  artery  Latex rubber  the  water. ends  The  as d e s c r i b e d i n General bags were f a s h i o n e d  of  24  initiated.  PROTOCOL AND MEASUREMENTS  :  these  animals  detail  the g i l l  bags  from slits  probes  t o measure t h e  were r e c o v e r e d i n a ' D e l t a  described i n General in  h  the d o r s a l  E l e c t r o m a g n e t i c flow  of  ( F i g . 1)  in  into  g l o v e s and a t t a c h e d o v e r  expired  ventilation  and  c a n n u l a was i n s e r t e d  Heisler  (1978).  experimental  Materials  There  was a  procedures  were  26  F i g u r e 1. conduct in the details.  Diagram of experimental apparatus used to respiratory and acid-base regulatory experiments shark, Scvliorhinus stellar is. See text for  28  After tension After  3 was  a  various  levels  The  at  each  PC02  h  in  and  and  Blood  from  7  Blood  o f a c i d o s e s and HCO3  to  HCC>3~  were c a r r i e d identical 0.3  the cannula  with  protocol,  level.  The  t h e water  natural  by i n f u s i o n  mmHg.  in  were General  a t 30 min, 1 30 m i n d u r i n g  exposure  the f i r s t ,  additional  44  the cannula  described  s a m p l e s were t a k e n  the changes  levels.  -  concentrations  o f h y p e r o x i a and every  were  r a i s e d to  measurement o f b l o o d pH,  t h e methods  experiments  minimize  supplemented  to  procedures  through to  a range and  ranged  the onset  In  P02  pressure.  was a l s o  PC02  f o r the d i r e c t  Methods.  additional  experiment.  order  water  pH,  hyperoxia/ hypercapnia  sampling  infused  time  according  and 2 h a f t e r  Two  o f atmospheric  t o induce  blood  Tco2-  the combined  mmHg  t h e ambient  700 u l o f b l o o d was removed t h r o u g h t  and  Materials  fish  levels  sample  calculated  500  taken,  t h e ambient  f o r each  hypercapnia  were  o n l y t h e o x y g e n t e n s i o n was i n c r e a s e d ,  of  range  Approximately  to  where  tension  consequent  samples  elevated  period  PC02  the  control  M  out.  to that  The p r o t o c o l f o r t h e above  NaHCC>3 o r 0.6 M HC1 was  each  elevation  i n blood  pH.  i n Pco i n 2  I n the  second  PC02 was e l e v a t e d t o o n l y one  accumulation o f NaHCC>3 t h r o u g h  of  blood  HCO3  -  was  the cannula.  STATISTICS :  Correlation  analyses  were  used  to  describe  the  29  relationships  between  data  sets.  RESULTS  EXPERIMENT IA.  All  animals  respect  to  pressure  of  heads  overlap heads  of  8  The  +  animals  with  mean  plasma  pressure  levels in  for fish.  Van  Dam  heads  shown  by  pump r a t h e r volume  (Van Dam  various  Blood  boxes  there  1938;  lactate  were  also  and d i d n o t  was  significantly considerable  n e u t r a l and  others, than  a  pressure.  effected  volumes, a l t h o u g h  As  with  adrenaline  experiencing  t h e c h a n g e s i n head  buccal  state  +. 0.02(means +, 1S.E.) mM,  ventilation heads  steady  0.42(mean + 1S.E.) n a n o m o l e s / 1 ,  resting  0.58  1).  the  adjust  in  t h e r a n g e s o f Vg a t n e g a t i v e ,  (Table  pressure  2.6  ventilation in  volume  fish  frequency  positive  varied  stroke  of breathing  ( F i g . 2) i n t h e f a c e o f imposed Davis  and Cameron 1971;  Randall  J o n e s 1973). The  inverse the  low,  imposed  different  and  of  and  samples from 5 f i s h  with  significantly The  to  11  consistent  consistently  quiescent  exchange.  was  concentrations  vary  were  gas  concentration  value  : ROLE OF VENTILATION IN ACID-BASE REGULATION  relationship power  reverse  between  function  relationship.  Vg  ( F i g . 3).  and  arterial  Pco2  Vg and a r t e r i a l  was  an  P02 showed  30  Table  1.  pressures  Gill  ventilation  i n a Van Dam  volumes  RANGE OF HEAD  RANGE OF Vg  PRESSURES(mm)  (ml/min)  -8 t o 0 0 0  t o +21  (Vg) o f f i s h e x p e r i e n c i n g v a r i o u s head  apparatus. MEANS + STD.ERR.  26.8 t o 131.3  56.28 + 6 . 9 5  42.2 t o 139.9  75.13 + 12.27  48.4 t o 342.4  145.92 + 19.39  n.s.  not s i g n i f i c a n t l y d i f f e r e n t  sig.  significantly different  by ANOVA  by same t e s t  i  ; P<.05  ~1 "1 n. s. =j =j  7...  ssi'c ig.  J  31  F i g u r e 2. The relationship between breathing frequency (f), t h e volume o f water pumped each b r e a t h or s t r o k e volume ( V ) and t h e volume o f water f l o w i n g o v e r t h e gills (Vg) o f t r o u t w i t h a Van Dam a p p a r a t u s . Best f i t linear regression lines : s v  Vsv Vsv  = 0.0892 + 0.1456 * Vg; r = 288.145 - 0.7093 * f ; r  2 2  = 0.9211; N = 42 = 0.0025; N = 39  (ml/min)  33  F i g u r e 3. The relationship between arterial PC02 and ventilation volume (Vg) over the range of v e n t i l a t i o n volumes imposed on trout i n this study. Black dots represent f i s h w h i c h ram v e n t i l a t e d . B e s t f i t power c u r v e : Pco = 8.95*Vg~°•209. _ 0.484; N = 43. 2  r  34  35  Pco Po There Pco  was  a  2  negative  with  and  2  mean £  significant  with  Pco  respiratory  1S.E.).  yielded  gas  2 9 2  arterial  with  Pco  sufficient  results  2  and  r  HCO3  -  are  elevated  ventilation The changes  a  increase in  in  plasma  exponentially  as of  hypercapnia,  causes  and  Vg  Pco  2  and  ( F i g . 12) .  a  the  significant  : r = -0.333, N =  2  , V g / M c o , were 2  At  low  RQ,  was  volume.  0.87  40  associatd  l e v e l s o f Vg,  ventilation  both The  + 0.04(means  of these  IN  parameters  VENTILATION  of  environmental  acidosis.  Blood  consequence  pH  (Fig.  pH  is  7,  pH  8  best &  Pco  2  and  drops;  gill  correlated  with  9).  decreases  change  during  a considerable decrease Vg  but  elevation  ventilation pH  weak  well.  plasma  elimination  between  as  pH  an  plasma  and  i n c r e a s e s as  and  2  = 0.637 ( F i g . 6 ) .  2  oxygen,  in  31  analysis  EXPERIMENT IB. ROLE OF  Given  r = 0.469, N =  gill  regression  a s l o p e o f 0.012  43  Pco  exchange r a t i o ,  Linear  r = 0.484, N =  a  ( F i g . 5).  decreased  2  9  requirements  values  2  0  showed  2  convection  2  ( F i g . 4).  correlation  Mco  -  n e g a t i v e c o r r e l a t i o n between P o  Mco ,  lower  0  0  excretion,  Higher  -  = 27.66 * V g -  2  : r = -0.784, N = 31  2  C0  Mo  * vg  = 8.95  2  (Fig.  IDas  well  Vg (Fig.  this i n the as  increases 10).  exposure  The to  correlation  between  [HC03~3  36  F i g u r e 4. The relationship between arterial Pco2 and arterial P02 over the range of ventilation volumes imposed on trout i n this study. PC02 = 5.660 0.019*Po ; r = 0.6150; N = 31. 2  2  co (mmHg) r a  2  3  Pa  0 2  (mmHg)  38  F i g u r e 5. The relationship between the convection requirement f o r CO2 (Vg/Mco2) and arterial PC02 over the range of ventilation v o l u m e s imposed i n t h i s s t u d y . Vg/Mco = 3 . 1 6 5 * P c o ~ ° ' ; r = 0.333; N = 4 0 ) . 4 7 7  2  2  40  F i g u r e 6. The relationship between CO2 excretion (HC02) and O2 uptake (M02) a t d i f f e r e n t ventilation volumes. The g a s exchange r a t i o was 0.87 + 0.04. M02 = 0.298 + 1.012*Mco ; r = 0.637; N = 42. 2  2  5  0  1  M  2 0 2  3  4  (mM/h/kg)  5  42  F i g u r e 7. The relationship b e t w e e n v e n t i l a t o r y volume and plasma pH (pHpl) i n Scvliorhinus stellaris undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. Ventilatory volume (Vg a c t . / V g c o n t r . ) i s the ratio of t h e measured Vg d i v i d e d by t h e Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the b e s t f i t l i n e f o r a l l the experimental data f o r an i n d i v i d u a l f i s h . The d a t a set shown includes two experiments. One i s the combination o f environmental hyperoxia and hypercapnia exposure alone. The other has t h e added t r e a t m e n t o f NaHC03 o r HC1 infusion t o k e e p t h e pH ' c o n s t a n t , o r t o m i n i m i z e t h e change i n pH. 1  Coefficients Vg  line  a c t . / Vg c o n t r . = a * e a  5. 3996 0. 2600 5. 7770 2. 2580 4. 7401 2. 3955 1. 3504 2. 2187 1. 1901 2. 2668 2. 8936 2. 2743 3. 1797  f o r each  b * * * * * * * * * * * * *  1 15 IO 10 0  9  6  ioio  IO" 1021 IO" IO" 1013 IO" 1014  ion  -4.9322 -2.4640 -2.1344 -3.2478 -3.3712 -3.8865 -6.5133 -3.4635 -3.4600 -4.2010 -3.5686 -4.4154 -3.6072  (  b  : * P> H  r  2  0. 950 0. 841 0. 792 0.636 0.696 0.963 0. 934 0. 846 0. 830 0.978 0.833 0.956 0.860  43  44  F i g u r e 8. The relationship between v e n t i l a t o r y volume and plasma pH (pHpl) in Scvliorhinus stellaris undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. Ventilatory volume (Vg a c t . / V g c o n t r . ) i s the ratio of the measured Vg d i v i d e d by t h e Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the b e s t f i t l i n e f o r a l l the experimental d a t a f o r an i n d i v i d u a l f i s h . T h i s graph shows only the d a t a f o r the experiment which i n v o l v e d the e x p o s u r e t o e n v i r o n m e n t a l h y p e r o x i a and h y p e r c a p n i a .  45  46  F i g u r e 9. The relationship between v e n t i l a t o r y volume and plasma pH (pHpl) i n Scvliorhinus stellaris undergoing simultaneous exposure to environmental hyperoxia and hypercapnia. Ventilatory volume (Vg a c t . / V g c o n t r . ) i s the ratio of the measured Vg d i v i d e d by t h e Vg d u r i n g hyperoxia. Each l i n e r e p r e s e n t s the b e s t f i t l i n e f o r a l l the experimental d a t a f o r an i n d i v i d u a l f i s h . T h i s graph shows only the d a t a f o r the experiment which i n v o l v e d the exposure to environmental hyperoxia and h y p e r c a p n i a and where NaHCC>3 o r HC1 was i n f u s e d to minimize changes i n pH due t o t h e f i r s t t r e a t m e n t ( F i g u r e 7 ) .  48  F i g u r e 10. The best f i t l i n e f o r the aggregate data s e t f o r both data sets d e s c r i b e d i n F i g u r e 7. a b o v e . E a c h symbol r e p r e s e n t s an i n d i v i d u a l f i s h . Coefficients  f o r the l i n e  Vg a c t . / Vg c o n t r . a  5.0476 * 1 0  = a * e < b  1 0  -3.353  b  : * P> H  r2  0.875  49  50  F i g u r e 11. The r e l a t i o n s h i p between v e n t i l a t o r y volume (Vg act./Vg contr.) and a r t e r i a l Pco2 i n shark, s u b j e c t e d t o three e x p e r i m e n t a l p r o t o c o l s : 1.exposure t o e n v i r o n m e n t a l hyperoxia and h y p e r c a p n i a ( + ) ; 2 . e x p o s u r e t o e n v i r o n m e n t a l hyperoxia and h y p e r c a p n i a and i n f u s e d w i t h NaHC03 o r HC1 to m i n i m i z e c h a n g e s i n pH ( o ) ; 3 . e x p o s u r e t o e n v i r o n m e n t a l hyperoxia and h y p e r c a p n i a and i n f u s e d with NaHCC>3 t o l e v e l s beyond t h o s e a c c u m u l a t e d by t h e a n i m a l s ( x ) .  51  52  F i g u r e 12. As ventilatory [ H C O 3 ] i n mM -  i n 11. e x c e p t t h a t t h e r e l a t i o n s h i p between volume (Vg a c t . / V g contr.) and arterial f o r the three experimental prolocols.  53  x  X  X X X X  + • •+ «• •  +  + •  I r-  I  1  U  o  I  I  o u  X  +  x  O  •  I  I  I  .  s»x~  in o  —• o  54  Shark  accumulate  in  plasma  to  be  HCO3  maximum beyond  ventilation  level  this  to  to  -  pH due t o e n v i r o n m e n t a l  a  NaHCC>3  plasma  hypercapnia  this  maximum  compensate  and t h e r e  accumulation.  level  produces  the  fall seems  Infusion of  no  change  in  ( F i g . 11 & 1 2 ) .  DISCUSSION  The  animals  steady  state  concerned. in  is  levels Pcx>2  or  in  Vg  levels  range  by  Mazeaud  by  is  a  and  42.2  to  constant  were  RQ a n d t h e  concentrations to  (1981)  and H e i s l e r  i n blood  i n an e l e v a t i o n o f  P02.  At n e u t r a l heads,  head p r e s s u r e s ,  139.9ml/min.  the observed  The l o w e r  end o f t h i s  i n t h e r e g i o n where Vg e v i d e n t l y l i m i t s g a s e x c h a n g e gills  and  levels  have  no  apparent  reaction  Mazeaud  lOOml/min r e s u l t  decrease  the  diffusing  the  and i n a  parameters  and l a c t a t e  t h e a b s e n c e o f any imposed were  unstressed  fish.  below a b o u t  and  were  a l l measured  supported  across  gases  as  IA  catecholamine  (1984) f o r r e s t i n g  blood  far  the  reported  Vg  experiment  as This  agreement those  in  capacity  of  velocities  presumably  P02 a t t h e h i g h  affects  have  effect  the  in  blood  gill  blood  gas t e n s i o n s .  Vg  on b l o o d  gas t e n s i o n s .  to these  gases and / o r the  and water w i t h  the major determing  Vg l e v e l s .  Higher  The  r e s p e c t t o these  effect  on  PC02  and  55  These which  observations  have  shown  associated  with  Jackson increase  in  between  blood  without  which  mmHg.  the  The  decrease  the  was  not  2  and  obvious  P02  in  study confirms  the  from  with  and  gill  delivery,  therefore  to  CO2  that estimate Vg  a  that  limiting  by as  resulted  ventilation  were i n s t e a d y  unsteady  ventilation of  and  O2  arterial  oxygen  and  P02  however,  both  CO2,  i n the  utilization  P02 Mo  2  There and  and  reduction in aerobic  may  decreased  oxygen g r a d i e n t . arterial  states,  volumes  (1978) o b s e r v e d  tissue  between  The  oxygen  the lowered  production  correlation  Vg.  increase  these animals  : tissue  with  an  differences  r a t e s a t t h e low  blood  experiment,  in  Pco2-  Randall  that  and  i n c r e a s e i n b l o o d PC02  ventilation  suggested  this  decreased  with  and  is  (Wood  (1980) e s t i m a t e d  than r e f l e c t i n g  and  Vg,  results  can  component  since  rates  Burggren  by  Jackson  transfer  uptake  rates  determined  this  Rather  fish  i n an  Pco2  PC02  excretion  excretion  transfer  tissue  uptake  uptake  CO2  regard.  declining  sturgeon  no  result  expected  respectively. 0  would  increases i n blood  in this  reflect  CO2  convective  from  the  maintaining  Wood and  r e d u c t i o n i n gas  levels state  hypoxia,  reduces  which  on  Thus  a  in  i n comparable The  water.  data  which  arterial  effect  increased  be  in  with previous s t u d i e s  hypercapnia, no  alkalosis.  could  excretion 2  and  during  respiratory there  increase  has  causing  consistent hyperoxia,  whereas Vg,  ventilation  that  an  1980)  are  was was  oxygen arterial  metabolism  56  could  not  because head  be  high  and  clear  Vg  low  The  increase  low  These 1974)  data  that  increased  by  1971;  gill  that  effort  effort  pressure  there  is  e x p e n d e d by  compensated  lactate  Carbon  i n blood water  The  no the  by  an  concentrations  water  and Jones  1974;  cannot  above n o r m a l  ventilation conditions  be  levels.  could  i n the  only blood  i n f i s h e s u n d e r a wide r a n g e  of  are  reported  of  of is  well  §_t  1980,  ventilation  1977),  Janssen  and  and  aj,..  large  i s stimulated 1976;  infusion Randall  in  fish  1971,  Itazawa  and  1982).  It  Jones  in fish  acid  1 9 7 5 ) , and  to  (Saunders  H o g l u n d and of  body  Cameron  1977,  S m i t h and  (Hargis  al..  a  documented  Davis  Dejours  Jackson  in  ventilation  1967,  exposure to a c i d waters et.  decreasing  excretion  flow  increase  flow  Randall  Wood and  only  Eddy  Pco2-  content  1973,  can  i n t r o u t by  dioxide  gill  ( S o i v i o 1981,  flow  level  sensitivity  oxygen  Dively  studies  correcting alkalotic  known t h a t  (Cunningham  the  water  PC02  conditions  Holeton  1978,  a positive  not  adjustments of g i l l  in gill  environmental  Takeda  was  previous  blood  in  literature.  also  in  that  environmental  and  with  the  m e t a b o l i s m as  increasing  increases  Randall  in respiratory  head, such  and  M02  agree with  the  effective  1962,  Vg  in  volume.  Changes  reduction  a negative  between  changes  implies  through  a  in a l l fish.  ventilation  with  with  reduction  significantly  is  Vg  to  often associated  i n anaerobic  remained  be  was  correlation  fish.  This  related  stressed Persson or  base  hypercapnia  57  (Van  Dam  1975;  1938;  Eddy  increase  Randall  and  Jones  Smith  and  Jones  1976; in  stimulation  blood of  conditions  externally through  Furthermore,  Neville  is  or  dehydration  (1979a,b)  not stimulated  would  i n acid  Randall  I t seems t h a t  is  Exposure  internally  the  J a n s s e n and  1982).  tensions  ventilation.  hypercapnia  trout  Pco2  1973;  important of  in  fish  an the  to  acid  c a u s e some d e g r e e o f of  the  has shown t h a t  HCO3  ion.  -  ventilation in  conditions unless  accompanied  by h y p e r c a p n i a . A  large  portion  o f the r e p o r t e d  between  ventilation  volumes  to  orientation  of  the  maintenance  of  blood  review  have  (see a  relatively  compared Acid  to  CO2  capacity  for  shown  ,to  1937;  trout,  exposed Randall  O2  exist  Eddy to  by  and  1971,  Jones  hypercapnia  (Babak  of fishes Eddy  1973)  ventilation and Smith  and with  o f the  reduce  the  1973). carrying  w h i c h have  is  Boutilier reduced  (Smith  there  is  P e y r a u d and  and  (1982)  Irving 1986;  when f i s h Jones  only  a superimposed  been  e_t al..  and  Dedek 1907; Jones  i n fishes  ( c a r p . B l a c k and  1971,  hyperoxia  capacity  Dejours  Root e f f e c t s  Ventilation  environmental  of  and  to the  Oxygen seems t o  1973;  would  point  flow  on v e n t i l a t i o n  Jones  blood  t h e Bohr  1973).  1972).  influence  i n the b l o o d  stimulation  Dejours  the  Cameron  and  water  S h e l t o n e_t al.. 1986).  greater  in  of g i l l  u p t a k e and c a r r y i n g  (Randall  conditions  tench,  by  relationship  and a c i d - b a s e d i s t u r b a n c e s  the c o n t r o l  the oxygen  d a t a on t h e  a  are 1982;  minimal  condition Serfaty  showed  that  of  1964; the  58  stimulation  of  ventilation  environmental  hypercapnia  conditions.  There  trend  in  exhibit  and  to  may  supported ventilation to  the  The  to  the  blood  addition  to  of  The  second  against  the  acidotic first changes  some  while  other  fishes  respiratory while  stimulation Johansen  of  (1966)  shifts. than  O2  carrying  i n the elasmobranch i s IB.  The  sensitivity  v e r t e b r a t e s than  regulation  sensors  i n the  of  closely  including blood,  the  related  given of  those  by  PC02  this can  always  be  sensitivity  to  attenuated  Randall  and  in  blood  the by  Cameron  scope o f a d j u s t i n g b l o o d and  with  remains.  The  low  ignored. and  substantiated  is  be  in  Bohr  is  ventilation  status  compartment  which e x h i b i t  show  response t o CO2  teleosts.  o f the a c i d - b a s e  the oxygen s e n s o r s cannot  response  reason  experiment.  a  and  Bohr o r Root  other  their  conditions  in  other  fishes,  oxygen, a r e s i d u a l  Lenfant  of experiment  i n p u t s from  and  emerging  Furthermore,  shows  ventilation  i n p u t s from  in  1973).  factors  hyperoxic  to t h i s  little  mild  PC02 and e s p e c i a l l y pH a r e more s i m i l a r  in  or  shifts  hypercapnia  that  blood  wide r a n g e o f  Root  showed no  driving  situation  show  sucklevi,  the r e s u l t s  possibility  of  A  be  by  (Dejours  to  blood of C y p r i n i d  but  hypercapnia,  possibility  capacity  exposed  abolished with  The  shifts  Saualus  showed t h a t i t s b l o o d The  be  data.  Root  with  trout  a r e , however, e x c e p t i o n s  hypercapnia  dogfish,  ventilation  could  published  Bohr  stimulus the  the  in  only  the  to result  (1973) correct of  the  PC02 t h r o u g h  i n the range o f  2-3  59  mmHg. and on  Further  C0 the  not  as  2  well  as  ventilatory  only  those  exhibit  a  acid-base species  experiments  the acid-base response  animals  sensitivity status  of  as  those  contributions  to  this  in  fishes. sets,  the While  lacking in  of 0  2  p a r a m e t e r s o f pH and P c o  2  in fish  a r e needed.  Bohr  and  ventilation  the blood,  such  exist  that d e l i n e a t e the e f f e c t s  knowledge  of  clear  trends  the e x c e p t i o n a r e e q u a l l y  Root s h i f t s changes  experiments o f t h i s  i n the C y p r i n i d a e field  to  Since  of fish  that  i n the nature  would make  physiology.  i t is  on  valuable  Great  gaps  the r e g u l a t i o n o f v e n t i l a t i o n i n seem t o be e m e r g i n g among obvious.  data  SECTION  2.  TRANSEPITHELIAL ION  FLUXES  FOR ACID-BASE REGULATION IN FISHES  61  INTRODUCTION  Under gain  steady  water through  urine. This  Body loss,  (Na ) lamellae  fish  chloride  as  they  loss  1977,  and  (Cl~)  are  small,  the  ions  out  The  o f the blood  excretion.  uptake  of  across  the  major  way  transepithelial 1982,  i n which ion  1984).  secondary  developed  and  o f the few  systems a r e a l s o  fish  and a l s o  Na  +  and  in  poorly  to those o f diffusively  through  the gut  the o s m o t i c a l l y d r i v e n  excess  water  C l ~ i s actively  by t h e c h l o r i d e c e l l s  located  sodium  The c h l o r i d e c e l l s  the g i l l  e t al.. 1979, M a r s h a l l 1982)  i n the  i n this  In saltwater,  to r e p l a c e  between the s e c o n d a r y  1980,  by  osmotically  e x c e s s water  lost  poorly  saltwater.  water gill.  Scheffey  A  also  and t u b u l a r  C l ~ through  Foskett  and  are  and Payan 1977a).  in  drink  at  pumped  +  and e x c r e t e  fish  A l l these c h a r a c t e r i s t i c s are opposite cells  Na  freshwater  counteracted  The m i t o c h o n d r i a  developed.  gain  is  chloride  freshwater  gills  electrolytes  (Girard  number.  their  however,  and  +  and  state conditions,  ( K a r n a k y §_t a l .  and N i s h i o k a  on t h e e p i t h e l i u m  1980,  Foskett  a t the base o f  lamellae. fish  exchange Since  regulate across  Krogh  acid-base the  gill  (1939) f i r s t  status  i s by  (see H e i s l e r suggested  that  62  the  active  coupled NH4  uptake  to  the  for  +  Na  of  Na  movement and  +  and  +  of  HCO3  f o r Cl~",  -  the e x i s t e n c e o f these  evidence  for  1964;  Kerstetter  1978;  Maetz  and  the  Maetz et  +  Perry  C1~/HC03~  (Maetz  and  circumstantial  composition  extracellular 2).  pH  water  exposed  with  Vancouver provide  a  to  hypercapnia, production Antony  a  in  1977; P a y a n  DeRenzis  evidence water  gill.  that  affects  suggests the  of of  animals  that the  regulation of  to acid-base disturbances (Table t h e r e c o v e r y o f p l a s m a pH hypercapnia  was g r e a t e r i n  One o f t h e aims o f t h i s  S e c t i o n was t o  evidence on  +  for this  experiments  which  can occur  relationship. where  c o n d i t i o n s by e x p o s u r e  combined  and  i n the ambient  reports  fresh  Romeu  than  condition  photosynthesis  particles  and  fish  water with and  as w e l l  naturally  a s from  respiration bacteria  o r g a n i c d e b r i s a t depths  were  to environmental  by s u r f a c e p l a n t s s u c h a s h y a c i n t h mats  1973)  glycolysis  1964;  i n the f i s h  to environmental  acidotic  have  i s experimental  (Maetz  Romeu  (1981) showed t h a t  experimental  Section  subjected  the  water.  further  This  of  +  1981; Wood et_ al.. 1984)  higher concentration of Na  tap  studies  H ,  1981; P e r r y et_ a l . 1981; H o l e t o n  i n response  P e r r y et. al.. trout  There +  and R a n d a l l  a l . 1983) e x c h a n g e p r o c e s s e s  ionic  +  was  r e l e v a n t ions  §_t. §JL.. 1970; Cameron 1976; E v a n s  1973;  is  links.  goldfish  many  Na /H (NH4 )  1973; P e r r y a n d R a n d a l l  There  in  the  the  the acid-base  confirmed  both  C l ~ in  from CO2  ( U l t s c h and  the absence o f and  attached  anaerobic  to  falling  o f 50-100m i n s e a water  63  Table  2.  Time  course  for  pH  compensation  in  fish  s t r e s s e d with  environmental hypercapnia. SPECIES  HATER  Pco  2  mmHg  Conoer conaer Scvliorhinus Ictalurus  stellaris  Dunctatus  Salmo a a i r d n e r i  TIME  REFERENCES  h  8  8-10  8  8-10°  Heisler  10  24P  Cameron  15  22P  Eddy e_t a l -  1977  5.2  72  J a n s s e n and  Randall  Toews eJt al.-  c  c  1983  ai.. 1976 1980  1975  c H  complete +  ion  compensation;  concentration  Vancouver  tap  was  t o 3 itiM.  raised  water;  7.5  24P(52%)*  7.8  24P(88%)**  p  partial  P e r r y g £ al.. 1981  compensation;  * Percent recovery of  before  hypercapnia  was  **  * e x c e p t Na  ion concentration  As  +  imposed,  i n dechlorinated i n the water  64  (Harvey  1974).  disturbance  It  would  epithelium  and  treatments  would  n a t u r e and The  waters  on  the  and pH  CI  mechanism o f  the  i f there  these  ions  carp  during  a  regulatory  strength  should  of  Na the  of  of  trout  the  3)  acclimated  were compared  concentrations the  were  Na  +  blood  movement  of  respectively.  -  directional  of  r e g u l a t i o n of  HCO3 , Cl~  gill  additional  transepithelial  and  +  the  processes.  (Table  and  +  determine  recovery  the  acid-base  understanding  the  affect  H (NH4 )  isotopes to  better  altering  +  this  across  performance of  ionic  water  to  fluxes  i o n exchange  i s a dependence o f  Radioactive into  in  that  imposing  super i m p o s i t i o n  these  of d i f f e r e n t  in  that ion  the  result  hypothesis -  hoped  stimulate that  acid-base  to  was  also  fluxes of  injected these  ions  from a c i d o s i s .  MATERIALS AND  EXPERIMENT 2A.  SALINITY  METHODS  - TROUT - HYPERCAPNIA  ANIMALS :  Rainbow 1535  g  indoors large  trout,  were under glass  Salmo  obtained  from  ambient aquaria  g a i r d n e r i , w e i g h i n g b e t w e e n 800  light at  a  a  commercial conditions.  density  o f about  hatchery  and  and held  They were h e l d 150  in  1 / f i s h which  65  Table  3.  Mean  water  Na  +  and  Cl  concentrations  for the two  experiments i n t h i s section. A l l concentrations i n mM.  Trout - Hypercapnia - 3 S a l i n i t i e s SAMPLING TIME  [Na ]  [Cl ]  [Na+]  EC1-  [Na ]  [ci-  CONTROL  2.32  3. 29  97. 72  113. 82  320. 72  333. 82  + . 25h + .5h  2.61  3. 32 3. 25  94. 2  113. 2 113., 1  330. 0  332. 75  93. 8  331. 17  +lh  2.19  3. 33  98. 2  114. 2  323.,5 314..0  +2h +4h  2.42 2.54 2.28  3. 42 3. 38 3. 58  100 .6 94. 4 97. 8  113. 9  332. 25 327.,42 324..92  2.41 2. 38  3. 58  96. 6  111.,4 113.,0 110,.5  318., 17 318.,67  3. 46  96. 0  110.,5  333,.0  332., 33 335.,4  + . 25h  2.37  3.,55  92. 75  114,. 88  325,.5  330,. 25  + .5h  2.18  3. 58  95. 6  2.17  3.,45  94. 2  110 ,,9 108.. 8  325,.8 315..0  332,, 2  +lh +5h  2.45 2.47  3.,58  97. 4  113,.9  338 .4  334 . 1  +10h  3. 25  99. 4  114..6  330..6  333,.8  +20h + 24h  2.21 2.67  3.,41  95. 4 96. 75  114 .8 117,.0  313,.8  3., 2  336,.0 337.. 1  +  -  +  HYPERCAPNIA  +8h +20h +24h  2.45  332. 58  323,,5 319,.0  RECOVERY  331..7  335,.25  Conger - Hypercapnia - 6 S a l n i t i e s SAMPLING TIME  [Cl~]  [Cl~]  [Cl~]  [Cl ]  [Cl ]  initial  < 3  40  80  140  360  tCl~) determ.  -  -  [Cl ] -  540  66  received The  decholrinated  fish  were  water  were f e d t o s a t i a t i o n  acclimated  to  prior  to  fresh  dechlorinated  about  3,  salts  used  A.l.  tap  3  water  experimentation.  100  and  300  several  times  salinities Sea  water  a t 10 +_ 3°C (mean +_ S . E . ) .  to  mM.  a day.  forat least  s a l t s were added  a c h i e v e NaCl The c h e m i c a l  A l l fish 1 month  t o ambient  concentrations of composition o f the  t o make up t h e v a r i o u s s a l i n i t i e s  i s g i v e n i n Table  o f Appendix I .  SURGERY AND APPARATUS :  in  All  animals  the  dorsal  described which in  in  the  shown  and  Experimental  with c h r o n i c indwelling  The s a l i n i t y  was a c c l i m a t e d was m a i n t a i n e d  was  The  Methods  procedures  to  i n s u r g e r y and  procedures.  recovered  13.  cannulas  e t al_. (1972) a s  M a t e r i a l s and Methods.  experimental  Figure  Materials  surgical  General  animal  in  fitted  a o r t a by t h e method o f S o i v i o  animal  subsequent Each  were  as  in  the  system  is  a  were  'Delta initiated  experimental  chamber  described i n General Bicarbonate at  least  System'. 24 h a f t e r  procedures.  PROTOCOL :  The acclimated  experimental at  the  protocol above  consisted salinities  of to  exposing 24  h  of  fish 1  %  67  F i g u r e 13. Experimental a p p a r a t u s f o r e x p e r i m e n t 2A.: T r o u t S a l i n i t y - H y p e r c a p n i a . A l l components o f t h i s d i a g r a m are explained i n G e n e r a l M a t e r i a l s and Methods under the descriptions of the 'Delta Bicarbonate S y s t e m ' and t h e apparatus f o r measuring t r a n s e p i t h e l i a l p o t e n t i a l s (TEP).  co  69  environmental this  hypercapnia  exposure  eliminated. min,  for  Blood  24  and t h e n o b s e r v i n g t h e r e c o v e r y  h  after  the  h i g h Pco2 t e n s i o n s were  and water s a m p l e s were c o l l e c t e d  1 h, 2 h, 4 h, 8 h, 20 h, and 24 h a f t e r  the  hypercapnia  exposure  h, a n d 24 h a f t e r The time.  procedure  was c a r r i e d  5 ml o f w a t e r was removed  aortic  cannula  contact  with  potential  was  or  a  physiological so  to  KCl/agar  measurements  the  saline that  volume and  contact  Contact  with  of  sampling  the  a  from a  1 h, 5 h, 10 h, 20 exposure.  o u t f o r each  the system.  the  then with  KCl/agar  sampling  The d o r s a l  •T* p i e c e w h i c h  bridge  enabled  for transepithelial  (see General  was  15 m i n , 30  the beginning o f  syringe f o r blood c o l l e c t i o n .  collected;  turned  connected  either  (TEP)  Methods) was  and 15min, 30min,  t h e end o f t h e h y p e r c a p n i a  following  from  replaced  Materials  and  One ml o f b l o o d with  heparinized  t h e v a l v e on t h e •T' p i e c e was the  KCl/agar  b r i d g e was made.  b r i d g e was m a i n t a i n e d  times, a l l o w i n g near  continuous  between most recording of  TEP d u r i n g t h e e x p e r i m e n t a l p e r i o d s . Water continuous Materials  HC03~ basis and  concentration with  Methods.  the  was  also  apparatus  recorded  described  in  on  a  General  F o r t h i s and TEP r e c o r d i n g s , v a l u e s  were r e a d o f f t h e c h a r t a t t h e s a m p l i n g  times  listed  above.  MEASUREMENTS :  Whole  blood  pH,  total  CO2  (TC02  with  the  Capni-Con  70  III)  and  General  hematocrit Materials  centrifuged aliquot  Cl~  and  Methods.  p l a s m a was  and  the  by  as  CMT10  Methods).  plasma  micro-osmometer. concentrations TC02 v a l u e s  were  samples  was  concentrations in  were  i n General  recordings Net body  flux  of  water  rates  of  method same  measured in  ions  -  and  the  the  way  and  Na  pH  plasma  and  Cl~  but  electrode  Cl~  method  T o t a l ammonia  which were  and  i n an  Methods.  continuous determined.  were c a l c u l a t e d i n u n i t s o f  * volume) o f t h e p a r t i c u l a r i o n .  -  concentration  +  r a t e s o f change  a  HCO3  and  +  Materials  concentrations  and  Methods.  Methods.  in  the  with  an ammonia e l e c t r o d e  General  describes HCO3  for  with  with  w e i g h t / m i n by c a l c u l a t i n g  (concentration  as  Na  was  with  measured  for  for  Sodium  measured  M a t e r i a l s and  analysed  Materials  described also  was  the  automated  Section  osmolarity  and  +  Materials  using  were  That  General  An  ammonia  for Na  measured  calculated  concentrations way  (see  determined  General  C l ~ was  was  plasma.  osmolarity.  tensions  the  blood  analyzed  PC02  The  determination  for and  were  concentrations.  was  Plasma  as d e s c r i b e d  Water  described  as  titrator  Total  remaining  and f r o z e n f o r t o t a l  plasma  well  t h e methods i n  to o b t a i n separated  spectrophotometry  Radiometer  according The  acidified  remaining  concentrations  measured  measured  i n Eppendorf v i a l s  of  analysis  were  i n the  umol/Kg contents  71  STATISTICS :  Analysis  of  significance  among  salinities.  the  Paired  significance any  variance  was  used  means  of  Student's  any test  discern  statistical  parameter was  at  used  regression  analysis  certain  was  data  value  used  sets.  describe  The  level  test  v a l u e and  a t any one s a l i n i t y .  to  three  to  o f d i f f e r e n c e s between t h e mean c o n t r o l  subsequent experimental  between  t  to  Linear  the r e l a t i o n s h i p  of r e j e c t i o n i na l l  c a s e s was 5 %.  EXPERIMENT 2B. CONGER - SALINITY - HYPERCAPNIA  ANIMALS :  Conger  e e l . Conger c o n g e r , w e i g h i n g between 800 t o 1500 g  were  caught  the  laboratory  fiberglass surgery fed  varied  where  tanks  and  once  i n t h e Bay o f N a p l e s , they  supplied  Italy.  were with  held  were  in  the  at  ambient  subsequent e x p e r i m e n t a t i o n .  they  They were b r o u g h t t o  laboratory,  19°C  sea  in  water  200  1  before  The a n i m a l s were n o t a time p e r i o d  which  from 48 h t o 2 weeks.  SURGERY AND APPARATUS :  All  animals  were  fitted  with  chronic  indwelling  dorsal  72  aortic  cannulas  method  of  animals  similar  to the  Materials  conducted.  The  acclimation  water  An  initiating  8  or  in  were r e c o v e r e d  General  and  Methods  until  in  24  experimental  procedures.  were  three  periods  change  and  M a t e r i a l s and water  the experiment  o f t h e w a t e r was  identical  was  to  the  c h a n g e s d e s c r i b e d below  was  recovery  The  System' d e s c r i b e d i n  which  the s a l i n i t y  h  artery.  in a recirculating  'Delta Bicarbonate  Water t e m p e r a t u r e to  intestinal  described  chemistry  initiated.  20°C.  gastric  is  The  General  were  a  operation  Methods. system  via  maintained  period  was  constant  allowed  at  before  PROTOCOL :  There salinity hours the  excluding salinity  which the  were fish  Sampling  periods  1  h  h,  min, was  2 1 h,  and 2 h,  lower  than  exposed  to  were as  In the  first  t o one  of six  sea water. %  In the  t h e s t e p change  5 h after  control, lasted  three  8  hours  salinities  following  environmental  follows : 2 control  3 h after 4 h and  changed  1  experiment;  Each experiment  period.  o f t h e water was  were  the  hypercapnia.  the c o n t r o l  all  in  5 h,  hypercapnia.  samples;  in salinity;  the e n v i r o n m e n t a l  30  min,  and  30  hypercapnia  imposed. At  through below.  each the  sample p e r i o d , 500 cannula  and  u l o f whole b l o o d was  analysed  f o r the parameters  withdrawn described  73  MEASUREMENTS :  Whole the  blood  pH,  Capni-Con  tension  and  determined described  hematocrit  I I I ) , plasma  plasma  and  HCO3  total  i n General  using  (TC02  with  and  PC02  concentration  -  C l " concentrations  by t i t r a t i o n  CO2  were  calculated  or  t h e R a d i o m e t e r CMT10 t i t r a t o r  as  M a t e r i a l s and M e t h o d s .  STATISTICS :  Correlation describe  and  linear  regression  r e l a t i o n s h i p s between d a t a  a n a l y s i s were u s e d t o  sets.  EXPERIMENT 2C. CARP-HYPERCAPNIA-ISOTOPE  ANIMALS : Carp, obtained  Cvprinus from  a  carpio, commercial  glass  indoors  aquaria,  under  natural  light  water.  Fish  pelleted  carp  month p r i o r  SUGERY AND  weighing  were  hatchery  and  receiving  Fish  2000  and m a i n t a i n e d  15°C  g  were  i n large  100 1 p e r f i s h ,  dechlorinated  a day t o s a t i a t i o n  were a c c l i m a t e d  t o use i n e x p e r i m e n t s .  APPARATUS  to  a t a d e n s i t y o f about  f e d s e v e r a l times  feed.  1500  tap  with  a  f o r a t l e a s t one  74  All  fish  were f i t t e d  cannulas  using  recovered  in  in  detail  the  a  by  with c h r o n i c i n d w e l l i n g  method  and  recovery  period  Siovio  et  aortic  a l . . ( 1972)  and  ' D e l t a B i c a r b o n a t e S y s t e m ' ( F i g . 14) d e s c r i b e d Claiborne  protocol  of  dorsal  and  measurements after  Heisler  were  (1984).  carried  out  The  following  after  a  24 h  surgery.  PROTOCOL :  One Cl~35  hundered were  procedures samples 48 h  were  recovery  after  h,  were  out.  +  cannula  After  and  20 u C i o f  and t h e f o l l o w i n g  3 pre-exposure  control  after  the  h y p e r c a p n i a and d u r i n g a 21  high  CO2  tension  was  turned  s a m p l e s were t a k e n a t 0, 30 min, 1 h, 2 h, 4 h, 8 h,  29 h, 33 h, 45 h, 46 h, 49 h, 53 h and 69 h  regime.  every  o n l y 48 o f t h e 69  Water s a m p l e s were c o l l e c t e d  times as f o r the b l o o d e x c e p t  taken  sampling  the  Na 24  o n s e t o f h y p e r c a p n i a which l a s t e d  sampling  sampling  isotope  to 5 % environmental  25  the  the  through  carried  period  Blood 24  of  t a k e n , water and b l o o d s a m p l e s were t a k e n d u r i n g  h exposure  h,  h  injected  were  off.  uCi  4 h whenever p o s s i b l e  a t t h e same  that a d d i t i o n a l  samples  between t h e 53 and 69h  times.  MEASUREMENTS :  Water  pH  at  a  set  Pcx>2  as  well  as  Na  +  and  Cl~  75  F i g u r e 14. Experimental apparatus f o r e x p e r i m e n t 2C.: C a r p Isotope Hypercapnia. A l l components o f t h i s d i a g r a m are explained i n G e n e r a l M a t e r i a l s and Methods u n d e r the description of the 'Delta Bicarbonate System'. The experimental apparatus f o r e x p e r i m e n t 2B. was s i m i l a r t o t h i s apparatus.  lonmeter Digital Printer  Computer Power on-off  Temp.  - 5  mm  Control  Thermostat "^_30°C  Power on - off  —  Heater  NH^-Electrode  Cooler  W-f—f  PH  Amplifier • Recorder  Valve  Na.Cl'  <r—j> Plasma Na .Cl*. Na*,Cl" L  i  nmp  n  Temp. Probe  Water Na" .Cl  36  Ft!' —  1% C0 in N 2  2  30 °C  Filter,  Air or C0 /Air 2  77  concentrations appropriate  were  reference  System',  The  various  measured  NaCl  calculated  Na  the  concentrations  of  spectrophotometry  electrodes  Water  Bicarbonate  HCO3  concentrations  -  above measurements and methods. Na  and  with  C l ~ e l e c t r o d e s were c a l i b r a t e d w i t h  solutions.  using  glass  e l e c t r o d e s w i t h i n the 'Delta and  +  by  and  +  C l ~  by t i t r a t i o n  were  using  were Plasma  determined  the Radiometer  by CMT10  titrator respectively. Net  fluxes  of  determined  by  changes  each  of  injected  'cold' The  system  first.  +  a  of  that  minute  were  with  Cl~35  specific  and  of  of  fluxes  o f each  activities in  sample  with  the  o f the ions. Gamma and B e t a was  counted  3 weeks, a p e r i o d e x c e e d i n g t h e  isotope,  the  Cl 3g  activity  _  f o r decay, background, c o u n t i n g  automated  were  concentration  together  each  water  t h e water and p l a s m a  were c o u n t e d  activity  period  Corrections  efficiency  and  and  unidirectional  and  +  blood  volume  determined  Na 24  Na 24  After  counted.  water  o f the isotopes  The  half-life  between  The  were  concentrations  activities  ion  ion.  for  counters.  per  the  isotope  activities  each  in  the machines.  was  t i m e and  Disintigrations  (dpm) were t h e n c a l c u l a t e d .  STATISTICS :  Student's fluxes  of  t  these  tests ions  were  used  t o compare  t h e mean c o n t r o l  t o t h e mean f l u x e s a f t e r  the treatments  78  were  imposed.  The 5 % l e v e l  o f r e j e c t i o n was a p p l i e d  in all  cases.  RESULTS  EXPERIMENT 2A & 2B. EFFECTS OF WATER  SALINITY  ON ACID-BASE REGULATION IN FISHES  I.  Acid  - Base  An  elevation to  PC02  In  trout  rise  to  There  on  was  both  accumulation accumulation greater positive  than  times  over  H  +  the  positive  and water  in  those  water  drop  i n pHe i n d u c e d by  was  compensated  salinity  in  period  ( F i g . 17,18 & 1 9 ) .  between  the  degree  of  effected  by  ( F i g . 18 & 2 0 ) .  HC03~  acclimated  of  (Fig. from  pHe  (Fig. to  t o 3mM  between  changed  plasma  time c o u r s e o f t h e exposure  acclimated  i n the conger  caused  v a l u e s ( F i g . 15 & 1 6 ) .  hypercapnia  correlation  plasma  flux  control  compensation  trout  pressures  the i n i t i a l  salinity  species of  Pco2  the  external  correlation  accumulation Net  water  environmental  the  a  compensation In  4-6  degrees  depending  of  and i n t h e conger,  exposure varying  Regulation.  water  21  was &  HCO3  22).  -  100 and 300mM N a C l was ( F i g . 2 1 ) . T h e r e was a salinity  and  HC03~  23). an  influx  during  control  79  F i g u r e 15. Means + one standard error (S.E.) o f plasma PC02 i n rainbow trout acclimated t o 3, 100 and 300 mM NaCl and e x p o s e d to environmental hypercapnia. Time course shown includes control, 1 % hypercapnia and recovery periods. A l l u n i t s i n mmHg.  ( D H U J U I )  '"'-'d  81  g u r e 16. Plasma PC02 i n conger d u r i n g c o n t r o l , salinitychange and exposure to 1 % environmental hypercapnia. Each line represents an i n d i v i d u a l f i s h . Control period was i n sea water. S a l i n i t i e s were c h a n g e d i n one s t e p t o those noted and fish were e x p o s e d t o t h e h y p e r c a p n i a a t those s a l i n i t i e s .  Plasma  Pco  2  f o r Individual  Cummulatlve Time (h)  Fish  83  g u r e 17. Means +_ S.E. o f plasma pH i n rainbow t r o u t acclimated t o 3, 100 a n d 300 mM NaCl and e x p o s e d t o environmental hypercapnia. Time course shown i n c l u d e s c o n t r o l , 1 % h y p e r c a p n i a and r e c o v e r y p e r i o d s .  85  u r e 18. As Fig. 17 except that a l l values were referenced to the average value during the control period. Means.  •  3mM  Cummulative Time (h) + 100 mM o  300 mM  oo 0^1  87  g u r e 19. Plasma pH c h a n g e s from a v e r a g e c o n t r o l v a l u e s i n conger during salinity change and exposure to 1 % hypercapnia. Each line represents an i n d i v i d u a l f i s h . Actual average control v a l u e s f o r pH were : 360mM=7.829, 140mM=7.736, 155mM=7.847, 0.005mM=8.843, 2.6mM=7.816, 80mM=7.783(mean o f 6 ) , 40mM=7.835(mean o f 2 ) .  Cumm. Time (h)  co CO  89  g u r e 20. Effect of water salinity, represented [Cl~], on pH recovery i n conger. Percent recovery the pH value measured prior to exposure to 1 environmental hypercapnia was c a l c u l a t e d by d i v i d i n g pH value at the end of the exposure period by pre-exposure value and multiplying by 100. Results l i n e a r r e g r e s s i o n a n a l y s i s shown.  by to % the the of  Effect 100 - i  of Water Salinity on  pH  (post—/pre—hypercapnia) * 100 —  Recovery ,  P  0  200  400 [Cl"]water  (mM)  600  91  F i g u r e 21. Means +_ S.E. o f plasma HCO3 c o n c e n t r a t i o n s in rainbow t r o u t a c c l i m a t e d t o 3, 100 and 300 mM NaCl and exposed to environmental hypercapnia. Time c o u r s e shown includes control, 1 % h y p e r c a p n i a and r e c o v e r y p e r i o d s . A l l u n i t s i n mM. -  92  93  F i g u r e 22. Plasma HCO3 changes from mean control values i n c o n g e r d u r i n g s a l i n i t y change and e x p o s u r e t o 1 % hypercapnia. Each l i n e r e p r e s e n t s an i n d i v i d u a l f i s h . Actual average control values for HCO3 were : 0.005mM=3.68, 2.6mM=3.52, 40mM=2.92(mean of 2), 80mM=3.15(mean o f 5 ) , 140mM=3.07, 155=2.63, 360=3.40. A l l v a l u e s i n mM. -  -  Delta  0  [HCO"]  2  f o r Individual  4  6  Fish  8  Cummulatlve Time (h) vo  95  F i g u r e 23. Effect of water salinity, represented by [Cl~], on H C O 3 accumulation in conger. Calculations as i n F i g . 20 e x c l u d i n g m u l t i p l i c a t i o n by 100. R e s u l t s o f l i n e a r r e g r e s s i o n a n a l y s i s shown. -  Effect  of Water  Salinity on  [HCO~]pl  (post—/pre—hypercapnia)  1  0  r  1  100  1  1  200 [Cr]water  —r  1  300  1  1  400  (mM) vo  97  conditions  to an e f f l u x  hypercapnia among HCO3  in  groups  a l l groups  of  efflux  -  during  fish  was  the p e r i o d o f exposure  of trout  were  ( F i g . 24.).  not d i f f e r e n t .  resumed  at  the  end  Differences  Net H of  t o water  i n f l u x or  +  the  recovery  period.  II.  Plasma  Ions.  Plasma and  C l  conger  compared after  ( F i g . 26)  lOh  300mM  (Fig.  of  decreased  exposure  were  NaCl  both  27).  Plasma  experiment.  Plasma  osmolarity  compared  to  were  control no  +  in  lOh o f exposure  during trends  28)  III.  A c i d - B a s e and I o n C o r r e l a t i o n s :  plasma trout  were (Na  at  +  weak -  but  C l ~ ) and  a l l three  plasma  salinities  +  t o 100  were n o t measured i n  t o 3mM was  exposure  significant  Na  i n the t r o u t  correlations  HCC>3~  reduced  to hypercapnia.  i n t h e 100 and 300mM  (Fig.  There  period  Plasma  i n the t r o u t acclimated  trout acclimated  significant  ( F i g . 25)  the exposure  trout.  concentrations  values  trout  These c h a n g e s were marked  the  the f i r s t  Na  the  during  in  increased  during  the c o n g e r  There  in  to control concentrations.  concentrations and  concentrations  -  groups  between  concentrations  ( F i g . 29 a . b . c ) .  in  The r e a s o n  98  for  using  the  parameter  transepithelial  flux  accumulation  of  C1~/HC03~ fluxes  of  Na  due  the  Na /H . changes  in  plasma  HCO3  between (Fig.  the  that  plasma  31).  changes  in  to  HCO3 .  +  be  these  Cl~  was  plasma  HC03  ( F i g . 30). with  the  also  a  C l ~ " and  and  s i n c e the  in  opposite (Na  combined  -  the  correlated  T h e r e was changes  no  change  in  negative  plasma  largely  than  were  +  fluxes.  was  rather  plasma  the  +  Therefore  -  effect  in  There  Na /H  subtracted  have  of  that  have r e s u l t e d i n t h e  t h e c o r r e l a t i o n above  associated  +  was  via  were  effect  C1~/HCC>3~  in Na  (Fig.  -  would  Increases  +  with  CI  net  a n a l y s i s showed to  +  the  Cl~)  HC03~ ions  -  -  +  ions could  The  accumulate  represents  Further  IV.  and  +  to  both  plasma  exchanges.  directions Cl~)  of  (Na  plasma  correlation  HCO3  -  i n conger  32).  Transepithelial Potentials  TEP  values  acclimated  to  respectively. sampling generally groups  as  during  the  variability.  were n e g a t i v e , 3,  100  There  periods  (TEP)  in  increased well. recovery  was fish during  There  and  near zero 300  and p o s i t i v e i n t r o u t  mM  (Fig.  a d e p o l a r i z i n g trend in  a l l  33  a.b.c),  i n the  initial  salinities.  TEP  the exposure to h y p e r c a p n i a was  periods  a trend although  towards c o n t r o l there  was  values in a l l values  considerable  99  g u r e 24. Means +. S.E. of net H f l u x i n rainbow t r o u t acclimated to 3, 100 and 300mM NaCl and e x p o s e d t o 1 % Time course shown i n c l u d e s environmental hypercapnia. A l l values control, 1 % h y p e r c a p n i a and r e c o v e r y p e r i o d s . i n umol/Kg/min. +  1  0  0  101  F i g u r e 25. Plasma Cl~ concentrations i n rainbow trout acclimated t o 3, 100 and 300mM NaCl and e x p o s e d t o 1 % environmental hypercapnia. Time c o u r s e shown i n c l u d e s control, 1 % h y p e r c a p n i a and r e c o v e r y p e r i o d s . A l l values i n mM. Means + S.E.  102  O  (WUJ) gaicJOIHO  VlrMSVHd  103  g u r e 26. Changes in plasma C l ~ concentrations from average control values i n c o n g e r d u r i n g s a l i n i t y change and exposure to 1 % e n v i r o n m e n t a l h y p e r c a p n i a . Each l i n e represents an i n d i v i d u a l f i s h . S a l i n i t y l e v e l s shown n e x t to each line as C l ~ c o n c e n t r a t i o n . A c t u a l a v e r a g e c o n t r o l [CI ] were : 0.005=150.25, 2.6=148.75, 80=151.88(mean o f 2), 140=165.25, 155=155.25, 360=153.75. A l lvalues i n mM. -  Delta  0  [CI  ]pl vs Time  2  4  f o r Ind.  Cummulatlve Time (h)  6  Fish  8  105  F i g u r e 27. Means +. S.E. o f plasma Na concentrations i n rainbow trout acclimated t o 3, 100 and 300mM N a C l and exposed to 1 % environmental hypercapnia. Time c o u r s e shown includes control, 1 % hypercapnia and recovery periods. A l l v a l u e s i n mM. +  PLASMA  901  SODIUM  (mM)  107  F i g u r e 28. Means +_ S.E. o f plasma O s m o l a r i t y i n rainbow trout acclimated t o 3, 100 and 300mM NaCl and e x p o s e d t o 1% e n v i r o n m e n t a l hypercapnia f o r 24h and r e c o v e r e d f o r 24h. A l l v a l u e s i n mOsmol.  TIME  (h)  109  Figure 29.a.b.c. Relationship o f plasma ([Na ]-[Cl~]) to plasma [HCO3 ] during exposure and r e c o v e r y from 1 % hypercapnia i n rainbow trout acclimated t o 3, 100 and 300mM NaCl, respectively. A l l v a l u e s i n mM. Best f i t r e g r e s s i o n l i n e s shown. +  -  Correlation coefficient r  Coefficients ([Na ]-[Cl ])=a a +  n  _  for best-fit line + b ([HC0 ]) b -  3  3mM  0.5281  64  0.9430  26. 62  lOOmM  0.3676  57  0.7222  34.66  300mM  0.3503  85  1.4437  11. 27  110  Ill  Figure 30.a.b.c. Relationship o f changes i n plasma C l ~ concentrations to corresponding changes in plasma HCC>3~ c o n c e n t r a t i o n s during exposure and r e c o v e r y f r o m 1 % h y p e r c a p n i a i n r a i n b o w t r o u t a c c l i m a t e d t o 3, 100 and 300 mM NaCl, respectively. A l l v a l u e s i n mM. Best f i t r e g r e s s i o n l i n e shown.  Correlation coefficient r  Coefficients ([Na ]-[Cl~])=a a +  n  f o rbest-fit line + b ([HCO3-]) b  3mM  0.5084  58  -0.8470  -1.0584  lOOmM  0.3986  48  -0.5439  -0.0905  300mM  0.4139  74  -0.7463  -0.4684  112  (WUi)  [-10] V  113  Figure 31.a.b.c. R e l a t i o n s h i p o f changes i concentrations to corresponding changes HCO3 concentrations during exposure and 1 % hypercapnia i n rainbow t r o u t a c c l i m a t e d 300mM NaCl, respectively. A l l values in r e g r e s s i o n l i n e shown. -  Correlation coefficient r  Coefficients ([Na ]-[Cl 3)=a a +  n  -  n  plasma Na in plasma r e c o v e r y from t o 3, 100 and mM. Best f i t +  for best-fit line + b ([HC0 ]) b -  3  3mM  0.0541  61  -0.2275  -1.0536  lOOmM  0.0255  59  -0.0424  0.1704  300mM  0.0361  74  0.0960  -0.3126  114  -40  - T  -8  T  -4  1  O  A H C O ; 1  ,  4  8  — ,  12  3  115  g u r e 32. Relationship of changes in plasma C l concentrations to corresponding changes in plasma HCO3 concentrations in conger exposed to salinity changes (filled c i r c l e s ) a n d exposure to 1 % environmental hypercapnia. A l l v a l u e s i n mM.  -  -  116  20,  3mM  a.  •  i  r-  20-,  lOOmM o o  10.  ^ <co  °  °  cP° °  b.  . oo-o  o  -10 -  O <  -20-  -30.  1  1  r  1  -i  1  r  20-  300mM 10  -I  -10  -I  c.  -20A  -30.  —r -4  -  i  r  4  12  AHCO3 (mM)  117  Figure 33.a.b.c. Means £ S . E . o f t r a n s - e p i t h e l i a l p o t e n t i a l (TEP) v a l u e s d u r i n g c o n t r o l , e x p o s u r e and r e c o v e r y f r o m 1% environmental hypercapnia i n rainbow t r o u t a c c l i m a t e d to 3, 100 and 300mM N a C l , r e s p e c t i v e l y . A l l v a l u e s i n mV.  118  119  Hematocrit hypercapnia groups  and  of  variable  increased  then d e c l i n e d  fish.  of f i s h  EXPERIMENT 2C.  during  was  a  the  net  values  of  the  a e r a t i o n w i t h a i r was  experiment There relative  to  to  the  control  (Fig.  36).  hypercapnia  +  environmental the  C0  ( F i g . 35).  over  was  2  There  the c o u r s e o f fish  t r e n d over  course of  the e n t i r e  t r e n d from a b o u t hypercapnia.  the  24h  Na  +  efflux  as through  of C l ~  was  mark, d u r i n g  This trend continued  as w e l l  the  ( F i g . 35). efflux  Unidirectional  period  water  i n the u n i d i r e c t i o n a l  net  uptake  flux results from  the  c a n o n l y be  water  environmental  h y p e r c a p n i a and  the  period  water  of Na  the  the  lower  exposure througout  the r e c o v e r y  period  36).  The Cl~  flux  5%  resumed  change  no  more  ( F i g . 34).  after  was  environmental  (Fig.  the net  reversed  to the t r e n d f o r c o n t r o l  the c o n t r o l  the  was  to  relative  experiments than  trend  in  ina l l  and  the b l o o d to  fish  turned  change  higher  of  CARP ISOTOPE EXPERIMENT  This  no  onset  with sampling  a c c l i m a t e d i n 3mM  hypercapnia.  was  the  were  f l u x o f C l ~ from  exposure  o f f and  with  thereafter  Hematocrit  i n the group  There  initially  recovery returned  to  in  response  to the exposure  the a c i d - b a s e d i s t u r b a n c e .  started, control  e x p l a i n e d by r e d u c t i o n o f  the u p t a k e  levels.  rate of C I  With  -  from  to  Once the  the y e t unchanged  120  g u r e 34. Means £ S.E. o f p l a s m a h e m a t o c r i t v a l u e s ( H c t i n %) of trout acclimated to 3, 100 and 300 mM NaCl and exposed to 1% e n v i r o n m e n t a l h y p e r c a p n i a f o r 24h and t h e n r e c o v e r e d f o r 24h.  121  122  F i g u r e 35. Means + « fluxes of Na (filled circles) and C l ~ ( o p e n circles) i n carp d u r i n g exposure to 5% environmental hypercapnia f o r 48h f o l l o w e d by 24h recovery. Fluxes are referenced to the c o n t r o l p o i n t (half filled circle). Control n e t f l u x e s f o r N a and Cl are shown by the and t h e lines respectively and a r e e x t r a p o l a t i o n s o f n e t f l u x t r e n d s prior t o the exposure p e r i o d extended over the e n t i r e time course o f the experiment. A l l d a t a shown a r e f o r t h e water. Therefore p o s i t i v e v a l u e s i n d i c a t e e f f l u x from the f i s h and n e g a t i v e v a l u e s i n d i c a t e u p t a k e by t h e f i s h . S  E  o  f  n  e  t  +  +  -  NET FLUX o — o  Na  +  Na conirol +  •—• c r  ANa+  Cl~ control  ACl" (mmol/ kg fish)  A i  \  A /  No  Na  J  0  20  +  L  AO  T  .  f u l  Time (h)  60  t\5 CO  124  F i g u r e 36. Means +. S.E. o f e f f l u x rates for Na (filled circles) and C l ~ ( o p e n circles) i n carp during exposure to 5% environmental hypercapnia f o r 48h f o l l o w e d by 24h recovery. Fluxes are referenced to the c o n t r o l p o i n t (half filled circle). Control effluxes for Na and Cl are shown by the and the lines respectively and a r e e x t r a p o l a t i o n s o f e f f l u x t r e n d s p r i o r to the exposure period extended over the e n t i r e time course o f the experiment. +  +  -  EFFLUX  15 Na •••• Na control - • Cl" — Cl" control —o  +  +  10 ANa  +  i  ACI"  Na  (mmol/ kg fish) 4cr  0  o  20  AO  Time (h)  60  +  126  efflux  of  observed from  Cl~  from  decline  the  reduced  n  water  blood,  net f l u x  must  efflux  control  the  in  have  order  trends could  this  would  from b l o o d also  that  been an  result  to water. reduced  unchanged  Na  i n the uptake  +  t o match net  flux  the from  result.  DISCUSSION  Environmental trout  and  followed  hypercapnia  conger by  a  and  trend of  characteristic  of fish  water  Janssen 1967,  plasma  Randall  Cameron  and  Cross  1976,1980,  Randall  fall  in  acidosis plasma  with  an  These  -  in  pH  was  associated trends  are  to e n v i r o n m e n t a l h y p e r c a p n i a i n and H e i s l e r  Eddy  e £ al..  1972,  et. a l . . 1969;  Randall  plasma  recovery  exposed  1975,  a  HCO3 .  (carp, Claiborne  and  (dogfish.  initial  towards  accumulation  fresh  the  caused  Eddy  spotted  e t a l . 1976;  coho  1984; 1977,  rainbow Lloyd  1976)  dogfish,  and  and  trout, White  s e a water  Heisler  et. a l .  s a l m o n , B u b i e n and Meade  1979). Water acidoses in  salinity in  marine  supports  trout  conger the  had  a  positive  acclimated eel  correlation  between  correction  of  the  acid-base  to higher  conditioned  circumstantial ionic  effect  i n correcting  salinities  to d i l u t e  evidence content  disturbances  for  as w e l l  waters. the  as  This  positive  o f t h e w a t e r and from  the  exposure  the to  127  environmental  hypercapnia.  Because  concentration,  and  so  were n e a r l y e q u i v a l e n t a t t h e t h r e e  that  the  they  data  was  concentrations The  analysed i n the  in  plays  acidosis  in  1973, and  Heisler  (1984) in  Evans  the  important  trout of HCO3  -  and  a in  ion  (Maetz Renzis  and  1975,  1979).  the  net  this  exchange  leak  from  plasma  present  link,  C l  -  Kerstetter  and  in  but  This  by  the  exchange  -  a  of  the  exposure  between  C l  to and  -  b e e n documented 1964,  and  De  Renzis  Kirshner  (1976) and  1972,  Claiborne  C1~/HCC>3~  o f p l a s m a pH these  plays during  studies to  and  exchange  carp, respectively,  also  play  a  i o n c o n c e n t r a t i o n s i n the  d i d not  pH  i n conger.  has  exchanges  in  with  Garcia-Romeu  While  changes  process  caused  that  +  consequent was  and  +  compensation  compensation  r e p o r t e d here  exhange the  +  the  Cameron  grayling  in  and  movements  arctic  role,  controlled  correlated  -  The  shown  +  -  salinities,  Na  C1 /HCC>3  conger  Na /H (NH4 )  experiment this  that  hypercapnia.  demonstrated  the  was  -  trout  have  role  environmental  significant  De  to  i n both  role  and  studies  Kormanik  an  major  transepithelial  Maetz  process  -  HCO3  hypercapnia.  numerous  and  C l  trout  environmental  in  a  regard  plasma  suggests  mechanism  in  of  plasma  relationship  with  i n t h e w a t e r was  HCO3  water.  accumulation  reductions  HC03~  thus b u f f e r i n g ,  the  show a s i g n i f i c a n t accumulation  recovery.  of  plasma  It i s possible  not d e t e c t e d because o f a  t o w a t e r w h i c h would have had  t o be  role  that Na  +  roughly  128  equivalent the  to  efflux  plasma to  of  [Na ]  H in  +  with in  reduction  in  maintaining the  higher The  rates  not  known,  effect  the  stimulated of  Cl~  efflux  a  which  -  of  from  effected  the e q u i v a l e n t  fluxes  pH  by a while  of course, pertains  is  since  to  in fish  While u n r e l a t e d  fish,  The available  exchange  accumulation.  -  Qpsanus  process to  fish  accumulation.  process  Chloride  efflux  in  the  process.  accumulation  of in  It is  i s modulated could  be a d e c r e a s e i n t h e p a s s i v e  result  exchange  toad  -  HCO3  t o any  (1979) d e m o n s t r a t e d  HCO3 .  therefore  this  o f net  i s o b s c u r e d by t h e h i g h  marine  for  nature at higher  the r e s o l u t i o n  and E v a n s  the  mechanism  would  of this  ions.  water  there could  HCC>3~ t h r o u g h t h i s  This,  changes  Kormanik  however, how HCO3  associated  the c o r r e c t i o n o f plasma  technical  increasing  or  in  rates.  concentrations  as  to the  Cl~~ uptake from the water  concentration  exchange  water  of  are p a r t i a l l y  Cl~  salt  correlated  concentrations  to i n v e s t i g a t i o n s  increased  C1~/HC03~  not  exposure  a r e unknown.  disturbance,  by  Cl~  i n f r e s h w a t e r and  acid-base  beta,  were  hours a f t e r  t o e n v i r o n m e n t a l h y p e r c a p n i a was  limitations  background  plasma  normal e f f l u x  through  the e a r l y  changes  accumulation  salinities  salinities fluxes  in  the  condition  W h i l e t h e r e were c h a n g e s i n  -  HCO3-  c a r p exposed  during  i o n i n exchange f o r  HCO3 .  reduction  the  uptake o f t h i s  to the water.  +  these  i n plasma  The  active  trout  hypercapnia,  changes  at  the  to be  influx  o f plasma  129  There  may  uptake  of  uptake  from  to  ions  a  the  from  acid  and  uptake  rates  and  are  of  the  HCO3""  existence  of  (Kerstetter  an and  mammalian  pancreas  al..  brush  border  1977,  Kinne-Saffran the and  localization  to  at  Shaw 1960; Ye,  that  while  during  Na  least  Cl~  +  uptake  freshwater  +  Na  +  exposed  McWilliams unpublished normal C l ~  recovery from  from  the  membranes  Utida  of  Necturus  1976)  of this  include  regulation.  ATPase  water  in  1974,  gills  Liang  1979, and  anion  (Kerstetter  exchanges  the  the f i s h  intestinal  implicates  vivo  showing  Van  The and  gill 1977,  found i n and  renal  Os et. a l .  Sacktor  ( W i e b e l h a u s e t a_l..  i n trout  in  and B o r n a n c i n  and c r a b (Lee 1982).  enzyme  accumulate  enzymes have b e e n  e t aj.. 1972),  Kinne  to  studies  (Humphreys et. al..  and  step  provides  DeRenzis  Similar  (Simon  carp  in. v i t r o  1974,  1980).  gills  uptake  stimulated  and c r a b (Lee 1982)  acid-base  was  reduced  Kirschner  et,  1974)  active  1972;  resumed  previous  anion  Bornancin  (Morisawa  of  Na  exposure  observed i n trout  Dunson  active in  supporting  in  seems t h a t  r e d u c e d upon  Wood 1985;  probably  i t  active  during recovery.  plasma  and  been  here  hypercapnia,  Modulation  evidence  Packer  also  reducing  Inhibition  has a l s o and  of  because  i n c a r p was  irregularity  environmental  the  water  (Wright  1978; The  phenomenon  hypercapnia.  t h e water  data).  continued  the  water  waters  Potts  general  from  environmental  uptake to  be  1976),  1971), e e l microsomal Kirschner  i t s functional  related  to i o n o -  role and  130  The  lack  during  c o n s i s t e n t change  hypercapnia  exhanges were  of  were  at  et_  dogfish  and  constant  hypercapnia the  The  reduction  in  i n the  onset  of hypercapnia  TEP  This  the  which  catechlamines  are  released  environmental  hypercapnia.  et  al..  the  furthermore,  (1984)  observed  catecholamines  of  the  to  ions  since  ionic  ionic  the  to  charges  findings of spotted remained  environmental  i n t r o u t exposed i n the  if ion  the  ionic  to  changes  depolarizing  increased  the  reduction  of  Perry  periods  An  and  i n the  water  observed  concentration  the  effects  during  that  shown  that  to exposure  Heustis  (1984)  s w e l l i n g which Isaia  increase  after  the  has  to and  could  e t al.. ( 1 9 7 8 ) ,  water p e r m e a b i l i t y  increase of  to  present  (1986)  red c e l l  i n membranes.  trend  been  hematocrit.  movement  the d e p o l a r i z i n g  have b e e n due  Nikinmma  in fish.  channels  and  i n t r o u t i n response  in  g i l l s s u g g e s t s an  the  water.  the  osmolarity  sampling  have  reported  increase  to  values  initial  may  likely  through  that  imbalance  i n t r o u t may  is  explain  trout  (1983) f o r the  exposure  hematocrit  during  catecholamines  Baroin  with  of  experiment.  increase  time.  that  f a r as  i n osmolarity  s u g g e s t s an  trend  of  as  consistent  during  osmolarity  suggests  Toews e t §JL.  fish  i n 3 mM  mM  respectively,  these The  300  i n balance  is  (1976) and  conger,  hypercapnia.  during  This  aJU  in  and  taking place  concerned.  Heisler  100  i n the  i n response  i n water  permeability  general  permeability  molecules are Given these  h e r e m i g h t be gradients  occurring  observations, expected  between b l o o d  from and  131  Reports  of  the  general  the  acclimation  TEP  values  trend  of  i n the l i t e r a t u r e  increasing values  medium  is  consistent  (Kerstetter  et, al_. 1970; P o t t s  1974;  1975;  Eddy  also  increase  values  during  The ionic  strength  uptake  1984.).  has  been  This  is  undergoing known.  the an  of a  fish  wide  isotonic  values  a  during  exposure to  range o f s a l i n i t i e s ,  and  hypertonic  dominant of  role  plasma  media. in  HCO3 . -  this This  shown t o be a m o d u l a t i o n o f t h e a c t i v e freshwater  contrast  and K e r s t e t t e r and M i z e in  These  S e c t i o n demonstrates that the  accumulation  in in  1978).  and r e t u r n t o w a r d s c o n t r o l  plays  the  process  over  near  -  accumulation  studies  water has a p o s i t i v e i n f l u e n c e o n t h e  C1 /HC03~  through  reported  1973; House and Maetz  Potts  performance  hypotonic,  regulation  not  the  the s a l i n i t y o f  hypercapnia.  hypercapnia  Additionally,  change  of  and  set i n this  regulatory  including  (1981)  data  from  with  and Eddy  hypercapnia  recovery  environmental  Cl~  during  entire  acid-base  McWilliams  with  are v a r i a b l e but  influx  acidosis.  (Cameron 1976; Wood e_t a l .  to the r e s u l t s (1976) who  rates  of  The r e a s o n s  Na  +  of Perry  f o u n d no and  for this  e_t a l .  significant  Cl~  in  trout  discrepency are  SECTION  3.  FURTHER ANALYSIS OF  THE  TROUT-SALINITY-HYPERCAPNIA EXPERIMENT  INTRODUCTION  This  Section  presents  concentration  and  the  Salinity  Trout  It  was  -  the  the  water  transepithelial  general  understanding  from  environmental  were  carried  blood for  and the  three  observed  HCO3  implied  that  played trout  an  C1 /HC03 exposure  to by  type  determine  passive or a c t i v e Second,  the  i n S e c t i o n 2. to  gain  more  d u r i n g exposure  t o and r e c o v e r y  Accordingly, three  analyses  of  role of  of  in  plasma  exposure  to  Data  was  The  the the  process  result  aim o f t h i s  carp  -  active  inhibited to  and  pH r e g u l a t i o n i n  that  hypercapnia  -  hypercapnia  from  being  The  C l  exchange  extracellular  indicated  manner  the experiment.  C1~/HC03~  acidosis.  process  these  a  in  in a qualitative  changes  during  accumulation.  whether  from  o f i o n s between  between  environmental  HCO3""  (TEP) d a t a  analyses  was a n a l y s e d  experiment  to  ionic  distribution  conditions  modulations  exchange  _  -  accumulation  -  the  t h e e l e c t r o c h e m i c a l g r a d i e n t between  salinity  this  hypercapnia  observed  for C l  important  in  _  First,  of  experiment  these  i n trout  correlation  plasma  potential  hypercapnia.  out.  water  of  the observed  blood  analysis  - Hypercapnia aim  about  and  further  during in  the  analysis  was  i o n g r a d i e n t s were b e i n g  maintained  processes.  apparent  p e r m e a b i l i t y o f the g i l l  epithelium  134  to  Na  and  +  variables data  Cl~  in  sets,  the  determined  Goldman e q u a t i o n  one  f o r each  concentrations the  was  of  Na  Cl~  expected of  the  according  Comparisons plasma  Nernst  of  to  these  of  active  concentrations  of  to e n v i r o n m e n t a l  their  The  the  enabled  and based  or  in  Cl~  by  which the  those  to  based A  of  the  during  and  measured  the  relative  the  observed  after  exposure  CI  value  i n the  Materials  p a r a m e t e r s were  values:  the  permeability of g i l l  The  of  S e c t i o n were  new  -  on  in this  been d e s c r i b e d  (PCI )  -  used  Several  and  Nernst  concentration  concentration  to  gradients.  METHODS  values  have  HCC>3~.  measured  equation.  of  and  and  distribution  maintaining  ion before,  calculated  some  (PNa+)  a passive  indications  processes  Methods o f S e c t i o n 2. on  a p p l i e d to c a l c u l a t e the  hypercapnia.  methods  measured  the  three  and  water  electrochemical  MATERIALS AND  +  these  The  i n c l u d e d TEP  concentrations  gave  each  was  b a s e d on  expected  concentrations  magnitude  Na ,  1943).  f o r p l a s m a and  equation  plasma c o n c e n t r a t i o n s  ions  +  which  for  solution. Finally,  Na  solving  (Goldman  salinity,  and  +  by  o f an  the Ratio  is  i o n d i v i d e d by  calculations 1.0,  Nernst  using  derived  Ratio  the  ratio  the the  t h e r e f o r e , means t h a t t h e  to for of  expected Nernst ion i s  135 distributed  according  to  the  existing  electrochemical  gradients. The were  equations  used  HCO3  to  ions  -  presented  calculate as  well  in  Appendix  the  Nersnt  as  the  I I and A p p e n d i x I I I  Ratio  f o r N a , C l ~ and +  relative  permeabilities,  PNa+/PHC0 -, P C I - / P H C O 3 - and P N a / P C l " . +  3  RESULTS  I.  ELECTROCHEMICAL GRADIENT ANALYSIS OF C I . -  Trout to  1% h y p e r c a p n i a  towards pH than  acclimated  those  There  were c h a n g e s  a  pH o v e r  t h e 24h e x p o s u r e  in  3mM.  i n plasma  plasma  correlated  was  ( F i g . 37 from  possible  blood  period.  i n t h e 100 and 300mM a c c l i m a t e d  in  changes  concentrations  of  greater  exposed  p l a s m a a c i d o s i s and t r e n d s  The d e g r e e o f r e c o v e r y  to the degree o f plasma H C O 3  Increases  gradient  plasma  acclimated  the  strongly  in  was  proportional  with  showed a n i n i t i a l  recovery  recovery  t o 3, 100 and 300mM N a C l and t h e n  in  plasma  t o water  in  At  3mM  were  most  plasma  C l ~  NaCl,  ( F i g . 38 a . ) .  exchange  associated  concentrations.  -  concentrations  a.b.c).  C1~/HC03~  HCO3  decreases  was  accumulation.  ion concentrations  HCO3"" to  -  fish  process,  the C l ~  On t h e b a s i s the  passive  136  Figure 37.a. R e l a t i o n s h i p o f plasma HCO3 t o p l a s m a (Na C l ) f o r three s a l i n i t i e s . Best f i t r e g r e s s i o n l i n e s represent data f o r t r o u t d u r i n g h y p e r c a p n i a and r e c o v e r y periods. Figure 37.b. Relationship of delta HC0 to d e l t a C l f o r p l a s m a d a t a from h y p e r c a p n i a and r e c o v e r y p e r i o d s . Figure 37.c. Relationship of delta HC0 to delta Na f o r p l a s m a d a t a from h y p e r c a p n i a and r e c o v e r y p e r i o d s . For Figures 37a.b.c. : 3 mM; 100 mM; 300 mM. A l l u n i t s i n mM. -  +  -  -  -  3  -  +  3  Correlation coefficient  Coefficients for best-fit Y = a + b * X a.  r  n  a  b  3mM  0.5281  64  0.9430  26.62  lOOmM  0.3676  57  0.7222  34.66  300mM  0.3503  85  1.4437  11. 27  b. r  n  a  b  3mM  0.5084  58  -0 .8470  -1.0584  lOOmM  0.3986  48  -0.5439  -0.0905  300mM  0.4139  74  -0.7463  -0.4684  c. r  n  a  b  3mM  0.0541  61  -0.2275  -1.0536  lOOmM  0.0255  59  -0.0424  0.1704  300mM  0.0361  74  0.0960  -0 .3126  line  138  F i g u r e 38. Hypothetical active and p a s s i v e C l ~ movements across the trout gill a t three salinities and t h e associated transepithelial potential (TEP) and plasma HCO3 accumulation characteristics. A l l data from trout exposed t o e n v i r o n m e n t a l h y p e r c a p n i a a t t h r e e water salinities. The symbols w and b r e p r e s e n t t h e water and blood compartments, respectively. S o l i d and b r o k e n l i n e s with a r r o w s r e p r e s e n t t h e n e t d i r e c t i o n a l movement o f i o n s across the g i l l epithelium which i s r e p r e s e n t e d by t h e double v e r t i c a l l i n e s . -  139  1Q0mM  cr  Cl"  .... _^HCO:  Cl"  NEAR ZERO  HIGH  POSITIVE  HIGH  300mM  t  > Passive  +—..W.~.~Z...  Active  140  efflux  of  blood  accumulation also  would  lOOmM, in  of  gradient  could  efflux have  HCO3 .  at  the  and  C l ~  the  passive  ( F i g . 38 b . ) .  the  this  of  mechanism,  related  inside-positive  that  analysis  the  CI  the  data  CI  and  -  lOOmM  trend  of  active fish  of  On  consequent a  HC03~  processes  exposed Although  of C l ~ .  At  o f plasma  At  the g i l l  to blood and  a  lost  300mM,  the  ( F i g . 38 c ) .  C1~/HC03~ instead  On  exchange o f gained.  would have e n h a n c e d C l ~ u p t a k e  possible  movements  accumulation  -  of  C l ~ shows  in  the  basis  of  passive  CI  -  p r e d i c t the g r e a t e s t r e d u c t i o n HCO3  accumulation  -  of HCO3  accumulation must  gradient  fish  300mM c a n n o t be a t t r i b u t e d t o p a s s i v e  the  loss  have  to hypercapnia the f i s h  concentration  p o t e n t i a l across  been  HCO3  and  s e t would  and  values  b a s i s as w e l l .  the  of  100  movements.  -  in  to  water  potential  degree  acclimated  TEP  efflux  movements.  -  have  from t h e w a t e r o n a p a s s i v e This  observed  would have had a m i n o r e f f e c t i n  movements  would  -  negative  large accumulation  zero  HCO3 from  passive  HCO3  the  I t i s u n l i k e l y that the  i t s small  near  salinity  f o r C l ~ was  basis  The  the  e f f e c t e d the observed  epithelium  gradient  The  -  C l ~ along  Similarly,  -  caused  f o r p l a s m a C l ~ , i n t h e same d i r e c t i o n a s  small  of  have  HCO3 .  enhanced  3 mM, was v e r y  passive  could  plasma  have  the  C l ~  -  in  i n 300mM.  was  in  3mM,  Since  i n plasma the  least  the observed  the opposite  accumulated  movements,  direction  the plasma H C O 3 i n -  a t 100 and 300mM.  i n 3mM c o u l d  have a c c u m u l a t e d  t h e plasma  141  HCO3  through  -  the  passive  studying  a  efflux  the  environmental processes  C1~/HCC>3 of  Cl~,  acid-base  fish  process  experiment  showed  linked  2 C , which  regulation  hypercapnia,  i n that  exchange  -  in  that  carp  active  i n freshwater e f f e c t e d  to  involved  exposed  to  i o n exchange  the accumulation  o f p l a s m a HC03~.  II.  PERMEABILITY  OF  THE  GILL  TO  Na+  (PNa )  AND C l "  +  (PC1~).  PNa  was  +  about  0.2  of  procedures  were  environmental relative  of in  -  initiated  hypercapnia  To  +  after  data the  values  beginning  were  changed  then  sampling  from  changed  large  to  about  was  greatest  to  changes i n just  after  v a l u e s a t t h e end o f t h e 24h similar  t r a n s i e n t s were  for  -  3  changes  seen  was s t a r t e d .  PC1 /PHC0 -  hypercapnia  control  Exposure  transient  to hypercapnia and  PC1~  experimental  a.b.).  which  -  of  39  slight  3  s e t showed  before  caused  to c o n t r o l  whereas  -  trout  a s m a l l e r degree,  PNa /PHC0  3-300mM  PHCO3  (Fig.  r e c o v e r y from e x p o s u r e  Both  within  (Fig.  values to large 4 times c o n t r o l  39  the  t h e f i r s t 2h a.b.).  Both  negative values values a t the l h  period.  PNa /PHC03~ +  and  PHCO3  and d e c r e a s e d  exposure.  and  0.4  permeabilities  exposure  after  about  100-300mM  calculated showed  for  similar  the values  data as  sets: well  as  3-100mM trends  142  Figure 39.a. and b. P e r m e a b i l i t e s of Na (PNa /PHCC>3 ) and C l (PC1~/PHC03~) relative to HC0 for the gill of trout during and a f t e r exposure to environmental hypercapnia. These two p a r a m e t e r s were derived by s i m u l t a n e o u s s o l u t i o n o f t h e Goldman e q u a t i o n for three unique pairs of data from t h e t h r e e water salinities investigated. The t h r e e s e t s o f s o l u t i o n s from this analysis a r e r e p r e s e n t e d by t h e t h r e e l i n e s on e a c h graph. +  +  -  -  _  3  143  CUMMULATIVE TIME (h)  144  throughout  the  experiment  values  derived  from  during  exposure  to  compared  lowest  They  were  when  decreased  data than  There  was  They were  higher  from lower  during recovery  sets  3-300mM  those an  data  in  the  ( F i g . 39 b . ) .  which  were  gradually  D e v i a t i o n s from t h e  hypercapnia  3-100mM  data  for  were  i n the  set.  During  the  100-300  and  i n v a l u e and t r e n d a n d were f o r t h e 3-300mM d a t a s e t .  PC1 /PHCC>3 -  values  -  i n the  f o r t h e 3-100mM d a t a s e t . values  averaged  ( F i g . 40 a . b . ) .  exposure  values  The d i f f e r e n c e s  period.  values c a l c u l a t e d  +  period  the  -  s e t was u s e d and  hypercapnia  similar  increase  data  set.  calculated  were  PC1~/PHCC>3  s e t was u s e d  during  for  PNa /PCl~  after  to  about  This ratio  environmental  occurred during hypercapnia  2.5  f o r the  was c h a n g e d d u r i n g  hypercapnia.  Changes  were a b o l i s h e d by t h e e n d o f  24h e x p o s u r e . The  and  values  hour o f r e c o v e r y  The  the  the  PC1~/PHC03~  lower  which  set.  hypercapnia,  during  values  3  and  and  t h e 24 h e x p o s u r e  control  PC1~/PHC0 ~ recovery,  when  equal  over  of  to  f o r t h e 100-300mM  approximately  control  data  t h e 3-100mM d a t a  highest  intermediate  first  3-300mM  hypercapnia  exposure  were  3-300mM  the  These d i f f e r e d  t o 3-300mM v a l u e s .  During  range  ( F i g . 39 a . ) .  ratios  3-300mM  hypercapnia average  of  PNa /PCl~  were  similar  +  period.  control  in  Those  values  f o r the their  values  after  data  sets:  response decreased  onset  of  100-300mM  during relative  hypercapnia  the to and  145  Figure 40.a. Permeability o f Na r e l a t i v e to C T f o r the trout gill during and a f t e r exposure to environmental hypercapnia. Calculated by d i v i s i o n of PNa /PHC03~ by PC1~/PHCC>3"; see figure legends 39a. and 39b. above. Figure 40.b. D a t a o f 40.a. r e f e r e n c e d t o t h e a v e r a g e c o n t r o l values. Exptl.(PNa /PCl )-Ave. Control(PNa /PCl ). +  -  +  +  _  +  -  146  CUMMULATIVE TIME (h)  147  increased time.  gradually In  increased  contrast,  relative  hypercapnia  by  t h e 24h s a m p l i n g  of  and  100-300mM  and  than  control  was  caused  +  PNa /PCl~ recovery  from  +  PNa /PCl~  this  the  hypercapnia  than  this  values  set.  The  values  The e n d f o r the higher This  relative  period.  stayed  change,  The  sampling  to  lower  periods  relative near  PNa /PCl~  the  recovery ratio  control  ratios  +  +  near  levels.  values  PNa /PCl~  to  between 2-4h when  in  f o r t h e 3-100 mM d a t a s e t was due t o  sharply 1 h after  increased  onset  o f the experiment.  initial  which  during  set  values  t o i n c r e a s e and remain  _  in  after  ratios  +  PC1 /PHC03~  for  data  back t o c o n t r o l  control  f o r the remainder  values  -  decreased it  to near  the  lower  3-100mM  f o r a period  PC1~/PHCC>3~  PNa /PHCC>3  control  decline  t h e 24h s a m p l i n g  immediately  declined  time e x c e p t  levels  increased  Other  levels  3-300mM d a t a s e t s  values  +  data  generally  values  -  control  by  f o r the  +  caused  by  PNa /PHCC>3  levels  PNa /PCl~  was a t r a n s i e n t hypercapnia  control  to  of  there  to  period  to levels.  remained  f o r t h e 3-100 mM  f o r t h e 3-300 mM d a t a s e t  the end o f h y p e r c a p n i a a f t e r  control  near  levels  f o r the remainder  which o f the  experiment. The mM  difference  in  s e t and  the  data  suggests over of  that  mM.  +  range  values  other data sets,  the apparent  the s a l i n i t y 3-100  PNa /PCl~  between  3-300 a n d 100-300 mM,  p e r m e a b i l i t y to these  investigated  The s i m i l a r i t y  t h e 3-100  i o n s changed  and o c c u r r e d i n t h e range  i n t r e n d s i n d a t a whenever t h e  148  300  mM  that on  data  some  s e t was  aspect  the parameters  III.  period and  averaged  about  34, 1.3 and 0.7 f o r f i s h  respectively in  this  and  period  average  and  0.58  a.b.c).  f o r 3mM,  hypercapnia  were  variability  masked  the  significant which end  there of  the  was  a  24h.  average  i n 3mM,  to hypercapnia recovery  in all  p e r i o d were  except  f o r the  300mM g r o u p o f f i s h  towards c o n t r o l  ratios  values.  f o r p l a s m a C l ~ were a b o u t 20,  lOOmM and 300mM r e s p e c t i v e l y change  i n this  periods.  value  ratio  The r a t i o s  higher  during  than  during  a t lOOmM d u r i n g  control  values but  T h e r e was a g e n e r a l  trend  recovery.  was a  Nernst  There  in  recovery  back t o t h e c o n t r o l  ratio  The e n d o f h y p e r c a p n i a d e c l i n e d t o the c o n t r o l ratios  ( F i g . 42  a t 3mM  decline  Nernst  lOOmM  T h e r e was a g e n e r a l  the  the d i f f e r e n c e s .  initial  control  no c o n s i s t e n t d i f f e r e n c e from  generally  control  which s u b s e q u e n t l y The  recovery  the  showed  T h e r e was l i t t l e and  during  trend  Nernst  hypercapnia  during  +  during exposure  Values  where t h e r e was a c l e a r  Na  ( F i g . 41 a . b . c ) .  ratio  variable  hypercapnia  towards  calculations.  f o r plasma  generally  0.85  had a s t r o n g i n f l u e n c e  ANALYSIS  salinities.  The  salinity  ratio  increase  the  high  Nernst  300mM  three  of this i n these  NERNST RATIO  The  included i n the c a l c u l a t i o n s i n d i c a t e s  a t 300mM value  caused  after by t h e  an i n c r e a s e  value.  f o r plasma  HCO3  -  were  about  149  Figure 41.a.b.c. Means + S.E. of ratios o f measured t o expected plasma Na concentrations f o r t r o u t d u r i n g and after exposure t o e n v i r o n m e n t a l h y p e r c a n i a a t t h r e e water salinities. 'Expected' v a l u e s c a l c u l a t e d from the Nernst e q u a t i o n . F i l l e d c i r c l e s a r e the a v e r a g e c o n t r o l p o i n t s . +  150  151  Figure 42.a.b.c. Means + S.E. of ratios o f measured to expected plasma C l ~ concentrations f o r t r o u t d u r i n g and after exposure to e n v i r o n m e n t a l h y p e r c a n i a a t t h r e e water salinities. 'Expected' v a l u e s c a l c u l a t e d from the N e r n s t e q u a t i o n . F i l l e d c i r c l e s a r e the a v e r a g e c o n t r o l p o i n t s .  152  153  Figure 43.a.b.c. Means +_ S.E. of ratios o f measured t o expected plasma HC0~3 concentrations for trout during and after exposure to environmental hypercania at three water salinities. ' E x p e c t e d ' v a l u e s c a l c u l a t e d from the Nernst equation. Filled circles are the average c o n t r o l points.  154  155  8,  13,  and  a.b.c).  Unlike  Nernst both  13 f o r 3mM,  ratios  the  times  and  during  near-control  ratios  f o r HCO3  hypercapnia  4-5  lOOmM and 300mM r e s p e c t i v e l y  showed  -  +  and C l ~ , t r e n d s  consistent  recovery periods.  the  levels  for Na  hypercapnia  ( F i g . 43  trends  The r a t i o s  period  and  i n the during  increased  returned  to  by t h e e n d o f t h e r e c o v e r y p e r i o d .  DISCUSSION  The  analyses  of  qualitative  of  Na  to  and  +  three  i n this  statements  Section provide a basis for a series about the nature  C l ~ between water  o f the d i s t r i b u t i o n  b l o o d and w a t e r  salinities  and  i n trout  exposed  to  acclimated  environmental  hypercapnia. Active HCO3  -  HCO3  -  all  rather  exchange in  of  showed  that  regulation but  the  of  also  higher  only  the  to  1%  accumulation environmental  investigated. hypercapnia was t h i s  involving  This study  f o r plasma in  process salinities  this  HCO3  of  Section  of this  suggest  was s t i m u l a t e d i n t r o u t in  order  to  hypercapnia a t with the  S e c t i o n 2 which important i n  process in  that  i n carp t h a t was  fresh  the  acclimated  accumulate  plasma  acidosis  accumulation  -  of  exchange p r o c e s s  i t was t h e i n h i b i t i o n  a C l ~ for  agrees  p l a s m a pH d u r i n g a h y p e r c a p n i c  that  analyses  exchange  carp  not  responsible The  exposed  salinities  results  passive processes  effected  trout  three  than  water.  C1~/HC03~ t o t h e two  t h e p l a s m a HCO3-";  156  that  is,  an  from  blood  to  water  from  water  to  blood.  C1 /HC03~  active  step and  exchange  -  arcticus  played  an  hypercapnia  The  ATPase  enzyme i n t h i s deviations  conclusion  accumulation acclimated during were  of at  when  The  an  of  the  t o a s t e p change  at  a l l sampling  the  Nernst  that  HCO3  fish  active  regulation  of  subjected  to  in  temperature, in Section  of  HCO3  as  well  HC03~  reported  accumulation  was of  an the  ratios  100  and  for fish  300  in  in fish  trout  for  HCO3""  fish 2.  NaCl,  both  consistent at  the  mM  with three  Plasma  a c c l i m a t e d a t 100  were g r e a t e r t h a n  periods  a c c l i m a t e d to 3  accumulation Section  mM  in  in  for experimental  are a l s o  in  support  involved  hypercapnia  These r e s u l t s  similar which  as  also  -  were  Nernst  a c c l i m a t e d to  times.  for HCO3  during  -  salinities.  salinities  w h i c h would be  processes  were g r e a t e r t h a n r a t i o s  degree  gill  -  Thvmallus  discussion  ratios  active  plasma  for fish  which  both  in  balance  in  periods  of  NaCl,  role  gill  of  case.  of  all  control similar  the  the  of C l ~  the p u b l i s h e d i n f o r m a t i o n f o r the e x i s t e n c e o f  stimulated  regulated  the  in  or  uptake  (1976) showed t h a t  w h i c h d i s t u r b e d p l a s m a pH.  presented  anion  i n the l i n k e d  important  environmental  2  also  process  acid-base  of  i n v o l v e d i n the e f f l u x  Cameron  extracellular  both  was  HCO3""  and  300  the a c c u m u l a t i o n  mM  at 3  mM. The mechanism  proposed in  fish  active  nature  acclimated  of to  the the  C1~/HCG"3~ three  exchange  salinities  is  157  further  supported  The  Nernst  acclimated and  300  hand, for  in  another  ratios  for  Cl~  near  unity  3 mM  mM.  lowest  those  and  The  were  Nernst  by  Nernst f o r the  a s p e c t o f the N e r n s t  a c c l i m a t e d a t the is  defined  as  concentration  of  an  over  passive  distribution  proposed  link  exchange ion  The  for  that  t h e r e were two between  acclimated  t o 3 mM  the  consisted  other  both  of  Nernst were  which  passive  300  mM  close NaCl  less  an  active f o r each  HCO3  One  group  positive  a c c l i m a t e d t o 100 near  at  than  100  t o one  showed one.  higher  one.  than  in and  fish 300  relative  Nernst The  mM,  all  -  Both  was  fish  ratios  ratios  that  300  Na  and C l ~  +  expected  acclimated  t o 3 mM.  fish  for  reciprocal  Na  ratios  and  ions the  existing For  of Na  a t 3 mM. +  mM,  on  to  Nernst  and  and  according  to the  the  with r e g a r d to i o n  water.  distribution  gradients  acclimated were  ratios  plasma  water,  and  of f i s h  the  a c c o r d i n g to a  by  -  Cl~  w h i c h showed l a r g e  maintained  electrochemical  Cl~  Because  Na , +  fish  highest  o f t h e measured  HCO3  100  other  showed t h a t p l a s m a c o n c e n t r a t i o n s o f t h e s e  actively of  and  results  and  o f the  showed  ratios  basis  NaCl  the  the o b s e r v e d  groups  blood  on  -  expected  and  predict  ratios  HCO3 ,  i o n b e t w e e n b l o o d and  other.  distribution  were  that  w h i c h complemented e a c h Nernst  fish  high s a l i n i t i e s .  Cl~  would  for  the r a t i o  the  between  process  suggest  fish  of  f o r the  a c c l i m a t e d t o 3 mM  two  ratio  highest  data  f o r those a c c l i m a t e d a t  ratios fish  ion  were  ratio  +  Fish  and in  C l ~ which  o f the Nernst  ratios  158  during  the  control  concentrations than  While and least  even  pumping  exchange to  100  is  synthesis shown  300  in  in  ion the  by  fish  Lee  a a  There  i s , however, no  rate  times  of  a  CT~ lower  passive  evidence against  acclimated  in  time  course  these  plasma  of  An  in  100  such  processes.  ions,  in  the  fish  acclimated to  body  accumulation  as  have b e e n shown t o  expected  as  of  i s the The  order  to  develop  to a  the  stimulus  experiment.  response. cellular  C1~/HC03  mM  regulate  would be  in this  involvement  300  Callinectes C1~/HCC>3~  response  f o r such a  active  and  -  ion  the  exception  in  for  f u n c t i o n to  be  several  hypertonic  HCO3  at  recruitment  crab. in  cells  mM.  are  ATPase  First,  would n o t  i n 100  cells,  there  blue  w a t e r s and  disturbance  fish  mM,  i s not  development  argument  chloride  of c e l l u l a r  chloride  they  recruitment  in  and  +  s t i m u l a t i o n of C l ~  waters,  which  similar  their  acid-base  of  to  i n 300  dilute  i n hypertonic  an  molecular  basis  (1982) i n t h e  medium  acclimated of  Another  Na  1.72  anion-stimulated  Since  from  changes  new  concentrations  possibility  uptake  -  involvement  to  mM.  fish  and  the  i n t h i s experiment.  NaCl,  fish  in  of  observed mM  1.43  a s c r i b e the  acclimated  acclimated  showed  body  to  HCO3  fish  fluids,  develop  tempting  t h a t argue a g a i n s t  sapidus,  on  plasma  ions.  the  as  that  about  expected  associated  for  reasons  of  it  the  show  were m a i n t a i n e d  concentrations  distribution  or  periods  exchange  in  difference in  the  time of  and  -  course hours,  of  net  strongly  159  suggests  that  mechanisms  rather  molecular days.  there  was  than  structures, It  is  possible  present  sufficient  to  both  salinities.  HCO3  in  -  the  salinity  the  requirement of  The  conger  for  more  plasma were  Na  +  and  _ 3  sets  for  when  as  spans  transport  graded  the t r a n s p o r t  i n 100 mM  observed  -  NaCl  o f plasma  Section  2, a c c o r d i n g t o  possibility  which p r e c l u d e s  cell  C l ~ and  HCO3  development -  to e f f e c t the  concentration  changes.  a c c l i m a t e d t o t h e new s a l i n i t i e s a  was  i n fish at  accumulation  in  and  i n the order o f  of  fish  HCO3  chloride  the  period  times  shown  less  too  short  permeable  relative  levels  f o r such  by  exposure  derive  these  g r e a t e r than  data  d a t a s e t show t h a t PC1~/PHCC>3~  large  fish  when  -  of in  different  values.  the average  s e t was u s e d .  explained.  HCO3  t o h y p e r c a p n i a and  i n these p e r m e a b i l i t i e s be  in  the d i f f e r e n c e s  to  than  permeabilites  differed  _ 3  during  slightly  cannot  and  are The  3-300mM  fluctuations  PNa /PHCC>3~  Cl~  used  were  t h e 3-300 mM +  plasma  PC1-/PHC0  were  permeabilities  sampling  of  reported  salinities  recovery  values  gill  The  gill.  different  data  time  stimulation  hours,  and  +  the  PNa /PHC0  large  three  existing  r e c r u i t m e n t to take p l a c e .  across  during  not  of  development o f c e l l u l a r  supports t h i s  for  than  Both  the  the  conger,  water  cellular  in  that  accumulate  the  degree  possible  which r e q u i r e  mechanisms  high  stimulation  control  The r e a s o n s f o r  d u r i n g the i n i t i a l  The  PNa /PCl~ +  fluctuations  occurred  Both  in  data  i n both  concert;  160  i.e.  PNa /PCl~  values  +  PC1~/PHC03~ used.  was  While  it  permeabilities these  data  reduced  when  is  possible  were  were  not changed  useful  trout  than  to  gill  in  C l  permeability  under  -  and  Masoni  Potts  (1978)  studies  gill  Na the  equal  in  distilled larger Na  the  Eddy  water  (1975) Na  +  than  implies  While  that  ions  and  + +  in  ions  than in  the  this  study.  estimate  of  passive  a c t i v e p r o c e s s e s under  leak  steady  of state  The +  Na  +  was  (1976)  and  ( M g ) had + +  a  greater  to  mainly  the  gill.  more p e r m e a b l e for  about  Na  +  of  being  conditions.  were  1.5-2.0 f o r  agreement o f  PNa /PCl~  by  fish  water  knwon  ratios  f o r C l " by  the  suggests a  t o be  and  approximately  c h a r a c t e r i s t i c s of  Nernst  those  of  fresh  are  +  by  supported  Masoni  t o be  This  -  Na  McWilliams  magnesium  PCI .  The  -  by  I s a i a and  f o u n d brown t r o u t g i l l s  range  the  Ca  to  have b e e n r e p o r t e d  anquilla,  divalent  Cl .  the  Relative  permeability  these  permeability  greater  with  of  than  to  salinity  estimate  by  also  aspects of  These r e s u l t s a r e  Cl~.  PNa  ion  individual  conditions.  e e l and  greater  in  since  was  observed r e s u l t s ,  some  unity the  Anguilla  +  how  set  t i m e s more p e r m e a b l e  a  changes on  passive  consistently the  eel,  effect  affect  to  say the  state  near  permeabilities  permeability  +  to  than  +  give  2.5  for  showing  to  data  +  f o r brown t r o u t .  previous  showed  (1976)  3-100mM  little.  PNa /PCl~.  steady of  very  explaining  about  estimates  Isaia  to  is  the  to  subsequently c a l c u l a t e d r a t i o The  changed  about  this 2.5  compensated  161  The  relative  changed  with  recovery.  salinity  An  from  Section and  of  these  therefore  observed +  may  lead  the  water  with  not  value,  -  to  PNa /PCl~ in  either  can  be  in  the  is  -  0  control values slightly  i n this  3-100  +  to  a  +  in  values  mM  Na  how  toward this i s  may be i n c r e a s i n g  +  leak of Na  the a c t i v e  +  be v e r y  uptake s t e p i s  sensitive  PC1 /PHC0 3  level  early  low  in  data the  The  t o changes  values.  -  from  This s e t which  hypercapnia  because  the  increase  in  greater  than  the  increase  in  were  therefore  only  based  on  parameter.  data  compared  reduced  freshwater  concentrations  the p a s s i v e  to  are  consistent  trends  It is  An i n c r e a s e i n t h e fish  of  while  or  Conclusions  +  PNa /PCl~  for  leak  tend  PNa /PHC03~.  The  place.  i n t h e two p o i n t s i n t h e 3-100 mM  Those  PC1~/PHCC>3  also 3  period.  Cl~.  m i g h t be o c c u r r i n g s i m u l t a n e o u s l y .  +  below  and  +  mechanisms by w h i c h t h e  plasma N a  rates,  PNa /PHC0 -  seen  i s the c o n s t a n t  permeabilities i n this  the  example,  passive  events  values  +  Na  and d u r i n g  represent apparent p e r m e a b i l i t i e s ,  t o w a t e r may be c o n s t a n t or both  +  to hypercapnia  relative  o f i o n s took  uptake  +  PNa /PCl~,  However, i t d o e s n o t s p e c i f y  The Na  gill,  calculations  indicate  reductions  the  both  of  for  values.  normal  reduced  fall  do  place.  blood  to  -  terms  distribution  PNa /PHCC>3  taking  HCO3  i n these  calculations  that  of  when e x p o s e d  assumption  permeability clear  permeability  set to  showed  control  PC1~/PHC03~  generally values.  values  higher  T h i s was due relative  to  162  corresponding Cl  +  permeability  -  set  by  the  experiment, in  PNa /PHCC>3  trout  can  assuming  Cl  been  may  any  have  reduced  at  that  in  similar 3  the  fluxes  carp  and  exposed  basis,  the  apparent  to the reduced  permeability of  uptake  o f C l ~ without  The  mM  data  PNa /PCl~  reduction  PC1~/PHC03~  that  data  set.  over  the  values  aspect Cl~  this  contrast, 3  hypercapnia  300  would  result  in  over  PNa /PHC03~  mM  to  i t  such  result was  in  such  influenced  was  It  was  the  compared  to  the  the  100-300  in  -  low  as t h e a c t i v e  case  -  PNa /PCl~ +  salinity.  this  mM  PC1 /PHC03  In spite  to s p e c u l a t e t h a t  apparent the  b o t h showed  increased  resulted  i s tempting  decreased as  values  i n t h e 3-300 mM  salinity,  water,  the  i t  which  +  blood  +  +  and 3-300 mM  hypercapnia.  PNa /PHC03~  PNa /PHC0 ~  the  from  during  produced  differences,  of  data s e t .  in  In  during  these  100-300  values  +  greater  sets,  hypercapnia  to environmental  b l o o d t o water.  two  data  would have o c c u r r e d  mM  due  apparent  salinity  change i n t h e p a s s i v e l e a k from  lower  of  observed  that  acclimated On  This  be e x p l a i n e d f o r t h e 3 mM  C l ~ flux  hypercapnia. -  values.  -  some  efflux of  parameter.  This  PC1~/PHC03~  value  for  the  300  mM  SECTION  CATECHOLAMINE  RELEASE  IN  4.  ACID  INFUSED  TROUT  164  INTRODUCTION  An  increase  the  acute  and  can  and  Fromm  1975; and  in  stress have  circulating  response  varied  1969;  i n fish  effects  Bergman,  P a y a n and G i r a r d  Olson  Perry  gill  (Isaia  ejt a l _ . 1978).  have  to t h i s  1986)  Fromm  1978),  1967)  (Richards  1974; Wood 1974,  ionic  fluxes  (Girard  1981), and g e n e r a l p e r m e a b i l i t y o f t h e Acid-base  d i s t u r b a n c e s a r e no  phenomenon and p l a s m a c a t e c h o l a m i n e s  been observed  (Primmett  flow p a t t e r n s  and  1977; B o o t h  1977;  exception  (Nakano and T o m l i n s o n  on b l o o d  Payan  fish  catecholamines c h a r a c t e r i z e s  to increase i n response  ejt a l _ . 1986;  to severe  i n fish metabolic  Ye, i n p r e p . ) and r e s p i r a t o r y  (Perry  acidoses.  Several  effects  of  catecholamines  processes  across  of  a r e known.  In freshwater,  C1~/HC0 ~  exchange  fishes  inhibits  Girard  t h e e r y t h r o c y t e membrane  3  stimulates  Na /H (NH4 ) +  and  +  Payan  modulation  of  acid-base  balance  fluids. increased (Cossins  The in  on  1977;  these  Payan  and g i l l  e_t  the  1978).  vitro  and R i c h a r d s o n  with  pH  of  the  1985).  et_  The  intracellular  intracellular  epithelium  a l . . 1984)  (Payan  i o n exchange p r o c e s s e s of  transfer  beta receptor s t i m u l a t i o n  (Perry  exchange  +  ion  the  addition  This serves  and  a l . . 1975;  result  of  the  i s to r e s t o r e the and  trout of  extracellular red  cell is  catecholamines  to o f f s e t  t h e Bohr  165  and  Root  oxygen  shifts  that  carrying  shown  in,  which  capacity  vivo  for  causes  are  a  of  trout  plasma  e_t al.. (1983) a l s o  similar  magnitude  to  It  is  release  in  response  experiments  was  other  of  aspect  The  two  tested trout  during  the  thus  of  the  a  i n the of  the  to to  This  e f f e c t has  (Primmett that  et. al..  epithelium  the  acid-base  metabolic the  been  exercise  al_.  1986).  a metabolic acidosis  Primmett  gill  et  the  of  (1986) trout  induced that  disturbance the  acidoses  of  was  in  the  catecholamine in  the  acid-base disturbance  or  above to  some  exercise.  vivo  hypothesis  significance and  the  thus p r o t e c t s  c l e a r , however, whether  due  in.  the  increase  not  blood.  showed  with c o r r e c t i o n of  blood.  and  a f t e r a bout of a n a e r o b i c  that  i o n exchanges a c r o s s  associated  the  acidosis  Holeton  net  exhibited  bout  experiments that of  a c i d load this  reported  catecholamines  anaerobic of  in  the  release  blood  are  exercise and  this  that  Section  released  in  i n response  to  the  functional  i s to m a i n t a i n e r y t h r o c y t i c  oxygen c a r r y i n g c a p a c i t y  of  i n face  pH  the  blood  of  of  experiments  with  measurements  and  plasma a c i d o s i s .  MATERIALS AND  This identical slightly  Section  describes  treatment different  but sampling  two with  METHODS  series  different  times.  The  first  experiment  was  166  designed release  to with  regulation had  on  investigate acid  and  the  infusion  the second  possibility and  i t s role  investigated  of  catecholamine  in  effects  red  this  cell  pH  treatment  ionoregulation.  EXPERIMENT 4A.  CATECHOLAMINE RELEASE AND  RED  C E L L pH  REGULATION :  ANIMALS :  Rainbow g  were  in  trout,  obtained  fiberglass  Salmo g a i r d n e r i . from a c o m m e r c i a l  tanks  Vancouver  tap  water  Fish  fed  ad.  were  trout  pellets.  supplied (8-10°C;  221.5 t o 460  h a t c h e r y and h e l d  with pH  from  flowing  6.9-7.1;  outdoors  dechlorinated CaC03  4  ppm).  from  self  feeders containing dry  In the l a b o r a t o r y ,  fish  were k e p t  at  1ibitum  weighing  perspex  aquaria  10°C  and  starved  surgical  o p e r a t i o n s and e x p e r i m e n t a t i o n .  for  48  i n blackened h  prior  to  SURGERY :  The  dorsal  chronically Smith  aorta  of  a l l experimental  animals  were  c a n n u l a t e d w i t h PE50 c a n n u l a s by t h e p r o c e d u r e s o f  and B e l l  (1964)  167  PROTOCOL :  Fouteen cannula made an  animals  with  up  5  served  as  were  controls. blood  infusion  and  time  for  oxygen  the  remainder  500  periods  Portions  total  of  of  anaerobically.  The  resulting  chilled  syringe  and  transferred  storage  at  catecholamines.  blood  c e n t r i f u g a t i o n was u s e d  solution  treated i n alone  and  about 5 minutes.  60  before  the  and  120 min  sample were  analyzed  sample plasma to  an  red  was  was  p l a s m a was  The  aortic  and h e m o g l o b i n c o n c e n t r a t i o n ;  the  for  fish  taken  30,  each b l o o d  content  Stored  were 5,  N HC1  the s a l i n e  averaged  ul  of  -40°C.  the d o r s a l  Five  i n f u s e d with  of  post-infusion. pHe,  saline.  I n f u s i o n times  samples at  through  body w e i g h t o f a 0.05  physiological  fashion  Arterial  infused  ml*Kg~l  i n 120 mM  identical  were  taken  up i n t o  Eppendorf subsequently  cell  for intracellular  centrifuged a  vial for analyzed  fraction  after  pH measurement.  ROOT EFFECT DETERMINATION :  Blood dorsal  samples  aortic  immediately  cannulas  pooled,  tonometer,  and  containing  either  Wosthoff  were  pumps).  drawn f r o m q u i e s c e n t as d e s c r i b e d above.  fish  Blood  fitted  with  samples  were  t r a n s f e r r e d t o an i n t e r m i t t e n t l y  equilibrated 0.2  or  After  against 1.0  30-40  %  C0 min  h u m i d i f i e d gas in  2  of  air  rotating mixtures  (mixed  tonometry  with  a t 10°C,  168  blood  was  syringe cell  taken  (Hamilton) pH,  into  and  a  positive  and measurements  hemoglobin  Adrenaline the  up  displacement  were made o f b l o o d  concentration  noradrenaline  and  stages  oxygen  and r e d content.  c o n c e n t r a t i o n s were measured i n  p l a s m a o f a 1 ml sample o f t h e b l o o d  initial  gas-tight  o f the e q u i l i b r a t i o n  pool  taken  during the  procedure.  ANALYTICAL PROCEDURES :  The  procedures  determinations described  in  concentrations bulletin the  no.  as  whole  well  General were  as  blood for  Materials  determined  525).  Lex-02~Cont  USA).  for  Blood  apparatus  Pc02  and  using high  pressure  described  by Woodward  red  cell  measurements  Methods.  pH are  Hemoglobin  s p e c t r o p h o t o m e t r i c a l l y (Sigma  oxygen c o n t e n t s (Lexington  P l a s m a a d r e n a l i n e and n o r a d r e n a l i n e  measured  and  liquid  were measured  Instruments,  Mass.,  concentrations  c h r o m a t o g r a p h y by a  with  were  method  (1982).  S T A T I S T I C A L PROCEDURES :  The was with  Student's  used a  described  to 5  %  t test  discern level  by l i n e a r  (paired  statistical  of rejection.  and u n p a i r e d  as a p p r o p r i a t e )  significance Various  regression analysis.  data  between means s e t s were  also  169  EXPERIMENT 4B. ION REGULATION AFTER ACID INFUSON :  to  The  Materials  and Methods f o r t h i s  that  f o r t h e e x p e r i m e n t 4A.  Only  e x p e r i m e n t were s i m i l a r  the d i f f e r e n c e s a r e noted  below.  ANIMALS AND PREPARATION :  The 378  Rainbow t r o u t , Salmo g a i r d n e r i , w e i g h e d between 280 t o  g.  All fish  catheters  through  recirculating were  made  sphincter the  for  a  system  tied  was  carried  waste.  fish  constituted ions  the  The  entire  of  the f i s h .  o f the catheters  j u s t past the  i t i n place.  As a n a d d i t i o n a l  head r e l a t i v e  t o t h e two f l a p s w h i c h t h e c a t h e r and was  positioned  to i t s o r i g i n .  chamber c o n s i s t e d  and  out  to the outside o f  The end o f t h e c a t h e t e r  a  urinary  was a p p l i e d  were a n c h o r e d  connected  with  The u r i n a r y  p a p i l l a e and wrapped a r o u n d  negative  single  glue  secure  sutures  experimental  in  to  i n place.  non-volatile water  system  to  urinary  a slight The  urine  Tissue  wall  cotton  the  firmly  which  fitted  o f PE 60 w h i c h were l e d up t h e u r e t e r muscle.  security,  at  water  catheter  cover  i n t h i s e x p e r i m e n t were  of a black  p e r s p e x box  t o a pump and a r e s e r v o i r .  recirculating chemicals.  An  closed  system  aeration  stone  The for mixed  r e s e r v o i r and e q u i l i b r a t e d the system w i t h a i r .  water volume was a p p r o x i m a t e l y I t was hoped  that  this  10 t i m e s  the weight  s y s t e m would be s e n s i t i v e  170  enough  to  water. not  reflect  The  during  t h e n e t t r a n s f e r s o f i o n s between f i s h and  s y s t e m w a t e r was renewed s e v e r a l t i m e s  the s h o r t experimental  protocol described  a day b u t below.  PROTOCOL :  Eight  animals  injected  with  connected  1  collected  the  saline.  the  and  and  dorsal  bridge  was  5  aortic  for a  were sham  cannula  was o b t a i n e d  b r o k e n a n d a 300 u l whole  which  served  was  transepithelial  f o r t h e measurements b e l o w .  infusion,  a t time  HC1  Once s t a b l e r e a d i n g  connection  before  with  The  KCl/agar  was c o l l e c t e d  value,  infused  (TEP) r e a d i n g .  min), the  sample  the  to  potential  were  (<  blood  Samples were  as another  control  p e r i o d s o f 5, 15, 40, 60 a n d 120 min a f t e r  infusion of either  acid  or saline  was c o m p l e t e .  MEASUREMENTS :  Plasma  ion  concentrations  Mg  were  determined  and NH4  concentration  +  Solarzano Ion sample were  + +  Na ,  C l  +  -  , K ,  spectrophotometry.  measured  by  a  Ca  +  + +  Plasma  modification  o f the  method. concentrations by  above  were  spectrophotometry.  determined  (Wosthoff  was  by  of  pump)  by and  equilibrating determinig  measured  Water  HCO3  each the  f o r each -  sample  TC02  water  concentrations with  content  1 % CO2 of  that  171  sample. these  Water values  and  concentration  HCO3  the  solubility  was  constant  calculated  using  from B o u t i l i e r  §_t a l .  (1985). TEP  values  described as  in  described  were  determined  General  according  M a t e r i a l s and  in Section  3.  to  Methods and  the  methods  Nernst  were c a l c u l a t e d f o r p l a s m a  ratios  ions.  STATISTICS :  Paired  and  statistical rejection  unpaired  S t u d e n t ' s t t e s t s were u s e d  significance  between  means  using  5  to  discern  %  as  the  level.  RESULTS  EXPERIMENT 4A.  The  CATECHOLAMINE RELEASE & RED  maximum  sampling  period  Plasma  pH  fell  levels  at  that  adrenaline  in  which 0.167 time  was  pH  declined  of  the  fish,  red c e l l  the pH  occurred  REGULATION  i n the  first  i n f u s i o n o f a c i d ended.  that of p r e - i n f u s i o n c o n t r o l  a).  At  increased only  at  pH  5min a f t e r  ( F i g . 44  increased slightly  plasma  u n i t s below  concentrations  noradrenaline  14  change  C E L L pH  the by  slightly  same t i m e as  6 - f o l d whereas p l a s m a ( F i g . 44  5min mark. ( F i g . 44  increased  plasma  relative  d).  Red  b) .  In 4  cell out  to p r e - i n f u s i o n  172  F i g u r e 44. Means +_ S.E. o f a. A r t e r i a l p l a s m a pH ( p H e ) , b. red c e l l pH (pHi), c . mis 0 bound p e r gram o f haemoglobin (mis 0 *gHg ) and d. plasma catecholamine concentrations following intra-arterial infusion of a 5 ml*Kg body w e i g h t 0.05N HC1 s o l u t i o n of 120mM s a l i n e i n 14 r a i n b o w t r o u t . Shaded v e r t i c a l b a r represents the i n f u s i o n time period. Shaded h o r i z o n t a l bars (labelled shams) r e p r e s e n t £ 1 standar e r r o r o f the combined mean d a t a o f f i v e c o n t r o l e x p e r i m e n t s d e s i g n e d t o examine the i n f l u e n c e o f the s a l i n e v e h i c l e alone ( i . e . 5 ml*Kg o f 120mM saline injections). The s h a d i n g i s representative o f +_ 1 S.E.M. f o r both a d r e n a l i n e and noradrenaline levels in the bottom panel. C = p r e - i n f u s i o n c o n t r o l . (N = 1 4 ) . 2  - 1  2  _ 1  - 1  173  Time  (min)  174  levels  in  control  the  face  animals  of a f a l l  there  was  i n p l a s m a pH.  no  change  Relative  i n O2  to  c o n t e n t per  gram  of  hemoglobin  throughout  44  c).  2h  following  t h e 5min mark, o f maximum change i n  parameters,  p l a s m a pH  and  these  In the  returned  toward  control  fish  any  the  of  values  showed  no  in  present greater  circulating  to  changes  pH  caused  a  trout in  vitro  hemoglobin  The  decreased  acidification  by  the  as  catecholamine  c o n c e n t r a t i o n s are infused  fish  a Root s h i f t .  pH  levels  of  the  i n pHe,  the  c o n c e n t r a t i o n ( F i g . 45). Reduction  w i t h h i g h CO2  i n the absence  amount  of  well.  addition  infused  pre-infusion  g r e a t e r the decrease  equilibration  levels.  Saline  to t h e i r  in acid  exhibit  by  gradually  4).  pHe  reduction of red c e l l  catecholamine  elevated  blood  a.d).  catecholamine  The  levels  (Fig.  p o s t - i n f u s i o n changes i n  relative  increase i n catecholamine  Rainbow plasma  ( F i g . 44  Table  in  experiment. the  catecholamine  significant  ( F i g . 44 a . b . c . d . and  proportional  It  of acid  and  tensions  of increased  oxygen is  bound  assumed  to that  i n the absence  would have had  of  t h e same  of  effect  46).  EXPERIMENT 4B.  PLASMA AND HC1  As  values  measured v a r i a b l e s  Changes  (Fig.  control  the c o u r s e o f t h e e x p e r i m e n t s  the  in  the  WATER IONS AND  TEP  CHANGES AFTER  INFUSION  previous  experiment,  p l a s m a pH  also  declined  175  Table O2  4.  Means + S.E. o f a r t e r i a l plasma pH(pHe), r e d c e l l  bound  per  gram  noradrenaline([NA]) infusion the  of  means  pre-infusion  a  5  of  values  are  before  120mM s a l i n e  pHe  pHi  1  and  solution  significantly  following i n five  different  intra-arterial  animals.  from  those  None o f of  + 5min  + 30min  + 60min  + 120min  7.948  7.963  7.954  7.947  7. 947  + 0.034  + 0.033  + 0.028  + 0.026  + 0.030  7.413  7. 418  7.400  7. 382  7. 415  + 0.027  + 0.032  + 0.018  + 0.019  + 0.029  1.04  1.04  1.03  1.03  1. 04  + 0.03  + 0.03  + 0.04  + 0.05  + 0.04  0. 31  0.45  0.34  0.76  0.74  + 0.11  + 0.12  + 0.11  + 0.36  + 0.17  [NA]  0. 27  0.23  0. 22  0.17  0. 23  nmol*L~l  + 0.04  + 0.05  + 0.03  + 0.03  + 0.03  -  the  POST-INFUSION  nmol*L l  [A]  and  controls.  CONTROL  2  1  PRE-INFUSION  PARAMETER  mls0 *gHb-  _1  concentrations ml*Kg~  p H ( p H i ) , mis  hemoglobin(mls02*gHb ), adrenaline([A])  176  F i g u r e 45. Relationship between the changes i n p l a s m a pH and the corresponding changes i n plasma adrenaline concentrations between pre-infusion control s a m p l e s and the + 5min post-infusion s a m p l e s f o r e a c h o f 14 a n i m a l s contributing to the mean data i n F i g . 2.1. Linear regression analysis of the data points were used to generate the best f i t l i n e . D e l t a [ A d r e n a l i n e ] = 42.46 2 . 2 8 * D e l t a pHe, r = 0.92. 2  177  178  g u r e 46. R e l a t i o n s h i p between 0 capacity p e r gram o f haemoglobin a n d r e d b l o o d c e l l pH i n r a i n b o w t r o u t b l o o d equilibrated in. v i t r o (10°C) a g a i n s t g a s m i x t u r e s h a v i n g an oxygen partial p r e s s u r e o f 152 mmHg a n d a C0 partial pressure o f 2.5 mmHg. D a t a a r e i n d i v i d u a l measurements o n a single blood pool from three animals. Haemoglobin concentration o f the blood p o o l = 8.4 g H b * 1 0 0 m l ~ l . The line o f best f i t was g e n e r a t e d by l i n e a r regression analysis where mis 0 * g [ H b ] = -5.005 + 0.800*pHi ( r = 0.94). 2  2  _ 1  2  179  Red c e l l  pH  180  about  0.17  (Fig.  47  fish  a).  and  transient  at  40  and  pH  infused  fish  was  infused  Plasma  HCO3  variable  and  HCO3  -  -  5min  HCO3  not  significantly level  in  -  for  and  acid  experiment. throughout  was  during  occurred. pH  infused  P l a s m a pH  value  control which  There of  the  120min, a t  different  levels.  was sham  the  end  fish  showed  from  their  of  the  acid  d i f f e r e n t from v a l u e s  in  ( F i g . 47  HC1  saline  of  the  for  the  fish  infused  pre-infusion  difference  infused Values  with  fish in  Hct  throughout  experiment  groups (Fig.  increased  toward  experiment.  While  fish  pre-infusion  s a l i n e , the  control  i n the  both  the  Plasma in  acid-infused  d i f f e r e n t from e i t h e r  15  declined  gradually  course of  5,  however,  120min mark.  fish  fish  the  Mean v a l u e s ,  infused  the  infused  trends during  b).  l e v e l s by  concentration  no  the  periods  By  not  i n f u s i o n and over  0.56mM l o w e r t h a n t h e There  sham  plasma  increasing  control  levels  120min  the  were  infused  saline  significantly  periods  to  the  or  a c i d and  showed  following  pre-infusion  in  concentrations  concentrations  first  fish  control  fish.  and  close  increase  control  not  40min s a m p l i n g  declined  sampling  infused  which  pre-infusion  saline  were  acid  pre-infusion  for saline  early  both  values  respective  the  60min p o s t - i n f u s i o n .  experiment,  plasma  the  i n the  i n the  controls the  change  to  values  changes  slight  of  relative  P l a s m a pH  d i d not  maximum a  units  the  was level  mean v a l u e  was  value. v a l u e s between the  course  declined 48).  with  There  saline of  the  sampling were  no  181  Figure 47. Means + S.E. o f a. Plasma pH and b. p l a s m a HCO3 concentrations i n rainbow trout i n f u s e d w i t h HC1 and saline i n the d o r s a l a o r t a v i a a c h r o n i c i n d w e l l i n g catheter ( a t t h e 0 time mark w h i c h r e p r e s e n t s t h e end o f a 5min infusion routine) over the time course o f the e x p e r i m e n t s . [HCC>3~] i n mM. -  182  TIME  (min)  183  g u r e 48. Means + S.E. o f a. H e m a t o c r i t (%) o f r a i n b o w trout infused w i t h HC1 and s a l i n e i n t h e d o r s a l a o r t a v i a a chronic indwelling catheter ( a t t h e 0 t i m e mark w h i c h represents t h e end o f a 5min i n f u s i o n r o u t i n e ) o v e r t h e time c o u r s e o f the experiments.  184  27,  0  30  70  TIME (min)  110  185  significant the or  trends  duration acid  observed either  in  infused  fish  in  of  any  saline or acid  saline  Relative  and  to  TEP  sampling  7 ,  experiment ratio  in values  b).  values  NH +,  fish  were h i g h l y throughout control  fish  the experiment. there  was a  15min f o l l o w i n g  fluctuated  were  about  reminder not  acid values  o f the  significantly  mean ( F i g . 4 9 ) .  ratios + +  ,  calculated and  f o r Na , C l , +  Mg++  for  -  each  v a r i a b l e and no s i g n i f i c a n t t r e n d s  were o b s e r v e d f o r  above  throughout  variable for  value,  f o r the  values  Ca  4  in  The r e s u l t s were  K  and  +  the  first  remained + +  the  Mg  pre-infusion  ( F i g . 50  Ca at  Nernst  were  the experiment.  -  increased  50  3  HCO3 ,  -  the  shows  period  generally Cl ,  fish  infused  latter  trends  ion concentrations  i n the f i r s t  infused  These  HC0  +  for acid  saline  (Table 6 ) .  pre-infusion TEP  for either  No c o n s i s t e n t  values  from t h e p r e - i n f u s i o n  Table K ,  fish  infused  in  saline  experiment. different  infused  i t s own  infusion.  5).  t h e measured water  acid  increase  the  (Table  potential  transient  for  ion concentrations for  o f the course o f the experiment  Transepthelial both  measured p l a s m a  a).  + +  control  acid  and  throughout the i n NH4  +  which  t o t h e end o f t h e e x p e r i m e n t  (Fig.  120min  Nernst r a t i o s f o r a l l ions  ratios  infusion after  Nernst r a t i o s d e c l i n e d 60  values  Na+  T h e r e was a r a p i d d e c l i n e  15min f o l l o w i n g stable  ratios.  t o l e v e l s below  sampling  are not equal  periods to unity.  control  (Fig. 51).  186  Table  5.  Means  +. S.E.  o f plasma i o n c o n c e n t r a t i o n s  with  ION  CONTROL  + 5min  + 15min  + 40min  + 60min  + 120min  143.52 + 3.94  144.68 + 5.56  149.97 + 5.81  145.90 + 5.27  147.12 + 5.64  149.03 + 4.16  s-Na+  119.76 + 9.62  120.10 + 8.02  121.07 + 8.83  116.41 + 9.42  120.32 + 7.88  118.57 + 7.64  a-Cl~  114.15 + 6.30  114.86 + 6. 27  111.16 + 5.23  116.17 + 6.59  115.78 + 6.21  111.16 + 5. 27  s-Cl  107.94 + 12.25  110.41 + 12.78  110.20 + 12.30  106.28 + 12.69  114.03 + 12.01  113.12 + 12.34  3.69 + 0. 35  3.48 + 0. 22  3. 92 + 0.53  3. 38 + 0. 15  3.46 + 0. 15  3.92 + 0.53  4.93 + 0.38  4.14 + 0. 37  4.44 + 0.54  4.97 + 1.27  4. 16 + 0.40  4. 15 + 0.39  a-Na  a-K s-K  +  -  +  +  a-HCKN = 8) and s - s a l i n e (N = 5 ) .  f o r rainbow  infused  A l l concentrations  a-Ca  + +  1.24 + 0. 17  1.08 + 0.10  0.91 + 0.10  1.03 + 0.11  1.05 + 0.14  0. 91 + 0.10  s-Ca  + +  1.57 + 0.41  1.35 + 0.26  1.21 + 0.24  1.13 + 0.29  1. 18 + 0.26  1. 16 + 0.25  a-Mg  ++  0.57 + 0.08  0.56 + 0.06  0.52 + 0.09  0.59 + 0.09  0.58 + 0.07  0.52 + 0.09  s-Mg  ++  0.60 + 0.09  0.55 + 0.05  0.54 + 0.05  0.54 + 0.06  0.55 + 0.04  0.59 0.04  +  0.04 + 0.01  0.04 + 0.01  0.03 + 0.01  0.03 + 0.01  0.04 + 0.01  0.03 + 0.01  a-NH  4  trout i n mM.  187  Table  6.  Means  +_  of  water i o n c o n c e n t r a t i o n s f o r rainbow  infused  with  ION  CONTROL  + 5min  + 15min  + 40min  + 60min  + 120min  a-HC0 "  0.61 + 0.11  0.52 + 0.11  0.57 + 0.11  0.57 + 0.10  0.51 + 0.12  0.57 + 0.11  s-HC0 -  0. 27 + 0. 12  0. 22 + 0.05  0.43 + 0.23  0. 34 + 0. 14  0. 34 + 0.12  0.63 + 0. 32  »-Na  +  0.89 + 0.06  0. 84 + 0.04  0.77 + 0.04  0.79 + 0.03  0.83 + 0.04  0.77 + 0.04  s-Na+  0.90 + 0.07  0.85 + 0.08  0.84 + 0.07  0.80 + 0.07  0. 85 + 0.08  0.84 + 0.07  a-Cl  -  0.92 + 0.04  0.91 + 0.04  0.90 + 0.05  0. 89 + 0.04  0.09 + 0.04  0.90 + 0.05  s-Cl"  0. 88 + 0.03  0. 85 + 0.02  0.85 + 0.04  0. 85 + 0.05  0.85 + 0.05  0.85 + 0.04  a-K +  0.03 + 0.01  0.03 + 0.01  0.04 + 0.01  0.04 + 0.01  0.04 + 0.01  0.04 + 0.01  5-K+  0.04 + 0.01  0.05 + 0.01  0.04 + 0.01  0.05 + 0.01  0.05 + 0.01  0.05 + 0.01  + +  0.03 + 0.01  0.03 + 0.01  0.03 + 0.01  0.03 + 0.01  0.03 + 0.01  0.03 + 0.01  + +  0.02 + 0.01  0.02 + 0.01  0.02 + 0.01  0.01 + 0.01  0.02 + 0.01  0.02 + 0.01  0.01  0.01  0.01  0.01  0.01 0.01  0.35 + 0.15  0.36 + 0.15  3  3  »-Ca s-Ca  a-HCKN  S.E.  = 8) and s - s a l i n e ( N = 5 ) .  a-Mg  0.01 0.01 0.01 +. v a r i a b i l i t y u n d e t e c t a b l e  s-Mg ++  0.01 0.01 £ v a r i a b i l i t y undetectable  a-NH 0.03 +0.13 +  4  0.32 +0.14  0.36 +0.15  0.31 + 0.13  trout  A l l c o n c e n t r a t i o n s i n mM.  188  Table  7.  Means + S.E. o f plasma r a t i o s o f : Measured  [ i o n ] by t h e N e r n s t  equation f o r trout  infused  with  [ i o n ] / Expected  HC1.  ION  CTRL.  +5min  +15min  +40min  +60min  +120min  Na  82.682  84.475  95.660  90.071  100.221  108.297  + 10.861  + 8.113  + 9.187  + 6.662  + 11.696  + 11.712  226.910  261.204  254.701  271.775  266.697  250.564  + 28.901  + 21.257  + 25.862  + 34.589  + 39.165  + 31.741  60.181  66.312  71.870  73.194  79.919  94.506  + 24.384  + 21.032  + 18.538  + 21.961  + 22.469  + 54.794  +  CI"  K  +  HC0 3  NH4+  Ca  Mg  + +  +  +  24.934  29.144  33.043  30.043  22.956  28.542  +4.548  +4.970  + 5.312  + 6.694  + 4.379  + 7.212  0.108  0.080  0.060  0.060  0.064  0.061  + 0.020  + 0.020  + 0.010  + 0.010  + 0.010  + 0.009  10.075  12.262  12.518  9.095  6.905  7.640  + 2.398  + 2.304  + 2.111  + 0.963  + 1.354  + 1.043  15.948  14.318  17.086  17.890  11.219  14.829  +1.266  +2.094  +4.272  +5.664  +2.915  +4.266  189  F i g u r e 49. Means + S.E. o f t r a n s - e p i t h e l i a l potentials (TEP) i n rainbow t r o u t i n f u s e d w i t h 5ml*Kg body w e i g h t of a 0.05 N HC1 s o l u t i o n made up i n 120mM p h y s i o l o g i c a l s a l i n e and t h e s a l i n e a l o n e . x  TRANSEPITHELIAL POTENTIALS (TEP)  191  F i g u r e 50. Means +. S.E. of a. nernst ratio f o r plasma Na in trout infused with 5ml*Kg body w e i g h t 0.05 N HC1. Filled circle is the p r e - i n f u s i o n c o n t r o l p o i n t , b. As a. e x c e p t t h a t i t p e r t a i n s t o NH/j"". +  - 1  1  192  193  F i g u r e 51. Means +. S.E. of the nernst r a t i o f o r plasma Ca i n trout infused with 5 m l * K g ^ body w e i g h t 0.05 N HC1. F i l l e d c i r c l e i s the p r e - i n f u s i o n c o n t r o l p o i n t . + +  -  194  195  DISCUSSION  These  acid  increased  acid  exercise  release  excess  load  +  reduces maintains  a significant  release  the  of  the  recent  in fish i s , at  in  02~Hb  that  of  trout.  study  phenomenon  the  in  increase i n  and  of fishes  this  in  This  in. v i v o  evidence  supports  and R i c h a r d s o n  degree  The  was due  circulating  i n p l a s m a pH from c o n t r o l functional  maintenance need  values  significance of  o f blood for  (1985)  shown by t h e b l o o d  r e l a t i o n s h i p between  apparent  the  turn  and s a t u r a t i o n o f t h e b l o o d i n  m a i n t e n a n c e o f 02~Hb a f f i n i t y  capacity. the  This  (eg.  due t o t h e  extracellular fluid pH  that the  to acidoses  partially,  by C o s s i n s  and t h e change  the  of anaerobic  suggests  i n response  erythrocytic  affinity  this  highlights  when  evidence  o f t h e Bohr and Root s h i f t s  catecholamines  bout  that  o f the catecholamines,  least  in  The p r o p o r t i o n a l  carrying  a  the c i r c u l a t o r y system.  in. v i t r o  attenuation  greatest  This  plasma a c i d o s i s .  showed  further  in  change  to  and  during  concentration  the  demonstrate  trout  a l . . 1986)  catecholamine  this  in  and n o r a d r e n a l i n e .  ejb H  which  experiments  of catecholamines  Primmett  the  load  elicits  adrenaline  face  infusion  this  0 -Hb 2  affinity  protection i s  o f a c i d o s i s , o r d e l t a pHe,  i s also  maximum. As  expected,  concentration  is  the nature o f t h i s a  increase  t r a n s i e n t phenomenon.  i n catecholamine  This  i s consistent  196  with  the  1967) in  acute  and  stress  also  with a v a i l a b l e  catecholamine  during  a  burst  swim in  conditions  of  acidosis  resting in  (Nakano and  Tomlinson  d a t a where t r a n s i e n t  increases  have  (Primmett  IA  and  in fish  concentrations  experiment  volume  response  et. al..  Section l . , i n 24h  response  also  after  to  an  suggests  1986).  The  which a n i m a l s  levels  that  acid-base  i n trout  results  i n steady  an a l t e r a t i o n  i n which c a t e c h o l a m i n e  values,  been o b s e r v e d  state  in ventilatory  were c o n s i s t e n t  increase i n  disturbance  of  with  catecholamines  is  a  transient  phenomenon. While in  H o l e t o n et. al..  plasma  metabolic changes  and  ion  experiments. this  Section  Primmett  et.  order  recorded to  concentrations  concentrations acidosis  were  induced  about  by  other  studies  al..  1986)  where  induced;  was of  for  0.5  and  to 0.3  the H o l e t o n  exhaustion  and  (Primmett  e t al_.  may  have  been  too  magnitude  that  could  observed i n t h e two  one  half  metabolic the  those units  from  trout  change al..  to  induce  detected  The  insult  net  ion  in  the  been  acid-base i n pH  values were  to  and  have  Changes  swum  in  i n pH  1983  of  the c o n t r o l  were  present  experiments  acidoses  studies.  to  significant  i n the  pattern  1986), r e s p e c t i v e l y .  be  no  s t u d y above where t r o u t  where  mild  et_  changes  response  the  (Holeton  although  similar  in  i n excercised trout,  represent  experimentally disturbance  ion  induced  The  demonstrated  the  water  acidosis in  (1983) showed a number o f n e t  in  were  prodded  exhaustion i n our  study  changes  in a  water, g i v e n  the  197  fish/water  volume  acid-base  disturbance  transepithelial compensatory treatment, The  ratio. that  ion  ion  exchanges  in  uptake  Na  of there  blood  were  suggests  increased  to  proposed load  Na /NH4 +  the  suggested  1964,  that  NH4  transport  stimulation  has  adrenaline  1978).  (Payan The  being  existing  been  the  e£  +  al..  epithelium  the  excess  Na /H (NH4 ) +  +  is  H  +  exchange  +  evidence  fish  This  for  a  P a y a n 1978) and Maetz  (1973)  compete  for  for similar  for  trout  stimulation  of  Na .  Such  +  in  sites  response  beta  a to  adrenergic  G i r a r d and P a y a n 1977; P a y a n  ratio  actively  pumped  out  through  reduce  exchange  1975;  +  and  may  in  for of  gradients active  d u r i n g the  +  (Maetz  Nernst  this  for Na  o f t h e i o n pump.  in  reduced  that  catecholamines.  o f the g i l l  There  reported  electrochemical  possibility stimulated  vehicle  through  receptors  H  the  i n [ N a ] i n the  a  1977,  and  of  n e t changes  activity  process  +  aspect  other  b a s i s , the f a c t  through  Evans  by  On t h i s  permeability  epithelium.  exchange  +  the  was a s t i m u l a t i o n o f t h e a c t i v e  theoretically  plasma  gill  some  ratios  infusion.  increased  could  the  Garcia-Romeu  was  that  offset  in circulating  significant  match  the  a  acid  no  process  of  across  on  with  +  that  e f f e c t e d changes i n  were by  Nernst  there  possible  induced  caused  the  indicate that  also  which  such as the i n c r e a s e  increases  is  was  fluxes  experiment  that  It  NH4  +  means t h a t i t  the animal and  exchange  i t  supports  process  the a c t i o n o f catecholamines  a g a i n s t the  was  the being  i n response to  198  the  acid infusion. Two  conditions  active the  Na /H (NH4 ) +  +  reduction  infusion; and  influx  the  of  gill  exchange  Na  The  -  increased,  leak  of C l  was  hypothesis, the  increase  may  it  this of  have in  had  possible  to  i n the  Na  or  to  increase  +  an  increase  in  in Section  3.  -  s t i m u l a t i o n of  across the  between  to C l  this  -  in  catecholamines.  than C l  the  cation  increase PNa  +  and  would a l s o  have  i o n t o compensate minimal g i v e n  the  the mild  G i v e n the  lack of  evidence  balance  in this  proposed  Ca  + +  m i g h t have b e e n  i t s electrochemical  c a t i o n balance  in  to  anion  an  analyses  be  charge that  was  The  gill  general. for  o f an  general  differential the  an  after acid  assuming a g e n e r a l  a c t i v e uptake of  involved is  proposed  role  permeability  a  permeable  taken place,  water a g a i n s t  negative  more  of  electrical neutrality.  there  was  well.  The  and  the  that  major trout  efflux  i n response  there  have  of  to m a i n t a i n  as  a  in gill  i n the  t o water  was  +  acidosis  -  increase  permeability but  increased  from  the  epithelium.  could  PCI .  that  blood  the  cation  permeability  degree  from  +  ions  that  above h y p o t h e s i s  playing  that  to  the  exchange  permeability  suggested  accompany  (1978) d e m o n s t r a t e d  permeability  the  is  suggests  trout  H  another  gill  in  of  concomitant  e i . al..  This  +  that  the  Isaia  must  blood  gradient  (Fig.  51).  to  taken  balance  GENERAL  DISCUSSION  200  GENERAL DISCUSSION  Fish  regulate  disturbance returned  (see  treatment  hypercapnia. exchange  A  of  disturbances several  catecholamines 1986;  Perry  confirm fish  to  status of  the  a  swim  The  following a  as  w e l l as  ion  Acid-base  s t r e s s response  in fish  the  and  levels  of  (Primmett e t a l .  studies i n this  regarding and  the  a  environmental  conditions.  levels  statements  in  is  e p i t h e l i u m c h a r a c t e r i s e the  results of  result  i s , pH  or d u r i n g  to  ventilation  generalized  disturbances  the  describe  thesis  response  some  r e s t o r a t i o n o f the  of  specific acid-base  blood.  data  from  suggest  that  there  status  the  between  regulating  the  ventilation  volume.  experiments  i s a greater  of  processes  While  burst  acid-base  That  time e i t h e r  exposure  gill  resting  general  The  acid-base  a  of  an  c o n d i t i o n s have b e e n shown t o r a i s e  1986).  which  over  the  to  review).  acidotic  elicit  acid-base  processes  the  to  above  these  for  as  as  across  also  acidotic  such  such  fishes  response  levels  stimulation  processes  response  in  1985  resting  perturbation  prolonged  pH  Heisler  toward  short-term  blood  the  blood  blood  Pco2  ventilation  tension  reported  potential  in fishes  and of  increases  water the  in  in this  f o r r e g u l a t i n g the  through rather  blood  thesis  by  i o n exchange than  through  adjustments  response  to  of  acid  201  conditions, in  the  the  blood  limited reduces  disturbances. in  steady  about  2  Pco2  The state  mmHg  occur  during  CO2  H  of  a  HCO3  to  -  circumstances case  of  PC02  HCO3  tension  values might  by  limiting  CO2  Given  functional  uptake of  by  of is  the  load  of  increase  PC02  stimulation  limited  to  metabolism.  in  that  case  reducing  to  blood  from  flow.  will  titrate  +  water by  the  by  levels.  This  excess  also  that  increased  e n d o g e n o u s , s u c h as H  reduced  An  thereby  is  showed  h y p e r c a p n i a due  will  role  Pcx>2  be  acid-base  below n o r m a l  blood.  and  2  gill  limited the  acid  r u l e has  in  range  toward  Alkalotic  of  those in  the The  normal  conditions  v e n t i l a t i o n and  of  becomes  the  conditions  fish  shifts oxygen  many e x c e p t i o n s .  under  acid  the  2  thereby  excretion  attractive  stimulation  is  Root to  C 0  for adjusting  possibility  t o o f f s e t Bohr and  ventilation  only  1.  tension  excretion.  ventilation  simple  be  significance to  to  Pco2  acidosis.  conditions  corrected  this  ventilation,  response  the  might  be  C0  titration  -  any  origin  acidotic  increased  also  water  functional  where  can  ventilation  metabolic  in  in Section  Pco2  in  the  in correcting  to environmental  from  The  ventilation  experiment  rise  molecular  tensions.  for adjusting  potential  reducing  exposure  of  ions  first  probably  diffusion +  its  conditions,  by  tensions  range  of  that  While  i s well  the  of  oxygen  sensitivity  documented,  Elasmobranchs which  conditions  have b l o o d  the  ventilation in  enhancement .  by  this  increase  which does  not  202  exhibit  either  reported  to  show  hypercapnia has  also  Bohr  while been  carrying  capacity  which  (Boutilier prep.).  et  al_.  in  showing capacity  in  in  spite in  attributed  the  1986,  fish  Primmett which  an  (Smith acidotic The that blood  Jones  pH.  equivocal ventilatory Root  shifts  sensitivity  Given nature  of  of  clearly capacity  studies  have  conditions  sensitivity  o f the d o g f i s h ventilation  lack  o f Bohr  of  the  direct  teleost  ventilation  carrying  not  hyperoxic  residual  by  stimulation of  While i n v i v o with  i s the  in. v i v o  oxygen  The  in  acid infusion  oxygen c a r r y i n g  the  by  is offset  blood  i n Section  was  to the  blood,  1 showed  most s e n s i t i v e t o  and R o o t s h i f t s relationship  s t i m u l a t i o n under a c i d c o n d i t i o n s shown  1986,  exist.  study  stimulation  Richardson  Perry  The  by  conditions  i s , therefore,  effect  some  c o n d i t i o n seems t o  the  in  i n the blood  of this  and  It  oxygen  be o f f s e t  effect  a plasma a c i d o s i s .  1982),  ventilation  effect  this  reduction  to r e d u c t i o n s  and  been  shifts.  such  cytosol.  acidotic conditions  attenuation  Cossins under  this  demonstrated  m i g h t be d e m o n s t r a t e d i n v i t r o .  shown  have  blood  can  §_t al.. 1986,  the e r y t h r o c y t e  of  in  conditions  in  by  and R o o t  reduction  §_t a l _ . 1986,  released  4.  Cyprinids  shows Bohr  acidotic  significant  ventilation  as  of  Section  no  blood  that  mechanism  alkalinization study  their  shifts.  v e n t i l a t o r y response to exposure to  (Boutilier are  The  Root  little  shown  catecholamines 1986)  or  and  and  between  t h e Bohr  i t i s possible  to the a c t u a l acid-base  the  and  that a  status of  203  the  blood  in  published  teleosts  data  question  is  attenuate  must,  why the  exists.  Some i n c o n s i s t e n c i e s  however,  raising  the  increased  be  reconciled.  oxygen  ventilation  hypercapnic  conditions  shown  c a t e c h o l a m i n e s o f f s e t the  that  vivo  (Boutilier  vitro  (Cossins  are  released  hypercapnia The  changes this  ion  1984,  experiments situations  of  Heisler  characterizes in  in  exposed  1982)  when i t has  Bohr and  Root  been  shifts  in  1986)  that  catecholamines  to  environmental  and  exposed  acid-base  conditions  thesis  in  et. al..  gill  is  and  in  disturbances reported  clearly  and  blood  this  a  water  (Cameron 1976,  carp  ion concentrations  et. thesis)  al..  1983,  change  water a  few  1976)  net  experiment  (Claiborne  conger in  and  Mize  the  through  With  and  in  in  greater  flow.  1981b., K e r s t e t t e r  transfer rates  and  fish  in  and  trout various  T r a n s e p i t h e l i a l i o n t r a n s f e r s seem  renal  1985). the  to  §_t a l . . 1986)  correcting  acidotic  acidoses.  over  would  Primmett  species  Holeton in  predominate  for  changes  thesis)  Heisler  water  i o n e x c h a n g e p r o c e s s e s between b l o o d  (Perry in  Jones  obvious  content of trout  the  1986).  this  of  through  exceptions  by  that  in  adjustments than  in  the  experiments  1985;  Richardson  (Perry  and  al.  and  potential  general  in  et:  ( S m i t h and  One  in  function The  recovery  in this  accumulation  of blood  pH  regard of  a f t e r the  to  (see  review  blood  HC03~  initial  fall  acidotic conditions. In  the  trout,  conger  and  carp,  active  C1 /HC03~ -  204  exchange to  processes  reduce  between  blood  C l  concentrations  i n response  is  with  consistent  have  been  White Janssen  and  Randall  e£  1980;  Cross  al..  It  rather  between  in  similar  rapidly  this  s t u d i e s where  fish  ( L l o y d and  and R a n d a l l 1975;  1976; B o r j e s o n 1976;  These  gill  tempered concluded  in  processes  were  HCO3  water  -  with  ionic  through C l  -  .  this  uptake  -  from  to  exchanges  over  thesis  from  that  i n v o l v e d i n the  exchange  processes  The u n i d i r e c t i o n a l i o n  hypercapnia.  water a  few  It  i n conger  is  occurred  only the  three  under  fresh  This i n h i b i t i o n can  h o u r s and s i n c e t h i s  and Payan 1 9 7 7 ) , even  in  the  hours  in  fresh  in  Section  experiments  o f plasma that  and t r o u t  conditions.  the net  likely  mechanism o f  r e p o r t e d l y p r e s e n t on the r e s p i r a t o r y  have  for  values.  t h e water which e f f e c t e d  were p r e s e n t  fresh  is  §_t a l .  t h e c a r p showed t h a t i t was a n i n h i b i t i o n o f  epithelium (Girard  could  experiments  passive  and  near  uptake  the  plasma  processes  occur  the  This  Cameron  1984).  -  hypercapnia.  hypercapnia  e t al..  HCO3  and Meade 1979; H e i s l e r  Heisler  response  or  -  Bubien  blood  t h a t i o n and t h e a s s o c i a t e d a c c u m u l a t i o n  water  Cl  1969;  1975; H e i s l e r  from  of  C l  in  -  o f other  aJL.  and  with  active  changes HCO3  results  et  than  blood  study  the  to environmental  environmental  1976;  clear  accumulation  flux  increase  observed  i n p l a s m a pH v a l u e s r e t u r n i n g t o w a r d s c o n t r o l is  active  and  -  to  Randall  Claiborne  resulted  the  exposed  1967;  t h e b l o o d and w a t e r were  cells of  i t is likely  conger water.  which It  2 on t r o u t  that were was that  205  active  processes  HCC>3~  in  seemed  most  efflux  cell  protein  according active  to  Nernst  were  300  mM  higher  than  While observed  blood from a was  at  those  net  ratio  changes  the pH.  H  +  than  synthesis  process.  mechanisms  involvement  of  new  This hypothesis e e l which a l s o  from  salinity.  hypercapnic  exchange  at  acidosis  f o r these  ions.  The N e r n s t  mM  than those  ratios  for fish  f o r HCO3  -  f o r the  a l l three  s t u d y was s e e n  3  might  showed a  Further support  C1~/HC03~  of  transport  trout  in  infused  i n the  ratios for  i n 100 mM a n d  was l o w e r  i n fish  i o n c o n c e n t r a t i o n s were n o t  with a c i d ,  of  the  a s r e p o r t e d i n S e c t i o n 4,  that  exchange  +  load  plasma  suggested  Na /H (NH4 ) +  the s t i m u l a t i o n o f C l ~ transport  the conger  It  f o r 100 mM a n d 300 mM.  analysis  +  300 mM N a C l .  the  Nernst  i n trout  active  or  in  analysis  whereas  3 mM  reduce  investigated  and  rather  recovery  the  mM  through  for this  of  a c c u m u l a t i o n o f plasma  involved  gill  environmental  ratio  Cl~  the  in  100  this  to include  nature  salinities  to  recruitment  response  effected  uptake  -  on  be e x t e n d e d  Nernst  that  specifically  graded  at  HCO3  present  chloride  have  acclimated  likely  for  already  also  fish  must  t h e r e might mechanism  plasma,  I t i s hypothesized that  have b e e n a n working  and t h e r e b y  this  active  to  correcting  uptake  of Na  +  w a t e r t o b l o o d was matched w i t h a n e q u i v a l e n t i n c r e a s e i n  passive  leak  not observed There  for this  ion, since a net accumulation o f Na  +  i n t h e plasma.  was g e n e r a l a g r e e m e n t between p e r m e a b i l i t y e s t i m a t e s  206  of  the  gill  Cl~.  and  Under  steady  exposure  to  agreement  between  Nernst those  ratios  for  Cl~  3-100  in  to  influx  the  observed  also  water,  rates net  conditions  Primmett  et_  al..  release  in  catecholamines  the  all  three  Section in  acid-base  be  4,  which  systems.  An  also to  include  inhibition  the  Na  -  for  exposure in  +  to  carp  i n response  to  r a t e s , would r e s u l t  in  concentration. i n t r o u t i n response by  Perry  et. aJL-  the also  (1986) and The  to  i n the  an  by  increased  pointed  change  perturbs  (1986) f o r  t r o u t exposed  showed  C1 /HC03~  than  in  cardiovascular of  the  that  to the  trout,  increase  in  plasma  pH.  in  since a s i g n i f i c a n t  blood  to  decrease  in  the  good  The  Both the  i n f u s i o n of a c i d  expected  status of  functions  reduced  to  levels.  observation  exercise.  salinities  proportion  r e s u l t s should  was  burst  The  than  the  Boutilier  trend  was  i n trout during  reported  for  of catecholamines.  reported  These  (1986)  there  salinity  hypercapnia.  by  prior  2.5  and  +  times g r e a t e r  lower  influx  depolarizing  at  was  been  hypercapnia,  hypercapnia  three  i n plasma C l ~  has  to  and  2.0  of catecholamines  exposure  hematocrit  to  Na  trout  +  normal e f f l u x  reduction release  1.5  range  Cl~  given  in  for  PNa /PCl~ of  agreed with  environmental  i n vivo  acid  Cl~  salinity  This  fresh  the  The  mM  for  all  of  flux  hypercapnia,  estimate  at  net  conditions  w h i c h was  +  permeability  exposure  to  the Na  hypercapnia. that  active  environmental  for  the  of  state  ratio  apparent over  degree  change  in  many p h y s i o l o g i c a l and  ion  exchange  transport (Perry  et_  207  al.  1984)  through and  and  beta  Payan  gill. in  1977;  under  an  of  acid-infused  trout  of  shown  to  Section  present  Na  significantly  mM  with  blood to  during  and  at  these  accumulation  Another of beta and  Na  +  was  Na  hypercapnia t i m e s and  +  in  +  specifically  the  and 300 +  flux  C l ~ would have b e e n  PCI  Nernst  i n the PNa /PCl~  of net Na  over  study i t was  trout.  a t a l l sampling  c l e a r agreement  -  levels  that  e s p e c i a l l y a t t h e 100 mM  +  for  i n the t r o u t  to environmental  component of  While  suggested  acid-infused  stimulation Na  species  reported.  is a possibility  salinities,  +  been  i n that  -  blood  between expected  due  i n order  to  an  t o mask  as a r e s u l t o f  uptake.  +  known  and  effect  C l ~ fluxes  receptor Girard  PNa  to hypercapnia,  where c a t e c h o l a m i n e  unity  no  The  any  Na  the  from  possible  in  increased  in  was  increase net  the  There  hypercapnia,  water.  increase  4  i n t r o u t exposed  There this  not  f o r the f i s h  C1~/HCC>3~ e x c h a n g e  exposed  such a s t i m u l a t i o n  as  deviated  levels.  data  +  of  exchange  +  exchange  +  i n catecholamines  Section  be h i g h ,  masked  for  increased  in  +  ( P a y a n e t a l _ . 1975; G i r a r d  water  has  2 was n o t s e e n .  and  ratios  increase  +  +  has been r e p o r t e d  fresh  Na /H  Na /H (NH4 )  inhibition  conditions  stimulation  were  in  actual  similar  1978)  explain  carp  in  stimulation  Payan  may  the  although  increase  receptor  This  seen  an  stimulation  o f catecholamines from  chloride  i s the i n h i b i t i o n  cells  by a n a l p h a and  ( P i c ejt al.. 1975; G i r a r d  1978; S h u t t l e w o r t h  1976; Payan  1978; Degnan §_t_ ai.. 1977).  This  208  supports Cl~  one  from  conclusion  of  to  the  to  water  in  trout at  100  recruitment  elevated  rather  than  therefore,  at  inhibition  of  -  either  anion-stimulated the  besides  It  should  biological  reported that  (cAMP)  possible  are  the  that  stimulating  had  Cl~ to  out,  that  other  than the  the  are  rat  actions the  Cl~  efflux  and  and  this effect  This  also  for  the  reported  ion  transport  one C l  -  by by  For  example,  catecholamines Halm e t aJL..  is  a §_t  to  act  has  al..  of  1983).  (1983) b u t  colonic While  in  the  as  beta AMP CI~  i t is  a s e c o n d a r y messenger s u c h as transport  the been  through c y c l i c  stimulant  on  across  r e s u l t seems a n o m a l o u s s i n c e  (Foster of  was  r e s u l t s of experiments on  believed  cAMP  that  mechanisms f o r a c t i v e  consistent.  colon  that  This  exist.  not  rabbit  presence  overcome  locations  that other  transport  exogenous in  or  sites  noted  Cl~ the  mechanisms  are  catecholamines  agonists  and  transport  is  a l s o be  author points  adrenergic  of  concentration.  on  not  salinities.  increased  there  was  inhibited C l ~ efflux  high  have  likely  i n plasma c h o r i d e  chloride cells  of for  the  would  The  have  -  salinities  chloride cells  cell  of  mM  HCO3  the  from the  barriers  inhibition  for  ATPase  effects  300  set  a c t i v e e f f l u x of  effected  the  the  that  chloride  efflux  the  that  efflux  reduction  suggests  on  i t  uptake  Cl~  the  of chloride c e l l s .  responsible  net  and  stimulated  HCO3  the  mM  would  consequent  2 that  that  catecholamines  suggests  actually  Section  blood  accumulation due  of  presence  Ca  + +  of  209  exogenous  cAMP  the r e s u l t s While NH-4  are not the  movements  are  -  and  by  these  groups  which  regulation  are  regulation range. expansion and  ion  further regulation  of The of  ions  affected  the  the  transfer  from  movements  occur.  animal  obvious  also  d a t a base r e g a r d i n g the r o l e  research would a l s o  into be  and  t h e p o i n t where i o n i c  i n acid-base the  most  possible  desirable.  on  on  the  the  acid-base Acid-base  and  osmotic  i t s adaptive  true. of  regulation controls  of  linkage of  demands  i s compromised beyond  processes  into  ion transfers.  is  these  descriptive  The  osmoregulation  relationship  and  +  i n the e f f e c t  processes,  these  Na  Studies  exchange  by  these  determining  as  and  -  acid-base of  understood.  places  beyond  opposite  that  movements  made, s u c h  i n that both  occur  shows  factors  well  t o be  these  of  processes  cannot  effect,  HC03  +  recovery the  the  of H ,  f i s h e s have b e e n l a r g e l y  these  regulatory  balance  to  not  in  on  mechanisms two  during  documented, are  movements  epithelium  link  are s t a r t i n g  catecholamines  the b e t a a d r e n e r g i c  the  gill  their  regulation  inroads  on  fish  processes  acid-base and  set  modulated  well  exchange  masking  definitive.  the  are  disturbances  thus  data  across  +  Cl  and  While  ventilation i s needed, over  this  210  REFERENCES  211  REFERENCES Annio, J.S. 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A. and C M . Wood. 1985. a n a n a l y s i s o f b r a n c h i a l ammonia e x c r e t i o n i n t h e freshwater rainbow t r o u t : e f f e c t s of environmental pH c h a n g e and s o d i u m u p t a k e b l o c k a d e . J . Exp. B i o l . 114, 329-353. Zeidler, R. a n d Kim, H.D. 1977. P r e f e r e n t i a l h e m o l y s i s o f postnatal calf red cells induced by internal a l k a l i n i z a t i o n . J . Gen. P h y s i o l . 70, 385-401.  222  APPENDICES  223  APPENDIX I .  Table  A.l.  various  salinities  purchased F.R.G.  Ionic  from  c o n c e n t r a t i o i is in  experimen  Wiegandt  A l l concentrations  GmbH  for 2A.  & Co.,  the s e a s a l t in  Section  Sterkenhofweg  used t o make up the 2.  These s a l t s  13, D-4150 K r e f e l d 1,  i n mM.  ION  CONCENTRATION  ION  CONCENTRATION  Na  468.0  Cl"  545.7  10.0  Br  0.8  53.3  I~  10.4  S0 -  Sr  0.2  HCO3-  2.4  F  0.07  H3BO3  25  K  +  +  Mg Ca  + +  + +  -  0.003 4  were  28.1  224  APPENDIX I I . NERNST RATIO CALCULATIONS  RT TEP = —  TEP i n v o l t s ; calculate  given  or [ a n i o n l p l  [cationlpl  or  * In  ZF  To  [cationlw  what  Concentrations  [anionlw i n moles; I n = n a t u r a l l o g  the plasma c a t i o n c o n c e n t r a t i o n ,  f o r example,  t h e TEP and [ c a t i o n l w and assuming t h e c o n d i t i o n s  f o r the  equation,  e  -TEP/(RT/ZF)  [cationlpl*  = [cationlw  = (cationlw  NERNST RATIO = [ c a t i o n l p l  / [cation]pl  / ~  measured  e  T E P /  (RT/2F)  / I c a t i o n ] p l expected*  would be Nernst  225  APPENDIX I I I . PERMEABILITY CALCULATIONS  RT PNa [Na ]w + P C l - [ C l ] p l + P H C 0 [ H C 0 ~ J p l TEP = — * In ZF PNa [Na ]pl + P C I [Cl ]w + PHC0 [HC0 ]w +  +  -  _  3  +  +  -  3  _  -  -  3  C = e*<  TEp  / / RT  ZF,  3  >  C (PNa [Na ] p l + P C l [ C l ) i » + P H C 0 [ H C 0 ) w ) * (PNa tNa ]w + P C l - t C l " ] p l + P H C 0 ~ l H C 0 ] p l ) +  +  _  -  _  _  3  +  3  +  -  3  d i v i d e by P H C 0  3  3  C ( P N a / P H C 0 ~ [ N a ] p l + PCl /PHC0 -{Cl-]w + [HC0 -Jw) = (PNa /PHC0 -[Na ]w + P C l - / P H C 0 - [ C l ] p l + [ H C 0 ~ ] p l ) +  _  +  3  3  +  3  +  _  3  3  3  set  to 0  P N a / P H C 0 ( C [ N a ] p l - [Na+]w) + PCl-/PHC0 -(C[Cl-]w - t C l - ] p l ) + (C[HC0 "]w - [ H C 0 - ] p D - 0 +  _  +  3  3  3  3  P N a / P H C 0 ( C [ N a ] p l - [Na ]tO + PCl /PHC0 -(CfCl-Ji» - [Cl-Jpl) - (C[HC0 ]w - ( H C 0 - J p l ) +  _  +  +  3  _  3  -  3  SOLVED FOR P N a / P H C 0 +  3  3  & PC1 /PHC0 -  _ 3  FOR 2 SALINITIES  3 EXPERIMENTAL SALINITIES ALLOWED 3 UNIQUE SALINITY PAIRS WHICH ALLOWED 3 ESTIMATES OF EACH OF THESE PARAMETERS PNa /PCl +  -  •> P N a / P H C 0 +  _ 3  / PC1 /PHC0 -  _ 3  THESE ESTIMATES WERE MADE FOR EACH SAMPLING TIME THROUGH THE EXPERIMENT  PUBLICATIONS Heming, T.A., D.J. R a n d a l l , R.G. B o u t i l i e r , G.K. Iwama and D.N. Primmett. 1985. I o n i c equilibria i n r e d blood cells of rainbow t r o u t . In Press i n R e s p i r a t i o n Physiology. Iwama, G.K., G.L. Greer and D . J . R a n d a l l . 1986. Changes i n selected hematological parameters i n j u v e n i l e Chinook, s a l m o n subjected to a b a c t e r i a l c h a l l e n g e and a t o x i c a n t . J. Fish B i o l . 28, 563-572. Boutilier, R.G., G.K. Iwama and D . J . R a n d a l l . 1986. A c u t e extracellular acidoses promote catecholamine release i n rainbow trout (Salmo gairdneri) : i n t e r a c t i o n s between r e d cell pH and 02~Hb carrying capacity. J . Exp. B i o l . 123, 145-157. Boutilier, R.G., G.K. Iwama, T.A. Heming and D . J . R a n d a l l . 1985. The a p p a r e n t pK o f c a r b o n i c a c i d i n r a i n b o w t r o u t b l o o d p l a s m a between 5 and 15 C. Resp. P h y s i o l . 61, 237-254. Boutilier, R.G., T.A. Heming and G.K. Iwama. 1985. Physicochemical parameters for use i n f i s h respiratory physiology. In Fish Physiology V o l . XA A p p e n d i x , e d . W.S. Hoar and D . J . R a n d a l l , pg.403-430. A c a d e m i c P r e s s . N.Y. Iwama, G.K. and G.L. G r e e r . 1982. M o r t a l i t y i n j u v e n i l e c h i n o o k salmon exposed t o sodium pentachlorophenate and u n d e r g o i n g progressive symptomatic b a c t e r i a l kidney disease. Can. T e c h . Rep. F i s h . A q u a t . S c i . No. 1100, 9p. Iwama, G.K. and A.F. T a u t z . 1981. A s i m p l e g r o w t h model f o r salmonids i n hatcheries. Can. J . F i s h . Aquat. S c i . 38, 649-656. Iwama, G.K. 1980. I n c u b a t i o n t i m e s r e s u l t i n g f r o m e x p e r i m e n t a l injections o f kidney disease bacteria into juvenile coho salmon. P r o g . F i s h . C u l t . 4 2 ( 2 ) , 182-183. Iwama, G.K. and G.L. Greer. 1980. E f f e c t of a bacterial infection on t h e t o x i c i t y o f sodium pentachlorophenate to j u v e n i l e coho s a l m o n . T r a n s . Am. F i s h . S o c . 1 0 9 ( 3 ) , 290-292. Iwama, G.K. 1981. Comment on " S i m p l e g r o w t h model f o r s a l m o n i d s in hatcheries. Can. J . F i s h e r i e s A q u a t . S c i . 39(8), 1220-1221. Iwama, G.K. and G.L. Greer. 1979. T o x i c i t y o f sodium pentachlorophenate t o j u v e n i l e c h i n o o k salmon under c o n d i t i o n s of high loading d e n s i t y and c o n t i n u o u s f l o w e x p o s u r e . Bull. E n v i r o n . Contam. T o x i c o l . 23, 711-716. Iwama, G.K., G.L. G r e e r and P.A. L a r k i n . 1976. Changes i n some hematological characteristics o f coho salmon (Oncorhvnchus kisutch) i n response to acute exposure t o d e h y d r o a b i e t i c a c i d DHAA at different exercise levels. J . F i s h . Res. B o a r d Can. 33, 285-289. Iwama, G.K. 1979. "One-Eye", a d i s e a s e o f r a i n b o w t r o u t (Salmo gairdneri) a t the Kootenay T r o u t Hatchery, B r i t i s h Columbia. B r i t . C o l . F i s h and W i l d l i g f e B r . T e c h . C i r . No. 42, l i p . Iwama, G.K., C.Y. Cho and J . D . Hynes ( E d i t o r s ) . 1981. Handbook of Fish Culture. Government of Ontario Publ. Ontario Ministry of Natural R e s o u r c e s . ISBN 0-7743-6343-6.  

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