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The effect of intravenous salt loading on osmoregulation of hydrated glaucous-winged gulls, Larus glaucescens Raveendran, Lethika 1987

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THE EFFECT OF INTRAVENOUS SALT LOADING ON OSMOREGULATION OF HYDRATED GLAUCOUS-WINGED GULLS, Larus a l a u c e s c e n s . by LETHIKA RAVEENDRAN B.Sc., NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE, 1982 .Sc.(Hons), NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1987 (c) L e t h i k a Raveendran, 1987 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 Z o o l o g y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 „ , 14 Oct 1987 Date DE-6G/81) ABSTRACT Renal f u n c t i o n of f r e s h water a c c l i m a t e d Glaucous-winged G u l l s , L a r u s qlaucescens. was s t u d i e d d u r i n g i n f u s i o n of h y p o t o n i c and h y p e r t o n i c NaCl. Two experimental p r o t o c o l s were f o l l o w e d . In one, the c l o s e d u r i n e c o l l e c t i o n system (CCS), u r e t e r a l u r i n e was c o l l e c t e d u s i n g c a t h e t e r s g l u e d over u r e t e r a l openings of a supine, p r e v i o u s l y a n e s t h e t i z e d g u l l . In the other, the open u r i n e c o l l e c t i o n system (OCS), u r e t e r a l u r i n e was c o l l e c t e d through a f u n n e l p l a c e d i n t h e urodeum o f a s t a n d i n g , u n a n e s t h e t i z e d b i r d . In both p r o t o c o l s , t h e r e was continuous s a l i n e i n f u s i o n of hypotonic (hydration) and h y p e r t o n i c (LOAD) s a l i n e a t 0.286 m l - m i n - 1 . Glomerular f i l t r a t i o n r a t e (GFR) and e f f e c t i v e r e n a l plasma flow (ERPF), m l ( k g * m i n ) - 1 , were determined by 1 4 C - p o l y e t h y l e n e g l y c o l (PEG) and 3H-para-aminohippuric a c i d (PAH) c l e a r a n c e s . Plasma v a s o t o c i n (P^VT' pg*ml - 1) was measured. At the end o f 4 h h y d r a t i o n w i t h 0.02 M NaCl, u r i n e flow was h i g h but matched i n f u s i o n r a t e o n l y i n CCS b i r d s (CCS, 0.29 + 0.05; OCS, 0.17 + 0.03 rnl-min"1), GFR (CCS, 5.56 + 0.85; OCS, 5.36 + 0.77) and ERPF (CCS, 15.80 + 1.60; OCS, 14.35 + 1.65) were h i g h ; u r i n e sodium ( U N a + ) c o n c e n t r a t i o n was low (CCS, 15.0 + 7.3; OCS, 36.4 + 6.0 m E q * l _ 1 ) , U N a + e x c r e t i o n was low (CCS, 6.38 + 4.2; OCS, 5.19 uEq-min - 1) ; urine/plasma PEG r a t i o ( U / P P E G ) was h i g h (CCS, 22.4 + 4.4, OCS, 39.6 + 8.5); f r e e water c l e a r a n c e (C H 2 o ) w a s p o s i t i v e (CCS, 0.143 + 0.011; OCS, i i i 0.052 + 0.019 ml'min - 1) , and P^VT w a s l o w ( c c s» 14.7 + 7.4; OCS, 1 6 . 1 + 2 . 4 ) i n both groups. Immediately f o l l o w i n g i n f u s i o n o f 5 M NaCl, GFR, ERPF and u r i n e flow i n c r e a s e d f o r about 10 mins. F i f t e e n minutes l a t e r , the GFR o f CCS g u l l s f e l l t o 70% o f p r e - l o a d v a l u e s (P < 0.05) and i n OCS g u l l s , GFR and ERPF f e l l t o 64% (P < 0.01) and 61% (P < 0.05). E i g h t y mins a f t e r i n f u s i o n o f 5 M NaCl, the GFR and ERPF of CCS g u l l s r e t u r n e d t o pre-LOAD l e v e l s , but remained low i n OCS g u l l s . Twenty-five minutes a f t e r s a l t l o a d , u r i n e flow had f a l l e n t o 49% (P < 0.05) and remained low. In OCS g u l l s , u r i n e flow had f a l l e n t o 13% (P < 0.001) a f t e r 185 mins. In both CCS and OCS g u l l s , U N a + c o n c e n t r a t i o n and e x c r e t i o n i n c r e a s e d s i g n i f i c a n t l y . S i x t y minutes a f t e r s a l t l o a d , U N a + e x c r e t i o n r e t u r n e d t o pre-LOAD l e v e l s but U N a + c o n c e n t r a t i o n remained h i g h i n CCS (111.7 + 57.5) and OCS (132.8 + 12.5) g u l l s . U / P P E G a t t a i n e d 134.3 + 26.5 i n CCS and 181.2 + 32.4 i n OCS g u l l s . C H 2 o f e l l s i g n i f i c a n t l y (P < 0.05) i n CCS g u l l s but remained unchanged i n OCS g u l l s . Mean P^VT i n c r e a s e d t o 122.5 + 5.5 i n CCS and 96.0 + 12.6 i n OCS g u l l s . In both CCS and OCS g u l l s , s a l t g l a n d s e c r e t i o n was i n i t i a t e d but ceased 60 mins a f t e r 5M NaCl i n f u s i o n , although 60% of the l o a d was r e t a i n e d i n the g u l l . i v TABLE OF CONTENTS ABSTRACT i i LIST OF ABBREVIATIONS V LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 MATERIALS AND METHODS 6 RESULTS 24 GULLS BEFORE INFUSION 24 INFUSED GULLS 24 BLOOD PRESSURE EXPERIMENTS 46 DISCUSSION 52 LITERATURE CITED 76 V LIST OF ABBREVIATIONS AVP - A r g i n i n e V a s o p r e s s i n AVT - A r g i n i n e V a s o t o c i n BSA - Bovine Serum Albumin CCS - Cl o s e d C o l l e c t i o n System C H 2 o ~ Free Water Clearance cosm ~ Osmolar Clearance ECF - E x t r a c e l l u l a r F l u i d ERPF - E f f e c t i v e Renal Plasma Flow GFR - Glomerular F i l t r a t i o n Rate Hct - Hematocrit MT - Mesotocin NRS - Normal Rabbit Serum OCS - Open C o l l e c t i o n System OXY - Oxy t o c i n PAH - Para-aminohippuric a c i d PAVT ~ Plasma AVT P d - - Plasma C h l o r i d e PEG - P o l y e t h y l e n e g l y c o l Pj^ + - Plasma Potassium P N a + - Plasma Sodium pOsm ~ Plasma O s m o l a l i t y RIA - Radioimmunoassay TFA - T r i f l u o r o a c e t i c a c i d U C i _ - U r i n e C h l o r i d e U K + - U r i n e Potassium v i U N a + - U r i n e Sodium uOsm ~ U r i n e O s m o l a l i t y u/ pOsm ~ Urine/Plasma O s m o l a l i t y R a t i o U / P P E G - Urine/Plasma PEG R a t i o v i i LIST OF TABLES 1. E f f e c t o f s a l t l o a d on plasma AVT and plasma osmotic c o n c e n t r a t i o n s 29 2. E f f e c t o f s a l t l o a d on u r i n e i o n i c and osmotic c o n c e n t r a t i o n s 38 3. E f f e c t o f s a l t l o a d on urine/plasma PEG r a t i o , p e r c e n t sodium r e a b s o r p t i o n , and water and osmolar c l e a r a n c e 44 v i i i LIST OF FIGURES 1. Schematic drawing of experimental p r o t o c o l 9 2. E f f e c t of p r i o r e x t r a c t i o n of plasma on immunoreactive AVT measurements 15 3. Standard curves of AVT, showing i m m u n o r e a c t i v i t i e s of AVP, AVT, OXY and MT i n the RIA system 20 4. E f f e c t of s a l t l o a d on h e m a t o c r i t 25 5. E f f e c t of s a l t l o a d on plasma e l e c t r o l y t e c o n c e n t r a t i o n 27 6. E f f e c t of s a l t l o a d on plasma AVT c o n c e n t r a t i o n s i n t h r e e CCS g u l l s 31 7. E f f e c t of hypotonic s a l i n e i n f u s i o n on g l o m e r u l a r f i l t r a t i o n r a t e and u r i n e flow 34 8 E f f e c t of s a l t l o a d on g lomerular f i l t r a t i o n r a t e , e f f e c t i v e r e n a l plasma flow and u r i n e flow 36 9. E f f e c t o f s a l t l o a d on r a t e of r e n a l e x c r e t i o n 40 10. E f f e c t of s a l t l o a d on urine/plasma PEG r a t i o 42 11. E f f e c t of s a l t l o a d on mean a r t e r i a l p r e s s u r e i n b l o o d p r e s s u r e experiments 47 12. E f f e c t of s a l t l o a d on plasma e l e c t r o l y t e c o n c e n t r a t i o n i n b l o o d p r e s s u r e experiments 49 13. The r e l a t i o n s h i p between plasma o s m o l a l i t y and plasma AVT c o n c e n t r a t i o n 66 i x ACKNOWLEDGEMENTS I would l i k e t o g i v e a s p e c i a l v o t e o f thanks t o Dr. Maryanne R. Hughes f o r her guidance and a s s i s t a n c e i n p l a n n i n g and e x e c u t i n g t h i s p r o j e c t . I a l s o thank Drs. Nadine Wilson, David R. Jones and Anthony M. Perks f o r t h e i r a d v i c e and f o r r e v i e w i n g t h i s manuscript. T h i s r e s e a r c h was supported by N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l Grant A-3442 of Canada t o Maryanne R. Hughes. 1 INTRODUCTION B i r d s i n h a b i t a wide range of h a b i t a t s , both t e r r e s t r i a l and o c e a n i c , and e x h i b i t osmoregulatory c a p a c i t i e s t o d e a l with osmotic environments ra n g i n g from d e s e r t s and oceans, where f r e e water i s almost t o t a l l y l a c k i n g , t o f r e s h water l a k e s and streams, where i t i s i n excess. Marine b i r d s , i n p a r t i c u l a r , f r e q u e n t l y encounter osmotic s t r e s s e s . T h e i r d i e t i n c l u d e s marine i n v e r t e b r a t e s , which are o s m o t i c a l l y e q u i v a l e n t t o sea water. The degree of s a l t s t r e s s normally f a c e d by a marine b i r d would be l e t h a l t o most t e r r e s t r i a l b i r d s . The key t o the success of marine b i r d s l i e s i n t h e i r a b i l i t y t o o b t a i n s o l u t e f r e e water from sea water through the remarkably e f f i c i e n t e x t r a r e n a l e x c r e t i o n o f NaCl v i a the s a l t glands. T h e r e have been many s t u d i e s of the osmoregulatory c a p a c i t i e s of t e r r e s t r i a l s p e c i e s , p a r t i c u l a r l y the domesticated s p e c i e s , s i n c e they are e a s i l y a c c e s s i b l e and have o n l y one route f o r water and i o n e x c r e t i o n , the kidneys. There are fewer s t u d i e s of osmoregulation i n marine b i r d s (Skadhauge, 1981) because t h e i r osmoregulatory processes are more complicated, due t o e x t r a r e n a l e x c r e t i o n by the s a l t glands. Osmotic s t r e s s may be induced by the l o s s of body water or the a d d i t i o n of o s m o t i c a l l y a c t i v e m a t e r i a l s t o the body water. Both mechanisms occur i n marine b i r d s , but i n g e s t i o n of food and water, h i g h i n NaCl content, i s probably the more s i g n i f i c a n t method. Marine b i r d s such as cormorants, Phalacrocorax a u r i t u s  a u r i t u s (Schmidt-Nielsen, JBrgensen and Osaki, 1958), h e r r i n g 2 g u l l s , Larus arqentatus (Douglas, 1970) , glaucous-winged g u l l s , L a r u s q l a u c e s c e n s (Hughes, 1970a) and k e l p g u l l s , Larus  dominicanus (Gray and Erasmus, unpublished) can m a i n t a i n the osmotic c o n c e n t r a t i o n o f t h e i r plasma a t about one t h i r d t h a t of the food and water they consume. The h i g h l y e f f i c i e n t s a l t glands, by which they accomplish t h i s , have been observed t o be a p r e f e r r e d r o u t e o f e x c r e t i o n even i n o s m o t i c a l l y u n s t r e s s e d marine b i r d s (Hughes, 1970a). Although the c o n t r i b u t i o n o f the s a l t glands t o osmoregulation i n marine b i r d s i s w e l l s t u d i e d , the f u n c t i o n a l r e l a t i o n s h i p between r e n a l and e x t r a r e n a l pathways i n marine b i r d s i s p o o r l y understood. The simultaneous f u n c t i o n o f the kidneys d u r i n g e x t r a r e n a l s a l t e x c r e t i o n , has been s t u d i e d o n l y i n domestic ducks, Anas  p i a t y r h y n c h o s (Simon-Oppermann, Simon, Deutsch, Mohring and Schoun, 1980; Simon-Oppermann and Simon, 1982; Gers t b e r g e r , Simon and Gray, 1984). E x t r a r e n a l s a l t s e c r e t i o n i n ducks i s poor compared t o marine s p e c i e s and they are unable t o m a i n t a i n body mass o r plasma c o n c e n t r a t i o n when g i v e n even d i l u t e seawater t o d r i n k (Roberts and Hughes, 1984). The e x t r a r e n a l system i n ducks r e q u i r e s s a l i n e a c c l i m a t i o n b e f o r e i t becomes f u n c t i o n a l l y e f f i c i e n t . The i n t e g r a t i o n o f osmoregulatory f u n c t i o n between r e n a l and e x t r a r e n a l organs has not been e v a l u a t e d i n a marine s p e c i e s capable o f m a i n t a i n i n g a con s t a n t i n t e r n a l environment when kept under normal hyperosmotic environmental c o n d i t i o n s . The a v i a n kidney possesses two types o f nephrons, r e p t i l i a n -type nephrons, which l a c k the loops o f Henle and cannot 3 c o n c e n t r a t e u r i n e , and mammalian-type nephrons w i t h have loops of Henle and can c o n c e n t r a t e u r i n e . U s i n g mammalian-type nephrons, b i r d s l i k e mammals, are a b l e t o produce hyperosmotic u r i n e (Braun and D a n t z l e r , 1972). The savannah marsh sparrow, P a s s e r c u l u s  sandwichensis. produces u r i n e about f o u r times the o s m o l a l i t y of the b l o o d (Poulson and Bartholomew (1962). Most o t h e r s p e c i e s , however, have a more l i m i t e d c o n c e n t r a t i n g a b i l i t y . In g e n e r a l , a v i a n s p e c i e s w i t h s a l t glands have l a r g e r kidneys than those t h a t l a c k them (Hughes, 1970b) and g u l l (Larus o c c i d e n t a l i s ) kidneys have a l a r g e r number of mammalian-type nephrons r e l a t i v e t o body s i z e than any o t h e r a v i a n s p e c i e s known (Braun, 1984). The u r e t e r a l u r i n e of glaucous-winged g u l l s i s u s u a l l y a l s o s t r o n g l y h y p e r t o n i c t o plasma, but c o n t a i n s low sodium, c h l o r i d e and potassium c o n c e n t r a t i o n s ( G o l d s t e i n , Hughes and Braun, 1986). Even f o l l o w i n g stomach l o a d i n g w i t h sea water, the u r i n e of both cormorants and h e r r i n g g u l l s , has a low sodium c o n c e n t r a t i o n (Schmidt-Nielsen e t a l . , 1958; Douglas, 1970). These s t u d i e s suggest t h a t r e n a l f u n c t i o n of t e r r e s t r i a l and marine b i r d s i s d i s s i m i l a r and t h a t marine b i r d s do not r e l y on the kidneys f o r i o n e x c r e t i o n . The p r o d u c t i o n of u r i c a c i d , as the major form o f n i t r o g e n e x c r e t i o n , i s a l s o an a v i a n a d a p t a t i o n t o conserve water. U r i c a c i d , b e i n g h i g h l y i n s o l u b l e i n water, f r e e s water f o r the e x c r e t i o n of o t h e r wastes. The a s s o c i a t i o n of c a t i o n s w i t h u r i c a c i d t o form u r a t e s produces more o s m o t i c a l l y f r e e water. As much 4 as 98% of e x c r e t e d Na has been found t o be a s s o c i a t e d w i t h u r i c a c i d (Braun, 1978). The r e n a l response t o s a l t l o ads have been e v a l u a t e d i n s t a r l i n g s , Sturnus v u l g a r i s (Braun, 1978), c h i c k e n s , G a l l u s  domesticus ( D a n t z l e r , 1966) and q u a i l s , Lophortyx g a m b e l i i (Braun and D a n t z l e r , 1974). A l l these b i r d s l a c k s a l t glands. In response t o s a l t s t r e s s , s t a r l i n g s attempted t o lower i n c r e a s i n g l e v e l s o f plasma o s m o l a l i t y by m a i n t a i n i n g r e n a l f u n c t i o n (glomerular f i l t r a t i o n r a te) and i n c r e a s i n g u r i n e flow, thereby produ c i n g a more co n c e n t r a t e d u r i n e a t the expense of body water. In c o n t r a s t , the g a l l i n a c e o u s b i r d s , chickens and d e s e r t q u a i l s , reduced r e n a l f u n c t i o n and t o l e r a t e d h i g h e r plasma o s m o l a l i t y r a t h e r than l o s e body water. The i n c r e a s e d plasma sodium and o s m o l a l i t y a s s o c i a t e d w i t h environmental c o n d i t i o n s of s a l t s t r e s s may be s i m u l a t e d by i n t r a v e n o u s NaCl i n f u s i o n . An i n c r e a s e i n plasma o s m o l a l i t y has been shown t o be c o r r e l a t e d w i t h an i n c r e a s e i n the l e v e l of plasma AVT, the a v i a n a n t i d i u r e t i c hormone (Mohring, Schoun, Simon-Oppermann and Simon, 1980; S t a l l o n e and Braun, 1986). AVT a f f e c t s both glomerular f i l t r a t i o n ( D a n t z l e r and Braun 1974, 1980) and t u b u l a r t r a n s p o r t ( S t a l l o n e and Braun, 1985) t o conserve water. In c h i c k e n s , i n f u s i o n of AVT produces e f f e c t s mimicking those of s a l t l o a d i n g , c o n f i r m i n g the a n t i d i u r e t i c r o l e of AVT (Ames, Steven and Skadhauge, 1971; Skadhauge, 1964, 1968). The e f f e c t s of AVT on s a l t g l a n d s e c r e t i o n are q u e s t i o n a b l e . These i n c l u d e a r e d u c t i o n of N a + c o n c e n t r a t i o n but i n c r e a s e of 5 s a l t g l a n d s e c r e t i o n r a t e i n the goose, Anser anser (Peaker, 1970) and a t h i g h doses, AVT can induce s e c r e t i o n , i n the absence of a s a l t l o a d , although a t a low r a t e . G i l l and B u r f o r d (1969) showed t h a t v a s o p r e s s i n , the mammalian a n t i d i u r e t i c hormone, reduced s a l t g l a n d s e c r e t i o n r a t e and sodium c o n c e n t r a t i o n i n s a l t loaded ducks and geese. Hughes (1962), however, found t h a t mammalian a n t i d i u r e t i c hormones n e i t h e r s t i m u l a t e d s a l t gland s e c r e t i o n nor a f f e c t e d the r a t e o f s e c r e t i o n i n g u l l s . T h i s study was done t o examine i f s a l t l o a d i n g had an e f f e c t on s a l t g l a n d s e c r e t i o n and r e n a l f u n c t i o n o f hydrated glaucous-winged g u l l s . j 6 MATERIALS AND METHODS Experimental animals. Glaucous-winged g u l l s (Larus qlaucescens) used i n these s t u d i e s were 2-3 years o l d and had been c o l l e c t e d as n e s t l i n g s on C h r i s t i e I s l e t , i n Howe Sound, B r i t i s h Columbia (49° 30'N; 123° 18'W). They were r a i s e d i n l a r g e outdoor e n c l o s u r e s a t South Campus Animal Care F a c i l i t y , U n i v e r s i t y of B r i t i s h Columbia. The mean body mass of g u l l s used i n experiments employing the c l o s e d and open u r i n e c o l l e c t i o n systems, was 936.7 + 40.7 g and 865.6 + 32.5 g r e s p e c t i v e l y . G u l l s were f e d p r e v i o u s l y - f r o z e n h e r r i n g (Hughes, 1972a) supplemented w i t h a l i q u i d v i t a m i n mixture (Paramette, A y e r s t L a b o r a t o r i e s , Montreal) and f r e s h water ad l i b i t u m . To reduce f e c a l m a t e r i a l and u r i c a c i d content o f u r i n e , b i r d s were f a s t e d f o r 18 h b e f o r e the experiment, but had f r e e access t o d r i n k i n g water. General experimental procedure C a t h e t e r s (Delmed I-Cath, Delmed Inc. Canton, US) were i n s e r t e d i n t o the l e f t b r a c h i a l v e i n f o r the i n f u s i o n o f the s a l i n e s o l u t i o n s and r i g h t b r a c h i a l v e i n f o r the c o l l e c t i o n o f bl o o d samples. Both c a t h e t e r s were f i l l e d w i t h h e p a r i n i z e d (10 mU'ml - 1) i s o t o n i c s a l i n e between samplings t o ensure patency. The b i r d ' s wings were l i g h t l y bound t o i t s body w i t h p l a s t i c tape and the b i r d was r e s t r a i n e d a p p r o p r i a t e l y , depending on the method of u r i n e c o l l e c t i o n . 7 Experiments were conducted u s i n g two d i f f e r e n t p r o t o c o l s and employing two d i f f e r e n t d e v i c e s f o r u r e t e r a l u r i n e c o l l e c t i o n . 1) Closed Collection System (CCS). The CCS technique o f u r e t e r a l u r i n e c o l l e c t i o n i s s i m i l a r t o t h a t , o r i g i n a l l y developed by Sperber (1948) and m o d i f i e d by Cooke and Young (1970). A c o l l e c t i o n f u n n e l was c o n s t r u c t e d by f l a r i n g one end of a 3 cm p i e c e o f p o l y e t h y l e n e t u b i n g (O.D. 3.50 mm; I.D. 2.69 mm) and a h o l e was p i e r c e d i n the stem, where i t abutted the f l a r e d p o r t i o n , t o minimise p r e s s u r e d i f f e r e n c e s t h a t might impede u r i n e flow i n the tube. To reduce s t r e s s d u r i n g the attachment o f the c o l l e c t i n g f u n n e l s , the b i r d was a n e s t h e t i z e d w i t h 0.3 ml S o m n o t o l ( s o d i u m p e n t o b a r b i t o l , M.T.C. P h a r m a c e u t i c a l s , M i s s i s s a u g a O n t a r i o ) . The urodeum was e v e r t e d manually and a fu n n e l was g l u e d over each u r e t e r a l opening w i t h c y a n o a c r y l a t e adhesive (Krazy g l u e ) . The b i r d was p l a c e d i n a supine p o s i t i o n and secured t o a wooden board w i t h p l a s t i c tape. To f a c i l i t a t e u r i n e c o l l e c t i o n i n t o preweighed p o l y s t y r e n e tubes, the board was t i l t e d e l e v a t i n g the b i r d ' s head t o an angle o f 30°C. The b i r d was allowed t o re c o v e r from the a n e s t h e s i a b e f o r e the s a l t l o a d was g i v e n . P r o t o c o l ( F i g . 1) : The CCS b i r d s were i n f u s e d f o r 4 h with 0.02 M NaCl, f o l l o w e d by 13 mins of 5 M NaCl, f o l l o w e d by 4 h of 0.02 M NaCl. A l l s o l u t i o n s were i n f u s e d a t 0.286 m l ' m i n - 1 . 2) Open Collection System (OCS). The OCS used a d e v i c e which ensured the f r e e flow o f u r i n e . I t was made by g l u i n g the bottom 30 mm of a p o l y s t y r e n e t e s t - t u b e (diameter 14 mm) t o a s h o r t 8 p l a s t i c f u n n e l o f the same diameter. An opening, s l i g h t l y l a r g e r than the area encompassing the u r e t e r a l p a p i l l a e , was burnt i n t o the s i d e o f the tube. The s t a n d i n g b i r d was r e s t r a i n e d i n a s p e c i a l l y designed perspex s l i n g . One ml o f l o c a l a n e s t h e s i a ( X y l o c a i n e 2%, L i d o c a i n e H y d r o c h l o r i d e I n j e c t i o n USP, A s t r a Pharmaceuticals Canada L t d , M i s s i s s a u g a Ontario) was i n j e c t e d under the s k i n around the c l o a c a l opening. The e x t e r i o r o f the d e v i c e was l u b r i c a t e d w i t h V a s e l i n e petroleum j e l l y and i n s e r t e d i n t o the urodeum. U r i n e dropped d i r e c t l y i n t o the f u n n e l and was c a r r i e d t o a preweighed 5 ml p o l y s t y r e n e tube. P r o t o c o l ( F i g . 1) : Same as CCS except t h a t 30 mins a f t e r s a l t l o a d i n g , the r a t e o f i n f u s i o n was reduced t o 0.029 m l * m i n - 1 f o r t he remaining 3 1/2 h. P r e - s a l t l o a d i n f u s i o n p e r i o d . An i n i t i a l b l o o d sample (2.5 ml) was taken b e f o r e i n f u s i o n of 0.02 M NaCl. T h i r t y minutes a f t e r i n i t i a t i o n o f i n f u s i o n , 0.2 ml i s o t o n i c s a l i n e , c o n t a i n i n g 2 u C i 1 4 C - p o l y e t h y l e n e g l y c o l (PEG, MW 4000, New England Nuclear) and 10.4 u l 0.01 N HCl c o n t a i n i n g 10 u C i 3H-para-aminohippuric a c i d (PAH, MW 194.2, NEN), were g i v e n as bol u s i n j e c t i o n s through the i n f u s i o n c a t h e t e r . T h i s was f o l l o w e d by 3 1/2 h i n f u s i o n o f 0.02 M NaCl c o n t a i n i n g PEG (0.019 u C i - m l - 1 ) and PAH (0.212 u C i ' m l " 1 ) . During the f i n a l 30 mins, t h r e e 10 min u r i n e c o l l e c t i o n s were made with a b l o o d sample (0.5 ml, t h i r d sample 2.5 ml) c o l l e c t e d i n t o a nonheparinzed s y r i n g e a t the midpoint o f each u r i n e c o l l e c t i o n . 9 F i g u r e l . Schematic drawing of experimental p r o t o c o l . B i r d s were c o n t i n o u s l y i n f u s e d : 4 h of 0.02 M NaCl (unshaded) a t 0.286 ml'min"*, f o l l o w e d by 13 mins o f 5 M NaCl (shaded), f o l l o w e d by 4 h of 0.02 M NaCl a t 0.286 ml • m i n - 1 , except f o r the f i n a l 0.02 M NaCl i n f u s i o n i n OCS g u l l s , i n which i n f u s i o n and u r i n e formation was matched (0.029 ml'min--1-) . A p r e - i n f u s i o n b l o o d sample (arrow) was taken. Subsequent b l o o d samples were taken a t the midpoint o f u r i n e c o l l e c t i o n s . Three b l o o d samples were taken a t 25, 15 and 5 mins b e f o r e s a l t l o a d i n g (mean pre-LOAD v a l u e , d e s i g n a t e d as -15 mins); one b l o o d sample was taken a t the midpoint of s a l t l o a d i n g (LOAD v a l u e , d e s i g n a t e d as 0 mins) and o t h e r b l o o d samples were taken a t the midpoint of timed u r i n e c o l l e c t i o n s (post-LOAD v a l u e s ) . (CCS, c l o s e d c o l l e c t i o n system; OCS, open c o l l e c t i o n system) 210 mins 0 286 ml-min-' C C S G U L L S 210 mins 30 13 30 mins mins mins INFUSION RATE 0-286 ml-min-' 1 I t 0 029 D O C S G U L L S ml-min-' * M J \ \ \ PRE -INFUSION P R E - L O A D L O A D P O S T - L O A D 11 The mean hemat o c r i t and plasma c o n c e n t r a t i o n s o f these samples are d e s i g n a t e d as the p r e - l o a d v a l u e s (time = -15 mins). S a l t l o a d and p o s t s a l t l o a d i n f u s i o n p e r i o d . A f t e r 4 h of 0.02 M NaCl i n f u s i o n , the NaCl c o n c e n t r a t i o n of the p e r f u s a t e was changed t o 5 M NaCl (LOAD) f o r 13 mins. Then the i n f u s i o n of 0.02 M NaCl i n f u s a t e was resumed and continued a t 0.286 m l - m i n - 1 f o r 4 h i n CCS experiments o r reduced a f t e r 30 mins t o 0.029 m l - m i n - 1 i n OCS b i r d s ( F i g . 1). In both methods, wit h the onset of a n t i d i u r e s i s , u r i n e c o l l e c t i o n was made over 3-6 i n t e r v a l s each 30-60 mins. About o n e - h a l f h b e f o r e 5M NaCl i n f u s i o n , the g u l l ' s head was secured t o a s p e c i a l h o l d e r . A few minutes b e f o r e t h i s i n f u s i o n , a preweighed v i a l was p l a c e d over the upper beak. S a l t g l a n d s e c r e t i o n was c o l l e c t e d over s i m i l a r time i n t e r v a l s as the u r i n e . Plasma of CCS g u l l s was assayed f o r plasma AVT measurements without p r i o r e x t r a c t i o n . OCS g u l l plasma was e x t r a c t e d u s i n g Sep-Pak c a r t r i d g e s . Mean a r t e r i a l p r e s s u r e Four experiments t o observe the e f f e c t of the s a l t l o a d on mean a r t e r i a l p r e s s u r e were c a r r i e d out on two OCS g u l l s (mean body mass, 945 + 125 g) c h r o n i c a l l y c a t h e t e r i z e d two days b e f o r e the experiment. Under a s e p t i c c o n d i t i o n s and l o c a l a n e s t h e s i a , a c a t h e t e r (Intravenous medical v i n y l t u b i n g #3, Bolab Inc., Lake Havasu C i t y , Arizona) was i n s e r t e d i n t o the b r a c h i a l a r t e r y so t h a t the c a t h e t e r t i p was p o s i t i o n e d near the h e a r t . The f r e e end 12 o f t he c a t h e t e r was secured t o the s k i n w i t h adhesive tape and tucked under the wing, which was l i g h t l y taped t o the body. During the p o s t - s u r g e r y r e c o v e r y p e r i o d , the c a t h e t e r was f l u s h e d w i t h h e p a r i n i z e d i s o t o n i c s a l i n e (50 U/ml) d a i l y t o ma i n t a i n patency. On the morning of the experiment, a c a t h e t e r (Delmed I-Cath, Delmed Inc. Canton, US), f o r the i n f u s i o n o f s a l i n e , was i n s e r t e d i n t o the b r a c h i a l v e i n o f the unoperated wing. Blood sampling and pr e s s u r e r e c o r d i n g s were made v i a the a r t e r i a l c a t h e t e r . The p r o t o c o l f o r OCS g u l l s was f o l l o w e d except t h a t o n l y two bl o o d samples were taken b e f o r e s a l t l o a d i n g , which was f o l l o w e d by o n l y 50 mins 0.02 M NaCl i n f u s i o n . The mean a r t e r i a l p r e s s u r e was determined from b l o o d p r e s s u r e r e c o r d i n g s o b t a i n e d u s i n g a s t r a i n - g a u g e p r e s s u r e t r a n s d u c e r ( B i o t e c , BT-70, Pasadena, C a l i f o r n i a ) and r e c o r d e r (Harvard, Mass.). C o l l e c t i o n and h a n d l i n g o f blood, u r i n e and s a l t g l a n d s e c r e t i o n  samples. Blood samples (0.5 ml, samples f o r plasma sodium, c h l o r i d e , potassium, o s m o l a l i t y , r a d i o a c t i v i t y , h e m a t o c r i t ; 2.0 ml, samples f o r AVT) were c o l l e c t e d i n t o c l e a n nonheparinized s y r i n g e s . The 0.5 ml b l o o d sample was t r a n s f e r r e d t o a 1.5 ml dry, p r e v i o u s l y h e p a r i n i z e d (0.5 U/tube) Eppendorf c e n t r i f u g e tube. Strumia h e m a t o c r i t tubes were f i l l e d and c e n t r i f u g e d a t 15,600 g f o r 3 mins f o r hema t o c r i t (Hct) d e t e r m i n a t i o n . The remaining b l o o d was c e n t r i f u g e d a t 15,600 g f o r 3 mins. The plasma was removed and s t o r e d a t 4°C f o r a n a l y s i s the next day. 13 The pre-weighed tubes c o n t a i n i n g u r i n e and s a l t gland s e c r e t i o n were reweighed. U r i n e samples were c e n t r i f u g e d and the supernatant f l u i d was decanted and s t o r e d a t room temperature (22-24°C, t o prevent u r i c a c i d p r e c i p i t a t i o n ) f o r a n a l y s i s the next day. For some u r i n e samples from t h r e e CCS and t h r e e OCS g u l l s , the u r i n e p e l l e t was a i r - d r i e d and s t o r e d a t -20°C f o r the a n a l y s i s of u r i c a c i d . A n a l y t i c a l procedures E l e c t r o l y t e c o n c e n t r a t i o n s . Plasma (50 u l ) , u r i n e (50 u l ) and s a l t g l a n d s e c r e t i o n (10 u l ) were taken f o r d e t e r m i n a t i o n of [Na +] and [K +] u s i n g an i n t e r n a l standard flame photometer (Model 143, Instrumentation Laboratory Inc., Boston). The [Na +] of d i s s o l v e d u r i c a c i d samples were analysed u s i n g the s o l v e n t l i t h i u m carbonate as the i n t e r n a l standard. The [ C l ~ ] was determined u s i n g a Buchler D i g i t a l Chloridometer. Osmotic p r e s s u r e . U r i n e o s m o l a l i t y (U 0 s i l l) and plasma o s m o l a l i t y ( p0sm) were measured i n CCS g u l l s by f r e e z i n g p o i n t d e p r e s s i o n (Advanced Osmometer, Model 31LAS) and i n OCS g u l l s u s i n g a Wescor Model 5500 Vapour Pressure Osmometer. R a d i o a c t i v i t y . A l i q u o t s (50 u l ) of plasma and u r i n e samples were p l a c e d i n t o 10 ml of Aquasol-2 (NEN, Canada) and assayed f o r 1 4 C and 3H u s i n g a d u a l l a b e l l i q u i d s c i n t i l l a t i o n c o u n t i n g program (Beckman model LS9000 s c i n t i l l a t i o n c o u n t e r ) . U r i c a c i d . The s o l i d f r a c t i o n of u r i n e was weighed, p u l v e r i z e d and d i s s o l v e d u s i n g a m o d i f i c a t i o n of the method o f Bose (1944). 14 One ml o f l i t h i u m carbonate (0.5% w/v) was added f o r every 2.5 mg p r e c i p i t a t e and the mixture was shaken o v e r n i g h t a t 70°C. More s o l v e n t was added and the sample was shaken u n t i l a l l the p r e c i p i t a t e was d i s s o l v e d . U r i c a c i d was a n a l y s e d u s i n g a Technicon Auto-Analyser. Radioimmunoassay (RIA) of A r g i n i n e v a s o t o c i n (AVT)  Methods of c o l l e c t i n g and e x t r a c t i n g b l o o d . Blood samples (2.0 ml) f o r RIA were t r a n s f e r r e d i n t o c h i l l e d EDTA t u b e s ( V a c u t a i n e r , B e c t o n - D i c k i n s o n Canada Ltd) and c e n t r i f u g e d a t 4° a t 600 g. Plasma was removed and s t o r e d i n s e a l e d 12 x 75 mm p o l y e t h y l e n e tubes a t -20°C. The AVT c o n c e n t r a t i o n of u n e x t r a c t e d plasma was compared t o t h a t of plasma e x t r a c t e d by 2 d i f f e r e n t methods 1) acetone / petroleum e t h e r e x t r a c t i o n and 2) Sep-Pak c a r t r i d g e e x t r a c t i o n . I t was found ( F i g . 2) t h a t 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 PAVT between the acetone e x t r a c t e d plasma and the u n e x t r a c t e d plasma, however, Sep-Pak e x t r a c t e d plasma y i e l d e d s i g n i f i c a n t l y h i g h e r P^VT than both the u n e x t r a c t e d plasma (P < 0.05) and the ether-acetone e x t r a c t e d plasma (P < 0.05, p a i r e d t - t e s t ) . 1) Acetone/petroleum e x t r a c t i o n . Plasma was e x t r a c t e d w i t h o r g a n i c s o l v e n t s as d e s c r i b e d by Robertson, Mahr, Athar and Sinha (1973) w i t h m o d i f i c a t i o n s by Ledsome, Wilson and Ngsee (1982). One ml plasma samples, i n c l u d i n g 1 q u a l i t y c o n t r o l ( r e f e r e n c e plasma used i n a l l assays) and 1 r e c o v e r y sample (plasma wi t h known amount o f 1 2 5 I - A V T ) were p i p e t t e d i n t o pre-weighed 13x100 mm s i l i c o n i z e d t e s t - t u b e s , mixed wi t h 3 ml s p e c t r a l grade acetone 15 F i g u r e 2. E f f e c t o f p r i o r e x t r a c t i o n o f plasma on immunoreactive AVT measurements of non-infused g u l l s . Measurements are means + SE ( number of b i r d s = 9 ) 60 cn Q 50 -> < < < 40 " 30 -A C E T O N E S E P - P A K U N E X T R A C T E D E X T R A C T E D 17 (Eastman Kodak Co.) and w e l l mixed (vortex a g i t a t o r ) f o r 1 min and c e n t r i f u g e d (60 mins) a t 500 g a t 4°C i n a r e f r i g e r a t e d c e n t r i f u g e ( I n t e r n a t i o n a l R e f r i g e r a t e d C e n t r i f u g e , I n t e r n a t i o n a l Equipment Co., Mass., USA). The supernatant f l u i d was decanted i n t o pre-weighed 16x150 mm s i l i c o n i z e d t e s t - t u b e s . The p e l l e t was resuspended by mixing i n 1 ml c o l d acetone, c e n t r i f u g e d and decanted as d e s c r i b e d e a r l i e r . The supernatant f l u i d s were pooled and 7 mis o f c o l d petroleum e t h e r (BDH Chemicals) was added and the mixture was g e n t l y mixed f o r 1 min. S e p a r a t i o n of the mixture i n t o two phases o c c u r r e d when the tubes were l e f t u n d i s t u r b e d f o r 1 h r a t 4°C. The upper (petroleum ether) phase was a s p i r a t e d and d i s c a r d e d . One hundred u l 0.2 M a c e t i c a c i d ( A l l i e d F i s h e r S c i e n t i f i c ) was added t o a l l tubes except r e c o v e r y . The tubes were kept on an i c e bath w h i l e the acetone was evaporated under n i t r o g e n . The r e m a i n i n g plasma volume was determined by reweighing tubes. Samples were mixed, s e a l e d w i t h p a r a f i l m and s t o r e d a t -20°C f o r RIA the next day. Recovery of s y n t h e t i c AVT added t o plasma samples was 85.4 + 2.4% a f t e r e x t r a c t i o n . R e s u l t s were not c o r r e c t e d f o r r e c o v e r y . 2) Sep-Pak e x t r a c t i o n . AVT was e x t r a c t e d from plasma by a d s o r p t i o n onto prepacked o c t a d e c a s i l y l - s i l i c a c a r t r i d g e s (Sep-Pak C18, Waters) a c c o r d i n g t o a method adapted from H a r t t e r (1986). Each c a r t r i d g e was used f o r o n l y one sample. Before each use, c a r t r i d g e s were s e q u e n t i a l l y washed wi t h 4 ml 100% methanol ( g l a s s d i s t i l l e d , OmniSolv, BDH Chemicals); 0.5% t r i f l u o r o a c e t i c a c i d (TFA, HPLC/Spectro Grade, P i e r c e Chemical Co.) i n 4 ml 90% 18 methanol; and 8 ml d i s t i l l e d water. One ml of thawed plasma was s l o w l y (over 1 min) passed through the c a r t r i d g e and the plasma-loaded c a r t r i d g e was r i n s e d w i t h 8 ml d i s t i l l e d water. The AVT was e l u t e d w i t h 3.4 ml 90% methanol - 0.5% TFA. The e l u a t e was evaporated under n i t r o g e n and l y o p h i l i z e d t o dryness i n a vacuum evaporator (Speed Vac Concentrator, Savant, N.Y., USA). The d r i e d e x t r a c t e d samples were s e a l e d w i t h P a r a f i l m and s t o r e d a t -20°C f o r RIA the next day. Q u a l i t y c o n t r o l plasma and r e c o v e r y plasma samples were t r e a t e d i n the same way as e x p e r i m e n t a l l y obtained plasma samples. Recovery o f s y n t h e t i c AVT added t o plasma samples averaged 93.1 + 0.8% a f t e r e x t r a c t i o n . R e s u l t s were not c o r r e c t e d f o r re c o v e r y . R a d i o i o d i n a t i o n of AVT. I o d i n a t i o n was performed u s i n g the method d e s c r i b e d by Ledsome e t a l . (1982). Ten u l 0.05 M a c e t i c a c i d , and 20 u l phosphate b u f f e r (0.5 M, pH 7.4) were added t o 10 ug o f l y o p h i l i z e d p e p t i d e (Sigma Chemical, no. V-0130 l o t 95F-5865, c o r r e c t e d f o r % p e p t i d e content) . T h i s was f o l l o w e d by a d d i t i o n o f 10 u l chloramine T (Eastman Kodak 2A2, 1 mg-ml - 1) , and 15 u l o f N a 1 2 5 I (Amersham R a d i o c h e m i c a l s ) . The r e a c t i o n was allowed t o proceed f o r 50 sec. Bovine serum albumin (BSA) s o l u t i o n (100 u l 0.25 m g - u l - 1 i s o t o n i c s a l i n e ; Pentex, M i l e s Labs Inc., l o t 71) was added, f o l l o w e d by 50 mg AGI-X10 (Biorad, no. 9995) i n 200 u l water. The mixture was c e n t r i f u g e d f o r 3 mins, and the supernatant f l u i d was t r a n s f e r r e d t o a CM Sephadex C-25 column a t 4°C. P u r i f i e d i o d i n a t e d m a t e r i a l was 19 s t o r e d a t 4°C. The s p e c i f i c a c t i v i t y of i o d i n a t e d AVT (1446 uCi-ug" 1) was c a l c u l a t e d , u s i n g the s e l f - d i s p l a c e m e n t method of M o r r i s (1976) . Radioimmunoassay (RIA). Antiserum (#GP 7 11-15) was r a i s e d a g a i n s t AVT i n guinea p i g s . The r e l a t i v e c r o s s - r e a c t i v i t i e s of AVP, mesotocin (MT) (the o t h e r n e u r a l lobe p e p t i d e i n b i r d s ) and o x y t o c i n (OXY) were computed a g a i n s t [125I]AVT and AVT a t 50% displacement and amounted t o 61.7%, 1.3% and 29% r e s p e c t i v e l y ( F i g . 3 ). Antiserum was d i l u t e d t o 1:10,000 u s i n g phosphate b u f f e r (0.15 M, pH 7.2) c o n t a i n i n g 0.35% normal r a b b i t serum (NRS) and lOOul of the d i l u t e d antiserum was used i n each tube f o r a f i n a l d i l u t i o n o f 1:100,000 u s i n g b u f f e r c o n t a i n i n g 0.35% NRS. Plasma samples r e q u i r e d b u f f e r c o n t a i n i n g 0.35% NRS and b u f f e r used i n standard tubes c o n t a i n e d 0.1% BSA i n a d d i t i o n t o NRS. RIA was c a r r i e d out i n 12 x 75 mm s i l i c o n i z e d g l a s s tubes. Tubes without AVT (maximum b i n d i n g ) ; tubes without AVT or a n t i s e r u m ( f o r determining n o n s p e c i f i c binding) and tubes c o n t a i n i n g known c o n c e n t r a t i o n s of AVT (standard c u r v e ) , were analyzed c o n c u r r e n t l y and i n t r i p l i c a t e . Plasma samples were anal y s e d i n t r i p l i c a t e f o r s p e c i f i c b i n d i n g and i n d u p l i c a t e f o r n o n - s p e c i f i c b i n d i n g . L y o p h i l i z e d , e x t r a c t e d p l a s m a , r e c o n s t i t u t e d i n b u f f e r , was c o n c e n t r a t e d or d i l u t e d depending on e s t i m a t e d hormone c o n c e n t r a t i o n t o ensure t h a t lOOul of plasma (volume used i n each tube) had an AVT c o n c e n t r a t i o n t h a t f e l l i n the l i n e a r p o r t i o n of the standard curve i e . the area of g r e a t e s t 20 F i g u r e 3. Standard curves of a r g i n i n e v a s o t o c i n (AVT) showing i m m u n o r e a c t i v i t i e s of a r g i n i n e v a s o p r e s s i n (AVP), AVT, o x y t o c i n (OXY) and mesotocin (MT) w i t h GP 7 11-15 antiserum. B/F = B - [ D (B + F) ] F where B D F = bound t r a c e r counts, = n o n - s p e c i f i c b i n d i n g counts = f r e e t r a c e r counts 22 accuracy. For each RIA a s e t of standard tubes c o n t a i n i n g 0.5, 1, 2.5, 5, 10, 25, 50 and 100 pg/lOOul i n b u f f e r was prepared from a s e p a r a t e l y f r o z e n (-20°C) a l i q u o t of the AVT s t o c k standard s o l u t i o n (50 n g ' m l - 1 0.1M a c e t i c a c i d ) . A l l tubes were made up t o 900 u l w i t h the a p p r o p r i a t e b u f f e r , mixed and incubated f o r 48 h a t 4°C. Then 100 u l [1251]-monoiodo-AVT (approx 2,000 cpm) was added t o each tube ( f i n a l i n c u b a t i o n volume 1.0 ml). The tubes were w e l l mixed and incubated f o r a f u r t h e r 96 h a t 4°C. To s e p a r a t e bound from f r e e hormone, 250 u l c h a r c o a l - d e x t r a n mixture (0.167g Dextran T-70; 30 ml phosphate b u f f e r ; 20 ml 1.6% g - g l o b u l i n and 835g ch a r c o a l ) was added t o each tube. The tubes were w e l l mixed and c e n t r i f u g e d a t 500 g f o r 45 mins a t 4°C. The bound hormone (supernatant f l u i d ) was decanted i n t o a c l e a n tube and was counted s e p a r a t e l y from the f r e e hormone ( c h a r c o a l p e l l e t ) u s i n g an Automatic gamma counter, LKB Wallac RiaGamma 1274. Data r e d u c t i o n was performed u s i n g the program, 232-COM ( I n f o r m a t i c a Information Systems, Vancouver). The mid-point of the l i n e a r p o r t i o n of the standard curve (50% binding) was 14.0 + 0.2 p g - t u b e - 1 (n=3) . The mean i n t e r -assay and i n t r a - a s s a y v a r i a t i o n was 12% and 5% r e s p e c t i v e l y . 23 C a l c u l a t i o n s Glomerular f i l t r a t i o n r a t e (GFR), GFR = U r i n e 1 4C-PEG (dpm-ml - 1) x U r i n e Flow (ml-min - 1) Plasma 1 4C-PEG (dpm-ml" 1) E f f e c t i v e Renal Plasma Flow (ERPF), ERPF = U r i n e 1 4C-PAH (dpm-ml - 1) x U r i n e Flow (ml-min" 1) Plasma 3H-PAH (dpm-ml - 1) Osmolar Clearance ( C 0 s m ) , cOsm = U r i n e O s m o l a l i t y (mOsm-kg - 1) X U r i n e Flow (ml-min - 1) Plasma O s m o l a l i t y (mOsm-kg - 1) Free water c l e a r a n c e ( C H 2 o ) / C H 2 o = U r i n e Flow (ml-min - 1) - C 0 s m (ml-min - 1) S t a t i s t i c a l e v a l u a t i o n The data are presented as means wit h standard e r r o r (SE). To t e s 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 s between c o r r e s p o n d i n g time p e r i o d s i n CCS and OCS experiments unpaired t - t e s t s were used. P a i r e d t - t e s t s were used t o d e t e c t the f i r s t s i g n i f i c a n t change a f t e r i n f u s i o n o f LOAD w i t h i n e i t h e r CCS or OCS experiments. RESULTS I. GULLS BEFORE INFUSION Measurements are presented as mean + se o f 8 CCS and 6 OCS g u l l s . Mean Hematocrit (Hct) and Plasma C o n c e n t r a t i o n P r i o r t o i n f u s i o n , the CCS g u l l s had s i g n i f i c a n t l y (P < 0.05) h i g h e r Posm (CCS, 332.2 + 4.3; OCS, 316.2 + 3.6 mOsm-kg - 1) and lower plasma c h l o r i d e ( P e l - ) c o n c e n t r a t i o n (CCS, 111.3 + 1.2; OCS, 115.9 +1.4 mEq-1"1) than OCS g u l l s . N e i t h e r the Hct (CCS, 44.8 + 1.2; OCS, 44.3 + 1.7%, F i g . 4 ), P N a + (CCS, 147.3 + 3.2; OCS, 149.0 + 1.4) nor P K + (CCS, 4.0 + 0.2; OCS, 3.6 + 0.2, F i g . 5) were s i g n i f i c a n t l y d i f f e r e n t between CCS and OCS g u l l s . Mean Plasma AVT C o n c e n t r a t i o n S i n c e P^VT measurements of CCS g u l l s ( F i g . 6) were ob t a i n e d u s i n g u n e x t r a c t e d plasma w h i l e those of OCS g u l l s (Table 1) were obtained u s i n g Sep-Pak e x t r a c t e d plasma, comparison between CCS and OCS g u l l s i s not v a l i d . The i n i t i a l P^VT w a s 5 8 • 7 ± 25.8 and 50.2 + 7.5 p g - m l - 1 i n CCS and OCS g u l l s . I I . INFUSED GULLS Mean Hematocrit and Plasma C o n c e n t r a t i o n A f t e r 0.02 M NaCl i n f u s i o n , CCS g u l l s had h i g h e r Posm ( p < ° « 0 1 ) (CCS, 329.1 + 6.7; OCS, 301.2 + 2.6 mOsm-kg"1) and P K + (P < 0.05) (CCS, 3.5 + 0.1; OCS, 2.8 + 0.3 mEq'l - 1) but lower u r i n e c o n c e n t r a t i o n and h i g h e r u r i n e flow r a t e than OCS g u l l s . 25 F i g u r e 4. Comparison of Hct v a l u e s a t p r e - i n f u s i o n ( f i r s t v a l u e ) , a f t e r h y d r a t i o n (-15 mins), s a l t l o a d (0 mins, arrow) and p o s t - l o a d p e r i o d s i n CCS ( c l o s e d c i r c l e s ) and OCS (open c i r c l e s ) g u l l s . *P < 0.05, **P < 0.01, ***P < 0.001 open * i n d i c a t e s s i g n i f i c a n c e i n comparison between p r e - i n f u s i o n and pre-LOAD v a l u e s ; c l o s e d * i n d i c a t e s s i g n i f i c a n c e i n comparison between pre-LOAD and p o s t -LOAD v a l u e . Values are means + SE (n i s i n d i c a t e d a t each mean po i n t ) 26 ( % ) l l U 0 O l V l r N 3 V H 27 F i g u r e 5. Comparison of plasma e l e c t r o l y t e c o n c e n t r a t i o n s a t p r e -i n f u s i o n ( f i r s t v a l u e ) , a f t e r h y d r a t i o n (-15 mins), s a l t l o a d (0 mins) and p o s t - l o a d p e r i o d i n CCS ( c l o s e d symbols) and OCS (open symbols) g u l l s . S i n g l e symbols r e p r e s e n t i n d i v i d u a l b i r d s . (n i n d i c a t e d a t each mean p o i n t , u n l e s s i d e n t i c a l t o p r e c e d i n g graph) ( P r o t o c o l as i n F i g . 1, * d e t a i l s as i n F i g . 4) P L A S M A CONCENTRATION ( m E q - m l " 1 ) P O T A S S I U M C H L O R I D E S O D I U M w ui r 10 6 0J 6 ro O OJ O O Ul O o 01 o 0) o o oo Table 1. Effect of infusion of acute s a l t load (5 H NaCl, LOAD) on plasma AVT (following Sep-Pak extraction) and plasma osmotic concentrations. Samples were taken before (pre-INF) and a f t e r (pre-LOAD) hydration using 0.02 M NaCl infusion for 4 h at 0.286 ml "min"1; a f t e r infusion of 5 M NaCl (LOAD) for 13 mins and at 10, 25 and 60 mins a f t e r loading. Time of Infusion (mins) Concentrations pre-INF -15 0 JK) 25 60 (pg-ml"') (pre-LOAD) (LOAD) (nOsm.kg - 1 ) PAVT OCS (6) 50.2 t 7.5 (6) 16.1 t 2 . 1b (5) 36.1 t T . M (5) 81.5 1 11.3 (6) 96.0 t 12.6 P03m CCS (8) 332.2 1 1.3 ( U 329.1 t 6 . 7 a (2) 368.6 t 8.1 (2) 388.5 1 1.1 (2) 350.2 ± 8 . 3 (3) 373.5 i 16.2 OCS (6) 316.2 t 3.6* (6) 301.2 t 2 . 6 C " (5) 331.8 t 2 . 7 * " (5) 316.0 1 2.1 (6) 337.8 ± 3.2 (1) 339.0 *P < 0.05; * * *P < 0.001, l - t es t for paired comparison between pre-LOAD value and r i r s t s i g n i f i c a n t change. *P < 0.05; **P < 0.01, t - tes t for comparison between groups. a H < 0.05; 6 P < 0.01; C P < 0.001, t - tes t for paired comparison between pre-INF and pre-LOAD values. ( ) Indicates nunber of b i rds . Means are given t SE. 30 CCS A f t e r 0.02 M NaCl i n f u s i o n , t h e r e were s m a l l but s i g n i f i c a n t (P < 0.05) decreases i n Hct ( F i g . 4), Posm (Table 1), P N a + and P K+ ( F i g . 5). F o l l o w i n g 5 M NaCl i n f u s i o n , t h e r e was a s i g n i f i c a n t decrease i n Hct (P < 0.01, F i g . 4) and P K + (P < 0.05,Fig. 5) and i n c r e a s e (P < 0.001) i n Pfla+ a n d P C 1 - ( F : L9- 5 ) • OCS A f t e r 0.02 M NaCl i n f u s i o n , t h e r e were s i g n i f i c a n t decreases i n Po Sm ( p < 0.001, Table 1), PN s+ ( p < 0.01) and P c i -(P < 0.05, F i g . 5). F o l l o w i n g 5 M NaCl i n f u s i o n , t h e r e was a s i g n i f i c a n t (P < 0.05) decrease i n Hct ( F i g . 4) and i n c r e a s e (P < 0.001) i n pOsm (Table 1), P N a + , P K + and P c l _ ( F i g . 5). Plasma AVT C o n c e n t r a t i o n (PAVT) CCS S i n c e o n l y plasma from 3 b i r d s were analysed, the PAVT l e v e l s i n the i n d i v i d u a l b i r d s were p l o t t e d ( F i g . 6). A f t e r 0.02 M NaCl i n f u s i o n , mean P^VT decreased from 58.7 + 25.8 t o 14.7 + 7.4 p g - m l - 1 . A f t e r the 5 M NaCl i n f u s i o n , PAVT i n c r e a s e d t o 122.5 + 5.5. OCS A f t e r 0.02 M NaCl i n f u s i o n , mean P^VT w a s s i g n i f i c a n t l y decreased (P < 0.01) from 50.2 + 7.5 t o 16.1 + 2.4 p g * m l - 1 (Table 1). A f t e r 5 M NaCl i n f u s i o n , P^VT i n c r e a s e d t o 96.0 + 12.6 (P < 0.05). H y d r a t i o n p e r i o d In a p r e l i m i n a r y experiment i n f u s i o n of i s o t o n i c s a l i n e i n one b i r d f a i l e d t o i n c r e a s e u r i n e flow r a t e . Subsequently, 0.08 M NaCl and 0.02 M NaCl were i n f u s e d i n t o CCS g u l l s a t 2 i n f u s i o n r a t e s so as t o determine the c o n c e n t r a t i o n of 31 F i g u r e 6. Comparison of P A W u s i n 9 Sep-Pak e x t r a c t e d plasma, a t p r e - i n f u s i o n ( f i r s t v a l u e ) , a f t e r h y d r a t i o n (-15 mins), s a l t l o a d (0 mins) and p o s t - l o a d p e r i o d i n t h r e e CCS g u l l s . ( P r o t o c o l as i n F i g . 1) 32 s a l i n e and r a t e o f i n f u s i o n needed t o induce a u r i n e flow r a t e t h a t would match the i n f u s i o n r a t e , thus a t t a i n i n g u r i n e i n p u t -output balance ( F i g . 7). Though GFR d i d not appear t o be a f f e c t e d , u r i n e flow tended t o i n c r e a s e w i t h time of d i l u t e s a l i n e i n f u s i o n , w i t h 0.02 M NaCl showing the h i g h e s t v a l u e s . Glomerular F i l t r a t i o n Rate (GFR). E f f e c t i v e Renal Plasma  Flow (ERPF) GFR and ERPF of CCS and OCS g u l l 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 ( F i g . 8) . Due t o the s i g n i f i c a n t i n d i v i d u a l v a r i a t i o n the GFR and ERPF are expressed as pe r c e n t of the mean v a l u e d u r i n g the 30 min p e r i o d p r i o r t o the s a l t l o a d i n g (pre-LOAD v a l u e ) . CCS The mean pre-LOAD GFR and ERPF were 5.56 + 0.85 and 15.8 + 1.73 m l * ( k g ' m i n ) - 1 r e s p e c t i v e l y . F o l l o w i n g 5 M NaCl i n f u s i o n , t h e r e was a s i g n i f i c a n t (P < 0.05) i n c r e a s e i n ERPF t o 138 + 14% of pre-LOAD v a l u e and decrease i n GFR t o 70 + 10% of pre-LOAD v a l u e ( F i g . 8 ) . OCS A f t e r h y d r a t i o n , the mean GFR and ERPF were 5.36 + 0.77 and 14.35 + 1.81 ml•(kg'min)~ 1 r e s p e c t i v e l y . A f t e r 5 M NaCl i n f u s i o n , t h e r e was a s i g n i f i c a n t decrease i n ERPF t o 63 + 15% of pre-LOAD v a l u e (P < 0.05) and GFR t o 64 + 6% of pre-LOAD (P < 0.01, F i g . 8). U r i n e Flow and C o n c e n t r a t i o n H y d r a t e d CCS g u l l s h a d s i g n i f i c a n t l y lower u r i n e U 0 s m (P < 0.01) and lower u r i n e c h l o r i d e (U c^_) c o n c e n t r a t i o n than OCS g u l l s (Table 2 ) . CCS The mean pre-LOAD u r i n e flow was 0.29 + 0.05 m l ' m i n - 1 . A f t e r i n f u s i o n o f 5 M NaCl, u r i n e flow decreased s i g n i f i c a n t l y 34 F i g u r e 7. E f f e c t o f 0.02 M and 0.08 M NaCl i n f u s i o n on GFR and u r e t e r a l u r i n e flow u s i n g the CCS c o l l e c t i o n i n g u l l s . The c o l l e c t i o n s were begun approximately 220 mins a f t e r i n i t i a t i o n o f h y d r a t i o n , (n i s i n d i c a t e d a t each mean v a l u e ) . 35 I i I i 1 1 1— 2 2 0 3 0 0 3 8 0 4 6 0 TIME ( mins ) 36 F i g u r e 8. Comparison of GFR, ERPF and u r i n e flow, a l l expressed as p e r c e n t o f p r e - l o a d , a f t e r h y d r a t i o n (-15 mins), s a l t l o a d (0 mins) and p o s t - l o a d p e r i o d i n CCS ( c l o s e d symbols) and OCS (open symbols) g u l l s . Open t r i a n g l e s r e p r e s e n t a s i n g l e OCS g u l l measurement. ( P r o t o c o l as i n F i g . 1) * 323 °/c * 597 'Ic T a b l e 2 . E f f e c t o f a c u t e s a l t l o a d (5 M N a C l , LOAD) o n u r i n e i o n i c a n d o s m o t i c c o n c e n t r a t i o n s . T ine o f I n f u s i o n (B ins )  Concent ra t ions -15 2 IP. £ 5 60 \00 i i i (nOM.kg"') (pre-LOAD) (LOAD) (poat-LOAD) ( u E q . n l " ' ) U0SB CCS (5) 100.3 l 17.0 (5) 286.9 i 5 3 . 9 » (5) 502 .5 t 62 .7 (1) 567.5 i 101.3 OCS 16) 221 .3 i 17 .3 * ' ' (6) 339.7 i 38.0" (5) 111 .1 1 19 .2 (6) 505.3 i 73 .0 .Sodium CCS (8) 15.0 t 7 .3 (7) 100.0 1 13.1"" (8) 181 .6 i 20 .0 (7) 166.2 1 19.2 (5) 86.3 4 17.5 (1) 105.0 t 26.0 (2) H I . .7 t 57.5 OCS (6) 36.1 i 6 .0 (5) 137.7 t 2 9 . 1 » (6) 190 . 1 t 17.1 (6) 176.1 t 27 .5 (6) I 3 L 5 1 12.3 (5) 128.9 1 12.3 (1) 132. 8 t 12.5 C h l o r i d e CCS (8) 9.3 1 1.1 (7) 93.1 i 1 5 . 6 " <8) 196 .6 t 22.7 (7) 237.7 t 28.2 (5) 177.7 1 39.7 (1) 171.1 t 22.8 (2) 69. .7 » 19.3 OCS (6) 26.5 t 1 .5* (5) 131.9 t 27 .5 * (6) 191 .3 1 18.6 (6) 21 3.0 1 32.9 (6) 177.2 t 25 . 1 (5) 151.7 1 30.1 (1) 110. 3 t 12.9 Polasaium CCS (8) 2 .5 1 0.1 (7) 2.6 1 0.5 (8) 7 .0 1 2.2 (7) 20.8 t 7.5 (5) 12.1 i 6.0" (1) 21.0 i 7.3 (2) 7. .0 t 1.9 OCS <6> 1.6 t 1 .0 (5) 3.7 t 1.7 (6) 1 .2 t 1.5 (6) 7.1 1 1.8 (6) 9.6 i 2 .0 (5) 13.0 1 1.0 (1) 27. .2 t 10.7 •P <• 0.0^; i f P < 0.01. t-test r or paired comparison between pre-LOAD value and f i rs t significant change. *P < 0.05, t-test for comparison between groups. ( ) Indicates number of birds. Means are given t NE. CO 0 0 39 (P < 0.05) t o 6 + I I of pre-LOAD ( F i g . 8 ) . There were s i g n i f i c a n t (P < 0.01) i n c r e a s e s i n u r i n e sodium ( U N a + ) and c h l o r i d e ( U c i _ ) c o n c e n t r a t i o n (Table 2) and e x c r e t i o n r a t e ( F i g . 9) . The U 0 s m (Table 2) and u r i n e potassium ( U K + ) e x c r e t i o n r a t e ( F i g . 9) were s i g n i f i c a n t l y i n c r e a s e d (P < 0.05). OCS The mean pre-LOAD u r i n e flow was 0.17 + 0.03 m l - m i n - 1 . A f t e r 5 M NaCl i n f u s i o n , u r i n e flow decreased s i g n i f i c a n t l y (P < 0.001) t o 13 + 5% of pre-LOAD v a l u e ( F i g . 8). The c o n c e n t r a t i o n and r a t e o f e x c r e t i o n o f U N a + ( F i g . 9) were s i g n i f i c a n t l y i n c r e a s e d (P < 0.05). U 0 sm and UQI- w e r e i n c r e a s e d s i g n i f i c a n t l y (P < 0.05, Table 2 ) . Urine/Plasma O s m o l a l i t y R a t i o (U/Ppsm) The mean pre-LOAD uOsm o f c c s g u l l s was s i g n i f i c a n t l y (P < 0.01) lower U / P 0 s m than OCS g u l l s . CCS The mean pre-LOAD U / P 0 s m was 0.34 + 0.06. Ten minutes a f t e r s a l t l o a d i n g , U / P 0 s m i n c r e a s e d t o 1.36 + 0.01. OCS The mean pre-LOAD U / P 0 s m was 0.75 + 0.06. Ten minutes a f t e r s a l t l o a d i n g , U/P 0 s i n i n c r e a s e d t o 1.19 + 0.16. Urine/Plasma PEG R a t i o ( U / P P E G ) I n d i v i d u a l b i r d U / P P E G changes are shown i n F i g . 10. CCS A f t e r 5 M NaCl i n f u s i o n , U / P P E G decreased (P < 0.05) from 22.4 + 4.4 but 60 mins a f t e r s a l t l o a d , i n c r e a s e d s i g n i f i c a n t l y (P < 0.05) t o 134.3 + 26.5 (Table 3). OCS A f t e r 5 M NaCl i n f u s i o n , U / P P E G i n c r e a s e d s i g n i f i c a n t l y (P < 0.05) from 39.6 + 8.5 t o 181.2 + 32.4 (Table 3). F i g u r e 9. Comparison of r a t e of r e n a l e x c r e t i o n a f t e r h y d r a t i o n (-15 mins), s a l t l o a d (0 mins) and p o s t - l o a d p e r i o d i n CCS ( c l o s e d c i r c l e s ) and OCS (open c i r c l e s ) g u l l s . ( P r o t o c o l as i n F i g . 1) RATE OF RENAL EXCRETION ( uEqmin" 1 ) 42 F i g u r e 10. Comparison of U / P P E G of hydrated (-15 mins), s a l t loaded (0 mins) and po s t s a l t - l o a d p e r i o d s i n i n d i v i d u a l CCS and OCS g u l l s . ( P r o t o c o l as i n F i g . 1) T a b l e 3 . E f f e c t o f a c u t e s a l t l o a d i n g o n U r i n e / P l a s m a PEG r a t i o , p e r c e n t s o d i u m r e a b s o r p t i o n a n d w a t e r a n d o s m o l a r c l e a r a n c e ( C H 2 o a n d C 0 s m , m l ' m i n " 1 I . Time o f In fus ion (Bins) Parameters -15 ( p r e - L O A D ) 0 ( L O A D ) 10 ( p o s t - L O A D ) 25 60 100 1B-> U / P P E G C C S (7) 22.1 1 1.1 (7) 26.2 1 5 .8 (7) 12.1 1 1 . 1 " (6) 51 .5 1 30.8 (5) 131.3 » 26 .5* (3) 221.1 t 66.8 (2) 62.5 t j i . i O C S (6) 39.6 t 8 .5 (5) 25.8 i 12.9 (6) 17.0 t 10.1 (6) 28 .9 i 9 .3 (6) 79.1 * 25.2 (5) 126.1 1 53.6 (1) 181.2 j 32.1" 1 Na reabsorbed C C S (7) 98 .6 1 1.1 (7) 95.9 i 1 . 3 " (7) 87 .1 t 3.0 (6) 96 . 3 1 1.1 (1) 99.1 t 0.2 (3) 99.5 1 0 .3 (2) 99.0 t 0.1 O C S (6) 99.3 t 0.1 (5) 91.7 t 2 .7 * (6) 73.1 t 6 .8 (6) 89.9 l 1.5 (6) 91 . 3 t 1.1 (5) 91.1 1 1.8 I D 99.6 t 0.1 C H ? 0 < B l . " l n " ' ) C C S (3) 0.113 « 0.011 (2) -0 .016 t 0.006* (2) -0 .077 1 o.ooa (1) - 0 .023 O C S (6) 0.052 t 0.019* (5) -0 .001 l 0.013 (5) -0 .033 i 0.011 (6) -0 .016 i 0.029 C O S B ( o l - » l n " ' ) C C S (3) 0.090 l 0.017 (2) 0.159 t 0.010 (2) 0.293 i 0.028 (1) 0 .011 O C S (6) 0.111 t 0.011 (5) 0.115 1 0.126 (5) 0.598 1 0.198 (6) 0.173 t 0 .032 •P < 0 .0b; f i P < 0 . 0 1 , t - t e s t fo r pa i red comparison between pre-LOAD value and f i r s t s i g n i f i c a n t change. *P < 0 .05 , t - t e s t fo r comparison between groups. ( ) Ind ica tes number of b i r d s . Means are g iven t S E . 45 Percent Na Reabsorbed. Free Water Clearance (C H20^ a n d  Osmolar Clearance (C 0 = I t l) CCS g u l l s had s i g n i f i c a n t l y (P < 0.05) h i g h e r C H 2 0 (CCS, 0.143 + 0.011; OCS, 0.052 + 0.019 ml-min" 1) than OCS g u l l s (Table 3 ) . C C S A f t e r 5 M NaCl i n f u s i o n , % Na reabsorbed decreased s i g n i f i c a n t l y (P < 0.01) from 98.6 + 1.1 t o 95.9 + 1.3 and C H 2 o decreased s i g n i f i c a n t l y (P < 0.05) t o -0.016 + 0.006. C 0 s m was not s i g n i f i c a n t l y i n c r e a s e d (Table 3 ) . OCS A f t e r 5 M NaCl i n f u s i o n , % Na reabsorbed decreased s i g n i f i c a n t l y (P < 0.05) from 99.3 + 0.1 t o 91.7 + 2.7. The i n c r e a s e i n C 0 s i n and decrease i n C H 2 Q was not s i g n i f i c a n t . U r i c a c i d e x c r e t i o n There were 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 u r i c a c i d e x c r e t i o n between CCS and OCS g u l l s . A f t e r 5 M NaCl i n f u s i o n , u r i c a c i d c o n c e n t r a t i o n and e x c r e t i o n o f Na + a s s o c i a t e d w i t h u r i c a c i d was not s t a t i s t i c a l l y d i f f e r e n t . C C S The mean pre-LOAD u r i c a c i d c o n c e n t r a t i o n was 0.94 + 0.53 mg'ml - 1. The r a t e o f e x c r e t i o n o f Na + a s s o c i a t e d w i t h u r i c a c i d was 0.23 + 0.13 uEq-min" 1. A f t e r 5 M NaCl i n f u s i o n , u r i c a c i d c o n c e n t r a t i o n was 11.97 + 6.97 nig * m l - 1 . The r a t e o f e x c r e t i o n o f N a + a s s o c i a t e d w i t h u r i c a c i d was 1.70 + 0.25 uEq-min - 1. OCS The mean pre-LOAD u r i c a c i d c o n c e n t r a t i o n was 1.09 + 1.01 mg*ml - 1. The r a t e o f e x c r e t i o n o f Na + a s s o c i a t e d w i t h u r i c a c i d was 0.35 + 0.15 uEq-min - 1. 46 A f t e r 5 M NaCl i n f u s i o n , u r i c a c i d c o n c e n t r a t i o n was 0. 50 + 0.20 nig*ml - 1. The r a t e o f e x c r e t i o n o f N a + a s s o c i a t e d w i t h u r i c a c i d was 0.10 + 0.08 uEq*min _ 1. S a l t Gland S e c r e t i o n Both the time t o i n i t i a t i o n o f s a l t g l a n d s e c r e t i o n and the volume of s e c r e t i o n formed by CCS and OCS g u l l s i n response t o 1. v. i n f u s i o n o f 5 M NaCl were the same. CCS. The time t o i n i t i a t i o n o f s e c r e t i o n was 9.0 + 1.0 mins and the volume of s e c r e t i o n was 7.3 + 1.3 ml. At 25 mins, 92.8% of t o t a l s a l t g l a n d s e c r e t i o n had been formed, c o n t a i n i n g 23.8% of the a d m i n i s t e r e d sodium. OCS. The time t o i n i t i a t i o n o f s e c r e t i o n was 9.0 + 1.5 mins and the volume of s e c r e t i o n was 5.8 + 1.8 ml. At 25 mins, 78.1% o f t o t a l s a l t g l a n d s e c r e t i o n , c o n t a i n i n g 21.2% of the ad m i n i s t e r e d sodium. I I I . BLOOD PRESSURE EXPERIMENTS Mean A r t e r i a l P r e ssure. The mean pre-LOAD a r t e r i a l p r e s s u r e was 146.7 + 6.0 mmHg. In 3 experiments performed on the 2 b i r d s , immediately f o l l o w i n g 5 M NaCl i n f u s i o n , the mean a r t e r i a l p r e s s u r e r o s e s l i g h t l y (3%) and decreased by 10% about 40-50 mins a f t e r s a l t l o a d ( F i g . 11). Mean Hct and Plasma C o n c e n t r a t i o n s The mean pre-LOAD Hct 30.6 + 4% was l e s s than the p r e - i n f u s i o n Hct 34.7 + 4%. Pfla+ a n d P c i - decreased i n both b i r d s ( F i g 12) . Posm remained unchanged (308 + 0.04 t o 308 + 7.0 mOsm-kg"1). A f t e r 5 M NaCl i n f u s i o n , Hct 47 F i g u r e 11. Comparison of mean a r t e r i a l p r e s s u r e o f hydrated (-10 mins), s a l t loaded (0 mins) and p o s t s a l t - l o a d p e r i o d s i n 2 g u l l s . Open symbols i n d i c a t e one experiment with one b i r d and c l o s e d symbols i n d i c a t e t h r e e experiments w i t h a second b i r d . ( I n f u s i o n p r o t o c o l as i n F i g . 1) M E A N A R T E R I A L P R E S S U R E ( m m H g ) 8fr 49 F i g u r e 12. Comparison of plasma e l e c t r o l y t e c o n c e n t r a t i o n of non-i n f u s e d ( f i r s t v a l u e ) , hydrated (-10 mins), s a l t loaded (0 mins) and post s a l t - l o a d p e r i o d s i n 2 OCS g u l l s . Open symbols i n d i c a t e one experiment w i t h one b i r d and c l o s e d symbols i n d i c a t e t h r e e experiments w i t h a second b i r d . ( I n f u s i o n p r o t o c o l as i n F i g . 1) PLASMA CONCENTRATION ( mEg-ml"' ) P O T A S S ' U M C H L O - D E S O O . ? W - - _ -O A =• W W * Ul K , n ^ was 28.9 + 5%. There was an i n c r e a s e i n Posm t o 3 5 1 ± °*° mosm'kg - 1. I n f u s i o n of 5 M NaCl caused an i n c r e a s e i n Pfla+ a r*d P C 1 - c o n c e n t r a t i o n s i n a l l the experiments ( F i g 12). Plasma a r g i n i n e v a s o t o c i n . (PAyip) The p r e - i n f u s i o n P^VT o f Sep-Pak e x t r a c t e d plasma was 13.7 + 1.7 p g ^ m l - 1 . The pre-LOAD PAVT w a s 1 1 - 1 ± 2.9 p g * m l - 1 . A f t e r i n f u s i o n of 5 M NaCl, PAVT l e v e l s was 80.3 + 16.7 p g * m l _ 1 . S a l t Gland S e c r e t i o n The time t o i n i t i a t i o n o f s a l t g l a n d s e c r e t i o n i n response t o i n f u s i o n of 5 M NaCl was 6.5 + 2.1 mins. Volume of s e c r e t i o n averaged 10.40 + 3.45 ml. At 50 mins p o s t - l o a d , 41.1% and 15.9% of the s a l t - l o a d was e x c r e t e d by the s a l t glands and the kidneys r e s p e c t i v e l y . 52 DISCUSSION The p r e s e n t study i s the f i r s t t o d e s c r i b e r e n a l f u n c t i o n i n a marine b i r d . In these experiments, g u l l GFR and ERPF were measured d u r i n g s t a t e s of h y d r a t i o n and s a l t l o a d i n g . Previous s t u d i e s o f the e f f e c t of s a l t l o a d i n g on r e n a l f u n c t i o n i n b i r d s have been c a r r i e d out on t e r r e s t r i a l b i r d s such as the c h i c k e n ( D a n t z l e r , 1966; B a i l e y and Nishimura, 1984), d e s e r t q u a i l (Braun and D a n t z l e r , 1972) and s t a r l i n g (Braun, 1978), which l a c k s a l t glands, or the domestic duck, (Simon-Oppermann and Simon, 1982), which has r a t h e r i n e f f e c t u a l s a l t g lands. Domestic ducks are unable t o s u s t a i n body mass or plasma c o n c e n t r a t i o n when g i v e n d i l u t e seawater t o d r i n k (Roberts and Hughes 1984), s i n c e t h e i r s e c r e t i o n i s o n l y as c o n c e n t r a t e d as seawater. The s a l t glands of d u c k l i n g s r e q u i r e s a l i n e a c c l i m a t i o n t o become f u n c t i o n a l and ducks are not thought t o use e x t r a r e n a l s a l t s e c r e t i o n i n the absence o f s a l i n e s t r e s s (Simon 1982), whereas g u l l s hatch w i t h f u l l y f u n c t i o n a l glands (Hughes, unpublished observation) and u t i l i z e e x t r a r e n a l s e c r e t i o n even when g i v e n freshwater t o d r i n k (Hughes, 1970a). Although g u l l s a l t glands s e c r e t e NaCl v e r y e f f i c i e n t l y , a l l o t h e r m a t e r i a l s must l e a v e the body through the k i d n e y s . The p r e s e n t i n v e s t i g a t i o n s were undertaken t o determine the GFR and ERPF o f g u l l s and the f u n c t i o n a l r e l a t i o n s h i p between the two e x c r e t o r y pathways, the kidneys and the s a l t g lands. URINE COLLECTION U r i n e c o l l e c t i o n i n b i r d s p r e s e n t s problems not encountered i n mammals. B i r d s l a c k b l a d d e r s and u r i n e and f e a c e s are r e t a i n e d 53 i n a common chamber, the c l o a c a . The c l o a c a l c o n t e n t s may move backwards i n t o the i n t e s t i n e s where p o s t - r e n a l m o d i f i c a t i o n of u r i n e can occur (Skadhauge 1981). To study the e f f e c t o f s a l t l o a d i n g on k i d n e y f u n c t i o n , i t i s necessary t o c o l l e c t uncontaminated u r e t e r a l u r i n e . In p r e l i m i n a r y experiments, u r i n e was c o l l e c t e d by i n s e r t i n g cannulae i n t o u r e t e r s ( D a n t z l e r , 1966; Braun, 1978). T h i s method was unusable because the c a t h e t e r s f r e q u e n t l y became b l o c k e d by u r i c a c i d d e p o s i t s and flow r a t e s were u n r e l i a b l e . T h i s c o l l e c t i o n technique was m o d i f i e d by p r e p a r i n g c a t h e t e r s t h a t were g l u e d over the u r e t e r a l o r i f i c e s ( L i d a h l and Sperber, 1956; Campbell, 1960). T h i s i s c a l l e d the c l o s e d c o l l e c t i o n system (CCS). T h i s procedure d i d not t o t a l l y e r a d i c a t e the problems, but, because the c a t h e t e r s were not i n s e r t e d i n the u r e t e r s , u r e t e r a l p e r i s t a l s i s was not d i s r u p t e d (Wideman and Braun, 1982). T h i s method o f c a t h e t e r i z a t i o n adds t o the normal u r e t e r a l back-p r e s s u r e , w h i c h may a l l o w i n c r e a s e d t u b u l a r i s o s m o t i c r e a b s o r p t i o n and decrease u r i n e flow r a t e ( K i i l and Auckland, 1961; Hughes, i n press) and i n c r e a s e u r i n e osmotic c o n c e n t r a t i o n (Hughes, i n press) . L a t e r a d e v i c e was made t o be i n s e r t e d i n t o the c l o a c a t o c o l l e c t u r i n e as i t was e x p e l l e d from the u r e t e r (open c o l l e c t i o n system, OCS). T h i s d e v i c e was s i m i l a r t o t h a t used by Simon-Oppermann and Simon (1982) t o o b t a i n duck u r i n e . They used a p e r f o r a t e d bulb i n s e r t e d i n t o the c l o a c a and withdrew u r i n e by continuous s u c t i o n . With the c o l l e c t o r used f o r g u l l s , u r i n e never c o n t a c t e d the e p i t h e l i a l l i n i n g o f the c l o a c a (as d i d 54 the duck's u r i n e ) , so t h a t no m o d i f i c a t i o n of u r i n e volume or c o n c e n t r a t i o n would have been p o s s i b l e . The g u l l s s t u d i e d by the CCS method were g i v e n a g e n e r a l a n e s t h e t i c f o r the a p p l i c a t i o n of the u r e t e r a l f u n n e l s , w h i l e the OCS g u l l s r e c e i v e d o n l y a l o c a l i n j e c t i o n o f a n e s t h e t i c around the c l o a c a l s p h i n c t e r . Although CCS g u l l s r e g a i n e d consciousness an hour b e f o r e i n f u s i o n of 5 M NaCl, b i r d s sometimes appeared drowsy, and the a n e s t h e t i c may have a f f e c t e d N a + t r a n s p o r t and water p e r m e a b i l i t y , as s p e c u l a t e d by Skadhauge (1967,1968) i n the lower i n t e s t i n e s . The p h y s i c a l p o s i t i o n of the b i r d s i n the two procedures was a l s o d i f f e r e n t . The CCS b i r d s were supine, w h i l e OCS b i r d s were s t a n d i n g d u r i n g the experiments. In p r e v i o u s p u b l i s h e d accounts o f s a l t l o a d i n g s t u d i e s (except those of Simon-Oppermann and Simon (1982) u s i n g ducks), b i r d s were s t u d i e d i n a supine p o s i t i o n as i n the CCS g u l l s . The r e t u r n of venous blood, p a r t i c u l a r l y through the r e n a l p o r t a l system, may have been d i f f e r e n t i n the two c o l l e c t i o n methods (Sperber 1948). T h i s was not measured. The i n f u s i o n r a t e of CCS g u l l s was con s t a n t throughout the experiment, w h i l e t h a t of OCS g u l l s was matched t o the u r i n e flow r a t e a f t e r s a l t l o a d i n g . S i n c e u r i n e flow r a t e decreased s i g n i f i c a n t l y 30 mins a f t e r i n f u s i o n of 5 M NaCl, the continued i n f u s i o n o f 0.02 M NaCl a t 0.286 ml* m i n - 1 , r e s u l t e d i n an i n c r e a s e i n e x t r a c e l l u l a r f l u i d (ECF) volume t h a t may have a f f e c t e d r e n a l f u n c t i o n i n CCS g u l l s . In the subsequent 55 experiments on OCS g u l l s , i n f u s i o n and e x c r e t i o n r a t e s d u r i n g the post-LOAD p e r i o d were matched. Thus, the r e n a l responses i n OCS g u l l s should be a t t r i b u t a b l e p r i m a r i l y t o the s a l t l o a d . Hydrated G u l l s Plasma c o n c e n t r a t i o n The Hct v a l u e s of noninfused glaucous-winged g u l l s , 44 + 2%, were comparable t o those of t h i s s p e c i e s i n p r e v i o u s s t u d i e s (45.6 + 1.0, Hughes 1977; 42.6 + 1.4, Roberts and Hughes, 1984) and k e l p g u l l s (41 + 1%, Gray and Erasmus, unpublished data) i n d i c a t i n g the b i r d s were i n a normal s t a t e of h y d r a t i o n a t the commencement o f the experiments. The Posm v a l u e s o b t a i n e d , 316.2 + 3.6 mOsnrkg - 1, were a l s o comparable t o those o f glaucous-winged g u l l s , 327.0 + 3.0 (Roberts and Hughes, 1984) and k e l p g u l l s , 315.5 + 1.0 mOsnrkg - 1 (Gray and Erasmus, u n p u b l i s h e d ) . The CCS g u l l s had h i g h e r Posm (332.2 + 4.3 mOsnrkg - 1) compared t o OCS g u l l s p r i o r t o the i n i t i a t i o n of 0.02 M NaCl i n f u s i o n , s u g g e s t i n g t h a t the CCS g u l l s had g r e a t e r e v a p o r a t i v e water l o s s d u r i n g the l o n g e r time r e q u i r e d t o a t t a c h the cannulae and f o r the b i r d t o rouse from the e f f e c t s of the a n e s t h e t i c (one t o two hours) than o c c u r r e d d u r i n g the few minutes needed f o r the i n s e r t i o n o f the c o l l e c t i n g d e v i c e i n OCS g u l l s . H y d r a t i o n In a p r e l i m i n a r y experiment (one g u l l ) t o determine the i n f u s a t e NaCl c o n c e n t r a t i o n t h a t would induce a u r i n e flow r a t e equal t o i n f u s i o n r a t e , i s o t o n i c s a l i n e was i n f u s e d f o r 3 h a t 0.192 m l - m i n - 1 . T h i s d i d not i n c r e a s e the u r i n e flow r a t e . Simon-56 Oppermann and Simon (1982) were a b l e t o induce d i u r e s i s i n Pekin ducks by c o n t i n u o u s l y i n f u s i n g a t 1-2 ml•min" 1 o f e i t h e r hyposmotic (200 mOsnrkg" 1) g l u c o s e s o l u t i o n or i s o s m o t i c NaCl s o l u t i o n f o r 3-4 h. G u l l s r e q u i r e much more d i l u t e s a l i n e than ducks t o induce t u r n o v e r . Subsequent experiments u s i n g 0.08 M NaCl and 0.02 M NaCl ( F i g . 7) a t v a r y i n g r a t e s , showed t h a t i t was necessary t o i n f u s e 0.02 M NaCl (0.286 ml-min - 1) f o r 4 h t o achieve a balance between i n f u s i o n and r e n a l output. S t u d i e s u s i n g s t a r l i n g s (Braun, 1978), chickens ( D a n t z l e r , 1966) and q u a i l s (Braun and D a n t z l e r , 1975) employed 2.5% mannitol i n f u s i o n a t 0.4 ml * ( k g * m i n ) - 1 f o r 1 h b e f o r e c l e a r a n c e and s a l t l o a d i n g s t u d i e s were performed. In these s t u d i e s , i n f u s i o n and u r i n e flow r a t e s d i d not match. A f t e r i n f u s i o n of 0.02 M NaCl f o r 4 h, r e t e n t i o n of f l u i d i n the body i s shown by r e d u c t i o n o f Hct, plasma e l e c t r o l y t e and osmotic c o n c e n t r a t i o n s and P^VT ( F ; l9* 4 ' 5 and 6, T a b l e 1) . The h i g h e r i n i t i a l Posm observed i n CCS g u l l s p e r s i s t e d even a f t e r i n f u s i o n o f 0.02 M NaCl. The accompanying h i g h e r P K + observed i n CCS b i r d s i s s i m i l a r t o t h a t observed by B a i l e y and Nishimura (1984) i n domestic fowls a f t e r time c o n t r o l (no i n f u s i o n ) . The K + probably o r i g i n a t e d from the i n t r a c e l l u l a r p o o l but the cause of i t s r e l e a s e i s not known. The decrease i n P^VT a f t e r i n f u s i o n o f 0.02 M NaCl, was observed i n Pekin ducks responding t o a water-load (Simon-Oppermann, Gray, Szczepanska-Sadowska and Simon, 1984). These o b s e r v a t i o n s t o g e t h e r w i t h those o f S t a l l o n e (1984), S t a l l o n e and Braun (1985) and Koike ( i n press) , suggest t h a t ECF volume and 57 osmotic c o n c e n t r a t i o n i n t e r a c t t o r e g u l a t e AVT r e l e a s e , as i s the case w i t h AVP (Robertson, 1977). I n f u s i o n w i t h 0.02 M NaCl reduced Posm a n d pNa+ t o t n e s a m e e x t e n t as d i d Na-free 2.5% mannitol i n f u s i o n i n chickens ( D a n t z l e r , 1966) and s t a r l i n g s (Braun, 1978). Renal f u n c t i o n of the two groups of hydrated g u l l 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 except f o r h i g h e r Cjj20 i n c c s g u l l s , which may be a t t r i b u t e d t o the l o n g e r time u r i n e was exposed t o kidney t u b u l e s or, p o s s i b l y , t o the a n e s t h e t i c . Skadhauge (1967, 1968) found a lower r a t e of water a b s o r p t i o n i n the lower i n t e s t i n e s of c h i c k e n s compared t o a study by Anderson and Braun (1985) and proposed t h a t a n e s t h e s i a might a l t e r Na + t r a n s p o r t and water p e r m e a b i l i t y i n the lower i n t e s t i n e s . GFR (CCS, 5.56 + 0.85; OCS, 5.36 + 0.77 ml• ( k g ' m i n ) _ 1 ) v a l u e s were comparable t o those i n s t a r l i n g s i n f u s e d w i t h 2.5% mannitol i n f u s i o n or i s o t o n i c s a l i n e , 7.4 + 0.28, ( C l a r k , Braun and Wideman, 1976) but they were h i g h e r than those i n 2.5% m a n n i t o l - i n f u s e d c h i c k e n s , 1.23 + 0.04, 2.85 + 0.13 and 1.023 + 0.085, (Da n t z l e r , 1966; S t a l l o n e and Braun, 1983; B a i l e y and Nishimura, 1984), s t a r l i n g s , 2.822 + 0.080, (Braun, 1978) and water-loaded q u a i l s , 1.390 + 0.221, (Braun and D a n t z l e r , 1975). T h i s may simply r e f l e c t the g r e a t e r i n f u s i o n volume and d u r a t i o n used i n the p r e s e n t study compared t o o t h e r s t u d i e s (Roberts, Baudinette and Wheldrake, 1985). U r i n e flow of g u l l s was h i g h e r than i n m a n n i t o l - i n f u s e d 58 ch i c k e n s , 0.25 + 0.007, 0.162 + 0.013, (D a n t z l e r , 1966; B a i l e y and Nishimura, 1984) and s t a r l i n g s , 0.181 + 0.009, (Braun, 1978). U r i n e c o n c e n t r a t i o n s were comparable t o those i n the l i q u i d p o r t i o n o f the u r e t e r a l u r i n e o f m a n n i t o l - i n f u s e d s t a r l i n g s (Braun, 1978), although lower than i n q u a i l s (Braun and D a n t z l e r , 1972) . However, due t o the h i g h e r i n i t i a l u r i n e flow r a t e i n these g u l l s than s t a r l i n g s (Braun, 1978), the r a t e o f e x c r e t i o n o f N a + was h i g h e r i n the g u l l s . The d i u r e t i c s t a t e o f the g u l l s i n the p r e s e n t study i s r e f l e c t e d by lower U / P 0 s m compared t o s t a r l i n g s (Braun, 1978) e x c r e t i n g hyperosmotic u r i n e , and q u a i l s (Braun and D a n t z l e r , 1972) e x c r e t i n g i s o s m o t i c u r i n e . The U / P 0 s m of g u l l s was comparable t o t h a t o f chickens ( D a n t z l e r , 1966). pOsm w a s maintained b e t t e r i n CCS g u l l s than i n OCS g u l l s . T h i s was due t o the g r e a t e r u r i n a r y water l o s s o f CCS g u l l s , which produced u r i n e w i t h o n e - h a l f the o s m o l a l i t y o f OCS g u l l s . The OCS g u l l s continued t o produce u r i n e w i t h c o n s i d e r a b l e osmotic a c t i v i t y (224 + 17 mOsnrkg - 1; mean U / P 0 s m r a t i o was about 0.74) d e s p i t e the prolonged i n f u s i o n o f 0.02 M NaCl. The g u l l U / P 0 s m was s i m i l a r t o t h a t o f m a n n i t o l - i n f u s e d c h i c k e n s , 0.78 (D a n t z l e r 1966). The CCS g u l l s had lower u r i n e o s m o l a l i t y than the g u l l s , which had u n r e s t r i c t e d u r i n e flow (OCS g u l l s ) , s u g g e s t i n g t h a t more NaCl was reabsorbed d u r i n g the slower passage through the r e n a l t u b u l e s o f CCS g u l l s . However, i n both groups o f b i r d s , t h e r e was almost complete r e a b s o r p t i o n o f NaCl d u r i n g t h i s p e r i o d (Table 3), so the d i f f e r e n c e i n u r i n e o s m o l a l i t y between the two groups was not due t o i t s NaCl 59 content. The p e r c e n t o f Na+ reabsorbed was s i m i l a r t o water-loaded d e s e r t q u a i l (Braun and D a n t z l e r , 1975) and m a n n i t o l -i n f u s e d s t a r l i n g s (Braun, 1978). The U/Pp eg r a t i o s o f g u l l s i n f u s e d w i t h 0.02M NaCl are h i g h (CCS, 22.4+4.4; OCS, 39.6+8.5) d e s p i t e the d i l u t e i n f u s i o n . A s i m i l a r U/P occurs i n mannitol i n f u s e d s t a r l i n g s (Braun 1978), but i s 4-5 times h i g h e r than t h a t o f c h i c k e n s . The h i g h U/Pp eg r a t i o i n d i c a t e s h i g h water r e a b s o r p t i o n d u r i n g the p e r i o d o f water l o a d i n g . The u r i c a c i d c o n c e n t r a t i o n (9.8 + 0.46 mg*ml _ 1) i n u r e t e r a l u r i n e o f m a n n i t o l - i n f u s e d s t a r l i n g s was much h i g h e r than t h a t observed i n g u l l u r i n e (CCS, 0.94 + 0.53; OCS, 1.09 + 1.01), but was a s s o c i a t e d w i t h a much lower u r i n e flow r a t e (0.015 ml-min - 1) (Braun 1978). EFFECT OF 5 M NaCl LOADING During the 13 mins of s a l t l o a d i n g , 18.6 mEq of NaCl was g i v e n t o the g u l l . In oth e r s t u d i e s loads were g r e a t e r , but ad m i n i s t e r e d s l o w l y (1.0 M NaCl a t 0.4-0.5 mEq-min - 1) amounting t o 50 mEq*kg - 1 i n chickens ( D a n t z l e r , 1966), 44 mEq*kg - 1 i n q u a i l s (Braun and D a n t z l e r , 1972) and 32 mEq'kg - 1 i n s t a r l i n g s (Braun, 1978) . The NaCl l o a d used i n the p r e s e n t study on g u l l s i s e q u i v a l e n t t o the s a l t l o a d accumulated a f t e r approximately 45 mins i n the oth e r s t u d i e s . The s a l t l o a d g i v e n t o the g u l l s has been g i v e n as a bol u s i n j e c t i o n t o e l i c i t s a l t gland s e c r e t i o n as i n p e l i c a n s (Schmidt-Nielsen and Fange, 1958) and g r e a t e r b o l u s l o a d s (35 mEq*Kg"1) have been a d m i n i s t e r e d o r a l l y 60 t o e l i c i t s e c r e t i o n i n ducks ( P h i l l i p s , Holmes and B u t l e r , 1961). The o b j e c t i v e of the p r e s e n t study was t o study the r e n a l f u n c t i o n d u r i n g s a l t g l a n d a c t i v i t y i n response t o a known NaCl l o a d . S i n c e g u l l s were w e l l hydrated, a s t r o n g l y h y p e r t o n i c NaCl s o l u t i o n was r e q u i r e d t o i n i t i a t e a response. In a p r e l i m i n a r y experiment, the s a l t glands of a s i m i l a r l y hydrated g u l l d i d not s e c r e t e when the b i r d was i n f u s e d w i t h 1 M NaCl s o l u t i o n f o r 13 mins. The i n f u s i o n of 5 M NaCl, caused marked r i s e s i n Pfla+ a n c * P C1_ ( F i g . 5) and Posm (Table 1) i n OCS g u l l s . The 15% i n c r e a s e i n Pfla+ and 10% i n c r e a s e i n Posm w e r e lower than those i n the c h i c k e n (18%, 20%), q u a i l (30%, 12%) and s t a r l i n g (26%, 16%). The i n c r e a s e i n Posm w a s n ° t s i g n i f i c a n t i n CCS g u l l s . T h i s may be r e l a t e d t o the i n i t i a l l y h i g h Posm v a l u e s p r i o r t o s a l t l o a d i n g . The s a l t - l o a d was accompanied by a drop i n Hct of about 17% i n both groups, i n d i c a t i n g a s h i f t o f f l u i d i n t o the v a s c u l a r space. Marked i n c r e a s e s i n PAVT l e v e l s ( F i g . 6, Table 1) p a r a l l e l e d i n c r e a s e s i n P^a+ and Posm* Renal f u n c t i o n The i n f u s i o n o f 5 M NaCl caused an i n i t i a l i n c r e a s e i n GFR ( F i g . 8) and u r i n e flow, due t o the expansion o f the i n t r a v a s c u l a r volume brought about by a s h i f t o f c e l l u l a r f l u i d i n t o the e x t r a c e l l u l a r space ( i n d i c a t e d by a sharp drop i n Hct, F i g . 4) i n response t o the h i g h l y hyperosmotic i n f u s a t e . A f t e r 25 mins, GFR decreased t o 70% and 43% and u r i n e flow r a t e decreased t o 6% and 13% of pre-LOAD l e v e l s i n CCS and OCS g u l l s . A decrease i n GFR and u r i n e flow r a t e i n response t o l e s s c o n c e n t r a t e d NaCl loads had been demonstrated i n the d e s e r t q u a i l (Braun and D a n t z l e r , 1972) and domestic fowl ( D a n t z l e r , 1966). In c o n t r a s t , the s t a r l i n g showed no changes i n GFR, but g r e a t l y i n c r e a s e d u r i n e flow (Braun, 1978), when i n f u s e d w i t h 5.8% (1 M) NaCl. GFR r e t u r n e d t o normal v a l u e s i n CCS g u l l s a f t e r 80 mins. T h i s i s p r o b a b l y due t o expansion of ECF volume caused by the h i g h p o s t - l o a d i n f u s i o n r a t e . The decrease i n GFR i n OCS g u l l s , i n which the p o s t s a l t l o a d i n f u s i o n r a t e matched the u r i n e flow r a t e , l a s t e d 185 mins. The decrease i n GFR i n OCS i s probably due t o glomerular i n t e r m i t t e n c y , which i s the c e s s a t i o n of f i l t e r i n g by some r e p t i l i a n - t y p e nephrons induced by s p e c i f i c v a s o c o n s t r i c t o r a c t i o n s of AVT on the r e n a l v a s c u l a t u r e , probably a t the l e v e l of the a f f e r e n t a r t e r i o l e s . Glomerular i n t e r m i t t e n c y has been d e s c r i b e d i n c h i c k e n ( D a n t z l e r , 1966) and q u a i l GFR (Braun and D a n t z l e r , 1972,1974; Braun, 1976) under s i m i l a r experimental c o n d i t i o n s . D a n t z l e r (1966) confirmed changes i n i n t e r m i t t e n c y by a c o r r e s p o n d i n g decrease i n ERPF, which was a l s o observed i n the p r e s e n t study. In Gambel's q u a i l , the AVT-induced r e d u c t i o n i n GFR was q u a n t i t a t i v e l y accounted f o r by r e d u c t i o n i n the number of f i l t e r i n g r e p t i l i a n type nephrons, which l e d Braun and D a n t z l e r (1974) t o suggest t h a t AVT-induced r e d u c t i o n i n GFR, which would reduce volume flow through the medullary c o l l e c t i n g d u c t s, might be more important than i n c r e a s e d t u b u l a r water 62 p e r m e a b i l i t y i n enhancing the c o n c e n t r a t i n g a b i l i t y o f the kidney. However, Wideman, S a t n i c k , M i t s o s , Bennet and Smith (1987) found t h a t acute o r c h r o n i c r e d u c t i o n i n GFR i n response t o u n i l a t e r a l sodium i n f u s i o n through the r e n a l p o r t a l system i n chi c k e n s was not due t o r e d u c t i o n i n the number o f f i l t e r i n g nephrons, although they c o u l d not r u l e out p u l s a t i l e changes i n GFR. Reduction i n GFR has been mimicked by a d m i n i s t r a t i o n o f exogenous AVT i n q u a i l s (Braun and D a n t z l e r , 1974), chickens (Skadhauge, 1964; D a n t z l e r , 1966; Ames e t a l . , 1971; S t a l l o n e and Braun, 1985) and ducks (Kaul, Gerstberger, Gray and Simon, 1983). In these s t u d i e s , the decrease i n GFR f o l l o w i n g AVT i n f u s i o n was h i g h l y v a r i a b l e , ranging from 0-90%. T h i s range may r e f l e c t p h y s i o l o g i c a l d i f f e r e n c e s among the s p e c i e s , but may a l s o be due t o d i f f e r e n c e s i n experimental technique. These i n c l u d e the use of a n e s t h e t i c s , d i f f e r e n t AVT doses and d i f f e r e n t modes of AVT a d m i n i s t r a t i o n (bolus i n j e c t i o n o r constant i n f u s i o n ) . Mohring e t a l . (1980) and S t a l l o n e and Braun (1985) used co n s t a n t i n f u s i o n of AVT t o show an ac c u r a t e dose-response r e l a t i o n s h i p between PAVT and GFR i n conscious Pekin ducks and fowls a t steady s t a t e p h y s i o l o g i c a l l e v e l s . In g u l l s , P^VT i n c r e a s e d d r a m a t i c a l l y on s a l t l o a d i n g (Table 1, F i g . 6) and may be the c a u s a t i v e agent f o r GFR decrease, but t h i s was not confirmed by the a d m i n i s t r a t i o n o f exogenous AVT. The c o m p a r i s o n o f g l o m e r u l a r t o t u b u l a r mechanisms c o n t r i b u t i n g t o a n t i d i u r e s i s can be e s t a b l i s h e d by AVT dose-63 dependency s t u d i e s . Braun and D a n t z l e r (1974) suggested t h a t AVT induced r e d u c t i o n o f GFR was the more important determinant of u r i n e flow. Dose-dependency s t u d i e s i n ducks (Mohring e t a l . , 1980) and ch i c k e n s ( S t a l l o n e and Braun, 1985), i n d i c a t e t h a t a t p h y s i o l o g i c a l P A V T l e v e l s , t u b u l a r mechanisms a re o f primary importance and glomerular mechanisms o f secondary importance i n water c o n s e r v a t i o n . R e n a l t u b u l a r r e s p o n s e s , q u a l i t a t i v e l y s i m i l a r t o those seen i n g u l l s , have been r e p o r t e d i n s t u d i e s o f oth e r AVT i n f u s e d b i r d s p e c i e s . S i n c e the p r e s e n t study d i d not i n c l u d e dose-dependency s t u d i e s , i t remains t o be determined whether t u b u l a r o r g l o m e r u l a r mechanisms are of g r e a t e r importance i n r e n a l f u n c t i o n o f g u l l s . However, the observed time l a g between the immediate r e d u c t i o n i n C H 2 o a n d the delayed r e d u c t i o n i n GFR i n t h i s study suggests the primary importance o f t u b u l a r mechanisms. Mean a r t e r i a l p r e s s u r e The mean a r t e r i a l p r e s s u r e measured i n g u l l s i s i n the 150-180 mmHg range r e p o r t e d i n ducks (Ringer, Weiss and S t u r k i e , 1955; Szczepanska-Sadowska, Simon-Oppermann, Gray and Simon, 1985) and h i g h e r than the approximately 100 mmHg v a l u e s r e p o r t e d f o r c h i c k e n s (Ames e t a l . , 1971; B a i l e y and Nishimura, 1984) and d e s e r t q u a i l (Braun and Da n t z l e r , 1974). In t he bl o o d p r e s s u r e experiments, the i n f u s i o n o f s a l t appeared t o be a s s o c i a t e d w i t h a gradual d e c l i n e i n mean a r t e r i a l p r e s s u r e (10%, F i g . 11). Szczepanska-Sadowska e t a l . (1985) found t h a t s a l i n e a c c l i m a t i o n r a i s e d Posm a n d PAVT l e v e l s but lowered 64 a r t e r i a l b l o o d p r e s s u r e , s u g g e s t i n g t h a t a c e n t r a l a c t i o n o f AVT may c o n t r i b u t e t o the lower b l o o d p r e s s u r e i n s a l t - a d a p t e d ducks. S t a l l o n e and Braun (1986) i n f u s e d AVT a t p h y s i o l o g i c a l l e v e l s i n c h i c k e n s and found no e f f e c t on a r t e r i a l b l o o d p r e s s u r e , s u g g e s t i n g t h a t AVT does not e x e r t systemic vasodepressor a c t i v i t y a t p h y s i o l o g i c a l l e v e l s as does the mammalian a n t i d i u r e t i c hormone, AVP (Robertson, 1977). Doses of AVT a t s u p r a p h y s i o l o g i c a l l e v e l s f a i l e d t o a f f e c t b l o o d p r e s s u r e i n ducks (Gerstberger e t a l . , 1984), w h i l e p h a r m a c o l o g i c a l doses exceeding 100 n g * k g _ 1 were found t o decrease b l o o d p r e s s u r e i n c h i c k e n s (Ames e t a l . , 1971) and i n c r e a s e b l o o d p r e s s u r e i n d e s e r t q u a i l s (Braun and D a n t z l e r , 1974). The study of the i n t e r r e l a t i o n s h i p s between the c a r d i o v a s c u l a r system and AVT i n b i r d s has been hampered by the l i m i t e d knowledge o f n e u r a l mechanisms t h a t r e g u l a t e b l o o d p r e s s u r e . Whether hypotensive e f f e c t o f c h r o n i c s a l t a d a p t a t i o n i n ducks (Szczepanska-Sadowska e t a l . , 1985) can be a t t r i b u t e d t o the p e r i p h e r a l a c t i o n of AVT i s not c l e a r . In c h i c k e n s , both n e u r a l lobe p e p t i d e s , mesotocin (MT) and AVT, have vasodepressor a c t i o n s (Boissonnas, Buttmann, Berde and Konzett, 1961). Whether the v a s o a c t i v e p r o p e r t i e s of AVT and MT i n b i r d s are r e l a t e d t o p e r i p h e r a l r e g u l a t i o n of c a r d i o v a s c u l a r f u n c t i o n i s not known (Koike i n p r e s s ) . In the domestic fowl, acute removal of 10 t o 50% of the b l o o d volume (Nouwen, Decuypere, Kuhn, M i c h e l s , H a l l and Chadwick, 1984; S t a l l o n e and Braun, 1986) and i s o s m o t i c volume expansion ( S t a l l o n e and Braun, 1986) f a i l e d t o i n f l u e n c e AVT 65 l e v e l s . The Pekin duck, however, responded d i f f e r e n t l y . Removal of 10% o f the b l o o d volume, a nonhypotensive hemorrhage, caused plasma AVT l e v e l s t o i n c r e a s e more than 30% (Szczepanska-Sadowska e t a l . , 1985). Removal of 20% of b l o o d volume from glaucous-winged g u l l s caused a t w o - f o l d i n c r e a s e i n P&VT (Hughes, G o l d s t e i n and Thomas, unpublished d a t a ) . Plasma A r g i n i n e v a s o t o c i n (P^VT) • PAVT l e v e l s of glaucous-winged g u l l s were h i g h e r than those r e p o r t e d i n k e l p g u l l s (Gray and Erasmus, unpublished d a t a ) , c h i c k e n s (Arad and Skadhauge, 1984; S t a l l o n e and Braun, 1986) and ducks (Mohring e t a l . , 1980; Gray and Simon, 1983,1987). The P^VT i n k e l p g u l l s was measured u s i n g a c e t o n e - e x t r a c t e d plasma, which i n g l a u c o u s - w i n g e d g u l l s y i e l d e d lower PAVT than Sep-Pak e x t r a c t e d plasma ( F i g . 13). Thus the h i g h e r P^VT recorded i n the p r e s e n t seems reasonable. I t a l s o suggests t h a t these w i l d b i r d s p e c i e s may be e a s i l y t r a u m a t i z e d . The h i g h P^VT p r e s e n t i n non-infused glaucous-winged g u l l s , suggests t h a t AVT i s r e l e a s e d by the neurohypophysis a t a h i g h r a t e i n g u l l s , even i n the absence of s a l i n e s t r e s s . The P^VT o r b i r d s used i n the b l o o d p r e s s u r e experiments was lower than t h a t of OCS g u l l s , although the p r o t o c o l used was s i m i l a r , except the b i r d s were handled l e s s . Simon-Oppermann (per s o n a l communication) has observed an i n c r e a s e i n duck P^VT due t o h a n d l i n g . 66 F i g u r e 13. The R e l a t i o n s h i p between Posm a n d PAVT' u s i n g Sep-Pak e x t r a c t e d plasma i n g u l l s . O N O N - I N F U S E D 2 8 0 3 0 0 3 2 0 3 4 0 P L A S M A O S M O L A L I T Y ( m O s m - k g - 1 ) 68 The plasma of CCS g u l l s was not e x t r a c t e d p r i o r t o RIA. The v a r i o u s procedures f o l l o w e d i n e x t r a c t i n g plasma f o r RIA account f o r some l o s s of hormone, t h e r e f o r e , RIA measurement of immunoreactive AVT i n u n e x t r a c t e d plasma i s d e s i r a b l e . E x t r a c t i o n of g u l l plasma was unnecessary s i n c e the RIA s e n s i t i v i t y and plasma hormone c o n c e n t r a t i o n s were h i g h and changes i n P^VT o b t a i n e d were comparable t o o t h e r s t u d i e s (Skadhauge, 1981). With the l a t e r experiments on OCS g u l l s , the plasma was e x t r a c t e d t o a l l o w comparison of the g u l l data w i t h v a l u e s p u b l i s h e d from s t u d i e s i n which RIA was performed on e x t r a c t e d plasma: b e n t o n i t e e x t r a c t e d (Koike, Pryor and Neldon, 1979); acetone e x t r a c t e d (Mohring e t a l . , 1980; Arnason, R i c e , Chadwick and Skadhauge, 1986) ; Sep-Pak e x t r a c t e d ( S t a l l o n e and Braun, 1986); acetone and Sep-Pak e x t r a c t e d (Nouwen and Kuhn, 1983). O s m o l a l i t y of the ECF compartment has been shown t o be one o f the most s e n s i t i v e c o n t r o l l i n g f a c t o r s f o r AVT r e l e a s e (Koike e t a l . , 1979; Simon-Oppermann e t a l . , 1980; Mohring e t a l . , 1980; Arad and Skadhauge, 1984). S t a l l o n e and Braun (1986) confirmed t h a t Posm ^ s a primary determinant o f AVT s e c r e t i o n by the neurohypophysis of the domestic fowl. The r e l a t i o n s h i p between PAVT a n d p0sm o f glaucous-winged g u l l s i s shown i n F i g . 13. Although the t h r e s h o l d o s m o l a l i t y f o r AVT r e l e a s e i n glaucous-winged g u l l s (282 mOsnrkg""1) i s i n the range (273-296 mOsnrkg" - 1) found i n chickens (Koike e t a l . , 1979; Arnason, R i c e and Skadhauge, 1982; Arad and Skadhauge, 1984; Arad, Arnason, Chadwick and Skadhauge, 1985; S t a l l o n e and Braun, 1986) and ducks 69 (Mohring e t a l . , 1980; Gray and Simon, 1983), the s e n s i t i v i t y f o r AVT r e l e a s e i s h i g h e r . The range o f s e n s i t i v i t y o f r e l e a s e ( i n d i c a t e d by the s l o p e o f the r e g r e s s i o n l i n e ) i n the domestic fowl and duck ranged between 0.23-0.39 p g * m l - 1 plasma per mOsnrkg - 1 plasma. The s l o p e s from glaucous-winged g u l l s and k e l p g u l l s (Gray and Erasmus, unpublished data) were 1.25 and 0.87 p g ' m l - 1 plasma per mOsnrkg - 1 plasma. T h i s suggests t h a t the s e n s i t i v i t y o f r e l e a s e i n marine b i r d s may be g r e a t e r than i n t e r r e s t r i a l b i r d s . There was a weak c o r r e l a t i o n between P^VT a n d P0sm i n t n e combined data of non-infused and h y p o t o n i c a l l y i n f u s e d g u l l s (r 2=0.25) but no c o r r e l a t i o n i n h y p e r t o n i c a l l y i n f u s e d g u l l s (r 2=0.01) ( F i g . 13). T h i s c o n t r a s t s w i t h the o b s e r v a t i o n o f S t a l l o n e and Braun (1986) t h a t P^VT a n d pOsm w e r e more c l o s e l y c o r r e l a t e d (r 2=0.89) i n h y p e r t o n i c a l l y i n f u s e d than h y p o t o n i c a l l y i n f u s e d c h i c k e n s (r 2=0.33). Renal water e x c r e t i o n In the pr e s e n t study, i n f u s i o n o f 5 M NaCl r e s u l t e d i n a marked r i s e i n P^VT' which was a s s o c i a t e d w i t h a r e d u c t i o n i n r e n a l water e x c r e t i o n ( F i g . 8), % sodium reabsorbed and neg a t i v e C H20 (Table 3) , r e s u l t i n g i n i n c r e a s e d U 0 s i n and U / P 0 s m r a t i o . The r i s e i n U 0 s m was g r e a t e r i n the g u l l than the c h i c k e n ( D a n t z l e r , 1966) o r s t a r l i n g (Braun, 1978). S a l t - l o a d e d q u a i l d i d not i n c r e a s e U 0 s : m . I n the s t a r l i n g , U / P 0 s m r a t i o i n c r e a s e d due t o the i n c r e a s e i n u r i n e flow (Braun 1978). In g u l l s , f o l l o w i n g a b r i e f p e r i o d o f d i u r e s i s a f t e r s a l t l o a d i n g , t h e r e was a marked 70 decrease i n u r i n e flow. In CCS g u l l s , 1 h a f t e r s a l t l o a d i n g ( F i g . 8) , GFR r e t u r n e d t o normal, however, r e a b s o r p t i o n o f water remained h i g h up t o 100 minutes post-LOAD. In OCS g u l l s , GFR remained low and the h i g h r e a b s o r p t i o n o f water p e r s i s t e d throughout post-LOAD i n f u s i o n i n OCS g u l l s so t h a t u r i n e flow decreased markedly. The CCS g u l l s c o n t i n u e d t o f i l t e r l a r g e amounts of water and NaCl and r e t r i e v e almost a l l o f i t . The r e n a l t u b u l a r r e a b s o r p t i o n o f sodium decreased d u r i n g the i n f u s i o n o f a s a l t - l o a d as observed i n the q u a i l (Braun and Da n t z l e r , 1974) and ch i c k e n ( D a n t z l e r , 1966). The U/P Na f e r r o c y a n i d e d i d not i n c r e a s e i n the c h i c k e n ( D a n t z l e r 1966) and q u a i l (Braun and D a n t z l e r 1972), but i n c r e a s e d s l i g h t l y from the i n i t i a l l y h i g h v a l u e s o f 18 t o maximum v a l u e s o f 25 i n the s t a r l i n g . The U / P P E G v a l u e s i n the g u l l ( F i g . 10) were a l s o i n i t i a l l y h i g h (16.4) but i n c r e a s e d d r a m a t i c a l l y t o 474 wit h s a l t l o a d i n g . T h i s suggests the water r e a b s o r p t i v e c a p a c i t i e s o f the g u l l kidney g r e a t l y exceed those o f even the d e s e r t q u a i l . U r i c a c i d e x c r e t i o n S i n c e c a t i o n s i n t e r a c t w i t h u r i c a c i d , c a t i o n e x c r e t i o n w i t h u r a t e s i n a v i a n u r i n e may c o n t r i b u t e s i g n i f i c a n t l y t o sodium and potassium e x c r e t i o n . The NaCl i n f u s i o n d i d not appear t o have any e f f e c t on u r i c a c i d e x c r e t i o n . The sm a l l sample s i z e t e s t e d p r o b a b l y accounted f o r the l a c k o f s t a t i s t i c a l s i g n i f i c a n c e i n the d a t a . S i n c e the s o l u b i l i t y o f the a c i d form o f u r a t e i s v e r y low i n water, i t i s v e r y u n l i k e l y t h a t a s i g n i f i c a n t amount o f u r a t e a c t u a l l y e x i s t e d i n t h i s form i n the l i q u i d p o r t i o n o f the u r i n e ( D a n t z l e r , 1978). P r e l i m i n a r y measurements confirmed t h a t the l i q u i d p o r t i o n c o n t a i n e d o n l y 5% o f t o t a l u r i c a c i d . Only the p r e c i p i t a t e d p o r t i o n o f u r e t e r a l u r i n e was a n a l y s e d i n t h i s study. Braun (1978) measured u r i c a c i d i n both l i q u i d and p r e c i p i t a t e d p o r t i o n s of u r e t e r a l u r i n e of s t a r l i n g s i n f u s e d w i t h 2.5% m a n n i t o l . He found u r i c a c i d l e v e l s 10 times g r e a t e r than those i n the p r e s e n t study. U r i n e flow r a t e o f s t a r l i n g s was, however, twenty times lower. When the s t a r l i n g s were ad m i n i s t e r e d 32 mEq'kg - 1 body weight s a l t l o a d , u r i n e flow i n c r e a s e d w i t h u r i c a c i d c o n c e n t r a t i o n s f a l l i n g t o those i n the p r e s e n t study. Thus, the h i g h u r i n e flow r a t e i n the p r e s e n t study probably accounted f o r the low u r i c a c i d c o n c e n t r a t i o n . In the hydrated g u l l s , u r a t e content o f the u r i n e was low w i t h few c a t i o n s trapped i n t h i s p o r t i o n of the u r i n e . Less than 10% o f t o t a l r e n a l Na + e x c r e t i o n was found i n the p r e c i p i t a t e s . T h i s i s i n marked c o n t r a s t t o the s t u d i e s by Braun (1978) i n s t a r l i n g s and Hughes (1972b) i n k i t t i w a k e s , R i s s a t r i d a c t v l a . i n which a t l e a s t 50% of Na + was a s s o c i a t e d w i t h u r i c a c i d . The low u r i c a c i d c o n c e n t r a t i o n c o u l d a l s o be a t t r i b u t e d t o the n u t r i t i o n a l s t a t e of the animal. F r a c t i o n a l e x c r e t i o n of u r i c a c i d - u r a t e s , h i g h e r i n f e d than f a s t e d b i r d s , i s augmented by a h i g h p r o t e i n content of the d i e t (Skadhauge, 1981) . The b i r d s 72 used i n the p r e s e n t study, u n l i k e the s t a r l i n g s used by Braun (1978), were f a s t e d f o r 18 hr p r i o r t o the experiment. Under normal c o n d i t i o n s , u r e t e r a l u r i n e may be r e g u r g i t a t e d i n t o the lower i n t e s t i n e (Wiener 1902), where r e a b s o r p t i o n may occur. When the u r i n e - f e a c e s mixture of c o n s c i o u s , u n f a s t e d , normally hydrated d e s e r t q u a i l entered the lower i n t e s t i n e , 47% water, 62% sodium and 49% potassium was reabsorbed (Anderson and Braun, 1985). They suggested t h a t p o s t - r e n a l m o d i f i c a t i o n i n the lower i n t e s t i n e i s f i n e l y tuned t o the osmotic requirements o f the b i r d . The u r i n e samples i n the p r e s e n t study, were not s u b j e c t e d t o p o s t - r e n a l m o d i f i c a t i o n . Much of the water and sodium e x c r e t e d d u r i n g the i n i t i a l d i u r e s i s i n response t o 5 M NaCl i n f u s i o n , might have been r e t r i e v e d i f the u r i n e had undergone p o s t r e n a l m o d i f i c a t i o n i n the hind-gut. The reabsorbed sodium might have been e x c r e t e d more e f f i c i e n t l y by the s a l t glands thus c o n s e r v i n g water. In s a l t - a d a p t e d glaucous-winged g u l l s , G o l d s t e i n e t a l . (1986) found the e p i t h e l i u m of the lower i n t e s t i n e , though of s m a l l s u r f a c e area, had h i g h sodium and water t r a n s p o r t c a p a c i t i e s . Percentage o f s a l t l o a d e l i m i n a t e d In response t o the 5M NaCl l o a d , s a l t g l a n d NaCl s e c r e t i o n of t h e same c o n c e n t r a t i o n was i n i t i a t e d i n the same l e n g t h of time i n both CCS and OCS g u l l s , i n d i c a t i n g t h e r e was no a n e s t h e t i c i n h i b i t i o n of s a l t g l a n d f u n c t i o n i n CCS g u l l s . S a l t g l a n d NaCl s e c r e t i o n ceased 25 mins a f t e r the s a l t i n f u s i o n , 73 l e a v i n g a major p o r t i o n o f s a l t l o a d (CCS, 58%; OCS, 56%) i n the g u l l s . A t the end of the experiment, Pfl a+ l e v e l s were e l e v a t e d over p r e - l o a d l e v e l s i n OCS g u l l s . In CCS g u l l s , Pfl a+ w a s n o t e l e v a t e d and Hct was lowered probably due t o r e t e n t i o n o f d i l u t e i n f u s a t e d u r i n g the p e r i o d o f a n t i d i u r e s i s . Although about 60% of the s a l t l o a d was r e t a i n e d i n a l l g u l l s , i t i s p u z z l i n g t h a t s a l t g l a n d s e c r e t i o n ceased completely 1 h a f t e r i n f u s i o n o f s a l t l o a d i n CCS and OCS g u l l s . Using e s t i m a t e d ECF volume (Ruch and Hughes 1975) and Posm measurements, i t i s p o s s i b l e t o s p e c u l a t e on the f a t e o f the excess sodium i n the body f l u i d s . In a g u l l weighing 815 g, the ECF volume (38% o f body mass, Ruch and Hughes 1975) would be 310 ml b e f o r e h y d r a t i o n . A f t e r 4 h of 0.02 M NaCl i n f u s i o n , Hct f e l l by 9%. Assuming t h a t t h i s r e f l e c t s an i n c r e a s e i n ECF volume, the ECF volume would be 338 ml. At t h i s time Posm w a s 2 9 5 niOsm-l" 1 so the t o t a l ECF o s m o l a l i t y would have been 99,700 uOsm b e f o r e s a l t l o a d i n g . I n f u s i o n o f 5 M NaCl (0.286 m l * m i n _ 1 f o r 13 mins) would add 18,590 uEq Na + t o the ECF volume. At 25 mins p o s t - l o a d , the t o t a l N a + output by the s a l t glands was 5,915.5 uEq and the output by the kidneys was 1,089 uEq amounting t o a t o t a l o f 7,004 uEq Na + e x c r e t e d , l e a v i n g 11,585.5 uEq Na + s t i l l i n the b i r d . S i n c e NaCl i s i o n i c , t he e q u i v a l e n t osmotic l o a d works out t o be twice t h i s , namely 23,171 uOsm. T h i s added t o 99,750 uOsm a l r e a d y p r e s e n t i n the g u l l , g i v e s a t o t a l of 122,843 uOsm a t 25 mins p o s t - l o a d . 74 The i n f u s i o n o f s a l t l o a d caused a f u r t h e r 9.8% drop i n Hct. U s i n g the p r e v i o u s assumption, the ECF volume was i n c r e a s e d t o 371 ml. C o n s i d e r i n g volume changes, the combined volume o f u r i n e and s a l t s e c r e t i o n was 14.11 ml, w h i l e the t o t a l volume o f f l u i d c o n t a i n i n g s a l t l o a d (3.72 ml) and subsequent 25 mins of i n f u s i o n of h y p o t o n i c s a l i n e (7.15 ml) was 10.87 ml. The volume debt i n c u r r e d (3.24 ml) reduced the ECF volume t o 368 ml. With a t o t a l o f 122,843 uOsm and an estimated ECF volume o f 368 ml, Posm o f the g u l l was p r e d i c t e d t o be 333.9 u O s i r r m l - 1 . At 25 mins p o s t -l o a d , the Posm w a s f o u n d t o be 323 u O s n r m l - 1 . The d i f f e r e n c e , approximately 20% of s a l t l o a d , or 3% t o t a l o s m o l a l i t y c a l c u l a t e d t o be i n the ECF volume was not found t h e r e , s u g g e s t i n g t h a t Na+ might not be d i s t r i b u t e d s o l e l y i n the ECF compartment i n g u l l s . Gray and Simon (1985) and Gray, Kaul, Brummermann and Simon (1987) found t h a t , although 2 2 N a + space was i d e n t i c a l i n both f r e s h water and sea water ducks, the d i s t r i b u t i o n space of 3H i n u i i n , assumed t o remain i n the ECF, was found t o be lower i n sea water ducks. They s p e c u l a t e d t h a t a n o n - e x t r a c e l l u l a r Na + p o o l e x i s t s i n s a l t a c c l i m a t e d ducks, which i s probably the case i n the p r e s e n t study, a c c o u n t i n g f o r the r e t e n t i o n o f s a l t . In summary Glaucous-winged g u l l s responded t o the s a l t l o a d by d e c r e a s i n g Hct, GFR, ERPF and u r i n e flow, i n c r e a s i n g PNa+' PAVT and U / P P E G and i n i t i a t i n g s a l t g l a n d s e c r e t i o n w i t h no change i n mean a r t e r i a l p r e s s u r e . At the end of the experiment, though almost 60% o f s a l t l o a d was r e t a i n e d i n the b i r d s , s a l t g l a n d s e c r e t i o n had ceased and i n OCS g u l l s GFR, ERPF and u r i n e flow showed no r e c o v e r y t o v a l u e s b e f o r e s a l t l o a d i n g . In CCS g u l l s , however, GFR and ERPF r e t u r n e d t o p r e - l o a d l e v e l s a f t e r 60 mins, probably i n response t o the con t i n u e d i n f u s i o n o f hypotonic s a l i n e a f t e r s a l t l o a d i n g . 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