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Effects of circulating catecholamines on diving in ducks (Anas platyrhynchos) Lacombe, A. M. A., 1990

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EFFECTS OF CIRCULATING CATECHOLAMINES ON DIVING IN DUCKS (Anas platvrhvnchos) by A.M.A. LACOMBE B.Sc, Universite Louis Pasteur FRANCE, 1982. M.Sc, Universite de Bretagne Occidentale FRANCE, 1985. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Zoology. We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1990 © Agnes M.A. LACOMBE, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available 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 department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 20OLQ<^-\ The University of British Columbia Vancouver, Canada Date 3 0 A P * \ ^ W Q DE-6 (2/88) ABSTRACT. Plasma catecholamines have been measured i n chronically adrenalectomised (ADX) ducks, in chronically adrenal denervat-ed ducks (DNX), i n t h e i r respective sham-operated controls (SH-adx, SH-dnx) as well as i n intact ducks a f t e r 3 minutes forced submergence. The results showed that 100% of the plasma Epinephrine (EP) and 40 to 80% of plasma Norepinephrine (NE) released during the dive came from the adrenal glands. 20 to 60% of plasma NE came from endings of the autonomic vascular sympathetic nerves which are strongly stimulated during div-ing. Adrenal catecholamines were released by nerve activation only; non neural mechanisms did not play any role i n t h e i r release. Maximum dive times (MDT) in chronically adrenalectomised ducks (ADX: 5 min. 19 + 20 sec.) and in chro n i c a l l y adrenal denervated ducks (DNX: 7 min. 10 + 13 sec.) were si g n i f i c a n t -ly lower than in sham-operated controls (respectively SH-adx: 9 min. 58 + 45 s e c , SH-dnx: 12 min. 10 + 28 s e c ) . Venous i n f u s i o n of catecholamines i n ADX and DNX during the dive increased MDT: MDT of DNX ducks perfused with catecholamines (9 min. 46 + 20 sec.) were s i g n i f i c a n t l y higher than i n DNX perfused with saline (7 min. 21 +_ 17 s e c ) , but did not reach the MDT observed in the SH-dnx: other adrenal products must be involved. Diving heart rates of ADX and DNX (at 4 min. dive respectively: 62 + 16 and 3 1 + 2 beats/min.) were sig n i f i c a n t -ly higher than i n t h e i r sham-operated controls (23 + 3 and 17 +_ 2 beats/min.) . Blood pressure during the dive was s i g n i f i -i i j c a n t l y lower i n ADX and DNX (at 4 min. d i v e r e s p e c t i v e l y : 93 + 8 and 9 8 + 4 mmHg) compared w i t h t h e i r sham-operated c o n t r o l s (131 + 12 and 118 + 6 mmHg). I n f u s i o n o f catecholamines i n DNX r a i s e d b l o o d p r e s s u r e towards SH-dnx v a l u e s , but t h e r e was no change i n h e a r t r a t e . PaC>2, CaC>2, pHa and l a c t a t e l e v e l s i n DNX ( r e s p e c t i v e l y : 4 2 + 2 mmHg, 4.5 + 0.8 ml 02 /100ml b l o o d , 7.233 + 0.016, 3.1 + 0.3 mM) were s i g n i f i c a n t l y lower than i n SH-dnx a f t e r 5 minutes submergence (53 + 1 mmHg, 6.8 + 0.4 ml 02 /100 ml bl o o d , 7.301 + 0.007, 4.8 + 0.4 mM). There was a l s o a s i g n i f i c a n t i n c r e a s e o f plasma N a + (+ 5.4 + 1.7 mEq/L) i n SH-dnx a f t e r 5 minutes submergence, but t h i s was not the case i n DNX where i t was K + ( + 1 . 1 + 0 . 4 mEq/L) w h i c h i n c r e a s e d . T h i s suggested t h a t a d r e n a l c a t e c h o l a m i n e s i n c r e a s e t o l e r a n c e t o underwater submersion by enhancing p e r i p h e r a l v a s o c o n s t r i c -t i o n , t h u s p r e s e r v i n g t h e C>2 s t o r e s f o r t h e h e a r t and b r a i n . Moreover, t h e y may a f f e c t t h e a c i d - b a s e e q u i l i b r i u m d u r i n g d i v i n g by i n c r e a s i n g t h e a c t i v i t y o f t h e Na +/K + pump and may a l s o have a d i r e c t e f f e c t on the r a t e o f g l y c o g e n o l y s i s . P r e v e n t i n g the a c t i o n s of catecholamines on the h e a r t by i n j e c t i n g b e t a - b l o c k e r d u r i n g f o r c e d submersion d i d not de-c r e a s e MDT; however t h e c a r d i o v a s c u l a r response was markedly a f f e c t e d . D uring beta-blockade, d i v i n g h e a r t r a t e rose s t e a d i -l y f r o m 2 4 + 6 b e a t s / m i n u t e a f t e r 2 m i n u t e s t o 52 + 8 beats/minute a f t e r 6 minutes d i v i n g . I n c o n t r a s t , h e a r t r a t e s remained c l o s e t o the l e v e l s reached a t 2 minutes ( 1 7 + 3 and 19 ± 4 beats/minute) throughout the c o n t r o l d i v e s . Perfusion pressure and blood flow have been recorded simultaneously in both hind limbs of ducks. One leg was per-fused with d i f f e r e n t blood mixtures devoid of catecholamines (Test leg) and compared with the other, perfused with the ducks'own blood (autoperfused l e g ) . This showed that hyper-capnia has a depressant effect on the neural component of the p e r i p h e r a l v a s o c o n s t r i c t i o n . P erfusion of t e s t legs with hypoxic-hypercapnic blood to which catecholamines were added, showed that c i r c u l a t i n g catecholamines are needed to increase peripheral vasoconstriction during diving. In summary, during forced submergence c i r c u l a t i n g ca-techolamines, released mainly by the adrenal glands, compen-sate for the depressant action of hypercapnia on the neural component of peripheral vasoconstriction. Maintenance of t h i s peripheral vasoconstriction during forced diving ensures that O2 stores are not wasted on p e r i p h e r a l t i s s u e s , and t h i s explains how MDT i s prolonged. iv TABLE OF CONTENTS ABSTRACT p . i i LIST OP TABLES p.ix LIST OF FIGURES p.x ACKNOWLEDGEMENTS p . x i i i GENERAL INTRODUCTION p . l 1- Oxygen stores and blood properties in diving birds and mammals p.7 2- Diving response: apnoea and cardiovascular adjustments p. 15 3- I n i t i a t i o n and maintenance of the diving response p. 18 4- Metabolism p.22 5- Why study forced-dives in Pekin ducks? p.31 6- Circulating catecholamines and maximum dive time (MDT) p. 35 CHAPTER 1: The source of ci r c u l a t i n g catecholamines i n forced dived Pekin ducks p.41 INTRODUCTION p. 42 MATERIAL AND METHODS p.46 1- Animals p.46 2- Major surgical procedures p.46 3- Diving protocol p.51 4- Measurement of physiological variables . p.52 v 5- A n a l y s i s of data p.54 RESULTS p.55 DISCUSSION p. 65 CHAPTER 2: Influence of adrenal catecholamines on maximum dive time p.69 INTRODUCTION p.70 MATERIAL AND METHODS p. 72 1- Animals p.72 2- O p e r a t i v e and post o p e r a t i v e procedures p.72 3- Minor surgery and e x p e r i m e n t a l procedure p.72 4- Measurement and a n a l y s i s of p h y s i o l o g i c a l v a r i a b l e s p.76 5- A n a l y s i s o f data p.77 RESULTS p.79 1- E f f e c t s o f adrenalectomy and a d r e n a l d e n e r v a t i o n on maximum d i v e time p.7 9 2- E f f e c t s o f adrenalectomy and a d r e n a l d e n e r v a t i o n on h e a r t r a t e and b l o o d p r e s s u r e p. 86 3- E f f e c t s o f adrenalectomy and a d r e n a l d e n e r v a t i o n on b l o o d gas and pH p. 93 4- E f f e c t s o f adrenalectomy and a d r e n a l d e n e r v a t i o n on s t r o n g i o n s and b l o o d g l u c o s e p.101 DISCUSSION p. 109 v i CHAPTER 3: Influence of beta-adrenoceptors blockade on maximum dive time p.118 INTRODUCTION p.119 MATERIAL AND METHODS p. 121 1- Animals p.121 2- Minor surgery and experimental procedure p. 121 3- Analysis of data p. 122 RESULTS p.123 1- Influence of propranolol on maximum dive time p. 123 2- Effects of propranolol on heart rate and blood pressure p. 123 DISCUSSION . p. 129 CHAPTER 4: Respective roles of sympathetic nerves and sympathetic humoral agents on the vascular resistance of the hind limb of pekin ducks during diving .. p.134 INTRODUCTION p. 135 MATERIAL AND METHODS p.138 1- Animals p.138 2- Surgery and experimental procedure p.138 3- Measurements and analysis of the physiological variables p. 143 4- Analysis of data p.148 v i i RESULTS p. 150 1- Heart r a t e and mean a r t e r i a l b l o o d p r e s s u r e p. 150 2- E f f e c t s o f hyp o x i c and / or hy p e r c a p n i c b l o o d d e v o i d of catecholamines on the v a s c u l a r r e s i s t a n c e of h i n d limbs d u r i n g d i v i n g . p.150 3- E f f e c t s o f c i r c u l a t i n g catecholamines on the v a s c u l a r conductance (1 / r e s i s t a n c e ) o f h i n d l i m b d u r i n g d i v i n g p. 169 DISCUSSION p. 172 GENERAL DISCUSSION p.178 BIBLIOGRAPHY p. 192 v i i i LIST OP TABLES. TABLE 1: Survey o f t h e l o n g e s t underwater submergences p e r -formed by d i v i n g and n o n - d i v i n g b i r d s and mammals . p.4 TABLE 2: S u r v e y o f t h e b l o o d oxygen s t o r e s , m u s c l e oxygen s t o r e s and r e s p i r a t o r y p r o p e r t i e s o f t h e b l o o d , i n d i v i n g and n o n - d i v i n g mammals p.10-11-12 TABLE 3: L e v e l s o f p l a s m a c a t e c h o l a m i n e s d u r i n g d i v e s i n a d r e n a l denervated ducks i n f u s e d w i t h s a l i n e o r w i t h c a t e c h o -lamines and i n t h e i r sham-operated c o n t r o l s p.85 TABLE 4: P r e - d i v e and d i v e v a l u e s o f Pa02, Pac02/ pHa, and plasma l e v e l s o f l a c t a t e and gluco s e i n ad r e n a l e c t o m i s e d ducks and t h e i r sham-operated c o n t r o l s p. 98 TABLE 5: P r e - d i v e and d i v e v a l u e s o f a r t e r i a l plasma l e v e l s o f g l u c o s e , l a c t a t e " , N a + , K +, CI and S t r o n g Ions D i f f e r e n c e (SID) i n a d r e n a l d e n e r v a t e d ducks and t h e i r s h a m - o p e r a t e d c o n t r o l s p. 105 i x LIST OP FIGURES. FIGURE 3.: C a r d i o v a s c u l a r r e s p o n s e s t o f o r c e d submersion o f a P e k i n duck p. 17 FIGURE 2: Heart r a t e d u r i n g a f o r c e d d i v e and v o l u n t a r y dabble i n P e k i n duck p.33 FIGURE 3: I n c r e a s e i n n o r e p i n e p h r i n e and e p i n e p h r i n e d u r i n g f o r c e d d i v e s i n ducks p.37 FIGURE 4: Schematic view o f catecholamines r e l e a s e . p.44 FIGURE 5: V e n t r a l view o f the r i g h t a d r e n a l g l a n d o f the P e k i n duck p.49 FIGURE 6: Plasma l e v e l s o f n o r e p i n e p h r i n e and e p i n e p h r i n e i n i n t a c t , sham-operated and a d r e n a l e c t o m i s e d ducks b e f o r e and a f t e r 3 minutes of f o r c e d d i v i n g p.57 FIGURE 7_: Plasma l e v e l s o f n o r e p i n e p h r i n e and e p i n e p h r i n e i n i n t a c t , sham-operated, and a d r e n a l denervated ducks b e f o r e and a f t e r 3 minutes o f f o r c e d d i v i n g p.59 FIGURE 8: Heart r a t e d u r i n g a 3 minute f o r c e d d i v e s i n i n t a c t , s h am-adrenalectomised, a d r e n a l e c t o m i s e d , sham-adrenal dener-v a t e d , a d r e n a l denervated P e k i n ducks p.61 FIGURE 9: Mean a r t e r i a l b l o o d p r e s s u r e b e f o r e and a f t e r 2 minutes f o r c e d d i v i n g i n i n t a c t , sham-adrenalectomised, adre-n a l e c t o m i s e d , s h a m - a d r e n a l d e n e r v a t e d , a d r e n a l d e n e r v a t e d P e k i n ducks p. 64 FIGURE 10: Sample c h a r t r e c o r d i n g from an experiment t o d e t e r -m i n e maximum e n d u r a n c e t o f o r c e d s u b m e r s i o n i n P e k i n ducks p.75 FIGURE 11: Maximum d i v e t i m e i n i n t a c t , s h a m - o p e r a t e d and adr e n a l e c t o m i s e d ducks w i t h and wit h o u t i n f u s i o n o f c a t e c h o l a -mines p.81 FIGURE 12: Maximum d i v e time i n the i n t a c t , sham-operated and a d r e n a l denervated ducks w i t h and wit h o u t i n f u s i o n o f catecho-lamines p. 83 FIGURE 13: Heart r a t e and mean a r t e r i a l b l o o d p r e s s u r e d u r i n g d i v i n g i n a d r e n a l e c t o m i s e d d u c k s and t h e i r s h a m - o p e r a t e d c o n t r o l s p. 88 x FIGURE 14: Heart rate and mean a r t e r i a l blood pressure during div i n g i n adrenal denervated ducks and t h e i r sham-operated controls p. 90 FIGURE 15: Heart rate and mean a r t e r i a l blood pressure during d i v i n g i n adrenal denervated ducks infused with s a l i n e or catecholamines and in sham-operated ducks p. 92 FIGURE 16: PaC^ and CaO? in adrenal denervated ducks and their sham-operated controls during diving p. 96 FIGURE 17: A r t e r i a l content of 02 as a function of Pa02 i n denervated ducks and in sham denervated ducks p.100 FIGURE 18: PaC0 2 and pHa i n adrenal denervated ducks and i n their sham-operated controls p. 103 FIGURE 19: Absolute changes (dive values - predive values) of [H +]a, PaC02 and a r t e r i a l l e v e l s of ions a f t e r 5 minutes submergence in adrenal denervated ducks p.108 FIGURE 20: Maximum dive time i n ducks injected with proprano-l o l and saline p. 125 FIGURE 21: Heart rate and mean a r t e r i a l blood pressure during diving in ducks injected with saline or with propranolol p.127 FIGURE 22: Diagram of the experimental set up used for the hind limb perfusion experiments p. 142 FIGURE 23: Pressure regulator for the blood chamber p.147 FIGURE 24: Heart rate and central mean a r t e r i a l blood pressure during diving in ducks whose legs were perfused with hypoxic-hypercapnic blood, with hyperoxic-hypocapnic blood, with hypoxic-hypocapnic, with hyperoxic-hypercapnic blood and with hypoxic-hypercapnic blood with catecholamines p. 152 FIGURE 25: Blood gas tensions and pH of blood perfusing autop-erfused and t e s t legs at the beginning and at the end of dives p. 154 FIGURE 26: Perfusion pressure and blood flow in hind limbs of ducks whose legs during diving were perfused with t h e i r own blood, and with hypoxic-hypercapnic blood p.156 FIGURE 27: Perfusion pressure and blood flow in hind limbs of ducks whose legs during diving were perfused with t h e i r own blood and with hyperoxic-hypocapnic blood p. 158 xi FIGURE 28: P e r f u s i o n p r e s s u r e and b l o o d f l o w i n h i n d l i m b s o f ducks whose l e g s d u r i n g d i v i n g were p e r f u s e d w i t h t h e i r own b l o o d and w i t h hypoxic-hypocapnic b l o o d p. 161 FIGURE 29: P e r f u s i o n p r e s s u r e and b l o o d f l o w i n h i n d l i m b s o f ducks whose l e g s d u r i n g d i v i n g were p e r f u s e d w i t h t h e i r own b l o o d and w i t h h y p e r o x i c - h y p e r c a p n i c b l o o d p.163 FIGURE 30: Examples o f t r a c e s r e c o r d e d d u r i n g 2 d i v e s . In t h e f i r s t d i v e o n l y one l e g was p e r f u s e d w i t h the duck's own b l o o d and t h e o t h e r was p e r f u s e d by h y p o x i c h y p e r c a p n i c b l o o d w i t h -out catecholamines. In the second d i v e both l e g s were autoper-f u s e d p.165 FIGURE 3 1 : R e l a t i o n s h i p between t h e a b s o l u t e i n c r e a s e i n v a s c u l a r r e s i s t a n c e d u r i n g d i v i n g i n t h e t e s t l e g s and i n aut o p e r f u s e d l e g s p. 168 FIGURE 32: V a s c u l a r conductance i n h i n d l i m b s p e r f u s e d w i t h h y p o x i c and hyp e r c a p n i c b l o o d w i t h o u t , and w i t h catecholamines d u r i n g d i v i n g p. 171 FIGURE 33: The r e l a t i o n s h i p o f h e a r t r a t e t o b i l a t e r a l s t i m u -l a t i o n of t h e d i s t a l c u t ends o f t h e vagus and c a r d i a c sympa-t h e t i c nerves p. 185 FIGURE 34: Heart r a t e , mean a r t e r i a l b l o o d p r e s s u r e , p e r f u s i o n p r e s s u r e and b l o o d f l o w i n t h e a u t o p e r f u s e d l e g , i n a duck d u r i n g f o r c e d d i v e and re c o v e r y p.189 x i i A C K N O W L E D G E M E N T S . I thank my advisor, Professor David R. Jones, who provid-ed me with the opportunity to pursue a Ph.D. i n his research laboratory. I am most grateful for his advice and kindness. Dr. Jones' indomitable s p i r i t was as much of an inspiration to me as was his invaluable s c i e n t i f i c guidance. I also thank those i n the Department of Zoology who helped me and allowed me to use t h e i r equipment: M. Hughes, D. Randall, W. Milsom, P. Hochachka, A. Perks and D. Mense. The pressure-flow con-t r o l l e r in Chapter IV was designed with the help of R. Deane and I am grateful for his contribution. I especially thank my colleagues who helped me with my work, gave me advice and put up with my English. Among them are M. Hedrick, C. Kasserra, M. Lutcavage, P. Bushnell, R. Stephenson and l a s t , but certainly not least, G. Gabbott, who made t h i s thesis bearable for the reader. I thank my former colleague, F. Smith, who introduced me to the mysteries of electronics. The people working in the workshop, the administration, the Zoology stores and at South Campus were always very kind and helpful and I thank them very much. For t h e i r unrelenting support and love, I thank my par-ents and my sister, who in spite of their worry, never demand-ed of me to follow another road. My thanks too, to Frederic, for being with them during the hard times. Lastly, I thank my husband Geoff for a l l his love, kindness and understanding during the completion of this degree. x i i i GENERAL INTRODUCTION. 1 Paul Bert (1870) was the f i r s t to attempt an explanation for the remarkable a b i l i t y of diving homeothermic vertebrates to survive periods of prolonged submersion. He suggested that ducks dive longer than chickens because they have a higher ra t i o of blood volume to body weight and thus a larger oxygen store. Even though t h i s explanation of the phenomenon has proven to be i n s u f f i c i e n t , Bert pioneered the f i e l d of diving physiology by d e f i n i n g the problem c l e a r l y : a i r - b r e a t h i n g diving animals can tole r a t e underwater submersion far better than t h e i r non-diving counterparts (Table 1), and yet, a l -though some d i v i n g animals may have evolved morphological adaptations to aquatic l i f e , they do not possess any special respiratory organs which would allow them to extract oxygen from water. From that time on, the search has continued for the solution which permits such remarkable tolerance to under-water submergence. In 1899 Bert's explanation was challenged by Charles Richet who thought that the extra oxygen store in ducks was not s u f f i c i e n t to meet the requirements of a long dive. He found that oxygen consumption decreased markedly 2 TABLE 1: Survey of the longest underwater submersions per-formed by diving and non-diving birds and mammals. 3 BIIPS -MM Diras-Pigeon (Coluibia) Ben (Gallas doaesticas) -Divas-Dccestie dack (Ants pUthyftunchos) Red head duck (Aythya aiericana) Canada goose (Branta canadensis) Conwrant (Phalacrocorai pelaticas) Puffin (Morton fratercala) Pengains (Eadyptes chrysolophas) (Pygocelis papaa) (Pygocelis adeliae) (Aptenodytes forsteri) Gaillewts (ilria grylle) (Uria troile) MMALi -MM DITEXS-Khite rat (Ratlas rattus) labbit (Oryctolagas cannicalas) Cat (Felis catus) Dog (Canis faiiliaris) Van (How sapiens) -DIYBS-Huskrat (Ondatra libethica) Beaver (Castor canadensis) Sea otter (Enhydra htris) Florida ianatee (Trichechas latirostris) Gray seal (Balicboeras grypas) Northern far seal (Callorhinas arsinas) Northern elephant seals (Hirounga angastirostris) leddell seal (Leotonychotes leddelUO flooded seal (Cystophora cristata) Coawn porpoise (Phocaena phocaena) Bottlenosed dolphin (Tarsiop trancatas) Spena ihale (Physeter catodon) Blae whale (Balaenoptera lascalas) Pin thale (Balaenoptera plapalis) Finback ihale (Balaenoptera physalas) Gray ihale (Eschrichtias robastas) Bottlenosed ihale (Hyperoodon rostratus) LONGEST DIVES (unite) 1 (Andersen 1966) 3 (Andersen 1966) 21.8 (Lacoabe and Jones 1987) 25 (lichet 1839) g (Parilla and Jones 1984) 4 (Jones 1984) 4 (Jones 1984) 4 (Andersen 1966) 5 (Andersen 1966) I (Andersen 1966) 6 (Kooyian 1975) 18 (looyun e l aL 1971) I! (Andersen 1966) 12 (Andersen 1966) 2 (Andersen 1966) 3 (Andersen 1966) 3 (Andersen 1966) 4 (Andersen 1966) 3 (Cross 1965) 12 (Andersen 1966) 9 (Druawnd 1980) 15 (Andersen 1966) 4.42 (Loughlin 1980) IS (Andersen 1966) 18 (Andersen 1966) 5.4 (looywn elai . 1976) 47.7 (Leboeuf et al, 1988) 72 (looyun etal, 1980) 18 (Andersen 1966) 12 (Scholander 1940) 4.75 (lidpiy ej.al. 1969) 60 (Lockyer 1977) 75 (Andersen 1966) 50 (Andersen 1966) 30 (Andersen 1966) 10 (lay e t a ! 1978) 30 (Andersen 1966) 16.53 (Evans 1974) 120 (Andersen 1966) 4 d u r i n g d i v i n g which, he suggested, may be caused by p h y s i o l o g -i c a l a d j u s t m e n t s t a k i n g p l a c e d u r i n g d i v i n g ( L a n g l o i s and R i c h e t 1898, a and b; R i c h e t 1899). Bohr (1897) suggested t h a t energy c o u l d be d e r i v e d from an i n c r e a s e i n a n a e r o b i c metabo-l i s m , and t h a t t h i s would account f o r t h e d i m i n i s h e d oxygen consumption. L i t t l e more p r o g r e s s was made f o r t h e nex t few decades. Then i n t h e 1930s a b r e a k t h r o u g h came from t h e s t u d -i e s o f L a u r e n c e I r v i n g . The e s s e n c e o f h i s work remains t o t h i s day as the g r e a t e s t c o n t r i b u t i o n t o d i v i n g p h y s i o l o g y . I r v i n g (1934) p o s t u l a t e d t h a t the b r a i n , and p o s s i b l y the h e a r t i n homeothermic v e r t e b r a t e s , a re not a b l e t o cope w i t h oxygen d e p r i v a t i o n . He c o n t r a s t e d t h i s w i t h t h e a b i l i t y o f muscle t i s s u e t o s u s t a i n i s c h e m i a f o r extended p e r i o d s o f time w i t h o u t s u f f e r i n g from i r r e v e r s i b l e damage. N e i t h e r p h y s i c a l nor chemical p r o c e s s e s , he f e l t , c o u l d ever c o m p l e t e l y e x p l a i n t h e a b i l i t y o f t h e d i v e r t o cope w i t h p r o l o n g e d submergence and he a d v o c a t e d t h e s t u d y o f c a r d i o v a s c u l a r r e f l e x e s w h i c h a d j u s t b l o o d f l o w d u r i n g d i v i n g . He p r o p o s e d t h a t d u r i n g d i v i n g , oxygen s t o r e s would be c o n s e r v e d e x c l u s i v e l y f o r t h e b r a i n and h e a r t . T h i s c o u l d be a c h i e v e d by r e d i r e c t i n g b l o o d f l o w away from t h e p e r i p h e r a l organs and t i s s u e s t o w a r d t h e h e a r t and b r a i n . He support e d t h i s h y p o t h e s i s by demonstrat-i n g t h a t d u r i n g r e s p i r a t o r y a r r e s t an i n c r e a s e i n b l o o d f l o w t o t h e b r a i n does i n d e e d o c c u r and t h a t f l o w t o t h e muscles d e c r e a s e s ( I r v i n g 1938) . S c h o l a n d e r (1940) c o r r o b o r a t e d I r -v i n g ' s t h e o r y by showing t h a t a l t h o u g h plasma l a c t a t e doubled, or t r i p l e d , d u r i n g d i v i n g , i t i n c r e a s e d even more (10 t i m e s t h e p r e - d i v e v a l u e s ) a f t e r e m e r s i o n . These r e s u l t s s u g g e s t e d 5 t h a t b l o o d f l o w does not p e r f u s e muscles d u r i n g d i v i n g . Not o n l y do t h e muscles undergo a n a e r o b i c metabolism but a l s o t h e end p r o d u c t o f t h i s m e t a b o l i s m , l a c t a t e , cannot be removed f r o m t h e m u s c l e s d u r i n g d i v i n g . O n l y upon e m e r s i o n , when b l o o d f l o w i s r e e s t a b l i s h e d , i s l a c t a t e f l u s h e d out o f t h e s e p e r i p h e r a l t i s s u e s . I t then appears i n t h e c i r c u l a t i o n , caus-i n g t h e huge p o s t - d i v e peak i n plasma l a c t a t e l e v e l s ( I r v i n g e t a l . 1942; Scholander et a l j . 1942) . In an attempt t o u n d e r s t a n d mechanisms a l l o w i n g such an a d a p t a t i o n t o underwater submersion, r e s e a r c h has f o c u s e d on the f o l l o w i n g f a c t o r s : 1) the oxygen s t o r e s a v a i l a b l e d u r i n g d i v e s , 2) the a b i l i t y of b l o o d t o l o a d and u n l o a d oxygen at the l ungs and t i s s u e s r e s p e c t i v e l y , and t o b u f f e r t h e excess CO2 and l a c t i c a c i d , 3) t h e a b i l i t y t o c o n s e r v e , as e f f i c i e n t l y as p o s s i b l e , oxygen s t o r e s f o r the h e a r t and b r a i n , 4) s p e c i a l m e t a b o l i c a d a p t a t i o n s o f t i s s u e s t o oxygen d e p r i v a t i o n , 5) t h e a b i l i t y t o c o n t r o l t h e r e s p i r a t o r y d r i v e d u r i n g d i v i n g i n order t o m a i n t a i n apnoea. 6 1- OXYGEN STORES AND BLOOD PROPERTIES IN DIVING BIRDS AND MAMMALS: Gas exchange i s not p o s s i b l e d u r i n g d i v i n g and c o n s e -q u e n t l y , a n i m a l s must r e l y on t h e i r o xygen s t o r e s and on an a e r o b i c metabolism t o s u r v i v e d u r i n g submergence. Oxygen i s s t o r e d i n the b l o o d , i n the muscles, and i n the lungs (and a i r sacs i n b i r d s ) . Oxygen s t o r e s and b l o o d p r o p e r t i e s o f d i v i n g mammals and b i r d s have been e x t e n s i v e l y r e v i e w e d by L e n f a n t ( 1 9 6 9 ) ; B u t l e r and Jones ( 1 9 8 2 ) ; S n yder ( 1 9 8 3 ) ; J ones and F u r i l l a ( 1 9 8 7 ) ; S t e p h e n s o n and J o n e s (1989) and Kooyman (1989) . I t i s not easy t o e s t a b l i s h t h e i m p o r t a n c e o f t h e l u n g s (and a i r sacs) as oxygen s t o r e s d u r i n g d i v i n g , because i t i s not known i f d i v e s occur on i n s p i r a t i o n o r e x p i r a t i o n . D i v i n g on i n s p i r a t i o n i n c r e a s e s oxygen s t o r e s , but a l s o i n c r e a s e s buoyancy and f o r deep d i v e r s , r a i s e s t h e r i s k o f n i t r o g e n r e l a t e d a c c i d e n t s (such as n a r c o s i s and bends). Snyder (1983) s u g g e s t e d t h a t t h e lu n g s a r e not a s i t e o f oxygen s t o r a g e i n l o n g d u r a t i o n d i v e r s , s u c h as s e a l s and w h a l e s : t h e s m a l l lungs o f whales and the lungs o f s e a l s are l i k e l y t o c o l l a p s e d u r i n g d i v i n g . Short d u r a t i o n d i v e r s such as rod e n t s , d o l p h i n s and p o r p o i s e s a r e thou g h t t o d i v e on i n s p i r a t i o n and t o use l u n g oxygen d u r i n g d i v i n g (Snyder 1983). Some d i v i n g b i r d s such as t u f t e d ducks and pochards d i v e on e x p i r a t i o n (Woakes and B u t l e r 1975; B u t l e r and Woakes 1976; 1979), w h i l e o t h e r s , such as emperor penguins d i v e on i n s p i r a t i o n (Kooyman e t a l . 1971a). I n P e k i n ducks, t h e oxygen s t o r e s o f t h e l u n g and a i r sacs make up h a l f o f the oxygen a v a i l a b l e d u r i n g f o r c e d - d i v i n g (Hudson and Jones 1986). 7 The s i z e o f t h e b l o o d oxygen s t o r e depends on t h e b l o o d volume per u n i t o f body mass and on the hemoglobin c o n c e n t r a -t i o n . A wide range of v a l u e s f o r b l o o d oxygen s t o r e s have been r e c o r d e d f o r a v a r i e t y o f d i v i n g mammals, from t h e same t o o v e r 3 t i m e s t h e l e v e l s f o u n d i n t e r r e s t r i a l mammals. The l a r g e r oxygen s t o r e s a r e due t o h i g h e r c o n c e n t r a t i o n s o f hemoglobin and l a r g e r b l o o d volumes (Table 2 ) . The h i g h e s t v a l u e s r e c o r d e d are tho s e of the p i n n i p e d s which are known t o be l o n g - d u r a t i o n d i v e r s ( T a b l e 2; B u t l e r and J o n e s 1982; Snyder 1983; Kooyman et a l . 1989) . A q u a t i c b i r d s t e n d t o have b l o o d volumes and hemoglobin c o n c e n t r a t i o n s i n t h e upper end of the range f o r a l l b i r d s (Jones and F u r i l l a 1987; Stephenson and Jones 1989; Kooyman 1989). The rec e n t o b s e r v a t i o n by Q v i s t et al- (1986) o f an i n c r e a s e i n hemoglobin c o n c e n t r a t i o n of a s e a l d u r i n g d i v i n g , suggests t h a t t h e s p l e e n may be r e s p o n s i -b l e f o r r e l e a s i n g s t o r e d r e d b l o o d c e l l s d u r i n g d i v e s and thus adding another component t o b l o o d oxygen s t o r e s . S k e l e t a l muscle myoglobin c o n c e n t r a t i o n s i n d i v i n g mam-mals and i n some d i v i n g b i r d s , such as a d u l t penguins, can be as much as one o r d e r o f magnitude h i g h e r than t e r r e s t r i a l a n i mals (Table 2; C a s t e l l i n i and Somero 1981; Kooyman 1989). In ducks and geese, t h e c o n c e n t r a t i o n s o f m y o g l o b i n are i n t h e upper range o f v a l u e s u s u a l l y o b s e r v e d f o r b i r d s ( K e i j e r and B u t l e r 1982; Snyder et a l . 1984). 8 TABLE 2: Survey o f t h e b l o o d oxygen s t o r e s ( H e m a t o c r i t i n %; hemoglobin i n g/lOOml; 0 2 c a p a c i t y i n ml/lOOml) , of t h e b l o o d volume (% of body mass), of the muscle oxygen s t o r e s (g/lOOg) and of the r e s p i r a t o r y p r o p e r t i e s of whole b l o o d (P50 i - n ntmHg; Bohr e f f e c t ; H i l l c o e f f i c i e n t ; Haldane e f f e c t i n mM/L; and b u f f e r c a p a c i t y i n mM HCC>3~/L.pH u n i t ) o f d i v i n g (Table 2-A) and n o n - d i v i n g (Table 2-B) mammals. The r e s p i r a t o r y p r o p e r t i e s o f t h e b l o o d were measured at a pH of 7.4 and at temperatures o f 37-38°C. A-^ , Andersen (1966); Blt B i n k l e y 1980; B 2, Bryden and Lim (1969); B 3 , B l e s s i n g (1972); B 4, B l e s s i n g and Hartschen-Niemeyer (1969); B 5 , B i o -l o g i c a l Handbook 1971); C-j_, Clausen and E r s l a n d (1968); C 2, C a s t e l l i n i and Somero (1981); C 3, Clausen and E r s l a n d (1969); nlr Horvath e t a l . (1968); K 1, Kooyman (1968); L 1 # L e n f a n t e t a l . (1970); L 2 , L e n f a n t (1969); L3, Len-f a n t e t a l . (1969); L 4 , Lane et a l . (1972); L 5 , Lenfant e t a l . (1968); L 6 , Lechner (1976); P l 7 P a r e r and M e t c a l f e (1967); R-^ , Ridgway and Johnson (1966) ; Slf S c h o l a n d e r (1940); S 2, Simpson e t a l . (1970); S 3, S l e e t e t a h (1981) . 9 TABLE 2-A: D I V O S H Q I A - H E M O - 02 TOCRIT G L O B I N CAPACITY P L A T Y P U S (Ornithorhynchas a o i t i n u s ) 21 Um V O L E ( A r v i c o l a t e r r e s t r i s ) 19 K I S H A T (Ondatra l i b e t h i c a ) BEAVER (C a s t o r f i b e r ) 18 1! 16 S E A O T T E R (Eohydra l u t r i s ) (8 17 22 M A N A T E E ( T r i c h e c h u s u n a U s l a t i r o s t r i ) 47 15 17 DUQONC (Dugong dugong) 16 WALRUS (Odobenus rosaarus) 4! 16 21 NORTHERN FUR S E A L ( C a l l o r h i n i s i r s i n i s ) 49 17 20 C A L I F O R N I A S E A L I O N ( Z a l o p h i s c a l i f o r n i a n u s ) 50 15 20;23 NOtTHEIN S E A L I O N ( E m e t o p i a s jubata) 40 16 20 H A I K U S E A L (Pboca v i t i l i o a ) S8;S1 21; 20 29;26 C O W O N S E A L (Pboca f i s c i a t i ) 67 25 14 R I B B O N S E A L ( H i s t r i o p h o c a ( a s c i a t a ) G R A Y S E A L ( H a l i c h o e r i s grypus) 24 W E D D E L L S E A L (Leptonycbotes l e d d e l l i i ) S8;6l !4 16;12;11 SOUTHERN E L E P H A N T S E A L (Mirovnga l e o n i n a ) 59 11 40;!1 NORTHERN E L E P H A N T S E A L (Mironnga a n g u s t i r o s t r i s ) 21 27;1I HOODED S E A L (Cystophora c r i s t a t a ) (3 26 27;16 BLOOD UYO- P50 BOHR N HALDANE BUFFER REFERENCES VOLUME GLOBIN ( a H g ) EFFECT EFFECT CAPACITY DHOLE BLOOD 27 0.56 P, \ h h L , ; U ; C , vh Wh 18 0.54 14 1.1 1.2 21 0.56 27 9 1.1;2.9 11 0.46 18 1.5 17 11 1 I I 3.5; 1.9 10 0.48 17 1.2 10 0.66 2.5 1 12 2.4 12 0.8 2.6 2.5 15 11 5.5;4.4 11 0.51 2.6 4.2 40 11 8.1 8.1 (.1 11 2.9 15 4.5 29 0.61 20 16 22 5.1 11 0.61 2.6 4.2 (2 4.1 24;I0 0.66 2.4 16 DIVERS HBIA- HEUO-TOCRIT GLOBlti 02 BLOOD UYO- PSD B01IR N BALDANE BUFFER REFERENCES CAPACITY VOLUME GLOBIN (mBt) EFFECT EFFECT CAPACITY-WHOLE BLOOD AMAZON DOLPHIN (Inia geoffrensis) PACIFIC DOLPHIN (Laeenorhynchus obliqiidens) 49; KILLER WHALE (Orcinus orca) 44 PILOT WHALE (Globicepbala scawmi) 40 DALL'S PORPOISE (Phocoenoides dalli) S7 HARBOR PORPOISE (Pbocoena pbocoena) SPOTTED DOLPHIN (Stenella atteouata) BOTTLENOSED DOLPHIN (Tursiops truncatus) 45;41 (Tursiops truncatus) S2;43 (Tursiops truncatus gilli) RISSO DOLPHIN (Craipus griseus) SPERM WHALE (Physeter catodonj 42;52 PIGMY SPERM WHALE (Kogia breviceps) GRAY WHALE (Eschrichtius gibbons) FIN WHALE (Balaenoptera physalus) BLUE WHALE (Balaenoptera lusculus) BOTTLENOSED WHALE (Hyperoodon rostratus) 14 19 25 17;19 26;25 25 16;IS 23;22 31 15 20;22 31 20 27 14 19 24; 21 4.1 25 2.5 20; 14 33;19;22 7.1;7.4 3.3 Z5;27 16 18-20;2l 18 21; 24 26 26 34 31 15; 18 29;2(;19 211 5.7 27 16 32 n 17 30 20-25 3.7 9;10 2l;14 6.3 0.59 2.6 30 h 0.72 2.4 2.9 43 h 0.74 2.6 3.4 43 0.62 2.6 2.8 36 h 0.6 0.66 2.1 2.5 2.5 43 V<Hi h LjiRpHjiBj 0.71 2.5 3.1 41 L2 4 0.97 2.6 4.4 49 0.48 0.46 2.6 2.7 h h s, TABLE 2-B: NON-DIVERS SRREI ( C r o c i d i r i r a s s i l a ) HOUSE (Bus m s c u l u s ) HAMSTER ( M e s o e r i c e t i s a i r a t i s ) HEDGEHOG ( E r i n a c e u s earopaeus) RATTUS ( R a t U s r a t t a s ) GUINEA FIG ( C a m p r o c e l l u s ) RABBIT ( O r y c t o l a g i s c i n n i c i l u ) CAT ( P e l i s c a t o s ) DOG ( C a n i s f a i i l i a r i s ) TASHAN1AM DEVIL ( S a r c o p b i l u s b i r r i s i i ) IANGAIOO (Macropis sp.) PIG (Sns s c r o f a ) ANTEATEI (T a c h y g l o s s a s s e t o s u s ) SHEEP ( O t i s a r i e s ) GOAT (Capra h i r c i s ) VICUNA ( L l a u v i c u n a ) LLAMA ( L l u a g l a n a ) YAI (Poephagus grunniens) CATTLE (Bos sp.) HOtSE (Kquus cabal1 us) AXIS DEAR ( C e r v i s nippon) CAMEL ( C a i e l u s b a c t r i a n u s ) ELEPHANT ( L o i o d o n t a a f r i c a n a ) SQUIREL MONKEY ( S a i i i r i s c i u r e a ) MACAQUE (Macaca n l a t t a ) BABOON (Pap i o leucophaeus) ORANG OUTANG (Pongo p y g u e i s ) CHIMPANZEE (Pan t r o g l o d y t e s ) GORILLA ( G o r i l l a g o r i l l a ) MAN ( B o n s a p i e n s ) HFJ1A- HEUO- 0! TOCIIT GL061N CAPACin 22 14.8 19 49 16 19 19 46 14.8 21 4! 14.4 14 4! 11.9 16 4! 11.9 17 46 14.8 14 27 41 24 19 11.7 22 22 1! 10.9 15 11 10.S 11 41 25 25 21 40 11.5 18 11 11 25 22 28 21 16 18 42 12.6 17 15 17 42 12.1 17 16 47 16 20 BLOOD MYO- P50 BOHR N HALDANE BUFFER REFERENCES VOLUME GLOBIN (aflg) EFFECT EFFECT CAPACITY WHOLE BLOOD 17 0.61 Be 14 0.61 Bt 28 0.41 Be 16 0.49 B s 6.4 0.1 15 BejLj 7.5 27 0.47 Be 5.6 0.04 11 0.45 fcjC, 5.5 16 0.54 8j 8.6 0.6 29 0.50 28 BejCj 41 0.47 BJ 8.8 28 0.54 Be 6.5 0.17 11 0.42 BjjCj 26 0.49 Be 6.6 12-17 0.12-0.48 2.7;2.8 Be. 7.1 10 0.5 2.8 Be Be 21 0.42 2.4 47 Be 26 0.5 B s 5.7 0.45 11 0.49 24 B S ; C , 7.6 Ij 28 0.56 29 Be 8.1 24 0.49 27 Be. 22 0.4 B5 16 0.54 Be 5.4 12 2.6 Be 17 0.55 BS 24 0.55 Bs 26 0 59 B s 25 0.47 B s 8.4 27 0.48 29 Be B l o o d oxygen l o a d i n g and u n l o a d i n g p r o p e r t i e s , a t t h e lu n g s and t i s s u e s r e s p e c t i v e l y , a re i m p o r t a n t f o r underwater s u r v i v a l : B l o o d w i t h a h i g h a f f i n i t y f o r oxygen would f a v o u r f u l l u t i l i z a t i o n o f oxygen i n t h e l u n g , and b l o o d w i t h a low a f f i n i t y would a l l o w a more complete oxygen u n l o a d i n g a t t h e t i s s u e s . However, t h e r e i s no c l e a r r e l a t i o n s h i p between P^Q and d i v i n g a b i l i t y (Table 2 ) . Snyder (1983) has suggested t h a t t h e b l o o d oxygen a f f i n i t y o f d i v i n g mammals may be c o r r e l a t e d w i t h t h e i r l u n g oxygen s t o r e s . F o r i n s t a n c e , l a r g e c e t a c e a n s and p i n n i p e d s whose lungs c o l l a p s e d u r i n g d i v i n g have a lower b l o o d oxygen a f f i n i t y t h a n t e r r e s t r i a l mammals o f e q u i v a l e n t weight, and t h i s would t e n d t o promote the d i f f u s i o n o f oxygen i n t o the t i s s u e s . However, r o d e n t s and s m a l l cetaceans have a h i g h e r oxygen a f f i n i t y t h a n t h e i r t e r r e s t r i a l e q u i v a l e n t s , which may promote oxygen l o a d i n g a t the l u n g s . Another f a c t o r which promotes the u n l o a d i n g of b l o o d oxygen i n the t i s s u e s of d i v i n g mammals such as p i n n i p e d s and c e t a c e a n s i s t h e i r en-hanced Bohr e f f e c t ( T a b l e 2 ) . H i l l c o e f f i c i e n t s o f marine mammals a r e not d i f f e r e n t from t h o s e o f t e r r e s t r i a l mammals (Table 2 ) . A q u a t i c mammals cannot e x p e l t h e end p r o d u c t s o f m e t a b o l i s m w h i c h a c c u m u l a t e i n t h e body d u r i n g d i v i n g . I n p i n n i p e d s and c e t a c e a n s , t h e h i g h e r b u f f e r i n g c a p a c i t y o f t h e i r b l o o d as w e l l as t h e l a r g e r Haldane e f f e c t a r e a b l e t o reduce pH and CC>2 f l u c t u a t i o n s (Table 2) . Less data are a v a i l -a b l e f o r d i v i n g b i r d s . B l o o d oxygen a f f i n i t y and t h e Bohr e f f e c t i n b i r d s do n o t seem t o be c o r r e l a t e d w i t h d i v i n g a b i l i t y (Jones and F u r i l l a 1987/ Stephenson and Jones 1989). The b u f f e r i n g c a p a c i t y of A d e l i e penguin b l o o d (a good d i v i n g b i r d ) i s h i g h e r than t h a t o f t e r r e s t r i a l b i r d s (Lenfant et a l . 1969)/ and t h e c a p a c i t y o f duck b l o o d i s no d i f f e r e n t from c h i c k e n b l o o d ( N i g h t i n g a l e and Fedde 1972; S c h e i p e r e t a l . 1975) . The s t o r e d oxygen a v a i l a b l e f o r d i v e s by Weddell s e a l s i s 60 ml oxygen K g - 1 (Kooyman 1989) or 1339 mmoles f o r a 500 Kg a n i m a l . The m e t a b o l i c r a t e o f a 500 Kg W e d d e l l s e a l i s 83 mmole.min - 1 (Hochachka 1981). T h e r e f o r e , i f we suppose t h a t d i v e s performed by s e a l s are s t r i c t l y a e r o b i c , a 500 Kg i n d i -v i d u a l c o u l d s t a y u n d e r w a t e r f o r 16 m i n u t e s . Most o f t h e observed d i v e s range between 5 and 25 minutes, and t h u s , t h e y a r e l i k e l y t o have been p e r f o r m e d a e r o b i c a l l y . T h i s i s con-f i r m e d by t h e l a c k o f plasma l a c t a t e d u r i n g and a f t e r t h e s e d i v e s . However, some o f t h e d i v e s r e c o r d e d have r e a c h e d 50 minutes and even 1.2 hours (Kooyman et a l . 1980). These d i v e s c o u l d not have been p e r f o r m e d p u r e l y a e r o b i c a l l y , and i t i s c l e a r t h a t o t h e r mechanisms a r e i n v o l v e d i n t h e s e f e a t s o f submergence. By u s i n g a s i m i l a r c a l c u l a t i o n f o r a e r o b i c metabolism i n P e k i n d u c k s , i t c a n be e s t i m a t e d t h a t t h e s e d u c k s c a n n o t p e r f o r m d i v e s o f more than 2.5 minutes d u r a t i o n (O2 s t o r e s = 57 ml f o r 2.5 Kg ducks, Hudson and Jones 1986; and whole body metabolism = 11.3 x 2 . 5 ° * 7 2 3 = 21.9 m l . m i n - 1 , S c h m i d t - N i e l s e n 1979). I t was, t h e r e f o r e , a s t o n i s h i n g t o d i s c o v e r t h a t t h e t o l e r a n c e o f a P e k i n duck t o f o r c e d submersion i s c o n s i d e r a b l y l o n g e r : a 2.5 P e k i n duck can w i t h s t a n d 22 m i n u tes o f submer-s i o n w i t h o u t s u s t a i n i n g p h y s i o l o g i c a l damage (Pe r s o n a l d a t a ) . These simple c a l c u l a t i o n s c o n f i r m , beyond any doubt, t h a t 14 i n a d d i t i o n t o an i n c r e a s e o f oxygen s t o r e s , o t h e r s p e c i a l a d a p t a t i o n s a r e n e c e s s a r y f o r a q u a t i c a n i m a l s t o t o l e r a t e p r o l o n g e d underwater submergence. 2- DIVING RESPONSE: APNQEA AND CARDIOVASCULAR ADJUSTMENTS: As I r v i n g and S c h o l a n d e r p r o p o s e d , i n a d d i t i o n t o t h e apnoea e x h i b i t e d by a q u a t i c mammals and b i r d s d u r i n g f o r c e d submersion, an i n c r e a s e of v a s c u l a r r e s i s t a n c e i n many p e r i p h -e r a l t i s s u e s and organs must o c c u r ( F i g u r e 1; Andersen 1959; Johansen 1964; B u t l e r and Jones 1971; Daly 1972; Jones et a l . 197 9; Zapol et a l . 197 9; McKean 1982; H e i e i s and Jones 1988). T h i s p e r i p h e r a l v a s o c o n s t r i c t i o n i s mediated by the sympathet-i c b r a n c h o f t h e autonomic nervous system (Kobinger and Oda 1969; B u t l e r and J o n e s 1971; A n d e r s e n and B l i x 1 9 7 4 ) . As I r v i n g suspected, c e r e b r a l b l o o d f l o w i s unchanged (Kerem and E i s n e r 1973; Z a p o l e t a l . 1979) o r may even i n c r e a s e d u r i n g d i v i n g (Jones e t a l . 1979; H e i e i s and Jones 1988) . L i k e w i s e , c o r o n a r y b l o o d f l o w remains unchanged i n ducks (Jones e t a l . 1979) and i n s e a l s , i t appears t o d e c r e a s e p r o p o r t i o n a t e l y w i t h t h e r e d u c t i o n i n c a r d i a c o utput ( B l i x e t al . 1 9 7 6; Zapol e t a l . 1979). R e s i s t a n c e o f t h e pulmonary v a s c u l a r bed i n -c r e a s e s d u r i n g d i v i n g ; however, t h e i n c r e a s e i s c o n s i d e r a b l y l e s s t h a n i n t h e p e r i p h e r a l organs (Jones and H o l e t o n 1972; S i n n e t t e t a l . 1978). A p r o f o u n d d e c r e a s e o f c a r d i a c o u t p u t accompanies t h e s e v a s c u l a r changes, t h u s p r e v e n t i n g any undue r i s e o f a r t e r i a l b l o o d p r e s s u r e . T h i s d e c r e a s e o f c a r d i a c o u t p u t i s caused p r i m a r i l y by an extreme r e d u c t i o n i n h e a r t r a t e ( F i g u r e 1) mediated by the vagus nerves ( B u t l e r and Jones 1968; 1971). Measurement o f s t r o k e volume i n d i v i n g mammals FIGURE 1_: Cardiovascular responses to forced submersion of a Pekin duck (Anas platyrhynchos). Peripheral resistance refers to the r e s i s t a n c e to blood flow i n one l e g . Blood oxygen tension°was measured i n the brachiocephalic a r t e r y , (from Gabbott 1985). 16 air flow • 3 r ' f f l (L/min ) . 3 400 II heart rate 200 (beats/min) peripheral resistance (pru) 0 20 0 U arterial blood pressure (mm Hg) blood oxygen tension (mm Hg) 230 115 0 L 150 75 0 L m^4 dive surface 30sec 17 and b i r d s has proven t o be d i f f i c u l t and th e da t a are co n f u s -i n g . Some s t u d i e s have r e c o r d e d t h a t s t r o k e volume d e c r e a s e s ( S i n n e t t e t a l . 1978; Z a p o l e t a l . 1979; B l i x and F o l k o w 1 9 8 3 ) , w h i l e o t h e r s c o n c l u d e d t h a t i t r e m a i n s unchanged ( E i s n e r e t a l . 1964; B l i x e t a l . 1976; Folkow e t a l . 1967; Jone s and H o l e t o n 1 9 7 2 ) . The o v e r a l l c onsequence o f t h e s e c a r d i o v a s c u l a r changes d u r i n g d i v i n g i s , as I r v i n g suggested, a p r e f e r e n t i a l r e d i s t r i b u t i o n o f t h e c i r c u l a t i n g b l o o d f l o w , s o . t h a t oxygen s u p p l i e s are c o n f i n e d almost e x c l u s i v e l y t o the c e n t r a l nervous system and the h e a r t . 3- INITIATION AND MAINTENANCE OF THE DIVING RESPONSE: In o r d e r t o s u r v i v e l o n g p e r i o d s o f submersion, c a r d i o -v a s c u l a r adjustments have t o be s e t i n p l a c e q u i c k l y , as soon as t h e d i v e b e g i n s , i n o r d e r t o p r e v e n t oxygen s t o r e s from b e i n g p r e m a t u r e l y d e p l e t e d by t h e p e r i p h e r a l t i s s u e s . More-over, once these c a r d i o v a s c u l a r adjustments are i n p l a c e , they must be ma i n t a i n e d throughout t h e d i v e . I t has been d i f f i c u l t t o d e f i n e the p r e c i s e mechanisms by which submersion i n i t i a t e s and m a i n t a i n s the c a r d i o v a s c u l a r adjustments d e s c r i b e d above. T h i s i s m a i n l y because o f t h e many d i f f e r e n t s e n s o r y i n p u t s which may be i n v o l v e d , and the f a c t t h a t i n t e r s p e c i f i c d i f f e r -ences e x i s t . D i v i n g mammals, as w e l l as some d i v i n g b i r d s , respond t o f a c e immersion w i t h an i n s t a n t a n e o u s b r a d y c a r d i a w h i c h sug-g e s t s a r e f l e x a c t i v i t y t r i g g e r e d by f a c i a l or n a s a l r e c e p t o r s (Angell-James and Da l y 1969; Folkow e t a l . 1971; Dykes 1974; Drummond and Jones 197 9; C a t l e t t and Jo h n s t o n 1974; Mangalam 18 and Jones 1984). F u r i l l a and Jones (1986) d i v e d redhead ducks (Aythya americana) a f t e r h a v i n g s p r a y e d x y l o c a i n e (a l o c a l a n e s t h e t i c ) i n t o t h e i r nares. They observed t h a t the immediate b r a d y c a r d i a a s s o c i a t e d w i t h submersion was v i r t u a l l y e l i m i -n a t e d , which c o n f i r m e d t h e r o l e o f t h e n a s a l r e c e p t o r s as an i n i t i a t i n g mechanism. When h e a r t r a t e i s p l o t t e d a g a i n s t t i m e , t h e p r o f i l e o f d i v i n g b r a d y c a r d i a i n d o m e s t i c ducks (Anas p l a t v r h y n c h o s ) shows a g r a d u a l f a l l i n h e a r t r a t e s e v e r a l seconds a f t e r t h e s t a r t o f the d i v e . T h i s d i f f e r s from the i n s t a n t a n e o u s brady-c a r d i a observed i n redhead ducks, and argues a g a i n s t a r e f l e x t r i g g e r e d by a water s t i m u l u s . D e n e r v a t i o n o f the c a r o t i d body chemoreceptors i n P e k i n ducks suppresses t h e d i v i n g b r a d y c a r d -i a almost c o m p l e t e l y (Jones and Purves 1970). T h i s work sup-p o r t s t h e h y p o t h e s i s t h a t t h e p r o g r e s s i v e h y p o x i a and hyper-c a p n i a , which develops as a consequence of d i v i n g apnoea, are t h e s t i m u l i w h i c h t r i g g e r t h e c a r d i o v a s c u l a r a d j u s t m e n t s . Jones e t a l . (1982) e s t i m a t e d t h a t a p p r o x i m a t e l y 85% o f t h e t o t a l b r a d y c a r d i a and 67% o f t h e h i n d l i m b v a s o c o n s t r i c t i o n were due t o s t i m u l a t i o n o f p e r i p h e r a l c hemoreceptors; 30% o f t h e h i n d l i m b v a s o c o n s t r i c t i o n was due t o s t i m u l a t i o n o f c e n t r a l chemoreceptors and t h e r e s t was due t o s t i m u l a t i o n o f b a r o c e p t o r s and o t h e r u n i d e n t i f i e d i n p u t s . I n d i v i n g ducks, where b r a d y c a r d i a i s i n i t i a t e d by t h e c o n t a c t o f w a t e r on n a s a l r e c e p t o r s , c a r o t i d body c h e m o r e c e p t o r s may a l s o be p a r t i a l l y i n v o l v e d i n t h e maintenance o f t h e c a r d i o v a s c u l a r adjustments d u r i n g l o n g d i v e s (Stephenson and Jones 1989). 19 In spite of the huge changes in heart rate and peripheral vascular resistance during diving, there i s l i t t l e change i n mean a r t e r i a l blood pressure. This could imply a r o l e for a r t e r i a l baroreceptors i n the i n i t i a t i o n and maintenance of the c a r d i o v a s c u l a r adjustments. Anderson and B l i x (1974) suggested that diving bradycardia i s merely the expression of a b a r o r e f l e x which responds to an i n c i p i e n t r i s e of mean a r t e r i a l blood pressure, caused by a chemoreceptor driven vasoconstriction. However, Jones (1973) did not observe any changes in the diving bradycardia of chronically baroreceptor denervated ducks compared with t h e i r sham-operated controls. After noticing a f a l l i n mean a r t e r i a l blood pressure and a reduced peripheral vasoconstriction i n barodenervated ducks, he advanced the idea that i t i s peripheral vasoconstriction which i s the expression of a b a r o r e f l e x . The increase i n peripheral resistance was caused by the drop of blood pressure t r i g g e r e d by a chemoreceptor driven bradycardia. However, recent work (Jones et a l . 1982/ 1983/ Smith 1987) has shown that during diving, bradycardia and peripheral vasoconstric-tion are both mediated by the chemoreceptors. Within the f i r s t minute of submersion, a r t e r i a l baroreceptor a c t i v i t y helps to balance cardiac output against vasoconstriction. As submersion continues, there i s a progressive attenuation of the barore-fl e x caused by a negative feedback of the chemoreflex on the baroreflex. Several other sensory inputs have been thought to play a role i n the i n i t i a t i o n of the diving response. For instance, postural changes, such as those executed by voluntarily diving animals, may also contribute to the development of d i v i n g responses (Huxley 1913; Paton 1913). Furthermore, there i s no doubt that the a c t i v i t y from diverse receptor groups must i n t e r a c t ; for example, hypoxia and hypercapnia have to be associated with a cessation of respiratory a c t i v i t y i n order for bradycardia and vasoconstriction to develop (Butler and Taylor 1973). In Pekin ducks apnoea i s i n i t i a t e d by contact of water on receptors in the nares, the g l o t t i s and around the base of the beak (Bamford and Jones 1974). The maintenance of this apnoea i s crucial to the maintenance of the dive. Curiously, trigemi-nal and glossopharyngeal inputs a r i s i n g from these receptors are ineffective after 100 seconds of diving, and yet, despite thi s , apnoea continues (Bamford and Jones 1974). Moreover, the progressive hypoxia and hypercapnia developing during the dive should steadily increase the respiratory drive. Since i t has been shown that a 3 kg Pekin duck, for example, can endure submersion for at least 13 minutes (Hudson and Jones 1986), t h i s i n c r e a s i n g r e s p i r a t o r y drive must be suppressed. The mechanisms causing t h i s suppression are not known but they involve a marked change i n r e s p i r a t o r y s e n s i t i v i t y to C0 2 l i n k e d to the withdrawal of rhythmic pulmonary a f f e r e n t informations during asphyxia (Cohen 1964; Bamford and Jones 1976 a and b). It has been suggested that the cardiovascular response to forced diving i s nothing more than a modification of the Type II fear reaction, or freezing response, which i s t y p i c a l l y 21 characterized by bradycardia, hypotension and a decrease of v e n t i l a t i o n (Gabrielsen et a l . 1977; Smith et a l . 1981 a and b) .. This proposal finds some support i n the discovery of a huge increase i n plasma catecholamine l e v e l s during forced diving (Hudson and Jones 1982). The hypothesis was put forward that the fear response i s triggered by the manipulation of the animal for the dive i t s e l f (Gaunt and Gans 1969; Kanwisher et al 1981). Although there may be certain apparent s i m i l a r i t i e s i n behavior, there are fundamental differences between the d i v i n g response and the fear r e a c t i o n . The f o l l o w i n g two examples alone provide a strong argument for the rejection of the fear hypothesis: 1) i f Pekin ducks breathe pure oxygen before forced dives, the bradycardia and plasma catecholamine l e v e l s are considerably reduced ( F u r i l l a and Jones 1986; Mangalam et al_. 1987) even though the handling of the animal i s the same; and 2) when a l l brain tissue above the mesenceph-alon i s removed, including the hypothalamus (which mediates the fear response: Evans 1976), the response to forced submer-sion i s exactly the same as that i n i n t a c t ducks (Gabbott 1985). 4- METABOLISM: When compared with non-divers, diving mammals and birds do not e x h i b i t any exceptional adaptations f o r anaerobic metabolism which would explain t h e i r remarkable underwater tolerance. Although the b r a i n s of s e a l s have been shown to be s l i g h t l y more tolerant to hypoxia than those of t e r r e s t r i a l mammals, t h i s could be explained by a higher cerebral c a p i l -22 l a r y d e n s i t y , a l a r g e r g l y c o g e n s t o r e , and a h i g h e r l e v e l o f l a c t a t e dehydrogenase a c t i v i t y ( E i s n e r e t a l . 1970; Kerem and E i s n e r 1973; Kerem et a l . 1973; Murphy et a l . 1980; C a s t e l l i n i e t a l . 1981). In t h e case o f m a l l a r d ducks, however, t h e i r b r a i n t i s s u e appears t o be no more r e s i s t a n t t o h y p o x i a t h a n t h a t o f c h i c k e n s (Bryan and Jones 1980). Weddell s e a l h e a r t s a l s o possess u n u s u a l l y h i g h glycogen s t o r e s , which may be used as f u e l d u r i n g l o n g d i v e s (Kerem et a l . 1973). The a c t i v i t y of l a c t a t e dehydrogenase i n s e a l h e a r t s i s h i g h e r than i n t e r r e s -t r i a l mammals, and t h i s p r e s u m a b l y p r o v i d e s them w i t h t h e a b i l i t y t o use l a c t a t e as a f u e l d u r i n g d i v e s (Murphy e t a l . 1980; C a s t e l l i n i e t a l . 1981; Hochachka 1981), p r e s e r v i n g b l o o d g l u c o s e f o r t h e b r a i n . L a c t a t e i s a l s o used as f u e l by the lungs d u r i n g d i v i n g i n s e a l s (Murphy e t a l . 1980) . U n l i k e o t h e r body t i s s u e s , b r a i n and h e a r t m e t a b o l i s m i n s e a l s r e -mains a e r o b i c d u r i n g p r o l o n g e d d i v e s or f o r c e d submersions. W i t h t h e e x c e p t i o n o f h i g h e r b u f f e r i n g c a p a c i t y , t h e s k e l e t a l muscle of d i v i n g b i r d s and mammals as a g e n e r a l r u l e does n o t seem t o be any more a d a p t e d t o h y p o x i a t h a n t h e muscle o f t e r r e s t r i a l a n i m a l s (Kerem e t a l . 1973; S t o r e y and Hochachka 1974; C a s t e l l i n i e t a l . 1981; C a s t e l l i n i and Somero 1981) . I t s h o u l d be p o s s i b l e t o c a l c u l a t e t h e t h e o r e t i c a l maxi-mum d i v e time (MDT) of ducks from measures of the oxygen s t o r e and t h e r a t e o f oxygen c o n s u m p t i o n by t h e c e n t r a l n e r v o u s s y s t e m (CNS), t h e h e a r t and t h e l u n g s . Thus, f o r a 1 Kg d u c k : MDT = Oxygen s t o r e s Q>2 uptake by the CNS, he a r t and lungs The only oxygen available to heart and brain i s stored in the respiratory system and the blood (Hudson and Jones 1986): 0 2 stores = 19.2 ml If brain weight i s 4.9 g (Hudson and Jones 1986) and i f the brain metabolic rate estimated for the chicken i s used (Mink et a l . 1981), then: oxygen uptake by the brain = 4.9 g x 0.06 ml.g - 1 = 0.29 ml.min - 1 If the spinal cord i s approximately half of the weight of the brain, and i f i t s weight s p e c i f i c metabolic rate i s half that of the brain (Mink et a l . 1981), then: oxygen uptake by the spinal cord = 2.45 g x 0.03 ml.g - 1 = 0 . 0 7 ml.min - 1 The weight of the lungs i s about 12 g for a 1 Kg duck (Lasiewski and Calder 1 9 7 1 ) . As estimates for the metabolic rate of avian lung tissue are unavailable, values obtained for mammals can be used. Metabolic rates for lung t i s s u e range from 0.004 to 0.02 ml.g - 1.min - 1 (Krebs 1950/ Wallace et a l . 1 9 7 4 ) . Hence the lung could use anywhere from 0.048 to 0.24 — l — l ml.g .mm . However, since blood flow to the lungs i s strongly reduced during diving (Jones et a l 1979), perhaps the lowest metabolic rate should be chosen. 24 Power output of the heart = cardiac output x a r t e r i a l blood pressure, as defined by Jones and Johansen (1972) , and cardiac output = heart rate x stroke volume Thus i f stroke volume i s estimated at 1.5 ml during the dive (Jones and Holeton 1972), and mean a r t e r i a l blood pressure and heart rate are 118 mmHg and 29 beats.minute - 1 r e s p e c t i v e l y (Hudson and Jones 1986), then: power output ( l e f t v e n t r i c l e ) = 1 1 8 x 1 3 3 3 x 1.5 x 29 = 6.8 x 10 6 erg.min - 1 I f mean blood pressure i n the pulmonary artery i s 60 mmHg (Jones personal communication), then: power output ( r i g h t ventricle) = 6 0 x 1 3 3 3 x 1 - 5 x 2 9 = 3.5 x 10^ erg.min - 1 Therefore, the t o t a l power output of the heart i s : c cardiac power output = (6.8 + 3.5) x 10 = 10.3 x 10 6 erg.min - 1 Assuming that the u t i l i z a t i o n of 1 ml of oxygen releases the Q c a l o r i f i c equivalent of 2.2 x 10 ergs, and that the e f f i c i e n -cy of the heart i s 10%, the t o t a l uptake of oxygen by the heart i s : 0 2 uptake = 10.3 x 10 6 x (100/10) x 10 - 8/2.2 = 0.47 ml 0 2.min - 1 Given the above calculations, the oxygen consumed during the dive by the brain, sp i n a l cord, lungs and heart equals 0.88 ml.min - 1, thus: 25 MDT = 19.2 / 0.88 = 22 minutes. However the MDT determined for a 1 kg duck (using EEG to i n d i c a t e the dive l i m i t ) was only 6.6 minutes (Hudson and Jones 1986). It takes 1 minute for a 1 Kg duck to e s t a b l i s h the cardiovascular adjustments to d i v i n g . The increase i n peripheral vasoconstriction i s i n d i r e c t l y r e f l e c t e d by the drop i n heart rate i n the f i r s t minute of a dive. From t h i s , i t can be estimated that h a l f as much blood flows to the peripheral organs during the f i r s t minute of the dive. There-fore, the oxygen consumption during the f i r s t minute would be: 0 2 consumption =11.3 M 0 ' 7 2 3 X 1/2 = 5.7 ml.min - 1 and MDT, thus corrected, would be: MDT = [ (19.2 - 5.7) / 0.88] + 1 = 16.3 minutes This c a l c u l a t e d value i s s t i l l much greater than the measured value of 6.6 minutes. For the MDT to be only 6.6 minutes, the oxygen uptake i n a 1 Kg duck during diving would have to be: MDT = [(19.2 - 5.7) / V0 2 d i v e ] + 1 (VC>2 d i v e i s oxygen consumed a f t e r the f i r s t minute of the dive) 6.6 = [13.5 / V0 2 d i v e ] + 1 _ i V0 2 dive = 2 , 4 ml.min 26 The value of 2.4 ml.min - 1 represents the oxygen consumption rate of the duck afte r the f i r s t minute of diving. However, since only 0.88 ml.min - 1 i s consumed by the CNS, lung and heart, what accounts for t h i s extra 1.52 ml.min - 1 of 0 2 up-take? It has been shown that blood flow decreases considerably in peripheral tissues during diving; however, i t does not stop completely. Thus, the remaining flow could account for some of the extra oxygen (1.52 ml.min - 1) consumed during the dive. For example, blood flow dropped to 7-9% of the pre-dive value in the kidney, to 11-13% i n the l e g and to 4% and 2 1 % i n the gizzard and the i n t e s t i n e s r e s p e c t i v e l y . Furthermore, when blood flow i s maintained to the brain, as described above, flow also continues to other tissues of the head, such as the c r a n i a l muscles and skin (Johansen et a l . 1964; Butler and Jones 1971; Jones et a l . 1979). Hochachka (1981) calculated that i f the MDT i n Weddell seals was 1.2 hours (Kooyman et a l . 1980) and i f oxygen stores were consumed exclusively by the brain, heart, and lungs, then the oxygen store would be depleted by only 25%, and thus, could not be the factor l i m i t i n g the length of the dive. The blood glucose l e v e l drops during d i v i n g i n Weddell seals (Murphy et a l . 1980) and so Hochachka suggested instead, that the f a c t o r l i m i t i n g the dive length was the rate of blood glucose u t i l i z a t i o n . However, the above calculation determines the maximum underwater tolerance and i t i s not c e r t a i n that the longest free dive observed (1.2 hours) represents the maximal dive time of Weddell seals. Again, although blood flow i n p e r i p h e r a l t i s s u e s i s g r e a t l y decreased, i t i s not stopped e n t i r e l y : a p p r o x i m a t e l y 42% o f t h e c a r d i a c o u t p u t d u r i n g d i v i n g p e r f u s e s t h e p e r i p h e r a l t i s s u e s ( Z a p o l e t a l . 1979) . T h i s f a c t o r s hould be taken i n t o c o n s i d e r a t i o n i n the c a l c u l a -t i o n o f oxygen s t o r e d e p l e t i o n and c o u l d a l s o c o n t r i b u t e t o a r e p l e n i s h m e n t o f b l o o d g l u c o s e by the l i v e r d u r i n g d i v i n g . In o r d e r t o d i s c o v e r t h e f a c t o r s l i m i t i n g t h e l e n g t h o f d i v e s , the end-dive v a l u e s o f the b l o o d gases (PaC>2, PaC0 2, pHa) and m e t a b o l i t e s ( g l u c o s e , l a c t a t e ) s h o u l d be c o r r e l a t e d w i t h t h e end o f a maximum f o r c e d d i v e , as d e f i n e d by Hudson and Jones (1986). By f o l l o w i n g such a p r o c e d u r e i t has been shown t h a t b l o o d oxygen s t o r e s , not b l o o d g l u c o s e l e v e l s , seem t o be the l i m i t i n g f a c t o r o f MDT i n f o r c e d d i v e d P e k i n ducks (Hudson and Jones 1986). I n 1899, R i c h e t o b s e r v e d t h a t 3 and 8 day o l d ducks d i d not d i v e as l o n g as a d u l t s . R e c e n t l y , Hudson and Jones (1986) a l s o observed a g r e a t v a r i a b i l i t y i n MDT among ducks, and they e s t a b l i s h e d t h a t MDT i s r e l a t e d t o body mass (M) , t h u s : MDT = 6.6 M 0' 6 4. As i t i s demonstrated above, MDT can be o b t a i n e d by d i v i d i n g 0 2 s t o r e s a v a i l a b l e f o r t h e CNS, l u n g and h e a r t by t h e 0 2 c o n s u m p t i o n o f t h e s e organs d u r i n g d i v i n g . I n d ucks, t h e s e oxygen s t o r e s a r e p r o p o r t i o n a l t o M4- • ^ (Hudson and Jones 1986). The s p i n a l c o r d and t h e l u n g s consume v e r y l i t t l e o f the oxygen d u r i n g d i v i n g , and they were consequently n e g l e c t e d i n Hudson and Jones'(1986) c a l c u l a t i o n s . The combined mass of 28 the heart and b r a i n i n ducks, over 0.5 Kg, i s p r o p o r t i o n a l t o M0.85 (Hudson and Jones 198 6), and the metabolic r a t e can be ob t a i n e d by m u l t i p l y i n g the mass exponent by the m e t a b o l i c exponent (0.723) (Schmidt-Nielsen 1979). Therefore, MDT i s p r o p o r t i o n a l to M1.19 / ( M0.85)0.723 = M0.61 This c a l c u l a t e d value of M^*^1 i s q u i t e c l o s e to the meas-ured value of M1-1'^4, and suggests that MDT increases with body mass because the 0 2 stores increase with body mass f a s t e r than oxygen uptake by the heart and b r a i n . During f o r c e d d i v i n g , a e r o b i c metabolism i n a 1 kg duck — 1 drops to 2.4 ml.min , about 20% of the p r e - d i v e v a l u e (see c a l c u l a t i o n above), and anaerobic metabolism occurs i n pe r i p h -e r a l t i s s u e s (Scholander 1940) . Moreover, not only does aero-b i c metabolism decrease d u r i n g f o r c e d d i v e s , but a drop of t o t a l m e t a b o l i c r a t e i n d i v i n g b i r d s , as w e l l as d i v i n g mam-mals, seems to occur. P i c k w e l l (1968), using d i r e c t calorime-t r y , c a l c u l a t e d that forced dived Pekin ducks may experience a red u c t i o n i n t o t a l energy metabolism by up to 90% of pre- d i v e r e s t i n g l e v e l s . Moreover, drops i n temperature of the b r a i n (by 2.5°C), abdomen and muscles (by 1°C) of the bladdernose s e a l were observed by Scholander and coworkers (1942) d u r i n g 15 minute for c e d d i v e s . A d e c l i n e of 1-2 °C i n r e c t a l tempera-tu r e was observed during a forc e d dive i n the harbor s e a l , but t h i s was not accompanied by any changes i n b r a i n temperature (Eisner et a l . 1975). Decreases i n core temperature have a l s o been recorded i n f r e e d i v i n g Weddell s e a l s . Most f r e e d i v e s by seals are less than 25 minutes long and are aerobic; howev-er, occasional exploratory dives may be considerably longer, ranging from 25 minutes to 1.2 hours (Kooyman et a l . 1 9 8 0 ) . During these longer dives, lactate production (indicative of anaerobic metabolism) increased and body temperature dropped. In a 53 minute dive, body temperature dropped from 38°C to 3 4 . 9 ° C (Kooyman et a l . 1 9 8 0 ) . Qvist et a l . (1986) observed that the central blood temperature i n Weddell seals varied from a maximum of 38.6 °C at r e s t to 35.1 °C during free diving. Also the oxygen debt measured afte r a forced dive i n ducks was less than the predicted d e f i c i t (Scholander 1940) . These r e s u l t s are s t r i k i n g because they suggest that these animals may be able to lower t h e i r basal metabolism i n order to achieve maximum underwater tolerance. This could obviously c o n t r i b u t e to the a b i l i t y of d i v i n g animals to sustain long dives by decreasing the rate of oxygen and glu-cose consumption. A decrease i n metabolic rate has a further advantage i n that i t would reduce the accumulation of end products, such as CO2 and lactate, during diving. This would decrease the recovery period (the time which has to be spent on the surface) a f t e r long dives, such as the exploratory free dives observed i n Weddell seals (Kooyman et a_l. 1980) . The mechanisms allowing such a metabolic feat during diving are s t i l l unknown. 30 5- WHY STUDY FORCED-DIVES IN PEKIN DUCKS?: E v e n t h o u g h t h e y h a v e b e e n o b s e r v e d t o d i v e f o r f o o d ( F u r i l l a a nd J o n e s 1987b) and f o r e s c a p i n g c a p t u r e ( p e r s o n a l o b s e r v a t i o n s ) , P e k i n d u c k s (Anas p l a t y r h y n c h o s ) a r e m a i n l y d a b b l e r s . T h e i r s t r a t e g y , when f o r a g i n g u n d e r w a t e r f o r f o o d , c o n s i s t s m a i n l y o f immersing o n l y t h e upper p a r t o f t h e i r body f o r 9 se c o n d s a t most (Jones and F u r i l l a 1987). In s u c h s h o r t d i v e s , ducks a r e e s s e n t i a l l y a e r o b i c and have l i t t l e need f o r any d e f e n s e a g a i n s t a s p h y x i a . Thus, i t i s a l l t h e more s u r -p r i s i n g t h a t t h e s e a n i m a l s a r e a b l e t o e x e c u t e s u c h p r o f o u n d c a r d i o v a s c u l a r c h a n g e s a n d c a n s u r v i v e f o r more t h a n 20 mi n u t e s underwater ( F i g u r e 2) w i t h o u t any p h y s i o l o g i c a l damage (Huds o n a n d J o n e s 1 9 8 6 ) . T h i s d u a l i t y o f d i v i n g b e h a v i o r ( a e r o b i c f r e e d i v e s w i t h l i t t l e c a r d i o v a s c u l a r change v e r s u s a n a e r o b i c f o r c e d d i v e s w i t h s t r o n g b r a d y c a r d i a ) o b s e r v e d i n P e k i n d u c k s ( F i g u r e 2 ) , i s a l s o f o u n d i n t r u e d i v i n g b i r d s ( A y t h y a a m e r i c a n a ) a n d i n d i v i n g mammals ( W e d d e l l s e a l s ) (Kooyman and C a m p b e l l 1972; Kooyman e t al. 1980; L i g g i n s e t a l . 1980; B u t l e r and Jo n e s 1982; F u r i l l a and Jo n e s 1986; H i l l e t a l . 1987). A l t h o u g h t h e p a t t e r n o f r e s p o n s e e x h i b i t e d by f o r c e d d i v e d a n i m a l s i s n o t n o r m a l l y s e e n i n t h e s h o r t d i v e s o f v o l u n t a r i l y d i v i n g d u c k s , t h i s p a t t e r n has b e e n o b s e r v e d i n d u c k s t h a t have been s u d d e n l y t r a p p e d u n d e r w a t e r ( S t e p h e n s o n e t a l . 1986; F u r i l l a and Jones 1987a). In f r e e d i v i n g mammals a l s o , t h e f o r c e d d i v e p a t t e r n o f r e s p o n s e has sometimes been o b s e r v e d d u r i n g l o n g e x p l o r a t o r y d i v e s ( i . e . e x c e e d i n g 20 m i n u t e s : Kooyman and C a m p b e l l 1972; Kooyman e t a_l. 1980). I t FIGURE 2 : Heart rate during A: Forced dive in Pekin B: Free dabble in Pekin Head in ( { ) and out of ( f ) duck (pe r s o n a l d a t a ) , duck (from F u r i l l a & Jones 1986). water. 32 33 i s l i k e l y that divers have evolved an important corticolimbic c o n t r o l of t h e i r c a r d i o v a s c u l a r bulbar r e f l e x responses (Gabbott 1985; F u r i l l a and Jones 1987a; McCulloch 1989) which could explain the great difference i n magnitude of the diving responses observed. This enables animals to adjust t h e i r asphyxic defense according to the anticipated challenge of the dive, and allows them to cope with unexpected events happening during the dive (Blix 1987). In forced dives, the f u l l poten-t i a l of t h e i r defense against asphyxia i s activated and thus the most extreme cardiovascular responses are observed. Speculation aside, the diving responses of forced dived Pekin ducks are si m i l a r to those observed i n diving mammals and birds during long exploratory free dives and during forced dives. Furthermore, unlike mammals, ducks possess only one set of a r t e r i a l baroreceptors (located in the wall of the ascend-ing aorta) which i s anatomically separated from the peripheral chemoreceptors (located i n the c a r o t i d bodies) (Jones and Purves 1970; Jones 1973). This anatomical p e c u l i a r i t y has permitted study of the d i f f e r e n t i a t i o n between the roles of peripheral chemoreceptors and the roles of baroreceptors i n the i n i t i a t i o n and maintenance of the dive response (Jones and Purvis 1970; Jones 1973; L i l l o and Jones 1982; Jones et a l . 1983). The many and extensive studies of the diving responses of Pekin ducks have provided invaluable insights into the f u l l p o t e n t i a l of one of the most e f f i c i e n t defenses against ap-noeic asphyxia which has evolved in diving mammals and birds. 34 6- CIRCULATING CATECHOLAMINES AND MDT: The mechanisms underlying survival for such long periods of apnoeic asphyxia are s t i l l not very well known. For exam-ple, the question of whether or not adrenal catecholamines have a si g n i f i c a n t role in forced diving of birds and mammals has been debated without having been d i r e c t l y investigated (Hance et a l . 1982; Hudson & Jones 1982; Mangalam et a l 1987). This study has been undertaken in this thesis. , A certain amount of evidence i s beginning to accumulate which suggests that catecholamines may play a v i t a l role i n the diving response. For instance, i n contrast to the s i t u a -t i o n i n peripheral tissues, the blood supply to the adrenal glands continues during forced submersion. In fact, i t may even increase i n ducks (Johansen 1964; Jones et a l . 1 9 7 9 ) , although i t i s unchanged in nutria (McKean 1982) and decreases by 40% in Weddell seals (Zapol et a l . 1979). Another observa-t i o n i s that forced diving i s associated with a s i g n i f i c a n t increase i n c i r c u l a t i n g catecholamines (epinephrine, EP; and norepinephrine, NE). In ducks, plasma catecholamine concentra-tions double after a dive of one minute and increase 1000 fold after 10 to 16 minutes of diving (Huang et a l . 1974; Hudson & Jones 1982; Figure 3 ) . Four to 30 fold increases in catechola-mine levels have also been observed in the Harbor seal after 4 to 6 minutes of submergence (Hance et a l . 1982) . It has been recently noted that catecholamine secretion may be triggered by stimuli known to be c r u c i a l for invoking diving responses, such as a variation in blood gas tensions (Jones et a l . 1982; Mangalam et a l . 1987; Rose e_t a l . 1983) or s t i m u l a t i o n of 35 FIGURE 3.: Increase i n Norepinephrine (open symbols) and Epine-p h r i n e ( c l o s e d symbols) d u r i n g f o r c e d d i v e s i n d u c k s . A l l d i v e s s t a r t e d a t d i v e t i m e 0 and ended when t h e l a s t sample was t a k e n . R e c o v e r y t i m e 0 r e p r e s e n t s when each a n i m a l was a l l o w e d t o s u r f a c e , (from Hudson and Jones 1982). 36 PLASMA CATECHOLAMINES (nono moles liter*1) nasal receptors by water ( A l l i s o n & Powis 1971; Drummond & Jones 1979). Kobinger and Oda (1969), Andersen and Blix (1974) and Butler and Jones (1968; 1971) have demonstrated the impor-tance of the sympathetic branch of the autonomic nervous system in the i n i t i a t i o n and maintenance of peripheral vaso-c o n s t r i c t i o n during forced-diving i n ducks. This was accom-plished with the use of adrenergic blockade or catecholamine depleting agents; however, none of these studies d i f f e r e n t i a t -ed between the r o l e of sympathetic nerves and the r o l e of adrenal catecholamines. As sympathetic nerve a c t i v i t y and plasma catecholamines are known to engender s i m i l a r p h y s i o l o g i c a l responses, the question raised i s : What are the relative roles of the sympa-t h e t i c nerves and of these enormous l e v e l s of c i r c u l a t i n g catecholamines during diving? It i s reasonable to consider that t h i s remarkable increase of c i r c u l a t i n g catecholamines plays an important role in the diving response because catech-olamines are known to influence a v a r i e t y of p h y s i o l o g i c a l processes indispensable to d i v i n g . Gooden (1980) suggested that c i r c u l a t i n g catecholamines could enhance p e r i p h e r a l vasoconstriction and could counteract the depressant action of hypoxia on neural vasoconstriction. Hypoxia also has a depres-sant action on myocardial c o n t r a c t i l i t y (McKean 1984; Nakhja-van et a l . 1971; Tyberg et a l . 1970), and t h i s could be com-pensated for by c i r c u l a t i n g catecholamines, which have a well known positive inotropic effect on the myocardium. Circulating catecholamines have been shown to protect the brain against 38 ischemic damage (Koide et a l . 1986). They may also enhance the e x c i t a t i o n of chemoreceptors (Milsom and Sadig 1983), and consequently increase the i n t e n s i t y of the dive response. C i r c u l a t i n g catecholamines could compensate for the drop of blood oxygen a f f i n i t y caused by a c i d o s i s (Nikinmaa 1983) during dives and thus, ducks could maximize the u t i l i z a t i o n of pulmonary oxygen stores. During diving, anaerobic metabolism increases, and by promoting g l y c o g e n o l y s i s , c i r c u l a t i n g catecholamines could also help s a t i s f y the substrate require-ments. Throughout the hist o r y of diving physiology, there has been l i t t l e e f f o r t made to study the humoral control of the diving response (Robin et a l . 1981; Mangalam et a l . 1987). As has been shown in t h i s review, the primary emphasis has been the neural control of the diving response. This work w i l l show that i n addition to the autonomic nervous system, hor-mones, such as adrenal catecholamines, play an important role in the defense against apnoeic asphyxia. In the f i r s t part of t h i s thesis, I determined the r e l a -t i v e importance of the adrenal glands in the release of circu-l a t i n g catecholamines during forced d i v i n g of Pekin ducks. Then I explored the influence of adrenal catecholamines on the maximum tolerance to forced submersion. Ducks in which catech-olamine release from adrenal glands had been prevented, were forced dived and maximum dive times were compared with times measured with control ducks. To understand the mechanisms involved in the process, heart rate, blood pressure, blood gas 39 values as well as blood metabolites and ions were -measured. In order to estimate the role of c i r c u l a t i n g catecholamines on peripheral vasoconstriction and on cardiac function during diving, two sets of experiments were conducted on Pekin ducks. One set involved hind limb perfusion while the second set involved beta-blockade. 40 CHAPTER 1: THE SOURCE OF CIRCULATING CATECHOLAMINES IN FORCED DIVED PEKIN DUCKS. 41 I N T R O D U C T I O N . A c t i v a t i o n of the sympathetic branch of the autonomic nervous system can be triggered (Figure 4) by the contact of water on the nasal receptors ( A l l i s o n and Powis 1971), by variations of blood gas tension at the chemoreceptors (Rose et a l . 1983), by a drop of blood pressure (Ito et. a l . 1984; Engeland et a l . 1981), by hypothermia and hypoglycemia (Young et a l . 1984) and by stress (Archer 1979). Circulating catecho-lamines can be released by the sympathetic nerve endings and by the adrenal glands (Figure 4) . Mangalam e_t a l . (1987) showed that catecholamine release was t r i g g e r e d mostly by hypoxia during diving. Acidosis, hypercapnia and even a stress component related to the handling of the animal for the dive i t s e l f , had l i t t l e effect on catecholamine release. By compar-ing adrenal denervated ducks with sham operated ducks, Manga-lam et a l . (1987) showed that h a l f of the plasma norepine-phrine (NE) and 90% of plasma epinephrine (EP) released during dives came from neural activation of the adrenal glands. They thought that non-neural releasing mechanisms might also have t r i g g e r e d catecholamine release from the adrenals during FIGURE 4.: Schematic view of catecholamines release (for expla-nations see text). 43 STRESS HYPOGLYCEMIA HYPOTHERMIA WATER CONTACT" nasal receptors HYPO 0» HYPER C0 4 chemoreceptors baro receptors 1 | B L O O D " PRESSURE ACTH CORTEX (corticosteroids) SYMPATHETIC NERVES f plasma renin 1 f angiotensin II MEDULLA _ (catocholaminss) ADRENAL GLANDS EP NE release of catecholamine from nerve endings H Y P O X I A A C I D O S I S 44 f o r c e d d i v e s . N o n - n e u r a l r e l e a s i n g mechanisms c o u l d i n v o l v e i n h e r e n t c h e m o s e n s i t i v i t y of t h e a d r e n a l g l a n d i t s e l f ( F i g u r e 4: Comline and S i l v e r 1961; Nahas e t a l . 1967 S t e i n s l a n d e t  a l . , 1970; Jones and R o b i n s o n 1975) o r h u m o r a l mechanisms secondary t o the e f f e c t s of a s p h y x i a ( F i g u r e 4 ) . For example, h y p o x i a causes a r e l e a s e o f a d r e n o c o r t i c o t r o p i c hormone which mediates catecholamine r e l e a s e ( C r i t c h l e y e t a l . , 1982). A l s o , d u r i n g f o r c e d d i v i n g , v a s o c o n s t r i c t i o n o f the r e n a l bed (Jones e t a l . , 1979) a s s o c i a t e d w i t h h y p o x i a and h y p e r c a p n i a (Drum-mond and Lindheimer 1982; Rose et a l . , 1984) would s t i m u l a t e the r e n i n - a n g i o t e n s i n system c a u s i n g r e l e a s e of catecholamines from t h e a d r e n a l s ( F e l d e r g and Lewis 1965; W i l s o n and B u t l e r 1983a; C o r w i n et. a l , 1985) . Hence, i n t h e p r e s e n c e o f non-n e u r a l mechanisms, th e importance o f t h e a d r e n a l g l a n d s com-p a r e d w i t h c o n t r i b u t i o n s from autonomic s y m p a t h e t i c v a s c u l a r nerve e n d i n g s , t o t h e i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s d u r i n g a d i v e remains a matter o f s p e c u l a t i o n . In order t o o b t a i n an e s t i m a t e of the r e l a t i v e impor-tance of the a d r e n a l glands and autonomic v a s c u l a r sympathetic nerves t o t h e catecholamine i n c r e a s e observed d u r i n g a f o r c e d d i v e , I compared t h e p l a s m a l e v e l s o f f r e e c a t e c h o l a m i n e s r e l e a s e d i n 3 minute d i v e s by a d r e n a l e c t o m i s e d ducks (ADX) w i t h t h e i r sham-operated c o n t r o l s (SH-adx). F u r t h e r , t o e s t i -mate t h e importance o f n o n - n e u r a l mechanisms s t i m u l a t i n g t h e r e l e a s e o f c a t e c h o l a m i n e s from t h e a d r e n a l g l a n d s , I a l s o compared t h e s e v a l u e s w i t h t h o s e o b t a i n e d i n a d r e n a l dener-v a t e d ducks (DNX) and t h e i r sham operated c o n t r o l s (SH-dnx) . 45 MATERIALS AND METHODS. 1- ANIMALS: The 35 ducks used i n t h i s study were 2-3 month o l d , male, w h i t e P e k i n s (Anas p l a t v r h y n c h o s ) r a n g i n g i n mass from 2 t o 3.5 k g . M a l e s were u s e d b e c a u s e a d r e n a l e c t o m y and a d r e n a l d e n e r v a t i o n a r e e a s i e r t o p e r f o r m t h a n on f e m a l e s . I n female b i r d s , o v a r i e s p r e v e n t a c c e s s t o a d r e n a l g l a n d s . Stock b i r d s were grouped i n an outdoor f i e l d w i t h f r e e access t o water and f o o d ( B u c k e r f i e l d ' s 16% l a y e r p e l l e t s , A b b o t s f o r d B . C . ) . D u r i n g t h e e x p e r i m e n t s , t h e y were ke p t i n d o o r s i n i n d i v i d u a l cages (55 X 55 X 60 cm) a t 22°C on a 12 hour l i g h t / d a r k c y c l e w i t h f o o d and w a t e r s u p p l i e d ad l i b i t u m . Seven ducks were a d r e n a l e c t o m i s e d (ADX) and 6 were sham operated f o l l o w i n g t he procedure used f o r adrenalectomy (SH-adx). I n 8 ducks a d r e n a l glands were denervated (DNX) w h i l e i n 7 o t h e r s sham o p e r a t i o n was performed f o l l o w i n g t h e procedure f o r a d r e n a l d e n e r v a t i o n (SH-dnx). Seven ducks were l e f t i n t a c t : t h ey were not submit-t e d t o su r g e r y . 2- MAJOR SURGICAL PROCEDURES: The s u r g i c a l t e c h n i q u e s used f o r a d r e n a l e c t o m y and sham o p e r a t i o n were e s s e n t i a l l y t h o s e d e s c r i b e d by Thomas and P h i l l i p s ( 1 9 7 5 ) . A maj o r d i f f e r e n c e was t h a t t h e i n t e r v a l between t h e two l a p a r o t o m i e s was one week i n s t e a d o f 1 or 2 d a y s . One hour b e f o r e t h e s e c o n d l a p a r o t o m y , 2 mg/kg body w e i g h t o f p r e d n i s o n e was g i v e n o r a l l y t o t h e ducks. A l s o , a deeper plane of anaesthesia was used (10 mg/kg body weight i . v . of sodium pentobarbital (Somnotol, MTC) followed by 8 mg/kg every 1/2 hour) . F i n a l l y , the b i r d was t i d a l l y v e n t i l a t e d with pure 0 2 as surgery v i o l a t e d the a i r sacs and may cause a decrease in respiratory efficiency. When the ducks were surg i c a l l y anaesthetised, they were secured on one side, with t h e i r upper leg extended backward to draw the sartorius muscle away from the s i t e of i n c i s i o n which was plucked and d i s i n f e c t e d with alcohol. An i n c i s i o n was made between the s i x t h and seventh v e r t e b r a l r i b s . The r i b s were retracted very carefully in order not to tear the lung tissue present at the dorsal border of the i n c i s i o n , and a small hole was made in the anterodorsal portion of the abdominal a i r sac to gain access to the adrenal glands which were surrounded by the vena cava, the aorta, lungs, kidney and t e s t i s (Figure 5) . Adrena-lectomy i t s e l f was performed under a dissecting microscope (X 5; Carl Zeiss, Germany). The adrenal glands had to be removed as far as possible i n one piece. Extensive use of ligatures prevented bleeding and kept the operative f i e l d clear so that small fragments of gland could be seen and removed. At t h i s time, only one gland was removed. After the gland was removed, the area was carefully inspected for residual adrenal tissue, and the ligatures on the adrenal blood vessels were consoli-dated with tissue cement (Histoacryl; B. Braun Melsungen AG, Melsungen, W., Germany). The abdominal a i r sac was then closed with surgical s i l k . The ribs were approximated with ligatures, and the skin wound sutured with surgical s i l k . One week later, a second laparotomy was performed on the other side following the same procedure, to remove the other adrenal gland. Sham-FIGURE 5: Ventral view of the r i g h t adrenal gland (in s i t u X15) of the Pekin duck (Anas platyrhynchos). 48 49 o p e r a t i o n c o n s i s t e d o f a l l t h e f o r e g o i n g p r o c e d u r e s , e x c e p t t h a t t h e g l a n d s were l e f t u n d i s t u r b e d w i t h t h e e x c e p t i o n o f f r e e i n g t h e lu m b a r v e i n as t h o u g h i t was g o i n g t o be l i g a -t u r e d . S h a m - o p e r a t e d d u c k s w e r e l e f t o p e n 4 h o u r s on t h e s u r g i c a l t a b l e , m a t c h i n g t h e t i m e needed t o p e r f o r m t h e remov-a l o f t h e a d r e n a l g l a n d s . R e c o v e r y a f t e r t h e e n d o f t h e s e c o n d s u r g e r y was a t l e a s t two weeks, w h i c h a l l o w e d t h e b i r d t o r e g a i n p r e - s u r g i c a l body w e i g h t . The av e r a g e body masses o f a d r e n a l e c t o m i s e d (ADX) a n d s h a m - a d r e n a l e c t o m i s e d (SH-adx) d u c k s were 2.9 + 0.2 kg and 2,8 +_ 0.3 kg b e f o r e s u r g e r y r e -s p e c t i v e l y , and 2.6 + 0.3 kg and 2.7 + 0.2 k g a t t h e e n d o f t h e r e c o v e r y p e r i o d . H a e m a t o c r i t s were 37.2 + 0.5% f o r SH-adx, and 35.8 + 0.8% f o r ADX a t t h e e n d o f t h e r e c o v e r y p e r i o d . Because c o r t i c a l t i s s u e was a l s o removed, ADX ducks were g i v e n p r e d n i s o n e o r a l l y (2 mg/kg body w e i g h t d a i l y ) , and s a l t w a t e r (0.8% NaCl) t o d r i n k a f t e r t h e s e c o n d l a p a r o t o m y . The i n n e r v a t i o n o f t h e a d r e n a l g l a n d s has been d e s c r i b e d i n t h e f o w l by Freedman ( 1 9 6 8 ) . The s u r g i c a l t e c h n i q u e u s e d f o r a d r e n a l g l a n d d e n e r v a t i o n and sham- o p e r a t i o n s was t h a t d e s c r i b e d a b o v e f o r a d r e n a l e c t o m y . I n o r d e r t o f a c i l i t a t e a c c e s s t o t h e n e r v e s , t h e lumbar v e i n was l i g a t e d and c u t i n b o t h a d r e n a l d e n e r v a t e d (DNX) a n d S h a m - o p e r a t e d (SH-dnx) d u c k s . To make c e r t a i n t h a t a l l n e r v e s h a d b e e n s e c t i o n e d , t h e a d r e n a l g l a n d s were f r e e d f r o m t h e s u r r o u n d i n g t i s s u e ( c o n n e c t i v e t i s s u e , v e n a c a v a , a o r t a and t e s t e s ) , and o n l y m a i n t a i n e d i n p l a c e by t h e a d r e n a l v e i n s and a r t e r i e s . Sham-o p e r a t i o n c o n s i s t e d o f a l l t h e f o r e g o i n g p r o c e d u r e , b u t t h e a d r e n a l n e r v e s were n o t s e c t i o n e d . The t i m e f o r r e c o v e r y a f t e r s u r g e r y was 2 t o 3 weeks. The average body weight of DNX and SH-dnx ducks were, 2.6 + 0.1 kg and 2.7 + 0.2 kg b e f o r e s u r -g e r y r e s p e c t i v e l y , and 2.6 + 0.1 kg and 2.6 +. 0.1 kg a t t h e end o f t h e r e c o v e r y p e r i o d . At t h i s t i m e , t h e h a e m a t o c r i t was 37.2 + 0.9% f o r DNX, and 36.3 + 1.9% f o r SH-dnx. A l l the ducks were k i l l e d w i t h an overdose of a n a e s t h e t i c a t t h e end o f t h e s e r i e s o f e x p e r i m e n t s e x c e p t f o r one ADX b i r d w h i c h was m a i n t a i n e d on f o o d and s a l t w a t e r o n l y and k i l l e d 2 y e a r s l a t e r . The s u c c e s s f u l removal o f t h e a d r e n a l g l a n d s i n ADX and s e c t i o n o f a d r e n a l n e r v e s i n 6 DNX was checked post mortem. 3-DIVING PROTOCOL: C a n n u l a t i o n o f a b r a c h i a l a r t e r y was done under l o c a l a n a e s t h e s i a ( L i d o c a i n e h y d r o c h l o r i d e ; X y l o c a i n e 2% A s t r a ) at l e a s t one day b e f o r e any d i v e s . The t i p of the a r t e r i a l cannu-l a (P.E. 90; I n t r a m e d i c P o l y e t h y l e n e t u b i n g , C l a y Adams) was advanced u n t i l i t l a y near the j u n c t i o n of the b r a c h i o c e p h a l i c a r t e r y and a o r t a . The b i r d was p l a c e d i n t h e s i t t i n g p o s i t i o n and s e c u r e d w i t h t a p e t o an o p e r a t i n g t a b l e . The e l e c t r o c a r d i o g r a m l e a d s were i n s e r t e d s u b c u t a n e o u s l y , one above t h e l e f t t h i g h , t h e o t h e r below t h e r i g h t s h o u l d e r , w h i l e a ground l e a d was a t -t a c h e d t o t h e r i g h t f o o t . The a r t e r i a l c a n n u l a was used t o measure b l o o d p r e s s u r e and t o t a k e b l o o d samples b e f o r e and at 3 m i n u tes submergence, w h i c h i n t e r r u p t e d b l o o d p r e s s u r e r e -c o r d i n g . The duck was l e f t u n d i s t u r b e d b e h i n d a s c r e e n f o r 15 51 m i n u t e s b e f o r e an e x p e r i m e n t s t a r t e d . Ducks were d i v e d by l o w e r i n g t h e i r head g e n t l y i n t o a beaker o f water (16-20°C). Samples o f a r t e r i a l b l o o d were t a k e n a n a e r o b i c a l l y i n 1 ml c h i l l e d h e p a r i n i z e d p l a s t i c s y r i n g e s , and i m m e d i a t e l y put i n i c e . Plasma was s e p a r a t e d w i t h i n 5 minutes a f t e r b l o o d sam-p l i n g , and was s t o r e d at -80°C. 4-MEASUREMENT OF PHYSIOLOGICAL VARIABLES: Free c a t e c h o l a m i n e s i n t h e plasma were measured by HPLC, f o l l o w i n g the tech n i q u e d e s c r i b e d by Mangalam e t a l (1987). I n a 1.5 ml p o l y p r o p y l e n e v i a l , 0.5 ml plasma, 0.5 ml i c e d 3.5 M T r i s b u f f e r (pH 8.6), 5 0 u l o f d i h y d r o x y b e n z y l a m i n e (DHBA, a s y n t h e t i c c a t e c h o l a m i n e used as an i n t e r n a l s t a n d a r d , from Sigma), and 14 mg o f ch r o m a t o g r a p h i c grade a c t i v a t e d a l u m i n a (BDH) were combined. C a t e c h o l a m i n e s t a n d a r d s were p r e p a r e d u s i n g 0.1 M N a 2 P 0 4 b u f f e r (pH 7.0) (0.5 m l ) , t h e s o l u t i o n s t a n d a r d o f n o r e p i n e p h r i n e (NE) and e p i n e p h r i n e (EP) (50 u l ) , t h e t r i s b u f f e r (0.5 m l ) , DHBA (50 ug) and a l s o a l u m i n a (14 mg). Standards and plasma were p r o c e s s e d t o g e t h e r and kept on i c e when not b e i n g m a n i p u l a t e d . The v i a l s were shaken f o r 5 minutes and a f t e r t h e alumina s e t t l e d , t h e supernatant plasma was a s p i r a t e d . About 1 ml o f d e i o n i z e d water was added, t h e v i a l s were shaken f o r 2 minutes, and the supernatant a s p i r a t e d a g a i n ; t h i s was re p e a t e d . A f t e r t h e f i n a l s h a k i n g , t h e super-n a t a n t was a s p i r a t e d , u n t i l near d r y n e s s o f t h e a l u m i n a . 50 u l of 0.1 M HCIO^ w e r e added t o the v i a l s t o e l u t e the c a t e c h -o l a m i n e s f r o m t h e a l u m i n a . The v i a l s were v o r t e x e d , l e f t s t a n d i n g f o r 5 m i n u t e s on i c e , t h e n v o r t e x e d a g a i n . B r i e f c e n t r i f u g a t i o n c o n c e n t r a t e d t h e a l u m i n a and 20 u l o f t h e 52 supernatant was i n j e c t e d i n t o the HPLC. A Spectra P h y s i c s SP 8700 HPLC c o n t r o l l e r and pump were used t o p r o v i d e an i s o c r a t i c f low of 2.0 ml/min th r o u g h a Beckman 10 x 0.46 cm column of lOum ODS U l t r a s p h e r e r e v e r s e phase packing. The mobile phase c o n s i s t e d of 50mM c i t r a t e , 100 mM sodium a c e t a t e , 40 mM a c e t i c a c i d , and about 1 uM sodium heptane s u l f o n i c a c i d added as an i o n p a i r i n g agent t o i n -crease separation. The d e t e c t i o n system used was a B i o a n a l y t i -c a l Systems BAS LC-4A e l e c t r o c h e m i c a l d e t e c t o r , u s i n g a BAS P l e x i g l a s TL-3 e l e c t r o d e , with +0.67V to +0.7V a p p l i e d across the s i l i c o n e grease/graphite a c t i v e surface. The catecholamine peaks were measured and concentrations were c a l c u l a t e d by r e l a t i n g the r a t i o s of catecholamine peaks to DHBA peak i n the standard to the r a t i o s i n the plasma samples. The l i m i t of s e n s i t i v i t y ( s i g n a l : n o i s e >30) of the system was about 1 femtomole f o r NE and EP. There was a l i n e a r r a t i o response over the range of .5 nM to 500 nM. The c o e f f i c i e n t of recovery was between 63 and 75% .Plasma l e v e l s of dopamine do not change s i g n i f i c a n t l y d u r i n g t h e d i v e s (Mangalam et aJL 1987), so only the values f o r NE and EP are reported. A f t e r the p o s t - o p e r a t i v e recovery p e r i o d plasma Na + and K + c o n c e n t r a t i o n s were ana l y s e d i n ADX ducks and t h e i r sham operated c o n t r o l s , u s i n g a flame photometer (Instrumentation L a b o r a t o r y I n c o r p o r a t e d , B o s t o n M a s s . ) . Plasma C l ~ was measured by i s o m e t r i c t i t r a t i o n w i t h a B u c k l e r d i g i t a l c h l o r i d o m e t e r (4-2500, F o r t Lee, New J e r s e y ) , and plasma glu c o s e u s i n g an enzymatic assay k i t (# 16-UV Sigma Chemical co., St L o u i s , MO). A r t e r i a l b l o o d pH (pHa) was determined 53 u s i n g an I n s t r u m e n t a t i o n L a b o r a t o r y 813 PH/ B l o o d g a s a n a l y z e r . ECG and a r t e r i a l b l o o d p r e s s u r e s i g n a l s were d i s p l a y e d on a c h a r t r e c o r d e r (Physiograph 6, E&M Instrument Co INC Houston Texas) . 5-ANALYSIS OF DATA: C o m p a r i s o n s were made between i n t a c t d u c k s and sham-o p e r a t e d c o n t r o l s t o e s t i m a t e t h e e f f e c t s o f s u r g i c a l and p o s t - s u r g i c a l trauma. Comparison between SH-adx and ADX was made t o e s t i m a t e t h e e f f e c t s of t h e adrenalectomy p e r se, and comparison between SH-dnx and DNX t o e s t a b l i s h t h e e f f e c t s o f t h e a d r e n a l d e n e r v a t i o n p e r se. A p a i r e d t - t e s t was used t o t e s t t h e d i f f e r e n c e between p r e - d i v e and 2 minute d i v e b l o o d p r e s s u r e s . F o r comparisons between more t h a n two v a l u e s , an ANOVA, and Newman-Keuls t e s t were used. A s i g n i f i c a n t d i f f e r -ence between two v a l u e s was assumed i f p<0.05. I n t h e t e x t and graphs v a l u e s a re r e p r e s e n t e d by t h e i r means + s t a n d a r d e r r o r o f the mean. 54 R E S U L T S . The main differences in pre-dive levels of plasma catech-olamines among the 5 groups of ducks were the absence of plasma Epinephrine (EP) in ADX (Figure 6,A), and s i g n i f i c a n t l y lower plasma l e v e l s of Norepinephrine (NE) and Epinephrine (EP) i n DNX (Figure 7,A). A s i g n i f i c a n t increase i n plasma catecholamine l e v e l s was observed after 3 minutes diving i n a l l the 5 groups (Figures 6,B & 7,B) except for plasma EP i n DNX ducks. Furthermore, no EP was detectable i n the plasma of ADX ducks a f t e r 3 minutes submergence as was the case pre-dive. During the dive, levels of catecholamines in ADX and DNX were s i g n i f i c a n t l y lower than those found i n t h e i r sham-operated controls. After 3 minutes diving, the l e v e l s of NE and EP i n SH-adx were s i g n i f i c a n t l y higher than in the intact ducks, but no differences were observed between SH-dnx and i n t a c t s . No s i g n i f i c a n t differences i n the diving levels of NE and EP were observed between ADX and DNX. No difference i n diving levels of plasma NE was observed between SH-adx and SH-dnx, but diving levels of plasma EP i n SH-adx were higher than in SH-dnx. The heart rate dropped s i g n i f i c a n t l y during the dive i n a l l 5 groups (Figure 8 ) . There was no s i g n i f i c a n t difference in pre-dive or dive heart rate between intact, SH-adx, SH-dnx and DNX. However pre-dive and dive heart rate of ADX were s i g n i f i c a n t l y higher than those of intact, SH-adx, SH-dnx and DNX (Figure 8) . 55 FIGURE 6: Plasma l e v e l s of n o r e p i n e p h r i n e (NE) and e p i n e p h r i n e (EP) i n i n t a c t (open, n=5), sham-operated ( f i l l e d , n=5), and a d r e n a l e c t o m i s e d ( c r o s s - h a t c h e d , n=6) ducks b e f o r e (A) and a f t e r 3 minutes o f f o r c e d d i v i n g (B). S i g n i f i c a n t l y d i f f e r e n t from i n t a c t ducks: o, from SH-adx: •, from ADX: *, from p r e - d i v e v a l u e : +. 56 PLASMA CATECHOLAMINES (nanomoles per liter) m ro -+-cn 73 m I o < m oo > m m CD 57 FIGURE 2 : Plasma l e v e l s of norepinephrine (NE) and epine-phrine (EP) i n i n t a c t (open, n=5), sham-operated ( f i l l e d , n=7), and adrenal denervated (cross-hatched, n=8) ducks before (A) and after 3 minutes of forced diving (B). Sig n i f i c a n t l y different from intact ducks: o, from SH-dnx: •, from DNX: *, from pre-dive value: +. 58 PLASMA CATECHOLAMINES (nanomoles per liter) o K> -F- CD CO 59 FIGURE 8.: Heart rate during forced dives. A- Intact (open c i r c l e , n=7), sham-adrenalectomised (open triangle, n=6), adrenalectomised (open square, n=7). B- Intact (open c i r c l e , n=7), sham-adrenal denervated ( f i l l e d triangle, n=7), adrenal denervated ( f i l l e d square, n=8). S i g n i f i c a n t l y d i f f e r e n t from the other groups: *, from pre-dive value: +. 60 In a l l 5 groups t h e mean a r t e r i a l b l o o d p r e s s u r e dropped s i g n i f i c a n t l y a f t e r 2 minutes o f d i v i n g . No s i g n i f i c a n t d i f -f e r e n c e s i n t h e p r e - d i v e o r d i v e v a l u e s were o b s e r v e d among the 5 groups (F i g u r e 9 ) . There was no d i f f e r e n c e between ADX and SH-adx i n plasma l e v e l s o f N a + (138 + 1.4; 137 + 0 . 9 meq/L r e s p e c t i v e l y ) , K + (3.7 + 0.2; 3.1 + 0.2 meq/L r e s p e c t i v e l y ) , g l u c o s e (155 + 18; 197 + 10 mg/lOOml r e s p e c t i v e l y ) o r i n a r t e r i a l pH (7.38 + 0.02; 7.42 + 0.02 r e s p e c t i v e l y ) . However, t h e plasma l e v e l o f C l ~ i n ADX (105 +0.9 meq/L) was s i g n i f i c a n t l y h i g h e r than i n SH-adx (102 +0.9 meq/L). 62 FIGURE _9: Mean a r t e r i a l b l o o d p r e s s u r e b e f o r e and a f t e r 2 minutes f o r c e d d i v i n g . A- I n t a c t (open, n=5), Sham-operated ( f i l l e d , n=6), and adre-n a l e c t o m i s e d ( c r o s s - h a t c h e d , n=7) d u c k s b e f o r e and a t 2 minutes f o r c e d d i v i n g . B- I n t a c t (open, n=5), Sham-operated ( f i l l e d , n=7), and adre-n a l d e n e r v a t e d ( c r o s s - h a t c h e d , n=8) ducks b e f o r e and a t 2 minutes f o r c e d d i v i n g . S i g n i f i c a n t l y d i f f e r e n t from p r e - d i v e v a l u e : +. 63 DISCUSSION. Plasma levels of catecholamines measured in adrenalecto-mised ducks (ADX) showed that during the dive 100% of EP o r i g i n a t e from the adrenal glands. The NE released during d i v i n g i n ADX on the other hand must have a r i s e n from the discharge of autonomic sympathetic nerves innervating the vasculature, which are strongly stimulated during the dive (Jones et a l . 1 9 7 9 ) . Adrenal denervation also s i g n i f i c a n t l y decreased plasma EP levels during dives by 98%. Since adrenal denervated ducks (DNX) did not release more NE than ADX ducks during the dive, and because there was no increase of EP i n the DNX ducks during dives, then adrenal catecholamine re-lease during dives must be mediated only by a neural mecha-nism. Non-neural mechanisms did not appear to play any role. Evaluating the proportion of NE released by the adrenal glands during diving i s more d i f f i c u l t . Comparisons between i n t a c t and sham-operated ducks allow an estimation of the effects of surgical and post surgical procedure per se on the adrenal release of catecholamines. Surgery did not appear to have damaged the adrenal glands because at rest and during diving plasma catecholamines i n sham-operated ducks were not below those of intact ducks. The higher catecholamines levels in sham-operated ducks, compared with intact ducks, may have been an e f f e c t of post s u r g i c a l s t r e s s which enhances the o v e r a l l release of c i r c u l a t i n g catecholamines by the adrenal glands during diving. Such an e f f e c t cannot occur in ADX and 65 DNX because either there i s no adrenal glands or there i s no way to cause adrenal catecholamine release. If this i s so, i t may not be accurate to compare catecholamine l e v e l s of ADX with those of SH-adx i n order to determine the proportion of catecholamines released by the adrenal glands during "diving" per se. However, i t i s surprising that an increase of plasma catecholamines caused by post s u r g i c a l stress was not also observed before the dive in these sham-operated ducks. Hudson and Jones (1982) recorded huge individual variations in plasma catecholamine levels among ducks during diving. This variation could contribute importantly, i f not explain, the heterogenei-ty of diving catecholamine levels in Intact, SH-adx and SH-dnx ducks. When compared with Intact and SH-adx the percentage drop of NE i n ADX was 42% and 70% r e s p e c t i v e l y . A s i m i l a r comparison between Intact, SH-dnx and DNX revealed values of 70% and 80% which are quite close. Thus, the percentage of NE release by the adrenal glands i s variable and seems to fluctu-ate between 42 and 80%. Plasma EP and NE i n DNX decreased by 98% and 7 0 - 8 0 % which was greater than the 90% and 50% reduction in EP and NE found by Mangalam et a l . (1987). This discrepancy could be due to a regrowth of adrenal nerve f i b e r s i n Mangalam's adrenal denervated ducks. This would trigger a small release of adre-nal catecholamines during forced diving. In order to prevent any regrowth in the present study, a 3 to 5 mm section of the adrenal nerves was removed. Plasma levels of NE and EP meas-ured at rest and aft e r 3 minutes submergence i n intact ducks were s i m i l a r to values found i n r e s t i n g and d i v i n g i n t a c t 66 ducks by o t h e r s ( S t u r k i e e t a l . , 1970; Huang e t a l . , 1974; Hudson and Jones 1982; W i l s o n and B u t l e r 1983a; Mangalam e t  a l . , 1987). A f t e r a d r e n a l e c t o m y EP was not d e t e c t e d i n t h e plasma at r e s t , which agrees w i t h r e s u l t s o f B u t l e r and Wilson (1985) and Wilson and B u t l e r (1983b,c). D u r i n g f o r c e d d i v e s , a l l groups o f ducks showed b r a d y -c a r d i a and h y p o t e n s i o n . C e r t a i n l y i t i s r e m a r k a b l e t h a t , d u r i n g d i v e s , such l a r g e d i f f e r e n c e s i n plasma c a t e c h o l a m i n e s between DNX and SH-dnx were not r e f l e c t e d by any marked d i f -f e r e n c e s i n t h e i r c a r d i o v a s c u l a r p e r f o r m a n c e . F u r t h e r m o r e , d i v i n g h e a r t r a t e of ADX was s i g n i f i c a n t l y h i g h e r than t h a t o f DNX a l t h o u g h t h e i r d i v i n g l e v e l s o f c a t e c h o l a m i n e s were s i m i -l a r . Consequently, autonomic nerves p l a y t h e main r o l e i n the e s t a b l i s h m e n t and maintenance o f the c a r d i o v a s c u l a r responses t o d i v i n g ( K o b i n g e r and Oda 1968; B u t l e r and J o n e s 1971; W i l s o n and West 1985). A d r e n a l c a t e c h o l a m i n e s do not seem t o p a r t i c i p a t e i n c a r d i o v a s c u l a r adjustments, at l e a s t not d u r i n g s h o r t d i v e s . However, P e k i n ducks can be f o r c e d d i v e d f o r 20 minutes w i t h o u t any p h y s i o l o g i c a l damage (Jones and F u r i l l a 1987) and a p o s s i b l e r o l e o f a d r e n a l catecholamines on maximal underwater t o l e r a n c e has yet t o be i n v e s t i g a t e d . The d i f f e r e n c e i n c a r d i o v a s c u l a r adjustments between ADX and t h e 4 o t h e r groups o f ducks d u r i n g d i v i n g c o u l d be due t o t h e l a c k o f endogenous c o r t i c o s t e r o i d . Heart r a t e i n r e s t i n g ADX was t w i c e t h a t o f SH-adx. The e f f e c t s o f c o r t i c o s t e r o i d s on the c a r d i o v a s c u l a r system are not f u l l y u n d e r s t o o d (Fowler and Chou, 1960; L e f e r e t a l . , 1968; Sevy e t a l . , 1974; Rovet-67 t o , 1974; L e f e r , 1974). C a r d i o v a s c u l a r c o l l a p s e , one conse-quence o f adr e n a l e c t o m y , can be a r e s u l t o f h y p o t e n s i o n r e -s u l t i n g f r om d e c r e a s e d N a + r e a b s o r p t i o n i n t h e k i d n e y s , caused by a l a c k o f m i n e r a l o c o r t i c o i d s . However, t h e l e v e l o f N a + (and o t h e r plasma i o n s ) and t h e p r e - d i v e mean a r t e r i a l b l o o d p r e s s u r e i n ADX and SH-adx were s i m i l a r and i n the range of v a l u e s u s u a l l y r e c o r d e d i n ducks ( B u t l e r and W i l s o n 1985; R o b e r t s and Hughes, 1984; S h i m i z u and J o n e s , 1987) so t h i s d i f f e r e n c e i n h e a r t r a t e has t o i n v o l v e some o t h e r mechanism. The e f f e c t o f ad r e n a l e c t o m y on t h e c a r d i o v a s c u l a r system o f ducks has been s t u d i e d by B u t l e r and W i l s o n ( B u t l e r 1985; B u t l e r and W i l s o n 1985). They showed t h a t i n ADX t h e r e was a s i g n i f i c a n t i n c r e a s e i n h e a r t r a t e a s s o c i a t e d w i t h a decrease i n b l o o d p r e s s u r e and s t r o k e volume. They d i d not observe any a l t e r a t i o n i n p e r i p h e r a l r e s i s t a n c e or b l o o d and plasma v o l -umes. I n j e c t i o n o f be t a m e t h a s o n e p r e v e n t e d h y p o t e n s i o n i n t h e i r ADX, as d i d p r e d n i s o n e i n our s t u d y . However, t h e s e a u t h o r s d i d n o t g i v e any i n f o r m a t i o n about h e a r t r a t e and s t r o k e volume i n t h e i r ADX betamethasone i n j e c t e d ducks. An i n c r e a s e i n h e a r t r a t e , a s s o c i a t e d w i t h a normal mean a r t e r i a l b l o o d p r e s s u r e , may r e f l e c t an a d a p t a t i o n o f t h e c a r d i o v a s c u -l a r system t o a de c r e a s e i n s t r o k e volume (due t o a de c r e a s e o f m y o c a r d i a l performance ( L e f e r et: a l . , 1968; Sevy e t a l . , 1974; R o v e t t o 1974)) i n c h r o n i c a l l y a d r e n a l e c t o m i s e d ducks supplemented w i t h exogenous c o r t i c o s t e r o i d such as prednisone. I n o t h e r words, even though p r e d n i s o n e m a i n t a i n s t h e i o n i c b a l a n c e i t s t i l l may not be an adequate r e p l a c e m e n t t h e r a p y f o r c o r t i c o s t e r o n e and a l d o s t e r o n e , the endogenous c o r t i c o s t -e r o i d s . 68 CHAPTER 2: INFLUENCE OF ADRENAL CATECHOLAMINES ON MAXIMUM DIVE TIME. 69 I N T R O D U C T I O N . The previous chapter showed that c i r c u l a t i n g catechola-mines do not seem to have any s i g n i f i c a n t role during diving. However, Pekin ducks were forced dived for 3 minutes only. Ducks such as these (2 to 3.5 kg) could e a s i l y survive 10 to 14 minutes of submergence (Hudson and Jones 1986). Three minutes may have been too short a dive to register differences between ducks whose release of c i r c u l a t i n g catecholamines had been prevented, and their controls. The role of ci r c u l a t i n g catecholamines in determining the maximal tolerance to underwater submergence i s investigated in t h i s chapter. It was not possible to use adrenergic blockade for t h i s study, because t h i s would not allow discrimination between the roles of the sympathetic nerves and adrenal ca-techolamines. Preventing catecholamine release from the adre-nal glands by putting blood flow occluders around the adrenal veins or arteries was abandoned because the adrenal glands are c l o s e l y applied to the. aorta and vena cava and there i s no place to f i t an occluder around adrenal a r t e r i e s and veins. 70 Moreover, because c o r t i c a l and medullary tissues are intermin-gled i n birds, adrenal demedullation cannot be done. Conse-quently maximum dive times (MDT) were compared between ducks whose adrenal catecholamine release had been prevented (by adrenalectomy "ADX" or by denervation of adrenal glands "DNX") and t h e i r sham-operated controls (SH-adx, SH-dnx). These MDT values were also compared with the MDT of ADX and DNX perfused with synthetic catecholamines. Heart rate, blood pressure, blood gas l e v e l s and concentrations of lactate, glucose and ions were also measured. 71 MATERIAL AND METHODS. 1- ANIMALS: The experiments were done on 39, two to three month old male Pekin ducks (Anas platyrhynchos) ranging in mass from 2 to 3.5 Kgs. They were kept i n conditions s i m i l a r to those described i n chapter I. Eight ducks were adrenalectomised (ADX) and 6 were sham-operated (SH-adx) following the proce-dure used for adrenalectomy. In 9 ducks, adrenal glands were denervated (DNX) while i n 9 others, sham-operation was per-formed following the procedure for adrenal denervation (SH-dnx) . Seven ducks were intact. 2- OPERATIVE AND POST-OPERATIVE PROCEDURES: The s u r g i c a l techniques and post-operative procedures used for adrenalectomy and adrenal denervation are described in chapter I. 3- MINOR SURGERY AND EXPERIMENTAL PROCEDURE: Cannulation of the brachial artery and vein was done with P.E. 90 (Intramedic Polyethylene tubing, Clay Adams) tubing, under local anaesthesia (Lidocaine hydrochloride; Xylocaine 2% Astra). The birds were given one day to recover. The position of the cannulae was checked at post mortem: the t i p of the a r t e r i a l cannula was positioned at the junction of the brachi-72 o c e p h a l i c a r t e r y and a o r t a ; the t i p o f t h e venous cannula was p o s i t i o n e d i n the vena cava. The b i r d was p l a c e d i n a s i t t i n g p o s i t i o n and s e c u r e d w i t h t a p e t o an o p e r a t i n g t a b l e as d e s c r i b e d i n c h a p t e r I . Only one d i v e was p e r f o r m e d each day, and a t l e a s t 48 hours e l a p s e d between d i v e s . Maximum underwater t o l e r a n c e , w i t h o u t any r i s k o f perma-nent p h y s i o l o g i c a l damage or d i s t r e s s t o t h e a n i m a l , i s i n d i -c a t e d by a f l a t t e n i n g o f t h e EEG and a sudden r i s e i n h e a r t r a t e t o w a r d s p r e - d i v e l e v e l s ( F i g u r e 10; Hudson & J o n e s 1986). I n t h e p r e s e n t e x p e r i m e n t s , t h i s v e r y c h a r a c t e r i s t i c i n c r e a s e o f h e a r t r a t e was t a k e n as an i n d i c a t i o n t h a t t h e maximum d i v e time had been reached. The v e n o u s c a n n u l a was c o n n e c t e d t o a H a r v a r d i n f u s i o n / w i t h d r a w a l pump (Model 901, M i l l i s , Mass.) f o r con-t i n u o u s i n f u s i o n of catecholamines (Norepinephrine b i t a r t r a t e : L e v o p h e d : W i n t h r o p , A u r o r a , O n t a r i o , Canada; E p i n e p h r i n e c h l o r i d e : P a r k - D a v i s , Scarborough, O n t a r i o , Canada) or s a l i n e i n t o ADX and DNX ducks d u r i n g f o r c e d d i v e s , b e g i n n i n g as soon as t h e d u c k ' s head was u n d e r w a t e r . D u r i n g t h e d i v e , t h e p e r f u s i o n r a t e was s e t t o generate the plasma l e v e l o f c a t e c h -o l a m i n e s measured i n d i v i n g P e k i n ducks by Hudson and Jones (1982). I n t h i s s e t o f e x p e r i m e n t s , f o r each duck f i v e d i v e s were c a r r i e d out, and i n the f i r s t and f i f t h d i v e , the p e r f u -s i o n was done w i t h the c a r r i e r ( s a l i n e ) o n l y . Seven DNX ducks and two ADX ducks were p e r f u s e d w i t h catecholamines. 73 FIGURE 10: Sample chart recording from an experiment to deter-mine maximum endurance to forced submersion i n Pekin ducks (from Hudson and Jones 198 6). Symbols are: HR, heart rate; ABP, a r t e r i a l blood pressure; EEK, electroencephalogram. The s o l i d diamond indicates the beginning of head submersion; the hollow diamond dive termina-tion. 74 DIVE TIME (minutes) 4-MEASUREMENT AND ANALYSIS OF THE PHYSIOLOGICAL VARIABLES: ECG and a r t e r i a l blood pressure signals were amplified and displayed on a chart recorder (Physiograph 6, E&M Instru-ment Co, Houston Texas). A r t e r i a l blood samples (on average: 1.5 ml) were taken anaerobically before the dive, at 3 and 5 minutes in the dive and at the end of the dive. 0.3 ml was used to measure Pa02, PaC02 and pHa using an Instrumentation L a b o r a t o r i e s 813 pH/Blood gas analyser (Lexington MA). 0.1 ml was used to determine Ca02 following the method developed by Tucker (1967) using a radiometer gas analyser (Radiometer PHM71 MK2, Copen-hagen, Denmark). The rest of the blood was centrifuged and the plasma was c o l l e c t e d and used to determine lactate and glu-cose. Lactate and glucose levels were measured using enzymat-i c assay k i t s (# 826-UV; # 16-UV, Sigma, St Louis, MO). Other a r t e r i a l blood samples (1 ml) were taken before the dive and at 5 minutes into the dive to determine plasma levels of Na +, K + and C l ~ . Plasma levels of free catecholamines were meas-ured in 1 ml of blood taken anaerobically before the dive and at 3, 5 and 10 minutes i n the dive ( i f the dive lasted long enough) and at the end of the dive. Plasma Na + and K + were analysed using a flame photometer (Instrumentation Laboratory Incorporated Boston Mass.). Plasma C l ~ was measured by isomet-r i c t i t r a t i o n with a Buckler d i g i t a l chloridometer (4-2500, Fort Lee, New Jersey). Plasma free catecholamines were meas-ured by HPLC following the technique described i n chapter I. Plasma levels of dopamine do not change s i g n i f i c a n t l y during dives (Mangalam et a l . 1987), so only the values for norepine-76 phrine (NE) and epinephrine (EP) are reported. Fourteen ml of a r t e r i a l b l o o d were taken from 3 i n t a c t ducks at r e s t . Two a l i q u o t s of 7 ml were e q u i l i b r a t e d simulta-neously at 40°C wi t h a hypoxic and h y p e r c a p n i c gas mixture (P02=54 mm Hg; PC02=64 mmHg) i n a two chamber tonometer. A f t e r 30 minutes, P a 0 2 , PaC0 2, pHa and C a 0 2 were measured. One microgram of both NE and EP were added t o one chamber, and an e q u i v a l e n t volume of s a l i n e (100 ul) was added t o the other. Pa0 2, PaC0 2, pHa and Ca0 2 were then measured a f t e r 30 seconds, 2 and 5 minutes. 5-ANALYSIS OF DATA: Comparisons were made between i n t a c t ducks and sham-operated c o n t r o l s to estimate the e f f e c t s of s u r g i c a l trauma. Comparison between SH-adx and ADX was made t o e s t i m a t e the e f f e c t s of the adrenalectomy per se, and comparison between SH-dnx and DNX to e s t a b l i s h the e f f e c t s of the adrenal dener-v a t i o n per se. S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were based on r e s u l t s of Student's t t e s t , or a n a l y s i s of v a r i a n c e f o l l o w e d by the Newman-Keuls t e s t . The c r i t i c a l l i m i t f o r s i g n i f i c a n t d i f f e r e n c e was s e t a t a p r o b a b i l i t y o f 5% (p<0.05). In the t e x t and graphs, values are given as mean +_ standard e r r o r of the mean (SE). N i s the number of ducks, and n i s the number of dives i n a given group. In a l l the d i v e s , h e a r t r a t e and mean a r t e r i a l b l o o d pressure were recorded f o r every minute u n t i l 1 minute before the end of the dive. Because of d i f f e r e n c e s i n MDT among ducks of the same group, fewer v a l u e s were recorded f o r the longer dives. In other words, because the length of the dive for each animal i s different, the sampling time for n decreases. Conse-quently, the mean and SE for values recorded during the dive are only included as data for a n a l y s i s when the number of dives remaining i s more than, or equal to, half of the o r i g i -nal number of dives. However, mean + SE of heart rate and blood pressure were calculated for a l l ducks at the end of a l l dives, and one minute before the end of these dives. These values are also reported graphically. S t a t i s t i c a l l y s i g n i f i -cant differences among values at the end of the dive and at one minute before the end of the dive were also based on results of Student's t test, or analysis of variance followed by the Newman-Keuls test, and the c r i t i c a l l i m i t for s i g n i f i -cant difference was set at a probability of 5% . 78 RESULTS. 1-EFFECTS OF ADRENALECTOMY AND ADRENAL DENERVATION ON MAXIMUM  DIVE TIME (MDT): Maximum dive time (MDT) of adrenalectomised ducks (ADX: 5 min 19 + 20 sec) was s i g n i f i c a n t l y below that of their sham-operated controls (SH-adx: 9 min 58 + 45 sec). The MDT of SH-adx was also s i g n i f i c a n t l y lower than i n the i n t a c t ducks (Intact: 12 min 14 + 53 sec), showing that surgery affected underwater endurance (Figure 11). In contrast, MDT of SH-dnx was not s i g n i f i c a n t l y d i f f e r e n t from MDT of i n t a c t ducks (Figure 12). MDT of adrenal denervated ducks (DNX: 7 min 10 + 13 sec) was s i g n i f i c a n t l y reduced compared with sham-operated controls (SH-dnx ducks: 12 min 10 + 28 sec) (Figure 12). MDT of DNX ducks perfused with saline (7 min 21 + 17 sec) was not s i g n i f i c a n t l y different from that of non-perfused DNX (Figure 12). In DNX ducks perfused with catecholamines, MDT increased s i g n i f i c a n t l y (9 min 46 + 20 sec, Figure 12). Howev-er, MDT was s t i l l s i g n i f i c a n t l y below the values observed i n SH-dnx ducks. Two ADX ducks perfused with catecholamines showed a similar increase in MDT (Figure 11). The levels of catecholamines generated during these dives are given in table 3. Perfusion of catecholamines i n DNX s i g n i f i c a n t l y increased diving l e v e l s of plasma catecholamines almost to the values found in their sham-operated controls which had levels similar to those previously recorded during forced dives by Hudson and Jones (1982) (Table 3). FIGURE 11: Maximal dive time i n i n t a c t , sham-operated and adrenalectomised ducks with and without infusion of catechola-mines . (I: i n t a c t ducks, N=7, n=14; SH-adx: sham-operated ducks, N=6, n=12; ADX: adrenalectomised ducks, . N=8, n=16; ADX+sal.: ad r e n a l e c t o m i s e d ducks i n f u s e d with s a l i n e , N=2, n=2; ADX+cat.: adrenalectomised ducks infused with catecholamines, N=2, n=2.) I, S, A, s i g n i f i c a n t l y different from I, SH-ADX, and ADX ducks respectively. 80 MAXIMUM DIVE TIME ( m i n u t e s ) w , > Q + O — X FIGURE 12: Maximal dive time i n the intact, sham-operated and adrenal denervated ducks with and without infusion of catecho-lamines . (I: i n t a c t ducks, N=7, n=14; SH-dnx: sham-operated ducks, N=9, n=18; DNX: ad r e n a l denervated ducks, N=9, n = 18; DNX+sal.: adrenal denervated ducks infused with saline, N=7, n=14; DNX+cat.: adrenal denervated ducks infused with catech-olamines, N=7, n=21.) I, S, D, Ds, Dc, s i g n i f i c a n t l y different from I, SH-dnx, DNX, DNX+sal., and DNX+cat. ducks respectively. 82 MAXIMUM DIVE TIME (minutes TABLE 3: Levels of plasma catecholamines during dives in adre nal denervated ducks infused with saline (DNX+sal.), adrenal denervated ducks infused with catecholamines (DNX+cat.), and their sham-operated controls which were unperfused (SH-dnx). 0, *, •, s i g n i f i c a n t l y d i f f e r e n t from SH-dnx, DNX+sal. and DNX+cat. respectively. 84 NOREPINEPHRINE Cnano»oles per l i t e r ) PRE DIVE 3min. 5»in. lCVnin. END DIVE N 7 7 3 3 4 RVG 5. 1 31B 1131 5049 5158 SE 0.8 78 579 1309 619 MAX B.O 651 2288 6960 7012 MIN 2.9 135 531 2545 4451 **m m K N 6 6 6 6 DNX+sal. RVG 2.0 63 246 192 SE 0.4 11 35 24 MRX 3.4 114 361 274 MIN 1.0 39 141 116 o o om o» N 6 5 6 5 DNX+cat. RVG 2.9 224 1363 1755 SE 0.4 41 159 448 MAX 4.4 355 1776 3272 MIN 1.6 134 827 467 O K Ko EPINEPHRINE (nanonoles per liter> PRE DIVE 3min. 5m in. 10min. END DIVE N 7 SH-dnx RVG 1.3 SE 0.3 MRX 2.3 MIN 0 7 3 71 447 15 317 118 1073 19 51 3 4 2312 2355 713 260 3396 3088 968 1866 N 6 6 6 6 DNX+sal. RVG D.9 2.1 8.7 8.2 SE 0.5 0.8 2.7 1.8 MAX 2.6 5.4 17.1 12.9 MIN O 0.5 1.2 1.9 om om m om N 6 5 6 5 DNX+cat. RVG 1.8 121 990 1344 SE 0.7 24 133 403 MHX 4.8 170 1389 2766 MIN O 58 553 268 MO **o **o KO 85 2-EFFECTS OF ADRENALECTOMY AND ADRENAL DENERVATION ON HEART  RATE (HR) AND BLOOD PRESSURE (BP): Mean pre-dive heart rate of ADX ducks was s i g n i f i c a n t l y above that of SH-adx ducks. However, pre-dive mean a r t e r i a l blood pressure was not s i g n i f i c a n t l y d i f f e r e n t . During the dive, ADX ducks had s i g n i f i c a n t l y higher heart rates than SH-adx ducks, yet blood pressure i n the ADX ducks f e l l s i g n i f i -cantly lower (Figure 13). Diving heart rates in ADX and SH-adx were s i g n i f i c a n t l y d i f f e r e n t from t h e i r respective pre-dive heart rates. Blood pressure during diving in ADX was s i g n i f i -cantly d i f f e r e n t from i t s pre-dive value, which was not the case for SH-adx. Mean pre-dive heart rate and blood pressure of DNX and SH-dnx ducks were not s i g n i f i c a n t l y d i f f e r e n t . During the dive, however, heart rates i n DNX ducks were s i g n i f i c a n t l y higher than in SH-dnx ducks. The drop i n mean blood pressure o c c u r r i n g during dives was greater i n DNX ducks, and was s i g n i f i c a n t l y below that of SH-dnx (Figure 14). Diving heart rates i n DNX and SH-dnx were s i g n i f i c a n t l y d i f f e r e n t from t h e i r pre-dive values. Diving blood pressure in DNX was s i g -n i f i c a n t l y lower than the pre-dives value. From 2 to 7 minutes into the dive, blood pressure in SH-dnx was also si g n i f i c a n t l y lower than i t s pre-dive value. There were no d i f f e r e n c e s i n pre-dive heart rate and blood pressure among SH-dnx ducks, DNX ducks which were to be perfused with saline and DNX ducks to be perfused with catech-olamines (Figure 15) . Diving heart rates i n a l l those ducks 86 FIGURE 13: Heart r a t e and mean a r t e r i a l b l o o d p r e s s u r e d u r i n g d i v i n g i n a d r e n a l e c t o m i s e d d u c k s (ADX, N=8, n=8, h o l l o w squares) and t h e i r sham-operated c o n t r o l s (SH-adx, N=6, n=6, h o l l o w t r i a n g l e s ) . *, s i g n i f i c a n t l y d i f f e r e n t from SH-adx. 87 MEAN ARTERIAL BLOOD P R E S S U R E (mm Hg) HEART RATE (beats per minute) 01 o o o ai o o o o o b=LT-H>H * I—OH I—> > O X Q CO O H FIGURE 14: Heart rate and mean a r t e r i a l blood pressure during diving i n adrenal denervated ducks (DNX, N=9, n=18, f i l l e d squares) and th e i r sham-operated controls (SH-dnx, N=9, n=18, f i l l e d triangles) . *, s i g n i f i c a n t l y different from SH-dnx. 89 MEAN ARTERIAL BLOOD P R E S S U R E (mm Hg) HEART RATE (beats per minute) O FIGURE 15: Heart rate and mean a r t e r i a l blood pressure during d i v i n g i n adrenal denervated ducks i n f u s e d with s a l i n e (DNX+sal., N=7, n=14 f o r heart rate, N=5, n=10 f o r blood pressure, f i l l e d c i r c l e s ) , or catecholamines (DNX+cat., N=7, N=21 for heart rate, N=5, n=15 for blood pressure, hollow c i r c l e s ) , and i n sham-operated ducks (SH-dnx, N=9, n=18, f i l l e d t r i angles). *, s i g n i f i c a n t l y different from the other groups. 91 DIVE TIME (minutes) were s i g n i f i c a n t l y below t h e i r r e s p e c t i v e p r e - d i v e v a l u e s . T h e i r b l o o d p r e s s u r e s d u r i n g d i v i n g were a l s o s i g n i f i c a n t l y lower than t h e i r r e s p e c t i v e p r e - d i v e v a l u e s , except i n DNX p e r f u s e d with catecholamines i n which d i v i n g b l o o d p r e s s u r e s at 6 and 7 minutes d i v i n g and at one minute before the end of the dive were not s i g n i f i c a n t l y d i f f e r e n t from t h e i r pre-dive v a l u e . Heart r a t e of DNX ducks d u r i n g the d i v e remained s i g -n i f i c a n t l y higher than i n the SH-DNX ducks d e s p i t e p e r f u s i o n with catecholamines (Figure 15). Blood pressure of DNX ducks perfused with catecholamines was s i g n i f i c a n t l y higher than i n DNX ducks p e r f u s e d w i t h s a l i n e a f t e r 2 minutes and d i d not d i f f e r from the values of SH-dnx ducks (Figure 15). Towards the end of a d i v e , a l l ducks showed an i n c r e a s e i n h e a r t r a t e ( F i g u r e 13, 14, 15). Sham-operated c o n t r o l ducks, a l s o showed a r i s e i n b l o o d p r e s s u r e toward the p r e -d i v e v a l u e s . However at the end of d i v e s , b l o o d p r e s s u r e i n ADX, DNX, and DNX perfused with s a l i n e remained s i g n i f i c a n t l y below t h e i r p r e - d i v e v a l u e s ( F i g u r e 13, 14, 15), and b l o o d p r e s s u r e of DNX p e r f u s e d with catecholamines rose toward the pre-dive values and then f e l l s i g n i f i c a n t l y below the pre-dive value (Figure 15). 3-EFFECTS OF ADRENALECTOMY AND ADRENAL DENERVATION ON BLOOD  GAS AND pH: P r e - d i v e Pa0 2 and Ca0 2 were not s i g n i f i c a n t l y d i f f e r e n t i n DNX and SH-dnx ducks. During the d i v e Pa0 2 dropped f a s t e r i n DNX than i n SH-dnx ducks, and the mean values were s i g n i f -93 i c a n t l y d i f f e r e n t a f t e r b o t h 3 minutes and 5 minutes submer-gence. P a 0 2 r e c o r d e d i n DNX ducks and i n SH-dnx ducks a t t h e end of d i v e s were not s i g n i f i c a n t l y d i f f e r e n t (Figure 16). The f a l l i n Ca0 2 i n DNX and SH-dnx ducks f o l l o w e d the same p a t t e r n as P a 0 2 ( F i g u r e 16). I n ADX a l s o , P a 0 2 dropped f a s t e r than i n SH-adx (Table 4A). Moreover, P a 0 2 r e c o r d e d b e f o r e and a t t h e end o f t h e d i v e s were s i g n i f i c a n t l y h i g h e r i n ADX t h a n i n t h e i r sham-operated c o n t r o l s . P l o t t i n g p r e - d i v e and d i v e v a l u e s o f a r t e r i a l 0 2 content a g a i n s t t h e i r a s s o c i a t e d P 0 2 ( F i g u r e 17), shows t h a t i n DNX, the 0 2 a f f i n i t y o f the b l o o d d u r i n g the d i v e was not d i f f e r e n t from b l o o d 0 2 a f f i n i t y i n SH-dnx i n which c i r c u l a t i n g catecho-l a m i n e s r o s e s p e c t a c u l a r l y . There was no d i f f e r e n c e i n P a 0 2 , C a 0 2 and pHa between DNX a f t e r 3 m i n u t e s d i v i n g and SH-dnx a f t e r 5 minutes d i v i n g , however PaC0 2 was s i g n i f i c a n t l y h i g h e r i n SH-dnx. At t h e end o f d i v e s , no d i f f e r e n c e i n P a 0 2 , C a 0 2 and pHa between DNX and SH-dnx were o b s e r v e d , but PaC0 2 was s i g n i f i c a n t l y h i g h e r i n SH-dnx. R e s t i n g P a 0 2 , PaC0 2 pHa, Ca0 2 i n t h e 3 ducks used i n the tonometry s t u d y were 7 4 + 1 mmHg, 3 2 + 1 mmHg, 7.51 + 0.01, and 13.3 + 0.9 ml p e r 100 ml o f b l o o d r e s p e c t i v e l y . A f t e r e q u i l i b r a t i o n by tonometry, no s i g n i f i c a n t d i f f e r e n c e s were observed b e f o r e (Pa0 2 = 5 4 + 2 mmHg, PaC0 2 = 6 4 + 3 mmHg, pHa = 7.30 + 0.01, C a 0 2 = 62 + 7%) o r a f t e r t h e a d d i t i o n o f s a l i n e (Pa0 2 = 5 4 + 3 mmHg, PaC0 2 = 6 4 + 2 mmHg, pHa = 7.29 + 0.02, Ca0 2 a f t e r 30 sec, 2 min, 5 min = 63 + 6 %, 59 + 7 %, 61 + 6 % ) . Because t h e h e m a t o c r i t i n one o f th e ducks was lower (33%) t h a n i n t h e o t h e r ducks ( 4 5 % ) , C a 0 2 i s e x p r e s s e d as a 94 FIGURE 16; Pa0 2 and Ca0 2 i n adrenal denervated ducks (DNX, N=8, n=16, f i l l e d squares) and t h e i r sham-operated controls (SH-dnx, N=6, n=12, f i l l e d triangles) during diving. +, *, s i g n i f i c a n t l y different from pre-dive value and from SH-dnx value respectively. 95 DIVE TIME (minutes) TABLE A : Pre-dive and dive values of (A) P a 0 2 / Paco 2/ pHa, and (B) plasma levels of lactate and glucose i n adrenalectomised ducks (ADX) and their sham-operated controls (SH-adx). +/ *, s i g n i f i c a n t l y different from pre-dive value and from SH-adx value respectively. 97 H: BLOOD GRS RND PH PaD2 mmHg PaCQ2 mmHg pHa SH-adx ADX SH-adx ADX SH-adx ADX N=6, n=6 N=7, n=7 N=6, n=6 N=6, n=6 N=6, n=6 N=7, n=7 PRE-DIVE MEAN 101 108 x 30 29 7.421 7.377 SE 1 2 1 1 0.020 0.022 3 m i n u t e MEAN 58 + 53 + 47 + 41 + 7.251 + 7.206 + SE 3 3 3 3 0.031 0.025 DIVE 5 minute MEAN 51 + 40 52 + 52 + 7.191 + 7. 1Q3 +*f SE 4 2 2 3 0.029 0.025 END-DIVE MEAN 33 + 38 +** 65 + 54 +* 7.020 + 7.080 + SE 1 2 3 4 0.030 O.Q36 B: BLOOD GLUCOSE AND LACTATE PLASMA GLUCOSE PLASMA LACTATE m i l 1 i m o l e s p e r 1 i t e r mi 11 1moles p e r 1 i t e r SH-adx ADX SH-adx ADX N=6, n=6 N=6, n=6 N=6, n=6 N=6, r PRE-DIVE MEAN ID.4 8.4 2.4 1.8 SE 0.6 0.7 O.S 0.2 3 mi n u t e MEAN 9.4 8.7 4.5 2.6 * SE 0.7 l.O 0.6 0.4 DIVE 5 mi n u t e MEAN 9. 1 10.4 5.7 + 4.6 + SE 0.6 0.5 0.7 0.4 END-DIVE MEAN 9.7 12. O +* 8.7 + 5.6 + SE 0.6 0.4 1. 1 0.9 FIGURE 17: A r t e r i a l content of 02 as a function of Pa0 2 i n denervated ducks (DNX: N=8, n=16, f i l l e d squares) and i n sham denervated ducks (SH-dnx: N=6, n=12, f i l l e d triangles) . PaC02 (mmHg), and pHa for each of these points are written on the graph: on the right side for DNX, on the l e f t side for SH-dnx. 99 18 14 10 8 6 4 2 0 SH-dnx DNX 32±0.6 • 7.49±0.01 " 31±0.6 7.49±0.01 45±0.8 _ 7.34±0.01 A 49±1.1 ^ 45±1 „ 7.3010.01 ^ 7.31 ±0.01 i 51±1.3 i 7.23±0.02 - 66±2.2 r _ 57± 1.4 7.1±0.03 7.15±0.02 1 1 i i i i 0 20 40 60 80 100 120 ARTERIAL PARTIAL P R E S S U R E O F 02 ( m m Hg) 100 percentage o f t h e r e s t i n g v a l u e . No d i f f e r e n c e s were observed e i t h e r b e f o r e (Pa0 2 = 5 3 + 3 mmHg, PaC0 2 = 6 3 + 3 mmHg, pHa = 7.28 + 0.02, Ca0 2 = 60 + 7 %) or a f t e r t he a d d i t i o n o f c a t e c h -olamines (Pa0 2 = 5 3 + 3 mmHg, PaC0 2 = 6 5 + 2 mmHg, pHa = 7.28 + 0.02, Ca0 2 a f t e r 30 sec, 2 min, 5 min = 58 + 7 %, 59 + 8 %, 59 + 9 %) . No d i f f e r e n c e i n PaC0 2 between DNX and SH-dnx d u r i n g t h e d i v e was observed, except at t h e end when PaC0 2 i n DNX ducks was s i g n i f i c a n t l y l o wer t h a n i n t h e i r SH-dnx ( F i g u r e 1 8 ) . No d i f f e r e n c e i n pHa was observed except at f i v e minutes i n t o the d i v e when pHa i n DNX was s i g n i f i c a n t l y l o w e r t h a n i n SH-dnx ( F i g u r e 1 8 ) . S i m i l a r r e s u l t s were o b s e r v e d i n SH-adx and ADX (Table 4A) . 4-EFFECTS OF ADRENALECTOMY AND ADRENAL DENERVATION ON STRONG  IONS AND BLOOD GLUCOSE: I n DNX, p r e - d i v e v a l u e s o f plas m a l a c t a t e and g l u c o s e were not s i g n i f i c a n t l y d i f f e r e n t from t h e i r r e s p e c t i v e sham-operated c o n t r o l s (Table 5 ) , n e i t h e r were the plasma l e v e l s o f l a c t a t e and g l u c o s e i n ADX and SH-adx ( T a b l e 4 B ) . A f t e r 5 minutes d i v i n g i n DNX, plasma l e v e l s o f g l u c o s e were s i g n i f i -c a n t l y h i g h e r and plasma l e v e l s o f l a c t a t e s i g n i f i c a n t l y lower t h a n i n SH-dnx ( T a b l e 5 ) . T h i s i s s i m i l a r t o t h e t r e n d ob-serve d i n the ADX and SH-adx (Table 4B). However, d u r i n g d i v i n g , d i f f e r e n c e s i n p H a between ADX and SH-adx as w e l l as between DNX and SH-dnx d i d not always match t h e a s s o c i a t e d d i f f e r e n c e s i n PaC0 2 and plasma l a c t a t e 101 FIGURE 18: PaC0 2 and pHa i n adrenal denervated ducks (DNX, N=8, n=16, f i l l e d squares) and i n their sham-operated controls (SH-dnx, N=6, n=12, f i l l e d triangles) during diving. +, *, s i g n i f i c a n t l y different from pre-dive value and from SH-dnx value respectively. 102 CM o O b _ O Ld DT CO to LxJ or Q_ or < 0_ < or Ld h-or < CL or Ld \— or < 12 14 DIVE TIME (minutes) 103 TABLE 5_: Pre-dive and dive values of a r t e r i a l plasma levels of glucose, l a c t a t e - , Na +, K +, C l ~ and Strong Ions Difference (SID) in adrenal denervated ducks (DNX) and their sham-operat-ed controls (SH-dnx). +, *, s i g n i f i c a n t l y different from pre-dive value and from SH-dnx value. 104 BLOOD GLUCOSE AND STRONG IONS PLASMA GLUCOSE PLASMA LACTATE aillimoles per l i t e r otillimoles per l i t e r SH-dnx DNX SH-dnx DHX N=8, n=8 N=8, n~3 N=8, n=8 N=8, n=8 PRE-OIVE MEAN lO. 1 9.8 1.8 1.4 SE 0.3 Q-2 0.3 O.l DIVE 5 niriute MEAN 8.8 + 11.7 * 4-8 • 3. 1 +* SE 0-3 O.B Q.4 Q.3 PLASMA Na+ 11111mo1es per l i t e r SH-dnx DNX N=8, n=8 N=7, n=7 PLASMA K+ tilliiaoles per l i t e r SH-dnx DHX N=8, n=B N=7, n=7 PRE-OIVE MEAN SE 135 2 139 0.5 3.6 O. 1 3.5 O.l DIVE 5 minute MEAN SE 141 + 1 140 1 3.5 O. 1 4.6 +*« 0.4 PLASMA C l - PLASMA SIO m i l l mo 1 es per l i t e r milliiaoles per l i t e r SH-dnx DNX SH-dnx DNX N=8, n=8 N=7, n=7 N=8, n=8 N=7, n=7 PRE—DIVE MEAN 103 105 34.0 36.4 SE 1 1 1.4 0.5 DIVE 5 ainute MEAN 103 104 36.6 + 37.0 SE 1 1 1.1 0.7 levels (Table 4, Figure 18 and Table 5). For example, pH a of DNX after 5 minutes diving was s i g n i f i c a n t l y lower than that of SH-dnx even though i t s PaC02 was not different from that of SH-dnx, and plasma l a c t a t e was s i g n i f i c a n t l y lower. In an attempt to c l a r i f y this problem, plasma ions were measured i n SH-dnx and DNX before and at 5 minutes into the dive. In DNX, pre-dive values of plasma Na +, K,+, Cl~, were not signifi c a n t -l y d i f f e r e n t from t h e i r respective sham-operated controls (Table 5) . In SH-dnx, plasma levels of Na + and lactate after 5 minutes d i v i n g were s i g n i f i c a n t l y higher than pre-dive values. In DNX, plasma l e v e l s of K + and of l a c t a t e a f t e r 5 minutes diving were s i g n i f i c a n t l y higher than pre-dive values (Table 5). An approximate strong ion difference (SID = plasma levels of Na + and K + - plasma levels of Lactate - and Cl~) was calculated and i t s variations during diving were compared with variations of pH a and PC0 2 in DNX and SH-dnx. The increase of plasma Na +, lactate and SID at 5 minute submergence was s i g -n i f i c a n t l y higher i n SH-dnx than i n DNX (Figure 19), but the absolute change i n plasma K +, H + and PC0 2 a f t e r 5 minutes diving was s i g n i f i c a n t l y higher in DNX (Figure 19). 106 FIGURE 19: Absolute changes (Dive values - predive values) of [H +]a, PaC02 and a r t e r i a l l e v e l s of ions a f t e r 5 minutes submergence in adrenal denervated ducks (DNX: N=7, n=7; except for l a c t a t e : N=8, n=8) and t h e i r sham-operated controls (SH-dnx: N=8, n=8). +, *, s i g n i f i c a n t l y different from pre-dive value and from SH-dnx value respectively. 107 CHANGE IN ARTERIAL LEVELS OF IONS (milliequivalents per liter) I ro o ro > ai oo I 1 1 1 1 1 CHANGE IN [H+l (nanoequivalents per liter) DISCUSSION. The average maximum dive time (MDT) i n ADX and DNX ducks was s i g n i f i c a n t l y below the MDT of t h e i r respective sham-operated controls. In ADX ducks MDT decreased by 47% (relative to SH-adx) while in DNX ducks MDT f e l l by 41% (relative to SH-dnx) . Heart rate during the dive i n ADX and DNX ducks was s i g n i f i c a n t l y higher than i n t h e i r sham-operated controls, while t h e i r blood pressures dropped s i g n i f i c a n t l y lower. Previous work has shown that adrenalectomy reduces plasma levels of c i r c u l a t i n g NE and EP by 70% and 100% respectively compared with t h e i r sham-operated c o n t r o l s during d i v i n g (Chapter I ) . Diving lev e l s of NE and EP were reduced by 80% and 98% respectively in DNX compared with SH-dnx (Table 1 and Chapter I ) . When catecholamines were infused i n ADX and DNX ducks, MDT increased. In DNX ducks there was a s i g n i f i c a n t increase i n MDT during i n f u s i o n of catecholamines, but MDT remained s i g n i f i c a n t l y below that of SH-dnx ducks. Infusing catecholamines i n DNX ducks during dives s i g n i f i c a n t l y i n -creased blood pressure to values s i m i l a r to those i n SH-dnx ducks. Although heart rate decreased s l i g h t l y , i t was not si g n i f i c a n t l y different from that of DNX infused with saline. Thus, c i r c u l a t i n g catecholamines released by the adrenals do increase MDT. However, since MDT of DNX infused with catecho-lamines never reached MDT of t h e i r sham-controls, catechola-mines may not be the only adrenal hormones involved in promot-ing MDT. In mammals, a small release of C o r t i s o l (0.5 to 3 times) has been recorded a f t e r one hour of hypoxia (Jones et a l . 1988/ Nishijima et a l . 1989/ Paulick et a l . 1987). Even though Ringer (197 6) stated that in birds, nerve fibers do not enter the c o r t i c a l tissue, there i s some evidence of an innervation of c o r t i c a l c e l l s (Unsicker 1973). The action of such nerves on the r e g u l a t i o n of a d r e n o c o r t i c a l functions i s not well known (Holzwarth et a l . 1987), but Engeland et a l . (1985) showed that splanchnic nerves were not involved i n C o r t i s o l release during hemorrhage. The release of c o r t i c o s t e r o i d s i s triggered humorally, and a neural release of corticosteroids seems unlikely. In DNX ducks,, the humoral regulation of c o r t i -costeroid release was not prevented, but i t did not seem to play a major role i n promoting underwater endurance because the drop i n MDT was s i m i l a r i n ADX and DNX (respectively 47 and 41%). This also implies that the pre-experimental d i f f e r -ences due to a lack of endogenous c o r t i c o s t e r o i d (e.q. pre-dive heart rate, C l ~ , PaC^ (Chapter I)) do not a f f e c t the underwater performance of ADX ducks. Other adrenal products such as enkephalines may contribute to the prolongation of MDT and may lower heart rate during dives. In mammals, enkephalins are found in the adrenal medulla, i n the same c e l l s and v e s i -c l e s as the catecholamines, and are co-secreted with them (Hanbauer et a l . 1982/ Viveros & Wilson 1983/ Wilson et a l . 1982). Furthermore, enkephalins have also been shown to cause bradycardia and apnoea (Fennessy & Rattray 1971/ Sapru et. a l . 1981). 110 The h i g h e r h e a r t r a t e and lower b l o o d p r e s s u r e observed in ADX and DNX during diving suggests a lower stroke volume and/or lower peripheral resistance than in their sham-operated controls. Some authors have shown that adrenal ca-techolamines play an important role in the cardiac response to hypoxia compared with the sympathetic nerves (Baugh et. a l . 1959; Chiong & Hatcher 1972; Lee et a l . 1980; Nahas et a l . 1954), and others have shown the opposite (Achtel 1972; Kahler et a l . 1962; Kontos & Lower 1969; Korner & White 1966). Hypox-i a per se has a d i r e c t depressant action on myocardial con-t r a c t i l i t y (McKean 1984; Nakhjavan et a l . 1971; Tyberg et a l . 1970) . So, catecholamines, which have a well known po s i t i v e inotropic e f f e c t on the myocardium, may compensate for t h i s depressant action, and maintain the myocardial c o n t r a c t i l i t y during the dive. Oxygen stores, reflected by p a r t i a l pressure and content of a r t e r i a l oxygen, dropped faster in ADX and DNX animals than i n t h e i r respective shams. This suggests that a decrement i n peripheral vasoconstriction during dives i n ADX and DNX causes an increase i n blood flow and oxygen consump-tion in the peripheral tissues leading to a more rapid u t i l i -zation of oxygen stores. This could be the reason for a reduc-t i o n of MDT in ADX and DNX ducks. It has been observed that adrenalectomy perturbs the vascular responses to hypoxia (Baugh et a l . 1959; Kahler et a l . 1962). Circulating catecho-lamines released by the adrenal medulla contribute to vasomo-t o r responses i n the hind limb, renal and mesenteric beds (Allison & Powis 1971, 1976; Berecek & Brody 1982; Powis 1974; Yardley and Hilton 1987) and would appear to be more e f f i c i e n t for maintaining vasoconstriction in hypoxia, since neurogenic vasoconstriction i s more susceptible to i n h i b i t i o n by l o c a l hypoxia in blood vessels (Gooden 1980). Lower levels of plasma lactate in DNX suggest an increase in the ra t i o of aerobic to anaerobic metabolism compared with SH-dnx. This also supports the hypothesis of a lower peripher-a l r e s i s t a n c e i n DNX during the dive. Increase of aerobic versus anaerobic metabolism in DNX compared with SH-dnx, also implies increased CO2 production during diving. DNX showed a s l i g h t l y larger increase i n PCO2 during d i v i n g than SH-dnx (Figure 20), but the levels of PC0 2 reached by DNX were not as high as might be expected (Figure 19). Blood i n DNX, being more deoxygenated than in SH-dnx, would carry more C02 at any given PC02 (haldane e f f e c t ; Scheipers et a l . 1975) . However t h i s does not account f o r much and may not s a t i s f a c t o r i l y explain why PC0 2 in ADX and DNX doesn't r i s e any faster. The increase i n heart rate i n ADX and DNX towards the end of dives was not associated with an increase i n blood pressure to pre-dive values. This suggests that when ADX and DNX reach t h e i r maximal underwater tolerance, p e r i p h e r a l vasoconstriction i s not maintained, in contrast to their sham-operated c o n t r o l s . Consequently, the r e s u l t i n g release of lactate from peripheral tissues into the central c i r c u l a t i o n i n ADX could explain the lack of s i g n i f i c a n t difference i n plasma l e v e l s of lactate at the end of the dive between ADX and SH-adx whose peripheral cir c u l a t i o n i s s t i l l shut down. 112 It has been shown in certain teleost fishes that c i r c u -l a t i n g catecholamines increase blood a f f i n i t y for 0 2 during acidosis by maintaining the i n t r a c e l l u l a r pH of the erythro-cytes higher than that of the plasma (Nikinmaa 1983). The tonometry experiment showed that ci r c u l a t i n g catecholamines do not have any effect on oxygen a f f i n i t y in ducks during hypoxia and acidosis, supporting Nikinmaa e_t a l . (1984) who showed that catecholamines do not have any e f f e c t on erythrocyte pH in birds. Also, there were no significant differences in Pa0 2, CaC>2 and pHa between DNX at 3 minutes diving and SH-dnx at 5 minutes diving or between DNX and SH-dnx at the end of dives. This supports the contention that c i r c u l a t i n g catecholamines do not have any action on the a f f i n i t y of the blood for oxy-gen. It i s possible that in v i t r o an action of adrenal catech-olamines on blood a f f i n i t y for 0 2 could be masked by an i n -verse change in a f f i n i t y due to the difference in PC02 between DNX and SH-dnx. However, such a difference in PC0 2 at con-stant pH does not r e s u l t i n a s i g n i f i c a n t change i n oxygen a f f i n i t y (Isaacks et a l . 1986). During dives the a c t i o n of even a large increase i n c i r c u l a t i n g catecholamines on hepatic glycogenolysis must be insignificant because of the r e s t r i c t i o n of blood flow through peripheral tissues and l i v e r (Jones et a l . 1979; Zapol et. a l . 1979). Blood glucose levels stay constant during forced dives (Mangalam et a l . 1987; Robin et a l . 1981) or even decrease (Murphy et a l . 1980). Therefore, during forced diving, the major glucose sources a v a i l a b l e to the t i s s u e s are l o c a l glycogen stores for the peripheral tissues and lo c a l glycogen stores and blood glucose for the heart, brain and lungs. The higher plasma glucose i n diving ADX and DNX compared with the drop i n t h e i r sham-operated controls may be due to a release of glucose by the l i v e r caused by the weaker peripheral vaso-constriction observed in these ducks. An approximate plasma SID (strong ion d i f f e r e n c e = sum of cations - sum of anions) was calculated from l a c t a t e - , Na +, K + and C l ~ . In SH-dnx, SID showed a s i g n i f i c a n t increase during the dive (+2.6 mEq/L) which was also observed by Shimi-zu and Jones (1987). No change was observed i n DNX which explains the bigger drop i n a r t e r i a l pH i n DNX compared with SH-dnx, during the dive despite the smaller increases i n plasma lactate and PaC0 2. Plasma levels of l a c t a t e - and Na + in SH-dnx increased during the dive. This was also observed by Shimizu and Jones (1987) . However in DNX, i t was the plasma levels of l a c t a t e - and K + which increased during the dive. If the increase of plasma lactate observed during forced dives in DNX and t h e i r sham-operated controls was a consequence of anaerobic metabolism, the increases of Na + in SH-dnx and K + in DNX are harder to e x p l a i n . However, an hypothesis can be proposed based on two sets of experiments, one which analyzes the effects of ischemia on skeletal muscle and myocardium and the other which analyzes the effects of c i r c u l a t i n g catechola-mines on these muscles. When ske l e t a l muscles are subjected to ischemia (Irwing and Noakes 1985), and s i m i l a r l y when the myocardium i s sub-jected to anoxia or ischemia (Rau et a l . 1977; Wiegand et a l . 114 1979; H i l l and Gettes 1980; Shine 1981) they rapidly lose some of th e i r i n t r a c e l l u l a r potassium into the extracellular space and to the c i r c u l a t i o n . Most of these studies have been done on the heart. Weiss and Shine (1981) showed that this release i s biphasic: i t reaches i t s maximum after 5 minutes of expo-sure, then l e v e l s o f f for about 30 minutes, and then r i s e s again. This l a t e r phase, which i s i r r e v e r s i b l e , has been linked to c e l l necrosis (Conrad et a l 1979). The early phase which i s reversible on reoxygenation (Rau et a l . 1977; Weiss and Shine 1981) i s not due to a decrease of K + influx into the c e l l s , but to an increase of efflux (Kleber 1983; Shine 1981; Rau et a l . 1977). This increase of net K + e f f l u x out of the c e l l s i s not due to an i n h i b i t i o n of the Na +/K + pump (Kleber 1983; Rau et a l . 1977), and i s dissociated from the energetic state of the myocytes (Rau and Langer 1978). Efflux of anions such as l a c t a t e occur at the same time as e f f l u x of K + i n ischemic heart and s k e l e t a l muscles (Matur and Case 1973; Irwing and Noakes 1985). Crake et al (1987) showed that potas-sium loss from c e l l s i s secondary to the extrusion of lactate (end product of anaerobic metabolism) from the myocyte, i n order to maintain e l e c t r i c a l neutrality across the sarcolemma. Intravenous i n j e c t i o n of adrenaline leads to a short l i v e d i n i t i a l release of K + by the l i v e r (Vick et a l . 1972), mediated by the alpha-adrenergic receptors (Todd and Vick 1971) followed by a prolonged decrease i n the plasma K + (Vick et a l . 1972; Struthers and Reid 1984) due to a net uptake of potassium i n s k e l e t a l muscles and myocardium (Vick et. a l . 1972; Clausen and Flatman 1977; Sejersted et a l . 1985) mediat-ed mostly by beta2~adrenergic receptors (Clausen and Flatman 1980; Clausen 1983; S t r u t h e r s and R e i d 1984; E l l i n g s e n 1987). Moreover i n t r a c e l l u l a r sodium a c t i v i t y i n c a r d i a c c e l l s de-c r e a s e s d u r i n g p e r f u s i o n w i t h n o r e p i n e p h r i n e ( C l a u s e n and Flatman 1977; Wasserstrom et a l . 1982; Lee and V a s s a l l e 1983). T h i s i n f l u x o f K + i n t o t h e c e l l s and t h i s d e c r e a s e o f i n t r a -c e l l u l a r N a + a r e due t o an i n c r e a s e o f t h e N a + / K + ATPase a c t i v i t y by catecholamines (Clausen and Flatman 1977; S e j e r s t -ed e t a l . 1985; Lee and V a s s a l e 1983). Based on the s e r e s u l t s , i t can be proposed t h a t p o t a s s i -um, t o g e t h e r w i t h l a c t a t e , l e a v e s t h e i n t r a - c e l l u l a r compart-ment d u r i n g d i v i n g . Because of the i n c r e a s e o f ca t e c h o l a m i n e s which enhances the a c t i v i t y of the Na +/K + pump, K + i s r e t u r n e d t o t h e i n t r a c e l l u l a r compartment, and t h e mat c h i n g e f f l u x o f Na + causes t h e i n c r e a s e o f plasma N a + observed d u r i n g d i v i n g . I f t h i s i n c r e a s e i n c i r c u l a t i n g c a t e c h o l a m i n e s i s p r e v e n t e d (as i n DNX ducks) plasma l e v e l s o f K + r a t h e r t h a n N a + s h o u l d i n c r e a s e , which i s p r e c i s e l y what has been observed. In f o r c e d d i v e d ducks, t h e i n c r e a s e i n p e r i p h e r a l v a s o -c o n s t r i c t i o n i s due t o the a c t i v i t y of t h e sy m p a t h e t i c branch o f t h e a u t o n o m i c n e r v o u s s y s t e m . K o b i n g e r and Oda (1969) observed changes i n c a r d i o v a s c u l a r adjustments i n ducks f o r c e d d i v e d f o r 90 s e c o n d s , a f t e r i n j e c t i o n o f a n t i - a d r e n e r g i c agents such as r e s e r p i n e , b r e t y l i u m , g u a n e t h i d i n e , methyldopa and c a t a p r e s . They c o n c l u d e d t h a t t h e changes o b s e r v e d were due t o an a l t e r a t i o n of the p e r i p h e r a l v a s o c o n s t r i c t i o n d u r i n g d i v i n g due t o an i m p a i r m e n t o f t h e a u t o n o m i c s y m p a t h e t i c nervous system. However t h e y d i d not check t h e e x t e n t o f t h e 116 depletion of the catecholamines stores i n sympathetic nerves, adrenal glands, and central nervous system after injection of the adrenergic neuron blocking agents. So, i t was impossible for them to d i f f e r e n t i a t e the role of peripheral sympathetic nerves, from the role of adrenal glands on the cardiovascular response to forced dives. Andersen and B l i x (1974) injected adrenergic alpha-receptor blockers (phentolamines), as well as reserpine before dives i n ducks which were normally able to endure forced submersion for more than 10 minutes. MDT were reduced considerably, r e s p e c t i v e l y to 150 seconds and 45 seconds (Andersen & Blix 1974). The effectiveness of phento-lamine and reserpine was not controlled, and so i t i s impossi-ble to conclude i f the reduction of MDT observed i n ducks dived with reserpine i s due to a depletion of catecholamines stored i n sympathetic nerves only or i n both sympathetic nerves and adrenal glands. The work done in t h i s chapter differe n t i a t e s between the role of sympathetic nerves and the role of adrenal catechola-mines on the cardiovascular adjustments to forced diving'. I suggest that sympathetic nerves i n i t i a t e the peripheral vaso-constriction at the beginning of the dive, and also p a r t i a l l y maintain i t during the dive. However, peripheral vasocon-s t r i c t i o n c o n t r o l l e d by sympathetic nerves i s not strong enough to eliminate blood flow from peripheral tissues. Adre-nal catecholamines seem to enhance and maintain peripheral v a s o c o n s t r i c t i o n during the dive and thus would prevent 0 2 stores ( i n d i s p e n s i b l e f o r the b r a i n and heart) from being depleted by peripheral tissues. Therefore tolerance to under-water submersion i s increased. 117 CHAPTER 3: INFLUENCE OF PROPANOLOL ON MAXIMUM DIVE TIME. 118 INTRODUCTION. The cardiovascular p e c u l i a r i t i e s observed during diving in adrenalectomised (ADX) and adrenal denervated ducks (DNX) (higher heart rate associated with a lower a r t e r i a l blood pressure) suggested a reduction in peripheral vasoconstriction and consequently a faster drop i n oxygen stores leading to a decrease of maximum dive time (MDT). However, as I mentioned i n chapter 2, a decrease i n stroke volume could also have occurred in ADX and adrenal DNX, due to a depressant effect of hypoxia (McKean 1984; Nakhjavan et a l . 1971; Tyberg et a l . 1970) or / and acidosis (Rau et a l . 1977; Gonzalez and Clancy 1975; Cingolani et a l . 1970) per se on myocardial c o n t r a c t i l -i t y . A decrease i n cardiac output might cause a drop in MDT because of a lack of an adequate cerebral and cardiac blood flow and pressure. The hypothesis that c i r c u l a t i n g catechola-mines could counteract the depressant action of hypoxia, and acidosis on the heart and thus enhance MDT has to be i n v e s t i -gated. 119 By injecting propranolol (beta 1 and 2 adrenergic block-ade) in Pekin ducks at the beginning of the dive, most of the actions of catecholamines on the myocardium can be prevented. In t h i s chapter, the MDT from such dives are compared with control dives in which ducks were injected with saline. This preliminary experiment established whether or not catechola-mines, by their action on the myocardium, enhanced MDT. This experiment did not differentiate between the action of catech-olamines from sympathetic nerves, and the action of catecho-lamines from the adrenal glands. On the other hand, i n the absence of any s i g n i f i c a n t effect on MDT, i t would be l i k e l y that the enhancement of MDT by adrenal catecholamines i s not achieved through myocardial actions v i a beta-adrenergic recep-tors . 120 MATERIAL AND METHODS. 1- ANIMALS: These experiments were performed on 10 two to three month old male Pekin ducks ranging i n mass from 2.5 to 3 Kgs. They were kept in conditions similar to those described in Chapter I. 2- MINOR SURGERY AND EXPERIMENTAL PROCEDURE: Cannulation of the b r a c h i a l a r t e r y and vein was done under l o c a l anesthesia (Lidocaine hydrochloride, Xylocaine 2% Astra) as described i n chapter I I . The birds were given one day to recover from surgery. The position of the cannula was checked at post mortem: the t i p of the a r t e r i a l cannula was positioned at the junction of the brachiocephalic artery and aorta; the t i p of the venous cannula was positioned i n the vena cava. Birds were placed in a s i t t i n g position and secured with tape to an operating table. The electrocardiogram leads were inserted subcutaneously, one above the l e f t thigh, the other below the right shoulder, and the ground lead to the web of the right foot. The a r t e r i a l cannula was connected to a blood pressure transducer. The ducks were l e f t undisturbed behind a screen for 15 minutes to s e t t l e down before an experiment started. The head of the duck was then gently lowered into the water. During the 121 f i r s t 10 seconds of the d i v e w i t h beta-blockade, 4 mg/Kg of p r o p r a n o l o l (Sigma) d i s s o l v e d i n 1 ml of s a l i n e was i n j e c t e d i n t o the duck v i a the venous cannula. The cannula was f l u s h e d with 2 ml. of s a l i n e . T h i s dose of p r o p r a n o l o l was enough to prevent, f o r 20 minutes, the t a c h y c a r d i a f o l l o w i n g an i n j e c -t i o n of 50 ug of i s o p r o t e r e n o l h y d r o c h l o r i d e (Winthrop l a b . O n t a r i o ) . In c o n t r o l d i v e s , 3 ml of s a l i n e was i n j e c t e d f o l -lowing the same procedure as t h a t f o r d i v e s w i t h b e t a - b l o c k -ade. Three dives were performed on each duck with 48 hours of r e c o v e r y between d i v e s . In the f i r s t and t h i r d d i v e s , o n l y s a l i n e was i n j e c t e d ( c o n t r o l d i v e s ) . In t h e second d i v e , p r o p r a n o l o l was i n j e c t e d . Maximum d i v e time was measured f o l l o w i n g the c r i t e r i a e s t a b l i s h e d i n chapter I I . 3 - A N A L Y S I S O F D A T A : Heart r a t e and mean a r t e r i a l blood pressure were sampled every minute, u n t i l 30 seconds before the end of the d i v e . In the t e x t and graphs, v a l u e s are g i v e n as mean + s t a n d a r d e r r o r of the mean (SE). Because the l e n g t h of the d i v e f o r each animal i s d i f f e r e n t , the sampling time f o r n (number of dives) decreases. The mean and SE f o r values recorded during the d i v e a r e o n l y i n c l u d e d as d a t a f o r a n a l y s i s when the number of d i v e s remaining i s more than, or equal t o , h a l f of the o r i g i n a l number of d i v e s . Mean and SE of values at the end of d i v e s are a l s o i n c l u d e d on graphs. S t a t i s t i c a l l y s i g n i f i -cant d i f f e r e n c e s were based on r e s u l t s of a n a l y s i s of variance f o l l o w e d by the Newman-Keuls t e s t . A s i g n i f i c a n t d i f f e r e n c e was assumed i f p<0.05. 122 RESULTS. 1- EFFECTS OF PROPRANOLOL ON MAXIMUM DIVE TIME (MDT): In the f i r s t and t h i r d dives, i n which the ducks were injected with saline, mean MDT was 13 min. 53 sec. +_ 1 min. 11 s e c , and 12 min. 55 sec. + 1 min. 16 sec respectively . MDT i n the second dive, i n which ducks were i n j e c t e d with propranolol was 12 min. 5 sec. + 1 min. 1 sec. There were no sig n i f i c a n t differences among the mean MDT i n the three dives (Figure 20) . 2- EFFECTS OF PROPRANOLOL ON HEART RATE (HR) AND BLOOD PRESSURE  (BP) : There were no s i g n i f i c a n t differences i n pre-dive heart rate among dives. Diving heart rates, in the three dives were sig n i f i c a n t l y lower than their respective pre-dive values with exception of those at end dive (Figure 21). In the f i r s t and t h i r d dives, a strong bradycardia de-veloped. Heart rate dropped from 154 + 7 beats/minute to 17 + 3 beats/minute at 2 minutes into the dive during the f i r s t dive, and from 159 + 11 to 19 + 4 beats/minute i n the t h i r d dive. Diving heart rates i n the f i r s t and t h i r d dive were not s i g n i f i c a n t l y d i f f e r e n t and remained close to the l e v e l s reached at 2 minutes throughout the dive (Figure 21). The heart rate p r o f i l e during the second dive, in which ducks were i n j e c t e d with propranolol was quite d i f f e r e n t . Heart rate dropped from 150 + 10 beats/minute to 24 + 6 after 123 FIGURE 20: Maximum dive time in ducks injected with propranolol and saline. ( 1 - s a l . : f i r s t dive s a l i n e i n j e c t e d ; 2-prop.: second dive propranolol injected; 3-sal.: t h i r d dive saline injected) 124 M A X I M U M DIVE TIME ( m i n u t e s ) ho -f-CT) 00 - f - o ro CD FIGURE 21: Heart r a t e and mean a r t e r i a l b l o o d p r e s s u r e d u r i n g d i v i n g i n ducks i n j e c t e d w i t h s a l i n e ( 1 s t d i v e : h o l l o w t r i a n -g l e s , n=10 f o r h e a r t r a t e and b l o o d p r e s s u r e ; 3 r d d i v e : h o l l o w c i r c l e s , n=8 f o r h e a r t r a t e and n=6 f o r b l o o d p r e s s u r e ) or w i t h p r o p r a n o l o l ( 2 n d d i v e : f i l l e d square, n=10 f o r h e a r t r a t e and n=9 f o r b l o o d p r e s s u r e ) . *: s i g n i f i c a n t l y d i f f e r e n t from both c o n t r o l d i v e s . +: s i g n i f i c a n t l y d i f f e r e n t from the f i r s t c o n t r o l d i v e . 126 250 DIVE TIME (minutes) 127 2 minutes d i v i n g , but a f t e r 3 minutes i t had i n c r e a s e d and was s i g n i f i c a n t l y h i g h e r t h a n d u r i n g t h e s a l i n e i n j e c t e d d i v e s ( F i g u r e 2 1 ) . A f t e r 6 minutes submergence, h e a r t r a t e s i n t h e f i r s t and t h i r d d i v e were 1 8 + 2 and 21 ± 4 beats/minute but i n the b e t a - b l o c k e d d i v e , h e a r t r a t e was 52 ± 8 beats/minute. No s i g n i f i c a n t d i f f e r e n c e s i n p r e - d i v e b l o o d p r e s s u r e were o b s e r v e d among t h e t h r e e d i v e s ( F i g u r e 2 1 ) . B l o o d p r e s -s u r e s d u r i n g d i v i n g i n t h e f i r s t and t h i r d d i v e s were n o t s i g n i f i c a n t l y d i f f e r e n t . I n p r o p r a n o l o l i n j e c t e d d i v e s , b l o o d p r e s s u r e s dropped s i g n i f i c a n t l y below p r e - d i v e l e v e l s a f t e r 1 m i n u t e d i v i n g and s t a y e d l o w e r t h a n t h e p r e - d i v e v a l u e t h r o u g h o u t t h e d i v e . T h i s was n o t t h e c a s e i n t h e s a l i n e d i v e s : d i v i n g b l o o d p r e s s u r e was ne v e r s i g n i f i c a n t l y l o w e r t h a n t h e p r e - d i v e v a l u e s . B l o o d p r e s s u r e s s i g n i f i c a n t l y dropped from 157 _+ 4 t o 83 + 7 mmHg a f t e r 6 minutes d i v i n g i n p r o p r a n o l o l d i v e s . In the f i r s t d i v e , b l o o d p r e s s u r e was 170 + 8 mmHg b e f o r e t h e d i v e and 126 + 12 mmHg a t 6 m i n u t e i n t o d i v e s and i n the t h i r d d i v e , i t was 155 + 10 and 120 + 20 mmHg r e s p e c t i v e l y ( F i g u r e 21) . 128 DISCUSSION: Preventing the cardiac actions of catecholamines did not decrease MDT even though the c a r d i o v a s c u l a r responses to d i v i n g were st r o n g l y a l t e r e d . The increase i n heart rate observed in the beta-blockade dives was not associated with an increase of blood pressure. This suggests that a decrease of peripheral vasoconstriction or a decrease i n stroke volume occurred during the beta-blocked dives. The fact that MDT was not shorter during the beta-blocked dives implies that the oxygen stores were not depleted faster than during the control dives. That, plus the fact that propranolol i f anything r e i n -forced vasoconstriction by blocking the vaso d i l a t o r beta-2 receptors, suggests that a decrease of stroke volume (not peripheral vasoconstriction) occurred i n beta-blocked dives and was accompanied by an increase of heart rate. Since the beta actions of catecholamines on the heart were suppressed, and alpha effects are not expected to be great (see next para graph) these changes must be a consequence of the d i r e c t effects of vagal a c t i v i t y , hypoxia, hypercapnia, and acidosis imposed on the heart during diving. It has long been known that i n mammals, the p o s i t i v e chronotropic, dromotropic, and inotropic effects of catechola-mines on the heart are mediated through beta-adrenoceptors (Berne and Levy 1981), and this i s also true for birds (Bolton 1967, Bolton and Bowman 1969; Tummons and Sturkie 1970). However, r e c e n t s t u d i e s on mammals have d e m o n s t r a t e d t h e e x i s t e n c e o f a l p h a - a d r e n o c e p t o r s i n t h e h e a r t . I t was n o t p o s s i b l e t o b l o c k t h e s e a l p h a - adrenoceptors d u r i n g t h e d i v e w i t h o u t s u p p r e s s i n g as w e l l t h e p e r i p h e r a l v a s o c o n s t r i c t i o n and, consequently, the d i v e response. However, beta-adrenocep-t o r s seem t o be t h e main r e c e p t o r s t h r o u g h which t h e c a r d i a c a c t i o n o f c a t e c h o l a m i n e s i s e x p r e s s e d i n a d u l t mammals and b i r d s . B r i s t o w e t a l . (1988) e s t a b l i s h e d t h a t t h e r e l a t i v e p r o p o r t i o n o f alpha and be t a adrenoceptors i n a human he a r t i s 15% and 85%. A l s o , Skomedal e t a l . (1988) showed t h a t i n t h e i s o l a t e d p a p i l l a r y muscles o f r a t h e a r t , the c a r d i a c i n o t r o p i c response, e l i c i t e d a f t e r NE s t i m u l a t i o n decreased by 75% a f t e r p r o p r a n o l o l blockade. The p h y s i o l o g i c a l s i g n i f i c a n c e o f a l p h a -a d r e n o c e p t o r s i n c a r d i a c r e g u l a t i o n i s not w e l l e s t a b l i s h e d and a p p e a r s m i n o r compared t o b e t a - a d r e n o c e p t o r s . A l p h a a d r e n o c e p t o r s have a d i r e c t p o s i t i v e i n o t r o p i c e f f e c t on t h e heart (Aass et a l . 1983/ Bruckner e t a l . 1984; Skomedal e t a l . 1985), t h e y d e c r e a s e t h e n e u r a l r e l e a s e o f NE ( S t a r k e 1972; Yamaguchi e t a l . 1977) and i n c r e a s e NE uptake by both adrener-g i c nerve t e r m i n a l s and e x t r a n e u r o n a l t i s s u e s ( S t a r k e e t a l . 1971; I v e r s e n 1973). They a l s o d e c r e a s e n e u r a l r e l e a s e o f a c e t y l c h o l i n e ( W e t z e l e t a l . 1985; M c G r a t t a n e t a_l 1986; McDonough e t a l . 198 6), although t h i s has not been observed i n b i r d s ( L o f f e l h o l z e t a l . 1984). Furthermore, B o l t o n (1967) and B o l t o n and Bowman (1969) i n f a c t f a i l e d t o d e m o n s t r a t e t h e e x i s t e n c e o f any alpha-adrenoceptors i n b i r d s . A f t e r adding NE t o t h e h e a r t , t h e y l o o k e d a t t h e c a r d i a c i n o t r o p i c changes when a l p h a - a d r e n o c e p t o r b l o c k e r s were added. The f a c t t h a t t h e y d i d not f i n d any a l p h a - a d r e n o c e p t o r s c o u l d be because 130 t h e i r a c t i v i t y was masked by that of cardiac beta-receptors: they should have added NE f i r s t , then a beta-1 and 2 receptor blocking agent and then the alpha blocking agent (Skomedal et a l . 1988). In chickens, the effect of sympathetic nerve stimu-l a t i o n or NE on heart rate was nearly abolished after i n j e c -tion of propranolol (Bolton 1967; Tummons and Sturkie 1970). Thus i t i s l i k e l y that, by i n j e c t i n g propranolol i n Pekin ducks at the beginning of the dive, I prevented most of the action of catecholamines on the heart. In birds as i n mammals, stimulation of parasympathetic nerves causes bradycardia ( F u r i l l a and Jones 1987; Lang and Levy 1989) as well as a negative inotropic effect (Folkow and Yonce 1967; Furnival et a l . 1973; Lang and Levy 1989). The main consequence of the direct action of hypoxia and acidosis on the heart i s a decrease i n c o n t r a c t i l i t y . Ng et a l . (1966) showed that the eff e c t of hypoxia on cardiac con-t r a c t i l i t y depends on the severity of the hypoxia. Moderate hypoxia increases v e n t r i c u l a r c o n t r a c t i l i t y , while severe hypoxia decreases ventricular c o n t r a c t i l i t y in mammals (Shine 1981; McKean 1984; McKean and Landon 1982; Rau et a l . 1977; England and Krause 1987) as well as i n ducks (Abati 1975) . Acidosis also has a di r e c t negative inotropic e f f e c t on the heart (Rau et a l . 1977; Gonzalez and Clancy 1975; Cingolani et a l . 1970), respiratory being more potent than metabolic acido-si s (Steenbergen e_t a l . 1977) . Acidosis potentiates the depres-sant effect of hypoxia on myocardial functions (Downing et a l . 1966). Negative i n o t r o p i c e f f e c t s have also been observed 131 during ischaemia (Katz and Hetcht 1969; Steenbergen et a l . 1977) . As a result of their positive inotropic effects, catecho-lamines can compensate for the d e b i l i t a t i n g effects of hypox-ia , hypercapnia, acidosis, ischaemia (England and Krause 1987; Nakanishi et. a l . 1987) and vagal a c t i v i t y on cardiac contrac-t i l i t y . The prevention of the action of catecholamines during diving would cause a decrease i n cardiac c o n t r a c t i l i t y and stroke volume. The resulting decrease in cardiac output could be compensated for, in this particular case, by an increase of heart rate invoked by the baroreceptor reflex. However, chemo-receptors during the dive tend to i n h i b i t the b a r o r e f l e x (Smith 1987). Nevertheless, the cardiac action of catechola-mines i s prevented by i n j e c t i o n of beta-blockade, so the increase of heart rate can only be due to a decrease of vagal a c t i v i t y on the heart. Previous work has been done on the effects of propranolol during d i v i n g (Butler and Jones 1968, 1971; Folkow et a l . 1967; Gooden et aJL 1974), but no increase of heart rate was observed because the dive times used i n these experiments did not exceed 2 minutes and, as I have shown, propranolol affects cardiovascular responses later in the dive. However, Ferrante and Opdyke (1969) working on Nutria submerged for 1 minute demonstrated that vagotomy or atropinization caused a marked increase i n v e n t r i c u l a r c o n t r a c t i l i t y to more than 30% above control l e v e l s and that propranolol administrated to these vagotomised or atropinised animals greatly reduced or abol-132 ished t h i s increase. This shows that very early i n the dive, there i s only a small e f f e c t of catecholamines on cardiac c o n t r a c t i l i t y which i s masked by the vagal a c t i v i t y . This explains why injection of propranolol did not alter the cardi-ovascular adjustments. Sympathetic a c t i v i t y on the heart i n creases throughout the dive ( i e . increase i n c i r c u l a t i n g catecholamines). Moreover, hypoxia, hypercapnia and acidosis develop and reach the l e v e l s which would depress cardi a c c o n t r a c t i l i t y (after 3 minutes into diving) i f catecholamines were not there. During diving the heart i s controlled by powerful nega-tive inotropic and chronotropic influences (the vagus, hypox-i a , hypercapnia and a c i d o s i s ) . This work demonstrates a s i g -n i f i c a n t effect of the sympathetic system whose strong posi-t i v e i n o t r o p i c action mediated through beta-adrenoceptors, counterbalance these depressant influences exerted on cardiac c o n t r a c t i l i t y . At 2 minutes in t o the dive i n Pekin ducks, stroke volume i s not s i g n i f i c a n t l y different from i t s pre-dive value (Jones and Holeton 1972). This and the fact that catech-olamines prevent stroke volume dropping l a t e r i n the dive, suggest that catecholamines maintain stroke volume at i t s pre-dive values throughout the dive. However, the cardiac effect of the sympathetic system had no bearing on MDT. 133 CHAPTER 4: RESPECTIVE ROLES OF SYMPATHETIC NERVES AND SYMPATHETIC HUMORAL AGENTS ON THE VASCULAR RESISTANCE OF THE HIND LIMB OF PEKIN DUCKS DURING DIVING. 134 INTRODUCTION. It has been suggested i n Chapter II that the drop i n maximum dive time (MDT) observed in adrenalectomised (ADX) and adrenal denervated (DNX) ducks was caused i n part by a decrease in peripheral vasoconstriction due to the absence of adrenal catecholamines during diving. Peripheral vasoconstric-t i o n could be enhanced by adrenal catecholamines exerting their effects d i r e c t l y on the sympathetic nerves and/or vascu-la r smooth muscle. Indeed, Gooden (1980) suggests that a role for c i r c u l a t i n g catecholamines could be to sustain vasocon-s t r i c t i o n during prolonged dives when neurally mediated vaso-constriction i s depressed by hypoxia. In t h i s chapter I w i l l examine the following questions: 1) Are the ef f e c t s of c i r c u l a t i n g catecholamines and sympa-thetic nerve activation on peripheral vasomotor tone additive? 2) Do hypoxia, hypercapnia and acidosis reduce the degree of v a s o c o n s t r i c t i o n produced by sympathetic nerve a c t i v a t i o n during diving? 135 In order to d i r e c t l y assess whether or not c i r c u l a t i n g catecholamines can enhance peripheral vasoconstriction during a dive, a system was developed to monitor the changes i n vascular resistance simultaneously i n both hind limbs. One leg was perfused with the duck's own blood (autoperfused leg) and the other with d i f f e r e n t blood mixtures necessitated by the experimental protocol (test leg). Because of the enormous decrease of hind limb blood flow (from 40 - 50 ml/min to 2 - 0 ml/min) and v a r i a t i o n i n blood pressure during a dive (180 mmHg to 120 mmHg), I chose not to employ c l a s s i c a l methods of perfusion which use constant flow or pressure. Before and during the dive, the autoperfused leg was perfused at variable pressure and flows matching those normally observed i n a duck under these conditions. The t e s t leg was perfused with the same flow as the autoperfused leg. Differences i n perfusion pressure between autoperfused and test legs indicated d i f f e r -ences in their vascular resistances during the dive. To estimate the respective roles of sympathetic nerves and humoral factors on the vascular resistance during a dive, the test leg was perfused with blood obtained from the resting animal. The gas 0 2 and C0 2 tensions of t h i s blood were ad-justed before perfusion to match those found during the dive. The " r e s t i n g " blood, e q u i l i b r a t e d with d i f f e r e n t gas mixtures, was used i n a further set of perfusions in order to determine whether or not peripheral vasoconstriction, mediated by sympathetic nerves, i s affected by hypoxia and/or hypercap-nia. 136 Norepinephrine (NE) and epinephrine (E) were added to hypoxic-hypercapnic blood perfusing the test leg to match the catecholamine lev e l s measured during diving (Hudson & Jones 1982). This demonstrated the role of c i r c u l a t i n g catechola-mines on the peripheral vasoconstriction during the dive. 137 MATERIAL AND METHODS. 1- ANIMALS: Male Pekin ducks 3 to 5 months old and 3 to 3.5 Kgs i n mass were kept under conditions similar to those described i n chapter I. Twenty six ducks were used for perfusion. Twenty six ducks served as blood donors. 2- SURGERY AND EXPERIMENTAL PROCEDURE: In donor ducks, the b r a c h i a l vein was cannulated with P.E. 190 tubing (Intramedic Polyethylene tubing, Clay Adam), under l o c a l anesthesia, following the procedure described i n chapter II. In the experimental ducks, a l l surgery was performed at least one day before a perfusion experiment. The b r a c h i a l vein and artery were cannulated with P.E. 190 tubing (Intra-medic Polyethylene tubing, Clay Adam), following the procedure described i n chapter I I . In order to prevent any leg impair-ments, and degradation of the vascular system, non-occlusive cannulations were performed on the i s c h i a t i c a r t e r i e s under l o c a l a n e sthesia (Lidocaine h y d r o c h l o r i d e ; Xylocaine 2% Astra). The skin was cut along the caudal side of the femur, exposing the biceps femoris. An i n c i s i o n was made i n t h i s muscle adjacent to the femur, 1.5 cm from the hip j o i n t to expose the i s c h i a t i c artery which was clamped i n two places 1 cm apart. An i n c i s i o n was made in i t s wall between the clamps 138 and the ends of a single catheter (15 cm length, 3 and 3.7 mm i n s i d e and outside diameters) were i n s e r t e d 1.5 cm i n the vessels proximally and d i s t a l l y , to form a loop i n which the blood flowed fre e l y when the clamp was removed. The catheter was t i e d into the vessel, and muscle and skin were sutured, leaving part of the loop exposed outside the animal. The catether was made of Polyvinyl chloride (PVC, size V-11, BOLAB, Lake Havasu City, Arizona). It was treated with a heparin-containing solution (TD-MAC; 25%; Polysciences Inc.; Warrington Pennsylvania) to bind heparin onto the tubing wall in order to prevent blood c l o t t i n g . The length of the tubing was f i l l e d with a 1:1 mixture of TD-MAC solution and toluene for 4 minutes. Then, the mixture was removed and the tubing was rinsed with 20 ml. of saline containing 500 IU of heparin per ml. The tubing was a i r dried for at least 4 hours. The effectiveness of t h i s procedure was such, that blood could flow freely through the catheter for up to 4 weeks without the need to flush i t or inject heparin into the animal. The donor duck was placed on i t s back on an operating table and the perfused duck was placed i n a s i t t i n g p o s ition on another operating table. Ten ml of saline was injected into the venous cannula of the donor, and immediately after, 10 ml of blood was withdrawn. This procedure was repeated 6 times, removing 60 ml of venous blood from a donor duck without creating a hypotension which might have lead to release of catecholamines. In order to minimize cross r e a c t i v i t y , 10 ml of donor blood was i n j e c t e d into the venous cannula of the duck to be used in the perfusion experiment, and 10 ml venous blood was extracted. This was repeated 6 times and 60 ml of venous blood was extracted. This blood was then poured i n t o a blood gas chamber maintained at 41 °C (T: Figure 22). The blood was s t i r r e d very gently to equilibrate i t with the inflow gas mixtures necessi-tated by the experimental protocol. S t i r r i n g did not cause any hemolysis. The EEG was recorded as described i n chapter I. The i s c h i a t i c loops were cut, the bra c h i a l a r t e r i a l cannula, as well as the d i s t a l and proximal parts of the loops were then connected up as described i n Figure 22. The duck (D: Figure 22) was l e f t undisturbed for 5 to 10 minutes while both legs were perfused with their own blood. Ducks were dived by lower-ing t h e i r head gently into a beaker of cold water (16-20°C). Then, the test leg was connected to a given blood gas mixture and perfused with i t throughout the dive. The dive was termi-nated when the blood chamber was empty or maximum dive time was reached. MDT was determined following the characteristics established in Chapter II. Seven ducks had t h e i r t e s t l e g perfused with hypoxic-hypercapnic blood, 5 with hyperoxic-hypocapnic blood, 8 with hyperoxic-hypercapnic blood and 6 with hypoxic-hypocapnic blood. The effects of acidosis on the neurogenic component of peripheral vasoconstriction during diving are secondary to the effects of hypercapnia because the drop of pH observed in the blood during diving i s due to an increase of PaCC^ and not to a drop of SID (Shimizu and Jones 1987; Chapter I I ) . Conse-140 FIGURE 22: Diagram of the experimental set up used for the hind limb perfusion experiments (for explanations see text). 141 Chart racordtr q u e n t l y , t h e e f f e c t s o f t h e d i v i n g a c i d o s i s on t h e n e u r a l v a s o c o n s t r i c t i o n s h o u l d be t h e same as t h e e f f e c t s o f hyper-c a p n i a . R e s t i n g b l o o d was u s e d i n t h e s e e x p e r i m e n t s , so c i r c u l a t i n g catecholamines d i d not exceed r e s t i n g l e v e l s . In f i v e other ducks, t e s t l e g s were p e r f u s e d w i t h hypox-i c - h y p e r c a p n i c b l o o d i n t o which NE (Norepinephrine b i t a r t r a t e : Levophed W i n t h r o p , A u r o r a , O n t a r i o , Canada) and EP ( E p i n e -p h r i n e c h l o r i d e : P a r k - D a v i s , S c a r b o r o u g h , O n t a r i o , Canada) were i n f u s e d t o y i e l d l e v e l s m a t c h i n g t h o s e r e c o r d e d by Hudson and Jones (1982). The p r o c e d u r e u s e d f o r t h i s group d i f f e r e d from t h a t d e s c r i b e d above, because o n l y t h e t e s t l e g was can n u l a t e d . The d i s t a l and p r o x i m a l p a r t s of the loop were c o n n e c t e d as d e s c r i b e d i n F i g u r e 22 f o r t h e t e s t l e g . The downstream c a t h e t e r was c o n n e c t e d t h r o u g h a s i d e arm, t o a Ha r v a r d i n f u s i o n pump (model 901 M i l l i s Mass.; I : F i g u r e 22) i n f u s i n g a t a r a t e o f 0.136 ml/min. S a l i n e was i n f u s e d i n t o t h e b l o o d f l o w o f t h e t e s t l e g f o r two m i n u t e s b e f o r e t h e d i v e . Three minutes a f t e r t h e d i v e had commenced, t h e s a l i n e i n f u s i o n was r e p l a c e d by catecholamines (0.136 ml/min i n f u s i n g 0.34 ug o f NE and 0.34ug o f EP p e r mi n u t e ) and m a i n t a i n e d u n t i l the end o f d i v e . 3-MEASUREMENTS AND ANALYSIS OF THE PHYSIOLOGICAL VARIABLES: The set-up used f o r the p e r f u s i o n experiments i s shown i n F i g u r e 22. P e r f u s i o n o f t h e t e s t and a u t o p e r f u s e d l e g was ma i n t a i n e d w i t h a p e r i s t a l t i c pump (Watson-Marlow; H.R. Flow i n d u c e r ; C o r n w a l l , England; P: F i g u r e 22). Although the autop-143 erfused leg was perfused with the duck's own blood before and throughout the dive, the test leg was only perfused with the duck'own blood before the dive. At the beginning of the dive, the stopcocks (S: Figure 22) were turned to perfuse the test leg with the blood from the thermostated chamber (T: Figure 22). In order to keep blood volume constant, an equivalent volume of blood was pumped out of the animal i n t o a waste container (W: Figure 22). The autoperfused leg was perfused before and during the dive with variable pressures and flows matching the levels normally observed in ducks in these condi-t i o n s . This was done using a pressure flow : c o n t r o l l e r (C: Figure 22). This controller compared the perfusion pressure of the autoperfused leg with the mean a r t e r i a l blood pressure of the duck and increased or decreased the flow of the pump i n order to maintain perfusion pressure i d e n t i c a l to the mean a r t e r i a l blood pressure. For example, i f the vascular r e s i s t -ance increased in the autoperfused leg flow decreased in order to keep perfusion pressure s i m i l a r to mean a r t e r i a l blood pressure. The test leg was perfused at the same flow as the autoperfused l e g although i t s p e r f u s i o n pressure was not con t r o l l e d (Figure 22). The blood used for perfusion of the test leg was stored and kept at 41°C i n the chamber through which desired gas mixtures were c i r c u l a t e d (T: Figure 22). The length of the tubing was the same i n the c i r c u i t s perfus-ing the two legs and was kept to a minimum in order to prevent c o o l i n g of blood. Because the tubing i n s i d e the pump was expansible, pressures upstream of the pump had to be the same in both c i r c u i t s in order to keep the flow in each leg identi-c a l . Consequently, the blood chamber (T: figure 22) was pres-surized at mean a r t e r i a l blood pressure (+5 mm Hg). The pres-sure inside the chamber was checked throughout the dive with a mercury manometer (M: figure 22). The pressurization system consisted of a regulator (A: Figure 22; Figure 23) connected to the gas outflow of the blood chamber. A flowmeter (F: Figure 22) was used to set the pressure i n the blood chamber to mean a r t e r i a l blood pressure and the pr e s s u r i z a t i o n was checked over a range of 50 to 250 mm Hg before beginning the experiment. The experimental set up for the catecholamine experiment d i f f e r e d from the previous d e s c r i p t i o n as only one leg was cannulated. In this case, the pressure flow controller simply compared the perfusion pressure of the test leg with the mean a r t e r i a l blood pressure. ECG, mean a r t e r i a l blood pressure, perfusion pressure of autoperfused and tes t legs, as well as blood flow i n these legs were recorded on a chart recorder (Watanabe III, Type WTR 281, Japan; R: Figure 22). The difference between vascular resistance of the autoperfused and test legs was reflected by any difference i n perfusion pressure. Vascular resistance was r are the average values of perfusion pressure, blood flow and vascular resistance over a 1 minute (t+l-t) i n t e r v a l . Venous pressure was ignored i n t h i s c a l c u l a t i o n because i t did not sig n i f i c a n t l y change the overall results (Jones 1973). calculated by the formulae and 145 FIGURE 23: Pressure regulator for the blood chamber. If the pressure in the chamber was less than the mean a r t e r i a l blood pressure (MAP), the e l a s t i c membrane (m) bowed to ob-struct the gas outflow tubing. Pressure insi d e the chamber then rose to exceed MAP and the membrane was reflected allow-ing the gas to escape. 146 MAP GAS OUTFLOW GAS INFLOW ( f r o a the b l o o d cb b e r ) 147 At the beginning and at the end of a dive, 1 ml of blood was taken i n a i r t i g h t syringes from the duck and from the blood chamber i n order to measure blood gas values and pH using an Instrumentation Laboratories 813 pH/Blood gas analyz-er (Lexington MA). In 14 random t r i a l s , blood was taken from the gas chamber and catecholamine levels were measured follow-ing the technique described in chapter I. 4-ANALYSIS OF DATA: Heart rate, mean a r t e r i a l blood pressure as well as perfusion pressure and blood flow i n both legs were averaged over one minute inter v a l s before and throughout dives for up to 8 minutes. Si g n i f i c a n t differences i n heart rate and mean a r t e r i a l blood pressure among the 5 groups of ducks were established using an analysis of variance, followed i n the case of s i g -nificant differences by the Newman-Keuls test. S i g n i f i c a n t differences i n perfusion pressure and blood flow between test and autoperfused legs, were established by using a paired Student's t - t e s t . Comparisons of perfusion pressure and blood flow among the 4 groups of ducks whose tes t legs were perfused with blood mixtures devoid of catecholamines, were done using an analysis of variance, followed in the case of significant differences by the Newman-Keuls test. In order to compare the e f f e c t s that these d i f f e r e n t blood mixtures have on the p e r i p h e r a l v a s o c o n s t r i c t i o n , l i n e a r regression between the changes of vascular resistance i n the autoperfused leg and in the associated test leg during diving 148 were calculated in the 4 groups of ducks. Significant d i f f e r -ences among t h e i r slopes were obtained using an analysis of covariance followed by the Newman-Keuls t e s t . S t a t i s t i c a l l y s i g n i f i c a n t differences i n vascular conductance between test legs perfused with hypoxic-hyperoxic blood with or without infusion of catecholamines were established by using unpaired Student's t-test. The f i d u c i a l l i m i t for a sig n i f i c a n t difference was set at a pr o b a b i l i t y of 5% (p < 0.05). In the text and graphs, values are given as mean + standard error of the mean. When perfusion pressure in autoperfused and test legs at rest differed by 15% and more, the ducks were not included i n the experiment. Two ducks were dismissed on this c r i t e r i o n . 149 RESULTS. 1- HEART RATES (HR) AND MEAN ARTERIAL BLOOD PRESSURES (MABP): A l l ducks i n t h e 5 groups p r e s e n t e d the c l a s s i c d i v i n g brady-c a r d i a ( F i g u r e 2 4 ) . There was no s i g n i f i c a n t d i f f e r e n c e i n p r e - d i v e and d i v e h e a r t r a t e s or mean a r t e r i a l b l o o d p r e s s u r e s among the 5 groups of ducks. 2- EFFECTS OF HYPOXIC AND / OR HYPERCAPNIC BLOOD DEVOID OF CATECHOLAMINES ON THE VASCULAR RESISTANCE OF HIND LIMBS DURING  DIVING: PO2, PCO2 and pH o f the b l o o d p e r f u s i n g t e s t and autoper-f u s e d l e g s of ducks were measured at the b e g i n n i n g and at the end o f a l l d i v e s ( f i g u r e 2 5 ) . To a s c e r t a i n t h a t t h e method used t o remove 60 ml o f b l o o d from ducks d i d not t r i g g e r a r e l e a s e o f c a t e c h o l a m i n e s , l e v e l s o f NE and EP were measured i n t h e b l o o d p e r f u s i n g t h e t e s t l e g . These v a l u e s (2.8 + 0.5 nM f o r NE and 0.3 +_ 0.1 nM f o r EP) were below t h e r e s t i n g plasma l e v e l s measured i n ducks i n chapters I and I I . A f t e r 2 m i n u t e s d i v i n g , p e r f u s i o n p r e s s u r e i n t h e l e g p e r f u s e d w i t h h y p o x i c - h y p e r c a p n i c b l o o d dropped s i g n i f i c a n t l y below t h a t o f the a u t o p e r f u s e d l e g , and remained s i g n i f i c a n t l y l o w e r t h r o u g h o u t t h e d i v e ( F i g u r e 2 6 ) . D u r i n g d i v i n g , t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n p e r f u s i o n p r e s s u r e between a u t o p e r f u s e d and t e s t l e g s o f ducks p e r f u s e d w i t h h y p e r o x i c -hypocapnic b l o o d ( F i g u r e 27) or w i t h h y p o x i c - h y p o c a p n i c b l o o d 150 FIGURE 24: Heart rate and central mean a r t e r i a l blood pressure during diving in ducks whose legs were perfused with hypoxic and hypercapnic blood (open t r i a n g l e s ) , with hyperoxic and hypocapnic blood (open diamonds), with hypoxic and hypocapnic (open squares), with hyperoxic and hypercapnic blood (open circles) and with hypoxic and hypercapnic blood with catecho-lamines (inverse open triangles) . 151 MEAN ARTERIAL BLOOD PRESSURE (mm Hg) o HEART BEATS ( b e a t s / minute) o o cn o -f-M O O -+-cn o o -+ O O O cn o ho o o cn o -+ fo CO oo --l/Y / J J L i \ \v \ lllOlflM Hh>h v\ FIGURE 25: Blood gas tensions and pH of blood perfusing autop-erfused and test legs at the beginning (hollow bars) and at the end ( f i l l e d bars) of dives. Group with hypoxic and hypercapnic hind limb perfusion (A), hyperoxic and hypercapnic perfusion (B), hypoxic and hypocap-nic perfusion (C), hyperoxic and hypocapnic perfusion (D) , hypoxic and hypercapnic perfusion with catecholamines (E). 153 X e E CN o CL 1001 60-X E E O O a. x Q. B D CONTROL LEGS EXPERIMENTAL LEGS 154 FIGURE 2 6: Perfusion pressure and blood flow i n hind limbs of ducks whose legs during diving were perfused with t h e i r own blood (autoperfused leg, hollow t r i a n g l e s ) , and with hypoxic and hypercapnic blood (test leg, f i l l e d t riangles). +: s i g n i f i c a n t l y different from control legs. In the lower graph, the f i l l e d t r i a n g l e s overlap the open triangles. 155 TEST LEG: HYPOXIA AND HYPERCAPNIA WITHOUT CATECHOLAMINES 200--150-1 0 0 -DIVE TIME (minute) 156 FIGURE 27: Perfusion pressure and blood flow in hind limbs of ducks whose legs during diving were perfused with t h e i r own blood (autoperfused leg, hollow diamonds), and with hyperoxic and hypocapnic blood (test leg, f i l l e d diamonds) . In the lower graph, the f i l l e d diamonds overlap the open diamonds. 157 TEST LEG: HYPEROXIA AND HYPOCAPNIA WITHOUT CATECHOLAMINES 250 + 200 + 150 4-100 + 50 DIVE TIME (minute) 158 ( F i g u r e 2 8 ) . However, a f t e r 2 m i n u t e s o f d i v i n g , p e r f u s i o n w i t h h y p e r o x i c - h y p e r c a p n i c b l o o d caused p e r f u s i o n p r e s s u r e i n t e s t l e g t o f a l l s i g n i f i c a n t l y below t h a t o f t h e a u t o p e r f u s e d l e g ( Figure 29) . B e f o r e d i v i n g , t h e p e r f u s i o n p r e s s u r e i n a u t o p e r f u s e d l e g s was not s i g n i f i c a n t l y d i f f e r e n t from t h a t i n t h e t e s t l e g s ( F i g u r e s 26, 27, 28, 2 9 ) . No d i f f e r e n c e s i n b l o o d f l o w were o b s e r v e d b e f o r e and d u r i n g d i v i n g between t h e a u t o p e r -f u s e d l e g and t e s t l e g s i n any o f t h e 4 g r o u p s o f d u c k s ( F i g u r e s 26, 27, 28, 2 9 ) , except b e f o r e d i v i n g i n ducks p e r -fused w i t h hypoxic-hypocapnic b l o o d (Figure 28). Hind l i m b b l o o d f l o w d u r i n g d i v i n g dropped d r a s t i c a l l y i n a l l ducks ( F i g u r e s 26, 27, 28, 2 9 ) . No d i f f e r e n c e s i n b l o o d f l o w were observed b e f o r e and d u r i n g d i v e s among the 4 groups of ducks, except f o r t h e ducks p e r f u s e d w i t h h y p e r o x i c - h y p o -c a p n i c b l o o d which a t 3 minutes i n t o t h e d i v e had a s i g n i f i -c a n t l y h i g h e r b l o o d f l o w t h a n ducks p e r f u s e d w i t h h y p o x i c -h y p e r c a p n i c b l o o d ( F i g u r e s 26, 27) . B e f o r e d i v i n g , p e r f u s i o n p r e s s u r e i n t e s t l e g s was t h e same i n a l l 4 groups o f ducks ( F i g u r e s 26, 27, 28, 2 9 ) . P e r f u s i o n p r e s s u r e i n a u t o p e r f u s e d l e g s b e f o r e and d u r i n g d i v i n g was not s i g n i f i c a n t l y d i f f e r e n t among the 4 groups o f ducks (Figures 26, 27, 28, 29). An example o f t r a c e s r e c o r d e d d u r i n g a d i v e when the duck was p e r f u s e d w i t h h y p o x i c - h y p e r c a p n i c b l o o d i s g i v e n i n f i g u r e 30A. P e r f u s i o n p r e s s u r e i n t h e t e s t l e g was lower than i n the aut o p e r f u s e d l e g . Another d i v e was completed 30 minutes l a t e r FIGURE 28: Perfusion pressure and blood flow in hind limbs of ducks whose legs during diving were perfused with t h e i r own blood (autoperfused leg, hollow squares), and with hypoxic and hypocapnic blood (test leg, f i l l e d squares). +: s i g n i f i c a n t l y different from control legs. In the lower graph, the f i l l e d squares overlap the open square. 160 TEST LEG: HYPOXIA HYPOCAPNIA WITHOUT CATECHOLAMINES DIVE TIME (minute) 161 FIGURE 29: Perfusion pressure and blood flow in hind limbs of ducks whose legs during diving were perfused with t h e i r own blood (autoperfused leg, hollow c i r c l e s ) , and with hyperoxic and hypercapnic blood (test leg, f i l l e d c i r c l e s ) . +: s i g n i f i c a n t l y different from control legs. In the lower graph, the f i l l e d c i r c l e s overlap the open c i r -cles . 162 TEST LEG: HYPEROXIA AND HYPERCAPNIA WITHOUT CATECHOLAMINES 163 FIGURE 30: Exemples of traces recorded during 2 dives: mean a r t e r i a l blood pressure (MAB), hind limb perfusion pressure and blood flow (Pp e rf/ F p e r f ^ * I n t h e f ^ r s t dive (A), only one leg (autoperfused leg) was perfused with the duck's own blood and the other (test leg) was perfused by hypoxic hypercapnic blood without catecholamines. In the second dive (B), both legs were autoperfused. 164 but i n t h i s case b o t h l e g s were a u t o p e r f u s e d ( F i g u r e 30B) and no d i f f e r e n c e s o c c u r r e d between the p e r f u s i o n p r e s s u r e s d u r i n g t h i s d i v e . R e s t i n g v a s c u l a r r e s i s t a n c e f o r a u t o p e r f u s e d and t e s t l e g s were 4 + 0.2 and 4 + 0 . 2 PRU ( P e r i p h e r a l R e s i s t a n c e U n i t s i n mmHg x min x m l - 1 ) r e s p e c t i v e l y f o r h y p o x i c - h y p e r c a p n i c p e r f u s i o n , 4 +_ 0.4 and 4 + 0.5 PRU f o r h y p e r o x i c - h y p o c a p n i c p e r f u s i o n , 3.8 +_ 0.2 and 4 + 0 . 1 PRU f o r hypoxic-hypocapnic p e r f u s i o n and 4.8 + 0.3 and 4.9 +_ 0.2 f o r h y p e r o x i c - h y p e r c a p n i c p e r f u s i o n . The r e l a t i o n s h i p between the a b s o l u t e i n c r e a s e i n v a s c u l a r r e s i s t -ance d u r i n g d i v i n g ( r e s i s t a n c e d u r i n g t h e d i v e - r e s i s t a n c e d u r i n g p r e - d i v e ) i n t h e t e s t l e g and t h e a b s o l u t e i n c r e a s e o f v a s c u l a r r e s i s t a n c e d u r i n g d i v i n g i n t h e a u t o p e r f u s e d l e g i s shown i n F i g u r e 31. The a b s o l u t e i n c r e a s e i n v a s c u l a r r e s i s t -ance i n t e s t l e g s was p o s i t i v e l y c o r r e l a t e d w i t h t h a t i n a u t o p e r f u s e d l e g s . The r e g r e s s i o n l i n e f o r d u c k s p e r f u s e d w i t h h y p o x i c -h y p e r c a p n i c b l o o d (A: F i g u r e 31) was not s i g n i f i c a n t l y d i f f e r -e n t from t h a t o f ducks p e r f u s e d w i t h h y p e r o x i c - h y p e r c a p n i c b l o o d (C: F i g u r e 3 1 ) . F u r t h e r m o r e , t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the r e g r e s s i o n l i n e f o r ducks p e r f u s e d w i t h h y p e r o x i c - h y p o c a p n i c b l o o d (B: F i g u r e 31) and t h e r e g r e s s i o n l i n e f o r ducks p e r f u s e d w i t h h y p o x i c - h y p o c a p n i c b l o o d (D: F i g u r e 31). However, the r e g r e s s i o n l i n e s of the two groups of ducks p e r f u s e d w i t h h y p e r c a p n i c b l o o d (A and C: F i g u r e 31) are s i g n i f i c a n t l y d i f f e r e n t from r e g r e s s i o n l i n e s o f ducks p e r -f u s e d w i t h hypocapnic b l o o d (B and D: F i g u r e 31). 166 FIGURE 31: R e l a t i o n s h i p between the absolute increase i n vascular r e s i s t a n c e during d i v i n g i n the t e s t legs and i n autoperfused legs. Ducks perfused with hypoxic and hypercapnic blood (A: f i l l e d t r i a n g l e s ) , hyperoxic and hypocapnic blood (B: hollow d i a -monds), hyperoxic hypercapnic blood (C: f i l l e d c i r c l e s ) , and hypoxic and hypocapnic blood (D: hollow squares). The l i n e a r r e g r e s s i o n equation, the standard e r r o r of the slope (in parentheses) and the coefficient of determination are shown on each graph. The slopes of the regression i n A and C are s i g n i f i c n t l y different from the slopes in B and D. 167 0 50 100 150.200.250.300 350 400 0 50 100 150 200 250 300 350 400 C D CHANGES IM VASCULAR RESISTANCE (P.EM1.): CONTROL LEG 168 2-EFFECTS OF CIRCULATING CATECHOLAMINES ON HIND LIMB VASCULAR  CONDUCTANCE (1 / RESISTANCE) DURING DIVING: V a s c u l a r conductance was measured i n l e g s p e r f u s e d w i t h h y p o x i c and h y p e r c a p n i c b l o o d d u r i n g d i v i n g . S a l i n e (0.136 — i ml.min ) i n f u s e d i n t o t h e b l o o d p e r f u s a t e b e f o r e and d u r i n g d i v i n g d i d not have any s i g n i f i c a n t e f f e c t on v a s c u l a r con-d u c t a n c e ( F i g u r e 3 2 ) . However i n f u s i n g s a l i n e c o n t a i n i n g c a t e c h o l a m i n e s a f t e r 3 minutes submergence caused a s i g n i f i -cant drop i n conductance (Figure 32) w i t h i n 5 minutes. Because b l o o d f l o w d u r i n g d i v i n g was v e r y low (0-2 ml/min.), at l e a s t 1 minute was r e q u i r e d f o r t h e i n f u s e d c a t e c h o l a m i n e s t o reac h the v a s c u l a r bed of the h i n d l i m b . 169 FIGURE 32: Vascular conductance i n hind limbs perfused with hypoxic and hypercapnic blood without ( f i l l e d t r i a n g l e s ) , and with catecholamines (inverse f i l l e d triangles) during diving. : switching from saline infusion to catecholamine infusion. * : s i g n i f i c a n t l y d i f f e r e n t from hind limbs perfused without catecholamines. 170 o o VASCULAR CONDUCTANCE (ml / min.mmHg) o b o o o i .i i 1 1 1 1 1 1 •< • -i i i 1 1 1 H 1 H • * 4 ^ « DISCUSSION. Comparison of the results of perfusion of hind limbs with hypoxic and hypercapnic blood with and without catecholamines demonstrated that c i r c u l a t i n g catecholamines played an impor-tant r o l e i n increasing peripheral vasoconstriction during diving. This supports the findings in chapter II: circulating catecholamines increase MDT by enhancing peripheral vasocon-s t r i c t i o n . Perfusion with hyperoxic and hypocapnic blood demonstrated that sympathetic nerves were effective enough to achieve the normal level of peripheral vasoconstriction during diving. Vascular resistance of hind limbs perfused with hyper-capnic blood during dives was always lower than those of control legs. This was not the case when perfusion was done with hypoxic mixtures; perfusion with hypoxic and hypocapnic blood did not a f f e c t vascular resistance during dives. Fur-thermore, the slope of the l i n e a r regression for hypoxic and hypercapnic perfusion was not s i g n i f i c a n t l y below that of hyperoxic and hypercapnic perfusion. This suggests that cir c u -l a t i n g catecholamines are needed to increase peripheral vascu-l a r resistance during diving because of the depressant effect of hypercapnia on the neural component of peripheral vasocon-s t r i c t i o n . Consequently they would enhance MDT by preventing the peripheral tissue from having access to the 0 2 stores. 172 Blood flow and vascular resistance measured i n autoper-fused legs before diving and at 2 minutes afte r submergence were si m i l a r to the values recorded with electromagnetic or u l t r a s o n i c flow probes (Butler and Jones 1971; Jones 1973; Smith 1987). This confirms that the present preparation did not affect the physiological values observed at rest or inter-fere with cardiovascular adjustment during the dive. This preparation presents advantages compared with t r a d i t i o n a l methods of perfusion which use constant flow or pressure. By allowing blood flow and p e r f u s i o n pressure to be kept at physiological values, this preparation i s adequate to perform any perfusions involving strong variations of vascular r e s i s t -ances, without creating any a r t i f a c t i n the measurement of vascular resistance, or causing any discomfort for the unanes-thetised animal. The results obtained in t h i s chapter contrast with those of Gooden (1980) who suggested that hypoxia i n h i b i t e d neuro-genic vasoconstriction during diving. The experiments done by Gooden were very different from those described in this chap-t e r : duckling and chicken mesenteric a r t e r i e s were isolated, perfused and exposed to intravascular and extravascular hypox-i a for 45 minutes i n order to mimic diving. He observed that hypoxia depressed vasoconstriction mediated by intravascular i n j e c t i o n of NE or mediated by nerve stimulation. However, nervously mediated vasoconstriction was depressed to a s i g n i f -icantly greater extent, and surprisingly did not appear to be more resistant to hypoxia in duckling than in chicken. Gooden speculated that c i r c u l a t i n g catecholamines during d i v i n g 173 sustain vasoconstriction and thus compensate for the strong depression by hypoxia of the neurally mediated vasoconstric-t i o n . However, since 45 minutes of hypoxic exposure i s far longer than any MDT observed i n Pekin ducks, the r e s u l t s obtained i n these experiments may not be representative of physiological responses that occur in forced dived ducks. Peripheral vasoconstriction i s mediated by the sympa-t h e t i c branch of the autonomic nervous system (Butler and Jones 1971; Andersen and Blix 1974). Peripheral vasoconstric-t i o n during diving i s extremely powerful because i t can shut down circulation in the peripheral organs in order to spare 0 2 stores for the heart and brain (Jones et a l . 1979; Heieis and Jones 1988) . Folkow et a l . (1966; 1971) demonstrated that peripheral vasoconstriction in diving birds (ducks) and mam-mals (nutria) i s far more e f f i c i e n t than that of non-divers such as turkeys and cats. In ducks, the larger arteries s i t u -ated outside the muscles are narrower and t h e i r sympathetic innervation i s f a r more abundant than i n non-divers which explains t h e i r strong vasoconstrictor c a p a b i l i t i e s (Folkow et a l . 1966). Moreover, i n sp i t e of production of vas o d i l a t o r metabolites by peripheral tissues during diving, vasoconstric-t i o n i s maintained i n ducks. Folkow et a l . (1966) induced a p e r i p h e r a l v a s o c o n s t r i c t i o n i n anaesthetised vagotomised ducks, comparable to that observed i n i n t a c t ducks during forced dives, by giving them a hypercapnic-hyperoxic mixture to breath combined with controlled bleeding. They observed not only that blood flow to the leg almost stopped during vasocon-174 s t r i c t i o n , but also that exercising the leg during intense vasoconstriction did not increase blood flow. These re s u l t s contrast with those obtained with cats, where v a s o d i l a t a t i o n produced by exercise overcame the intense neurogenic c o n s t r i c t i o n (Kjellmer 1965; Folkow et a l . 1966). Folkow et a l . (1966) suggested that ascending vasodilatation i s not observed i n ducks during d i v i n g because an intense vasoconstriction occurs i n the larger and r i c h l y innervated arteries which are situated outside the skeletal muscles, and hence out of reach of v a s o d i l a t o r metabolites produced by these muscles. Folkow et a l . concluded that the vasocon-s t r i c t i o n observed i n ducks was neurogenic only, and they did not attribute any p a r t i c i p a t i o n of adrenal catecholamines to the peripheral vasoconstriction even though hypercapnia and hemorrhage could have triggered a release of catecholamines by the adrenal glands (Engeland et aJL. 1981; Mangalam et a l . 1987). My experiments d i f f e r e n t i a t e d between neurogenic and humoral v a s o c o n s t r i c t i o n , and showed that the neurogenic vasoconstriction i s not maintained against the vasodilating action of CC>2. The a b i l i t y of the peripheral vasoconstriction to withstand the metabolic d i l a t o r influence in ducks appears to reside primarily i n the action of c i r c u l a t i n g catechola-mines di r e c t l y at the s i t e of the vasoconstriction. It has been shown that hypercapnia can depress vasocon-s t r i c t i o n induced by the sympathetic branch of the autonomic nervous system. Hypercapnia acts on i s o l a t e d blood vessels inducing a s l i g h t r e l a x a t i o n whereas hypocapnia causes a slight contraction (Roger et aJL. 1965; Kontos 1971; Edvinsson 175 and Sercombe 1976). These e f f e c t s a r e m a r k e d l y e x a g g e r a t e d when the a l p h a - a d r e n e r g i c r e c e p t o r s i n the v a s c u l a r w a l l s are a c t i v a t e d by c a t e c h o l a m i n e s : h y p e r c a p n i a d e p r e s s e s and hypo-c a p n i a p o t e n t i a t e s t h e v a s o c o n s t r i c t o r a c t i o n o f c i r c u l a t i n g c a t echolamines or of s y m p a t h e t i c nerves (Bygdeman 1963; Roger et a l . 1965; E d v i n s s o n and Sercombe 1976; Hjemdahl and F r e d -holm 1976). In f a c t , i t i s the changes of b l o o d pH r e s u l t i n g from h y p e r c a p n i a and h y p o c a p n i a w h i c h reduce or p o t e n t i a t e t h e vasomotor a c t i o n o f c a t e c h o l a m i n e s (Roger e t a_l. 1965; Edvinsson and Sercombe 197 6) . T h i s means t h a t more c a t e c h o l a -mines a r e needed f o r a g i v e n i n t e n s i t y o f v a s o c o n s t r i c t i o n when v e s s e l s a r e s u b j e c t e d t o h y p e r c a p n i a . The s y m p a t h e t i c nerves appear t o be unable t o supply the e x t r a c a t e c h o l a m i n e s r e q u i r e d f o r t h e maintenance of t h e p e r i p h e r a l v a s o c o n s t r i c -t i o n d u r i n g d i v i n g . T h i s e x t r a dose o f c a t e c h o l a m i n e s i s s u p p l i e d by the a d r e n a l g l a n d s . S e v e r a l mechanisms c o u l d be p r o p o s e d t o e x p l a i n t h e d e p r e s s i o n o f s y m p a t h e t i c v a s o c o n s t r i c t i o n by hydrogen i o n s . 1) A c i d o s i s a c t s by r e d u c i n g t h e r e l e a s e o f NE by t h e sympa-t h e t i c n e r v e e n d i n g s ( S h e p h e r d and V a n h o u t t e 1 9 8 5 ) . 2) A c i d o s i s c o u l d i n c r e a s e t h e a c t i v i t y o f C a t e c h o l - O -M e t h y l t r a n s f e r a s e , and t h e r e f o r e t h e r e m o v a l o f NE f r o m e x t r a n e u r o n a l s i t e s would be i n c r e a s e d . However, t h e o p t i m a l pH f o r t h i s enzyme i s between 7.5 and 8.2 ( A x e l r o d and Tom-c h i c k 1958) which does not l e n d credence t o t h i s h y p o t h e s i s . 3) Gende et al.. (1985) showed t h a t H + d e c r e a s e s t h e a f f i n i t y o f c a r d i a c b e t a - a d r e n o r e c e p t o r s f o r a g o n i s t s . T h i s may a l s o o c c u r i n t h e a l p h a r e c e p t o r s i n b l o o d v e s s e l s when s u b m i t t e d 176 to acidosis. 4) Increased concentrations of hydrogen ion can i n t e r f e r e with exitation-contraction coupling (Nakamaru and Swartz 1972; Inesi and H i l l 1983). Therefore, c o n t r a c t i l i t y of the vascular smooth muscle would.decrease due to muscle f a -tigue . 177 GENERAL DISCUSSION. 178 This work demonstrates the importance of the adrenal glands i n aquatic a i r breathing animals during forced submer-sion, and the following mechanism of action of adrenal catech-olamines i s proposed. As soon as a Pekin duck's head i s submerged, Pa0 2 and pHa drop, and PaC02 increases. This stimu-lates the chemoreceptors and t r i g g e r s a drop i n heart rate, mediated by the vagus, and an increase of peripheral r e s i s t -ance mediated by the sympathetic nerves. These cardiovascular adjustments allow a r e d i s t r i b u t i o n of 0 2 blood stores toward the heart and brain. As the dive continues, the increase of PaC0 2 (which i s responsible for the large drop of pHa observed during diving: Shimizu and Jones 1987) depresses the neurogen-i c component of the vasoconstriction. Without the release of catecholamines from the adrenal glands (as in adrenalectomised (ADX) and adrenal denervated (DNX) ducks), the p e r i p h e r a l t i s s u e s would have access to the 0 2 blood stores, and the maximum dive time (MDT) would be reduced. However, in intact ducks, catecholamines released by the adrenal glands compen-sate for the depressant action of hypercapnia on the neural 179 component of the peripheral vasoconstriction. Thus, peripheral resistance i s maintained during diving i n sp i t e of the i n -crease of hypercapnia. Oxygen stores are not depleted by the peripheral tissues which explains how MDT i s enhanced. The function of the sympathetic nerves could be to i n i t i a t e a rapid increase i n peripheral resistance at the beginning of dives, i n order to prevent 0 2 stores from being prematurely depleted by peripheral tissues. Several intriguing questions, some of which have been discussed i n the previous chapters, have been raised by the results obtained in this work. Adrenal catecholamines, which comprise 100% of the epine-phrine and 40 to 80% of the norepinephrine found in the blood during diving, promote MDT but they do not seem to act alone. Other adrenal products whose release i s also triggered by the adrenal nerves may be implicated. These substances have not ..yet been i d e n t i f i e d but i t was suggested in Chapter 2 that adrenal opiates are the most l i k e l y contenders. Adrenal catecholamines do not promote MDT by protecting the heart against the depressant effects of hypoxia, hypercap-nia or acidosis, or even by maximizing pulmonary uptake of 0 2 by the blood by increasing blood a f f i n i t y for 0 2 during acido-s i s . Instead adrenal catecholamines enhance MDT primarily by i n c r e a s i n g p e r i p h e r a l r e s i s t a n c e during d i v i n g . They act dir e c t l y on the site of vasoconstriction i t s e l f and compensate for the depressant e f f e c t of C0 2 on the neurogenic component of the vasoconstriction e l i c i t e d during diving. However, two other mechanisms by which adrenal catecholamines could enhance 180 peripheral vasoconstriction during diving, must also be con-sidered: 1) In studies on rats, c i r c u l a t i n g catecholamines appear to p r o t e c t the b r a i n against ischemic damage by a d i r e c t action on the brain i t s e l f (Koide et al,. 1986) . The mechanism of such an action i s not known. If the r i s e of catecholamine l e v e l s i s prevented during diving, normal functions of the cardiovascular centers may be adversely affected i n hypoxic conditions and consequently peripheral vasoconstriction would be impaired. 2) It i s known that c i r c u l a t i n g catecholamines increase the a c t i v i t y of the ca r o t i d chemoreceptor nerve fibers (Fol-gering et a l . 1982; Milsom and Sadig 1 9 8 3 ) , and that the d i v i n g response of Pekin ducks i s mainly t r i g g e r e d by the hypoxic stimulus on the peripheral chemoreceptors (Jones et  a l . 1 9 8 2 ) . This suggests that during d i v i n g , c i r c u l a t i n g catecholamines may potentiate the c a r o t i d body response to hypoxia and consequently may enhance the peripheral vasocon-s t r i c t i o n . I f the release of c i r c u l a t i n g catecholamines i s suppressed, a weaker diving response would occur, resulting in a shortened MDT. The action of c i r c u l a t i n g catecholamines on ca r o t i d chemoreceptors, however, i s mediated by beta-adreno-ceptors (Folgering et a l . 1982; Milsom and Sadig 1983). Since no s i g n i f i c a n t decrease of MDT was observed i n beta-receptor blocked dives compared with c o n t r o l dives, the e f f e c t of c i r c u l a t i n g catecholamines on MDT would not appear to be mediated through an additional excitation of the chemorecep-181 t o r s d u r i n g d i v e s . The p o s s i b i l i t y t h a t t h e a c t i o n o f c i r c u -l a t i n g c a t e c h o l a m i n e s on chemoreceptors i s mediated by a l p h a -a d r e n e r g i c r e c e p t o r s has not been s t u d i e d . However, t h i s does not seem v e r y l i k e l y because M i l s o m and S a d i g (1983) demon-s t r a t e d t h a t t h e e f f e c t s o f n o r e p i n e p h r i n e on chemoreceptors were c o m p l e t e l y a b o l i s h e d by b e t a - b l o c k a d e . The main r o l e o f t h e s y m p a t h e t i c nerves and o f t h e adre-n a l g l a n d s d u r i n g d i v i n g a p p e a r s t o be t o p r o v i d e a s t r o n g i n c r e a s e o f p e r i p h e r a l v a s c u l a r r e s i s t a n c e i n o r d e r t o p r e -s e r v e C>2 s t o r e s f o r t h e h e a r t and b r a i n . Some o f t h e e x p e r i -ments i n t h i s t h e s i s a l s o suggest t h a t t h e s y m p a t h e t i c nerves and / o r t h e a d r e n a l g l a n d s c o u n t e r b a l a n c e t h e d e p r e s s a n t i n f l u e n c e s e x e r t e d on c a r d i a c c o n t r a c t i l i t y by v a g a l a c t i v i t y , h y p o x i a , h y p e r c a p n i a and a c i d o s i s . C o n s e q u e n t l y one o f t h e a c t i o n o f catecholamines appear t o m a i n t a i n s t r o k e volume near th e p r e - d i v e v a l u e s throughout t h e d i v e . I s the maintenance o f s t r o k e volume an a d a p t a t i v e advan-t a g e f o r c o o p i n g w i t h a p n o e i c a s p h y x i a ; o r i s i t s i m p l y an i n d i r e c t e f f e c t o f t h e h i g h l e v e l s o f c i r c u l a t i n g c a t e c h o l a -mines whose p r i m e f u n c t i o n i s t o enhance p e r i p h e r a l r e s i s t -ance? From t h e s e r i e s o f experiments d e s c r i b e d i n Chapter 4, i t w o u l d seem t h a t t h e second s u g g e s t i o n i s more l i k e l y be-cause p r e v e n t i n g t h e a c t i o n o f catecholamines on the h e a r t d i d n o t d i m i n i s h t h e ducks' a b i l i t y t o t o l e r a t e l o n g p e r i o d s o f submersion. 182 The effects of the sympathetic and parasympathetic sys-tems on heart rate have long been studied i n mammals (Rosen-blueth and Simeone 1934; Samaan 1935; Levy and Zieske 1969; Warner and Russell 1969). It has been established that t h e i r antagonistic e f f e c t s are not additive, but that interactions take place between the two systems (Levy 1971, 1984; Berne and Levy 1981; Chassaing et a l . 1 9 8 3 ) . F u r i l l a and Jones (1987) examined the relationship of heart rate to b i l a t e r a l stimula-tion of the cardiac parasympathetic and sympathetic nerves i n Pekin ducks (Figure 3 3 ) . By comparing heart rate during d i v i n g i n c o n t r o l and beta blocked ducks from the present study with values obtained by these authors, an estimation of the r e l a t i v e contributions of the two components of the auto-nomic nervous system can be evaluated. During the propranolol dives there was l i t t l e or no sympathetic a c t i v i t y : the e s t i -mated l e v e l of vagal a c t i v i t y decreased from 1 0 0 % at 2 minutes into the dive (A: f i g u r e 33; when bradycardia was f u l l y developed) to approximately 63% a f t e r 10 minutes (B: figure 3 3 ) . However during the control dives, heart rates at 10 minutes into the dive were not si g n i f i c a n t l y different from those at 2 minutes, and sympathetic a c t i v i t y was at i t s maximum. (100%) because of the huge release of c i r c u l a t i n g catecholamines. But, according to figure 33, such low heart rates cannot be produced with t h i s degree of sympathetic s t i m u l a t i o n . Three hypotheses are o f f e r e d to exp l a i n the divergence between my results and those of F u r i l l a and Jones. 1) The maximum l e v e l of stimulation of parasympathetic nerves may not have been reached during F u r i l l a and Jones' 183 FIGURE 33: The relationship of heart rate to b i l a t e r a l stimula-t i o n of the d i s t a l cut ends of the vagus and cardiac sympa-t h e t i c nerves. 100% represents the frequency of stimulation above which no further changes i n heart rate occurred with increases i n stimulation frequency (from F u r i l l a and Jones 1987a). Heart rate and cardiac sympathetic and parasympathetic nerves a c t i v i t i e s in propranolol injected ducks at 2 minutes (A) and 10 minutes (B) into diving. 184 Heort Rate (min"1) (^UIUJ) ejoy 1JD9H 185 (1987) experiments. 2) There i s no c a r d i a c s y m p a t h e t i c c h r o n o t r o p i c a c t i v i t y d u r i n g d i v i n g (the a c t i o n o f the c a r d i a c sympathetic nerves i s m i n i m a l ) , and the a c t i o n of c i r c u l a t i n g catecholamines i s o n l y s e l e c t i v e l y i n o t r o p i c . . H o w e v e r , i t i s d i f f i c u l t t o see by what mechanism c i r c u l a t i n g c a t e c h o l a m i n e s c o u l d be c o m p l e t e l y prevented from a c t i n g on the s i n o a t r i a l node. 3) H y p o x i a and i s c h a e m i a a l s o d e p r e s s t h e s i n o a t r i a l n o dal r a t e and c o n d u c t i o n ( B i l l e t t e e t a l . 1973; B e l l a r d i n e l l i e t a l . 1980, 1981; Weiss and S h i n e 1981; McKean and Landon 1982; Stowe et a l . 1985). Thus, d u r i n g d i v i n g , t h e a c t i o n o f t h e vagus on t h e h e a r t r a t e c o u l d be p o t e n t i a t e d by h y p o x i a and a c i d o s i s ( C o u r t i c e et a l . . 1983; P o t t e r e t a l . 1986). This c o u l d e x p l a i n the d i s c r e p a n c y between v a l u e s i n c o n t r o l d i v e s , and v a l u e s i n f i g u r e 33 because i n t h e i r experiments, F u r i l l a and Jones d i d not s u b j e c t t h e ducks' h e a r t s t o c o n d i t i o n s o f hypoxia, hypercapnia and a c i d o s i s s i m i l a r t o those encountered d u r i n g d i v i n g . I n o r d e r t o f u l l y u n d e r s t a n d t h e r e g u l a t i o n o f h e a r t d u r i n g d i v i n g , t h e r e l a t i o n s h i p o f h e a r t r a t e , s t r o k e volume and c a r d i a c c o n t r a c t i l i t y t o b i l a t e r a l s t i m u l a t i o n o f c a r d i a c s y m p a t h e t i c and p a r a s y m p a t h e t i c nerves s h o u l d be s t u d i e d when t h e h e a r t i s p e r f u s e d w i t h b l o o d o f d i f f e r e n t gas t e n s i o n s , w i t h and w i t h o u t c i r c u l a t i n g catecholamines. There i s a steady i n c r e a s e of c i r c u l a t i n g c a t e c h o l a m i n e s d u r i n g d i v i n g , t o over 1000 times p r e - d i v e l e v e l s . One minute a f t e r the end o f the d i v e the l e v e l s of c i r c u l a t i n g c a t e c h o l a -186 mines are s t i l l h i g h : a p p r o x i m a t e l y 100 t i m e s p r e - d i v e l e v e l s (Hudson and Jones 198 6 ) . T h i s r a i s e s some q u e s t i o n s about the a b i l i t y o f the b l o o d f l o w t o resume i n the p e r i p h e r a l t i s s u e s a f t e r t h e d i v e . Some o f t h e ducks i n Chapter 4 were d i v e d u n t i l they reached t h e i r maximum l i m i t o f underwater t o l e r a n c e c h a r a c t e r i s e d by an i n c r e a s e o f t h e i r h e a r t r a t e s toward p r e -d i v e v a l u e s ( F i g u r e 3 4 ) . At t h e end o f d i v e s and d u r i n g t h e f i r s t minute of post d i v e r e c o v e r y , the i n c r e a s e i n h e a r t r a t e was not a s s o c i a t e d w i t h an i n c r e a s e o f h i n d l i m b b l o o d f l o w ( F i g u r e 3 4 ) . Thus, c i r c u l a t i n g c a t e c h o l a m i n e s appeared t o m a i n t a i n p e r i p h e r a l v a s o c o n s t r i c t i o n t h r o u g h o u t t h e d i v e and c o n t i n u e d t o do so, even a f t e r e m ersion, i n t h e e a r l y s t a g e s of the recovery p e r i o d . I t i s q u i t e c l e a r t h a t the i n c r e a s e o f hea r t r a t e at the end o f d i v i n g i s not caused by a c o l l a p s e of t h e p e r i p h e r a l v a s o c o n s t r i c t i o n . An h y p o t h e s i s can be ad-vanced t o e x p l a i n why h e a r t r a t e i n c r e a s e s when t h e maximum underwater t o l e r a n c e i s reached. The f l a t t e n i n g o f t h e EEG seen when maximum unde r w a t e r t o l e r a n c e i s r e a c h e d (Hudson and Jones 1986), i n d i c a t e s t h a t b r a i n f u n c t i o n has d e t e r i o r a t e d . C e n t r a l c a r d i o v a s c u l a r n u c l e i s h o u l d a l s o be a f f e c t e d and co n s e q u e n t l y t h e a c t i v i t y of the s y m p a t h e t i c and p a r a s y m p a t h e t i c n e r v e s may a l s o be i m p a i r e d . T h i s c o u l d e x p l a i n why t h e b r a d y c a r d i a i s n o t m a i n t a i n e d anymore. P e r i p h e r a l v a s o c o n s t r i c t i o n a t MDT would be main-t a i n e d , however by the c i r c u l a t i n g c a t e c h o l a m i n e s . A s s o c i a t e d w i t h t h i s r i s e of h e a r t r a t e , a decrease of s t r o k e volume must o c c u r b e c a u s e , a t MDT mean a r t e r i a l b l o o d p r e s s u r e i s n o t s i g n i f i c a n t l y h i g h e r than i t was p r e - d i v e , even though p e r i p h -e r a l v a s o c o n s t r i c t i o n i s a t i t s maximum. Thus, d e t e r i o r a t i o n 187 FIGURE 34: Heart rate (HR), mean a r t e r i a l blood pressure (MAB), hind limb perfusion pressure and blood flow ( P p e r f ; Fperf) ^ n a duck during forced dive and recovery. 188 Dt/E TIME (minute) 189 of brain function as well as cardiac f a i l u r e caused by the strong hypoxemia and acidosis may determine the maximum under-water tolerance. Under these circumstances, the heart and brain could be protected by the c i r c u l a t i n g catecholamines which continue to maintain peripheral vasoconstriction, even after emersion, i n the early stages of the recovery period. This would allow the heart and brain to recover before being h i t by the discharge of lactate flushed from the peripheral tissues. After 3 minutes of recovery, hind limb blood flow and vascular resistance are back to t h e i r pre-dive values. The high l e v e l of heart rate associated with a lower blood pres-sure indicate however that stroke volume i s s t i l l low and that the heart has not f u l l y recovered yet. To prevent oxygen stores from being prematurely depleted, vasoconstriction of the peripheral tissues i s quickly estab-l i s h e d at the s t a r t of the forced dive by the sympathetic nerves. As the dive progresses, vasoconstriction may slacken owing to the depressant action of hypercapnia; however, t h i s e f f e c t i s counteracted by the release of adrenal catechola-mines which serve to maintain vasoconstriction. This action d i f f e r s from that which happens in most voluntary dives. The stimuli which lead to a release of c i r c u l a t i n g catechola-mines (such as the v a r i a t i o n i n blood gas tension and the stimulation of nasoreceptors) are triggered during voluntary diving (see General Introduction). Most of the dives observed for free swimming birds and mammals are aerobic (see General Introduction) and consequently, are without much need of 190 peripheral vasoconstriction, nor of a large increase in circu-l a t i n g catecholamines. This raises some questions: i s there, or i s there not, a release of adrenal catecholamines during voluntary aerobic dives? If there i s , how could i t be prevent-ed from causing peripheral vasoconstriction? If there i s n ' t , what are the mechanisms which prevent t h i s release? In free diving mammals, the forced dive pattern of responses has been observed during long anaerobic exploratory dives (see General Introduction). 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