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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.Sc, University of Alberta, 1963 M.Sc., University of Alberta, 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1969 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Lib 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 for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Depart ment or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of p-g>\ r=> '<* \ \ The Unive r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date i ABSTRACT Respiratory and c i r c u l a t o r y changes which accompany submergence were studied i n Amphiuma tridactylum, an aquatic urodele. A l l experimentation was performed at 15° C with the exception of oxygen consumption recording taken at 25° C. The lungs, systemic arches, pulmonary a r t e r i e s , pulmonary veins, post caval vein and dorsal aorta were canaulated. S e r i a l sampling procedures enabled P O 2 and P C O 2 measurements to be made through several breathing cycles on a l l animals. Oxygen consumption i n Amphiuma at 15° C was the lowest recorded for any amphibian at a comparable temp-erature. I t was found that the lungs were the primary resp i r a t o r y exchange surface for oxygen consumption and were used very l i t t l e f o r carbon dioxide elimination. Oxygen tensions i n the major vessels showed large f l u c t u a t i o n s which were rel a t e d to the breathing cycle of the animal. Amphiuma breathed about once every hour at 15° C and i t was j u s t a f t e r a breath that oxygen tensions i n the lungs and major vessels were the highest. There was a d e f i n i t e gradient between the pulmonary artery and systemic arch which pers i s t e d throughout the breathing c y c l e . Termination of 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 detection mechanism. * • 1 1 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 brought about a b r e a t h i n g response whereas there was no r e l a t i o n -s h i p between the carbon d i o x i d e l e v e l s i n the body and the b r e a t h i n g response. The pulmonary a r t e r y had a lower d i a s t o l i c b l o o d p r e s s u r e than d i d the systemic a r c h . There was a s l i g h t p u l s e l a g i n the systemic a r c h when compared to the p r e s s u r e r i s e i n the pulmonary a r t e r y such t h a t a p r e s s u r e r i s e was n o t rec o r d e d i n the systemic a r c h u n t i l the b l o o d p r e s s u r e i n the two c i r c u i t s became e q u a l . I t was suggested t h a t the p u l s e l a g c o u l d account f o r deoxy-genated b l o o d b e i n g p r i m a r i l y shunted to the pulmonary c i r c u i t , and oxygenated b l o o d being shunted to the systemic c i r c u i t . • • * 1 1 1 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 i v DISCUSSION 94 GENERAL SUMMARY 101 LITERATURE CITED 104 V LIST OF TABLES Pages I . Types of cannulae and numbers of animals used. 19 I I Oxygen consumption measured i n f i f t e e n Amphiuma at 15°and 25° C 39 I I I Extension of the time between breaths by i n j e c t i n g atmospheric a i r into the lungs.. 59 IV A i r removal from the lungs during i n s p i r a t i o n 60 V I n j e c t i o n of nitrogen into the lungs of Amphiuma which were about t o s u r f a c e to breathe 61 VI Carbon dioxide i n j e c t i o n s into the lungs of Amphiuma 65 VII Nitrogen i n j e c t i o n s into the lungs of Amphiuma 70 v i LIST OF FIGURES Pages Figure 1 A drawing of the techniques used i n the cannulation of Amphiuma tridactylum 8 Figure 2 The major blood vessels, lungs and heart of Amphiuma tridactylum 10 Figure 3 Apparatus used for the measurement of oxygen consumption i n Amphiuma tridactylum 14 Figure 4 PO2 decrease i n the lungs of four Amphiuma tridactylum 21 Figure 5 Alveolar "PO2-PCO2 diagram" for six Amphiuma 23 Figure 6 The r e l a t i o n s h i p between lung volume and body weight i n Amphiuma 25 Figure 7 PO2 and PCO2 changes which occurred i n the lungs and dorsal aorta i n one animal during four breathing cycles 27 Figure 8 PO2 gradient between the dorsal aorta or systemic arch and the lung 28 Figure 9 Changes which occurred i n the pulmonary vein and lung P02 and PCO2 level s i n one animal during f i v e breathing cycles . 29 Figure 10 POo gradient between the pulmonary vein and lung i n Amphiuma 31 Figure 11 PO2 and PCO2 changes which occurred i n the pulmonary artery, systemic arch and lung i n one animal during one complete breathing cycle 32 Figure 12 PO2 gradient between the pulmonary artery and systemic arch i n eight Amphiuma 33 Figure 13 PO2 l e v e l s i n the systemic arch and p u l -monary artery (from F i g . 12) converted to per cent saturation 35 v i i Pages Figure 14 PO2 and PCO2 gradient between the post caval v e i n and dorsal aorta i n f i v e Amphiuma 36 Figure 15 "The Standard Amphiuma". A reconstruct-ion of the probable P02 fluctuations i n the lungs and major blood vessels during three breathing cycles 38 Figure 16 Rates of oxygen consumption at 15° C i n ten Amphiuma measured by a volume change technique 40 Figure 17 The probable mode of gas exchange i n Arrphiuma tridactylum 53 Figure 18 The removal of high concentrations of carbon dioxide and normal l e v e l s of oxygen from the lungs of four Amphiuma ..... 63 Figure 19 A r t e r i a l PO2 le v e l s through several breathing cycles i n Amphiuma 67 Figure 20 A r t i f i c i a l increase i n tank water PO2 and the corresponding increase i n dorsal aorta PO2 69 Figure 21 P02 le v e l s i n the dorsal aorta and systemic arch i n f i v e Amphiuma at the time o f breathing ...... 72 Figure 22 Diving bradycardia or breathing tachycar-d i a i n f i v e Amphiuma 87 Figure 23 Breathing 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 artery (PA) and systemic arch (SA) 88 Figure 24 Injections of high concentrations of carbon dioxide into the lungs and the associated pressure changes i n the systemic arch 90 Figure 25 Simultaneous pressure recordings i n the pulmonary artery and systemic arch show-ing the pulse lag i n the systemic arch 91 v i i i Page Figure 26 Pressure relationships i n the pul-monary artery and systemic arch i n ; a. slowly beating heart. b. superimposed pressure recordings from the pulmonary and systemic arches i n another Amphiuma 92 ACKNOWLEDGEMENTS I wish to thank Dr. David Randall, Chairman of my committee, for his guidance and enthusiasm through-out the study. I also thank Dr. W. S. Hoar, Dr. A. M. Perks., Dr. J . E. P h i l l i p s , Dr. D. R. Jonet and Dr. G. Shelton f o r t h e i r c r i t i c a l reviews of the manuscript. I also wish to thank my wife, Lorette, for her constant encouragement throughout the study. I am g r a t e f u l also to the following persons: Dr. J . R. Ledsome, Physiology Department, U.B.C., for discussions pertaining to the Third section of this thesis; Dr. G. Shelton, Biology Department, The University of East Anglia, f o r his useful advice and assistance during the summer of 1968; and my colleagues, who provided not only useful ideas throughout my study, but also made my stay at the University of B r i t i s h Columbia an enjoyable one. F i n a n c i a l support was provided by two Teaching Assistantships from the University of B r i t i s h Columbia and a bursary from the Fi s h e r i e s Research Board of Canada. The research was supported by grants from the National Research •Council, The Fi s h e r i e s Research Board of Canada and the B r i t i s h Columbia Heart Foundation. GENERAL INTRODUCTION Interest i n the respiratory and c i r c u l a t o r y dynamics i n the Amphibia dates back to the 19th century when Brucke (1852) i n Germany and l a t e r Sabatier (1873) i n France examined, with p r i m i t i v e techniques by our standards, the anatomy and physiology of frogs and other vertebrates. They postulated what i s now known as the " C l a s s i c a l Hypothesis" of amphibian double c i r c u l a t i o n . In b r i e f , the hypothesis states that deoxygen-ated blood from the r i g h t atrium enters the v e n t r i c l e and remains on the r i g h t side whereas oxygenated blood from the l e f t atrium enters and remains on the l e f t side of the v e n t r i c l e . Second, there i s very l i t t l e mixing of the two types of blood as a r e s u l t of the trabeculate nature of the v e n t r i c l e . Third, the s p i r a l valve i n the conus d i r e c t s the deoxygenated blood to the pulmonary c i r c u i t , aided by a lower pressure i n the pulmonary vessels. The r e s u l t of this channeling i s that the pulmonary c i r c u i t contains primarily deoxygenated blood and the systemic c i r c u i t oxygenated blood. The points of agreement today with this e a r l i e r hypothesis are that there i s a separation of the two types of blood i n the v e n t r i c l e and that the blood which flows to the pulmonary c i r c u i t appears to be r e l a t i v e l y ueoxygenated and comes from the r i g h t atrium. The oxygenated blood from the l e f t atrium i s thought to be primarily shunted to the systemic c i r c u i t . Recent work i n t h i s area has been done by Foxon (1947 and 1964), Hazelhoff (1952), De Graaf (1957), Sharma (1957), Simons (1959), DeLong (1962), Johansen (1963) and Johansen and D i t a d i (1966). The actual mechanism whereby oxygenated blood enters the systemic c i r c u i t and deoxygenated blood shunted to the pulmonary c i r c u i t i s obscure. The amphibian, Amphiuma tridactylum, used i n thi s study i s an aquatic urodele found i n freshwater drainage ditches, ponds and slow moving streams i n the southeastern United States. The reasons for choosing thi s animal are that i t i s large, the blood vessels are large and accessible for cannulation and minimal maintenance i s required to keep these animals i n the laboratory. Recovery from operations i s rapid and complete. The animal breathes very infrequently (at 15° C breathing occurs every 45 to 75 minutes (Darnell 1948)), and therefore changes i n blood gas tensions between breaths can be e a s i l y monitored. Many amphibians take up oxygen through th e i r skins as well as th e i r lungs but Amphiuma, although aquatic and unable to survive on land, i s dependent upon access to the atmosphere and cannot remain submerged for more than four to f i v e hours (Darnell 1948). This amphibian therefore probably takes up most of i t s oxygen v i a the lungs. The lungs are large, elongated sacs which extend at l e a s t 4/5 of the way along the body ca 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 lack of a cutaneous branch from the pulmonary artery and the absence of the ductus B o t a l l i (Baker 1949). The nuleated red blood c e l l s are the largest known i n any animal (70 to 80 microns i n diameter). In the f i r s t section of t h i s study, oxygen and carbon dioxide tensions i n the major blood vessels and lungs were measured i n free-moving, unanaesthetized animals. Oxygen consumption at 15 and 25°C was also measured i n several animals. In the second section the animals' breathing responses to the i n j e c t i o n s of various oxygen and carbon dioxide concentrations into the lungs were determined. Termination of i n s p i r a t i o n and the various st i m u l i i n -volved were also studied. In the t h i r d section experiments were performed to determine the mechanism whereby oxygenated blood was sent to the systemic c i r c u i t and deoxygenated blood to the pulmonary c i r c u i t . Experiments were also designed 4 to show the ef f e c t s of i n s p i r a t i o n and expiration on the c i r c u l a t o r y system. In summary, the objectivas of this study were to obtain a more complete picture of the physiology of gas exchange i n one species of amphibian and attempt to elucidate the mechanisms involved i n the associated respiratory and c i r c u l a t o r y processes. GENERAL MATERIALS AND METHODS A. ANIMALS The amphibians used i n this study, Amphiuma  tridactylum, were obtained every four to s i x weeks from the North Carolina B i o l o g i c a l Supply Company i n Durham, North Carolina, U.S.A. A l l animals were maintained at the University of B r i t i s h Columbia i n t h i r t y gallon aquaria containing fresh dechlorinated water at 12-18°C. The animals were used for experiments within s i x weeks of a r r i v a l and feeding was not necessary. Survival was good and the animals did not show any sign of furunculo-s i s i f the water i n which they were maintained was allowed to become somewhat "swampy", sim i l a r to the i r normal habitat. A l l of the animals used were adults, ranging i n weight from 250-1000 g and i n length from 50-100 cm. Since actual metabolic rate was not an important parameter i n the majority of experiments, male and female animals were used at random. B . OPERATIONS AND CANNULATIONS A l l operations were performed a f t e r animals had been anaesthetized by t o t a l immersion i n a solut i o n of MS 222 ( t r i c a i n e methanesulphonate, Sandoz) at con-centrations of 15 g/1. Anaesthesia occurred within 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 of an hour a t t h i s dosage. Care was taken to keep the s k i n m o i s t throughout an o p e r a t i o n . A f t e r the oper-a t i o n the animals r e c o v e r e d f u l l y i n f r e s h water w i t h i n 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 r i g h t lung was can n u l a t e d and lungs were a r t i f i c i a l l y v e n t i l a t e d w i t h atmospheric a i r to speed up r e c o v e r y . V a r i o u s v e i n s and a r t e r i e s were ca n n u l a t e d and i n a l l cases i n d w e l l i n g , c h r o n i c , p o l y e t h y l e n e cannulae were used. Bl o o d v e s s e l and lung cannulae were a l l made from Clay-Adams P.E. 50 tubing (I.D. 0.023 i n c h e s , O.D. 0.038 i n c h e s ) . Blood cannulae were f i l l e d w i t h h e p a r i n -i z e d s a l i n e (250 IU per ml) to prevent c l o t t i n g . Depending upon the type o f c a n n u l a t i o n d e s i r e d , a 5 cm i n c i s i o n was made i n the v e n t r a l musculature and body w a l l e i t h e r a t a p o i n t midway between the a n t e r i o r appendages ( f o r pulmonary a r t e r y , systemic a r c h and pulmonary v e i n c a n n u l a t i o n s ) or 5 cm a n t e r i o r to the vent ( t i p o f the lung, p o s t c a v a l v e i n or d o r s a l a o r t i c c a n n u l a t i o n s ) . A f t e r the o p e r a t i o n the p e r i c a r d i u m was sewn up and the i n c i s i o n i n the body w a l l c l o s e d w i t h Clay-Adams 9 mm wound c l i p s . C a n n u l a t i o n of the pulmonary a r t e r y , d o r s a l aorta and systemic arch are r e l a t i v e l y e a s i l y performed by sharpening a 55-65 cm length of P.E. 50 tubing, f i l l i n g i t with heparinized s a l i n e , attaching a 1 ml hypodermic syringe to the unsharpened end and i n s e r t i n g the sharp-ened end into the artery ( F i g . 1). I t was important that cannulae inserted into the pulmonary artery and systemic arch were of a s i m i l a r length such that meaningful com-parisons of blood pressure i n the two vessels could be made. After the artery had been exposed and a free 0.5 cm portion of the vessel clamped, a 23 gauge needle was used to puncture a small hole i n the artery. The sharp-ened portion of the cannula was then forced into the v e s s e l and the clamps removed. The muscular nature of the artery forms a t i g h t seal around the cannula and there was no leakage of blood from the point of entry i n t o the v e s s e l . The s i z e of the indwelling cannulae was never more than one t h i r d the s i z e of the vessel, minimizing the e f f e c t of the cannulae on the pattern of blood flow i n the v e s s e l . A f t e r i n s e r t i o n the cannula was t i e d i n t o place, usually to a piece of strong tissue beside the v e s s e l . At the point of e x i t from the body, the cannula was also sutured to the skin of the animal so that minor movements of the animal would not r e s u l t i n the cannula being pulled from the v e s s e l . 8 Figure 1 A drawing of the techniques used i n the cannulation of Amphiuma tridactylum. a. a r t e r i a l cannulation b. venous cannulation. A . A R T E R I A L ligature including part of vein to prevent leakage B. V E N O U S 9 Venous cannulations, on the other hand, were more d i f f i c u l t . The pulmonary vein was cannulated at a point where the v e i n leaves the lung and before i t fuses to the sinus venosus. Cannulation procedure was s i m i l a r to that described for a r t e r i a l cannulation with the exception that venous cannulae did not remain firm l y i n the vessel unless the cannula were sutured to the v e s s e l ( F i g . 1). In the cannulation of the post caval vein i t was possible to enter the larger vessel by f o r c -ing the cannula through a small c o l l a t e r a l v e in. Small c o l l a t e r a l veins used i n this technique were ones which were draining small f a t bodies and i t was f e l t that this would i n no way disturb the general c i r c u l a t i o n of the blood. By cannulation of the veins and a r t e r i e s using the above mentioned techniques, i t was possible to obtain s e r i a l samples of blood at any time desired with minimal disturbance to the animal. Figure 2 i l l u s t r a t e s the major vessels cannulated and t h e i r s p a c i a l relationships with the heart. The d i s t a l t i p of the r i g h t lung of each animal was cannulated by i n s e r t i n g the f l a r e d end of a 50 cm length of P.E. 50 tubing into a small i n c i s i o n i n the lung and then f i r m l y suturing the lung around the cannula. I t was possible to obtain small samples of alveolar gases 10. Figure 2 The major blood vessels, lungs and heart of Amphiuma tridactylum. 4 —carotid arch "i--systemic arch truncus a. conus a. ventricle common carotid aortic arches pulmonary arteries --pulmonary a. spiral^ _ valve l.&f. atria -lung pulmonary v. sinus yenosus 11„ (through the lung cannula) at any time during the exper-iments with minimal disturbance to the animal. C. ANALYTICAL PROCEDURES 1. Blood Gases Blood oxygen and carbon dioxide tensions were measured on a Radiometer Acid-Base Analyzer Type PMH 71. The PO2 and PCO2 electrodes were c a l i b r a t e d with saline e q u i l i b r a t e d at the desired p a r t i a l pressures. Blood was allowed to flow from the animals through the cannulae i n t o the PO2 and PCO2 electrode systems; the t o t a l amount of blood i n the cannulae from one sample never exceeded 0.20 ml. After the reading had been made (3-5 minutes) the blood was gently forced back into the animal, a n e g l i g i b l e amount of blood was l o s t and the t o t a l blood volume of the animal was probably not disturbed. 2. Alveolar Gases Alveolar gas samples of no larger than 0.20 ml were analyzed for oxygen, nitrogen and carbon dioxide on a Varian Aerograph Gas Chromatograph Series 200. The separation columns used i n the chromatograph were S i l i c a - g e l (screen si z e 42/60) and Molecular Sieve 5A (screen s i z e 42/60). The columns were arranged i n series with the thermal conductivity detector. Standard gas samples used to c a l i b r a t e the chromatograph were obtained 12 from The Matheson Company of Canada, Whitby, Ontario and the Canadian L i q u i d A i r Company, Vancouver, B.C. The t o t a l amount of time required for the analysis of one sample was six minutes. Gas samples were taken from the lungs i n a 0.25 ml Hamilton Syringe (Gastight # 1750 with a Chaney adaptor) and injected into the gas chromatograph. 3. Blood Pressure To record blood pressure:, the cannulae were connected to Statham 23AA ( a r t e r i s l ) , 23BB (venous), or 23 Db (small volume a r t e r i a l ) pressure transducers and displayed on either a Beckman Type R Dynograph or a G i l s o n Polygraph. The pressure transducers were c a l -ibrated with a column of saline p r i o r to and during a l l experiments. A square wave pressure change was applied to the complete system (cannulae, transducers, amplifier and pen recorder) i n a method described by Shelton and Jones (1965a) and i t was found that the response time of the equipment was 0.2-0.25 msec, ten times fa s t e r than any pressure change recorded from the experimental animals. D. MEASUREMENT OF OXYGEN CONSUMPTION Oxygen consumption i n Amphiuma tridactylum 13 at 15*C was measured i n two d i f f e r e n t ways. In the f i r s t method an animal was put into a 4,850 ml Erlenmeyer f l a s k and immersed i n a water bath at 15°C. The f l a s k was then : : i l l e d with water, also at 15°C, and sealed with a rubber stopper perforated with a 5 ml pipette ( F i g . 3). The t i p of the pipette extended into the animal chamber and was also f i l l e d with water. I t was assumed from info:onation provided by Krogh (1904), Winterstein et. a l . (1944), Jones (1967) and myself (obtained from the f i r s t section of this thesis) that very l i t t l e carbon dioxide i s released into the lungs of amphibians. Hence, a drop i n the water l e v e l i n the pipette would be i n d i c a t -ive of a decrease i n lung volume and therefore the animals volume and would represent the amount of oxygen consumed plus any nitrogen d i f f u s i n g out of the lung. The animal was allowed to breathe once every hour at which time the f l a s k was sealed again and the water l e v e l i n the pipette set at zero. Oxygen and carbon dioxide tensions i n the water surrounding the animal were continually monitored by pumping water from the chamber through s i l a s t i c tubing, past the oxygen and carbon dioxide electrodes, and back in t o the animal chamber. In the second method of oxygen consumption the animal was placed into a 4,850 ml Erlenmeyer f l a s k 14 F i g u r e 3 Apparatus used f o r the measurement of oxygen consumption i n Amphiuma t r i d a c t y l u m . a. volume change method. b. m o d i f i e d Scholander method. A . V O L U M E C H A N G E 15 containing 1,000 ml of fresh water. A i r samples were taken every hour from the chamber through a t i g h t rubber seal at the top of the f l a s k and analyzed for oxygen, nitrogen and carbon dioxide concentrations on a gas chro-matograph ( F i g . 3). The Erlenmeyer f l a s k was immersed i n water at 15°C. E. ANIMAL SURVIVAL Animals with chronic indwelling cannulae sur-vived for periods i n excess of sevan days. Death usually resulted from some v i o l e n t movement which pulled 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 per-formed, i n order to measure lung volume and the physical dimensions of the animal. 1 6 PART I . GAS TENSIONS IN THE LUNGS AND MAJOR BLOOD VESSELS IN A FREE MOVING AMPHIBIAN, AMPHIUMA TRIDACTYLUM. INTRODUCTION In the majority of investigations amphibian blood gas tensions and contents have been determined from terminal blood samples (DeLong, 1962; Johansen, 1963). S e r i a l blood samples for gas analysis have been taken i n only a few instances (Len.cant and Johansen, 1967; Shelton, personal communication on unpublished data). In the experiments reported here, s e r i a l blood samples were obtained for blood gas analysis from free moving, unanaesthetized animals. DeLong (1962) found that i n analysis of the oxygen content of terminal blood samples i n several Rana pipiens "the carotids receive primarily l e f t a t r i a l blood, whj.ch i s highly oxygenated, whereas the pulmo-cutaneous vessels receive blood almost exclusively from the r i g h t atrium". He also found that there was con-siderable mixing of oxygenated and deoxygenated blood. Johansen (1963) found i n h i s analysis of the oxygen content of terminal samples i n the major vessels of Amphiuma tridactylum that i n most animals sampled, the a o r t i c arch received blood of a higher oxygen content than did the pulmonary arch. He also found that the oxygen content i n the pulmonary vein was s i m i l a r to that found i n the a o r t i c arch. The r e s u l t s he obtained however, could possibly be quite abnormal i n that i n most exper-iments pure oxygen was injected i n t o the lungs p r i o r to sampling. Johansen and D i t a d i (1966), working on the giant toad, Bufo paracnemis, obtained r e s u l t s s i m i l a r to those of Johansen (1963). Terminal blood sampling i s acceptable i f only one blood sample from the animal i s required or i f the experimental animal i s too small to take more than one sample. Terminal determinations have the disadvantage that the state of blood i s known only at one p a r t i c u l a r time. Lengthy times between breaths must c e r t a i n l y af-f e c t the oxygen and carbon dioxide lev e l s i n the blood 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 blood. Determination of the oxygen or carbon dioxide content of blood i s indeed useful for a complete under-standing of the respiratory physiology of an amphibian; however, i f content determinations are desired blood must be permanently removed from the body and s e r i a l sampling on one animal could d r a s t i c a l l y upset the nor-mal physiology by lowering the t o t a l blood volume. P a r t i a l 18 pressure determinations have the advantage that micro blood samples are required and can be returned to the body after the determination has been made. Content can then be calculated from the blood d i s s o c i a t i o n curves. S e r i a l blood gas determinations have been car-r i e d out by Lenfant and Johansen (1967). They subjected three species of amphibians (Necturus maculosus, Amphiuma  tridactylum and Rana catesbeiana) to prolonged periods i n the a i r or under water. Although the e f f e c t s on i n d i v -i d u a l aniirals were not shown, they found that i n Amphiuma tridactylum and Rana catesbeiana the o v e r a l l e f f e c t of submergence was to lower the blood oxygen tensions and to s l i g h t l y r a i s e the blood carbon dioxide tensions and by keeping the aquatic Necturus maculosus exposed to the a i r , they found that blood oxygen tensions dropped and the carbon dioxide rose s l i g h t l y . The present experiments were designed to study the changes i n gas tensions of oxygen and carbon dioxide i n the major vessels and lungs of Amphiuma tridactylum during normal breathing cycles, and to determine the re-lationships between these l e v e l s . Oxygen consumption was also measured. 19 MATERIALS AND METHODS These experiments were ca r r i e d out on f i f t y -four Amphiuma weighing between 250 and 1000 g. Animals were not used for p h y s i o l o g i c a l experimentation i f they had been at the u n i v e r s i t y f o r a period longer than six weeks. The water temperature for a l l experiments other than oxygen consumption determinations was 15-0.05°C. The Amphiuma were anaesthetized and cannulated as previously described. No more than three cannulae were inserted into the animal at any one time. In a l l cases a minimum of two hours was allowed to elapse be-fore the s t a r t of the experiment proper. Table I i n d i c -ates the types of cannulae used and the number of exper-iments done, contributing to the experimental re s u l t s of t h i s section. Table I. Types of cannulae and numbers of animals used. Type of cannulation number of animals lung alone 6 lung and dorsal aorta 9 lung & systemic arch & pulmonary vein 16 lung & dorsal aorta & post caval vein 5 lung & dorsal aorta & pulmonary vein 2 lung & pulmonary artery & pulmonary vein 1 Blood and lung gas tensions were measured as described i n the General Materials and Methods. Oxygen consumption of f i f t e e n Amphiuma at 15°C was measured using the volume change technique (10 Amphiuma) and modified Scholander technique (5 Amphiuma). The ten animals measured at 15°C by the volume change method were also measured at 25°C using the same method. RESULTS A. ALVEOLAR GASES Amphiuma tridactylum remains submerged f o r long periods of time and surfaces to breathe. The Amphiuma i n these experiments had a breathing i n t e r v a l of 55 i 3 (S.E.) minutes at 15°C. Although c r i t i c a l temperature experiments were not done the one experiment performed at 8°C extended the breathing i n t e r v a l to 285 minutes. P02 i n the lungs declined between breaths, f a l l i n g from 101 - 4.8 to 44 - 2.9 mm Hg. The highest P02's are recorded immediately a f t e r a breath and the lowest immediately p r i o r to a breath ( F i g . 4). Where s e r i a l samples were taken c l o s e l y enough to observe the shape of the PO2 disappearance curve, i t was observed that oxygen concentrations dropped i n a s l i g h t l y reversed 2 1 Figure 4 P02 decrease i n the lungs of four Amphiuma, The highest PO2 l e v e l s occur immediately a f t e r a breath and the lowest lev e l s immediately p r i o r to a breath. 22 sigmoid-shaped curve. More simply, the rate of oxygen decrease from the lungs was not as rapid i n the f i r s t f i f t e e n minutes as i t was for most of the remaining time. Figure 4 also i l l u s t r a t e s the ef f e c t s of a low temperature on the nature of the lung oxygen disappearance curve. At 8 C°the PC*2 reached 23 mm Hg within 285 minutes, when the animal breathed. Alveolar PCO2 varied l i t t l e throughout the breathing cycle i n a l l experiments. The f i r s t lung sample, taken immediately a f t e r a breath, tended to be s l i g h t l y lower i n carbon dioxide concentration than that found during the rest of the breathing i n t e r v a l . Within f i v e minutes the aveolar PCO2 returned to the mean l e v e l of 14.9 mm Hg af t e r the recorded drop of about 16% (the percentage drop i s a mean value calculated from 20 animals through 68 breathing cycles) and did not fluctuate ap-preciably thereafter. In only one out of twenty-two experiments In which the lung carbon dioxide was measured did the le v e l s increase to any extent as the animal remained submerged. A "PO2 and PCO2 diagram" was constructed for breathing cycles of six Amphiuma ( F i g . 5). The d i a -gram describes the changes i n PO2 and PCO2 i n the lungs during a breathing cy c l e . The R l i n e which would des-c r i b e PO2 and PCO2 changes i n the Amphiuma lung would 23 Figure 5 Alveolar "PO2-PCO2 diagram" for six Amphiuma The diagram i l l u s t r a t e s the rel a t i o n s h i p be-tween alveolar PO2 and PCO2. 24 e s s e n t i a l l y be equal to zero during the majority of the breathing i n t e r v a l . I t was found that there was an inverse r e l a -tionship between the alveolar P0£ and PN£ such that i f there was no increase i n the alveolar PCO2 over a period of time, the decrease i n PO2 would r e s u l t i n a sim i l a r increase i n PN2. Tota l lung volume i n a l l animals was a function of the weight of the animal ( F i g . 6). Lung volume i n any animal was determined by successive f i l l i n g and removal of a i r through the indwelling cannula with a c a l i b r a t e d syringe. The accuracy of this method was confirmed by several autopsies performed i n which the lungs were re-moved and the volume measured. I t was not possible to damage the lung i n an animal by i n j e c t i o n s of a i r , i n that, as soon as the lungs were f u l l , the animal would open the spiracles on the an t e r o - l a t e r a l part of the body and release excess a i r . T i d a l volume was estimated using an i n d i r e c t method. Knowing that the mean alveolar PO2 p r i o r to a breath i s 44 mm Hg and immediately a f t e r a breath i s 101 mm Hg, and that the PO2 of the ins p i r e d a i r i s about 160 mm Hg (dependent upon the atmospheric pressure of the day), the t i d a l volume, based on a p r i n c i p l e of Figure 6 The re 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% of t o t a l lung volume. B. BLOOD GAS CONCENTRATIONS 1. Dorsal Aorta Figure 7 i l l u s t r a t e s the changes which occurred i n the lung and dorsal aorta i n one animal during four breathing cycles. As the oxygen tensions i n the lung f e l l , the PO2 le v e l s i n the dorsal aorta also f e l l . The rapid r i s e i n the lung oxygen tensions immediately a f t e r a breath resulted i n an almost simultaneous r i s e i n the dorsal aorta PO2. The gradient established between the dorsal aorta and lung oxygen tensions was r e l a t i v e l y constant within any animal and did not change markedly before, during or af t e r a breath once the gradient had been established (Fig.8). Dorsal aorta PCO2 lev e l s were always 1-10 mm Hg lower than lung PC02 l e v e l s . This phenomenon was observed i n a l l experiments i n which the lung and dorsal aorta PCO2 was monitored. 2. Pulmonary Vein Because of the d i f f i c u l t y of pulmonary vein cannulation, the number of determinations of gas tensions i n blood from this vessel was l i m i t e d . Nevertheless, i n a l l experiments the pulmonary ve i n PO2 lev e l s were very close to those found i n the lung (Figure 9 i s an 27 Figure 7 PO2 and PCO2 changes which occurred i n the lungs and dorsal aorta i n one animal during four breathing cycles. V e r t i c a l arrows at the top of the diagram indicate breathing times. T I M E (min) 28 Figure 8 PO2 gradient between the dorsal aorta or systemic arch and the lung. The % time submerged on the abscissa of the graph equates a l l breathing i n t e r v a l s , i . e . 1007o = i n t e r v a l between breaths Means are given - S.E. for each 20% of the i n t e r v a l . Each mean represents not fewer than 10 gradient measurements. D A o r S A - L U N G GRADIENT mm Hg 40 30 h 1 20 40 60 80 100 b TIME SUBMERGED 29 Figure 9 Changes which occurred i n the pulmonary vein and lung PO2 and PCO2 l e v e l s i n one animal during f i v e breathing cycles. V e r t i c a l arrows at the top of the diagram indicate breathing times. example of f i v e breathing cycles i n one animal). Immed-i a t e l y a f t e r a breath the PO2 gradient between the lung and pulmonary vein was very small but increased as the animal renained submerged ( F i g . 10). Pulmonary vein PCO2 le v e l s were, i n almost every sample (one exception at 73 minutes, F i g . 9), lower than PCO2 i n the lungs. 3. Pulmonary Artery PO2 le v e l s i n the pulmonary artery are highest immediately a f t e r a breath and drop to the lowest lev e l s j u s t before a breath ( F i g . 11). PO2 gradients (the term gradient i n thi s instance i s e s s e n t i a l l y a PO2 difference between two vessels) between the dorsal aorta and pulmon-ary a r t e r j were found i n a l l experiments where these two vessels were cannulated*. The blood i n the systemic c i r -c u i t (systemic arch or dorsal aorta) i s always more highly oxygenated than the blood flowing to the lungs v i a the pulmonary artery. Figure 12 i l l u s t r a t e s the PO2 differences between these two c i r c u i t s i n eight d i f f e r e n t Amphiuma. The gradient, immediatel)' a f t e r a breath, de-creased as the length of submerged time increased. The PO2 gradient was i n i t i a l l y as large as 25-30 mm Hg and f e l l to 1-5 mm Hg ( F i g . 12). * From the point of view of blood gas tensions, no d i s t i n c t i o n was made between the dorsal aorta, systemic arch or ascending aorta and the terms w i l l be used interchangeably. 31 Figure 1 0 PO£ gradient between the pulmonary vein and lung i n Amphiuma. Gradient measurements were made at each point a PO2 determination was made i n either the lung or pulmonary vein. "]lhe % time submerged on the abscissa of the g;raph equates a l l breathing i n t e r v a l s i n a l l emimals, i . e . 1 0 0 % = i n t e r v a l between breaths. I^he l i n e was f i t t e d to the data by eye. 32 Figure 11 PO2 and PCQ2 changes which occurred i n the pulmonary artery, systemic arch and lung i n one animal during a complete breathing c y c l e . V e r t i c a l arrows at the top of the diagram indicate breathing times. 33 Figure 12 PO2 gradient between the pulmonary artery and systemic arch i n eight Amphiuma. Graphic presentation i s the same as i n F i g . 10. 34 Using an oxygen d i s s o c i a t i o n curve for Amphiuma  tridactylum, derived from data of Lenfant and Johansen (1967) and Scott (1931), the P0 2 tensions i n the systemic arch and pulmonary artery shown i n Figure 11 were conv-erted to % saturation ( F i g . 13). This was done to des-cr i b e the pulmonary artery-systemic arch gradient i n terms of oxygen content as well as p a r t i a l pressure d i f -ferences. Afte r a breath the gradient i n this case ( F i g . 13) was about 257a and f e l l i n 50 minutes to 107o saturation difference. There was no v i s i b l e PCO;? gradient between the pulmonary artery and systemic arch i n any experiment. The mean systemic arch or dorsal aorta PCO2 of ten Amphiuma was 11.9 mm Hg and from the same animals the mean pulmon-ary artery PCO2 was 12.2 mm Hg. 4. Post Caval Vein -From simultaneous cannulations of the dorsal aorta and the post caval vein i t was possible to determine the PO2 difference between these two vessels through sev-e r a l breathing cycles. The composite of this data i s shown i n Figure 14. Immediately a f t e r a breath the PO2 difference i s quite large (17-30 mm Hg), a f t e r which the gradient decreases as the length of time the animal remains under the water increases (1-10 mm Hg^ immediately before 35. Figure 13 PO2 l e v e l s i n the systemic arch and pulmonary artery from Figure 12 converted to percent saturation. 100 i — i - O L U <f < CO 80 60 40 20 80 systemic arch pulmonary artery 120 160 TIME (min) 200 36 Figure 14 PO2 and PCO2 gradient between the post caval v e i n and dorsal aorta i n f i v e Amphiuma. Graphic presentation i s the same as i n F i g . 10. breathing). Oxygen tensions i n the post caval vein are always lower than those recorded i n the dorsal aorta. Mean PCO2 l e v e l s i n the post caval vein of f i v e Amphiuma (46 samples) was 12.6 mm Hg. In the same animals, the dorsal aorta PCO2 tensions had a mean of 11.6 mm Hg ( i n d i v i d u a l samples from the two vessels were taken within ten minutes of each other). 5. Standard Animal I t was impossible to obtain blood from a l l major vessels and lung gas measurements simultaneously. I t was however possible to construct a generalized out-l i n e of the probable oxygen tensions i n the major vessels during a breathing cycle. Knowing the pulmonary vein-lung gradient, the dorsal aorta-lung gradient, the pul-monary artery-systemic arch gradient and the post caval vein-dorsal aorta gradient, as well as knowing the mean oxygen tensions i n the lungs and vessels immediately before ar.d a f t e r a breath, i t was possible to combine thi s information to form a representative or "standard animal" ( F i g . 15) . P O 2 f e l l most ra p i d l y between breaths i n the pulmonary vein ( F i g . 15) and lea s t r a p i d l y i n the pul-monary artery and post caval vein (which f a l l at the 38 Figure 15 "The Standard Amphiuma". A reconstruction of the probable P O 2 fluctuations i n the lungs and major blood vessels during three breathing cyc l e s . Estimates obtained from avai l a b l e gradient data and mean blood and lung measure-ments . TIME (min) 39 same r a t e ) . Rapid r i s e s i n P O 2 occurred i n the blood 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 described e a r l i e r . Table II shows the mean consumption for f i f t e e n animals used. Table I I . Oxygen consumption measured i n f i f t e e n Amphiuma at 15 and 25°C. Method Number of animals Temp°C Mean oxygen Time meas-consumption ured per ul/am/hr animal Volume change 10 15 6.67*0.14 (S.E) 3 hours Modified Scholander 5 15 6.89-0.039 (S.E.) 22-28 hours Volume Change 10 (Same animals as at 15°C) 25 19.0*0.23 (S.E.) 3 hours Using two respirometry techniques, the mean oxygen consumption d i f f e r e d very l i t t l e . The rate at which oxygen was consumed over a one hour period was not regular. Figure 16 i l l u s t r a t e s the mean oxygen consump-ti o n for ten animals for a period of one hour measured at f i v e minute i n t e r v a l s . Each experiment was repeated 40 Figure 16 Rates of oxygen consumption at 15°C i n ten Amphiuma measured by a volume change technique. Means are given for each f i v e minute i n t e r v a l . three times on each animal. The most rapid oxygen consump ti o n occurred within the f i r s t f i f t e e n minutes of sub-mergence; compared to t h i s , consumption within the l a s t f o r t y - f i v e minutes i s very slow. Oxygen consumption i n Amphiuma measured at 25°C increased to 19.0 ul/gm/hr, a l -most a threefold increase from the consumption at 15°C. . 42 DISCUSSION Amphiuma tridactylum breathes about once every hour almost completely replacing a l l a i r within the lung at each breath. Before taking a breath, 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 surface of the water and w i l l remove most of the residual a i r from the lungs by lowering the f l o o r of the mouth with the exter-nal nares closed. The f l o o r of the mouth i s then raised and the external nares opened and the a i r i n the mouth i s expelled into the a i r above the water surface. This type of breathing 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 expired a i r i s removed above the water surface such that bubbling does not occur i n the water. Lung volume i n Amphiuma i s related to the size of the i n d i v i d u a l animal and represents about 6-7% of the t o t a l volume of the animal. For comparison, the t o t a l lung volume of man represents 7-8% of the body volume (Lambertsen, 1961). An Amphiuma weighing 500-700 gm would have, on average, a lung volume between 30-40 ml ( F i g . 6). The removal of a i r from the lungs i s not com-plete because oxygen tensions do not approach atmospheric 43 a i r tensions. In f a c t , t i d a l volume would appear to be about 72% i n Amphiuma. This i s much higher than i n man ( t i d a l volume of about 12%, at rest) but the ra-ther infrequent method of breathing would necessitate an almost complete change of a i r within the lungs. The amount of a i r taken into the lungs i s re-l a t e d to the length of time the animal remained at the surface, the amount of a i r previously removed from the lungs anc the lev e l s to which the a r t e r i a l l e v e l s dropped during the time submerged. Quantification of the r e s u l t s were not possible but i t did appear, from observations, that the lower the a r t e r i a l PO2 l e v e l s were, the longer the animal remained at the surface breathing and the more "gulps" of a i r swallowed. Most a i r samples were taken from the posterior portion of the lung. I n i t i a l l y , a f t e r a breath, P0£ levels i n the lung did not change rapid l y even though the animal was consuming oxygen at a high rate compared with that occurring towards the end of a breathing cycle. PO2 l e v e l s i n posterior portions of the lung may have been maintained by mixing a i r of a higher PO2, from the more anterior portions of the lung, with that i n the posterior portions of the lung. This mixing could have been aided by contractions of the lung and movements of the animal. 44 In support of this statement, Czopek (1962) has shown i n Amphiuma means, a c l o s e l y related species to A. tridactylum that there are a great number of smooth-muscle c e l l s i n the pulmonary walls and ridges and suggests that "the lungs, are able to contract and thereby promote aeration". The oxygen and carbon dioxide concentrations i n the lung i n Amphiuma are such that gas exchange i s d i f f i c u l t to analyze using the O2-CO2 diagram. Hughes (1966) suggests that oxygen and carbon dioxide i n the lung of Rana catesbiana, when plotted on an O2-CO2 dia-gram, would f a l l on the R=0.4 l i n e . In Amphiuma the f i r s t one or two lung samples a f t e r a breath may f a l l on the R=0.4-0.5 l i n e but as the animal remains submerged the R l i n e e s s e n t i a l l y becomes zero and the usefulness of the u2~^ u2 diagram i n describing gas exchange i n this amphib-ian becomes l i m i t e d . I t i s quite possible that i f Hughes had continued the study to show the gas concentrations i n the lung a f t e r the animal had been submerged for a longer period of time ( c h a r a c t e r i s t i c of the species (Noble 1931)), the R l i n e might have decreased consider-ably such as that found i n Amphiuma. Oxygen consumption i n Araphiuma tridactylum i s the lowest recorded of any amphibian measured at a comparable temperature. (Brown, 1964; Jones, 1967). 45 The rate of oxygen uptake of a submerged animal between breaths decreases rapidly a f t e r the large i n i t i a l con-sumption within the f i r s t 5-10 minutes a f t e r a breath of 2-3.5 ul/gm body weight. The rate of decrease i n oxygen consumption i s sim i l a r to the drop i n alveolar PO2 be-tween breaths. In the volume change experiments on oxygen consumption, the water PO2 was monitored continuously throughout the three hour experimental period. The P0 2 drop i n the water over this period was never more than 1-2 mm Hg. This drop i n PO2 would account for no more than a 0.2-0.4 ml loss i n oxygen i n the whole system. I t i s clear from this evidence that the animals re l y very l i t t l e on oxygen i n the water to supply or supplement metabolic de-mands. Since oxygen consumption measurements using the two techniques were almost i d e n t i c a l , i t appears that a volume change i n Amphiuma while submerged i s i n d i c a t i v e of the amount of oxygen being consumed, and a l l oxygen i s taken up v i a the lungs. By the same reasoning, i f the two methods give similar r e s u l t s , nitrogen must not leave the lung as the animal consumes oxygen. I f nitrogen, which b u i l t up i n the lung, was removed into the blood and water, there should have been a much larger volume change than was ac t u a l l y measured. The lack of increase i n lung PCO2 between 46 breaths i s i n t e r e s t i n g when one considers the volume changes which must occur i n the lung as oxygen diffuses i n t o the blood. As a i r enters the lung during a breath the lung ECO2 i s s l i g h t l y d i l u t e d but returns to normal within 4-6 minutes. The resultant increase i n lung PCO2 af t e r a breath must r e s u l t from carbon dioxide entering the lung from the blood. I f this i n i t i a l amount of carbon dioxide were to remain i n the lung, the lung PCO2 should increase by 2-4 mm Hg simply as a r e s u l t of the concentrat ing effect, of oxygen leaving the lung. This however was not observed and the conclusion can be made that carbon dioxide which entered the lung from the blood immediately a f t e r a breath, must i n part return to the blood between breaths. The r e s u l t of this phenomenon then i s to have lung PCO2 concentrations higher than PCO2 level s i n any of the major blood vessels. Once the PCO2 gradient i s established between the blood and the lungs, no more carbon dioxide enters the lungs unless blood PCO2 level s increase s.harply. The oxygen gradient between a i r and blood across the lung wall i s i n i t i a l l y small but Increases with time between breaths. The calculated volume of oxygen leaving the lung decreases with time a f t e r a breath. The transfer factor of the lung (V02/aP02) i s therefore f a l l i n g 47 during the i n t e r v a l between breaths. The change i n transfer factor could be related to many factors i n c l u d -ing the volume and pattern of blood flow to the lungs, and the d i s t r i b u t i o n of a i r within the lungs. A change i n transfer factor indicates that one or more of these para-meters i s alte r e d and i s a f f e c t i n g gas transfer rates across the lung. The high PO2 le v e l s i n the pulmonary vein immediately a f t e r a breath would indicate, for a short period, of time at lea s t , the blood leaving the lung i s f u l l y saturated with oxygen. Using an oxygen d i s s o c i a t i o n curve for Amphiuma tridactylum blood constructed by Lenfant and Johansen (1967), 100% saturation occurs at PO2 leve l s above 90 mm Hg. P0£ i n the pulmonary vein often exceeded this l e v e l j u s t a f t e r a breath, i n d i c a t i n g that during this period, blood leaving the lungs was f u l l y saturated with oxygen. Somewhat more i n t e r e s t i n g i s the P0£ gradient established between the pulmonary artery and the system-i c arch. The problem of whether the single v e n t r i c l e of an amphibian can maintain a divided stream of oxygenated and deoxygenated blood and somehow d i r e c t the deoxygen-ated blood to the lungs and the oxygenated blood to the systemic c i r c u l a t i o n has been a topic of discussion and experimentation since the mid-19th century. The exper-iments i n this study show that there i s a d e f i n i t e oxy-gen gradient between these two c i r c u i t s over a long per-iod of time, even during long i n t e r v a l s between breaths. DeLong (1962), Johansen (1963) and Johansen and D i t a d i (1966) have a l l made measurements of oxygen and carbon dioxide contents i n the two vessels but did not obtain s e r i a l samples over a long period of time. In the three published works j u s t mentioned, oxygen content gradients were found to ex i s t between the body and lung c i r c u i t i n Rana pipiens (DeLong, 1962), Bufo paracnemis (Johansen and D i t a d i , 1966) and Amphiuma tridactylum (Johansen, 1963). In this i n v e s t i g a t i o n i t was shown that the gradient between the pulmonary arch and the systemic arch Is present throughout the breathing cycle. The gradient decreases as the animal remains submerged, but does not disappear completely. The gradient i s definately greater immediately a f t e r the animal has taken a breath (20-25 mm Hg) and this i s probably because the pulmonary vein (oxygenated blood) and the venous return (primarily post caval vein, deoxygenated blood) gradient i s greatest at th i s time. Although actual PO2 gradients were not meas-ured between the pulmonary vein and the post caval v e i n i t can be seen from the reconstruction of the standard 49 •. animal ( F i g . 15) that the PO2 gradient between the vessels becomes smaller as the length of time submerged increases. This i n turn appears to r e s u l t from the increase i n the lung-pulmonary vein gradient as the time a f t e r breathing increases. Therefore, the decrease i n the pulmonary artery-systemic arch gradient between breaths i s a function of the decrease i n the pulmonary vein-post caval vein (venous re-turn) gradient and probably not a loss i n the separation c a p a b i l i t i e s of the heart and associated vessels. I t i s important to point out that oxygen tensions i n the pulmonary artery and systemic arches are usually below 70 mm Hg. The changes i n blood PO2 w i l l cause marked changes i n percent saturation as i t i s i n this portion of the Amphiuma d i s s o c i a t i o n curve that changes i n PO2 r e s u l t i n large changes i n percent saturation (Compare Figs. 11 and 13). As has been described e a r l i e r (see General Materials and Methods), continuous; measurements i n the venous return ( i n this case the post caval vein) are d i f f i c u l t i n that cannulation procedures are much more complex. Nevertheless, s u f f i c i e n t data was obtained to ascertain the level s of PO2 i n the venous return i n r e l a t i o n to the le v e l s i n the dorsal aorta. The post 50 caval vein-dorsal aorta PO2 gradient was between 20-30 mm Hg immediately following a breath and decreased to between 5-10 mm Hg immediately before a breath. Rather than r e f l e c t i n g an increase i n tissue u t i l i z a t i o n of oxygen, this decrease i n gradient simply r e f l e c t s the decrease i n t o t a l oxygen entering the c i r c u l a t i o n as the lung PO2 drops, i . e . , dorsal aorta l e v e l f a l l s , VO2 f a l l s between breaths. A l l of the fluctuations i n blood PO2 lev e l s i n a l l the major vessels occurred i n free-moving Amphiuma with access to the surface. The fluctuations are definate-l y r e l a t e d to the intermittent type of breathing which this animal e x h i b i t s . Lenfant and Johansen (1967) suggest, on data c o l l e c t e d from six Amphiuma, that normal a r t e r i a l PO2 lev e l s vary from 72-100 mm Hg (9 samples) and do not mention i n d i v i d u a l v a r i a t i o n withi.n one animal between breaths. I f their animals were "prevented from surfacing" for 43 minutes, they found that a r t e r i a l PO2 dropped some-what, although i t i s impossible to ascertain from their data the e f f e c t of submersion on the i n d i v i d u a l animal. The implication i s made by them that fluctuations i n a r t e r i a l PO2 are not normal i n Amphiuma. This i s i n t o t a l disagree-ment with the present study. Lenfant and Johansen (1967) have also shown 51 that i f Amphiuma were prevented from surfacing for periods of 43 minutes the PCC*2 i n a r t e r i a l blood rose. In a l l of the experiments i n the present study, where a r t e r i a l or venous PCO2 le v e l s were monitored, there was no i n d i c a t -ion that submergence resulted i n an increase i n blood PCO2 l e v e l s . A l b e i t , Lenfant and Johansen 1s experiments were performed at 20*C (5°C higher than the present study), but i t i s doubtful that t h i s increase i n exper-imental temperature would r e s u l t i n this difference i n response to submergence. Because PCO2 level s do not increase i n either the lungs (with the exception of the s l i g h t lung PCO2 r i s e shortly a f t e r a breath) or the blood between breaths, we can conclude that carbon dioxide produced by metabol-ism i s removed v i a the skin into the surrounding water. This concept i s not new and has been shown as early as 1904 by Krogh (on anuran amphibians). Carbon dioxide transfer from the blood to water w i l l depend upon the dimensions of the exchange surface, i . e . , the surface area of the skin, the volume and pattern of blood flow through the skin, and the concentration gradients between the blood and the water. There i s a much larger PCO2 gradient e x i s t i n g between the blood and the water than between the blood and the lungs. Carbon 52 dioxide w i l l enter the lung u n t i l the gradient i s small enough to e f f e c t i v e l y eliminate further passage of carbon dioxide into the lungs, whereas the amount of carbon dioxide which can pass into the water i s large because of the high s o l u b i l i t y of the gas i n water as well as the f a c t that the volume of water surrounding an animal i s very large. The presence of carbonic anhydrase i n blood increases the rate of formation of free carbon dioxide from bicarbonate and w i l l help to maintain high carbon dioxide l e v e l s i n the blood as carbon dioxide diffuses into the water. Carbonic anhydrase leve l s have not been measured i n Amphiuma, but i t i s known that this enzyme i s absent i n the skin of Rana climatans and Rana Cates-biana (Maren, 1967). Hence, i n the presence of low or non-existant l e v e l s of this enzyme, the rate of formation of carbon dioxide from bicarbonate may l i m i t the rate of carbon dioxide transfer to the water across the skin. The Haldane e f f e c t i s small i n Amphiuma (Len-fant and Johansen, 1967) and therefore i s not very import-ant i n augmenting the removal of carbon dioxide from the blood into the lungs. Figure 17 i l l u s t r a t e s the probable mode of gas exchange i n Amphiuma tridactylum. The bucco-pharyngeal mucosa of this species was not considered to • 53 Figure 17 The probable mode of gas exchange i n Amphiuma tridactylum. BODY CIRCUIT systemic arch WATER venous return VERY LOW 0 2 LUNG CIRCUIT pulmonary artery INCOMPLETE DOUBLE CIRCULATION pulmonary vein be a s i g n i f i c a n t respiratory exchange surface primarily because of i t s small size i n comparison to the t o t a l body size and secondly, Czopek (1902) found that i n a cl o s e l y related species, Amphiuma means, vascular supply to the mouth region was poorly developed. PART I I RESPIRATORY CONTROL IN AI4PHIUMA TRIDACTYLUM INTRODUCTION Work i n the f i e l d o f amphibian r e s p i r a t o r y -c o n t r o l has not been e x t e n s i v e . N e i l and Zotterman (1950) and de Marneffe-Foulon (1962) i n v e s t i g a t e d the neu r o p h y s i . o l o g i c a l b a s i s o f some r e s p i r a t o r y r e f l e x e s i n the f r o g . Jones and S h e l t o n (1964) and Jones (1966) were p a r t i c u l a r l y 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 rec o v e r y jfrom d i v i n g b r a d y c a r d i a a:id how submergence i n f l u e n c e d h e a r t r a t e i n the f r o g . Jones (1966), work-i n g on a v a r i e t y of f r o g s (Bufo bu£o, Rana p i p i e n s and Rana te m p o r a r i a ) , allowed them to s u r f a c e i n t o d i f f e r e n t c o n c e n t r a t i o n s o f oxygen, carbon d i o x i d e and n i t r o g e n , and suggested " t h a t the f r o g i s s e n s i t i v e to oxygen l a c k b o t h d u r i n g development of b r a d y c a r d i a and prolonged r e -covery from i t " . A l t hough Jones has shown that d i v i n g b r a d y c a r d i a and re c o v e r y from d i v i n g b r a d y c a r d i a are more s e n s i t i v e to oxygen l a c k than to the presence of carbon d i o x i d e , the q u e s t i o n o f what were 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 the c o n t r o l of b r e a t h i n g r e -mained unanswered. T a g l i e t t i and C a s e l l a (1966) have presented n e u r o p h y s i o l o g i c a l evidence demonstrating t h a t s t r e t c h r e c e p t o r s i n f r o g ' s lungs are i n v o l v e d i n the t e r m i n a t i o n 56 of the lung f i l l i n g process. More recently (1968) they have also produced evidence for the presence of d e f l a t -ion receptors i n frogs' lungs. I t i s possible, from this evidence at l e a s t , that the stimulus for i n f l a t i o n and the termination of i n f l a t i o n could be related to the volume of a i r which the frog has i n i t s lungs. Since Amphiuma tridactylum remains submerged for extended periods of time (about 54 minutes at 15°C), i t was convenient to design experiments i n which not only gas tensions i n the blood and lungs were monitored but also to i n j e c t varied concentrations of oxygen, nitrogen and carbon dioxide into the lungs and to observe the breathing responses of the animal. Experiments designed to determine the animal's a b i l i t y to detect volume changes i n the lungs were also c a r r i e d out. 57 MATERIALS AND METHODS Twenty-two Amphiuma t r i d a c t y l u m were used f o r e x p e r i m e n t a t i o n i n t h i s s e c t i o n . A d d i t i o n a l i n f o r -n a t i o n was; o b t a i n e d from 17 animals used i n P a r t I of t h i s t h e s i s . Experiments to determine the animals response to v a r i e d gas c o n c e n t r a t i o n s were of f o u r types; i , the oxygen t e n s i o n i n the lungs was lowered by i n j e c t i n g q u a n t i t i e s of n i t r o g e n i n t o the lungs; i i , the carbon d i o x i d e t e n s i o n i n the lungs was r a i s e d by i n j e c t i n g q u a n t i t i e s of carbon d i o x i d e i n t o the lungs; i i i , the carbon d i o x i d e t e n s i o n i n the surrounding water was r a i s e d and the c o r r e s p o n d i n g e f f e c t s on b l o o d and lung carbon d i o x i d e observed, and i v , the oxygen t e n s i o n i n the lungs was r a i s e d by i n j e c t i n g pure oxygen i n t o the l u n g s . Gases were i n j e c t e d i n t o the lungs through a cannula (P.E. 50) i n s e r t e d i n t o the lungs as p r e v i o u s l y d e s c r i b e d . T e r m i n a t i o n of i n s p i r a t i o n experiments were performed by e i t h e r i n j e c t i n g n i t r o g e n i n t o the lung as the animal was attempting to s u r f a c e or to remove a i r from the lungs as the animal was b r e a t h i n g . The b r e a t h i n g response was recorded a f t e r the n i t r o g e n i n -j e c t i o n and the amount of a i r "swallowed" was measured i f a i r was mechanically being removed from the lung dur-ing i n s p i r a t i o n . Lung gas concentrations were measured on a Varian Aerograph Gas Chromatograph. Water and blood PO2 and PCO2 were determined using a Radiometer Acid-Base Analyzer. Standardized gas mixtures used for lung i n j e c t ions were obtained from The Matheson Company of Canada and the Canadian Liquid.Air Company. Prior to i n j e c t i o n of gas mixtures into the lung, the gases were saturated with 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 glass aquarium contain-ing ten l i t r e s of water. The water temperature i n a l l ex periments was maintained at 15°C-0.5°C. RESULTS A. BREATHING RATES In a l l experiments i n which blood and lung oxygen and carbon dioxide tensions were measured, d i s -turbances to the normal breathing cycle ( i . e . movement of tank, loud low frequency noises, or rapid movements above the water surface) were minimal. In these exper-iments the mean i n t e r v a l between breaths was 55-3 min-. utes, the range from 26 to 120 minutes. An extension of the time between breaths could \ 59 be accomplished by i n j e c t i n g atmospheric a i r into the lungs. Table III i l l u s t r a t e s experiments on four d i f f e r e n t animals. Table I I I . Extension of the time between breaths by i n j e c t i n g atmospheric a i r into the lungs. Animal No. Freq. of a i r Time under Time from l a s t i n j e c t i o n water 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 3 , 20 ml/20 min 247 min 52 min 4 20 ml/20-24 min 345 min 62 min In animals 1, 3 and 4 there were no apparent signs of a g i t a t i o n or attempts to surface u n t i l i n j e c t i o n s had been stopped. In animal 2 the time between in j e c t i o n s was varied from 20-40 minutes and occasionally a f t e r 35-40 minutes had elapsed the animal moved around the bottom of the tank as though i t was about to breathe. When these movements occurred, a i r was injected into the 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 submerged. I f the volume of continuous a i r i n j e c t i o n s exceeded the t o t a l lung volume, the animal removed the excess a i r as 60 bubbles through the s p i r a c u l a r openings w h i l e submerged. An e x t e n s i o n of the time between b r e a t h s c o u l d a l s o be accomplished by i n j e c t i n g pure oxygen i n t o the lun g s . Twenty ml of pure oxygen per animal, was i n j e c t e d i n t o the lungs o f f i v e Amphiuma. The mean time between breaths i n these animals was i n c r e a s e d to 135-6 minutes, the t o t a l range extending from 108-170 minutes. B. TERMINATION OF INSPIRATION I n a free-moving Amphiuma, i n s p i r a t i o n i s term-i n a t e d a f t e r 3-5 l a r g e " g u l p s " of a i r have been swallowed. A f t e r t h i s p e r i o d the animal w i l l submerge and r e t u r n to the bottom of the tank. I t was found t h a t i f a l l the a i r en-t e r i n g the. lungs w h i l e b r e a t h i n g was si m u l t a n e o u s l y removed, w i t h a s y r i n g e through the lung cannula, the animal would c o n t i n u e b r e a t h i n g f o r an extended p e r i o d of time. Table IV i l l u s t r a t e s f i v e such experiments performed where the a c t u a l amounts of a i r removed from the lung was measured b e f o r e i n -s p i r a t i o n was terminated. T a b l e IV. A i r removal from the lungs d u r i n g i n s p i r a t i o n . Animal No. Normal Lung Volume Volume removed b e f o r e t e r m i n a t i o n of i n s p i r -. • a t i o n  1 40 ml 78 ml 2 47 ml 70 ml 3 34 ml 76 ml 4 37 ml 106 ml 5 45 ml 95 ml 61 The alternate experiment was performed on sever-a l other /cmphiuma. As an animal raised i t s neck and snout towards the water surface to breathe, pure nitrogen was i n -jected into the lungs before the animal reached the surface. I t was found that a f t e r an i n j e c t i o n of nitrogen, the anim-a l did not breathe, but returned to the bottom of the tank for a short period of time and then resurfaced to breathe. Table V shows the amount of nitrogen injected as four anim-als surfaced and the time elapsed u n t i l they resurfaced to breathe. I f more than One experiment was performed on one Table V. Inje c t i o n of nitrogen into the lungs of Amphiuma which were about to surface to breathe. Animal No Amount of N 2 injected as animal was surfacing Time u n t i l breath 1 20 ml 40 ml 50 ml 60 ml 5 min 11 min • 9 min 5 min 2 40 ml 20 ml 5% min 2% min 3 40 ml 6 min 4 40 ml 5 min animal, at l e a s t two hours were a].lowed between injec t i o n s of nitrogen. Any experiment i n which the animals snout act u a l l y came above the water surface before or during 62 the i n j e c t i o n of nitrogen was disregarded i n that oxygen may have entered the lung. C. BREATHING ONSET AND ITS RELATION TO BODY PC02 LEVELS Figure 18 represents a series of experiments i n which carbon dioxid e / a i r mixtures were injected into the lungs of four animals and the rate of removal of these gases from the lungs monitored u n t i l the time of breathing. The removal of carbon dioxide from the lung, which was present i n i t i a l l y i n concentrations 3-5 times that of normal l e v e l s , was s l i g h t l j ' faster than that of oxygen. I t was assumed that, since the animals were under water, the carbon dioxide was being removed from the lung, int o the blood and then into the surrounding water. In a l l cases the lung PCO2 le v e l s ( F i g . 18) had returned to a base l e v e l (15-25 mm Hg) 30-70 minutes before the animal breathed. Various concentrations of carbon dioxide i n a i r were injected into the lungs. After i n j e c t i o n , the time to onset of breathing was noted, and i n several i n -stances the concentration of gases i n the lung, as near to the time of breathing as possible, were also measured. In these experiments a minimum of three hours was allowed between i n j e c t i o n s i f the same animal was being used. Figure 18 The removal of high concentrations of carbon dioxide and normal l e v e l s of oxygen from the lungs of four Amphiuma. Breathing times are marked with v e r t i c a l ar-rows and the abbreviation " b r . n . Table VI i l l u s t r a t e s the res u l t s obtained. The breathing times a f t e r i n j e c t i o n of 10% carbon dioxide i n a i r were quite v a r i a b l e , ranging from 1 minute to 83 minutes with a mean of 43.1-7.4 minutes. After i n j e c t i o n s of 15% carbon dioxide i n a i r , the response was less v a r i a b l e , the time u n t i l breathing ranged from 41 to 72 minutes with a mean value of 51.8-4.1 minutes. Afte r i n j e c t i o n of 20% carbon dioxide i n a i r , the mean breathing time dropped to 20.6 minutes. The number of experiments done i n which more than 20%, carbon dioxide was injected were fewer i n -.lumber and i t can only be said that i n j e c t i o n s of high concentrations of carbon dioxide i n the lungs (over 207o CO2) resulted i n the animal coming to the surface to breathe within a short time (less than 29 minutes). I t was impossible to ascribe any relat i o n s h i p between the onset of breathing and the blood PCO2 l e v e l s . Figure 19 i l l u s t r a t e s the re s u l t s from four animals i n which the systemic arch PCO2 le v e l s were followed through several breathing cycles. I t cannot even be said that breathing occurred as the blood PCO2 rose, for i n fac t breathing sometimes occurred as the blood PCO2 f e l l . I f the blood PCO2 le v e l s i n an animal were a r t i f i c i a l l y raised by increasing the PCO2 i n the 65 Table VI. Carbon dioxide i n j e c t i o n s into the lungs of Amphiuma. Time since Lung PO2 Lung PCO2 Type of Time afte r l a s t breath p r i o r to p r i o r to i n j e c t i o n i n j e c t i o n u n t i l i n j e c t i o n i n j e c t i o n next breath 1 min 60 ml 10% C0 2 63 min 40 min 27 .5 ( 13 ) * 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 52 min — — — 60 ml 10% C0 2 43 min 4 min 53 .6 (9) 20 .6 (9) 20 ml 10% C0 2 30 min 5 min 92 .7 (47) 22 .8 (47) 40 ml 10% c o 2 83 min 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 8 min 50 ml 10% C0 2 1 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 64 min 31 .1 (4) 23 .9 (4) 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 3 min 30 ml 15% C02 48 min 4 min 100 ml 15% c o 2 55 min 8 min 33 .7 (4) 21 .9 (4) 30 ml 15% c o 2 50 min * Bracketed numbers indicate the time (minutes) that had elapsed between the i n j e c t i o n of CO2 and the lung meas-urements . 66 Table VI. (Continued) Time since Lung P0 2 Lung PC0 2 Type of Time afte r l a s t breath p r i o r to p r i o r to i n j e c t i o n i n j e c t i o n u n t i l i n j e c t i o n i n j e c t i o n next breath 59 min - - 60 ml 20% C0 2 20 min 40 min 60 ml 20% C0 2 17 min 3 min 60 ml 20% C0 2 25 min 8 min — / 10 ml 30% C0 2 1 min 51 min 10 ml 40% C0 2 29 min 40 min 10 ml 50% C0 2 3 min 23 min 65.0 (9) 16.0 (9) 10 ml pure C0 2 6 min 30 min --• 10 ml pure C02 2 min 57 min • 20 ml pure C0 2 3 min 3 min 20 ml pure C0 2 5 min 67 Figure' 19 A r t e r i a l PCO2 lev e l s through several breathing cycles i n Amphiuma. V e r t i c a l arrows extending above the graphed l i n e for a p a r t i c u l a r animal, indicate the time of breath. J — — I I I I _i I I ' ' 1 20 40 60 80 100 120 140 160 180 200 220 T I M E ( m i n ) surrounding water, i t can be seen ( F i g . 2 0 ) that as the PCO2 of the water rose to and remained at 80 mm Hg, the dorsal aorta PCO2 rose to a l e v e l between 40-45 mm Hg. When the water was replaced (low P C O 2 ) the dorsal aorta PCO2 f e l l , P0£ fluctuations i n the dorsal aorta did not appear to be affected by the increase i n dorsal aorta PCO l e v e l s ( F i g . 2 0 ) . A s i m i l a r experiment was performed on another animal and i t was found that although the dorsal aorta P C 0 - , rose to 40 mm Hg the animal did not breathe u n t i l the oxygen tension i n the dorsal aorta dropped to b tween 30-40 mm Hg (followed through three breathing cycle varying i n length from 42-61 minutes). D. BREATHING ONSET AND ITS RELATION TO BODY PO? LEVELS Removal of oxygen from the lungs was accomplished by flushing large amounts of nitrogen through the lungs v i a the lung cannulae. In Table VII, eighteen experiments per formed on f i v e animals are shown to i l l u s t r a t e the e f f e c t of oxygen removal from the lung on breathing rate i n Amphiuma. Since only pure nitrogen was being injected into the animal, i t was important to consider the time spent submerged before the nitrogen i n j e c t i o n was made. I f the time elapsed between the previous breath and the i n j e c t i o n time was not considered, the mean time to breathing was 9.0*1.22 minutes. I f i n j e c t i o n s were made 69 Figure 2 0 A r t i f i c i a l increase i n tank water PCO2 and the corresponding increase i n dorsal aorta P C O 2 . V e r t i c a l arrows at the top of the diagram indicate the breathing times. 70 Table VII. Nitrogen i n j e c t i o n s into the lungs of Amphiuma. Time since Lung PO2 Lung PCO2 Type of Time afte r l a s t breath p r i o r to p r i o r to i n j e c t i o n i n j e c t i o n u n t i l i n j e c t i o n i n j e c t i o n next breath 1 min 7 .7 (3)* 10 .2 (3) 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 13 min 7 .8 (5) 8 .9 (5) 140 ml N 2 16 min 14 min 50 ml N 2 7 min 2 min 3 .8 (1) 4 .9 (1) 100 ml N 2 8 min 15 min 13 .0 (10) 16 .0 (10) 60 ml N 2 17 min 15 min 8 .1 (1) 6 .6 (I) 80 ml N 2 3 min 17 min 100 ml N 2 13 min 20 min 50 ml N 2 5 min 27 min 8 •14 (1) 10 .0 (1) 75 ml N 2 3 min 37 min 60 ml N, 5 min 46 min 16 .6 (6) 14 .8 (6) 15 ml N 2 13 min * Bracketed numbers indicate the time (minutes) that had elapsed between the i n j e c t i o n of N 2 and the lung meas-urements . 71 within 15 minutes a f t e r a breath, the mean time between breaths was 15.3 minutes, or on the average 9.4 minutes a f t e r the i n j e c t i o n . I f i n j e c t i o n s of nitrogen were made 15 minutes or more af t e r the animal had taken a breath, the mean time to breathe a f t e r the i n j e c t i o n was 8.4 minutes (the times of 9.4 and 8.4 minutes are not s i g -n i f i c a n t l y d i f f e r e n t at the 5% l e v e l ) . Using data from Part I of: this thesis, P0£ l e v e l s i n the dorsal aorta and systemic arch at the time of breathing were obtained from f i v e d i f f e r e n t an-imals ( F i g . 21). As i t was not possible to obtain blood PO2 measurements at the exact time of breathing on a l l occasions, i t was necessary to extrapolate "breathing values" by extending the removal curve for oxygen i n the blood to the time of breathing. When i l l u s t r a t e d i n this manner ( F i g . 21) there was as much as 14 mm Hg difference i n a r t e r i a l breathing values during f i v e breathing cycles i n one animal and as l i t t l e as 1 mm Hg through three breathing cycles i n another animal.. 72 Figure 21 PCX-, l e v e l s i n the dorsal aorta and systemic arch i n f i v e Amphiuma at the time of breathing. TIME (min) DISCUSSION Amphiuma tridactylum breathes about once every hour (55 - 3 minutes). The animal becomes agitated, r i s e s to the surface and f i l l s i t s lungs.. I t then returns to the bottom of the tank. The mean time between breaths, re-corded i n this study, i s i n agreement with data reported by Darnel]. (1948). I t i s apparent (Part I of this thesis) that the primary function of surfacing i n Amphiuma i s to replenish the a i r i n the lungs. What sti m u l i determine the cessation of i n s p i r a t i o n ? In four experiments i n which a i r was withdrawn from the I.ungs as the animal was breathing, lack of term-i n a t i o n of. the i n s p i r a t o r y process indicates that f i l l i n g the lungs i n Amphiuma terminates breathing. Termination 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 of a i r had passed through the lungs and had been removed through the can-nula, was probably because the speed of extraction of a i r d i d not equal the rate of lung i n f l a t i o n by the animal. The important observation i s not the exact amount of a i r removed from the lungs but that, as a i r was withdrawn from the Lungs, more was forced into the lungs by the an-imal than would normally f i l l the lungs. The i n s p i r a t o r y event i s not based on a c e r t a i n number of "gulps" of a i r but rather on the volume of a i r i n the lungs which i n turn probably triggers s t r e t c h receptors i n the lung. These have been shown to be present: i n other amphibians (Neil e t . a l . , 1950; P a i n t a l , 1963; Widdicombe, 1964; T a g l i e t t i and Casella, 1966; Shimac.a, 1966; Jones, 1966). The nature of the gas mixture seems to be un-important i n regulating the termination of i n s p i r a t i o n . The behavior of the animal p r i o r to i n s p i r a t i o n i s very c h a r a c t e r i s t i c and i t was possible to i n f l a t e the lungs with nitrogen during this period. B i l l i n g the lungs with nitrogen was s u f f i c i e n t to terminate the i n s p i r a t o r y response and the animal returned to the bottom of the tank for a short while. This "appeasement" period was sho r t l i v e d and i n a l l cases the animal returned to the surface to breathe within 2^-11 minutes. A si m i l a r type of response was recorded by Jones (1966). He found that frogs surfacing into nitrogen nearly recovered from diving bradycardia, however only lung i n f l a t i o n s with a i r and "release of anoxia" brought about complete re-covery. Because of the d i f f i c u l t y of recording an "E.C.G." through the thick skin and musculature of Amphiuma, heart rate was not monitored i n most of the experiments i n t h i s part of tha study. I t i s possible however, that there might be a r e l a t i o n s h i p between heart rate and breathing 75 since (as w i l l be shown i n Part I I I of this study) there i s a gradual slowing down of the heart rate as the animal remains submerged. The major question to be considered i n this part of the study i s what s t i m u l i are involved i n deter-mining the onset of breathing i n Amphiuma. Continuous i n j e c t i o n s of a i r into 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 surface. Submerged times i n Excess of four hours c l e a r l y indicate that the s t i m u l i or stimulus involved i n triggering the animals breathing response could be overridden or negated by per-i o d i c a i r i n j e c t i o n s . Such lengthy times beneath the water surface also indicate, a. p r i o r i , that carbon dioxide must be s a t i s f a c t o r i l y eliminated by respiratory surfaces other than the lungs. There i s the possibly however, that carbon dioxide was removed as a i r bubbled out of the lungs at the time of a i r i n j e c t i o n . I n j e c t i o n of pure oxygen into the lungs of Am-phiuma also extended the i n t e r v a l between breaths. Injection of 20 ml pure oxygen extended the breathing i n t e r v a l to s l i g h t l y more than twice the normal breathing i n t e r v a l . Since the pure oxygen/air mixture i n the lungs would not exceed the normal lung capacity, the lengthy submergence time would necessarily decrease the volume of a i r i n the lungs more than would occur i f normal breathing had taken place. This alone would indicate that d e f l a t i o n receptors, found by T a g l i e t t i and C a s e l l a ( 1 9 6 8 ) i n the frog lung, did not provide the breathing stimulus i n Amphiuma. The chemical regulation of r e s p i r a t i o n by car-bon dioxide i n mammals i s a well documented phenomenon (review by Hornbein, 1 9 6 5 ) and i t was thought that carbon dioxide might play an important role i n the regulation of breathing i n Amphiuma. Carbon dioxide/air mixtures were injected into the lungs. The r e s u l t s of these i n j e c t i o n s were i n no way conclusive. For example the mean breathing time a f t e r i n j e c t i o n of 1 5 7 o carbon dioxide was 8 - 9 min-utes longer than i f 1 0 % carbon dioxide were inj e c t e d . In-j e c t i o n of 2 0 7 o carbon dioxide resulted i n the animal breathing within 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 breathing. In this p a r t i c u l a r instance the amount of oxygen i n the injected sample would be lowered to 1 6 - 1 7 % simply by the presence of the carbon dioxide i n the gas mixture. I t seems doubt-f u l however, that a 5 % drop i n oxygen concentration i n the lung would shorten the i n t e r v a l between i n j e c t i o n and the next breath to 2 1 minutes. Higher concentrations of carbon dioxide ( 3 0 % - p u r e C O 2 ) injected into the lungs pro-77 duces an even shorter i n t e r v a l between breaths. Amphiuma tridactylum, under normal environ-mental conditions, would never encounter carbon dioxide concentrations as high as those injected into the lungs. Mammalian alveolar concentrations r a r e l y exceed 10% carbon dioxide (76 mm Hg S.T.P.) and i t has been shown e a r l i e r in t h i s study that the mean alveolar PC0 2 for Amphiuma i s about 15 mm Hg. In conclusion, i t appears that Amphiuma has some detection mechanism whereby breathing onset w i l l occur more r a p i d l y i f unphysiological doses of carbon dioxide are injected into the lungs. If the a r t e r i a l PCC^ i s raised to twice the normal l e v e l by increasing the PCO^ of the surrounding water, no change i n the normal breathing pattern r e s u l t s . In normal, free-moving Amphiuma i n fresh water, no r e l a t i o n -ship was found between the PCC^ levels in the blood and lungs and the onset of breathing (data used from Part I of t h i s study). PCC^ i n the major vessels might either be f a l l i n g s l i g h t l y , r i s i n g s l i g h t l y or more frequently, be constant at the time of breathing. Elimination of carbon dioxide from the body i s very rapid i n Amphiuma tridactylum. Carbon dioxide injected into the lungs, r a i s i n g alveolar PCC^ to 3-5 times .the normal l e v e l s , is removed from the lungs within 78 ten minutes a f t e r i n j e c t i o n . The rapid removal of carbon dioxide from the lungs, i f lung P C O 2 i s raised, and the rapid r i s e i n blood P C O 2 , i f water P C 0 2 l e v e l s are i n -creased, indicate that the Amphiuma skin i s capable of rap i d l y transfering large amounts of carbon dioxide across th i s respiratory surface. Injections of nitrogen into the lungs of Amphiuma, thereby lowering the alveolar P O 2 l e v e l s , i s somewhat more ph y s i o l o g i c a l i n that when an animal has remained submerged for periods up to an hour, the lung i s normally f i l l e d with .90-95% nitrogen. The e f f e c t of lowering alveolar P 0 2 i s that the times between breaths i s definately shortened. The actual response time i s va r i a b l e and i s related to the fact that, i f one postulates the presence of an oxygen chemo-receptor (which triggers the breathing response), inj e c t i o n s of nitrogen into the lung w i l l lower blood P O 2 at a rate which i s related to the oxygen reserves (hemoglobin bound) i n the body and the metabolic rate of the animal. The rate of oxygen consumption of Amphiuma, as shown e a r l i e r , i s very low and i s related to the blood oxygen content. The mean time to breathing of nine minutes afte r i n j e c t i o n i s not an unreasonable length of time for blood P O 2 l e v e l s to drop to l e v e l s similar to those i n the lung. I t seems quite 79 reasonable to suggest at this point that the onset of breathing i s much more se n s i t i v e to oxygen deprivation than to increases i n carbon dioxide concentrations. These observations indicate that oxygen chemo-receptors may be involved i n the i n i t i a t i o n of breathing i n Amphiuma. Relative consistency between the PO2 i n the dorsal aorta and systemic arch and the onset of breathing indicates that the receptor s i t e s might be located i n the a r t e r i a l c i r c u l a t i o n . In an o s c i l l a t i n g system, such as breathing i n Amphiuma, i t does not seem unreasonable to postulate a control mechanism triggered by another o s c i l -l a t i n g parameter (body PO2 l e v e l s ) rather than a system i n which o s c i l l a t i o n s do not normally occur (body PCO2 l e v e l s ) . High carbon dioxide lev e l s however, do a l t e r the i n t e r v a l between breaths. Carbon dioxide may have either a d i r e c t or an i n d i r e c t e f f e c t on breathing i n Amphiuma. Increased lev e l s of carbon dioxide may stim-ulate oxygen consumption, which would shorten the breath-ing i n t e r v a l . Other alternatives might be that carbon dioxide may e f f e c t the re l a t i o n s h i p between P0£ and breath-ing d i r e c t l y or perhaps carbon dioxide simply has a d i r e c t e f f e c t on breathing 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. Shelton and Jones (1965 a,b and 1968) and Johansen and Hanson (1968) provide more recent accounts of research being done i n this area. In view of the exten-sive reviews provided, only a brie:! resume on subjects pertinent to the present study w i l l be given here. The " c l a s s i c a l hypothesis" of blood flow through the amphibian heart was f i r s t put forward by Brucke (1852) and l a t e r modified by Sabatier (1873). They stated that oxy genated and deoxygenated blood remained unmixed i n the vent r i c l e , the. oxygenated blood positioned on the rig h t side of the v e n t r i c l e was the f i r s t to leave the heart upon ventric u l a r contraction. The d i r e c t i o n of flow of deoxygenated blood through the conus was aided by the s p i r a l valve and because of the lower pressure i n the pulmonary c i r c u i t , deoxygenated blood p r e f e r e n t i a l l y flowed into the lung c i r -c u i t . As pressure i n the pulmonary and systemic c i r c u i t s became equal the s p i r a l valve was then thought to shut o f f flow to the pulmonary c i r c u i t and the oxygen r i c h blood 81 leaving the v e n t r i c l e would enter the systemic and c a r o t i d v e ssels. Since Brucke and Sabatier, Vandervael (1933) completely discarded the c l a s s i c a l hypothesis and stated that blood i n the v e n t r i c l e and major vessels was com-p l e t e l y mixed. Noble (1925), Acolat: (1931, 1938), Foxon (1951), Simons and Michaelis (1953), de Graaf (1957), Simons (1959), DeLong (1962), Sharma (1957), Johansen (1963), Jchansen and D i t a d i (1966) and Shelton (pers. comm.) have shown, by a v a r i e t y of techniques on several species of amphibians, that there i s . a s e l e c t i v e d i s t r i b -ution of oxygenated and deoxygenated blood to the pulmon-ary and systemic c i r c u i t s . In discussion of this topic the two points of agreement (with the exception of Vandervael) are: i . the trabeculate nature of the amphibian v e n t r i c l e does enable the blood to remain r e l a t i v e l y unmixed, i i . blood from the r i g h t atrium (l e a s t oxygen-ated) i s found i n the r i g h t side of the vent-r i c l e , closer to the semilunar valves than the oxygenated blood from the l e f t atrium. Much disagreement occurs i n the l i t e r a t u r e on the sequence 82 of events after v e n t r i c u l a r systole that f a c i l i t a t e the movements of deoxygenated blood to the lungs. 'Differences i n the pulse pressure between the pulmonary and systemic c i r c u i t s has been recorded by sev-e r a l people. Acolat (1938) found a 1-3 mm Hg lower d i a s t o l i c pressure i n the pulmocutaneous branch i n eight species of anurans. De Graaf (1957), working on Xenopus l a e v i s , found that d i a s t o l i c pressures i n the pulmocutaneous artery were on the average 7 mm Hg lower than i n the other two arches. DeGraaf also found that there was a " l a g " i n the r i s e i n pressure i n the systemic c i r c u i t , i n that the pressure i n the pulmocutaneous c i r c u i t rose 0.3.0-0.15 seconds before the pressure increased i n the systemic c i r c u i t . Johansen (1963) cannulating vessels at a greater distance from the heart i n Amphiuma tridactylum recorded lower d i a s t o l i c pressures i n the pulmonary artery than i n the systemic arch and also recorded a " s l i g h t l y e a r l i e r pressure r i s e i n the pulmonary artery". Neither de Graaf nor Johansen attached any s i g n i f i c a n c e to the pressure lag i n the sys-temic c i r c u i t . Shelton and Jones (1968)., working on three species of Anura and one urodele, found consistently lower pressures i n the anuran pulmocutaneous artery than were recorded simultaneously i n the syscemic arch, but found 83 si m i l a r sj'stolic and d i a s t o l i c pressures i n the urodele. Similar pulse pressures i n the urodele were thought to occur because of the ductus B o t a l l i i n this p a r t i c u l a r animal. Shelton (pers. comm.), i n recent blood flow studies on Xenopus l a e v i s , has evidence for a s l i g h t increase i n blood flow to the pulmonary c i r c u i t p r i o r to flow i n the systemic. Shelton and Jones (pers. comm.) have data which suggests chat i n some anurans there i s an increase i n blood flow'to the lungs for a period of time following a breath bu; that blood flow to the two c i r c u i t s i s equal throughout the greater portion of the breathing cycle. Johansen (1963) was the f i r s t to examine the cardiovascular dynamics i n Amphiuma tridactylum and he off e r s a p a r t i a l explanation to the blood flow patterns i n the major vessels i n this animal. In general he re-corded blood pressure and blood flow i n the major vessels and also recorded ci n e f l u o r o g r a p h i c a l l y , the movement of blood through the Amphiuma heart into the a r t e r i a l c i r -c u l a t i o n . He stated that shunting of deoxygenated blood to the pulmonary c i r c u i t and oxygenated blood to the sys-temic c i r c u i t was accomplished by "laminar outflow patterns from the v e n t r i c l e with a right-hand s p i r a l movement through the undivided bulbus co r d i s " . Johansen was also 84 aware that: d i a s t o l i c and s y s t o l i c pressure changes i n the major vessels could a l t e r the s e l e c t i v e passage of blood, but s p e c i f i c d e t a i l s with regards to this phenomenon were not given The objectives i n this part of the study were to record blood pressures simultaneously i n the body and lung c i r c u i t s of Amphiuma tridactylum and to determine i f there were any pulse pressure differences. The possible presence of pressure "lags" i n the systemic c i r c u i t and the effects of breathing and a i r i n j e c t i o n s upon blood pressure i n the major a r t e r i e s was also investigated. MATERIALS AND METHODS The experiments i n this study were performed on 23 adult Amphiuma tridactylum. The animals were cannulated by a method described previously i n this thesis (General Materials and Methods). Blood pressure was monitored with Statham 23AA, 23BB or 23 Db pressure transducers which were i n turn con-nected to a Beckman Type R Dynograph. Pressure transducers were ca l i b r a t e d with a column of s a l i n e . The response time of the pressure recording equipment was 0.20-0.25 msec. The experiments were c a r r i e d out i n a 20 l i t r e glass aquarium which was p a r t i a l l y f i l l e d with 10 l i t r e o f . f r e s h water held at 15°C. A l l animals were free-moving and unanaesthetized; records were not taken u n t i l 4-6 hours aft e r the operation. RESULTS A. HEART HATE Heart rate 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 heart rate recorded at 15° C was 5 beats/min and the highest 19 beats/min. In general there was a tachy-cardia immediately following a breath and a gradual slow-ing down of the heart rate as the submerged time increased. 86 The mean f l u c t u a t i o n i n heart rate between breaths was 5.1 beats/min. Figure 22 i l l u s t r a t e s the "diving bradycardia" or "breathing tachycardia" i n several Amphiuma. B • L U N G FILLING AND CIRCULATORY CHANGES The tachycardia, which occurred when Amphiuma breathed was, i n most cases, associated with a s l i g h t drop i n blood pressure i n both the pulmonary and systemic c i r -c u i t s . Figure 23a i l l u s t r a t e s the breathing tachycardia as well as the pressure drop i n the two c i r c u i t s . I t i s import-ant to note that the pulse pressure i n the pulmonary c i r c u i t does not decrease to the same extent as does the pulse pres-sure i n the systemic c i r c u i t . A similar phenomenon could be observed by a r t i f i c i a l l y f i l l i n g the lung with a i r or nitrogen ( F i g . 23b). Figure 23c further i l l u s t r a t e s the extent to which lung volume a f f e c t s blood and pulse pres-sures. Injections of a i r into the lung raised the sys-temic d i a s t o l i c pressure by about 3 mm Hg. After a short period of time the a i r was removed and the d i a s t o l i c pres-sure i n the systemic arch returned to the o r i g i n a l l e v e l . Injections of low concentrations of carbon dio-xide (5-15%) produced e f f e c t s on blood pressure and heart rate similar to those of 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 2 2 Diving bradycardia or breathing tachycardia i n f i v e Amphiuma. A l l animals breathed at time " 0 " and breathed again at a point marked by a v e r t i c a l arrow. 88 F i g u r e 23 B r e a t h i n g and lung i n f l e c t i o n and d e f l a t i o n e f f e c t s on the pulmonar}' a r t e r y (PA) and systemic a r c h (SA). Recordings o b t a i n e d s i m u l t a n e o u s l y . a. B r e a t h i n g t a c h y c a r d i a and the a s s o c i a t e d b l o o d p r e s s u r e drop i n the pulmonary and systemic b l o o d c i r c u i t s . b. A r t i f i c i a l f i l l i n g of the lungs w i t h n i t r o g e n c. The e f f e c t s of lung d e f l a t i o n on p u l s e p r e s s u r e . A. B. CO co LU ££ CL Q O o co 30 r-PA 15 U) r £ o 30 SA 15 "AAAAAj 10ml N . J • • » i ' ' ' WWWWWIM j i J i i . i c . 301-PA 15 30 r-•\ lung deflation — / 10ml SA 15 wvwwwvw . AAAAA/WWW 5 mm interval mX ' t i l l | L -I I I 1 L . J TIME (lOsec. interval) animal was i n a state of bradycardia, a breathing type bradycardia would r e s u l t , d i a s t o l i c pressures would f a l l more i n the pulmonary artery than i n the systemic arch and there was not a general decrease i n blood pressure. Injections of very high concentrations of carbon dioxide (25, 50 and 100%) into the lungs produced an almost im-mediate drop i n blood pressure and heart rate ( F i g . 24). C. THE LAG PHENOMENON D i a s t o l i c pressures i n the pulmonary a r t e r i e s were on the average 3-5 mm Hg lower than those recorded simultaneously i n the systemic arch at a point 1 cm an-t e r i o r to the v e n t r i c l e . Figure 25 i l l u s t r a t e s this plus the f a c t that there was a d e f i n i t e lag i n blood pressure r i s e i n the systemic arch. The pressure r i s e i n the pul-monary arch was very rapid at the s t a r t of v e n t r i c u l a r systole and i t was only at the point where the blood pres-sure i n the two c i r c u i t s was equal that the pressure rose i n the systemic c i r c u i t . In the animal described i n Figure 24, the lag was calculated to be 0.18-0.20 seconds i n duration. Figure 26a shows the pressure relationships i n the two main arches i n a very slowly beating heart (about 8 beats/min). The calculated lag i s 0.20-0.25 sec 90 Figure 24 Injections of high concentrations of carbon dioxide into the lungs and the associated pressure changes i n the systemic arch. 25 X CO, 30 20 10 0 J 1 I I I I I I I I I ^ L U < r v " O t/> < LU o r t ^ G£ 4? 20 -E X o o o CO 30 r 10 0 50 # CO, - i I I I I I I K. I 1 I 1 1 I I 30 20 101-0 lOO^CO, * * i i i i i i i — J 1 — i 1 — i — J 1 1 — u TIME (lO sec. interval) 91 Figure 25 Simultaneous pressure recordings i n the pul-monary artery (PA) and systemic arch (SA) showing the pulse lag i n the systemic arch. a. slow chart drive on recorder. b. rapid chart drive o;i recorder. PA 2Q O 4 0 SA 20 TIME INTERVAL—Isec 92 Figure 26 Pressure relationships i n the pulmonary artery (PA) and systemic: arch (SA) i n a. slowly beating heart b. Superimposed pressure recordings from the pulmonary and systemic arches i n another Amphiuma. 6|_g LULU a a n s s a a d a o o i a 93 and the d i a s t o l i c pulse pressure difference between the two arches i s 3.8 mm Hg. Figure 26b shows the difference i n outflow pattern i n the two c i r c u i t s . There i s a more rapid f a l L i n blood pressure aft e r i n c i s u r a i n the pul-monary artery than i n the systemic c i r c u i t . Peak s y s t o l i c pressures i n this 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. S y s t o l i c 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 arches. 94 DISCUSSION Jones and Shelton (1964) and Jones (1966; 1968) have discussed diving bradycardia i n several species of anuran amphibians. They have shown that diving bradycardia i s very pronounced i n the Anura. Amphiuma tridactylum, an aquatic urodele, does not show a rapid drop i n heart rate upon submergence, which may either r e f l e c t the f a c t that submergence was "voluntary" or that, because a submerged habitat is-normal for Amphiuma, the diving bradycardia i s not very pronounced. The s t i m u l i involved i n bringing about diving bradycardia i n amphibians i s s t i l l unclear. Leivestad (1960), working on the toad, Bufo bufo,. has shown that submergence for two hours and the resultant diving brady-cardia doss not r e s u l t i n an oxygen debt being b u i l t up. Jones (1957) has shown that during submergence i n three species o£ anuran amphibians, the rel a t i o n s h i p between heart rate and oxygen uptake i s simply; " i f one i s low then the other i s generally low". In Amphiuma the r e l a t -ionship i s also that lower oxygen consumption during the l a t t e r part of the submerged period usually coincides with lower heart rates. When Amphiuma breathes, or the lungs are a r t i f -i c i a l l y i n f l a t e d with a i r , there i s a greater increase i n 95 pulse pressure i n the systemic arch than i n the pulmonary artery. This i s very i n d i c a t i v e of an increased flow to the pulmonary c i r c u i t for a short period of time aft e r a breath. Recent blood flow studies done by Shelton (pers. comm.) on Xenopus laevis and Jones (pers. comm.) on Rana  pipiens, indicate that for a short period of time aft e r a breath there i s increased blood flow to the pulmonary c i r -c u i t . A f t e r the i n i t i a l increase the blood flow to the pulmocutaneous dropped and i n Rana pipiens there was less flow to the pulmocutaneous than the systemic arches and i n Xenopus la e v i s the blood flow to the two arches was more or less equal. Therefore, i n Amphiuma, i f pressure f a l l s as flow increases i n the pulmonary artery, there must be a substantial f a l l i n lung peripheral resistance during breathing. To account for this phenomenon there must be increased vasoconstriction during the submerged period and vas o d i l a t i o n during the breathing process and for a short period of time thereafter. Injections of nitrogen into the lungs produced the same eff e c t s as those described by Jones (1966) i n that there i s a "release of the bradycardia" but the ef-fects on blood pressure of nitrogen i n j e c t i o n are different: 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 pressure increase i n the pulmonary 96 a r t e r y b u t r e t u r n t o n o r m a l w i t h i n 1-2 m i n u t e s . S y s t o l i c a n d d i a s t o l i c p r e s s u r e s i n c r e a s e i n t h e s y s t e m i c a r c h f o r 1-2 m i n u t e s b u t t h e d i a s t o l i c p r e s s u r e s r e m a i n e l e v a t e d , r e s u l t i n g i n a d e c r e a s e i n p u l s e p r e s s u r e o v e r a l l . T h e e x -p l a n a t i o n f o r t h i s m i g h t b e t h a t w i t h n i t r o g e n i n j e c t i o n 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 o r v a s o d i l a t o r y r e s p o n s e s a n d t h e d e c r e a s e i n p u l s e p r e s s u r e m i g h t b e d u e t o t h e i n c r e a s e i n h e a r t r a t e o r p o s s i b l y t h e i n c r e a s e i n r e s i s t -a n c e i n t h e b o d y c i r c u i t a s a r e s u l t o f t h e p h y s i c a l d i s -p l a c e m e n t i n t h e b o d y c a u s e d b y l u n g i n f l a t i o n . T h e r a p i d d r o p i n h e a r t r a t e a n d b l o o d p r e s s u r e a f t e r i n j e c t i o n s o f h i g h c o n c e n t r a t i o n s o f c a r b o n d i o x i d e i n t o t h e l u n g i s d i f f i c u l t t o e x p l a i n w i t h o u t some f u r t h e r e x p e r i m e n t a t i o n i n t h i s a r e a . E x p l a n a t i o n s o f t h i s p h e n o -m e n o n a r e c o m p l i c a t e d b y t h e f a c t t h a t t o e l l i c i t s u c h a r e s p o n s e , e x t r e m e l y h i g h , u n p h y s i o l o g i c a l c o n c e n t r a t i o n s o f c a r b o n d i o x i d e w e r e n e c e s s a r y . P e a k s y s t o l i c p r e s s u r e s r e c o r d e d s i m u l t a n e o u s l y i n t h e p u l m o n a r y a n d s y s t e m i c a r c h e s a r e , i n t h e m a j o r i t y o f c a s e s , e q u a l a n d o c c u r a t t h e same t i m e . I f t h e f r e q u e n c y r e s p o n s e t i m e i n t h e r e c o r d i n g s y s t e m i s a d e q u a t e ( t h i s h a s a l r e a d y t e e n s h o w n t o b e t r u e i n t h i s s t u d y ) a n d p r e s s u r e s a r e r e c o r d e d w i t h i n 2 cm o f t h e v e n t r i c l e , p r e s s u r e p u l s e s 97 from the v e n t r i c l e w i l l a r r i v e at the recording s i t e s sim-ultaneously (Shelton and Jones,(1968); Womersley,(1955)). D i a s t o l i c pressures and the rate of runoff are on the other hand related to peripheral resistance, compliance and heart rate. Runoff has been shown by Shelton and Jones (1968) and de Graaf (1957) to be more rapid i n the pulmonary arch and i t was: suggested by de Graaf that this was a r e s u l t of the lower resistance i n the lung c a p i l l a r y beds. The pulse lag recorded i n the present study, between the pressure r i s e i n the pulmonary artery and systemic arch, could not occur unless there was some occlusion to flow i n the sys-temic c i r c u i t for a short period of time. In an attempt to elucidate the anatomical function-ing of the s p i r a l valve and associated structures, ten Am-phiuma we::e anaesthetized a f t e r normal experimentation and the v e n t r i c l e , conus, truncus and pulmonary portions of the c i r c u l a t o r y system were dissected free of the body. In s a l -ine solutions, i n c i s i o n s or "windows" were made i n the ant-e r i o r portion of the conus and posterior portion of the truncus such that the functioning of the s p i r a l valve could be observed with a dissecting microscope. I t appeared that the s p i r a l valve i n Amphiuma was a triangular ridge of muscular tissue extending the length of the conus and pro-98 truding into the conus lumen to the extent of 2/3 of the conus diameter. At the anterior end of the conus the t r i -angular r:-.dge becomes a round, rather lobular structure which appears to occlude the entrance to the systemic c i r -c u i t during the i n i t i a l v e n t r i c u l a r outflow. This tissue could occlude the vessel b r i e f l y as the blood pressure r i s e s or :Lt could simply be forced into this p o s i t i o n as the conus i s mechanically elongated as i t becomes turgid with blood. Of the two a l t e r n a t i v e s , I would prefer the "occlusion as a blood pressure phenomenon" i n that the pressure lag i n the systemic c i r c u i t i s very b r i e f and a pulse pressure i s recorded there at the same time as the pressures i n the two c i r c u i t s are equal. A pulse lag 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 heart beat which extends for 5-6 seconds. However, i f one considers that v e n t r i c u l a r output occurs from the s t a r t of systole to the i n c i s u r a , the lag 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 primarily deoxygenated blood i s flowing to the pulmonary c i r c u i t , be long enough to set up the gradients between the two c i r c u i t s reported e a r l i e r i n this study (Part 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 that, i f the 99 f i r s t 10-15% of the blood leaving the v e n t r i c l e was mixed venous and entered the pulmonary c i r c u i t , the gradients be-tween the pulmonary artery and systemic arch i n Amphiuma could have been obtained by this phenomenon. Shelton and Jones (1968) have shown i n the uro-dele Salamandra salamandra that there are no pulse pressure differences between the pulmonary and systemic c i r c u i t s , and suggest that the presence of a ductus B o t a l l i equalizes, pressure i n the two c i r c u i t s . Simons (1959), working on Triturus c r i s t a t u s and Salamandra maculosa, found that there was no d i f f e r e n t i a l d i s t r i b u t i o n of blood 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 anim-als possess a ductus B o t a l l i . This embryonic blood vessel i s retained i n most urodeles; Amphiuma provides one excep-t i o n . I t follows then, i f a l l t e r r e s t r i a l amphibians de-pendent upon pulmonary r e s p i r a t i o n , lack a ductus B o t a l l i (to my knowledge a l l adult anurans lack this vessel) the pressure difference created by having a separate pulmonary and systemic c i r c u i t could be strongly implicated i n the separation of the two types of blood. I t has been shown that there are pulse pressure differences i n the two c i r c u i t s , a possible explanation 100 has been given as to how the d i a s t o l i c pressure d i f f e r -ences i n the two c i r c u i t s could r e s u l t i n blood from the body bein;* i n part sent to the lung c i r c u i t and the reason for such a phenomenon I think i s best stated by Foxon (196^ ) when he said, "perhaps evolutionary s e l e c t i o n has acted i n favour of those animals which possessed, not some hy-po t h e t i c a l mechanism for the se l e c t i o n of blood for the head region, but some mechanism which prevented blood which had returned from the lungs from being immediately sent there again". 101 SUMMARY 1. The experimental animal of this study was Amphiuma tridactylum, an aquatic u::odele. Amphiuma breathed about once every hour and almost completely replaced a l l a i r within the lungs at each breath. 2. While Amphiuma remained submerged between breaths, oxygen was removed from the lungs but carbon dioxide lev e l s did not increase. The R l i n e for alveolar gases i n this animal was therefore zero. 3. Oxygen consumption i n Amphiuma at 15°C was the lowest recorded for any amphibian at a comparable temperature. Most of the oxygen was consumed within the f i r s t f i f t e e n minutes of submergence. The primary r e s p i r -atory surface for oxygen consumption was the lungs. 4. Oxygen tensions i n the major vessels o s c i l -l a ted with the breathing cycles. There was a d e f i n i t e gradient between the pulmonary artery and systemic arch which pers i s t e d throughout the breathing cycle. The grad-i e n t decreased with time submerged, being caused by the decrease i n gradient between the oxygen tensions i n the pulmonary ve i n and venous return. 5. Afte r each breath i n Amphiuma the oxygen tensions, i n a l l the vessels studied rose r a p i d l y , the tensions i n the pulmonary vein increased to le v e l s found 102 i n the lungs, and were usually completely saturated. 6. Termination of i n s p i r a t i o n was shown to be con t r o l l e d by a volume detection mechanism. Animals were shown to continue the breathing process i f a i r was simult-aneously removed from the lungs through a lung cannula. Injections of nitrogen into the lungs terminated i n s p i r -a t i o n for a short time but breathing occurred a short time a f t e r . 7. Carbon dioxide 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 did not r e s u l t i n the onset of breathing i f the oxygen tensions were s u f f i c i e n t l y high. Very high concentrations of carbon dioxide i n the lungs resulted i n a shortening of the time between breaths. Increased l e v e l s of carbon dioxide i n the dorsal aorta did not bring about the breathing response. 8. Removal of oxygen from the lungs brought about a rapid breathing response. The presence of an a r t e r i a l oxygen chemoreceptor was postulated as a mechanism for c o n t r o l l i n g breathing i n Amphiuma. 9. The diving bradycardia response i n Amphiuma was not very pronounced and was quite i r r e g u l a r . 10. When Amphiuma breathed there was a greater increase i n pulse pressure i n the systemic arch than i n 103 the pulmonary artery. I f pressure f e l l as flow increased, there must have been a substantial f a l l i n lung peripheral resistance when the animal breathed. 11. There was a lower d i a s t o l i c pressure i n the pulmonary artery than i n the systemic arch. Pulse pressure was generally greatest i n the pulmonary artery. There was a s l i g h t pressure lag i n the systemic arch compared to the pressure cise i n the pulmonary artery. I t was suggested that the f i r s t blood to leave the v e n t r i c l e would flow to the pulmonary artery i n i t i a l l y because of the lower pressure i n the lung c i r c u i t and possibly because the entrance to the systemic c i r c u i t appeared to be blocked during the i n i t i a l phase of v e n t r i c u l a r output. The pressure lag phenomenon i n the systemic arch was thought to account for the PO2 difference between the pulmonary artery and the systemic arch. 104 LITERATURE CITED Acolat, M.L. 1931. Recherch.es anat;omiques r e l a t i v e s a l a separation du sang veineux et du sang a r t e r i a l dans l a coer de l a Grenouille. C.R. Acad. S c i . P a r i s . 192: 767-769. 1938. Etude compare de l a pression sanguine dans l e c i r c u i t pulmonaire et dans l e c i r c u i t general chez lez Batracien et les R e p t i l e s . C.R. Acad. S c i . P a r i s . 206: 207-209. Baker, C. L. 1945. The natural h i s t o r y and morphology of Amphiumae. J . Tennessee Acad. S c i . 2_0: 55-91. Brown, G.Vii.Jr. 1964. The metabolism of Amphibia. In Physiology of the Amphibia. Edited by J.A. Moore. Academic Press, New York and London, pp. 1-98. Brucke, E. von. 1852. Beitrage zur vergleichanden anatomie und physiologie des gefass-systems der amphibien. Denkschr. Akad. Wiss. Wien. 3_: 335-367. Czopek, J . 1962. Va s c u l a r i z a t i o n of respiratory surfaces i n some Caudata. Copeia. 1962: 576-587. Darn e l l , R. M. 1948. Environmental factors which deter-mine the habitat of Amphiuma. J . Tennessee Acad. S c i . 23: 3-12. DeLong, K. T. 1962. Quantative analysis of blood c i r -c u l a t i o n through the frog heart. S c i . 138: 693-69 105 Foxon, G.E.H. 1964. Blood and r e s p i r a t i o n . In Physiology of the Amphibia. Ed. J.A. Moore. Academic Press, New York and London, pp. 151-209. de Graaf, A. R. 1957. Investigations into the d i s t r i b u -t i o n of blood i n the heart and a o r t i c arches of Xenopus l a e v i s . J . Exp. B i o l . 34: 143-176. Hazelhoff, E. H. 1952. Die trennung der blutmassen mit verscheidenem sauerstoffgehalt im froscherzen. Experientia. £5: 77. Hornbein, T. F. 1965. The chemical regulation of v e n t i l -a t i o n . In Physiology and Biophysics. Ed. T. C. Ruch and H. D. Patton. W„B. Saunders Co. Philadelphia and London, pp. 803-819. Hughes, G. M. 1966. Species v a r i a t i o n i n gas exchange. Proc. Roy. Soc. Med. 59: 494-500. Johansen, K. 1963. Cardiovascular dynamics i n the amphibian, Amphiuma tridactylum, Cuvier. Acta. Physiol. 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 giant toad Bufo paracnemis. Physiol. Zool. 39: 140-150. Johansen, K. and D. Hanson. 1968. Functional anatomy of the hearts of lungfishes and amphibians Am. Zoologist. 8: 191-210. 106 Jones, D. R. 1966. Factors a f f e c t i n g the recovery from diving bradycardia i n the frog. J. Exp. B i o l . 44:. 397-411. i 1967. Oxygen consumption and heart rate of several species of Anuran Amphibia during sub-mergence. Comp. Biochem. Physiol. 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 of diving bradycardia i n Anuran Amphibia. Comp. Biochem. Physiol. 25_: 821-834. Jones, D.R. and G. Shelton. 1964. facto r s influencing submergence and the heart rate i n the frog. J . Exp. B i o l . 41: 417-431. Krogh, A. 1904. On the cutaneous and pulmonary resp-i r a t i o n of the frog. Skand. Arch. Physiol. 15: 328-419. Lambertsen, C. J . 1961. Respiration. In Medical Physiology, Part V. Ed. P. Bard. C.V. Mosby Co., St. Louis, pp. 559-710. Leivestad., H. 1960. The e f f e c t of prolonged submersion on the metabolism and the heart rate i n the toad. Arbok. Univ. Bergen. (Mat.-nat. serie) 5_: 1-15. Lenfant, C. and K. Johansen. 1967. Respiratory adaptations i n selected amphibians. Resp. Physiol. 2: 247-260, 107 Maren, T. H. 1967. Carbonic anhydrase: chemistry, physiology and i n h i b i t i o n . P hysiol. Rev. 47: 595-781. de Marnefi:e-Foulon, C. 1962. Contribution a l'etude du mechanisme et du controle des movements r e s p i r a t o i r e s chez Rana. Ann. Soc. Zool. Belg. 92: 81-132. N e i l , E., L. Strom and Y. Zotterman. 1950. Action pot- ; e n t i a l of afferent f i b r e s i n the IX and X c r a n i a l nerves of the frog. Acta. Physiol. Scand. 20: 338-350. . • 1950. Cardiac vagal afferent f i b r e s i n the cat and frog. Acta. P h y s i o l . Scand. 20: 160-165. Noble, G. K. 1925. The integumentary, pulmonary and cardiac modifications correlated with increased cutaneous r e s p i r a t i o n i n the Amphibia: a solut i o n to the "hairy f r o g " problem. J . Morph. 4fJ: 341-416. Noble, G. K. 1931. The Biology of the Amphibia McGraw H i l l , New York. P a i n t a l , A. S. 1963. Vagal afferent f i b r e s . Ergebn. Phys i o l . 52: 74-156. Sabatier, A. 1873. Etudes sur l a coeur et l a c i r c u l a t i o n centrale dans l a se r i e des Vertebres. Montpellier, P a r i s . 108 Scott, W. J . 1931. Oxygen and carbon dioxide transport by the blood of the urodele, Amphiuma tridactylum. B i o l . B u l l . 61: 211-222. Sharma, H, W, 1957. The anatomy and mode of action of the heart of the frog, Rana t i g r i n a . J. Morph. 100: 313-344. Shelton, G. and D. R. Jones. 1965a. Pressure and volume rel a t i o n s h i p s i n the v e n t r i c l e , conus and a r t -e r i a l arches of the frog heart. J . Exp. B i o l . 43: 479-488. ' 1965b. Central blood pressure and heart output i n surfaced and submerged frogs. J . Exp. B i o l . 42: 339-357. Shelton, G. and D. R. Jones. 1968. A comparative study of c e n t r a l blood pressures i n f i v e amphibians. J . Exp. B i o l . 49: 631-643. Shimada, K . 1966. Mechanical properties of the smooth muscle i n the lung of the toad. Acta. Med. B i o l . (Niigata) 14: 23-33. Simons, J . R. 1959. The d i s t r i b u t i o n of the blood from the heart i n some Amphibia. Proc. Zool. Soc. Lond. 132: 51-64. Simons, J . R. and A.R. Michaelis. 1953. A cinematograph-i c technique using u l t r a - v i o l e t i l l u m i n a t i o n for amphibian blood c i r c u l a t i o n . Nature. Lond. 171:•801 109 T a g l i e t t i , V. and C. C a s e l l a . 1966. Stretch receptors stimulation i n frog's lungs. Pflugers Arch. ges. Physiol. 292: 297-308. T a g l i e t t i , V. and C. C a s e l l a . 1968. De f l a t i o n receptors i n frog's lungs. Pflugers Arch. ges. Physiol. 304: 81-89. Vandervael, F. 1933. Recherches sur l e mechanisme de l a c i r c u l a t i o n du sang dans l a coeur des amphibiens Anoures. Arch. B i o l . P a r i s . 44: 571-606. Widdicom.be, J . G. 1964. Respiration r e f l e x e s . In Handbook of Physiology, sec. 3, v o l . 1. Amer. Physiol. S o c , Washington, pp. 585-630. Winterstein, H., F. Alpdogan, and M. Basoglu. 1944. Untersuchungen uber die a l v e o l a r l u f t des frosches Istanb. Univ. Fen. Fak. Mecm. 9.: 171-180. Womersley, J . R. 1955. Method for the c a l c u l a t i o n of v e l o c i t y , rate of flow c.nd viscous drag i n a r t e r i e s when the pressure gradient i s known. J. P h y s i o l . 127: 553-562. 

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