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Respiration and circulation in Amphiuma Tridactylum Toews, Daniel Peter 1969

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RESPIRATION AND CIRCULATION IN AMPHIUMA  TRIDACTYLUM  by DANIEL PETER TOEWS B . S c , U n i v e r s i t y o f A l b e r t a , 1963 M.Sc., U n i v e r s i t y o f A l b e r t a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the department of ZOOLOGY We accept t h i s t h e s i s as conforming required  to the  standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1969  In presenting  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 that  the L i b r a r y  s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  Study. I f u r t h e r agree that 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 g r a n t e d by the Head o f my Depart ment or by h i s r e p r e s e n t a t i v e s .  I t i s understood that copying or  p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain w i t h o u t my w r i t t e n  permission.  Department o f  p-g>\ r=> '<* \ \  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date  s h a l l n o t be allowed  i  ABSTRACT R e s p i r a t o r y and c i r c u l a t o r y changes which  accompany submergence were s t u d i e d i n Amphiuma t r i d a c t y l u m , an a q u a t i c u r o d e l e . A l l experimentation  was  performed  a t 1 5 ° C w i t h the e x c e p t i o n o f oxygen consumption r e c o r d i n g taken a t 2 5 ° C. The  l u n g s , systemic a r c h e s , pulmonary  a r t e r i e s , pulmonary v e i n s , p o s t c a v a l v e i n and d o r s a l a o r t a were c a n a u l a t e d . S e r i a l sampling P O 2  and  P C O 2  procedures  enabled  measurements to be made through s e v e r a l  b r e a t h i n g c y c l e s on a l l a n i m a l s . Oxygen consumption i n Amphiuma a t 15° C was  the  lowest r e c o r d e d f o r any amphibian a t a comparable tempe r a t u r e . I t was  found  t h a t the lungs were the primary  r e s p i r a t o r y exchange s u r f a c e f o r oxygen consumption  and  were used v e r y l i t t l e f o r carbon d i o x i d e e l i m i n a t i o n . Oxygen t e n s i o n s i n the major v e s s e l s showed l a r g e f l u c t u a t i o n s which were r e l a t e d to the b r e a t h i n g c y c l e of the a n i m a l . Amphiuma breathed about once every hour a t 15° C and i t was  j u s t a f t e r a b r e a t h t h a t oxygen  t e n s i o n s i n the lungs and major v e s s e l s were the h i g h e s t . There was a r t e r y and breathing  a d e f i n i t e g r a d i e n t between the pulmonary systemic a r c h which p e r s i s t e d throughout  the  cycle. T e r m i n a t i o n o f i n s p i r a t i o n i n Amphiuma  was  shown to be c o n t r o l l e d by a volume d e t e c t i o n mechanism.  * •  11  I t was  found  t h a t low oxygen t e n s i o n s i n the b l o o d  about a b r e a t h i n g s h i p between breathing  response  the carbon  whereas  t h e r e was  dioxide levels  slight  pulse  the p r e s s u r e  than d i d the systemic  l a g i n the systemic rise  r i s e was  not recorded  suggested  genated blood being circuit, circuit.  a r c h when compared  primarily  such  i n the systemic  i n t h e two c i r c u i t s  t h a t the p u l s e  diastolic  a r c h . T h e r e was  i n the pulmonary a r t e r y  the b l o o d p r e s s u r e was  and t h e  response.  pressure  pressure  relation-  i n t h e body  The p u l m o n a r y a r t e r y h a d a l o w e r blood  no  brought  and o x y g e n a t e d b l o o d b e i n g  to  that a arch  until  became e q u a l . I t  l a g c o u l d account shunted  a  f o r deoxy-  to the pulmonary  shunted  to the  systemic  • • *  111  TABLE OF CONTENTS INTRODUCTION  1  GENERAL MATERIALS AND METHODS  5  A. ANIMALS  5  B. OPERATIONS AND CANNULATIONS  5  C. ANALYTICAL PROCEDURES  11  D. MEASUREMENT OF OXYGEN CONSUMPTION  12  E . ANIMAL SURVIVAL  15  PART I . GAS TENSIONS IN THE LUNGS AND MAJOR BLOOD VESSELS IN A FREE-MOVING AMPHIBIAN, AMPHIUMA TRIDACTYLUM. INTRODUCTION  16  MATERIALS AND METHODS  19  RESULTS  20  DISCUSSION  42  PART I I . RESPIRATORY CONTROL IN AMPHIUMA TRIDACTYLUM INTRODUCTION  55  MATERIALS AND METHODS  57  RESULTS  58  DISCUSSION  73  PART I I I . SOME FEATURES OF.THE CIRCULATION IN AMPHIUMA TRIDACTYLUM  ; -';, v  INTRODUCTION  80  MATERIALS AND METHODS  85  RESULTS  85  iv DISCUSSION  94  GENERAL SUMMARY  101  LITERATURE CITED  104  V  LIST OF TABLES I  . Types o f cannulae and numbers o f animals used.  Pages 19  II  Oxygen consumption measured i n f i f t e e n Amphiuma a t 15°and 25° C  39  III  E x t e n s i o n o f the time between breaths by i n j e c t i n g atmospheric a i r i n t o the l u n g s . .  59  IV  A i r removal from the lungs d u r i n g inspiration  60  V  I n j e c t i o n o f n i t r o g e n i n t o the lungs o f Amphiuma which were about t o s u r f a c e to breathe  61  VI  Carbon d i o x i d e i n j e c t i o n s i n t o the lungs o f Amphiuma  65  VII  N i t r o g e n i n j e c t i o n s i n t o the lungs o f Amphiuma  70  vi LIST OF FIGURES Figure 1 Figure 2 Figure 3  Figure 4 Figure 5 Figure 6 Figure 7  Figure 8 Figure 9  F i g u r e 10 F i g u r e 11  F i g u r e 12 F i g u r e 13  Pages  A drawing o f the techniques used i n the c a n n u l a t i o n o f Amphiuma t r i d a c t y l u m  8  The major b l o o d v e s s e l s , lungs and h e a r t o f Amphiuma t r i d a c t y l u m  10  Apparatus used f o r the measurement o f oxygen consumption i n Amphiuma tridactylum  14  PO2 decrease i n the lungs o f f o u r Amphiuma t r i d a c t y l u m  21  A l v e o l a r "PO2-PCO2 diagram" f o r s i x Amphiuma  23  The r e l a t i o n s h i p between lung volume and body weight i n Amphiuma  25  PO2 and PCO2 changes which o c c u r r e d i n the lungs and d o r s a l a o r t a i n one animal during four breathing cycles  27  PO2 g r a d i e n t between the d o r s a l a o r t a or systemic a r c h and the lung  28  Changes which o c c u r r e d i n the pulmonary v e i n and lung P02 and PCO2 l e v e l s i n one animal d u r i n g f i v e b r e a t h i n g c y c l e s  .  29  POo g r a d i e n t between the pulmonary v e i n and lung i n Amphiuma  31  PO2 and PCO2 changes which o c c u r r e d i n the pulmonary a r t e r y , systemic a r c h and lung i n one animal d u r i n g one complete breathing cycle  32  PO2 g r a d i e n t between the pulmonary a r t e r y and systemic a r c h i n e i g h t Amphiuma  33  PO2 l e v e l s i n the systemic a r c h and p u l monary a r t e r y (from F i g . 12) converted to p e r cent s a t u r a t i o n  35  vii Pages F i g u r e 14  F i g u r e 15  F i g u r e 16  F i g u r e 17 F i g u r e 18  F i g u r e 19  PO2 and PCO2 g r a d i e n t between the p o s t c a v a l v e i n and d o r s a l a o r t a i n f i v e Amphiuma  36  "The Standard Amphiuma". A r e c o n s t r u c t i o n o f the probable P02 f l u c t u a t i o n s i n the lungs and major b l o o d v e s s e l s d u r i n g three b r e a t h i n g c y c l e s  38  Rates o f oxygen consumption a t 15° C i n ten Amphiuma measured by a volume change technique  40  The probable mode o f gas exchange i n Arrphiuma t r i d a c t y l u m  53  The removal o f h i g h c o n c e n t r a t i o n s o f carbon d i o x i d e and normal l e v e l s o f oxygen from the lungs o f f o u r Amphiuma  .....  63  A r t e r i a l PO2 l e v e l s through s e v e r a l b r e a t h i n g c y c l e s i n Amphiuma  67  A r t i f i c i a l i n c r e a s e i n tank water PO2 and the corresponding i n c r e a s e i n d o r s a l a o r t a PO2  69  P02 l e v e l s i n the d o r s a l a o r t a and systemic a r c h i n f i v e Amphiuma a t the time o f breathing ......  72  F i g u r e 22  Diving bradycardia or breathing d i a i n f i v e Amphiuma  87  F i g u r e 23  B r e a t h i n g and lung i n f l a t i o n and d e f l a t i o n e f f e c t s on the pulmonary a r t e r y (PA) and systemic a r c h (SA)  88  Injections of high concentrations of carbon d i o x i d e i n t o the lungs and the a s s o c i a t e d p r e s s u r e changes i n the systemic a r c h  90  F i g u r e 20  F i g u r e 21  F i g u r e 24  F i g u r e 25  tachycar-  Simultaneous p r e s s u r e r e c o r d i n g s i n the pulmonary a r t e r y and systemic a r c h showi n g the p u l s e l a g i n the systemic a r c h  91  viii Page F i g u r e 26  P r e s s u r e r e l a t i o n s h i p s i n the p u l monary a r t e r y and systemic a r c h i n ; a. s l o w l y b e a t i n g h e a r t . b. superimposed p r e s s u r e r e c o r d i n g s from the pulmonary and systemic arches i n another Amphiuma  92  ACKNOWLEDGEMENTS I wish to thank D r . D a v i d R a n d a l l , o f my committee,  Chairman  f o r h i s guidance and enthusiasm through-  out the study. I a l s o thank D r . W. S. Hoar, D r . A. M. Perks., Dr. J . E . P h i l l i p s , D r . D. R. Jonet and D r . G. S h e l t o n f o r t h e i r c r i t i c a l reviews o f the m a n u s c r i p t . I a l s o wish to thank my w i f e , L o r e t t e , f o r her c o n s t a n t encouragement throughout the study. I am g r a t e f u l a l s o to the f o l l o w i n g p e r s o n s : Dr. J . R. Ledsome, P h y s i o l o g y Department,  U.B.C., f o r  d i s c u s s i o n s p e r t a i n i n g to the T h i r d s e c t i o n o f t h i s t h e s i s ; D r . G. S h e l t o n , B i o l o g y Department,  The U n i v e r s i t y  o f E a s t A n g l i a , f o r h i s u s e f u l a d v i c e and a s s i s t a n c e d u r i n g the summer o f 1968; and my c o l l e a g u e s , who p r o v i d e d n o t only u s e f u l i d e a s throughout my study, b u t a l s o made my s t a y a t the U n i v e r s i t y o f B r i t i s h Columbia an e n j o y a b l e one. F i n a n c i a l support was p r o v i d e d by two Teaching A s s i s t a n t s h i p s from the U n i v e r s i t y o f B r i t i s h Columbia and a b u r s a r y from the F i s h e r i e s Research Board o f Canada. The r e s e a r c h was supported by g r a n t s from the N a t i o n a l Research •Council, The F i s h e r i e s Research Board o f Canada and the B r i t i s h Columbia H e a r t F o u n d a t i o n .  GENERAL INTRODUCTION I n t e r e s t i n the r e s p i r a t o r y and  circulatory  dynamics i n the Amphibia dates back to the 19th  century  when Brucke (1852) i n Germany and l a t e r S a b a t i e r (1873) i n France standards,  examined, w i t h p r i m i t i v e techniques by  our  the anatomy and p h y s i o l o g y of f r o g s and  v e r t e b r a t e s . They p o s t u l a t e d what i s now  known as  " C l a s s i c a l H y p o t h e s i s " o f amphibian double In b r i e f ,  the h y p o t h e s i s  other the  circulation.  s t a t e s t h a t deoxygen-  a t e d b l o o d from the r i g h t a t r i u m e n t e r s the v e n t r i c l e and remains on the r i g h t s i d e whereas oxygenated b l o o d from the l e f t  a t r i u m e n t e r s and remains on the l e f t  of  the v e n t r i c l e . Second, there i s v e r y l i t t l e  of  the two  side  mixing  types of b l o o d as a r e s u l t of the t r a b e c u l a t e  n a t u r e of the v e n t r i c l e . T h i r d , the s p i r a l v a l v e i n the conus d i r e c t s the deoxygenated b l o o d to the pulmonary c i r c u i t , a i d e d by a lower p r e s s u r e i n the pulmonary v e s s e l s . The r e s u l t of t h i s c h a n n e l i n g i s t h a t the pulmonary c i r c u i t c o n t a i n s p r i m a r i l y deoxygenated b l o o d and  the systemic c i r c u i t oxygenated b l o o d . The p o i n t s o f agreement today w i t h t h i s  h y p o t h e s i s are t h a t there i s a s e p a r a t i o n of the types o f b l o o d i n the v e n t r i c l e and  earlier two  t h a t the b l o o d which  flows to the pulmonary c i r c u i t appears to be  relatively  ueoxygenated  and comes from the r i g h t a t r i u m . The  oxygenated b l o o d from the l e f t a t r i u m i s thought to be p r i m a r i l y shunted to the systemic c i r c u i t . Recent work i n t h i s a r e a has been done by Foxon (1947 and  1964),  H a z e l h o f f (1952), De Graaf (1957), Sharma (1957), Simons (1959), DeLong (1962), Johansen  (1963) and Johansen  D i t a d i (1966). The a c t u a l mechanism whereby  oxygenated  b l o o d e n t e r s the systemic c i r c u i t and deoxygenated shunted to the pulmonary c i r c u i t  and  blood  i s obscure.  The amphibian, Amphiuma t r i d a c t y l u m , used i n t h i s study i s an a q u a t i c u r o d e l e found i n freshwater d r a i n a g e d i t c h e s , ponds and slow moving streams i n the s o u t h e a s t e r n U n i t e d S t a t e s . The reasons f o r choosing t h i s animal a r e t h a t i t i s l a r g e , the b l o o d v e s s e l s are  l a r g e and a c c e s s i b l e f o r c a n n u l a t i o n and minimal  maintenance  i s r e q u i r e d to keep these animals i n the  l a b o r a t o r y . Recovery  from o p e r a t i o n s i s r a p i d  and  complete. The animal breathes v e r y i n f r e q u e n t l y ( a t 15° C b r e a t h i n g occurs every 45 to 75 minutes  ( D a r n e l l 1948)),  and t h e r e f o r e changes i n b l o o d gas t e n s i o n s between b r e a t h s can be e a s i l y monitored. Many amphibians  take up  oxygen through t h e i r s k i n s as w e l l as t h e i r lungs but Amphiuma, a l t h o u g h a q u a t i c and unable to s u r v i v e on l a n d , i s dependent  upon access to the atmosphere and  cannot  remain submerged f o r more than f o u r to f i v e hours ( D a r n e l l 1948). T h i s amphibian  t h e r e f o r e probably takes  up most o f i t s oxygen v i a the l u n g s . The lungs a r e l a r g e , e l o n g a t e d sacs which extend a t l e a s t 4/5  of the way  along  the body c a v i t y . The c i r c u l a t o r y system i s t y p i c a l l y amphibian  except f o r the l a c k o f a cutaneous  branch  from the pulmonary a r t e r y and the absence of the ductus Botalli  (Baker 1949). The n u l e a t e d r e d b l o o d c e l l s  a r e the l a r g e s t known i n any animal (70 to 80 microns i n diameter). In the f i r s t  s e c t i o n o f t h i s study, oxygen  and carbon d i o x i d e t e n s i o n s i n the major b l o o d v e s s e l s and lungs were measured i n free-moving, u n a n a e s t h e t i z e d a n i m a l s . Oxygen consumption  a t 15 and 25°C was  also  measured i n s e v e r a l a n i m a l s . I n the second s e c t i o n the animals' b r e a t h i n g responses to the i n j e c t i o n s o f v a r i o u s oxygen and  carbon  d i o x i d e c o n c e n t r a t i o n s i n t o the lungs were determined. T e r m i n a t i o n o f i n s p i r a t i o n and the v a r i o u s s t i m u l i i n v o l v e d were a l s o  studied.  I n the t h i r d s e c t i o n experiments were performed t o determine the mechanism whereby oxygenated b l o o d was sent to the systemic c i r c u i t and deoxygenated  b l o o d to  the pulmonary c i r c u i t . Experiments were a l s o designed  4  to show the e f f e c t s o f i n s p i r a t i o n and e x p i r a t i o n on the circulatory  system.  I n summary, the o b j e c t i v a s o f t h i s study were to o b t a i n a more complete p i c t u r e o f the p h y s i o l o g y  of  gas exchange i n one s p e c i e s o f amphibian and attempt to e l u c i d a t e the mechanisms i n v o l v e d i n the a s s o c i a t e d r e s p i r a t o r y and c i r c u l a t o r y p r o c e s s e s .  GENERAL MATERIALS AND METHODS A. ANIMALS The amphibians  used i n t h i s study, Amphiuma  t r i d a c t y l u m , were o b t a i n e d every f o u r to s i x weeks from the N o r t h C a r o l i n a B i o l o g i c a l Supply Company i n Durham, N o r t h C a r o l i n a , U.S.A. A l l animals were m a i n t a i n e d a t the U n i v e r s i t y o f B r i t i s h Columbia i n t h i r t y  gallon  a q u a r i a c o n t a i n i n g f r e s h d e c h l o r i n a t e d water a t 12-18°C. The animals were used f o r experiments w i t h i n s i x weeks o f a r r i v a l and f e e d i n g was n o t n e c e s s a r y . S u r v i v a l was good and the animals d i d n o t show any s i g n o f f u r u n c u l o s i s i f the water i n which they were m a i n t a i n e d was a l l o w e d to become somewhat "swampy", s i m i l a r to t h e i r normal  habitat. A l l o f the animals used were a d u l t s , r a n g i n g  i n weight from 250-1000 g and i n l e n g t h from 50-100 cm. S i n c e a c t u a l m e t a b o l i c r a t e was n o t an important parameter i n the m a j o r i t y o f experiments, male and female animals were used a t random. B . OPERATIONS AND CANNULATIONS A l l o p e r a t i o n s were performed a f t e r animals had been a n a e s t h e t i z e d by t o t a l immersion i n a s o l u t i o n of MS 222 ( t r i c a i n e methanesulphonate,  Sandoz) a t con-  c e n t r a t i o n s o f 15 g/1. A n a e s t h e s i a o c c u r r e d w i t h i n 20-40  6 minutes and the animal  remained a n a e s t h e t i z e d  f o r % to  3/4  o f an hour a t t h i s  d o s a g e . C a r e was t a k e n  t o keep  the  s k i n moist  ation  throughout an o p e r a t i o n . A f t e r the oper-  the animals  recovered  fully  i n f r e s h water  within  one  hour. I n a l l experiments which r e q u i r e d an o p e r a t i o n ,  the  right  l u n g was c a n n u l a t e d  ventilated with  atmospheric  Various in  a l l cases  veins  and lungs  a i r t o s p e e d up  recovery.  a n d a r t e r i e s were c a n n u l a t e d a n d  v e s s e l and lung  f r o m C l a y - A d a m s P.E. 50 t u b i n g 0.038 i n c h e s ) . B l o o d saline  artificially  indwelling, chronic, polyethylene  were used. B l o o d  ized  were  cannulae  c a n n u l a e were a l l made  (I.D.  0.023 i n c h e s ,  c a n n u l a e were f i l l e d w i t h  (250 I U p e r m l ) t o p r e v e n t  Depending upon t h e type  O.D.  heparin-  clotting.  of cannulation  desired,  a 5 cm i n c i s i o n was made i n t h e v e n t r a l m u s c u l a t u r e a n d body w a l l e i t h e r appendages  a t a p o i n t midway b e t w e e n t h e a n t e r i o r  ( f o r pulmonary a r t e r y , systemic  pulmonary v e i n c a n n u l a t i o n s ) vent  ( t i p o f the lung, post  o r 5 cm a n t e r i o r t o t h e caval vein or dorsal  cannulations). A f t e r the operation sewn up a n d t h e i n c i s i o n C l a y - A d a m s 9 mm wound Cannulation  a r c h and  aortic  t h e p e r i c a r d i u m was  i n t h e body w a l l c l o s e d  with  clips. o f t h e pulmonary a r t e r y , d o r s a l  a o r t a and by  systemic a r c h are r e l a t i v e l y e a s i l y performed  sharpening a 55-65 cm  i t with heparinized syringe ened end  length  of P.E.  s a l i n e , attaching  to the unsharpened end i n t o the a r t e r y  and  50  tubing,  a 1 ml  hypodermic  i n s e r t i n g the  ( F i g . 1 ) . I t was  cm  that  systemic  such that meaningful com-  p a r i s o n s of b l o o d p r e s s u r e i n the two made. A f t e r the a r t e r y had  sharp-  important  cannulae i n s e r t e d i n t o the pulmonary a r t e r y and a r c h were o f a s i m i l a r l e n g t h  filling  vessels  been exposed and  could  a free  be 0.5  p o r t i o n of the v e s s e l clamped, a 23 gauge n e e d l e  was  used to puncture a s m a l l h o l e i n the a r t e r y . The  sharp-  ened p o r t i o n o f the cannula was  the  then f o r c e d i n t o  v e s s e l and  the clamps removed. The muscular n a t u r e of  the a r t e r y  forms a t i g h t s e a l around the cannula  t h e r e was  no  leakage o f b l o o d from the p o i n t of  i n t o the v e s s e l . The was  of was  the  entry  cannulae  t h i r d the s i z e o f the  the e f f e c t o f the cannulae on  vessel, pattern  b l o o d f l o w i n the v e s s e l . A f t e r i n s e r t i o n the tied into place,  b e s i d e the v e s s e l . At the cannula was so  s i z e of the i n d w e l l i n g  never more than one  minimizing  and  usually  cannula  to a p i e c e of strong  tissue  the p o i n t o f e x i t from the body,  a l s o s u t u r e d to the s k i n of the  animal  t h a t minor movements o f the animal would not  result  i n the cannula being p u l l e d from the  vessel.  8  Figure 1  A drawing o f the techniques used i n the c a n n u l a t i o n o f Amphiuma t r i d a c t y l u m . a. a r t e r i a l b . venous  cannulation  cannulation.  A.  ARTERIAL  ligature including part of vein to prevent leakage  B.  V E N O U S  9 Venous c a n n u l a t i o n s , on the other hand, were more d i f f i c u l t . The pulmonary v e i n was  cannulated  at a  p o i n t where the v e i n l e a v e s the lung and b e f o r e i t f u s e s to the s i n u s venosus. C a n n u l a t i o n  procedure was  to t h a t d e s c r i b e d f o r a r t e r i a l c a n n u l a t i o n w i t h  similar the  e x c e p t i o n t h a t venous cannulae d i d not remain f i r m l y i n the v e s s e l u n l e s s the cannula were sutured to the v e s s e l ( F i g . 1 ) . I n the c a n n u l a t i o n of the p o s t c a v a l v e i n i t was  p o s s i b l e to enter the l a r g e r v e s s e l by  i n g the cannula  forc-  through a s m a l l c o l l a t e r a l v e i n . Small  c o l l a t e r a l v e i n s used i n t h i s technique were ones which were d r a i n i n g s m a l l f a t bodies and would i n no way  i t was  felt  d i s t u r b the g e n e r a l c i r c u l a t i o n o f  b l o o d . By c a n n u l a t i o n of the v e i n s and a r t e r i e s the above mentioned techniques, s e r i a l samples of b l o o d a t any disturbance  that t h i s  i t was  and  using  p o s s i b l e to o b t a i n  time d e s i r e d w i t h minimal  to the animal. F i g u r e 2 i l l u s t r a t e s  major v e s s e l s cannulated  the  the  their spacial relationships  w i t h the h e a r t . The d i s t a l t i p of the r i g h t lung of each was  cannulated  l e n g t h o f P.E. lung and I t was  by i n s e r t i n g the f l a r e d end o f a 50  animal cm  50 tubing i n t o a s m a l l i n c i s i o n i n the  then f i r m l y s u t u r i n g the lung around the  cannula.  p o s s i b l e to o b t a i n s m a l l samples o f a l v e o l a r gases  10.  Figure 2  The major b l o o d v e s s e l s , lungs and o f Amphiuma  tridactylum.  heart  4 —carotid arch  "i--systemic arch  common carotid  aortic arches  truncus a. conus a.  pulmonary arteries  --pulmonary a.  ventricle spiral^ _ valve  l.&f. atria  -lung pulmonary v. sinus yenosus  11„ (through  the lung cannula) a t any time d u r i n g the exper-  iments w i t h minimal  d i s t u r b a n c e to the animal.  C. ANALYTICAL PROCEDURES 1. Blood Gases B l o o d oxygen and carbon d i o x i d e t e n s i o n s were measured on a Radiometer Acid-Base A n a l y z e r Type PMH 71. The PO2 and PCO2 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  saline  e q u i l i b r a t e d a t the d e s i r e d p a r t i a l p r e s s u r e s . Blood was a l l o w e d to f l o w from the animals  through  the cannulae  i n t o the PO2 and PCO2 e l e c t r o d e systems; the t o t a l amount o f b l o o d i n the cannulae  from one sample never exceeded  0.20 m l . A f t e r the r e a d i n g had been made (3-5 minutes) the b l o o d was g e n t l y f o r c e d back i n t o the animal, a n e g l i g i b l e amount o f b l o o d was l o s t and the t o t a l b l o o d volume o f the animal was p r o b a b l y n o t d i s t u r b e d . 2. A l v e o l a r Gases A l v e o l a r gas samples o f no l a r g e r than 0.20 ml were a n a l y z e d f o r oxygen, n i t r o g e n and carbon d i o x i d e on a V a r i a n Aerograph Gas Chromatograph S e r i e s 200. The s e p a r a t i o n columns used i n the chromatograph were S i l i c a - g e l ( s c r e e n s i z e 42/60) and M o l e c u l a r Sieve 5A ( s c r e e n s i z e 42/60). The columns were arranged i n s e r i e s w i t h the thermal c o n d u c t i v i t y d e t e c t o r . Standard gas samples used  to c a l i b r a t e the chromatograph were o b t a i n e d  12 from The Matheson Company o f Canada, Whitby, O n t a r i o and  the Canadian L i q u i d A i r Company, Vancouver,  The  t o t a l amount o f time r e q u i r e d f o r the a n a l y s i s of  one  sample was  s i x minutes. Gas  from the lungs i n a 0.25  B.C.  samples were taken  ml Hamilton  Syringe (Gastight  # 1750 w i t h a Chaney adaptor) and i n j e c t e d i n t o the gas chromatograph. 3. B l o o d  Pressure  To r e c o r d b l o o d pressure:, the cannulae were connected or  23 Db  to Statham 23AA ( a r t e r i s l ) ,  23BB (venous),  ( s m a l l volume a r t e r i a l ) p r e s s u r e t r a n s d u c e r s  and d i s p l a y e d on e i t h e r a Beckman Type R Dynograph or a G i l s o n Polygraph. The p r e s s u r e t r a n s d u c e r s were  cal-  i b r a t e d w i t h a column of s a l i n e p r i o r to and d u r i n g a l l experiments. A square wave p r e s s u r e change was a p p l i e d to  the complete system (cannulae, t r a n s d u c e r s , a m p l i f i e r  and pen r e c o r d e r ) i n a method d e s c r i b e d by S h e l t o n and Jones (1965a) and i t was of  the equipment was  found  t h a t the response  0.2-0.25 m s e c , ten times  than any p r e s s u r e change recorded from the  time  faster  experimental  animals. D. MEASUREMENT OF OXYGEN CONSUMPTION Oxygen consumption i n Amphiuma t r i d a c t y l u m  13 at  15*C  was measured i n two d i f f e r e n t ways. I n the  method an animal was  put i n t o a 4,850 ml  Erlenmeyer  f l a s k and immersed i n a water b a t h a t 15°C. The was  first  then : : i l l e d w i t h water, a l s o a t 15°C, and  flask  sealed  w i t h a rubber stopper p e r f o r a t e d w i t h a 5 ml p i p e t t e (Fig.  3 ) . The t i p o f the p i p e t t e extended i n t o the animal  chamber and was  a l s o f i l l e d w i t h water. I t was  from info:onation p r o v i d e d by Krogh  assumed  (1904), W i n t e r s t e i n  et.  a l . (1944), Jones (1967) and myself ( o b t a i n e d from  the  first  s e c t i o n of t h i s t h e s i s ) t h a t v e r y l i t t l e carbon  d i o x i d e i s r e l e a s e d i n t o the lungs o f amphibians. Hence, a drop i n the water ive  l e v e l i n the p i p e t t e would be  indicat-  o f a decrease i n lung volume and t h e r e f o r e the animals  volume and would r e p r e s e n t the amount o f oxygen consumed p l u s any n i t r o g e n d i f f u s i n g out o f the l u n g . The animal was  a l l o w e d to b r e a t h e once every hour a t which time the  f l a s k was set  s e a l e d a g a i n and the water l e v e l i n the p i p e t t e  a t z e r o . Oxygen and carbon d i o x i d e t e n s i o n s i n the  water  surrounding the animal were c o n t i n u a l l y monitored  by pumping water from the chamber through s i l a s t i c t u b i n g , p a s t the oxygen and carbon d i o x i d e e l e c t r o d e s , and back i n t o the animal chamber. In the  the second method o f oxygen  animal was  consumption  p l a c e d i n t o a 4,850 ml Erlenmeyer  flask  14  Figure  3  A p p a r a t u s u s e d f o r t h e measurement o f o x y g e n c o n s u m p t i o n i n Amphiuma a.  volume change method.  b. modified  Scholander  method.  tridactylum.  A.VOLUME  CHANGE  15 c o n t a i n i n g 1,000  ml o f f r e s h water. A i r samples were  taken every hour from the chamber through a t i g h t rubber s e a l a t the top o f the f l a s k and a n a l y z e d f o r oxygen, n i t r o g e n and carbon d i o x i d e c o n c e n t r a t i o n s on a gas c h r o matograph ( F i g . 3 ) . The Erlenmeyer f l a s k was  immersed  i n water a t 15°C. E . ANIMAL SURVIVAL Animals w i t h c h r o n i c i n d w e l l i n g cannulae s u r v i v e d f o r p e r i o d s i n excess o f sevan days. Death u s u a l l y r e s u l t e d from some v i o l e n t movement which p u l l e d the cannulae from the v e s s e l . In most cases however, the animal was  k i l l e d , a f t e r the experiments had been p e r -  formed, i n order to measure lung volume and the p h y s i c a l dimensions of the a n i m a l .  16  PART I .  GAS TENSIONS IN THE LUNGS AND MAJOR BLOOD VESSELS IN A FREE MOVING AMPHIBIAN, AMPHIUMA TRIDACTYLUM. INTRODUCTION I n the m a j o r i t y o f i n v e s t i g a t i o n s amphibian  b l o o d gas t e n s i o n s and contents have been  determined  from t e r m i n a l b l o o d samples (DeLong, 1962; Johansen, 1963). S e r i a l b l o o d samples f o r gas a n a l y s i s have been taken i n o n l y a few i n s t a n c e s (Len.cant and Johansen, 1967;  S h e l t o n , p e r s o n a l communication on u n p u b l i s h e d  d a t a ) . I n the experiments  r e p o r t e d here, s e r i a l b l o o d  samples were o b t a i n e d f o r b l o o d gas a n a l y s i s from moving, u n a n a e s t h e t i z e d  free  animals.  DeLong (1962) found  t h a t i n a n a l y s i s o f the  oxygen content o f t e r m i n a l b l o o d samples i n s e v e r a l Rana p i p i e n s "the c a r o t i d s r e c e i v e p r i m a r i l y l e f t  atrial  b l o o d , whj.ch i s h i g h l y oxygenated, whereas the pulmocutaneous v e s s e l s r e c e i v e b l o o d almost e x c l u s i v e l y  from  the r i g h t a t r i u m " . He a l s o found t h a t there was cons i d e r a b l e mixing o f oxygenated and deoxygenated b l o o d . Johansen (1963) found i n h i s a n a l y s i s o f the oxygen content o f t e r m i n a l samples i n the major v e s s e l s o f Amphiuma t r i d a c t y l u m t h a t i n most animals  sampled,  the a o r t i c a r c h r e c e i v e d b l o o d o f a h i g h e r oxygen c o n t e n t  than d i d the pulmonary a r c h . He a l s o found oxygen content i n the pulmonary v e i n was  t h a t the  s i m i l a r to t h a t  found i n the a o r t i c a r c h . The r e s u l t s he o b t a i n e d however, c o u l d p o s s i b l y be q u i t e abnormal i n t h a t i n most iments  pure oxygen was  sampling.  i n j e c t e d i n t o the lungs p r i o r  Johansen and D i t a d i (1966), working on  g i a n t toad, Bufo paracnemis, to those of Johansen  exper-  obtained r e s u l t s  to  the  similar  (1963).  Terminal b l o o d sampling  i s acceptable i f only  one b l o o d sample from the animal i s r e q u i r e d or i f the e x p e r i m e n t a l animal i s too s m a l l to take more than sample. T e r m i n a l d e t e r m i n a t i o n s have the  one  disadvantage  t h a t the s t a t e o f b l o o d i s known o n l y a t one  particular  time. Lengthy times between breaths must c e r t a i n l y a f f e c t the oxygen and carbon d i o x i d e l e v e l s i n the b l o o d and i t i s d i f f i c u l t  to determine  from one  sample the  normal gas l e v e l s i n the b l o o d . D e t e r m i n a t i o n of the oxygen or carbon d i o x i d e c o n t e n t o f b l o o d i s indeed u s e f u l f o r a complete unders t a n d i n g o f the r e s p i r a t o r y p h y s i o l o g y o f an amphibian; however, i f content d e t e r m i n a t i o n s are d e s i r e d b l o o d must be permanently removed from the body and sampling  serial  on one animal c o u l d d r a s t i c a l l y upset the nor-  mal p h y s i o l o g y by lowering the t o t a l b l o o d volume. P a r t i a l  18 p r e s s u r e d e t e r m i n a t i o n s have the advantage t h a t micro b l o o d samples a r e r e q u i r e d and can be r e t u r n e d to the body a f t e r  the d e t e r m i n a t i o n has been made. Content  then be c a l c u l a t e d from the b l o o d d i s s o c i a t i o n  can  curves.  S e r i a l b l o o d gas d e t e r m i n a t i o n s have been c a r r i e d out by L e n f a n t and Johansen (1967). They s u b j e c t e d t h r e e s p e c i e s of amphibians (Necturus maculosus, Amphiuma t r i d a c t y l u m and Rana c a t e s b e i a n a ) to prolonged i n the a i r or under water. Although  periods  the e f f e c t s on  i d u a l aniirals were n o t shown, they found  indiv-  that i n  Amphiuma t r i d a c t y l u m and Rana c a t e s b e i a n a the o v e r a l l e f f e c t of submergence was t e n s i o n s and  to lower  to s l i g h t l y r a i s e the b l o o d carbon d i o x i d e  t e n s i o n s and by keeping  the a q u a t i c Necturus maculosus  exposed to the a i r , they found dropped and  the b l o o d oxygen  t h a t b l o o d oxygen t e n s i o n s  the carbon d i o x i d e r o s e  slightly.  The p r e s e n t experiments were designed the changes i n gas  t e n s i o n s o f oxygen and carbon  i n the major v e s s e l s and  to study dioxide  lungs of Amphiuma t r i d a c t y l u m  d u r i n g normal b r e a t h i n g c y c l e s , and  to determine the r e -  l a t i o n s h i p s between these l e v e l s . Oxygen consumption was  a l s o measured.  19 MATERIALS AND These experiments  METHODS  were c a r r i e d out on  f o u r Amphiuma weighing between 250 and 1000  g.  fiftyAnimals  were n o t used f o r p h y s i o l o g i c a l e x p e r i m e n t a t i o n i f they had been a t the u n i v e r s i t y f o r a p e r i o d longer than s i x weeks. The water temperature  f o r a l l experiments  than oxygen consumption d e t e r m i n a t i o n s was  other  15-0.05°C.  The Amphiuma were a n a e s t h e t i z e d and  cannulated  as p r e v i o u s l y d e s c r i b e d . No more than three cannulae were i n s e r t e d i n t o the animal a t any one  time. I n a l l  cases a minimum of two hours was  allowed to e l a p s e be-  f o r e the s t a r t of the experiment  proper. Table I i n d i c -  a t e s the types of cannulae used and the number of iments done, c o n t r i b u t i n g of this Table I .  to the experimental  exper-  results  section. Types o f cannulae and numbers of animals  used.  Type o f c a n n u l a t i o n  number o f animals  lung alone  6  l u n g and d o r s a l a o r t a  9  lung & systemic a r c h & pulmonary v e i n  16  lung & d o r s a l aorta & post caval v e i n  5  lung & d o r s a l a o r t a & pulmonary v e i n  2  l u n g & pulmonary a r t e r y & pulmonary v e i n  1  B l o o d and lung gas t e n s i o n s were measured as d e s c r i b e d i n the G e n e r a l M a t e r i a l s and Methods. Oxygen consumption  of f i f t e e n Amphiuma a t 15°C was  measured  u s i n g the volume change technique (10 Amphiuma) and m o d i f i e d Scholander technique (5 Amphiuma). The  ten  animals measured a t 15°C by the volume change method were a l s o measured a t 25°C u s i n g the same method. RESULTS A. ALVEOLAR GASES Amphiuma t r i d a c t y l u m remains  submerged f o r  long p e r i o d s o f time and s u r f a c e s to b r e a t h e .  The  Amphiuma i n these experiments had a b r e a t h i n g i n t e r v a l o f 55 i 3 (S.E.) minutes temperature performed 285  a t 15°C. Although  critical  experiments were n o t done the one  a t 8°C extended  experiment  the b r e a t h i n g i n t e r v a l to  minutes. P02  i n the lungs d e c l i n e d between b r e a t h s ,  f a l l i n g from 101  - 4.8  to 44 - 2.9 mm  Hg. The h i g h e s t  P02's are r e c o r d e d immediately a f t e r a b r e a t h and lowest immediately p r i o r  to a b r e a t h ( F i g . 4 ) . Where  s e r i a l samples were taken c l o s e l y the shape of the PO2  the  enough to observe  disappearance curve, i t was  t h a t oxygen c o n c e n t r a t i o n s dropped  observed  i n a s l i g h t l y reversed  2 1  Figure 4  P02  decrease i n the lungs o f f o u r Amphiuma,  The h i g h e s t PO2  l e v e l s occur immediately  a b r e a t h and the lowest l e v e l s prior  to a b r e a t h .  after  immediately  22 sigmoid-shaped  c u r v e . More simply, the r a t e o f oxygen  decrease from the lungs was n o t as r a p i d i n the f i r s t f i f t e e n minutes  as i t was f o r most o f the remaining  Figure 4 also i l l u s t r a t e s  time.  the e f f e c t s o f a low temperature  on the n a t u r e o f the lung oxygen disappearance c u r v e . At  8 C°the PC*2 reached 23 mm Hg w i t h i n 285 minutes, when  the animal b r e a t h e d . A l v e o l a r PCO2 v a r i e d l i t t l e throughout the b r e a t h i n g c y c l e i n a l l experiments. The f i r s t  lung sample,  taken immediately a f t e r a b r e a t h , tended to be s l i g h t l y lower i n carbon d i o x i d e c o n c e n t r a t i o n than that found d u r i n g the r e s t o f the b r e a t h i n g i n t e r v a l . W i t h i n f i v e minutes  the a v e o l a r PCO2 r e t u r n e d to the mean l e v e l o f  14.9 mm Hg a f t e r the r e c o r d e d drop o f about 16% ( t h e percentage drop i s a mean v a l u e c a l c u l a t e d from 20 animals through 68 b r e a t h i n g c y c l e s ) and d i d n o t f l u c t u a t e app r e c i a b l y t h e r e a f t e r . I n o n l y one o u t o f twenty-two experiments did  the l e v e l s i n c r e a s e to any extent as the animal  remained for  I n which the lung carbon d i o x i d e was measured  submerged. A "PO2 and PCO2 diagram" was c o n s t r u c t e d  b r e a t h i n g c y c l e s o f s i x Amphiuma ( F i g . 5 ) . The d i a -  gram d e s c r i b e s the changes i n PO2 and PCO2 i n the lungs d u r i n g a b r e a t h i n g c y c l e . The R l i n e which would desc r i b e PO2 and PCO2 changes i n the Amphiuma lung would  23  Figure 5  Alveolar  "PO2-PCO2 diagram" f o r s i x Amphiuma  The diagram i l l u s t r a t e s tween a l v e o l a r  the r e l a t i o n s h i p be-  PO2 and PCO2.  24  e s s e n t i a l l y be equal t o zero d u r i n g the m a j o r i t y o f the  breathing i n t e r v a l . I t was found t h a t t h e r e was an i n v e r s e  rela-  t i o n s h i p between the a l v e o l a r P0£ and PN£ such that i f t h e r e was no i n c r e a s e i n the a l v e o l a r PCO2 over a p e r i o d of  time, the decrease i n PO2 would r e s u l t i n a s i m i l a r  i n c r e a s e i n PN2. T o t a l lung volume i n a l l animals was a f u n c t i o n of  the weight o f the animal ( F i g . 6 ) . Lung volume i n any  animal was determined by s u c c e s s i v e f i l l i n g air  and removal o f  through the i n d w e l l i n g c a n n u l a w i t h a c a l i b r a t e d  s y r i n g e . The accuracy o f t h i s method was confirmed by s e v e r a l a u t o p s i e s performed i n which the lungs were r e moved and the volume measured. I t was n o t p o s s i b l e to damage the  lung i n an animal by i n j e c t i o n s o f a i r ,  as soon as the lungs were f u l l ,  i n that,  the animal would open the  s p i r a c l e s on the a n t e r o - l a t e r a l p a r t o f the body and r e l e a s e excess a i r . T i d a l volume was e s t i m a t e d u s i n g an i n d i r e c t method. Knowing that the mean a l v e o l a r PO2 p r i o r t o a b r e a t h i s 44 mm Hg and immediately a f t e r a b r e a t h i s 101 mm Hg, and t h a t the PO2 o f the i n s p i r e d a i r i s about 160 mm Hg (dependent upon the atmospheric p r e s s u r e o f the day),  the t i d a l volume, based on a p r i n c i p l e o f  Figure 6  The r e l a t i o n s h i p between lung volume and body weight i n Amphiuma.  10001  900  800  BODY WEIGHT (gm) 700 «*  600  5001 30  40  50  LUNG VOLUME (ml)  26 / gas d i l u t i o n , would be about 72% o f t o t a l lung volume. B. BLOOD GAS CONCENTRATIONS 1. D o r s a l A o r t a F i g u r e 7 i l l u s t r a t e s the changes which o c c u r r e d in  the lung and d o r s a l a o r t a i n one animal  during  four  b r e a t h i n g c y c l e s . As the oxygen t e n s i o n s i n the lung the PO2 l e v e l s i n the d o r s a l a o r t a a l s o f e l l .  fell,  The r a p i d  r i s e i n t h e lung oxygen t e n s i o n s immediately a f t e r a breath r e s u l t e d i n an almost simultaneous r i s e i n the d o r s a l aorta PO2.  The g r a d i e n t e s t a b l i s h e d between the d o r s a l a o r t a and  lung oxygen t e n s i o n s was r e l a t i v e l y constant w i t h i n any animal  and d i d n o t change markedly b e f o r e , d u r i n g o r a f t e r  a b r e a t h once the g r a d i e n t had been e s t a b l i s h e d ( F i g . 8 ) . D o r s a l a o r t a PCO2 l e v e l s were always 1-10 mm Hg lower than lung PC02 l e v e l s . T h i s phenomenon was observed in was  a l l experiments i n which the lung and d o r s a l a o r t a PCO2 monitored. 2. Pulmonary V e i n Because o f the d i f f i c u l t y o f pulmonary v e i n  c a n n u l a t i o n , the number o f determinations  o f gas t e n s i o n s  i n b l o o d from t h i s v e s s e l was l i m i t e d . N e v e r t h e l e s s , i n all  experiments the pulmonary v e i n PO2 l e v e l s were v e r y  c l o s e t o those found i n the lung ( F i g u r e 9 i s an  27  Figure 7  PO2 and PCO2 changes which o c c u r r e d lungs and  d o r s a l a o r t a i n one  i n the  animal d u r i n g  f o u r b r e a t h i n g c y c l e s . V e r t i c a l arrows a t the top o f the diagram i n d i c a t e b r e a t h i n g times.  T  I  M  E  (min)  28  Figure 8  PO2 g r a d i e n t between the d o r s a l a o r t a or systemic  a r c h and the l u n g .  The % time submerged on the a b s c i s s a of the graph equates a l l b r e a t h i n g i n t e r v a l s , i . e . 1007o = i n t e r v a l between breaths Means a r e g i v e n - S.E. f o r each 20% o f the i n t e r v a l . Each mean r e p r e s e n t s n o t fewer 10 g r a d i e n t measurements.  than  DAorSA-LUNG  1  40  GRADIENT mm Hg 30  h  20  40  60  b TIME SUBMERGED  80  100  29  Figure 9  Changes which o c c u r r e d  i n the pulmonary v e i n  and lung P O 2 and PCO2 l e v e l s i n one animal d u r i n g f i v e b r e a t h i n g c y c l e s . V e r t i c a l arrows a t the top of the diagram i n d i c a t e b r e a t h i n g times.  example o f f i v e b r e a t h i n g c y c l e s i n one a n i m a l ) . Immedi a t e l y a f t e r a b r e a t h the PO2 g r a d i e n t between the lung and pulmonary v e i n was v e r y s m a l l b u t i n c r e a s e d as the animal  renained  submerged ( F i g . 10). Pulmonary v e i n  PCO2 l e v e l s were, i n almost every at  sample (one e x c e p t i o n  73 minutes, F i g . 9 ) , lower than PCO2 i n the l u n g s . 3. Pulmonary A r t e r y PO2 l e v e l s i n the pulmonary a r t e r y a r e h i g h e s t  immediately  a f t e r a b r e a t h and drop to the lowest  levels  j u s t b e f o r e a b r e a t h ( F i g . 1 1 ) . PO2 g r a d i e n t s (the term g r a d i e n t i n t h i s i n s t a n c e i s e s s e n t i a l l y a PO2 d i f f e r e n c e between two v e s s e l s ) between the d o r s a l a o r t a and pulmona r y a r t e r j were found  i n a l l experiments where these two  v e s s e l s were c a n n u l a t e d * . cuit  The b l o o d i n the systemic  cir-  (systemic a r c h or d o r s a l a o r t a ) i s always more  h i g h l y oxygenated than the b l o o d f l o w i n g to the lungs via  the pulmonary a r t e r y . F i g u r e 12 i l l u s t r a t e s  the PO2  d i f f e r e n c e s between these two c i r c u i t s i n e i g h t d i f f e r e n t Amphiuma. The g r a d i e n t , immediatel)' a f t e r a b r e a t h , dec r e a s e d as the l e n g t h o f submerged time i n c r e a s e d . The PO2 g r a d i e n t was i n i t i a l l y fell *  as l a r g e as 25-30 mm Hg and  to 1-5 mm Hg ( F i g . 1 2 ) .  From the p o i n t o f view o f b l o o d gas t e n s i o n s , no d i s t i n c t i o n was made between the d o r s a l a o r t a , systemic a r c h or ascending a o r t a and the terms w i l l be used interchangeably.  31  Figure 10  PO£ g r a d i e n t between the pulmonary v e i n and lung i n Amphiuma. G r a d i e n t measurements were made a t each p o i n t a PO2 d e t e r m i n a t i o n was made i n e i t h e r the lung or pulmonary v e i n . "]lhe % time submerged on the a b s c i s s a o f the g;raph equates a l l b r e a t h i n g  intervals i n a l l  emimals, i . e . 1 0 0 % = i n t e r v a l between b r e a t h s . I^he  l i n e was f i t t e d  to the data by eye.  32  F i g u r e 11 PO2  and PCQ2 changes which o c c u r r e d i n the  pulmonary a r t e r y , i n one  systemic  a r c h and  lung  animal d u r i n g a complete b r e a t h i n g  c y c l e . V e r t i c a l arrows a t the top o f diagram i n d i c a t e b r e a t h i n g  times.  the  33  F i g u r e 12 PO2 g r a d i e n t between the pulmonary a r t e r y and  systemic  Graphic  a r c h i n e i g h t Amphiuma.  p r e s e n t a t i o n i s the same as i n F i g . 10.  34 U s i n g an oxygen d i s s o c i a t i o n curve f o r Amphiuma t r i d a c t y l u m , d e r i v e d from data o f L e n f a n t and Johansen (1967) and S c o t t (1931),  the P 0  2  t e n s i o n s i n the systemic  a r c h and pulmonary a r t e r y shown i n F i g u r e 11 were conve r t e d to % s a t u r a t i o n ( F i g . 13). T h i s was done to desc r i b e the pulmonary a r t e r y - s y s t e m i c a r c h g r a d i e n t i n terms o f oxygen content as w e l l as p a r t i a l p r e s s u r e f e r e n c e s . A f t e r a b r e a t h the g r a d i e n t i n t h i s  dif-  case  ( F i g . 13) was about 257 and f e l l i n 50 minutes to 107 a  saturation  o  difference. There was no v i s i b l e PCO;? g r a d i e n t between the  pulmonary a r t e r y and systemic a r c h i n any experiment.  The  mean systemic a r c h or d o r s a l a o r t a PCO2 o f t e n Amphiuma was 11.9 mm Hg and from the same animals  the mean pulmon-  ary a r t e r y PCO2 was 12.2 mm Hg. 4. Post C a v a l V e i n From simultaneous  c a n n u l a t i o n s o f the d o r s a l  a o r t a and the p o s t c a v a l v e i n i t was p o s s i b l e to determine the PO2 d i f f e r e n c e between these two v e s s e l s through e r a l b r e a t h i n g c y c l e s . The composite  sev-  o f t h i s data i s shown  i n F i g u r e 14. Immediately a f t e r a b r e a t h the PO2 d i f f e r e n c e i s q u i t e l a r g e (17-30 mm Hg), a f t e r which the g r a d i e n t decreases  as the l e n g t h o f time the animal remains under  the water i n c r e a s e s (1-10 mm Hg^ immediately  before  35.  F i g u r e 13 PO2  l e v e l s i n the systemic a r c h and pulmonary  a r t e r y from F i g u r e 12 converted to p e r c e n t saturation.  100 i —  80  systemic arch  60  i - O LU  <f  < CO  40  20  pulmonary artery  80  120  160  TIME (min)  200  36  F i g u r e 14 PO2 and PCO2 g r a d i e n t between the p o s t c a v a l v e i n and d o r s a l a o r t a i n f i v e Amphiuma. Graphic  p r e s e n t a t i o n i s the same as i n F i g . 10.  b r e a t h i n g ) . Oxygen t e n s i o n s i n the post c a v a l v e i n a r e always lower than those recorded  i n the d o r s a l a o r t a .  Mean P C O 2 l e v e l s i n the p o s t c a v a l v e i n o f f i v e Amphiuma (46 samples) was 12.6 mm Hg. I n the same animals, the d o r s a l a o r t a PCO2 tensions had a mean o f 11.6 mm Hg ( i n d i v i d u a l samples from the two v e s s e l s were taken w i t h i n t e n minutes o f each o t h e r ) . 5. Standard Animal It  was i m p o s s i b l e  to o b t a i n blood  major v e s s e l s and lung gas measurements It  from a l l  simultaneously.  was however p o s s i b l e to c o n s t r u c t a g e n e r a l i z e d out-  l i n e o f the p r o b a b l e oxygen t e n s i o n s during a breathing lung g r a d i e n t ,  i n the major v e s s e l s  c y c l e . Knowing the pulmonary v e i n -  the d o r s a l a o r t a - l u n g g r a d i e n t ,  monary a r t e r y - s y s t e m i c  the p u l -  a r c h g r a d i e n t and the post  caval  v e i n - d o r s a l a o r t a g r a d i e n t , as w e l l as knowing the mean oxygen t e n s i o n s  i n the lungs and v e s s e l s  b e f o r e ar.d a f t e r a b r e a t h , this information  immediately  i t was p o s s i b l e to combine  to form a r e p r e s e n t a t i v e or "standard  a n i m a l " ( F i g . 15) . PO2  f e l l most r a p i d l y between breaths  i n the  pulmonary v e i n ( F i g . 15) and l e a s t r a p i d l y i n the p u l monary a r t e r y and p o s t c a v a l v e i n (which f a l l  a t the  38  F i g u r e 15 "The Standard Amphiuma". A r e c o n s t r u c t i o n o f the probable P O 2  f l u c t u a t i o n s i n the lungs  and major b l o o d v e s s e l s during three b r e a t h i n g c y c l e s . Estimates o b t a i n e d from a v a i l a b l e g r a d i e n t data and mean b l o o d and lung measurements .  TIME (min)  39 same r a t e ) .  Rapid r i s e s  i n P O 2 o c c u r r e d i n the b l o o d  and lungs immediately a f t e r  a breath.  C. OXYGEN CONSUMPTION IN AMPHIUMA. TRIDACTYLUM Oxygen consumption was measured  i n Amphiuma  by two methods d e s c r i b e d e a r l i e r . Table I I shows the mean consumption f o r f i f t e e n animals used. Table I I . Oxygen consumption measured  in fifteen  Amphiuma a t 15 and 25°C. Method Volume change Modified Scholander  Number o f animals  Temp°C  Mean oxygen consumption ul/am/hr  Time measu r e d per animal  10  15  6.67*0.14 (S.E)  5  15  6.89-0.039 (S.E.) 22-28 hours  25  19.0*0.23 (S.E.) 3 hours  Volume Change 10 (Same animals as a t 15°C)  3 hours  U s i n g two r e s p i r o m e t r y techniques, the mean oxygen consumption d i f f e r e d which oxygen was  very l i t t l e .  The r a t e a t  consumed over a one hour p e r i o d was not  r e g u l a r . F i g u r e 16 i l l u s t r a t e s the mean oxygen consumpt i o n f o r ten animals f o r a p e r i o d o f one hour a t f i v e minute i n t e r v a l s .  measured  Each experiment was r e p e a t e d  40  F i g u r e 16 Rates o f oxygen consumption a t 15°C i n ten Amphiuma measured  by a volume change  Means a r e g i v e n f o r each f i v e minute  technique. interval.  three times on each a n i m a l . The most r a p i d oxygen consump t i o n o c c u r r e d w i t h i n the f i r s t f i f t e e n minutes o f submergence; compared  to t h i s , consumption w i t h i n the l a s t  f o r t y - f i v e minutes i s very Amphiuma measured  slow. Oxygen consumption i n  a t 25°C i n c r e a s e d  to 19.0 ul/gm/hr, a l -  most a t h r e e f o l d i n c r e a s e from the consumption a t 15°C. .  42 DISCUSSION Amphiuma t r i d a c t y l u m breathes about once every hour almost completely r e p l a c i n g a l l a i r w i t h i n the lung a t each b r e a t h . B e f o r e t a k i n g a b r e a t h , the animal w i l l r a i s e i t s snout s l i g h t l y above the s u r f a c e of the water and w i l l remove most of the r e s i d u a l a i r from the lungs by l o w e r i n g the f l o o r of the mouth w i t h the e x t e r n a l nares c l o s e d . The f l o o r o f the mouth i s then r a i s e d and the e x t e r n a l nares opened and the a i r i n the mouth i s expelled  i n t o the a i r above the water  s u r f a c e . T h i s type  of b r e a t h i n g movement i s c h a r a c t e r i s t i c of  amphibians  which u t i l i z e pulmonary r e s p i r a t i o n as a method of gas exchange. Under normal circumstances a l l of the e x p i r e d a i r i s removed above the water  s u r f a c e such that b u b b l i n g  does not occur i n the water. Lung volume i n Amphiuma i s r e l a t e d to the s i z e of the i n d i v i d u a l animal and r e p r e s e n t s about 6-7% t o t a l volume of the a n i m a l . For comparison, lung volume of man (Lambertsen,  r e p r e s e n t s 7-8%  o f the  the t o t a l  o f the body volume  1961). An Amphiuma weighing 500-700 gm  would have, on average, a lung volume between 30-40 ml (Fig. 6). The removal of a i r from the lungs i s not comp l e t e because  oxygen t e n s i o n s do not approach  atmospheric  43 air  t e n s i o n s . In f a c t ,  t i d a l volume would appear to  be about 72% i n Amphiuma. T h i s i s much h i g h e r i n man  than  ( t i d a l volume of about 12%, a t r e s t ) but  the r a -  t h e r i n f r e q u e n t method of b r e a t h i n g would n e c e s s i t a t e an almost complete change of a i r w i t h i n the The lated  lungs.  amount of a i r taken i n t o the lungs i s r e -  to the l e n g t h of time the animal remained at  the  s u r f a c e , the amount of a i r p r e v i o u s l y removed from  the  lungs anc during  the l e v e l s to which the a r t e r i a l  l e v e l s dropped  the time submerged. Q u a n t i f i c a t i o n of the  were not p o s s i b l e but i t d i d appear, from t h a t the lower the a r t e r i a l  PO2  results  observations,  l e v e l s were, the  the animal remained a t the s u r f a c e b r e a t h i n g and  longer the more  " g u l p s " of a i r swallowed. Most a i r samples were taken from the p o s t e r i o r p o r t i o n of the l u n g . I n i t i a l l y , a f t e r a b r e a t h , P0£  levels  i n the lung d i d not change r a p i d l y even though the animal was  consuming oxygen a t a h i g h r a t e compared w i t h  occurring  towards the end  of a b r e a t h i n g  cycle.  l e v e l s i n p o s t e r i o r p o r t i o n s of the lung may maintained  by mixing a i r of a h i g h e r PO2,  a n t e r i o r p o r t i o n s of the lung, w i t h  that PO2  have been  from the more  t h a t i n the p o s t e r i o r  p o r t i o n s of the l u n g . T h i s mixing c o u l d have been aided by c o n t r a c t i o n s of the lung and movements of the  animal.  44 I n support  of t h i s statement, Czopek (1962) has  shown i n  Amphiuma means, a c l o s e l y r e l a t e d s p e c i e s to A. t r i d a c t y l u m t h a t t h e r e are a g r e a t number of smooth-muscle c e l l s i n the pulmonary w a l l s and  r i d g e s and  a r e a b l e to c o n t r a c t and The in  suggests  thereby promote a e r a t i o n " .  oxygen and carbon d i o x i d e c o n c e n t r a t i o n s  the lung i n Amphiuma are such t h a t gas  difficult  t h a t "the lungs,  exchange i s  to a n a l y z e using the O2-CO2 diagram. Hughes  (1966) suggests  t h a t oxygen and carbon d i o x i d e i n the  lung of Rana c a t e s b i a n a , when p l o t t e d on an O2-CO2 d i a gram, would f a l l one  or two  on the R=0.4 l i n e . In Amphiuma the  lung samples a f t e r a b r e a t h may  R=0.4-0.5 l i n e but as the animal  on  the  remains submerged  the  R l i n e e s s e n t i a l l y becomes zero and u  2 ~ ^ 2 diagram i n d e s c r i b i n g gas u  fall  first  the u s e f u l n e s s of  the  exchange i n t h i s amphib-  i a n becomes l i m i t e d . I t i s q u i t e p o s s i b l e t h a t i f Hughes had in  continued  the study  the lung a f t e r  to show the gas  concentrations  the animal had been submerged f o r a  longer p e r i o d of time ( c h a r a c t e r i s t i c of the (Noble  species  1931)), the R l i n e might have decreased  a b l y such as t h a t found  consider-  i n Amphiuma.  Oxygen consumption i n Araphiuma t r i d a c t y l u m is  the lowest recorded  of any  amphibian measured a t a  comparable temperature. (Brown, 1964;  Jones, 1967).  45 The r a t e o f oxygen uptake o f a submerged animal between b r e a t h s decreases r a p i d l y a f t e r sumption w i t h i n the f i r s t  the l a r g e i n i t i a l con-  5-10 minutes a f t e r a b r e a t h of  2-3.5 ul/gm body weight. The r a t e of decrease i n oxygen consumption i s s i m i l a r to the drop i n a l v e o l a r PO2 between b r e a t h s . I n the volume change experiments on oxygen consumption, the water PO2 was monitored c o n t i n u o u s l y throughout the three hour experimental p e r i o d . The P 0 in  2  the water over t h i s p e r i o d was never more than 1-2  drop mm  Hg. T h i s drop i n PO2 would account f o r no more than a 0.20.4 ml l o s s i n oxygen i n the whole system. I t i s c l e a r from t h i s evidence that the animals r e l y v e r y l i t t l e on oxygen i n the water to supply or supplement m e t a b o l i c demands. Since oxygen consumption measurements u s i n g the two techniques were almost i d e n t i c a l , i t appears that a volume change i n Amphiuma w h i l e submerged i s i n d i c a t i v e of  the amount o f oxygen being consumed, and a l l oxygen i s  taken up v i a the l u n g s . By the same r e a s o n i n g , i f the two methods g i v e s i m i l a r r e s u l t s , n i t r o g e n must not leave the lung as the animal consumes oxygen. I f n i t r o g e n , which b u i l t up i n the lung, was removed i n t o the b l o o d and water, there should have been a much l a r g e r volume change than was a c t u a l l y measured. The l a c k o f i n c r e a s e i n lung PCO2 between  46 breaths  i s i n t e r e s t i n g when one c o n s i d e r s the volume  changes which must occur i n the lung as oxygen d i f f u s e s i n t o the b l o o d . As a i r enters the lung d u r i n g a b r e a t h the lung ECO2 i s s l i g h t l y d i l u t e d b u t r e t u r n s to normal w i t h i n 4-6 minutes. The r e s u l t a n t i n c r e a s e i n lung PCO2 a f t e r a b r e a t h must r e s u l t from carbon d i o x i d e e n t e r i n g the lung from the b l o o d . I f t h i s i n i t i a l  amount o f carbon  d i o x i d e were to remain i n the lung, the lung PCO2 should i n c r e a s e by 2-4 mm Hg simply as a r e s u l t of the c o n c e n t r a t ing  effect, o f oxygen l e a v i n g the l u n g . T h i s however was  n o t observed  and the c o n c l u s i o n can be made t h a t carbon  d i o x i d e which entered  the lung from the b l o o d  immediately  a f t e r a b r e a t h , must i n p a r t r e t u r n to the b l o o d between b r e a t h s . The r e s u l t o f t h i s phenomenon then i s to have lung PCO2 c o n c e n t r a t i o n s h i g h e r of  than PCO2 l e v e l s i n any  the major b l o o d v e s s e l s . Once the PCO2 g r a d i e n t i s  e s t a b l i s h e d between the b l o o d and the lungs, no more carbon d i o x i d e e n t e r s the lungs u n l e s s b l o o d PCO2 l e v e l s i n c r e a s e s.harply. The oxygen g r a d i e n t between a i r and b l o o d the lung w a l l i s i n i t i a l l y  s m a l l b u t Increases w i t h  across time  between b r e a t h s . The c a l c u l a t e d volume o f oxygen l e a v i n g the lung decreases  w i t h time a f t e r a b r e a t h . The t r a n s f e r  f a c t o r o f the lung (V02/aP02) i s t h e r e f o r e f a l l i n g  47 d u r i n g the i n t e r v a l between b r e a t h s . The change i n t r a n s f e r f a c t o r c o u l d be r e l a t e d  to many f a c t o r s  includ-  i n g the volume and p a t t e r n o f b l o o d flow to the lungs, and  the d i s t r i b u t i o n of a i r w i t h i n the lungs. A change i n  t r a n s f e r f a c t o r i n d i c a t e s t h a t one or more of these parameters i s a l t e r e d and i s a f f e c t i n g gas t r a n s f e r r a t e s a c r o s s the l u n g . The h i g h PO2 l e v e l s i n the pulmonary v e i n immediately  a f t e r a b r e a t h would i n d i c a t e , f o r a s h o r t  period, o f time a t l e a s t , fully  the blood l e a v i n g the lung i s  s a t u r a t e d w i t h oxygen. Using an oxygen d i s s o c i a t i o n  curve f o r Amphiuma t r i d a c t y l u m b l o o d c o n s t r u c t e d by L e n f a n t and Johansen (1967), 100% s a t u r a t i o n occurs a t PO2 l e v e l s above 90 mm Hg. P0£ i n the pulmonary v e i n o f t e n exceeded t h i s l e v e l j u s t a f t e r a b r e a t h ,  indicating  t h a t d u r i n g t h i s p e r i o d , b l o o d l e a v i n g the lungs was fully  s a t u r a t e d w i t h oxygen. Somewhat more i n t e r e s t i n g i s the P 0 £ g r a d i e n t  e s t a b l i s h e d between the pulmonary a r t e r y and the systemi c a r c h . The problem o f whether the s i n g l e v e n t r i c l e o f an amphibian can m a i n t a i n a d i v i d e d stream o f oxygenated and deoxygenated blood and somehow d i r e c t the deoxygena t e d b l o o d to the lungs and the oxygenated b l o o d to the systemic c i r c u l a t i o n has been a t o p i c o f d i s c u s s i o n and  experimentation iments  s i n c e the mid-19th c e n t u r y . The  exper-  i n t h i s study show that there i s a d e f i n i t e oxy-  gen g r a d i e n t between these two c i r c u i t s over a long peri o d of time, even d u r i n g long i n t e r v a l s between b r e a t h s . DeLong (1962), Johansen (1963) and Johansen and  Ditadi  (1966) have a l l made measurements of oxygen and  carbon  d i o x i d e contents i n the two v e s s e l s but d i d not o b t a i n s e r i a l samples over a long p e r i o d of time. I n the three p u b l i s h e d works j u s t mentioned, oxygen content g r a d i e n t s were found  to e x i s t between the body and  lung  circuit  i n Rana p i p i e n s (DeLong, 1962), Bufo paracnemis and D i t a d i , 1966)  (Johansen  and Amphiuma t r i d a c t y l u m (Johansen,  I n t h i s i n v e s t i g a t i o n i t was g r a d i e n t between the pulmonary a r c h and Is p r e s e n t throughout  1963).  shown that the the systemic  arch  the b r e a t h i n g c y c l e . The g r a d i e n t  decreases as the animal remains submerged, but does not d i s a p p e a r c o m p l e t e l y . The g r a d i e n t i s d e f i n a t e l y g r e a t e r immediately mm  Hg)  and  (oxygenated  after  the animal has  taken a b r e a t h (20-25  t h i s i s probably because the pulmonary v e i n b l o o d ) and  the venous r e t u r n ( p r i m a r i l y  post  c a v a l v e i n , deoxygenated b l o o d ) g r a d i e n t i s g r e a t e s t a t t h i s time. Although a c t u a l PO2 g r a d i e n t s were not measured between the pulmonary v e i n and  the post c a v a l v e i n  i t can be seen from the r e c o n s t r u c t i o n of the standard  49 •. animal ( F i g . 15) that the PO2 g r a d i e n t between the v e s s e l s becomes s m a l l e r as the l e n g t h o f time submerged  increases.  T h i s i n t u r n appears to r e s u l t from the i n c r e a s e i n the lung-pulmonary v e i n g r a d i e n t increases. Therefore,  as the time a f t e r  breathing  the decrease i n the pulmonary a r t e r y -  systemic a r c h g r a d i e n t between b r e a t h s i s a f u n c t i o n o f the decrease i n the pulmonary v e i n - p o s t turn) gradient  c a v a l v e i n (venous r e -  and probably n o t a l o s s i n the s e p a r a t i o n  c a p a b i l i t i e s o f the h e a r t and a s s o c i a t e d I t i s important to point  vessels.  out t h a t oxygen  tensions  i n the pulmonary a r t e r y and systemic arches a r e u s u a l l y below 70 mm Hg. The changes i n blood changes i n percent  PO2 w i l l cause marked  s a t u r a t i o n as i t i s i n t h i s p o r t i o n o f  the Amphiuma d i s s o c i a t i o n curve t h a t changes i n PO2 r e s u l t i n l a r g e changes i n percent  s a t u r a t i o n (Compare F i g s . 11  and 1 3 ) . As has been d e s c r i b e d  earlier  (see General  M a t e r i a l s and Methods), continuous; measurements i n the venous r e t u r n ( i n t h i s case the post c a v a l v e i n ) a r e d i f f i c u l t i n that cannulation complex. N e v e r t h e l e s s ,  procedures a r e much more  s u f f i c i e n t data was  obtained  to a s c e r t a i n the l e v e l s o f PO2 i n the venous r e t u r n i n r e l a t i o n to the l e v e l s i n the d o r s a l a o r t a . The post  50 c a v a l v e i n - d o r s a l a o r t a PO2 mm  Hg immediately  between 5-10  mm  g r a d i e n t was  between 20-30  f o l l o w i n g a b r e a t h and decreased to  Hg immediately b e f o r e a b r e a t h . Rather  than r e f l e c t i n g an i n c r e a s e i n t i s s u e u t i l i z a t i o n of oxygen, t h i s decrease i n g r a d i e n t simply r e f l e c t s decrease i n t o t a l oxygen e n t e r i n g lung PO2  the  the c i r c u l a t i o n as the  drops, i . e . , d o r s a l a o r t a l e v e l f a l l s , VO2  falls  between b r e a t h s . All all  of the f l u c t u a t i o n s i n b l o o d PO2  levels i n  the major v e s s e l s o c c u r r e d i n free-moving Amphiuma  w i t h access to the s u r f a c e . The f l u c t u a t i o n s are d e f i n a t e l y r e l a t e d to the i n t e r m i t t e n t type of b r e a t h i n g which t h i s animal e x h i b i t s . L e n f a n t and Johansen  (1967) suggest,  on data c o l l e c t e d from s i x Amphiuma, that normal PO2  l e v e l s v a r y from 72-100 mm  mention  Hg  (9 samples) and do not  i n d i v i d u a l v a r i a t i o n withi.n one animal between  b r e a t h s . I f t h e i r animals were "prevented from for  arterial  43 minutes,  they found that a r t e r i a l PO2  surfacing"  dropped  what, a l t h o u g h i t i s i m p o s s i b l e to a s c e r t a i n from data the e f f e c t of submersion  some-  their  on the i n d i v i d u a l animal. The  i m p l i c a t i o n i s made by them t h a t f l u c t u a t i o n s i n a r t e r i a l PO2  are not normal  i n Amphiuma. T h i s i s i n t o t a l d i s a g r e e -  ment w i t h the p r e s e n t study. L e n f a n t and Johansen  (1967) have a l s o shown  51 t h a t i f Amphiuma were prevented  from s u r f a c i n g f o r p e r i o d s  o f 43 minutes the PCC*2 i n a r t e r i a l blood r o s e . I n a l l of the experiments i n the present study, where a r t e r i a l or venous PCO2 l e v e l s were monitored,  there was  no  indicat-  i o n t h a t submergence r e s u l t e d i n an i n c r e a s e i n blood PCO2 l e v e l s . A l b e i t , L e n f a n t and Johansen s experiments 1  were performed a t 20*C (5°C h i g h e r than the p r e s e n t s t u d y ) , but i t i s d o u b t f u l that t h i s i n c r e a s e i n experi m e n t a l temperature would r e s u l t i n t h i s d i f f e r e n c e i n response  to submergence. Because PCO2 l e v e l s do not i n c r e a s e i n e i t h e r  the lungs ( w i t h the e x c e p t i o n of the s l i g h t lung PCO2 r i s e s h o r t l y a f t e r a b r e a t h ) or the blood between b r e a t h s , we  can conclude  t h a t carbon d i o x i d e produced by metabol-  ism i s removed v i a the s k i n i n t o the surrounding T h i s concept 1904  i s not new  by Krogh (on anuran  water.  and has been shown as e a r l y  as  amphibians).  Carbon d i o x i d e t r a n s f e r from the b l o o d to water w i l l depend upon the dimensions of the exchange s u r f a c e , i.e.,  the s u r f a c e area of the s k i n , the volume and p a t t e r n  o f b l o o d flow through  the s k i n , and  g r a d i e n t s between the blood and  the c o n c e n t r a t i o n  the water. There i s a  much l a r g e r PCO2 g r a d i e n t e x i s t i n g between the blood and  the water than between the b l o o d and  the lungs. Carbon  52 d i o x i d e w i l l enter the lung u n t i l the g r a d i e n t i s s m a l l enough to e f f e c t i v e l y e l i m i n a t e f u r t h e r passage of d i o x i d e i n t o the lungs, whereas the amount of  carbon  carbon  d i o x i d e which can pass i n t o the water i s l a r g e because o f the h i g h s o l u b i l i t y of the gas fact  i n water as w e l l as  t h a t the volume of water surrounding  the  an animal i s  very large. The presence  of c a r b o n i c anhydrase i n b l o o d  i n c r e a s e s the r a t e of f o r m a t i o n of f r e e carbon from b i c a r b o n a t e and w i l l h e l p to m a i n t a i n h i g h  dioxide carbon  d i o x i d e l e v e l s i n the b l o o d as carbon d i o x i d e d i f f u s e s i n t o the water. Carbonic  anhydrase l e v e l s have not been  measured i n Amphiuma, but i t i s known that t h i s enzyme i s absent  i n the s k i n of Rana c l i m a t a n s and Rana Cates-  b i a n a (Maren, 1967). Hence, i n the presence  of low or  non-  e x i s t a n t l e v e l s of t h i s enzyme, the r a t e of f o r m a t i o n of carbon  d i o x i d e from b i c a r b o n a t e may  l i m i t the r a t e of  carbon  d i o x i d e t r a n s f e r to the water a c r o s s the  skin.  The Haldane e f f e c t i s small i n Amphiuma f a n t and Johansen, 1967)  and  t h e r e f o r e i s not v e r y  (Lenimport-  ant i n augmenting the removal of carbon d i o x i d e from the b l o o d i n t o the l u n g s . F i g u r e 17 i l l u s t r a t e s mode o f gas  the  probable  exchange i n Amphiuma t r i d a c t y l u m . The bucco-  pharyngeal mucosa of t h i s s p e c i e s was  not c o n s i d e r e d to  •  F i g u r e 17  The  probable mode of gas  Amphiuma  tridactylum.  exchange i n  53  LUNG CIRCUIT  BODY CIRCUIT  pulmonary artery  systemic arch  WATER  VERY LOW 0  venous return  2  INCOMPLETE DOUBLE CIRCULATION  pulmonary vein  be a s i g n i f i c a n t r e s p i r a t o r y exchange s u r f a c e p r i m a r i l y because o f i t s small s i z e i n comparison to the t o t a l body s i z e and secondly,  Czopek (1902) found that i n a  c l o s e l y r e l a t e d s p e c i e s , Amphiuma means, v a s c u l a r to the mouth r e g i o n was p o o r l y  developed.  supply  PART I I  RESPIRATORY CONTROL I N AI4PHIUMA TRIDACTYLUM INTRODUCTION Work i n t h e f i e l d  o f amphibian respiratory-  c o n t r o l has n o t been e x t e n s i v e . (1950) and de M a r n e f f e - F o u l o n neurophysi.ological in  recovery  heart  concentrations suggested  both during  (1966)  allowed  "that  submergence (1966), work-  ( B u f o b u £ o , Rana p i p i e n s and them t o s u r f a c e  o f oxygen, c a r b o n d i o x i d e the f r o g  into  and n i t r o g e n , lack  and p r o l o n g e d r e -  A l t h o u g h J o n e s h a s shown t h a t  and r e c o v e r y  different  i s s e n s i t i v e t o oxygen  development o f b r a d y c a r d i a  from i t " .  bradycardia  a:id how  r a t e i n the f r o g . Jones  on a v a r i e t y o f f r o g s  covery  (1964) and J o n e s  i n t e r e s t e d i n f a c t o r s a f f e c t i n g the  Rana t e m p o r a r i a ) ,  and  o f some r e s p i r a t o r y r e f l e x e s  jfrom d i v i n g b r a d y c a r d i a  influenced  and Z o t t e r m a n  (1962) i n v e s t i g a t e d t h e  t h e f r o g . J o n e s and S h e l t o n  were p a r t i c u l a r l y  ing  basis  Neil  diving  from d i v i n g b r a d y c a r d i a a r e  more s e n s i t i v e t o o x y g e n l a c k t h a n t o t h e p r e s e n c e o f carbon dioxide, (or  stimulus)  mained  the question  involved  o f what were t h e s t i m u l i  i n the c o n t r o l o f b r e a t h i n g r e -  unanswered. Taglietti  neurophysiological receptors  i n frog's  a n d C a s e l l a (1966) h a v e evidence demonstrating lungs a r e i n v o l v e d  presented  that  stretch  i n the t e r m i n a t i o n  56 o f the lung f i l l i n g  p r o c e s s . More r e c e n t l y (1968) they  have a l s o produced evidence f o r the presence  of d e f l a t -  i o n r e c e p t o r s i n f r o g s ' l u n g s . I t i s p o s s i b l e , from t h i s evidence a t l e a s t , t h a t the stimulus f o r i n f l a t i o n  and  the t e r m i n a t i o n of i n f l a t i o n c o u l d be r e l a t e d to the volume of a i r which the f r o g has i n i t s lungs. S i n c e Amphiuma t r i d a c t y l u m remains submerged f o r extended p e r i o d s of time i t was gas  convenient  (about 54 minutes a t 15°C),  to d e s i g n experiments i n which not only  t e n s i o n s i n the b l o o d and  lungs were monitored  but  a l s o to i n j e c t v a r i e d c o n c e n t r a t i o n s of oxygen, n i t r o g e n and carbon d i o x i d e i n t o the lungs and b r e a t h i n g responses  to observe  of the animal. Experiments  to determine the animal's  ability  i n the lungs were a l s o c a r r i e d  the  designed  to d e t e c t volume changes  out.  57 MATERIALS AND Twenty-two Amphiuma for  experimentation i n this  METHODS t r i d a c t y l u m were  section. Additional  n a t i o n was; o b t a i n e d f r o m 17 a n i m a l s u s e d this  used infor-  i n Part  I of  thesis. Experiments  to v a r i e d gas  to determine  c o n c e n t r a t i o n s were o f f o u r  o x y g e n t e n s i o n i n t h e l u n g s was quantities dioxide  of n i t r o g e n  into  carbon  dioxide  raised  and  tension  t h e l u n g s was  into  injecting  the l u n g s ; i i i ,  by  and  was lung  i v , the oxygen t e n s i o n i n  injecting  pure  oxygen i n t o  into  the lungs  cannula  into  t h e l u n g s as  inserted  the  on b l o o d and  l u n g s . G a s e s were i n j e c t e d ( P . E . 50)  carbon  i n the surrounding water  observed, raised  the  r a i s e d by  the c o r r e s p o n d i n g e f f e c t s  carbon dioxide  injecting  the l u n g s ; i i ,  of carbon dioxide  response  types; i , the  l o w e r e d by  t e n s i o n i n t h e l u n g s was  quantities  the animals  through  the a  previously  described. Termination of i n s p i r a t i o n performed as air  by  either  t h e a n i m a l was from  attempting  t h e l u n g s as  breathing jection  injecting  r e s p o n s e was  and  experiments  nitrogen into  were  the lung  t o s u r f a c e o r t o remove  t h e a n i m a l was recorded after  b r e a t h i n g . The the n i t r o g e n i n -  t h e amount o f a i r " s w a l l o w e d "  was  measured  i f a i r was m e c h a n i c a l l y being removed from the lung during  inspiration. Lung gas c o n c e n t r a t i o n s were measured on a  V a r i a n Aerograph Gas Chromatograph.  Water and b l o o d  and PCO2 were determined u s i n g a Radiometer  PO2  Acid-Base  A n a l y z e r . S t a n d a r d i z e d gas mixtures used f o r lung i n j e c t i o n s were o b t a i n e d from The Matheson Company of Canada and the Canadian L i q u i d . A i r Company. P r i o r to i n j e c t i o n o f gas m i x t u r e s i n t o the lung, the gases were s a t u r a t e d w i t h water. A l l experiments were performed on animals f r e e l y moving i n a twenty  l i t r e g l a s s aquarium  i n g ten l i t r e s of water. The water periments was m a i n t a i n e d a t  contain-  temperature i n a l l ex  15°C-0.5°C.  RESULTS A. BREATHING RATES I n a l l experiments i n which b l o o d and lung oxygen and carbon d i o x i d e tensions were measured, d i s turbances to the normal b r e a t h i n g c y c l e ( i . e . movement o f tank, loud low frequency n o i s e s , or r a p i d movements above the water  s u r f a c e ) were minimal. I n these exper-  iments the mean i n t e r v a l between b r e a t h s was  55-3  min-.  u t e s , the range from 26 to 120 minutes. An e x t e n s i o n o f the time between b r e a t h s c o u l d  \  59  be accomplished by i n j e c t i n g atmospheric a i r i n t o  the l u n g s .  Table I I I i l l u s t r a t e s experiments on f o u r d i f f e r e n t  Table I I I .  E x t e n s i o n of the time between b r e a t h s by injecting  Animal No.  atmospheric a i r i n t o  F r e q . of a i r injection  the lungs.  Time under water  Time from l a s t i n j e c t i o n to breath  1  20 ml/20-25 min  356 min  56 min  2  , 20 ml/20-40 min  303 min  41 min  20 ml/20 min  247 min  52 min  20 ml/20-24 min  345 min  62 min  3  ,  4  In  animals 1, 3 and 4 there were no apparent s i g n s of  agitation  or attempts  to s u r f a c e u n t i l i n j e c t i o n s  been stopped. I n animal 2 the time between was v a r i e d  had  injections  from 20-40 minutes and o c c a s i o n a l l y  after  35-40 minutes had e l a p s e d the animal moved around bottom of the tank as though i t was  the  about to b r e a t h e .  When these movements o c c u r r e d , a i r was the  animals.  injected  into  lungs and the animal c h a r a c t e r i s t i c a l l y c o i l e d i t -  s e l f on the bottom of the tank and remained I f the volume o f continuous a i r i n j e c t i o n s  submerged. exceeded  the  t o t a l lung volume, the animal removed the excess a i r as  60 bubbles  through An  the s p i r a c u l a r  openings  submerged.  e x t e n s i o n o f t h e time between b r e a t h s  a l s o be a c c o m p l i s h e d by i n j e c t i n g Twenty m l o f p u r e  pure  oxygen i n t o  o x y g e n p e r animal, was i n j e c t e d  l u n g s o f f i v e Amphiuma. The mean t h e s e a n i m a l s was i n c r e a s e d range  while  extending from  could the lungs.  i n t o the  time between b r e a t h s i n  t o 135-6 m i n u t e s ,  the t o t a l  108-170 m i n u t e s .  B . TERMINATION OF INSPIRATION I n a f r e e - m o v i n g Amphiuma, inated After  after this  the bottom tering  3-5  period  large  " g u l p s " o f a i r have been  the animal w i l l  swallowed.  t h a t i f a l l the a i r en-  the. l u n g s w h i l e b r e a t h i n g was s i m u l t a n e o u s l y removed, through  t h e l u n g c a n n u l a , the a n i m a l would  c o n t i n u e b r e a t h i n g f o r an extended illustrates  five  such experiments  amounts o f a i r removed f r o m s p i r a t i o n was  Table IV. A n i m a l No.  period  o f time. T a b l e IV  performed  where t h e a c t u a l  t h e l u n g was m e a s u r e d b e f o r e i n -  terminated.  A i r removal  from  the lungs during  N o r m a l L u n g Volume  •  1 2 3 4 5  i s term-  submerge a n d r e t u r n t o  o f t h e t a n k . I t was f o u n d  with a syringe  .  inspiration  40 47 34 37 45  ml ml ml ml ml  inspiration.  Volume removed b e f o r e termination of inspiration 78 m l 70 m l 76 m l 106 m l 95 m l  61 The a l t e r n a t e experiment was  performed on s e v e r -  a l o t h e r /cmphiuma. As an animal r a i s e d i t s neck and towards  the water  snout  s u r f a c e to breathe, pure n i t r o g e n was i n -  j e c t e d i n t o the lungs b e f o r e the animal reached the s u r f a c e . I t was  found that a f t e r an i n j e c t i o n o f n i t r o g e n , the anim-  a l d i d not b r e a t h e , but r e t u r n e d to the bottom o f the tank f o r a s h o r t p e r i o d o f time and then r e s u r f a c e d to b r e a t h e . Table V shows the amount of n i t r o g e n i n j e c t e d as f o u r animals  s u r f a c e d and the time e l a p s e d u n t i l  b r e a t h e . I f more than One experiment was  T a b l e V.  they r e s u r f a c e d to performed on  one  I n j e c t i o n of n i t r o g e n i n t o the lungs of Amphiuma which were about to s u r f a c e to b r e a t h e .  Animal  No  Amount o f N 2 i n j e c t e d as animal was s u r f a c i n g ml ml ml ml  Time u n t i l breath 5 11 • 9 5  min min min min  1  20 40 50 60  2  40 ml 20 ml  5% min 2% min  3  40 ml  6 min  4  40 ml  5 min  animal, a t l e a s t two hours were a].lowed between i n j e c t i o n s of n i t r o g e n . Any  experiment i n which the animals snout  a c t u a l l y came above the water  s u r f a c e b e f o r e or d u r i n g  62 the  i n j e c t i o n o f n i t r o g e n was  may  have e n t e r e d the l u n g .  C. BREATHING ONSET AND  d i s r e g a r d e d i n t h a t oxygen  ITS RELATION TO BODY PC02 LEVELS  F i g u r e 18 r e p r e s e n t s a s e r i e s of  experiments  i n which carbon d i o x i d e / a i r mixtures were i n j e c t e d the  into  lungs o f f o u r animals and the r a t e o f removal of  these gases from the lungs monitored u n t i l  the time of  b r e a t h i n g . The removal of carbon d i o x i d e from the lung, which was  present i n i t i a l l y  t h a t of normal l e v e l s , was oxygen. I t was water,  i n c o n c e n t r a t i o n s 3-5  times  s l i g h t l j ' f a s t e r than that of  assumed t h a t , s i n c e the animals were under  the carbon d i o x i d e was  b e i n g removed from the lung,  i n t o the b l o o d and then i n t o the surrounding water. I n all  cases the lung PCO2 l e v e l s  a base l e v e l (15-25 mm  ( F i g . 18) had r e t u r n e d to  Hg) 30-70 minutes b e f o r e the animal  breathed. V a r i o u s c o n c e n t r a t i o n s of carbon d i o x i d e i n air  were i n j e c t e d i n t o the l u n g s . A f t e r i n j e c t i o n ,  the  time to onset o f b r e a t h i n g was noted, and i n s e v e r a l i n stances the c o n c e n t r a t i o n o f gases i n the lung, as near to  the time o f b r e a t h i n g as p o s s i b l e , were a l s o measured.  In  these experiments a minimum o f t h r e e hours was  allowed  between i n j e c t i o n s i f the same animal was b e i n g used.  F i g u r e 18  The removal o f h i g h c o n c e n t r a t i o n s of carbon d i o x i d e and normal l e v e l s of oxygen from the lungs o f f o u r Amphiuma. Breathing rows and  times are marked w i t h v e r t i c a l a r the a b b r e v i a t i o n " b r . . n  Table VI i l l u s t r a t e s  the r e s u l t s o b t a i n e d .  The b r e a t h i n g  times a f t e r i n j e c t i o n o f 10% carbon d i o x i d e i n a i r were q u i t e v a r i a b l e , ranging  from 1 minute to 83 minutes w i t h  a mean o f 43.1-7.4 minutes. A f t e r i n j e c t i o n s o f 15% carbon d i o x i d e i n a i r , the response was l e s s v a r i a b l e , the time u n t i l b r e a t h i n g  ranged from 41 to 72 minutes  w i t h a mean v a l u e o f 51.8-4.1 minutes. A f t e r i n j e c t i o n o f 20% carbon d i o x i d e i n a i r , the mean b r e a t h i n g  time  dropped to 20.6 minutes. The number o f experiments done i n which more than 20%, carbon d i o x i d e was i n j e c t e d were fewer i n -.lumber and i t can only be s a i d t h a t i n j e c t i o n s of high concentrations  o f carbon d i o x i d e i n the lungs  (over 207o CO2) r e s u l t e d i n the animal coming to the s u r f a c e t o breathe w i t h i n a s h o r t time ( l e s s than 29 minutes). I t was i m p o s s i b l e between the onset  o f b r e a t h i n g and the b l o o d PCO2 l e v e l s .  F i g u r e 19 i l l u s t r a t e s which the systemic several breathing  to a s c r i b e any r e l a t i o n s h i p  the r e s u l t s from f o u r animals i n  a r c h PCO2 l e v e l s were f o l l o w e d c y c l e s . I t cannot even be s a i d  through that  b r e a t h i n g o c c u r r e d as the b l o o d PCO2 r o s e , f o r i n f a c t breathing  sometimes o c c u r r e d as the b l o o d PCO2 f e l l . If  artificially  the b l o o d PCO2 l e v e l s i n an animal were r a i s e d by i n c r e a s i n g the PCO2 i n the  65 Table V I . Time s i n c e l a s t breath  Carbon d i o x i d e i n j e c t i o n s Lung PO2 p r i o r to injection  Lung PCO2 p r i o r to injection  1 min  into  the lungs o f Amphiuma.  Type o f Time a f t e r injection injection until next b r e a t h  60 ml 10% C0  2  63 min  40 min  27 .5 ( 1 3 ) * 20 .4 (13)  60 ml 10% c o  2  15 min  41 min  28 .0 (13)  14 .0 (13)  60 ml 10% C0  2  1 min  — — —  60 ml 10% C0  2  43 min  (9)  20 ml 10% C0  2  30 min  2  83 min  52 min 4 min  53 .6  5 min  92 .7 (47)  22 .8 (47)  40 ml 10% c o  10 min  25 .8  (1)  21 .4  (1)  20 ml 10% CO2  43 min  4 min  146 .6  (3)  42 .7  (3)  60 ml 10% C02  6 miti  50 ml 10% C0  2  1 min  (9)  20 .6  8 min 7 min  33 .6  (6)  17 .4  (6)  70 ml 10% c o  2  85 min  10 min  30 •6  (5)  19 .7  (5)  80 ml 10% C0  2  62 min  5 min  20 ml 10% c o  2  61 min  3 min  30 ml 10% C0  2  46 min  15 min  60 ml 10% C0  2  64 min  60 ml 15% C02  72 min  41 min  15 ml 15% c o  2  45 min  57 min  30 ml 15% c o  2  41 min  64 min  31 .1  (4)  23 .9  (4)  30 ml 15% C02  3 min 4 min 8 min *  33 .7  (4)  21 .9  (4)  48 min  100 ml 15% c o  2  55 min  30 ml 15% c o  2  50 min  Bracketed numbers i n d i c a t e the time (minutes) t h a t had e l a p s e d between the i n j e c t i o n o f CO2 and the lung measurements .  66 Table VI.  Time s i n c e l a s t breath  59 min  (Continued)  Lung P 0 p r i o r to injection  Lung P C 0 p r i o r to injection  2  2  2  20 min  40 min  60 ml 20% C 0  2  17 min  3 min  60 ml 20% C 0  2  25 min  10 ml 30% C 0  2  1 min  51 min  10 ml 40% C 0  2  29 min  40 min  10 ml 50% C 0  2  3 min  —  23 min  65.0  30 min  --•  57 min 3 min  -  Time a f t e r injection until next b r e a t h  60 ml 20% C 0  8 min  -  Type o f injection  /  (9)  16.0  (9)  •  10 ml pure C 0  2  6 min  10 ml pure C02  2 min  20 ml pure C 0  2  3 min  20 ml pure C 0  2  5 min  67  F i g u r e ' 19  A r t e r i a l PCO2 l e v e l s through s e v e r a l b r e a t h i n g c y c l e s i n Amphiuma. V e r t i c a l arrows extending above the graphed l i n e f o r a p a r t i c u l a r animal, i n d i c a t e the time of b r e a t h .  J — — I 20  40  I 60  I 80  I  _i  100  120  T I M E  (min)  I  I  '  '  140  160  180  200  1  220  surrounding  seen ( F i g . 2 0 )  water, i t can be  t h a t as  P C O 2 of the water r o s e  to and  d o r s a l aorta PCO2 rose  to a l e v e l between 40-45 mm  When the water was PCO2 f e l l ,  replaced  the Hg.  (low P C O 2 ) the d o r s a l a o r t a  the i n c r e a s e i n d o r s a l a o r t a  ( F i g . 2 0 ) . A s i m i l a r experiment was  another animal and a o r t a PC0-, rose until  Hg,  P0£ f l u c t u a t i o n s i n the d o r s a l a o r t a d i d not  appear t o be a f f e c t e d by levels  remained a t 80 mm  the  i t was  to 40 mm  performed  found t h a t although Hg  PCO on  the d o r s a l  the animal d i d not  breathe  the oxygen t e n s i o n i n the d o r s a l a o r t a dropped to b  tween 30-40 mm  Hg  ( f o l l o w e d through three b r e a t h i n g  v a r y i n g i n l e n g t h from 42-61 D. BREATHING ONSET AND  minutes).  ITS RELATION TO BODY PO?  Removal of oxygen from the lungs was f l u s h i n g l a r g e amounts of n i t r o g e n  experiments per  o f oxygen removal from the lung on b r e a t h i n g Amphiuma. S i n c e o n l y pure n i t r o g e n was  spent submerged b e f o r e  the  effect  rate i n  being i n j e c t e d  important to c o n s i d e r  the  the n i t r o g e n i n j e c t i o n was  time made.  I f the time e l a p s e d between the p r e v i o u s b r e a t h and i n j e c t i o n time was b r e a t h i n g was  not c o n s i d e r e d ,  by  through the lungs v i a  formed on f i v e animals are shown to i l l u s t r a t e  i t was  LEVELS  accomplished  the lung cannulae. In Table V I I , e i g h t e e n  i n t o the animal,  cycle  the  the mean time to  9.0*1.22 minutes. I f i n j e c t i o n s were made  69  Figure 20  A r t i f i c i a l i n c r e a s e i n tank water PCO2 and the corresponding increase i n d o r s a l aorta P C O 2 . V e r t i c a l arrows a t the top of the diagram i n d i c a t e the b r e a t h i n g  times.  70 Table V I I . N i t r o g e n i n j e c t i o n s Time s i n c e l a s t breath  Lung PO2 p r i o r to injection  into  Lung PCO2 p r i o r to injection  7 .7 ( 3 ) * 10 .2  the lungs o f Amphiuma. Type o f injection  Time a f t e r injection until next b r e a t h  40 ml N  2  11 min  2 min  100 ml N  2  8 min  3 min  100 ml N  2  12 min  4 min  40 ml N  2  4 min  3 min  100 ml N  2  8 min  5 min  60 ml N  2  21 min  6 min  40 ml N  2  5.5 min  12 min  60 ml N  2  3 min  140 ml N  2  16 min  50 ml N  2  7 min  (1)  100 ml N  2  8 min  16 .0 (10)  60 ml N  2  17 min  80 ml N  2  3 min  17 min  100 ml N  2  13 min  20 min  50 ml N  2  5 min  75 ml N  2  3 min  1 min  13 min  7 .8  (5)  8 .9  (3)  (5)  14 min 2 min 15 min 15 min  27 min  3 .8  (1)  13 .0 (10) 8 .1  (1)  8 •14 (1)  4 .9  6 .6  10 .0  (I)  (1)  60 ml N,  37 min 46 min  16 .6  (6)  14 .8  (6)  15 ml N  2  5 min 13 min  * Bracketed numbers i n d i c a t e the time (minutes) that had e l a p s e d between the i n j e c t i o n o f N and the lung measurements . 2  71 w i t h i n 15 minutes a f t e r a b r e a t h , breaths after  the mean time between  was 15.3 minutes, or on the average 9.4 minutes  the i n j e c t i o n . I f i n j e c t i o n s  15 minutes or more a f t e r  o f n i t r o g e n were made  the animal had taken a b r e a t h ,  the mean time to breathe a f t e r  the i n j e c t i o n was 8.4  minutes ( t h e times o f 9.4 and 8.4 minutes a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t the 5% l e v e l ) . Using levels  data from P a r t I of: t h i s t h e s i s , P 0 £  i n the d o r s a l a o r t a and systemic  time o f b r e a t h i n g were o b t a i n e d imals  from f i v e d i f f e r e n t an-  ( F i g . 21). As i t was n o t p o s s i b l e to o b t a i n b l o o d  PO2 measurements a t the exact occasions,  i t was necessary  v a l u e s " by extending blood  a r c h a t the  time o f b r e a t h i n g on a l l  to e x t r a p o l a t e  the removal curve  "breathing  f o r oxygen i n the  to the time o f b r e a t h i n g . When i l l u s t r a t e d i n t h i s  manner ( F i g . 21) there was as much as 14 mm Hg d i f f e r e n c e i n a r t e r i a l breathing values during f i v e breathing i n one animal and as l i t t l e as 1 mm Hg through breathing  c y c l e s i n another  animal..  cycles  three  72  F i g u r e 21  PCX-, l e v e l s i n the d o r s a l a o r t a and  systemic  a r c h i n f i v e Amphiuma a t the time of b r e a t h i n g .  TIME (min)  DISCUSSION Amphiuma t r i d a c t y l u m breathes about once every hour (55 - 3 m i n u t e s ) . The animal becomes a g i t a t e d , to the s u r f a c e and f i l l s  rises  i t s lungs.. I t then r e t u r n s to  the bottom of the tank. The mean time between b r e a t h s , r e corded i n t h i s study, i s i n agreement w i t h data r e p o r t e d by Darnel]. (1948). I t i s apparent  ( P a r t I of t h i s  thesis)  t h a t the primary f u n c t i o n of s u r f a c i n g i n Amphiuma i s to r e p l e n i s h the a i r i n the l u n g s . What s t i m u l i determine  the  c e s s a t i o n of i n s p i r a t i o n ? I n f o u r experiments  i n which a i r was  from the I.ungs as the animal was  withdrawn  b r e a t h i n g , l a c k of  i n a t i o n of. the i n s p i r a t o r y process i n d i c a t e s t h a t  term-  filling  the lungs i n Amphiuma terminates b r e a t h i n g . T e r m i n a t i o n of i n s p i r a t i o n , a f t e r a c e r t a i n volume o f a i r had through the lungs and had been removed through n u l a , was  passed  the can-  probably because the speed of e x t r a c t i o n of a i r  d i d n o t equal the r a t e of lung i n f l a t i o n by the a n i m a l . The important o b s e r v a t i o n i s not the exact amount of a i r removed from the lungs but t h a t , as a i r was from the Lungs, more was  withdrawn  f o r c e d i n t o the lungs by the an-  i m a l than would n o r m a l l y f i l l  the l u n g s . The  inspiratory  event i s not based on a c e r t a i n number of " g u l p s " o f a i r b u t r a t h e r on the volume of a i r i n the lungs which i n  turn probably  t r i g g e r s s t r e t c h r e c e p t o r s i n the  These have been shown to be present: i n other (Neil et.al.,  1950;  Paintal,  T a g l i e t t i and C a s e l l a , 1966; The nature important  1963;  Shimac.a, 1966;  of the gas mixture  of the animal  c h a r a c t e r i s t i c and  i t was  amphibians  Widdicombe,  1964;  Jones, 1966).  seems to be  i n r e g u l a t i n g the t e r m i n a t i o n of  The b e h a v i o r  lung.  un-  inspiration.  p r i o r to i n s p i r a t i o n i s very p o s s i b l e to i n f l a t e the  with n i t r o g e n during this p e r i o d . B i l l i n g n i t r o g e n was  s u f f i c i e n t to terminate  response and  the animal  the  lungs  the lungs  with  inspiratory  r e t u r n e d to the bottom of  the  tank f o r a s h o r t w h i l e . This "appeasement" p e r i o d  was  s h o r t l i v e d and  i n a l l cases  the animal  s u r f a c e to breathe w i t h i n 2^-11 of response was  returned  to the  minutes. A s i m i l a r  recorded by Jones (1966). He  found  f r o g s s u r f a c i n g i n t o n i t r o g e n n e a r l y recovered  that  from  d i v i n g b r a d y c a r d i a , however only lung i n f l a t i o n s a i r and  type  with  " r e l e a s e of a n o x i a " brought about complete r e -  covery. Because of the d i f f i c u l t y of r e c o r d i n g an "E.C.G." through the t h i c k s k i n and musculature of Amphiuma, h e a r t r a t e was  not monitored i n most of the experiments i n t h i s  p a r t of tha study. I t i s p o s s i b l e however, t h a t there might be a r e l a t i o n s h i p between h e a r t r a t e and  breathing  75 s i n c e (as w i l l be shown i n P a r t I I I of t h i s study) there i s a g r a d u a l slowing down of the h e a r t r a t e as the animal remains  submerged. The major q u e s t i o n to be c o n s i d e r e d i n t h i s  p a r t o f the study i s what s t i m u l i are i n v o l v e d i n d e t e r mining the onset of b r e a t h i n g i n Amphiuma. Continuous i n j e c t i o n s of a i r i n t o the lungs r e s u l t i n animals being " s a t i s f i e d " to remain below the water  s u r f a c e . Submerged  times i n Excess of f o u r hours c l e a r l y i n d i c a t e that the s t i m u l i or s t i m u l u s i n v o l v e d i n t r i g g e r i n g the animals b r e a t h i n g response c o u l d be o v e r r i d d e n or negated by peri o d i c a i r i n j e c t i o n s . Such lengthy times beneath the water s u r f a c e a l s o i n d i c a t e , a. p r i o r i , be s a t i s f a c t o r i l y  that carbon d i o x i d e must  e l i m i n a t e d by r e s p i r a t o r y s u r f a c e s other  than the lungs. There i s the p o s s i b l y however, that carbon d i o x i d e was  removed as a i r bubbled out of the lungs a t the  time of a i r i n j e c t i o n . I n j e c t i o n o f pure oxygen i n t o the lungs of phiuma a l s o extended  Am-  the i n t e r v a l between b r e a t h s . I n j e c t i o n  o f 20 ml pure oxygen extended  the b r e a t h i n g i n t e r v a l to  s l i g h t l y more than twice the normal b r e a t h i n g i n t e r v a l . S i n c e the pure oxygen/air mixture i n the lungs would not exceed the normal lung c a p a c i t y , the lengthy submergence time would n e c e s s a r i l y decrease the volume of a i r i n the  lungs more than would occur  i f normal b r e a t h i n g had  p l a c e . T h i s alone would i n d i c a t e t h a t d e f l a t i o n found by  T a g l i e t t i and C a s e l l a  d i d not p r o v i d e The bon  the b r e a t h i n g  chemical  ( 1 9 6 8 )  stimulus  taken  receptors,  i n the f r o g lung, i n Amphiuma.  r e g u l a t i o n of r e s p i r a t i o n by  car-  d i o x i d e i n mammals i s a w e l l documented phenomenon  (review by Hornbein, 1 9 6 5 ) and d i o x i d e might p l a y an important breathing  i t was  thought t h a t carbon  r o l e i n the r e g u l a t i o n of  i n Amphiuma. Carbon d i o x i d e / a i r mixtures were  i n j e c t e d i n t o the lungs. The were i n no way  r e s u l t s of these i n j e c t i o n s  c o n c l u s i v e . For example the mean b r e a t h i n g  time a f t e r i n j e c t i o n of 1 5 7 o carbon d i o x i d e was utes longer j e c t i o n of  8 - 9 min-  than i f 1 0 % carbon d i o x i d e were i n j e c t e d . I n 207  o  carbon d i o x i d e r e s u l t e d i n the animal  b r e a t h i n g w i t h i n about 2 1 minutes, a d e f i n i t e  shortening  of the i n t e r v a l between i n j e c t i o n and b r e a t h i n g . In particular  this  i n s t a n c e the amount of oxygen i n the i n j e c t e d  sample would be  lowered to  1 6 - 1 7 %  simply by  o f the carbon d i o x i d e i n the gas m i x t u r e .  the presence  I t seems doubt-  f u l however, that a 5 % drop i n oxygen c o n c e n t r a t i o n i n the lung would shorten the next b r e a t h  the i n t e r v a l between i n j e c t i o n  to 2 1 minutes. Higher c o n c e n t r a t i o n s  carbon d i o x i d e ( 3 0 % - p u r e C O 2 ) i n j e c t e d i n t o the lungs  and of pro-  77 duces an even s h o r t e r i n t e r v a l between  breaths.  Amphiuma t r i d a c t y l u m , under normal  environ-  mental c o n d i t i o n s , would never encounter carbon d i o x i d e concentrations  as high as those i n j e c t e d i n t o the lungs.  Mammalian a l v e o l a r c o n c e n t r a t i o n s carbon d i o x i d e earlier  r a r e l y exceed 10%  (76 mm Hg S.T.P.) and i t has been shown  i n t h i s study t h a t the mean a l v e o l a r PC0 f o r 2  Amphiuma i s about 15 mm Hg.  In c o n c l u s i o n ,  i t appears  t h a t Amphiuma has some d e t e c t i o n mechanism whereby onset w i l l  breathing  occur more r a p i d l y i f u n p h y s i o l o g i c a l doses  o f carbon d i o x i d e a r e i n j e c t e d i n t o the lungs. I f the a r t e r i a l  PCC^ i s r a i s e d to twice the  normal l e v e l by i n c r e a s i n g the PCO^ o f the surrounding water, no change i n the normal b r e a t h i n g  pattern  results.  In normal, free-moving Amphiuma i n f r e s h water, no r e l a t i o n s h i p was found between the PCC^ l e v e l s lungs and the onset o f b r e a t h i n g of t h i s s t u d y ) . be  falling  be  constant  i n the blood and  (data used from Part I  PCC^ i n the major v e s s e l s might e i t h e r  slightly,  r i s i n g s l i g h t l y or more f r e q u e n t l y ,  a t the time o f b r e a t h i n g . E l i m i n a t i o n o f carbon d i o x i d e from the body  i s v e r y r a p i d i n Amphiuma t r i d a c t y l u m . injected  i n t o the lungs,  times .the normal l e v e l s ,  Carbon d i o x i d e  r a i s i n g a l v e o l a r PCC^ to 3-5 i s removed from the lungs w i t h i n  78 ten minutes a f t e r i n j e c t i o n . The r a p i d removal o f carbon d i o x i d e from the lungs, i f lung P C O 2 rapid r i s e i n blood P C O 2 ,  i f water P C 0  c r e a s e d , i n d i c a t e t h a t the Amphiuma rapidly  transfering  i s r a i s e d , and the 2  l e v e l s are i n -  s k i n i s capable o f  l a r g e amounts o f carbon d i o x i d e a c r o s s  this respiratory surface. I n j e c t i o n s o f n i t r o g e n i n t o the lungs o f Amphiuma, thereby  lowering  the a l v e o l a r P O 2 l e v e l s , i s somewhat more  p h y s i o l o g i c a l i n t h a t when an animal has remained submerged f o r p e r i o d s up to an hour, the lung i s normally  filled  with  .90-95% n i t r o g e n . The e f f e c t o f lowering a l v e o l a r P 0 i s that 2  the times between breaths a c t u a l response  i s d e f i n a t e l y shortened. The  time i s v a r i a b l e and i s r e l a t e d to the f a c t  t h a t , i f one p o s t u l a t e s the presence r e c e p t o r (which  o f an oxygen chemo-  t r i g g e r s the b r e a t h i n g response),  o f n i t r o g e n i n t o the lung w i l l  injections  lower b l o o d P O 2 a t a r a t e  which i s r e l a t e d to the oxygen r e s e r v e s (hemoglobin bound) i n the body and the m e t a b o l i c  r a t e o f the animal. The r a t e  o f oxygen consumption o f Amphiuma, as shown e a r l i e r , i s v e r y low and i s r e l a t e d to the b l o o d oxygen c o n t e n t . The mean time  to b r e a t h i n g o f n i n e minutes a f t e r i n j e c t i o n i s  not an unreasonable  l e n g t h o f time f o r b l o o d P O 2 l e v e l s to  drop to l e v e l s s i m i l a r to those i n the l u n g . I t seems q u i t e  79 reasonable breathing  to suggest a t t h i s p o i n t t h a t the onset o f i s much more s e n s i t i v e to oxygen d e p r i v a t i o n  than to i n c r e a s e s i n carbon d i o x i d e These o b s e r v a t i o n s  concentrations.  i n d i c a t e t h a t oxygen chemo-  r e c e p t o r s may be i n v o l v e d i n the i n i t i a t i o n o f b r e a t h i n g i n Amphiuma. R e l a t i v e c o n s i s t e n c y between the P O 2 i n the d o r s a l a o r t a and systemic  a r c h and the onset  i n d i c a t e s t h a t the r e c e p t o r  s i t e s might be l o c a t e d i n the  a r t e r i a l c i r c u l a t i o n . I n an o s c i l l a t i n g breathing  of breathing  system, such as  i n Amphiuma, i t does n o t seem unreasonable to  p o s t u l a t e a c o n t r o l mechanism t r i g g e r e d by another  oscil-  l a t i n g parameter (body P O 2 l e v e l s ) r a t h e r than a system i n which o s c i l l a t i o n s do n o t normally  occur  (body PCO2  l e v e l s ) . High carbon d i o x i d e l e v e l s however, do a l t e r the i n t e r v a l between b r e a t h s . Carbon d i o x i d e may have e i t h e r a d i r e c t or an i n d i r e c t e f f e c t on b r e a t h i n g i n Amphiuma. Increased  l e v e l s o f carbon d i o x i d e may  u l a t e oxygen consumption, which would shorten  stim-  the b r e a t h -  i n g i n t e r v a l . Other a l t e r n a t i v e s might be t h a t carbon d i o x i d e may e f f e c t  the r e l a t i o n s h i p between P0£ and b r e a t h -  i n g d i r e c t l y or perhaps carbon d i o x i d e simply has a d i r e c t e f f e c t on b r e a t h i n g  i n the c l a s s i c a l mammalian sense.  80 PART I I I .  SOME FEATURES OF THE CIRCULATION IN AMPHIUMA TRIDACTYLUM INTRODUCTION Noble (1931) and Foxon (1964) have reviewed the  work done up to f i v e years ago on the c i r c u l a t o r y  dynamics  of Amphibia. S h e l t o n and Jones (1965 a,b and 1968) and Johansen and Hanson (1968) p r o v i d e more r e c e n t  accounts  of r e s e a r c h b e i n g done i n t h i s a r e a . I n view o f the extens i v e reviews p r o v i d e d , only a brie:! resume on s u b j e c t s p e r t i n e n t to the p r e s e n t study w i l l be g i v e n The  here.  " c l a s s i c a l h y p o t h e s i s " o f b l o o d flow  through  the amphibian h e a r t was f i r s t put forward by Brucke (1852) and  l a t e r m o d i f i e d by S a b a t i e r (1873).  They s t a t e d that oxy  genated and deoxygenated b l o o d remained unmixed i n the vent ricle,  the. oxygenated b l o o d p o s i t i o n e d on the r i g h t s i d e o f  the v e n t r i c l e was the f i r s t  to l e a v e the h e a r t upon v e n t r i c  u l a r c o n t r a c t i o n . The d i r e c t i o n o f flow o f deoxygenated b l o o d through  the conus was a i d e d by the s p i r a l v a l v e and  because of the lower p r e s s u r e i n the pulmonary deoxygenated b l o o d p r e f e r e n t i a l l y flowed i n t o  circuit, the lung  cir-  c u i t . As p r e s s u r e i n the pulmonary and systemic c i r c u i t s became equal the s p i r a l v a l v e was then thought  to shut o f f  f l o w t o the pulmonary c i r c u i t and the oxygen r i c h b l o o d  81 l e a v i n g the v e n t r i c l e would e n t e r the systemic and  carotid  vessels. S i n c e Brucke and S a b a t i e r , Vandervael (1933) completely d i s c a r d e d the c l a s s i c a l h y p o t h e s i s and  stated  t h a t blood i n the v e n t r i c l e and major v e s s e l s was  com-  p l e t e l y mixed. Noble  (1925), Acolat: (1931, 1938), Foxon  (1951), Simons and M i c h a e l i s  (1953), de Graaf  Simons (1959), DeLong (1962), Sharma (1957),  (1957), Johansen  (1963), Jchansen and D i t a d i (1966) and S h e l t o n ( p e r s . comm.) have shown, by a v a r i e t y of techniques on  several  s p e c i e s of amphibians,  distrib-  u t i o n o f oxygenated ary  that t h e r e i s . a s e l e c t i v e  and deoxygenated  b l o o d to the pulmon-  and systemic c i r c u i t s . In  d i s c u s s i o n o f t h i s t o p i c the two p o i n t s of  agreement ( w i t h the e x c e p t i o n o f Vandervael) a r e : i.  the t r a b e c u l a t e n a t u r e of the  amphibian  v e n t r i c l e does enable the b l o o d to remain r e l a t i v e l y unmixed, ii.  b l o o d from the r i g h t a t r i u m ( l e a s t oxygenated) i s found i n the r i g h t s i d e o f the v e n t ricle, the  c l o s e r to the semilunar v a l v e s than  oxygenated b l o o d from the l e f t a t r i u m .  Much disagreement occurs i n the l i t e r a t u r e on the  sequence  82 of  events  a f t e r v e n t r i c u l a r s y s t o l e that f a c i l i t a t e the  movements of deoxygenated b l o o d to the  lungs.  'Differences i n the p u l s e p r e s s u r e between the pulmonary and systemic c i r c u i t s has  been recorded by  e r a l people. A c o l a t (1938) found a 1-3 mm p r e s s u r e i n the pulmocutaneous branch anurans. De Graaf that d i a s t o l i c  Hg  lower  sev-  diastolic  i n e i g h t s p e c i e s of  (1957), working on Xenopus l a e v i s ,  found  p r e s s u r e s i n the pulmocutaneous a r t e r y were  on the average 7 mm DeGraaf a l s o found  Hg  lower  than i n the other two  t h a t there was  arches.  a " l a g " i n the r i s e i n  p r e s s u r e i n the systemic c i r c u i t , i n that the p r e s s u r e i n the pulmocutaneous c i r c u i t r o s e 0.3.0-0.15 seconds b e f o r e the p r e s s u r e i n c r e a s e d i n the systemic c i r c u i t . Johansen (1963) c a n n u l a t i n g v e s s e l s a t a g r e a t e r d i s t a n c e from the h e a r t i n Amphiuma t r i d a c t y l u m recorded lower  diastolic  p r e s s u r e s i n the pulmonary a r t e r y than i n the  systemic  a r c h and a l s o recorded a " s l i g h t l y e a r l i e r p r e s s u r e i n the pulmonary a r t e r y " . N e i t h e r de Graaf nor a t t a c h e d any temic  rise  Johansen  s i g n i f i c a n c e to the p r e s s u r e l a g i n the s y s -  circuit. S h e l t o n and Jones (1968)., working on  three  s p e c i e s of Anura and one u r o d e l e , found c o n s i s t e n t l y  lower  p r e s s u r e s i n the anuran pulmocutaneous a r t e r y than were recorded s i m u l t a n e o u s l y i n the syscemic  a r c h , but  found  83 s i m i l a r s j ' s t o l i c and d i a s t o l i c p r e s s u r e s i n the u r o d e l e . S i m i l a r p u l s e p r e s s u r e s i n the u r o d e l e were thought to occur because o f the ductus B o t a l l i i n t h i s  particular  animal. S h e l t o n ( p e r s . comm.), i n r e c e n t b l o o d flow s t u d i e s on Xenopus l a e v i s , has evidence  f o r a slight increase i n  b l o o d flow to the pulmonary c i r c u i t p r i o r  to flow i n the  systemic. S h e l t o n and Jones ( p e r s . comm.) have data which suggests  chat i n some anurans there i s an i n c r e a s e i n  b l o o d f l o w ' t o the lungs f o r a p e r i o d of time f o l l o w i n g a b r e a t h b u ; that b l o o d flow to the two c i r c u i t s i s equal throughout the g r e a t e r p o r t i o n o f the b r e a t h i n g Johansen (1963) was the f i r s t  cycle.  to examine the  c a r d i o v a s c u l a r dynamics i n Amphiuma t r i d a c t y l u m and he o f f e r s a p a r t i a l e x p l a n a t i o n to the b l o o d flow p a t t e r n s in  the major v e s s e l s i n t h i s animal. I n g e n e r a l he r e -  corded b l o o d p r e s s u r e and b l o o d flow i n the major v e s s e l s and a l s o recorded c i n e f l u o r o g r a p h i c a l l y , the movement o f b l o o d through the Amphiuma h e a r t i n t o the a r t e r i a l  cir-  c u l a t i o n . He s t a t e d t h a t shunting o f deoxygenated b l o o d to  the pulmonary c i r c u i t and oxygenated b l o o d to the s y s -  temic c i r c u i t was accomplished  by "laminar outflow p a t t e r n s  from the v e n t r i c l e w i t h a r i g h t - h a n d s p i r a l movement through the u n d i v i d e d bulbus c o r d i s " . Johansen was a l s o  84 aware that: d i a s t o l i c and s y s t o l i c p r e s s u r e changes i n the major v e s s e l s c o u l d a l t e r  the s e l e c t i v e passage o f blood,  but s p e c i f i c d e t a i l s w i t h regards  to t h i s phenomenon were  not g i v e n The o b j e c t i v e s i n t h i s p a r t of the study were to r e c o r d b l o o d p r e s s u r e s s i m u l t a n e o u s l y i n the body and lung c i r c u i t s o f Amphiuma t r i d a c t y l u m and to determine i f there were any p u l s e p r e s s u r e d i f f e r e n c e s . The p o s s i b l e presence o f p r e s s u r e " l a g s " i n the systemic c i r c u i t and the e f f e c t s o f b r e a t h i n g and a i r i n j e c t i o n s upon b l o o d p r e s s u r e i n the major a r t e r i e s was a l s o i n v e s t i g a t e d .  MATERIALS AND METHODS The  experiments  i n t h i s study were performed  on  23 a d u l t Amphiuma t r i d a c t y l u m . The animals were cannulated by a method d e s c r i b e d p r e v i o u s l y i n t h i s t h e s i s  (General  M a t e r i a l s and Methods). B l o o d p r e s s u r e was monitored  w i t h Statham 23AA,  23BB or 23 Db p r e s s u r e t r a n s d u c e r s which were i n t u r n connected to a Beckman Type R Dynograph. P r e s s u r e  transducers  were c a l i b r a t e d w i t h a column of s a l i n e . The response of  time  the p r e s s u r e r e c o r d i n g equipment was 0.20-0.25 msec. The  experiments  were c a r r i e d out i n a 20  litre  g l a s s aquarium which was p a r t i a l l y f i l l e d w i t h 10 l i t r e o f . f r e s h water h e l d a t 15°C. A l l animals were  free-moving  and u n a n a e s t h e t i z e d ; r e c o r d s were not taken u n t i l  4-6  hours a f t e r the o p e r a t i o n .  RESULTS A. HEART HATE Heart r a t e i n any p a r t i c u l a r animal was v a r i a b l e . The  lowesc h e a r t r a t e recorded a t 15° C was 5 beats/min  the h i g h e s t 19 beats/min. c a r d i a immediately ing  and  I n g e n e r a l there was a tachy-  f o l l o w i n g a b r e a t h and a g r a d u a l slow-  down o f the h e a r t r a t e as the submerged time i n c r e a s e d .  86 The mean f l u c t u a t i o n i n h e a r t r a t e between breaths was 5.1 beats/min. F i g u r e 22 i l l u s t r a t e s or " b r e a t h i n g B  •  L  U  N  the " d i v i n g b r a d y c a r d i a "  t a c h y c a r d i a " i n s e v e r a l Amphiuma.  FILLING AND CIRCULATORY CHANGES  G  The breathed  t a c h y c a r d i a , which o c c u r r e d when Amphiuma  was, i n most cases, a s s o c i a t e d w i t h a s l i g h t  i n b l o o d p r e s s u r e i n both the pulmonary and systemic c u i t s . F i g u r e 23a i l l u s t r a t e s the b r e a t h i n g  drop cir-  t a c h y c a r d i a as  w e l l as the p r e s s u r e drop i n the two c i r c u i t s . I t i s important to note t h a t the p u l s e p r e s s u r e i n the pulmonary c i r c u i t does n o t decrease  to the same extent as does the p u l s e p r e s -  sure i n the systemic be observed  c i r c u i t . A s i m i l a r phenomenon c o u l d  by a r t i f i c i a l l y  filling  the lung w i t h a i r or  n i t r o g e n ( F i g . 23b). F i g u r e 23c f u r t h e r i l l u s t r a t e s the extent  to which lung volume a f f e c t s b l o o d and p u l s e p r e s -  s u r e s . I n j e c t i o n s o f a i r i n t o the lung r a i s e d the s y s temic d i a s t o l i c  p r e s s u r e by about 3 mm Hg. A f t e r a s h o r t  p e r i o d of time the a i r was removed and the d i a s t o l i c sure i n the systemic  a r c h r e t u r n e d to the o r i g i n a l  pres-  level.  I n j e c t i o n s o f low c o n c e n t r a t i o n s o f carbon d i o x i d e (5-15%) produced e f f e c t s on b l o o d p r e s s u r e and h e a r t r a t e s i m i l a r to those o f a i r i n j e c t i o n s . I f the p a r t i c u l a r  87  Figure 22  Diving bradycardia  or b r e a t h i n g  tachycardia  i n f i v e Amphiuma. A l l animals breathed time " 0 " and breathed marked by a v e r t i c a l  again at a point arrow.  at  88  F i g u r e 23  Breathing effects  and  on  systemic  lung  inflection  and  t h e pulmonar}' a r t e r y  arch  (SA). Recordings  deflation (PA)  and  obtained  simultaneously. a. B r e a t h i n g blood  t a c h y c a r d i a and  pressure  systemic  blood  b. A r t i f i c i a l c . The  effects  pressure.  the a s s o c i a t e d  drop i n the pulmonary  and  circuits.  filling  of  the  lungs with  of lung d e f l a t i o n  on  nitrogen  pulse  A.  B. 30 r-  PA  CO  co  LU ££ CL  U)  o  r  Q £ O  o  co  15  30  "AAAAAj  WWWWWIM  10ml N .  SA 15 J  •  •  »  i  '  '  j  '  i  J  i  i. i  c. 301-  PA  •\  15  lung deflation 30 r-  wvwwwvw .  SA  — /  10ml  AAAAA/WWW  5 mm interval  15  mX  '  t  i  l  l  |  L  -I  I  I  1  TIME (lOsec. interval)  L.  J  animal was i n a s t a t e o f b r a d y c a r d i a , a b r e a t h i n g  type  b r a d y c a r d i a would r e s u l t , d i a s t o l i c p r e s s u r e s would more i n the pulmonary a r t e r y than i n the systemic and  there was n o t a g e n e r a l decrease  fall  arch  i n blood pressure.  I n j e c t i o n s o f v e r y h i g h c o n c e n t r a t i o n s o f carbon  dioxide  (25, 50 and 100%) i n t o the lungs produced an almost immediate drop i n b l o o d p r e s s u r e and h e a r t r a t e ( F i g . 24). C. THE LAG PHENOMENON D i a s t o l i c p r e s s u r e s i n the pulmonary a r t e r i e s were on the average 3-5 mm Hg lower  than those  recorded  s i m u l t a n e o u s l y i n the systemic a r c h a t a p o i n t 1 cm ant e r i o r to the v e n t r i c l e . F i g u r e 25 i l l u s t r a t e s the f a c t  that there was a d e f i n i t e l a g i n b l o o d  this plus pressure  r i s e i n the systemic a r c h . The p r e s s u r e r i s e i n the p u l monary a r c h was v e r y r a p i d a t the s t a r t o f v e n t r i c u l a r s y s t o l e and i t was only a t the p o i n t where the b l o o d p r e s sure i n the two c i r c u i t s was equal t h a t the p r e s s u r e  rose  i n the systemic c i r c u i t . I n the animal d e s c r i b e d i n F i g u r e 24,  the l a g was c a l c u l a t e d to be 0.18-0.20 seconds i n  duration. F i g u r e 26a shows the p r e s s u r e i n the two main arches (about  8 beats/min).  relationships  i n a very slowly beating heart  The c a l c u l a t e d l a g i s 0.20-0.25 sec  90  F i g u r e 24  I n j e c t i o n s of h i g h c o n c e n t r a t i o n s of carbon d i o x i d e i n t o the lungs and  the a s s o c i a t e d  p r e s s u r e changes i n the systemic  arch.  25 X CO, 30 20 10 0  <  ^  J  1  I  I  I  I  I  I  I  I  I  I  K.  I  1  I  i  i  i—J  LU  50 # CO, rv  O < ^  " t/> LU G£  X o o o  30 r  4? 20 ort  E  10  0  -i  I  I  I  I  I  1  1  I  I  CO  lOO^CO, 30 20 1010  *  *  i  i  i  i  1—i  1 — i — J  TIME (lO sec. interval)  1  1—u  91  F i g u r e 25  Simultaneous p r e s s u r e r e c o r d i n g s i n the p u l monary a r t e r y (PA) and  systemic  arch  showing the p u l s e l a g i n the systemic a.  slow c h a r t d r i v e on r e c o r d e r .  b.  r a p i d c h a r t d r i v e o;i r e c o r d e r .  (SA) arch.  PA  2Q O 40  SA  20  TIME I N T E R V A L — I s e c  92  F i g u r e 26  Pressure  r e l a t i o n s h i p s i n the pulmonary  a r t e r y (PA) and  systemic: a r c h (SA) i n  a.  slowly beating  heart  b.  Superimposed p r e s s u r e r e c o r d i n g s from the pulmonary and another Amphiuma.  systemic  arches  in  6|_g  aanssaad  LULU  aooia  93 and  the d i a s t o l i c p u l s e p r e s s u r e d i f f e r e n c e between the  two arches i s 3.8 mm Hg. F i g u r e 26b shows the d i f f e r e n c e i n o u t f l o w p a t t e r n i n the two c i r c u i t s . There i s a more r a p i d f a l L i n b l o o d p r e s s u r e a f t e r i n c i s u r a i n the p u l monary a r t e r y than i n the systemic c i r c u i t . Peak s y s t o l i c p r e s s u r e s i n t h i s p a r t i c u l a r animal were reached  simult-  aneously  i n the two arches, which was always the case.  Systolic  pressures r a r e l y d i f f e r e d by more than 0.0-  1.0 mm Hg i n the two a r c h e s .  94 DISCUSSION Jones and S h e l t o n (1964) and Jones (1966; 1968) have d i s c u s s e d d i v i n g b r a d y c a r d i a i n s e v e r a l s p e c i e s o f anuran amphibians. They have shown t h a t d i v i n g b r a d y c a r d i a i s v e r y pronounced i n the Anura. Amphiuma t r i d a c t y l u m , an a q u a t i c u r o d e l e , does n o t show a r a p i d drop i n h e a r t r a t e upon submergence,  which may e i t h e r r e f l e c t  the f a c t  that  submergence was " v o l u n t a r y " or t h a t , because a submerged h a b i t a t i s - n o r m a l f o r Amphiuma, the d i v i n g b r a d y c a r d i a i s not very  pronounced. The s t i m u l i i n v o l v e d i n b r i n g i n g about d i v i n g  b r a d y c a r d i a i n amphibians i s s t i l l  unclear. Leivestad  (1960), working on the toad, Bufo bufo,. has shown t h a t submergence f o r two hours and the r e s u l t a n t d i v i n g bradyc a r d i a doss n o t r e s u l t i n an oxygen debt b e i n g b u i l t up. Jones (1957) has shown t h a t d u r i n g submergence i n three s p e c i e s o£ anuran amphibians, the r e l a t i o n s h i p between h e a r t r a t e and oxygen uptake i s simply;  " i f one i s low  then the other i s g e n e r a l l y low". I n Amphiuma the r e l a t i o n s h i p i s a l s o t h a t lower oxygen consumption d u r i n g the l a t t e r p a r t o f the submerged p e r i o d u s u a l l y c o i n c i d e s w i t h lower h e a r t r a t e s . When Amphiuma b r e a t h e s , icially  or the lungs a r e a r t i f -  i n f l a t e d w i t h a i r , there i s a g r e a t e r i n c r e a s e i n  95 p u l s e p r e s s u r e i n the systemic a r c h than i n the pulmonary a r t e r y . T h i s i s v e r y i n d i c a t i v e o f an i n c r e a s e d flow to the pulmonary c i r c u i t f o r a s h o r t p e r i o d o f time a f t e r a b r e a t h . Recent b l o o d flow s t u d i e s done by S h e l t o n ( p e r s . comm.) on Xenopus l a e v i s and Jones ( p e r s . comm.) on Rana p i p i e n s , i n d i c a t e t h a t f o r a s h o r t p e r i o d o f time a f t e r a b r e a t h there i s i n c r e a s e d b l o o d flow to the pulmonary  cir-  c u i t . A f t e r the i n i t i a l i n c r e a s e the blood flow to the pulmocutaneous dropped and i n Rana p i p i e n s there was l e s s f l o w to the pulmocutaneous than the systemic arches and i n Xenopus l a e v i s the b l o o d flow to the two arches was more or l e s s e q u a l . T h e r e f o r e , i n Amphiuma, i f p r e s s u r e  falls  as f l o w i n c r e a s e s i n the pulmonary a r t e r y , there must be a s u b s t a n t i a l f a l l i n lung p e r i p h e r a l r e s i s t a n c e d u r i n g b r e a t h i n g . To account  f o r t h i s phenomenon there must be  i n c r e a s e d v a s o c o n s t r i c t i o n d u r i n g the submerged p e r i o d and v a s o d i l a t i o n d u r i n g the b r e a t h i n g process and f o r a s h o r t p e r i o d o f time t h e r e a f t e r . I n j e c t i o n s o f n i t r o g e n i n t o the lungs produced the same e f f e c t s as those d e s c r i b e d by Jones (1966) i n t h a t there i s a " r e l e a s e o f the b r a d y c a r d i a " b u t the e f f e c t s on blood p r e s s u r e o f n i t r o g e n i n j e c t i o n are d i f f e r e n t : from those which occur when the animal breathes  normally.  S y s t o l i c and d i a s t o l i c p r e s s u r e i n c r e a s e i n the pulmonary  96 artery  but  return  and d i a s t o l i c 1-2  pressures  minutes but  resulting  in  planation  for  to normal w i t h i n  the  increase  diastolic  a decrease this  in  in  the  decrease  increase ance  in  in  heart  the body  placement  in The  after into  in  lung  rate  the body rapid of  is  experimentation  or  circuit  injections the  pulse  that  concentrations  area.  extremely  of  dioxide  high,  were  cases,  response already are  teen  recorded  shown within  to  the  increase  in  resist-  the p h y s i c a l  dis-  rate  and b l o o d of  pressure  carbon  dioxide  some  further  Explanations  this  pheno-  fact  that  to  of  ellicit  unphysiological  and s y s t e m i c  the  responses  explain without  pressures  e q u a l and occur time i n  injection  such  a  concentrations  necessary.  Peak s y s t o l i c  of  the  ex-  inflation.  high  response,  the pulmonary  lung  The  due  of  in heart  c o m p l i c a t e d by  in  as a r e s u l t  drop  menon a r e  carbon  the  for  elevated,  vasodilatory  possibly  to  arch  overall.  m i g h t be  c a u s e d by  this  remain  with nitrogen or  Systolic  systemic  pressure  difficult in  the  pressure  t h e r e may b e n o v a s o c o n s t r i c t i v e and  minutes.  pressures  pulse  m i g h t be  1-2  at  arches the  recording  to be  true  2 cm o f  recorded are,  in  same t i m e .  system i s in  simultaneously  this  the If  the  adequate  study)  the v e n t r i c l e ,  majority  and  frequency (this  has  pressures  pressure  pulses  97 from the v e n t r i c l e w i l l a r r i v e a t the r e c o r d i n g ultaneously  ( S h e l t o n and  D i a s t o l i c pressures  sim-  Jones,(1968); Womersley,(1955)). the r a t e o f r u n o f f are on the  other  hand r e l a t e d to p e r i p h e r a l r e s i s t a n c e , compliance and  heart  r a t e . Runoff has  and  sites  been shown by  Shelton  and  Jones (1968)  and  de G r a a f (1957) to be more r a p i d i n the pulmonary  and  i t was: suggested by de Graaf t h a t t h i s was  a r e s u l t of  the lower r e s i s t a n c e i n the lung c a p i l l a r y beds. The l a g recorded  i n the p r e s e n t  study, between the  r i s e i n the pulmonary a r t e r y and occur u n l e s s  there was  temic c i r c u i t  arch  pulse  pressure  systemic a r c h , c o u l d  some o c c l u s i o n to flow i n the  f o r a s h o r t p e r i o d of  not sys-  time.  I n an attempt to e l u c i d a t e the a n a t o m i c a l f u n c t i o n i n g o f the s p i r a l v a l v e and phiuma we::e  anaesthetized  associated  s t r u c t u r e s , ten  Am-  a f t e r normal e x p e r i m e n t a t i o n  and  the v e n t r i c l e , conus, truncus and circulatory  pulmonary p o r t i o n s  of  system were d i s s e c t e d f r e e of the body. I n  the sal-  i n e s o l u t i o n s , i n c i s i o n s or "windows" were made i n the ante r i o r p o r t i o n of the conus and p o s t e r i o r p o r t i o n of  the  truncus such that the f u n c t i o n i n g of the s p i r a l v a l v e  could  be observed w i t h a d i s s e c t i n g microscope. I t appeared  that  the s p i r a l v a l v e i n Amphiuma was  a t r i a n g u l a r r i d g e of  muscular t i s s u e extending the l e n g t h of the conus and  pro-  98 t r u d i n g i n t o the conus lumen to the extent of 2/3  of the  conus diameter. A t the a n t e r i o r end of the conus the  tri-  angular r:-.dge becomes a round, r a t h e r l o b u l a r s t r u c t u r e which appears  to occlude the entrance to the systemic  cir-  c u i t d u r i n g the i n i t i a l v e n t r i c u l a r outflow. T h i s t i s s u e c o u l d o c c l u d e the v e s s e l b r i e f l y as the b l o o d p r e s s u r e r i s e s or :Lt c o u l d simply be f o r c e d i n t o t h i s p o s i t i o n as the conus i s m e c h a n i c a l l y elongated as i t becomes t u r g i d w i t h b l o o d . Of the two a l t e r n a t i v e s , I would p r e f e r the " o c c l u s i o n as a b l o o d p r e s s u r e phenomenon" i n that the p r e s s u r e lag i n the systemic c i r c u i t i s v e r y b r i e f  and  a p u l s e p r e s s u r e i s recorded there a t the same time as  the  p r e s s u r e s i n the two c i r c u i t s are e q u a l . A p u l s e l a g of 0.2  seconds does not s t r i k e  one  as being s i g n i f i c a n t when compared to a h e a r t beat which extends  f o r 5-6  seconds. However, i f one c o n s i d e r s that  v e n t r i c u l a r output occurs from the i n c i s u r a ,  the s t a r t of s y s t o l e to  the l a g time can amount to 10-15% of the  v e n t r i c u l a r output  time. Could the amount of time, which  p r i m a r i l y deoxygenated b l o o d i s f l o w i n g to the pulmonary c i r c u i t , be long enough to s e t up two  the g r a d i e n t s between the  c i r c u i t s r e p o r t e d e a r l i e r i n t h i s study  ( P a r t 1)2  Spec-  u l a t i n g on the data a v a i l a b l e i t would appear t h a t , i f the  99 first  10-15% of the b l o o d l e a v i n g the v e n t r i c l e was  venous and  mixed  entered the pulmonary c i r c u i t , the g r a d i e n t s  tween the pulmonary a r t e r y and  be-  systemic a r c h i n Amphiuma  c o u l d have been o b t a i n e d by t h i s phenomenon. S h e l t o n and Jones (1968) have shown i n the u r o d e l e Salamandra salamandra t h a t there are no p u l s e p r e s s u r e d i f f e r e n c e s between the pulmonary and and  suggest  t h a t the presence  p r e s s u r e i n the two  no d i f f e r e n t i a l  circuits,  of a ductus B o t a l l i equalizes,  c i r c u i t s . Simons (1959), working  T r i t u r u s c r i s t a t u s and was  systemic  Salamandra maculosa, found  on  t h a t there  d i s t r i b u t i o n of b l o o d demonstratable  by  i n j e c t i o n of dye. I n t e r e s t i n g l y enough, both of these anima l s possess  a ductus B o t a l l i . This embryonic b l o o d v e s s e l  i s r e t a i n e d i n most u r o d e l e s ; Amphiuma p r o v i d e s one t i o n . I t f o l l o w s then, i f a l l t e r r e s t r i a l amphibians pendent upon pulmonary r e s p i r a t i o n , l a c k a ductus (to my  excepde-  Botalli  knowledge a l l a d u l t anurans l a c k t h i s v e s s e l ) the  p r e s s u r e d i f f e r e n c e c r e a t e d by having a separate pulmonary and  systemic c i r c u i t c o u l d be  s e p a r a t i o n of the two  s t r o n g l y i m p l i c a t e d i n the  types of b l o o d .  I t has been shown t h a t there are p u l s e p r e s s u r e d i f f e r e n c e s i n the two c i r c u i t s , a p o s s i b l e e x p l a n a t i o n  100 has been g i v e n as to how the d i a s t o l i c ences i n the two c i r c u i t s  pressure  differ-  c o u l d r e s u l t i n b l o o d from the  body bein;* i n p a r t sent to the lung c i r c u i t and the reason for  such a phenomenon I t h i n k i s b e s t s t a t e d by Foxon (196^ )  when he s a i d , "perhaps e v o l u t i o n a r y s e l e c t i o n has a c t e d i n favour o f those animals which possessed,  n o t some hy-  p o t h e t i c a l mechanism f o r the s e l e c t i o n o f b l o o d f o r the head r e g i o n , b u t some mechanism which prevented which had r e t u r n e d from the lungs from b e i n g sent there a g a i n " .  blood  immediately  101 SUMMARY 1. The experimental animal o f t h i s study was Amphiuma t r i d a c t y l u m , an a q u a t i c u::odele. Amphiuma b r e a t h e d about once every hour and almost  completely  r e p l a c e d a l l a i r w i t h i n the lungs a t each b r e a t h . 2. While Amphiuma remained  submerged between  b r e a t h s , oxygen was removed from the lungs but carbon d i o x i d e l e v e l s d i d n o t i n c r e a s e . The R l i n e f o r a l v e o l a r gases  i n t h i s animal was t h e r e f o r e z e r o . 3. Oxygen consumption i n Amphiuma a t 15°C  was the lowest r e c o r d e d f o r any amphibian temperature. first  a t a comparable  Most o f the oxygen was consumed w i t h i n the  f i f t e e n minutes o f submergence. The primary  respir-  a t o r y s u r f a c e f o r oxygen consumption was the l u n g s . 4. Oxygen t e n s i o n s i n the major v e s s e l s o s c i l l a t e d w i t h the b r e a t h i n g c y c l e s . There was a d e f i n i t e g r a d i e n t between the pulmonary a r t e r y and systemic a r c h which p e r s i s t e d throughout  the b r e a t h i n g c y c l e . The grad-  i e n t decreased w i t h time submerged, b e i n g caused by the decrease i n g r a d i e n t between the oxygen t e n s i o n s i n the pulmonary v e i n and venous r e t u r n . 5. A f t e r each b r e a t h i n Amphiuma the oxygen t e n s i o n s , i n a l l the v e s s e l s s t u d i e d r o s e r a p i d l y , the t e n s i o n s i n the pulmonary v e i n i n c r e a s e d to l e v e l s  found  102 i n the lungs, and were u s u a l l y completely 6. T e r m i n a t i o n  saturated.  o f i n s p i r a t i o n was shown to be  c o n t r o l l e d by a volume d e t e c t i o n mechanism. Animals were shown to c o n t i n u e aneously  the b r e a t h i n g process  removed from the lungs  i f a i r was s i m u l t -  through a lung  I n j e c t i o n s o f n i t r o g e n i n t o the lungs  cannula.  terminated  inspir-  a t i o n f o r a s h o r t time but b r e a t h i n g o c c u r r e d a s h o r t time a f t e r . 7. Carbon d i o x i d e i n the. lungs i n doses 3-5 times  the normal l e v e l s were removed from the lungs  r a p i d l y and d i d n o t r e s u l t i n the onset of b r e a t h i n g i f the oxygen t e n s i o n s were s u f f i c i e n t l y h i g h . Very h i g h c o n c e n t r a t i o n s of carbon d i o x i d e i n the lungs r e s u l t e d i n a s h o r t e n i n g o f the time between b r e a t h s .  Increased  l e v e l s of carbon d i o x i d e i n the d o r s a l a o r t a d i d n o t b r i n g about the b r e a t h i n g  response.  8. Removal o f oxygen from the lungs brought about a r a p i d b r e a t h i n g response.  The presence of an  a r t e r i a l oxygen chemoreceptor was p o s t u l a t e d as a mechanism f o r c o n t r o l l i n g b r e a t h i n g i n Amphiuma. 9. The d i v i n g b r a d y c a r d i a response i n Amphiuma was n o t v e r y pronounced and was q u i t e i r r e g u l a r . 10. When Amphiuma breathed  there was a g r e a t e r  i n c r e a s e i n p u l s e p r e s s u r e i n the systemic  a r c h than i n  103 the pulmonary a r t e r y . I f p r e s s u r e f e l l as flow i n c r e a s e d , t h e r e must have been a s u b s t a n t i a l f a l l r e s i s t a n c e when the animal 11. There was  i n lung p e r i p h e r a l  breathed.  a lower  diastolic  p r e s s u r e i n the  pulmonary a r t e r y than i n the systemic a r c h . P u l s e p r e s s u r e was  g e n e r a l l y g r e a t e s t i n the pulmonary a r t e r y . There  was  a slight  p r e s s u r e l a g i n the systemic a r c h compared to the  pressure  c i s e i n the pulmonary a r t e r y . I t was  t h a t the f i r s t  b l o o d to l e a v e the v e n t r i c l e would flow to  the pulmonary a r t e r y i n i t i a l l y p r e s s u r e i n the lung c i r c u i t entrance  suggested  because of the  lower  and p o s s i b l y because the  to the systemic c i r c u i t appeared to be  d u r i n g the i n i t i a l  phase of v e n t r i c u l a r output.  p r e s s u r e l a g phenomenon i n the systemic a r c h was to account  f o r the PO2  a r t e r y and  the systemic  blocked The thought  d i f f e r e n c e between the pulmonary arch.  104 LITERATURE CITED A c o l a t , M.L.  1931. Recherch.es anat;omiques r e l a t i v e s a l a  s e p a r a t i o n du sang veineux e t du sang dans l a coer de l a G r e n o u i l l e . C.R.  arterial  Acad. S c i .  P a r i s . 192: 767-769. 1938. Etude compare de l a p r e s s i o n sanguine dans l e c i r c u i t pulmonaire e t dans l e c i r c u i t general C.R. Baker, C. L .  chez l e z B a t r a c i e n  Acad. S c i . 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P h y s i o l . Scand. Suppl. 217: 1-82. Johansen, K. and A.S.F. D i t a d i . 1966. Double c i r c u l a t i o n i n the g i a n t toad Bufo paracnemis. P h y s i o l . Z o o l . 39: 140-150. Johansen, K. and D. Hanson. 1968. F u n c t i o n a l anatomy o f the h e a r t s o f l u n g f i s h e s and amphibians Am.  Z o o l o g i s t . 8: 191-210.  106 Jones, D. R. 1966. F a c t o r s a f f e c t i n g  the r e c o v e r y from  d i v i n g b r a d y c a r d i a i n the f r o g . J . Exp. B i o l . 44:. 397-411. i  1967. Oxygen consumption and h e a r t r a t e o f s e v e r a l s p e c i e s o f Anuran Amphibia d u r i n g submergence. Comp. Biochem. P h y s i o l . 2fJ: 691-707. 1968. S p e c i f i c and seasonal v a r i a t i o n s i n development o f d i v i n g b r a d y c a r d i a i n Anuran Amphibia. Comp. Biochem. P h y s i o l . 25_: 821-834.  Jones, D.R. and G. S h e l t o n . 1964. f a c t o r s  influencing  submergence and the h e a r t r a t e i n the f r o g . J . 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