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The adrenal gland and the diving response in ducks (Anas platyrhynchos) Mangalam, Harry Joseph 1984

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THE ADRENAL GLAND AND THE DIVING RESPONSE IN DUCKS (ANAS PLATYRHYNCHOS) by HARRY J MANGALAM / it?-B.Sc. (Hon), The U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the FACULTY OF GRADUATE STUDIES Department of Zoology Ne accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1984 ©Harry Joseph Mangalam, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^ooio The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date I V . I 2\ W DE-6 £3/81) A b s t r a c t The extreme e l e v a t i o n in plasma l e v e l s of norepinephrine (NE) and epinephrine (EP) which occurs during f o r c e d d i v i n g of ducks (Anas platyrhynchos) was s t u d i e d before and a f t e r denervation of the adrenal glands. E l e v a t e d PaCO a, decreased a r t e r i a l pH, decreased blood glucose as w e l l as low PaO-^ have been d e s c r i b e d as c a u s a l factors in t h i s response. These v a r i a b l e s , as w e l l as blood pressure, heart r a t e and b r e a t h i n g frequency were measured in ducks dived a f t e r b r e a t h i n g a i r or pure 0X to c l a r i f y and q u a n t i f y the mechanism i n v o l v e d and i t s p h y s i o l o g i c a l f u n c t i o n . Both NE and EP c o n c e n t r a t i o n i n c r e a s e d by up to 2 orders of magnitude in the 4 minute dive p e r i o d , but by a s i g n i f i c a n t l y l e s s e r amount i f the duck breathed 0^, before the d i v e . N h i l e pH and PaCO^ were w e l l c o r r e l a t e d with the changes in plasma NE and EP l e v e l s d u r i n g both a i r and 0 A d i v e s , both pH and PaCO z changed more in the 0 Z t r i a l s , i n d i c a t i n g that they are not the primary cause of the response. Plasma glucose l e v e l s were v a r i a b l e . PaO a values l e s s than normal c o r r e l a t e d w e l l with i n c r e a s i n g NE and EP c o n c e n t r a t i o n s , but at high PaO^s, there was no c o r r e l a t i o n , suggesting that hypoxia i s the p e r m i s s i v e s t a t e f o r the f u l l response. Compared w i t h b r e a t h i n g a i r , b r e a t h i n g Ox b e f o r e the d i v e a t t e n u a t e d the d i v i n g b r a d y c a r d i a , e l i m i n a t e d the decrease i n b l o o d p r e s s u r e n o r m a l l y observed d u r i n g d i v e s , and caused more extreme c h a n g e s - i n pH, PaC0 1, and of c o u r s e , PaO^. D e n e r v a t i n g the a d r e n a l s d e c r e a s e d the amounts of both c a t e c h o l a m i n e s r e l e a s e d d u r i n g d i v e s a f t e r b r e a t h i n g a i r and 0^, EP s i g n i f i c a n t l y more than NE. A d r e n a l d e n e r v a t i o n per se d i d not cause a s i g n i f i c a n t change i n h e a r t r a t e , b l o o d p r e s s u r e , a r t e r i a l gas t e n s i o n s , pH, or plasma g l u c o s e changes d u r i n g d i v e s a l t h o u g h the o p e r a t i o n caused i n c r e a s e d v a r i a t i o n i n some of the parameters. In ducks, the cause f o r the c a t e c h o l a m i n e r e l e a s e i s d e c r e a s i n g Pa^a.? and f u l l e x p r e s s i o n of the response i s dependent on i n t a c t i n n e r v a t i o n of the a d r e n a l g l a n d , a l t h o u g h t h e r e i s a component t h a t i s u n a f f e c t e d by d e n e r v a t i o n . N h i l e p o s s i b l e r o l e s f o r t h i s response are d i s c u s s e d , the t r u e p h y s i o l o g i c a l f u n c t i o n of t h i s r esponse remains c a s t i n shadows. - i v-TABLE OF CONTENTS Page LIST OF FIGURES v LIST OF TABLES v i ACKNOHLEDGEMENTS v i i INTRODUCTION 1 MATERIALS AND METHODS General 5 Surgery • 5 Fhysi o l o g i c a l V a r i a b l e s 7 HPLC Methods 8 Experimental p r o t o c o l 10 A n a l y s i s of r e s u l t s • 1 2 RESULTS Plasma Catecholamines 13 Heart Rates 1 5 Blood Pressure 16 Plasma Glucose • 1 ? A r t e r i a l pH • • I 8 A r t e r i a l POj. 1 8 A r t e r i a l PC0 Z I 9 B r e a t h i n g Frequency ...20 DISCUSSION 2 1 REFERENCES 3 1 -, v-LIST OF FIGURES Page F i g u r e 1 - Plasma Catecholamine Concentrations Adrenal Denervates 13a F i g u r e 2 - Plasma Catecholamine Concentrations Sham Adrenal Denervates 13c F i g u r e 3 - Catecholamines as a f u n c t i o n of blood gases and pH • 13d Fi g u r e 4 - Heart Rate 15a F i g u r e 5 - Blood Pressure 16a F i g u r e 6 - Plasma Glucose Concentrations 17a F i g u r e 7 - A r t e r i a l pH 18a F i g u r e 8 - Blood Gases ....18b F i g u r e 9 - Breathing Frequency 20a -v i -LIST OF TABLES Page T a b l e 1 - L i n e s of best f i t and r e g r e s s i o n c o e f f i c i e n t s 13c ACKNOWLEDGEMENTS I would l i k e to express s p e c i a l thanks to my a d v i s o r , Dr. David R. Jones, and al s o to the f a c u l t y of the Dept. of Zoology f o r i n t r o d u c i n g me to some of the c h a l l e n g e s of s c i e n c e . One of the s e v e r a l l e s s o n s that I le a r n e d was that an i n v e s t i g a t i o n can not proceed in a vacuum; a simple adage, but one that I found to be too t r u e . Good s c i e n c e r e q u i r e s a give and take, a constant exchange of ideas, the sounding board of c o l l e a g u e s , in the same f i e l d and o u t s i d e of i t . I would l i k e to thank those at UBC who helped me in t h i s way, e s p e c i a l l y the l a d s in the l a b , Geoff Gabbott, Frank Smith, the Ma g n i f i c e n t Marks, H e i e i s and Douse, and Bob F u r i l l a , a l l of whom not only c o n t r i b u t e d ideas but helped me r e s u s i t a t e ducks and a m p l i f i e r s , decode pH meters and tape r e c o r d e r s , clean cages, and f i x my b i k e . I am b e t t e r f o r knowing them. 1 I n t r o d u c t i o n The d i v i n g r e f l e x i s a s e t of p h y s i o l o g i c a l r e s p o n s e s e x h i b i t e d to v a r i o u s e x t e n t s by a l l t e r r e s t r i a l v e r t e b r a t e s when immersed i n water and by some a q u a t i c v e r t e b r a t e s when wi t h d r a w n f r o m w a t e r . T h e s e r e s p o n s e s i n c l u d e a r e d i s t r i b u t i o n of b l o o d f l o w to t h o s e t i s s u e s most s e n s i t i v e to h y p o x i a , t h e h e a r t and b r a i n . The l u n g s a r e u n a v o i d a b l y i n c l u d e d i n homeotherms but t h e r e may be c o n s i d e r a b l e d i v e r s i o n of b l o o d t h r o u g h n o n - n u t r i t i v e s h u n t s . A l s o i n c l u d e d i n the s h o r t l i s t of p r e f e r e n t i a l l y p e r f u s e d o r g a n s i s the a d r e n a l g l a n d ( Z a p o l e_t a l ( 1 9 7 9 ) , J o h a n s e n ( 1 9 6 4 ) , McKean ( 1 9 8 2 ) , J o n e s e t a l ( 1 9 7 9 ) ) . I t has l o n g been known t h a t t h e r e i s a p o s i t i v e r e l a t i o n s h i p between t h e c o n d i t i o n s e n d u r e d i n a s p h y x i a and t h e r e l e a s e of a d r e n a l s e c r e t i o n s , n o t a b l y 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 t h e mammal (Cannon and H o s k i n s , 1911) and more r e c e n t l y i n the b i r d (Huang e_t a l , 1976, Hudson and J o n e s , 1 9 8 2 ) . The duck, e s p e c i a l l y , e x h i b i t s r e m a r k a b l e l e v e l s of c a t e c h o l a m i n e s (CAs) d u r i n g a f o r c e d d i v e , up to 1000 t i m e s h i g h e r than p r e d i v e r e s t i n g v a l u e s . The p r i m a r y c a u s e f o r t h i s i n c r e a s e i n a d r e n a l s e c r e t i o n i s s t i l l u n c e r t a i n . I t has been c o r r e l a t e d w i t h h y p o x i a , h y p e r c a p n e a , and i n c r e a s e d a c i d i t y (Cannon and H o s k i n s ( 1 9 1 1 ) , Cantu e_t a l ( 1 9 6 6 ) , C o m l i n e e_t a_l ( 1 9 6 5 ) ) and t h e r e i s s p e c u l a t i o n t h a t a s w e l l as b e i n g n e u r a l l y m e d i a t e d , t h e g l a n d may have some i n h e r e n t c h e m o s e n s i t i v i ty ( C o m l i n e and S i l v e r ( 1 9 6 1 ) , S t e i n s l a n d e_t a l ( 1 9 7 0 ) ) . The l i t e r a t u r e a l s o c o n t a i n s r e f e r e n c e s to e l e v a t e d l e v e l s of pla s m a CAs i n n e o n a t a l sheep and humans ( L a g e r c r a n t z and B i s t o l e t t i ( 1 9 7 3 ) , H olden e t a l ( 1 9 7 2 ) ) , i m m e d i a t e l y a f t e r b i r t h 2 and i n f e t a l sheep s u b j e c t e d to h y p o x i a j_n u t e r o (Cohen e_t a l (198 2 ) , Lewis e_t a l ( 1 9 8 2 ) ) . The d i v i n g r e f l e x and t h i s f e t a l r e s p onse to h y p o x i a a r e known to be s i m i l a r i n many r e s p e c t s ; t h i s CA response i s yet another s i m i l a r i t y . The enormous amount of e p i n e p h r i n e r e l e a s e d i n f o r c e d d i v e s f u r t h e r i m p l i e s t h a t the a d r e n a l g l a n d s have an important r o l e i n the d i v i n g r e s p o s e , but t h i s r o l e i s not un d e r s t o o d . Indeed, these h i g h l e v e l s of CAs are c u r i o u s f o r a number of r e a s o n s . CA r e l e a s e i s g e n e r a l l y a r e s u l t of s y m p a t h e t i c s t i m u l a t i o n - the a c t i v a t i o n of the f i g h t or f l i g h t r e s p o n s e . In t h i s r e s p o n s e , h e a r t r a t e and b l o o d p r e s s u r e i n c r e a s e and m e t a b o l i c changes i n c l u d e i n c r e a s e s i n g l y c o g e n o l y s i s and g l u c o n e o g e n e s i s , and i n glucagon and f r e e f a t t y a c i d r e l e a s e . C o n c u r r e n t l y , i n s u l i n r e l e a s e and h e p a t i c uptake of g l u c o s e a r e i n h i b i t e d . The s p l a n c h n i c and cutaneous c i r c u l a t i o n s a r e c o n s t r i c t e d and e x t r a b l o o d i s d i v e r t e d through the s k e l e t a l and c a r d i a c beds. T h i s g e n e r a l a c t i v a t i o n i s accompanied by an i n c r e a s e d m e t a b o l i c r a t e , a r e s u l t not p a r t i c u l a r l y d e s i r a b l e to an organism s t a r v e d f o r oxygen. N h i l e the f i g h t or f l i g h t response and the d i v i n g response s h a r e many c h a r a c t e r i s t i c s , n o t a b l y the s y m p a t h e t i c a c t i v a t i o n , i n the d i v i n g r e sponse h e a r t r a t e drops under v a g a l i n f l u e n c e , but b l o o d p r e s s u r e d e c r e a s e s o n l y s l i g h t l y as most of the p e r i p h e r y i s c o n s t r i c t e d ( S c h o l a n d e r e_t a l (1942), Andersen, ( 1 9 6 6 ) ) . By r e d u c i n g the mass of p e r f u s e d t i s s u e s , m e t abolism drops markedly and the CAs might p l a y a r o l e i n h e l p i n g to m o b i l i z e g l u c o s e and ot h e r s u b s t r a t e s t h a t c o u l d be used to m a i n t a i n metabolism a n a e r o b i c a l l y d u r i n g the p e r i o d of h y p o x i a . There i s qrowinq e v i d e n c e from t e l e m e t r i c ( B u t l e r and Noakes, 3 (1982a,b), Kanwisher e_t ad (1981)) and m e t a b o l i c s t u d i e s (Kooyman et a l ( 1 9 8 0 ) , C a s t e l l i n i et. a l . (1981)) on f r e e l y d i v i n g b i r d s and mammals t h a t the c l a s s i c a l d i v i n g response i s not invoked i n the v a s t m a j o r i t y of u n r e s t r a i n e d d i v e s . I t seems c l e a r t h a t the f u l l d i v i n g response i s e x h i b i t e d very rarely i n the w i l d , but i t may p l a y a c r i t i c a l p a r t i n extreme s i t u a t i o n s such as e s c a p i n g a p r e d a t o r or s e a r c h i n g f o r a new b r e a t h i n g h o l e . As w e l l , the r e f l e x i t s e l f i s of c o n s i d e r a b l e i n t e r e s t , as the sympatho-adrenal a x i s i s a major h o m e o s t a t i c mechanism i n v e r t e b r a t e s . P r e v i o u s t e s t s f o r CAs have been e i t h e r by b i o a s s a y or by r a d i o e n z y m a t i c assay (Passon and P e u l l e r , 1973), both of which can be very s e n s i t i v e , but are time-consuming and not h i g h l y a c c u r a t e , e s p e c i a l l y at h i g h CA c o n c e n t r a t i o n s . In t h i s s t u d y , High P r e s s u r e L i q u i d Chromatography (HPLC) w i t h e l e c t r o c h e m i c a l d e t e c t i o n p r o v i d e d q u i c k and a c c u r a t e d e t e r m i n a t i o n s of plasma CAs w i t h s u f f i c i e n t s e n s i t i v i t y f o r d e t e c t i n g b a s a l l e v e l s ( F a l c o n e r et_ al_ (1982), Woodward et a l ( 1 9 8 2 ) ) . The purpose of t h i s study was to c o n f i r m r e c e n t l y r e p o r t e d v a l u e s , u s i n g the above mentioned HLPC system and to determine whether CA r e l e a s e was due to h y p o x i a per se. or to o t h e r s t i m u l i . To t h i s end, not o n l y were the s t a n d a r d b l o o d gas parameters measured (PaO^, PaCO z, pHa) w h i l e the b i r d s were b e i n g d i v e d under two l e v e l s of oxygen, but a l s o plasma g l u c o s e , as g l u c o s e i s one of the main m e t a b o l i c c u r r e n c i e s , e s p e c i a l l y i n h y p o x i a . The q u e s t i o n of how a d r e n a l s e c r e t i o n was mediated was a l s o examined. Comline and S i l v e r (1961) s t a t e d t h a t a d r e n a l s e c r e t i o n was not e n t i r e l y under n e u r a l c o n t r o l and perhaps t h e r e was an i n h e r e n t c h e m o s e n s i t i v i t y i n the g l a n d i t s e l f . T h i s p o i n t was examined by d e n e r v a t i n g the a d r e n a l s b i l a t e r a l l y , and t e s t i n g the 4 ducks' r e s p o n s e s to f o r c e d d i v i n g . Plasma CAs were used as a measure of the change i n the a d r e n a l response and h e a r t r a t e and b l o o d p r e s s u r e were m o n i t o r e d to e v a l u a t e the e f f e c t of t h a t change on the d i v i n g r e s p o n s e . 5 Mater i a l s and Methods: General: The ten white Pekin ducks (Anas platyrhynchos) used in these experiments were kept in open a i r pens with food and water s u p p l i e d ad l i b . F o l l o w i n g surgery, they were kept indoors at 22* C on a 12 h o u r l i g h t / d a r k c y c l e , a l s o with food and u- = t e r s u p p l i e d ad l i b . Only two females were u s e d as the o v a r i e s r e s t r i c t access to the ad r e n a l s . The average weight of the denervates at the beginning of the experiment was 3.2 +0.4 kg; at the end, i t was 2.8 + 0.3 kg. The sham denervates s t a r t e d at an average of 3.5 + 0.4 kg and ended at 2.9 + 0.5 kg. Hematocrit also decreased through the experiment, in s p i t e of r e t u r n i n g red c e l l s to the b i r d s a f t e r plasma s e p a r a t i o n . Average hematocrits at the beginning of the experiments were 0.40 + 0.03 f o r the denervates and 0.42 + 0.05 f o r the shams. These values decreased to 0.29 + 0.0? and 0.33 + 0.06 at the end of the experiment, r e s p e c t i v e l y . Surgery: Cannulations of the b r a c h i a l a r t e r y were done under l o c a l a n a e s t h e t i c ( X y l o c a i n e , A s t r a ) . Denervations of the adrenal gland and sham o p e r a t i o n s were done with the animal under general a n a e s t h e s i a (sodium p e n t o b a r b i t a l i . a . (Somnotol, MTC) 10 mg/kg i n i t i a l l y , f o l l o w e d by 8 mg/kg every h a l f hour u n t i l the end of the 3 - 4 hr o p e r a t i o n ) . For these o p e r a t i o n s , the b i r d was intu b a t e d and t i d a l l y v e n t i l a t e d with pure 0 1 ? as surgery v i o l a t e d 6 the a i r s a c s and caused a decrease i n r e s p i r a t o r y e f f i c i e n c y . Surgery was not a s e p t i c , but i n s t r u m e n t s were s t e r i l i z e d b e f o r e use, the a r e a of i n c i s i o n was f r e e d of f e a t h e r s and d i s i n f e c t e d w i t h 75% e t h a n o l and c a r e was taken to keep the ar e a of i n , c i s i o n f r e e from c o n t a m i n a t i o n . A c c e s s to the a d r e n a l g l a n d was gain e d as d e s c r i b e d by Thomas and P h i l l i p s (1975). The a d r e n a l g l a n d c o u l d be seen as a p a l e y e l l o w body l y i n g m e d i a l l y and d o r s a l l y beneath the t i s s u e sheet of the v i s e e r o p e r i t o n e a l s a c . Long f o r c e p s were used to t e a r a l o n g the d o r s a l border of the sac where i t l a y apposed to the l u n g . Great c a r e was taken not to damage the l u n g i t s e l f . The d i f f u s e network of c o n n e c t i v e t i s s u e beneath the sac was c l e a r e d and the nerves-s u p p l y i n g the g l a n d were exposed and s e c t i o n e d . In the case of the shams, the ne r v e s were i d e n t i f i e d , but not s e c t i o n e d . The r i b s were s u t u r e d t o g e t h e r and the s k i n c l o s e d so t h a t i t b a l l o o n e d but d i d not l e a k w i t h the a r t i f i c i a l v e n t i l a t i o n . The o p e r a t i o n was re p e a t e d on the oth e r s i d e d u r i n g the same o p e r a t i o n . The b i r d s were a l l o w e d 5 - 7 days to r e c o v e r . S e v e r a l d i s s e c t i o n s were made i n p r e p a r a t i o n f o r t h i s study and the a d r e n a l g l a n d s from these a n i m a l s were c a r e f u l l y e x c i s e d , b l o t t e d d r y , and weighed. Some of these samples were homogenized i n p e r c h l o r i c a c i d ( H C 1 0 * + ) to e x t r a c t the CAs to determine p e r c e n t a g e c o n t e n t . P h y s i o l o g i c a l V a r i a b l e s : B l o o d p r e s s u r e (BP) was r e c o r d e d through a PE 160 c a n n u l a connected to a B i o Tec BT-70 t r a n s d u c e r . The c a n n u l a was i n s e r t e d i n t o a b r a c h i a l a r t e r y and advanced so t h a t the t i p was near the j u n c t i o n w i t h the a o r t a . To determine h e a r t r a t e (HR), a ratemeter was t r i g g e r e d by the ECG, which was measured u s i n g 1" needle e l e c t r o d e s i n s e r t e d s u b c u t a n e o u s l y , j u s t o f f the m i d l i n e i n the back and c h e s t . B r e a t h i n g f r e q u e n c y ( f r e s p ) was measured u s i n g an impedance c o n v e r t e r (Biocom model 991), w i t h the remote t h e r m i s t o r probe taped over one n a r i s . A l l v a r i a b l e s were d i s p l a y e d on a T e c h n i - r i t e 888 c h a r t r e c o r d e r and r e c o r d e d on a Hewlett Packard 3907C 1/2" tape system. In a d d i t i o n , BP and HR s i g n a l s were d i g i t i z e d by a 12 b i t a n a l o g to d i g i t a l (A/D) c o n v e r t e r (an ADY11-A i n a MINC 11 c o n f i g u r a t i o n (LSI 11/23 p r o c e s s o r , D i g i t a l Equipment)) at 256Hz. The v a l u e s were averaged over 2 s i n t e r v a l s , the r e s u l t s p l o t t e d on a d i g i t a l p l o t t e r and s t o r e d on d i s c f o r l a t e r a n a l y s i s . Samples f o r b l o o d gas, pH, CA, and g l u c o s e a n a l y s i s were taken a n a e r o b i c a l l y i n 1 ml 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 , capped, and i m m e d i a t e l y i c e d . B l o o d gases and pH were a n a l y z e d i n l e s s than 7 m i n u tes a f t e r s a m p l i n g 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 i e s 813 pH/Blood Gas A n a l y z e r . To compensate f o r the b i r d s ' 41'C body temp e r a t u r e , the bath temperature was i n c r e a s e d to 41*C and v a l u e s of c a l i b r a t i o n b u f f e r s and gases were c o r r e c t e d to the h i g h e r t i p e r a t u r e . Gas e l e c t r o d e membranes and e l e c t r o l y t e s were changed weekly. The h e x o k i n a s e method was used to determine plasma g l u c o s e c o n c e n t r a t i o n , u s i n g Sigma k i t #15. Plasma a l i q u o t s of 0.1 ml were 8 froze n at -80*C w i t h i n 15 minutes of sampling and were s t o r e d f o r up to 40 days before a n a l y s i s . Blanks c o n t a i n i n g s a l i n e i n s t e a d of hexokinase s o l u t i o n were used to c o n t r o l f o r hemolyred samples as per Sigma T e c h n i c a l B u l l e t i n No. 15-UV. Glucose turnover was not measured. HPLC Methods: Plasma was separated w i t h i n 7 - 1 0 minutes of sampling and the red c e l l s were returned to the duck. The f o l l o w i n g were combined i n a 1.5 ml polypropylene 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), 50 u l of 100 ng/ml dihydroxybenzylamine (DHBA, a s y n t h e t i c CA used a s an i n t e r n a l standard, from Sigma), and 14 mg of chromatographic grade a c t i v a t e d alumina (BDH). CA standards were prepared using 0.1 M NaPOi j b u f f e r (pH 7.0) i n s t e a d of plasma and 50 jul of a s o l u t i o n c o n t a i n i n g 100 ng/ml of norepinephrine ( N E ) , epinephrine (EP), and dopamine (DA) ( a l l from Sigma) d i s s o l v e d in 0.1 M HClOij a s w e l l a s the T r i s b u f f e r and alumina. A l l s o l u t i o n s were made up using HPLC-grade water. T r i a l s in which 50 u l of 0.1 M HCIO^ was added to the plasma to c o n t r o l f o r the a c i d i f y i n g e f f e c t s of the CA standard s o l u t i o n showed that there was no d i f f e r e n c e between samples in which there was an a c i d a l i q u o t and those in which there was not; the a d d i t i o n a l a c i d was t h e r e f o r e omitted. Standards and plasma samples were processed together, kept on i c e when not being manipulated. The v i a l s were shaken f o r 5 minutes and a f t e r the alumina s e t t l e d , the supernatant plasma was a s p i r a t e d . About 1 ml of d i s t i l l e d , d e i o n i z e d water was added, the v i a l s were shaken f o r 2 minutes, and the supernatant a s p i r a t e d again; t h i s was repeated. A f t e r the f i n a l shaking, the alumina was a s p i r a t e d to near dryness. 50 ^ i l of 0.1 M HClOi^ were added to the v i a l s to e l u t e the CAs from the alumina. The v i a l s were vortexed, l e f t s t a n d i n g f o r 5 minutes on i c e , then vortexed again. B r i e f c e n t r i f u g a t i o n concentrated the alumina and up to 20 u l of the a c i d i c supernatant CA s o l u t i o n was i n j e c t e d i n t o the HPLC. P e r c h l o r i c a c i d e x t r a c t s of adrenal gland were c e n t r i f u g e d f o r 5 minutes in an Eppendorf M i c r o c e n t r i f u g e to remove p a r t i c u l a t e s , d i l u t e d with HCIOJJ , then f i l t e r e d through M i l l i p o r e Type HA (0.45 um) c e l l u l o s e f i l t e r s . The f i l t r a t e was i n j e c t e d i n t o the H P L C . A S p e c t r a P h y s i c s SP 8700 HPLC c o n t r o l l e r and pump were used to provide an i s o c r a t i c flow of 2.0 ml/min through a Beckman 10x0.46 cm column of 10 um ODS U l t r a s p h e r e reverse phase packing. The mobile phase c o n s i s t e d of 50 mM 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 ion p a i r i n g agent to i n c r e a s e s e p a r a t i o n (Woodward, 1982). The mobile phase was r e c i r c u l a t e d f o r up to a' month with l i t t l e change in s e n s i t i v i t y . A l l CA standards and mobile phases were made up using HPLC grade water. 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 , using a BAS p l e x i g l a s T L - 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 g r e a s e / g r a p hite a c t i v e s u r f a c e . The e l e c t r o d e packing was r e p l a c e d about every 20 days or when response dropped o f f n o t i c i b l y . 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 > 3) of the system was about 0.6 10 femtomole i n j e c t e d f o r NE, about 1.0 femtomole f o r EP and DA. B l a n k s of 0.1 M HCIO^ were i n j e c t e d to determine the t r u e b a s e l i n e and s p u r i o u s peaks. The CA peaks were measured by hand and a c t u a l c o n c e n t r a t i o n s were c a l c u l a t e d by a computer program t h a t r e l a t e d the r a t i o s of CA peaks / DHBA peak i n the s t a n d a r d to the r a t i o s i n the plasma samples. N h i l e the r a t i o response f l a t t e n e d out at h i g h CA l e v e l s , t h e r e was a l i n e a r r a t i o response over the range 0.5 nM - 600 nM f o r a l l CAs measured ( i e , at very h i g h l e v e l s , t h i s method would u n d e r e s t i m a t e CA c o n c e n t r a t i o n s u n l e s s c o r r e c t e d to a c a l i b r a t i o n c u r v e ) . V a r i a t i o n between c o n t r o l samples of i d e n t i c a l CA c o n c e n t r a t i o n which were p r o c e s s e d i n p a r a l l e l , was l e s s than 15%. E x p e r i m e n t a l P r o t o c o l : The b i r d was p l a c e d i n the s i t t i n g p o s i t i o n and s e c u r e d to an o p e r a t i n g c r a d l e w i t h tape. A f t e r p o s i t i o n i n g the ECG and t h e r m i s t o r l e a d s , the BP t r a n s d u c e r was connected to the b r a c h i a l a r t e r y c a n n u l a and the b i r d was l e f t f o r about 2 hours to s e t t l e down b e f o r e the experiment s t a r t e d . P r e d i v e r e s t i n g v a l u e s of a l l v a r i a b l e s were measured w i t h the b i r d b r e a t h i n g a i r f r e e l y w i t h i t s head i n the u p r i g h t p o s i t i o n . BP, f r e s p , and HR were measured over an 80 s time p e r i o d ; b l o o d gases, pH, and CA v a l u e s were det e r m i n e d from a sample of b l o o d taken a f t e r t h i s 80 s p e r i o d . Each b i r d was d i v e d t w i c e b e f o r e the s u r g e r y and t w i c e a f t e r w a r d s (each t i m e , a f t e r b r e a t h i n g a i r and a f t e r b r e a t h i n g 0 , ) . The p r o t o c o l f o r the c o n t r o l d i v e s was i d e n t i c a l to that of 11 the d i v e s f o l l o w i n g the denervation or sham o p e r a t i o n s . The p r o t o c o l f o r the d i v e s before which a i r was breathed ( a i r d i v e s ) was as f o l l o w s : The head was r e s t r a i n e d in a rubber s l i n g and h e l d in an empty funnel through which a stream of a i r flowed to prevent CO-L accumulation. A f t e r at l e a s t 10 minutes, the chart and tape r e c o r d e r s and A/D conversion were s t a r t e d . A f t e r 80 s, the funnel was f i l l e d w ith 14 - 16 C water to i n i t i a t e the d i v e . At 2 minutes (D2) and 4 minutes (D4) into the d i v e , a 1.1 ml blood sample f o r CA, glucose, and blood gas a n a l y s i s was taken a n a e r o b i c a l l y and i c e d . Immediately a f t e r the l a s t dive blood sample, the b i r d ' s head was r a i s e d from the funnel and subsequent blood samples were taken 1, 5, and 10 minutes into recovery ( R l , R5, and RIO). Continuous r e c o r d i n g of the c a r d i o v a s c u l a r and r e s p i r a t o r y v a r i a b l e s continued f o r 5 minutes a f t e r the d i v e . The p r o t o c o l f o r d i v e s before which 0 a was breathed (0^ dives) was s i m i l a r , with the f o l l o w i n g e x c e p t i o n s : 0Z was d e l i v e r e d to the duck at about 8 1/min v i a a p l a s t i c bag f a s t e n e d around the f u n n e l , s l i n g , and neck of the b i r d . The funnel was f i l l e d before the d i v e sequence and the duck's head, although lowered r e l a t i v e to i t s r e s t i n g posture, was not completely in the f u n n e l . The d i v e was i n i t i a t e d by lowering the head into the water. At the end of the d i v e , the head was r a i s e d but remained in the bag, b r e a t h i n g 0j_. For the f i r s t minute a f t e r the d i v e , the flow of 0^ was turned up to about 12 1/min to assure that l i t t l e C0^ would be rebreathed; i t was then r e t u r n e d to 8 1/min f o r the remainder of the recovery p e r i o d . It was random as to whether a i r or 0^ d i v e s were performed f i r s t , in both c o n t r o l and post-o p e r a t i v e d i v e s . 12 A n a l y s i s of R e s u l t s : D i g i t i z e d v a l u e s of HR and BP were p l o t t e d on a video s c r e e n u s i n g an i n t e r a c t i v e g r a p h i n g program and the s t a r t and end p o i n t s of a l l the d i v e s a l i g n e d u s i n g event markers and resumption of b r e a t h i n g as cues. A p r e d e t e r m i n e d number of v a l u e s b r a c k e t i n g these p o i n t s were s e l e c t e d by the program and w r i t t e n to a f i l e f o r a n a l y s i s . B r e a t h i n g f r e q u e n c y was determined by c o u n t i n g the r e s p i r a t o r y c y c l e s over a p e r i o d of time and c o n v e r t i n g to b r e a t h s / m i n u t e . One minute p e r i o d s were used when the b i r d s were r e s t i n g ; the f i r s t s of r e c o v e r y was d i v i d e d i n t o 10 s p e r i o d s , the next minute i n t o 20 s p e r i o d s and the l a s t 3 p e r i o d s were each 1 minute l o n g . For some c o n t r o l v a l u e s , d a t a from o t h e r ducks -' c o n t r o l d i v e s were i n c l u d e d i n orde r to b e t t e r e s t i m a t e the p o p u l a t i o n r e s p o n s e . These d a t a a r e noted i n the t e x t and i n legends i f i n c l u d e d . Some da t a were a n a l y z e d u s i n g t h e ONEWAY and TWOWAY proc e d u r e s from the UCSD i m p l e m e n t a t i o n of the M IN ITAB s t a t i s t i c s package. S t u d e n t ' s t t e s t was used to compare p a i r e d d a t a between t r e a t m e n t s , s i g n i f i c a n c e b e i n g s e t at the 35% c o n f i d e n c e l e v e l f o r a l l t e s t s . I used a Hewlett P a c k a r d - s u p p l i e d program f o r an HP-41 s e r i e s c a l c u l a t o r to f i t best l i n e s to d a t a . V a l u e s r e p o r t e d a re means + one s t a n d a r d d e v i a t i o n . 13 RESULTS: Plasma C a t e c h o l a m i n e s : CA v a l u e s a r e g i v e n i n nM. The m o l e c u l a r w e i g h t s of the CAs a r e : NE - 169.2, EP - 183.2, and DA - 153.6 g/mole. Co n v e r s i o n from nM to pg/ml i s t h e r e f o r e : 1 nM NE = 169.2 pg/ml, e t c . The a b b r e v i a t i o n "nsd" r e f e r s to "no s i g n i f i c a n t d i f f e r e n c e " or "not s i g n i f i c a n t l y d i f f e r e n t " . The c o n t r o l r e s u l t s o b t a i n e d from a i r d i v e s a r e d e s c r i b e d f i r s t , f o l l o w e d by any d i f f e r e n c e s noted between groups. Plasma NE and EP l e v e l s i n a l l ducks i n c r e a s e d through the d i v e , then f e l l r a p i d l y a f t e r emergence and were nsd from the p r e d i v e l e v e l s a f t e r 14 minutes ( F i g s . 1,2). Plasma DA changes were v a r i a b l e ( F i g s . 1,2), but they d i d not change s i g n i f i c a n t l y d u r i n g the d i v e . The average r e s t i n g v a l u e of NE was 2.5 + 0.4 nM, EP was 5.3 + 1.3 nM, and DA was 4.6 + 2.6 nM. The average maximum NE l e v e l o bserved was 295 + 147 nM; the maximum f o r EP was 253 + 82. B r e a t h i n g 0^ b e f o r e the d i v e d i d not s i g n i f i c a n t l y change the r e s t i n g CA v a l u e s , but s i g n i f i c a n t l y d e c r e a s e d the amounts r e l e a s e d i n the d i v e . Peak NE and EP d e c r e a s e d to 42 + 22 nM and 52 + 37 nM, r e s p e c t i v e l y . Plama NE and EP c o r r e l a t e d w e l l w i t h PaO^, PaCO^, and pHa ( F i g . 3, T a b l e 1 ) . In c o n t r o l a i r d i v e s (N=14, v a l u e s from other groups i n c l u d e d ) , the l o w e s t r e g r e s s i o n c o e f f i c i e n t o b t a i n e d was |rl=0.714, f o r EP vs PaCO^ (DA was not c o n s i d e r e d ) . In 0^ c o n t r o l d i v e s (N=14, v a l u e s from o t h e r groups i n c l u d e d ) , the r c o e f f i c i e n t worsened c o n s i d e r a b l y i n the CA vs PaO^ r e l a t i o n s h i p s . The r F i gure 1. Plasma Catecholamine C o n c e n t r a t i o n s : Adrenal Denervate Group. The p l o t s are semilog, hence the unequal e r r o r bars. Those p o i n t s with a downward arrow f o r the lower e r r o r bar have s t d . dev.'s that extend below zero and are t h e r e f o r e undefined on a l o g s c a l e . The d i v e i s 4 minutes long; 5, 9, and 14 represent p o s t - d i v e v a l u e s . For a l l p a n e l s : Squares - Norepinephrine C i r c l e s - Epinephrine T r i a n g l e s - Dopamine Panel A - C o n t r o l s Panel B - Denervates Values are mean + 1 s t d . dev. (N=5, n=l) 13b AIR TRIALS OXYGEN TRIALS TIME COURSE OF DIVE AND RECOVERY (MIN) F i g u r e 2. Plasma Catech o l a m i n e C o n c e n t r a t i o n s : Sham Denervate Group. The p l o t s a re s e m i l o g , hence the unequal e r r o r b a r s . Those p o i n t s w i t h a downward arrow f o r the lower e r r o r bar have s t d . dev.'s t h a t e x t e n d below ze r o and a r e t h e r e f o r e u n d e f i n e d on a l o g s c a l e . The d i v e i s 4 minutes l o n g ; 5, 9, and 14 r e p r e s e n t p o s t - d i v e v a l u e s . For a l l p a n e l s : Squares - N o r e p i n e p h r i n e C i r c l e s - E p i n e p h r i n e T r i a n g l e s - Dopamine Pan e l A - C o n t r o l s P a n e l B - Sham Denervates V a l u e s a r e mean + 1 s t d . dev. (N=5, n = l ) . 1 3 o l TIME COURSE OF DIVE AND RECOVERY (MIN) I 3 e Table 1. Best Lines and Regression C o e f f i c i e n t s : The best l i n e was f i t t e d to the f o l l o w i n g equation according to the l e a s t squares method: bx y = ae where a = y i n t e r c e p t ^ b = measure of sl o p e pHa was coded by s u b t r a c t i n g 7.0 from the a c t u a l pHa; thus 7.345 = 0.345. T h i s was done because the expon e n t i a l r e l a t i o n s h i p between pH and the CAs made the y i n t e r c e p t equal to about 10 b 8. A y i n t e r c e p t at 7 makes more sense, p h y s i o l o g i c a l l y a = nM b = nM/torr f o r Pa0 2, PaCO b = nM/pH un i t f o r pHa Table 1 A I R Pooled Con t r o l s (N-14) Pre-Denerva-t i on (N=5) (N=5) NE EP NE EP a= 4330. a= 2230 . a= 87.8 a= 289. P a 0 2 b= -6.7? x i o x b= -6.16 x i o x b= -5.05 x i o 3 b= -9.41 x i o 3 r = -0.938 r = -0.846 r = -0.282 r = -0.351 pHa a= b= 16,1-50 . -2.16 x i o 4 a= b= 10,099 -2.08 x 10 M a= b= 58.62 -8.33 x 10* a= b= 46.08 -7.24 x 10* r = -0.925 r = -0.883 r = -0.903 r = -0.618 a= 2.27 x i d 1 a= 5.05 x i o z a= 0 .507 a= 0 .996 PaCOj b= 0 .170 b= 0 .146 b= 5.14 x b= 3.97 x 10*" r = 0 .840 r = 0 .714 r = 0 .849 r = 0 .517 a= 2,600 . — r a= 1,250. a= 253. a= 99.0 PaO a b= -6.29 x 10* b= -4.9? x io3- b= -9.60 x 10* b= -5.37 x 10* r = -0.960 r = -0.918 r = -0 .395 r = -0.253 a= 28,700 a= 3,800 a= 67. 8 a= 76.8 a. pHa b= -2.41 x 10 M b= -1.96 x ^ ~ - 9 . 4 2 x 10* b= -7.75 x 10* r = -0.955 r = -0.940 r = -0.958 r = -0.902 a= 4.9? x i o 3 a= 3.52 x i o r a= 0 .251 a= 0 .597 PaCO a b= 0 .198 b= 0 .158 b= 6.37 x IO* b= 5.66 x i o 2 " r = 0 . 955 r = r = 0 .917 r = 0 .934 PaO a a= b= 2,340. -6.60 x l o 3 " a= b= 249. -4.23 x iox a= b= 15.1 -3.19 x a = b= 32.2 -5.37 x 10* r = -0.936 r = -0.816 r = -0.21? r = -0.451 es a= 6690 a= 565. i d * a = 28.1 a= 15.7 pHa b= -1.94 x 10* b= -1.29 x b= -7.88 x 10 3 b= -4.93 x 10* r = -0.925 r = -0.838 r = -0.946 r = -0.728 PaCO^ a= b= 1.07 x 0 .163 10* a= b= 0.103 0.103 a= b= 0 .383 4.11 x i o x a= b= 1 .22 2 .36 x i o 1 r = 0 .885 r = 0 .758 r = 0 .895 r = 0 .631 V3g F i g u r e 3. Catecholamines as a f u n c t i o n of blood gas tensions and pH in pooled c o n t r o l s (N=14, n=l). For a l l panels: Squares - Norepinephrine C i r c l e s - Epinephrine Panel A - Norepinephrine and epinephrine as a f u n c t i o n of PaOj, duri n g a i r d i v e s . Panel B - Norepinephrine and epinephrine as a f u n c t i o n of PaC^ during 0^ d i v e s . Panel C - Norepinephrine and epinephrine as a f u n c t i o n of PaCO^ during a i r d i v e s . Panel D - Norepinephrine and epinephrine as a f u n c t i o n of PaCO^ during 0 ^ d i v e s . Panel E - Norepinephrine and epinephrine as a f u n c t i o n of pHa during a i r d i v e s . Panel F - Norepinephrine and epinephrine as a f u n c t i o n of pHa during 0^ d i v e s . 1 3 h AIR TRIALS OXYGEN TRIALS B ARTERIAL Po, (torr) b.S.K X 1tfJ ? 0 5 0 60 70 80 90 100 110 120 130 ARTERIAL Pco, (torr) •—I—• 1 • 1 • i — * — — i — 2 0 30 40 50 6 0 70 o.2.:? x i s 2 b. 0.170 r . O J l l 0.5.05 X 18J o.o.us C.0.7U 80 90 < a. z o 10 3 10 2 2 irf Q. UJ o z < 10° ioo 3 1 '6 ' 20 30 40 SO — I — 6 0 70 O-0.J07 , r . O J K o. 0.996 . b.3.97xlfl' r. 0.517 80 i 9 0 100 i E 1 „K F ON • 7.0 0.16200. r.-0.925 0.10.100. 0.-20J r . - U I ) ARTERIAL pH — r -7.1 7.2 7.3 — i — 7.4 — i 7.5 a .it* , . -0.903 14 v a l u e s f o r the o t h e r r e l a t i o n s h i p s remained about the same or worsened s l i g h t l y . The same s i t u a t i o n was seen i n the d e n e r v a t e t r i a l s , c l o s e r c o r r e l a t i o n d u r i n g a i r d i v e s than d u r i n g the 0 Z t r i a l s . The s l o p e of the r e l a t i o n s h i p d e c r e a s e d markedly i n the d e n e r v a t e s compared to the i n t a c t b i r d s , w i t h EP more a f f e c t e d than NE. Both NE and EP c o r r e l a t e d w e l l to pH i n a i r and 0 Z d i v e s , much more c l o s e l y than to PaO^ i n the l a t t e r ; NE a l s o c o r r e l a t e d w e l l to PaCO x ( T a b l e 1 ) . In both a i r and 0^ c o n t r o l t r i a l s , the amounts of plasma NE and EP were nsd at any p o i n t i n the d i v e or r e c o v e r y . The r a t i o of peak NE:EP r e l e a s e d d u r i n g d i v e s changed from 1.19 + 0.49 : 1 ( a i r d i v e s ) and 0.945 + 0.503 : 1 ( 0 Z d i v e s ) when i n t a c t to 3.4 + 1.04 : 1 ( a i r d i v e s ) and 1.63 + 0.33 : 1 ( 0 t d i v e s ) , a f t e r d e n e r v a t i o n . D e n e r v a t i n g the a d r e n a l s d e c r e a s e d the amount of NE and EP r e l e a s e d i n both a i r and Q% d i v e s , EP by a s i g n i f i c a n t amount. In the a i r d i v e s , the average peak NE and EP c o n c e n t r a t i o n s d e c r e a s e d to 130 + 22 nM and 43 + 21 nM from 295 + 147 nM and 253 + 82 nM r e s p e c t i v e l y a f t e r d e n e r v a t i o n . D e n e r v a t i o n a l s o changed the p r o p o r t i o n of c a t e c h o l a m i n e s r e l e a s e d . B e f o r e d e n e r v a t i o n , NE and EP l e v e l s were nsd from each o t h e r ; a f t e r d e n e r v a t i o n , NE l e v e l s at D4 were s i g n i f i c a n t l y h i g h e r . A f t e r the sham o p e r a t i o n , peak average l e v e l s of both NE and EP were s l i g h t l y h i g h e r but nsd from b e f o r e . 15 Adrenal Catecholamines: The average weights of the adrenal glands, determined from 11 size-matched broodmates, were 46.7 + 11.3 mg/kg body weight f o r the l e f t adrenal and 50.3 + 15.8 mg/kg f o r the r i g h t . Adrenal CAs were determined from 5 ducks of comparable weight from the same brood. NE was measured at 11.5 + 2.1 |jmol/g wet adrenal t i s s u e from the l e f t adrenal and 16.4 + 2.9 jjmol/g from the r i g h t . EP was 9.1 + 2. jjmol/g from the l e f t and 13.5 + 3.2 |imol/g from the r i g h t and DA was 4.1 + 1.9 jjmol/g from the l e f t and 5.2 + l . i from the r i g h t . O v e r a l l in the ad r e n a l , the r a t i o of NE:EP:DA was 1.14 + 0.12 : 1 : 0.37 + 0.11. Heart Rates: The development of b r a d y c a r d i a in the f o r c e d dived duck has-been w e l l d e s c r i b e d (Andersen (1963), Jones and Purves (1970), B u t l e r and Jones (1971)). B r i e f l y , the HR i n c r e a s e s t r a n s i e n t l y as the d i v e begins, then f a l l s as the di v e proceeds u n t i l about 50 -60 s, when i t reaches a basal l e v e l that i s maintained u n t i l the end of the d i v e . Upon emergence, the HR i n c r e a s e s to values w e l l above the pr e d i v e values w i t h i n seconds, then f a l l s to r e s t i n g l e v e l s over the course of some minutes. B r e a t h i n g 0-^  before the di v e had no e f f e c t on the r e s t i n g HR but s i g n i f i c a n t l y slowed the development of d i v i n g b r a d y c a r d i a in a l l b i r d s , r e g a r d l e s s of other treatments ( F i g . 4 ) . The d i f f e r e n c e in HRs between 0« and a i r d i v e s was g r e a t e s t about 40 s a f t e r F i g u r e 4. Heart Rate. For a l l p a n e l s : V a l u e s a re mean + 1 s t d . dev. (N=5, n = l ) . The blank space s e p a r a t i n g the b e g i n n i n g and the end of the d i v e i s due to the a l i g n i n g of those two p o i n t s , r e q u i r i n g a maximum adjustment of 5 s. The d i v e i s i n d i c a t e d by the downward d e f l e c t i o n i n the l i n e below the time s c a l e . Upward d e f l e c t i o n i n d i c a t e s the end of the d i v e . E x p r i m e n t a l Group: Pa n e l A - C o n t r o l v a l u e s P a n e l B - Denervates Sham Group: P a n e l D - C o n t r o l v a l u e s P a n e l E - Sham Denervates 16 submergence. At t h i s p o i n t , i n the c o n t r o l s , HR was 135 + 26 beats/min i n the 0 a d i v e and 70 + 25 beats/min i n the a i r d i v e . Near the end of the 4 minute d i v e , however, the HRs were nsd. A f t e r the d i v e , HRs i n both 0% and a i r t r i a l s i n c r e a s e d r a p i d l y to w e l l above r e s t i n g v a l u e s , but w h i l e peak t a c h y c a r d i a i n the a i r t r i a l s were reached w i t h i n 5 - 10 s, i t was d e l a y e d 20 - 50 s i n the 0 Z t r i a l s . A l s o , average p o s t d i v e t a c h y c a r d i a was c o n s i s t e n t l y h i g h e r i n the 0^ t r i a l s . HR was 327 + 43 beats/min a f t e r b r e a t h i n g 0^ and 293 + 67 beats/min a f t e r b r e a t h i n g a i r i n the c o n t r o l t r i a l s . A f t e r t h i s abrupt t a c h y c a r d i a , average HR i n the a i r t r i a l s g e n e r a l l y f e l l f o r 40 - 50 s, then l e v e l l e d o f f above the p r e d i v e r e s t i n g v a l u e s , or even i n c r e a s e d s l i g h t l y . In 0^ t r i a l s d u r i n g the same p e r i o d , average HR f e l l c o n t i n u o u s l y but by 5 minutes p o s t d i v e , i t had not r e t u r n e d to r e s t i n g v a l u e s . Average r e s t i n g and r e c o v e r y HRs i n c r e a s e d s i g n i f i c a n t l y a f t e r s u r g e r y i n both the d e n e r v a t e s and shams. A d r e n a l d e n e r v a t i o n i n c r e a s e d the v a r i a b i l i t y i n HR more than d i d the sham o p e r a t i o n , but the means were nsd. Bl o o d p r e s s u r e : Average r e s t i n g BP i n the c o n t r o l s was 150 - 160 t o r r ( F i g . 5 ) . W h i l e head r e s t r a i n t d i d not change BP, the b e g i n n i n g of both a i r and 0-^  d i v e s was a s s o c i a t e d w i t h an a b r u p t , i f t r a n s i e n t i n c r e a s e of about 20 t o r r . In a i r d i v e s , t h i s i n c r e a s e was f o l l o w e d by a decrease so that BP f e l l below the p r e d i v e r e s t i n g 164 F i g u r e 5. Blood Pressure. For a l l panels: The middle t r a c i n g of each panel i s the mean value, the b r a c k e t i n g l i n e s d e f i n e p l u s and minus one s t d . dev. (N=5, n=l). The blank space s e p a r a t i n g the beginning and the end of the dive i s due to the a l i g n i n g of those two p o i n t s , r e q u i r i n g a maximum adjustment of 5 s. The l a r g e b l i p s seen at 2, 4, and 5 minutes into the sequence are due to blood sampling f o r m e t a b o l i t e s . The dive i s i n d i c a t e d by the downward d e f l e c t i o n in the l i n e below the time s c a l e . Upward d e f l e c t i o n i n d i c a t e s the end of the d i v e . Exprimental Group: Panel A - C o n t r o l values Panel B - Denervates Sham Group: Panel D - C o n t r o l value Panel E - Sham Denervat 5 AIR TRIALS OXYGEN TRIALS 17 v a l u e s w i t h i n a minute. The average BP c o n t i n u e d to drop u n t i l 2.3 minutes i n t o the d i v e , then s t a b i l i z e d at t h i s lower v a l u e u n t i l the end. The v a r i a t i o n a s s o c i a t e d w i t h the mean BP i n c r e a s e d u n t i l mean BP s t a b i l i z e d ; t h e r e a f t e r i t remained c o n s t a n t u n t i l the end of the d i v e . In Oj_ d i v e s , the i n i t i a l i n c r e a s e was a l s o f o l l o w e d by a de c r e a s e , but the de c r e a s e was slower and the average BP r a r e l y f e l l below the p r e d i v e r e s t i n g v a l u e . A f t e r the d i v e , BP i n c r e a s e d w i t h i n 3 s to w e l l above p r e d i v e r e s t i n g v a l u e s , then f e l l w i t h i n a minute to s l i g h t l y below p r e d i v e v a l u e s , and f i n a l l y r o s e s l o w l y , a p p r o a c h i n g p r e d i v e v a l u e s 1.5 - 2.0 minutes p o s t d i v e . G e n e r a l l y , t h e r e was l e s s v a r i a t i o n i n BP i n an 0 t d i v e , both over the c o u r s e of the d i v e and compared to the oth e r b i r d s i n the ser i es. A f t e r both d e n e r v a t i o n and sham o p e r a t i o n s , r e s t i n g BP dropped from 155 - 160 t o r r to 135 - 140 t o r r . There were nsd's between c o n t r o l and de n e r v a t e d i v e BPs; w h i l e the v a r i a b i l i t y i n a i r d i v e s was s l i g h t l y g r e a t e r than i n c o n t r o l s , i t was not c o n s i s t e n t l y so. BP i n 0^ d i v e s were as t i g h t l y r e g u l a t e d as i n con t r o l s . Plasma G l u c o s e : Plasma g l u c o s e v a r i e d g r e a t l y among b i r d s and between groups. The d e n e r v a t e c o n t r o l group had a r e s t i n g l e v e l of 7.8 + 1 . 9 mM (1 mM = 18 mg/dl) i n the a i r t r i a l s w h i l e the sham c o n t r o l group had a mean of 4.2 + 1.9 mM ( F i g . 6 ) . Plasma g l u c o s e l e v e l s d i d not change s i g n i f i c a n t l y through the a i r or 0j_ d i v e s . The h i g h e s t F i gure 6. Plasma g l u c o s e c o n c e n t r a t i o n . 1 7 a P a n e l A - V a l u e s from e x p e r i m e n t a l group. Squares - C o n t r o l v a l u e s C i r c l e s - Denervates P a n e l B - V a l u e s from sham group. Squares - C o n t r o l v a l u e s C i r c l e s - Sham Denervates Nb: In the a i r t r i a l , 3 d e v i a t i o n b a r s exceed the upper bound of the graph. A = 12.6, B = 14.2, C = 12.3. V a l u e s a r e mean + 1 s t d . dev. (N=5, n = l ) . "R" on a b s c i s s a r e p r e s e n t s r e s t i n g v a l u e s ; d i v e s a r e 4 minutes i n l o n g . 17b 18 v a l u e s i n a i r d i v e s were reached one minute a f t e r emergence, 9.9 + 1.6 mM i n the c o n t r o l a i r d i v e s and they d e c r e a s e d i n the p e r i o d f o l l o w i n g i n a l l c a s e s but one. In 0^ t r i a l s , not o n l y was the i n c r e a s e a f t e r the d i v e s m a l l e r , but the v a l u e was not always the h i g h e s t of the d i v e . The average plasma g l u c o s e c o n c e n t r a t i o n at RIO was h i g h e r than b e f o r e the d i v e i n a l l cases but one. A d r e n a l d e n e r v a t i o n di'd not a f f e c t e i t h e r r e s t i n g or peak v a l u e s of g l u c o s e a l t h o u g h i t d i d have a s i g n i f i c a n t e f f e c t on plasma CA c o n c e n t r a t i o n s . A r t e r i a l pH: There was a c h a r a c t e r i s t i c d e c r ease i n a r t e r i a l pH i n a l l d i v e s . From r e s t i n g v a l u e s , the pH dropped through the 4 minute d i v e , d e c r e a s i n g more over the f i r s t 2 minute p e r i o d than over the second. Once the d i v e ended, average pH r o s e i n a l l cases but one ( F i g . 7 ) , but i n no c a s e s d i d the pH r e g a i n r e s t i n g v a l u e s by one minute a f t e r the d i v e . From an average p r e d i v e pH of 7.39 + 0.02 b r e a t h i n g a i r , pH d e c r e a s e d through the d i v e to 7.20 + 0.02. In 0X t r i a l s , r e s t i n g pH was d e p r e s s e d , r e l a t i v e to the a i r t r i a l s , to 7.36 + 0.03 and was lower at both s a m p l i n g t i m e s ; the minimum pH a t t a i n e d was 7.07 + 0.04. The p o s t d i v e i n c r e a s e i n pH was g r e a t e r i n the 0^ t r i a l s so t h a t at one minute a f t e r the d i v e , the pH of the 0 X t r i a l s was g r e a t e r than, or e q u a l to t h a t i n the a i r t r i a l s , even though pH i n the 0- d i v e s was lower b e f o r e emergence. \8CA F i g u r e 7. A r t e r i a l pH. Lar g e , open symbols r e p r e s e n t v a l u e s from oxygen t r i a l s . S m a l l , s o l i d symbols r e p r e s e n t v a l u e s from a i r t r i a l s . P a n e l A - V a l u e s from e x p e r i m e n t a l group. Squares - C o n t r o l v a l u e s C i r c l e s - Denervates P a n e l B - V a l u e s from sham group. Squares - C o n t r o l v a l u e s C i r c l e s - Sham Denervates "R" on a b s c i s s a r e p r e s e n t s r e s t i n g v a l u e s ; d i v e s a re 4 minutes l o n g . V a l u e s a r e mean + 1 s t d . dev. (N=5, n = l ) . TIME COURSE OF DIVE AND RECOVERY (MIN) F i g u r e 8. Blood gases. " l8c For panels from Experimental Group: Squares - C o n t r o l values C i r c l e s - Denervates For panels from Sham Group: Squares - C o n t r o l values C i r c l e s - Sham Denervates Panel A l : P0X values obtained during oxygen d i v e s . Panel A2: PO^ values obtained during a i r d i v e s . Panel B: PCO^ values obtained from both a i r and oxygen d i v e s : Small, s o l i d symbols i n d i c a t e values obtained d u r i n g a i r d i v e s . Large, open symbols i n d i c a t e values obtained d u r i n g oxygen d i v e s . "R" on a b s c i s s a r e p r e s e n t s r e s t i n g v alues; d i v e s are 4 minutes long. Values are mean + 1 s t d . dev. (N=5, n=l). 19 A r t e r i a l PO^: B r e a t h i n g a i r before the dive r e s u l t e d in a r e s t i n g P a 0 i . of 111 + 7 t o r r which decreased to 4 3 + 6 t o r r at 4 minutes i n t o the d i v e ( F i g . 8 ) . L i k e pH, PaO x in the a i r t r i a l s decreased more in the f i r s t two minutes than in the second, but t h i s was not seen in the 0^ d i v e s . One minute a f t e r the d i v e , PaO z had i n c r e a s e d so that i t was higher (120 + 9 t o r r ) than before the d i v e . Breathing 0^ p r e d i v e i n c r e a s e d r e s t i n g PaO^ to 338 + 57 t o r r , s i g n i f i c a n t l y over that seen in the a i r t r i a l s and average PaO^ never f e l l below 300 t o r r at any point in the d i v e s . The r e s t i n g Pa0 z values in a i r c o n t r o l s were s l i g h t l y higner than in those that had surgery and in 0^ d i v e s , P a 0 x was s l i g h t l y , but not s i g n i f i c a n t l y higher in c o n t r o l s than in the denervates or shams through the whole d i v e . There were nsd's in r e s t i n g and d i v i n g PaO^s between denervates and shams or between c o n t r o l s and denervates or shams. A r t e r i a l PC0 x: Rest i n g PaCO^ in a i r t r i a l s was 31.6 +1.7 t o r r ; b r e a t h i n g 0^ r a i s e d i t s i g n i f i c a n t l y to 36.2 + 1.1 t o r r ( F i g . 8). During the d i v e , i t i n c r e a s e d more during the f i r s t two minute p e r i o d than during the second, c o n s i s t e n t with the changes seen in PaO^. Maximum PaCO^ measured in the d i v e s was 54. + 4.2 t o r r in the a i r t r i a l s , s i g n i f i c a n t l y higher (77.8 + 4.3) in the 0j_ t r i a l s . One minute a f t e r the d i v e ended, PaCO^s were nsd from r e s t i n g values in both a i r and 0, t r i a l s . L i k e the PaQ-s, there were nsds between 20 c o n t r o l s and denervates or shams. Brea t h i n g Frequency: In a l l cases, the average predive r e s t i n g f r e s p was nsd between when the b i r d was b r e a t h i n g a i r and when i t was br e a t h i n g 0 X ( F i g . 9 ) . Average p r e d i v e f r e s p decreased c o n s i s t e n t l y by about 5 breaths/min a f t e r the b i r d s ' heads were r e s t r a i n e d . P o s t d i v e f r e s p decreased e x p o n e n t i a l l y ( i e f r e s p = fn(ke * ) over the f i r s t 4 minutes p o s t d i v e . A f t e r a i r and O x d i v e s , f r e s p ' s were q u i t e s i m i l a r immediately a f t e r the d i v e , but the d i f f e r e n c e s became more pronounced as recovery progressed. During recovery, f r e s p decreased from an i n i t i a l value of 34 + 5.4 breaths/min to 20 + 5.7 breaths/min in 5 minutes a f t e r b r e a t h i n g a i r , and from 32 + 6.8 breaths/min to 16 + 3.7 breaths/min a f t e r 0^. A f t e r surgery, the average p o s t - a i r dive f r e s p i n c r e a s e d by about 5 breaths/min over c o n t r o l s during the f i r s t minute of recovery. N e i t h e r the f r e s p nor the v a r i a t i o n in i t in c r e a s e d s i g n i f i c a n t l y a f t e r b r e a t h i n g 0^ p r e d i v e . A f t e r b r e a t h i n g a i r , however, the v a r i a t i o n in f r e s p was much greater in the denervates over t h i s f i r s t minute p o s t d i v e . F i g u r e 9. B r e a t h i n g Frequency. 20a For a l l p a n e l s : T r i a n g l e s - V a l u e s o b t a i n e d d u r i n g a i r dives-C i r c l e s - V a l u e s o b t a i n e d d u r i n g oxygen d i v e s E x p e r i m e n t a l Group: Pa n e l A - C o n t r o l v a l u e s P a n e l B - Denervates Sham Group: Pa n e l D - C o n t r o l v a l u e s P a n e l E - Sham Denervates On a b s c i s s a , "F" i n d i c a t e s t h a t the b i r d ' s head was f r e e ; "R" i n d i c a t e s t h a t the head was r e s t r a i n e d . V a l u e s a r e mean + 1 s t d . dev. (N=5, n = l ) . BREATHING FREQUENCY (BREATHS- MIN"1) —* r*o N J cu O O w O tn o tn — ' 1 1 • • o • tn i T I J m o o e 7 J to m < m > o m o o < m ; o -< C P -cr> -•»o -OD -so o tn o • N > tn tn o tn •P. 7) ] tn ->j -oo -10 J BREATHING F R E Q U E N C Y ( B R E A T H S • MIN"1) o —* K> fvj tn o cn —i 1 i_ co cn o ^02 21 Di s c u s s i on: T h i s study has c o n f i r m e d p r e v i o u s l y r e p o r t e d v a l u e s of plasma CAs i n ducks f o r c e d to d i v e a f t e r b r e a t h i n g a i r (Hudson and Jones, 1982). These r e s u l t s a l s o agree w i t h r e s t i n g v a l u e s f o r ducks p u b l i s h e d by o t h e r a u t h o r s ( W i l s o n and B u t l e r ( 1 9 8 3 c ) , S t u r k i e e_t a l (1970)) . A s u b s t a n t i a l p a r t of the a d r e n a l response i s mediated v i a nervous c o n t r o l . When the s p l a n c h n i c and s u b s i d i a r y nerve s u p p l i e s to the a d r e n a l s were c u t , t h e r e was a s i g n i f i c a n t d e c r e a s e i n the b l o o d CA l e v e l s d u r i n g d i v i n g . Peak plasma EP c o n c e n t r a t i o n , a measure of m e d u l l a r y c o n t r i b u t i o n , d e c r e a s e d by 80% a f t e r d e n e r v a t i o n ; not o n l y d i d i t decrease but the amount r e l a t i v e to NE d e c r e a s e d as w e l l . I t cannot be s a i d , however, what p a r t of t h i s i s due to d e c r e a s e d r e l e a s e and what p a r t to decreased s y n t h e s i s as the d e n e r v a t i o n may have i n t e r f e r e d w i t h normal p r o d u c t i o n of CAs (Linger and P h i l l i p s , 1983) and a d r e n a l s were not a n a l y z e d f o r CA c o n t e n t a f t e r d e n e r v a t i o n . There was, however, enough CA s y n t h e s i s to m a i n t a i n b a s a l l e v e l s of NE, EP, and DA and to permit a s i g n i f i c a n t response i n the d i v e s . There i s a n o n - n e u r a l component to t h i s response as nerve s e c t i o n d e c r e a s e s i t , but does not c o m p l e t e l y b l o c k i t . S i m i l a r p a r t i a l independence of n e u r a l mechanisms have been shown to occur i n sheep (Comline and S i l v e r , 1961), cows (Bloom e_t a l , 1976), and r a b b i t s (Korner and White, 1966). Of the f a c t o r s measured i n t h i s e x p e r i m e n t , h y p o x i a r a t h e r than h y p e r c a p n i a , low pH, or h y p o g l y c e m i a , i s best c a u s a l l y r e l a t e d to CA r e l e a s e i n f o r c e d d i v e s . In the a i r d i v e s , CAs 22 i n c r e a s e d with d e c r e a s i n g PaO^ in a w e l l d e f i n e d manner; in d i v e s , they d i d not, i n d i c a t i n g that there i s a value above which PaOj_ does not s t r o n g l y a f f e c t CAs r e l e a s e . Above t h i s p o i n t , other f a c t o r s are more important. A r t e r i a l pH and PaCO are w e l l c o r r e l a t e d to CA r e l e a s e , in both a i r and 0X d i v e s , and indeed, t h i s may be the reason that they have been*postulated as causal f a c t o r s , but i f pH ( S t e i n s l a n d et a l (1970) in dogs, Jones and Robinson (1975) in sheep) or PaCO (Cantu ejt a l , 1966, in dogs) were the primary determining f a c t o r s in ducks, the i n c r e a s e in CAs would have been greater in the 0^ d i v e s where the pHa and PaCO^ changes were more pronounced than in the a i r d i v e s . The s m a l l e r , but s t i l l s i g n i f i c a n t i n c r e a s e in CAs observed dur i n g the 0X d i v e s suggests that part of the response i s independent of hypoxia. T h i s component may be r e g u l a t e d by P a C O ^ or pHa a c t i n g through c e n t r a l or p e r i p h e r a l chemoreceptors; i t i s probably due to an i n t e g r a t i o n of many f a c t o r s , as in the c a r o t i d body ( E y z a g u i r r e and Koyano, 1965). It i s c l e a r , however, that pH and/or PaCO^ are not the primary determinants of plasma CAs in d i v i n g ducks. The adrenal medulla i s composed of chromaffin t i s s u e , which i s e m b r y o l o g i c a l l y d e r i v e d from rhombencephalic neural c r e s t , the same t i s s u e that forms the c a r o t i d body chemoreceptors and i s h i s t o l o g i c a l l y s i m i l a r to that s t r u c t u r e (Pearse e_t al., 1973). There have been r e p o r t s (Comline and S i l v e r (1961), S t e i n s l a n d e_t a l (1970), Jones and Robinson (1975)) that there i s a two part response to hypoxia, the f i r s t part due to s t i m u l a t i o n v i a the s p l a n c h n i c nerve, the second e x h i b i t e d in severe hypoxia due to the c h e m o s e n s i t i v i t y of the gland i t s e l f . S t u d i e s attempting to 23 show t h i s d i r e c t l y , by r e c o r d i n g from the s p l a n c h n i c n e r v e s , or s e l e c t i v e p e r f u s i o n of the g l a n d and measuring CAs are r a r e ( a l t h o u g h see Comline and S i l v e r , 1961) and t h i s may be an i n t e r e s t i n g avenue of i n v e s t i g a t i o n . D u r i n g an a i r d i v e , the CAs do not s t a r t i n c r e a s i n g d r a m a t i c a l l y u n t i l a f t e r 1 - 2 mi n u t e s ; by t h i s time the p e r i p h e r y i s f u l l y c o n s t r i c t e d and t h e r e i s a much reduced exchange between p e r i p h e r a l and c e n t r a l b l o o d p o o l s (Murphy e_t a l , 1980). The end d i v e CA l e v e l s s h o u l d t h e r e f o r e be almost w h o l l y from the a d r e n a l s . EP can be c o n s i d e r e d to be almost e n t i r e l y of a d r e n a l o r i g i n and t h e r e f o r e i s a meter of a d r e n a l a c t i v i t y . Assuming an u n a f f e c t e d s y n t h e s i s r a t e , these r e s u l t s showed t h a t 80% of the a d r e n a l c o n t r i b u t i o n i s dependent on i n n e r v a t i o n , l e a v i n g 20% mediated by o t h e r mechanisms. T h i s assumption i s somewhat c o n t r o v e r s i a l , and a n a l y s i s of the a d r e n a l g l a n d s s h o u l d have been performed at auto p s y . There a r e 2 s o u r c e s of these CAs, the a d r e n a l g l a n d s and s y m p a t h e t i c nerve t e r m i n a l s . ( T r a d i t i o n a l l y , the s y m p a t h e t i c t r a n s m i t t e r was thought to be o n l y NE, but t h e r e i s some e v i d e n c e (Berecek and Brody, 1982) t h a t EP may a l s o be a t r a n s m i t t e r . ) The r e l a t i v e c o n t r i b u t i o n of these two s o u r c e s cannot be d e l i n e a t e d except by removing one of them though adrenalectomy or sympathectomy. The former was t r i e d , but I was o n l y a b l e to s u c c e s s f u l l y a d r e n a l e c t o m i z e and m a i n t a i n a s i n g l e duck. C e r t a i n l y the t e c h n i q u e i s p o s s i b l e (Thomas and P h i l i p s ( 1 9 75), N i l s o n and B u t l e r (1982a,b)) and t h i s approach may be worth t r y i n g a g a i n . Chemical sympathectomy, u s i n g g u a n e t h i d i n e which s p a r e s a d r e n a l CAs i s much e a s i e r (Fronek, 1980), but i t s use i n ducks has not been w e l l documented and the s i d e e f f e c t s are not w e l l 24 known (Kobinger and Oda, 1969). The denervates were t r e a t e d with guanethidine to see i f BP c o n t r o l would c o l l a p s e in a d i v e a f t e r b l o c k i n g the sympathetic system. A f t e r a d m i n i s t e r i n g i t to ducks, blood NE l e v e l s i n c r e a s e d d u r i n g d i v i n g and there was no s i g n i f i c a n t p e r t u r b a t i o n in BP beyond that caused by the d e n e r v a t i o n . Whether t h i s was a r e s u l t of a compensatory i n c r e a s e from the adrenal gland or a r e s u l t of a longer recovery p e r i o d a f t e r surgery, I am unable to say. Reserpine i s a CA d e p l e t e r that a f f e c t s both sympathetic nerve t e r m i n a l s and the adrenal gland. Although i t s use in humans-i s a s s o c i a t e d with myriad s i d e e f f e c t s , o v e r a l l i t may be a l e s s d r a s t i c way of p r e v e n t i n g the CA response to d i v i n g than the s u r g i c a l procedure. It i s p o s s i b l e that the CA response observed d u r i n g the dive i s not a primary e f f e c t of the hypoxia, but a secondary one in response to other s t i m u l i that i n c r e a s e at the same time. Recent evidence suggests that i t may be secondary to hypoxic v a s o c o n s t r i c t i o n of the r e n a l blood supply. Wilson and B u t l e r (1983c) found that plasma NE l e v e l s i n c r e a s e d , sometimes up to 40 times by i . v . a d m i n i s t r a t i o n of a n g i o t e n s i n (ANG) I I , a c i r c u l a t i n g p e p t i d e 50 times more potent a pressor than NE; i t a l s o p o t e n t i a t e s the r e l e a s e of NE from both sympathetic nerve t e r m i n a l s and the adrenal medulla. A c c o r d i n g to the data of Jones e_t al_ (1979), r e n a l blood flow drops to 1/15 the p r e d i v e value in a f o r c e d d i v e in ducks. T h i s would be a potent s t i m u l u s f o r the a c t i v a t i o n of the renin-ANGII system, which might help e x p l a i n the dramatic i n c r e a s e in CAs. Indeed, Drummond and Lindheimer (1982) found that plasma r e n i n 25 a c t i v i t y , a measure of the s t a t e of a c t i v a t i o n of the renin-ANGII system, was found to be e l e v a t e d during hypoxia in the f e t a l sheep. CAs have p o s i t i v e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s on the heart, i n c r e a s e BP and i n c r e a s e blood c o n c e n t r a t i o n s of such s u b s t r a t e s as glucose, f r e e f a t t y a c i d s and l a c t a t e (Goodman and Gilman, 1980). They have v a r i a b l e e f f e c t s on blood flow and have been i m p l i c a t e d in the a c t i v a t i o n of phosphofructokinase in rat heart (Clark and Patten, 1981). However, the c a r d i a c e f f e c t s of CAs are overridden by vagal c o n t r o l and blood glucose l e v e l s do not change in a c o n v i n c i n g manner during a d i v e . The f l u s h of CAs through the l i v e r at the end of the d i v e , though, would account f o r the i n c r e a s e in glucose c o n c e n t r a t i o n observed at the end of a i r d i v e s . CAs a l s o mediate glucagon r e l e a s e and i n h i b i t i o n of i n s u l i n r e l e a s e which would p o t e n t i a t e the i n c r e a s e in blood glucose and account f o r the glucose remaining e l e v a t e d 10 minutes a f t e r the d i v e . Changes s i m i l a r to those seen in t h i s study also occur in f o r c e d dived s e a l s (Robin e_t a l (1982), Murphy e_t a l (1980), Kooyman et a l (1980)); glucose decreased d u r i n g , then i n c r e a s e d a f t e r the d i v e . NE and EP c o n s t r i c t c e r t a i n p e r i p h e r a l v a s c u l a r beds in ducks and i t might be thought that the high l e v e l s of CAs play a r o l e in blood pressure r e g u l a t i o n , but in both r a b b i t s (Korner and N h i t e , 1966) and ducks (Wilson and B u t l e r , 1983a,b), blood pressure was w e l l maintained when the animals were adrenalectomized. The ot a d r e n e r g i c a c t i v a t i o n of phosphof ructok i nase in c a r d i a c muscle (Clark and Patten, 1981), i f a p p l i c a b l e to ducks, might enable the heart to u t i l i z e endogenous s t o r e s of glycogen more e f f i c i e n t l y , thereby s p a r i n g c i r c u l a t i n g glucose f o r the b r a i n . However, heart 26 muscle, e s p e c i a l l y in avian s p e c i e s , metabolizes only a small amount of glucose, r e l y i n g mostly on f r e e f a t t y a c i d s f o r s u b s t r a t e . CAs are not the only product of the adrenal and a p o s s i b i l i t y e x i s t s that the reason that the blood supply to the adrenal i s continued i s to allow access to the adrenal s t e r o i d s . These s t e r o i d s are "found to i n c r e a s e in the same c o n d i t i o n s that cause CA r e l e a s e , in s e a l s ( L i g g i n s et a l (1379), P s O b i n e_t a l (1381)) and c a l v e s (Bloom e_t al_, 1976), but i t i s d i f f i c u l t to assign a meaningful r o l e to them duri n g short term hypoxic s t r e s s , as s t e r o i d s act b y r e g u l a t i n g p r o t e i n s y n t h e s i s which has a much longer time cours= than a i l but the most extreme d i v e s . Within the past f i v e years, the i d e n t i f i c a t i o n of adrenal o p i o i d s , c o s e c r e t e d with CAs, has added another l a y e r of complexity to t h i s response. Dynorphin and l e u - and met-enkephalin have been i d e n t i f i e d in a c i d e x t r a c t s of bovine, human, and dog adrenal glands (Kimura e_t a l (1980), Lemaire e_t al_ (1981)). These o p i o i d s , because of t h e i r short b i o l o g i c a l h a l f l i f e in the c i r c u l a t i o n , probably help c o o r d i n a t e responses between neighboring c e l l s or groups of c e l l s (Bloom, 1984) but as yet, no d e f i n i t e r o l e has been a s c r i b e d to them. Although the CA response i s dramatic, when the ducks were prevented from r e l e a s i n g the normal amounts of CAs in hypoxia (NE decreased by h a l f , EP by 5/6), they d i d not s u f f e r unduly. These l e v e l s are s t i l l enormously i n c r e a s e d over r e s t i n g l e v e l s and could s t i l l be s a t u r a t i n g the mechanism being invoked. C e r t a i n l y , the adrenal denervates showed a reasonably normal c a r d i a c response, when the e f f e c t s of the ope r a t i o n were discounted. 2? The l i t e r a t u r e s u g g e s t s that t h i s CA response i s common i n d i v i n g v e r t e b r a t e s . I t has been observed i n s e a l s (Hance e_t a l . 1982), muskrats (Mangalam, u n p u b l . ) , and beavers (Mangalam, unpubl.) a l t h o u g h to a l e s s e r degree than i n ducks. The a d r e n a l g l a n d i s one of the few t i s s u e s t h a t c o n t i n u e s to be p e r f u s e d i n a f o r c e d d i v e ; w h i l e a b s o l u t e b l o o d f l o w may d e c r e a s e , the pe r c e n t a g e of c a r d i a c output r e c e i v e d d u r i n g these f o r c e d d i v e s i n c r e a s e s . The a b s o l u t e b l o o d f l o w may be more s i g n i f i c a n t , however, because as b l o o d f l o w d e c r e a s e s , 0-j_ d e l i v e r y w i l l d e c r e a s e p r o p o r t i o n a t e l y and i f the a d r e n a l has chemoreceptor a c t i v i t y , the 0^ l a c k w i l l s t i m u l a t e i t . The i n c l u s i o n of the a d r e n a l i n the c e n t r a l c i r c u l a t i o n d u r i n g h y p o x i c e p i s o d e s i n d i c a t e s an imp o r t a n t r o l e f o r a d r e n a l p r o d u c t s i n f e t a l and neonate h y p o x i a , as w e l l as i n d i v i n g . In e v o l u t i o n , t r a i t s t h a t a r e even s l i g h t l y d e l e t e r i o u s to the s p e c i e s a r e q u i c k l y e l i m i n a t e d or become r e c e s s i v e , yet t h i s r e sponse i s p h y l o g e n e t i c a l l y w i d e s p r e a d , d e s p i t e at l e a s t one n o t a b l e d e l e t e r i o u s e f f e c t . High l e v e l s of CAs have c l e a r l y been l i n k e d to m y o c a r d i a l damage due to the g e n e r a t i o n of oxygen f r e e r a d i c a l s i n the c o u r s e of t h e i r m etabolism ( S i n g a l e_t ad, 1982). At the CA c o n c e n t r a t i o n s observed i n the ducks, t h i s may be a s i g n i f i c a n t e f f e c t , e s p e c i a l l y a f t e r r e p e a t e d d i v e s . The p u t a t i v e r o l e of CAs i n f a c i l i t a t i n g gas t r a n s p o r t a c r o s s RBC membranes may have an o f f s e t t i n g advantage, but t h i s would not e x p l a i n the enormous l e v e l s o b s e r v e d . Duck hemoglobin (Hb) i s n o r m a l l y about 90-95% s a t u r a t e d ( S c h e i p e r s e_t a l _ , 1975). B r e a t h i n g pure 0% t r i p l e s PaO^ and s h o u l d t h e r e f o r e cause n e a r l y 100% s a t u r a t i o n of Hb, as w e l l as t r i p l i n g the s m a l l amount of 0 o c a r r i e d i n s o l u t i o n . T h i s e x t r a 5-10% b l o o d 28 0^ c o n t e n t a l l o w s the b i r d to repay the 0X debt i n c u r r e d through a d i v e i n s i g n i f i c a n t l y l e s s time than i t c o u l d i f b r e a t h i n g a i r . T h i s can be seen i n the much f a s t e r r e t u r n to r e s t i n g f r e s p ' s a f t e r a 0% d i v e than a f t e r an a i r d i v e . In the i n t a c t b i r d s , t h e r e was nsd between f r e s p ' s a f t e r b r e a t h i n g 0/j_ or a i r d u r i n g the f i r s t minute a f t e r the d i v e , d e s p i t e the l a r g e d i f f e r e n c e i n PaO^s. L a t e r i n the r e c o v e r y , however, the d i f f e r e n c e i n f r e s p ' s was more pronounced, even though 1 minute p o s t - d i v e , the PaO^ of the b i r d s b r e a t h i n g a i r was h i g h e r than b e f o r e the d i v e . I f p o s t - d i v e hyperpnea were r e g u l a t e d s o l e l y by chemoreceptor i n p u t , f r e s p s h o u l d r e t u r n to p r e d i v e l e v e l s w i t h i n 30 s. O b v i o u s l y more f a c t o r s a re i n v o l v e d , the s t i m u l u s f o r b r e a t h i n g b e i n g d etermined by c e n t r a l i n t e g r a t i o n of a number of sensory i n p u t s ( c e n t r a l chemoreceptors, mechanoreceptors, pulmonary r e c e p t o r s , e t c ) , not j u s t c a r o t i d chemoreceptor s t i m u l a t i o n . L i l l o and Jones (1982) showed t h a t p o s t d i v e hyperpnea was p a r t l y independent of a r t e r i a l b l o o d gases, as ducks t h a t were a r t i f i c i a l l y v e n t i l a t e d through a d i v e s t i l l e x h i b i t e d a hyperpnea a f t e r the d i v e . There a r e s e v e r a l r e a s o n s f o r the more r a p i d r e t u r n to r e s t i n g f r e s p a f t e r b r e a t h i n g Ox. Not o n l y can the b l o o d c a r r y 5-10% more a f t e r the d i v e , but due to the s e v e r a l m i l l i l i t r e s of 0/^ r e m a i n i n g i n the l u n g , the PaO^ i s so h i g h that the b l o o d i s n e a r l y 100% s a t u r a t e d d u r i n g the d i v e . T h i s c o n t i n u e d h i g h PaO^ through the d i v e a l l o w s more p e r i p h e r a l p e r f u s i o n r e s u l t i n g i n more a e r o b i c m e t abolism and l e s s of an 0^ debt p o s t d i v e . T h i s c o n t i n u e d a e r o b i c metabolism produces more CO^ (seen i n the s i g n i f i c a n t l y h i g h e r PaCO^ v a l u e s f o r the 0j_ d i v e s ) and l e s s 29 l a c t a t e p r o d u c t i o n . Whatever l a c t a t e i s produced i s a v a i l a b l e c e n t r a l l y and t h e r e f o r e c o n t r i b u t e s to decreased pH. (In an a i r di v e , although there i s more l a c t a t e produced, i t remains in the periphery u n t i l the end of the di v e (Murphy e_t a l , 1980). Another e f f e c t of d i v i n g a f t e r b r e a t h i n g 0j_ i s that there i s more p e r f u s i o n of the periphery and the plasma glucose should be more t i g h t l y r e g u l a t e d ; the sharp i n c r e a s e in glucose seen in the a i r t r i a l s should be absent. In f a c t , w h i l e plasma glucose a f t e r 02_ t r i a l s may reach values comparable to those a f t e r a i r t r i a l s , the l a r g e changes one minute a f t e r the dive are not seen. In the usual d e s c r i p t i o n s of the d i v i n g response in ducks, the b r a d y c a r d i a and 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 are often d e s c r i b e d as having c a n c e l l i n g e f f e c t s . T h i s has been shown not to be the case ( B u t l e r and Jones, 1971) and i t i s c l e a r in t h i s work that there i s a d e f i n i t e drop in BP in the course of a i r d i v e s which i s not seen in O x d i v e s . There have been r e p o r t s that c e n t r a l i n t e r a c t i o n between chemoreceptors and ba r o r e c e p t o r s ( H e i s t a d et a l , 1974, Cr u s c i elewsk i e_t al_, 1981), causes the a c t i v a t i o n of one and i n h i b i t s the i n f l u e n c e of the other. T h i s appears to be the case here; in the a i r d i v e s , the p r o g r e s s i v e i n c r e a s e in hypoxia and hypercapnia i s a s s o c i a t e d with a decrease in BP which r e t u r n s to r e s t i n g values only a f t e r the duck begins to breathe again. In the 0^ d i v e , the PaO^ remains 3 times greater than normal through the e n t i r e d i v e , and even though the PaCO^ in c r e a s e s d r a m a t i c a l l y , the o v e r a l l chemoreceptor s t i m u l a t i o n w i l l be l e s s than in an a i r di v e (Jones e_t al_, 1982). In t h i s case, the BP i s much b e t t e r r e g u l a t e d than in the the a i r dive and r a r e l y f a l l s below r e s t i n g l e v e l s . 30 C o n t r o l HR changes through the d i v e were comparable to o t h e r s r e p o r t e d under s i m i l a r c o n d i t i o n s of gases (Mangalam, 1980). C a r o t i d body chemoreceptor c o n t r o l of d i v i n g b r a d y c a r d i a i s w e l l documented i n the P e k i n duck (Jones and P u r v e s , 1970), and thus i t i s no s u r p r i s e t h a t the b r a d y c a r d i a i n the 0^ d i v e s was r e t a r d e d . The e v e n t u a l development of b r a d y c a r d i a can be a t t r i b u t e d to the h i g h v a l u e s of' PaCO^ a t t a i n e d d u r i n g the 0^ d i v e s . N h i l e t h i s study has demonstrated t h a t pH and PaCO^ are not primary causes of a d r e n a l s e c r e t i o n i n d i v i n g ducks and that a l a r g e p a r t of t h a t s e c r e t i o n i s dependent on i n t a c t i n n e r v a t i o n , i t has not r e s o l v e d the main p o i n t . What do the CAs do? Nhy are they r e l e a s e d ? Why i n such h i g h c o n c e n t r a t i o n s ? I f I may end by p l a n t i n g a s i g n post or two: the a d r e n a l s , w i t h t h e i r involvement i n both n e u r a l and e n d o c r i n e f u n c t i o n a re a major c o n t r o l p o i n t i n hom e o s t a s i s . I t would be a s e r i o u s m i s t a k e , e s p e c i a l l y i n view of so much t a n t a l i z i n g ( i f c i r c u m s t a n t i a l ) e v i d e n c e to n e g l e c t thern when c o n s i d e r i n g the c o n t r o l of the d i v i n g r e s p o n s e . 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